Publicação
Development of a cellular model of oculopharyngeal muscular dystrophy
| Resumo: | A significant number of debilitating human diseases are associated with the deposition of protein aggregates. These include Alzheimer’s disease, Huntington’s disease and Parkinson’s disease, among others. Under pathological conditions, proteins or peptides that are abnormally soluble undergo aggregation and generate stable insoluble fibers that accumulate in a variety of organs. Oculopharyngeal Muscular Dystrophy (OPMD; OMIM 164300) is a dominant late-onset disease, characterized by progressive ptosis, dysphagia and proximal limb weakness. The OPMD mutation consists of short expansions of a (GCN)10 repeat coding for a polyalanine tract at the N-terminus of the polly(A)-binding protein nuclear 1 (PABPN1) (Brais et al., 1998). This results in an alanine-expanded form of PABPN1, which aggregates and forms dense, insoluble intranuclear inclusion bodies in skeletal muscle of OPMD patients. PABPN1 is ubiquitously expressed and is involved in poly(A) tail synthesis and poly(A) tail length control (Kuhn and Wahle, 2004). The molecular mechanisms leading from PABPN1 mutation to OPMD phenotype and the role of aggregate formation in the progression of the disease are poorly understood. In the present study we proposed to contribute to the understanding of OPMD pathophysiology. Our first goal was to elucidate the mechanisms involved in the formation of OPMD nuclear inclusions. We developed a cellular model of PABPN1 aggregation based on the exogenous over-expression of different protein variants in both HeLa and myogenic C2 cells. We were able to show that normal PABPN1 is inherently aggregation-prone when overexpressed. The formation of insoluble aggregates was observed upon expression of PABPN1 variants containing either a polyalanine expansion or a complete deletion of the polyalanine tract, indicating that the OPMD-causing mutation is not essential for the formation of inclusions. Conversely, PABPN1 variants containing deletions or point mutations in any of the domains involved in PABPN1 function in polyadenylation fail to form aggregates, strongly suggesting that the in vivo aggregation of PABN1 is tightly coupled to its function in polyadenylation. We monitored the dynamics of PABPN1 interactions within the inclusions by photobleaching analysis and observed that both normal and alanine-expanded PABPN1 molecules are not irreversibly sequestered into aggregates, they are in constant Exchange between the inclusions and the nucleoplasm. Taken together, these observations raise serious questions concerning OPMD models based on the over-expression of PABPN1 and argue against a pathological role of the aggregates by the irreversible trapping of other nuclear components. Our next goal was to identify mechanisms of cellular dysfunction underlying OPMD. We developed a cellular model of the disease based on the stable expression of normal (10 alanines) and mutant (17 alanines) PABN1 in myogenic C2 cells. Gene expression profiling of our cellular model identified Evi3 as a consistently up-regulated gene in muscle cells expressing mutant PABPN1. The human homolog to mouse Evi3, ZNF521, encodes a transcription factor which is known to interact with Smad proteins and to participate in BMPdependent transcriptional activation (Bond et al., 2004). We tested the BMP-dependent transcription of a reporter gene in our cellular model of OPMD. Whereas cell lines expressing normal PABPN1 displayed strong transcriptional activation in response to BMP, cell lines expressing mutant PABPN1 failed to activate transcription of this BMP-responsive element. The differential expression of the human homolog to Evi3 was validated in primary muscle cell lines derived from OPMD patients. Analysis of ZNF521 mRNA levels in OPMD and control sternocleidomastoideus muscle corroborated the up-regulation observed in the celular model. In contrast, we observed a down-regulation of ZNF521 mRNA in cricopharyngeus muscle from OPMD patients when compared to control. This controversial result may reflect the specificity of the cricopharyngeal muscle, which renders it more susceptible to OPMD dysfunction. Morphological mitochondrial abnormalities in OPMD muscle biopsies were reported several times (Julien et al., 1974; Pauzner et al., 1991; Pratt and Meyers, 1986; Wonget al., 1996) but tended to be considered as a non-specific secondary phenomenon of the disease. We proposed to address the role of mitochondrial dysfunction in OPMD through the evaluation of mitochondrial alterations in muscle biopsies from patients and in the celular model of the disease. We found that muscle biopsies from OPMD patients display na increased number of cytochrome c oxidase (COX)-negative fibers when compared to similarly aged controls. In addition, analysis of our cellular model revealed that cell lines. expressing mutant PABPN1 produce higher protein levels of the 39 kDa subunit from the.mitochondrial respiratory chain complex I. Although requiring further investigation, these.results are indicative of a role for mitochondrial dysfunction in the progression of OPMD. In summary, our work produced important information concerning the role of PABPN1 aggregates in OPMD. In addition, we identified altered BMP-dependent transcription as a candidate mechanism for OPMD pathology. Finally, we provided evidence that mitochondrial alterations may contribute to the disease phenotype. Hopefully, the further investigation of these lines of work will prove valuable to the full understanding of OPMD pathophysiology. |
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| Autores principais: | Calado, Patrícia Ramalhete Mendes da Silva, 1974- |
| Assunto: | Células Distrofia muscular oculofaríngea Patologia Fisiologia Teses de doutoramento - 2007 |
| Ano: | 2007 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | A significant number of debilitating human diseases are associated with the deposition of protein aggregates. These include Alzheimer’s disease, Huntington’s disease and Parkinson’s disease, among others. Under pathological conditions, proteins or peptides that are abnormally soluble undergo aggregation and generate stable insoluble fibers that accumulate in a variety of organs. Oculopharyngeal Muscular Dystrophy (OPMD; OMIM 164300) is a dominant late-onset disease, characterized by progressive ptosis, dysphagia and proximal limb weakness. The OPMD mutation consists of short expansions of a (GCN)10 repeat coding for a polyalanine tract at the N-terminus of the polly(A)-binding protein nuclear 1 (PABPN1) (Brais et al., 1998). This results in an alanine-expanded form of PABPN1, which aggregates and forms dense, insoluble intranuclear inclusion bodies in skeletal muscle of OPMD patients. PABPN1 is ubiquitously expressed and is involved in poly(A) tail synthesis and poly(A) tail length control (Kuhn and Wahle, 2004). The molecular mechanisms leading from PABPN1 mutation to OPMD phenotype and the role of aggregate formation in the progression of the disease are poorly understood. In the present study we proposed to contribute to the understanding of OPMD pathophysiology. Our first goal was to elucidate the mechanisms involved in the formation of OPMD nuclear inclusions. We developed a cellular model of PABPN1 aggregation based on the exogenous over-expression of different protein variants in both HeLa and myogenic C2 cells. We were able to show that normal PABPN1 is inherently aggregation-prone when overexpressed. The formation of insoluble aggregates was observed upon expression of PABPN1 variants containing either a polyalanine expansion or a complete deletion of the polyalanine tract, indicating that the OPMD-causing mutation is not essential for the formation of inclusions. Conversely, PABPN1 variants containing deletions or point mutations in any of the domains involved in PABPN1 function in polyadenylation fail to form aggregates, strongly suggesting that the in vivo aggregation of PABN1 is tightly coupled to its function in polyadenylation. We monitored the dynamics of PABPN1 interactions within the inclusions by photobleaching analysis and observed that both normal and alanine-expanded PABPN1 molecules are not irreversibly sequestered into aggregates, they are in constant Exchange between the inclusions and the nucleoplasm. Taken together, these observations raise serious questions concerning OPMD models based on the over-expression of PABPN1 and argue against a pathological role of the aggregates by the irreversible trapping of other nuclear components. Our next goal was to identify mechanisms of cellular dysfunction underlying OPMD. We developed a cellular model of the disease based on the stable expression of normal (10 alanines) and mutant (17 alanines) PABN1 in myogenic C2 cells. Gene expression profiling of our cellular model identified Evi3 as a consistently up-regulated gene in muscle cells expressing mutant PABPN1. The human homolog to mouse Evi3, ZNF521, encodes a transcription factor which is known to interact with Smad proteins and to participate in BMPdependent transcriptional activation (Bond et al., 2004). We tested the BMP-dependent transcription of a reporter gene in our cellular model of OPMD. Whereas cell lines expressing normal PABPN1 displayed strong transcriptional activation in response to BMP, cell lines expressing mutant PABPN1 failed to activate transcription of this BMP-responsive element. The differential expression of the human homolog to Evi3 was validated in primary muscle cell lines derived from OPMD patients. Analysis of ZNF521 mRNA levels in OPMD and control sternocleidomastoideus muscle corroborated the up-regulation observed in the celular model. In contrast, we observed a down-regulation of ZNF521 mRNA in cricopharyngeus muscle from OPMD patients when compared to control. This controversial result may reflect the specificity of the cricopharyngeal muscle, which renders it more susceptible to OPMD dysfunction. Morphological mitochondrial abnormalities in OPMD muscle biopsies were reported several times (Julien et al., 1974; Pauzner et al., 1991; Pratt and Meyers, 1986; Wonget al., 1996) but tended to be considered as a non-specific secondary phenomenon of the disease. We proposed to address the role of mitochondrial dysfunction in OPMD through the evaluation of mitochondrial alterations in muscle biopsies from patients and in the celular model of the disease. We found that muscle biopsies from OPMD patients display na increased number of cytochrome c oxidase (COX)-negative fibers when compared to similarly aged controls. In addition, analysis of our cellular model revealed that cell lines. expressing mutant PABPN1 produce higher protein levels of the 39 kDa subunit from the.mitochondrial respiratory chain complex I. Although requiring further investigation, these.results are indicative of a role for mitochondrial dysfunction in the progression of OPMD. In summary, our work produced important information concerning the role of PABPN1 aggregates in OPMD. In addition, we identified altered BMP-dependent transcription as a candidate mechanism for OPMD pathology. Finally, we provided evidence that mitochondrial alterations may contribute to the disease phenotype. Hopefully, the further investigation of these lines of work will prove valuable to the full understanding of OPMD pathophysiology. |
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