Abstract

Activation of caspase-6 in the striatum of both presymptomatic and affected persons with Huntington's disease (HD) is an early event in the disease pathogenesis. However, little is known about the role of caspase-6 outside the central nervous system (CNS) and whether caspase activation might play a role in the peripheral phenotypes, such as muscle wasting observed in HD.

We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A. Using mouse embryonic fibroblasts (MEFs) from YAC128 mice, we show that this increase in caspase-6 activity can be mitigated by pifithrin-α (pifα), an inhibitor of p53 transcriptional activity, but not through the inhibition of p53′s mitochondrial pro-apoptotic function. Remarkably, the p53-mediated increase in caspase-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase-6. Furthermore, these results suggest that this pathway is activated both within and outside the CNS in HD and may contribute to both loss of CNS neurons and muscle atrophy.

INTRODUCTION

Huntington disease (HD) is a neurodegenerative disorder that is caused by an elongation of the polyglutamine tract in the ubiquitously expressed huntingtin protein (Htt). HD arises predominantly from a gain of toxic function with increasing numbers of glutamines leading to earlier onset of disease (1). Proteolytic cleavage of the mutant Htt (mHtt) protein is an important pathogenic process that leads to N-terminal fragments containing the polyglutamine tract (2), which impairs numerous cellular pathways such as transcription (3). Caspase-6, one of the proteases cleaving Htt, is activated in striatal tissue from both presymptomatic and symptomatic HD subjects as well as early in the disease course in the YAC128 mouse model of HD (4). Caspase-6 cleaves Htt at amino acid 586 and preventing proteolysis at this site rescues the HD phenotype in mice expressing mHtt resistant to caspase-6 cleavage (C6R mice). These mice are fully protected from motor, cognitive and neuropathologic changes observed in the YAC128 HD model (5–7).

The mechanism for the early activation of caspase-6 remains unknown. However, upregulation of the enzyme at the mRNA level has been shown in vitro (4). Caspase-6 has previously been identified as a transcriptional target of the tumour suppressor protein p53 (8), and the caspase-6 promoter contains a p53-binding site (9). Direct transcriptional regulation by p53 has been demonstrated by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs) using the wild-type caspase-6 promoter as well as mutations that abolish p53 binding (9). p53 protein levels and activity are increased in brain tissue from HD patients as well as in animal models of HD (10–13), which has been attributed to increased activation of its upstream activating kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR) (14–16).

Loss or mutation of p53 is a primary cause of different cancers (17) and an increase in p53 activity conversely might be postulated to protect cells from malignant transformation. Consistent with this hypothesis, three European studies found a lower-than-expected incidence of cancer in HD patients compared to controls (18–20). The incidence of cancers was significantly lower for all cancers of most major tissues and organs, which suggests a systemic effect rather than being confined to the central nervous system (CNS). Indeed, it has become apparent that HD is not only a disease of the CNS. In addition to the core neurological symptoms, a number of other phenotypes are observed in HD patients, including weight loss, osteoporosis, testicular degeneration, endocrine disturbances, metabolic dysfunction and skeletal muscle wasting (21). Several studies suggest that the peripheral features of HD may be independent of and occur in parallel to the changes in the CNS (22–26), since the dysfunction of peripheral cells also occurs when they are isolated and not under the influence of the CNS (22–25), which suggests a direct detrimental effect of peripherally expressed mHtt. For an example, it has been shown that myoblast, fibroblast and lymphoblast cultures from HD patients have mitochondrial abnormalities and increased susceptibility to a variety of stressors (27–29), as well as an increase in the proteolytic cleavage of Htt and the generation of nuclear N-terminal mHtt fragments (30), indicating that the same pathways that lead to neurodegeneration might also be activated in peripheral tissues. The peripheral manifestations of HD are thought to be a contributor to the morbidity and mortality in patients (21). Muscle wasting in particular is a well-known phenotype, where the skeletal muscles undergo atrophy despite being highly active as a result of hyperkinesia (31). However, the processes leading to muscle degeneration remain unknown.

Here, we show that caspase-6 activity is not only upregulated in the CNS in HD, but also in muscle tissue from HD patients as well as the HD mouse models YAC128 and R6/2. In addition, we demonstrate that p53 protein levels are increased in both primary neurons after stress and at baseline in muscle tissue from HD mouse models, which is associated with increased caspase-6 transcription and activation. Using mouse embryonic fibroblasts (MEFs) from the YAC128 HD mouse model, we show that p53 transcriptionally induces caspase-6 expression and that this mechanism accounts in part for the amplified caspase-6 activation in the presence of mHtt. Finally, we show that inhibition of p53 transcriptional activity attenuates both the aberrant caspase-6 expression and activation. Our data show that caspase-6 activation in HD is not confined to the CNS and suggest that p53 and caspase-6 may not only contribute to mHtt-mediated neuronal dysfunction and death, but also have a significant impact on the peripheral symptoms observed in HD. The increase in caspase-6 activity seen in skeletal muscle also suggests that inhibition of caspase-6 or interference with pathways leading to its activation may not only correct the neuronal degeneration, but also alleviate peripheral symptoms of HD, and that peripheral tissues could be used to assess the efficacy of such interventions. These findings suggest that p53 activity is an important upstream regulator of caspase-6 activity in HD.

RESULTS

Caspase-6 activity is increased in muscle tissue from HD mouse models and HD patients

Caspase-6 is widely expressed in the brain (4,32,33) as well as in peripheral tissues with high expression levels in heart, lung, kidney and muscle (34). It has been shown that caspase-6 plays a central role under several conditions, including neurodegenerative disorders, ischaemic stroke, as well as renal ischaemia (34). However, little is known about caspase-6 activity in muscle wasting seen in HD (35–37).

There are several reports on intramuscular mHtt inclusion formation and muscle atrophy in the R6/2 mouse model (36,38–40), and in the YAC128 mouse model of HD, muscle grip strength is reduced (41), which may suggest muscular atrophy. However, biochemical changes in skeletal muscle tissue have not yet been reported in YAC128 mice, prompting us to investigate caspase-6 activity in muscle tissue in different mouse models. To this end, we quantified the amount of cleaved lamin A in those tissues with a Mesoscale enzyme-linked immunosorbent assay (ELISA) method, since we and others have demonstrated previously that lamin A is a highly specific substrate of caspase-6 (42–44).

We first assessed caspase-6 activity in skeletal hindlimb muscle from YAC128 mice and their wild-type (wt) littermates at 12 months of age, as well as from R6/2 mice and their wt littermates at 12 weeks of age. While YAC128 mice develop the HD phenotype slowly over the course of 12 months, the R6/2 model expresses a short N-terminal fragment of the mHtt protein and shows a very rapid disease progression with premature death occurring between 10–13 weeks of age (45). In both mouse models, we found that muscular caspase-6 activity was significantly increased in animals expressing mHtt when compared to their wt littermates (Fig. 1A and B). This parallels our previous results showing increased caspase-6 activity in the brain of YAC128 mice (4).

Figure 1.

Caspase-6 activity is increased in muscle tissue from HD patients and mouse models. The presence of lamin A fragments generated by caspase-6 cleavage was assessed by ELISA. Increased levels of cleaved lamin A were found in skeletal hindlimb muscle tissue from R6/2 mice at 12 weeks of age (A) (R6/2: n = 8, wt: n = 8) and YAC128 mice at 12 months of age (B) (YAC128: n = 13, wt: n = 12) when compared with their wt littermates. Increased levels of cleaved lamin A were also detected in skeletal muscle tissue from human HD patients when compared to age- and sex-matched controls (C) (HD: n = 5, control: n = 4). Statistical significance was determined by Student's t-test, * P < 0.05, *** P < 0.001.

Figure 1.

Caspase-6 activity is increased in muscle tissue from HD patients and mouse models. The presence of lamin A fragments generated by caspase-6 cleavage was assessed by ELISA. Increased levels of cleaved lamin A were found in skeletal hindlimb muscle tissue from R6/2 mice at 12 weeks of age (A) (R6/2: n = 8, wt: n = 8) and YAC128 mice at 12 months of age (B) (YAC128: n = 13, wt: n = 12) when compared with their wt littermates. Increased levels of cleaved lamin A were also detected in skeletal muscle tissue from human HD patients when compared to age- and sex-matched controls (C) (HD: n = 5, control: n = 4). Statistical significance was determined by Student's t-test, * P < 0.05, *** P < 0.001.

To validate our findings and determine whether the aberrant activation of caspase-6 and cleavage of caspase-6 substrates is also part of the skeletal muscle pathology seen in HD patients, we obtained post mortem tissue samples from age-matched HD patients and non-affected control individuals and assessed lysates from skeletal muscle tissue for the presence of cleaved lamin A. Significantly increased levels of this processed caspase-6 substrate were observed in skeletal muscle from HD patients, indicating that muscle wasting in these individuals is accompanied by caspase-6 activation (Fig. 1C).

Protein levels of p53 are increased in YAC128 and R6/2 muscle tissue and associated with increased caspase-6 transcription

Previous studies have provided some evidence for the role of p53 in the pathogenesis of HD. The protein levels of p53 and its transcriptional activity are increased in human HD brain samples as well as in several HD models (10,46,47), where it seems to play a central role in the observed mitochondria-associated cellular dysfunction and behavioural abnormalities. Interestingly, caspase-6 can be transcriptionally regulated by p53, a feature that is shared with caspase-7 but not caspase-3 (9). To further elucidate the pathway of caspase-6 activation in skeletal muscle, we investigated whether caspase-6 mRNA levels were altered in muscle tissue from HD mouse models. Caspase-6 mRNA levels were increased in both YAC128 and R6/2 mice as assessed by qPCR (Fig. 2A and B). As a reference gene, we chose Pgk1 for these experiments, since it was the most stable among the 10 genes tested (Supplementary Material, Fig. S1). Increased p53 levels have been associated with increased transcription of its target genes (48). We therefore measured the p53 protein levels in muscle tissue, which were significantly increased in both YAC128 and R6/2 mice when compared with their wt littermates (Fig. 2C and D). All western blots used for quantification and raw quantification data are shown in Supplementary Material, Fig. S2.

Figure 2.

Caspase-6 expression is increased in YAC128 and R6/2 muscle tissue and associated with increased p53 levels. Caspase-6 mRNA levels were measured by qPCR analyses in skeletal hindlimb muscle tissue from 12 month old YAC128 (A) (YAC128: n = 5, wt: n = 5) and 12 week old R6/2 mice (B) (R6/2: n = 8, wt: n = 8). mRNA levels of the target gene caspase-6 was normalized to the reference gene Pgk1 (Phosphoglycerate kinase 1). mRNA levels were then normalized to control samples. Increased levels of p53 protein were found by western blot in 12-month-old YAC128 (C) (YAC128: n = 19, wt: n = 16) and 12-week-old R6/2 mice (D) (R6/2: n = 8, wt: n = 8) when compared to their wt littermates. Expression is normalized to actin, statistical significance was determined by Student's t-test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 2.

Caspase-6 expression is increased in YAC128 and R6/2 muscle tissue and associated with increased p53 levels. Caspase-6 mRNA levels were measured by qPCR analyses in skeletal hindlimb muscle tissue from 12 month old YAC128 (A) (YAC128: n = 5, wt: n = 5) and 12 week old R6/2 mice (B) (R6/2: n = 8, wt: n = 8). mRNA levels of the target gene caspase-6 was normalized to the reference gene Pgk1 (Phosphoglycerate kinase 1). mRNA levels were then normalized to control samples. Increased levels of p53 protein were found by western blot in 12-month-old YAC128 (C) (YAC128: n = 19, wt: n = 16) and 12-week-old R6/2 mice (D) (R6/2: n = 8, wt: n = 8) when compared to their wt littermates. Expression is normalized to actin, statistical significance was determined by Student's t-test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Primary neurons from YAC128 mice show increased p53 levels, caspase-6 expression and activation

To compare our findings from muscle tissue with previously reported changes in the CNS, we established primary cortical neuronal cultures from YAC128 and wt mice to assess the levels of p53 protein, caspase-6 expression and activation. At baseline we did not find any differences between the two genotypes, whereas treatment with camptothecin, which is known to acutely activate p53, resulted in increased p53 levels that were exacerbated in neurons derived from the YAC128 mice (Fig. 3A). The increase in p53 protein is paralleled by increased expression of caspase-6 (Fig. 3B) as well as increased caspase-6 activity in stressed YAC128 neurons when compared with wt neurons (Fig. 3C). These findings parallel the results obtained for skeletal muscle tissue and support the idea that increased transcription of caspase-6 ultimately leads to changes in its enzymatic activity, and that mHtt initiates similar pathogenic pathways in peripheral tissues as well as in neurons.

Figure 3.

YAC128 neurons show increased p53 levels, caspase-6 expression and activation similar to muscle tissue. Primary cortical neuron cultures were stressed with 5 µm camptothecin for 30 h and analysed for p53 and caspase-6 levels. Results are expressed as percentage of DMSO control treatment. p53 protein levels increase after camptothecin stress, and this increase is exacerbated in neurons derived from the YAC128 mouse model of HD (A). Treatment with camptothecin leads to a significant increase in caspase-6 mRNA levels, with neurons derived from YAC128 mice showing a higher increase than neurons derived from wt mice (B). mRNA expression of the target gene caspase-6 was normalized to RNA input. mRNA expression was normalized to control samples. Cleavage of the caspase-6 specific substrate lamin A by protein extracts from neuronal cultures shows an increase in caspase-6 activity in camptothecin-stressed cultures that is exacerbated in samples derived from YAC128 mice (C). Statistical significance was assessed by two-way ANOVA and post hoc Bonferroni comparisons, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

YAC128 neurons show increased p53 levels, caspase-6 expression and activation similar to muscle tissue. Primary cortical neuron cultures were stressed with 5 µm camptothecin for 30 h and analysed for p53 and caspase-6 levels. Results are expressed as percentage of DMSO control treatment. p53 protein levels increase after camptothecin stress, and this increase is exacerbated in neurons derived from the YAC128 mouse model of HD (A). Treatment with camptothecin leads to a significant increase in caspase-6 mRNA levels, with neurons derived from YAC128 mice showing a higher increase than neurons derived from wt mice (B). mRNA expression of the target gene caspase-6 was normalized to RNA input. mRNA expression was normalized to control samples. Cleavage of the caspase-6 specific substrate lamin A by protein extracts from neuronal cultures shows an increase in caspase-6 activity in camptothecin-stressed cultures that is exacerbated in samples derived from YAC128 mice (C). Statistical significance was assessed by two-way ANOVA and post hoc Bonferroni comparisons, *P < 0.05, **P < 0.01, ***P < 0.001.

Caspase-6 activation is attenuated by p53 inhibition

We have previously shown that caspase-6 is activated in MEFs after induction of DNA damage with camptothecin (42). Using this system, we found that p53 protein levels were increased shortly (4 h) after the addition of camptothecin and remained high until the last time point investigated (16 h, see Fig. 4A). In parallel, the levels of the active form of caspase-6 increased dramatically after 16 h of stress (Fig. 4A), whereas treatment of the cells with the p53 inhibitor pifα before addition of camptothecin reduced the accumulation of the active form of caspase-6 (Fig. 4A). Under the same conditions, p53 protein levels were still increased, indicating that pifα does not prevent the accumulation of p53 but instead specifically suppressed its transcriptional activity (49). To confirm that the processing of caspase-6 correlates with its activity, we measured the cleavage of its endogenous substrate lamin A (42). In agreement with the western blot data, we observed increased cleavage of lamin A following the addition of camptothecin (16 h, see Fig. 4B). Pre-treatment with pifα significantly suppressed this effect. Further confirmation for the involvement of p53 in caspase-6 activation after camptothecin stress was obtained using MEF cells disrupted in the p53 gene. Using these cells, we found significantly lower levels of active caspase-6 16 h after camptothecin treatment than in MEFs generated from wt mice (Fig. 4C).

Figure 4.

Caspase-6 activation is attenuated by p53 inhibition. MEFs derived from wt mice were treated with camptothecin and the p53 inhibitor pifα and levels of p53, pro- and active caspase-6 were analysed by western blot (A). Camptothecin leads to a strong increase in active caspase-6 after 16 h of treatment, and this increase is mitigated by treatment with the p53-inhibitor pifα (A). The increase in active caspase-6 is paralleled by increased cleavage of lamin A (B), confirming the presence of active enzyme. Lamin A cleavage was reduced when MEFs were treated with both camptothecin and pifα (B). Caspase-6 is not activated after camptothecin in immortalized MEFs derived from p53−/− mice, while it is present in the corresponding wt cells after treatment with 5 µm camptothecin for 16 h (C). Statistical significance was assessed by two-way ANOVA and post hoc Bonferroni comparisons, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Caspase-6 activation is attenuated by p53 inhibition. MEFs derived from wt mice were treated with camptothecin and the p53 inhibitor pifα and levels of p53, pro- and active caspase-6 were analysed by western blot (A). Camptothecin leads to a strong increase in active caspase-6 after 16 h of treatment, and this increase is mitigated by treatment with the p53-inhibitor pifα (A). The increase in active caspase-6 is paralleled by increased cleavage of lamin A (B), confirming the presence of active enzyme. Lamin A cleavage was reduced when MEFs were treated with both camptothecin and pifα (B). Caspase-6 is not activated after camptothecin in immortalized MEFs derived from p53−/− mice, while it is present in the corresponding wt cells after treatment with 5 µm camptothecin for 16 h (C). Statistical significance was assessed by two-way ANOVA and post hoc Bonferroni comparisons, *P < 0.05, **P < 0.01, ***P < 0.001.

Aberrant caspase-6 expression and activation in YAC128 MEFs are ameliorated by p53 inhibition

Having established that p53 protein levels as well as caspase-6 expression and activation are elevated in both primary cortical neurons and skeletal muscle tissue from HD mouse models, we wanted to determine if these alterations could be modulated by specifically inhibiting p53 activity. To this end, we took advantage of MEFs generated from the YAC128 mouse model of HD (50) and corresponding wild-type littermate mice. When these cells were treated with camptothecin, we observed an increase in p53 protein levels that was exacerbated in YAC128 MEFs (Fig. 5A). p53 displayed a predominantly nuclear localization, which is necessary for its transcriptional activity. Increased nuclear p53 levels were paralleled by an increase in the expression of caspase-6 mRNA (Fig. 5B). The increase in caspase-6 transcription after camptothecin stress was mitigated by treatment with the p53 inhibitor pifα (Fig. 5B). Furthermore, the observed increase in caspase-6 activity following its transcription after camptothecin stress was reduced following the addition of pifα (Fig. 5C and D). While mRNA levels of caspase-6 were statistically not different between YAC128 and wt samples after treatment with pifα, the p53 inhibitor did not completely rescue caspase-6 activity levels (Fig. 5C and D), indicating that additional pathways might contribute to the activation of the enzyme in YAC128 MEFs. Similar results were obtained in primary cortical neurons (Supplementary Material, Fig. S3), indicating that the regulation of caspase-6 expression by p53 is conserved between mitotic and post-mitotic cells. Overall, these data suggest that p53 activity is exacerbated in the presence of mHtt resulting in increased transcription of caspase-6. Increased caspase-6 protein expression and activation was further mitigated by the p53 inhibitor pifα.

Figure 5.

Increased caspase-6 expression and activity in YAC128 MEFs are dependent on p53. MEFs derived from YAC128 mice and their wt littermates were stressed with 5 µm camptothecin for 40 h analysed for p53 by western blot. Increased levels of p53 were found in nuclear-enriched fractions after camptothecin treatment, indicating activation of p53 (A). MEFs derived from YAC128 mice showed a significant increase in p53 levels compared to their wt littermates (A). p53 protein expression was normalized to the nuclear transcription factor TFIIB. Treatment with camptothecin leads to an increase in caspase-6 mRNA levels in MEFs, and this increase is mitigated by concomitant treatment with the p53 inhibitor pifα (B). mRNA levels of the target gene, caspase-6, was normalized to the reference gene, RPL13a. mRNA levels were then normalized to control samples. In YAC128 MEFs, caspase-6 mRNA levels are significantly increased over wt after camptothecin treatment, whereas no significant difference was observed when cells were treated with pifα (B). Increased levels of active caspase-6 were observed in camptothecin treated MEFs by western blot (C), an effect that was exacerbated in YAC128 MEFs. Treatment with pifα reduced the levels of active caspase-6 back to baseline (C). Levels of cleaved lamin A were measured by ELISA in MEFs and parallel the levels of active caspase-6, confirming the presence of the active enzyme after camptothecin stress and its reduction upon pifα treatment (D). Statistical significance was assessed by two-way ANOVA and post-hoc Bonferroni comparisons, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Increased caspase-6 expression and activity in YAC128 MEFs are dependent on p53. MEFs derived from YAC128 mice and their wt littermates were stressed with 5 µm camptothecin for 40 h analysed for p53 by western blot. Increased levels of p53 were found in nuclear-enriched fractions after camptothecin treatment, indicating activation of p53 (A). MEFs derived from YAC128 mice showed a significant increase in p53 levels compared to their wt littermates (A). p53 protein expression was normalized to the nuclear transcription factor TFIIB. Treatment with camptothecin leads to an increase in caspase-6 mRNA levels in MEFs, and this increase is mitigated by concomitant treatment with the p53 inhibitor pifα (B). mRNA levels of the target gene, caspase-6, was normalized to the reference gene, RPL13a. mRNA levels were then normalized to control samples. In YAC128 MEFs, caspase-6 mRNA levels are significantly increased over wt after camptothecin treatment, whereas no significant difference was observed when cells were treated with pifα (B). Increased levels of active caspase-6 were observed in camptothecin treated MEFs by western blot (C), an effect that was exacerbated in YAC128 MEFs. Treatment with pifα reduced the levels of active caspase-6 back to baseline (C). Levels of cleaved lamin A were measured by ELISA in MEFs and parallel the levels of active caspase-6, confirming the presence of the active enzyme after camptothecin stress and its reduction upon pifα treatment (D). Statistical significance was assessed by two-way ANOVA and post-hoc Bonferroni comparisons, *P < 0.05, **P < 0.01, ***P < 0.001.

Inhibition of p53-mediated transcriptional activity, but not mitochondrial activity, leads to a reduction in caspase-6 activation

In addition to regulating the transcription of numerous genes, it has been demonstrated that p53 can migrate to the mitochondria in response to multiple apoptotic stimuli, where it interacts with Bcl2 family members causing mitochondrial membrane permeabilization, cytochrome C release and activation of executioner caspases (51,52). We therefore wanted to investigate whether inhibition of p53′s mitochondrial pro-apoptotic function would affect caspase-6 activation using pifithrin-μ (pifµ, a small molecule that inhibits the interaction between p53 and Bcl2 proteins (53,54)). While cytoplasmic p53 is visible by immunofluorescence in MEFs treated with camptothecin alone, the concomitant treatment with camptothecin and pifµ confines p53 to the nucleus, preventing any potential interaction with the mitochondria (Supplementary Material, Fig. S4). We found that pifµ does not mitigate the observed increase in caspase-6 transcription after camptothecin treatment (Fig. 6A), which is consistent with previous studies demonstrating that pifµ mostly inhibits the direct effects of p53 on mitochondria and not its transcriptional activity (54). Next, we wanted to determine whether pifµ would affect the activation of caspase-6 after camptothecin stress by preventing p53-mediated mitochondrial depolarization. While the increased levels of active caspase-6 and cleaved lamin A after camptothecin stress (Figs. 5C and D, 6B and C) was reduced to baseline by pifα, we did not observe any reduction in caspase-6 activity after treatment with pifµ at similar doses. These findings suggest that the increased activation of caspase-6 in HD model systems is not due to p53′s pro-apoptotic function at the mitochondria, but rather mediated through the transcriptional upregulation of caspase-6 and other pro-apoptotic p53 target genes.

Figure 6.

Inhibition of p53′s mitochondrial activity does not decrease caspase-6 activity or expression levels. p53 can localize to the mitochondria and directly lead to mitochondrial depolarization, a process that is inhibited by the compound pifµ. Treatment of wt or YAC128 MEFs with camptothecin and pifµ does not reduce the levels of caspase-6 mRNA, confirming that the inhibitor does not influence p53′s transcriptional activity (A). mRNA levels of the target gene, caspase-6, was normalized to the control gene, RPL13a. mRNA levels were then normalized to control samples. Concomitant treatment with pifµ and camptothecin does not reduce the levels of active caspase-6 (B) or cleaved lamin A (C) in wt or YAC128 MEFs, confirming that the mitochondrial pro-apoptotic role of p53 is not necessary for these processes. Statistical significance was assessed by two-way ANOVA and post hoc Bonferroni comparisons, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

Inhibition of p53′s mitochondrial activity does not decrease caspase-6 activity or expression levels. p53 can localize to the mitochondria and directly lead to mitochondrial depolarization, a process that is inhibited by the compound pifµ. Treatment of wt or YAC128 MEFs with camptothecin and pifµ does not reduce the levels of caspase-6 mRNA, confirming that the inhibitor does not influence p53′s transcriptional activity (A). mRNA levels of the target gene, caspase-6, was normalized to the control gene, RPL13a. mRNA levels were then normalized to control samples. Concomitant treatment with pifµ and camptothecin does not reduce the levels of active caspase-6 (B) or cleaved lamin A (C) in wt or YAC128 MEFs, confirming that the mitochondrial pro-apoptotic role of p53 is not necessary for these processes. Statistical significance was assessed by two-way ANOVA and post hoc Bonferroni comparisons, *P < 0.05, **P < 0.01, ***P < 0.001.

DISCUSSION

In this study, we demonstrate that activation of caspase-6 in cells expressing mHtt is not exclusive to the brain, but also occurs in skeletal muscle derived from HD patients and mouse models of HD demonstrating that peripheral and CNS pathogenic processes share important features. Myocytes, similar to neurons, are post-mitotic, long-lived cells, and muscle is a tissue that is affected in HD patients (21).

Our data suggest that caspase-6 activation in muscle as well as in neurons could be due to aberrant accumulation and activation of p53, leading to increased expression of caspase-6 as well as other pro-apoptotic p53 target genes such as Bax and PUMA, thus lowering the apoptotic threshold in cells expressing mHtt. This notion is consistent with a previous study proposing such a mechanism to explain the sensitization of tumour cells to apoptosis-inducing agents after overexpression of p53, since the resulting increase in caspase-6 protein levels amplifies incoming cell death signals (8).

Caspase-6 is classified as an executioner caspase, since it has a short pro-domain and is activated by cleavage, which is thought to be catalysed by initiator caspases (−8, −9 or −10) (55). In HD, caspase-6 activation seems to be a chronic process that is initiated even at presymptomatic stages, in the absence of overt neuronal cell death (4). While p53 activity induces the expression of the pro-form of caspase-6, its activation could take place either through auto-activation that can occur when pro-form levels surpass a certain threshold or through the intrinsic apoptotic pathway involving mitochondrial depolarization through the action of Bax, PUMA and Noxa (p53 target genes), cytochrome c release and the activation of an initiator caspase such as caspase-9 (Fig. 7).

Figure 7.

p53-mediated caspase-6 activation in HD. A number of pathways have been described that can lead to p53 activation in HD: The expression of mHtt is known to induce excitotoxicity can directly induce DNA damage and mHtt also shows an aberrant direct interaction with p53. Excitotoxicity can cause the activation of p53 either through the activation of calpains, or through the generation of ROS, subsequent DNA damage and induction of p53 through the ATM/ATR pathway. Once activated, p53 fulfills its transcriptional role in the nucleus leading to the increased expression of caspase-6 as well as other pro-apoptotic proteins, such as Bax and Puma. These two proteins are known to cause mitochondrial depolarization and cytochrome c release, which leads to the activation of caspases. This activation of downstream p53 targets can be blocked by pifα treatment. Active caspase-6 in turn can cleave mHtt, providing a toxic feedback loop. p53 can also translocate to the mitochondria and directly cause mitochondrial depolarization through its interaction with Bcl2, a mechanism which can be blocked by the inhibitor pifµ. Our data suggest that this pathway is not the primary cause of caspase-6 activation in an HD context.

Figure 7.

p53-mediated caspase-6 activation in HD. A number of pathways have been described that can lead to p53 activation in HD: The expression of mHtt is known to induce excitotoxicity can directly induce DNA damage and mHtt also shows an aberrant direct interaction with p53. Excitotoxicity can cause the activation of p53 either through the activation of calpains, or through the generation of ROS, subsequent DNA damage and induction of p53 through the ATM/ATR pathway. Once activated, p53 fulfills its transcriptional role in the nucleus leading to the increased expression of caspase-6 as well as other pro-apoptotic proteins, such as Bax and Puma. These two proteins are known to cause mitochondrial depolarization and cytochrome c release, which leads to the activation of caspases. This activation of downstream p53 targets can be blocked by pifα treatment. Active caspase-6 in turn can cleave mHtt, providing a toxic feedback loop. p53 can also translocate to the mitochondria and directly cause mitochondrial depolarization through its interaction with Bcl2, a mechanism which can be blocked by the inhibitor pifµ. Our data suggest that this pathway is not the primary cause of caspase-6 activation in an HD context.

Increased p53 activity is associated with a phenotype that is consistent with accelerated aging in vivo (56). Mice expressing mutant p53 that augments the activity of wt p53 show reduced longevity, osteoporosis, generalized organ atrophy and diminished stress tolerance (56). Intriguingly, these mice also exhibit severe muscle atrophy, whereas p53−/− mice on the other hand are resistant to muscle wasting and tumour-induced cachexia (57,58), indicating that increased p53 activity could also confer a degenerative phenotype to muscle tissue in HD patients. Another interesting link to muscular degeneration is provided by a study that reports severe muscular dystrophy in a mouse disrupted in the gene encoding the caspase-6-specific substrates lamin A and C (59). It is tempting to speculate that cleavage of lamins A and C by caspase-6 could lead to a similar, but progressive degenerative muscle phenotype in HD.

p53 can be activated in response to numerous types of stress, which include but are not limited to DNA damage, oxidative stress, and excitotoxicity (60). While normal synaptic activity is thought to have a protective effect through the down-regulation of p53- and the induction of CREB-mediated gene transcription (61), p53 activation in HD may be triggered by the aberrant activation of extra-synaptic NMDA receptors. Excitotoxicity leads to the generation of reactive oxygen species (ROS) causing DNA damage (62–64) and stimulates calpain activity through the excessive influx of Ca2+ (65). Both of these pathways lead to activation of p53 (Fig. 7). In addition, the mHtt protein itself has been proposed as a source of double-strand DNA breaks through its interaction with the DNA repair protein Ku70 (66). This interaction was shown to impair Ku70 function in neurons (67), and overexpression of Ku70 ameliorated the phenotype in HD mouse and Drosophila models (66,68). The DNA damage response in the context of mHtt can lead to activation of the ATM/ATR pathways causing phosphorylation of p53 at Serine 15 and 46 (16,69), which in turn induces structural changes in p53 and its subsequent activation (70). Intriguingly, caffeine, a well-known inhibitor of ATM/ATR activities, has been shown to effectively prevent mHtt-dependent toxicity in vitro by reducing p53 phosphorylation.

It is unclear whether the full-length mHtt protein or proteolytic fragments mediate DNA damage. However, N-terminal mHtt fragments are abundant in the nucleus and also the fragment generated by caspase-6 cleavage has a nuclear localization (71). Therefore, it is possible that an amplification loop may exist, where mHtt leads to DNA damage, p53 is stabilized and increases transcription of caspase-6, which in turn increases the likelihood of caspase-6 activation and the generation of further mHtt fragments that enter the nucleus (Fig. 7). Interestingly, it has been shown that N-terminal fragments of mHtt directly interact with p53 increasing its nuclear localization as well as its transcriptional activity (10,13), which suggests that mHtt may directly influence the transcription of p53 target genes.

In the experimental paradigms used in our study, DNA damage induced by treatment with camptothecin was used to unmask differences in p53 levels and apoptotic signalling between HD and control cells. The acute stress with camptothecin might thereby exacerbate DNA damage and oxidative stress that otherwise accumulates slowly and leads to degenerative processes over time. Using this stress paradigm, we demonstrate that inhibition of p53-mediated transcription can decrease the activation of caspase-6, which suggests that targeting p53 activity and reducing caspase-6 transcription could be an approach to reduce the cleavage of caspase-6 substrates such as mHtt. Interestingly, Htt itself has been reported to be a transcriptional target of p53 (46) and in a mouse model of HD, deficiency of p53 led to a reduction of mHtt expression (47). In these mice, hyperactivity, clasping of hind limbs, impairment of prepulse inhibition and deficit on the rotarod motor task were ameliorated upon loss of p53, indicating that p53 and its downstream target genes play a role in modulating the severity of HD phenotype (10).

p53 is best known for its role as a tumour suppressor, and there is increasing evidence that HD patients are protected from a wide range of cancers (18,72), raising the intriguing possibility that increased activity of p53 might offer protection against the proliferation of pre-neoplastic cells in HD. The findings in our study might provide a biochemical rationale and mechanism for the reduced cancer incidence in HD. However, when considering p53 as a potential therapeutic target in HD it is important to recall that p53−/− mice have a high incidence of cancers (73). Therefore, it becomes very apparent that regulation of p53 activity has benefits that however may be associated with liabilities depending on the degree of p53 suppression. The therapeutic goal in HD would therefore be to decrease the activity of p53 to the normal range and not to suppress it completely.

It has become increasingly clear that p53 target genes have a wide variety of expression patterns, and mechanisms such as post-translational modification of p53 (74), the nature of the chromatin landscape around the target gene promoter (75), as well as the core promoter architecture of p53 target genes significantly influence its activity (76). Additionally, p53-interacting proteins such as Hzf (hemapoietic zinc finger) can cause p53 to favour the promoters of certain cell cycle arrest genes while preventing the binding to those of pro-apoptotic genes (77). Further mechanisms that have been identified include unequal turnover of p53-target gene mRNA, promoter DNA methylation and miRNA processing between different cell types, causing cell-type specific responses to active p53 (78). It is therefore important to both define the pool of target genes that are upregulated in response to p53 overactivation, and to elucidate the exact mechanism of p53 dysfunction in HD. Such an analysis will show whether therapeutic targets with a low risk of neoplastic side effects can be identified.

HD research mostly focuses on the brain and neuronal systems, since the striatum is the primary site of pathology (79). However, mHtt and many other proteins implicated in the pathogenesis of HD, such as caspase-6 or p53, are expressed ubiquitously and peripheral tissues are also affected (21). While there is no effective treatment for HD available today, targeted therapies are under development, and studies on potential deregulation of the target proteins in peripheral tissues such as skeletal muscle could provide valuable information about biological changes that track disease progression. The idea that the effects of mHtt in peripheral cells can be used as biomarkers is appealing, since sampling is minimally invasive and inexpensive. Validated biomarkers can be used to assess the response to a therapeutic intervention and would be of considerable value in the development of drugs and critical for making clinical trials more efficient. Ultimately, a combination of both clinical, neuroimaging and biochemical biomarkers would most likely be required to secure reliable tracking of disease onset and progression.

MATERIALS AND METHODS

The cleaved lamin A antibody for Mesoscale ELISA and p53 antibody for western blotting were from Cell Signaling technology, and actin and calnexin antibodies were from Sigma. Pure lamin A protein was obtained from Abcam, the antibody against caspase-6 (full-length and active forms) was raised in rabbit against a peptide derived from the p20 subunit of active caspase-6 (HDVPVVPLDMVD). Camptothecin was obtained from Sigma, pifα and pifµ from Enzo Life Sciences, and cell culture reagents were purchased from Gibco.

Skeletal muscle tissue

Human skeletal muscle tissue was obtained from the Huntington Disease BioBank at the University of British Columbia and the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD. Ethical approval for the collection, storage and usage of samples for research was obtained from the University of British Columbia/Children's and Women's Health Centre of British Columbia Research Ethics Board (UBC C&W REB H06-70467 and H06-70410). Tissue from 5 male HD patients (CAG sizes 41–50, mean disease duration 17.2 years, mean age at death 60.8 years) and 4 male controls (mean age at death 59.25 years) was analysed.

All mouse experiments were carried out in accordance with protocols (Animal protocol A07-0106) approved by the UBC Committee on Animal Care and the Canadian Council on Animal Care. Experiments were performed in accordance with the Danish Animal Experiments Inspectorate's guidelines (permit no. 2007/561-1345), the Danish Working Environment Authority (permit no. 20070033239/4) and the EC Directive 86/609/EEC for animal experiments. YAC128 (line HD53) mice were 12 months of age when used for the analysis of muscle tissues. These mice are on a FVB/N background and were of mixed gender. R6/2 mice, transgenic for exon 1 of the human HD gene encompassing approximately 144 CAG repeat units (45), originated from the Jackson Laboratory (Bar Harbor, Maine) and were maintained by backcrossing males to CBA/J × B6 females (Taconic, Denmark). Twelve-week-old animals of both sexes were used. Mouse skeletal hindlimb muscle was dissected and snap-frozen on dry ice for protein analyses. Tissue that was used for mRNA analysis was stored in RNA later (Invitrogen) according to manufacturer's instructions.

Generation and treatment of MEF cell lines

The protocol for the generation of MEF cells is described in detail elsewhere (42). Briefly, Day 12.5 embryos resulting from timed-pregnant heterozygous breedings of YAC128 mice (line 53) were used and tissues not used for culture were genotyped. Cultures were established from mice positive for the YAC128 transgene and their wt littermates, and primary cells were used for all experiments to avoid artefacts caused by immortalization. p53 was activated by treating cultured MEFs with 5 µm camptothecin for 40 h, control samples were treated with an equivalent volume of the solvent DMSO only. The p53 inhibitors pifα and pifµ were added to the cell culture medium 4 h before the addition of camptothecin at a final concentration of 10 µm. Cells were harvested by scraping in the medium, pellets were lysed in SDP+ lysis buffer (50 mm Tris pH8, 150 mm NaCl, 40 mm β-glycerophosphate, 10 mm NaF and 1% Igepal with 1 mm PMSF, 2 mm Na vanadate, 5 µm zVAD and ‘Complete’ protease inhibitor cocktail (Roche)) on ice, and protein concentrations were determined in the cleared lysates after centrifugation. MEF cultures from p53−/− mice and their wt littermates were obtained by the same protocol. Due to the rapid growth of p53−/− cultures, these cells were compared with an immortalized wt MEF line that was transfected with SV40 large T antigen and had a similar growth rate.

Generation and treatment of primary neuronal cultures

Primary cortical neuronal cultures were prepared from Day 16.5 YAC128 (line 55) and wt (FVB/N) mouse embryos as described (6). In short, cortices were micro-dissected from Day 16.5 embryos in ice-cold Hank's balanced salt solution (HBSS+; Gibco), then diced and dissociated with 0.05% trypsin-EDTA (Gibco). Trypsin was neutralized with 10% fetal calf serum in neuro basal medium (NBM+) and treated with DNAse I (153U/μl). Tissue was triturated with pipette five to six times. Cells were plated on poly-d-lysine coated 6-well plates with 2 ml of Neurobasal media (Gibco #21103-049), B27 (Gibco #17504-044), 100 U/ml penicillin-streptomycin (PS) (Gibco), 0.5 mml-glutamine and maintained at 37C, 5% CO2 with humidity. Cells were fed with 200 μl fresh medium every fifth day. Cells were stressed by the addition of 5 µm camptothecin to the medium after 10 days in culture and harvested 30 h after treatment as described (42).

Western blotting

Western blots were performed on collected samples lysed in SDP+ lysis buffer. Protein concentration was measured by a DC protein assay kit (Bio Rad, USA) and equal amounts were separated on 4–12% Bis-Tris gels (Invitrogen, USA). Protein was transferred to PVDF Immobilon-FL membranes by electroblotting and membranes were developed with primary antibodies in 5% bovine serum albumin/phosphate buffered saline. The following antibodies were used for immunoblotting: mouse-anti-p53 (1:1000) from Cell signalling (#2524), rabbit-anti-Actin (1:10 000) from Sigma (A2103), rabbit-anti-calnexin (1:5000) from Sigma (C4731) and rabbit-anti-TFIIB (1:1000) from Santa Cruz (sc225). Fluorescently labelled secondary antibodies goat-anti-mouse or goat-anti-rabit conjugated to either 700 or 800 IR dye (1:5000; Rockland, USA) and the LiCor Odyssey Infrared Imaging system were used for detection.

Quantitative real-time PCR (qPCR) analysis

Total RNA was isolated using an RNeasy Mini kit (Qiagen) according to manufacturer's instructions. RNA was treated with DNase I (Invitrogen) and RNA was reverse transcribed using SuperScript III (Invitrogen) and oligo-dT primers according to manufacturer's instructions to generate complementary DNA (cDNA) for RT-qPCR. Primers were designed using Primer3 software and validated by evaluating efficiency (E > 1.90) and one-peak melting curve. Ten reference genes from independent cellular pathways, including Pgk1, Eif4a, Tnnc1, Rpl27a, Canx, Atp5a, Sdha, Yhwaz, Rpl13 and Actb, were tested and validated for stability in muscle tissue collected from 12 month old FVB and YAC128 mice. The expression of the reference genes were normalized to RNA input and based on the lowest variation and difference in the mean expression Pgk1 was selected as the best reference gene out of the panel (see Supplementary Material, Fig. S1A). Subsequently, Pgk1 was tested on muscle tissue collected from 11-week-old wt and R6/2 mice. Pgk1 showed low variation and no genotypic difference and was confirmed as reference gene for R6/2 (see Supplementary Material, Fig. S1B). In neuronal cultures, all 10 reference genes were affected by the camptothecin treatment and therefore, RNA input was used for normalization (see Supplementary Material, Fig. S5). RNA quality was confirmed to be sufficient by determining the RNA integrity number (80) and the S28/S18 rRNA ratio with an Agilent 2100 bioanalyzer (Supplementary Material, Fig. S6). For the MEFs, we used Rpl13a as reference gene, which showed no changes after camptothecin treatment and was not affected by the genotype of the cell line (data not shown). For primers used in this analysis, see Supplementary Material, Table S1.

The qPCR was run with an SYBR Green Power master mix (Applied Biosystems) on the ABI Prism 7500 Sequence Detection System. The primers were purchased from IDT. Gene expression was calculated using the delta–delta Ct method.

Mesoscale ELISA for detection of cleaved lamin A

This method is described in detail for MEF as well as primary neuronal samples elsewhere (42). Muscle tissue samples were lysed in 50 mm HEPES pH 7.4, 100 mm NaCl, 1% Igepal, 1 mm EDTA and 10% glycerol with 4.2 mm pefabloc and ‘Complete’ protease inhibitor cocktail (Roche) with an electric homogenizer on ice, and protein concentrations were determined in the cleared lysates after centrifugation. Samples were adjusted to 2 µg/µl in lysis buffer and further diluted to 0.2 µg/µl in PBS. 5 µl aliquots were spotted onto a Mesoscale ELISA plate and processed as described in (42).

Immunofluorescence analysis

Cells were grown on coverslips and fixed with 4% paraformaldehyde in PBS for 1 h at room temperature. After permeabilization with 0.3% Triton X-100 in PBS, cells were stained with antibodies against p53 and COX IV (both from Cell Signaling Technologies) and secondary antibodies coupled to Alexa-586 and Alexa-488 dyes (Invitrogen). Coverslips were mounted on microscopy slides using mounting medium containing Hoechst dye (Invitrogen) and visualized on a fluorescence microscope.

Statistical analysis

Data are expressed as means ± standard error and as box and whisker plot with median, maximal and minimal values. Where appropriate, the results were analysed using one-way ANOVA with the Tukey post hoc test or two-way ANOVA with the Bonferroni post hoc test. Pairwise comparisons between genotypes were assessed with a Student's t-test. Analysis for P-values was carried out with GraphPad Prism Ver.5. Differences were considered statistically significant when P < 0.05: *P < 0.05; **P < 0.01; ***P < 0.001.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

Conflict of Interest statement. None declared.

FUNDING

This work was supported by a grant from the Canadian Institute of Health Research (CIHR) [CGD-85375]. M.R.H. is a Killam University Professor and holds a Canada Research Chair in Human Genetics. D.E.E. was supported by a CIHR postdoctoral fellowship. N.H.S. was supported by the Ph.D. School of Genetic Medicine, University of Copenhagen and a Ripples of Hope post-doctoral fellowship. S.L. was supported by a CIHR Doctoral Award. B.R.L. is supported by the Michael Smith Foundation for Health Research. The R6/2 mouse work was supported by a grant from Stadslæge Svend Ahrendt Larsen and Grosserer Jon Johannesens Foundation. We thank Lili Liu for the preparation of primary neuronal cultures and Yu-zhou Yang for the generation of the caspase-6 antibody. Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD. The role of the NICHD Brain and Tissue Bank is to distribute tissue, and therefore, cannot endorse the studies performed or the interpretation of results.

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Author notes

The authors wish it to be known that, in their opinion, the first 2 authors should be regarded as joint first Authors

Supplementary data