Molecular mechanisms underlying presynaptic plasticity: characterization of the RIM1α and SV2A interactome

Synaptic plasticity encompasses various cellular mechanisms, which confer synapses the ability to react and adapt to ongoing changes in network activity. Some of the suggested mechanisms include remodelling and/or assembly of active zones (AZ), and modulation of neurotransmitter release. At the molecular level posttranslational modifications of proteins, e.g. phosphorylation, have been reported to be associated with these events. Two components of the release machinery, RIM1α and synaptic vesicle protein 2A (SV2A) were shown to be actively involved in presynaptic plasticity. However, the impact of posttranslational modifications, like phosphorylation, on the function of these proteins is not well understood. Therefore, the goals of this thesis were to examine the impact of phosphorylation on the binding properties of RIM1α and to identify and analyse novel binding partners for SV2A. We found that the distribution of RIM1α at synapses is altered after globally increasing the level of phosphorylation, while the total level remained unchanged, suggesting that the association of RIM1α with the CAZ is controlled by its phosphorylation status. Affinity purification and MS revealed that alterations in the phosphorylation status of RIM1α affected its affinity to specific binding partners. Out of the identified proteins, four candidates with a potential functional link were chosen to be further analysed in binding assays: two kinases (unc-51-like kinase 1/2, serine arginine protein kinase 2), one calcium-binding protein (Copine VI), and proteins involved in trafficking (vesicle-associated membrane protein (VAMP) associated-protein A/B). Interestingly, RIM1α may represent the first AZ substrate for ULKs and SRPK2, which in D.melanogaster have already been linked to the assembly of AZs. This may support the hypothesis that both ULKs and SRPK2 could be actively involved in controlling not only RIM1α’s function but also its association with the CAZ. VAP proteins, by specifically binding the C2A-domain of RIM1α, may contribute to control the trafficking of RIM1α to the synapse. Copine VI may regulate the function of RIM1α in a calcium-dependent manner. Further analysis will reveal if these novel interactions may have any functional relevance for the function of RIM1α. The last part of the study was dedicated to another presynaptic protein, SV2A. To date the role played by SV2A in SV priming is not fully elucidated. Therefore, to gain insight into the enigmatic function of SV2A identification of novel binding partners was pursued. Different affinity purification strategies coupled to MS were performed in order to identify the SV2A proteome. However, none of these approaches resulted in the identification of novel interacting proteins, which could be further verified in biochemical assays. Taken together, the findings of this thesis may form the basis for further functional studies in order to decipher the molecular mechanisms underlying the function of RIM1α and in consequence, the role of RIM1α in presynaptic plasticity

A presynaptic phosphosignaling hub for lasting homeostatic plasticity

Stable function of networks requires that synapses adapt their strength to levels of neuronal activity, and failure to do so results in cognitive disorders. How such homeostatic regulation may be implemented in mammalian synapses remains poorly understood. Here we show that the phosphorylation status of several positions of the active-zone (AZ) protein RIM1 are relevant for synaptic glutamate release. Position RIMS1045 is necessary and sufficient for expression of silencing-induced homeostatic plasticity and is kept phosphorylated by serine arginine protein kinase 2 (SRPK2). SRPK2-induced upscaling of synaptic release leads to additional RIM1 nanoclusters and docked vesicles at the AZ and is not observed in the absence of RIM1 and occluded by RIMS1045E. Our data suggest that SRPK2 and RIM1 represent a presynaptic phosphosignaling hub that is involved in the homeostatic balance of synaptic coupling of neuronal networks

Rab Interacting Molecules 2 and 3 Directly Interact with the Pore-Forming CaV1.3 Ca2+ Channel Subunit and Promote Its Membrane Expression

Rab interacting molecules (RIMs) are multi-domain proteins that positively regulate the number of Ca2+ channels at the presynaptic active zone (AZ). Several molecular mechanisms have been demonstrated for RIM-binding to components of the presynaptic Ca2+ channel complex, the key signaling element at the AZ. Here, we report an interaction of the C2B domain of RIM2α and RIM3γ with the C-terminus of the pore-forming α–subunit of CaV1.3 channels (CaV1.3α1), which mediate stimulus-secretion coupling at the ribbon synapses of cochlear inner hair cells (IHCs). Co-expressing full-length RIM2α with a Ca2+ channel complex closely resembling that of IHCs (CaV1.3α1-CaVß2a) in HEK293 cells doubled the Ca2+-current and shifted the voltage-dependence of Ca2+ channel activation by approximately +3 mV. Co-expression of the short RIM isoform RIM3γ increased the CaV1.3α1-CaVß2a-mediated Ca2+-influx in HEK293 cells, but disruption of RIM3γ in mice left Ca2+-influx in IHCs and hearing intact. In conclusion, we propose that RIM2α and RIM3γ directly interact with the C-terminus of the pore-forming subunit of CaV1.3 Ca2+ channels and positively regulate their plasma membrane expression in HEK293 cells