Molecular Mechanisms for Active Zone Assembly at Vertebrate Synapses

Abstract

Neurons are unique for their ability to transmit a presynaptic signal to postsynaptic target cells on a sub-millisecond timescale. This rapid and precise exchange of information relies on the ability of presynaptic nerve terminals to temporally and spatially control the exocytosis of neurotransmitter-packed vesicles in response to membrane depolarization from an action potential. Because neurotransmitter release is largely restricted to specialized areas of the presynaptic plasma membrane called active zones, it is thought that these active zones provide the necessary machinery to orchestrate rapid synaptic transmission. Active zones are evolutionarily conserved protein networks that couple fusion competent synaptic vesicles to the core membrane fusion machinery, exactly opposite to postsynaptic receptors. Decades of research have identified many protein families specifically associated with these specialized release sites. At vertebrate synapses, these protein families include Munc13, RIM, ELKS, RIM-BP, piccolo/bassoon, and Liprin-α. While much work has been done to dissect the specific role each individual protein family plays in synaptic transmission, the role of the active zone as a complex macromolecular structure and the molecular mechanisms of its assembly are not well understood. Thus the goal of this dissertation is to address two fundamental questions: 1) whether the active zone as a whole is necessary for synapse formation and synaptic vesicle exocytosis, and 2) what the molecular mechanisms that contribute to active zone assembly are. To answer these questions, I generated the strongest active zone disrupted mutant to date by using mouse genetics to simultaneously remove two core protein families, RIMs and ELKS, in neuronal cultures. Using a combination of light and electron microscopy and molecular biology, I show that while the active zone is necessary for docking synaptic vesicles to the presynaptic plasma membrane, synapse formation is unaffected and a pool of releasable vesicles persists. Subsequent rescue experiments reveal RIM as the major organizer of active zone scaffolding and identify Liprin-α as a candidate upstream of RIM and ELKS in active zone assembly. Finally, I present a working model for vertebrate active zone assembly.