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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Continuous remodeling of adult extraocular muscles as an explanation for selective craniofacial vulnerability in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular mystrophy (OPMD) is an inherited disorder caused by mutations of the polyadenylate binding protein nuclear 1 (PABPN1) gene. While a pathogenic hypothesis has been formulated that links the genetic and molecular abnormalities to cellular abnormalities, there is no proven explanation for the targeting of the craniofacial muscles. We propose a hypothesis that bridges this gap. It is based on the phenomenon of continuous remodeling of normal adult extraocular muscles (EOMs). Unlike the EOMs, the myonuclei of other skeletal muscles are postmitotic in the adult unless the muscles are injured. Continuous myofiber remodeling most likely requires upregulation of genes involved in cell cycling, and in protein degradation and synthesis. PABPN1 is a nuclear protein that performs the essential function of controlling polyadenylation of mRNA and the fidelity of protein synthesis. In OPMD, the ongoing production of mutant PABPN1 in muscles undergoing continuous remodeling could result in a failure of accurate production of mRNA required for the maintenance of the myocytes. Over many years, this would lead to cumulative myonuclear loss and finally to myofiber loss. This hypothesis offers an explanation for the selective involvement of extraocular muscles affected in OPMD and the onset of symptoms in adulthood.     

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.     

A case of rare recessive oculopharyngeal muscular dystrophy (OPMD) coexisting with hereditary neuropathy with liability to pressure palsies (HNPP)

Oculopharyngeal muscular dystrophy (OPMD) is typically inherited in an autosomal dominant fashion and is characterized by late onset proximal muscle weakness, ptosis and difficulty swallowing. It is caused by expansion mutations in the PABPN1 gene on chromosome 14q11. There is also a rare recessive form of the disease caused by homozygosity of a very small expansion mutation in the same gene. Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder characterized by recurrent peripheral monofocal neuropathies. In this report a patient with both recessive OPMD and HNPP is described. The presence of two genetically unlinked neurological diagnoses in the same individual is a rare event and may have delayed the diagnoses.     

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.     

Anti-prion Drugs Targeting the Protein Folding Activity of the Ribosome Reduce PABPN1 Aggregation

Prion diseases are caused by the propagation of PrP^Sc, the pathological conformation of the PrP^C prion protein. The molecular mechanisms underlying PrP^Sc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrP^Sc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrP^Sc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00992-6.Key Words: Prions, PrP^Sc, drug repositioning, PFAR, OPMD, PABPN1     

Oculopharyngeal Muscular Dystrophy: Phenotypic and Genotypic Studies in a Chinese Population

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset neuromuscular degenerative disease characterized by ptosis, dysphagia, and proximal muscle weakness. The genetic basis has been identified as an abnormal (GCN) expansion encoding the polyalanine tract in exon 1 of the polyadenylate-binding protein nuclear 1 gene (PABPN1). OPMD is worldwide distributed, but has rarely been reported in East Asians. In this study, we summarized the clinical and genetic characteristics of 34 individuals from 13 unrelated families in Chinese population. In our cohort, the mean age at onset was 47.2 years. Dysphagia, rather than ptosis, was the most common initial symptom. Genetically, we identified seven genotypes in our patients, including one compound heterozygote of (GCN)₁₁/(GCN)₁₂. The genetic heterogeneity implies that there is no single founder effect in Chinese population, and our data also support that the (GCN)₁₁ polymorphism may have a disease-modifying effect. Additionally, the clinical features showed homogeneity within families, which suggests that other genetic factors apart from the already known genotype also play a role in modifying the phenotype.     

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.     

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.     

Homozygotes for oculopharyngeal muscular dystrophy have a severe form of the disease.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) usually begins with ptosis or dysphagia during the fifth or sixth decade of life. We studied 7 patients with OPMD symptoms starting before the age of 36 years. All were found to be homozygotes for the dominant (GCG)9 OPMD mutation. On average, disease onset was 18 years earlier than in heterozygotes, and patients had a significantly larger number of muscle nuclei containing intranuclear inclusions (INIs) (9.4 vs 4.9%).