Michael Wilson - US grants

The regulated expression of presynaptic nerve terminal proteins is critical both in developing the circuitry and in controlling neurotransmitter signaling that underlies nervous system function and ultimately behavior. The long-term goal is to define the molecular mechanisms governing the regulation and interactions between these synaptic proteins, and how this contributes to the diversity and plasticity of normal synaptic transmission, and how dysregulation of these processes leads to neurophysiological deficits. Specifically, this investigation addresses the function of SNAP-25, a protein that plays a key role in vesicle docking and regulated exocytosis of neurotransmitters. The mouse mutant, coloboma, bearing a contiguous gene defect and deficient in SNAP-25 expression, implicates dysregulation of SNAP-25 in spontaneous hyperactivity, and with delayed neurobehavorial development, deficits in learning and memory, and at the cellular level in abnormal hippocampal physiology and neurotransmitter release. Mutations affecting SNAP-25 expression may serve as effective models of hyperkinesis, a prominent component of Attention Deficit Hyperactivity Disorder, Tourette syndrome and other neurophysiological disorders. The proposed studies will test the hypothesis that SNAP-25 is involved in these abnormalities, and that two developmentally regulated isoforms of the protein have specialized roles that contribute differently to neural development and mature physiology of synaptic transmission. Towards this goal an integrated approach incorporating the following Specific Aims is proposed: 1) to determine if the phenotypic effects ascribed to the coloboma mutation are specific to SNAP-25. These studies will use Snap gene "rescued" and homologous recombinant null mutants to characterize deficits in neurobehavioral development and learning, in hippocampal electrophysiology, including long-term potentiation and theta EEG activity, and in transmitter release using in vitro synaptosomal preparations. 2) to determine the molecular specificity of SNAP-25 isoforms. Experiments using yeast expression systems and in vitro protein binding assays will characterize the differential interactions of SNAP- 25a and b isoforms with syntaxin and other presynaptic proteins involved in regulated vesicular exocytosis. 3) to define the specific role of the SNAP-25b isoform in neurotransmission. SNAP-25b will be over-expressed in neural cell lines deficient in this isoform, and the cells will be assayed for synaptic vesicle cycling and acetylcholine release as indexes of SNAP-25b function in synaptic transmission. 4) to establish the function of SNAP-25b in the intact nervous system, a homologous recombination strategy will be used to generate mutant mice that are limited to SNAP-25a isoform expression. Analysis of these partial loss- of-function mutants at behaviorial, electrophysiological and neurochemical levels will determine if deficits in mature neurophysiology and behavior result from deficiencies in specialized functions of SNAP- 25b. Through these studies, a better understanding of the molecular processes of neurotransmission will be achieved and, importantly, novel well-defined animal models will be established for the design and characterization of therapeutics targeted to hyperactivity in human neuropsychiatric disorders.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The development, maintenance and control of neurotransmitter secretion at chemical synapses underlie the neuronal communication necessary for the proper function of the nervous system. Deficits in synaptic transmission and connectivity, resulting from genetic or epigenetic abnormalities revealed during development, are likely to contribute to impaired brain function. In particular, regulated exocytosis of classical neurotransmitters and neuropeptides is a key factor that underlies the necessary presynaptic input for effective communication between neurons. The overall goal of the research of our laboratory is to gain a better understanding of the molecular, cellular and developmental mechanisms which control neuroexocytosis. Our hypothesis is that the developmental regulation of the composition of the molecular machinery responsible for membrane trafficking and vesicular fusion for neurotransmission is instrumental in developing how neurons communicate during the initial contact and formation of synapses, and ultimately in sculpting the synaptic physiology of the mature nervous system. To address this hypothesis, we have focused on the protein SNAP-25, a key element of the SNARE complex mediating membrane fusion for transmitter release. We propose that the transition from a constitutively expressed homologue, SNAP-23, to the regulated expression of SNAP-25 isoforms plays a key role in mediating vesicle fusion for growing axons to functional synapses capable of evoked neurotransmitter release and synaptic activity. Our investigations make use of Snap25 gene mutants in the mouse developed through previous funding of this grant and a conditional mutation to be generated during the present period. In Specific Aim 1, experiments are designed to examine the role of spontaneous neurotransmitter release in cultured cells and fetal brain development using Snap25 null mutants. In Specific Aim 2, we will examine whether the switch in SNAP-25 isoforms is required for maturation of synaptic transmission during postnatal development using a mutation that prolongs overexpression of the SNAP-25a isoform. Finally in Specific Aim 3, we will extend our analysis to study later steps of synaptogenesis and synaptic maturation that occur after birth, and in the long-term neurodegenerative processes, by investigating synaptic functions of a conditional Snap25 gene mutation controlled by inducible expression of Cre recombinase. If successful, these investigations will shed light on the roles of SNAP-25 and its isoforms during development and for synaptic plasticity required for normal brain function, and how abnormalities in presynaptic mechanisms of neurotransmission are involved in neuropsychiatric disease. [unreadable] [unreadable]

This IGERT award supports a broadly-based education and training program at the University of New Mexico that will enable students from various disciplines to develop new nanoscale imaging and sensing tools and to apply them in investigations of fundamental problems in cell biology and neuroscience. By infusing training in cell biology and neuroscience with training in the technological basis of chemistry, electrical engineering, and physics, the university will create an exciting learning environment in which to prepare students for the challenges and opportunities presented by the nanotechnology revolution. The program will include internships at national laboratories and industry, travel to national and international meetings, international collaboration combined with internship placements, and a web-based dissemination. The research component of the program will generate improved understanding of important and relatively unexplored biological processes, enabled by creation of new imaging and sensing instruments. To realize this goal, the program will capitalize on the existing strengths to develop new quantum-dot-based technologies coupled with hyperspectral and multiphoton microscopy that carry the potential to revolutionize research on genomic, cellular, and synaptic activities. The educational component of the program will emphasize collaboration across traditional academic disciplines, with co-advisors from cell biology/neuroscience and engineering/science fields, rotations between various laboratories with different research styles, and active interactions among students with different academic backgrounds. New courses developed under this project will lead to a Graduate Certificate Program in Biomedical Science and Engineering with Concentration in Nanotechnology, to be followed by a new PhD program in Systems Biology and Bioengineering. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

DESCRIPTION (provided by applicant): Tissue invasion and metastasis, one of the six purported capabilities acquired by human cancers, accounts for more than 90% of cancer deaths and represents an understudied but promising area for future therapeutic developments. The long-term goal of our program is to understand how malignant tumor cells acquire the invasive and metastatic phenotype. In the next five years, we plan to focus on the microenvironment of tumor cell surface and test the hypothesis that cell surface proteolysis regulates the invasive and metastatic properties of malignant tumors. Current evidence suggest that proteinases contribute to tumor invasion and metastasis by not only degrading the extracellular matrix as a barrier, but also functioning to regulate pathways controlling cell growth, migration and apoptosis through releasing latent growth factors or cleaving various receptors and their ligands for both activation and inactivation. Yet, efforts targeting tumor proteinases especially the MMPs have not achieved any clinical success so far. Several outstanding reviews have recently been published to address this apparent gap between "scientific success and clinical failure" for the MMP field. We would like to argue that one neglected area is proteolvsis on tumor cell surface. Our evidence both in vitro and in vivo suggests that the same proteinase behaves differently when it is tethered on cell surface or secreted. We hypothesize that the membrane-bound MMPs are more efficient for proteolysis and harder to inhibit than soluble ones, thus, enabling tumor invasion and metastasis. To test this idea, we designed three specific aims: 1) Characterize the invasive and metastatic phenotype conferred by MT1-MMP expressed on tumor cell surface both in vitro and in vivo;2) Determine the contributions of the hemopexin- and catalytic- domains of MT1-MMP towards the invasive and metastatic phenotype;and 3) Characterize the microenvironment on tumor cell surface that enables MT1-MMP to mediate invasion and metastasis. Accomplishment of these aims may empower the design of a new generation of MMP inhibitors targeting the tumor cell surface and provide model systems to test the efficacies of these potential therapeutics.