Molecular mechanisms of synaptic vesicle recycling with a focus on Endophilin A and Rabconnectin-3a

Abstract

Synaptic vesicle (SV) recycling at the synapse is fundamental to neurotransmission and it sustains unimpaired neuronal communication. It is a complex cell biological process, where the molecular steps are finely coordinated and intensively studied in the last three decades. The basic principles and the regulatory mechanisms underlying key steps like exocytosis and endocytosis have been broadly understood. Yet, there are several missing links in SV recycling process. Some of the open questions that are not well understood include the mechanism of how exocytosis is coupled with endocytic retrieval, how a newly formed vesicle (through endocytosis) is transformed into a fusion-competent vesicle for the next round of events, and how the SV recycling process is regulated. Several major molecular players have been identified and their roles characterized in specific steps of the recycling process. But, how specific proteins or factors are made available at the right time for the precise step in the process is not fully understood. In this study, we explore some of the missing links in the SV recycling process. The first study reports a new role for an endocytic protein called endophilin-A (henceforth endophilin) in exocytosis and potential link to the coupling mechanism between exocytosis and endocytosis. In the second study, we characterized the role of a protein called rabconnectin-3a in SV recycling process and how it potentially acts as a link between several steps: endocytosis, vesicle acidification and maturation of newly formed vesicles. Endophilin is a well-studied endocytic adaptor protein. We found a direct role for endophilin in large dense-core vesicle (LDCV) exocytosis, independent of its role in endocytosis, using the neurosecretory adrenal chromaffin cell and mouse model lacking all three endophilins. The lack of endophilin affects the vesicle recruitment, priming and fusion during the exocytic process. These exocytic defects could be rescued by both endophilin A1 and endophilin A2. The reduced exocytosis was not due to a reduction in the number of vesicles, since the LDCV number per cell, or cell area, was unaltered in the absence of endophilin. The absence of endophilin did not majorly affect protein and membrane recycling by endocytic mechanisms. Further, none of the major exocytic or endocytic factors were affected in its absence. SH3 domain of endophilin was important for its function in exocytosis. The role of endophilin in vesicle recruitment and release, at least in part was mediated through the interaction with a scaffolding protein, called intersectin. This study offers an example of how proteins can assist several steps in vesicle recycling process and how protein-protein interactions help to keep endocytic proteins compartmentalized, to make them available at the right moment for their action in the vesicle cycle. In the second study, we focused on how the process of endocytosis is linked to vesicle acidification and refilling of neurotransmitters (NT) to form a fusion-competent vesicle. Rabconnectin-3a (Rbcn-3a) is a putative regulator of v-ATPase, the proton pump on the vesicle that is responsible for acidification process. We characterized the role of Rbcn-3a in SV recycling in neurons. Rbcn-3a is present on both synaptic vesicles, as well as clathrin-coated vesicles. Although the mice without Rbcn-3a die in the early embryonic stage, neurons developed normally and formed synapses. Yet, in the absence of Rbcn-3a, the number of SVs was reduced at synapses and neuronal activity was impaired. Presynaptic activity monitored using pHluorin tagged to vGLUT1 showed that the recovery after stimulation is delayed in the absence of Rbcn-3a. This delay was not due to a fault in endocytic retrieval, but due to a defective acidification of the vesicles. As a consequence of defective recycling, recruitment of Rab3 on SVs was reduced and its stability on the vesicles was impaired in Rbcn-3a knock-out neurons. Neurons also accumulated lysosome-like structure, suggesting a role for Rbcn-3a in membrane trafficking pathways in neurons. This study reveals a role for Rbcn-3a in regulation of vesicle acidification and how it links endocytosis to acidification and recruitment of peripheral proteins for vesicle maturation process during recycling. The findings reported in my doctoral thesis work offer some information on the missing links in the vesicle recycling pathway. It also reinstates how the recycling process is tightly coordinated: the molecular players can participate in more than one step of the recycling process and link them.