Nuclear envelope dysfunctions observed in patients with myotonic dystrophy type 1

• Autor(a) principal: Viegas, Diana Ferreira
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/30799
• topic: Muscular dystrophies; Myotonic dystrophies; Nuclear envelope; Fibroblasts; Western blot; Immunocytochemistry

Muscular dystrophies are a very heterogeneous group of inherited diseases that are characterized by muscle weakness, loss of muscle mass and, in some cases, alterations in nervous system. To date, more than 30 different forms of muscular dystrophies are known, including myotonic dystrophy (DM). Myotonic dystrophy has two distinct variants, myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2), which result from an abnormal expansion of the CTG trinucleotide and CCTG tetranucleotide, respectively. DM1 results from an abnormal expansion of CTG in the 3 'untranslated region of the DMPK gene. The mutant DMPK mRNA accumulates in the nucleus as ribonuclear foci compromising the normal nuclear function. In recent years, nuclear envelope (NE) proteins have been associated with muscular dystrophies and specifically with DM1, as being an essential component in the response of cells to external mechanical stresses and in maintaining the integrity of muscle cells. To date, only few studies have shown that fibroblasts and myoblasts/myotubes from patients with DM1 have a compromised nuclear structure, together with altered location and intracellular levels of some NE proteins. Despite this, the role of NE proteins in DM1 is remain unclear and further studies are needed. Therefore, in present work, we analysed fibroblasts from an apparently healthy control and from patients with DM1 with approximately 1000 and 2000 CTG repeat length (representing the adult and congenital phenotypes, respectively) using the techniques western blotting and immunocytochemistry to evaluate NE protein levels and subcellular localization, respectively. Concerning intracellular levels of NE proteins, we observed an increase in LAP1, SUN1, lamin A/C and lamin B1 intracellular levels, in patients with DM1 when compared with controls. In the case of nesprin-1, a decrease in the intracellular was observed. The intracellular levels of nesprin-2 were decreased only with DM1_2000 fibroblasts when compared with controls. Finally, the emerin proteins levels were similar between the DM1-derived fibroblasts and controls. In the study of immunocytochemistry, we found that the nuclei of DM1-derived fibroblasts, when marked only with DAPI, presented an increase in deformed nuclei, accompanied by a significant increase in nuclear area (DM1_2000). In addition, we found that the nuclei of DM1-derived fibroblasts, positive for lamin A/C proteins. emerin, LAP1 and nesprin-1 showed also an increase in the number of deformed nuclei and nuclear inclusions. In the case of the lamin A/C, emerin and LAP1, there was also an increase in the immunolabelling of the NE and the nucleoplasm. In summary, we demonstrated that DM1 patients tend to have changes in the intracellular levels of NE proteins and that these are accompanied by changes in the nuclear architecture. Thus, further studies are needed to elucidate the contribution of this structure to the pathological mechanisms of DM1.