Human Molecular Genetics Advance Access originally published online on January 4, 2008
Human Molecular Genetics 2008 17(8):1097-1108; doi:10.1093/hmg/ddm382
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Wild-type PABPN1 is anti-apoptotic and reduces toxicity of the oculopharyngeal muscular dystrophy mutation
Department of Medical Genetics, University of Cambridge, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, UK
* To whom correspondence should be addressed. Tel: +44 1223762608; Fax: +44 1223331206; Email: dcr1000{at}hermes.cam.ac.uk
Received October 17, 2007; Revised December 7, 2007; Accepted December 26, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, progressive disease caused by the abnormal expansion of a polyalanine tract-encoding (GCG)n trinucleotide repeat in the poly-(A) binding protein nuclear 1 (PABPN1) gene. OPMD is generally inherited as an autosomal dominant disorder and the polyalanine expansion mutation is thought to confer a toxic gain-of-function on mutant PABPN1 which forms aggregates within skeletal myocyte nuclei. Here we describe a novel beneficial function of wild-type PABPN1. Wild-type PABPN1 over-expression can reduce mutant PABPN1 toxicity in both cell and mouse models of OPMD. In addition, wild-type PABPN1 provides some protection to cells against pro-apoptotic insults distinct from the OPMD mutation such as staurosporine treatment and Bax expression. Conversely, PABPN1 knockdown (which itself is not toxic) makes cells more susceptible to apoptotic stimuli. The protective effect of wild-type PABPN1 is mediated by its regulation of X-linked inhibitor of apoptosis (XIAP) protein translation. This normal activity of PABPN1 is partially lost for mutant PABPN1; elevated levels of XIAP are seen in mice expressing a wild-type but not a mutant PABPN1 transgene. This raises the possibility that a compromise of the anti-apoptotic function of PABPN1 might contribute to the disease mechanism of OPMD.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disorder caused by the abnormal expansion of a (GCG)n trinucleotide repeat in the coding region of the poly-(A) binding protein nuclear 1 (PABPN1) gene (1). In unaffected individuals, (GCG)6 codes for the first six alanines in a homopolymeric stretch of ten alanines. In most patients this (GCG)6 repeat is expanded to (GCG)8–13, leading to a stretch of 12–17 alanines in mutant PABPN1. The polyalanine expansion mutation is thought to confer a toxic gain-of-function on mutant PABPN1 (2,3) and PABPN1 with an expanded polyalanine tract forms aggregates consisting of tubular filaments within the nuclei of skeletal muscle fibres (4–6).
OPMD patients typically present in their fifth or sixth decades with initial symptoms of dysphagia and ptosis caused by weakening of the pharyngeal and ocular muscles, respectively (2). OPMD is a progressive myopathy and the disease can proceed to affect all voluntary muscles leading to proximal muscle weakness.
We have modelled OPMD in mice using the human skeletal actin promoter to drive high level, muscle-specific expression of a mutant PABPN1 transgene encoding a 17 alanine repeat (A17; sufficient to cause human disease) (7). All mice were normal at birth. However, mice expressing the mutant PABPN1 transgene developed a progressive muscle weakness accompanied by the formation of aggregates containing mutant PABPN1 in skeletal myocyte nuclei (a feature of the human disease), in the absence of lymphocytic infiltration and inflammation (7). This phenotype was not seen in mice expressing a control, wild-type PABPN1 transgene (A10) at similar levels. A17 mice have increased numbers of TUNEL-positive nuclei and elevated levels of BAX compared with mice expressing a wild-type PABPN1 transgene at equivalent levels or non-transgenic mice (7). In a cell model of OPMD, we see activation of the intrinsic cell death pathway as shown by an increased number of abnormal, apoptotic nuclei, elevated caspase 3 activity and cytochrome c release from the mitochondria to cytosol (7,8). Apoptotic markers, seen in both cell and mouse models of OPMD, were reversed by doxycycline treatment (7,9), which is thought to exert its effect by both reducing aggregate load and by distinct anti-apoptotic effects. We have also shown that lithium decreases toxicity induced by the expression of a fusion protein comprising a polyalanine expansion tagged to green fluorescent protein in a Drosophila model, at least partially via activation of the anti-apoptotic Wnt pathway (10). Importantly, the phenotype induced by the expression of a mutant PABPN1 transgene in a Drosophila model of OPMD was suppressed by the viral anti-apoptotic protein P35 (11). These data are compatible with the possibility that the enhanced apoptosis seen in these OPMD models may be causally influencing the phenotype.
Wild-type PABPN1 is a ubiquitously expressed protein that binds specifically and with a high affinity to poly(A) tails at the 3’ ends of mRNAs (12). PABPN1 forms a polyadenylation complex with cleavage and polyadenylation specificity factor (CPSF) and poly(A) polymerase (PAP), stimulates PAP-mediated polyadenylation and controls the length of poly(A) tails to 
250 nucleotides. PABPN1 contains a nuclear localization sequence (13) and is predominantly nuclear at steady state levels. However, it is also thought to have a role in the nuclear export of mRNA, shuttling between the nucleus and cytosol by a carrier-mediated mechanism (13). More recently, an essential role for PABPN1 in cytoplasmic poly(A) tail length control during Drosophila early development has been proposed (14). Here we have discovered that over-expression of wild-type PABPN1 could reduce toxicity caused by the expression of mutant PABPN1 in both our cell model and mouse model of OPMD. Furthermore, wild-type PABPN1 can protect against pro-apoptotic insults distinct from the OPMD mutation such as BAX transfection and staurosporine treatment, while knock-down of endogenous PABPN1 makes cells more susceptible to these insults. This protective effect of wild-type PABPN1 may be mediated by its upregulation of X-linked inhibitor of apoptosis (XIAP) at the translational level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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We initially used our previously described cell model of OPMD (8) to explore the protective effects of wild-type PABPN1. COS-7 cells have increased levels of cell death (abnormal apoptotic nuclei) when transfected with EGFP-tagged mutant PABPN1 (EGFPA17; 17 alanines) compared with cells expressing otherwise identical constructs with wild-type (10 alanines) repeats (8). We used COS-7 cells as they express PABPN1 (a ubiquitously expressed protein), give a good readout of cell death due to mutant PABPN1 expression compared with other cell lines we have tried and can be efficiently transfected with over-expression constructs and siRNA. COS-7 cells express HA- and EGFP-tagged A10 and A17 constructs at similar levels that are much higher than levels of endogenous PABPN1 (Supplementary Material, Fig. S1A). Examples of cell phenotypes that can be induced by mutant PABPN1 expression and that are scored in this paper are shown in Supplementary Material, Figure S1B–D. Fewer EGFPA17-expressing cells had an abnormal, apoptotic nuclear morphology when co-transfected with HA-tagged wild-type PABPN1 (HA-A10), compared with co-transfection with empty vector (HA; Fig. 1A). This rescue affect was independent of aggregate load; the number of EGFP-positive aggregates was not altered when cells were co-transfected with EGFPA17 and HA-A10 compared with EGFPA17 and empty vector (HA) (Fig. 1B). Thus, wild-type PABPN1 does not appear to modulate toxicity by reducing oligomerization or aggregation of EGFPA17. We investigated if wild-type PABPN1 could protect against apoptotic insults distinct from the OPMD mutation and induced cell death both chemically (staurosporine treatment) and genetically (BAX over-expression). BAX specifically commits the cell to apoptosis by permeabilizing the outer mitochondrial membrane, resulting in the release of proteins like cytochrome c into the cytosol, which, in turn, leads to caspase activation. COS-7 cells transfected with EGFPA10 were more resistant to staurosporine treatment (as shown by abnormal, apoptotic nuclei) than cells transfected with empty vector (EGFPC1), cell death was reduced from 54.8% in staurosporine treated, EGFPC1 transfected cells to 42.8% in staurosporine treated, EGFPA10 transfected cells (a 22% reduction, Fig. 1C). Likewise, wild-type PABPN1 reduced BAX-induced apoptosis by
25% (Fig. 1D). We wanted to see if reducing levels of endogenous PABPN1 in COS-7 cells could affect susceptibility to apoptosis and knocked down PABPN1 using siRNA. PABPN1 protein levels were reduced in cells transfected with siRNA directed against PABPN1 compared with cells transfected with a control, scrambled siRNA (which targets no specific sequence) at 72 and 96 h post-transfection (Fig. 1E). PABPN1 knock-down itself was not obviously toxic to COS-7 cells in the time period examined (Fig. 1F,G,I,J). However, cells transfected with siRNA targeting PABPN1 were more susceptible to apoptosis induced by both staurosporine treatment (Fig. 1F,G,H) and BAX over-expression (Fig. 1I and J) than cells transfected with control siRNA.
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We tested if wild-type PABPN1 could protect against mutant PABPN1 toxicity in vivo, using a previously described mouse model of OPMD (7). A10 mice express a wild-type PABPN1 transgene with 10 alanines and A17 mice have a mutant PABPN1 transgene with 17 alanines; both transgenes are expressed at similar levels on the same FvB background (7). A17 mice are undistinguishable from non-transgenic mice at birth and even at 2 months of age no significant difference in muscle strength can be seen between mice expressing the A17 transgene and their non-transgenic littermates. A17 mice develop a progressive muscle weakness that can be seen from
4 months of age—early enough to allow studies of chemical and genetic modifiers (7). Hemizygous A10 and A17 mice [lines A10-1 and A17-1 (7), which express high transgene levels that are comparable] were crossed to produce litters comprising double transgenic mice expressing both A10 and A17 transgenes (A10xA17), mice expressing A10 alone, mice expressing A17 alone and non-transgenic mice. The OPMD mutation results in muscle weakness in mice as the primary phenotype (7). Double transgenic A10xA17 mice were stronger than littermates expressing the mutant A17 transgene alone, as assessed by fore-limb grip strength (Fig. 2A) and grip strength from all limbs (Fig. 2B). No difference in grip strength between A10 mice and non-transgenic mice was observed (Fig. 2A and B; 4). We also saw reduced levels of apoptosis (TUNEL-positive nuclei) in biceps sections of 6-month-old A10xA17 mice, compared with A17 mice (Fig. 2C). However, we did not observe a reduction in the number of nuclei containing PABPN1-labelled aggregates in biceps sections from A10xA17, compared with A17 mice and actually saw a slight increase in aggregate load (Fig. 2D). Wild-type PABPN1 itself can form aggregates, although at much lower rates than mutant PABPN1 (8,15) and the higher number of nuclei containing aggregates in double transgenic mice may be a reflection of this. Alternatively, the A10 transgene may protect aggregate-containing nuclei from apoptosis and thus increase aggregate number in skeletal muscle fibres by increasing survival of cells with increased mutant PABPN1 accumulation. We also compared the histology of muscle sections from A17 and A10xA17 mice and observed a significant but slight decrease in the number of centralized nuclei (as a percentage of the total myoblast nuclei scored) in haematoxylin and eosin stained biceps sections from 6-month-old A10xA17 mice compared with A17 mice (Fig. 2E). Centralized nuclei are thought to represent a regenerative process occurring in muscle. However, it is uncertain what the real biological relevance of this is in the context of OPMD.
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We used our OPMD cell model to start investigating the mechanism by which wild-type PABPN1 was exerting its anti-apoptotic effect. Caspase 3 is activated in cells transfected with mutant EGFPA17 and levels of active caspase 3 (as shown by western blot) were reduced in cells co-transfected with HA-A10 compared with cells co-transfected with empty vector (HA; Fig. 3A and B). These effects of wild-type PABPN1 over-expression are compatible with siRNA data in Fig. 1F–I. Likewise, expression of wild-type PABPN1 (EGFPA10) reduced staurosporine-induced caspase 3 activation when compared with expression of empty vector (EGFPC1; Fig. 3C–E). However, wild-type PABPN1 did not reduce the number of cells with cytochrome c release (faint diffuse cytochrome c immuno-labelling as opposed to the bright punctuate labelling of mitochondria seen in unaffected cells) in response to either staurosporine treatment (Fig. 3E) or A17 expression (Fig. 3F). This suggested that wild-type PABPN1 was acting downstream of cytochrome c release in the apoptotic pathway and inhibiting caspase activation. We considered X-linked inhibitor of apoptosis (XIAP) as a candidate, as it can directly inhibit caspase activation and thus prevent apoptosis (16,17). XIAP is a potent anti-apoptotic molecule and its over-expression is sufficient to protect against cell death in a variety of in vivo paradigms (18–20). Western blotting of biceps muscle lysates from 6-month-old mice showed an increase in XIAP protein levels in both A10 and A10xA17 mice compared with non-transgenic littermates (Fig. 3G and H). Conversely, levels of XIAP were reduced in COS-7 cells transfected with siRNA against PABPN1 compared with cells transfected with control siRNA (Fig. 3I and J). A17 mice did not have elevated levels of XIAP in biceps muscle samples, when compared with non-transgenic littermates (Fig. 3G and H). The A10 and A17 transgenes are expressed at similar levels (7) and this suggests that mutant PABPN1 is unable to regulate levels of XIAP to the same extent as wild-type PABPN1 does.
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Wild-type PABPN1 was likely to be exerting its anti-apoptotic effect through the regulation of XIAP levels and we were interested to see if XIAP over-expression itself could protect from mutant PABPN1 toxicity. Cells co-transfected with EGFPA17 and an XIAP expression vector showed reduced levels of cell death compared with cells co-transfected with EGFPA17 and empty vector, assessed by western blotting for levels of active caspase 3 (Fig. 4A) and scoring of abnormal apoptotic nuclei (Fig. 4B). In addition, the protective effect of wild-type PABPN1 against staurosporine treatment was abolished by XIAP knockdown (Fig. 4C–F).
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Levels of XIAP are tightly regulated in response to a variety of cellular stimuli and can be regulated at the transcriptional level (17,21,22), at the translational level (23) or through XIAP ubiquitination and proteasome degradation (24). Quantitative RT–PCR showed that, despite an increase in XIAP protein levels, A10 mice did not have elevated levels of XIAP RNA compared with non-transgenic mice (Fig. 5A). Similarly, levels of XIAP RNA were unaltered in cells transfected with siRNA against PABPN1, compared with cells transfected with control siRNA (Fig. 5B). Thus, an effect of wild-type PABPN1 on the transcriptional regulation of XIAP or the degradation of XIAP mRNA transcripts could be discounted. Wild-type PABPN1 is altering XIAP protein levels independently of RNA levels, most likely through translation of XIAP mRNA transcripts or degradation of XIAP protein. To test if wild-type PABPN1 is altering the degradation or translation of XIAP, we first carried out an experiment using the protein synthesis inhibitor cycloheximide. This cycloheximide pulse-chase protocol tests if the degradation of XIAP is modulated by wild-type PABPN1. Cells transfected with siRNA targeting PABPN1 had reduced protein levels of XIAP compared with cells transfected with control siRNA (Fig. 5C). The degradation of XIAP in the cycloheximide chase period was not enhanced by PABPN1 knock-down (Fig. 5C and D), suggesting that we should test if the effect was at the levels of translation. To test if wild-type PABPN1 was affecting the translation of XIAP, we carried out a 35S-methionine pulse experiment. COS-7 cells transfected with either control or PABPN1 siRNA were pulsed with 35S-methionine for 1 h prior to immuno-precipitation with an XIAP antibody. There was reduced incorporation of 35S-methionine into XIAP (as a function of incorporation into total protein synthesis) in PABPN1 knock-down cells compared with control cells, corresponding to a reduction in XIAP translation (Fig. 5E and F).
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We have excluded some possible mechanisms by which PABPN1 may be regulating XIAP at the translational level. XIAP has a long 5'-UTR that contains a large number of out-of-frame initiation sites, short open-reading frames and a high degree of predicted secondary structure (25,26). Therefore it is inefficiently translated. The 5'-UTR of XIAP contains an internal ribosomal entry site (IRES) thought to translate the protein under stress conditions, when cap-dependent translation is inhibited (23). Using a bicistronic construct (23), we have shown that PABPN1 has no effect on XIAP-IRES activity (data not shown). However, given the problems and caveats associated with this experimental approach (27,28) an effect of PABPN1 on XIAP-IRES activity cannot be completely discounted. Increasing or decreasing the efficiency of translation [e.g. by altering eIF4E levels (29) or the phosphorylation of 4E-BP1 (30)] has an effect on cell death and this may be mediated by XIAP (31,32). PABPN1 does not alter levels of eIF4E or the phosphorylation status of 4E-BP1 (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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Here we have described a novel, anti-apoptotic function for wild-type PABPN1 that is mediated via translational regulation of XIAP. We have shown that wild-type PABPN1 levels can modulate protein levels of XIAP both in COS-7 cells and in muscle from transgenic mice. Importantly, the protective effects of wild-type PABPN1 in cells are not seen when XIAP is knocked down, suggesting that this may be the predominant mediator of this beneficial function, at least in cells. Wild-type PABPN1 over-expression is anti-apoptotic in cells and improves muscle strength in OPMD model mice. It may be likely that elevated XIAP levels mediated by wild-type PABPN1 over-expression in mice will have anti-apoptotic effects in vivo. XIAP is a potent anti-apoptotic molecule (16,17) that has been shown to be protective in a variety of disease models (18–20). This raises the possibility that wild-type PABPN1 is protecting against the muscle-phenotype-induced mutant protein via an anti-apoptotic route. Apoptosis has been proposed to be an important contributor to muscle fibre loss in some muscular dystrophies (33,34). Markers of apoptosis are increased in the muscle of certain muscular dystrophies (34–38) and strategies to reduce apoptosis have been shown to improve muscle strength in some transgenic models of muscular dystrophy such as limb-girdle muscular dystrophy (39,40). Levels of apoptosis have not been assessed in human OPMD muscle biopsies. It may prove difficult to detect apoptotic nuclei above background levels in a late-onset, progressive disease if the clearance of apoptotic nuclei keeps pace with their formation. It may be much easier to see apoptotic cell death in mouse models of OPMD where a more severe, earlier-onset form of the disease is induced. Indeed, increased levels of TUNEL-positive nuclei and apoptotic markers have been seen in two independent mouse models of OPMD (7,41) and the phenotype induced by the expression of a mutant PABPN1 transgene in a Drosophila model of OPMD was suppressed by the viral anti-apoptotic protein P35 (11). Levels of TUNEL-stained nuclei seen in A17 mice were decreased by over-expression wild-type PABPN1. Thus, we believe that it will be important to test in future whether specific anti-apoptotic strategies alleviate mutant PABPN1-induced muscle weakness in mice, analogous to what has been seen in limb-girdle dystrophy (39,40). In mice, mutant PABPN1 over-expression induces lower levels of XIAP than wild-type PABPN1 over-expression, raising the possibility that this normal function of PABN1 is partially lost for mutant PABPN1. In other words, the OPMD mutation may cause disease primarily via a gain-of-function mechanism which may be exacerbated to some extent by the partial loss of anti-apoptotic, wild-type PABPN1 function. There is currently no PABPN1 knock-out mouse available. In future, heterozygous null animals may be a useful tool to assess the effects of a partial loss of PABPN1 function in vivo and to test whether a compromise of the anti-apoptotic function of PABPN1 contributes to OPMD disease mechanism. However, our cell culture data suggest that any strategies aiming to knock down mutant PABN1 expression to alleviate symptoms of this disease will need to avoid perturbing wild-type PABN1 levels.
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MATERIALS AND METHODS |
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Generation of HA tagged PABPN1 construct
pEGFPC1-PABPN1A10 (8) was digested with BamHI and blunt-ended with Klenow before cutting with KpnI to release the wild-type PABPN1 cDNA fragment. The HA-tag vector pHM6 (Roche Diagnostics) was digested with EcoRI and blunt-ended before digestion with KpnI. The PABPN1 cDNA fragment was ligated into the blunt and EcoRI sites of pHM6 to produce a vector encoding HA-tagged wild-type PABPN1 (HA-A10; pHM6-PABPN1-A10).
Cell culture
COS-7 (African green monkey kidney) were maintained in Dulbecco's modified medium (Sigma Aldrich) supplemented with 10% fetal bovine serum, 100 U/ml1 penicillin/streptomycin, 2 mM l-glutamine and 1 mM sodium pyruvate at 37°C in 5% carbon dioxide. Cells were transiently co-transfected with plasmids encoding EGFP-tagged A17 [pEGFPC1-PABPN1-A17 (8)], EGFP-tagged A10 [pEGFPC1-PABPN1-A10 (8)], HA-tagged A10 (pHM6-PABPN1-A10; described above), EGFPC1 (Clontech), pHM6 (empty HA-tag vector; Roche) and XIAP (clone TC19404 in pCMV6-XL5; OriGene Technologies Inc.) using lipofectAMINE reagent (Invitrogen) and following the manufacturers protocol. All co-transfections were carried out with plasmids at a molar ratio of 1:1 and spiked with equal amounts of dsRed2 (Clontech) as a transfection control. Cells were incubated for 48 h before collection.
siRNA against PABPN1 (Pre-designed siRNA AM16704; Ambion Inc.) or its control siRNA (Silencer negative control siRNA; Ambion Inc.) and siRNA targeting XIAP (siGENOME smart pool siRNA; Dharmacon Inc.) or control siRNA (siCONTROL non-targeting pool; Dharmacon Inc.) was transfected into cells at 50 nM using lipofectamine 2000 (Invitrogen) following manufacturers instructions. Knockdown of both endogenous PABPN1 and XIAP was seen at 72 h.
To induce apoptosis, staurosporine (3 µM; Sigma Aldrich) was added to the cell media for the final 6 h post-transfection. In an alternative model of cell death, GFP-BAX (42) (a kind gift from R.J. Youle, NIH, Bethesda, MD) was transfected into cells 16 h before collection.
For the cycloheximide pulse-chase experiments, cells were transfected with siRNA (as above), incubated for 48 h and then treated with cyclohexamide (10 µg/ml) for a further 24 h before collection.
Transgenic mice
OPMD transgenic mice have been previously described (7). All studies and procedures were carried out following UK Home Office regulations and animals were caged under standard conditions (12 h light, 12 h dark; food and water available ad libitum). Mice heterozygous for mutant (line A17-1) or wild-type (line A10-1) PABPN1 transgenes were crossed to produce double transgenics (A10xA17) and compared with littermates expressing a single A10 or A17 transgene and non-transgenic littermates. Genotype was determined by Southern blot; the A10 and A17 transgenes differ by only 7 alanine codons, so were distinguished from each other using a restriction enzyme that cuts once within the transgene and in the DNA sequence surrounding the transgene. The resulting restriction pattern differs between the different OPMD transgenic mice lines and is dependent on integration site. We isolated genomic DNA from tail biopsies by lysis in 0.25 mg/ml proteinase K in 1% SDS, 100 mM NaCl, 100 mM EDTA, 50 mM Tris, pH 8 at 55°C overnight followed by salt extraction and ethanol precipitation. Ten micrograms of genomic DNA was digested with EcoRI, run on a 0.8% (w/v) agarose gel and transferred to positively charged nylon membrane (Amersham Biosciences). Membranes were hybridized with [
32P]dATP-labelled probes generated using the Prime-a-Gene labelling system (Promega) and a 1 kb dsDNA template derived from SacI digestion of pBUDCE4-HSA-PABPBwt (plasmid used in the production of A10 mice). Bands were visualized by autoradiography.
Grip strength was assessed using a grip strength meter (Bioseb). Mice were assessed with the investigator blind to genotype. Mice were given alphanumeric identities that provided no clue to genotype. We analysed grip strength meter data from each treatment time point with unpaired t-tests and the overall effect from all treatment time points with repeated-measures ANOVA (STATVIEW software, version 4.53; Abacus Concepts).
Western blotting
Nuclear extracts were prepared using a standard kit (Active Motif) and whole cell lysates prepared by homogenizing tissue or cell pellets in 50 mM Tris–HCl pH7.4, 0.5% Triton X-100 with protease inhibitor cocktail (Complete; Roche Diagnostics). Proteins were separated on 10% SDS–polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond ECL membrane; Amersham Biosciences), which were blocked by incubation in 5% dried milk in 0.1 M PBS, 0.1% Tween-20, pH 7.6. Membranes were probed with primary antibodies raised against PABPN1 (a kind gift from Prof Elmar Wahle, Halle, Germany; 1:5000), Histone H3 (loading control for nuclear extracts; Cell Signalling Technology; 1:1000), caspase 3 (Cell Signalling Technology; 1:1000), XIAP (Cell Signalling Technology; 1:1000), EGFP (Clontech; 1:5000), dsRed (BD Pharmigen; 1:500) or actin (loading control for whole tissue or cell extracts; Sigma Aldrich; 1:5000). HRP-conjugated antibodies (Amersham Biosciences; 1:5000) were then added to the blots. Immuno-reactive bands were detected with enhanced chemiluminescence reagent (ECL; Amersham Biosciences) and signal visualized by the exposing membrane to ECL Hyperfilm (Amersham Biosciences). Densitometric analysis of blots was carried out using ImageJ software (NIH). At least three separate experiments were analysed, band intensities were normalized to actin band intensity and the control condition was set to 100%. Significance was determined using unpaired t-tests.
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde in 0.1 M PBS pH 7.6, immunolabelled with antibodies raised against HA (Covance; 1:500), cytochrome c (Pharmingen; 1:500) or active caspase 3 (Promega; 1:250) and fluorophore-conjugated secondary antibodies (Alexa Fluor 488 goat anti-rabbit or Alexa Fluor 488 goat anti-mouse; 1:1000; Molecular Probes) and mounted in citifluor (Citifluor Ltd) containing 4',6-diamidino-2-phenylindole (DAPI; 3 µg/ml; Sigma-Aldrich Ltd) to visualize nuclei. Transfected cells were scored for abnormal apoptotic nuclei, aggregates, cytochrome c release from the mitochondria into the cytosol (diffuse cytochrome c immuno-labelling as opposed to the bright punctuate labelling of mitochondria seen in unaffected cells) and active caspase 3 (bright, fluorescent immuno-labelling using the active caspase 3 antibody). Experiments were carried out in sextuplicate and 200 cells per coverslip were scored with the investigator blind to the identity of the sample. Pooled estimates were calculated as odds ratios (OR; the ratios of the proportion of aggregate containing/normal (or TUNEL positive/TUNEL negative) nuclei in different experimental conditions) with 95% confidence intervals, as described previously. OR and P-values were determined by unconditional logistical regression analysis using the general log linear analysis option of SPSS Version 6.1 (SPSS, Chicago, IL).
Histology
Tissue was snap frozen in liquid nitrogen-cooled isopentane and 10 µm sections were cut on a cryostat (Leica Microsystems) to poly-L-lysine coated slides. Sections were fixed in acetone. For immuno-labelling, slides were blocked with 1% normal goat serum in 0.1 M PBS, 0.1% Triton X100 and then incubated, at 4°C overnight, in primary antibody (anti-PABPN1; a kind gift from Prof Elmar Wahle, Halle, Germany) diluted (1:500) in 1% normal goat serum in 0. 1M PBS, 0.1% Triton X100. Slides were washed in 0.1 M PBS, 0.1% Triton X100 and incubated in fluorophore-conjugated secondary antibody (Alexa Fluor 488 goat anti-rabbit; 1:1000; Molecular Probes) for 2 h at room temperature in the dark. Slides were washed again and sections mounted in citifluor (Citifluor Ltd) containing 4',6-diamidino-2-phenylindole (DAPI; 3 µg/ml; Sigma-Aldrich Ltd) to visualize nuclei. To remove soluble proteins, sections were incubated in 1 M KCl, 30 mM HEPES, 65 mM PIPES, 10 mM EDTA, 2 mM MgCl2, pH 6.9 for 1 h at room temperature prior to immuno-labelling. Aggregates are resistant to this KCl treatment (7). Fluorescent DNA fragmentation (TUNEL; terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay was carried out on skeletal muscle sections using a standard kit (ApoAlert DNA fragmentation assay kit; BD Biosciences). For histological analysis of muscle morphology and pathology, sections were stained with haemotoxylin and eosin (H&E). Nuclei that contained aggregates, TUNEL positive nuclei and centralized nuclei were scored. Three samples per group and 200 nuclei per sample were scored with the viewer blind to the identity of the slide. Pooled estimates were calculated as odds ratios as described above.
Quantitative RT–PCR
RNA was isolated from tissue or cell pellets using Trizol reagent (Invitrogen) following the manufacturers instructions. One microgram of RNA was treated with DNase1 (Invitrogen) to remove any contaminating genomic DNA and reverse transcribed using a Superscript III first strand synthesis kit (Invitrogen) following the manufacturers instructions. cDNA produced was subjected to quantitative PCR using Taqman pre-designed primers and probes for XIAP (Human, Hs00236913; mouse, Mm00776565; Applied Biosystems), mouse GAPDH (Applied Biosystems), human 18S (Applied Biosystems) in the presence of Taqman Universal PCR master mix (Applied Biosystems) on a Applied Biosystems 7900HT fast real-time PCR machine. As a control, an additional set of reactions omitting reverse transcriptase were performed.
35S-methionine pulse labelling
COS-7 cells were transfected with siRNA as above and incubated for 72 h. Cells incubated in methionine-free media (Invitrogen) for 2 h prior to pulsing with media containing 35S-labelled methionine (100 µci/ml for 1 h; GE Healthcare). Cells were washed in PBS, collected and lysed in buffer comprising 20 mM Tris–HCl pH 7.2, 2 mM MgCl2, 150 mM NaCl and 0.5% (v/v) NP-40 with protease inhibitor cocktail (Complete; Roche Diagnostics). Lysates were immuno-precipitated with antibody against XIAP (1:200; Cell Signalling Technology) and protein G conjugated agarose beads (Santa Cruz Biotechnology Inc.). Twenty microlitres of total cell lysate was kept as a control for 35S-methionine incorporation into total protein synthesis (Input). Immuno-precipitates and input were run on SDS–PAGE. Gels were opposed to a phosphorimager screen that was developed using a Storm Phosphorimager (GE Healthcare). Band intensities were quantified using ImageQuant software (GE Healthcare). This experiment was carried out nine times.
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SUPPLEMENTARY MATERIAL |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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Supplementary Material is available at HMG Online.
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FUNDING |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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This work was funded by the Wellcome Trust (Senior Fellowship to D.C.R.) and the Muscular Dystrophy Campaign, UK.
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ACKNOWLEDGEMENTS |
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The authors would like to thank E.Wahle for the kind gift of PABPN1 antibody, R.J. Youle for the EGFP-Bax construct, O. Sadiq for technical assistance and Drs M. Futter, F. Menzies, B. Ravikumar and S. Luo for helpful comments and discussions.
Conflict of Interest statement. None declared.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALFUNDINGREFERENCES |
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