Research ArticleHuntington’s Disease

PPARδ activation by bexarotene promotes neuroprotection by restoring bioenergetic and quality control homeostasis

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Science Translational Medicine  06 Dec 2017:
Vol. 9, Issue 419, eaal2332
DOI: 10.1126/scitranslmed.aal2332

Defeating neurotoxicity with a repurposed drug

PPARδ is a permissive nuclear receptor that heterodimerizes with the retinoid X receptor (RXR) to activate target genes. Interference with transcription of PPARδ target genes contributes to neurodegeneration in Huntington’s disease (HD). In new work, Dickey et al. evaluated the RXR agonist bexarotene in cellular models of HD and in an HD mouse model. They determined that bexarotene was effective at countering HD neurotoxicity in mouse primary neurons, human HD patient stem cell–derived neurons, and the BAC-HD mouse model. The authors then examined the basis for PPARδ’s neuroprotective effect and found that treatment with RXR/PPARδ agonists enhanced oxidative metabolism, promoted mitochondrial quality control, and boosted protein homeostasis by activating autophagy.

Abstract

Neurons must maintain protein and mitochondrial quality control for optimal function, an energetically expensive process. The peroxisome proliferator–activated receptors (PPARs) are ligand-activated transcription factors that promote mitochondrial biogenesis and oxidative metabolism. We recently determined that transcriptional dysregulation of PPARδ contributes to Huntington’s disease (HD), a progressive neurodegenerative disorder resulting from a CAG-polyglutamine repeat expansion in the huntingtin gene. We documented that the PPARδ agonist KD3010 is an effective therapy for HD in a mouse model. PPARδ forms a heterodimer with the retinoid X receptor (RXR), and RXR agonists are capable of promoting PPARδ activation. One compound with potent RXR agonist activity is the U.S. Food and Drug Administration–approved drug bexarotene. We tested the therapeutic potential of bexarotene in HD and found that bexarotene was neuroprotective in cellular models of HD, including medium spiny-like neurons generated from induced pluripotent stem cells (iPSCs) derived from patients with HD. To evaluate bexarotene as a treatment for HD, we treated the N171-82Q mouse model with the drug and found that bexarotene improved motor function, reduced neurodegeneration, and increased survival. To determine the basis for PPARδ neuroprotection, we evaluated metabolic function and noted markedly impaired oxidative metabolism in HD neurons, which was rescued by bexarotene or KD3010. We examined mitochondrial and protein quality control in cellular models of HD and observed that treatment with a PPARδ agonist promoted cellular quality control. By boosting cellular activities that are dysfunctional in HD, PPARδ activation may have therapeutic applications in HD and potentially other neurodegenerative diseases.

INTRODUCTION

Huntington’s disease (HD) is a progressive autosomal dominant neurodegenerative disorder in which patients develop motor and cognitive impairment (1). HD pathology is defined by degeneration and death of medium spiny neurons of the striatum, as well as cortical pyramidal neurons that project to the striatum (2, 3). In 1993, a CAG trinucleotide repeat expansion mutation in the coding region of the huntingtin (htt) gene was identified as the cause of HD (4). As observed in other polyglutamine (polyQ) repeat diseases, htt glutamine tracts that exceed a certain length threshold (~37 repeats in HD) adopt a new pathogenic confirmation and are resistant to the normal processes of protein turnover, leading to cellular toxicity and neurodegeneration (5). The length of the mutant htt polyQ expansion inversely correlates with the age of disease onset and rate of disease progression in HD patients.

Neurons in the brain require continued production of high-energy compounds by mitochondria. We and others have linked mitochondrial dysfunction in HD to transcriptional dysregulation of peroxisome proliferator–activated receptor (PPAR) gamma coactivator-1 alpha (PGC-1α), a coactivator that coordinates transcriptional programs that culminate in mitochondrial biogenesis and enhanced oxidative metabolism (68). The importance of PGC-1α for HD pathogenesis is underscored by the observation that PGC-1α overexpression is sufficient to rescue motor dysfunction, prevent accumulation of misfolded htt protein, and stave off neurodegeneration in HD mice (9). To determine the mechanistic basis for PGC-1α transcription interference in HD, we performed an unbiased screen that showed PPARs to be htt interactors and documented an interaction between PPARδ and htt in non-neuronal cells, striatal-like neurons, and the cerebral cortex of HD mice (10). We noted that PPARδ is highly expressed in neurons of the central nervous system (CNS) and demonstrated that expression of dominant-negative PPARδ in CNS is sufficient to produce motor phenotypes, neurodegeneration, mitochondrial defects, and transcriptional abnormalities that closely parallel HD disease phenotypes (10). We then evaluated a selective, potent PPARδ agonist, KD3010, and after confirming that it crosses the blood-brain barrier to up-regulate expression of PPARδ target genes in the cortex and striatum, we tested KD3010 in N171-82Q transgenic mice, a rodent model of HD. This study established the efficacy of KD3010 PPARδ agonist therapy as a potential therapeutic approach for HD (10).

One facet of PPARδ biology with relevance to therapy development is that PPARδ forms a heterodimer with retinoid X receptor (RXR), and the resulting “permissive” PPARδ-RXR heterodimer is subject to dual ligand regulation, meaning that RXR agonists can promote PPARδ activation (11). One drug compound with potent RXR agonist activity is bexarotene, a synthetic product structurally similar to retinoic acid compounds, known endogenous RXR ligands. Bexarotene (Targretin) is U.S. Food and Drug Administration–approved for use in patients with T cell cutaneous lymphoma. One provocative study reported that bexarotene administration to a mouse model of Alzheimer’s disease (AD) yielded a marked rescue of cognitive, social, and olfactory deficits, accompanied by improved neural circuit function and enhanced clearance of soluble Aβ oligomers (12). The mechanistic basis for this effect was proposed to involve increased PPARγ activation (13). Because PPARδ is highly expressed in CNS neurons, more so than PPARγ (14), the mechanistic basis for the therapeutic action of bexarotene in AD deserves reconsideration, in light of our discovery of a role for PPARδ in maintaining normal nervous system function (10) and recent work demonstrating the neuroprotective effect of PPARδ agonist treatment in AD mice (15).

Here, we considered the neurotherapeutic potential of bexarotene in HD and found that bexarotene was neuroprotective in multiple cellular models of HD, ranging from mouse striatal and cortical neurons to medium spiny neurons generated from induced pluripotent stem cells (iPSCs) derived from patients with HD. We then treated the N171-82Q HD mouse model with bexarotene and observed improved neuron survival and motor function. To determine the molecular basis for PPARδ agonist therapy, we evaluated metabolic dysfunction in HD and documented markedly impaired oxidative metabolism in HD neurons, which was rescued by bexarotene or KD3010. We examined mitochondrial and protein quality control in cellular models of HD and observed that PPARδ agonist therapy achieved neuroprotection by promoting quality control function.

RESULTS

Bexarotene promotes PPARδ activation resulting in neuroprotection

To evaluate the effect of bexarotene on PPARδ activation, we cotransfected PPARδ and a 3x–PPAR response element luciferase reporter construct into primary cortical neurons derived from a bacterial artificial chromosome transgenic HD (BAC-HD) mouse model containing the full-length htt gene with 97 glutamine repeats or into primary cortical neurons derived from littermate control [wild-type (WT)] mice. We noted marked induction of PPARδ activation upon bexarotene treatment (Fig. 1A). Bexarotene promotion of PPARδ activation was comparable to PPARδ activation using a PPARδ-selective agonist GW501516 (Fig. 1A). We also observed a marked increase in PPARδ activation when we boosted RXR expression (fig. S1A), corroborating the permissive nature of PPARδ activation upon heterodimerization with RXR (11). To determine whether bexarotene-induced PPARδ activation countered mutant htt neurotoxicity, we assessed the effect of bexarotene treatment on mitochondrial dysfunction and cell death in BAC-HD neurons. When we measured mitochondrial membrane potential using two different fluorescent probes, we noted significantly reduced mitochondrial membrane potential in BAC-HD neurons, which was rescued upon bexarotene treatment (Fig. 1B and fig. S1B). Similarly, bexarotene treatment yielded a significant reduction in the frequency of BAC-HD neurons that displayed caspase-3 activation, an indicator of impending cellular demise (Fig. 1C). Previous studies of bexarotene neuroprotection in an AD mouse model have proposed that bexarotene neuroprotection relies upon activation of PPARγ (12). To determine the basis for bexarotene neuroprotection in HD neurons, we repeated bexarotene treatment of BAC-HD primary neurons along with concurrent knockdown of either PPARα, PPARδ, or PPARγ, which was accomplished by coexpression of short hairpin RNA (shRNA) vectors directed against each of the different PPARs (fig. S1C). When we measured mitochondrial membrane potential in BAC-HD neurons treated with bexarotene and subjected to knockdown of either PPARα, PPARδ, or PPARγ, we found that bexarotene rescue of mitochondrial membrane potential occurred despite PPARα or PPARγ knockdown but was prevented by concurrent knockdown of PPARδ (Fig. 1D). Bexarotene neuroprotection was not limited to BAC-HD primary cortical neurons because bexarotene treatment also improved mitochondrial function and cell survival of WT neurons; enhanced WT neuron function upon exposure to bexarotene also depended upon PPARδ (fig. S1, D and E). We then assayed bexarotene rescue of BAC-HD neuronal cell death and noted that PPARδ knockdown eliminated any benefit from bexarotene treatment, whereas PPARα or PPARγ knockdown did not significantly affect bexarotene neuroprotection (Fig. 1E). Finally, to determine whether bexarotene treatment of BAC-HD primary neurons promoted increased PPARδ activation, we measured the RNA expression of PPARδ targets previously shown to respond to PPARδ agonist treatment pharmacodynamically in mice (10). We documented bexarotene-induced expression of these PPARδ target genes (Fig. 1F). We noted that combined bexarotene + GW501516 treatment often yielded even greater increases in PPARδ target gene expression (Fig. 1F).

Fig. 1. Bexarotene promotes PPARδ activation to ameliorate the neurotoxicity of mutant huntingtin.

(A) We measured 3x–PPAR response element luciferase reporter activity in primary cortical neurons from WT control mice or bacterial artificial chromosome transgenic Huntington’s disease (BAC-HD) mice cotransfected with Renilla luciferase vector and treated with bexarotene (500 nM), GW501516 (100 nM), or vehicle. Peroxisome proliferator–activated receptor δ (PPARδ) activation in BAC-HD mouse neurons was repressed at baseline compared to wild-type (WT) mouse neurons. **P < 0.01, Student’s t test. Bexarotene and GW501516 treatment promoted PPARδ activation in BAC-HD mouse neurons. **P < 0.01, analysis of variance (ANOVA) with post-hoc Tukey test. n = 3 biological replicates; n = 3 technical replicates. Results were normalized to WT mouse neurons at baseline. (B) Mitochondrial membrane potential of primary cortical neurons from WT and BAC-HD mice, treated with vehicle or bexarotene (500 nM), was determined from the ratio of mitochondrial to cytosolic JC-1 fluorescence. *P < 0.05, Student’s t test. n = 3 biological replicates; n = 3 technical replicates. Results were normalized to WT mouse neurons at baseline. Similar results were obtained using tetramethylrhodamine methyl ester (TMRM) as the fluorescent probe (fig. S1B). (C) We quantified active caspase-3 immunostaining of primary cortical neurons from WT and BAC-HD mice, treated with vehicle or bexarotene (500 nM) for 24 hours and H2O2 (25 μM) for 4 hours. *P < 0.05, **P < 0.01, Student’s t test. n = 3 biological replicates; 30 to 50 cells were counted per experiment. (D) Mitochondrial membrane potential was measured in BAC-HD mouse primary cortical neurons, transfected with the indicated short hairpin RNA (shRNA) expression vector (control = scrambled shRNA), and treated with vehicle or bexarotene (500 nM). Mitochondrial membrane potential was determined from the ratio of mitochondrial to cytosolic JC-1 fluorescence. *P < 0.05, ANOVA with post-hoc Tukey test. n = 3 biological replicates; n = 3 technical replicates. Results were normalized to WT mouse neurons at baseline as in (B). (E) We quantified active caspase-3 immunostaining of BAC-HD mouse primary cortical neurons, transfected with the indicated shRNA expression vectors, and treated with vehicle or bexarotene (500 nM) for 24 hours and H2O2 (25 μM) for 4 hours. *P < 0.05, ANOVA with post-hoc Tukey test. n = 3 biological replicates; 30 to 50 cells were counted per experiment. (F) We performed reverse transcription polymerase chain reaction (RT-PCR) analysis of RNA expression of the PPARδ target genes pyruvate dehydrogenase kinase isoform 4 (PDK4), angiopoietin-like 4 (Angptl4), and uncoupling protein 2 (UCP2) in BAC-HD mouse primary cortical neurons, treated as indicated. **P < 0.01, ANOVA with post-hoc Tukey test. n = 6 independent experiments. Error bars represent SEM.

To further evaluate bexarotene neuroprotection in HD, we pursued experiments in different models of mutant htt neurotoxicity that aim to recapitulate HD neuropathology. First, we transfected cocultured mouse cortical and striatal neurons with N-terminal htt containing 90 amino acids with either an 8-glutamine repeat (Nt-90-8Q) or a 73-glutamine repeat expansion (Nt-90-73Q) and treated cocultured cortical and striatal neurons with increasing bexarotene concentrations. We observed a significant increase (P < 0.05 or P < 0.01) in neuron survival in a dose-dependent manner (Fig. 2, A and B). We then tested the effect of bexarotene in primary cortical neurons transfected with N-terminal htt containing 586 amino acids with an 82-glutamine repeat expansion (Nt-586-82Q) and noted a dose-dependent reduction (P < 0.01 or P < 0.001) in neuronal cell death (Fig. 2C). We also differentiated human HD iPSCs into striatal medium spiny-like neurons and transferred them to brain-derived neurotrophic factor (BDNF)–free neural induction medium because withdrawal of neurotrophic factor support promoted neuronal cell death. To evaluate rescue, we supplemented the media with either bexarotene or BDNF, which robustly prevented neuronal cell death in this system. We observed marked protection of HD medium spiny-like neurons from cell death upon either bexarotene or BDNF treatment (Fig. 2D). These findings indicate that bexarotene can ameliorate mutant htt neurotoxicity.

Fig. 2. Bexarotene is neuroprotective in mouse and human HD neurons in vitro.

(A) We measured cell survival in mouse CD1 cortical neuron-striatal neuron cocultures transfected with the indicated Htt expression vector and treated with bexarotene at the indicated concentrations. Total numbers counted for fluorescently transfected neurons for each condition were normalized to Htt Nt-90-8Q–transfected neurons at baseline, with neuron survival arbitrarily set to 1. *P < 0.05, **P < 0.01, Student’s t test. n = 3 independent experiments. (B) We measured survival of striatal neurons in cortical neuron-striatal neuron cocultures transfected with the indicated Htt expression vector and treated with bexarotene at the indicated concentration. Total numbers counted for fluorescently transfected neurons for each condition were normalized to Htt Nt-90-8Q–transfected neurons at baseline, with survival arbitrarily set to 1. *P < 0.05, **P < 0.01, Student’s t test. n = 3 independent experiments. (C) We quantified cell death in mouse primary cortical neurons, transfected with the indicated Htt expression vector, and treated with vehicle or bexarotene at the indicated concentration. **P < 0.01, ***P < 0.001, Student’s t test. n = 6 independent experiments. (D) We quantified cell death in medium spiny-like neurons differentiated from an induced pluripotent stem cell line derived from a patient with HD carrying a 60Q allele in the huntingtin gene and treated with bexarotene (1.0 μM) or brain-derived neurotrophic factor (BDNF; 20 ng/ml). *P < 0.05, **P <0.01, Student’s t test. n = 3 independent experiments. Error bars represent SEM.

Bexarotene treatment improves motor function and rescues neurodegeneration in HD mice

Because bexarotene displayed neuroprotection against mutant htt toxicity and is already approved for use in humans, we investigated bexarotene treatment in N171-82Q mice, which recapitulate HD-like motor phenotypes and neurodegeneration within a time frame of 5 to 6 months (16). Bexarotene is a lipophilic molecule that readily crosses the blood-brain barrier in rodents (17, 18). To establish the dosage for a preclinical trial, we performed a pharmacodynamics study by delivering bexarotene via intraperitoneal injection, at either 10 or 30 mg/kg for 3 days/week for 1 week, to 6-week-old WT C57BL/6J mice and then measuring PPARδ target gene expression in the striatum. We observed comparable increases in PPARδ target gene expression in the brains of mice on the dosage regimen (10 mg/kg) and the dosage regimen (30 mg/kg) (fig. S2). However, we noted that the bexarotene dosage regimen (30 mg/kg) was not well tolerated, causing significant morbidity. The bexarotene dosage (10 mg/kg) regimen did not cause weight loss or visible side effects based on a neurological screening exam, nor did we detect any evidence of organ toxicity at necropsy.

Next, we injected N171-82Q mice with either bexarotene or vehicle (10 mg/kg per day), three times per week, beginning at 6 weeks of age. We adhered to recommended preclinical trial guidelines, intended to avoid spurious results (19, 20). We tracked the progression of disease phenotypes in vehicle- and bexarotene-treated HD mice by performing a composite neurological examination (21) and rotarod analysis at monthly intervals. Bexarotene treatment rescued neurological dysfunction and improved motor function in HD mice, as compared to vehicle-treated HD mice (Fig. 3, A and B, and fig. S3, A to D). Bexarotene also extended life span in HD mice by an average of 9.8 days (Fig. 3C). Neuropathology analysis further indicated that bexarotene treatment yielded a reduction in htt protein aggregates and prevented neuronal cell loss in the striatum of HD mice (Fig. 3, D to F).

Fig. 3. Bexarotene is neuroprotective in the N171-82Q mouse model of HD.

(A) We recorded neurological dysfunction scores (0 to 12, where 0 is normal and 12 is severely affected) in cohorts (n = 10 to 19 mice per group) of nontransgenic control mice (Non-Tg), vehicle-treated HD mice, and bexarotene-treated HD mice at monthly intervals, beginning at the initiation of the treatment. *P < 0.05, **P < 0.01, ANOVA with post-hoc Tukey test. (B) We measured times for the latency to fall on the rotarod test in cohorts (n = 12 to 24 mice per group) of nontransgenic control mice, vehicle-treated HD mice, and bexarotene-treated HD mice at monthly intervals, beginning at the initiation of the treatment. *P < 0.05, **P < 0.01, ANOVA with post-hoc Tukey test. (C) We measured survival of vehicle-treated HD mice (n = 24) and bexarotene-treated HD mice (n = 26) over time. Bexarotene-treated HD mice lived longer than did vehicle-treated HD mice. P < 0.05, log-rank test. (D) Sections of striatum from 18-week-old nontransgenic control mice, vehicle-treated HD mice, and bexarotene-treated HD mice (n = 5 to 7 mice per group) were immunostained for the neuronal marker NeuN and with the anti-polyglutamine antibody EM48. Scale bar, 20 μm. (E) We quantified EM48 puncta from the data in (D). *P < 0.05, ANOVA with post-hoc Tukey test. n = 5 to 8 mice per group. (F) We quantified neuron numbers from data in (D). *P < 0.05, ANOVA with post-hoc Tukey test. n = 5 to 8 mice per group. Error bars represent SEM.

Bexarotene rescues PPARδ target gene expression in both CNS and skeletal muscle of HD mice

To confirm that bexarotene treatment elicited induction of PPARδ activation in HD mice, we measured the expression of the PPARδ target genes angiopoietin-like 4 (Angptl4) and uncoupling protein 2 (Ucp2) in the cortex and striatum of bexarotene-treated mice and documented marked increases in Angptl4 and Ucp2 expression in comparison to vehicle-treated HD mice (Fig. 4, A and B). In human patients, sampling of CNS tissues was not feasible. Because PPARδ is highly expressed in skeletal muscle, a practical alternative strategy would be to measure PPARδ target gene expression in this highly accessible tissue in the HD mice. We thus assayed the expression of five PPARδ target genes, Ucp2, adipose differentiation–related protein (Adfp), lipoprotein lipase (Lpl), pyruvate dehydrogenase kinase isoform 4 (PDK4), and stearoyl–coenzyme A desaturase 1 (Scd1), in quadriceps muscle samples obtained from WT controls and bexarotene- and vehicle-treated HD mice. For all tested PPARδ target genes except Lpl, we observed rescue of gene expression; for Pdk4 and Scd1, we noted marked up-regulation of expression at three- to fivefold that of WT HD mice (Fig. 4C). To assess the rapidity of productive PPARδ target engagement in mouse skeletal muscle, we assembled two additional cohorts of HD N171-82Q mice, and we treated one HD cohort with vehicle and the other HD cohort with bexarotene at 10 mg/kg for 1 week. Induction of PPARδ target gene expression in quadriceps muscle was detected for three of the five targets in HD mice treated with bexarotene for just 1 week (fig. S4). These results indicate that PPARδ target gene expression in mouse skeletal muscle could serve as a marker of response to PPARδ agonist treatment.

Fig. 4. Bexarotene promotes PPARδ activation of target genes in mouse brain and muscle.

(A) We performed RT-PCR analysis to measure RNA expression of the PPARδ target genes Angptl4 and UCP2 in the cortex of 18-week-old nontransgenic control mice, vehicle-treated HD mice, and bexarotene-treated HD mice. **P < 0.01, ANOVA with post-hoc Tukey test. n = 9 to 12 mice per group. (B) We performed RT-PCR analysis of RNA expression of Angptl4 and UCP2 in the striatum of 18-week-old nontransgenic control mice, vehicle-treated HD mice, and bexarotene-treated HD mice. **P < 0.01, ANOVA with post-hoc Tukey test. n = 9 to 12 mice per group. (C) We performed RT-PCR analysis of RNA expression of five PPARδ target genes in the quadriceps muscle of 18-week-old nontransgenic control mice, vehicle-treated HD mice, and bexarotene-treated HD mice. ADFP, adipose differentiation–related protein; LPL, lipoprotein lipase; SCD1, stearoyl–coenzyme A desaturase 1. *P < 0.05, **P < 0.01, ANOVA with post-hoc Tukey test. n = 9 mice per group. Error bars represent SEM.

PPARδ agonist treatment restores oxidative metabolic function in HD mice

Our bexarotene preclinical trial in HD mice, together with a recent preclinical trial of the PPARδ agonist KD3010 (10), suggests that PPARδ agonists may be a potential treatment for HD. How does PPARδ agonist treatment achieve in vivo neuroprotection? PPARδ has been shown to improve bioenergetics function by promoting mitochondrial adenosine triphosphate (ATP) generation in skeletal muscle (22, 23). Mitochondrial dysfunction is a key feature of HD pathogenesis (24). To determine whether PPARδ agonist treatment affected mitochondrial function in HD, we performed a bioenergetics profile of HD mouse primary cortical neurons in comparison to WT mouse primary cortical neurons and evaluated the effects of bexarotene and KD3010. Extracellular flux analysis revealed that HD mouse neurons displayed a markedly reduced oxygen consumption rate (OCR) at baseline compared to WT neurons and that the spare respiratory capacity of HD neurons, reflected by the maximal OCR, was also markedly reduced in comparison to WT neurons (Fig. 5A). Because PPARγ was shown to drive ATP generation through mitochondrial oxidation of fatty acids (25), we supplied WT and HD mouse primary cortical neurons with palmitate, but this substrate did not produce any further differences in oxidative metabolism between WT and HD neurons. Treatment with bexarotene or KD3010 yielded a marked improvement in both basal OCR and maximal OCR in HD neurons (Fig. 5, A to C), restoring OCR to that of WT neurons. In addition to measuring OCR, we also recorded rates of glycolysis by assaying the extracellular acidification rate (ECAR) and confirmed that HD neurons produce ATP primarily via glycolysis but that bexarotene or KD3010 treatment reverts HD neurons to an oxidative mode of energy production (Fig. 5D). Given the potency of PPARδ agonist treatment for boosting oxidative metabolism in HD neurons, we measured the effect of bexarotene or KD3010 treatment on WT neurons and noted significant increases in basal OCR (P < 0.01) and maximal OCR (P < 0.05) (fig. S5A), indicating that PPARδ activation is also capable of boosting bioenergetics function in WT neurons. To determine the relevance of this bioenergetics profile of HD primary neurons to the effects of bexarotene treatment in vivo, we performed quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis on striatal RNAs for subsets of PPARδ target genes whose protein products function in the oxidative phosphorylation, glycolysis, or gluconeogenesis pathways. We documented restoration of a gene expression pattern matching the WT oxidative profile for bexarotene-treated mice (Fig. 5E). When we extended this analysis to skeletal muscle from bexarotene- and KD3010-treated HD mice, we observed a similar phenomenon (fig. S5B).

Fig. 5. PPARδ agonist treatment reverses impaired oxidative metabolism in mouse HD neurons.

(A) Mitochondrial respiratory states were assessed by extracellular flux analysis in primary cortical neurons obtained from BAC-HD mice (HD) and littermate controls (WT) and treated with vehicle (Veh), bexarotene (Bex; 500 nM), or KD3010 (100 nM). (B) We quantified basal oxygen consumption rate (OCR) in neurons from (A). **P < 0.01, ANOVA with post-hoc Tukey test. n = 6 to 7 samples per condition. (C) We quantified maximal OCR in neurons from (A). **P < 0.01, ANOVA with post-hoc Tukey test. n = 6 to 7 samples per condition. (D) We calculated OCR/extracellular acidification rate (ECAR) in neurons from (B). *P < 0.05, ANOVA with post-hoc Tukey test. n = 6 to 7 samples per condition. (E) We performed RT-PCR analysis for RNA expression of the PPARδ target genes Ascl3, Ascl1, Hk4, Hk2, Pck1, and Pcx (representative of different metabolic pathways) in the striatum of 18-week-old vehicle-treated nontransgenic control mice (WT), vehicle-treated HD mice, and bexarotene-treated HD mice. PAL, palmitate; Olig, oligomycin; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; A/R, Antimycin A/Rotenone B. *P < 0.05, **P < 0.01, ANOVA with post-hoc Tukey test. n = 3 mice per group. Error bars represent SEM.

PPARδ improves mitochondrial and protein quality control to achieve neuroprotection

Neurons are placed under a constant demand for energy and are in a continuous battle to maintain mitochondrial quality control and protein quality control. Decompensation of proteostasis and mitochondrial quality control are defining features of neurodegenerative diseases, including HD (24, 26). Because quality control processes are extremely energy intensive, and PGC-1α genetic rescue prevents protein aggregation and boosts mitochondrial function (9), we reasoned that PPARδ agonist treatment may ameliorate defects in mitochondrial quality control and protein quality control through its rescue of metabolic function. To test this hypothesis, we examined the mitochondrial morphology of striatal-like neurons carrying homozygous CAG-111 repeat expansion mutations in the mouse htt gene (ST-Hdh Q111/Q111) (27). Comparison of ST-Hdh Q111/Q111 cells with control ST-Hdh Q7/Q7 cells revealed an increase in mitochondrial fragmentation in Q111/Q111 cells (Fig. 6, A and B). Bexarotene treatment of ST-Hdh Q111/Q111 striatal-like cells yielded a significant reduction (P < 0.05 or P < 0.01) in mitochondrial fragmentation in a dose-dependent fashion (Fig. 6B). Because PPARδ agonist treatment boosted metabolic function in normal neurons (fig. S4A), we tested the effect of PPARδ activation on ST-Hdh Q7/Q7 cells and observed an increase in mitochondrial length; we detected a reduction in mitochondrial length upon PPARδ knockdown (Fig. 6C). Oxidative stress impairs mitochondrial quality control, promoting fragmentation (28). When we treated ST-Hdh Q7/Q7 striatal-like cells with hydrogen peroxide, we observed a marked reduction in mitochondrial length, and this mitochondrial length reduction could be ameliorated by combined PPARδ overexpression and bexarotene treatment (Fig. 6D). Similarly, hydrogen peroxide yielded markedly greater mitochondrial fragmentation in ST-Hdh Q111/Q111 striatal-like cells, which was rescued by combined PPARδ overexpression and bexarotene agonist treatment (Fig. 6E). To assess the in vivo relevance of these findings, we quantified mitochondrial genomic DNA (mitoDNA) and nuclear genomic DNA (nDNA) in the striatum of bexarotene-treated HD mice. We documented an increase in the mitoDNA/nDNA ratio, which is an index of the abundance of mitochondrial biomass, in HD mice treated with bexarotene (Fig. 6F).

Fig. 6. Bexarotene activation of PPARδ rescues altered mitochondrial morphology and protein quality control in HD mouse neurons.

(A) Representative images of striatal-like cells from ST-Hdh Q7/Q7 (control) and ST-Hdh Q111/Q111 (HD) knock-in mice immunostained with Tom20 antibody are shown. Note the tubular appearance of mitochondrial network in the Q7/Q7 cells compared to fragmented appearance of mitochondrial network in the Q111/Q111 cells. Scale bars, 20 and 5 μm (inset). (B) We classified the mitochondrial network of ST-Hdh cells as tubular or fragmented as in (A) and then determined the percentage of cells with fragmented mitochondria. About 43% of Q111/Q111 cells contained a fragmented mitochondrial network, whereas only ~3% of Q7/Q7 cells contained a fragmented mitochondrial network. **P < 0.01, Student’s t test. Treatment of Q111/Q111 cells with bexarotene reduced mitochondrial fragmentation in a dose-dependent manner. *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA with post-hoc Tukey test. n = 51 to 87 cells per plate, six to nine plates per cell line. (C) We measured average mitochondrial length in ST-Hdh Q7/Q7 striatal-like cells immunostained with Tom20 antibody as in (A). We transfected these cells with a PPARδ expression vector and then treated them with GW501516 or transfected them with a PPARδ shRNA expression construct. **P < 0.01, ANOVA with post-hoc Tukey test. n = 42 to 56 cells per plate, three plates per condition. (D) We measured average mitochondrial length in ST-Hdh Q7/Q7 striatal-like cells immunostained with Tom20 antibody as in (A). We treated these cells (with or without PPARδ expression vector transfection) with hydrogen peroxide with or without GW501516 treatment. **P < 0.01, ANOVA with post-hoc Tukey test. n = 38 to 56 cells per plate, three plates per condition. (E) We measured average mitochondrial length in ST-Hdh Q111/Q111 striatal-like cells immunostained with Tom20 antibody as in (A). We treated these cells (with or without PPARδ expression vector transfection) with hydrogen peroxide with or without GW501516 treatment. **P < 0.01, ANOVA with post-hoc Tukey test. n = 35 to 47 cells per replicate, three replicates per condition. (F) We performed quantitative PCR analysis of a mitochondrial genomic amplicon and a nuclear genomic amplicon and determined the ratio of mitochondrial DNA to nuclear DNA in the striatum of 18-week-old nontransgenic control mice, vehicle-treated HD mice, and bexarotene-treated HD mice. **P < 0.01, ANOVA with post-hoc Tukey test. n = 9 to 12 mice per group. Error bars represent SEM.

To determine whether PPARδ activation status affects protein quality control, we used a Neuro2a cell culture model of mutant htt aggregation, where transfection of N-terminal htt protein with 104 glutamine repeats yields marked aggregate formation in Neuro2a cells subjected to oxidative stress (9). Using this system, we found that treatment with the PPARδ agonist GW501516 or bexarotene elicited a marked reduction in htt-Q104 protein aggregation (Fig. 7A) and confirmed that bexarotene-mediated turnover of mutant htt protein was RXR-dependent (fig. S6). To establish which arm of the proteostasis pathway was responsible for the bexarotene-mediated reduction in htt aggregation, we performed htt-104Q transfection of Neuro2a cells in the presence of bexarotene, in combination with either lactacystin or spautin-1. We found that spautin-1 inhibition of autophagy blunted the reduction of htt aggregation achieved with bexarotene treatment, but lactacystin, an inhibitor of the ubiquitin-proteasome system, had no significant effect (Fig. 7B). Knockdown of the autophagy-related 7 (Atg7) gene similarly yielded a significant blunting of bexarotene-mediated htt aggregate rescue in this system (Fig. 7B). To directly evaluate the effect of PPARδ activation on autophagy, we assayed autophagy flux in Neuro2a cells subjected to PPARδ shRNA knockdown or PPARδ agonist activation and noted a reduction in autophagy flux upon PPARδ knockdown and a significant increase in autophagy flux with PPARδ agonist activation (Fig. 7C). This increase in autophagy flux could also be achieved with bexarotene activation of RXR (Fig. 7D). Transcription factor EB (TFEB) is a master regulator of autophagy (29), and upon PGC-1α induction, TFEB expression increases, thereby promoting autophagy (9). To assess the role of TFEB in PPARδ activation of autophagy, we measured autophagy flux in HeLa cells deficient in TFEB (fig. S7, A and B). We detected an increase in autophagy flux upon PPARδ agonist treatment that was comparable to the increased autophagy flux observed in WT HeLa cells treated with a PPARδ agonist (fig. S7, C and D), thereby ruling out a role for TFEB in PPARδ-mediated autophagy activation. To determine whether bexarotene amelioration of mutant htt protein aggregation depended upon PPARδ activation, we quantified htt-Q104 aggregation in Neuro2a cells treated with bexarotene in the presence or absence of a specific PPARδ inhibitor (GSK3787) and noted that PPARδ inhibition abrogated bexarotene amelioration of htt protein aggregation (Fig. 7E). These findings indicate that PPARδ activation achieves neuroprotection by improving mitochondrial and protein quality control pathways.

Fig. 7. Bexarotene activation of PPARδ promotes proteostasis by inducing the autophagy pathway in mouse neurons.

(A) We quantified the percentage of Neuro2a cells containing htt protein aggregates, when transfected with a htt-104Q expression vector, treated for 24 hours with GW501516 (500 nM) or bexarotene (1 μM), and exposed to H2O2 (25 μM) for 4 hours. **P < 0.01, ANOVA with post-hoc Tukey test. n = 30 to 50 cells per sample, nine samples per condition. (B) We quantified the percentage of Neuro2a cells containing htt protein aggregates, when transfected with a htt-104Q expression vector, treated for 24 hours with bexarotene (1 μM), spautin-1 (10 nM), or lactacystin (5 nM), and exposed to H2O2 (25 μM) for 4 hours. **P < 0.01, ANOVA with post-hoc Tukey test. n = 30 to 50 cells per sample, 9 to 12 samples per condition. ATG7, autophagy-related 7 gene. (C) We performed microtubule-associated protein 1A/1B-light chain 3 (LC3) immunoblot analysis of Neuro2a cells cultured in normal media in the presence or absence of bafilomycin, transfected with a PPARδ shRNA vector or a PPARδ expression vector, and treated with GW501516 (100 nM). β-Actin served as a loading control. (D) We performed densitometry analysis of the LC3 immunoblotting results shown in (C) to determine autophagy flux. *P < 0.05, **P < 0.01, ANOVA with post-hoc Tukey test. n = 3 independent experiments. (E) We quantified the percentage of Neuro2a cells containing htt protein aggregates, when transfected with a htt-104Q expression vector, treated with bexarotene (500 nM) alone, or bexarotene (500 nM) plus the PPARδ inhibitor GSK3787 (200 nM), and exposed to H2O2 (25 μM) for 4 hours. **P < 0.01, ANOVA with post-hoc Tukey test. n = 30 to 50 cells per sample, nine samples per condition. Error bars represent SEM.

DISCUSSION

HD and other neurodegenerative disorders, including AD and Parkinson’s disease (PD), share two key defining cellular pathologies: mitochondrial dysfunction and impaired protein-organelle quality control. We and others previously linked mitochondrial dysfunction and transcriptional dysregulation in HD to interference with the transcription coactivator PGC-1α (6, 8, 30). Because mutant htt does not directly interact with PGC-1α to blunt its function, we pursued the mechanistic basis for this transcription interference and identified the nuclear hormone receptor PPARδ as a direct target of mutant htt neurotoxicity (10). Because PPARδ heterodimerizes with RXR to activate its target genes, and the resulting permissive PPARδ-RXR heterodimer is subject to dual ligand regulation, RXR agonists are capable of promoting PPARδ activation (11). Here, we examined bexarotene in various in vitro cellular models of HD and observed robust neuroprotection, suggesting the neurotherapeutic potential of bexarotene in HD. We investigated bexarotene treatment in HD N171-82Q mice using a study design that adhered to guidelines for rigor and reproducibility (19, 20). We documented improvements in motor function, htt protein aggregation, striatal neurodegeneration, and mouse survival.

Bexarotene (also called Targretin) is approved for use in humans for T cell cutaneous lymphoma but is also currently in clinical trials in AD patients based on previous preclinical trial work in an AD mouse model (12). The mechanistic basis for the therapeutic efficacy of bexarotene was proposed to be increased activation of PPARγ, supporting a presumed role for enhanced Aβ clearance by PPARγ-expressing microglia (13). Because PPARδ is highly expressed in CNS neurons (14), and bexarotene can also potently activate PPARδ, we sought the basis for bexarotene-mediated neuroprotection in BAC-HD primary neurons by concurrently knocking down the expression of PPARα, PPARδ, or PPARγ. For all tested readouts, we found that PPARδ is required for amelioration of mutant htt neurotoxicity. Our findings thus suggested that bexarotene neuroprotection involved PPARδ activation and that enhanced PPARδ activation could be contributing to the beneficial effects of bexarotene in AD (12, 31). Although bexarotene readily crosses the blood-brain barrier in rodents (17, 18), it does not efficiently cross the blood-brain barrier in healthy human subjects (32), implying that bexarotene therapeutic responses observed in neurodegenerative disease patients may stem from the peripheral benefits of RXR and PPARδ activation or from alteration of blood-brain barrier function in human patients (33).

An important question that we sought to answer in this investigation was how PPARδ achieved neuroprotection. Numerous studies have examined the role of PPARδ in skeletal muscle, and these efforts led to the realization that PPARδ strongly favors oxidative metabolism, resulting in an increase in ATP (22, 23). Increased PPARδ activation in skeletal muscle is sufficient to yield profound changes in muscle physiology and endurance exercise performance. These changes have been linked to altered gene expression that enhances the function of the tricarboxylic acid cycle and oxidative phosphorylation pathway (22, 23), preservation of glucose concentrations (34), and boosting of PGC-1α (35). We recently surveyed the expression of the PPARs and found that only PPARδ is highly expressed in CNS neurons (10). We further found that transgenic expression of a dominant-negative PPARδ mutant in mice resulted in marked neurodegeneration in the context of diminished electron transport chain activity and greatly reduced ATP (10). We therefore directly assayed metabolic function by performing extracellular flux analysis on cortical neurons from BAC-HD mice and observed marked reductions in OCR. These metabolic defects were reversed when we treated HD neurons with bexarotene or the PPARδ agonist KD3010, indicating that PPARδ agonist therapy, whether achieved with a PPARδ agonist or RXR agonist, is capable of reverting HD neurons from glycolytic metabolism to oxidative metabolism.

Neurons are unique because they are postmitotic, have high energy requirements, and are exquisitely vulnerable to misfolded protein stress and defects in organelle quality control. Misfolded proteins, or peptide fragments thereof, are a defining feature of neurodegenerative disorders, including HD, PD, and AD (36). Neurons thus require energy not only for synaptic neurotransmission and transport of materials back and forth along their dendrites and axons but also for maintaining protein and organelle quality control. We reasoned that impaired oxidative metabolism in HD likely deprives neurons of the energy required to maintain mitochondrial quality control and proteostasis, especially given that altered mitochondrial dynamics and proteostasis are well-established characteristics of HD pathology. Evidence from patient material, cell culture models, and BAC-HD mice indicates that HD neurons contain highly fragmented mitochondria (3739). Because excessive mitochondrial fission occurs in HD, likely because of altered regulation of the fission regulatory proteins Drp1 and Fis1 (38, 40, 41), dysregulation of mitochondrial dynamics in HD implies that maintenance of normal mitochondrial morphology in HD requires an even greater expenditure of energy. Support for this view comes from ultrastructural analysis of mice that recapitulate HD phenotypes upon expressing dominant-negative PPARδ in striatal neurons, whose mitochondria appear highly fragmented (10). After confirming that excessive mitochondrial fragmentation occurs in ST-Hdh Q111/Q111 mouse striatal-like cells, we treated Q111/Q111 cells with bexarotene and observed reductions in mitochondrial fragmentation. We further found that improved energy production can counter mitochondrial fragmentation induced by oxidative stress in normal striatal-like cells, suggesting that PPARδ activation may be capable of supporting mitochondrial quality control in different stress situations.

In our bexarotene HD mouse study and in a previous study of PGC-1α overexpression (9), these interventions yielded reductions in htt protein aggregation in the brains of HD mice. To determine how bexarotene activation of PPARδ promotes proteostasis, we tested the effect of proteasome inhibition or autophagy inhibition on the bexarotene-mediated reduction of htt aggregates and found that autophagy inhibition sharply countered htt aggregate reduction in bexarotene-treated cells. We documented markedly increased autophagy flux when we transfected cells with PPARδ in the presence of an agonist but noted diminished autophagy flux when we performed PPARδ knockdown. Hence, our results indicate that PPARδ activation can up-regulate autophagy. This finding agrees with previous work where PPARδ agonist treatment of cardiomyocytes yielded increased light chain 3 (LC3)–II, suggestive of autophagy induction, and where an analysis of PPARδ knockout mice revealed reductions in autophagy markers in the heart (42). Because PGC-1α can promote increased expression of TFEB (9), a master regulator of autophagy, we evaluated the effect of PPARδ modulation in TFEB knockout cells and observed that PPARδ activation yielded increased autophagy flux in the absence of TFEB, indicating that PPARδ up-regulation of autophagy is not TFEB-dependent.

A frequent observation in neurodegenerative diseases is the failure of mitochondrial energy production to keep up with CNS demand for ATP, coupled with an inability to maintain protein quality control and organellar homeostasis. Energy production and quality control function are inextricably linked because neurons require energy for proteostasis and organelle quality control, and protein and organelle quality control must operate efficiently if a neuron is to retain a complement of fully functional mitochondria to carry out the task of ATP generation. If one arm of this homeostasis loop is disrupted, the other arm of the loop will inevitably become dysfunctional, and worsening dyshomeostasis will ensue because the altered process will act as a positive feedback loop. In HD, we and others have discovered a central role for impaired mitochondrial energy production and quality control (6, 9, 30), and we have homed in on PPARδ as a regulatory factor capable of promoting oxidative metabolism to yield energy. Because energy is necessary for promoting mitochondrial quality control at the level of mitochondrial dynamics, we tested whether PPARδ activation could rescue mitochondrial fragmentation in HD and found that PPARδ activation prevented mitochondrial fragmentation in HD cells. In HD, as in other neurodegenerative diseases, there is selective vulnerability of certain types of neurons. In the case of HD, it is striatal medium spiny neurons that are preferentially lost. Although the basis for this selective vulnerability is unknown, certain studies have found that striatal neurons are prone to mitochondrial fragmentation due to increased expression of the Drp1 receptor Fis1 (43), a pro-fission effect of dopaminergic signaling (40), or increased S-nitrosylation of Drp-1 (41). Thus, in HD, the energy demands of medium spiny neurons, together with a predisposition to fragmentation of the mitochondrial network, may explain why PPARδ transcription interference contributes to HD pathogenesis and why PPARδ promotion of mitochondrial fusion in HD is neuroprotective.

Here, we considered an alternate approach for PPARδ activation based on its formation of permissive heterodimers with RXR and found that bexarotene treatment counters mutant htt toxicity both in vitro and in vivo in HD. Our findings thus indicate that bexarotene deserves consideration as a potential therapy for HD. However, because this study only evaluated bexarotene in an HD mouse model featuring expression of truncated protein, further study of bexarotene in an HD mouse model featuring expression of full-length htt protein is warranted. Furthermore, although HD mouse models recapitulate many aspects of the human disease, their predictive value for gauging the potential utility of a therapeutic intervention remains uncertain. Performing parallel studies in cell culture, mouse primary neurons, and human stem cell models is necessary to corroborate evidence for an agent’s neuroprotection. Although this strategy was used here, it is important to recognize that these systems also have their limitations. Although bexarotene is approved for use in humans, its use is associated with side effects that can be dose-limiting (44). We have previously shown that the PPARδ agonist KD3010 is capable of robust neuroprotection in HD (10). Although KD3010 was found to be safe in humans in a phase 1b clinical trial, its dosage range for chronic, long-term use has yet to be established. Because bexarotene and KD3010 act on different transcription factors, one appealing approach would be to use combinatorial therapy in which the dosages of each compound could be reduced to limit their respective side effects while achieving an additive or perhaps even synergistic treatment response in HD patients.

MATERIALS AND METHODS

Study design

The primary objective of this study was to determine whether the RXR agonist bexarotene was an effective treatment for HD. Bexarotene was evaluated in primary mouse neurons and human patient stem cell–derived neurons in experiments that used quantitative real-time PCR and assays of mitochondrial function and cell death. Bexarotene was tested in a preclinical trial in a mouse model of HD, with motor function, neuropathology, and survival as the outcome measures. The preclinical trial, which was approved by and performed in accordance with the University of California, San Diego (UCSD) Institutional Animal Care and Use Committee (IACUC), adhered to a protocol where we arbitrarily divided littermates and balanced genders between experimental groups, with behavioral testing performed by investigators blinded to the treatment group of the mice. We based our group sizes on power analysis to achieve 80% likelihood of detection of a 30% rescue of motor phenotypes. In the second half of this study, we sought the mechanisms by which PPARδ agonists achieved neuroprotection in our HD mouse model using experiments that used quantitative real-time PCR, in vitro assays, immunohistochemistry, and Western blotting. For all experiments, replicate numbers are stated in the figure legends.

Cell culture and primary neuron studies

ST-Hdh cells were cultured as described previously (45). Primary cortical neurons from BAC-HD and WT mice were prepared as described previously (10, 46). Cotransfection with indicated constructs [as described previously (10)] was done with Lipofectamine 3000 as per the manufacturer’s protocol (Invitrogen). Lentiviral transduction was used to induce gene expression or knockdown in primary neurons, with infection achieved by adding 1 × 107 titer units of lentivirus to the culture media. For reporter assays, cells were drug-treated 24 hours after reporter transfection, harvested 24 hours later, and subjected to analysis using the Dual-Luciferase Reporter Assay System (Promega). Mitochondrial membrane potential was measured via live cell loading with a potential-sensitive dye, either JC-1 or tetramethylrhodamine methyl ester (TMRM), using the Tecan M200 PRO Reader. JC-1 was used for preliminary tests, followed by more extensive mitochondrial characterization by Seahorse metabolism analysis. Analysis of cell death with immunofluorescence to activated caspase-3 was performed as described (47). H2O2 treatment was 25 μM for 4 hours. Glucose starvation was done for 2 hours in Dulbecco’s modified Eagle’s medium without glucose and 10% dialyzed fetal bovine serum. Treatments with PPARδ antagonist GSK3787 (GSK), lactacystin (L6785, Sigma-Aldrich), spautin-1 (SML0440, Sigma-Aldrich), and bafilomycin A1 (B1793, Sigma-Aldrich) were as indicated for individual experiments. In all experiments, the investigator was blinded to culture conditions and cell treatments.

Primary mouse cortical neurons were prepared from CD1 mice as described previously (48). Cotransfection was performed at days in vitro 5 with the N586 fragment of Htt containing either 22Q (N586-Htt 22Q) or 82Q (N586-Htt 82Q) and enhanced green fluorescent protein (GFP) (10:1 ratio) with Lipofectamine 2000 according to the manufacturer’s protocol. Bexarotene treatment (1 μM) was done at the time of transfection. After 48 hours of expression, cells were fixed with 4% paraformaldehyde (PFA) for 30 min, and nuclei were stained with Hoechst 33258 (bis-benzimide, Sigma-Aldrich). Image acquisition was done using the AxioVision imaging software on an Axiovert 100 inverted microscope (Carl Zeiss). Analysis and quantification were performed using Volocity (PerkinElmer). Nuclear staining intensity of GFP-positive cells was measured, and neurons with a nuclear intensity of up to 200% of the intensity of healthy control were considered viable.

Primary rat cortical and striatal neurons were isolated from E18 Sprague-Dawley rat brains as described previously (49). Briefly, Nt-90-8Q and Nt-90-73Q constructs were transfected into separately isolated cortical and striatal neurons using electroporation. Cortical and striatal neurons were also cotransfected with yellow fluorescent protein (YFP) or mCherry, respectively, as separate viability markers, and then cocultured on previously established glial cell beds for 5 days before automated counting of YFP or mCherry neurons (49). Total numbers counted for fluorescently transfected neurons for each condition were normalized to Htt Nt-90-8Q–transfected neurons at baseline, with survival arbitrarily set to 1.

Animal studies and preclinical trial

All animal experimentation adhered to National Institutes of Health (NIH) guidelines and was approved by and performed in accordance with the UCSD IACUC. Cohort sizes were designated on the basis of power analysis for threshold effects of at least 25% difference. After genotyping, we performed motor baseline assessment before group assignments and divided littermates and balanced genders between experimental groups, in accordance with guidelines intended to avoid spurious results (19, 20). After group assignment, we initiated Monday-Wednesday-Friday intraperitoneal injections of bexarotene (10 mg/kg per day) as a suspension in corn oil (3 mg/ml) at 6 weeks of age. Blinded observers visually inspected mice for obvious neurological signs, examined mice with a composite neurological evaluation tool as described previously (21), and also examined motor phenotypes by performing rotarod testing as described previously (10). For neuropathology experiments, brains were harvested, and histopathology, volume measurements, and stereology analysis were performed as described previously (10). In all cases, the scorer was blinded to the genotype status and treatment condition of the mice.

Real-time RT-PCR analysis

RNA samples were isolated using TRIzol (Life Technologies). Genomic DNA was removed using RNase-free DNase (Ambion). mRNA quantification was performed using the 7500 Real-Time PCR System (Applied Biosystems) with Applied Biosystems Assays-on-Demand primers and TaqMan-based probes (50) or using the SYBR Green system (51). Applied Biosystems TaqMan primer and probe set designations are available upon request. 18S or β-actin RNA was used as internal controls. Relative expression levels were calculated via the ΔΔCt method.

Western blot analysis

Proteins were run on 10% bis-tris gels (Invitrogen) and transferred to polyvinylidene difluoride membranes (Millipore) before blocking in Odyssey Blocking Buffer (LI-COR Biosciences). Membranes were incubated with antibodies as indicated: LC3 (NB100-2220, Fisher); TFEB (4240S, Cell Signaling), p-S6K (9234, Cell Signaling), and p-S6 (2215, Cell Signaling); or β-actin (ab8226, Abcam); and imaged on the Odyssey System (LI-COR Biosciences).

Mitochondrial studies

The OCR and ECAR of primary neurons grown in Seahorse plates were measured using an Extracellular Flux Analyzer (Seahorse Bioscience), following the manufacturer’s instructions. The Seahorse values were normalized by protein mass, which was determined by bicinchoninic acid protein assay (Thermo Fisher Scientific) after the measurement.

ST-Hdh cells were transfected with the indicated constructs [as previously described (10)] with Lipofectamine 3000 as per the manufacturer’s protocol (Invitrogen) and treated with compounds as indicated: H2O2 (25 μM, 4 hours); GW501516 was at 100 nM for 24 hours. Cells were fixed with 4% PFA and stained for translocase of outer mitochondrial membrane 20 (TOM20) to delineate mitochondria. Cells were imaged at 63× on a Zeiss 780 confocal microscope. Images were analyzed with the NIH ImageJ program using a written script from Dickey and Strack (52).

Statistical analysis

All data were prepared for analysis with a standard spreadsheet software (Microsoft Excel). Statistical analysis was done using Microsoft Excel, Prism 4.0 (GraphPad), or the VassarStats website (http://vassarstats.net/). For analysis of variance (ANOVA), if statistical significance (P < 0.05) was achieved, we performed post hoc analysis to account for multiple comparisons. All t tests were two-tailed unless otherwise indicated, and the level of significance (α) was always set at 0.05.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/9/419/eaal2332/DC1

Fig. S1. Bexarotene promotes neuroprotection by activating PPARδ.

Fig. S2. Bexarotene pharmacodynamics analysis of PPARδ target gene activation in CNS yields a suitable dosage and delivery scheme for preclinical trial testing.

Fig. S3. Bexarotene treatment of HD mice ameliorates motor function decline.

Fig. S4. Bexarotene promotes PPARδ activation of target genes in skeletal muscle after 1 week of treatment.

Fig. S5. PPARδ activation enhances oxidative function in neurons and restores an oxidative gene expression pattern in the CNS of HD mice.

Fig. S6. Bexarotene-mediated htt protein aggregate reduction is RXR-dependent.

Fig. S7. PPARδ activation of autophagy does not require TFEB.

REFERENCES AND NOTES

Acknowledgments: We thank E. Lopez for technical support and Y. Matsuoka for providing compound GSK3787. Funding: This work was supported by the Hereditary Disease Foundation, the Cure Huntington’s Disease Initiative, and grants from the NIH (R01 NS065874 and R01 AG033082 to A.R.L.S. and National Research Service Award F32 NS081964 to A.S.D.). R.M.E. is an Investigator of the Howard Hughes Medical Institute at the Salk Institute for Biological Studies and March of Dimes Chair in Molecular and Developmental Biology. Author contributions: A.S.D. and A.R.L.S. provided the conceptual framework for the study. A.S.D. and A.R.L.S. designed all the experiments, with assistance from W.F. and R.M.E. for extracellular flux analysis, D.C.L. for the cortico-striatal neuron coculture experiments, C.A.R. for the human stem cell–derived neuron studies, and E.M. for EM48 immunohistochemistry and neuropathology analysis. A.S.D., D.N.S., M.A., K.R.S., N.A., S.A., M.J.V.K., K.O., S.K.G.-H., A.L.F., J.M.N., N.L., C.L.H., and D.C.L. performed the experiments. A.S.D. and A.R.L.S. wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the study are present in the paper and the Supplementary Materials. Requests for HD stem cell materials, available through a materials transfer agreement, should be directed to A.R.L.S.
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