Analysis of Fig4 interfered cells and its role on the endocytic pathway

• Autor(a) principal: Vieira, Tiago Pinto
• 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/10773/30940
• topic: Phosphoinositides; Fig4; Charcot-Marie-Tooth type 4J; Endocytic pathway

Mutations in the inositol phosphatase Fig4 were firstly associated with Charcot-Marie-Tooth 4J (CMT4J) neuropathy, a rare recessive demyelinating form of CMT with highly variable onset and characterized by severe motor dysfunction and involvement of motor and sensory neurons. Interestingly, frameshift and other missense mutations have been reported to be responsible of Yunis-Varon syndrome and familial epilepsy with polymicrogyria extending the spectrum of phenotypes associated with FIG4 mutations. Fig4 dephosphorylates the endolysosome enriched PI(3,5)P2 to generate PI(3)P. Enlarged LAMP2 positive vacuoles with watery appearance or filled with electron dense material (depending on cell type) are found in neurons, muscle and cartilage of Fig4 null mice, suggesting a dysfunction of these compartments. However, the pathogenic mechanism(s) still remain elusive. Strikingly, the endosomal system, which is a crossroad of distinct intracellular pathways, has emerged and is still emerging as key player in various neurological disorders. This research aimed to understand the role of Fig4 in regulating the endosomal pathway in order to elucidate the pathomechanisms of these neurological disorders. Moreover, these analyses also intended to contribute to a better comprehension of phosphoinositide as regulators of membrane trafficking. To this purpose, the effects of the knockdown of Fig4 expression on the endosomal pathway were analyzed. By western blot analyses and immunofluoescence assays the homeostasis and dynamics of endosomal compartments were evaluated. Furthermore, the impact of Fig4 knockdown on cell viability was studied by performing growth curves. Fig4 knockdown was found to drastically alter the whole endo-lysosome axis: lysosomes appear as large dots and also late and early endosomes are numerous and enlarged. In addition, the levels of endocytic resistant proteins are increased, suggesting an alteration of their dynamics. These results indicate that Fig4 activity is crucial for the homeostasis and function of endosomal compartments in different cells types. The dysfunction of these pathways might underlie the pathogenesis of Fig4-associated diseases.