Joshua M. Kaplan - US grants

DESCRIPTION (provided by applicant): The long-term goal of this project is to characterize the post-synaptic mechanisms regulating synaptic transmission, particularly those that occur during homeostatic plasticity. At central synapses, changes in expression of post-synaptic receptors are thought to occur during activity-dependent plasticity, including homeostatic plasticity. We developed C. elegans as a model to study trafficking of an ionotropic glutamate receptor (GluR) GLR-1 at central synapses. We showed that GLR-1 levels are up regulated during activity blockade, a model for homeostatic plasticity, that clathrin-mediated endocytosis is required for this compensation, and that GLR-1 is sorted into either of two post-endocytic trafficking pathways (recycling versus degradation). Ubiquitylation of GLR-1 promotes its trafficking into the degradation pathway whereas RAB-2 promotes sorting into the recycling pathway. At neuromuscular junctions (NMJs), retrograde signals from muscles regulate the growth, stability, and strength of their motor neuron inputs. We identified a novel retrograde signaling pathway whereby increased muscle activity induces a muscle transcription factor (MEF-2), and MEF-2 in turn induces a retrograde signal that decreases presynaptic release of neurotransmitter. Here, we propose three aims to characterize these two forms of homeostatic regulation. First, we will determine which Rab proteins regulate GLR-1 trafficking. Rab GTPases regulate specific steps in protein transport; consequently, Rab mutant phenotypes will allow us to experimentally define discrete steps in GLR-1 trafficking. We are particularly interested in endosomal Rabs, because changes in endosomal trafficking have been implicated in activity dependent plasticity. Second, we will determine how the MEF-2- dependent retrograde message is regulated by muscle activity. We will test whether changes in endogenous muscle activity alter MEF-2 transcriptional activity, whether this is mediated by activity-dependent dephosphorylation of MEF-2 by calcineurin, and if MEF-2 activity is specifically coupled to nicotinic acetylcholine receptors. Third, we will identify genes that act downstream of MEF-2, and are required for the MEF-2-dependent retrograde message. One such gene (aex-1) was identified in preliminary studies. In summary, our experiments will provide new insights into the specific post-synaptic mechanisms underlying two forms of homeostatic regulation. Given the strong conservation of these pathways across phylogeny, it is likely that our experiments will provide new insights into the general mechanisms underlying these fundamental aspects of synaptic cell biology.

? DESCRIPTION (provided by applicant): The goal of this project is to identify factors that regulate neurotransmitter release kinetics. The motivation for this project is two-fold. First, slowed release of synaptic vesicles (SVs) prolongs post-synaptic currents, which can change post-synaptic excitability and action potential firing patterns. Thus, release kinetics has profoun effects on circuit development and cognition. Second, mutations linked to Autism alter post-synaptic response kinetics by various mechanisms. The SVs available for release comprise multiple functionally distinct pools, which fuse with different kinetics and release probabilities. Synchronous (or fast) release occurs over a few milliseconds while delayed (or slow) release occurs over 10-100 ms. The detailed mechanisms regulating release kinetics have not been determined. We propose to identify factors and mechanisms that tune release kinetics, using C. elegans as a model system. In preliminary studies, we identified three syntaxin-binding proteins that dictate release kinetics at neuromuscular junctions (NMJs). We showed that UNC-13L promotes fast release, UNC-13S promotes slow release, and Tomosyn (a third syntaxin-binding protein) inhibits slow release. Based on these preliminary results, we will determine how UNC-13L accelerates release, how the different UNC-13 proteins produce differences in spontaneous release, and how SVs are coupled to calcium channels. These studies should provide new insights into the biochemical mechanisms regulating release kinetics.

The extracellular calcium-sensing receptor (CaSR) most likely plays a critical role in modulating the kidney's response to calcium and its processing of other electrolytes, but its exact function and mechanisms of action in the kidney have yet to be elucidated. To determine the role of CaSR in the kidney, a transgenic mouse model over expressing the CaSR in the distal tubules of the kidney will be developed and the effect of over expression and subsequent increased activation of CaSR on whole animal physiology and cellular physiology examined. The effect of activation of CaSR in the distal tubules of the kidney on renal calcium reabsorption, uniary concentrating ability, apical potassium channel activity, and cyclic-AMP generation in response to vasopressin will be determined. The potential clinical significance of this work is far-reaching. Determine the role of CaSR in the kidney will certainly shed light on the rare genetic disorders resulting from mutations in CaSR, but it should also improve understanding of primary processes in nephrolithiasis, osteoporosis, parathyroid disorders, and possibly even essential hypertension.

DESCRIPTION (provided by applicant): GLUTAMATE RECEPTOR TRAFFICKING IN C. ELEGANS. Recent work has shown that changes in the abundance of AMPA-type glutamate receptors at synapses are an important potential mechanism for expressing activity-dependent changes in synaptic activity. We have undertaken a comprehensive analysis of the trafficking of a C. elegans AMPA-type glutamate receptor (GLR-1). In the prior funding period, we identified a scaffolding protein (LIN-10/Mintl) required for synaptic targeting of GLR-1, we showed that anterograde trafficking of GLR-1 is regulated by voltage-activate calcium channels and CAMKII, and we showed that formation of ubiquitin-GLR-1 conjugates triggers endocytosis and post-endocytic degradation of GLR-1. In preliminary studies reported here, we identify four new genes that regulate synaptic abundance of GLR-1, one of which encodes a subunit of an SCF ubiquitin ligase. We also show that the synaptic abundance of GLR-1 is regulated by a homeostatic mechanism similar to synaptic scaling. We propose three new aims to define the biochemical mechanisms regulating GLR-1 abundance at synapses. First, we will identify the physiologically relevant targets of the SCF ubiquitin ligase and will determine how it alters the function of GLR-1 synapses. Second, we will determine what aspect of GLR-1 trafficking is regulated by genes discovered in our screen for GLR-1 mislocalization mutants and whether these defects are an indirect consequence of a failure to localize other proteins. Third, we will determine whether synaptic scaling is bi-directional, and whether it is triggered by glutamate, postsynaptic depolarization, or by neuropeptides. And we will determine what aspects of GLR-1 trafficking are regulated by synaptic scaling. In summary, changes in the synaptic abundance of AMPA receptors has been proposed as a mechanism for producing activity-dependent changes in synaptic strength, and hence in learning and memory. Given the strong conservation of these pathways across phylogeny, it is likely that our experiments will provide new insights into the mechanisms underlying these fundamental aspects of synaptic cell biology.

[unreadable] DESCRIPTION (provided by applicant): The "Cell Biology of the Neuron" Gordon Conference, to be held for the eighth time in June 2008, has become the premier international meeting for discussing progress in the rapidly moving field of cellular neurobiology. The focus of the meeting will be on the cell biological aspects of neural function. The meeting will encompass a diverse set of interdisciplinary approaches, including genetic, molecular, cellular, biophysical, and behavioral approaches to understanding the brain. A diverse set of model systems will be represented at the meeting, ranging from C. elegans to humans. This year the following themes will be covered: synapse development, pre-synaptic mechanisms, post-synaptic signaling, axon and dendrite morphogenesis, cortical connectivity, imaging circuits, and synaptic plasticity. The chosen speakers are among the most active in the field and are also articulate thinkers who should generate useful discussion by integrating their work and ideas with those in other fields. The format consists of 20 minute talks followed by a discussion of 10 minutes or more. The meeting roster contains a mixture of promising young scientists near the beginning of their careers and established scientists. Nine of the speakers are women and five are from outside the US. In addition, eight slots are being kept open to select speakers for short talks on exciting, late-breaking developments. Most participants (except speakers) will present posters on their work, ensuring maximum communication and exchange of ideas among participants. With its interdisciplinary focus on neural mechanisms, this conference is of special interest for understanding affective and behavioral disorders, hereditary defects in brain development, mental health and drug addiction. The emphasis on neural development and plasticity has relevance for child health and development. The speakers include investigators who are specifically studying the molecular basis of drug tolerance and addiction, affective and cognitive disorders (e.g. anxiety and autism), hereditary defects in brain development, and diseases of aging such as Alzheimer's and Parkinson,s diseases. The conference should be of great potential interest to the missions of institutes including NIMH, NINDS, NICHD, NIA, NIDA and NIAAA. This proposal requests fund to support the "Cell Biology of the Neuron" Gordon Research Conference, to be held in June 2008. This conference brings together a diverse set of neurobiologists, all of whom study the fundamental building blocks of the brain, neurons and synapses. In particular, this meeting will highlight recent discoveries in how the brain develops its complex set of synaptic connections, and how these connections are modified during development, or in different disease states. Thus, this meeting will further the mission of several institutes, including NIGMS, NINDS, NIMH, NIDA, NEI, and NIAAA. . [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this project is to identify factors that regulate secretion of neuropeptides generally, and to determine how these secreted peptides regulate behavior. The motivation for this project is two-fold. First, insulin secretion, and its misregulation, plays a pivotal role in aging, diabetes, and obesity. Second, while a great deal has been learned about mechanisms regulating secretion of classical neurotransmitters, far less is known about those regulating secretion of neuropeptides and hormones. Classical neurotransmitters are packaged in synaptic vesicles (SVs), which are clustered at active zones. Neuropeptides are packaged into large dense core vesicles (DCVs), and are distributed throughout axons and dendrites. Secretion of SVs occurs at active zones, in a rapid, phasic manner in response to single action potentials. Secretion of DCVs occurs typically after trains of depolarization, fusion events occur far from active zones, and they occur relatively slowly following depolarization. Following exocytosis, the SV pool is rapidly reconstituted at nerve terminals by endocytic recycling of SV components, and refilling with neurotransmitters. By contrast, the releasable pool of DCVs must be reconstituted by anterograde transport of immature secretory granules from the soma. Relatively little is known about the biochemical basis for these differences. We propose to identify factors that are required for or that regulate DCV secretion, using C. elegans as a model system. In preliminary studies, we identify a neuropeptide that regulates arousal from a sleep-like state. Here we propose to determine how secretion of this peptide is regulated, and the mechanism by which this peptide causes arousal. These studies should provide new insights into the cellular mechanisms regulating secretion of neuropeptides sleep, and arousal.

? DESCRIPTION (provided by applicant): The goal of this project is to identify cellular defects that are caused by mutations in genes linked to Autism spectrum disorders (ASD), using the C. elegans neuromuscular junction as a model. In particular, we will test two prominent models for pathophysiological mechanisms in ASD. First, we test the idea that ASD linked genes play a direct role in regulating activity induced gene expression. Second, we will determine if ASD linked genes alter the synaptic targeting of GABAA receptors, thereby altering inhibitory synaptic strength. A critical feature of the genetics of ASD is that mutations conferring risk are nearly always heterozygous in affected individuals, implying that the majority of ASD linked genes are dose sensitive. Thus, we will ask if activity-induced gene expression and synaptic targeting of GABAA receptors are sensitive to copy number variations in ASD linked genes. In addition to testing their potential importance in the pathophysiology of ASD, our Aims address basic mechanisms controlling nervous system development and function.

Analysis of embryonic brain wiring in C. elegans The goal of this project is to investigate how neural circuits are assembled in C. elegans embryos. We develop assays for an embryonic behavior (a sleep-like quiescent state) and for the neurons controlling this behavior. This embryonic sleep state is analogous to the quiescence that occurs during larval molts. We will determine if quiescence is triggered by cuticle synthesis. We will image synapse formation in embryos and will determine if sleep circuit development is controlled by heterochronic genes (which control larval skin development). We will also ask if circuit development is altered by mutations that prevent sleep or by those linked to Autism spectrum disorders (ASD). These Aims address basic questions about the genetic and physiological mechanisms controlling early brain development.