%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 147 0 R 269 0 R 315 0 R 330 0 R 357 0 R 371 0 R ] /Count 7 /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 /F6 271 0 R >> /XObject << /I1 13 0 R /I2 14 0 R /I3 284 0 R /I4 321 0 R /I5 336 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:20170830034624+00'00') /ModDate (D:20170830034624+00'00') /Title (Caspase-6 does not contribute to the proteolysis of mutant huntingtin in the HdhQ150 knock-in 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 ] /Contents 7 0 R >> endobj 7 0 obj << /Length 28973 >> 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 [(Caspase-6 does not contribute to the proteolysis of mutant )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(huntingtin in the HdhQ150 knock-in 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 [(July 16, 2012)] TJ ET BT 72.681 650.140 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 77.556 650.140 Td /F3 9.8 Tf [(HD Models)] TJ ET BT 26.250 638.299 Td /F1 9.8 Tf [(Christian Landles)] TJ ET 0.271 0.267 0.267 rg BT 101.569 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 106.990 638.299 Td /F1 9.8 Tf [(Andreas Weiss)] TJ ET 0.271 0.267 0.267 rg BT 172.549 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 177.970 638.299 Td /F1 9.8 Tf [(Sophie Franklin)] TJ ET 0.271 0.267 0.267 rg BT 245.703 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 251.124 638.299 Td /F1 9.8 Tf [(David Howland)] TJ ET 0.271 0.267 0.267 rg BT 316.683 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 322.104 638.299 Td /F1 9.8 Tf [(Gill Bates)] TJ ET 0.271 0.267 0.267 rg BT 26.250 626.394 Td /F1 9.8 Tf [(Landles C, Weiss A, Franklin S, Howland D, Bates G. Caspase-6 does not contribute to the proteolysis of mutant huntingtin in )] TJ ET BT 26.250 614.490 Td /F1 9.8 Tf [(the HdhQ150 knock-in mouse model of Huntingtons disease. PLOS Currents Huntington Disease. 2012 Jul 16 . Edition 1. doi: )] TJ ET BT 26.250 602.585 Td /F1 9.8 Tf [(10.1371/4fd085bfc9973.)] TJ ET q 15.000 30.177 577.500 570.027 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 a late-onset progressive neurodegenerative disorder characterised by irrepressible motor )] TJ ET BT 26.250 541.623 Td /F1 9.8 Tf [(dysfunction, cognitive decline and psychiatric disturbances for which there is no effective disease-modifying treatment. The )] TJ ET BT 26.250 529.719 Td /F1 9.8 Tf [(proteolytic cleavage of huntingtin \(HTT\) to generate N-terminal fragments has been proposed to be a key aspect of HD )] TJ ET BT 26.250 517.814 Td /F1 9.8 Tf [(pathogenesis. In particular, it has been shown that HTT can be cleaved at amino acid 586 by caspase-6 \(CASP6\) and that )] TJ ET BT 26.250 505.909 Td /F1 9.8 Tf [(prevention of cleavage at this site is neuroprotective and can rescue HD-related phenotypes in YAC transgenic HD mouse )] TJ ET BT 26.250 494.004 Td /F1 9.8 Tf [(models. To determine the role that CASP6 plays in HTT proteolysis, we evaluated the effects of the genetic ablation of Casp6 in )] TJ ET BT 26.250 482.100 Td /F1 9.8 Tf [(the HdhQ150 knock-in mouse model of HD. Here we show that the loss of CASP6 had no effect on the proteolysis of HTT, and )] TJ ET BT 26.250 470.195 Td /F1 9.8 Tf [(did not modify the pattern of N-terminal HTT fragments that are present in the brains of these animals. Furthermore, we show )] TJ ET BT 26.250 458.290 Td /F1 9.8 Tf [(that CASP6 ablation does not influence the steady-state levels of soluble HTT in the brains of presymptomatic mice. Therefore, )] TJ ET BT 26.250 446.385 Td /F1 9.8 Tf [(we conclude that CASP6 is not necessary for HTT proteolysis in the HdhQ150 mouse model of HD, and that targeting CASP6 )] TJ ET BT 26.250 434.481 Td /F1 9.8 Tf [(as a therapeutic strategy should be approached with caution in the context of this complex disease.)] TJ ET BT 26.250 415.076 Td /F1 9.8 Tf [(* Corresponding author: Gillian P. Bates, Department of Medical and Molecular Genetics, Kings College London School of )] TJ ET BT 26.250 403.171 Td /F1 9.8 Tf [(Medicine, 8th Floor Tower Wing, Guys Hospital, London, SE1 9RT, United Kingdom. Phone: +44 20 7188 3722; Fax: +44 20 )] TJ ET BT 26.250 391.266 Td /F1 9.8 Tf [(7188 2585; Email: gillian.bates@kcl.ac.uk)] TJ ET BT 26.250 354.664 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 334.710 Td /F1 9.8 Tf [(This work was funded by the CHDI Foundation, a privately funded non-profit biomedical research organisation exclusively )] TJ ET BT 26.250 322.805 Td /F1 9.8 Tf [(dedicated to discovering and developing therapeutics that slow the progression of HD. For this study CHDI Foundation assisted )] TJ ET BT 26.250 310.900 Td /F1 9.8 Tf [(with the generation of Casp6-deficient mice.)] TJ ET BT 26.250 281.798 Td /F4 12.0 Tf [(INTRODUCTION)] TJ ET BT 26.250 261.843 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an autosomal-dominant neurodegenerative disorder with onset in midlife that is characterised by )] TJ ET BT 26.250 249.939 Td /F1 9.8 Tf [(irrepressible motor dysfunction, personality changes, and cognitive decline ultimately leading to death 15-20 years after disease )] TJ ET BT 26.250 238.034 Td /F1 9.8 Tf [(onset )] TJ ET 0.267 0.267 0.267 rg BT 52.809 239.541 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 57.628 238.034 Td /F1 9.8 Tf [(. HD belongs to a family of neurodegenerative diseases caused by mutations in which the expansion of a CAG )] TJ ET BT 26.250 226.129 Td /F1 9.8 Tf [(trinucleotide repeat results in an aberrantly long polyglutamine \(polyQ\) tract in the encoded protein. In HD, the CAG expansion )] TJ ET BT 26.250 214.224 Td /F1 9.8 Tf [(occurs within exon 1 of the )] TJ ET BT 144.391 214.224 Td /F5 9.8 Tf [(HTT)] TJ ET BT 163.345 214.224 Td /F1 9.8 Tf [( gene and leads to an elongated polyQ tract at the N-terminus of the HD protein huntingtin )] TJ ET BT 26.250 202.320 Td /F1 9.8 Tf [(\(HTT\), a protein of many diverse functions )] TJ ET 0.267 0.267 0.267 rg BT 211.022 203.827 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 215.841 202.320 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 221.262 203.827 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 226.081 202.320 Td /F1 9.8 Tf [(. Individuals with \(CAG\))] TJ ET BT 327.939 200.255 Td /F1 8.7 Tf [(35 )] TJ ET BT 339.986 202.320 Td /F1 9.8 Tf [(or less remain unaffected, whereas those with \(CAG\))] TJ ET BT 568.652 200.255 Td /F1 8.7 Tf [(40 )] TJ ET BT 26.250 190.415 Td /F1 9.8 Tf [(and above will develop HD within a normal lifespan. The length of the CAG expansion is inversely correlated with age of )] TJ ET BT 26.250 178.510 Td /F1 9.8 Tf [(disease onset, with repeats at the higher end of the spectrum causing the juvenile form of HD.)] TJ ET BT 26.250 159.105 Td /F1 9.8 Tf [(Numerous HD mouse models have been developed and are being used to study the pathogenic pathways involved in HD )] TJ ET 0.267 0.267 0.267 rg BT 551.921 160.613 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 556.740 159.105 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 147.201 Td /F1 9.8 Tf [(The )] TJ ET BT 45.760 147.201 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 63.641 147.201 Td /F1 9.8 Tf [(Q150 knock-in mouse carries ~150 CAG repeats which have been inserted into the mouse HD gene \()] TJ ET BT 501.231 147.201 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 519.112 147.201 Td /F1 9.8 Tf [(\) )] TJ ET 0.267 0.267 0.267 rg BT 525.070 148.708 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 529.888 147.201 Td /F1 9.8 Tf [(. It has )] TJ ET BT 26.250 135.296 Td /F1 9.8 Tf [(been proposed that the accumulation of a critical concentration of N-terminal HTT fragments represents a rate-limiting step in )] TJ ET BT 26.250 123.391 Td /F1 9.8 Tf [(the onset and progression of HD-related phenotypes in this model )] TJ ET 0.267 0.267 0.267 rg BT 312.929 124.898 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 317.748 123.391 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 323.169 124.898 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 327.988 123.391 Td /F1 9.8 Tf [(. Numerous studies suggest that the smallest N-terminal )] TJ ET BT 26.250 111.486 Td /F1 9.8 Tf [(HTT fragments are pivotal to the molecular pathogenesis of HD. Human post-mortem studies showed that only N-terminal )] TJ ET BT 26.250 99.582 Td /F1 9.8 Tf [(antibodies detect nuclear inclusions in patient brains )] TJ ET 0.267 0.267 0.267 rg BT 254.400 101.089 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 259.219 99.582 Td /F1 9.8 Tf [( and formic acid solubilisation released a small N-terminal fragment )] TJ ET 0.267 0.267 0.267 rg BT 551.806 101.089 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 556.625 99.582 Td /F1 9.8 Tf [(. In )] TJ ET BT 26.250 87.677 Td /F1 9.8 Tf [(cell models, HTT has been shown to be cleaved into smaller fragments called cp-A and cp-B )] TJ ET 0.267 0.267 0.267 rg BT 427.784 89.184 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 432.603 87.677 Td /F1 9.8 Tf [( or cp-1 and cp-2 )] TJ ET 0.267 0.267 0.267 rg BT 509.014 89.184 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 513.832 87.677 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 519.253 89.184 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 528.891 87.677 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 75.772 Td /F1 9.8 Tf [(Furthermore, using the )] TJ ET BT 127.582 75.772 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 145.463 75.772 Td /F1 9.8 Tf [(Q150 knock-in model we recently demonstrated that full-length HTT is cleaved into a number of N-)] TJ ET BT 26.250 63.867 Td /F1 9.8 Tf [(terminal HTT fragments, the smallest of which is an exon 1 HTT protein )] TJ ET 0.267 0.267 0.267 rg BT 336.729 65.375 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 341.548 63.867 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 44.463 Td /F1 9.8 Tf [(The ensuing search for sites of proteolytic cleavage of human HTT raised the hypothesis that the cleavage of HTT by proteases )] 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 [(Caspase-6 does not contribute to the proteolysis of mutant )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(huntingtin in the HdhQ150 knock-in 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 [(July 16, 2012)] TJ ET BT 72.681 650.140 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 77.556 650.140 Td /F3 9.8 Tf [(HD Models)] TJ ET BT 26.250 638.299 Td /F1 9.8 Tf [(Christian Landles)] TJ ET 0.271 0.267 0.267 rg BT 101.569 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 106.990 638.299 Td /F1 9.8 Tf [(Andreas Weiss)] TJ ET 0.271 0.267 0.267 rg BT 172.549 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 177.970 638.299 Td /F1 9.8 Tf [(Sophie Franklin)] TJ ET 0.271 0.267 0.267 rg BT 245.703 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 251.124 638.299 Td /F1 9.8 Tf [(David Howland)] TJ ET 0.271 0.267 0.267 rg BT 316.683 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 322.104 638.299 Td /F1 9.8 Tf [(Gill Bates)] TJ ET 0.271 0.267 0.267 rg BT 26.250 626.394 Td /F1 9.8 Tf [(Landles C, Weiss A, Franklin S, Howland D, Bates G. Caspase-6 does not contribute to the proteolysis of mutant huntingtin in )] TJ ET BT 26.250 614.490 Td /F1 9.8 Tf [(the HdhQ150 knock-in mouse model of Huntingtons disease. PLOS Currents Huntington Disease. 2012 Jul 16 . Edition 1. doi: )] TJ ET BT 26.250 602.585 Td /F1 9.8 Tf [(10.1371/4fd085bfc9973.)] TJ ET q 15.000 30.177 577.500 570.027 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 a late-onset progressive neurodegenerative disorder characterised by irrepressible motor )] TJ ET BT 26.250 541.623 Td /F1 9.8 Tf [(dysfunction, cognitive decline and psychiatric disturbances for which there is no effective disease-modifying treatment. The )] TJ ET BT 26.250 529.719 Td /F1 9.8 Tf [(proteolytic cleavage of huntingtin \(HTT\) to generate N-terminal fragments has been proposed to be a key aspect of HD )] TJ ET BT 26.250 517.814 Td /F1 9.8 Tf [(pathogenesis. In particular, it has been shown that HTT can be cleaved at amino acid 586 by caspase-6 \(CASP6\) and that )] TJ ET BT 26.250 505.909 Td /F1 9.8 Tf [(prevention of cleavage at this site is neuroprotective and can rescue HD-related phenotypes in YAC transgenic HD mouse )] TJ ET BT 26.250 494.004 Td /F1 9.8 Tf [(models. To determine the role that CASP6 plays in HTT proteolysis, we evaluated the effects of the genetic ablation of Casp6 in )] TJ ET BT 26.250 482.100 Td /F1 9.8 Tf [(the HdhQ150 knock-in mouse model of HD. Here we show that the loss of CASP6 had no effect on the proteolysis of HTT, and )] TJ ET BT 26.250 470.195 Td /F1 9.8 Tf [(did not modify the pattern of N-terminal HTT fragments that are present in the brains of these animals. Furthermore, we show )] TJ ET BT 26.250 458.290 Td /F1 9.8 Tf [(that CASP6 ablation does not influence the steady-state levels of soluble HTT in the brains of presymptomatic mice. Therefore, )] TJ ET BT 26.250 446.385 Td /F1 9.8 Tf [(we conclude that CASP6 is not necessary for HTT proteolysis in the HdhQ150 mouse model of HD, and that targeting CASP6 )] TJ ET BT 26.250 434.481 Td /F1 9.8 Tf [(as a therapeutic strategy should be approached with caution in the context of this complex disease.)] TJ ET BT 26.250 415.076 Td /F1 9.8 Tf [(* Corresponding author: Gillian P. Bates, Department of Medical and Molecular Genetics, Kings College London School of )] TJ ET BT 26.250 403.171 Td /F1 9.8 Tf [(Medicine, 8th Floor Tower Wing, Guys Hospital, London, SE1 9RT, United Kingdom. Phone: +44 20 7188 3722; Fax: +44 20 )] TJ ET BT 26.250 391.266 Td /F1 9.8 Tf [(7188 2585; Email: gillian.bates@kcl.ac.uk)] TJ ET BT 26.250 354.664 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 334.710 Td /F1 9.8 Tf [(This work was funded by the CHDI Foundation, a privately funded non-profit biomedical research organisation exclusively )] TJ ET BT 26.250 322.805 Td /F1 9.8 Tf [(dedicated to discovering and developing therapeutics that slow the progression of HD. For this study CHDI Foundation assisted )] TJ ET BT 26.250 310.900 Td /F1 9.8 Tf [(with the generation of Casp6-deficient mice.)] TJ ET BT 26.250 281.798 Td /F4 12.0 Tf [(INTRODUCTION)] TJ ET BT 26.250 261.843 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an autosomal-dominant neurodegenerative disorder with onset in midlife that is characterised by )] TJ ET BT 26.250 249.939 Td /F1 9.8 Tf [(irrepressible motor dysfunction, personality changes, and cognitive decline ultimately leading to death 15-20 years after disease )] TJ ET BT 26.250 238.034 Td /F1 9.8 Tf [(onset )] TJ ET 0.267 0.267 0.267 rg BT 52.809 239.541 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 57.628 238.034 Td /F1 9.8 Tf [(. HD belongs to a family of neurodegenerative diseases caused by mutations in which the expansion of a CAG )] TJ ET BT 26.250 226.129 Td /F1 9.8 Tf [(trinucleotide repeat results in an aberrantly long polyglutamine \(polyQ\) tract in the encoded protein. In HD, the CAG expansion )] TJ ET BT 26.250 214.224 Td /F1 9.8 Tf [(occurs within exon 1 of the )] TJ ET BT 144.391 214.224 Td /F5 9.8 Tf [(HTT)] TJ ET BT 163.345 214.224 Td /F1 9.8 Tf [( gene and leads to an elongated polyQ tract at the N-terminus of the HD protein huntingtin )] TJ ET BT 26.250 202.320 Td /F1 9.8 Tf [(\(HTT\), a protein of many diverse functions )] TJ ET 0.267 0.267 0.267 rg BT 211.022 203.827 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 215.841 202.320 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 221.262 203.827 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 226.081 202.320 Td /F1 9.8 Tf [(. Individuals with \(CAG\))] TJ ET BT 327.939 200.255 Td /F1 8.7 Tf [(35 )] TJ ET BT 339.986 202.320 Td /F1 9.8 Tf [(or less remain unaffected, whereas those with \(CAG\))] TJ ET BT 568.652 200.255 Td /F1 8.7 Tf [(40 )] TJ ET BT 26.250 190.415 Td /F1 9.8 Tf [(and above will develop HD within a normal lifespan. The length of the CAG expansion is inversely correlated with age of )] TJ ET BT 26.250 178.510 Td /F1 9.8 Tf [(disease onset, with repeats at the higher end of the spectrum causing the juvenile form of HD.)] TJ ET BT 26.250 159.105 Td /F1 9.8 Tf [(Numerous HD mouse models have been developed and are being used to study the pathogenic pathways involved in HD )] TJ ET 0.267 0.267 0.267 rg BT 551.921 160.613 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 556.740 159.105 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 147.201 Td /F1 9.8 Tf [(The )] TJ ET BT 45.760 147.201 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 63.641 147.201 Td /F1 9.8 Tf [(Q150 knock-in mouse carries ~150 CAG repeats which have been inserted into the mouse HD gene \()] TJ ET BT 501.231 147.201 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 519.112 147.201 Td /F1 9.8 Tf [(\) )] TJ ET 0.267 0.267 0.267 rg BT 525.070 148.708 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 529.888 147.201 Td /F1 9.8 Tf [(. It has )] TJ ET BT 26.250 135.296 Td /F1 9.8 Tf [(been proposed that the accumulation of a critical concentration of N-terminal HTT fragments represents a rate-limiting step in )] TJ ET BT 26.250 123.391 Td /F1 9.8 Tf [(the onset and progression of HD-related phenotypes in this model )] TJ ET 0.267 0.267 0.267 rg BT 312.929 124.898 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 317.748 123.391 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 323.169 124.898 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 327.988 123.391 Td /F1 9.8 Tf [(. Numerous studies suggest that the smallest N-terminal )] TJ ET BT 26.250 111.486 Td /F1 9.8 Tf [(HTT fragments are pivotal to the molecular pathogenesis of HD. Human post-mortem studies showed that only N-terminal )] TJ ET BT 26.250 99.582 Td /F1 9.8 Tf [(antibodies detect nuclear inclusions in patient brains )] TJ ET 0.267 0.267 0.267 rg BT 254.400 101.089 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 259.219 99.582 Td /F1 9.8 Tf [( and formic acid solubilisation released a small N-terminal fragment )] TJ ET 0.267 0.267 0.267 rg BT 551.806 101.089 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 556.625 99.582 Td /F1 9.8 Tf [(. In )] TJ ET BT 26.250 87.677 Td /F1 9.8 Tf [(cell models, HTT has been shown to be cleaved into smaller fragments called cp-A and cp-B )] TJ ET 0.267 0.267 0.267 rg BT 427.784 89.184 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 432.603 87.677 Td /F1 9.8 Tf [( or cp-1 and cp-2 )] TJ ET 0.267 0.267 0.267 rg BT 509.014 89.184 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 513.832 87.677 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 519.253 89.184 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 528.891 87.677 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 75.772 Td /F1 9.8 Tf [(Furthermore, using the )] TJ ET BT 127.582 75.772 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 145.463 75.772 Td /F1 9.8 Tf [(Q150 knock-in model we recently demonstrated that full-length HTT is cleaved into a number of N-)] TJ ET BT 26.250 63.867 Td /F1 9.8 Tf [(terminal HTT fragments, the smallest of which is an exon 1 HTT protein )] TJ ET 0.267 0.267 0.267 rg BT 336.729 65.375 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 341.548 63.867 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 44.463 Td /F1 9.8 Tf [(The ensuing search for sites of proteolytic cleavage of human HTT raised the hypothesis that the cleavage of HTT by proteases )] 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 [(Caspase-6 does not contribute to the proteolysis of mutant )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(huntingtin in the HdhQ150 knock-in 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 [(July 16, 2012)] TJ ET BT 72.681 650.140 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 77.556 650.140 Td /F3 9.8 Tf [(HD Models)] TJ ET BT 26.250 638.299 Td /F1 9.8 Tf [(Christian Landles)] TJ ET 0.271 0.267 0.267 rg BT 101.569 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 106.990 638.299 Td /F1 9.8 Tf [(Andreas Weiss)] TJ ET 0.271 0.267 0.267 rg BT 172.549 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 177.970 638.299 Td /F1 9.8 Tf [(Sophie Franklin)] TJ ET 0.271 0.267 0.267 rg BT 245.703 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 251.124 638.299 Td /F1 9.8 Tf [(David Howland)] TJ ET 0.271 0.267 0.267 rg BT 316.683 638.299 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 322.104 638.299 Td /F1 9.8 Tf [(Gill Bates)] TJ ET 0.271 0.267 0.267 rg BT 26.250 626.394 Td /F1 9.8 Tf [(Landles C, Weiss A, Franklin S, Howland D, Bates G. Caspase-6 does not contribute to the proteolysis of mutant huntingtin in )] TJ ET BT 26.250 614.490 Td /F1 9.8 Tf [(the HdhQ150 knock-in mouse model of Huntingtons disease. PLOS Currents Huntington Disease. 2012 Jul 16 . Edition 1. doi: )] TJ ET BT 26.250 602.585 Td /F1 9.8 Tf [(10.1371/4fd085bfc9973.)] TJ ET q 15.000 30.177 577.500 570.027 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 a late-onset progressive neurodegenerative disorder characterised by irrepressible motor )] TJ ET BT 26.250 541.623 Td /F1 9.8 Tf [(dysfunction, cognitive decline and psychiatric disturbances for which there is no effective disease-modifying treatment. The )] TJ ET BT 26.250 529.719 Td /F1 9.8 Tf [(proteolytic cleavage of huntingtin \(HTT\) to generate N-terminal fragments has been proposed to be a key aspect of HD )] TJ ET BT 26.250 517.814 Td /F1 9.8 Tf [(pathogenesis. In particular, it has been shown that HTT can be cleaved at amino acid 586 by caspase-6 \(CASP6\) and that )] TJ ET BT 26.250 505.909 Td /F1 9.8 Tf [(prevention of cleavage at this site is neuroprotective and can rescue HD-related phenotypes in YAC transgenic HD mouse )] TJ ET BT 26.250 494.004 Td /F1 9.8 Tf [(models. To determine the role that CASP6 plays in HTT proteolysis, we evaluated the effects of the genetic ablation of Casp6 in )] TJ ET BT 26.250 482.100 Td /F1 9.8 Tf [(the HdhQ150 knock-in mouse model of HD. Here we show that the loss of CASP6 had no effect on the proteolysis of HTT, and )] TJ ET BT 26.250 470.195 Td /F1 9.8 Tf [(did not modify the pattern of N-terminal HTT fragments that are present in the brains of these animals. Furthermore, we show )] TJ ET BT 26.250 458.290 Td /F1 9.8 Tf [(that CASP6 ablation does not influence the steady-state levels of soluble HTT in the brains of presymptomatic mice. Therefore, )] TJ ET BT 26.250 446.385 Td /F1 9.8 Tf [(we conclude that CASP6 is not necessary for HTT proteolysis in the HdhQ150 mouse model of HD, and that targeting CASP6 )] TJ ET BT 26.250 434.481 Td /F1 9.8 Tf [(as a therapeutic strategy should be approached with caution in the context of this complex disease.)] TJ ET BT 26.250 415.076 Td /F1 9.8 Tf [(* Corresponding author: Gillian P. Bates, Department of Medical and Molecular Genetics, Kings College London School of )] TJ ET BT 26.250 403.171 Td /F1 9.8 Tf [(Medicine, 8th Floor Tower Wing, Guys Hospital, London, SE1 9RT, United Kingdom. Phone: +44 20 7188 3722; Fax: +44 20 )] TJ ET BT 26.250 391.266 Td /F1 9.8 Tf [(7188 2585; Email: gillian.bates@kcl.ac.uk)] TJ ET BT 26.250 354.664 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 334.710 Td /F1 9.8 Tf [(This work was funded by the CHDI Foundation, a privately funded non-profit biomedical research organisation exclusively )] TJ ET BT 26.250 322.805 Td /F1 9.8 Tf [(dedicated to discovering and developing therapeutics that slow the progression of HD. For this study CHDI Foundation assisted )] TJ ET BT 26.250 310.900 Td /F1 9.8 Tf [(with the generation of Casp6-deficient mice.)] TJ ET BT 26.250 281.798 Td /F4 12.0 Tf [(INTRODUCTION)] TJ ET BT 26.250 261.843 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an autosomal-dominant neurodegenerative disorder with onset in midlife that is characterised by )] TJ ET BT 26.250 249.939 Td /F1 9.8 Tf [(irrepressible motor dysfunction, personality changes, and cognitive decline ultimately leading to death 15-20 years after disease )] TJ ET BT 26.250 238.034 Td /F1 9.8 Tf [(onset )] TJ ET 0.267 0.267 0.267 rg BT 52.809 239.541 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 57.628 238.034 Td /F1 9.8 Tf [(. HD belongs to a family of neurodegenerative diseases caused by mutations in which the expansion of a CAG )] TJ ET BT 26.250 226.129 Td /F1 9.8 Tf [(trinucleotide repeat results in an aberrantly long polyglutamine \(polyQ\) tract in the encoded protein. In HD, the CAG expansion )] TJ ET BT 26.250 214.224 Td /F1 9.8 Tf [(occurs within exon 1 of the )] TJ ET BT 144.391 214.224 Td /F5 9.8 Tf [(HTT)] TJ ET BT 163.345 214.224 Td /F1 9.8 Tf [( gene and leads to an elongated polyQ tract at the N-terminus of the HD protein huntingtin )] TJ ET BT 26.250 202.320 Td /F1 9.8 Tf [(\(HTT\), a protein of many diverse functions )] TJ ET 0.267 0.267 0.267 rg BT 211.022 203.827 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 215.841 202.320 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 221.262 203.827 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 226.081 202.320 Td /F1 9.8 Tf [(. Individuals with \(CAG\))] TJ ET BT 327.939 200.255 Td /F1 8.7 Tf [(35 )] TJ ET BT 339.986 202.320 Td /F1 9.8 Tf [(or less remain unaffected, whereas those with \(CAG\))] TJ ET BT 568.652 200.255 Td /F1 8.7 Tf [(40 )] TJ ET BT 26.250 190.415 Td /F1 9.8 Tf [(and above will develop HD within a normal lifespan. The length of the CAG expansion is inversely correlated with age of )] TJ ET BT 26.250 178.510 Td /F1 9.8 Tf [(disease onset, with repeats at the higher end of the spectrum causing the juvenile form of HD.)] TJ ET BT 26.250 159.105 Td /F1 9.8 Tf [(Numerous HD mouse models have been developed and are being used to study the pathogenic pathways involved in HD )] TJ ET 0.267 0.267 0.267 rg BT 551.921 160.613 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 556.740 159.105 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 147.201 Td /F1 9.8 Tf [(The )] TJ ET BT 45.760 147.201 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 63.641 147.201 Td /F1 9.8 Tf [(Q150 knock-in mouse carries ~150 CAG repeats which have been inserted into the mouse HD gene \()] TJ ET BT 501.231 147.201 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 519.112 147.201 Td /F1 9.8 Tf [(\) )] TJ ET 0.267 0.267 0.267 rg BT 525.070 148.708 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 529.888 147.201 Td /F1 9.8 Tf [(. It has )] TJ ET BT 26.250 135.296 Td /F1 9.8 Tf [(been proposed that the accumulation of a critical concentration of N-terminal HTT fragments represents a rate-limiting step in )] TJ ET BT 26.250 123.391 Td /F1 9.8 Tf [(the onset and progression of HD-related phenotypes in this model )] TJ ET 0.267 0.267 0.267 rg BT 312.929 124.898 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 317.748 123.391 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 323.169 124.898 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 327.988 123.391 Td /F1 9.8 Tf [(. Numerous studies suggest that the smallest N-terminal )] TJ ET BT 26.250 111.486 Td /F1 9.8 Tf [(HTT fragments are pivotal to the molecular pathogenesis of HD. Human post-mortem studies showed that only N-terminal )] TJ ET BT 26.250 99.582 Td /F1 9.8 Tf [(antibodies detect nuclear inclusions in patient brains )] TJ ET 0.267 0.267 0.267 rg BT 254.400 101.089 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 259.219 99.582 Td /F1 9.8 Tf [( and formic acid solubilisation released a small N-terminal fragment )] TJ ET 0.267 0.267 0.267 rg BT 551.806 101.089 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 556.625 99.582 Td /F1 9.8 Tf [(. In )] TJ ET BT 26.250 87.677 Td /F1 9.8 Tf [(cell models, HTT has been shown to be cleaved into smaller fragments called cp-A and cp-B )] TJ ET 0.267 0.267 0.267 rg BT 427.784 89.184 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 432.603 87.677 Td /F1 9.8 Tf [( or cp-1 and cp-2 )] TJ ET 0.267 0.267 0.267 rg BT 509.014 89.184 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 513.832 87.677 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 519.253 89.184 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 528.891 87.677 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 75.772 Td /F1 9.8 Tf [(Furthermore, using the )] TJ ET BT 127.582 75.772 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 145.463 75.772 Td /F1 9.8 Tf [(Q150 knock-in model we recently demonstrated that full-length HTT is cleaved into a number of N-)] TJ ET BT 26.250 63.867 Td /F1 9.8 Tf [(terminal HTT fragments, the smallest of which is an exon 1 HTT protein )] TJ ET 0.267 0.267 0.267 rg BT 336.729 65.375 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 341.548 63.867 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 44.463 Td /F1 9.8 Tf [(The ensuing search for sites of proteolytic cleavage of human HTT raised the hypothesis that the cleavage of HTT by proteases )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(1)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 8 0 obj << /Type /Font /Subtype /Type1 /Name /F1 /BaseFont /Helvetica /Encoding /WinAnsiEncoding >> endobj 9 0 obj << /Type /Font /Subtype /Type1 /Name /F2 /BaseFont /Times-Bold /Encoding /WinAnsiEncoding >> endobj 10 0 obj << /Type /Font /Subtype /Type1 /Name /F3 /BaseFont /Times-Italic /Encoding /WinAnsiEncoding >> endobj 11 0 obj << /Type /Font /Subtype /Type1 /Name /F4 /BaseFont /Helvetica-Bold /Encoding /WinAnsiEncoding >> endobj 12 0 obj << /Type /Font /Subtype /Type1 /Name /F5 /BaseFont /Helvetica-Oblique /Encoding /WinAnsiEncoding >> endobj 13 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 1 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceGray /BitsPerComponent 8 /Length 144>> stream x1 0 'ݲ؎"e{dzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAtlM0\ endstream endobj 14 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /SMask 13 0 R /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 3 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceRGB /BitsPerComponent 8 /Length 4223>> stream xە8=,8C#h!hGv#0=j$q1uaNĥT*J&a_Zaa0 a0 a0 H^UUUUX!0 3SY|^G0 ÌÔ{]wa_>"OeYaG!8e}a6̖{4M&d:"qʲ\QUUMS<ϛNld2l6Z a<=2B(2vK7M\.GW0 rrWWavߖe9C?Eq8Gqy~~}_qdϽ(neYʒGjK4EQNjͷm{X?|7m۶mqP:Ȳp8Kmu]lNzuZ,NTeY"jlUUQW\,aRiVbȗLkHkEsjd.L6=Iו P]1t7Sc2uIuMM; K5>A$Ǒ$I׋nY 0_Hat;aq䃰U$nR ,zO7bT mhhԫܻu1F*qm'P}ܙ<ϕALO6Lw3nB.T4co{߽.lV $laԀIu!gwChxJ]Ne (+M"d-Eqc=P ! 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Studies have identified active caspase-3 \(HTT-513 and HTT-552\) and caspase-6 \(HTT-)] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(586\) sites )] TJ ET 0.267 0.267 0.267 rg BT 71.227 757.079 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 80.864 755.571 Td /F1 9.8 Tf [(, calpain sites \(HTT-469 and HTT-536\) )] TJ ET 0.267 0.267 0.267 rg BT 250.456 757.079 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 260.093 755.571 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 265.514 757.079 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 275.151 755.571 Td /F1 9.8 Tf [(, and recently an MMP-10 site \(HTT-402\) )] TJ ET 0.267 0.267 0.267 rg BT 455.039 757.079 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 464.676 755.571 Td /F1 9.8 Tf [(. Additionally, a number of )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(less well-defined HTT cleavage fragments have been isolated from HD post-mortem brains )] TJ ET 0.267 0.267 0.267 rg BT 420.716 745.174 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 425.534 743.667 Td /F1 9.8 Tf [( and from a number of HD model )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(systems, for which the precise cleavage sites and proteases involved remain elusive )] TJ ET 0.267 0.267 0.267 rg BT 392.558 733.269 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 397.376 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 402.797 733.269 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 412.435 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 417.856 733.269 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 427.493 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 432.914 733.269 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 442.551 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 447.972 733.269 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 457.610 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 463.031 733.269 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 472.668 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 478.089 733.269 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 487.726 731.762 Td /F1 9.8 Tf [(. The prevention of )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(cleavage at the caspase-6 but not caspase-3 sites was found to be protective in the YAC128 HD mouse model )] TJ ET 0.267 0.267 0.267 rg BT 505.862 721.364 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 515.500 719.857 Td /F1 9.8 Tf [(. In that study, )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(mutation of the aspartate at amino acid 586 ameliorated HD-phenotypes, implying that the caspase-6 mediated cleavage of )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(HTT is critical in the pathogenesis of HD. The HTT-586 fragment could itself be pathogenic and / or cleavage at this site might )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(initiate further proteolytic events that generate smaller N-terminal fragments with detrimental effects: the cascade hypothesis. )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(Alternatively, the aspartate substitution may alter the conformation of HTT without altering cleavage, and it could be this )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(structural change that improves the phenotype of these mice.)] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(We previously mapped the location of the proteolytic cleavage sites required to generate the N-terminal HTT fragments that are )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(present in the )] TJ ET BT 88.036 629.024 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 105.917 629.024 Td /F1 9.8 Tf [(Q150 HD model, and this suggested that )] TJ ET BT 284.761 629.024 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 334.613 629.024 Td /F1 9.8 Tf [( might be generated by caspase-6 )] TJ ET 0.267 0.267 0.267 rg BT 484.734 630.531 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 489.553 629.024 Td /F1 9.8 Tf [(. To test this )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(hypothesis, we crossed caspase-6 \()] TJ ET BT 181.226 617.119 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 209.404 617.119 Td /F1 9.8 Tf [(\)-deficient \()] TJ ET BT 258.163 617.119 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 286.341 621.007 Td /F1 8.7 Tf [(/)] TJ ET BT 298.388 617.119 Td /F1 9.8 Tf [(\) mice with the )] TJ ET BT 363.947 617.119 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 381.828 617.119 Td /F1 9.8 Tf [(Q150 mouse model of HD and analysed the )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(pattern of N-terminal HTT fragments. This genetic approach would test whether CASP6 actually cleaves HTT at amino acid 586)] TJ ET BT 26.250 593.310 Td /F5 9.8 Tf [(in vivo)] TJ ET BT 53.882 593.310 Td /F1 9.8 Tf [(, and also assess the so called cascade hypothesis should cleavage at HTT-586 be inhibited. In concurrence with our )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(previous findings, we were able to identify the same 14 prominent N-terminal mutant HTT fragments in the brain of the )] TJ ET BT 537.793 581.405 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 26.250 569.500 Td /F1 9.8 Tf [(Q150 mouse. Interestingly, we showed that the ablation of CASP6 had no effect on the production of )] TJ ET BT 462.543 569.500 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 512.395 569.500 Td /F1 9.8 Tf [( or any other N-)] TJ ET BT 26.250 557.595 Td /F1 9.8 Tf [(terminal HTT proteolytic fragments. In addition, the ablation of CASP6 had no effect on the levels of the full-length HTT protein. )] TJ ET BT 26.250 545.691 Td /F1 9.8 Tf [(Therefore, CASP6 does not play a direct role in the molecular pathogenesis that occurs in )] TJ ET BT 415.889 545.691 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 433.771 545.691 Td /F1 9.8 Tf [(Q150 mice through the )] TJ ET BT 26.250 533.786 Td /F1 9.8 Tf [(modulation of HTT proteolysis or modifying the levels of mutant HTT.)] TJ ET BT 26.250 497.183 Td /F4 12.0 Tf [(MATERIALS AND METHODS)] TJ ET BT 26.250 477.229 Td /F4 9.8 Tf [(Mouse Breeding, Maintenance, Genotyping, and CAG Repeat Sizing.)] TJ ET BT 342.647 477.229 Td /F1 9.8 Tf [( All mouse experiments were performed under the )] TJ ET BT 26.250 465.324 Td /F1 9.8 Tf [(Animals Scientific Procedures Act \(1986\) under project and personal licences approved and issued by the Home Office. The )] TJ ET BT 26.250 453.420 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 44.132 453.420 Td /F1 9.8 Tf [(Q150 knock-in mice were maintained, genotyped, and CAG repeat-sized as previously described )] TJ ET 0.267 0.267 0.267 rg BT 464.639 454.927 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 474.277 453.420 Td /F1 9.8 Tf [( . The mean repeat size )] TJ ET BT 26.250 441.515 Td /F1 9.8 Tf [(was 179 7.6 \(S.D.\). The )] TJ ET BT 146.019 441.515 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 174.197 445.403 Td /F1 8.7 Tf [(/ )] TJ ET BT 188.653 441.515 Td /F1 9.8 Tf [(mouse was created by Taconic Biosciences and genotyped as previously described )] TJ ET 0.267 0.267 0.267 rg BT 552.269 443.022 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 561.906 441.515 Td /F1 9.8 Tf [( . )] TJ ET BT 26.250 429.610 Td /F1 9.8 Tf [(The )] TJ ET BT 45.760 429.610 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 73.937 429.610 Td /F1 9.8 Tf [( mice were maintained by backcrossing )] TJ ET BT 246.785 429.610 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 274.963 433.498 Td /F1 8.7 Tf [(+/ )] TJ ET BT 289.661 429.610 Td /F1 9.8 Tf [(males to C57BL/6J females \(Charles River, A003\). )] TJ ET BT 510.733 429.610 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 528.614 433.498 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 557.284 429.610 Td /F1 9.8 Tf [(::)] TJ ET BT 26.250 417.705 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 54.427 421.594 Td /F1 8.7 Tf [(/ )] TJ ET BT 68.883 417.705 Td /F1 9.8 Tf [(\(Dble\) and)] TJ ET BT 114.397 417.705 Td /F5 9.8 Tf [( Hdh)] TJ ET BT 134.988 421.594 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 163.658 417.705 Td /F1 9.8 Tf [(::)] TJ ET BT 169.079 417.705 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 197.256 421.594 Td /F1 8.7 Tf [(+/+ )] TJ ET BT 212.198 417.705 Td /F1 9.8 Tf [(\()] TJ ET BT 215.444 417.705 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 233.326 421.594 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 261.995 417.705 Td /F1 9.8 Tf [(\) mice were generated by crossing )] TJ ET BT 413.715 417.705 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 441.893 421.594 Td /F1 8.7 Tf [(+/ )] TJ ET BT 456.591 417.705 Td /F1 9.8 Tf [(males with C57BL/6J )] TJ ET BT 550.874 417.705 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 26.250 409.689 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 54.919 405.801 Td /F1 9.8 Tf [(::)] TJ ET BT 60.340 405.801 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 88.518 409.689 Td /F1 8.7 Tf [(+/ )] TJ ET BT 103.217 405.801 Td /F1 9.8 Tf [(females. Mouse brains were snap-frozen in liquid nitrogen and stored at 80C.)] TJ ET BT 26.250 386.396 Td /F4 9.8 Tf [(Immunoprecipitation.)] TJ ET BT 125.388 386.396 Td /F1 9.8 Tf [( Whole brains were homogenised in ice-cold HEPES buffer \(50 mM HEPES/NaOH \(pH7.0\), 150 mM )] TJ ET BT 26.250 374.491 Td /F1 9.8 Tf [(NaCl, 10 mM EDTA, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS\) with 10 mM DTT, 1 mM PMSF, and complete )] TJ ET BT 26.250 362.586 Td /F1 9.8 Tf [(protease inhibitor mixture \(Roche\). Lysates were processed for western blotting or immunoprecipitation as described )] TJ ET 0.267 0.267 0.267 rg BT 530.725 364.094 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 535.543 362.586 Td /F1 9.8 Tf [(. For )] TJ ET BT 26.250 350.682 Td /F1 9.8 Tf [(immunoprecipitation, either 2 g mIgG, 2B7, S830, or 3B5H10 was used. \(See Table 1 and 2 for antibody details\).)] TJ ET BT 26.250 331.277 Td /F4 9.8 Tf [(Western Immunoblotting.)] TJ ET BT 143.796 331.277 Td /F1 9.8 Tf [( For western blotting, 30 g of total protein or 6 l of immunoprecipitate in Laemmli buffer were )] TJ ET BT 26.250 319.372 Td /F1 9.8 Tf [(denatured at 75C for 10 min and separated by 8% or 10% SDS-PAGE, blotted onto nitrocellulose membranes \(Whatman\) and )] TJ ET BT 26.250 307.467 Td /F1 9.8 Tf [(immunoprobed as described )] TJ ET 0.267 0.267 0.267 rg BT 151.966 308.975 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 156.785 307.467 Td /F1 9.8 Tf [(. Primary antibodies: MW1 \(1:1,000, )] TJ ET 0.267 0.267 0.267 rg BT 315.008 308.975 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 324.646 307.467 Td /F1 9.8 Tf [(\); CASP6 \(1:125\); ?-ACTIN \(1:10,000\). Secondary )] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(antibodies: anti-mouse HRP \(1:3,000\); anti-rabbit HRP \(1:3,000\). \(See Table 1 and 2 for antibody details\).)] TJ ET BT 26.250 276.158 Td /F4 9.8 Tf [(TR-FRET.)] TJ ET BT 70.661 276.158 Td /F1 9.8 Tf [( Time-resolved Frster resonance energy transfer \(TR-FRET\) was performed as described previously )] TJ ET 0.267 0.267 0.267 rg BT 510.611 277.665 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 520.248 276.158 Td /F1 9.8 Tf [(. 2B7 )] TJ ET BT 26.250 264.253 Td /F1 9.8 Tf [(antibody was labelled with terbium cryptate donor fluorophore \(CisBio\). MW1 was labelled with D2 acceptor fluorophore )] TJ ET BT 26.250 252.348 Td /F1 9.8 Tf [(\(CisBio\). MAB2166 was labelled with Alexa-fluor \(A\)488 \(Invitrogen\).)] TJ ET Q q 15.000 34.049 577.500 742.951 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(contributes to the pathogenesis of HD. Studies have identified active caspase-3 \(HTT-513 and HTT-552\) and caspase-6 \(HTT-)] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(586\) sites )] TJ ET 0.267 0.267 0.267 rg BT 71.227 757.079 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 80.864 755.571 Td /F1 9.8 Tf [(, calpain sites \(HTT-469 and HTT-536\) )] TJ ET 0.267 0.267 0.267 rg BT 250.456 757.079 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 260.093 755.571 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 265.514 757.079 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 275.151 755.571 Td /F1 9.8 Tf [(, and recently an MMP-10 site \(HTT-402\) )] TJ ET 0.267 0.267 0.267 rg BT 455.039 757.079 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 464.676 755.571 Td /F1 9.8 Tf [(. Additionally, a number of )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(less well-defined HTT cleavage fragments have been isolated from HD post-mortem brains )] TJ ET 0.267 0.267 0.267 rg BT 420.716 745.174 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 425.534 743.667 Td /F1 9.8 Tf [( and from a number of HD model )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(systems, for which the precise cleavage sites and proteases involved remain elusive )] TJ ET 0.267 0.267 0.267 rg BT 392.558 733.269 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 397.376 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 402.797 733.269 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 412.435 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 417.856 733.269 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 427.493 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 432.914 733.269 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 442.551 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 447.972 733.269 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 457.610 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 463.031 733.269 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 472.668 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 478.089 733.269 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 487.726 731.762 Td /F1 9.8 Tf [(. The prevention of )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(cleavage at the caspase-6 but not caspase-3 sites was found to be protective in the YAC128 HD mouse model )] TJ ET 0.267 0.267 0.267 rg BT 505.862 721.364 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 515.500 719.857 Td /F1 9.8 Tf [(. In that study, )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(mutation of the aspartate at amino acid 586 ameliorated HD-phenotypes, implying that the caspase-6 mediated cleavage of )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(HTT is critical in the pathogenesis of HD. The HTT-586 fragment could itself be pathogenic and / or cleavage at this site might )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(initiate further proteolytic events that generate smaller N-terminal fragments with detrimental effects: the cascade hypothesis. )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(Alternatively, the aspartate substitution may alter the conformation of HTT without altering cleavage, and it could be this )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(structural change that improves the phenotype of these mice.)] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(We previously mapped the location of the proteolytic cleavage sites required to generate the N-terminal HTT fragments that are )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(present in the )] TJ ET BT 88.036 629.024 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 105.917 629.024 Td /F1 9.8 Tf [(Q150 HD model, and this suggested that )] TJ ET BT 284.761 629.024 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 334.613 629.024 Td /F1 9.8 Tf [( might be generated by caspase-6 )] TJ ET 0.267 0.267 0.267 rg BT 484.734 630.531 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 489.553 629.024 Td /F1 9.8 Tf [(. To test this )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(hypothesis, we crossed caspase-6 \()] TJ ET BT 181.226 617.119 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 209.404 617.119 Td /F1 9.8 Tf [(\)-deficient \()] TJ ET BT 258.163 617.119 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 286.341 621.007 Td /F1 8.7 Tf [(/)] TJ ET BT 298.388 617.119 Td /F1 9.8 Tf [(\) mice with the )] TJ ET BT 363.947 617.119 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 381.828 617.119 Td /F1 9.8 Tf [(Q150 mouse model of HD and analysed the )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(pattern of N-terminal HTT fragments. This genetic approach would test whether CASP6 actually cleaves HTT at amino acid 586)] TJ ET BT 26.250 593.310 Td /F5 9.8 Tf [(in vivo)] TJ ET BT 53.882 593.310 Td /F1 9.8 Tf [(, and also assess the so called cascade hypothesis should cleavage at HTT-586 be inhibited. In concurrence with our )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(previous findings, we were able to identify the same 14 prominent N-terminal mutant HTT fragments in the brain of the )] TJ ET BT 537.793 581.405 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 26.250 569.500 Td /F1 9.8 Tf [(Q150 mouse. Interestingly, we showed that the ablation of CASP6 had no effect on the production of )] TJ ET BT 462.543 569.500 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 512.395 569.500 Td /F1 9.8 Tf [( or any other N-)] TJ ET BT 26.250 557.595 Td /F1 9.8 Tf [(terminal HTT proteolytic fragments. In addition, the ablation of CASP6 had no effect on the levels of the full-length HTT protein. )] TJ ET BT 26.250 545.691 Td /F1 9.8 Tf [(Therefore, CASP6 does not play a direct role in the molecular pathogenesis that occurs in )] TJ ET BT 415.889 545.691 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 433.771 545.691 Td /F1 9.8 Tf [(Q150 mice through the )] TJ ET BT 26.250 533.786 Td /F1 9.8 Tf [(modulation of HTT proteolysis or modifying the levels of mutant HTT.)] TJ ET BT 26.250 497.183 Td /F4 12.0 Tf [(MATERIALS AND METHODS)] TJ ET BT 26.250 477.229 Td /F4 9.8 Tf [(Mouse Breeding, Maintenance, Genotyping, and CAG Repeat Sizing.)] TJ ET BT 342.647 477.229 Td /F1 9.8 Tf [( All mouse experiments were performed under the )] TJ ET BT 26.250 465.324 Td /F1 9.8 Tf [(Animals Scientific Procedures Act \(1986\) under project and personal licences approved and issued by the Home Office. The )] TJ ET BT 26.250 453.420 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 44.132 453.420 Td /F1 9.8 Tf [(Q150 knock-in mice were maintained, genotyped, and CAG repeat-sized as previously described )] TJ ET 0.267 0.267 0.267 rg BT 464.639 454.927 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 474.277 453.420 Td /F1 9.8 Tf [( . The mean repeat size )] TJ ET BT 26.250 441.515 Td /F1 9.8 Tf [(was 179 7.6 \(S.D.\). The )] TJ ET BT 146.019 441.515 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 174.197 445.403 Td /F1 8.7 Tf [(/ )] TJ ET BT 188.653 441.515 Td /F1 9.8 Tf [(mouse was created by Taconic Biosciences and genotyped as previously described )] TJ ET 0.267 0.267 0.267 rg BT 552.269 443.022 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 561.906 441.515 Td /F1 9.8 Tf [( . )] TJ ET BT 26.250 429.610 Td /F1 9.8 Tf [(The )] TJ ET BT 45.760 429.610 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 73.937 429.610 Td /F1 9.8 Tf [( mice were maintained by backcrossing )] TJ ET BT 246.785 429.610 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 274.963 433.498 Td /F1 8.7 Tf [(+/ )] TJ ET BT 289.661 429.610 Td /F1 9.8 Tf [(males to C57BL/6J females \(Charles River, A003\). )] TJ ET BT 510.733 429.610 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 528.614 433.498 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 557.284 429.610 Td /F1 9.8 Tf [(::)] TJ ET BT 26.250 417.705 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 54.427 421.594 Td /F1 8.7 Tf [(/ )] TJ ET BT 68.883 417.705 Td /F1 9.8 Tf [(\(Dble\) and)] TJ ET BT 114.397 417.705 Td /F5 9.8 Tf [( Hdh)] TJ ET BT 134.988 421.594 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 163.658 417.705 Td /F1 9.8 Tf [(::)] TJ ET BT 169.079 417.705 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 197.256 421.594 Td /F1 8.7 Tf [(+/+ )] TJ ET BT 212.198 417.705 Td /F1 9.8 Tf [(\()] TJ ET BT 215.444 417.705 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 233.326 421.594 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 261.995 417.705 Td /F1 9.8 Tf [(\) mice were generated by crossing )] TJ ET BT 413.715 417.705 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 441.893 421.594 Td /F1 8.7 Tf [(+/ )] TJ ET BT 456.591 417.705 Td /F1 9.8 Tf [(males with C57BL/6J )] TJ ET BT 550.874 417.705 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 26.250 409.689 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 54.919 405.801 Td /F1 9.8 Tf [(::)] TJ ET BT 60.340 405.801 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 88.518 409.689 Td /F1 8.7 Tf [(+/ )] TJ ET BT 103.217 405.801 Td /F1 9.8 Tf [(females. Mouse brains were snap-frozen in liquid nitrogen and stored at 80C.)] TJ ET BT 26.250 386.396 Td /F4 9.8 Tf [(Immunoprecipitation.)] TJ ET BT 125.388 386.396 Td /F1 9.8 Tf [( Whole brains were homogenised in ice-cold HEPES buffer \(50 mM HEPES/NaOH \(pH7.0\), 150 mM )] TJ ET BT 26.250 374.491 Td /F1 9.8 Tf [(NaCl, 10 mM EDTA, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS\) with 10 mM DTT, 1 mM PMSF, and complete )] TJ ET BT 26.250 362.586 Td /F1 9.8 Tf [(protease inhibitor mixture \(Roche\). Lysates were processed for western blotting or immunoprecipitation as described )] TJ ET 0.267 0.267 0.267 rg BT 530.725 364.094 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 535.543 362.586 Td /F1 9.8 Tf [(. For )] TJ ET BT 26.250 350.682 Td /F1 9.8 Tf [(immunoprecipitation, either 2 g mIgG, 2B7, S830, or 3B5H10 was used. \(See Table 1 and 2 for antibody details\).)] TJ ET BT 26.250 331.277 Td /F4 9.8 Tf [(Western Immunoblotting.)] TJ ET BT 143.796 331.277 Td /F1 9.8 Tf [( For western blotting, 30 g of total protein or 6 l of immunoprecipitate in Laemmli buffer were )] TJ ET BT 26.250 319.372 Td /F1 9.8 Tf [(denatured at 75C for 10 min and separated by 8% or 10% SDS-PAGE, blotted onto nitrocellulose membranes \(Whatman\) and )] TJ ET BT 26.250 307.467 Td /F1 9.8 Tf [(immunoprobed as described )] TJ ET 0.267 0.267 0.267 rg BT 151.966 308.975 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 156.785 307.467 Td /F1 9.8 Tf [(. Primary antibodies: MW1 \(1:1,000, )] TJ ET 0.267 0.267 0.267 rg BT 315.008 308.975 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 324.646 307.467 Td /F1 9.8 Tf [(\); CASP6 \(1:125\); ?-ACTIN \(1:10,000\). Secondary )] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(antibodies: anti-mouse HRP \(1:3,000\); anti-rabbit HRP \(1:3,000\). \(See Table 1 and 2 for antibody details\).)] TJ ET BT 26.250 276.158 Td /F4 9.8 Tf [(TR-FRET.)] TJ ET BT 70.661 276.158 Td /F1 9.8 Tf [( Time-resolved Frster resonance energy transfer \(TR-FRET\) was performed as described previously )] TJ ET 0.267 0.267 0.267 rg BT 510.611 277.665 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 520.248 276.158 Td /F1 9.8 Tf [(. 2B7 )] TJ ET BT 26.250 264.253 Td /F1 9.8 Tf [(antibody was labelled with terbium cryptate donor fluorophore \(CisBio\). MW1 was labelled with D2 acceptor fluorophore )] TJ ET BT 26.250 252.348 Td /F1 9.8 Tf [(\(CisBio\). MAB2166 was labelled with Alexa-fluor \(A\)488 \(Invitrogen\).)] TJ ET Q q 15.000 34.049 577.500 742.951 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(contributes to the pathogenesis of HD. Studies have identified active caspase-3 \(HTT-513 and HTT-552\) and caspase-6 \(HTT-)] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(586\) sites )] TJ ET 0.267 0.267 0.267 rg BT 71.227 757.079 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 80.864 755.571 Td /F1 9.8 Tf [(, calpain sites \(HTT-469 and HTT-536\) )] TJ ET 0.267 0.267 0.267 rg BT 250.456 757.079 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 260.093 755.571 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 265.514 757.079 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 275.151 755.571 Td /F1 9.8 Tf [(, and recently an MMP-10 site \(HTT-402\) )] TJ ET 0.267 0.267 0.267 rg BT 455.039 757.079 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 464.676 755.571 Td /F1 9.8 Tf [(. Additionally, a number of )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(less well-defined HTT cleavage fragments have been isolated from HD post-mortem brains )] TJ ET 0.267 0.267 0.267 rg BT 420.716 745.174 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 425.534 743.667 Td /F1 9.8 Tf [( and from a number of HD model )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(systems, for which the precise cleavage sites and proteases involved remain elusive )] TJ ET 0.267 0.267 0.267 rg BT 392.558 733.269 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 397.376 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 402.797 733.269 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 412.435 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 417.856 733.269 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 427.493 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 432.914 733.269 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 442.551 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 447.972 733.269 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 457.610 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 463.031 733.269 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 472.668 731.762 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 478.089 733.269 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 487.726 731.762 Td /F1 9.8 Tf [(. The prevention of )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(cleavage at the caspase-6 but not caspase-3 sites was found to be protective in the YAC128 HD mouse model )] TJ ET 0.267 0.267 0.267 rg BT 505.862 721.364 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 515.500 719.857 Td /F1 9.8 Tf [(. In that study, )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(mutation of the aspartate at amino acid 586 ameliorated HD-phenotypes, implying that the caspase-6 mediated cleavage of )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(HTT is critical in the pathogenesis of HD. The HTT-586 fragment could itself be pathogenic and / or cleavage at this site might )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(initiate further proteolytic events that generate smaller N-terminal fragments with detrimental effects: the cascade hypothesis. )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(Alternatively, the aspartate substitution may alter the conformation of HTT without altering cleavage, and it could be this )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(structural change that improves the phenotype of these mice.)] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(We previously mapped the location of the proteolytic cleavage sites required to generate the N-terminal HTT fragments that are )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(present in the )] TJ ET BT 88.036 629.024 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 105.917 629.024 Td /F1 9.8 Tf [(Q150 HD model, and this suggested that )] TJ ET BT 284.761 629.024 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 334.613 629.024 Td /F1 9.8 Tf [( might be generated by caspase-6 )] TJ ET 0.267 0.267 0.267 rg BT 484.734 630.531 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 489.553 629.024 Td /F1 9.8 Tf [(. To test this )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(hypothesis, we crossed caspase-6 \()] TJ ET BT 181.226 617.119 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 209.404 617.119 Td /F1 9.8 Tf [(\)-deficient \()] TJ ET BT 258.163 617.119 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 286.341 621.007 Td /F1 8.7 Tf [(/)] TJ ET BT 298.388 617.119 Td /F1 9.8 Tf [(\) mice with the )] TJ ET BT 363.947 617.119 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 381.828 617.119 Td /F1 9.8 Tf [(Q150 mouse model of HD and analysed the )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(pattern of N-terminal HTT fragments. This genetic approach would test whether CASP6 actually cleaves HTT at amino acid 586)] TJ ET BT 26.250 593.310 Td /F5 9.8 Tf [(in vivo)] TJ ET BT 53.882 593.310 Td /F1 9.8 Tf [(, and also assess the so called cascade hypothesis should cleavage at HTT-586 be inhibited. In concurrence with our )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(previous findings, we were able to identify the same 14 prominent N-terminal mutant HTT fragments in the brain of the )] TJ ET BT 537.793 581.405 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 26.250 569.500 Td /F1 9.8 Tf [(Q150 mouse. Interestingly, we showed that the ablation of CASP6 had no effect on the production of )] TJ ET BT 462.543 569.500 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 512.395 569.500 Td /F1 9.8 Tf [( or any other N-)] TJ ET BT 26.250 557.595 Td /F1 9.8 Tf [(terminal HTT proteolytic fragments. In addition, the ablation of CASP6 had no effect on the levels of the full-length HTT protein. )] TJ ET BT 26.250 545.691 Td /F1 9.8 Tf [(Therefore, CASP6 does not play a direct role in the molecular pathogenesis that occurs in )] TJ ET BT 415.889 545.691 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 433.771 545.691 Td /F1 9.8 Tf [(Q150 mice through the )] TJ ET BT 26.250 533.786 Td /F1 9.8 Tf [(modulation of HTT proteolysis or modifying the levels of mutant HTT.)] TJ ET BT 26.250 497.183 Td /F4 12.0 Tf [(MATERIALS AND METHODS)] TJ ET BT 26.250 477.229 Td /F4 9.8 Tf [(Mouse Breeding, Maintenance, Genotyping, and CAG Repeat Sizing.)] TJ ET BT 342.647 477.229 Td /F1 9.8 Tf [( All mouse experiments were performed under the )] TJ ET BT 26.250 465.324 Td /F1 9.8 Tf [(Animals Scientific Procedures Act \(1986\) under project and personal licences approved and issued by the Home Office. The )] TJ ET BT 26.250 453.420 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 44.132 453.420 Td /F1 9.8 Tf [(Q150 knock-in mice were maintained, genotyped, and CAG repeat-sized as previously described )] TJ ET 0.267 0.267 0.267 rg BT 464.639 454.927 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 474.277 453.420 Td /F1 9.8 Tf [( . The mean repeat size )] TJ ET BT 26.250 441.515 Td /F1 9.8 Tf [(was 179 7.6 \(S.D.\). The )] TJ ET BT 146.019 441.515 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 174.197 445.403 Td /F1 8.7 Tf [(/ )] TJ ET BT 188.653 441.515 Td /F1 9.8 Tf [(mouse was created by Taconic Biosciences and genotyped as previously described )] TJ ET 0.267 0.267 0.267 rg BT 552.269 443.022 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 561.906 441.515 Td /F1 9.8 Tf [( . )] TJ ET BT 26.250 429.610 Td /F1 9.8 Tf [(The )] TJ ET BT 45.760 429.610 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 73.937 429.610 Td /F1 9.8 Tf [( mice were maintained by backcrossing )] TJ ET BT 246.785 429.610 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 274.963 433.498 Td /F1 8.7 Tf [(+/ )] TJ ET BT 289.661 429.610 Td /F1 9.8 Tf [(males to C57BL/6J females \(Charles River, A003\). )] TJ ET BT 510.733 429.610 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 528.614 433.498 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 557.284 429.610 Td /F1 9.8 Tf [(::)] TJ ET BT 26.250 417.705 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 54.427 421.594 Td /F1 8.7 Tf [(/ )] TJ ET BT 68.883 417.705 Td /F1 9.8 Tf [(\(Dble\) and)] TJ ET BT 114.397 417.705 Td /F5 9.8 Tf [( Hdh)] TJ ET BT 134.988 421.594 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 163.658 417.705 Td /F1 9.8 Tf [(::)] TJ ET BT 169.079 417.705 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 197.256 421.594 Td /F1 8.7 Tf [(+/+ )] TJ ET BT 212.198 417.705 Td /F1 9.8 Tf [(\()] TJ ET BT 215.444 417.705 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 233.326 421.594 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 261.995 417.705 Td /F1 9.8 Tf [(\) mice were generated by crossing )] TJ ET BT 413.715 417.705 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 441.893 421.594 Td /F1 8.7 Tf [(+/ )] TJ ET BT 456.591 417.705 Td /F1 9.8 Tf [(males with C57BL/6J )] TJ ET BT 550.874 417.705 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 26.250 409.689 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 54.919 405.801 Td /F1 9.8 Tf [(::)] TJ ET BT 60.340 405.801 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 88.518 409.689 Td /F1 8.7 Tf [(+/ )] TJ ET BT 103.217 405.801 Td /F1 9.8 Tf [(females. Mouse brains were snap-frozen in liquid nitrogen and stored at 80C.)] TJ ET BT 26.250 386.396 Td /F4 9.8 Tf [(Immunoprecipitation.)] TJ ET BT 125.388 386.396 Td /F1 9.8 Tf [( Whole brains were homogenised in ice-cold HEPES buffer \(50 mM HEPES/NaOH \(pH7.0\), 150 mM )] TJ ET BT 26.250 374.491 Td /F1 9.8 Tf [(NaCl, 10 mM EDTA, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS\) with 10 mM DTT, 1 mM PMSF, and complete )] TJ ET BT 26.250 362.586 Td /F1 9.8 Tf [(protease inhibitor mixture \(Roche\). Lysates were processed for western blotting or immunoprecipitation as described )] TJ ET 0.267 0.267 0.267 rg BT 530.725 364.094 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 535.543 362.586 Td /F1 9.8 Tf [(. For )] TJ ET BT 26.250 350.682 Td /F1 9.8 Tf [(immunoprecipitation, either 2 g mIgG, 2B7, S830, or 3B5H10 was used. \(See Table 1 and 2 for antibody details\).)] TJ ET BT 26.250 331.277 Td /F4 9.8 Tf [(Western Immunoblotting.)] TJ ET BT 143.796 331.277 Td /F1 9.8 Tf [( For western blotting, 30 g of total protein or 6 l of immunoprecipitate in Laemmli buffer were )] TJ ET BT 26.250 319.372 Td /F1 9.8 Tf [(denatured at 75C for 10 min and separated by 8% or 10% SDS-PAGE, blotted onto nitrocellulose membranes \(Whatman\) and )] TJ ET BT 26.250 307.467 Td /F1 9.8 Tf [(immunoprobed as described )] TJ ET 0.267 0.267 0.267 rg BT 151.966 308.975 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 156.785 307.467 Td /F1 9.8 Tf [(. Primary antibodies: MW1 \(1:1,000, )] TJ ET 0.267 0.267 0.267 rg BT 315.008 308.975 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 324.646 307.467 Td /F1 9.8 Tf [(\); CASP6 \(1:125\); ?-ACTIN \(1:10,000\). Secondary )] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(antibodies: anti-mouse HRP \(1:3,000\); anti-rabbit HRP \(1:3,000\). \(See Table 1 and 2 for antibody details\).)] TJ ET BT 26.250 276.158 Td /F4 9.8 Tf [(TR-FRET.)] TJ ET BT 70.661 276.158 Td /F1 9.8 Tf [( Time-resolved Frster resonance energy transfer \(TR-FRET\) was performed as described previously )] TJ ET 0.267 0.267 0.267 rg BT 510.611 277.665 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 520.248 276.158 Td /F1 9.8 Tf [(. 2B7 )] TJ ET BT 26.250 264.253 Td /F1 9.8 Tf [(antibody was labelled with terbium cryptate donor fluorophore \(CisBio\). MW1 was labelled with D2 acceptor fluorophore )] TJ ET BT 26.250 252.348 Td /F1 9.8 Tf [(\(CisBio\). MAB2166 was labelled with Alexa-fluor \(A\)488 \(Invitrogen\).)] 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 149 0 obj << /Type /Annot /Subtype /Link /A 150 0 R /Border [0 0 0] /H /I /Rect [ 71.2268 756.2769 80.8641 765.0952 ] >> endobj 150 0 obj << /Type /Action >> endobj 151 0 obj << /Type /Annot /Subtype /Link /A 152 0 R /Border [0 0 0] /H /I /Rect [ 250.4556 756.2769 260.0929 765.0952 ] >> endobj 152 0 obj << /Type /Action >> endobj 153 0 obj << /Type /Annot /Subtype /Link /A 154 0 R /Border [0 0 0] /H /I /Rect [ 265.5139 756.2769 275.1513 765.0952 ] >> endobj 154 0 obj << /Type /Action >> endobj 155 0 obj << /Type /Annot /Subtype /Link /A 156 0 R /Border [0 0 0] /H /I /Rect [ 455.0388 756.2769 464.6761 765.0952 ] >> endobj 156 0 obj << /Type /Action >> endobj 157 0 obj << /Type /Annot /Subtype /Link /A 158 0 R /Border [0 0 0] /H /I /Rect [ 420.7155 744.3722 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0] /H /I /Rect [ 315.0082 308.1729 324.6455 316.9913 ] >> 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 [ 510.6105 276.8634 520.2478 285.6818 ] >> endobj 268 0 obj << /Type /Action >> endobj 269 0 obj << /Type /Page /Parent 3 0 R /Annots [ 272 0 R 274 0 R 276 0 R 278 0 R 280 0 R 282 0 R 285 0 R 287 0 R 289 0 R 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 ] /Contents 270 0 R >> endobj 270 0 obj << /Length 45215 >> stream 0.271 0.267 0.267 rg q 15.000 29.157 577.500 747.843 re W n q 26.250 763.264 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 766.011 Td /F1 9.8 Tf [(Table 1: Primary Antibodies)] TJ ET Q 1.000 1.000 1.000 rg 26.250 606.082 555.000 149.682 re f 0.965 0.965 0.965 rg 27.000 742.734 108.148 12.280 re f 0.267 0.267 0.267 rg 26.625 754.639 108.523 0.750 re f 26.625 742.359 0.750 13.030 re f 0.271 0.267 0.267 rg BT 31.500 745.490 Td /F4 9.8 Tf [(Antibody)] TJ ET 0.965 0.965 0.965 rg 135.148 742.734 139.176 12.280 re f 0.267 0.267 0.267 rg 135.148 754.639 139.176 0.750 re f 0.271 0.267 0.267 rg BT 139.648 745.490 Td /F4 9.8 Tf [(Epitope)] TJ ET 0.965 0.965 0.965 rg 274.324 742.734 118.517 12.280 re f 0.267 0.267 0.267 rg 274.324 754.639 118.517 0.750 re f 0.271 0.267 0.267 rg BT 278.824 745.490 Td /F4 9.8 Tf [(Description)] TJ ET 0.965 0.965 0.965 rg 392.841 742.734 187.659 12.280 re f 0.267 0.267 0.267 rg 392.841 754.639 188.034 0.750 re f 580.125 742.359 0.750 13.030 re f 0.271 0.267 0.267 rg BT 397.341 745.490 Td /F4 9.8 Tf [(Source)] TJ ET 0.267 0.267 0.267 rg 26.625 742.359 108.898 0.750 re f 26.625 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 732.504 Td /F1 9.8 Tf [(mIgG)] TJ ET 0.267 0.267 0.267 rg 134.773 742.359 139.926 0.750 re f 134.773 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 732.504 Td /F1 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg 273.949 742.359 119.267 0.750 re f 273.949 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 732.504 Td /F1 9.8 Tf [(Mouse IgG)] TJ ET 0.267 0.267 0.267 rg 392.466 742.359 188.409 0.750 re f 392.466 726.478 0.750 16.631 re f 580.125 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 732.504 Td /F1 9.8 Tf [(Alpha Diagnostic \(#20008-250\))] TJ ET 0.267 0.267 0.267 rg 26.625 726.478 108.898 0.750 re f 26.625 707.646 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 716.623 Td /F1 9.8 Tf [(2B7)] TJ ET 0.267 0.267 0.267 rg 134.773 726.478 139.926 0.750 re f 134.773 707.646 0.750 19.582 re f 0.271 0.267 0.267 rg BT 140.023 716.623 Td /F1 9.8 Tf [(HTT aa1-17)] TJ ET 0.267 0.267 0.267 rg 273.949 726.478 119.267 0.750 re f 273.949 707.646 0.750 19.582 re f 0.271 0.267 0.267 rg BT 279.199 716.623 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 726.478 188.409 0.750 re f 392.466 707.646 0.750 19.582 re f 580.125 707.646 0.750 19.582 re f BT 397.716 714.711 Td /F4 8.7 Tf [(25)] TJ ET 26.625 707.646 108.898 0.750 re f 26.625 688.814 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 697.791 Td /F1 9.8 Tf [(S830)] TJ ET 0.267 0.267 0.267 rg 134.773 707.646 139.926 0.750 re f 134.773 688.814 0.750 19.582 re f 0.271 0.267 0.267 rg BT 140.023 697.791 Td /F1 9.8 Tf [(Exon1 \(53Q\))] TJ ET 0.267 0.267 0.267 rg 273.949 707.646 119.267 0.750 re f 273.949 688.814 0.750 19.582 re f 0.271 0.267 0.267 rg BT 279.199 697.791 Td /F1 9.8 Tf [(Polyclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 707.646 188.409 0.750 re f 392.466 688.814 0.750 19.582 re f 580.125 688.814 0.750 19.582 re f BT 397.716 695.879 Td /F4 8.7 Tf [(26)] TJ ET 26.625 688.814 108.898 0.750 re f 26.625 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 678.959 Td /F1 9.8 Tf [(3B5H10)] TJ ET 0.267 0.267 0.267 rg 134.773 688.814 139.926 0.750 re f 134.773 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 678.959 Td /F1 9.8 Tf [(N171 \(65Q\))] TJ ET 0.267 0.267 0.267 rg 273.949 688.814 119.267 0.750 re f 273.949 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 678.959 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 688.814 188.409 0.750 re f 392.466 672.932 0.750 16.631 re f 580.125 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 678.959 Td /F1 9.8 Tf [(Sigma-Aldrich \(P1874\))] TJ ET 0.267 0.267 0.267 rg 26.625 672.932 108.898 0.750 re f 26.625 654.100 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 663.077 Td /F1 9.8 Tf [(MW1)] TJ ET 0.267 0.267 0.267 rg 134.773 672.932 139.926 0.750 re f 134.773 654.100 0.750 19.582 re f 0.271 0.267 0.267 rg BT 140.023 663.077 Td /F1 9.8 Tf [(PolyQ)] TJ ET 0.267 0.267 0.267 rg 273.949 672.932 119.267 0.750 re f 273.949 654.100 0.750 19.582 re f 0.271 0.267 0.267 rg BT 279.199 663.077 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 672.932 188.409 0.750 re f 392.466 654.100 0.750 19.582 re f 580.125 654.100 0.750 19.582 re f BT 397.716 661.166 Td /F4 8.7 Tf [(23)] TJ ET 26.625 654.100 108.898 0.750 re f 26.625 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 644.245 Td /F1 9.8 Tf [(MAB2166)] TJ ET 0.267 0.267 0.267 rg 134.773 654.100 139.926 0.750 re f 134.773 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 644.245 Td /F1 9.8 Tf [(HTT aa443-457)] TJ ET 0.267 0.267 0.267 rg 273.949 654.100 119.267 0.750 re f 273.949 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 644.245 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 654.100 188.409 0.750 re f 392.466 638.219 0.750 16.631 re f 580.125 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 644.245 Td /F1 9.8 Tf [(Millipore \(MAB2166\))] TJ ET 0.267 0.267 0.267 rg 26.625 638.219 108.898 0.750 re f 26.625 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 628.364 Td /F1 9.8 Tf [(CASP6)] TJ ET 0.267 0.267 0.267 rg 134.773 638.219 139.926 0.750 re f 134.773 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 628.364 Td /F1 9.8 Tf [(CASP6 cleavage site)] TJ ET 0.267 0.267 0.267 rg 273.949 638.219 119.267 0.750 re f 273.949 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 628.364 Td /F1 9.8 Tf [(Polyclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 638.219 188.409 0.750 re f 392.466 622.338 0.750 16.631 re f 580.125 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 628.364 Td /F1 9.8 Tf [(Cell Signalling \(#9762\))] TJ ET 0.267 0.267 0.267 rg 26.625 622.338 108.898 0.750 re f 26.625 606.457 108.898 0.750 re f 26.625 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 612.483 Td /F1 9.8 Tf [(Beta-ACTIN)] TJ ET 0.267 0.267 0.267 rg 134.773 622.338 139.926 0.750 re f 134.773 606.457 139.926 0.750 re f 134.773 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 612.483 Td /F1 9.8 Tf [(ACTIN aa1-15)] TJ ET 0.267 0.267 0.267 rg 273.949 622.338 119.267 0.750 re f 273.949 606.457 119.267 0.750 re f 273.949 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 612.483 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 622.338 188.409 0.750 re f 392.466 606.457 188.409 0.750 re f 392.466 606.457 0.750 16.631 re f 580.125 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 612.483 Td /F1 9.8 Tf [(Abcam \(ab6276\))] TJ ET q 26.250 547.345 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 550.093 Td /F1 9.8 Tf [(Table 2: Secondary Antibodies)] TJ ET Q 1.000 1.000 1.000 rg 26.250 494.303 555.000 45.542 re f 0.965 0.965 0.965 rg 27.000 526.816 187.143 12.280 re f 0.267 0.267 0.267 rg 26.625 538.720 187.518 0.750 re f 26.625 526.441 0.750 13.030 re f 0.271 0.267 0.267 rg BT 31.500 529.572 Td /F4 9.8 Tf [(Antibody)] TJ ET 0.965 0.965 0.965 rg 214.143 526.816 198.531 12.280 re f 0.267 0.267 0.267 rg 214.143 538.720 198.531 0.750 re f 0.271 0.267 0.267 rg BT 218.643 529.572 Td /F4 9.8 Tf [(Description)] TJ ET 0.965 0.965 0.965 rg 412.675 526.816 167.825 12.280 re f 0.267 0.267 0.267 rg 412.675 538.720 168.200 0.750 re f 580.125 526.441 0.750 13.030 re f 0.271 0.267 0.267 rg BT 417.175 529.572 Td /F4 9.8 Tf [(Source)] TJ ET 0.267 0.267 0.267 rg 26.625 526.441 187.893 0.750 re f 26.625 510.559 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 516.586 Td /F1 9.8 Tf [(Anti-mouse HRP)] TJ ET 0.267 0.267 0.267 rg 213.768 526.441 199.281 0.750 re f 213.768 510.559 0.750 16.631 re f 0.271 0.267 0.267 rg BT 219.018 516.586 Td /F1 9.8 Tf [(Polyclonal-rabbit)] TJ ET 0.267 0.267 0.267 rg 412.300 526.441 168.575 0.750 re f 412.300 510.559 0.750 16.631 re f 580.125 510.559 0.750 16.631 re f 0.271 0.267 0.267 rg BT 417.550 516.586 Td /F1 9.8 Tf [(DAKO \(P0260\))] TJ ET 0.267 0.267 0.267 rg 26.625 510.559 187.893 0.750 re f 26.625 494.678 187.893 0.750 re f 26.625 494.678 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 500.704 Td /F1 9.8 Tf [(Anti-rabbit HRP)] TJ ET 0.267 0.267 0.267 rg 213.768 510.559 199.281 0.750 re f 213.768 494.678 199.281 0.750 re f 213.768 494.678 0.750 16.631 re f 0.271 0.267 0.267 rg BT 219.018 500.704 Td /F1 9.8 Tf [(Polyclonal-swine)] TJ ET 0.267 0.267 0.267 rg 412.300 510.559 168.575 0.750 re f 412.300 494.678 168.575 0.750 re f 412.300 494.678 0.750 16.631 re f 580.125 494.678 0.750 16.631 re f 0.271 0.267 0.267 rg BT 417.550 500.704 Td /F1 9.8 Tf [(DAKO \(P0449\))] TJ ET BT 26.250 422.582 Td /F4 12.0 Tf [(RESULTS)] TJ ET BT 26.250 402.627 Td /F6 9.8 Tf [(Casp6)] TJ ET BT 55.510 406.516 Td /F6 8.7 Tf [(/)] TJ ET BT 67.556 402.627 Td /F4 9.8 Tf [(mice show no CASP6 expression in the brain.)] TJ ET BT 279.414 402.627 Td /F1 9.8 Tf [( Exons 2-5 of the )] TJ ET BT 355.835 402.627 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 384.012 402.627 Td /F1 9.8 Tf [( gene, which encodes the catalytic domain of )] TJ ET BT 26.250 390.723 Td /F1 9.8 Tf [(the caspase-6 \(CASP6\) protein, were removed to generate )] TJ ET BT 283.114 390.723 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 311.291 394.611 Td /F1 8.7 Tf [(/)] TJ ET BT 323.338 390.723 Td /F1 9.8 Tf [( mice. )] TJ ET BT 352.052 390.723 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 380.229 394.611 Td /F1 8.7 Tf [(/ )] TJ ET BT 394.685 390.723 Td /F1 9.8 Tf [(mice are viable and fertile, and do not )] TJ ET BT 26.250 378.818 Td /F1 9.8 Tf [(show any overt phenotypes )] TJ ET 0.267 0.267 0.267 rg BT 147.647 380.325 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 157.285 378.818 Td /F1 9.8 Tf [(. We began by performing western blots to ensure that these mice do not express CASP6 and to )] TJ ET BT 26.250 366.913 Td /F1 9.8 Tf [(verify the genotypes. Protein lysates were prepared from half brains of 2 month old wild type and )] TJ ET BT 445.149 366.913 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 473.327 370.801 Td /F1 8.7 Tf [(/ )] TJ ET BT 487.783 366.913 Td /F1 9.8 Tf [(mice and analysed )] TJ ET BT 26.250 355.008 Td /F1 9.8 Tf [(by western blotting using an anti-CASP6 antibody. As previously reported )] TJ ET 0.267 0.267 0.267 rg BT 345.436 356.516 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 355.073 355.008 Td /F1 9.8 Tf [(, we found that the )] TJ ET BT 438.007 355.008 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 466.184 358.897 Td /F1 8.7 Tf [(/ )] TJ ET BT 480.640 355.008 Td /F1 9.8 Tf [(mice constitutively )] TJ ET BT 26.250 343.104 Td /F1 9.8 Tf [(lacked CASP6 as compared to wild type littermates \(Fig. 1\).)] TJ ET 0.965 0.965 0.965 rg 26.250 167.609 555.000 165.614 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 333.223 m 581.250 333.223 l 581.250 332.473 l 26.250 332.473 l f 26.250 167.609 m 581.250 167.609 l 581.250 168.359 l 26.250 168.359 l f q 225.000 0 0 72.000 35.250 251.473 cm /I3 Do Q q 35.250 178.859 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 235.949 Td /F4 9.8 Tf [(Fig. 1: Figure 1. CASP6 expression in Casp6/and wild type brains)] TJ ET BT 35.250 216.579 Td /F1 9.8 Tf [(Western blotting of total brain lysates from 2 month old mice using an anti-CASP6 antibody shows that CASP6 was absent )] TJ ET BT 35.250 202.843 Td /F1 9.8 Tf [(in Casp6/ samples when compared to wild type \(Casp6+/+\) samples. Beta-ACTIN served as a loading control. ID = )] TJ ET BT 35.250 189.106 Td /F1 9.8 Tf [(Immunodetection.)] TJ ET Q BT 26.250 150.585 Td /F4 9.8 Tf [(Genetic ablation of )] TJ ET BT 116.730 150.585 Td /F6 9.8 Tf [(Casp6)] TJ ET BT 145.990 150.585 Td /F4 9.8 Tf [( does not modify HTT proteolysis in )] TJ ET BT 314.479 150.585 Td /F6 9.8 Tf [(Hdh)] TJ ET BT 333.433 150.585 Td /F4 9.8 Tf [(Q150 knock-in mice.)] TJ ET BT 427.726 150.585 Td /F1 9.8 Tf [( To identify N-terminal HTT )] TJ ET BT 26.250 138.681 Td /F1 9.8 Tf [(fragments in the brains of )] TJ ET BT 138.970 138.681 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 156.851 138.681 Td /F1 9.8 Tf [(Q150 knock-in mice, we previously used a combination of immunoprecipitation and western )] TJ ET BT 26.250 126.776 Td /F1 9.8 Tf [(blotting that identified 14 prominent N-terminal fragments, which in addition to the full-length protein, could be readily detected in )] TJ ET BT 26.250 114.871 Td /F1 9.8 Tf [(the cytoplasmic but not nuclear fractions. These fragments were present at all ages and did not arise as a consequence of the )] TJ ET BT 26.250 102.966 Td /F1 9.8 Tf [(pathogenic process. Furthermore, in an attempt to determine the source of some of the N-terminal HTT fragments, proteolytic )] TJ ET BT 26.250 91.062 Td /F1 9.8 Tf [(digests were performed with caspase enzymes which indicated that )] TJ ET BT 319.949 91.062 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 369.801 91.062 Td /F1 9.8 Tf [( may terminate at the caspase-6 HTT-586 )] TJ ET BT 26.250 79.157 Td /F1 9.8 Tf [(cleavage site )] TJ ET 0.267 0.267 0.267 rg BT 85.862 80.664 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 90.680 79.157 Td /F1 9.8 Tf [(. In order to corroborate this observation, we investigated whether the presence of this proteolytic fragment, )] TJ ET BT 26.250 67.252 Td /F1 9.8 Tf [(along with all other observed N-terminal HTT fragments, was modulated upon the genetic ablation of CASP6.)] TJ ET BT 496.619 67.252 Td /F5 9.8 Tf [( Casp6)] TJ ET BT 527.507 71.140 Td /F1 8.7 Tf [(+/ )] TJ ET BT 542.206 67.252 Td /F1 9.8 Tf [(males )] TJ ET BT 26.250 55.347 Td /F1 9.8 Tf [(were bred to )] TJ ET BT 83.151 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 101.032 59.236 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 129.702 55.347 Td /F1 9.8 Tf [(::)] TJ ET BT 135.123 55.347 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 163.300 59.236 Td /F1 8.7 Tf [(+/ )] TJ ET BT 177.999 55.347 Td /F1 9.8 Tf [(females to generate )] TJ ET BT 266.880 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 284.762 59.236 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 315.840 55.347 Td /F1 9.8 Tf [(mice on either a )] TJ ET BT 387.912 55.347 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 416.090 55.347 Td /F1 9.8 Tf [( wild type )] TJ ET BT 459.438 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 477.320 59.236 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 505.989 55.347 Td /F1 9.8 Tf [(::)] TJ ET BT 511.410 55.347 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 539.588 59.236 Td /F1 8.7 Tf [(+/+ )] TJ ET BT 554.529 55.347 Td /F1 9.8 Tf [(\()] TJ ET BT 557.776 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 26.250 47.331 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 54.919 43.443 Td /F1 9.8 Tf [(\) or )] TJ ET BT 72.255 43.443 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 100.432 43.443 Td /F1 9.8 Tf [( knock-out )] TJ ET BT 148.120 43.443 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 166.001 47.331 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 194.670 43.443 Td /F1 9.8 Tf [(::)] TJ ET BT 200.091 43.443 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 228.269 47.331 Td /F1 8.7 Tf [(/ )] TJ ET BT 242.725 43.443 Td /F1 9.8 Tf [(\(Dble\) background. We obtained at least 12 mice for each genotype and all )] TJ ET Q q 15.000 29.157 577.500 747.843 re W n q 26.250 763.264 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 766.011 Td /F1 9.8 Tf [(Table 1: Primary Antibodies)] TJ ET Q 1.000 1.000 1.000 rg 26.250 606.082 555.000 149.682 re f 0.965 0.965 0.965 rg 27.000 742.734 108.148 12.280 re f 0.267 0.267 0.267 rg 26.625 754.639 108.523 0.750 re f 26.625 742.359 0.750 13.030 re f 0.271 0.267 0.267 rg BT 31.500 745.490 Td /F4 9.8 Tf [(Antibody)] TJ ET 0.965 0.965 0.965 rg 135.148 742.734 139.176 12.280 re f 0.267 0.267 0.267 rg 135.148 754.639 139.176 0.750 re f 0.271 0.267 0.267 rg BT 139.648 745.490 Td /F4 9.8 Tf [(Epitope)] TJ ET 0.965 0.965 0.965 rg 274.324 742.734 118.517 12.280 re f 0.267 0.267 0.267 rg 274.324 754.639 118.517 0.750 re f 0.271 0.267 0.267 rg BT 278.824 745.490 Td /F4 9.8 Tf [(Description)] TJ ET 0.965 0.965 0.965 rg 392.841 742.734 187.659 12.280 re f 0.267 0.267 0.267 rg 392.841 754.639 188.034 0.750 re f 580.125 742.359 0.750 13.030 re f 0.271 0.267 0.267 rg BT 397.341 745.490 Td /F4 9.8 Tf [(Source)] TJ ET 0.267 0.267 0.267 rg 26.625 742.359 108.898 0.750 re f 26.625 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 732.504 Td /F1 9.8 Tf [(mIgG)] TJ ET 0.267 0.267 0.267 rg 134.773 742.359 139.926 0.750 re f 134.773 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 732.504 Td /F1 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg 273.949 742.359 119.267 0.750 re f 273.949 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 732.504 Td /F1 9.8 Tf [(Mouse IgG)] TJ ET 0.267 0.267 0.267 rg 392.466 742.359 188.409 0.750 re f 392.466 726.478 0.750 16.631 re f 580.125 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 732.504 Td /F1 9.8 Tf [(Alpha Diagnostic \(#20008-250\))] TJ ET 0.267 0.267 0.267 rg 26.625 726.478 108.898 0.750 re f 26.625 707.646 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 716.623 Td /F1 9.8 Tf [(2B7)] TJ ET 0.267 0.267 0.267 rg 134.773 726.478 139.926 0.750 re f 134.773 707.646 0.750 19.582 re f 0.271 0.267 0.267 rg BT 140.023 716.623 Td /F1 9.8 Tf [(HTT aa1-17)] TJ ET 0.267 0.267 0.267 rg 273.949 726.478 119.267 0.750 re f 273.949 707.646 0.750 19.582 re f 0.271 0.267 0.267 rg BT 279.199 716.623 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 726.478 188.409 0.750 re f 392.466 707.646 0.750 19.582 re f 580.125 707.646 0.750 19.582 re f BT 397.716 714.711 Td /F4 8.7 Tf [(25)] TJ ET 26.625 707.646 108.898 0.750 re f 26.625 688.814 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 697.791 Td /F1 9.8 Tf [(S830)] TJ ET 0.267 0.267 0.267 rg 134.773 707.646 139.926 0.750 re f 134.773 688.814 0.750 19.582 re f 0.271 0.267 0.267 rg BT 140.023 697.791 Td /F1 9.8 Tf [(Exon1 \(53Q\))] TJ ET 0.267 0.267 0.267 rg 273.949 707.646 119.267 0.750 re f 273.949 688.814 0.750 19.582 re f 0.271 0.267 0.267 rg BT 279.199 697.791 Td /F1 9.8 Tf [(Polyclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 707.646 188.409 0.750 re f 392.466 688.814 0.750 19.582 re f 580.125 688.814 0.750 19.582 re f BT 397.716 695.879 Td /F4 8.7 Tf [(26)] TJ ET 26.625 688.814 108.898 0.750 re f 26.625 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 678.959 Td /F1 9.8 Tf [(3B5H10)] TJ ET 0.267 0.267 0.267 rg 134.773 688.814 139.926 0.750 re f 134.773 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 678.959 Td /F1 9.8 Tf [(N171 \(65Q\))] TJ ET 0.267 0.267 0.267 rg 273.949 688.814 119.267 0.750 re f 273.949 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 678.959 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 688.814 188.409 0.750 re f 392.466 672.932 0.750 16.631 re f 580.125 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 678.959 Td /F1 9.8 Tf [(Sigma-Aldrich \(P1874\))] TJ ET 0.267 0.267 0.267 rg 26.625 672.932 108.898 0.750 re f 26.625 654.100 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 663.077 Td /F1 9.8 Tf [(MW1)] TJ ET 0.267 0.267 0.267 rg 134.773 672.932 139.926 0.750 re f 134.773 654.100 0.750 19.582 re f 0.271 0.267 0.267 rg BT 140.023 663.077 Td /F1 9.8 Tf [(PolyQ)] TJ ET 0.267 0.267 0.267 rg 273.949 672.932 119.267 0.750 re f 273.949 654.100 0.750 19.582 re f 0.271 0.267 0.267 rg BT 279.199 663.077 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 672.932 188.409 0.750 re f 392.466 654.100 0.750 19.582 re f 580.125 654.100 0.750 19.582 re f BT 397.716 661.166 Td /F4 8.7 Tf [(23)] TJ ET 26.625 654.100 108.898 0.750 re f 26.625 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 644.245 Td /F1 9.8 Tf [(MAB2166)] TJ ET 0.267 0.267 0.267 rg 134.773 654.100 139.926 0.750 re f 134.773 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 644.245 Td /F1 9.8 Tf [(HTT aa443-457)] TJ ET 0.267 0.267 0.267 rg 273.949 654.100 119.267 0.750 re f 273.949 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 644.245 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 654.100 188.409 0.750 re f 392.466 638.219 0.750 16.631 re f 580.125 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 644.245 Td /F1 9.8 Tf [(Millipore \(MAB2166\))] TJ ET 0.267 0.267 0.267 rg 26.625 638.219 108.898 0.750 re f 26.625 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 628.364 Td /F1 9.8 Tf [(CASP6)] TJ ET 0.267 0.267 0.267 rg 134.773 638.219 139.926 0.750 re f 134.773 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 628.364 Td /F1 9.8 Tf [(CASP6 cleavage site)] TJ ET 0.267 0.267 0.267 rg 273.949 638.219 119.267 0.750 re f 273.949 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 628.364 Td /F1 9.8 Tf [(Polyclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 638.219 188.409 0.750 re f 392.466 622.338 0.750 16.631 re f 580.125 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 628.364 Td /F1 9.8 Tf [(Cell Signalling \(#9762\))] TJ ET 0.267 0.267 0.267 rg 26.625 622.338 108.898 0.750 re f 26.625 606.457 108.898 0.750 re f 26.625 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 612.483 Td /F1 9.8 Tf [(Beta-ACTIN)] TJ ET 0.267 0.267 0.267 rg 134.773 622.338 139.926 0.750 re f 134.773 606.457 139.926 0.750 re f 134.773 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 612.483 Td /F1 9.8 Tf [(ACTIN aa1-15)] TJ ET 0.267 0.267 0.267 rg 273.949 622.338 119.267 0.750 re f 273.949 606.457 119.267 0.750 re f 273.949 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 612.483 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 622.338 188.409 0.750 re f 392.466 606.457 188.409 0.750 re f 392.466 606.457 0.750 16.631 re f 580.125 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 612.483 Td /F1 9.8 Tf [(Abcam \(ab6276\))] TJ ET q 26.250 547.345 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 550.093 Td /F1 9.8 Tf [(Table 2: Secondary Antibodies)] TJ ET Q 1.000 1.000 1.000 rg 26.250 494.303 555.000 45.542 re f 0.965 0.965 0.965 rg 27.000 526.816 187.143 12.280 re f 0.267 0.267 0.267 rg 26.625 538.720 187.518 0.750 re f 26.625 526.441 0.750 13.030 re f 0.271 0.267 0.267 rg BT 31.500 529.572 Td /F4 9.8 Tf [(Antibody)] TJ ET 0.965 0.965 0.965 rg 214.143 526.816 198.531 12.280 re f 0.267 0.267 0.267 rg 214.143 538.720 198.531 0.750 re f 0.271 0.267 0.267 rg BT 218.643 529.572 Td /F4 9.8 Tf [(Description)] TJ ET 0.965 0.965 0.965 rg 412.675 526.816 167.825 12.280 re f 0.267 0.267 0.267 rg 412.675 538.720 168.200 0.750 re f 580.125 526.441 0.750 13.030 re f 0.271 0.267 0.267 rg BT 417.175 529.572 Td /F4 9.8 Tf [(Source)] TJ ET 0.267 0.267 0.267 rg 26.625 526.441 187.893 0.750 re f 26.625 510.559 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 516.586 Td /F1 9.8 Tf [(Anti-mouse HRP)] TJ ET 0.267 0.267 0.267 rg 213.768 526.441 199.281 0.750 re f 213.768 510.559 0.750 16.631 re f 0.271 0.267 0.267 rg BT 219.018 516.586 Td /F1 9.8 Tf [(Polyclonal-rabbit)] TJ ET 0.267 0.267 0.267 rg 412.300 526.441 168.575 0.750 re f 412.300 510.559 0.750 16.631 re f 580.125 510.559 0.750 16.631 re f 0.271 0.267 0.267 rg BT 417.550 516.586 Td /F1 9.8 Tf [(DAKO \(P0260\))] TJ ET 0.267 0.267 0.267 rg 26.625 510.559 187.893 0.750 re f 26.625 494.678 187.893 0.750 re f 26.625 494.678 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 500.704 Td /F1 9.8 Tf [(Anti-rabbit HRP)] TJ ET 0.267 0.267 0.267 rg 213.768 510.559 199.281 0.750 re f 213.768 494.678 199.281 0.750 re f 213.768 494.678 0.750 16.631 re f 0.271 0.267 0.267 rg BT 219.018 500.704 Td /F1 9.8 Tf [(Polyclonal-swine)] TJ ET 0.267 0.267 0.267 rg 412.300 510.559 168.575 0.750 re f 412.300 494.678 168.575 0.750 re f 412.300 494.678 0.750 16.631 re f 580.125 494.678 0.750 16.631 re f 0.271 0.267 0.267 rg BT 417.550 500.704 Td /F1 9.8 Tf [(DAKO \(P0449\))] TJ ET BT 26.250 422.582 Td /F4 12.0 Tf [(RESULTS)] TJ ET BT 26.250 402.627 Td /F6 9.8 Tf [(Casp6)] TJ ET BT 55.510 406.516 Td /F6 8.7 Tf [(/)] TJ ET BT 67.556 402.627 Td /F4 9.8 Tf [(mice show no CASP6 expression in the brain.)] TJ ET BT 279.414 402.627 Td /F1 9.8 Tf [( Exons 2-5 of the )] TJ ET BT 355.835 402.627 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 384.012 402.627 Td /F1 9.8 Tf [( gene, which encodes the catalytic domain of )] TJ ET BT 26.250 390.723 Td /F1 9.8 Tf [(the caspase-6 \(CASP6\) protein, were removed to generate )] TJ ET BT 283.114 390.723 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 311.291 394.611 Td /F1 8.7 Tf [(/)] TJ ET BT 323.338 390.723 Td /F1 9.8 Tf [( mice. )] TJ ET BT 352.052 390.723 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 380.229 394.611 Td /F1 8.7 Tf [(/ )] TJ ET BT 394.685 390.723 Td /F1 9.8 Tf [(mice are viable and fertile, and do not )] TJ ET BT 26.250 378.818 Td /F1 9.8 Tf [(show any overt phenotypes )] TJ ET 0.267 0.267 0.267 rg BT 147.647 380.325 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 157.285 378.818 Td /F1 9.8 Tf [(. We began by performing western blots to ensure that these mice do not express CASP6 and to )] TJ ET BT 26.250 366.913 Td /F1 9.8 Tf [(verify the genotypes. Protein lysates were prepared from half brains of 2 month old wild type and )] TJ ET BT 445.149 366.913 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 473.327 370.801 Td /F1 8.7 Tf [(/ )] TJ ET BT 487.783 366.913 Td /F1 9.8 Tf [(mice and analysed )] TJ ET BT 26.250 355.008 Td /F1 9.8 Tf [(by western blotting using an anti-CASP6 antibody. As previously reported )] TJ ET 0.267 0.267 0.267 rg BT 345.436 356.516 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 355.073 355.008 Td /F1 9.8 Tf [(, we found that the )] TJ ET BT 438.007 355.008 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 466.184 358.897 Td /F1 8.7 Tf [(/ )] TJ ET BT 480.640 355.008 Td /F1 9.8 Tf [(mice constitutively )] TJ ET BT 26.250 343.104 Td /F1 9.8 Tf [(lacked CASP6 as compared to wild type littermates \(Fig. 1\).)] TJ ET 0.965 0.965 0.965 rg 26.250 167.609 555.000 165.614 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 333.223 m 581.250 333.223 l 581.250 332.473 l 26.250 332.473 l f 26.250 167.609 m 581.250 167.609 l 581.250 168.359 l 26.250 168.359 l f q 225.000 0 0 72.000 35.250 251.473 cm /I3 Do Q q 35.250 178.859 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 235.949 Td /F4 9.8 Tf [(Fig. 1: Figure 1. CASP6 expression in Casp6/and wild type brains)] TJ ET BT 35.250 216.579 Td /F1 9.8 Tf [(Western blotting of total brain lysates from 2 month old mice using an anti-CASP6 antibody shows that CASP6 was absent )] TJ ET BT 35.250 202.843 Td /F1 9.8 Tf [(in Casp6/ samples when compared to wild type \(Casp6+/+\) samples. Beta-ACTIN served as a loading control. ID = )] TJ ET BT 35.250 189.106 Td /F1 9.8 Tf [(Immunodetection.)] TJ ET Q BT 26.250 150.585 Td /F4 9.8 Tf [(Genetic ablation of )] TJ ET BT 116.730 150.585 Td /F6 9.8 Tf [(Casp6)] TJ ET BT 145.990 150.585 Td /F4 9.8 Tf [( does not modify HTT proteolysis in )] TJ ET BT 314.479 150.585 Td /F6 9.8 Tf [(Hdh)] TJ ET BT 333.433 150.585 Td /F4 9.8 Tf [(Q150 knock-in mice.)] TJ ET BT 427.726 150.585 Td /F1 9.8 Tf [( To identify N-terminal HTT )] TJ ET BT 26.250 138.681 Td /F1 9.8 Tf [(fragments in the brains of )] TJ ET BT 138.970 138.681 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 156.851 138.681 Td /F1 9.8 Tf [(Q150 knock-in mice, we previously used a combination of immunoprecipitation and western )] TJ ET BT 26.250 126.776 Td /F1 9.8 Tf [(blotting that identified 14 prominent N-terminal fragments, which in addition to the full-length protein, could be readily detected in )] TJ ET BT 26.250 114.871 Td /F1 9.8 Tf [(the cytoplasmic but not nuclear fractions. These fragments were present at all ages and did not arise as a consequence of the )] TJ ET BT 26.250 102.966 Td /F1 9.8 Tf [(pathogenic process. Furthermore, in an attempt to determine the source of some of the N-terminal HTT fragments, proteolytic )] TJ ET BT 26.250 91.062 Td /F1 9.8 Tf [(digests were performed with caspase enzymes which indicated that )] TJ ET BT 319.949 91.062 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 369.801 91.062 Td /F1 9.8 Tf [( may terminate at the caspase-6 HTT-586 )] TJ ET BT 26.250 79.157 Td /F1 9.8 Tf [(cleavage site )] TJ ET 0.267 0.267 0.267 rg BT 85.862 80.664 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 90.680 79.157 Td /F1 9.8 Tf [(. In order to corroborate this observation, we investigated whether the presence of this proteolytic fragment, )] TJ ET BT 26.250 67.252 Td /F1 9.8 Tf [(along with all other observed N-terminal HTT fragments, was modulated upon the genetic ablation of CASP6.)] TJ ET BT 496.619 67.252 Td /F5 9.8 Tf [( Casp6)] TJ ET BT 527.507 71.140 Td /F1 8.7 Tf [(+/ )] TJ ET BT 542.206 67.252 Td /F1 9.8 Tf [(males )] TJ ET BT 26.250 55.347 Td /F1 9.8 Tf [(were bred to )] TJ ET BT 83.151 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 101.032 59.236 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 129.702 55.347 Td /F1 9.8 Tf [(::)] TJ ET BT 135.123 55.347 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 163.300 59.236 Td /F1 8.7 Tf [(+/ )] TJ ET BT 177.999 55.347 Td /F1 9.8 Tf [(females to generate )] TJ ET BT 266.880 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 284.762 59.236 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 315.840 55.347 Td /F1 9.8 Tf [(mice on either a )] TJ ET BT 387.912 55.347 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 416.090 55.347 Td /F1 9.8 Tf [( wild type )] TJ ET BT 459.438 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 477.320 59.236 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 505.989 55.347 Td /F1 9.8 Tf [(::)] TJ ET BT 511.410 55.347 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 539.588 59.236 Td /F1 8.7 Tf [(+/+ )] TJ ET BT 554.529 55.347 Td /F1 9.8 Tf [(\()] TJ ET BT 557.776 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 26.250 47.331 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 54.919 43.443 Td /F1 9.8 Tf [(\) or )] TJ ET BT 72.255 43.443 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 100.432 43.443 Td /F1 9.8 Tf [( knock-out )] TJ ET BT 148.120 43.443 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 166.001 47.331 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 194.670 43.443 Td /F1 9.8 Tf [(::)] TJ ET BT 200.091 43.443 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 228.269 47.331 Td /F1 8.7 Tf [(/ )] TJ ET BT 242.725 43.443 Td /F1 9.8 Tf [(\(Dble\) background. We obtained at least 12 mice for each genotype and all )] TJ ET Q q 15.000 29.157 577.500 747.843 re W n q 26.250 763.264 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 766.011 Td /F1 9.8 Tf [(Table 1: Primary Antibodies)] TJ ET Q 1.000 1.000 1.000 rg 26.250 606.082 555.000 149.682 re f 0.965 0.965 0.965 rg 27.000 742.734 108.148 12.280 re f 0.267 0.267 0.267 rg 26.625 754.639 108.523 0.750 re f 26.625 742.359 0.750 13.030 re f 0.271 0.267 0.267 rg BT 31.500 745.490 Td /F4 9.8 Tf [(Antibody)] TJ ET 0.965 0.965 0.965 rg 135.148 742.734 139.176 12.280 re f 0.267 0.267 0.267 rg 135.148 754.639 139.176 0.750 re f 0.271 0.267 0.267 rg BT 139.648 745.490 Td /F4 9.8 Tf [(Epitope)] TJ ET 0.965 0.965 0.965 rg 274.324 742.734 118.517 12.280 re f 0.267 0.267 0.267 rg 274.324 754.639 118.517 0.750 re f 0.271 0.267 0.267 rg BT 278.824 745.490 Td /F4 9.8 Tf [(Description)] TJ ET 0.965 0.965 0.965 rg 392.841 742.734 187.659 12.280 re f 0.267 0.267 0.267 rg 392.841 754.639 188.034 0.750 re f 580.125 742.359 0.750 13.030 re f 0.271 0.267 0.267 rg BT 397.341 745.490 Td /F4 9.8 Tf [(Source)] TJ ET 0.267 0.267 0.267 rg 26.625 742.359 108.898 0.750 re f 26.625 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 732.504 Td /F1 9.8 Tf [(mIgG)] TJ ET 0.267 0.267 0.267 rg 134.773 742.359 139.926 0.750 re f 134.773 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 732.504 Td /F1 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg 273.949 742.359 119.267 0.750 re f 273.949 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 732.504 Td /F1 9.8 Tf [(Mouse IgG)] TJ ET 0.267 0.267 0.267 rg 392.466 742.359 188.409 0.750 re f 392.466 726.478 0.750 16.631 re f 580.125 726.478 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 732.504 Td /F1 9.8 Tf [(Alpha Diagnostic \(#20008-250\))] TJ ET 0.267 0.267 0.267 rg 26.625 726.478 108.898 0.750 re f 26.625 707.646 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 716.623 Td /F1 9.8 Tf [(2B7)] TJ ET 0.267 0.267 0.267 rg 134.773 726.478 139.926 0.750 re f 134.773 707.646 0.750 19.582 re f 0.271 0.267 0.267 rg BT 140.023 716.623 Td /F1 9.8 Tf [(HTT aa1-17)] TJ ET 0.267 0.267 0.267 rg 273.949 726.478 119.267 0.750 re f 273.949 707.646 0.750 19.582 re f 0.271 0.267 0.267 rg BT 279.199 716.623 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 726.478 188.409 0.750 re f 392.466 707.646 0.750 19.582 re f 580.125 707.646 0.750 19.582 re f BT 397.716 714.711 Td /F4 8.7 Tf [(25)] TJ ET 26.625 707.646 108.898 0.750 re f 26.625 688.814 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 697.791 Td /F1 9.8 Tf [(S830)] TJ ET 0.267 0.267 0.267 rg 134.773 707.646 139.926 0.750 re f 134.773 688.814 0.750 19.582 re f 0.271 0.267 0.267 rg BT 140.023 697.791 Td /F1 9.8 Tf [(Exon1 \(53Q\))] TJ ET 0.267 0.267 0.267 rg 273.949 707.646 119.267 0.750 re f 273.949 688.814 0.750 19.582 re f 0.271 0.267 0.267 rg BT 279.199 697.791 Td /F1 9.8 Tf [(Polyclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 707.646 188.409 0.750 re f 392.466 688.814 0.750 19.582 re f 580.125 688.814 0.750 19.582 re f BT 397.716 695.879 Td /F4 8.7 Tf [(26)] TJ ET 26.625 688.814 108.898 0.750 re f 26.625 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 678.959 Td /F1 9.8 Tf [(3B5H10)] TJ ET 0.267 0.267 0.267 rg 134.773 688.814 139.926 0.750 re f 134.773 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 678.959 Td /F1 9.8 Tf [(N171 \(65Q\))] TJ ET 0.267 0.267 0.267 rg 273.949 688.814 119.267 0.750 re f 273.949 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 678.959 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 688.814 188.409 0.750 re f 392.466 672.932 0.750 16.631 re f 580.125 672.932 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 678.959 Td /F1 9.8 Tf [(Sigma-Aldrich \(P1874\))] TJ ET 0.267 0.267 0.267 rg 26.625 672.932 108.898 0.750 re f 26.625 654.100 0.750 19.582 re f 0.271 0.267 0.267 rg BT 31.875 663.077 Td /F1 9.8 Tf [(MW1)] TJ ET 0.267 0.267 0.267 rg 134.773 672.932 139.926 0.750 re f 134.773 654.100 0.750 19.582 re f 0.271 0.267 0.267 rg BT 140.023 663.077 Td /F1 9.8 Tf [(PolyQ)] TJ ET 0.267 0.267 0.267 rg 273.949 672.932 119.267 0.750 re f 273.949 654.100 0.750 19.582 re f 0.271 0.267 0.267 rg BT 279.199 663.077 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 672.932 188.409 0.750 re f 392.466 654.100 0.750 19.582 re f 580.125 654.100 0.750 19.582 re f BT 397.716 661.166 Td /F4 8.7 Tf [(23)] TJ ET 26.625 654.100 108.898 0.750 re f 26.625 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 644.245 Td /F1 9.8 Tf [(MAB2166)] TJ ET 0.267 0.267 0.267 rg 134.773 654.100 139.926 0.750 re f 134.773 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 644.245 Td /F1 9.8 Tf [(HTT aa443-457)] TJ ET 0.267 0.267 0.267 rg 273.949 654.100 119.267 0.750 re f 273.949 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 644.245 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 654.100 188.409 0.750 re f 392.466 638.219 0.750 16.631 re f 580.125 638.219 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 644.245 Td /F1 9.8 Tf [(Millipore \(MAB2166\))] TJ ET 0.267 0.267 0.267 rg 26.625 638.219 108.898 0.750 re f 26.625 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 628.364 Td /F1 9.8 Tf [(CASP6)] TJ ET 0.267 0.267 0.267 rg 134.773 638.219 139.926 0.750 re f 134.773 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 628.364 Td /F1 9.8 Tf [(CASP6 cleavage site)] TJ ET 0.267 0.267 0.267 rg 273.949 638.219 119.267 0.750 re f 273.949 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 628.364 Td /F1 9.8 Tf [(Polyclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 638.219 188.409 0.750 re f 392.466 622.338 0.750 16.631 re f 580.125 622.338 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 628.364 Td /F1 9.8 Tf [(Cell Signalling \(#9762\))] TJ ET 0.267 0.267 0.267 rg 26.625 622.338 108.898 0.750 re f 26.625 606.457 108.898 0.750 re f 26.625 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 612.483 Td /F1 9.8 Tf [(Beta-ACTIN)] TJ ET 0.267 0.267 0.267 rg 134.773 622.338 139.926 0.750 re f 134.773 606.457 139.926 0.750 re f 134.773 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 140.023 612.483 Td /F1 9.8 Tf [(ACTIN aa1-15)] TJ ET 0.267 0.267 0.267 rg 273.949 622.338 119.267 0.750 re f 273.949 606.457 119.267 0.750 re f 273.949 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 279.199 612.483 Td /F1 9.8 Tf [(Monoclonal)] TJ ET 0.267 0.267 0.267 rg 392.466 622.338 188.409 0.750 re f 392.466 606.457 188.409 0.750 re f 392.466 606.457 0.750 16.631 re f 580.125 606.457 0.750 16.631 re f 0.271 0.267 0.267 rg BT 397.716 612.483 Td /F1 9.8 Tf [(Abcam \(ab6276\))] TJ ET q 26.250 547.345 555.000 13.736 re W n 0.271 0.267 0.267 rg BT 26.250 550.093 Td /F1 9.8 Tf [(Table 2: Secondary Antibodies)] TJ ET Q 1.000 1.000 1.000 rg 26.250 494.303 555.000 45.542 re f 0.965 0.965 0.965 rg 27.000 526.816 187.143 12.280 re f 0.267 0.267 0.267 rg 26.625 538.720 187.518 0.750 re f 26.625 526.441 0.750 13.030 re f 0.271 0.267 0.267 rg BT 31.500 529.572 Td /F4 9.8 Tf [(Antibody)] TJ ET 0.965 0.965 0.965 rg 214.143 526.816 198.531 12.280 re f 0.267 0.267 0.267 rg 214.143 538.720 198.531 0.750 re f 0.271 0.267 0.267 rg BT 218.643 529.572 Td /F4 9.8 Tf [(Description)] TJ ET 0.965 0.965 0.965 rg 412.675 526.816 167.825 12.280 re f 0.267 0.267 0.267 rg 412.675 538.720 168.200 0.750 re f 580.125 526.441 0.750 13.030 re f 0.271 0.267 0.267 rg BT 417.175 529.572 Td /F4 9.8 Tf [(Source)] TJ ET 0.267 0.267 0.267 rg 26.625 526.441 187.893 0.750 re f 26.625 510.559 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 516.586 Td /F1 9.8 Tf [(Anti-mouse HRP)] TJ ET 0.267 0.267 0.267 rg 213.768 526.441 199.281 0.750 re f 213.768 510.559 0.750 16.631 re f 0.271 0.267 0.267 rg BT 219.018 516.586 Td /F1 9.8 Tf [(Polyclonal-rabbit)] TJ ET 0.267 0.267 0.267 rg 412.300 526.441 168.575 0.750 re f 412.300 510.559 0.750 16.631 re f 580.125 510.559 0.750 16.631 re f 0.271 0.267 0.267 rg BT 417.550 516.586 Td /F1 9.8 Tf [(DAKO \(P0260\))] TJ ET 0.267 0.267 0.267 rg 26.625 510.559 187.893 0.750 re f 26.625 494.678 187.893 0.750 re f 26.625 494.678 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 500.704 Td /F1 9.8 Tf [(Anti-rabbit HRP)] TJ ET 0.267 0.267 0.267 rg 213.768 510.559 199.281 0.750 re f 213.768 494.678 199.281 0.750 re f 213.768 494.678 0.750 16.631 re f 0.271 0.267 0.267 rg BT 219.018 500.704 Td /F1 9.8 Tf [(Polyclonal-swine)] TJ ET 0.267 0.267 0.267 rg 412.300 510.559 168.575 0.750 re f 412.300 494.678 168.575 0.750 re f 412.300 494.678 0.750 16.631 re f 580.125 494.678 0.750 16.631 re f 0.271 0.267 0.267 rg BT 417.550 500.704 Td /F1 9.8 Tf [(DAKO \(P0449\))] TJ ET BT 26.250 422.582 Td /F4 12.0 Tf [(RESULTS)] TJ ET BT 26.250 402.627 Td /F6 9.8 Tf [(Casp6)] TJ ET BT 55.510 406.516 Td /F6 8.7 Tf [(/)] TJ ET BT 67.556 402.627 Td /F4 9.8 Tf [(mice show no CASP6 expression in the brain.)] TJ ET BT 279.414 402.627 Td /F1 9.8 Tf [( Exons 2-5 of the )] TJ ET BT 355.835 402.627 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 384.012 402.627 Td /F1 9.8 Tf [( gene, which encodes the catalytic domain of )] TJ ET BT 26.250 390.723 Td /F1 9.8 Tf [(the caspase-6 \(CASP6\) protein, were removed to generate )] TJ ET BT 283.114 390.723 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 311.291 394.611 Td /F1 8.7 Tf [(/)] TJ ET BT 323.338 390.723 Td /F1 9.8 Tf [( mice. )] TJ ET BT 352.052 390.723 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 380.229 394.611 Td /F1 8.7 Tf [(/ )] TJ ET BT 394.685 390.723 Td /F1 9.8 Tf [(mice are viable and fertile, and do not )] TJ ET BT 26.250 378.818 Td /F1 9.8 Tf [(show any overt phenotypes )] TJ ET 0.267 0.267 0.267 rg BT 147.647 380.325 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 157.285 378.818 Td /F1 9.8 Tf [(. We began by performing western blots to ensure that these mice do not express CASP6 and to )] TJ ET BT 26.250 366.913 Td /F1 9.8 Tf [(verify the genotypes. Protein lysates were prepared from half brains of 2 month old wild type and )] TJ ET BT 445.149 366.913 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 473.327 370.801 Td /F1 8.7 Tf [(/ )] TJ ET BT 487.783 366.913 Td /F1 9.8 Tf [(mice and analysed )] TJ ET BT 26.250 355.008 Td /F1 9.8 Tf [(by western blotting using an anti-CASP6 antibody. As previously reported )] TJ ET 0.267 0.267 0.267 rg BT 345.436 356.516 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 355.073 355.008 Td /F1 9.8 Tf [(, we found that the )] TJ ET BT 438.007 355.008 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 466.184 358.897 Td /F1 8.7 Tf [(/ )] TJ ET BT 480.640 355.008 Td /F1 9.8 Tf [(mice constitutively )] TJ ET BT 26.250 343.104 Td /F1 9.8 Tf [(lacked CASP6 as compared to wild type littermates \(Fig. 1\).)] TJ ET 0.965 0.965 0.965 rg 26.250 167.609 555.000 165.614 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 333.223 m 581.250 333.223 l 581.250 332.473 l 26.250 332.473 l f 26.250 167.609 m 581.250 167.609 l 581.250 168.359 l 26.250 168.359 l f q 225.000 0 0 72.000 35.250 251.473 cm /I3 Do Q q 35.250 178.859 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 235.949 Td /F4 9.8 Tf [(Fig. 1: Figure 1. CASP6 expression in Casp6/and wild type brains)] TJ ET BT 35.250 216.579 Td /F1 9.8 Tf [(Western blotting of total brain lysates from 2 month old mice using an anti-CASP6 antibody shows that CASP6 was absent )] TJ ET BT 35.250 202.843 Td /F1 9.8 Tf [(in Casp6/ samples when compared to wild type \(Casp6+/+\) samples. Beta-ACTIN served as a loading control. ID = )] TJ ET BT 35.250 189.106 Td /F1 9.8 Tf [(Immunodetection.)] TJ ET Q BT 26.250 150.585 Td /F4 9.8 Tf [(Genetic ablation of )] TJ ET BT 116.730 150.585 Td /F6 9.8 Tf [(Casp6)] TJ ET BT 145.990 150.585 Td /F4 9.8 Tf [( does not modify HTT proteolysis in )] TJ ET BT 314.479 150.585 Td /F6 9.8 Tf [(Hdh)] TJ ET BT 333.433 150.585 Td /F4 9.8 Tf [(Q150 knock-in mice.)] TJ ET BT 427.726 150.585 Td /F1 9.8 Tf [( To identify N-terminal HTT )] TJ ET BT 26.250 138.681 Td /F1 9.8 Tf [(fragments in the brains of )] TJ ET BT 138.970 138.681 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 156.851 138.681 Td /F1 9.8 Tf [(Q150 knock-in mice, we previously used a combination of immunoprecipitation and western )] TJ ET BT 26.250 126.776 Td /F1 9.8 Tf [(blotting that identified 14 prominent N-terminal fragments, which in addition to the full-length protein, could be readily detected in )] TJ ET BT 26.250 114.871 Td /F1 9.8 Tf [(the cytoplasmic but not nuclear fractions. These fragments were present at all ages and did not arise as a consequence of the )] TJ ET BT 26.250 102.966 Td /F1 9.8 Tf [(pathogenic process. Furthermore, in an attempt to determine the source of some of the N-terminal HTT fragments, proteolytic )] TJ ET BT 26.250 91.062 Td /F1 9.8 Tf [(digests were performed with caspase enzymes which indicated that )] TJ ET BT 319.949 91.062 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 369.801 91.062 Td /F1 9.8 Tf [( may terminate at the caspase-6 HTT-586 )] TJ ET BT 26.250 79.157 Td /F1 9.8 Tf [(cleavage site )] TJ ET 0.267 0.267 0.267 rg BT 85.862 80.664 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 90.680 79.157 Td /F1 9.8 Tf [(. In order to corroborate this observation, we investigated whether the presence of this proteolytic fragment, )] TJ ET BT 26.250 67.252 Td /F1 9.8 Tf [(along with all other observed N-terminal HTT fragments, was modulated upon the genetic ablation of CASP6.)] TJ ET BT 496.619 67.252 Td /F5 9.8 Tf [( Casp6)] TJ ET BT 527.507 71.140 Td /F1 8.7 Tf [(+/ )] TJ ET BT 542.206 67.252 Td /F1 9.8 Tf [(males )] TJ ET BT 26.250 55.347 Td /F1 9.8 Tf [(were bred to )] TJ ET BT 83.151 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 101.032 59.236 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 129.702 55.347 Td /F1 9.8 Tf [(::)] TJ ET BT 135.123 55.347 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 163.300 59.236 Td /F1 8.7 Tf [(+/ )] TJ ET BT 177.999 55.347 Td /F1 9.8 Tf [(females to generate )] TJ ET BT 266.880 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 284.762 59.236 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 315.840 55.347 Td /F1 9.8 Tf [(mice on either a )] TJ ET BT 387.912 55.347 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 416.090 55.347 Td /F1 9.8 Tf [( wild type )] TJ ET BT 459.438 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 477.320 59.236 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 505.989 55.347 Td /F1 9.8 Tf [(::)] TJ ET BT 511.410 55.347 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 539.588 59.236 Td /F1 8.7 Tf [(+/+ )] TJ ET BT 554.529 55.347 Td /F1 9.8 Tf [(\()] TJ ET BT 557.776 55.347 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 26.250 47.331 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 54.919 43.443 Td /F1 9.8 Tf [(\) or )] TJ ET BT 72.255 43.443 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 100.432 43.443 Td /F1 9.8 Tf [( knock-out )] TJ ET BT 148.120 43.443 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 166.001 47.331 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 194.670 43.443 Td /F1 9.8 Tf [(::)] TJ ET BT 200.091 43.443 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 228.269 47.331 Td /F1 8.7 Tf [(/ )] TJ ET BT 242.725 43.443 Td /F1 9.8 Tf [(\(Dble\) background. We obtained at least 12 mice for each genotype and all )] TJ ET Q q 225.000 0 0 72.000 35.250 251.473 cm /I3 Do 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 271 0 obj << /Type /Font /Subtype /Type1 /Name /F6 /BaseFont /Helvetica-BoldOblique /Encoding /WinAnsiEncoding >> endobj 272 0 obj << /Type /Annot /Subtype /Link /A 273 0 R /Border [0 0 0] /H /I /Rect [ 397.7158 713.9094 407.3531 722.7277 ] >> endobj 273 0 obj << /Type /Action >> endobj 274 0 obj << /Type /Annot /Subtype /Link /A 275 0 R /Border [0 0 0] /H /I /Rect [ 397.7158 695.0774 407.3531 703.8957 ] >> endobj 275 0 obj << /Type /Action >> endobj 276 0 obj << /Type /Annot /Subtype /Link /A 277 0 R /Border [0 0 0] /H /I /Rect [ 397.7158 660.3642 407.3531 669.1825 ] >> endobj 277 0 obj << /Type /Action >> endobj 278 0 obj << /Type /Annot /Subtype /Link /A 279 0 R /Border [0 0 0] /H /I /Rect [ 147.6472 379.5234 157.2846 388.3417 ] >> endobj 279 0 obj << /Type /Action >> endobj 280 0 obj << /Type /Annot /Subtype /Link /A 281 0 R /Border [0 0 0] /H /I /Rect [ 345.4357 355.7139 355.0731 364.5322 ] >> endobj 281 0 obj << /Type /Action >> endobj 282 0 obj << /Type /Annot /Subtype /Link /A 283 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 251.4727 260.2500 323.4727 ] >> endobj 283 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/Fig1-JPEG-300x961.jpg) >> endobj 284 0 obj << /Type /XObject /Subtype /Image /Width 300 /Height 96 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 8734>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   `," }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?R(A.w $vIOwgڬMw\Cc*4.aF03^XCiVW &E2#5c/v߇l<=cl]mjب؁m|Q'@#v@X𾟧Y~uGEkVAį$Q >$ kj_е\k:m"8TnYg-D:g>*;.xþ"ttmW73͢mKo)rޭBX> 95,1iM]OaxbxX[y!YV@HFwڷ)ꏧh?/"ЭE:h:i#M_JB^BX@1`8|?_ÏxNJKvBrm71cug=-+O4"v9rNs3O#|ikFKҦu-S8ÜH 2^ueo}sw;%@>5ן ~h_^f;hD3@s~Қ/xFߊm~V7z , $Ƭe}Cti6ڦkH$fd=I ={? 4o:{&HuX4LG,)@AuAK1Q¢ =>(4w4V AbE(#2xp `z18%m|sWzMcFju~"kQb7Pc>+7& 4}1mQk}\G,*`L8suW5-\?4kw7AEfxkZo4[m'I&lE}bǖ$IkN ;Eb5?kot ^jqC 3טw>^54]GD⺸DB l*3++{輻#9*& BfA}f/opOe,dax y MOмuƷYjr4,2E1P!G[ӼIM]edu$0>;㏆7o=wGmTU 9# !e$%'uKHF 0EP4V|Aӡ-aN0=tx ׼Gyͮc=?cBs~5ȵ|-+xOTs/,{ i-ڑFJpe(M⟅Aeyge'Y|PԖ52cq}3NH,V@w(>>0>)O?Z8*›ǨQuPH\5B!? zl " "sWˑ!si> 67Ҋն4LCl?"a']i,4)'oÿOOį+n+=uHWᦎ,4%߁Ҁ3?j|{>Qo([v i[G0{j 9xX@ğQ_GjFZ K/t4!yK#'v`IE} @2LT^`v%ۊ ~_Mx_D?W>ԡ[+oEOُ^>)>_-ьg֥hV?ï_Er>(:x[>t^Dqּ/mb rڟ%RE>aϴi'o"_ 31;bU% ~ [OMyt*%2ĭ $G . 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Mutant HTT was )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(immunoprecipitated with antibodies that recognize epitopes at the N-terminus \(2B7; S830; or 3B5H10\) \(Fig. 2B-C\) and )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(immunodetected with MW1, an antibody that recognizes the expanded polyQ tract and, therefore, does not detect wild type )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(HTT. A mouse IgG \(mIgG\) antibody was used as a negative control \(Fig. 2A\), and )] TJ ET BT 381.189 700.452 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 409.367 700.452 Td /F1 9.8 Tf [( genotypes were confirmed by western )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(blotting \(Fig. 2E\). In both )] TJ ET BT 135.723 688.548 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 153.605 692.436 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 182.274 688.548 Td /F1 9.8 Tf [( and Dble samples we were able to identify all of the N-terminal fragments identified )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(previously, including )] TJ ET BT 116.740 676.643 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 166.591 676.643 Td /F1 9.8 Tf [( \(the proposed caspase-6 HTT-586 fragment\) )] TJ ET 0.267 0.267 0.267 rg BT 365.462 678.150 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 370.281 676.643 Td /F1 9.8 Tf [(. Therefore, the proteolysis of full-length HTT, to )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(generate )] TJ ET BT 67.444 664.738 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 117.296 664.738 Td /F1 9.8 Tf [( or any other N-terminal HTT fragments was unaltered in the absence of CASP6.)] TJ ET 0.965 0.965 0.965 rg 26.250 83.771 555.000 571.086 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 654.857 m 581.250 654.857 l 581.250 654.107 l 26.250 654.107 l f 26.250 83.771 m 581.250 83.771 l 581.250 84.521 l 26.250 84.521 l f q 388.500 0 0 450.000 35.250 195.107 cm /I4 Do Q q 35.250 95.021 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 179.583 Td /F4 9.8 Tf [(Fig. 2: Figure 2. Analysis of N-terminal HTT fragments in Hdh+/Q150 and Dble mouse brains.)] TJ ET BT 35.250 160.213 Td /F1 9.8 Tf [(Immunoprecipitation of mutant HTT from Hdh+/Q150 or Dble samples at 2 months of age with either: \()] TJ ET BT 475.531 160.213 Td /F4 9.8 Tf [(A)] TJ ET BT 482.570 160.213 Td /F1 9.8 Tf [(\) mIgG \(control\), \()] TJ ET BT 559.498 160.213 Td /F4 9.8 Tf [(B)] TJ ET BT 35.250 146.477 Td /F1 9.8 Tf [(\) 2B7, \()] TJ ET BT 67.220 146.477 Td /F4 9.8 Tf [(C)] TJ ET BT 74.260 146.477 Td /F1 9.8 Tf [(\) S830, or \()] TJ ET BT 123.029 146.477 Td /F4 9.8 Tf [(D)] TJ ET BT 130.069 146.477 Td /F1 9.8 Tf [(\) 3B5H10, and immunodetection with MW1. Fourteen prominent N-terminal fragments can be )] TJ ET BT 35.250 132.741 Td /F1 9.8 Tf [(detected \(Fragments 1-14\), in addition to 3 weaker fragments \(Fragments a-c\), numbered and labelled in panel \(B\). \()] TJ ET BT 537.580 132.741 Td /F4 9.8 Tf [(E)] TJ ET BT 544.083 132.741 Td /F1 9.8 Tf [(\) )] TJ ET BT 35.250 119.005 Td /F1 9.8 Tf [(Casp6 genotypes were confirmed by western blotting, Beta-ACTIN served as a loading control. IP = Immunoprecipitation; )] TJ ET BT 35.250 105.268 Td /F1 9.8 Tf [(ID = Immunodetection; In = Interface between stacking and resolving gel; FL = full-length protein. \(n = 3/genotype\).)] TJ ET Q BT 26.250 66.747 Td /F4 9.8 Tf [(Steady state wild type and mutant HTT levels are not changed in the brains of presymptomatic )] TJ ET BT 466.199 66.747 Td /F6 9.8 Tf [(Hdh)] TJ ET BT 485.153 70.636 Td /F6 8.7 Tf [(+)] TJ ET BT 490.215 70.636 Td /F4 8.7 Tf [(/Q150)] TJ ET BT 513.823 66.747 Td /F4 9.8 Tf [( and Dble )] TJ ET BT 26.250 54.843 Td /F4 9.8 Tf [(mice.)] TJ ET BT 51.181 54.843 Td /F1 9.8 Tf [( To quantify the relative levels of mutant HTT with respect to total HTT, brain lysates from 2 month-old mice were )] TJ ET BT 26.250 42.938 Td /F1 9.8 Tf [(subjected to the highly sensitive TR-FRET assay \(Fig. 3\). This assay uses the labelled antibody pair: 2B7-Terbium cryptate and )] TJ ET Q q 15.000 28.652 577.500 748.348 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(groups were well matched for their CAG repeat size \()] TJ ET BT 255.453 767.476 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 273.334 771.364 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 304.413 767.476 Td /F1 9.8 Tf [(179 7.5 \(S.D\); \(Dble 179 9.5 \(S.D\)\).)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(To compare the pattern of HTT proteolytic fragments, we applied a combination of HTT immunoprecipitation and )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(immunodetection from whole brain lysates prepared from )] TJ ET BT 274.963 736.167 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 292.844 740.055 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 321.514 736.167 Td /F1 9.8 Tf [( and Dble mice aged 2 months \(Fig. 2\). Mutant HTT was )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(immunoprecipitated with antibodies that recognize epitopes at the N-terminus \(2B7; S830; or 3B5H10\) \(Fig. 2B-C\) and )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(immunodetected with MW1, an antibody that recognizes the expanded polyQ tract and, therefore, does not detect wild type )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(HTT. A mouse IgG \(mIgG\) antibody was used as a negative control \(Fig. 2A\), and )] TJ ET BT 381.189 700.452 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 409.367 700.452 Td /F1 9.8 Tf [( genotypes were confirmed by western )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(blotting \(Fig. 2E\). In both )] TJ ET BT 135.723 688.548 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 153.605 692.436 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 182.274 688.548 Td /F1 9.8 Tf [( and Dble samples we were able to identify all of the N-terminal fragments identified )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(previously, including )] TJ ET BT 116.740 676.643 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 166.591 676.643 Td /F1 9.8 Tf [( \(the proposed caspase-6 HTT-586 fragment\) )] TJ ET 0.267 0.267 0.267 rg BT 365.462 678.150 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 370.281 676.643 Td /F1 9.8 Tf [(. Therefore, the proteolysis of full-length HTT, to )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(generate )] TJ ET BT 67.444 664.738 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 117.296 664.738 Td /F1 9.8 Tf [( or any other N-terminal HTT fragments was unaltered in the absence of CASP6.)] TJ ET 0.965 0.965 0.965 rg 26.250 83.771 555.000 571.086 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 654.857 m 581.250 654.857 l 581.250 654.107 l 26.250 654.107 l f 26.250 83.771 m 581.250 83.771 l 581.250 84.521 l 26.250 84.521 l f q 388.500 0 0 450.000 35.250 195.107 cm /I4 Do Q q 35.250 95.021 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 179.583 Td /F4 9.8 Tf [(Fig. 2: Figure 2. Analysis of N-terminal HTT fragments in Hdh+/Q150 and Dble mouse brains.)] TJ ET BT 35.250 160.213 Td /F1 9.8 Tf [(Immunoprecipitation of mutant HTT from Hdh+/Q150 or Dble samples at 2 months of age with either: \()] TJ ET BT 475.531 160.213 Td /F4 9.8 Tf [(A)] TJ ET BT 482.570 160.213 Td /F1 9.8 Tf [(\) mIgG \(control\), \()] TJ ET BT 559.498 160.213 Td /F4 9.8 Tf [(B)] TJ ET BT 35.250 146.477 Td /F1 9.8 Tf [(\) 2B7, \()] TJ ET BT 67.220 146.477 Td /F4 9.8 Tf [(C)] TJ ET BT 74.260 146.477 Td /F1 9.8 Tf [(\) S830, or \()] TJ ET BT 123.029 146.477 Td /F4 9.8 Tf [(D)] TJ ET BT 130.069 146.477 Td /F1 9.8 Tf [(\) 3B5H10, and immunodetection with MW1. Fourteen prominent N-terminal fragments can be )] TJ ET BT 35.250 132.741 Td /F1 9.8 Tf [(detected \(Fragments 1-14\), in addition to 3 weaker fragments \(Fragments a-c\), numbered and labelled in panel \(B\). \()] TJ ET BT 537.580 132.741 Td /F4 9.8 Tf [(E)] TJ ET BT 544.083 132.741 Td /F1 9.8 Tf [(\) )] TJ ET BT 35.250 119.005 Td /F1 9.8 Tf [(Casp6 genotypes were confirmed by western blotting, Beta-ACTIN served as a loading control. IP = Immunoprecipitation; )] TJ ET BT 35.250 105.268 Td /F1 9.8 Tf [(ID = Immunodetection; In = Interface between stacking and resolving gel; FL = full-length protein. \(n = 3/genotype\).)] TJ ET Q BT 26.250 66.747 Td /F4 9.8 Tf [(Steady state wild type and mutant HTT levels are not changed in the brains of presymptomatic )] TJ ET BT 466.199 66.747 Td /F6 9.8 Tf [(Hdh)] TJ ET BT 485.153 70.636 Td /F6 8.7 Tf [(+)] TJ ET BT 490.215 70.636 Td /F4 8.7 Tf [(/Q150)] TJ ET BT 513.823 66.747 Td /F4 9.8 Tf [( and Dble )] TJ ET BT 26.250 54.843 Td /F4 9.8 Tf [(mice.)] TJ ET BT 51.181 54.843 Td /F1 9.8 Tf [( To quantify the relative levels of mutant HTT with respect to total HTT, brain lysates from 2 month-old mice were )] TJ ET BT 26.250 42.938 Td /F1 9.8 Tf [(subjected to the highly sensitive TR-FRET assay \(Fig. 3\). This assay uses the labelled antibody pair: 2B7-Terbium cryptate and )] TJ ET Q q 15.000 28.652 577.500 748.348 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(groups were well matched for their CAG repeat size \()] TJ ET BT 255.453 767.476 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 273.334 771.364 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 304.413 767.476 Td /F1 9.8 Tf [(179 7.5 \(S.D\); \(Dble 179 9.5 \(S.D\)\).)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(To compare the pattern of HTT proteolytic fragments, we applied a combination of HTT immunoprecipitation and )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(immunodetection from whole brain lysates prepared from )] TJ ET BT 274.963 736.167 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 292.844 740.055 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 321.514 736.167 Td /F1 9.8 Tf [( and Dble mice aged 2 months \(Fig. 2\). Mutant HTT was )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(immunoprecipitated with antibodies that recognize epitopes at the N-terminus \(2B7; S830; or 3B5H10\) \(Fig. 2B-C\) and )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(immunodetected with MW1, an antibody that recognizes the expanded polyQ tract and, therefore, does not detect wild type )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(HTT. A mouse IgG \(mIgG\) antibody was used as a negative control \(Fig. 2A\), and )] TJ ET BT 381.189 700.452 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 409.367 700.452 Td /F1 9.8 Tf [( genotypes were confirmed by western )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(blotting \(Fig. 2E\). In both )] TJ ET BT 135.723 688.548 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 153.605 692.436 Td /F1 8.7 Tf [(+/Q150)] TJ ET BT 182.274 688.548 Td /F1 9.8 Tf [( and Dble samples we were able to identify all of the N-terminal fragments identified )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(previously, including )] TJ ET BT 116.740 676.643 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 166.591 676.643 Td /F1 9.8 Tf [( \(the proposed caspase-6 HTT-586 fragment\) )] TJ ET 0.267 0.267 0.267 rg BT 365.462 678.150 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 370.281 676.643 Td /F1 9.8 Tf [(. Therefore, the proteolysis of full-length HTT, to )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(generate )] TJ ET BT 67.444 664.738 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 117.296 664.738 Td /F1 9.8 Tf [( or any other N-terminal HTT fragments was unaltered in the absence of CASP6.)] TJ ET 0.965 0.965 0.965 rg 26.250 83.771 555.000 571.086 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 654.857 m 581.250 654.857 l 581.250 654.107 l 26.250 654.107 l f 26.250 83.771 m 581.250 83.771 l 581.250 84.521 l 26.250 84.521 l f q 388.500 0 0 450.000 35.250 195.107 cm /I4 Do Q q 35.250 95.021 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 179.583 Td /F4 9.8 Tf [(Fig. 2: Figure 2. Analysis of N-terminal HTT fragments in Hdh+/Q150 and Dble mouse brains.)] TJ ET BT 35.250 160.213 Td /F1 9.8 Tf [(Immunoprecipitation of mutant HTT from Hdh+/Q150 or Dble samples at 2 months of age with either: \()] TJ ET BT 475.531 160.213 Td /F4 9.8 Tf [(A)] TJ ET BT 482.570 160.213 Td /F1 9.8 Tf [(\) mIgG \(control\), \()] TJ ET BT 559.498 160.213 Td /F4 9.8 Tf [(B)] TJ ET BT 35.250 146.477 Td /F1 9.8 Tf [(\) 2B7, \()] TJ ET BT 67.220 146.477 Td /F4 9.8 Tf [(C)] TJ ET BT 74.260 146.477 Td /F1 9.8 Tf [(\) S830, or \()] TJ ET BT 123.029 146.477 Td /F4 9.8 Tf [(D)] TJ ET BT 130.069 146.477 Td /F1 9.8 Tf [(\) 3B5H10, and immunodetection with MW1. Fourteen prominent N-terminal fragments can be )] TJ ET BT 35.250 132.741 Td /F1 9.8 Tf [(detected \(Fragments 1-14\), in addition to 3 weaker fragments \(Fragments a-c\), numbered and labelled in panel \(B\). \()] TJ ET BT 537.580 132.741 Td /F4 9.8 Tf [(E)] TJ ET BT 544.083 132.741 Td /F1 9.8 Tf [(\) )] TJ ET BT 35.250 119.005 Td /F1 9.8 Tf [(Casp6 genotypes were confirmed by western blotting, Beta-ACTIN served as a loading control. IP = Immunoprecipitation; )] TJ ET BT 35.250 105.268 Td /F1 9.8 Tf [(ID = Immunodetection; In = Interface between stacking and resolving gel; FL = full-length protein. \(n = 3/genotype\).)] TJ ET Q BT 26.250 66.747 Td /F4 9.8 Tf [(Steady state wild type and mutant HTT levels are not changed in the brains of presymptomatic )] TJ ET BT 466.199 66.747 Td /F6 9.8 Tf [(Hdh)] TJ ET BT 485.153 70.636 Td /F6 8.7 Tf [(+)] TJ ET BT 490.215 70.636 Td /F4 8.7 Tf [(/Q150)] TJ ET BT 513.823 66.747 Td /F4 9.8 Tf [( and Dble )] TJ ET BT 26.250 54.843 Td /F4 9.8 Tf [(mice.)] TJ ET BT 51.181 54.843 Td /F1 9.8 Tf [( To quantify the relative levels of mutant HTT with respect to total HTT, brain lysates from 2 month-old mice were )] TJ ET BT 26.250 42.938 Td /F1 9.8 Tf [(subjected to the highly sensitive TR-FRET assay \(Fig. 3\). This assay uses the labelled antibody pair: 2B7-Terbium cryptate and )] TJ ET Q q 388.500 0 0 450.000 35.250 195.107 cm /I4 Do 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 317 0 obj << /Type /Annot /Subtype /Link /A 318 0 R /Border [0 0 0] /H /I /Rect [ 365.4622 677.3484 370.2809 686.1667 ] >> endobj 318 0 obj << /Type /Action >> endobj 319 0 obj << /Type /Annot /Subtype /Link /A 320 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 195.1073 423.7500 645.1073 ] >> endobj 320 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/Fig2-JPEG115.jpg) >> endobj 321 0 obj << /Type /XObject /Subtype /Image /Width 518 /Height 600 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 58520>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?KK_B >g(٥{`]\oܩ>cQĈXd"e>jzNjmSŞ-o.b9֊(Z(֎h}h֊(֎h}h֊1@4Q@>sE֑SjZB8"?0~_.| \|YMk^xwU𥽮DKj"F|Hqk]~Z|/~0 b4 %2} hION+xXcHaBϭP (hǽsF 0}h@> ъ(Z9 QEsFw}h4PG>v>cފ(֊1@>b (4O&}7]guEPO,:woEMY]46u][-:Α$k92djď M]h\%Mo(@JGk;7o\U]Fkq.:d[[խ+i'NHyTY@J T>mɾ{X 4"m6|n S_G,%y,( R7`qx:O I =CTCmYƖȐE(LqaZv_~ih?|v |Enb7St 8Cxݘnm̳υQQObnp7z4ּEkws k fSdcg m8|Yh_gjdk<"`&R~b{}dRо/xOP ^CHfƖD6d~UݸGլcז !JTI3܂6f:!*%Eenxl~#>k-3 ]~4Si#hb=~n1C.ScyWإ5+o;S{]A2V٢^(;h+Vs%n%IEb'o8ȭn$_d u< 2YQrK>x;~&|5ҍ=vXo#Fv^"c F0 xA%o趌okZbĒT+ }}e{oYMͭk,SDDae# O^///|;օyGow[Lb@0\=+OP#h>SE;?E|)+ ž>Ǐ >#|E M*+ ccGc[RR4PQE-f^!= lFm%WqZ|¿tk˲nP~ucv*y񧘹17~Jo`y7è? ިF;W7᷄<;fc]Ӵ:kR0B7DUq`վ$wD=/dS i3†VedP;#W`Xr+GiNW#slB F &YrѫܤUx^=Hi:zꗹF" f21ϑ/Ҩk<1-Ե1odB&(YN  ,^럃nayRLY[J|A5"( Ş߼_zӾj S{8&2)uH/ d%mT?:ͳKio@Y}&+=T~-ZdS}o, kyhUM$|uy|s_ÞM&okb673 ]p}U x֕?vr[CH;9<&]ѳf h99@h|L/Fn;[ #dS/Dt],ˀF_߉>VyisK}l$%ecʑ$ʯFXFO#>C_|0#ߎ jZC Es4j \r9$~ /9owi=!kl$VU@e'U@==ZYV4yYT݌g9ǧ4XG ̪X '<J+N~ۼ_>Ci2Iiu<T&zS|)|)=6/u͆i&ij^-R.?uImѴY,)Z[0,KO3}/g:]iwo$.-ܼhb%kdޒ*ֈWP_ ztwo,Te;HKaUpkf 㺂9eE#eYH A1z:=GgZCOog ]F4fV$1D\U@.[9'&$Qm6 8z㏅>9ukGSDkqL Er(3g~ !Jtz,AuCp֌Uw2 rq`^Si2 SUV.ԮMnayvH2rQB[#րT5[=nof[xXI$XQ=ȫehY"crud{+ε/:攷:5[.:jث+$ry-e'Ϲt>SJ0=+>4'k#u8Ewg9Zy(׫m5Q>=J}=K!ycc#FI28Ma|O{k?w2cu4 ?~Rqd 50xYumFq{bwڕמ"K ZBdfvK zQPXZG[pF+O+K!`v%1$I=h:CHQfcMAjƛig $#z0 ddָ_8|G9 I^飲[h+-Fb\3[Gu +Xm fKlgmNB1,^1灒yMFOWaִ짖(]2[2F}.&umIZl;F|q=8r 9t:IVS וOTty5mBJCE?7N־+h pttd麼0̾M;u*U;;:0 Z67qgo8rY+$m$s@5V~ȷwa[N7P~ ; &)?I; '+ O93`k"ÿ1k""pI)MqЅЅ[1iOJ?I; &)Oy&+Ҍ{~ ; &)I; &+4P K?B ?I; &+?΀8RA OR &+’wB?OoR“wB?OoWkHMqD/ ÿ1{"Nv/ÿ1{"R &+<@OmwiNJVi`g3'־xG,U{.(P_.:7E'NmF!*FaUa Arѹ`)օi?,#[qך_Nngg_]]Ne[8bgFYfxrX|C HF@4tM:xM>HaGHcGvx@{K_|7ς<9qs6i$K۳%dy$A'j9'ŭwBUO֑C%bM8O^(kO –y3{"cmJ첢Fi2ת&Sހ9&1? 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QE P(JOƏƀixQH(J.xtپy͜k!El+E0#fvTW~%xWZ.m-cYQe@f3;8!MwA#&|'e\OZFmN.&JVݱHiܤ;jzVӵy(XWLksF\2G% ۺ7`B_o z۳mx^HD,HP3<2zξľ)Q.|uXmF44hTyg*(ڰ~!x@ x?  8K牤azqB;`n Rhn`Q\yr2+/:d`@@=~ 'յ:}J.-Yv_Bs_=4afu?g_Bq@4P~ Ҋ1~QPhh?J GGnvR~Š(AQڀQQPEIQ@ E ǥ-*;Xo!1\E I2 R~i OѴkXmcG1o ey9V5ML4j:}c]@EOU( % el\٢.Fa0U_D?]cjȹ쩾x.8ȭ_—׊YVVڅZ%2A%X歪P()h埇1ЯFo8\RAV$(9! ~u5֝wD;U4Y>s,zNQo3*mx.$g'#j,??erZŸk b?x^¾(tX#}??HqM/Qko^IK__=ωt5@ !]Бcx??CAK| #2if454n\m5/죷1$X Ό~ХKU h/t5@x @Ie;5Oo1W9omf+&ƪb~C)43@▵@t+J#-e#>CKH+Wn9?2ifW _rK@xyUBcvkM<#׊G)t5Vb5*=?$uس}Vu^!cIr6Լ/(x\Nݠ:قݤ@@, ?֊ҀހӭJ;Pzu}h}h?Z҃@Ah4~4vEZN(@G@ƏƁG~~4~4;t~4~4P(hh (?˟z%6N"KW!A9$0+pEj̶ |A|[ijXGO2,NzsN0]14^uiFz:S8C?⯇i20xšmgGq%:$W H pK*w ZéuqwE;;|2[Y,B%w43GeV֢լgw8VU$mqCXhaIbeE *+G?AYMsMwr+uM0 p[ ?ǔ{-" ){&-H# <<={a[Dn*ox Y" $jTo5OW|]A^ƀ8mo VoNKL2Z`o^!k xW|1?J-Myį~.]tK pB~fgހ9/t*M[!8˸_'$zm<~5_ƭ.)l;' 7A \P w1++nR3h&%1׍~^ׇ>i֞9[j50m^C|C~_N1`gmz36JNnq1JNk<~!;ﶆ?}5K𿋅u5 VG a΀>y!9¾'>o⃥ٙct׶Kk)9دˏۏĿ^{;Dum&xakE.GP@@bcImX߉^e dk{ßoǺK_YHe{OZJ<5_?lcr:q=b[g_@ !x=|3}Vu'4~4h4(EvҊ9R~4(?Z  (;P((@EPG@QEhQE(P~4zE J( 8((Š(VFjz~N[Ֆ[[Nh\28#Z_H]P;Rs υooՋ_ ;bů\:yuRo d- {.( ŏW>W'ÄRY袀IɌc uOrz (,Om1- ?m(v)^|al?]1mQ{lq~(V4煻umN/w\{tQ@X̗|ufxO;bQ@X <ͷTo~.|5< [tQ@X? c9Mb>ض袀{ƞǻv53][ މy> endobj 323 0 obj << /Type /Action >> endobj 324 0 obj << /Type /Annot /Subtype /Link /A 325 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 195.1073 423.7500 645.1073 ] >> endobj 325 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/Fig2-JPEG115.jpg) >> endobj 326 0 obj << /Type /Annot /Subtype /Link /A 327 0 R /Border [0 0 0] /H /I /Rect [ 365.4622 677.3484 370.2809 686.1667 ] >> endobj 327 0 obj << /Type /Action >> endobj 328 0 obj << /Type /Annot /Subtype /Link /A 329 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 195.1073 423.7500 645.1073 ] >> endobj 329 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/Fig2-JPEG115.jpg) >> endobj 330 0 obj << /Type /Page /Parent 3 0 R /Annots [ 332 0 R 334 0 R 337 0 R 339 0 R 341 0 R 343 0 R 345 0 R 347 0 R 349 0 R 351 0 R 353 0 R 355 0 R ] /Contents 331 0 R >> endobj 331 0 obj << /Length 22191 >> stream 0.271 0.267 0.267 rg q 15.000 33.379 577.500 743.621 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(MAB2166-Alexa488 to quantify levels of total HTT, or 2B7-Terbium cryptate and MW1-D2 to quantify levels of mutant HTT \(the )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(wild type mouse protein is not detected by MW1\) )] TJ ET 0.267 0.267 0.267 rg BT 239.209 757.079 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 248.847 755.571 Td /F1 9.8 Tf [(. There were no significant differences in the steady state levels of either )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(mutant HTT \(Fig. 3A\), or total HTT \(Fig. 3B\) between )] TJ ET BT 257.072 743.667 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 274.953 747.555 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 306.032 743.667 Td /F1 9.8 Tf [(and Dble samples, when wild type \()] TJ ET BT 459.380 743.667 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 477.261 747.555 Td /F1 8.7 Tf [(+/+)] TJ ET BT 489.793 743.667 Td /F1 9.8 Tf [(::)] TJ ET BT 495.214 743.667 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 523.392 747.555 Td /F1 8.7 Tf [(+/+)] TJ ET BT 535.924 743.667 Td /F1 9.8 Tf [(\) )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(fractions served as controls. Mutant HTT could not be detected in wild type fractions as expected \(Fig. 3A\), however, the 2B7-)] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(MAB2166 signal was lower in the )] TJ ET BT 172.559 719.857 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 190.440 723.745 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 221.519 719.857 Td /F1 9.8 Tf [(and Dble fractions than in wild type, largely because the 2B7-MAB2166 FRET is )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(weaker in the presence of highly expanded CAG-repeats present in the mutant HTT protein \(Fig. 3B\). Therefore these data )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(show that the ablation of CASP6 does not influence soluble levels of wild type or mutant HTT in the )] TJ ET BT 456.001 696.048 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 473.882 696.048 Td /F1 9.8 Tf [(Q150 brain.)] TJ ET 0.965 0.965 0.965 rg 26.250 287.912 555.000 398.255 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 686.167 m 581.250 686.167 l 581.250 685.417 l 26.250 685.417 l f 26.250 287.912 m 581.250 287.912 l 581.250 288.662 l 26.250 288.662 l f q 450.000 0 0 279.000 35.250 397.417 cm /I5 Do Q q 35.250 299.162 537.000 92.255 re W n 0.271 0.267 0.267 rg BT 35.250 381.893 Td /F4 9.8 Tf [(Fig. 3: Figure 3. Steady-state levels of mutant HTT and total HTT are unchanged between Hdh+/Q150and Dble )] TJ ET BT 35.250 369.988 Td /F4 9.8 Tf [(samples)] TJ ET BT 35.250 350.618 Td /F1 9.8 Tf [(TR-FRET was performed on total brain lysates prepared from Hdh+/Q150 or Dble samples at 2 months of age. \()] TJ ET BT 518.333 350.618 Td /F4 9.8 Tf [(A)] TJ ET BT 525.373 350.618 Td /F1 9.8 Tf [(\) 2B7-)] TJ ET BT 35.250 336.882 Td /F1 9.8 Tf [(MW1, to quantify levels of mutant HTT, \()] TJ ET BT 209.726 336.882 Td /F4 9.8 Tf [(B)] TJ ET BT 216.766 336.882 Td /F1 9.8 Tf [(\) 2B7-MAB2166, to quantify levels of total HTT. Wild type fractions served as )] TJ ET BT 35.250 323.146 Td /F1 9.8 Tf [(controls. The 2B7-MAB2166 signal was higher in wild type lysates than the Hdh+/Q150 or Dble lysates largely because the )] TJ ET BT 35.250 309.409 Td /F1 9.8 Tf [(TR-FRET signal is reduced in the presence of a long polyQ tract. \(n = 4/genotype\).)] TJ ET Q BT 26.250 253.691 Td /F4 12.0 Tf [(DISCUSSION)] TJ ET BT 26.250 233.736 Td /F1 9.8 Tf [(Numerous studies provided evidence to show that HTT can be cleaved by proteases, however, the relevance of this finding to )] TJ ET BT 26.250 221.832 Td /F1 9.8 Tf [(the pathogenesis of HD remains elusive. Specifically, there is evidence to indicate that protecting the CASP6 cleavage of HTT )] TJ ET BT 26.250 209.927 Td /F1 9.8 Tf [(at amino acid 586 is neuroprotective and can rescue HD-related phenotypes )] TJ ET 0.267 0.267 0.267 rg BT 357.906 211.434 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 367.543 209.927 Td /F1 9.8 Tf [(. To extend this analysis and test this hypothesis )] TJ ET BT 26.250 198.022 Td /F5 9.8 Tf [(in vivo)] TJ ET BT 53.882 198.022 Td /F1 9.8 Tf [(, we investigated the effects of the genetic ablation of )] TJ ET BT 285.853 198.022 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 314.031 198.022 Td /F1 9.8 Tf [( in the )] TJ ET BT 343.300 198.022 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 361.182 198.022 Td /F1 9.8 Tf [(Q150 mouse model of HD. In this study, we )] TJ ET BT 26.250 186.117 Td /F1 9.8 Tf [(showed that )] TJ ET BT 81.532 186.117 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 109.710 190.006 Td /F1 8.7 Tf [(/)] TJ ET BT 121.757 186.117 Td /F1 9.8 Tf [( mice do not express the CASP6 protein. We crossed the )] TJ ET BT 371.035 186.117 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 388.916 186.117 Td /F1 9.8 Tf [(Q150 mice to )] TJ ET BT 449.610 186.117 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 477.788 186.117 Td /F1 9.8 Tf [( mice and employed )] TJ ET BT 26.250 174.213 Td /F1 9.8 Tf [(our previously described unbiased immunoprecipitation and immunodetection strategy to detect N-terminal HTT proteolytic )] TJ ET BT 26.250 162.308 Td /F1 9.8 Tf [(cleavage fragments in the brains of )] TJ ET BT 180.700 162.308 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 198.581 166.196 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 229.660 162.308 Td /F1 9.8 Tf [(mice that did or did not express CASP6. In accordance with our previous )] TJ ET BT 26.250 150.403 Td /F1 9.8 Tf [(findings, we could detect the C-terminus of 14 prominent mutant HTT N-terminal fragments \()] TJ ET BT 423.972 150.403 Td /F5 9.8 Tf [(Fragments 1-14)] TJ ET BT 492.787 150.403 Td /F1 9.8 Tf [(\) and three )] TJ ET BT 26.250 138.498 Td /F1 9.8 Tf [(additional fragments \()] TJ ET BT 119.996 138.498 Td /F5 9.8 Tf [(Fragments a-c)] TJ ET BT 182.845 138.498 Td /F1 9.8 Tf [(\) in the brain at 2 months of age )] TJ ET 0.267 0.267 0.267 rg BT 323.216 140.006 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 328.034 138.498 Td /F1 9.8 Tf [(. We found that the ablation of CASP6 did not modify the )] TJ ET BT 26.250 126.594 Td /F1 9.8 Tf [(production of )] TJ ET BT 85.325 126.594 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 135.177 126.594 Td /F1 9.8 Tf [( \(the previously proposed caspase-6 HTT-586 fragment\), or any other N-terminal HTT fragment. Since )] TJ ET BT 26.250 114.689 Td /F1 9.8 Tf [(the ablation of CASP6 had no effect on the proteolysis of mutant HTT we were unable to test the cascade hypothesis to )] TJ ET BT 26.250 102.784 Td /F1 9.8 Tf [(determine whether the production of one fragment is essential for the generation of others.)] TJ ET BT 26.250 83.379 Td /F1 9.8 Tf [(Given that )] TJ ET BT 73.401 83.379 Td /F5 9.8 Tf [(Fragment 8 )] TJ ET BT 125.963 83.379 Td /F1 9.8 Tf [(is still present in the absence of CASP6, it must therefore be generated by an alternative protease that )] TJ ET BT 26.250 71.475 Td /F1 9.8 Tf [(cleaves either at the HTT-586 site or at a different position nearby. If the cleavage occurs at HTT-586, an alternative protease )] TJ ET BT 26.250 59.570 Td /F1 9.8 Tf [(also recognises the CASP6 recognition motif and therefore, redundancy would be expected to ensure that the site is cleaved )] TJ ET BT 26.250 47.665 Td /F1 9.8 Tf [(irrespective of whether CASP6 is present or absent. This could account for a compensatory mechanism in these constitutive )] TJ ET Q q 15.000 33.379 577.500 743.621 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(MAB2166-Alexa488 to quantify levels of total HTT, or 2B7-Terbium cryptate and MW1-D2 to quantify levels of mutant HTT \(the )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(wild type mouse protein is not detected by MW1\) )] TJ ET 0.267 0.267 0.267 rg BT 239.209 757.079 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 248.847 755.571 Td /F1 9.8 Tf [(. There were no significant differences in the steady state levels of either )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(mutant HTT \(Fig. 3A\), or total HTT \(Fig. 3B\) between )] TJ ET BT 257.072 743.667 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 274.953 747.555 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 306.032 743.667 Td /F1 9.8 Tf [(and Dble samples, when wild type \()] TJ ET BT 459.380 743.667 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 477.261 747.555 Td /F1 8.7 Tf [(+/+)] TJ ET BT 489.793 743.667 Td /F1 9.8 Tf [(::)] TJ ET BT 495.214 743.667 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 523.392 747.555 Td /F1 8.7 Tf [(+/+)] TJ ET BT 535.924 743.667 Td /F1 9.8 Tf [(\) )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(fractions served as controls. Mutant HTT could not be detected in wild type fractions as expected \(Fig. 3A\), however, the 2B7-)] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(MAB2166 signal was lower in the )] TJ ET BT 172.559 719.857 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 190.440 723.745 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 221.519 719.857 Td /F1 9.8 Tf [(and Dble fractions than in wild type, largely because the 2B7-MAB2166 FRET is )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(weaker in the presence of highly expanded CAG-repeats present in the mutant HTT protein \(Fig. 3B\). Therefore these data )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(show that the ablation of CASP6 does not influence soluble levels of wild type or mutant HTT in the )] TJ ET BT 456.001 696.048 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 473.882 696.048 Td /F1 9.8 Tf [(Q150 brain.)] TJ ET 0.965 0.965 0.965 rg 26.250 287.912 555.000 398.255 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 686.167 m 581.250 686.167 l 581.250 685.417 l 26.250 685.417 l f 26.250 287.912 m 581.250 287.912 l 581.250 288.662 l 26.250 288.662 l f q 450.000 0 0 279.000 35.250 397.417 cm /I5 Do Q q 35.250 299.162 537.000 92.255 re W n 0.271 0.267 0.267 rg BT 35.250 381.893 Td /F4 9.8 Tf [(Fig. 3: Figure 3. Steady-state levels of mutant HTT and total HTT are unchanged between Hdh+/Q150and Dble )] TJ ET BT 35.250 369.988 Td /F4 9.8 Tf [(samples)] TJ ET BT 35.250 350.618 Td /F1 9.8 Tf [(TR-FRET was performed on total brain lysates prepared from Hdh+/Q150 or Dble samples at 2 months of age. \()] TJ ET BT 518.333 350.618 Td /F4 9.8 Tf [(A)] TJ ET BT 525.373 350.618 Td /F1 9.8 Tf [(\) 2B7-)] TJ ET BT 35.250 336.882 Td /F1 9.8 Tf [(MW1, to quantify levels of mutant HTT, \()] TJ ET BT 209.726 336.882 Td /F4 9.8 Tf [(B)] TJ ET BT 216.766 336.882 Td /F1 9.8 Tf [(\) 2B7-MAB2166, to quantify levels of total HTT. Wild type fractions served as )] TJ ET BT 35.250 323.146 Td /F1 9.8 Tf [(controls. The 2B7-MAB2166 signal was higher in wild type lysates than the Hdh+/Q150 or Dble lysates largely because the )] TJ ET BT 35.250 309.409 Td /F1 9.8 Tf [(TR-FRET signal is reduced in the presence of a long polyQ tract. \(n = 4/genotype\).)] TJ ET Q BT 26.250 253.691 Td /F4 12.0 Tf [(DISCUSSION)] TJ ET BT 26.250 233.736 Td /F1 9.8 Tf [(Numerous studies provided evidence to show that HTT can be cleaved by proteases, however, the relevance of this finding to )] TJ ET BT 26.250 221.832 Td /F1 9.8 Tf [(the pathogenesis of HD remains elusive. Specifically, there is evidence to indicate that protecting the CASP6 cleavage of HTT )] TJ ET BT 26.250 209.927 Td /F1 9.8 Tf [(at amino acid 586 is neuroprotective and can rescue HD-related phenotypes )] TJ ET 0.267 0.267 0.267 rg BT 357.906 211.434 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 367.543 209.927 Td /F1 9.8 Tf [(. To extend this analysis and test this hypothesis )] TJ ET BT 26.250 198.022 Td /F5 9.8 Tf [(in vivo)] TJ ET BT 53.882 198.022 Td /F1 9.8 Tf [(, we investigated the effects of the genetic ablation of )] TJ ET BT 285.853 198.022 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 314.031 198.022 Td /F1 9.8 Tf [( in the )] TJ ET BT 343.300 198.022 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 361.182 198.022 Td /F1 9.8 Tf [(Q150 mouse model of HD. In this study, we )] TJ ET BT 26.250 186.117 Td /F1 9.8 Tf [(showed that )] TJ ET BT 81.532 186.117 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 109.710 190.006 Td /F1 8.7 Tf [(/)] TJ ET BT 121.757 186.117 Td /F1 9.8 Tf [( mice do not express the CASP6 protein. We crossed the )] TJ ET BT 371.035 186.117 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 388.916 186.117 Td /F1 9.8 Tf [(Q150 mice to )] TJ ET BT 449.610 186.117 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 477.788 186.117 Td /F1 9.8 Tf [( mice and employed )] TJ ET BT 26.250 174.213 Td /F1 9.8 Tf [(our previously described unbiased immunoprecipitation and immunodetection strategy to detect N-terminal HTT proteolytic )] TJ ET BT 26.250 162.308 Td /F1 9.8 Tf [(cleavage fragments in the brains of )] TJ ET BT 180.700 162.308 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 198.581 166.196 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 229.660 162.308 Td /F1 9.8 Tf [(mice that did or did not express CASP6. In accordance with our previous )] TJ ET BT 26.250 150.403 Td /F1 9.8 Tf [(findings, we could detect the C-terminus of 14 prominent mutant HTT N-terminal fragments \()] TJ ET BT 423.972 150.403 Td /F5 9.8 Tf [(Fragments 1-14)] TJ ET BT 492.787 150.403 Td /F1 9.8 Tf [(\) and three )] TJ ET BT 26.250 138.498 Td /F1 9.8 Tf [(additional fragments \()] TJ ET BT 119.996 138.498 Td /F5 9.8 Tf [(Fragments a-c)] TJ ET BT 182.845 138.498 Td /F1 9.8 Tf [(\) in the brain at 2 months of age )] TJ ET 0.267 0.267 0.267 rg BT 323.216 140.006 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 328.034 138.498 Td /F1 9.8 Tf [(. We found that the ablation of CASP6 did not modify the )] TJ ET BT 26.250 126.594 Td /F1 9.8 Tf [(production of )] TJ ET BT 85.325 126.594 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 135.177 126.594 Td /F1 9.8 Tf [( \(the previously proposed caspase-6 HTT-586 fragment\), or any other N-terminal HTT fragment. Since )] TJ ET BT 26.250 114.689 Td /F1 9.8 Tf [(the ablation of CASP6 had no effect on the proteolysis of mutant HTT we were unable to test the cascade hypothesis to )] TJ ET BT 26.250 102.784 Td /F1 9.8 Tf [(determine whether the production of one fragment is essential for the generation of others.)] TJ ET BT 26.250 83.379 Td /F1 9.8 Tf [(Given that )] TJ ET BT 73.401 83.379 Td /F5 9.8 Tf [(Fragment 8 )] TJ ET BT 125.963 83.379 Td /F1 9.8 Tf [(is still present in the absence of CASP6, it must therefore be generated by an alternative protease that )] TJ ET BT 26.250 71.475 Td /F1 9.8 Tf [(cleaves either at the HTT-586 site or at a different position nearby. If the cleavage occurs at HTT-586, an alternative protease )] TJ ET BT 26.250 59.570 Td /F1 9.8 Tf [(also recognises the CASP6 recognition motif and therefore, redundancy would be expected to ensure that the site is cleaved )] TJ ET BT 26.250 47.665 Td /F1 9.8 Tf [(irrespective of whether CASP6 is present or absent. This could account for a compensatory mechanism in these constitutive )] TJ ET Q q 15.000 33.379 577.500 743.621 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(MAB2166-Alexa488 to quantify levels of total HTT, or 2B7-Terbium cryptate and MW1-D2 to quantify levels of mutant HTT \(the )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(wild type mouse protein is not detected by MW1\) )] TJ ET 0.267 0.267 0.267 rg BT 239.209 757.079 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 248.847 755.571 Td /F1 9.8 Tf [(. There were no significant differences in the steady state levels of either )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(mutant HTT \(Fig. 3A\), or total HTT \(Fig. 3B\) between )] TJ ET BT 257.072 743.667 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 274.953 747.555 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 306.032 743.667 Td /F1 9.8 Tf [(and Dble samples, when wild type \()] TJ ET BT 459.380 743.667 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 477.261 747.555 Td /F1 8.7 Tf [(+/+)] TJ ET BT 489.793 743.667 Td /F1 9.8 Tf [(::)] TJ ET BT 495.214 743.667 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 523.392 747.555 Td /F1 8.7 Tf [(+/+)] TJ ET BT 535.924 743.667 Td /F1 9.8 Tf [(\) )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(fractions served as controls. Mutant HTT could not be detected in wild type fractions as expected \(Fig. 3A\), however, the 2B7-)] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(MAB2166 signal was lower in the )] TJ ET BT 172.559 719.857 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 190.440 723.745 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 221.519 719.857 Td /F1 9.8 Tf [(and Dble fractions than in wild type, largely because the 2B7-MAB2166 FRET is )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(weaker in the presence of highly expanded CAG-repeats present in the mutant HTT protein \(Fig. 3B\). Therefore these data )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(show that the ablation of CASP6 does not influence soluble levels of wild type or mutant HTT in the )] TJ ET BT 456.001 696.048 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 473.882 696.048 Td /F1 9.8 Tf [(Q150 brain.)] TJ ET 0.965 0.965 0.965 rg 26.250 287.912 555.000 398.255 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 686.167 m 581.250 686.167 l 581.250 685.417 l 26.250 685.417 l f 26.250 287.912 m 581.250 287.912 l 581.250 288.662 l 26.250 288.662 l f q 450.000 0 0 279.000 35.250 397.417 cm /I5 Do Q q 35.250 299.162 537.000 92.255 re W n 0.271 0.267 0.267 rg BT 35.250 381.893 Td /F4 9.8 Tf [(Fig. 3: Figure 3. Steady-state levels of mutant HTT and total HTT are unchanged between Hdh+/Q150and Dble )] TJ ET BT 35.250 369.988 Td /F4 9.8 Tf [(samples)] TJ ET BT 35.250 350.618 Td /F1 9.8 Tf [(TR-FRET was performed on total brain lysates prepared from Hdh+/Q150 or Dble samples at 2 months of age. \()] TJ ET BT 518.333 350.618 Td /F4 9.8 Tf [(A)] TJ ET BT 525.373 350.618 Td /F1 9.8 Tf [(\) 2B7-)] TJ ET BT 35.250 336.882 Td /F1 9.8 Tf [(MW1, to quantify levels of mutant HTT, \()] TJ ET BT 209.726 336.882 Td /F4 9.8 Tf [(B)] TJ ET BT 216.766 336.882 Td /F1 9.8 Tf [(\) 2B7-MAB2166, to quantify levels of total HTT. Wild type fractions served as )] TJ ET BT 35.250 323.146 Td /F1 9.8 Tf [(controls. The 2B7-MAB2166 signal was higher in wild type lysates than the Hdh+/Q150 or Dble lysates largely because the )] TJ ET BT 35.250 309.409 Td /F1 9.8 Tf [(TR-FRET signal is reduced in the presence of a long polyQ tract. \(n = 4/genotype\).)] TJ ET Q BT 26.250 253.691 Td /F4 12.0 Tf [(DISCUSSION)] TJ ET BT 26.250 233.736 Td /F1 9.8 Tf [(Numerous studies provided evidence to show that HTT can be cleaved by proteases, however, the relevance of this finding to )] TJ ET BT 26.250 221.832 Td /F1 9.8 Tf [(the pathogenesis of HD remains elusive. Specifically, there is evidence to indicate that protecting the CASP6 cleavage of HTT )] TJ ET BT 26.250 209.927 Td /F1 9.8 Tf [(at amino acid 586 is neuroprotective and can rescue HD-related phenotypes )] TJ ET 0.267 0.267 0.267 rg BT 357.906 211.434 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 367.543 209.927 Td /F1 9.8 Tf [(. To extend this analysis and test this hypothesis )] TJ ET BT 26.250 198.022 Td /F5 9.8 Tf [(in vivo)] TJ ET BT 53.882 198.022 Td /F1 9.8 Tf [(, we investigated the effects of the genetic ablation of )] TJ ET BT 285.853 198.022 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 314.031 198.022 Td /F1 9.8 Tf [( in the )] TJ ET BT 343.300 198.022 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 361.182 198.022 Td /F1 9.8 Tf [(Q150 mouse model of HD. In this study, we )] TJ ET BT 26.250 186.117 Td /F1 9.8 Tf [(showed that )] TJ ET BT 81.532 186.117 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 109.710 190.006 Td /F1 8.7 Tf [(/)] TJ ET BT 121.757 186.117 Td /F1 9.8 Tf [( mice do not express the CASP6 protein. We crossed the )] TJ ET BT 371.035 186.117 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 388.916 186.117 Td /F1 9.8 Tf [(Q150 mice to )] TJ ET BT 449.610 186.117 Td /F5 9.8 Tf [(Casp6)] TJ ET BT 477.788 186.117 Td /F1 9.8 Tf [( mice and employed )] TJ ET BT 26.250 174.213 Td /F1 9.8 Tf [(our previously described unbiased immunoprecipitation and immunodetection strategy to detect N-terminal HTT proteolytic )] TJ ET BT 26.250 162.308 Td /F1 9.8 Tf [(cleavage fragments in the brains of )] TJ ET BT 180.700 162.308 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 198.581 166.196 Td /F1 8.7 Tf [(+/Q150 )] TJ ET BT 229.660 162.308 Td /F1 9.8 Tf [(mice that did or did not express CASP6. In accordance with our previous )] TJ ET BT 26.250 150.403 Td /F1 9.8 Tf [(findings, we could detect the C-terminus of 14 prominent mutant HTT N-terminal fragments \()] TJ ET BT 423.972 150.403 Td /F5 9.8 Tf [(Fragments 1-14)] TJ ET BT 492.787 150.403 Td /F1 9.8 Tf [(\) and three )] TJ ET BT 26.250 138.498 Td /F1 9.8 Tf [(additional fragments \()] TJ ET BT 119.996 138.498 Td /F5 9.8 Tf [(Fragments a-c)] TJ ET BT 182.845 138.498 Td /F1 9.8 Tf [(\) in the brain at 2 months of age )] TJ ET 0.267 0.267 0.267 rg BT 323.216 140.006 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 328.034 138.498 Td /F1 9.8 Tf [(. We found that the ablation of CASP6 did not modify the )] TJ ET BT 26.250 126.594 Td /F1 9.8 Tf [(production of )] TJ ET BT 85.325 126.594 Td /F5 9.8 Tf [(Fragment 8)] TJ ET BT 135.177 126.594 Td /F1 9.8 Tf [( \(the previously proposed caspase-6 HTT-586 fragment\), or any other N-terminal HTT fragment. Since )] TJ ET BT 26.250 114.689 Td /F1 9.8 Tf [(the ablation of CASP6 had no effect on the proteolysis of mutant HTT we were unable to test the cascade hypothesis to )] TJ ET BT 26.250 102.784 Td /F1 9.8 Tf [(determine whether the production of one fragment is essential for the generation of others.)] TJ ET BT 26.250 83.379 Td /F1 9.8 Tf [(Given that )] TJ ET BT 73.401 83.379 Td /F5 9.8 Tf [(Fragment 8 )] TJ ET BT 125.963 83.379 Td /F1 9.8 Tf [(is still present in the absence of CASP6, it must therefore be generated by an alternative protease that )] TJ ET BT 26.250 71.475 Td /F1 9.8 Tf [(cleaves either at the HTT-586 site or at a different position nearby. If the cleavage occurs at HTT-586, an alternative protease )] TJ ET BT 26.250 59.570 Td /F1 9.8 Tf [(also recognises the CASP6 recognition motif and therefore, redundancy would be expected to ensure that the site is cleaved )] TJ ET BT 26.250 47.665 Td /F1 9.8 Tf [(irrespective of whether CASP6 is present or absent. This could account for a compensatory mechanism in these constitutive )] TJ ET Q q 450.000 0 0 279.000 35.250 397.417 cm /I5 Do 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 332 0 obj << /Type /Annot /Subtype /Link /A 333 0 R /Border [0 0 0] /H /I /Rect [ 239.2095 756.2769 248.8468 765.0952 ] >> endobj 333 0 obj << /Type /Action >> endobj 334 0 obj << /Type /Annot /Subtype /Link /A 335 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 397.4167 485.2500 676.4167 ] >> endobj 335 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/Fig3-JPEG1.jpg) >> endobj 336 0 obj << /Type /XObject /Subtype /Image /Width 600 /Height 372 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 33573>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   tX" }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?NxPMfӥ}| Yd\9?njaƫajEzn[yERw{WU$ϪncKn;UMMrx \=_|7χ4XRo541T*pN95Nhl?*Z9n@sI-Es@ QNiy(hi(hh旚(旚(旚(hsIQ/4QRsG4E'4E@4sG4fG4s@sG4QA( LI.i(旚J\~Tf9gKRR8桼NxS$LrKw46hx>2GѼ$`V[Pg 3+G? χvt 29m<mX0T?1S 5쯣PW|?q GƩ#"$T.iez3d~Y 6(֥:&n@o$墉HEP eb컦]|.]FM߂/QxnbPȖ cSǸPj\~叁<96.: BCo} {a^7o'[හkkKXum+'l+_ x/ݫ$e6A[^*IG> ѭAw_ h4v&ۆjno?g?]HOG"ek.Wk|9Zo[}h|sewaIkh1]eTK|qGl[Ao'~E/ Z[Al3Zb#6ae@!=7OvtkW˜"+,IPg^;n:foiqI1'DEUG('ezwHw`@ıں'_H I)EZ{ڰy!y56nlmnx+Ѩhi(hh(i{PIK~4RQOƏր?Z(vTj}ժMhDыrI<K\o.QD/5a27;r3dPxG?oZ,ĭ?P[1 YۥP|邲e<cζ|g1^#MXĶ?-ԍ~Le/,%>2vDkR~e3y쳹+go7?6 v0k;Rh`\I봟ڀ8ط>>7-WW 4>Y6G"l*^e;zMCWWƭ[x: MhQHO+c}c|>~ ^j3 "V42Xq"%ٓ{ldwS@gm4[-ߎ{? 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It would also confound therapeutic approaches directed at CASP6 inhibition. In concurrence with our findings, it )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(has recently been demonstrated that the ablation of CASP6 activity in an aged BACHD mouse model did not show a reduction )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(in the proteolysis of HTT at amino acid 586 )] TJ ET 0.267 0.267 0.267 rg BT 214.288 745.174 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 223.926 743.667 Td /F1 9.8 Tf [(, and our work now extends this observation to two different HD mouse models.)] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(The ablation of CASP6 in BACHD mice aged 13 months resulted in a reduction of both wild type and mutant HTT levels, )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(possibly due to the activation of protein clearance pathways )] TJ ET 0.267 0.267 0.267 rg BT 286.380 713.864 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 296.017 712.357 Td /F1 9.8 Tf [(. We performed TR-FRET to quantify levels of both mutant HTT )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(and total HTT and found that the absence of CASP6 had no effect on soluble HTT levels in presymptomatic mice. Such a )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(difference in the effect of ablating CASP6 on HTT levels in young \(2 month\) )] TJ ET BT 354.113 688.548 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 371.995 688.548 Td /F1 9.8 Tf [(Q150 and aged \(13 month\) BACHD mice is not )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(clear. It could be that protein clearance pathways are regulated by CASP6 only at later stages of disease, particularly when )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(toxic HTT aggregates are present.)] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(Preventing the formation of N-terminal HTT fragments is an attractive therapeutic strategy that needs to be understood. Our )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(data suggest that CASP6 is not necessary for the proteolysis of mutant HTT in )] TJ ET BT 366.574 633.429 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 384.455 633.429 Td /F1 9.8 Tf [(Q150 mice and by implication does not play )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(a direct role in HD pathogenesis in this mouse model via this mechanism. Our work highlights the importance of identifying the )] TJ ET BT 26.250 609.619 Td /F1 9.8 Tf [(source of HTT proteolytic fragments to allow the design of rational therapeutic strategies when considering how one should )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(combat the many HD-related phenotypes associated with this complex neurodegenerative disease.)] TJ ET BT 26.250 561.112 Td /F4 12.0 Tf [(COMPETING INTERESTS)] TJ ET BT 26.250 541.158 Td /F1 9.8 Tf [(AW is an employee of Novartis AG.)] TJ ET BT 26.250 504.555 Td /F4 12.0 Tf [(ACKNOWLEDGEMENTS)] TJ ET BT 26.250 484.601 Td /F1 9.8 Tf [(We thank Dr. Anna Bobrowska and members of the Neurogenetics Laboratory for critical advice on the manuscript.)] TJ ET BT 26.250 447.998 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 420.544 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 420.544 Td /F1 9.8 Tf [(Bates GP, Benn C \(2002\) The Polyglutamine Diseases. In: Bates GP, Harper PS, Jones AL, editors. Huntington's Disease. )] TJ ET BT 26.250 408.639 Td /F1 9.8 Tf [(3rd ed. Oxford: Oxford University Press. pp. 429-472.)] TJ ET BT 26.250 389.235 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 389.235 Td /F1 9.8 Tf [(Landles C, Bates GP \(2004\) Huntingtin and the molecular pathogenesis of Huntington's disease. Fourth in molecular )] TJ ET BT 26.250 377.330 Td /F1 9.8 Tf [(medicine review series. EMBO Rep 5: 958-963.)] TJ ET BT 26.250 357.925 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 357.925 Td /F1 9.8 Tf [(Harjes P, Wanker EE \(2003\) The hunt for huntingtin function: interaction partners tell many different stories. Trends Biochem )] TJ ET BT 26.250 346.020 Td /F1 9.8 Tf [(Sci 28: 425-433.)] TJ ET BT 26.250 326.616 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 326.616 Td /F1 9.8 Tf [(Lin CH, Tallaksen-Greene S, Chien WM, Cearley JA, Jackson WS, et al. \(2001\) Neurological abnormalities in a knock-in )] TJ ET BT 26.250 314.711 Td /F1 9.8 Tf [(mouse model of Huntington's disease. Hum Mol Genet 10: 137-144.)] TJ ET BT 26.250 295.306 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 295.306 Td /F1 9.8 Tf [(Woodman B, Butler R, Landles C, Lupton MK, Tse J, et al. \(2007\) The Hdh\(Q150/Q150\) knock-in mouse model of HD and )] TJ ET BT 26.250 283.401 Td /F1 9.8 Tf [(the R6/2 exon 1 model develop comparable and widespread molecular phenotypes. Brain Res Bull 72: 83-97.)] TJ ET BT 26.250 263.997 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 263.997 Td /F1 9.8 Tf [(Landles C, Sathasivam K, Weiss A, Woodman B, Moffitt H, et al. \(2010\) Proteolysis of mutant huntingtin produces an exon 1 )] TJ ET BT 26.250 252.092 Td /F1 9.8 Tf [(fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem 285: 8808-8823.)] TJ ET BT 26.250 232.687 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 232.687 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, et al. \(1997\) Aggregation of huntingtin in neuronal intranuclear )] TJ ET BT 26.250 220.782 Td /F1 9.8 Tf [(inclusions and dystrophic neurites in brain. Science 277: 1990-1993.)] TJ ET BT 26.250 201.378 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 201.378 Td /F1 9.8 Tf [(Lunkes A, Lindenberg KS, Ben-Haiem L, Weber C, Devys D, et al. \(2002\) Proteases acting on mutant huntingtin generate )] TJ ET BT 26.250 189.473 Td /F1 9.8 Tf [(cleaved products that differentially build up cytoplasmic and nuclear inclusions. Mol Cell 10: 259-269.)] TJ ET BT 26.250 170.068 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 170.068 Td /F1 9.8 Tf [(Ratovitski T, Nakamura M, D'Ambola J, Chighladze E, Liang Y, et al. \(2007\) N-terminal proteolysis of full-length mutant )] TJ ET BT 26.250 158.163 Td /F1 9.8 Tf [(huntingtin in an inducible PC12 cell model of Huntington's disease. Cell Cycle 6: 2970-2981.)] TJ ET BT 26.250 138.759 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 138.759 Td /F1 9.8 Tf [(Ratovitski T, Gucek M, Jiang H, Chighladze E, Waldron E, et al. \(2009\) Mutant huntingtin N-terminal fragments of specific )] TJ ET BT 26.250 126.854 Td /F1 9.8 Tf [(size mediate aggregation and toxicity in neuronal cells. J Biol Chem 284: 10855-10867.)] TJ ET BT 26.250 107.449 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 107.449 Td /F1 9.8 Tf [(Wellington CL, Ellerby LM, Gutekunst CA, Rogers D, Warby S, et al. \(2002\) Caspase cleavage of mutant huntingtin )] TJ ET BT 26.250 95.544 Td /F1 9.8 Tf [(precedes neurodegeneration in Huntington's disease. J Neurosci 22: 7862-7872.)] TJ ET BT 26.250 76.140 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 76.140 Td /F1 9.8 Tf [(Kim YJ, Yi Y, Sapp E, Wang Y, Cuiffo B, et al. \(2001\) Caspase 3-cleaved N-terminal fragments of wild-type and mutant )] TJ ET BT 26.250 64.235 Td /F1 9.8 Tf [(huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent )] TJ ET BT 26.250 52.330 Td /F1 9.8 Tf [(proteolysis. Proc Natl Acad Sci U S A 98: 12784-12789.)] TJ ET Q q 15.000 42.449 577.500 734.551 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(knock-out mice. It would also confound therapeutic approaches directed at CASP6 inhibition. In concurrence with our findings, it )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(has recently been demonstrated that the ablation of CASP6 activity in an aged BACHD mouse model did not show a reduction )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(in the proteolysis of HTT at amino acid 586 )] TJ ET 0.267 0.267 0.267 rg BT 214.288 745.174 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 223.926 743.667 Td /F1 9.8 Tf [(, and our work now extends this observation to two different HD mouse models.)] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(The ablation of CASP6 in BACHD mice aged 13 months resulted in a reduction of both wild type and mutant HTT levels, )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(possibly due to the activation of protein clearance pathways )] TJ ET 0.267 0.267 0.267 rg BT 286.380 713.864 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 296.017 712.357 Td /F1 9.8 Tf [(. We performed TR-FRET to quantify levels of both mutant HTT )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(and total HTT and found that the absence of CASP6 had no effect on soluble HTT levels in presymptomatic mice. Such a )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(difference in the effect of ablating CASP6 on HTT levels in young \(2 month\) )] TJ ET BT 354.113 688.548 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 371.995 688.548 Td /F1 9.8 Tf [(Q150 and aged \(13 month\) BACHD mice is not )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(clear. It could be that protein clearance pathways are regulated by CASP6 only at later stages of disease, particularly when )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(toxic HTT aggregates are present.)] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(Preventing the formation of N-terminal HTT fragments is an attractive therapeutic strategy that needs to be understood. Our )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(data suggest that CASP6 is not necessary for the proteolysis of mutant HTT in )] TJ ET BT 366.574 633.429 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 384.455 633.429 Td /F1 9.8 Tf [(Q150 mice and by implication does not play )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(a direct role in HD pathogenesis in this mouse model via this mechanism. Our work highlights the importance of identifying the )] TJ ET BT 26.250 609.619 Td /F1 9.8 Tf [(source of HTT proteolytic fragments to allow the design of rational therapeutic strategies when considering how one should )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(combat the many HD-related phenotypes associated with this complex neurodegenerative disease.)] TJ ET BT 26.250 561.112 Td /F4 12.0 Tf [(COMPETING INTERESTS)] TJ ET BT 26.250 541.158 Td /F1 9.8 Tf [(AW is an employee of Novartis AG.)] TJ ET BT 26.250 504.555 Td /F4 12.0 Tf [(ACKNOWLEDGEMENTS)] TJ ET BT 26.250 484.601 Td /F1 9.8 Tf [(We thank Dr. Anna Bobrowska and members of the Neurogenetics Laboratory for critical advice on the manuscript.)] TJ ET BT 26.250 447.998 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 420.544 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 420.544 Td /F1 9.8 Tf [(Bates GP, Benn C \(2002\) The Polyglutamine Diseases. In: Bates GP, Harper PS, Jones AL, editors. Huntington's Disease. )] TJ ET BT 26.250 408.639 Td /F1 9.8 Tf [(3rd ed. Oxford: Oxford University Press. pp. 429-472.)] TJ ET BT 26.250 389.235 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 389.235 Td /F1 9.8 Tf [(Landles C, Bates GP \(2004\) Huntingtin and the molecular pathogenesis of Huntington's disease. Fourth in molecular )] TJ ET BT 26.250 377.330 Td /F1 9.8 Tf [(medicine review series. EMBO Rep 5: 958-963.)] TJ ET BT 26.250 357.925 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 357.925 Td /F1 9.8 Tf [(Harjes P, Wanker EE \(2003\) The hunt for huntingtin function: interaction partners tell many different stories. Trends Biochem )] TJ ET BT 26.250 346.020 Td /F1 9.8 Tf [(Sci 28: 425-433.)] TJ ET BT 26.250 326.616 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 326.616 Td /F1 9.8 Tf [(Lin CH, Tallaksen-Greene S, Chien WM, Cearley JA, Jackson WS, et al. \(2001\) Neurological abnormalities in a knock-in )] TJ ET BT 26.250 314.711 Td /F1 9.8 Tf [(mouse model of Huntington's disease. Hum Mol Genet 10: 137-144.)] TJ ET BT 26.250 295.306 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 295.306 Td /F1 9.8 Tf [(Woodman B, Butler R, Landles C, Lupton MK, Tse J, et al. \(2007\) The Hdh\(Q150/Q150\) knock-in mouse model of HD and )] TJ ET BT 26.250 283.401 Td /F1 9.8 Tf [(the R6/2 exon 1 model develop comparable and widespread molecular phenotypes. Brain Res Bull 72: 83-97.)] TJ ET BT 26.250 263.997 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 263.997 Td /F1 9.8 Tf [(Landles C, Sathasivam K, Weiss A, Woodman B, Moffitt H, et al. \(2010\) Proteolysis of mutant huntingtin produces an exon 1 )] TJ ET BT 26.250 252.092 Td /F1 9.8 Tf [(fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem 285: 8808-8823.)] TJ ET BT 26.250 232.687 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 232.687 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, et al. \(1997\) Aggregation of huntingtin in neuronal intranuclear )] TJ ET BT 26.250 220.782 Td /F1 9.8 Tf [(inclusions and dystrophic neurites in brain. Science 277: 1990-1993.)] TJ ET BT 26.250 201.378 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 201.378 Td /F1 9.8 Tf [(Lunkes A, Lindenberg KS, Ben-Haiem L, Weber C, Devys D, et al. \(2002\) Proteases acting on mutant huntingtin generate )] TJ ET BT 26.250 189.473 Td /F1 9.8 Tf [(cleaved products that differentially build up cytoplasmic and nuclear inclusions. Mol Cell 10: 259-269.)] TJ ET BT 26.250 170.068 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 170.068 Td /F1 9.8 Tf [(Ratovitski T, Nakamura M, D'Ambola J, Chighladze E, Liang Y, et al. \(2007\) N-terminal proteolysis of full-length mutant )] TJ ET BT 26.250 158.163 Td /F1 9.8 Tf [(huntingtin in an inducible PC12 cell model of Huntington's disease. Cell Cycle 6: 2970-2981.)] TJ ET BT 26.250 138.759 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 138.759 Td /F1 9.8 Tf [(Ratovitski T, Gucek M, Jiang H, Chighladze E, Waldron E, et al. \(2009\) Mutant huntingtin N-terminal fragments of specific )] TJ ET BT 26.250 126.854 Td /F1 9.8 Tf [(size mediate aggregation and toxicity in neuronal cells. J Biol Chem 284: 10855-10867.)] TJ ET BT 26.250 107.449 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 107.449 Td /F1 9.8 Tf [(Wellington CL, Ellerby LM, Gutekunst CA, Rogers D, Warby S, et al. \(2002\) Caspase cleavage of mutant huntingtin )] TJ ET BT 26.250 95.544 Td /F1 9.8 Tf [(precedes neurodegeneration in Huntington's disease. J Neurosci 22: 7862-7872.)] TJ ET BT 26.250 76.140 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 76.140 Td /F1 9.8 Tf [(Kim YJ, Yi Y, Sapp E, Wang Y, Cuiffo B, et al. \(2001\) Caspase 3-cleaved N-terminal fragments of wild-type and mutant )] TJ ET BT 26.250 64.235 Td /F1 9.8 Tf [(huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent )] TJ ET BT 26.250 52.330 Td /F1 9.8 Tf [(proteolysis. Proc Natl Acad Sci U S A 98: 12784-12789.)] TJ ET Q q 15.000 42.449 577.500 734.551 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(knock-out mice. It would also confound therapeutic approaches directed at CASP6 inhibition. In concurrence with our findings, it )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(has recently been demonstrated that the ablation of CASP6 activity in an aged BACHD mouse model did not show a reduction )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(in the proteolysis of HTT at amino acid 586 )] TJ ET 0.267 0.267 0.267 rg BT 214.288 745.174 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 223.926 743.667 Td /F1 9.8 Tf [(, and our work now extends this observation to two different HD mouse models.)] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(The ablation of CASP6 in BACHD mice aged 13 months resulted in a reduction of both wild type and mutant HTT levels, )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(possibly due to the activation of protein clearance pathways )] TJ ET 0.267 0.267 0.267 rg BT 286.380 713.864 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 296.017 712.357 Td /F1 9.8 Tf [(. We performed TR-FRET to quantify levels of both mutant HTT )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(and total HTT and found that the absence of CASP6 had no effect on soluble HTT levels in presymptomatic mice. Such a )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(difference in the effect of ablating CASP6 on HTT levels in young \(2 month\) )] TJ ET BT 354.113 688.548 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 371.995 688.548 Td /F1 9.8 Tf [(Q150 and aged \(13 month\) BACHD mice is not )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(clear. It could be that protein clearance pathways are regulated by CASP6 only at later stages of disease, particularly when )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(toxic HTT aggregates are present.)] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(Preventing the formation of N-terminal HTT fragments is an attractive therapeutic strategy that needs to be understood. Our )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(data suggest that CASP6 is not necessary for the proteolysis of mutant HTT in )] TJ ET BT 366.574 633.429 Td /F5 9.8 Tf [(Hdh)] TJ ET BT 384.455 633.429 Td /F1 9.8 Tf [(Q150 mice and by implication does not play )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(a direct role in HD pathogenesis in this mouse model via this mechanism. Our work highlights the importance of identifying the )] TJ ET BT 26.250 609.619 Td /F1 9.8 Tf [(source of HTT proteolytic fragments to allow the design of rational therapeutic strategies when considering how one should )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(combat the many HD-related phenotypes associated with this complex neurodegenerative disease.)] TJ ET BT 26.250 561.112 Td /F4 12.0 Tf [(COMPETING INTERESTS)] TJ ET BT 26.250 541.158 Td /F1 9.8 Tf [(AW is an employee of Novartis AG.)] TJ ET BT 26.250 504.555 Td /F4 12.0 Tf [(ACKNOWLEDGEMENTS)] TJ ET BT 26.250 484.601 Td /F1 9.8 Tf [(We thank Dr. Anna Bobrowska and members of the Neurogenetics Laboratory for critical advice on the manuscript.)] TJ ET BT 26.250 447.998 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 420.544 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 420.544 Td /F1 9.8 Tf [(Bates GP, Benn C \(2002\) The Polyglutamine Diseases. In: Bates GP, Harper PS, Jones AL, editors. Huntington's Disease. )] TJ ET BT 26.250 408.639 Td /F1 9.8 Tf [(3rd ed. Oxford: Oxford University Press. pp. 429-472.)] TJ ET BT 26.250 389.235 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 389.235 Td /F1 9.8 Tf [(Landles C, Bates GP \(2004\) Huntingtin and the molecular pathogenesis of Huntington's disease. Fourth in molecular )] TJ ET BT 26.250 377.330 Td /F1 9.8 Tf [(medicine review series. EMBO Rep 5: 958-963.)] TJ ET BT 26.250 357.925 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 357.925 Td /F1 9.8 Tf [(Harjes P, Wanker EE \(2003\) The hunt for huntingtin function: interaction partners tell many different stories. Trends Biochem )] TJ ET BT 26.250 346.020 Td /F1 9.8 Tf [(Sci 28: 425-433.)] TJ ET BT 26.250 326.616 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 326.616 Td /F1 9.8 Tf [(Lin CH, Tallaksen-Greene S, Chien WM, Cearley JA, Jackson WS, et al. \(2001\) Neurological abnormalities in a knock-in )] TJ ET BT 26.250 314.711 Td /F1 9.8 Tf [(mouse model of Huntington's disease. Hum Mol Genet 10: 137-144.)] TJ ET BT 26.250 295.306 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 295.306 Td /F1 9.8 Tf [(Woodman B, Butler R, Landles C, Lupton MK, Tse J, et al. \(2007\) The Hdh\(Q150/Q150\) knock-in mouse model of HD and )] TJ ET BT 26.250 283.401 Td /F1 9.8 Tf [(the R6/2 exon 1 model develop comparable and widespread molecular phenotypes. Brain Res Bull 72: 83-97.)] TJ ET BT 26.250 263.997 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 263.997 Td /F1 9.8 Tf [(Landles C, Sathasivam K, Weiss A, Woodman B, Moffitt H, et al. \(2010\) Proteolysis of mutant huntingtin produces an exon 1 )] TJ ET BT 26.250 252.092 Td /F1 9.8 Tf [(fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem 285: 8808-8823.)] TJ ET BT 26.250 232.687 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 232.687 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, et al. \(1997\) Aggregation of huntingtin in neuronal intranuclear )] TJ ET BT 26.250 220.782 Td /F1 9.8 Tf [(inclusions and dystrophic neurites in brain. Science 277: 1990-1993.)] TJ ET BT 26.250 201.378 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 201.378 Td /F1 9.8 Tf [(Lunkes A, Lindenberg KS, Ben-Haiem L, Weber C, Devys D, et al. \(2002\) Proteases acting on mutant huntingtin generate )] TJ ET BT 26.250 189.473 Td /F1 9.8 Tf [(cleaved products that differentially build up cytoplasmic and nuclear inclusions. Mol Cell 10: 259-269.)] TJ ET BT 26.250 170.068 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 170.068 Td /F1 9.8 Tf [(Ratovitski T, Nakamura M, D'Ambola J, Chighladze E, Liang Y, et al. \(2007\) N-terminal proteolysis of full-length mutant )] TJ ET BT 26.250 158.163 Td /F1 9.8 Tf [(huntingtin in an inducible PC12 cell model of Huntington's disease. Cell Cycle 6: 2970-2981.)] TJ ET BT 26.250 138.759 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 138.759 Td /F1 9.8 Tf [(Ratovitski T, Gucek M, Jiang H, Chighladze E, Waldron E, et al. \(2009\) Mutant huntingtin N-terminal fragments of specific )] TJ ET BT 26.250 126.854 Td /F1 9.8 Tf [(size mediate aggregation and toxicity in neuronal cells. J Biol Chem 284: 10855-10867.)] TJ ET BT 26.250 107.449 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 107.449 Td /F1 9.8 Tf [(Wellington CL, Ellerby LM, Gutekunst CA, Rogers D, Warby S, et al. \(2002\) Caspase cleavage of mutant huntingtin )] TJ ET BT 26.250 95.544 Td /F1 9.8 Tf [(precedes neurodegeneration in Huntington's disease. J Neurosci 22: 7862-7872.)] TJ ET BT 26.250 76.140 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 76.140 Td /F1 9.8 Tf [(Kim YJ, Yi Y, Sapp E, Wang Y, Cuiffo B, et al. \(2001\) Caspase 3-cleaved N-terminal fragments of wild-type and mutant )] TJ ET BT 26.250 64.235 Td /F1 9.8 Tf [(huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent )] TJ ET BT 26.250 52.330 Td /F1 9.8 Tf [(proteolysis. Proc Natl Acad Sci U S A 98: 12784-12789.)] TJ ET 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 359 0 obj << /Type /Annot /Subtype /Link /A 360 0 R /Border [0 0 0] /H /I /Rect [ 214.2885 744.3722 223.9258 753.1905 ] >> endobj 360 0 obj << /Type /Action >> endobj 361 0 obj << /Type /Annot /Subtype /Link /A 362 0 R /Border [0 0 0] /H /I /Rect [ 286.3800 713.0627 296.0173 721.8810 ] >> endobj 362 0 obj << /Type /Action >> endobj 363 0 obj << /Type /Annot /Subtype /Link /A 364 0 R /Border [0 0 0] /H /I /Rect [ 214.2885 744.3722 223.9258 753.1905 ] >> endobj 364 0 obj << /Type /Action >> endobj 365 0 obj << /Type /Annot /Subtype /Link /A 366 0 R /Border [0 0 0] /H /I /Rect [ 286.3800 713.0627 296.0173 721.8810 ] >> endobj 366 0 obj << /Type /Action >> endobj 367 0 obj << /Type /Annot /Subtype /Link /A 368 0 R /Border [0 0 0] /H /I /Rect [ 214.2885 744.3722 223.9258 753.1905 ] >> endobj 368 0 obj << /Type /Action >> endobj 369 0 obj << /Type /Annot /Subtype /Link /A 370 0 R /Border [0 0 0] /H /I /Rect [ 286.3800 713.0627 296.0173 721.8810 ] >> endobj 370 0 obj << /Type /Action >> endobj 371 0 obj << /Type /Page /Parent 3 0 R /Contents 372 0 R >> endobj 372 0 obj << /Length 16285 >> stream 0.271 0.267 0.267 rg q 15.000 256.643 577.500 520.357 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Gafni J, Hermel E, Young JE, Wellington CL, Hayden MR, et al. \(2004\) Inhibition of calpain cleavage of huntingtin reduces )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(toxicity: accumulation of calpain/caspase fragments in the nucleus. J Biol Chem 279: 20211-20220.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Miller JP, Holcomb J, Al-Ramahi I, de Haro M, Gafni J, et al. \(2010\) Matrix metalloproteinases are modifiers of huntingtin )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(proteolysis and toxicity in Huntington's disease. Neuron 67: 199-212.)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Kim YJ, Sapp E, Cuiffo BG, Sobin L, Yoder J, et al. \(2006\) Lysosomal proteases are involved in generation of N-terminal )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(huntingtin fragments. Neurobiol Dis 22: 346-356.)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Schilling G, Klevytska A, Tebbenkamp AT, Juenemann K, Cooper J, et al. \(2007\) Characterization of huntingtin pathologic )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(fragments in human Huntington disease, transgenic mice, and cell models. J Neuropathol Exp Neurol 66: 313-320.)] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 634.738 Td /F1 9.8 Tf [(Sun B, Fan W, Balciunas A, Cooper JK, Bitan G, et al. \(2002\) Polyglutamine repeat length-dependent proteolysis of )] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(huntingtin. Neurobiol Dis 11: 111-122.)] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 603.429 Td /F1 9.8 Tf [(Tanaka Y, Igarashi S, Nakamura M, Gafni J, Torcassi C, et al. \(2006\) Progressive phenotype and nuclear accumulation of )] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(an amino-terminal cleavage fragment in a transgenic mouse model with inducible expression of full-length mutant huntingtin. )] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(Neurobiol Dis 21: 381-391.)] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 560.214 Td /F1 9.8 Tf [(Waldron-Roby E, Ratovitski T, Wang X, Jiang M, Watkin E, et al. \(2012\) Transgenic mouse model expressing the caspase 6 )] TJ ET BT 26.250 548.310 Td /F1 9.8 Tf [(fragment of mutant huntingtin. J Neurosci 32: 183-193.)] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 528.905 Td /F1 9.8 Tf [(Graham RK, Deng Y, Slow EJ, Haigh B, Bissada N, et al. \(2006\) Cleavage at the caspase-6 site is required for neuronal )] TJ ET BT 26.250 517.000 Td /F1 9.8 Tf [(dysfunction and degeneration due to mutant huntingtin. Cell 125: 1179-1191.)] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 497.595 Td /F1 9.8 Tf [(Sathasivam K, Lane A, Legleiter J, Warley A, Woodman B, et al. \(2010\) Identical oligomeric and fibrillar structures captured )] TJ ET BT 26.250 485.691 Td /F1 9.8 Tf [(from the brains of R6/2 and knock-in mouse models of Huntington's disease. Hum Mol Genet 19: 65-78.)] TJ ET BT 26.250 466.286 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 466.286 Td /F1 9.8 Tf [(Uribe V, Wong BK, Graham RK, Cusack CL, Skotte NH, et al. \(2012\) Rescue from excitotoxicity and axonal degeneration )] TJ ET BT 26.250 454.381 Td /F1 9.8 Tf [(accompanied by age-dependent behavioral and neuroanatomical alterations in caspase-6-deficient mice. Hum Mol Genet 21: )] TJ ET BT 26.250 442.476 Td /F1 9.8 Tf [(1954-1967.)] TJ ET BT 26.250 423.072 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 423.072 Td /F1 9.8 Tf [(Ko J, Ou S, Patterson PH \(2001\) New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its )] TJ ET BT 26.250 411.167 Td /F1 9.8 Tf [(binding proteins. Brain Res Bull 56: 319-329.)] TJ ET BT 26.250 391.762 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 391.762 Td /F1 9.8 Tf [(Weiss A, Abramowski D, Bibel M, Bodner R, Chopra V, et al. \(2009\) Single-step detection of mutant huntingtin in animal )] TJ ET BT 26.250 379.857 Td /F1 9.8 Tf [(and human tissues: a bioassay for Huntington's disease. Anal Biochem 395: 8-15.)] TJ ET BT 26.250 360.453 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 360.453 Td /F1 9.8 Tf [(Weiss A, Grueninger S, Abramowski D, Giorgio FP, Lopatin MM, et al. \(2011\) Microtiter plate quantification of mutant and )] TJ ET BT 26.250 348.548 Td /F1 9.8 Tf [(wild-type huntingtin normalized to cell count. Anal Biochem 410: 304-306.)] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 329.143 Td /F1 9.8 Tf [(Sathasivam K, Woodman B, Mahal A, Bertaux F, Wanker EE, et al. \(2001\) Centrosome disorganization in fibroblast cultures )] TJ ET BT 26.250 317.238 Td /F1 9.8 Tf [(derived from R6/2 Huntington's disease \(HD\) transgenic mice and HD patients. Hum Mol Genet 10: 2425-2435.)] TJ ET BT 26.250 297.834 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 297.834 Td /F1 9.8 Tf [(Gafni J, Papanikolaou T, Degiacomo F, Holcomb J, Chen S, et al. \(2012\) Caspase-6 Activity in a BACHD Mouse Modulates )] TJ ET BT 26.250 285.929 Td /F1 9.8 Tf [(Steady-State Levels of Mutant Huntingtin Protein But Is Not Necessary for Production of a 586 Amino Acid Proteolytic )] TJ ET BT 26.250 274.024 Td /F1 9.8 Tf [(Fragment. J Neurosci 32: 7454-7465.)] TJ ET Q q 15.000 256.643 577.500 520.357 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Gafni J, Hermel E, Young JE, Wellington CL, Hayden MR, et al. \(2004\) Inhibition of calpain cleavage of huntingtin reduces )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(toxicity: accumulation of calpain/caspase fragments in the nucleus. J Biol Chem 279: 20211-20220.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Miller JP, Holcomb J, Al-Ramahi I, de Haro M, Gafni J, et al. \(2010\) Matrix metalloproteinases are modifiers of huntingtin )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(proteolysis and toxicity in Huntington's disease. Neuron 67: 199-212.)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Kim YJ, Sapp E, Cuiffo BG, Sobin L, Yoder J, et al. \(2006\) Lysosomal proteases are involved in generation of N-terminal )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(huntingtin fragments. Neurobiol Dis 22: 346-356.)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Schilling G, Klevytska A, Tebbenkamp AT, Juenemann K, Cooper J, et al. \(2007\) Characterization of huntingtin pathologic )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(fragments in human Huntington disease, transgenic mice, and cell models. J Neuropathol Exp Neurol 66: 313-320.)] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 634.738 Td /F1 9.8 Tf [(Sun B, Fan W, Balciunas A, Cooper JK, Bitan G, et al. \(2002\) Polyglutamine repeat length-dependent proteolysis of )] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(huntingtin. Neurobiol Dis 11: 111-122.)] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 603.429 Td /F1 9.8 Tf [(Tanaka Y, Igarashi S, Nakamura M, Gafni J, Torcassi C, et al. \(2006\) Progressive phenotype and nuclear accumulation of )] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(an amino-terminal cleavage fragment in a transgenic mouse model with inducible expression of full-length mutant huntingtin. )] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(Neurobiol Dis 21: 381-391.)] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 560.214 Td /F1 9.8 Tf [(Waldron-Roby E, Ratovitski T, Wang X, Jiang M, Watkin E, et al. \(2012\) Transgenic mouse model expressing the caspase 6 )] TJ ET BT 26.250 548.310 Td /F1 9.8 Tf [(fragment of mutant huntingtin. J Neurosci 32: 183-193.)] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 528.905 Td /F1 9.8 Tf [(Graham RK, Deng Y, Slow EJ, Haigh B, Bissada N, et al. \(2006\) Cleavage at the caspase-6 site is required for neuronal )] TJ ET BT 26.250 517.000 Td /F1 9.8 Tf [(dysfunction and degeneration due to mutant huntingtin. Cell 125: 1179-1191.)] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 497.595 Td /F1 9.8 Tf [(Sathasivam K, Lane A, Legleiter J, Warley A, Woodman B, et al. \(2010\) Identical oligomeric and fibrillar structures captured )] TJ ET BT 26.250 485.691 Td /F1 9.8 Tf [(from the brains of R6/2 and knock-in mouse models of Huntington's disease. Hum Mol Genet 19: 65-78.)] TJ ET BT 26.250 466.286 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 466.286 Td /F1 9.8 Tf [(Uribe V, Wong BK, Graham RK, Cusack CL, Skotte NH, et al. \(2012\) Rescue from excitotoxicity and axonal degeneration )] TJ ET BT 26.250 454.381 Td /F1 9.8 Tf [(accompanied by age-dependent behavioral and neuroanatomical alterations in caspase-6-deficient mice. Hum Mol Genet 21: )] TJ ET BT 26.250 442.476 Td /F1 9.8 Tf [(1954-1967.)] TJ ET BT 26.250 423.072 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 423.072 Td /F1 9.8 Tf [(Ko J, Ou S, Patterson PH \(2001\) New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its )] TJ ET BT 26.250 411.167 Td /F1 9.8 Tf [(binding proteins. Brain Res Bull 56: 319-329.)] TJ ET BT 26.250 391.762 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 391.762 Td /F1 9.8 Tf [(Weiss A, Abramowski D, Bibel M, Bodner R, Chopra V, et al. \(2009\) Single-step detection of mutant huntingtin in animal )] TJ ET BT 26.250 379.857 Td /F1 9.8 Tf [(and human tissues: a bioassay for Huntington's disease. Anal Biochem 395: 8-15.)] TJ ET BT 26.250 360.453 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 360.453 Td /F1 9.8 Tf [(Weiss A, Grueninger S, Abramowski D, Giorgio FP, Lopatin MM, et al. \(2011\) Microtiter plate quantification of mutant and )] TJ ET BT 26.250 348.548 Td /F1 9.8 Tf [(wild-type huntingtin normalized to cell count. Anal Biochem 410: 304-306.)] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 329.143 Td /F1 9.8 Tf [(Sathasivam K, Woodman B, Mahal A, Bertaux F, Wanker EE, et al. \(2001\) Centrosome disorganization in fibroblast cultures )] TJ ET BT 26.250 317.238 Td /F1 9.8 Tf [(derived from R6/2 Huntington's disease \(HD\) transgenic mice and HD patients. Hum Mol Genet 10: 2425-2435.)] TJ ET BT 26.250 297.834 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 297.834 Td /F1 9.8 Tf [(Gafni J, Papanikolaou T, Degiacomo F, Holcomb J, Chen S, et al. \(2012\) Caspase-6 Activity in a BACHD Mouse Modulates )] TJ ET BT 26.250 285.929 Td /F1 9.8 Tf [(Steady-State Levels of Mutant Huntingtin Protein But Is Not Necessary for Production of a 586 Amino Acid Proteolytic )] TJ ET BT 26.250 274.024 Td /F1 9.8 Tf [(Fragment. J Neurosci 32: 7454-7465.)] TJ ET Q q 15.000 256.643 577.500 520.357 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Gafni J, Hermel E, Young JE, Wellington CL, Hayden MR, et al. \(2004\) Inhibition of calpain cleavage of huntingtin reduces )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(toxicity: accumulation of calpain/caspase fragments in the nucleus. J Biol Chem 279: 20211-20220.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Miller JP, Holcomb J, Al-Ramahi I, de Haro M, Gafni J, et al. \(2010\) Matrix metalloproteinases are modifiers of huntingtin )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(proteolysis and toxicity in Huntington's disease. Neuron 67: 199-212.)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Kim YJ, Sapp E, Cuiffo BG, Sobin L, Yoder J, et al. \(2006\) Lysosomal proteases are involved in generation of N-terminal )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(huntingtin fragments. Neurobiol Dis 22: 346-356.)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Schilling G, Klevytska A, Tebbenkamp AT, Juenemann K, Cooper J, et al. \(2007\) Characterization of huntingtin pathologic )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(fragments in human Huntington disease, transgenic mice, and cell models. J Neuropathol Exp Neurol 66: 313-320.)] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 634.738 Td /F1 9.8 Tf [(Sun B, Fan W, Balciunas A, Cooper JK, Bitan G, et al. \(2002\) Polyglutamine repeat length-dependent proteolysis of )] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(huntingtin. Neurobiol Dis 11: 111-122.)] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 603.429 Td /F1 9.8 Tf [(Tanaka Y, Igarashi S, Nakamura M, Gafni J, Torcassi C, et al. \(2006\) Progressive phenotype and nuclear accumulation of )] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(an amino-terminal cleavage fragment in a transgenic mouse model with inducible expression of full-length mutant huntingtin. )] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(Neurobiol Dis 21: 381-391.)] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 560.214 Td /F1 9.8 Tf [(Waldron-Roby E, Ratovitski T, Wang X, Jiang M, Watkin E, et al. \(2012\) Transgenic mouse model expressing the caspase 6 )] TJ ET BT 26.250 548.310 Td /F1 9.8 Tf [(fragment of mutant huntingtin. J Neurosci 32: 183-193.)] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 528.905 Td /F1 9.8 Tf [(Graham RK, Deng Y, Slow EJ, Haigh B, Bissada N, et al. \(2006\) Cleavage at the caspase-6 site is required for neuronal )] TJ ET BT 26.250 517.000 Td /F1 9.8 Tf [(dysfunction and degeneration due to mutant huntingtin. Cell 125: 1179-1191.)] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 497.595 Td /F1 9.8 Tf [(Sathasivam K, Lane A, Legleiter J, Warley A, Woodman B, et al. \(2010\) Identical oligomeric and fibrillar structures captured )] TJ ET BT 26.250 485.691 Td /F1 9.8 Tf [(from the brains of R6/2 and knock-in mouse models of Huntington's disease. Hum Mol Genet 19: 65-78.)] TJ ET BT 26.250 466.286 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 466.286 Td /F1 9.8 Tf [(Uribe V, Wong BK, Graham RK, Cusack CL, Skotte NH, et al. \(2012\) Rescue from excitotoxicity and axonal degeneration )] TJ ET BT 26.250 454.381 Td /F1 9.8 Tf [(accompanied by age-dependent behavioral and neuroanatomical alterations in caspase-6-deficient mice. Hum Mol Genet 21: )] TJ ET BT 26.250 442.476 Td /F1 9.8 Tf [(1954-1967.)] TJ ET BT 26.250 423.072 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 423.072 Td /F1 9.8 Tf [(Ko J, Ou S, Patterson PH \(2001\) New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its )] TJ ET BT 26.250 411.167 Td /F1 9.8 Tf [(binding proteins. Brain Res Bull 56: 319-329.)] TJ ET BT 26.250 391.762 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 391.762 Td /F1 9.8 Tf [(Weiss A, Abramowski D, Bibel M, Bodner R, Chopra V, et al. \(2009\) Single-step detection of mutant huntingtin in animal )] TJ ET BT 26.250 379.857 Td /F1 9.8 Tf [(and human tissues: a bioassay for Huntington's disease. Anal Biochem 395: 8-15.)] TJ ET BT 26.250 360.453 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 360.453 Td /F1 9.8 Tf [(Weiss A, Grueninger S, Abramowski D, Giorgio FP, Lopatin MM, et al. \(2011\) Microtiter plate quantification of mutant and )] TJ ET BT 26.250 348.548 Td /F1 9.8 Tf [(wild-type huntingtin normalized to cell count. Anal Biochem 410: 304-306.)] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 329.143 Td /F1 9.8 Tf [(Sathasivam K, Woodman B, Mahal A, Bertaux F, Wanker EE, et al. \(2001\) Centrosome disorganization in fibroblast cultures )] TJ ET BT 26.250 317.238 Td /F1 9.8 Tf [(derived from R6/2 Huntington's disease \(HD\) transgenic mice and HD patients. Hum Mol Genet 10: 2425-2435.)] TJ ET BT 26.250 297.834 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 297.834 Td /F1 9.8 Tf [(Gafni J, Papanikolaou T, Degiacomo F, Holcomb J, Chen S, et al. \(2012\) Caspase-6 Activity in a BACHD Mouse Modulates )] TJ ET BT 26.250 285.929 Td /F1 9.8 Tf [(Steady-State Levels of Mutant Huntingtin Protein But Is Not Necessary for Production of a 586 Amino Acid Proteolytic )] TJ ET BT 26.250 274.024 Td /F1 9.8 Tf [(Fragment. 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