Project Grant K99NS133484

IMMUNE REGULATION OF SPINAL CORD REGENERATION - PROJECT SUMMARY/ABSTRACT SPINAL CORD INJURY IN MAMMALS TRIGGERS A CASCADE OF CELLULAR EVENTS THAT LEAD TO THE LOSS OF SENSORY AND MOTOR FUNCTION CAUDAL TO THE SITE OF INJURY. FOLLOWING SPINAL CORD INJURY, IMMUNE CELLS, INCLUDING MICROGLIA AND MACROPHAGES, INFILTRATE INTO THE LESION SITE AND BECOME ACTIVATED. DEPLETING MICROGLIA AND MACROPHAGES IN MAMMALIAN SYSTEMS HAS SHOWN BOTH BENEFICIAL AND DETRIMENTAL EFFECTS POST-INJURY. IDENTIFYING THE SPECIFIC REGENERATIVE IMMUNE REQUIREMENTS IN MAMMALIAN SYSTEMS HAS PROVEN DIFFICULT DUE TO A COMPLEX COMBINATION OF ANTI-REGENERATIVE BARRIERS. IN CONTRAST, ZEBRAFISH SPONTANEOUSLY REGENERATE A FULLY SEVERED SPINAL CORD AND PROVIDE A PLATFORM FOR IDENTIFYING PATHWAYS NECESSARY FOR SPINAL CORD REGENERATION. THE ZEBRAFISH IMMUNE SYSTEM IS CONSERVED WITH MAMMALS, AND THEREFORE PROVIDES A UNIQUE SYSTEM TO IDENTIFY PRO-REGENERATIVE IMMUNE PATHWAYS. IN PRELIMINARY DATA, I FOUND MICROGLIA AND MACROPHAGES ARE NECESSARY FOR FUNCTIONAL AND ANATOMICAL RECOVERY POST-INJURY, BUT THE PATHWAYS DIRECTING MICROGLIA/MACROPHAGE-DEPENDENT SPINAL CORD REGENERATION ARE NOT KNOWN. MICROGLIA AND MACROPHAGES ARE HIGHLY PLASTIC CELLS, AND THEIR GENE EXPRESSION AND BEHAVIOR HAVE DIRECT IMPLICATIONS ON FUNCTIONAL OUTCOMES FOLLOWING NEURAL INJURY. THIS PROPOSAL WILL IDENTIFY MICROGLIA/ MACROPHAGE-SPECIFIC CELLULAR IDENTITIES, GENE EXPRESSION, AND PATHWAYS THAT ARE NECESSARY FOR SPINAL CORD REGENERATION IN THE ADULT ZEBRAFISH. FIRST, TWO OF THE MOST IMPORTANT FUNCTIONS OF MICROGLIA AND MACROPHAGES FOLLOWING SPINAL CORD INJURY ARE TO DIRECT THE HEALING OF INJURED TISSUE AND CLEAR THE LESION SITE OF CELLULAR DEBRIS. AIM 1 (K99 PHASE) WILL UTILIZE LOSS-OF-FUNCTION MUTANTS TO DEFINE GENES UPSTREAM OF WOUND HEALING THAT ARE NECESSARY FOR RE-ESTABLISHING IMMUNE PRIVILEGE OF THE SPINAL CORD AFTER INJURY. AIM 2 (K99/R00 PHASE) WILL MOVE FROM THE ADULT ZEBRAFISH SPINAL CORD TO A HUMAN CELL CULTURE SYSTEM TO VISUALIZE BEHAVIOR IN HUMAN IPSC-DERIVED MICROGLIA AND TEST THE CONSERVATION OF PRO-REGENERATIVE GENE FUNCTION IN HUMAN CELLS. LASTLY, THE ORIGIN OF IMMUNE CELLS WILL DICTATE THEIR CELLULAR FUNCTION AND EFFECT ON REGENERATION, AND THE ORIGINS OF PRO-REGENERATIVE MICROGLIA AND MACROPHAGES ARE UNKNOWN. IN AIM 3 (R00 PHASE), I WILL PERFORM LINEAGE TRACING IN THE ADULT ZEBRAFISH REGENERATING SPINAL CORD TO CHARACTERIZE THE ORIGIN OF EXPANDING IMMUNE CELLS POST-INJURY. THESE AIMS ARE DESIGNED TO APPLY MY STRENGTHS IN ZEBRAFISH GENETICS AND REGENERATION TO THE NEW FIELD OF NEUROIMMUNOLOGY. TO FACILITATE MY ABILITY TO CARRY OUT THESE PROPOSED EXPERIMENTS, I HAVE ASSEMBLED A TEAM OF ADVISORS AND COLLABORATORS, TAKING ADVANTAGE OF THE VIBRANT NEURAL INJURY AND NEUROIMMUNOLOGY COMMUNITIES AT WASHINGTON UNIVERSITY SCHOOL OF MEDICINE. THIS PROPOSAL WILL GENERATE NOVEL TOOLS AND PROTOCOLS TO MEASURE THE IMMUNE EVENTS DURING SPINAL CORD REGENERATION AND OFFERS A FOUNDATIONAL NICHE IN THE SPINAL CORD INJURY FIELD THROUGH WHICH I CAN LAUNCH A FUTURE TENURE-TRACK RESEARCH FACULTY POSITION. ADDITIONALLY, WORK PROPOSED HERE WILL IDENTIFY PRO-REGENERATIVE PATHWAYS THAT HAVE DIRECT RELEVANCE TO HUMAN HEALTH AND PROVIDE POTENTIAL THERAPIES FOR HUMAN SPINAL CORD INJURY PATIENTS.