Impaired PGC-1alpha function in muscle in Huntington's disease

Abstract

We investigated the role of PPAR gamma coactivator 1alpha (PGC-1alpha) in muscle dysfunction in Huntington's disease (HD). We observed reduced PGC-1alpha and target genes expression in muscle of HD transgenic mice. We produced chronic energy deprivation in HD mice by administering the catabolic stressor beta-guanidinopropionic acid (GPA), a creatine analogue that reduces ATP levels, activates AMP-activated protein kinase (AMPK), which in turn activates PGC-1alpha. Treatment with GPA resulted in increased expression of AMPK, PGC-1alpha target genes, genes for oxidative phosphorylation, electron transport chain and mitochondrial biogenesis, increased oxidative muscle fibers, numbers of mitochondria and motor performance in wild-type, but not in HD mice. In muscle biopsies from HD patients, there was decreased PGC-1alpha, PGC-1beta and oxidative fibers. Oxygen consumption, PGC-1alpha, NRF1 and response to GPA were significantly reduced in myoblasts from HD patients. Knockdown of mutant huntingtin resulted in increased PGC-1alpha expression in HD myoblast. Lastly, adenoviral-mediated delivery of PGC-1alpha resulted increased expression of PGC-1alpha and markers for oxidative muscle fibers and reversal of blunted response for GPA in HD mice. These findings show that impaired function of PGC-1alpha plays a critical role in muscle dysfunction in HD, and that treatment with agents to enhance PGC-1alpha function could exert therapeutic benefits. Furthermore, muscle may provide a readily accessible tissue in which to monitor therapeutic interventions.

Figure 1.

Huntington's disease transgenic mice display transcriptional impairment of PGC-1α and its downstream target genes, AMP kinase pathway and impaired energy metabolism in muscles. (A–C) Total RNA was isolated from the type I fibers enriched soleus, type II fibers enriched gastrocnemius (GM) and extensor digitorum longus (EDL) muscles from wild-type (WT; black bar) and NLS-N171-82Q Huntington's disease mice (HD; gray bar). Quantitative real-time PCR analysis was performed for relative mRNA expression of PGC-1α, PGC-1β, NRF-1 and Tfam and normalized to β-actin. In this and all other figures data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 (n = 5 in each group). (D) Analysis of PGC-1α protein levels by western blot in LDE, GM and soleus muscle of WT and HD mice. β-actin was used as loading control. (E–G) ATP and PCr levels are decreased in NLS-N171-82Q HD mice under chronic energy deprivation conditions. To create an artificial model of chronic energy deprivation conditions, WT and HD mice were treated for 10 weeks with the creatine analogue β-guanidinopropionic acid (GPA), which reduces the levels of high energy phosphate metabolites. After GPA treatment, HPLC measurement of high energy phosphate metabolites such as PCr, AMP, ADP and ATP was carried out in soleus muscles from WT and HD mice. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 5 in each group). (H) Relative mRNA expression of AMP-activated protein kinase (AMPK) in soleus muscles from WT and NLS-N171-82Q HD mice treated with NS or GPA. AMPK mRNA levels were normalized to β-actin. *P < 0.05, **P < 0.01 (n = 5 in each group). (I and J) Analysis of AMPK, phosphorylated AMPK (pAMPK) and LKB-1 protein levels by western blots in extracts of soleus muscles from WT and HD mice treated with NS or GPA. Relative density expressed after normalization with β-actin. n = 5 mice were used from each group for each protein, with the average value of the WT+NS group set to 1. Representative blots showing 2–3 samples from each group. *P < 0.05.

Figure 2.

Decreased proportion of oxidative type I fibers and oxidative capacity in soleus and gastrocnemius (GM) muscles from NLS-N171-82Q HD mice under chronic energy deprivation conditions. (A) Gross morphology of soleus and GM from WT and HD mice treated with NS or GPA. Muscles from WT mice showed a distinct red color characteristic of oxidative muscle, whereas HD muscles from HD mice are light red color under baseline conditions. GPA treatment caused a redder appearance in WT mice, but not in HD+GPA mice. (B) Histochemical staining for succinate dehydrogenase (SDH) in muscle sections from WT and HD mice treated with NS or GPA. Arrows indicate dark stained type I oxidative fibers, arrowheads mark to intermediate type IIA oxidative fibers and asterisks point to type IIB glycolytic fibers (Scale bar = 100 µm). (C) Analysis of myosin heavy chain (MHC) isoforms (type I, IIA and IIB) expression in soleus muscle from WT and HD mice using a specific antibody for MHC. Arrows indicate dark stained oxidative type I fibers, arrowheads mark to intermediate oxidative type IIA fibers and asterisks point to glycolytic type IIB fibers (Scale bar = 100 µm). (D) Quantification of MHC isoforms (type I, IIA and IIB) in soleus muscle. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 5 mice in each group). (E–G) Quantitative real-time PCR analysis of fiber type markers such as troponin 1 (fast), troponin 1 (slow) and myoglobin in soleus muscle of WT and HD mice treated with NS or GPA. Data are expressed as mean ± SEM. **P < 0.01, ***P < 0.001 (n = 5 mice in each group).

Figure 3.

Decreased mitochondrial area/number and rotarod performance in NLS-N171-82Q HD mice under energy deprivation conditions. (A) Transmission electron microscopic analysis of soleus muscle from WT and HD mice treated with NS or GPA. Upper panel shows the mitochondria on lower magnification (4800×) and lower panel shows mitochondria on higher magnification (19 000×). Arrow indicates mitochondria along Z-axis. M, mitochondria; Z, Z-axis; I, I band; A, A band. Inset depicts schematic diagram of muscle fiber anatomy. An altered morphology and alignment of mitochondria along the Z-axis was observed in HD mice under basal conditions when compared with WT mice, where mitochondria are well organized (Fig. 3A, upper). GPA treatment in WT and HD mice caused an altered orientation of mitochondria along the Z-axis, and varying size of mitochondria, while an increase in numbers of mitochondria was observed only in WT mice (Fig. 3A, upper). Higher magnification depicts normal shape and size of mitochondria and the presence of well assembled cristae in mitochondria of soleus muscle of WT mice (Fig. 3A, lower). Mitochondria in GPA-treated and untreated HD mice are slightly larger in size with irregular assembly of cristae. GPA treatment also caused an enlargement of mitochondria in WT mice (Scale bars = 0.2 µm for upper panel and 2 µm for lower panel). (B and C) Corresponding quantification of percentage mitochondria area fraction and mitochondria number from the transmission electron microscopic images of soleus muscles from WT and HD mice. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 (n = 8). (D) Rotarod performance was measured in GPA-treated WT and HD mice at the 10th week of GPA treatment. Mice were tested for latency to fall on accelerated rotarod in thrid consecutive trials per day for 4 days. Data are expressed as mean ± SEM. ns = non-significant versus HD + NS. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 10 mice per group).

Figure 4.

Affymetrix microarray gene expression profile and metabolic studies in soleus muscle revealed enrichment of pathways involved in mitochondrial biogenesis and muscle function in WT mice under chronic energy deprivation conditions. (A) We performed gene set enrichment analysis (GSEA) of gene expression profile in soleus muscles obtained from GPA-treated WT and HD mice. Four representatives gene set enrichment plots for pathways involved in the electron transport chain, glycolysis and gluconeogenesis, muscle fat and connective tissue-specific genes, and striated muscle contraction pathway are shown. These pathways are significantly enriched in WT mice treated with GPA when compared with HD+GPA mice. Upper part of the enrichment plot indicates the enrichment score on the y-axis and corresponding maximum peak. Middle part shows the location of genes in gene set as hits in a ranked list of genes. Middle left part indicates positively correlated genes in WT+GPA group when compared with negatively correlated genes in HD+GPA group. Lower part shows the histogram for the rank of genes in a 45 000 genes rank ordered dataset (n = 4 mice/group). (B) Cluster analysis of genes and corresponding heat map from enrichment plots show the expression levels of a subset of genes in metabolic pathways as described above (red, increased gene expression; blue, reduced gene expression).

Figure 5.

PGC-1α and its downstream target genes transcription in NLS-N171-82Q HD mice under chronic energy deprivation conditions. (A and B) We confirmed microarray gene expression data by quantitative real-time PCR. Relative mRNA expression levels of PGC-1α and its downstream target genes (PGC-1β, NRF-1, NRF-2, PPAR-α, PPAR-δ, CREB and ERR-α), genes involved in mitochondrial function (COX-II, COX-IV and CYTC) and mitochondrial biogenesis (Tfam) was measured in soleus muscles from GPA-treated and untreated WT and HD mice. Relative mRNA expression was normalized to β-actin. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 5 mice/group).

Figure 6.

Adenoviral vector-mediated delivery of PGC-1α increases the muscle oxidative capacity and reverses the blunted response for GPA in transgenic HD mice. (A) Expression of GFP labeled PGC-1α adenoviral vectors in the muscle of HD transgenic mice 4 weeks after injection. A vast majority of adenoviral transduced muscle fibers exhibiting green fluorescence of GFP. (B) Relative mRNA expression of PGC-1α and oxidative muscle fiber marker MHC-I in the muscles from PGC-1α adenoviral injected WT and HD mice. mRNA levels were normalized to β-actin. P < 0.05, *= versus control, # = versus AAV/GFP. (C) Histochemical staining for succinate dehydrogenase (SDH) in muscle sections from PGC-1α adenoviral injected WT and HD mice treated with NS or GPA. Overexpression of PGC-1α resulted in increased number of dark stained type I oxidative fibers in muscle of WT and HD mice. Arrows indicate dark stained type I oxidative fibers, arrowheads mark to intermediate type IIA oxidative fibers and asterisks point to type IIB glycolytic fibers (Scale bar = 100 µm).

Figure 7.

Impaired PGC-1α and target genes transcription, decreased oxidative capacity and cellular respiration in muscle biopsies and myoblasts from HD patients. (A) Quantitative real-time PCR analysis was performed in RNA isolated from muscle biopsies from symptomatic human HD patients and matched control subjects. Relative mRNA expression of PGC-1α and target genes and oxidative muscle markers (myoglobin and troponin I slow) was measured by normalizing values to β-actin. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01 (n = 9 control subjects and n = 13 HD patients). (B) We established muscle cell (myoblast) culture from HD patients and control subjects. To create chronic energy deprivation conditions, myoblast cultures were treated with GPA for 7 days. Immunofluorescence analysis of PGC-1α (green fluorescence), oxidative muscle marker myoglobin (red fluorescence) and DAPI (blue fluorescence) in myoblast cultures from HD patients and control subjects. Co-localization of PGC-1α and myoglobin is shown in merged images (yellow fluorescence). Arrowheads indicate immunoreactivity for PGC-1α and myoglobin. In HD myoblasts, decreased immunostaining and co-localization of PGC-1α and myoglobin is observed. Following GPA treatment, PGC-1α and myoglobin expression are increased in control myoblasts, but not in HD myoblasts. Scale bars = 50 µm. (C–E) Quantitative real-time PCR analysis was performed in RNA isolated from GPA-treated myoblast cultures from HD patients and control subjects. Relative mRNA expression of PGC-1α and target genes and mitochondrial function genes was measured by normalizing values to β-actin. Data are expressed as mean ± SEM. * and ^x = versus WT+NS, # = versus WT+GPA, *P < 0.05, **P < 0.01, ***P < 0.001 (n = 3 control subjects and n = 5 HD patients). (F) Measurement of cellular oxygen consumption in GPA-treated myoblast cultures from HD patients and control subjects. Oxygen consumption was recorded under basal conditions and after addition of pyruvate and the mitochondrial uncoupler dinitrophenol (DNP). Data are expressed as mean ± SEM. *P < 0.05 (n = 3 control subjects and n = 5 HD patients). (G) Increased PGC-1α mRNA expression in HD myoblasts by knockdown of mutant huntingtin. Control and HD myoblasts were transiently transfected with plasmid vector containing scramble and ShRNA target sequences A and D against mutant huntingtin. Knockdown of mutant huntingtin followed by GPA treatment caused a significant increase of PGC-1α mRNA expression in HD myoblasts. Data are expressed as mean ± SEM of two experiments. * = versus Control, ^x = versus scramble ShRNA. *, ^xP < 0.05.