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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

眼咽型肌营养不良汉族六家系的临床和遗传学特点

目的 初步总结我国汉族眼咽型肌营养不良(OPMD)患者的临床和多腺苷酸结合蛋白核1(PABPN1)基因改变特点.方法 6个OPMD家系共28例患者,男性13例,女性15例,发病年龄32～70岁,平均发病年龄49.7岁.在可确定首发症状的患者中,以吞咽困难或构音障碍首发的13例、眼睑下垂首发的4例、双下肢无力首发的1例.经过3～20年均出现眼睑下垂、吞咽困难和构音障碍,其中7例出现四肢近端无力.对6例先证者做肌肉活体组织检查,标本进行常规组织病理和电镜检查.对6个家系的先证者以及部分家庭成员进行PABPN1基因检查,并对6例先证者进行单体型分析.结果 6例先证者的肌肉活体组织检查均发现肌纤维直径轻度变异加大伴随肌纤维内镶边空泡形成,4例患者经电镜检查发现OPMD典型的核内栅栏样丝状包涵体.3个家系的PABPN1基因型为(GCG)9,另外3个家系的基因型分别为(GCG)6(GCA)1(GCG)3、(GCG)10和(GCG)8.2个携带(GCG)9突变的家系存在rs2239579(C)-(GCG)9-SNP2622(C)的单倍体型.结论 吞咽异常和眼睑下垂均是我国汉族OPMD患者的首发症状.肌纤维出现镶边空泡以及核内包涵体是我国患者的常见病理改变.PABPN1基因的(GCG)异常扩增和(GCA)插入突变均出现在我国患者,起源具有多源性.携带(GCG)9突变的部分家系可能来自共同祖先.     

眼咽型肌营养不良一家系临床、电生理、病理及基因分析

目的 报道1个出现神经源性骨骼肌损害的眼咽型肌营养不良(OPMD)家系的临床、骨骼肌病理和基因改变特点.方法 先证者为60岁男性,50岁时出现双下肢近端无力,53岁出现吞咽困难和构音障碍,57岁出现双眼睑下垂和眼球突出.肌酸激酶轻度增加,四肢肌电图为神经源性损伤,周围神经传导速度下降20%～43%.家系3代中除先证者外尚有5例在45岁后出现吞咽困难,4～20年后出现眼睑下垂,其中3例有肢体无力.对先证者左肱二头肌做肌肉活体组织检查,标本进行组织学、酶组织化学以及免疫组织化学染色(以抗结蛋白和泛素蛋白抗体作为一抗)和超微病理检查.对先证者和家系中另外18人进行多腺苷酸结合蛋白(PABPN1)基因检查.结果 先证者肌纤维内出现镶边空泡伴泛素阳性沉积物和成组分布的小角状萎缩肌纤维,个别肌纤维出现再生改变伴随结蛋白沉积,可见细胞色素C氧化酶阴性肌纤维.电镜检查发现约3%的肌纤维核内存在栅栏样细丝包涵体.先证者和另外11名家系成员的PABPN1基因存在(GCG)9异常扩增.结论 杂合(GCG).异常扩增性OPMD可先出现咽喉肌无力,伴随脱髓鞘性神经病.我国患者也存在肌纤维核内包涵体.     

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.     

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.     

Nuclear inclusions in innervated cultured muscle fibers from patients with oculopharyngeal muscular dystrophy

We established monolayer muscle fiber cultures from muscle biopsies of 3 patients with oculopharyngeal muscular dystrophy (OPMD) who had characteristic intranuclear inclusions (INI-A) in their muscle fibers. Aneural cultures had normal morphology, except for a few muscle fibers that contained small vacuoles. Innervated cultures had large cytoplasmic vacuoles in a number of muscle fibers. Those muscle fibers were breaking easily, and could not be maintained longer than 2 months. Electron microscopy showed unusual intranuclear inclusions (INI-B) not previously reported in aneurally cultured muscle fibers of OPMD or in any normal or disease-control aneural or innervated cultured human muscle fibers. They resembled, but were not identical to, the INI-A, and they occurred in both the cultured fibers and the original muscle biopsies of all 3 patients. Our study demonstrate that (1) nuclear inclusions in OPMD reflect an intrinsic genetic defect; and (2) neuronal influence, advanced maturation, or both, seem to be essential for their induction in muscle fibers.     

Nuclear accumulation of expanded PABP2 gene product in oculopharyngeal muscular dystrophy

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by (GCG) repeat expansions in exon 1 of the poly(A) binding protein 2 gene (PABP2). To elucidate the molecular mechanism underlying the disease, we raised an antiserum against a synthetic peptide fragment predicted from PABP2 cDNA. The peptide corresponded to amino acids 271-291 where a cluster of posttranslational arginine methylation occurs. We examined the subcellular localization of PABP2 in muscle specimens from five patients with OPMD, 14 patients with various neuromuscular disorders, and three normal controls. All Japanese patients with OPMD have been shown to have expanded (GCG)(8, 9, or 11) mutations in PABP2, as well as intranuclear tubulofilamentous inclusions (ITFI) of 8.5 nm. None of 50 separate Japanese control individuals were shown to have expanded (GCG) repeat in PABP2. Positive immunoreaction for polyclonal PABP2 was confined to the intranuclear aggregates of muscle fibers exclusively in patients with OPMD. Frequency of the nuclei positive for PABP2 (2%) was similar to that of ITFI detected by electron microscopy (2.5%). There was no apparent relationship between the frequency of PABP2-positive intranuclear aggregates and the severity of muscle fiber damage. In contrast, nuclear immunoreaction was not detected in any samples from normal controls or from other neuromuscular diseases. These results suggest the presence of molecular modification of the product of expanded (GCG) repeat in PABP2, since the synthetic antigen peptide may not recognize a highly dimethylated cluster of arginine residues of the native PABP2, but may recognize the mutated form. Nuclear accumulation of expanded PABP2 product implies a causative role for ITFI.     

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.     

Expanded polyglutamine impairs normal nuclear distribution of fused in sarcoma and poly (rC)‐binding protein 1 in Huntington's disease

Huntington's disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the huntingtin protein. Immunohistochemical studies using the 1C2 antibody for polyglutamine expansion have detected characteristic intranuclear inclusions (INIs) in affected neurons in HD. Further, in vitro and mouse models of HD have shown that the INIs recruit several proteins relating to RNA splicing and translation. In the present study, we immunohistochemically investigated the association of INIs with various heterogeneous nuclear ribonucleoproteins in the cerebral cortex of four autopsy cases of HD. Fused in sarcoma (FUS) was colocalized with 1C2‐positive nuclear inclusions in all examined cases. Localization of poly (rC)‐binding protein 1 (PCBP1) in 1C2‐positive nuclear inclusions was also observed. Double immunofluorescence revealed complete or partial loss of the normal, diffuse nuclear distribution of FUS or PCBP1 in neurons with 1C2‐positive nuclear inclusions. This maldistribution of FUS in cortical neurons suggests a severe disturbance of messenger RNA processing, which may be a common pathogenetic mechanism of FUS‐related familial amyotrophic lateral sclerosis.     

Intranuclear inclusions in oculopharyngeal muscular dystrophy contain poly(A) binding protein 2

Intranuclear inclusions are one of the ultrastructural hallmarks of oculopharyngeal muscular dystrophy (OPMD), a disorder caused by small polyalanine (GCG) expansions in the gene that codes for a ubiquitous nuclear protein called poly(A) binding protein 2 (PABP2). We studied OPMD skeletal muscle and found that 1.0 to 10.0% of myocyte nuclei contained discreet PABP2 immunoreactive intranuclear inclusions, providing the first direct evidence of the relation between the proposed gene for OPMD and the pathology of OPMD.     

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%).     

Oculopharyngeal muscular dystrophy

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease with worldwide distribution. It usually presents in the fifth or sixth decades with progressive dysphagia, eyelid ptosis, and proximal limb weakness. Unique intranuclear filament inclusions in skeletal muscle fibers are its morphological hallmark. Surgical correction of the ptosis and cricopharyngeal myotomy are the only therapies available. Autosomal dominant OPMD is caused by short (GCG)8-13 riplet-repeat expansions in the polyadenylation binding protein 2 (PABP2) gene, which is localized in chromosome 14q11. Autosomal recessive OPMD is caused by a double dose of a (GCG)7 PABP2 allele. The GCG expansions cause lengthening of a predicted polyalanine tract in the protein. The expanded polyalanine domains may cause polyalanine nuclear toxicity by accumulating as nondegradable nuclear filaments.