Research ArticleHuntington’s Disease

PGC-1α Rescues Huntington’s Disease Proteotoxicity by Preventing Oxidative Stress and Promoting TFEB Function

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Science Translational Medicine  11 Jul 2012:
Vol. 4, Issue 142, pp. 142ra97
DOI: 10.1126/scitranslmed.3003799


Huntington’s disease (HD) is caused by CAG repeat expansions in the (huntingtin htt) gene, yielding proteins containing polyglutamine repeats that become misfolded and resist degradation. Previous studies demonstrated that mutant htt interferes with transcriptional programs coordinated by the peroxisome proliferator–activated receptor γ (PPARγ) coactivator 1α (PGC-1α), a regulator of mitochondrial biogenesis and oxidative stress. We tested whether restoration of PGC-1α could ameliorate the symptoms of HD in a mouse model. We found that PGC-1α induction virtually eliminated htt protein aggregation and ameliorated HD neurodegeneration in part by attenuating oxidative stress. PGC-1α promoted htt turnover and the elimination of protein aggregates by activating transcription factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. TFEB alone was capable of reducing htt aggregation and neurotoxicity, placing PGC-1α upstream of TFEB and identifying these two molecules as important therapeutic targets in HD and potentially other neurodegenerative disorders caused by protein misfolding.


Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder in which patients develop involuntary movements (“chorea”), suffer cognitive decline, and experience psychiatric illness (1). The disorder is relentlessly progressive, and patients succumb to their disease usually 10 to 30 years after onset. Neuropathology studies established that a region of the midbrain, known as the striatum, is principally involved in HD (2). In classic HD, significant cerebral cortex degeneration and atrophy also occur, whereas cerebellar, thalamic, and spinal cord neuron populations are spared. HD displays anticipation, which is defined as an earlier age of onset and more rapid disease progression in successive generations within an affected pedigree. The cause of the disease is expansion of a CAG trinucleotide repeat within the first exon of the huntingtin (htt) gene (3). The CAG repeat is translated into an expanded polyglutamine (polyQ) tract in the amino terminal region of the htt protein, and once the polyQ tract is expanded, it misfolds to adopt a pathogenic conformation. HD is one of nine inherited neurodegenerative disorders that are all caused by CAG repeats located within the coding regions of their genes (4). A considerable body of work has shown that polyQ disease proteins undergo a conformational change when the glutamine tract exceeds a certain threshold length, typically 35 repeats or greater (5). Misfolding of the polyQ disease protein is the crux of its molecular pathology because polyQ expansion tracts from the different disease proteins can all be detected by specific antibodies, such as 1C2 (6). Although polyQ disease proteins undergo structural transformations driven by a common mutational motif, each disorder is characterized by a distinct pattern of neuropathology affecting different neuronal populations. Because the different polyQ disease proteins exhibit widespread and overlapping patterns of expression, the mechanistic basis of this selective vulnerability remains unclear (4).

Before the discovery of the HD gene, several lines of evidence implicated mitochondrial dysfunction in this disorder (7), including studies of the mitochondrial toxin 3-nitropropionic acid in rodents (8). Since the characterization of the htt gene, numerous studies have extended these findings. Weight loss, despite increased caloric intake, has been documented in HD patients and mouse models (9, 10), suggestive of a negative energy balance. Bioenergetics studies of striatal neurons from late-stage HD patients revealed reduced activities for key components of the oxidative phosphorylation pathway, including complexes II, III, and IV of the electron transport chain (11). Analysis of adenine nucleotide ratios strongly supports these findings, as adenosine 5′-triphosphate (ATP) production is decreased as a function of CAG repeat length in human HD lymphoblastoid cell lines (12).

As data for mitochondrial dysfunction in HD have accumulated, investigators have sought a mechanistic basis for these findings. Studies of mitochondria isolated from HD patients and mice indicated that HD mitochondria depolarize at decreased calcium ion levels, and mutant htt protein may directly interact with mitochondria to yield this effect (1315). However, after the discovery of the HD gene, investigators soon realized that the entry of mutant htt protein to the nucleus is a crucial step in disease pathogenesis and assembled considerable evidence for transcription dysregulation (16). While evaluating the N171-82Q mouse model of HD (which contains 82 polyQ repeats in an N-terminal truncated version of the htt protein) (17) for metabolic abnormalities, we uncovered a phenotype of profound hypothermia and deranged body temperature regulation (18). This finding led us to consider a role for the transcription factor peroxisome proliferator–activated receptor γ (PPARγ) coactivator 1α (PGC-1α) in HD because PGC-1α is principally responsible for coordinating the adaptive thermogenesis response in rodents (19). Furthermore, PGC-1α stimulates the expression of genes required for mitochondrial energy production while concomitantly inducing genes dedicated to countering reactive oxygen species (ROS) generated as by-products of oxidative metabolism (20, 21). PGC-1α is thus the key regulatory node in a complex network of transcription programs that culminate in mitochondrial biogenesis and enhanced mitochondrial function, making it a strong candidate for involvement in HD. Indeed, earlier work had shown that PGC-1α knockout mice develop neurological abnormalities and show prominent neurodegeneration (22, 23). On the basis of in vitro and in vivo studies of PGC-1α function in HD mice and striatal RNA expression array data from HD patients, we and others demonstrated a central role for PGC-1α transcription interference in HD (18, 24). Together, these studies and subsequent work done by another group (25) support a model for HD pathogenesis in which htt interference with PGC-1α transcription coactivation is a major contributor to mitochondrial dysfunction (26).

To test the hypothesis that PGC-1α is a major factor in HD neurological dysfunction and neurodegeneration, we set out to determine whether genetic overexpression of PGC-1α could compensate for the documented interference with PGC-1α function in an HD mouse model. We established an inducible transgenic system for overexpressing PGC-1α and used this approach to create HD transgenic mice that express increased levels of PGC-1α. We report here that not only does PGC-1α ameliorate HD neurological phenotypes, but PGC-1α also virtually eradicates htt protein aggregates in the brains of HD mice. We demonstrate that PGC-1α’s ability to coactivate the expression of ROS defense genes, thus diminishing oxidative stress, promotes ubiquitin-proteasome system (UPS) function, and this contributes to htt protein aggregate reduction. Further investigation of PGC-1α’s ability to enhance proteostasis led us to identify transcription factor EB (TFEB), a master regulator of the autophagy-lysosome pathway (27), as a key target of PGC-1α. When we examined the mechanistic basis of htt protein aggregate reduction, we determined that TFEB alone is capable of preventing htt aggregation even without PGC-1α induction, placing TFEB downstream of PGC-1α in the prevention of htt aggregation and neurotoxicity.


Induction of PGC-1α expression rescues neurological phenotypes in HD transgenic mice

To determine whether increased expression of PGC-1α could ameliorate HD, we developed a system to induce PGC-1α expression in transgenic mice. Because overexpression of PGC-1α during development can produce disease phenotypes and result in lethality, we obtained a tet-responsive element (TRE)-PGC-1α transgenic line (28). We also obtained a line of mice genetically engineered to contain the reverse–tet transactivator (rtTA) cDNA downstream of the Rosa26 gene promoter, with a floxed STOP cassette placed between the promoter and the rtTA coding sequence (29). After crossing the Rosa26-floxed STOP-rtTA line with mice carrying male germ line–specific protamine-1–Cre recombinase (30), we derived F1 males with the STOP cassette excised in haploid germ cells (that is, Rosa26-rtTA). These males were crossed to TRE-PGC-1α females to generate Rosa26-rtTA–TRE-PGC-1α bigenic mice. When the Rosa26-rtTA–TRE-PGC-1α bigenic mice receive doxycycline, the rtTA becomes activated and should promote the expression of PGC-1α (fig. S1A). To validate our induction system, we derived Rosa26-rtTA–TRE-PGC-1α bigenic mice. These mice were fed doxycycline for 6 weeks beginning at weaning, and then we measured the expression levels of PGC-1α by reverse transcription polymerase chain reaction (RT-PCR). We observed marked induction of PGC-1α in the cortex and the striatum, but only a moderate increase of PGC-1α in muscle and brown adipose tissue (Fig. 1A).

Fig. 1

PGC-1α expression rescues HD neurological phenotypes. (A) PGC-1α mRNA expression upon doxycycline induction in 10-week-old bigenic Rosa26-rtTA–TRE-PGC-1α mice (n = 6 per group). Fold increase is normalized to endogenous PGC-1α in control mice (*P < 0.05; ***P < 0.001). Error bars, SEM. BAT, brown adipose tissue. (B) The ledge test, a direct measure of coordination, was performed on cohorts (n = 10 to 15 per group) of littermate controls (CTRL), HD N171-82Q mice (HD), and HD N171-82Q mice induced to express PGC-1α (HD + PGC-1α) at 14 and 18 weeks of age (*P < 0.05). Error bars, SD. (C) Grip strength analysis in HD transgenic mice (n = 13 to 15 per group). HD mice display reduced forepaw grip strength (force) at 13 weeks of age (#P < 0.05), worsening further at 18 weeks of age (##P < 0.01). Induction of PGC-1α yields improvement at 13 and 18 weeks of age (*P < 0.05). Each score is the mean of three tests per time point per mouse. Error bars, SEM. (D) Rotarod analysis on 13-week-old cohorts (n = 10 to 12 per group), including a cohort (n = 3) of HD + PGC-1α mice that did not receive doxycycline (no dox). The HD + PGC-1α group performed comparably to controls but significantly better than the HD and HD + PGC-1α (no dox) groups (P < 0.01). Error bars, SD. (E) Rotarod analysis on 18-week-old cohorts (n = 10 to 12 per group). The HD + PGC-1α group performed comparably to controls but significantly better than the HD and HD + PGC-1α (no dox) groups (P < 0.01). Error bars, SD.

The N171-82Q mouse model recapitulates phenotype abnormalities representative of human HD (17). To test whether restoration of PGC-1α function could ameliorate neurological disease in HD, we crossed N171-82Q mice with inducible PGC-1α bigenic mice, utilizing a breeding scheme that yielded three different cohorts: triple-transgenic mice, HD mice (no rtTA and PGC-1α transgenes), and non-HD controls (fig. S1B). We subjected the cohorts to behavioral testing and noted that expression of PGC-1α, at levels consistent with previous induction, significantly improved performance on the ledge test once HD mice became symptomatic (Fig. 1B). PGC-1α expression improved forepaw grip strength in the HD mice (Fig. 1C) and rectified foreleg stride length distances on gait analysis (control, 50.0 ± 3.3 mm; HD, 42.2 ± 0.5 mm; and HD + PGC-1α, 48.5 ± 2.1 mm). PGC-1α expression also enabled HD triple-transgenic mice to perform comparably to control mice on the rotarod test; HD triple-transgenic mice not treated with doxycycline did as poorly as the HD mice on the rotarod test (Fig. 1, D and E).

PGC-1α prevents huntingtin protein aggregation and rescues HD neurodegeneration

The formation of protein aggregates, which are visible at the light microscope level, is an established pathological hallmark of HD. Although initial studies suggested that htt aggregates are toxic, it is now generally accepted that misfolded htt protein conformers, either in monomeric form or as soluble oligomers or fibrils, are the toxic species (31, 32). Nonetheless, although aggregates are not the toxic species, their production requires high concentrations of misfolded htt; hence, their elimination correlates with marked reductions in pathogenic htt protein (33). When we examined the brains of 18-week-old HD mice induced to express PGC-1α, we observed a marked reduction in htt protein aggregation in the hippocampus and cortex (Fig. 2, A to F). Quantification of neurons containing htt protein aggregates in HD, triple-transgenic, and control mice confirmed this observation and demonstrated that induction of PGC-1α in triple-transgenic mice is required to achieve this outcome (Fig. 2G). We also noted a threefold reduction in htt protein aggregation in the striatum (P < 0.05), although there were fewer cells with aggregates (7.5 to 11.6%) in HD mice not induced to express PGC-1α. We then evaluated the brains of younger mice and determined that prominent htt protein aggregation is apparent in ~7% of hippocampal neurons at 10 weeks of age but was reduced fivefold in HD mice induced to express PGC-1α. Filter trap assay is a widely used method to measure the level of SDS-insoluble misfolded proteins (34), and a variety of antibodies are available to detect different species of amyloidogenic proteins, including 1C2 (for expanded polyQ tracts), A11 (for prefibrillar oligomers), and OC (for fibrils) (35). Using each of these antibodies, we performed filter trap assays on protein lysates isolated from the striatum of HD, triple-transgenic, and control mice and noted a reduction in the level of SDS-insoluble polyQ-expanded htt protein, oligomeric htt protein, and fibrillar htt protein in HD mice induced to express PGC-1α (Fig. 2H). We measured the levels of SDS-insoluble polyQ-expanded htt protein, oligomeric htt protein, and fibrillar htt protein and also quantified the levels of htt transgene mRNA and soluble htt protein (fig. S2). The observed reductions in the levels of different misfolded, insoluble htt amyloidogenic species could not be attributed to an effect of PGC-1α on HD transgene expression because real-time RT-PCR analysis revealed similar levels of htt transgene mRNA in HD transgenic mice expressing PGC-1α and in HD transgenic mice lacking both transgenes (fig. S2). As expected, reductions in soluble htt protein levels corresponded to reductions in insoluble htt protein conformers in HD transgenic mice expressing PGC-1α (fig. S2). To determine whether improved behavior and reduced htt aggregation in HD triple-transgenic mice were accompanied by an amelioration of neurodegeneration, we completed a stereological assessment of the striatum and found that induction of PGC-1α increased both striatal volume and neuron number (Fig. 2, I and J, and fig. S3). We also charted survival, comparing HD mice induced to express PGC-1α with their HD littermates, and did not detect any extension in overall life span (fig. S4A). Closer examination of the Kaplan-Meier plot, however, revealed that there was an extension in life span for Rosa26-rtTA–HD–TRE-PGC-1α transgenic mice when the first 50% of deaths are considered (fig. S4A). Analysis of 20-week-old mice induced to express PGC-1α indicated reduced RNA expression, such that the level of PGC-1α in brown adipose tissue was roughly equivalent to the PGC-1α level in control littermates at this age. Because HD N171-82Q mice may die because of impaired thermoregulation (18), we measured the body temperature of HD mice induced to express PGC-1α and noted that body temperature did not improve in these mice (fig. S4B). However, because central nervous system (CNS)–restricted rescue of HD neurodegeneration can extend life span (36), we considered other possible explanations and found that after 20 weeks of age, HD triple-transgenic mice also exhibited a modest reduction (~25%) in PGC-1α induction in the brain. We thus attribute the lack of life span extension in Rosa26-rtTA–HD–TRE-PGC-1α transgenic mice to inadequate induction of PGC-1α in brown adipose tissue combined with diminished induction of PGC-1α in the CNS.

Fig. 2

PGC-1α expression prevents htt aggregate formation and rescues HD neurodegeneration. (A to F) Sections from the frontal cortex (A to C) and hippocampus CA3 region (D to F) of 18-week-old HD mice (A and D), non-HD littermate controls (B and E), and HD mice induced to express PGC-1α (C and F). Anti-htt antibody EM48 (green); DAPI (4′,6′-diamidino-2-phenylindole) nuclear stain (blue). (G) Quantification of htt aggregate formation in 18-week-old HD mice (*P < 0.05). Error bars, SD. (H) Filter trap assays were performed using different antibodies that detect alternative misfolded species of htt protein. 1C2 reveals a reduction in SDS-insoluble htt protein for 18-week-old HD transgenic mice expressing PGC-1α (green), compared to HD mice lacking the PGC-1α transgene (red). A11 reveals a reduction in oligomeric htt protein for 18-week-old HD transgenic mice expressing PGC-1α (green), compared to HD mice (red). OC reveals a reduction in fibrillar htt protein for 18-week-old HD transgenic mice expressing PGC-1α (green), compared to HD mice (red). Non-HD littermate controls do not exhibit appreciable levels of SDS-insoluble, oligomeric, or fibrillar htt protein (blue). (I) Mean striatum volume in 18-week-old non-HD (CTRL), HD transgenic (HD), and HD mice induced to express PGC-1α (HD + PGC-1α) (*P < 0.05). Error bars, SEM. (J) Number of neurons in 18-week-old non-HD (CTRL), HD transgenic (HD), and HD mice induced to express PGC-1α (HD + PGC-1α) (*P < 0.05). Error bars, SEM.

PGC-1α boosts mitochondrial function and inhibits oxidative damage in HD mice

PGC-1α is a master regulator of mitochondrial biogenesis and respiration and acts as a transcriptional coactivator that transduces physiological stimuli into specific metabolic programs (21). We and others have documented marked reductions in mitochondrial function in HD (18, 24, 25). To determine whether PGC-1α overexpression could rescue this transcription interference, we assayed the expression levels of PGC-1α target genes in the striatum of 13-week-old HD mice, non-HD littermates, and HD mice induced to express PGC-1α. RT-PCR analysis indicated that PGC-1α induction increases the expression of mitochondrial genes required for increased biogenesis and enhanced energy production (Fig. 3A). To measure ATP content, we performed high-performance liquid chromatography analysis of adenine nucleotides from extracts of striatum and cortex dissected from 13-week-old mice. Although the ATP/adenosine 5′-diphosphate (ADP) ratio was only slightly reduced in HD mice, the ATP/ADP ratio was markedly increased in the striatum and cortex of triple-transgenic mice (Fig. 3B). We then measured mitochondrial complex I and complex II activities, which were both decreased in HD mice, and found that PGC-1α induction rescued these enzymatic activities (Fig. 3, C to D), confirming that impaired mitochondrial function can be rescued by increased PGC-1α activation. We also quantified mitochondrial DNA copy number and found that PGC-1α increased mitochondrial genome number in HD mice induced to express PGC-1α (fig. S5A). Mitochondrial DNA copy number was reduced in ST-Hdh striatal-like cells heterozygous or homozygous for the Q111 htt allele (fig. S5B), but transfection of a PGC-1α expression construct into ST-HdhQ111/Q111 striatal-like cells yielded a marked increase in mitochondrial DNA copy number in the transfected cells (fig. S5C).

Fig. 3

PGC-1α expression restores mitochondrial function in HD transgenic mice. (A) Real-time RT-PCR analysis indicated that striatal PGC-1α target gene mRNAs, obtained from sets (n = 4 to 6 per group) of 13-week-old HD mice, are significantly decreased in HD mice, but all five mitochondrial PGC-1α target gene mRNAs are markedly increased in HD mice expressing PGC-1α (**P < 0.01; *P < 0.05). ACADM, acyl-coenzyme A dehydrogenase; Cox6a1, cytochrome c oxidase subunit VIa polypeptide 1; CYCS, cytochrome c, somatic; NDUFS3, NADH (reduced form of NAD+) dehydrogenase (ubiquinone) Fe-S protein 3; and TFAM, transcription factor A, mitochondrial. Error bars, SEM. (B) ATP/ADP ratios are elevated upon PGC-1α induction in the striatum (**P < 0.01) and cortex (*P < 0.05). Error bars, SEM. (C) We measured mitochondrial complex I activity in the striatum of 13-week-old HD mice by measuring the reduction of 2,6-dichloroindophenol (DCIP) mediated by complex I and observed a significant rescue of complex I activity in HD mice expressing PGC-1α (**P < 0.01). Error bars, SEM. (D) Mitochondrial complex II activity is markedly improved in the striatum of 13-week-old HD transgenic mice expressing PGC-1α as measured by reduction of DCIP mediated by complex II (*P < 0.05). Error bars, SEM.

In parallel with its induction of genes that promote mitochondrial biogenesis and respiration, PGC-1α concomitantly drives the expression of genes whose products counter oxidative stress (20). Thus, PGC-1α–regulated induction of ROS defense genes prevents oxidative stress in the face of increased mitochondrial respiration. An important set of questions includes whether oxidative stress contributes to the demise of neurons in HD and whether interference with PGC-1α transcriptional activation of target genes promotes oxidative stress in HD. To address these questions, we pursued a series of assays designed to measure oxidative protein damage, lipid peroxidation, and oxidative DNA damage. We began by performing immunoblot analysis to measure carbonyl adduct formation in striatum and noted high levels of protein carbonyls in HD mice (Fig. 4A). However, protein carbonyl content was dramatically reduced in striatal samples from HD mice induced to express PGC-1α (Fig. 4A). We then assessed lipid peroxidation by immunoblot analysis of 4-hydroxynonenal adduct formation on proteins isolated from the striatum of HD, triple-transgenic, and control mice. Western blot analysis revealed strong immunoreactivity for 4-hydroxynonenal–containing proteins in HD striatal samples but yielded weaker signals for HD mice induced to express PGC-1α and for non–HD controls (Fig. 4B). Quantification of thiobarbituric acid–reactive species in striatal samples from HD, triple-transgenic, and control mice corroborated the lipid peroxidation data obtained in the 4-hydroxynonenal assay. We examined DNA oxidative damage in HD mice by measuring the levels of 8-hydroxy-2-deoxyguanosine (8-OH-dG) and noted much higher levels of 8-OH-dG in the striatum of HD mice, which were reduced to normal in HD mice overexpressing PGC-1α (Fig. 4C). To determine whether the amelioration of oxidative stress observed in doxycycline-treated Rosa26-rtTA–HD–TRE-PGC-1α transgenic mice stems from an increase in the expression of PGC-1α–regulated ROS defense genes, we performed RT-PCR analysis and noted increased levels of expression for three of four target genes (Fig. 4D). These findings suggest that the brains of HD mice exhibit markedly increased oxidative stress, whereas induction of PGC-1α in triple-transgenic mice yields levels of oxidative stress akin to those in normal control mice. This protection against oxidative stress correlates with recovery of PGC-1α–regulated expression of ROS defense genes.

Fig. 4

PGC-1α expression protects against HD oxidative damage by inducing ROS defense genes. (A) Immunoblot analysis for protein carbonyl content in the striatum of 13-week-old HD transgenic mice was performed by preparing dinitrophenylhydrazine (DNPH)-derivatized protein lysates and comparing with nonderivatized protein lysates in alternate lanes. We reprobed for β-actin to confirm equivalent protein loading. (B) Immunoblot analysis for lipid peroxidation via 4-hydroxynonenal adduct formation in the striatum of 13-week-old HD transgenic mice. We reprobed for β-actin to confirm equivalent protein loading. (C) We measured 8-OH-dG in the striatum of 13-week-old HD mice and observed a significant reduction in 8-OH-dG levels in HD mice induced to express PGC-1α (*P < 0.05). Error bars, SEM. (D) Real-time RT-PCR analysis of striatal RNAs obtained from sets (n = 4 to 6 per group) of 13-week-old HD mice. The expressions of four key ROS defense genes—superoxide dismutase 1 and 2 (SOD1 and SOD2), glutathione peroxidase (GPX), and catalase—subject to PGC-1α regulation are increased in HD mice induced to express PGC-1α (*P < 0.05). Error bars, SEM.

PGC-1α amelioration of htt protein aggregation correlates with reduced oxidative stress

Because our studies of HD mice revealed a role for PGC-1α in reducing htt aggregate formation, we investigated whether PGC-1α mediates this effect by countering oxidative stress. To test this hypothesis, we began by examining the levels of oxidative stress in ST-Hdh striatal-like cells derived from a knock-in HD mouse model that features expression of full-length htt protein (37). Quantification of oxidative stress indicated that ST-HdhQ111/Q111 striatal-like cells from mice homozygous for an expanded CAG111 knock-in htt allele have increased ROS levels compared to ST-HdhQ7/Q7 striatal-like cells from mice homozygous for a normal CAG7 htt allele (Fig. 5A). Coexpression of PGC-1α completely normalized ROS levels in ST-HdhQ111/Q111 striatal-like cells (Fig. 5A). To gauge the ability of oxidative stress to promote htt aggregate formation, we transfected Neuro2a cells with a green fluorescent protein (GFP)–tagged exon 1-htt-104Q expression construct, along with a red fluorescent protein (RFP)–PGC-1α or RFP-empty vector. After differentiating Neuro2a cells into neuron-like cells, we supplemented the media with increasing concentrations of hydrogen peroxide and noted a concentration-dependent production of protein aggregates in htt-104Q–expressing cells (Fig. 5B). Coexpression of PGC-1α, however, blocked htt aggregate formation (Fig. 5, B to C). To determine whether inhibition of aggregate formation reflected reduced oxidative stress, we established a third set of Neuro2a cells in which we added the ROS scavenger N-acetylcysteine and observed a reduction in htt aggregates akin to the reduction achieved with PGC-1α (Fig. 5C). We examined the effect of various conditions and treatments on htt aggregation in transfected Neuro2a cells by filter trap assay, and found that oxidative stress promoted production of SDS-insoluble htt and that PGC-1α and N-acetylcysteine reduced the level of SDS-insoluble protein (Fig. 5D). Indeed, when expression of PGC-1α was combined with N-acetylcysteine treatment, we observed a marked reduction in insoluble htt protein (Fig. 5D). To rule out an effect of oxidative stress on cytomegalovirus (CMV) promoter–driven expression of htt-104Q, we measured htt RNA levels by real-time RT-PCR analysis under different conditions and noted levels of expression consistent with transfection efficiency.

Fig. 5

PGC-1α expression counters htt protein aggregate formation induced by oxidative stress. (A) At increasing hydrogen peroxide concentrations, ST-HdhQ111/Q111 striatal-like cells exhibited greater ROS formation compared to ST-HdhQ7/Q7 cells; however, overexpression of PGC-1α in ST-HdhQ111/Q111 cells prevented ROS formation (*P < 0.05). RFU, relative fluorescence units. (B) Neuro2a cells were transfected with an htt-104Q–eGFP expression construct cotransfected with either an empty vector (RFP-empty) or a PGC-1α expression construct (RFP–PGC-1α). As the hydrogen peroxide concentration increased, greater numbers of cells contained punctate htt-104Q staining. Coexpression of PGC-1α markedly diminished the frequency of cells containing aggregated htt as shown by a decrease in the number of cells exhibiting green punctum formation. (C) Quantification of the effect of PGC-1α on htt protein aggregate formation in Neuro2a cells exposed to oxidative stress. There is a reduction in the percentage of Neuro2a cells with htt aggregates upon PGC-1α overexpression or when cultured in the presence of the ROS scavenger N-acetylcysteine (NAC) (*P < 0.05). Error bars, SD. (D) Filter trap assay of Neuro2a cells expressing polyQ-expanded htt protein under different treatment conditions. Oxidative stress promoted the formation of insoluble htt protein, whereas PGC-1α expression or NAC supplementation reduced insoluble htt protein. Combining PGC-1α and NAC together yielded the greatest reduction in insoluble htt protein. (E) We measured htt protein aggregate formation in Neuro2a cells expressing htt-Q82 in 1 mM hydrogen peroxide in the absence or presence of PGC-1α, lactacystin, and 3-methyladenine (3-MA). Proteasome inhibition or macroautophagy inhibition due to lactacystin or 3-MA, respectively, prevented PGC-1α from reducing htt protein aggregation (*P < 0.05). Error bars, SEM. (F) In the absence of oxidative stress, chymotrypsin-like activity, which mediates cleavage of glutamine-glutamine peptide bonds, is identical for untreated Neuro2a cells and PGC-1α–expressing or NAC-treated Neuro2a cells. Oxidative stress yields marked reductions in chymotrypsin-like activity for untreated cells (#P < 0.0001), but under such oxidative stress conditions, Neuro2a cells that express PGC-1α or are exposed to NAC retain much higher levels of chymotrypsin-like activity, measured as fluorescence light units (FLU) upon cleavage of a fluorescent peptide substrate (**P < 0.01). Error bars, SEM.

PGC-1α amelioration of huntingtin neurotoxicity is dependent on protein turnover pathways

Given the dramatic PGC-1α–mediated reduction of htt protein aggregation in Neuro2a cells, an important question is whether decreased htt aggregate formation has any effect on polyQ-htt neurotoxicity. This issue is crucial because aggregate formation can be protective if associated with a reduction in the concentration of toxic htt monomers and oligomers (38). To address this, we measured the level of apoptotic cell death in Neuro2a cells by immunostaining for activated caspase-3 because apoptotic cell death is an accepted measure of polyQ neurotoxicity (39). When we compared Neuro2a cells expressing GFP–htt-25Q or GFP–htt-104Q, we observed numerous htt-104Q–expressing cells that exhibited caspase-3 activation, whereas almost all htt-25Q–expressing cells were negative for activated caspase-3 (fig. S6A). We then counted htt-104Q–expressing cells that were positive for activated caspase-3 with increasing levels of oxidative stress when treated with N-acetylcysteine or induced to coexpress PGC-1α. We found that PGC-1α overexpression or N-acetylcysteine treatment prevented htt-104Q–dependent cell death, most significantly under maximal oxidative stress conditions (fig. S6B). Because previous work has drawn a distinction between the toxicity of diffuse htt protein versus aggregated htt protein (31), we quantified the extent of caspase-3 activation in htt-104Q–expressing Neuro2a cells that lacked visible aggregates and in htt-104Q–expressing Neuro2a cells that contained visible aggregates. When we considered htt-104Q cells lacking visible aggregates, we noted protection against cell death by both PGC-1α and N-acetylcysteine (fig. S6C). We also observed protection against cell death by PGC-1α and N-acetylcysteine in htt-104Q–expressing cells that contained visible aggregates (fig. S6D), suggesting that reduced oxidative stress has neuroprotective effects, regardless of whether diffuse or aggregated forms of htt predominate.

The two major pathways for protein turnover in the CNS are the UPS and the macroautophagy pathway (hereinafter referred to as autophagy). To delineate the pathway by which PGC-1α promotes htt protein turnover, we examined the role of the UPS and the autophagy in htt protein aggregation. To do this, we quantified htt protein aggregation in Neuro2a cells exposed to oxidative stress and treated PGC-1α–transfected cells with lactacystin to inhibit the UPS or with 3-methyladenine to inhibit autophagosome formation. Htt-104Q–expressing Neuro2a cells treated with lactacystin or 3-methyladenine no longer displayed a reduction in htt aggregate formation when cotransfected with PGC-1α, indicating that PGC-1α–mediated htt aggregate elimination requires the UPS and the autophagy pathway to be functional (Fig. 5E). Numerous studies of polyQ neurotoxicity have shown that modulating the UPS can reduce polyQ aggregate formation and toxicity (40, 41). To test if PGC-1α–mediated aggregate reduction might involve modulation of UPS degradation function, we assayed the chymotrypsin-like activity of the proteasome in Neuro2a cells under baseline and oxidative stress conditions. Upon exposure to high concentrations of hydrogen peroxide, chymotrypsin-like activity markedly decreased; however, when Neuro2a cells expressed PGC-1α or were exposed to N-acetylcysteine, higher levels of chymotrypsin-like activity were retained (Fig. 5F). These findings suggest that PGC-1α–mediated mitigation of oxidative stress may promote htt protein turnover and aggregate reduction by supporting enhanced UPS activity.

PGC-1α induction of TFEB prevents htt protein aggregation

In light of the importance of autophagy for PGC-1α–mediated turnover of htt protein aggregates, we considered possible mechanisms by which PGC-1α might be promoting autophagy pathway function. On the basis of recent work, TFEB has emerged as a master regulator of both the autophagy pathway and the lysosomal biogenesis (27), leading us to test if TFEB gene expression is regulated by PGC-1α. We began our studies by measuring TFEB RNA levels and noted a reduction in TFEB expression in HD mice (Fig. 6A). This reduction in TFEB gene expression was strongly rescued in HD transgenic mice expressing PGC-1α (Fig. 6A). We obtained corroborating results when we transfected Neuro2a cells with htt-exon 1 expression constructs of different glutamine lengths, noting again that PGC-1α cotransfection rescued endogenous TFEB expression in the presence of htt-104Q (Fig. 6B). To delineate the basis of PGC-1α regulation of TFEB, we analyzed the TFEB promoter region and found that the TFEB gene possesses at least three different transcription start sites, giving rise to three different isoforms, all with the same coding exons but different 5′ untranslated regions (UTRs) (Fig. 6C). Chromatin immunoprecipitation (ChIP) analysis revealed that PGC-1α primarily occupies the proximal promoter region for the TFEB isoform with the most 3′ transcription start site (Fig. 6D). On the basis of this finding, we generated a TFEB promoter-reporter construct linked to luciferase and then performed transactivation assays in Neuro2a cells. We found that transfection of PGC-1α into Neuro2a cells expressing this reporter construct yielded a robust induction of luciferase activity (Fig. 6E), confirming the use of this TFEB promoter-reporter construct for measuring PGC-1α–regulated transcription. We then performed TFEB promoter-reporter assays in ST-Hdh striatal-like cells and observed reductions in TFEB transactivation in Q111/Q111 cells, compared to Q7/Q7 and Q7/Q111 cells (Fig. 6F). Q111/Q111 cells transfected with PGC-1α, however, displayed a pronounced rescue of luciferase activity (Fig. 6F), confirming a key transcription regulatory role for PGC-1α. Further studies in Neuro2a cells reinforced the importance of PGC-1α for transactivation of TFEB expression because PGC-1α knockdown under baseline conditions or with sucrose treatment to induce lysosomal stress yielded reductions in TFEB expression (fig. S7).

Fig. 6

PolyQ-expanded htt interferes with PGC-1α transactivation of TFEB expression. (A) Real-time RT-PCR analysis of striatal RNAs, obtained from sets (n = 4 to 6 per group) of 13-week-old HD mice, reveals that TFEB expression is decreased in HD mice (*P < 0.05) but markedly increased in HD mice expressing PGC-1α (**P < 0.01). Error bars, SEM. (B) TFEB expression is decreased in htt-104Q–expressing Neuro2a cells (*P < 0.05); however, cotransfection with a PGC-1α expression construct strongly rescues polyQ-htt repression of TFEB expression (**P < 0.01). Error bars, SEM. (C) Diagram of the TFEB transcription start sites and promoter region. Boxes represent exons (with coding exons filled), solid lines correspond to 5′ and 3′ UTRs, and tented lines indicate introns. Positions of amplicons employed in the ChIP analysis in panel D are shown, as is the ~2-kb fragment (red solid line) that was cloned into a luciferase reporter vector to yield the TFEB promoter-reporter construct used in panels E and F. (D) Results of ChIP analysis for PGC-1α occupancy of the TFEB promoter. Isolated DNAs were subjected to quantitative PCR analysis for a series of amplicons in the TFEB promoter to exon 1 region (panel C). PGC-1α occupancy was greatest for amplicon ‘E’ (**P < 0.01). IgG, immunoglobulin G. Error bars, SEM. (E) PGC-1α transactivation of TFEB gene expression using a TFEB luciferase promoter-reporter construct containing ~2 kb of the isoform 1 proximal promoter (panel C). Luciferase activity is reported as relative luminescence units (RLU), with “no PGC-1α” arbitrarily set to 1. (F) Htt polyQ length–dependent repression of TFEB transactivation in ST-Hdh striatal-like cells. We performed transactivation assays with the TFEB promoter-reporter construct and noted repression of TFEB transactivation in Q111/Q111 cells (*P < 0.05). However, when we cotransfected Q111/Q111 cells with the PGC-1α expression construct, we observed marked rescue (*P < 0.05). Luciferase activity is reported as RLUs. Error bars, SEM.

Because PGC-1α can positively regulate the expression of TFEB, we wondered if TFEB target gene expression would be altered in HD mice subject to PGC-1α rescue. To evaluate this hypothesis, we selected a subset of TFEB target genes and measured their RNA expression levels in the striatum of HD mice. We found that four of these five TFEB target genes displayed reduced expression in HD mice, and all five TFEB target genes were induced, most markedly, in HD mice expressing PGC-1α (Fig. 7A). Western blot analysis of cathepsin D, another TFEB target, indicated that HD mice expressing PGC-1α show increased protein levels of this TFEB target (Fig. 7B). Because TFEB has been proposed to be a master regulator of the autophagy-lysosome pathway, we next tested if TFEB overexpression could reduce htt protein aggregate formation in Neuro2a cells subjected to increasing levels of oxidative stress. These studies revealed that TFEB overexpression is capable of dramatically reducing htt protein aggregation (Fig. 7C), akin to what we had observed with PGC-1α overexpression (Fig. 5C). This finding raised an important question: Does PGC-1α depend upon TFEB induction to prevent htt protein aggregation? To address this, we again measured htt aggregation in Neuro2a cells exposed to oxidative stress, but transfected the htt-104Q–expressing Neuro2a cells with a PGC-1α expression construct, alone or in combination with a TFEB short hairpin RNA (shRNA) knockdown construct (Fig. 7D). In a separate set of experiments, htt-104Q–expressing Neuro2a cells were transfected with a TFEB expression construct, alone or in combination with a PGC-1α shRNA construct (Fig. 7D). As expected, overexpression of PGC-1α or TFEB reduced htt protein aggregation; however, whereas TFEB overexpression reduced htt aggregates despite coexpression of a PGC-1α shRNA, PGC-1α overexpression could not significantly reduce htt protein aggregation when combined with TFEB knockdown (Fig. 7D). These findings place PGC-1α upstream of TFEB and autophagy-lysosome pathway activation.

Fig. 7

PGC-1α promotes TFEB target gene induction and TFEB-mediated htt aggregate reduction. (A) Real-time RT-PCR analysis of striatal RNAs, obtained from sets (n = 4 to 6 per group) of 13-week-old HD mice, reveals that the expression levels of four of five TFEB target genes are significantly decreased in HD mice, whereas expression of all five TFEB target genes is markedly increased in HD mice expressing PGC-1α (*P < 0.05; **P < 0.01). CTSF, cathepsin F; GLA, galactosidase α; MCOLN1, mucolipin-1; TPI, tripeptidyl peptidase I; HEXA, hexosaminidase A. Error bars, SEM. (B) Western blot analysis of cathepsin D, a TFEB target gene product, reveals increased expression of the fully processed ~32-kD mature form in the striatum of HD mice expressing PGC-1α. Blots were reprobed for β-actin to permit normalization of band intensities (right) on analysis by the NIH ImageJ program. (C) Neuro2a cells were transfected with an htt-104Q–eGFP expression construct and cotransfected with either an empty vector or a TFEB expression construct. As the hydrogen peroxide (H2O2) concentration increased, we detected greater numbers of cells containing punctate htt-104Q staining. Coexpression of TFEB markedly diminished the percentage of cells containing aggregated htt (*P < 0.05; **P < 0.01). Error bars, SEM. (D) Neuro2a cells were cultured with 0.1 mM hydrogen peroxide, transfected with a htt-104Q–eGFP expression construct, and cotransfected with a PGC-1α expression construct (PGC-1α o.e.), a PGC-1α shRNA knockdown construct (PGC-1α k.d.), a TFEB expression construct (TFEB o.e.), or a TFEB shRNA knockdown construct (TFEB k.d.), as indicated. There was a reduction in htt aggregates upon PGC-1α overexpression, TFEB overexpression, and TFEB overexpression despite simultaneous PGC-1α knockdown (*P < 0.05). However, PGC-1α overexpression in the presence of TFEB knockdown did not reduce htt aggregate formation. NAC is a positive control for aggregate reduction (*P < 0.05). Error bars, SEM.


The recognition of protein misfolding as a shared feature of many neurodegenerative diseases, inherited or sporadic, represented a fundamental advance in our understanding of these disorders. HD is but one member of a large class of neurodegenerative proteinopathies that include Alzheimer’s disease, Parkinson’s disease (PD), prion diseases, amyotrophic lateral sclerosis, and tauopathies (42). These disorders are all characterized by an age-dependent process of neuronal dysfunction and demise, presumably initiated by the cell’s inability to deal with cellular stress induced by a specific misfolded protein. Why are neurons and other highly specialized CNS cell types exquisitely susceptible to degeneration in these different diseases? An answer to this question may lie in the fact that neurons and other specialized CNS cells are unique because such cells (i) are postmitotic, (ii) constantly demand high levels of energy, and (iii) need to maintain protein quality control throughout a bipolar elongated cell body. For these reasons, any process that disrupts mitochondrial function, either at the level of bioenergetics capacity or organelle/protein quality control, tends to preferentially compromise neurological function, resulting in neurodegeneration (43, 44).

HD is a neurodegenerative disorder characterized by selective vulnerability of the striatum and cortical projection neurons. An extensive literature has established that impaired energy metabolism and mitochondrial dysfunction are prominent features of HD pathogenesis (18, 24, 25, 45). Here, we evaluated the contribution of impaired PGC-1α function to HD pathogenesis by attempting a genetic rescue using an inducible bigenic PGC-1α expression system. After validating the system, we performed behavioral and neuropathological analyses of HD mice induced to express PGC-1α. Our results indicate that PGC-1α up-regulation can restore normal motor and coordination function and prevent neuron loss in the striatum. Analysis of PGC-1α–regulated genes and energy production revealed a recovery of target gene expression, mitochondrial complex I and II activities, and ATP generation. These compelling findings strongly support a role for PGC-1α transcription interference in HD and confirm that PGC-1α deserves to be considered as a therapeutic target in HD. Because PGC-1α boosted mitochondrial function and reduced oxidative stress, our results suggest that PGC-1α may also have therapeutic application in related neurodegenerative proteinopathies, such as PD. This view is supported by a study in which a meta-GSEA (gene set enrichment analysis) analysis of 17 microarray data sets from PD brains identified coordinate down-regulation of 425 PGC-1α target genes as a significantly shared feature in PD and showed that PGC-1α overexpression could mitigate α-synuclein and rotenone toxicity in neurons (46). Moreover, another group has documented diminished PGC-1α function in PD and linked the PGC-1α dysfunction to altered degradation of a novel parkin substrate known as PARIS (47).

Although we anticipated that PGC-1α expression might reverse HD neurological phenotypes and neurodegeneration, our analysis of HD neuropathology yielded an unexpected result that PGC-1α virtually eliminated htt protein aggregates. We hypothesized that one possible explanation for this effect is PGC-1α’s ability to reduce oxidative stress. Human HD brains exhibit signs of oxidative damage consistent with high levels of oxidative stress (48). To determine whether such oxidative damage is recapitulated in the striatum of HD N171-82Q mice, we measured protein carbonyl adduct formation, lipid peroxidation, and oxidative DNA damage and observed strong evidence of oxidative damage. In the striatum of HD mice induced to express PGC-1α, markers of oxidative damage were not increased but instead were similar to levels detected in non–HD controls. Real-time RT-PCR analysis indicated that the expression of ROS defense genes, subject to PGC-1α induction, was increased. We also measured ROS levels in ST-Hdh striatal-like cells from HD knock-in mice and observed a roughly threefold increase in ROS levels in ST-HdhQ111/Q111 cells. Transfection of a PGC-1α expression construct into ST-HdhQ111/Q111 cells normalized ROS levels, confirming that PGC-1α is capable of countering oxidative stress associated with htt neurotoxicity. Although reduced oxidative stress is likely to be neuroprotective, we tested if a connection exists between oxidative stress and htt protein aggregation. We observed a marked increase in htt-104Q aggregate formation in differentiated Neuro2a cells under conditions of increasing oxidative stress and noted that coexpression of PGC-1α prevented htt protein aggregation. When we tested if the ROS scavenger N-acetylcysteine could block oxidative stress-dependent htt-104Q aggregate formation, we observed strong inhibition of htt aggregation. Filter trap analysis corroborated these results and supports the conclusion that htt protein aggregation is favored by oxidative stress conditions. Because a preclinical trial of N-acetylcysteine in mice overexpressing mutant α-synuclein reduced α-synuclein accumulation and prevented loss of dopaminergic nerve terminals and striatal neurodegeneration (49), attenuation of oxidative stress may have the potential to ameliorate the symptoms of certain neurodegenerative proteinopathies.

One of the major challenges for postmitotic neurons is to maintain protein quality control. To eliminate misfolded proteins, neurons rely on properly functioning UPS and autophagy-lysosome pathway. If either of these protein turnover pathways is incapacitated, then neuronal dysfunction and neurodegeneration will result (50). When we evaluated the role of the UPS and the autophagy pathway in PGC-1α–dependent amelioration of htt protein aggregation, we found that fully functional UPS and autophagy pathway are necessary. Because oxidative stress can inhibit UPS function by inactivation of proteasome subunits through direct oxidative modification (5153), or impair the UPS by presenting it with an excessive load of oxidatively damaged substrates (54), we hypothesized that PGC-1α–mediated mitigation of oxidative stress might enhance UPS function, thereby contributing to htt protein turnover and aggregate reduction. When we directly studied the effect of oxidative stress on one key measure of UPS enzymatic activity, we noted that oxidative stress dramatically inhibited this proteasomal activity, but expression of PGC-1α or treatment with N-acetylcysteine provided a significant rescue of the enzymatic activity in Neuro2a cells.

Although our findings implicate reduced oxidative stress as a factor in the elimination of htt protein aggregates by PGC-1α, it seemed unlikely that this alone could account for PGC-1α’s profound antiaggregation effect. Because a normally functioning autophagy pathway is critical for maintaining protein quality control in the CNS (50), we hypothesized that a potential link might exist between PGC-1α transactivation and enhanced autophagy-lysosome pathway activity. In 2009, a transcription factor known as TFEB was identified as a key regulator of lysosome biogenesis (55). Further studies then demonstrated that TFEB promotes the expression of genes in the autophagy pathway in addition to genes encoding components of the lysosome (27), indicating that TFEB is a major node in the regulation of the entire autophagy-lysosome pathway. To evaluate the role of TFEB in PGC-1α–mediated htt protein turnover, we measured TFEB expression levels in HD in vitro and in vivo models and found that polyQ-expanded htt repressed TFEB gene expression, an effect that was rescued in both cases by PGC-1α. ChIP analysis of TFEB promoter regions associated with different transcription start sites localized PGC-1α occupancy to a proximal region of one of these promoters, enabling us to derive a TFEB promoter-reporter construct. When we studied TFEB transactivation in ST-Hdh striatal-like cells with this promoter-reporter construct, we observed repression of TFEB promoter activity in Q111/Q111 homozygous cells and again documented that PGC-1α expression could dramatically rescue this repression. We then tested if TFEB expression could prevent htt protein aggregation in Neuro2a cells expressing htt-104Q protein under conditions of oxidative stress and observed marked TFEB-dependent reductions in htt aggregate formation, consistent with earlier work indicating that TFEB promoted clearance of polyQ-expanded htt (55). To clarify the pathway relationship between PGC-1α and TFEB in the suppression of htt protein aggregation, we tested if PGC-1α required TFEB to limit htt aggregate formation or if TFEB required PGC-1α to limit htt aggregate formation. We found that TFEB is capable of reducing htt aggregation when PGC-1α is knocked down, but that PGC-1α in the presence of TFEB knockdown no longer reduced htt aggregation. Our findings indicate that PGC-1α promotes htt protein turnover and aggregate suppression by coactivating the expression of TFEB and places PGC-1α upstream of TFEB in the transcriptional regulation of the autophagy-lysosome pathway.

Why should PGC-1α coactivation be linked to enhanced autophagy pathway function? Considering that PGC-1α promotes mitochondrial biogenesis and increased mitochondrial metabolic activity, the up-regulation of mitochondrial number and mass required to achieve higher energy production likely results in a proportionately greater accumulation of damaged mitochondria that need to be turned over. Consequently, enhanced autophagy-lysosome pathway function would be required to accommodate this increased need for mitochondrial turnover via autophagy, a process known as mitophagy (56). Although the transcription factors that PGC-1α coactivates to promote autophagy-lysosome pathway function remain unknown, the PPARs are likely candidates, as they are potent inducers of mitochondrial biogenesis and mitochondrial activity in a wide range of cell types (57). Because neurons are continually on the edge for energy production and protein quality control, our findings highlight the importance of PGC-1α function and TFEB action in neurodegenerative disease and establish PGC-1α and TFEB as attractive therapeutic targets for developing new therapies to treat HD and certain other neurodegenerative disorders characterized by protein misfolding.

Materials and Methods

Mouse breeding and behavioral studies

All animal experiments adhered to National Institute of Health (NIH) guidelines and were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC) and the University of California, San Diego, IACUC. Rosa26-floxed STOP-rtTA mice and protamine-1–Cre transgenic mice were obtained from the Jackson Laboratory. The TRE-PGC-1α mice were obtained from the Kelly Lab (28). All transgenic and gene-targeted lines were crossed onto the C57BL/6J strain background for more than 10 generations before directed breeding experiments. Behavioral studies and survival analysis were performed as previously described (18, 58, 59).

Neuropathological analysis

Mice were euthanized and perfused as previously described (58, 59), and frozen parasagittal or coronal sections were cut at 10-μm thickness on a sliding microtome. After permeabilization with 0.05% Triton X/phosphate-buffered saline (PBS) for 10 min, slides were blocked with 5% normal goat serum (Vector Laboratories) and 1% fetal bovine serum (Sigma) for 1 hour and then incubated with primary antibody EM48 (1:50; Millipore) for 1 hour, washed with PBS three times, and incubated with secondary antibody for 1 hour. Nuclear staining was achieved with Hoechst 33342 (Molecular Probes). Confocal imaging analysis was performed on a Zeiss Axiovert 200 M inverted microscope (Carl Zeiss Inc.). Quantification of aggregation was performed by counting the number of cells in the field with punctate EM48 staining divided by the total number of cells in the field. For each brain region, we analyzed five sections per individual and studied at least six mice per cohort. For stereological analysis, we used the Cavalieri method (60). Briefly, the region of interest in each section was selected with a 4× objective on an Olympus BX55 microscope (Olympus). Each region was then divided into randomly selected squares by Stereo Investigator software (MBF Bioscience). An average of 25 squares per area in each section was used to count the total number of nucleolus-containing neurons with a 100× oil objective. The total number of neurons was calculated according to the optical fractionator (61). The volume of the striatum was determined from serial section analysis with point counting and Cavalieri’s rule. Images from sections were captured at ×12.5 magnification and projected onto a video monitor. Point counting was performed as above. Volume was computed separately for the right and left sides and corrected for shrinkage. For striatal volume, sections were stained with cresyl violet as previously described, and neurons were differentiated from glial cells by size (62). In all cases, the scorer was blinded to the cohort status of the mice.

Vector constructs

The htt expression constructs have been previously described (18). For expression of PGC-1α, we cloned a mouse PGC-1α cDNA (gift of D. E. Kelly) into the multiple cloning site of the pTAG-RFP vector (Evrogen). We then inserted the CMV promoter with a Kozak sequence between the stop codon of RFP and the PGC-1α cDNA. We confirmed RFP and PGC-1α expression by Western blot analysis. The TFEB expression construct was obtained from Origene. To derive the TFEB promoter-report construct, we PCR-amplified a 2-kb proximal promoter fragment from mouse BAC RP23-205M10 DNA (BACPAC Resources Center) and inserted this fragment into the Nhe I and Hind III restriction sites in the pGL3-Basic vector (Promega). For PGC-1α and TFEB knockdown, we screened a series of PGC-1α and TFEB shRNAs (MISSION shRNA, Sigma) and noted superior knockdown with PGC-1α shRNA TRCN0000095313 and TFEB shRNA TRCN0000085548, which were used for all subsequent experimentation.

Statistical analysis

All data were prepared for analysis with standard spreadsheet software (Microsoft Excel). Statistical analysis was done with Microsoft Excel, Prism 4.0 (GraphPad), or the VassarStats Web site ( Analysis of variance was performed for all experiments unless otherwise noted, and if statistical significance (P < 0.05) was achieved, we performed post hoc analysis to account for multiple comparisons. The level of significance (α) was set at 0.05.

Supplementary Materials

Materials and Methods

Fig. S1. Induction of PGC-1α expression in transgenic mice.

Fig. S2. Effect of PGC-1α overexpression on htt RNA and protein expression.

Fig. S3. Representative striatal sections for stereological analysis.

Fig. S4. Effect of PGC-1α overexpression on HD mouse survival and thermoregulation.

Fig. S5. Mitochondrial DNA copy number is reduced in HD mice and striatal-like cells.

Fig. S6. Effect of PGC-1α overexpression on cell death in htt-82Q–expressing cells.

Fig. S7. PGC-1α knockdown represses TFEB gene expression.

References and Notes

  1. Acknowledgments: We thank G. MacDonald, A. C. Smith, K. Saijo, C. K. Glass, and B. L. Sopher for technical assistance; D. E. Kelly for the PGC-1α expression construct and the TRE-PGC-1α transgenic mice; and C. G. Glabe for the A11 and OC antibodies. Funding: This work was supported by funding from the Hereditary Disease Foundation and the Cure Huntington’s Disease Initiative and grants from the NIH (R01 AG033082 and R01 NS065874 to A.R.L.S., P01 HL034322 to E.R.L., and R01 AG018440, R01 NS057096, and R01 AG022074 to E.M.). Author contributions: A.R.L.S. designed experiments, provided funding, and wrote the paper. T.T. designed and performed all the experiments and wrote the paper. T.D.A., K.R.S., and J.A. performed mouse behavioral studies; B.E.M. assisted with TFEB expression regulation assays; and R.A.V.R. assisted with the neuropathological analysis. V.A.D. assisted with mouse experiment performance and design. E.R.L. and E.M. performed experiments, analyzed the results, and contributed data. Competing interests: The authors declare that they have no competing interests.
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