Abstract
The yeast Saccharomyces cerevisiae is a powerful model to study the molecular mechanisms underlying α-synuclein (α-syn) cytotoxicity. This is due to the high degree of conservation of cellular processes with higher eukaryotes and the fact that yeast does not endogenously express α-synuclein. In this work, we focused specifically on the interplay between α-syn and intracellular Ca2+ homeostasis. Using temperature-sensitive SEC4 mutants and deletion strains for the vacuolar Ca2+ transporters Pmc1 and Vcx1, together with aequorin-based Ca2+ recordings, we show that overexpression of α-syn shifts the predominant temporal pattern of organellar Ca2+ release from a biphasic to a quasi-monophasic response. Fragmentation and vesiculation of vacuolar membranes in α-syn expressing cells can account for the faster release of vacuolar Ca2+. α-Syn further significantly reduced Ca2+ storage resulting in increased resting cytosolic Ca2+ levels. Overexpression of the vacuolar Ca2+ ATPase Pmc1 in wild-type cells prevented the α-syn-induced increase in resting Ca2+ and was able to restore growth. We propose that α-syn-induced disruptions in Ca2+ signaling might be an important step in initiating cell death.
Highlights
Α-Syn misfolding and aggregation are linked to Parkinson’s disease (PD) (Poewe et al, 2017)
Dopaminergic neurons in PD are vulnerable to disruptions in Ca2+ homeostasis due to their distinctive pacemaker activity, which is heavily reliant on Ca2+ entry (Pacelli et al, 2015). α-Syn probably interacts with components of the Ca2+ toolkit altering Ca2+ homeostasis and triggering downstream toxic effects
Since Ca2+ efflux across the plasma membrane in yeast may rely on vesicular exocytosis, we proposed that defects in vesicular trafficking may underlie α-syn-induced Ca2+ dysregulation
Summary
Α-Syn misfolding and aggregation are linked to Parkinson’s disease (PD) (Poewe et al, 2017). The high degree of conservation of protein folding and degradation, Ca2+ homeostasis and vesicle trafficking between yeast and higher eukaryotes and the fact that yeast does not express homologs of the human synuclein family allow to exploit yeast as a platform to study the molecular mechanisms underlying α-syn cytotoxicity (Franssens et al, 2010). Analysis of various yeast models for α-syn has shown that overexpression of wild-type or mutant α-syn results in growth inhibition and the formation of cytotoxic intracellular inclusions. Previous studies in mammalian cells have mainly focused on the effects of externally applied α-syn oligomers on Ca2+ homeostasis. Several studies have demonstrated that α-syn overexpression strongly inhibits vesicle trafficking, thereby disrupting normal processing and transport of proteins within the secretory pathway. Whereas α-syn overexpression and the formation of oligomers may inhibit SNARE and RAB/Sec functions leading to reduced secretory activity (Choi et al, 2013). We show that α-syn alters Ca2+ handling through two distinct mechanisms involving α-syn-mediated disruption of vesicle trafficking and vacuolar Ca2+ storage
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