Dataset Information

Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knockdown and replacement of mutant expanded PABPN1.

ABSTRACT: Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100?000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11-18 alanine tract that is misfolded and prone to form intranuclear inclusions, which are the hallmark of the disease. Other features of OPMD include muscle fibrosis and atrophy in affected muscles. Currently, no pharmacological treatments are available, and OPMD patients can only be referred to surgeons for cricopharyngeal myotomy or corrective surgery of extraocular muscles to ease ptosis. We recently tested a two-AAV `silence' and `replace' vector-based gene therapy treatment in a mouse model of OPMD. We demonstrate here that this gene therapy approach can revert already established insoluble aggregates and partially rescues the muscle from atrophy, which are both crucially important since in most cases OPMD patients already have an established disease when diagnosed. This strategy also prevents the formation of muscle fibrosis and stabilizes the muscle strength to the level of healthy muscles. Furthermore, we show here that similar results can be obtained using a single AAV vector incorporating both the `silence' and `replace' cassettes. These results further support the application of a gene therapy approach as a novel treatment for OPMD in humans.

SUBMITTER: Malerba A

PROVIDER: S-EPMC7343048 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knockdown and replacement of mutant expanded PABPN1.

Malerba Alberto A Klein Pierre P Lu-Nguyen Ngoc N Cappellari Ornella O Strings-Ufombah Vanessa V Harbaran Sonal S Roelvink Peter P Suhy David D Trollet Capucine C Dickson George G

Human molecular genetics 20191001 19

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100 000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11-18 alanine tract that is misfolded and prone to ...[more]

PMID: 31294444

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Project description:Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder associated with a premutation repeat expansion (55-200 CGG repeats) in the 5' noncoding region of the FMR1 gene. Solitary intranuclear inclusions within FXTAS neurons and astrocytes constitute a hallmark of the disorder, yet our understanding of how and why these bodies form is limited. Here, we have discovered that FXTAS inclusions emit a distinct autofluorescence spectrum, which forms the basis of a novel, unbiased method for isolating FXTAS inclusions by preparative fluorescence-activated cell sorting (FACS). Using a combination of autofluorescence-based FACS and liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based proteomics, we have identified more than two hundred proteins that are enriched within the inclusions relative to FXTAS whole nuclei. Whereas no single protein species dominates inclusion composition, highly enriched levels of conjugated small ubiquitin-related modifier 2 (SUMO 2) protein and p62/sequestosome-1 (p62/SQSTM1) protein were found within the inclusions. Many additional proteins involved with RNA binding, protein turnover, and DNA damage repair were enriched within inclusions relative to total nuclear protein. The current analysis has also allowed the first direct detection, through peptide sequencing, of endogenous FMRpolyG peptide, the product of repeat-associated non-ATG (RAN) translation of the FMR1 mRNA. However, this peptide was found only at extremely low levels and not within whole FXTAS nuclear preparations, raising the question whether endogenous RAN products exist at quantities sufficient to contribute to FXTAS pathogenesis. The abundance of the inclusion-associated ubiquitin- and SUMO-based modifiers supports a model for inclusion formation as the result of increased protein loads and elevated oxidative stress leading to maladaptive autophagy. These results highlight the need to further investigate FXTAS pathogenesis in the context of endogenous systems.

Project description:In eukaryote genomes, the polyadenylation site marks termination of mature RNA transcripts by a poly-adenine tail. The polyadenylation site is recognized by a dynamic protein complex, among which the poly-adenine-binding protein nuclear1 plays a key role. Reduced poly-adenine-binding protein nuclear1 levels are found in aged muscles and are even lower in oculopharyngeal muscular dystrophy patients. Oculopharyngeal muscular dystrophy is a rare, late onset autosomal dominant myopathy, and is caused by an alanine expansion mutation in poly-adenine-binding protein nuclear1. Mutant poly-adenine-binding protein nuclear1 forms insoluble nuclear aggregates leading to depletion of functional poly-adenine-binding protein nuclear1 levels. In oculopharyngeal muscular dystrophy models, increased utilization of proximal polyadenylation sites has been observed in tandem 3'-untranslated regions, and most often cause gene upregulation. However, global alterations in expression profiles canonly partly be explained by polyadenylation site switches within the most distal 3'-untranslated region. Most poly-adenine signals are found at the distal 3'-untranslated region, but a significant part is also found in internal gene regions, like introns, exons, and internal 3'-untranslated regions. Here, we investigated poly-adenine-binding protein nuclear1's role in polyadenylation site utilization in internal gene regions. In the quadriceps muscle of oculopharyngeal muscular dystrophy mice expressing expPABPN1 we found significant polyadenylation site switches between gene regions in 17% of genes with polyadenylation site in multiple regions (N?=?574; 5% False Discovery Rate). Polyadenylation site switches between gene regions were associated with differences in transcript expression levels and alterations in open reading frames. Transcripts ending at internal polyadenylation site were confirmed in tibialis anterior muscles from the same mice and in mouse muscle cell cultures overexpressing expPABPN1. The polyadenylation site switches were associated with nuclear accumulation of full-length transcripts. Our results provide further insights into the diverse roles of poly-adenine-binding protein nuclear1 in the post-transcriptional control of muscle gene expression and its relevance for oculopharyngeal muscular dystrophy pathology and muscle aging.

Project description:BackgroundMany cases of frontotemporal dementia (FTD) are familial, often with an autosomal dominant pattern of inheritance. Some are due to a mutation in the tau- encoding gene, on chromosome 17, and show an accumulation of abnormal tau in brain tissue (FTDP-17T). Most of the remaining familial cases do not exhibit tau pathology, but display neuropathology similar to patients with dementia and motor neuron disease, characterized by the presence of ubiquitin-immunoreactive (ub-ir), dystrophic neurites and neuronal cytoplasmic inclusions in the neocortex and hippocampus (FTLD-U). Recently, we described a subset of patients with familial FTD with autopsy-proven FTLD-U pathology and with the additional finding of ub-ir neuronal intranuclear inclusions (NII). NII are a characteristic feature of several other neurodegenerative conditions for which the genetic basis is abnormal expansion of a polyglutamine-encoding trinucleotide repeat region. The genetic basis of familial FTLD-U is currently not known, however the presence of NII suggests that a subset of cases may represent a polyglutamine expansion disease.MethodsWe studied DNA and post mortem brain tissue from 5 affected members of 4 different families with NII and one affected individual with familial FTLD-U without NII. Patient DNA was screened for CAA/CAG trinucleotide expansion in a set of candidate genes identified using a genome-wide computational approach. Genes containing CAA/CAG trinucleotide repeats encoding at least five glutamines were examined (n = 63), including the nine genes currently known to be associated with human disease. CAA/CAG tract sizes were compared with published normal values (where available) and with those of healthy controls (n = 94). High-resolution agarose gel electrophoresis was used to measure allele size (number of CAA/CAG repeats). For any alleles estimated to be equal to or larger than the maximum measured in the control population, the CAA/CAG tract length was confirmed by capillary electrophoresis. In addition, immunohistochemistry using a monoclonal antibody that recognizes proteins containing expanded polyglutamines (1C2) was performed on sections of post mortem brain tissue from subjects with NII.ResultsNo significant polyglutamine-encoding repeat expansions were identified in the DNA from any of our FTLD-U patients. NII in the FTLD-U cases showed no 1C2 immunoreactivity.ConclusionWe find no evidence to suggest that autosomal dominant FTLD-U with NII is a polyglutamine expansion disease.

Dataset Information

Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knockdown and replacement of mutant expanded PABPN1.

Publications

Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knockdown and replacement of mutant expanded PABPN1.

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