Project Grant F30AG099672

INVESTIGATING REDUCTIVE STRESS AS A MECHANISM OF NEURONAL VULNERABILITY TO AGE-RELATED NAD DECLINE - ABSTRACT NAD IS AN ESSENTIAL COFACTOR OF LIFE, YET IT DECLINES WITH AGE. NEURONS ARE PARTICULARLY VULNERABLE TO LOSS OF NAD, AND THUS NAD LOSS MAY BE A MECHANISTIC PRINCIPLE LINKING AGING TO NEURONAL VULNERABILITY IN AGE-ASSOCIATED NEURODEGENERATIVE DISEASE. MOST THERAPEUTIC STRATEGIES HAVE FOCUSED ON DIRECTLY BOOSTING NAD LEVELS, AS OPPOSED TO TARGETING THE DOWNSTREAM CONSEQUENCES OF NAD LOSS. THIS IS LIKELY FOR TWO REASONS: NAD LOSS DISRUPTS A WIDE RANGE OF CELLULAR PROCESSES, AND THE CONSEQUENCES OF NEURONAL NAD LOSS HAVE NOT BEEN FULLY IDENTIFIED. A CRISPR-CAS9 KNOCKOUT SCREEN WAS PERFORMED TO IDENTIFY THE MOST SIGNIFICANT PATHWAYS DOWNSTREAM OF NAD LOSS REGULATING NEURONAL VULNERABILITY. THIS SCREEN TOOK ADVANTAGE OF A GENETIC PARADIGM OF NEURONAL DEATH CAUSED BY THE NADASE SARM1. TO MIMIC CHRONIC, AGE-RELATED NAD DECLINE, VACOR, A PHARMACOLOGICAL TOOL INDUCING LOW-LEVEL SARM1 ACTIVATION, WAS USED. THE DRUG SURVIVAL SCREEN IDENTIFIED THE OXIDOREDUCTASE CYB5R4 AS A POTENT HIT. CYB5R4 KNOCKOUT (KO) ROBUSTLY PROTECTS AGAINST DEATH, WHILE CYB5R4 OVEREXPRESSION ENHANCES DEATH. IMPORTANTLY, CYB5R4 KO PROTECTS DOWNSTREAM OF NAD DEPLETION, INDICATING THAT LOSS OF NAD IS NOT SUFFICIENT FOR DEATH IN THIS MODEL. FURTHER METABOLOMIC AND GENOMIC EXPERIMENTS REVEALED A ROLE FOR REDUCTIVE STRESS, AKIN TO AND OPPOSING OXIDATIVE STRESS. I HYPOTHESIZE THAT REDUCTIVE STRESS, CATALYZED BY CYB5R4, PLAYS A CAUSAL ROLE IN MEDIATING NEURONAL VULNERABILITY TO AGE-RELATED NAD LOSS. TO TEST THIS HYPOTHESIS, I WILL USE IN VITRO AND IN SILICO METHODS. MY EXPERIMENTS PROPOSED IN AIM 1 WILL DISSECT THE MECHANISM BY WHICH NAD LOSS LEADS TO REDOX STRESS, USING INDUCED PLURIPOTENT STEM CELL-DERIVED HUMAN CORTICAL NEURONS (INEURONS). I WILL DEFINE SOURCES OF REDOX STRESS UPON LOSS OF NAD BY 1) LIVE-CELL IMAGING OF ORGANELLE-TARGETED SENSORS, AND 2) MASS-SPECTROMETRY-BASED METABOLOMICS, LEVERAGING THE PHENOTYPE OF CYB5R4 KO AND OVEREXPRESSION. AIM 2 WILL EXPLORE THE THERAPEUTIC VALUE OF TARGETING REDUCTIVE STRESS ACROSS VARIOUS MODELS OF NEURONAL NAD DECLINE. I WILL FIRST USE GENETIC TOOLS TO DIRECTLY INHIBIT REDUCTIVE STRESS AND ASSESS PROTECTION IN NADASE-DEPENDENT DEATH IN INEURONS. THEN, I WILL TEST FOR THE TRANSCRIPTOMIC PRESENCE OF REDUCTIVE STRESS IN ALZHEIMER'S DISEASE PATIENT BRAINS, EXPLORING PROTEINOPATHY AS A DOWNSTREAM OUTCOME OF REDUCTIVE STRESS IN NEURONS. THESE EXPERIMENTS WILL ELUCIDATE A NOVEL METABOLIC VULNERABILITY OF NEURONS BY IDENTIFYING THE CAUSES AND CONSEQUENCES OF REDUCTIVE STRESS IN THE CONTEXT OF NAD DECLINE. IF SUCCESSFUL, THIS PROJECT WILL PROVIDE INSIGHT INTO MECHANISMS DRIVING NEURODEGENERATION IN NATURAL AND PATHOLOGICAL AGING.