PGC-1α rescues Huntington's disease proteotoxicity by preventing oxidative stress and promoting TFEB function - PubMed (original) (raw)

PGC-1α rescues Huntington's disease proteotoxicity by preventing oxidative stress and promoting TFEB function

Taiji Tsunemi et al. Sci Transl Med. 2012.

Abstract

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Figure 1. PGC-1α expression rescues HD neurological phenotypes

A) PGC-1α RNA expression upon doxycycline induction in 10 week-old bigenic Rosa26-rtTA – TRE- PGC-1α mice (n = 6 / group). Fold increase is normalized to endogenous PGC-1α in controls. (* P < .05; *** P < .001). Error bars = s.e.m. B) The ledge test, a direct measure of coordination, was performed on cohorts (n = 10 – 15 / 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 < .05). Errors bar = s.d. C) Grip strength analysis in HD transgenic mice (n = 13 – 15 / group). HD mice display reduced forepaw grip strength at 13 weeks of age (# P < .05), worsening further at 18 weeks of age (## P < .01). Induction of PGC-1α yields a significant improvement at 13 and 18 weeks of age (* P < .05). Each score is the mean of three tests / time point per mouse, and errors bar = s.e.m. D) Rotarod analysis on 13 week-old cohorts (n = 10 – 12 / 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 < .01). Error bars = s.d. E) Rotarod analysis on 18 week-old cohorts (n = 10 – 12 / group). The HD + PGC-1α group performed comparably to controls, but significantly better than the HD and HD + PGC-1α (no dox) groups (P < .01). Error bars = s.d.

Figure 2

Figure 2. PGC-1α expression prevents htt aggregate formation and rescues HD neurodegeneration

A–F) Sections from the frontal cortex (A–C) and hippocampus CA3 region (D–F) of 18 week-old HD mice (A,D), non-HD littermate controls (B,E), and HD mice induced to express PGC-1α (C,F). Anti-htt antibody EM48 (green); DAPI (blue). G) Quantification of htt aggregate formation in 18 week-old HD mice (* P < 0.05). Error bars = s.d. 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 < .05). Error bars = s.e.m. 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 < .05). Error bars = s.e.m.

Figure 3

Figure 3. PGC-1α expression restores mitochondrial function in HD transgenic mice

A) Real-time RT-PCR analysis of striatal RNAs, obtained from sets (n = 4 – 6 / group) of 13 week-old HD mice, are significantly decreased in HD mice, but all five mitochondrial PGC-1α target genes are markedly increased in HD mice expressing PGC-1α (** P < .01; * P < .05). Error bars = s.e.m. B) ATP/ADP ratios are significantly elevated upon PGC-1α induction in the striatum (** P < .01) and cortex (* P < .05). Error bars = s.e.m. C) We measured mitochondrial complex I activity in the striatum of 13 week-old HD mice, and and observed a significant rescue of complex I activity in HD mice expressing PGC-1α (** P < .01). Error bars = s.e.m. D) Mitochondrial complex II activity is markedly improved in the striatum of 13 week-old HD transgenic mice expressing PGC-1α (* P < .05). Error bars = s.e.m.

Figure 4

Figure 4. PGC-1α expression protects against HD oxidative damage by inducing reactive oxygen species defense genes

A) Immunoblot analysis for protein carbonyl content in the striatum of 13 week-old HD transgenic mice was performed by preparing DNPH-derivatized protein lysates, and comparing with non-derivatized protein lysates in alternate lanes, as shown. We reprobed for β-actin to confirm equivalent protein loading. B) Immunoblot analysis for lipid peroxidation via 4-hydroxynonenal (4-HNE) 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 the level of 8-OH-deoxyguanosine (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 < .05). Error bars = s.e.m. D) Real-time RT-PCR analysis of striatal RNAs, obtained from sets (n = 4 – 6 / group) of 13 week-old HD mice. The expression levels of most key reactive oxygen species defense genes subject to PGC-1α regulation are significantly increased in HD mice induced to express PGC-1α (* P < .05). Error bars = s.e.m.

Figure 5

Figure 5. PGC-1α expression counters htt protein aggregate formation induced by oxidative stress

A) At increasing hydrogen peroxide concentrations, ST-Hdh Q111/Q111 cells exhibited significantly greater levels of ROS formation compared to ST-Hdh Q7/Q7 cells; however, over-expression of PGC-1α in ST-Hdh Q111/Q111 cells prevented ROS formation (* P < .05). B) Neuro2a cells were transfected with an htt-104Q-eGFP expression construct, co-transfected with either an empty vector (RFP-empty) or a PGC-1α expression construct (RFP-PGC-1α). As the hydrogen peroxide concentration increased, we noted greater numbers of cells containing punctate htt-104Q staining. Co-expression of PGC-1α markedly diminished the frequency of cells containing aggregated htt. C) Quantification of the effect of PGC-1α upon htt protein aggregate formation in Neuro2a cells exposed to oxidative stress. There is a significant reduction in the % of Neuro2a cells with htt aggregates upon PGC-1α over-expression, or when cultured in the presence of the ROS scavenger, N-acetylcysteine (NAC) (* P < .05). Error bars = s.d. D) Filter trap assay on Neuro2a cells expressing polyQ-expanded htt protein under different. Oxidative stress promoted the formation of insoluble htt protein, while 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 prevented PGC-1α from reducing htt protein aggregation (* P < .05). Error bars = s.e.m. F) In the absence of oxidative stress, chymotrypsin-like activity levels are identical for untreated Neuro2a cells and PGC-1α-expressing or NAC-treated Neuro2a cells. Significant oxidative stress yields marked reductions in chymotrypsin-like activity for untreated cells (# P < .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 (** P < .01). Error bars = s.e.m.

Figure 6

Figure 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 – 6 / group) of 13 week-old HD mice, reveals that TFEB expression is significantly decreased in HD mice (* P < .05), but markedly increased in HD mice expressing PGC-1α (** P < .01). Error bars = s.e.m. B) TFEB expression is significantly decreased in htt-104Q-expressing Neuro2a cells (* P < .05); however, co-transfection with a PGC-1α expression construct strongly rescues polyQ-htt repression of TFEB expression (** P < .01). Error bars = s.e.m. 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 chromatin immunoprecipitation (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 qPCR 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 < .01). Error bars = s.e.m. 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). 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 a significant repression of TFEB transactivation in Q111/Q111 cells (* P < .05). However, when we co-transfected Q111/Q111 cells with the PGC-1α expression construct, we observed a marked rescue (* P < .05). Error bars = s.e.m.

Figure 7

Figure 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 – 6 / group) of 13 week-old HD mice, reveals that the expression levels of four out of five TFEB target genes are significantly decreased in HD mice, while all five TFEB target genes are markedly increased in HD mice expressing PGC-1α (* P < .05; ** P < .01). Error bars = s.e.m. B) Western blot analysis of cathepsin D, a TFEB target gene, reveals an increased expression of the fully processed ~32 kDa mature form in the striatum of HD mice expressing PGC-1α. Blots were reprobed for β-actin to permit normalization upon Image J analysis of band intensities, which is shown to the right. C) Neuro2a cells were transfected with an htt-104Q-eGFP expression construct, and co-transfected with either an empty vector or a TFEB expression construct. As the hydrogen peroxide concentration increased, we detected greater numbers of cells containing punctate htt- 104Q staining. Co-expression of TFEB markedly diminished the % of cells containing aggregated htt (* P < .05; ** P < .01). Error bars = s.e.m. D) Neuro2a cells were cultured at 0.1 mM hydrogen peroxide, transfected with a htt-104Q-eGFP expression construct, and co-transfected with a PGC-1α expression construct (PGC-1α o.e.), PGC-1α shRNA knock-down construct (PGC-1α k.d), TFEB expression construct (TFEB o.e.), or TFEB shRNA knock-down construct (TFEB k.d.), as indicated. We noted a significant reduction in htt aggregates upon PGC-1α over-expression, TFEB over-expression, and TFEB over-expression despite simultaneous PGC-1α knock-down (* P < .05). However, PGC-1α over-expression in the presence of TFEB knock-down did not reduce htt aggregate formation. N-acetylcysteine (NAC) is a positive control for aggregate reduction (* P < .05). Error bars = s.e.m.

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