Project Grant R21NS138718

CONVERGING MOLECULAR MECHANISMS IN AXON DEGENERATION AND ASTROCYTE MORPHOGENESIS - SUMMARY ASTROCYTES ARE ONE OF THE MAJOR GLIAL SUBTYPES OF THE CENTRAL NERVOUS SYSTEM (CNS), EVOLUTIONARILY CONSERVED FROM DROSOPHILA TO HUMANS WITH REGARD TO THEIR MORPHOLOGY, MOLECULAR COMPOSITION, AND FUNCTION. ALTHOUGH THEIR MORPHOLOGICAL COMPLEXITY IS THOUGHT TO BE ESSENTIAL FOR ASTROCYTES TO PERFORM THEIR MYRIAD ROLES IN THE CNS BY PHYSICALLY CONTACTING AND INTERACTING WITH DIVERSE NEURAL STRUCTURES, INCLUDING SYNAPSES AND THE VASCULATURE, WE ONLY KNOW OF A HANDFUL OF PATHWAYS THAT CRITICALLY REGULATE ASTROCYTE MORPHOLOGY. FROM AN UNBIASED, FORWARD GENETIC SCREEN IN DROSOPHILA, WE SURPRISINGLY IDENTIFIED DSARM AND DWNK/FRAY, BOTH OF WHICH HAVE BEEN IMPLICATED IN AXON DEGENERATION, AS POTENTIAL NEW REGULATORS OF ASTROCYTE MORPHOLOGY. PRELIMINARY FINDINGS IN DROSOPHILA SHOW THAT DSARM/SARM1 AND DWNK/WNK WORK SYNERGISTICALLY TO REGULATE NOT ONLY AXON DEGENERATION BUT ALSO ASTROCYTE MORPHOLOGY. HERE, WE PROPOSE TO LEVERAGE THE LARVAL ZEBRAFISH SYSTEM TO ASK WHETHER THE ROLE OF SARM1 AND WNK IN REGULATING ASTROCYTE DEVELOPMENT IS EVOLUTIONARILY CONSERVED. IN THE AXON DEGENERATION PATHWAY, NAD+ DEPLETION IS THOUGHT TO BE UPSTREAM OF DSARM/SARM1 AND DWNK/WNK ACTIVATION, AND AXUNDEAD (AXED) TO BE DOWNSTREAM. WHEREAS THE IMPORTANCE OF NAD+ DEPLETION UPSTREAM OF DSARM/SARM1 AND DWNK/WNK HAS BEEN SHOWN ACROSS SPECIES, THE FUNCTIONAL ORTHOLOG OF AXED IS UNKNOWN IN VERTEBRATES. WE THEREFORE PROPOSE TO DETERMINE IF AND HOW THESE KNOWN AXON DEGENERATION PATHWAYS UP- AND DOWNSTREAM OF DSARM/SARM1 AND DWNK/WNK ALSO FUNCTION IN VERTEBRATE ASTROCYTES. BY HARNESSING THE PHARMACOLOGICAL AND GENETIC TOOLKITS AVAILABLE IN LARVAL ZEBRAFISH, WE WILL INTERROGATE THE INVOLVEMENT OF THE NAD+ BIOSYNTHETIC PATHWAY IN CONTROLLING PROCESS DYNAMICS AND MORPHOLOGY OF ASTROCYTES IN VIVO. WE WILL ADDITIONALLY PERFORM A TARGETED REVERSE GENETIC SCREEN TO IDENTIFY THE FUNCTIONAL VERTEBRATE ORTHOLOG OF AXED IN MEDIATING SARM1/WNK EFFECTS ON ASTROCYTE MORPHOLOGY. TOGETHER, THIS STUDY WILL DEFINE NEW ROLES FOR KEY MOLECULES IN AXON DEGENERATION IN THE REGULATION OF ASTROCYTE MORPHOGENESIS AND FURTHER OUR UNDERSTANDING OF THE SARM1 SIGNALING PATHWAY IN VIVO.