Increased thermotolerance in the absence of ataxin-3 in C. elegans

• Author(s): Carvalho, Andreia Alexandra Neves de
• Date: 2009
• Persistent ID: https://hdl.handle.net/1822/10854
• Origin: RepositóriUM - Universidade do Minho
• Document Type: Master thesis
• Language: English
• Contributor(s): Universidade do Minho; Maciel, P.; Rodrigues, Ana João Gomes

Description

Ataxin-3 is the protein involved in Machado-Joseph disease, one of the nine neurodegenerative disorders caused by a polyglutamine (polyQ) expansion. This polyQ expansion causes the appearance of misfolded protein species, aggregates, neuronal dysfunction and cell death. Ataxin-3 is a deubiquitylating (DUB) enzyme in vitro, is able to bind ubiquitin and ubiquitylated substrates and has been involved in the protein degradation by the ubiquitin-proteasome pathway (UPP). Accumulation of damaged or unneeded proteins due to the absence of a processing enzyme such as ATX-3 can disrupt cellular homeostasis. However, besides its putative role in the UPP, little is known about ataxin-3 cellular function. The possibility to carry out functional studies in a multicellular yet simple organism, and the availability of a C. elegans atx-3 knockout strain in our lab, led us to proceed our studies in this model. C. elegans lacking ATX-3 are viable and with no overt phenotype in basal conditions. However, considering the role of ataxin-3 in protein quality control we decided to analyze the effects of ataxin-3 absence in protein homeostasis stress conditions. In this work, we studied an ATX-3 knockout mutant after a heat stress insult that compromises the protein homeostasis of the whole organism. We showed that the mutant worms have an exacerbated stress response and survive significantly better than wild type animals upon a heat shock stimulus. The increased stress resistance is further enhanced by a previous mild heat shock. At a molecular level, ATX-3 mutants have a distinct profile with several molecular chaperones up-regulated at 25ºC (stress-threshold temperature), such as HSP-4 and HSP-16 family members – HSP-16.1 and HSP-16.49 – as analyzed by western blot and mRNA expression studies. These results suggested to us that the absence of ataxin-3 throughout C. elegans development might lead to a mild cellular stress, probably due to an imbalance of ATX-3 substrate degradation, having global consequences on protein homeostasis. To test this hypothesis, we used temperature-sensitive (ts) strains that constitute highly sensitive indicators of disruption in protein homeostasis, because ts mutant proteins are very dependent on the cellular folding environment. However, we have found no differences between wild type and mutant animals, in muscle and neuronal cells. Finally, we showed that the stress-resistance phenotype is dependent on the DAF-16 pathway, known to modulate aging. In summary, this work gathered significant insight into ataxin-3 biological function and pathways where it is involved.