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

Abstract

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. <br /> 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. <br /> 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. <br /> 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.