α-Synuclein Budding Yeast Model: Toxicity Enhanced by Impaired Proteasome and Oxidative Stress

Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein alpha-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T alpha-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, alpha-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed alpha-synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of alpha-synuclein. Both sen3-1and doa3-1 mutants exhibited synthetic lethality with alpha-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), alpha-synuclein was extremely lethal to cells that lacked manganese superoxide dismutase Mn-SOD (sod2Delta) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Delta). Despite the toxicity, sod2Delta cells never displayed intracellular aggregates of alpha-synuclein. We suggest that the toxic alpha-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve alpha-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate alpha-synuclein toxicity.

Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson’s disease models

“…By varying the copy number and integration loci of -syn, we found an unusually strong dose dependence between -syn expression levels and toxicity (Cooper et al, 2006;Gitler et al, 2008); this is highly reminiscent of the extreme dosage sensitivity of -syn observed with certain dominantly inherited forms of PD (Singleton et al, 2003;Ibanez et al, 2009). In this model, -syn toxicity causes lipid droplet accumulation (Outeiro and Lindquist, 2003), impairs proteasome-mediated protein degradation (Chen et al, 2005;Sharma et al, 2006) and elicits vesicle trafficking defects (Outeiro and Lindquist, 2003;Soper et al, 2008) with specific ERto-Golgi blockade (Cooper et al, 2006;Gitler et al, 2008). All of these phenotypes have been observed in neuronal models and implicated in the pathogenesis of PD.…”

Yeast unfolds the road map toward α-synuclein-induced cell death

“…37,46 However, later reports showed that a-syn toxicity is dependent on the genetic background as in certain yeast strains the cells can be loaded with a-syn inclusions without showing a significant growth defect. 38,47 Moreover, cells of the fission yeast Saccharomyces pombe show extensive a-syn inclusion formation without any toxicity. 48 As WT-syn and the A53T mutant do not target the plasma membrane in S. pombe cells and, as such, behave in a manner similar to the A30P mutant in S. cerevisiae cells, it was hypothesized that the a-syn-derived toxicity would correlate better with the membrane binding capacity rather than with the fibrillization rate of the protein.…”

Signaling pathways and posttranslational modifications of tau in Alzheimer's disease: the humanization of yeast cells

“…In fact, (over)expression of genes encoding key proteins involved in human dementias proved to be lethal to yeast cells, such as that for synuclein (depending on the genetic background of the yeast strain) and the protein TDP-43 involved in amyotrophic lateral sclerosis (ALS) [86,87]. In contrast, human tau overexpression from the GAL1/10 promoter in yeast, which lacks an endogenous tau homolog, does not produce any detectable phenotype in logarithmically growing wild-type cells [23,66].…”

“…2 have been successfully expressed, and in many cases shown to functionally complement the respective yeast deletion mutants provided by either of the deletion collections kept in Stanford/USA or the EUROSCARF collection in Frankfurt/Germany. However, it should be noted that caution as to the genetic background of the yeast strain applied for specific purposes may be required, as examplified for the variations in toxic effects of synuclein gene expression [87].…”