Robert Edwards - US grants

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.

Within recent years, the United States' excess electrical generating capacity has diminished. In order to provide a dependable supply of electricity in the face of less generating margin, responsiveness, availability, and reliability of existing power plants need to be improved. To provide this improvement, a Bailey NETWORK 90 distributed microprocessor based control system is requested which will be interfaced with other computer capabilities at Penn State in order to create a multi-user laboratory for research in the field of intelligent distributed control for electric power generation. The new and unique research laboratory will be attuned with the current evolutionary trend in commercial power plant dynamic control system organization. In addition to digital forms of classical control algorithms, the full capabilities of digital control systems depend on implementation of advanced software applications: automation of routine equipment monitoring, process fault diagnostics, effective and reliable communication, rule-based programming, and modern control algorithms. The research need is to fully develop these software capabilities in the distributed control environment in order to achieve the desired improvement in power plant performance. Advanced control research for electric power generation requires an interdisciplinary approach and researchers from the Electrical, Mechanical, Industrial, and Nuclear Engineering departments will be involved.

This award, of $390,000, is for research and curriculum development for power plant intelligent distributed control. Many new specialized skills in applying simulation, computer networking, and microprocessor-based controller programming have been acquired by the faculty and graduate students that have been choosing this research. In order to accelerate the achievement of the goal of improving power generation reliability, economy, and safety through the implementation of modern control technologies, an efficient mechanism is needed for teaching new graduate students and industry specialists the required skills for conducting applied research in this area. For this purpose, current research will be expanded to plant-wide system implementation and an introductory graduate level course will be developed over a three year period. The course will later be suitable for advanced undergraduates that are going to graduate school or will be employed in the power industry.

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.

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.

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.

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.

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.

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.

SUBPROJECT ABSTRACT NOT AVAILABLE

Substantial research suggests that the prevalence of persistent pain and sensitivity to certain laboratory pain stimuli increase with advancing age. These effects may be due to a decline in the efficacy of pain-inhibition in older adults. The goal of the proposed research is to characterize potential age-associated decrements in endogenous pain modulation. The specific aims of this project are: 1) to investigate the existence and magnitude of such decrements; 2) to examine potential physiological mechanisms; 3) to determine their clinical relevance; and 4) to elucidate the role of endogenous opioids in pain inhibition. Eighty subjects (40 younger, 40 older) will undergo assessment of diffuse noxious inhibitory controls (DNIC), a phenomenon which refers to the decrease in perceived pain from a noxious stimulus produced by concurrent application of a second noxious stimulus. Induction of DNIC is a frequently-used methodology for assessing the integrity of endogenous pain-modulatory systems in humans. DNIC will be assessed under conditions of double blind administration of naloxone (producing opioid blockade) or saline (placebo control). The proposed project will broaden an extant program of research investigating senescent alterations in pain responses and will provide important and novel information regarding age-related alterations in endogenous pain modulation.

DESCRIPTION (provided by applicant): The candidate's long-term career objectives relate to understanding the mechanisms underpinning psychosocial factors' impact on pain-related outcomes. This Career Development Award (K23) will provide the candidate the necessary training to develop this research program. Specific training aims of this K23 include development of expertise in the psychoneuroimmunology of pain and in-vivo assessment methods. The proposed research plan examines biopsychosocial aspects of pain in rheumatoid arthritis (RA), a chronically painful disorder with well-documented psychosocial and biological components. Among the most consistently important of the cognitive and affective variables impacting on the experience of pain is catastrophizing. While catastrophizing has been identified as an important predictor of chronic pain and disability, little is known regarding the mechanisms by which it produces its deleterious effects. Current theory suggests that 2 of the pathways through which catastrophizing operates are: (1) recruitment of social responses that reduce self-management of pain, and (2) direct negative effects on pain-regulatory pathways and other neurophysiological systems. Understanding catastrophizing's mechanisms of action will assist in the development of interventions designed to reduce its impact, improving the health-related quality of life of individuals with chronic pain. The proposed research plan involves parallel studies in patients with RA; a prospective observational study will examine the social dimensions of catastrophizing while a laboratory- based study will concurrently evaluate its psychophysiological and immunological consequences. The need for further research into catastrophizing's mechanisms of action is increasingly important in the context of an epidemic of chronic pain. The candidate's goal for the proposed project is to understand the pathways by which catastrophizing impacts pain-related outcomes in order to refine biopsychosocial models of pain and facilitate the development or enhancement of psychosocial interventions for chronic arthritis pain.

Abstract Not Provided.

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.

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.

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.

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.

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.

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.

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.

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.

? 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.

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.

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.

Chronic pain affects over 100 million American adults and >40% of older adults age ?65, with estimated costs of >$500 billion to $1 trillion annually. Older adults are at increased risk of having multiple painful conditions and are living longer with the negative impacts of chronic pain. Chronic pain, regardless of anatomy or diagnosis involved (e.g., back pain, migraine), is the leading cause of disability worldwide. Limited insights into whether common mechanisms underlie all pain conditions, regardless of diagnosis, has contributed to inadequate pain management options, and in turn, to the opioid epidemic. Health care providers who treat one pain condition (e.g., joint pain) typically do not manage symptoms in other parts of the body (e.g., abdominal pain), and patients are often referred from one specialist to another. Studying commonalities of various chronic overlapping pain conditions (COPC) in community-based cohorts unselected for pain conditions can provide novel insights into causes and consequences of chronic pain as a disease itself. For example, neurophysiologic alterations in pain processing such as pain sensitization (assessing ascending pain pathways) and conditioned pain modulation (CPM) (assessing descending pain modulation) may be common mechanisms underlying all chronic pain. Such knowledge would spur development of novel pain management approaches for all types of pain regardless of anatomy or diagnosis involved. Despite the substantial public health burden of chronic pain, little is known about the epidemiology and evolution of COPC in older adults, nor of the impact of COPC on physical function, risk of nursing home admission, and mortality in older adults. We propose to evaluate chronic pain in the upcoming study visit of a thoroughly-characterized community-based older adult cohort unselected for any pain complaints, the Framingham Heart Study (FHS) (N~1874, mean age 76, 84% ?age 70). We aim to understand the epidemiology of COPC, regardless of anatomy or diagnosis involved, the evolution of COPC over time, neurophysiologic alterations in pain processing as risk factors for COPC and its evolution, and consequences of COPC and altered pain processing in older adults. We propose acquiring objective quantitative sensory testing (QST) measures of pain sensitization and CPM, which are associated with pain severity and response to treatment in experimental settings of subjects selected for particular pain conditions. However, whether QST- assessed neurophysiologic alterations may be common underlying risk factors for all forms of chronic pain, and for functional limitations, institutionalization, and mortality in a community-based cohort of older adults unselected for pain is unknown. We will collect data regarding a multitude of chronic pain conditions, QST, self-report and performance-based physical function, and nursing home admissions during the next planned study visit, as well as two follow-up assessments to obtain longitudinal data, and leverage the ongoing FHS surveillance of mortality. Our work will address several knowledge gaps, and insights gained may ultimately facilitate new preventive and therapeutic approaches to relieving chronic pain and its consequences, regardless of underlying diagnosis.

Chronic pain affects over 100 million American adults, with an estimated cost of $560-635 billion annually, yet this major burden is relatively understudied in African Americans (AAs). Older adults are at increased risk of having multiple painful conditions and are living longer with the negative impacts of chronic pain. Chronic pain, regardless of anatomy or diagnosis involved (e.g., back pain, migraine), is the leading cause of disability worldwide. Limited insights into whether common mechanisms underlie all pain conditions, regardless of diagnosis, has contributed to inadequate pain management options. Health care providers who treat one pain condition (e.g., joint pain) typically do not manage symptoms in other parts of the body (e.g., abdominal pain), and patients are often referred from one specialist to another. Studying commonalities of various chronic overlapping pain conditions (COPC) in community-based cohorts unselected for pain conditions can provide novel insights into causes and consequences of chronic pain as a disease itself. For example, neurophysiologic alterations in pain processing such as pain sensitization (assessing ascending pain pathways) and conditioned pain modulation (CPM) (assessing descending pain modulation) may be common mechanisms underlying all chronic pain. Such knowledge would spur development of novel pain management approaches for all types of pain regardless of anatomy or diagnosis involved. Despite the substantial public health burden of chronic pain, little is known about the epidemiology and evolution of COPC in older AA adults, nor of the impact of COPC on physical or psychosocial function, risk of nursing home admission, and mortality in older AAs. We propose to evaluate chronic pain in the upcoming study visit of a thoroughly-characterized community-based older AA cohort unselected for any pain complaints, the Jackson Heart Study (JHS) (N~2540, two-thirds ?age 65). We aim to understand the epidemiology of COPC, regardless of anatomy or diagnosis involved, the evolution of COPC over time, neurophysiologic alterations in pain processing, healthy lifestyle factors and psychosocial factors as risk factors for COPC, and consequences of COPC and altered pain processing in older adults. We propose acquiring objective quantitative sensory testing (QST) measures of pain sensitization and CPM, which are associated with pain severity in experimental settings. Whether QST-assessed neurophysiologic alterations may be common underlying risk factors for all forms of chronic pain, and for poor physical and psychosocial functioning, institutionalization, and mortality in a community-based cohort of older AAs unselected for pain is unknown. We will collect data regarding a multitude of chronic pain conditions, QST, healthy lifestyle factors, physical and psychosocial function, and nursing home admissions during the next planned study visit, as well as two follow-up assessments to obtain longitudinal data, and leverage the ongoing JHS surveillance of mortality. Our work will address several knowledge gaps, and insights gained may ultimately facilitate new preventive and therapeutic approaches to relieving chronic pain and its consequences, regardless of underlying diagnosis.

Abstract Pain is the most common symptom leading patients to consult a physician in the United States, and its negative effects on quality of life can be substantial. Chronic pain is associated with both direct (e.g., health care) and indirect costs (e.g., lost wages, disability days) that have been estimated to range from $560-635 billion annually. Although considerable progress has been made in understanding the pathophysiologic mechanisms of pain, the most widely prescribed medications for acute and chronic pain ? non-steroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics ? have major drawbacks, including modest efficacy and significant risks that can limit long-term use. Consequently, there is a compelling public health need for the development of pain treatments with improved efficacy and safety. Millions of Americans also suffer from addiction or receive anesthesia for surgical procedures, and although there are efficacious treatments available in each of these additional therapeutic areas, many existing interventions have only modest efficacy or have incompletely characterized safety risks. The primary objective of the Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks (ACTTION) public-private partnership is to identify, prioritize, sponsor, coordinate, and promote innovative activities that will expedite the discovery and development of improved analgesic, anesthetic, and addiction treatments for the benefit of the public health. The major activities of the ACTTION partnership include conducting systematic reviews, consensus meetings, and methodologically-focused research studies as well as developing and qualifying novel clinical outcome assessments and biomarkers, with the aim of increasing the assay sensitivity and informativeness of clinical trials. The knowledge gained from these efforts will accelerate the development of novel medications and other treatments for pain, anesthesia, and addiction that are more effective and safer than existing treatments.

Chronic pain affects >100 million American adults with estimated costs of up to $1 trillion annually. Older adults are at increased risk of having multiple painful conditions and are living longer with the negative impacts of chronic pain. Chronic pain, regardless of anatomy or diagnosis involved (e.g., back pain, migraine), is the leading cause of disability worldwide. Limited insights into whether common mechanisms underlie all pain conditions, regardless of diagnosis, has contributed to inadequate pain management options, and in turn, to the opioid epidemic. Health care providers who treat one pain condition (e.g., joint pain) typically do not manage symptoms in other parts of the body (e.g., abdominal pain), and patients are often referred from one specialist to another. Studying commonalities of various chronic overlapping pain conditions (COPC) in community-based cohorts unselected for pain conditions can provide novel insights into causes of chronic pain as a disease itself, and into shared risk factors that may be promising targets to help ameliorate chronic pain regardless of diagnosis. Alterations in nociceptive signaling such as pain sensitization assessed by quantitative sensory testing (QST) may commonly underlie chronic pain, but why such alterations occur is not known, and whether such nociceptive signaling changes are heritable is also not known. Beyond nociception, there may be broader nervous system dysfunction underlying chronic pain. Generalized heightened sensitivity to external stimuli (e.g., light, sound) and impaired autonomic nervous system functioning, reflected by diminished heart rate variability (HRV), are associated with chronic pain, but it is unclear if they contribute to COPC, QST-assessed abnormalities, or development of chronic pain. Treatments targeting these potential risk factors could represent new avenues for pain management. Another untapped potential is in understanding whether positive factors such as resilience, sleep quality, and physical activity can be harnessed to alter risk of COPC or QST abnormalities. We propose evaluating COPC in the upcoming study visit of a community-based middle age and older adult cohort unselected for any pain complaints, the 3rd Generation of the Framingham Heart Study (FHS) (N~3374, mean age 60). We aim to understand the relation of multisensory sensitivity, autonomic function, resilience, sleep, and physical activity to COPC, QST-assessed pain processing and evolution of chronic pain over time; and to study heritability of QST abnormalities. Understanding the relation of these novel factors to COPC and QST would spur development of novel pain management approaches for all types of pain regardless of diagnosis involved. We will collect data regarding common chronic pain complaints, QST (to assess pain sensitization), and proposed risk factors, and conduct two follow-up assessments to obtain longitudinal data. We will leverage the ongoing FHS Offspring (2nd Generation) Exam in which we are collecting the same QST measures to assess heritability of pain processing abnormalities. Our work will address several knowledge gaps, and insights gained may facilitate new approaches to relieving chronic pain and its consequences, regardless of underlying diagnosis.