Molecular Dissection of Active Zone Functions in Neurotransmitter Release

Speed and precise regulation of synaptictransmission are critical for complex brain functions such as cognition and learning. Release ofneurotransmitters from a presynaptic nerve terminal is often impaired in neurological disorders,including autism, schizophrenia, addiction and neurodegeneration. Exact knowledge of the molecularmechanisms for neurotransmitter release is thus critical for understanding brain disease. The activezone of a presynaptic nerve terminal is the site of neurotransmitter release. An active zone consists of ahighly specialized network of proteins that organizes synaptic vesicles for fast Ca2+-triggering ofrelease, a central requirement for speed and precision of synaptic transmission. It is our over-archinggoal to understand how the protein machinery at the active zone operates. We approach this goal bydissecting the molecular functions of active zone components. ELKS proteins are highly enriched atactive zones, indicating that ELKS functions in neuronal exocytosis at the active zone. Before release,active zones dock and prime synaptic vesicles for exocytosis close to presynaptic Ca2+-channels. HowELKS operates during these processes to control release is not understood, maybe in part because nosystematic genetic approach has been taken in vertebrates to address ELKS function. We have nowgenerated conditional knockout mice for both mammalian ELKS genes, ELKS1 and ELKS2. Amplepreliminary data lead to our central hypothesis: ELKS proteins increase release probability thoughcontrolling presynaptic Ca2+-influx, and they modulate the size of the pool of readily releasable vesicles.We address separate components of this hypothesis in three specific aims, and we dissect theunderlying molecular mechanisms. In aim 1, we hypothesize that ELKS1 and ELKS2 proteins haveboth shared and distinct functions. We determine how each ELKS gene contributes to the functions ofactive zones in neurotransmitter release by systematically studying presynaptic phenotypes in thenewly generated conditional single knockout mice for ELKS1 and ELKS2, and in the ELKS1/2 doubleknockout mice. In preliminary experiments we find that ELKS proteins enhance presynaptic Ca2+-influx, and that individual and double ELKS deletions differentially affect the pool of readily releasable vesicles. In aim 2, we determine the mechanisms by which ELKS controls presynaptic Ca2+-influx. In aim 3, we propose a specific hypothesis that unifies effects on vesicle pools observed in ELKS mutants. We examine this hypothesis, determine the underlying molecular mechanisms and consider numerous alternative explanations. Our research is innovative because it addresses a novel hypothesis by a combination of genetic, biochemical and functional experiments of unique depth. Ultimately, this approach will lead to precise insights into the molecular control of neurotransmitter release, a key neuronal process that fails during various brain diseases