Post-Translational Protein Modifications involved in Exo- and Endocytosis of Synaptic Vesicles

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 TiO 2 -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 fieldstimulation evoked reproducible glutamate release that was significantly suppressed in the absence of Ca 2+ , 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 Ca 2+ -influx evoked by the plasma membrane depolarization.Comparison of phosphorylation events in synaptosomes stimulated in the presence of Ca 2+ , EGTA (0 net Ca 2+ ) 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 Ca 2+ -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 Ca 2+ -dependent sites are presumably regulated by CaMKII, which corroborates Ca 2+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 prote...