Cameron Gundersen - US grants

The goal of this project is to improve our understanding of the transmitter release process and the means of regulating presynaptic events in nerve cells. The approach entails the reconstitution of a neuronal transmitter release system using the oocytes of Xenopus laevis. These oocytes can be microinjected with a wide range of materials, and importantly, have been shown to translate exogenously supplied messenger RNA. The reconstruction of the release process involves three phases. First, oocytes will be injected with messenger RNA from electromotor neurons of Torpedo or Narcine. This step may be crucial for endowing oocytes with components needed to support the exocytotic discharge of transmitter. The oocyte will then be injected with synaptic vesicles isolated from the electroplax tissue of Torpedo or Narcine. Finally, the oocytes will be stimulated electrically or chemically to determine whether evoked release of transmitter (acetylcholine) can be detected. Success of this reconstitution effort will facilitate a detailed examination of the molecular components involved in transmitter secretion. The resulting observations may open new avenues to the study of biochemical correlates of synaptic plasticity. Moreover, a wide range of neurological deficits (eg., dementia, Parkinsonism, mental retardation) might be amenable to scrutiny in a new light.

The molecular events that culminate in exocytosis are unknown although recent work has suggested that small G-proteins regulate the movement and targeting of membranous structures and processes (membrane-trafficking) including exocytosis. Among the distinctive modifications of G-proteins that regulate cyclic interactions of these proteins with cellular membranes are polyisoprenylation and fatty-acylation of C-terminal Cys residues. Certain snake venom phospholipase A2 enzymes are extremely potent neurotoxins at the presynaptic terminal and block neurotransmitter release without structural damage to the nerve ending. The hypothesis to be tested is that these venoms may act to modify the small G-proteins and thereby block the exocytotic process of neurotransmitter release. This research has the potential for increasing understanding of exocytosis and providing molecular tools for the study of neurotransmission and exocytosis in a wide variety of cells and tissues.

The long-term goal of this project is to increase our understanding of synaptic communication in the nervous system. The molecular mechanism by which neurotransmitters are released at chemical synapses is still not resolved. However, it is known that calcium channels of nerve endings are vital for regulating this process. This work directly addresses questions concerning the structure and properties of presynaptic calcium channels, and it will lead to the production of reagents that should be of long-term utility for studying these proteins. Specifically, this project entails the production of monoclonal and polyclonal antibodies against a candidate subunit of a presynaptic calcium channel that was recently cloned by this laboratory. These antibodies will be used to purify this protein and other candidate calcium channel subunits. These antibodies will also be used for the immunolocalization of this antigen in tissues and subcellular fractions. These investigations should dramatically improve our knowledge of presynaptic calcium channels and how they contribute to normal synaptic function. This is a crucial prerequisite for applied studies of how these channels are modulated by environmental and genetic factors in health and disease.

An integrated microarraying facility will be initiated in the Department of Molecular and Medical Pharmacology (DMMP) in the Center for Health Sciences (CHS) at UCLA. The facility will consist of a DNA processing robot, a microarrayer, a scanner, a DNA/clone resource, and a computational facility. The microarray facility has been designed to provide the highest practicable throughput of this powerful technology.

The research will cover the whole range of modern biology including studies of retroviruses, differentiation of neural stem cells and other cells, the cellular response to metals, synaptic transmission, neural growth factors, nitrous oxide physiology, immune system differentiation, neural aspects of behavior and glucocorticoid signaling. An important part is to improve the bioinformatics of microarray technologies using the data provided by the facility.

This facility will provide researchers and their students the ability to visualize gene expression patterns for thousands of genes at once and have a major impact on research in cell and molecular biology at UCLA.

DESCRIPTION: (Applicant's Abstract) Cysteine-string proteins (csps) are a novel family of synaptic vesicle proteins that have been implicated in two important processes at nerve endings. First, recent evidence strongly supports the hypothesis that csps are part of a unique regulatory interaction that takes place between a docked synaptic vesicle and presynaptic calcium (Ca) channels. The functional consequence of this interaction is that the presynaptic Ca channels are rendered competent to open in response to membrane depolarization. However, the molecular and biophysical mechanisms of this interaction remain unknown. Because csps associate with a specific isoform of Hsp70 molecular chaperones, we have hypothesized that the csp-Ca channel link involves a specific isoform of Hsp70. Specific Aim 1 directly tests this hypothesis using both biochemical and physiological approaches. Second, we have postulated that csps participate in events that are necessary for the fusion of synaptic vesicles with the plasma membrane. The rationale for this proposal stems from a consideration of the unusual structure of csps. The cysteine string domain of these proteins contains as many as 11 consecutive cysteine residues. As far as we know, all of these cysteine residues are fatty acylated. A protein with this type of a hydrophobic domain can assume a conformation at membrane interfaces that can effectively cross-link adjacent membranes (a model of this interaction is presented). Specific Aim 2 tests the validity of this model of membrane cross-linking by studying the effect of csps on the aggregation, fusion or lysis of liposomes under different empirical conditions. Finally, Specific Aim 3 considers several inter-related structural studies of csp that are relevant to its presumed functions. Among the planned studies are efforts: (I) to resolve unequivocally the degree of fatty acylation of native csps; (ii) to examine the secondary structure of membrane-associated csps; and (iii) to use a protein palmitoylthioesterase to probe the role of membrane tethering and palmitoyl residues in csp function at nerve endings. In summary, these investigations will advance our knowledge of csps on several fronts that are germane to its function at synapses. Because csps are widely distributed at nerve endings and in other secretory cells, this work is of fundamental importance to our understanding of membrane trafficking events in normal and pathological circumstances.