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Master's Dissertation
DOI
https://doi.org/10.11606/D.41.2020.tde-09112020-111224
Document
Author
Full name
Jáina Araújo Reis
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2020
Supervisor
Committee
Ferrari, Merari de Fatima Ramires (President)
Borges, Maria Nathalia de Carvalho Magalhães Moraes Figueira
Gomes, Fernando
Ureshino, Rodrigo Portes
Title in Portuguese
Distribuição celular e função da proteína C9ORF72 em modelos celulares de Esclerose Lateral Amiotrófica
Keywords in Portuguese
1. C9ORF72
2. Proteínas Rab
3. Autofagia
4. Homeostase do RE
5. Neurodegeneração
Abstract in Portuguese
Estudos genéticos recentes apontaram uma mutação no gene C9ORF72 como a causa genética mais comum para Esclerose lateral amiotrófica (ELA) e Demência Frontotemporal (FTD), porém, sua contribuição para o surgimento dessas desordens neurodegenerativas permanece incerta e é dificultada pela falta de compreensão completa de sua função. Assim, desvendar as funções de C9ORF72 e as vias bioquímicas das quais participa é um passo fundamental para compreender completamente os mecanismos patogênicos associados à C9ELA/FTD. Neste sentido, o presente trabalho teve por objetivo avaliar aspectos da distribuição celular e expressão de C9ORF72, sua interação com proteínas Rabs e sua contribuição para a via autofágica e para a manutenção da homeostase do retículo endoplasmático (RE) em modelos celulares de ELA, como células primárias de camundongos SOD1G93A e da linhagem Neuro-2a. Os ensaios experimentais revelaram uma localização celular ampla para a isoforma longa de C9ORF72, presente no interior do núcleo, membrana nuclear, citoplasma, vesículas e membrana plasmática. A análise da expressão de proteínas envolvidas na autofagia mostrou que a presença de SOD1 mutante provoca disfunção autofágica, com redução nos níveis de Rab1, P62 e Beclina-1, e leva a translocação nuclear da isoforma longa de C9ORF72. Curiosamente a translocação de C9-L não interfere na sua interação citoplasmática com as proteínas Rab estudadas. De maneira semelhante, o silenciamento de C9ORF72 por lipofecção de siRNA levou a prejuízo da autofagia com acúmulo de autofagossomos e redução de Rab1, uma proteína indispensável para o tráfego RE-Golgi e formação de vesículas autofágicas, o que desencadeou remodelamento do retículo endoplasmático e alterações no transporte entre RE e Golgi, evidenciado pela redução de ATF6? clivada. Em conjunto, esses resultados sugerem que C9ORF72 atua na regulação da via autofágica e na manutenção da homeostase do RE, além de fornecerem indícios de um possível papel nuclear para a isoforma longa de C9ORF72, independente de sua função citoplasmática.
Title in English
Cellular distribution and function of C9ORF72 protein in cellular models of Amyotrophic Lateral Sclerosis
Keywords in English
1. C9ORF72
2. Rab proteins
3. Autophagy
4. ER homeostasis
5. Neurodegeneration
Abstract in English
Recent genetic studies have pointed to a mutation in the C9ORF72 gene as the most common genetic cause for Amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD), however, its contribution to the emergence of these neurodegenerative disorders remains uncertain and is hampered by a lack of complete understanding of its function. Thus, unraveling the functions of C9ORF72 and the biochemical pathways in which it participates is a pivotal step towards the fully knowledge of the pathogenic mechanisms associated with C9ALS/FTD. In view of this, the present study aimed to evaluate aspects of the cellular distribution and expression of C9ORF72, its interaction with Rabs proteins and its contribution to the autophagic pathway and to the maintenance of endoplasmic reticulum (ER) homeostasis in ALS cell models, such as primary neurons from SOD1G93A mice and Neuro-2a cell line. Experimental data revealed a wide cell location for the long C9ORF72 isoform, present inside the nucleus, nuclear membrane, cytoplasm, vesicles and plasma membrane. Analysis of the expression of proteins involved in autophagy showed that the presence of mutant SOD1 causes autophagic dysfunction, with a reduction in the levels of Rab1, P62 and Beclin-1, and leads to nuclear translocation of the long C9ORF72 isoform. Interestingly, C9-L translocation does not interfere with its cytoplasmic interaction with the studied Rab proteins. Similarly, silencing C9ORF72 by siRNA lipofection led to defective autophagy with accumulation of autophagosomes and reduction of Rab1, involved in ER-Golgi traffic, which triggered remodeling of the endoplasmic reticulum and impairment of transport between RE and Golgi, evidenced by the reduction of cleaved ATF6?. Together, these results suggest that C9ORF72 acts in the regulation of the autophagic pathway and in the maintenance of ER homeostasis, in addition to its possible nuclear role for the long isoform of C9ORF72, regardless of its cytoplasmic function.
 
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Publishing Date
2021-02-15
 
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