Project Grant F30EY036720

ELUCIDATING THE ROLE OF THE THOUSAND AND ONE KINASES IN RETINAL GANGLION CELL DEATH - ABSTRACT GLAUCOMA CAUSES IRREVERSIBLE, PROGRESSIVE VISION LOSS VIA AXON INJURY AND SUBSEQUENT CELL DEATH OF RETINAL GANGLION CELLS (RGCS). CURRENT TREATMENTS AIM TO LOWER INTRAOCULAR PRESSURE, ALTHOUGH THIS IS INSUFFICIENT IN SOME PATIENTS AND ASSOCIATED WITH COMPLICATIONS IN OTHERS. AS SUCH, IT IS PROPOSED HEREIN TO DEVELOP COMPLEMENTARY STRATEGIES TO MITIGATE AXON INJURY SIGNALING. FROM PREVIOUS HIGH-THROUGHPUT FUNCTIONAL GENOMIC SCREENS IN RGCS, DUAL LEUCINE ZIPPER KINASE (DLK) AND ITS PARALOG, LEUCINE ZIPPER KINASE (LZK), EMERGED AS CENTRAL PLAYERS IN THE CELL-AUTONOMOUS PATHWAY CAUSING CELL DEATH FOLLOWING AXON INJURY VIA UPSTREAM REGULATION OF JUN N-TERMINAL KINASE 1-3 (JNK1-3). DESPITE DLK/LZK'S ROLE AS MASTER REGULATORS OF RGC CELL DEATH FOLLOWING INJURY, THEIR REGULATION IS POORLY UNDERSTOOD. FURTHER INVESTIGATION OF DLK AND ITS INTERACTIONS HAS THE POTENTIAL TO REVEAL THE UPSTREAM REGULATORY MECHANISMS INVOLVED IN RGC DEATH AND THUS, ADDITIONAL THERAPEUTIC TARGETS. IN AN ADDITIONAL HIGH THROUGHPUT SCREEN OF THE ENTIRE KINOME IN MOUSE RGCS, THE THOUSAND AND ONE (TAO) KINASES, SPECIFICALLY TAOK1 AND TAOK2, SURFACED AS KEY MEDIATORS OF CELL DEATH. COMBINED KNOCKOUTS OF TAOK1 AND TAOK2 PROMOTED ROBUST AND SUSTAINED RGC SURVIVAL IN THE MOUSE OPTIC NERVE CRUSH (ONC) MODEL. THE CENTRAL HYPOTHESIS OF THIS PROPOSAL IS THAT TAOK1/2 WORK AS REGULATORS OF THE DLK/LZK/JNK SIGNALING AXIS AND MEDIATE NEURONAL CELL DEATH IN RESPONSE TO AXON INJURY. THIS HYPOTHESIS WILL BE STUDIED IN A RODENT MODEL AND IN HUMAN STEM CELL-DERIVED RGCS USING PHARMACOLOGICAL AND VIRAL-BASED TOOLS COMBINED WITH CRISPR/CAS9 GENE EDITING TO PRODUCE LOSS- AND GAIN-OF-FUNCTIONS. AIM 1 WILL ELUCIDATE THE RELATIONSHIP BETWEEN THE TAO KINASES AND DLK/LZK/JNK IN AXON INJURY SIGNALING, BY GENETICALLY PROBING THE UPSTREAM/DOWNSTREAM RELATIONSHIP OF THESE KEY KINASES IN VITRO AND IN VIVO IN MICE. AIM 2 WILL EXPLORE THE ROLE OF TAO KINASES IN CELL DEATH SIGNALING USING AN IN VITRO MODEL OF HUMAN RGCS. IF SUCCESSFUL, THE WORK PROPOSED HEREIN WILL SHED LIGHT ON THE ROLE OF THE TAO KINASES IN THE PATHWAY RESPONSIBLE FOR RGC DEATH IN A MOUSE MODEL AND IN HUMAN STEM CELL-DERIVED RGCS. THE NEUROPROTECTIVE POTENTIAL OF TAO KINASE INHIBITION HAS YET TO BE FULLY UNDERSTOOD AND COULD BE LEVERAGED TO MITIGATE RGC DEATH AND VISION LOSS IN GLAUCOMA. FURTHERMORE, WITH THE COMPLETION OF AIM 1, THE PRINCIPAL INVESTIGATOR WILL ACQUIRE NEW SKILLS IN ADENOVIRUS AND AAV PRODUCTION, HISTOLOGICAL AND FUNCTIONAL MEASURES OF RGC NEUROPROTECTION, AND VIRAL/CRISPR-BASED APPROACHES TO MANIPULATING GENE FUNCTION. WITH THE COMPLETION OF AIM 2, THE PRINCIPAL INVESTIGATOR WILL ACQUIRE NEW SKILLS IN HUMAN STEM CELL CULTURE AND RNA SEQUENCING. THE PRINCIPAL INVESTIGATOR WILL GREATLY BENEFIT FROM WORKING UNDER THE DIRECT SUPERVISION OF THE SPONSOR ON THIS PROPOSAL, DR. DEREK WELSBIE, AN EXPERT IN GLAUCOMA AND DLK/LZK SIGNALING, AND THE CO-SPONSOR, DR. KARL WAHLIN, AN EXPERT IN THE DIRECTED DIFFERENTIATION OF STEM CELLS INTO RETINAL TISSUE. ADVISEMENT FROM A COMMITTEE OF EXPERTS IN THE FIELD, CONTINUED COURSEWORK AND ACCESS TO CORE FACILITIES AT UC SAN DIEGO WILL ALSO BE A BOON TO HIS TRAINING AND EFFORTS.