Investigations of Synaptic Alterations in Models of Parkinson’s Disease
by Mohammed Al-Azzani
Date of Examination:2023-08-03
Date of issue:2024-07-02
Advisor:Prof. Dr. Tiago Fleming Outeiro
Referee:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Nils Brose
Referee:Prof. Dr. Christine Stadelmann-Nessler
Referee:Prof. Dr. Rubén Fernández-Busnadiego
Persistent Address:
http://dx.doi.org/10.53846/goediss-10585
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Abstract
English
Alpha-synuclein (aSyn) is a presynaptic protein implicated in physiological synaptic roles, though its precise function is largely unknown. In Parkinson's disease (PD), the initial pathological events often involve synaptic impairments and aSyn aggregation, even before evident neurodegeneration. Abnormal aSyn forms can cause synaptic dysfunctions, but the toxicity of different aSyn species in along aSyn aggregation pathway is still a matter of ongoing debate. In the first part of our study, we performed a comprehensive comparison of different exogenous aSyn (monomers, oligomers, and preformed fibrils (PFFs)) on primary hippocampal neurons. PFFs exposure at days in vitro (DIV) 7 mainly induced aggregation of endogenous aSyn associated with more neurotoxicity, and a decline in some synaptic protein levels at DIV 26. At DIV 21, the activity of recycling synaptic vesicle was impacted upon treatments with both oligomers and PFFs. Additionally, PFFs exposure at DIV 14 decreased neurons mean firing rate one-week post treatment. In the second part of our study, we investigated how differences in recombinant aSyn purification protocols could affect its aggregation properties, potentially affecting downstream applications. We found that aSyn extracted under “High salt“ conditions showed a faster and higher aggregation profile compared to “No salt“ extracted aSyn using in vitro Thioflavin T based aggregation assays. In the third part of our study, we studied aSyn interactions, particularly at synaptic vesicles isolated from adult mice brains, and performed aSyn pulldown assays. Proteomics data revealed many potential aSyn interactors. Gene ontology analysis linked these candidates to synaptic vesicle cycling, neurotransmitter release, and, interestingly, pathways associated with neurons and neuronal projections developments, suggesting aSyn has roles beyond traditional synaptic function roles. Using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) proteomics in HEK cells, we discovered potential interactors for endogenous aSyn in control cell lines. Many of these candidates have nuclear localization and are implicated in RNA and DNA processes, supporting aSyn roles in the nucleus. Finally, we explored the effects of aSyn glycation, a posttranslational modification involved in neurodegeneration. We exposed mouse hippocampal neurons to glycated and unglycated aSyn at DIV 07. The glycated aSyn showed more neurotoxicity at DIV 26, but the capacity to induce aggregation of endogenous aSyn was unchanged regardless of glycation. Furthermore, glycated aSyn exhibited comparable effects on the levels of synaptic proteins compared to unglycated aSyn and did not significantly affect spontaneous neuronal activity in our assays. In conclusion, our study revealed that aSyn PFFs induced more neurotoxicity and seeding potential than oligomeric species. Both forms, however, showed comparable synaptic vesicle recycling disruption, suggesting seeding-dependent and independent toxic mechanisms. We also found that different aSyn purification methods can affect its aggregation behavior, suggesting a need for standardized production protocols. Furthermore, we identified potential new aSyn interacting partners that could point towards novel physiological roles for aSyn. Lastly, while we did not observe synaptic effects for glycated aSyn, it did exhibit cytotoxicity, suggesting functional impacts may require higher concentrations.
Keywords: Parkinson's disease, Alpha-synuclein, neurodegeneration, synaptic vesicles, synaptic function, aggregation, glycation
