. This application is being submitted to PA-18-591 in accordance with NOT-OD-18-194. This work directly addresses INCLUDE research objective component 1, ?Targeted, high risk-high reward, basic science studies in areas highly relevant to Down syndrome (DS)?. The proposed basic science studies directly target a new area of high impact science that is likely to translate into new therapeutic approaches to DS. The topic of emphasis is metabolomic profiling and therapeutic development targeted to mitochondrial dysfunction in existing C. elegans DS invertebrate and novel zebrafish DS vertebrate animal models. Mitochondrial dysfunction causing activation of intracellular stress cascades has been implicated in the pathophysiology of many neurodevelopmental diseases, including Down Syndrome (DS). Indeed, DS patientderived cell lines and animal model studies have demonstrated that increased oxidative stress and defective mitochondrial energy metabolism occurs in DS. In particular, calcineurin/NFAT pathway genes DYRK1A and DSCR1/RCAN1 are major contributors to altered mitochondrial morphology and function in DS, and together serve as negative regulators of calcineurin gene expression. Existing DS model worm strains include two transgenic lines over-expressing DYRK1A(mbk-1) and DSCR1/RCAN1(rcn-1) that display slow growth, reproductive defects, small body size, and altered neurological behaviors, as well as two loss-of-function calcineurin subunit A (cna-1/tax-6(p675)) and calcineurin subunit B (cnb-1(jh103)) strains with defective development and healthspan. Interestingly, translational research studies in these DS worm models have suggested that therapies that improve mitochondrial function and reduce oxidative stress are among the most promising therapies for DS. Therefore, optimizing therapeutic strategies that target mitochondrial dysfunction in DS is essential to identify the safest and most potent drugs to prioritize for clinical trials aimed at improving mitochondrial health and multi-system outcomes in DS patients. Over the past 12 years, we have successfully developed multiple primary mitochondrial RC disease translational model animals using both C. elegans (worms) and D. rerio (zebrafish) to decipher the underlying molecular mechanisms and identify lead therapeutic targets for mitochondrial diseases. This has enabled us to pioneer the development of a robust cadre of phenotypic, biochemical and genetic methods to study, understand, and characterize their mitochondrial pathophysiology and therapeutic effects. In our parent NIGMS R01 research program, we have specifically identified 17 potent lead compounds in 3 major therapy classes (antioxidants, signalling modifiers, intermediary metabolic modifiers), as well as a combinatorial therapy of N-acetylcysteine, nicotinic acid, and glucose, that synergistically rescues the short lifespan of gas- 1(fc21) complex I NDUFS2 subunit mutant C. elegans and prevents brain death in a rotenone-based complex I disease zebrafish model. We hypothesize that these robust methodologies and potent therapeutic leads can now be readily applied to C. elegans and zebrafish DS models to better evaluate their altered mitochondrial physiology and prioritize lead therapeutic candidates. Here, we propose to extend our current knowledge of primary mitochondrial disease therapeutic approaches to perform high-throughput compound screens and mechanistic validation of lead therapies? tolerability and optimal dose in simple invertebrate (worms) and vertebrate (zebrafish) DS animal models. This work will be performed in two Specific Aims:
AIM 1 : To evaluate the therapeutic efficacy of lead mitochondrial disease single and combinatorial therapies in C. elegans DS models;
and AIM 2 : To evaluate mitochondrial disease therapies in a novel zebrafish DS model.
In AIM 1, we will utilize a series of four established C. elegans DS models in the calcineurin signaling pathway in which mitochondrial dysfunction has been previously demonstrated.
In AIM 2, we will use newer CRISPR-on methodologies to generate novel vertebrate DS models in which to screen their overall survival and mitochondrial pathophysiology in multiple organs, targeting both the same calcineurin pathway genes as will be studied in C. elegans, as well as several other genes in the Hsa21 critical region that are known to influence mitochondrial function and oxidative stress. In both DS model systems, we will screen our lead mitochondrial disease single and combinatorial therapies prior identified in primary mitochondrial disease complex I deficient worms and validated in complex I deficient zebrafish. C. elegans treatment efficacy and tolerability in 4 calcineurin pathway mutant worm models will be assessed on major outcomes at the level of gene expression (GFP-based transgenic high-throughput screen), physiologic indicators of DS physiology (growth, brood size, healthspan), neurologic indicators of DS pathology (chemoattractant sensitivity, serotonin-induced egg lay activity), and mitochondrial physiology (high-throughput fluorescence screens of mitochondrial mass, membrane potential, and unfolded protein response; glutathione levels). Zebrafish treatment efficacy and tolerability in a novel CRISPR-on DS model will be studied on larval survival, physiology (neurobehavioral responses, heart rate, activity), and mitochondrial physiology (mitochondrial morphology, mitochondrial mass, membrane potential, ETC enzyme activity, ATP production, oxidative stress) in whole fish and/or different organs (brain, heart, liver, muscle), using standard techniques in which we have demonstrated expertise. All model animal studies are anticipated to be completed in one year. Overall, we postulate that the proposed pre-clinical DS simple model animal screens will offer a robust means to optimize lead therapies that ultimately improve survival, function, or feeling as primary outcomes, which are prioritized by the Food and Drug Administration to pursue in clinical treatment trials in human DS subjects.
Mitochondrial respiratory chain dysfunction occurs in an astonishingly frequent, varied and largely untreatable group of multi-systemic genetic disorders that afflict all ages and ethnicities. Translational research investigations in Caenorhabditis elegans (invertebrate worms) and Danio rerio (vertebrate zebrafish) animal models of RC disease may demonstrate effective combinations of pharmacologic and nutritional therapies, and their specific mechanisms, that restore cell and organ function toward normal to improve overall animal health.
