%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 207 0 R 305 0 R 337 0 R 353 0 R 362 0 R 377 0 R 386 0 R 402 0 R 411 0 R 534 0 R 560 0 R 562 0 R ] /Count 13 /Resources << /ProcSet 4 0 R /Font << /F1 8 0 R /F2 9 0 R /F3 10 0 R /F4 11 0 R /F5 12 0 R >> /XObject << /I1 13 0 R /I2 14 0 R /I3 341 0 R /I4 344 0 R /I5 357 0 R /I6 368 0 R /I7 381 0 R /I8 390 0 R /I9 393 0 R /I10 406 0 R /I11 415 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:20180722215038+00'00') /ModDate (D:20180722215038+00'00') /Title (Protection by glia-conditioned medium in a cell model of Huntington disease PLOS Currents Huntington Disease) >> endobj 6 0 obj << /Type /Page /Parent 3 0 R /Annots [ 15 0 R 17 0 R 19 0 R 21 0 R 23 0 R 25 0 R 27 0 R 29 0 R 31 0 R 33 0 R 35 0 R 37 0 R 39 0 R 41 0 R 43 0 R 45 0 R 47 0 R 49 0 R 51 0 R 53 0 R 55 0 R 57 0 R 59 0 R 61 0 R 63 0 R 65 0 R 67 0 R 69 0 R 71 0 R 73 0 R 75 0 R 77 0 R 79 0 R 81 0 R 83 0 R 85 0 R 87 0 R 89 0 R 91 0 R 93 0 R 95 0 R 97 0 R 99 0 R 101 0 R 103 0 R 105 0 R 107 0 R 109 0 R 111 0 R 113 0 R 115 0 R 117 0 R 119 0 R 121 0 R 123 0 R 125 0 R 127 0 R 129 0 R 131 0 R 133 0 R 135 0 R 137 0 R 139 0 R 141 0 R 143 0 R 145 0 R 147 0 R 149 0 R 151 0 R 153 0 R 155 0 R 157 0 R 159 0 R 161 0 R 163 0 R 165 0 R 167 0 R 169 0 R 171 0 R 173 0 R 175 0 R 177 0 R 179 0 R 181 0 R 183 0 R 185 0 R 187 0 R 189 0 R 191 0 R 193 0 R 195 0 R 197 0 R 199 0 R 201 0 R 203 0 R 205 0 R ] /Contents 7 0 R >> endobj 7 0 obj << /Length 31100 >> stream q 375.000 0 0 39.000 222.000 738.000 cm /I2 Do Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Protection by glia-conditioned medium in a cell model of )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Huntington disease)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(July 2, 2012)] TJ ET BT 67.806 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 72.681 675.088 Td /F3 9.8 Tf [(Molecular/Cellular)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Carolina Ruiz)] TJ ET 0.271 0.267 0.267 rg BT 84.760 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 90.181 663.247 Td /F1 9.8 Tf [(Maria Jose Casarejos)] TJ ET 0.271 0.267 0.267 rg BT 184.453 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 189.874 663.247 Td /F1 9.8 Tf [(Ana Gomez)] TJ ET 0.271 0.267 0.267 rg BT 241.354 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 246.775 663.247 Td /F1 9.8 Tf [(Rosa Solano)] TJ ET 0.271 0.267 0.267 rg BT 302.594 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 308.015 663.247 Td /F1 9.8 Tf [(Justo Garcia de Yebenes)] TJ ET 0.271 0.267 0.267 rg BT 417.488 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 422.909 663.247 Td /F1 9.8 Tf [(Maria Angeles Mena)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Ruiz C, Casarejos MJ, Gomez A, Solano R, de Yebenes JG, Mena MA. Protection by glia-conditioned medium in a cell model )] TJ ET BT 570.290 651.342 Td /F1 9.8 Tf [(of )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Huntington disease. PLOS Currents Huntington Disease. 2012 Jul 2 . Edition 1. doi: 10.1371/4fbca54a2028b.)] TJ ET q 15.000 26.911 577.500 610.146 re W n 0.271 0.267 0.267 rg BT 26.250 610.335 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 590.381 Td /F1 9.8 Tf [(The physiological role of huntingtin and the pathogenic mechanisms that produce the disease are unknown. Mutant huntingtin )] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(changes its normal localization and produces cytoplasmic and intranuclear inclusions, changes gene transcription, alters )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(synaptic transmission, impairs mitochondrial activity and activates caspases and other pro-apoptotic molecules, promotes )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(excitotoxicity, energy deficits, synthesis and release reduction of neurotrophic factors and oxidative stress. Previous studies )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(confirm that the mutant huntingtin difficult neurotrophic function of astrocytes leading to neuronal dysfunction in Huntingtons )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(disease. Our objective was to study the neuroprotective potential role of glia-conditioned medium \(GCM\) in an in vitro model of )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(Huntingtons disease. We used conditionally-immortalized striatal neuronal progenitor cell lines \(STHdhQ7/Q7 and )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(STHdhQ111/Q111\) expressing endogenous levels of normal and mutant huntingtin with 7 and 111 glutamines, respectively. We )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(studied the protection of fetal and postnatal glia conditioned medium \(GCM\) on H2O2 \(2 M\), glutamate \(5 mM\) and 3-)] TJ ET BT 26.250 483.238 Td /F1 9.8 Tf [(nitropropionic acid \(2.5 mM\) related toxicity. We also compared the neuroprotective effects of GCM versus that of the growth )] TJ ET BT 26.250 471.333 Td /F1 9.8 Tf [(factors bFGF, BDNF and GDNF.)] TJ ET BT 26.250 459.429 Td /F1 9.8 Tf [(Fetal GCM protects from every toxin, reducing the cell death and increasing the cell survival. Fetal GCM reduces the caspases )] TJ ET BT 26.250 447.524 Td /F1 9.8 Tf [(fragmentation of the protein PARP, the expression of chaperone Hsp70 and the accumulation of ROS and polyubiquitinated )] TJ ET BT 26.250 435.619 Td /F1 9.8 Tf [(proteins. In addition, in Q111 striatal cells treated with H2O2 \(2 M\) for 24 hours, the intracellular GSH levels are higher in the )] TJ ET BT 26.250 423.714 Td /F1 9.8 Tf [(presence of GCM. Notably, the 13-day and 2-month postnatal GCM, totally protects from H2O2 induced cell death in mutant )] TJ ET BT 26.250 411.810 Td /F1 9.8 Tf [(striatal cells. GCM neuroprotective effects are more potent than those of the already identified neurotrophic factors.)] TJ ET BT 26.250 399.905 Td /F1 9.8 Tf [(We conclude that GCM protects Q111 cells from neuronal neurotoxins and the effects of GCM are more potent than those of )] TJ ET BT 26.250 388.000 Td /F1 9.8 Tf [(any known neurotrophic factor. GCM may contain new and more potent, as yet unidentified, neurotrophic molecules, potentially )] TJ ET BT 26.250 376.095 Td /F1 9.8 Tf [(useful in patients with Huntingtons disease.)] TJ ET BT 26.250 339.493 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 319.539 Td /F1 9.8 Tf [(This work has been supported by the Spanish Ministry of Health, FIS 2010/172, CIBERNED 2006/05/0059, CIBERNED PI2010-)] TJ ET BT 26.250 307.634 Td /F1 9.8 Tf [(6, MINICIN 2011 and CAM 2011/BMD-2308.)] TJ ET BT 26.250 278.531 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 258.577 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an autosomal dominant disorder characterized by progressive motor, cognitive and psychiatric )] TJ ET BT 26.250 246.672 Td /F1 9.8 Tf [(disturbances )] TJ ET 0.267 0.267 0.267 rg BT 84.233 248.180 Td /F4 8.7 Tf [(1)] TJ ET BT 89.052 248.180 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 93.871 246.672 Td /F1 9.8 Tf [( associated to predominant neuronal loss in the striatum and other brain areas )] TJ ET 0.267 0.267 0.267 rg BT 433.658 248.180 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 438.477 246.672 Td /F1 9.8 Tf [(. HD is caused by an expanded )] TJ ET BT 26.250 234.768 Td /F1 9.8 Tf [(chain of polyglutamines localized in the N-terminal region of the huntingtin protein)] TJ ET 0.267 0.267 0.267 rg BT 377.406 236.275 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 382.225 234.768 Td /F1 9.8 Tf [( . The physiological role of huntingtin and the )] TJ ET BT 26.250 222.863 Td /F1 9.8 Tf [(pathogenic mechanisms that produce the disease are unknown. Mutant huntingtin produces fibrillary deposits; changes its )] TJ ET BT 26.250 210.958 Td /F1 9.8 Tf [(normal localization, produces cytoplasmic and intranuclear inclusions; changes gene transcription; alters synaptic transmission; )] TJ ET BT 26.250 199.053 Td /F1 9.8 Tf [(impairs mitochondrial activity and activates caspases and other pro-apoptotic molecules )] TJ ET 0.267 0.267 0.267 rg BT 407.728 200.561 Td /F4 8.7 Tf [(5)] TJ ET BT 412.547 200.561 Td /F4 8.7 Tf [(6)] TJ ET BT 417.366 200.561 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 422.185 199.053 Td /F1 9.8 Tf [(. The fact that the localization of )] TJ ET BT 26.250 187.149 Td /F1 9.8 Tf [(huntingtin is ubiquitous, while there is a lesional gradient with more severe involvement of the striatum, suggests that some )] TJ ET BT 26.250 175.244 Td /F1 9.8 Tf [(regional factors, such as synaptic connections and local metabolic rates play a role in neuronal degeneration.)] TJ ET BT 26.250 155.839 Td /F1 9.8 Tf [(Glutamate is considered to play a pathogenic role in HD mediated by metabotropic, and perhaps other, receptors )] TJ ET 0.267 0.267 0.267 rg BT 515.602 157.346 Td /F4 8.7 Tf [(8)] TJ ET BT 520.421 157.346 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 525.240 155.839 Td /F1 9.8 Tf [(. Reactive )] TJ ET BT 26.250 143.934 Td /F1 9.8 Tf [(oxygen species \(ROS\) may play an important role in HD )] TJ ET 0.267 0.267 0.267 rg BT 271.160 145.442 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 280.798 143.934 Td /F1 9.8 Tf [( and may be the initial molecules leading to apoptosis. Hydrogen )] TJ ET BT 26.250 132.030 Td /F1 9.8 Tf [(peroxide \(H)] TJ ET BT 76.638 129.965 Td /F1 8.7 Tf [(2)] TJ ET BT 81.457 132.030 Td /F1 9.8 Tf [(O)] TJ ET BT 89.042 129.965 Td /F1 8.7 Tf [(2)] TJ ET BT 93.861 132.030 Td /F1 9.8 Tf [(\) is one of the major oxygen species which mediates cytotoxicity )] TJ ET 0.267 0.267 0.267 rg BT 372.925 133.537 Td /F4 8.7 Tf [(11)] TJ ET BT 382.563 133.537 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 392.200 132.030 Td /F1 9.8 Tf [(. Overproduction of ROS contributes to )] TJ ET BT 26.250 120.125 Td /F1 9.8 Tf [(deregulation of transmitter release by increasing the extracellular level of glutamate and monoamine transmitters, which act as )] TJ ET BT 26.250 108.220 Td /F1 9.8 Tf [(triggers of deleterious cellular events leading to neurodegeneration )] TJ ET 0.267 0.267 0.267 rg BT 317.268 109.727 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 326.905 108.220 Td /F1 9.8 Tf [(. In addition, mitochondria play a key role in the regulation )] TJ ET BT 26.250 96.315 Td /F1 9.8 Tf [(of neuronal aging and activity of proteins related to neuronal death )] TJ ET 0.267 0.267 0.267 rg BT 315.123 97.823 Td /F4 8.7 Tf [(14)] TJ ET BT 324.760 97.823 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 334.398 96.315 Td /F1 9.8 Tf [(. 3-Nitropropionic acid \(3-NP\) is a natural toxin that )] TJ ET BT 26.250 84.411 Td /F1 9.8 Tf [(irreversibly inhibits the succinate dehydrogenase enzyme, which is the main constituent of mitochondrial chain complex II. In )] TJ ET BT 26.250 72.506 Td /F1 9.8 Tf [(rodents, 3-NP produces striatal lesions, mostly involving medium-sized spiny projection neurons; a pattern of lesions very )] TJ ET BT 26.250 60.601 Td /F1 9.8 Tf [(similar to those of patients with HD )] TJ ET 0.267 0.267 0.267 rg BT 179.052 62.108 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 188.689 60.601 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 41.196 Td /F1 9.8 Tf [(Recent studies have revealed the role of glia in different neurodegenerative diseases, including HD )] TJ ET 0.267 0.267 0.267 rg BT 455.455 42.704 Td /F4 8.7 Tf [(12)] TJ ET BT 465.092 42.704 Td /F4 8.7 Tf [(16)] TJ ET BT 474.729 42.704 Td /F4 8.7 Tf [(17)] TJ ET BT 484.367 42.704 Td /F4 8.7 Tf [(18)] TJ ET BT 494.004 42.704 Td /F4 8.7 Tf [(19)] TJ ET BT 503.641 42.704 Td /F4 8.7 Tf [(20)] TJ ET BT 513.279 42.704 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 522.916 41.196 Td /F1 9.8 Tf [(. )] TJ ET BT 528.337 41.196 Td /F5 9.8 Tf [(In vitro)] TJ ET BT 557.597 41.196 Td /F1 9.8 Tf [(, )] TJ ET Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Protection by glia-conditioned medium in a cell model of )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Huntington disease)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(July 2, 2012)] TJ ET BT 67.806 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 72.681 675.088 Td /F3 9.8 Tf [(Molecular/Cellular)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Carolina Ruiz)] TJ ET 0.271 0.267 0.267 rg BT 84.760 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 90.181 663.247 Td /F1 9.8 Tf [(Maria Jose Casarejos)] TJ ET 0.271 0.267 0.267 rg BT 184.453 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 189.874 663.247 Td /F1 9.8 Tf [(Ana Gomez)] TJ ET 0.271 0.267 0.267 rg BT 241.354 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 246.775 663.247 Td /F1 9.8 Tf [(Rosa Solano)] TJ ET 0.271 0.267 0.267 rg BT 302.594 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 308.015 663.247 Td /F1 9.8 Tf [(Justo Garcia de Yebenes)] TJ ET 0.271 0.267 0.267 rg BT 417.488 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 422.909 663.247 Td /F1 9.8 Tf [(Maria Angeles Mena)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Ruiz C, Casarejos MJ, Gomez A, Solano R, de Yebenes JG, Mena MA. Protection by glia-conditioned medium in a cell model )] TJ ET BT 570.290 651.342 Td /F1 9.8 Tf [(of )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Huntington disease. PLOS Currents Huntington Disease. 2012 Jul 2 . Edition 1. doi: 10.1371/4fbca54a2028b.)] TJ ET q 15.000 26.911 577.500 610.146 re W n 0.271 0.267 0.267 rg BT 26.250 610.335 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 590.381 Td /F1 9.8 Tf [(The physiological role of huntingtin and the pathogenic mechanisms that produce the disease are unknown. Mutant huntingtin )] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(changes its normal localization and produces cytoplasmic and intranuclear inclusions, changes gene transcription, alters )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(synaptic transmission, impairs mitochondrial activity and activates caspases and other pro-apoptotic molecules, promotes )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(excitotoxicity, energy deficits, synthesis and release reduction of neurotrophic factors and oxidative stress. Previous studies )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(confirm that the mutant huntingtin difficult neurotrophic function of astrocytes leading to neuronal dysfunction in Huntingtons )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(disease. Our objective was to study the neuroprotective potential role of glia-conditioned medium \(GCM\) in an in vitro model of )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(Huntingtons disease. We used conditionally-immortalized striatal neuronal progenitor cell lines \(STHdhQ7/Q7 and )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(STHdhQ111/Q111\) expressing endogenous levels of normal and mutant huntingtin with 7 and 111 glutamines, respectively. We )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(studied the protection of fetal and postnatal glia conditioned medium \(GCM\) on H2O2 \(2 M\), glutamate \(5 mM\) and 3-)] TJ ET BT 26.250 483.238 Td /F1 9.8 Tf [(nitropropionic acid \(2.5 mM\) related toxicity. We also compared the neuroprotective effects of GCM versus that of the growth )] TJ ET BT 26.250 471.333 Td /F1 9.8 Tf [(factors bFGF, BDNF and GDNF.)] TJ ET BT 26.250 459.429 Td /F1 9.8 Tf [(Fetal GCM protects from every toxin, reducing the cell death and increasing the cell survival. Fetal GCM reduces the caspases )] TJ ET BT 26.250 447.524 Td /F1 9.8 Tf [(fragmentation of the protein PARP, the expression of chaperone Hsp70 and the accumulation of ROS and polyubiquitinated )] TJ ET BT 26.250 435.619 Td /F1 9.8 Tf [(proteins. In addition, in Q111 striatal cells treated with H2O2 \(2 M\) for 24 hours, the intracellular GSH levels are higher in the )] TJ ET BT 26.250 423.714 Td /F1 9.8 Tf [(presence of GCM. Notably, the 13-day and 2-month postnatal GCM, totally protects from H2O2 induced cell death in mutant )] TJ ET BT 26.250 411.810 Td /F1 9.8 Tf [(striatal cells. GCM neuroprotective effects are more potent than those of the already identified neurotrophic factors.)] TJ ET BT 26.250 399.905 Td /F1 9.8 Tf [(We conclude that GCM protects Q111 cells from neuronal neurotoxins and the effects of GCM are more potent than those of )] TJ ET BT 26.250 388.000 Td /F1 9.8 Tf [(any known neurotrophic factor. GCM may contain new and more potent, as yet unidentified, neurotrophic molecules, potentially )] TJ ET BT 26.250 376.095 Td /F1 9.8 Tf [(useful in patients with Huntingtons disease.)] TJ ET BT 26.250 339.493 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 319.539 Td /F1 9.8 Tf [(This work has been supported by the Spanish Ministry of Health, FIS 2010/172, CIBERNED 2006/05/0059, CIBERNED PI2010-)] TJ ET BT 26.250 307.634 Td /F1 9.8 Tf [(6, MINICIN 2011 and CAM 2011/BMD-2308.)] TJ ET BT 26.250 278.531 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 258.577 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an autosomal dominant disorder characterized by progressive motor, cognitive and psychiatric )] TJ ET BT 26.250 246.672 Td /F1 9.8 Tf [(disturbances )] TJ ET 0.267 0.267 0.267 rg BT 84.233 248.180 Td /F4 8.7 Tf [(1)] TJ ET BT 89.052 248.180 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 93.871 246.672 Td /F1 9.8 Tf [( associated to predominant neuronal loss in the striatum and other brain areas )] TJ ET 0.267 0.267 0.267 rg BT 433.658 248.180 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 438.477 246.672 Td /F1 9.8 Tf [(. HD is caused by an expanded )] TJ ET BT 26.250 234.768 Td /F1 9.8 Tf [(chain of polyglutamines localized in the N-terminal region of the huntingtin protein)] TJ ET 0.267 0.267 0.267 rg BT 377.406 236.275 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 382.225 234.768 Td /F1 9.8 Tf [( . The physiological role of huntingtin and the )] TJ ET BT 26.250 222.863 Td /F1 9.8 Tf [(pathogenic mechanisms that produce the disease are unknown. Mutant huntingtin produces fibrillary deposits; changes its )] TJ ET BT 26.250 210.958 Td /F1 9.8 Tf [(normal localization, produces cytoplasmic and intranuclear inclusions; changes gene transcription; alters synaptic transmission; )] TJ ET BT 26.250 199.053 Td /F1 9.8 Tf [(impairs mitochondrial activity and activates caspases and other pro-apoptotic molecules )] TJ ET 0.267 0.267 0.267 rg BT 407.728 200.561 Td /F4 8.7 Tf [(5)] TJ ET BT 412.547 200.561 Td /F4 8.7 Tf [(6)] TJ ET BT 417.366 200.561 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 422.185 199.053 Td /F1 9.8 Tf [(. The fact that the localization of )] TJ ET BT 26.250 187.149 Td /F1 9.8 Tf [(huntingtin is ubiquitous, while there is a lesional gradient with more severe involvement of the striatum, suggests that some )] TJ ET BT 26.250 175.244 Td /F1 9.8 Tf [(regional factors, such as synaptic connections and local metabolic rates play a role in neuronal degeneration.)] TJ ET BT 26.250 155.839 Td /F1 9.8 Tf [(Glutamate is considered to play a pathogenic role in HD mediated by metabotropic, and perhaps other, receptors )] TJ ET 0.267 0.267 0.267 rg BT 515.602 157.346 Td /F4 8.7 Tf [(8)] TJ ET BT 520.421 157.346 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 525.240 155.839 Td /F1 9.8 Tf [(. Reactive )] TJ ET BT 26.250 143.934 Td /F1 9.8 Tf [(oxygen species \(ROS\) may play an important role in HD )] TJ ET 0.267 0.267 0.267 rg BT 271.160 145.442 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 280.798 143.934 Td /F1 9.8 Tf [( and may be the initial molecules leading to apoptosis. Hydrogen )] TJ ET BT 26.250 132.030 Td /F1 9.8 Tf [(peroxide \(H)] TJ ET BT 76.638 129.965 Td /F1 8.7 Tf [(2)] TJ ET BT 81.457 132.030 Td /F1 9.8 Tf [(O)] TJ ET BT 89.042 129.965 Td /F1 8.7 Tf [(2)] TJ ET BT 93.861 132.030 Td /F1 9.8 Tf [(\) is one of the major oxygen species which mediates cytotoxicity )] TJ ET 0.267 0.267 0.267 rg BT 372.925 133.537 Td /F4 8.7 Tf [(11)] TJ ET BT 382.563 133.537 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 392.200 132.030 Td /F1 9.8 Tf [(. Overproduction of ROS contributes to )] TJ ET BT 26.250 120.125 Td /F1 9.8 Tf [(deregulation of transmitter release by increasing the extracellular level of glutamate and monoamine transmitters, which act as )] TJ ET BT 26.250 108.220 Td /F1 9.8 Tf [(triggers of deleterious cellular events leading to neurodegeneration )] TJ ET 0.267 0.267 0.267 rg BT 317.268 109.727 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 326.905 108.220 Td /F1 9.8 Tf [(. In addition, mitochondria play a key role in the regulation )] TJ ET BT 26.250 96.315 Td /F1 9.8 Tf [(of neuronal aging and activity of proteins related to neuronal death )] TJ ET 0.267 0.267 0.267 rg BT 315.123 97.823 Td /F4 8.7 Tf [(14)] TJ ET BT 324.760 97.823 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 334.398 96.315 Td /F1 9.8 Tf [(. 3-Nitropropionic acid \(3-NP\) is a natural toxin that )] TJ ET BT 26.250 84.411 Td /F1 9.8 Tf [(irreversibly inhibits the succinate dehydrogenase enzyme, which is the main constituent of mitochondrial chain complex II. In )] TJ ET BT 26.250 72.506 Td /F1 9.8 Tf [(rodents, 3-NP produces striatal lesions, mostly involving medium-sized spiny projection neurons; a pattern of lesions very )] TJ ET BT 26.250 60.601 Td /F1 9.8 Tf [(similar to those of patients with HD )] TJ ET 0.267 0.267 0.267 rg BT 179.052 62.108 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 188.689 60.601 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 41.196 Td /F1 9.8 Tf [(Recent studies have revealed the role of glia in different neurodegenerative diseases, including HD )] TJ ET 0.267 0.267 0.267 rg BT 455.455 42.704 Td /F4 8.7 Tf [(12)] TJ ET BT 465.092 42.704 Td /F4 8.7 Tf [(16)] TJ ET BT 474.729 42.704 Td /F4 8.7 Tf [(17)] TJ ET BT 484.367 42.704 Td /F4 8.7 Tf [(18)] TJ ET BT 494.004 42.704 Td /F4 8.7 Tf [(19)] TJ ET BT 503.641 42.704 Td /F4 8.7 Tf [(20)] TJ ET BT 513.279 42.704 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 522.916 41.196 Td /F1 9.8 Tf [(. )] TJ ET BT 528.337 41.196 Td /F5 9.8 Tf [(In vitro)] TJ ET BT 557.597 41.196 Td /F1 9.8 Tf [(, )] TJ ET Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Protection by glia-conditioned medium in a cell model of )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Huntington disease)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(July 2, 2012)] TJ ET BT 67.806 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 72.681 675.088 Td /F3 9.8 Tf [(Molecular/Cellular)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Carolina Ruiz)] TJ ET 0.271 0.267 0.267 rg BT 84.760 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 90.181 663.247 Td /F1 9.8 Tf [(Maria Jose Casarejos)] TJ ET 0.271 0.267 0.267 rg BT 184.453 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 189.874 663.247 Td /F1 9.8 Tf [(Ana Gomez)] TJ ET 0.271 0.267 0.267 rg BT 241.354 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 246.775 663.247 Td /F1 9.8 Tf [(Rosa Solano)] TJ ET 0.271 0.267 0.267 rg BT 302.594 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 308.015 663.247 Td /F1 9.8 Tf [(Justo Garcia de Yebenes)] TJ ET 0.271 0.267 0.267 rg BT 417.488 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 422.909 663.247 Td /F1 9.8 Tf [(Maria Angeles Mena)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Ruiz C, Casarejos MJ, Gomez A, Solano R, de Yebenes JG, Mena MA. Protection by glia-conditioned medium in a cell model )] TJ ET BT 570.290 651.342 Td /F1 9.8 Tf [(of )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Huntington disease. PLOS Currents Huntington Disease. 2012 Jul 2 . Edition 1. doi: 10.1371/4fbca54a2028b.)] TJ ET q 15.000 26.911 577.500 610.146 re W n 0.271 0.267 0.267 rg BT 26.250 610.335 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 590.381 Td /F1 9.8 Tf [(The physiological role of huntingtin and the pathogenic mechanisms that produce the disease are unknown. Mutant huntingtin )] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(changes its normal localization and produces cytoplasmic and intranuclear inclusions, changes gene transcription, alters )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(synaptic transmission, impairs mitochondrial activity and activates caspases and other pro-apoptotic molecules, promotes )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(excitotoxicity, energy deficits, synthesis and release reduction of neurotrophic factors and oxidative stress. Previous studies )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(confirm that the mutant huntingtin difficult neurotrophic function of astrocytes leading to neuronal dysfunction in Huntingtons )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(disease. Our objective was to study the neuroprotective potential role of glia-conditioned medium \(GCM\) in an in vitro model of )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(Huntingtons disease. We used conditionally-immortalized striatal neuronal progenitor cell lines \(STHdhQ7/Q7 and )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(STHdhQ111/Q111\) expressing endogenous levels of normal and mutant huntingtin with 7 and 111 glutamines, respectively. We )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(studied the protection of fetal and postnatal glia conditioned medium \(GCM\) on H2O2 \(2 M\), glutamate \(5 mM\) and 3-)] TJ ET BT 26.250 483.238 Td /F1 9.8 Tf [(nitropropionic acid \(2.5 mM\) related toxicity. We also compared the neuroprotective effects of GCM versus that of the growth )] TJ ET BT 26.250 471.333 Td /F1 9.8 Tf [(factors bFGF, BDNF and GDNF.)] TJ ET BT 26.250 459.429 Td /F1 9.8 Tf [(Fetal GCM protects from every toxin, reducing the cell death and increasing the cell survival. Fetal GCM reduces the caspases )] TJ ET BT 26.250 447.524 Td /F1 9.8 Tf [(fragmentation of the protein PARP, the expression of chaperone Hsp70 and the accumulation of ROS and polyubiquitinated )] TJ ET BT 26.250 435.619 Td /F1 9.8 Tf [(proteins. In addition, in Q111 striatal cells treated with H2O2 \(2 M\) for 24 hours, the intracellular GSH levels are higher in the )] TJ ET BT 26.250 423.714 Td /F1 9.8 Tf [(presence of GCM. Notably, the 13-day and 2-month postnatal GCM, totally protects from H2O2 induced cell death in mutant )] TJ ET BT 26.250 411.810 Td /F1 9.8 Tf [(striatal cells. GCM neuroprotective effects are more potent than those of the already identified neurotrophic factors.)] TJ ET BT 26.250 399.905 Td /F1 9.8 Tf [(We conclude that GCM protects Q111 cells from neuronal neurotoxins and the effects of GCM are more potent than those of )] TJ ET BT 26.250 388.000 Td /F1 9.8 Tf [(any known neurotrophic factor. GCM may contain new and more potent, as yet unidentified, neurotrophic molecules, potentially )] TJ ET BT 26.250 376.095 Td /F1 9.8 Tf [(useful in patients with Huntingtons disease.)] TJ ET BT 26.250 339.493 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 319.539 Td /F1 9.8 Tf [(This work has been supported by the Spanish Ministry of Health, FIS 2010/172, CIBERNED 2006/05/0059, CIBERNED PI2010-)] TJ ET BT 26.250 307.634 Td /F1 9.8 Tf [(6, MINICIN 2011 and CAM 2011/BMD-2308.)] TJ ET BT 26.250 278.531 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 258.577 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an autosomal dominant disorder characterized by progressive motor, cognitive and psychiatric )] TJ ET BT 26.250 246.672 Td /F1 9.8 Tf [(disturbances )] TJ ET 0.267 0.267 0.267 rg BT 84.233 248.180 Td /F4 8.7 Tf [(1)] TJ ET BT 89.052 248.180 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 93.871 246.672 Td /F1 9.8 Tf [( associated to predominant neuronal loss in the striatum and other brain areas )] TJ ET 0.267 0.267 0.267 rg BT 433.658 248.180 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 438.477 246.672 Td /F1 9.8 Tf [(. HD is caused by an expanded )] TJ ET BT 26.250 234.768 Td /F1 9.8 Tf [(chain of polyglutamines localized in the N-terminal region of the huntingtin protein)] TJ ET 0.267 0.267 0.267 rg BT 377.406 236.275 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 382.225 234.768 Td /F1 9.8 Tf [( . The physiological role of huntingtin and the )] TJ ET BT 26.250 222.863 Td /F1 9.8 Tf [(pathogenic mechanisms that produce the disease are unknown. Mutant huntingtin produces fibrillary deposits; changes its )] TJ ET BT 26.250 210.958 Td /F1 9.8 Tf [(normal localization, produces cytoplasmic and intranuclear inclusions; changes gene transcription; alters synaptic transmission; )] TJ ET BT 26.250 199.053 Td /F1 9.8 Tf [(impairs mitochondrial activity and activates caspases and other pro-apoptotic molecules )] TJ ET 0.267 0.267 0.267 rg BT 407.728 200.561 Td /F4 8.7 Tf [(5)] TJ ET BT 412.547 200.561 Td /F4 8.7 Tf [(6)] TJ ET BT 417.366 200.561 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 422.185 199.053 Td /F1 9.8 Tf [(. The fact that the localization of )] TJ ET BT 26.250 187.149 Td /F1 9.8 Tf [(huntingtin is ubiquitous, while there is a lesional gradient with more severe involvement of the striatum, suggests that some )] TJ ET BT 26.250 175.244 Td /F1 9.8 Tf [(regional factors, such as synaptic connections and local metabolic rates play a role in neuronal degeneration.)] TJ ET BT 26.250 155.839 Td /F1 9.8 Tf [(Glutamate is considered to play a pathogenic role in HD mediated by metabotropic, and perhaps other, receptors )] TJ ET 0.267 0.267 0.267 rg BT 515.602 157.346 Td /F4 8.7 Tf [(8)] TJ ET BT 520.421 157.346 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 525.240 155.839 Td /F1 9.8 Tf [(. Reactive )] TJ ET BT 26.250 143.934 Td /F1 9.8 Tf [(oxygen species \(ROS\) may play an important role in HD )] TJ ET 0.267 0.267 0.267 rg BT 271.160 145.442 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 280.798 143.934 Td /F1 9.8 Tf [( and may be the initial molecules leading to apoptosis. Hydrogen )] TJ ET BT 26.250 132.030 Td /F1 9.8 Tf [(peroxide \(H)] TJ ET BT 76.638 129.965 Td /F1 8.7 Tf [(2)] TJ ET BT 81.457 132.030 Td /F1 9.8 Tf [(O)] TJ ET BT 89.042 129.965 Td /F1 8.7 Tf [(2)] TJ ET BT 93.861 132.030 Td /F1 9.8 Tf [(\) is one of the major oxygen species which mediates cytotoxicity )] TJ ET 0.267 0.267 0.267 rg BT 372.925 133.537 Td /F4 8.7 Tf [(11)] TJ ET BT 382.563 133.537 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 392.200 132.030 Td /F1 9.8 Tf [(. Overproduction of ROS contributes to )] TJ ET BT 26.250 120.125 Td /F1 9.8 Tf [(deregulation of transmitter release by increasing the extracellular level of glutamate and monoamine transmitters, which act as )] TJ ET BT 26.250 108.220 Td /F1 9.8 Tf [(triggers of deleterious cellular events leading to neurodegeneration )] TJ ET 0.267 0.267 0.267 rg BT 317.268 109.727 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 326.905 108.220 Td /F1 9.8 Tf [(. In addition, mitochondria play a key role in the regulation )] TJ ET BT 26.250 96.315 Td /F1 9.8 Tf [(of neuronal aging and activity of proteins related to neuronal death )] TJ ET 0.267 0.267 0.267 rg BT 315.123 97.823 Td /F4 8.7 Tf [(14)] TJ ET BT 324.760 97.823 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 334.398 96.315 Td /F1 9.8 Tf [(. 3-Nitropropionic acid \(3-NP\) is a natural toxin that )] TJ ET BT 26.250 84.411 Td /F1 9.8 Tf [(irreversibly inhibits the succinate dehydrogenase enzyme, which is the main constituent of mitochondrial chain complex II. In )] TJ ET BT 26.250 72.506 Td /F1 9.8 Tf [(rodents, 3-NP produces striatal lesions, mostly involving medium-sized spiny projection neurons; a pattern of lesions very )] TJ ET BT 26.250 60.601 Td /F1 9.8 Tf [(similar to those of patients with HD )] TJ ET 0.267 0.267 0.267 rg BT 179.052 62.108 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 188.689 60.601 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 41.196 Td /F1 9.8 Tf [(Recent studies have revealed the role of glia in different neurodegenerative diseases, including HD )] TJ ET 0.267 0.267 0.267 rg BT 455.455 42.704 Td /F4 8.7 Tf [(12)] TJ ET BT 465.092 42.704 Td /F4 8.7 Tf [(16)] TJ ET BT 474.729 42.704 Td /F4 8.7 Tf [(17)] TJ ET BT 484.367 42.704 Td /F4 8.7 Tf [(18)] TJ ET BT 494.004 42.704 Td /F4 8.7 Tf [(19)] TJ ET BT 503.641 42.704 Td /F4 8.7 Tf [(20)] TJ ET BT 513.279 42.704 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 522.916 41.196 Td /F1 9.8 Tf [(. )] TJ ET BT 528.337 41.196 Td /F5 9.8 Tf [(In vitro)] TJ ET BT 557.597 41.196 Td /F1 9.8 Tf [(, )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(1)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 8 0 obj << /Type /Font /Subtype /Type1 /Name /F1 /BaseFont /Helvetica /Encoding /WinAnsiEncoding >> endobj 9 0 obj << /Type /Font /Subtype /Type1 /Name /F2 /BaseFont /Times-Bold /Encoding /WinAnsiEncoding >> endobj 10 0 obj << /Type /Font /Subtype /Type1 /Name /F3 /BaseFont /Times-Italic /Encoding /WinAnsiEncoding >> endobj 11 0 obj << /Type /Font /Subtype /Type1 /Name /F4 /BaseFont /Helvetica-Bold /Encoding /WinAnsiEncoding >> endobj 12 0 obj << /Type /Font /Subtype /Type1 /Name /F5 /BaseFont /Helvetica-Oblique /Encoding /WinAnsiEncoding >> endobj 13 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 1 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceGray /BitsPerComponent 8 /Length 144>> stream x1 0 'ݲ؎"e{dzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAtlM0\ endstream endobj 14 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /SMask 13 0 R /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 3 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceRGB /BitsPerComponent 8 /Length 4223>> stream xە8=,8C#h!hGv#0=j$q1uaNĥT*J&a_Zaa0 a0 a0 H^UUUUX!0 3SY|^G0 ÌÔ{]wa_>"OeYaG!8e}a6̖{4M&d:"qʲ\QUUMS<ϛNld2l6Z a<=2B(2vK7M\.GW0 rrWWavߖe9C?Eq8Gqy~~}_qdϽ(neYʒGjK4EQNjͷm{X?|7m۶mqP:Ȳp8Kmu]lNzuZ,NTeY"jlUUQW\,aRiVbȗLkHkEsjd.L6=Iו P]1t7Sc2uIuMM; K5>A$Ǒ$I׋nY 0_Hat;aq䃰U$nR ,zO7bT mhhԫܻu1F*qm'P}ܙ<ϕALO6Lw3nB.T4co{߽.lV $laԀIu!gwChxJ]Ne (+M"d-Eqc=P ! 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We chose the cell line )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(STHdh)] TJ ET BT 56.592 704.341 Td /F1 8.7 Tf [(Q111/111)] TJ ET BT 94.656 700.452 Td /F1 9.8 Tf [( \(Q111\), untreated or challenged with striatal neurotoxins, to model the molecular and cellular events that take )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(place in HD. These cells are striatal progenitor cells from knock-in mouse models of HD, expressing expanded human )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(huntingtin at baseline )] TJ ET 0.267 0.267 0.267 rg BT 121.098 678.150 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 130.735 676.643 Td /F1 9.8 Tf [(. These cells also reproduce several features observed in HD and in mouse models of this disease. )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(Q111 cells exhibit impaired energy metabolism, reduced ATP and ATP/ADP ratio )] TJ ET 0.267 0.267 0.267 rg BT 378.469 666.245 Td /F4 8.7 Tf [(25)] TJ ET BT 388.106 666.245 Td /F4 8.7 Tf [(26)] TJ ET BT 397.743 666.245 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 407.381 664.738 Td /F1 9.8 Tf [(, as well as increased sensitivity to 3-)] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(NP )] TJ ET 0.267 0.267 0.267 rg BT 42.503 654.341 Td /F4 8.7 Tf [(28)] TJ ET BT 52.141 654.341 Td /F4 8.7 Tf [(29)] TJ ET 0.271 0.267 0.267 rg BT 61.778 652.833 Td /F1 9.8 Tf [( and respiratory chain defects )] TJ ET 0.267 0.267 0.267 rg BT 192.379 654.341 Td /F4 8.7 Tf [(30)] TJ ET 0.271 0.267 0.267 rg BT 202.017 652.833 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(In this work, we have tested if fetal and postnatal mice GCM protected Q111 neurons from spontaneous cell death as well as )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(from exposure to glutamate, oxidative damage and mitochondrial toxins.)] TJ ET BT 26.250 602.119 Td /F4 9.8 Tf [(Material and methods)] TJ ET BT 26.250 582.714 Td /F4 9.8 Tf [(Culture Media. )] TJ ET BT 96.138 582.714 Td /F1 9.8 Tf [(Dulbeccos modified Eagles medium \(DMEM\) with high glucose \(4.5 g/liter\) was from Sigma \(Madrid, Spain\), )] TJ ET BT 26.250 570.810 Td /F1 9.8 Tf [(Hams F12 nutrient mixture, Eagles minimal essential medium \(EMEM\) with Earls salts, Leibovitzs L-15 medium; and )] TJ ET BT 26.250 558.905 Td /F1 9.8 Tf [(supplements as L-glutamine, fetal bovine serum \(FBS\), sodium pyruvate, geneticin and L-glutamine, purchased from Invitrogen )] TJ ET BT 26.250 547.000 Td /F1 9.8 Tf [(\(Madrid, Spain\). Glucose 45 %, trypsin-EDTA, insulin, putrescine, progesterone, and sodium selenite were from Sigma \(Madrid, )] TJ ET BT 26.250 535.095 Td /F1 9.8 Tf [(Spain\), and human transferrin, 30 % iron saturated, was from Boehringer- Mannheim \(Barcelona, Spain\).)] TJ ET BT 26.250 515.691 Td /F4 9.8 Tf [(Neuronal and glial cultures. )] TJ ET BT 156.832 515.691 Td /F1 9.8 Tf [(Conditionally immortalized, STHdh)] TJ ET BT 306.368 519.579 Td /F1 8.7 Tf [(Q7/Q7)] TJ ET BT 331.900 515.691 Td /F1 9.8 Tf [( \(Q7\), and Q111 striatal neuronal progenitor cell lines )] TJ ET BT 26.250 503.786 Td /F1 9.8 Tf [(expressing endogenous levels of huntingtin with 7 and 111 glutamines, respectively, were kindly provided by Prof. Alberch )] TJ ET BT 26.250 491.881 Td /F1 9.8 Tf [(\(Universitat de Barcelona, Spain\) and Prof. Marcy E. Macdonald \(Massachusetts General Hospital, Boston, USA\) and were )] TJ ET BT 26.250 479.976 Td /F1 9.8 Tf [(described previously by Trettel)] TJ ET 0.267 0.267 0.267 rg BT 158.460 481.484 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 168.097 479.976 Td /F1 9.8 Tf [( . Striatal cells were grown at 33 C in Dulbeccos Modified Eagles Medium \(DMEM\), )] TJ ET BT 26.250 468.072 Td /F1 9.8 Tf [(supplemented with 10 % fetal bovine serum \(FBS\), 1 % streptomycin-penicillin, 2 mM L-glutamine, 1 mM sodium pyruvate, and )] TJ ET BT 26.250 456.167 Td /F1 9.8 Tf [(400 ?g/ml geneticin \(G418\). Twenty-four hours after plating, the cells were changed to serum-free defined medium \(EF12\) as )] TJ ET BT 26.250 444.262 Td /F1 9.8 Tf [(previously reported )] TJ ET 0.267 0.267 0.267 rg BT 111.865 445.769 Td /F4 8.7 Tf [(31)] TJ ET BT 121.502 445.769 Td /F4 8.7 Tf [(32)] TJ ET 0.271 0.267 0.267 rg BT 131.139 444.262 Td /F1 9.8 Tf [(. EF12 consisted of a 1:1 \(v/v\) EMEM and nutrient mixture of Hams F-12, supplemented with D-)] TJ ET BT 26.250 432.357 Td /F1 9.8 Tf [(glucose \(6 mg/ml\), insulin \(25 g/ml\), transferrin \(100 g/ml\), putrescine \(60 M\), progesterone \(20 nM\), and sodium selenite )] TJ ET BT 26.250 420.453 Td /F1 9.8 Tf [(\(30 nM\).)] TJ ET BT 26.250 401.048 Td /F1 9.8 Tf [(Glial striatal cultures were obtained from 129SV/C57BL/6 wild-type \(WT\) mice as previously described )] TJ ET 0.267 0.267 0.267 rg BT 468.929 402.555 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 478.567 401.048 Td /F1 9.8 Tf [(. Glia-enriched striatal )] TJ ET BT 26.250 389.143 Td /F1 9.8 Tf [(cultures were obtained from sibling cells kept in culture from 1015 days to 3 months in DMEM-FBS. Positive staining with anti-)] TJ ET BT 26.250 377.238 Td /F1 9.8 Tf [(GFAP antibody identified the astrocytes in these cultures. After 1015 days in culture, the number of astrocytes was about )] TJ ET BT 26.250 365.334 Td /F1 9.8 Tf [(8090 % of total cells )] TJ ET 0.267 0.267 0.267 rg BT 121.634 366.841 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 131.272 365.334 Td /F1 9.8 Tf [(. The cultures were fed once per week. To obtain the glia conditioned medium \(GCM\), DMEM-FBS )] TJ ET BT 26.250 353.429 Td /F1 9.8 Tf [(medium was discarded, and the cells were washed out three times with Leibovitzs L-15 and subsequently cultured in serum )] TJ ET BT 26.250 341.524 Td /F1 9.8 Tf [(free defined medium. After 24 hours of culture under such conditions, the medium was collected and stored frozen. This )] TJ ET BT 26.250 329.619 Td /F1 9.8 Tf [(medium was considered GCM.)] TJ ET BT 26.250 310.215 Td /F4 9.8 Tf [(Antibodies. )] TJ ET BT 82.049 310.215 Td /F1 9.8 Tf [(The following antibodies were used: Anti-mouse IgG fluorescein from Jackson \(PA, USA\) and anti-rabbit IgG Alexa )] TJ ET BT 26.250 298.310 Td /F1 9.8 Tf [(Fluor from Molecular Probes \(Eugene, OR, USA\). Mouse monoclonal anti-HSP-70 and rabbit polyclonal anti-CHIP antibodies )] TJ ET BT 26.250 286.405 Td /F1 9.8 Tf [(were from Santa Cruz Biotechnology \(Heidelberg, Germany\). Mouse monoclonal anti-PARP was from Cell Signalling )] TJ ET BT 26.250 274.500 Td /F1 9.8 Tf [(Technology \(Denver, USA\). Mouse monoclonal anti-ubiquitin, anti-synaptophysin and anti--tubulin class III antibodies were )] TJ ET BT 26.250 262.596 Td /F1 9.8 Tf [(from Chemicon, Millipore \(Temecula, CA, USA\). Anti-mouse and anti-rabbit horseradish peroxidase secondary antibodies were )] TJ ET BT 26.250 250.691 Td /F1 9.8 Tf [(from Amersham. -Actin secondary antibody was an anti-mouse phosphatase alkaline conjugated from Sigma \(Madrid, Spain\).)] TJ ET BT 26.250 231.286 Td /F4 9.8 Tf [(Chemicals. )] TJ ET BT 80.441 231.286 Td /F1 9.8 Tf [(5,5-Dithio-bis-2-nitrobenzoic acid \(DTNB\), hydrogen peroxide, sodium glutamate, 3-nitropropionic acid, trypan blue )] TJ ET BT 26.250 219.381 Td /F1 9.8 Tf [(reagent and reduced glutathione \(GSH\) were from Sigma \(Madrid, Spain\). NADPH and GSH reductase \(GR\) were from )] TJ ET BT 26.250 207.477 Td /F1 9.8 Tf [(Boehringer-Mannheim \(Barcelona, Spain\). The cytotoxicity detection kit for lactate dehydrogenase \(LDH\) and the cell )] TJ ET BT 26.250 195.572 Td /F1 9.8 Tf [(proliferation kit I [3-\(4,5-dimethylthiazol-2-yl\)-2,5-diphenyl tetrazolium bromide \(MTT\)] were from Roche Diagnostics. The BCA )] TJ ET BT 26.250 183.667 Td /F1 9.8 Tf [(protein assay kit was from Pierce \(Rockford, Ill, USA\). All other reagents were of the highest purity commercially available from )] TJ ET BT 26.250 171.762 Td /F1 9.8 Tf [(Merck or Sigma.)] TJ ET BT 26.250 152.358 Td /F4 9.8 Tf [(Treatment of the cells)] TJ ET BT 127.026 152.358 Td /F1 9.8 Tf [(Experiments were performed in 12 and 24-well culture plates for western blot and for survival and death )] TJ ET BT 26.250 140.453 Td /F1 9.8 Tf [(assays, respectively. For the immunocytochemistry detection, the cells were cultured in 13 mm-diameter glass cover slips with a )] TJ ET BT 26.250 128.548 Td /F1 9.8 Tf [(density of 5000 cells. After 3 days in vitro \(DIV\) in the growth medium \(DMEM-FBS\), Q7 and Q111 cells reached confluence. At )] TJ ET BT 26.250 116.643 Td /F1 9.8 Tf [(this time, the medium was discarded and the cells were subsequently cultured and treated in a chemically serum-free defined )] TJ ET BT 26.250 104.739 Td /F1 9.8 Tf [(medium \(EF12\) or in striatal glia-conditioned medium \(GCM\) \(embryonic E16, postnatal of 13 days and postnatal of 2 months\).)] TJ ET BT 26.250 85.334 Td /F1 9.8 Tf [(In order to test the protection of fetal GCM against different neurotoxins, we added the following chemicals to Q7 and Q111 cells )] TJ ET BT 26.250 73.429 Td /F1 9.8 Tf [(cultured in defined medium, supplemented or not with fetal GCM: sodium glutamate \(5 mM\), H)] TJ ET BT 432.649 71.365 Td /F1 8.7 Tf [(2)] TJ ET BT 437.468 73.429 Td /F1 9.8 Tf [(O)] TJ ET BT 445.054 71.365 Td /F1 8.7 Tf [(2)] TJ ET BT 449.872 73.429 Td /F1 9.8 Tf [( \(2 M\), 3NP acid \(2.5 mM\) )] TJ ET BT 26.250 61.524 Td /F1 9.8 Tf [(for 24 hours. In order to study the protection of postnatal GCM against H)] TJ ET BT 338.406 59.460 Td /F1 8.7 Tf [(2)] TJ ET BT 343.225 61.524 Td /F1 9.8 Tf [(O)] TJ ET BT 350.810 59.460 Td /F1 8.7 Tf [(2)] TJ ET BT 355.629 61.524 Td /F1 9.8 Tf [( induced cell death in Q111 cells we used postnatal )] TJ ET BT 26.250 49.620 Td /F1 9.8 Tf [(striatal GCM, from 13-day and 2-month old mice and the experiments were performed as described in the previous paragraph.)] TJ ET Q q 15.000 39.739 577.500 737.261 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(glial cells release chemical substances into the media )] TJ ET 0.267 0.267 0.267 rg BT 260.338 768.983 Td /F4 8.7 Tf [(22)] TJ ET BT 269.975 768.983 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 279.612 767.476 Td /F1 9.8 Tf [(, including antioxidants and neurotrophic factors. Glia-conditioned )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(medium \(GCM\) is rich in antioxidants, namely: reduced glutathione \(GSH\) and ascorbic acid \(AA\); and peptidic growth factors: )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(glial cell linederived neurotrophic factor \(GDNF\), brain-derived neurotrophic factor \(BDNF\), nerve growth factor \(NGF\), and )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(basic fibroblast growth factor \(bFGF\) as well as novel neurotrophic proteins )] TJ ET 0.267 0.267 0.267 rg BT 352.992 733.269 Td /F4 8.7 Tf [(12)] TJ ET BT 362.629 733.269 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 372.267 731.762 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(The aim of this study was to test the neuroprotective effect of GCM in a cellular neuronal model of HD. We chose the cell line )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(STHdh)] TJ ET BT 56.592 704.341 Td /F1 8.7 Tf [(Q111/111)] TJ ET BT 94.656 700.452 Td /F1 9.8 Tf [( \(Q111\), untreated or challenged with striatal neurotoxins, to model the molecular and cellular events that take )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(place in HD. These cells are striatal progenitor cells from knock-in mouse models of HD, expressing expanded human )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(huntingtin at baseline )] TJ ET 0.267 0.267 0.267 rg BT 121.098 678.150 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 130.735 676.643 Td /F1 9.8 Tf [(. These cells also reproduce several features observed in HD and in mouse models of this disease. )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(Q111 cells exhibit impaired energy metabolism, reduced ATP and ATP/ADP ratio )] TJ ET 0.267 0.267 0.267 rg BT 378.469 666.245 Td /F4 8.7 Tf [(25)] TJ ET BT 388.106 666.245 Td /F4 8.7 Tf [(26)] TJ ET BT 397.743 666.245 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 407.381 664.738 Td /F1 9.8 Tf [(, as well as increased sensitivity to 3-)] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(NP )] TJ ET 0.267 0.267 0.267 rg BT 42.503 654.341 Td /F4 8.7 Tf [(28)] TJ ET BT 52.141 654.341 Td /F4 8.7 Tf [(29)] TJ ET 0.271 0.267 0.267 rg BT 61.778 652.833 Td /F1 9.8 Tf [( and respiratory chain defects )] TJ ET 0.267 0.267 0.267 rg BT 192.379 654.341 Td /F4 8.7 Tf [(30)] TJ ET 0.271 0.267 0.267 rg BT 202.017 652.833 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(In this work, we have tested if fetal and postnatal mice GCM protected Q111 neurons from spontaneous cell death as well as )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(from exposure to glutamate, oxidative damage and mitochondrial toxins.)] TJ ET BT 26.250 602.119 Td /F4 9.8 Tf [(Material and methods)] TJ ET BT 26.250 582.714 Td /F4 9.8 Tf [(Culture Media. )] TJ ET BT 96.138 582.714 Td /F1 9.8 Tf [(Dulbeccos modified Eagles medium \(DMEM\) with high glucose \(4.5 g/liter\) was from Sigma \(Madrid, Spain\), )] TJ ET BT 26.250 570.810 Td /F1 9.8 Tf [(Hams F12 nutrient mixture, Eagles minimal essential medium \(EMEM\) with Earls salts, Leibovitzs L-15 medium; and )] TJ ET BT 26.250 558.905 Td /F1 9.8 Tf [(supplements as L-glutamine, fetal bovine serum \(FBS\), sodium pyruvate, geneticin and L-glutamine, purchased from Invitrogen )] TJ ET BT 26.250 547.000 Td /F1 9.8 Tf [(\(Madrid, Spain\). Glucose 45 %, trypsin-EDTA, insulin, putrescine, progesterone, and sodium selenite were from Sigma \(Madrid, )] TJ ET BT 26.250 535.095 Td /F1 9.8 Tf [(Spain\), and human transferrin, 30 % iron saturated, was from Boehringer- Mannheim \(Barcelona, Spain\).)] TJ ET BT 26.250 515.691 Td /F4 9.8 Tf [(Neuronal and glial cultures. )] TJ ET BT 156.832 515.691 Td /F1 9.8 Tf [(Conditionally immortalized, STHdh)] TJ ET BT 306.368 519.579 Td /F1 8.7 Tf [(Q7/Q7)] TJ ET BT 331.900 515.691 Td /F1 9.8 Tf [( \(Q7\), and Q111 striatal neuronal progenitor cell lines )] TJ ET BT 26.250 503.786 Td /F1 9.8 Tf [(expressing endogenous levels of huntingtin with 7 and 111 glutamines, respectively, were kindly provided by Prof. Alberch )] TJ ET BT 26.250 491.881 Td /F1 9.8 Tf [(\(Universitat de Barcelona, Spain\) and Prof. Marcy E. Macdonald \(Massachusetts General Hospital, Boston, USA\) and were )] TJ ET BT 26.250 479.976 Td /F1 9.8 Tf [(described previously by Trettel)] TJ ET 0.267 0.267 0.267 rg BT 158.460 481.484 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 168.097 479.976 Td /F1 9.8 Tf [( . Striatal cells were grown at 33 C in Dulbeccos Modified Eagles Medium \(DMEM\), )] TJ ET BT 26.250 468.072 Td /F1 9.8 Tf [(supplemented with 10 % fetal bovine serum \(FBS\), 1 % streptomycin-penicillin, 2 mM L-glutamine, 1 mM sodium pyruvate, and )] TJ ET BT 26.250 456.167 Td /F1 9.8 Tf [(400 ?g/ml geneticin \(G418\). Twenty-four hours after plating, the cells were changed to serum-free defined medium \(EF12\) as )] TJ ET BT 26.250 444.262 Td /F1 9.8 Tf [(previously reported )] TJ ET 0.267 0.267 0.267 rg BT 111.865 445.769 Td /F4 8.7 Tf [(31)] TJ ET BT 121.502 445.769 Td /F4 8.7 Tf [(32)] TJ ET 0.271 0.267 0.267 rg BT 131.139 444.262 Td /F1 9.8 Tf [(. EF12 consisted of a 1:1 \(v/v\) EMEM and nutrient mixture of Hams F-12, supplemented with D-)] TJ ET BT 26.250 432.357 Td /F1 9.8 Tf [(glucose \(6 mg/ml\), insulin \(25 g/ml\), transferrin \(100 g/ml\), putrescine \(60 M\), progesterone \(20 nM\), and sodium selenite )] TJ ET BT 26.250 420.453 Td /F1 9.8 Tf [(\(30 nM\).)] TJ ET BT 26.250 401.048 Td /F1 9.8 Tf [(Glial striatal cultures were obtained from 129SV/C57BL/6 wild-type \(WT\) mice as previously described )] TJ ET 0.267 0.267 0.267 rg BT 468.929 402.555 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 478.567 401.048 Td /F1 9.8 Tf [(. Glia-enriched striatal )] TJ ET BT 26.250 389.143 Td /F1 9.8 Tf [(cultures were obtained from sibling cells kept in culture from 1015 days to 3 months in DMEM-FBS. Positive staining with anti-)] TJ ET BT 26.250 377.238 Td /F1 9.8 Tf [(GFAP antibody identified the astrocytes in these cultures. After 1015 days in culture, the number of astrocytes was about )] TJ ET BT 26.250 365.334 Td /F1 9.8 Tf [(8090 % of total cells )] TJ ET 0.267 0.267 0.267 rg BT 121.634 366.841 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 131.272 365.334 Td /F1 9.8 Tf [(. The cultures were fed once per week. To obtain the glia conditioned medium \(GCM\), DMEM-FBS )] TJ ET BT 26.250 353.429 Td /F1 9.8 Tf [(medium was discarded, and the cells were washed out three times with Leibovitzs L-15 and subsequently cultured in serum )] TJ ET BT 26.250 341.524 Td /F1 9.8 Tf [(free defined medium. After 24 hours of culture under such conditions, the medium was collected and stored frozen. This )] TJ ET BT 26.250 329.619 Td /F1 9.8 Tf [(medium was considered GCM.)] TJ ET BT 26.250 310.215 Td /F4 9.8 Tf [(Antibodies. )] TJ ET BT 82.049 310.215 Td /F1 9.8 Tf [(The following antibodies were used: Anti-mouse IgG fluorescein from Jackson \(PA, USA\) and anti-rabbit IgG Alexa )] TJ ET BT 26.250 298.310 Td /F1 9.8 Tf [(Fluor from Molecular Probes \(Eugene, OR, USA\). Mouse monoclonal anti-HSP-70 and rabbit polyclonal anti-CHIP antibodies )] TJ ET BT 26.250 286.405 Td /F1 9.8 Tf [(were from Santa Cruz Biotechnology \(Heidelberg, Germany\). Mouse monoclonal anti-PARP was from Cell Signalling )] TJ ET BT 26.250 274.500 Td /F1 9.8 Tf [(Technology \(Denver, USA\). Mouse monoclonal anti-ubiquitin, anti-synaptophysin and anti--tubulin class III antibodies were )] TJ ET BT 26.250 262.596 Td /F1 9.8 Tf [(from Chemicon, Millipore \(Temecula, CA, USA\). Anti-mouse and anti-rabbit horseradish peroxidase secondary antibodies were )] TJ ET BT 26.250 250.691 Td /F1 9.8 Tf [(from Amersham. -Actin secondary antibody was an anti-mouse phosphatase alkaline conjugated from Sigma \(Madrid, Spain\).)] TJ ET BT 26.250 231.286 Td /F4 9.8 Tf [(Chemicals. )] TJ ET BT 80.441 231.286 Td /F1 9.8 Tf [(5,5-Dithio-bis-2-nitrobenzoic acid \(DTNB\), hydrogen peroxide, sodium glutamate, 3-nitropropionic acid, trypan blue )] TJ ET BT 26.250 219.381 Td /F1 9.8 Tf [(reagent and reduced glutathione \(GSH\) were from Sigma \(Madrid, Spain\). NADPH and GSH reductase \(GR\) were from )] TJ ET BT 26.250 207.477 Td /F1 9.8 Tf [(Boehringer-Mannheim \(Barcelona, Spain\). The cytotoxicity detection kit for lactate dehydrogenase \(LDH\) and the cell )] TJ ET BT 26.250 195.572 Td /F1 9.8 Tf [(proliferation kit I [3-\(4,5-dimethylthiazol-2-yl\)-2,5-diphenyl tetrazolium bromide \(MTT\)] were from Roche Diagnostics. The BCA )] TJ ET BT 26.250 183.667 Td /F1 9.8 Tf [(protein assay kit was from Pierce \(Rockford, Ill, USA\). All other reagents were of the highest purity commercially available from )] TJ ET BT 26.250 171.762 Td /F1 9.8 Tf [(Merck or Sigma.)] TJ ET BT 26.250 152.358 Td /F4 9.8 Tf [(Treatment of the cells)] TJ ET BT 127.026 152.358 Td /F1 9.8 Tf [(Experiments were performed in 12 and 24-well culture plates for western blot and for survival and death )] TJ ET BT 26.250 140.453 Td /F1 9.8 Tf [(assays, respectively. For the immunocytochemistry detection, the cells were cultured in 13 mm-diameter glass cover slips with a )] TJ ET BT 26.250 128.548 Td /F1 9.8 Tf [(density of 5000 cells. After 3 days in vitro \(DIV\) in the growth medium \(DMEM-FBS\), Q7 and Q111 cells reached confluence. At )] TJ ET BT 26.250 116.643 Td /F1 9.8 Tf [(this time, the medium was discarded and the cells were subsequently cultured and treated in a chemically serum-free defined )] TJ ET BT 26.250 104.739 Td /F1 9.8 Tf [(medium \(EF12\) or in striatal glia-conditioned medium \(GCM\) \(embryonic E16, postnatal of 13 days and postnatal of 2 months\).)] TJ ET BT 26.250 85.334 Td /F1 9.8 Tf [(In order to test the protection of fetal GCM against different neurotoxins, we added the following chemicals to Q7 and Q111 cells )] TJ ET BT 26.250 73.429 Td /F1 9.8 Tf [(cultured in defined medium, supplemented or not with fetal GCM: sodium glutamate \(5 mM\), H)] TJ ET BT 432.649 71.365 Td /F1 8.7 Tf [(2)] TJ ET BT 437.468 73.429 Td /F1 9.8 Tf [(O)] TJ ET BT 445.054 71.365 Td /F1 8.7 Tf [(2)] TJ ET BT 449.872 73.429 Td /F1 9.8 Tf [( \(2 M\), 3NP acid \(2.5 mM\) )] TJ ET BT 26.250 61.524 Td /F1 9.8 Tf [(for 24 hours. In order to study the protection of postnatal GCM against H)] TJ ET BT 338.406 59.460 Td /F1 8.7 Tf [(2)] TJ ET BT 343.225 61.524 Td /F1 9.8 Tf [(O)] TJ ET BT 350.810 59.460 Td /F1 8.7 Tf [(2)] TJ ET BT 355.629 61.524 Td /F1 9.8 Tf [( induced cell death in Q111 cells we used postnatal )] TJ ET BT 26.250 49.620 Td /F1 9.8 Tf [(striatal GCM, from 13-day and 2-month old mice and the experiments were performed as described in the previous paragraph.)] TJ ET Q q 15.000 39.739 577.500 737.261 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(glial cells release chemical substances into the media )] TJ ET 0.267 0.267 0.267 rg BT 260.338 768.983 Td /F4 8.7 Tf [(22)] TJ ET BT 269.975 768.983 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 279.612 767.476 Td /F1 9.8 Tf [(, including antioxidants and neurotrophic factors. Glia-conditioned )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(medium \(GCM\) is rich in antioxidants, namely: reduced glutathione \(GSH\) and ascorbic acid \(AA\); and peptidic growth factors: )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(glial cell linederived neurotrophic factor \(GDNF\), brain-derived neurotrophic factor \(BDNF\), nerve growth factor \(NGF\), and )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(basic fibroblast growth factor \(bFGF\) as well as novel neurotrophic proteins )] TJ ET 0.267 0.267 0.267 rg BT 352.992 733.269 Td /F4 8.7 Tf [(12)] TJ ET BT 362.629 733.269 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 372.267 731.762 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(The aim of this study was to test the neuroprotective effect of GCM in a cellular neuronal model of HD. We chose the cell line )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(STHdh)] TJ ET BT 56.592 704.341 Td /F1 8.7 Tf [(Q111/111)] TJ ET BT 94.656 700.452 Td /F1 9.8 Tf [( \(Q111\), untreated or challenged with striatal neurotoxins, to model the molecular and cellular events that take )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(place in HD. These cells are striatal progenitor cells from knock-in mouse models of HD, expressing expanded human )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(huntingtin at baseline )] TJ ET 0.267 0.267 0.267 rg BT 121.098 678.150 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 130.735 676.643 Td /F1 9.8 Tf [(. These cells also reproduce several features observed in HD and in mouse models of this disease. )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(Q111 cells exhibit impaired energy metabolism, reduced ATP and ATP/ADP ratio )] TJ ET 0.267 0.267 0.267 rg BT 378.469 666.245 Td /F4 8.7 Tf [(25)] TJ ET BT 388.106 666.245 Td /F4 8.7 Tf [(26)] TJ ET BT 397.743 666.245 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 407.381 664.738 Td /F1 9.8 Tf [(, as well as increased sensitivity to 3-)] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(NP )] TJ ET 0.267 0.267 0.267 rg BT 42.503 654.341 Td /F4 8.7 Tf [(28)] TJ ET BT 52.141 654.341 Td /F4 8.7 Tf [(29)] TJ ET 0.271 0.267 0.267 rg BT 61.778 652.833 Td /F1 9.8 Tf [( and respiratory chain defects )] TJ ET 0.267 0.267 0.267 rg BT 192.379 654.341 Td /F4 8.7 Tf [(30)] TJ ET 0.271 0.267 0.267 rg BT 202.017 652.833 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(In this work, we have tested if fetal and postnatal mice GCM protected Q111 neurons from spontaneous cell death as well as )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(from exposure to glutamate, oxidative damage and mitochondrial toxins.)] TJ ET BT 26.250 602.119 Td /F4 9.8 Tf [(Material and methods)] TJ ET BT 26.250 582.714 Td /F4 9.8 Tf [(Culture Media. )] TJ ET BT 96.138 582.714 Td /F1 9.8 Tf [(Dulbeccos modified Eagles medium \(DMEM\) with high glucose \(4.5 g/liter\) was from Sigma \(Madrid, Spain\), )] TJ ET BT 26.250 570.810 Td /F1 9.8 Tf [(Hams F12 nutrient mixture, Eagles minimal essential medium \(EMEM\) with Earls salts, Leibovitzs L-15 medium; and )] TJ ET BT 26.250 558.905 Td /F1 9.8 Tf [(supplements as L-glutamine, fetal bovine serum \(FBS\), sodium pyruvate, geneticin and L-glutamine, purchased from Invitrogen )] TJ ET BT 26.250 547.000 Td /F1 9.8 Tf [(\(Madrid, Spain\). Glucose 45 %, trypsin-EDTA, insulin, putrescine, progesterone, and sodium selenite were from Sigma \(Madrid, )] TJ ET BT 26.250 535.095 Td /F1 9.8 Tf [(Spain\), and human transferrin, 30 % iron saturated, was from Boehringer- Mannheim \(Barcelona, Spain\).)] TJ ET BT 26.250 515.691 Td /F4 9.8 Tf [(Neuronal and glial cultures. )] TJ ET BT 156.832 515.691 Td /F1 9.8 Tf [(Conditionally immortalized, STHdh)] TJ ET BT 306.368 519.579 Td /F1 8.7 Tf [(Q7/Q7)] TJ ET BT 331.900 515.691 Td /F1 9.8 Tf [( \(Q7\), and Q111 striatal neuronal progenitor cell lines )] TJ ET BT 26.250 503.786 Td /F1 9.8 Tf [(expressing endogenous levels of huntingtin with 7 and 111 glutamines, respectively, were kindly provided by Prof. Alberch )] TJ ET BT 26.250 491.881 Td /F1 9.8 Tf [(\(Universitat de Barcelona, Spain\) and Prof. Marcy E. Macdonald \(Massachusetts General Hospital, Boston, USA\) and were )] TJ ET BT 26.250 479.976 Td /F1 9.8 Tf [(described previously by Trettel)] TJ ET 0.267 0.267 0.267 rg BT 158.460 481.484 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 168.097 479.976 Td /F1 9.8 Tf [( . Striatal cells were grown at 33 C in Dulbeccos Modified Eagles Medium \(DMEM\), )] TJ ET BT 26.250 468.072 Td /F1 9.8 Tf [(supplemented with 10 % fetal bovine serum \(FBS\), 1 % streptomycin-penicillin, 2 mM L-glutamine, 1 mM sodium pyruvate, and )] TJ ET BT 26.250 456.167 Td /F1 9.8 Tf [(400 ?g/ml geneticin \(G418\). Twenty-four hours after plating, the cells were changed to serum-free defined medium \(EF12\) as )] TJ ET BT 26.250 444.262 Td /F1 9.8 Tf [(previously reported )] TJ ET 0.267 0.267 0.267 rg BT 111.865 445.769 Td /F4 8.7 Tf [(31)] TJ ET BT 121.502 445.769 Td /F4 8.7 Tf [(32)] TJ ET 0.271 0.267 0.267 rg BT 131.139 444.262 Td /F1 9.8 Tf [(. EF12 consisted of a 1:1 \(v/v\) EMEM and nutrient mixture of Hams F-12, supplemented with D-)] TJ ET BT 26.250 432.357 Td /F1 9.8 Tf [(glucose \(6 mg/ml\), insulin \(25 g/ml\), transferrin \(100 g/ml\), putrescine \(60 M\), progesterone \(20 nM\), and sodium selenite )] TJ ET BT 26.250 420.453 Td /F1 9.8 Tf [(\(30 nM\).)] TJ ET BT 26.250 401.048 Td /F1 9.8 Tf [(Glial striatal cultures were obtained from 129SV/C57BL/6 wild-type \(WT\) mice as previously described )] TJ ET 0.267 0.267 0.267 rg BT 468.929 402.555 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 478.567 401.048 Td /F1 9.8 Tf [(. Glia-enriched striatal )] TJ ET BT 26.250 389.143 Td /F1 9.8 Tf [(cultures were obtained from sibling cells kept in culture from 1015 days to 3 months in DMEM-FBS. Positive staining with anti-)] TJ ET BT 26.250 377.238 Td /F1 9.8 Tf [(GFAP antibody identified the astrocytes in these cultures. After 1015 days in culture, the number of astrocytes was about )] TJ ET BT 26.250 365.334 Td /F1 9.8 Tf [(8090 % of total cells )] TJ ET 0.267 0.267 0.267 rg BT 121.634 366.841 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 131.272 365.334 Td /F1 9.8 Tf [(. The cultures were fed once per week. To obtain the glia conditioned medium \(GCM\), DMEM-FBS )] TJ ET BT 26.250 353.429 Td /F1 9.8 Tf [(medium was discarded, and the cells were washed out three times with Leibovitzs L-15 and subsequently cultured in serum )] TJ ET BT 26.250 341.524 Td /F1 9.8 Tf [(free defined medium. After 24 hours of culture under such conditions, the medium was collected and stored frozen. This )] TJ ET BT 26.250 329.619 Td /F1 9.8 Tf [(medium was considered GCM.)] TJ ET BT 26.250 310.215 Td /F4 9.8 Tf [(Antibodies. )] TJ ET BT 82.049 310.215 Td /F1 9.8 Tf [(The following antibodies were used: Anti-mouse IgG fluorescein from Jackson \(PA, USA\) and anti-rabbit IgG Alexa )] TJ ET BT 26.250 298.310 Td /F1 9.8 Tf [(Fluor from Molecular Probes \(Eugene, OR, USA\). Mouse monoclonal anti-HSP-70 and rabbit polyclonal anti-CHIP antibodies )] TJ ET BT 26.250 286.405 Td /F1 9.8 Tf [(were from Santa Cruz Biotechnology \(Heidelberg, Germany\). Mouse monoclonal anti-PARP was from Cell Signalling )] TJ ET BT 26.250 274.500 Td /F1 9.8 Tf [(Technology \(Denver, USA\). Mouse monoclonal anti-ubiquitin, anti-synaptophysin and anti--tubulin class III antibodies were )] TJ ET BT 26.250 262.596 Td /F1 9.8 Tf [(from Chemicon, Millipore \(Temecula, CA, USA\). Anti-mouse and anti-rabbit horseradish peroxidase secondary antibodies were )] TJ ET BT 26.250 250.691 Td /F1 9.8 Tf [(from Amersham. -Actin secondary antibody was an anti-mouse phosphatase alkaline conjugated from Sigma \(Madrid, Spain\).)] TJ ET BT 26.250 231.286 Td /F4 9.8 Tf [(Chemicals. )] TJ ET BT 80.441 231.286 Td /F1 9.8 Tf [(5,5-Dithio-bis-2-nitrobenzoic acid \(DTNB\), hydrogen peroxide, sodium glutamate, 3-nitropropionic acid, trypan blue )] TJ ET BT 26.250 219.381 Td /F1 9.8 Tf [(reagent and reduced glutathione \(GSH\) were from Sigma \(Madrid, Spain\). NADPH and GSH reductase \(GR\) were from )] TJ ET BT 26.250 207.477 Td /F1 9.8 Tf [(Boehringer-Mannheim \(Barcelona, Spain\). The cytotoxicity detection kit for lactate dehydrogenase \(LDH\) and the cell )] TJ ET BT 26.250 195.572 Td /F1 9.8 Tf [(proliferation kit I [3-\(4,5-dimethylthiazol-2-yl\)-2,5-diphenyl tetrazolium bromide \(MTT\)] were from Roche Diagnostics. The BCA )] TJ ET BT 26.250 183.667 Td /F1 9.8 Tf [(protein assay kit was from Pierce \(Rockford, Ill, USA\). All other reagents were of the highest purity commercially available from )] TJ ET BT 26.250 171.762 Td /F1 9.8 Tf [(Merck or Sigma.)] TJ ET BT 26.250 152.358 Td /F4 9.8 Tf [(Treatment of the cells)] TJ ET BT 127.026 152.358 Td /F1 9.8 Tf [(Experiments were performed in 12 and 24-well culture plates for western blot and for survival and death )] TJ ET BT 26.250 140.453 Td /F1 9.8 Tf [(assays, respectively. For the immunocytochemistry detection, the cells were cultured in 13 mm-diameter glass cover slips with a )] TJ ET BT 26.250 128.548 Td /F1 9.8 Tf [(density of 5000 cells. After 3 days in vitro \(DIV\) in the growth medium \(DMEM-FBS\), Q7 and Q111 cells reached confluence. At )] TJ ET BT 26.250 116.643 Td /F1 9.8 Tf [(this time, the medium was discarded and the cells were subsequently cultured and treated in a chemically serum-free defined )] TJ ET BT 26.250 104.739 Td /F1 9.8 Tf [(medium \(EF12\) or in striatal glia-conditioned medium \(GCM\) \(embryonic E16, postnatal of 13 days and postnatal of 2 months\).)] TJ ET BT 26.250 85.334 Td /F1 9.8 Tf [(In order to test the protection of fetal GCM against different neurotoxins, we added the following chemicals to Q7 and Q111 cells )] TJ ET BT 26.250 73.429 Td /F1 9.8 Tf [(cultured in defined medium, supplemented or not with fetal GCM: sodium glutamate \(5 mM\), H)] TJ ET BT 432.649 71.365 Td /F1 8.7 Tf [(2)] TJ ET BT 437.468 73.429 Td /F1 9.8 Tf [(O)] TJ ET BT 445.054 71.365 Td /F1 8.7 Tf [(2)] TJ ET BT 449.872 73.429 Td /F1 9.8 Tf [( \(2 M\), 3NP acid \(2.5 mM\) )] TJ ET BT 26.250 61.524 Td /F1 9.8 Tf [(for 24 hours. In order to study the protection of postnatal GCM against H)] TJ ET BT 338.406 59.460 Td /F1 8.7 Tf [(2)] TJ ET BT 343.225 61.524 Td /F1 9.8 Tf [(O)] TJ ET BT 350.810 59.460 Td /F1 8.7 Tf [(2)] TJ ET BT 355.629 61.524 Td /F1 9.8 Tf [( induced cell death in Q111 cells we used postnatal )] TJ ET BT 26.250 49.620 Td /F1 9.8 Tf [(striatal GCM, from 13-day and 2-month old mice and the experiments were performed as described in the previous paragraph.)] 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 209 0 obj << /Type /Annot /Subtype /Link /A 210 0 R /Border [0 0 0] /H /I /Rect [ 260.3378 768.1817 269.9751 777.0000 ] >> endobj 210 0 obj << /Type /Action >> endobj 211 0 obj << /Type /Annot /Subtype /Link /A 212 0 R /Border [0 0 0] /H /I /Rect [ 269.9751 768.1817 279.6124 777.0000 ] >> endobj 212 0 obj << /Type /Action >> endobj 213 0 obj << /Type /Annot /Subtype /Link /A 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the mechanisms of neuroprotection by fetal GCM, we studied the ubiquitination of proteins, and the expression of )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(death and chaperone proteins by Western blot assays. To compare the effects of GCM with the well-known neuronal growth )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(factors, we treated Q111 cells with fetal GCM versus different growth factors such as GDNF \(50 and 100 ng/ml\), bFGF \(10 and )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(20 ng/ml\) and BDNF \(50 and 100 ng/ml\). We initially studied the effects of each growth factor on Q111 cells and compared )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(them with the fetal GCM. Then, we studied the protection of the growth factors and fetal GCM on H)] TJ ET BT 452.198 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 457.017 719.857 Td /F1 9.8 Tf [(O)] TJ ET BT 464.602 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 469.421 719.857 Td /F1 9.8 Tf [( induced cell death in )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(Q111 cells.)] TJ ET BT 26.250 688.548 Td /F4 9.8 Tf [(Cell survival, proliferation assay and cell differentiation. )] TJ ET BT 287.969 688.548 Td /F1 9.8 Tf [(Necrotic cell death was measured according to LDH activity in the )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(culture medium and by trypan blue dye exclusion in cells. LDH activity was measured by using a cytotoxicity detection kit )] TJ ET 0.267 0.267 0.267 rg BT 548.616 678.150 Td /F4 8.7 Tf [(12)] TJ ET BT 558.253 678.150 Td /F4 8.7 Tf [(33)] TJ ET 0.271 0.267 0.267 rg BT 567.891 676.643 Td /F1 9.8 Tf [( . )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(Apoptotic cell death was measured by nuclei stained with bis-benzimide \(Hoechst 33342\). Mitochondrial activity was analyzed )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(with the 3-\(4,5-dimethylthiazol-2-yl\)-2,5-diphenyl tetrazolium bromide \(MTT\) assay in Q7 and Q111 cells. The MTT assay )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(determines the ability of cells to metabolize MTT. At the end of the cell treatment period, 300 l of culture medium were )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(removed from total 500 l of each well, and 20 l of MTT solution \(5 mg/ml\) added and incubated for 2 h. At this time, 200 l of )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(solubilization solution \(10 % SDS in 0.01 M HCl\) were then added to the wells, and, after 24 h of incubation at 37 C, 100 l )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(transferred into a 96-well microtiter plate. The absorption value at 540 nm was measured in an automatic microtiter reader )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(\(Spectra Fluor; Tecan, Mnnedorf, Switzerland\). To test the differentiation of the cells, we counted the number of multiprocess )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(cells as the number of cells with 3 or more prolongations.)] TJ ET BT 26.250 562.000 Td /F4 9.8 Tf [(Immunocytochemistry. )] TJ ET BT 135.694 562.000 Td /F1 9.8 Tf [(After the experimental treatments, the cells were fixed with 4 % paraformaldehyde. Then, cells were )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(postfixed and permeabilized in ethanol-acetic acid \(19:1\) for 10 min at -20 C and incubated in a blocking solution followed by )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(overnight incubation at 4 C with the cytoplasmatic class III -tubulin primary antibody \(1:300\) and with a protein of the )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(presynaptic vesicle exocytosis, synaptophysin primary antibody \(1:200\). Fluorescein and Alexa Fluor-conjugated secondary )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(antibodies were used to visualize positive cells under fluorescent microscopy. Nuclei were stained by bis-benzimide \(Hoechst )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(33342\) and immunostaining was visualized under fluorescent microscopy. The number of immunoreactive cells was counted in )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(predefined parallel strips.)] TJ ET BT 26.250 471.167 Td /F4 9.8 Tf [(Measurement of GSH and ROS levels.)] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(Total glutathione levels were measured by the method of Tietze )] TJ ET 0.267 0.267 0.267 rg BT 303.170 453.269 Td /F4 8.7 Tf [(34)] TJ ET 0.271 0.267 0.267 rg BT 312.807 451.762 Td /F1 9.8 Tf [(. Intracellular levels of ROS were detected using the non-)] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(fluorescence DCF dye, which is oxidized by intracellular ROS to form the highly fluorescence DCF. The cells were incubated )] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(with 5 M DCFH-DA 30 min before H)] TJ ET BT 186.618 425.888 Td /F1 8.7 Tf [(2)] TJ ET BT 191.437 427.953 Td /F1 9.8 Tf [(O)] TJ ET BT 199.022 425.888 Td /F1 8.7 Tf [(2)] TJ ET BT 203.841 427.953 Td /F1 9.8 Tf [( treatments. After washing the excess of DCF with a medium free of phenol red \(PBS )] TJ ET BT 26.250 416.048 Td /F1 9.8 Tf [(with 1mM glucose\), the fluorescence was measured in a laser-scanning confocal microscope \(Nikon eclipse Ti\) or in an )] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(automatic microtiter reader \(Spectra Fluor, Tecan\) at excitation and emission of 485 nm and 582 nm, respectively. Fluorescence )] TJ ET BT 26.250 392.238 Td /F1 9.8 Tf [(was quantified by automated image analysis with Image Pro software. For each section mean fluorescence was calculated from )] TJ ET BT 26.250 380.334 Td /F1 9.8 Tf [(7 separate single fields.)] TJ ET BT 26.250 360.929 Td /F4 9.8 Tf [(Western blot analysis and detection of ubiquitinated proteins.)] TJ ET BT 26.250 341.524 Td /F1 9.8 Tf [(Q111 extracts for western blot analysis were prepared in ice-cold extraction buffer consisting of 20 mM TrisHCl \(pH 7.4\), 10 )] TJ ET BT 26.250 329.619 Td /F1 9.8 Tf [(mM potassium acetate \(AcK\), 1 mM dithiothreitol \(DTT\), 0.25 % NP-40, 1 mM EDTA, 2 mM EGTA, 1 mM PMSF, protease )] TJ ET BT 26.250 317.715 Td /F1 9.8 Tf [(inhibitors cocktail \(SIGMA\) and a cocktail of phosphatase inhibitors \(100 mM sodium fluoride, 20 mM sodium molybdate and 20 )] TJ ET BT 26.250 305.810 Td /F1 9.8 Tf [(mM -glicerophosphate\). The samples were homogenized, centrifuged at 4 C and protein content was determined by the BCA )] TJ ET BT 26.250 293.905 Td /F1 9.8 Tf [(protein assay kit. Total protein \(30 g\) was electrophoresed in 10 % SDS-PAGE gels and transferred to 0.45 mm nitrocellulose )] TJ ET BT 26.250 282.000 Td /F1 9.8 Tf [(membranes \(Amersham\), as described previously)] TJ ET 0.267 0.267 0.267 rg BT 241.345 283.508 Td /F4 8.7 Tf [(12)] TJ ET BT 250.982 283.508 Td /F4 8.7 Tf [(33)] TJ ET 0.271 0.267 0.267 rg BT 260.619 282.000 Td /F1 9.8 Tf [(. After blocking, blots were incubated overnight at 4 C in 5 % non- fat )] TJ ET BT 26.250 270.096 Td /F1 9.8 Tf [(dried milk with primary antibodies: the chaperone anti-HSP-70 \(1/700\); anti-CHIP \(1/1000\) and anti-PARP \(1/1000\).)] TJ ET BT 26.250 250.691 Td /F1 9.8 Tf [(To determine changes in ubiquitination, cell cultures treated or untreated with H)] TJ ET BT 368.738 248.627 Td /F1 8.7 Tf [(2)] TJ ET BT 373.557 250.691 Td /F1 9.8 Tf [(O)] TJ ET BT 381.142 248.627 Td /F1 8.7 Tf [(2)] TJ ET BT 385.961 250.691 Td /F1 9.8 Tf [( \(2 M\) in serum-free defined medium and in )] TJ ET BT 26.250 238.786 Td /F1 9.8 Tf [(GCM for 24 h, were scraped in 150 l of lysis buffer [50mM TrisHCl, 150 mM NaCl, 20 mM EDTA, 1 % TritonX-100, 50 mM )] TJ ET BT 26.250 226.881 Td /F1 9.8 Tf [(sodium fluoride, 20 mM N-ethyl-maleimide, 100 M sodium ortovanadate, 1 mM PMSF and protease inhibitors cocktail )] TJ ET BT 26.250 214.977 Td /F1 9.8 Tf [(\(SIGMA\)] and boiled for 5 min. The lysates were centrifuged at 12 000 x g at 4 C for 30 min and 7.5 g of protein were )] TJ ET BT 26.250 203.072 Td /F1 9.8 Tf [(conducted to immunoblot assay with a rabbit polyclonal antibody to ubiquitin.)] TJ ET BT 26.250 183.667 Td /F1 9.8 Tf [(The secondary antibodies \(1/2000\) followed by ECL detection reagents \(Amersham\) were used for immunodetection. )] TJ ET BT 26.250 171.762 Td /F1 9.8 Tf [(Immunoblot of -actin diluted \(1/20000\) was performed to demonstrate equal protein loading. The blots were quantified by )] TJ ET BT 26.250 159.858 Td /F1 9.8 Tf [(computer-assisted video densitometry.)] TJ ET BT 26.250 140.453 Td /F4 9.8 Tf [(Characterization of defined and glia-conditioned medium protein profile. )] TJ ET BT 363.766 140.453 Td /F1 9.8 Tf [(Defined E12 and embryonic E16 glia-conditioned )] TJ ET BT 26.250 128.548 Td /F1 9.8 Tf [(media were lyophilized and concentrated in 5 % of initial volume. Protein concentration was determined by BCA method and 5 )] TJ ET BT 26.250 116.643 Td /F1 9.8 Tf [(g of protein from the supernatants of both media, were used for the electrophoresis in 4-20 % polyacrylamide gel. )] TJ ET BT 26.250 104.739 Td /F1 9.8 Tf [(Electrophoresis was carried out at 150V. After completion of electrophoresis, the gel was developed using silver staining to )] TJ ET BT 26.250 92.834 Td /F1 9.8 Tf [(observe protein profile.)] TJ ET BT 26.250 73.429 Td /F4 9.8 Tf [(Statistical analysis. )] TJ ET BT 118.924 73.429 Td /F1 9.8 Tf [(The statistical analysis was performed by one-way analysis of variance \(ANOVA\), followed by )] TJ ET BT 26.250 61.524 Td /F1 9.8 Tf [(NewmanKeuls multiple comparison test. Differences were considered statistically significant when p< 0.05. Analysis of data )] TJ ET BT 26.250 49.620 Td /F1 9.8 Tf [(was performed using the Graph Pad Software \(San Diego, CA\) Prism 4 software.)] TJ ET Q q 15.000 20.334 577.500 756.666 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(To investigate the mechanisms of neuroprotection by fetal GCM, we studied the ubiquitination of proteins, and the expression of )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(death and chaperone proteins by Western blot assays. To compare the effects of GCM with the well-known neuronal growth )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(factors, we treated Q111 cells with fetal GCM versus different growth factors such as GDNF \(50 and 100 ng/ml\), bFGF \(10 and )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(20 ng/ml\) and BDNF \(50 and 100 ng/ml\). We initially studied the effects of each growth factor on Q111 cells and compared )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(them with the fetal GCM. Then, we studied the protection of the growth factors and fetal GCM on H)] TJ ET BT 452.198 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 457.017 719.857 Td /F1 9.8 Tf [(O)] TJ ET BT 464.602 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 469.421 719.857 Td /F1 9.8 Tf [( induced cell death in )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(Q111 cells.)] TJ ET BT 26.250 688.548 Td /F4 9.8 Tf [(Cell survival, proliferation assay and cell differentiation. )] TJ ET BT 287.969 688.548 Td /F1 9.8 Tf [(Necrotic cell death was measured according to LDH activity in the )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(culture medium and by trypan blue dye exclusion in cells. LDH activity was measured by using a cytotoxicity detection kit )] TJ ET 0.267 0.267 0.267 rg BT 548.616 678.150 Td /F4 8.7 Tf [(12)] TJ ET BT 558.253 678.150 Td /F4 8.7 Tf [(33)] TJ ET 0.271 0.267 0.267 rg BT 567.891 676.643 Td /F1 9.8 Tf [( . )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(Apoptotic cell death was measured by nuclei stained with bis-benzimide \(Hoechst 33342\). Mitochondrial activity was analyzed )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(with the 3-\(4,5-dimethylthiazol-2-yl\)-2,5-diphenyl tetrazolium bromide \(MTT\) assay in Q7 and Q111 cells. The MTT assay )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(determines the ability of cells to metabolize MTT. At the end of the cell treatment period, 300 l of culture medium were )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(removed from total 500 l of each well, and 20 l of MTT solution \(5 mg/ml\) added and incubated for 2 h. At this time, 200 l of )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(solubilization solution \(10 % SDS in 0.01 M HCl\) were then added to the wells, and, after 24 h of incubation at 37 C, 100 l )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(transferred into a 96-well microtiter plate. The absorption value at 540 nm was measured in an automatic microtiter reader )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(\(Spectra Fluor; Tecan, Mnnedorf, Switzerland\). To test the differentiation of the cells, we counted the number of multiprocess )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(cells as the number of cells with 3 or more prolongations.)] TJ ET BT 26.250 562.000 Td /F4 9.8 Tf [(Immunocytochemistry. )] TJ ET BT 135.694 562.000 Td /F1 9.8 Tf [(After the experimental treatments, the cells were fixed with 4 % paraformaldehyde. Then, cells were )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(postfixed and permeabilized in ethanol-acetic acid \(19:1\) for 10 min at -20 C and incubated in a blocking solution followed by )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(overnight incubation at 4 C with the cytoplasmatic class III -tubulin primary antibody \(1:300\) and with a protein of the )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(presynaptic vesicle exocytosis, synaptophysin primary antibody \(1:200\). Fluorescein and Alexa Fluor-conjugated secondary )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(antibodies were used to visualize positive cells under fluorescent microscopy. Nuclei were stained by bis-benzimide \(Hoechst )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(33342\) and immunostaining was visualized under fluorescent microscopy. The number of immunoreactive cells was counted in )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(predefined parallel strips.)] TJ ET BT 26.250 471.167 Td /F4 9.8 Tf [(Measurement of GSH and ROS levels.)] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(Total glutathione levels were measured by the method of Tietze )] TJ ET 0.267 0.267 0.267 rg BT 303.170 453.269 Td /F4 8.7 Tf [(34)] TJ ET 0.271 0.267 0.267 rg BT 312.807 451.762 Td /F1 9.8 Tf [(. Intracellular levels of ROS were detected using the non-)] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(fluorescence DCF dye, which is oxidized by intracellular ROS to form the highly fluorescence DCF. The cells were incubated )] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(with 5 M DCFH-DA 30 min before H)] TJ ET BT 186.618 425.888 Td /F1 8.7 Tf [(2)] TJ ET BT 191.437 427.953 Td /F1 9.8 Tf [(O)] TJ ET BT 199.022 425.888 Td /F1 8.7 Tf [(2)] TJ ET BT 203.841 427.953 Td /F1 9.8 Tf [( treatments. After washing the excess of DCF with a medium free of phenol red \(PBS )] TJ ET BT 26.250 416.048 Td /F1 9.8 Tf [(with 1mM glucose\), the fluorescence was measured in a laser-scanning confocal microscope \(Nikon eclipse Ti\) or in an )] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(automatic microtiter reader \(Spectra Fluor, Tecan\) at excitation and emission of 485 nm and 582 nm, respectively. Fluorescence )] TJ ET BT 26.250 392.238 Td /F1 9.8 Tf [(was quantified by automated image analysis with Image Pro software. For each section mean fluorescence was calculated from )] TJ ET BT 26.250 380.334 Td /F1 9.8 Tf [(7 separate single fields.)] TJ ET BT 26.250 360.929 Td /F4 9.8 Tf [(Western blot analysis and detection of ubiquitinated proteins.)] TJ ET BT 26.250 341.524 Td /F1 9.8 Tf [(Q111 extracts for western blot analysis were prepared in ice-cold extraction buffer consisting of 20 mM TrisHCl \(pH 7.4\), 10 )] TJ ET BT 26.250 329.619 Td /F1 9.8 Tf [(mM potassium acetate \(AcK\), 1 mM dithiothreitol \(DTT\), 0.25 % NP-40, 1 mM EDTA, 2 mM EGTA, 1 mM PMSF, protease )] TJ ET BT 26.250 317.715 Td /F1 9.8 Tf [(inhibitors cocktail \(SIGMA\) and a cocktail of phosphatase inhibitors \(100 mM sodium fluoride, 20 mM sodium molybdate and 20 )] TJ ET BT 26.250 305.810 Td /F1 9.8 Tf [(mM -glicerophosphate\). The samples were homogenized, centrifuged at 4 C and protein content was determined by the BCA )] TJ ET BT 26.250 293.905 Td /F1 9.8 Tf [(protein assay kit. Total protein \(30 g\) was electrophoresed in 10 % SDS-PAGE gels and transferred to 0.45 mm nitrocellulose )] TJ ET BT 26.250 282.000 Td /F1 9.8 Tf [(membranes \(Amersham\), as described previously)] TJ ET 0.267 0.267 0.267 rg BT 241.345 283.508 Td /F4 8.7 Tf [(12)] TJ ET BT 250.982 283.508 Td /F4 8.7 Tf [(33)] TJ ET 0.271 0.267 0.267 rg BT 260.619 282.000 Td /F1 9.8 Tf [(. After blocking, blots were incubated overnight at 4 C in 5 % non- fat )] TJ ET BT 26.250 270.096 Td /F1 9.8 Tf [(dried milk with primary antibodies: the chaperone anti-HSP-70 \(1/700\); anti-CHIP \(1/1000\) and anti-PARP \(1/1000\).)] TJ ET BT 26.250 250.691 Td /F1 9.8 Tf [(To determine changes in ubiquitination, cell cultures treated or untreated with H)] TJ ET BT 368.738 248.627 Td /F1 8.7 Tf [(2)] TJ ET BT 373.557 250.691 Td /F1 9.8 Tf [(O)] TJ ET BT 381.142 248.627 Td /F1 8.7 Tf [(2)] TJ ET BT 385.961 250.691 Td /F1 9.8 Tf [( \(2 M\) in serum-free defined medium and in )] TJ ET BT 26.250 238.786 Td /F1 9.8 Tf [(GCM for 24 h, were scraped in 150 l of lysis buffer [50mM TrisHCl, 150 mM NaCl, 20 mM EDTA, 1 % TritonX-100, 50 mM )] TJ ET BT 26.250 226.881 Td /F1 9.8 Tf [(sodium fluoride, 20 mM N-ethyl-maleimide, 100 M sodium ortovanadate, 1 mM PMSF and protease inhibitors cocktail )] TJ ET BT 26.250 214.977 Td /F1 9.8 Tf [(\(SIGMA\)] and boiled for 5 min. The lysates were centrifuged at 12 000 x g at 4 C for 30 min and 7.5 g of protein were )] TJ ET BT 26.250 203.072 Td /F1 9.8 Tf [(conducted to immunoblot assay with a rabbit polyclonal antibody to ubiquitin.)] TJ ET BT 26.250 183.667 Td /F1 9.8 Tf [(The secondary antibodies \(1/2000\) followed by ECL detection reagents \(Amersham\) were used for immunodetection. )] TJ ET BT 26.250 171.762 Td /F1 9.8 Tf [(Immunoblot of -actin diluted \(1/20000\) was performed to demonstrate equal protein loading. The blots were quantified by )] TJ ET BT 26.250 159.858 Td /F1 9.8 Tf [(computer-assisted video densitometry.)] TJ ET BT 26.250 140.453 Td /F4 9.8 Tf [(Characterization of defined and glia-conditioned medium protein profile. )] TJ ET BT 363.766 140.453 Td /F1 9.8 Tf [(Defined E12 and embryonic E16 glia-conditioned )] TJ ET BT 26.250 128.548 Td /F1 9.8 Tf [(media were lyophilized and concentrated in 5 % of initial volume. Protein concentration was determined by BCA method and 5 )] TJ ET BT 26.250 116.643 Td /F1 9.8 Tf [(g of protein from the supernatants of both media, were used for the electrophoresis in 4-20 % polyacrylamide gel. )] TJ ET BT 26.250 104.739 Td /F1 9.8 Tf [(Electrophoresis was carried out at 150V. After completion of electrophoresis, the gel was developed using silver staining to )] TJ ET BT 26.250 92.834 Td /F1 9.8 Tf [(observe protein profile.)] TJ ET BT 26.250 73.429 Td /F4 9.8 Tf [(Statistical analysis. )] TJ ET BT 118.924 73.429 Td /F1 9.8 Tf [(The statistical analysis was performed by one-way analysis of variance \(ANOVA\), followed by )] TJ ET BT 26.250 61.524 Td /F1 9.8 Tf [(NewmanKeuls multiple comparison test. Differences were considered statistically significant when p< 0.05. Analysis of data )] TJ ET BT 26.250 49.620 Td /F1 9.8 Tf [(was performed using the Graph Pad Software \(San Diego, CA\) Prism 4 software.)] TJ ET Q q 15.000 20.334 577.500 756.666 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(To investigate the mechanisms of neuroprotection by fetal GCM, we studied the ubiquitination of proteins, and the expression of )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(death and chaperone proteins by Western blot assays. To compare the effects of GCM with the well-known neuronal growth )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(factors, we treated Q111 cells with fetal GCM versus different growth factors such as GDNF \(50 and 100 ng/ml\), bFGF \(10 and )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(20 ng/ml\) and BDNF \(50 and 100 ng/ml\). We initially studied the effects of each growth factor on Q111 cells and compared )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(them with the fetal GCM. Then, we studied the protection of the growth factors and fetal GCM on H)] TJ ET BT 452.198 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 457.017 719.857 Td /F1 9.8 Tf [(O)] TJ ET BT 464.602 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 469.421 719.857 Td /F1 9.8 Tf [( induced cell death in )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(Q111 cells.)] TJ ET BT 26.250 688.548 Td /F4 9.8 Tf [(Cell survival, proliferation assay and cell differentiation. )] TJ ET BT 287.969 688.548 Td /F1 9.8 Tf [(Necrotic cell death was measured according to LDH activity in the )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(culture medium and by trypan blue dye exclusion in cells. LDH activity was measured by using a cytotoxicity detection kit )] TJ ET 0.267 0.267 0.267 rg BT 548.616 678.150 Td /F4 8.7 Tf [(12)] TJ ET BT 558.253 678.150 Td /F4 8.7 Tf [(33)] TJ ET 0.271 0.267 0.267 rg BT 567.891 676.643 Td /F1 9.8 Tf [( . )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(Apoptotic cell death was measured by nuclei stained with bis-benzimide \(Hoechst 33342\). Mitochondrial activity was analyzed )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(with the 3-\(4,5-dimethylthiazol-2-yl\)-2,5-diphenyl tetrazolium bromide \(MTT\) assay in Q7 and Q111 cells. The MTT assay )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(determines the ability of cells to metabolize MTT. At the end of the cell treatment period, 300 l of culture medium were )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(removed from total 500 l of each well, and 20 l of MTT solution \(5 mg/ml\) added and incubated for 2 h. At this time, 200 l of )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(solubilization solution \(10 % SDS in 0.01 M HCl\) were then added to the wells, and, after 24 h of incubation at 37 C, 100 l )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(transferred into a 96-well microtiter plate. The absorption value at 540 nm was measured in an automatic microtiter reader )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(\(Spectra Fluor; Tecan, Mnnedorf, Switzerland\). To test the differentiation of the cells, we counted the number of multiprocess )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(cells as the number of cells with 3 or more prolongations.)] TJ ET BT 26.250 562.000 Td /F4 9.8 Tf [(Immunocytochemistry. )] TJ ET BT 135.694 562.000 Td /F1 9.8 Tf [(After the experimental treatments, the cells were fixed with 4 % paraformaldehyde. Then, cells were )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(postfixed and permeabilized in ethanol-acetic acid \(19:1\) for 10 min at -20 C and incubated in a blocking solution followed by )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(overnight incubation at 4 C with the cytoplasmatic class III -tubulin primary antibody \(1:300\) and with a protein of the )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(presynaptic vesicle exocytosis, synaptophysin primary antibody \(1:200\). Fluorescein and Alexa Fluor-conjugated secondary )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(antibodies were used to visualize positive cells under fluorescent microscopy. Nuclei were stained by bis-benzimide \(Hoechst )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(33342\) and immunostaining was visualized under fluorescent microscopy. The number of immunoreactive cells was counted in )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(predefined parallel strips.)] TJ ET BT 26.250 471.167 Td /F4 9.8 Tf [(Measurement of GSH and ROS levels.)] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(Total glutathione levels were measured by the method of Tietze )] TJ ET 0.267 0.267 0.267 rg BT 303.170 453.269 Td /F4 8.7 Tf [(34)] TJ ET 0.271 0.267 0.267 rg BT 312.807 451.762 Td /F1 9.8 Tf [(. Intracellular levels of ROS were detected using the non-)] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(fluorescence DCF dye, which is oxidized by intracellular ROS to form the highly fluorescence DCF. The cells were incubated )] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(with 5 M DCFH-DA 30 min before H)] TJ ET BT 186.618 425.888 Td /F1 8.7 Tf [(2)] TJ ET BT 191.437 427.953 Td /F1 9.8 Tf [(O)] TJ ET BT 199.022 425.888 Td /F1 8.7 Tf [(2)] TJ ET BT 203.841 427.953 Td /F1 9.8 Tf [( treatments. After washing the excess of DCF with a medium free of phenol red \(PBS )] TJ ET BT 26.250 416.048 Td /F1 9.8 Tf [(with 1mM glucose\), the fluorescence was measured in a laser-scanning confocal microscope \(Nikon eclipse Ti\) or in an )] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(automatic microtiter reader \(Spectra Fluor, Tecan\) at excitation and emission of 485 nm and 582 nm, respectively. Fluorescence )] TJ ET BT 26.250 392.238 Td /F1 9.8 Tf [(was quantified by automated image analysis with Image Pro software. For each section mean fluorescence was calculated from )] TJ ET BT 26.250 380.334 Td /F1 9.8 Tf [(7 separate single fields.)] TJ ET BT 26.250 360.929 Td /F4 9.8 Tf [(Western blot analysis and detection of ubiquitinated proteins.)] TJ ET BT 26.250 341.524 Td /F1 9.8 Tf [(Q111 extracts for western blot analysis were prepared in ice-cold extraction buffer consisting of 20 mM TrisHCl \(pH 7.4\), 10 )] TJ ET BT 26.250 329.619 Td /F1 9.8 Tf [(mM potassium acetate \(AcK\), 1 mM dithiothreitol \(DTT\), 0.25 % NP-40, 1 mM EDTA, 2 mM EGTA, 1 mM PMSF, protease )] TJ ET BT 26.250 317.715 Td /F1 9.8 Tf [(inhibitors cocktail \(SIGMA\) and a cocktail of phosphatase inhibitors \(100 mM sodium fluoride, 20 mM sodium molybdate and 20 )] TJ ET BT 26.250 305.810 Td /F1 9.8 Tf [(mM -glicerophosphate\). The samples were homogenized, centrifuged at 4 C and protein content was determined by the BCA )] TJ ET BT 26.250 293.905 Td /F1 9.8 Tf [(protein assay kit. Total protein \(30 g\) was electrophoresed in 10 % SDS-PAGE gels and transferred to 0.45 mm nitrocellulose )] TJ ET BT 26.250 282.000 Td /F1 9.8 Tf [(membranes \(Amersham\), as described previously)] TJ ET 0.267 0.267 0.267 rg BT 241.345 283.508 Td /F4 8.7 Tf [(12)] TJ ET BT 250.982 283.508 Td /F4 8.7 Tf [(33)] TJ ET 0.271 0.267 0.267 rg BT 260.619 282.000 Td /F1 9.8 Tf [(. After blocking, blots were incubated overnight at 4 C in 5 % non- fat )] TJ ET BT 26.250 270.096 Td /F1 9.8 Tf [(dried milk with primary antibodies: the chaperone anti-HSP-70 \(1/700\); anti-CHIP \(1/1000\) and anti-PARP \(1/1000\).)] TJ ET BT 26.250 250.691 Td /F1 9.8 Tf [(To determine changes in ubiquitination, cell cultures treated or untreated with H)] TJ ET BT 368.738 248.627 Td /F1 8.7 Tf [(2)] TJ ET BT 373.557 250.691 Td /F1 9.8 Tf [(O)] TJ ET BT 381.142 248.627 Td /F1 8.7 Tf [(2)] TJ ET BT 385.961 250.691 Td /F1 9.8 Tf [( \(2 M\) in serum-free defined medium and in )] TJ ET BT 26.250 238.786 Td /F1 9.8 Tf [(GCM for 24 h, were scraped in 150 l of lysis buffer [50mM TrisHCl, 150 mM NaCl, 20 mM EDTA, 1 % TritonX-100, 50 mM )] TJ ET BT 26.250 226.881 Td /F1 9.8 Tf [(sodium fluoride, 20 mM N-ethyl-maleimide, 100 M sodium ortovanadate, 1 mM PMSF and protease inhibitors cocktail )] TJ ET BT 26.250 214.977 Td /F1 9.8 Tf [(\(SIGMA\)] and boiled for 5 min. The lysates were centrifuged at 12 000 x g at 4 C for 30 min and 7.5 g of protein were )] TJ ET BT 26.250 203.072 Td /F1 9.8 Tf [(conducted to immunoblot assay with a rabbit polyclonal antibody to ubiquitin.)] TJ ET BT 26.250 183.667 Td /F1 9.8 Tf [(The secondary antibodies \(1/2000\) followed by ECL detection reagents \(Amersham\) were used for immunodetection. )] TJ ET BT 26.250 171.762 Td /F1 9.8 Tf [(Immunoblot of -actin diluted \(1/20000\) was performed to demonstrate equal protein loading. The blots were quantified by )] TJ ET BT 26.250 159.858 Td /F1 9.8 Tf [(computer-assisted video densitometry.)] TJ ET BT 26.250 140.453 Td /F4 9.8 Tf [(Characterization of defined and glia-conditioned medium protein profile. )] TJ ET BT 363.766 140.453 Td /F1 9.8 Tf [(Defined E12 and embryonic E16 glia-conditioned )] TJ ET BT 26.250 128.548 Td /F1 9.8 Tf [(media were lyophilized and concentrated in 5 % of initial volume. Protein concentration was determined by BCA method and 5 )] TJ ET BT 26.250 116.643 Td /F1 9.8 Tf [(g of protein from the supernatants of both media, were used for the electrophoresis in 4-20 % polyacrylamide gel. )] TJ ET BT 26.250 104.739 Td /F1 9.8 Tf [(Electrophoresis was carried out at 150V. After completion of electrophoresis, the gel was developed using silver staining to )] TJ ET BT 26.250 92.834 Td /F1 9.8 Tf [(observe protein profile.)] TJ ET BT 26.250 73.429 Td /F4 9.8 Tf [(Statistical analysis. )] TJ ET BT 118.924 73.429 Td /F1 9.8 Tf [(The statistical analysis was performed by one-way analysis of variance \(ANOVA\), followed by )] TJ ET BT 26.250 61.524 Td /F1 9.8 Tf [(NewmanKeuls multiple comparison test. Differences were considered statistically significant when p< 0.05. Analysis of data )] TJ ET BT 26.250 49.620 Td /F1 9.8 Tf [(was performed using the Graph Pad Software \(San Diego, CA\) Prism 4 software.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(3)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 307 0 obj << /Type /Annot /Subtype /Link /A 308 0 R /Border [0 0 0] /H /I /Rect [ 548.6160 677.3484 558.2533 686.1667 ] >> endobj 308 0 obj << /Type /Action >> endobj 309 0 obj << /Type /Annot /Subtype /Link /A 310 0 R /Border [0 0 0] /H /I /Rect [ 558.2533 677.3484 567.8907 686.1667 ] >> endobj 310 0 obj << /Type /Action >> endobj 311 0 obj << /Type /Annot /Subtype /Link /A 312 0 R /Border [0 0 0] /H /I /Rect [ 303.1695 452.4677 312.8068 461.2860 ] >> endobj 312 0 obj << /Type /Action >> endobj 313 0 obj << /Type /Annot /Subtype /Link /A 314 0 R /Border [0 0 0] /H /I /Rect [ 241.3448 282.7059 250.9821 291.5243 ] >> endobj 314 0 obj << /Type /Action >> endobj 315 0 obj << /Type /Annot /Subtype /Link /A 316 0 R /Border [0 0 0] /H /I /Rect [ 250.9821 282.7059 260.6194 291.5243 ] >> endobj 316 0 obj << /Type /Action >> endobj 317 0 obj << /Type /Annot /Subtype /Link /A 318 0 R /Border [0 0 0] /H /I /Rect [ 548.6160 677.3484 558.2533 686.1667 ] >> endobj 318 0 obj << /Type /Action >> endobj 319 0 obj << /Type /Annot /Subtype /Link /A 320 0 R /Border [0 0 0] /H /I /Rect [ 558.2533 677.3484 567.8907 686.1667 ] >> endobj 320 0 obj << /Type /Action >> endobj 321 0 obj << /Type /Annot /Subtype /Link /A 322 0 R /Border [0 0 0] /H /I /Rect [ 303.1695 452.4677 312.8068 461.2860 ] >> endobj 322 0 obj << /Type /Action >> endobj 323 0 obj << /Type /Annot /Subtype /Link /A 324 0 R /Border [0 0 0] /H /I /Rect [ 241.3448 282.7059 250.9821 291.5243 ] >> endobj 324 0 obj << /Type /Action >> endobj 325 0 obj << /Type /Annot /Subtype /Link /A 326 0 R /Border [0 0 0] /H /I /Rect [ 250.9821 282.7059 260.6194 291.5243 ] >> endobj 326 0 obj << /Type /Action >> endobj 327 0 obj << /Type /Annot /Subtype /Link /A 328 0 R /Border [0 0 0] /H /I /Rect [ 548.6160 677.3484 558.2533 686.1667 ] >> endobj 328 0 obj << /Type /Action >> endobj 329 0 obj << /Type /Annot /Subtype /Link /A 330 0 R /Border [0 0 0] /H /I /Rect [ 558.2533 677.3484 567.8907 686.1667 ] >> endobj 330 0 obj << /Type /Action >> endobj 331 0 obj << /Type /Annot /Subtype /Link /A 332 0 R /Border [0 0 0] /H /I /Rect [ 303.1695 452.4677 312.8068 461.2860 ] >> endobj 332 0 obj << /Type /Action >> endobj 333 0 obj << /Type /Annot /Subtype /Link /A 334 0 R /Border [0 0 0] /H /I /Rect [ 241.3448 282.7059 250.9821 291.5243 ] >> endobj 334 0 obj << /Type /Action >> endobj 335 0 obj << /Type /Annot /Subtype /Link /A 336 0 R /Border [0 0 0] /H /I /Rect [ 250.9821 282.7059 260.6194 291.5243 ] >> endobj 336 0 obj << /Type /Action >> endobj 337 0 obj << /Type /Page /Parent 3 0 R /Annots [ 339 0 R 342 0 R 345 0 R 347 0 R 349 0 R 351 0 R ] /Contents 338 0 R >> endobj 338 0 obj << /Length 10994 >> stream 0.271 0.267 0.267 rg q 15.000 119.497 577.500 657.503 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Results)] TJ ET BT 26.250 748.071 Td /F4 9.8 Tf [(Fetal striatal GCM prevents cell death produced by sodium glutamate in Q7 and more so in Q111 cells. )] TJ ET BT 503.074 748.071 Td /F1 9.8 Tf [(Sodium )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(glutamate, 5 mM x 24h, reduced cell proliferation and mitochondrial activity and increased cell death in Q7 and Q111 cells in )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(serum-free defined medium \(Fig 1A-C\). After treatment with GCM the number of high affinity GABA uptake sites, a marker of )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(striatal neurons, was reduced to around 75 % of controls \(Fig 1D\). Co-treatment, with fetal GCM completely reverted these )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(effects of glutamate in Q111 cells \(Fig 1A-D\).)] TJ ET 0.965 0.965 0.965 rg 26.250 398.581 555.000 291.991 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 690.571 m 581.250 690.571 l 581.250 689.821 l 26.250 689.821 l f 26.250 398.581 m 581.250 398.581 l 581.250 399.331 l 26.250 399.331 l f q 225.000 0 0 159.000 35.250 521.821 cm /I3 Do Q q 35.250 409.831 537.000 105.991 re W n 0.271 0.267 0.267 rg BT 35.250 506.298 Td /F4 9.8 Tf [(Fig. 1: Effects of fetal striatal gla-conditioned medium \(GCM\) on sodium glutamate induced toxicity in Q7 and )] TJ ET BT 35.250 494.393 Td /F4 9.8 Tf [(Q111 cells)] TJ ET BT 35.250 475.023 Td /F1 9.8 Tf [(\(A\) Representative micrograph of Q111 cells after 3DIV in GCM or DM during 24 hours. Cells were treated with Glut 5 mM )] TJ ET BT 35.250 461.287 Td /F1 9.8 Tf [(in DM or GCM \(scale bar = 50 ?m\). \(B\) Mitochondrial activity \(MTT assay\). \(C\) Cell death measured by LDH assay. \(D\) 3H-)] TJ ET BT 35.250 447.550 Td /F1 9.8 Tf [(GABA uptake in Q111 cells. The values are expressed as mean SEM \(n = 5-6 samples in each experimental group\). )] TJ ET BT 35.250 433.814 Td /F1 9.8 Tf [(Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. *p<0.05, ***p<0.001 vs control )] TJ ET BT 35.250 420.078 Td /F1 9.8 Tf [(group. +p<0.05, +++p<0.001 Q111 cells vs Q7 cells. )] TJ ET BT 263.975 425.798 Td /F1 8.7 Tf [(&&&)] TJ ET BT 281.317 420.078 Td /F1 9.8 Tf [(p<0.001 GCM + Glut vs Glut.)] TJ ET Q BT 26.250 381.557 Td /F4 9.8 Tf [(The fetal striatal GCM prevents cell death produced by 3-nitropropionic acid in Q111 cells. )] TJ ET BT 446.173 381.557 Td /F1 9.8 Tf [(The mitochondrial complex II )] TJ ET BT 26.250 369.652 Td /F1 9.8 Tf [(inhibitor \(3NP, 2.5 mM x 24h\) induced cell death in Q111 cells, as shown by reducing the neuronal area of the culture \(Fig 2A )] TJ ET BT 26.250 357.747 Td /F1 9.8 Tf [(and B\) and increasing the number of apoptotic cells of around 50 % more than in the untreated cultures \(Fig 2C and D\). Co-)] TJ ET BT 26.250 345.843 Td /F1 9.8 Tf [(treatment with fetal GCM increased the neuronal area of the cultures, expressed by the area of -tubulin \(Fig 2A and B\), and )] TJ ET BT 26.250 333.938 Td /F1 9.8 Tf [(reduced the levels of apoptosis not only to levels of the cultures untreated with 3NP, but to levels below 50 % of those of the )] TJ ET BT 26.250 322.033 Td /F1 9.8 Tf [(controls \(Fig 2C and D\).)] TJ ET 0.965 0.965 0.965 rg 26.250 119.497 555.000 192.655 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 312.152 m 581.250 312.152 l 581.250 311.402 l 26.250 311.402 l f q 225.000 0 0 159.000 35.250 143.402 cm /I4 Do Q q 35.250 119.497 537.000 17.905 re W n Q Q q 15.000 119.497 577.500 657.503 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Results)] TJ ET BT 26.250 748.071 Td /F4 9.8 Tf [(Fetal striatal GCM prevents cell death produced by sodium glutamate in Q7 and more so in Q111 cells. )] TJ ET BT 503.074 748.071 Td /F1 9.8 Tf [(Sodium )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(glutamate, 5 mM x 24h, reduced cell proliferation and mitochondrial activity and increased cell death in Q7 and Q111 cells in )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(serum-free defined medium \(Fig 1A-C\). After treatment with GCM the number of high affinity GABA uptake sites, a marker of )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(striatal neurons, was reduced to around 75 % of controls \(Fig 1D\). Co-treatment, with fetal GCM completely reverted these )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(effects of glutamate in Q111 cells \(Fig 1A-D\).)] TJ ET 0.965 0.965 0.965 rg 26.250 398.581 555.000 291.991 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 690.571 m 581.250 690.571 l 581.250 689.821 l 26.250 689.821 l f 26.250 398.581 m 581.250 398.581 l 581.250 399.331 l 26.250 399.331 l f q 225.000 0 0 159.000 35.250 521.821 cm /I3 Do Q q 35.250 409.831 537.000 105.991 re W n 0.271 0.267 0.267 rg BT 35.250 506.298 Td /F4 9.8 Tf [(Fig. 1: Effects of fetal striatal gla-conditioned medium \(GCM\) on sodium glutamate induced toxicity in Q7 and )] TJ ET BT 35.250 494.393 Td /F4 9.8 Tf [(Q111 cells)] TJ ET BT 35.250 475.023 Td /F1 9.8 Tf [(\(A\) Representative micrograph of Q111 cells after 3DIV in GCM or DM during 24 hours. Cells were treated with Glut 5 mM )] TJ ET BT 35.250 461.287 Td /F1 9.8 Tf [(in DM or GCM \(scale bar = 50 ?m\). \(B\) Mitochondrial activity \(MTT assay\). \(C\) Cell death measured by LDH assay. \(D\) 3H-)] TJ ET BT 35.250 447.550 Td /F1 9.8 Tf [(GABA uptake in Q111 cells. The values are expressed as mean SEM \(n = 5-6 samples in each experimental group\). )] TJ ET BT 35.250 433.814 Td /F1 9.8 Tf [(Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. *p<0.05, ***p<0.001 vs control )] TJ ET BT 35.250 420.078 Td /F1 9.8 Tf [(group. +p<0.05, +++p<0.001 Q111 cells vs Q7 cells. )] TJ ET BT 263.975 425.798 Td /F1 8.7 Tf [(&&&)] TJ ET BT 281.317 420.078 Td /F1 9.8 Tf [(p<0.001 GCM + Glut vs Glut.)] TJ ET Q BT 26.250 381.557 Td /F4 9.8 Tf [(The fetal striatal GCM prevents cell death produced by 3-nitropropionic acid in Q111 cells. )] TJ ET BT 446.173 381.557 Td /F1 9.8 Tf [(The mitochondrial complex II )] TJ ET BT 26.250 369.652 Td /F1 9.8 Tf [(inhibitor \(3NP, 2.5 mM x 24h\) induced cell death in Q111 cells, as shown by reducing the neuronal area of the culture \(Fig 2A )] TJ ET BT 26.250 357.747 Td /F1 9.8 Tf [(and B\) and increasing the number of apoptotic cells of around 50 % more than in the untreated cultures \(Fig 2C and D\). Co-)] TJ ET BT 26.250 345.843 Td /F1 9.8 Tf [(treatment with fetal GCM increased the neuronal area of the cultures, expressed by the area of -tubulin \(Fig 2A and B\), and )] TJ ET BT 26.250 333.938 Td /F1 9.8 Tf [(reduced the levels of apoptosis not only to levels of the cultures untreated with 3NP, but to levels below 50 % of those of the )] TJ ET BT 26.250 322.033 Td /F1 9.8 Tf [(controls \(Fig 2C and D\).)] TJ ET 0.965 0.965 0.965 rg 26.250 119.497 555.000 192.655 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 312.152 m 581.250 312.152 l 581.250 311.402 l 26.250 311.402 l f q 225.000 0 0 159.000 35.250 143.402 cm /I4 Do Q q 35.250 119.497 537.000 17.905 re W n Q Q q 15.000 119.497 577.500 657.503 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Results)] TJ ET BT 26.250 748.071 Td /F4 9.8 Tf [(Fetal striatal GCM prevents cell death produced by sodium glutamate in Q7 and more so in Q111 cells. )] TJ ET BT 503.074 748.071 Td /F1 9.8 Tf [(Sodium )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(glutamate, 5 mM x 24h, reduced cell proliferation and mitochondrial activity and increased cell death in Q7 and Q111 cells in )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(serum-free defined medium \(Fig 1A-C\). After treatment with GCM the number of high affinity GABA uptake sites, a marker of )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(striatal neurons, was reduced to around 75 % of controls \(Fig 1D\). Co-treatment, with fetal GCM completely reverted these )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(effects of glutamate in Q111 cells \(Fig 1A-D\).)] TJ ET 0.965 0.965 0.965 rg 26.250 398.581 555.000 291.991 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 690.571 m 581.250 690.571 l 581.250 689.821 l 26.250 689.821 l f 26.250 398.581 m 581.250 398.581 l 581.250 399.331 l 26.250 399.331 l f q 225.000 0 0 159.000 35.250 521.821 cm /I3 Do Q q 35.250 409.831 537.000 105.991 re W n 0.271 0.267 0.267 rg BT 35.250 506.298 Td /F4 9.8 Tf [(Fig. 1: Effects of fetal striatal gla-conditioned medium \(GCM\) on sodium glutamate induced toxicity in Q7 and )] TJ ET BT 35.250 494.393 Td /F4 9.8 Tf [(Q111 cells)] TJ ET BT 35.250 475.023 Td /F1 9.8 Tf [(\(A\) Representative micrograph of Q111 cells after 3DIV in GCM or DM during 24 hours. Cells were treated with Glut 5 mM )] TJ ET BT 35.250 461.287 Td /F1 9.8 Tf [(in DM or GCM \(scale bar = 50 ?m\). \(B\) Mitochondrial activity \(MTT assay\). \(C\) Cell death measured by LDH assay. \(D\) 3H-)] TJ ET BT 35.250 447.550 Td /F1 9.8 Tf [(GABA uptake in Q111 cells. The values are expressed as mean SEM \(n = 5-6 samples in each experimental group\). )] TJ ET BT 35.250 433.814 Td /F1 9.8 Tf [(Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. *p<0.05, ***p<0.001 vs control )] TJ ET BT 35.250 420.078 Td /F1 9.8 Tf [(group. +p<0.05, +++p<0.001 Q111 cells vs Q7 cells. )] TJ ET BT 263.975 425.798 Td /F1 8.7 Tf [(&&&)] TJ ET BT 281.317 420.078 Td /F1 9.8 Tf [(p<0.001 GCM + Glut vs Glut.)] TJ ET Q BT 26.250 381.557 Td /F4 9.8 Tf [(The fetal striatal GCM prevents cell death produced by 3-nitropropionic acid in Q111 cells. )] TJ ET BT 446.173 381.557 Td /F1 9.8 Tf [(The mitochondrial complex II )] TJ ET BT 26.250 369.652 Td /F1 9.8 Tf [(inhibitor \(3NP, 2.5 mM x 24h\) induced cell death in Q111 cells, as shown by reducing the neuronal area of the culture \(Fig 2A )] TJ ET BT 26.250 357.747 Td /F1 9.8 Tf [(and B\) and increasing the number of apoptotic cells of around 50 % more than in the untreated cultures \(Fig 2C and D\). Co-)] TJ ET BT 26.250 345.843 Td /F1 9.8 Tf [(treatment with fetal GCM increased the neuronal area of the cultures, expressed by the area of -tubulin \(Fig 2A and B\), and )] TJ ET BT 26.250 333.938 Td /F1 9.8 Tf [(reduced the levels of apoptosis not only to levels of the cultures untreated with 3NP, but to levels below 50 % of those of the )] TJ ET BT 26.250 322.033 Td /F1 9.8 Tf [(controls \(Fig 2C and D\).)] TJ ET 0.965 0.965 0.965 rg 26.250 119.497 555.000 192.655 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 312.152 m 581.250 312.152 l 581.250 311.402 l 26.250 311.402 l f q 225.000 0 0 159.000 35.250 143.402 cm /I4 Do Q q 35.250 119.497 537.000 17.905 re W n Q Q q 225.000 0 0 159.000 35.250 521.821 cm /I3 Do Q q 225.000 0 0 159.000 35.250 143.402 cm /I4 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(4)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 339 0 obj << /Type /Annot /Subtype /Link /A 340 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 521.8215 260.2500 680.8215 ] >> endobj 340 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure1.jpg) >> endobj 341 0 obj << /Type /XObject /Subtype /Image /Width 300 /Height 212 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 19888>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Qɣ4qGfhڎ(89( (ڎ(8j({QǵGq@PEQ5⿉>kzդ#E2B>b>8*a^>4{NuΔey#o2)s`#} Q9:~'kw4>:%wI6F (ÅzGaX[A@8a kFhڎ=8j98(j88 ZմO~6[]ծm/K{}1,?V:,&DĪ8#_~!Ov+uZ5՚[ʏtZȲKDh[u߁.{~ӯ5;WxX",AE_JвU<tMvr3$#F#!w|sր4|5 6hEneLRDa¶9zfhXYmXF@ڮq@G>ZRӞ.akD \)B V8ڀ)pi0h((9/>sI/IR'K9)N})9“h? 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(q" q|V? ڱ>/Ծ,xpi [i{$O> $ g8_߷>O͸Lƀ,fk'_ ux~[R[dI͹)`[`qkZ4*ץFU(ZrQj]m{\ԝ8T[ٴ>=WYt=/CZ ~6m@j{@S,Misoh'R1Sr|V7"ឹ1,xe{0p c##u¾Fxg2B!2{z_mJs)bʤM4Ii}*mH=U q'{y{}j<;PѴ[=' -f%<'=;u%|}xcLno%kC"eg ~Xlj#n_KMCEM0@y\V׉[MFW&5}*>r>c:kˣ*t*K#'4~իvTke]yKLԯ|uyi͵O4;AV Ik}j bKLan>P9ӥEPh3xbmij.IqkCgh:_E66$QR̹\(aTAH{i8˓][.xVQUOWhmyզihd4FWGʻM̜|{ ?س\o{{XѠ4&^+22OϵρwKa{{O"cCKH{Ww_߀~)V"Wk\3x$WXT.X%)Bhkߠ˫ai͸n)ͩV[ͲZ\ KW'-D:EБ佴Y.w§<=+ H.xizuٹӎkZs]EqٺuD2xjHG^u+t@͝2JP6H F1ӊO|0>WJ[%C3'=t | 6$g=#S鶲}J(mk0x}A^e J䒩ʤn]s-i *E씥ǫn\qR-.mS,^3HAòO#{Vke\ˬ[̲(H":t+oi-[R #{YD,m4msXdD  u>Y+WJ(B}J tk,*I9Wl~ܷj#4#I /t> 7Pah],4q 9I7)8ȯߎIn'UGrI~a3߄j)r~f]"UR*JyuzsIϵ.(4k?WZ=6k5n$Xzg=+C([VׯxdSOFB[j'{[Vд۝CR \H8u,ǀ* W,活.-]&wU6VA>HPmF :ѿ%p( {XoHXHPA¢4h%{-+LN-cFG $Ba+j6/Z5;@U8\ _ xs;NMm?"(9w,:6fQ.(ڼ#įZ]J J&KQo F]l|RmC{?^䅾$ly$S$0@vvF(}}9ր9><#-5XD7cC\r*9o5+1+L0k%Z^/<׹$?gECxSɁV?GFaAiW)unH˹Npø=+hrc/ G.tk_ƶW!^VB۞;T~~iȸjm#ǡ}(F9><5t^yk,jQ('wtkf3T_1"xx[xu|S-fI u,rYOQ\&eQεt|?|3AXxEmѴ[kLG+o_O>>uĪs}q st1'> >ptaVJ/ݾxL=JNs[;+G??Ut?ugB o}iAUZ\$ͩҧF*D՟G7?=<|0^OZ5ۭJ^{I5F dj4s@G`Q@GF(Ҁ—Ž=((ҀŒ0h((Œ9h9((qIEOPފ(4{EA\{~4Q@Pފ(APޏƊ(h?\{E%E(҃E endstream endobj 345 0 obj << /Type /Annot /Subtype /Link /A 346 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 521.8215 260.2500 680.8215 ] >> endobj 346 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure1.jpg) >> endobj 347 0 obj << /Type /Annot /Subtype /Link /A 348 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 143.4022 260.2500 302.4022 ] >> endobj 348 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-2.jpg) >> endobj 349 0 obj << /Type /Annot /Subtype /Link /A 350 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 521.8215 260.2500 680.8215 ] >> endobj 350 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure1.jpg) >> endobj 351 0 obj << /Type /Annot /Subtype /Link /A 352 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 143.4022 260.2500 302.4022 ] >> endobj 352 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-2.jpg) >> endobj 353 0 obj << /Type /Page /Parent 3 0 R /Annots [ 355 0 R 358 0 R 360 0 R ] /Contents 354 0 R >> endobj 354 0 obj << /Length 12738 >> stream q 15.000 222.989 577.500 554.011 re W n 0.965 0.965 0.965 rg 26.250 611.287 555.000 165.713 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 611.287 m 581.250 611.287 l 581.250 612.037 l 26.250 612.037 l f q 35.250 622.537 537.000 154.463 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 2: The gla-conditioned medium protects from 3-nitropropionic acid \(3NP\) toxicity in Q111 cells)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(\(A\) Photomicrographs from control \(DM\), 3NP \(2.5mM\) and fetal striatal GCM treated cells for 24 h at 3 DIV \(scale bar = 30 )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(?-tubulin\). \(C\) )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(Photomicrograph of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells. The arrows )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(indicate the apoptotic cells in 3NP treated group. \(D\) Percentage of chromatin condensed and fragmented nuclei in the )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(different experimental groups. The values are expressed as mean SEM \(n = 4-6 samples in each experimental group\). )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. **p<0.01, ***p<0.001 vs control )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(group. )] TJ ET BT 65.602 671.408 Td /F1 8.7 Tf [(&&)] TJ ET BT 77.163 665.689 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 112.673 671.408 Td /F1 8.7 Tf [(&&&)] TJ ET BT 130.015 665.689 Td /F1 9.8 Tf [(p<0.001 GCM + 3NP vs 3NP.)] TJ ET Q BT 26.250 594.263 Td /F4 9.8 Tf [(Fetal striatal GCM decreases H)] TJ ET BT 169.302 592.199 Td /F4 8.7 Tf [(2)] TJ ET BT 174.121 594.263 Td /F4 9.8 Tf [(O)] TJ ET BT 181.706 592.199 Td /F4 8.7 Tf [(2)] TJ ET BT 186.525 594.263 Td /F4 9.8 Tf [( induced cell death in Q111 cells. )] TJ ET BT 342.057 594.263 Td /F1 9.8 Tf [(The treatment with H)] TJ ET BT 432.547 592.199 Td /F1 8.7 Tf [(2)] TJ ET BT 437.365 594.263 Td /F1 9.8 Tf [(O)] TJ ET BT 444.951 592.199 Td /F1 8.7 Tf [(2)] TJ ET BT 449.769 594.263 Td /F1 9.8 Tf [(, 2M for 24 hours, )] TJ ET BT 26.250 582.358 Td /F1 9.8 Tf [(decreased the -tubulin area of the cultures to around 2/3 of the controls \(Fig 3A and B\), almost doubled the percentage of )] TJ ET BT 26.250 570.453 Td /F1 9.8 Tf [(apoptotic cells in the cultures \(Fig 3C\) and more than tripled the number of necrotic cells, as shown by trypan blue staining \(Fig )] TJ ET BT 26.250 558.549 Td /F1 9.8 Tf [(3D and E\). Co-treatment with fetal GCM completely prevented the toxic effects of H)] TJ ET BT 385.518 556.484 Td /F1 8.7 Tf [(2)] TJ ET BT 390.337 558.549 Td /F1 9.8 Tf [(O)] TJ ET BT 397.922 556.484 Td /F1 8.7 Tf [(2)] TJ ET BT 402.741 558.549 Td /F1 9.8 Tf [(, on -tubulin staining \(Fig 3A and B\) )] TJ ET BT 26.250 546.644 Td /F1 9.8 Tf [(and apoptosis \(Fig 3C\) and reduced the percentage of necrosis to levels even below those of the controls \(Fig 3D and E\).)] TJ ET BT 26.250 527.239 Td /F1 9.8 Tf [(The treatment with H)] TJ ET BT 116.740 525.175 Td /F1 8.7 Tf [(2)] TJ ET BT 121.558 527.239 Td /F1 9.8 Tf [(O)] TJ ET BT 129.144 525.175 Td /F1 8.7 Tf [(2)] TJ ET BT 133.963 527.239 Td /F1 9.8 Tf [(, induced greater accumulation of intracellular ROS in Q111 cells than in Q7 cells \(data not shown\). )] TJ ET BT 26.250 515.334 Td /F1 9.8 Tf [(The accumulation of ROS in Q111 cells was time \(Fig 3F and G\) and dose dependent \(Fig 3H\). Co-treatment with fetal GCM )] TJ ET BT 26.250 503.430 Td /F1 9.8 Tf [(completely prevented the elevation of ROS induced by H)] TJ ET BT 271.199 501.365 Td /F1 8.7 Tf [(2)] TJ ET BT 276.018 503.430 Td /F1 9.8 Tf [(O)] TJ ET BT 283.603 501.365 Td /F1 8.7 Tf [(2)] TJ ET BT 288.422 503.430 Td /F1 9.8 Tf [(\(Fig 3I and J\).)] TJ ET 0.965 0.965 0.965 rg 26.250 222.989 555.000 270.560 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 493.549 m 581.250 493.549 l 581.250 492.799 l 26.250 492.799 l f q 168.750 0 0 225.000 35.250 258.799 cm /I5 Do Q q 35.250 222.989 537.000 29.810 re W n Q Q q 15.000 222.989 577.500 554.011 re W n 0.965 0.965 0.965 rg 26.250 611.287 555.000 165.713 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 611.287 m 581.250 611.287 l 581.250 612.037 l 26.250 612.037 l f q 35.250 622.537 537.000 154.463 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 2: The gla-conditioned medium protects from 3-nitropropionic acid \(3NP\) toxicity in Q111 cells)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(\(A\) Photomicrographs from control \(DM\), 3NP \(2.5mM\) and fetal striatal GCM treated cells for 24 h at 3 DIV \(scale bar = 30 )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(?-tubulin\). \(C\) )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(Photomicrograph of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells. The arrows )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(indicate the apoptotic cells in 3NP treated group. \(D\) Percentage of chromatin condensed and fragmented nuclei in the )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(different experimental groups. The values are expressed as mean SEM \(n = 4-6 samples in each experimental group\). )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. **p<0.01, ***p<0.001 vs control )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(group. )] TJ ET BT 65.602 671.408 Td /F1 8.7 Tf [(&&)] TJ ET BT 77.163 665.689 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 112.673 671.408 Td /F1 8.7 Tf [(&&&)] TJ ET BT 130.015 665.689 Td /F1 9.8 Tf [(p<0.001 GCM + 3NP vs 3NP.)] TJ ET Q BT 26.250 594.263 Td /F4 9.8 Tf [(Fetal striatal GCM decreases H)] TJ ET BT 169.302 592.199 Td /F4 8.7 Tf [(2)] TJ ET BT 174.121 594.263 Td /F4 9.8 Tf [(O)] TJ ET BT 181.706 592.199 Td /F4 8.7 Tf [(2)] TJ ET BT 186.525 594.263 Td /F4 9.8 Tf [( induced cell death in Q111 cells. )] TJ ET BT 342.057 594.263 Td /F1 9.8 Tf [(The treatment with H)] TJ ET BT 432.547 592.199 Td /F1 8.7 Tf [(2)] TJ ET BT 437.365 594.263 Td /F1 9.8 Tf [(O)] TJ ET BT 444.951 592.199 Td /F1 8.7 Tf [(2)] TJ ET BT 449.769 594.263 Td /F1 9.8 Tf [(, 2M for 24 hours, )] TJ ET BT 26.250 582.358 Td /F1 9.8 Tf [(decreased the -tubulin area of the cultures to around 2/3 of the controls \(Fig 3A and B\), almost doubled the percentage of )] TJ ET BT 26.250 570.453 Td /F1 9.8 Tf [(apoptotic cells in the cultures \(Fig 3C\) and more than tripled the number of necrotic cells, as shown by trypan blue staining \(Fig )] TJ ET BT 26.250 558.549 Td /F1 9.8 Tf [(3D and E\). Co-treatment with fetal GCM completely prevented the toxic effects of H)] TJ ET BT 385.518 556.484 Td /F1 8.7 Tf [(2)] TJ ET BT 390.337 558.549 Td /F1 9.8 Tf [(O)] TJ ET BT 397.922 556.484 Td /F1 8.7 Tf [(2)] TJ ET BT 402.741 558.549 Td /F1 9.8 Tf [(, on -tubulin staining \(Fig 3A and B\) )] TJ ET BT 26.250 546.644 Td /F1 9.8 Tf [(and apoptosis \(Fig 3C\) and reduced the percentage of necrosis to levels even below those of the controls \(Fig 3D and E\).)] TJ ET BT 26.250 527.239 Td /F1 9.8 Tf [(The treatment with H)] TJ ET BT 116.740 525.175 Td /F1 8.7 Tf [(2)] TJ ET BT 121.558 527.239 Td /F1 9.8 Tf [(O)] TJ ET BT 129.144 525.175 Td /F1 8.7 Tf [(2)] TJ ET BT 133.963 527.239 Td /F1 9.8 Tf [(, induced greater accumulation of intracellular ROS in Q111 cells than in Q7 cells \(data not shown\). )] TJ ET BT 26.250 515.334 Td /F1 9.8 Tf [(The accumulation of ROS in Q111 cells was time \(Fig 3F and G\) and dose dependent \(Fig 3H\). Co-treatment with fetal GCM )] TJ ET BT 26.250 503.430 Td /F1 9.8 Tf [(completely prevented the elevation of ROS induced by H)] TJ ET BT 271.199 501.365 Td /F1 8.7 Tf [(2)] TJ ET BT 276.018 503.430 Td /F1 9.8 Tf [(O)] TJ ET BT 283.603 501.365 Td /F1 8.7 Tf [(2)] TJ ET BT 288.422 503.430 Td /F1 9.8 Tf [(\(Fig 3I and J\).)] TJ ET 0.965 0.965 0.965 rg 26.250 222.989 555.000 270.560 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 493.549 m 581.250 493.549 l 581.250 492.799 l 26.250 492.799 l f q 168.750 0 0 225.000 35.250 258.799 cm /I5 Do Q q 35.250 222.989 537.000 29.810 re W n Q Q q 15.000 222.989 577.500 554.011 re W n 0.965 0.965 0.965 rg 26.250 611.287 555.000 165.713 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 611.287 m 581.250 611.287 l 581.250 612.037 l 26.250 612.037 l f q 35.250 622.537 537.000 154.463 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 2: The gla-conditioned medium protects from 3-nitropropionic acid \(3NP\) toxicity in Q111 cells)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(\(A\) Photomicrographs from control \(DM\), 3NP \(2.5mM\) and fetal striatal GCM treated cells for 24 h at 3 DIV \(scale bar = 30 )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(?-tubulin\). \(C\) )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(Photomicrograph of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells. The arrows )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(indicate the apoptotic cells in 3NP treated group. \(D\) Percentage of chromatin condensed and fragmented nuclei in the )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(different experimental groups. The values are expressed as mean SEM \(n = 4-6 samples in each experimental group\). )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. **p<0.01, ***p<0.001 vs control )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(group. )] TJ ET BT 65.602 671.408 Td /F1 8.7 Tf [(&&)] TJ ET BT 77.163 665.689 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 112.673 671.408 Td /F1 8.7 Tf [(&&&)] TJ ET BT 130.015 665.689 Td /F1 9.8 Tf [(p<0.001 GCM + 3NP vs 3NP.)] TJ ET Q BT 26.250 594.263 Td /F4 9.8 Tf [(Fetal striatal GCM decreases H)] TJ ET BT 169.302 592.199 Td /F4 8.7 Tf [(2)] TJ ET BT 174.121 594.263 Td /F4 9.8 Tf [(O)] TJ ET BT 181.706 592.199 Td /F4 8.7 Tf [(2)] TJ ET BT 186.525 594.263 Td /F4 9.8 Tf [( induced cell death in Q111 cells. )] TJ ET BT 342.057 594.263 Td /F1 9.8 Tf [(The treatment with H)] TJ ET BT 432.547 592.199 Td /F1 8.7 Tf [(2)] TJ ET BT 437.365 594.263 Td /F1 9.8 Tf [(O)] TJ ET BT 444.951 592.199 Td /F1 8.7 Tf [(2)] TJ ET BT 449.769 594.263 Td /F1 9.8 Tf [(, 2M for 24 hours, )] TJ ET BT 26.250 582.358 Td /F1 9.8 Tf [(decreased the -tubulin area of the cultures to around 2/3 of the controls \(Fig 3A and B\), almost doubled the percentage of )] TJ ET BT 26.250 570.453 Td /F1 9.8 Tf [(apoptotic cells in the cultures \(Fig 3C\) and more than tripled the number of necrotic cells, as shown by trypan blue staining \(Fig )] TJ ET BT 26.250 558.549 Td /F1 9.8 Tf [(3D and E\). Co-treatment with fetal GCM completely prevented the toxic effects of H)] TJ ET BT 385.518 556.484 Td /F1 8.7 Tf [(2)] TJ ET BT 390.337 558.549 Td /F1 9.8 Tf [(O)] TJ ET BT 397.922 556.484 Td /F1 8.7 Tf [(2)] TJ ET BT 402.741 558.549 Td /F1 9.8 Tf [(, on -tubulin staining \(Fig 3A and B\) )] TJ ET BT 26.250 546.644 Td /F1 9.8 Tf [(and apoptosis \(Fig 3C\) and reduced the percentage of necrosis to levels even below those of the controls \(Fig 3D and E\).)] TJ ET BT 26.250 527.239 Td /F1 9.8 Tf [(The treatment with H)] TJ ET BT 116.740 525.175 Td /F1 8.7 Tf [(2)] TJ ET BT 121.558 527.239 Td /F1 9.8 Tf [(O)] TJ ET BT 129.144 525.175 Td /F1 8.7 Tf [(2)] TJ ET BT 133.963 527.239 Td /F1 9.8 Tf [(, induced greater accumulation of intracellular ROS in Q111 cells than in Q7 cells \(data not shown\). )] TJ ET BT 26.250 515.334 Td /F1 9.8 Tf [(The accumulation of ROS in Q111 cells was time \(Fig 3F and G\) and dose dependent \(Fig 3H\). Co-treatment with fetal GCM )] TJ ET BT 26.250 503.430 Td /F1 9.8 Tf [(completely prevented the elevation of ROS induced by H)] TJ ET BT 271.199 501.365 Td /F1 8.7 Tf [(2)] TJ ET BT 276.018 503.430 Td /F1 9.8 Tf [(O)] TJ ET BT 283.603 501.365 Td /F1 8.7 Tf [(2)] TJ ET BT 288.422 503.430 Td /F1 9.8 Tf [(\(Fig 3I and J\).)] TJ ET 0.965 0.965 0.965 rg 26.250 222.989 555.000 270.560 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 493.549 m 581.250 493.549 l 581.250 492.799 l 26.250 492.799 l f q 168.750 0 0 225.000 35.250 258.799 cm /I5 Do Q q 35.250 222.989 537.000 29.810 re W n Q Q q 168.750 0 0 225.000 35.250 258.799 cm /I5 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(5)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 355 0 obj << /Type /Annot /Subtype /Link /A 356 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 258.7987 204.0000 483.7987 ] >> endobj 356 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-3.jpg) >> endobj 357 0 obj << /Type /XObject /Subtype /Image /Width 225 /Height 300 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 28823>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?A|Ai:cyw3BEe_FZֲu :yYGuu'G g?ƥiYxQm9(#!Ug, I?aZfsb+<9ڿ+On[^CHMg2bGt1jv(yiQ@n'dңK]Fݩ py)f[km:|JQ~k׶Y=oKU{'n="h0{Ir6Ao߷:oQT+\XL1B8wWӏ/=9.ntIyJҔ͢[Zvo_%/__g5wAڜPG%u)Ÿۃ'~n&{oL--" WH9$gU kMU};kx| FW^vC o?n?5O泱I{KFُ'$s׏bcs` sWzd:UúFl!+F~lGx❶Ah]tqOjL#ن kv៷߈7 _"z:?:Lњ34ft~u}nh4lud 졎a0 \/< w{<șR<̑98$ia6A1C=3Fh( ]B!>)k{q F,]ђo%UF>_Oڗ̈́-NjuEhb*TRzI1QI^ÊsR_3쥰 e[c q\Ieezgk qKK}VЍJ]r? :-?Kn\r02I]5zLJ:宛=X=ƠZGm`|Jթ]+kv#Sy4ՖeV _Ť=oKdt7%<ӵGIiIj}\ nKn8+oAšn6ee,v>~7``r C$MX1b 1Xg,E[?U/6⵻w6[ #KO?TK']{_*scL4[˻ʔ{W6Fm]!cjI **+ǧֽԿl3saio&L+(v߫W?yem_^՟x7,I~ԄH<W#h kp+)闲Оy I JLABY ǶT|hg-KZ_-$'YI  QNYRBpNd^"ԡmjO"0ܞP˜_3┿ ~Px%%P/b_'*Z|v ?xzH$;[@9I?ſU+}ZMӢ{;km^T $ UK~ۓpy'0wm]hz=oiMyR6W8_0 y\ֿ%A^yJ k)fkJy"!U@e9o UJNOQx u{ eF/]F}M|)]:^dmmmFmb͜ w&)[q0M6>f~~lg`+-]4-J}^f3MvP73$ d>^8QQitz6u߻3 +ѷ[{T! omM"ekiLvt-wFu Bys2Y\cϭv7M TN (KUp!H@`mu{iqѪ򚦜Z.]k+6uZ u[O'C!e r0x֨xźMR.a61Y#d]0;gzgm x[Ök?IR/.\B38{0%Nor9+M i7ֺvumwcIմB[fp[?)ϙ7% %J;o}48 j)(ttҹ_5[<;h5( PqpHW g^֬m#]O4|>fbQ5V]厩ql5 XіQT-+`X~ ,[2&[r8I6A`{ |乭wh6(<ֻw+EU}kyݜF+[wS\g9>Z>"?=,ן%'-5;/Gif tR<Y_q;-ʃ} ݁=`b9c?׎<)c7N,-Zko"w!O ,؜: P lV8oG H.R9@r Q}?^):0K\iUuݔGnŰ pXa[xK MRm7Sf-Cрt-8_kc*Msj优 $*|+i6z//#e[Kv"(P}3yG G-ݺyb2(a0gi7{{itNoUԿx^ҭ`] KfZ;N13soR1$v0+H8 dRQO+>=zNqu 8*gYz^Bo8X`8F!%޽oյ:ua SM}zukuh׺>i4Z1ܺ"ۺL* kxĶI5kx|9A[}Nkέ|>A}[ZIymHo)mF9Kxkuμ |EԐ3])S9M]:y:iR:KMlG'5}F].Y\Eá <~\}u椗7Zi -$@*ל\˪ir}I.وf{U:}GJԬ,|x;YIœ䂭ӓֻ0TNG涱b :TkiӲt!_GukohTZ6ͽ7C1#prAx~,\C+I݃#(Bf`9v[qq>6YM#Iw {j ucvV {8dvC +63^9xw%:rn֎-ߢ&'J{=ںѶKDs7_KnVtD%WT:}06+:+N8.KCKmq!F0HǠnk>E?wڤRAJ~}sz?[A{_q4Z{{bHh;Ys_F(ӝKqG:iBQ:qqGwi+oc+4fczDŽ Q>ue4ZO6;2XLR\H?H/hy}>"ƘXZ|=JP{;^kYY-TgtoѾxsY5ԛR _ <$c5 vEmjZ.{L6-嵳vRM㛩㑵$L*l)` $<n\|[񝞉e4Ww$ח1`a*(Ag^U}NbEߞmυ|]xEK[mBHj9|m⫸/ᵍ&8oP!x0w]HXH\o|ֱg&G>_JKa@#%N͸ ǹQ ^ۦZYzi2}a7{Y-,4S?2h,&X%\ c998Nֿ_5, ImQk|BVk;ɨ{[V!oĂ6lnIR4mɏ)nPTw6v `c-_S̤4ϥ̦<+CoDq}uFVZZpGn+xz H7P#h~T$gv_Cbd># xkXؾ@g< zO]Mm#2ODrgC.4o7s>UK﫹Xgצ{xK㵆k2ZFE#1 5__,|AOhť͜%̓-p 1=ߗ۷Zfߔ qF('k4>xSQ`hs`PI`(sb|E%iؼI?FFH*YXpEt9bџâ(v$ΣmxLU.G_mھܞ!tVėr_\K&(ۍ1,9Qou9sY䩧Җ{V#rÜ15.b(7d$=xbXd((oNI]6 07 e獭8\t)FLbrX9*r'mukx&IZB rqp:rqz Рi ?7f2 ^xEVbxgkؑCd\r2k{RP_/-$F3g! 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cmP(Νo%;|ȝJH# Ao_-,kn2FXyǜ g/x6z)uG ʒK?9Tꇡx/Ï~'Qxml乒9 "."PB&4+ k\ZdFG-'vPxl+;EWagmsIkh 2EDg$c T(~.#oKkgE: ׈|o7JWoZi2I VRd|I> endobj 359 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-3.jpg) >> endobj 360 0 obj << /Type /Annot /Subtype /Link /A 361 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 258.7987 204.0000 483.7987 ] >> endobj 361 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-3.jpg) >> endobj 362 0 obj << /Type /Page /Parent 3 0 R /Annots [ 364 0 R 366 0 R 369 0 R 371 0 R 373 0 R 375 0 R ] /Contents 363 0 R >> endobj 363 0 obj << /Length 23610 >> stream q 15.000 136.225 577.500 640.775 re W n 0.965 0.965 0.965 rg 26.250 563.605 555.000 213.395 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 563.605 m 581.250 563.605 l 581.250 564.355 l 26.250 564.355 l f q 35.250 574.855 537.000 202.145 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: The gla-conditioned medium protects from cell death and intracellular ROS accumulation induced by H2O2 )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(in Q111 cells)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(\(A\) Photomicrographs from control, H)] TJ ET BT 196.710 735.053 Td /F1 8.7 Tf [(2)] TJ ET BT 201.529 736.201 Td /F1 9.8 Tf [(O)] TJ ET BT 209.114 735.053 Td /F1 8.7 Tf [(2)] TJ ET BT 213.933 736.201 Td /F1 9.8 Tf [( \(2 M\) and fetal striatal GCM treated cells for 24 h at 3 DIV \(scale bar = 30 m\) )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(?-tubulin\). \(C\) Percentage of )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(chromatin condensed and fragmented nuclei in the different experimental groups. \(D\) Photomicrographs of cell death by )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(trypan blue staining from the four experimental groups. \(E\) Percentage of necrotic cells stained with trypan blue in the four )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(experimental groups. \(F\) Confocal photomicrographs of time-dependently ROS generation-induced by 100 ?M H)] TJ ET BT 519.698 680.108 Td /F1 8.7 Tf [(2)] TJ ET BT 524.517 681.256 Td /F1 9.8 Tf [(O)] TJ ET BT 532.102 680.108 Td /F1 8.7 Tf [(2)] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(treatment \(scale = 50 ?m\). \(G\) DCF fluorescence was measured with a reader at 485 nm of excitation and 582 nm emission )] TJ ET BT 35.250 653.784 Td /F1 9.8 Tf [(and expressed in optical density units \(O.D\). \(H\) Quantification of dose-dependent ROS generation by H)] TJ ET BT 484.491 652.635 Td /F1 8.7 Tf [(2)] TJ ET BT 489.310 653.784 Td /F1 9.8 Tf [(O)] TJ ET BT 496.895 652.635 Td /F1 8.7 Tf [(2)] TJ ET BT 501.714 653.784 Td /F1 9.8 Tf [( \(12.5, 25, 50, )] TJ ET BT 35.250 640.048 Td /F1 9.8 Tf [(100 and 200 ?M\) for 10 min and expressed in O.D. \(I\) Photomicrographs with fluorescence microscope of pre-treatment )] TJ ET BT 35.250 626.311 Td /F1 9.8 Tf [(with GCM 30 min before 100 ?M H)] TJ ET BT 185.341 625.163 Td /F1 8.7 Tf [(2)] TJ ET BT 190.160 626.311 Td /F1 9.8 Tf [(O)] TJ ET BT 197.746 625.163 Td /F1 8.7 Tf [(2)] TJ ET BT 202.564 626.311 Td /F1 9.8 Tf [( for 10 min prevented the ROS generation. \(J\) Quantification of intracellular ROS )] TJ ET BT 35.250 612.575 Td /F1 9.8 Tf [(generation in the four experimental groups. The values are expressed as mean SEM \(n = 4-6 samples in each )] TJ ET BT 35.250 598.839 Td /F1 9.8 Tf [(experimental group\). \(Scale bar = 30 ?m\). Statistical analysis was performed by one-way ANOVA followed by Newman-)] TJ ET BT 35.250 585.103 Td /F1 9.8 Tf [(Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control group. )] TJ ET BT 290.261 590.822 Td /F1 8.7 Tf [(&&)] TJ ET BT 301.823 585.103 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 337.332 590.822 Td /F1 8.7 Tf [(&&&)] TJ ET BT 354.674 585.103 Td /F1 9.8 Tf [(p<0.001 GCM + H)] TJ ET BT 433.795 583.954 Td /F1 8.7 Tf [(2)] TJ ET BT 438.614 585.103 Td /F1 9.8 Tf [(O2 vs H)] TJ ET BT 473.831 583.954 Td /F1 8.7 Tf [(2)] TJ ET BT 478.650 585.103 Td /F1 9.8 Tf [(O)] TJ ET BT 486.235 583.954 Td /F1 8.7 Tf [(2)] TJ ET BT 491.054 585.103 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 546.582 Td /F4 9.8 Tf [(Mechanisms implicated in the GCM neuroprotection in Q111 cells)] TJ ET BT 26.250 527.177 Td /F4 9.8 Tf [(Free radicals. )] TJ ET BT 91.829 527.177 Td /F1 9.8 Tf [(In order to study the mechanisms implicated in the GCM neuroprotection from H)] TJ ET BT 436.998 525.113 Td /F1 8.7 Tf [(2)] TJ ET BT 441.817 527.177 Td /F1 9.8 Tf [(O)] TJ ET BT 449.402 525.113 Td /F1 8.7 Tf [(2)] TJ ET BT 454.221 527.177 Td /F1 9.8 Tf [( induced cell death, we )] TJ ET BT 26.250 515.272 Td /F1 9.8 Tf [(investigated the differential pattern of proteins between defined medium \(DM\) and fetal GCM and the differences in the free )] TJ ET BT 26.250 503.367 Td /F1 9.8 Tf [(radical scavengers. Gel electrophoresis of GCM and DM revealed the presence of seven bands in the GCM, which were absent )] TJ ET BT 26.250 491.463 Td /F1 9.8 Tf [(or much weaker in DM. These bands have a relative molecular weight of 250, 123, 38, 36, 33, 29 and 12 KDa \(Fig 4A\). GSH )] TJ ET BT 26.250 479.558 Td /F1 9.8 Tf [(levels were many folds greater in fetal GCM \(0.58 0.04\) than in DM \(0.013 0.01\). This may explain, at least in part, the GCM )] TJ ET BT 26.250 467.653 Td /F1 9.8 Tf [(protective effects \(Fig 4B\). Treatment with H)] TJ ET BT 216.980 465.589 Td /F1 8.7 Tf [(2)] TJ ET BT 221.798 467.653 Td /F1 9.8 Tf [(O)] TJ ET BT 229.384 465.589 Td /F1 8.7 Tf [(2)] TJ ET BT 234.202 467.653 Td /F1 9.8 Tf [(, 2M, decreased the intracellular levels of GSH and co-treatment with fetal )] TJ ET BT 26.250 455.748 Td /F1 9.8 Tf [(GCM increased these levels even above the levels of the controls \(Fig 4C\).)] TJ ET BT 26.250 436.344 Td /F4 9.8 Tf [(Apoptotic proteins. )] TJ ET BT 118.339 436.344 Td /F1 9.8 Tf [(The cleavage of PARP by caspases is recognized as a hallmark of apoptosis )] TJ ET 0.267 0.267 0.267 rg BT 452.695 437.851 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 462.333 436.344 Td /F1 9.8 Tf [( . The H)] TJ ET BT 497.014 434.279 Td /F1 8.7 Tf [(2)] TJ ET BT 501.832 436.344 Td /F1 9.8 Tf [(O)] TJ ET BT 509.418 434.279 Td /F1 8.7 Tf [(2)] TJ ET BT 514.236 436.344 Td /F1 9.8 Tf [( activated the )] TJ ET BT 26.250 424.439 Td /F1 9.8 Tf [(accumulation of this cleavage and the fetal GCM prevented this effect. H)] TJ ET BT 338.942 422.375 Td /F1 8.7 Tf [(2)] TJ ET BT 343.761 424.439 Td /F1 9.8 Tf [(O)] TJ ET BT 351.346 422.375 Td /F1 8.7 Tf [(2)] TJ ET BT 356.165 424.439 Td /F1 9.8 Tf [(, 2M x 24h, tripled the ratio PARP cleaved/full, an )] TJ ET BT 26.250 412.534 Td /F1 9.8 Tf [(index of apoptosis, in Q111 cells and co-treatment with GCM greatly diminished this index \(Fig 4D\).)] TJ ET BT 26.250 393.129 Td /F4 9.8 Tf [(Chaperones. )] TJ ET BT 88.016 393.129 Td /F1 9.8 Tf [(The chaperone HSP 70 levels were increased by treatment with H)] TJ ET BT 373.048 391.065 Td /F1 8.7 Tf [(2)] TJ ET BT 377.866 393.129 Td /F1 9.8 Tf [(O)] TJ ET BT 385.452 391.065 Td /F1 8.7 Tf [(2)] TJ ET BT 390.271 393.129 Td /F1 9.8 Tf [(, 2M x 24h \(Fig 4E\) and these effects )] TJ ET BT 26.250 381.225 Td /F1 9.8 Tf [(were prevented by fetal GCM \(Fig 4E\). The levels of CHIP were not changed by treatment with H)] TJ ET BT 444.038 379.160 Td /F1 8.7 Tf [(2)] TJ ET BT 448.856 381.225 Td /F1 9.8 Tf [(O)] TJ ET BT 456.442 379.160 Td /F1 8.7 Tf [(2)] TJ ET BT 461.260 381.225 Td /F1 9.8 Tf [(, 2M x 24h, GCM or both )] TJ ET BT 26.250 369.320 Td /F1 9.8 Tf [(\(data not shown\).)] TJ ET BT 26.250 349.915 Td /F4 9.8 Tf [(Polyubiquitinated proteins)] TJ ET BT 149.227 349.915 Td /F1 9.8 Tf [(H)] TJ ET BT 156.266 347.851 Td /F1 8.7 Tf [(2)] TJ ET BT 161.085 349.915 Td /F1 9.8 Tf [(O)] TJ ET BT 168.670 347.851 Td /F1 8.7 Tf [(2)] TJ ET BT 173.489 349.915 Td /F1 9.8 Tf [(, 2M x 24 hours, induced the accumulation of polyubiquitinated proteins in Q111 cells \(Fig )] TJ ET BT 26.250 338.010 Td /F1 9.8 Tf [(4F and G\). Co-treatment with GCM reverted this accumulation \(Fig 4F and G\).)] TJ ET 0.965 0.965 0.965 rg 26.250 136.225 555.000 191.905 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 328.129 m 581.250 328.129 l 581.250 327.379 l 26.250 327.379 l f q 225.000 0 0 158.250 35.250 160.129 cm /I6 Do Q q 35.250 136.225 537.000 17.905 re W n Q Q q 15.000 136.225 577.500 640.775 re W n 0.965 0.965 0.965 rg 26.250 563.605 555.000 213.395 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 563.605 m 581.250 563.605 l 581.250 564.355 l 26.250 564.355 l f q 35.250 574.855 537.000 202.145 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: The gla-conditioned medium protects from cell death and intracellular ROS accumulation induced by H2O2 )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(in Q111 cells)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(\(A\) Photomicrographs from control, H)] TJ ET BT 196.710 735.053 Td /F1 8.7 Tf [(2)] TJ ET BT 201.529 736.201 Td /F1 9.8 Tf [(O)] TJ ET BT 209.114 735.053 Td /F1 8.7 Tf [(2)] TJ ET BT 213.933 736.201 Td /F1 9.8 Tf [( \(2 M\) and fetal striatal GCM treated cells for 24 h at 3 DIV \(scale bar = 30 m\) )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(?-tubulin\). \(C\) Percentage of )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(chromatin condensed and fragmented nuclei in the different experimental groups. \(D\) Photomicrographs of cell death by )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(trypan blue staining from the four experimental groups. \(E\) Percentage of necrotic cells stained with trypan blue in the four )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(experimental groups. \(F\) Confocal photomicrographs of time-dependently ROS generation-induced by 100 ?M H)] TJ ET BT 519.698 680.108 Td /F1 8.7 Tf [(2)] TJ ET BT 524.517 681.256 Td /F1 9.8 Tf [(O)] TJ ET BT 532.102 680.108 Td /F1 8.7 Tf [(2)] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(treatment \(scale = 50 ?m\). \(G\) DCF fluorescence was measured with a reader at 485 nm of excitation and 582 nm emission )] TJ ET BT 35.250 653.784 Td /F1 9.8 Tf [(and expressed in optical density units \(O.D\). \(H\) Quantification of dose-dependent ROS generation by H)] TJ ET BT 484.491 652.635 Td /F1 8.7 Tf [(2)] TJ ET BT 489.310 653.784 Td /F1 9.8 Tf [(O)] TJ ET BT 496.895 652.635 Td /F1 8.7 Tf [(2)] TJ ET BT 501.714 653.784 Td /F1 9.8 Tf [( \(12.5, 25, 50, )] TJ ET BT 35.250 640.048 Td /F1 9.8 Tf [(100 and 200 ?M\) for 10 min and expressed in O.D. \(I\) Photomicrographs with fluorescence microscope of pre-treatment )] TJ ET BT 35.250 626.311 Td /F1 9.8 Tf [(with GCM 30 min before 100 ?M H)] TJ ET BT 185.341 625.163 Td /F1 8.7 Tf [(2)] TJ ET BT 190.160 626.311 Td /F1 9.8 Tf [(O)] TJ ET BT 197.746 625.163 Td /F1 8.7 Tf [(2)] TJ ET BT 202.564 626.311 Td /F1 9.8 Tf [( for 10 min prevented the ROS generation. \(J\) Quantification of intracellular ROS )] TJ ET BT 35.250 612.575 Td /F1 9.8 Tf [(generation in the four experimental groups. The values are expressed as mean SEM \(n = 4-6 samples in each )] TJ ET BT 35.250 598.839 Td /F1 9.8 Tf [(experimental group\). \(Scale bar = 30 ?m\). Statistical analysis was performed by one-way ANOVA followed by Newman-)] TJ ET BT 35.250 585.103 Td /F1 9.8 Tf [(Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control group. )] TJ ET BT 290.261 590.822 Td /F1 8.7 Tf [(&&)] TJ ET BT 301.823 585.103 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 337.332 590.822 Td /F1 8.7 Tf [(&&&)] TJ ET BT 354.674 585.103 Td /F1 9.8 Tf [(p<0.001 GCM + H)] TJ ET BT 433.795 583.954 Td /F1 8.7 Tf [(2)] TJ ET BT 438.614 585.103 Td /F1 9.8 Tf [(O2 vs H)] TJ ET BT 473.831 583.954 Td /F1 8.7 Tf [(2)] TJ ET BT 478.650 585.103 Td /F1 9.8 Tf [(O)] TJ ET BT 486.235 583.954 Td /F1 8.7 Tf [(2)] TJ ET BT 491.054 585.103 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 546.582 Td /F4 9.8 Tf [(Mechanisms implicated in the GCM neuroprotection in Q111 cells)] TJ ET BT 26.250 527.177 Td /F4 9.8 Tf [(Free radicals. )] TJ ET BT 91.829 527.177 Td /F1 9.8 Tf [(In order to study the mechanisms implicated in the GCM neuroprotection from H)] TJ ET BT 436.998 525.113 Td /F1 8.7 Tf [(2)] TJ ET BT 441.817 527.177 Td /F1 9.8 Tf [(O)] TJ ET BT 449.402 525.113 Td /F1 8.7 Tf [(2)] TJ ET BT 454.221 527.177 Td /F1 9.8 Tf [( induced cell death, we )] TJ ET BT 26.250 515.272 Td /F1 9.8 Tf [(investigated the differential pattern of proteins between defined medium \(DM\) and fetal GCM and the differences in the free )] TJ ET BT 26.250 503.367 Td /F1 9.8 Tf [(radical scavengers. Gel electrophoresis of GCM and DM revealed the presence of seven bands in the GCM, which were absent )] TJ ET BT 26.250 491.463 Td /F1 9.8 Tf [(or much weaker in DM. These bands have a relative molecular weight of 250, 123, 38, 36, 33, 29 and 12 KDa \(Fig 4A\). GSH )] TJ ET BT 26.250 479.558 Td /F1 9.8 Tf [(levels were many folds greater in fetal GCM \(0.58 0.04\) than in DM \(0.013 0.01\). This may explain, at least in part, the GCM )] TJ ET BT 26.250 467.653 Td /F1 9.8 Tf [(protective effects \(Fig 4B\). Treatment with H)] TJ ET BT 216.980 465.589 Td /F1 8.7 Tf [(2)] TJ ET BT 221.798 467.653 Td /F1 9.8 Tf [(O)] TJ ET BT 229.384 465.589 Td /F1 8.7 Tf [(2)] TJ ET BT 234.202 467.653 Td /F1 9.8 Tf [(, 2M, decreased the intracellular levels of GSH and co-treatment with fetal )] TJ ET BT 26.250 455.748 Td /F1 9.8 Tf [(GCM increased these levels even above the levels of the controls \(Fig 4C\).)] TJ ET BT 26.250 436.344 Td /F4 9.8 Tf [(Apoptotic proteins. )] TJ ET BT 118.339 436.344 Td /F1 9.8 Tf [(The cleavage of PARP by caspases is recognized as a hallmark of apoptosis )] TJ ET 0.267 0.267 0.267 rg BT 452.695 437.851 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 462.333 436.344 Td /F1 9.8 Tf [( . The H)] TJ ET BT 497.014 434.279 Td /F1 8.7 Tf [(2)] TJ ET BT 501.832 436.344 Td /F1 9.8 Tf [(O)] TJ ET BT 509.418 434.279 Td /F1 8.7 Tf [(2)] TJ ET BT 514.236 436.344 Td /F1 9.8 Tf [( activated the )] TJ ET BT 26.250 424.439 Td /F1 9.8 Tf [(accumulation of this cleavage and the fetal GCM prevented this effect. H)] TJ ET BT 338.942 422.375 Td /F1 8.7 Tf [(2)] TJ ET BT 343.761 424.439 Td /F1 9.8 Tf [(O)] TJ ET BT 351.346 422.375 Td /F1 8.7 Tf [(2)] TJ ET BT 356.165 424.439 Td /F1 9.8 Tf [(, 2M x 24h, tripled the ratio PARP cleaved/full, an )] TJ ET BT 26.250 412.534 Td /F1 9.8 Tf [(index of apoptosis, in Q111 cells and co-treatment with GCM greatly diminished this index \(Fig 4D\).)] TJ ET BT 26.250 393.129 Td /F4 9.8 Tf [(Chaperones. )] TJ ET BT 88.016 393.129 Td /F1 9.8 Tf [(The chaperone HSP 70 levels were increased by treatment with H)] TJ ET BT 373.048 391.065 Td /F1 8.7 Tf [(2)] TJ ET BT 377.866 393.129 Td /F1 9.8 Tf [(O)] TJ ET BT 385.452 391.065 Td /F1 8.7 Tf [(2)] TJ ET BT 390.271 393.129 Td /F1 9.8 Tf [(, 2M x 24h \(Fig 4E\) and these effects )] TJ ET BT 26.250 381.225 Td /F1 9.8 Tf [(were prevented by fetal GCM \(Fig 4E\). The levels of CHIP were not changed by treatment with H)] TJ ET BT 444.038 379.160 Td /F1 8.7 Tf [(2)] TJ ET BT 448.856 381.225 Td /F1 9.8 Tf [(O)] TJ ET BT 456.442 379.160 Td /F1 8.7 Tf [(2)] TJ ET BT 461.260 381.225 Td /F1 9.8 Tf [(, 2M x 24h, GCM or both )] TJ ET BT 26.250 369.320 Td /F1 9.8 Tf [(\(data not shown\).)] TJ ET BT 26.250 349.915 Td /F4 9.8 Tf [(Polyubiquitinated proteins)] TJ ET BT 149.227 349.915 Td /F1 9.8 Tf [(H)] TJ ET BT 156.266 347.851 Td /F1 8.7 Tf [(2)] TJ ET BT 161.085 349.915 Td /F1 9.8 Tf [(O)] TJ ET BT 168.670 347.851 Td /F1 8.7 Tf [(2)] TJ ET BT 173.489 349.915 Td /F1 9.8 Tf [(, 2M x 24 hours, induced the accumulation of polyubiquitinated proteins in Q111 cells \(Fig )] TJ ET BT 26.250 338.010 Td /F1 9.8 Tf [(4F and G\). Co-treatment with GCM reverted this accumulation \(Fig 4F and G\).)] TJ ET 0.965 0.965 0.965 rg 26.250 136.225 555.000 191.905 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 328.129 m 581.250 328.129 l 581.250 327.379 l 26.250 327.379 l f q 225.000 0 0 158.250 35.250 160.129 cm /I6 Do Q q 35.250 136.225 537.000 17.905 re W n Q Q q 15.000 136.225 577.500 640.775 re W n 0.965 0.965 0.965 rg 26.250 563.605 555.000 213.395 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 563.605 m 581.250 563.605 l 581.250 564.355 l 26.250 564.355 l f q 35.250 574.855 537.000 202.145 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: The gla-conditioned medium protects from cell death and intracellular ROS accumulation induced by H2O2 )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(in Q111 cells)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(\(A\) Photomicrographs from control, H)] TJ ET BT 196.710 735.053 Td /F1 8.7 Tf [(2)] TJ ET BT 201.529 736.201 Td /F1 9.8 Tf [(O)] TJ ET BT 209.114 735.053 Td /F1 8.7 Tf [(2)] TJ ET BT 213.933 736.201 Td /F1 9.8 Tf [( \(2 M\) and fetal striatal GCM treated cells for 24 h at 3 DIV \(scale bar = 30 m\) )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(?-tubulin\). \(C\) Percentage of )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(chromatin condensed and fragmented nuclei in the different experimental groups. \(D\) Photomicrographs of cell death by )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(trypan blue staining from the four experimental groups. \(E\) Percentage of necrotic cells stained with trypan blue in the four )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(experimental groups. \(F\) Confocal photomicrographs of time-dependently ROS generation-induced by 100 ?M H)] TJ ET BT 519.698 680.108 Td /F1 8.7 Tf [(2)] TJ ET BT 524.517 681.256 Td /F1 9.8 Tf [(O)] TJ ET BT 532.102 680.108 Td /F1 8.7 Tf [(2)] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(treatment \(scale = 50 ?m\). \(G\) DCF fluorescence was measured with a reader at 485 nm of excitation and 582 nm emission )] TJ ET BT 35.250 653.784 Td /F1 9.8 Tf [(and expressed in optical density units \(O.D\). \(H\) Quantification of dose-dependent ROS generation by H)] TJ ET BT 484.491 652.635 Td /F1 8.7 Tf [(2)] TJ ET BT 489.310 653.784 Td /F1 9.8 Tf [(O)] TJ ET BT 496.895 652.635 Td /F1 8.7 Tf [(2)] TJ ET BT 501.714 653.784 Td /F1 9.8 Tf [( \(12.5, 25, 50, )] TJ ET BT 35.250 640.048 Td /F1 9.8 Tf [(100 and 200 ?M\) for 10 min and expressed in O.D. \(I\) Photomicrographs with fluorescence microscope of pre-treatment )] TJ ET BT 35.250 626.311 Td /F1 9.8 Tf [(with GCM 30 min before 100 ?M H)] TJ ET BT 185.341 625.163 Td /F1 8.7 Tf [(2)] TJ ET BT 190.160 626.311 Td /F1 9.8 Tf [(O)] TJ ET BT 197.746 625.163 Td /F1 8.7 Tf [(2)] TJ ET BT 202.564 626.311 Td /F1 9.8 Tf [( for 10 min prevented the ROS generation. \(J\) Quantification of intracellular ROS )] TJ ET BT 35.250 612.575 Td /F1 9.8 Tf [(generation in the four experimental groups. The values are expressed as mean SEM \(n = 4-6 samples in each )] TJ ET BT 35.250 598.839 Td /F1 9.8 Tf [(experimental group\). \(Scale bar = 30 ?m\). Statistical analysis was performed by one-way ANOVA followed by Newman-)] TJ ET BT 35.250 585.103 Td /F1 9.8 Tf [(Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control group. )] TJ ET BT 290.261 590.822 Td /F1 8.7 Tf [(&&)] TJ ET BT 301.823 585.103 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 337.332 590.822 Td /F1 8.7 Tf [(&&&)] TJ ET BT 354.674 585.103 Td /F1 9.8 Tf [(p<0.001 GCM + H)] TJ ET BT 433.795 583.954 Td /F1 8.7 Tf [(2)] TJ ET BT 438.614 585.103 Td /F1 9.8 Tf [(O2 vs H)] TJ ET BT 473.831 583.954 Td /F1 8.7 Tf [(2)] TJ ET BT 478.650 585.103 Td /F1 9.8 Tf [(O)] TJ ET BT 486.235 583.954 Td /F1 8.7 Tf [(2)] TJ ET BT 491.054 585.103 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 546.582 Td /F4 9.8 Tf [(Mechanisms implicated in the GCM neuroprotection in Q111 cells)] TJ ET BT 26.250 527.177 Td /F4 9.8 Tf [(Free radicals. )] TJ ET BT 91.829 527.177 Td /F1 9.8 Tf [(In order to study the mechanisms implicated in the GCM neuroprotection from H)] TJ ET BT 436.998 525.113 Td /F1 8.7 Tf [(2)] TJ ET BT 441.817 527.177 Td /F1 9.8 Tf [(O)] TJ ET BT 449.402 525.113 Td /F1 8.7 Tf [(2)] TJ ET BT 454.221 527.177 Td /F1 9.8 Tf [( induced cell death, we )] TJ ET BT 26.250 515.272 Td /F1 9.8 Tf [(investigated the differential pattern of proteins between defined medium \(DM\) and fetal GCM and the differences in the free )] TJ ET BT 26.250 503.367 Td /F1 9.8 Tf [(radical scavengers. Gel electrophoresis of GCM and DM revealed the presence of seven bands in the GCM, which were absent )] TJ ET BT 26.250 491.463 Td /F1 9.8 Tf [(or much weaker in DM. These bands have a relative molecular weight of 250, 123, 38, 36, 33, 29 and 12 KDa \(Fig 4A\). GSH )] TJ ET BT 26.250 479.558 Td /F1 9.8 Tf [(levels were many folds greater in fetal GCM \(0.58 0.04\) than in DM \(0.013 0.01\). This may explain, at least in part, the GCM )] TJ ET BT 26.250 467.653 Td /F1 9.8 Tf [(protective effects \(Fig 4B\). Treatment with H)] TJ ET BT 216.980 465.589 Td /F1 8.7 Tf [(2)] TJ ET BT 221.798 467.653 Td /F1 9.8 Tf [(O)] TJ ET BT 229.384 465.589 Td /F1 8.7 Tf [(2)] TJ ET BT 234.202 467.653 Td /F1 9.8 Tf [(, 2M, decreased the intracellular levels of GSH and co-treatment with fetal )] TJ ET BT 26.250 455.748 Td /F1 9.8 Tf [(GCM increased these levels even above the levels of the controls \(Fig 4C\).)] TJ ET BT 26.250 436.344 Td /F4 9.8 Tf [(Apoptotic proteins. )] TJ ET BT 118.339 436.344 Td /F1 9.8 Tf [(The cleavage of PARP by caspases is recognized as a hallmark of apoptosis )] TJ ET 0.267 0.267 0.267 rg BT 452.695 437.851 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 462.333 436.344 Td /F1 9.8 Tf [( . The H)] TJ ET BT 497.014 434.279 Td /F1 8.7 Tf [(2)] TJ ET BT 501.832 436.344 Td /F1 9.8 Tf [(O)] TJ ET BT 509.418 434.279 Td /F1 8.7 Tf [(2)] TJ ET BT 514.236 436.344 Td /F1 9.8 Tf [( activated the )] TJ ET BT 26.250 424.439 Td /F1 9.8 Tf [(accumulation of this cleavage and the fetal GCM prevented this effect. H)] TJ ET BT 338.942 422.375 Td /F1 8.7 Tf [(2)] TJ ET BT 343.761 424.439 Td /F1 9.8 Tf [(O)] TJ ET BT 351.346 422.375 Td /F1 8.7 Tf [(2)] TJ ET BT 356.165 424.439 Td /F1 9.8 Tf [(, 2M x 24h, tripled the ratio PARP cleaved/full, an )] TJ ET BT 26.250 412.534 Td /F1 9.8 Tf [(index of apoptosis, in Q111 cells and co-treatment with GCM greatly diminished this index \(Fig 4D\).)] TJ ET BT 26.250 393.129 Td /F4 9.8 Tf [(Chaperones. )] TJ ET BT 88.016 393.129 Td /F1 9.8 Tf [(The chaperone HSP 70 levels were increased by treatment with H)] TJ ET BT 373.048 391.065 Td /F1 8.7 Tf [(2)] TJ ET BT 377.866 393.129 Td /F1 9.8 Tf [(O)] TJ ET BT 385.452 391.065 Td /F1 8.7 Tf [(2)] TJ ET BT 390.271 393.129 Td /F1 9.8 Tf [(, 2M x 24h \(Fig 4E\) and these effects )] TJ ET BT 26.250 381.225 Td /F1 9.8 Tf [(were prevented by fetal GCM \(Fig 4E\). The levels of CHIP were not changed by treatment with H)] TJ ET BT 444.038 379.160 Td /F1 8.7 Tf [(2)] TJ ET BT 448.856 381.225 Td /F1 9.8 Tf [(O)] TJ ET BT 456.442 379.160 Td /F1 8.7 Tf [(2)] TJ ET BT 461.260 381.225 Td /F1 9.8 Tf [(, 2M x 24h, GCM or both )] TJ ET BT 26.250 369.320 Td /F1 9.8 Tf [(\(data not shown\).)] TJ ET BT 26.250 349.915 Td /F4 9.8 Tf [(Polyubiquitinated proteins)] TJ ET BT 149.227 349.915 Td /F1 9.8 Tf [(H)] TJ ET BT 156.266 347.851 Td /F1 8.7 Tf [(2)] TJ ET BT 161.085 349.915 Td /F1 9.8 Tf [(O)] TJ ET BT 168.670 347.851 Td /F1 8.7 Tf [(2)] TJ ET BT 173.489 349.915 Td /F1 9.8 Tf [(, 2M x 24 hours, induced the accumulation of polyubiquitinated proteins in Q111 cells \(Fig )] TJ ET BT 26.250 338.010 Td /F1 9.8 Tf [(4F and G\). Co-treatment with GCM reverted this accumulation \(Fig 4F and G\).)] TJ ET 0.965 0.965 0.965 rg 26.250 136.225 555.000 191.905 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 328.129 m 581.250 328.129 l 581.250 327.379 l 26.250 327.379 l f q 225.000 0 0 158.250 35.250 160.129 cm /I6 Do Q q 35.250 136.225 537.000 17.905 re W n Q Q q 225.000 0 0 158.250 35.250 160.129 cm /I6 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 364 0 obj << /Type /Annot /Subtype /Link /A 365 0 R /Border [0 0 0] /H /I /Rect [ 452.6955 437.0492 462.3328 445.8675 ] >> endobj 365 0 obj << /Type /Action >> endobj 366 0 obj << /Type /Annot /Subtype /Link /A 367 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 160.1295 260.2500 318.3795 ] >> endobj 367 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-4.jpg) >> endobj 368 0 obj << /Type /XObject /Subtype /Image /Width 300 /Height 211 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 17542>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Qϵ.ESl/쭮ŕ.Gf@uRc@F}?pj7^ Ѯ$,̎#rnY {It5UBDHb p~lNyNNxA BL"g+ F`SpPUu)E 7BW$q۽y~yw[9n&8Ycd/8' M={W6??uFԯcl`5<\Qsg+<|KҼOƿūhl>o 10"pK9Mv~2 C**PrQՏqDwmgq]L!Yw2}G'@$rqӿ k,5ʤ1sC?|acdtC42#)ڬ@ W<ܞ4"6Դ>A0!®UH8-Ӂ XaYkKxa,0J:\UVuuoB_gtDg0[zE0>fpXgiCSc:7ƒAnXz@y>߭.Eϵȣ"4f"LQj\2(3E.E>ԹO6$Ӯ"Y,\D$lĢi =Iȣ PfRQ@ j3KFE&hϵ.E>ԹdPfQ@ jt^>x l^3H]eHH@: p+W>ζ7&+ڈWMS0#x :Ye6h,XudnV;x@'+<_Tc޿?h۩gޤn,0{ޱ /3WG C54htDgA%ydGCKi%@܂x?y!9TgTnc.Uz~y q8+T3\Ff2#=Z\dВ`܁#(%r1W#_et 4x1Ld?$@pYaz}oD:?:<5]g5fx-w$$/3I22;Q@(Gϵ,QΛ1NmRHѰ@# 3Zϵǽϵ/~["wB. 1~zw5-"Sӧui*RA}E^ϵǽϵϵexogtt"Z|*TpFppV=뙓o6RxCKva 7ٷvwoq#h'ڌR=ǽ&}ϵ.=ǽ!>gڗbj3Kz|C&7JɼH2*'$D>gڳ~)WPmRŤ!si*trUZ8ϵǽ&^*wws,MB=zu\שX/^c|+Ӯ-/DрpC0?Y ,G( |Nx*?EH' |qoԚ-Kw\4AGJq8r~o3 >yv[j[1F6;0v󃃎 URTQ 8)V[r^-GƝ|:}oGKH8'i`)@$+#MYᓮjMf%X[ RA;^:u?[Z޶u B;s5ͭ-220*\H>?G|; wG]Ep?|;wNhϵ.Ep7?:Vu~ۋI"2CN%XpN w5j?/t745{V-sk (/~@dk@ŸMqdmZG$5ut?}/h=\MsK,Zm xhZ9U]HdaʎV俳 __=z;")3Fh8nXI3T`JCҼ[UmGTLӓq,[h H`A k#}yP6:ȷF̸I1)]J0]{W~ VZ8̏#HJb0xe!j­cXaϊ8P@ ︯?n t;x̍^|jercI$5Pnh`xoï \kIg A#$Lƪ(k/W^#PI hz7hR'Sy;ejo{S1-kȿ?w4mB4kG״)L2ENX ĩuz*M߄te<+1$89e q?SW?<O'0tJ A_zPnh~4&Eq@&[JCʒa"x*QI|CYLzgmt?{v nrF0zu??Y[8[Y/rNC|۷p9Y WP$.oKy8MJ.SL+G╎vm>+Y@ Z_phŎsl+WrK+dKǕ3Z}OJ̝6\;98ӷz 3%.|z{WE|Fi%}%ji3etoK?Fݭ>z&Vt,QUs>ו|$ԥk P-"oR@»>VOyc ė>kW8?J>o6OkZwXӮe)$*$hq&7q2+yG;habdd @ nB: C u89 |}|>]. 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Gel electrophoresis of defined medium and fetal striatal glia-conditioned )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(medium \(GCM\) and their corresponding densitometric scanning. Numbers 1 to 7 \(250, 123, 38, 36, 33, 29 and 12 KDa )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(respectively\) on right panel indicate the seven bands only present in GCM. \(B\) Levels of glutathione in defined medium and )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(fetal GCM. \(C\) Intracellular glutathione levels in Q111 cells treated with H)] TJ ET BT 351.150 705.749 Td /F1 8.7 Tf [(2)] TJ ET BT 355.969 706.897 Td /F1 9.8 Tf [(O)] TJ ET BT 363.554 705.749 Td /F1 8.7 Tf [(2)] TJ ET BT 368.373 706.897 Td /F1 9.8 Tf [( \(2 M\) and GCM for 24 h. \(D\) Expression of )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(the full and cleaved, activated, forms of PARP in cells treated with H2O2 in DM or GCM. \(E\) Expression of chaperone )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(Hsp70. \(F\) Accumulation of polyubiquitinated proteins induced by H)] TJ ET BT 326.248 678.276 Td /F1 8.7 Tf [(2)] TJ ET BT 331.067 679.425 Td /F1 9.8 Tf [(O)] TJ ET BT 338.653 678.276 Td /F1 8.7 Tf [(2)] TJ ET BT 343.471 679.425 Td /F1 9.8 Tf [( in Q111 cells and \(G\) their corresponding )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(densitometric analysis. The values are expressed as mean SEM \(n = 4-6 samples in each experimental group\). Statistical )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(analysis was performed by one-way ANOVA followed by Newman-Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control )] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(group. )] TJ ET BT 65.602 643.936 Td /F1 8.7 Tf [(&)] TJ ET BT 71.382 638.216 Td /F1 9.8 Tf [(p<0.05, )] TJ ET BT 106.892 643.936 Td /F1 8.7 Tf [(&&)] TJ ET BT 118.453 638.216 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 153.963 643.936 Td /F1 8.7 Tf [(&&&)] TJ ET BT 171.305 638.216 Td /F1 9.8 Tf [(p<0.001 GCM + H)] TJ ET BT 250.426 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 255.245 638.216 Td /F1 9.8 Tf [(O)] TJ ET BT 262.830 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 267.649 638.216 Td /F1 9.8 Tf [( vs H)] TJ ET BT 289.859 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 294.678 638.216 Td /F1 9.8 Tf [(O)] TJ ET BT 302.264 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 307.082 638.216 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 566.790 Td /F4 9.8 Tf [(Postnatal GCM prevents cell death produced by H)] TJ ET BT 257.598 564.726 Td /F4 8.7 Tf [(2)] TJ ET BT 262.417 566.790 Td /F4 9.8 Tf [(O)] TJ ET BT 270.002 564.726 Td /F4 8.7 Tf [(2)] TJ ET BT 274.821 566.790 Td /F4 9.8 Tf [( in Q111 cells. )] TJ ET BT 342.574 566.790 Td /F1 9.8 Tf [(We have also studied the protective effects of )] TJ ET BT 26.250 554.886 Td /F1 9.8 Tf [(postnatal GCM, of 13 days and 2 months in the huntingtin mutant cells \(Fig 5\). Postnatal GCM prevents H)] TJ ET BT 481.994 552.821 Td /F1 8.7 Tf [(2)] TJ ET BT 486.813 554.886 Td /F1 9.8 Tf [(O)] TJ ET BT 494.398 552.821 Td /F1 8.7 Tf [(2)] TJ ET BT 499.217 554.886 Td /F1 9.8 Tf [( induced cell )] TJ ET BT 26.250 542.981 Td /F1 9.8 Tf [(death in Q111 cells. GCM prevented the H)] TJ ET BT 209.959 540.917 Td /F1 8.7 Tf [(2)] TJ ET BT 214.778 542.981 Td /F1 9.8 Tf [(O)] TJ ET BT 222.364 540.917 Td /F1 8.7 Tf [(2)] TJ ET BT 227.182 542.981 Td /F1 9.8 Tf [(-induced reduction of the -tubulin area and the increased apoptosis in Q111 )] TJ ET BT 26.250 531.076 Td /F1 9.8 Tf [(cells \(F 5A-H\).)] TJ ET 0.965 0.965 0.965 rg 26.250 80.787 555.000 440.408 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 521.195 m 581.250 521.195 l 581.250 520.445 l 26.250 520.445 l f 26.250 80.787 m 581.250 80.787 l 581.250 81.537 l 26.250 81.537 l f q 168.750 0 0 225.000 35.250 286.445 cm /I7 Do Q q 35.250 92.037 537.000 188.408 re W n 0.271 0.267 0.267 rg BT 35.250 270.921 Td /F4 9.8 Tf [(Fig. 5: The 13-day and 2-month postnatal striatal glial conditioned medium protects from H2O2 toxicity in Q111 )] TJ ET BT 35.250 259.017 Td /F4 9.8 Tf [(cells)] TJ ET BT 35.250 239.647 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, H)] TJ ET BT 185.341 238.498 Td /F1 8.7 Tf [(2)] TJ ET BT 190.160 239.647 Td /F1 9.8 Tf [(O)] TJ ET BT 197.746 238.498 Td /F1 8.7 Tf [(2)] TJ ET BT 202.564 239.647 Td /F1 9.8 Tf [( \(2 M\) and 13 day postnatal striatal GCM treated cells for 24 h at 3 DIV \(scale bar = )] TJ ET BT 35.250 225.910 Td /F1 9.8 Tf [(30 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-tubulin\). \(C\) )] TJ ET BT 35.250 212.174 Td /F1 9.8 Tf [(Photomicrograph of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells. The arrows )] TJ ET BT 35.250 198.438 Td /F1 9.8 Tf [(indicate the apoptotic cells produced by the H)] TJ ET BT 231.976 197.289 Td /F1 8.7 Tf [(2)] TJ ET BT 236.794 198.438 Td /F1 9.8 Tf [(O)] TJ ET BT 244.380 197.289 Td /F1 8.7 Tf [(2)] TJ ET BT 249.199 198.438 Td /F1 9.8 Tf [( toxicity. \(D\) Percentage of chromatin condensed and fragmented nuclei in )] TJ ET BT 35.250 184.702 Td /F1 9.8 Tf [(the different experimental groups. \(E\) Photomicrographs of control, H)] TJ ET BT 333.288 183.553 Td /F1 8.7 Tf [(2)] TJ ET BT 338.107 184.702 Td /F1 9.8 Tf [(O)] TJ ET BT 345.692 183.553 Td /F1 8.7 Tf [(2)] TJ ET BT 350.511 184.702 Td /F1 9.8 Tf [( \(2 M\) and 2 month postnatal striatal GCM )] TJ ET BT 35.250 170.965 Td /F1 9.8 Tf [(treated cells for 24 h at 3 DIV \(scale bar = 30 ?m\) and \(F\) their corresponding quantification of total neurons expressed as )] TJ ET BT 35.250 157.229 Td /F1 9.8 Tf [(microtubule protein -tubulin. \(G\) Photomicrographs of total nuclei stained with bis-benzimide in the four experimental )] TJ ET BT 35.250 143.493 Td /F1 9.8 Tf [(groups of Q111 cells. The arrows indicate the apoptotic cells produced by the H)] TJ ET BT 378.284 142.344 Td /F1 8.7 Tf [(2)] TJ ET BT 383.103 143.493 Td /F1 9.8 Tf [(O)] TJ ET BT 390.688 142.344 Td /F1 8.7 Tf [(2)] TJ ET BT 395.507 143.493 Td /F1 9.8 Tf [( toxicity \(H\) Percentage of chromatin-)] TJ ET BT 35.250 129.757 Td /F1 9.8 Tf [(condensed and fragmented nuclei in the different experimental groups. The values are expressed as mean SEM \(n = 4-6 )] TJ ET BT 35.250 116.020 Td /F1 9.8 Tf [(samples in each experimental group\). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls )] TJ ET BT 35.250 102.284 Td /F1 9.8 Tf [(test. *p<0.05, **p<0.01 vs control group. )] TJ ET BT 210.857 108.004 Td /F1 8.7 Tf [(&)] TJ ET BT 216.638 102.284 Td /F1 9.8 Tf [(p<0.05, )] TJ ET BT 252.147 108.004 Td /F1 8.7 Tf [(&&)] TJ ET BT 263.709 102.284 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 299.218 108.004 Td /F1 8.7 Tf [(&&&)] TJ ET BT 316.560 102.284 Td /F1 9.8 Tf [(p<0.001 GCM + H)] TJ ET BT 395.682 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 400.500 102.284 Td /F1 9.8 Tf [(O)] TJ ET BT 408.086 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 412.904 102.284 Td /F1 9.8 Tf [( vs H)] TJ ET BT 435.115 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 439.934 102.284 Td /F1 9.8 Tf [(O)] TJ ET BT 447.519 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 452.338 102.284 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 63.763 Td /F4 9.8 Tf [(Comparative effects of neurotrophic factors and GCM on Q111 cells)] TJ ET BT 26.250 44.358 Td /F4 9.8 Tf [(The growth factor bFGF has beneficial effects in Q111 cells but fewer than fetal GCM. )] TJ ET BT 423.397 44.358 Td /F1 9.8 Tf [(Basic FGF 10 and 20 ng/ml, did not )] TJ ET Q q 15.000 30.073 577.500 746.927 re W n 0.965 0.965 0.965 rg 26.250 583.814 555.000 193.186 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 583.814 m 581.250 583.814 l 581.250 584.564 l 26.250 584.564 l f q 35.250 595.064 537.000 181.936 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 4: Mechanisms of GCM neuroprotection in Q111 cells)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(\(A\) Pattern of band proteins in GCM and DM. Gel electrophoresis of defined medium and fetal striatal glia-conditioned )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(medium \(GCM\) and their corresponding densitometric scanning. Numbers 1 to 7 \(250, 123, 38, 36, 33, 29 and 12 KDa )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(respectively\) on right panel indicate the seven bands only present in GCM. \(B\) Levels of glutathione in defined medium and )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(fetal GCM. \(C\) Intracellular glutathione levels in Q111 cells treated with H)] TJ ET BT 351.150 705.749 Td /F1 8.7 Tf [(2)] TJ ET BT 355.969 706.897 Td /F1 9.8 Tf [(O)] TJ ET BT 363.554 705.749 Td /F1 8.7 Tf [(2)] TJ ET BT 368.373 706.897 Td /F1 9.8 Tf [( \(2 M\) and GCM for 24 h. \(D\) Expression of )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(the full and cleaved, activated, forms of PARP in cells treated with H2O2 in DM or GCM. \(E\) Expression of chaperone )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(Hsp70. \(F\) Accumulation of polyubiquitinated proteins induced by H)] TJ ET BT 326.248 678.276 Td /F1 8.7 Tf [(2)] TJ ET BT 331.067 679.425 Td /F1 9.8 Tf [(O)] TJ ET BT 338.653 678.276 Td /F1 8.7 Tf [(2)] TJ ET BT 343.471 679.425 Td /F1 9.8 Tf [( in Q111 cells and \(G\) their corresponding )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(densitometric analysis. The values are expressed as mean SEM \(n = 4-6 samples in each experimental group\). Statistical )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(analysis was performed by one-way ANOVA followed by Newman-Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control )] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(group. )] TJ ET BT 65.602 643.936 Td /F1 8.7 Tf [(&)] TJ ET BT 71.382 638.216 Td /F1 9.8 Tf [(p<0.05, )] TJ ET BT 106.892 643.936 Td /F1 8.7 Tf [(&&)] TJ ET BT 118.453 638.216 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 153.963 643.936 Td /F1 8.7 Tf [(&&&)] TJ ET BT 171.305 638.216 Td /F1 9.8 Tf [(p<0.001 GCM + H)] TJ ET BT 250.426 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 255.245 638.216 Td /F1 9.8 Tf [(O)] TJ ET BT 262.830 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 267.649 638.216 Td /F1 9.8 Tf [( vs H)] TJ ET BT 289.859 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 294.678 638.216 Td /F1 9.8 Tf [(O)] TJ ET BT 302.264 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 307.082 638.216 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 566.790 Td /F4 9.8 Tf [(Postnatal GCM prevents cell death produced by H)] TJ ET BT 257.598 564.726 Td /F4 8.7 Tf [(2)] TJ ET BT 262.417 566.790 Td /F4 9.8 Tf [(O)] TJ ET BT 270.002 564.726 Td /F4 8.7 Tf [(2)] TJ ET BT 274.821 566.790 Td /F4 9.8 Tf [( in Q111 cells. )] TJ ET BT 342.574 566.790 Td /F1 9.8 Tf [(We have also studied the protective effects of )] TJ ET BT 26.250 554.886 Td /F1 9.8 Tf [(postnatal GCM, of 13 days and 2 months in the huntingtin mutant cells \(Fig 5\). Postnatal GCM prevents H)] TJ ET BT 481.994 552.821 Td /F1 8.7 Tf [(2)] TJ ET BT 486.813 554.886 Td /F1 9.8 Tf [(O)] TJ ET BT 494.398 552.821 Td /F1 8.7 Tf [(2)] TJ ET BT 499.217 554.886 Td /F1 9.8 Tf [( induced cell )] TJ ET BT 26.250 542.981 Td /F1 9.8 Tf [(death in Q111 cells. GCM prevented the H)] TJ ET BT 209.959 540.917 Td /F1 8.7 Tf [(2)] TJ ET BT 214.778 542.981 Td /F1 9.8 Tf [(O)] TJ ET BT 222.364 540.917 Td /F1 8.7 Tf [(2)] TJ ET BT 227.182 542.981 Td /F1 9.8 Tf [(-induced reduction of the -tubulin area and the increased apoptosis in Q111 )] TJ ET BT 26.250 531.076 Td /F1 9.8 Tf [(cells \(F 5A-H\).)] TJ ET 0.965 0.965 0.965 rg 26.250 80.787 555.000 440.408 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 521.195 m 581.250 521.195 l 581.250 520.445 l 26.250 520.445 l f 26.250 80.787 m 581.250 80.787 l 581.250 81.537 l 26.250 81.537 l f q 168.750 0 0 225.000 35.250 286.445 cm /I7 Do Q q 35.250 92.037 537.000 188.408 re W n 0.271 0.267 0.267 rg BT 35.250 270.921 Td /F4 9.8 Tf [(Fig. 5: The 13-day and 2-month postnatal striatal glial conditioned medium protects from H2O2 toxicity in Q111 )] TJ ET BT 35.250 259.017 Td /F4 9.8 Tf [(cells)] TJ ET BT 35.250 239.647 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, H)] TJ ET BT 185.341 238.498 Td /F1 8.7 Tf [(2)] TJ ET BT 190.160 239.647 Td /F1 9.8 Tf [(O)] TJ ET BT 197.746 238.498 Td /F1 8.7 Tf [(2)] TJ ET BT 202.564 239.647 Td /F1 9.8 Tf [( \(2 M\) and 13 day postnatal striatal GCM treated cells for 24 h at 3 DIV \(scale bar = )] TJ ET BT 35.250 225.910 Td /F1 9.8 Tf [(30 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-tubulin\). \(C\) )] TJ ET BT 35.250 212.174 Td /F1 9.8 Tf [(Photomicrograph of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells. The arrows )] TJ ET BT 35.250 198.438 Td /F1 9.8 Tf [(indicate the apoptotic cells produced by the H)] TJ ET BT 231.976 197.289 Td /F1 8.7 Tf [(2)] TJ ET BT 236.794 198.438 Td /F1 9.8 Tf [(O)] TJ ET BT 244.380 197.289 Td /F1 8.7 Tf [(2)] TJ ET BT 249.199 198.438 Td /F1 9.8 Tf [( toxicity. \(D\) Percentage of chromatin condensed and fragmented nuclei in )] TJ ET BT 35.250 184.702 Td /F1 9.8 Tf [(the different experimental groups. \(E\) Photomicrographs of control, H)] TJ ET BT 333.288 183.553 Td /F1 8.7 Tf [(2)] TJ ET BT 338.107 184.702 Td /F1 9.8 Tf [(O)] TJ ET BT 345.692 183.553 Td /F1 8.7 Tf [(2)] TJ ET BT 350.511 184.702 Td /F1 9.8 Tf [( \(2 M\) and 2 month postnatal striatal GCM )] TJ ET BT 35.250 170.965 Td /F1 9.8 Tf [(treated cells for 24 h at 3 DIV \(scale bar = 30 ?m\) and \(F\) their corresponding quantification of total neurons expressed as )] TJ ET BT 35.250 157.229 Td /F1 9.8 Tf [(microtubule protein -tubulin. \(G\) Photomicrographs of total nuclei stained with bis-benzimide in the four experimental )] TJ ET BT 35.250 143.493 Td /F1 9.8 Tf [(groups of Q111 cells. The arrows indicate the apoptotic cells produced by the H)] TJ ET BT 378.284 142.344 Td /F1 8.7 Tf [(2)] TJ ET BT 383.103 143.493 Td /F1 9.8 Tf [(O)] TJ ET BT 390.688 142.344 Td /F1 8.7 Tf [(2)] TJ ET BT 395.507 143.493 Td /F1 9.8 Tf [( toxicity \(H\) Percentage of chromatin-)] TJ ET BT 35.250 129.757 Td /F1 9.8 Tf [(condensed and fragmented nuclei in the different experimental groups. The values are expressed as mean SEM \(n = 4-6 )] TJ ET BT 35.250 116.020 Td /F1 9.8 Tf [(samples in each experimental group\). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls )] TJ ET BT 35.250 102.284 Td /F1 9.8 Tf [(test. *p<0.05, **p<0.01 vs control group. )] TJ ET BT 210.857 108.004 Td /F1 8.7 Tf [(&)] TJ ET BT 216.638 102.284 Td /F1 9.8 Tf [(p<0.05, )] TJ ET BT 252.147 108.004 Td /F1 8.7 Tf [(&&)] TJ ET BT 263.709 102.284 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 299.218 108.004 Td /F1 8.7 Tf [(&&&)] TJ ET BT 316.560 102.284 Td /F1 9.8 Tf [(p<0.001 GCM + H)] TJ ET BT 395.682 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 400.500 102.284 Td /F1 9.8 Tf [(O)] TJ ET BT 408.086 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 412.904 102.284 Td /F1 9.8 Tf [( vs H)] TJ ET BT 435.115 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 439.934 102.284 Td /F1 9.8 Tf [(O)] TJ ET BT 447.519 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 452.338 102.284 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 63.763 Td /F4 9.8 Tf [(Comparative effects of neurotrophic factors and GCM on Q111 cells)] TJ ET BT 26.250 44.358 Td /F4 9.8 Tf [(The growth factor bFGF has beneficial effects in Q111 cells but fewer than fetal GCM. )] TJ ET BT 423.397 44.358 Td /F1 9.8 Tf [(Basic FGF 10 and 20 ng/ml, did not )] TJ ET Q q 15.000 30.073 577.500 746.927 re W n 0.965 0.965 0.965 rg 26.250 583.814 555.000 193.186 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 583.814 m 581.250 583.814 l 581.250 584.564 l 26.250 584.564 l f q 35.250 595.064 537.000 181.936 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 4: Mechanisms of GCM neuroprotection in Q111 cells)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(\(A\) Pattern of band proteins in GCM and DM. Gel electrophoresis of defined medium and fetal striatal glia-conditioned )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(medium \(GCM\) and their corresponding densitometric scanning. Numbers 1 to 7 \(250, 123, 38, 36, 33, 29 and 12 KDa )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(respectively\) on right panel indicate the seven bands only present in GCM. \(B\) Levels of glutathione in defined medium and )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(fetal GCM. \(C\) Intracellular glutathione levels in Q111 cells treated with H)] TJ ET BT 351.150 705.749 Td /F1 8.7 Tf [(2)] TJ ET BT 355.969 706.897 Td /F1 9.8 Tf [(O)] TJ ET BT 363.554 705.749 Td /F1 8.7 Tf [(2)] TJ ET BT 368.373 706.897 Td /F1 9.8 Tf [( \(2 M\) and GCM for 24 h. \(D\) Expression of )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(the full and cleaved, activated, forms of PARP in cells treated with H2O2 in DM or GCM. \(E\) Expression of chaperone )] TJ ET BT 35.250 679.425 Td /F1 9.8 Tf [(Hsp70. \(F\) Accumulation of polyubiquitinated proteins induced by H)] TJ ET BT 326.248 678.276 Td /F1 8.7 Tf [(2)] TJ ET BT 331.067 679.425 Td /F1 9.8 Tf [(O)] TJ ET BT 338.653 678.276 Td /F1 8.7 Tf [(2)] TJ ET BT 343.471 679.425 Td /F1 9.8 Tf [( in Q111 cells and \(G\) their corresponding )] TJ ET BT 35.250 665.689 Td /F1 9.8 Tf [(densitometric analysis. The values are expressed as mean SEM \(n = 4-6 samples in each experimental group\). Statistical )] TJ ET BT 35.250 651.952 Td /F1 9.8 Tf [(analysis was performed by one-way ANOVA followed by Newman-Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control )] TJ ET BT 35.250 638.216 Td /F1 9.8 Tf [(group. )] TJ ET BT 65.602 643.936 Td /F1 8.7 Tf [(&)] TJ ET BT 71.382 638.216 Td /F1 9.8 Tf [(p<0.05, )] TJ ET BT 106.892 643.936 Td /F1 8.7 Tf [(&&)] TJ ET BT 118.453 638.216 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 153.963 643.936 Td /F1 8.7 Tf [(&&&)] TJ ET BT 171.305 638.216 Td /F1 9.8 Tf [(p<0.001 GCM + H)] TJ ET BT 250.426 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 255.245 638.216 Td /F1 9.8 Tf [(O)] TJ ET BT 262.830 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 267.649 638.216 Td /F1 9.8 Tf [( vs H)] TJ ET BT 289.859 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 294.678 638.216 Td /F1 9.8 Tf [(O)] TJ ET BT 302.264 637.068 Td /F1 8.7 Tf [(2)] TJ ET BT 307.082 638.216 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 566.790 Td /F4 9.8 Tf [(Postnatal GCM prevents cell death produced by H)] TJ ET BT 257.598 564.726 Td /F4 8.7 Tf [(2)] TJ ET BT 262.417 566.790 Td /F4 9.8 Tf [(O)] TJ ET BT 270.002 564.726 Td /F4 8.7 Tf [(2)] TJ ET BT 274.821 566.790 Td /F4 9.8 Tf [( in Q111 cells. )] TJ ET BT 342.574 566.790 Td /F1 9.8 Tf [(We have also studied the protective effects of )] TJ ET BT 26.250 554.886 Td /F1 9.8 Tf [(postnatal GCM, of 13 days and 2 months in the huntingtin mutant cells \(Fig 5\). Postnatal GCM prevents H)] TJ ET BT 481.994 552.821 Td /F1 8.7 Tf [(2)] TJ ET BT 486.813 554.886 Td /F1 9.8 Tf [(O)] TJ ET BT 494.398 552.821 Td /F1 8.7 Tf [(2)] TJ ET BT 499.217 554.886 Td /F1 9.8 Tf [( induced cell )] TJ ET BT 26.250 542.981 Td /F1 9.8 Tf [(death in Q111 cells. GCM prevented the H)] TJ ET BT 209.959 540.917 Td /F1 8.7 Tf [(2)] TJ ET BT 214.778 542.981 Td /F1 9.8 Tf [(O)] TJ ET BT 222.364 540.917 Td /F1 8.7 Tf [(2)] TJ ET BT 227.182 542.981 Td /F1 9.8 Tf [(-induced reduction of the -tubulin area and the increased apoptosis in Q111 )] TJ ET BT 26.250 531.076 Td /F1 9.8 Tf [(cells \(F 5A-H\).)] TJ ET 0.965 0.965 0.965 rg 26.250 80.787 555.000 440.408 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 521.195 m 581.250 521.195 l 581.250 520.445 l 26.250 520.445 l f 26.250 80.787 m 581.250 80.787 l 581.250 81.537 l 26.250 81.537 l f q 168.750 0 0 225.000 35.250 286.445 cm /I7 Do Q q 35.250 92.037 537.000 188.408 re W n 0.271 0.267 0.267 rg BT 35.250 270.921 Td /F4 9.8 Tf [(Fig. 5: The 13-day and 2-month postnatal striatal glial conditioned medium protects from H2O2 toxicity in Q111 )] TJ ET BT 35.250 259.017 Td /F4 9.8 Tf [(cells)] TJ ET BT 35.250 239.647 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, H)] TJ ET BT 185.341 238.498 Td /F1 8.7 Tf [(2)] TJ ET BT 190.160 239.647 Td /F1 9.8 Tf [(O)] TJ ET BT 197.746 238.498 Td /F1 8.7 Tf [(2)] TJ ET BT 202.564 239.647 Td /F1 9.8 Tf [( \(2 M\) and 13 day postnatal striatal GCM treated cells for 24 h at 3 DIV \(scale bar = )] TJ ET BT 35.250 225.910 Td /F1 9.8 Tf [(30 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-tubulin\). \(C\) )] TJ ET BT 35.250 212.174 Td /F1 9.8 Tf [(Photomicrograph of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells. The arrows )] TJ ET BT 35.250 198.438 Td /F1 9.8 Tf [(indicate the apoptotic cells produced by the H)] TJ ET BT 231.976 197.289 Td /F1 8.7 Tf [(2)] TJ ET BT 236.794 198.438 Td /F1 9.8 Tf [(O)] TJ ET BT 244.380 197.289 Td /F1 8.7 Tf [(2)] TJ ET BT 249.199 198.438 Td /F1 9.8 Tf [( toxicity. \(D\) Percentage of chromatin condensed and fragmented nuclei in )] TJ ET BT 35.250 184.702 Td /F1 9.8 Tf [(the different experimental groups. \(E\) Photomicrographs of control, H)] TJ ET BT 333.288 183.553 Td /F1 8.7 Tf [(2)] TJ ET BT 338.107 184.702 Td /F1 9.8 Tf [(O)] TJ ET BT 345.692 183.553 Td /F1 8.7 Tf [(2)] TJ ET BT 350.511 184.702 Td /F1 9.8 Tf [( \(2 M\) and 2 month postnatal striatal GCM )] TJ ET BT 35.250 170.965 Td /F1 9.8 Tf [(treated cells for 24 h at 3 DIV \(scale bar = 30 ?m\) and \(F\) their corresponding quantification of total neurons expressed as )] TJ ET BT 35.250 157.229 Td /F1 9.8 Tf [(microtubule protein -tubulin. \(G\) Photomicrographs of total nuclei stained with bis-benzimide in the four experimental )] TJ ET BT 35.250 143.493 Td /F1 9.8 Tf [(groups of Q111 cells. The arrows indicate the apoptotic cells produced by the H)] TJ ET BT 378.284 142.344 Td /F1 8.7 Tf [(2)] TJ ET BT 383.103 143.493 Td /F1 9.8 Tf [(O)] TJ ET BT 390.688 142.344 Td /F1 8.7 Tf [(2)] TJ ET BT 395.507 143.493 Td /F1 9.8 Tf [( toxicity \(H\) Percentage of chromatin-)] TJ ET BT 35.250 129.757 Td /F1 9.8 Tf [(condensed and fragmented nuclei in the different experimental groups. The values are expressed as mean SEM \(n = 4-6 )] TJ ET BT 35.250 116.020 Td /F1 9.8 Tf [(samples in each experimental group\). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls )] TJ ET BT 35.250 102.284 Td /F1 9.8 Tf [(test. *p<0.05, **p<0.01 vs control group. )] TJ ET BT 210.857 108.004 Td /F1 8.7 Tf [(&)] TJ ET BT 216.638 102.284 Td /F1 9.8 Tf [(p<0.05, )] TJ ET BT 252.147 108.004 Td /F1 8.7 Tf [(&&)] TJ ET BT 263.709 102.284 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 299.218 108.004 Td /F1 8.7 Tf [(&&&)] TJ ET BT 316.560 102.284 Td /F1 9.8 Tf [(p<0.001 GCM + H)] TJ ET BT 395.682 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 400.500 102.284 Td /F1 9.8 Tf [(O)] TJ ET BT 408.086 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 412.904 102.284 Td /F1 9.8 Tf [( vs H)] TJ ET BT 435.115 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 439.934 102.284 Td /F1 9.8 Tf [(O)] TJ ET BT 447.519 101.136 Td /F1 8.7 Tf [(2)] TJ ET BT 452.338 102.284 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 63.763 Td /F4 9.8 Tf [(Comparative effects of neurotrophic factors and GCM on Q111 cells)] TJ ET BT 26.250 44.358 Td /F4 9.8 Tf [(The growth factor bFGF has beneficial effects in Q111 cells but fewer than fetal GCM. )] TJ ET BT 423.397 44.358 Td /F1 9.8 Tf [(Basic FGF 10 and 20 ng/ml, did not )] TJ ET Q q 168.750 0 0 225.000 35.250 286.445 cm /I7 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 379 0 obj << /Type /Annot /Subtype /Link /A 380 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 286.4452 204.0000 511.4452 ] >> endobj 380 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-5.jpg) >> endobj 381 0 obj << /Type /XObject /Subtype /Image /Width 225 /Height 300 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 34242>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?#/Ok>_0XI4#+jȱ,zF$%' Ec|𖭨iĝ M,.*Mt#T zŝ"@ds~ ~ 49hY!u[2@Sk s?ҸqXx^_i睌Rr_׻/ۋ60▁q), >Z صc9x5SuZ鴃U]F+(n%P2vq'i|/V=)83@*:_F~AzqƱMAuoFΤUMӃJVݓzo K6]KFI@@)WV>qfPĮ2Fq7>2|PҴ{.VΪtO*16 957Ť^޳ ̗`+ Rđ?0>ƸfSn6me >vEIMٮfV_A*~,pt~>xx4o.$J"FI";}}Aoa"nj'<~կൎYe޻s_-i7 ^9G;+~تCGpj+_֛_CjgNEz'n bIT! 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Both doses of bFGF decreased the percentage of apoptotic cells \(Fig 6 C and D\) and increased the cell differentiation )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(measured by the number of cells with multiprocesses \(Fig 6 E and F\), but the effects of bFGF, at the doses used in these )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(experiments, were less potent than those of GCM.)] TJ ET 0.965 0.965 0.965 rg 26.250 320.850 555.000 401.031 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 721.881 m 581.250 721.881 l 581.250 721.131 l 26.250 721.131 l f 26.250 320.850 m 581.250 320.850 l 581.250 321.600 l 26.250 321.600 l f q 168.750 0 0 225.000 35.250 487.131 cm /I8 Do Q q 35.250 332.100 537.000 149.031 re W n 0.271 0.267 0.267 rg BT 35.250 471.607 Td /F4 9.8 Tf [(Fig. 6: The effects of growth factor bFGF and the fetal striatal glia-conditioned in Q111 cells)] TJ ET BT 35.250 452.237 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, fetal striatal GCM, bFGF \(10 ng/mL\) and bFGF \(20 ng/mL\) treated cells for 24 h at 3 DIV )] TJ ET BT 35.250 438.501 Td /F1 9.8 Tf [(\(scale bar = 30 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-tubulin\). )] TJ ET BT 35.250 424.765 Td /F1 9.8 Tf [(\(C\) Photomicrographs of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells \(scale bar = )] TJ ET BT 35.250 411.028 Td /F1 9.8 Tf [(30 m\). The arrows indicate the apoptotic cells. \(D\) Percentage of chromatin condensed and fragmented nuclei in the )] TJ ET BT 35.250 397.292 Td /F1 9.8 Tf [(different experimental groups. \(E\) Photomicrographs of synaptophysin staining \(scale bar = 30 m\). A portion of this )] TJ ET BT 35.250 383.556 Td /F1 9.8 Tf [(micrograph has been magnified in the picture on the right. \(F\) Percentage of cells with multiprocesses, cells with more than )] TJ ET BT 35.250 369.820 Td /F1 9.8 Tf [(3 neurite arborization, to test the cell differentiation. The values are expressed as mean SEM \(n = 6 samples in each )] TJ ET BT 35.250 356.083 Td /F1 9.8 Tf [(experimental group\). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. *p<0.05, )] TJ ET BT 35.250 342.347 Td /F1 9.8 Tf [(**p<0.01, ***p<0.001 vs control group. ^p<0.05 bFGF vs fetal striatal GCM.)] TJ ET Q BT 26.250 303.826 Td /F4 9.8 Tf [(The growth factor GDNF has beneficial effects in Q111 cells but fewer than fetal GCM. )] TJ ET BT 425.561 303.826 Td /F1 9.8 Tf [(GDNF, 50 and 100 ng/ml, did not )] TJ ET BT 26.250 291.921 Td /F1 9.8 Tf [(increase, as GCM does, the -tubulin area of the cultures \(Fig 7A and B\) and had a significant but very mild protective effect on )] TJ ET BT 26.250 280.017 Td /F1 9.8 Tf [(apoptosis, which is strongly suppressed by GCM \(Fig 7C and D\). GDNF 50 ng/ml failed to increase the percentage of cells with )] TJ ET BT 26.250 268.112 Td /F1 9.8 Tf [(multiple processes, GDNF, 100ng/ml increased it by around 25 %; however, GCM almost doubled it \(Fig 7E and F\). As a )] TJ ET BT 26.250 256.207 Td /F1 9.8 Tf [(consequence, GCM was a much more potent neurotrophic agent for Q111 cells than GDNF at doses used in this study.)] TJ ET 0.965 0.965 0.965 rg 26.250 5.576 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 246.326 m 581.250 246.326 l 581.250 245.576 l 26.250 245.576 l f q 168.000 0 0 225.000 35.250 11.576 cm /I9 Do Q q 35.250 5.576 537.000 0.000 re W n Q Q q 15.000 5.576 577.500 771.424 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(change the neuronal area of the cultures, as measured by the -tubulin staining, a parameter which is increased by GCM \(Fig 6 )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(A and B\). Both doses of bFGF decreased the percentage of apoptotic cells \(Fig 6 C and D\) and increased the cell differentiation )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(measured by the number of cells with multiprocesses \(Fig 6 E and F\), but the effects of bFGF, at the doses used in these )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(experiments, were less potent than those of GCM.)] TJ ET 0.965 0.965 0.965 rg 26.250 320.850 555.000 401.031 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 721.881 m 581.250 721.881 l 581.250 721.131 l 26.250 721.131 l f 26.250 320.850 m 581.250 320.850 l 581.250 321.600 l 26.250 321.600 l f q 168.750 0 0 225.000 35.250 487.131 cm /I8 Do Q q 35.250 332.100 537.000 149.031 re W n 0.271 0.267 0.267 rg BT 35.250 471.607 Td /F4 9.8 Tf [(Fig. 6: The effects of growth factor bFGF and the fetal striatal glia-conditioned in Q111 cells)] TJ ET BT 35.250 452.237 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, fetal striatal GCM, bFGF \(10 ng/mL\) and bFGF \(20 ng/mL\) treated cells for 24 h at 3 DIV )] TJ ET BT 35.250 438.501 Td /F1 9.8 Tf [(\(scale bar = 30 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-tubulin\). )] TJ ET BT 35.250 424.765 Td /F1 9.8 Tf [(\(C\) Photomicrographs of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells \(scale bar = )] TJ ET BT 35.250 411.028 Td /F1 9.8 Tf [(30 m\). The arrows indicate the apoptotic cells. \(D\) Percentage of chromatin condensed and fragmented nuclei in the )] TJ ET BT 35.250 397.292 Td /F1 9.8 Tf [(different experimental groups. \(E\) Photomicrographs of synaptophysin staining \(scale bar = 30 m\). A portion of this )] TJ ET BT 35.250 383.556 Td /F1 9.8 Tf [(micrograph has been magnified in the picture on the right. \(F\) Percentage of cells with multiprocesses, cells with more than )] TJ ET BT 35.250 369.820 Td /F1 9.8 Tf [(3 neurite arborization, to test the cell differentiation. The values are expressed as mean SEM \(n = 6 samples in each )] TJ ET BT 35.250 356.083 Td /F1 9.8 Tf [(experimental group\). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. *p<0.05, )] TJ ET BT 35.250 342.347 Td /F1 9.8 Tf [(**p<0.01, ***p<0.001 vs control group. ^p<0.05 bFGF vs fetal striatal GCM.)] TJ ET Q BT 26.250 303.826 Td /F4 9.8 Tf [(The growth factor GDNF has beneficial effects in Q111 cells but fewer than fetal GCM. )] TJ ET BT 425.561 303.826 Td /F1 9.8 Tf [(GDNF, 50 and 100 ng/ml, did not )] TJ ET BT 26.250 291.921 Td /F1 9.8 Tf [(increase, as GCM does, the -tubulin area of the cultures \(Fig 7A and B\) and had a significant but very mild protective effect on )] TJ ET BT 26.250 280.017 Td /F1 9.8 Tf [(apoptosis, which is strongly suppressed by GCM \(Fig 7C and D\). GDNF 50 ng/ml failed to increase the percentage of cells with )] TJ ET BT 26.250 268.112 Td /F1 9.8 Tf [(multiple processes, GDNF, 100ng/ml increased it by around 25 %; however, GCM almost doubled it \(Fig 7E and F\). As a )] TJ ET BT 26.250 256.207 Td /F1 9.8 Tf [(consequence, GCM was a much more potent neurotrophic agent for Q111 cells than GDNF at doses used in this study.)] TJ ET 0.965 0.965 0.965 rg 26.250 5.576 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 246.326 m 581.250 246.326 l 581.250 245.576 l 26.250 245.576 l f q 168.000 0 0 225.000 35.250 11.576 cm /I9 Do Q q 35.250 5.576 537.000 0.000 re W n Q Q q 15.000 5.576 577.500 771.424 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(change the neuronal area of the cultures, as measured by the -tubulin staining, a parameter which is increased by GCM \(Fig 6 )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(A and B\). Both doses of bFGF decreased the percentage of apoptotic cells \(Fig 6 C and D\) and increased the cell differentiation )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(measured by the number of cells with multiprocesses \(Fig 6 E and F\), but the effects of bFGF, at the doses used in these )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(experiments, were less potent than those of GCM.)] TJ ET 0.965 0.965 0.965 rg 26.250 320.850 555.000 401.031 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 721.881 m 581.250 721.881 l 581.250 721.131 l 26.250 721.131 l f 26.250 320.850 m 581.250 320.850 l 581.250 321.600 l 26.250 321.600 l f q 168.750 0 0 225.000 35.250 487.131 cm /I8 Do Q q 35.250 332.100 537.000 149.031 re W n 0.271 0.267 0.267 rg BT 35.250 471.607 Td /F4 9.8 Tf [(Fig. 6: The effects of growth factor bFGF and the fetal striatal glia-conditioned in Q111 cells)] TJ ET BT 35.250 452.237 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, fetal striatal GCM, bFGF \(10 ng/mL\) and bFGF \(20 ng/mL\) treated cells for 24 h at 3 DIV )] TJ ET BT 35.250 438.501 Td /F1 9.8 Tf [(\(scale bar = 30 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-tubulin\). )] TJ ET BT 35.250 424.765 Td /F1 9.8 Tf [(\(C\) Photomicrographs of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells \(scale bar = )] TJ ET BT 35.250 411.028 Td /F1 9.8 Tf [(30 m\). The arrows indicate the apoptotic cells. \(D\) Percentage of chromatin condensed and fragmented nuclei in the )] TJ ET BT 35.250 397.292 Td /F1 9.8 Tf [(different experimental groups. \(E\) Photomicrographs of synaptophysin staining \(scale bar = 30 m\). A portion of this )] TJ ET BT 35.250 383.556 Td /F1 9.8 Tf [(micrograph has been magnified in the picture on the right. \(F\) Percentage of cells with multiprocesses, cells with more than )] TJ ET BT 35.250 369.820 Td /F1 9.8 Tf [(3 neurite arborization, to test the cell differentiation. The values are expressed as mean SEM \(n = 6 samples in each )] TJ ET BT 35.250 356.083 Td /F1 9.8 Tf [(experimental group\). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. *p<0.05, )] TJ ET BT 35.250 342.347 Td /F1 9.8 Tf [(**p<0.01, ***p<0.001 vs control group. ^p<0.05 bFGF vs fetal striatal GCM.)] TJ ET Q BT 26.250 303.826 Td /F4 9.8 Tf [(The growth factor GDNF has beneficial effects in Q111 cells but fewer than fetal GCM. )] TJ ET BT 425.561 303.826 Td /F1 9.8 Tf [(GDNF, 50 and 100 ng/ml, did not )] TJ ET BT 26.250 291.921 Td /F1 9.8 Tf [(increase, as GCM does, the -tubulin area of the cultures \(Fig 7A and B\) and had a significant but very mild protective effect on )] TJ ET BT 26.250 280.017 Td /F1 9.8 Tf [(apoptosis, which is strongly suppressed by GCM \(Fig 7C and D\). GDNF 50 ng/ml failed to increase the percentage of cells with )] TJ ET BT 26.250 268.112 Td /F1 9.8 Tf [(multiple processes, GDNF, 100ng/ml increased it by around 25 %; however, GCM almost doubled it \(Fig 7E and F\). As a )] TJ ET BT 26.250 256.207 Td /F1 9.8 Tf [(consequence, GCM was a much more potent neurotrophic agent for Q111 cells than GDNF at doses used in this study.)] TJ ET 0.965 0.965 0.965 rg 26.250 5.576 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 246.326 m 581.250 246.326 l 581.250 245.576 l 26.250 245.576 l f q 168.000 0 0 225.000 35.250 11.576 cm /I9 Do Q q 35.250 5.576 537.000 0.000 re W n Q Q q 168.750 0 0 225.000 35.250 487.131 cm /I8 Do Q q 168.000 0 0 225.000 35.250 11.576 cm /I9 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(8)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 388 0 obj << /Type /Annot /Subtype /Link /A 389 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 487.1310 204.0000 712.1310 ] >> endobj 389 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-6.jpg) >> endobj 390 0 obj << /Type /XObject /Subtype /Image /Width 225 /Height 300 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 31457>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?5׋c6iq[P ӕ$p$ VizaXAqaoմl:Gj܅DmA_U "m~..XX0I8OOzVuTdk^]6*'Ԅcn3du^w^u8D$72p̸'o$:W?~>>3s˫==Ԭ4Qs$ʅT*E%U\er@$| yM6 &C8\s ,Jgje&ͮ^zJQpVGݻ[K?Ht ZB)״2u P1g_ h:vjW֗fOYD-ݼ@12 qzt-IRզ$?"B(Va?b|Ì1ӅT^wty_ O0ޭ]ik]uh-gʎ)%v$=qWU w,ILІ$Ȱ$ĜLקBY,D4o]Œh_ΏΓ4f h4&h3Fh:?:Lњâ(w5z_ڋFo=.-hs^erF1pyCӭ~_X ][|~P4;$˘}ÌwsOJS3Z1k7e~|_xY/`yH˱s`c'w,f_Zn;.$ #޽7A_CA7F~h-u,\fu ʇ⼃F}ki~]C<L*!5 1'88ʿ$~i+;ݮ[;G0} %i)5Ů٧~uK  )- (nHIcA-m-b:w[]M4ˍҤ"F<p3oF"-%pϻ/&|~[ L `yY|c8#!|>Zf$BT#[ #!oAWoJԭ4-'^ Zrg@Qe&Sck4|s4x`9w? h 5? kǥrYO1D-HV0I[۷˨Ӎ:99;]zEU k>c[ (XsH"s䢌x7φw,5]NywA%@bp M3n|G--\W,[?:V#XO mjGn? }}+Pvw_Q*mr/zkⵟ<-NbէrY\T!WSF ; h:N[Ykmv5>ra7L f=mf/4KG(miԅYy+eۛ4Dk:ơ{,3 |:5NQienfxz)=tֵc¼TZ~"DSŖ:`!%nq""Cpv#x{=[qG rB4fO6O&x7iZ%Vue\32}!渏-Ҿ)5˗mmR8hfdVpsNyᰯUiUA+?Dצ,:SJT.^{_ ZKHft[~]ּEg{ xj9ż?vR#9_Iz?i&:M年@r .;Q }(}SLsJsf/ڱTc mj<\NlGyD({*։$m(⼦}'.>Hm/-O%rQH Lx,'ƟL^ zX;kcw_|⛋ |<5:klQݼ\uYׁ=ռ v._T$3:Kq}?o Z:vo^\c7W-`9feG<xxv_^CwmrG2{e}<7zT^MsTNMf\f$9I5t?<NT6o~n v&>O|[a%փwǐ"sDQ 6l`tu{}ù,ͫO Rg^6#*+ Q@)|1.Q=9i$Si]_Tk Zszxmuq0rDz$9qȯ30mRRɫs2\Rm{_Ms)M|ksk]̲/2xp}2ڣ[E-],ݼ餞(%M2@c#Ð>.NJ!K}hD.# cCYr=Nk.Ch^Skaتl\-v"RRݜ]}v*U87g}vOikhZ>~?jt/;i-qe,Ubɮ_۸[wi-"6"߼m #OėP]A`Jmbᣅdf a=z+^J5IU0'?qAyQJ kE&:/(ђw+'mz[]5IW.Xh_ohgim\|Üո}7I_>#-X5Kח X7a6kyo-|.bu\[A,ebNH5τmO$/RdGz7F[ӺZf9jfiMӚjIj]4֚y}G/_SSĚzNg LDO$hQUp9U)Zk.qTcB|.-Ark^H{ B|AQh[kZ| H;Owۄ.[RHγ1, TaynL;5M?&b5 cUZ2N6Fҿ>5ߋux4n[.4kl9yz c֍x94~k ]9TW3J|ֻ3>-{ڗ$ڊ]\xJ?z*@6D\^[[7>? 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The arrows indicate the apoptotic cells. \(D\) Percentage of chromatin condensed and fragmented nuclei )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(in the different experimental groups. \(E\) Photomicrographs of synaptophysin staining \(scale bar = 30 m\). \(F\) Percentage of )] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(cells multiprocesses, cells with more than 3 neurite arborization, to check the cell differentiation. The values are expressed )] TJ ET BT 35.250 653.784 Td /F1 9.8 Tf [(as mean SEM \(n = 6 samples in each experimental group\). Statistical analysis was performed by one-way ANOVA )] TJ ET BT 35.250 640.048 Td /F1 9.8 Tf [(followed by Newman-Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control group. ^^p<0.01, ^^^p<0.001 GDNF vs fetal )] TJ ET BT 35.250 626.311 Td /F1 9.8 Tf [(striatal GCM.)] TJ ET Q BT 26.250 587.790 Td /F4 9.8 Tf [(Effects of growth factor BDNF and fetal GCM in Q111 cells. )] TJ ET BT 301.473 587.790 Td /F1 9.8 Tf [(BDNF, 50 and 100 ng/ml, did not increase, as GCM does, the -)] TJ ET BT 26.250 575.886 Td /F1 9.8 Tf [(tubulin area of the cultures \(Fig 8A and B\) and had a significant but smaller protective effect on apoptosis than GCM \(Fig 8C )] TJ ET BT 26.250 563.981 Td /F1 9.8 Tf [(and D\). BDNF, 50 ng/ml, failed to increase the number of differentiated cells with multiple processes. BDNF, 100 ng/ml, )] TJ ET BT 26.250 552.076 Td /F1 9.8 Tf [(increased this number significantly, with respect to control, but was less potent than GCM \(Fig 8E and F\). As a consequence, )] TJ ET BT 26.250 540.171 Td /F1 9.8 Tf [(GCM was a much more potent neurotrophic agent for Q111 cells than BDNF at doses used in this study.)] TJ ET 0.965 0.965 0.965 rg 26.250 131.091 555.000 399.200 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 530.290 m 581.250 530.290 l 581.250 529.540 l 26.250 529.540 l f 26.250 131.091 m 581.250 131.091 l 581.250 131.841 l 26.250 131.841 l f q 168.750 0 0 225.000 35.250 295.540 cm /I10 Do Q q 35.250 142.341 537.000 147.200 re W n 0.271 0.267 0.267 rg BT 35.250 280.017 Td /F4 9.8 Tf [(Fig. 8: The growth factor BDNF and the fetal striatal glia-conditioned medium protect from H2O2 induced cell death )] TJ ET BT 35.250 268.112 Td /F4 9.8 Tf [(in Q111 cells)] TJ ET BT 35.250 248.742 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, fetal striatal GCM, BDNF \(50 ng/ml\) and BDNF \(100 ng/ml\) treated cells for 24 h at 3 DIV )] TJ ET BT 35.250 235.006 Td /F1 9.8 Tf [(\(scale bar = 10 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-tubulin\). )] TJ ET BT 35.250 221.269 Td /F1 9.8 Tf [(\(C\) Photomicrograph of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells \(scale bar = 30 )] TJ ET BT 35.250 207.533 Td /F1 9.8 Tf [(m\). The arrows indicate the apoptotic cells. \(D\) Percentage of chromatin-condensed and fragmented nuclei in the different )] TJ ET BT 35.250 193.797 Td /F1 9.8 Tf [(experimental groups. \(E\) Photomicrograph of synaptophysin staining \(scale bar = 30 m\). \(F\) Percentage of multiprocess )] TJ ET BT 35.250 180.061 Td /F1 9.8 Tf [(cells, \(with more than 3 neurite arborization\) to test the cell differentiation. The values are expressed as mean SEM \(n = 6 )] TJ ET BT 35.250 166.324 Td /F1 9.8 Tf [(samples in each experimental group\). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls )] TJ ET BT 35.250 152.588 Td /F1 9.8 Tf [(test. *p<0.05, **p<0.01, ***p<0.001 vs control group. ^p<0.05, ^^p<0.01, ^^^p<0.001 BDNF vs fetal striatal GCM.)] TJ ET Q BT 26.250 114.067 Td /F4 9.8 Tf [(Comparative effects of growth factors and fetal striatal GCM on H)] TJ ET BT 329.095 112.003 Td /F4 8.7 Tf [(2)] TJ ET BT 333.913 114.067 Td /F4 9.8 Tf [(O)] TJ ET BT 341.499 112.003 Td /F4 8.7 Tf [(2)] TJ ET BT 346.318 114.067 Td /F4 9.8 Tf [( induced cell death. )] TJ ET BT 439.518 114.067 Td /F1 9.8 Tf [(We compared the effects of )] TJ ET BT 26.250 102.162 Td /F1 9.8 Tf [(bFGF, GDNF, BDNF and fetal GCM on H)] TJ ET BT 205.046 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 209.864 102.162 Td /F1 9.8 Tf [(O)] TJ ET BT 217.450 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 222.268 102.162 Td /F1 9.8 Tf [( induced toxicity. BDNF and bFGF failed to revert the H)] TJ ET BT 460.695 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 465.514 102.162 Td /F1 9.8 Tf [(O)] TJ ET BT 473.099 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 477.918 102.162 Td /F1 9.8 Tf [( induced reduction of )] TJ ET BT 26.250 90.258 Td /F1 9.8 Tf [(the -tubulin area of the cultures while GDNF and GCM did \(Fig 9A and B\). BDNF, GDNF and GCM prevented the H)] TJ ET BT 529.096 88.193 Td /F1 8.7 Tf [(2)] TJ ET BT 533.915 90.258 Td /F1 9.8 Tf [(O)] TJ ET BT 541.501 88.193 Td /F1 8.7 Tf [(2)] TJ ET BT 26.250 78.353 Td /F1 9.8 Tf [(induced apoptosis, being GCM the most effective \(Fig 9C\) while bFGF failed to decrease apoptosis. None of the neurotrophic )] TJ ET BT 26.250 66.448 Td /F1 9.8 Tf [(factors, at the doses used in this study, reverted the reduction of differentiated cells induced by H)] TJ ET BT 444.086 64.384 Td /F1 8.7 Tf [(2)] TJ ET BT 448.905 66.448 Td /F1 9.8 Tf [(O)] TJ ET BT 456.490 64.384 Td /F1 8.7 Tf [(2)] TJ ET BT 461.309 66.448 Td /F1 9.8 Tf [(, but GCM completely )] TJ ET BT 26.250 54.543 Td /F1 9.8 Tf [(normalized it \(Fig 9D and E\). As a consequence, GCM was a more potent neurotrophic agent for Q111 cells than any of the )] TJ ET BT 26.250 42.639 Td /F1 9.8 Tf [(neurotrophic factors at the doses used in this study.)] TJ ET Q q 15.000 32.758 577.500 744.242 re W n 0.965 0.965 0.965 rg 26.250 604.814 555.000 172.186 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 604.814 m 581.250 604.814 l 581.250 605.564 l 26.250 605.564 l f q 35.250 616.064 537.000 160.936 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 7: The growth factor GDNF and the fetal striatal glia-conditioned medium protect from H2O2 induced cell death )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(in Q111 cells)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, fetal striatal GCM, GDNF \(50 ng/mL\) and GDNF \(100 ng/mL\) treated cells for 24 h at 3 )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(DIV \(scale bar = 10 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-)] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(tubulin\). \(C\) Photomicrographs of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(\(scale bar = 30 m\). The arrows indicate the apoptotic cells. \(D\) Percentage of chromatin condensed and fragmented nuclei )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(in the different experimental groups. \(E\) Photomicrographs of synaptophysin staining \(scale bar = 30 m\). \(F\) Percentage of )] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(cells multiprocesses, cells with more than 3 neurite arborization, to check the cell differentiation. The values are expressed )] TJ ET BT 35.250 653.784 Td /F1 9.8 Tf [(as mean SEM \(n = 6 samples in each experimental group\). Statistical analysis was performed by one-way ANOVA )] TJ ET BT 35.250 640.048 Td /F1 9.8 Tf [(followed by Newman-Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control group. ^^p<0.01, ^^^p<0.001 GDNF vs fetal )] TJ ET BT 35.250 626.311 Td /F1 9.8 Tf [(striatal GCM.)] TJ ET Q BT 26.250 587.790 Td /F4 9.8 Tf [(Effects of growth factor BDNF and fetal GCM in Q111 cells. )] TJ ET BT 301.473 587.790 Td /F1 9.8 Tf [(BDNF, 50 and 100 ng/ml, did not increase, as GCM does, the -)] TJ ET BT 26.250 575.886 Td /F1 9.8 Tf [(tubulin area of the cultures \(Fig 8A and B\) and had a significant but smaller protective effect on apoptosis than GCM \(Fig 8C )] TJ ET BT 26.250 563.981 Td /F1 9.8 Tf [(and D\). BDNF, 50 ng/ml, failed to increase the number of differentiated cells with multiple processes. BDNF, 100 ng/ml, )] TJ ET BT 26.250 552.076 Td /F1 9.8 Tf [(increased this number significantly, with respect to control, but was less potent than GCM \(Fig 8E and F\). As a consequence, )] TJ ET BT 26.250 540.171 Td /F1 9.8 Tf [(GCM was a much more potent neurotrophic agent for Q111 cells than BDNF at doses used in this study.)] TJ ET 0.965 0.965 0.965 rg 26.250 131.091 555.000 399.200 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 530.290 m 581.250 530.290 l 581.250 529.540 l 26.250 529.540 l f 26.250 131.091 m 581.250 131.091 l 581.250 131.841 l 26.250 131.841 l f q 168.750 0 0 225.000 35.250 295.540 cm /I10 Do Q q 35.250 142.341 537.000 147.200 re W n 0.271 0.267 0.267 rg BT 35.250 280.017 Td /F4 9.8 Tf [(Fig. 8: The growth factor BDNF and the fetal striatal glia-conditioned medium protect from H2O2 induced cell death )] TJ ET BT 35.250 268.112 Td /F4 9.8 Tf [(in Q111 cells)] TJ ET BT 35.250 248.742 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, fetal striatal GCM, BDNF \(50 ng/ml\) and BDNF \(100 ng/ml\) treated cells for 24 h at 3 DIV )] TJ ET BT 35.250 235.006 Td /F1 9.8 Tf [(\(scale bar = 10 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-tubulin\). )] TJ ET BT 35.250 221.269 Td /F1 9.8 Tf [(\(C\) Photomicrograph of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells \(scale bar = 30 )] TJ ET BT 35.250 207.533 Td /F1 9.8 Tf [(m\). The arrows indicate the apoptotic cells. \(D\) Percentage of chromatin-condensed and fragmented nuclei in the different )] TJ ET BT 35.250 193.797 Td /F1 9.8 Tf [(experimental groups. \(E\) Photomicrograph of synaptophysin staining \(scale bar = 30 m\). \(F\) Percentage of multiprocess )] TJ ET BT 35.250 180.061 Td /F1 9.8 Tf [(cells, \(with more than 3 neurite arborization\) to test the cell differentiation. The values are expressed as mean SEM \(n = 6 )] TJ ET BT 35.250 166.324 Td /F1 9.8 Tf [(samples in each experimental group\). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls )] TJ ET BT 35.250 152.588 Td /F1 9.8 Tf [(test. *p<0.05, **p<0.01, ***p<0.001 vs control group. ^p<0.05, ^^p<0.01, ^^^p<0.001 BDNF vs fetal striatal GCM.)] TJ ET Q BT 26.250 114.067 Td /F4 9.8 Tf [(Comparative effects of growth factors and fetal striatal GCM on H)] TJ ET BT 329.095 112.003 Td /F4 8.7 Tf [(2)] TJ ET BT 333.913 114.067 Td /F4 9.8 Tf [(O)] TJ ET BT 341.499 112.003 Td /F4 8.7 Tf [(2)] TJ ET BT 346.318 114.067 Td /F4 9.8 Tf [( induced cell death. )] TJ ET BT 439.518 114.067 Td /F1 9.8 Tf [(We compared the effects of )] TJ ET BT 26.250 102.162 Td /F1 9.8 Tf [(bFGF, GDNF, BDNF and fetal GCM on H)] TJ ET BT 205.046 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 209.864 102.162 Td /F1 9.8 Tf [(O)] TJ ET BT 217.450 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 222.268 102.162 Td /F1 9.8 Tf [( induced toxicity. BDNF and bFGF failed to revert the H)] TJ ET BT 460.695 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 465.514 102.162 Td /F1 9.8 Tf [(O)] TJ ET BT 473.099 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 477.918 102.162 Td /F1 9.8 Tf [( induced reduction of )] TJ ET BT 26.250 90.258 Td /F1 9.8 Tf [(the -tubulin area of the cultures while GDNF and GCM did \(Fig 9A and B\). BDNF, GDNF and GCM prevented the H)] TJ ET BT 529.096 88.193 Td /F1 8.7 Tf [(2)] TJ ET BT 533.915 90.258 Td /F1 9.8 Tf [(O)] TJ ET BT 541.501 88.193 Td /F1 8.7 Tf [(2)] TJ ET BT 26.250 78.353 Td /F1 9.8 Tf [(induced apoptosis, being GCM the most effective \(Fig 9C\) while bFGF failed to decrease apoptosis. None of the neurotrophic )] TJ ET BT 26.250 66.448 Td /F1 9.8 Tf [(factors, at the doses used in this study, reverted the reduction of differentiated cells induced by H)] TJ ET BT 444.086 64.384 Td /F1 8.7 Tf [(2)] TJ ET BT 448.905 66.448 Td /F1 9.8 Tf [(O)] TJ ET BT 456.490 64.384 Td /F1 8.7 Tf [(2)] TJ ET BT 461.309 66.448 Td /F1 9.8 Tf [(, but GCM completely )] TJ ET BT 26.250 54.543 Td /F1 9.8 Tf [(normalized it \(Fig 9D and E\). As a consequence, GCM was a more potent neurotrophic agent for Q111 cells than any of the )] TJ ET BT 26.250 42.639 Td /F1 9.8 Tf [(neurotrophic factors at the doses used in this study.)] TJ ET Q q 15.000 32.758 577.500 744.242 re W n 0.965 0.965 0.965 rg 26.250 604.814 555.000 172.186 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 604.814 m 581.250 604.814 l 581.250 605.564 l 26.250 605.564 l f q 35.250 616.064 537.000 160.936 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 7: The growth factor GDNF and the fetal striatal glia-conditioned medium protect from H2O2 induced cell death )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(in Q111 cells)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, fetal striatal GCM, GDNF \(50 ng/mL\) and GDNF \(100 ng/mL\) treated cells for 24 h at 3 )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(DIV \(scale bar = 10 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-)] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(tubulin\). \(C\) Photomicrographs of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(\(scale bar = 30 m\). The arrows indicate the apoptotic cells. \(D\) Percentage of chromatin condensed and fragmented nuclei )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(in the different experimental groups. \(E\) Photomicrographs of synaptophysin staining \(scale bar = 30 m\). \(F\) Percentage of )] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(cells multiprocesses, cells with more than 3 neurite arborization, to check the cell differentiation. The values are expressed )] TJ ET BT 35.250 653.784 Td /F1 9.8 Tf [(as mean SEM \(n = 6 samples in each experimental group\). Statistical analysis was performed by one-way ANOVA )] TJ ET BT 35.250 640.048 Td /F1 9.8 Tf [(followed by Newman-Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control group. ^^p<0.01, ^^^p<0.001 GDNF vs fetal )] TJ ET BT 35.250 626.311 Td /F1 9.8 Tf [(striatal GCM.)] TJ ET Q BT 26.250 587.790 Td /F4 9.8 Tf [(Effects of growth factor BDNF and fetal GCM in Q111 cells. )] TJ ET BT 301.473 587.790 Td /F1 9.8 Tf [(BDNF, 50 and 100 ng/ml, did not increase, as GCM does, the -)] TJ ET BT 26.250 575.886 Td /F1 9.8 Tf [(tubulin area of the cultures \(Fig 8A and B\) and had a significant but smaller protective effect on apoptosis than GCM \(Fig 8C )] TJ ET BT 26.250 563.981 Td /F1 9.8 Tf [(and D\). BDNF, 50 ng/ml, failed to increase the number of differentiated cells with multiple processes. BDNF, 100 ng/ml, )] TJ ET BT 26.250 552.076 Td /F1 9.8 Tf [(increased this number significantly, with respect to control, but was less potent than GCM \(Fig 8E and F\). As a consequence, )] TJ ET BT 26.250 540.171 Td /F1 9.8 Tf [(GCM was a much more potent neurotrophic agent for Q111 cells than BDNF at doses used in this study.)] TJ ET 0.965 0.965 0.965 rg 26.250 131.091 555.000 399.200 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 530.290 m 581.250 530.290 l 581.250 529.540 l 26.250 529.540 l f 26.250 131.091 m 581.250 131.091 l 581.250 131.841 l 26.250 131.841 l f q 168.750 0 0 225.000 35.250 295.540 cm /I10 Do Q q 35.250 142.341 537.000 147.200 re W n 0.271 0.267 0.267 rg BT 35.250 280.017 Td /F4 9.8 Tf [(Fig. 8: The growth factor BDNF and the fetal striatal glia-conditioned medium protect from H2O2 induced cell death )] TJ ET BT 35.250 268.112 Td /F4 9.8 Tf [(in Q111 cells)] TJ ET BT 35.250 248.742 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, fetal striatal GCM, BDNF \(50 ng/ml\) and BDNF \(100 ng/ml\) treated cells for 24 h at 3 DIV )] TJ ET BT 35.250 235.006 Td /F1 9.8 Tf [(\(scale bar = 10 m\) and \(B\) their corresponding quantification of total neurons expressed as microtubule protein \(-tubulin\). )] TJ ET BT 35.250 221.269 Td /F1 9.8 Tf [(\(C\) Photomicrograph of total nuclei stained with bis-benzimide in the four experimental groups of Q111 cells \(scale bar = 30 )] TJ ET BT 35.250 207.533 Td /F1 9.8 Tf [(m\). The arrows indicate the apoptotic cells. \(D\) Percentage of chromatin-condensed and fragmented nuclei in the different )] TJ ET BT 35.250 193.797 Td /F1 9.8 Tf [(experimental groups. \(E\) Photomicrograph of synaptophysin staining \(scale bar = 30 m\). \(F\) Percentage of multiprocess )] TJ ET BT 35.250 180.061 Td /F1 9.8 Tf [(cells, \(with more than 3 neurite arborization\) to test the cell differentiation. The values are expressed as mean SEM \(n = 6 )] TJ ET BT 35.250 166.324 Td /F1 9.8 Tf [(samples in each experimental group\). Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls )] TJ ET BT 35.250 152.588 Td /F1 9.8 Tf [(test. *p<0.05, **p<0.01, ***p<0.001 vs control group. ^p<0.05, ^^p<0.01, ^^^p<0.001 BDNF vs fetal striatal GCM.)] TJ ET Q BT 26.250 114.067 Td /F4 9.8 Tf [(Comparative effects of growth factors and fetal striatal GCM on H)] TJ ET BT 329.095 112.003 Td /F4 8.7 Tf [(2)] TJ ET BT 333.913 114.067 Td /F4 9.8 Tf [(O)] TJ ET BT 341.499 112.003 Td /F4 8.7 Tf [(2)] TJ ET BT 346.318 114.067 Td /F4 9.8 Tf [( induced cell death. )] TJ ET BT 439.518 114.067 Td /F1 9.8 Tf [(We compared the effects of )] TJ ET BT 26.250 102.162 Td /F1 9.8 Tf [(bFGF, GDNF, BDNF and fetal GCM on H)] TJ ET BT 205.046 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 209.864 102.162 Td /F1 9.8 Tf [(O)] TJ ET BT 217.450 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 222.268 102.162 Td /F1 9.8 Tf [( induced toxicity. BDNF and bFGF failed to revert the H)] TJ ET BT 460.695 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 465.514 102.162 Td /F1 9.8 Tf [(O)] TJ ET BT 473.099 100.098 Td /F1 8.7 Tf [(2)] TJ ET BT 477.918 102.162 Td /F1 9.8 Tf [( induced reduction of )] TJ ET BT 26.250 90.258 Td /F1 9.8 Tf [(the -tubulin area of the cultures while GDNF and GCM did \(Fig 9A and B\). BDNF, GDNF and GCM prevented the H)] TJ ET BT 529.096 88.193 Td /F1 8.7 Tf [(2)] TJ ET BT 533.915 90.258 Td /F1 9.8 Tf [(O)] TJ ET BT 541.501 88.193 Td /F1 8.7 Tf [(2)] TJ ET BT 26.250 78.353 Td /F1 9.8 Tf [(induced apoptosis, being GCM the most effective \(Fig 9C\) while bFGF failed to decrease apoptosis. None of the neurotrophic )] TJ ET BT 26.250 66.448 Td /F1 9.8 Tf [(factors, at the doses used in this study, reverted the reduction of differentiated cells induced by H)] TJ ET BT 444.086 64.384 Td /F1 8.7 Tf [(2)] TJ ET BT 448.905 66.448 Td /F1 9.8 Tf [(O)] TJ ET BT 456.490 64.384 Td /F1 8.7 Tf [(2)] TJ ET BT 461.309 66.448 Td /F1 9.8 Tf [(, but GCM completely )] TJ ET BT 26.250 54.543 Td /F1 9.8 Tf [(normalized it \(Fig 9D and E\). As a consequence, GCM was a more potent neurotrophic agent for Q111 cells than any of the )] TJ ET BT 26.250 42.639 Td /F1 9.8 Tf [(neurotrophic factors at the doses used in this study.)] TJ ET Q q 168.750 0 0 225.000 35.250 295.540 cm /I10 Do 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 404 0 obj << /Type /Annot /Subtype /Link /A 405 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 295.5405 204.0000 520.5405 ] >> endobj 405 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-8.jpg) >> endobj 406 0 obj << /Type /XObject /Subtype /Image /Width 225 /Height 300 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 31242>> 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 $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?s~}K LjfI,myծ6'FF"$:+ FFA.ko~ڃ>#wk/uER馚Ob RpےtTvym< 'R{eN/?s" w_P#Ԣ3#I93o$4yq֬6=ś]nPm!GPo(cz sxv>gm&g׺ۣ?z(~-j?t 2YZgoپL̻Lvv eqYZuVFk 3+b"AQ3ƨGߏu _)v[M<'YHFkN(h6m>P^W}m:vt?x(~ x|mb Fkw,i'wDg)y$4Ý> [WwxQ:_= So38#VrFh䯜~%u'۫ dR_I]aqi2T1L;vYԯ5oķwr-$b @+eWUa15!}ϥ(֑j2k;@Iź]=Cmj2+3sǞx9k?Z3Fh/GI3@i:Lњ_ΏΓ4f0袊~_`Eo [xU畔FR w ~>+?-z䵅ZQnn{xߕjֿ&"nAbWo[˃ qS|{=ZYF$`\Nۃ<= ro$@%XhqZ-՞ecٛ{Ti8aFx=zWd> RN**Sݧ'/v}4'}RxM Ӭ.kq+K͍[ v鷞$w4. 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V|1[Z̃z ҽ:IoM #'O0H?RCuxm9\1$`p8VӜEkYpjژЌE'(N]dlgXK+Ly6i#qCT~Z5DU#+O~:Տ :mN6 JY(äs3&aO+ A{샥`MZ(՛-\IM>Kķ 2[@.k !"ΉN~B{7'𭿭߯0JtUnM7uklbZӔ;\f>mե#ŷw u->} -l!Iv 'xnYEa۵Η{\_j:^c I$8UU' 8*O@ xu3~)wT|5' `q")ఐ m_y~|F?o =M6ՎqpO@joEwx'Q{a iۥF ?18'!xG_GĚcfعwbBj8QH߇~~5jjOR 1{(r*#pqZ30Q2j:x!V\J܎i%^MSNCiۦW 9=㢫%? 6ο{0F*5ĭcM r@ט1[T%Ju[yhӭ\)䌁K^3->T4mŅW7L r~T8+l\f0袊8f׉,UMr r w LvZkiuX`;d}kQ_O!{ai_x_nG4sbkYap}N\-JPkA5 K}lm>yzo&G8 ͌Jsd!#8P01x^\on"rէif wcgdžΦD10@'/& ,QC ]ܮy9~rƒNROYMMVWŗH+C:w"dRl3nU|A ߊI¶߱I4w "m p a_W sHaՌGeM7ImEgoal a Kz(qMNW<,ul5zxj\$In({яz8cހ1> endobj 408 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-8.jpg) >> endobj 409 0 obj << /Type /Annot /Subtype /Link /A 410 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 295.5405 204.0000 520.5405 ] >> endobj 410 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-8.jpg) >> endobj 411 0 obj << /Type /Page /Parent 3 0 R /Annots [ 413 0 R 416 0 R 418 0 R 420 0 R 422 0 R 424 0 R 426 0 R 428 0 R 430 0 R 432 0 R 434 0 R 436 0 R 438 0 R 440 0 R 442 0 R 444 0 R 446 0 R 448 0 R 450 0 R 452 0 R 454 0 R 456 0 R 458 0 R 460 0 R 462 0 R 464 0 R 466 0 R 468 0 R 470 0 R 472 0 R 474 0 R 476 0 R 478 0 R 480 0 R 482 0 R 484 0 R 486 0 R 488 0 R 490 0 R 492 0 R 494 0 R 496 0 R 498 0 R 500 0 R 502 0 R 504 0 R 506 0 R 508 0 R 510 0 R 512 0 R 514 0 R 516 0 R 518 0 R 520 0 R 522 0 R 524 0 R 526 0 R 528 0 R 530 0 R 532 0 R ] /Contents 412 0 R >> endobj 412 0 obj << /Length 33911 >> stream 0.271 0.267 0.267 rg q 15.000 41.658 577.500 735.342 re W n 0.965 0.965 0.965 rg 26.250 443.800 555.000 333.200 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 777.000 m 581.250 777.000 l 581.250 776.250 l 26.250 776.250 l f 26.250 443.800 m 581.250 443.800 l 581.250 444.550 l 26.250 444.550 l f q 225.000 0 0 159.000 35.250 608.250 cm /I11 Do Q q 35.250 455.050 537.000 147.200 re W n 0.271 0.267 0.267 rg BT 35.250 592.726 Td /F4 9.8 Tf [(Fig. 9: Comparison of bFGF, BDNF, GDNF and the fetal striatal GCM, neuroprotection from H2O2 induced cell )] TJ ET BT 35.250 580.821 Td /F4 9.8 Tf [(death in Q111 cells)] TJ ET BT 35.250 561.451 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, H)] TJ ET BT 185.341 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 190.160 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 197.746 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 202.564 561.451 Td /F1 9.8 Tf [( \(2 M\), bFGF 10 ng/ml + H)] TJ ET BT 322.041 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 326.860 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 334.445 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 339.264 561.451 Td /F1 9.8 Tf [(, BDNF 50 ng/ml + H)] TJ ET BT 429.480 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 434.299 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 441.885 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 446.703 561.451 Td /F1 9.8 Tf [(, GDNF 50 ng/ml + H)] TJ ET BT 538.002 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 542.821 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 550.406 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 555.225 561.451 Td /F1 9.8 Tf [(, )] TJ ET BT 35.250 547.715 Td /F1 9.8 Tf [(GCM + H)] TJ ET BT 76.151 546.567 Td /F1 8.7 Tf [(2)] TJ ET BT 80.970 547.715 Td /F1 9.8 Tf [(O)] TJ ET BT 88.555 546.567 Td /F1 8.7 Tf [(2)] TJ ET BT 93.374 547.715 Td /F1 9.8 Tf [( treated cells for 24 h at 3 DIV \(scale bar = 30 m\) and \(B\) their corresponding quantification of total neurons )] TJ ET BT 35.250 533.979 Td /F1 9.8 Tf [(expressed as microtubule protein \(-tubulin\). \(C\) Percentage of chromatin condensed and fragmented nuclei in the different )] TJ ET BT 35.250 520.243 Td /F1 9.8 Tf [(experimental groups. \(D\) Photomicrograph of synaptophysin staining \(red\) and Hoechst staining \(blue\) \(scale bar = 15 m\). )] TJ ET BT 35.250 506.506 Td /F1 9.8 Tf [(\(E\) Percentage of multiprocess cells, \(with more than 3 neurite arborization\) to test the cell differentiation. The values are )] TJ ET BT 35.250 492.770 Td /F1 9.8 Tf [(expressed as mean SEM \(n = 4 samples in each experimental group\). Statistical analysis was performed by one-way )] TJ ET BT 35.250 479.034 Td /F1 9.8 Tf [(ANOVA followed by Newman-Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control group. )] TJ ET BT 420.297 484.754 Td /F1 8.7 Tf [(&)] TJ ET BT 426.078 479.034 Td /F1 9.8 Tf [(p<0.05, )] TJ ET BT 461.587 484.754 Td /F1 8.7 Tf [(&&)] TJ ET BT 473.149 479.034 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 508.658 484.754 Td /F1 8.7 Tf [(&&&)] TJ ET BT 526.000 479.034 Td /F1 9.8 Tf [(p<0.001 )] TJ ET BT 35.250 465.298 Td /F1 9.8 Tf [(growth factor or GCM + H)] TJ ET BT 146.595 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 151.414 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 158.999 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 163.818 465.298 Td /F1 9.8 Tf [( vs H)] TJ ET BT 186.028 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 190.847 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 198.433 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 203.251 465.298 Td /F1 9.8 Tf [(,; ^^^p<0.001 growth factor + H)] TJ ET BT 337.830 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 342.649 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 350.235 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 355.053 465.298 Td /F1 9.8 Tf [( vs fetal striatal GCM + H)] TJ ET BT 463.688 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 468.506 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 476.092 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 480.911 465.298 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 426.777 Td /F4 9.8 Tf [(Discussion)] TJ ET BT 26.250 407.372 Td /F1 9.8 Tf [(These studies reveal that fetal and postnatal striatal GCM protects from spontaneous degeneration and cell death induced by )] TJ ET BT 26.250 395.467 Td /F1 9.8 Tf [(exposure to glutamate, 3-NP acid and H)] TJ ET BT 199.663 393.403 Td /F1 8.7 Tf [(2)] TJ ET BT 204.482 395.467 Td /F1 9.8 Tf [(O)] TJ ET BT 212.068 393.403 Td /F1 8.7 Tf [(2)] TJ ET BT 216.886 395.467 Td /F1 9.8 Tf [( in striatal cells with polyglutamines expansions of huntingtin which could be )] TJ ET BT 26.250 383.562 Td /F1 9.8 Tf [(considered as an in vitro models of HD. GCM is rich in small antioxidants, like GSH and ascorbic acid, and peptidic neurotrophic )] TJ ET BT 26.250 371.658 Td /F1 9.8 Tf [(factors, such as GDNF, BDNF, neurotrophin-3 \(NT-3\), NGF, and bFGF as well as novel DA neurotrophic factors )] TJ ET 0.267 0.267 0.267 rg BT 511.195 373.165 Td /F4 8.7 Tf [(22)] TJ ET BT 520.833 373.165 Td /F4 8.7 Tf [(36)] TJ ET BT 530.470 373.165 Td /F4 8.7 Tf [(37)] TJ ET BT 540.108 373.165 Td /F4 8.7 Tf [(38)] TJ ET BT 549.745 373.165 Td /F4 8.7 Tf [(39)] TJ ET BT 559.382 373.165 Td /F4 8.7 Tf [(40)] TJ ET BT 569.020 373.165 Td /F4 8.7 Tf [(41)] TJ ET BT 26.250 361.260 Td /F4 8.7 Tf [(42)] TJ ET BT 35.887 361.260 Td /F4 8.7 Tf [(43)] TJ ET 0.271 0.267 0.267 rg BT 45.525 359.753 Td /F1 9.8 Tf [(. The effects of GCM observed in these experiments are, however, more potent than those of any known neuroprotective or )] TJ ET BT 26.250 347.848 Td /F1 9.8 Tf [(neurotrophic factor ever tested in vitro models of HD.)] TJ ET BT 26.250 328.443 Td /F1 9.8 Tf [(All the mechanisms of neurotoxicity triggered by the neurotoxins used in these experiments, including abnormal excitatory )] TJ ET BT 26.250 316.539 Td /F1 9.8 Tf [(neurotransmission, abnormal mitochondrial function, and excessive oxidative stress have been demonstrated in patients with )] TJ ET BT 26.250 304.634 Td /F1 9.8 Tf [(HD and animal models of this disease )] TJ ET 0.267 0.267 0.267 rg BT 192.605 306.141 Td /F4 8.7 Tf [(44)] TJ ET BT 202.242 306.141 Td /F4 8.7 Tf [(45)] TJ ET BT 211.879 306.141 Td /F4 8.7 Tf [(46)] TJ ET 0.271 0.267 0.267 rg BT 221.517 304.634 Td /F1 9.8 Tf [(. Glutamate is a major excitatory amino acid neurotransmitter in the central )] TJ ET BT 26.250 292.729 Td /F1 9.8 Tf [(nervous system. A high concentration of glutamate induces neuronal cell damage under in vitro conditions )] TJ ET 0.267 0.267 0.267 rg BT 485.797 294.236 Td /F4 8.7 Tf [(47)] TJ ET BT 495.434 294.236 Td /F4 8.7 Tf [(48)] TJ ET BT 505.071 294.236 Td /F4 8.7 Tf [(49)] TJ ET 0.271 0.267 0.267 rg BT 514.709 292.729 Td /F1 9.8 Tf [(. Striatal )] TJ ET BT 26.250 280.824 Td /F1 9.8 Tf [(medium spiny neurons, which receive a strong excitatory input in their dendritic spines from cortical neurons, through the cortico )] TJ ET BT 26.250 268.920 Td /F1 9.8 Tf [(striatal pathways, are neurons of projection mostly to the pallidum, which contain GABA and other neurotransmitters, and are )] TJ ET BT 26.250 257.015 Td /F1 9.8 Tf [(particularly vulnerable to HD. Their progressive depletion is in strong correlation with symptom severity )] TJ ET 0.267 0.267 0.267 rg BT 471.094 258.522 Td /F4 8.7 Tf [(50)] TJ ET 0.271 0.267 0.267 rg BT 480.731 257.015 Td /F1 9.8 Tf [(. Our data shows that )] TJ ET BT 26.250 245.110 Td /F1 9.8 Tf [(the GCM reverted cell death produced by glutamate and increased the levels of GABA uptake sites, a marker of GABA neurons, )] TJ ET BT 26.250 233.205 Td /F1 9.8 Tf [(in STHdhQ111/111 cells.)] TJ ET BT 26.250 213.801 Td /F1 9.8 Tf [(In HD there is a mitochondrial dysfunction that produces a significant decrease in the activity of mitochondrial respiratory chain )] TJ ET BT 26.250 201.896 Td /F1 9.8 Tf [(complexes \(MCC\) II, III and IV, among other factors)] TJ ET 0.267 0.267 0.267 rg BT 248.959 203.403 Td /F4 8.7 Tf [(51)] TJ ET 0.271 0.267 0.267 rg BT 258.597 201.896 Td /F1 9.8 Tf [(. 3-Nitropropionic acid is a natural toxin that irreversibly inhibits the )] TJ ET BT 26.250 189.991 Td /F1 9.8 Tf [(succinate dehydrogenase enzyme, which is the main constituent of MCC II. Administered to rodents, 3-NP reproduces the brain )] TJ ET BT 26.250 178.086 Td /F1 9.8 Tf [(lesions observed in HD patients, which consist of degeneration of the striatum with particular impact on medium-sized spiny )] TJ ET BT 26.250 166.182 Td /F1 9.8 Tf [(projection neurons )] TJ ET 0.267 0.267 0.267 rg BT 109.164 167.689 Td /F4 8.7 Tf [(52)] TJ ET 0.271 0.267 0.267 rg BT 118.801 166.182 Td /F1 9.8 Tf [(. In Q111 cells, we have observed that the 3-NP produces a decrease in cell survival and an increase in )] TJ ET BT 26.250 154.277 Td /F1 9.8 Tf [(necrotic and apoptotic cell death. This study shows the protection by GCM of 3-NP induced cell death.)] TJ ET BT 26.250 134.872 Td /F1 9.8 Tf [(We challenged Q111 cells with H)] TJ ET BT 169.302 132.808 Td /F1 8.7 Tf [(2)] TJ ET BT 174.121 134.872 Td /F1 9.8 Tf [(O)] TJ ET BT 181.706 132.808 Td /F1 8.7 Tf [(2)] TJ ET BT 186.525 134.872 Td /F1 9.8 Tf [( to study the effects of the fetal and postnatal GCM on free radical metabolism and )] TJ ET BT 26.250 122.967 Td /F1 9.8 Tf [(ubiquitinated proteins in Q111 cells. H)] TJ ET BT 190.450 120.903 Td /F1 8.7 Tf [(2)] TJ ET BT 195.268 122.967 Td /F1 9.8 Tf [(O)] TJ ET BT 202.854 120.903 Td /F1 8.7 Tf [(2)] TJ ET BT 207.673 122.967 Td /F1 9.8 Tf [( produces an accumulation of free radicals and polyubiquitinated proteins in Q111 )] TJ ET BT 26.250 111.063 Td /F1 9.8 Tf [(cells and fetal GCM reverts both nearly to control levels. Fetal GCM also prevents the H)] TJ ET BT 404.472 108.998 Td /F1 8.7 Tf [(2)] TJ ET BT 409.291 111.063 Td /F1 9.8 Tf [(O)] TJ ET BT 416.876 108.998 Td /F1 8.7 Tf [(2)] TJ ET BT 421.695 111.063 Td /F1 9.8 Tf [( induced cell death in Q111 cells. )] TJ ET BT 26.250 99.158 Td /F1 9.8 Tf [(The cleavage of PARP by caspases is recognized as a hallmark of apoptosis )] TJ ET 0.267 0.267 0.267 rg BT 360.607 100.665 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 370.244 99.158 Td /F1 9.8 Tf [(. H)] TJ ET BT 382.705 97.094 Td /F1 8.7 Tf [(2)] TJ ET BT 387.523 99.158 Td /F1 9.8 Tf [(O)] TJ ET BT 395.109 97.094 Td /F1 8.7 Tf [(2)] TJ ET BT 399.927 99.158 Td /F1 9.8 Tf [( activated the accumulation of this )] TJ ET BT 26.250 87.253 Td /F1 9.8 Tf [(cleavage and fetal GCM prevented this effect. The chaperone HSP70 is increased in the presence of H)] TJ ET BT 470.635 85.189 Td /F1 8.7 Tf [(2)] TJ ET BT 475.454 87.253 Td /F1 9.8 Tf [(O)] TJ ET BT 483.040 85.189 Td /F1 8.7 Tf [(2)] TJ ET BT 487.858 87.253 Td /F1 9.8 Tf [( as a marker of cell )] TJ ET BT 26.250 75.348 Td /F1 9.8 Tf [(stress but the fetal GCM reduced it. Fetal GCM increased the levels of the antioxidant GSH in Q111 cells and prevented the H)] TJ ET BT 569.247 73.284 Td /F1 8.7 Tf [(2)] TJ ET BT 26.250 63.444 Td /F1 9.8 Tf [(O)] TJ ET BT 33.835 61.379 Td /F1 8.7 Tf [(2)] TJ ET BT 38.654 63.444 Td /F1 9.8 Tf [( induced depletion of GSH. The 13-day and 2-month postnatal GCM also decreased the H)] TJ ET BT 427.221 61.379 Td /F1 8.7 Tf [(2)] TJ ET BT 432.040 63.444 Td /F1 9.8 Tf [(O)] TJ ET BT 439.625 61.379 Td /F1 8.7 Tf [(2)] TJ ET BT 444.444 63.444 Td /F1 9.8 Tf [( induced cell death in this in )] TJ ET BT 26.250 51.539 Td /F1 9.8 Tf [(vitro model of HD.)] TJ ET Q q 15.000 41.658 577.500 735.342 re W n 0.965 0.965 0.965 rg 26.250 443.800 555.000 333.200 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 777.000 m 581.250 777.000 l 581.250 776.250 l 26.250 776.250 l f 26.250 443.800 m 581.250 443.800 l 581.250 444.550 l 26.250 444.550 l f q 225.000 0 0 159.000 35.250 608.250 cm /I11 Do Q q 35.250 455.050 537.000 147.200 re W n 0.271 0.267 0.267 rg BT 35.250 592.726 Td /F4 9.8 Tf [(Fig. 9: Comparison of bFGF, BDNF, GDNF and the fetal striatal GCM, neuroprotection from H2O2 induced cell )] TJ ET BT 35.250 580.821 Td /F4 9.8 Tf [(death in Q111 cells)] TJ ET BT 35.250 561.451 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, H)] TJ ET BT 185.341 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 190.160 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 197.746 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 202.564 561.451 Td /F1 9.8 Tf [( \(2 M\), bFGF 10 ng/ml + H)] TJ ET BT 322.041 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 326.860 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 334.445 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 339.264 561.451 Td /F1 9.8 Tf [(, BDNF 50 ng/ml + H)] TJ ET BT 429.480 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 434.299 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 441.885 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 446.703 561.451 Td /F1 9.8 Tf [(, GDNF 50 ng/ml + H)] TJ ET BT 538.002 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 542.821 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 550.406 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 555.225 561.451 Td /F1 9.8 Tf [(, )] TJ ET BT 35.250 547.715 Td /F1 9.8 Tf [(GCM + H)] TJ ET BT 76.151 546.567 Td /F1 8.7 Tf [(2)] TJ ET BT 80.970 547.715 Td /F1 9.8 Tf [(O)] TJ ET BT 88.555 546.567 Td /F1 8.7 Tf [(2)] TJ ET BT 93.374 547.715 Td /F1 9.8 Tf [( treated cells for 24 h at 3 DIV \(scale bar = 30 m\) and \(B\) their corresponding quantification of total neurons )] TJ ET BT 35.250 533.979 Td /F1 9.8 Tf [(expressed as microtubule protein \(-tubulin\). \(C\) Percentage of chromatin condensed and fragmented nuclei in the different )] TJ ET BT 35.250 520.243 Td /F1 9.8 Tf [(experimental groups. \(D\) Photomicrograph of synaptophysin staining \(red\) and Hoechst staining \(blue\) \(scale bar = 15 m\). )] TJ ET BT 35.250 506.506 Td /F1 9.8 Tf [(\(E\) Percentage of multiprocess cells, \(with more than 3 neurite arborization\) to test the cell differentiation. The values are )] TJ ET BT 35.250 492.770 Td /F1 9.8 Tf [(expressed as mean SEM \(n = 4 samples in each experimental group\). Statistical analysis was performed by one-way )] TJ ET BT 35.250 479.034 Td /F1 9.8 Tf [(ANOVA followed by Newman-Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control group. )] TJ ET BT 420.297 484.754 Td /F1 8.7 Tf [(&)] TJ ET BT 426.078 479.034 Td /F1 9.8 Tf [(p<0.05, )] TJ ET BT 461.587 484.754 Td /F1 8.7 Tf [(&&)] TJ ET BT 473.149 479.034 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 508.658 484.754 Td /F1 8.7 Tf [(&&&)] TJ ET BT 526.000 479.034 Td /F1 9.8 Tf [(p<0.001 )] TJ ET BT 35.250 465.298 Td /F1 9.8 Tf [(growth factor or GCM + H)] TJ ET BT 146.595 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 151.414 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 158.999 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 163.818 465.298 Td /F1 9.8 Tf [( vs H)] TJ ET BT 186.028 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 190.847 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 198.433 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 203.251 465.298 Td /F1 9.8 Tf [(,; ^^^p<0.001 growth factor + H)] TJ ET BT 337.830 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 342.649 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 350.235 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 355.053 465.298 Td /F1 9.8 Tf [( vs fetal striatal GCM + H)] TJ ET BT 463.688 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 468.506 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 476.092 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 480.911 465.298 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 426.777 Td /F4 9.8 Tf [(Discussion)] TJ ET BT 26.250 407.372 Td /F1 9.8 Tf [(These studies reveal that fetal and postnatal striatal GCM protects from spontaneous degeneration and cell death induced by )] TJ ET BT 26.250 395.467 Td /F1 9.8 Tf [(exposure to glutamate, 3-NP acid and H)] TJ ET BT 199.663 393.403 Td /F1 8.7 Tf [(2)] TJ ET BT 204.482 395.467 Td /F1 9.8 Tf [(O)] TJ ET BT 212.068 393.403 Td /F1 8.7 Tf [(2)] TJ ET BT 216.886 395.467 Td /F1 9.8 Tf [( in striatal cells with polyglutamines expansions of huntingtin which could be )] TJ ET BT 26.250 383.562 Td /F1 9.8 Tf [(considered as an in vitro models of HD. GCM is rich in small antioxidants, like GSH and ascorbic acid, and peptidic neurotrophic )] TJ ET BT 26.250 371.658 Td /F1 9.8 Tf [(factors, such as GDNF, BDNF, neurotrophin-3 \(NT-3\), NGF, and bFGF as well as novel DA neurotrophic factors )] TJ ET 0.267 0.267 0.267 rg BT 511.195 373.165 Td /F4 8.7 Tf [(22)] TJ ET BT 520.833 373.165 Td /F4 8.7 Tf [(36)] TJ ET BT 530.470 373.165 Td /F4 8.7 Tf [(37)] TJ ET BT 540.108 373.165 Td /F4 8.7 Tf [(38)] TJ ET BT 549.745 373.165 Td /F4 8.7 Tf [(39)] TJ ET BT 559.382 373.165 Td /F4 8.7 Tf [(40)] TJ ET BT 569.020 373.165 Td /F4 8.7 Tf [(41)] TJ ET BT 26.250 361.260 Td /F4 8.7 Tf [(42)] TJ ET BT 35.887 361.260 Td /F4 8.7 Tf [(43)] TJ ET 0.271 0.267 0.267 rg BT 45.525 359.753 Td /F1 9.8 Tf [(. The effects of GCM observed in these experiments are, however, more potent than those of any known neuroprotective or )] TJ ET BT 26.250 347.848 Td /F1 9.8 Tf [(neurotrophic factor ever tested in vitro models of HD.)] TJ ET BT 26.250 328.443 Td /F1 9.8 Tf [(All the mechanisms of neurotoxicity triggered by the neurotoxins used in these experiments, including abnormal excitatory )] TJ ET BT 26.250 316.539 Td /F1 9.8 Tf [(neurotransmission, abnormal mitochondrial function, and excessive oxidative stress have been demonstrated in patients with )] TJ ET BT 26.250 304.634 Td /F1 9.8 Tf [(HD and animal models of this disease )] TJ ET 0.267 0.267 0.267 rg BT 192.605 306.141 Td /F4 8.7 Tf [(44)] TJ ET BT 202.242 306.141 Td /F4 8.7 Tf [(45)] TJ ET BT 211.879 306.141 Td /F4 8.7 Tf [(46)] TJ ET 0.271 0.267 0.267 rg BT 221.517 304.634 Td /F1 9.8 Tf [(. Glutamate is a major excitatory amino acid neurotransmitter in the central )] TJ ET BT 26.250 292.729 Td /F1 9.8 Tf [(nervous system. A high concentration of glutamate induces neuronal cell damage under in vitro conditions )] TJ ET 0.267 0.267 0.267 rg BT 485.797 294.236 Td /F4 8.7 Tf [(47)] TJ ET BT 495.434 294.236 Td /F4 8.7 Tf [(48)] TJ ET BT 505.071 294.236 Td /F4 8.7 Tf [(49)] TJ ET 0.271 0.267 0.267 rg BT 514.709 292.729 Td /F1 9.8 Tf [(. Striatal )] TJ ET BT 26.250 280.824 Td /F1 9.8 Tf [(medium spiny neurons, which receive a strong excitatory input in their dendritic spines from cortical neurons, through the cortico )] TJ ET BT 26.250 268.920 Td /F1 9.8 Tf [(striatal pathways, are neurons of projection mostly to the pallidum, which contain GABA and other neurotransmitters, and are )] TJ ET BT 26.250 257.015 Td /F1 9.8 Tf [(particularly vulnerable to HD. Their progressive depletion is in strong correlation with symptom severity )] TJ ET 0.267 0.267 0.267 rg BT 471.094 258.522 Td /F4 8.7 Tf [(50)] TJ ET 0.271 0.267 0.267 rg BT 480.731 257.015 Td /F1 9.8 Tf [(. Our data shows that )] TJ ET BT 26.250 245.110 Td /F1 9.8 Tf [(the GCM reverted cell death produced by glutamate and increased the levels of GABA uptake sites, a marker of GABA neurons, )] TJ ET BT 26.250 233.205 Td /F1 9.8 Tf [(in STHdhQ111/111 cells.)] TJ ET BT 26.250 213.801 Td /F1 9.8 Tf [(In HD there is a mitochondrial dysfunction that produces a significant decrease in the activity of mitochondrial respiratory chain )] TJ ET BT 26.250 201.896 Td /F1 9.8 Tf [(complexes \(MCC\) II, III and IV, among other factors)] TJ ET 0.267 0.267 0.267 rg BT 248.959 203.403 Td /F4 8.7 Tf [(51)] TJ ET 0.271 0.267 0.267 rg BT 258.597 201.896 Td /F1 9.8 Tf [(. 3-Nitropropionic acid is a natural toxin that irreversibly inhibits the )] TJ ET BT 26.250 189.991 Td /F1 9.8 Tf [(succinate dehydrogenase enzyme, which is the main constituent of MCC II. Administered to rodents, 3-NP reproduces the brain )] TJ ET BT 26.250 178.086 Td /F1 9.8 Tf [(lesions observed in HD patients, which consist of degeneration of the striatum with particular impact on medium-sized spiny )] TJ ET BT 26.250 166.182 Td /F1 9.8 Tf [(projection neurons )] TJ ET 0.267 0.267 0.267 rg BT 109.164 167.689 Td /F4 8.7 Tf [(52)] TJ ET 0.271 0.267 0.267 rg BT 118.801 166.182 Td /F1 9.8 Tf [(. In Q111 cells, we have observed that the 3-NP produces a decrease in cell survival and an increase in )] TJ ET BT 26.250 154.277 Td /F1 9.8 Tf [(necrotic and apoptotic cell death. This study shows the protection by GCM of 3-NP induced cell death.)] TJ ET BT 26.250 134.872 Td /F1 9.8 Tf [(We challenged Q111 cells with H)] TJ ET BT 169.302 132.808 Td /F1 8.7 Tf [(2)] TJ ET BT 174.121 134.872 Td /F1 9.8 Tf [(O)] TJ ET BT 181.706 132.808 Td /F1 8.7 Tf [(2)] TJ ET BT 186.525 134.872 Td /F1 9.8 Tf [( to study the effects of the fetal and postnatal GCM on free radical metabolism and )] TJ ET BT 26.250 122.967 Td /F1 9.8 Tf [(ubiquitinated proteins in Q111 cells. H)] TJ ET BT 190.450 120.903 Td /F1 8.7 Tf [(2)] TJ ET BT 195.268 122.967 Td /F1 9.8 Tf [(O)] TJ ET BT 202.854 120.903 Td /F1 8.7 Tf [(2)] TJ ET BT 207.673 122.967 Td /F1 9.8 Tf [( produces an accumulation of free radicals and polyubiquitinated proteins in Q111 )] TJ ET BT 26.250 111.063 Td /F1 9.8 Tf [(cells and fetal GCM reverts both nearly to control levels. Fetal GCM also prevents the H)] TJ ET BT 404.472 108.998 Td /F1 8.7 Tf [(2)] TJ ET BT 409.291 111.063 Td /F1 9.8 Tf [(O)] TJ ET BT 416.876 108.998 Td /F1 8.7 Tf [(2)] TJ ET BT 421.695 111.063 Td /F1 9.8 Tf [( induced cell death in Q111 cells. )] TJ ET BT 26.250 99.158 Td /F1 9.8 Tf [(The cleavage of PARP by caspases is recognized as a hallmark of apoptosis )] TJ ET 0.267 0.267 0.267 rg BT 360.607 100.665 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 370.244 99.158 Td /F1 9.8 Tf [(. H)] TJ ET BT 382.705 97.094 Td /F1 8.7 Tf [(2)] TJ ET BT 387.523 99.158 Td /F1 9.8 Tf [(O)] TJ ET BT 395.109 97.094 Td /F1 8.7 Tf [(2)] TJ ET BT 399.927 99.158 Td /F1 9.8 Tf [( activated the accumulation of this )] TJ ET BT 26.250 87.253 Td /F1 9.8 Tf [(cleavage and fetal GCM prevented this effect. The chaperone HSP70 is increased in the presence of H)] TJ ET BT 470.635 85.189 Td /F1 8.7 Tf [(2)] TJ ET BT 475.454 87.253 Td /F1 9.8 Tf [(O)] TJ ET BT 483.040 85.189 Td /F1 8.7 Tf [(2)] TJ ET BT 487.858 87.253 Td /F1 9.8 Tf [( as a marker of cell )] TJ ET BT 26.250 75.348 Td /F1 9.8 Tf [(stress but the fetal GCM reduced it. Fetal GCM increased the levels of the antioxidant GSH in Q111 cells and prevented the H)] TJ ET BT 569.247 73.284 Td /F1 8.7 Tf [(2)] TJ ET BT 26.250 63.444 Td /F1 9.8 Tf [(O)] TJ ET BT 33.835 61.379 Td /F1 8.7 Tf [(2)] TJ ET BT 38.654 63.444 Td /F1 9.8 Tf [( induced depletion of GSH. The 13-day and 2-month postnatal GCM also decreased the H)] TJ ET BT 427.221 61.379 Td /F1 8.7 Tf [(2)] TJ ET BT 432.040 63.444 Td /F1 9.8 Tf [(O)] TJ ET BT 439.625 61.379 Td /F1 8.7 Tf [(2)] TJ ET BT 444.444 63.444 Td /F1 9.8 Tf [( induced cell death in this in )] TJ ET BT 26.250 51.539 Td /F1 9.8 Tf [(vitro model of HD.)] TJ ET Q q 15.000 41.658 577.500 735.342 re W n 0.965 0.965 0.965 rg 26.250 443.800 555.000 333.200 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 777.000 m 581.250 777.000 l 581.250 776.250 l 26.250 776.250 l f 26.250 443.800 m 581.250 443.800 l 581.250 444.550 l 26.250 444.550 l f q 225.000 0 0 159.000 35.250 608.250 cm /I11 Do Q q 35.250 455.050 537.000 147.200 re W n 0.271 0.267 0.267 rg BT 35.250 592.726 Td /F4 9.8 Tf [(Fig. 9: Comparison of bFGF, BDNF, GDNF and the fetal striatal GCM, neuroprotection from H2O2 induced cell )] TJ ET BT 35.250 580.821 Td /F4 9.8 Tf [(death in Q111 cells)] TJ ET BT 35.250 561.451 Td /F1 9.8 Tf [(\(A\) Photomicrographs of control, H)] TJ ET BT 185.341 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 190.160 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 197.746 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 202.564 561.451 Td /F1 9.8 Tf [( \(2 M\), bFGF 10 ng/ml + H)] TJ ET BT 322.041 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 326.860 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 334.445 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 339.264 561.451 Td /F1 9.8 Tf [(, BDNF 50 ng/ml + H)] TJ ET BT 429.480 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 434.299 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 441.885 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 446.703 561.451 Td /F1 9.8 Tf [(, GDNF 50 ng/ml + H)] TJ ET BT 538.002 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 542.821 561.451 Td /F1 9.8 Tf [(O)] TJ ET BT 550.406 560.303 Td /F1 8.7 Tf [(2)] TJ ET BT 555.225 561.451 Td /F1 9.8 Tf [(, )] TJ ET BT 35.250 547.715 Td /F1 9.8 Tf [(GCM + H)] TJ ET BT 76.151 546.567 Td /F1 8.7 Tf [(2)] TJ ET BT 80.970 547.715 Td /F1 9.8 Tf [(O)] TJ ET BT 88.555 546.567 Td /F1 8.7 Tf [(2)] TJ ET BT 93.374 547.715 Td /F1 9.8 Tf [( treated cells for 24 h at 3 DIV \(scale bar = 30 m\) and \(B\) their corresponding quantification of total neurons )] TJ ET BT 35.250 533.979 Td /F1 9.8 Tf [(expressed as microtubule protein \(-tubulin\). \(C\) Percentage of chromatin condensed and fragmented nuclei in the different )] TJ ET BT 35.250 520.243 Td /F1 9.8 Tf [(experimental groups. \(D\) Photomicrograph of synaptophysin staining \(red\) and Hoechst staining \(blue\) \(scale bar = 15 m\). )] TJ ET BT 35.250 506.506 Td /F1 9.8 Tf [(\(E\) Percentage of multiprocess cells, \(with more than 3 neurite arborization\) to test the cell differentiation. The values are )] TJ ET BT 35.250 492.770 Td /F1 9.8 Tf [(expressed as mean SEM \(n = 4 samples in each experimental group\). Statistical analysis was performed by one-way )] TJ ET BT 35.250 479.034 Td /F1 9.8 Tf [(ANOVA followed by Newman-Keuls test. *p<0.05, **p<0.01, ***p<0.001 vs control group. )] TJ ET BT 420.297 484.754 Td /F1 8.7 Tf [(&)] TJ ET BT 426.078 479.034 Td /F1 9.8 Tf [(p<0.05, )] TJ ET BT 461.587 484.754 Td /F1 8.7 Tf [(&&)] TJ ET BT 473.149 479.034 Td /F1 9.8 Tf [(p<0.01, )] TJ ET BT 508.658 484.754 Td /F1 8.7 Tf [(&&&)] TJ ET BT 526.000 479.034 Td /F1 9.8 Tf [(p<0.001 )] TJ ET BT 35.250 465.298 Td /F1 9.8 Tf [(growth factor or GCM + H)] TJ ET BT 146.595 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 151.414 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 158.999 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 163.818 465.298 Td /F1 9.8 Tf [( vs H)] TJ ET BT 186.028 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 190.847 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 198.433 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 203.251 465.298 Td /F1 9.8 Tf [(,; ^^^p<0.001 growth factor + H)] TJ ET BT 337.830 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 342.649 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 350.235 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 355.053 465.298 Td /F1 9.8 Tf [( vs fetal striatal GCM + H)] TJ ET BT 463.688 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 468.506 465.298 Td /F1 9.8 Tf [(O)] TJ ET BT 476.092 464.149 Td /F1 8.7 Tf [(2)] TJ ET BT 480.911 465.298 Td /F1 9.8 Tf [(.)] TJ ET Q BT 26.250 426.777 Td /F4 9.8 Tf [(Discussion)] TJ ET BT 26.250 407.372 Td /F1 9.8 Tf [(These studies reveal that fetal and postnatal striatal GCM protects from spontaneous degeneration and cell death induced by )] TJ ET BT 26.250 395.467 Td /F1 9.8 Tf [(exposure to glutamate, 3-NP acid and H)] TJ ET BT 199.663 393.403 Td /F1 8.7 Tf [(2)] TJ ET BT 204.482 395.467 Td /F1 9.8 Tf [(O)] TJ ET BT 212.068 393.403 Td /F1 8.7 Tf [(2)] TJ ET BT 216.886 395.467 Td /F1 9.8 Tf [( in striatal cells with polyglutamines expansions of huntingtin which could be )] TJ ET BT 26.250 383.562 Td /F1 9.8 Tf [(considered as an in vitro models of HD. GCM is rich in small antioxidants, like GSH and ascorbic acid, and peptidic neurotrophic )] TJ ET BT 26.250 371.658 Td /F1 9.8 Tf [(factors, such as GDNF, BDNF, neurotrophin-3 \(NT-3\), NGF, and bFGF as well as novel DA neurotrophic factors )] TJ ET 0.267 0.267 0.267 rg BT 511.195 373.165 Td /F4 8.7 Tf [(22)] TJ ET BT 520.833 373.165 Td /F4 8.7 Tf [(36)] TJ ET BT 530.470 373.165 Td /F4 8.7 Tf [(37)] TJ ET BT 540.108 373.165 Td /F4 8.7 Tf [(38)] TJ ET BT 549.745 373.165 Td /F4 8.7 Tf [(39)] TJ ET BT 559.382 373.165 Td /F4 8.7 Tf [(40)] TJ ET BT 569.020 373.165 Td /F4 8.7 Tf [(41)] TJ ET BT 26.250 361.260 Td /F4 8.7 Tf [(42)] TJ ET BT 35.887 361.260 Td /F4 8.7 Tf [(43)] TJ ET 0.271 0.267 0.267 rg BT 45.525 359.753 Td /F1 9.8 Tf [(. The effects of GCM observed in these experiments are, however, more potent than those of any known neuroprotective or )] TJ ET BT 26.250 347.848 Td /F1 9.8 Tf [(neurotrophic factor ever tested in vitro models of HD.)] TJ ET BT 26.250 328.443 Td /F1 9.8 Tf [(All the mechanisms of neurotoxicity triggered by the neurotoxins used in these experiments, including abnormal excitatory )] TJ ET BT 26.250 316.539 Td /F1 9.8 Tf [(neurotransmission, abnormal mitochondrial function, and excessive oxidative stress have been demonstrated in patients with )] TJ ET BT 26.250 304.634 Td /F1 9.8 Tf [(HD and animal models of this disease )] TJ ET 0.267 0.267 0.267 rg BT 192.605 306.141 Td /F4 8.7 Tf [(44)] TJ ET BT 202.242 306.141 Td /F4 8.7 Tf [(45)] TJ ET BT 211.879 306.141 Td /F4 8.7 Tf [(46)] TJ ET 0.271 0.267 0.267 rg BT 221.517 304.634 Td /F1 9.8 Tf [(. Glutamate is a major excitatory amino acid neurotransmitter in the central )] TJ ET BT 26.250 292.729 Td /F1 9.8 Tf [(nervous system. A high concentration of glutamate induces neuronal cell damage under in vitro conditions )] TJ ET 0.267 0.267 0.267 rg BT 485.797 294.236 Td /F4 8.7 Tf [(47)] TJ ET BT 495.434 294.236 Td /F4 8.7 Tf [(48)] TJ ET BT 505.071 294.236 Td /F4 8.7 Tf [(49)] TJ ET 0.271 0.267 0.267 rg BT 514.709 292.729 Td /F1 9.8 Tf [(. Striatal )] TJ ET BT 26.250 280.824 Td /F1 9.8 Tf [(medium spiny neurons, which receive a strong excitatory input in their dendritic spines from cortical neurons, through the cortico )] TJ ET BT 26.250 268.920 Td /F1 9.8 Tf [(striatal pathways, are neurons of projection mostly to the pallidum, which contain GABA and other neurotransmitters, and are )] TJ ET BT 26.250 257.015 Td /F1 9.8 Tf [(particularly vulnerable to HD. Their progressive depletion is in strong correlation with symptom severity )] TJ ET 0.267 0.267 0.267 rg BT 471.094 258.522 Td /F4 8.7 Tf [(50)] TJ ET 0.271 0.267 0.267 rg BT 480.731 257.015 Td /F1 9.8 Tf [(. Our data shows that )] TJ ET BT 26.250 245.110 Td /F1 9.8 Tf [(the GCM reverted cell death produced by glutamate and increased the levels of GABA uptake sites, a marker of GABA neurons, )] TJ ET BT 26.250 233.205 Td /F1 9.8 Tf [(in STHdhQ111/111 cells.)] TJ ET BT 26.250 213.801 Td /F1 9.8 Tf [(In HD there is a mitochondrial dysfunction that produces a significant decrease in the activity of mitochondrial respiratory chain )] TJ ET BT 26.250 201.896 Td /F1 9.8 Tf [(complexes \(MCC\) II, III and IV, among other factors)] TJ ET 0.267 0.267 0.267 rg BT 248.959 203.403 Td /F4 8.7 Tf [(51)] TJ ET 0.271 0.267 0.267 rg BT 258.597 201.896 Td /F1 9.8 Tf [(. 3-Nitropropionic acid is a natural toxin that irreversibly inhibits the )] TJ ET BT 26.250 189.991 Td /F1 9.8 Tf [(succinate dehydrogenase enzyme, which is the main constituent of MCC II. Administered to rodents, 3-NP reproduces the brain )] TJ ET BT 26.250 178.086 Td /F1 9.8 Tf [(lesions observed in HD patients, which consist of degeneration of the striatum with particular impact on medium-sized spiny )] TJ ET BT 26.250 166.182 Td /F1 9.8 Tf [(projection neurons )] TJ ET 0.267 0.267 0.267 rg BT 109.164 167.689 Td /F4 8.7 Tf [(52)] TJ ET 0.271 0.267 0.267 rg BT 118.801 166.182 Td /F1 9.8 Tf [(. In Q111 cells, we have observed that the 3-NP produces a decrease in cell survival and an increase in )] TJ ET BT 26.250 154.277 Td /F1 9.8 Tf [(necrotic and apoptotic cell death. This study shows the protection by GCM of 3-NP induced cell death.)] TJ ET BT 26.250 134.872 Td /F1 9.8 Tf [(We challenged Q111 cells with H)] TJ ET BT 169.302 132.808 Td /F1 8.7 Tf [(2)] TJ ET BT 174.121 134.872 Td /F1 9.8 Tf [(O)] TJ ET BT 181.706 132.808 Td /F1 8.7 Tf [(2)] TJ ET BT 186.525 134.872 Td /F1 9.8 Tf [( to study the effects of the fetal and postnatal GCM on free radical metabolism and )] TJ ET BT 26.250 122.967 Td /F1 9.8 Tf [(ubiquitinated proteins in Q111 cells. H)] TJ ET BT 190.450 120.903 Td /F1 8.7 Tf [(2)] TJ ET BT 195.268 122.967 Td /F1 9.8 Tf [(O)] TJ ET BT 202.854 120.903 Td /F1 8.7 Tf [(2)] TJ ET BT 207.673 122.967 Td /F1 9.8 Tf [( produces an accumulation of free radicals and polyubiquitinated proteins in Q111 )] TJ ET BT 26.250 111.063 Td /F1 9.8 Tf [(cells and fetal GCM reverts both nearly to control levels. Fetal GCM also prevents the H)] TJ ET BT 404.472 108.998 Td /F1 8.7 Tf [(2)] TJ ET BT 409.291 111.063 Td /F1 9.8 Tf [(O)] TJ ET BT 416.876 108.998 Td /F1 8.7 Tf [(2)] TJ ET BT 421.695 111.063 Td /F1 9.8 Tf [( induced cell death in Q111 cells. )] TJ ET BT 26.250 99.158 Td /F1 9.8 Tf [(The cleavage of PARP by caspases is recognized as a hallmark of apoptosis )] TJ ET 0.267 0.267 0.267 rg BT 360.607 100.665 Td /F4 8.7 Tf [(35)] TJ ET 0.271 0.267 0.267 rg BT 370.244 99.158 Td /F1 9.8 Tf [(. H)] TJ ET BT 382.705 97.094 Td /F1 8.7 Tf [(2)] TJ ET BT 387.523 99.158 Td /F1 9.8 Tf [(O)] TJ ET BT 395.109 97.094 Td /F1 8.7 Tf [(2)] TJ ET BT 399.927 99.158 Td /F1 9.8 Tf [( activated the accumulation of this )] TJ ET BT 26.250 87.253 Td /F1 9.8 Tf [(cleavage and fetal GCM prevented this effect. The chaperone HSP70 is increased in the presence of H)] TJ ET BT 470.635 85.189 Td /F1 8.7 Tf [(2)] TJ ET BT 475.454 87.253 Td /F1 9.8 Tf [(O)] TJ ET BT 483.040 85.189 Td /F1 8.7 Tf [(2)] TJ ET BT 487.858 87.253 Td /F1 9.8 Tf [( as a marker of cell )] TJ ET BT 26.250 75.348 Td /F1 9.8 Tf [(stress but the fetal GCM reduced it. Fetal GCM increased the levels of the antioxidant GSH in Q111 cells and prevented the H)] TJ ET BT 569.247 73.284 Td /F1 8.7 Tf [(2)] TJ ET BT 26.250 63.444 Td /F1 9.8 Tf [(O)] TJ ET BT 33.835 61.379 Td /F1 8.7 Tf [(2)] TJ ET BT 38.654 63.444 Td /F1 9.8 Tf [( induced depletion of GSH. The 13-day and 2-month postnatal GCM also decreased the H)] TJ ET BT 427.221 61.379 Td /F1 8.7 Tf [(2)] TJ ET BT 432.040 63.444 Td /F1 9.8 Tf [(O)] TJ ET BT 439.625 61.379 Td /F1 8.7 Tf [(2)] TJ ET BT 444.444 63.444 Td /F1 9.8 Tf [( induced cell death in this in )] TJ ET BT 26.250 51.539 Td /F1 9.8 Tf [(vitro model of HD.)] TJ ET Q q 225.000 0 0 159.000 35.250 608.250 cm /I11 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 413 0 obj << /Type /Annot /Subtype /Link /A 414 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 608.2500 260.2500 767.2500 ] >> endobj 414 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2012/06/figure-9.jpg) >> endobj 415 0 obj << /Type /XObject /Subtype /Image /Width 300 /Height 212 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 32448>> 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 ?!_5} +KI60}4rd`=Bpkx?S[1=k^+>Z]Z"[[Cq W$8W}s[i[[ʂZaלp'䫉r~:+T\^m &5#Jpm-]&a7mUk:44)&8\$9 =~ |mW?& %K̘9V?8jcY~4L%Qs88O _C}+ZZۓܪ0rq߰K{~[{o7~E~"XB?h|XKTI=%d 6cp@kWCExouk;6R=EYZa&}rzqxZOC'F2U:l᧙ӫ' ^;4~Q_/7xSN?5CHI'+(H "&}"r2xo~?Ѽgi w)= $10F+`/݄V̬&cci+]ZIi}?a9(|G~:^\G`xR-"M;O\䆹q2c~}4Y HU!M˛`|tdEJ5}NhO1<҄k:M5}Ֆ}~W53Wּ?Z쿴Ee]\F&\]$LFQHfF+w>,߆1:qo9]&vҬI!GbF %A=+zu vҮJI螎ߑZu }wRXıN ݝrg G̯7 =GZᾏyԖ2hXdծЕXԠؖ$):WgXI65Uo;A9|e[֌T?5l`QW+Y{Gٺk-Con,3Åٻ[Gy~4ԃ_WlP$@ |⽻Qdž4]`kmv.ÕBY [ ^w⟀wSV}Y[4m(Ypp9T1=[_]?&y9va)ԩ ʽ9.X$+YvVYj}ڦ5}2˦kinY*6/3a… '~"񝖵mYRu;*`, `>bC&}M2o@MT?x׷x[R}v+Iҵ+M&)VO]XjZt=B5i˖sh{]zhld1+ybVRT1'Re͒00*n!*Y_xZwo-Sn%29<+  ^eފx 3g!|~jۏA[Bv%lmFRqջ}?=KO^-L]*lb |z/N+^`^ڝž v!rNAÇ u)۟-ml56<(h=2p+m~/bkknk[Y 5:F kJR'=K/>ZQi'[44v7 xgBӵēwvq@<2cw@+ХVkx냃>-x.W𞙧ͪ͢܋gEF7&%%*ǭc|:&~ !P$^$ 0;P=HJٽݯgK-iq%ϟV0Q9) X4K|Av-ml币 D덑z܁U״+Nj y5J!+tr@,H`ts wѢAKk_Dѵ]ju۶ӧPZUن+@bX0a0:f4O iߊgd{H/t[, 8,XrzUzmGXӴM.iz~uxB7GJ?#`Wȿ&<]y%N҆jEMmaJӧn릶tӾO¯z}TQ\I|4ú^#Yy@ Dl\ eu>]Y,tȬ`(Z@Ȓ0p?Ӏ3["X|SJo٭u)D-4AH8պ !>RJ:%4–gK5lԢeV7 f#LK82N, XI\ƬO9S[d*@2ןֽRf63-_ٳ%&I=:VĽ 潢[hH wK{ >&EOuHkb04>&v=/᰿{kӲ['Yx_bg呃7%oe"0ٔcSI6@9둎¿mcGȏ^87ϧI43G Ll1_ҧ[T}"[{BVhU]<_v3 l6jtKEL <%zJ-GEѫl-oƺ;$tˋo:{2Ć@΄I%pJ#9, tO?ŚGג 5F i 5"H/$ݴ8/ۍKr2i,-k<.x zoOVu4AtS[ L \H \=7iifu-7ub^XH=Fz׭h4MKLOf*0eaw^]{Z,|O$5|H|](+'H֮$宣YKsg=H-e2݇0^(nmuy< 6OB0'%|f!y'z)-L#R"brO~%|tg wSas Q[+~fX,tp7ǟſMaZޙ[VydsI$LҌF@N(>|vo<|7&af`2NPk+Ǡ#e'~3e/h>nwB>f|u{cI7O9L2yS[6UF;VxEvF6̤?ipw{☼׾!)ukQDʖV:մQr6C*;屓Ұ(ǵ. & |`i:Rȃ.O Gy]7מ~+UlR;DFYTA ֌Zt[v'UBf^ss.#qh%1ʲ+emq߽z"~uC_k YEq AVDeۀA1^*H<%w{aCnEb1Q[%@\9 [ZO_Sy|+: DNO kDRBIke'ӗmX|TR^I-lhM^+*c}[]z☝1R#2Җ*ČF+_KmkvkhOT7z޴|q/#אlZMŭ^o;oKbMUGkG9-jz4i8, @9r=:9Q^G¼N3%rzo-}kb^Ӵ9^m700Y;*W|=_MjS67tJȳ:,Ծ7>N ` /:.ul?O,w< gl( .Ҽ+􋿇_ |Gw3M%9r eCV\rZ]Y-mJuDܓ՛vmi}oJZ~wð\r=-^B* `O'bY;9/{W*qp4Ρi:fˍ." qU6X|+ɂk]}Zxm U"xna8WO^ %mb WQJ c[&Ӽ9⸚R=kRI"C*3FA9 ?xZ\dI'?#ۍ734c}*X yxg`w[Qn5UPQZ,>]Y[R+w}o/:<1^kfkM].$9Brs1_oFCAL~<3mr?=c]IkW"^\*B { pxk;}Cšud<T'Nu*M[~]{ynխRmi˵}(5ƹ^4tuk=/MM(umQcg [ę8pnku{>Kh':Ծ5_ihIXz`cJ%Wj!AFhD1Xx⻟xn-jCύɦjQ0ON85SzGÝžҥ%hTrkw\9lPp8POְ| "1lb\dZ]Ca,&(wXeֶ]0#NvwV\]?uLyPĶ1}ƑZcZĺuϛu՟ ȱפ=?=VW kuހ!$U&tsW?S:U׶tߐ{ļ.<~mƵ iְt{Ľ/'o[oӇOzZJ> ծ-le{k(A$)rÃ޿(A I'R;p<9l.QIm@v>O?_hoع.@Qs$+w4=) 5%Ry+r`p{W:p/sՎ"T}ՖWvg}xῈGƚ֦$*ˍ"'5Koϋ<х HI߹?JӜg~-?'!6fX2PS%r$,ӵ[P;dcp|?+qEKHYnI t!nT[O&0tmt[ik᎐.#hiF2EA|!P⹝ɎB*b8"'/u 'ߥpl"=G%ܭ,/ gk/\{Cj֚4㺂;M7KHgGYc5 nTC Bi{Hog*ɲRKO7IK}:m ϊ-".[L#g}/F=2 5ouؒ+6gCyv3lkĞoM {mF(`Hʿ6cPI[C`Œ*Qn[>¼:g(-4=_/14#--oPn8q3]g aĖbXc7J<"Xz9u߈u%ӭm!Bc?,",c>XrcC9>R%Z G8`<Â3-_Oɞ~qQTjS{gCM>73Ee{ai/$F@8<I g4v:s-nwZa1F'3?xŶ:e ]|8>9HUKGr~bZk'Յ'&X*d}wv@PUh4S{ShX*a;^jzoM'<_lLZ ,W~vU<1¿ 뻘 !o9RiV >-⽒;$nC((厸1"񕾏;cPHܤ< kl#LœVҪF[wvJ1nmV]z%ӣw߄Z+m:[<;J!ĝrFz`⵮G3|P K1%`"99esW% NhE5XD >u c~֬|=VKƷ{YtFapqg*JUSRQZ}w߾ Qu58]}V{>i >.Ei8е3]K i8Ee[kq 8b]0Nӌc:yҴ'tB;]u./HJ `^ⶻ {s\sa/Py؃돥yhkZ.^[kzyxj_N|M u[ӥf->ফcGo$w;OsW㟆 #%Ó:vV'oU?60G9=csxAM}kyjwBhv39]K7~'|gRᯊm|)j3<j$#Fo"0ħtn.Vgm~J_e)7f+Kiϛeg_]Zthqj3bNe܅889ɯq\ZXVnf@H݇Hsp{+g-+|+} }gT> 8U( :sk`{y:KotS^X,dna$nzxm* (nԭi:VxX(i^Ji۩xÞ oKu5\5m|+vב.C^r p+Dq&##m{NJ/kj4D9$ȑ`9 Z6aiXmfn1e'7nedϫlqe6x4Au╿@<9}'r㉑ X9 $x>qO)~j`JFE)%@ >i+V&V)e0D?[,=+%xAuFhlfc>Uoh|F%TL@b|D4H|Ko7I|7)2l ^N4]VdvtO屴cQ2IpXxLWOuxHgkqou5ƣ:%x0_:d8l>'Ԥӯ7m;C|=uxNK{47VѬ^t]  'hH,rJč@32v#IA_M>~/,`kNUA³/# 9Qɮg:O!lg Ŗ^I7; $-:ר^xᾟP8fM>{wIT&_XcU%>JMMm['Vi$ҽmoס%Keommb!2(kcIN9|! 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rich in neurotrophic factors we investigated whether the effects of GCM could be related to the presence of these )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(factors. We compared the effects of GCM and bFGF, GDNF and BDNF, at different concentrations, on one of the models of )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(toxicity for Q111 cells used in this study; the challenge with H)] TJ ET BT 290.153 741.602 Td /F1 8.7 Tf [(2)] TJ ET BT 294.972 743.667 Td /F1 9.8 Tf [(O)] TJ ET BT 302.557 741.602 Td /F1 8.7 Tf [(2)] TJ ET BT 307.376 743.667 Td /F1 9.8 Tf [(. We found that GCM was more potent than any of the above )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(mentioned factors for the rescue of Q111 cells, the reduction of apoptosis and the induction of arborisation, after treatment with )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(H)] TJ ET BT 33.290 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 38.108 719.857 Td /F1 9.8 Tf [(O)] TJ ET BT 45.694 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 50.512 719.857 Td /F1 9.8 Tf [(. However, glia-expressing mutant huntingtin can also secret toxic molecules )] TJ ET 0.267 0.267 0.267 rg BT 383.221 721.364 Td /F4 8.7 Tf [(53)] TJ ET BT 392.859 721.364 Td /F4 8.7 Tf [(54)] TJ ET BT 402.496 721.364 Td /F4 8.7 Tf [(55)] TJ ET BT 412.133 721.364 Td /F4 8.7 Tf [(56)] TJ ET 0.271 0.267 0.267 rg BT 26.250 700.452 Td /F1 9.8 Tf [(We conclude that the GCM is a powerful neuroprotective agent for Q111 striatal cells. GCM should be tested on animal models )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(of HD and, if the results obtained in this study are confirmed, on patients with HD.)] TJ ET BT 26.250 651.945 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 631.991 Td /F1 9.8 Tf [(Corresponding author: Dr. Maria Angeles Mena Department of Neurobiology, Hospital Ramn y Cajal Crta. de Colmenar Viejo )] TJ ET BT 26.250 620.086 Td /F1 9.8 Tf [(Km 9,100. Madrid, 28034. Spain Phone: +34- 91-3368384. e-mail: maria.a.mena@hrc.es This study has been supported in part )] TJ ET BT 26.250 608.181 Td /F1 9.8 Tf [(by grants from the Spanish Ministry of Health, FIS 2007/275, FIS 2010/172, CIBERNED 2006/05/0059, CIBERNED PI2010-6, )] TJ ET BT 26.250 596.277 Td /F1 9.8 Tf [(CAM 2011/BMD-2308 and MINCIN 2011. We are indebted to Laura Rodriguez, Izaskun Rodal and MPaz Muoz for excellent )] TJ ET BT 26.250 584.372 Td /F1 9.8 Tf [(technical assistance and Mrs. Claire Marsden for editorial help.)] TJ ET BT 26.250 555.269 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 527.815 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 527.815 Td /F1 9.8 Tf [(Bamford KA, Caine ED, Kido DK et al. \(1989\) Clinical-pathologic correlation in Huntington's disease: a neuropsychological )] TJ ET BT 26.250 515.910 Td /F1 9.8 Tf [(and computed tomography study. Neurology 39:796-801)] TJ ET BT 26.250 496.506 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 496.506 Td /F1 9.8 Tf [(Caine ED, Hunt RD, Weingartner H, Ebert MH \(1978\) Huntington's dementia. Clinical and neuropsychological features. Arch )] TJ ET BT 26.250 484.601 Td /F1 9.8 Tf [(Gen Psychiatry 35:377-384)] TJ ET BT 26.250 465.196 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 465.196 Td /F1 9.8 Tf [(Vonsattel JP, Keller C, Cortes Ramirez EP \(2011\) Huntington's disease - neuropathology. Handb Clin Neurol 100:83-100)] TJ ET BT 26.250 445.791 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 445.791 Td /F1 9.8 Tf [(Li SH, Li XJ \(2004\) Huntingtin-protein interactions and the pathogenesis of Huntington's disease. Trends Genet 20:146-154)] TJ ET BT 26.250 426.387 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 426.387 Td /F1 9.8 Tf [(Zuccato C, Valenza M, Cattaneo E \(2010\) Molecular mechanisms and potential therapeutical targets in Huntington's disease. )] TJ ET BT 26.250 414.482 Td /F1 9.8 Tf [(Physiol Rev 90:905-981)] TJ ET BT 26.250 395.077 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 395.077 Td /F1 9.8 Tf [(Rubio I, Rodriguez-Navarro JA, Tomas-Zapico C et al. \(2009\) Effects of partial suppression of parkin on huntingtin mutant )] TJ ET BT 26.250 383.172 Td /F1 9.8 Tf [(R6/1 mice. Brain Res 1281:91-100)] TJ ET BT 26.250 363.768 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 363.768 Td /F1 9.8 Tf [(Han I, You Y, Kordower JH et al. \(2010\) Differential vulnerability of neurons in Huntington's disease: the role of cell type-)] TJ ET BT 26.250 351.863 Td /F1 9.8 Tf [(specific features. J Neurochem113:1073-1091)] TJ ET BT 26.250 332.458 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 332.458 Td /F1 9.8 Tf [(Ribeiro FM, Paquet M, Ferreira LT et al. \(2010\) Metabotropic glutamate receptor-mediated cell signaling pathways are )] TJ ET BT 26.250 320.553 Td /F1 9.8 Tf [(altered in a mouse model of Huntington's disease. J Neurosci 30:316-324)] TJ ET BT 26.250 301.149 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 301.149 Td /F1 9.8 Tf [(Ribeiro FM, Pires RG, Ferguson SS \(2011\) Huntington's disease and Group I metabotropic glutamate receptors. Mol )] TJ ET BT 26.250 289.244 Td /F1 9.8 Tf [(Neurobiol 43:1-11)] TJ ET BT 26.250 269.839 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 269.839 Td /F1 9.8 Tf [(Duran R, Barrero FJ, Morales B et al. \(2010\) Oxidative stress and plasma aminopeptidase activity in Huntington's disease. J )] TJ ET BT 26.250 257.934 Td /F1 9.8 Tf [(Neural Transm 117:325-332)] TJ ET BT 26.250 238.530 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 238.530 Td /F1 9.8 Tf [(Lander HM \(1997\) An essential role for free radicals and derived species in signal transduction. Faseb J 11:118-124)] TJ ET BT 26.250 219.125 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 219.125 Td /F1 9.8 Tf [(Solano RM, Casarejos MJ, Menendez-Cuervo J et al. \(2008\) Glial dysfunction in parkin null mice: effects of aging. J )] TJ ET BT 26.250 207.220 Td /F1 9.8 Tf [(Neurosci 28:598-611)] TJ ET BT 26.250 187.815 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 187.815 Td /F1 9.8 Tf [(Marangolo M, McGee MM, Tipton KF et al. \(2001\) Oxidative stress induces apoptosis in C6 glioma cells: involvement of )] TJ ET BT 26.250 175.911 Td /F1 9.8 Tf [(mitogen-activated protein kinases and nuclear factor kappa B. Neurotox Res 3:397-409)] TJ ET BT 26.250 156.506 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 156.506 Td /F1 9.8 Tf [(Lin MT, Beal MF \(2006\) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443:787-795)] TJ ET BT 26.250 137.101 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 137.101 Td /F1 9.8 Tf [(Oliveira JM \(2010\) Nature and cause of mitochondrial dysfunction in Huntington's disease: focusing on huntingtin and the )] TJ ET BT 26.250 125.196 Td /F1 9.8 Tf [(striatum. J Neurochem 114:1-12)] TJ ET BT 26.250 105.792 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 105.792 Td /F1 9.8 Tf [(Politis M, Pavese N, Tai YF et al. \(2011\) Microglial activation in regions related to cognitive function predicts disease onset )] TJ ET BT 26.250 93.887 Td /F1 9.8 Tf [(in Huntington's disease: a multimodal imaging study. Hum Brain Mapp 32:258-270)] TJ ET BT 26.250 74.482 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 74.482 Td /F1 9.8 Tf [(Mena MA, Garcia de Yebenes J \(2008\) Glial cells as players in parkinsonism: the "good," the "bad," and the "mysterious" )] TJ ET BT 26.250 62.577 Td /F1 9.8 Tf [(glia. Neuroscientist 14:544-560)] TJ ET Q q 15.000 52.697 577.500 724.303 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Since GCM is rich in neurotrophic factors we investigated whether the effects of GCM could be related to the presence of these )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(factors. We compared the effects of GCM and bFGF, GDNF and BDNF, at different concentrations, on one of the models of )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(toxicity for Q111 cells used in this study; the challenge with H)] TJ ET BT 290.153 741.602 Td /F1 8.7 Tf [(2)] TJ ET BT 294.972 743.667 Td /F1 9.8 Tf [(O)] TJ ET BT 302.557 741.602 Td /F1 8.7 Tf [(2)] TJ ET BT 307.376 743.667 Td /F1 9.8 Tf [(. We found that GCM was more potent than any of the above )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(mentioned factors for the rescue of Q111 cells, the reduction of apoptosis and the induction of arborisation, after treatment with )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(H)] TJ ET BT 33.290 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 38.108 719.857 Td /F1 9.8 Tf [(O)] TJ ET BT 45.694 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 50.512 719.857 Td /F1 9.8 Tf [(. However, glia-expressing mutant huntingtin can also secret toxic molecules )] TJ ET 0.267 0.267 0.267 rg BT 383.221 721.364 Td /F4 8.7 Tf [(53)] TJ ET BT 392.859 721.364 Td /F4 8.7 Tf [(54)] TJ ET BT 402.496 721.364 Td /F4 8.7 Tf [(55)] TJ ET BT 412.133 721.364 Td /F4 8.7 Tf [(56)] TJ ET 0.271 0.267 0.267 rg BT 26.250 700.452 Td /F1 9.8 Tf [(We conclude that the GCM is a powerful neuroprotective agent for Q111 striatal cells. GCM should be tested on animal models )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(of HD and, if the results obtained in this study are confirmed, on patients with HD.)] TJ ET BT 26.250 651.945 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 631.991 Td /F1 9.8 Tf [(Corresponding author: Dr. Maria Angeles Mena Department of Neurobiology, Hospital Ramn y Cajal Crta. de Colmenar Viejo )] TJ ET BT 26.250 620.086 Td /F1 9.8 Tf [(Km 9,100. Madrid, 28034. Spain Phone: +34- 91-3368384. e-mail: maria.a.mena@hrc.es This study has been supported in part )] TJ ET BT 26.250 608.181 Td /F1 9.8 Tf [(by grants from the Spanish Ministry of Health, FIS 2007/275, FIS 2010/172, CIBERNED 2006/05/0059, CIBERNED PI2010-6, )] TJ ET BT 26.250 596.277 Td /F1 9.8 Tf [(CAM 2011/BMD-2308 and MINCIN 2011. We are indebted to Laura Rodriguez, Izaskun Rodal and MPaz Muoz for excellent )] TJ ET BT 26.250 584.372 Td /F1 9.8 Tf [(technical assistance and Mrs. Claire Marsden for editorial help.)] TJ ET BT 26.250 555.269 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 527.815 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 527.815 Td /F1 9.8 Tf [(Bamford KA, Caine ED, Kido DK et al. \(1989\) Clinical-pathologic correlation in Huntington's disease: a neuropsychological )] TJ ET BT 26.250 515.910 Td /F1 9.8 Tf [(and computed tomography study. Neurology 39:796-801)] TJ ET BT 26.250 496.506 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 496.506 Td /F1 9.8 Tf [(Caine ED, Hunt RD, Weingartner H, Ebert MH \(1978\) Huntington's dementia. Clinical and neuropsychological features. Arch )] TJ ET BT 26.250 484.601 Td /F1 9.8 Tf [(Gen Psychiatry 35:377-384)] TJ ET BT 26.250 465.196 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 465.196 Td /F1 9.8 Tf [(Vonsattel JP, Keller C, Cortes Ramirez EP \(2011\) Huntington's disease - neuropathology. Handb Clin Neurol 100:83-100)] TJ ET BT 26.250 445.791 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 445.791 Td /F1 9.8 Tf [(Li SH, Li XJ \(2004\) Huntingtin-protein interactions and the pathogenesis of Huntington's disease. Trends Genet 20:146-154)] TJ ET BT 26.250 426.387 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 426.387 Td /F1 9.8 Tf [(Zuccato C, Valenza M, Cattaneo E \(2010\) Molecular mechanisms and potential therapeutical targets in Huntington's disease. )] TJ ET BT 26.250 414.482 Td /F1 9.8 Tf [(Physiol Rev 90:905-981)] TJ ET BT 26.250 395.077 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 395.077 Td /F1 9.8 Tf [(Rubio I, Rodriguez-Navarro JA, Tomas-Zapico C et al. \(2009\) Effects of partial suppression of parkin on huntingtin mutant )] TJ ET BT 26.250 383.172 Td /F1 9.8 Tf [(R6/1 mice. Brain Res 1281:91-100)] TJ ET BT 26.250 363.768 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 363.768 Td /F1 9.8 Tf [(Han I, You Y, Kordower JH et al. \(2010\) Differential vulnerability of neurons in Huntington's disease: the role of cell type-)] TJ ET BT 26.250 351.863 Td /F1 9.8 Tf [(specific features. J Neurochem113:1073-1091)] TJ ET BT 26.250 332.458 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 332.458 Td /F1 9.8 Tf [(Ribeiro FM, Paquet M, Ferreira LT et al. \(2010\) Metabotropic glutamate receptor-mediated cell signaling pathways are )] TJ ET BT 26.250 320.553 Td /F1 9.8 Tf [(altered in a mouse model of Huntington's disease. J Neurosci 30:316-324)] TJ ET BT 26.250 301.149 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 301.149 Td /F1 9.8 Tf [(Ribeiro FM, Pires RG, Ferguson SS \(2011\) Huntington's disease and Group I metabotropic glutamate receptors. Mol )] TJ ET BT 26.250 289.244 Td /F1 9.8 Tf [(Neurobiol 43:1-11)] TJ ET BT 26.250 269.839 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 269.839 Td /F1 9.8 Tf [(Duran R, Barrero FJ, Morales B et al. \(2010\) Oxidative stress and plasma aminopeptidase activity in Huntington's disease. J )] TJ ET BT 26.250 257.934 Td /F1 9.8 Tf [(Neural Transm 117:325-332)] TJ ET BT 26.250 238.530 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 238.530 Td /F1 9.8 Tf [(Lander HM \(1997\) An essential role for free radicals and derived species in signal transduction. Faseb J 11:118-124)] TJ ET BT 26.250 219.125 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 219.125 Td /F1 9.8 Tf [(Solano RM, Casarejos MJ, Menendez-Cuervo J et al. \(2008\) Glial dysfunction in parkin null mice: effects of aging. J )] TJ ET BT 26.250 207.220 Td /F1 9.8 Tf [(Neurosci 28:598-611)] TJ ET BT 26.250 187.815 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 187.815 Td /F1 9.8 Tf [(Marangolo M, McGee MM, Tipton KF et al. \(2001\) Oxidative stress induces apoptosis in C6 glioma cells: involvement of )] TJ ET BT 26.250 175.911 Td /F1 9.8 Tf [(mitogen-activated protein kinases and nuclear factor kappa B. Neurotox Res 3:397-409)] TJ ET BT 26.250 156.506 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 156.506 Td /F1 9.8 Tf [(Lin MT, Beal MF \(2006\) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443:787-795)] TJ ET BT 26.250 137.101 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 137.101 Td /F1 9.8 Tf [(Oliveira JM \(2010\) Nature and cause of mitochondrial dysfunction in Huntington's disease: focusing on huntingtin and the )] TJ ET BT 26.250 125.196 Td /F1 9.8 Tf [(striatum. J Neurochem 114:1-12)] TJ ET BT 26.250 105.792 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 105.792 Td /F1 9.8 Tf [(Politis M, Pavese N, Tai YF et al. \(2011\) Microglial activation in regions related to cognitive function predicts disease onset )] TJ ET BT 26.250 93.887 Td /F1 9.8 Tf [(in Huntington's disease: a multimodal imaging study. Hum Brain Mapp 32:258-270)] TJ ET BT 26.250 74.482 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 74.482 Td /F1 9.8 Tf [(Mena MA, Garcia de Yebenes J \(2008\) Glial cells as players in parkinsonism: the "good," the "bad," and the "mysterious" )] TJ ET BT 26.250 62.577 Td /F1 9.8 Tf [(glia. Neuroscientist 14:544-560)] TJ ET Q q 15.000 52.697 577.500 724.303 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Since GCM is rich in neurotrophic factors we investigated whether the effects of GCM could be related to the presence of these )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(factors. We compared the effects of GCM and bFGF, GDNF and BDNF, at different concentrations, on one of the models of )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(toxicity for Q111 cells used in this study; the challenge with H)] TJ ET BT 290.153 741.602 Td /F1 8.7 Tf [(2)] TJ ET BT 294.972 743.667 Td /F1 9.8 Tf [(O)] TJ ET BT 302.557 741.602 Td /F1 8.7 Tf [(2)] TJ ET BT 307.376 743.667 Td /F1 9.8 Tf [(. We found that GCM was more potent than any of the above )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(mentioned factors for the rescue of Q111 cells, the reduction of apoptosis and the induction of arborisation, after treatment with )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(H)] TJ ET BT 33.290 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 38.108 719.857 Td /F1 9.8 Tf [(O)] TJ ET BT 45.694 717.793 Td /F1 8.7 Tf [(2)] TJ ET BT 50.512 719.857 Td /F1 9.8 Tf [(. However, glia-expressing mutant huntingtin can also secret toxic molecules )] TJ ET 0.267 0.267 0.267 rg BT 383.221 721.364 Td /F4 8.7 Tf [(53)] TJ ET BT 392.859 721.364 Td /F4 8.7 Tf [(54)] TJ ET BT 402.496 721.364 Td /F4 8.7 Tf [(55)] TJ ET BT 412.133 721.364 Td /F4 8.7 Tf [(56)] TJ ET 0.271 0.267 0.267 rg BT 26.250 700.452 Td /F1 9.8 Tf [(We conclude that the GCM is a powerful neuroprotective agent for Q111 striatal cells. GCM should be tested on animal models )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(of HD and, if the results obtained in this study are confirmed, on patients with HD.)] TJ ET BT 26.250 651.945 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 631.991 Td /F1 9.8 Tf [(Corresponding author: Dr. Maria Angeles Mena Department of Neurobiology, Hospital Ramn y Cajal Crta. de Colmenar Viejo )] TJ ET BT 26.250 620.086 Td /F1 9.8 Tf [(Km 9,100. Madrid, 28034. Spain Phone: +34- 91-3368384. e-mail: maria.a.mena@hrc.es This study has been supported in part )] TJ ET BT 26.250 608.181 Td /F1 9.8 Tf [(by grants from the Spanish Ministry of Health, FIS 2007/275, FIS 2010/172, CIBERNED 2006/05/0059, CIBERNED PI2010-6, )] TJ ET BT 26.250 596.277 Td /F1 9.8 Tf [(CAM 2011/BMD-2308 and MINCIN 2011. 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Neuroscientist 14:544-560)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(11)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 536 0 obj << /Type /Annot /Subtype /Link /A 537 0 R /Border [0 0 0] /H /I /Rect [ 383.2213 720.5627 392.8587 729.3810 ] >> endobj 537 0 obj << /Type /Action >> endobj 538 0 obj << /Type /Annot /Subtype /Link /A 539 0 R /Border [0 0 0] /H /I /Rect [ 392.8587 720.5627 402.4960 729.3810 ] >> endobj 539 0 obj << /Type /Action >> endobj 540 0 obj << /Type /Annot /Subtype /Link /A 541 0 R /Border [0 0 0] /H /I /Rect [ 402.4960 720.5627 412.1333 729.3810 ] >> endobj 541 0 obj << /Type /Action >> endobj 542 0 obj << /Type /Annot /Subtype /Link /A 543 0 R /Border [0 0 0] /H /I /Rect [ 412.1333 720.5627 421.7707 729.3810 ] >> endobj 543 0 obj << /Type /Action >> endobj 544 0 obj << /Type /Annot /Subtype /Link /A 545 0 R /Border [0 0 0] /H /I /Rect [ 383.2213 720.5627 392.8587 729.3810 ] >> endobj 545 0 obj << /Type /Action >> endobj 546 0 obj << /Type /Annot /Subtype /Link /A 547 0 R /Border [0 0 0] /H /I /Rect [ 392.8587 720.5627 402.4960 729.3810 ] >> endobj 547 0 obj << /Type /Action >> endobj 548 0 obj << /Type /Annot /Subtype /Link /A 549 0 R /Border [0 0 0] /H /I /Rect [ 402.4960 720.5627 412.1333 729.3810 ] >> endobj 549 0 obj << /Type /Action >> endobj 550 0 obj << /Type /Annot /Subtype /Link /A 551 0 R /Border [0 0 0] /H /I /Rect [ 412.1333 720.5627 421.7707 729.3810 ] >> endobj 551 0 obj << /Type /Action >> endobj 552 0 obj << /Type /Annot /Subtype /Link /A 553 0 R /Border [0 0 0] /H /I /Rect [ 383.2213 720.5627 392.8587 729.3810 ] >> endobj 553 0 obj << /Type /Action >> endobj 554 0 obj << /Type /Annot /Subtype /Link /A 555 0 R /Border [0 0 0] /H /I /Rect [ 392.8587 720.5627 402.4960 729.3810 ] >> endobj 555 0 obj << /Type /Action >> endobj 556 0 obj << /Type /Annot /Subtype /Link /A 557 0 R /Border [0 0 0] /H /I /Rect [ 402.4960 720.5627 412.1333 729.3810 ] >> endobj 557 0 obj << /Type /Action >> endobj 558 0 obj << /Type /Annot /Subtype /Link /A 559 0 R /Border [0 0 0] /H /I /Rect [ 412.1333 720.5627 421.7707 729.3810 ] >> endobj 559 0 obj << /Type /Action >> endobj 560 0 obj << /Type /Page /Parent 3 0 R /Contents 561 0 R >> endobj 561 0 obj << /Length 22739 >> stream 0.271 0.267 0.267 rg q 15.000 37.477 577.500 739.523 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Cicchetti F, Soulet D, Freeman TB \(2011\) Neuronal degeneration in striatal transplants and Huntington's disease: potential )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(mechanisms and clinical implications. Brain 134:641-652)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Bradford J, Shin JY, Roberts M et al. \(2009\) Expression of mutant huntingtin in mouse brain astrocytes causes age-)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(dependent neurological symptoms. Proc Natl Acad Sci U S A 106:22480-22485)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Besson MT, Dupont P, Fridell YW, Lievens JC \(2010\) Increased energy metabolism rescues glia-induced pathology in a )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(Drosophila model of Huntington's disease. Hum Mol Genet 19:3372-3382)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(McGeer PL, McGeer EG \(2008\) Glial reactions in Parkinson's disease. Mov Disord 23:474-483)] TJ ET BT 26.250 646.643 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 646.643 Td /F1 9.8 Tf [(Mena MA, Casarejos MJ, Carazo A et al. \(1997\) Glia protect fetal midbrain dopamine neurons in culture from L-DOPA )] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(toxicity through multiple mechanisms. J Neural Transm 104:317-328)] TJ ET BT 26.250 615.333 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 615.333 Td /F1 9.8 Tf [(de Bernardo S, Canals S, Casarejos MJ et al. \(2003\) Glia-conditioned medium induces de novo synthesis of tyrosine )] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(hydroxylase and increases dopamine cell survival by differential signaling pathways. J Neurosci Res 73:818-830)] TJ ET BT 26.250 584.024 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 584.024 Td /F1 9.8 Tf [(Mena MA, Casarejos MJ, Estrada C, de Yebenes JG \(1994\) Effects of retinoic acid on NB 69 human neuroblastoma cells )] TJ ET BT 26.250 572.119 Td /F1 9.8 Tf [(and fetal rat mid brain neurons. J Neural Transm Park Dis Dement Sect 8:85-97)] TJ ET BT 26.250 552.714 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 552.714 Td /F1 9.8 Tf [(Trettel F, Rigamonti D, Hilditch-Maguire P et al. \(2000\) Dominant phenotypes produced by the HD mutation in )] TJ ET BT 26.250 540.810 Td /F1 9.8 Tf [(STHdh\(Q111\) striatal cells. Hum Mol Genet 9:2799-2809)] TJ ET BT 26.250 521.405 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 521.405 Td /F1 9.8 Tf [(Seong IS, Ivanova E, Lee JM et al. \(2005\) HD CAG repeat implicates a dominant property of huntingtin in mitochondrial )] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(energy metabolism. Hum Mol Genet 14:2871-2880)] TJ ET BT 26.250 490.095 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 490.095 Td /F1 9.8 Tf [(Gines S, Seong IS, Fossale E et al. \(2003\) Specific progressive cAMP reduction implicates energy deficit in presymptomatic )] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(Huntington's disease knock-in mice. Hum Mol Genet 12:497-508)] TJ ET BT 26.250 458.786 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 458.786 Td /F1 9.8 Tf [(Ruan Q, Lesort M, MacDonald ME, Johnson GV \(2004\) Striatal cells from mutant huntingtin knock-in mice are selectively )] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(vulnerable to mitochondrial complex II inhibitor-induced cell death through a non-apoptotic pathway. Hum Mol Genet 13:669-681)] TJ ET BT 26.250 427.476 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 427.476 Td /F1 9.8 Tf [(Mao Z, Choo YS, Lesort M \(2006\) Cystamine and cysteamine prevent 3-NP-induced mitochondrial depolarization of )] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(Huntington's disease knock-in striatal cells. Eur J Neurosci 23:1701-1710)] TJ ET BT 26.250 396.167 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 396.167 Td /F1 9.8 Tf [(Milakovic T, Johnson GV \(2005\) Mitochondrial respiration and ATP production are significantly impaired in striatal cells )] TJ ET BT 26.250 384.262 Td /F1 9.8 Tf [(expressing mutant huntingtin. J Biol Chem 280:30773-30782)] TJ ET BT 26.250 364.857 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 364.857 Td /F1 9.8 Tf [(Mena MA, Pardo B, Paino CL, De Yebenes JG \(1993\) Levodopa toxicity in foetal rat midbrain neurones in culture: )] TJ ET BT 26.250 352.953 Td /F1 9.8 Tf [(modulation by ascorbic acid. Neuroreport 4:438-440)] TJ ET BT 26.250 333.548 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 333.548 Td /F1 9.8 Tf [(Pardo B, Paino CL, Casarejos MJ, Mena MA \(1997\) Neuronal-enriched cultures from embryonic rat ventral mesencephalon )] TJ ET BT 26.250 321.643 Td /F1 9.8 Tf [(for pharmacological studies of dopamine neurons. 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J Neurosci Res 30:359-371)] TJ ET BT 26.250 51.762 Td /F1 9.8 Tf [(41.)] TJ ET BT 43.553 51.762 Td /F1 9.8 Tf [(Mena MA, de Bernardo S, Casarejos MJ et al. \(2002\) The role of astroglia on the survival of dopamine neurons. Mol )] TJ ET Q q 15.000 37.477 577.500 739.523 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Cicchetti F, Soulet D, Freeman TB \(2011\) Neuronal degeneration in striatal transplants and Huntington's disease: potential )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(mechanisms and clinical implications. Brain 134:641-652)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Bradford J, Shin JY, Roberts M et al. \(2009\) Expression of mutant huntingtin in mouse brain astrocytes causes age-)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(dependent neurological symptoms. Proc Natl Acad Sci U S A 106:22480-22485)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Besson MT, Dupont P, Fridell YW, Lievens JC \(2010\) Increased energy metabolism rescues glia-induced pathology in a )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(Drosophila model of Huntington's disease. Hum Mol Genet 19:3372-3382)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(McGeer PL, McGeer EG \(2008\) Glial reactions in Parkinson's disease. Mov Disord 23:474-483)] TJ ET BT 26.250 646.643 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 646.643 Td /F1 9.8 Tf [(Mena MA, Casarejos MJ, Carazo A et al. \(1997\) Glia protect fetal midbrain dopamine neurons in culture from L-DOPA )] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(toxicity through multiple mechanisms. J Neural Transm 104:317-328)] TJ ET BT 26.250 615.333 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 615.333 Td /F1 9.8 Tf [(de Bernardo S, Canals S, Casarejos MJ et al. \(2003\) Glia-conditioned medium induces de novo synthesis of tyrosine )] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(hydroxylase and increases dopamine cell survival by differential signaling pathways. J Neurosci Res 73:818-830)] TJ ET BT 26.250 584.024 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 584.024 Td /F1 9.8 Tf [(Mena MA, Casarejos MJ, Estrada C, de Yebenes JG \(1994\) Effects of retinoic acid on NB 69 human neuroblastoma cells )] TJ ET BT 26.250 572.119 Td /F1 9.8 Tf [(and fetal rat mid brain neurons. J Neural Transm Park Dis Dement Sect 8:85-97)] TJ ET BT 26.250 552.714 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 552.714 Td /F1 9.8 Tf [(Trettel F, Rigamonti D, Hilditch-Maguire P et al. \(2000\) Dominant phenotypes produced by the HD mutation in )] TJ ET BT 26.250 540.810 Td /F1 9.8 Tf [(STHdh\(Q111\) striatal cells. Hum Mol Genet 9:2799-2809)] TJ ET BT 26.250 521.405 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 521.405 Td /F1 9.8 Tf [(Seong IS, Ivanova E, Lee JM et al. \(2005\) HD CAG repeat implicates a dominant property of huntingtin in mitochondrial )] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(energy metabolism. Hum Mol Genet 14:2871-2880)] TJ ET BT 26.250 490.095 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 490.095 Td /F1 9.8 Tf [(Gines S, Seong IS, Fossale E et al. \(2003\) Specific progressive cAMP reduction implicates energy deficit in presymptomatic )] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(Huntington's disease knock-in mice. Hum Mol Genet 12:497-508)] TJ ET BT 26.250 458.786 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 458.786 Td /F1 9.8 Tf [(Ruan Q, Lesort M, MacDonald ME, Johnson GV \(2004\) Striatal cells from mutant huntingtin knock-in mice are selectively )] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(vulnerable to mitochondrial complex II inhibitor-induced cell death through a non-apoptotic pathway. Hum Mol Genet 13:669-681)] TJ ET BT 26.250 427.476 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 427.476 Td /F1 9.8 Tf [(Mao Z, Choo YS, Lesort M \(2006\) Cystamine and cysteamine prevent 3-NP-induced mitochondrial depolarization of )] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(Huntington's disease knock-in striatal cells. Eur J Neurosci 23:1701-1710)] TJ ET BT 26.250 396.167 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 396.167 Td /F1 9.8 Tf [(Milakovic T, Johnson GV \(2005\) Mitochondrial respiration and ATP production are significantly impaired in striatal cells )] TJ ET BT 26.250 384.262 Td /F1 9.8 Tf [(expressing mutant huntingtin. J Biol Chem 280:30773-30782)] TJ ET BT 26.250 364.857 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 364.857 Td /F1 9.8 Tf [(Mena MA, Pardo B, Paino CL, De Yebenes JG \(1993\) Levodopa toxicity in foetal rat midbrain neurones in culture: )] TJ ET BT 26.250 352.953 Td /F1 9.8 Tf [(modulation by ascorbic acid. Neuroreport 4:438-440)] TJ ET BT 26.250 333.548 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 333.548 Td /F1 9.8 Tf [(Pardo B, Paino CL, Casarejos MJ, Mena MA \(1997\) Neuronal-enriched cultures from embryonic rat ventral mesencephalon )] TJ ET BT 26.250 321.643 Td /F1 9.8 Tf [(for pharmacological studies of dopamine neurons. Brain Res Brain Res Protoc 1:127-132)] TJ ET BT 26.250 302.238 Td /F1 9.8 Tf [(33.)] TJ ET BT 43.553 302.238 Td /F1 9.8 Tf [(Casarejos MJ, Solano RM, Menendez J et al. \(2005\) Differential effects of l-DOPA on monoamine metabolism, cell survival )] TJ ET BT 26.250 290.334 Td /F1 9.8 Tf [(and glutathione production in midbrain neuronal-enriched cultures from parkin knockout and wild-type mice. J Neurochem )] TJ ET BT 26.250 278.429 Td /F1 9.8 Tf [(94:1005-1014)] TJ ET BT 26.250 259.024 Td /F1 9.8 Tf [(34.)] TJ ET BT 43.553 259.024 Td /F1 9.8 Tf [(Tietze F \(1969\) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: )] TJ ET BT 26.250 247.119 Td /F1 9.8 Tf [(applications to mammalian blood and other tissues. Anal Biochem 27:502-522)] TJ ET BT 26.250 227.715 Td /F1 9.8 Tf [(35.)] TJ ET BT 43.553 227.715 Td /F1 9.8 Tf [(Tong WM, Cortes U, Wang ZQ \(2001\) Poly\(ADP-ribose\) polymerase: a guardian angel protecting the genome and )] TJ ET BT 26.250 215.810 Td /F1 9.8 Tf [(suppressing tumorigenesis. Biochim Biophys Acta 1552:27-37)] TJ ET BT 26.250 196.405 Td /F1 9.8 Tf [(36.)] TJ ET BT 43.553 196.405 Td /F1 9.8 Tf [(Lin LF, Doherty DH, Lile JD et al. \(1993\) GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic )] TJ ET BT 26.250 184.500 Td /F1 9.8 Tf [(neurons. Science 260:1130-1132)] TJ ET BT 26.250 165.096 Td /F1 9.8 Tf [(37.)] TJ ET BT 43.553 165.096 Td /F1 9.8 Tf [(Hoffer BJ, Hoffman A, Bowenkamp K et al. \(1994\) Glial cell line-derived neurotrophic factor reverses toxin-induced injury to )] TJ ET BT 26.250 153.191 Td /F1 9.8 Tf [(midbrain dopaminergic neurons in vivo. Neurosci Lett 182:107-111)] TJ ET BT 26.250 133.786 Td /F1 9.8 Tf [(38.)] TJ ET BT 43.553 133.786 Td /F1 9.8 Tf [(Takeshima T, Johnston JM, Commissiong JW \(1994\) Mesencephalic type 1 astrocytes rescue dopaminergic neurons from )] TJ ET BT 26.250 121.881 Td /F1 9.8 Tf [(death induced by serum deprivation. J Neurosci 14:4769-4779)] TJ ET BT 26.250 102.477 Td /F1 9.8 Tf [(39.)] TJ ET BT 43.553 102.477 Td /F1 9.8 Tf [(Muller HW, Junghans U, Kappler J \(1995\) Astroglial neurotrophic and neurite-promoting factors. Pharmacol Ther 65:1-18)] TJ ET BT 26.250 83.072 Td /F1 9.8 Tf [(40.)] TJ ET BT 43.553 83.072 Td /F1 9.8 Tf [(Engele J, Schubert D, Bohn MC \(1991\) Conditioned media derived from glial cell lines promote survival and differentiation )] TJ ET BT 26.250 71.167 Td /F1 9.8 Tf [(of dopaminergic neurons in vitro: role of mesencephalic glia. J Neurosci Res 30:359-371)] TJ ET BT 26.250 51.762 Td /F1 9.8 Tf [(41.)] TJ ET BT 43.553 51.762 Td /F1 9.8 Tf [(Mena MA, de Bernardo S, Casarejos MJ et al. \(2002\) The role of astroglia on the survival of dopamine neurons. Mol )] TJ ET Q q 15.000 37.477 577.500 739.523 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Cicchetti F, Soulet D, Freeman TB \(2011\) Neuronal degeneration in striatal transplants and Huntington's disease: potential )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(mechanisms and clinical implications. Brain 134:641-652)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Bradford J, Shin JY, Roberts M et al. \(2009\) Expression of mutant huntingtin in mouse brain astrocytes causes age-)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(dependent neurological symptoms. Proc Natl Acad Sci U S A 106:22480-22485)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Besson MT, Dupont P, Fridell YW, Lievens JC \(2010\) Increased energy metabolism rescues glia-induced pathology in a )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(Drosophila model of Huntington's disease. Hum Mol Genet 19:3372-3382)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(McGeer PL, McGeer EG \(2008\) Glial reactions in Parkinson's disease. Mov Disord 23:474-483)] TJ ET BT 26.250 646.643 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 646.643 Td /F1 9.8 Tf [(Mena MA, Casarejos MJ, Carazo A et al. \(1997\) Glia protect fetal midbrain dopamine neurons in culture from L-DOPA )] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(toxicity through multiple mechanisms. J Neural Transm 104:317-328)] TJ ET BT 26.250 615.333 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 615.333 Td /F1 9.8 Tf [(de Bernardo S, Canals S, Casarejos MJ et al. \(2003\) Glia-conditioned medium induces de novo synthesis of tyrosine )] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(hydroxylase and increases dopamine cell survival by differential signaling pathways. J Neurosci Res 73:818-830)] TJ ET BT 26.250 584.024 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 584.024 Td /F1 9.8 Tf [(Mena MA, Casarejos MJ, Estrada C, de Yebenes JG \(1994\) Effects of retinoic acid on NB 69 human neuroblastoma cells )] TJ ET BT 26.250 572.119 Td /F1 9.8 Tf [(and fetal rat mid brain neurons. J Neural Transm Park Dis Dement Sect 8:85-97)] TJ ET BT 26.250 552.714 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 552.714 Td /F1 9.8 Tf [(Trettel F, Rigamonti D, Hilditch-Maguire P et al. \(2000\) Dominant phenotypes produced by the HD mutation in )] TJ ET BT 26.250 540.810 Td /F1 9.8 Tf [(STHdh\(Q111\) striatal cells. Hum Mol Genet 9:2799-2809)] TJ ET BT 26.250 521.405 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 521.405 Td /F1 9.8 Tf [(Seong IS, Ivanova E, Lee JM et al. \(2005\) HD CAG repeat implicates a dominant property of huntingtin in mitochondrial )] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(energy metabolism. Hum Mol Genet 14:2871-2880)] TJ ET BT 26.250 490.095 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 490.095 Td /F1 9.8 Tf [(Gines S, Seong IS, Fossale E et al. \(2003\) Specific progressive cAMP reduction implicates energy deficit in presymptomatic )] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(Huntington's disease knock-in mice. Hum Mol Genet 12:497-508)] TJ ET BT 26.250 458.786 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 458.786 Td /F1 9.8 Tf [(Ruan Q, Lesort M, MacDonald ME, Johnson GV \(2004\) Striatal cells from mutant huntingtin knock-in mice are selectively )] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(vulnerable to mitochondrial complex II inhibitor-induced cell death through a non-apoptotic pathway. Hum Mol Genet 13:669-681)] TJ ET BT 26.250 427.476 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 427.476 Td /F1 9.8 Tf [(Mao Z, Choo YS, Lesort M \(2006\) Cystamine and cysteamine prevent 3-NP-induced mitochondrial depolarization of )] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(Huntington's disease knock-in striatal cells. Eur J Neurosci 23:1701-1710)] TJ ET BT 26.250 396.167 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 396.167 Td /F1 9.8 Tf [(Milakovic T, Johnson GV \(2005\) Mitochondrial respiration and ATP production are significantly impaired in striatal cells )] TJ ET BT 26.250 384.262 Td /F1 9.8 Tf [(expressing mutant huntingtin. J Biol Chem 280:30773-30782)] TJ ET BT 26.250 364.857 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 364.857 Td /F1 9.8 Tf [(Mena MA, Pardo B, Paino CL, De Yebenes JG \(1993\) Levodopa toxicity in foetal rat midbrain neurones in culture: )] TJ ET BT 26.250 352.953 Td /F1 9.8 Tf [(modulation by ascorbic acid. Neuroreport 4:438-440)] TJ ET BT 26.250 333.548 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 333.548 Td /F1 9.8 Tf [(Pardo B, Paino CL, Casarejos MJ, Mena MA \(1997\) Neuronal-enriched cultures from embryonic rat ventral mesencephalon )] TJ ET BT 26.250 321.643 Td /F1 9.8 Tf [(for pharmacological studies of dopamine neurons. Brain Res Brain Res Protoc 1:127-132)] TJ ET BT 26.250 302.238 Td /F1 9.8 Tf [(33.)] TJ ET BT 43.553 302.238 Td /F1 9.8 Tf [(Casarejos MJ, Solano RM, Menendez J et al. \(2005\) Differential effects of l-DOPA on monoamine metabolism, cell survival )] TJ ET BT 26.250 290.334 Td /F1 9.8 Tf [(and glutathione production in midbrain neuronal-enriched cultures from parkin knockout and wild-type mice. J Neurochem )] TJ ET BT 26.250 278.429 Td /F1 9.8 Tf [(94:1005-1014)] TJ ET BT 26.250 259.024 Td /F1 9.8 Tf [(34.)] TJ ET BT 43.553 259.024 Td /F1 9.8 Tf [(Tietze F \(1969\) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: )] TJ ET BT 26.250 247.119 Td /F1 9.8 Tf [(applications to mammalian blood and other tissues. Anal Biochem 27:502-522)] TJ ET BT 26.250 227.715 Td /F1 9.8 Tf [(35.)] TJ ET BT 43.553 227.715 Td /F1 9.8 Tf [(Tong WM, Cortes U, Wang ZQ \(2001\) Poly\(ADP-ribose\) polymerase: a guardian angel protecting the genome and )] TJ ET BT 26.250 215.810 Td /F1 9.8 Tf [(suppressing tumorigenesis. Biochim Biophys Acta 1552:27-37)] TJ ET BT 26.250 196.405 Td /F1 9.8 Tf [(36.)] TJ ET BT 43.553 196.405 Td /F1 9.8 Tf [(Lin LF, Doherty DH, Lile JD et al. \(1993\) GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic )] TJ ET BT 26.250 184.500 Td /F1 9.8 Tf [(neurons. Science 260:1130-1132)] TJ ET BT 26.250 165.096 Td /F1 9.8 Tf [(37.)] TJ ET BT 43.553 165.096 Td /F1 9.8 Tf [(Hoffer BJ, Hoffman A, Bowenkamp K et al. \(1994\) Glial cell line-derived neurotrophic factor reverses toxin-induced injury to )] TJ ET BT 26.250 153.191 Td /F1 9.8 Tf [(midbrain dopaminergic neurons in vivo. Neurosci Lett 182:107-111)] TJ ET BT 26.250 133.786 Td /F1 9.8 Tf [(38.)] TJ ET BT 43.553 133.786 Td /F1 9.8 Tf [(Takeshima T, Johnston JM, Commissiong JW \(1994\) Mesencephalic type 1 astrocytes rescue dopaminergic neurons from )] TJ ET BT 26.250 121.881 Td /F1 9.8 Tf [(death induced by serum deprivation. J Neurosci 14:4769-4779)] TJ ET BT 26.250 102.477 Td /F1 9.8 Tf [(39.)] TJ ET BT 43.553 102.477 Td /F1 9.8 Tf [(Muller HW, Junghans U, Kappler J \(1995\) Astroglial neurotrophic and neurite-promoting factors. Pharmacol Ther 65:1-18)] TJ ET BT 26.250 83.072 Td /F1 9.8 Tf [(40.)] TJ ET BT 43.553 83.072 Td /F1 9.8 Tf [(Engele J, Schubert D, Bohn MC \(1991\) Conditioned media derived from glial cell lines promote survival and differentiation )] TJ ET BT 26.250 71.167 Td /F1 9.8 Tf [(of dopaminergic neurons in vitro: role of mesencephalic glia. J Neurosci Res 30:359-371)] TJ ET BT 26.250 51.762 Td /F1 9.8 Tf [(41.)] TJ ET BT 43.553 51.762 Td /F1 9.8 Tf [(Mena MA, de Bernardo S, Casarejos MJ et al. \(2002\) The role of astroglia on the survival of dopamine neurons. Mol )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(12)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 562 0 obj << /Type /Page /Parent 3 0 R /Contents 563 0 R >> endobj 563 0 obj << /Length 14660 >> stream 0.271 0.267 0.267 rg q 15.000 280.453 577.500 496.547 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Neurobiol 25:245-263)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(42.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Panchision DM, Martin-DeLeon PA, Takeshima T et al. \(1998\) An immortalized, type-1 astrocyte of mesencephalic origin )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(source of a dopaminergic neurotrophic factor. J Mol Neurosci 11:209-221)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(43.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(Reuss B, Unsicker K \(2000\) Survival and differentiation of dopaminergic mesencephalic neurons are promoted by )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(dopamine-mediated induction of FGF-2 in striatal astroglial cells. Mol Cell Neurosci 16:781-792)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(44.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Chen CM \(2011\) Mitochondrial dysfunction, metabolic deficits, and increased oxidative stress in Huntington's disease. )] TJ ET BT 26.250 673.548 Td /F1 9.8 Tf [(Chang Gung Med J 34:135-152)] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(45.)] TJ ET BT 43.553 654.143 Td /F1 9.8 Tf [(Browne SE, Bowling AC, MacGarvey U et al. \(1997\) Oxidative damage and metabolic dysfunction in Huntington's disease: )] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(selective vulnerability of the basal ganglia. Ann Neurol 41:646-653)] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(46.)] TJ ET BT 43.553 622.833 Td /F1 9.8 Tf [(Polidori MC, Mecocci P, Browne SE et al. \(1999\) Oxidative damage to mitochondrial DNA in Huntington's disease parietal )] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(cortex. Neurosci Lett 272:53-56)] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(47.)] TJ ET BT 43.553 591.524 Td /F1 9.8 Tf [(Li N, Liu B, Dluzen DE, Jin Y \(2007\) Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity in PC12 )] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(cells. J Ethnopharmacol 111:458-463)] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(48.)] TJ ET BT 43.553 560.214 Td /F1 9.8 Tf [(Ankarcrona M, Dypbukt JM, Bonfoco E et al. \(1995\) Glutamate-induced neuronal death: a succession of necrosis or )] TJ ET BT 26.250 548.310 Td /F1 9.8 Tf [(apoptosis depending on mitochondrial function. Neuron 15:961-973)] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(49.)] TJ ET BT 43.553 528.905 Td /F1 9.8 Tf [(Choi DW \(1987\) Ionic dependence of glutamate neurotoxicity. J Neurosci 7:369-379)] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(50.)] TJ ET BT 43.553 509.500 Td /F1 9.8 Tf [(Myers RH, Vonsattel JP, Stevens TJ et al. \(1988\) Clinical and neuropathologic assessment of severity in Huntington's )] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(disease. Neurology 38:341-347)] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(51.)] TJ ET BT 43.553 478.191 Td /F1 9.8 Tf [(Gil JM, Rego AC \(2008\) Mechanisms of neurodegeneration in Huntington's disease. Eur J Neurosci 27:2803-2820)] TJ ET BT 26.250 458.786 Td /F1 9.8 Tf [(52.)] TJ ET BT 43.553 458.786 Td /F1 9.8 Tf [(Beal MF, Brouillet E, Jenkins BG et al. \(1993\) Neurochemical and histologic characterization of striatal excitotoxic lesions )] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(produced by the mitochondrial toxin 3-nitropropionic acid. J Neurosci 13:4181-4192)] TJ ET BT 26.250 427.476 Td /F1 9.8 Tf [(53.)] TJ ET BT 43.553 427.476 Td /F1 9.8 Tf [(Shin, J.Y., Fang, Z.H., Yu, Z.X., Wang, C.E., Li, S.H. and Li, X.J \(2005\) Expression of mutant huntingtin in glial cells )] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(contributes to neuronal excitotoxicity. J Cell Biol. 171:1001-1012)] TJ ET BT 26.250 396.167 Td /F1 9.8 Tf [(54.)] TJ ET BT 43.553 396.167 Td /F1 9.8 Tf [(Sapp, E., Kegel, K.B., Aronin, N., Hashikawa, T., Uchiyama, Y., Tohyama, K., Bhide, P.G., Vonsattel, J.P. and DiFiglia, M )] TJ ET BT 26.250 384.262 Td /F1 9.8 Tf [(\(2001\) Early and progressive accumulation of reactive microglia in the Huntington disease brain. J Neuropathol Exp Neurol. 60: )] TJ ET BT 26.250 372.357 Td /F1 9.8 Tf [(161-172)] TJ ET BT 26.250 352.953 Td /F1 9.8 Tf [(55.)] TJ ET BT 43.553 352.953 Td /F1 9.8 Tf [(Chou, S.Y., Weng, J.Y., Lai, H.L., Liao, F., Sun, S.H., Tu, P.H., Dickson, D.W. and Chern, Y \(2008\) Expanded-)] TJ ET BT 26.250 341.048 Td /F1 9.8 Tf [(polyglutamine huntingtin protein suppresses the secretion and production of a chemokine \(CCL5/RANTES\) by astrocytes. J )] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(Neurosci. 28:3277-90)] TJ ET BT 26.250 309.738 Td /F1 9.8 Tf [(56.)] TJ ET BT 43.553 309.738 Td /F1 9.8 Tf [(Bradford, J., Shin, J.Y., Roberts, M., Wang, C.E., Sheng, G., Li, S. and Li, X.J \(2010\) Mutant huntingtin in glial cells )] TJ ET BT 26.250 297.834 Td /F1 9.8 Tf [(exacerbates neurological symptoms of Huntington disease mice. J Biol Chem. 285:10653-61.)] TJ ET Q q 15.000 280.453 577.500 496.547 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Neurobiol 25:245-263)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(42.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Panchision DM, Martin-DeLeon PA, Takeshima T et al. \(1998\) An immortalized, type-1 astrocyte of mesencephalic origin )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(source of a dopaminergic neurotrophic factor. J Mol Neurosci 11:209-221)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(43.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(Reuss B, Unsicker K \(2000\) Survival and differentiation of dopaminergic mesencephalic neurons are promoted by )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(dopamine-mediated induction of FGF-2 in striatal astroglial cells. Mol Cell Neurosci 16:781-792)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(44.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Chen CM \(2011\) Mitochondrial dysfunction, metabolic deficits, and increased oxidative stress in Huntington's disease. )] TJ ET BT 26.250 673.548 Td /F1 9.8 Tf [(Chang Gung Med J 34:135-152)] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(45.)] TJ ET BT 43.553 654.143 Td /F1 9.8 Tf [(Browne SE, Bowling AC, MacGarvey U et al. \(1997\) Oxidative damage and metabolic dysfunction in Huntington's disease: )] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(selective vulnerability of the basal ganglia. Ann Neurol 41:646-653)] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(46.)] TJ ET BT 43.553 622.833 Td /F1 9.8 Tf [(Polidori MC, Mecocci P, Browne SE et al. \(1999\) Oxidative damage to mitochondrial DNA in Huntington's disease parietal )] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(cortex. Neurosci Lett 272:53-56)] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(47.)] TJ ET BT 43.553 591.524 Td /F1 9.8 Tf [(Li N, Liu B, Dluzen DE, Jin Y \(2007\) Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity in PC12 )] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(cells. J Ethnopharmacol 111:458-463)] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(48.)] TJ ET BT 43.553 560.214 Td /F1 9.8 Tf [(Ankarcrona M, Dypbukt JM, Bonfoco E et al. \(1995\) Glutamate-induced neuronal death: a succession of necrosis or )] TJ ET BT 26.250 548.310 Td /F1 9.8 Tf [(apoptosis depending on mitochondrial function. Neuron 15:961-973)] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(49.)] TJ ET BT 43.553 528.905 Td /F1 9.8 Tf [(Choi DW \(1987\) Ionic dependence of glutamate neurotoxicity. J Neurosci 7:369-379)] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(50.)] TJ ET BT 43.553 509.500 Td /F1 9.8 Tf [(Myers RH, Vonsattel JP, Stevens TJ et al. \(1988\) Clinical and neuropathologic assessment of severity in Huntington's )] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(disease. Neurology 38:341-347)] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(51.)] TJ ET BT 43.553 478.191 Td /F1 9.8 Tf [(Gil JM, Rego AC \(2008\) Mechanisms of neurodegeneration in Huntington's disease. Eur J Neurosci 27:2803-2820)] TJ ET BT 26.250 458.786 Td /F1 9.8 Tf [(52.)] TJ ET BT 43.553 458.786 Td /F1 9.8 Tf [(Beal MF, Brouillet E, Jenkins BG et al. \(1993\) Neurochemical and histologic characterization of striatal excitotoxic lesions )] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(produced by the mitochondrial toxin 3-nitropropionic acid. J Neurosci 13:4181-4192)] TJ ET BT 26.250 427.476 Td /F1 9.8 Tf [(53.)] TJ ET BT 43.553 427.476 Td /F1 9.8 Tf [(Shin, J.Y., Fang, Z.H., Yu, Z.X., Wang, C.E., Li, S.H. and Li, X.J \(2005\) Expression of mutant huntingtin in glial cells )] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(contributes to neuronal excitotoxicity. J Cell Biol. 171:1001-1012)] TJ ET BT 26.250 396.167 Td /F1 9.8 Tf [(54.)] TJ ET BT 43.553 396.167 Td /F1 9.8 Tf [(Sapp, E., Kegel, K.B., Aronin, N., Hashikawa, T., Uchiyama, Y., Tohyama, K., Bhide, P.G., Vonsattel, J.P. and DiFiglia, M )] TJ ET BT 26.250 384.262 Td /F1 9.8 Tf [(\(2001\) Early and progressive accumulation of reactive microglia in the Huntington disease brain. J Neuropathol Exp Neurol. 60: )] TJ ET BT 26.250 372.357 Td /F1 9.8 Tf [(161-172)] TJ ET BT 26.250 352.953 Td /F1 9.8 Tf [(55.)] TJ ET BT 43.553 352.953 Td /F1 9.8 Tf [(Chou, S.Y., Weng, J.Y., Lai, H.L., Liao, F., Sun, S.H., Tu, P.H., Dickson, D.W. and Chern, Y \(2008\) Expanded-)] TJ ET BT 26.250 341.048 Td /F1 9.8 Tf [(polyglutamine huntingtin protein suppresses the secretion and production of a chemokine \(CCL5/RANTES\) by astrocytes. J )] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(Neurosci. 28:3277-90)] TJ ET BT 26.250 309.738 Td /F1 9.8 Tf [(56.)] TJ ET BT 43.553 309.738 Td /F1 9.8 Tf [(Bradford, J., Shin, J.Y., Roberts, M., Wang, C.E., Sheng, G., Li, S. and Li, X.J \(2010\) Mutant huntingtin in glial cells )] TJ ET BT 26.250 297.834 Td /F1 9.8 Tf [(exacerbates neurological symptoms of Huntington disease mice. J Biol Chem. 285:10653-61.)] TJ ET Q q 15.000 280.453 577.500 496.547 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Neurobiol 25:245-263)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(42.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Panchision DM, Martin-DeLeon PA, Takeshima T et al. \(1998\) An immortalized, type-1 astrocyte of mesencephalic origin )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(source of a dopaminergic neurotrophic factor. J Mol Neurosci 11:209-221)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(43.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(Reuss B, Unsicker K \(2000\) Survival and differentiation of dopaminergic mesencephalic neurons are promoted by )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(dopamine-mediated induction of FGF-2 in striatal astroglial cells. Mol Cell Neurosci 16:781-792)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(44.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Chen CM \(2011\) Mitochondrial dysfunction, metabolic deficits, and increased oxidative stress in Huntington's disease. )] TJ ET BT 26.250 673.548 Td /F1 9.8 Tf [(Chang Gung Med J 34:135-152)] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(45.)] TJ ET BT 43.553 654.143 Td /F1 9.8 Tf [(Browne SE, Bowling AC, MacGarvey U et al. \(1997\) Oxidative damage and metabolic dysfunction in Huntington's disease: )] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(selective vulnerability of the basal ganglia. Ann Neurol 41:646-653)] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(46.)] TJ ET BT 43.553 622.833 Td /F1 9.8 Tf [(Polidori MC, Mecocci P, Browne SE et al. \(1999\) Oxidative damage to mitochondrial DNA in Huntington's disease parietal )] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(cortex. Neurosci Lett 272:53-56)] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(47.)] TJ ET BT 43.553 591.524 Td /F1 9.8 Tf [(Li N, Liu B, Dluzen DE, Jin Y \(2007\) Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity in PC12 )] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(cells. J Ethnopharmacol 111:458-463)] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(48.)] TJ ET BT 43.553 560.214 Td /F1 9.8 Tf [(Ankarcrona M, Dypbukt JM, Bonfoco E et al. \(1995\) Glutamate-induced neuronal death: a succession of necrosis or )] TJ ET BT 26.250 548.310 Td /F1 9.8 Tf [(apoptosis depending on mitochondrial function. Neuron 15:961-973)] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(49.)] TJ ET BT 43.553 528.905 Td /F1 9.8 Tf [(Choi DW \(1987\) Ionic dependence of glutamate neurotoxicity. J Neurosci 7:369-379)] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(50.)] TJ ET BT 43.553 509.500 Td /F1 9.8 Tf [(Myers RH, Vonsattel JP, Stevens TJ et al. \(1988\) Clinical and neuropathologic assessment of severity in Huntington's )] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(disease. Neurology 38:341-347)] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(51.)] TJ ET BT 43.553 478.191 Td /F1 9.8 Tf [(Gil JM, Rego AC \(2008\) Mechanisms of neurodegeneration in Huntington's disease. Eur J Neurosci 27:2803-2820)] TJ ET BT 26.250 458.786 Td /F1 9.8 Tf [(52.)] TJ ET BT 43.553 458.786 Td /F1 9.8 Tf [(Beal MF, Brouillet E, Jenkins BG et al. \(1993\) Neurochemical and histologic characterization of striatal excitotoxic lesions )] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(produced by the mitochondrial toxin 3-nitropropionic acid. J Neurosci 13:4181-4192)] TJ ET BT 26.250 427.476 Td /F1 9.8 Tf [(53.)] TJ ET BT 43.553 427.476 Td /F1 9.8 Tf [(Shin, J.Y., Fang, Z.H., Yu, Z.X., Wang, C.E., Li, S.H. and Li, X.J \(2005\) Expression of mutant huntingtin in glial cells )] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(contributes to neuronal excitotoxicity. J Cell Biol. 171:1001-1012)] TJ ET BT 26.250 396.167 Td /F1 9.8 Tf [(54.)] TJ ET BT 43.553 396.167 Td /F1 9.8 Tf [(Sapp, E., Kegel, K.B., Aronin, N., Hashikawa, T., Uchiyama, Y., Tohyama, K., Bhide, P.G., Vonsattel, J.P. and DiFiglia, M )] TJ ET BT 26.250 384.262 Td /F1 9.8 Tf [(\(2001\) Early and progressive accumulation of reactive microglia in the Huntington disease brain. J Neuropathol Exp Neurol. 60: )] TJ ET BT 26.250 372.357 Td /F1 9.8 Tf [(161-172)] TJ ET BT 26.250 352.953 Td /F1 9.8 Tf [(55.)] TJ ET BT 43.553 352.953 Td /F1 9.8 Tf [(Chou, S.Y., Weng, J.Y., Lai, H.L., Liao, F., Sun, S.H., Tu, P.H., Dickson, D.W. and Chern, Y \(2008\) Expanded-)] TJ ET BT 26.250 341.048 Td /F1 9.8 Tf [(polyglutamine huntingtin protein suppresses the secretion and production of a chemokine \(CCL5/RANTES\) by astrocytes. J )] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(Neurosci. 28:3277-90)] TJ ET BT 26.250 309.738 Td /F1 9.8 Tf [(56.)] TJ ET BT 43.553 309.738 Td /F1 9.8 Tf [(Bradford, J., Shin, J.Y., Roberts, M., Wang, C.E., Sheng, G., Li, S. and Li, X.J \(2010\) Mutant huntingtin in glial cells )] TJ ET BT 26.250 297.834 Td /F1 9.8 Tf [(exacerbates neurological symptoms of Huntington disease mice. 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