Title: Rosiglitazone Treatment Prevents Mitochondrial Dysfunction in Mutant Huntingtin-expressing Cells
Abstract: Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the PPAR family of transcription factors. Synthetic PPARγ agonists are used as oral anti-hyperglycemic drugs for the treatment of non-insulin-dependent diabetes. However, emerging evidence indicates that PPARγ activators can also prevent or attenuate neurodegeneration. Given these previous findings, the focus of this report is on the potential neuroprotective role of PPARγ activation in preventing the loss of mitochondrial function in Huntington disease (HD). For these studies we used striatal cells that express wild-type (STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin protein at physiological levels. Treatment of mutant cells with thapsigargin resulted in a significant decrease in mitochondrial calcium uptake, an increase in reactive oxygen species production, and a significant decrease in mitochondrial membrane potential. PPARγ activation by rosiglitazone prevented the mitochondrial dysfunction and oxidative stress that occurred when mutant striatal cells were challenged with pathological increases in calcium. The beneficial effects of rosiglitazone were likely mediated by activation of PPARγ, as all protective effects were prevented by the PPARγ antagonist GW9662. Additionally, the PPARγ signaling pathway was significantly impaired in the mutant striatal cells with decreases in PPARγ expression and reduced PPARγ transcriptional activity. Treatment with rosiglitazone increased mitochondrial mass levels, suggesting a role for the PPARγ pathway in mitochondrial function in striatal cells. Altogether, this evidence indicates that PPARγ activation by rosiglitazone attenuates mitochondrial dysfunction in mutant huntingtin-expressing striatal cells, and this could be an important therapeutic avenue to ameliorate the mitochondrial dysfunction that occurs in HD. Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the PPAR family of transcription factors. Synthetic PPARγ agonists are used as oral anti-hyperglycemic drugs for the treatment of non-insulin-dependent diabetes. However, emerging evidence indicates that PPARγ activators can also prevent or attenuate neurodegeneration. Given these previous findings, the focus of this report is on the potential neuroprotective role of PPARγ activation in preventing the loss of mitochondrial function in Huntington disease (HD). For these studies we used striatal cells that express wild-type (STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin protein at physiological levels. Treatment of mutant cells with thapsigargin resulted in a significant decrease in mitochondrial calcium uptake, an increase in reactive oxygen species production, and a significant decrease in mitochondrial membrane potential. PPARγ activation by rosiglitazone prevented the mitochondrial dysfunction and oxidative stress that occurred when mutant striatal cells were challenged with pathological increases in calcium. The beneficial effects of rosiglitazone were likely mediated by activation of PPARγ, as all protective effects were prevented by the PPARγ antagonist GW9662. Additionally, the PPARγ signaling pathway was significantly impaired in the mutant striatal cells with decreases in PPARγ expression and reduced PPARγ transcriptional activity. Treatment with rosiglitazone increased mitochondrial mass levels, suggesting a role for the PPARγ pathway in mitochondrial function in striatal cells. Altogether, this evidence indicates that PPARγ activation by rosiglitazone attenuates mitochondrial dysfunction in mutant huntingtin-expressing striatal cells, and this could be an important therapeutic avenue to ameliorate the mitochondrial dysfunction that occurs in HD. Huntington disease (HD) 2The abbreviations used are: HDHuntington diseasePPARperoxisome proliferator-activated receptorTZDsthiazolidinedionesKRHKrebs-Ringer-HepesTMRMtetramethyl rhodamine methyl esterAββ-amyloidROSreactive oxygen speciesPTPpermeability transition poreANOVAanalysis of variance2,7-DCF5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl esterPBSphosphate-buffered salineDNdominant negative. 2The abbreviations used are: HDHuntington diseasePPARperoxisome proliferator-activated receptorTZDsthiazolidinedionesKRHKrebs-Ringer-HepesTMRMtetramethyl rhodamine methyl esterAββ-amyloidROSreactive oxygen speciesPTPpermeability transition poreANOVAanalysis of variance2,7-DCF5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl esterPBSphosphate-buffered salineDNdominant negative. is a neurodegenerative disease that is inherited in an autosomal dominant manner, and is caused by the pathological elongation of the CAG repeats in exon one of the huntingtin gene (1The Huntington Disease Col Res Group Cell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (6890) Google Scholar). The pathogenesis of HD is manifested by dysfunction and severe loss of striatal neurons in the initial stages, and subsequently involves the cortex and other brain regions in the later stages of the disease (2Brandt J. Bylsma F.W. Gross R. Stine O.C. Ranen N. Ross C.A. Neurology. 1996; 46: 527-531Crossref PubMed Scopus (139) Google Scholar). Transcriptional deregulation (3Cui L. Jeong H. Borovecki F. Parkhurst C.N. Tanese N. Krainc D. Cell. 2006; 127: 59-69Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar) and proteasome dysfunction (4Diaz-Hernandez M. Hernandez F. Martin-Aparicio E. Gomez-Ramos P. Moran M.A. Castano J.G. Ferrer I. Avila J. Lucas J.J. J. Neurosci. 2003; 23: 11653-11661Crossref PubMed Google Scholar) have been suggested to be significant contributors to the pathogenic processes in HD. Additionally, calcium homeostasis deregulation (5Panov A.V. Gutekunst C.A. Leavitt B.R. Hayden M.R. Burke J.R. Strittmatter W.J. Greenamyre J.T. Nat. Neurosci. 2002; 5: 731-736Crossref PubMed Scopus (841) Google Scholar) and mitochondrial dysfunction (5Panov A.V. Gutekunst C.A. Leavitt B.R. Hayden M.R. Burke J.R. Strittmatter W.J. Greenamyre J.T. Nat. Neurosci. 2002; 5: 731-736Crossref PubMed Scopus (841) Google Scholar, 6Milakovic T. Johnson G.V. J. Biol. Chem. 2005; 280: 30773-30782Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) also have been strongly implicated in the pathogenesis of HD. Huntington disease peroxisome proliferator-activated receptor thiazolidinediones Krebs-Ringer-Hepes tetramethyl rhodamine methyl ester β-amyloid reactive oxygen species permeability transition pore analysis of variance 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester phosphate-buffered saline dominant negative. Huntington disease peroxisome proliferator-activated receptor thiazolidinediones Krebs-Ringer-Hepes tetramethyl rhodamine methyl ester β-amyloid reactive oxygen species permeability transition pore analysis of variance 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester phosphate-buffered saline dominant negative. Abnormalities in mitochondrial function have been observed in postmortem HD brains (7Stahl W.L. Swanson P.D. Neurology. 1974; 24: 813-819Crossref PubMed Google Scholar, 8Mann V.M. Cooper J.M. Javoy-Agid F. Agid Y. Jenner P. Schapira A.H. Lancet. 1990; 336: 749Abstract PubMed Scopus (114) Google Scholar, 9Browne S.E. Bowling A.C. MacGarvey U. Baik M.J. Berger S.C. Muqit M.M. Bird E.D. Beal M.F. Ann. Neurol. 1997; 41: 646-653Crossref PubMed Scopus (732) Google Scholar). More recent findings have provided compelling evidence that mitochondrial dysfunction is central to the pathogenesis of HD. Lymphoblasts derived from HD patients exhibit alterations in mitochondrial membrane potential in response to mitochondrial toxins and lower calcium loads in comparison to control lymphoblasts (5Panov A.V. Gutekunst C.A. Leavitt B.R. Hayden M.R. Burke J.R. Strittmatter W.J. Greenamyre J.T. Nat. Neurosci. 2002; 5: 731-736Crossref PubMed Scopus (841) Google Scholar, 10Sawa A. Wiegand G.W. Cooper J. Margolis R.L. Sharp A.H. Lawler Jr., J.F. Greenamyre J.T. Snyder S.H. Ross C.A. Nat. Med. 1999; 5: 1194-1198Crossref PubMed Scopus (312) Google Scholar). In addition, mitochondrial respiration and ATP production are significantly impaired in striatal cells expressing mutant huntingtin (6Milakovic T. Johnson G.V. J. Biol. Chem. 2005; 280: 30773-30782Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family of ligand-activated transcription factors (11Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1063) Google Scholar). To date, three mammalian PPAR subtypes have been isolated and termed PPARα, PPARβ, and PPARγ. PPARα is highly expressed in several tissues and PPARβ is an APC-regulated target of non-steroidal anti-inflammatory drugs (12He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar). PPARγ is a ligand-activated nuclear receptor implicated in several significant human pathologies, including cancer, atherosclerosis, and inflammation (13Berger J. Moller D.E. Annu. Rev. Med. 2002; 53: 409-435Crossref PubMed Scopus (2041) Google Scholar). PPARγ is the target of the insulin-sensitizing thiazolidinediones (TZDs) drugs, used to treat type II diabetes. Recent studies suggest that treatment of insulin resistance with a PPARγ agonist retards the development of Alzheimer disease (AD) (14Watson G.S. Craft S. 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Des. 2006; 12: 93-109Crossref PubMed Scopus (168) Google Scholar, 19Heneka M.T. Landreth G.E. Biochem. Biophys. Acta. 2007; 1771: 1031-1045Crossref PubMed Scopus (258) Google Scholar). However, activation of PPARγ by three different TZDs protected rat hippocampal neurons against β-amyloid (Aβ)-induced damage (20Inestrosa N.C. Godoy J.A. Quintanilla R.A. Koenig C.S. Bronfman M. Exp. Cell Res. 2005; 304: 91-104Crossref PubMed Scopus (171) Google Scholar), and the TZD rosiglitazone protects human neuroblastoma SH-SY5Y cells against acetaldehyde-induced cytotoxicity (21Jung T.W. Lee J.Y. Shim W.S. Kang E.S. Kim S.K. Ahn C.W. Lee H.C. Cha B.S. Biochem. Biophys. Res. Commun. 2006; 340: 221-227Crossref PubMed Scopus (38) Google Scholar). In addition, PPARγ activation by rosiglitazone up-regulates the Bcl-2 protective pathway and prevents neuronal degeneration induced by both oxidative stress and treatment with Aβ fibrils, with a concomitant increase in mitochondrial viability (22Fuenzalida K. Quintanilla R.A. Ramos P. Piderit D. Fuentealba R.A. Martinez G. Inestrosa N.C. Bronfman M. J. Biol. Chem. 2007; 282: 37006-37015Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Recent studies have also provided evidence that the expression of PGC-1α, a potent co-activator of PPARγ, is repressed by mutant huntingtin expression, and when PGC-1α knock-out (KO) mice are crossed with HD knockin mice, this resulted in increased neurodegeneration of striatal neurons and motor abnormalities in the HD mice (2Brandt J. Bylsma F.W. Gross R. Stine O.C. Ranen N. Ross C.A. Neurology. 1996; 46: 527-531Crossref PubMed Scopus (139) Google Scholar). At the same time, there is evidence suggesting that PPARγ agonists are neuroprotective and increase mitochondrial function (23Schutz B. Reimann J. Dumitrescu-Ozimek L. Kappes-Horn K. Landreth G.E. Schurmann B. Zimmer A. Heneka M.T. J. Neurosci. 2005; 25: 7805-7812Crossref PubMed Scopus (187) Google Scholar, 24Hunter R.L. Dragicevic N. Seifert K. Choi D.Y. Liu M. Kim H.C. Cass W.A. Sullivan P.G. Bing G. J. Neurochem. 2007; 100: 1375-1386Crossref PubMed Scopus (270) Google Scholar). It was also demonstrated that oral treatment with rosiglitazone induced mitochondrial biogenesis in mouse brain (25Strum J.C. Shehee R. Virley D. Richardson J. Mattie M. Selley P. Ghosh S. Nock C. Saunders A. Roses A. J. Alzheimers Dis. 2007; 11: 45-51Crossref PubMed Scopus (158) Google Scholar). Therefore, in this study, we explored the possibility of using PPARγ activation to ameliorate mutant huntingtin-induced mitochondrial dysfunction (5Panov A.V. Gutekunst C.A. Leavitt B.R. Hayden M.R. Burke J.R. Strittmatter W.J. Greenamyre J.T. Nat. Neurosci. 2002; 5: 731-736Crossref PubMed Scopus (841) Google Scholar, 6Milakovic T. Johnson G.V. J. Biol. Chem. 2005; 280: 30773-30782Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 26Oliveira J.M. Chen S. Almeida S. Riley R. Goncalves J. Oliveira C.R. Hayden M.R. Nicholls D.G. Ellerby L.M. Rego A.C. J. Neurosci. 2006; 26: 11174-11186Crossref PubMed Scopus (115) Google Scholar). Our results indicate that there are significant defects in the PPARγ signaling pathway in mutant huntingtin-expressing cells in comparison with cells that express wild-type huntingtin protein. In addition, pretreatment of mutant huntingtin-expressing cells with the PPARγ agonist rosiglitazone prevented the loss of mitochondrial potential, mitochondrial calcium deregulation, and oxidative stress overproduction in response to intracellular calcium overload. These findings suggest that activation of the PPARγ signaling pathway could ameliorate the mitochondrial function deficits that occur in HD. Reagents—Chemicals, culture media, and serum were obtained from Sigma-Aldrich, Roche Applied Sciences, Alexis Biochemical, and Invitrogen. Fluo-3 AM, Rhod-2 AM, 4-BrA23187, Calsein AM, Mitotracker Green™ (MitoGreen), tetramethyl rhodamine methyl ester (TMRM), Mitotracker Red® CM-H2XRos (MitoRed), 4′,6-diamidino-2-phenylindole (DAPI), and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (2,7-DCF) were obtained from Molecular Probes (Eugene, OR). Cell Culture—In this study, conditionally immortalized striatal progenitor cell lines STHdhQ7/Q7 (referred to in the text as wild-type cells) expressing endogenous wild-type huntingtin, and STHdhQ111/Q111 (referred to in the text as mutant cells) expressing comparable levels of mutant huntingtin with 111 glutamines were used (27Trettel F. Rigamonti D. Hilditch-Maguire P. Wheeler V.C. Sharp A.H. Persichetti F. Cattaneo E. MacDonald M.E. Hum. Mol. Genet. 2000; 9: 2799-2809Crossref PubMed Scopus (487) Google Scholar). These cell lines were a generous gift from Dr. M. E. MacDonald and were prepared from wild-type mice and homozygous HdhQ111/Q111 knockin mice as described previously (27Trettel F. Rigamonti D. Hilditch-Maguire P. Wheeler V.C. Sharp A.H. Persichetti F. Cattaneo E. MacDonald M.E. Hum. Mol. Genet. 2000; 9: 2799-2809Crossref PubMed Scopus (487) Google Scholar). Culturing conditions were the same as described in our previous studies (28Milakovic T. Quintanilla R.A. Johnson G.V. J. Biol. Chem. 2006; 281: 34785-34795Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Intracellular ROS Measurements—Clonal striatal cells were grow on polylysine-coated coverslips (30,000 cells/coverslip) and treated with 1 μm thapsigargin, 20 μm rosiglitazone, and/or 40 μm GW9662, as indicated. After treatment, the cells were incubated with the fluorescent probe 2,7-DCF at 10 μm for 30 min in Krebs-Ringer-Hepes (KRH) buffer supplemented with 5 mm glucose (29Ruan Q. Quintanilla R.A. Johnson G.V. J. Neurochem. 2007; 102: 25-36Crossref PubMed Scopus (19) Google Scholar). The coverslips were washed two times with PBS and fixed with 4% p-formaldehyde for 5 min. Cells were photographed using a Nikon fluorescence microscope integrated with a Spot digital camera (Diagnostic Instruments). All photographs were taken using the same exposure time and gain to minimize the photobleaching of 2,7-DCF. Images were quantified using Image-Pro Plus 6 software. Results in intensity units were expressed as average of fluorescence signal (F) minus background fluorescence (F0) in every image (29Ruan Q. Quintanilla R.A. Johnson G.V. J. Neurochem. 2007; 102: 25-36Crossref PubMed Scopus (19) Google Scholar, 30Santos M.J. Quintanilla R.A. Toro A. Grandy R. Dinamarca M.C. Godoy J.A. Inestrosa N.C. J. Biol. Chem. 2005; 280: 41057-41068Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Mitochondrial Potential Determination in Live Cells—Mitochondria membrane potential was determined using Mitotracker® Red CM-H2XRos (MitoRed) or TMRM (22Fuenzalida K. Quintanilla R.A. Ramos P. Piderit D. Fuentealba R.A. Martinez G. Inestrosa N.C. Bronfman M. J. Biol. Chem. 2007; 282: 37006-37015Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 28Milakovic T. Quintanilla R.A. Johnson G.V. J. Biol. Chem. 2006; 281: 34785-34795Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 30Santos M.J. Quintanilla R.A. Toro A. Grandy R. Dinamarca M.C. Godoy J.A. Inestrosa N.C. J. Biol. Chem. 2005; 280: 41057-41068Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Striatal cells were grown on poly-l-lysine-coated plates and cultured for 4 days. The cells were then loaded for 30 min with MitoRed or TMRM (100 nm) in KRH buffer supplemented with 5 mm glucose and containing 0.02% pluronic acid, then washed, and allowed to equilibrate for 20 min. Cells cultured in 35-mm dishes were then mounted on the stage of a confocal laser scanning microscope (Leica SP2, Germany), and the fluorescence changes were determined using a 40× water immersion objective. MitoRed and TMRM fluorescence were detected exciting with a 563-nm He-Ne laser very heavily attenuated (30% laser power), and the emission was collected at >570 nm for every dye per separate measure. Signal from control cells and cells treated with different stimuli were compared using identical settings for laser power, and detector sensitivity for each separate experiment. The images were analyzed with LCS Leica confocal software and recorded as mean MitoRed or TMRM fluorescence signal per live cell. Estimation of fluorescence intensities were presented as the pseudoratio (ΔF/Fo), which was calculated using the following formula: ΔF/Fo = (F - Fbase)/(Fbase - B), where F is the measured fluorescence intensity of the indicator, Fbase is the fluorescence intensity before the stimulation, and B is the background signal determined from the average of areas adjacent to the cells (22Fuenzalida K. Quintanilla R.A. Ramos P. Piderit D. Fuentealba R.A. Martinez G. Inestrosa N.C. Bronfman M. J. Biol. Chem. 2007; 282: 37006-37015Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 28Milakovic T. Quintanilla R.A. Johnson G.V. J. Biol. Chem. 2006; 281: 34785-34795Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 30Santos M.J. Quintanilla R.A. Toro A. Grandy R. Dinamarca M.C. Godoy J.A. Inestrosa N.C. J. Biol. Chem. 2005; 280: 41057-41068Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Cytosolic and Mitochondrial Calcium Measurements—Cells grown on poly-l-lysine-coated 35-mm dishes were loaded for 30 min (37 °C) with 5 μm Fluo-3 AM, and 10 μm Rhod-2 AM in KRH-glucose containing 0.02% pluronic acid. The fluorescence changes determined by Fluo-3 represent the cytoplasmic calcium changes (30Santos M.J. Quintanilla R.A. Toro A. Grandy R. Dinamarca M.C. Godoy J.A. Inestrosa N.C. J. Biol. Chem. 2005; 280: 41057-41068Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 31Quintanilla R.A. Munoz F.J. Metcalfe M.J. Hitschfeld M. Olivares G. Godoy J.A. Inestrosa N.C. J. Biol. Chem. 2005; 280: 11615-11625Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), and Rhod-2 fluorescence indicate calcium changes in the mitochondria (32Zhu L.P. Yu X.D. Ling S. Brown R.A. Kuo T.H. Cell Calcium. 2000; 28: 107-117Crossref PubMed Scopus (82) Google Scholar, 33Collins T.J. Lipp P. Berridge M.J. Bootman M.D. J. Biol. Chem. 2001; 276: 26411-26420Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 34Darios F. Muriel M.P. Khondiker M.E. Brice A. Ruberg A. J. Neurosci. 2005; 25: 4159-4168Crossref PubMed Scopus (43) Google Scholar). To estimate Rhod-2 fluorescence pattern in live mitochondria, we used Mitotracker Green™ dye (MitoGreen) (33Collins T.J. Lipp P. Berridge M.J. Bootman M.D. J. Biol. Chem. 2001; 276: 26411-26420Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). MitoGreen accumulates in the lipophilic environment of live mitochondria, and the signal is independent of the mitochondrial potential (33Collins T.J. Lipp P. Berridge M.J. Bootman M.D. J. Biol. Chem. 2001; 276: 26411-26420Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 35Mironov S.L. Ivannikov M.V. Johansson M. J. Biol. Chem. 2005; 280: 715-721Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Cells were washed three times and left in KRH-glucose for 10 min until cell fluorescence had equilibrated. Fluorescence was imaged with a confocal laser scanning microscope (Leica TCS SP2) using a 40× water immersion lens, as described previously (28Milakovic T. Quintanilla R.A. Johnson G.V. J. Biol. Chem. 2006; 281: 34785-34795Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Images were acquired using a 488-nm Argon laser to excite Fluo-3 fluorescence and a 563 nm He-Ne laser to excite Rhod-2 fluorescence. The signals were collected at 505-530 nm (Fluo-3) and at 590 nm (Rhod-2). The fluorescence background signal was subtracted from cell fluorescence measurements in every experiment. The fluorescence intensity variation was recorded from 10-20 cells on average per experiment. Estimation of fluorescence intensities of Fluo-3 and Rhod-2 were presented as a pseudoratio (ΔF/Fo), as described previously (30Santos M.J. Quintanilla R.A. Toro A. Grandy R. Dinamarca M.C. Godoy J.A. Inestrosa N.C. J. Biol. Chem. 2005; 280: 41057-41068Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 31Quintanilla R.A. Munoz F.J. Metcalfe M.J. Hitschfeld M. Olivares G. Godoy J.A. Inestrosa N.C. J. Biol. Chem. 2005; 280: 11615-11625Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Immunofluorescence Staining—Striatal cells plated on polylysine-coated coverslips (25,000 cells/coverslip) were double immunostained using the rabbit polyclonal anti-PPARγ antibody (Cell Signaling, Boston, MA) (1:500), and a mouse monoclonal anti-actin antibody (Sigma). The secondary antibodies used were 488 Alexa anti-mouse (Molecular Probes), for detection of actin and 593 Alexa anti-rabbit (Molecular Probes) for PPARγ. Coverslips were mounted and analyzed using a Zeiss fluorescence microscope (Carl Zeiss, Thornwood, NY) integrated with an Axiocam CCD camera (Carl Zeiss). Reverse Transcription and Real-time PCR—Total RNA was extracted from wild-type or mutant striatal cells using TRIzol (Invitrogen) as described in the manufacturer's protocol. Extracted total RNA was treated with RNase free-DNase I, Amplification Grade, (Invitrogen) to remove contaminating DNA, heat-treated to inactivate DNase I, and precipitated with ethanol to clean up the reaction. 2 μg of total RNA was subjected to reverse transcription using SuperScriptIII reverse transcriptase (Invitrogen) following the manufacturer's protocol. The real-time PCR reaction was performed in triplicate in a real-time PCR system (Bio-Rad) using SYBR GreenER qPCR SuperMix (Invitrogen) in a final volume of 25 μl. Amplification conditions consisted of an initial hot start at 95 °C for 10 min followed by amplification of 45 cycles (95 °C for 15 s, 60 °C for 20 s, and 72 °C for 40 s). Melting curve analysis was performed immediately after amplification from 55 to 95 °C. The threshold cycle (CT) of PPARγ was normalized to the CT value of TBP, and the relative amounts of mRNA are shown. Western Blotting—Cells were washed with ice-cold PBS and lysed in a modified radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.4% SDS, 0.2% sodium deoxycholate, 5% glycerol, 1 mm EDTA, 20 mm NaF, 2 mm Na3VO4) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml pepstatin). The lysates were sonicated, cleared from cellular debris by centrifugation, and assayed to determine protein concentration using the BCA assay (Pierce). Proteins (10-100 μg) were separated by 10% SDS-PAGE and transferred to the nitrocellulose membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) and incubated with the primary rabbit antibody against PPARγ (1:500 dilution, Cell Signaling) in TBST containing 2% bovine serum albumin at 4 °C overnight. After washing three times, horseradish peroxidase-conjugated secondary antibody against rabbit (1:3000 dilution) in TBST containing 5% skim milk was added, followed by incubation, rinsing, and detection of the immunoreactive bands, by chemiluminescence. Luciferase Assays—4 × 104 wild-type striatal cells or 8 × 104 mutant striatal cells were plated into each well of a 24-well plate. The following day, a reporter plasmid ((PPRE) X3-TK-Luc) (Addgene, Cambridge, MA)) (36Kim J.B. Wright H.M. Wright M. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4333-4337Crossref PubMed Scopus (540) Google Scholar) was transiently cotransfected with the other constructs as indicated (such as mPPARγ1 and/or dominant negative (DN)-mPPARγ1 (22Fuenzalida K. Quintanilla R.A. Ramos P. Piderit D. Fuentealba R.A. Martinez G. Inestrosa N.C. Bronfman M. J. Biol. Chem. 2007; 282: 37006-37015Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar)), using Lipofectamine 2000 (Invitrogen). phRL-TK (Promega, Madison, WI) was cotransfected to normalize the transfection efficiency for all experiments. 12-16 h after transfection, cells were given fresh medium and treated as indicated. 24 h after treatment, cells were washed with cold PBS, lysed with Passive lysis buffer (Promega), and collected. Cell lysates were put through one cycle of freeze-thaw and centrifuged. Luciferase activity was then measured using the Dual-Luciferase Reporter Assay System (Promega). Statistical Analysis—Results were expressed as mean ± S.E., and were analyzed using Student's t test, an unpaired Student's t test, or one-way ANOVA followed by Student-Newman-Keuls multiple comparisons test as indicated. Differences were considered significant when p < 0.05. Mutant Huntingtin Expression Significantly Affects the PPARγ Signaling Pathway in Striatal Cells—We previously have shown that PPARγ activation protects against Aβ and oxidative stress damage in hippocampal neurons (20Inestrosa N.C. Godoy J.A. Quintanilla R.A. Koenig C.S. Bronfman M. Exp. Cell Res. 2005; 304: 91-104Crossref PubMed Scopus (171) Google Scholar, 22Fuenzalida K. Quintanilla R.A. Ramos P. Piderit D. Fuentealba R.A. Martinez G. Inestrosa N.C. Bronfman M. J. Biol. Chem. 2007; 282: 37006-37015Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Additionally, one of the most interesting things that we observed was that the PPARγ-induced protection correlated with improved mitochondrial function (22Fuenzalida K. Quintanilla R.A. Ramos P. Piderit D. Fuentealba R.A. Martinez G. Inestrosa N.C. Bronfman M. J. Biol. Chem. 2007; 282: 37006-37015Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). PPARγ activation has been proposed to increase mitochondrial biogenesis in vivo (25Strum J.C. Shehee R. Virley D. Richardson J. Mattie M. Selley P. Ghosh S. Nock C. Saunders A. Roses A. J. Alzheimers Dis. 2007; 11: 45-51Crossref PubMed Scopus (158) Google Scholar), and treatment with PPARγ agonists has been proven successful in ameliorating neurodegenerative damage in ischemia and ALS (16Kiaei M. Kipiani K. Chen J. Calingasan N.Y. Beal M.F. Exp. Neurol. 2005; 191: 331-336Crossref PubMed Scopus (188) Google Scholar, 37Collino M. Aragno M. Mastrocola R. Gallicchio M. Rosa A.C. Dianzani C. Danni O. Thiemermann C. Fantozi R. 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