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. 2016 Jan;22(1):37-45.
doi: 10.1038/nm.4003. Epub 2015 Dec 7.

PPAR-δ Is Repressed in Huntington's Disease, Is Required for Normal Neuronal Function and Can Be Targeted Therapeutically

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Free PMC article

PPAR-δ Is Repressed in Huntington's Disease, Is Required for Normal Neuronal Function and Can Be Targeted Therapeutically

Audrey S Dickey et al. Nat Med. .
Free PMC article

Abstract

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene, which encodes a polyglutamine tract in the HTT protein. We found that peroxisome proliferator-activated receptor delta (PPAR-δ) interacts with HTT and that mutant HTT represses PPAR-δ-mediated transactivation. Increased PPAR-δ transactivation ameliorated mitochondrial dysfunction and improved cell survival of neurons from mouse models of HD. Expression of dominant-negative PPAR-δ in the central nervous system of mice was sufficient to induce motor dysfunction, neurodegeneration, mitochondrial abnormalities and transcriptional alterations that recapitulated HD-like phenotypes. Expression of dominant-negative PPAR-δ specifically in the striatum of medium spiny neurons in mice yielded HD-like motor phenotypes, accompanied by striatal neuron loss. In mouse models of HD, pharmacologic activation of PPAR-δ using the agonist KD3010 improved motor function, reduced neurodegeneration and increased survival. PPAR-δ activation also reduced HTT-induced neurotoxicity in vitro and in medium spiny-like neurons generated from stem cells derived from individuals with HD, indicating that PPAR-δ activation may be beneficial in HD and related disorders.

Figures

Figure 1
Figure 1. Huntingtin and PPARδ physically interact
(a) Immunoprecipitation with GFP antibody and immunoblot analysis of GFP-htt-25Q or GFP-htt-104Q and Flag-tagged PPARα, -δ, or -γ. GFP-empty-transfected HEK293 cells served as a negative control. (b) Immunoprecipitation of ST-Hdh striatal-like neurons of the indicated genotypes was performed with Htt antibody, followed by immunoblot analysis of PPARδ. (c) Immunoprecipitation of protein lysates from the cortex of 8 month-old BAC-HD mice and non-transgenic (Non-Tg) controls with Htt antibody, and immunoblot analysis of PPARδ. (d) Immunoprecipitation of protein lysates from the cortex of 8 month-old BAC-HD mice and non-transgenic (Non-Tg) controls with PPARδ antibody, and immunoblot analysis of Htt. Note detection of both transgenic polyQ-Htt protein and endogenous mouse Htt protein in BAC-HD samples, and detection of endogenous mouse Htt protein in Non-Tg control. (e) Immunoprecipitation of in vitro transcription-coupled translation of GFP-tagged htt-Q25 and PPARδ with GFP antibody, followed by immunoblot analysis of PPARδ. For (b)(e), immunoprecipitation with IgG only served as a negative control.
Figure 2
Figure 2. PPARδ activation rescues transcriptional repression, mitochondrial membrane depolarization, and neurotoxicity in HD neurons
(a) 3X-PPRE luciferase reporter activity in primary cortical neurons from BAC-HD or non-transgenic (Non-Tg) control mice, co-transfected with Renilla luciferase vector, and PPARδ and/or PGC-1α expression constructs, as indicated, and treated with GW501516 (100 nM) or vehicle. Results were normalized to Non-Tg neurons at baseline. *P < .05; t-test. (b) Mitochondrial membrane potential of primary cortical neurons from BAC-HD and Non-Tg mice, treated as indicated, was determined from the ratio of mitochondrial to cytosolic JC-1 fluorescence. Results were normalized to Non-Tg neurons at baseline, and CCCP treatment served as a positive control for depolarization. *P < .05, **P < .01; t-test. (c) Immunofluorescence of active caspase-3 (red) and microtubule-associated protein 2 (green) in primary cortical neurons from BAC-HD and Non-Tg mice, treated as indicated. Scale bar = 20 μm. (d) Quantification of active caspase-3 staining shown in (e). Results were normalized to Non-Tg neurons at baseline. *P < .05, **P < .01; t-test. (e) Cortex from Non-Tg mice immunostained with the indicated PPAR antibody (green) and NeuN antibody (red). Merged images reveal expression of indicated PPAR. Scale bar = 50 μm. (f) Immunofluorescence of active caspase-3 in primary cortical neurons from BAC-HD mice, transfected with the indicated shRNA construct for 3 days, or treated with fenofibrate 100 nM (PPARα agonist), GW501516 100 nM (PPARδ agonist), or pioglitazone 20 nM (PPARγ agonist) for 24 h, prior to exposure to 25 μM H2O2. *P < .05, **P < .01; ANOVA with post-hoc Tukey. Error bars = s.e.m. All experiments were performed with 3 biological replicates and 9 technical replicates per condition.
Figure 3
Figure 3. PPARδ transcriptional interference yields neuron dysfunction, and induces neurological phenotypes and mitochondrial abnormalities in transgenic mice
(a) RT-PCR analysis of PPARδ target gene expression (left) and ChIP analysis of PPARδ occupancy at the promoters of PPARδ target genes (right) in ST-Hdh wild-type (Q7/Q7) and homozygous HD mutant (Q111/Q111) striatal-like cells (n = 3 / genotype; 6 – 9 technical replicates). **P < .01, *P < .05; t-test. (b) Nestin-Cre;PPARδ-E411P mice weigh less than littermate control mice. **P < .01; t-test. (c) Nestin-Cre;PPARδ-E411P mice cannot easily dismount from the cage ledge . **P < .01; t-test. (d) Nestin-Cre;PPARδ-E411P mice exhibit a prominent clasping phenotype. ***P < .001; t-test. (e) Nestin-Cre;PPARδ-E411P mice display kyphosis. **P < .01; t-test. (f) Nestin-Cre;PPARδ-E411P mice attained significantly worse ‘latency to fall’ times on the accelerating rotarod . **P < .01, *P < .05; ANOVA with post-hoc Tukey test. (g) Decreases in mean stride length and reduced combined mesh and triangle grip strength were observed for Nestin-Cre;PPARδ-E411P mice. Grip strength is given in arbitrary units with littermate control performance set to 1. *P < .05; t-test. For (b)(g), cohort age was 8 months, and group sizes were 8 – 9. (h) Electron micrographs of mitochondria in striatum neurons of 8 month-old mice of indicated genotypes. Scale bar = 1 μm. (i) Quantification of neuron mitochondrial size in the striatum and cortex, as assessed by mitochondrial area and perimeter (n = 4 mice / group). *P < .05; t-test. (j) HPLC measurement of ATP concentrations in the striatum and cortex of 8 month-old mice of indicated genotypes (n = 3 mice / group). *P < .05; t-test. For all experiments, error bars = s.e.m.
Figure 4
Figure 4. Expression of dominant-negative PPARδ in neurons results in widespread neurodegeneration
(a) Dissection of whole brain reveals dramatic global atrophy in Nestin-Cre;PPARδ-E411P mice. Cortex volume (b) and basal ganglia volume (c) are markedly reduced in Nestin-Cre;PPARδ-E411P mice. (d) Sections of whole brain stained for NeuN (left), and quantification of neuron number (right). Scale bar = 250 μm. (e) Substantia nigra stained for tyrosine hydroxylase (TH) (left), and quantification of TH+ neurons (right). Scale bar = 10 μm. (f) Frontal cortex and hippocampus stained for microtubule-associated protein 2 (MAP2) (left), and quantification of MAP2 immunoreactivity as a percentage of overall neuropil (right). Scale bar (low power) = 100 μm. Scale bar (high power) = 10 μm. (g) Cortex and basal ganglia stained for GFAP (left), and quantification of overall GFAP immunoreactivity (right). Scale bar = 100 μm. For all experiments, *P < .05; t-test, cohort age was 10 months, group sizes were 4 – 6, and error bars = s.e.m.
Figure 5
Figure 5. Dominant-negative PPARδ expression in the striatum recapitulates HD-like motor dysfunction and transcriptional pathology
(a) Rgs9-Cre;PPARδ-E411P mice cannot easily dismount from the cage ledge. *P < .05; t-test. (b) Rgs9-Cre;PPARδ-E411P mice exhibit a prominent clasping phenotype. *P < .05; t-test. (c) Rgs9-Cre;PPARδ-E411P mice display kyphosis. *P < .05; t-test. (d) Rgs9-Cre;PPARδ-E411P mice exhibit impaired motor coordination on the accelerating rotarod. *P < .05; ANOVA with post-hoc Tukey test. (e) Mesh grip strength analysis. Grip strength is given in arbitrary units with littermate control performance set to 1. *P < .05; t-test. For (a)(e), cohort age was 9 months, and group sizes were 7. (f) Sections of striatum from 10 month-old Rgs9-Cre;PPARδ-E411P mice and littermate controls were immunostained for NeuN. Representative insets indicate reduced striatal neuron number in Rgs9-Cre;PPARδ-E411P mice. Scale bar (low power) = 200 μm; Scale bar (inset) = 20 μm. (g) Quantification of neuron number from (f). *P < .05; t-test. n = 4 mice / group. (h) RNA-Seq analysis on striatum from 10 month-old Nestin-Cre;PPARδ-E411P mice and littermate control mice (n = 3 / group). Pie charts indicate the percentage of genes for the highest ranked altered ‘Biofunctions’ pathways in microarray expression data from human HD caudate (left) and RNA-Seq analysis of PPARδ-E411P mouse striatum (right). Error bars = s.e.m.
Figure 6
Figure 6. Treatment with the PPARδ agonist KD3010 improves motor function, neurodegeneration, and survival in HD mice, and rescues neurotoxicity in human HD neurons
Composite neurological examination (a) and accelerating rotarod analysis (b) of HD mice, receiving vehicle or KD3010, and non-transgenic (Non-Tg) littermate controls, at indicated ages. ***P < .001, **P < .01, *P < .05; ANOVA with post-hoc Tukey test. Cohort sizes were as follows: 9 Non-Tg controls, 13 HD mice on vehicle, and 12 HD mice on KD3010. (c) Evaluation of neuropathology in striatum. Quantification of striatal choline acetyltransferase (ChAT) immunoreactivity (left), striatal htt aggregation (EM48) (center), and striatal volume by stereology (right) for 18 week-old HD mice receiving vehicle or KD3010 (n = 4 – 5 mice/group). **P < .01, *P < .05; t-test. (d) Kaplan-Meier plot reveals extended lifespan in HD mice receiving KD3010. *P < .05, Log-rank test. (e) Quantification of neuron cell death for medium spiny-like neurons differentiated from iPSC lines derived from a HD individual with a 60Q allele, and treated with KD3010 at the indicated concentrations, BDNF at 20 ng/ml (positive control), or the inactive enantiomer of KD3010 at 1 μM (negative control). Untreated neurons served as another negative control (Ctrl). **P < .01, *P < .05; ANOVA with post-hoc Bonferroni test. Error bars = s.e.m.

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