Pascal S. Kaeser - US grants

DESCRIPTION (provided by applicant): Speed and precise regulation of synaptic transmission are critical for complex brain functions such as cognition and learning. Release of neurotransmitters from a presynaptic nerve terminal is often impaired in neurological disorders, including autism, schizophrenia, addiction and neurodegeneration. Exact knowledge of the molecular mechanisms for neurotransmitter release is thus critical for understanding brain disease. The active zone of a presynaptic nerve terminal is the site of neurotransmitter release. An active zone consists of a highly specialized network of proteins that organizes synaptic vesicles for fast Ca2+-triggering of release, a central requirement for speed and precision of synaptic transmission. It is our over-arching goal to understand how the protein machinery at the active zone operates. We approach this goal by dissecting the molecular functions of active zone components. ELKS proteins are highly enriched at active 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. How ELKS operates during these processes to control release is not understood, maybe in part because no systematic genetic approach has been taken in vertebrates to address ELKS function. We have now generated conditional knockout mice for both mammalian ELKS genes, ELKS1 and ELKS2. Ample preliminary data lead to our central hypothesis: ELKS proteins increase release probability though controlling 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 the underlying molecular mechanisms. In aim 1, we hypothesize that ELKS1 and ELKS2 proteins have both shared and distinct functions. We determine how each ELKS gene contributes to the functions of active zones in neurotransmitter release by systematically studying presynaptic phenotypes in the newly generated conditional single knockout mice for ELKS1 and ELKS2, and in the ELKS1/2 double knockout 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.

Summary Dopamine is an important neuromodulator and pathologies in dopamine signaling are a hallmark of brain diseases such as neurodegeneration, substance abuse, and schizophrenia. Despite these important roles for dopamine, remarkably little is known about the molecular mechanisms of its release. Because dopamine acts as a volume transmitter, it is not clear whether dopamine release involves molecular machinery that warrants spatial and temporal precision for release. Alternatively, dopamine release could be spread over the surface of an axon, which is consistent with volume transmission. The release of classical transmitters relies on an active zone, a highly organized protein structure that contains scaffolding proteins such as RIM and ELKS and determines the precise localization, speed and accuracy of synaptic vesicle exocytosis. The active zone also provides mechanisms for regulation of release during plasticity. Our preliminary experiments reveal that the presynaptic scaffolding protein RIM is absolutely required for dopamine release in the mouse striatum, but that ELKS is dispensable for dopamine release. This is different from classical fast synapses, where knockout of either protein family leads to a reduction of 50-80% of release. We thus hypothesize that dopamine release necessitates mechanistically specialized release sites. This hypothesis is bolstered by superresolution microscopy in striatal brain slices, which shows that several release site scaffolding proteins are clustered inside dopamine axons. We pursue a two-pronged approach to address this central hypothesis. In aim one, we use rigorous conditional mouse genetics and electrophysiology in acute brain slices of the mouse striatum to systematically address the necessity of scaffolding proteins, priming proteins and Ca2+ channel tethers in dopamine release and in co-release of GABA and glutamate from dopamine neurons. This is the first study on the requirements of molecular scaffolds for dopamine secretion and it will lead to a comprehensive assessment of the dopamine release machinery. In aim two, we assess whether scaffolding proteins mediate dopamine secretion as soluble release factors, or whether they are assembled in clustered release sites to target dopamine release to specific membrane domains. The latter possibility is strongly supported by our preliminary data. We will combine superresolution microscopy, subcellular fractionation, electron microscopy and mouse genetics to study the existence and composition of dopamine release sites in the mouse striatum. We will assess how dopamine release sites are associated with vesicle clusters, with receptors for dopamine and for the co-transmitters GABA and glutamate, and with cholinergic innervation, which powerfully triggers dopamine release. These experiments will establish the existence, appearance and composition of dopamine release sites and their structural arrangement into striatal synaptic microcircuits. Our approach is the first comprehensive approach to dissect the secretory pathway for dopamine. We expect to identify new mechanisms that support dopamine release and to uncover general principles for neuromodulation.

Project Summary Neurotransmitter release critically depends on the precise assembly of the secretory machine. Within a presynaptic nerve terminal, synaptic vesicles exclusively fuse at the active zone, a protein scaffold that forms release sites opposed to postsynaptic receptors. This scaffold consists of RIM, ELKS, Liprin-? and other active zone specific proteins. It also contains many proteins that are important for secretion and synaptic structure, but that are not restricted to the active zone. In the past two decades, research from many laboratories has started to provide deep insight into the functions of individual proteins at the active zone. However, much less is known about the assembly mechanisms of this key protein scaffold. This is particularly true for vertebrate synapses, perhaps because no genetic mutation to date has strongly disrupted the active zone scaffold. We here overcome this limitation by generating conditional knockout mice to simultaneously delete RIM and ELKS in hippocampal neurons. This mutation leads to massive disruption of the presynaptic active zone scaffold with loss of most of its vital components and of vesicle docking. Based on extensive preliminary data, we hypothesize that RIM and ELKS are redundantly required for release site assembly and function, and that these scaffolding proteins are recruited to the active zone by Liprin-?. We designed three specific aims to address our overarching hypothesis. In the first aim, we rigorously test synaptic structure and synaptic vesicle docking in these active zone disrupted neurons. We propose rescue experiments with individual proteins or protein domains to evaluate the molecular hierarchy of the recruitment of active zone proteins and to dissect mechanisms for vesicle docking. In aim 2, we use the mutants with disrupted active zones to test two models that are prominent in the field: we will determine whether the active zone and docking are required for synaptic vesicle release and we will test whether the active zone targets release to the membrane domain opposed to postsynaptic receptors. Our preliminary data reveal that fusion competent vesicles persist upon disruption of the active zone and loss of vesicle docking, which is surprising given the dogma that fusion competent vesicles are docked. In aim 3, we address molecular mechanisms of active zone assembly upstream of RIM and ELKS. The most parsimonious interpretation of the literature and our preliminary data is that Liprin-? recruits RIM and ELKS for active zone scaffolding. We systematically test this hypothesis in newly generated Liprin-? knockout mice. This is the first study that addresses vertebrate Liprin-? function using a rigorous genetic approach. This grant application will generate new knowledge on the mechanisms of vertebrate active zone assembly and function. Human genetic studies have identified mutations in many active zone proteins, including in RIM and in ELKS, which contribute to neurological disease. Thus, precise knowledge of active zone assembly is important for understanding synaptic function in health and disease.

Abstract This is an application for an administrative diversity supplement to R01 MH113349, ?Dissecting the assembly of vertebrate neurotransmitter release sites? to support the research training and experience of Christopher Minasi. Christopher is a recent graduate of Florida Atlantic University. Christopher is Hispanic/Latino, a group that is underrepresented in biomedical sciences. He has a B.S. in Neuroscience and is highly interested in pursuing a career as a neuroscientist. Currently, he is participating in a post-baccalaureate program at Harvard, preparatory to applying to graduate school for matriculation in the fall of 2020. Christopher's work will elucidate three scientific questions in three phases. In the first phase, Christopher will determine whether ELKS can be recruited to the presynaptic plasma membrane upon disruption of the active zone protein complex, and preliminary data support that this is possible. In the second phase, Christopher will assess the mechanisms through which ELKS is recruited to synapses. In the third phase, Christopher will answer what active zone proteins are recruited by ELKS, and he will determine the underlying mechanisms. This research plan is structured such that in the early phases, Christopher learns experimental designs and techniques, and the key concepts in the field. In the later phases, he will lead the project intellectually by formulating hypotheses, by designing experiments to address these hypotheses, and by reaching scientific conclusions. This research experience is accompanied by multiple levels of mentoring, a focused plan to learn skills in scientific presentation, and guidance in developing a career in the neurosciences. It is also complemented with classwork to learn key concepts in molecular neuroscience. Together, the research and training plans are aimed at preparing Christopher for an academic career in neuroscience, with the concrete goal of admission to a top neuroscience graduate program by 2020.