%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 170 0 R 250 0 R 260 0 R 310 0 R 355 0 R 370 0 R 391 0 R 507 0 R 521 0 R 530 0 R 539 0 R ] /Count 12 /Resources << /ProcSet 4 0 R /Font << /F1 8 0 R /F2 9 0 R /F3 10 0 R /F4 11 0 R /F5 252 0 R >> /XObject << /I1 12 0 R /I2 13 0 R /I3 255 0 R /I4 326 0 R /I5 361 0 R /I6 378 0 R /I7 525 0 R /I8 534 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:20180721063939+00'00') /ModDate (D:20180721063939+00'00') /Title (Pitfalls in the detection of cholesterol in Huntingtons disease models PLOS Currents Huntington Disease) >> endobj 6 0 obj << /Type /Page /Parent 3 0 R /Annots [ 14 0 R 16 0 R 18 0 R 20 0 R 22 0 R 24 0 R 26 0 R 28 0 R 30 0 R 32 0 R 34 0 R 36 0 R 38 0 R 40 0 R 42 0 R 44 0 R 46 0 R 48 0 R 50 0 R 52 0 R 54 0 R 56 0 R 58 0 R 60 0 R 62 0 R 64 0 R 66 0 R 68 0 R 70 0 R 72 0 R 74 0 R 76 0 R 78 0 R 80 0 R 82 0 R 84 0 R 86 0 R 88 0 R 90 0 R 92 0 R 94 0 R 96 0 R 98 0 R 100 0 R 102 0 R 104 0 R 106 0 R 108 0 R 110 0 R 112 0 R 114 0 R 116 0 R 118 0 R 120 0 R 122 0 R 124 0 R 126 0 R 128 0 R 130 0 R 132 0 R 134 0 R 136 0 R 138 0 R 140 0 R 142 0 R 144 0 R 146 0 R 148 0 R 150 0 R 152 0 R 154 0 R 156 0 R 158 0 R 160 0 R 162 0 R 164 0 R 166 0 R 168 0 R ] /Contents 7 0 R >> endobj 7 0 obj << /Length 30659 >> stream q 375.000 0 0 39.000 222.000 738.000 cm /I2 Do Q q 15.000 662.316 577.500 75.684 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Pitfalls in the detection of cholesterol in Huntingtons disease )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(models)] TJ ET 0.271 0.267 0.267 rg BT 15.000 671.524 Td /F2 13.5 Tf [(Technical considerations in the detection of cholesterol in Huntingtons disease samples)] TJ ET Q BT 15.000 653.050 Td /F3 9.8 Tf [(October 11, 2012)] TJ ET BT 88.388 653.050 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 93.263 653.050 Td /F3 9.8 Tf [(HD Models)] TJ ET BT 26.250 641.209 Td /F1 9.8 Tf [(Manuela Marullo)] TJ ET 0.271 0.267 0.267 rg BT 98.312 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 103.733 641.209 Td /F1 9.8 Tf [(Marta Valenza)] TJ ET 0.271 0.267 0.267 rg BT 166.591 645.097 Td /F1 8.7 Tf [(1)] TJ ET BT 171.410 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 176.831 641.209 Td /F1 9.8 Tf [(Valerio Leoni)] TJ ET 0.271 0.267 0.267 rg BT 233.732 645.097 Td /F1 8.7 Tf [(2)] TJ ET BT 238.551 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 243.972 641.209 Td /F1 9.8 Tf [(Claudio Caccia)] TJ ET 0.271 0.267 0.267 rg BT 309.531 645.097 Td /F1 8.7 Tf [(3)] TJ ET BT 314.350 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 319.771 641.209 Td /F1 9.8 Tf [(Chiara Scarlatti)] TJ ET 0.271 0.267 0.267 rg BT 386.412 645.097 Td /F1 8.7 Tf [(4)] TJ ET BT 391.230 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 396.651 641.209 Td /F1 9.8 Tf [(Agnese De Mario)] TJ ET 0.271 0.267 0.267 rg BT 471.970 645.097 Td /F1 8.7 Tf [(4)] TJ ET BT 476.789 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 482.210 641.209 Td /F1 9.8 Tf [(Chiara Zuccato)] TJ ET 0.271 0.267 0.267 rg BT 548.315 645.097 Td /F1 8.7 Tf [(5)] TJ ET BT 553.134 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 26.250 629.304 Td /F1 9.8 Tf [(Stefano Di Donato)] TJ ET 0.271 0.267 0.267 rg BT 105.917 633.193 Td /F1 8.7 Tf [(6)] TJ ET BT 110.736 629.304 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 116.157 629.304 Td /F1 9.8 Tf [(Ernesto Carafoli)] TJ ET 0.271 0.267 0.267 rg BT 186.055 633.193 Td /F1 8.7 Tf [(4)] TJ ET BT 190.873 629.304 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 196.294 629.304 Td /F1 9.8 Tf [(Elena Cattaneo)] TJ ET 0.271 0.267 0.267 rg BT 26.250 618.132 Td /F4 9.0 Tf [(1)] TJ ET BT 31.254 618.132 Td /F1 9.0 Tf [( Universit degli Studi di Milano, )] TJ ET BT 161.799 618.132 Td /F4 9.0 Tf [(2)] TJ ET BT 166.803 618.132 Td /F1 9.0 Tf [( Foundation IRCCS Insitute of Neurology Carlo Besta, )] TJ ET BT 384.396 618.132 Td /F4 9.0 Tf [(3)] TJ ET BT 389.400 618.132 Td /F1 9.0 Tf [( IRCCS-Istituto Nazionale Neurologico Carlo )] TJ ET BT 26.250 607.143 Td /F1 9.0 Tf [(Besta, )] TJ ET BT 54.267 607.143 Td /F4 9.0 Tf [(4)] TJ ET BT 59.271 607.143 Td /F1 9.0 Tf [( Venetian Institute for Molecular Medicine, )] TJ ET BT 229.344 607.143 Td /F4 9.0 Tf [(5)] TJ ET BT 234.348 607.143 Td /F1 9.0 Tf [( University of Milan, )] TJ ET BT 315.366 607.143 Td /F4 9.0 Tf [(6)] TJ ET BT 320.370 607.143 Td /F1 9.0 Tf [( IRCCS Istituto Neurologico Carlo Besta)] TJ ET BT 26.250 595.422 Td /F1 9.8 Tf [(Marullo M, Valenza M, Leoni V, Caccia C, Scarlatti C, De Mario A, Zuccato C, Di Donato S, Carafoli E, Cattaneo E. Pitfalls in )] TJ ET BT 26.250 583.517 Td /F1 9.8 Tf [(the detection of cholesterol in Huntingtons disease models: Technical considerations in the detection of cholesterol in )] TJ ET BT 26.250 571.612 Td /F1 9.8 Tf [(Huntingtons disease samples. PLOS Currents Huntington Disease. 2012 Oct 11 . Edition 1. doi: 10.1371/505886e9a1968.)] TJ ET q 15.000 23.014 577.500 546.218 re W n 0.271 0.267 0.267 rg BT 26.250 542.510 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 522.555 Td /F1 9.8 Tf [(Background)] TJ ET BT 26.250 510.651 Td /F1 9.8 Tf [(Abnormalities in brain cholesterol homeostasis have been reported in Huntingtons disease \(HD\), an adult-onset )] TJ ET BT 26.250 498.746 Td /F1 9.8 Tf [(neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin \(HTT\) gene. However, the )] TJ ET BT 26.250 486.841 Td /F1 9.8 Tf [(results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and )] TJ ET BT 26.250 474.936 Td /F1 9.8 Tf [(mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of )] TJ ET BT 26.250 463.032 Td /F1 9.8 Tf [(several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, )] TJ ET BT 26.250 451.127 Td /F1 9.8 Tf [(colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods )] TJ ET BT 26.250 439.222 Td /F1 9.8 Tf [(to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein.)] TJ ET BT 26.250 419.817 Td /F1 9.8 Tf [(Results)] TJ ET BT 26.250 407.913 Td /F1 9.8 Tf [(Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical )] TJ ET BT 26.250 396.008 Td /F1 9.8 Tf [(method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality )] TJ ET BT 26.250 384.103 Td /F1 9.8 Tf [(also influenced the detection of intracellular cholesterol.)] TJ ET BT 26.250 364.698 Td /F1 9.8 Tf [(Conclusions)] TJ ET BT 26.250 352.794 Td /F1 9.8 Tf [(Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more )] TJ ET BT 26.250 340.889 Td /F1 9.8 Tf [(sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using )] TJ ET BT 26.250 328.984 Td /F1 9.8 Tf [(isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ )] TJ ET BT 26.250 317.079 Td /F1 9.8 Tf [(mutation in a constitutive or inducible manner.)] TJ ET BT 26.250 297.675 Td /F1 9.8 Tf [(*Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it)] TJ ET BT 26.250 261.072 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 241.118 Td /F1 9.8 Tf [(This work was supported by the Ministero della Salute and Fondi per giovani Ricercatori \(Italy; GR-2008-1145270\) to VL and )] TJ ET BT 26.250 229.213 Td /F1 9.8 Tf [(MV; STEM-HD \(EU, FP6\) and by the CHDI Foundation \(NY, USA\) to ECatt; by an ERANet-Neuron \(nEUROsyn\) Grant, and by )] TJ ET BT 26.250 217.308 Td /F1 9.8 Tf [(a Grant of the CARIPARO Foundation to ECar.)] TJ ET BT 26.250 188.206 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 168.252 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an adult-onset neurodegenerative disorder caused by an expansion in the CAG repeat in the 5 )] TJ ET BT 26.250 156.347 Td /F1 9.8 Tf [(terminus of the HD gene )] TJ ET 0.267 0.267 0.267 rg BT 134.631 157.854 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 139.450 156.347 Td /F1 9.8 Tf [(. In the Huntingtin protein \(HTT\), which is a ubiquitously expressed protein with beneficial roles in )] TJ ET BT 26.250 144.442 Td /F1 9.8 Tf [(brain neurons, the CAG repeat is translated into a polyglutamine \(polyQ\) tract )] TJ ET 0.267 0.267 0.267 rg BT 362.771 145.949 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 367.590 144.442 Td /F1 9.8 Tf [(. HD pathology involves multi-faceted )] TJ ET BT 26.250 132.537 Td /F1 9.8 Tf [(mechanisms that affect nearly all aspects of cellular physiology )] TJ ET 0.267 0.267 0.267 rg BT 300.440 134.045 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 305.258 132.537 Td /F1 9.8 Tf [(. Among these mechanisms, abnormalities in cholesterol )] TJ ET BT 26.250 120.633 Td /F1 9.8 Tf [(metabolism have been reported in HD cellular and animal models and in tissues from HD patients )] TJ ET 0.267 0.267 0.267 rg BT 449.449 122.140 Td /F4 8.7 Tf [(3)] TJ ET BT 454.267 122.140 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 459.086 120.633 Td /F1 9.8 Tf [(. In particular, mRNA levels )] TJ ET BT 26.250 108.728 Td /F1 9.8 Tf [(of key genes involved in cholesterol biosynthesis are reported to be reduced in clonal striatal-derived cells that overexpress )] TJ ET BT 26.250 96.823 Td /F1 9.8 Tf [(mutant HTT \(muHTT\) )] TJ ET 0.267 0.267 0.267 rg BT 122.132 98.330 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 126.950 96.823 Td /F1 9.8 Tf [( as well as in brain samples from HD mice and in post-mortem HD cerebral specimens )] TJ ET 0.267 0.267 0.267 rg BT 501.877 98.330 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 506.695 96.823 Td /F1 9.8 Tf [(. Biochemical )] TJ ET BT 26.250 84.918 Td /F1 9.8 Tf [(and mass spectrometry analyses confirmed reduced levels of cholesterol precursors early in the disease process in brains from )] TJ ET BT 26.250 73.014 Td /F1 9.8 Tf [(R6/2, YAC46, YAC72 and YAC128 knock-in mice and transgenic rats. At later time points, the same animals showed decreased )] TJ ET BT 26.250 61.109 Td /F1 9.8 Tf [(levels of sterols/cholesterol )] TJ ET 0.267 0.267 0.267 rg BT 146.000 62.616 Td /F4 8.7 Tf [(6)] TJ ET BT 150.818 62.616 Td /F4 8.7 Tf [(7)] TJ ET BT 155.637 62.616 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 160.456 61.109 Td /F1 9.8 Tf [(. Brain cholesterol homeostasis is also affected in humans in the early stages of the disease )] TJ ET 0.267 0.267 0.267 rg BT 559.309 62.616 Td /F4 8.7 Tf [(9)] TJ ET BT 564.127 62.616 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 573.765 61.109 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 49.204 Td /F1 9.8 Tf [(One possible mechanism that could explain this is a reduction in the nuclear translocation of SREBP, a master transcriptional )] TJ ET BT 26.250 37.299 Td /F1 9.8 Tf [(activator of several cholesterogenic genes )] TJ ET 0.267 0.267 0.267 rg BT 211.042 38.807 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 215.860 37.299 Td /F1 9.8 Tf [(. A recent report confirmed a reduced cholesterol level in neural stem \(NS\) cell lines )] TJ ET Q q 15.000 662.316 577.500 75.684 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Pitfalls in the detection of cholesterol in Huntingtons disease )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(models)] TJ ET 0.271 0.267 0.267 rg BT 15.000 671.524 Td /F2 13.5 Tf [(Technical considerations in the detection of cholesterol in Huntingtons disease samples)] TJ ET Q BT 15.000 653.050 Td /F3 9.8 Tf [(October 11, 2012)] TJ ET BT 88.388 653.050 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 93.263 653.050 Td /F3 9.8 Tf [(HD Models)] TJ ET BT 26.250 641.209 Td /F1 9.8 Tf [(Manuela Marullo)] TJ ET 0.271 0.267 0.267 rg BT 98.312 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 103.733 641.209 Td /F1 9.8 Tf [(Marta Valenza)] TJ ET 0.271 0.267 0.267 rg BT 166.591 645.097 Td /F1 8.7 Tf [(1)] TJ ET BT 171.410 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 176.831 641.209 Td /F1 9.8 Tf [(Valerio Leoni)] TJ ET 0.271 0.267 0.267 rg BT 233.732 645.097 Td /F1 8.7 Tf [(2)] TJ ET BT 238.551 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 243.972 641.209 Td /F1 9.8 Tf [(Claudio Caccia)] TJ ET 0.271 0.267 0.267 rg BT 309.531 645.097 Td /F1 8.7 Tf [(3)] TJ ET BT 314.350 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 319.771 641.209 Td /F1 9.8 Tf [(Chiara Scarlatti)] TJ ET 0.271 0.267 0.267 rg BT 386.412 645.097 Td /F1 8.7 Tf [(4)] TJ ET BT 391.230 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 396.651 641.209 Td /F1 9.8 Tf [(Agnese De Mario)] TJ ET 0.271 0.267 0.267 rg BT 471.970 645.097 Td /F1 8.7 Tf [(4)] TJ ET BT 476.789 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 482.210 641.209 Td /F1 9.8 Tf [(Chiara Zuccato)] TJ ET 0.271 0.267 0.267 rg BT 548.315 645.097 Td /F1 8.7 Tf [(5)] TJ ET BT 553.134 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 26.250 629.304 Td /F1 9.8 Tf [(Stefano Di Donato)] TJ ET 0.271 0.267 0.267 rg BT 105.917 633.193 Td /F1 8.7 Tf [(6)] TJ ET BT 110.736 629.304 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 116.157 629.304 Td /F1 9.8 Tf [(Ernesto Carafoli)] TJ ET 0.271 0.267 0.267 rg BT 186.055 633.193 Td /F1 8.7 Tf [(4)] TJ ET BT 190.873 629.304 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 196.294 629.304 Td /F1 9.8 Tf [(Elena Cattaneo)] TJ ET 0.271 0.267 0.267 rg BT 26.250 618.132 Td /F4 9.0 Tf [(1)] TJ ET BT 31.254 618.132 Td /F1 9.0 Tf [( Universit degli Studi di Milano, )] TJ ET BT 161.799 618.132 Td /F4 9.0 Tf [(2)] TJ ET BT 166.803 618.132 Td /F1 9.0 Tf [( Foundation IRCCS Insitute of Neurology Carlo Besta, )] TJ ET BT 384.396 618.132 Td /F4 9.0 Tf [(3)] TJ ET BT 389.400 618.132 Td /F1 9.0 Tf [( IRCCS-Istituto Nazionale Neurologico Carlo )] TJ ET BT 26.250 607.143 Td /F1 9.0 Tf [(Besta, )] TJ ET BT 54.267 607.143 Td /F4 9.0 Tf [(4)] TJ ET BT 59.271 607.143 Td /F1 9.0 Tf [( Venetian Institute for Molecular Medicine, )] TJ ET BT 229.344 607.143 Td /F4 9.0 Tf [(5)] TJ ET BT 234.348 607.143 Td /F1 9.0 Tf [( University of Milan, )] TJ ET BT 315.366 607.143 Td /F4 9.0 Tf [(6)] TJ ET BT 320.370 607.143 Td /F1 9.0 Tf [( IRCCS Istituto Neurologico Carlo Besta)] TJ ET BT 26.250 595.422 Td /F1 9.8 Tf [(Marullo M, Valenza M, Leoni V, Caccia C, Scarlatti C, De Mario A, Zuccato C, Di Donato S, Carafoli E, Cattaneo E. Pitfalls in )] TJ ET BT 26.250 583.517 Td /F1 9.8 Tf [(the detection of cholesterol in Huntingtons disease models: Technical considerations in the detection of cholesterol in )] TJ ET BT 26.250 571.612 Td /F1 9.8 Tf [(Huntingtons disease samples. PLOS Currents Huntington Disease. 2012 Oct 11 . Edition 1. doi: 10.1371/505886e9a1968.)] TJ ET q 15.000 23.014 577.500 546.218 re W n 0.271 0.267 0.267 rg BT 26.250 542.510 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 522.555 Td /F1 9.8 Tf [(Background)] TJ ET BT 26.250 510.651 Td /F1 9.8 Tf [(Abnormalities in brain cholesterol homeostasis have been reported in Huntingtons disease \(HD\), an adult-onset )] TJ ET BT 26.250 498.746 Td /F1 9.8 Tf [(neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin \(HTT\) gene. However, the )] TJ ET BT 26.250 486.841 Td /F1 9.8 Tf [(results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and )] TJ ET BT 26.250 474.936 Td /F1 9.8 Tf [(mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of )] TJ ET BT 26.250 463.032 Td /F1 9.8 Tf [(several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, )] TJ ET BT 26.250 451.127 Td /F1 9.8 Tf [(colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods )] TJ ET BT 26.250 439.222 Td /F1 9.8 Tf [(to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein.)] TJ ET BT 26.250 419.817 Td /F1 9.8 Tf [(Results)] TJ ET BT 26.250 407.913 Td /F1 9.8 Tf [(Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical )] TJ ET BT 26.250 396.008 Td /F1 9.8 Tf [(method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality )] TJ ET BT 26.250 384.103 Td /F1 9.8 Tf [(also influenced the detection of intracellular cholesterol.)] TJ ET BT 26.250 364.698 Td /F1 9.8 Tf [(Conclusions)] TJ ET BT 26.250 352.794 Td /F1 9.8 Tf [(Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more )] TJ ET BT 26.250 340.889 Td /F1 9.8 Tf [(sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using )] TJ ET BT 26.250 328.984 Td /F1 9.8 Tf [(isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ )] TJ ET BT 26.250 317.079 Td /F1 9.8 Tf [(mutation in a constitutive or inducible manner.)] TJ ET BT 26.250 297.675 Td /F1 9.8 Tf [(*Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it)] TJ ET BT 26.250 261.072 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 241.118 Td /F1 9.8 Tf [(This work was supported by the Ministero della Salute and Fondi per giovani Ricercatori \(Italy; GR-2008-1145270\) to VL and )] TJ ET BT 26.250 229.213 Td /F1 9.8 Tf [(MV; STEM-HD \(EU, FP6\) and by the CHDI Foundation \(NY, USA\) to ECatt; by an ERANet-Neuron \(nEUROsyn\) Grant, and by )] TJ ET BT 26.250 217.308 Td /F1 9.8 Tf [(a Grant of the CARIPARO Foundation to ECar.)] TJ ET BT 26.250 188.206 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 168.252 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an adult-onset neurodegenerative disorder caused by an expansion in the CAG repeat in the 5 )] TJ ET BT 26.250 156.347 Td /F1 9.8 Tf [(terminus of the HD gene )] TJ ET 0.267 0.267 0.267 rg BT 134.631 157.854 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 139.450 156.347 Td /F1 9.8 Tf [(. In the Huntingtin protein \(HTT\), which is a ubiquitously expressed protein with beneficial roles in )] TJ ET BT 26.250 144.442 Td /F1 9.8 Tf [(brain neurons, the CAG repeat is translated into a polyglutamine \(polyQ\) tract )] TJ ET 0.267 0.267 0.267 rg BT 362.771 145.949 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 367.590 144.442 Td /F1 9.8 Tf [(. HD pathology involves multi-faceted )] TJ ET BT 26.250 132.537 Td /F1 9.8 Tf [(mechanisms that affect nearly all aspects of cellular physiology )] TJ ET 0.267 0.267 0.267 rg BT 300.440 134.045 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 305.258 132.537 Td /F1 9.8 Tf [(. Among these mechanisms, abnormalities in cholesterol )] TJ ET BT 26.250 120.633 Td /F1 9.8 Tf [(metabolism have been reported in HD cellular and animal models and in tissues from HD patients )] TJ ET 0.267 0.267 0.267 rg BT 449.449 122.140 Td /F4 8.7 Tf [(3)] TJ ET BT 454.267 122.140 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 459.086 120.633 Td /F1 9.8 Tf [(. In particular, mRNA levels )] TJ ET BT 26.250 108.728 Td /F1 9.8 Tf [(of key genes involved in cholesterol biosynthesis are reported to be reduced in clonal striatal-derived cells that overexpress )] TJ ET BT 26.250 96.823 Td /F1 9.8 Tf [(mutant HTT \(muHTT\) )] TJ ET 0.267 0.267 0.267 rg BT 122.132 98.330 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 126.950 96.823 Td /F1 9.8 Tf [( as well as in brain samples from HD mice and in post-mortem HD cerebral specimens )] TJ ET 0.267 0.267 0.267 rg BT 501.877 98.330 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 506.695 96.823 Td /F1 9.8 Tf [(. Biochemical )] TJ ET BT 26.250 84.918 Td /F1 9.8 Tf [(and mass spectrometry analyses confirmed reduced levels of cholesterol precursors early in the disease process in brains from )] TJ ET BT 26.250 73.014 Td /F1 9.8 Tf [(R6/2, YAC46, YAC72 and YAC128 knock-in mice and transgenic rats. At later time points, the same animals showed decreased )] TJ ET BT 26.250 61.109 Td /F1 9.8 Tf [(levels of sterols/cholesterol )] TJ ET 0.267 0.267 0.267 rg BT 146.000 62.616 Td /F4 8.7 Tf [(6)] TJ ET BT 150.818 62.616 Td /F4 8.7 Tf [(7)] TJ ET BT 155.637 62.616 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 160.456 61.109 Td /F1 9.8 Tf [(. Brain cholesterol homeostasis is also affected in humans in the early stages of the disease )] TJ ET 0.267 0.267 0.267 rg BT 559.309 62.616 Td /F4 8.7 Tf [(9)] TJ ET BT 564.127 62.616 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 573.765 61.109 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 49.204 Td /F1 9.8 Tf [(One possible mechanism that could explain this is a reduction in the nuclear translocation of SREBP, a master transcriptional )] TJ ET BT 26.250 37.299 Td /F1 9.8 Tf [(activator of several cholesterogenic genes )] TJ ET 0.267 0.267 0.267 rg BT 211.042 38.807 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 215.860 37.299 Td /F1 9.8 Tf [(. A recent report confirmed a reduced cholesterol level in neural stem \(NS\) cell lines )] TJ ET Q q 15.000 662.316 577.500 75.684 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Pitfalls in the detection of cholesterol in Huntingtons disease )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(models)] TJ ET 0.271 0.267 0.267 rg BT 15.000 671.524 Td /F2 13.5 Tf [(Technical considerations in the detection of cholesterol in Huntingtons disease samples)] TJ ET Q BT 15.000 653.050 Td /F3 9.8 Tf [(October 11, 2012)] TJ ET BT 88.388 653.050 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 93.263 653.050 Td /F3 9.8 Tf [(HD Models)] TJ ET BT 26.250 641.209 Td /F1 9.8 Tf [(Manuela Marullo)] TJ ET 0.271 0.267 0.267 rg BT 98.312 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 103.733 641.209 Td /F1 9.8 Tf [(Marta Valenza)] TJ ET 0.271 0.267 0.267 rg BT 166.591 645.097 Td /F1 8.7 Tf [(1)] TJ ET BT 171.410 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 176.831 641.209 Td /F1 9.8 Tf [(Valerio Leoni)] TJ ET 0.271 0.267 0.267 rg BT 233.732 645.097 Td /F1 8.7 Tf [(2)] TJ ET BT 238.551 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 243.972 641.209 Td /F1 9.8 Tf [(Claudio Caccia)] TJ ET 0.271 0.267 0.267 rg BT 309.531 645.097 Td /F1 8.7 Tf [(3)] TJ ET BT 314.350 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 319.771 641.209 Td /F1 9.8 Tf [(Chiara Scarlatti)] TJ ET 0.271 0.267 0.267 rg BT 386.412 645.097 Td /F1 8.7 Tf [(4)] TJ ET BT 391.230 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 396.651 641.209 Td /F1 9.8 Tf [(Agnese De Mario)] TJ ET 0.271 0.267 0.267 rg BT 471.970 645.097 Td /F1 8.7 Tf [(4)] TJ ET BT 476.789 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 482.210 641.209 Td /F1 9.8 Tf [(Chiara Zuccato)] TJ ET 0.271 0.267 0.267 rg BT 548.315 645.097 Td /F1 8.7 Tf [(5)] TJ ET BT 553.134 641.209 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 26.250 629.304 Td /F1 9.8 Tf [(Stefano Di Donato)] TJ ET 0.271 0.267 0.267 rg BT 105.917 633.193 Td /F1 8.7 Tf [(6)] TJ ET BT 110.736 629.304 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 116.157 629.304 Td /F1 9.8 Tf [(Ernesto Carafoli)] TJ ET 0.271 0.267 0.267 rg BT 186.055 633.193 Td /F1 8.7 Tf [(4)] TJ ET BT 190.873 629.304 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 196.294 629.304 Td /F1 9.8 Tf [(Elena Cattaneo)] TJ ET 0.271 0.267 0.267 rg BT 26.250 618.132 Td /F4 9.0 Tf [(1)] TJ ET BT 31.254 618.132 Td /F1 9.0 Tf [( Universit degli Studi di Milano, )] TJ ET BT 161.799 618.132 Td /F4 9.0 Tf [(2)] TJ ET BT 166.803 618.132 Td /F1 9.0 Tf [( Foundation IRCCS Insitute of Neurology Carlo Besta, )] TJ ET BT 384.396 618.132 Td /F4 9.0 Tf [(3)] TJ ET BT 389.400 618.132 Td /F1 9.0 Tf [( IRCCS-Istituto Nazionale Neurologico Carlo )] TJ ET BT 26.250 607.143 Td /F1 9.0 Tf [(Besta, )] TJ ET BT 54.267 607.143 Td /F4 9.0 Tf [(4)] TJ ET BT 59.271 607.143 Td /F1 9.0 Tf [( Venetian Institute for Molecular Medicine, )] TJ ET BT 229.344 607.143 Td /F4 9.0 Tf [(5)] TJ ET BT 234.348 607.143 Td /F1 9.0 Tf [( University of Milan, )] TJ ET BT 315.366 607.143 Td /F4 9.0 Tf [(6)] TJ ET BT 320.370 607.143 Td /F1 9.0 Tf [( IRCCS Istituto Neurologico Carlo Besta)] TJ ET BT 26.250 595.422 Td /F1 9.8 Tf [(Marullo M, Valenza M, Leoni V, Caccia C, Scarlatti C, De Mario A, Zuccato C, Di Donato S, Carafoli E, Cattaneo E. Pitfalls in )] TJ ET BT 26.250 583.517 Td /F1 9.8 Tf [(the detection of cholesterol in Huntingtons disease models: Technical considerations in the detection of cholesterol in )] TJ ET BT 26.250 571.612 Td /F1 9.8 Tf [(Huntingtons disease samples. PLOS Currents Huntington Disease. 2012 Oct 11 . Edition 1. doi: 10.1371/505886e9a1968.)] TJ ET q 15.000 23.014 577.500 546.218 re W n 0.271 0.267 0.267 rg BT 26.250 542.510 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 522.555 Td /F1 9.8 Tf [(Background)] TJ ET BT 26.250 510.651 Td /F1 9.8 Tf [(Abnormalities in brain cholesterol homeostasis have been reported in Huntingtons disease \(HD\), an adult-onset )] TJ ET BT 26.250 498.746 Td /F1 9.8 Tf [(neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin \(HTT\) gene. However, the )] TJ ET BT 26.250 486.841 Td /F1 9.8 Tf [(results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and )] TJ ET BT 26.250 474.936 Td /F1 9.8 Tf [(mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of )] TJ ET BT 26.250 463.032 Td /F1 9.8 Tf [(several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, )] TJ ET BT 26.250 451.127 Td /F1 9.8 Tf [(colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods )] TJ ET BT 26.250 439.222 Td /F1 9.8 Tf [(to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein.)] TJ ET BT 26.250 419.817 Td /F1 9.8 Tf [(Results)] TJ ET BT 26.250 407.913 Td /F1 9.8 Tf [(Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical )] TJ ET BT 26.250 396.008 Td /F1 9.8 Tf [(method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality )] TJ ET BT 26.250 384.103 Td /F1 9.8 Tf [(also influenced the detection of intracellular cholesterol.)] TJ ET BT 26.250 364.698 Td /F1 9.8 Tf [(Conclusions)] TJ ET BT 26.250 352.794 Td /F1 9.8 Tf [(Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more )] TJ ET BT 26.250 340.889 Td /F1 9.8 Tf [(sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using )] TJ ET BT 26.250 328.984 Td /F1 9.8 Tf [(isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ )] TJ ET BT 26.250 317.079 Td /F1 9.8 Tf [(mutation in a constitutive or inducible manner.)] TJ ET BT 26.250 297.675 Td /F1 9.8 Tf [(*Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it)] TJ ET BT 26.250 261.072 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 241.118 Td /F1 9.8 Tf [(This work was supported by the Ministero della Salute and Fondi per giovani Ricercatori \(Italy; GR-2008-1145270\) to VL and )] TJ ET BT 26.250 229.213 Td /F1 9.8 Tf [(MV; STEM-HD \(EU, FP6\) and by the CHDI Foundation \(NY, USA\) to ECatt; by an ERANet-Neuron \(nEUROsyn\) Grant, and by )] TJ ET BT 26.250 217.308 Td /F1 9.8 Tf [(a Grant of the CARIPARO Foundation to ECar.)] TJ ET BT 26.250 188.206 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 168.252 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an adult-onset neurodegenerative disorder caused by an expansion in the CAG repeat in the 5 )] TJ ET BT 26.250 156.347 Td /F1 9.8 Tf [(terminus of the HD gene )] TJ ET 0.267 0.267 0.267 rg BT 134.631 157.854 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 139.450 156.347 Td /F1 9.8 Tf [(. In the Huntingtin protein \(HTT\), which is a ubiquitously expressed protein with beneficial roles in )] TJ ET BT 26.250 144.442 Td /F1 9.8 Tf [(brain neurons, the CAG repeat is translated into a polyglutamine \(polyQ\) tract )] TJ ET 0.267 0.267 0.267 rg BT 362.771 145.949 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 367.590 144.442 Td /F1 9.8 Tf [(. HD pathology involves multi-faceted )] TJ ET BT 26.250 132.537 Td /F1 9.8 Tf [(mechanisms that affect nearly all aspects of cellular physiology )] TJ ET 0.267 0.267 0.267 rg BT 300.440 134.045 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 305.258 132.537 Td /F1 9.8 Tf [(. Among these mechanisms, abnormalities in cholesterol )] TJ ET BT 26.250 120.633 Td /F1 9.8 Tf [(metabolism have been reported in HD cellular and animal models and in tissues from HD patients )] TJ ET 0.267 0.267 0.267 rg BT 449.449 122.140 Td /F4 8.7 Tf [(3)] TJ ET BT 454.267 122.140 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 459.086 120.633 Td /F1 9.8 Tf [(. In particular, mRNA levels )] TJ ET BT 26.250 108.728 Td /F1 9.8 Tf [(of key genes involved in cholesterol biosynthesis are reported to be reduced in clonal striatal-derived cells that overexpress )] TJ ET BT 26.250 96.823 Td /F1 9.8 Tf [(mutant HTT \(muHTT\) )] TJ ET 0.267 0.267 0.267 rg BT 122.132 98.330 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 126.950 96.823 Td /F1 9.8 Tf [( as well as in brain samples from HD mice and in post-mortem HD cerebral specimens )] TJ ET 0.267 0.267 0.267 rg BT 501.877 98.330 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 506.695 96.823 Td /F1 9.8 Tf [(. Biochemical )] TJ ET BT 26.250 84.918 Td /F1 9.8 Tf [(and mass spectrometry analyses confirmed reduced levels of cholesterol precursors early in the disease process in brains from )] TJ ET BT 26.250 73.014 Td /F1 9.8 Tf [(R6/2, YAC46, YAC72 and YAC128 knock-in mice and transgenic rats. At later time points, the same animals showed decreased )] TJ ET BT 26.250 61.109 Td /F1 9.8 Tf [(levels of sterols/cholesterol )] TJ ET 0.267 0.267 0.267 rg BT 146.000 62.616 Td /F4 8.7 Tf [(6)] TJ ET BT 150.818 62.616 Td /F4 8.7 Tf [(7)] TJ ET BT 155.637 62.616 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 160.456 61.109 Td /F1 9.8 Tf [(. Brain cholesterol homeostasis is also affected in humans in the early stages of the disease )] TJ ET 0.267 0.267 0.267 rg BT 559.309 62.616 Td /F4 8.7 Tf [(9)] TJ ET BT 564.127 62.616 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 573.765 61.109 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 49.204 Td /F1 9.8 Tf [(One possible mechanism that could explain this is a reduction in the nuclear translocation of SREBP, a master transcriptional )] TJ ET BT 26.250 37.299 Td /F1 9.8 Tf [(activator of several cholesterogenic genes )] TJ ET 0.267 0.267 0.267 rg BT 211.042 38.807 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 215.860 37.299 Td /F1 9.8 Tf [(. 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26.250 747.555 Td /F1 8.7 Tf [(140/7Q)] TJ ET BT 54.677 743.667 Td /F1 9.8 Tf [(\) )] TJ ET 0.267 0.267 0.267 rg BT 60.634 745.174 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 70.271 743.667 Td /F1 9.8 Tf [(. In contrast, other groups reported cholesterol accumulation, not cholesterol reduction, in mouse and human HD )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(tissues and cell cultures )] TJ ET 0.267 0.267 0.267 rg BT 132.457 733.269 Td /F4 8.7 Tf [(12)] TJ ET BT 142.094 733.269 Td /F4 8.7 Tf [(13)] TJ ET BT 151.731 733.269 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 161.369 731.762 Td /F1 9.8 Tf [(. In particular, it was reported that muHTT could interact with caveolin-1 and cause an )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(accumulation of intracellular cholesterol in primary neurons and in brains from the YAC72 transgenic mouse model of HD )] TJ ET 0.267 0.267 0.267 rg BT 550.235 721.364 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 559.872 719.857 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(Further, primary rat neurons that overexpress muHTT fragments showed sterol accumulation, and SIRT2 inhibition reduced )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(sterol levels via decreased nuclear trafficking of SREBP2 )] TJ ET 0.267 0.267 0.267 rg BT 274.972 697.555 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 284.610 696.048 Td /F1 9.8 Tf [(. Lastly, muHTT expression resulted in the accumulation of )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(cholesterol in HD cellular and murine models and in HD-affected human brains, and its altered cellular distribution contributed to )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(NMDA-mediated excitotoxicity )] TJ ET 0.267 0.267 0.267 rg BT 158.987 673.745 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 168.624 672.238 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(These conflicting data, which might be the result of the use of different techniques and experimental materials in the different )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(studies, raise the questions of whether cholesterol levels increase or decrease in HD and how this affects cellular and brain )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(dysfunction in the disease. A tentative unifying hypothesis suggests that decreased cholesterol biosynthesis in HD may be the )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(consequence of sterol accumulation )] TJ ET 0.267 0.267 0.267 rg BT 184.493 618.626 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 189.311 617.119 Td /F1 9.8 Tf [(, but further clarification regarding cholesterol level changes in HD is needed.)] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(In this study, we used several methods to measure sterols and cholesterol levels in immortalized knock-in cells derived from the )] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(embryonic striatum of mice carrying the endogenous HTT gene with 7Q \(ST Kin)] TJ ET BT 369.791 589.698 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 388.581 585.810 Td /F1 9.8 Tf [(\) or 109Q inserted into the mouse locus \(ST )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(Kin)] TJ ET BT 40.339 577.793 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 78.403 573.905 Td /F1 9.8 Tf [(\). We found different results depending on the technique adopted and that definitive methodology, such as gas )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(chromatography-mass spectrometry \(GC-MS\), should be favoured. Changes in cholesterol levels have been linked to NMDA )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(receptor activity )] TJ ET 0.267 0.267 0.267 rg BT 97.230 551.603 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 106.867 550.095 Td /F1 9.8 Tf [(, but we could not detect this receptor when analysing the same cells used in the earlier study. Our data also )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(reveal that sample preparation, degree of confluence and clonal properties strongly influence the results and, in particular, the )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(level of intracellular cholesterol that is detected. A further confounding factor may stem from changes in the properties or )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(technical issues related to the distribution and/or culture of target cell lines carrying the HD gene.)] TJ ET BT 26.250 477.779 Td /F4 12.0 Tf [(Results and discussion)] TJ ET BT 26.250 457.824 Td /F4 9.8 Tf [(Comparison of different methods that measure sterol content)] TJ ET BT 26.250 438.420 Td /F1 9.8 Tf [(A number of methods are available for measuring lipid and cholesterol content in cells. Among the commercially available kits, )] TJ ET BT 26.250 426.515 Td /F1 9.8 Tf [(the enzymatic method, which is based on cholesterol esterase and oxidase, has been used to measure cholesterol in HD )] TJ ET BT 26.250 414.610 Td /F1 9.8 Tf [(samples )] TJ ET 0.267 0.267 0.267 rg BT 65.260 416.117 Td /F4 8.7 Tf [(6)] TJ ET BT 70.078 416.117 Td /F4 8.7 Tf [(13)] TJ ET BT 79.716 416.117 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 89.353 414.610 Td /F1 9.8 Tf [(. This method is not specific for cholesterol but all sterols can be measured since the ?-OH group involved in the )] TJ ET BT 26.250 402.705 Td /F1 9.8 Tf [(enzymatic reaction mediated by cholesterol oxidase is common to many sterols. To quantify the extent of this possible )] TJ ET BT 26.250 390.801 Td /F1 9.8 Tf [(confounding effect, we loaded ST Kin)] TJ ET BT 187.759 394.689 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 206.548 390.801 Td /F1 9.8 Tf [( cells with increasing concentrations of exogenous cholesterol and then subjected the )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(cellular preparations to lipid detection by the enzymatic method.)] TJ ET BT 26.250 359.491 Td /F1 9.8 Tf [(Specifically, ST Kin)] TJ ET BT 109.691 363.379 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 128.480 359.491 Td /F1 9.8 Tf [( cells were cultured in normal growth medium and incubated with increasing concentrations of )] TJ ET BT 26.250 347.586 Td /F1 9.8 Tf [(cholesterol \(0, 5, 15, 30, 50 and 80 g/ml\) for 16 hours \(Fig. 1\). Lipids were then purified by solvent-based extraction and )] TJ ET BT 26.250 335.682 Td /F1 9.8 Tf [(measured with the Amplex Red Enzyme Assay \(Invitrogen\). Figure 1b shows that the enzymatic method detected the )] TJ ET BT 26.250 323.777 Td /F1 9.8 Tf [(accumulation of exogenous cholesterol in ST Kin)] TJ ET BT 237.055 327.665 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 255.844 323.777 Td /F1 9.8 Tf [( cells loaded with 5 g/ml and 15 g/ml cholesterol, while linearity was lost )] TJ ET BT 26.250 311.872 Td /F1 9.8 Tf [(at 30 g/ml. Higher doses of cholesterol were toxic, as evident in the phase contrast pictures of cultured cells treated with 50 )] TJ ET BT 26.250 299.967 Td /F1 9.8 Tf [(g/ml and 80 g/ml cholesterol \(Fig. 1a\).)] TJ ET BT 26.250 280.563 Td /F1 9.8 Tf [(These findings confirmed that the enzymatic method could reliably detect accumulation of exogenously supplied cholesterol in )] TJ ET BT 26.250 268.658 Td /F1 9.8 Tf [(cultured cells \(up to 15 g/ml\). In addition, the enzymatic detection may give misleading results when measuring subtle changes )] TJ ET BT 26.250 256.753 Td /F1 9.8 Tf [(in endogenous cholesterol levels as this assay reflects the total sterol rather than the total cholesterol content, as noted )] TJ ET BT 26.250 244.848 Td /F1 9.8 Tf [(previously )] TJ ET 0.267 0.267 0.267 rg BT 72.845 246.356 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 82.483 244.848 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 225.444 Td /F1 9.8 Tf [(Colorimetric methods \(filipin staining and Nile Red staining\) and gas chromatography-mass spectrometry \(GC-MS\) have also )] TJ ET BT 26.250 213.539 Td /F1 9.8 Tf [(been used to detect cholesterol levels. The colorimetric methods are based on fluorescent molecules that bind to lipids and )] TJ ET BT 26.250 201.634 Td /F1 9.8 Tf [(sterols with high affinity, while GC-MS is an analytical method that allows detection of cholesterol after its chromatographic )] TJ ET BT 26.250 189.729 Td /F1 9.8 Tf [(separation from other sterols and by isotope dilution with deuterium labelled internal standard. To compare the analytical )] TJ ET BT 26.250 177.825 Td /F1 9.8 Tf [(sensibility of these methods, we performed cholesterol loading or depletion of ST Kin)] TJ ET BT 391.495 181.713 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 412.693 177.825 Td /F1 9.8 Tf [(cells and then measured the )] TJ ET BT 26.250 165.920 Td /F1 9.8 Tf [(cholesterol content using both methods.)] TJ ET BT 26.250 146.515 Td /F1 9.8 Tf [(In the first set of experiments, ST Kin)] TJ ET BT 186.111 150.403 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 207.310 146.515 Td /F1 9.8 Tf [(cells were incubated with 0 \(vehicle\), 3, 5, 7 and 10 g/ml of cholesterol for 16 hours )] TJ ET BT 26.250 134.610 Td /F1 9.8 Tf [(and then analysed by filipin staining or GC-MS. As shown in Figure 1c, visual inspection of the stained cultures revealed a )] TJ ET BT 26.250 122.706 Td /F1 9.8 Tf [(difference in the filipin staining of cells cultured with 10 g/ml of cholesterol compared to control, but we could not detect any )] TJ ET BT 26.250 110.801 Td /F1 9.8 Tf [(differences in cultures treated with <7 g/ml cholesterol \(Fig. 1c; Suppl. Fig. 1a\). Notably, the fluorescence intensity varied from )] TJ ET BT 26.250 98.896 Td /F1 9.8 Tf [(field to field for the 10 g/ml culture, leading to a large SD \(Suppl. Fig. 1a\). GC-MS analysis showed a gradual dose-dependent )] TJ ET BT 26.250 86.991 Td /F1 9.8 Tf [(increase of cholesterol content in ST Kin)] TJ ET BT 200.746 90.880 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 221.944 86.991 Td /F1 9.8 Tf [(cells \(Fig. 1d\).)] TJ ET BT 26.250 67.587 Td /F1 9.8 Tf [(Next we exposed cultures of ST Kin)] TJ ET BT 180.690 71.475 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 201.889 67.587 Td /F1 9.8 Tf [(cells to increasing concentrations of methyl-beta-cyclodextrin \(M?CD; 0, 1, 5, 10 and )] TJ ET BT 26.250 55.682 Td /F1 9.8 Tf [(25 mM\) for 1 hour in order to deplete cells of cholesterol. The cholesterol content was then measured by filipin staining or by )] TJ ET BT 26.250 43.777 Td /F1 9.8 Tf [(GC-MS. Figure 1e and 1f show that the cholesterol depletion was detectable by both methods. However, GC-MS was more )] TJ ET Q q 15.000 29.492 577.500 747.508 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(developed from embryonic brains of wild-type and knock-in HD mice expressing full-length endogenous normal HTT \(Hdh)] TJ ET BT 549.679 771.364 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 568.468 767.476 Td /F1 9.8 Tf [(\) )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(or muHTT \(Hdh)] TJ ET BT 93.964 759.460 Td /F1 8.7 Tf [(140/140Q)] TJ ET BT 132.028 755.571 Td /F1 9.8 Tf [(\) but not in NS cell lines derived from embryonic stem cells of heterozygous knock-in HD mice \(Hdh)] TJ ET BT 26.250 747.555 Td /F1 8.7 Tf [(140/7Q)] TJ ET BT 54.677 743.667 Td /F1 9.8 Tf [(\) )] TJ ET 0.267 0.267 0.267 rg BT 60.634 745.174 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 70.271 743.667 Td /F1 9.8 Tf [(. In contrast, other groups reported cholesterol accumulation, not cholesterol reduction, in mouse and human HD )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(tissues and cell cultures )] TJ ET 0.267 0.267 0.267 rg BT 132.457 733.269 Td /F4 8.7 Tf [(12)] TJ ET BT 142.094 733.269 Td /F4 8.7 Tf [(13)] TJ ET BT 151.731 733.269 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 161.369 731.762 Td /F1 9.8 Tf [(. In particular, it was reported that muHTT could interact with caveolin-1 and cause an )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(accumulation of intracellular cholesterol in primary neurons and in brains from the YAC72 transgenic mouse model of HD )] TJ ET 0.267 0.267 0.267 rg BT 550.235 721.364 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 559.872 719.857 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(Further, primary rat neurons that overexpress muHTT fragments showed sterol accumulation, and SIRT2 inhibition reduced )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(sterol levels via decreased nuclear trafficking of SREBP2 )] TJ ET 0.267 0.267 0.267 rg BT 274.972 697.555 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 284.610 696.048 Td /F1 9.8 Tf [(. Lastly, muHTT expression resulted in the accumulation of )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(cholesterol in HD cellular and murine models and in HD-affected human brains, and its altered cellular distribution contributed to )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(NMDA-mediated excitotoxicity )] TJ ET 0.267 0.267 0.267 rg BT 158.987 673.745 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 168.624 672.238 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(These conflicting data, which might be the result of the use of different techniques and experimental materials in the different )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(studies, raise the questions of whether cholesterol levels increase or decrease in HD and how this affects cellular and brain )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(dysfunction in the disease. A tentative unifying hypothesis suggests that decreased cholesterol biosynthesis in HD may be the )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(consequence of sterol accumulation )] TJ ET 0.267 0.267 0.267 rg BT 184.493 618.626 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 189.311 617.119 Td /F1 9.8 Tf [(, but further clarification regarding cholesterol level changes in HD is needed.)] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(In this study, we used several methods to measure sterols and cholesterol levels in immortalized knock-in cells derived from the )] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(embryonic striatum of mice carrying the endogenous HTT gene with 7Q \(ST Kin)] TJ ET BT 369.791 589.698 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 388.581 585.810 Td /F1 9.8 Tf [(\) or 109Q inserted into the mouse locus \(ST )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(Kin)] TJ ET BT 40.339 577.793 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 78.403 573.905 Td /F1 9.8 Tf [(\). We found different results depending on the technique adopted and that definitive methodology, such as gas )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(chromatography-mass spectrometry \(GC-MS\), should be favoured. Changes in cholesterol levels have been linked to NMDA )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(receptor activity )] TJ ET 0.267 0.267 0.267 rg BT 97.230 551.603 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 106.867 550.095 Td /F1 9.8 Tf [(, but we could not detect this receptor when analysing the same cells used in the earlier study. Our data also )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(reveal that sample preparation, degree of confluence and clonal properties strongly influence the results and, in particular, the )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(level of intracellular cholesterol that is detected. A further confounding factor may stem from changes in the properties or )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(technical issues related to the distribution and/or culture of target cell lines carrying the HD gene.)] TJ ET BT 26.250 477.779 Td /F4 12.0 Tf [(Results and discussion)] TJ ET BT 26.250 457.824 Td /F4 9.8 Tf [(Comparison of different methods that measure sterol content)] TJ ET BT 26.250 438.420 Td /F1 9.8 Tf [(A number of methods are available for measuring lipid and cholesterol content in cells. Among the commercially available kits, )] TJ ET BT 26.250 426.515 Td /F1 9.8 Tf [(the enzymatic method, which is based on cholesterol esterase and oxidase, has been used to measure cholesterol in HD )] TJ ET BT 26.250 414.610 Td /F1 9.8 Tf [(samples )] TJ ET 0.267 0.267 0.267 rg BT 65.260 416.117 Td /F4 8.7 Tf [(6)] TJ ET BT 70.078 416.117 Td /F4 8.7 Tf [(13)] TJ ET BT 79.716 416.117 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 89.353 414.610 Td /F1 9.8 Tf [(. This method is not specific for cholesterol but all sterols can be measured since the ?-OH group involved in the )] TJ ET BT 26.250 402.705 Td /F1 9.8 Tf [(enzymatic reaction mediated by cholesterol oxidase is common to many sterols. To quantify the extent of this possible )] TJ ET BT 26.250 390.801 Td /F1 9.8 Tf [(confounding effect, we loaded ST Kin)] TJ ET BT 187.759 394.689 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 206.548 390.801 Td /F1 9.8 Tf [( cells with increasing concentrations of exogenous cholesterol and then subjected the )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(cellular preparations to lipid detection by the enzymatic method.)] TJ ET BT 26.250 359.491 Td /F1 9.8 Tf [(Specifically, ST Kin)] TJ ET BT 109.691 363.379 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 128.480 359.491 Td /F1 9.8 Tf [( cells were cultured in normal growth medium and incubated with increasing concentrations of )] TJ ET BT 26.250 347.586 Td /F1 9.8 Tf [(cholesterol \(0, 5, 15, 30, 50 and 80 g/ml\) for 16 hours \(Fig. 1\). Lipids were then purified by solvent-based extraction and )] TJ ET BT 26.250 335.682 Td /F1 9.8 Tf [(measured with the Amplex Red Enzyme Assay \(Invitrogen\). Figure 1b shows that the enzymatic method detected the )] TJ ET BT 26.250 323.777 Td /F1 9.8 Tf [(accumulation of exogenous cholesterol in ST Kin)] TJ ET BT 237.055 327.665 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 255.844 323.777 Td /F1 9.8 Tf [( cells loaded with 5 g/ml and 15 g/ml cholesterol, while linearity was lost )] TJ ET BT 26.250 311.872 Td /F1 9.8 Tf [(at 30 g/ml. Higher doses of cholesterol were toxic, as evident in the phase contrast pictures of cultured cells treated with 50 )] TJ ET BT 26.250 299.967 Td /F1 9.8 Tf [(g/ml and 80 g/ml cholesterol \(Fig. 1a\).)] TJ ET BT 26.250 280.563 Td /F1 9.8 Tf [(These findings confirmed that the enzymatic method could reliably detect accumulation of exogenously supplied cholesterol in )] TJ ET BT 26.250 268.658 Td /F1 9.8 Tf [(cultured cells \(up to 15 g/ml\). In addition, the enzymatic detection may give misleading results when measuring subtle changes )] TJ ET BT 26.250 256.753 Td /F1 9.8 Tf [(in endogenous cholesterol levels as this assay reflects the total sterol rather than the total cholesterol content, as noted )] TJ ET BT 26.250 244.848 Td /F1 9.8 Tf [(previously )] TJ ET 0.267 0.267 0.267 rg BT 72.845 246.356 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 82.483 244.848 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 225.444 Td /F1 9.8 Tf [(Colorimetric methods \(filipin staining and Nile Red staining\) and gas chromatography-mass spectrometry \(GC-MS\) have also )] TJ ET BT 26.250 213.539 Td /F1 9.8 Tf [(been used to detect cholesterol levels. The colorimetric methods are based on fluorescent molecules that bind to lipids and )] TJ ET BT 26.250 201.634 Td /F1 9.8 Tf [(sterols with high affinity, while GC-MS is an analytical method that allows detection of cholesterol after its chromatographic )] TJ ET BT 26.250 189.729 Td /F1 9.8 Tf [(separation from other sterols and by isotope dilution with deuterium labelled internal standard. To compare the analytical )] TJ ET BT 26.250 177.825 Td /F1 9.8 Tf [(sensibility of these methods, we performed cholesterol loading or depletion of ST Kin)] TJ ET BT 391.495 181.713 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 412.693 177.825 Td /F1 9.8 Tf [(cells and then measured the )] TJ ET BT 26.250 165.920 Td /F1 9.8 Tf [(cholesterol content using both methods.)] TJ ET BT 26.250 146.515 Td /F1 9.8 Tf [(In the first set of experiments, ST Kin)] TJ ET BT 186.111 150.403 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 207.310 146.515 Td /F1 9.8 Tf [(cells were incubated with 0 \(vehicle\), 3, 5, 7 and 10 g/ml of cholesterol for 16 hours )] TJ ET BT 26.250 134.610 Td /F1 9.8 Tf [(and then analysed by filipin staining or GC-MS. As shown in Figure 1c, visual inspection of the stained cultures revealed a )] TJ ET BT 26.250 122.706 Td /F1 9.8 Tf [(difference in the filipin staining of cells cultured with 10 g/ml of cholesterol compared to control, but we could not detect any )] TJ ET BT 26.250 110.801 Td /F1 9.8 Tf [(differences in cultures treated with <7 g/ml cholesterol \(Fig. 1c; Suppl. Fig. 1a\). Notably, the fluorescence intensity varied from )] TJ ET BT 26.250 98.896 Td /F1 9.8 Tf [(field to field for the 10 g/ml culture, leading to a large SD \(Suppl. Fig. 1a\). GC-MS analysis showed a gradual dose-dependent )] TJ ET BT 26.250 86.991 Td /F1 9.8 Tf [(increase of cholesterol content in ST Kin)] TJ ET BT 200.746 90.880 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 221.944 86.991 Td /F1 9.8 Tf [(cells \(Fig. 1d\).)] TJ ET BT 26.250 67.587 Td /F1 9.8 Tf [(Next we exposed cultures of ST Kin)] TJ ET BT 180.690 71.475 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 201.889 67.587 Td /F1 9.8 Tf [(cells to increasing concentrations of methyl-beta-cyclodextrin \(M?CD; 0, 1, 5, 10 and )] TJ ET BT 26.250 55.682 Td /F1 9.8 Tf [(25 mM\) for 1 hour in order to deplete cells of cholesterol. The cholesterol content was then measured by filipin staining or by )] TJ ET BT 26.250 43.777 Td /F1 9.8 Tf [(GC-MS. Figure 1e and 1f show that the cholesterol depletion was detectable by both methods. However, GC-MS was more )] TJ ET Q q 15.000 29.492 577.500 747.508 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(developed from embryonic brains of wild-type and knock-in HD mice expressing full-length endogenous normal HTT \(Hdh)] TJ ET BT 549.679 771.364 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 568.468 767.476 Td /F1 9.8 Tf [(\) )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(or muHTT \(Hdh)] TJ ET BT 93.964 759.460 Td /F1 8.7 Tf [(140/140Q)] TJ ET BT 132.028 755.571 Td /F1 9.8 Tf [(\) but not in NS cell lines derived from embryonic stem cells of heterozygous knock-in HD mice \(Hdh)] TJ ET BT 26.250 747.555 Td /F1 8.7 Tf [(140/7Q)] TJ ET BT 54.677 743.667 Td /F1 9.8 Tf [(\) )] TJ ET 0.267 0.267 0.267 rg BT 60.634 745.174 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 70.271 743.667 Td /F1 9.8 Tf [(. In contrast, other groups reported cholesterol accumulation, not cholesterol reduction, in mouse and human HD )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(tissues and cell cultures )] TJ ET 0.267 0.267 0.267 rg BT 132.457 733.269 Td /F4 8.7 Tf [(12)] TJ ET BT 142.094 733.269 Td /F4 8.7 Tf [(13)] TJ ET BT 151.731 733.269 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 161.369 731.762 Td /F1 9.8 Tf [(. In particular, it was reported that muHTT could interact with caveolin-1 and cause an )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(accumulation of intracellular cholesterol in primary neurons and in brains from the YAC72 transgenic mouse model of HD )] TJ ET 0.267 0.267 0.267 rg BT 550.235 721.364 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 559.872 719.857 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(Further, primary rat neurons that overexpress muHTT fragments showed sterol accumulation, and SIRT2 inhibition reduced )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(sterol levels via decreased nuclear trafficking of SREBP2 )] TJ ET 0.267 0.267 0.267 rg BT 274.972 697.555 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 284.610 696.048 Td /F1 9.8 Tf [(. Lastly, muHTT expression resulted in the accumulation of )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(cholesterol in HD cellular and murine models and in HD-affected human brains, and its altered cellular distribution contributed to )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(NMDA-mediated excitotoxicity )] TJ ET 0.267 0.267 0.267 rg BT 158.987 673.745 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 168.624 672.238 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(These conflicting data, which might be the result of the use of different techniques and experimental materials in the different )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(studies, raise the questions of whether cholesterol levels increase or decrease in HD and how this affects cellular and brain )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(dysfunction in the disease. A tentative unifying hypothesis suggests that decreased cholesterol biosynthesis in HD may be the )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(consequence of sterol accumulation )] TJ ET 0.267 0.267 0.267 rg BT 184.493 618.626 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 189.311 617.119 Td /F1 9.8 Tf [(, but further clarification regarding cholesterol level changes in HD is needed.)] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(In this study, we used several methods to measure sterols and cholesterol levels in immortalized knock-in cells derived from the )] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(embryonic striatum of mice carrying the endogenous HTT gene with 7Q \(ST Kin)] TJ ET BT 369.791 589.698 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 388.581 585.810 Td /F1 9.8 Tf [(\) or 109Q inserted into the mouse locus \(ST )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(Kin)] TJ ET BT 40.339 577.793 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 78.403 573.905 Td /F1 9.8 Tf [(\). We found different results depending on the technique adopted and that definitive methodology, such as gas )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(chromatography-mass spectrometry \(GC-MS\), should be favoured. Changes in cholesterol levels have been linked to NMDA )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(receptor activity )] TJ ET 0.267 0.267 0.267 rg BT 97.230 551.603 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 106.867 550.095 Td /F1 9.8 Tf [(, but we could not detect this receptor when analysing the same cells used in the earlier study. Our data also )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(reveal that sample preparation, degree of confluence and clonal properties strongly influence the results and, in particular, the )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(level of intracellular cholesterol that is detected. A further confounding factor may stem from changes in the properties or )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(technical issues related to the distribution and/or culture of target cell lines carrying the HD gene.)] TJ ET BT 26.250 477.779 Td /F4 12.0 Tf [(Results and discussion)] TJ ET BT 26.250 457.824 Td /F4 9.8 Tf [(Comparison of different methods that measure sterol content)] TJ ET BT 26.250 438.420 Td /F1 9.8 Tf [(A number of methods are available for measuring lipid and cholesterol content in cells. Among the commercially available kits, )] TJ ET BT 26.250 426.515 Td /F1 9.8 Tf [(the enzymatic method, which is based on cholesterol esterase and oxidase, has been used to measure cholesterol in HD )] TJ ET BT 26.250 414.610 Td /F1 9.8 Tf [(samples )] TJ ET 0.267 0.267 0.267 rg BT 65.260 416.117 Td /F4 8.7 Tf [(6)] TJ ET BT 70.078 416.117 Td /F4 8.7 Tf [(13)] TJ ET BT 79.716 416.117 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 89.353 414.610 Td /F1 9.8 Tf [(. This method is not specific for cholesterol but all sterols can be measured since the ?-OH group involved in the )] TJ ET BT 26.250 402.705 Td /F1 9.8 Tf [(enzymatic reaction mediated by cholesterol oxidase is common to many sterols. To quantify the extent of this possible )] TJ ET BT 26.250 390.801 Td /F1 9.8 Tf [(confounding effect, we loaded ST Kin)] TJ ET BT 187.759 394.689 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 206.548 390.801 Td /F1 9.8 Tf [( cells with increasing concentrations of exogenous cholesterol and then subjected the )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(cellular preparations to lipid detection by the enzymatic method.)] TJ ET BT 26.250 359.491 Td /F1 9.8 Tf [(Specifically, ST Kin)] TJ ET BT 109.691 363.379 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 128.480 359.491 Td /F1 9.8 Tf [( cells were cultured in normal growth medium and incubated with increasing concentrations of )] TJ ET BT 26.250 347.586 Td /F1 9.8 Tf [(cholesterol \(0, 5, 15, 30, 50 and 80 g/ml\) for 16 hours \(Fig. 1\). Lipids were then purified by solvent-based extraction and )] TJ ET BT 26.250 335.682 Td /F1 9.8 Tf [(measured with the Amplex Red Enzyme Assay \(Invitrogen\). Figure 1b shows that the enzymatic method detected the )] TJ ET BT 26.250 323.777 Td /F1 9.8 Tf [(accumulation of exogenous cholesterol in ST Kin)] TJ ET BT 237.055 327.665 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 255.844 323.777 Td /F1 9.8 Tf [( cells loaded with 5 g/ml and 15 g/ml cholesterol, while linearity was lost )] TJ ET BT 26.250 311.872 Td /F1 9.8 Tf [(at 30 g/ml. Higher doses of cholesterol were toxic, as evident in the phase contrast pictures of cultured cells treated with 50 )] TJ ET BT 26.250 299.967 Td /F1 9.8 Tf [(g/ml and 80 g/ml cholesterol \(Fig. 1a\).)] TJ ET BT 26.250 280.563 Td /F1 9.8 Tf [(These findings confirmed that the enzymatic method could reliably detect accumulation of exogenously supplied cholesterol in )] TJ ET BT 26.250 268.658 Td /F1 9.8 Tf [(cultured cells \(up to 15 g/ml\). In addition, the enzymatic detection may give misleading results when measuring subtle changes )] TJ ET BT 26.250 256.753 Td /F1 9.8 Tf [(in endogenous cholesterol levels as this assay reflects the total sterol rather than the total cholesterol content, as noted )] TJ ET BT 26.250 244.848 Td /F1 9.8 Tf [(previously )] TJ ET 0.267 0.267 0.267 rg BT 72.845 246.356 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 82.483 244.848 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 225.444 Td /F1 9.8 Tf [(Colorimetric methods \(filipin staining and Nile Red staining\) and gas chromatography-mass spectrometry \(GC-MS\) have also )] TJ ET BT 26.250 213.539 Td /F1 9.8 Tf [(been used to detect cholesterol levels. The colorimetric methods are based on fluorescent molecules that bind to lipids and )] TJ ET BT 26.250 201.634 Td /F1 9.8 Tf [(sterols with high affinity, while GC-MS is an analytical method that allows detection of cholesterol after its chromatographic )] TJ ET BT 26.250 189.729 Td /F1 9.8 Tf [(separation from other sterols and by isotope dilution with deuterium labelled internal standard. To compare the analytical )] TJ ET BT 26.250 177.825 Td /F1 9.8 Tf [(sensibility of these methods, we performed cholesterol loading or depletion of ST Kin)] TJ ET BT 391.495 181.713 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 412.693 177.825 Td /F1 9.8 Tf [(cells and then measured the )] TJ ET BT 26.250 165.920 Td /F1 9.8 Tf [(cholesterol content using both methods.)] TJ ET BT 26.250 146.515 Td /F1 9.8 Tf [(In the first set of experiments, ST Kin)] TJ ET BT 186.111 150.403 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 207.310 146.515 Td /F1 9.8 Tf [(cells were incubated with 0 \(vehicle\), 3, 5, 7 and 10 g/ml of cholesterol for 16 hours )] TJ ET BT 26.250 134.610 Td /F1 9.8 Tf [(and then analysed by filipin staining or GC-MS. As shown in Figure 1c, visual inspection of the stained cultures revealed a )] TJ ET BT 26.250 122.706 Td /F1 9.8 Tf [(difference in the filipin staining of cells cultured with 10 g/ml of cholesterol compared to control, but we could not detect any )] TJ ET BT 26.250 110.801 Td /F1 9.8 Tf [(differences in cultures treated with <7 g/ml cholesterol \(Fig. 1c; Suppl. Fig. 1a\). Notably, the fluorescence intensity varied from )] TJ ET BT 26.250 98.896 Td /F1 9.8 Tf [(field to field for the 10 g/ml culture, leading to a large SD \(Suppl. Fig. 1a\). GC-MS analysis showed a gradual dose-dependent )] TJ ET BT 26.250 86.991 Td /F1 9.8 Tf [(increase of cholesterol content in ST Kin)] TJ ET BT 200.746 90.880 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 221.944 86.991 Td /F1 9.8 Tf [(cells \(Fig. 1d\).)] TJ ET BT 26.250 67.587 Td /F1 9.8 Tf [(Next we exposed cultures of ST Kin)] TJ ET BT 180.690 71.475 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 201.889 67.587 Td /F1 9.8 Tf [(cells to increasing concentrations of methyl-beta-cyclodextrin \(M?CD; 0, 1, 5, 10 and )] TJ ET BT 26.250 55.682 Td /F1 9.8 Tf [(25 mM\) for 1 hour in order to deplete cells of cholesterol. The cholesterol content was then measured by filipin staining or by )] TJ ET BT 26.250 43.777 Td /F1 9.8 Tf [(GC-MS. Figure 1e and 1f show that the cholesterol depletion was detectable by both methods. However, GC-MS was more )] 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 172 0 obj << /Type /Annot /Subtype /Link /A 173 0 R /Border [0 0 0] /H /I /Rect [ 60.6339 744.3722 70.2713 753.1905 ] >> endobj 173 0 obj << /Type /Action >> endobj 174 0 obj << /Type /Annot /Subtype /Link /A 175 0 R /Border [0 0 0] /H /I /Rect [ 132.4567 732.4674 142.0941 741.2858 ] >> endobj 175 0 obj << /Type /Action >> endobj 176 0 obj << /Type /Annot /Subtype /Link /A 177 0 R /Border [0 0 0] /H /I /Rect [ 142.0941 732.4674 151.7314 741.2858 ] >> endobj 177 0 obj << /Type /Action >> endobj 178 0 obj << /Type /Annot /Subtype /Link /A 179 0 R /Border [0 0 0] /H /I /Rect [ 151.7314 732.4674 161.3688 741.2858 ] >> endobj 179 0 obj << /Type /Action >> endobj 180 0 obj << /Type /Annot /Subtype /Link /A 181 0 R /Border [0 0 0] /H /I /Rect [ 550.2345 720.5627 559.8718 729.3810 ] >> endobj 181 0 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Specifically, when cells were treated with 1 mM M?CD, the decrease in cholesterol content )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(was detectable by GC-MS but not by filipin staining. We concluded that GC-MS, but not filipin staining, could detect increased )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(cholesterol levels in vitro on the order of a few g/ml. In addition, filipin staining resulted in variable fluorescence within the same )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(sample. GC-MS was also more sensible for detecting decreased cholesterol levels in vitro, because GC-MS, but not filipin )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(staining, detected a decrease in cholesterol after acute treatment with >1 mM M?CD. The higher M?CD doses decreased cell )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(survival. We concluded that although filipin can detect free cholesterol, it is not quantitative and may be more suitable for )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(detection of cholesterol )] TJ ET BT 129.220 696.048 Td /F5 9.8 Tf [(in situ)] TJ ET BT 154.687 696.048 Td /F1 9.8 Tf [(. In contrast, GC-MS is reliable when appropriate standards are used.)] TJ ET 0.965 0.965 0.965 rg 26.250 202.512 555.000 483.655 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 q 319.500 0 0 450.000 35.250 226.417 cm /I3 Do Q q 35.250 202.512 537.000 17.905 re W n Q Q q 15.000 202.512 577.500 574.488 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(sensible compared to filipin staining. Specifically, when cells were treated with 1 mM M?CD, the decrease in cholesterol content )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(was detectable by GC-MS but not by filipin staining. We concluded that GC-MS, but not filipin staining, could detect increased )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(cholesterol levels in vitro on the order of a few g/ml. In addition, filipin staining resulted in variable fluorescence within the same )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(sample. GC-MS was also more sensible for detecting decreased cholesterol levels in vitro, because GC-MS, but not filipin )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(staining, detected a decrease in cholesterol after acute treatment with >1 mM M?CD. The higher M?CD doses decreased cell )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(survival. We concluded that although filipin can detect free cholesterol, it is not quantitative and may be more suitable for )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(detection of cholesterol )] TJ ET BT 129.220 696.048 Td /F5 9.8 Tf [(in situ)] TJ ET BT 154.687 696.048 Td /F1 9.8 Tf [(. In contrast, GC-MS is reliable when appropriate standards are used.)] TJ ET 0.965 0.965 0.965 rg 26.250 202.512 555.000 483.655 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 q 319.500 0 0 450.000 35.250 226.417 cm /I3 Do Q q 35.250 202.512 537.000 17.905 re W n Q Q q 15.000 202.512 577.500 574.488 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(sensible compared to filipin staining. Specifically, when cells were treated with 1 mM M?CD, the decrease in cholesterol content )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(was detectable by GC-MS but not by filipin staining. We concluded that GC-MS, but not filipin staining, could detect increased )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(cholesterol levels in vitro on the order of a few g/ml. In addition, filipin staining resulted in variable fluorescence within the same )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(sample. GC-MS was also more sensible for detecting decreased cholesterol levels in vitro, because GC-MS, but not filipin )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(staining, detected a decrease in cholesterol after acute treatment with >1 mM M?CD. The higher M?CD doses decreased cell )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(survival. We concluded that although filipin can detect free cholesterol, it is not quantitative and may be more suitable for )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(detection of cholesterol )] TJ ET BT 129.220 696.048 Td /F5 9.8 Tf [(in situ)] TJ ET BT 154.687 696.048 Td /F1 9.8 Tf [(. 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|^w,D C^)/7-tm;R]]ɡ2Ğ[y# P粼K1z h ;*,$EbQPVSd;iWtmERc>TH4Uyȫ'M%_\{z71,Vx^pOՎ4v; OZ_ZOZ_ZCSPGCJzQEQ@ ރQE NN(E-PzhҗP)PQ@Pw PP(ZZ(t!Q@ {hZEZPzP:QEGz(QE'/PE endstream endobj 256 0 obj << /Type /Annot /Subtype /Link /A 257 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 226.4167 354.7500 676.4167 ] >> endobj 257 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/10/fig1-chol-methods_11-10-12.jpg) >> endobj 258 0 obj << /Type /Annot /Subtype /Link /A 259 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 226.4167 354.7500 676.4167 ] >> endobj 259 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/10/fig1-chol-methods_11-10-12.jpg) >> endobj 260 0 obj << /Type /Page /Parent 3 0 R /Annots [ 262 0 R 264 0 R 266 0 R 268 0 R 270 0 R 272 0 R 274 0 R 276 0 R 278 0 R 280 0 R 282 0 R 284 0 R 286 0 R 288 0 R 290 0 R 292 0 R 294 0 R 296 0 R 298 0 R 300 0 R 302 0 R 304 0 R 306 0 R 308 0 R ] /Contents 261 0 R >> endobj 261 0 obj << /Length 34501 >> stream q 15.000 24.163 577.500 752.837 re W n 0.965 0.965 0.965 rg 26.250 473.924 555.000 303.076 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 473.924 m 581.250 473.924 l 581.250 474.674 l 26.250 474.674 l f q 35.250 485.174 537.000 291.826 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: Cholesterol levels in ST Kin7/7Q cells loaded with or depleted of cholesterol.)] TJ ET BT 35.250 748.106 Td /F4 9.8 Tf [(a\))] TJ ET BT 43.918 748.106 Td /F1 9.8 Tf [( Phase contrast images of ST Kin)] TJ ET BT 188.608 753.826 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 207.397 748.106 Td /F1 9.8 Tf [( cells loaded with increasing concentrations of cholesterol \(5, 15, 30, 50 and 80 )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(g/ml\) show that high concentrations of cholesterol \(5080 g/ml\) are toxic to the cells and lead to dramatic morphological )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(changes. 10X magnification. )] TJ ET BT 160.440 720.634 Td /F4 9.8 Tf [(b\))] TJ ET BT 169.644 720.634 Td /F1 9.8 Tf [( Cholesterol levels, as measured by enzymatic method a, in the same ST Kin)] TJ ET BT 501.817 726.353 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 520.606 720.634 Td /F1 9.8 Tf [( cells )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(shown in \(a\), show that cholesterol accumulation is detected in ST-Kin7/7Q cells loaded with 5 and 15 g/ml cholesterol, )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(while linearity is lost at 30 g/ml. In the cells loaded with the highest amounts of cholesterol, i.e. 50 and 80 g/ml, we could )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(not measure cholesterol accumulation because these doses led to cell death, as shown in the phase contrast images. )] TJ ET BT 544.668 679.425 Td /F4 9.8 Tf [(c-d\))] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(Comparison of cholesterol quantification using filipin staining \(c\) and GC-MS \(d\) in ST Kin)] TJ ET BT 421.048 671.408 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 439.837 665.689 Td /F1 9.8 Tf [( cells loaded with increasing )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(concentrations of cholesterol \(3, 5, 7 and 10 g/ml\) suggests that filipin staining is less sensitive than GC-MS. The images )] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(shown were cropped from 20X magnification images. )] TJ ET BT 267.173 638.216 Td /F4 9.8 Tf [(e-f\))] TJ ET BT 282.334 638.216 Td /F1 9.8 Tf [( Comparison of filipin staining \(e\) and GC-MS \(f\) quantification of )] TJ ET BT 35.250 624.480 Td /F1 9.8 Tf [(cholesterol in ST Kin)] TJ ET BT 124.657 630.200 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 143.447 624.480 Td /F1 9.8 Tf [( cells depleted of cholesterol by exposing them to increasing concentrations of M?CD \(1, 5, 10 and )] TJ ET BT 35.250 610.744 Td /F1 9.8 Tf [(25 mM\). The images shown were cropped from 20X magnification images. Cells were plated at the same densities in 6-well )] TJ ET BT 35.250 597.007 Td /F1 9.8 Tf [(plates for cholesterol detection by the enzymatic method and by GC-MS and in 12-well plates for filipin staining. Two )] TJ ET BT 35.250 583.271 Td /F1 9.8 Tf [(independent experiments were performed in which the cells were processed in parallel for filipin staining and for cholesterol )] TJ ET BT 35.250 569.535 Td /F1 9.8 Tf [(quantification by GC-MS. The graphs \(d, f\) represent the mean SEM as determined from the analysis of two independent )] TJ ET BT 35.250 555.799 Td /F1 9.8 Tf [(experiments in which each sample was processed in duplicate. Statistics: One-way ANOVA and the Newman-Keuls )] TJ ET BT 35.250 542.062 Td /F1 9.8 Tf [(Multiple Comparison Test were performed. Significant differences are indicated by asterisks: *p < 0.05; **p < 0.01; ***p < )] TJ ET BT 35.250 528.326 Td /F1 9.8 Tf [(0.001\).)] TJ ET Q BT 26.250 456.900 Td /F4 9.8 Tf [(Sterol content in striatal immortalized knock-in cells expressing muHTT)] TJ ET BT 26.250 437.496 Td /F1 9.8 Tf [(To investigate the consistency of colorimetric staining, enzymatic methods and GC-MS for detecting sterol changes in HD cell )] TJ ET BT 26.250 425.591 Td /F1 9.8 Tf [(models, we evaluated intracellular lipid and cholesterol levels in ST Kin)] TJ ET BT 331.864 429.479 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 353.062 425.591 Td /F1 9.8 Tf [(cells and in ST Kin cells expressing muHTT with )] TJ ET BT 26.250 413.686 Td /F1 9.8 Tf [(109Q \(ST Kin)] TJ ET BT 85.316 417.574 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 123.380 413.686 Td /F1 9.8 Tf [(\) )] TJ ET 0.267 0.267 0.267 rg BT 129.337 415.193 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 138.974 413.686 Td /F1 9.8 Tf [(. We first used Nile Red, a vital lipophilic dye that is used to label neutral lipids \(i.e. triacylglycerols or )] TJ ET BT 26.250 401.781 Td /F1 9.8 Tf [(cholesteryl esters\) )] TJ ET 0.267 0.267 0.267 rg BT 108.062 403.289 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 117.700 401.781 Td /F1 9.8 Tf [(, to detect the accumulation of intracellular lipid droplets in ST Kin)] TJ ET BT 400.030 405.670 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 418.820 401.781 Td /F1 9.8 Tf [( cells and in two ST Kin)] TJ ET BT 520.151 405.670 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 558.215 401.781 Td /F1 9.8 Tf [( cell )] TJ ET BT 26.250 389.877 Td /F1 9.8 Tf [(clones \(clone #2 and clone #6\). As shown in Figure 2a, low-magnification fluorescent images did not show differences in lipid )] TJ ET BT 26.250 377.972 Td /F1 9.8 Tf [(accumulation between wild-type and mutant cells. Notably, the staining was heterogeneous within each cell line; thus, high-)] TJ ET BT 26.250 366.067 Td /F1 9.8 Tf [(magnification fluorescent analysis of a few cells or single cells was avoided, as this would lead to misleading results.)] TJ ET BT 26.250 346.662 Td /F1 9.8 Tf [(In addition to Nile Red, we also tried filipin staining. We did not find any significant differences between the staining of ST Kin)] TJ ET BT 26.250 338.646 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 45.039 334.758 Td /F1 9.8 Tf [( cells and the staining of ST Kin)] TJ ET BT 181.608 338.646 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 219.672 334.758 Td /F1 9.8 Tf [( clones #2 or #6 \(Fig. 2a\). Filipin staining was heterogeneous in that some cells )] TJ ET BT 26.250 322.853 Td /F1 9.8 Tf [(showed a punctuate signal and some cells showed more diffuse staining. In addition, there was high variability in the fluorescent )] TJ ET BT 26.250 310.948 Td /F1 9.8 Tf [(intensity in different fields of the same well \(Suppl. Fig. 1b\). This variability was more evident within the different clones )] TJ ET BT 26.250 299.043 Td /F1 9.8 Tf [(compared to in experiments in which cholesterol was added or removed \(Fig. 1c, e\). Of note, we found that filipin exhibited rapid )] TJ ET BT 26.250 287.139 Td /F1 9.8 Tf [(photo-bleaching and only modest natural fluorescence under UV excitation, which introduces another confounding factor in the )] TJ ET BT 26.250 275.234 Td /F1 9.8 Tf [(detection of cholesterol using this method.)] TJ ET BT 26.250 255.829 Td /F1 9.8 Tf [(These results using two qualitative colorimetric methods, i.e. Nile Red and filipin staining, indicated that the levels of neutral )] TJ ET BT 26.250 243.924 Td /F1 9.8 Tf [(lipids and free cholesterol were similar in wild-type and muHTT cell lines, conflicting with the results reported by other )] TJ ET BT 26.250 232.020 Td /F1 9.8 Tf [(researchers that have used the same cell lines )] TJ ET 0.267 0.267 0.267 rg BT 230.006 233.527 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 239.643 232.020 Td /F1 9.8 Tf [(. Indeed, in the study by Del Toro et al. \(2010\), the authors detected increased )] TJ ET BT 26.250 220.115 Td /F1 9.8 Tf [(Nile Red and filipin staining, as well as cholesterol accumulation as detected by the enzymatic method, in a clone of ST Kin)] TJ ET BT 26.250 212.098 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 64.314 208.210 Td /F1 9.8 Tf [( cells compared to ST Kin)] TJ ET BT 175.396 212.098 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 194.185 208.210 Td /F1 9.8 Tf [( cells )] TJ ET 0.267 0.267 0.267 rg BT 219.106 209.717 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 228.743 208.210 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 188.805 Td /F1 9.8 Tf [(We are aware that filipin staining cannot be considered a quantitative method for measuring sterol/cholesterol content in cells or )] TJ ET BT 26.250 176.901 Td /F1 9.8 Tf [(tissues, so we rechecked the levels of intracellular sterols in ST Kin)] TJ ET BT 316.147 180.789 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 334.936 176.901 Td /F1 9.8 Tf [( cells and ST Kin)] TJ ET BT 408.090 180.789 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 446.154 176.901 Td /F1 9.8 Tf [( cells by the enzymatic )] TJ ET BT 26.250 164.996 Td /F1 9.8 Tf [(method used by Del Toro et al. \(2010\) )] TJ ET 0.267 0.267 0.267 rg BT 193.160 166.503 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 202.798 164.996 Td /F1 9.8 Tf [( and in our previous study )] TJ ET 0.267 0.267 0.267 rg BT 317.146 166.503 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 321.964 164.996 Td /F1 9.8 Tf [(. Notably, different protocols for the preparation of the )] TJ ET BT 26.250 153.091 Td /F1 9.8 Tf [(samples were used in the two studies. The protocol used by Del Toro et al. \(2010\) )] TJ ET 0.267 0.267 0.267 rg BT 382.281 154.598 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 391.918 153.091 Td /F1 9.8 Tf [(was based on a protein lysate preparation )] TJ ET BT 26.250 141.186 Td /F1 9.8 Tf [(followed by cholesterol esterase and oxidase incubation \(method a\). In Valenza et al. \(2005\) )] TJ ET 0.267 0.267 0.267 rg BT 426.195 142.694 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 431.014 141.186 Td /F1 9.8 Tf [(, a solvent-based extraction was )] TJ ET BT 26.250 129.282 Td /F1 9.8 Tf [(used to obtain a lipid fraction from the cells \(method b\).)] TJ ET BT 26.250 109.877 Td /F1 9.8 Tf [(In agreement with the filipin staining results shown in Figure 2a, the sterol content as measured by method a was the same in )] TJ ET BT 26.250 97.972 Td /F1 9.8 Tf [(ST Kin)] TJ ET BT 55.510 101.860 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 74.299 97.972 Td /F1 9.8 Tf [( and in one clone of ST Kin)] TJ ET BT 191.367 101.860 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 229.431 97.972 Td /F1 9.8 Tf [( cells \(clone #2, Fig. 2B\) but was increased in the second ST Kin)] TJ ET BT 507.969 101.860 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 546.033 97.972 Td /F1 9.8 Tf [( cell )] TJ ET BT 26.250 86.067 Td /F1 9.8 Tf [(clone \(clone #6\). Because sterols are lipids, it is better to measure sterols in a cellular lipid extract rather than in a cellular )] TJ ET BT 26.250 74.163 Td /F1 9.8 Tf [(protein extract. To evaluate whether the preparation of the sample influenced cholesterol/sterol detection, we also measured )] TJ ET BT 26.250 62.258 Td /F1 9.8 Tf [(sterol levels after lipid extraction from the same samples \(method b; Fig. 2\). As shown in Figure 2c, method b, but not method a, )] TJ ET BT 26.250 50.353 Td /F1 9.8 Tf [(detected similar sterol levels in ST Kin)] TJ ET BT 190.966 54.241 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 209.756 50.353 Td /F1 9.8 Tf [( cells and in the two ST Kin)] TJ ET BT 327.351 54.241 Td /F1 8.7 Tf [(109/109Q )] TJ ET BT 367.824 50.353 Td /F1 9.8 Tf [(cell clones \(Fig. 2B\). In Del Toro et al. \(2010\), )] TJ ET BT 26.250 38.448 Td /F1 9.8 Tf [(the finding of an increased level of cholesterol at the plasma membrane was linked to increased sensitivity of the muHTT cells )] TJ ET Q q 15.000 24.163 577.500 752.837 re W n 0.965 0.965 0.965 rg 26.250 473.924 555.000 303.076 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 473.924 m 581.250 473.924 l 581.250 474.674 l 26.250 474.674 l f q 35.250 485.174 537.000 291.826 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: Cholesterol levels in ST Kin7/7Q cells loaded with or depleted of cholesterol.)] TJ ET BT 35.250 748.106 Td /F4 9.8 Tf [(a\))] TJ ET BT 43.918 748.106 Td /F1 9.8 Tf [( Phase contrast images of ST Kin)] TJ ET BT 188.608 753.826 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 207.397 748.106 Td /F1 9.8 Tf [( cells loaded with increasing concentrations of cholesterol \(5, 15, 30, 50 and 80 )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(g/ml\) show that high concentrations of cholesterol \(5080 g/ml\) are toxic to the cells and lead to dramatic morphological )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(changes. 10X magnification. )] TJ ET BT 160.440 720.634 Td /F4 9.8 Tf [(b\))] TJ ET BT 169.644 720.634 Td /F1 9.8 Tf [( Cholesterol levels, as measured by enzymatic method a, in the same ST Kin)] TJ ET BT 501.817 726.353 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 520.606 720.634 Td /F1 9.8 Tf [( cells )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(shown in \(a\), show that cholesterol accumulation is detected in ST-Kin7/7Q cells loaded with 5 and 15 g/ml cholesterol, )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(while linearity is lost at 30 g/ml. In the cells loaded with the highest amounts of cholesterol, i.e. 50 and 80 g/ml, we could )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(not measure cholesterol accumulation because these doses led to cell death, as shown in the phase contrast images. )] TJ ET BT 544.668 679.425 Td /F4 9.8 Tf [(c-d\))] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(Comparison of cholesterol quantification using filipin staining \(c\) and GC-MS \(d\) in ST Kin)] TJ ET BT 421.048 671.408 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 439.837 665.689 Td /F1 9.8 Tf [( cells loaded with increasing )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(concentrations of cholesterol \(3, 5, 7 and 10 g/ml\) suggests that filipin staining is less sensitive than GC-MS. The images )] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(shown were cropped from 20X magnification images. )] TJ ET BT 267.173 638.216 Td /F4 9.8 Tf [(e-f\))] TJ ET BT 282.334 638.216 Td /F1 9.8 Tf [( Comparison of filipin staining \(e\) and GC-MS \(f\) quantification of )] TJ ET BT 35.250 624.480 Td /F1 9.8 Tf [(cholesterol in ST Kin)] TJ ET BT 124.657 630.200 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 143.447 624.480 Td /F1 9.8 Tf [( cells depleted of cholesterol by exposing them to increasing concentrations of M?CD \(1, 5, 10 and )] TJ ET BT 35.250 610.744 Td /F1 9.8 Tf [(25 mM\). The images shown were cropped from 20X magnification images. Cells were plated at the same densities in 6-well )] TJ ET BT 35.250 597.007 Td /F1 9.8 Tf [(plates for cholesterol detection by the enzymatic method and by GC-MS and in 12-well plates for filipin staining. Two )] TJ ET BT 35.250 583.271 Td /F1 9.8 Tf [(independent experiments were performed in which the cells were processed in parallel for filipin staining and for cholesterol )] TJ ET BT 35.250 569.535 Td /F1 9.8 Tf [(quantification by GC-MS. The graphs \(d, f\) represent the mean SEM as determined from the analysis of two independent )] TJ ET BT 35.250 555.799 Td /F1 9.8 Tf [(experiments in which each sample was processed in duplicate. Statistics: One-way ANOVA and the Newman-Keuls )] TJ ET BT 35.250 542.062 Td /F1 9.8 Tf [(Multiple Comparison Test were performed. Significant differences are indicated by asterisks: *p < 0.05; **p < 0.01; ***p < )] TJ ET BT 35.250 528.326 Td /F1 9.8 Tf [(0.001\).)] TJ ET Q BT 26.250 456.900 Td /F4 9.8 Tf [(Sterol content in striatal immortalized knock-in cells expressing muHTT)] TJ ET BT 26.250 437.496 Td /F1 9.8 Tf [(To investigate the consistency of colorimetric staining, enzymatic methods and GC-MS for detecting sterol changes in HD cell )] TJ ET BT 26.250 425.591 Td /F1 9.8 Tf [(models, we evaluated intracellular lipid and cholesterol levels in ST Kin)] TJ ET BT 331.864 429.479 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 353.062 425.591 Td /F1 9.8 Tf [(cells and in ST Kin cells expressing muHTT with )] TJ ET BT 26.250 413.686 Td /F1 9.8 Tf [(109Q \(ST Kin)] TJ ET BT 85.316 417.574 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 123.380 413.686 Td /F1 9.8 Tf [(\) )] TJ ET 0.267 0.267 0.267 rg BT 129.337 415.193 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 138.974 413.686 Td /F1 9.8 Tf [(. We first used Nile Red, a vital lipophilic dye that is used to label neutral lipids \(i.e. triacylglycerols or )] TJ ET BT 26.250 401.781 Td /F1 9.8 Tf [(cholesteryl esters\) )] TJ ET 0.267 0.267 0.267 rg BT 108.062 403.289 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 117.700 401.781 Td /F1 9.8 Tf [(, to detect the accumulation of intracellular lipid droplets in ST Kin)] TJ ET BT 400.030 405.670 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 418.820 401.781 Td /F1 9.8 Tf [( cells and in two ST Kin)] TJ ET BT 520.151 405.670 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 558.215 401.781 Td /F1 9.8 Tf [( cell )] TJ ET BT 26.250 389.877 Td /F1 9.8 Tf [(clones \(clone #2 and clone #6\). As shown in Figure 2a, low-magnification fluorescent images did not show differences in lipid )] TJ ET BT 26.250 377.972 Td /F1 9.8 Tf [(accumulation between wild-type and mutant cells. Notably, the staining was heterogeneous within each cell line; thus, high-)] TJ ET BT 26.250 366.067 Td /F1 9.8 Tf [(magnification fluorescent analysis of a few cells or single cells was avoided, as this would lead to misleading results.)] TJ ET BT 26.250 346.662 Td /F1 9.8 Tf [(In addition to Nile Red, we also tried filipin staining. We did not find any significant differences between the staining of ST Kin)] TJ ET BT 26.250 338.646 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 45.039 334.758 Td /F1 9.8 Tf [( cells and the staining of ST Kin)] TJ ET BT 181.608 338.646 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 219.672 334.758 Td /F1 9.8 Tf [( clones #2 or #6 \(Fig. 2a\). Filipin staining was heterogeneous in that some cells )] TJ ET BT 26.250 322.853 Td /F1 9.8 Tf [(showed a punctuate signal and some cells showed more diffuse staining. In addition, there was high variability in the fluorescent )] TJ ET BT 26.250 310.948 Td /F1 9.8 Tf [(intensity in different fields of the same well \(Suppl. Fig. 1b\). This variability was more evident within the different clones )] TJ ET BT 26.250 299.043 Td /F1 9.8 Tf [(compared to in experiments in which cholesterol was added or removed \(Fig. 1c, e\). Of note, we found that filipin exhibited rapid )] TJ ET BT 26.250 287.139 Td /F1 9.8 Tf [(photo-bleaching and only modest natural fluorescence under UV excitation, which introduces another confounding factor in the )] TJ ET BT 26.250 275.234 Td /F1 9.8 Tf [(detection of cholesterol using this method.)] TJ ET BT 26.250 255.829 Td /F1 9.8 Tf [(These results using two qualitative colorimetric methods, i.e. Nile Red and filipin staining, indicated that the levels of neutral )] TJ ET BT 26.250 243.924 Td /F1 9.8 Tf [(lipids and free cholesterol were similar in wild-type and muHTT cell lines, conflicting with the results reported by other )] TJ ET BT 26.250 232.020 Td /F1 9.8 Tf [(researchers that have used the same cell lines )] TJ ET 0.267 0.267 0.267 rg BT 230.006 233.527 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 239.643 232.020 Td /F1 9.8 Tf [(. Indeed, in the study by Del Toro et al. \(2010\), the authors detected increased )] TJ ET BT 26.250 220.115 Td /F1 9.8 Tf [(Nile Red and filipin staining, as well as cholesterol accumulation as detected by the enzymatic method, in a clone of ST Kin)] TJ ET BT 26.250 212.098 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 64.314 208.210 Td /F1 9.8 Tf [( cells compared to ST Kin)] TJ ET BT 175.396 212.098 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 194.185 208.210 Td /F1 9.8 Tf [( cells )] TJ ET 0.267 0.267 0.267 rg BT 219.106 209.717 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 228.743 208.210 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 188.805 Td /F1 9.8 Tf [(We are aware that filipin staining cannot be considered a quantitative method for measuring sterol/cholesterol content in cells or )] TJ ET BT 26.250 176.901 Td /F1 9.8 Tf [(tissues, so we rechecked the levels of intracellular sterols in ST Kin)] TJ ET BT 316.147 180.789 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 334.936 176.901 Td /F1 9.8 Tf [( cells and ST Kin)] TJ ET BT 408.090 180.789 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 446.154 176.901 Td /F1 9.8 Tf [( cells by the enzymatic )] TJ ET BT 26.250 164.996 Td /F1 9.8 Tf [(method used by Del Toro et al. \(2010\) )] TJ ET 0.267 0.267 0.267 rg BT 193.160 166.503 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 202.798 164.996 Td /F1 9.8 Tf [( and in our previous study )] TJ ET 0.267 0.267 0.267 rg BT 317.146 166.503 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 321.964 164.996 Td /F1 9.8 Tf [(. Notably, different protocols for the preparation of the )] TJ ET BT 26.250 153.091 Td /F1 9.8 Tf [(samples were used in the two studies. The protocol used by Del Toro et al. \(2010\) )] TJ ET 0.267 0.267 0.267 rg BT 382.281 154.598 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 391.918 153.091 Td /F1 9.8 Tf [(was based on a protein lysate preparation )] TJ ET BT 26.250 141.186 Td /F1 9.8 Tf [(followed by cholesterol esterase and oxidase incubation \(method a\). In Valenza et al. \(2005\) )] TJ ET 0.267 0.267 0.267 rg BT 426.195 142.694 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 431.014 141.186 Td /F1 9.8 Tf [(, a solvent-based extraction was )] TJ ET BT 26.250 129.282 Td /F1 9.8 Tf [(used to obtain a lipid fraction from the cells \(method b\).)] TJ ET BT 26.250 109.877 Td /F1 9.8 Tf [(In agreement with the filipin staining results shown in Figure 2a, the sterol content as measured by method a was the same in )] TJ ET BT 26.250 97.972 Td /F1 9.8 Tf [(ST Kin)] TJ ET BT 55.510 101.860 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 74.299 97.972 Td /F1 9.8 Tf [( and in one clone of ST Kin)] TJ ET BT 191.367 101.860 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 229.431 97.972 Td /F1 9.8 Tf [( cells \(clone #2, Fig. 2B\) but was increased in the second ST Kin)] TJ ET BT 507.969 101.860 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 546.033 97.972 Td /F1 9.8 Tf [( cell )] TJ ET BT 26.250 86.067 Td /F1 9.8 Tf [(clone \(clone #6\). Because sterols are lipids, it is better to measure sterols in a cellular lipid extract rather than in a cellular )] TJ ET BT 26.250 74.163 Td /F1 9.8 Tf [(protein extract. To evaluate whether the preparation of the sample influenced cholesterol/sterol detection, we also measured )] TJ ET BT 26.250 62.258 Td /F1 9.8 Tf [(sterol levels after lipid extraction from the same samples \(method b; Fig. 2\). As shown in Figure 2c, method b, but not method a, )] TJ ET BT 26.250 50.353 Td /F1 9.8 Tf [(detected similar sterol levels in ST Kin)] TJ ET BT 190.966 54.241 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 209.756 50.353 Td /F1 9.8 Tf [( cells and in the two ST Kin)] TJ ET BT 327.351 54.241 Td /F1 8.7 Tf [(109/109Q )] TJ ET BT 367.824 50.353 Td /F1 9.8 Tf [(cell clones \(Fig. 2B\). In Del Toro et al. \(2010\), )] TJ ET BT 26.250 38.448 Td /F1 9.8 Tf [(the finding of an increased level of cholesterol at the plasma membrane was linked to increased sensitivity of the muHTT cells )] TJ ET Q q 15.000 24.163 577.500 752.837 re W n 0.965 0.965 0.965 rg 26.250 473.924 555.000 303.076 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 473.924 m 581.250 473.924 l 581.250 474.674 l 26.250 474.674 l f q 35.250 485.174 537.000 291.826 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: Cholesterol levels in ST Kin7/7Q cells loaded with or depleted of cholesterol.)] TJ ET BT 35.250 748.106 Td /F4 9.8 Tf [(a\))] TJ ET BT 43.918 748.106 Td /F1 9.8 Tf [( Phase contrast images of ST Kin)] TJ ET BT 188.608 753.826 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 207.397 748.106 Td /F1 9.8 Tf [( cells loaded with increasing concentrations of cholesterol \(5, 15, 30, 50 and 80 )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(g/ml\) show that high concentrations of cholesterol \(5080 g/ml\) are toxic to the cells and lead to dramatic morphological )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(changes. 10X magnification. )] TJ ET BT 160.440 720.634 Td /F4 9.8 Tf [(b\))] TJ ET BT 169.644 720.634 Td /F1 9.8 Tf [( Cholesterol levels, as measured by enzymatic method a, in the same ST Kin)] TJ ET BT 501.817 726.353 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 520.606 720.634 Td /F1 9.8 Tf [( cells )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(shown in \(a\), show that cholesterol accumulation is detected in ST-Kin7/7Q cells loaded with 5 and 15 g/ml cholesterol, )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(while linearity is lost at 30 g/ml. In the cells loaded with the highest amounts of cholesterol, i.e. 50 and 80 g/ml, we could )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(not measure cholesterol accumulation because these doses led to cell death, as shown in the phase contrast images. )] TJ ET BT 544.668 679.425 Td /F4 9.8 Tf [(c-d\))] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(Comparison of cholesterol quantification using filipin staining \(c\) and GC-MS \(d\) in ST Kin)] TJ ET BT 421.048 671.408 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 439.837 665.689 Td /F1 9.8 Tf [( cells loaded with increasing )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(concentrations of cholesterol \(3, 5, 7 and 10 g/ml\) suggests that filipin staining is less sensitive than GC-MS. The images )] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(shown were cropped from 20X magnification images. )] TJ ET BT 267.173 638.216 Td /F4 9.8 Tf [(e-f\))] TJ ET BT 282.334 638.216 Td /F1 9.8 Tf [( Comparison of filipin staining \(e\) and GC-MS \(f\) quantification of )] TJ ET BT 35.250 624.480 Td /F1 9.8 Tf [(cholesterol in ST Kin)] TJ ET BT 124.657 630.200 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 143.447 624.480 Td /F1 9.8 Tf [( cells depleted of cholesterol by exposing them to increasing concentrations of M?CD \(1, 5, 10 and )] TJ ET BT 35.250 610.744 Td /F1 9.8 Tf [(25 mM\). The images shown were cropped from 20X magnification images. Cells were plated at the same densities in 6-well )] TJ ET BT 35.250 597.007 Td /F1 9.8 Tf [(plates for cholesterol detection by the enzymatic method and by GC-MS and in 12-well plates for filipin staining. Two )] TJ ET BT 35.250 583.271 Td /F1 9.8 Tf [(independent experiments were performed in which the cells were processed in parallel for filipin staining and for cholesterol )] TJ ET BT 35.250 569.535 Td /F1 9.8 Tf [(quantification by GC-MS. The graphs \(d, f\) represent the mean SEM as determined from the analysis of two independent )] TJ ET BT 35.250 555.799 Td /F1 9.8 Tf [(experiments in which each sample was processed in duplicate. Statistics: One-way ANOVA and the Newman-Keuls )] TJ ET BT 35.250 542.062 Td /F1 9.8 Tf [(Multiple Comparison Test were performed. Significant differences are indicated by asterisks: *p < 0.05; **p < 0.01; ***p < )] TJ ET BT 35.250 528.326 Td /F1 9.8 Tf [(0.001\).)] TJ ET Q BT 26.250 456.900 Td /F4 9.8 Tf [(Sterol content in striatal immortalized knock-in cells expressing muHTT)] TJ ET BT 26.250 437.496 Td /F1 9.8 Tf [(To investigate the consistency of colorimetric staining, enzymatic methods and GC-MS for detecting sterol changes in HD cell )] TJ ET BT 26.250 425.591 Td /F1 9.8 Tf [(models, we evaluated intracellular lipid and cholesterol levels in ST Kin)] TJ ET BT 331.864 429.479 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 353.062 425.591 Td /F1 9.8 Tf [(cells and in ST Kin cells expressing muHTT with )] TJ ET BT 26.250 413.686 Td /F1 9.8 Tf [(109Q \(ST Kin)] TJ ET BT 85.316 417.574 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 123.380 413.686 Td /F1 9.8 Tf [(\) )] TJ ET 0.267 0.267 0.267 rg BT 129.337 415.193 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 138.974 413.686 Td /F1 9.8 Tf [(. We first used Nile Red, a vital lipophilic dye that is used to label neutral lipids \(i.e. triacylglycerols or )] TJ ET BT 26.250 401.781 Td /F1 9.8 Tf [(cholesteryl esters\) )] TJ ET 0.267 0.267 0.267 rg BT 108.062 403.289 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 117.700 401.781 Td /F1 9.8 Tf [(, to detect the accumulation of intracellular lipid droplets in ST Kin)] TJ ET BT 400.030 405.670 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 418.820 401.781 Td /F1 9.8 Tf [( cells and in two ST Kin)] TJ ET BT 520.151 405.670 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 558.215 401.781 Td /F1 9.8 Tf [( cell )] TJ ET BT 26.250 389.877 Td /F1 9.8 Tf [(clones \(clone #2 and clone #6\). As shown in Figure 2a, low-magnification fluorescent images did not show differences in lipid )] TJ ET BT 26.250 377.972 Td /F1 9.8 Tf [(accumulation between wild-type and mutant cells. Notably, the staining was heterogeneous within each cell line; thus, high-)] TJ ET BT 26.250 366.067 Td /F1 9.8 Tf [(magnification fluorescent analysis of a few cells or single cells was avoided, as this would lead to misleading results.)] TJ ET BT 26.250 346.662 Td /F1 9.8 Tf [(In addition to Nile Red, we also tried filipin staining. We did not find any significant differences between the staining of ST Kin)] TJ ET BT 26.250 338.646 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 45.039 334.758 Td /F1 9.8 Tf [( cells and the staining of ST Kin)] TJ ET BT 181.608 338.646 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 219.672 334.758 Td /F1 9.8 Tf [( clones #2 or #6 \(Fig. 2a\). Filipin staining was heterogeneous in that some cells )] TJ ET BT 26.250 322.853 Td /F1 9.8 Tf [(showed a punctuate signal and some cells showed more diffuse staining. In addition, there was high variability in the fluorescent )] TJ ET BT 26.250 310.948 Td /F1 9.8 Tf [(intensity in different fields of the same well \(Suppl. Fig. 1b\). This variability was more evident within the different clones )] TJ ET BT 26.250 299.043 Td /F1 9.8 Tf [(compared to in experiments in which cholesterol was added or removed \(Fig. 1c, e\). Of note, we found that filipin exhibited rapid )] TJ ET BT 26.250 287.139 Td /F1 9.8 Tf [(photo-bleaching and only modest natural fluorescence under UV excitation, which introduces another confounding factor in the )] TJ ET BT 26.250 275.234 Td /F1 9.8 Tf [(detection of cholesterol using this method.)] TJ ET BT 26.250 255.829 Td /F1 9.8 Tf [(These results using two qualitative colorimetric methods, i.e. Nile Red and filipin staining, indicated that the levels of neutral )] TJ ET BT 26.250 243.924 Td /F1 9.8 Tf [(lipids and free cholesterol were similar in wild-type and muHTT cell lines, conflicting with the results reported by other )] TJ ET BT 26.250 232.020 Td /F1 9.8 Tf [(researchers that have used the same cell lines )] TJ ET 0.267 0.267 0.267 rg BT 230.006 233.527 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 239.643 232.020 Td /F1 9.8 Tf [(. Indeed, in the study by Del Toro et al. \(2010\), the authors detected increased )] TJ ET BT 26.250 220.115 Td /F1 9.8 Tf [(Nile Red and filipin staining, as well as cholesterol accumulation as detected by the enzymatic method, in a clone of ST Kin)] TJ ET BT 26.250 212.098 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 64.314 208.210 Td /F1 9.8 Tf [( cells compared to ST Kin)] TJ ET BT 175.396 212.098 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 194.185 208.210 Td /F1 9.8 Tf [( cells )] TJ ET 0.267 0.267 0.267 rg BT 219.106 209.717 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 228.743 208.210 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 188.805 Td /F1 9.8 Tf [(We are aware that filipin staining cannot be considered a quantitative method for measuring sterol/cholesterol content in cells or )] TJ ET BT 26.250 176.901 Td /F1 9.8 Tf [(tissues, so we rechecked the levels of intracellular sterols in ST Kin)] TJ ET BT 316.147 180.789 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 334.936 176.901 Td /F1 9.8 Tf [( cells and ST Kin)] TJ ET BT 408.090 180.789 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 446.154 176.901 Td /F1 9.8 Tf [( cells by the enzymatic )] TJ ET BT 26.250 164.996 Td /F1 9.8 Tf [(method used by Del Toro et al. \(2010\) )] TJ ET 0.267 0.267 0.267 rg BT 193.160 166.503 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 202.798 164.996 Td /F1 9.8 Tf [( and in our previous study )] TJ ET 0.267 0.267 0.267 rg BT 317.146 166.503 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 321.964 164.996 Td /F1 9.8 Tf [(. Notably, different protocols for the preparation of the )] TJ ET BT 26.250 153.091 Td /F1 9.8 Tf [(samples were used in the two studies. The protocol used by Del Toro et al. \(2010\) )] TJ ET 0.267 0.267 0.267 rg BT 382.281 154.598 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 391.918 153.091 Td /F1 9.8 Tf [(was based on a protein lysate preparation )] TJ ET BT 26.250 141.186 Td /F1 9.8 Tf [(followed by cholesterol esterase and oxidase incubation \(method a\). In Valenza et al. \(2005\) )] TJ ET 0.267 0.267 0.267 rg BT 426.195 142.694 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 431.014 141.186 Td /F1 9.8 Tf [(, a solvent-based extraction was )] TJ ET BT 26.250 129.282 Td /F1 9.8 Tf [(used to obtain a lipid fraction from the cells \(method b\).)] TJ ET BT 26.250 109.877 Td /F1 9.8 Tf [(In agreement with the filipin staining results shown in Figure 2a, the sterol content as measured by method a was the same in )] TJ ET BT 26.250 97.972 Td /F1 9.8 Tf [(ST Kin)] TJ ET BT 55.510 101.860 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 74.299 97.972 Td /F1 9.8 Tf [( and in one clone of ST Kin)] TJ ET BT 191.367 101.860 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 229.431 97.972 Td /F1 9.8 Tf [( cells \(clone #2, Fig. 2B\) but was increased in the second ST Kin)] TJ ET BT 507.969 101.860 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 546.033 97.972 Td /F1 9.8 Tf [( cell )] TJ ET BT 26.250 86.067 Td /F1 9.8 Tf [(clone \(clone #6\). Because sterols are lipids, it is better to measure sterols in a cellular lipid extract rather than in a cellular )] TJ ET BT 26.250 74.163 Td /F1 9.8 Tf [(protein extract. To evaluate whether the preparation of the sample influenced cholesterol/sterol detection, we also measured )] TJ ET BT 26.250 62.258 Td /F1 9.8 Tf [(sterol levels after lipid extraction from the same samples \(method b; Fig. 2\). As shown in Figure 2c, method b, but not method a, )] TJ ET BT 26.250 50.353 Td /F1 9.8 Tf [(detected similar sterol levels in ST Kin)] TJ ET BT 190.966 54.241 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 209.756 50.353 Td /F1 9.8 Tf [( cells and in the two ST Kin)] TJ ET BT 327.351 54.241 Td /F1 8.7 Tf [(109/109Q )] TJ ET BT 367.824 50.353 Td /F1 9.8 Tf [(cell clones \(Fig. 2B\). In Del Toro et al. \(2010\), )] TJ ET BT 26.250 38.448 Td /F1 9.8 Tf [(the finding of an increased level of cholesterol at the plasma membrane was linked to increased sensitivity of the muHTT cells )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(4)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 262 0 obj << /Type /Annot /Subtype /Link /A 263 0 R /Border [0 0 0] /H /I /Rect [ 129.3368 414.3917 138.9741 423.2100 ] >> endobj 263 0 obj << /Type /Action >> endobj 264 0 obj << /Type /Annot /Subtype /Link /A 265 0 R /Border [0 0 0] /H /I /Rect [ 108.0623 402.4869 117.6996 411.3052 ] >> endobj 265 0 obj << /Type /Action >> endobj 266 0 obj << /Type /Annot /Subtype /Link /A 267 0 R /Border [0 0 0] /H /I /Rect [ 230.0055 232.7252 239.6428 241.5435 ] >> endobj 267 0 obj << /Type /Action >> endobj 268 0 obj << /Type /Annot /Subtype /Link /A 269 0 R /Border [0 0 0] /H /I /Rect [ 219.1061 208.9157 228.7434 217.7340 ] >> endobj 269 0 obj << /Type /Action >> endobj 270 0 obj << /Type /Annot /Subtype /Link /A 271 0 R /Border [0 0 0] /H /I /Rect [ 193.1602 165.7014 202.7976 174.5197 ] >> endobj 271 0 obj << /Type /Action >> endobj 272 0 obj << /Type /Annot /Subtype /Link /A 273 0 R /Border [0 0 0] /H /I /Rect [ 317.1456 165.7014 321.9643 174.5197 ] >> 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 [ 382.2810 153.7967 391.9183 162.6150 ] >> 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 [ 426.1950 141.8919 431.0137 150.7102 ] >> 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 [ 129.3368 414.3917 138.9741 423.2100 ] >> 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 [ 108.0623 402.4869 117.6996 411.3052 ] >> 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 [ 230.0055 232.7252 239.6428 241.5435 ] >> endobj 283 0 obj << /Type /Action >> endobj 284 0 obj << /Type /Annot /Subtype /Link /A 285 0 R /Border [0 0 0] /H /I /Rect [ 219.1061 208.9157 228.7434 217.7340 ] >> endobj 285 0 obj << /Type /Action >> endobj 286 0 obj << /Type /Annot /Subtype /Link /A 287 0 R /Border [0 0 0] /H /I /Rect [ 193.1602 165.7014 202.7976 174.5197 ] >> endobj 287 0 obj << /Type /Action >> endobj 288 0 obj << /Type /Annot /Subtype /Link /A 289 0 R /Border [0 0 0] /H /I /Rect [ 317.1456 165.7014 321.9643 174.5197 ] >> endobj 289 0 obj << /Type /Action >> endobj 290 0 obj << /Type /Annot /Subtype /Link /A 291 0 R /Border [0 0 0] /H /I /Rect [ 382.2810 153.7967 391.9183 162.6150 ] >> endobj 291 0 obj << /Type /Action >> endobj 292 0 obj << /Type /Annot /Subtype /Link /A 293 0 R /Border [0 0 0] /H /I /Rect [ 426.1950 141.8919 431.0137 150.7102 ] >> endobj 293 0 obj << /Type /Action >> endobj 294 0 obj << /Type /Annot /Subtype /Link /A 295 0 R /Border [0 0 0] /H /I /Rect [ 129.3368 414.3917 138.9741 423.2100 ] >> endobj 295 0 obj << /Type /Action >> endobj 296 0 obj << /Type /Annot /Subtype /Link /A 297 0 R /Border [0 0 0] /H /I /Rect [ 108.0623 402.4869 117.6996 411.3052 ] >> endobj 297 0 obj << /Type /Action >> endobj 298 0 obj << /Type /Annot /Subtype /Link /A 299 0 R /Border [0 0 0] /H /I /Rect [ 230.0055 232.7252 239.6428 241.5435 ] >> endobj 299 0 obj << /Type /Action >> endobj 300 0 obj << /Type /Annot /Subtype /Link /A 301 0 R /Border [0 0 0] /H /I /Rect [ 219.1061 208.9157 228.7434 217.7340 ] >> endobj 301 0 obj << /Type /Action >> endobj 302 0 obj << /Type /Annot /Subtype /Link /A 303 0 R /Border [0 0 0] /H /I /Rect [ 193.1602 165.7014 202.7976 174.5197 ] >> endobj 303 0 obj << /Type /Action >> endobj 304 0 obj << /Type /Annot /Subtype /Link /A 305 0 R /Border [0 0 0] /H /I /Rect [ 317.1456 165.7014 321.9643 174.5197 ] >> endobj 305 0 obj << /Type /Action >> endobj 306 0 obj << /Type /Annot /Subtype /Link /A 307 0 R /Border [0 0 0] /H /I /Rect [ 382.2810 153.7967 391.9183 162.6150 ] >> endobj 307 0 obj << /Type /Action >> endobj 308 0 obj << /Type /Annot /Subtype /Link /A 309 0 R /Border [0 0 0] /H /I /Rect [ 426.1950 141.8919 431.0137 150.7102 ] >> endobj 309 0 obj << /Type /Action >> endobj 310 0 obj << /Type /Page /Parent 3 0 R /Annots [ 312 0 R 314 0 R 316 0 R 318 0 R 320 0 R 322 0 R 324 0 R 327 0 R 329 0 R 331 0 R 333 0 R 335 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 ] /Contents 311 0 R >> endobj 311 0 obj << /Length 12270 >> stream 0.271 0.267 0.267 rg q 15.000 130.619 577.500 646.381 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(to NMDA-mediated excitotoxicity )] TJ ET 0.267 0.267 0.267 rg BT 169.828 768.983 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 179.466 767.476 Td /F1 9.8 Tf [(. However, we failed to detect the activity of NMDA or AMPA receptors, as judged by Ca2+ )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(efflux \(Suppl. Fig. 2a, b\) and by a lack of change in the expression of the NMDA 2B, GLUR1 and GLUR5 genes by qualitative )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(PCR \(data not shown\) in ST Kin)] TJ ET BT 164.427 747.555 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 183.216 743.667 Td /F1 9.8 Tf [( cells. Moreover, we also failed to detect the expression of the NMDA subunit NR1, as )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(judged by real time qPCR, in ST Kin)] TJ ET BT 182.309 735.650 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 201.098 731.762 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 252.042 735.650 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 290.106 731.762 Td /F1 9.8 Tf [( cells \(Fig. 2S c\).)] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(To further verify the level of cholesterol in ST Kin)] TJ ET BT 236.499 716.245 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 255.288 712.357 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 306.232 716.245 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 344.296 712.357 Td /F1 9.8 Tf [( cells, we analysed our cellular preparations by )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(isotope dilution GC-MS. The data in Figure 2d confirmed the results obtained using the enzymatic method on lipid extracts )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(\(method b\) and previous data from an immortalized striatal cell line with inducible expression of the N-terminal fragment of )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(muHTT )] TJ ET 0.267 0.267 0.267 rg BT 61.457 678.150 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 66.276 676.643 Td /F1 9.8 Tf [(. Specifically, we detected no changes in cholesterol levels in this cell system growing in serum-containing medium.)] TJ ET BT 26.250 657.238 Td /F1 9.8 Tf [(Taken together, these findings suggest that differences in sample preparation methods, such as the use of lipid vs. protein )] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(extracts, can lead to differences in the detection of changes in cholesterol levels. This may account for the inconsistent results )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(from different laboratories when different methods are used. Our results also highlighted the importance of measuring )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(cholesterol in cells and tissues with the most appropriate method; specifically, GC-MS appears to be the most sensitive and )] TJ ET BT 26.250 609.619 Td /F1 9.8 Tf [(reliable method. In agreement with this finding, all results obtained by GC-MS in different studies show decreases in cholesterol )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(levels in HD cells and tissues )] TJ ET 0.267 0.267 0.267 rg BT 155.203 599.222 Td /F4 8.7 Tf [(7)] TJ ET BT 160.022 599.222 Td /F4 8.7 Tf [(8)] TJ ET BT 164.841 599.222 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 174.478 597.714 Td /F1 9.8 Tf [(. Finally, although outside the scope of the present study, our inability to detect the NMDA-)] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(AMPA receptor in ST Kin cell lines made it almost impossible to study the possible link between cholesterol changes and )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(increased sensitivity to NMDA-mediated excitotoxicity of these muHTT cells, as proposed previously )] TJ ET 0.267 0.267 0.267 rg BT 460.262 575.412 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 469.899 573.905 Td /F1 9.8 Tf [(.)] TJ ET 0.965 0.965 0.965 rg 26.250 130.619 555.000 433.405 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 564.024 m 581.250 564.024 l 581.250 563.274 l 26.250 563.274 l f q 450.000 0 0 399.750 35.250 154.524 cm /I4 Do Q q 35.250 130.619 537.000 17.905 re W n Q Q q 15.000 130.619 577.500 646.381 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(to NMDA-mediated excitotoxicity )] TJ ET 0.267 0.267 0.267 rg BT 169.828 768.983 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 179.466 767.476 Td /F1 9.8 Tf [(. However, we failed to detect the activity of NMDA or AMPA receptors, as judged by Ca2+ )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(efflux \(Suppl. Fig. 2a, b\) and by a lack of change in the expression of the NMDA 2B, GLUR1 and GLUR5 genes by qualitative )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(PCR \(data not shown\) in ST Kin)] TJ ET BT 164.427 747.555 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 183.216 743.667 Td /F1 9.8 Tf [( cells. Moreover, we also failed to detect the expression of the NMDA subunit NR1, as )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(judged by real time qPCR, in ST Kin)] TJ ET BT 182.309 735.650 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 201.098 731.762 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 252.042 735.650 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 290.106 731.762 Td /F1 9.8 Tf [( cells \(Fig. 2S c\).)] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(To further verify the level of cholesterol in ST Kin)] TJ ET BT 236.499 716.245 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 255.288 712.357 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 306.232 716.245 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 344.296 712.357 Td /F1 9.8 Tf [( cells, we analysed our cellular preparations by )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(isotope dilution GC-MS. The data in Figure 2d confirmed the results obtained using the enzymatic method on lipid extracts )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(\(method b\) and previous data from an immortalized striatal cell line with inducible expression of the N-terminal fragment of )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(muHTT )] TJ ET 0.267 0.267 0.267 rg BT 61.457 678.150 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 66.276 676.643 Td /F1 9.8 Tf [(. Specifically, we detected no changes in cholesterol levels in this cell system growing in serum-containing medium.)] TJ ET BT 26.250 657.238 Td /F1 9.8 Tf [(Taken together, these findings suggest that differences in sample preparation methods, such as the use of lipid vs. protein )] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(extracts, can lead to differences in the detection of changes in cholesterol levels. This may account for the inconsistent results )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(from different laboratories when different methods are used. Our results also highlighted the importance of measuring )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(cholesterol in cells and tissues with the most appropriate method; specifically, GC-MS appears to be the most sensitive and )] TJ ET BT 26.250 609.619 Td /F1 9.8 Tf [(reliable method. In agreement with this finding, all results obtained by GC-MS in different studies show decreases in cholesterol )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(levels in HD cells and tissues )] TJ ET 0.267 0.267 0.267 rg BT 155.203 599.222 Td /F4 8.7 Tf [(7)] TJ ET BT 160.022 599.222 Td /F4 8.7 Tf [(8)] TJ ET BT 164.841 599.222 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 174.478 597.714 Td /F1 9.8 Tf [(. Finally, although outside the scope of the present study, our inability to detect the NMDA-)] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(AMPA receptor in ST Kin cell lines made it almost impossible to study the possible link between cholesterol changes and )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(increased sensitivity to NMDA-mediated excitotoxicity of these muHTT cells, as proposed previously )] TJ ET 0.267 0.267 0.267 rg BT 460.262 575.412 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 469.899 573.905 Td /F1 9.8 Tf [(.)] TJ ET 0.965 0.965 0.965 rg 26.250 130.619 555.000 433.405 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 564.024 m 581.250 564.024 l 581.250 563.274 l 26.250 563.274 l f q 450.000 0 0 399.750 35.250 154.524 cm /I4 Do Q q 35.250 130.619 537.000 17.905 re W n Q Q q 15.000 130.619 577.500 646.381 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(to NMDA-mediated excitotoxicity )] TJ ET 0.267 0.267 0.267 rg BT 169.828 768.983 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 179.466 767.476 Td /F1 9.8 Tf [(. However, we failed to detect the activity of NMDA or AMPA receptors, as judged by Ca2+ )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(efflux \(Suppl. Fig. 2a, b\) and by a lack of change in the expression of the NMDA 2B, GLUR1 and GLUR5 genes by qualitative )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(PCR \(data not shown\) in ST Kin)] TJ ET BT 164.427 747.555 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 183.216 743.667 Td /F1 9.8 Tf [( cells. Moreover, we also failed to detect the expression of the NMDA subunit NR1, as )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(judged by real time qPCR, in ST Kin)] TJ ET BT 182.309 735.650 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 201.098 731.762 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 252.042 735.650 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 290.106 731.762 Td /F1 9.8 Tf [( cells \(Fig. 2S c\).)] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(To further verify the level of cholesterol in ST Kin)] TJ ET BT 236.499 716.245 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 255.288 712.357 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 306.232 716.245 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 344.296 712.357 Td /F1 9.8 Tf [( cells, we analysed our cellular preparations by )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(isotope dilution GC-MS. The data in Figure 2d confirmed the results obtained using the enzymatic method on lipid extracts )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(\(method b\) and previous data from an immortalized striatal cell line with inducible expression of the N-terminal fragment of )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(muHTT )] TJ ET 0.267 0.267 0.267 rg BT 61.457 678.150 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 66.276 676.643 Td /F1 9.8 Tf [(. Specifically, we detected no changes in cholesterol levels in this cell system growing in serum-containing medium.)] TJ ET BT 26.250 657.238 Td /F1 9.8 Tf [(Taken together, these findings suggest that differences in sample preparation methods, such as the use of lipid vs. protein )] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(extracts, can lead to differences in the detection of changes in cholesterol levels. This may account for the inconsistent results )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(from different laboratories when different methods are used. Our results also highlighted the importance of measuring )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(cholesterol in cells and tissues with the most appropriate method; specifically, GC-MS appears to be the most sensitive and )] TJ ET BT 26.250 609.619 Td /F1 9.8 Tf [(reliable method. In agreement with this finding, all results obtained by GC-MS in different studies show decreases in cholesterol )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(levels in HD cells and tissues )] TJ ET 0.267 0.267 0.267 rg BT 155.203 599.222 Td /F4 8.7 Tf [(7)] TJ ET BT 160.022 599.222 Td /F4 8.7 Tf [(8)] TJ ET BT 164.841 599.222 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 174.478 597.714 Td /F1 9.8 Tf [(. Finally, although outside the scope of the present study, our inability to detect the NMDA-)] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(AMPA receptor in ST Kin cell lines made it almost impossible to study the possible link between cholesterol changes and )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(increased sensitivity to NMDA-mediated excitotoxicity of these muHTT cells, as proposed previously )] TJ ET 0.267 0.267 0.267 rg BT 460.262 575.412 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 469.899 573.905 Td /F1 9.8 Tf [(.)] TJ ET 0.965 0.965 0.965 rg 26.250 130.619 555.000 433.405 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 564.024 m 581.250 564.024 l 581.250 563.274 l 26.250 563.274 l f q 450.000 0 0 399.750 35.250 154.524 cm /I4 Do Q q 35.250 130.619 537.000 17.905 re W n Q Q q 450.000 0 0 399.750 35.250 154.524 cm /I4 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 312 0 obj << /Type /Annot /Subtype /Link /A 313 0 R /Border [0 0 0] /H /I /Rect [ 169.8285 768.1817 179.4658 777.0000 ] >> endobj 313 0 obj << /Type /Action >> endobj 314 0 obj << /Type /Annot /Subtype /Link /A 315 0 R /Border [0 0 0] /H /I /Rect [ 61.4573 677.3484 66.2759 686.1667 ] >> endobj 315 0 obj << /Type /Action >> endobj 316 0 obj << /Type /Annot /Subtype /Link /A 317 0 R /Border [0 0 0] /H /I /Rect [ 155.2035 598.4199 160.0222 607.2382 ] >> endobj 317 0 obj << /Type /Action >> endobj 318 0 obj << /Type /Annot /Subtype /Link /A 319 0 R /Border [0 0 0] /H /I /Rect [ 160.0222 598.4199 164.8408 607.2382 ] >> endobj 319 0 obj << /Type /Action >> endobj 320 0 obj << /Type /Annot /Subtype /Link /A 321 0 R /Border [0 0 0] /H /I /Rect [ 164.8408 598.4199 174.4782 607.2382 ] >> endobj 321 0 obj << /Type /Action >> endobj 322 0 obj << /Type /Annot /Subtype /Link /A 323 0 R /Border [0 0 0] /H /I /Rect [ 460.2615 574.6104 469.8988 583.4287 ] >> 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 154.5240 485.2500 554.2740 ] >> endobj 325 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/10/fig2-chol-methods_11-10-12.jpg) >> endobj 326 0 obj << /Type /XObject /Subtype /Image /Width 600 /Height 533 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 66082>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?,/񇉴 YC>x$-K ' |iඟ4еnoPei|*/>'*5֙euϬZ=ժE-$6l`0,/l>-敩QO.M>\J"$?$(+|M_? _LxR_m_Zx%X$FH[GnjnV&ާBkW>K\_5S,!͢ eNwqdH"y$u4(I>^IU|1'n5 ~JVH㙠 E @?eU{_[xRMW⎧jZM֋v5Nr6K#d@<+f(K|'dnFEta'8߱3g53gg.:3ks j F>VZ88zƬ۰LPE~T~TPhQ@PMQE(3(QEQFhEPGQ@gEQPhQ@Q@u (ڌE~TQGG@P? 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Similarly, filipin staining does not show any differences between the )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(cell lines. 20X magnification. b-d\) Comparison of two enzymatic methods and GC-MS for measuring cholesterol levels in ST )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(Kin)] TJ ET BT 49.339 712.617 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 68.128 706.897 Td /F1 9.8 Tf [( and ST K-in)] TJ ET BT 122.319 712.617 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 160.383 706.897 Td /F1 9.8 Tf [( cell lines. The two methods differ in the sample preparation method that is used prior to )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(cholesterol detection. The cells were processed to obtain either a protein lysate, as described in Del Toro 2010 \(method a\), )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(or a lipid extract, as described in Valenza et al. 2005 \(method b\), and both preparations were subjected to cholesterol )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(esterase and oxidase assay. Using enzymatic method a, an increase in sterol levels was detected in ST Kin)] TJ ET BT 499.116 671.408 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 537.180 665.689 Td /F1 9.8 Tf [( clone )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(#6 compared to wild-type ST Kin)] TJ ET BT 176.137 657.672 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 194.927 651.952 Td /F1 9.8 Tf [( cells, while similar levels of sterols were found in ST Kin)] TJ ET BT 438.745 657.672 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 476.809 651.952 Td /F1 9.8 Tf [( clone #2 and ST Kin)] TJ ET BT 35.250 643.936 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 54.039 638.216 Td /F1 9.8 Tf [( cells \(b\). Sterol quantification by method b does not reveal any differences in sterol content in ST Kin)] TJ ET BT 490.284 643.936 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 509.073 638.216 Td /F1 9.8 Tf [( versus ST )] TJ ET BT 35.250 624.480 Td /F1 9.8 Tf [(Kin)] TJ ET BT 49.339 630.200 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 87.403 624.480 Td /F1 9.8 Tf [( clonal cells \(c\). Similarly, GC-MS showed no differences between the wild-type and HD cell lines \(d\). Each )] TJ ET BT 35.250 610.744 Td /F1 9.8 Tf [(sample was analysed in duplicate, and the graphs show the mean SEM of three independent experiments. Statistics: One-)] TJ ET BT 35.250 597.007 Td /F1 9.8 Tf [(way ANOVA and the Newman-Keuls Multiple Comparison Test were performed. Significant differences are represented by )] TJ ET BT 35.250 583.271 Td /F1 9.8 Tf [(asterisks: *p < 0.05.)] TJ ET Q BT 26.250 511.845 Td /F4 9.8 Tf [(Levels of cholesterol precursors in ST Kin cells that express muHTT)] TJ ET BT 26.250 492.441 Td /F1 9.8 Tf [(To verify the decrease of the cholesterol levels in the HD cells used in this study, we used isotope dilution GC-MS to measure )] TJ ET BT 26.250 480.536 Td /F1 9.8 Tf [(cholesterol precursors, including lanosterol, lathosterol, desmosterol and 7-dehydroxycholesterol \(7DHC\). Cells were collected )] TJ ET BT 26.250 468.631 Td /F1 9.8 Tf [(when they reached 8090% confluence. Figure 3 shows that the levels of lanosterol and lathosterol in both ST kin)] TJ ET BT 516.168 472.519 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 26.250 456.726 Td /F1 9.8 Tf [(clones were unchanged compared to control cells, while the levels of desmosterol and 7DHC were significantly decreased in )] TJ ET BT 26.250 444.822 Td /F1 9.8 Tf [(both ST Kin)] TJ ET BT 77.194 448.710 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 115.258 444.822 Td /F1 9.8 Tf [( clones compared to control cells \(Fig. 3 c-d\). These results indicate a reduction in the availability of some, )] TJ ET BT 26.250 432.917 Td /F1 9.8 Tf [(but not all, cholesterol precursors in these HD cells.)] TJ ET BT 26.250 413.512 Td /F1 9.8 Tf [(Cholesterol homeostasis )] TJ ET BT 135.704 413.512 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 164.417 413.512 Td /F1 9.8 Tf [( might be influenced by the culture conditions, including cell over-confluence )] TJ ET 0.267 0.267 0.267 rg BT 495.527 415.019 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 505.165 413.512 Td /F1 9.8 Tf [(. To evaluate )] TJ ET BT 26.250 401.607 Td /F1 9.8 Tf [(whether cell density affected cholesterol detection, we measured the levels of cholesterol and cholesterol precursors in ST Kin)] TJ ET BT 26.250 393.591 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 45.039 389.703 Td /F1 9.8 Tf [( cells and in both clones of ST Kin)] TJ ET BT 191.904 393.591 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 229.968 389.703 Td /F1 9.8 Tf [( cells that were grown to over-confluence. For these experiments, rather than )] TJ ET BT 26.250 377.798 Td /F1 9.8 Tf [(splitting the cells after they were confluent, they were left for another 2 days in culture, leading to ~4050% over-confluence. In )] TJ ET BT 26.250 365.893 Td /F1 9.8 Tf [(contrast to findings obtained in sub-confluent growth conditions \(Fig. 2d\), cholesterol was found to be increased in ST Kin)] TJ ET BT 26.250 357.877 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 64.314 353.988 Td /F1 9.8 Tf [( clone #6 cells compared to ST Kin)] TJ ET BT 214.961 357.877 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 233.751 353.988 Td /F1 9.8 Tf [( cells \(Fig. 4a\). Conversely, the cholesterol content was similar in ST Kin)] TJ ET BT 545.848 357.877 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 26.250 342.084 Td /F1 9.8 Tf [(cells and ST Kin)] TJ ET BT 96.694 345.972 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 134.758 342.084 Td /F1 9.8 Tf [( clone #2 cells \(Fig. 4a\). The lanosterol level was unchanged in both clones \(Fig. 4b\), while lathosterol )] TJ ET BT 26.250 330.179 Td /F1 9.8 Tf [(was significantly increased only in ST Kin)] TJ ET BT 204.519 334.067 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 242.583 330.179 Td /F1 9.8 Tf [( clone #6 cells compared to ST Kin)] TJ ET BT 393.230 334.067 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 414.429 330.179 Td /F1 9.8 Tf [(cells \(p<0.01\) \(Fig. 4c\). Moreover, the )] TJ ET BT 26.250 318.274 Td /F1 9.8 Tf [(levels of desmosterol and 7DHC were significantly increased in ST Kin)] TJ ET BT 330.226 322.162 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 368.290 318.274 Td /F1 9.8 Tf [( #6 compared to ST Kin)] TJ ET BT 470.714 322.162 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 508.778 318.274 Td /F1 9.8 Tf [( clone #2 cells )] TJ ET BT 26.250 306.369 Td /F1 9.8 Tf [(\(p<0.01\) and compared to ST Kin)] TJ ET BT 170.677 310.258 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 191.875 306.369 Td /F1 9.8 Tf [(cells \(Fig. 4d-e\). These results suggest that growth conditions may affect the levels of )] TJ ET BT 26.250 294.465 Td /F1 9.8 Tf [(different sterols in HD clones in different ways and that these effects are unlikely to be linked to the presence of muHTT.)] TJ ET 0.965 0.965 0.965 rg 26.250 -181.166 555.000 465.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 284.584 m 581.250 284.584 l 581.250 283.834 l 26.250 283.834 l f q 440.250 0 0 450.000 35.250 -175.166 cm /I5 Do Q q 35.250 -181.166 537.000 0.000 re W n Q Q q 15.000 -181.166 577.500 958.166 re W n 0.965 0.965 0.965 rg 26.250 528.869 555.000 248.131 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 528.869 m 581.250 528.869 l 581.250 529.619 l 26.250 529.619 l f q 35.250 540.119 537.000 236.881 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 2: Neutral lipids and sterols in ST Kin 7/7Q and in ST Kin109/109Q cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(a\) Staining with Nile Red, a marker of neutral lipids, does not show any differences between wild-type cells \(ST Kin)] TJ ET BT 529.438 753.826 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 548.228 748.106 Td /F1 9.8 Tf [(\) )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(and HD cells \(ST Kin)] TJ ET BT 125.730 740.090 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 163.794 734.370 Td /F1 9.8 Tf [(, clone #2 and clone #6\). Similarly, filipin staining does not show any differences between the )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(cell lines. 20X magnification. b-d\) Comparison of two enzymatic methods and GC-MS for measuring cholesterol levels in ST )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(Kin)] TJ ET BT 49.339 712.617 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 68.128 706.897 Td /F1 9.8 Tf [( and ST K-in)] TJ ET BT 122.319 712.617 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 160.383 706.897 Td /F1 9.8 Tf [( cell lines. The two methods differ in the sample preparation method that is used prior to )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(cholesterol detection. The cells were processed to obtain either a protein lysate, as described in Del Toro 2010 \(method a\), )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(or a lipid extract, as described in Valenza et al. 2005 \(method b\), and both preparations were subjected to cholesterol )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(esterase and oxidase assay. Using enzymatic method a, an increase in sterol levels was detected in ST Kin)] TJ ET BT 499.116 671.408 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 537.180 665.689 Td /F1 9.8 Tf [( clone )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(#6 compared to wild-type ST Kin)] TJ ET BT 176.137 657.672 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 194.927 651.952 Td /F1 9.8 Tf [( cells, while similar levels of sterols were found in ST Kin)] TJ ET BT 438.745 657.672 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 476.809 651.952 Td /F1 9.8 Tf [( clone #2 and ST Kin)] TJ ET BT 35.250 643.936 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 54.039 638.216 Td /F1 9.8 Tf [( cells \(b\). Sterol quantification by method b does not reveal any differences in sterol content in ST Kin)] TJ ET BT 490.284 643.936 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 509.073 638.216 Td /F1 9.8 Tf [( versus ST )] TJ ET BT 35.250 624.480 Td /F1 9.8 Tf [(Kin)] TJ ET BT 49.339 630.200 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 87.403 624.480 Td /F1 9.8 Tf [( clonal cells \(c\). Similarly, GC-MS showed no differences between the wild-type and HD cell lines \(d\). Each )] TJ ET BT 35.250 610.744 Td /F1 9.8 Tf [(sample was analysed in duplicate, and the graphs show the mean SEM of three independent experiments. Statistics: One-)] TJ ET BT 35.250 597.007 Td /F1 9.8 Tf [(way ANOVA and the Newman-Keuls Multiple Comparison Test were performed. Significant differences are represented by )] TJ ET BT 35.250 583.271 Td /F1 9.8 Tf [(asterisks: *p < 0.05.)] TJ ET Q BT 26.250 511.845 Td /F4 9.8 Tf [(Levels of cholesterol precursors in ST Kin cells that express muHTT)] TJ ET BT 26.250 492.441 Td /F1 9.8 Tf [(To verify the decrease of the cholesterol levels in the HD cells used in this study, we used isotope dilution GC-MS to measure )] TJ ET BT 26.250 480.536 Td /F1 9.8 Tf [(cholesterol precursors, including lanosterol, lathosterol, desmosterol and 7-dehydroxycholesterol \(7DHC\). Cells were collected )] TJ ET BT 26.250 468.631 Td /F1 9.8 Tf [(when they reached 8090% confluence. Figure 3 shows that the levels of lanosterol and lathosterol in both ST kin)] TJ ET BT 516.168 472.519 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 26.250 456.726 Td /F1 9.8 Tf [(clones were unchanged compared to control cells, while the levels of desmosterol and 7DHC were significantly decreased in )] TJ ET BT 26.250 444.822 Td /F1 9.8 Tf [(both ST Kin)] TJ ET BT 77.194 448.710 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 115.258 444.822 Td /F1 9.8 Tf [( clones compared to control cells \(Fig. 3 c-d\). These results indicate a reduction in the availability of some, )] TJ ET BT 26.250 432.917 Td /F1 9.8 Tf [(but not all, cholesterol precursors in these HD cells.)] TJ ET BT 26.250 413.512 Td /F1 9.8 Tf [(Cholesterol homeostasis )] TJ ET BT 135.704 413.512 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 164.417 413.512 Td /F1 9.8 Tf [( might be influenced by the culture conditions, including cell over-confluence )] TJ ET 0.267 0.267 0.267 rg BT 495.527 415.019 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 505.165 413.512 Td /F1 9.8 Tf [(. To evaluate )] TJ ET BT 26.250 401.607 Td /F1 9.8 Tf [(whether cell density affected cholesterol detection, we measured the levels of cholesterol and cholesterol precursors in ST Kin)] TJ ET BT 26.250 393.591 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 45.039 389.703 Td /F1 9.8 Tf [( cells and in both clones of ST Kin)] TJ ET BT 191.904 393.591 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 229.968 389.703 Td /F1 9.8 Tf [( cells that were grown to over-confluence. For these experiments, rather than )] TJ ET BT 26.250 377.798 Td /F1 9.8 Tf [(splitting the cells after they were confluent, they were left for another 2 days in culture, leading to ~4050% over-confluence. In )] TJ ET BT 26.250 365.893 Td /F1 9.8 Tf [(contrast to findings obtained in sub-confluent growth conditions \(Fig. 2d\), cholesterol was found to be increased in ST Kin)] TJ ET BT 26.250 357.877 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 64.314 353.988 Td /F1 9.8 Tf [( clone #6 cells compared to ST Kin)] TJ ET BT 214.961 357.877 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 233.751 353.988 Td /F1 9.8 Tf [( cells \(Fig. 4a\). Conversely, the cholesterol content was similar in ST Kin)] TJ ET BT 545.848 357.877 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 26.250 342.084 Td /F1 9.8 Tf [(cells and ST Kin)] TJ ET BT 96.694 345.972 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 134.758 342.084 Td /F1 9.8 Tf [( clone #2 cells \(Fig. 4a\). The lanosterol level was unchanged in both clones \(Fig. 4b\), while lathosterol )] TJ ET BT 26.250 330.179 Td /F1 9.8 Tf [(was significantly increased only in ST Kin)] TJ ET BT 204.519 334.067 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 242.583 330.179 Td /F1 9.8 Tf [( clone #6 cells compared to ST Kin)] TJ ET BT 393.230 334.067 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 414.429 330.179 Td /F1 9.8 Tf [(cells \(p<0.01\) \(Fig. 4c\). Moreover, the )] TJ ET BT 26.250 318.274 Td /F1 9.8 Tf [(levels of desmosterol and 7DHC were significantly increased in ST Kin)] TJ ET BT 330.226 322.162 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 368.290 318.274 Td /F1 9.8 Tf [( #6 compared to ST Kin)] TJ ET BT 470.714 322.162 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 508.778 318.274 Td /F1 9.8 Tf [( clone #2 cells )] TJ ET BT 26.250 306.369 Td /F1 9.8 Tf [(\(p<0.01\) and compared to ST Kin)] TJ ET BT 170.677 310.258 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 191.875 306.369 Td /F1 9.8 Tf [(cells \(Fig. 4d-e\). These results suggest that growth conditions may affect the levels of )] TJ ET BT 26.250 294.465 Td /F1 9.8 Tf [(different sterols in HD clones in different ways and that these effects are unlikely to be linked to the presence of muHTT.)] TJ ET 0.965 0.965 0.965 rg 26.250 -181.166 555.000 465.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 284.584 m 581.250 284.584 l 581.250 283.834 l 26.250 283.834 l f q 440.250 0 0 450.000 35.250 -175.166 cm /I5 Do Q q 35.250 -181.166 537.000 0.000 re W n Q Q q 15.000 -181.166 577.500 958.166 re W n 0.965 0.965 0.965 rg 26.250 528.869 555.000 248.131 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 528.869 m 581.250 528.869 l 581.250 529.619 l 26.250 529.619 l f q 35.250 540.119 537.000 236.881 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 2: Neutral lipids and sterols in ST Kin 7/7Q and in ST Kin109/109Q cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(a\) Staining with Nile Red, a marker of neutral lipids, does not show any differences between wild-type cells \(ST Kin)] TJ ET BT 529.438 753.826 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 548.228 748.106 Td /F1 9.8 Tf [(\) )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(and HD cells \(ST Kin)] TJ ET BT 125.730 740.090 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 163.794 734.370 Td /F1 9.8 Tf [(, clone #2 and clone #6\). Similarly, filipin staining does not show any differences between the )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(cell lines. 20X magnification. b-d\) Comparison of two enzymatic methods and GC-MS for measuring cholesterol levels in ST )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(Kin)] TJ ET BT 49.339 712.617 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 68.128 706.897 Td /F1 9.8 Tf [( and ST K-in)] TJ ET BT 122.319 712.617 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 160.383 706.897 Td /F1 9.8 Tf [( cell lines. The two methods differ in the sample preparation method that is used prior to )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(cholesterol detection. The cells were processed to obtain either a protein lysate, as described in Del Toro 2010 \(method a\), )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(or a lipid extract, as described in Valenza et al. 2005 \(method b\), and both preparations were subjected to cholesterol )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(esterase and oxidase assay. Using enzymatic method a, an increase in sterol levels was detected in ST Kin)] TJ ET BT 499.116 671.408 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 537.180 665.689 Td /F1 9.8 Tf [( clone )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(#6 compared to wild-type ST Kin)] TJ ET BT 176.137 657.672 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 194.927 651.952 Td /F1 9.8 Tf [( cells, while similar levels of sterols were found in ST Kin)] TJ ET BT 438.745 657.672 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 476.809 651.952 Td /F1 9.8 Tf [( clone #2 and ST Kin)] TJ ET BT 35.250 643.936 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 54.039 638.216 Td /F1 9.8 Tf [( cells \(b\). Sterol quantification by method b does not reveal any differences in sterol content in ST Kin)] TJ ET BT 490.284 643.936 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 509.073 638.216 Td /F1 9.8 Tf [( versus ST )] TJ ET BT 35.250 624.480 Td /F1 9.8 Tf [(Kin)] TJ ET BT 49.339 630.200 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 87.403 624.480 Td /F1 9.8 Tf [( clonal cells \(c\). Similarly, GC-MS showed no differences between the wild-type and HD cell lines \(d\). Each )] TJ ET BT 35.250 610.744 Td /F1 9.8 Tf [(sample was analysed in duplicate, and the graphs show the mean SEM of three independent experiments. Statistics: One-)] TJ ET BT 35.250 597.007 Td /F1 9.8 Tf [(way ANOVA and the Newman-Keuls Multiple Comparison Test were performed. Significant differences are represented by )] TJ ET BT 35.250 583.271 Td /F1 9.8 Tf [(asterisks: *p < 0.05.)] TJ ET Q BT 26.250 511.845 Td /F4 9.8 Tf [(Levels of cholesterol precursors in ST Kin cells that express muHTT)] TJ ET BT 26.250 492.441 Td /F1 9.8 Tf [(To verify the decrease of the cholesterol levels in the HD cells used in this study, we used isotope dilution GC-MS to measure )] TJ ET BT 26.250 480.536 Td /F1 9.8 Tf [(cholesterol precursors, including lanosterol, lathosterol, desmosterol and 7-dehydroxycholesterol \(7DHC\). Cells were collected )] TJ ET BT 26.250 468.631 Td /F1 9.8 Tf [(when they reached 8090% confluence. Figure 3 shows that the levels of lanosterol and lathosterol in both ST kin)] TJ ET BT 516.168 472.519 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 26.250 456.726 Td /F1 9.8 Tf [(clones were unchanged compared to control cells, while the levels of desmosterol and 7DHC were significantly decreased in )] TJ ET BT 26.250 444.822 Td /F1 9.8 Tf [(both ST Kin)] TJ ET BT 77.194 448.710 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 115.258 444.822 Td /F1 9.8 Tf [( clones compared to control cells \(Fig. 3 c-d\). These results indicate a reduction in the availability of some, )] TJ ET BT 26.250 432.917 Td /F1 9.8 Tf [(but not all, cholesterol precursors in these HD cells.)] TJ ET BT 26.250 413.512 Td /F1 9.8 Tf [(Cholesterol homeostasis )] TJ ET BT 135.704 413.512 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 164.417 413.512 Td /F1 9.8 Tf [( might be influenced by the culture conditions, including cell over-confluence )] TJ ET 0.267 0.267 0.267 rg BT 495.527 415.019 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 505.165 413.512 Td /F1 9.8 Tf [(. To evaluate )] TJ ET BT 26.250 401.607 Td /F1 9.8 Tf [(whether cell density affected cholesterol detection, we measured the levels of cholesterol and cholesterol precursors in ST Kin)] TJ ET BT 26.250 393.591 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 45.039 389.703 Td /F1 9.8 Tf [( cells and in both clones of ST Kin)] TJ ET BT 191.904 393.591 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 229.968 389.703 Td /F1 9.8 Tf [( cells that were grown to over-confluence. For these experiments, rather than )] TJ ET BT 26.250 377.798 Td /F1 9.8 Tf [(splitting the cells after they were confluent, they were left for another 2 days in culture, leading to ~4050% over-confluence. In )] TJ ET BT 26.250 365.893 Td /F1 9.8 Tf [(contrast to findings obtained in sub-confluent growth conditions \(Fig. 2d\), cholesterol was found to be increased in ST Kin)] TJ ET BT 26.250 357.877 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 64.314 353.988 Td /F1 9.8 Tf [( clone #6 cells compared to ST Kin)] TJ ET BT 214.961 357.877 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 233.751 353.988 Td /F1 9.8 Tf [( cells \(Fig. 4a\). Conversely, the cholesterol content was similar in ST Kin)] TJ ET BT 545.848 357.877 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 26.250 342.084 Td /F1 9.8 Tf [(cells and ST Kin)] TJ ET BT 96.694 345.972 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 134.758 342.084 Td /F1 9.8 Tf [( clone #2 cells \(Fig. 4a\). The lanosterol level was unchanged in both clones \(Fig. 4b\), while lathosterol )] TJ ET BT 26.250 330.179 Td /F1 9.8 Tf [(was significantly increased only in ST Kin)] TJ ET BT 204.519 334.067 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 242.583 330.179 Td /F1 9.8 Tf [( clone #6 cells compared to ST Kin)] TJ ET BT 393.230 334.067 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 414.429 330.179 Td /F1 9.8 Tf [(cells \(p<0.01\) \(Fig. 4c\). Moreover, the )] TJ ET BT 26.250 318.274 Td /F1 9.8 Tf [(levels of desmosterol and 7DHC were significantly increased in ST Kin)] TJ ET BT 330.226 322.162 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 368.290 318.274 Td /F1 9.8 Tf [( #6 compared to ST Kin)] TJ ET BT 470.714 322.162 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 508.778 318.274 Td /F1 9.8 Tf [( clone #2 cells )] TJ ET BT 26.250 306.369 Td /F1 9.8 Tf [(\(p<0.01\) and compared to ST Kin)] TJ ET BT 170.677 310.258 Td /F1 8.7 Tf [(7/7Q )] TJ ET BT 191.875 306.369 Td /F1 9.8 Tf [(cells \(Fig. 4d-e\). These results suggest that growth conditions may affect the levels of )] TJ ET BT 26.250 294.465 Td /F1 9.8 Tf [(different sterols in HD clones in different ways and that these effects are unlikely to be linked to the presence of muHTT.)] TJ ET 0.965 0.965 0.965 rg 26.250 -181.166 555.000 465.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 284.584 m 581.250 284.584 l 581.250 283.834 l 26.250 283.834 l f q 440.250 0 0 450.000 35.250 -175.166 cm /I5 Do Q q 35.250 -181.166 537.000 0.000 re W n Q Q q 440.250 0 0 450.000 35.250 -175.166 cm /I5 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(6)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 357 0 obj << /Type /Annot /Subtype /Link /A 358 0 R /Border [0 0 0] /H /I /Rect [ 495.5272 414.2177 505.1646 423.0360 ] >> endobj 358 0 obj << /Type /Action >> endobj 359 0 obj << /Type /Annot /Subtype /Link /A 360 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 -175.1663 475.5000 274.8337 ] >> endobj 360 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/10/fig3-chol-methods_11-10-121.jpg) >> endobj 361 0 obj << /Type /XObject /Subtype /Image /Width 587 /Height 600 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 39583>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   XK" }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Q4 γo6Ԃ8FYݏ&4(7пi/~'@eC#FOv~ ZJ >{If$hY2I?D{|+-3VIK6v 0 T84HhPKHQրLQڀJ1@ EI@ E%(cZ)1ւ9;AZ( (Lq@ E%PAQ( ;F)(hP)=(i(RQހJɠPIc-EE-[KbjL |GyV74}-XHK 8xg>xkn<^&5QC %u@ vW fx|M Ubk[ i^c}{r=C,;'B"" r8u xn]?غ}.LB^D/%T|a^_}/Bj>*S˨\iSFl&),BѓeA4^ӢNJ7jse$nLo߷<ϢqF+?ej~+SXRgIup̋}@^@ \QE-PcڗPh@ 1KEQ4[Oh}KY1RѺa'^!gOxw[4՛gw k6:/X&gk /./$qNg$(P6+> |ewc|1EpxrCKMX;]X.e lEV:z}+]o|7i$h+lרѐ'FxFStWΟ|gxO[]bmP\uu :H Aoqwu.cHג]mC\;$~_-vmr~ŲiXQRƱ9L s\De?3;PxyօzķֳjϬ <\5?v,2P8sZ?ٟu&mX> M%Ѓɓnvo;cx__ xwEISM2K42H6Oں=h}(( (Q((((? 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XfahՃ 区ۺ 1Z=oä^/.&E5)QȂ29 '+#g J_f7u,dּM:Id3^isdOhb)Oc't"BLӬ{H('' [@Q@We 쥚Zx?to2Cm rH|Jc_Hޕ$oO)ċB,®C5?$_ڣzot#' tiZ?.yL{wwUESDǍ$J[^)dY`X|253,ee$wb hm% Q(TE8€:D"r-M,Rč,++(7Oݦڤ$>i%0[ZjZybIhcBr)Rk;"͞%4ZxHOo1 hA4qKUx WCu+mnB,FJ&|bH76UpMuPEPEQEsEPEPEPA(RR(@w\'ůz /NTOqFR0T ebAG\h,d.meN ,j}X/Q  KQdiVzIc1}1ЛRm jO-3@&77g?[4ɧ\ksIb@84|ghlVZA2Fit-đƪq,p@'[ ~oe|m[yƲ7 >5ӵ/)<7q$_1jZwl R+ǰ\@.r:}+mm V$ 8P81@( (E~-_YZ]ȰA{GvlHh|wCB&FTvk <)]jInyF)#`rvW*9>axc^4ִ.^(ίyṞ=GLjֲC|fB8,E8g?|EkỿOawRXnT [ )b)e_e(4@Q@Q@Q(4QE ))x" ;~TcZ(E@Pw }n5WZԶPLfѤ0$x0NrI@(@ck~1Ѽ9f} pj'j~U$dVW/P3c&u-նea!2.<(?:gG{A񍆕-t˦aHyQΎGNQE-Mc+wAokAwx/eVإT{`BAv放 }ytUY6!_= \D_(8&9/  >Kx6hp<dž ̰`]R݊QF(@q^0LO;ў#1I%s4N or˻9?WF1C6ⴻ #"=/J?4O/xyuk{仹o wnaE9ISXl߆+t%]m?ċENPsIm0 _<x{Oi ĒJ,JXfX;Q ⴴiZNZu"K{s*\Ei {+F ( ( ( (4PEΌ})1@ E~tRR@E%)()i?ZZ(4PҁEQE(4P(/ i0n}{JִqaۤL7Xd;WxMZkLNᔍ.$j8sS  ( |w >W4}3Y @W8$˹rH8 n__)%ݤ:z 8m", 0!!gff$8*D(8 (EEQFh(((((@Q@yZŝ.&4]Q,.uyo 1RVH1(XQOU&>5C뺮j$f[ɬ,i qF"!Qugf%{(3EQFh˾*||mYJ1 œD-OOD,TX]9$f5|- ˨]\jzE%u#\A8P2X䞾(((((EPE4QEQEK@ GzJ(h4f PEfE(fEPGj(@Q@g(=hQހƊ)(hEPEP?QEQE~4fJ(Fh(Q((Fh((3@Q@((@Q(ƀ ( (Ɗ(%袀(Ew(4QEQ@(PEPGj((h(((((( ( (EPGj(((((((((((((((֊( endstream endobj 362 0 obj << /Type /Annot /Subtype /Link /A 363 0 R /Border [0 0 0] /H /I /Rect [ 495.5272 414.2177 505.1646 423.0360 ] >> endobj 363 0 obj << /Type /Action >> endobj 364 0 obj << /Type /Annot /Subtype /Link /A 365 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 -175.1663 475.5000 274.8337 ] >> endobj 365 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/10/fig3-chol-methods_11-10-121.jpg) >> endobj 366 0 obj << /Type /Annot /Subtype /Link /A 367 0 R /Border [0 0 0] /H /I /Rect [ 495.5272 414.2177 505.1646 423.0360 ] >> endobj 367 0 obj << /Type /Action >> endobj 368 0 obj << /Type /Annot /Subtype /Link /A 369 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 -175.1663 475.5000 274.8337 ] >> endobj 369 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/10/fig3-chol-methods_11-10-121.jpg) >> endobj 370 0 obj << /Type /Page /Parent 3 0 R /Annots [ 372 0 R 374 0 R 376 0 R 379 0 R 381 0 R 383 0 R 385 0 R 387 0 R 389 0 R ] /Contents 371 0 R >> endobj 371 0 obj << /Length 11342 >> stream q 15.000 59.962 577.500 717.038 re W n 0.965 0.965 0.965 rg 26.250 671.664 555.000 105.336 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 671.664 m 581.250 671.664 l 581.250 672.414 l 26.250 672.414 l f q 35.250 682.914 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Levels of cholesterol precursors in ST Kin7/7Q and ST Kin109/109Q cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Isotopic dilution GC-MS performed on ST Kin)] TJ ET BT 230.318 753.826 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 249.108 748.106 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 300.051 753.826 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 338.115 748.106 Td /F1 9.8 Tf [( cells grown in standard fetal bovine serum-)] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(containing medium shows that a-b\) lanosterol and lathosterol are moderately reduced in clone # 2 and #6 compared to wild-)] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(type cells, c-d\) while downstream cholesterol precursors \(desmosterol and 7dhc\) are significantly reduced in the muHTT )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(clones compared to wtHTT cells. The results are shown as the mean SEM of three independent experiments. Statistics: )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(One-way ANOVA and the Newman-Keuls Multiple Comparison Test were performed.)] TJ ET Q BT 26.250 654.640 Td /F1 9.8 Tf [(Because we obtained different results in ST Kin)] TJ ET BT 231.097 658.528 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 269.162 654.640 Td /F1 9.8 Tf [( clone #2 and clone #6 cells, we next used mass spectrometry to )] TJ ET BT 26.250 642.735 Td /F1 9.8 Tf [(measure the levels of cholesterol and cholesterol precursors in immortalized striatal cells that express the N-terminal fragment )] TJ ET BT 26.250 630.831 Td /F1 9.8 Tf [(of muHTT upon doxycycline administration )] TJ ET 0.267 0.267 0.267 rg BT 213.187 632.338 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 218.005 630.831 Td /F1 9.8 Tf [(. This model allows us to measure the levels in the same cellular background with )] TJ ET BT 26.250 618.926 Td /F1 9.8 Tf [(or without the expression of muHTT and to investigate differences that are linked to muHTT expression. Importantly, this )] TJ ET BT 26.250 607.021 Td /F1 9.8 Tf [(circumvents clone-dependent effects. In agreement with results obtained previously using the enzymatic method )] TJ ET 0.267 0.267 0.267 rg BT 512.882 608.528 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 517.701 607.021 Td /F1 9.8 Tf [(, we found )] TJ ET BT 26.250 595.116 Td /F1 9.8 Tf [(that the cholesterol level did not vary significantly in the uninduced cells compared to cells that were induced to express muHTT )] TJ ET BT 26.250 583.212 Td /F1 9.8 Tf [(\(33.602.14 ?g chol/mg prot vs. 33.861.76 ?g chol/mg prot, respectively\). These cells were grown in serum-containing )] TJ ET BT 26.250 571.307 Td /F1 9.8 Tf [(medium, and the exogenous cholesterol sources present in the medium may have helped the cells maintain a constant level of )] TJ ET BT 26.250 559.402 Td /F1 9.8 Tf [(cellular cholesterol. On the other hand, lathosterol, which is an indicator of cholesterol biosynthesis, was reduced in cells )] TJ ET BT 26.250 547.497 Td /F1 9.8 Tf [(expressing muHTT compared to cells cultured in uninduced conditions \(1.6470.1048 ng latho/mg prot vs. 2.8410.2988 ng )] TJ ET BT 26.250 535.593 Td /F1 9.8 Tf [(latho/mg prot, respectively; p<0.01\).)] TJ ET 0.965 0.965 0.965 rg 26.250 59.962 555.000 465.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 525.712 m 581.250 525.712 l 581.250 524.962 l 26.250 524.962 l f q 323.250 0 0 450.000 35.250 65.962 cm /I6 Do Q q 35.250 59.962 537.000 0.000 re W n Q Q q 15.000 59.962 577.500 717.038 re W n 0.965 0.965 0.965 rg 26.250 671.664 555.000 105.336 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 671.664 m 581.250 671.664 l 581.250 672.414 l 26.250 672.414 l f q 35.250 682.914 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Levels of cholesterol precursors in ST Kin7/7Q and ST Kin109/109Q cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Isotopic dilution GC-MS performed on ST Kin)] TJ ET BT 230.318 753.826 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 249.108 748.106 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 300.051 753.826 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 338.115 748.106 Td /F1 9.8 Tf [( cells grown in standard fetal bovine serum-)] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(containing medium shows that a-b\) lanosterol and lathosterol are moderately reduced in clone # 2 and #6 compared to wild-)] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(type cells, c-d\) while downstream cholesterol precursors \(desmosterol and 7dhc\) are significantly reduced in the muHTT )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(clones compared to wtHTT cells. The results are shown as the mean SEM of three independent experiments. Statistics: )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(One-way ANOVA and the Newman-Keuls Multiple Comparison Test were performed.)] TJ ET Q BT 26.250 654.640 Td /F1 9.8 Tf [(Because we obtained different results in ST Kin)] TJ ET BT 231.097 658.528 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 269.162 654.640 Td /F1 9.8 Tf [( clone #2 and clone #6 cells, we next used mass spectrometry to )] TJ ET BT 26.250 642.735 Td /F1 9.8 Tf [(measure the levels of cholesterol and cholesterol precursors in immortalized striatal cells that express the N-terminal fragment )] TJ ET BT 26.250 630.831 Td /F1 9.8 Tf [(of muHTT upon doxycycline administration )] TJ ET 0.267 0.267 0.267 rg BT 213.187 632.338 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 218.005 630.831 Td /F1 9.8 Tf [(. This model allows us to measure the levels in the same cellular background with )] TJ ET BT 26.250 618.926 Td /F1 9.8 Tf [(or without the expression of muHTT and to investigate differences that are linked to muHTT expression. Importantly, this )] TJ ET BT 26.250 607.021 Td /F1 9.8 Tf [(circumvents clone-dependent effects. In agreement with results obtained previously using the enzymatic method )] TJ ET 0.267 0.267 0.267 rg BT 512.882 608.528 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 517.701 607.021 Td /F1 9.8 Tf [(, we found )] TJ ET BT 26.250 595.116 Td /F1 9.8 Tf [(that the cholesterol level did not vary significantly in the uninduced cells compared to cells that were induced to express muHTT )] TJ ET BT 26.250 583.212 Td /F1 9.8 Tf [(\(33.602.14 ?g chol/mg prot vs. 33.861.76 ?g chol/mg prot, respectively\). These cells were grown in serum-containing )] TJ ET BT 26.250 571.307 Td /F1 9.8 Tf [(medium, and the exogenous cholesterol sources present in the medium may have helped the cells maintain a constant level of )] TJ ET BT 26.250 559.402 Td /F1 9.8 Tf [(cellular cholesterol. On the other hand, lathosterol, which is an indicator of cholesterol biosynthesis, was reduced in cells )] TJ ET BT 26.250 547.497 Td /F1 9.8 Tf [(expressing muHTT compared to cells cultured in uninduced conditions \(1.6470.1048 ng latho/mg prot vs. 2.8410.2988 ng )] TJ ET BT 26.250 535.593 Td /F1 9.8 Tf [(latho/mg prot, respectively; p<0.01\).)] TJ ET 0.965 0.965 0.965 rg 26.250 59.962 555.000 465.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 525.712 m 581.250 525.712 l 581.250 524.962 l 26.250 524.962 l f q 323.250 0 0 450.000 35.250 65.962 cm /I6 Do Q q 35.250 59.962 537.000 0.000 re W n Q Q q 15.000 59.962 577.500 717.038 re W n 0.965 0.965 0.965 rg 26.250 671.664 555.000 105.336 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 671.664 m 581.250 671.664 l 581.250 672.414 l 26.250 672.414 l f q 35.250 682.914 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Levels of cholesterol precursors in ST Kin7/7Q and ST Kin109/109Q cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Isotopic dilution GC-MS performed on ST Kin)] TJ ET BT 230.318 753.826 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 249.108 748.106 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 300.051 753.826 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 338.115 748.106 Td /F1 9.8 Tf [( cells grown in standard fetal bovine serum-)] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(containing medium shows that a-b\) lanosterol and lathosterol are moderately reduced in clone # 2 and #6 compared to wild-)] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(type cells, c-d\) while downstream cholesterol precursors \(desmosterol and 7dhc\) are significantly reduced in the muHTT )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(clones compared to wtHTT cells. The results are shown as the mean SEM of three independent experiments. Statistics: )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(One-way ANOVA and the Newman-Keuls Multiple Comparison Test were performed.)] TJ ET Q BT 26.250 654.640 Td /F1 9.8 Tf [(Because we obtained different results in ST Kin)] TJ ET BT 231.097 658.528 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 269.162 654.640 Td /F1 9.8 Tf [( clone #2 and clone #6 cells, we next used mass spectrometry to )] TJ ET BT 26.250 642.735 Td /F1 9.8 Tf [(measure the levels of cholesterol and cholesterol precursors in immortalized striatal cells that express the N-terminal fragment )] TJ ET BT 26.250 630.831 Td /F1 9.8 Tf [(of muHTT upon doxycycline administration )] TJ ET 0.267 0.267 0.267 rg BT 213.187 632.338 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 218.005 630.831 Td /F1 9.8 Tf [(. This model allows us to measure the levels in the same cellular background with )] TJ ET BT 26.250 618.926 Td /F1 9.8 Tf [(or without the expression of muHTT and to investigate differences that are linked to muHTT expression. Importantly, this )] TJ ET BT 26.250 607.021 Td /F1 9.8 Tf [(circumvents clone-dependent effects. In agreement with results obtained previously using the enzymatic method )] TJ ET 0.267 0.267 0.267 rg BT 512.882 608.528 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 517.701 607.021 Td /F1 9.8 Tf [(, we found )] TJ ET BT 26.250 595.116 Td /F1 9.8 Tf [(that the cholesterol level did not vary significantly in the uninduced cells compared to cells that were induced to express muHTT )] TJ ET BT 26.250 583.212 Td /F1 9.8 Tf [(\(33.602.14 ?g chol/mg prot vs. 33.861.76 ?g chol/mg prot, respectively\). These cells were grown in serum-containing )] TJ ET BT 26.250 571.307 Td /F1 9.8 Tf [(medium, and the exogenous cholesterol sources present in the medium may have helped the cells maintain a constant level of )] TJ ET BT 26.250 559.402 Td /F1 9.8 Tf [(cellular cholesterol. On the other hand, lathosterol, which is an indicator of cholesterol biosynthesis, was reduced in cells )] TJ ET BT 26.250 547.497 Td /F1 9.8 Tf [(expressing muHTT compared to cells cultured in uninduced conditions \(1.6470.1048 ng latho/mg prot vs. 2.8410.2988 ng )] TJ ET BT 26.250 535.593 Td /F1 9.8 Tf [(latho/mg prot, respectively; p<0.01\).)] TJ ET 0.965 0.965 0.965 rg 26.250 59.962 555.000 465.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 525.712 m 581.250 525.712 l 581.250 524.962 l 26.250 524.962 l f q 323.250 0 0 450.000 35.250 65.962 cm /I6 Do Q q 35.250 59.962 537.000 0.000 re W n Q Q q 323.250 0 0 450.000 35.250 65.962 cm /I6 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(7)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 372 0 obj << /Type /Annot /Subtype /Link /A 373 0 R /Border [0 0 0] /H /I /Rect [ 213.1867 631.5362 218.0054 640.3545 ] >> endobj 373 0 obj << /Type /Action >> endobj 374 0 obj << /Type /Annot /Subtype /Link /A 375 0 R /Border [0 0 0] /H /I /Rect [ 512.8822 607.7267 517.7009 616.5450 ] >> endobj 375 0 obj << /Type /Action >> endobj 376 0 obj << /Type /Annot /Subtype /Link /A 377 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 65.9617 358.5000 515.9617 ] >> endobj 377 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/10/fig4-chol-methods_11-10-12.jpg) >> endobj 378 0 obj << /Type /XObject /Subtype /Image /Width 431 /Height 600 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 35429>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?'^^NyeaF5V(22d5en;Hx!Z+mgRA^RcOvw.AoWf˩闷]ۉ+k{!t<0`hq^m|d -thXr aue/*//[D'Vk-.wTRm!=:߅ĐkS}`{ZDr 9Y #|XUk++tQɝZ -zg+ @4*+VԮe$..4R5]ct+F1y7b> |37&Ч֮T5]p7r: 9=رu}ihϽ-->~4hihϽ-~4-@ ϭ-hih>4PsGK@ GKG@ ϭ-'>~4@ ѓKAϽtPgގ}ih?3Jh@='G@ j1IG(qGJO—cڏʏ€~cڗ~Ҧ5REaPH!L Is=+tc^)SR+YV9~f;A:>8x'Fդ~swͳp(0LHqF=_ wQ|aAmQFl;I YK喗4Y5ncrIۀŻ7@&+ vxtjskH~'(ϵ.)^~~ԸJ? 1KOʀ Rⓧj3(.)? ? \QO€b(.(&}/@ QIQ@)i?G@ E&(((:Nq@~|[F[^xBeS6 ^ɸev[yqȮ_ ^#[mOXF%ͽv|$R[F1p@(?:_Γ֔@ K'RtqI c8ΓykkXYKZ[ib#H mXgk61]P[~ڮhwIibQ7 $9fcXH.th^#N𽡱.nckKk.Am(;8Zz 3G/%5=F+ l4ɯvWs@JfֿcAƐI!%y"7j6 Jy1 8<,MxXԵHdkVaMv=ҹuKn_QihμTҴ4\Yr^lðUU_ rw`})ǭQ'F/@IڗZL ?:?:QG8:8P~tQQ'KPGq@ Gu8:8OΗZ8'KPPIKE'E- z_Iݮ-D[HL|YK(8 k}S㗌{g e" #jԐ]C֕Y9~Ͼ;otj|^8?J:i_i }]gEkJR{G ?*wy"!=+#bNUzh(4f )yRׇ|KoZRXeI ʑ#+)y0_0|F񿏭t\xRtRkTqBl DSr|֍A}VԳծlRiv2㺷p8dxNB?7Q^;kơxTĄ[Pk$܃d`WȒ5TM04QKI(4f (4QKI(4f /4򢖊J)sG4Q^h)xE-QKI(&4R@ E;ZLQ/GTKJ7̟fMIȹ>ٮ3+?3ajrB(~H$rrEz\׊~$xg7}[W#qD.7l.]FrEt>MjVW֐X\4MdЩs)vJ) zIJ]6[/[5OSK=0CVʀ _־h?nׄŏOE[=o{ 3}2u;W_KqG__?h'N_}zq@UN_}TK+5Q]^ҕPX(+8쯎4|?rC?'/>W(ʿ8 O+ ^(<+ ?8 OU(ʿ8 Of-;gýV)x_WIr+j,H&R2Z(Z)8_֓('q@ zzqGq@ ?ZN)x(Z?ZL\?Z?ZN(Rq@ zzёIdP'RP'Q@ GIK@ΥI*@^w Bg^-KDҡ涅tLS=f2;cl%*:YH?Jx~@wk-(i<˧"fe@cVCgLzoҔRW|u?540MuR,""a'>Y0a6;Oؓv^^ ][#jhӼB.op< ^::)(J(J(:?:J(h O|@a<#Oom.o..X6āPn1s^_ߴW/_ _'DS1Ʀ6yGorwgj_Γ4P~tQ@ E%%P_ΊJ;wP~t%-cIK@ KI@ 'Ji(qIPtP⒖󢖀Ί)9Z1@\ҖP1#|4xqߦ|E!y"׼^G7ĝoK+?7ᾏ-䕡6 yEeG<פW~3>5kMׇ=ZvX RB829!8 (LQ?J(1_cx >0{Kyu<_Bt[ǒw q(%z(8bo5=J]B ޡ+w> rOh4b(1F()h1F( sF( P:(c(cERb sF( PP1GEQEQb( RIEQ? _€b(QG@ QIQPRQ@ Wf_^ M|+}"9fTe"]wީ@GֹO|*u=c[u V#ܛX1@AJ(ae?E K~E#3df_і9g -oN$]WP~𢒊_’_’_Š?*LPGIQ@ QRQ@ QRQ@ GIE/GIPh ? 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$|ˬ#H Am漛]#f_MJUh,]J[H(X]<ԟ;ֿ*=W9*n{UIGgZPI }rYXPhf ǿ^k_G3o=/5JU֎kʿ᛼3AyUֿ*=[ k*m麅j:$׳Ism8>tvsÿ|3AyUy_ x_Z[<נ5F,bWY@ComH"%2;T0ҚZEzխ樱sqenFGvkmg FsK3@&)qI1KI1@=i1KNԴ:h1۾}ob.f[h īf'lɠ ^53?WZU-[:0ˁI= &|MnUm7PѧXX&#̆,Qm, hџ1J q`]B?u?&$_jjcW /t fqkuwin=ʌ!AiYçkXRKa@<z`R'ծIA0ۖI@dV\ՙFY >'i_ tKp3yBB] D@',@>C!}7Y^R,dy,.\2j%̖C"9yL)=^ZR;vRoIq-Chċ9Y9\P &]GRVڅq(x/F]KI1I1@ &+żmۯӶc0}wc~(2јLb#pk+DUECMeᷝ@[ Ij!-y27k:ZK9.!jg9BD);bV潯[_La; j|n* B(+KH,ma qETP01LQZLqF(qF(.&.)1@ яzLQ1KbOχa=:Uk(Dw;[BA+r >(Q"Ҵ-J4Co&dMl#eSx >=ej޽j*Y< 7rA/mʉg]; ~%^$LkQG#.#3Es$e` ~&=WJ.o|)uzmn\ β*K qꮭxS_MӦu9bnm F4F2N_8.~oc]ft-n^qm] %%ٞ4znDѨ(dJeVI I䌞YI oLeqieb8-(ߒI$rI$I&p=iqI\ZhS꺽Vl:$ F9<$Ii^ _Q`ā+9< *3xUVb@n]/ie^y#B-П i1HK6wS$4|E]KNTQwwLea&s8HX `bS%W> | ~}^x^3I4KUso |ƉG;b}3Cm|;iUm㵁*OsW@G&.&)q@IF( )sFh(.h%.h{ƾ<|{fɷ8kvl8՝f;TU .Юuj߂~/]Cgң-.A_b\U,2č]_6t<3qf:h{wG1`p9C_KmͽeE$\ f$i[g k +F-a Whڋ~@d{\њJ)sT5XUvwŽ ae\lvU,x$ (]OSѴ,lm2ss(( f' M||'>/)䱝?{EmgSv$ rcx-cڇ[&SG2Empl0<+bJCE.1[xBkH"kME.]W6378g2zHGmoV$D8B(qRR挊LE.Exό*6OK0Hz,`?-%`OSn+xEԣg˕#GWh $YX)a9|1ɉRi([ʅ_b Ab⣅U|{x[ZgziR45G,S Ѫ%h%}'(QK3@&isI(4K3@ FisFhυ<-uc/" m sIoh%!@ #*3'c*rGi?ǂ,[i|<-q47@[mŬ=v`J6>/ϖo5l$ %Ɯc(%Xݾ`9^<93JKın.|҄@ݻII9MrhZ>)H4TG @cZf44f~x=.`[[mBO wh@d4 K&51khhZ-Z֮- $B]21A!gv~ ԥ׵a\v@+-$Dʼn-&G#OMf5+h-L!B^KUI8Gܷp3 KY€9ր 3K3@ H9$TB)c8*'>,A]igKM"VUY X\`FWaC^,4m*mt(Vb^9=ORI&K_j J|+ $!qI$1*Lњ3@ )sFh3E.E4QLQZ)1@RbPc1@ E&(swgR\E ȆY!79 "B$5~Nj>" 1x\d[_Ï4m 󐏥lDpi3,Hbj>ԭ&e)$3tu=C)c\ǂi /5 WR;v> endobj 380 0 obj << /Type /Action >> endobj 381 0 obj << /Type /Annot /Subtype /Link /A 382 0 R /Border [0 0 0] /H /I /Rect [ 512.8822 607.7267 517.7009 616.5450 ] >> endobj 382 0 obj << /Type /Action >> endobj 383 0 obj << /Type /Annot /Subtype /Link /A 384 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 65.9617 358.5000 515.9617 ] >> endobj 384 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/10/fig4-chol-methods_11-10-12.jpg) >> endobj 385 0 obj << /Type /Annot /Subtype 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R 493 0 R 495 0 R 497 0 R 499 0 R 501 0 R 503 0 R 505 0 R ] /Contents 392 0 R >> endobj 392 0 obj << /Length 32833 >> stream q 15.000 25.630 577.500 751.370 re W n 0.965 0.965 0.965 rg 26.250 616.719 555.000 160.281 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 616.719 m 581.250 616.719 l 581.250 617.469 l 26.250 617.469 l f q 35.250 627.969 537.000 149.031 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 4: Levels of cholesterol precursors in over-confluent ST Kin7/7Q and ST Kin109/109Q cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(The same mass spectrometry analysis of cholesterol and cholesterol precursors reported in Figure 2 was also performed in )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(cells harvested at over-confluence. a\) Cholesterol levels are similar in ST Kin)] TJ ET BT 366.857 740.090 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 404.921 734.370 Td /F1 9.8 Tf [( clone #2 and ST Kin)] TJ ET BT 495.431 740.090 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 514.220 734.370 Td /F1 9.8 Tf [( but are )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(increased in ST Kin)] TJ ET BT 119.782 726.353 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 157.847 720.634 Td /F1 9.8 Tf [( clone #6 compared to ST Kin)] TJ ET BT 286.283 726.353 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 305.073 720.634 Td /F1 9.8 Tf [(. b\) Lanosterol was not significantly different in the mHTT )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(clones and wild-type cells, while lathosterol \(c\) was higher in ST Kin)] TJ ET BT 327.301 712.617 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 365.366 706.897 Td /F1 9.8 Tf [( clone #6 compared to wild-type and ST Kin)] TJ ET BT 35.250 698.881 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 73.314 693.161 Td /F1 9.8 Tf [( clone #2. d-e\) Similar changes are observed in the downstream cholesterol precursors desmosterol and 7dhc. )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(Although it does not reach the significance, desmosterol and 7dhc are increased in ST Kin)] TJ ET BT 423.817 685.145 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 461.881 679.425 Td /F1 9.8 Tf [( clone #6 compared to )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(ST Kin)] TJ ET BT 64.510 671.408 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 83.299 665.689 Td /F1 9.8 Tf [(. There are no significant differences between ST Kin)] TJ ET BT 312.531 671.408 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 350.595 665.689 Td /F1 9.8 Tf [( clone #6 and ST Kin)] TJ ET BT 441.105 671.408 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 459.894 665.689 Td /F1 9.8 Tf [( cells. The results are )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(shown as the mean SEM from analyses in two independent experiments. Statistics: One-way ANOVA and the Newman-)] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(Keuls Multiple Comparison Test were performed.)] TJ ET Q BT 26.250 599.695 Td /F1 9.8 Tf [(Taken together, these results illustrate the complexity of detecting cholesterol and its precursors )] TJ ET BT 442.429 599.695 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 471.142 599.695 Td /F1 9.8 Tf [( and show that clonal )] TJ ET BT 26.250 587.790 Td /F1 9.8 Tf [(variability and differences in cell density can lead to considerably different results.)] TJ ET BT 26.250 551.188 Td /F4 12.0 Tf [(Conclusions)] TJ ET BT 26.250 531.234 Td /F1 9.8 Tf [(Cholesterol is a structural component of biological membranes and is the precursor of numerous signalling molecules. In the )] TJ ET BT 26.250 519.329 Td /F1 9.8 Tf [(brain, cholesterol is involved in neurite outgrowth, myelination and synaptic activity, and its importance in brain physiology and )] TJ ET BT 26.250 507.424 Td /F1 9.8 Tf [(pathology is clear from neurological diseases in which there are changes in its level or distribution )] TJ ET 0.267 0.267 0.267 rg BT 449.459 508.931 Td /F4 8.7 Tf [(3)] TJ ET BT 454.277 508.931 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 459.096 507.424 Td /F1 9.8 Tf [(. Changes in sterol levels in )] TJ ET BT 26.250 495.519 Td /F1 9.8 Tf [(cellular and animal models of HD have been detected in previous studies, but the direction of these changes remains a matter )] TJ ET BT 26.250 483.615 Td /F1 9.8 Tf [(of debate. Specifically, while some studies report a decrease in cholesterol levels in HD models )] TJ ET 0.267 0.267 0.267 rg BT 439.699 485.122 Td /F4 8.7 Tf [(6)] TJ ET BT 444.517 485.122 Td /F4 8.7 Tf [(7)] TJ ET BT 449.336 485.122 Td /F4 8.7 Tf [(8)] TJ ET BT 454.155 485.122 Td /F4 8.7 Tf [(11)] TJ ET BT 463.792 485.122 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 473.429 483.615 Td /F1 9.8 Tf [(, others report the )] TJ ET BT 26.250 471.710 Td /F1 9.8 Tf [(opposite )] TJ ET 0.267 0.267 0.267 rg BT 65.816 473.217 Td /F4 8.7 Tf [(12)] TJ ET BT 75.453 473.217 Td /F4 8.7 Tf [(13)] TJ ET BT 85.090 473.217 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 94.728 471.710 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 452.305 Td /F1 9.8 Tf [(In this study, we investigated factors that could underlie these conflicting findings.)] TJ ET BT 26.250 432.900 Td /F1 9.8 Tf [(As we expected, the methods used to detect cholesterol, sample preparation methods, cell line clonal properties and cell growth )] TJ ET BT 26.250 420.996 Td /F1 9.8 Tf [(conditions all influenced the levels of cholesterol that were detected. It is critical to be aware of the limitations and artefacts that )] TJ ET BT 26.250 409.091 Td /F1 9.8 Tf [(arise from using less sensitive methods or from inappropriate sample preparation. Colorimetric methods such as filipin staining )] TJ ET BT 26.250 397.186 Td /F1 9.8 Tf [(should be avoided when the aim is quantitative detection of cholesterol levels in cells or tissue samples. Cholesterol levels may )] TJ ET BT 26.250 385.281 Td /F1 9.8 Tf [(be different due to the existence of variants of what is believed to be the same cell line. In this study, we found that two clones )] TJ ET BT 26.250 373.377 Td /F1 9.8 Tf [(of ST Kin cells, which were derived years ago in our laboratory at the University of Milan )] TJ ET 0.267 0.267 0.267 rg BT 408.265 374.884 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 417.902 373.377 Td /F1 9.8 Tf [(, were not, in fact, identical. The )] TJ ET BT 26.250 361.472 Td /F1 9.8 Tf [(clones have been distributed by us and by collaborating laboratories to many other laboratories, as well as by direct transfer )] TJ ET BT 26.250 349.567 Td /F1 9.8 Tf [(from a recipient lab to yet another lab. Different laboratory practices and mislabelling may have resulted in the propagation of )] TJ ET BT 26.250 337.662 Td /F1 9.8 Tf [(cells with properties that are different from expected or that differ from lab to lab. Contrary to reports from other groups )] TJ ET BT 538.340 337.662 Td /F1 9.8 Tf [(14, )] TJ ET BT 554.603 337.662 Td /F1 9.8 Tf [(19)] TJ ET BT 565.445 337.662 Td /F1 9.8 Tf [(, )] TJ ET BT 26.250 325.758 Td /F1 9.8 Tf [(we found no NMDAr or AMPAr activity and no expression of the NMDA subunit NR1 mRNA in ST-Kin cells. Genetic-based )] TJ ET BT 26.250 313.853 Td /F1 9.8 Tf [(assays for the identification of cell lines )] TJ ET 0.267 0.267 0.267 rg BT 197.489 315.360 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 207.127 313.853 Td /F1 9.8 Tf [( and the presence of a dedicated biorepository that controls the quality and )] TJ ET BT 26.250 301.948 Td /F1 9.8 Tf [(distribution )] TJ ET 0.267 0.267 0.267 rg BT 76.102 303.455 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 85.739 301.948 Td /F1 9.8 Tf [( of cell lines would reduce the risk of laboratory errors; such issues have already caused major damage in other )] TJ ET BT 26.250 290.043 Td /F1 9.8 Tf [(fields )] TJ ET 0.267 0.267 0.267 rg BT 51.717 291.551 Td /F4 8.7 Tf [(22)] TJ ET BT 61.354 291.551 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 70.992 290.043 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 270.639 Td /F1 9.8 Tf [(When a novel disease target emerges, prior to embarking in drug development initiatives, it is essential to validate the target in )] TJ ET BT 26.250 258.734 Td /F1 9.8 Tf [(mammalian models of the disease. In the case of targets associated with changes in cholesterol synthesis, a reliable, sensitive )] TJ ET BT 26.250 246.829 Td /F1 9.8 Tf [(detection method is key for ensuring that the results can be interpreted with confidence. When considering cholesterol )] TJ ET BT 26.250 234.924 Td /F1 9.8 Tf [(homeostasis, interpretation may be even more cumbersome given the multiplicity of cellular regulatory steps and compensatory )] TJ ET BT 26.250 223.020 Td /F1 9.8 Tf [(mechanisms at play; indeed, such mechanisms can present a challenge in experimental conditions. Small variations in cell )] TJ ET BT 26.250 211.115 Td /F1 9.8 Tf [(culture conditions, or other confounding factors introduced in the experimental setting, may affect the results of cholesterol level )] TJ ET BT 26.250 199.210 Td /F1 9.8 Tf [(determination. One study found a link between SIRT2 and cholesterol in worm, fly and cell culture HD models )] TJ ET 0.267 0.267 0.267 rg BT 500.402 200.717 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 510.040 199.210 Td /F1 9.8 Tf [(. This link )] TJ ET BT 26.250 187.305 Td /F1 9.8 Tf [(should be validated using a more sensitive method to detect cholesterol )] TJ ET 0.267 0.267 0.267 rg BT 337.860 188.813 Td /F4 8.7 Tf [(24)] TJ ET BT 347.497 188.813 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 357.135 187.305 Td /F1 9.8 Tf [(, especially since others failed to confirm )] TJ ET BT 534.867 187.305 Td /F5 9.8 Tf [(in vivo, )] TJ ET BT 567.920 187.305 Td /F1 9.8 Tf [(in )] TJ ET BT 26.250 175.401 Td /F1 9.8 Tf [(mouse models, SIRT2 inhibition as a therapeutic strategy in HD )] TJ ET 0.267 0.267 0.267 rg BT 303.150 176.908 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 312.787 175.401 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 155.996 Td /F1 9.8 Tf [(To conclude, the hydrophobicity of different lipids, their susceptibility to oxidation in different experimental conditions, the )] TJ ET BT 26.250 144.091 Td /F1 9.8 Tf [(specific extraction procedures used to obtain the desired species and the technological limitations of the methods used for )] TJ ET BT 26.250 132.186 Td /F1 9.8 Tf [(measuring lipid levels, all suggest that the HD scientific community should use caution in interpreting results. In addition, it may )] TJ ET BT 26.250 120.282 Td /F1 9.8 Tf [(be helpful to consult with lipid experts. Our findings make it clear that the most sensitive and reliable analytical techniques, )] TJ ET BT 26.250 108.377 Td /F1 9.8 Tf [(along with appropriate sample preparation, should be used for unambiguous and unbiased measurement and interpretation of )] TJ ET BT 26.250 96.472 Td /F1 9.8 Tf [(cholesterol dysfunction in the context of HD.)] TJ ET BT 26.250 59.870 Td /F4 12.0 Tf [(Methods)] TJ ET BT 26.250 39.915 Td /F4 9.8 Tf [(Reagents.)] TJ ET BT 72.845 39.915 Td /F1 9.8 Tf [( Filipin, Nile Red, cholesterol, M?CD and simvastatin were purchased from Sigma Chemicals Co. \(St. Louis, MO, )] TJ ET Q q 15.000 25.630 577.500 751.370 re W n 0.965 0.965 0.965 rg 26.250 616.719 555.000 160.281 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 616.719 m 581.250 616.719 l 581.250 617.469 l 26.250 617.469 l f q 35.250 627.969 537.000 149.031 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 4: Levels of cholesterol precursors in over-confluent ST Kin7/7Q and ST Kin109/109Q cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(The same mass spectrometry analysis of cholesterol and cholesterol precursors reported in Figure 2 was also performed in )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(cells harvested at over-confluence. a\) Cholesterol levels are similar in ST Kin)] TJ ET BT 366.857 740.090 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 404.921 734.370 Td /F1 9.8 Tf [( clone #2 and ST Kin)] TJ ET BT 495.431 740.090 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 514.220 734.370 Td /F1 9.8 Tf [( but are )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(increased in ST Kin)] TJ ET BT 119.782 726.353 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 157.847 720.634 Td /F1 9.8 Tf [( clone #6 compared to ST Kin)] TJ ET BT 286.283 726.353 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 305.073 720.634 Td /F1 9.8 Tf [(. b\) Lanosterol was not significantly different in the mHTT )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(clones and wild-type cells, while lathosterol \(c\) was higher in ST Kin)] TJ ET BT 327.301 712.617 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 365.366 706.897 Td /F1 9.8 Tf [( clone #6 compared to wild-type and ST Kin)] TJ ET BT 35.250 698.881 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 73.314 693.161 Td /F1 9.8 Tf [( clone #2. d-e\) Similar changes are observed in the downstream cholesterol precursors desmosterol and 7dhc. )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(Although it does not reach the significance, desmosterol and 7dhc are increased in ST Kin)] TJ ET BT 423.817 685.145 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 461.881 679.425 Td /F1 9.8 Tf [( clone #6 compared to )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(ST Kin)] TJ ET BT 64.510 671.408 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 83.299 665.689 Td /F1 9.8 Tf [(. There are no significant differences between ST Kin)] TJ ET BT 312.531 671.408 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 350.595 665.689 Td /F1 9.8 Tf [( clone #6 and ST Kin)] TJ ET BT 441.105 671.408 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 459.894 665.689 Td /F1 9.8 Tf [( cells. The results are )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(shown as the mean SEM from analyses in two independent experiments. Statistics: One-way ANOVA and the Newman-)] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(Keuls Multiple Comparison Test were performed.)] TJ ET Q BT 26.250 599.695 Td /F1 9.8 Tf [(Taken together, these results illustrate the complexity of detecting cholesterol and its precursors )] TJ ET BT 442.429 599.695 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 471.142 599.695 Td /F1 9.8 Tf [( and show that clonal )] TJ ET BT 26.250 587.790 Td /F1 9.8 Tf [(variability and differences in cell density can lead to considerably different results.)] TJ ET BT 26.250 551.188 Td /F4 12.0 Tf [(Conclusions)] TJ ET BT 26.250 531.234 Td /F1 9.8 Tf [(Cholesterol is a structural component of biological membranes and is the precursor of numerous signalling molecules. In the )] TJ ET BT 26.250 519.329 Td /F1 9.8 Tf [(brain, cholesterol is involved in neurite outgrowth, myelination and synaptic activity, and its importance in brain physiology and )] TJ ET BT 26.250 507.424 Td /F1 9.8 Tf [(pathology is clear from neurological diseases in which there are changes in its level or distribution )] TJ ET 0.267 0.267 0.267 rg BT 449.459 508.931 Td /F4 8.7 Tf [(3)] TJ ET BT 454.277 508.931 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 459.096 507.424 Td /F1 9.8 Tf [(. Changes in sterol levels in )] TJ ET BT 26.250 495.519 Td /F1 9.8 Tf [(cellular and animal models of HD have been detected in previous studies, but the direction of these changes remains a matter )] TJ ET BT 26.250 483.615 Td /F1 9.8 Tf [(of debate. Specifically, while some studies report a decrease in cholesterol levels in HD models )] TJ ET 0.267 0.267 0.267 rg BT 439.699 485.122 Td /F4 8.7 Tf [(6)] TJ ET BT 444.517 485.122 Td /F4 8.7 Tf [(7)] TJ ET BT 449.336 485.122 Td /F4 8.7 Tf [(8)] TJ ET BT 454.155 485.122 Td /F4 8.7 Tf [(11)] TJ ET BT 463.792 485.122 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 473.429 483.615 Td /F1 9.8 Tf [(, others report the )] TJ ET BT 26.250 471.710 Td /F1 9.8 Tf [(opposite )] TJ ET 0.267 0.267 0.267 rg BT 65.816 473.217 Td /F4 8.7 Tf [(12)] TJ ET BT 75.453 473.217 Td /F4 8.7 Tf [(13)] TJ ET BT 85.090 473.217 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 94.728 471.710 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 452.305 Td /F1 9.8 Tf [(In this study, we investigated factors that could underlie these conflicting findings.)] TJ ET BT 26.250 432.900 Td /F1 9.8 Tf [(As we expected, the methods used to detect cholesterol, sample preparation methods, cell line clonal properties and cell growth )] TJ ET BT 26.250 420.996 Td /F1 9.8 Tf [(conditions all influenced the levels of cholesterol that were detected. It is critical to be aware of the limitations and artefacts that )] TJ ET BT 26.250 409.091 Td /F1 9.8 Tf [(arise from using less sensitive methods or from inappropriate sample preparation. Colorimetric methods such as filipin staining )] TJ ET BT 26.250 397.186 Td /F1 9.8 Tf [(should be avoided when the aim is quantitative detection of cholesterol levels in cells or tissue samples. Cholesterol levels may )] TJ ET BT 26.250 385.281 Td /F1 9.8 Tf [(be different due to the existence of variants of what is believed to be the same cell line. In this study, we found that two clones )] TJ ET BT 26.250 373.377 Td /F1 9.8 Tf [(of ST Kin cells, which were derived years ago in our laboratory at the University of Milan )] TJ ET 0.267 0.267 0.267 rg BT 408.265 374.884 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 417.902 373.377 Td /F1 9.8 Tf [(, were not, in fact, identical. The )] TJ ET BT 26.250 361.472 Td /F1 9.8 Tf [(clones have been distributed by us and by collaborating laboratories to many other laboratories, as well as by direct transfer )] TJ ET BT 26.250 349.567 Td /F1 9.8 Tf [(from a recipient lab to yet another lab. Different laboratory practices and mislabelling may have resulted in the propagation of )] TJ ET BT 26.250 337.662 Td /F1 9.8 Tf [(cells with properties that are different from expected or that differ from lab to lab. Contrary to reports from other groups )] TJ ET BT 538.340 337.662 Td /F1 9.8 Tf [(14, )] TJ ET BT 554.603 337.662 Td /F1 9.8 Tf [(19)] TJ ET BT 565.445 337.662 Td /F1 9.8 Tf [(, )] TJ ET BT 26.250 325.758 Td /F1 9.8 Tf [(we found no NMDAr or AMPAr activity and no expression of the NMDA subunit NR1 mRNA in ST-Kin cells. Genetic-based )] TJ ET BT 26.250 313.853 Td /F1 9.8 Tf [(assays for the identification of cell lines )] TJ ET 0.267 0.267 0.267 rg BT 197.489 315.360 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 207.127 313.853 Td /F1 9.8 Tf [( and the presence of a dedicated biorepository that controls the quality and )] TJ ET BT 26.250 301.948 Td /F1 9.8 Tf [(distribution )] TJ ET 0.267 0.267 0.267 rg BT 76.102 303.455 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 85.739 301.948 Td /F1 9.8 Tf [( of cell lines would reduce the risk of laboratory errors; such issues have already caused major damage in other )] TJ ET BT 26.250 290.043 Td /F1 9.8 Tf [(fields )] TJ ET 0.267 0.267 0.267 rg BT 51.717 291.551 Td /F4 8.7 Tf [(22)] TJ ET BT 61.354 291.551 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 70.992 290.043 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 270.639 Td /F1 9.8 Tf [(When a novel disease target emerges, prior to embarking in drug development initiatives, it is essential to validate the target in )] TJ ET BT 26.250 258.734 Td /F1 9.8 Tf [(mammalian models of the disease. In the case of targets associated with changes in cholesterol synthesis, a reliable, sensitive )] TJ ET BT 26.250 246.829 Td /F1 9.8 Tf [(detection method is key for ensuring that the results can be interpreted with confidence. When considering cholesterol )] TJ ET BT 26.250 234.924 Td /F1 9.8 Tf [(homeostasis, interpretation may be even more cumbersome given the multiplicity of cellular regulatory steps and compensatory )] TJ ET BT 26.250 223.020 Td /F1 9.8 Tf [(mechanisms at play; indeed, such mechanisms can present a challenge in experimental conditions. Small variations in cell )] TJ ET BT 26.250 211.115 Td /F1 9.8 Tf [(culture conditions, or other confounding factors introduced in the experimental setting, may affect the results of cholesterol level )] TJ ET BT 26.250 199.210 Td /F1 9.8 Tf [(determination. One study found a link between SIRT2 and cholesterol in worm, fly and cell culture HD models )] TJ ET 0.267 0.267 0.267 rg BT 500.402 200.717 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 510.040 199.210 Td /F1 9.8 Tf [(. This link )] TJ ET BT 26.250 187.305 Td /F1 9.8 Tf [(should be validated using a more sensitive method to detect cholesterol )] TJ ET 0.267 0.267 0.267 rg BT 337.860 188.813 Td /F4 8.7 Tf [(24)] TJ ET BT 347.497 188.813 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 357.135 187.305 Td /F1 9.8 Tf [(, especially since others failed to confirm )] TJ ET BT 534.867 187.305 Td /F5 9.8 Tf [(in vivo, )] TJ ET BT 567.920 187.305 Td /F1 9.8 Tf [(in )] TJ ET BT 26.250 175.401 Td /F1 9.8 Tf [(mouse models, SIRT2 inhibition as a therapeutic strategy in HD )] TJ ET 0.267 0.267 0.267 rg BT 303.150 176.908 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 312.787 175.401 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 155.996 Td /F1 9.8 Tf [(To conclude, the hydrophobicity of different lipids, their susceptibility to oxidation in different experimental conditions, the )] TJ ET BT 26.250 144.091 Td /F1 9.8 Tf [(specific extraction procedures used to obtain the desired species and the technological limitations of the methods used for )] TJ ET BT 26.250 132.186 Td /F1 9.8 Tf [(measuring lipid levels, all suggest that the HD scientific community should use caution in interpreting results. In addition, it may )] TJ ET BT 26.250 120.282 Td /F1 9.8 Tf [(be helpful to consult with lipid experts. Our findings make it clear that the most sensitive and reliable analytical techniques, )] TJ ET BT 26.250 108.377 Td /F1 9.8 Tf [(along with appropriate sample preparation, should be used for unambiguous and unbiased measurement and interpretation of )] TJ ET BT 26.250 96.472 Td /F1 9.8 Tf [(cholesterol dysfunction in the context of HD.)] TJ ET BT 26.250 59.870 Td /F4 12.0 Tf [(Methods)] TJ ET BT 26.250 39.915 Td /F4 9.8 Tf [(Reagents.)] TJ ET BT 72.845 39.915 Td /F1 9.8 Tf [( Filipin, Nile Red, cholesterol, M?CD and simvastatin were purchased from Sigma Chemicals Co. \(St. Louis, MO, )] TJ ET Q q 15.000 25.630 577.500 751.370 re W n 0.965 0.965 0.965 rg 26.250 616.719 555.000 160.281 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 616.719 m 581.250 616.719 l 581.250 617.469 l 26.250 617.469 l f q 35.250 627.969 537.000 149.031 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 4: Levels of cholesterol precursors in over-confluent ST Kin7/7Q and ST Kin109/109Q cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(The same mass spectrometry analysis of cholesterol and cholesterol precursors reported in Figure 2 was also performed in )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(cells harvested at over-confluence. a\) Cholesterol levels are similar in ST Kin)] TJ ET BT 366.857 740.090 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 404.921 734.370 Td /F1 9.8 Tf [( clone #2 and ST Kin)] TJ ET BT 495.431 740.090 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 514.220 734.370 Td /F1 9.8 Tf [( but are )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(increased in ST Kin)] TJ ET BT 119.782 726.353 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 157.847 720.634 Td /F1 9.8 Tf [( clone #6 compared to ST Kin)] TJ ET BT 286.283 726.353 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 305.073 720.634 Td /F1 9.8 Tf [(. b\) Lanosterol was not significantly different in the mHTT )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(clones and wild-type cells, while lathosterol \(c\) was higher in ST Kin)] TJ ET BT 327.301 712.617 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 365.366 706.897 Td /F1 9.8 Tf [( clone #6 compared to wild-type and ST Kin)] TJ ET BT 35.250 698.881 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 73.314 693.161 Td /F1 9.8 Tf [( clone #2. d-e\) Similar changes are observed in the downstream cholesterol precursors desmosterol and 7dhc. )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(Although it does not reach the significance, desmosterol and 7dhc are increased in ST Kin)] TJ ET BT 423.817 685.145 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 461.881 679.425 Td /F1 9.8 Tf [( clone #6 compared to )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(ST Kin)] TJ ET BT 64.510 671.408 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 83.299 665.689 Td /F1 9.8 Tf [(. There are no significant differences between ST Kin)] TJ ET BT 312.531 671.408 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 350.595 665.689 Td /F1 9.8 Tf [( clone #6 and ST Kin)] TJ ET BT 441.105 671.408 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 459.894 665.689 Td /F1 9.8 Tf [( cells. The results are )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(shown as the mean SEM from analyses in two independent experiments. Statistics: One-way ANOVA and the Newman-)] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(Keuls Multiple Comparison Test were performed.)] TJ ET Q BT 26.250 599.695 Td /F1 9.8 Tf [(Taken together, these results illustrate the complexity of detecting cholesterol and its precursors )] TJ ET BT 442.429 599.695 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 471.142 599.695 Td /F1 9.8 Tf [( and show that clonal )] TJ ET BT 26.250 587.790 Td /F1 9.8 Tf [(variability and differences in cell density can lead to considerably different results.)] TJ ET BT 26.250 551.188 Td /F4 12.0 Tf [(Conclusions)] TJ ET BT 26.250 531.234 Td /F1 9.8 Tf [(Cholesterol is a structural component of biological membranes and is the precursor of numerous signalling molecules. In the )] TJ ET BT 26.250 519.329 Td /F1 9.8 Tf [(brain, cholesterol is involved in neurite outgrowth, myelination and synaptic activity, and its importance in brain physiology and )] TJ ET BT 26.250 507.424 Td /F1 9.8 Tf [(pathology is clear from neurological diseases in which there are changes in its level or distribution )] TJ ET 0.267 0.267 0.267 rg BT 449.459 508.931 Td /F4 8.7 Tf [(3)] TJ ET BT 454.277 508.931 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 459.096 507.424 Td /F1 9.8 Tf [(. Changes in sterol levels in )] TJ ET BT 26.250 495.519 Td /F1 9.8 Tf [(cellular and animal models of HD have been detected in previous studies, but the direction of these changes remains a matter )] TJ ET BT 26.250 483.615 Td /F1 9.8 Tf [(of debate. Specifically, while some studies report a decrease in cholesterol levels in HD models )] TJ ET 0.267 0.267 0.267 rg BT 439.699 485.122 Td /F4 8.7 Tf [(6)] TJ ET BT 444.517 485.122 Td /F4 8.7 Tf [(7)] TJ ET BT 449.336 485.122 Td /F4 8.7 Tf [(8)] TJ ET BT 454.155 485.122 Td /F4 8.7 Tf [(11)] TJ ET BT 463.792 485.122 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 473.429 483.615 Td /F1 9.8 Tf [(, others report the )] TJ ET BT 26.250 471.710 Td /F1 9.8 Tf [(opposite )] TJ ET 0.267 0.267 0.267 rg BT 65.816 473.217 Td /F4 8.7 Tf [(12)] TJ ET BT 75.453 473.217 Td /F4 8.7 Tf [(13)] TJ ET BT 85.090 473.217 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 94.728 471.710 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 452.305 Td /F1 9.8 Tf [(In this study, we investigated factors that could underlie these conflicting findings.)] TJ ET BT 26.250 432.900 Td /F1 9.8 Tf [(As we expected, the methods used to detect cholesterol, sample preparation methods, cell line clonal properties and cell growth )] TJ ET BT 26.250 420.996 Td /F1 9.8 Tf [(conditions all influenced the levels of cholesterol that were detected. It is critical to be aware of the limitations and artefacts that )] TJ ET BT 26.250 409.091 Td /F1 9.8 Tf [(arise from using less sensitive methods or from inappropriate sample preparation. Colorimetric methods such as filipin staining )] TJ ET BT 26.250 397.186 Td /F1 9.8 Tf [(should be avoided when the aim is quantitative detection of cholesterol levels in cells or tissue samples. Cholesterol levels may )] TJ ET BT 26.250 385.281 Td /F1 9.8 Tf [(be different due to the existence of variants of what is believed to be the same cell line. In this study, we found that two clones )] TJ ET BT 26.250 373.377 Td /F1 9.8 Tf [(of ST Kin cells, which were derived years ago in our laboratory at the University of Milan )] TJ ET 0.267 0.267 0.267 rg BT 408.265 374.884 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 417.902 373.377 Td /F1 9.8 Tf [(, were not, in fact, identical. The )] TJ ET BT 26.250 361.472 Td /F1 9.8 Tf [(clones have been distributed by us and by collaborating laboratories to many other laboratories, as well as by direct transfer )] TJ ET BT 26.250 349.567 Td /F1 9.8 Tf [(from a recipient lab to yet another lab. Different laboratory practices and mislabelling may have resulted in the propagation of )] TJ ET BT 26.250 337.662 Td /F1 9.8 Tf [(cells with properties that are different from expected or that differ from lab to lab. Contrary to reports from other groups )] TJ ET BT 538.340 337.662 Td /F1 9.8 Tf [(14, )] TJ ET BT 554.603 337.662 Td /F1 9.8 Tf [(19)] TJ ET BT 565.445 337.662 Td /F1 9.8 Tf [(, )] TJ ET BT 26.250 325.758 Td /F1 9.8 Tf [(we found no NMDAr or AMPAr activity and no expression of the NMDA subunit NR1 mRNA in ST-Kin cells. Genetic-based )] TJ ET BT 26.250 313.853 Td /F1 9.8 Tf [(assays for the identification of cell lines )] TJ ET 0.267 0.267 0.267 rg BT 197.489 315.360 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 207.127 313.853 Td /F1 9.8 Tf [( and the presence of a dedicated biorepository that controls the quality and )] TJ ET BT 26.250 301.948 Td /F1 9.8 Tf [(distribution )] TJ ET 0.267 0.267 0.267 rg BT 76.102 303.455 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 85.739 301.948 Td /F1 9.8 Tf [( of cell lines would reduce the risk of laboratory errors; such issues have already caused major damage in other )] TJ ET BT 26.250 290.043 Td /F1 9.8 Tf [(fields )] TJ ET 0.267 0.267 0.267 rg BT 51.717 291.551 Td /F4 8.7 Tf [(22)] TJ ET BT 61.354 291.551 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 70.992 290.043 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 270.639 Td /F1 9.8 Tf [(When a novel disease target emerges, prior to embarking in drug development initiatives, it is essential to validate the target in )] TJ ET BT 26.250 258.734 Td /F1 9.8 Tf [(mammalian models of the disease. In the case of targets associated with changes in cholesterol synthesis, a reliable, sensitive )] TJ ET BT 26.250 246.829 Td /F1 9.8 Tf [(detection method is key for ensuring that the results can be interpreted with confidence. When considering cholesterol )] TJ ET BT 26.250 234.924 Td /F1 9.8 Tf [(homeostasis, interpretation may be even more cumbersome given the multiplicity of cellular regulatory steps and compensatory )] TJ ET BT 26.250 223.020 Td /F1 9.8 Tf [(mechanisms at play; indeed, such mechanisms can present a challenge in experimental conditions. Small variations in cell )] TJ ET BT 26.250 211.115 Td /F1 9.8 Tf [(culture conditions, or other confounding factors introduced in the experimental setting, may affect the results of cholesterol level )] TJ ET BT 26.250 199.210 Td /F1 9.8 Tf [(determination. One study found a link between SIRT2 and cholesterol in worm, fly and cell culture HD models )] TJ ET 0.267 0.267 0.267 rg BT 500.402 200.717 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 510.040 199.210 Td /F1 9.8 Tf [(. This link )] TJ ET BT 26.250 187.305 Td /F1 9.8 Tf [(should be validated using a more sensitive method to detect cholesterol )] TJ ET 0.267 0.267 0.267 rg BT 337.860 188.813 Td /F4 8.7 Tf [(24)] TJ ET BT 347.497 188.813 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 357.135 187.305 Td /F1 9.8 Tf [(, especially since others failed to confirm )] TJ ET BT 534.867 187.305 Td /F5 9.8 Tf [(in vivo, )] TJ ET BT 567.920 187.305 Td /F1 9.8 Tf [(in )] TJ ET BT 26.250 175.401 Td /F1 9.8 Tf [(mouse models, SIRT2 inhibition as a therapeutic strategy in HD )] TJ ET 0.267 0.267 0.267 rg BT 303.150 176.908 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 312.787 175.401 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 155.996 Td /F1 9.8 Tf [(To conclude, the hydrophobicity of different lipids, their susceptibility to oxidation in different experimental conditions, the )] TJ ET BT 26.250 144.091 Td /F1 9.8 Tf [(specific extraction procedures used to obtain the desired species and the technological limitations of the methods used for )] TJ ET BT 26.250 132.186 Td /F1 9.8 Tf [(measuring lipid levels, all suggest that the HD scientific community should use caution in interpreting results. In addition, it may )] TJ ET BT 26.250 120.282 Td /F1 9.8 Tf [(be helpful to consult with lipid experts. Our findings make it clear that the most sensitive and reliable analytical techniques, )] TJ ET BT 26.250 108.377 Td /F1 9.8 Tf [(along with appropriate sample preparation, should be used for unambiguous and unbiased measurement and interpretation of )] TJ ET BT 26.250 96.472 Td /F1 9.8 Tf [(cholesterol dysfunction in the context of HD.)] TJ ET BT 26.250 59.870 Td /F4 12.0 Tf [(Methods)] TJ ET BT 26.250 39.915 Td /F4 9.8 Tf [(Reagents.)] TJ ET BT 72.845 39.915 Td /F1 9.8 Tf [( Filipin, Nile Red, cholesterol, M?CD and simvastatin were purchased from Sigma Chemicals Co. \(St. Louis, MO, )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(8)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 393 0 obj << /Type /Annot /Subtype /Link /A 394 0 R /Border [0 0 0] /H /I /Rect [ 449.4585 508.1297 454.2772 516.9480 ] >> endobj 394 0 obj << /Type /Action >> endobj 395 0 obj << /Type /Annot /Subtype /Link /A 396 0 R /Border [0 0 0] 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Filipin is unstable in solution, so a 50 mg/ml stock was prepared in DMSO and stored in aliquots at -80C to avoid )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(freezing/thawing. The aliquots were always protected from exposure to light. Simvastatin was prepared as an 11.94 mM stock )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(solution in DMSO. M?CD was added to the medium to achieve the final indicated concentration.)] TJ ET BT 26.250 724.262 Td /F4 9.8 Tf [(Cell cultures.)] TJ ET BT 87.480 724.262 Td /F1 9.8 Tf [( ST Kin)] TJ ET BT 119.450 728.150 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 138.240 724.262 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 189.183 728.150 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 227.247 724.262 Td /F1 9.8 Tf [( cells, and inducible immortalized striatal cells expressing the first 548 amino )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(acids of muHTT with 128 CAG repeats after doxycycline administration, were grown at 33C in high glucose DMEM \(Euroclone\) )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(supplemented with 10% Fetal Bovine Serum \(FBS\), penicillin \(100 U/ml\) and streptomycin \(100 ?g /ml\) plus 2 mM L-glutamine )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(as described elsewhere )] TJ ET 0.267 0.267 0.267 rg BT 131.375 690.055 Td /F4 8.7 Tf [(5)] TJ ET BT 136.193 690.055 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 145.831 688.548 Td /F1 9.8 Tf [(. In the case of inducible HD cells, FBS was used that was tested to ensure that it was tetracycline-)] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(free.)] TJ ET BT 26.250 657.238 Td /F4 9.8 Tf [(Cholesterol and M?CD treatment.)] TJ ET BT 180.641 657.238 Td /F1 9.8 Tf [( Cells were counted and seeded in 6-well multiwell plates \(for cholesterol detection with the )] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(enzymatic method or by GC-MS\) or in 12-well multiwell plates \(for colorimetric methods\) and incubated with different )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(concentrations of cholesterol \(stock solution, 10 mg/ml in ethanol\) for 16 hours or with media containing 1, 5, 10 or 25 mM )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(M?CD for 1 hour.)] TJ ET BT 26.250 602.119 Td /F4 9.8 Tf [(Immunofluorescence staining and microscopy analysis.)] TJ ET BT 285.795 602.119 Td /F1 9.8 Tf [( For Nile Red and filipin staining, the cells were plated on coverslips )] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(and fixed in 4% paraformaldehyde for 15 min. For Nile Red staining, the cells were incubated with a solution containing 10 g/ml )] TJ ET BT 26.250 578.310 Td /F1 9.8 Tf [(Nile Red for 15 min. For filipin staining, the cells were washed with 50 mM NH)] TJ ET BT 361.640 576.245 Td /F1 8.7 Tf [(4)] TJ ET BT 366.459 578.310 Td /F1 9.8 Tf [(Cl in PBS \(2 x 5 min\) to quench PFA, washed )] TJ ET BT 26.250 566.405 Td /F1 9.8 Tf [(once with PBS and incubated with filipin solution \(100 g/ml in PBS\) for 30 min with mild agitation and protected from the light. )] TJ ET BT 26.250 554.500 Td /F1 9.8 Tf [(After a wash with PBS, the cells were incubated in H)] TJ ET BT 252.762 552.436 Td /F1 8.7 Tf [(2)] TJ ET BT 257.581 554.500 Td /F1 9.8 Tf [(O \(5 sec\) to remove PBS salts and mounted with Mowiol. The stained )] TJ ET BT 26.250 542.595 Td /F1 9.8 Tf [(cells were examined, and images were acquired with either a Leica DMI 6000B inverted microscope or a Leica 4000B upright )] TJ ET BT 26.250 530.691 Td /F1 9.8 Tf [(microscope with LAS-AF imaging software. The images were processed using Adobe Photoshop.)] TJ ET BT 26.250 511.286 Td /F4 9.8 Tf [(Measurement of total cholesterol by the enzymatic method.)] TJ ET BT 300.937 511.286 Td /F1 9.8 Tf [( The measurement of total sterol levels using the enzymatic )] TJ ET BT 26.250 499.381 Td /F1 9.8 Tf [(method were performed on a lipid fraction isolated with solvent extraction as described by Valenza et al., 2005 and on protein )] TJ ET BT 26.250 487.476 Td /F1 9.8 Tf [(lysate that was isolated as described previously by Del Toro et al., 2010. Briefly, for lipid extraction the cells in each well were )] TJ ET BT 26.250 475.572 Td /F1 9.8 Tf [(washed twice with 1 ml cold PBS and 1 ml of hexane/isopropanol \(3:2, v/v\) was added to the well. After rocking at room )] TJ ET BT 26.250 463.667 Td /F1 9.8 Tf [(temperature for 1 hour to extract the lipids, the liquid in each well was transferred to a glass tube, the solvents were removed )] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(using a stream of nitrogen at room temperature and the lipid pellets were solubilised. The protein lysate for sterol )] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(measurements was prepared with the same buffer as in Del Toro et al., 2010: ice-cold 10 mM Tris, pH 7.5, 150 mM NaCl, 5 mM )] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(EDTA, 1 mM PMSF, 2 mM orthovanadate, 10 ug/ml aprotinin and 1 ug/ml leupeptin. Total cholesterol was assayed in lipid )] TJ ET BT 26.250 416.048 Td /F1 9.8 Tf [(extracts and protein lysates using the Amplex Red Enzyme Assay \(Invitrogen\) according to the manufacturers instructions )] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(and normalized by protein concentration. The latter was determined using a BCA protein assay kit \(Pierce, Rockford, IL, USA\).)] TJ ET BT 26.250 384.738 Td /F4 9.8 Tf [(GC-MS and isotopic-dilution mass spectrometry.)] TJ ET BT 252.167 384.738 Td /F1 9.8 Tf [( Cell homogenates were prepared in PBS, and 80 ?l of homogenate was )] TJ ET BT 26.250 372.834 Td /F1 9.8 Tf [(added to a screw-capped vial sealed with a Teflon-lined septum along with 5 g of 2H6-cholesterol and 100 ng of 2H4-)] TJ ET BT 26.250 360.929 Td /F1 9.8 Tf [(lathosterol \(CDN Isotopes\) as internal standards. To prevent auto-oxidation, 25 ?l of butylated hydroxytoluene \(BHT\) \(5 g/l\) and )] TJ ET BT 26.250 349.024 Td /F1 9.8 Tf [(25 ?l of EDTA \(10 g/l\) were added to each vial, and vials were flushed with argon to remove air. Alkaline hydrolysis was allowed )] TJ ET BT 26.250 337.119 Td /F1 9.8 Tf [(to proceed at room temperature \(22C\) for 1 h in the presence of 1 M ethanolic KOH solution with magnetic stirring. After )] TJ ET BT 26.250 325.215 Td /F1 9.8 Tf [(hydrolysis sterols \(cholesterol, lathosterol, lanosterol, desmosterol and 7-dehydrocholesterol\) were extracted two times with 5 ml )] TJ ET BT 26.250 313.310 Td /F1 9.8 Tf [(of cyclohexane plus 5 mL of ethyl-acetate. The organic solvents were evaporated under a gentle stream of argon and converted )] TJ ET BT 26.250 301.405 Td /F1 9.8 Tf [(into trimethylsilyl ethers [BSTFA + TMCS 1%]. GC-MS was performed on a Perkin Elmer Clarus 600C gas chromatographer- )] TJ ET BT 26.250 289.500 Td /F1 9.8 Tf [(mass selective detector. The GC was equipped with a DB-XLB \(30 m 0.25 mm i.d. 0.25 ?m film; J & W Scientific, Palo Alto, )] TJ ET BT 26.250 277.596 Td /F1 9.8 Tf [(CA, USA\), and the injection was performed in the splitless injection mode using helium \(1 ml/min\) as a carrier gas. The initial )] TJ ET BT 26.250 265.691 Td /F1 9.8 Tf [(temperature of 180C was maintained for 1 min, increased by 20C/min up to 270C, then increased by 5C/min to the final )] TJ ET BT 26.250 253.786 Td /F1 9.8 Tf [(temperature of 290C, which was maintained for 10 min. The mass spectrometer was used in selected ion-monitoring mode, )] TJ ET BT 26.250 241.881 Td /F1 9.8 Tf [(and the neutral sterols were monitored as their TMSi derivatives using the following masses: 2H6-cholesterol at m/z 464 \(M+-)] TJ ET BT 26.250 229.977 Td /F1 9.8 Tf [(OTMSi\); cholesterol at m/z 458 \(M+-OTMSi\); 2H4-lathosterol at 462 m/z \(M+-OTMSi\), lathosterol at 458 m/z \(M+-OTMSi\), )] TJ ET BT 26.250 218.072 Td /F1 9.8 Tf [(desmosterol at m/z 343 \(M+-OTMSi\); 7-dehydrocholesterol at m/z 325 \(M+-OTMSi\) and lanosterol at 393 and 498 m/z \(M+-)] TJ ET BT 26.250 206.167 Td /F1 9.8 Tf [(OTMSi\). Peak integration was performed manually, and the sterols were quantified from the selected-ion monitoring analyses )] TJ ET BT 26.250 194.262 Td /F1 9.8 Tf [(by comparison with internal standards using standard curves. The identity of all of the sterols was verified by comparison with )] TJ ET BT 26.250 182.358 Td /F1 9.8 Tf [(the full-scan mass spectra of authentic compounds. Additional qualifier ions \(characteristic fragment ions\) were used for )] TJ ET BT 26.250 170.453 Td /F1 9.8 Tf [(structural identification.)] TJ ET BT 26.250 151.048 Td /F4 9.8 Tf [(Ca2+ measurements with recombinant aequorin.)] TJ ET BT 251.377 151.048 Td /F1 9.8 Tf [( Before transfection, cells were seeded onto 13-mm cover glass slips for )] TJ ET BT 26.250 139.143 Td /F1 9.8 Tf [(the aequorin \(AEQ\) measurements and allowed to grow to 7080% confluence. The cells were transformed using the Ca2+-)] TJ ET BT 26.250 127.239 Td /F1 9.8 Tf [(phosphate technique in the presence of 1.5 ?g AEQ cDNA. The Ca2+ measurements were carried out 48 hours after )] TJ ET BT 26.250 115.334 Td /F1 9.8 Tf [(transfection. To reconstitute AEQ, the coverslips with the transfected ST Kin)] TJ ET BT 354.103 119.222 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 372.893 115.334 Td /F1 9.8 Tf [( cells were incubated for 65 minutes with 5 mM )] TJ ET BT 26.250 103.429 Td /F1 9.8 Tf [(coelenterazine in Krebs-Ringer buffer \(KRB\) medium with 1 mM CaCl2 and then transferred to the perfusion chamber of a low-)] TJ ET BT 26.250 91.524 Td /F1 9.8 Tf [(noise photomultiplier with a built-in amplifier-discriminator \(Thorn-EMI photon counting board\). All the AEQ measurements were )] TJ ET BT 26.250 79.620 Td /F1 9.8 Tf [(carried out at 37C in modified KRB \(135 mM NaCl, 5 mM KCl, 0.4 mM KH)] TJ ET BT 348.195 77.555 Td /F1 8.7 Tf [(2)] TJ ET BT 353.014 79.620 Td /F1 9.8 Tf [(PO)] TJ ET BT 367.102 77.555 Td /F1 8.7 Tf [(4)] TJ ET BT 371.921 79.620 Td /F1 9.8 Tf [(, 20 mM HEPES, 0.1% glucose; pH 7.4\). Each )] TJ ET BT 26.250 67.715 Td /F1 9.8 Tf [(experiment began when the cells were perfused with 1 mM CaCl2. After about 30 s, cells were perfused with Mg)] TJ ET BT 510.152 71.603 Td /F1 8.7 Tf [(2+)] TJ ET BT 520.032 67.715 Td /F1 9.8 Tf [(-free KRB )] TJ ET BT 26.250 55.810 Td /F1 9.8 Tf [(containing CaCl)] TJ ET BT 95.066 53.746 Td /F1 8.7 Tf [(2)] TJ ET BT 99.884 55.810 Td /F1 9.8 Tf [( \(1 mM\), glutamate \(100 ?M\) and glycine \(100 ?M\) or NMDA 100 ?M \(Invitrogen\). After 120 s, ATP \(100 ?M\) )] TJ ET BT 26.250 43.905 Td /F1 9.8 Tf [(was added to elicit transient Ca)] TJ ET BT 161.726 47.794 Td /F1 8.7 Tf [(2+)] TJ ET BT 171.606 43.905 Td /F1 9.8 Tf [( efflux. At the end of each experiment, cells were lysed by adding 100 ?M digitonin in a )] TJ ET Q q 15.000 29.620 577.500 747.380 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(USA\). Filipin is unstable in solution, so a 50 mg/ml stock was prepared in DMSO and stored in aliquots at -80C to avoid )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(freezing/thawing. The aliquots were always protected from exposure to light. Simvastatin was prepared as an 11.94 mM stock )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(solution in DMSO. M?CD was added to the medium to achieve the final indicated concentration.)] TJ ET BT 26.250 724.262 Td /F4 9.8 Tf [(Cell cultures.)] TJ ET BT 87.480 724.262 Td /F1 9.8 Tf [( ST Kin)] TJ ET BT 119.450 728.150 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 138.240 724.262 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 189.183 728.150 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 227.247 724.262 Td /F1 9.8 Tf [( cells, and inducible immortalized striatal cells expressing the first 548 amino )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(acids of muHTT with 128 CAG repeats after doxycycline administration, were grown at 33C in high glucose DMEM \(Euroclone\) )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(supplemented with 10% Fetal Bovine Serum \(FBS\), penicillin \(100 U/ml\) and streptomycin \(100 ?g /ml\) plus 2 mM L-glutamine )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(as described elsewhere )] TJ ET 0.267 0.267 0.267 rg BT 131.375 690.055 Td /F4 8.7 Tf [(5)] TJ ET BT 136.193 690.055 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 145.831 688.548 Td /F1 9.8 Tf [(. In the case of inducible HD cells, FBS was used that was tested to ensure that it was tetracycline-)] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(free.)] TJ ET BT 26.250 657.238 Td /F4 9.8 Tf [(Cholesterol and M?CD treatment.)] TJ ET BT 180.641 657.238 Td /F1 9.8 Tf [( Cells were counted and seeded in 6-well multiwell plates \(for cholesterol detection with the )] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(enzymatic method or by GC-MS\) or in 12-well multiwell plates \(for colorimetric methods\) and incubated with different )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(concentrations of cholesterol \(stock solution, 10 mg/ml in ethanol\) for 16 hours or with media containing 1, 5, 10 or 25 mM )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(M?CD for 1 hour.)] TJ ET BT 26.250 602.119 Td /F4 9.8 Tf [(Immunofluorescence staining and microscopy analysis.)] TJ ET BT 285.795 602.119 Td /F1 9.8 Tf [( For Nile Red and filipin staining, the cells were plated on coverslips )] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(and fixed in 4% paraformaldehyde for 15 min. For Nile Red staining, the cells were incubated with a solution containing 10 g/ml )] TJ ET BT 26.250 578.310 Td /F1 9.8 Tf [(Nile Red for 15 min. For filipin staining, the cells were washed with 50 mM NH)] TJ ET BT 361.640 576.245 Td /F1 8.7 Tf [(4)] TJ ET BT 366.459 578.310 Td /F1 9.8 Tf [(Cl in PBS \(2 x 5 min\) to quench PFA, washed )] TJ ET BT 26.250 566.405 Td /F1 9.8 Tf [(once with PBS and incubated with filipin solution \(100 g/ml in PBS\) for 30 min with mild agitation and protected from the light. )] TJ ET BT 26.250 554.500 Td /F1 9.8 Tf [(After a wash with PBS, the cells were incubated in H)] TJ ET BT 252.762 552.436 Td /F1 8.7 Tf [(2)] TJ ET BT 257.581 554.500 Td /F1 9.8 Tf [(O \(5 sec\) to remove PBS salts and mounted with Mowiol. The stained )] TJ ET BT 26.250 542.595 Td /F1 9.8 Tf [(cells were examined, and images were acquired with either a Leica DMI 6000B inverted microscope or a Leica 4000B upright )] TJ ET BT 26.250 530.691 Td /F1 9.8 Tf [(microscope with LAS-AF imaging software. The images were processed using Adobe Photoshop.)] TJ ET BT 26.250 511.286 Td /F4 9.8 Tf [(Measurement of total cholesterol by the enzymatic method.)] TJ ET BT 300.937 511.286 Td /F1 9.8 Tf [( The measurement of total sterol levels using the enzymatic )] TJ ET BT 26.250 499.381 Td /F1 9.8 Tf [(method were performed on a lipid fraction isolated with solvent extraction as described by Valenza et al., 2005 and on protein )] TJ ET BT 26.250 487.476 Td /F1 9.8 Tf [(lysate that was isolated as described previously by Del Toro et al., 2010. Briefly, for lipid extraction the cells in each well were )] TJ ET BT 26.250 475.572 Td /F1 9.8 Tf [(washed twice with 1 ml cold PBS and 1 ml of hexane/isopropanol \(3:2, v/v\) was added to the well. After rocking at room )] TJ ET BT 26.250 463.667 Td /F1 9.8 Tf [(temperature for 1 hour to extract the lipids, the liquid in each well was transferred to a glass tube, the solvents were removed )] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(using a stream of nitrogen at room temperature and the lipid pellets were solubilised. The protein lysate for sterol )] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(measurements was prepared with the same buffer as in Del Toro et al., 2010: ice-cold 10 mM Tris, pH 7.5, 150 mM NaCl, 5 mM )] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(EDTA, 1 mM PMSF, 2 mM orthovanadate, 10 ug/ml aprotinin and 1 ug/ml leupeptin. Total cholesterol was assayed in lipid )] TJ ET BT 26.250 416.048 Td /F1 9.8 Tf [(extracts and protein lysates using the Amplex Red Enzyme Assay \(Invitrogen\) according to the manufacturers instructions )] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(and normalized by protein concentration. The latter was determined using a BCA protein assay kit \(Pierce, Rockford, IL, USA\).)] TJ ET BT 26.250 384.738 Td /F4 9.8 Tf [(GC-MS and isotopic-dilution mass spectrometry.)] TJ ET BT 252.167 384.738 Td /F1 9.8 Tf [( Cell homogenates were prepared in PBS, and 80 ?l of homogenate was )] TJ ET BT 26.250 372.834 Td /F1 9.8 Tf [(added to a screw-capped vial sealed with a Teflon-lined septum along with 5 g of 2H6-cholesterol and 100 ng of 2H4-)] TJ ET BT 26.250 360.929 Td /F1 9.8 Tf [(lathosterol \(CDN Isotopes\) as internal standards. To prevent auto-oxidation, 25 ?l of butylated hydroxytoluene \(BHT\) \(5 g/l\) and )] TJ ET BT 26.250 349.024 Td /F1 9.8 Tf [(25 ?l of EDTA \(10 g/l\) were added to each vial, and vials were flushed with argon to remove air. Alkaline hydrolysis was allowed )] TJ ET BT 26.250 337.119 Td /F1 9.8 Tf [(to proceed at room temperature \(22C\) for 1 h in the presence of 1 M ethanolic KOH solution with magnetic stirring. After )] TJ ET BT 26.250 325.215 Td /F1 9.8 Tf [(hydrolysis sterols \(cholesterol, lathosterol, lanosterol, desmosterol and 7-dehydrocholesterol\) were extracted two times with 5 ml )] TJ ET BT 26.250 313.310 Td /F1 9.8 Tf [(of cyclohexane plus 5 mL of ethyl-acetate. The organic solvents were evaporated under a gentle stream of argon and converted )] TJ ET BT 26.250 301.405 Td /F1 9.8 Tf [(into trimethylsilyl ethers [BSTFA + TMCS 1%]. GC-MS was performed on a Perkin Elmer Clarus 600C gas chromatographer- )] TJ ET BT 26.250 289.500 Td /F1 9.8 Tf [(mass selective detector. The GC was equipped with a DB-XLB \(30 m 0.25 mm i.d. 0.25 ?m film; J & W Scientific, Palo Alto, )] TJ ET BT 26.250 277.596 Td /F1 9.8 Tf [(CA, USA\), and the injection was performed in the splitless injection mode using helium \(1 ml/min\) as a carrier gas. The initial )] TJ ET BT 26.250 265.691 Td /F1 9.8 Tf [(temperature of 180C was maintained for 1 min, increased by 20C/min up to 270C, then increased by 5C/min to the final )] TJ ET BT 26.250 253.786 Td /F1 9.8 Tf [(temperature of 290C, which was maintained for 10 min. The mass spectrometer was used in selected ion-monitoring mode, )] TJ ET BT 26.250 241.881 Td /F1 9.8 Tf [(and the neutral sterols were monitored as their TMSi derivatives using the following masses: 2H6-cholesterol at m/z 464 \(M+-)] TJ ET BT 26.250 229.977 Td /F1 9.8 Tf [(OTMSi\); cholesterol at m/z 458 \(M+-OTMSi\); 2H4-lathosterol at 462 m/z \(M+-OTMSi\), lathosterol at 458 m/z \(M+-OTMSi\), )] TJ ET BT 26.250 218.072 Td /F1 9.8 Tf [(desmosterol at m/z 343 \(M+-OTMSi\); 7-dehydrocholesterol at m/z 325 \(M+-OTMSi\) and lanosterol at 393 and 498 m/z \(M+-)] TJ ET BT 26.250 206.167 Td /F1 9.8 Tf [(OTMSi\). Peak integration was performed manually, and the sterols were quantified from the selected-ion monitoring analyses )] TJ ET BT 26.250 194.262 Td /F1 9.8 Tf [(by comparison with internal standards using standard curves. The identity of all of the sterols was verified by comparison with )] TJ ET BT 26.250 182.358 Td /F1 9.8 Tf [(the full-scan mass spectra of authentic compounds. Additional qualifier ions \(characteristic fragment ions\) were used for )] TJ ET BT 26.250 170.453 Td /F1 9.8 Tf [(structural identification.)] TJ ET BT 26.250 151.048 Td /F4 9.8 Tf [(Ca2+ measurements with recombinant aequorin.)] TJ ET BT 251.377 151.048 Td /F1 9.8 Tf [( Before transfection, cells were seeded onto 13-mm cover glass slips for )] TJ ET BT 26.250 139.143 Td /F1 9.8 Tf [(the aequorin \(AEQ\) measurements and allowed to grow to 7080% confluence. The cells were transformed using the Ca2+-)] TJ ET BT 26.250 127.239 Td /F1 9.8 Tf [(phosphate technique in the presence of 1.5 ?g AEQ cDNA. The Ca2+ measurements were carried out 48 hours after )] TJ ET BT 26.250 115.334 Td /F1 9.8 Tf [(transfection. To reconstitute AEQ, the coverslips with the transfected ST Kin)] TJ ET BT 354.103 119.222 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 372.893 115.334 Td /F1 9.8 Tf [( cells were incubated for 65 minutes with 5 mM )] TJ ET BT 26.250 103.429 Td /F1 9.8 Tf [(coelenterazine in Krebs-Ringer buffer \(KRB\) medium with 1 mM CaCl2 and then transferred to the perfusion chamber of a low-)] TJ ET BT 26.250 91.524 Td /F1 9.8 Tf [(noise photomultiplier with a built-in amplifier-discriminator \(Thorn-EMI photon counting board\). All the AEQ measurements were )] TJ ET BT 26.250 79.620 Td /F1 9.8 Tf [(carried out at 37C in modified KRB \(135 mM NaCl, 5 mM KCl, 0.4 mM KH)] TJ ET BT 348.195 77.555 Td /F1 8.7 Tf [(2)] TJ ET BT 353.014 79.620 Td /F1 9.8 Tf [(PO)] TJ ET BT 367.102 77.555 Td /F1 8.7 Tf [(4)] TJ ET BT 371.921 79.620 Td /F1 9.8 Tf [(, 20 mM HEPES, 0.1% glucose; pH 7.4\). Each )] TJ ET BT 26.250 67.715 Td /F1 9.8 Tf [(experiment began when the cells were perfused with 1 mM CaCl2. After about 30 s, cells were perfused with Mg)] TJ ET BT 510.152 71.603 Td /F1 8.7 Tf [(2+)] TJ ET BT 520.032 67.715 Td /F1 9.8 Tf [(-free KRB )] TJ ET BT 26.250 55.810 Td /F1 9.8 Tf [(containing CaCl)] TJ ET BT 95.066 53.746 Td /F1 8.7 Tf [(2)] TJ ET BT 99.884 55.810 Td /F1 9.8 Tf [( \(1 mM\), glutamate \(100 ?M\) and glycine \(100 ?M\) or NMDA 100 ?M \(Invitrogen\). After 120 s, ATP \(100 ?M\) )] TJ ET BT 26.250 43.905 Td /F1 9.8 Tf [(was added to elicit transient Ca)] TJ ET BT 161.726 47.794 Td /F1 8.7 Tf [(2+)] TJ ET BT 171.606 43.905 Td /F1 9.8 Tf [( efflux. At the end of each experiment, cells were lysed by adding 100 ?M digitonin in a )] TJ ET Q q 15.000 29.620 577.500 747.380 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(USA\). Filipin is unstable in solution, so a 50 mg/ml stock was prepared in DMSO and stored in aliquots at -80C to avoid )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(freezing/thawing. The aliquots were always protected from exposure to light. Simvastatin was prepared as an 11.94 mM stock )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(solution in DMSO. M?CD was added to the medium to achieve the final indicated concentration.)] TJ ET BT 26.250 724.262 Td /F4 9.8 Tf [(Cell cultures.)] TJ ET BT 87.480 724.262 Td /F1 9.8 Tf [( ST Kin)] TJ ET BT 119.450 728.150 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 138.240 724.262 Td /F1 9.8 Tf [( and ST Kin)] TJ ET BT 189.183 728.150 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 227.247 724.262 Td /F1 9.8 Tf [( cells, and inducible immortalized striatal cells expressing the first 548 amino )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(acids of muHTT with 128 CAG repeats after doxycycline administration, were grown at 33C in high glucose DMEM \(Euroclone\) )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(supplemented with 10% Fetal Bovine Serum \(FBS\), penicillin \(100 U/ml\) and streptomycin \(100 ?g /ml\) plus 2 mM L-glutamine )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(as described elsewhere )] TJ ET 0.267 0.267 0.267 rg BT 131.375 690.055 Td /F4 8.7 Tf [(5)] TJ ET BT 136.193 690.055 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 145.831 688.548 Td /F1 9.8 Tf [(. In the case of inducible HD cells, FBS was used that was tested to ensure that it was tetracycline-)] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(free.)] TJ ET BT 26.250 657.238 Td /F4 9.8 Tf [(Cholesterol and M?CD treatment.)] TJ ET BT 180.641 657.238 Td /F1 9.8 Tf [( Cells were counted and seeded in 6-well multiwell plates \(for cholesterol detection with the )] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(enzymatic method or by GC-MS\) or in 12-well multiwell plates \(for colorimetric methods\) and incubated with different )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(concentrations of cholesterol \(stock solution, 10 mg/ml in ethanol\) for 16 hours or with media containing 1, 5, 10 or 25 mM )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(M?CD for 1 hour.)] TJ ET BT 26.250 602.119 Td /F4 9.8 Tf [(Immunofluorescence staining and microscopy analysis.)] TJ ET BT 285.795 602.119 Td /F1 9.8 Tf [( For Nile Red and filipin staining, the cells were plated on coverslips )] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(and fixed in 4% paraformaldehyde for 15 min. For Nile Red staining, the cells were incubated with a solution containing 10 g/ml )] TJ ET BT 26.250 578.310 Td /F1 9.8 Tf [(Nile Red for 15 min. For filipin staining, the cells were washed with 50 mM NH)] TJ ET BT 361.640 576.245 Td /F1 8.7 Tf [(4)] TJ ET BT 366.459 578.310 Td /F1 9.8 Tf [(Cl in PBS \(2 x 5 min\) to quench PFA, washed )] TJ ET BT 26.250 566.405 Td /F1 9.8 Tf [(once with PBS and incubated with filipin solution \(100 g/ml in PBS\) for 30 min with mild agitation and protected from the light. )] TJ ET BT 26.250 554.500 Td /F1 9.8 Tf [(After a wash with PBS, the cells were incubated in H)] TJ ET BT 252.762 552.436 Td /F1 8.7 Tf [(2)] TJ ET BT 257.581 554.500 Td /F1 9.8 Tf [(O \(5 sec\) to remove PBS salts and mounted with Mowiol. The stained )] TJ ET BT 26.250 542.595 Td /F1 9.8 Tf [(cells were examined, and images were acquired with either a Leica DMI 6000B inverted microscope or a Leica 4000B upright )] TJ ET BT 26.250 530.691 Td /F1 9.8 Tf [(microscope with LAS-AF imaging software. The images were processed using Adobe Photoshop.)] TJ ET BT 26.250 511.286 Td /F4 9.8 Tf [(Measurement of total cholesterol by the enzymatic method.)] TJ ET BT 300.937 511.286 Td /F1 9.8 Tf [( The measurement of total sterol levels using the enzymatic )] TJ ET BT 26.250 499.381 Td /F1 9.8 Tf [(method were performed on a lipid fraction isolated with solvent extraction as described by Valenza et al., 2005 and on protein )] TJ ET BT 26.250 487.476 Td /F1 9.8 Tf [(lysate that was isolated as described previously by Del Toro et al., 2010. Briefly, for lipid extraction the cells in each well were )] TJ ET BT 26.250 475.572 Td /F1 9.8 Tf [(washed twice with 1 ml cold PBS and 1 ml of hexane/isopropanol \(3:2, v/v\) was added to the well. After rocking at room )] TJ ET BT 26.250 463.667 Td /F1 9.8 Tf [(temperature for 1 hour to extract the lipids, the liquid in each well was transferred to a glass tube, the solvents were removed )] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(using a stream of nitrogen at room temperature and the lipid pellets were solubilised. The protein lysate for sterol )] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(measurements was prepared with the same buffer as in Del Toro et al., 2010: ice-cold 10 mM Tris, pH 7.5, 150 mM NaCl, 5 mM )] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(EDTA, 1 mM PMSF, 2 mM orthovanadate, 10 ug/ml aprotinin and 1 ug/ml leupeptin. Total cholesterol was assayed in lipid )] TJ ET BT 26.250 416.048 Td /F1 9.8 Tf [(extracts and protein lysates using the Amplex Red Enzyme Assay \(Invitrogen\) according to the manufacturers instructions )] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(and normalized by protein concentration. The latter was determined using a BCA protein assay kit \(Pierce, Rockford, IL, USA\).)] TJ ET BT 26.250 384.738 Td /F4 9.8 Tf [(GC-MS and isotopic-dilution mass spectrometry.)] TJ ET BT 252.167 384.738 Td /F1 9.8 Tf [( Cell homogenates were prepared in PBS, and 80 ?l of homogenate was )] TJ ET BT 26.250 372.834 Td /F1 9.8 Tf [(added to a screw-capped vial sealed with a Teflon-lined septum along with 5 g of 2H6-cholesterol and 100 ng of 2H4-)] TJ ET BT 26.250 360.929 Td /F1 9.8 Tf [(lathosterol \(CDN Isotopes\) as internal standards. To prevent auto-oxidation, 25 ?l of butylated hydroxytoluene \(BHT\) \(5 g/l\) and )] TJ ET BT 26.250 349.024 Td /F1 9.8 Tf [(25 ?l of EDTA \(10 g/l\) were added to each vial, and vials were flushed with argon to remove air. Alkaline hydrolysis was allowed )] TJ ET BT 26.250 337.119 Td /F1 9.8 Tf [(to proceed at room temperature \(22C\) for 1 h in the presence of 1 M ethanolic KOH solution with magnetic stirring. After )] TJ ET BT 26.250 325.215 Td /F1 9.8 Tf [(hydrolysis sterols \(cholesterol, lathosterol, lanosterol, desmosterol and 7-dehydrocholesterol\) were extracted two times with 5 ml )] TJ ET BT 26.250 313.310 Td /F1 9.8 Tf [(of cyclohexane plus 5 mL of ethyl-acetate. The organic solvents were evaporated under a gentle stream of argon and converted )] TJ ET BT 26.250 301.405 Td /F1 9.8 Tf [(into trimethylsilyl ethers [BSTFA + TMCS 1%]. GC-MS was performed on a Perkin Elmer Clarus 600C gas chromatographer- )] TJ ET BT 26.250 289.500 Td /F1 9.8 Tf [(mass selective detector. The GC was equipped with a DB-XLB \(30 m 0.25 mm i.d. 0.25 ?m film; J & W Scientific, Palo Alto, )] TJ ET BT 26.250 277.596 Td /F1 9.8 Tf [(CA, USA\), and the injection was performed in the splitless injection mode using helium \(1 ml/min\) as a carrier gas. The initial )] TJ ET BT 26.250 265.691 Td /F1 9.8 Tf [(temperature of 180C was maintained for 1 min, increased by 20C/min up to 270C, then increased by 5C/min to the final )] TJ ET BT 26.250 253.786 Td /F1 9.8 Tf [(temperature of 290C, which was maintained for 10 min. The mass spectrometer was used in selected ion-monitoring mode, )] TJ ET BT 26.250 241.881 Td /F1 9.8 Tf [(and the neutral sterols were monitored as their TMSi derivatives using the following masses: 2H6-cholesterol at m/z 464 \(M+-)] TJ ET BT 26.250 229.977 Td /F1 9.8 Tf [(OTMSi\); cholesterol at m/z 458 \(M+-OTMSi\); 2H4-lathosterol at 462 m/z \(M+-OTMSi\), lathosterol at 458 m/z \(M+-OTMSi\), )] TJ ET BT 26.250 218.072 Td /F1 9.8 Tf [(desmosterol at m/z 343 \(M+-OTMSi\); 7-dehydrocholesterol at m/z 325 \(M+-OTMSi\) and lanosterol at 393 and 498 m/z \(M+-)] TJ ET BT 26.250 206.167 Td /F1 9.8 Tf [(OTMSi\). Peak integration was performed manually, and the sterols were quantified from the selected-ion monitoring analyses )] TJ ET BT 26.250 194.262 Td /F1 9.8 Tf [(by comparison with internal standards using standard curves. The identity of all of the sterols was verified by comparison with )] TJ ET BT 26.250 182.358 Td /F1 9.8 Tf [(the full-scan mass spectra of authentic compounds. Additional qualifier ions \(characteristic fragment ions\) were used for )] TJ ET BT 26.250 170.453 Td /F1 9.8 Tf [(structural identification.)] TJ ET BT 26.250 151.048 Td /F4 9.8 Tf [(Ca2+ measurements with recombinant aequorin.)] TJ ET BT 251.377 151.048 Td /F1 9.8 Tf [( Before transfection, cells were seeded onto 13-mm cover glass slips for )] TJ ET BT 26.250 139.143 Td /F1 9.8 Tf [(the aequorin \(AEQ\) measurements and allowed to grow to 7080% confluence. The cells were transformed using the Ca2+-)] TJ ET BT 26.250 127.239 Td /F1 9.8 Tf [(phosphate technique in the presence of 1.5 ?g AEQ cDNA. The Ca2+ measurements were carried out 48 hours after )] TJ ET BT 26.250 115.334 Td /F1 9.8 Tf [(transfection. To reconstitute AEQ, the coverslips with the transfected ST Kin)] TJ ET BT 354.103 119.222 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 372.893 115.334 Td /F1 9.8 Tf [( cells were incubated for 65 minutes with 5 mM )] TJ ET BT 26.250 103.429 Td /F1 9.8 Tf [(coelenterazine in Krebs-Ringer buffer \(KRB\) medium with 1 mM CaCl2 and then transferred to the perfusion chamber of a low-)] TJ ET BT 26.250 91.524 Td /F1 9.8 Tf [(noise photomultiplier with a built-in amplifier-discriminator \(Thorn-EMI photon counting board\). All the AEQ measurements were )] TJ ET BT 26.250 79.620 Td /F1 9.8 Tf [(carried out at 37C in modified KRB \(135 mM NaCl, 5 mM KCl, 0.4 mM KH)] TJ ET BT 348.195 77.555 Td /F1 8.7 Tf [(2)] TJ ET BT 353.014 79.620 Td /F1 9.8 Tf [(PO)] TJ ET BT 367.102 77.555 Td /F1 8.7 Tf [(4)] TJ ET BT 371.921 79.620 Td /F1 9.8 Tf [(, 20 mM HEPES, 0.1% glucose; pH 7.4\). Each )] TJ ET BT 26.250 67.715 Td /F1 9.8 Tf [(experiment began when the cells were perfused with 1 mM CaCl2. After about 30 s, cells were perfused with Mg)] TJ ET BT 510.152 71.603 Td /F1 8.7 Tf [(2+)] TJ ET BT 520.032 67.715 Td /F1 9.8 Tf [(-free KRB )] TJ ET BT 26.250 55.810 Td /F1 9.8 Tf [(containing CaCl)] TJ ET BT 95.066 53.746 Td /F1 8.7 Tf [(2)] TJ ET BT 99.884 55.810 Td /F1 9.8 Tf [( \(1 mM\), glutamate \(100 ?M\) and glycine \(100 ?M\) or NMDA 100 ?M \(Invitrogen\). After 120 s, ATP \(100 ?M\) )] TJ ET BT 26.250 43.905 Td /F1 9.8 Tf [(was added to elicit transient Ca)] TJ ET BT 161.726 47.794 Td /F1 8.7 Tf [(2+)] TJ ET BT 171.606 43.905 Td /F1 9.8 Tf [( efflux. At the end of each experiment, cells were lysed by adding 100 ?M digitonin in a )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(9)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 509 0 obj << /Type /Annot /Subtype /Link /A 510 0 R /Border [0 0 0] /H /I /Rect [ 131.3745 689.2532 136.1932 698.0715 ] >> endobj 510 0 obj << /Type /Action >> endobj 511 0 obj << /Type /Annot /Subtype /Link /A 512 0 R /Border [0 0 0] /H /I /Rect [ 136.1932 689.2532 145.8305 698.0715 ] >> endobj 512 0 obj << /Type /Action >> endobj 513 0 obj << /Type /Annot /Subtype /Link /A 514 0 R /Border [0 0 0] /H /I /Rect [ 131.3745 689.2532 136.1932 698.0715 ] >> endobj 514 0 obj << /Type /Action >> endobj 515 0 obj << /Type /Annot /Subtype /Link /A 516 0 R /Border [0 0 0] /H /I /Rect [ 136.1932 689.2532 145.8305 698.0715 ] >> endobj 516 0 obj << /Type /Action >> endobj 517 0 obj << /Type /Annot /Subtype /Link /A 518 0 R /Border [0 0 0] /H /I /Rect [ 131.3745 689.2532 136.1932 698.0715 ] >> endobj 518 0 obj << /Type /Action >> endobj 519 0 obj << /Type /Annot /Subtype /Link /A 520 0 R /Border [0 0 0] /H /I /Rect [ 136.1932 689.2532 145.8305 698.0715 ] >> endobj 520 0 obj << /Type /Action >> endobj 521 0 obj << /Type /Page /Parent 3 0 R /Annots [ 523 0 R 526 0 R 528 0 R ] /Contents 522 0 R >> endobj 522 0 obj << /Length 11610 >> stream 0.271 0.267 0.267 rg q 15.000 43.586 577.500 733.414 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(hypotonic Ca2+-rich solution \(10 mM CaCl2\) to discharge the remaining unused AEQ pool. The light signal was collected by the )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(photomultiplier and stored on an IBM-compatible computer and off-line calibrated into Ca2+ concentration values. )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(Luminescence was calibrated off-line into Ca2+ concentration values using a computer algorithm based on the Ca2+ response )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(curve of wild-type aequorin.)] TJ ET BT 26.250 712.357 Td /F4 9.8 Tf [(RNA isolation and PCR measurements.)] TJ ET BT 207.746 712.357 Td /F1 9.8 Tf [( Twenty-four hours after plating, AEQ-transfected and untransfected cells were )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(harvested for RNA isolation using Trizol \(Invitrogen\). RNA was isolated, purified and reverse transcribed using a commercially )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(available kit \(Invitrogen\). The cDNA was subjected to real time PCR amplification \(CFX96, Biorad\) as described in Valenza et )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(al., 2010. PCR was carried out with the same quantity of cDNA using the following primers: NMDA subunit NR1 for: )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(GCCTCCAGCTTCAAGAGACGT; rev: TGTGTTCCCGTCATAGGGAGAG. mGLUR1 for: GGTCCCTTCTGACACTTTGC; rev: )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(CATTCCACTCTCGCCGTAAT. mGlUR5 for: GCCATGGTAGACATAGTGAAG; rev: TAAGAGTGGGCGATGCAAAT. NMDA 2B )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(for: ACGGCAGCAAATCCTACTTCT; rev: ACCACTGGCTTATTGGTGACA. GAPDH for: CAAGGTCATCCATGACAACTT; rev: )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(GGGCCATCCACAGTCTTCTG.)] TJ ET BT 26.250 609.619 Td /F4 9.8 Tf [(Statistical analysis.)] TJ ET BT 116.213 609.619 Td /F1 9.8 Tf [( Graphpad Prism v.4.0 \(Graphpad software, San Diego, CA, USA\) software was used to perform one-way )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(ANOVA and Newman-Keuls Multiple Comparison post-hoc testing.)] 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 [(The authors have declared that no competing interests exist.)] TJ ET BT 26.250 504.555 Td /F4 12.0 Tf [(Authors contributions)] TJ ET BT 26.250 484.601 Td /F1 9.8 Tf [(MM and MV performed most of the experiments and contributed to the conception and design of the experiments and the )] TJ ET BT 26.250 472.696 Td /F1 9.8 Tf [(acquisition, analysis and interpretation of data; VL and CC performed all the experiments involved mass spectrometry, and VL )] TJ ET BT 26.250 460.791 Td /F1 9.8 Tf [(and SDD contributed to analysis and interpretation of mass spectrometry data; CS, ADM, ECar performed all the experiments )] TJ ET BT 26.250 448.887 Td /F1 9.8 Tf [(related to NMDA and contributed to analysis and interpretation of data; ECatt coordinated the study, made contribution to )] TJ ET BT 26.250 436.982 Td /F1 9.8 Tf [(conception and design, to analysis and interpretation of data. MV, MM and ECatt wrote the paper.)] TJ ET BT 26.250 417.577 Td /F1 9.8 Tf [(*MM and MV contributed equally to this work.)] TJ ET BT 26.250 380.975 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 361.020 Td /F1 9.8 Tf [(We acknowledge a financial contribution from an anonymous donor.)] TJ ET BT 26.250 331.918 Td /F4 12.0 Tf [(APPENDIX 1)] TJ ET BT 26.250 294.766 Td /F4 12.0 Tf [(Supplementary Figures)] TJ ET 0.965 0.965 0.965 rg 26.250 43.586 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 284.336 m 581.250 284.336 l 581.250 283.586 l 26.250 283.586 l f q 168.750 0 0 225.000 35.250 49.586 cm /I7 Do Q q 35.250 43.586 537.000 0.000 re W n Q Q q 15.000 43.586 577.500 733.414 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(hypotonic Ca2+-rich solution \(10 mM CaCl2\) to discharge the remaining unused AEQ pool. The light signal was collected by the )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(photomultiplier and stored on an IBM-compatible computer and off-line calibrated into Ca2+ concentration values. )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(Luminescence was calibrated off-line into Ca2+ concentration values using a computer algorithm based on the Ca2+ response )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(curve of wild-type aequorin.)] TJ ET BT 26.250 712.357 Td /F4 9.8 Tf [(RNA isolation and PCR measurements.)] TJ ET BT 207.746 712.357 Td /F1 9.8 Tf [( Twenty-four hours after plating, AEQ-transfected and untransfected cells were )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(harvested for RNA isolation using Trizol \(Invitrogen\). RNA was isolated, purified and reverse transcribed using a commercially )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(available kit \(Invitrogen\). The cDNA was subjected to real time PCR amplification \(CFX96, Biorad\) as described in Valenza et )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(al., 2010. PCR was carried out with the same quantity of cDNA using the following primers: NMDA subunit NR1 for: )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(GCCTCCAGCTTCAAGAGACGT; rev: TGTGTTCCCGTCATAGGGAGAG. mGLUR1 for: GGTCCCTTCTGACACTTTGC; rev: )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(CATTCCACTCTCGCCGTAAT. mGlUR5 for: GCCATGGTAGACATAGTGAAG; rev: TAAGAGTGGGCGATGCAAAT. NMDA 2B )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(for: ACGGCAGCAAATCCTACTTCT; rev: ACCACTGGCTTATTGGTGACA. GAPDH for: CAAGGTCATCCATGACAACTT; rev: )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(GGGCCATCCACAGTCTTCTG.)] TJ ET BT 26.250 609.619 Td /F4 9.8 Tf [(Statistical analysis.)] TJ ET BT 116.213 609.619 Td /F1 9.8 Tf [( Graphpad Prism v.4.0 \(Graphpad software, San Diego, CA, USA\) software was used to perform one-way )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(ANOVA and Newman-Keuls Multiple Comparison post-hoc testing.)] 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 [(The authors have declared that no competing interests exist.)] TJ ET BT 26.250 504.555 Td /F4 12.0 Tf [(Authors contributions)] TJ ET BT 26.250 484.601 Td /F1 9.8 Tf [(MM and MV performed most of the experiments and contributed to the conception and design of the experiments and the )] TJ ET BT 26.250 472.696 Td /F1 9.8 Tf [(acquisition, analysis and interpretation of data; VL and CC performed all the experiments involved mass spectrometry, and VL )] TJ ET BT 26.250 460.791 Td /F1 9.8 Tf [(and SDD contributed to analysis and interpretation of mass spectrometry data; CS, ADM, ECar performed all the experiments )] TJ ET BT 26.250 448.887 Td /F1 9.8 Tf [(related to NMDA and contributed to analysis and interpretation of data; ECatt coordinated the study, made contribution to )] TJ ET BT 26.250 436.982 Td /F1 9.8 Tf [(conception and design, to analysis and interpretation of data. MV, MM and ECatt wrote the paper.)] TJ ET BT 26.250 417.577 Td /F1 9.8 Tf [(*MM and MV contributed equally to this work.)] TJ ET BT 26.250 380.975 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 361.020 Td /F1 9.8 Tf [(We acknowledge a financial contribution from an anonymous donor.)] TJ ET BT 26.250 331.918 Td /F4 12.0 Tf [(APPENDIX 1)] TJ ET BT 26.250 294.766 Td /F4 12.0 Tf [(Supplementary Figures)] TJ ET 0.965 0.965 0.965 rg 26.250 43.586 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 284.336 m 581.250 284.336 l 581.250 283.586 l 26.250 283.586 l f q 168.750 0 0 225.000 35.250 49.586 cm /I7 Do Q q 35.250 43.586 537.000 0.000 re W n Q Q q 15.000 43.586 577.500 733.414 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(hypotonic Ca2+-rich solution \(10 mM CaCl2\) to discharge the remaining unused AEQ pool. The light signal was collected by the )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(photomultiplier and stored on an IBM-compatible computer and off-line calibrated into Ca2+ concentration values. )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(Luminescence was calibrated off-line into Ca2+ concentration values using a computer algorithm based on the Ca2+ response )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(curve of wild-type aequorin.)] TJ ET BT 26.250 712.357 Td /F4 9.8 Tf [(RNA isolation and PCR measurements.)] TJ ET BT 207.746 712.357 Td /F1 9.8 Tf [( Twenty-four hours after plating, AEQ-transfected and untransfected cells were )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(harvested for RNA isolation using Trizol \(Invitrogen\). RNA was isolated, purified and reverse transcribed using a commercially )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(available kit \(Invitrogen\). The cDNA was subjected to real time PCR amplification \(CFX96, Biorad\) as described in Valenza et )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(al., 2010. PCR was carried out with the same quantity of cDNA using the following primers: NMDA subunit NR1 for: )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(GCCTCCAGCTTCAAGAGACGT; rev: TGTGTTCCCGTCATAGGGAGAG. mGLUR1 for: GGTCCCTTCTGACACTTTGC; rev: )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(CATTCCACTCTCGCCGTAAT. mGlUR5 for: GCCATGGTAGACATAGTGAAG; rev: TAAGAGTGGGCGATGCAAAT. NMDA 2B )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(for: ACGGCAGCAAATCCTACTTCT; rev: ACCACTGGCTTATTGGTGACA. GAPDH for: CAAGGTCATCCATGACAACTT; rev: )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(GGGCCATCCACAGTCTTCTG.)] TJ ET BT 26.250 609.619 Td /F4 9.8 Tf [(Statistical analysis.)] TJ ET BT 116.213 609.619 Td /F1 9.8 Tf [( Graphpad Prism v.4.0 \(Graphpad software, San Diego, CA, USA\) software was used to perform one-way )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(ANOVA and Newman-Keuls Multiple Comparison post-hoc testing.)] 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 [(The authors have declared that no competing interests exist.)] TJ ET BT 26.250 504.555 Td /F4 12.0 Tf [(Authors contributions)] TJ ET BT 26.250 484.601 Td /F1 9.8 Tf [(MM and MV performed most of the experiments and contributed to the conception and design of the experiments and the )] TJ ET BT 26.250 472.696 Td /F1 9.8 Tf [(acquisition, analysis and interpretation of data; VL and CC performed all the experiments involved mass spectrometry, and VL )] TJ ET BT 26.250 460.791 Td /F1 9.8 Tf [(and SDD contributed to analysis and interpretation of mass spectrometry data; CS, ADM, ECar performed all the experiments )] TJ ET BT 26.250 448.887 Td /F1 9.8 Tf [(related to NMDA and contributed to analysis and interpretation of data; ECatt coordinated the study, made contribution to )] TJ ET BT 26.250 436.982 Td /F1 9.8 Tf [(conception and design, to analysis and interpretation of data. MV, MM and ECatt wrote the paper.)] TJ ET BT 26.250 417.577 Td /F1 9.8 Tf [(*MM and MV contributed equally to this work.)] TJ ET BT 26.250 380.975 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 361.020 Td /F1 9.8 Tf [(We acknowledge a financial contribution from an anonymous donor.)] TJ ET BT 26.250 331.918 Td /F4 12.0 Tf [(APPENDIX 1)] TJ ET BT 26.250 294.766 Td /F4 12.0 Tf [(Supplementary Figures)] TJ ET 0.965 0.965 0.965 rg 26.250 43.586 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 284.336 m 581.250 284.336 l 581.250 283.586 l 26.250 283.586 l f q 168.750 0 0 225.000 35.250 49.586 cm /I7 Do Q q 35.250 43.586 537.000 0.000 re W n Q Q q 168.750 0 0 225.000 35.250 49.586 cm /I7 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(10)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 523 0 obj << /Type /Annot /Subtype /Link /A 524 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 49.5855 204.0000 274.5855 ] >> endobj 524 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/09/Diapositiva5.jpg) >> endobj 525 0 obj << /Type /XObject /Subtype /Image /Width 225 /Height 300 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 20462>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Siqi95يG~ _;q†)[hW"FQCpKъ|Gb5+L7qGH*!XV% X/E7Sj0/{Kyv s3mX0Ѹ9}FPh>"'HU0|30GoprHѿ NP~P~P~_“w(Ғ^()((Ғ^(ғbJ8zQ%%qE&(Rgޖ/|5|A>ЗSٯ7N%ry Q~VڋZ jS2py(ɟ:e7lW=ǵdx?68--V+f 9ES8#ҵ*ٯMN@{⿁e2Mkp\}YY&+dBi6LΧ*]d]܀۳ݧg4s@Qe6x^PǗd cIa7?Lkt[L*FIH=N 6NkOٓvSZۤsLN;  ,4]"@ҭ5mGg>@Kߵ!=k!__76W!L[p3#1@QQQQ~T~TQ@GG4s@GEcE4PיFƭψ> t5u;g{_?q^~Ιz]yO9u?fO^_!kx.G?tZxXi6:L7([i=si7g'9誺WWGcs G0u}M}lڬ,|FXZۭJ ё|R>|SM|^Lu,$JGi\'En5ƘMBO~ jQx[Xw #⦪"6dHˀx*F9"G51ԵPR1,$I$.8 o٤csY)]/^WPRQ@ E%RQ@KIE-%RQ@}qE{cÈV]ON7<$Z0np˼yk3Z PcX.B̧t_+0ap>_nxz1mo,qyգdRnTss^g~ZןXZL-s<#ŎM{,1^ZM[Ǘ}f;O|~ 7wɋcv|IH߀D_\k-ne?kҰ}Mfѱ[…tbY&81GҴ~%{g}_{1`A7ǼY>. 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VB75'/1?Hd@InTܴx`2:{W" 5o,)jvm,mUDĩ0 |$vq@0W2Ý.[k>Ҽ=lu-8|Oc'$rg.VL$GR&U]T#W__uxWQ׆dE%Ԑkiz7LokH|Cyqr]Z+ح#1<žƟKi M^[Y>`W-|V<o x/P[ s:FNz+?؟J?_[Xhw{cQ\KYo>AK jPg!u "H`!9b~5~l5MnĤ9rF6󸞜j n mm✫R:kſMEtCѱ3-VAֻ++ IbD8~"5DڻJބ䜐 ߏxYZ7Z&+\~u2O FÏëx6~P]QQM"; jwR}n * rHxWDu6oc,1 u$pG,QA$8PO]i/Eh톃?Mt^x_I_W.?kz~uo[zg񷍥eTš?IhG=+ös#V'8_ >7C>.oXm3FI!?"xUjKndi=@ z_iK[$oqN qF6>\}/E%~TQ@ IRj(!I@(4~T~TJOʏʎԴ(ʏʊZOʎݩi(){~T~T-'GE~T~TPcE&h endstream endobj 526 0 obj << /Type /Annot /Subtype /Link /A 527 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 49.5855 204.0000 274.5855 ] >> endobj 527 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/09/Diapositiva5.jpg) >> endobj 528 0 obj << /Type /Annot /Subtype /Link /A 529 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 49.5855 204.0000 274.5855 ] >> endobj 529 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/09/Diapositiva5.jpg) >> endobj 530 0 obj << /Type /Page /Parent 3 0 R /Annots [ 532 0 R 535 0 R 537 0 R ] /Contents 531 0 R >> endobj 531 0 obj << /Length 15490 >> stream q 15.000 43.096 577.500 733.904 re W n 0.965 0.965 0.965 rg 26.250 685.400 555.000 91.600 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 685.400 m 581.250 685.400 l 581.250 686.150 l 26.250 686.150 l f q 35.250 696.650 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: \(Fig. S1\) Quantification of images of filipin staining in ST Kin cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Quantification of filipin staining was performed using imageJ software. a\) Ten fields were analysed for each condition. The )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(graph represent the average of the quantifications SEM. b\) Representative fluorescent images of filipin-stained cells )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(showing the high variability in the fluorescent intensity in different fields of the same well for each cell line. The images )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(shown were cropped from 20X magnification images.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 290.605 555.000 387.295 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 677.900 m 581.250 677.900 l 581.250 677.150 l 26.250 677.150 l f 26.250 290.605 m 581.250 290.605 l 581.250 291.355 l 26.250 291.355 l f q 168.750 0 0 225.000 35.250 443.150 cm /I8 Do Q q 35.250 301.855 537.000 135.295 re W n 0.271 0.267 0.267 rg BT 35.250 427.626 Td /F4 9.8 Tf [(Fig. 2: \(Fig. S2\) NMDA activity in ST Kin cells.)] TJ ET BT 35.250 408.256 Td /F1 9.8 Tf [(Ca2+ transients in the cytosol of ST Kin)] TJ ET BT 206.226 413.976 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 225.015 408.256 Td /F1 9.8 Tf [( cells were induced by stimulation with the purinergic receptor agonist ATP. Cell )] TJ ET BT 35.250 394.520 Td /F1 9.8 Tf [(culture and transfection protocols and methods for monitoring cytosolic Ca2+ using AEQ are described in the Materials and )] TJ ET BT 35.250 380.784 Td /F1 9.8 Tf [(Methods. a\) Effects of adding glutamate \(GLU\) and ATP. b\) Effects of adding NMDA and ATP. The opening of the InsP3 )] TJ ET BT 35.250 367.048 Td /F1 9.8 Tf [(receptor, which is induced by stimulation of the purinergic receptors, elicits the expected Ca2+ transient, whereas no )] TJ ET BT 35.250 353.311 Td /F1 9.8 Tf [(transient is elicited by the addition of glutamate or NMDA. c\) Quantification of NR1 mRNA levels in striatal knock-in cells. )] TJ ET BT 35.250 339.575 Td /F1 9.8 Tf [(The NMDA subunit NR1 is not expressed in ST Kin)] TJ ET BT 255.785 345.295 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 274.575 339.575 Td /F1 9.8 Tf [( cells \(0.00009 0.00004\) or ST K-in)] TJ ET BT 434.172 345.295 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 472.236 339.575 Td /F1 9.8 Tf [( cells \(clone #2, )] TJ ET BT 35.250 325.839 Td /F1 9.8 Tf [(0.00038 0.00013; clone #6, 0.00043 0.00016\). The grey column represents the expression of NR1 mRNA as a positive )] TJ ET BT 35.250 312.103 Td /F1 9.8 Tf [(control \(levels in brains from 6-week-old mice: 27.21472 9.12243\).)] TJ ET Q BT 26.250 256.384 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 228.930 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 228.930 Td /F1 9.8 Tf [(A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The )] TJ ET BT 26.250 217.025 Td /F1 9.8 Tf [(Huntington's Disease Collaborative Research Group. Cell. 1993 Mar 26;72\(6\):971-83.)] TJ ET BT 26.250 197.620 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 197.620 Td /F1 9.8 Tf [(Zuccato C, Valenza M, Cattaneo E. Molecular mechanisms and potential therapeutical targets in Huntington's disease. )] TJ ET BT 26.250 185.715 Td /F1 9.8 Tf [(Physiol Rev. 2010 Jul;90\(3\):905-81.)] TJ ET BT 26.250 166.311 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 166.311 Td /F1 9.8 Tf [(Valenza M, Cattaneo E. Emerging roles for cholesterol in Huntington's disease. Trends Neurosci. 2011 Sep;34\(9\):474-86.)] TJ ET BT 26.250 146.906 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 146.906 Td /F1 9.8 Tf [(Karasinska JM, Hayden MR. Cholesterol metabolism in Huntington disease. Nat Rev Neurol. 2011 Oct;7\(10\):561-72.)] TJ ET BT 26.250 127.501 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 127.501 Td /F1 9.8 Tf [(Sipione S, Rigamonti D, Valenza M, Zuccato C, Conti L, Pritchard J, et al. Early transcriptional profiles in huntingtin-inducible )] TJ ET BT 26.250 115.596 Td /F1 9.8 Tf [(striatal cells by microarray analyses. Hum Mol Genet. 2002 Aug 15;11\(17\):1953-65.)] TJ ET BT 26.250 96.192 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 96.192 Td /F1 9.8 Tf [(Valenza M, Rigamonti D, Goffredo D, Zuccato C, Fenu S, Jamot L, et al. Dysfunction of the cholesterol biosynthetic pathway )] TJ ET BT 26.250 84.287 Td /F1 9.8 Tf [(in Huntington's disease. J Neurosci. 2005 Oct 26;25\(43\):9932-9.)] TJ ET BT 26.250 64.882 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 64.882 Td /F1 9.8 Tf [(Valenza M, Carroll JB, Leoni V, Bertram LN, Bjorkhem I, Singaraja RR, et al. Cholesterol biosynthesis pathway is disturbed in )] TJ ET BT 26.250 52.977 Td /F1 9.8 Tf [(YAC128 mice and is modulated by huntingtin mutation. Hum Mol Genet. 2007 Sep 15;16\(18\):2187-98.)] TJ ET Q q 15.000 43.096 577.500 733.904 re W n 0.965 0.965 0.965 rg 26.250 685.400 555.000 91.600 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 685.400 m 581.250 685.400 l 581.250 686.150 l 26.250 686.150 l f q 35.250 696.650 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: \(Fig. S1\) Quantification of images of filipin staining in ST Kin cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Quantification of filipin staining was performed using imageJ software. a\) Ten fields were analysed for each condition. The )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(graph represent the average of the quantifications SEM. b\) Representative fluorescent images of filipin-stained cells )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(showing the high variability in the fluorescent intensity in different fields of the same well for each cell line. The images )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(shown were cropped from 20X magnification images.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 290.605 555.000 387.295 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 677.900 m 581.250 677.900 l 581.250 677.150 l 26.250 677.150 l f 26.250 290.605 m 581.250 290.605 l 581.250 291.355 l 26.250 291.355 l f q 168.750 0 0 225.000 35.250 443.150 cm /I8 Do Q q 35.250 301.855 537.000 135.295 re W n 0.271 0.267 0.267 rg BT 35.250 427.626 Td /F4 9.8 Tf [(Fig. 2: \(Fig. S2\) NMDA activity in ST Kin cells.)] TJ ET BT 35.250 408.256 Td /F1 9.8 Tf [(Ca2+ transients in the cytosol of ST Kin)] TJ ET BT 206.226 413.976 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 225.015 408.256 Td /F1 9.8 Tf [( cells were induced by stimulation with the purinergic receptor agonist ATP. Cell )] TJ ET BT 35.250 394.520 Td /F1 9.8 Tf [(culture and transfection protocols and methods for monitoring cytosolic Ca2+ using AEQ are described in the Materials and )] TJ ET BT 35.250 380.784 Td /F1 9.8 Tf [(Methods. a\) Effects of adding glutamate \(GLU\) and ATP. b\) Effects of adding NMDA and ATP. The opening of the InsP3 )] TJ ET BT 35.250 367.048 Td /F1 9.8 Tf [(receptor, which is induced by stimulation of the purinergic receptors, elicits the expected Ca2+ transient, whereas no )] TJ ET BT 35.250 353.311 Td /F1 9.8 Tf [(transient is elicited by the addition of glutamate or NMDA. c\) Quantification of NR1 mRNA levels in striatal knock-in cells. )] TJ ET BT 35.250 339.575 Td /F1 9.8 Tf [(The NMDA subunit NR1 is not expressed in ST Kin)] TJ ET BT 255.785 345.295 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 274.575 339.575 Td /F1 9.8 Tf [( cells \(0.00009 0.00004\) or ST K-in)] TJ ET BT 434.172 345.295 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 472.236 339.575 Td /F1 9.8 Tf [( cells \(clone #2, )] TJ ET BT 35.250 325.839 Td /F1 9.8 Tf [(0.00038 0.00013; clone #6, 0.00043 0.00016\). The grey column represents the expression of NR1 mRNA as a positive )] TJ ET BT 35.250 312.103 Td /F1 9.8 Tf [(control \(levels in brains from 6-week-old mice: 27.21472 9.12243\).)] TJ ET Q BT 26.250 256.384 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 228.930 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 228.930 Td /F1 9.8 Tf [(A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The )] TJ ET BT 26.250 217.025 Td /F1 9.8 Tf [(Huntington's Disease Collaborative Research Group. Cell. 1993 Mar 26;72\(6\):971-83.)] TJ ET BT 26.250 197.620 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 197.620 Td /F1 9.8 Tf [(Zuccato C, Valenza M, Cattaneo E. Molecular mechanisms and potential therapeutical targets in Huntington's disease. )] TJ ET BT 26.250 185.715 Td /F1 9.8 Tf [(Physiol Rev. 2010 Jul;90\(3\):905-81.)] TJ ET BT 26.250 166.311 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 166.311 Td /F1 9.8 Tf [(Valenza M, Cattaneo E. Emerging roles for cholesterol in Huntington's disease. Trends Neurosci. 2011 Sep;34\(9\):474-86.)] TJ ET BT 26.250 146.906 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 146.906 Td /F1 9.8 Tf [(Karasinska JM, Hayden MR. Cholesterol metabolism in Huntington disease. Nat Rev Neurol. 2011 Oct;7\(10\):561-72.)] TJ ET BT 26.250 127.501 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 127.501 Td /F1 9.8 Tf [(Sipione S, Rigamonti D, Valenza M, Zuccato C, Conti L, Pritchard J, et al. Early transcriptional profiles in huntingtin-inducible )] TJ ET BT 26.250 115.596 Td /F1 9.8 Tf [(striatal cells by microarray analyses. Hum Mol Genet. 2002 Aug 15;11\(17\):1953-65.)] TJ ET BT 26.250 96.192 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 96.192 Td /F1 9.8 Tf [(Valenza M, Rigamonti D, Goffredo D, Zuccato C, Fenu S, Jamot L, et al. Dysfunction of the cholesterol biosynthetic pathway )] TJ ET BT 26.250 84.287 Td /F1 9.8 Tf [(in Huntington's disease. J Neurosci. 2005 Oct 26;25\(43\):9932-9.)] TJ ET BT 26.250 64.882 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 64.882 Td /F1 9.8 Tf [(Valenza M, Carroll JB, Leoni V, Bertram LN, Bjorkhem I, Singaraja RR, et al. Cholesterol biosynthesis pathway is disturbed in )] TJ ET BT 26.250 52.977 Td /F1 9.8 Tf [(YAC128 mice and is modulated by huntingtin mutation. Hum Mol Genet. 2007 Sep 15;16\(18\):2187-98.)] TJ ET Q q 15.000 43.096 577.500 733.904 re W n 0.965 0.965 0.965 rg 26.250 685.400 555.000 91.600 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 685.400 m 581.250 685.400 l 581.250 686.150 l 26.250 686.150 l f q 35.250 696.650 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: \(Fig. S1\) Quantification of images of filipin staining in ST Kin cells.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Quantification of filipin staining was performed using imageJ software. a\) Ten fields were analysed for each condition. The )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(graph represent the average of the quantifications SEM. b\) Representative fluorescent images of filipin-stained cells )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(showing the high variability in the fluorescent intensity in different fields of the same well for each cell line. The images )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(shown were cropped from 20X magnification images.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 290.605 555.000 387.295 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 677.900 m 581.250 677.900 l 581.250 677.150 l 26.250 677.150 l f 26.250 290.605 m 581.250 290.605 l 581.250 291.355 l 26.250 291.355 l f q 168.750 0 0 225.000 35.250 443.150 cm /I8 Do Q q 35.250 301.855 537.000 135.295 re W n 0.271 0.267 0.267 rg BT 35.250 427.626 Td /F4 9.8 Tf [(Fig. 2: \(Fig. S2\) NMDA activity in ST Kin cells.)] TJ ET BT 35.250 408.256 Td /F1 9.8 Tf [(Ca2+ transients in the cytosol of ST Kin)] TJ ET BT 206.226 413.976 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 225.015 408.256 Td /F1 9.8 Tf [( cells were induced by stimulation with the purinergic receptor agonist ATP. Cell )] TJ ET BT 35.250 394.520 Td /F1 9.8 Tf [(culture and transfection protocols and methods for monitoring cytosolic Ca2+ using AEQ are described in the Materials and )] TJ ET BT 35.250 380.784 Td /F1 9.8 Tf [(Methods. a\) Effects of adding glutamate \(GLU\) and ATP. b\) Effects of adding NMDA and ATP. The opening of the InsP3 )] TJ ET BT 35.250 367.048 Td /F1 9.8 Tf [(receptor, which is induced by stimulation of the purinergic receptors, elicits the expected Ca2+ transient, whereas no )] TJ ET BT 35.250 353.311 Td /F1 9.8 Tf [(transient is elicited by the addition of glutamate or NMDA. c\) Quantification of NR1 mRNA levels in striatal knock-in cells. )] TJ ET BT 35.250 339.575 Td /F1 9.8 Tf [(The NMDA subunit NR1 is not expressed in ST Kin)] TJ ET BT 255.785 345.295 Td /F1 8.7 Tf [(7/7Q)] TJ ET BT 274.575 339.575 Td /F1 9.8 Tf [( cells \(0.00009 0.00004\) or ST K-in)] TJ ET BT 434.172 345.295 Td /F1 8.7 Tf [(109/109Q)] TJ ET BT 472.236 339.575 Td /F1 9.8 Tf [( cells \(clone #2, )] TJ ET BT 35.250 325.839 Td /F1 9.8 Tf [(0.00038 0.00013; clone #6, 0.00043 0.00016\). The grey column represents the expression of NR1 mRNA as a positive )] TJ ET BT 35.250 312.103 Td /F1 9.8 Tf [(control \(levels in brains from 6-week-old mice: 27.21472 9.12243\).)] TJ ET Q BT 26.250 256.384 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 228.930 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 228.930 Td /F1 9.8 Tf [(A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The )] TJ ET BT 26.250 217.025 Td /F1 9.8 Tf [(Huntington's Disease Collaborative Research Group. Cell. 1993 Mar 26;72\(6\):971-83.)] TJ ET BT 26.250 197.620 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 197.620 Td /F1 9.8 Tf [(Zuccato C, Valenza M, Cattaneo E. Molecular mechanisms and potential therapeutical targets in Huntington's disease. )] TJ ET BT 26.250 185.715 Td /F1 9.8 Tf [(Physiol Rev. 2010 Jul;90\(3\):905-81.)] TJ ET BT 26.250 166.311 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 166.311 Td /F1 9.8 Tf [(Valenza M, Cattaneo E. Emerging roles for cholesterol in Huntington's disease. Trends Neurosci. 2011 Sep;34\(9\):474-86.)] TJ ET BT 26.250 146.906 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 146.906 Td /F1 9.8 Tf [(Karasinska JM, Hayden MR. Cholesterol metabolism in Huntington disease. Nat Rev Neurol. 2011 Oct;7\(10\):561-72.)] TJ ET BT 26.250 127.501 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 127.501 Td /F1 9.8 Tf [(Sipione S, Rigamonti D, Valenza M, Zuccato C, Conti L, Pritchard J, et al. Early transcriptional profiles in huntingtin-inducible )] TJ ET BT 26.250 115.596 Td /F1 9.8 Tf [(striatal cells by microarray analyses. Hum Mol Genet. 2002 Aug 15;11\(17\):1953-65.)] TJ ET BT 26.250 96.192 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 96.192 Td /F1 9.8 Tf [(Valenza M, Rigamonti D, Goffredo D, Zuccato C, Fenu S, Jamot L, et al. Dysfunction of the cholesterol biosynthetic pathway )] TJ ET BT 26.250 84.287 Td /F1 9.8 Tf [(in Huntington's disease. J Neurosci. 2005 Oct 26;25\(43\):9932-9.)] TJ ET BT 26.250 64.882 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 64.882 Td /F1 9.8 Tf [(Valenza M, Carroll JB, Leoni V, Bertram LN, Bjorkhem I, Singaraja RR, et al. Cholesterol biosynthesis pathway is disturbed in )] TJ ET BT 26.250 52.977 Td /F1 9.8 Tf [(YAC128 mice and is modulated by huntingtin mutation. Hum Mol Genet. 2007 Sep 15;16\(18\):2187-98.)] TJ ET Q q 168.750 0 0 225.000 35.250 443.150 cm /I8 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(11)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 532 0 obj << /Type /Annot /Subtype /Link /A 533 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 443.1502 204.0000 668.1502 ] >> endobj 533 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/09/Diapositiva6.jpg) >> endobj 534 0 obj << /Type /XObject /Subtype /Image /Width 225 /Height 300 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 10664>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Siqi95يG~ _;q†)[hW"FQCpKъ|Gb5+L7qGH*!XV% X/E7Sj0/{Kyv s3mX0Ѹ9}FPXxxʹ1cӛRx].sكޗv (Ka}SE/GIE/GIE/GH(()1Gz_Ž=)(’_Ž=)(=)1F(xLQ@ ǥRQ@ QRQ@Rbu&}k]C_Kv$\1n3 1@Oj Լ9ef;ԖF #!6A0e"%m~:њ"tC`y>6/~Qx~PQ3\u__xk<{{[ax}Ǖo9@NhrWAyW@yu9<) (%e=O)xmC÷ )fGR2& 4fMWT񮋨>t 7Zva!e2?1n7ww&h/@ 3E4&h-&h/I3KPE/E'K'>xF O%k͖ r_J"Fاc6N `*'~J߽Ib WvR ]*}z(9?*?*OjΟk;Kum5DNpHF3xb':xUh]I.gI1}*?*( #C#lʊ@,@3PyɌ~cb _pq_ŗ`tg[Q y xBw1hZ[BC@`:Ut_^5ts^7 ^j@?*?*9ʏʊ99(h }(&Z)1F('^' 6> 7QKmOjd9Irp+cOu]l VHv}e`.UyAO[8n5IrNNX};`{s@u<MV̷2GKp S~:f}>Ѭ-?퓞p+M6i$Kxyw<.n:m@ E!RK/hduЦ㵦$]s^ M픚`nnR wD%mXú[]ѴIx}:}6*f>)euxPE|Y,({"p!Im=8,T`I[+񝎏V\j)BMrJn_/p=IUrύ&vou7.kJN6H `.UyA/E%RQ@ E%~PRQ@ E%gGPGK'4xF O%kjrBz]>N O?k}OV%ڦP6ü&}OJV5|IԼ蚆`k'rJQaQElSG},Q춄# [;\`SxJj^sű|8maV FzhY|2ݞggS[(mwwl=g x ^0Q^6߯^^vsu_(gt.#J-.ivK.OL8U$y07^=?3ںbF pˌ"8J| <3|ZlR[7OpZR_Y?_./5x>ũ[^CYFwYq"d;XsO5)#>U輪˾i<K{MM'ĐG5Ŝ)>cƠ]rҌHUD#P}I~⺗h+NYi$6of 7amUldɼO:M|68+6`n.t`FL@=L{uZӡ{)'KR3\.|7l,Ckjtʳ1$# #LsW5i?w^}/Y]Y]KJa,?)J$2cZ^/tk'Sh%T %E,=<ށ?FmFYk=I6.-&I~ms$͏DqiV}KK~B٘ i;-𽍏n-9+[], ]k̇h0s_BכAh ΣoFb%^-|٤֎Hw#S##I#pW9KJ)^ڙ^7 Zn[B{y#fY5$(F tۚ(J)yJ?J^h^h^h^h^h} יᆹoN t2f6Yp,-< :o%_S<⮝x>_N%jR<2jl, ͙і7%&< q#F\m (e$@Z#40Iڊ9'Ԓd6t=@s2Px rR {B˟1@4i&P.4)燺Fڍ$kYuyws1ߚ$^njfm8sc5 Z/.z<mpEj#EU@U{m*k,ปdĪ~bO$k-?bARys(5 .VC}iJۄw+8pG^OV+Wq.𮆃[]DSKE V:chͬ@"TnN& KI< \*wkK,Jxd21>@PEPGE-%PEPQEgQK(gğ5Kgy+kSq`kğ5Ky+oS4ש͙p^1Wç1ϴcoLyZ_gx}Kd`nn0AF 1EbK@WŽ>-K1cY[*}»A\aH?vMNN= Ҽc&&>frn^[r3-x# d+»ҿ羧+.:;(4ѬQfX?5 5ƩoIn}nSUn]_SI J ?xX%BFH=c֑dHNI'5 J ??]_S@,]SG45놄/RD?wn(.M_Ğ NԾyqgΫmRvXZ ;-$m_u|[5{dd-»ҿ羧+.:+Ht8-m[¨XxoM5+B"9`I/y8 J ??]_S@4Zib#<B:*W3 J ??]_S@6gu$a?;|sqz%' }CK3O a&pQe .8÷ QzzQzPzzzQzPzzzQzPzzzQzP(J(kY:Z\߬l#= خaH>6| PgI1K&kD΃KɭtVh_p܍= Sr nI4fu-444g\Rb4f Rb4f Q3Fh1KLb4f Q3Fh1(㧍VF Ii=·9Kt[3|Rx9G 0 Z]Oi&m8l쉞=[d*)T}^(> )5%iu{2aZ(k`wػj Y_ X2\A{Hٮf)Fݥ32'sɫ>D%d2:"߂;k亇Eᾲrehs<tC9dceՍW:3hK9D±i>,_VmƄG,G#ZͥǛ52rx/'`hgu,ՙ}+K'no ӧvԒH0C\ό&`m;AWxn fpBW#8[OI[C<|qS^ Pt`Ǟuk"#\x^Kcw|x9lX8%װ_H {žwxsB H%ᔌҩoB--6^O?['Oɣ!w4WPrW=*TsK."_.>85KK=oL-!V-fvaaqoI*F{7_tִBݢܐc}/U8Pg͵5X0Id2|ǍW?/5XEHLe&#h$W9;]fiۭ"8my:kY!%/$0UE$z;;#-Wyĥy:at[?K4O0r?:/ dC,L]O 8 =p?"GA]>(KL :b4fR8.((b4QE?f4k济mE.U%#Ku\(CryǼfh72捨D;yr[HYr;psXѴ"N-fO5H "5fO-F5f[n@]~e9ḣ%W5ǯԏ.Nn̜ɯFht EXy $+6>#zBC4u`ko|';V4ڄjS pW]\)>Sq=luiCY%oFsH^]8YwB? 7ެ +5#jvKjѢgnVrI@k\KElC8:)A' ߜd=пgwV㯂!Y'[HgDq;Vb GP(4Q3Q3@fE4fњ3@E4P^CO\G_^DG+#{)#W^1S5[{̐(s) AXc%AOVZE;הjo.ai դkxa.+0dHqH`=b)h@ .1zW {;8<84SǪi,ZX2Pow|GGPGGP1GG@ K&)qGG@>J}wj=k_k6\Wנ~?^_Eq@X~tQ@ я%-я%~t~tQրZ1Ί8.gOfqjJ]gT3|nV86\0)siv2_gQ >R9טះ=j-͛|%3N$ nll#(0G8<WޥJQ׷RvΖR?F||*$=NPHh4RQ@ E%RQހ=iO/5Cm_J}wjjK߀H+.??D褢J;J(hJ(sE'E.k3\ΏxXluhs$I}lnPؚhM @ NJ GR| bs(P$z}ySj:l|7{;XIa (RHbY`mzo45]W,Ktg|2oOTnQ۟-}4 T00 ppsFk4<$iUүaПv@>hDևo;LʆntŪj (B:ʱ" &to!lˊK~5u'5\M-!i_᧋YF2#}5ۓ.D_Ls_ߴ{_!S:n/eqq+7ψ7]K>}<--咱 k g#F y?+C>|QKo&?kـ@~sa|U𶽩xP]?OY;&`m4cu3ITFmN>u8bMٌvs^M&L?@ K?Gh4~&uqhbFh4 ?xEԏRHlcGA g$a=Ϋ^j X* :vLF՘)͉~MyDfOٳE;+`67k>#|Du-Y<-9o"o/b),,+Vdg?RxeO>P.U4 #cl0#Cfs7nhrb53FvU'8=Kq{8-K:fڼ($k&*xkJQԮe啂acWдˋ*XoEQݏ@$y&yc5_[]++ .vDq"Wr2`W]s7{Jf|-O!e7CH`CYNii}"%(T` OgLW#㿉ZgPNwFoe}O|@857xDRfuuWT.x*y09 {>}clj&_[KY$l1snROG.g/%6Sj JH oPR~` ڞ(b(1EP1EbQE8=撀 ('yOkv!t=&{x~VI\ݗ‚F:šNs5Վmes9-!p8,8'k(i _|Lյ5F!J dKdudᶴdFW oJӴm&m $ArI$IIɫ ^P=} QMgG l tf@;cƀʖj(Ɗ(hhhʊ?(i?*Q@@w4=(4~Tʏʔ@ QQږ֗%PQQK@ Q۵-%%/zOʏʏʏʖL}( endstream endobj 535 0 obj << /Type /Annot /Subtype /Link /A 536 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 443.1502 204.0000 668.1502 ] >> endobj 536 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/09/Diapositiva6.jpg) >> endobj 537 0 obj << /Type /Annot /Subtype /Link /A 538 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 443.1502 204.0000 668.1502 ] >> endobj 538 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/09/Diapositiva6.jpg) >> endobj 539 0 obj << /Type /Page /Parent 3 0 R /Contents 540 0 R >> endobj 540 0 obj << /Length 18833 >> stream 0.271 0.267 0.267 rg q 15.000 179.024 577.500 597.976 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 759.976 Td /F1 9.8 Tf [(Valenza M, Leoni V, Tarditi A, Mariotti C, Bjorkhem I, Di Donato S, et al. Progressive dysfunction of the cholesterol )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(biosynthesis pathway in the R6/2 mouse model of Huntington's disease. Neurobiol Dis. 2007 Oct;28\(1\):133-42.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 728.667 Td /F1 9.8 Tf [(Leoni V, Mariotti C, Tabrizi SJ, Valenza M, Wild EJ, Henley SM, et al. Plasma 24S-hydroxycholesterol and caudate MRI in )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(pre-manifest and early Huntington's disease. Brain. 2008 Nov;131\(Pt 11\):2851-9.)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Leoni V, Mariotti C, Nanetti L, Salvatore E, Squitieri F, Bentivoglio AR, et al. Whole body cholesterol metabolism is impaired )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(in Huntington's disease. Neurosci Lett. 2011 May 2;494\(3\):245-9.)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Ritch JJ, Valencia A, Alexander J, Sapp E, Gatune L, Sangrey GR, et al. Multiple phenotypes in Huntington disease mouse )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(neural stem cells. Mol Cell Neurosci. 2012 May;50\(1\):70-81.)] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 634.738 Td /F1 9.8 Tf [(Trushina E, Singh RD, Dyer RB, Cao S, Shah VH, Parton RG, et al. Mutant huntingtin inhibits clathrin-independent )] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(endocytosis and causes accumulation of cholesterol in vitro and in vivo. Hum Mol Genet. 2006 Dec 15;15\(24\):3578-91.)] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 603.429 Td /F1 9.8 Tf [(Luthi-Carter R, Taylor DM, Pallos J, Lambert E, Amore A, Parker A, et al. SIRT2 inhibition achieves neuroprotection by )] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(decreasing sterol biosynthesis. Proc Natl Acad Sci U S A. 2010 Apr 27;107\(17\):7927-32.)] TJ ET BT 26.250 572.119 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 572.119 Td /F1 9.8 Tf [(Del Toro D, Xifro X, Pol A, Humbert S, Saudou F, Canals JM, et al. Altered cholesterol homeostasis contributes to )] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(enhanced excitotoxicity in Huntington's disease. J Neurochem. 2010 Oct;115\(1\):153-67.)] TJ ET BT 26.250 540.810 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 540.810 Td /F1 9.8 Tf [(Valenza M, Leoni V, Tarditi A, Mariotti C, Bjorkhem I, Di Donato S, et al. Progressive dysfunction of the cholesterol )] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(biosynthesis pathway in the R6/2 mouse model of Huntington's disease. Neurobiol Dis. 2007 Oct;28\(1\):133-42.)] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 509.500 Td /F1 9.8 Tf [(Trettel F, Rigamonti D, Hilditch-Maguire P, Wheeler VC, Sharp AH, Persichetti F, et al. Dominant phenotypes produced by )] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(the HD mutation in STHdh\(Q111\) striatal cells. Hum Mol Genet. 2000 Nov 22;9\(19\):2799-809.)] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 478.191 Td /F1 9.8 Tf [(Greenspan P, Mayer EP, Fowler SD. Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol. 1985 )] TJ ET BT 26.250 466.286 Td /F1 9.8 Tf [(Mar;100\(3\):965-73.)] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 446.881 Td /F1 9.8 Tf [(Corvera S, DiBonaventura C, Shpetner HS. Cell confluence-dependent remodeling of endothelial membranes mediated by )] TJ ET BT 26.250 434.976 Td /F1 9.8 Tf [(cholesterol. J Biol Chem. 2000 Oct 6;275\(40\):31414-21.)] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 415.572 Td /F1 9.8 Tf [(Gines S, Ivanova E, Seong IS, Saura CA, MacDonald ME. Enhanced Akt signaling is an early pro-survival response that )] TJ ET BT 26.250 403.667 Td /F1 9.8 Tf [(reflects N-methyl-D-aspartate receptor activation in Huntington's disease knock-in striatal cells. J Biol Chem. 2003 Dec )] TJ ET BT 26.250 391.762 Td /F1 9.8 Tf [(12;278\(50\):50514-22.)] TJ ET BT 26.250 372.357 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 372.357 Td /F1 9.8 Tf [(Demichelis F, Greulich H, Macoska JA, Beroukhim R, Sellers WR, Garraway L, et al. SNP panel identification assay \(SPIA\): )] TJ ET BT 26.250 360.453 Td /F1 9.8 Tf [(a genetic-based assay for the identification of cell lines. Nucleic Acids Res. 2008 Apr;36\(7\):2446-56.)] TJ ET BT 26.250 341.048 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 341.048 Td /F1 9.8 Tf [(Diaferia GR, Cardano M, Cattaneo M, Spinelli CC, Dessi SS, DeBlasio P, et al. The science of stem cell biobanking: )] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(investing in the future. J Cell Physiol. 2012 Jan;227\(1\):14-9.)] TJ ET BT 26.250 309.738 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 309.738 Td /F1 9.8 Tf [(Masters JR. False cell lines: The problem and a solution. Cytotechnology. 2002 Jul;39\(2\):69-74.)] TJ ET BT 26.250 290.334 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 290.334 Td /F1 9.8 Tf [(Lacroix M. Persistent use of "false" cell lines. Int J Cancer. 2008 Jan 1;122\(1\):1-4.)] TJ ET BT 26.250 270.929 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 270.929 Td /F1 9.8 Tf [(Valenza M, Cattaneo E. Neuroprotection and brain cholesterol biosynthesis in Huntington's disease. Proc Natl Acad Sci U S )] TJ ET BT 26.250 259.024 Td /F1 9.8 Tf [(A. 2010 Sep 14;107\(37\):E143; author reply 4.)] TJ ET BT 26.250 239.619 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 239.619 Td /F1 9.8 Tf [(Luthi-Carter R, Kazantsev A. Reply to Valenza and Cattaneo: SIRT2-mediated neuroprotection and cholesterol )] TJ ET BT 26.250 227.715 Td /F1 9.8 Tf [(dyshomeostasis in Huntingtons disease. Proc Natl Acad Sci U S A. 2010;107\(37\):E144.)] TJ ET BT 26.250 208.310 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 208.310 Td /F1 9.8 Tf [(Bobrowska A, Donmez G, Weiss A, Guarente L, Bates G. SIRT2 ablation has no effect on tubulin acetylation in brain, )] TJ ET BT 26.250 196.405 Td /F1 9.8 Tf [(cholesterol biosynthesis or the progression of Huntington's disease phenotypes in vivo. PLoS One. 2012;7\(4\):e34805.)] TJ ET Q q 15.000 179.024 577.500 597.976 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 759.976 Td /F1 9.8 Tf [(Valenza M, Leoni V, Tarditi A, Mariotti C, Bjorkhem I, Di Donato S, et al. Progressive dysfunction of the cholesterol )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(biosynthesis pathway in the R6/2 mouse model of Huntington's disease. Neurobiol Dis. 2007 Oct;28\(1\):133-42.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 728.667 Td /F1 9.8 Tf [(Leoni V, Mariotti C, Tabrizi SJ, Valenza M, Wild EJ, Henley SM, et al. Plasma 24S-hydroxycholesterol and caudate MRI in )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(pre-manifest and early Huntington's disease. Brain. 2008 Nov;131\(Pt 11\):2851-9.)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Leoni V, Mariotti C, Nanetti L, Salvatore E, Squitieri F, Bentivoglio AR, et al. Whole body cholesterol metabolism is impaired )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(in Huntington's disease. Neurosci Lett. 2011 May 2;494\(3\):245-9.)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Ritch JJ, Valencia A, Alexander J, Sapp E, Gatune L, Sangrey GR, et al. Multiple phenotypes in Huntington disease mouse )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(neural stem cells. Mol Cell Neurosci. 2012 May;50\(1\):70-81.)] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 634.738 Td /F1 9.8 Tf [(Trushina E, Singh RD, Dyer RB, Cao S, Shah VH, Parton RG, et al. Mutant huntingtin inhibits clathrin-independent )] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(endocytosis and causes accumulation of cholesterol in vitro and in vivo. Hum Mol Genet. 2006 Dec 15;15\(24\):3578-91.)] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 603.429 Td /F1 9.8 Tf [(Luthi-Carter R, Taylor DM, Pallos J, Lambert E, Amore A, Parker A, et al. SIRT2 inhibition achieves neuroprotection by )] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(decreasing sterol biosynthesis. Proc Natl Acad Sci U S A. 2010 Apr 27;107\(17\):7927-32.)] TJ ET BT 26.250 572.119 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 572.119 Td /F1 9.8 Tf [(Del Toro D, Xifro X, Pol A, Humbert S, Saudou F, Canals JM, et al. Altered cholesterol homeostasis contributes to )] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(enhanced excitotoxicity in Huntington's disease. J Neurochem. 2010 Oct;115\(1\):153-67.)] TJ ET BT 26.250 540.810 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 540.810 Td /F1 9.8 Tf [(Valenza M, Leoni V, Tarditi A, Mariotti C, Bjorkhem I, Di Donato S, et al. Progressive dysfunction of the cholesterol )] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(biosynthesis pathway in the R6/2 mouse model of Huntington's disease. Neurobiol Dis. 2007 Oct;28\(1\):133-42.)] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 509.500 Td /F1 9.8 Tf [(Trettel F, Rigamonti D, Hilditch-Maguire P, Wheeler VC, Sharp AH, Persichetti F, et al. Dominant phenotypes produced by )] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(the HD mutation in STHdh\(Q111\) striatal cells. Hum Mol Genet. 2000 Nov 22;9\(19\):2799-809.)] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 478.191 Td /F1 9.8 Tf [(Greenspan P, Mayer EP, Fowler SD. Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol. 1985 )] TJ ET BT 26.250 466.286 Td /F1 9.8 Tf [(Mar;100\(3\):965-73.)] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 446.881 Td /F1 9.8 Tf [(Corvera S, DiBonaventura C, Shpetner HS. Cell confluence-dependent remodeling of endothelial membranes mediated by )] TJ ET BT 26.250 434.976 Td /F1 9.8 Tf [(cholesterol. J Biol Chem. 2000 Oct 6;275\(40\):31414-21.)] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 415.572 Td /F1 9.8 Tf [(Gines S, Ivanova E, Seong IS, Saura CA, MacDonald ME. Enhanced Akt signaling is an early pro-survival response that )] TJ ET BT 26.250 403.667 Td /F1 9.8 Tf [(reflects N-methyl-D-aspartate receptor activation in Huntington's disease knock-in striatal cells. J Biol Chem. 2003 Dec )] TJ ET BT 26.250 391.762 Td /F1 9.8 Tf [(12;278\(50\):50514-22.)] TJ ET BT 26.250 372.357 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 372.357 Td /F1 9.8 Tf [(Demichelis F, Greulich H, Macoska JA, Beroukhim R, Sellers WR, Garraway L, et al. SNP panel identification assay \(SPIA\): )] TJ ET BT 26.250 360.453 Td /F1 9.8 Tf [(a genetic-based assay for the identification of cell lines. Nucleic Acids Res. 2008 Apr;36\(7\):2446-56.)] TJ ET BT 26.250 341.048 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 341.048 Td /F1 9.8 Tf [(Diaferia GR, Cardano M, Cattaneo M, Spinelli CC, Dessi SS, DeBlasio P, et al. The science of stem cell biobanking: )] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(investing in the future. J Cell Physiol. 2012 Jan;227\(1\):14-9.)] TJ ET BT 26.250 309.738 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 309.738 Td /F1 9.8 Tf [(Masters JR. False cell lines: The problem and a solution. Cytotechnology. 2002 Jul;39\(2\):69-74.)] TJ ET BT 26.250 290.334 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 290.334 Td /F1 9.8 Tf [(Lacroix M. Persistent use of "false" cell lines. Int J Cancer. 2008 Jan 1;122\(1\):1-4.)] TJ ET BT 26.250 270.929 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 270.929 Td /F1 9.8 Tf [(Valenza M, Cattaneo E. Neuroprotection and brain cholesterol biosynthesis in Huntington's disease. Proc Natl Acad Sci U S )] TJ ET BT 26.250 259.024 Td /F1 9.8 Tf [(A. 2010 Sep 14;107\(37\):E143; author reply 4.)] TJ ET BT 26.250 239.619 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 239.619 Td /F1 9.8 Tf [(Luthi-Carter R, Kazantsev A. Reply to Valenza and Cattaneo: SIRT2-mediated neuroprotection and cholesterol )] TJ ET BT 26.250 227.715 Td /F1 9.8 Tf [(dyshomeostasis in Huntingtons disease. Proc Natl Acad Sci U S A. 2010;107\(37\):E144.)] TJ ET BT 26.250 208.310 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 208.310 Td /F1 9.8 Tf [(Bobrowska A, Donmez G, Weiss A, Guarente L, Bates G. SIRT2 ablation has no effect on tubulin acetylation in brain, )] TJ ET BT 26.250 196.405 Td /F1 9.8 Tf [(cholesterol biosynthesis or the progression of Huntington's disease phenotypes in vivo. PLoS One. 2012;7\(4\):e34805.)] TJ ET Q q 15.000 179.024 577.500 597.976 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 759.976 Td /F1 9.8 Tf [(Valenza M, Leoni V, Tarditi A, Mariotti C, Bjorkhem I, Di Donato S, et al. Progressive dysfunction of the cholesterol )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(biosynthesis pathway in the R6/2 mouse model of Huntington's disease. Neurobiol Dis. 2007 Oct;28\(1\):133-42.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 728.667 Td /F1 9.8 Tf [(Leoni V, Mariotti C, Tabrizi SJ, Valenza M, Wild EJ, Henley SM, et al. Plasma 24S-hydroxycholesterol and caudate MRI in )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(pre-manifest and early Huntington's disease. Brain. 2008 Nov;131\(Pt 11\):2851-9.)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Leoni V, Mariotti C, Nanetti L, Salvatore E, Squitieri F, Bentivoglio AR, et al. Whole body cholesterol metabolism is impaired )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(in Huntington's disease. Neurosci Lett. 2011 May 2;494\(3\):245-9.)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Ritch JJ, Valencia A, Alexander J, Sapp E, Gatune L, Sangrey GR, et al. Multiple phenotypes in Huntington disease mouse )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(neural stem cells. Mol Cell Neurosci. 2012 May;50\(1\):70-81.)] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 634.738 Td /F1 9.8 Tf [(Trushina E, Singh RD, Dyer RB, Cao S, Shah VH, Parton RG, et al. Mutant huntingtin inhibits clathrin-independent )] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(endocytosis and causes accumulation of cholesterol in vitro and in vivo. Hum Mol Genet. 2006 Dec 15;15\(24\):3578-91.)] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 603.429 Td /F1 9.8 Tf [(Luthi-Carter R, Taylor DM, Pallos J, Lambert E, Amore A, Parker A, et al. SIRT2 inhibition achieves neuroprotection by )] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(decreasing sterol biosynthesis. Proc Natl Acad Sci U S A. 2010 Apr 27;107\(17\):7927-32.)] TJ ET BT 26.250 572.119 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 572.119 Td /F1 9.8 Tf [(Del Toro D, Xifro X, Pol A, Humbert S, Saudou F, Canals JM, et al. Altered cholesterol homeostasis contributes to )] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(enhanced excitotoxicity in Huntington's disease. J Neurochem. 2010 Oct;115\(1\):153-67.)] TJ ET BT 26.250 540.810 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 540.810 Td /F1 9.8 Tf [(Valenza M, Leoni V, Tarditi A, Mariotti C, Bjorkhem I, Di Donato S, et al. Progressive dysfunction of the cholesterol )] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(biosynthesis pathway in the R6/2 mouse model of Huntington's disease. Neurobiol Dis. 2007 Oct;28\(1\):133-42.)] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 509.500 Td /F1 9.8 Tf [(Trettel F, Rigamonti D, Hilditch-Maguire P, Wheeler VC, Sharp AH, Persichetti F, et al. Dominant phenotypes produced by )] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(the HD mutation in STHdh\(Q111\) striatal cells. Hum Mol Genet. 2000 Nov 22;9\(19\):2799-809.)] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 478.191 Td /F1 9.8 Tf [(Greenspan P, Mayer EP, Fowler SD. Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol. 1985 )] TJ ET BT 26.250 466.286 Td /F1 9.8 Tf [(Mar;100\(3\):965-73.)] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 446.881 Td /F1 9.8 Tf [(Corvera S, DiBonaventura C, Shpetner HS. Cell confluence-dependent remodeling of endothelial membranes mediated by )] TJ ET BT 26.250 434.976 Td /F1 9.8 Tf [(cholesterol. J Biol Chem. 2000 Oct 6;275\(40\):31414-21.)] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 415.572 Td /F1 9.8 Tf [(Gines S, Ivanova E, Seong IS, Saura CA, MacDonald ME. Enhanced Akt signaling is an early pro-survival response that )] TJ ET BT 26.250 403.667 Td /F1 9.8 Tf [(reflects N-methyl-D-aspartate receptor activation in Huntington's disease knock-in striatal cells. J Biol Chem. 2003 Dec )] TJ ET BT 26.250 391.762 Td /F1 9.8 Tf [(12;278\(50\):50514-22.)] TJ ET BT 26.250 372.357 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 372.357 Td /F1 9.8 Tf [(Demichelis F, Greulich H, Macoska JA, Beroukhim R, Sellers WR, Garraway L, et al. SNP panel identification assay \(SPIA\): )] TJ ET BT 26.250 360.453 Td /F1 9.8 Tf [(a genetic-based assay for the identification of cell lines. Nucleic Acids Res. 2008 Apr;36\(7\):2446-56.)] TJ ET BT 26.250 341.048 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 341.048 Td /F1 9.8 Tf [(Diaferia GR, Cardano M, Cattaneo M, Spinelli CC, Dessi SS, DeBlasio P, et al. The science of stem cell biobanking: )] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(investing in the future. J Cell Physiol. 2012 Jan;227\(1\):14-9.)] TJ ET BT 26.250 309.738 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 309.738 Td /F1 9.8 Tf [(Masters JR. False cell lines: The problem and a solution. Cytotechnology. 2002 Jul;39\(2\):69-74.)] TJ ET BT 26.250 290.334 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 290.334 Td /F1 9.8 Tf [(Lacroix M. Persistent use of "false" cell lines. Int J Cancer. 2008 Jan 1;122\(1\):1-4.)] TJ ET BT 26.250 270.929 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 270.929 Td /F1 9.8 Tf [(Valenza M, Cattaneo E. Neuroprotection and brain cholesterol biosynthesis in Huntington's disease. Proc Natl Acad Sci U S )] TJ ET BT 26.250 259.024 Td /F1 9.8 Tf [(A. 2010 Sep 14;107\(37\):E143; author reply 4.)] TJ ET BT 26.250 239.619 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 239.619 Td /F1 9.8 Tf [(Luthi-Carter R, Kazantsev A. Reply to Valenza and Cattaneo: SIRT2-mediated neuroprotection and cholesterol )] TJ ET BT 26.250 227.715 Td /F1 9.8 Tf [(dyshomeostasis in Huntingtons disease. Proc Natl Acad Sci U S A. 2010;107\(37\):E144.)] TJ ET BT 26.250 208.310 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 208.310 Td /F1 9.8 Tf [(Bobrowska A, Donmez G, Weiss A, Guarente L, Bates G. SIRT2 ablation has no effect on tubulin acetylation in brain, )] TJ ET BT 26.250 196.405 Td /F1 9.8 Tf [(cholesterol biosynthesis or the progression of Huntington's disease phenotypes in vivo. 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