Characterization of the nuclear envelope alterations in Myotonic Dystrophy Type 1

• Autor(a) principal: Basílio, Ana Cláudia Miquelino
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/28409
• topic: Myotonic dystrophy type 1; Nuclear envelope proteins; Nuclear functions; Human fibroblasts

The myotonic dystrophy type 1 (DM1) is a multisystem genetic disorder mainly associated with degeneration of skeletal muscle and caused by a CTG repeat expansion in the 3′ untranslated region of the DMPK gene, accumulating as nuclear foci and compromising the nuclear function. In the last few years, some studies associate nuclear envelope proteins as a significant component in the response of cells to mechanical stress, helping to maintain muscle cell integrity, demonstrating a clear association to DM1 pathology. In fact, it has been demonstrated that DM1 patient’s fibroblasts presents an impaired nuclear structure and an altered localization of some NE proteins. However, the contribution of the NE for DM1 was not fully elucidated. In this work, we performed an ATR-FTIR spectroscopy study of human control and DM1 fibroblasts to identify spectral differences. The human patients’ fibroblasts included in this study have 1000 CTG and 2000 CTG repeats, representing the adult and congenital DM1 types, respectively. DM1 fibroblasts present differences at the molecular level, mainly in protein and lipids structures: DM1 donors can be distinguished from controls by a larger lipidic stretching, more saturated lipidic stretching and presence of protein aggregates. These differences can be explained by the dysregulation of lipins in DM1, affecting the lipid metabolism, and RNA and polyglutamine expansion proteins accumulation as nuclear aggregates, respectively. Moreover, the nuclear profile in human fibroblasts derived from DM1 patients was evaluated, in order to characterize some particular nuclear features, namely the nuclear area, circularity, deformations (blebs and misshaped nuclei) and micronucleus presence. Our results evidenced a tendency to increase the nuclear area, the number of cells with micronuclei and the % of deformed nuclei in DM1 donors, comparing to the control. Further, in order to unravel some nuclear envelope proteins alterations in DM1, namely in lamin A/C, emerin, SUN1 and LAP1, we performed immunocytochemistry analysis in human fibroblasts. Regarding lamin A/C, emerin and LAP1, these proteins showed an altered localization in nuclear envelope, in both DM1 fibroblasts. Also, both lamin A/C and emerin are highly present in protein positive-nuclear inclusions whereas SUN1 and LAP1 are highly present in protein positive-nuclear invaginations. To conclude, spectral differences have been identified between controls and DM1 fibroblasts. Moreover, the nuclear architecture and nuclear envelope proteins seems to be altered in DM1 donors. Therefore, further studies should be performed in order to elucidate nuclear envelope proteins contribution to the disease.