Mitochondrial Dysfunction Reveals the Role of mRNA Poly(A) Tail Regulation in Oculopharyngeal Muscular Dystrophy Pathogenesis

Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.     

A Drosophila model of oculopharyngeal muscular dystrophy reveals intrinsic toxicity of PABPN1

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of particular muscles. OPMD is caused by short GCG repeat expansions within the gene encoding the nuclear poly(A)-binding protein 1 (PABPN1) that extend an N-terminal polyalanine tract in the protein. Mutant PABPN1 aggregates as nuclear inclusions in OMPD patient muscles. We have created a Drosophila model of OPMD that recapitulates the features of the human disorder: progressive muscle degeneration, with muscle defects proportional to the number of alanines in the tract, and formation of PABPN1 nuclear inclusions. Strikingly, the polyalanine tract is not absolutely required for muscle degeneration, whereas another domain of PABPN1, the RNA-binding domain and its function in RNA binding are required. This demonstrates that OPMD does not result from polyalanine toxicity, but from an intrinsic property of PABPN1. We also identify several suppressors of the OPMD phenotype. This establishes our OPMD Drosophila model as a powerful in vivo test to understand the disease process and develop novel therapeutic strategies.     

Lithium chloride attenuates cell death in oculopharyngeal muscular dystrophy by perturbing Wnt/β-catenin pathway

Expansion of polyalanine tracts causes at least nine inherited human diseases. Among these, a polyalanine tract expansion in the poly (A)-binding protein nuclear 1 (_expPABPN1) causes oculopharyngeal muscular dystrophy (OPMD). So far, there is no treatment for OPMD patients. Developing drugs that efficiently sustain muscle protection by activating key cell survival mechanisms is a major challenge in OPMD research. Proteins that belong to the Wnt family are known for their role in both human development and adult tissue homeostasis. A hallmark of the Wnt signaling pathway is the increased expression of its central effector, beta-catenin (β-catenin) by inhibiting one of its upstream effector, glycogen synthase kinase (GSK)3β. Here, we explored a pharmacological manipulation of a Wnt signaling pathway using lithium chloride (LiCl), a GSK-3β inhibitor, and observed the enhanced expression of β-catenin protein as well as the decreased cell death normally observed in an OPMD cell model of murine myoblast (C2C12) expressing the expanded and pathogenic form of the _expPABPN1. Furthermore, this effect was also observed in primary cultures of mouse myoblasts expressing _expPABPN1. A similar effect on β-catenin was also observed when lymphoblastoid cells lines (LCLs) derived from OPMD patients were treated with LiCl. We believe manipulation of the Wnt/β-catenin signaling pathway may represent an effective route for the development of future therapy for patients with OPMD.     

PABPN1 polyalanine tract deletion and long expansions modify its aggregation pattern and expression

Expansions of a (GCN)10/polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) cause autosomal dominant oculopharyngeal muscular dystrophy (OPMD). In OPMD muscles, as in models, PABPN1 accumulates in intranuclear inclusions (INIs) whereas in other diseases caused by similar polyalanine expansions, the mutated proteins have been shown to abnormally accumulate in the cytoplasm. This study presents the impact on the subcellular localization of PABPN1 produced by large expansions or deletion of its polyalanine tract. Large tracts of more than 24 alanines result in the nuclear accumulation of PABPN1 in SFRS2-positive functional speckles and a significant decline in cell survival. These large expansions do not cause INIs formation nor do they lead to cytoplasmic accumulation. Deletion of the polyalanine tract induces the formation of aggregates that are located on either side and cross the nuclear membrane, highlighting the possible role of the N-terminal polyalanine tract in PABPN1 nucleo-cytoplasmic transport. We also show that even though five other proteins with polyalanine tracts tend to aggregate when over-expressed they do not co-aggregate with PABPN1 INIs. This study presents the first experimental evidence that there may be a relative loss of function in OPMD by decreasing the availability of PABPN1 through an INI-independent mechanism.     

Hsp70 Chaperones and Type I PRMTs Are Sequestered at Intranuclear Inclusions Caused by Polyalanine Expansions in PABPN1

Genomic instability at loci with tandem arrays of simple repeats is the cause for many neurological, neurodegenerative and neuromuscular diseases. When located in coding regions, disease-associated expansions of trinucleotide repeats are translated into homopolymeric amino acid stretches of glutamine or alanine. Polyalanine expansions in the poly(A)-binding protein nuclear 1 (PABPN1) gene causes oculopharyngeal muscular dystrophy (OPMD). To gain novel insight into the molecular pathophysiology of OPMD, we studied the interaction of cellular proteins with normal and expanded PABPN1. Pull-down assays show that heat shock proteins including Hsp70, and type I arginine methyl transferases (PRMT1 and PRMT3) associate preferentially with expanded PABPN1. Immunofluorescence microscopy further reveals accumulation of these proteins at intranuclear inclusions in muscle from OPMD patients. Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure. Taken together, our results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones. This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity.     

Oculopharyngeal muscular dystrophy: potential therapies for an aggregate-associated disorder

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, autosomal dominant disease caused by the abnormal expansion of a polyalanine tract within the coding region of poly(A) binding protein nuclear 1 (PABPN1). The resultant mutant PABPN1 forms aggregates within the nuclei of skeletal muscle fibres. The mechanism by which the polyalanine expansion mutation in PABN1 causes disease is unclear. However, the mutation is thought to confer a toxic gain-of-function on the protein. Despite controversy over the role of aggregates, it has been consistently shown that agents that reduce aggregate load in cell models of OPMD also reduce levels of cell death. Recently generated animal models of OPMD will help elucidate the mechanism of disease and allow the trial of potential therapeutics. Indeed, administration of known anti-aggregation drugs attenuated muscle weakness in an OPMD mouse model. This suggests that anti-aggregation therapies may be beneficial in OPMD.     

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.     

Doxycycline attenuates and delays toxicity of the oculopharyngeal muscular dystrophy mutation in transgenic mice

The muscular dystrophies are a heterogeneous group of disorders for which there are currently no cures. Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset, progressive disease that generally presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is 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. In unaffected individuals, (GCG)6 codes for the first six alanines in a homopolymeric stretch of ten alanines. In most individuals with OPMD this (GCG)6 repeat is expanded to (GCG)8-13, leading to a stretch of 12-17 alanines in mutant PABPN1. PABPN1 with an expanded polyalanine tract forms aggregates consisting of tubular filaments within the nuclei of skeletal muscle fibers. We have developed a transgenic mouse model of OPMD that manifests progressive muscle weakness accompanied by intranuclear aggregates and TUNEL-stained nuclei in skeletal muscle fibers. The onset and severity of these abnormalities were substantially delayed and attenuated by doxycycline treatment, which may exert its therapeutic effect by reducing aggregates and by distinct antiapoptotic properties. Doxycycline may represent a safe and feasible therapeutic for this disease.     

The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein

Background

Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear.

Results

In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells.

Conclusions

Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD.     

Oculopharyngeal muscular dystrophy (OPMD): analysis of the PABPN1 gene expansion sequence in 86 patients reveals 13 different expansion types and further evidence for unequal recombination as the mutational mechanism

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset neuromuscular degenerative disease characterised by proximal muscle weakness, ptosis and swallowing difficulty. The causative genetic abnormality is an expansion consisting of 2–7 additional base triplets in a repeat sequence in exon 1 of the PABPN1 (PABP2) gene and results in an increase in length of the polyalanine tract in the PABPN1 protein from 10 to 12–17 residues. The expansions are stable through meiosis and mitosis suggesting a different mechanism of mutation from that of most other triplet repeat mutations. Most reports describe OPMD expansions as consisting of multiples of a GCG sequence. However, some studies have detected GCA interspersions. We have analysed 86 OPMD patients with a PABPN1 gene expansion, including three compound heterozygotes, and have identified 13 different types of expansion mutation, six of which contain GCA and GCG and almost all of which are consistent with a mutational mechanism of unequal recombination.     

PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin–proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.     

The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

Although the canonical transforming growth factor β signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin–dependent process, whereas increased BMP–Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders.     

Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO₄, and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40, and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein.     

Myostatin promotes the wasting of human myoblast cultures through promoting ubiquitin-proteasome pathway-mediated loss of sarcomeric proteins

Myostatin is a negative regulator of skeletal muscle growth and in fact acts as a potent inducer of "cachectic-like" muscle wasting in mice. The mechanism of action of myostatin in promoting muscle wasting has been predominantly studied in murine models. Despite numerous reports linking elevated levels of myostatin to human skeletal muscle wasting conditions, little is currently known about the signaling mechanism(s) through which myostatin promotes human skeletal muscle wasting. Therefore, in this present study we describe in further detail the mechanisms behind myostatin regulation of human skeletal muscle wasting using an in vitro human primary myotube atrophy model. Treatment of human myotube populations with myostatin promoted dramatic myotubular atrophy. Mechanistically, myostatin-induced myotube atrophy resulted in reduced p-AKT concomitant with the accumulation of active dephosphorylated Forkhead Box-O (FOXO1) and FOXO3. We further show that addition of myostatin results in enhanced activation of atrogin-1 and muscle-specific RING finger protein 1 (MURF1) and reduced expression of both myosin light chain (MYL) and myosin heavy chain (MYH). In addition, we found that myostatin-induced loss of MYL and MYH proteins is dependent on the activity of the proteasome and mediated via SMAD3-dependent regulation of FOXO1 and atrogin-1. Therefore, these data suggest that the mechanism through which myostatin promotes muscle wasting is very well conserved between species, and that myostatin-induced human myotube atrophy is mediated through inhibition of insulin-like growth factor (IGF)/phosphoinositide 3-kinase (PI3-K)/AKT signaling and enhanced activation of the ubiquitin-proteasome pathway and elevated protein degradation.