Project Grant R21AG088501

IN VIVO SCREENING METHODS TARGETING LEVELS OF TAU PROTEIN WITHIN MOUSE BRAINS - PROJECT SUMMARY/ABSTRACT NEURODEGENERATIVE DISEASES ARE DEVASTATING, FATAL DISEASES THAT AFFECT MILLIONS OF PEOPLE WORLDWIDE AND HAVE NO DEFINITIVE TREATMENT. A WELL-KNOWN NEURODEGENERATIVE DISEASE, ALZHEIMER'S DISEASE (AD), IS THE THIRD MOST COMMON LEADING CAUSE OF DEATH, WITH THE AD ASSOCIATION IN 2022 REPORTING MORE THAN 6 MILLION PEOPLE AFFECTED IN US. ALTHOUGH THERE ARE NO CLEAR UNDERLYING MECHANISMS OF PATHOGENESIS, ACCUMULATION OF TOXIC PROTEIN SPECIES, SUCH AS TAU PROTEIN IN AD, DRIVES A LARGE AND DIVERSE GROUP OF NEURODEGENERATIVE DISEASES, BOTH GENETIC AND SPORADIC. MODULATING LEVELS OF DISEASE-DRIVING PROTEINS HAS IMPROVED DISEASE PHENOTYPES, HIGHLIGHTING A PROMISING STRATEGY FOR THERAPEUTIC DEVELOPMENT. LARGE-SCALE CELL-BASED GENETIC PERTURBATION SCREENS ARE CANONICAL AND CRITICAL TOOLS USED TO FIND ESSENTIAL PROTEIN REGULATORS THAT CONTROL LEVELS OF DISEASE- DRIVING PROTEINS. HOWEVER, SINCE THESE 2D MODELS CONSIST OF ACTIVELY DIVIDING CELLS WITH VASTLY DIFFERENT PROPERTIES THAT LACK PHYSIOLOGICAL AUTHENTICITY COMPARED TO IN VIVO CELLS, IMPORTANT BRAIN-SPECIFIC REGULATORS ARE NOT PRESENT AND THUS NOT DETECTED AND FALSE POSITIVES ARE MORE LIKELY TO BE DETECTED. THIS RESULTS IN AN INEFFICIENT PROCESS REQUIRING MULTIPLE ROUNDS OF LABOR-INTENSIVE IN VITRO ASSAYS FOLLOWED BY VALIDATION USING IN VIVO MODELS BEFORE ANIMAL STUDIES CAN BE PERFORMED THAT WILL HOPEFULLY LEAD TO VALID THERAPEUTIC TARGETS. THE GOAL OF THIS PROPOSAL IS TO BYPASS CELL-BASED SCREENS BY DEVELOPING A FEASIBLE AND HIGH- THROUGHPUT GENOME-WIDE IN VIVO CRISPR/CAS9 SCREENING METHOD (AIM 1). FURTHERMORE, TO ENHANCE TRANSLATION AND AS A MEANS OF VALIDATION, THIS METHOD WILL BE USED TO FIND REGULATORS OF TAU PROTEIN LEVELS IN THE MOUSE BRAIN (AIM 2). AIM 1 WILL SET THE PARAMETERS FOR IN VIVO SCREENING USING INTRACEREBROVENTRICULAR (ICV) INJECTION OF THE ADENO-ASSOCIATED VIRUS (AAV) HARBORING GRNA/CAS9 INTO NEONATAL MICE TO BROADLY PERTURB TARGET GENES IN THE BRAIN. FLUORESCENCE ACTIVATED CELL SORTING (FACS) ANALYSIS WILL THEN BE UTILIZED TO TRACK PROTEIN LEVELS IN EACH DISSOCIATED SINGLE CELL IN A HIGH THROUGHPUT MANNER. AIM 2 WILL EXPAND THE IN VIVO SCREENING METHOD INTO A TAUOPATHY MOUSE MODEL, WHICH RECAPITULATES AD-RELATED PHENOTYPES AND INVOLVES THE PATHOGENIC ACCUMULATION OF TAU PROTEIN IN TWO BRAIN REGIONS (CEREBRAL CORTEX AND STRIATUM) VULNERABLE TO NEURODEGENERATION. THE IMPACT OF THIS PROPOSAL IS MANIFOLD: PROVIDING A POWERFUL TOOL TO BYPASS LABOR-INTENSIVE IN VITRO PROCESSES FOR THE IDENTIFICATION OF NEW THERAPEUTIC TARGETS FOR NUMEROUS NEURODEGENERATIVE DISEASES; PROVIDING COMPREHENSIVE INSIGHT INTO THE REGULATION OF TAU AND REVEAL CANDIDATE THERAPEUTIC TARGETS FOR TAUOPATHIES; ENHANCING OUR UNDERSTANDING OF HOW DISEASE-CAUSING PROTEINS ARE DIFFERENTLY REGULATED DEPENDING ON THE BRAIN REGION, IN NORMAL PHYSIOLOGICAL CONDITIONS VERSUS DISEASE CONDITION, WHICH WILL PROVIDE INSIGHT INTO REGIONAL VULNERABILITY/RESILIENCE TO NEURODEGENERATION. TAKEN TOGETHER, COMPLETION OF THIS ENDEAVOR WILL PROVIDE A NOVEL AND POWERFUL TOOL TO UNDERSTAND THE REGULATION OF DISEASE-DRIVING PROTEINS AND WILL HELP US DISCOVER PROMISING THERAPEUTIC TARGETS THAT ARE VALIDATED IN THE BRAIN, MAKING THE STRATEGY APPLICABLE TO MANY NEUROLOGICAL DISEASES.