Establishment of skin-derived fibroblast cell lines for the study of protein phosphorylation in Myotonic Dystrophy type 1

• Autor(a) principal: Costa, Adriana Emília Amaral
• 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/10773/31035
• topic: Skin biopsies; Primary cultures; Myotonic dystrophy type 1; Muscular dystrophies; Neuromuscular disorders; Fibroblasts; Protein phosphorylation; Immunocytochemistry

Muscular dystrophies are a molecularly, genetically, and clinically heterogeneous group of disorders characterized mainly by progressive muscle weakness and degeneration. Within this group, the most common muscular dystrophy in adults is Myotonic Dystrophy type 1 (DM1), an inherited autosomal dominant disorder caused by an expansion of the (CTG)n trinucleotide repeats on the 3’ untranslated region of the DMPK gene. Patients with DM1 not only present muscular symptoms such as myotonia and muscle wasting, but also extramuscular symptoms such as cataracts, cardiac conduction abnormalities and insulin resistance. In DM1, the increased length of triplet expansions leads to the accumulation of (CUG)n mRNA, that forms hairpin-like structures in the nucleus, leading to a toxic “gain-of-function” that deregulates RNA binding proteins such as MBNL1 and CUGBP1. This consequently affects alternative splicing of different mRNAs, which damages the normal function of different signalling pathways regulated through phosphorylation, an important regulatory mechanism. To understand the different impaired phosphorylation signalling pathways affected in DM1 we firstly conducted a systematic review about protein phosphorylation in DM1. The results provided a compilation of the altered protein phosphorylation events, namely the signalling pathways which regulate key cellular events. Some main findings are the underactivation of signalling pathways, such as AKT/mTOR and AMPK upon insulin signalling and starvation conditions, respectively. Also, myoblast differentiation was impaired since, during differentiation, there was an increase of activity of signalling pathways that stimulate cell proliferation (e.g. MEK/ERK, PKR/PERK) and a decrease of important proteins for muscle development, such as DMPK. In order to be able to study the mechanisms underlying the impaired signalling pathways described in the systematic review, it is necessary to establish DM1 cell models. For that reason, fibroblasts have been widely used for the study of this disorder due to its versatility and easy manipulation. We then successfully established skin-derived human fibroblast cell lines from patients with DM1 through a skin punch biopsy explant. These cell lines were subsequently characterized through indirect immunocytochemistry using a fibroblast-specific marker TE-7. The intracellular levels and localization of DMPK were also evaluated. It was possible to detect differences, although not statistically significant, of a reduced expression of DMPK in DM1-derived fibroblasts from late and juvenile onset with controls. To conclude, these fibroblasts can be an important cell model for the study of phosphorylation pathways and other mechanisms, such as nuclear envelope alterations and being an important research tool for DM1. As future perspectives, these cell models can be used to study protein phosphatases, such as PP1 and PP2, since there is not enough evidence of how these are altered in DM1 and could highly contribute to unravel new molecular mechanisms.