2011 — 2021 |
Grill, Brock |
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. |
Mechanisms of Synapse Formation and Axon Termination in C. Elegans
DESCRIPTION (provided by applicant): Synapses are the connections that transmit information flow in the brain. Several diseases and conditions that result in a loss of synapses or synaptic function can be thought of as diseases of the synapse including neurodegenerative diseases, such as Alzheimer's disease, and trauma to the central nervous system from stroke. While disease-specific therapies will be helpful, broad based therapies such as those that trigger new synapse formation or stabilize existing synapses will also be extremely valuable potentially slowing disease progression, or improving recovery following trauma or disease onset. To our knowledge there is no pharmaceutical that specifically triggers new synapse formation or stabilizes existing connections. Achieving this milestone remains a primary, pressing and urgent goal of the medical community. The first step in achieving this goal is to understand how nature builds a synapse, allowing the identification of the best therapeutic targets. The long-term goal of our research program is to identify and understand the molecular players that orchestrate synapse formation, and integrate synapse formation with other key neurodevelopmental processes, such as axon termination. Importantly, synapse formation is an evolutionarily conserved process that occurs in simple invertebrates, such as the worm C. elegans, through human beings. Thus, molecules that are critical to synapse formation will also be evolutionarily conserved. Using C. elegans as a model system, we aim to rapidly and efficiently identify conserved molecules that function in synapse formation and axon termination. While we are a long way from fully understanding how a synapse is built and maintained, it is important to emphasize that many of the molecules that are known to regulate this process were identified using C. elegans. One such molecule that regulates synapse formation, as well as axon termination, guidance and regeneration is the Regulator of Presynaptic Morphology (RPM)-1. While its key and central role as a neurodevelopmental regulatory protein potentially makes RPM-1 an ideal therapeutic target, we still have very limited knowledge on how RPM-1 functions. To gain insight into RPM-1's mechanism of action, we have recently performed a proteomic screen to identify proteins that bind to RPM-1. In this proposal, we aim to study two novel, conserved RPM-1 binding proteins that we identified in our proteomic screen, NPP-17 and T23F11.1. We will use transgenics, genetics and cell biology in C. elegans to determine if NPP-17 and T23F11.1 function in synapse formation and axon termination. We will also determine if NPP-17 and T23F11.1 mediate RPM-1 function, and how NPP-17 and T23F11.1 relate to pathways that are known to act downstream of RPM-1. Importantly, both T23F11.1 and NPP-17 are conserved molecules with no known function in neurons. Thus, understanding the neuronal function and mechanisms of action for these molecules will bring us significantly closer to the goal of understanding how to build a synapse, and the ultimate goal of pharmacologically manipulating this process for maximum therapeutic impact.
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2011 — 2016 |
Grill, Brock |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Molecular and Proteomic Analysis of Neurodevelopmental Signaling in C. Elegans @ University of Minnesota-Twin Cities
At present, we have a relatively limited understanding of how the human nervous system develops. The complexity of our brains, which are composed of billions of neurons that make trillions of cellular connections called synapses, has made this a very important, but difficult problem to understand. The simpler nervous systems of invertebrates, such as flies and nematodes, allow us to study the basic and evolutionarily conserved mechanisms responsible for nervous system development in a less complex and more manageable model system. The nematode Caenorhabditis elegans has emerged as an exceptionally powerful system for studying how neurons develop and form their intercellular connections (synapses). Our proposal uses biochemical, genetic and proteomic approaches to study the molecular processes that regulate neuronal and synaptic development. Specifically, we will utilize a previously characterized presynaptic protein called rpm-1 to identify additional molecules that interact with rpm-1 to facilitate neuronal and synaptic development. The successful completion of our project will lead to new insights into the molecular mechanisms that control development of the nervous system. Given that the molecules we study are evolutionarily conserved from C. elegans to human beings, the knowledge we gain will also be highly applicable to our understanding the mechanisms that drive development of the human brain. Because C. elegans is a non-parasitic nematode, is small in size, and is easy to manipulate, it is an ideal organism for teaching students about nervous systems function. Our research will be directly integrated with educating the community about neuroscience through outreach efforts directed at elementary, secondary and post-secondary students.
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2015 — 2016 |
Grill, Brock Martemyanov, Kirill A. |
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.) |
A Transgenic Model of Opioid Tolerance and Drug Discovery
? DESCRIPTION (provided by applicant): Opioid drugs are the most widely used analgesics in medicine, and are also some of the most widely abused substances. Tolerance to opioids is a major medical problem resulting in reduced efficacy of these compounds for treatment of chronic pain, and increasing the potential for addiction and abuse. In this proposal, we detail an innovative, potentially transformative approach to reveal insight into the mechanisms of opioid tolerance, and propose to develop a humanized, transgenic platform for discovery of small molecules that block or reduce tolerance. Our approach aims to find drugs and drug targets that block tolerance, thereby greatly increasing the efficacy of existing blockbuster pharmaceuticals. Blocking or reducing tolerance to opioids would also be a major step towards preventing dependence. We propose to use the nematode, C. elegans, as a genetic model system to study signaling by a G protein coupled receptor (GPCR) that mediates analgesic and addictive responses to opioids, the µ-opioid receptor (MOR). We will generate transgenic C. elegans that express human MOR and respond to its stimulation by opioids with behavioral changes. By using a series of cell-specific promoters, we propose to analyze two major types of behavior: chemosensation and locomotion. Further, we will develop assays to study behavioral tolerance caused by prolonged exposure to MOR agonists. In this proposal, we take aim at developing a humanized, transgenic platform to study opioid-induced tolerance with the potential to provide significant and unbiased insight into the mechanisms of tolerance and its chemical attenuation. Because our proposal lets the biology of a living organism provide a solution to the problem of tolerance, our approach is a significant advance over previous efforts that have focused on specific molecular targets using cell-based assays that have, unfortunately, had limited success. The microscopic size of C. elegans and the scalability of the assays we propose to develop are simply not possible in mammalian or vertebrate whole organismal systems further supporting our choice of model system. The successful implementation of our proposal has the potential to be transformative to how drug addiction is studied. Successful implementation of this proposal will lay the ground for the discovery of novel small molecules and genes that regulator of tolerance. The success of our work will encourage the development of humanized C. elegans high throughput behavioral platforms for many other addiction targets including cocaine, methamphetamine, and nicotine.
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2019 — 2021 |
Grill, Brock Martemyanov, Kirill A. |
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. |
Molecular Genetic Mechanisms of Opioid Receptor Signaling
Summary Opioid drugs are the most widely used analgesics in clinic, and are also some of the most widely abused substances. The adverse actions of these drugs, including peripheral side effects, dependence and tolerance, severely limit their utility as prescription analgesics for long term pain management. The µ-opioid receptor (MOR) is the primary target of the analgesic and rewarding effects of opioids. Thus, efforts aimed at developing safer and more effective opioid treatments will require a much deeper understanding of MOR signaling. Our long-term goal is to use unbiased forward genetics to dissect the molecular organization of the MOR signaling network using whole-animal behavioral responses to opioids as a phenotypic readout. Towards this goal, we developed a transgenic MOR model (tgMOR), in which mammalian MOR is expressed in the nervous system of the nematode C. elegans. We found that tgMOR animals gain the ability to respond to opioids, and exhibit all the cardinal behavioral hallmarks of opioid responses seen in higher organisms including acute depressant effects, desensitization and tolerance. We further demonstrated key known molecular players that control opioid responsiveness in mammals play conserved functions in tgMOR worms. Taking advantage of this model, we completed an unbiased, forward genetic screen for modifiers of behavioral opioid sensitivity, and isolated a large number of mutants with altered opioid responses. We have developed a pipeline for discovery, identification and validation of genes responsible for phenotypes using a combination of whole genome sequencing, mapping and targeted CRISPR/Cas9 gene editing. Using this approach, we uncovered several known and novel genes that regulate opioid responsiveness in worms, and confirmed their effects on MOR signaling using cell-based assays with cultured mammalian cells. Our findings suggest an elaborate, largely unknown, network of players exists to regulate MOR signaling. Thus, the main effort of this project focuses on identifying and characterizing these players by analyzing tgMOR mutants isolated from our unbiased, forward genetic screen. Our first aim will be to identify the genes responsible for 1) hypersensitivity, 2) hyposensitivity, and 3) impaired tolerance by pursuing a subsets of mutants from each phenotypic category. In the second aim, we will validate and perform mechanistic studies on identified, conserved regulators of MOR signaling using a comprehensive platform of cell-based assays that monitor various aspects of MOR signaling. The third aim will focus on exploring the pharmacogenomics by which MOR impacts behavior. To do so, we analyze interactions between genetic MOR variants found naturally in the human population, FDA-approved opioid drugs, and different genetic backgrounds using a humanized tgMOR C. elegans platform. It is anticipated that these studies will advance our understanding of how opioids act thereby paving the way to the development of safer opioid therapeutics.
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