To achieve understanding of how neural circuits function, we need to unravel the neuronal integration of incoming signals and modulation of synaptic transmission strength over time. This is a process known as synaptic plasticity and is an essential mechanism behind memory and learning. While focus has largely been on post-synaptic plasticity, pre-synaptic plasticity has been widely neglected. An important part of pre-synaptic plasticity is modulation of the synaptic vesicle cycle, which governs vesicle availability/localization – and thereby neurotransmitter release potential.

Protein phosphorylation modulates several aspects of the synaptic vesicle cycle and is thus a potential widespread mode of presynaptic plasticity. We have isolated presynaptic nerve terminals (synaptosomes) from rats and depolarized them with KCl to investigate the role of activity-dependent phospho-signaling in neurotransmission up to 15 min after depolarization. Performing enzymatic fluorescence-based assays of glutamate release, we show that this treatment induces synaptic depression. We have quantified >5000 phosphorylation sites in synaptosomes using dimethylation, phospho-peptide enrichment and fractionation via TiO2, Sequential elution from IMAC and HILIC (TiSH) followed by nanoLC-MS analysis. We observe prolonged reprogramming of the presynaptic phosphoproteome after depolarization. Changes in regulatory phosphosites on phosphatase regulators – key integrators of neuronal signaling networks – suggest a likely link between depolarisation and long-term global presynaptic phosphorylation changes. Importantly, the protein machinery of the active zone controlling the release of neurotransmitter-containing vesicles appears to be regulated by activity-dependent phosphorylation. Amongst these proteins, the key synaptic plasticity protein Rab3 interacting molecule (RIM1), which links calcium influx to neurotransmitter release, contained several regulated phosphorylation sites without known functions. This establishes RIM1 as a prime candidate for being the link between activity-dependent phospho-regulation and presynaptic plasticity.

MS-based protein pulldown experiments using phospho-deficient and -mimetic mutants of RIM1 are currently underway and will reveal the phospho-dependent mediators of presynaptic plasticity. Primary hippocampal neurons will be transfected with these constructs to validate the functional effect of the RIM1 phosphorylation sites using dye uptake/release assays.