Karl J. Clark, Ph.D. - US grants

DESCRIPTION (provided by applicant): It is essential for a living organism to interact with and adjust to the environment by generating responses at molecular, cellular, and system levels. In some cases, these changes result in life-long or even multi- generational changes to physiology and behavior. Understanding the interactions between our genes and the environment is vital to our understanding of addiction. Therefore, molecular and genetic tools to dissect and discover these interactions need to be developed. Of critical importance is the bridge between genetics and behavior that is gated by the stress response. A stress response is a physiological and/or behavioral response to a real or perceived threat or "stressor" that helps maintain or restore normal balance or homeostasis. When a stressor threatens homeostasis, a diverse suite of neuronal, endocrine, and autonomic response mechanisms can be utilized to regain the status quo. In addition to the immediate changes in response to a stressor, long-term changes in the neural network or epigenetic transcriptome can record the molecular memory of this event and thereby alter future responses to similar stressors. The clinical significance of stress-aggravated disorders is extremely high, and can occur when a stressor is too strong (acute), occurs too often (chronic), or is recorded to the epigenome incorrectly and/or during critical developmental periods. The stress response, particularly glucocorticoids, affects the onset, severity, withdrawal, and relapse of addiction. Smoking and thereby nicotine addiction, is the number one preventable cause of death in the world. This proposal will use a genetic model organism, zebrafish (Danio rerio), to study the interaction of the stress response and behavioral sensitization to a psychostimulant- nicotine. Specifically this application will characterize glucocorticoid receptor activation following exposure to nicotine;characterize changes in behavioral sensitization to nicotine caused by stressors, exogenous corticosteroids, or antagonists to the stress response;and perform a screen for modifiers of the stress response. The goal of this proposal is to build a foundation of data to accelerate the use of the genetically tractable, high-throughput zebrafish model to study the interaction of stress and addiction. Innovative elements of this grant use genetic sensor fish to permit spatio-temporal visualization of glucocorticoid receptor activation in all brain tissues in response to drug exposure or other stressors, and a unique mutant library resource of transposon-based insertional mutant zebrafish to do the first forward genetic screen for modifiers of the stress response in a vertebrate. ! PUBLIC HEALTH RELEVANCE: The stress response is vital for adaptation to our environment. Unfortunately, stress can influence many aspects of the addiction process including onset, severity, withdrawal, and relapse. The adaptation of a genetically tractable, high throughput model vertebrate organism, like zebrafish, could lead to the discovery of new genetic pathways that contribute to vulnerability or resilience to addiction and stress.

Abstract Mitochondria have critical normal roles in metabolism, organ homeostasis, apoptosis and aging. Mitochondria play important but still largely mysterious roles in human physiology. Mutations in both nuclear DNA associated with proteins imported into mitochondria as well as mitochondrial DNA (mtDNA) are pathogenic. Despite this clear association of genotype with disease, there are no current treatments for patients with mitochondrial disease. Mitochondria represent a unique cellular compartment with different DNA and RNA repair and editing rules. For example, DNA nucleases that introduce double strand breaks and subsequent repair in nuclear DNA induce the degradation of mtDNA. Indeed, none of the common repair pathways found in the nucleus are active in mitochondria. This proposal uses the well-established TALE-based programmable DNA binding system for targeting mtDNA and the single-tube FUSX TALE assembly system to rapidly generate any protein-based genome engineering reagents. Similarly, we use a new, protein-based and programmable RNA binding system based on PPR proteins, a class of naturally occurring, mitochondrially localized RNA editors from plants. This application harnesses the unique environment of mitochondria to generate a new toolbox to expand the repertoire of tools to edit the human genome (RFA-RM-18-017). To develop these new molecular reagents for mtDNA and mtRNA editing of somatic cells, we will conduct the following aims: I. Develop new classes of mtDNA editing tools. Enhanced approaches to the use of mitoTALENs for preferential degradation of pathogenic mtDNA variants for MELAS and KSS will be developed, including novel nuclearly encoded reporters to detect non-mitochondrial off-targeting gene editing events. A new class of TALE mitochondrial base editors will be developed to directly edit mtDNA for pathogenic variants. II. mtRNA editing tools will be generated through harnessing the PPR family of naturally occurring programmable RNA editors. We will use our new FUSR assembly system to rapidly develop optimal RNA binding reagents, including the fusion to a set of test RNA nuclease or editing protein domains. Errant fusion transcripts in mtDNA deletion or single base variants in heteroplasmic cells will be used as the test paradigm for potential RNA editing platform development with the potential use as a therapeutic. Milestones for initial stages include the establishment of novel mtDNA heteroplasmy converting mitoTALENs against MELAS and KSS followed by testing of the new mtDNA base editor. For mtRNA editing, establishing PPR scaffolding rules for mtRNA binding followed by new programmable RNA nucleases and editors will be established. Deliverables include these novel mtDNA and mtRNA editing systems as well as humans cells with matched nuclearly encoded off-target reporter cassettes for use by any mitochondrial gene editing therapeutic system.

Abstract Intense acute stress or prolonged stress that overwhelms the body's stress response (SR) system is detrimental to an organism's health and associated with the onset or aggravation of a broad spectrum of health outcomes, including psychiatric disorders. To devise effective therapeutic strategies for stress-aggravated disorders, it is essential to advance our understanding regarding the pathways and genes that regulate our body's response to stress. The prevailing thought is that glucocorticoids, like cortisol or corticosterone, primarily act through genomic actions of their cognate receptors, mineralocorticoid (MR) and glucocorticoid receptors (GR), by effecting transcription. However, appreciation of the role glucocorticoids play in rapid non-genomic responses has led to a push to better understand how these non-genomic responses contribute to stress responses, overall stress system regulation, and contributions to health and disease. Identifying and studying gene products that regulate or modify rapid, non-genomic stress responses will significantly impact our understanding of how SR regulation contributes to health, potentially providing new diagnostics and therapeutics to protect against or treat disease. Zebrafish are genetically tractable vertebrates with conserved SR signaling pathways?a combination of properties that make them an ideal model for discovering genetic modifiers of vertebrate- specific SR signaling. In this proposal we intend to clarify the contribution of key regulators of SR signaling, looking at their role in rapid non-genomic signaling as well as their potential to influence development of the SR in vertebrates. We will also follow up on the discovery of novel genes linked to rapid stress responsive behaviors and use a unique resource of zebrafish mutants to discover more genes that influence the vertebrate SR.

The overarching goal of this application is to create tools and efficient methods to define genes that can promote human health. While a tremendous amount of data has been cataloged on gene mutation and changes in gene expression associated with complex human disease, our understanding of those genes that could be co-opted to restore patient health is lacking. To address this need and test for genes that when restored to wild type function promote health, we propose develop mutagenic, revertible and conditional alleles that provide spatial and temporal control of gene expression. The ability to make site-specific, untagged mutant alleles in zebrafish and other models has been greatly advanced by custom nucleases that include TALENs and CRISPR/Cas9 systems. These systems operate on the same principle: they are designed to bind to specific sequences in the genome and create a double strand break. The goals of this proposal leverage the activities of TALEN and CRISPR/Cas9 technologies to make site-specific double strand breaks. These tools and techniques will have direct implications for providing precise gene editing techniques to assess the roles of genes in disease and their ability to promote health following disease progression. While we will develop these methodologies in zebrafish due to their ease of gene delivery, we anticipate these methodologies will not only enhance the efficiency of gene editing but will be readily adaptable for use in other model organisms and large animals. In our opinion, this will have important implications for modeling human disease and health in animal systems by greatly enhancing the ability to make predictable alleles, small nucleotide polymorphisms similar to those associated with human disease, and conditional alleles to test for the ability of a gene to restore health.

Project Summary/Abstract In this Administrative Supplement application, we propose to apply our targeted integration strategy to build a novel in vivo zebrafish model of microglia inflammasome signaling in neuroinflammation. Chronic neuroinflammation driven by microglia activation underlies the pathophysiology of Alzheimer?s Disease (AD) and (PD) and related dementia. a-synuclein secreted by neurons in Parkinson?s patients form aggregates that are taken up by microglia, however, our understanding of the downstream intracellular mechanisms that signal microglia inflammasome activation is incomplete. We will build on our expertise in zebrafish site directed genome engineering and mouse models of neuroinflammation to generate a zebrafish model of conditional active fyn kinase that will allow temporal and spatial induction of microglia inflammasome signaling and activation. Our model will provide an in vivo platform for phenotypic chemical screens that recapitulates the cellular complexity of neuroinflammation. The new tools we develop for microglia specific Cre expression and conditional control of inflammasome signaling will provide an important resource for zebrafish researchers investigating immune function in disease pathogenesis as well as development of translational strategies.