Project Grant F31NS145625

ASTROCYTE CONNEXIN 43 PHOSPHORYLATION IN SEIZURE SUSCEPTIBILITY AFTER TRAUMATIC BRAIN INJURY - PROJECT SUMMARY TRAUMATIC BRAIN INJURY (TBI) AFFECTS ROUGHLY 69 MILLION PEOPLE ANNUALLY AND ABOUT 2.56 MILLION SUBSEQUENTLY DEVELOP POST-TRAUMATIC EPILEPSY (PTE), YET NO THERAPY PREVENTS PTE, HIGHLIGHTING THE NEED TO IDENTIFY EARLY, TARGETABLE MECHANISMS THAT DRIVE EPILEPTOGENESIS. ASTROCYTES MAINTAIN EXTRACELLULAR ION BALANCE AND SUPPLY METABOLIC SUPPORT AND, THROUGH BRAIN-WIDE COUPLING VIA CONNEXIN-43 (CX43) GAP JUNCTIONS, ARE POSITIONED TO SHAPE CIRCUIT EXCITABILITY AFTER INJURY. DESPITE EXTENSIVE STUDY, THE ROLE OF ASTROCYTE COUPLING IN PTE REMAINS CONTROVERSIAL. SOME WORK CLAIMS COUPLING RESTRAINS SEIZURES BY BUFFERING EXTRACELLULAR POTASSIUM, WHEREAS OTHER STUDIES CLAIM COUPLING SPREADS PRO-CONVULSIVE METABOLITES. HOWEVER, THESE PRIOR WORKS DID NOT ASSESS COUPLING'S EFFECTS ON NEURONAL ACTIVITY IN MODELS THAT REPRODUCE TBI-SPECIFIC PATHOLOGY. AT THE MOLECULAR LEVEL, CX43 FUNCTION IS MEDIATED BY PHOSPHORYLATION, AND AFTER TBI OUR LABORATORY OBSERVES INCREASED PHOSPHORYLATION AT SERINE-368 (PS368), A MODIFICATION THAT IN HEART AND KIDNEY CLOSES CX43 CHANNELS AND PRECEDES THEIR INTERNALIZATION AND DEGRADATION. CONSISTENT WITH A PATHOLOGICAL ROLE FOR THIS MODIFICATION, PRELIMINARY DATA REVEALS MICE HARBORING A NON-PHOSPHORYLATABLE S368A SUBSTITUTION ARE PROTECTED FROM TBI- INDUCED SEIZURE SUSCEPTIBILITY EVEN THOUGH THEY DISPLAY FEWER CX43 PLAQUES, A STRUCTURAL READOUT THAT DOES NOT REPORT WHETHER CHANNELS ARE OPEN OR CLOSED; THUS, IT REMAINS UNKNOWN WHETHER PROTECTION REFLECTS PRESERVED FUNCTIONAL COUPLING, ALTERED CX43 TURNOVER, OR BOTH. THIS PROPOSAL TESTS THE HYPOTHESIS THAT TBI PROMOTES SEIZURE SUSCEPTIBILITY THROUGH PS368-MEDIATED ASTROCYTE UNCOUPLING. AIM 1 WILL TEST HOW SELECTIVE CLOSURE OF ASTROCYTIC CX43 GAP JUNCTIONS SHAPES SEIZURE SUSCEPTIBILITY AND PTE. I WILL EXPRESS A TET-ON-INDUCIBLE, DOMINANT-NEGATIVE CX43T154A VIA ASTROCYTE-TROPIC AAV-PHP.EB TO CLOSE CX43 GAP JUNCTIONS IN ADULT C57BL/6 MICE. SEIZURE SUSCEPTIBILITY WILL BE QUANTIFIED WITH A REPEATED SUBCONVULSIVE PENTYLENETETRAZOL PARADIGM AND BEHAVIORAL RACINE SCORING, AND PTE INCIDENCE, LATENCY, AND BURDEN WILL BE MEASURED BY LONG-TERM ELECTROENCEPHALOGRAPHY AFTER DIFFUSE WEIGHT-DROP TBI. AIM 2 WILL DEFINE HOW TBI-INDUCED CX43 PS368 REGULATES ASTROCYTE COUPLING AND CX43 TURNOVER BY COMBINING S368A KNOCK-IN MICE WITH DYE-FILLING ASSAYS OF FUNCTIONAL COUPLING AND BIOCHEMICAL AND IMAGING-BASED MEASURES OF CX43 INTERNALIZATION AND DEGRADATION. TOGETHER, THESE STUDIES WILL ESTABLISH WHETHER S368 PHOSPHORYLATION-MEDIATED ASTROCYTE UNCOUPLING DRIVES POST-TRAUMATIC HYPEREXCITABILITY AND PTE AND WILL EVALUATE CX43 PS368 AND ASTROCYTE NETWORK COUPLING AS MECHANISTIC TARGETS FOR PREVENTING POST-TRAUMATIC EPILEPSY.