STUDY OF α-SYNUCLEIN AGGREGATION IN A HUMANIZED YEAST MODEL

Parkinson’s Disorder (PD) is one of the major age-related neurodegenerative disorders that mainly affects the motor system, leading to the loss of dopaminergic neurons. Although PD is a progressive neurodegenerative disorder that primarily affects individuals over the age of 60; mutations of genes associated with hereditary forms of early-onset PD have been identified. This project focuses on the SNCA gene that encodes α-synuclein (α-syn), the main component of Lewy bodies which are found in the substantia nigra pars compacta of PD patients. Missense mutations in SNCA (also known as PARK1) were the first to be identified in familial cases of PD. Furthermore, duplications and triplications of SNCA have a gene dosage effect on the severity of the disorder. Currently, there is no cure for PD, but treatments that reduce the symptoms and improve the patient’s quality of life are available. Levodopa is the first-line drug in the treatment of the disorder and the more recent therapy of deep brain stimulation (DBS) effectively improves the patient’s movement. This doctoral project aimed to investigate the mechanisms underlying a-syn toxicity by studying the protein in yeast, a simple and comprehensive model organism. The resulting findings define molecular pathways that in the future may be targeted for the treatment of synucleinopathies.
This work started with the observation that cobalt, nickel and deferoxamine can rescue the toxicity induced by α-syn aggregation in yeast cells. These compounds were identified as promising candidates in a phenotypic high-throughput screening of an FDA-approved drug library on yeast engineered to overexpress human α-syn fused to GFP. After confirming that the three highlighted compounds do not directly decrease the expression level of the human protein, we concluded that the observed protective effect must involve a more complex molecular mechanism. To uncover this mechanism, we performed different analyses. Fluorescence microscopy revealed that treatment with Cobalt, Nickel, and Deferoxamine significantly reduced the number of α-syn aggregates in yeast cells. The formation and clearance of the inclusions were quantitatively monitored by time-lapse imagining in cells grown in a microfluidic system. The three treatments reduced the number and size of aggregates through different kinetics. Cobalt, in particular, eliminated all aggregates within the first 6 hours and prevented the formation of new ones.
We also observed a rescue effect by increasing the oxygenation level of the media. To assess the importance of oxygen, we cultivated the cells in different formats with decreased oxygenation until we reached complete anoxia. We were able to observe the toxic phenotype only when the cells were grown under hypoxia conditions.
To identify the key mechanism involved, we performed transcriptomic profiling coupled with pathway enrichment analysis on the strains without and with Cobalt and Deferoxamine treatment. Our analyses showed significant changes in fatty acid metabolism, ergosterol homeostasis, iron metabolism, and, more unexpectedly, in the thiamine biosynthesis pathway. The importance of the thiamine biosynthetic process was further investigated by generating a-syn strains overexpressing the enzymes THI4, THI5, THI11, THI12 and THI13. A moderate growth rescue effect was observed for some of these overexpression strains.
Following a multi-omics approach, we conducted metabolomic and lipidomic profiling to assess the impact of a-syn expression on the cell metabolic processes and on the membrane composition with a focus on phospholipids, which were also quantified through 31P Nuclear Magnetic Resonance (31P-NMR). We found that α-syn expression decreases the levels of TCA cycle and one-carbon metabolism intermediates and alters the phospholipid profile. In particular, we noticed an increased level of intermediates involved in phosphatidylinositol biosynthesis in the strain that overexpresses α-syn. This result supports the recent literature on the initiator role of Phosphatidylinositol 3-phosphate (PIP3) in the aggregation of α-syn. Interestingly, Cobalt treatment inverted the observed trend, reducing the levels of phosphoinositide.
Our study highlights that hypoxia triggers a-syn aggregation and toxicity, suggesting that oxygen availability plays an important role in neurodegenerative processes. Furthermore, pathological α-syn expression compromises central metabolic processes, potentially altering membrane lipid composition and further promoting protein aggregation in a self-perpetuating cycle. Small molecules able to modulate the hypoxia response, most likely at the level of lipid and iron metabolism, can protect against α-syn toxicity. The results offer important insights into the mechanisms of α-syn toxicity and potential therapeutic interventions for neurodegenerative diseases involving protein aggregation.