HnRNP A1 and A/B interaction with PABPN1 in oculopharyngeal muscular dystrophy

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive ptosis, dysphagia and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)6 repeat to (GCG) in the PABPN1 gene. The mutations lead to the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of PABPN1. The mutated PABPN1 (mPABPN1) induces the formation of intranuclear filamentous inclusions that sequester poly(A) RNA and are associated with cell death. METHODS: Human fetal brain cDNA library was used to look for PABPNI binding proteins using yeast two-hybrid screen. The protein interaction was confirmed by GST pull-down and co-immunoprecipitation assays. Oculopharyngeal muscular dystrophy cellular model and OPMD patient muscle tissue were used to check whether the PABPN1 binding proteins were involved in the formation of OPMD intranuclear inclusions. RESULTS: We identify two PABPNI interacting proteins, hnRNP A1 and hnRNP A/B. When co-expressed with mPABPN1 in COS-7 cells, predominantly nuclear protein hnRNP A1 and A/B co-localize with mPABPN1 in the insoluble intranuclear aggregates. Patient studies showed that hnRNP A1 is sequestered in OPMD nuclear inclusions. CONCLUSIONS: The hnRNP proteins are involved in mRNA processing and mRNA nucleocytoplasmic export, sequestering of hnRNPs in OPMD intranuclear aggregates supports the view that OPMD intranuclear inclusions are "poly(A) RNA traps", which would interfere with RNA export, and cause muscle cell death.     

Progressive myopathy in an inducible mouse model of oculopharyngeal muscular dystrophy

The genetic basis of oculopharyngeal muscular dystrophy (OPMD) is a short expansion of a polyalanine tract (normal allele: 10 alanines, mutant allele: 11-17 alanines) in the nuclear polyadenylate binding protein PABPN1 which is essential for controlling poly(A) tail length in messenger RNA. Mutant PABPN1 forms nuclear inclusions in OPMD muscle. To investigate the pathogenic role of mutant PABPN1 in vivo, we generated a ligand-inducible transgenic mouse model by using the mifepristone-inducible gene expression system. Induction of ubiquitous expression of mutant PABPN1 resulted in skeletal and cardiac myopathy. Histological changes of degenerative myopathy were preceded by nuclear inclusions of insoluble PABPN1. Downregulation of mutant PABPN1 expression attenuated the myopathy and reduced the nuclear burden of insoluble PABPN1. These results support association between mutant PABPN1 accumulation and degenerative myopathy in mice. Resolution of myopathy in mice suggests that the disease process in OPMD patients may be treatable.     

Premature proliferative arrest of cricopharyngeal myoblasts in oculo-pharyngeal muscular dystrophy: Therapeutic perspectives of autologous myoblast transplantation

Cultures of myoblasts isolated from cricopharyngeal muscles from patients with oculopharyngeal muscular dystrophy (OPMD) have been performed to study the effect of the expanded (GCG)8-13 repeat, located on the poly(A) binding protein nuclear-1 (PABPN1), on satellite cell phenotype. Cell cultures exhibited a reduced myogenicity, as well as a rapid decrease in proliferative lifespan, as compared to controls. The incorporation of BrdU decreased during the proliferative lifespan, due to a progressive accumulation of non-dividing cells. A lower fusion index was also observed, but myoblasts were able to form large myotubes when OPMD cultures were purified, although a rapid loss of myogenicity during successive passages was also observed. Myoblasts isolated from unaffected muscles did not show the defects observed in cricopharyngeal muscle cultures. The PABPN1 was predominantly located in nuclei of myoblasts and in both the nuclei and cytoplasm of myotubes in OPMD cultures. In vivo analysis of OPMD muscles showed that the number of satellite cells was slightly higher than that observed in age matched controls. Mutation of the PABPN1 in OPMD provokes premature senescence in dividing myoblasts, that may be due to intranuclear toxic aggregates. These results suggest that myoblast autografts, isolated from unaffected muscles, and injected into the dystrophic pharyngeal muscles, may be a useful therapeutic strategy to restore muscular function. Its tolerance and feasibility has been preclinically demonstrated in the dog.     

PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.     

Executive functions are impaired in heterozygote patients with oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disorder caused by a small expansion of a short polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). It presents with adult onset of progressive eyelid drooping, swallowing difficulties and proximal limb weakness, usually without involvement of central nervous system (CNS). However, cognitive decline with relevant behavioural and psychological symptoms has been recently described in homozygous patients. In this study, we performed for the first time an extensive neuropsychological and neuropsychiatric evaluation on 11 OPMD heterozygote patients. We found that they were less efficient than a matched control sample on several tests, particularly those tapping executive functions. Moreover, the presence of negative correlation between GCN expansion size and some neuropsychological scores raises the issue that CNS involvement might be linked to the genetic defect, being worse in patients with larger expansion. Our results might be consistent with the toxic gain-of-function theory in the pathogenesis of OPMD and hint at a possible direct role of PABPN1 in the CNS also in heterozygote patients.     

Identification of a novel mutation in a Korean patient with oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disorder characterized by progressive dysphagia and bilateral ptosis. Mutations in the polyadenylate binding protein nuclear 1 (PABPN1) gene have been found to cause OPMD. The typical mutation is a stable trinucleotide repeat expansion in the first exon of the PABPN1 gene, in which (GCG)(6) is the normal repeat length. We investigated a Korean patient with OPMD and identified a novel mutation: a heterozygous insertion of a 9-bp sequence [(GCG)(GCA)(GCA); c.27_28insGCGGCAGCA] instead of the (GCG) repeat expansion, resulting in an in-frame insertion of three alanines (p.A10insAAA). To the best of our knowledge, this is the first report of a genetically confirmed case of OPMD in Korea.     

The dynamism of PABPN1 nuclear inclusions during the cell cycle

Oculopharyngeal muscular dystrophy (OPMD) is caused by expansion of a (GCN)10 to a (GCN)11-17 repeat coding for a polyalanine domain at the N-terminal part of poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by the presence of intranuclear inclusions (INIs) in skeletal muscle fibers of patients. The formation of GFP-b13AlaPABPN1 INIs and their fate through the cell cycle were followed by time-lapse imaging. Our observations demonstrated that the GFP-b13AlaPABPN1 INIs are dynamic structures that can disassemble during mitosis. However, their presence in cells occasionally led to apoptosis. The length of the polyalanine tail or the overexpression of PABPN1 did not significantly affect the percentage of soluble PABPN1 in vitro. Moreover, overexpression of either the wild type (wt) or mutant (mut) forms of PABPN1 slowed down the cell proliferation. The slowing down of proliferation together with the occasional occurrence of apoptosis could contribute in vivo to the late onset of this disease.     

A late-onset case of oculopharyngeal muscular dystrophy carrying a (GCG)8 repeat expansion in the PAPBN1 gene]

We report a sporadic case of a female patient with oculopharyngeal muscular dystrophy (OPMD). Her father died at age 86 and mother at age 74. There was no familial occurrence of the disease. The patient initially developed a nasal voice at age 66. Neurological examinations on admission at age 72 revealed bilateral ptosis, a limitation of ocular movement without diplopia, dysphagia, and proximal muscle weakness. Serum creatine kinase level was slightly increased. Biopsied muscle specimens showed variation in fiber size as well as the occasional presence of rimmed vacuoles. On the basis of these clinical and laboratory findings, we suspected a diagnosis of OPMD, although a family history was absent. To confirm the diagnosis of OPMD, we performed a gene analysis for poly A binding protein, nuclear 1 (PABPN1; PABP2), which revealed a mild expansion of GCG repeat (8 repeats) as a heterozygous state. Clinical features of the patient were consistent with those in a previous literature reporting that patients carrying (GCG)8 repeat as a heterozygous state show a relatively late onset and a mild phenotype. The case of this patient emphasizes the importance of the PABPN1 gene analysis for patients showing muscular weakness involving oculopharyngeal and proximal limb muscles even when a familial occurrence of the disease is not apparent.     

Soluble expanded PABPN1 promotes cell death in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by the expansion of a polyalanine repeat (GCG)(8-13) in exon 1 of the PABPN1 gene. Skeletal muscle fibers nuclei from OPMD patients contain insoluble polyalanine expanded PABPN1 (expPABPN1) nuclear aggregates that sequester different cellular components. Whether these aggregates are pathogenic, or the consequence of a molecular defense mechanism, remains controversial in the field of neurodegenerative disorders and OPMD. Our cellular model shows that interfering with the formation of expPABPN1-induced large nuclear aggregates increases the availability of nuclear expPABPN1 and significantly exacerbates cell death. Live microscopy reveals that cells harboring an increased amount of the soluble forms of expPABPN1 are significantly more prone to toxicity than those with nuclear aggregates. This is the first report directly indicating that nuclear aggregation in OPMD may reflect an active process by which cells sequester and inactivate the soluble toxic form of expPABPN1.     

Variability of the recessive oculopharyngeal muscular dystrophy phenotype

Oculopharyngeal muscular dystrophy (OPMD) is usually transmitted as an autosomal-dominant trait and characterized by an expansion from 6 to 8 or more GCG/GCA repeats in the poly-(A) binding protein nuclear 1 (PABPN1) gene on chromosome 14q11. Autosomal-recessive OPMD with a homozygous (GCG)7 expansion of PABPN1 has only been described in two Canadian patients, who showed a comparably mild phenotype, suggesting that it is less severe than the dominant form. We clinically and genetically characterized the first two reported cases of autosomal-recessive OPMD in Europe. Remarkably, both patients revealed severe and diverse phenotypes, with an unusual onset and atypical clinical course in one patient. Former studies found a 1%-2% frequency of the (GCG)7 allele, which theoretically produces an incidence of 1:10,000 of autosomal-recessive OPMD in the general population. We conclude that the apparent rarity of the autosomal-recessive form of OPMD may be due to the fact that genetic testing is generally administered only to patients with typical clinical features or a positive family history.     

Oculopharyngeal muscular dystrophy: A polyalanine myopathy

It has been 10 years since the identification of the first PABPN1 gene (GCN)_n/polyalanine mutations responsible for oculopharyngeal muscular dystrophy (OPMD). These mutations have been found in most cases of OPMD diagnosed in more than 35 countries. Sequence analyses have shown that such mutations have occurred numerous times in human history. Although PABPN1 was found early on to be a component of the classic filamentous intranuclear inclusions (INIs), mRNA and other proteins also have been found to coaggregate in the INIs. It is still unclear if the INIs play a pathologic or a protective role. The generation of numerous cell and animal models of OPMD has led to greater insight into its complex molecular pathophysiology and identified the first candidate therapeutic molecules. This paper reviews basic and clinical research on OPMD, with special emphasis on recent developments in the understanding of its pathophysiology.     

Transgenic expression of an expanded (GCG)13 repeat PABPN1 leads to weakness and coordination defects in mice

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder caused by a (GCG)n trinucleotide repeat expansion in the poly(A) binding protein nuclear-1 (PABPN1) gene, which in turn leads to an expanded polyalanine tract in the protein. We generated transgenic mice expressing either the wild type or the expanded form of human PABPN1, and transgenic animals with the expanded form showed clear signs of abnormal limb clasping, muscle weakness, coordination deficits, and peripheral nerves alterations. Analysis of mitotic and postmitotic tissues in those transgenic animals revealed ubiquitinated PABPN1-positive intranuclear inclusions (INIs) in neuronal cells. This latter observation led us to test and confirm the presence of similar INIs in postmortem brain sections from an OPMD patient. Our results indicate that expanded PABPN1, presumably via the toxic effects of its polyalanine tract, can lead to inclusion formation and neurodegeneration in both the mouse and the human.     

Extraocular muscles have fundamentally distinct properties that make them selectively vulnerable to certain disorders

While skeletal muscles generally perform specific limited roles, extraocular muscles (EOMs) have to be responsive over a wider dynamic range. As a result, EOMs have fundamentally distinct structural, functional, biochemical and immunological properties compared to other skeletal muscles. While these properties enable high fatigue resistance and the rapid and precise control of extraocular motility, they might also explain why EOMs are selectively involved in certain disorders, such as chronic progressive external ophthalmoplegia (CPEO), myasthenia gravis and Graves' ophthalmopathy. This review first gives an overview of the novel myofibre classification in EOMs and then focuses on those properties that might explain why ophthalmoplegia should be so prominent in these disorders.     

Progress in understanding the pathogenesis of oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping (ptosis), swallowing difficulties (dysphagia), and proximal limb weakness. The autosomal dominant form of this disease is caused by expansions of a (GCG)6 repeat to (GCG)8-13 in the PABPN1 gene. These mutations lead to the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminal domain of PABPN1. Mutated PABPN1 (mPABPN1) induces the formation of muscle intranuclear inclusions that are thought to be the hallmark of this disease. In this review, we discuss: 1) OPMD genetics and PABPN I function studies; 2) diseases caused by polyalanine expansions and cellular polyalanine toxicity; 3) mPABPN1-induced intranuclear inclusion toxicity; 4) role of oligomerization of mPABPNI in the formation and toxicity of OPMD intranuclear inclusions and; 5) recruitment of subcellular components to the OPMD inclusions. We present a potential molecular mechanism for OPMD pathogenesis that accounts for these observations.     

Mutation and haplotype analysis of oculopharyngeal muscular dystrophy in Thai patients

Oculopharyngeal muscular dystrophy (OPMD) is an inherited neuromuscular disease associated with a short trinucleotide repeat expansion in Exon 1 of the PABPN1 gene. OPMD is uncommon in East Asian populations, and there have been no previous reports of Thai patients. We studied clinical and molecular genetic features of six unrelated Thai patients with autosomal dominant OPMD. All patients had expansions of the guanine-cytosine-guanine (GCG) repeat ranging from three to seven additional repeats in the PABPN1 gene. Haplotype analysis showed that these mutations might have originated independently. Analysis of the size of the GCG repeat in the PABPN1 gene in 200 Thai control patients showed that 0.5% of the control subjects possessed (GCG)₇, thereby suggesting that the prevalence of autosomal recessive OPMD in the Thai population was approximately 1 in 160,000. In conclusion, our data suggest that OPMD in Thailand may be more common than previously thought.     

Oculopharyngeal muscular dystrophy with PABPN1 mutation in a Chinese Malaysian woman

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disorder of middle age presenting as progressive dysphagia and eyelid ptosis, due to short expansions of the GCG trinucleotide repeat (from GCG6 to GCG8-13) in the polyadenylate binding-protein nuclear 1 (PABPN1) gene. OPMD is rarely seen in Asians and morphologically and/or genetically confirmed cases have been reported in Japanese kindreds only. We report a 64 year old Chinese-Malaysian woman who presented with progressive dysphagia and bilateral ptosis for about 6 years. Her mother and elder brother (both deceased) were believed to be affected. Muscle histopathology revealed angulated fibres with rimmed vacuoles. Genetic analysis showed repeat expansion in one allele to (GCG)9 while normal in the other (GCG)6. This is the first non-Japanese Asian family with genetically confirmed OPMD.     

Genetic heterogeneity in 30 German patients with oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is due to short elongations of a polyalanine tract in the poly(A) binding protein nuclear 1 (PABPN1) gene. Originally GCG expansions in which (GCG)₆ is extended to (GCG)_7–13 were found. Subsequently five further genotypes with additional GCA– and GCG–trinucleotides were identified in single OPMD patients. This indicated larger genetic heterogeneity and showed that unequal crossing–over and not replication slippage must be the underlying mechanism of elongation.We performed sequencing of the PABPN1 gene in 30 German OPDM index patients to determine the exact genotype. The original GCG expansion ranging from (GCG)₈ to (GCG)₁₁ was found in 22 patients. In 8 patients, however, three different elongated alleles other than classical (GCG)_7–13 were observed. Two of these genotypes had already been identified in Japanese patients. One genotype was recently identified showing (GCG)₆ followed by inserted (GCA)₃GCG in four unrelated patients. This study further supports the theory of unequal crossing over as the molecular mechanism leading to elongation. It shows that other genotypes than classical (GCG)_7–13 are rather common in German OPMD patients. The data imply that there is no single founder effect in German OPMD patients. Drs. Müller and Deschauer contributed equally to this work.     

Oculopharyngeal muscular dystrophy: clinical and morphological follow-up study reveals mitochondrial alterations and unique nuclear inclusions in a severe autosomal recessive type

Oculopharyngeal muscular dystrophy (OPMD) is usually a late onset, autosomal dominant dystrophy that affects extraocular eye muscles, pharyngeal muscles, and the trunk and limb musculature. In the present presumably recessively inherited case, with a clinical history of oculopharyngeal myopathy and distal weakness, paracrystalline mitochondrial inclusions and unique nuclear inclusions were found. In a biopsy obtained from the erector spinae muscle, marked muscle fibre atrophy and hypertrophy, occasional muscle fibre necrosis, and considerable fibrosis of the endomysium were noted. Similar signs of a chronic myopathy could already be detected in a biopsy from the anterior tibial muscle that had been obtained 10 years before. In both muscles, nuclear inclusions were seen in numerous severely affected, atrophic muscle fibres. These inclusions consisted of straight or helically wound 2–4 nm filaments. The outer diameter of the double helix was 12–1 5 nm and the periodicity of its repeats was about 15 rim, The filaments were often accumulated in clusters with a paracrystalline arrangement. No nuclear inclusions consisting of 8.5 nm tubular filaments, typically found in cases of OPMD, were detected. In addition, paracrystalline inclusions were present in a large number of mitochondria in several muscle fibres of the erector spinae muscle indicating that mitochondria could be primarily involved in the disease.     

Intrinsic laryngeal muscles are spared from myonecrosis in the mdx mouse model of Duchenne muscular dystrophy

Intrinsic laryngeal muscles share many anatomical and physiological properties with extraocular muscles, which are unaffected in both Duchenne muscular dystrophy and mdx mice. We hypothesized that intrinsic laryngeal muscles are spared from myonecrosis in mdx mice and may serve as an additional tool to understand the mechanisms of muscle sparing in dystrophinopathy. Intrinsic laryngeal muscles and tibialis anterior (TA) muscle of adult and aged mdx and control C57Bl/10 mice were investigated. The percentage of central nucleated fibers, as a sign of muscle fibers that had undergone injury and regeneration, and myofiber labeling with Evans blue dye, as a marker of myofiber damage, were studied. Except for the cricothyroid muscle, none of the intrinsic laryngeal muscles from adult and old mdx mice showed signs of myofiber damage or Evans blue dye labeling, and all appeared to be normal. Central nucleation was readily visible in the TA of the same mdx mice. A significant increase in the percentage of central nucleated fibers was observed in adult cricothyroid muscle compared to the other intrinsic laryngeal muscles, which worsened with age. Thus, we have shown that the intrinsic laryngeal muscles are spared from the lack of dystrophin and may serve as a useful model to study the mechanisms of muscle sparing in dystrophinopathy.