The emerging role of autophagy/mitophagy in α-synuclein-induced toxicity: studies on the yeast chronological aging model

• Autor(es): Marques, Belém Sampaio
• Data: 2012
• Identificador Persistente: https://hdl.handle.net/1822/22938
• Origem: RepositóriUM - Universidade do Minho
• Tipo de Documento: Tese de doutoramento
• Idioma: Inglês
• Contribuidor(es): Universidade do Minho; Ludovico, Paula; Maciel, P.

Descrição

α-Synuclein (α-syn) misfolding and aggregation is strongly associated with both idiopathic and familial forms of Parkinson disease (PD). Evidence suggests that α-syn has an impact on cell clearance routes and protein quality control systems, such as the ubiquitin-proteasome system (UPS) and autophagy. Recent advances in the key role of the autosomal recessive PARK2/PARKIN and PINK1 genes in mitophagy, highlighted this process as a prominent new pathogenic mechanism. Nevertheless, the role of autophagy/mitophagy in the pathogenesis of sporadic and autosomal dominant familial forms of PD is still enigmatic. The yeast Saccharomyces cerevisiae is a powerful “empty room” model that has been exploited to clarify different molecular aspects associated with α-syn toxicity, which combines the advantage of being an established system for aging research. Here, we showed that the heterologous expression of α-syn WT or the A53T mutant form induced toxicity, additionally the toxic phenotype was more aggravated when the α-syn expression was induced at the stationary cells growth phase. At this growth phase, it was demonstrated that the induction of the toxic α-syn variants led to the stimulation of the unfolded protein response (UPR), by the activation of the Ire-Hac1 signaling pathway. The activation of this pathway is probably responsible by the induction of the ATG6 and ATG8 mRNA levels, linked with autophagy and ATG32, ATG33 and DNM1 mRNA levels, which are genes specifically associated with mitophagy, suggesting that autophagy and mitophagy are stimulated under α-syn-induced toxicity. In fact modulation of autophagy, by its inhibition with chloroquine or 3 methyladenine, reverted the α-syn toxic phenotype detected by an extension of in the chronological life span (CLS) of cells expressing the α-syn WT or A53T mutant forms. Additionally, with the specific method to quantify the autophagy or mitophagy activities, the alkaline phosphatase assay, it was confirmed that the toxicity due to the heterologous expression of the toxic α-syn variants was accompanied by exacerbated stimulation of both autophagy and mitophagy. Modulation of mitophagy, by its impairment, achieved by deletion of ATG11 or ATG32 resulted in a CLS extension, accompanied by physiologic autophagic levels, further implicating mitophagy in the α-syn toxicity. It is established that reactive oxygen species, particularly superoxide anion (O22-), are associated with autophagy induction. In the experimented conditions, the stimulation of autophagy/mitophagy under α-syn expression was accompanied with O22- accumulation. Nevertheless, cells with impaired mitophagy display lower levels of O22-. Thus, the findings do not rule out O22- as inducer of autophagy in these conditions. Other molecules that have been associated with the regulation of autophagy in higher eukaryotic cells are the sirtuins, particularly the SIRT1. Deletion of SIR2, the yeast homologue of SIRT1, essential for α-syn-induced toxicity, abolished autophagy and mitophagy, thereby rescuing cells from α-syn toxicity. These data show that Sir2 functions as a regulator of autophagy, like its mammalian homologue, SIRT1, but also of mitophagy, mediating the ATG32 mRNA levels. Caloric restriction (CR) is a physiologic intervention known to promote life span extension, by reducing the activity of various signal transduction pathways either directly or through the decrease in the activity of nutrient-sensing pathways. Wild type cells submitted to CR experienced a CLS extension accompanied by physiological levels of autophagy/mitophagy, independently of the expression of α-syn toxic variants. In fact, in α-syn-induced toxicity conditions, autophagy/mitophagy induction was significant lower in comparison with the same cells submitted to non-CR conditions, supporting the hypothesis that induction of autophagy/mitophagy are directly related with the α-syn-induced toxicity. CR, like mitophagy abrogation, has the capacity to reduce α-syn-induced toxicity. The observed high CLS of atg11 and atg32 cells expressing α-syn WT or A53T mutant form in comparison with the wild type cells submitted to the same conditions, suggested that CR controls autophagy/mitophagy independently, or at least partially independent, of the pathways regulating mitophagy . Similar results were observed in sir2 cells, in which CR is able to further increase the CLS of sir2 cells expressing α-syn toxic variants, suggesting that under CR conditions, the activated signaling pathways are independent of Sir2. Altogether our work highlights that α-syn expression is associated with autophagy, particularly mitophagy stimulation mediated by the regulation of ATG32 by Sir2, an important phenomenon linked to α-syn-induced toxicity during aging. These findings open new insights to the study of the mechanism associated with PD pathogenesis in higher eukaryote organisms.