%PDF-1.3 1 0 obj << /Type /Catalog /Outlines 2 0 R /Pages 3 0 R >> endobj 2 0 obj << /Type /Outlines /Count 0 >> endobj 3 0 obj << /Type /Pages /Kids [6 0 R 159 0 R 263 0 R 289 0 R 339 0 R 443 0 R 596 0 R 612 0 R 634 0 R 643 0 R 652 0 R 654 0 R 656 0 R 658 0 R ] /Count 14 /Resources << /ProcSet 4 0 R /Font << /F1 8 0 R /F2 9 0 R /F3 10 0 R /F4 11 0 R /F5 12 0 R >> /XObject << /I1 13 0 R /I2 14 0 R /I3 495 0 R /I4 600 0 R /I5 603 0 R /I6 618 0 R /I7 621 0 R /I8 638 0 R /I9 647 0 R >> >> /MediaBox [0.000 0.000 612.000 792.000] >> endobj 4 0 obj [/PDF /Text /ImageC ] endobj 5 0 obj << /Creator (DOMPDF) /CreationDate (D:20180721142301+00'00') /ModDate (D:20180721142301+00'00') /Title (Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in a Lentiviral Mouse Model of Huntingtons Disease PLOS Currents Huntington Disease) >> endobj 6 0 obj << /Type /Page /Parent 3 0 R /Annots [ 15 0 R 17 0 R 19 0 R 21 0 R 23 0 R 25 0 R 27 0 R 29 0 R 31 0 R 33 0 R 35 0 R 37 0 R 39 0 R 41 0 R 43 0 R 45 0 R 47 0 R 49 0 R 51 0 R 53 0 R 55 0 R 57 0 R 59 0 R 61 0 R 63 0 R 65 0 R 67 0 R 69 0 R 71 0 R 73 0 R 75 0 R 77 0 R 79 0 R 81 0 R 83 0 R 85 0 R 87 0 R 89 0 R 91 0 R 93 0 R 95 0 R 97 0 R 99 0 R 101 0 R 103 0 R 105 0 R 107 0 R 109 0 R 111 0 R 113 0 R 115 0 R 117 0 R 119 0 R 121 0 R 123 0 R 125 0 R 127 0 R 129 0 R 131 0 R 133 0 R 135 0 R 137 0 R 139 0 R 141 0 R 143 0 R 145 0 R 147 0 R 149 0 R 151 0 R 153 0 R 155 0 R 157 0 R ] /Contents 7 0 R >> endobj 7 0 obj << /Length 27725 >> stream q 375.000 0 0 39.000 222.000 738.000 cm /I2 Do Q q 15.000 659.406 577.500 78.594 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Neuronal Atrophy in a Lentiviral Mouse Model of )] TJ ET BT 15.000 668.146 Td /F2 21.0 Tf [(Huntingtons Disease)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 650.140 Td /F3 9.8 Tf [(November 6, 2015)] TJ ET BT 91.635 650.140 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 96.510 650.140 Td /F3 9.8 Tf [(Huntington Disease)] TJ ET BT 26.250 638.299 Td /F1 9.8 Tf [(Jonathan Fox)] TJ ET 0.271 0.267 0.267 rg BT 85.325 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 90.746 638.299 Td /F1 9.8 Tf [(Zhen Lu)] TJ ET 0.271 0.267 0.267 rg BT 126.519 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 131.940 638.299 Td /F1 9.8 Tf [(Lorraine Barrows)] TJ ET 0.271 0.267 0.267 rg BT 26.250 626.394 Td /F1 9.8 Tf [(Fox J, Lu Z, Barrows L. Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in a Lentiviral Mouse )] TJ ET BT 26.250 614.490 Td /F1 9.8 Tf [(Model of Huntingtons Disease. PLOS Currents Huntington Disease. 2015 Nov 6 . Edition 1. doi: )] TJ ET BT 26.250 602.585 Td /F1 9.8 Tf [(10.1371/currents.hd.b966ec2eca8e2d89d2bb4d020be4351e.)] TJ ET q 15.000 28.739 577.500 571.465 re W n 0.271 0.267 0.267 rg BT 26.250 573.482 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 553.528 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is caused by a trinucleotide CAG repeat in the huntingtin gene \(HTT\) that results in expression of a )] TJ ET BT 26.250 541.623 Td /F1 9.8 Tf [(polyglutamine-expanded mutant huntingtin protein \(mHTT\). N-terminal fragments of mHTT accumulate in brain neurons and glia )] TJ ET BT 26.250 529.719 Td /F1 9.8 Tf [(as soluble monomeric and oligomeric species as well as insoluble protein aggregates and drive the disease process. )] TJ ET BT 26.250 517.814 Td /F1 9.8 Tf [(Decreasing mHTT levels in brain provides protection and reversal of disease signs in HD mice making mHTT a prime target for )] TJ ET BT 26.250 505.909 Td /F1 9.8 Tf [(disease modification. There is evidence for aberrant thiol oxidation within mHTT and other proteins in HD models. Based on )] TJ ET BT 26.250 494.004 Td /F1 9.8 Tf [(this, we hypothesized that a specific thiol-disulfide oxidoreductase exists that decreases mHTT levels in cells and provides )] TJ ET BT 26.250 482.100 Td /F1 9.8 Tf [(protection in HD mice. We undertook an in-vitro genetic screen of key thiol-disulfide oxidoreductases then completed secondary )] TJ ET BT 26.250 470.195 Td /F1 9.8 Tf [(screens to identify those with mHTT decreasing properties. Our in-vitro experiments identified thioredoxin 1 and thioredoxin-)] TJ ET BT 26.250 458.290 Td /F1 9.8 Tf [(related transmembrane protein 3 as proteins that decrease soluble mHTT levels in cultured cells. Using a lentiviral mouse )] TJ ET BT 26.250 446.385 Td /F1 9.8 Tf [(model of HD we tested the effect of these proteins in striatum. Both proteins decreased mHTT-induced striatal neuronal atrophy. )] TJ ET BT 26.250 434.481 Td /F1 9.8 Tf [(Findings provide evidence for a role of dysregulated protein-thiol homeostasis in the pathogenesis of HD.)] TJ ET BT 26.250 397.878 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 377.924 Td /F1 9.8 Tf [(Funding was provided by NIH/NINDS R21NS072372. Zhen Lu was supported in part by NIH COBRE 5P20RR015640.)] TJ ET BT 26.250 348.821 Td /F4 12.0 Tf [(Notice of Correction)] TJ ET BT 26.250 328.867 Td /F4 9.8 Tf [(7 January 2016: )] TJ ET BT 102.124 328.867 Td /F1 9.8 Tf [(PLOS Currents -. Correction: Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in )] TJ ET BT 26.250 316.962 Td /F1 9.8 Tf [(a Lentiviral Mouse Model of Huntingtons Disease. PLOS Currents Huntington Disease. 2016 Jan 7 . Edition 1. doi: )] TJ ET BT 26.250 305.058 Td /F1 9.8 Tf [(10.1371/currents.hd.30bdfed3d88457fd605cb8f95fba29b5. )] TJ ET 0.267 0.267 0.267 rg BT 283.182 305.058 Td /F1 9.8 Tf [(View Correction.)] TJ ET 0.271 0.267 0.267 rg BT 26.250 268.455 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 248.501 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is a progressive autosomal dominant neurodegenerative disorder caused by a trinucleotide CAG )] TJ ET BT 26.250 236.596 Td /F1 9.8 Tf [(repeat expansion in exon-1 of the huntingtin gene \()] TJ ET BT 246.298 236.596 Td /F5 9.8 Tf [(HTT)] TJ ET BT 265.252 236.596 Td /F1 9.8 Tf [(\))] TJ ET 0.267 0.267 0.267 rg BT 268.499 238.103 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 273.317 236.596 Td /F1 9.8 Tf [(. Disease onset is typically in early to mid-adult life with a range from )] TJ ET BT 26.250 224.691 Td /F1 9.8 Tf [(childhood to advanced age. Clinical signs of HD include involuntary movements, psychiatric problems and cognitive decline. )] TJ ET BT 26.250 212.787 Td /F1 9.8 Tf [(Motor signs of HD result largely from dysfunction and loss of neurons in striatum and cerebral cortex)] TJ ET 0.267 0.267 0.267 rg BT 458.116 214.294 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 462.935 216.675 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 465.344 214.294 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 470.163 212.787 Td /F1 9.8 Tf [(. Treatments that delay )] TJ ET BT 26.250 200.882 Td /F1 9.8 Tf [(onset or progression of human HD have yet to be developed.)] TJ ET BT 26.250 181.477 Td /F1 9.8 Tf [(Trinucleotide-repeat expansion within the )] TJ ET BT 206.703 181.477 Td /F5 9.8 Tf [(HTT)] TJ ET BT 225.657 181.477 Td /F1 9.8 Tf [( gene results in expression of a polyglutamine-expanded mutant huntingtin )] TJ ET BT 26.250 169.572 Td /F1 9.8 Tf [(protein \(mHTT\). Full-length soluble mHTT undergoes enzymatic cleavage to generate soluble N-terminal mHTT polyglutamine )] TJ ET BT 26.250 157.668 Td /F1 9.8 Tf [(containing fragments)] TJ ET 0.267 0.267 0.267 rg BT 116.749 159.175 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 121.568 161.556 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 123.978 159.175 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 128.796 157.668 Td /F1 9.8 Tf [(. Mutant huntingtin N-terminal fragments exist as monomers, soluble oligomers and larger insoluble )] TJ ET BT 26.250 145.763 Td /F1 9.8 Tf [(aggregates)] TJ ET 0.267 0.267 0.267 rg BT 75.029 147.270 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 79.848 145.763 Td /F1 9.8 Tf [(. Soluble N-terminal mHTT fragments are thought to be the main drivers of disease progression)] TJ ET 0.267 0.267 0.267 rg BT 490.586 147.270 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 495.405 145.763 Td /F1 9.8 Tf [( and mouse )] TJ ET BT 26.250 133.858 Td /F1 9.8 Tf [(models of HD that express these fragments have a rapidly progressive phenotype)] TJ ET 0.267 0.267 0.267 rg BT 378.488 135.365 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 383.307 133.858 Td /F1 9.8 Tf [(. N-terminal mHTT results in HD through )] TJ ET BT 26.250 121.953 Td /F1 9.8 Tf [(accumulation in cells and aberrant interactions with numerous proteins)] TJ ET 0.267 0.267 0.267 rg BT 330.245 123.461 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 335.064 125.842 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 337.473 123.461 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 347.111 121.953 Td /F1 9.8 Tf [( and possibly direct production of reactive oxygen )] TJ ET BT 26.250 110.049 Td /F1 9.8 Tf [(species)] TJ ET 0.267 0.267 0.267 rg BT 59.303 111.556 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 68.940 110.049 Td /F1 9.8 Tf [(. Genetic therapeutic approaches that decrease mHTT levels offer the possibility of inhibiting downstream disease )] TJ ET BT 26.250 98.144 Td /F1 9.8 Tf [(pathways, reversing the disease process, and are a promising approach for future treatment of human HD)] TJ ET 0.267 0.267 0.267 rg BT 483.077 99.651 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 492.714 102.032 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 495.123 99.651 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 504.761 102.032 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 507.170 99.651 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 516.807 102.032 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 519.216 99.651 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 528.854 98.144 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 78.739 Td /F1 9.8 Tf [(We have previously reported that the toxic N-terminal 171 fragment of mHTT is prone to thiol oxidation resulting in the formation )] TJ ET BT 26.250 66.834 Td /F1 9.8 Tf [(of soluble oligomeric mHTT; furthermore, these oligomers are degraded more slowly than monomeric mHTT)] TJ ET 0.267 0.267 0.267 rg BT 492.212 68.342 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 497.031 66.834 Td /F1 9.8 Tf [(. Therefore, thiol )] TJ ET BT 26.250 54.930 Td /F1 9.8 Tf [(oxidation within mHTT may contribute to cellular accumulation and toxicity. There is accumulating evidence for the )] TJ ET BT 26.250 43.025 Td /F1 9.8 Tf [(dysregulation of thiol homeostasis in HD. For example, S-nitrosylation of dynamin-related protein 1, a GTPase that mediates )] TJ ET Q q 15.000 659.406 577.500 78.594 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Neuronal Atrophy in a Lentiviral Mouse Model of )] TJ ET BT 15.000 668.146 Td /F2 21.0 Tf [(Huntingtons Disease)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 650.140 Td /F3 9.8 Tf [(November 6, 2015)] TJ ET BT 91.635 650.140 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 96.510 650.140 Td /F3 9.8 Tf [(Huntington Disease)] TJ ET BT 26.250 638.299 Td /F1 9.8 Tf [(Jonathan Fox)] TJ ET 0.271 0.267 0.267 rg BT 85.325 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 90.746 638.299 Td /F1 9.8 Tf [(Zhen Lu)] TJ ET 0.271 0.267 0.267 rg BT 126.519 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 131.940 638.299 Td /F1 9.8 Tf [(Lorraine Barrows)] TJ ET 0.271 0.267 0.267 rg BT 26.250 626.394 Td /F1 9.8 Tf [(Fox J, Lu Z, Barrows L. Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in a Lentiviral Mouse )] TJ ET BT 26.250 614.490 Td /F1 9.8 Tf [(Model of Huntingtons Disease. PLOS Currents Huntington Disease. 2015 Nov 6 . Edition 1. doi: )] TJ ET BT 26.250 602.585 Td /F1 9.8 Tf [(10.1371/currents.hd.b966ec2eca8e2d89d2bb4d020be4351e.)] TJ ET q 15.000 28.739 577.500 571.465 re W n 0.271 0.267 0.267 rg BT 26.250 573.482 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 553.528 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is caused by a trinucleotide CAG repeat in the huntingtin gene \(HTT\) that results in expression of a )] TJ ET BT 26.250 541.623 Td /F1 9.8 Tf [(polyglutamine-expanded mutant huntingtin protein \(mHTT\). N-terminal fragments of mHTT accumulate in brain neurons and glia )] TJ ET BT 26.250 529.719 Td /F1 9.8 Tf [(as soluble monomeric and oligomeric species as well as insoluble protein aggregates and drive the disease process. )] TJ ET BT 26.250 517.814 Td /F1 9.8 Tf [(Decreasing mHTT levels in brain provides protection and reversal of disease signs in HD mice making mHTT a prime target for )] TJ ET BT 26.250 505.909 Td /F1 9.8 Tf [(disease modification. There is evidence for aberrant thiol oxidation within mHTT and other proteins in HD models. Based on )] TJ ET BT 26.250 494.004 Td /F1 9.8 Tf [(this, we hypothesized that a specific thiol-disulfide oxidoreductase exists that decreases mHTT levels in cells and provides )] TJ ET BT 26.250 482.100 Td /F1 9.8 Tf [(protection in HD mice. We undertook an in-vitro genetic screen of key thiol-disulfide oxidoreductases then completed secondary )] TJ ET BT 26.250 470.195 Td /F1 9.8 Tf [(screens to identify those with mHTT decreasing properties. Our in-vitro experiments identified thioredoxin 1 and thioredoxin-)] TJ ET BT 26.250 458.290 Td /F1 9.8 Tf [(related transmembrane protein 3 as proteins that decrease soluble mHTT levels in cultured cells. Using a lentiviral mouse )] TJ ET BT 26.250 446.385 Td /F1 9.8 Tf [(model of HD we tested the effect of these proteins in striatum. Both proteins decreased mHTT-induced striatal neuronal atrophy. )] TJ ET BT 26.250 434.481 Td /F1 9.8 Tf [(Findings provide evidence for a role of dysregulated protein-thiol homeostasis in the pathogenesis of HD.)] TJ ET BT 26.250 397.878 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 377.924 Td /F1 9.8 Tf [(Funding was provided by NIH/NINDS R21NS072372. Zhen Lu was supported in part by NIH COBRE 5P20RR015640.)] TJ ET BT 26.250 348.821 Td /F4 12.0 Tf [(Notice of Correction)] TJ ET BT 26.250 328.867 Td /F4 9.8 Tf [(7 January 2016: )] TJ ET BT 102.124 328.867 Td /F1 9.8 Tf [(PLOS Currents -. Correction: Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in )] TJ ET BT 26.250 316.962 Td /F1 9.8 Tf [(a Lentiviral Mouse Model of Huntingtons Disease. PLOS Currents Huntington Disease. 2016 Jan 7 . Edition 1. doi: )] TJ ET BT 26.250 305.058 Td /F1 9.8 Tf [(10.1371/currents.hd.30bdfed3d88457fd605cb8f95fba29b5. )] TJ ET 0.267 0.267 0.267 rg BT 283.182 305.058 Td /F1 9.8 Tf [(View Correction.)] TJ ET 0.271 0.267 0.267 rg BT 26.250 268.455 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 248.501 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is a progressive autosomal dominant neurodegenerative disorder caused by a trinucleotide CAG )] TJ ET BT 26.250 236.596 Td /F1 9.8 Tf [(repeat expansion in exon-1 of the huntingtin gene \()] TJ ET BT 246.298 236.596 Td /F5 9.8 Tf [(HTT)] TJ ET BT 265.252 236.596 Td /F1 9.8 Tf [(\))] TJ ET 0.267 0.267 0.267 rg BT 268.499 238.103 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 273.317 236.596 Td /F1 9.8 Tf [(. Disease onset is typically in early to mid-adult life with a range from )] TJ ET BT 26.250 224.691 Td /F1 9.8 Tf [(childhood to advanced age. Clinical signs of HD include involuntary movements, psychiatric problems and cognitive decline. )] TJ ET BT 26.250 212.787 Td /F1 9.8 Tf [(Motor signs of HD result largely from dysfunction and loss of neurons in striatum and cerebral cortex)] TJ ET 0.267 0.267 0.267 rg BT 458.116 214.294 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 462.935 216.675 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 465.344 214.294 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 470.163 212.787 Td /F1 9.8 Tf [(. Treatments that delay )] TJ ET BT 26.250 200.882 Td /F1 9.8 Tf [(onset or progression of human HD have yet to be developed.)] TJ ET BT 26.250 181.477 Td /F1 9.8 Tf [(Trinucleotide-repeat expansion within the )] TJ ET BT 206.703 181.477 Td /F5 9.8 Tf [(HTT)] TJ ET BT 225.657 181.477 Td /F1 9.8 Tf [( gene results in expression of a polyglutamine-expanded mutant huntingtin )] TJ ET BT 26.250 169.572 Td /F1 9.8 Tf [(protein \(mHTT\). Full-length soluble mHTT undergoes enzymatic cleavage to generate soluble N-terminal mHTT polyglutamine )] TJ ET BT 26.250 157.668 Td /F1 9.8 Tf [(containing fragments)] TJ ET 0.267 0.267 0.267 rg BT 116.749 159.175 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 121.568 161.556 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 123.978 159.175 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 128.796 157.668 Td /F1 9.8 Tf [(. Mutant huntingtin N-terminal fragments exist as monomers, soluble oligomers and larger insoluble )] TJ ET BT 26.250 145.763 Td /F1 9.8 Tf [(aggregates)] TJ ET 0.267 0.267 0.267 rg BT 75.029 147.270 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 79.848 145.763 Td /F1 9.8 Tf [(. Soluble N-terminal mHTT fragments are thought to be the main drivers of disease progression)] TJ ET 0.267 0.267 0.267 rg BT 490.586 147.270 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 495.405 145.763 Td /F1 9.8 Tf [( and mouse )] TJ ET BT 26.250 133.858 Td /F1 9.8 Tf [(models of HD that express these fragments have a rapidly progressive phenotype)] TJ ET 0.267 0.267 0.267 rg BT 378.488 135.365 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 383.307 133.858 Td /F1 9.8 Tf [(. N-terminal mHTT results in HD through )] TJ ET BT 26.250 121.953 Td /F1 9.8 Tf [(accumulation in cells and aberrant interactions with numerous proteins)] TJ ET 0.267 0.267 0.267 rg BT 330.245 123.461 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 335.064 125.842 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 337.473 123.461 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 347.111 121.953 Td /F1 9.8 Tf [( and possibly direct production of reactive oxygen )] TJ ET BT 26.250 110.049 Td /F1 9.8 Tf [(species)] TJ ET 0.267 0.267 0.267 rg BT 59.303 111.556 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 68.940 110.049 Td /F1 9.8 Tf [(. Genetic therapeutic approaches that decrease mHTT levels offer the possibility of inhibiting downstream disease )] TJ ET BT 26.250 98.144 Td /F1 9.8 Tf [(pathways, reversing the disease process, and are a promising approach for future treatment of human HD)] TJ ET 0.267 0.267 0.267 rg BT 483.077 99.651 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 492.714 102.032 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 495.123 99.651 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 504.761 102.032 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 507.170 99.651 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 516.807 102.032 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 519.216 99.651 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 528.854 98.144 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 78.739 Td /F1 9.8 Tf [(We have previously reported that the toxic N-terminal 171 fragment of mHTT is prone to thiol oxidation resulting in the formation )] TJ ET BT 26.250 66.834 Td /F1 9.8 Tf [(of soluble oligomeric mHTT; furthermore, these oligomers are degraded more slowly than monomeric mHTT)] TJ ET 0.267 0.267 0.267 rg BT 492.212 68.342 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 497.031 66.834 Td /F1 9.8 Tf [(. Therefore, thiol )] TJ ET BT 26.250 54.930 Td /F1 9.8 Tf [(oxidation within mHTT may contribute to cellular accumulation and toxicity. There is accumulating evidence for the )] TJ ET BT 26.250 43.025 Td /F1 9.8 Tf [(dysregulation of thiol homeostasis in HD. For example, S-nitrosylation of dynamin-related protein 1, a GTPase that mediates )] TJ ET Q q 15.000 659.406 577.500 78.594 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Neuronal Atrophy in a Lentiviral Mouse Model of )] TJ ET BT 15.000 668.146 Td /F2 21.0 Tf [(Huntingtons Disease)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 650.140 Td /F3 9.8 Tf [(November 6, 2015)] TJ ET BT 91.635 650.140 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 96.510 650.140 Td /F3 9.8 Tf [(Huntington Disease)] TJ ET BT 26.250 638.299 Td /F1 9.8 Tf [(Jonathan Fox)] TJ ET 0.271 0.267 0.267 rg BT 85.325 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 90.746 638.299 Td /F1 9.8 Tf [(Zhen Lu)] TJ ET 0.271 0.267 0.267 rg BT 126.519 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 131.940 638.299 Td /F1 9.8 Tf [(Lorraine Barrows)] TJ ET 0.271 0.267 0.267 rg BT 26.250 626.394 Td /F1 9.8 Tf [(Fox J, Lu Z, Barrows L. Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in a Lentiviral Mouse )] TJ ET BT 26.250 614.490 Td /F1 9.8 Tf [(Model of Huntingtons Disease. PLOS Currents Huntington Disease. 2015 Nov 6 . Edition 1. doi: )] TJ ET BT 26.250 602.585 Td /F1 9.8 Tf [(10.1371/currents.hd.b966ec2eca8e2d89d2bb4d020be4351e.)] TJ ET q 15.000 28.739 577.500 571.465 re W n 0.271 0.267 0.267 rg BT 26.250 573.482 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 553.528 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is caused by a trinucleotide CAG repeat in the huntingtin gene \(HTT\) that results in expression of a )] TJ ET BT 26.250 541.623 Td /F1 9.8 Tf [(polyglutamine-expanded mutant huntingtin protein \(mHTT\). N-terminal fragments of mHTT accumulate in brain neurons and glia )] TJ ET BT 26.250 529.719 Td /F1 9.8 Tf [(as soluble monomeric and oligomeric species as well as insoluble protein aggregates and drive the disease process. )] TJ ET BT 26.250 517.814 Td /F1 9.8 Tf [(Decreasing mHTT levels in brain provides protection and reversal of disease signs in HD mice making mHTT a prime target for )] TJ ET BT 26.250 505.909 Td /F1 9.8 Tf [(disease modification. There is evidence for aberrant thiol oxidation within mHTT and other proteins in HD models. Based on )] TJ ET BT 26.250 494.004 Td /F1 9.8 Tf [(this, we hypothesized that a specific thiol-disulfide oxidoreductase exists that decreases mHTT levels in cells and provides )] TJ ET BT 26.250 482.100 Td /F1 9.8 Tf [(protection in HD mice. We undertook an in-vitro genetic screen of key thiol-disulfide oxidoreductases then completed secondary )] TJ ET BT 26.250 470.195 Td /F1 9.8 Tf [(screens to identify those with mHTT decreasing properties. Our in-vitro experiments identified thioredoxin 1 and thioredoxin-)] TJ ET BT 26.250 458.290 Td /F1 9.8 Tf [(related transmembrane protein 3 as proteins that decrease soluble mHTT levels in cultured cells. Using a lentiviral mouse )] TJ ET BT 26.250 446.385 Td /F1 9.8 Tf [(model of HD we tested the effect of these proteins in striatum. Both proteins decreased mHTT-induced striatal neuronal atrophy. )] TJ ET BT 26.250 434.481 Td /F1 9.8 Tf [(Findings provide evidence for a role of dysregulated protein-thiol homeostasis in the pathogenesis of HD.)] TJ ET BT 26.250 397.878 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 377.924 Td /F1 9.8 Tf [(Funding was provided by NIH/NINDS R21NS072372. Zhen Lu was supported in part by NIH COBRE 5P20RR015640.)] TJ ET BT 26.250 348.821 Td /F4 12.0 Tf [(Notice of Correction)] TJ ET BT 26.250 328.867 Td /F4 9.8 Tf [(7 January 2016: )] TJ ET BT 102.124 328.867 Td /F1 9.8 Tf [(PLOS Currents -. Correction: Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in )] TJ ET BT 26.250 316.962 Td /F1 9.8 Tf [(a Lentiviral Mouse Model of Huntingtons Disease. PLOS Currents Huntington Disease. 2016 Jan 7 . Edition 1. doi: )] TJ ET BT 26.250 305.058 Td /F1 9.8 Tf [(10.1371/currents.hd.30bdfed3d88457fd605cb8f95fba29b5. )] TJ ET 0.267 0.267 0.267 rg BT 283.182 305.058 Td /F1 9.8 Tf [(View Correction.)] TJ ET 0.271 0.267 0.267 rg BT 26.250 268.455 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 248.501 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is a progressive autosomal dominant neurodegenerative disorder caused by a trinucleotide CAG )] TJ ET BT 26.250 236.596 Td /F1 9.8 Tf [(repeat expansion in exon-1 of the huntingtin gene \()] TJ ET BT 246.298 236.596 Td /F5 9.8 Tf [(HTT)] TJ ET BT 265.252 236.596 Td /F1 9.8 Tf [(\))] TJ ET 0.267 0.267 0.267 rg BT 268.499 238.103 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 273.317 236.596 Td /F1 9.8 Tf [(. Disease onset is typically in early to mid-adult life with a range from )] TJ ET BT 26.250 224.691 Td /F1 9.8 Tf [(childhood to advanced age. Clinical signs of HD include involuntary movements, psychiatric problems and cognitive decline. )] TJ ET BT 26.250 212.787 Td /F1 9.8 Tf [(Motor signs of HD result largely from dysfunction and loss of neurons in striatum and cerebral cortex)] TJ ET 0.267 0.267 0.267 rg BT 458.116 214.294 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 462.935 216.675 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 465.344 214.294 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 470.163 212.787 Td /F1 9.8 Tf [(. Treatments that delay )] TJ ET BT 26.250 200.882 Td /F1 9.8 Tf [(onset or progression of human HD have yet to be developed.)] TJ ET BT 26.250 181.477 Td /F1 9.8 Tf [(Trinucleotide-repeat expansion within the )] TJ ET BT 206.703 181.477 Td /F5 9.8 Tf [(HTT)] TJ ET BT 225.657 181.477 Td /F1 9.8 Tf [( gene results in expression of a polyglutamine-expanded mutant huntingtin )] TJ ET BT 26.250 169.572 Td /F1 9.8 Tf [(protein \(mHTT\). Full-length soluble mHTT undergoes enzymatic cleavage to generate soluble N-terminal mHTT polyglutamine )] TJ ET BT 26.250 157.668 Td /F1 9.8 Tf [(containing fragments)] TJ ET 0.267 0.267 0.267 rg BT 116.749 159.175 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 121.568 161.556 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 123.978 159.175 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 128.796 157.668 Td /F1 9.8 Tf [(. Mutant huntingtin N-terminal fragments exist as monomers, soluble oligomers and larger insoluble )] TJ ET BT 26.250 145.763 Td /F1 9.8 Tf [(aggregates)] TJ ET 0.267 0.267 0.267 rg BT 75.029 147.270 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 79.848 145.763 Td /F1 9.8 Tf [(. Soluble N-terminal mHTT fragments are thought to be the main drivers of disease progression)] TJ ET 0.267 0.267 0.267 rg BT 490.586 147.270 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 495.405 145.763 Td /F1 9.8 Tf [( and mouse )] TJ ET BT 26.250 133.858 Td /F1 9.8 Tf [(models of HD that express these fragments have a rapidly progressive phenotype)] TJ ET 0.267 0.267 0.267 rg BT 378.488 135.365 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 383.307 133.858 Td /F1 9.8 Tf [(. N-terminal mHTT results in HD through )] TJ ET BT 26.250 121.953 Td /F1 9.8 Tf [(accumulation in cells and aberrant interactions with numerous proteins)] TJ ET 0.267 0.267 0.267 rg BT 330.245 123.461 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 335.064 125.842 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 337.473 123.461 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 347.111 121.953 Td /F1 9.8 Tf [( and possibly direct production of reactive oxygen )] TJ ET BT 26.250 110.049 Td /F1 9.8 Tf [(species)] TJ ET 0.267 0.267 0.267 rg BT 59.303 111.556 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 68.940 110.049 Td /F1 9.8 Tf [(. Genetic therapeutic approaches that decrease mHTT levels offer the possibility of inhibiting downstream disease )] TJ ET BT 26.250 98.144 Td /F1 9.8 Tf [(pathways, reversing the disease process, and are a promising approach for future treatment of human HD)] TJ ET 0.267 0.267 0.267 rg BT 483.077 99.651 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 492.714 102.032 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 495.123 99.651 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 504.761 102.032 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 507.170 99.651 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 516.807 102.032 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 519.216 99.651 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 528.854 98.144 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 78.739 Td /F1 9.8 Tf [(We have previously reported that the toxic N-terminal 171 fragment of mHTT is prone to thiol oxidation resulting in the formation )] TJ ET BT 26.250 66.834 Td /F1 9.8 Tf [(of soluble oligomeric mHTT; furthermore, these oligomers are degraded more slowly than monomeric mHTT)] TJ ET 0.267 0.267 0.267 rg BT 492.212 68.342 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 497.031 66.834 Td /F1 9.8 Tf [(. Therefore, thiol )] TJ ET BT 26.250 54.930 Td /F1 9.8 Tf [(oxidation within mHTT may contribute to cellular accumulation and toxicity. There is accumulating evidence for the )] TJ ET BT 26.250 43.025 Td /F1 9.8 Tf [(dysregulation of thiol homeostasis in HD. 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stream 0.271 0.267 0.267 rg q 15.000 28.182 577.500 748.818 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(mitochondria fission, has been shown to promote degeneration in HD models)] TJ ET 0.267 0.267 0.267 rg BT 358.978 768.983 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 368.616 767.476 Td /F1 9.8 Tf [(. Another study in cultured HD cells showed )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(increased thiol oxidation of peroxiredoxin 1, a protein involved in removal of hydrogen and lipid peroxides)] TJ ET 0.267 0.267 0.267 rg BT 478.748 757.079 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 488.385 755.571 Td /F1 9.8 Tf [(. Furthermore, there )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(are increased levels of copper and iron in mouse HD brain, which in unbound form, can promote thiol oxidation)] TJ ET 0.267 0.267 0.267 rg BT 503.123 745.174 Td /F4 8.7 Tf [(18)] TJ ET BT 512.760 745.174 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 522.397 747.555 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 524.807 745.174 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 534.444 743.667 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(Thiol-disulfide oxidoreductase enzymes mediate protein repair and folding processes. They share a common C-X-X-C catalytic )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(sequence within a thioredoxin-type domain that is required for enzymatic activity and they reduce disulfides by forming a )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(catalytic-site disulfide which is then reduced by an external electron donor)] TJ ET 0.267 0.267 0.267 rg BT 344.344 701.960 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 353.981 700.452 Td /F1 9.8 Tf [(. These enzymes differ in cell location, protein )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(substrates and mechanism of reactivation. Thioredoxins are small thiol-disulfide oxidoreductases involved in repair of oxidatively )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(damaged thiols and redox regulation of cell-signaling pathways via thiol switches)] TJ ET 0.267 0.267 0.267 rg BT 373.603 678.150 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 383.241 680.531 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 385.650 678.150 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 395.288 676.643 Td /F1 9.8 Tf [(. Glutaredoxins have thiol reductase and )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(deglutathioylation activity; they are required for normal mitochondrial function and protection against neurodegenerative )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(processes)] TJ ET 0.267 0.267 0.267 rg BT 70.681 654.341 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 80.318 656.722 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 82.727 654.341 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 92.365 656.722 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 94.774 654.341 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 104.411 652.833 Td /F1 9.8 Tf [(. Protein-disulfide isomerases are another group of enzymes with a thioredoxin domain; they are located in )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(the endoplasmic reticulum \(ER\) and other compartments where they regulate a large number of processes via disulfide )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(exchange reactions)] TJ ET 0.267 0.267 0.267 rg BT 110.792 630.531 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 120.430 629.024 Td /F1 9.8 Tf [(. Collectively, these oxidoreductases regulate protein folding in the cell secretory pathway, modulation of )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(activity within cell signaling pathways, and repair of oxidatively-damaged nuclear, cytoplasmic and mitochondrial proteins )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(\(reviewed)] TJ ET 0.267 0.267 0.267 rg BT 68.507 606.722 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 78.144 609.103 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 80.553 606.722 Td /F4 8.7 Tf [(28)] TJ ET 0.271 0.267 0.267 rg BT 90.191 605.214 Td /F1 9.8 Tf [(\).)] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(Mutant huntingtin undergoes a number of post-translational modifications. Phosphorylation status of serine 13 and 16 within the )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(N-terminus of huntingtin protein is a critical determinant of HD)] TJ ET 0.267 0.267 0.267 rg BT 292.298 575.412 Td /F4 8.7 Tf [(29)] TJ ET 0.271 0.267 0.267 rg BT 301.936 573.905 Td /F1 9.8 Tf [(. Furthermore, acetylation of mHTT lysine 444, down-stream of )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(the glutamine expansion, promotes mHTT clearance by increasing trafficking to the autophagosome)] TJ ET 0.267 0.267 0.267 rg BT 457.063 563.507 Td /F4 8.7 Tf [(30)] TJ ET 0.271 0.267 0.267 rg BT 466.701 562.000 Td /F1 9.8 Tf [(. These post-translational )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(modifications suggest potential therapeutic approaches for modifying HD proximally at the level of mHTT. We have reasoned )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(that there may be a thiol-disulfide oxidoreductase that has protective effects by decreasing levels of N171 mHTT, possibly by )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(direct activity on mHTT protein. We therefore undertook a study to seek a thiol-disulfide oxidoreductase with mHTT lowering )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(effects in HD cells that we could test in HD mice. We found that thioredoxin 1 \(TRX1\) and thioredoxin-related transmembrane )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(protein 3 \(TMX3\) both decreased levels of mHTT in cells but did not find evidence for a direct interaction with mHTT. Using a )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(lentiviral mouse model expressing N171 mHTT)] TJ ET 0.267 0.267 0.267 rg BT 228.884 492.079 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 238.522 490.572 Td /F1 9.8 Tf [(, we found that TRX1 and TMX3 decreased striatal neuronal atrophy. Findings )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(support a modulatory role of TRX1 and TMX3 in these HD model systems.)] TJ ET BT 26.250 442.064 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 422.110 Td /F4 9.8 Tf [(Materials:)] TJ ET BT 71.763 422.110 Td /F1 9.8 Tf [( Mouse anti-huntingtin \(HTT\) \(MAB5374\) was from Chemicon, and mouse monoclonal anti-?-actin antibody \(AC40\) )] TJ ET BT 26.250 410.205 Td /F1 9.8 Tf [(from Sigma. Unless otherwise stated all chemicals were from Sigma.)] TJ ET BT 26.250 390.801 Td /F4 9.8 Tf [(Primary screen for mutant HTT protein lowering thiol-disulfide oxidoreductases:)] TJ ET BT 397.910 390.801 Td /F1 9.8 Tf [( COS-1 cells were grown in DMEM )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(supplemented with 10% fetal bovine serum \(FBS\), 1% L-glutamine and 1% penicillin and streptomycin at 37?C and 5% CO2. )] TJ ET BT 26.250 366.991 Td /F1 9.8 Tf [(For experiments cells were grown in 12-well plates and transfected with plasmid\(s\) using lipofectamine 2000 \(Life Technologies\) )] TJ ET BT 26.250 355.086 Td /F1 9.8 Tf [(and reduced serum medium \(OPTI-MEM-1; Life Technologies\) at 70-80% confluency using standard procedures. N171-40Q )] TJ ET BT 26.250 343.182 Td /F1 9.8 Tf [(was in pcDNA1 vector and those encoding thiol-disulfide oxidoreductases were in pQE-TRiSystem vector \(Qiagen\) and )] TJ ET BT 26.250 331.277 Td /F1 9.8 Tf [(expressed with a polyhistidine tag. Gene accession numbers are: NM_001118890.1, NM_016066.4, NM_006541.4, )] TJ ET BT 26.250 319.372 Td /F1 9.8 Tf [(NM_016417.2, NM_001080476.2, NM_001080516.1, NM_001164478.1, NM_003329.1, NM_012473.3, NM_019022.3, )] TJ ET BT 26.250 307.467 Td /F1 9.8 Tf [(NM_021156.3, NM_005742.3, NM_015051.2, NM_004261.3 and NM_080430.2. For plasmids encoding N171-40Q and the thiol-)] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(disulfide oxidoreductases 830 ng of each plasmid DNA was used. We co-transfected with plasmids encoding N171-40Q and )] TJ ET BT 26.250 283.658 Td /F1 9.8 Tf [(GFP as a control. Cells were lysed 48 hours after transfection and levels of soluble N171-40Q and actin were measured by )] TJ ET BT 26.250 271.753 Td /F1 9.8 Tf [(Western blot analysis \(see below\). Actin normalized values were then determined.)] TJ ET BT 26.250 252.348 Td /F4 9.8 Tf [(Secondary screen for mutant HTT protein lowering thiol-disulfide oxidoreductase:)] TJ ET BT 406.032 252.348 Td /F1 9.8 Tf [( To provide a robust control for each )] TJ ET BT 26.250 240.444 Td /F1 9.8 Tf [(candidate proteins enzymatic activity, we expressed as a control the same protein but with mutation of active-site residues that )] TJ ET BT 26.250 228.539 Td /F1 9.8 Tf [(blocks activity. Thiol-disulfide oxidoreducases have a C-X-X-C active-site and share a common catalytic mechanism. PDIA6 has )] TJ ET BT 26.250 216.634 Td /F1 9.8 Tf [(two C-X-X-C motifs while the others have one. In one study mutation of the N-terminal cysteine within this motif completely )] TJ ET BT 26.250 204.729 Td /F1 9.8 Tf [(blocked enzymatic activity while mutation of the lower cysteine inhibited activity by 90% [32]. Therefore, we replaced the N-)] TJ ET BT 26.250 192.825 Td /F1 9.8 Tf [(terminal active-site cysteine with serine to generate enzymatically inactive control proteins. We generated constructs expressing )] TJ ET BT 26.250 180.920 Td /F1 9.8 Tf [(enzymatically inactive TRX1, TMX3, GLRX1, PDIA6 and FLJ44606 using QuikChange site-directed mutagenesis kit )] TJ ET BT 26.250 169.015 Td /F1 9.8 Tf [(\(Stratagene\) for use as a specific control for each candidate test protein. Constructs were verified by DNA sequencing. COS1 )] TJ ET BT 26.250 157.110 Td /F1 9.8 Tf [(cells were transfected with plasmids encoding active or inactive thiol-disulfide oxidoreductase, and then N171-40Q levels )] TJ ET BT 26.250 145.206 Td /F1 9.8 Tf [(determined by Western blot analysis.)] TJ ET BT 26.250 125.801 Td /F4 9.8 Tf [(Western blot analysis:)] TJ ET BT 129.737 125.801 Td /F1 9.8 Tf [( For cell culture experiments, cells were washed in cold PBS then lysed directly in lysis buffer [20 mM )] TJ ET BT 26.250 113.896 Td /F1 9.8 Tf [(TRIS \(pH 7.4\), 1 mM EDTA, 0.15 M NaCl, 0.1% Triton-x100 and protease inhibitor cocktail]. Thirty g protein samples were )] TJ ET BT 26.250 101.991 Td /F1 9.8 Tf [(resolved by reducing SDS-PAGE. Proteins were transferred to PVDF, blocked with 5% non-fat milk in Tris-buffered saline )] TJ ET BT 26.250 90.087 Td /F1 9.8 Tf [(containing 0.1% Tween \(TBS-T\) at room temperature for 1 hour then incubated with anti-HTT \(MAB5492 1:2000 dilution\) and )] TJ ET BT 26.250 78.182 Td /F1 9.8 Tf [(anti-actin \(AC40 1:2000\) overnight at 4?C. After the primary incubation, membranes were washed 4 times for 10 minutes in )] TJ ET BT 26.250 66.277 Td /F1 9.8 Tf [(TBS-T and incubated with goat polyclonal to mouse IgG HRP \(Abcam, 1:2000 dilution\) at room temperature for an hour. Then )] TJ ET BT 26.250 54.372 Td /F1 9.8 Tf [(membranes were washed again and placed in Western Blotting Luminol Reagent \(Santa Cruz Biotechnology, Inc.\) before )] TJ ET BT 26.250 42.468 Td /F1 9.8 Tf [(imaging with a CP1000 Film Processor \(AGFA\). Image J software \(NIH\) was used to quantify band density. Total HTT protein )] TJ ET Q q 15.000 28.182 577.500 748.818 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(mitochondria fission, has been shown to promote degeneration in HD models)] TJ ET 0.267 0.267 0.267 rg BT 358.978 768.983 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 368.616 767.476 Td /F1 9.8 Tf [(. Another study in cultured HD cells showed )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(increased thiol oxidation of peroxiredoxin 1, a protein involved in removal of hydrogen and lipid peroxides)] TJ ET 0.267 0.267 0.267 rg BT 478.748 757.079 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 488.385 755.571 Td /F1 9.8 Tf [(. Furthermore, there )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(are increased levels of copper and iron in mouse HD brain, which in unbound form, can promote thiol oxidation)] TJ ET 0.267 0.267 0.267 rg BT 503.123 745.174 Td /F4 8.7 Tf [(18)] TJ ET BT 512.760 745.174 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 522.397 747.555 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 524.807 745.174 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 534.444 743.667 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(Thiol-disulfide oxidoreductase enzymes mediate protein repair and folding processes. They share a common C-X-X-C catalytic )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(sequence within a thioredoxin-type domain that is required for enzymatic activity and they reduce disulfides by forming a )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(catalytic-site disulfide which is then reduced by an external electron donor)] TJ ET 0.267 0.267 0.267 rg BT 344.344 701.960 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 353.981 700.452 Td /F1 9.8 Tf [(. These enzymes differ in cell location, protein )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(substrates and mechanism of reactivation. Thioredoxins are small thiol-disulfide oxidoreductases involved in repair of oxidatively )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(damaged thiols and redox regulation of cell-signaling pathways via thiol switches)] TJ ET 0.267 0.267 0.267 rg BT 373.603 678.150 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 383.241 680.531 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 385.650 678.150 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 395.288 676.643 Td /F1 9.8 Tf [(. Glutaredoxins have thiol reductase and )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(deglutathioylation activity; they are required for normal mitochondrial function and protection against neurodegenerative )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(processes)] TJ ET 0.267 0.267 0.267 rg BT 70.681 654.341 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 80.318 656.722 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 82.727 654.341 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 92.365 656.722 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 94.774 654.341 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 104.411 652.833 Td /F1 9.8 Tf [(. Protein-disulfide isomerases are another group of enzymes with a thioredoxin domain; they are located in )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(the endoplasmic reticulum \(ER\) and other compartments where they regulate a large number of processes via disulfide )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(exchange reactions)] TJ ET 0.267 0.267 0.267 rg BT 110.792 630.531 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 120.430 629.024 Td /F1 9.8 Tf [(. Collectively, these oxidoreductases regulate protein folding in the cell secretory pathway, modulation of )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(activity within cell signaling pathways, and repair of oxidatively-damaged nuclear, cytoplasmic and mitochondrial proteins )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(\(reviewed)] TJ ET 0.267 0.267 0.267 rg BT 68.507 606.722 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 78.144 609.103 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 80.553 606.722 Td /F4 8.7 Tf [(28)] TJ ET 0.271 0.267 0.267 rg BT 90.191 605.214 Td /F1 9.8 Tf [(\).)] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(Mutant huntingtin undergoes a number of post-translational modifications. Phosphorylation status of serine 13 and 16 within the )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(N-terminus of huntingtin protein is a critical determinant of HD)] TJ ET 0.267 0.267 0.267 rg BT 292.298 575.412 Td /F4 8.7 Tf [(29)] TJ ET 0.271 0.267 0.267 rg BT 301.936 573.905 Td /F1 9.8 Tf [(. Furthermore, acetylation of mHTT lysine 444, down-stream of )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(the glutamine expansion, promotes mHTT clearance by increasing trafficking to the autophagosome)] TJ ET 0.267 0.267 0.267 rg BT 457.063 563.507 Td /F4 8.7 Tf [(30)] TJ ET 0.271 0.267 0.267 rg BT 466.701 562.000 Td /F1 9.8 Tf [(. These post-translational )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(modifications suggest potential therapeutic approaches for modifying HD proximally at the level of mHTT. We have reasoned )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(that there may be a thiol-disulfide oxidoreductase that has protective effects by decreasing levels of N171 mHTT, possibly by )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(direct activity on mHTT protein. We therefore undertook a study to seek a thiol-disulfide oxidoreductase with mHTT lowering )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(effects in HD cells that we could test in HD mice. We found that thioredoxin 1 \(TRX1\) and thioredoxin-related transmembrane )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(protein 3 \(TMX3\) both decreased levels of mHTT in cells but did not find evidence for a direct interaction with mHTT. Using a )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(lentiviral mouse model expressing N171 mHTT)] TJ ET 0.267 0.267 0.267 rg BT 228.884 492.079 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 238.522 490.572 Td /F1 9.8 Tf [(, we found that TRX1 and TMX3 decreased striatal neuronal atrophy. Findings )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(support a modulatory role of TRX1 and TMX3 in these HD model systems.)] TJ ET BT 26.250 442.064 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 422.110 Td /F4 9.8 Tf [(Materials:)] TJ ET BT 71.763 422.110 Td /F1 9.8 Tf [( Mouse anti-huntingtin \(HTT\) \(MAB5374\) was from Chemicon, and mouse monoclonal anti-?-actin antibody \(AC40\) )] TJ ET BT 26.250 410.205 Td /F1 9.8 Tf [(from Sigma. Unless otherwise stated all chemicals were from Sigma.)] TJ ET BT 26.250 390.801 Td /F4 9.8 Tf [(Primary screen for mutant HTT protein lowering thiol-disulfide oxidoreductases:)] TJ ET BT 397.910 390.801 Td /F1 9.8 Tf [( COS-1 cells were grown in DMEM )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(supplemented with 10% fetal bovine serum \(FBS\), 1% L-glutamine and 1% penicillin and streptomycin at 37?C and 5% CO2. )] TJ ET BT 26.250 366.991 Td /F1 9.8 Tf [(For experiments cells were grown in 12-well plates and transfected with plasmid\(s\) using lipofectamine 2000 \(Life Technologies\) )] TJ ET BT 26.250 355.086 Td /F1 9.8 Tf [(and reduced serum medium \(OPTI-MEM-1; Life Technologies\) at 70-80% confluency using standard procedures. N171-40Q )] TJ ET BT 26.250 343.182 Td /F1 9.8 Tf [(was in pcDNA1 vector and those encoding thiol-disulfide oxidoreductases were in pQE-TRiSystem vector \(Qiagen\) and )] TJ ET BT 26.250 331.277 Td /F1 9.8 Tf [(expressed with a polyhistidine tag. Gene accession numbers are: NM_001118890.1, NM_016066.4, NM_006541.4, )] TJ ET BT 26.250 319.372 Td /F1 9.8 Tf [(NM_016417.2, NM_001080476.2, NM_001080516.1, NM_001164478.1, NM_003329.1, NM_012473.3, NM_019022.3, )] TJ ET BT 26.250 307.467 Td /F1 9.8 Tf [(NM_021156.3, NM_005742.3, NM_015051.2, NM_004261.3 and NM_080430.2. For plasmids encoding N171-40Q and the thiol-)] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(disulfide oxidoreductases 830 ng of each plasmid DNA was used. We co-transfected with plasmids encoding N171-40Q and )] TJ ET BT 26.250 283.658 Td /F1 9.8 Tf [(GFP as a control. Cells were lysed 48 hours after transfection and levels of soluble N171-40Q and actin were measured by )] TJ ET BT 26.250 271.753 Td /F1 9.8 Tf [(Western blot analysis \(see below\). Actin normalized values were then determined.)] TJ ET BT 26.250 252.348 Td /F4 9.8 Tf [(Secondary screen for mutant HTT protein lowering thiol-disulfide oxidoreductase:)] TJ ET BT 406.032 252.348 Td /F1 9.8 Tf [( To provide a robust control for each )] TJ ET BT 26.250 240.444 Td /F1 9.8 Tf [(candidate proteins enzymatic activity, we expressed as a control the same protein but with mutation of active-site residues that )] TJ ET BT 26.250 228.539 Td /F1 9.8 Tf [(blocks activity. Thiol-disulfide oxidoreducases have a C-X-X-C active-site and share a common catalytic mechanism. PDIA6 has )] TJ ET BT 26.250 216.634 Td /F1 9.8 Tf [(two C-X-X-C motifs while the others have one. In one study mutation of the N-terminal cysteine within this motif completely )] TJ ET BT 26.250 204.729 Td /F1 9.8 Tf [(blocked enzymatic activity while mutation of the lower cysteine inhibited activity by 90% [32]. Therefore, we replaced the N-)] TJ ET BT 26.250 192.825 Td /F1 9.8 Tf [(terminal active-site cysteine with serine to generate enzymatically inactive control proteins. We generated constructs expressing )] TJ ET BT 26.250 180.920 Td /F1 9.8 Tf [(enzymatically inactive TRX1, TMX3, GLRX1, PDIA6 and FLJ44606 using QuikChange site-directed mutagenesis kit )] TJ ET BT 26.250 169.015 Td /F1 9.8 Tf [(\(Stratagene\) for use as a specific control for each candidate test protein. Constructs were verified by DNA sequencing. COS1 )] TJ ET BT 26.250 157.110 Td /F1 9.8 Tf [(cells were transfected with plasmids encoding active or inactive thiol-disulfide oxidoreductase, and then N171-40Q levels )] TJ ET BT 26.250 145.206 Td /F1 9.8 Tf [(determined by Western blot analysis.)] TJ ET BT 26.250 125.801 Td /F4 9.8 Tf [(Western blot analysis:)] TJ ET BT 129.737 125.801 Td /F1 9.8 Tf [( For cell culture experiments, cells were washed in cold PBS then lysed directly in lysis buffer [20 mM )] TJ ET BT 26.250 113.896 Td /F1 9.8 Tf [(TRIS \(pH 7.4\), 1 mM EDTA, 0.15 M NaCl, 0.1% Triton-x100 and protease inhibitor cocktail]. Thirty g protein samples were )] TJ ET BT 26.250 101.991 Td /F1 9.8 Tf [(resolved by reducing SDS-PAGE. Proteins were transferred to PVDF, blocked with 5% non-fat milk in Tris-buffered saline )] TJ ET BT 26.250 90.087 Td /F1 9.8 Tf [(containing 0.1% Tween \(TBS-T\) at room temperature for 1 hour then incubated with anti-HTT \(MAB5492 1:2000 dilution\) and )] TJ ET BT 26.250 78.182 Td /F1 9.8 Tf [(anti-actin \(AC40 1:2000\) overnight at 4?C. After the primary incubation, membranes were washed 4 times for 10 minutes in )] TJ ET BT 26.250 66.277 Td /F1 9.8 Tf [(TBS-T and incubated with goat polyclonal to mouse IgG HRP \(Abcam, 1:2000 dilution\) at room temperature for an hour. Then )] TJ ET BT 26.250 54.372 Td /F1 9.8 Tf [(membranes were washed again and placed in Western Blotting Luminol Reagent \(Santa Cruz Biotechnology, Inc.\) before )] TJ ET BT 26.250 42.468 Td /F1 9.8 Tf [(imaging with a CP1000 Film Processor \(AGFA\). Image J software \(NIH\) was used to quantify band density. Total HTT protein )] TJ ET Q q 15.000 28.182 577.500 748.818 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(mitochondria fission, has been shown to promote degeneration in HD models)] TJ ET 0.267 0.267 0.267 rg BT 358.978 768.983 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 368.616 767.476 Td /F1 9.8 Tf [(. Another study in cultured HD cells showed )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(increased thiol oxidation of peroxiredoxin 1, a protein involved in removal of hydrogen and lipid peroxides)] TJ ET 0.267 0.267 0.267 rg BT 478.748 757.079 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 488.385 755.571 Td /F1 9.8 Tf [(. Furthermore, there )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(are increased levels of copper and iron in mouse HD brain, which in unbound form, can promote thiol oxidation)] TJ ET 0.267 0.267 0.267 rg BT 503.123 745.174 Td /F4 8.7 Tf [(18)] TJ ET BT 512.760 745.174 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 522.397 747.555 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 524.807 745.174 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 534.444 743.667 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(Thiol-disulfide oxidoreductase enzymes mediate protein repair and folding processes. They share a common C-X-X-C catalytic )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(sequence within a thioredoxin-type domain that is required for enzymatic activity and they reduce disulfides by forming a )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(catalytic-site disulfide which is then reduced by an external electron donor)] TJ ET 0.267 0.267 0.267 rg BT 344.344 701.960 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 353.981 700.452 Td /F1 9.8 Tf [(. These enzymes differ in cell location, protein )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(substrates and mechanism of reactivation. Thioredoxins are small thiol-disulfide oxidoreductases involved in repair of oxidatively )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(damaged thiols and redox regulation of cell-signaling pathways via thiol switches)] TJ ET 0.267 0.267 0.267 rg BT 373.603 678.150 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 383.241 680.531 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 385.650 678.150 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 395.288 676.643 Td /F1 9.8 Tf [(. Glutaredoxins have thiol reductase and )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(deglutathioylation activity; they are required for normal mitochondrial function and protection against neurodegenerative )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(processes)] TJ ET 0.267 0.267 0.267 rg BT 70.681 654.341 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 80.318 656.722 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 82.727 654.341 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 92.365 656.722 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 94.774 654.341 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 104.411 652.833 Td /F1 9.8 Tf [(. Protein-disulfide isomerases are another group of enzymes with a thioredoxin domain; they are located in )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(the endoplasmic reticulum \(ER\) and other compartments where they regulate a large number of processes via disulfide )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(exchange reactions)] TJ ET 0.267 0.267 0.267 rg BT 110.792 630.531 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 120.430 629.024 Td /F1 9.8 Tf [(. Collectively, these oxidoreductases regulate protein folding in the cell secretory pathway, modulation of )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(activity within cell signaling pathways, and repair of oxidatively-damaged nuclear, cytoplasmic and mitochondrial proteins )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(\(reviewed)] TJ ET 0.267 0.267 0.267 rg BT 68.507 606.722 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 78.144 609.103 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 80.553 606.722 Td /F4 8.7 Tf [(28)] TJ ET 0.271 0.267 0.267 rg BT 90.191 605.214 Td /F1 9.8 Tf [(\).)] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(Mutant huntingtin undergoes a number of post-translational modifications. Phosphorylation status of serine 13 and 16 within the )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(N-terminus of huntingtin protein is a critical determinant of HD)] TJ ET 0.267 0.267 0.267 rg BT 292.298 575.412 Td /F4 8.7 Tf [(29)] TJ ET 0.271 0.267 0.267 rg BT 301.936 573.905 Td /F1 9.8 Tf [(. Furthermore, acetylation of mHTT lysine 444, down-stream of )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(the glutamine expansion, promotes mHTT clearance by increasing trafficking to the autophagosome)] TJ ET 0.267 0.267 0.267 rg BT 457.063 563.507 Td /F4 8.7 Tf [(30)] TJ ET 0.271 0.267 0.267 rg BT 466.701 562.000 Td /F1 9.8 Tf [(. These post-translational )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(modifications suggest potential therapeutic approaches for modifying HD proximally at the level of mHTT. We have reasoned )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(that there may be a thiol-disulfide oxidoreductase that has protective effects by decreasing levels of N171 mHTT, possibly by )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(direct activity on mHTT protein. We therefore undertook a study to seek a thiol-disulfide oxidoreductase with mHTT lowering )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(effects in HD cells that we could test in HD mice. We found that thioredoxin 1 \(TRX1\) and thioredoxin-related transmembrane )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(protein 3 \(TMX3\) both decreased levels of mHTT in cells but did not find evidence for a direct interaction with mHTT. Using a )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(lentiviral mouse model expressing N171 mHTT)] TJ ET 0.267 0.267 0.267 rg BT 228.884 492.079 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 238.522 490.572 Td /F1 9.8 Tf [(, we found that TRX1 and TMX3 decreased striatal neuronal atrophy. Findings )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(support a modulatory role of TRX1 and TMX3 in these HD model systems.)] TJ ET BT 26.250 442.064 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 422.110 Td /F4 9.8 Tf [(Materials:)] TJ ET BT 71.763 422.110 Td /F1 9.8 Tf [( Mouse anti-huntingtin \(HTT\) \(MAB5374\) was from Chemicon, and mouse monoclonal anti-?-actin antibody \(AC40\) )] TJ ET BT 26.250 410.205 Td /F1 9.8 Tf [(from Sigma. Unless otherwise stated all chemicals were from Sigma.)] TJ ET BT 26.250 390.801 Td /F4 9.8 Tf [(Primary screen for mutant HTT protein lowering thiol-disulfide oxidoreductases:)] TJ ET BT 397.910 390.801 Td /F1 9.8 Tf [( COS-1 cells were grown in DMEM )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(supplemented with 10% fetal bovine serum \(FBS\), 1% L-glutamine and 1% penicillin and streptomycin at 37?C and 5% CO2. )] TJ ET BT 26.250 366.991 Td /F1 9.8 Tf [(For experiments cells were grown in 12-well plates and transfected with plasmid\(s\) using lipofectamine 2000 \(Life Technologies\) )] TJ ET BT 26.250 355.086 Td /F1 9.8 Tf [(and reduced serum medium \(OPTI-MEM-1; Life Technologies\) at 70-80% confluency using standard procedures. N171-40Q )] TJ ET BT 26.250 343.182 Td /F1 9.8 Tf [(was in pcDNA1 vector and those encoding thiol-disulfide oxidoreductases were in pQE-TRiSystem vector \(Qiagen\) and )] TJ ET BT 26.250 331.277 Td /F1 9.8 Tf [(expressed with a polyhistidine tag. Gene accession numbers are: NM_001118890.1, NM_016066.4, NM_006541.4, )] TJ ET BT 26.250 319.372 Td /F1 9.8 Tf [(NM_016417.2, NM_001080476.2, NM_001080516.1, NM_001164478.1, NM_003329.1, NM_012473.3, NM_019022.3, )] TJ ET BT 26.250 307.467 Td /F1 9.8 Tf [(NM_021156.3, NM_005742.3, NM_015051.2, NM_004261.3 and NM_080430.2. For plasmids encoding N171-40Q and the thiol-)] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(disulfide oxidoreductases 830 ng of each plasmid DNA was used. We co-transfected with plasmids encoding N171-40Q and )] TJ ET BT 26.250 283.658 Td /F1 9.8 Tf [(GFP as a control. Cells were lysed 48 hours after transfection and levels of soluble N171-40Q and actin were measured by )] TJ ET BT 26.250 271.753 Td /F1 9.8 Tf [(Western blot analysis \(see below\). Actin normalized values were then determined.)] TJ ET BT 26.250 252.348 Td /F4 9.8 Tf [(Secondary screen for mutant HTT protein lowering thiol-disulfide oxidoreductase:)] TJ ET BT 406.032 252.348 Td /F1 9.8 Tf [( To provide a robust control for each )] TJ ET BT 26.250 240.444 Td /F1 9.8 Tf [(candidate proteins enzymatic activity, we expressed as a control the same protein but with mutation of active-site residues that )] TJ ET BT 26.250 228.539 Td /F1 9.8 Tf [(blocks activity. Thiol-disulfide oxidoreducases have a C-X-X-C active-site and share a common catalytic mechanism. PDIA6 has )] TJ ET BT 26.250 216.634 Td /F1 9.8 Tf [(two C-X-X-C motifs while the others have one. In one study mutation of the N-terminal cysteine within this motif completely )] TJ ET BT 26.250 204.729 Td /F1 9.8 Tf [(blocked enzymatic activity while mutation of the lower cysteine inhibited activity by 90% [32]. Therefore, we replaced the N-)] TJ ET BT 26.250 192.825 Td /F1 9.8 Tf [(terminal active-site cysteine with serine to generate enzymatically inactive control proteins. We generated constructs expressing )] TJ ET BT 26.250 180.920 Td /F1 9.8 Tf [(enzymatically inactive TRX1, TMX3, GLRX1, PDIA6 and FLJ44606 using QuikChange site-directed mutagenesis kit )] TJ ET BT 26.250 169.015 Td /F1 9.8 Tf [(\(Stratagene\) for use as a specific control for each candidate test protein. Constructs were verified by DNA sequencing. COS1 )] TJ ET BT 26.250 157.110 Td /F1 9.8 Tf [(cells were transfected with plasmids encoding active or inactive thiol-disulfide oxidoreductase, and then N171-40Q levels )] TJ ET BT 26.250 145.206 Td /F1 9.8 Tf [(determined by Western blot analysis.)] TJ ET BT 26.250 125.801 Td /F4 9.8 Tf [(Western blot analysis:)] TJ ET BT 129.737 125.801 Td /F1 9.8 Tf [( For cell culture experiments, cells were washed in cold PBS then lysed directly in lysis buffer [20 mM )] TJ ET BT 26.250 113.896 Td /F1 9.8 Tf [(TRIS \(pH 7.4\), 1 mM EDTA, 0.15 M NaCl, 0.1% Triton-x100 and protease inhibitor cocktail]. Thirty g protein samples were )] TJ ET BT 26.250 101.991 Td /F1 9.8 Tf [(resolved by reducing SDS-PAGE. Proteins were transferred to PVDF, blocked with 5% non-fat milk in Tris-buffered saline )] TJ ET BT 26.250 90.087 Td /F1 9.8 Tf [(containing 0.1% Tween \(TBS-T\) at room temperature for 1 hour then incubated with anti-HTT \(MAB5492 1:2000 dilution\) and )] TJ ET BT 26.250 78.182 Td /F1 9.8 Tf [(anti-actin \(AC40 1:2000\) overnight at 4?C. After the primary incubation, membranes were washed 4 times for 10 minutes in )] TJ ET BT 26.250 66.277 Td /F1 9.8 Tf [(TBS-T and incubated with goat polyclonal to mouse IgG HRP \(Abcam, 1:2000 dilution\) at room temperature for an hour. Then )] TJ ET BT 26.250 54.372 Td /F1 9.8 Tf [(membranes were washed again and placed in Western Blotting Luminol Reagent \(Santa Cruz Biotechnology, Inc.\) before )] TJ ET BT 26.250 42.468 Td /F1 9.8 Tf [(imaging with a CP1000 Film Processor \(AGFA\). Image J software \(NIH\) was used to quantify band density. Total HTT protein )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(2)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 161 0 obj << /Type /Annot /Subtype /Link /A 162 0 R /Border [0 0 0] /H /I /Rect [ 358.9785 768.1817 368.6158 777.0000 ] >> endobj 162 0 obj << /Type /Action >> endobj 163 0 obj << /Type /Annot /Subtype /Link /A 164 0 R /Border [0 0 0] /H /I /Rect [ 478.7475 756.2769 488.3848 765.0952 ] >> endobj 164 0 obj << /Type /Action >> endobj 165 0 obj << /Type /Annot /Subtype /Link /A 166 0 R /Border [0 0 0] /H /I /Rect [ 503.1225 744.3722 512.7598 753.1905 ] >> endobj 166 0 obj << /Type /Action >> endobj 167 0 obj << /Type /Annot /Subtype /Link /A 168 0 R /Border [0 0 0] /H /I /Rect [ 512.7598 744.3722 522.3972 753.1905 ] >> endobj 168 0 obj << /Type /Action >> endobj 169 0 obj << /Type /Annot /Subtype /Link /A 170 0 R /Border [0 0 0] /H /I /Rect [ 524.8065 744.3722 534.4438 753.1905 ] >> endobj 170 0 obj 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Td /F1 9.8 Tf [(levels were determined by the ratios of the values for HTT and ?-actin. For mouse studies, mice were anesthetized and )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(perfused with cold-heparinized 0.9% \(w/v\) saline. Brains were sectioned frontally at a 1 mm interval then two sections were )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(taken at the level of the injection site, striatum dissected and then frozen on dry ice before storing at -80?C. Brains were )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(homogenized in lysis buffer which were then incubated on ice for 5 minutes then centrifuged for 10 minutes at 16 000 x g and )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(4?C. Protein concentrations were determined then Western blot analysis performed as described above.)] TJ ET BT 26.250 700.452 Td /F4 9.8 Tf [(N171 HTT protein TRX1 interaction experiment:)] TJ ET BT 245.128 700.452 Td /F1 9.8 Tf [( We used a previously described approach to test whether N171 mHTT is a )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(direct substrate of TRX1)] TJ ET 0.267 0.267 0.267 rg BT 131.375 690.055 Td /F4 8.7 Tf [(33)] TJ ET 0.271 0.267 0.267 rg BT 141.012 692.436 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 143.421 690.055 Td /F4 8.7 Tf [(34)] TJ ET 0.271 0.267 0.267 rg BT 153.059 688.548 Td /F1 9.8 Tf [(. In brief, a variant of TRX1 with a mutation of the lower cysteine residue within the CXXC active-)] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(site motif is used to trap the catalytic disulfide-linked heterodimer with the substrate protein. Heterodimers can be detected by )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(non-reducing Western blot analysis. DNA encoding N171-40Q, TRX1 and mutant TRX1 \(mTRX1\) was sub-cloned into the )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(bacterial expression vector pGEX-6P-1 \(GE Healthcare\). Proteins were expressed in bacteria, purified using a GST column, )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(then GST removed by cleavage with PreScission Protease \(GE Healthcare\) as previously described)] TJ ET 0.267 0.267 0.267 rg BT 457.034 642.436 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 461.853 640.929 Td /F1 9.8 Tf [( . The purified protein was )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(buffer exchanged into 50 mM Tris \(pH 7.0\) and 150 mM NaCl. N171-40Q was incubated with TXN or mutant TXN at room )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(temperature for one hour; 50 mM N-ethylmaleimide was then added to block free thiols and the samples were then resolved by )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(reducing or non-reducing SDS-PAGE and proteins detected by western blot analysis.)] TJ ET BT 26.250 585.810 Td /F4 9.8 Tf [(Mouse husbandry:)] TJ ET BT 112.927 585.810 Td /F1 9.8 Tf [( All procedures and euthanasia methods were approved by the University of Wyoming Institutional Animal )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(Care and Use Committee and were also in accordance with NIH guidelines. We used female B6/C3H F1 mice purchased from )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(the Jackson Laboratory. Mice were maintained under standard conditions of housing and lighting. They were fed a standard )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(cereal-based rodent chow and had ad-libitum access to acidified water \(pH 3-4\).)] TJ ET BT 26.250 530.691 Td /F4 9.8 Tf [(Mouse study experimental design:)] TJ ET BT 186.082 530.691 Td /F1 9.8 Tf [( C57BL/6 x C3H F1 female mice were purchased at 5-6 weeks of age. Pre-treatment )] TJ ET BT 26.250 518.786 Td /F1 9.8 Tf [(wheel analysis was at 7 weeks of age. Surgeries were at ~8 weeks of age and mice were sacrificed 6-weeks later based on the )] TJ ET BT 26.250 506.881 Td /F1 9.8 Tf [(studies of de Almeida et al)] TJ ET 0.267 0.267 0.267 rg BT 140.598 508.388 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 150.235 506.881 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 487.476 Td /F4 9.8 Tf [(Lentiviral synthesis:)] TJ ET BT 120.532 487.476 Td /F1 9.8 Tf [( We utilized a four-plasmid system for generation of lentivirus expressing N171-18Q and N171-82Q )] TJ ET BT 26.250 475.572 Td /F1 9.8 Tf [(\(kindly provided by Dr. Deglon\). We subcloned DNA encoding functionally active and inactive versions of TRX1 and TMX3 from )] TJ ET BT 26.250 463.667 Td /F1 9.8 Tf [(pQE-Tri plasmids into the SIN-pGK which uses the phosphoglycerate kinase promoter to drive gene expression. For each virus, )] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(the four plasmids were transfected in the molar ratio of 1:1:1:3 for pMDG, CMV?8.92, pRSV-Rev and SIN-pGK, respectively. )] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(For each T-150 sized flask we used 3.3, 7.2, 2.2 and 7.2 g plasmid. 293T cells were grown to ~60% confluency in 10% FBS-)] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(DMEM and 2 mM glutamine. They were then transfected using jetPrime transfection reagent \(Polyplus 114-07\) according to the )] TJ ET BT 26.250 416.048 Td /F1 9.8 Tf [(manufacturers instructions. For each T-150 flask we used 1 ml of jetPrime buffer, 20 g combined plasmids, 50 jetPrime )] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(reagent and 36 mls of cell culture medium. Cells were transfected for four hours, washed twice in PBS then the medium )] TJ ET BT 26.250 392.238 Td /F1 9.8 Tf [(replaced. Cells were incubated for a further 72 hours prior to virus purification. Medium was harvested and placed into sterile )] TJ ET BT 26.250 380.334 Td /F1 9.8 Tf [(tubes on wet ice then filtered using a 0.22 m filter. The filtered supernatant was centrifuged in a SW28 swinging bucket rotor )] TJ ET BT 26.250 368.429 Td /F1 9.8 Tf [(on a Beckman L8-80 centrifuge at 141k x g for 2 hours at 4 degrees C. Supernatant was decanted then tubes were inverted for )] TJ ET BT 26.250 356.524 Td /F1 9.8 Tf [(4 minutes. The pellet was re-suspended in 300-500 l of sterile PBS then transferred to a siliconized tube. Samples were then )] TJ ET BT 26.250 344.619 Td /F1 9.8 Tf [(centrifuged at 19000 x g for 60 min at 4 degrees C to concentrate then the pellet resuspended in 50-150 l PBS by gentle )] TJ ET BT 26.250 332.715 Td /F1 9.8 Tf [(pipetting. The samples were then mixed gently overnight using a tilted horizontal shaker at 4 degrees C. Lentiviruses were )] TJ ET BT 26.250 320.810 Td /F1 9.8 Tf [(quantified in duplicate using a p24 ELISA \(Zeptometrix\) according to the manufacturers instructions. Lentiviral samples were )] TJ ET BT 26.250 308.905 Td /F1 9.8 Tf [(diluted to 4.0 ng p24 / l in PBS, stored at 4 degrees C in siliconized tubes and used within 2 weeks of preparation.)] TJ ET BT 26.250 289.500 Td /F4 9.8 Tf [(Stereotaxic surgery:)] TJ ET BT 120.532 289.500 Td /F1 9.8 Tf [( Mice were anesthetized with 1.75 mg ketamine and 0.25 mg xylazine per 20 grams body weight. After )] TJ ET BT 26.250 277.596 Td /F1 9.8 Tf [(mounting in a stereotaxic frame they were injected at the following coordinates: AP =+0.4; DV= -3.9, then pull up to -3.7 before )] TJ ET BT 26.250 265.691 Td /F1 9.8 Tf [(injection; ML= \(all to right\), +2.0 if body weight ?22.5 g, 1.95 if body weight = 20.5-22.4 g, 1.85 if body weight = 19.0-20.4 g, and )] TJ ET BT 26.250 253.786 Td /F1 9.8 Tf [(1.75 if body weight = 17.5-18.9 g. The needle size used was inch, 31 gauge, and with a 30 degree bevel. Virus was delivered )] TJ ET BT 26.250 241.881 Td /F1 9.8 Tf [(with a peristaltic pump at a rate of 200 nl per minute \(2.5 l over 12.5 minutes\). The needle was then left in place an additional )] TJ ET BT 26.250 229.977 Td /F1 9.8 Tf [(10 minutes before slow removal. Each mouse was injected with 10 ng of p24 equivalents of virus. For testing paired viral )] TJ ET BT 26.250 218.072 Td /F1 9.8 Tf [(injections, different combinations of virus were injected; we used 5 ng of p24 equivalents of each virus which were pre-mixed )] TJ ET BT 26.250 206.167 Td /F1 9.8 Tf [(prior to injection.)] TJ ET BT 26.250 186.762 Td /F4 9.8 Tf [(Wheel activity analysis:)] TJ ET BT 135.713 186.762 Td /F1 9.8 Tf [( Spontaneous wheel activity was measured before and after surgery. Mice were placed individually in )] TJ ET BT 26.250 174.858 Td /F1 9.8 Tf [(cages containing running wheels for 4 days, running times in both light and dark cycles \(12 hours each\) were recorded. The first )] TJ ET BT 26.250 162.953 Td /F1 9.8 Tf [(day was used to familiarize mice with the instrument. Data from days 2-4 were used for analysis.)] TJ ET BT 26.250 143.548 Td /F4 9.8 Tf [(Immunofluorescence staining and brain stereology:)] TJ ET BT 266.266 143.548 Td /F1 9.8 Tf [( Mice were sacrificed by an intra-peritoneal overdose of phenytoin and )] TJ ET BT 26.250 131.643 Td /F1 9.8 Tf [(pentobarbital solution. This was followed by a 2 minute perfusion in heparinized 0.9% saline immediately followed by perfusion )] TJ ET BT 26.250 119.739 Td /F1 9.8 Tf [(of 200 mls of freshly prepared 4% paraformaldehyde \(in 0.1 M phosphate buffer, pH 7.5\). Brains were removed 1-4 hours later )] TJ ET BT 26.250 107.834 Td /F1 9.8 Tf [(and immersion fixed for a further 24 hours before being transferred to cryo-preservant \(10% glycerol in 0.1 M phosphate buffer, )] TJ ET BT 26.250 95.929 Td /F1 9.8 Tf [(pH 7.5\). Brains were sectioned frontally at 40 ?m and stored in 0.1 M phosphate buffer \(pH 7.5\) containing 0.05% azide at 4C. )] TJ ET BT 26.250 84.024 Td /F1 9.8 Tf [(Striatal sections at the level of the anterior commissure were incubated with primary anti-huntingtin antibody EM48 \(Chemicon\) )] TJ ET BT 26.250 72.120 Td /F1 9.8 Tf [(at 1:1000 dilution in PBS-0.1% tween-10% goat serum for 3 days at 4?C, then followed by a 24 hour incubation with Alexa-fluor )] TJ ET BT 26.250 60.215 Td /F1 9.8 Tf [(488 labeled secondary antibody at 1:500 dilution \(Life Technologies\). Sections were washed in PBS three times for 10 minutes )] TJ ET BT 26.250 48.310 Td /F1 9.8 Tf [(and stained with fluorescent Nissl \(Life Technologies\) for 1 hour at room temperature \(1:100 dilution in PBS\), followed by )] TJ ET BT 26.250 36.405 Td /F1 9.8 Tf [(washing in PBS twice for 15 minutes and mounting on slides using Fluoromount G \(Southern Biotech\). Slides were air-dried )] TJ ET Q q 15.000 22.120 577.500 754.880 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(levels were determined by the ratios of the values for HTT and ?-actin. For mouse studies, mice were anesthetized and )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(perfused with cold-heparinized 0.9% \(w/v\) saline. Brains were sectioned frontally at a 1 mm interval then two sections were )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(taken at the level of the injection site, striatum dissected and then frozen on dry ice before storing at -80?C. Brains were )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(homogenized in lysis buffer which were then incubated on ice for 5 minutes then centrifuged for 10 minutes at 16 000 x g and )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(4?C. Protein concentrations were determined then Western blot analysis performed as described above.)] TJ ET BT 26.250 700.452 Td /F4 9.8 Tf [(N171 HTT protein TRX1 interaction experiment:)] TJ ET BT 245.128 700.452 Td /F1 9.8 Tf [( We used a previously described approach to test whether N171 mHTT is a )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(direct substrate of TRX1)] TJ ET 0.267 0.267 0.267 rg BT 131.375 690.055 Td /F4 8.7 Tf [(33)] TJ ET 0.271 0.267 0.267 rg BT 141.012 692.436 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 143.421 690.055 Td /F4 8.7 Tf [(34)] TJ ET 0.271 0.267 0.267 rg BT 153.059 688.548 Td /F1 9.8 Tf [(. In brief, a variant of TRX1 with a mutation of the lower cysteine residue within the CXXC active-)] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(site motif is used to trap the catalytic disulfide-linked heterodimer with the substrate protein. Heterodimers can be detected by )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(non-reducing Western blot analysis. DNA encoding N171-40Q, TRX1 and mutant TRX1 \(mTRX1\) was sub-cloned into the )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(bacterial expression vector pGEX-6P-1 \(GE Healthcare\). Proteins were expressed in bacteria, purified using a GST column, )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(then GST removed by cleavage with PreScission Protease \(GE Healthcare\) as previously described)] TJ ET 0.267 0.267 0.267 rg BT 457.034 642.436 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 461.853 640.929 Td /F1 9.8 Tf [( . The purified protein was )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(buffer exchanged into 50 mM Tris \(pH 7.0\) and 150 mM NaCl. N171-40Q was incubated with TXN or mutant TXN at room )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(temperature for one hour; 50 mM N-ethylmaleimide was then added to block free thiols and the samples were then resolved by )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(reducing or non-reducing SDS-PAGE and proteins detected by western blot analysis.)] TJ ET BT 26.250 585.810 Td /F4 9.8 Tf [(Mouse husbandry:)] TJ ET BT 112.927 585.810 Td /F1 9.8 Tf [( All procedures and euthanasia methods were approved by the University of Wyoming Institutional Animal )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(Care and Use Committee and were also in accordance with NIH guidelines. We used female B6/C3H F1 mice purchased from )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(the Jackson Laboratory. Mice were maintained under standard conditions of housing and lighting. They were fed a standard )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(cereal-based rodent chow and had ad-libitum access to acidified water \(pH 3-4\).)] TJ ET BT 26.250 530.691 Td /F4 9.8 Tf [(Mouse study experimental design:)] TJ ET BT 186.082 530.691 Td /F1 9.8 Tf [( C57BL/6 x C3H F1 female mice were purchased at 5-6 weeks of age. Pre-treatment )] TJ ET BT 26.250 518.786 Td /F1 9.8 Tf [(wheel analysis was at 7 weeks of age. Surgeries were at ~8 weeks of age and mice were sacrificed 6-weeks later based on the )] TJ ET BT 26.250 506.881 Td /F1 9.8 Tf [(studies of de Almeida et al)] TJ ET 0.267 0.267 0.267 rg BT 140.598 508.388 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 150.235 506.881 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 487.476 Td /F4 9.8 Tf [(Lentiviral synthesis:)] TJ ET BT 120.532 487.476 Td /F1 9.8 Tf [( We utilized a four-plasmid system for generation of lentivirus expressing N171-18Q and N171-82Q )] TJ ET BT 26.250 475.572 Td /F1 9.8 Tf [(\(kindly provided by Dr. Deglon\). We subcloned DNA encoding functionally active and inactive versions of TRX1 and TMX3 from )] TJ ET BT 26.250 463.667 Td /F1 9.8 Tf [(pQE-Tri plasmids into the SIN-pGK which uses the phosphoglycerate kinase promoter to drive gene expression. For each virus, )] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(the four plasmids were transfected in the molar ratio of 1:1:1:3 for pMDG, CMV?8.92, pRSV-Rev and SIN-pGK, respectively. )] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(For each T-150 sized flask we used 3.3, 7.2, 2.2 and 7.2 g plasmid. 293T cells were grown to ~60% confluency in 10% FBS-)] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(DMEM and 2 mM glutamine. They were then transfected using jetPrime transfection reagent \(Polyplus 114-07\) according to the )] TJ ET BT 26.250 416.048 Td /F1 9.8 Tf [(manufacturers instructions. For each T-150 flask we used 1 ml of jetPrime buffer, 20 g combined plasmids, 50 jetPrime )] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(reagent and 36 mls of cell culture medium. Cells were transfected for four hours, washed twice in PBS then the medium )] TJ ET BT 26.250 392.238 Td /F1 9.8 Tf [(replaced. Cells were incubated for a further 72 hours prior to virus purification. Medium was harvested and placed into sterile )] TJ ET BT 26.250 380.334 Td /F1 9.8 Tf [(tubes on wet ice then filtered using a 0.22 m filter. The filtered supernatant was centrifuged in a SW28 swinging bucket rotor )] TJ ET BT 26.250 368.429 Td /F1 9.8 Tf [(on a Beckman L8-80 centrifuge at 141k x g for 2 hours at 4 degrees C. Supernatant was decanted then tubes were inverted for )] TJ ET BT 26.250 356.524 Td /F1 9.8 Tf [(4 minutes. The pellet was re-suspended in 300-500 l of sterile PBS then transferred to a siliconized tube. Samples were then )] TJ ET BT 26.250 344.619 Td /F1 9.8 Tf [(centrifuged at 19000 x g for 60 min at 4 degrees C to concentrate then the pellet resuspended in 50-150 l PBS by gentle )] TJ ET BT 26.250 332.715 Td /F1 9.8 Tf [(pipetting. The samples were then mixed gently overnight using a tilted horizontal shaker at 4 degrees C. Lentiviruses were )] TJ ET BT 26.250 320.810 Td /F1 9.8 Tf [(quantified in duplicate using a p24 ELISA \(Zeptometrix\) according to the manufacturers instructions. Lentiviral samples were )] TJ ET BT 26.250 308.905 Td /F1 9.8 Tf [(diluted to 4.0 ng p24 / l in PBS, stored at 4 degrees C in siliconized tubes and used within 2 weeks of preparation.)] TJ ET BT 26.250 289.500 Td /F4 9.8 Tf [(Stereotaxic surgery:)] TJ ET BT 120.532 289.500 Td /F1 9.8 Tf [( Mice were anesthetized with 1.75 mg ketamine and 0.25 mg xylazine per 20 grams body weight. After )] TJ ET BT 26.250 277.596 Td /F1 9.8 Tf [(mounting in a stereotaxic frame they were injected at the following coordinates: AP =+0.4; DV= -3.9, then pull up to -3.7 before )] TJ ET BT 26.250 265.691 Td /F1 9.8 Tf [(injection; ML= \(all to right\), +2.0 if body weight ?22.5 g, 1.95 if body weight = 20.5-22.4 g, 1.85 if body weight = 19.0-20.4 g, and )] TJ ET BT 26.250 253.786 Td /F1 9.8 Tf [(1.75 if body weight = 17.5-18.9 g. The needle size used was inch, 31 gauge, and with a 30 degree bevel. Virus was delivered )] TJ ET BT 26.250 241.881 Td /F1 9.8 Tf [(with a peristaltic pump at a rate of 200 nl per minute \(2.5 l over 12.5 minutes\). The needle was then left in place an additional )] TJ ET BT 26.250 229.977 Td /F1 9.8 Tf [(10 minutes before slow removal. Each mouse was injected with 10 ng of p24 equivalents of virus. For testing paired viral )] TJ ET BT 26.250 218.072 Td /F1 9.8 Tf [(injections, different combinations of virus were injected; we used 5 ng of p24 equivalents of each virus which were pre-mixed )] TJ ET BT 26.250 206.167 Td /F1 9.8 Tf [(prior to injection.)] TJ ET BT 26.250 186.762 Td /F4 9.8 Tf [(Wheel activity analysis:)] TJ ET BT 135.713 186.762 Td /F1 9.8 Tf [( Spontaneous wheel activity was measured before and after surgery. Mice were placed individually in )] TJ ET BT 26.250 174.858 Td /F1 9.8 Tf [(cages containing running wheels for 4 days, running times in both light and dark cycles \(12 hours each\) were recorded. The first )] TJ ET BT 26.250 162.953 Td /F1 9.8 Tf [(day was used to familiarize mice with the instrument. Data from days 2-4 were used for analysis.)] TJ ET BT 26.250 143.548 Td /F4 9.8 Tf [(Immunofluorescence staining and brain stereology:)] TJ ET BT 266.266 143.548 Td /F1 9.8 Tf [( Mice were sacrificed by an intra-peritoneal overdose of phenytoin and )] TJ ET BT 26.250 131.643 Td /F1 9.8 Tf [(pentobarbital solution. This was followed by a 2 minute perfusion in heparinized 0.9% saline immediately followed by perfusion )] TJ ET BT 26.250 119.739 Td /F1 9.8 Tf [(of 200 mls of freshly prepared 4% paraformaldehyde \(in 0.1 M phosphate buffer, pH 7.5\). Brains were removed 1-4 hours later )] TJ ET BT 26.250 107.834 Td /F1 9.8 Tf [(and immersion fixed for a further 24 hours before being transferred to cryo-preservant \(10% glycerol in 0.1 M phosphate buffer, )] TJ ET BT 26.250 95.929 Td /F1 9.8 Tf [(pH 7.5\). Brains were sectioned frontally at 40 ?m and stored in 0.1 M phosphate buffer \(pH 7.5\) containing 0.05% azide at 4C. )] TJ ET BT 26.250 84.024 Td /F1 9.8 Tf [(Striatal sections at the level of the anterior commissure were incubated with primary anti-huntingtin antibody EM48 \(Chemicon\) )] TJ ET BT 26.250 72.120 Td /F1 9.8 Tf [(at 1:1000 dilution in PBS-0.1% tween-10% goat serum for 3 days at 4?C, then followed by a 24 hour incubation with Alexa-fluor )] TJ ET BT 26.250 60.215 Td /F1 9.8 Tf [(488 labeled secondary antibody at 1:500 dilution \(Life Technologies\). Sections were washed in PBS three times for 10 minutes )] TJ ET BT 26.250 48.310 Td /F1 9.8 Tf [(and stained with fluorescent Nissl \(Life Technologies\) for 1 hour at room temperature \(1:100 dilution in PBS\), followed by )] TJ ET BT 26.250 36.405 Td /F1 9.8 Tf [(washing in PBS twice for 15 minutes and mounting on slides using Fluoromount G \(Southern Biotech\). Slides were air-dried )] TJ ET Q q 15.000 22.120 577.500 754.880 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(levels were determined by the ratios of the values for HTT and ?-actin. For mouse studies, mice were anesthetized and )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(perfused with cold-heparinized 0.9% \(w/v\) saline. Brains were sectioned frontally at a 1 mm interval then two sections were )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(taken at the level of the injection site, striatum dissected and then frozen on dry ice before storing at -80?C. Brains were )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(homogenized in lysis buffer which were then incubated on ice for 5 minutes then centrifuged for 10 minutes at 16 000 x g and )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(4?C. Protein concentrations were determined then Western blot analysis performed as described above.)] TJ ET BT 26.250 700.452 Td /F4 9.8 Tf [(N171 HTT protein TRX1 interaction experiment:)] TJ ET BT 245.128 700.452 Td /F1 9.8 Tf [( We used a previously described approach to test whether N171 mHTT is a )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(direct substrate of TRX1)] TJ ET 0.267 0.267 0.267 rg BT 131.375 690.055 Td /F4 8.7 Tf [(33)] TJ ET 0.271 0.267 0.267 rg BT 141.012 692.436 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 143.421 690.055 Td /F4 8.7 Tf [(34)] TJ ET 0.271 0.267 0.267 rg BT 153.059 688.548 Td /F1 9.8 Tf [(. In brief, a variant of TRX1 with a mutation of the lower cysteine residue within the CXXC active-)] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(site motif is used to trap the catalytic disulfide-linked heterodimer with the substrate protein. Heterodimers can be detected by )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(non-reducing Western blot analysis. DNA encoding N171-40Q, TRX1 and mutant TRX1 \(mTRX1\) was sub-cloned into the )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(bacterial expression vector pGEX-6P-1 \(GE Healthcare\). Proteins were expressed in bacteria, purified using a GST column, )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(then GST removed by cleavage with PreScission Protease \(GE Healthcare\) as previously described)] TJ ET 0.267 0.267 0.267 rg BT 457.034 642.436 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 461.853 640.929 Td /F1 9.8 Tf [( . The purified protein was )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(buffer exchanged into 50 mM Tris \(pH 7.0\) and 150 mM NaCl. N171-40Q was incubated with TXN or mutant TXN at room )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(temperature for one hour; 50 mM N-ethylmaleimide was then added to block free thiols and the samples were then resolved by )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(reducing or non-reducing SDS-PAGE and proteins detected by western blot analysis.)] TJ ET BT 26.250 585.810 Td /F4 9.8 Tf [(Mouse husbandry:)] TJ ET BT 112.927 585.810 Td /F1 9.8 Tf [( All procedures and euthanasia methods were approved by the University of Wyoming Institutional Animal )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(Care and Use Committee and were also in accordance with NIH guidelines. We used female B6/C3H F1 mice purchased from )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(the Jackson Laboratory. Mice were maintained under standard conditions of housing and lighting. They were fed a standard )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(cereal-based rodent chow and had ad-libitum access to acidified water \(pH 3-4\).)] TJ ET BT 26.250 530.691 Td /F4 9.8 Tf [(Mouse study experimental design:)] TJ ET BT 186.082 530.691 Td /F1 9.8 Tf [( C57BL/6 x C3H F1 female mice were purchased at 5-6 weeks of age. Pre-treatment )] TJ ET BT 26.250 518.786 Td /F1 9.8 Tf [(wheel analysis was at 7 weeks of age. Surgeries were at ~8 weeks of age and mice were sacrificed 6-weeks later based on the )] TJ ET BT 26.250 506.881 Td /F1 9.8 Tf [(studies of de Almeida et al)] TJ ET 0.267 0.267 0.267 rg BT 140.598 508.388 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 150.235 506.881 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 487.476 Td /F4 9.8 Tf [(Lentiviral synthesis:)] TJ ET BT 120.532 487.476 Td /F1 9.8 Tf [( We utilized a four-plasmid system for generation of lentivirus expressing N171-18Q and N171-82Q )] TJ ET BT 26.250 475.572 Td /F1 9.8 Tf [(\(kindly provided by Dr. Deglon\). We subcloned DNA encoding functionally active and inactive versions of TRX1 and TMX3 from )] TJ ET BT 26.250 463.667 Td /F1 9.8 Tf [(pQE-Tri plasmids into the SIN-pGK which uses the phosphoglycerate kinase promoter to drive gene expression. For each virus, )] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(the four plasmids were transfected in the molar ratio of 1:1:1:3 for pMDG, CMV?8.92, pRSV-Rev and SIN-pGK, respectively. )] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(For each T-150 sized flask we used 3.3, 7.2, 2.2 and 7.2 g plasmid. 293T cells were grown to ~60% confluency in 10% FBS-)] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(DMEM and 2 mM glutamine. They were then transfected using jetPrime transfection reagent \(Polyplus 114-07\) according to the )] TJ ET BT 26.250 416.048 Td /F1 9.8 Tf [(manufacturers instructions. For each T-150 flask we used 1 ml of jetPrime buffer, 20 g combined plasmids, 50 jetPrime )] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(reagent and 36 mls of cell culture medium. Cells were transfected for four hours, washed twice in PBS then the medium )] TJ ET BT 26.250 392.238 Td /F1 9.8 Tf [(replaced. Cells were incubated for a further 72 hours prior to virus purification. Medium was harvested and placed into sterile )] TJ ET BT 26.250 380.334 Td /F1 9.8 Tf [(tubes on wet ice then filtered using a 0.22 m filter. The filtered supernatant was centrifuged in a SW28 swinging bucket rotor )] TJ ET BT 26.250 368.429 Td /F1 9.8 Tf [(on a Beckman L8-80 centrifuge at 141k x g for 2 hours at 4 degrees C. Supernatant was decanted then tubes were inverted for )] TJ ET BT 26.250 356.524 Td /F1 9.8 Tf [(4 minutes. The pellet was re-suspended in 300-500 l of sterile PBS then transferred to a siliconized tube. Samples were then )] TJ ET BT 26.250 344.619 Td /F1 9.8 Tf [(centrifuged at 19000 x g for 60 min at 4 degrees C to concentrate then the pellet resuspended in 50-150 l PBS by gentle )] TJ ET BT 26.250 332.715 Td /F1 9.8 Tf [(pipetting. The samples were then mixed gently overnight using a tilted horizontal shaker at 4 degrees C. Lentiviruses were )] TJ ET BT 26.250 320.810 Td /F1 9.8 Tf [(quantified in duplicate using a p24 ELISA \(Zeptometrix\) according to the manufacturers instructions. Lentiviral samples were )] TJ ET BT 26.250 308.905 Td /F1 9.8 Tf [(diluted to 4.0 ng p24 / l in PBS, stored at 4 degrees C in siliconized tubes and used within 2 weeks of preparation.)] TJ ET BT 26.250 289.500 Td /F4 9.8 Tf [(Stereotaxic surgery:)] TJ ET BT 120.532 289.500 Td /F1 9.8 Tf [( Mice were anesthetized with 1.75 mg ketamine and 0.25 mg xylazine per 20 grams body weight. After )] TJ ET BT 26.250 277.596 Td /F1 9.8 Tf [(mounting in a stereotaxic frame they were injected at the following coordinates: AP =+0.4; DV= -3.9, then pull up to -3.7 before )] TJ ET BT 26.250 265.691 Td /F1 9.8 Tf [(injection; ML= \(all to right\), +2.0 if body weight ?22.5 g, 1.95 if body weight = 20.5-22.4 g, 1.85 if body weight = 19.0-20.4 g, and )] TJ ET BT 26.250 253.786 Td /F1 9.8 Tf [(1.75 if body weight = 17.5-18.9 g. The needle size used was inch, 31 gauge, and with a 30 degree bevel. Virus was delivered )] TJ ET BT 26.250 241.881 Td /F1 9.8 Tf [(with a peristaltic pump at a rate of 200 nl per minute \(2.5 l over 12.5 minutes\). The needle was then left in place an additional )] TJ ET BT 26.250 229.977 Td /F1 9.8 Tf [(10 minutes before slow removal. Each mouse was injected with 10 ng of p24 equivalents of virus. For testing paired viral )] TJ ET BT 26.250 218.072 Td /F1 9.8 Tf [(injections, different combinations of virus were injected; we used 5 ng of p24 equivalents of each virus which were pre-mixed )] TJ ET BT 26.250 206.167 Td /F1 9.8 Tf [(prior to injection.)] TJ ET BT 26.250 186.762 Td /F4 9.8 Tf [(Wheel activity analysis:)] TJ ET BT 135.713 186.762 Td /F1 9.8 Tf [( Spontaneous wheel activity was measured before and after surgery. Mice were placed individually in )] TJ ET BT 26.250 174.858 Td /F1 9.8 Tf [(cages containing running wheels for 4 days, running times in both light and dark cycles \(12 hours each\) were recorded. The first )] TJ ET BT 26.250 162.953 Td /F1 9.8 Tf [(day was used to familiarize mice with the instrument. Data from days 2-4 were used for analysis.)] TJ ET BT 26.250 143.548 Td /F4 9.8 Tf [(Immunofluorescence staining and brain stereology:)] TJ ET BT 266.266 143.548 Td /F1 9.8 Tf [( Mice were sacrificed by an intra-peritoneal overdose of phenytoin and )] TJ ET BT 26.250 131.643 Td /F1 9.8 Tf [(pentobarbital solution. This was followed by a 2 minute perfusion in heparinized 0.9% saline immediately followed by perfusion )] TJ ET BT 26.250 119.739 Td /F1 9.8 Tf [(of 200 mls of freshly prepared 4% paraformaldehyde \(in 0.1 M phosphate buffer, pH 7.5\). Brains were removed 1-4 hours later )] TJ ET BT 26.250 107.834 Td /F1 9.8 Tf [(and immersion fixed for a further 24 hours before being transferred to cryo-preservant \(10% glycerol in 0.1 M phosphate buffer, )] TJ ET BT 26.250 95.929 Td /F1 9.8 Tf [(pH 7.5\). Brains were sectioned frontally at 40 ?m and stored in 0.1 M phosphate buffer \(pH 7.5\) containing 0.05% azide at 4C. )] TJ ET BT 26.250 84.024 Td /F1 9.8 Tf [(Striatal sections at the level of the anterior commissure were incubated with primary anti-huntingtin antibody EM48 \(Chemicon\) )] TJ ET BT 26.250 72.120 Td /F1 9.8 Tf [(at 1:1000 dilution in PBS-0.1% tween-10% goat serum for 3 days at 4?C, then followed by a 24 hour incubation with Alexa-fluor )] TJ ET BT 26.250 60.215 Td /F1 9.8 Tf [(488 labeled secondary antibody at 1:500 dilution \(Life Technologies\). Sections were washed in PBS three times for 10 minutes )] TJ ET BT 26.250 48.310 Td /F1 9.8 Tf [(and stained with fluorescent Nissl \(Life Technologies\) for 1 hour at room temperature \(1:100 dilution in PBS\), followed by )] TJ ET BT 26.250 36.405 Td /F1 9.8 Tf [(washing in PBS twice for 15 minutes and mounting on slides using Fluoromount G \(Southern Biotech\). Slides were air-dried )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(3)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 265 0 obj << /Type /Annot /Subtype /Link /A 266 0 R /Border [0 0 0] /H /I /Rect [ 131.3745 689.2532 141.0118 698.0715 ] >> endobj 266 0 obj << /Type /Action >> endobj 267 0 obj << /Type /Annot /Subtype /Link /A 268 0 R /Border [0 0 0] /H /I /Rect [ 143.4212 689.2532 153.0585 698.0715 ] >> endobj 268 0 obj << /Type /Action >> endobj 269 0 obj << /Type /Annot /Subtype /Link /A 270 0 R /Border [0 0 0] /H /I /Rect [ 457.0342 641.6342 461.8529 650.4525 ] >> endobj 270 0 obj << /Type /Action >> endobj 271 0 obj << /Type /Annot /Subtype /Link /A 272 0 R /Border [0 0 0] /H /I /Rect [ 140.5980 507.5867 150.2353 516.4050 ] >> endobj 272 0 obj << /Type /Action >> endobj 273 0 obj << /Type /Annot /Subtype /Link /A 274 0 R /Border [0 0 0] /H /I /Rect [ 131.3745 689.2532 141.0118 698.0715 ] >> endobj 274 0 obj << /Type /Action >> endobj 275 0 obj << /Type /Annot /Subtype /Link /A 276 0 R /Border [0 0 0] /H /I /Rect [ 143.4212 689.2532 153.0585 698.0715 ] >> endobj 276 0 obj << /Type /Action >> endobj 277 0 obj << /Type /Annot /Subtype /Link /A 278 0 R /Border [0 0 0] /H /I /Rect [ 457.0342 641.6342 461.8529 650.4525 ] >> endobj 278 0 obj << /Type /Action >> endobj 279 0 obj << /Type /Annot /Subtype /Link /A 280 0 R /Border [0 0 0] /H /I /Rect [ 140.5980 507.5867 150.2353 516.4050 ] >> endobj 280 0 obj << /Type /Action >> endobj 281 0 obj << /Type /Annot /Subtype /Link /A 282 0 R /Border [0 0 0] /H /I /Rect [ 131.3745 689.2532 141.0118 698.0715 ] >> endobj 282 0 obj << /Type /Action >> endobj 283 0 obj << /Type /Annot /Subtype /Link /A 284 0 R /Border [0 0 0] /H /I /Rect [ 143.4212 689.2532 153.0585 698.0715 ] >> endobj 284 0 obj << /Type /Action >> endobj 285 0 obj << /Type /Annot /Subtype /Link /A 286 0 R /Border [0 0 0] /H /I /Rect [ 457.0342 641.6342 461.8529 650.4525 ] >> endobj 286 0 obj << /Type /Action >> endobj 287 0 obj << /Type /Annot /Subtype /Link /A 288 0 R /Border [0 0 0] /H /I /Rect [ 140.5980 507.5867 150.2353 516.4050 ] >> endobj 288 0 obj << /Type /Action >> endobj 289 0 obj << /Type /Page /Parent 3 0 R /Annots [ 291 0 R 293 0 R 295 0 R 297 0 R 299 0 R 301 0 R 303 0 R 305 0 R 307 0 R 309 0 R 311 0 R 313 0 R 315 0 R 317 0 R 319 0 R 321 0 R 323 0 R 325 0 R 327 0 R 329 0 R 331 0 R 333 0 R 335 0 R 337 0 R ] /Contents 290 0 R >> endobj 290 0 obj << /Length 29545 >> stream 0.271 0.267 0.267 rg q 15.000 32.587 577.500 744.413 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(overnight in the dark and then stored at 4?C. Nissl stain was used to detect neuronal cell bodies at 640/660 nm. Images were )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(captured using a Zeiss 710 confocal microscope. We stained several striatal sections per mouse to identify sections containing )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(expressed protein. Within each region of positive mHTT staining 2-3 image stacks were obtained using a 60x objective \(z-stack )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(interval = 0.46 m\). Within these images we quantified all striatal neurons regardless of whether there was mHTT staining as )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(suppression of mHTT levels by TRX1 or TMX3 could result in loss of detectable staining. Striatal neuronal volumes were )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(estimated using the confocal module in Stereoinvestigator software \(MicroBrightField, Williston, VT\) and the five-ray nucleator )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(method.)] TJ ET BT 26.250 676.643 Td /F4 9.8 Tf [(Quantitative PCR for TMX3 and TXN1 in N171-82Q HD mice:)] TJ ET BT 302.019 676.643 Td /F1 9.8 Tf [( We used essentially the same method as previously described)] TJ ET 0.267 0.267 0.267 rg BT 26.250 666.245 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 35.887 664.738 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 645.333 Td /F4 9.8 Tf [(Statistical analyses:)] TJ ET BT 119.460 645.333 Td /F1 9.8 Tf [( Data was analyzed with SAS software version 9. Students t-test were used for the analysis of the )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(secondary screens. One-way ANOVA was used for the analysis striatal neuronal volumes. Repeated-measures ANOVA was )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(used to analyze spontaneous wheel-running data. P-values less than 0.05 were considered significant.)] TJ ET BT 26.250 584.921 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 564.967 Td /F4 9.8 Tf [(Identification of TRX1 and TMX3 as candidate mHTT decreasing proteins in HD)] TJ ET BT 26.250 545.562 Td /F1 9.8 Tf [(We studied a broad range of human thiol-disulfide oxidoreductase genes. This group includes all the well-studied thiol-disulfide )] TJ ET BT 26.250 533.658 Td /F1 9.8 Tf [(oxidoreductases to include the glutaredoxin, thioredoxin and thioredoxin reductase gene families. There are a growing number )] TJ ET BT 26.250 521.753 Td /F1 9.8 Tf [(of poorly characterized proteins considered to have a thioredoxin-like domain and probable thiol-disulfide oxidoreductase )] TJ ET BT 26.250 509.848 Td /F1 9.8 Tf [(activity. We studied several of these proteins to include the thioredoxin-related transmembrane proteins and thioredoxin-like )] TJ ET BT 26.250 497.943 Td /F1 9.8 Tf [(proteins. COS1 cells were co-transfected with plasmids encoding the N171-40Q fragment of mHTT protein together with a )] TJ ET BT 26.250 486.039 Td /F1 9.8 Tf [(plasmid encoding the test thiol-disulfide oxidoreductase. Co-transfection with plasmids encoding N171-40Q mHTT and GFP )] TJ ET BT 26.250 474.134 Td /F1 9.8 Tf [(were used as controls. Cells were lysed 24 hours post-transfection and analyzed for soluble mHTT levels. For each gene )] TJ ET BT 26.250 462.229 Td /F1 9.8 Tf [(studied, N171-40Q expression was normalized to actin then the result normalized to the N171-40Q/GFP control \(100%\). As )] TJ ET BT 26.250 450.324 Td /F1 9.8 Tf [(shown in Fig. 1A, we found markedly different effects of test thiol-disulfide oxidoreductases on soluble N171-40Q mHTT levels. )] TJ ET BT 26.250 438.420 Td /F1 9.8 Tf [(However, we subsequently found that co-transfection with GFP significantly suppressed N171-40Q protein levels \(not shown\). )] TJ ET BT 26.250 426.515 Td /F1 9.8 Tf [(Therefore, rather than comparing test genes with the N171-40Q/GFP baseline, we qualitatively selected genes for more detailed )] TJ ET BT 26.250 414.610 Td /F1 9.8 Tf [(testing based on them resulting in low or high N171-40Q expression levels, compared to other candidates. Thioredoxin 1 )] TJ ET BT 26.250 402.705 Td /F1 9.8 Tf [(\(TRX1\) and thioredoxin-related transmembrane protein 3 \(TMX3\) were chosen as candidates that may decrease N171-40Q )] TJ ET BT 26.250 390.801 Td /F1 9.8 Tf [(levels. Glutaredoxin 1 \(GLX1\), protein disulfide isomerase family A, member 6 \(PDIA6\) and FLJ44606 were chosen as )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(candidates that may increase mHTT. Sel-M and Sel-15 are selenoproteins that were also mHTT decreasing candidates; they )] TJ ET BT 26.250 366.991 Td /F1 9.8 Tf [(were not included in subsequent investigations. To provide a more rigorous assessment of the selected candidates we )] TJ ET BT 26.250 355.086 Td /F1 9.8 Tf [(developed a secondary screening assay to further validate results. The candidate proteins share a common thioredoxin-domain )] TJ ET BT 26.250 343.182 Td /F1 9.8 Tf [(structure with the same catalytic mechanism that involves a C-X-X-C motif. Further, mutation of the first cysteine residue of this )] TJ ET BT 26.250 331.277 Td /F1 9.8 Tf [(site blocks enzymatic activity)] TJ ET 0.267 0.267 0.267 rg BT 150.865 332.784 Td /F4 8.7 Tf [(32)] TJ ET 0.271 0.267 0.267 rg BT 160.502 331.277 Td /F1 9.8 Tf [(. To specifically assess the effect of thiol-disulfide oxidoreductase enzymatic activity on mHTT )] TJ ET BT 26.250 319.372 Td /F1 9.8 Tf [(levels in our secondary screen we compared the effect of active protein with protein in which enzymatic activity had been )] TJ ET BT 26.250 307.467 Td /F1 9.8 Tf [(blocked by replacement of the critical cysteine with serine \(see methods\). As shown in Fig. 1B-D GLX1 \(1B\) increased mHTT )] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(\(p<0.05\); TRX1 \(1C\) decreased mHTT \(p<0.05\); and TMX3 \(1D\) decreased mHTT \(p<0.01\) consistent with the findings from )] TJ ET BT 26.250 283.658 Td /F1 9.8 Tf [(the primary screen \(Fig. 1A\). However, active LJ44606 \(1E\) and PDIA6 \(1F\) had no effect on mHTT levels \(p>0.05\). GLX1 )] TJ ET BT 26.250 271.753 Td /F1 9.8 Tf [(increased N171-40Q protein levels \(1B\) suggesting that GLX1 inhibitors may decrease mHTT. However, while GLX1 knockout )] TJ ET BT 26.250 259.848 Td /F1 9.8 Tf [(is not lethal in mice)] TJ ET 0.267 0.267 0.267 rg BT 109.154 261.356 Td /F4 8.7 Tf [(36)] TJ ET 0.271 0.267 0.267 rg BT 118.792 259.848 Td /F1 9.8 Tf [( the protein is required for normal mitochondrial function)] TJ ET 0.267 0.267 0.267 rg BT 359.929 261.356 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 369.566 259.848 Td /F1 9.8 Tf [(. As GLX1 inhibitors would be predicted to be )] TJ ET BT 26.250 247.944 Td /F1 9.8 Tf [(toxic, we excluded it from further investigation.)] TJ ET BT 26.250 228.539 Td /F1 9.8 Tf [(To further elucidate mechanisms for decreasing mHTT levels in cells we used two approaches. First, we tested to determine if )] TJ ET BT 26.250 216.634 Td /F1 9.8 Tf [(TRX1 and TMX3 can decrease levels of a N171-40Q variant that lacks thiol-containing cysteine residues and does not form )] TJ ET BT 26.250 204.729 Td /F1 9.8 Tf [(reduction-sensitive oligomers. We used a plasmid encoding N171-40Q-4CA in which all four cysteine residues are mutated to )] TJ ET BT 26.250 192.825 Td /F1 9.8 Tf [(block formation of thiol-dependent oligomers)] TJ ET 0.267 0.267 0.267 rg BT 218.081 194.332 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 222.900 192.825 Td /F1 9.8 Tf [(. In co-transfection experiments there was no effect of TRX1 and TMX3 on )] TJ ET BT 26.250 180.920 Td /F1 9.8 Tf [(decreasing N171-40Q-4CA as compared to mutant TRX1 and mutant TMX3 controls \(Fig. 2A-B\). Infact, functional TMX3 )] TJ ET BT 26.250 169.015 Td /F1 9.8 Tf [(increased N171-40Q-4CA compared to mutant TMX3 control \(p<0.05\) \(Fig. 2B\). The reason for this is unclear. However, the )] TJ ET BT 26.250 157.110 Td /F1 9.8 Tf [(lack of decrease of N171-40Q-4CA by active TXN1 and TMX3 suggests that the decreasing effects of these thiol-disulfide )] TJ ET BT 26.250 145.206 Td /F1 9.8 Tf [(oxidoreductase on N171-40Q levels depends on the presence of N171 HTT thiols. TRX1 is a cytosolic and nuclear protein and )] TJ ET BT 26.250 133.301 Td /F1 9.8 Tf [(could therefore have direct contact with HTT)] TJ ET 0.267 0.267 0.267 rg BT 217.535 134.808 Td /F4 8.7 Tf [(37)] TJ ET 0.271 0.267 0.267 rg BT 227.173 137.189 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 229.582 134.808 Td /F4 8.7 Tf [(38)] TJ ET 0.271 0.267 0.267 rg BT 239.219 133.301 Td /F1 9.8 Tf [(. We therefore addressed if N171-40Q mHTT is a direct substrate of TRX1. )] TJ ET BT 26.250 121.396 Td /F1 9.8 Tf [(We used a previously described method that can trap TRX1 with its substrate in a catalytic intermediate state [34] . As shown in )] TJ ET BT 26.250 109.491 Td /F1 9.8 Tf [(Fig. 3 we found no evidence that N171-40Q mHTT is a direct substrate of TRX1 under cell-free conditions. Similar experiments )] TJ ET BT 26.250 97.587 Td /F1 9.8 Tf [(using transfected COS cells also failed to find evidence of a direct interaction between N171 HTT and TRX1 \(not shown\). TMX3 )] TJ ET BT 26.250 85.682 Td /F1 9.8 Tf [(is a transmembrane protein with its active site within the endoplasmic reticulum)] TJ ET 0.267 0.267 0.267 rg BT 367.071 87.189 Td /F4 8.7 Tf [(39)] TJ ET 0.271 0.267 0.267 rg BT 376.708 85.682 Td /F1 9.8 Tf [(. As there is no evidence that HTT is an )] TJ ET BT 26.250 73.777 Td /F1 9.8 Tf [(endoplasmic reticulum luminal protein we did not test for a direct TMX3 HTT interaction. However, as both TRX1 and TMX3 )] TJ ET BT 26.250 61.872 Td /F1 9.8 Tf [(could be protective by effects on non-HTT targets we tested them in mouse HD.)] TJ ET Q q 15.000 32.587 577.500 744.413 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(overnight in the dark and then stored at 4?C. Nissl stain was used to detect neuronal cell bodies at 640/660 nm. Images were )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(captured using a Zeiss 710 confocal microscope. We stained several striatal sections per mouse to identify sections containing )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(expressed protein. Within each region of positive mHTT staining 2-3 image stacks were obtained using a 60x objective \(z-stack )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(interval = 0.46 m\). Within these images we quantified all striatal neurons regardless of whether there was mHTT staining as )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(suppression of mHTT levels by TRX1 or TMX3 could result in loss of detectable staining. Striatal neuronal volumes were )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(estimated using the confocal module in Stereoinvestigator software \(MicroBrightField, Williston, VT\) and the five-ray nucleator )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(method.)] TJ ET BT 26.250 676.643 Td /F4 9.8 Tf [(Quantitative PCR for TMX3 and TXN1 in N171-82Q HD mice:)] TJ ET BT 302.019 676.643 Td /F1 9.8 Tf [( We used essentially the same method as previously described)] TJ ET 0.267 0.267 0.267 rg BT 26.250 666.245 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 35.887 664.738 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 645.333 Td /F4 9.8 Tf [(Statistical analyses:)] TJ ET BT 119.460 645.333 Td /F1 9.8 Tf [( Data was analyzed with SAS software version 9. Students t-test were used for the analysis of the )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(secondary screens. One-way ANOVA was used for the analysis striatal neuronal volumes. Repeated-measures ANOVA was )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(used to analyze spontaneous wheel-running data. P-values less than 0.05 were considered significant.)] TJ ET BT 26.250 584.921 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 564.967 Td /F4 9.8 Tf [(Identification of TRX1 and TMX3 as candidate mHTT decreasing proteins in HD)] TJ ET BT 26.250 545.562 Td /F1 9.8 Tf [(We studied a broad range of human thiol-disulfide oxidoreductase genes. This group includes all the well-studied thiol-disulfide )] TJ ET BT 26.250 533.658 Td /F1 9.8 Tf [(oxidoreductases to include the glutaredoxin, thioredoxin and thioredoxin reductase gene families. There are a growing number )] TJ ET BT 26.250 521.753 Td /F1 9.8 Tf [(of poorly characterized proteins considered to have a thioredoxin-like domain and probable thiol-disulfide oxidoreductase )] TJ ET BT 26.250 509.848 Td /F1 9.8 Tf [(activity. We studied several of these proteins to include the thioredoxin-related transmembrane proteins and thioredoxin-like )] TJ ET BT 26.250 497.943 Td /F1 9.8 Tf [(proteins. COS1 cells were co-transfected with plasmids encoding the N171-40Q fragment of mHTT protein together with a )] TJ ET BT 26.250 486.039 Td /F1 9.8 Tf [(plasmid encoding the test thiol-disulfide oxidoreductase. Co-transfection with plasmids encoding N171-40Q mHTT and GFP )] TJ ET BT 26.250 474.134 Td /F1 9.8 Tf [(were used as controls. Cells were lysed 24 hours post-transfection and analyzed for soluble mHTT levels. For each gene )] TJ ET BT 26.250 462.229 Td /F1 9.8 Tf [(studied, N171-40Q expression was normalized to actin then the result normalized to the N171-40Q/GFP control \(100%\). As )] TJ ET BT 26.250 450.324 Td /F1 9.8 Tf [(shown in Fig. 1A, we found markedly different effects of test thiol-disulfide oxidoreductases on soluble N171-40Q mHTT levels. )] TJ ET BT 26.250 438.420 Td /F1 9.8 Tf [(However, we subsequently found that co-transfection with GFP significantly suppressed N171-40Q protein levels \(not shown\). )] TJ ET BT 26.250 426.515 Td /F1 9.8 Tf [(Therefore, rather than comparing test genes with the N171-40Q/GFP baseline, we qualitatively selected genes for more detailed )] TJ ET BT 26.250 414.610 Td /F1 9.8 Tf [(testing based on them resulting in low or high N171-40Q expression levels, compared to other candidates. Thioredoxin 1 )] TJ ET BT 26.250 402.705 Td /F1 9.8 Tf [(\(TRX1\) and thioredoxin-related transmembrane protein 3 \(TMX3\) were chosen as candidates that may decrease N171-40Q )] TJ ET BT 26.250 390.801 Td /F1 9.8 Tf [(levels. Glutaredoxin 1 \(GLX1\), protein disulfide isomerase family A, member 6 \(PDIA6\) and FLJ44606 were chosen as )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(candidates that may increase mHTT. Sel-M and Sel-15 are selenoproteins that were also mHTT decreasing candidates; they )] TJ ET BT 26.250 366.991 Td /F1 9.8 Tf [(were not included in subsequent investigations. To provide a more rigorous assessment of the selected candidates we )] TJ ET BT 26.250 355.086 Td /F1 9.8 Tf [(developed a secondary screening assay to further validate results. The candidate proteins share a common thioredoxin-domain )] TJ ET BT 26.250 343.182 Td /F1 9.8 Tf [(structure with the same catalytic mechanism that involves a C-X-X-C motif. Further, mutation of the first cysteine residue of this )] TJ ET BT 26.250 331.277 Td /F1 9.8 Tf [(site blocks enzymatic activity)] TJ ET 0.267 0.267 0.267 rg BT 150.865 332.784 Td /F4 8.7 Tf [(32)] TJ ET 0.271 0.267 0.267 rg BT 160.502 331.277 Td /F1 9.8 Tf [(. To specifically assess the effect of thiol-disulfide oxidoreductase enzymatic activity on mHTT )] TJ ET BT 26.250 319.372 Td /F1 9.8 Tf [(levels in our secondary screen we compared the effect of active protein with protein in which enzymatic activity had been )] TJ ET BT 26.250 307.467 Td /F1 9.8 Tf [(blocked by replacement of the critical cysteine with serine \(see methods\). As shown in Fig. 1B-D GLX1 \(1B\) increased mHTT )] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(\(p<0.05\); TRX1 \(1C\) decreased mHTT \(p<0.05\); and TMX3 \(1D\) decreased mHTT \(p<0.01\) consistent with the findings from )] TJ ET BT 26.250 283.658 Td /F1 9.8 Tf [(the primary screen \(Fig. 1A\). However, active LJ44606 \(1E\) and PDIA6 \(1F\) had no effect on mHTT levels \(p>0.05\). GLX1 )] TJ ET BT 26.250 271.753 Td /F1 9.8 Tf [(increased N171-40Q protein levels \(1B\) suggesting that GLX1 inhibitors may decrease mHTT. However, while GLX1 knockout )] TJ ET BT 26.250 259.848 Td /F1 9.8 Tf [(is not lethal in mice)] TJ ET 0.267 0.267 0.267 rg BT 109.154 261.356 Td /F4 8.7 Tf [(36)] TJ ET 0.271 0.267 0.267 rg BT 118.792 259.848 Td /F1 9.8 Tf [( the protein is required for normal mitochondrial function)] TJ ET 0.267 0.267 0.267 rg BT 359.929 261.356 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 369.566 259.848 Td /F1 9.8 Tf [(. As GLX1 inhibitors would be predicted to be )] TJ ET BT 26.250 247.944 Td /F1 9.8 Tf [(toxic, we excluded it from further investigation.)] TJ ET BT 26.250 228.539 Td /F1 9.8 Tf [(To further elucidate mechanisms for decreasing mHTT levels in cells we used two approaches. First, we tested to determine if )] TJ ET BT 26.250 216.634 Td /F1 9.8 Tf [(TRX1 and TMX3 can decrease levels of a N171-40Q variant that lacks thiol-containing cysteine residues and does not form )] TJ ET BT 26.250 204.729 Td /F1 9.8 Tf [(reduction-sensitive oligomers. We used a plasmid encoding N171-40Q-4CA in which all four cysteine residues are mutated to )] TJ ET BT 26.250 192.825 Td /F1 9.8 Tf [(block formation of thiol-dependent oligomers)] TJ ET 0.267 0.267 0.267 rg BT 218.081 194.332 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 222.900 192.825 Td /F1 9.8 Tf [(. In co-transfection experiments there was no effect of TRX1 and TMX3 on )] TJ ET BT 26.250 180.920 Td /F1 9.8 Tf [(decreasing N171-40Q-4CA as compared to mutant TRX1 and mutant TMX3 controls \(Fig. 2A-B\). Infact, functional TMX3 )] TJ ET BT 26.250 169.015 Td /F1 9.8 Tf [(increased N171-40Q-4CA compared to mutant TMX3 control \(p<0.05\) \(Fig. 2B\). The reason for this is unclear. However, the )] TJ ET BT 26.250 157.110 Td /F1 9.8 Tf [(lack of decrease of N171-40Q-4CA by active TXN1 and TMX3 suggests that the decreasing effects of these thiol-disulfide )] TJ ET BT 26.250 145.206 Td /F1 9.8 Tf [(oxidoreductase on N171-40Q levels depends on the presence of N171 HTT thiols. TRX1 is a cytosolic and nuclear protein and )] TJ ET BT 26.250 133.301 Td /F1 9.8 Tf [(could therefore have direct contact with HTT)] TJ ET 0.267 0.267 0.267 rg BT 217.535 134.808 Td /F4 8.7 Tf [(37)] TJ ET 0.271 0.267 0.267 rg BT 227.173 137.189 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 229.582 134.808 Td /F4 8.7 Tf [(38)] TJ ET 0.271 0.267 0.267 rg BT 239.219 133.301 Td /F1 9.8 Tf [(. We therefore addressed if N171-40Q mHTT is a direct substrate of TRX1. )] TJ ET BT 26.250 121.396 Td /F1 9.8 Tf [(We used a previously described method that can trap TRX1 with its substrate in a catalytic intermediate state [34] . As shown in )] TJ ET BT 26.250 109.491 Td /F1 9.8 Tf [(Fig. 3 we found no evidence that N171-40Q mHTT is a direct substrate of TRX1 under cell-free conditions. Similar experiments )] TJ ET BT 26.250 97.587 Td /F1 9.8 Tf [(using transfected COS cells also failed to find evidence of a direct interaction between N171 HTT and TRX1 \(not shown\). TMX3 )] TJ ET BT 26.250 85.682 Td /F1 9.8 Tf [(is a transmembrane protein with its active site within the endoplasmic reticulum)] TJ ET 0.267 0.267 0.267 rg BT 367.071 87.189 Td /F4 8.7 Tf [(39)] TJ ET 0.271 0.267 0.267 rg BT 376.708 85.682 Td /F1 9.8 Tf [(. As there is no evidence that HTT is an )] TJ ET BT 26.250 73.777 Td /F1 9.8 Tf [(endoplasmic reticulum luminal protein we did not test for a direct TMX3 HTT interaction. However, as both TRX1 and TMX3 )] TJ ET BT 26.250 61.872 Td /F1 9.8 Tf [(could be protective by effects on non-HTT targets we tested them in mouse HD.)] TJ ET Q q 15.000 32.587 577.500 744.413 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(overnight in the dark and then stored at 4?C. Nissl stain was used to detect neuronal cell bodies at 640/660 nm. Images were )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(captured using a Zeiss 710 confocal microscope. We stained several striatal sections per mouse to identify sections containing )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(expressed protein. Within each region of positive mHTT staining 2-3 image stacks were obtained using a 60x objective \(z-stack )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(interval = 0.46 m\). Within these images we quantified all striatal neurons regardless of whether there was mHTT staining as )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(suppression of mHTT levels by TRX1 or TMX3 could result in loss of detectable staining. Striatal neuronal volumes were )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(estimated using the confocal module in Stereoinvestigator software \(MicroBrightField, Williston, VT\) and the five-ray nucleator )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(method.)] TJ ET BT 26.250 676.643 Td /F4 9.8 Tf [(Quantitative PCR for TMX3 and TXN1 in N171-82Q HD mice:)] TJ ET BT 302.019 676.643 Td /F1 9.8 Tf [( We used essentially the same method as previously described)] TJ ET 0.267 0.267 0.267 rg BT 26.250 666.245 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 35.887 664.738 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 645.333 Td /F4 9.8 Tf [(Statistical analyses:)] TJ ET BT 119.460 645.333 Td /F1 9.8 Tf [( Data was analyzed with SAS software version 9. Students t-test were used for the analysis of the )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(secondary screens. One-way ANOVA was used for the analysis striatal neuronal volumes. Repeated-measures ANOVA was )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(used to analyze spontaneous wheel-running data. P-values less than 0.05 were considered significant.)] TJ ET BT 26.250 584.921 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 564.967 Td /F4 9.8 Tf [(Identification of TRX1 and TMX3 as candidate mHTT decreasing proteins in HD)] TJ ET BT 26.250 545.562 Td /F1 9.8 Tf [(We studied a broad range of human thiol-disulfide oxidoreductase genes. This group includes all the well-studied thiol-disulfide )] TJ ET BT 26.250 533.658 Td /F1 9.8 Tf [(oxidoreductases to include the glutaredoxin, thioredoxin and thioredoxin reductase gene families. There are a growing number )] TJ ET BT 26.250 521.753 Td /F1 9.8 Tf [(of poorly characterized proteins considered to have a thioredoxin-like domain and probable thiol-disulfide oxidoreductase )] TJ ET BT 26.250 509.848 Td /F1 9.8 Tf [(activity. We studied several of these proteins to include the thioredoxin-related transmembrane proteins and thioredoxin-like )] TJ ET BT 26.250 497.943 Td /F1 9.8 Tf [(proteins. COS1 cells were co-transfected with plasmids encoding the N171-40Q fragment of mHTT protein together with a )] TJ ET BT 26.250 486.039 Td /F1 9.8 Tf [(plasmid encoding the test thiol-disulfide oxidoreductase. Co-transfection with plasmids encoding N171-40Q mHTT and GFP )] TJ ET BT 26.250 474.134 Td /F1 9.8 Tf [(were used as controls. Cells were lysed 24 hours post-transfection and analyzed for soluble mHTT levels. For each gene )] TJ ET BT 26.250 462.229 Td /F1 9.8 Tf [(studied, N171-40Q expression was normalized to actin then the result normalized to the N171-40Q/GFP control \(100%\). As )] TJ ET BT 26.250 450.324 Td /F1 9.8 Tf [(shown in Fig. 1A, we found markedly different effects of test thiol-disulfide oxidoreductases on soluble N171-40Q mHTT levels. )] TJ ET BT 26.250 438.420 Td /F1 9.8 Tf [(However, we subsequently found that co-transfection with GFP significantly suppressed N171-40Q protein levels \(not shown\). )] TJ ET BT 26.250 426.515 Td /F1 9.8 Tf [(Therefore, rather than comparing test genes with the N171-40Q/GFP baseline, we qualitatively selected genes for more detailed )] TJ ET BT 26.250 414.610 Td /F1 9.8 Tf [(testing based on them resulting in low or high N171-40Q expression levels, compared to other candidates. Thioredoxin 1 )] TJ ET BT 26.250 402.705 Td /F1 9.8 Tf [(\(TRX1\) and thioredoxin-related transmembrane protein 3 \(TMX3\) were chosen as candidates that may decrease N171-40Q )] TJ ET BT 26.250 390.801 Td /F1 9.8 Tf [(levels. Glutaredoxin 1 \(GLX1\), protein disulfide isomerase family A, member 6 \(PDIA6\) and FLJ44606 were chosen as )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(candidates that may increase mHTT. Sel-M and Sel-15 are selenoproteins that were also mHTT decreasing candidates; they )] TJ ET BT 26.250 366.991 Td /F1 9.8 Tf [(were not included in subsequent investigations. To provide a more rigorous assessment of the selected candidates we )] TJ ET BT 26.250 355.086 Td /F1 9.8 Tf [(developed a secondary screening assay to further validate results. The candidate proteins share a common thioredoxin-domain )] TJ ET BT 26.250 343.182 Td /F1 9.8 Tf [(structure with the same catalytic mechanism that involves a C-X-X-C motif. Further, mutation of the first cysteine residue of this )] TJ ET BT 26.250 331.277 Td /F1 9.8 Tf [(site blocks enzymatic activity)] TJ ET 0.267 0.267 0.267 rg BT 150.865 332.784 Td /F4 8.7 Tf [(32)] TJ ET 0.271 0.267 0.267 rg BT 160.502 331.277 Td /F1 9.8 Tf [(. To specifically assess the effect of thiol-disulfide oxidoreductase enzymatic activity on mHTT )] TJ ET BT 26.250 319.372 Td /F1 9.8 Tf [(levels in our secondary screen we compared the effect of active protein with protein in which enzymatic activity had been )] TJ ET BT 26.250 307.467 Td /F1 9.8 Tf [(blocked by replacement of the critical cysteine with serine \(see methods\). As shown in Fig. 1B-D GLX1 \(1B\) increased mHTT )] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(\(p<0.05\); TRX1 \(1C\) decreased mHTT \(p<0.05\); and TMX3 \(1D\) decreased mHTT \(p<0.01\) consistent with the findings from )] TJ ET BT 26.250 283.658 Td /F1 9.8 Tf [(the primary screen \(Fig. 1A\). However, active LJ44606 \(1E\) and PDIA6 \(1F\) had no effect on mHTT levels \(p>0.05\). GLX1 )] TJ ET BT 26.250 271.753 Td /F1 9.8 Tf [(increased N171-40Q protein levels \(1B\) suggesting that GLX1 inhibitors may decrease mHTT. However, while GLX1 knockout )] TJ ET BT 26.250 259.848 Td /F1 9.8 Tf [(is not lethal in mice)] TJ ET 0.267 0.267 0.267 rg BT 109.154 261.356 Td /F4 8.7 Tf [(36)] TJ ET 0.271 0.267 0.267 rg BT 118.792 259.848 Td /F1 9.8 Tf [( the protein is required for normal mitochondrial function)] TJ ET 0.267 0.267 0.267 rg BT 359.929 261.356 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 369.566 259.848 Td /F1 9.8 Tf [(. As GLX1 inhibitors would be predicted to be )] TJ ET BT 26.250 247.944 Td /F1 9.8 Tf [(toxic, we excluded it from further investigation.)] TJ ET BT 26.250 228.539 Td /F1 9.8 Tf [(To further elucidate mechanisms for decreasing mHTT levels in cells we used two approaches. First, we tested to determine if )] TJ ET BT 26.250 216.634 Td /F1 9.8 Tf [(TRX1 and TMX3 can decrease levels of a N171-40Q variant that lacks thiol-containing cysteine residues and does not form )] TJ ET BT 26.250 204.729 Td /F1 9.8 Tf [(reduction-sensitive oligomers. We used a plasmid encoding N171-40Q-4CA in which all four cysteine residues are mutated to )] TJ ET BT 26.250 192.825 Td /F1 9.8 Tf [(block formation of thiol-dependent oligomers)] TJ ET 0.267 0.267 0.267 rg BT 218.081 194.332 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 222.900 192.825 Td /F1 9.8 Tf [(. In co-transfection experiments there was no effect of TRX1 and TMX3 on )] TJ ET BT 26.250 180.920 Td /F1 9.8 Tf [(decreasing N171-40Q-4CA as compared to mutant TRX1 and mutant TMX3 controls \(Fig. 2A-B\). Infact, functional TMX3 )] TJ ET BT 26.250 169.015 Td /F1 9.8 Tf [(increased N171-40Q-4CA compared to mutant TMX3 control \(p<0.05\) \(Fig. 2B\). The reason for this is unclear. However, the )] TJ ET BT 26.250 157.110 Td /F1 9.8 Tf [(lack of decrease of N171-40Q-4CA by active TXN1 and TMX3 suggests that the decreasing effects of these thiol-disulfide )] TJ ET BT 26.250 145.206 Td /F1 9.8 Tf [(oxidoreductase on N171-40Q levels depends on the presence of N171 HTT thiols. TRX1 is a cytosolic and nuclear protein and )] TJ ET BT 26.250 133.301 Td /F1 9.8 Tf [(could therefore have direct contact with HTT)] TJ ET 0.267 0.267 0.267 rg BT 217.535 134.808 Td /F4 8.7 Tf [(37)] TJ ET 0.271 0.267 0.267 rg BT 227.173 137.189 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 229.582 134.808 Td /F4 8.7 Tf [(38)] TJ ET 0.271 0.267 0.267 rg BT 239.219 133.301 Td /F1 9.8 Tf [(. We therefore addressed if N171-40Q mHTT is a direct substrate of TRX1. )] TJ ET BT 26.250 121.396 Td /F1 9.8 Tf [(We used a previously described method that can trap TRX1 with its substrate in a catalytic intermediate state [34] . As shown in )] TJ ET BT 26.250 109.491 Td /F1 9.8 Tf [(Fig. 3 we found no evidence that N171-40Q mHTT is a direct substrate of TRX1 under cell-free conditions. Similar experiments )] TJ ET BT 26.250 97.587 Td /F1 9.8 Tf [(using transfected COS cells also failed to find evidence of a direct interaction between N171 HTT and TRX1 \(not shown\). TMX3 )] TJ ET BT 26.250 85.682 Td /F1 9.8 Tf [(is a transmembrane protein with its active site within the endoplasmic reticulum)] TJ ET 0.267 0.267 0.267 rg BT 367.071 87.189 Td /F4 8.7 Tf [(39)] TJ ET 0.271 0.267 0.267 rg BT 376.708 85.682 Td /F1 9.8 Tf [(. As there is no evidence that HTT is an )] TJ ET BT 26.250 73.777 Td /F1 9.8 Tf [(endoplasmic reticulum luminal protein we did not test for a direct TMX3 HTT interaction. However, as both TRX1 and TMX3 )] TJ ET BT 26.250 61.872 Td /F1 9.8 Tf [(could be protective by effects on non-HTT targets we tested them in mouse HD.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(4)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 291 0 obj << /Type /Annot /Subtype /Link /A 292 0 R /Border [0 0 0] /H /I /Rect [ 26.2500 665.4437 35.8873 674.2620 ] >> endobj 292 0 obj << /Type /Action >> endobj 293 0 obj << /Type /Annot /Subtype /Link /A 294 0 R /Border [0 0 0] /H /I /Rect [ 150.8648 331.9824 160.5021 340.8008 ] >> endobj 294 0 obj << /Type /Action >> endobj 295 0 obj << /Type /Annot /Subtype /Link /A 296 0 R /Border [0 0 0] /H /I /Rect [ 109.1543 260.5539 118.7916 269.3723 ] >> endobj 296 0 obj << /Type /Action >> endobj 297 0 obj << /Type /Annot /Subtype /Link /A 298 0 R /Border [0 0 0] /H /I /Rect [ 359.9286 260.5539 369.5659 269.3723 ] >> endobj 298 0 obj << /Type /Action >> endobj 299 0 obj 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HD)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Lentiviral vectors have been used to model HD in rats and macaques)] TJ ET 0.267 0.267 0.267 rg BT 324.298 749.579 Td /F4 8.7 Tf [(40)] TJ ET 0.271 0.267 0.267 rg BT 333.935 748.071 Td /F1 9.8 Tf [(. We used a four plasmid lentiviral system previously )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(described by de Almeida et al in rats)] TJ ET 0.267 0.267 0.267 rg BT 183.400 737.674 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 193.038 736.167 Td /F1 9.8 Tf [( to model HD in wild-type mice. This system drives expression of the N171 HTT fragment )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(under the control of the phosphoglycerate kinase promoter providing neuronal expression at physiologically relevant protein )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(levels. Mice were injected with virus at ~8 weeks and sacrificed as ~14 weeks of age. Lentiviral-mediated expression of N171-)] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(18Q and 82Q HTT resulted in detectable expression by immunofluorescence staining of brain sections \(Fig. 4A\). However, )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(neuronal expression was only found immediately around the needle tract. Western blot analysis failed to detect N171-18Q but )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(detected N171-82Q \(Fig. 4B\). Therefore, both wild-type and mutant N171 protein were expressed in brain; higher expression of )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(mHTT may be related to its accumulation as part of the disease process)] TJ ET 0.267 0.267 0.267 rg BT 336.748 666.245 Td /F4 8.7 Tf [(41)] TJ ET 0.271 0.267 0.267 rg BT 346.386 664.738 Td /F1 9.8 Tf [(. We chose to use striatal neuronal cell body volume )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(as our main outcome as cell atrophy is a consistent manifestation of mHTT expression in neurons)] TJ ET 0.267 0.267 0.267 rg BT 446.748 654.341 Td /F4 8.7 Tf [(42)] TJ ET 0.271 0.267 0.267 rg BT 456.385 652.833 Td /F1 9.8 Tf [(. In this preliminary study, )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(while differences were not statistically significant \(p=0.2202\) striatal neuronal cell body volume means were lower in the N171-)] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(82Q \(n=11\) versus N171-18Q \(n=5\) group \(520 53 and 639 78 m3, respectively\).)] TJ ET BT 26.250 609.619 Td /F4 9.8 Tf [(TRX1 and TMX3 decrease neuronal atrophy in mouse HD)] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(We sub-cloned cDNA-encoding TMX3 and TRX1 into the same lentiviral expression system used for N171 expression)] TJ ET 0.267 0.267 0.267 rg BT 534.517 591.722 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 544.155 590.214 Td /F1 9.8 Tf [( then )] TJ ET BT 26.250 578.310 Td /F1 9.8 Tf [(generated enzymatically inactive variants by point mutagenesis for use as gene-specific controls for enzymatic activity of the )] TJ ET BT 26.250 566.405 Td /F1 9.8 Tf [(test protein. We removed the histidine tags during the sub-cloning process. While we did not attempt to show protein expression )] TJ ET BT 26.250 554.500 Td /F1 9.8 Tf [(in brain, we did demonstrate in-vivo transcript expression of TMX3 and TRX1 in liver 6 weeks after intra-venous injection of )] TJ ET BT 26.250 542.595 Td /F1 9.8 Tf [(neonatal mice \(not shown\). We then undertook experiments in which we compared the effects of N171-18Q, N171-82Q, and )] TJ ET BT 26.250 530.691 Td /F1 9.8 Tf [(N171-82Q with TMX3 or TRX1 following mouse striatal injection. To control for the total level of lentiviral delivery to striatum we )] TJ ET BT 26.250 518.786 Td /F1 9.8 Tf [(co-injected virus encoding mutant \(inactive\) TMX3 or TRX1 in the N171-18Q and N171-82Q treatment groups. Striatal injections )] TJ ET BT 26.250 506.881 Td /F1 9.8 Tf [(were at ~8 weeks and mice were sacrificed at ~14 weeks of age. We completed confocal stereology to quantify striatal neuronal )] TJ ET BT 26.250 494.976 Td /F1 9.8 Tf [(cell body volume. As our candidate treatments were chosen based on their ability to decrease mHTT it is possible that mHTT )] TJ ET BT 26.250 483.072 Td /F1 9.8 Tf [(would not be detected in an infected cell by immunofluorescence and that the transduced cell would not be quantified. )] TJ ET BT 26.250 471.167 Td /F1 9.8 Tf [(Therefore, we changed our methodologic approach. We stained brain sections for mHTT then captured confocal images in )] TJ ET BT 26.250 459.262 Td /F1 9.8 Tf [(regions where there was some neuronal mHTT staining. We then quantified cell body volume for all neurons within an image )] TJ ET BT 26.250 447.357 Td /F1 9.8 Tf [(stack. As shown in Fig. 5 \(TMX3 experiment\) and Fig. 6 \(TRX1 experiment\) we found that striatal neuronal cell body volumes )] TJ ET BT 26.250 435.453 Td /F1 9.8 Tf [(were significantly decreased in N171-82Q versus N171-18Q expressing mice \(p-values: 0.0350 and 0.0035, respectively\). )] TJ ET BT 26.250 423.548 Td /F1 9.8 Tf [(Functionally active TMX3 and TRX1 both decreased this effect of mHTT on striatal neuronal atrophy \(p-values: 0.0387 and )] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(0.0046, respectively\). Due to the presence of detectable mHTT staining only around the needle tract quantification of soluble )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(mHTT levels by Western blot analysis following brain dissection would not have been reliable therefore we did not attempt this. )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(Finally, we studied TRX1 and TMX3 transcript levels by qPCR in 14-week old N171-82Q transgenic HD mice \(equivalent to )] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(early-advanced disease)] TJ ET 0.267 0.267 0.267 rg BT 129.210 377.436 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 138.847 375.929 Td /F1 9.8 Tf [(\); these mice express the N171-82Q HTT fragment under the control of the prion promoter)] TJ ET 0.267 0.267 0.267 rg BT 527.385 377.436 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 532.204 375.929 Td /F1 9.8 Tf [( \(Fig. 7\). )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(There was no evidence of decreased expression of both genes in striatum and cerebral cortex; in fact, in striatal TRX1 transcript )] TJ ET BT 26.250 352.119 Td /F1 9.8 Tf [(expression was significantly increased in HD mice \(p<0.01\) \(Fig. 7A\). We additionally completed searches of publically available )] TJ ET BT 26.250 340.215 Td /F1 9.8 Tf [(HD micro-array data sets using GeoProfiles. In the R6/1 mouse model of HD there was an increase in cerebral TXN1 transcript )] TJ ET BT 26.250 328.310 Td /F1 9.8 Tf [(from 22-27 weeks of age with 1 of 2 probes; TMX3 transcript changes were not found)] TJ ET 0.267 0.267 0.267 rg BT 394.215 329.817 Td /F4 8.7 Tf [(43)] TJ ET 0.271 0.267 0.267 rg BT 403.852 328.310 Td /F1 9.8 Tf [(. Micro-array analysis in 12 and 24 )] TJ ET BT 26.250 316.405 Td /F1 9.8 Tf [(month old full-length mHTT expressing YAC128 HD and wild-type litter-mate striata did not reveal expression differences for )] TJ ET BT 26.250 304.500 Td /F1 9.8 Tf [(TXN1 or TMX3)] TJ ET 0.267 0.267 0.267 rg BT 91.263 306.008 Td /F4 8.7 Tf [(44)] TJ ET 0.271 0.267 0.267 rg BT 100.900 304.500 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 267.898 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 247.944 Td /F1 9.8 Tf [(Abnormal redox homeostasis and oxidative stress are consistent features of human HD and cell-based and animal models)] TJ ET 0.267 0.267 0.267 rg BT 553.510 249.451 Td /F4 8.7 Tf [(45)] TJ ET 0.271 0.267 0.267 rg BT 563.148 251.832 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 565.557 249.451 Td /F4 8.7 Tf [(46)] TJ ET 0.271 0.267 0.267 rg BT 575.195 247.944 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 236.039 Td /F1 9.8 Tf [(Identification of appropriate targets for modulation of redox homeostasis could provide novel therapeutic approaches for treating )] TJ ET BT 26.250 224.134 Td /F1 9.8 Tf [(HD. Protein thiols are an important site of post-translational modification involved in the regulation of redox responsive cell )] TJ ET BT 26.250 212.229 Td /F1 9.8 Tf [(signaling processes)] TJ ET 0.267 0.267 0.267 rg BT 111.865 213.737 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 121.502 212.229 Td /F1 9.8 Tf [(. Oxidative stress can result in increased protein thiol oxidation and disruption of these homeostatic )] TJ ET BT 26.250 200.325 Td /F1 9.8 Tf [(processes, potentially contributing to cell dysfunction and degeneration. Transgenic mice expressing the N171 mHTT protein )] TJ ET BT 26.250 188.420 Td /F1 9.8 Tf [(fragment develop a phenotype similar to human HD including striatal atrophy)] TJ ET 0.267 0.267 0.267 rg BT 356.804 189.927 Td /F4 8.7 Tf [(47)] TJ ET 0.271 0.267 0.267 rg BT 366.442 188.420 Td /F1 9.8 Tf [(. We have shown that the N171 fragment of HTT )] TJ ET BT 26.250 176.515 Td /F1 9.8 Tf [(can form thiol-dependent oligomers which are degraded more slowly than a N171 protein variant that lacks thiols and is unable )] TJ ET BT 26.250 164.610 Td /F1 9.8 Tf [(to oligomerize)] TJ ET 0.267 0.267 0.267 rg BT 86.934 166.118 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 91.753 164.610 Td /F1 9.8 Tf [(. Numerous proteins with thiol-disulfide oxidoreductase activity exist that facilitate the reduction of oxidized )] TJ ET BT 26.250 152.706 Td /F1 9.8 Tf [(protein thiols in cells)] TJ ET 0.267 0.267 0.267 rg BT 114.029 154.213 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 123.667 152.706 Td /F1 9.8 Tf [(. Here we sought to identify if there are thiol-disulfide oxidoreductase enzymes that can decrease mHTT )] TJ ET BT 26.250 140.801 Td /F1 9.8 Tf [(levels in cells and provide protection against neuronal atrophy in HD mice. We tested a representative set of thiol-disulfide )] TJ ET BT 26.250 128.896 Td /F1 9.8 Tf [(oxidoreductases for mHTT decreasing effects. We used primary and secondary cell-based screens to identify candidate genes )] TJ ET BT 26.250 116.991 Td /F1 9.8 Tf [(for testing in HD mice \(Fig. 1\). Based on our previous findings)] TJ ET 0.267 0.267 0.267 rg BT 292.308 118.499 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 297.127 116.991 Td /F1 9.8 Tf [( enzymatic conversion of mHTT oligomer to monomer by a thiol-)] TJ ET BT 26.250 105.087 Td /F1 9.8 Tf [(disulfide oxidoreductase is expected to result in increased monomer degradation and potentially no change in monomer to )] TJ ET BT 26.250 93.182 Td /F1 9.8 Tf [(oligomer ratio; we therefore quantified total soluble mHTT levels by reducing SDS-PAGE in our cell-based studies. To enable )] TJ ET BT 26.250 81.277 Td /F1 9.8 Tf [(timely progression to in-vivo testing we utilized a lentiviral system to drive expression of N171 mHTT and test our candidate )] TJ ET BT 26.250 69.372 Td /F1 9.8 Tf [(genes in mouse brain \(Figs. 5-6\).)] TJ ET BT 26.250 49.968 Td /F1 9.8 Tf [(Consistent with reports of lentivirus-induced HD in rats showing no behavioral changes)] TJ ET 0.267 0.267 0.267 rg BT 400.143 51.475 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 409.780 49.968 Td /F1 9.8 Tf [(, effects of striatal expression of N171-)] TJ ET BT 26.250 38.063 Td /F1 9.8 Tf [(82Q mHTT in mice on spontaneous wheel running activity were not found \(Figs. 5-6\). N171 HTT expression was found mainly )] TJ ET Q q 15.000 23.777 577.500 753.223 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Validation of a lentiviral mouse model of HD)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Lentiviral vectors have been used to model HD in rats and macaques)] TJ ET 0.267 0.267 0.267 rg BT 324.298 749.579 Td /F4 8.7 Tf [(40)] TJ ET 0.271 0.267 0.267 rg BT 333.935 748.071 Td /F1 9.8 Tf [(. We used a four plasmid lentiviral system previously )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(described by de Almeida et al in rats)] TJ ET 0.267 0.267 0.267 rg BT 183.400 737.674 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 193.038 736.167 Td /F1 9.8 Tf [( to model HD in wild-type mice. This system drives expression of the N171 HTT fragment )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(under the control of the phosphoglycerate kinase promoter providing neuronal expression at physiologically relevant protein )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(levels. Mice were injected with virus at ~8 weeks and sacrificed as ~14 weeks of age. Lentiviral-mediated expression of N171-)] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(18Q and 82Q HTT resulted in detectable expression by immunofluorescence staining of brain sections \(Fig. 4A\). However, )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(neuronal expression was only found immediately around the needle tract. Western blot analysis failed to detect N171-18Q but )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(detected N171-82Q \(Fig. 4B\). Therefore, both wild-type and mutant N171 protein were expressed in brain; higher expression of )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(mHTT may be related to its accumulation as part of the disease process)] TJ ET 0.267 0.267 0.267 rg BT 336.748 666.245 Td /F4 8.7 Tf [(41)] TJ ET 0.271 0.267 0.267 rg BT 346.386 664.738 Td /F1 9.8 Tf [(. We chose to use striatal neuronal cell body volume )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(as our main outcome as cell atrophy is a consistent manifestation of mHTT expression in neurons)] TJ ET 0.267 0.267 0.267 rg BT 446.748 654.341 Td /F4 8.7 Tf [(42)] TJ ET 0.271 0.267 0.267 rg BT 456.385 652.833 Td /F1 9.8 Tf [(. In this preliminary study, )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(while differences were not statistically significant \(p=0.2202\) striatal neuronal cell body volume means were lower in the N171-)] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(82Q \(n=11\) versus N171-18Q \(n=5\) group \(520 53 and 639 78 m3, respectively\).)] TJ ET BT 26.250 609.619 Td /F4 9.8 Tf [(TRX1 and TMX3 decrease neuronal atrophy in mouse HD)] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(We sub-cloned cDNA-encoding TMX3 and TRX1 into the same lentiviral expression system used for N171 expression)] TJ ET 0.267 0.267 0.267 rg BT 534.517 591.722 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 544.155 590.214 Td /F1 9.8 Tf [( then )] TJ ET BT 26.250 578.310 Td /F1 9.8 Tf [(generated enzymatically inactive variants by point mutagenesis for use as gene-specific controls for enzymatic activity of the )] TJ ET BT 26.250 566.405 Td /F1 9.8 Tf [(test protein. We removed the histidine tags during the sub-cloning process. While we did not attempt to show protein expression )] TJ ET BT 26.250 554.500 Td /F1 9.8 Tf [(in brain, we did demonstrate in-vivo transcript expression of TMX3 and TRX1 in liver 6 weeks after intra-venous injection of )] TJ ET BT 26.250 542.595 Td /F1 9.8 Tf [(neonatal mice \(not shown\). We then undertook experiments in which we compared the effects of N171-18Q, N171-82Q, and )] TJ ET BT 26.250 530.691 Td /F1 9.8 Tf [(N171-82Q with TMX3 or TRX1 following mouse striatal injection. To control for the total level of lentiviral delivery to striatum we )] TJ ET BT 26.250 518.786 Td /F1 9.8 Tf [(co-injected virus encoding mutant \(inactive\) TMX3 or TRX1 in the N171-18Q and N171-82Q treatment groups. Striatal injections )] TJ ET BT 26.250 506.881 Td /F1 9.8 Tf [(were at ~8 weeks and mice were sacrificed at ~14 weeks of age. We completed confocal stereology to quantify striatal neuronal )] TJ ET BT 26.250 494.976 Td /F1 9.8 Tf [(cell body volume. As our candidate treatments were chosen based on their ability to decrease mHTT it is possible that mHTT )] TJ ET BT 26.250 483.072 Td /F1 9.8 Tf [(would not be detected in an infected cell by immunofluorescence and that the transduced cell would not be quantified. )] TJ ET BT 26.250 471.167 Td /F1 9.8 Tf [(Therefore, we changed our methodologic approach. We stained brain sections for mHTT then captured confocal images in )] TJ ET BT 26.250 459.262 Td /F1 9.8 Tf [(regions where there was some neuronal mHTT staining. We then quantified cell body volume for all neurons within an image )] TJ ET BT 26.250 447.357 Td /F1 9.8 Tf [(stack. As shown in Fig. 5 \(TMX3 experiment\) and Fig. 6 \(TRX1 experiment\) we found that striatal neuronal cell body volumes )] TJ ET BT 26.250 435.453 Td /F1 9.8 Tf [(were significantly decreased in N171-82Q versus N171-18Q expressing mice \(p-values: 0.0350 and 0.0035, respectively\). )] TJ ET BT 26.250 423.548 Td /F1 9.8 Tf [(Functionally active TMX3 and TRX1 both decreased this effect of mHTT on striatal neuronal atrophy \(p-values: 0.0387 and )] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(0.0046, respectively\). Due to the presence of detectable mHTT staining only around the needle tract quantification of soluble )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(mHTT levels by Western blot analysis following brain dissection would not have been reliable therefore we did not attempt this. )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(Finally, we studied TRX1 and TMX3 transcript levels by qPCR in 14-week old N171-82Q transgenic HD mice \(equivalent to )] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(early-advanced disease)] TJ ET 0.267 0.267 0.267 rg BT 129.210 377.436 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 138.847 375.929 Td /F1 9.8 Tf [(\); these mice express the N171-82Q HTT fragment under the control of the prion promoter)] TJ ET 0.267 0.267 0.267 rg BT 527.385 377.436 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 532.204 375.929 Td /F1 9.8 Tf [( \(Fig. 7\). )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(There was no evidence of decreased expression of both genes in striatum and cerebral cortex; in fact, in striatal TRX1 transcript )] TJ ET BT 26.250 352.119 Td /F1 9.8 Tf [(expression was significantly increased in HD mice \(p<0.01\) \(Fig. 7A\). We additionally completed searches of publically available )] TJ ET BT 26.250 340.215 Td /F1 9.8 Tf [(HD micro-array data sets using GeoProfiles. In the R6/1 mouse model of HD there was an increase in cerebral TXN1 transcript )] TJ ET BT 26.250 328.310 Td /F1 9.8 Tf [(from 22-27 weeks of age with 1 of 2 probes; TMX3 transcript changes were not found)] TJ ET 0.267 0.267 0.267 rg BT 394.215 329.817 Td /F4 8.7 Tf [(43)] TJ ET 0.271 0.267 0.267 rg BT 403.852 328.310 Td /F1 9.8 Tf [(. Micro-array analysis in 12 and 24 )] TJ ET BT 26.250 316.405 Td /F1 9.8 Tf [(month old full-length mHTT expressing YAC128 HD and wild-type litter-mate striata did not reveal expression differences for )] TJ ET BT 26.250 304.500 Td /F1 9.8 Tf [(TXN1 or TMX3)] TJ ET 0.267 0.267 0.267 rg BT 91.263 306.008 Td /F4 8.7 Tf [(44)] TJ ET 0.271 0.267 0.267 rg BT 100.900 304.500 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 267.898 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 247.944 Td /F1 9.8 Tf [(Abnormal redox homeostasis and oxidative stress are consistent features of human HD and cell-based and animal models)] TJ ET 0.267 0.267 0.267 rg BT 553.510 249.451 Td /F4 8.7 Tf [(45)] TJ ET 0.271 0.267 0.267 rg BT 563.148 251.832 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 565.557 249.451 Td /F4 8.7 Tf [(46)] TJ ET 0.271 0.267 0.267 rg BT 575.195 247.944 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 236.039 Td /F1 9.8 Tf [(Identification of appropriate targets for modulation of redox homeostasis could provide novel therapeutic approaches for treating )] TJ ET BT 26.250 224.134 Td /F1 9.8 Tf [(HD. Protein thiols are an important site of post-translational modification involved in the regulation of redox responsive cell )] TJ ET BT 26.250 212.229 Td /F1 9.8 Tf [(signaling processes)] TJ ET 0.267 0.267 0.267 rg BT 111.865 213.737 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 121.502 212.229 Td /F1 9.8 Tf [(. Oxidative stress can result in increased protein thiol oxidation and disruption of these homeostatic )] TJ ET BT 26.250 200.325 Td /F1 9.8 Tf [(processes, potentially contributing to cell dysfunction and degeneration. Transgenic mice expressing the N171 mHTT protein )] TJ ET BT 26.250 188.420 Td /F1 9.8 Tf [(fragment develop a phenotype similar to human HD including striatal atrophy)] TJ ET 0.267 0.267 0.267 rg BT 356.804 189.927 Td /F4 8.7 Tf [(47)] TJ ET 0.271 0.267 0.267 rg BT 366.442 188.420 Td /F1 9.8 Tf [(. We have shown that the N171 fragment of HTT )] TJ ET BT 26.250 176.515 Td /F1 9.8 Tf [(can form thiol-dependent oligomers which are degraded more slowly than a N171 protein variant that lacks thiols and is unable )] TJ ET BT 26.250 164.610 Td /F1 9.8 Tf [(to oligomerize)] TJ ET 0.267 0.267 0.267 rg BT 86.934 166.118 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 91.753 164.610 Td /F1 9.8 Tf [(. Numerous proteins with thiol-disulfide oxidoreductase activity exist that facilitate the reduction of oxidized )] TJ ET BT 26.250 152.706 Td /F1 9.8 Tf [(protein thiols in cells)] TJ ET 0.267 0.267 0.267 rg BT 114.029 154.213 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 123.667 152.706 Td /F1 9.8 Tf [(. Here we sought to identify if there are thiol-disulfide oxidoreductase enzymes that can decrease mHTT )] TJ ET BT 26.250 140.801 Td /F1 9.8 Tf [(levels in cells and provide protection against neuronal atrophy in HD mice. We tested a representative set of thiol-disulfide )] TJ ET BT 26.250 128.896 Td /F1 9.8 Tf [(oxidoreductases for mHTT decreasing effects. We used primary and secondary cell-based screens to identify candidate genes )] TJ ET BT 26.250 116.991 Td /F1 9.8 Tf [(for testing in HD mice \(Fig. 1\). Based on our previous findings)] TJ ET 0.267 0.267 0.267 rg BT 292.308 118.499 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 297.127 116.991 Td /F1 9.8 Tf [( enzymatic conversion of mHTT oligomer to monomer by a thiol-)] TJ ET BT 26.250 105.087 Td /F1 9.8 Tf [(disulfide oxidoreductase is expected to result in increased monomer degradation and potentially no change in monomer to )] TJ ET BT 26.250 93.182 Td /F1 9.8 Tf [(oligomer ratio; we therefore quantified total soluble mHTT levels by reducing SDS-PAGE in our cell-based studies. To enable )] TJ ET BT 26.250 81.277 Td /F1 9.8 Tf [(timely progression to in-vivo testing we utilized a lentiviral system to drive expression of N171 mHTT and test our candidate )] TJ ET BT 26.250 69.372 Td /F1 9.8 Tf [(genes in mouse brain \(Figs. 5-6\).)] TJ ET BT 26.250 49.968 Td /F1 9.8 Tf [(Consistent with reports of lentivirus-induced HD in rats showing no behavioral changes)] TJ ET 0.267 0.267 0.267 rg BT 400.143 51.475 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 409.780 49.968 Td /F1 9.8 Tf [(, effects of striatal expression of N171-)] TJ ET BT 26.250 38.063 Td /F1 9.8 Tf [(82Q mHTT in mice on spontaneous wheel running activity were not found \(Figs. 5-6\). N171 HTT expression was found mainly )] TJ ET Q q 15.000 23.777 577.500 753.223 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Validation of a lentiviral mouse model of HD)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Lentiviral vectors have been used to model HD in rats and macaques)] TJ ET 0.267 0.267 0.267 rg BT 324.298 749.579 Td /F4 8.7 Tf [(40)] TJ ET 0.271 0.267 0.267 rg BT 333.935 748.071 Td /F1 9.8 Tf [(. We used a four plasmid lentiviral system previously )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(described by de Almeida et al in rats)] TJ ET 0.267 0.267 0.267 rg BT 183.400 737.674 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 193.038 736.167 Td /F1 9.8 Tf [( to model HD in wild-type mice. This system drives expression of the N171 HTT fragment )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(under the control of the phosphoglycerate kinase promoter providing neuronal expression at physiologically relevant protein )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(levels. Mice were injected with virus at ~8 weeks and sacrificed as ~14 weeks of age. Lentiviral-mediated expression of N171-)] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(18Q and 82Q HTT resulted in detectable expression by immunofluorescence staining of brain sections \(Fig. 4A\). However, )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(neuronal expression was only found immediately around the needle tract. Western blot analysis failed to detect N171-18Q but )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(detected N171-82Q \(Fig. 4B\). Therefore, both wild-type and mutant N171 protein were expressed in brain; higher expression of )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(mHTT may be related to its accumulation as part of the disease process)] TJ ET 0.267 0.267 0.267 rg BT 336.748 666.245 Td /F4 8.7 Tf [(41)] TJ ET 0.271 0.267 0.267 rg BT 346.386 664.738 Td /F1 9.8 Tf [(. We chose to use striatal neuronal cell body volume )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(as our main outcome as cell atrophy is a consistent manifestation of mHTT expression in neurons)] TJ ET 0.267 0.267 0.267 rg BT 446.748 654.341 Td /F4 8.7 Tf [(42)] TJ ET 0.271 0.267 0.267 rg BT 456.385 652.833 Td /F1 9.8 Tf [(. In this preliminary study, )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(while differences were not statistically significant \(p=0.2202\) striatal neuronal cell body volume means were lower in the N171-)] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(82Q \(n=11\) versus N171-18Q \(n=5\) group \(520 53 and 639 78 m3, respectively\).)] TJ ET BT 26.250 609.619 Td /F4 9.8 Tf [(TRX1 and TMX3 decrease neuronal atrophy in mouse HD)] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(We sub-cloned cDNA-encoding TMX3 and TRX1 into the same lentiviral expression system used for N171 expression)] TJ ET 0.267 0.267 0.267 rg BT 534.517 591.722 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 544.155 590.214 Td /F1 9.8 Tf [( then )] TJ ET BT 26.250 578.310 Td /F1 9.8 Tf [(generated enzymatically inactive variants by point mutagenesis for use as gene-specific controls for enzymatic activity of the )] TJ ET BT 26.250 566.405 Td /F1 9.8 Tf [(test protein. We removed the histidine tags during the sub-cloning process. While we did not attempt to show protein expression )] TJ ET BT 26.250 554.500 Td /F1 9.8 Tf [(in brain, we did demonstrate in-vivo transcript expression of TMX3 and TRX1 in liver 6 weeks after intra-venous injection of )] TJ ET BT 26.250 542.595 Td /F1 9.8 Tf [(neonatal mice \(not shown\). We then undertook experiments in which we compared the effects of N171-18Q, N171-82Q, and )] TJ ET BT 26.250 530.691 Td /F1 9.8 Tf [(N171-82Q with TMX3 or TRX1 following mouse striatal injection. To control for the total level of lentiviral delivery to striatum we )] TJ ET BT 26.250 518.786 Td /F1 9.8 Tf [(co-injected virus encoding mutant \(inactive\) TMX3 or TRX1 in the N171-18Q and N171-82Q treatment groups. Striatal injections )] TJ ET BT 26.250 506.881 Td /F1 9.8 Tf [(were at ~8 weeks and mice were sacrificed at ~14 weeks of age. We completed confocal stereology to quantify striatal neuronal )] TJ ET BT 26.250 494.976 Td /F1 9.8 Tf [(cell body volume. As our candidate treatments were chosen based on their ability to decrease mHTT it is possible that mHTT )] TJ ET BT 26.250 483.072 Td /F1 9.8 Tf [(would not be detected in an infected cell by immunofluorescence and that the transduced cell would not be quantified. )] TJ ET BT 26.250 471.167 Td /F1 9.8 Tf [(Therefore, we changed our methodologic approach. We stained brain sections for mHTT then captured confocal images in )] TJ ET BT 26.250 459.262 Td /F1 9.8 Tf [(regions where there was some neuronal mHTT staining. We then quantified cell body volume for all neurons within an image )] TJ ET BT 26.250 447.357 Td /F1 9.8 Tf [(stack. As shown in Fig. 5 \(TMX3 experiment\) and Fig. 6 \(TRX1 experiment\) we found that striatal neuronal cell body volumes )] TJ ET BT 26.250 435.453 Td /F1 9.8 Tf [(were significantly decreased in N171-82Q versus N171-18Q expressing mice \(p-values: 0.0350 and 0.0035, respectively\). )] TJ ET BT 26.250 423.548 Td /F1 9.8 Tf [(Functionally active TMX3 and TRX1 both decreased this effect of mHTT on striatal neuronal atrophy \(p-values: 0.0387 and )] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(0.0046, respectively\). Due to the presence of detectable mHTT staining only around the needle tract quantification of soluble )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(mHTT levels by Western blot analysis following brain dissection would not have been reliable therefore we did not attempt this. )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(Finally, we studied TRX1 and TMX3 transcript levels by qPCR in 14-week old N171-82Q transgenic HD mice \(equivalent to )] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(early-advanced disease)] TJ ET 0.267 0.267 0.267 rg BT 129.210 377.436 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 138.847 375.929 Td /F1 9.8 Tf [(\); these mice express the N171-82Q HTT fragment under the control of the prion promoter)] TJ ET 0.267 0.267 0.267 rg BT 527.385 377.436 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 532.204 375.929 Td /F1 9.8 Tf [( \(Fig. 7\). )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(There was no evidence of decreased expression of both genes in striatum and cerebral cortex; in fact, in striatal TRX1 transcript )] TJ ET BT 26.250 352.119 Td /F1 9.8 Tf [(expression was significantly increased in HD mice \(p<0.01\) \(Fig. 7A\). We additionally completed searches of publically available )] TJ ET BT 26.250 340.215 Td /F1 9.8 Tf [(HD micro-array data sets using GeoProfiles. In the R6/1 mouse model of HD there was an increase in cerebral TXN1 transcript )] TJ ET BT 26.250 328.310 Td /F1 9.8 Tf [(from 22-27 weeks of age with 1 of 2 probes; TMX3 transcript changes were not found)] TJ ET 0.267 0.267 0.267 rg BT 394.215 329.817 Td /F4 8.7 Tf [(43)] TJ ET 0.271 0.267 0.267 rg BT 403.852 328.310 Td /F1 9.8 Tf [(. Micro-array analysis in 12 and 24 )] TJ ET BT 26.250 316.405 Td /F1 9.8 Tf [(month old full-length mHTT expressing YAC128 HD and wild-type litter-mate striata did not reveal expression differences for )] TJ ET BT 26.250 304.500 Td /F1 9.8 Tf [(TXN1 or TMX3)] TJ ET 0.267 0.267 0.267 rg BT 91.263 306.008 Td /F4 8.7 Tf [(44)] TJ ET 0.271 0.267 0.267 rg BT 100.900 304.500 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 267.898 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 247.944 Td /F1 9.8 Tf [(Abnormal redox homeostasis and oxidative stress are consistent features of human HD and cell-based and animal models)] TJ ET 0.267 0.267 0.267 rg BT 553.510 249.451 Td /F4 8.7 Tf [(45)] TJ ET 0.271 0.267 0.267 rg BT 563.148 251.832 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 565.557 249.451 Td /F4 8.7 Tf [(46)] TJ ET 0.271 0.267 0.267 rg BT 575.195 247.944 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 236.039 Td /F1 9.8 Tf [(Identification of appropriate targets for modulation of redox homeostasis could provide novel therapeutic approaches for treating )] TJ ET BT 26.250 224.134 Td /F1 9.8 Tf [(HD. Protein thiols are an important site of post-translational modification involved in the regulation of redox responsive cell )] TJ ET BT 26.250 212.229 Td /F1 9.8 Tf [(signaling processes)] TJ ET 0.267 0.267 0.267 rg BT 111.865 213.737 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 121.502 212.229 Td /F1 9.8 Tf [(. Oxidative stress can result in increased protein thiol oxidation and disruption of these homeostatic )] TJ ET BT 26.250 200.325 Td /F1 9.8 Tf [(processes, potentially contributing to cell dysfunction and degeneration. Transgenic mice expressing the N171 mHTT protein )] TJ ET BT 26.250 188.420 Td /F1 9.8 Tf [(fragment develop a phenotype similar to human HD including striatal atrophy)] TJ ET 0.267 0.267 0.267 rg BT 356.804 189.927 Td /F4 8.7 Tf [(47)] TJ ET 0.271 0.267 0.267 rg BT 366.442 188.420 Td /F1 9.8 Tf [(. We have shown that the N171 fragment of HTT )] TJ ET BT 26.250 176.515 Td /F1 9.8 Tf [(can form thiol-dependent oligomers which are degraded more slowly than a N171 protein variant that lacks thiols and is unable )] TJ ET BT 26.250 164.610 Td /F1 9.8 Tf [(to oligomerize)] TJ ET 0.267 0.267 0.267 rg BT 86.934 166.118 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 91.753 164.610 Td /F1 9.8 Tf [(. Numerous proteins with thiol-disulfide oxidoreductase activity exist that facilitate the reduction of oxidized )] TJ ET BT 26.250 152.706 Td /F1 9.8 Tf [(protein thiols in cells)] TJ ET 0.267 0.267 0.267 rg BT 114.029 154.213 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 123.667 152.706 Td /F1 9.8 Tf [(. Here we sought to identify if there are thiol-disulfide oxidoreductase enzymes that can decrease mHTT )] TJ ET BT 26.250 140.801 Td /F1 9.8 Tf [(levels in cells and provide protection against neuronal atrophy in HD mice. We tested a representative set of thiol-disulfide )] TJ ET BT 26.250 128.896 Td /F1 9.8 Tf [(oxidoreductases for mHTT decreasing effects. We used primary and secondary cell-based screens to identify candidate genes )] TJ ET BT 26.250 116.991 Td /F1 9.8 Tf [(for testing in HD mice \(Fig. 1\). Based on our previous findings)] TJ ET 0.267 0.267 0.267 rg BT 292.308 118.499 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 297.127 116.991 Td /F1 9.8 Tf [( enzymatic conversion of mHTT oligomer to monomer by a thiol-)] TJ ET BT 26.250 105.087 Td /F1 9.8 Tf [(disulfide oxidoreductase is expected to result in increased monomer degradation and potentially no change in monomer to )] TJ ET BT 26.250 93.182 Td /F1 9.8 Tf [(oligomer ratio; we therefore quantified total soluble mHTT levels by reducing SDS-PAGE in our cell-based studies. To enable )] TJ ET BT 26.250 81.277 Td /F1 9.8 Tf [(timely progression to in-vivo testing we utilized a lentiviral system to drive expression of N171 mHTT and test our candidate )] TJ ET BT 26.250 69.372 Td /F1 9.8 Tf [(genes in mouse brain \(Figs. 5-6\).)] TJ ET BT 26.250 49.968 Td /F1 9.8 Tf [(Consistent with reports of lentivirus-induced HD in rats showing no behavioral changes)] TJ ET 0.267 0.267 0.267 rg BT 400.143 51.475 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 409.780 49.968 Td /F1 9.8 Tf [(, effects of striatal expression of N171-)] TJ ET BT 26.250 38.063 Td /F1 9.8 Tf [(82Q mHTT in mice on spontaneous wheel running activity were not found \(Figs. 5-6\). N171 HTT expression was found mainly )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(5)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 341 0 obj << /Type /Annot /Subtype /Link /A 342 0 R /Border [0 0 0] /H /I /Rect [ 324.2978 748.7769 333.9351 757.5952 ] >> endobj 342 0 obj << /Type /Action >> endobj 343 0 obj << /Type /Annot /Subtype /Link /A 344 0 R /Border [0 0 0] /H /I /Rect [ 183.4005 736.8722 193.0378 745.6905 ] >> endobj 344 0 obj << /Type /Action >> endobj 345 0 obj << /Type /Annot /Subtype /Link /A 346 0 R /Border [0 0 0] /H /I /Rect [ 336.7485 665.4437 346.3858 674.2620 ] >> endobj 346 0 obj << /Type /Action >> endobj 347 0 obj << /Type /Annot /Subtype /Link /A 348 0 R /Border [0 0 0] /H /I /Rect [ 446.7480 653.5389 456.3853 662.3573 ] >> endobj 348 0 obj << /Type /Action >> endobj 349 0 obj << /Type /Annot /Subtype /Link /A 350 0 R /Border [0 0 0] /H /I /Rect [ 534.5175 590.9199 544.1548 599.7382 ] >> 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506 0 R 508 0 R 510 0 R 512 0 R 514 0 R 516 0 R 518 0 R 520 0 R 522 0 R 524 0 R 526 0 R 528 0 R 530 0 R 532 0 R 534 0 R 536 0 R 538 0 R 540 0 R 542 0 R 544 0 R 546 0 R 548 0 R 550 0 R 552 0 R 554 0 R 556 0 R 558 0 R 560 0 R 562 0 R 564 0 R 566 0 R 568 0 R 570 0 R 572 0 R 574 0 R 576 0 R 578 0 R 580 0 R 582 0 R 584 0 R 586 0 R 588 0 R 590 0 R 592 0 R 594 0 R ] /Contents 444 0 R >> endobj 444 0 obj << /Length 36766 >> stream 0.271 0.267 0.267 rg q 15.000 -348.107 577.500 1125.107 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(around the needle tract suggesting transduction of a small percentage of the overall striatal volume potentially explaining the )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(lack of a behavioral phenotype. However, by characterizing somal volume of neurons we were able to obtain a measure of the )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(effect of mHTT and test proteins TRX1 and TMX3. Neuronal atrophy is a consistent morphologic feature of HD and is frequently )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(used as a marker of therapeutic effect )] TJ ET 0.267 0.267 0.267 rg BT 192.624 733.269 Td /F4 8.7 Tf [(42)] TJ ET 0.271 0.267 0.267 rg BT 202.261 731.762 Td /F1 9.8 Tf [(. Based on this outcome, we provide evidence that both TRX1 and TMX3 have )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(protective effects in the lentiviral mouse HD system tested \(Figs. 5-6\).)] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(TRX1 is a well-characterized cytoplasmic and nuclear thiol-disulfide oxidoreductase that has previously been demonstrated to )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(have protective effects in models of acute and chronic neurodegeneration. TRX1 transgenic mice demonstrate increased )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(resistance to neuronal degeneration induced by transient focal ischemia)] TJ ET 0.267 0.267 0.267 rg BT 336.232 678.150 Td /F4 8.7 Tf [(48)] TJ ET 0.271 0.267 0.267 rg BT 345.869 676.643 Td /F1 9.8 Tf [(. TRX1 interacting protein \(TXNIP\) is an endogenous )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(inhibitor of TRX1 and is expressed in brain)] TJ ET 0.267 0.267 0.267 rg BT 209.950 666.245 Td /F4 8.7 Tf [(49)] TJ ET 0.271 0.267 0.267 rg BT 219.587 664.738 Td /F1 9.8 Tf [(. Furthermore, TXNIP inhibitors provide protection in a rodent model of )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(thromboembolic stroke)] TJ ET 0.267 0.267 0.267 rg BT 124.862 654.341 Td /F4 8.7 Tf [(50)] TJ ET 0.271 0.267 0.267 rg BT 134.499 652.833 Td /F1 9.8 Tf [(. TRX1 has also been shown to promote neurogenesis and cognitive recovery following cerebral )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(ischemia in mice)] TJ ET 0.267 0.267 0.267 rg BT 98.302 642.436 Td /F4 8.7 Tf [(51)] TJ ET 0.271 0.267 0.267 rg BT 107.940 640.929 Td /F1 9.8 Tf [(. DJ-1 is an anti-oxidant protein; mutations in DJ-1 cause autosomal recessive early-onset Parkinsons )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(disease)] TJ ET 0.267 0.267 0.267 rg BT 59.849 630.531 Td /F4 8.7 Tf [(52)] TJ ET 0.271 0.267 0.267 rg BT 69.486 629.024 Td /F1 9.8 Tf [(. In one study, it was shown that DJ-1 mediates its neuroprotective effects by stimulating Nrf2-mediated upregulation )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(of TRX1)] TJ ET 0.267 0.267 0.267 rg BT 62.013 618.626 Td /F4 8.7 Tf [(53)] TJ ET 0.271 0.267 0.267 rg BT 71.650 617.119 Td /F1 9.8 Tf [(. The protective effect of 17?-estradiol in the tumor necrosis factor model of optic neuropathy is also mediated by )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(TRX1)] TJ ET 0.267 0.267 0.267 rg BT 51.171 606.722 Td /F4 8.7 Tf [(54)] TJ ET 0.271 0.267 0.267 rg BT 60.808 605.214 Td /F1 9.8 Tf [(. Protective effects of TRX1 in disparate models of neuronal degeneration are consistent with its key role in redox )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(regulation of signaling pathways and repair of oxidatively-modified thiols within diverse proteins. The current findings )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(demonstrate that protective effects of TRX1 also extend to a model of mouse HD.)] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(To determine if the effect of TRX1 on decreasing N171 mHTT \(Fig. 1\) is the result of a direct effect of TRX1 on N171-40Q we )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(used a previously reported approach that utilizes a TRX1 variant to trap the intermediate catalytic state of TRX1 di-sulfide linked )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(to its substrate protein as a heterodimer)] TJ ET 0.267 0.267 0.267 rg BT 198.035 539.698 Td /F4 8.7 Tf [(34)] TJ ET 0.271 0.267 0.267 rg BT 207.673 538.191 Td /F1 9.8 Tf [(. We used purified N171-40Q HTT and TRX1 or mutant TRX1 in a cell-free assay to )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(maximize the chances of finding an interaction. Despite this, we found no evidence that N171-40Q HTT is a direct substrate of )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(TRX1 \(Fig. 3\). As this result could be because the proteins expressed in bacteria failed to fold properly, we undertook similar )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(experiments in transfected COS cells but also failed to find evidence for a disulfide-linked heterodimer species \(not shown\). )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(Therefore, while we cannot fully exclude the possibility, we have no data to indicate that N171 HTT disulfides)] TJ ET 0.267 0.267 0.267 rg BT 495.001 492.079 Td /F4 8.7 Tf [(55)] TJ ET 0.271 0.267 0.267 rg BT 504.638 490.572 Td /F1 9.8 Tf [( may be a direct )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(substrate of TXN1.)] TJ ET BT 26.250 459.262 Td /F1 9.8 Tf [(TRX1 has many substrate proteins)] TJ ET 0.267 0.267 0.267 rg BT 176.351 460.769 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 185.989 463.150 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 188.398 460.769 Td /F4 8.7 Tf [(56)] TJ ET 0.271 0.267 0.267 rg BT 198.035 459.262 Td /F1 9.8 Tf [(. Therefore, the neuronal atrophy decreasing effect of TRX1 that we observed \(Fig. 6\) )] TJ ET BT 26.250 447.357 Td /F1 9.8 Tf [(may be the result of effects on non-HTT targets. Peroxiredoxins are a family of redox proteins that regulate hydrogen and lipid )] TJ ET BT 26.250 435.453 Td /F1 9.8 Tf [(peroxide levels by oxidation of catalytic cysteine thiols then subsequent reductive re-activation. Thioredoxins activate oxidized )] TJ ET BT 26.250 423.548 Td /F1 9.8 Tf [(peroxiredoxins)] TJ ET 0.267 0.267 0.267 rg BT 89.644 425.055 Td /F4 8.7 Tf [(57)] TJ ET 0.271 0.267 0.267 rg BT 99.282 423.548 Td /F1 9.8 Tf [(. Importantly, it has been shown in a rat cell model of HD that there is increased thiol oxidization of )] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(peroxiredoxins 1, 2 and 4 implying a functionally inactive state. Further, treatment of this cell line with a dithiol compound )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(protected against mHTT-induced toxicity and decreased the level of peroxiredoxin 1 oxidation)] TJ ET 0.267 0.267 0.267 rg BT 428.886 401.246 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 438.523 399.738 Td /F1 9.8 Tf [(. Apoptosis signal-regulating )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(kinase \(ASK1\) is a mitogen-activated protein kinase kinase kinase and an important regulator of oxidative and ER stress-)] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(induced apoptosis)] TJ ET 0.267 0.267 0.267 rg BT 104.835 377.436 Td /F4 8.7 Tf [(58)] TJ ET 0.271 0.267 0.267 rg BT 114.472 375.929 Td /F1 9.8 Tf [(. Inhibition of ASK1 using intra-cerebral infusion of an antibody has protective effects in mouse HD, )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(decreasing ER stress and resulting in behavioral improvements)] TJ ET 0.267 0.267 0.267 rg BT 299.348 365.531 Td /F4 8.7 Tf [(59)] TJ ET 0.271 0.267 0.267 rg BT 308.985 364.024 Td /F1 9.8 Tf [(. TRX1 is a negative regulator of ASK1)] TJ ET 0.267 0.267 0.267 rg BT 476.441 365.531 Td /F4 8.7 Tf [(60)] TJ ET 0.271 0.267 0.267 rg BT 486.078 364.024 Td /F1 9.8 Tf [(; therefore, this is )] TJ ET BT 26.250 352.119 Td /F1 9.8 Tf [(another potential mechanism of protection in our model. Therefore, while a weakness of this study is that the mechanism of )] TJ ET BT 26.250 340.215 Td /F1 9.8 Tf [(protection by TRX1 in our HD mouse model is undetermined, there are several substrate proteins that are in HD-associated )] TJ ET BT 26.250 328.310 Td /F1 9.8 Tf [(pathways and that could be mediating protective effects.)] TJ ET BT 26.250 308.905 Td /F1 9.8 Tf [(TMX3, in contrast to TRX1, has not previously been linked to neuroprotection for any brain disorder. However, mutations in the )] TJ ET BT 26.250 297.000 Td /F1 9.8 Tf [(TMX3 gene have been linked with microphthalmia and retinal developmental anomalies)] TJ ET 0.267 0.267 0.267 rg BT 403.409 298.508 Td /F4 8.7 Tf [(61)] TJ ET 0.271 0.267 0.267 rg BT 413.047 297.000 Td /F1 9.8 Tf [(. TMX3 is a single domain )] TJ ET BT 26.250 285.096 Td /F1 9.8 Tf [(transmembrane protein. It is primarily located in the endoplasmic reticulum \(ER\), with its catalytic domain in the ER lumen)] TJ ET 0.267 0.267 0.267 rg BT 550.205 286.603 Td /F4 8.7 Tf [(62)] TJ ET 0.271 0.267 0.267 rg BT 559.843 285.096 Td /F1 9.8 Tf [( but )] TJ ET BT 26.250 273.191 Td /F1 9.8 Tf [(is also present in the mitochondrial-associated membrane)] TJ ET 0.267 0.267 0.267 rg BT 275.509 274.698 Td /F4 8.7 Tf [(63)] TJ ET 0.271 0.267 0.267 rg BT 285.146 273.191 Td /F1 9.8 Tf [(. Protein substrates of TMX3 have not been reported. Huntingtin )] TJ ET BT 26.250 261.286 Td /F1 9.8 Tf [(protein is associated with ER membranes and has a role in intra-cellular trafficking between the Golgi and extracellular space)] TJ ET 0.267 0.267 0.267 rg BT 564.879 262.793 Td /F4 8.7 Tf [(64)] TJ ET 0.271 0.267 0.267 rg BT 574.516 261.286 Td /F1 9.8 Tf [(; )] TJ ET BT 26.250 249.381 Td /F1 9.8 Tf [(however, it has not been shown to be present within the ER lumen. As the TMX3 catalytic domain and N171-40Q mHTT would )] TJ ET BT 26.250 237.477 Td /F1 9.8 Tf [(not be expected to be present within the same cell compartment it is improbable that the N171 fragment is a direct substrate of )] TJ ET BT 26.250 225.572 Td /F1 9.8 Tf [(TMX3. However, mHTT expression does induce ER stress)] TJ ET 0.267 0.267 0.267 rg BT 278.736 227.079 Td /F4 8.7 Tf [(65)] TJ ET 0.271 0.267 0.267 rg BT 288.373 225.572 Td /F1 9.8 Tf [(. Increased expression of TMX3 in striatum may protect against )] TJ ET BT 26.250 213.667 Td /F1 9.8 Tf [(mHTT-induced ER pathology.)] TJ ET BT 26.250 194.262 Td /F1 9.8 Tf [(In summary, we have identified TRX1 and TMX3 as proteins that decrease both mHTT levels in cultured cells and mHTT-)] TJ ET BT 26.250 182.358 Td /F1 9.8 Tf [(induced striatal neuronal atrophy in HD mice. These findings support a role of thiol stress in the pathogenesis of HD. While the )] TJ ET BT 26.250 170.453 Td /F1 9.8 Tf [(findings of this study are novel there are some limitations. First, lentiviral protein expression in brain was only found surrounding )] TJ ET BT 26.250 158.548 Td /F1 9.8 Tf [(the needle tract with less spread than has been reported previously in rats)] TJ ET 0.267 0.267 0.267 rg BT 345.445 160.055 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 355.083 158.548 Td /F1 9.8 Tf [(. While the findings from the morphometric analysis )] TJ ET BT 26.250 146.643 Td /F1 9.8 Tf [(of neuronal cell-body size indicate protective effects, selection bias in sampling offsets the strengths of the stereologic method )] TJ ET BT 26.250 134.739 Td /F1 9.8 Tf [(used. Second, while neuronal atrophy is an important feature of HD neurodegeneration)] TJ ET 0.267 0.267 0.267 rg BT 401.274 136.246 Td /F4 8.7 Tf [(42)] TJ ET 0.271 0.267 0.267 rg BT 410.911 134.739 Td /F1 9.8 Tf [( this was the only outcome for which )] TJ ET BT 26.250 122.834 Td /F1 9.8 Tf [(we found an effect of mHTT expression in our lentiviral model. Despite the weaknesses, the findings suggest that specific )] TJ ET BT 26.250 110.929 Td /F1 9.8 Tf [(modulation of thiol homeostasis has beneficial effects in HD models. Future studies could address if increased expression of )] TJ ET BT 26.250 99.024 Td /F1 9.8 Tf [(TRX1 and TMX3 globally in mouse HD brain provides protection against multiple measures of neurodegeneration.)] TJ ET 0.965 0.965 0.965 rg 26.250 -348.107 555.000 437.250 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 89.143 m 581.250 89.143 l 581.250 88.393 l 26.250 88.393 l f q 450.000 0 0 421.500 35.250 -342.107 cm /I3 Do Q q 35.250 -348.107 537.000 0.000 re W n Q Q q 15.000 -348.107 577.500 1125.107 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(around the needle tract suggesting transduction of a small percentage of the overall striatal volume potentially explaining the )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(lack of a behavioral phenotype. However, by characterizing somal volume of neurons we were able to obtain a measure of the )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(effect of mHTT and test proteins TRX1 and TMX3. Neuronal atrophy is a consistent morphologic feature of HD and is frequently )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(used as a marker of therapeutic effect )] TJ ET 0.267 0.267 0.267 rg BT 192.624 733.269 Td /F4 8.7 Tf [(42)] TJ ET 0.271 0.267 0.267 rg BT 202.261 731.762 Td /F1 9.8 Tf [(. Based on this outcome, we provide evidence that both TRX1 and TMX3 have )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(protective effects in the lentiviral mouse HD system tested \(Figs. 5-6\).)] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(TRX1 is a well-characterized cytoplasmic and nuclear thiol-disulfide oxidoreductase that has previously been demonstrated to )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(have protective effects in models of acute and chronic neurodegeneration. TRX1 transgenic mice demonstrate increased )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(resistance to neuronal degeneration induced by transient focal ischemia)] TJ ET 0.267 0.267 0.267 rg BT 336.232 678.150 Td /F4 8.7 Tf [(48)] TJ ET 0.271 0.267 0.267 rg BT 345.869 676.643 Td /F1 9.8 Tf [(. TRX1 interacting protein \(TXNIP\) is an endogenous )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(inhibitor of TRX1 and is expressed in brain)] TJ ET 0.267 0.267 0.267 rg BT 209.950 666.245 Td /F4 8.7 Tf [(49)] TJ ET 0.271 0.267 0.267 rg BT 219.587 664.738 Td /F1 9.8 Tf [(. Furthermore, TXNIP inhibitors provide protection in a rodent model of )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(thromboembolic stroke)] TJ ET 0.267 0.267 0.267 rg BT 124.862 654.341 Td /F4 8.7 Tf [(50)] TJ ET 0.271 0.267 0.267 rg BT 134.499 652.833 Td /F1 9.8 Tf [(. TRX1 has also been shown to promote neurogenesis and cognitive recovery following cerebral )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(ischemia in mice)] TJ ET 0.267 0.267 0.267 rg BT 98.302 642.436 Td /F4 8.7 Tf [(51)] TJ ET 0.271 0.267 0.267 rg BT 107.940 640.929 Td /F1 9.8 Tf [(. DJ-1 is an anti-oxidant protein; mutations in DJ-1 cause autosomal recessive early-onset Parkinsons )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(disease)] TJ ET 0.267 0.267 0.267 rg BT 59.849 630.531 Td /F4 8.7 Tf [(52)] TJ ET 0.271 0.267 0.267 rg BT 69.486 629.024 Td /F1 9.8 Tf [(. In one study, it was shown that DJ-1 mediates its neuroprotective effects by stimulating Nrf2-mediated upregulation )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(of TRX1)] TJ ET 0.267 0.267 0.267 rg BT 62.013 618.626 Td /F4 8.7 Tf [(53)] TJ ET 0.271 0.267 0.267 rg BT 71.650 617.119 Td /F1 9.8 Tf [(. The protective effect of 17?-estradiol in the tumor necrosis factor model of optic neuropathy is also mediated by )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(TRX1)] TJ ET 0.267 0.267 0.267 rg BT 51.171 606.722 Td /F4 8.7 Tf [(54)] TJ ET 0.271 0.267 0.267 rg BT 60.808 605.214 Td /F1 9.8 Tf [(. Protective effects of TRX1 in disparate models of neuronal degeneration are consistent with its key role in redox )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(regulation of signaling pathways and repair of oxidatively-modified thiols within diverse proteins. The current findings )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(demonstrate that protective effects of TRX1 also extend to a model of mouse HD.)] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(To determine if the effect of TRX1 on decreasing N171 mHTT \(Fig. 1\) is the result of a direct effect of TRX1 on N171-40Q we )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(used a previously reported approach that utilizes a TRX1 variant to trap the intermediate catalytic state of TRX1 di-sulfide linked )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(to its substrate protein as a heterodimer)] TJ ET 0.267 0.267 0.267 rg BT 198.035 539.698 Td /F4 8.7 Tf [(34)] TJ ET 0.271 0.267 0.267 rg BT 207.673 538.191 Td /F1 9.8 Tf [(. We used purified N171-40Q HTT and TRX1 or mutant TRX1 in a cell-free assay to )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(maximize the chances of finding an interaction. Despite this, we found no evidence that N171-40Q HTT is a direct substrate of )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(TRX1 \(Fig. 3\). As this result could be because the proteins expressed in bacteria failed to fold properly, we undertook similar )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(experiments in transfected COS cells but also failed to find evidence for a disulfide-linked heterodimer species \(not shown\). )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(Therefore, while we cannot fully exclude the possibility, we have no data to indicate that N171 HTT disulfides)] TJ ET 0.267 0.267 0.267 rg BT 495.001 492.079 Td /F4 8.7 Tf [(55)] TJ ET 0.271 0.267 0.267 rg BT 504.638 490.572 Td /F1 9.8 Tf [( may be a direct )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(substrate of TXN1.)] TJ ET BT 26.250 459.262 Td /F1 9.8 Tf [(TRX1 has many substrate proteins)] TJ ET 0.267 0.267 0.267 rg BT 176.351 460.769 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 185.989 463.150 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 188.398 460.769 Td /F4 8.7 Tf [(56)] TJ ET 0.271 0.267 0.267 rg BT 198.035 459.262 Td /F1 9.8 Tf [(. Therefore, the neuronal atrophy decreasing effect of TRX1 that we observed \(Fig. 6\) )] TJ ET BT 26.250 447.357 Td /F1 9.8 Tf [(may be the result of effects on non-HTT targets. Peroxiredoxins are a family of redox proteins that regulate hydrogen and lipid )] TJ ET BT 26.250 435.453 Td /F1 9.8 Tf [(peroxide levels by oxidation of catalytic cysteine thiols then subsequent reductive re-activation. Thioredoxins activate oxidized )] TJ ET BT 26.250 423.548 Td /F1 9.8 Tf [(peroxiredoxins)] TJ ET 0.267 0.267 0.267 rg BT 89.644 425.055 Td /F4 8.7 Tf [(57)] TJ ET 0.271 0.267 0.267 rg BT 99.282 423.548 Td /F1 9.8 Tf [(. Importantly, it has been shown in a rat cell model of HD that there is increased thiol oxidization of )] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(peroxiredoxins 1, 2 and 4 implying a functionally inactive state. Further, treatment of this cell line with a dithiol compound )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(protected against mHTT-induced toxicity and decreased the level of peroxiredoxin 1 oxidation)] TJ ET 0.267 0.267 0.267 rg BT 428.886 401.246 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 438.523 399.738 Td /F1 9.8 Tf [(. Apoptosis signal-regulating )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(kinase \(ASK1\) is a mitogen-activated protein kinase kinase kinase and an important regulator of oxidative and ER stress-)] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(induced apoptosis)] TJ ET 0.267 0.267 0.267 rg BT 104.835 377.436 Td /F4 8.7 Tf [(58)] TJ ET 0.271 0.267 0.267 rg BT 114.472 375.929 Td /F1 9.8 Tf [(. Inhibition of ASK1 using intra-cerebral infusion of an antibody has protective effects in mouse HD, )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(decreasing ER stress and resulting in behavioral improvements)] TJ ET 0.267 0.267 0.267 rg BT 299.348 365.531 Td /F4 8.7 Tf [(59)] TJ ET 0.271 0.267 0.267 rg BT 308.985 364.024 Td /F1 9.8 Tf [(. TRX1 is a negative regulator of ASK1)] TJ ET 0.267 0.267 0.267 rg BT 476.441 365.531 Td /F4 8.7 Tf [(60)] TJ ET 0.271 0.267 0.267 rg BT 486.078 364.024 Td /F1 9.8 Tf [(; therefore, this is )] TJ ET BT 26.250 352.119 Td /F1 9.8 Tf [(another potential mechanism of protection in our model. Therefore, while a weakness of this study is that the mechanism of )] TJ ET BT 26.250 340.215 Td /F1 9.8 Tf [(protection by TRX1 in our HD mouse model is undetermined, there are several substrate proteins that are in HD-associated )] TJ ET BT 26.250 328.310 Td /F1 9.8 Tf [(pathways and that could be mediating protective effects.)] TJ ET BT 26.250 308.905 Td /F1 9.8 Tf [(TMX3, in contrast to TRX1, has not previously been linked to neuroprotection for any brain disorder. However, mutations in the )] TJ ET BT 26.250 297.000 Td /F1 9.8 Tf [(TMX3 gene have been linked with microphthalmia and retinal developmental anomalies)] TJ ET 0.267 0.267 0.267 rg BT 403.409 298.508 Td /F4 8.7 Tf [(61)] TJ ET 0.271 0.267 0.267 rg BT 413.047 297.000 Td /F1 9.8 Tf [(. TMX3 is a single domain )] TJ ET BT 26.250 285.096 Td /F1 9.8 Tf [(transmembrane protein. It is primarily located in the endoplasmic reticulum \(ER\), with its catalytic domain in the ER lumen)] TJ ET 0.267 0.267 0.267 rg BT 550.205 286.603 Td /F4 8.7 Tf [(62)] TJ ET 0.271 0.267 0.267 rg BT 559.843 285.096 Td /F1 9.8 Tf [( but )] TJ ET BT 26.250 273.191 Td /F1 9.8 Tf [(is also present in the mitochondrial-associated membrane)] TJ ET 0.267 0.267 0.267 rg BT 275.509 274.698 Td /F4 8.7 Tf [(63)] TJ ET 0.271 0.267 0.267 rg BT 285.146 273.191 Td /F1 9.8 Tf [(. Protein substrates of TMX3 have not been reported. Huntingtin )] TJ ET BT 26.250 261.286 Td /F1 9.8 Tf [(protein is associated with ER membranes and has a role in intra-cellular trafficking between the Golgi and extracellular space)] TJ ET 0.267 0.267 0.267 rg BT 564.879 262.793 Td /F4 8.7 Tf [(64)] TJ ET 0.271 0.267 0.267 rg BT 574.516 261.286 Td /F1 9.8 Tf [(; )] TJ ET BT 26.250 249.381 Td /F1 9.8 Tf [(however, it has not been shown to be present within the ER lumen. As the TMX3 catalytic domain and N171-40Q mHTT would )] TJ ET BT 26.250 237.477 Td /F1 9.8 Tf [(not be expected to be present within the same cell compartment it is improbable that the N171 fragment is a direct substrate of )] TJ ET BT 26.250 225.572 Td /F1 9.8 Tf [(TMX3. However, mHTT expression does induce ER stress)] TJ ET 0.267 0.267 0.267 rg BT 278.736 227.079 Td /F4 8.7 Tf [(65)] TJ ET 0.271 0.267 0.267 rg BT 288.373 225.572 Td /F1 9.8 Tf [(. Increased expression of TMX3 in striatum may protect against )] TJ ET BT 26.250 213.667 Td /F1 9.8 Tf [(mHTT-induced ER pathology.)] TJ ET BT 26.250 194.262 Td /F1 9.8 Tf [(In summary, we have identified TRX1 and TMX3 as proteins that decrease both mHTT levels in cultured cells and mHTT-)] TJ ET BT 26.250 182.358 Td /F1 9.8 Tf [(induced striatal neuronal atrophy in HD mice. These findings support a role of thiol stress in the pathogenesis of HD. While the )] TJ ET BT 26.250 170.453 Td /F1 9.8 Tf [(findings of this study are novel there are some limitations. First, lentiviral protein expression in brain was only found surrounding )] TJ ET BT 26.250 158.548 Td /F1 9.8 Tf [(the needle tract with less spread than has been reported previously in rats)] TJ ET 0.267 0.267 0.267 rg BT 345.445 160.055 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 355.083 158.548 Td /F1 9.8 Tf [(. While the findings from the morphometric analysis )] TJ ET BT 26.250 146.643 Td /F1 9.8 Tf [(of neuronal cell-body size indicate protective effects, selection bias in sampling offsets the strengths of the stereologic method )] TJ ET BT 26.250 134.739 Td /F1 9.8 Tf [(used. Second, while neuronal atrophy is an important feature of HD neurodegeneration)] TJ ET 0.267 0.267 0.267 rg BT 401.274 136.246 Td /F4 8.7 Tf [(42)] TJ ET 0.271 0.267 0.267 rg BT 410.911 134.739 Td /F1 9.8 Tf [( this was the only outcome for which )] TJ ET BT 26.250 122.834 Td /F1 9.8 Tf [(we found an effect of mHTT expression in our lentiviral model. Despite the weaknesses, the findings suggest that specific )] TJ ET BT 26.250 110.929 Td /F1 9.8 Tf [(modulation of thiol homeostasis has beneficial effects in HD models. Future studies could address if increased expression of )] TJ ET BT 26.250 99.024 Td /F1 9.8 Tf [(TRX1 and TMX3 globally in mouse HD brain provides protection against multiple measures of neurodegeneration.)] TJ ET 0.965 0.965 0.965 rg 26.250 -348.107 555.000 437.250 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 89.143 m 581.250 89.143 l 581.250 88.393 l 26.250 88.393 l f q 450.000 0 0 421.500 35.250 -342.107 cm /I3 Do Q q 35.250 -348.107 537.000 0.000 re W n Q Q q 15.000 -348.107 577.500 1125.107 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(around the needle tract suggesting transduction of a small percentage of the overall striatal volume potentially explaining the )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(lack of a behavioral phenotype. However, by characterizing somal volume of neurons we were able to obtain a measure of the )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(effect of mHTT and test proteins TRX1 and TMX3. Neuronal atrophy is a consistent morphologic feature of HD and is frequently )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(used as a marker of therapeutic effect )] TJ ET 0.267 0.267 0.267 rg BT 192.624 733.269 Td /F4 8.7 Tf [(42)] TJ ET 0.271 0.267 0.267 rg BT 202.261 731.762 Td /F1 9.8 Tf [(. Based on this outcome, we provide evidence that both TRX1 and TMX3 have )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(protective effects in the lentiviral mouse HD system tested \(Figs. 5-6\).)] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(TRX1 is a well-characterized cytoplasmic and nuclear thiol-disulfide oxidoreductase that has previously been demonstrated to )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(have protective effects in models of acute and chronic neurodegeneration. TRX1 transgenic mice demonstrate increased )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(resistance to neuronal degeneration induced by transient focal ischemia)] TJ ET 0.267 0.267 0.267 rg BT 336.232 678.150 Td /F4 8.7 Tf [(48)] TJ ET 0.271 0.267 0.267 rg BT 345.869 676.643 Td /F1 9.8 Tf [(. TRX1 interacting protein \(TXNIP\) is an endogenous )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(inhibitor of TRX1 and is expressed in brain)] TJ ET 0.267 0.267 0.267 rg BT 209.950 666.245 Td /F4 8.7 Tf [(49)] TJ ET 0.271 0.267 0.267 rg BT 219.587 664.738 Td /F1 9.8 Tf [(. Furthermore, TXNIP inhibitors provide protection in a rodent model of )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(thromboembolic stroke)] TJ ET 0.267 0.267 0.267 rg BT 124.862 654.341 Td /F4 8.7 Tf [(50)] TJ ET 0.271 0.267 0.267 rg BT 134.499 652.833 Td /F1 9.8 Tf [(. TRX1 has also been shown to promote neurogenesis and cognitive recovery following cerebral )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(ischemia in mice)] TJ ET 0.267 0.267 0.267 rg BT 98.302 642.436 Td /F4 8.7 Tf [(51)] TJ ET 0.271 0.267 0.267 rg BT 107.940 640.929 Td /F1 9.8 Tf [(. DJ-1 is an anti-oxidant protein; mutations in DJ-1 cause autosomal recessive early-onset Parkinsons )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(disease)] TJ ET 0.267 0.267 0.267 rg BT 59.849 630.531 Td /F4 8.7 Tf [(52)] TJ ET 0.271 0.267 0.267 rg BT 69.486 629.024 Td /F1 9.8 Tf [(. In one study, it was shown that DJ-1 mediates its neuroprotective effects by stimulating Nrf2-mediated upregulation )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(of TRX1)] TJ ET 0.267 0.267 0.267 rg BT 62.013 618.626 Td /F4 8.7 Tf [(53)] TJ ET 0.271 0.267 0.267 rg BT 71.650 617.119 Td /F1 9.8 Tf [(. The protective effect of 17?-estradiol in the tumor necrosis factor model of optic neuropathy is also mediated by )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(TRX1)] TJ ET 0.267 0.267 0.267 rg BT 51.171 606.722 Td /F4 8.7 Tf [(54)] TJ ET 0.271 0.267 0.267 rg BT 60.808 605.214 Td /F1 9.8 Tf [(. Protective effects of TRX1 in disparate models of neuronal degeneration are consistent with its key role in redox )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(regulation of signaling pathways and repair of oxidatively-modified thiols within diverse proteins. The current findings )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(demonstrate that protective effects of TRX1 also extend to a model of mouse HD.)] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(To determine if the effect of TRX1 on decreasing N171 mHTT \(Fig. 1\) is the result of a direct effect of TRX1 on N171-40Q we )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(used a previously reported approach that utilizes a TRX1 variant to trap the intermediate catalytic state of TRX1 di-sulfide linked )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(to its substrate protein as a heterodimer)] TJ ET 0.267 0.267 0.267 rg BT 198.035 539.698 Td /F4 8.7 Tf [(34)] TJ ET 0.271 0.267 0.267 rg BT 207.673 538.191 Td /F1 9.8 Tf [(. We used purified N171-40Q HTT and TRX1 or mutant TRX1 in a cell-free assay to )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(maximize the chances of finding an interaction. Despite this, we found no evidence that N171-40Q HTT is a direct substrate of )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(TRX1 \(Fig. 3\). As this result could be because the proteins expressed in bacteria failed to fold properly, we undertook similar )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(experiments in transfected COS cells but also failed to find evidence for a disulfide-linked heterodimer species \(not shown\). )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(Therefore, while we cannot fully exclude the possibility, we have no data to indicate that N171 HTT disulfides)] TJ ET 0.267 0.267 0.267 rg BT 495.001 492.079 Td /F4 8.7 Tf [(55)] TJ ET 0.271 0.267 0.267 rg BT 504.638 490.572 Td /F1 9.8 Tf [( may be a direct )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(substrate of TXN1.)] TJ ET BT 26.250 459.262 Td /F1 9.8 Tf [(TRX1 has many substrate proteins)] TJ ET 0.267 0.267 0.267 rg BT 176.351 460.769 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 185.989 463.150 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 188.398 460.769 Td /F4 8.7 Tf [(56)] TJ ET 0.271 0.267 0.267 rg BT 198.035 459.262 Td /F1 9.8 Tf [(. Therefore, the neuronal atrophy decreasing effect of TRX1 that we observed \(Fig. 6\) )] TJ ET BT 26.250 447.357 Td /F1 9.8 Tf [(may be the result of effects on non-HTT targets. Peroxiredoxins are a family of redox proteins that regulate hydrogen and lipid )] TJ ET BT 26.250 435.453 Td /F1 9.8 Tf [(peroxide levels by oxidation of catalytic cysteine thiols then subsequent reductive re-activation. Thioredoxins activate oxidized )] TJ ET BT 26.250 423.548 Td /F1 9.8 Tf [(peroxiredoxins)] TJ ET 0.267 0.267 0.267 rg BT 89.644 425.055 Td /F4 8.7 Tf [(57)] TJ ET 0.271 0.267 0.267 rg BT 99.282 423.548 Td /F1 9.8 Tf [(. Importantly, it has been shown in a rat cell model of HD that there is increased thiol oxidization of )] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(peroxiredoxins 1, 2 and 4 implying a functionally inactive state. Further, treatment of this cell line with a dithiol compound )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(protected against mHTT-induced toxicity and decreased the level of peroxiredoxin 1 oxidation)] TJ ET 0.267 0.267 0.267 rg BT 428.886 401.246 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 438.523 399.738 Td /F1 9.8 Tf [(. Apoptosis signal-regulating )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(kinase \(ASK1\) is a mitogen-activated protein kinase kinase kinase and an important regulator of oxidative and ER stress-)] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(induced apoptosis)] TJ ET 0.267 0.267 0.267 rg BT 104.835 377.436 Td /F4 8.7 Tf [(58)] TJ ET 0.271 0.267 0.267 rg BT 114.472 375.929 Td /F1 9.8 Tf [(. Inhibition of ASK1 using intra-cerebral infusion of an antibody has protective effects in mouse HD, )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(decreasing ER stress and resulting in behavioral improvements)] TJ ET 0.267 0.267 0.267 rg BT 299.348 365.531 Td /F4 8.7 Tf [(59)] TJ ET 0.271 0.267 0.267 rg BT 308.985 364.024 Td /F1 9.8 Tf [(. TRX1 is a negative regulator of ASK1)] TJ ET 0.267 0.267 0.267 rg BT 476.441 365.531 Td /F4 8.7 Tf [(60)] TJ ET 0.271 0.267 0.267 rg BT 486.078 364.024 Td /F1 9.8 Tf [(; therefore, this is )] TJ ET BT 26.250 352.119 Td /F1 9.8 Tf [(another potential mechanism of protection in our model. Therefore, while a weakness of this study is that the mechanism of )] TJ ET BT 26.250 340.215 Td /F1 9.8 Tf [(protection by TRX1 in our HD mouse model is undetermined, there are several substrate proteins that are in HD-associated )] TJ ET BT 26.250 328.310 Td /F1 9.8 Tf [(pathways and that could be mediating protective effects.)] TJ ET BT 26.250 308.905 Td /F1 9.8 Tf [(TMX3, in contrast to TRX1, has not previously been linked to neuroprotection for any brain disorder. However, mutations in the )] TJ ET BT 26.250 297.000 Td /F1 9.8 Tf [(TMX3 gene have been linked with microphthalmia and retinal developmental anomalies)] TJ ET 0.267 0.267 0.267 rg BT 403.409 298.508 Td /F4 8.7 Tf [(61)] TJ ET 0.271 0.267 0.267 rg BT 413.047 297.000 Td /F1 9.8 Tf [(. TMX3 is a single domain )] TJ ET BT 26.250 285.096 Td /F1 9.8 Tf [(transmembrane protein. It is primarily located in the endoplasmic reticulum \(ER\), with its catalytic domain in the ER lumen)] TJ ET 0.267 0.267 0.267 rg BT 550.205 286.603 Td /F4 8.7 Tf [(62)] TJ ET 0.271 0.267 0.267 rg BT 559.843 285.096 Td /F1 9.8 Tf [( but )] TJ ET BT 26.250 273.191 Td /F1 9.8 Tf [(is also present in the mitochondrial-associated membrane)] TJ ET 0.267 0.267 0.267 rg BT 275.509 274.698 Td /F4 8.7 Tf [(63)] TJ ET 0.271 0.267 0.267 rg BT 285.146 273.191 Td /F1 9.8 Tf [(. Protein substrates of TMX3 have not been reported. Huntingtin )] TJ ET BT 26.250 261.286 Td /F1 9.8 Tf [(protein is associated with ER membranes and has a role in intra-cellular trafficking between the Golgi and extracellular space)] TJ ET 0.267 0.267 0.267 rg BT 564.879 262.793 Td /F4 8.7 Tf [(64)] TJ ET 0.271 0.267 0.267 rg BT 574.516 261.286 Td /F1 9.8 Tf [(; )] TJ ET BT 26.250 249.381 Td /F1 9.8 Tf [(however, it has not been shown to be present within the ER lumen. As the TMX3 catalytic domain and N171-40Q mHTT would )] TJ ET BT 26.250 237.477 Td /F1 9.8 Tf [(not be expected to be present within the same cell compartment it is improbable that the N171 fragment is a direct substrate of )] TJ ET BT 26.250 225.572 Td /F1 9.8 Tf [(TMX3. However, mHTT expression does induce ER stress)] TJ ET 0.267 0.267 0.267 rg BT 278.736 227.079 Td /F4 8.7 Tf [(65)] TJ ET 0.271 0.267 0.267 rg BT 288.373 225.572 Td /F1 9.8 Tf [(. Increased expression of TMX3 in striatum may protect against )] TJ ET BT 26.250 213.667 Td /F1 9.8 Tf [(mHTT-induced ER pathology.)] TJ ET BT 26.250 194.262 Td /F1 9.8 Tf [(In summary, we have identified TRX1 and TMX3 as proteins that decrease both mHTT levels in cultured cells and mHTT-)] TJ ET BT 26.250 182.358 Td /F1 9.8 Tf [(induced striatal neuronal atrophy in HD mice. These findings support a role of thiol stress in the pathogenesis of HD. While the )] TJ ET BT 26.250 170.453 Td /F1 9.8 Tf [(findings of this study are novel there are some limitations. First, lentiviral protein expression in brain was only found surrounding )] TJ ET BT 26.250 158.548 Td /F1 9.8 Tf [(the needle tract with less spread than has been reported previously in rats)] TJ ET 0.267 0.267 0.267 rg BT 345.445 160.055 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 355.083 158.548 Td /F1 9.8 Tf [(. While the findings from the morphometric analysis )] TJ ET BT 26.250 146.643 Td /F1 9.8 Tf [(of neuronal cell-body size indicate protective effects, selection bias in sampling offsets the strengths of the stereologic method )] TJ ET BT 26.250 134.739 Td /F1 9.8 Tf [(used. Second, while neuronal atrophy is an important feature of HD neurodegeneration)] TJ ET 0.267 0.267 0.267 rg BT 401.274 136.246 Td /F4 8.7 Tf [(42)] TJ ET 0.271 0.267 0.267 rg BT 410.911 134.739 Td /F1 9.8 Tf [( this was the only outcome for which )] TJ ET BT 26.250 122.834 Td /F1 9.8 Tf [(we found an effect of mHTT expression in our lentiviral model. Despite the weaknesses, the findings suggest that specific )] TJ ET BT 26.250 110.929 Td /F1 9.8 Tf [(modulation of thiol homeostasis has beneficial effects in HD models. Future studies could address if increased expression of )] TJ ET BT 26.250 99.024 Td /F1 9.8 Tf [(TRX1 and TMX3 globally in mouse HD brain provides protection against multiple measures of neurodegeneration.)] TJ ET 0.965 0.965 0.965 rg 26.250 -348.107 555.000 437.250 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 89.143 m 581.250 89.143 l 581.250 88.393 l 26.250 88.393 l f q 450.000 0 0 421.500 35.250 -342.107 cm /I3 Do Q q 35.250 -348.107 537.000 0.000 re W n Q Q q 450.000 0 0 421.500 35.250 -342.107 cm /I3 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(6)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 445 0 obj << /Type /Annot /Subtype /Link /A 446 0 R /Border [0 0 0] /H /I /Rect [ 192.6240 732.4674 202.2613 741.2858 ] >> endobj 446 0 obj << /Type /Action >> endobj 447 0 obj << /Type /Annot /Subtype /Link /A 448 0 R /Border [0 0 0] /H /I /Rect [ 336.2317 677.3484 345.8691 686.1667 ] >> endobj 448 0 obj << 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Twenty-four hours later cells were lysed and analyzed by reducing SDS-PAGE and Western blot )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(analysis to quantify total soluble N171 mhtt. N171-40Q mutant huntingtin levels \(MAB5492 Millipore\) were normalized to )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(actin then to co-transfection control. TRX1 and TMX3 were chosen as targets that may decrease mhtt levels; glutaredoxin 1 )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(\(GLX1\), PDIA6 and FLJ44606 were chosen as targets that may increase mhtt. See results for more details. Shown are )] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(mean SEM. n=3, B-F. Secondary screen. COS1 cells were co-transfected with plasmids encoding N171-40Q and wild-)] TJ ET BT 35.250 653.784 Td /F1 9.8 Tf [(type or enzymatically non-functional \(control\) human thiol-disulfide oxidoreductases. Twenty-four hours later cells were )] TJ ET BT 35.250 640.048 Td /F1 9.8 Tf [(lysed for reducing SDS-PAGE and western blot analysis. Candidate gene expression was confirmed by PCR. The )] TJ ET BT 35.250 626.311 Td /F1 9.8 Tf [(letter/number codes above the right western blot lanes are the substitution of the mutant inactive protein. n=3-5, B. )] TJ ET BT 35.250 612.575 Td /F1 9.8 Tf [(Glutaredoxin 1 \(GLX1\) increases soluble N171-40Q mhtt levels. n=5, C. Thioredoxin 1 \(TRX1\) decreases soluble N171-)] TJ ET BT 35.250 598.839 Td /F1 9.8 Tf [(40Q mhtt levels. n=5, D. TMX3 decreases soluble N171-40Q mhtt levels. n=4, E. FLJ44606 has no effect on N171-40Q )] TJ ET BT 35.250 585.103 Td /F1 9.8 Tf [(mhtt levels. n=4, F. Protein disulfide isomerase A6 \(PDIA6\) has no effect on N171-40Q mhtt levels. n=5. P-values: *<0.05 )] TJ ET BT 35.250 571.366 Td /F1 9.8 Tf [(and **<0.01.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 260.269 555.000 282.100 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 542.369 m 581.250 542.369 l 581.250 541.619 l 26.250 541.619 l f 26.250 260.269 m 581.250 260.269 l 581.250 261.019 l 26.250 261.019 l f q 276.750 0 0 174.750 35.250 357.869 cm /I4 Do Q q 35.250 271.519 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 342.345 Td /F4 9.8 Tf [(Fig. 2: TRX1 and TMX3 do not decrease levels of thiol-blocked N171-40Q mutant huntingtin)] TJ ET BT 35.250 322.975 Td /F1 9.8 Tf [(A-B. COS1 cells were co-transfected with plasmids encoding mhtt with cysteines mutated to alanines \(N171-40Q-4CA\) and )] TJ ET BT 35.250 309.239 Td /F1 9.8 Tf [(TRX1 / TMX3. The letter/number codes above the right western blot lanes are the substitution of the mutant inactive )] TJ ET BT 35.250 295.503 Td /F1 9.8 Tf [(protein. A. TRX1 does not decrease soluble N171-40Q-4CA levels. B. TMX3 increases N171-40Q-4CA levels. n=4. P-)] TJ ET BT 35.250 281.767 Td /F1 9.8 Tf [(values: *<0.05.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 -104.981 555.000 357.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 252.769 m 581.250 252.769 l 581.250 252.019 l 26.250 252.019 l f q 450.000 0 0 342.000 35.250 -98.981 cm /I5 Do Q q 35.250 -104.981 537.000 0.000 re W n Q Q q 15.000 -104.981 577.500 881.981 re W n 0.965 0.965 0.965 rg 26.250 549.869 555.000 227.131 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 549.869 m 581.250 549.869 l 581.250 550.619 l 26.250 550.619 l f q 35.250 561.119 537.000 215.881 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: Primary and secondary screens for thiol-disulfide oxidoreductases that change total soluble mutant )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(huntingtin protein levels)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(COS1 cells were transfected with plasmids encoding human thiol-disulfide oxidoreductase proteins and N171-40Q mutant )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(huntingtin protein \(mhtt\). Twenty-four hours later cells were lysed and analyzed by reducing SDS-PAGE and Western blot )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(analysis to quantify total soluble N171 mhtt. N171-40Q mutant huntingtin levels \(MAB5492 Millipore\) were normalized to )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(actin then to co-transfection control. TRX1 and TMX3 were chosen as targets that may decrease mhtt levels; glutaredoxin 1 )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(\(GLX1\), PDIA6 and FLJ44606 were chosen as targets that may increase mhtt. See results for more details. Shown are )] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(mean SEM. n=3, B-F. Secondary screen. COS1 cells were co-transfected with plasmids encoding N171-40Q and wild-)] TJ ET BT 35.250 653.784 Td /F1 9.8 Tf [(type or enzymatically non-functional \(control\) human thiol-disulfide oxidoreductases. Twenty-four hours later cells were )] TJ ET BT 35.250 640.048 Td /F1 9.8 Tf [(lysed for reducing SDS-PAGE and western blot analysis. Candidate gene expression was confirmed by PCR. The )] TJ ET BT 35.250 626.311 Td /F1 9.8 Tf [(letter/number codes above the right western blot lanes are the substitution of the mutant inactive protein. n=3-5, B. )] TJ ET BT 35.250 612.575 Td /F1 9.8 Tf [(Glutaredoxin 1 \(GLX1\) increases soluble N171-40Q mhtt levels. n=5, C. Thioredoxin 1 \(TRX1\) decreases soluble N171-)] TJ ET BT 35.250 598.839 Td /F1 9.8 Tf [(40Q mhtt levels. n=5, D. TMX3 decreases soluble N171-40Q mhtt levels. n=4, E. FLJ44606 has no effect on N171-40Q )] TJ ET BT 35.250 585.103 Td /F1 9.8 Tf [(mhtt levels. n=4, F. Protein disulfide isomerase A6 \(PDIA6\) has no effect on N171-40Q mhtt levels. n=5. P-values: *<0.05 )] TJ ET BT 35.250 571.366 Td /F1 9.8 Tf [(and **<0.01.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 260.269 555.000 282.100 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 542.369 m 581.250 542.369 l 581.250 541.619 l 26.250 541.619 l f 26.250 260.269 m 581.250 260.269 l 581.250 261.019 l 26.250 261.019 l f q 276.750 0 0 174.750 35.250 357.869 cm /I4 Do Q q 35.250 271.519 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 342.345 Td /F4 9.8 Tf [(Fig. 2: TRX1 and TMX3 do not decrease levels of thiol-blocked N171-40Q mutant huntingtin)] TJ ET BT 35.250 322.975 Td /F1 9.8 Tf [(A-B. COS1 cells were co-transfected with plasmids encoding mhtt with cysteines mutated to alanines \(N171-40Q-4CA\) and )] TJ ET BT 35.250 309.239 Td /F1 9.8 Tf [(TRX1 / TMX3. The letter/number codes above the right western blot lanes are the substitution of the mutant inactive )] TJ ET BT 35.250 295.503 Td /F1 9.8 Tf [(protein. A. TRX1 does not decrease soluble N171-40Q-4CA levels. B. TMX3 increases N171-40Q-4CA levels. n=4. P-)] TJ ET BT 35.250 281.767 Td /F1 9.8 Tf [(values: *<0.05.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 -104.981 555.000 357.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 252.769 m 581.250 252.769 l 581.250 252.019 l 26.250 252.019 l f q 450.000 0 0 342.000 35.250 -98.981 cm /I5 Do Q q 35.250 -104.981 537.000 0.000 re W n Q Q q 276.750 0 0 174.750 35.250 357.869 cm /I4 Do Q q 450.000 0 0 342.000 35.250 -98.981 cm /I5 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(7)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 598 0 obj << /Type /Annot /Subtype /Link /A 599 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 357.8692 312.0000 532.6192 ] >> endobj 599 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2015/08/Fig2.jpg) >> endobj 600 0 obj << /Type /XObject /Subtype /Image /Width 369 /Height 233 /ColorSpace /DeviceCMYK /Decode [1 0 1 0 1 0 1 0] /Filter /DCTDecode /BitsPerComponent 8 /Length 131131>> 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OgAj|prN?JUsӚ Zt$ MHRGJrFRrI }(An)2hBG-i18Sh\JWnWZ0H@JcF3  Qq-ӧ6S c"`aɠwN]Xc8ǰ~w]5Vր@p}1JIN+0289(4⤌c[QJ秥5 s5 ޜ0sA\&: @델$zS GN~(.[{P̽F1R`?*C@ rhrOqx%F}ypH9}\|g)8PFߥ#xI QAs gC9#R]7S@PӚMpcchc B9{ Er2(;lpqPz6#>]XNSNO*VJ`y?Z8҆'ӭ !Fiflyx:_3 šXpC~)H\G+LӃ(ݐ{i ۱CtbŁμ~4Sl7pzy9?N(j(ǽ-%-C^3:#aIwq"F2ƊYU =5hm x]#]ћڈn셤$Yc,]TÅnyQ^_6~C.+ck 5G_Z %wE̐ 0׭FSIGg%1D-pz˿N rO:'? vÛ4Kī;eV7p+u ' ƀ5O mO:%&JP1p]qҼc_owxS:k1\O%8$e'8kĿbM^^-q|LE<;=" #}H2Ks` HˀIfo3ĢQ@ ^7V)=ӡM^ǁ^?Svt?ɨ#qy,gi\KmTld["0n 1Xړ1OHaW^2(zj cPtFr?:/aG̍n+>8^ md$cy}kY~"HIBq/a$š񜯘SO9>*x$ u)0;l],ÊW35G.#O8"t%40eH@`*nO=Wu%ݹ,4mNkH,؂Xgہƾ`ocT.X!5wl0GM}, 8_;~qkV͗)0PœοΊ]1= Ƚ>PRPQE-%PKIKހ“(-%-%s^qk&4]?w$/ǿ /ʊ0:WKK@e',)4]KQ^iz}y[Dk, 2WxANhiuilW̊D( AP\Ӽd!HK `D"Tt1+Wn \4)59eu-5+I5i\8bILWQڀ8? ~EVqxFٴmգmѼ-VC)95q/?tM2O5iΣ]^\ΎVY&Ƙ-``q^K@~ϟ |Cjzo jZ܍5އk,99,Kzj3%> zץ@h?f?J|-fτw\[.dH@VF #k("~tl?ɫFkiŇ>AD1*d\S:SP[e*@`isW#Rp:ހ怠{֜BOҀ0u Uƞ4BK 8;Y, HhB=y䝣־6J njXR@? u*mp6_kQ=`8_zBs ~ Vmτ,m>SVk6T2gT@}qR~FOMӎ-WP Xq_R|\O3< )w:Vݭp;p(~,z7hpڤZŜryE}өi#r+վߟihֵ+v Z_b;z_H\|yk t?cnЪ|s@?૿]j0gOGbs_Rv;ӣ,^b$5Ǧ8-DOQP?m%A(8aۊz?uXrOE}]4~bssm%`$">6kas zͬ[n8.Ӥf܊Oӭ'*84#ހ>OP֣?g[œjǐUqrDc\V[6Fz$к61fL Qx}+Oib]̣g$'0{V>gpe]FcW|ryI ?^bf$7+br@5 p8`k!GϨ5$|>H? {K 9XF3ӌk{–R0-p+lqN|?k)UnmnYLhNEKB=( ( J(PE(;EQE/j(9((Q@yG1P ?mj(FsJHA¾:r Pٳ yG=sV#9!W! FycMa"8b0(8 (느ٹ*PM8FQI1(Z7t<{Toj۰Sb( - q늊[d#c.(ؘ V̀()k!9۞1PL!zT-jQE0Z9iACFvPw,@;p?$E!["Y@ ?(?ʿR}984Q@Eg o)rEnLl3)l?!MPX扈>Ƣm \|墊h'Kbzm +m2U?4׉ߕPEørO~wi7(F_-3L9pWqE^͈+F]ACZw_( ?tVν( endstream endobj 604 0 obj << /Type /Annot /Subtype /Link /A 605 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 357.8692 312.0000 532.6192 ] >> endobj 605 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2015/08/Fig2.jpg) >> endobj 606 0 obj << /Type /Annot /Subtype /Link /A 607 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 -98.9805 485.2500 243.0195 ] >> endobj 607 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2015/10/Fig3rb.jpg) >> endobj 608 0 obj << /Type /Annot /Subtype /Link /A 609 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 357.8692 312.0000 532.6192 ] >> endobj 609 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2015/08/Fig2.jpg) >> endobj 610 0 obj << /Type /Annot /Subtype /Link /A 611 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 -98.9805 485.2500 243.0195 ] >> endobj 611 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2015/10/Fig3rb.jpg) >> endobj 612 0 obj << /Type /Page /Parent 3 0 R /Annots [ 614 0 R 616 0 R 619 0 R 622 0 R 624 0 R 626 0 R 628 0 R 630 0 R 632 0 R ] /Contents 613 0 R >> endobj 613 0 obj << /Length 9824 >> stream q 15.000 -85.895 577.500 862.895 re W n 0.965 0.965 0.965 rg 26.250 657.928 555.000 119.072 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 657.928 m 581.250 657.928 l 581.250 658.678 l 26.250 658.678 l f q 35.250 669.178 537.000 107.822 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: TRX1 does not interact directly with N171-40Q mutant huntingtin in a cell-free assay)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(TRX1, mutant TRX1 \(C35S\) and N171-40Q were expressed and purified from bacteria. N171-40Q was incubated with )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(TRX1 or mTRX1 at 250 C for 1 hour then free thiols blocked with 50 mM N-ethylmaleimide. Samples were then analyzed by )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(SDS-PAGE \(see methods\). Lanes: 1=N171-40Q htt alone; 2=TRX1 alone; 3=mTRX1 alone; 4-5=N171-40Q and TRX1; and )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(6-7= N171-40Q and mTRX1. Mutant TRX1 \(C35S\) is predicted to trap substrate proteins as heterodimers [33]. There is no )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(evidence of a heterodimer band \(estimated mass = 46 kDa\) in the non-reduced N171-40Q and mTRX1 group \(lane 6\). The )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(band migrating at ~65 kDa \(most prominent in lanes 4 and 6\) is dimeric N171-40Q as previously reported)] TJ ET 0.267 0.267 0.267 rg BT 487.991 682.397 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 492.810 679.425 Td /F1 9.8 Tf [(.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 319.855 555.000 330.572 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 650.428 m 581.250 650.428 l 581.250 649.678 l 26.250 649.678 l f 26.250 319.855 m 581.250 319.855 l 581.250 320.605 l 26.250 320.605 l f q 450.000 0 0 195.750 35.250 444.928 cm /I6 Do Q q 35.250 331.105 537.000 107.822 re W n 0.271 0.267 0.267 rg BT 35.250 429.404 Td /F4 9.8 Tf [(Fig. 4: Characterization of huntingtin expression in mouse brain following lentiviral delivery)] TJ ET BT 35.250 410.034 Td /F1 9.8 Tf [(A-B. Lentivirus encoding N171 htt fragments was injected intra-striatally into 8-week-old mice; sacrifice was at 14-weeks of )] TJ ET BT 35.250 396.298 Td /F1 9.8 Tf [(age. A. Detection of N171 htt in mouse striata following stereotaxic injection. Brains sections at the level of the stereotaxic )] TJ ET BT 35.250 382.561 Td /F1 9.8 Tf [(injections were stained for N171 htt using MAB5492 \(Chemicon\) and with a fluorescent Nissl staining. Top left shows )] TJ ET BT 35.250 368.825 Td /F1 9.8 Tf [(detection of 171-18Q; top right shows detection of N171-82Q; bottom show fluorescent Nissl staining. B. Detection of htt )] TJ ET BT 35.250 355.089 Td /F1 9.8 Tf [(expression by Western blot analysis. Striata were dissected then homogenized to extract protein for Western blot analysis )] TJ ET BT 35.250 341.353 Td /F1 9.8 Tf [(using MAB5492. N171-18Q is not detected. N171-82Q is detected.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 -85.895 555.000 398.250 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 312.355 m 581.250 312.355 l 581.250 311.605 l 26.250 311.605 l f q 450.000 0 0 382.500 35.250 -79.895 cm /I7 Do Q q 35.250 -85.895 537.000 0.000 re W n Q Q q 15.000 -85.895 577.500 862.895 re W n 0.965 0.965 0.965 rg 26.250 657.928 555.000 119.072 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 657.928 m 581.250 657.928 l 581.250 658.678 l 26.250 658.678 l f q 35.250 669.178 537.000 107.822 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: TRX1 does not interact directly with N171-40Q mutant huntingtin in a cell-free assay)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(TRX1, mutant TRX1 \(C35S\) and N171-40Q were expressed and purified from bacteria. N171-40Q was incubated with )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(TRX1 or mTRX1 at 250 C for 1 hour then free thiols blocked with 50 mM N-ethylmaleimide. Samples were then analyzed by )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(SDS-PAGE \(see methods\). Lanes: 1=N171-40Q htt alone; 2=TRX1 alone; 3=mTRX1 alone; 4-5=N171-40Q and TRX1; and )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(6-7= N171-40Q and mTRX1. Mutant TRX1 \(C35S\) is predicted to trap substrate proteins as heterodimers [33]. There is no )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(evidence of a heterodimer band \(estimated mass = 46 kDa\) in the non-reduced N171-40Q and mTRX1 group \(lane 6\). The )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(band migrating at ~65 kDa \(most prominent in lanes 4 and 6\) is dimeric N171-40Q as previously reported)] TJ ET 0.267 0.267 0.267 rg BT 487.991 682.397 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 492.810 679.425 Td /F1 9.8 Tf [(.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 319.855 555.000 330.572 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 650.428 m 581.250 650.428 l 581.250 649.678 l 26.250 649.678 l f 26.250 319.855 m 581.250 319.855 l 581.250 320.605 l 26.250 320.605 l f q 450.000 0 0 195.750 35.250 444.928 cm /I6 Do Q q 35.250 331.105 537.000 107.822 re W n 0.271 0.267 0.267 rg BT 35.250 429.404 Td /F4 9.8 Tf [(Fig. 4: Characterization of huntingtin expression in mouse brain following lentiviral delivery)] TJ ET BT 35.250 410.034 Td /F1 9.8 Tf [(A-B. Lentivirus encoding N171 htt fragments was injected intra-striatally into 8-week-old mice; sacrifice was at 14-weeks of )] TJ ET BT 35.250 396.298 Td /F1 9.8 Tf [(age. A. Detection of N171 htt in mouse striata following stereotaxic injection. Brains sections at the level of the stereotaxic )] TJ ET BT 35.250 382.561 Td /F1 9.8 Tf [(injections were stained for N171 htt using MAB5492 \(Chemicon\) and with a fluorescent Nissl staining. Top left shows )] TJ ET BT 35.250 368.825 Td /F1 9.8 Tf [(detection of 171-18Q; top right shows detection of N171-82Q; bottom show fluorescent Nissl staining. B. Detection of htt )] TJ ET BT 35.250 355.089 Td /F1 9.8 Tf [(expression by Western blot analysis. Striata were dissected then homogenized to extract protein for Western blot analysis )] TJ ET BT 35.250 341.353 Td /F1 9.8 Tf [(using MAB5492. N171-18Q is not detected. N171-82Q is detected.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 -85.895 555.000 398.250 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 312.355 m 581.250 312.355 l 581.250 311.605 l 26.250 311.605 l f q 450.000 0 0 382.500 35.250 -79.895 cm /I7 Do Q q 35.250 -85.895 537.000 0.000 re W n Q Q q 15.000 -85.895 577.500 862.895 re W n 0.965 0.965 0.965 rg 26.250 657.928 555.000 119.072 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 657.928 m 581.250 657.928 l 581.250 658.678 l 26.250 658.678 l f q 35.250 669.178 537.000 107.822 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: TRX1 does not interact directly with N171-40Q mutant huntingtin in a cell-free assay)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(TRX1, mutant TRX1 \(C35S\) and N171-40Q were expressed and purified from bacteria. N171-40Q was incubated with )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(TRX1 or mTRX1 at 250 C for 1 hour then free thiols blocked with 50 mM N-ethylmaleimide. Samples were then analyzed by )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(SDS-PAGE \(see methods\). Lanes: 1=N171-40Q htt alone; 2=TRX1 alone; 3=mTRX1 alone; 4-5=N171-40Q and TRX1; and )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(6-7= N171-40Q and mTRX1. Mutant TRX1 \(C35S\) is predicted to trap substrate proteins as heterodimers [33]. There is no )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(evidence of a heterodimer band \(estimated mass = 46 kDa\) in the non-reduced N171-40Q and mTRX1 group \(lane 6\). The )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(band migrating at ~65 kDa \(most prominent in lanes 4 and 6\) is dimeric N171-40Q as previously reported)] TJ ET 0.267 0.267 0.267 rg BT 487.991 682.397 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 492.810 679.425 Td /F1 9.8 Tf [(.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 319.855 555.000 330.572 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 650.428 m 581.250 650.428 l 581.250 649.678 l 26.250 649.678 l f 26.250 319.855 m 581.250 319.855 l 581.250 320.605 l 26.250 320.605 l f q 450.000 0 0 195.750 35.250 444.928 cm /I6 Do Q q 35.250 331.105 537.000 107.822 re W n 0.271 0.267 0.267 rg BT 35.250 429.404 Td /F4 9.8 Tf [(Fig. 4: Characterization of huntingtin expression in mouse brain following lentiviral delivery)] TJ ET BT 35.250 410.034 Td /F1 9.8 Tf [(A-B. Lentivirus encoding N171 htt fragments was injected intra-striatally into 8-week-old mice; sacrifice was at 14-weeks of )] TJ ET BT 35.250 396.298 Td /F1 9.8 Tf [(age. A. Detection of N171 htt in mouse striata following stereotaxic injection. Brains sections at the level of the stereotaxic )] TJ ET BT 35.250 382.561 Td /F1 9.8 Tf [(injections were stained for N171 htt using MAB5492 \(Chemicon\) and with a fluorescent Nissl staining. Top left shows )] TJ ET BT 35.250 368.825 Td /F1 9.8 Tf [(detection of 171-18Q; top right shows detection of N171-82Q; bottom show fluorescent Nissl staining. B. Detection of htt )] TJ ET BT 35.250 355.089 Td /F1 9.8 Tf [(expression by Western blot analysis. Striata were dissected then homogenized to extract protein for Western blot analysis )] TJ ET BT 35.250 341.353 Td /F1 9.8 Tf [(using MAB5492. N171-18Q is not detected. N171-82Q is detected.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 -85.895 555.000 398.250 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 312.355 m 581.250 312.355 l 581.250 311.605 l 26.250 311.605 l f q 450.000 0 0 382.500 35.250 -79.895 cm /I7 Do Q q 35.250 -85.895 537.000 0.000 re W n Q Q q 450.000 0 0 195.750 35.250 444.928 cm /I6 Do Q q 450.000 0 0 382.500 35.250 -79.895 cm /I7 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(8)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 614 0 obj << /Type /Annot /Subtype /Link /A 615 0 R /Border [0 0 0] /H /I /Rect [ 487.9912 681.5957 492.8099 690.4140 ] >> endobj 615 0 obj << /Type /Action >> endobj 616 0 obj << /Type /Annot /Subtype /Link /A 617 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 444.9277 485.2500 640.6777 ] >> endobj 617 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2015/10/Fig4rb.jpg) >> endobj 618 0 obj << /Type /XObject /Subtype /Image /Width 600 /Height 261 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 30173>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   X" }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?$xQд ǘQ Xǻ6{6>?[_ x@MtR#i,2(r[ \/s~6ߍ5ŽxNghՙQa\s|>/ە%e6ވ1uq5:jFsn9(cclaZ[Ii4uN̄zVbcڀRcڌ{PRǵ(ǵ.(&=(qF)? ? \QL{QPRb€bcڀbcڀbcڀRcڌ{PR~~{Qj\U=^&T;[= U'[ǵe1.s>3ѿlOz_x'}ɖM.m-D7G++0;>\!0FFE~Oit=Uoz48tky_]%9'NU#?t׶V+OtF eǝ3 8c4ǵǵ.(&=ǵ.(&=(qF)1F=qF)((qF)? 1@ 1Ij1@ 1IQPRcڏ€bbcڀbcڀR~P4 n|amtI|Gmhj3nV"FY-5+_i'$g j9<3LӼ-h7Wroc$B @p?@+RbPъL{Q@ E!PnoOK*K/V)v:`i.;1\da77;vkNPnMBO]UT[h#uʍu61|-]ʾVuqrm t%ّbrza_5˹4Cmqy-+\U[T99'$rx?1ú gO5gmyQaifϴ$agDǷ>.fЮ|=$3\$'5ɔʌʍy5r/#|IWu:iZY;)-+,`@w'2kvv~$&Ԗ:sC gml+ Űm#Hk9nsͼ4d E ( Ac xw>5KPӴh\i-m]_0 *ܯv]ߋ~ xT5jwZlZ?d9-1~< y/]OZ+Ӯ],m u/i7dh;ԔԀ=Cĺ7 "ĺ帆Oo:xhb d* Maw^g-Ok~.ԴwY֥ iн}qًJ݁d,IQ@ E&(-RQ@M@x| /w=eyx! 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Mice were injected with lentiviral combinations \(methods\) at 8-weeks of age and sacrificed at 14-weeks. Brain sections )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(were stained for Nissl substance and mhtt. Confocal images were collected in the region where htt staining was detected. )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(Neuronal cell body volumes were determined using the nucleator method on confocal z-stack images \(methods\). A. )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(Representative images of fluorescent Nissl stained neurons in the region of the striatal injection site. Numbers below )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(images represent the number of mice per treatment group; numbers in parenthesis represent the minimum and maximum )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(number of neurons counted per mouse within the group. B. Striatal neuronal cell body volume. Mice expressing N171-82Q )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(and mutant \(inactive\) TMX3 \(mTMX3\) have significantly smaller neuronal cell bodies than mice expressing N171-82Q and )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(active TMX3. The main effect p-value is 0.0346; pair-wise comparison p-values are on graph. n=9-10. C. Spontaneous )] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(wheel running activity is not altered by N171-18/82Q and / or TMX3 expression. See methods for experimental details. n=10.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 37.719 555.000 571.500 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 609.219 m 581.250 609.219 l 581.250 608.469 l 26.250 608.469 l f q 450.000 0 0 555.750 35.250 43.719 cm /I8 Do Q q 35.250 37.719 537.000 0.000 re W n Q Q q 15.000 37.719 577.500 739.281 re W n 0.965 0.965 0.965 rg 26.250 616.719 555.000 160.281 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 616.719 m 581.250 616.719 l 581.250 617.469 l 26.250 617.469 l f q 35.250 627.969 537.000 149.031 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 5: TMX3 expression decreases striatal neuronal atrophy in HD mice)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(A-B. Mice were injected with lentiviral combinations \(methods\) at 8-weeks of age and sacrificed at 14-weeks. Brain sections )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(were stained for Nissl substance and mhtt. Confocal images were collected in the region where htt staining was detected. )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(Neuronal cell body volumes were determined using the nucleator method on confocal z-stack images \(methods\). A. )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(Representative images of fluorescent Nissl stained neurons in the region of the striatal injection site. Numbers below )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(images represent the number of mice per treatment group; numbers in parenthesis represent the minimum and maximum )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(number of neurons counted per mouse within the group. B. Striatal neuronal cell body volume. Mice expressing N171-82Q )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(and mutant \(inactive\) TMX3 \(mTMX3\) have significantly smaller neuronal cell bodies than mice expressing N171-82Q and )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(active TMX3. The main effect p-value is 0.0346; pair-wise comparison p-values are on graph. n=9-10. C. Spontaneous )] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(wheel running activity is not altered by N171-18/82Q and / or TMX3 expression. See methods for experimental details. n=10.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 37.719 555.000 571.500 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 609.219 m 581.250 609.219 l 581.250 608.469 l 26.250 608.469 l f q 450.000 0 0 555.750 35.250 43.719 cm /I8 Do Q q 35.250 37.719 537.000 0.000 re W n Q Q q 15.000 37.719 577.500 739.281 re W n 0.965 0.965 0.965 rg 26.250 616.719 555.000 160.281 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 616.719 m 581.250 616.719 l 581.250 617.469 l 26.250 617.469 l f q 35.250 627.969 537.000 149.031 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 5: TMX3 expression decreases striatal neuronal atrophy in HD mice)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(A-B. Mice were injected with lentiviral combinations \(methods\) at 8-weeks of age and sacrificed at 14-weeks. Brain sections )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(were stained for Nissl substance and mhtt. Confocal images were collected in the region where htt staining was detected. )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(Neuronal cell body volumes were determined using the nucleator method on confocal z-stack images \(methods\). A. )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(Representative images of fluorescent Nissl stained neurons in the region of the striatal injection site. Numbers below )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(images represent the number of mice per treatment group; numbers in parenthesis represent the minimum and maximum )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(number of neurons counted per mouse within the group. B. Striatal neuronal cell body volume. Mice expressing N171-82Q )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(and mutant \(inactive\) TMX3 \(mTMX3\) have significantly smaller neuronal cell bodies than mice expressing N171-82Q and )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(active TMX3. The main effect p-value is 0.0346; pair-wise comparison p-values are on graph. n=9-10. C. Spontaneous )] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(wheel running activity is not altered by N171-18/82Q and / or TMX3 expression. 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Experimental design was as described for figure 5. A. Representative images of fluorescent Nissl stained neurons in )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(the region of the striatal injection site. Numbers below images represent the number of mice per treatment group; numbers )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(in parenthesis represent the minimum and maximum number of neurons counted per mouse within the group. B. Striatal )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(neuronal cell body volume. Mice expressing N171-82Q and mutant \(inactive\) TRX1 \(mTRX1\) have significantly smaller )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(neuronal cell bodies than mice expressing N171-82Q and active TRX1. The main effect p-value is 0.0545; pair-wise )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(comparison p-values are on graph. n=5-7, C. Spontaneous wheel running activity is not altered by N171-18/82Q and / or )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(TXN1 expression. See methods for experimental details. n=7-9.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 337.342 555.000 299.350 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 636.691 m 581.250 636.691 l 581.250 635.941 l 26.250 635.941 l f 26.250 337.342 m 581.250 337.342 l 581.250 338.092 l 26.250 338.092 l f q 450.000 0 0 192.000 35.250 434.941 cm /I9 Do Q q 35.250 348.592 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 419.418 Td /F4 9.8 Tf [(Fig. 7: TRX1 and TMX3 transcript levels in transgenic N171-82Q HD mouse brain)] TJ ET BT 35.250 400.048 Td /F1 9.8 Tf [(A. Transcript-encoding TRX1 in HD mice is significant higher than WT littermates in striatum, but not cortex. B. Transcript-)] TJ ET BT 35.250 386.311 Td /F1 9.8 Tf [(encoding TMX3 in HD mice is not altered compared to WT littermates in striatum and cortex. All analyses were in 14-week-)] TJ ET BT 35.250 372.575 Td /F1 9.8 Tf [(old mice corresponding to early-advanced disease. Values are normalized to actin. Shown are means 95% confidence )] TJ ET BT 35.250 358.839 Td /F1 9.8 Tf [(interval. n=9-10, p=value: **<0.01.)] TJ ET Q BT 26.250 303.120 Td /F4 12.0 Tf [(List of abbreviations)] TJ ET BT 26.250 283.166 Td /F1 9.8 Tf [(Mutant huntingtin protein, mhtt; Huntingtons disease, HD; endoplasmic reticulum, ER; sodium dodecyl sulfate, SDS; thioredoxin-)] TJ ET BT 26.250 271.261 Td /F1 9.8 Tf [(related transmembrane protein 3, TMX3; thioredoxin 1, TRX1; protein disulfide isomerase family A, member 6, PDIA6.)] TJ ET BT 26.250 234.659 Td /F4 12.0 Tf [(Competing interest statement)] TJ ET BT 26.250 214.704 Td /F1 9.8 Tf [(I have read the journals policy and have the following conflict. Application serial number 13/854,809 filed with US patent office.)] TJ ET BT 26.250 178.102 Td /F4 12.0 Tf [(Author contributions)] TJ ET BT 26.250 158.148 Td /F1 9.8 Tf [(ZL carried out cell culture studies, synthesized plasmid constructs, and assisted with the mouse experiments and writing of the )] TJ ET BT 26.250 146.243 Td /F1 9.8 Tf [(paper. LB carried out the viral synthesis and mouse experiments. JF conceived, designed and coordinated the study and wrote )] TJ ET BT 26.250 134.338 Td /F1 9.8 Tf [(the paper. All authors read and approved the final manuscript.)] TJ ET BT 26.250 97.736 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 77.781 Td /F1 9.8 Tf [(We thank Dr. Nicole Dglon for kindly providing the four-plasmid lentiviral system expressing N171-18Q and 82Q huntingtin )] TJ ET BT 26.250 65.877 Td /F1 9.8 Tf [(fragments.)] TJ ET Q q 15.000 26.344 577.500 750.656 re W n 0.965 0.965 0.965 rg 26.250 644.191 555.000 132.809 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 644.191 m 581.250 644.191 l 581.250 644.941 l 26.250 644.941 l f q 35.250 655.441 537.000 121.559 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 6: TRX1 expression decreases striatal neuronal atrophy in HD mice)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(A-B. Experimental design was as described for figure 5. A. Representative images of fluorescent Nissl stained neurons in )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(the region of the striatal injection site. Numbers below images represent the number of mice per treatment group; numbers )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(in parenthesis represent the minimum and maximum number of neurons counted per mouse within the group. B. Striatal )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(neuronal cell body volume. Mice expressing N171-82Q and mutant \(inactive\) TRX1 \(mTRX1\) have significantly smaller )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(neuronal cell bodies than mice expressing N171-82Q and active TRX1. The main effect p-value is 0.0545; pair-wise )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(comparison p-values are on graph. n=5-7, C. Spontaneous wheel running activity is not altered by N171-18/82Q and / or )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(TXN1 expression. See methods for experimental details. n=7-9.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 337.342 555.000 299.350 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 636.691 m 581.250 636.691 l 581.250 635.941 l 26.250 635.941 l f 26.250 337.342 m 581.250 337.342 l 581.250 338.092 l 26.250 338.092 l f q 450.000 0 0 192.000 35.250 434.941 cm /I9 Do Q q 35.250 348.592 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 419.418 Td /F4 9.8 Tf [(Fig. 7: TRX1 and TMX3 transcript levels in transgenic N171-82Q HD mouse brain)] TJ ET BT 35.250 400.048 Td /F1 9.8 Tf [(A. Transcript-encoding TRX1 in HD mice is significant higher than WT littermates in striatum, but not cortex. B. Transcript-)] TJ ET BT 35.250 386.311 Td /F1 9.8 Tf [(encoding TMX3 in HD mice is not altered compared to WT littermates in striatum and cortex. All analyses were in 14-week-)] TJ ET BT 35.250 372.575 Td /F1 9.8 Tf [(old mice corresponding to early-advanced disease. Values are normalized to actin. Shown are means 95% confidence )] TJ ET BT 35.250 358.839 Td /F1 9.8 Tf [(interval. n=9-10, p=value: **<0.01.)] TJ ET Q BT 26.250 303.120 Td /F4 12.0 Tf [(List of abbreviations)] TJ ET BT 26.250 283.166 Td /F1 9.8 Tf [(Mutant huntingtin protein, mhtt; Huntingtons disease, HD; endoplasmic reticulum, ER; sodium dodecyl sulfate, SDS; thioredoxin-)] TJ ET BT 26.250 271.261 Td /F1 9.8 Tf [(related transmembrane protein 3, TMX3; thioredoxin 1, TRX1; protein disulfide isomerase family A, member 6, PDIA6.)] TJ ET BT 26.250 234.659 Td /F4 12.0 Tf [(Competing interest statement)] TJ ET BT 26.250 214.704 Td /F1 9.8 Tf [(I have read the journals policy and have the following conflict. Application serial number 13/854,809 filed with US patent office.)] TJ ET BT 26.250 178.102 Td /F4 12.0 Tf [(Author contributions)] TJ ET BT 26.250 158.148 Td /F1 9.8 Tf [(ZL carried out cell culture studies, synthesized plasmid constructs, and assisted with the mouse experiments and writing of the )] TJ ET BT 26.250 146.243 Td /F1 9.8 Tf [(paper. LB carried out the viral synthesis and mouse experiments. JF conceived, designed and coordinated the study and wrote )] TJ ET BT 26.250 134.338 Td /F1 9.8 Tf [(the paper. All authors read and approved the final manuscript.)] TJ ET BT 26.250 97.736 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 77.781 Td /F1 9.8 Tf [(We thank Dr. Nicole Dglon for kindly providing the four-plasmid lentiviral system expressing N171-18Q and 82Q huntingtin )] TJ ET BT 26.250 65.877 Td /F1 9.8 Tf [(fragments.)] TJ ET Q q 15.000 26.344 577.500 750.656 re W n 0.965 0.965 0.965 rg 26.250 644.191 555.000 132.809 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 644.191 m 581.250 644.191 l 581.250 644.941 l 26.250 644.941 l f q 35.250 655.441 537.000 121.559 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 6: TRX1 expression decreases striatal neuronal atrophy in HD mice)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(A-B. Experimental design was as described for figure 5. A. Representative images of fluorescent Nissl stained neurons in )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(the region of the striatal injection site. Numbers below images represent the number of mice per treatment group; numbers )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(in parenthesis represent the minimum and maximum number of neurons counted per mouse within the group. B. Striatal )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(neuronal cell body volume. Mice expressing N171-82Q and mutant \(inactive\) TRX1 \(mTRX1\) have significantly smaller )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(neuronal cell bodies than mice expressing N171-82Q and active TRX1. The main effect p-value is 0.0545; pair-wise )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(comparison p-values are on graph. n=5-7, C. Spontaneous wheel running activity is not altered by N171-18/82Q and / or )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(TXN1 expression. See methods for experimental details. n=7-9.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 337.342 555.000 299.350 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 636.691 m 581.250 636.691 l 581.250 635.941 l 26.250 635.941 l f 26.250 337.342 m 581.250 337.342 l 581.250 338.092 l 26.250 338.092 l f q 450.000 0 0 192.000 35.250 434.941 cm /I9 Do Q q 35.250 348.592 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 419.418 Td /F4 9.8 Tf [(Fig. 7: TRX1 and TMX3 transcript levels in transgenic N171-82Q HD mouse brain)] TJ ET BT 35.250 400.048 Td /F1 9.8 Tf [(A. Transcript-encoding TRX1 in HD mice is significant higher than WT littermates in striatum, but not cortex. B. Transcript-)] TJ ET BT 35.250 386.311 Td /F1 9.8 Tf [(encoding TMX3 in HD mice is not altered compared to WT littermates in striatum and cortex. All analyses were in 14-week-)] TJ ET BT 35.250 372.575 Td /F1 9.8 Tf [(old mice corresponding to early-advanced disease. Values are normalized to actin. Shown are means 95% confidence )] TJ ET BT 35.250 358.839 Td /F1 9.8 Tf [(interval. n=9-10, p=value: **<0.01.)] TJ ET Q BT 26.250 303.120 Td /F4 12.0 Tf [(List of abbreviations)] TJ ET BT 26.250 283.166 Td /F1 9.8 Tf [(Mutant huntingtin protein, mhtt; Huntingtons disease, HD; endoplasmic reticulum, ER; sodium dodecyl sulfate, SDS; thioredoxin-)] TJ ET BT 26.250 271.261 Td /F1 9.8 Tf [(related transmembrane protein 3, TMX3; thioredoxin 1, TRX1; protein disulfide isomerase family A, member 6, PDIA6.)] TJ ET BT 26.250 234.659 Td /F4 12.0 Tf [(Competing interest statement)] TJ ET BT 26.250 214.704 Td /F1 9.8 Tf [(I have read the journals policy and have the following conflict. Application serial number 13/854,809 filed with US patent office.)] TJ ET BT 26.250 178.102 Td /F4 12.0 Tf [(Author contributions)] TJ ET BT 26.250 158.148 Td /F1 9.8 Tf [(ZL carried out cell culture studies, synthesized plasmid constructs, and assisted with the mouse experiments and writing of the )] TJ ET BT 26.250 146.243 Td /F1 9.8 Tf [(paper. LB carried out the viral synthesis and mouse experiments. JF conceived, designed and coordinated the study and wrote )] TJ ET BT 26.250 134.338 Td /F1 9.8 Tf [(the paper. All authors read and approved the final manuscript.)] TJ ET BT 26.250 97.736 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 77.781 Td /F1 9.8 Tf [(We thank Dr. Nicole Dglon for kindly providing the four-plasmid lentiviral system expressing N171-18Q and 82Q huntingtin )] TJ ET BT 26.250 65.877 Td /F1 9.8 Tf [(fragments.)] TJ ET Q q 450.000 0 0 192.000 35.250 434.941 cm /I9 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(10)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 645 0 obj << /Type /Annot /Subtype /Link /A 646 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 434.9415 485.2500 626.9415 ] >> endobj 646 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2015/10/Fig7rb.jpg) >> endobj 647 0 obj << /Type /XObject /Subtype /Image /Width 600 /Height 256 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 26841>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   X" }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?6_ҼZ}|# WpZ߬f7mA8-|}C}S/hqkqlT&q( |3_Ix}Kd{q.>qLPj( (^i(EQK@ii){QEQEQGE/z);ڀRv 製Q=((Q@j;Gz)i=h7?AkO:N{ RK}q,1>dM! -Q㓚7',~ H5OH8gUr~>g+4~ ӵO.!4J=ׯ$Q0uax8,>g5|{&w|ZhvIx[,$;9EǹG GQڊ;P04wvQGz(Z;@(QEPh@E@Q@vޒ ;RE(@QEWnYm|qt|YVjq(Z.]]X vSG=⮅okntYmѶ[l0z@VׂoU V [dE6^twQn81 |E^7+t_zLZBEMr)ho#e!W}𷉴m+NNnӲo'(W$a `Vsl|K5h cn@#. ) >o"W׺W:W$°XԐ$L$VfhUgYږygjPlCm>b#0#|#߇?ر]pt$%XF3a!+Sn߁-[ůjz&}[ͨo 4, !.ʻ1Ծ^S!,M&(բQ%!bvM)յKcIlnirPIɵc<?¿]#:UZ"=6],X,ʧz!`aF Z5cGdb2'IҬ`FVByן|c+h?>%x.ԵiO÷W–v5$y$m/TKFvHwbFK1'hRv@;Gj);P(4whPڊ;Q@z(@Rv4QEzڀ (E'm<%-$iYY4k % ý^gAZ&X,5V󿴴Ƴh扢QC!rCe?vʆ= QUecf]:C]č-ϱBCv^_3?|]+K׵c: 敧Ok浥h"Hhm4J'(4ObD^uObD^디RE%/z 'AEQA (v@Ev((EQEvGz(ZJ;QGa@/zJ;ڼibrצי~m??SZ @{T?*h))EPڊPڃ@E;RPڎQ@;PEw֎((^oFˡCS-[KE0"okqiP!dy!\r6[k;WYմGQ,{egUy#3pG$P[ |[RP$ 2(HVUx2Dz\S`I &jo5?70xF5[MDrY=̤Kϣ~ѺtO0 GFKqr[[8eg U&XI=(ھ0v#jZ5oj6ktZj"{w;9 heÅAg2G?HuϳīmSRY4B@:e:#;ۆqH?HK޼WpϦc[cqx=WmwEq9<cGh?;Vψ,o`{ fGkjROЀz5KFjZM\̲ WRAp{U =hրwQE5_A+ &q[m(Ac1жU-d}p@nA X4m?\n8`Fy<?g{ūatmZ1z+ss Bb-X6:9:Md[6]D> |D}nDc2KCVaA O a@!E–< om{O}c4pE F5>` Z_|^m|Z}mC&dt{0Ȉ?t|mϚbpƾ_xa>4ϥCe~Y?< 75ӭ#"UTnt$fm^kdρ̖vƏ]&TtTicm?8{SmeeOW20[[I%UEQ!,Đk^KMxT|kgile+vdۗUZ;Qހ (埴etȮ5]kH:݋isxzk(1*$G6 ~+w{fHIDcnQoq ;_Zvjj [ssv^#+DcPb$|(k>xv;m RC"%\񎵏ޡ3'1أkkB99YKQGz*E@z;;;(((h= QQ@y x G͟5%eP-R˵A0#LWv2xcUHn2uݿ5V0!lnҀ9_.u ً dO (iX *J6?|i]_ķ׆@ҠҼ7Y(7V v>'gT\ )SrT8]P??%7ZףQdGqο6_xFt}KVL4.adp8 Kcz|/v_l*YtW߳ אЍ}_~ϿF<%^Cq2=pu](Ki>ͧ]k:|C/LA#w o?=޴Kojpy ;(fvbX ՜i޷do_c~E`DߪS#j[GjZNPQގwQEb3EVoEVghv Gj(Qڀ ((@Q@j>;׾:ta Y"7ZcMavkW^E+O9#xTӭy lm0 ý+ɠ b+ؼSLŖq[b{MEp/!Wc A *߉<9|iu׷_YťkR{yoe}- oT,|t}*DlLJwZm.![U^3$W; ,n~oeM< bɒVo|yb?\H"?/zJZJ|}?z5=2Znh,nh'$! {1=NScZ#_8 'eh>.@g]z) 17eZ־ta/6Z7|]>y5w$c_?#N&uvGEg0wDߪX]wE/74Tږvv:W;˨ak Q- Tn R(O_l~2r-{J5+J Kf[eM$Xfܻp{}]ߥQEyy&_?k6גԮ<歺8o?C"ػ4hxX?~Jdo]WN)`L9k$Su5M꺞fUM" ǩ5F-WY)[WA~ص,u-WY)g6kMXdtQwQ@:Ky=AS¾6|R-Shf@$)Aa{mq97XPEow\&H*#*f8e5x|Xuw>;4K9 8&?Ip_V/ieT1@4W ^ePɴXOUIk{W~m??SZ @{T1]?E]?_k\u?kOk*sU|;![tbHTog\~{H4 >+2N>]KiI¨d~*/vk_ew~3ȗmaNUD[~-Z؜?KZܪ -QڎVw~ (Zl7zƿbIkO>EVg`voxlAƭhbDW 85󇇿i//_T)繸yݬb 8?]W/o?tf|1ĺ%YXGq*Igp+۽k?}3ms!nCI[r$]W/o1sǹ|wSWU}cy-Heb# u#@(x_/ /Gy:$zTcLI Bݏ~^?i2h:-.yIoFc(P ( ^/>kZFؽ$@̐ᔹgx'Ͽh :;m Bկ U1*DDVFD)L tҼ=?F43J[KM/ŚŕGl[2a$88${x G:/t 5kB_rp"Hl#{5@yvSؑzy$vSؑz%-%|}gsW=NSd+U-ؔwta/6Z|/v_k=k7|]>yk}//Ӊ]4QEY{F/74VzK26uVO< )y$($wk$Pu?P|+o. 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A novel gene containing a trinucleotide repeat that is expanded )] TJ ET BT 26.250 710.919 Td /F1 9.8 Tf [(and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell. )] TJ ET BT 26.250 699.015 Td /F1 9.8 Tf [(1993;72\(6\):971-83. PubMed PMID: 8458085.)] TJ ET BT 26.250 679.610 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 679.610 Td /F1 9.8 Tf [(Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, Richardson EP, Jr. Neuropathological classification of )] TJ ET BT 26.250 667.705 Td /F1 9.8 Tf [(Huntington's disease. Journal of neuropathology and experimental neurology. 1985;44\(6\):559-77. PubMed PMID: 2932539.)] TJ ET BT 26.250 648.300 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 648.300 Td /F1 9.8 Tf [(Wichmann T, Delong MR. Basal ganglia circuits in movement and movement disorders. In: Kultas-Ilinsky K, Ilinsky IA, )] TJ ET BT 26.250 636.396 Td /F1 9.8 Tf [(editors. Basal Ganglia and Thalamus in Health and Movement Disorders2001. p. 11-25.)] TJ ET BT 26.250 616.991 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 616.991 Td /F1 9.8 Tf [(Wellington CL, Ellerby LM, Gutekunst CA, Rogers D, Warby S, Graham RK, et al. Caspase cleavage of mutant huntingtin )] TJ ET BT 26.250 605.086 Td /F1 9.8 Tf [(precedes neurodegeneration in Huntington's disease. The Journal of neuroscience : the official journal of the Society for )] TJ ET BT 26.250 593.181 Td /F1 9.8 Tf [(Neuroscience. 2002;22\(18\):7862-72. PubMed PMID: 12223539.)] TJ ET BT 26.250 573.777 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 573.777 Td /F1 9.8 Tf [(Gafni J, Hermel E, Young JE, Wellington CL, Hayden MR, Ellerby LM. Inhibition of calpain cleavage of huntingtin reduces )] TJ ET BT 26.250 561.872 Td /F1 9.8 Tf [(toxicity: accumulation of calpain/caspase fragments in the nucleus. The Journal of biological chemistry. 2004;279\(19\):20211-20. )] TJ ET BT 26.250 549.967 Td /F1 9.8 Tf [(PubMed PMID: 14981075.)] TJ ET BT 26.250 530.562 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 530.562 Td /F1 9.8 Tf [(Fox JH, Connor T, Stiles M, Kama J, Lu Z, Dorsey K, et al. Cysteine Oxidation within N-terminal Mutant Huntingtin Promotes )] TJ ET BT 26.250 518.658 Td /F1 9.8 Tf [(Oligomerization and Delays Clearance of Soluble Protein. The Journal of biological chemistry. 2011;286\(20\):18320-30. PubMed )] TJ ET BT 26.250 506.753 Td /F1 9.8 Tf [(PMID: 21454633.)] TJ ET BT 26.250 487.348 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 487.348 Td /F1 9.8 Tf [(Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin )] TJ ET BT 26.250 475.443 Td /F1 9.8 Tf [(and the risk of neuronal death. Nature. 2004;431\(7010\):805-10. PubMed PMID: 15483602.)] TJ ET BT 26.250 456.039 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 456.039 Td /F1 9.8 Tf [(Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA, et al. Intranuclear inclusions and neuritic aggregates in )] TJ ET BT 26.250 444.134 Td /F1 9.8 Tf [(transgenic mice expressing a mutant N-terminal fragment of huntingtin. Human molecular genetics. 1999;8\(3\):397-407. PubMed )] TJ ET BT 26.250 432.229 Td /F1 9.8 Tf [(PMID: 9949199.)] TJ ET BT 26.250 412.824 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 412.824 Td /F1 9.8 Tf [(Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, Hersch SM, et al. Sp1 and TAFII130 transcriptional activity disrupted )] TJ ET BT 26.250 400.920 Td /F1 9.8 Tf [(in early Huntington's disease. Science. 2002;296\(5576\):2238-43. PubMed PMID: 11988536.)] TJ ET BT 26.250 381.515 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 381.515 Td /F1 9.8 Tf [(Steffan JS, Kazantsev A, Spasic-Boskovic O, Greenwald M, Zhu YZ, Gohler H, et al. The Huntington's disease protein )] TJ ET BT 26.250 369.610 Td /F1 9.8 Tf [(interacts with p53 and CREB-binding protein and represses transcription. Proceedings of the National Academy of Sciences of )] TJ ET BT 26.250 357.705 Td /F1 9.8 Tf [(the United States of America. 2000;97\(12\):6763-8. PubMed PMID: 10823891.)] TJ ET BT 26.250 338.301 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 338.301 Td /F1 9.8 Tf [(Hands S, Sajjad MU, Newton MJ, Wyttenbach A. In vitro and in vivo aggregation of a fragment of huntingtin protein directly )] TJ ET BT 26.250 326.396 Td /F1 9.8 Tf [(causes free radical production. The Journal of biological chemistry. 2011;286\(52\):44512-20. doi: 10.1074/jbc.M111.307587. )] TJ ET BT 26.250 314.491 Td /F1 9.8 Tf [(PubMed PMID: 21984825; PubMed Central PMCID: PMC3247975.)] TJ ET BT 26.250 295.086 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 295.086 Td /F1 9.8 Tf [(Yamamoto A, Lucas JJ, Hen R. Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's )] TJ ET BT 26.250 283.182 Td /F1 9.8 Tf [(disease. Cell. 2000;101\(1\):57-66. doi: 10.1016/S0092-8674\(00\)80623-6. PubMed PMID: 10778856.)] TJ ET BT 26.250 263.777 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 263.777 Td /F1 9.8 Tf [(Aronin N, DiFiglia M. Huntingtin-lowering strategies in Huntington's disease: antisense oligonucleotides, small RNAs, and )] TJ ET BT 26.250 251.872 Td /F1 9.8 Tf [(gene editing. Mov Disord. 2014;29\(11\):1455-61. doi: 10.1002/mds.26020. PubMed PMID: 25164989.)] TJ ET BT 26.250 232.467 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 232.467 Td /F1 9.8 Tf [(McBride JL, Pitzer MR, Boudreau RL, Dufour B, Hobbs T, Ojeda SR, et al. Preclinical safety of RNAi-mediated HTT )] TJ ET BT 26.250 220.563 Td /F1 9.8 Tf [(suppression in the rhesus macaque as a potential therapy for Huntington's disease. Mol Ther. 2011;19\(12\):2152-62. doi: )] TJ ET BT 26.250 208.658 Td /F1 9.8 Tf [(10.1038/mt.2011.219. PubMed PMID: 22031240; PubMed Central PMCID: PMCPMC3242667.)] TJ ET BT 26.250 189.253 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 189.253 Td /F1 9.8 Tf [(Kordasiewicz HB, Stanek LM, Wancewicz EV, Mazur C, McAlonis MM, Pytel KA, et al. Sustained therapeutic reversal of )] TJ ET BT 26.250 177.348 Td /F1 9.8 Tf [(Huntington's disease by transient repression of huntingtin synthesis. Neuron. 2012;74\(6\):1031-44. doi: )] TJ ET BT 26.250 165.444 Td /F1 9.8 Tf [(10.1016/j.neuron.2012.05.009. PubMed PMID: 22726834; PubMed Central PMCID: PMCPMC3383626.)] TJ ET BT 26.250 146.039 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 146.039 Td /F1 9.8 Tf [(Haun F, Nakamura T, Shiu AD, Cho DH, Tsunemi T, Holland EA, et al. S-nitrosylation of dynamin-related protein 1 )] TJ ET BT 26.250 134.134 Td /F1 9.8 Tf [(mediates mutant huntingtin-induced mitochondrial fragmentation and neuronal injury in Huntington's disease. Antioxidants & )] TJ ET BT 26.250 122.229 Td /F1 9.8 Tf [(redox signaling. 2013;19\(11\):1173-84. doi: 10.1089/ars.2012.4928. PubMed PMID: 23641925; PubMed Central PMCID: )] TJ ET BT 26.250 110.325 Td /F1 9.8 Tf [(PMC3785802.)] TJ ET BT 26.250 90.920 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 90.920 Td /F1 9.8 Tf [(Pitts A, Dailey K, Newington JT, Chien A, Arseneault R, Cann T, et al. Dithiol-based compounds maintain expression of )] TJ ET BT 26.250 79.015 Td /F1 9.8 Tf [(antioxidant protein peroxiredoxin 1 that counteracts toxicity of mutant huntingtin. The Journal of biological chemistry. )] TJ ET BT 26.250 67.110 Td /F1 9.8 Tf [(2012;287\(27\):22717-29. doi: 10.1074/jbc.M111.334565. PubMed PMID: 22577145; PubMed Central PMCID: PMC3391089.)] TJ ET BT 26.250 47.706 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 47.706 Td /F1 9.8 Tf [(Chen J, Marks E, Lai B, Zhang Z, Duce JA, Lam LQ, et al. Iron accumulates in Huntington's disease neurons: protection by )] TJ ET Q q 15.000 33.420 577.500 743.580 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 722.824 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 722.824 Td /F1 9.8 Tf [(The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded )] TJ ET BT 26.250 710.919 Td /F1 9.8 Tf [(and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell. )] TJ ET BT 26.250 699.015 Td /F1 9.8 Tf [(1993;72\(6\):971-83. PubMed PMID: 8458085.)] TJ ET BT 26.250 679.610 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 679.610 Td /F1 9.8 Tf [(Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, Richardson EP, Jr. Neuropathological classification of )] TJ ET BT 26.250 667.705 Td /F1 9.8 Tf [(Huntington's disease. Journal of neuropathology and experimental neurology. 1985;44\(6\):559-77. PubMed PMID: 2932539.)] TJ ET BT 26.250 648.300 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 648.300 Td /F1 9.8 Tf [(Wichmann T, Delong MR. Basal ganglia circuits in movement and movement disorders. In: Kultas-Ilinsky K, Ilinsky IA, )] TJ ET BT 26.250 636.396 Td /F1 9.8 Tf [(editors. Basal Ganglia and Thalamus in Health and Movement Disorders2001. p. 11-25.)] TJ ET BT 26.250 616.991 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 616.991 Td /F1 9.8 Tf [(Wellington CL, Ellerby LM, Gutekunst CA, Rogers D, Warby S, Graham RK, et al. Caspase cleavage of mutant huntingtin )] TJ ET BT 26.250 605.086 Td /F1 9.8 Tf [(precedes neurodegeneration in Huntington's disease. The Journal of neuroscience : the official journal of the Society for )] TJ ET BT 26.250 593.181 Td /F1 9.8 Tf [(Neuroscience. 2002;22\(18\):7862-72. PubMed PMID: 12223539.)] TJ ET BT 26.250 573.777 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 573.777 Td /F1 9.8 Tf [(Gafni J, Hermel E, Young JE, Wellington CL, Hayden MR, Ellerby LM. Inhibition of calpain cleavage of huntingtin reduces )] TJ ET BT 26.250 561.872 Td /F1 9.8 Tf [(toxicity: accumulation of calpain/caspase fragments in the nucleus. The Journal of biological chemistry. 2004;279\(19\):20211-20. )] TJ ET BT 26.250 549.967 Td /F1 9.8 Tf [(PubMed PMID: 14981075.)] TJ ET BT 26.250 530.562 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 530.562 Td /F1 9.8 Tf [(Fox JH, Connor T, Stiles M, Kama J, Lu Z, Dorsey K, et al. Cysteine Oxidation within N-terminal Mutant Huntingtin Promotes )] TJ ET BT 26.250 518.658 Td /F1 9.8 Tf [(Oligomerization and Delays Clearance of Soluble Protein. The Journal of biological chemistry. 2011;286\(20\):18320-30. PubMed )] TJ ET BT 26.250 506.753 Td /F1 9.8 Tf [(PMID: 21454633.)] TJ ET BT 26.250 487.348 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 487.348 Td /F1 9.8 Tf [(Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin )] TJ ET BT 26.250 475.443 Td /F1 9.8 Tf [(and the risk of neuronal death. Nature. 2004;431\(7010\):805-10. PubMed PMID: 15483602.)] TJ ET BT 26.250 456.039 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 456.039 Td /F1 9.8 Tf [(Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA, et al. Intranuclear inclusions and neuritic aggregates in )] TJ ET BT 26.250 444.134 Td /F1 9.8 Tf [(transgenic mice expressing a mutant N-terminal fragment of huntingtin. Human molecular genetics. 1999;8\(3\):397-407. PubMed )] TJ ET BT 26.250 432.229 Td /F1 9.8 Tf [(PMID: 9949199.)] TJ ET BT 26.250 412.824 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 412.824 Td /F1 9.8 Tf [(Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, Hersch SM, et al. Sp1 and TAFII130 transcriptional activity disrupted )] TJ ET BT 26.250 400.920 Td /F1 9.8 Tf [(in early Huntington's disease. Science. 2002;296\(5576\):2238-43. PubMed PMID: 11988536.)] TJ ET BT 26.250 381.515 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 381.515 Td /F1 9.8 Tf [(Steffan JS, Kazantsev A, Spasic-Boskovic O, Greenwald M, Zhu YZ, Gohler H, et al. The Huntington's disease protein )] TJ ET BT 26.250 369.610 Td /F1 9.8 Tf [(interacts with p53 and CREB-binding protein and represses transcription. Proceedings of the National Academy of Sciences of )] TJ ET BT 26.250 357.705 Td /F1 9.8 Tf [(the United States of America. 2000;97\(12\):6763-8. PubMed PMID: 10823891.)] TJ ET BT 26.250 338.301 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 338.301 Td /F1 9.8 Tf [(Hands S, Sajjad MU, Newton MJ, Wyttenbach A. In vitro and in vivo aggregation of a fragment of huntingtin protein directly )] TJ ET BT 26.250 326.396 Td /F1 9.8 Tf [(causes free radical production. The Journal of biological chemistry. 2011;286\(52\):44512-20. doi: 10.1074/jbc.M111.307587. )] TJ ET BT 26.250 314.491 Td /F1 9.8 Tf [(PubMed PMID: 21984825; PubMed Central PMCID: PMC3247975.)] TJ ET BT 26.250 295.086 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 295.086 Td /F1 9.8 Tf [(Yamamoto A, Lucas JJ, Hen R. Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's )] TJ ET BT 26.250 283.182 Td /F1 9.8 Tf [(disease. Cell. 2000;101\(1\):57-66. doi: 10.1016/S0092-8674\(00\)80623-6. PubMed PMID: 10778856.)] TJ ET BT 26.250 263.777 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 263.777 Td /F1 9.8 Tf [(Aronin N, DiFiglia M. Huntingtin-lowering strategies in Huntington's disease: antisense oligonucleotides, small RNAs, and )] TJ ET BT 26.250 251.872 Td /F1 9.8 Tf [(gene editing. Mov Disord. 2014;29\(11\):1455-61. doi: 10.1002/mds.26020. PubMed PMID: 25164989.)] TJ ET BT 26.250 232.467 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 232.467 Td /F1 9.8 Tf [(McBride JL, Pitzer MR, Boudreau RL, Dufour B, Hobbs T, Ojeda SR, et al. Preclinical safety of RNAi-mediated HTT )] TJ ET BT 26.250 220.563 Td /F1 9.8 Tf [(suppression in the rhesus macaque as a potential therapy for Huntington's disease. Mol Ther. 2011;19\(12\):2152-62. doi: )] TJ ET BT 26.250 208.658 Td /F1 9.8 Tf [(10.1038/mt.2011.219. PubMed PMID: 22031240; PubMed Central PMCID: PMCPMC3242667.)] TJ ET BT 26.250 189.253 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 189.253 Td /F1 9.8 Tf [(Kordasiewicz HB, Stanek LM, Wancewicz EV, Mazur C, McAlonis MM, Pytel KA, et al. Sustained therapeutic reversal of )] TJ ET BT 26.250 177.348 Td /F1 9.8 Tf [(Huntington's disease by transient repression of huntingtin synthesis. Neuron. 2012;74\(6\):1031-44. doi: )] TJ ET BT 26.250 165.444 Td /F1 9.8 Tf [(10.1016/j.neuron.2012.05.009. PubMed PMID: 22726834; PubMed Central PMCID: PMCPMC3383626.)] TJ ET BT 26.250 146.039 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 146.039 Td /F1 9.8 Tf [(Haun F, Nakamura T, Shiu AD, Cho DH, Tsunemi T, Holland EA, et al. S-nitrosylation of dynamin-related protein 1 )] TJ ET BT 26.250 134.134 Td /F1 9.8 Tf [(mediates mutant huntingtin-induced mitochondrial fragmentation and neuronal injury in Huntington's disease. Antioxidants & )] TJ ET BT 26.250 122.229 Td /F1 9.8 Tf [(redox signaling. 2013;19\(11\):1173-84. doi: 10.1089/ars.2012.4928. PubMed PMID: 23641925; PubMed Central PMCID: )] TJ ET BT 26.250 110.325 Td /F1 9.8 Tf [(PMC3785802.)] TJ ET BT 26.250 90.920 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 90.920 Td /F1 9.8 Tf [(Pitts A, Dailey K, Newington JT, Chien A, Arseneault R, Cann T, et al. Dithiol-based compounds maintain expression of )] TJ ET BT 26.250 79.015 Td /F1 9.8 Tf [(antioxidant protein peroxiredoxin 1 that counteracts toxicity of mutant huntingtin. The Journal of biological chemistry. )] TJ ET BT 26.250 67.110 Td /F1 9.8 Tf [(2012;287\(27\):22717-29. doi: 10.1074/jbc.M111.334565. PubMed PMID: 22577145; PubMed Central PMCID: PMC3391089.)] TJ ET BT 26.250 47.706 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 47.706 Td /F1 9.8 Tf [(Chen J, Marks E, Lai B, Zhang Z, Duce JA, Lam LQ, et al. Iron accumulates in Huntington's disease neurons: protection by )] TJ ET Q q 15.000 33.420 577.500 743.580 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 722.824 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 722.824 Td /F1 9.8 Tf [(The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded )] TJ ET BT 26.250 710.919 Td /F1 9.8 Tf [(and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell. )] TJ ET BT 26.250 699.015 Td /F1 9.8 Tf [(1993;72\(6\):971-83. PubMed PMID: 8458085.)] TJ ET BT 26.250 679.610 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 679.610 Td /F1 9.8 Tf [(Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, Richardson EP, Jr. Neuropathological classification of )] TJ ET BT 26.250 667.705 Td /F1 9.8 Tf [(Huntington's disease. Journal of neuropathology and experimental neurology. 1985;44\(6\):559-77. PubMed PMID: 2932539.)] TJ ET BT 26.250 648.300 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 648.300 Td /F1 9.8 Tf [(Wichmann T, Delong MR. Basal ganglia circuits in movement and movement disorders. In: Kultas-Ilinsky K, Ilinsky IA, )] TJ ET BT 26.250 636.396 Td /F1 9.8 Tf [(editors. Basal Ganglia and Thalamus in Health and Movement Disorders2001. p. 11-25.)] TJ ET BT 26.250 616.991 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 616.991 Td /F1 9.8 Tf [(Wellington CL, Ellerby LM, Gutekunst CA, Rogers D, Warby S, Graham RK, et al. Caspase cleavage of mutant huntingtin )] TJ ET BT 26.250 605.086 Td /F1 9.8 Tf [(precedes neurodegeneration in Huntington's disease. The Journal of neuroscience : the official journal of the Society for )] TJ ET BT 26.250 593.181 Td /F1 9.8 Tf [(Neuroscience. 2002;22\(18\):7862-72. PubMed PMID: 12223539.)] TJ ET BT 26.250 573.777 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 573.777 Td /F1 9.8 Tf [(Gafni J, Hermel E, Young JE, Wellington CL, Hayden MR, Ellerby LM. Inhibition of calpain cleavage of huntingtin reduces )] TJ ET BT 26.250 561.872 Td /F1 9.8 Tf [(toxicity: accumulation of calpain/caspase fragments in the nucleus. The Journal of biological chemistry. 2004;279\(19\):20211-20. )] TJ ET BT 26.250 549.967 Td /F1 9.8 Tf [(PubMed PMID: 14981075.)] TJ ET BT 26.250 530.562 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 530.562 Td /F1 9.8 Tf [(Fox JH, Connor T, Stiles M, Kama J, Lu Z, Dorsey K, et al. Cysteine Oxidation within N-terminal Mutant Huntingtin Promotes )] TJ ET BT 26.250 518.658 Td /F1 9.8 Tf [(Oligomerization and Delays Clearance of Soluble Protein. The Journal of biological chemistry. 2011;286\(20\):18320-30. PubMed )] TJ ET BT 26.250 506.753 Td /F1 9.8 Tf [(PMID: 21454633.)] TJ ET BT 26.250 487.348 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 487.348 Td /F1 9.8 Tf [(Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin )] TJ ET BT 26.250 475.443 Td /F1 9.8 Tf [(and the risk of neuronal death. Nature. 2004;431\(7010\):805-10. PubMed PMID: 15483602.)] TJ ET BT 26.250 456.039 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 456.039 Td /F1 9.8 Tf [(Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA, et al. Intranuclear inclusions and neuritic aggregates in )] TJ ET BT 26.250 444.134 Td /F1 9.8 Tf [(transgenic mice expressing a mutant N-terminal fragment of huntingtin. Human molecular genetics. 1999;8\(3\):397-407. PubMed )] TJ ET BT 26.250 432.229 Td /F1 9.8 Tf [(PMID: 9949199.)] TJ ET BT 26.250 412.824 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 412.824 Td /F1 9.8 Tf [(Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, Hersch SM, et al. Sp1 and TAFII130 transcriptional activity disrupted )] TJ ET BT 26.250 400.920 Td /F1 9.8 Tf [(in early Huntington's disease. Science. 2002;296\(5576\):2238-43. PubMed PMID: 11988536.)] TJ ET BT 26.250 381.515 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 381.515 Td /F1 9.8 Tf [(Steffan JS, Kazantsev A, Spasic-Boskovic O, Greenwald M, Zhu YZ, Gohler H, et al. The Huntington's disease protein )] TJ ET BT 26.250 369.610 Td /F1 9.8 Tf [(interacts with p53 and CREB-binding protein and represses transcription. Proceedings of the National Academy of Sciences of )] TJ ET BT 26.250 357.705 Td /F1 9.8 Tf [(the United States of America. 2000;97\(12\):6763-8. PubMed PMID: 10823891.)] TJ ET BT 26.250 338.301 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 338.301 Td /F1 9.8 Tf [(Hands S, Sajjad MU, Newton MJ, Wyttenbach A. In vitro and in vivo aggregation of a fragment of huntingtin protein directly )] TJ ET BT 26.250 326.396 Td /F1 9.8 Tf [(causes free radical production. The Journal of biological chemistry. 2011;286\(52\):44512-20. doi: 10.1074/jbc.M111.307587. )] TJ ET BT 26.250 314.491 Td /F1 9.8 Tf [(PubMed PMID: 21984825; PubMed Central PMCID: PMC3247975.)] TJ ET BT 26.250 295.086 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 295.086 Td /F1 9.8 Tf [(Yamamoto A, Lucas JJ, Hen R. Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's )] TJ ET BT 26.250 283.182 Td /F1 9.8 Tf [(disease. Cell. 2000;101\(1\):57-66. doi: 10.1016/S0092-8674\(00\)80623-6. PubMed PMID: 10778856.)] TJ ET BT 26.250 263.777 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 263.777 Td /F1 9.8 Tf [(Aronin N, DiFiglia M. Huntingtin-lowering strategies in Huntington's disease: antisense oligonucleotides, small RNAs, and )] TJ ET BT 26.250 251.872 Td /F1 9.8 Tf [(gene editing. Mov Disord. 2014;29\(11\):1455-61. doi: 10.1002/mds.26020. PubMed PMID: 25164989.)] TJ ET BT 26.250 232.467 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 232.467 Td /F1 9.8 Tf [(McBride JL, Pitzer MR, Boudreau RL, Dufour B, Hobbs T, Ojeda SR, et al. Preclinical safety of RNAi-mediated HTT )] TJ ET BT 26.250 220.563 Td /F1 9.8 Tf [(suppression in the rhesus macaque as a potential therapy for Huntington's disease. Mol Ther. 2011;19\(12\):2152-62. doi: )] TJ ET BT 26.250 208.658 Td /F1 9.8 Tf [(10.1038/mt.2011.219. PubMed PMID: 22031240; PubMed Central PMCID: PMCPMC3242667.)] TJ ET BT 26.250 189.253 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 189.253 Td /F1 9.8 Tf [(Kordasiewicz HB, Stanek LM, Wancewicz EV, Mazur C, McAlonis MM, Pytel KA, et al. Sustained therapeutic reversal of )] TJ ET BT 26.250 177.348 Td /F1 9.8 Tf [(Huntington's disease by transient repression of huntingtin synthesis. Neuron. 2012;74\(6\):1031-44. doi: )] TJ ET BT 26.250 165.444 Td /F1 9.8 Tf [(10.1016/j.neuron.2012.05.009. PubMed PMID: 22726834; PubMed Central PMCID: PMCPMC3383626.)] TJ ET BT 26.250 146.039 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 146.039 Td /F1 9.8 Tf [(Haun F, Nakamura T, Shiu AD, Cho DH, Tsunemi T, Holland EA, et al. S-nitrosylation of dynamin-related protein 1 )] TJ ET BT 26.250 134.134 Td /F1 9.8 Tf [(mediates mutant huntingtin-induced mitochondrial fragmentation and neuronal injury in Huntington's disease. Antioxidants & )] TJ ET BT 26.250 122.229 Td /F1 9.8 Tf [(redox signaling. 2013;19\(11\):1173-84. doi: 10.1089/ars.2012.4928. PubMed PMID: 23641925; PubMed Central PMCID: )] TJ ET BT 26.250 110.325 Td /F1 9.8 Tf [(PMC3785802.)] TJ ET BT 26.250 90.920 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 90.920 Td /F1 9.8 Tf [(Pitts A, Dailey K, Newington JT, Chien A, Arseneault R, Cann T, et al. Dithiol-based compounds maintain expression of )] TJ ET BT 26.250 79.015 Td /F1 9.8 Tf [(antioxidant protein peroxiredoxin 1 that counteracts toxicity of mutant huntingtin. The Journal of biological chemistry. )] TJ ET BT 26.250 67.110 Td /F1 9.8 Tf [(2012;287\(27\):22717-29. doi: 10.1074/jbc.M111.334565. PubMed PMID: 22577145; PubMed Central PMCID: PMC3391089.)] TJ ET BT 26.250 47.706 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 47.706 Td /F1 9.8 Tf [(Chen J, Marks E, Lai B, Zhang Z, Duce JA, Lam LQ, et al. Iron accumulates in Huntington's disease neurons: protection by )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(11)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 654 0 obj << /Type /Page /Parent 3 0 R /Contents 655 0 R >> endobj 655 0 obj << /Length 23549 >> stream 0.271 0.267 0.267 rg q 15.000 39.263 577.500 737.737 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(deferoxamine. PloS one. 2013;8\(10\):e77023. doi: 10.1371/journal.pone.0077023. PubMed PMID: 24146952; PubMed Central )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(PMCID: PMC3795666.)] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 736.167 Td /F1 9.8 Tf [(Fox JH, Kama JA, Lieberman G, Chopra R, Dorsey K, Chopra V, et al. Mechanisms of copper ion mediated Huntington's )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(disease progression. PloS one. 2007;2\(3\):e334. doi: 10.1371/journal.pone.0000334. PubMed PMID: 17396163; PubMed )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(Central PMCID: PMC1828629.)] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 692.952 Td /F1 9.8 Tf [(Dexter DT, Carayon A, Javoy-Agid F, Agid Y, Wells FR, Daniel SE, et al. Alterations in the levels of iron, ferritin and other )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(trace metals in Parkinson's disease and other neurodegenerative diseases affecting the basal ganglia. Brain. 1991;114 \( Pt )] TJ ET BT 26.250 669.143 Td /F1 9.8 Tf [(4\):1953-75. PubMed PMID: 1832073.)] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 649.738 Td /F1 9.8 Tf [(Holmgren A. Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a )] TJ ET BT 26.250 637.833 Td /F1 9.8 Tf [(disulfide. Structure. 1995;3\(3\):239-43. PubMed PMID: 7788289.)] TJ ET BT 26.250 618.429 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 618.429 Td /F1 9.8 Tf [(Benhar M, Forrester MT, Hess DT, Stamler JS. Regulated protein denitrosylation by cytosolic and mitochondrial )] TJ ET BT 26.250 606.524 Td /F1 9.8 Tf [(thioredoxins. Science. 2008;320\(5879\):1050-4. doi: 10.1126/science.1158265. PubMed PMID: 18497292; PubMed Central )] TJ ET BT 26.250 594.619 Td /F1 9.8 Tf [(PMCID: PMC2754768.)] TJ ET BT 26.250 575.214 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 575.214 Td /F1 9.8 Tf [(Wu C, Jain MR, Li Q, Oka SI, Li W, Kong AN, et al. Identification of Novel Nuclear Targets of Human Thioredoxin 1. )] TJ ET BT 26.250 563.310 Td /F1 9.8 Tf [(Molecular & cellular proteomics : MCP. 2014. doi: 10.1074/mcp.M114.040931. PubMed PMID: 25231459.)] TJ ET BT 26.250 543.905 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 543.905 Td /F1 9.8 Tf [(Hanschmann EM, Godoy JR, Berndt C, Hudemann C, Lillig CH. Thioredoxins, glutaredoxins, and peroxiredoxins--molecular )] TJ ET BT 26.250 532.000 Td /F1 9.8 Tf [(mechanisms and health significance: from cofactors to antioxidants to redox signaling. Antioxidants & redox signaling. )] TJ ET BT 26.250 520.095 Td /F1 9.8 Tf [(2013;19\(13\):1539-605. doi: 10.1089/ars.2012.4599. PubMed PMID: 23397885; PubMed Central PMCID: PMC3797455.)] TJ ET BT 26.250 500.691 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 500.691 Td /F1 9.8 Tf [(Johnson WM, Yao C, Siedlak SL, Wang W, Zhu X, Caldwell GA, et al. Glutaredoxin deficiency exacerbates )] TJ ET BT 26.250 488.786 Td /F1 9.8 Tf [(neurodegeneration in C. elegans models of Parkinson's disease. Human molecular genetics. 2014. doi: 10.1093/hmg/ddu542. )] TJ ET BT 26.250 476.881 Td /F1 9.8 Tf [(PubMed PMID: 25355420.)] TJ ET BT 26.250 457.476 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 457.476 Td /F1 9.8 Tf [(Kenchappa RS, Ravindranath V. Glutaredoxin is essential for maintenance of brain mitochondrial complex I: studies with )] TJ ET BT 26.250 445.572 Td /F1 9.8 Tf [(MPTP. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2003;17\(6\):717-9. )] TJ ET BT 26.250 433.667 Td /F1 9.8 Tf [(PubMed PMID: 12594173.)] TJ ET BT 26.250 414.262 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 414.262 Td /F1 9.8 Tf [(Grek C, Townsend DM. Protein Disulfide Isomerase Superfamily in Disease and the Regulation of Apoptosis. Endoplasmic )] TJ ET BT 26.250 402.357 Td /F1 9.8 Tf [(reticulum stress in diseases. 2014;1\(1\):4-17. doi: 10.2478/ersc-2013-0001. PubMed PMID: 25309899; PubMed Central PMCID: )] TJ ET BT 26.250 390.453 Td /F1 9.8 Tf [(PMC4192724.)] TJ ET BT 26.250 371.048 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 371.048 Td /F1 9.8 Tf [(Borges CR, Lake DF. Oxidative protein folding: nature's knotty challenge. Antioxidants & redox signaling. 2014;21\(3\):392-5. )] TJ ET BT 26.250 359.143 Td /F1 9.8 Tf [(doi: 10.1089/ars.2014.5946. PubMed PMID: 24766375.)] TJ ET BT 26.250 339.738 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 339.738 Td /F1 9.8 Tf [(Gu X, Greiner ER, Mishra R, Kodali R, Osmand A, Finkbeiner S, et al. Serines 13 and 16 are critical determinants of full-)] TJ ET BT 26.250 327.834 Td /F1 9.8 Tf [(length human mutant huntingtin induced disease pathogenesis in HD mice. Neuron. 2009;64\(6\):828-40. doi: )] TJ ET BT 26.250 315.929 Td /F1 9.8 Tf [(10.1016/j.neuron.2009.11.020. PubMed PMID: 20064390; PubMed Central PMCID: PMCPMC2807408.)] TJ ET BT 26.250 296.524 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 296.524 Td /F1 9.8 Tf [(Jeong H, Then F, Melia TJ, Jr., Mazzulli JR, Cui L, Savas JN, et al. Acetylation targets mutant huntingtin to )] TJ ET BT 26.250 284.619 Td /F1 9.8 Tf [(autophagosomes for degradation. Cell. 2009;137\(1\):60-72. PubMed PMID: 19345187.)] TJ ET BT 26.250 265.215 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 265.215 Td /F1 9.8 Tf [(de Almeida LP, Ross CA, Zala D, Aebischer P, Deglon N. Lentiviral-mediated delivery of mutant huntingtin in the striatum of )] TJ ET BT 26.250 253.310 Td /F1 9.8 Tf [(rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein )] TJ ET BT 26.250 241.405 Td /F1 9.8 Tf [(length. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2002;22\(9\):3473-83. doi: 20026337. )] TJ ET BT 26.250 229.500 Td /F1 9.8 Tf [(PubMed PMID: 11978824.)] TJ ET BT 26.250 210.096 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 210.096 Td /F1 9.8 Tf [(Kikuchi M, Doi E, Tsujimoto I, Horibe T, Tsujimoto Y. Functional analysis of human P5, a protein disulfide isomerase )] TJ ET BT 26.250 198.191 Td /F1 9.8 Tf [(homologue. Journal of biochemistry. 2002;132\(3\):451-5. PubMed PMID: 12204115.)] TJ ET BT 26.250 178.786 Td /F1 9.8 Tf [(33.)] TJ ET BT 43.553 178.786 Td /F1 9.8 Tf [(Verdoucq L, Vignols F, Jacquot JP, Chartier Y, Meyer Y. In vivo characterization of a thioredoxin h target protein defines a )] TJ ET BT 26.250 166.881 Td /F1 9.8 Tf [(new peroxiredoxin family. The Journal of biological chemistry. 1999;274\(28\):19714-22. PubMed PMID: 10391912.)] TJ ET BT 26.250 147.477 Td /F1 9.8 Tf [(34.)] TJ ET BT 43.553 147.477 Td /F1 9.8 Tf [(Tao K. Subcellular localization and in vivo oxidation-reduction kinetics of thiol peroxidase in Escherichia coli. FEMS )] TJ ET BT 26.250 135.572 Td /F1 9.8 Tf [(microbiology letters. 2008;289\(1\):41-5. doi: 10.1111/j.1574-6968.2008.01372.x. PubMed PMID: 19054092.)] TJ ET BT 26.250 116.167 Td /F1 9.8 Tf [(35.)] TJ ET BT 43.553 116.167 Td /F1 9.8 Tf [(Lu Z, Marks E, Chen J, Moline J, Barrows L, Raisbeck M, et al. Altered selenium status in Huntington's disease: )] TJ ET BT 26.250 104.262 Td /F1 9.8 Tf [(neuroprotection by selenite in the N171-82Q mouse model. Neurobiology of disease. 2014;71:34-42. doi: )] TJ ET BT 26.250 92.358 Td /F1 9.8 Tf [(10.1016/j.nbd.2014.06.022. PubMed PMID: 25014023.)] TJ ET BT 26.250 72.953 Td /F1 9.8 Tf [(36.)] TJ ET BT 43.553 72.953 Td /F1 9.8 Tf [(Ho YS, Xiong Y, Ho DS, Gao J, Chua BH, Pai H, et al. Targeted disruption of the glutaredoxin 1 gene does not sensitize )] TJ ET BT 26.250 61.048 Td /F1 9.8 Tf [(adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia. Free radical biology & medicine. 2007;43\(9\):1299-)] TJ ET BT 26.250 49.143 Td /F1 9.8 Tf [(312. PubMed PMID: 17893043.)] TJ ET Q q 15.000 39.263 577.500 737.737 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(deferoxamine. PloS one. 2013;8\(10\):e77023. doi: 10.1371/journal.pone.0077023. PubMed PMID: 24146952; PubMed Central )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(PMCID: PMC3795666.)] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 736.167 Td /F1 9.8 Tf [(Fox JH, Kama JA, Lieberman G, Chopra R, Dorsey K, Chopra V, et al. Mechanisms of copper ion mediated Huntington's )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(disease progression. PloS one. 2007;2\(3\):e334. doi: 10.1371/journal.pone.0000334. PubMed PMID: 17396163; PubMed )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(Central PMCID: PMC1828629.)] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 692.952 Td /F1 9.8 Tf [(Dexter DT, Carayon A, Javoy-Agid F, Agid Y, Wells FR, Daniel SE, et al. Alterations in the levels of iron, ferritin and other )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(trace metals in Parkinson's disease and other neurodegenerative diseases affecting the basal ganglia. Brain. 1991;114 \( Pt )] TJ ET BT 26.250 669.143 Td /F1 9.8 Tf [(4\):1953-75. PubMed PMID: 1832073.)] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 649.738 Td /F1 9.8 Tf [(Holmgren A. Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a )] TJ ET BT 26.250 637.833 Td /F1 9.8 Tf [(disulfide. Structure. 1995;3\(3\):239-43. PubMed PMID: 7788289.)] TJ ET BT 26.250 618.429 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 618.429 Td /F1 9.8 Tf [(Benhar M, Forrester MT, Hess DT, Stamler JS. Regulated protein denitrosylation by cytosolic and mitochondrial )] TJ ET BT 26.250 606.524 Td /F1 9.8 Tf [(thioredoxins. Science. 2008;320\(5879\):1050-4. doi: 10.1126/science.1158265. PubMed PMID: 18497292; PubMed Central )] TJ ET BT 26.250 594.619 Td /F1 9.8 Tf [(PMCID: PMC2754768.)] TJ ET BT 26.250 575.214 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 575.214 Td /F1 9.8 Tf [(Wu C, Jain MR, Li Q, Oka SI, Li W, Kong AN, et al. Identification of Novel Nuclear Targets of Human Thioredoxin 1. )] TJ ET BT 26.250 563.310 Td /F1 9.8 Tf [(Molecular & cellular proteomics : MCP. 2014. doi: 10.1074/mcp.M114.040931. PubMed PMID: 25231459.)] TJ ET BT 26.250 543.905 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 543.905 Td /F1 9.8 Tf [(Hanschmann EM, Godoy JR, Berndt C, Hudemann C, Lillig CH. Thioredoxins, glutaredoxins, and peroxiredoxins--molecular )] TJ ET BT 26.250 532.000 Td /F1 9.8 Tf [(mechanisms and health significance: from cofactors to antioxidants to redox signaling. Antioxidants & redox signaling. )] TJ ET BT 26.250 520.095 Td /F1 9.8 Tf [(2013;19\(13\):1539-605. doi: 10.1089/ars.2012.4599. PubMed PMID: 23397885; PubMed Central PMCID: PMC3797455.)] TJ ET BT 26.250 500.691 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 500.691 Td /F1 9.8 Tf [(Johnson WM, Yao C, Siedlak SL, Wang W, Zhu X, Caldwell GA, et al. Glutaredoxin deficiency exacerbates )] TJ ET BT 26.250 488.786 Td /F1 9.8 Tf [(neurodegeneration in C. elegans models of Parkinson's disease. Human molecular genetics. 2014. doi: 10.1093/hmg/ddu542. )] TJ ET BT 26.250 476.881 Td /F1 9.8 Tf [(PubMed PMID: 25355420.)] TJ ET BT 26.250 457.476 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 457.476 Td /F1 9.8 Tf [(Kenchappa RS, Ravindranath V. Glutaredoxin is essential for maintenance of brain mitochondrial complex I: studies with )] TJ ET BT 26.250 445.572 Td /F1 9.8 Tf [(MPTP. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2003;17\(6\):717-9. )] TJ ET BT 26.250 433.667 Td /F1 9.8 Tf [(PubMed PMID: 12594173.)] TJ ET BT 26.250 414.262 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 414.262 Td /F1 9.8 Tf [(Grek C, Townsend DM. Protein Disulfide Isomerase Superfamily in Disease and the Regulation of Apoptosis. Endoplasmic )] TJ ET BT 26.250 402.357 Td /F1 9.8 Tf [(reticulum stress in diseases. 2014;1\(1\):4-17. doi: 10.2478/ersc-2013-0001. PubMed PMID: 25309899; PubMed Central PMCID: )] TJ ET BT 26.250 390.453 Td /F1 9.8 Tf [(PMC4192724.)] TJ ET BT 26.250 371.048 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 371.048 Td /F1 9.8 Tf [(Borges CR, Lake DF. Oxidative protein folding: nature's knotty challenge. Antioxidants & redox signaling. 2014;21\(3\):392-5. )] TJ ET BT 26.250 359.143 Td /F1 9.8 Tf [(doi: 10.1089/ars.2014.5946. PubMed PMID: 24766375.)] TJ ET BT 26.250 339.738 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 339.738 Td /F1 9.8 Tf [(Gu X, Greiner ER, Mishra R, Kodali R, Osmand A, Finkbeiner S, et al. Serines 13 and 16 are critical determinants of full-)] TJ ET BT 26.250 327.834 Td /F1 9.8 Tf [(length human mutant huntingtin induced disease pathogenesis in HD mice. Neuron. 2009;64\(6\):828-40. doi: )] TJ ET BT 26.250 315.929 Td /F1 9.8 Tf [(10.1016/j.neuron.2009.11.020. PubMed PMID: 20064390; PubMed Central PMCID: PMCPMC2807408.)] TJ ET BT 26.250 296.524 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 296.524 Td /F1 9.8 Tf [(Jeong H, Then F, Melia TJ, Jr., Mazzulli JR, Cui L, Savas JN, et al. Acetylation targets mutant huntingtin to )] TJ ET BT 26.250 284.619 Td /F1 9.8 Tf [(autophagosomes for degradation. Cell. 2009;137\(1\):60-72. PubMed PMID: 19345187.)] TJ ET BT 26.250 265.215 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 265.215 Td /F1 9.8 Tf [(de Almeida LP, Ross CA, Zala D, Aebischer P, Deglon N. Lentiviral-mediated delivery of mutant huntingtin in the striatum of )] TJ ET BT 26.250 253.310 Td /F1 9.8 Tf [(rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein )] TJ ET BT 26.250 241.405 Td /F1 9.8 Tf [(length. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2002;22\(9\):3473-83. doi: 20026337. )] TJ ET BT 26.250 229.500 Td /F1 9.8 Tf [(PubMed PMID: 11978824.)] TJ ET BT 26.250 210.096 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 210.096 Td /F1 9.8 Tf [(Kikuchi M, Doi E, Tsujimoto I, Horibe T, Tsujimoto Y. Functional analysis of human P5, a protein disulfide isomerase )] TJ ET BT 26.250 198.191 Td /F1 9.8 Tf [(homologue. Journal of biochemistry. 2002;132\(3\):451-5. PubMed PMID: 12204115.)] TJ ET BT 26.250 178.786 Td /F1 9.8 Tf [(33.)] TJ ET BT 43.553 178.786 Td /F1 9.8 Tf [(Verdoucq L, Vignols F, Jacquot JP, Chartier Y, Meyer Y. In vivo characterization of a thioredoxin h target protein defines a )] TJ ET BT 26.250 166.881 Td /F1 9.8 Tf [(new peroxiredoxin family. The Journal of biological chemistry. 1999;274\(28\):19714-22. PubMed PMID: 10391912.)] TJ ET BT 26.250 147.477 Td /F1 9.8 Tf [(34.)] TJ ET BT 43.553 147.477 Td /F1 9.8 Tf [(Tao K. Subcellular localization and in vivo oxidation-reduction kinetics of thiol peroxidase in Escherichia coli. FEMS )] TJ ET BT 26.250 135.572 Td /F1 9.8 Tf [(microbiology letters. 2008;289\(1\):41-5. doi: 10.1111/j.1574-6968.2008.01372.x. PubMed PMID: 19054092.)] TJ ET BT 26.250 116.167 Td /F1 9.8 Tf [(35.)] TJ ET BT 43.553 116.167 Td /F1 9.8 Tf [(Lu Z, Marks E, Chen J, Moline J, Barrows L, Raisbeck M, et al. Altered selenium status in Huntington's disease: )] TJ ET BT 26.250 104.262 Td /F1 9.8 Tf [(neuroprotection by selenite in the N171-82Q mouse model. Neurobiology of disease. 2014;71:34-42. doi: )] TJ ET BT 26.250 92.358 Td /F1 9.8 Tf [(10.1016/j.nbd.2014.06.022. PubMed PMID: 25014023.)] TJ ET BT 26.250 72.953 Td /F1 9.8 Tf [(36.)] TJ ET BT 43.553 72.953 Td /F1 9.8 Tf [(Ho YS, Xiong Y, Ho DS, Gao J, Chua BH, Pai H, et al. Targeted disruption of the glutaredoxin 1 gene does not sensitize )] TJ ET BT 26.250 61.048 Td /F1 9.8 Tf [(adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia. Free radical biology & medicine. 2007;43\(9\):1299-)] TJ ET BT 26.250 49.143 Td /F1 9.8 Tf [(312. PubMed PMID: 17893043.)] TJ ET Q q 15.000 39.263 577.500 737.737 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(deferoxamine. PloS one. 2013;8\(10\):e77023. doi: 10.1371/journal.pone.0077023. PubMed PMID: 24146952; PubMed Central )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(PMCID: PMC3795666.)] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 736.167 Td /F1 9.8 Tf [(Fox JH, Kama JA, Lieberman G, Chopra R, Dorsey K, Chopra V, et al. Mechanisms of copper ion mediated Huntington's )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(disease progression. PloS one. 2007;2\(3\):e334. doi: 10.1371/journal.pone.0000334. PubMed PMID: 17396163; PubMed )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(Central PMCID: PMC1828629.)] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 692.952 Td /F1 9.8 Tf [(Dexter DT, Carayon A, Javoy-Agid F, Agid Y, Wells FR, Daniel SE, et al. Alterations in the levels of iron, ferritin and other )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(trace metals in Parkinson's disease and other neurodegenerative diseases affecting the basal ganglia. Brain. 1991;114 \( Pt )] TJ ET BT 26.250 669.143 Td /F1 9.8 Tf [(4\):1953-75. PubMed PMID: 1832073.)] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 649.738 Td /F1 9.8 Tf [(Holmgren A. Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a )] TJ ET BT 26.250 637.833 Td /F1 9.8 Tf [(disulfide. Structure. 1995;3\(3\):239-43. PubMed PMID: 7788289.)] TJ ET BT 26.250 618.429 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 618.429 Td /F1 9.8 Tf [(Benhar M, Forrester MT, Hess DT, Stamler JS. Regulated protein denitrosylation by cytosolic and mitochondrial )] TJ ET BT 26.250 606.524 Td /F1 9.8 Tf [(thioredoxins. Science. 2008;320\(5879\):1050-4. doi: 10.1126/science.1158265. PubMed PMID: 18497292; PubMed Central )] TJ ET BT 26.250 594.619 Td /F1 9.8 Tf [(PMCID: PMC2754768.)] TJ ET BT 26.250 575.214 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 575.214 Td /F1 9.8 Tf [(Wu C, Jain MR, Li Q, Oka SI, Li W, Kong AN, et al. Identification of Novel Nuclear Targets of Human Thioredoxin 1. )] TJ ET BT 26.250 563.310 Td /F1 9.8 Tf [(Molecular & cellular proteomics : MCP. 2014. doi: 10.1074/mcp.M114.040931. PubMed PMID: 25231459.)] TJ ET BT 26.250 543.905 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 543.905 Td /F1 9.8 Tf [(Hanschmann EM, Godoy JR, Berndt C, Hudemann C, Lillig CH. Thioredoxins, glutaredoxins, and peroxiredoxins--molecular )] TJ ET BT 26.250 532.000 Td /F1 9.8 Tf [(mechanisms and health significance: from cofactors to antioxidants to redox signaling. Antioxidants & redox signaling. )] TJ ET BT 26.250 520.095 Td /F1 9.8 Tf [(2013;19\(13\):1539-605. doi: 10.1089/ars.2012.4599. PubMed PMID: 23397885; PubMed Central PMCID: PMC3797455.)] TJ ET BT 26.250 500.691 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 500.691 Td /F1 9.8 Tf [(Johnson WM, Yao C, Siedlak SL, Wang W, Zhu X, Caldwell GA, et al. Glutaredoxin deficiency exacerbates )] TJ ET BT 26.250 488.786 Td /F1 9.8 Tf [(neurodegeneration in C. elegans models of Parkinson's disease. Human molecular genetics. 2014. doi: 10.1093/hmg/ddu542. )] TJ ET BT 26.250 476.881 Td /F1 9.8 Tf [(PubMed PMID: 25355420.)] TJ ET BT 26.250 457.476 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 457.476 Td /F1 9.8 Tf [(Kenchappa RS, Ravindranath V. Glutaredoxin is essential for maintenance of brain mitochondrial complex I: studies with )] TJ ET BT 26.250 445.572 Td /F1 9.8 Tf [(MPTP. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2003;17\(6\):717-9. )] TJ ET BT 26.250 433.667 Td /F1 9.8 Tf [(PubMed PMID: 12594173.)] TJ ET BT 26.250 414.262 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 414.262 Td /F1 9.8 Tf [(Grek C, Townsend DM. Protein Disulfide Isomerase Superfamily in Disease and the Regulation of Apoptosis. Endoplasmic )] TJ ET BT 26.250 402.357 Td /F1 9.8 Tf [(reticulum stress in diseases. 2014;1\(1\):4-17. doi: 10.2478/ersc-2013-0001. PubMed PMID: 25309899; PubMed Central PMCID: )] TJ ET BT 26.250 390.453 Td /F1 9.8 Tf [(PMC4192724.)] TJ ET BT 26.250 371.048 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 371.048 Td /F1 9.8 Tf [(Borges CR, Lake DF. Oxidative protein folding: nature's knotty challenge. Antioxidants & redox signaling. 2014;21\(3\):392-5. )] TJ ET BT 26.250 359.143 Td /F1 9.8 Tf [(doi: 10.1089/ars.2014.5946. PubMed PMID: 24766375.)] TJ ET BT 26.250 339.738 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 339.738 Td /F1 9.8 Tf [(Gu X, Greiner ER, Mishra R, Kodali R, Osmand A, Finkbeiner S, et al. Serines 13 and 16 are critical determinants of full-)] TJ ET BT 26.250 327.834 Td /F1 9.8 Tf [(length human mutant huntingtin induced disease pathogenesis in HD mice. Neuron. 2009;64\(6\):828-40. doi: )] TJ ET BT 26.250 315.929 Td /F1 9.8 Tf [(10.1016/j.neuron.2009.11.020. PubMed PMID: 20064390; PubMed Central PMCID: PMCPMC2807408.)] TJ ET BT 26.250 296.524 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 296.524 Td /F1 9.8 Tf [(Jeong H, Then F, Melia TJ, Jr., Mazzulli JR, Cui L, Savas JN, et al. Acetylation targets mutant huntingtin to )] TJ ET BT 26.250 284.619 Td /F1 9.8 Tf [(autophagosomes for degradation. Cell. 2009;137\(1\):60-72. PubMed PMID: 19345187.)] TJ ET BT 26.250 265.215 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 265.215 Td /F1 9.8 Tf [(de Almeida LP, Ross CA, Zala D, Aebischer P, Deglon N. Lentiviral-mediated delivery of mutant huntingtin in the striatum of )] TJ ET BT 26.250 253.310 Td /F1 9.8 Tf [(rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein )] TJ ET BT 26.250 241.405 Td /F1 9.8 Tf [(length. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2002;22\(9\):3473-83. doi: 20026337. )] TJ ET BT 26.250 229.500 Td /F1 9.8 Tf [(PubMed PMID: 11978824.)] TJ ET BT 26.250 210.096 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 210.096 Td /F1 9.8 Tf [(Kikuchi M, Doi E, Tsujimoto I, Horibe T, Tsujimoto Y. Functional analysis of human P5, a protein disulfide isomerase )] TJ ET BT 26.250 198.191 Td /F1 9.8 Tf [(homologue. Journal of biochemistry. 2002;132\(3\):451-5. PubMed PMID: 12204115.)] TJ ET BT 26.250 178.786 Td /F1 9.8 Tf [(33.)] TJ ET BT 43.553 178.786 Td /F1 9.8 Tf [(Verdoucq L, Vignols F, Jacquot JP, Chartier Y, Meyer Y. In vivo characterization of a thioredoxin h target protein defines a )] TJ ET BT 26.250 166.881 Td /F1 9.8 Tf [(new peroxiredoxin family. The Journal of biological chemistry. 1999;274\(28\):19714-22. PubMed PMID: 10391912.)] TJ ET BT 26.250 147.477 Td /F1 9.8 Tf [(34.)] TJ ET BT 43.553 147.477 Td /F1 9.8 Tf [(Tao K. Subcellular localization and in vivo oxidation-reduction kinetics of thiol peroxidase in Escherichia coli. FEMS )] TJ ET BT 26.250 135.572 Td /F1 9.8 Tf [(microbiology letters. 2008;289\(1\):41-5. doi: 10.1111/j.1574-6968.2008.01372.x. PubMed PMID: 19054092.)] TJ ET BT 26.250 116.167 Td /F1 9.8 Tf [(35.)] TJ ET BT 43.553 116.167 Td /F1 9.8 Tf [(Lu Z, Marks E, Chen J, Moline J, Barrows L, Raisbeck M, et al. Altered selenium status in Huntington's disease: )] TJ ET BT 26.250 104.262 Td /F1 9.8 Tf [(neuroprotection by selenite in the N171-82Q mouse model. Neurobiology of disease. 2014;71:34-42. doi: )] TJ ET BT 26.250 92.358 Td /F1 9.8 Tf [(10.1016/j.nbd.2014.06.022. PubMed PMID: 25014023.)] TJ ET BT 26.250 72.953 Td /F1 9.8 Tf [(36.)] TJ ET BT 43.553 72.953 Td /F1 9.8 Tf [(Ho YS, Xiong Y, Ho DS, Gao J, Chua BH, Pai H, et al. Targeted disruption of the glutaredoxin 1 gene does not sensitize )] TJ ET BT 26.250 61.048 Td /F1 9.8 Tf [(adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia. Free radical biology & medicine. 2007;43\(9\):1299-)] TJ ET BT 26.250 49.143 Td /F1 9.8 Tf [(312. PubMed PMID: 17893043.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(12)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 656 0 obj << /Type /Page /Parent 3 0 R /Contents 657 0 R >> endobj 657 0 obj << /Length 23942 >> stream 0.271 0.267 0.267 rg q 15.000 34.858 577.500 742.142 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(37.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase K, Schwarz C, Meloni A, Young C, et al. Huntingtin is a cytoplasmic protein associated with )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(vesicles in human and rat brain neurons. Neuron. 1995;14\(5\):1075-81. PubMed PMID: 7748555.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(38.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Gutekunst CA, Li SH, Yi H, Mulroy JS, Kuemmerle S, Jones R, et al. Nuclear and neuropil aggregates in Huntington's )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(disease: relationship to neuropathology. The Journal of neuroscience : the official journal of the Society for Neuroscience. )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(1999;19\(7\):2522-34. PubMed PMID: 10087066.)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(39.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Haugstetter J, Maurer MA, Blicher T, Pagac M, Wider G, Ellgaard L. Structure-function analysis of the endoplasmic )] TJ ET BT 26.250 673.548 Td /F1 9.8 Tf [(reticulum oxidoreductase TMX3 reveals interdomain stabilization of the N-terminal redox-active domain. The Journal of )] TJ ET BT 26.250 661.643 Td /F1 9.8 Tf [(biological chemistry. 2007;282\(46\):33859-67. doi: 10.1074/jbc.M706442200. PubMed PMID: 17881353.)] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(40.)] TJ ET BT 43.553 642.238 Td /F1 9.8 Tf [(Ruiz M, Deglon N. Viral-mediated overexpression of mutant huntingtin to model HD in various species. Neurobiology of )] TJ ET BT 26.250 630.333 Td /F1 9.8 Tf [(disease. 2012;48\(2\):202-11. doi: 10.1016/j.nbd.2011.08.023. PubMed PMID: 21889981.)] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(41.)] TJ ET BT 43.553 610.929 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, et al. Aggregation of huntingtin in neuronal intranuclear )] TJ ET BT 26.250 599.024 Td /F1 9.8 Tf [(inclusions and dystrophic neurites in brain. Science. 1997;277\(5334\):1990-3. PubMed PMID: 9302293.)] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(42.)] TJ ET BT 43.553 579.619 Td /F1 9.8 Tf [(Chopra V, Fox JH, Lieberman G, Dorsey K, Matson W, Waldmeier P, et al. A small-molecule therapeutic lead for )] TJ ET BT 26.250 567.714 Td /F1 9.8 Tf [(Huntington's disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse. Proceedings of the National )] TJ ET BT 26.250 555.810 Td /F1 9.8 Tf [(Academy of Sciences of the United States of America. 2007;104\(42\):16685-9. PubMed PMID: 17925440.)] TJ ET BT 26.250 536.405 Td /F1 9.8 Tf [(43.)] TJ ET BT 43.553 536.405 Td /F1 9.8 Tf [(Hodges A, Hughes G, Brooks S, Elliston L, Holmans P, Dunnett SB, et al. Brain gene expression correlates with changes in )] TJ ET BT 26.250 524.500 Td /F1 9.8 Tf [(behavior in the R6/1 mouse model of Huntington's disease. Genes Brain Behav. 2008;7\(3\):288-99. doi: 10.1111/j.1601-)] TJ ET BT 26.250 512.595 Td /F1 9.8 Tf [(183X.2007.00350.x. PubMed PMID: 17696994.)] TJ ET BT 26.250 493.191 Td /F1 9.8 Tf [(44.)] TJ ET BT 43.553 493.191 Td /F1 9.8 Tf [(Becanovic K, Pouladi MA, Lim RS, Kuhn A, Pavlidis P, Luthi-Carter R, et al. Transcriptional changes in Huntington disease )] TJ ET BT 26.250 481.286 Td /F1 9.8 Tf [(identified using genome-wide expression profiling and cross-platform analysis. Hum Mol Genet. 2010;19\(8\):1438-52. doi: )] TJ ET BT 26.250 469.381 Td /F1 9.8 Tf [(10.1093/hmg/ddq018. PubMed PMID: 20089533; PubMed Central PMCID: PMCPMC2846159.)] TJ ET BT 26.250 449.976 Td /F1 9.8 Tf [(45.)] TJ ET BT 43.553 449.976 Td /F1 9.8 Tf [(Valencia A, Sapp E, Kimm JS, McClory H, Reeves PB, Alexander J, et al. Elevated NADPH oxidase activity contributes to )] TJ ET BT 26.250 438.072 Td /F1 9.8 Tf [(oxidative stress and cell death in Huntington's disease. Hum Mol Genet. 2013;22\(6\):1112-31. doi: 10.1093/hmg/dds516. )] TJ ET BT 26.250 426.167 Td /F1 9.8 Tf [(PubMed PMID: 23223017; PubMed Central PMCID: PMC3578411.)] TJ ET BT 26.250 406.762 Td /F1 9.8 Tf [(46.)] TJ ET BT 43.553 406.762 Td /F1 9.8 Tf [(Browne SE, Bowling AC, MacGarvey U, Baik MJ, Berger SC, Muqit MM, et al. Oxidative damage and metabolic dysfunction )] TJ ET BT 26.250 394.857 Td /F1 9.8 Tf [(in Huntington's disease: selective vulnerability of the basal ganglia. Annals of neurology. 1997;41\(5\):646-53. PubMed PMID: )] TJ ET BT 26.250 382.953 Td /F1 9.8 Tf [(9153527.)] TJ ET BT 26.250 363.548 Td /F1 9.8 Tf [(47.)] TJ ET BT 43.553 363.548 Td /F1 9.8 Tf [(Cheng Y, Peng Q, Hou Z, Aggarwal M, Zhang J, Mori S, et al. Structural MRI detects progressive regional brain atrophy and )] TJ ET BT 26.250 351.643 Td /F1 9.8 Tf [(neuroprotective effects in N171-82Q Huntington's disease mouse model. NeuroImage. 2011;56\(3\):1027-34. PubMed PMID: )] TJ ET BT 26.250 339.738 Td /F1 9.8 Tf [(21320608.)] TJ ET BT 26.250 320.334 Td /F1 9.8 Tf [(48.)] TJ ET BT 43.553 320.334 Td /F1 9.8 Tf [(Zhou F, Gomi M, Fujimoto M, Hayase M, Marumo T, Masutani H, et al. Attenuation of neuronal degeneration in thioredoxin-)] TJ ET BT 26.250 308.429 Td /F1 9.8 Tf [(1 overexpressing mice after mild focal ischemia. Brain research. 2009;1272:62-70. doi: 10.1016/j.brainres.2009.03.023. )] TJ ET BT 26.250 296.524 Td /F1 9.8 Tf [(PubMed PMID: 19328186.)] TJ ET BT 26.250 277.119 Td /F1 9.8 Tf [(49.)] TJ ET BT 43.553 277.119 Td /F1 9.8 Tf [(Kim GS, Jung JE, Narasimhan P, Sakata H, Chan PH. Induction of thioredoxin-interacting protein is mediated by oxidative )] TJ ET BT 26.250 265.215 Td /F1 9.8 Tf [(stress, calcium, and glucose after brain injury in mice. Neurobiology of disease. 2012;46\(2\):440-9. doi: )] TJ ET BT 26.250 253.310 Td /F1 9.8 Tf [(10.1016/j.nbd.2012.02.008. PubMed PMID: 22366181; PubMed Central PMCID: PMC3323710.)] TJ ET BT 26.250 233.905 Td /F1 9.8 Tf [(50.)] TJ ET BT 43.553 233.905 Td /F1 9.8 Tf [(Ishrat T, Mohamed IN, Pillai B, Soliman S, Fouda AY, Ergul A, et al. Thioredoxin-Interacting Protein: a Novel Target for )] TJ ET BT 26.250 222.000 Td /F1 9.8 Tf [(Neuroprotection in Experimental Thromboembolic Stroke in Mice. Molecular neurobiology. 2014. doi: 10.1007/s12035-014-8766-)] TJ ET BT 26.250 210.096 Td /F1 9.8 Tf [(x. PubMed PMID: 24939693.)] TJ ET BT 26.250 190.691 Td /F1 9.8 Tf [(51.)] TJ ET BT 43.553 190.691 Td /F1 9.8 Tf [(Tian L, Nie H, Zhang Y, Chen Y, Peng Z, Cai M, et al. Recombinant human thioredoxin-1 promotes neurogenesis and )] TJ ET BT 26.250 178.786 Td /F1 9.8 Tf [(facilitates cognitive recovery following cerebral ischemia in mice. Neuropharmacology. 2014;77:453-64. doi: )] TJ ET BT 26.250 166.881 Td /F1 9.8 Tf [(10.1016/j.neuropharm.2013.10.027. PubMed PMID: 24212059.)] TJ ET BT 26.250 147.477 Td /F1 9.8 Tf [(52.)] TJ ET BT 43.553 147.477 Td /F1 9.8 Tf [(Taira T, Saito Y, Niki T, Iguchi-Ariga SM, Takahashi K, Ariga H. DJ-1 has a role in antioxidative stress to prevent cell death. )] TJ ET BT 26.250 135.572 Td /F1 9.8 Tf [(EMBO reports. 2004;5\(2\):213-8. doi: 10.1038/sj.embor.7400074. PubMed PMID: 14749723; PubMed Central PMCID: )] TJ ET BT 26.250 123.667 Td /F1 9.8 Tf [(PMC1298985.)] TJ ET BT 26.250 104.262 Td /F1 9.8 Tf [(53.)] TJ ET BT 43.553 104.262 Td /F1 9.8 Tf [(Im JY, Lee KW, Woo JM, Junn E, Mouradian MM. DJ-1 induces thioredoxin 1 expression through the Nrf2 pathway. Human )] TJ ET BT 26.250 92.358 Td /F1 9.8 Tf [(molecular genetics. 2012;21\(13\):3013-24. doi: 10.1093/hmg/dds131. PubMed PMID: 22492997; PubMed Central PMCID: )] TJ ET BT 26.250 80.453 Td /F1 9.8 Tf [(PMC3373246.)] TJ ET BT 26.250 61.048 Td /F1 9.8 Tf [(54.)] TJ ET BT 43.553 61.048 Td /F1 9.8 Tf [(Kitaoka Y, Munemasa Y, Hayashi Y, Kuribayashi J, Koseki N, Kojima K, et al. Axonal protection by 17beta-estradiol through )] TJ ET BT 26.250 49.143 Td /F1 9.8 Tf [(thioredoxin-1 in tumor necrosis factor-induced optic neuropathy. Endocrinology. 2011;152\(7\):2775-85. doi: 10.1210/en.2011-)] TJ ET Q q 15.000 34.858 577.500 742.142 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(37.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase K, Schwarz C, Meloni A, Young C, et al. Huntingtin is a cytoplasmic protein associated with )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(vesicles in human and rat brain neurons. Neuron. 1995;14\(5\):1075-81. PubMed PMID: 7748555.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(38.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Gutekunst CA, Li SH, Yi H, Mulroy JS, Kuemmerle S, Jones R, et al. Nuclear and neuropil aggregates in Huntington's )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(disease: relationship to neuropathology. The Journal of neuroscience : the official journal of the Society for Neuroscience. )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(1999;19\(7\):2522-34. PubMed PMID: 10087066.)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(39.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Haugstetter J, Maurer MA, Blicher T, Pagac M, Wider G, Ellgaard L. Structure-function analysis of the endoplasmic )] TJ ET BT 26.250 673.548 Td /F1 9.8 Tf [(reticulum oxidoreductase TMX3 reveals interdomain stabilization of the N-terminal redox-active domain. The Journal of )] TJ ET BT 26.250 661.643 Td /F1 9.8 Tf [(biological chemistry. 2007;282\(46\):33859-67. doi: 10.1074/jbc.M706442200. PubMed PMID: 17881353.)] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(40.)] TJ ET BT 43.553 642.238 Td /F1 9.8 Tf [(Ruiz M, Deglon N. Viral-mediated overexpression of mutant huntingtin to model HD in various species. Neurobiology of )] TJ ET BT 26.250 630.333 Td /F1 9.8 Tf [(disease. 2012;48\(2\):202-11. doi: 10.1016/j.nbd.2011.08.023. PubMed PMID: 21889981.)] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(41.)] TJ ET BT 43.553 610.929 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, et al. Aggregation of huntingtin in neuronal intranuclear )] TJ ET BT 26.250 599.024 Td /F1 9.8 Tf [(inclusions and dystrophic neurites in brain. Science. 1997;277\(5334\):1990-3. PubMed PMID: 9302293.)] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(42.)] TJ ET BT 43.553 579.619 Td /F1 9.8 Tf [(Chopra V, Fox JH, Lieberman G, Dorsey K, Matson W, Waldmeier P, et al. A small-molecule therapeutic lead for )] TJ ET BT 26.250 567.714 Td /F1 9.8 Tf [(Huntington's disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse. Proceedings of the National )] TJ ET BT 26.250 555.810 Td /F1 9.8 Tf [(Academy of Sciences of the United States of America. 2007;104\(42\):16685-9. PubMed PMID: 17925440.)] TJ ET BT 26.250 536.405 Td /F1 9.8 Tf [(43.)] TJ ET BT 43.553 536.405 Td /F1 9.8 Tf [(Hodges A, Hughes G, Brooks S, Elliston L, Holmans P, Dunnett SB, et al. Brain gene expression correlates with changes in )] TJ ET BT 26.250 524.500 Td /F1 9.8 Tf [(behavior in the R6/1 mouse model of Huntington's disease. Genes Brain Behav. 2008;7\(3\):288-99. doi: 10.1111/j.1601-)] TJ ET BT 26.250 512.595 Td /F1 9.8 Tf [(183X.2007.00350.x. PubMed PMID: 17696994.)] TJ ET BT 26.250 493.191 Td /F1 9.8 Tf [(44.)] TJ ET BT 43.553 493.191 Td /F1 9.8 Tf [(Becanovic K, Pouladi MA, Lim RS, Kuhn A, Pavlidis P, Luthi-Carter R, et al. Transcriptional changes in Huntington disease )] TJ ET BT 26.250 481.286 Td /F1 9.8 Tf [(identified using genome-wide expression profiling and cross-platform analysis. Hum Mol Genet. 2010;19\(8\):1438-52. doi: )] TJ ET BT 26.250 469.381 Td /F1 9.8 Tf [(10.1093/hmg/ddq018. PubMed PMID: 20089533; PubMed Central PMCID: PMCPMC2846159.)] TJ ET BT 26.250 449.976 Td /F1 9.8 Tf [(45.)] TJ ET BT 43.553 449.976 Td /F1 9.8 Tf [(Valencia A, Sapp E, Kimm JS, McClory H, Reeves PB, Alexander J, et al. Elevated NADPH oxidase activity contributes to )] TJ ET BT 26.250 438.072 Td /F1 9.8 Tf [(oxidative stress and cell death in Huntington's disease. Hum Mol Genet. 2013;22\(6\):1112-31. doi: 10.1093/hmg/dds516. )] TJ ET BT 26.250 426.167 Td /F1 9.8 Tf [(PubMed PMID: 23223017; PubMed Central PMCID: PMC3578411.)] TJ ET BT 26.250 406.762 Td /F1 9.8 Tf [(46.)] TJ ET BT 43.553 406.762 Td /F1 9.8 Tf [(Browne SE, Bowling AC, MacGarvey U, Baik MJ, Berger SC, Muqit MM, et al. Oxidative damage and metabolic dysfunction )] TJ ET BT 26.250 394.857 Td /F1 9.8 Tf [(in Huntington's disease: selective vulnerability of the basal ganglia. Annals of neurology. 1997;41\(5\):646-53. PubMed PMID: )] TJ ET BT 26.250 382.953 Td /F1 9.8 Tf [(9153527.)] TJ ET BT 26.250 363.548 Td /F1 9.8 Tf [(47.)] TJ ET BT 43.553 363.548 Td /F1 9.8 Tf [(Cheng Y, Peng Q, Hou Z, Aggarwal M, Zhang J, Mori S, et al. Structural MRI detects progressive regional brain atrophy and )] TJ ET BT 26.250 351.643 Td /F1 9.8 Tf [(neuroprotective effects in N171-82Q Huntington's disease mouse model. NeuroImage. 2011;56\(3\):1027-34. PubMed PMID: )] TJ ET BT 26.250 339.738 Td /F1 9.8 Tf [(21320608.)] TJ ET BT 26.250 320.334 Td /F1 9.8 Tf [(48.)] TJ ET BT 43.553 320.334 Td /F1 9.8 Tf [(Zhou F, Gomi M, Fujimoto M, Hayase M, Marumo T, Masutani H, et al. Attenuation of neuronal degeneration in thioredoxin-)] TJ ET BT 26.250 308.429 Td /F1 9.8 Tf [(1 overexpressing mice after mild focal ischemia. Brain research. 2009;1272:62-70. doi: 10.1016/j.brainres.2009.03.023. )] TJ ET BT 26.250 296.524 Td /F1 9.8 Tf [(PubMed PMID: 19328186.)] TJ ET BT 26.250 277.119 Td /F1 9.8 Tf [(49.)] TJ ET BT 43.553 277.119 Td /F1 9.8 Tf [(Kim GS, Jung JE, Narasimhan P, Sakata H, Chan PH. Induction of thioredoxin-interacting protein is mediated by oxidative )] TJ ET BT 26.250 265.215 Td /F1 9.8 Tf [(stress, calcium, and glucose after brain injury in mice. Neurobiology of disease. 2012;46\(2\):440-9. doi: )] TJ ET BT 26.250 253.310 Td /F1 9.8 Tf [(10.1016/j.nbd.2012.02.008. PubMed PMID: 22366181; PubMed Central PMCID: PMC3323710.)] TJ ET BT 26.250 233.905 Td /F1 9.8 Tf [(50.)] TJ ET BT 43.553 233.905 Td /F1 9.8 Tf [(Ishrat T, Mohamed IN, Pillai B, Soliman S, Fouda AY, Ergul A, et al. Thioredoxin-Interacting Protein: a Novel Target for )] TJ ET BT 26.250 222.000 Td /F1 9.8 Tf [(Neuroprotection in Experimental Thromboembolic Stroke in Mice. Molecular neurobiology. 2014. doi: 10.1007/s12035-014-8766-)] TJ ET BT 26.250 210.096 Td /F1 9.8 Tf [(x. PubMed PMID: 24939693.)] TJ ET BT 26.250 190.691 Td /F1 9.8 Tf [(51.)] TJ ET BT 43.553 190.691 Td /F1 9.8 Tf [(Tian L, Nie H, Zhang Y, Chen Y, Peng Z, Cai M, et al. Recombinant human thioredoxin-1 promotes neurogenesis and )] TJ ET BT 26.250 178.786 Td /F1 9.8 Tf [(facilitates cognitive recovery following cerebral ischemia in mice. Neuropharmacology. 2014;77:453-64. doi: )] TJ ET BT 26.250 166.881 Td /F1 9.8 Tf [(10.1016/j.neuropharm.2013.10.027. PubMed PMID: 24212059.)] TJ ET BT 26.250 147.477 Td /F1 9.8 Tf [(52.)] TJ ET BT 43.553 147.477 Td /F1 9.8 Tf [(Taira T, Saito Y, Niki T, Iguchi-Ariga SM, Takahashi K, Ariga H. DJ-1 has a role in antioxidative stress to prevent cell death. )] TJ ET BT 26.250 135.572 Td /F1 9.8 Tf [(EMBO reports. 2004;5\(2\):213-8. doi: 10.1038/sj.embor.7400074. PubMed PMID: 14749723; PubMed Central PMCID: )] TJ ET BT 26.250 123.667 Td /F1 9.8 Tf [(PMC1298985.)] TJ ET BT 26.250 104.262 Td /F1 9.8 Tf [(53.)] TJ ET BT 43.553 104.262 Td /F1 9.8 Tf [(Im JY, Lee KW, Woo JM, Junn E, Mouradian MM. DJ-1 induces thioredoxin 1 expression through the Nrf2 pathway. Human )] TJ ET BT 26.250 92.358 Td /F1 9.8 Tf [(molecular genetics. 2012;21\(13\):3013-24. doi: 10.1093/hmg/dds131. PubMed PMID: 22492997; PubMed Central PMCID: )] TJ ET BT 26.250 80.453 Td /F1 9.8 Tf [(PMC3373246.)] TJ ET BT 26.250 61.048 Td /F1 9.8 Tf [(54.)] TJ ET BT 43.553 61.048 Td /F1 9.8 Tf [(Kitaoka Y, Munemasa Y, Hayashi Y, Kuribayashi J, Koseki N, Kojima K, et al. Axonal protection by 17beta-estradiol through )] TJ ET BT 26.250 49.143 Td /F1 9.8 Tf [(thioredoxin-1 in tumor necrosis factor-induced optic neuropathy. Endocrinology. 2011;152\(7\):2775-85. doi: 10.1210/en.2011-)] TJ ET Q q 15.000 34.858 577.500 742.142 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(37.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase K, Schwarz C, Meloni A, Young C, et al. Huntingtin is a cytoplasmic protein associated with )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(vesicles in human and rat brain neurons. Neuron. 1995;14\(5\):1075-81. PubMed PMID: 7748555.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(38.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Gutekunst CA, Li SH, Yi H, Mulroy JS, Kuemmerle S, Jones R, et al. Nuclear and neuropil aggregates in Huntington's )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(disease: relationship to neuropathology. The Journal of neuroscience : the official journal of the Society for Neuroscience. )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(1999;19\(7\):2522-34. PubMed PMID: 10087066.)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(39.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Haugstetter J, Maurer MA, Blicher T, Pagac M, Wider G, Ellgaard L. Structure-function analysis of the endoplasmic )] TJ ET BT 26.250 673.548 Td /F1 9.8 Tf [(reticulum oxidoreductase TMX3 reveals interdomain stabilization of the N-terminal redox-active domain. The Journal of )] TJ ET BT 26.250 661.643 Td /F1 9.8 Tf [(biological chemistry. 2007;282\(46\):33859-67. doi: 10.1074/jbc.M706442200. PubMed PMID: 17881353.)] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(40.)] TJ ET BT 43.553 642.238 Td /F1 9.8 Tf [(Ruiz M, Deglon N. Viral-mediated overexpression of mutant huntingtin to model HD in various species. Neurobiology of )] TJ ET BT 26.250 630.333 Td /F1 9.8 Tf [(disease. 2012;48\(2\):202-11. doi: 10.1016/j.nbd.2011.08.023. PubMed PMID: 21889981.)] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(41.)] TJ ET BT 43.553 610.929 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, et al. Aggregation of huntingtin in neuronal intranuclear )] TJ ET BT 26.250 599.024 Td /F1 9.8 Tf [(inclusions and dystrophic neurites in brain. Science. 1997;277\(5334\):1990-3. PubMed PMID: 9302293.)] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(42.)] TJ ET BT 43.553 579.619 Td /F1 9.8 Tf [(Chopra V, Fox JH, Lieberman G, Dorsey K, Matson W, Waldmeier P, et al. A small-molecule therapeutic lead for )] TJ ET BT 26.250 567.714 Td /F1 9.8 Tf [(Huntington's disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse. Proceedings of the National )] TJ ET BT 26.250 555.810 Td /F1 9.8 Tf [(Academy of Sciences of the United States of America. 2007;104\(42\):16685-9. PubMed PMID: 17925440.)] TJ ET BT 26.250 536.405 Td /F1 9.8 Tf [(43.)] TJ ET BT 43.553 536.405 Td /F1 9.8 Tf [(Hodges A, Hughes G, Brooks S, Elliston L, Holmans P, Dunnett SB, et al. Brain gene expression correlates with changes in )] TJ ET BT 26.250 524.500 Td /F1 9.8 Tf [(behavior in the R6/1 mouse model of Huntington's disease. Genes Brain Behav. 2008;7\(3\):288-99. doi: 10.1111/j.1601-)] TJ ET BT 26.250 512.595 Td /F1 9.8 Tf [(183X.2007.00350.x. PubMed PMID: 17696994.)] TJ ET BT 26.250 493.191 Td /F1 9.8 Tf [(44.)] TJ ET BT 43.553 493.191 Td /F1 9.8 Tf [(Becanovic K, Pouladi MA, Lim RS, Kuhn A, Pavlidis P, Luthi-Carter R, et al. Transcriptional changes in Huntington disease )] TJ ET BT 26.250 481.286 Td /F1 9.8 Tf [(identified using genome-wide expression profiling and cross-platform analysis. Hum Mol Genet. 2010;19\(8\):1438-52. doi: )] TJ ET BT 26.250 469.381 Td /F1 9.8 Tf [(10.1093/hmg/ddq018. PubMed PMID: 20089533; PubMed Central PMCID: PMCPMC2846159.)] TJ ET BT 26.250 449.976 Td /F1 9.8 Tf [(45.)] TJ ET BT 43.553 449.976 Td /F1 9.8 Tf [(Valencia A, Sapp E, Kimm JS, McClory H, Reeves PB, Alexander J, et al. Elevated NADPH oxidase activity contributes to )] TJ ET BT 26.250 438.072 Td /F1 9.8 Tf [(oxidative stress and cell death in Huntington's disease. Hum Mol Genet. 2013;22\(6\):1112-31. doi: 10.1093/hmg/dds516. )] TJ ET BT 26.250 426.167 Td /F1 9.8 Tf [(PubMed PMID: 23223017; PubMed Central PMCID: PMC3578411.)] TJ ET BT 26.250 406.762 Td /F1 9.8 Tf [(46.)] TJ ET BT 43.553 406.762 Td /F1 9.8 Tf [(Browne SE, Bowling AC, MacGarvey U, Baik MJ, Berger SC, Muqit MM, et al. Oxidative damage and metabolic dysfunction )] TJ ET BT 26.250 394.857 Td /F1 9.8 Tf [(in Huntington's disease: selective vulnerability of the basal ganglia. Annals of neurology. 1997;41\(5\):646-53. PubMed PMID: )] TJ ET BT 26.250 382.953 Td /F1 9.8 Tf [(9153527.)] TJ ET BT 26.250 363.548 Td /F1 9.8 Tf [(47.)] TJ ET BT 43.553 363.548 Td /F1 9.8 Tf [(Cheng Y, Peng Q, Hou Z, Aggarwal M, Zhang J, Mori S, et al. Structural MRI detects progressive regional brain atrophy and )] TJ ET BT 26.250 351.643 Td /F1 9.8 Tf [(neuroprotective effects in N171-82Q Huntington's disease mouse model. NeuroImage. 2011;56\(3\):1027-34. PubMed PMID: )] TJ ET BT 26.250 339.738 Td /F1 9.8 Tf [(21320608.)] TJ ET BT 26.250 320.334 Td /F1 9.8 Tf [(48.)] TJ ET BT 43.553 320.334 Td /F1 9.8 Tf [(Zhou F, Gomi M, Fujimoto M, Hayase M, Marumo T, Masutani H, et al. Attenuation of neuronal degeneration in thioredoxin-)] TJ ET BT 26.250 308.429 Td /F1 9.8 Tf [(1 overexpressing mice after mild focal ischemia. Brain research. 2009;1272:62-70. doi: 10.1016/j.brainres.2009.03.023. )] TJ ET BT 26.250 296.524 Td /F1 9.8 Tf [(PubMed PMID: 19328186.)] TJ ET BT 26.250 277.119 Td /F1 9.8 Tf [(49.)] TJ ET BT 43.553 277.119 Td /F1 9.8 Tf [(Kim GS, Jung JE, Narasimhan P, Sakata H, Chan PH. Induction of thioredoxin-interacting protein is mediated by oxidative )] TJ ET BT 26.250 265.215 Td /F1 9.8 Tf [(stress, calcium, and glucose after brain injury in mice. Neurobiology of disease. 2012;46\(2\):440-9. doi: )] TJ ET BT 26.250 253.310 Td /F1 9.8 Tf [(10.1016/j.nbd.2012.02.008. PubMed PMID: 22366181; PubMed Central PMCID: PMC3323710.)] TJ ET BT 26.250 233.905 Td /F1 9.8 Tf [(50.)] TJ ET BT 43.553 233.905 Td /F1 9.8 Tf [(Ishrat T, Mohamed IN, Pillai B, Soliman S, Fouda AY, Ergul A, et al. Thioredoxin-Interacting Protein: a Novel Target for )] TJ ET BT 26.250 222.000 Td /F1 9.8 Tf [(Neuroprotection in Experimental Thromboembolic Stroke in Mice. Molecular neurobiology. 2014. doi: 10.1007/s12035-014-8766-)] TJ ET BT 26.250 210.096 Td /F1 9.8 Tf [(x. PubMed PMID: 24939693.)] TJ ET BT 26.250 190.691 Td /F1 9.8 Tf [(51.)] TJ ET BT 43.553 190.691 Td /F1 9.8 Tf [(Tian L, Nie H, Zhang Y, Chen Y, Peng Z, Cai M, et al. Recombinant human thioredoxin-1 promotes neurogenesis and )] TJ ET BT 26.250 178.786 Td /F1 9.8 Tf [(facilitates cognitive recovery following cerebral ischemia in mice. Neuropharmacology. 2014;77:453-64. doi: )] TJ ET BT 26.250 166.881 Td /F1 9.8 Tf [(10.1016/j.neuropharm.2013.10.027. PubMed PMID: 24212059.)] TJ ET BT 26.250 147.477 Td /F1 9.8 Tf [(52.)] TJ ET BT 43.553 147.477 Td /F1 9.8 Tf [(Taira T, Saito Y, Niki T, Iguchi-Ariga SM, Takahashi K, Ariga H. DJ-1 has a role in antioxidative stress to prevent cell death. )] TJ ET BT 26.250 135.572 Td /F1 9.8 Tf [(EMBO reports. 2004;5\(2\):213-8. doi: 10.1038/sj.embor.7400074. PubMed PMID: 14749723; PubMed Central PMCID: )] TJ ET BT 26.250 123.667 Td /F1 9.8 Tf [(PMC1298985.)] TJ ET BT 26.250 104.262 Td /F1 9.8 Tf [(53.)] TJ ET BT 43.553 104.262 Td /F1 9.8 Tf [(Im JY, Lee KW, Woo JM, Junn E, Mouradian MM. DJ-1 induces thioredoxin 1 expression through the Nrf2 pathway. Human )] TJ ET BT 26.250 92.358 Td /F1 9.8 Tf [(molecular genetics. 2012;21\(13\):3013-24. doi: 10.1093/hmg/dds131. PubMed PMID: 22492997; PubMed Central PMCID: )] TJ ET BT 26.250 80.453 Td /F1 9.8 Tf [(PMC3373246.)] TJ ET BT 26.250 61.048 Td /F1 9.8 Tf [(54.)] TJ ET BT 43.553 61.048 Td /F1 9.8 Tf [(Kitaoka Y, Munemasa Y, Hayashi Y, Kuribayashi J, Koseki N, Kojima K, et al. Axonal protection by 17beta-estradiol through )] TJ ET BT 26.250 49.143 Td /F1 9.8 Tf [(thioredoxin-1 in tumor necrosis factor-induced optic neuropathy. Endocrinology. 2011;152\(7\):2775-85. doi: 10.1210/en.2011-)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(13)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 658 0 obj << /Type /Page /Parent 3 0 R /Contents 659 0 R >> endobj 659 0 obj << /Length 14894 >> stream 0.271 0.267 0.267 rg q 15.000 298.548 577.500 478.452 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(0046. PubMed PMID: 21586560.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(55.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Fox JH, Connor T, Stiles M, Kama J, Lu Z, Dorsey K, et al. Cysteine oxidation within N-terminal mutant huntingtin promotes )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(oligomerization and delays clearance of soluble protein. J Biol Chem. 2011;286\(20\):18320-30. doi: 10.1074/jbc.M110.199448. )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(PubMed PMID: 21454633; PubMed Central PMCID: PMCPMC3093904.)] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(56.)] TJ ET BT 43.553 704.857 Td /F1 9.8 Tf [(Lee S, Kim SM, Lee RT. Thioredoxin and thioredoxin target proteins: from molecular mechanisms to functional significance. )] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(Antioxidants & redox signaling. 2013;18\(10\):1165-207. doi: 10.1089/ars.2011.4322. PubMed PMID: 22607099; PubMed Central )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(PMCID: PMC3579385.)] TJ ET BT 26.250 661.643 Td /F1 9.8 Tf [(57.)] TJ ET BT 43.553 661.643 Td /F1 9.8 Tf [(Chae HZ, Kim HJ, Kang SW, Rhee SG. Characterization of three isoforms of mammalian peroxiredoxin that reduce )] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(peroxides in the presence of thioredoxin. Diabetes research and clinical practice. 1999;45\(2-3\):101-12. PubMed PMID: )] TJ ET BT 26.250 637.833 Td /F1 9.8 Tf [(10588361.)] TJ ET BT 26.250 618.429 Td /F1 9.8 Tf [(58.)] TJ ET BT 43.553 618.429 Td /F1 9.8 Tf [(Hattori K, Naguro I, Runchel C, Ichijo H. The roles of ASK family proteins in stress responses and diseases. Cell )] TJ ET BT 26.250 606.524 Td /F1 9.8 Tf [(communication and signaling : CCS. 2009;7:9. doi: 10.1186/1478-811X-7-9. PubMed PMID: 19389260; PubMed Central )] TJ ET BT 26.250 594.619 Td /F1 9.8 Tf [(PMCID: PMC2685135.)] TJ ET BT 26.250 575.214 Td /F1 9.8 Tf [(59.)] TJ ET BT 43.553 575.214 Td /F1 9.8 Tf [(Cho KJ, Lee BI, Cheon SY, Kim HW, Kim HJ, Kim GW. Inhibition of apoptosis signal-regulating kinase 1 reduces )] TJ ET BT 26.250 563.310 Td /F1 9.8 Tf [(endoplasmic reticulum stress and nuclear huntingtin fragments in a mouse model of Huntington disease. Neuroscience. )] TJ ET BT 26.250 551.405 Td /F1 9.8 Tf [(2009;163\(4\):1128-34. doi: 10.1016/j.neuroscience.2009.07.048. PubMed PMID: 19646509.)] TJ ET BT 26.250 532.000 Td /F1 9.8 Tf [(60.)] TJ ET BT 43.553 532.000 Td /F1 9.8 Tf [(Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, et al. Mammalian thioredoxin is a direct inhibitor of )] TJ ET BT 26.250 520.095 Td /F1 9.8 Tf [(apoptosis signal-regulating kinase \(ASK\) 1. The EMBO journal. 1998;17\(9\):2596-606. doi: 10.1093/emboj/17.9.2596. PubMed )] TJ ET BT 26.250 508.191 Td /F1 9.8 Tf [(PMID: 9564042; PubMed Central PMCID: PMC1170601.)] TJ ET BT 26.250 488.786 Td /F1 9.8 Tf [(61.)] TJ ET BT 43.553 488.786 Td /F1 9.8 Tf [(Chao R, Nevin L, Agarwal P, Riemer J, Bai X, Delaney A, et al. A male with unilateral microphthalmia reveals a role for )] TJ ET BT 26.250 476.881 Td /F1 9.8 Tf [(TMX3 in eye development. PloS one. 2010;5\(5\):e10565. doi: 10.1371/journal.pone.0010565. PubMed PMID: 20485507; )] TJ ET BT 26.250 464.976 Td /F1 9.8 Tf [(PubMed Central PMCID: PMC2868029.)] TJ ET BT 26.250 445.572 Td /F1 9.8 Tf [(62.)] TJ ET BT 43.553 445.572 Td /F1 9.8 Tf [(Haugstetter J, Blicher T, Ellgaard L. Identification and characterization of a novel thioredoxin-related transmembrane protein )] TJ ET BT 26.250 433.667 Td /F1 9.8 Tf [(of the endoplasmic reticulum. The Journal of biological chemistry. 2005;280\(9\):8371-80. doi: 10.1074/jbc.M413924200. PubMed )] TJ ET BT 26.250 421.762 Td /F1 9.8 Tf [(PMID: 15623505.)] TJ ET BT 26.250 402.357 Td /F1 9.8 Tf [(63.)] TJ ET BT 43.553 402.357 Td /F1 9.8 Tf [(Lynes EM, Bui M, Yap MC, Benson MD, Schneider B, Ellgaard L, et al. Palmitoylated TMX and calnexin target to the )] TJ ET BT 26.250 390.453 Td /F1 9.8 Tf [(mitochondria-associated membrane. The EMBO journal. 2012;31\(2\):457-70. doi: 10.1038/emboj.2011.384. PubMed PMID: )] TJ ET BT 26.250 378.548 Td /F1 9.8 Tf [(22045338; PubMed Central PMCID: PMC3261551.)] TJ ET BT 26.250 359.143 Td /F1 9.8 Tf [(64.)] TJ ET BT 43.553 359.143 Td /F1 9.8 Tf [(Strehlow AN, Li JZ, Myers RM. Wild-type huntingtin participates in protein trafficking between the Golgi and the extracellular )] TJ ET BT 26.250 347.238 Td /F1 9.8 Tf [(space. Human molecular genetics. 2007;16\(4\):391-409. doi: 10.1093/hmg/ddl467. PubMed PMID: 17189290.)] TJ ET BT 26.250 327.834 Td /F1 9.8 Tf [(65.)] TJ ET BT 43.553 327.834 Td /F1 9.8 Tf [(Vidal R, Caballero B, Couve A, Hetz C. Converging pathways in the occurrence of endoplasmic reticulum \(ER\) stress in )] TJ ET BT 26.250 315.929 Td /F1 9.8 Tf [(Huntington's disease. Current molecular medicine. 2011;11\(1\):1-12. PubMed PMID: 21189122.)] TJ ET Q q 15.000 298.548 577.500 478.452 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(0046. PubMed PMID: 21586560.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(55.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Fox JH, Connor T, Stiles M, Kama J, Lu Z, Dorsey K, et al. Cysteine oxidation within N-terminal mutant huntingtin promotes )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(oligomerization and delays clearance of soluble protein. J Biol Chem. 2011;286\(20\):18320-30. doi: 10.1074/jbc.M110.199448. )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(PubMed PMID: 21454633; PubMed Central PMCID: PMCPMC3093904.)] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(56.)] TJ ET BT 43.553 704.857 Td /F1 9.8 Tf [(Lee S, Kim SM, Lee RT. Thioredoxin and thioredoxin target proteins: from molecular mechanisms to functional significance. )] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(Antioxidants & redox signaling. 2013;18\(10\):1165-207. doi: 10.1089/ars.2011.4322. PubMed PMID: 22607099; PubMed Central )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(PMCID: PMC3579385.)] TJ ET BT 26.250 661.643 Td /F1 9.8 Tf [(57.)] TJ ET BT 43.553 661.643 Td /F1 9.8 Tf [(Chae HZ, Kim HJ, Kang SW, Rhee SG. Characterization of three isoforms of mammalian peroxiredoxin that reduce )] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(peroxides in the presence of thioredoxin. Diabetes research and clinical practice. 1999;45\(2-3\):101-12. PubMed PMID: )] TJ ET BT 26.250 637.833 Td /F1 9.8 Tf [(10588361.)] TJ ET BT 26.250 618.429 Td /F1 9.8 Tf [(58.)] TJ ET BT 43.553 618.429 Td /F1 9.8 Tf [(Hattori K, Naguro I, Runchel C, Ichijo H. The roles of ASK family proteins in stress responses and diseases. Cell )] TJ ET BT 26.250 606.524 Td /F1 9.8 Tf [(communication and signaling : CCS. 2009;7:9. doi: 10.1186/1478-811X-7-9. PubMed PMID: 19389260; PubMed Central )] TJ ET BT 26.250 594.619 Td /F1 9.8 Tf [(PMCID: PMC2685135.)] TJ ET BT 26.250 575.214 Td /F1 9.8 Tf [(59.)] TJ ET BT 43.553 575.214 Td /F1 9.8 Tf [(Cho KJ, Lee BI, Cheon SY, Kim HW, Kim HJ, Kim GW. Inhibition of apoptosis signal-regulating kinase 1 reduces )] TJ ET BT 26.250 563.310 Td /F1 9.8 Tf [(endoplasmic reticulum stress and nuclear huntingtin fragments in a mouse model of Huntington disease. Neuroscience. )] TJ ET BT 26.250 551.405 Td /F1 9.8 Tf [(2009;163\(4\):1128-34. doi: 10.1016/j.neuroscience.2009.07.048. PubMed PMID: 19646509.)] TJ ET BT 26.250 532.000 Td /F1 9.8 Tf [(60.)] TJ ET BT 43.553 532.000 Td /F1 9.8 Tf [(Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, et al. Mammalian thioredoxin is a direct inhibitor of )] TJ ET BT 26.250 520.095 Td /F1 9.8 Tf [(apoptosis signal-regulating kinase \(ASK\) 1. The EMBO journal. 1998;17\(9\):2596-606. doi: 10.1093/emboj/17.9.2596. PubMed )] TJ ET BT 26.250 508.191 Td /F1 9.8 Tf [(PMID: 9564042; PubMed Central PMCID: PMC1170601.)] TJ ET BT 26.250 488.786 Td /F1 9.8 Tf [(61.)] TJ ET BT 43.553 488.786 Td /F1 9.8 Tf [(Chao R, Nevin L, Agarwal P, Riemer J, Bai X, Delaney A, et al. A male with unilateral microphthalmia reveals a role for )] TJ ET BT 26.250 476.881 Td /F1 9.8 Tf [(TMX3 in eye development. PloS one. 2010;5\(5\):e10565. doi: 10.1371/journal.pone.0010565. PubMed PMID: 20485507; )] TJ ET BT 26.250 464.976 Td /F1 9.8 Tf [(PubMed Central PMCID: PMC2868029.)] TJ ET BT 26.250 445.572 Td /F1 9.8 Tf [(62.)] TJ ET BT 43.553 445.572 Td /F1 9.8 Tf [(Haugstetter J, Blicher T, Ellgaard L. Identification and characterization of a novel thioredoxin-related transmembrane protein )] TJ ET BT 26.250 433.667 Td /F1 9.8 Tf [(of the endoplasmic reticulum. The Journal of biological chemistry. 2005;280\(9\):8371-80. doi: 10.1074/jbc.M413924200. PubMed )] TJ ET BT 26.250 421.762 Td /F1 9.8 Tf [(PMID: 15623505.)] TJ ET BT 26.250 402.357 Td /F1 9.8 Tf [(63.)] TJ ET BT 43.553 402.357 Td /F1 9.8 Tf [(Lynes EM, Bui M, Yap MC, Benson MD, Schneider B, Ellgaard L, et al. Palmitoylated TMX and calnexin target to the )] TJ ET BT 26.250 390.453 Td /F1 9.8 Tf [(mitochondria-associated membrane. The EMBO journal. 2012;31\(2\):457-70. doi: 10.1038/emboj.2011.384. PubMed PMID: )] TJ ET BT 26.250 378.548 Td /F1 9.8 Tf [(22045338; PubMed Central PMCID: PMC3261551.)] TJ ET BT 26.250 359.143 Td /F1 9.8 Tf [(64.)] TJ ET BT 43.553 359.143 Td /F1 9.8 Tf [(Strehlow AN, Li JZ, Myers RM. Wild-type huntingtin participates in protein trafficking between the Golgi and the extracellular )] TJ ET BT 26.250 347.238 Td /F1 9.8 Tf [(space. Human molecular genetics. 2007;16\(4\):391-409. doi: 10.1093/hmg/ddl467. PubMed PMID: 17189290.)] TJ ET BT 26.250 327.834 Td /F1 9.8 Tf [(65.)] TJ ET BT 43.553 327.834 Td /F1 9.8 Tf [(Vidal R, Caballero B, Couve A, Hetz C. Converging pathways in the occurrence of endoplasmic reticulum \(ER\) stress in )] TJ ET BT 26.250 315.929 Td /F1 9.8 Tf [(Huntington's disease. Current molecular medicine. 2011;11\(1\):1-12. PubMed PMID: 21189122.)] TJ ET Q q 15.000 298.548 577.500 478.452 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(0046. PubMed PMID: 21586560.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(55.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Fox JH, Connor T, Stiles M, Kama J, Lu Z, Dorsey K, et al. Cysteine oxidation within N-terminal mutant huntingtin promotes )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(oligomerization and delays clearance of soluble protein. J Biol Chem. 2011;286\(20\):18320-30. doi: 10.1074/jbc.M110.199448. )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(PubMed PMID: 21454633; PubMed Central PMCID: PMCPMC3093904.)] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(56.)] TJ ET BT 43.553 704.857 Td /F1 9.8 Tf [(Lee S, Kim SM, Lee RT. Thioredoxin and thioredoxin target proteins: from molecular mechanisms to functional significance. )] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(Antioxidants & redox signaling. 2013;18\(10\):1165-207. doi: 10.1089/ars.2011.4322. PubMed PMID: 22607099; PubMed Central )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(PMCID: PMC3579385.)] TJ ET BT 26.250 661.643 Td /F1 9.8 Tf [(57.)] TJ ET BT 43.553 661.643 Td /F1 9.8 Tf [(Chae HZ, Kim HJ, Kang SW, Rhee SG. Characterization of three isoforms of mammalian peroxiredoxin that reduce )] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(peroxides in the presence of thioredoxin. Diabetes research and clinical practice. 1999;45\(2-3\):101-12. PubMed PMID: )] TJ ET BT 26.250 637.833 Td /F1 9.8 Tf [(10588361.)] TJ ET BT 26.250 618.429 Td /F1 9.8 Tf [(58.)] TJ ET BT 43.553 618.429 Td /F1 9.8 Tf [(Hattori K, Naguro I, Runchel C, Ichijo H. The roles of ASK family proteins in stress responses and diseases. Cell )] TJ ET BT 26.250 606.524 Td /F1 9.8 Tf [(communication and signaling : CCS. 2009;7:9. doi: 10.1186/1478-811X-7-9. PubMed PMID: 19389260; PubMed Central )] TJ ET BT 26.250 594.619 Td /F1 9.8 Tf [(PMCID: PMC2685135.)] TJ ET BT 26.250 575.214 Td /F1 9.8 Tf [(59.)] TJ ET BT 43.553 575.214 Td /F1 9.8 Tf [(Cho KJ, Lee BI, Cheon SY, Kim HW, Kim HJ, Kim GW. Inhibition of apoptosis signal-regulating kinase 1 reduces )] TJ ET BT 26.250 563.310 Td /F1 9.8 Tf [(endoplasmic reticulum stress and nuclear huntingtin fragments in a mouse model of Huntington disease. Neuroscience. )] TJ ET BT 26.250 551.405 Td /F1 9.8 Tf [(2009;163\(4\):1128-34. doi: 10.1016/j.neuroscience.2009.07.048. PubMed PMID: 19646509.)] TJ ET BT 26.250 532.000 Td /F1 9.8 Tf [(60.)] TJ ET BT 43.553 532.000 Td /F1 9.8 Tf [(Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, et al. Mammalian thioredoxin is a direct inhibitor of )] TJ ET BT 26.250 520.095 Td /F1 9.8 Tf [(apoptosis signal-regulating kinase \(ASK\) 1. The EMBO journal. 1998;17\(9\):2596-606. doi: 10.1093/emboj/17.9.2596. PubMed )] TJ ET BT 26.250 508.191 Td /F1 9.8 Tf [(PMID: 9564042; PubMed Central PMCID: PMC1170601.)] TJ ET BT 26.250 488.786 Td /F1 9.8 Tf [(61.)] TJ ET BT 43.553 488.786 Td /F1 9.8 Tf [(Chao R, Nevin L, Agarwal P, Riemer J, Bai X, Delaney A, et al. A male with unilateral microphthalmia reveals a role for )] TJ ET BT 26.250 476.881 Td /F1 9.8 Tf [(TMX3 in eye development. PloS one. 2010;5\(5\):e10565. doi: 10.1371/journal.pone.0010565. PubMed PMID: 20485507; )] TJ ET BT 26.250 464.976 Td /F1 9.8 Tf [(PubMed Central PMCID: PMC2868029.)] TJ ET BT 26.250 445.572 Td /F1 9.8 Tf [(62.)] TJ ET BT 43.553 445.572 Td /F1 9.8 Tf [(Haugstetter J, Blicher T, Ellgaard L. Identification and characterization of a novel thioredoxin-related transmembrane protein )] TJ ET BT 26.250 433.667 Td /F1 9.8 Tf [(of the endoplasmic reticulum. The Journal of biological chemistry. 2005;280\(9\):8371-80. doi: 10.1074/jbc.M413924200. PubMed )] TJ ET BT 26.250 421.762 Td /F1 9.8 Tf [(PMID: 15623505.)] TJ ET BT 26.250 402.357 Td /F1 9.8 Tf [(63.)] TJ ET BT 43.553 402.357 Td /F1 9.8 Tf [(Lynes EM, Bui M, Yap MC, Benson MD, Schneider B, Ellgaard L, et al. Palmitoylated TMX and calnexin target to the )] TJ ET BT 26.250 390.453 Td /F1 9.8 Tf [(mitochondria-associated membrane. The EMBO journal. 2012;31\(2\):457-70. doi: 10.1038/emboj.2011.384. PubMed PMID: )] TJ ET BT 26.250 378.548 Td /F1 9.8 Tf [(22045338; PubMed Central PMCID: PMC3261551.)] TJ ET BT 26.250 359.143 Td /F1 9.8 Tf [(64.)] TJ ET BT 43.553 359.143 Td /F1 9.8 Tf [(Strehlow AN, Li JZ, Myers RM. Wild-type huntingtin participates in protein trafficking between the Golgi and the extracellular )] TJ ET BT 26.250 347.238 Td /F1 9.8 Tf [(space. Human molecular genetics. 2007;16\(4\):391-409. doi: 10.1093/hmg/ddl467. PubMed PMID: 17189290.)] TJ ET BT 26.250 327.834 Td /F1 9.8 Tf [(65.)] TJ ET BT 43.553 327.834 Td /F1 9.8 Tf [(Vidal R, Caballero B, Couve A, Hetz C. Converging pathways in the occurrence of endoplasmic reticulum \(ER\) stress in )] TJ ET BT 26.250 315.929 Td /F1 9.8 Tf [(Huntington's disease. Current molecular medicine. 2011;11\(1\):1-12. PubMed PMID: 21189122.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(14)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj xref 0 660 0000000000 65535 f 0000000008 00000 n 0000000073 00000 n 0000000119 00000 n 0000000543 00000 n 0000000580 00000 n 0000000862 00000 n 0000001470 00000 n 0000029248 00000 n 0000029355 00000 n 0000029463 00000 n 0000029574 00000 n 0000029687 00000 n 0000029803 00000 n 0000030191 00000 n 0000034672 00000 n 0000034799 00000 n 0000035010 00000 n 0000035137 00000 n 0000035348 00000 n 0000035475 00000 n 0000035686 00000 n 0000035813 00000 n 0000035929 00000 n 0000036055 00000 n 0000036156 00000 n 0000036283 00000 n 0000036377 00000 n 0000036505 00000 n 0000036601 00000 n 0000036729 00000 n 0000036817 00000 n 0000036945 00000 n 0000036981 00000 n 0000037109 00000 n 0000037145 00000 n 0000037273 00000 n 0000037309 00000 n 0000037437 00000 n 0000037473 00000 n 0000037601 00000 n 0000037637 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