Interplay between alpha-synuclein induced toxicity in Parkinson’s disease and cellular proteostasis in yeast

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Protein homeostasis is an essential factor for maintenance of normal cellular function. It is achieved by a delicate balance between protein synthesis and disposal. Deficits in proteostasis are linked to various neurodegenerative diseases, including Parkinson’s disease (PD). Characteristic for PD is the aggregation of alpha-synuclein into protein inclusions, indicating significant dysfunction in cellular proteostasis. Accumulation of alpha-synuclein is accompanied with highly elevated phosphorylation at S129, suggesting that this posttranslational modification is linked to pathogenicity and alpha-synuclein turnover. In the previous funding period, we have characterized the interplay between essential proteostasis pathways and alpha-synuclein toxicity, aggregation and turnover using the toolbox of yeast to model cellular basic mechanisms of PD. Expression of alpha-synuclein in yeast leads to toxicity, which is recognized as significant growth reduction and inclusion formation similar to neurons. Genome-wide screens with yeast strain collections identified multiple modulators of alpha-synuclein toxicity and aggregation. The most prominent categories were connected with protein homeostasis, including protein synthesis and ubiquitin-dependent degradation. We found that expression of alpha-synuclein reduces the abundance of 26S proteasome subunits and alters the ubiquitin conjugates. The proteostasis dysfunction correlates with alpha-synuclein turnover and depends on S129 phosphorylation. The aim of this research project is to understand the complex interplay between alpha-synuclein toxicity and cellular proteostasis in yeast, as well as the role of S129 phosphorylation in this process. The main goal will be assessed by two specific objectives: (i) We will determine, how alpha-synuclein and the phosphorylation deficient S129A variant affect protein dynamics in yeast, which proteins change their stability and what are the consequences for the cellular proteostasis. The main focus will be on the proteasomal chaperone Rpn14/PAAF1 identified in preliminary experiments for this proposal. (ii) We will decipher the impact of alpha-synuclein and S129A expression on the 26S proteasome homeostasis. We aim to elucidate, how these proteins affect the degradation of the 26S proteasomes by proteaphagy and their sorting into the protective proteasome storage granules under different stress conditions. Our goal is to obtain an integrative picture of the key components of the proteostasis network affected by alpha-synuclein accumulation and aggregation that will contribute to our understanding of the molecular mechanisms in aging and disease.