David E. Krantz, MD, PhD - US grants

neurotransmitter transport; phosphorylation; dopamine; synaptic vesicles; membrane activity; acetylcholine; amphetamines; protein localization; neuropharmacology; enzyme inhibitors; serine; neurochemistry; protein sequence; protein kinase; PC12 cells; laboratory mouse; genetically modified animals; tissue /cell culture; mutant; transfection;

DESCRIPTION (provided by applicant): Monoamine neurotransmitters including histamine regulate multiple physiological processes, including gastric acid secretion in humans. In Drosophila, histamine mediates photoreceptor synaptic transmission. In both organisms, monoamine transmitters are released from secretory vesicles during exocytosis. Since transmitter synthesis occurs in the cytoplasm, exocytotic release requires active transport into the vesicle lumen. Specific vesicular monoamine transporters (VMATs) mediate this activity and include the products of two distinct human VMAT genes (VMAT1 in adrenal and VMAT2 in neurons), and one predicted gene in Drosophila. Previously, we have studied the regulation of rat VMAT2, and have identified a dileucine-like signal encoded in the carboxy terminus of VMATs that mediates efficient endocytosis. In addition we have identified two motifs that regulate transporter sorting to secretory vesicles. These include two acidic residues at the -4 and -5 position relative to the dileucine motif, and a distinct cluster of acidic residues at the extreme carboxy terminus of VMAT2. To initiate studies of VMAT in Drosophila, we recently cloned and sequenced cDNAs encoding Drosophila VMAT (dVMAT). Sequence analysis predicts at least two alternatively spliced dVMAT varaints (A and B) that contain different predicted carboxy-terminal cytoplasmic domains. dVMAT-A contains sequences strikingly similar to the dileucine motif and acidic cluster that regulates membrane trafficking of rat VMAT2. The other variant, dVMAT-B, lacks these signals. To test whether alternative splicing of membrane trafficking signals represents a new mechanism for regulating VMAT function, we will now determine the localization of the two variants invivo, and test the function of the potential dileucine motif in dVMAT-A. To determine how changes in dVMAT localization affect transmitter release invivo, we will also screen for dVMAT mutant flies. In future experiments, we will use a dVMAT mutant as a null background to express VMAT variants showing different patterns of intracellular trafficking. Using vision as a behavioral readout, we will determine how changes in dVMAT trafficking affect histamine storage and release. I will also use VMAT mutants in Drosophila as a sensitized background for future genetic screens to identify molecules that regulate VMAT function.

DESCRIPTION (provided by applicant): Monoamine neurotransmitters including histamine regulate multiple physiological processes, including gastric acid secretion in humans. In Drosophila, histamine mediates photoreceptor synaptic transmission. In both organisms, monoamine transmitters are released from secretory vesicles during exocytosis. Since transmitter synthesis occurs in the cytoplasm, exocytotic release requires active transport into the vesicle lumen. Specific vesicular monoamine transporters (VMATs) mediate this activity and include the products of two distinct human VMAT genes (VMAT1 in adrenal and VMAT2 in neurons), and one predicted gene in Drosophila. Previously, we have studied the regulation of rat VMAT2, and have identified a dileucine-like signal encoded in the carboxy terminus of VMATs that mediates efficient endocytosis. In addition we have identified two motifs that regulate transporter sorting to secretory vesicles. These include two acidic residues at the -4 and -5 position relative to the dileucine motif, and a distinct cluster of acidic residues at the extreme carboxy terminus of VMAT2. To initiate studies of VMAT in Drosophila, we recently cloned and sequenced cDNAs encoding Drosophila VMAT (dVMAT). Sequence analysis predicts at least two alternatively spliced dVMAT varaints (A and B) that contain different predicted carboxy-terminal cytoplasmic domains. dVMAT-A contains sequences strikingly similar to the dileucine motif and acidic cluster that regulates membrane trafficking of rat VMAT2. The other variant, dVMAT-B, lacks these signals. To test whether alternative splicing of membrane trafficking signals represents a new mechanism for regulating VMAT function, we will now determine the localization of the two variants invivo, and test the function of the potential dileucine motif in dVMAT-A. To determine how changes in dVMAT localization affect transmitter release invivo, we will also screen for dVMAT mutant flies. In future experiments, we will use a dVMAT mutant as a null background to express VMAT variants showing different patterns of intracellular trafficking. Using vision as a behavioral readout, we will determine how changes in dVMAT trafficking affect histamine storage and release. I will also use VMAT mutants in Drosophila as a sensitized background for future genetic screens to identify molecules that regulate VMAT function.

DESCRIPTION (provided by applicant): Both genes and environmental toxins may act as risk factors for Parkinson's disease (PD), but their potential interplay remains poorly understood. This proposal seeks to investigate gene-environment interactions that are potentially relevant to PD using the model organism Drosophila melanogaster. Attention will be directed on genes and toxins associated with PD that affect mitochondrial dysfunction, protein ubiquitination and dopamine homeostasis. Over-expression of the Drosophila vesicular monoamine transporter (DVMAT) has been shown to protect against the neurotoxic effects of at least one mutant gene associated with PD (parkin), and at least one pesticide (rotenone) that selectively kills dopaminergic neurons. This study will therefore test the hypothesis that the neuroprotective effects of VMAT will extend to a different toxin, paraquat, thought to act by a different mechanism than rotenone, and whether this requires its localization to synaptic vesicles and if it is required at a specific time relative to neurotoxic insults. Studies here have shown that over-expression of mutant forms of parkin can cause dopaminergic cell degeneration in flies. If mutations in parkin function as a risk factor for PD, then additional environmental agents may enhance this risk. This study will test this hypothesis using rotenone, paraquat and other agents. Parallel experiments for gene-environment interactions will be performed with the PD related gene, pink1. Finally, already established pink mutant phenotypes will be used to screen for agents that rescue the neurotoxic effects of mitochondrial dysfunction.

[unreadable] DESCRIPTION (provided by applicant): The vesicular monoamine transporter (VMAT) is responsible for the storage of all monoamine neurotransmitters in the nervous system and inhibition of VMAT by the drug reserpine causes a behavioral state resembling depression. Intracellular trafficking has been proposed to regulate VMAT by controlling its localization to two types of secretory vesicles: synaptic vesicles (SVs) that cluster at the active zone of nerve terminals, and large dense core vesicles (LDCVs) that perform a neuromodulatory role. Although trafficking motifs for VMAT have been identified in culture, the signals required for its localization to SVs and LDCVs in vivo remain unclear. Furthermore, it is not known how trafficking signals in either VMAT or any other neurotransmitter transporter effects either localization or function in an intact animal. We propose to use the model genetic organism Drosophila melanogaster to explore these questions. We have characterized a mutation in the endogenous dVMAT gene, which provides a useful background for the analysis of transgenes containing trafficking mutants. We also have identified two splice variants (DVMAT-A and B) that contain divergent C-terminal trafficking domains and a motif in the C-terminus of DVMAT-A required for endocytosis in vitro. We will now determine how this motif and other potential endocytosis signals contribute to the localization of DVMAT-A to SVs in vivo. We also will identify signals required to sort DVMAT-A to LDCVs. To determine whether the C-terminal trafficking domain of DVMAT-A is required for its function in vivo, we will compare the ability of DVMAT-A, DVMAT-B and a C-terminal truncation to rescue defects in dVMAT mutants that depend on the function of DVMAT-A. Mutation of dVMAT also prevents histamine storage in subretinal glia that express DVMAT-B, thus providing an assay to study the function of this unusual isoform in vivo. We will perform genetic rescue experiments to determine whether the C-terminus of DVMAT-B is required for its function in vivo. To further investigate how the novel trafficking domain in DVMAT-B contributes to its function in vivo, we will determine its subcellular localization in the subretinal glia. These experiments will help determine how a neurotransmitter transporter linked to neuropsychiatric illness is regulated in vivo. The results may aid the development of novel treatment strategies for depression. [unreadable] [unreadable]

The goal of the UCLA-CGEP is to investigate the hypothesis that the cellular mechanisms of action identified for Putative Environmental Toxicants (PETs) contribute to a significant increase in PD risk;this project (Project 2) willfocus on investigations in Drosophila to Investigate the mechanisms of PET action and their interaction with .genetic lesions in the same cellular pathways. Epidemiological and in vitro data from our. group-have shown that exposure to PETs increases the risk of PD and suggested several potential mechanisms by which PETs may exert toxjc effects in dopaminergic.(DA) neurons: the proteaspme, microtubule function, and detoxification by aldehyde dehydrogenase (ALDH): We also have shown that altered expression of the vesicular mohoamine transporter (VMAT) affects the vulnerability of DA neurons to . neurodegeneration. The three Drosophila labs collaborating on Project 2 have extensive experience using Drosophila genetics to model neurodegenerative disorders and the contribution of environmental insults. Here, we propose to use Drosophila genetics to investigate: 1) which of the known biochemical activities of the PETs contribute to DA cell death, 2) whether PETs and genetic lesions in the same biochemical pathway can combine to increase DA cell death, and 3) how manipulation of VMAT affects the neurotoxicity of the PETs. Drugs and toxins that inhibit these processes have pleiotropic effects, and.we propose to precisely define the contribution of each pathway using molecular genetic mimics to inhibit the proteasome, microtubule function, and aldehyde dehydrogenase. The molecular genetic reagents we will use are either already available or readily made and will include RNA interference to knockdown expression of the ubiquitin activating enzyme (E1), misexpression of two well characterized dominant negative mutations.in 20S proteasome subunits, expression in DA neurons of the longest isoform of human tau, and loss of function mutations in Drosophila ALDH. The results of Project 2 and Project 1 will be used to help develop rodent models in Project 3, and^help determine biochemical pathways to be emphasized in the human genetic studies of Project 4. Fly models for PET exposure also will enable us in future aims to evaluate potential neuroprotective strategies.

DESCRIPTION (provided by applicant) This is a renewal of the training grant Training in Molecular Toxicology at UCLA that was first funded in 2008. The training grant has catalyzed the expansion and consolidation of molecular toxicological research and training at UCLA and has stimulated interactions and collaborations among the participating faculty and the pre- and postdoctoral trainees. During the current funding period, the number of training grant faculty members increased from nine to fourteen. The faculty has primary appointments in nine different departments at UCLA. Four of the new appointees are Assistant Professors, and represent potential future leaders of the program. Seven of the faculty members have M.D. degrees. These faculty provides a potential avenue for the recruitment of physician-scientists to postdoctoral (and perhaps also pre-doctoral) positions in the training grant. The training grant requests support for five years and for four pre-doctoral students in the Molecular Toxicology Interdepartment Doctoral Program (IDP) at UCLA, and two postdoctoral trainees in the laboratories of the training grant faculty. Pre-doctoral trainees wil usually be appointed for three years, and postdoctoral trainees will be appointed for up to two years. In general, only pre-doctoral trainees who have completed their first year (during which most do rotations) will be awarded positions on the training grant. This number of trainee positions requested is justified by the increase in the number of our faculty, their substantial NIEHS and non-NIEHS funding, the investigators' previous success in recruiting excellent pre-doctoral and postdoctoral trainees, and the excellent publication record of the trainees. The faculty has a common interest in the mechanisms whereby environmental toxins cause disease. There are four major foci of research interest: i) The role of pesticides in the etiology of Parkinson's disease, ii) genetic damage (including carcinogenesis) caused by environmental agents, (iii) the toxic effects of secondhand cigarette smoke and of fossil fuels and their combustion products, and iv) nanotoxicology. Other areas of research pursued by individual faculty include metal toxicity and the analysis of transcription networks in toxicity. The UCLA Vice Chancellor for Research very recently announced a Grand Challenge initiative. One of the six Challenges focuses on preventing diseases associated with energy, pesticides and environmental technologies. The training grant faculty and trainees are poised to make major contributions to this endeavor. The UCLA Academic Senate undertook an eight year review of the Molecular Toxicology IDP in 2010, and an External Advisory Committee reviewed the training grant in 2011. The Committees were clearly impressed with the IDP and the attainments of the training grant. UCLA has committed considerable resources to the Molecular Toxicology Program, and institutional support is very strong. Over the next five years the NIEHS training grant will further enhance and stimulate molecular toxicological research and training at UCLA, should encourage more students and postdoctoral fellows to pursue research in toxicology, and will signal the increasing importance of toxicology as a discipline at UCLA.

? DESCRIPTION (provided by applicant): Vesicular monoamine transporters (VMATs) are required for the storage and exocytotic release of all aminergic neurotransmitters. The mechanisms by which VMATs target to secretory vesicles remain unclear and the potential behavioral consequences of disrupting their localization are unknown. We are using the model organism Drosophila melanogaster to address these questions. The mutations we have generated thus far decrease the localization of Drosophila VMAT (DVMAT) to Synaptic Vesicles (SVs) and increase its localization to Large Dense Core Vesicles (LDCVs). The behavioral sequelae of these mutations provide some of the first information on the function of amine release from SVs versus LDCVs. We will now generate additional mutations to decrease the localization of DVMAT to LDCVs. These mutants will be useful for further behavioral tests and also help to define fundamental trafficking mechanisms in neurons. Additional experiments will use these mutants to define the poorly understood differences between trafficking in aminergic versus non-aminergic neurons and to determine how changes in amine release affect pre- and post-synaptic function in aminergic circuits. Further behavioral experiments will explore the affects of altered amine release in more complex behaviors and the response to aminergic drugs. The results of these experiments will be significant because they examine processes relevant to conserved neuromodulatory processes and the clinical effects of aminergic drugs. They are innovative because they exploit several new assays and because no other lab has examined the in vivo effects of mis-trafficking for a vesicular transporter, or the behavioral effects of changing the way neurotransmitters are released from particular vesicle types.

? DESCRIPTION (provided by applicant): Similar to the mammalian hippocampus, Drosophila mushroom bodies (MBs) are critical for learning and memory. Surprisingly, the neurotransmitter(s) stored and released by the intrinsic neurons of the MBs, the Kenyon cells (KCs) are not known. We recently identified a novel vesicular neurotransmitter transporter, portabella (prt) that is expressed in KCs. We propose to use prt as a tool to identity the neurotransmitter stored in KCs. We will use two complementary methods. In Aim 1, we will perform in vitro transport assays using cells that express prt. In Aim 1A, we will test transport o radiolabeled candidates. Aim 1B describes a backup plan using a technique that does not require radiolabeled compounds. Since 1B is more risky than 1A it will only be employed if both Aim 1A and Aim 2 fail. In Aim 2 we will assay the lumenal contents of secretory vesicles using electrochemical detection and mass spectrometry. In Aim 2A we will biochemically fractionate secretory vesicles. We hypothesize that the small molecule stored by prt in wt flies will be reduced in vesicles derived from the prt mutant. As a backup plan (Aim 2B), we will assay the exocytotically released contents of secretory vesicles from cultured KCs. We hypothesize that a small molecule released into the media by wt KCs will not be released by prt mutant neurons. Candidate molecules identified in Aim 2 (or 1B) will be directly tested in transport assays as in Aim 1A. The results of these experiments will determine the prt substrate. Since prt is the only vesicular neurotransmitter that is expressed in KCs, these experiments will identify the neurotransmitter(s) that is stored in KCs.

ABSTRACT We propose to use a model circuit in Drosophila to study aminergic neuromodulation. The relatively small number of presynaptic cells combined with easy access to both pre- and post-synaptic sites will allow us to quantify the activity of each aminergic neuron, their functional relationships to post-synaptic targets and how their activities are coordinated. Recently developed molecular genetic tools will allow us to map all of the relevant receptors for the first time. New methods of preparing the tissue will allow us to identify the function of both direct and indirect regulatory pathways. Using RNAi transgenes as well as classical mutations we will define the function of all of the aminergic and glutamatergic receptors in the circuit and how they interact. Preliminary Data have uncovered unexpected complexities in this relatively simple circuit that will be relevant to a variety of other more complex circuits less amenable to the detailed analysis we propose here.