Caracterización del papel regulador en la agregación de cuerpos de inclusión de poliglutaminas del gen unc-1/stomatin-like en C. elegans

Abstract

[ES] En el laboratorio hemos generado mutantes que modulan la dinámica de agregación de péptidos que contienen expansiones de 40 glutaminas en gusanos nematodos C. elegans (Cepa AM141; genotipo-rmIs133[unc-54p::40Q::YFP] Morley et al., PNAS 2002). Esta cepa presente un fenotipo de agregación de las poliglutaminas dependiente de la edad. Los individuos jóvenes apenas presentan agregados, mientras que los adultos los producen en abundancia. En el presente trabajo caracterizaremos el papel modulador de la agregación de estas poliglutaminas del gen unc-1 del gusano, que tiene homología con el gen humano stomatin-like protein 3 (STOML3). Gusanos portadores de un alelo de pérdida total de función de este gen (unc-1(vlt10)) tienen agravado el fenotipo de agregación de péptidos 40Q::YFP. Este fenotipo es cuantificable porque los agregados colapsan en cuerpos de inclusión que son fácilmente observados en un estereoscopio equipado con fluorescencia. Para demostrar que la pérdida de función de unc-1 produce este fenotipo introduciremos en AM141 otros alelos caracterizados del gen, y disponibles en el repositorio de cepas de gusano (Caenorhabditis Genetics Center). Además emplearemos RNAi para silenciar el gen y dar más robustez a los experimentos descritos arriba. Todo esto sentará las bases para una futura búsqueda del mecanismo por el cual el gen modula la agregación de poliglutaminas.

[EN] Huntington’s disease (HD) is a rare dominant neurodegenerative disease of genetic heritance. Patients of HD suffer coordination problems and chorea and progressive damage of the cognitive function. HD is caused by the presence of an abnormally long CAG expansion, encoding polyglutamines (polyQs), in the first exon of the huntingtin (htt) gene. When carriers have 39 or more triplets huntingtin acquires a toxic gain of function, which impairs many cellular functions. Although this gene is ubiquitously expressed, mutant huntingtin (mHtt) affects particularly neurons, inducing neuronal degeneration that leads to cell death. mHtt is very prone to aggregation and there is a vivid debate about whether aggregation of mHtt is a cause or a consequence of HD. In this regard it is essential to understand the molecules and mechanisms involved in the dynamics of aggregation of polyQ-containing proteins, such as mHtt. We use C. elegans to screen for genes that modify the dynamics of aggregation of polyQ-containing proteins. We isolated a number of mutants from the AM141 strain, which contains a transgene that induces the expression of a tandem of 40 glutamines (40Q) fused in frame with YFP. This strain shows an age-dependent aggregation pattern that can be easily followed using a dissecting microscope equipped with fluorescence. We sequenced by NGS means the whole genome of several mutant worms, and we have identified, using bioinformatics, a mutation in the unc-1/stomatinlike protein gene, vlt10, which enhances the aggregation phenotype of AM141. To verify the role of unc-1 in the dynamics of aggregation, we have introduced different unc-1 alleles within AM141 which confirmed that unc-1 is an enhancer of aggregation. We also show that ARNi against this gene also increases the speed of the aggregation pattern of AM141. We are currently working to find out the mechanism by which this gene modulates aggregation dynamics of polyglutamin-containing molecules.