Bing Zhang - US grants

A unique feature of the nervous system is the vast number of cell-cell connections called synapses. At synapses, nerve cells communicate with each other and with other target cells. At chemical synapses, and upon activation, nerve cells release transmitter from small synaptic vesicles (SVs) and then recycle these vesicles for reuse. Hence, SV recycling plays a major role in neuronal function. Clathrin and its associated molecules are critical players in vesicle recycling. However, the molecular mechanisms by which SVs are recycled are not fully understood. This project is directed towards understanding how SV proteins are recruited and recycled into newly formed vesicles. Genetic and biochemical studies from Dr. Zhang's laboratory and others suggest that the clathrin-accessory protein AP180 regulates both the rate and the fidelity of clathrin-mediated recycling of SVs. This project will test the hypothesis that AP180 also plays a role in helping retrieve vesicle proteins during vesicle recycling. They will be examining whether and how the composition of SVs is altered in mutant fruit flies (Drosophila) deficient of AP180 and also will investigate the mode by which AP180 might interact with vesicular proteins. These studies are expected to advance the understanding of SV recycling and nerve cell function. This project also closely integrates research with education, training, and outreach to local communities. He has trained and mentored undergraduates, graduates, and postdoctoral fellows, including women and students from underrepresented groups. Eleven undergraduates were co-authors with him on a prior NSF-sponsored project, including one as first author. During this funding period, he will continue his educational efforts in student mentoring and course development. In addition, he will be reach out to local schools and train elementary science teachers through a summer program organized by the Sam Noble Oklahoma Museum of Natural History at OU.

DESCRIPTION (provided by applicant): The cognitive function of the brain depends critically on the synaptic connections formed between neurons and their target cells. Inappropriate formation and development of synaptic connections can cause neuronal dysfunction and neurological disorders. Hence, understanding synaptic development and function remains a major goal for neuroscientists. As part of our long-term research interests, we aim to study the in vivo role of epsin and its signaling pathways in synaptic development at the neuromuscular junction (NMJ) of the fruit fly, Drosophila melanogaster. Our studies show that the Drosophila epsin (called liquid facets or lqf) gene plays a novel role in synaptic growth at NMJ. lqf mutations alter bouton shape, increase bouton size, and reduce synaptic transmission. Furthermore, we show that Lqf acts as a specific substrate of the deubiquitinating enzyme Faf to promote synaptic growth. Finally, we show that the level of MAP1B as well tubulin is reduced on microtubules (MTs) in a considerable number of synaptic boutons in the lqf mutant. In contrast, overexpression of Lqf in neurons appears to induce MT bundling in synaptic boutons. Through genetic screens, we have isolated a number of lqf modifiers, including the MAP1B mutant futsch and the lissencephaly 1 (dlis1) mutant affecting the MT cytoskeleton. These observations prompted us to hypothesize that Lqf regulates synaptic growth and function in an MT- and ubiquitin-dependent fashion. To test this hypothesis and to gain further insights into Lqf and Lis1 function, we propose three specific aims. In the 1st Aim, we will examine how lqf mutations and overexpression of Lqf affect the MT cytoskeleton using immuocytochemistry, transmission electron microscopy, and live imaging. We will also determine the structural domain that mediates Lqf function at synapses. In the 2nd Aim, we will determine the synaptic role of Lis1 and the genetic and biochemical relationship between Lqf and Lis1. Finally, we propose to characterize one of the novel lqf modifiers to further dissect the genetic pathway mediating the synaptic function of the Lqf-Lis1 complex. Lqf (epsin) and its partners (Futsch and DLis1) are highly conserved between flies and mammals. Further, the synaptic roles for epsin and DLis1 remain poorly understood. Thus, information derived from the proposed studies is expected to have broad biological and clinical significance. PUBLIC HEALTH RELEVANCE: The cognitive function and mental health depends critically on the synaptic connections formed between neurons and their target cells. This proposal studies Lqf, Lis1 and related proteins in synapse development and function using the fruit fly as a model genetic organism. Because many genes and protein functions are highly conserved from flies to humans, the findings to be obtained from this proposal are expected to provide insights into the cellular and molecular mechanisms of synapse development. These studies could also potentially advance the understanding of a major human developmental mental retardation disease called lissencephaly (smooth brain).

ABSTRACT PI (Zhang) Proposal # IOS-1025556

The nervous system controls human and animal behavior. From behavioral analysis, neuroscientists can gain insights into brain function such as learning, memory, decision-making and other cognitive activities. Yet, the neural mechanisms by which behavior is produced and regulated remain poorly understood. Dr. Zhang and his associate propose to develop a new molecular genetic method to non-invasively dissect the neural circuitry underlying behavior in the fruit fly, Drosophila melanogaster. They will be developing and refining a genetic method that allows for restrictive expression of genes in subsets of neurons in the fly brain. They will then examine the behavioral consequence following the perturbation of these neurons or a neural circuit. This brain-behavior mapping effort is expected to significantly advance the understanding of brain function. Another important aspect of this proposal is to develop new genetic tools. Dr. Zhang has begun and will continue to make these reagents freely available to the fly community. These reagents will be deposited in a public domain (such as the Bloomington Drosophila Stock Center). The strategy developed in the proposed work is also expected to be applicable to other genetic model organisms. This proposal has strong outreach and broader impacts. Building upon the past record of excellence, Dr. Zhang will actively participate in education, training, and outreach to local communities. This includes mentoring undergraduate and graduate students, training a postdoctoral fellow, involving a local high school science teacher in summer research, and participation of science outreach activities in local schools and museums.

Cells release various products to the outside through a process called exocytosis. In the nervous system, exocytosis mediates the release of chemicals including neurotransmitters, and a number of highly conserved proteins have been shown to control this process. In this project, a hypothesis will be tested that one protein originally discovered in yeast, VSM-1, may regulate exocytosis in the nervous system of multicellular animals. To test this hypothesis, the impact of mutating the gene coding for VSM-1 will be examined by studying the resulting changes at the level of molecules, cells, and whole organisms in C. elegans. At the cellular level, it will be determined if communication between neurons is impaired in mutants lacking the gene. Finally, whole mutant animals will be examined using pharmacological tests and fluorescent microscopy. Preliminary unpublished data showed that C. elegans strains containing the mutation in the VSM-1 gene are hyperactive and have more nerve endings than wild type animals. Insights into the mechanism of VSM-1 from this proposal will advance understanding of the nervous system of higher animals, including humans, because there are no reports yet on the function of VSM-1 in multicellular animals. The proposed research will also significantly enhance and promote students' training in the field of neuroscience since it will primarily be conducted by undergraduate students enrolled at Southwestern Oklahoma State University (SWOSU), a teaching institution in rural western Oklahoma with a growing commitment to research.

This Major Research Instrumentation award funds the acquisition of a multi-photon laser scanning confocal microscope for research and training in interdisciplinary fields on the University of Oklahoma (OU) Norman campus. The new instrument has higher sensitivity and faster speed and will significantly boost the capabilities for live and deep-tissue imaging by a diversity of life science and engineering researchers on the OU campus and at nearby research institutions. This new confocal microscope will be used by a number of NSF-sponsored laboratories that are already active in cell and molecular imaging as well as by new users who will incorporate confocal imaging in their research projects. The research topics include, but they are not limited to, neural crest migration in the primitive vertebrate lamprey, neurite outgrowth and synaptic vesicle trafficking in fruit flies, calcium imaging of deep nerve tissues, temporal and spatial patterns of protein complex formation in plants, and biofilm growth and morphology during corrosion of metallic surface. The new microscope will be an important part of education and research integration; students will be trained initially through a new upper undergraduate and graduate student course, Applied Confocal Imaging, taught collectively by the PIs. In addition, the PIs and their students will collaborate with rural and urban Oklahoma K-12 teachers in developing and implementing cutting-edge outreach and classroom projects. The results of these research, teaching, and outreach efforts will be broadly disseminated through participation of students and faculty at professional meetings and through peer-reviewed publications.