| dc.description.abstracteng | 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. | de |