Project Grant R01EY036028

CAMKII: RETINAL GANGLION CELL NEUROPROTECTION AND AXON REGENERATION - EXPRESSION OF ACTIVATED FORMS OF THE MULTIFUNCTIONAL TYPE II CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE (CAMKII) WERE RECENTLY DEMONSTRATED TO CONFER ROBUST RETINAL GANGLION CELL (RGC) NEUROPROTECTION IN MOUSE MODELS OF OPTIC NERVE INJURY AND NMDA EXCITOTOXICITY, MAKING CAMKII A LEADING TARGET FOR THERAPEUTIC INTERVENTION IN GLAUCOMA. HOWEVER, NEW PRELIMINARY DATA SHOW THAT EXPRESSION OF CONSTITUTIVELY ACTIVE CAMKII (CACAMKII) NOT ONLY PROMOTES RGC SURVIVAL AFTER OPTIC NERVE CRUSH (ONC) INJURY, BUT ALSO SUPPRESSES AXON REGENERATION. AS AXON REGENERATION WILL BE A NECESSARY FEATURE OF ANY POTENTIAL TREATMENT FOR GLAUCOMA THAT WOULD RESTORE VISION, A BETTER UNDERSTANDING OF THE MECHANISMS HOW CACAMKII CONFERS NEUROPROTECTION WILL BE IMPORTANT FOR THE SUCCESSFUL DEVELOPMENT OF CAMKII-DIRECTED THERAPEUTICS. WE HYPOTHESIZE THAT THE PHOSPHORYLATED EFFECTORS DRIVING CAMKII-DEPENDENT NEUROPROTECTION AND AXON GROWTH REPRESSION ARE DIFFERENT. IN THIS PROJECT, WE WILL DERIVE STRATEGIES THAT PROMOTE RGC SURVIVAL, BUT NOT PREVENT AXON REGENERATION BY INVESTIGATING HOW DIFFERENTIAL COMPARTMENTATION AND ACTIVATION CONFER CAMKII PHOSPHORYLATION OF RELEVANT EFFECTORS AND THEIR RESPECTIVE CELLULAR PROCESSES. IN PARTICULAR, WE HYPOTHESIZE THAT INDUCTION OF CREB AND/OR NF-KB GENE EXPRESSION BY NUCLEAR CAMKII AND POTENTIALLY CAMKIV IS REQUIRED FOR RGC SURVIVAL, WHILE AS AN OFF-TARGET EFFECT, CACAMKII EXPRESSION ELSEWHERE IN THE CELL SUPPRESSES AXON GROWTH. SPECIFIC AIM 1: THE ROLE OF ENDOGENOUS CAMKII IN RGC NEUROPROTECTION AND AXON SUPPRESSION. IN THIS AIM, WE WILL USE KNOCK-OUT MICE TO TEST WHETHER ENDOGENOUS CAMKII AND CAMKIV EXPRESSION IS REQUIRED FOR MAINTENANCE OF BASAL RGC SURVIVAL OR FOR NEUROPROTECTION AFTER ONC. IN ADDITION, WE WILL TEST WHETHER CAMKII KNOCK-OUT WILL PROMOTE AXON REGENERATION. BY AAV-MEDIATED EXPRESSION OF CAMKII-ACTIVATING PEPTIDES, WE WILL DIFFERENTIATE BETWEEN ALTERNATIVE MECHANISMS FOR CACAMKII'S NEUROPROTECTIVE EFFECTS: REPLACEMENT OF INSUFFICIENTLY EXPRESSED CAMKII OR ACTIVATION OF INSUFFICIENTLY ACTIVE ENDOGENOUS CAMKII. IN PARALLEL TO ASSAY FOR NEUROPROTECTION, WE WILL STUDY THE ACTIVITY OF CAMKII IN VIVO BY TRANSPUPILLARY IMAGING OF A CAMKII FLUORESCENT REPORTER. SPECIFIC AIM 2: COMPARTMENT-SPECIFIC CACAMKII NEUROPROTECTION AND SUPPRESSION OF AXON REGENERATION. WE WILL TEST WHETHER EXOGENOUSLY EXPRESSED CACAMKIIA FUSION PROTEINS LOCALIZED TO DIFFERENT INTRACELLULAR DOMAINS SELECTIVELY PROMOTE NEUROPROTECTION OR SUPPRESS AXON REGENERATION AFTER OPTIC NERVE INJURY. CONVERSELY, WE WILL TEST WHETHER LOCALIZED EXPRESSION OF CAMKII INHIBITORY PEPTIDES WILL RESCUE CACAMKII-SUPPRESSED AXON REGENERATION WITHOUT INHIBITING NEUROPROTECTION. SPECIFIC AIM 3: CAMKII-DEPENDENT RGC GENE EXPRESSION CONFERRING NEUROPROTECTION. WE HYPOTHESIZE THAT ACTIVATION OF CAMKII SIGNALING CONFERS NEUROPROTECTION BY INDUCTION OF CREB- AND NF-KB-DEPENDENT GENE EXPRESSION. SCRNA-SEQ AND SCATAC-SEQ WILL BE USED TO IDENTIFY GENES AND REGULATORY ELEMENTS ASSOCIATED WITH NEUROPROTECTION, WHILE CREB1 AND RELA RGC-SPECIFIC KNOCK-OUT MICE WILL BE STUDIED FOR RGC SURVIVAL AND AXON REGENERATION.