1985 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
The Selective Expression of Different Neural Growth (Rg) @ University of California San Francisco |
1 |
1986 — 1989 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
K08Activity Code Description: To provide the opportunity for promising medical scientists with demonstrated aptitude to develop into independent investigators, or for faculty members to pursue research aspects of categorical areas applicable to the awarding unit, and aid in filling the academic faculty gap in these shortage areas within health profession's institutions of the country. |
Role of Multiple Ngf Precursors in Neural Development @ University of California San Francisco
The proposed program will analyze the biological role of multiple Nerve Growth Factor (NGF) precursors in neural development. Mature NGF protein supports the survival of sympathetic and sensory neurons. NGF mRNA has however been found in several tissues not innervated by sympathetic or sensory neurons (e.g., brain, placenta). These findings suggest additional roles for NGF, or for other proteins encoded by the NGF gene. The original mouse NGF cDNA clones predict that this biologically active molecule results from the processing of a larger precursor protein; the mature NGF protein is located at the carboxy-terminus of the predicted precursor. Little is known about the function of the non-NGF moiety of the precursor. Our discovery of different RNA species, which encode different NGF precurosrs, raises the possibility of complex and novel regulatory strategies associated with this hormone. The long NGF mRNA contains two potential initiation codons and the second precedes the only substantial hydrophobic sequence, presumably the signal sequence for secretion, in the precursor protein. A short NGF transcript contains only the 2nd AUG, the first having been spliced out, and thus encodes a protein truncated at the amino-terminus, with the putative signal peptide directly following the initiator methionine. We postulate that the differences between the two NGF transcripts affect the cellular localization of NGF (e.g. membrane-bound in the larger mRNA vs secreted in the shorter), or change the biological activity of the precursor or of its cleavage products. This would account for the expression of NGF in supporting cells (glia, fibroblasts) in the case of a membrane-associated form and in target cells in the case of the secreted protein. Using an antibody to the NGF precursor expressed as a fusion protein in bacteria (no such antibody is yet available), we will identify the cellular localization and processing of the NGF-associated polypeptides in mammalian cells transfected with the different NGF cDNAs. We will purify the precursors and characterize their biological activity. Last, transgenic animals made to overexpress NGF in specific tissues will facilitate an analysis of the full role played by the NGF and non-NGF moieties of the precursor in development. The results of this study will elucidate aspects of normal neural development in molecular detail. It will also potentially indicate the locus of the primary defect in a developmental or degenerative neurological disorder and may well suggest avenues for intervention in many human conditions through the enhancement of regeneration.
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1 |
1993 — 2005 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
K02Activity Code Description: Undocumented code - click on the grant title for more information. R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transport of Neurotransmitters Into Synaptic Vesicles @ University of California San Francisco
Neurotransmitters are stored in synaptic vesicles so that their release may be regulated by neural activity. For classical transmitters, vesicular storage requires transport from the cytoplasm, where many are synthesized or appear after reuptake from the synapse. However, little is known about the molecular basis for vesicular transport, its potential for regulation or its role in synaptic transmission. The long-term objectives of this proposal are to understand how the transport of neurotransmitters into synaptic vesicles influences the processing of neural information and contributes to human neuropsychiatric disease. The proposal focuses on the vesicular transport of monoamines in light of their role in mental illness, drug abuse and Parkinson's disease. We originally used the neurotoxin MPP+ to isolate the first cDNA for a vesicular neurotransmitter transporter, a vesicular transporter for monoamines. The similarity of the predicted protein sequence to bacterial antibiotic resistance proteins supported a role in protecting against neural degeneration such as the form that occurs in Parkinson's disease. The sequence also defined a novel mammalian gene family and we have since isolated the cDNAs for a second vesicular monoamine transporter and a putative vesicular transporter for acetylcholine. The strategy of the proposal is to determine the mechanism, regulation and cell biology of the vesicular transporters by expression of the cloned cDNAs in heterologous systems, then extrapolate this information to their role in behavior and disease using transgenic models and human genetics. The first specific aim addresses the structural basis for transport activity, focusing on substrate recognition, the mechanism of active transport, efflux and the interaction with drugs. Second, we will characterize the regulation of vesicular amine transport by phosphorylation. Third, we will study the intracellular trafficking of the vesicular transporters to understand the mechanisms that determine the sites of transmitter storage. Fourth, we will target disruption of the gene encoding vesicular amine transport int he brain and assess the role of its inheritance in familial Parkinson's disease. Last, we will extend our study to the related transporter for acetylcholine and the unrelated synaptic vesicle proteoglycan SV2 which shows strong homology to nutrient transporters but whose function in synaptic transmission remains unknown. Correlation of the molecular and cellular analysis with the biological models and genetic studies will then indicate the role of vesicular neurotransmitter transport in information processing, behavior and human neuropsychiatric disease.
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1 |
1994 — 1995 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transport of Neurotransmitters Into Synaptic Vesicle @ University of California San Francisco
Neurotransmitters are stored in synaptic vesicles so that their release may be regulated by neural activity. For classical neurotransmitters, vesicular storage requires transport from the cytoplasm, where many of the transmitters are synthesized. However, little is known about neurotransmitter transport into synaptic vesicles or its potential for regulation, which would affect quantal size and hence the efficacy of synaptic transmission. The long-term objectives of this proposal are to understand how the transport of neurotransmitters into synaptic vesicles influences the processing of neural information and contributes to human disease. The proposal focuses on the vesicular transport of biogenic amines because these transmitters are involved in major mental illnesses and Parkinson's disease. The strategy is to isolate cDNA clones for the vesicular transport proteins, reconstitute their activity in a heterologous system and use this system to characterize the structural basis for transport function and regulation. We have recently isolated a cDNA clone for the chromaffin granule amine transporter using selection in the neurotoxin MPP+. The first specific aim of the proposal is to generate antibodies to this transporter and use these to confirm its suspected intracellular location. The second aim is to isolate related cDNAs and use the pattern of expression to suggest their function. The third specific aim is to characterize the functional properties of the transporters expressed in a heterologous system. Mutant and chimeric proteins will be used to dissect the structural basis for transport activity, specificity and interaction with pharmacologic agents. Heterologous expression in a neural cell line will identify the potential for regulation. Correlation of the molecular analysis with biological models and genetic information can then be used to determine the role of vesicular neurotransmitter transport in information processing and neuropsychiatric disease.
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1 |
1995 — 2016 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Presynaptic Mechanisms of Neural Plasticity @ University of California San Francisco
Drug abuse involves the pharmacologic activation of neural pathways involved in reward. Dopamine mediates these pathways and drugs of abuse bypass the normal stimuli for reward by increasing dopamine release more directly. This non-physiological activation then produces changes in synaptic transmission that presumably underlie tolerance, physical dependence and drug craving. Drug abuse therefore provides a model for neural plasticity in the reward pathway that has clear relevance for behavior. The long-term goal of this proposal is to understand how drugs of abuse alter synaptic transmission. The strategy is to focus on the membrane trafficking of proteins involved in neurotransmitter release and signal transduction, their regulation and the physiological significance of this regulation for the release of dopamine. Dr. Sulzer will use direct, amperometric methods to study the kinetic features of quantal dopamine release and in continued collaboration with Dr. Edwards, the role of vesicular monoamine transporter 2 (VMAT2) and D2 autoreceptors in the regulation of quantal size and the readily releasable pool of synaptic vesicles. To assess the potential for presynaptic regulation of quantal size, Dr. Edwards will study the membrane trafficking of VMAT2. Previous work has demonstrated the phosphorylation of V MAT2 and the closely related vesicular ACh transporter, and he will now address the role of two particular motifs in the trafficking of these proteins to large dense core vesicles (LDCVs) as well as synaptic vesicles, extend the analysis to primary neuronal culture and dopamine release with Dr. Sulzer, and use the new information about sorting signals to characterize the cytosolic sorting machinery. Dr. Kelly will explore two pathways involved in synaptic vesicle formation, using both pharmacologic and genetic approaches. In particular, he will examine the role of a brefeldin A-sensitive pathway in the recycling of proteins derived from LDCVs (such as VMAT2 and VAChT), and use mocha mice deficient in the adaptor protein AP-3 to assess the physiological significance of this pathway in collaboration with Dr. Sulzer. Dr. von Zastrow will address the role of membrane trafficking in the regulation of opiate receptors, and dissect the mechanisms responsible for their internalization after activation by ligand. In addition, he will characterize trafficking of the delta-opiate receptor to LDCVs in collaboration with the Edwards and Kelly groups. He will also test using knock-in mice the novel hypothesis that morphine's susceptibility to abuse derives from its failure to induce mu-opiate receptor internalization by altering the sorting sequences in vivo.
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1 |
1995 — 1999 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Role of Neurotransmitter Packaging in Neural Plasticity @ University of California San Francisco
Release of the neurotransmitter dopamine mediates the reward for such adaptive activities as eating and sex. Drugs of abuse circumvent the normal stimulation of this pathway and induce dopamine release more directly. The development of drug tolerance, physical dependence and craving therefore provides a paradigm to examine the more general question of neural plasticity, its role in the reward pathway and its relationship to behavior. Amphetamines induce the efflux of dopamine into the synapse and appear to interact with the vesicular amine transporter. The behavioral effects of amphetamines further suggest that regulation of these transport activities may have a role in the normal function of reqard pathways and in behavior. Indeed, Dr. Sulzer has used voltammetry to show that stimulation of dopamine D2-like receptors reduces quantal size. Activity-dependent changes in neurotransmitter transport may also account for drug tolerance, dependence and craving. The long-term objectives of this program are to understand how the transport of neurotransmitters into synaptic vesicles influences dopamine release and the function of the reqard pathway. We originally used the neurotoxin MPP+_ to isolate the first cDNA for a vesicular neurotransmitter transporter, a transporter for monoamines. The sequences defines a novel mammalian gene family and now includes two vesicular amine and one vesicular acetylcholine transporter. The strategy of the proposal is to study the mechanism, regulation and cell biology of the cloned neuronal transporter (VMAT2) by expression of the cloned cDNAs in heterologous systems, then determine the relationship to dopamine release. Specific Aim 1: Establish a role for transporter reversal in vesicular efflux, exchange and amphetamine action. Specific Aim 2: Examine the regulation of VMAT2. Specific Aim 3: Determine the effect of activity on intracellular trafficking of VMAT2. We will then extend these studies to primary neuronal cultures and in collaboration with Dr. Sulzer, use voltammetry to determine the role of VMAT2 in regulating quantal size.
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1 |
1996 — 2020 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Transport of Neurotransmitter Into Synaptic Vesicles @ University of California San Francisco
Specific membrane proteins catalyze the transport of classical neurotransmitters into secretory vesicles in preparation for release by regulated exocytosis. The long-term objectives of this proposal are to understand how this transport activity influences transmitter release, information processing and behavior. The program focuses on the vesicular transport of monoamines because these transmitters have a role in major mental illnesses and drug abuse. We have used selection in the neurotoxin MPP+ to isolate a cDNA encoding the vesicular monoamine transporter expressed in the adrenal gland (VMAT1), further implicating this activity in the form of neural degeneration that occurs in Parkinson's disease. The sequences define a novel mammalian gene family that now includes a second vesicular monoamine transporter expressed in the brain (VMAT2) and a vesicular transporter for acetylcholine (rVAChT). This program uses the cloned cDNAs for vesicular neurotransmitter transporters to understand aspects of their function that have relevance for synaptic transmission and human health. The first specific aim is to use an assay that we have recently developed to characterize the role of the transporter in vesicular amine efflux exchange and the action of amphetamines. The second specific aim is to determine the structural basis for substrate recognition, coupling to the driving force for transport delta/mu/H+ and drug interaction using VMAT1/VMAT2 chimeras and site-directed mutagenesis together with flux and drug binding assays. We will also assess whether VMAT2 functions as a monomer. The third specific aim is to determine the role of phosphorylation in the control of VMAT2 activity. The fourth specific aim extends the analysis to the vesicular ACh transporter and addresses issues that may influence its function such as its intracellular localization and posttranslational modification. These studies will both help to understand the molecular mechanism for neurotransmitter packaging and begin to reveal its role in quantal size, drug abuse and neural degeneration.
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1 |
1997 — 2007 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Vesicular Neurotransmitter Storage/Transport in Synapse Function and Development @ University of California San Francisco
Many transporters including neurotransmitter transporters use the movement of coupled ions and charge to drive the accumulation of substrate against a concentration gradient. Conversely, the movement of coupled ions and charge can be used to identify the substrates for orphan transporters. We have recently used pH imaging and electrophysiology to show that several orphan transporters exhibit the properties of classical amino acid transport systems N and A. The functional characteristics and location of these proteins further suggest a role in the glutamine-glutamate cycle that replenishes glutamate (and GABA) released from nerve terminals during synaptic transmission. The coupling of system N to H+exchange and the electrogenic nature of system A have also suggested that pH imaging and electrophysiology might be used to study intracellular transporters, which often couple to H+. Indeed, we have recently observed pH changes produced by substrates in cells expressing a lysosomal transporter mislocalized to the plasma membrane. The long-term objective of this proposal is to understand the function of intracellular transport proteins implicated in synaptic transmission and development. The strategy is to mislocalize these proteins on the cell surface so that we can use pH imaging and electrophysiology to determine their function. In terms of specific aims, we propose to 1) Knock out glutamine synthetase in the nervous system. Although the glutamine-glutamate cycle was proposed decades ago, its role in transmitter release remains unclear. We will therefore use cre recombinase to knock out the gene for glutamine synthetase specifically in the nervous system, and determine the effect on synaptic transmission. 2) Identify the substrates for synaptic vesicle protein SV2A. Among the first synaptic vesicle antigens to be identified, SV2 proteins strongly resemble a large family of transporters. Knock-out mice for SV2A alone show a severe phenotype. However, the function of SV2 remains unknown. We will now use biophysical approaches to characterize the function of SV2 mislocalized at the plasma membrane. 3) Determine the biochemical and cellular role of Diphthong (Dpth). Work from G. Davis' lab implicates the polytopic membrane protein Dpth in synaptic homeostasis. The sequence of Dpth predicts a polytopic membrane with similarity to transporters, and we will now determine its subcellular location in neurons, and identify its substrates as described for Aim 2.
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1 |
2000 — 2002 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Core--Cellular Phenotyping @ University of California San Francisco
SUBPROJECT ABSTRACT NOT AVAILABLE
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1 |
2003 |
Edwards, Robert H |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
New Perspectives in Transporter Biology-Faseb Conference @ Federation of Amer Soc For Exper Biology
DESCRIPTION (provided by applicant): This application requests support of a conference to be held under the auspices of the Federation of American Societies of Experimental Biology (FASEB) entitled "New Perspectives in Transporter Biology." This is the fourth bi-annual FASEB conference on the subject and is scheduled to be held from July 5 - July 10, 2003 at the Omni Tucson, in Tucson Arizona. Recent developments will make the meeting of great interest to a wide range of investigators, and we expect attendance by approximately 160 scientists. This conference will focus on the role of transport proteins in a number of biological systems, from renal physiology to synaptic transmission. The principles elucidated in one system have already begun to help illuminate the role of membrane transport in others. In addition, the meeting will address the function of transport proteins at multiple levels, from protein structure to behavior of the organism. Biophysical studies at high resolution have indeed already suggested novel roles for various transport proteins not anticipated from classical measurements of radiotracer flux. The meeting will thus provide an unusual opportunity for interaction between many different groups, including physiologists, molecular and cell biologists, biochemists, biophysicists, and structural biologists. The range of biological questions addressed will also encourage interactions between diverse groups. In addition to the role of transporters in normal physiology, the meeting will consider their role in disease (e.g., drug abuse and neural degeneration) and in drug delivery. The conference will consist of nine oral scientific session over five days (morning and evening), and afternoon poster sessions. The topics include epithelial transport, transport in disease, astrocyte-neuron interactions, regulation of transporters by membrane trafficking and changes in intrinsic transport activity, their role in signalling, intracellular transporters, transport mechanism and protein structure. Each oral session will be followed by a panel discussion of the topic. To promote the advancement of junior faculty, the most exciting poster submissions will be selected for short oral presentations during the regular oral sessions.
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0.916 |
2004 — 2008 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Hek Expression @ University of California San Francisco |
1 |
2006 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Id of Cause For Alpha-Synuclein Mobility Shift @ University of California San Francisco |
1 |
2006 — 2010 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Regulation of Neurotransmitter Transport Into Secretory Vesicles @ University of California, San Francisco
Dopamine provides reward for adaptive behaviors such as food and sexual activity, but drugs of abuse release dopamine directly, bypassing the normal regulatory mechanisms and producing long-term changes that result in tolerance, physical dependence and drug craving. The long-term objective of this proposal is to understand how dopamine release is regulated, and how changes in release influence behavior and contribute to drug abuse. The strategy is to use vesicular monoamine transporter 2 (VMAT2) as a tool to characterize the exocytotic release of monoamines, and to study it as a locus for regulation. Unlike most other classical neurotransmitters, monoamines including dopamine undergo release from cell body and dendrites as well as the axpn terminal. Importantly, somatodendritic dopamine release is required to induce behavioral sensitization, a model for drug-seeking. However, the mechanism of somatodendritic dopamine release remains unclear, with some evidence favoring vesicular release and other reverse flux through the dopamine transporter. In Specific Aim 1, we will assess the potential for exocytotic release by studying the trafficking of VMAT2, the carrier required for loading vesicles with monoamine. Preliminary data suggests that somatodendritic vesicles containing VMAT2 undergo regulated exocytosis, and we will now use both fixed cells and live cell imaging to characterize the exocytosis and endocytosis of VMAT2 in live neurons, and to identify the signals responsible for sorting VMAT2 to this pathway. We will then use this information to manipulate the localization of VMAT2 in vivo, test unambiguously the mechanism of somatodendritic dopamine release, and determine the role of somatodendritic release in the plasticity of monoamine systems in general. In Specific Aim 2, we will use VMAT2 to address basic questions about the relationship between vesicle filling and the recycling of membrane in the synaptic vesicle cycle. Exploring the biochemical basis for a genetic interaction identified in C. elegans, we have found that the v-SNARE synaptobrevin required for regulated exocytosis inhibits the activity of VMAT2, and this effect does not reflect changes in transporter expression or trafficking. Rather, the v-SNARE appears to inhibit VMAT2 directly. We will now characterize the mechanism for this inhibition, and use this information to determine the role of this interaction in dopamine release. The results will provide physiologically relevant information about a novel mechanism regulating vesicular neurotransmitter transport that may contribute to the long-term changes in dopamine release that accompany behavioral sensitization.
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1 |
2006 — 2010 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Communication @ University of California, San Francisco
DESCRIPTION (provided by applicant): Under normal conditions, adaptive behaviors such as eating and sex activate the reward system, and plasticity in the system serves to reinforce and promote these behaviors. Drugs of abuse bypass the normal requirements for stimulation and confer reward directly, altering the properties of the reward system to produce tolerance, physical dependence and drug craving. Drug addiction thus provides a dramatic example of plasticity in the reward pathway. The neurotransmitter dopamine has a crucial role in the reward pathway. Many drugs of abuse increase extracellular dopamine, and repeated intermittent administration can produce larger behavioral responses to subsequent drug exposure, a phenomenon known as behavioral sensitization that has been considered a model for the core features of addiction. Indeed, sensitization to psychostimulants requires the somatodendritic release of dopamine in the ventral tegmental area to induce this form of plasticity, and involves increased dopamine release in the nucleus accumbens in the expression of this plasticity. Together, these observations indicate that dopamine release and its regulation have an important role in the adaptation to drug use. The long-term objective of this proposal is to understand how drugs of abuse alter dopamine release. The strategy is to focus on the membrane trafficking of proteins involved in dopamine release and signal transduction and their regulation. In Project 1, Dr. Sulzer will use amperometry to understand the regulation of dopamine release through a flickering fusion pore, and other biophysical methods to assess the chronic effects of amphetamine on vesicle acidification. He will also embark on a newer line of investigation to study the mechanism and physiological role of structural changes in dopamine release sites. Dr. von Zastrow (Project 2) will examine the mechanisms involved in opioid and dopamine receptor internalization and recycling by real time imaging in primary neuronal culture. Dr. Edwards (Project 3) will investigate the role of regulated exocytosis in the somatodendritic release of dopamine required for behavioral sensitization. In addition, he will explore the relationship between vesicle filling and membrane recycling by studying the functional interaction of VMAT2 with synaptobrevin. In Project 4, Dr. Ryan will characterize the exocytosis and recycling of SVs in dopamine neurons, working with Dr. Sulzer to resolve differences in the nature of release (full fusion versus kiss-and-run), and with Drs. von Zastrow and Edwards on real-time imaging in live neurons, comparing the properties of regulated exocytosis in axon terminals and dendrites.
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1 |
2007 — 2010 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Communication Core @ University of California, San Francisco
This Program Project contains four distinct projects with overlapping goals and methodology. Dr. Sulzer uses primary neuronal culture and a variety of biophysical methods including amperometry, electrophysiology and capacitance to assess the release of dopamine and its regulation by modulation of exocytosis, vesicle acidification and synaptic growth. The regulation of monoamine release and in particular vesicle filling originally led to the assembly of this group around a strong bond between the laboratories of Drs. Sulzer and Edwards. In addition, Drs. von Zastrow and Edwards have now extended their work in cell lines to transfection of primary hippocampal, striatal and cortical cultures, largely as a result of this Program Project, and interactions between themselves and Dr. Sulzer. The proximity of von Zastrow and Edwards labs has also encouraged an interest in the role of presynaptic receptors in the regulation of dopamine release, and its potential for plasticity in drug abuse. Since Dr. Kelly has assumed many administrative duties and accordingly reduced the size of his lab, he will no longer participate in this Program Project. In his place, we have added Dr. Ryan for his expertise with vesicle recycling. As a result of the methods they have used, Dr. Ryan has a very different perspective on transmitter release from Dr. Sulzer, and we expect that through the interaction supported by this Program, they will work together to resolve the very interesting conflicts. In addition, Dr. Ryan has a particular interest in dopamine neurons since it seems likely that they recycle synaptic vesicle membrane differently from other neuronal populations. Overall, the physiological perspective of Drs. Sulzer and Ryan will also help to guide the molecular analysis by Drs. Von Zastrow and Edwards. Conversely, Drs. Von Zastrow and Edwards will generate hypotheses about transmitter release and dopamine receptor regulation that Dr. Sulzer can test directly.
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1 |
2009 — 2014 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Alpha-Synuclein and the Synaptic Vesicle Cycle @ University of California, San Francisco
DESCRIPTION (provided by applicant): Considerable evidence has implicated the protein ?-synuclein in the pathogenesis of Parkinson's disease (PD). Point mutations in ?-synuclein can cause autosomal dominant PD, and ?-synuclein accumulates in the Lewy bodies and dystrophic neurites of sporadic PD, suggesting a role for the protein in most forms of the disorder. Importantly, an increased dose of the wild type gene can also cause PD, indicating a pathogenic role for the normal protein and perhaps its normal function. However, the role of ?-synuclein in PD and its normal function remain poorly understood. Studies in yeast and mammalian systems have suggested a role for ?-synuclein in membrane trafficking. The protein interacts with membranes in vitro and localizes primarily to the axon terminal in neurons, but its effects on synaptic vesicle exocytosis and recycling remain controversial and poorly understood. The long-term objective of this proposal is to elucidate the function of ?-synuclein in its physiologically relevant context, at the nerve terminal. Since PD seems to involve an increase in ?-synuclein, the strategy has been to over-express the protein in primary neuronal culture, and assess its effects on the synaptic vesicle cycle by optical imaging of live cells. In preliminary experiments using a combination of optical imaging, electrophysiology, biochemistry and electron microscopy, we have found that over-expression ?-synuclein impairs neurotransmitter release by reducing the size of the synaptic vesicle recycling pool, providing some of the first unambiguous evidence for its role in neurons. We now propose to: 1) determine whether the inhibition of transmitter release by synuclein involves a gain in its normal function, by studying triple synuclein knock-out mice;2) characterize the effect of ?-synuclein on dispersion and reclustering of synaptic vesicle protein and membrane after exocytosis;3) assess the relationship between synuclein, synapsins and synphilin in synaptic vesicle mobilization;and 4) determine whether the effects of synuclein on neurotransmitter release contribute to degeneration. The results will extend previous work on ?-synuclein to its appropriate biological context in neurons, and provide a crucial physiological framework to understand the function of ?-synuclein. In the process, they will address a mechanism that may give rise to the degeneration observed in Parkinson's disease, and suggest therapeutic strategies to reverse functional disability due to the impaired release of transmitter from neurons that survive. PUBLIC HEALTH RELEVANCE: Current therapy for Parkinson's disease ameliorates symptoms without affecting the progressive neural degeneration that eventually results in severe disability. To develop more effective treatment, we are studying ?-synuclein, a protein of unknown function that has a central role in the pathogenesis of Parkinson's. We have recently found that synuclein inhibits neurotransmitter release by impairing synaptic vesicle mobilization, and will now characterize the mechanism responsible. In addition to exploring a mechanism to prevent the degeneration observed in Parkinson's, the experiments will suggest therapeutic strategies to improve the function of neurons that remain.
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1 |
2010 — 2012 |
Edwards, Robert H |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
2010 and 2012 Membrane Transport Proteins @ Gordon Research Conferences
DESCRIPTION (provided by applicant): All living organisms depend on the transport of inorganic ions and organic solutes across cellular membranes. An extremely diverse collection of membrane transport proteins together account for the flux of a wide range of molecules, including sugars, amino acids, metabolites, peptides, neurotransmitters, toxins and drugs. Further, membrane transport proteins not only contribute to physiologically important processes from nutrient absorption to nitrogen metabolism, renal function and synaptic transmission, but also play a key role in many human diseases from cancer to mental illness. However, technical limitations associated with the analysis of membrane proteins and transporters in particular has previously limited progress in our understanding of these fascinating and important proteins. On the other hand, recent advances in structural biology and functional analysis now enable investigators to address the molecular basis of transport in a physiological context, spurring rapid growth in the field. The long-term objective of this conference is to increase our understanding of the molecular mechanism, physiological role and contribution to disease of membrane transport proteins. This application seeks partial support for the 7th and 8th Gordon Research Conference (GRC) on Membrane Transport Proteins to be held in 2010 and 2012, respectively. To include a large European community focusing on transporters, it has been decided to alternate between European and US venues: the conference will return to the University of New England in 2010 (August 15- 20) and tentatively to Il Ciocco, Italy in 2012. This conference brings together researchers working on many different systems with a wide variety of experimental approaches. In addition to providing exposure to recent research, it will thus foster the exchange of ideas and promote the development of collaborations across fields and methodological disciplines. The sessions planned will cover a broad spectrum of membrane transport proteins including ion pumps, ABC (ATP binding cassette) transporters, amino acid transporters, intracellular transporters, neurotransmitter transporters and ion channels. In addition, the speakers will describe a diversity of techniques and approaches, from structural biology and biophysics to animal models and genetics. Two evening poster sessions will allow all the participants to share the results of their work. The conferences will conclude with a plenary lecture that integrates work on the molecular and cellular analysis of membrane transport proteins with physiology and disease. PUBLIC HEALTH RELEVANCE: This application requests partial support for 2 five-day Gordon Research Conferences in 2010 and 2012, which will focus on membrane transport proteins in normal physiology and disease. To promote our understanding of these fascinating and important but enigmatic proteins, the conferences will bring together investigators from a wide range of fields with diverse experimental approaches. As a result, the conferences will disseminate recent developments in the field, and foster collaboration between investigators who might not otherwise intersect.
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0.915 |
2012 — 2016 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Formation of the Regulated Secretory Pathway @ University of California, San Francisco
DESCRIPTION (provided by applicant): The regulated release of peptide hormones, neural peptides, growth factors and monoamines depends on their storage inside large dense core vesicles (LDCVs) capable of regulated exocytosis. However, we still understand remarkably little about how proteins sort into this regulated secretory pathway (RSP) rather than the constitutive secretory pathway that confers the immediate release of most newly synthesized proteins. In the trans-Golgi network (TGN), proteins destined for LDCVs aggregate to form a dense core, suggesting that lumenal or possibly membrane interactions drive LDCV biogenesis, with proteins destined for other organelles removed during the subsequent process of LDCV maturation. However, we have previously identified a cytoplasmic motif required for the sorting of vesicular monoamine transporter VMAT2 into LDCVs, suggesting a role for cytosolic machinery. Mutations in this motif increase cell surface expression of the transporter, apparently by diverting it from the regulated to the constitutive pathway. Reasoning that a defect in LDCV biogenesis should phenocopy the effect of these mutations in VMAT2, we screened for increased cell surface expression of the transporter in Drosophila S2 cells, which are highly susceptible to RNAi. We find that S2 cells express an RSP and remarkably, Drosophila VMAT (dVMAT) contains the same sorting motif as the mammalian transporter, with mutations in this motif also increasing cell surface expression in S2 cells. Screening 7000 Drosophila sequences conserved to mammals by flow cytometry for increased expression of wild type dVMAT, we identified a small number of genes that affect regulated protein secretion. Focusing on the heterotetrameric adaptor protein AP-3 because two of the subunits scored positive in the screen, we found that loss of AP-3 also dysregulates secretion in mammalian cells. Although LDCVs still form in the absence of AP-3, we find that they lack the proteins such as synaptotagmin required for regulated release. In the first two aims, we will determine how AP-3 contributes to formation of the RSP by testing the hypothesis that AP-3 functions to segregate cargo destined for the RSP, and in its absence, the two secretory pathways mix. We have also found that knockdown of the AP-3-interacting protein VPS41 dysregulates protein secretion, and in the third aim, will test the hypothesis that VPS41 functions as a coat protein for the AP-3 adaptor. We will also extend the analysis to mice lacking AP-3 and VPS41. The results will provide a foundation for future work on the molecular mechanisms involved in LDCV formation and the consequences for physiology, development and disease.
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1 |
2012 — 2016 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] Kreitzer, Anatol (co-PI) [⬀] Sulzer, David Vonzastrow, Mark E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core: Communication @ University of California, San Francisco
Why does this program need a communication/administration core? The original request for proposals (RFP) specifically mandated investigators at different institutions. Indeed, the RFP stipulated that the number of investigators from one institution could not exceed two. For this reason, the original proposal included R. Edwards, R.B. Kelly (both UCSF) and D. Sulzer (Columbia). We were forced to exclude M. von Zastrow (UCSF) because he would have been the third person at UCSF. Since these restrictions were dropped on resubmission, we added M. von Zastrow, but then replaced R.B. Kelly with T. Ryan (Cornell Med), and thus had two investigators in New York City as well as the two in San Francisco, With three independent institutions involved, and two different departments even at UCSF, the program has presented a significant challenge to communication and administration. Although we are now replacing T. Ryan with a UCSF-affiliated investigator, A. Kreitzer holds a position in the private Gladstone Institutes, and his project thus also requires an outside contract, with the associated complications.
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1 |
2012 — 2016 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] Kreitzer, Anatol (co-PI) [⬀] Sulzer, David Vonzastrow, Mark E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 3 - the Exocytotic Release of Dopamine @ University of California, San Francisco
The reward pathway links adaptive behavior to the appropriate environmental cues through changes in the release of dopamine. Drugs of abuse bypass the requirement for adaptive behavior by increasing dopamine directly, triggering plasticity that results in addiction. However, the mechanisms that control dopamine release remain poorly understood. Unlike other classical transmitters, dopamine acts as a neuromodulator, and it has remained unclear how it can convey a signal with the temporal resolution required to associate environmental cues with reward. In addition, dopamine undergoes release from dendrite as well as axon, and dendritic dopamine release has been implicated in plasticity of the reward system- The long-term objective of this proposal is thus to understand how the regulation of dopamine release contributes to the role of dopamine neurons in physiology and behavior. The strategy is to characterize the mechanisms that regulate dopamine release: 1) Determine the relationship between dopamine and glutamate release by midbrain dopamine neurons. Dopamine neurons form glutamate synapses in vitro, suggesting a mechanism for rapid, precise, synaptic signaling by these cells. Using VGLUT2 conditional knock-out mice, we have now demonstrated a dual role for glutamate co-release in vivo in dopamine storage and as an independent signal for postsynaptic neurons. We will now use live imaging of cultured midbrain dopamine neurons to characterize further the relationship between release ofthe two transmitters. Since VGLUT2 is highly expressed by ventral tegmental area (VTA) dopamine neurons early in development, we will also determine how the VGLUT2 cKO influences monoamine release and synapse formation in vitro, and extend the analysis in vivo using brain slices. 2) Characterize the properties of dendritic dopamine release. Using a pHluorin-based reporter for the vesicular monoamine transporter VMAT2, we have obsen/ed exocytotic events in the dendrites of cultured midbrain dopamine neurons. We will now use imaging to assess the role of release from dense core vesicles and endosomes, and its regulation by action potentials, calcium and synaptic input We will also examine the signals on VMAT2 responsible for its trafficking. The analysis of glutamate co-release by dopamine neurons will explore novel exocytotic pathways relevant to the role of dopamine neurons in reward, but also to many other neuronal populations which have recently been shown to mediate co-release. Similarly, the analysis of dendritic dopamine release will illuminate a process that may contribute to retrograde signaling and plasticity at many synapses.
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1 |
2013 — 2014 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Proteomic Analysis of Synaptic Vesicle Pools @ University of California, San Francisco
DESCRIPTION (provided by applicant): Classical studies in the field of synaptic transmission have assumed that neurotransmitter release occurs from a biochemically homogeneous population of synaptic vesicles, but considerable work from many experimental systems has shown that synaptic vesicles belong to pools that differ in their response to stimulation. These observations have given rise to two competing hypotheses, one that the pools are biochemically the same, with differences in behavior strictly stochastic, or extrinsic, reflecting differential association with the cytoskeleton or prior history rather than any intrinsic differences in composition. Alternatively, differences in molecular composition underlie the behavior of different synaptic vesicle pools, and recent work has suggested that the pools may retain their identity after recycling. Although controversial, we have recently shown that different synaptic vesicle proteins respond differently to stimulation, providing some of the first evidence that synaptic vesicle pools differ in composition. However, these experiments involved optical imaging of individual reporter constructs, and understanding how membrane protein composition determines the properties of synaptic vesicles requires a more systematic approach. We will thus label specific synaptic vesicle pools with magnetic nanoparticles strictly on the basis of their response to activity, and determine their composition by quantitative proteomic analysis: Aim 1: Optimize synaptic vesicle recovery from highly purified synaptoneurosomes. Standard procedures fail to recover synaptic vesicles associated with the plasma membrane, so we will optimize synaptic vesicle recovery from synaptoneurosomes using a combination of physical and chemical approaches. Aim 2: Optimize isolation of synaptic vesicles labeled with magnetic nanoparticles during stimulation. We will synthesize small magnetic nanoparticles, and optimize the labeling of different synaptic vesicle pools by different patterns of stimulation. Aim 3: Determine the composition of recycling and resting synaptic vesicle pools by quantitative proteomics using isobaric tag for relative and absolute quantitation (iTRAQ) or stable isotope labeling in mammals (SILAM). Identifying the molecular composition of different synaptic vesicle pools will provide a foundation for future work to explore the functin of the identified components in transmitter release, synapse development and plasticity.
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1 |
2015 — 2021 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] Stroud, Robert M (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structural Basis of Vesicular Neurotransmitter Transport @ University of California, San Francisco
? DESCRIPTION (provided by applicant): The transport of all classical transmitters into synaptic vesicles depends on an outwardly directed H+ electrochemical driving force (?µH+) produced by the vacuolar H+-ATPase. However, vesicular glutamate transport differs from the vesicular transport of other classical transmitters, and relies almost entirely on the electrical component o this gradient (??) rather than the chemical gradient (?pH). Indeed, it remains unclear whethe the vesicular glutamate transporters (VGLUTs) mediate H+ exchange at all. They may simply catalyze facilitated diffusion, or even function as anion channels. In contrast, the closely relate transporter sialin catalyzes the electroneutral cotransport of H+ with sialic acid, and it remains unknown how two members of the SLC17 family can mediate such apparently different activities. However, sialin has also been reported to mediate vesicular glutamate transport, suggesting that the two different activities reflect a common underlying mechanism. The long-term objective of this program is to understand how the SLC17 family confers both ??-driven diffusion and H+ cotransport. The strategy is to determine the structure of proteins in this family and use this information to guide studies of mechanism. Screening a number of bacterial proteins related to the VGLUTs, we have identified one that can be crystallized under a number of different conditions, and that diffracts to 3.7 Å in the lipidic cubic phase. We have also reconstituted the recombinant protein into artificial membranes and shown that it catalyzes the cotransport of an organic anion with H+, similar to sialin. We now propose to 1) refine the structure of DgoT at atomic resolution; 2) determine the structure of DgoT in different functional states, including substrate-bound; 3) test the role of specific residues implicated by the structur in substrate recognition and H+ movement; and 4) determine the structure of a metazoan VGLUT. The results will help us to understand how one class of transport proteins and perhaps even one protein can couple in apparently different ways to the H+ electrochemical driving force. At the same time, structural analysis should illuminate the mechanism for allosteric regulation of the VGLUTs by chloride, which remains poorly understood, and by H+, which we have recently discovered. The identification of mutants with altered properties also provides us with tools to test the physiological role of these properties by genetic manipulation in vitro and n vivo.
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1 |
2017 — 2021 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neurotransmitter Corelease @ University of California, San Francisco
Accumulating evidence shows that many neurons release two classical neurotransmitters, but fundamental questions remain about the cellular basis for corelease, with important implications for its physiological role. In this proposal, we use the vesicular neurotransmitter transporters to elucidate the mechanisms involved in corelease. In previous work, we showed that glutamate corelease by midbrain dopamine neurons serves two distinct roles, one in vesicle filling with dopamine and the other as an independent signal. Although the effects on vesicle filling require colocalization of the vesicular monoamine transporter VMAT2 and vesicular glutamate transporter VGLUT2 on the same synaptic vesicles, anatomy has suggested some segregation as well, but with unclear physiological consequences. We now find that dopamine neurons release glutamate and dopamine with different properties. Release of the two transmitters differs in short-term depression and depends on different presynaptic Ca++ channels. Synaptic vesicles belong to pools that differ in response to stimulation but these differences have been attributed to extrinsic factors such as cytoskeletal association. We now show that they also differ in composition because they contain different transmitters and release them with different properties. Through this mechanism, a single neuron can deconvolve its input into two distinct outputs. The long-term objectives of this project are to elucidate the cellular and molecular basis for neurotransmitter corelease and determine its role in information processing. The strategy is to use the vesicular transporters to characterize the different vesicle populations. Specifically, we will 1) compare monoamine and glutamate release by imaging VMAT2 and VGLUT2 in live neurons; 2) determine how VMAT2 and VGLUT2 target to distinct vesicle populations; 3) characterize the composition of monoamine and glutamate SVs by proteomics. The results will provide basic information about the organization of neurons, with direct relevance for corelease by other cells, but also for release by all neurons.
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1 |
2019 — 2021 |
Edwards, Robert H [⬀] Edwards, Robert H [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Function of Synuclein @ University of California, San Francisco
Signaling in the nervous system depends on the regulated exocytosis of specialized secretory vesicles. Membrane fusion initiates the process of release, but behavior of the pore formed by fusion can control the rate, extent and identity of what is released. Indeed, the fusion pore can reseal before full vesicle collapse into the plasma membrane, potentially trapping unreleased cargo in a form of exocytosis known as `kiss-and-run', a regulatory mechanism well-established for large dense core vesicles (LDCVs), which release neuromodulators. However, the mechanisms that regulate behavior of the fusion pore have remained unclear and its role in the release of classical neurotransmitters from synaptic vesicles (SVs) has been controversial. The actin cytoskeleton and its associated proteins have been suggested to influence pore behavior but the role has remained unclear. The presynaptic protein ?-synuclein has a central role in the pathogenesis of Parkinson's disease (PD). Human genetics shows that mutations in synuclein can cause the disease and the protein accumulates in all patients with the idiopathic disorder. Like other proteins important for neurodegeneration, however, its normal function has remained unknown. Using knockout mice and imaging by light and electron microscopy, we have found that endogenous synuclein normally regulates the fusion pore formed by both LDCVs and SVs, thus influencing the mode of release. The long-term objectives of this program are to understand how synuclein cooperates with other cellular factors to promote fusion pore dilation and how a disturbance in this activity contributes to disease. Since the available methods have limited analysis of the fusion pore and vesicle collapse, we have developed new methods to image the full scope of exocytosis by individual vesicles. Using these, we will study the role of synuclein in pore dilation and membrane collapse by both large dense core vesicles and synaptic vesicles. Specifically, we propose to 1) Characterize the role of synuclein in exocytosis by imaging at high resolution with several complementary methods, including false fluorescent neurotransmitters and Alexa dye entry. 2) Assess the interaction of synuclein with the actin cytoskeleton. Observations in multiple systems have implicated the actin cystoskeleton in exocytosis including pore dilation and we will determine whether synuclein acts through a common or independent mechanism. 3) Determine how the structure of ?-synuclein contributes to its role in exocytosis. We will determine how the N- terminal repeats and C-terminus contribute to normal function. We will test the role of established phosphorylation sites since they may contribute to idiopathic disease by mimicking inherited mutations.
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1 |