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• W4213007368 abstract "Neurotransmitter release is a key step that enables information flow between the pre- and post-synapse. However, regulation of the neurotransmitter release remains an intricate and widely unexplored matter despite recent advances in the understanding of the neurotransmitter release machinery and the analysis of the synaptic proteome and protein modifications. Indeed, post-translational protein modifications such as phosphorylation are suitable to quickly fine-tune the neurotransmitter release “in place” via affecting tertiary protein structures and protein-protein interactions, and globally, via modulating signaling pathways. Here, the investigations were focused on the dependence of protein phosphorylation in synaptosomes on the synaptic vesicle (SV) cycling, determining kinase-substrate interactions, and modulatory effects of selected sites on exo- and endocytosis. The analysis of synaptic phosphoproteome was conducted using TiO2-based enrichment of phosphorylated peptides with subsequent chemical labeling by isobaric mass tags (TMT) and a mass spectrometry-based quantification. Synaptosomes were employed as a functional model of a synapse as they contain the required neurotransmitter release machinery and respond to stimulation. First, the applicability of electrical stimulation was tested. The field- stimulation evoked reproducible glutamate release that was significantly suppressed in the absence of Ca2+, though it remained uncertain, to which degree the release is governed by exocytosis. Therefore, another approach using a KCl-induced depolarization and treatment with botulinum neurotoxins (BoNTs) was used to identify phosphorylation events that depend on SV cycling. BoNTs cleave specifically SNARE proteins and thus block exocytosis and SV cycling, but do not impede Ca2+-influx evoked by the plasma membrane depolarization. Comparison of phosphorylation events in synaptosomes stimulated in the presence of Ca2+, EGTA (0 net Ca2+) or pre-treated with BoNTs identified sites that were differentially phosphorylated following BoNT treatment, i.e., SV-cycling-dependent sites, and sites that were differentially phosphorylated when comparing Ca and EGTA conditions, but did not change under BoNT treatment, i.e., primarily Ca2+-dependent sites. Further differential expression analysis revealed that BoNT-treatment mostly caused de-phosphorylation of synaptic proteins. A kinase-substrate analysis showed that >25% of BoNT-responsive sites are predicted MAPK substrates and <9% are putative CaMKII targets. In contrast, >20% of primarily Ca2+-dependent sites are presumably regulated by CaMKII, which corroborates Ca2+- dependence of these phosphorylation events. SV-cycling-dependent phosphorylation sites on syntaxin-1 (T21/T23-Stx1), synaptobrevin (S75-Vamp2), and cannabinoid receptor-1 (S314/T322-Cnr1) were further investigated for their impact on exo- and endocytosis. In collaboration with Dr. Eugenio Fornasiero and Prof. Dr. Silvio O. Rizzoli, corresponding phosphomimetic and non-phosphorylatable variants of the proteins were expressed in cultured hippocampal neurons. Imaging of the pH-sensor pHluorine coupled to synaptobrevin-2 revealed that the expression of phosphomimetic and non-phosphorylatable sites affected exo- and endocytosis in neurons. This work is first to investigate the electrical stimulation in relation to the Ca2+-dependent neurotransmitter release and exocytosis in synaptosomes. It further provides a comprehensive draft of synaptosomal phosphoproteome and is first to demonstrate its global dependence on an active SV cycling. The analysis of cultured hippocampal neurons expressing non-phosphorylatable and phosphomimetic mutants of pre-synaptic proteins syntaxin-1, synaptobrevin-2, and cannabinoid receptor-1 further demonstrates that the identified SV-cycling-dependent sites affect exo- and endocytosis." @default.
• W4213007368 created "2022-02-24" @default.
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• W4213007368 date "2022-02-21" @default.
• W4213007368 modified "2023-09-27" @default.
• W4213007368 title "Post-Translational Protein Modifications involved in Exo- and Endocytosis of Synaptic Vesicles" @default.
• W4213007368 doi "https://doi.org/10.53846/goediss-8895" @default.
• W4213007368 hasPublicationYear "2022" @default.
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