Regis B. Kelly - US grants

A scheme is described that should allow us to select mutations in synaptic transmission. The shibire mutation of Drosophila melanogaster is a temperature-sensitive paralytic mutant that causes nerve terminals to be depleted of synaptic vesicles. When flies are held at a temperature just below their paralytic temperature, a small reduction in the efficiency of transmitter release will cause paralysis. This screen has been used to isolate 93 mutants, enhancer of shibire or E(shi), that display this phenotype when heterozygous. We wish to screen these mutants biochemically to identify those that affect synaptic vesicle biogenesis or function. Synaptic vesicles, isolated from adult Drosophila brain, will be identified by their homogeneous size, their content of synaptic vesicle marker proteins and their absence from shibire flies held at non-permissive temperatures. The ability of our synaptic vesicle assay to recognize defects in endocytosis will be tested using alleles of shibire with different temperature thresholds for paralysis. The enhancer mutations will then be screened to identify mutations that affect recycling of synaptic vesicles from the plasma membrane, or the probability of synaptic vesicle release. If the rationale is correct, selection of shibire enhancers will allow us for the first time to select mutations in regulated secretion.

DESCRIPTION: (adapted from applicant's abstract) The function of a nerve terminal is to release transmitter molecules at the right time and the right place. Transmitter is stored inside synaptic vesicles in nerve terminals until vesicles fuse with the plasma membrane, releasing their contents. After release, new synaptic vesicles re-appear within seconds. Using cell-free reconstitution assays, the applicants have discovered that there are two pathways of synaptic vesicle formation. Naturally occurring mutants in mice lack one of these pathways, the one that utilizes the adaptor complex AP3. Such mice have major behavioral abnormalities, resembling aberrant behavior found in humans. This proposal aims at understanding the basic molecular mechanisms in the AP3 pathway, in order to understand the behavior and lay the basis for developing novel drug therapies. It also compares the two pathways of vesicle biogenesis, looking for differences in molecular mechanism, physiological relevance and development sequence.

The Gordon Conference on The Cell Biology of the Neuron will provide an informal but unique forum for scientists working in this rapidly developing field. Sessions will be devoted to, among others, the following topics: the release of neurotransmitter, the biogenesis of the synaptic vesicle, the mechanisms of transport along axons, the extension of growth cones and the specific adhesion of neurons to their correct targets. These processes are tightly regulated in neurons; hence considerable attention will be addressed to the regulation of neuronal function and development by signalling mechanisms. Recent data on the storing of neurotransmitter will lead to discussion of ways to select mutants in synaptic transmission. Disease states of the neuron and possible therapeutic approaches will be a significant concern of the meeting. For example, we will discuss aberrations in neuronal endocytosis pathways that appear in Alzheimer's disease, the role of intermediate filament proteins in neurodegenerative diseases, the understanding of Parkinson's disease that comes from research on norephinephrine uptake and recent clarifications of how the neurotoxins act.

Deficiencies in a single gene can cause dramatic effects in brain function and so behavior. Linking the known genetic defect with the observed brain disorder has proven to be challenging. Here we describe experiments to link the neurological properties of mocha mice, its aberrant balance, overanxiety, and sensitivity to anesthetics to its underlying molecular deficiency, the absence of the heterotrimeric adaptor protein nAP-3. We have established already that nAP-3 is required for the formation of synaptic vesicles from endosomes, by a pathway sensitive to brefeldin A and the casein kinase inhibitor CKI-7. We use these pharmacological agents to mimic the mocha phenotype in the neuroendocrine cell line PC12. We ask if the presence of "mocha-like" conditions reveals or induces the expression of a different class of synaptic vesicles. Because secretory granule membranes are recycled to synaptic vesicles by an nAP-3 dependent mechanism, we examine the regulated secretory pathway in "mocha-like" PC12 cells. Finally, we initiate studies on mocha mice to determine if their properties are consistent with the findings from PC12 cells and if they are a valid model for the human disease, Panic Disorder Syndrome, which is a major portal to drug addiction.

The ultimate goal of this award is to improve the University of California, San Francisco's (UCSF) protections for human subjects. The challenges of expanding activity in human subjects research, an increasingly complex regulatory environment, and limited resources have hampered our ability to keep our educational programs, policies and infrastructure up to date. To address this, programs are proposed that will increase researchers' knowledge, reduce unnecessary correspondence and revision due to lack of knowledge, allow more efficient review and processing of applications, ensure that regulatory and ethical questions are addressed more clearly and completely, revise processes to incorporate recent changes in regulations and guidelines, and allow more detailed and accurate tracking. Specifically,(1) New educational modules will be developed which will improve the ability of the Committee on Human Research (CHR), UCSF's IRB, to deliver training; (2) all existing guidelines and policies will be updated, posted to the CHR web site, and made available as small brochures and booklets; and (3) a fully on-line application process will be developed that allows researchers to submit protocols electronically, correlates protocol types to training requirements, tracks adverse events, and tracks other compliance-related data. These activities will result in a reduction of time spent on routine submission, review, and tracking procedures and allow researchers and IRB members to focus on more substantive subject protection issues. IRB review letters will give less attention to correcting routine omissions and greater attention to larger issues. In order to assess the effectiveness of these activities, samples of letters will be evaluated before and after the grant period to see if the type of content has shifted. Process metrics will be tracked on IRB review cycles and other operational issues. Sample research records will be evaluated for the consistent use of IRB-approved consent forms. Feedback from knowledgeable faculty and staff who are aware of consent processes and subject satisfaction in multiple studies will be sought.