Scintillon Institute

CELLULAR/MOLECULAR PATHOPHYSIOLOGY OF MENTAL RETARDATION

RAMAN FLOW CYTOMETRY FOR DIAGNOSTICS AND DRUG DISCOVERY

THE ROLE OF GENETIC RISK FACTORS AND IMMUNE RESPONSE ON NVU FUNCTION IN POST-TBI COGNITIVE IMPAIRMENT.

LA JOLLA INTERDISCIPLINARY NEUROSCIENCE CENTER CORES

DEFINING THE FUNCTIONAL ORGANIZATION OF CEREBELLAR OUTPUT CIRCUITS THAT CONTROL FEEDING BEHAVIOR - PROJECT SUMMARY IN ADDITION TO MOTOR AND CLASSICAL CONDITIONING FUNCTIONS, THE CEREBELLUM CONTRIBUTES TO MOTIVATION AND REWARD PROCESSES THAT UNDERLIE COMPLEX BEHAVIORS. TO INFLUENCE NON-MOTOR PROCESSES, SUCH AS FEEDING AND FOOD- SEEKING BEHAVIORS, IT IS THOUGHT THAT THE CEREBELLUM MODULATES CORTICAL AND SUBCORTICAL FEEDING CENTERS. THE ONLY PATH THROUGH WHICH THE CEREBELLUM CAN INFLUENCE FEEDING CONTROL IS THROUGH CEREBELLAR OUTPUT CIRCUITS IN THE DEEP CEREBELLAR NUCLEI (DCN). YET LITTLE IS KNOWN ABOUT HOW DCN CIRCUITS ARE ORGANIZED AND WHETHER DISTINCT PATHWAYS ARE DEDICATED TO FEEDING AND FOOD-SEEKING BEHAVIORS. THE RECENT IDENTIFICATION OF DISCRETE SUBSETS OF DCN NEURONS THAT PROJECT TO THALAMIC, SUBTHALAMIC AND HYPOTHALAMIC BRAIN REGIONS INDICATES THE EXISTENCE OF NEURAL SUBTYPE ORGANIZATION TO CEREBELLAR OUTPUT. BASED ON PUBLISHED AND PRELIMINARY DATA, THE PRIMARY HYPOTHESES OF THIS PROPOSAL ARE THAT: 1) DISTINCT DCN-MEDIATED PATHWAYS PROJECT TO KNOWN FEEDING CENTERS TO INFLUENCE FOOD INTAKE; AND 2) THESE FEATURES IDENTIFY DISTINCT DCN CIRCUITS ESSENTIAL FOR FEEDING AND/OR METABOLISM; AND FINALLY, 3) DEDICATED DCN-MEDIATED PATHWAYS ARE ENGAGED DURING FEEDING, AND INFLUENCE THE NEURAL ACTIVITY OF SPECIFIC NEURONAL SUBTYPES IN KEY FEEDING CENTERS. THIS PROPOSAL WILL TEST THESE HYPOTHESES THROUGH THREE AIMS. AIM 1 DELINEATES DISTINCTIONS IN TARGET SELECTIVITY OF SPECIFIC DCN CIRCUITS. WE WILL EMPLOY CONDITIONAL VIRAL TRACING, AND GENETIC FATE-MAPPING METHODS TO DEFINE THE OUTPUT CONNECTIVITY OF DCN SUBPOPULATIONS TO FEEDING CENTERS (PARAVENTRICULAR NUCLEUS, LATERAL HYPOTHALAMUS, ARCUATE NUCLEUS AND ZONA INCERTA), WHICH WE HYPOTHESIZE INFLUENCE FEEDING BEHAVIOR. ADDITIONALLY, WE WILL DETERMINE IF MAJOR SUBCLASSES OF ARCUATE NEURONS (E.G. POMC OR AGRP) ARE LINKED TO THE DCN WITH SPECIFIC CRE-LINES AND TRANS-SYNAPTIC RABIES VIRUS. IN AIM 2, WE WILL DEFINE THE ROLE OF DCN CIRCUITS IN FEEDING CONTROL THROUGH OPTOGENETIC ACTIVATION AND SILENCING OF DISCRETE NEURONAL SUBPOPULATIONS IN THE DCN. SPECIFICALLY, WE WILL EXAMINE HOW SELECTIVE NEURAL MANIPULATION OF ANATOMICALLY- DEFINED DCN PATHWAYS INFLUENCES FOOD INTAKE AND METABOLISM, AND DISSOCIATE OUTPUT PATHWAYS FOR MOTOR CONTROL. FINALLY, THE EXPERIMENTS IN AIM 3 WILL DETERMINE THE ACTIVITY PROFILE OF DISCRETE DCN NEURONAL SUBPOPULATIONS, AND HOW ACTIVITY IN THESE SUBPOPULATIONS CHANGES NEURAL ACTIVITY OF KNOWN FEEDING CIRCUITS IN FREELY MOVING MICE DURING FOOD INTAKE USING DEEP-BRAIN IMAGING. BY DEFINING THE ANATOMICAL AND FUNCTIONAL ORGANIZATION OF CEREBELLAR OUTPUT PATHWAYS, AND THEIR ACTIVITY DYNAMICS INVOLVED IN FEEDING BEHAVIOR, THESE AIMS PROVIDE INSIGHT INTO MORE GENERAL MECHANISMS OF HOW CEREBELLUM CONTROLS MOTIVATION AND REWARD CIRCUITS, AND ESTABLISH A FRAMEWORK FOR EXPLORING THE MORE ENIGMATIC COGNITIVE ROLES OF THE CEREBELLUM. A MORE COMPREHENSIVE UNDERSTANDING OF CEREBELLAR FUNCTION WILL PROVIDE GREATER INSIGHT INTO HOW NEUROLOGICAL DISORDERS AND INJURIES DISRUPT FOOD INTAKE, AND LAY THE GROUNDWORK FOR DEVELOPMENT OF NOVEL TREATMENT STRATEGIES FOR OBESITY AND EATING DISORDERS.

EXPANDING THE SET OF GENETICALLY ENCODED TOOLS FOR COMPARTMENT-SPECIFIC MANIPULATION OF REDOX METABOLISM IN LIVING CELLS - ABSTRACT THE METABOLIC ENVIRONMENT THAT CELLS FACE HAS PROFOUND EFFECTS ON CELLULAR BEHAVIOR. THIS IS ESPECIALLY TRUE FOR THE REDUCTION-OXIDATION (REDOX) ENVIRONMENT, BUT MANY ASPECTS OF HOW REDOX METABOLISM IS REGULATED AND HOW IT DIRECTS CELLULAR DECISIONS ARE POORLY UNDERSTOOD. IN ORDER TO SYSTEMATICALLY ADDRESS THESE PRESSING QUESTIONS, IT IS NECESSARY TO HAVE TOOLS WITH WHICH KEY CONTRIBUTORS TO THE CELLULAR REDOX ENVIRONMENT CAN BE SAFELY AND DIRECTLY MODULATED WITH SPATIAL AND, MOST IMPORTANTLY, TEMPORAL RESOLUTION. WE PREVIOUSLY USED A H2O-FORMING NADH OXIDASE FROM LACTOBACILLUS BREVIS (LBNOX) TO DECREASE THE NADH/NAD+ RATIO WHEN ECTOPICALLY EXPRESSED IN CYTOPLASM OR MITOCHONDRIA OF MAMMALIAN CELLS. FURTHERMORE, WE ENGINEERED A VARIANT OF THIS ENZYME WITH STRICT SPECIFICITY TOWARDS NADPH (TPNOX). WE SUBSEQUENTLY EMPLOYED BOTH LBNOX AND TPNOX AS GENETICALLY ENCODED TOOLS TO SHOW THAT NAD+ REGENERATION BUT NOT ATP PRODUCTION IS A CRITICAL REQUIREMENT OF PROLIFERATION OF MAMMALIAN CELLS. IN OUR ORIGINAL MIRA ESI APPLICATION, WE PLAN TO CONTINUE DEVELOPMENT OF EVOLUTION-INSPIRED, GENETICALLY ENCODED TOOLS FOR SPATIOTEMPORAL MODULATION OF KEY CELLULAR REDOX PARAMETERS. IN PROJECT 1, WE PLAN TO EXPAND OUR TOOLKIT BY DEVELOPING A GENETICALLY ENCODED TOOL FOR THE DIRECT MODULATION OF NADH REDUCTIVE STRESS (I.E. INCREASED NADH/NAD+ RATIO). IN PROJECT 2, WE WILL ELUCIDATE THE METABOLIC AND CELLULAR CONSEQUENCES OF THE NADH REDUCTIVE STRESS IN VARIOUS BACKGROUNDS. WE WILL USE DROSOPHILA FLIES TO DIRECTLY TEST WHETHER REDOX MODULATION IN EITHER THE OXIDATIVE OR REDUCTIVE DIRECTION IS CORRELATED WITH STRESS RESISTANCE, HEALTHSPAN AND LIFESPAN. IN PROJECT 3 WE WILL COMBINE PROTEIN ENGINEERING AND IMAGING TECHNIQUES TO DEVELOP VERSIONS OF OUR TOOLS WHERE THE CORRESPONDING ENZYMATIC ACTIVITY IS CONTROLLED BY SMALL MOLECULE OR LIGHT STIMULATION TO ACHIEVE TEMPORAL CONTROL OF THE CORRESPONDING REDOX PAIRS. USING OUR TOOLS, WE WILL ALSO ILLUMINATE THE ROLE OF VARIOUS REDOX ACTIVE SMALL MOLECULES, INCLUDING SYSTEMIC MITOCHONDRIAL COMPLEX I INHIBITION AND ASSOCIATED REDOX IMBALANCE, IN THE PROGRESSION OF NEURONAL LOSS IN PARKINSON’S DISEASE (PD). THIS ADMINISTRATIVE SUPPLEMENT REQUESTS THE ACQUISITION OF A BIOTEK CYTATION C10 CONFOCAL IMAGING READER, WHICH WOULD ALLOW US TO USE AUTOMATED MICROSCOPY TO QUANTIFY MULTIPLE CELL PARAMETERS SIMULTANEOUSLY, INCLUDING CELLULAR SIZE AND SHAPE, MORPHOLOGICAL AND FUNCTIONAL CHANGES IN SUBCELLULAR STRUCTURES, INTER-ORGANELLE COMMUNICATION AND TO IMAGE FLUORESCENCE-BASED BIOSENSORS. IN SUMMARY, ACCESS TO A BIOTEK CYTATION C10 INSTRUMENT WILL SIGNIFICANTLY ACCELERATE EXPERIMENTS DESCRIBED IN PROJECTS 1-3.

CAR T CELLS ENGINEERED TO KILL HIV-INFECTED CELLS WHILE SECRETING BROADLY NEUTRALIZING ANTIBODIES

CENTRAL THERMOREGULATORY MECHANISMS DURING HEAT STRESS - ABSTRACT THERMOREGULATION IS CONTROLLED IN THE CNS WHERE PERIPHERAL THERMAL INFORMATION IS INTEGRATED AND THERMOREGULATORY RESPONSES ARE ACTIVATED. THE CORE THERMOREGULATORY NETWORK COMPRISES THE LATERAL PARABRACHIAL NUCLEUS, THE PREOPTIC AREA, THE DORSOMEDIAL HYPOTHALAMUS AND THE ROSTRAL RAPHE PALLIDUS AND IS INVOLVED IN THE MOST MECHANISMS OF ADAPTIVE THERMOREGULATION AND IN THE FEVER RESPONSE. THE THERMOREGULATORY MECHANISMS ACTIVATED DURING EXTREME HEAT EXPOSURE, WHEN CORE BODY TEMPERATURE (CBT) REACHES 40.5 C OR MORE, ARE NOT FULLY UNDERSTOOD. WE HAVE IDENTIFIED BRAIN REGIONS THAT ARE SPECIFICALLY ACTIVATED ONLY IN SUCH CONDITIONS AND OUR PRELIMINARY DATA INDICATE THAT THESE NEURONS ARE THERMOSENSITIVE AND ARE PROJECTING TO PREOPTIC THERMOREGULATORY NEURONS. OUR PRELIMINARY STUDIES HAVE ALSO REVEALED THAT ACTIVATION OF THESE POPULATION OF NEURONS INDUCES A POTENT HYPOTHERMIA. THE OVERARCHING HYPOTHESIS OF THIS PROPOSAL IS THAT A SPECIFIC POPULATION OF NEURONS ARE ACTIVATED AT HIGH CORE BODY TEMPERATURE AND THAT THEY STIMULATE DOWNSTREAM THERMOREGULATORY PREOPTIC NEURONS TRIGGERING HEAT LOSS MECHANISMS. IN SPECIFIC AIM 1 WE WILL IDENTIFY, USING GENETIC TOOLS, THE NEURONS ACTIVATED DURING HEAT STRESS AND CHARACTERIZE THEIR PHYSIOLOGICAL AND NEUROCHEMICAL PROPERTIES. IN SPECIFIC AIM 2 WE WILL STUDY USING TRANSGENIC MODELS, VIRAL VECTORS AND OPTOGENETICS THE NETWORK CONNECTIVITY OF THESE NEURONS AND THEIR ROLE IN THERMOREGULATION. BY EMPLOYING PHARMACOLOGICAL TOOLS, ELECTROPHYSIOLOGY AND TRANSGENIC MODELS WE WILL THEN STUDY THE ION CHANNELS INVOLVED IN THE THERMOSENSITIVE FIRING OF THE NEURONS ACTIVATED AT HIGH CBT AND WILL DETERMINE THEIR ROLE IN THERMOREGULATION (SPECIFIC AIM 3). STUDYING THE THERMOREGULATORY NEURONAL NETWORKS AND CELLULAR MECHANISMS ACTIVATED BY EXTREME HEAT EXPOSURE MAY LEAD TO BETTER THERAPIES FOR HEAT STROKE AND PROVIDE CENTRAL PHARMACOLOGICAL TARGETS FOR THE DEVELOPMENT OF HYPOTHERMIC AGENTS.

REPURPOSING AN FDA APPROVED DRUG, B-RAF KINASE INHIBITOR DABRAFENIB FOR PROTECTION FROM CISPLATIN- AND NOISE- INDUCED HEARING LOSS - ABSTRACT HEARING LOSS CAUSED BY NOISE, AGING AND CHEMOTHERAPY AFFECTS SEVEN HUNDRED MILLION PEOPLE WORLDWIDE, BUT THERE ARE NO FDA-APPROVED DRUGS TO PREVENT IT. THIS RESEARCH WILL TEST THE POTENTIAL TO REPURPOSE A SMALL MOLECULE BRAF INHIBITOR, DABRAFENIB (TAFINLAR), AN FDA-APPROVED DRUG FOR SEVERAL CANCERS, FOR NEW USE IN PREVENTING CISPLATIN-INDUCED HEARING LOSS. DABRAFENIB WAS A TOP HIT IN OUR UNBIASED HIGH-THROUGHPUT SCREENS OF 4,385 BIOACTIVE COMPOUNDS AND 187 SPECIFIC KINASE INHIBITORS FOR CISPLATIN-INDUCED CELL-DEATH PROTECTION IN AN INNER EAR CELL LINE. WE FOUND THAT DABRAFENIB FULLY PROTECTED THE OUTER HAIR CELLS AGAINST CISPLATIN TOXICITY IN MOUSE COCHLEAR EXPLANTS WITH IC50 OF 30 NM AND AN EXCELLENT THERAPEUTIC INDEX (LD50/IC50) OF >2000. MECHANISTICALLY, WE IDENTIFIED DABRAFENIB AND THREE ADDITIONAL BRAF INHIBITORS, TWO MEK1/2 INHIBITORS, AND AN ERK1/2 INHIBITOR IMMEDIATELY DOWNSTREAM OF BRAF IN THE CELLULAR PATHWAY, SUPPORTING THE ROLE OF BRAF IN CISPLATIN-INDUCED HAIR CELL DEATH. CISPLATIN TREATMENT OF THE INNER EAR CELL LINE CAUSED UPREGULATION OF PHOSPHO- BRAF, PHOSPHO-MEK1/2 AND PHOSPHO-ERK1/2 THAT WAS INHIBITED BY CO-TREATMENT WITH DABRAFENIB. MOREOVER, CISPLATIN TREATMENT OF COCHLEAR EXPLANTS OR NOISE EXPOSURE IN VIVO CAUSED UP-REGULATION OF PHOSPHO-ERK1/2 SHORT TIME AFTER DAMAGE IN SUPPORTING CELLS (INNER PHALANGEAL AND DEITERS’ CELLS) THAT WAS MITIGATED BY DABRAFENIB TREATMENT. FURTHERMORE, AT 100 NM DABRAFENIB PROTECTED ZEBRAFISH LATERAL LINE NEUROMASTS FROM CISPLATIN-INDUCED DEATH IN VIVO AND, IMPORTANTLY, SIGNIFICANT PROTECTION WAS ACHIEVED WITH ORAL DELIVERY OF DABRAFENIB FOR THREE CONSECUTIVE DAYS IN MOUSE MODELS AGAINST CISPLATIN-INDUCED HEARING LOSS. THE DAILY DOSE OF DABRAFENIB ADMINISTERED TO THE MICE WAS IN THE RANGE APPROVED FOR LONG-TERM HUMAN TREATMENT. IN THIS PROPOSAL, WE WILL TEST THE PROTECTION PROVIDED BY DABRAFENIB FOR CISPLATIN-INDUCED HEARING LOSS IN A MULTIPLE LOW DOSE CISPLATIN REGIMEN THAT MIMICS CLOSELY THE CISPLATIN TREATMENT OF CANCER PATIENTS IN THE CLINIC. FUNCTIONAL AUDITORY PERFORMANCE AND INNER EAR MORPHOLOGY WILL BE ASSESSED. TWO DOSES OF DABRAFENIB WILL BE TESTED TO EVALUATE THE THERAPEUTIC WINDOW OF THE DRUG IN VIVO. WE WILL ALSO DETERMINE DABRAFENIB’S INTERFERENCE WITH CISPLATIN TUMOR KILLING EFFICACY IN TUMOR CELL LINES AND MOUSE TUMOR MODELS IN WHICH CISPLATIN IS THE STANDARD TREATMENT, NEUROBLASTOMA AND LUNG CANCER. WE WILL CONFIRM BRAF KINASE IS THE MOLECULAR TARGET OF DABRAFENIB IN CISPLATIN-INDUCED HEARING LOSS BY GENERATING A SUPPORTING CELL SPECIFIC CONDITIONAL KNOCKOUT MOUSE IN WHICH BRAF IS SPECIFICALLY DELETED IN THE SUPPORTING CELLS OF THE INNER EAR STARTING AT POSTNATAL DAY 28 AND TESTING ITS RESISTANCE TO CISPLATIN. SUPPORTING CELLS’ ERK PHOSPHORYLATION CAN SERVE AS AN IN VIVO BIOMARKER FOR CISPLATIN DAMAGE AND EVALUATING TREATMENT WITH BRAF INHIBITORS AND CAN BE UTILIZED TO DETERMINE DABRAFENIB’S PK/PD PROPERTIES. OUR STUDY WILL REVEAL A NEW CELLULAR PATHWAY AND MOLECULAR TARGET BRAF KINASE FOR OTOPROTECTION AND WILL PROVIDE THE CRUCIAL DATA NEEDED FOR ADVANCING DABRAFENIB TO CLINICAL TRIALS IN HUMANS FOR PREVENTION OF CISPLATIN-INDUCED HEARING LOSS.

PEPTIDERGIC MODULATION OF THERMOREGULATION AND ENERGY EXPENDITURE

MODELING PARKINSON'S DISEASE WITH ISOGENIC HIPSC-DERIVED DOPAMINERGIC NEURONS

CEREBELLAR-STRIATAL CIRCUITS FOR CONTROL OF DOPAMINE ACTIVITY AND FOOD INTAKE - PROJECT SUMMARY RECENTLY, WE IDENTIFIED THE CEREBELLUM AS A NOVEL SATIATION CENTER IN THE BRAIN. YET, LITTLE IS KNOWN ABOUT THE PATHWAYS AND CIRCUIT MECHANISMS THROUGH WHICH THE CEREBELLUM REGULATES FOOD INTAKE. THE MAJOR RESEARCH GOAL OF THIS PROPOSAL IS TO DEFINE CEREBELLAR À STRIATAL CIRCUITS THAT REGULATE FOOD INTAKE AND BETTER UNDERSTAND THE PHYSIOLOGICAL CHANGES MEDIATED BY ACTIVITY IN THIS CIRCUIT. WE HAVE RECENTLY IDENTIFIED A SUBSET OF MOLECULARLY AND TOPOGRAPHICALLY-DISTINCT NEURONS IN THE LATERAL NUCLEUS OF THE MOUSE ANTERIOR DEEP CEREBELLAR NUCLEI (ADCN-LAT) THAT ARE ACTIVATED BY FOOD INTAKE (LOW ET AL., 2021). OUR FUNCTIONAL ASSESSMENT OF THESE NEURONS DEMONSTRATES THAT ACTIVATION OF ADCN-LAT NEURONS DRAMATICALLY DECREASES FOOD INTAKE BY REDUCING MEAL SIZE WITHOUT COMPENSATORY CHANGES TO METABOLIC RATE. WE DISCOVERED THAT ACTIVITY IN ADCN-LAT NEURONS REDUCES THE PHASIC DOPAMINE RESPONSE TO ADDITIONAL FOOD, LIKELY CURBING THE URGE TO EAT BY REDUCING THE REWARD VALUE OF ADDITIONAL FOOD (LOW ET AL., 2021). BASED ON PUBLISHED AND PRELIMINARY DATA, THE CENTRAL HYPOTHESES OF THIS PROPOSAL ARE THAT: 1) FOOD- AND NUTRIENT-SENSING ADCN-LAT NEURONS ARE POLYSYNAPTICALLY LINKED TO THE STRIATUM THROUGH SUBCORTICAL PATHWAYS TO INFLUENCE STRIATAL DOPAMINE LEVELS, AND THESE NEURONS HAVE A UNIQUE MOLECULAR PROFILE THAT CHANGES DURING OBESITY; 2) ADCN-LAT MEDIATED DOPAMINE CHANGES INFLUENCE NEURAL ACTIVITY IN A SUBSET OF STRIATAL NEURONS AND REGULATE AFFECTIVE AND MOTIVATIONAL DESIRE FOR FOOD; AND, 3) ADCN-STRIATAL CIRCUITS AND DA SIGNALING ARE DISRUPTED IN OBESITY. THIS PROPOSAL WILL TEST THESE HYPOTHESES THROUGH THREE AIMS. AIM 1 DELINEATES THE PATHWAYS THROUGH WHICH ADCN-LAT NEURONS CAN ACT ON STRIATAL DOPAMINE AND DEFINES TRANSCRIPT CHANGES IN ADCN-LAT NEURONS OF DIET-INDUCED OBESE MICE. WE WILL USE VIRAL TOOLS (AAVRETRO AND RABIES) TO DETERMINE THE INTERMEDIATE TARGETS OF ADCN-LAT NEURONS (HYPOTHESIZED TO BE LINKED BY VENTRAL TEGMENTAL AREA AND SUBSTANTIA NIGRA) AND SINGLE NUCLEI RNA-SEQUENCING TO IDENTIFY CHANGES IN TRANSCRIPTS OF ADCN-LAT NEURONS IN OBESE MICE. IN AIM 2, WE WILL EXAMINE THE INFLUENCE OF ADCN-MEDIATED CHANGES IN STRIATAL DA LEVELS ON THE NEURAL ACTIVITY OF SPECIFIC STRIATAL NEURON SUBTYPES (D1R AND D2R). THESE EXPERIMENTS WILL ALLOW US TO EXAMINE HOW CEREBELLAR ACTIVITY INFLUENCES THE STRIATAL RESPONSE TO FOOD ANTICIPATION, CONSUMPTION AND GASTRIC NUTRIENTS. FINALLY, THE EXPERIMENTS IN AIM 3 WILL ASSESS HOW DIET-INDUCED OBESITY IMPACTS ADCN-MEDIATED CHANGES IN STRIATAL NEURAL ACTIVITY AT A SINGLE CELL LEVEL, AND MORE IMPORTANTLY TEST THE HYPOTHESIS THAT SILENCING THE DCN LEADS TO AN ‘OBESITY- LIKE’ PATTERN OF NEURAL ACTIVITY IN THE STRIATUM. BY DEFINING CEREBELLAR-STRIATAL PATHWAYS, HOW CEREBELLAR ACTIVITY CONTROLS STRIATAL NEURON FUNCTION, AND HOW THIS CIRCUITRY MIGHT BE DISRUPTED IN OBESITY, THIS WORK WILL REVEAL CIRCUIT MECHANISMS THAT REGULATE FOOD REWARD PROCESSING WITH ADDED TRANSLATIONAL VALUE OF GUIDING THE DEVELOPMENT OF NON-INVASIVE CEREBELLAR STIMULATION AND CELL-TYPE SPECIFIC PHARMACOLOGICAL MANIPULATION OF STRIATAL NEURONS FOR LONG-TERM BODY WEIGHT MANAGEMENT.

FLUORESCENT PROTEINS FOR SUPERRESOLUTION IMAGING

MIR REGULATION OF THE NEUROVASCULATURE FUNCTION IN HEALTH AND DISEASE

TESTING THE UTILITY OF MIBIOAGE AS A PERSONALIZED AGING BIOMARKER - PROJECT SUMMARY AGING IS HIGHLY INDIVIDUAL PHENOMENON PROCEEDING AT DIFFERENT SPEED IN CHRONOLOGICALLY IDENTICAL PEOPLE. THESE DIFFERENCES KINDLE THE NOTION OF BIOLOGICAL AGE FOR WHICH CANDIDATE BIOMARKERS INCLUDE SERUM ANALYTES AND FRAILTY INDICES AND MORE RECENTLY DNA METHYLATION. LONGITUDINAL PROFILING IN HUMANS REVEALED THAT ORGANS AND TISSUES AGE WITH DIFFERENT SPEEDS RESULTING IN HIGHLY INDIVIDUAL AGEOTYPE. FURTHER, MEASUREMENTS AT THE SINGLE CELL RESOLUTION SIGNIFICANTLY IMPROVE THE INSIGHTS INTO HUMAN AGING PROCESS. THESE AND OTHER STUDIES UNDERSCORE THE NEED FOR PREDICTIVE BIOMARKERS OF AGING AT THE MOLECULAR LEVEL PREFERABLY WITH SINGLE CELL RESOLUTION TO UNRAVEL THE COMPLEXITY OF ORGANISMAL AGING AND TO PROVIDE TISSUE-SPECIFIC QUANTITATIVE SIGNATURES OF FUNCTIONAL AGE. TO BE INFORMATIVE SUCH MOLECULAR SIGNATURES MUST BE ANCHORED IN THE FUNCTIONAL READOUTS OF AGING SUCH AS METABOLIC, PHYSICAL, COGNITIVE, AND IMMUNE FUNCTIONS PREFERABLY AT THE LEVEL OF INDIVIDUAL ORGANISMS. THE TERSKIKH LABORATORY HAS DEVELOPED A NOVEL TECHNIQUE ROOTED IN THE ANALYSIS OF EPIGENOME TOPOGRAPHY AT THE SINGLE CELL LEVEL TO QUANTITATE CHANGES IN CHROMATIN LANDSCAPE. WE CAPTURE PATTERNS OF NUCLEAR STAINING OF EPIGENETIC MARKS (E.G. ACETYLATED AND METHYLATED HISTONES) AND EMPLOY AUTOMATED MICROSCOPY AND MACHINE LEARNING TO DETERMINE MULTIPARAMETRIC SIGNATURE OF CELLULAR STATE. APPLICATION OF THIS TECHNIQUE TO AGING, TERMED MICROSCOPIC IMAGING OF BIOLOGICAL AGE (MIBIOAGE), REVEALED ROBUST SEPARATION OF YOUNG AND OLD FRESHLY ISOLATED MOUSE AND HUMAN TISSUES, AND CORRELATED WITH CHRONOLOGICAL AGE WITHOUT LINEAR REGRESSION. A RECENTLY FUNDED CLINICAL TRIAL (U01 AG07694) WILL DETERMINE WHETHER THE MTOR INHIBITOR EVEROLIMUS SAFELY PROMOTES HEALTHSPAN IN HUMANS. WE PROPOSE TO TAKE ADVANTAGE OF A UNIQUE SET OF HUMAN SAMPLES ORIGINATING FROM U01 AG076941 CLINICAL TRIAL, TO ASSOCIATE (USING HYPERBOLIC EMBEDDING AND MACHINE LEARNING) INDIVIDUAL MIBIOAGE SIGNATURES IN PBMC AND SKELETAL MUSCLES WITH MULTIPLE FUNCTIONAL READOUTS.

INTERSECTION OF NEURO-IMMUNE-VASCULAR DYNAMICS IN PRECLINICAL NEUROHIV MODEL - SUMMARY FOLLOWING SYSTEMIC INFECTION, HIV-1 ENTERS THE CNS BY A “TROJAN HORSE” MECHANISM, USING INFECTED MONOCYTES OR T-CELLS FROM THE PERIPHERY TO CROSS THE BLOOD-BRAIN BARRIER (BBB) AND INFECT CNS CELLS. WITHIN THE CNS, MICROGLIA (MG), RESIDENT MACROPHAGES (MΦ), SERVE AS RESERVOIRS FOR HIV-1. ALTHOUGH NEURONS ARE NOT DIRECTLY INFECTED, THEY APPEAR HIGHLY SUSCEPTIBLE TO DYSFUNCTION DUE TO THE PRESENCE OF THE VIRUS IN THE CNS. HIV-1 INFECTION LEADS TO BBB DYSFUNCTION, EXACERBATING VIRAL REPLICATION WITHIN THE CNS. INFLAMMATORY FACTORS RELEASED BY INFECTED MG/MΦ CONTRIBUTE TO SELECTIVE SYNAPTIC DAMAGE IN NEURONS, GRADUALLY RESULTING IN CNS PATHOLOGY. THIS NEURONAL DYSFUNCTION MANIFESTS AS IMPAIRED COGNITIVE FUNCTION, KNOWN AS HAND, AFFECTING 30- 50% OF HIV-1 INDIVIDUALS REGARDLESS OF ANTIRETROVIRAL TREATMENT. THE GOAL OF THIS PROPOSAL IS TO DEVELOP NEW INSIGHTS INTO HOW HIV-1 AFFECTS THE BRAIN, RECAPITULATE ASPECTS OF BBB AND CNS PATHOLOGY IN A HIGHLY CONTROLLABLE MANNER AND DEFINE THE PATHOGENIC STATE OF CNS HIV-1 INFECTION. WE WILL DEVELOP A NEXT GENERATION PRECLINICAL MODEL OF NEUROHIV, THAT BETTER RECAPITULATE NEURO-IMMUNE-VASCULAR SYSTEM INTERACTIONS IN THE CONTEXT OF ART-MEDIATED HIV SUPPRESSION. WE WILL INCORPORATE PERIPHERAL IMMUNE CELLS THAT SUPPORT A NATURAL ROUTE OF HIV ENTRY ACROSS THE BBB INTO THE CNS, GLIAL CELLS RELEVANT TO THE NEUROPATHOGENESIS OF HIV INFECTION, NEURONS, BRAIN ENDOTHELIAL CELLS AND BBB SUPPORTING CELL TYPES. WE PROPOSE THE FOLLOWING AIMS TO ACHIEVE THE GOALS. AIM 1 (R61), GENERATE, CHARACTERIZE AND VALIDATE IPSC-DERIVED CNS CELLS AND IMMUNE CELLS. AIM 2 (R61), ESTABLISH A PERFUSED HIV-1 BLOOD-BBB/CNS MODEL. MILESTONES OF THE R61 PHASE WILL BE TO VALIDATE IPSC TYPES, OPTIMIZE PROTOCOLS AND DEMONSTRATE RELIABLE HIV-1 INFECTION OF MG/MΦ, ESTABLISHMENT OF AT A HIV-CNS 3D MODEL WITH FLOW, TEST MΦ’S BBB TRANSMIGRATION AND FINALLY TO OPTIMIZE PROTOCOLS TO TRACK THE SPREAD OF HIV-1GFP INFECTION WITHIN THE CNS COMPARTMENT. FINALLY AIM 3 (R33), WE WILL CREATE A 3D BLOOD- BBB/CNS MODEL WITH FLOW, WHICH DYNAMICALLY EXAMINES NEURO-IMMUNE-VASCULAR “CIRCUIT RESPONSES” IN THE CONTEXT OF ART-SUPPRESSED HIV-1 INFECTION. BY CREATING THE FIRST 3D HUMAN HIV-1 BLOOD-BBB/CNS MODEL THAT ALLOWS FOR SINGLE-CELL IMAGING AND TRANSCRIPTOMIC ANALYSIS, WE HAVE A UNIQUE OPPORTUNITY TO RECAPITULATE THE CLINICAL PICTURE OF NEUROHIV, INCLUDING NEUROLOGICAL ENDPOINT MEASURES. THIS MODEL WILL ENABLE US TO STUDY HOW HIV EXPRESSION DRIVE PATHOGENIC NEURO-IMMUNE-VASCULAR “CIRCUIT RESPONSES” IN THE CONTEXT OF AN ART- SUPPRESSED INFECTION AND TO EXAMINE THE POTENTIAL NEUROTOXIC EFFECTS OF ART THERAPIES THEMSELVES. THE PROPOSAL ADDRESSES SIGNIFICANT KNOWLEDGE GAPS RELATED TO THE DISEASE ETIOLOGY OF NEUROHIV IN CLINICAL POPULATIONS ON ART, WITH A HIGH POTENTIAL FOR OVERALL IMPACT.

ENGINEERED FLAVIN-DEPENDENT ENZYMES FOR PROBING REDOX ENVIRONMENT AND REGULATION

CAR T CELLS ENCODING ALPHA-CHAIN-ANCHORED AND SOLUBLE BNABS FOR HIV LATENCY

PHOSPHOPROTEOMIC TOOLS FOR SYNAPTIC VS. EXTRASYNAPTIC NMDA RECEPTOR SIGNALING

TESTING ENERGY EXPENDITURES AS BIOMARKERS OF AGING USING THERMAL IMAGING IN A CONTROLLED ENVIRONMENT - PROJECT ABSTRACT RODENT ANIMAL MODELS ARE KEY TO UNDERSTANDING THE FUNDAMENTAL BIOLOGICAL MECHANISM AND TO ADVANCING CLINICALLY RELEVANT TESTING, INCLUDING AGING AND AGE-RELATED DISEASES. INTIMATELY LINKED TO TEMPERATURE, TOTAL ENERGY EXPENDITURE (TEE) IS AN IMPORTANT CONCEPT TO UNDERSTAND HOW THE WEIGHT-REDUCED ORGANISMAL STATE IMPROVES METABOLISM OR HOW PHYSICAL ACTIVITY AFFECTS ENERGY EXPENDITURE. INTRIGUINGLY, AN AGE-RELATED INCREASE IN THE ENERGETIC COST OF PHYSICAL ACTIVITY IN MICE AND AGE-RELATED CHANGES IN PHYSICAL ACTIVITY ENERGY EXPENDITURE (PAEE) IN HUMANS SUGGEST THE UTILITY OF THESE METRICS AS BIOMARKERS OF AGING. WE PROPOSE TO DETERMINE AGE-RELATED CHANGES IN PAEE AND/OR OTHER TEE CONSTITUENTS AND THEIR UTILITY AS PHYSIOLOGICAL BIOMARKERS OF AGING. CLASSICAL METABOLIC CAGES RECORD ONLY ONE MOUSE PER CAGE, LIMITING THE STUDY OF SOCIAL BEHAVIORS, AND COMMERCIAL ACTIVITY MONITORING PLATFORMS DO NOT OFFER CONTINUOUS THERMAL IMAGING, ARE VERY EXPENSIVE, AND REQUIRE DEDICATED FACILITIES AND PERSONNEL. TO ADDRESS THESE SHORTCOMINGS, WE ARE DEVELOPING A COMPREHENSIVE PLATFORM FOR ENDURING THERMAL IMAGING IN A CONTROLLED ENVIRONMENT (ENTICE). BASED ON CONVENTIONAL HOME CAGES, ENTICE HAS THERMAL AND FAR-RED CAMERAS, A CONTROLLED FOOD DISPENSER, AN IN-CAGE MOUSE WEIGHING PLATFORM, AND A VOLUNTEER RUNNING WHEEL WITH DIRECT RECORDING OF ENERGY GENERATED BY AN ANIMAL. THERMAL AND FAR-RED MAGES ARE ANALYZED ON THE FLY, ENABLING REAL-TIME ANALYSIS OF ANIMAL BEHAVIORS. HERE, WE PROPOSE TO LONGITUDINALLY RECORD AND ANALYZE GROUPS OF YOUNG, MIDDLE-AGED, AND OLD MICE TO DETERMINE AGE-RELATED CHANGES IN PAEE AND/OR OTHER CONSTITUENT PARTS OF TEE AND TO UNCOVER COMMON DETERMINANTS AND INDIVIDUAL ASPECTS (HETEROGENEITY) OF MOUSE AGING. TO ENABLE THE STUDY OF THE SOCIAL ASPECT OF AGING, WE WILL DEVELOP A DEEP THERMAL IDENTIFICATION (DEEPT-ID) MODEL THAT COMBINES DEEP LEARNING NEURAL NETWORKS WITH CLASSICAL IMAGE TEXTURE FEATURES FOR RAPID AND ROBUST THERMAL IMAGE ANALYSIS AND MOUSE IDENTIFICATION THAT ENABLE DISTINGUISHING, SIMULTANEOUS TRACKING, AND ANALYSIS OF SEVERAL ANIMALS IN THE SAME CAGE. ENTICE REPRESENTS A COMPACT, AFFORDABLE (UNDER 10K USD) PLATFORM THAT COULD BE INSTALLED AND OPERATED IN AN INDIVIDUAL LABORATORY. WE PROVIDE A COMPREHENSIVE ASSEMBLY TUTORIAL AND A USER-FRIENDLY PYTHON-BASED SOFTWARE PACKAGE TO MAKE BEHAVIORAL STUDIES ACCESSIBLE TO ALL INVESTIGATORS WORKING WITH MICE. WE BELIEVE THAT THE DEVELOPMENT AND VALIDATION OF THIS NOVEL THERMAL IMAGING-BASED SCALABLE, COST-EFFECTIVE PLATFORM WILL HAVE A SIGNIFICANT IMPACT, ENABLING THE TESTING OF CANDIDATE DRUGS AND INTERVENTIONS IN VARIOUS MOUSE MODELS OF AGING AND AGE-RELATED DISEASES AND CAPTURING COMPREHENSIVE BEHAVIORAL AND PHYSIOLOGICAL PHENOTYPES.

NAD(P)H QUINONE OXIDOREDUCTASE 1 (NQO1)-MEDIATED BYPASS OF MITOCHONDRIAL ELECTRON TRANSPORT CHAIN WITH ARTIFICIAL AND ENDOGENOUS SUBSTRATES - ABSTRACT A WIDE RANGE OF RARE AND COMMON DISEASES ARE LINKED TO MITOCHONDRIAL DYSFUNCTION AND ASSOCIATED REDOX IMBALANCE. RESTORATION OF THE UNDERLYING REDOX IMBALANCE BY DECREASING THE CELLULAR NADH/NAD+ RATIO COULD BE SEEN AS AN EXTREMELY USEFUL GENERALIZABLE STRATEGY IN THE CONTEXT OF MULTIPLE DISEASE STATES. NAD(P)H:QUINONE OXIDOREDUCTASE 1 (NQO1) IS A SOLUBLE CYTOPLASMIC ENZYME THAT HAS BEEN MOSTLY VIEWED AS A XENOBIOTIC- METABOLIZING ENZYME, OR A BIOACTIVATOR OF CANCER DRUGS AT THE EXPENSE OF REDUCING EQUIVALENTS OF NAD(P)H. INTERESTINGLY, SOME OF NQO1 ARTIFICIAL SUBSTRATES, MOSTLY NAPHTHOQUINONES, WHEN REDUCED ARE CAPABLE OF SUBSEQUENTLY DONATING THEIR ELECTRONS TO THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN DOWNSTREAM OF COMPLEX I. THIS NQO1-MEDIATED ALTERNATIVE ELECTRON TRANSFER IS THEREFORE AN ATTRACTIVE STRATEGY TO ALLEVIATE REDUCTIVE STRESS AND SUPPORT ATP HOMEOSTASIS AS IT DEPENDS ON AN ENDOGENOUS ENZYME AND ONLY REQUIRES ADDITION OF RESPECTIVE NAPHTHOQUINONES. HOWEVER, NAPHTHOQUINONES CAPABLE OF BEING REDUCED BY NQO1 ARE EITHER NATURAL PRODUCTS OR SYNTHETIC REDOX SCAFFOLDS (E.G. IDEBENONE), AND WE CURRENTLY LACK INFORMATION ON ENDOGENOUS SUBSTRATES OF NQO1 AND ITS PLACE IN CELLULAR REDOX METABOLISM. TO CLOSE THIS KNOWLEDGE GAP, WE WILL USE ACTIVITY-BASED METABOLOMIC PROFILING WITH RECOMBINANT NQO1 TO IDENTIFY CELLULAR ENDOGENOUS METABOLITES THAT ARE INTERCONVERTED BY THIS ENZYME. NEXT, WE WILL RECONSTITUTE THE NQO1-MEDIATED ELECTRON TRANSFER WITH VARIOUS NAPHTHOQUINONES IN ISOLATED MITOCHONDRIA AND WILL STUDY THE BIOENERGETICS OF THIS NON-CANONICAL POINT OF ENTRY OF REDUCING EQUIVALENTS. THIS WILL ALLOW US TO RIGOROUSLY CHARACTERIZE NAPHTHOQUINONES AND RELATED REDOX-ACTIVE MOLECULES FOR THEIR ABILITY TO SAFELY BYPASS A CORRUPTED MITOCHONDRIAL ELECTRON TRANSPORT CHAIN WITHOUT INDUCING OXIDATIVE STRESS. OUR CURRENT APPROACH WILL, FOR THE FIRST TIME, ALLOW US TO IDENTIFY PHYSIOLOGICAL NQO1 SUBSTRATES AND HELP US BETTER RECONSTRUCT THE NQO1-MEDIATED ELECTRON TRANSFER. THIS WORK WILL ULTIMATELY PAVE THE WAY FOR DEVELOPING THERAPEUTIC MODALITIES THAT ARE BASED ON REDOX-ACTIVE SMALL MOLECULES THAT CAN ALLEVIATE REDUCTIVE STRESS.

USING A XENOTOPIC APPROACH TO MANIPULATE REDOX METABOLISM IN A TISSUE- AND COMPARTMENT-SPECIFIC MANNER TO STUDY AGING - ABSTRACT A STEADY DECLINE IN CELLULAR AND PHYSIOLOGICAL FUNCTION IS A RECOGNIZED ASPECT OF THE AGING PROCESS. IT IS NOW WIDELY ACCEPTED THAT IN HUMANS, AGING IS THE MAJOR RISK FACTOR FOR MULTIPLE DISEASES, INCLUDING CANCER, CARDIOVASCULAR DISEASES, AND NEURODEGENERATION. THUS, THE RESEARCH COMMUNITY HAS BECOME INTERESTED IN DEVELOPING SPECIFIC METHODS AND STRATEGIES TO MODULATE AGING-ASSOCIATED METABOLIC CHANGES — INCLUDING THOSE IN CELLULAR ENERGY METABOLISM — TO BETTER UNDERSTAND THE BASIC MECHANISMS OF AGING. IF SUCCESSFUL, INSIGHTS FROM SUCH APPROACHES COULD LEAD TO AN IMPROVED HEALTHSPAN IN HUMANS BY POTENTIALLY ENHANCING RESISTANCE TO AGING- ASSOCIATED DISEASES. CURRENTLY, IT IS NOT POSSIBLE TO MODULATE REDOX IMBALANCE OFTEN ASSOCIATED WITH MITOCHONDRIAL DYSFUNCTION, WHICH FURTHER PREVENTS OUR ABILITY TO ASSESS HOW IT PROMOTES AGING-ASSOCIATED METABOLIC CHANGES. IN PARTICULAR, IT IS CURRENTLY NOT FEASIBLE TO MODULATE LEVELS OF REDOX COFACTORS NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD) AND RELATED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADP), KEY CONTRIBUTORS TO THE CELLULAR REDOX ENVIRONMENT, WITH TISSUE- AND COMPARTMENT-SPECIFIC PRECISION. THE MAIN OBJECTIVE OF THIS GRANT PROPOSAL IS TO DEVELOP AND VALIDATE NOVEL GENETICALLY ENCODED TOOLS THAT DIRECTLY MODULATE BOTH NADH/NAD+ AND NADPH/NADP+ RATIOS WHEN EXPRESSED IN DIFFERENT TISSUES AND CELLULAR COMPARTMENTS OF DROSOPHILA. WE WILL USE THESE TOOLS TO TEST OUR HYPOTHESIS THAT DIRECT TARGETING OF REDOX METABOLISM EXTENDS HEALTH- AND LIFESPAN IN A TISSUE- AND ORGANELLE SPECIFIC MANNER. IN AIM 1, WE WILL DETERMINE THE SPECIFIC TISSUES RESPONSIBLE FOR THE BENEFICIAL EFFECTS OF TARGETING NAD METABOLISM WITH A WATER-FORMING NADH OXIDASE LBNOX ON LIFESPAN AND SLEEP IN DROSOPHILA. WE ALREADY DEMONSTRATED USING TWO TRANSGENIC DROSOPHILA LINES EXPRESSING LBNOX THAT A SEX- DEPENDENT PRO-OXIDATIVE SHIFT (A DECREASE) IN THE NADH/NAD+ RATIO CO-OCCURS WITH A PRO-REDUCTIVE SHIFT (AN INCREASE) IN THE NADPH/NADP+ RATIO IN THESE FLIES. MOST IMPORTANTLY, WE ESTABLISHED THAT CYTOSOLIC LBNOX EXPRESSION CAN LEAD TO MULTIPLE BENEFICIAL EFFECTS, SUCH AS LIFESPAN EXTENSION, RESISTANCE TO OXIDATIVE STRESS, IMPROVED NEUROMUSCULAR FUNCTION AND SLEEP. IN ADDITION, WE DEMONSTRATED THAT LBNOX WAS MUCH MORE EFFECTIVE FOR RESCUING PARAQUAT-INDUCED OXIDATIVE STRESS WHEN LOCALIZED TO THE MUSCLE COMPARED TO WHOLE-BODY OR NEURONAL-SPECIFIC EXPRESSION. BUILDING ON OUR PRELIMINARY RESULTS, WE WILL DETERMINE WHICH DROSOPHILA TISSUES MEDIATE THE BENEFICIAL EFFECTS OF LBNOX EXPRESSION ON AGING AND SLEEP AND WHETHER IMPROVED SLEEP IS REQUIRED FOR THE EXTENSION OF LIFESPAN. BECAUSE OUR PRELIMINARY EXPERIMENTS SHOWED THAT MODULATION OF NAD+ REGENERATION LED TO AN INCREASE IN NADP(H) LEVELS AND NADPH/NADP+ RATIO, AIM 2 WILL FOCUS ON ADDRESSING WHETHER THE ANTI- AGING EFFECTS AND AGING-RELATED SIGNATURES OF TARGETING NAD METABOLISM WITH LBNOX ARE MEDIATED BY THE CONVERSION OF NAD+ TO NADP+. FOR THIS AIM, WE WILL EXPLORE IF UBIQUITOUS AND/OR TISSUE-SPECIFIC BENEFICIAL EFFECTS OF LBNOX IN DROSOPHILA DEPEND ON NADK (AN ENZYME WHICH CONVERTS NAD+ INTO NADP+). IN SUMMARY, THE GROWING ARSENAL OF ENZYME-BASED GENETICALLY ENCODED TOOLS WE ARE DEVELOPING FOR TISSUE- AND COMPARTMENT- SPECIFIC MODULATION OF REDOX METABOLISM WILL ESTABLISH A TRANSFORMATIVE BIOCHEMICAL AND GENETIC DISCOVERY PIPELINE THAT CAN BE USED TO SYSTEMATICALLY IDENTIFY REDOX PATHWAYS THAT CONTRIBUTE TO THE PATHOBIOLOGY OF AGING.

DECIPHERING THE DICHOTOMY OF OXYGEN METABOLISM IN RENAL CANCER USING A NOVEL GENETICALLY ENCODED OXYGEN BIOSENSOR - ABSTRACT MOLECULAR OXYGEN (O2) PLAYS A CRUCIAL ROLE IN SHAPING EARTH'S BIOSPHERE. IT IS GENERALLY ACCEPTED THAT THE RISE OF OXYGEN IN THE ATMOSPHERE LED LIVING SYSTEMS TO EVOLVE A SOPHISTICATED RESPIRATORY CHAIN, WHICH ALLOWED ORGANISMS TO HARVEST ENERGY BY OXIDATIVE PHOSPHORYLATION. OXYGEN IS AN INDISPENSABLE ASPECT OF THE EUKARYOTIC LIFE AND IS A REACTANT IN MANY ENZYMATIC REACTIONS BEYOND OXIDATIVE PHOSPHORYLATION. HOWEVER, IT HAS FREQUENTLY BEEN AN OVERLOOKED VARIABLE IN BIOCHEMICAL AND CELLULAR STUDIES. RECENTLY, THERE IS INCREASING EVIDENCE THAT OXYGEN TENSION IS A CRUCIAL PARAMETER OF TISSUE, CELLULAR AND EVEN SUBCELLULAR METABOLISM. OXYGEN (AS O2) CONCENTRATIONS DIRECTLY INFLUENCE PHYSIOLOGY, REDOX SIGNALING, THE MANIFESTATION OF A WIDE RANGE OF PATHOLOGIES, AS WELL AS DEVELOPMENTAL AND DIFFERENTIATION PROGRAMS IN EUKARYOTIC CELLS AND TISSUES. A SIGNIFICANT OBSTACLE TO A COMPREHENSIVE UNDERSTANDING OF HOW DIFFERENT O2 TENSIONS CONTROL AND MODULATE THESE PHENOTYPES IS THE ABSENCE OF VERSATILE AND RELIABLE TECHNOLOGY TO MONITOR OXYGEN GRADIENTS IN LIVING CELLS. THIS PROPOSAL AIMS TO ADDRESS THIS TECHNOLOGICAL GAP BY DEVELOPING A PROTEIN-BASED FLUORESCENCE BIOSENSOR TO MONITOR O2 LEVELS IN MAMMALIAN CELLS. BASED ON CURRENTLY AVAILABLE BIOCHEMICAL AND STRUCTURAL INFORMATION, WE WILL ENGINEER CONJUGATES BETWEEN NATURALLY OCCURRING HEME-BASED PROTEIN SCAFFOLDS THAT BIND OXYGEN REVERSIBLY (SENSING UNIT) AND A FLUORESCENT PROTEIN UNAG (REPORTER UNIT). THE SENSOR CONSTRUCTS WILL BE DESIGNED SO THAT THE UNAG-BASED REPORTING UNIT RELAYS AND VISUALIZES CONFORMATIONAL AND ABSORPTION CHANGES OCCURRING UPON O2 BINDING WITHIN THE HEME-BASED SENSING UNIT. OUR APPROACH TAKES ADVANTAGE OF THE FACT THAT MATURATION OF THE CHROMOPHORE OF UNAG IS NOT OXYGEN DEPENDENT AND THEREFORE UNAG CAN BE USED AS A REPORTER UNIT AT VARIOUS OXYGEN TENSIONS. BIOSENSOR CANDIDATES WILL BE RIGOROUSLY CHARACTERIZED USING BIOCHEMICAL, SPECTROSCOPIC, AND IMAGING APPROACHES, BOTH AS PURIFIED RECOMBINANT PROTEINS AS WELL AS IN MAMMALIAN CELL CULTURE SYSTEMS. FINALLY, WE WILL APPLY OUR GENETICALLY ENCODED BIOSENSOR TO ELUCIDATE INTRACELLULAR O2 CONCENTRATION DEPENDENT METABOLIC REMODELING IN RENAL CANCER CELL LINES WITH EITHER INTACT OR DEFICIENT VON HIPPEL–LINDAU TUMOR SUPPRESSOR (VHL) GENE. OVERALL, THE TECHNOLOGY WE ARE DEVELOPING WILL PROVIDE TRANSFORMATIVE OPPORTUNITIES FOR UNDERSTANDING THE ROLE OF O2 METABOLISM IN CANCER AND BEYOND BY DIRECTLY VISUALIZING OXYGEN GRADIENTS AT SUB-ORGANELLAR RESOLUTION WITHIN LIVING CELLS.

DEFINING AND TARGETING THE COMPARTMENTALIZATION OF REDOX METABOLISM IN AGING USING NOVEL GENETICALLY ENCODED TOOLS