Kathryn E. McCormick, Ph.D. - US grants

DESCRIPTION (provided by applicant): Actions can be adaptive or maladaptive. Thus, deciding which action to pursue often has a significant impact on health and well-being. Not surprisingly, therefore, the nervous system has evolved the means to evaluate actions, enhancing the adaptive ones and suppressing the maladaptive ones. But the neural mechanisms underlying action selection are poorly understood. The proposed research addresses this problem in the context of foraging behavior in the nematode C. elegans for two main reasons. First, this widely used model organism offers many unusual experimental advantages including a compact nervous system of only 302 neurons, a complete neuroanatomical wiring diagram, and a wide range of electrophysiological and optophysiological techniques for linking the activity of identified neurons to behaviors. Second, C. elegans foraging presents in simplified form one of the most fundamental issues in action selection, namely the question of how the nervous system associates the value of the outcome of an action with the particular behavior that caused it, without also reinforcing other behaviors at the same time. The findings from these studies are likely to accelerate the analysis of action selection in other organisms, including humans, by revealing simple neural circuits that will serve as road maps for more complex ones. PUBLIC HEALTH RELEVANCE: The long-term objective of the proposed research is to understand how the nervous system combines sensory information with internal knowledge of ongoing behavior to promote beneficial actions. This is a basic research question with significant implications for health because mental illness frequently manifests as a deficit in action selection and other forms of decision making. Approximately 65% of all human disease genes have a counterpart in C. elegans, including genes implicated in psychiatric conditions that effect decision making such as schizophrenia and bipolar disorder. Thus, findings from the proposed research are likely to provide insights into the molecular mechanisms of action selection.

Project Summary / Abstract Genome wide DNA sequencing is now being adopted in clinical practice and an increasing number of variants are identified in epilepsy-associated genes, yet the clinical interpretation of the new variants is challenging. Some of the variants are known to be either pathological or benign, yet a majority of the gene variations remain unknown for their functional consequence. A large number of Variant of Uncertain Significance (VUS) are becoming commonplace in genes for human diseases, providing a significant barrier in making diagnoses and implementing therapies. Bioinformatic approaches can provide some insight into pathogenic probability of VUS alleles, but functional studies in animal model systems are often needed to make definitive of pathogenicity assignments. The expense and long timelines of mouse model production make the use of alternative small animal models attractive. In this proposal, the ?C. elegans?nematode is used as an alternative model capable of fast high-throughput production and screening. Human genes are installed as gene-swap replacements of the native disease-gene homologs. In preliminary work, gene-swap humanization of STXBP1 in the ?unc-18?locus rescued severe locomotion and behavior defects present in the gene knock-out animals. Pathogenic variants into the STXBP1??gene-swap loci leads to significant disruption of activity. In this proposal, significant and novel improvements are made to our existing pipeline for the functional analysis of variants in vivo. In Aim 1, the relevance and extensibility of the C. elegans model system for studying human disease is improved through simultaneous humanization of multiple related loci. In Aim 2, new methods are developed for molecular phenotyping, improving the resolution of inputs pathogenicity determination algorithms, and yielding mechanism-of-action level readouts to variant manipulations. In Aim 3, the improvements to the pipeline are tested to quantify gains in pathogenicity determination on a test set of variants.

Summary This supplement funding request is relevant for the development of chemical medical countermeasures to neurotoxin exposure. Specifically, we use a humanized animal model to provide greater relevance and translation of drugs as antidotes to the neurotoxic effects of exposure to aldicarb, a pesticide which causes potentially lethal buildup of acetylcholine (ACh) at the synapse. In the parent SBIR award, we are creating a polygenic humanization of C. elegans wherein three human genes involved in vesicular release of ACh (SNAP25, STXBP1 and STX1A) replace their orthologous loci (ric-4, unc-18 and unc-64) in a single transgenic line. The resulting polygenic humanized animal will be used as a platform for functional categorization of pathogenic genetic variants when patient-derived variants are installed. As a supplementary project, the double-humanized hSTXBP1/hSTX1A humanized animals, which we have already created, and a tiple-humanized hSNAP25/hSTXBP1/hSTX1A will be used as a platform for discovery of drugs that act as antidotes to aldicarb neurotoxicity. The human proteins functionally replace the worm orthologs, restoring regulated neurotransmitter release from the synapse. Treatment with aldicarb inhibits synaptic ACh esterase (AChE) and leads to build up of ACh at cholinergic synapses. This paralytic effect of aldicarb is well documented in prior C. elegans work. Yet, most common antidotes to aldicarb work via a diverse set of mechanisms, which have not been well characterized in C. elegans. As a result, we focus on four distinct mechanisms: (1) acting upstream to decrease presynaptic release of ACh (botulinum toxin A - Botox, (2) acting downstream to inhibit ACh receptors (atropine), (3) acting directly on the aldicarb-blocked AChE by promoting enzyme reactivation (pralidoxime), and (4) reducing GABA transmission which alleviates convulsive symptoms (diazepam). In this work to establish an aldicarb antidote discovery system, two aims are sought. The first aim will be to establish aldicarb paralytic activity that can be easily detected in the humanized animal models. The second aim will be to measure the antidote effects of four drugs (atropine, pralidoxime, diazepam and Botox) on aldicarb-induced paralysis. Success of the project occurs when an assay protocol is found capable of detecting the effects of an antidote in reducing aldicarb toxicity. In future work, this humanized system can be used as a screening platform for aldicarb antidotes as well as a broad range of neurotoxic agents that threaten human health.