Yishi Jin - US grants

Chemical synapses are composed of specialized subcellular structures. Presynaptic terminals contain an organized cytoskeletal architecture to facilitate neurotransmitter release. Most known presynaptic proteins are associated with synaptic vesicles and function in vesicle exocytosis and endocytosis. Few proteins are shown to play roles in organizing presynaptic structure. Work in this laboratory has focused on the molecular events controlling the differentiation of GABAergic motor neurons in the nematode C. elegans. A genetic screen identified several syd genes (for synapse defective) that affect the morphology and organization of the presynaptic terminals in these neurons. Our analysis has suggested that signaling via small GTPases and receptor protein tyrosine phosphatases may function at different steps in the presynaptic structural formation. This proposal focuses on dissecting the signaling pathway involving syd-3, a putative guanine nucleotide exchanger with a Ring-H2 finger. Specifically, we will clone syd-5 and syd-8, both exhibit similar mutant phenotypes as syd-3, suggesting that they may function in the same pathway. We will systematically analyze the ultrastructural defects in selected syd mutants using transmission electron microscopy. This study is the key for understanding how syd genes function in synapse formation. Genes that are involved in the same pathway may be identified as genetic suppressors or enhancers. We have isolated six semi-dominant suppressors of syd-3 and will continue their molecular and genetic characterization. In addition, we will perform a dominant enhancer screen on a temperature sensitive syd-3 mutation, which may identify genes that are sensitive to the dosage of syd-3. GABAergic neurons are essential for brain function. The use of model organisms is invaluable to the studies of the human nervous system. Our results may provide insights into the understanding of synapse formation in all organisms, and may shed light to the search of causes for cancer and other diseases that are associated with synapse malformation.

DESCRIPTION (provided by applicant): A molecular description of synaptogenesis remains a key research goal in understanding the development and function of the nervous system. By characterizing C. elegans genes that function in synapse formation, our work has contributed to the discovery of several signaling pathways instructing different aspects of presynaptic differentiation. Central to this application is the rpm-1 gene (for regulator of presynaptic morphology). Loss of function in rpm-1 causes disorganized presynaptic architecture and disrupts axonal patterning in a neuron-type specific manner. RPM-1 is a member of the conserved PHR protein family that includes mammalian Pam and Phr1, and Drosophila Highwire. PHR proteins are large molecules containing multiple functional domains, including an RCC1 guanine exchange factor homology domain and a Ring-finger E3 ubiquitin ligase domain. During the current funding period, we demonstrated that RPM-1 functions as an E3 ubiquitin ligase for the conserved MAP kinase kinase kinase DLK-1. DLK-1 activates two downstream kinases, a MAPKK MKK-4 and a p38 MAPK PMK-3. Down-regulation of this MAP kinase cascade by RPM-1 is essential for normal synapse formation. In parallel to the genetic approaches, we used biochemical methods to identify RPM-1 associated proteins, and discovered that RPM-1 positively regulates late endo-lysosomal trafficking via the RabGEF GLO-4. The overall goals of the present application are to define the targets of the RPM-1/MAPK cascade, and to understand how the cascade is regulated. We have identified two new genes, mak-2, the C. elegans ortholog of MAP kinase activated kinase 2, and uev-3, a protein containing an inactive ubiquitin conjugating enzyme domain. Loss of function in either gene behaves in a manner similar to that of inactivating the MAPK cascade. Our preliminary studies suggest that MAK-2 and UEV-3 act downstream of DLK-1 and MKK-4. In aim-1, we will examine the regulation of MAK-2 by the MAP kinases and identify the targets of MAK-2. In aim- 2, we will analyze the interaction between UEV-3 and the MAP kinases. In aim-3, we will explore a novel pathway in synapse development. Loss of function in individual synaptogenic pathways has mild effects on synaptic development and function, indicating a high degree of functional redundancy in synaptic signaling. Using genetic modifier screens to search for other synapse development genes, we have identified the gene sydn-1 (for syd enhancer), which appears to define a nuclear pathway that may regulate axonal pruning in a synaptogenesis dependent manner. We will investigate the cellular and molecular functions of SYDN-1 and its candidate interacting genes. Successful completion of our aims will elucidate how the PHR/MAPK pathway functions. Regulation of synapse stability is one of the major pathogenesis events associated with dementia and ageing. This study will contribute to the understanding of the basic mechanisms that build and maintain synapses, and may also provide insights into the pathogenesis of synapse dysfunction. PUBLIC HEALTH RELEVANCE: This application investigates the signal transduction pathway of a p38 MAP kinase in synapse development. It will determine the molecular, biochemical, and cellular interactions of a MAPKAP protein and a UEV-domain containing protein with the MAP kinase cascade. It will provide insights into the basic mechanisms controlling synapse stability and into the pathogenesis process underlying synapse dysfunction in diseases.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The active zones at the presynaptic terminals are located at the center of the synaptic vesicle exocytic zone, promote synaptic vesicles docking, and provide the precise registry between pre- and postsynaptic specializations. Active zones display morphologically distinct ultrastructures. By screening for C. elegans mutants with altered synaptic vesicles distribution, we discovered that the conserved protein SYD-2 is a key regulator of active zone assembly. In syd-2 lost-of-function mutants, active zones exhibit a fragmented appearance, causing diffused localization of synaptic vesicles. Recently, we uncovered an unusual syd-2 gain-of-function mutation that is able to promote synapse assembly in the absence of several upstream regulators. Using this mutation, we have dissected the complex interactions among active zone proteins. This study provides insight into the molecular network of presynaptic active zone assembly.

DESCRIPTION: The Neurosciences Graduate Program (NGP) at the University of California, San Diego (UCSD) is committed to training the next generation of neuroscience researchers, clinician-scientists and academicians. Over the past 20 years, the UCSD NGP has become one of the top neuroscience graduate programs in the country, ranked 4th in the nation in the 2010 National Research Council ranking. This training grant supports the first- and second-year students in the program, and is endorsed by strong institutional support from the participating departments at UCSD, the Salk Institute, The Scripps Research Institute and the Sanford-Burnham Medical Research Institute. These institutions are world-class research centers on the Torrey Pines Mesa, with the UCSD campus as the home academic institution. The NGP provides the broad umbrella that unites neuroscientists from all these institutions. The NGP provides trainees with a rich curriculum covering a broad spectrum of sub-disciplines in neurosciences, mentored research in the individual laboratories of outstanding investigators, and collaborative opportunities across different programs. The NGP responds to emerging areas of interest; a new formal specialization that expands the scope of training is Computational Neuroscience, added in the past few years. The NGP's training plan is structured such that the students form close interactions with each other and with the faculty upon entry to the program. Incoming students receive intensive hands-on laboratory training through the NGP Boot Camp, which also gives the students a unique bonding experience and initial exposure to the breadth of NGP research options. Following the core courses and three research lab rotations, students choose their dissertation thesis labs at the end of the first year. Each student's progress is monitored through an integrated series of cohesive formal evaluations. All students take a required course for scientific conduct and ethics. Students are enriched through a variety of activities that facilitate and enhance the interactions between students and training faculty. Career advising and mentorship are in place at each successive year. Vertical interactions among students from different years are facilitated through journal club, research rounds, and a prestigious seminar series organized and run by the NGP students, and the annual recruitment and retreat activities. Recruitment and admission to NGP is highly competitive. The program makes dedicated efforts to improve the recruitment and retention of under-represented students; the NGP ranks the top in representation of URM population among the UCSD graduate programs for STEM (Science, Technology, Engineering and Math). This training grant is central to the success of the UCSD neurosciences graduate training. The research productivity of the trainees is outstanding, and a large fraction of former trainees continue in scientific research and higher education. Over the next five years, the UCSD School of Medicine has set a goal to increase the size of the program through enhanced institutional support, with a strong commitment to improving the program's diversity.

DESCRIPTION (provided by applicant): The overall goal of this project is to use the genetically tractable model organism C. elegans to dissect the molecular basis of axon regeneration after injury. The small size, transparent body and simple anatomy of C. elegans allows single axons to be severed in vivo and their regrowth studied in depth. In the prior funding period we used large- scale genetic screens in C. elegans to discover conserved genes and pathways that play regrowth-promoting or regrowth-inhibiting roles in vivo. Many of these pathways are distinct from those involved in developmental axon outgrowth. Our large scale screens and analysis of genetic interactions have led to models for the function of these regrowth factors that we will test mechanistically in this proposal. We will define the roles of ne genes that affect regrowth via axonal microtubule dynamics. We will investigate the role of membrane trafficking regulators in axon regrowth. Results from this work will elucidate intrinsic mechanisms that allow mature axons to regrow after damage. In vertebrates, peripheral nerves are capable of regrowth, yet recovery after peripheral nerve trauma is often incomplete. Improved knowledge of regrowth mechanisms could also inform our understanding of why other neurons do not regrow. The mammalian CNS is only minimally capable of regeneration after injury, reflecting the combined effects of an inhibitory environment and of reduced intrinsic regrowth capacity. Our work addresses intrinsic mechanisms that promote or inhibit axon regrowth, a high priority for this field. Some signaling pathways have conserved roles in axon regrowth, suggesting analysis of C. elegans axon regrowth has implications for understanding axon repair mechanisms in medically relevant situations.

? DESCRIPTION (provided by applicant): The overall goal of this project is to use the genetically tractable model organism C. elegans to dissect the molecular basis of axon regeneration after injury. The small size, transparent body, and simple anatomy of C. elegans allows single axons to be severed in vivo and their regrowth studied in depth. In the prior funding period we used large- scale genetic screens in C. elegans to discover conserved genes and pathways that play regrowth-promoting or regrowth-inhibiting roles in vivo. Many of these pathways are distinct from those involved in developmental axon outgrowth. Our large scale screens and analyses of genetic interactions have led to models for the function of these regrowth factors that we will test mechanistically in this proposal. We will dissect a signaling pathway that inhibits axon regrowth via axonal microtubule dynamics. We will investigate the role of membrane trafficking regulators in axon extension. Results from this work will elucidate intrinsic mechanisms that allow mature axons to regrow after damage. In vertebrates, peripheral nerves are capable of regrowth, yet recovery after peripheral nerve trauma is often slow and incomplete. The mammalian CNS undergoes minimal regeneration after injury, reflecting the combined effects of an inhibitory environment and of reduced intrinsic regrowth capacity. Improved knowledge of regrowth mechanisms will also inform our understanding of why CNS neurons do not regrow. Our work addresses intrinsic mechanisms that promote or inhibit axon regrowth, a high priority for this field. Many signaling pathways have conserved roles in axon regrowth, suggesting analysis of C. elegans axon regrowth has implications for understanding axon repair mechanisms in medically relevant situations.

Throughout a lifetime of an organism synapse addition and elimination is on-going to ensure proper function of neuronal circuits. Growing evidence have revealed complex interactions involving intrinsic and extrinsic factors in synapse refinement with temporal and neuronal-type specificity. Studies using C. elegans have continued to expand the understanding of molecular and genetic pathways with single- synapse resolution. The locomotor circuit consists of several classes of excitatory cholinergic motor neurons and two classes of GABAergic motor neurons, and is a highly tractable system to discover mechanisms underlying synapse formation and refinement. Each neuron forms stereotyped pattern and number of synapses, providing an accurate readout to examine how synapses are dynamically regulated. Moreover, the development of the mature locomotor circuit involves a precisely timed remodeling of the embryonically born GABAergic neurons, known as ?DD synapse remodeling?, in the absence of axonal morphological changes. We developed the first in vivo visualization approach to examine DD synapse remodeling. In our recent studies, we have defined critical roles of microtubule dynamics in promoting cargo and motor interaction in the formation of new synapses in DD remodeling. Our findings underscore the concept that microtubules are not passive tracks but play an active role in cellular signaling. In the specific Aim 1 of this renewal application we will leverage our expertise in genetic pathway dissection with in vivo imaging of microtubule components to dissect the roles of a novel kinase in DD synapse remodeling. In parallel, we have investigated the mechanisms regulating the cholinergic neuron synapses, and have uncovered roles of inter-tissue interaction mediated by a IgSF transmembrane domain protein ZIG-10. Our studies show that ZIG-10 regulates phagocytotic pathway via a SRC kinase in the adjacent non-neuronal tissues. In specific Aim 2, we will tackle the cellular action and the physiological impact of this pathway using innovative technologies. We will further examine how neuronal activity regulates this pathway. In Aim 3, we will investigate the role of a conserved MAGUK protein that may link the ZIG-10 pathway to phospholipid biosynthesis in synapse maintenance. Genetic mutations of homologous molecules in human have been linked to various neurological diseases. Together our findings will provide important insights to the underlying signaling network and advance our knowledge in the understanding of human diseases.

The goal of this work is to define how axons regrow and reconnect after injury, focusing on molecular regulators acting within individual axons. Our model system is the simple animal C. elegans, in which single axons can be severed and regrow in vivo in a generally permissive environment. We have used large-scale genetic screens to discover conserved genes that promote or repress axon regrowth, most of which are not involved in developmental axon outgrowth. We propose to examine in depth the roles and interactions of three new regrowth-inhibiting pathways revealed from screening. First, we will dissect the roles of a conserved regulator of axonal sprouting that may regulate neuronal lipid metabolism. Second, we will examine how a highly conserved kinase pathway inhibits axon regrowth. Finally, we will elucidate the role of mRNA decay regulators in axonal regrowth and their potential link to mitochondrial function. Results from this work will elucidate intrinsic mechanisms that allow mature axons to respond to injury and regrow after damage. In vertebrates, peripheral nerves are capable of regrowth, yet recovery after peripheral nerve trauma is often slow and incomplete. The human CNS undergoes minimal regeneration after injury, reflecting the combined effects of an inhibitory environment and of reduced intrinsic regrowth capacity. Improved knowledge of regrowth mechanisms in organisms with high intrinsic regrowth capacity will also inform our understanding of why CNS neurons do not regrow. Many C. elegans pathways have been found to have conserved roles in axon regrowth, indicating the mechanisms underlying C. elegans axon regrowth will continue to yield insights into general principles of neuronal repair.

Project Summary PACS1 Syndrome is a de novo neurogenetic disorder affecting young children, characterized by distinct dysmorphic facial features, intellectual disability and developmental delays. In all cases identified throughout the world, individuals have the same exact amino acid substitution, p.R203W, in a highly conserved position of the PACS1 protein. PACS1 (Phosphofurin Acidic Cluster Sorting Protein 1) was first identified by its interaction with the proprotein convertase furin, and subsequent studies have revealed its role in secretary pathway, particularly the trans-Golgi network. PACS1 is conserved in all animals, and humans express an additional ortholog, PACS2. All PACS proteins contain multiple functional domains including the furin-binding region, in which the PACS1 syndrome variant resides. Currently, most understanding of PACS1 function is from cultured cells. A key question that must be addressed is how R203W changes PACS1 function and results in syndrome symptoms. The nematode C. elegans is a tractable model for studying human disease genes. A single C. elegans pacs-1 gene encodes a protein that shares all conserved domains, and the furin-binding region is especially well conserved between C. elegans and humans (45% identical/70% similar) with the disease variant site, R203, denoted as R116 in C. elegans. Here, we have generated a cePACS-1(R116W) model, using genome-editing. Additionally, using drugs to probe neuronal function, we found that pacs-1(R116W) animals are resistant to the paralyzing effect of the choline esterase inhibitor aldicarb. Consistent with its neuronal function, we also found that endogenous PACS-1 is expressed in the nervous system. Our C. elegans pacs-1(R116W) provides the first germline-expressed model of PACS1 syndrome. In this R03 application, we propose to complete a chemical compound screen, using the LOPAC chemical library, for behavioral effects on our cePACS1 model. We will then test top candidate hits using additional cell biology and functional assays. The project goal is within the feasibility and guideline of R03 application. The outcome will be informative for the development of designer-strategies in treatment of PACS1 syndrome, and also aid further characterization of physiological mechanisms underlying PACS1 syndrome.