Salk Institute For Biological Studies

CENTER FOR MULTIOMIC HUMAN BRAIN CELL ATLAS - ABSTRACT UNDERSTANDING CELL IDENTITIES AND THEIR SPATIAL DISTRIBUTIONS THROUGHOUT DIFFERENT REGIONS OF THE HUMAN BRAIN IS A FUNDAMENTAL STEP WHEN TRYING TO INTEGRATE PHYSIOLOGICAL, BEHAVIORAL, NEUROCHEMICAL AND MOLECULAR DATA. AT PRESENT, ALTHOUGH MAJOR CATEGORIES OF THE CELL-TYPES PRESENT IN THE HUMAN BRAIN HAVE BEEN DEFINED MOLECULARLY, THE DIFFERENT SUBTYPES WITHIN THESE CATEGORIES ALONG WITH THEIR LOCATIONS ARE FAR FROM UNDERSTOOD. GENE EXPRESSION DRIVES CELL PROGRAMS AND STATES THAT UNDERLIE DISTINCT BRAIN FUNCTIONS. OPEN CHROMATIN AND MODIFIED HISTONES MARK GENE-REGULATORY ELEMENTS THAT CONTROL CELL TYPE-SPECIFIC GENE EXPRESSION PATTERNS. CYTOSINE DNA METHYLATION (MC) IS A STABLE EPIGENOMIC SIGNATURE THAT PERSISTS IN POST-MITOTIC CELLS THROUGHOUT THEIR LIFETIME, DEFINING THEIR CELLULAR IDENTITY. SINGLE-CELL GENE EXPRESSION, OPEN CHROMATIN PROFILES AND DNA METHYLATION ASSAYS HAVE BEEN SUCCESSFULLY USED TO IDENTIFY DISTINCT CELL TYPES IN AN UNBIASED FASHION IN HETEROGENEOUS TISSUES INCLUDING THE HUMAN BRAIN. THIS UM1 PROPOSAL BUILDS ON OUR EARLIER SUCCESSES IN MOUSE BRAIN MAPPING TO PRODUCE DETAILED SINGLE-CELL MULTIMOMIC AND SPATIAL CELL MAPS AT THE SINGLE-CELL LEVEL ACROSS 100 ANATOMICALLY DEFINED REGIONS IN THE HUMAN BRAIN. PROFILING HUMAN BRAIN SAMPLES WILL PERMIT THE DISCOVERY OF UNIQUE GENE EXPRESSION PATTERNS FOR EACH MOLECULARLY-DEFINED CELL TYPE, IDENTIFY THEIR SPATIAL ORGANIZATION, AND THEIR SPECIFIC NON-CODING DNA REGULATORY REGIONS. MULTI-MODAL INTEGRATION BETWEEN EPIGENOMIC AND TRANSCRIPTOMIC SIGNATURES WILL ALLOW THE IDENTIFICATION OF NEW CELL TYPES AND UNIQUE CELL-TYPE MARKERS THAT WILL RAPIDLY BE MADE AVAILABLE TO THE ENTIRE COMMUNITY. THIS UM1 PROJECT WILL ALSO PROVIDE NEW TOOLS FOR GENETIC ACCESS OF PREVIOUSLY INACCESSIBLE BRAIN REGIONS AS WELL AS FACILITATE THE FUNCTIONAL ANALYSIS OF GENETIC VARIANTS ASSOCIATED WITH NEUROPSYCHIATRIC AND NEUROLOGICAL DISORDERS.

CANCER CENTER SUPPORT GRANT

CENTER FOR EPIGENOMICS OF THE MOUSE BRAIN ATLAS (CEMBA)

BIOLOGY OF NEUROENDOCRINE PEPTIDES

SPINAL CIRCUITS FOR THE CONTROL OF DEXTROUS MOVEMENT

OVERCOMING MECHANISMS OF THERAPEUTIC RESISTANCE IN PANCREATIC DUCTAL ADENOCARCINOMA - PROJECT SUMMARY – OVERALL WHILE MORTALITY RATES FOR MANY CANCERS ARE DECLINING, PANCREATIC DUCTAL ADENOCARCINOMA (PDA) REMAINS A HIGHLY LETHAL MALIGNANCY WITH THE WORST 5-YEAR SURVIVAL RATE OF THE COMMON MALIGNANCIES. UNFORTUNATELY, CURRENT THERAPIES ARE LARGELY INEFFECTIVE IN PDA, AN OUTCOME ATTRIBUTED IN LARGE PART TO THERAPEUTIC RESISTANCE. THE HIGHLY FIBROTIC AND POORLY VASCULARIZED TUMOR MICROENVIRONMENT (TME) RESTRICTS BOTH NUTRIENT AVAILABILITY AND DRUG DELIVERY, AND PROVIDES PRO-SURVIVAL AND IMMUNOSUPPRESSIVE CUES. THESE LIMITATIONS CREATE ENERGY STRESSES THAT DRIVE METABOLIC ADAPTATIONS TO SUPPORT TUMOR GROWTH AND THERAPEUTIC RESISTANCE. MOREOVER, THE HYPERACTIVATION OF PRO-SURVIVAL AND RESISTANCE PATHWAYS IN TUMOR AND STROMAL CELL TYPES RESULTS IN AN INTEGRATED RESISTANCE NETWORK. THE INDUCTION OF THESE PATHWAYS IS ORCHESTRATED BY CELL-TO-CELL COMMUNICATIONS WITHIN THE TME, INCLUDING ABERRANT GLYCOSYLATION (CA-19-9) AND THE SECRETION OF IMMUNOSUPPRESSIVE AND PRO-SURVIVAL PARACRINE FACTORS, SUCH AS LEUKEMIA INHIBITORY FACTOR (LIF) FROM CANCER-ASSOCIATED FIBROBLASTS (CAFS). IN ADDITION, CELLS ADAPT TO THE HYPOXIC, NUTRIENT-DEPLETED TME BY UPREGULATING CELL TYPE-SPECIFIC SURVIVAL PROGRAMS, INCLUDING AUTOPHAGY. ULTIMATELY, TO SUPPORT AND RESPOND TO THESE SIGNALING AND METABOLIC PROGRAMS, BOTH THE TUMOR AND STROMAL CELL EPIGENOMES ARE REPROGRAMMED, LEADING TO CELLULAR HETEROGENEITY AND PLASTICITY THAT RESTRICTS DURABLE THERAPY RESPONSES. EACH OF THESE PROGRAMS PROMOTE RESISTANCE TO A BROAD RANGE OF THERAPEUTICS, INCLUDING CHEMOTHERAPIES, TARGETED THERAPIES, AND IMMUNE CHECKPOINT INHIBITORS. THE CENTRAL HYPOTHESIS OF THIS PROGRAM IS THAT PANCREATIC CANCER HAS CO-OPTED AN INTEGRATED NETWORK OF EPIGENETIC PROGRAMS, PARACRINE SIGNALING PATHWAYS, AND METABOLIC ADAPTATIONS TO PROMOTE TUMOR SURVIVAL AND THERAPEUTIC RESISTANCE. BUILDING UPON THE INVESTIGATORS’ COMPLEMENTARY EXPERTISE IN EPIGENETICS, CELL SIGNALING, AND METABOLIC ADAPTATIONS, AS WELL AS COMMON INTERESTS IN PANCREATIC CANCER, THIS PROGRAM SEEKS TO UNDERSTAND THE INTERACTIONS THAT HINDER PDA THERAPEUTIC RESPONSES, WITH THE ULTIMATE GOAL OF IDENTIFYING VULNERABILITIES THAT CAN BE EXPLOITED AND TARGETED TO OVERCOME DRUG RESISTANCE. IMPORTANTLY, THE PROGRAM WILL UTILIZE ADVANCED MONO- AND CO-CULTURE ORGANOID SYSTEMS, CUTTING-EDGE MOUSE MODELS, NOVEL THERAPEUTICS, SINGLE-CELL APPROACHES, AND HUMAN CLINICAL SPECIMENS TO DELINEATE THE CONTRIBUTIONS OF BOTH TUMOR CELLS AND THEIR STROMAL SUPPORT NETWORK TO THERAPEUTIC RESISTANCE. MOREOVER, PROPOSED COOPERATIVE AND INNOVATIVE APPROACH WILL REVEAL HOW THESE RESISTANCE NODES ARE INTEGRATED AND CAN BE TARGETED TO IMPROVE THERAPEUTIC OUTCOMES IN PDA.

CIRCUIT-SPECIFIC CELL TYPES IN AGING AND ALZHEIMER'S DISEASE - ABSTRACT THE LONG-TERM GOAL OF THIS PROJECT IS TO DEFINE AND IDENTIFY CIRCUIT-SPECIFIC CELL TYPES–CELLULAR SCALE CONNECTOME– THAT ARE SELECTIVELY VULNERABLE TO LOSS OF CELL BODIES OR AXONAL CONNECTIONS OR CHANGE OF TRANSCRIPTOMIC SIGNATURES OF INDIVIDUAL NEURONS DURING THE PROGRESSION OF HEALTHY AGING AND ALZHEIMER'S DISEASE (AD). EVIDENCE SUGGESTS THAT KNOWLEDGE ON THE CHANGE OF CELLULAR SCALE CONNECTOMES–CELL TYPE-SPECIFIC CIRCUITS BY COUPLING SINGLE CELL TRANSCRIPTOME WITH BRAIN CONNECTIVITY– IS NEEDED FOR HOLISTIC UNDERSTANDING OF AGING AND AD AND PROVIDES AN EXPERIMENTALLY TRACTABLE BASIS TO ADDRESS LONGITUDINAL CHANGES. THESE AGING- AND AD- ASSOCIATED CHANGES MAY INCLUDE LOSS OF CELL TYPES, CONNECTIVITY OR ALTERATIONS IN TRANSCRIPTOMIC SIGNATURES. THIS APPROACH EMPLOYED HERE IS TO TEST THE HYPOTHESIS THAT THERE ARE AGING- OR AD STATE-SPECIFIC NEURAL AND MOLECULAR CIRCUITS THAT DRIVE THE PROGRESSION OF AGING AND AD. A LARGE BODY OF EVIDENCE DEMONSTRATES THAT AD IS A HETEROGENEOUS, MULTIFACTORIAL DISEASE THAT SELECTIVELY AFFECTS CERTAIN BRAIN REGIONS, E.G. THE ENTORHINAL CORTEX (EC), WHILE OTHER AREAS, SUCH AS THE CEREBELLUM, REMAIN UNAFFECTED. RECENT STUDIES ON THE STAGING OF AD NEUROPATHOLOGY SHOWED AD-RELATED NEUROPATHOLOGY BEGINS IN THE LOCUS COERULEUS (LC) OR THE EC, FOLLOWED BY THE HIPPOCAMPUS (HC) AND THEN THE PREFRONTAL CORTEX (PFC). THE LC CONTAINS BOTH ADRENERGIC (NA) AND NON- NORADRENERGIC NEURONS AND PROVIDES THE MAJOR NA INPUTS THROUGHOUT THE ENTIRE BRAIN. NEUROPATHOLOGICAL STAGING HAS SHOWN THAT TANGLES FIST APPEAR IN THE LC AND NA ACTIVATION HAS BEEN SHOWN TO AMELIORATE AD DEFICITS. THE EC PROVIDES KEY CORTICAL INPUTS TO THE HC, WHICH IS ESSENTIAL IN LEARNING MEMORY. THE PFC PROVIDES THE TOP-DOWN REGULATION ON VARIOUS HIGHER ORDER FUNCTIONS. BUT CELL TYPES-BASED INPUT AND/OR OUTPUT NETWORKS THAT ARE SELECTIVELY VULNERABLE AT THE SINGLE NEURONS LEVEL ARE NOT WELL UNDERSTOOD. AS AGING IS A MAJOR RISK FACTOR FOR AD, IT IS IMPORTANT TO UNDERSTAND WHETHER THERE ARE DISTINCT, SIMILAR OR OVERLAPPING SELECTIVELY VULNERABLE CIRCUIT-SPECIFIC CELL TYPES BETWEEN AGING AND AD. THIS PROJECT IS TO COMBINE RETROGRADE LABELING WITH MULTIOMIC SN-RNASEQ AND SN-ATACSEQ TO LINK TRANSCRIPTOMIC AND EPIGENOMIC PROPERTIES OF CELL TYPES TO NEURONAL PROJECTIONS AND INVESTIGATE CIRCUIT-SPECIFIC CHANGES ASSOCIATED WITH PROGRESSION OF AGING AND AD IN FOUR BRAIN REGIONS, NAMELY THE LC, EC, HC AND PFC, IN BOTH MALE AND FEMALE CONTROL AND AD MICE. FOR AD MICE, THE APPNLF MOUSE LINE–THAT CARRIES KNOCKIN HUMAN MUTATIONS IN THE AMYLOID PRECURSOR PROTEIN GENE AND, IMPORTANTLY, EXPRESSES PHYSIOLOGICAL LEVELS OF ASS, MIMICKING LATE ONSET AD–WILL BE USED. THE DATA FROM THIS PROJECT WILL PROVIDE NOVEL INSIGHTS ON THE TYPES OF NEURONS VULNERABLE TO DEGENERATION AND/OR ALTERATIONS OF MOLECULAR/SIGNALING SIGNATURE NETWORKS IN A SPATIAL AND TEMPORAL FASHION AND THE CORRELATION WITH NEUROPATHOLOGY AND COGNITIVE IMPAIRMENT. THIS APPROACH IS A MAJOR STEP TOWARD ESTABLISHING MULTISCALE MODELS THAT WILL HELP TO FILL THE GAP BETWEEN THE EFFECTS OF GENETIC VARIANTS (E.G., APP, AOPE OR TREM2) ON BRAIN TOPOLOGY WITH MOLECULAR NETWORKS IN AGING AND AD.

ASTROCYTE MODULATION OF NEURAL CIRCUIT FUNCTION AND BEHAVIOR - PROJECT SUMMARY: OVERALL “WHAT IS THE FUNCTION OF GLIAL CELLS IN NEURAL CENTERS? THE ANSWER IS STILL NOT KNOWN, AND IT MAY REMAIN UNSOLVED FOR MANY YEARS TO COME UNTIL SCIENTISTS FIND DIRECT METHODS TO ATTACK IT.” (RAMON Y CAJAL, 1901). THIS PROPHECY TURNED OUT TO BE ACCURATE. ASTROCYTES, ONE OF THE MOST ABUNDANT CELL TYPES IN THE BRAIN, HAVE LONG BEEN THOUGHT OF AS PRIMARILY PASSIVE SUPPORT CELLS. OVER THE PAST TWO DECADES, STUDIES INDICATE THAT ASTROCYTES PLAY PIVOTAL ROLES IN NERVOUS SYSTEM DEVELOPMENT, FUNCTION, AND DISEASES. HOWEVER, A MAJOR UNRESOLVED ISSUE IN NEUROSCIENCE IS HOW ASTROCYTES INTEGRATE DIVERSE NEURONAL SIGNALS UNDER HEALTHY CONDITIONS, MODULATE NEURAL CIRCUIT STRUCTURE AND FUNCTION AT MULTIPLE TEMPORAL AND SPATIAL SCALES, AND HOW ABERRANT EXCITATION AND MOLECULAR OUTPUT INFLUENCES SENSORIMOTOR BEHAVIOR AND CONTRIBUTES TO DISEASE. THE OVERALL GOAL OF THIS U19 TEAM-RESEARCH BRAIN CIRCUIT PROGRAM PROPOSAL IS TO ADDRESS THIS FUNDAMENTAL ISSUE BY DEVELOPING A DEEPER MECHANISTIC UNDERSTANDING OF ASTROCYTES’ ROLES IN NEURAL CIRCUIT OPERATION, COMPLEX BEHAVIORS, AND BRAIN COMPUTATION THEORIES. TWO OVERARCHING QUESTIONS WILL BE ADDRESSED: 1) HOW DO ASTROCYTES TEMPORALLY AND SPATIALLY INTEGRATE MOLECULAR SIGNALS FROM THE DIVERSE TYPES OF LOCAL AND PROJECTION NEURONS ACTIVATED DURING SENSORIMOTOR BEHAVIORS. 2) HOW DO ASTROCYTES CONVERT THIS INFORMATION INTO FUNCTIONAL OUTPUTS THAT MODULATE NEURAL CIRCUIT STRUCTURE AND FUNCTION AT DIFFERENT SPATIAL AND TEMPORAL SCALES. A MULTIDISCIPLINARY, COMPREHENSIVE EFFORT IS PROPOSED TO ADDRESS THESE QUESTIONS THAT CAN ONLY BE COMPLETED THROUGH CLOSE COLLABORATION BETWEEN RESEARCHERS WITH UNIQUE AND COMPLEMENTARY EXPERTISE. AN INNOVATIVE MULTI-SCALE APPROACH INTEGRATING FUNCTIONAL, ANATOMICAL, AND GENETIC ANALYSES WITH THEORETICAL MODELING WILL BE LEVERAGED. THIS APPROACH INVOLVES QUANTITATIVE BEHAVIORAL ASSAYS, LARGE-SCALE IMAGING OF CELLULAR AND MOLECULAR DYNAMICS, TARGETED CELL-TYPE-SPECIFIC MANIPULATIONS, HIGH- THROUGHPUT OMIC TECHNIQUES, GENETIC PROFILING, PROTEIN ENGINEERING, MACHINE LEARNING, AND COMPUTATIONAL MODELING. BY INTEGRATING EXPERIMENTAL AND THEORETICAL APPROACHES, MOLECULAR, CELLULAR, AND CIRCUIT MECHANISMS WILL BE DETERMINED THROUGH WHICH ASTROCYTES INFLUENCE NEURAL CIRCUITS AND CONTRIBUTE TO COMPLEX BEHAVIORS AND BRAIN COMPUTATION THEORIES. THE EXPERIMENTAL AND DATA ANALYSIS TOOLS DEVELOPED AS PART OF THIS PROJECT WILL BE INVALUABLE FOR THE BROADER NEUROSCIENCE COMMUNITY.

DECODING AND TARGETING THE LKB1-AMPK SIGNALING PATHWAY IN CANCER

HORMONAL REGULATION OF MAMMALIAN GENE EXPRESSION

SPINAL CIRCUITS FOR MECHANICAL ITCH AND LIGHT TOUCH

ATLAS FOR NEURONAL AND GLIAL CELL TYPES SELECTIVELY VULNERABLE TO PROTEINOPATHIES DURING PROGRESSION OF ALZHEIMER'S DISEASE - ABSTRACT THE LONG-TERM GOAL OF THIS PROJECT IS TO ELUCIDATE MULTIMODAL MECHANISMS UNDERLYING SELECTIVE VULNERABILITY OF NEURONAL AND NON-NEURONAL CELLS TO PROTEINOPATHIES DURING THE PROGRESSION OF ALZHEIMER'S DISEASE (AD). THE MOST NOTABLE FEATURE OF AD IS ITS STRIKINGLY AGE- AND SEX-DEPENDENT REGIONAL ONSET AND PROGRESSION–SELECTIVE VULNERABILITY–WHICH IS MANIFEST IN DISTINCT CLINICAL PRESENTATIONS, E.G., MEMORY IMPAIRMENT IN AD AND PATTERNS OF BRAIN DEGENERATION. RECENT STUDIES ON THE STAGING OF AD NEUROPATHOLOGY SHOWED AD-RELATED TAUOPATHY BEGINS IN THE LOCUS COERULEUS (LC), FOLLOWED BY NEUROFIBRILLARY TANGLES IN THE ENTORHINAL CORTEX (EC), FOLLOWED BY HIPPOCAMPAL (HC), AND THEN NEOCORTEX, E.G., PREFRONTAL CORTEX (PFC). ONE HYPOTHESIS FOR SELECTIVE VULNERABILITY IS THAT MAJOR AD RISK GENES ARE REGIONALLY RESTRICTED, BUT THE RESULTS SHOW THEY ARE RATHER BROADLY EXPRESSED. A BODY OF EVIDENCE SUPPORT THAT GLIA PLAY MULTIPLE ESSENTIAL ROLES AT DIFFERENT BRAIN REGION IN AD PATHOGENESIS. THESE DATA SUGGEST THAT SELECTIVE VULNERABILITY IS LIKELY A RESULT OF INTERPLAY OF INTRINSIC PROPERTIES OF NEURONS AND GLIAL CELLS IN THEIR RESPONSE TO PROTEINOPATHIES IN A DYNAMIC AND SPATIOTEMPORAL MANNER AT VULNERABLE BRAIN REGIONS. FOR EXAMPLE, EC NEURONS PROJECT MULTIPLE BRAIN REGIONS INCLUDING THE EC, PFC AND CEREBELLUM. YET, DEGENERATION OF EC AND PFC OCCURS AT DIFFERENT STAGES. IN CONTRAST, THE CEREBELLUM IS LARGELY NOT AFFECTED. THERE IS EMERGING EVIDENCE THAT SUBPOPULATIONS OF MICROGLIA TREAT EXCITATORY AND INHIBITORY NEURONS DIFFERENTLY. COULD DIFFERENT GLIAL CELL TYPES OR DIFFERENTIAL RESPONSE TO PROTEINOPATHIES IN DIFFERENT PROJECTING SOURCES AND/OR TARGETS CONTRIBUTE TO SELECTIVE VULNERABILITY? THESE RESULTS UNDERSCORE THE CENTRAL IMPORTANCE OF UNDERSTANDING THE DIVERSITY OF CELL TYPES AND MOLECULAR SIGNATURE DIFFERENCES IN BOTH SEXES AT SPATIOTEMPORAL, SINGLE-CELL RESOLUTION AND MULTIMODAL SCALES BETWEEN VULNERABLE AND LESS VULNERABLE NEURONAL AND GLIAL POPULATIONS SUSCEPTIBLE TO PROTEINOPATHIES IN AD. MAPTS305N;INT10+3 AND APPNL-F KNOCK-IN (KI) AD MICE DISPLAYING TAU AND ASS PROTEINOPATHIES, RESPECTIVELY, WILL BE USED TO ACHIEVE THIS GOAL. PRELIMINARY RESULTS WERE OBTAINED TO SUPPORT THE PROJECT. FIRST, BARCODED SINGLE-NEURONS BRAIN-WIDE PROJECTION MAPPING IN CONTROLS DEMONSTRATES SEX- AND AGE- DEPENDENT DIFFERENCES IN PROJECTIONS OF LC NEURONS TO SELECTIVE TARGETS. THE RESULTS SHOWED RECIPROCAL INNERVATIONS AMONG THESE FOUR BRAIN REGIONS. SECOND, THE ANALYSIS OF 16-MONTH-OLD CONTROL AND APPNL-F MICE SHOWED ABERRANT INNERVATION PATTERNS BY NOREPINEPHRINE (NE) NEURONS. THIRD, IMPAIRED PREPULSE INHIBITION (PPI) HAS BEEN SUGGESTED TO BE A BIOMARKER FOR PRODROMAL AD. IMPORTANTLY, PPI IMPAIRMENT WAS OBSERVED IN THE 5-MONTH-OLD APPNL-F MICE. FINALLY, SINGLE NUCLEI (SN)-RNASEQ OF THE EC FROM 16-MONTH-OLD CONTROL AND APPNL-F MICE SHOWED SELECTIVE LOSS OF NEURONAL AND NON-NEURONAL CELL TYPES AND DIFFERENTIAL EXPRESSION OF A LIST OF GENES, INCLUDING THOSE ASSOCIATED WITH NEURONAL AND GLIAL FUNCTION AND IMPAIRED PPI. SN-TRANSCRIPTOMICS AND-EPIGENOMICS WILL BE EMPLOYED TO IDENTIFY VULNERABLE CELL TYPES FOLLOWED BY SPATIAL TRANSCRIPTOMICS TO INVESTIGATE SPATIOTEMPORAL CORRELATION BETWEEN PROTEINOPATHIES AND VULNERABLE CELL TYPES AND GENE NETWORKS.

ORGANIZATION AND FUNCTION OF MOUSE VISUAL CORTICAL AREAS

COMBINING SINGLE CELL APPROACHES AND A DEVELOPMENTAL PERSPECTIVE TO DISCOVER STEM CELL CONTROL CIRCUITS AND THE CELLULAR AND MOLECULAR BASES OF CANCE

SOMATIC CELL CYCLE REGULATION BY PHOSPHORYLATION

CORE GRANT FOR VISION RESEARCH

THIS PROJECT PLATFORM WILL MITIGATE CURRENT DRUG-BASED TREATMENTS' LIMITATIONS BY AVOIDING OFF-TARGET AND SYSTEMIC SIDE EFFECTS WHILE PROVIDING A NOVEL, SCALABLE APPROACH TO CLINICAL APPLICATIONS WITH UNPARALLELED PRECISION AND SAFETY. A SONOGENETIC METHOD WILL BE USED TO ALLEVIATE SYMPTOMS OF A CLINICALLY RELEVANT INDICATION IN MICE AND LARGE ANIMAL MODELS.

HISTIDINE PHOSPHORYLATION AS A NEW TARGET FOR CANCER THERAPY

SIGNAL TRANSDUCTION BY TYROSINE PHOSPHORYLATION

CELL REGULATION, DIFFERENTIATION AND CANCER

HOST-MICROBE INTERACTIONS: HARNESSING CO-EVOLUTION TO TREAT DISEASE

SINGLE-CELL APPROACHES TO REVEAL HOW JUMPING GENES INDIVIDUALIZE NEURAL CIRCUITS

LOCAL CONNECTIONS OF INHIBITORY CORTICAL NEURONS

NEURAL CIRCUIT MECHANISMS OF SOCIAL HOMEOSTASIS IN INDIVIDUALS AND SUPRAORGANISMAL SOCIAL GROUPS

REGULATION OF HEPATIC GLUCONEOGENESIS BY THE CREB:TORC2 PATHWAY

MOLECULAR DETERMINANTS OF NEURAL DEVELOPMENT

SAN DIEGO NATHAN SHOCK CENTER

TRAVELING WAVES IN NEOCORTICAL CIRCUITS: MECHANISMS, COMPUTATIONAL ROLES IN SENSORY PROCESSING, AND IMPACT ON SENSORY PERCEPTION - PROJECT SUMMARY/ABSTRACT AN IMPORTANT LONGSTANDING GOAL IN NEUROSCIENCE RESEARCH IS TO UNDERSTAND HOW LARGE-SCALE SPATIOTEMPORAL PATTERNS OF NEURAL ACTIVITY EMERGE IN THE BRAIN AND WHETHER THEY HAVE A DIRECT ROLE IN SHAPING THE BRAIN’S COMPUTATIONAL PROCESSES AND THEREBY MAMMALIAN BEHAVIOR. NOTABLY, TRAVELING WAVES OF NEURAL ACTIVITY HAVE BEEN IMPLICATED IN SENSORY, COGNITIVE, AND MOTOR BEHAVIORS, AND THIS TEAM RECENTLY FOUND THAT INTRINSIC TRAVELING WAVES (ITWS) IN VISUAL CORTICAL AREAS PREDICT THE MAGNITUDE OF EVOKED SPIKING ACTIVITY AND VISUAL PERCEPTUAL SENSITIVITY IN AWAKE, BEHAVING NON-HUMAN PRIMATES. HOWEVER, IT REMAINS UNCLEAR WHETHER ITWS REFLECT AN UNDERLYING FUNDAMENTAL MECHANISM OF CORTICAL FUNCTION OR WHETHER THEY MIGHT SIMPLY BE EPIPHENOMENAL. THIS PROJECT WILL EXAMINE THE ROLE OF NEOCORTICAL ITWS IN VISUAL PERCEPTION AND IS GUIDED BY A THEORETICAL MODEL OF ITW ACTIVITY THAT MAKES SPECIFIC TESTABLE AND FALSIFIABLE PREDICTIONS ABOUT THE MECHANISMS THAT GIVE RISE TO THE SPATIOTEMPORAL FEATURES OF ITWS AND THEIR ROLES IN SENSORY BEHAVIOR. THE MODEL IS BASED ON A NETWORK OF SPIKING NEURONS AND PREDICTS THAT ITWS EMERGE FROM THE ANATOMICAL ORGANIZATION OF HORIZONTAL CORTICAL FIBERS AND RESULT IN SHIFTS IN THE BALANCE OF EXCITATORY (E) AND INHIBITORY (I) POPULATION ACTIVITY THAT TRAVEL AS WAVES, THE MODEL FURTHER PREDICTS THAT ITWS ARE RETINOTOPICALLY COORDINATED ACROSS VISUAL CORTICAL AREAS, AND THAT THIS COORDINATION IS THE MECHANISM BY WHICH ITWS IMPROVE PERCEPTION. HOWEVER, THESE PREDICTIONS COULD BE WRONG. EMPIRICAL TESTS OF THE MODEL’S PREDICTIONS WILL REQUIRE ACTIVITY MEASUREMENTS FROM SPECIFIC NEURON-TYPES, ACROSS WIDE FIELDS- OF-VIEW, AND WITH EXCELLENT SPATIAL AND TEMPORAL RESOLUTION IN AWAKE BEHAVING ANIMALS. TO PERFORM SUCH MEASUREMENTS, THE TEAM RECENTLY INVENTED SEVERAL NEW TECHNOLOGIES: (1) GENETICALLY ENCODED FLUORESCENT VOLTAGE INDICATORS (GEVIS) THAT THEY WILL TARGET TO SPECIFIC E OR I NEURAL POPULATIONS; (2) A CUSTOM-BUILT DUAL-COLOR FLUORESCENCE MESOSCOPE TO TRACK THE SUBTHRESHOLD POPULATION VOLTAGE DYNAMICS OF 2 NEURON-TYPES AT ONCE, ACROSS A 8-MM FIELD-OF-VIEW SPANNING MULTIPLE CORTICAL AREAS IN MARMOSET AND MOUSE CORTEX; (3) TRANSPARENT ELECTRODE ARRAYS THAT ALLOW MEASUREMENTS OF LFPS TO BE PERFORMED ACROSS THE CORTEX CONCURRENTLY WITH VOLTAGE IMAGING STUDIES OF E AND I POPULATION ACTIVITY; (4) TRANSPARENT LAMINAR ELECTRODE ARRAYS TO MEASURE THE SPIKING ACTIVITY OF CELLS ACROSS THE DIFFERENT CORTICAL LAYERS. BY COMBINING THE USE OF THESE FOUR INNOVATIONS IN MARMOSETS AND MICE PERFORMING A VISUAL DETECTION TASK, THE TEAM WILL TEST THE PREDICTIONS OF THE THEORETICAL MODEL AND LEARN HOW DIFFERENT CORTICAL NEURON-TYPES IMPACT ITW DYNAMICS AND PERCEPTUAL SENSITIVITY. AIM 1 TESTS THE HYPOTHESIS THAT E/I POPULATION ACTIVITY TRAVELS AS ITWS IN MICE AND MARMOSETS. AIM 2 TESTS THE PREDICTION THAT ITWS ARE COORDINATED RETINOTOPICALLY ACROSS CORTICAL AREAS, AND THAT THIS COORDINATION ENHANCES PERCEPTUAL SENSITIVITY. AIM 3 WILL ACCOUNT MECHANISTICALLY FOR THE RESULTS OF AIMS 1 AND 2 IN A THEORETICALLY GROUNDED SPIKING NETWORK MODEL. OVERALL, BY USING INNOVATIVE NEW TECHNOLOGIES THIS INTERDISCIPLINARY TEAM WILL PROVIDE KEY INSIGHTS INTO LONGSTANDING CONCEPTUAL ISSUES OF PROFOUND IMPORTANCE TO BRAIN RESEARCH.

CIRCADIAN PHOTOENTRAINMENT IN MAMMALS

TAM RECEPTORS AND FLAVIVIRUS INFECTION

GENETIC MODELS OF SPORADIC ALZHEIMERS DISEASE IN THE MARMOSET - ABSTRACT THE LONG-TERM GOAL OF THIS PROJECT IS TO DEVELOP, CHARACTERIZE, AND VALIDATE GENETICALLY MODIFIED MARMOSET MODELS OF SPORADIC ALZHEIMER'S DISEASE (AD) THAT WILL SERVE AS TOOLS FOR INVESTIGATING MOLECULAR AND CELLULAR DISEASE MECHANISMS, AND FOR IDENTIFYING THERAPEUTIC TARGETS. AD IS THE MOST COMMON CAUSE OF DEMENTIA, WITH THE MAJORITY OF CASES (~95%) APPEARING TO BE SPORADIC, WHICH IS CAUSED BY COMPLEX INTERACTIONS BETWEEN MULTIPLE GENE VARIANTS AND ENVIRONMENTAL FACTORS. NUMEROUS MODELS OF AD HAVE BEEN DEVELOPED (E.G., MICE); THESE MODELS ARE PRIMARILY FOCUSED ON FAMILIAL FORMS OF AD AND HAVE ENABLED SIGNIFICANT PROGRESS TOWARD UNDERSTANDING AD, BUT THEY FAIL TO RECAPITULATE THE FULL SPECTRUM OF MOLECULAR, CELLULAR, BEHAVIORAL, AND COGNITIVE PATHOLOGIES OBSERVED IN AD AND PROVIDE POOR PREDICTIVE VALUE WHEN TRYING TO TRANSLATE FINDINGS TO HUMAN CLINICAL TRIALS. SEVERAL LINES OF EVIDENCE SUGGEST THAT MARMOSETS MAY EFFECTIVELY BRIDGE THE GAP BETWEEN MICE AND HUMANS FOR BOTH BASIC AND TRANSLATIONAL NEUROSCIENCE RESEARCH. FIRST, MARMOSETS AND HUMANS HAVE VERY SIMILAR BRAIN STRUCTURES, COGNITIVE/SOCIAL BEHAVIORAL REPERTOIRES, METABOLISM, AND IMMUNE FUNCTIONS. SECOND, COMPARED TO OTHER PRIMATES, MARMOSETS HAVE SHORT LIFESPAN, SMALL BODY SIZE, AND HIGH REPRODUCTIVE POWER. FINALLY, GENE EDITING TOOLS ARE NOW AVAILABLE TO GENERATE VARIOUS TYPES OF GENETICALLY MODIFIED MARMOSETS. APOLIPOPROTEIN E (APOE) IS THE STRONGEST RISK FACTOR FOR LATE-ONSET, SPORADIC AD AND ALSO INCREASES THE AGE- DEPENDENT RISK OF MONOGENIC FAMILIAL AD AND INCIDENCE OF AD IN WOMEN. THERE ARE THREE APOE ALLELES IN HUMANS WITH THE APOE*E4 ALLELE CONFERRING INCREASED RISK AND THE APOE*E2 ALLELE CONFERRING DECREASED RISK RELATIVE TO THE COMMON APOE*E3 ALLELE. APOE ISOFORMS DIFFERENTIALLY MODULATE BOTH AMYLOID-SS (ASS)-DEPENDENT AND ASS-INDEPENDENT PATHWAYS TO AFFECT BRAIN HOMEOSTASIS, INCLUDING TAU-MEDIATED NEURODEGENERATION, MICROGLIAL INFLAMMATION, LIPID TRANSPORT, SYNAPTIC INTEGRITY, GLUCOSE METABOLISM AND CEREBROVASCULAR FUNCTION. THESE THREE APOE ALLELES DIFFER WITH REGARD TO CYSTEINE (C) AND ARGININE (R) AMINO ACIDS AT POSITIONS 112 AND 158 (C112/C158 IN APOE2; C112/R158 IN APOE3; AND R112/R158 IN APOE4). SEVERAL LINES OF EVIDENCE DEMONSTRATE THAT R61 IS CRITICAL FOR APOE4-MEDIATED AD RISK. MARMOSET APOE (MAPOE) IS ENCODED BY A SINGLE ALLELE AND CONTAINS THE EQUIVALENT OF R112 AND R158, BUT LACKS THE CRITICAL R61 EQUIVALENT (IT CONTAINS T INSTEAD), SUGGESTING THAT IT BEHAVES LIKE HUMAN APOE3. TO TEST THIS HYPOTHESIS, MAPOE T61R MUTANT PROTEIN WAS GENERATED TO TEST THE EFFECT ON INFLAMMATORY RESPONSES INDUCED BY LIPOPOLYSACCHARIDES IN MICROGLIAL CELLS. THE MAPOE T61R VARIANT RESULTED IN A MORE ROBUST RESPONSE COMPARED TO WILD TYPE MAPOE. SIMILAR RESULTS WERE FOUND WHEN HUMAN APOE4 WAS USED (APOE4>APOE3). TAKEN TOGETHER, THESE PRELIMINARY RESULTS INDICATE THAT MAPOE T61R IS FUNCTIONALLY EQUIVALENT TO HUMAN APOE4. FURTHER, SEVERAL PAIRS OF CRISPR GRNAS FOR GENERATING AN APOE NULL MUTATION HAVE BEEN IDENTIFIED. TO INVESTIGATE THE ROLE OF APOE IN SPORADIC AD IN THE MARMOSET, APOE NULL AND T61R MUTANT MARMOSETS WILL BE GENERATED AND CHARACTERIZED.

THE DISCOVERY OF HUMAN PEPTIDE ENCODING GENES

EPIGENOMIC CELL-TYPE CLASSIFICATION AND REGULATORY ELEMENT IDENTIFICATION IN THE HUMAN BRAIN

REGULATION OF MEMORY CD8 T CELL DEVELOPMENT

3/3-SCHIZOPHRENIA GENETICS AND BRAIN SOMATIC MOSAICISM

GENERATION OF FUNCTIONAL HUMAN ORGANS AND TISSUES USING INTERSPECIFIC BLASTOCYST COMPLEMENTATION

GENETIC FACTORS CONTROLLING THE INTENSITY OF SOCIAL BEHAVIOR

REGULATORY T CELL LINEAGE STABILITY CONTROLLED BY FOXP3 CNS2

(PQ5) MITOCHONDRIAL HETEROGENEITY IN MELANOMA TUMOR AND IMMUNE RESPONSES

TOLERANCE DEFENSES IN HOST-MICROBIOTA INTERACTIONS

DISSECTING NEURAL MECHANISMS INTEGRATING MULTIPLE INPUTS IN C. ELEGANS

SINGLE CELL ANALYSIS FOR FORENSIC EPIGENETICS (SAFE)

HIGH-THROUGHPUT MAPPING OF SELECTIVELY VULNERABLE CELL TYPES AND PROJECTIONS IN AGING AND ALZHEIMER'S DISEASE

SOLVING THE VALENCE ASSIGNMENT PROBLEM - PROJECT SUMMARY THE ABILITY TO DISCRIMINATE BETWEEN WHAT IS “GOOD” AND “BAD” IS TERMED VALENCE PROCESSING, AND PATHOLOGICAL PERTURBATIONS IN VALENCE PROCESSING CAN EXPLAIN MENTAL HEALTH DISORDERS RANGING FROM ANXIETY AND DEPRESSION TO COMPULSIVITY AND BIPOLAR DISORDER. THE KEY TO DEVELOPING MORE EFFECTIVE TREATMENTS FOR MENTAL HEALTH DISORDERS WITH FEWER SIDE-EFFECTS WILL BE IN THE SYNAPTIC, CELLULAR, AND CIRCUIT MECHANISMS. THIS PROPOSAL NOT ONLY LAYS OUT A VERY SPECIFIC RESEARCH PLAN TO PROBE THE FUNCTIONAL ROLE OF A NEUROPEPTIDE, NEUROTENSIN (NT), BUT IT ALSO LAYS OUT A COMPREHENSIVE, SYSTEMATIC APPROACH TO INVESTIGATING NEUROMODULATORY SYSTEMS. THE GENERAL APPROACH INCLUDES: 1) IDENTIFY A CIRCUIT THAT PLAYS A CAUSAL AND CRITICAL ROLE IN VALENCE PROCESSING, 2) PROFILE THE TRANSCRIPTOME OF THIS CIRCUIT COMPONENT, 3) SELECT FOR SURFACE RECEPTORS OR OTHER DRUGGABLE TARGETS, 4) DETERMINE THE CONTRIBUTION OF THIS NEUROMODULATORY SIGNAL ON PLASTICITY, 5) ESTABLISH THE INPUT-OUTPUT ARCHITECTURE OF THE NEUROMODULATORY INNERVATION AND POSTSYNAPTIC, DOWNSTREAM TARGETS, AND 6) ESTABLISH A CAUSAL ROLE FOR THIS PARTICULAR NEUROMODULATORY SIGNAL IN NEURAL ACTIVITY AND BEHAVIOR ASSOCIATED WITH VALENCE PROCESSING. THE SPECIFIC HYPOTHESES INCLUDED IN THIS PROPOSAL ARE: THAT NT SERVES TO GUIDE PLASTICITY TO THE APPROPRIATE TARGET, THAT THERE ARE PARALLEL CIRCUITS THAT HAVE SOME LOCAL INTERACTION IN THE BLA, AND THAT NT ALTERS THE CODING DYNAMICS BY INCREASING SIGNAL-TO-NOISE RATIO BY AMPLIFYING SIGNAL BY MODULATION OF GLUTAMATERGIC TRANSMISSION. A SUCCESSFUL OUTCOME OF THIS PROJECT WILL PROVIDE A SPECIFIC MODEL FOR HOW A NEUROMODULATORY SIGNAL SUCH AS NT CAN SOLVE THE “VALENCE ASSIGNMENT PROBLEM.”

UNBIASED EPIGENOMIC AND TRANSCRIPTOMIC PROFILING OF NON-NEURONAL CELLS IN THE MOUSE BRAIN

REGULATION OF MEMORY CD8 T CELL DEVELOPMENT

EPIGENOME-BASED CELL CENSUS AND REGULATORY ELEMENT DISCOVERY IN THE AGING MOUSE BRAIN

MECHANISMS GOVERNING DEVELOPMENT OF VISUAL CONNECTIONS

EXTRAMURAL RESEARCH FACILITIES IMPROVEMENT PROGRAM DATA*

DEVELOPMENT OF METHODS FOR MULTI-OMIC ANALYSIS OF DNA METHYLATION AND CHROMATIN ARCHITECTURE IN SINGLE CELLS

ASTROCYTE REGULATION OF NEURONAL AMPA GLUTAMATE RECEPTORS

SALK INSTITUTE FOR BIOLOGICAL STUDIES NINDS CENTER CORE GRANT

PEPTIDERGIC NEUROMODULATION OF MICROCIRCUITS THAT CONTROL CHEMOSENSATION-INDUCED BEHAVIORS

ARABIDOPSIS 2010: TOOLS AND TECHNOLOGIES TO ENABLE GENOME-WIDE SCREENS IN ARABIDOPSIS

DEFINING CRITICAL P53 THERAPEUTIC TARGETS AND MECHANISMS

MECHANISM OF BETA-CATENIN AND APC-REGULATED TRANSCRIPTION OF WNT TARGET GENES

ARABIDOPSIS 2010: REGULATORY NETWORKS CONTROLLING HORMONE-MEDIATED GROWTH

SPATIAL REGULATION OF DEVELOPMENTAL GENE EXPRESSION

THE ROLE OF BAF RELATED COMPLEXES IN REGULATORY T CELL DEVELOPMENT AND FUNCTION

USING GEROSCIENCE TO UNDERSTAND AND TREAT ALZHEIMER'S DISEASE

IMPACT OF TIME-RESTRICTED FEEDING IN REDUCING CANCER RISK THROUGH OPTIMIZING MITOCHONDRIA FUNCTION - PROJECT SUMMARY THIS APPLICATION, IN RESPONSE TO RFA-CA-004 “RESEARCH ANSWERS TO NATIONAL CANCER INSTITUTE'S (NCI) PROVOCATIVE QUESTIONS (R01 CLINICAL TRIAL OPTIONAL),” WILL ADDRESS “PQ2: HOW DOES INTERMITTENT FASTING AFFECT CANCER INCIDENCE, TREATMENT RESPONSE, OR OUTCOME?” OBESITY AND AGE ARE TWO MAJOR RISK FACTORS FOR CANCER DEVELOPMENT. THUS, THERAPEUTIC INTERVENTIONS THAT PREVENT OR DELAY THE DEVELOPMENT OF EXCESSIVE WEIGHT GAIN AND/OR AGE-ASSOCIATED PHYSIOLOGICAL DYSFUNCTION HOLD GREAT PROMISE FOR REDUCING CANCER RISK IN THE INCREASINGLY OBESE AND ELDERLY GLOBAL POPULATION. ONE SUCH INTERVENTION IS TIME-RESTRICTED EATING (TRE), A PRAGMATIC FORM OF INTERMITTENT FASTING IN WHICH DAILY CALORIC INTAKE IS CONSTRAINED TO A CONSISTENT WINDOW OF 8–12 HOURS WITHOUT EXPLICITLY REDUCING TOTAL CALORIC INTAKE. IN YOUNG MALE MICE, TIME-RESTRICTED FEEDING (TRF) REDUCES CANCER RISK BY PREVENTING OBESITY AND METABOLIC DISEASES. TRF HAS ALSO BEEN SHOWN TO REDUCE BREAST CANCER XENOGRAFT PROGRESSION IN OBESE MICE. IN HUMANS, SHORT-TERM CLINICAL STUDIES OF TRE HAVE REVEALED METABOLIC IMPROVEMENTS THAT PREDICT REDUCED CANCER RISK, AND EPIDEMIOLOGICAL EVIDENCE SUGGESTS THAT PROLONGED NIGHTLY FASTING CAN REDUCE THE RISK OF CANCER, INDEPENDENT OF CHANGES IN BODY WEIGHT. THIS PROMISING PRELIMINARY EVIDENCE SUGGESTS THAT TRE MAY BE AN EFFECTIVE INTERVENTION FOR REDUCING CANCER RISK. HOWEVER, THE EFFECTS OF TRF IN AGED ANIMALS AND IN THE CONTEXT OF AN OBESOGENIC WESTERN DIET HAVE NOT YET BEEN ESTABLISHED, AND THE MECHANISMS BY WHICH TRF REDUCES CANCER RISK REMAIN UNKNOWN. THIS APPLICATION BUILDS UPON PROMISING PRELIMINARY DATA AND LEVERAGES THE COMPLEMENTARY SKILLS OF THE RESEARCH TEAM TO ADDRESS THESE CRITICAL GAPS IN KNOWLEDGE. BOTH OBESITY AND AGING ARE ASSOCIATED WITH MITOCHONDRIAL DYSFUNCTION AND THE PRODUCTION OF PRO-TUMORIGENIC MITOCHONDRIAL METABOLITES. PROPOSED EXPERIMENTS TEST THE HYPOTHESIS THAT TRF OPTIMIZES MITOCHONDRIA FUNCTION THROUGH BOTH CELL-AUTONOMOUS AND SYSTEMIC MECHANISMS, THEREBY REDUCING CANCER RISK. IN AIM 1, THE IMPACT OF TRF ON MITOCHONDRIA FUNCTION AND RELATED PHYSIOLOGIES WILL BE ESTABLISHED IN AGED MICE. NUTRIENT METABOLISM, ENERGY CONSUMPTION, AND MITOCHONDRIA FUNCTION WILL BE ASSESSED IN THESE MICE. IN AIM 2, AN INNOVATIVE COMBINATION OF METABOLOMICS AND MITOCHONDRIA RESPIRATION ASSAYS WILL BE USED TO TEST THE IMPACT OF TRF ON MITOCHONDRIA FUNCTION IN NORMAL AND CANCER CELLS (ASSESSING BOTH CELL-AUTONOMOUS AND NON-CELL-AUTONOMOUS MECHANISMS). THE EFFECTS OF TRF ON TUMOR INCIDENCE WILL BE ASSESSED BY SUBJECTING TUMOR-PRONE MICE TO TRF. IN AIM 3, PLASMA COLLECTED FROM A RECENTLY CONCLUDED HUMAN TRE INTERVENTION STUDY WILL BE USED TO TEST THE EFFECT OF TRE ON MITOCHONDRIA FUNCTION AND CANCER RISK IN HUMANS. THE PROPOSED COMPARATIVE ANALYSIS OF TRE IN HUMANS AND MICE WILL PROVIDE CRITICAL MECHANISTIC INSIGHT INTO HOW ONE FORM OF INTERMITTENT FASTING CAN HELP PREVENT CANCER ONSET AND IMPROVE TREATMENT OUTCOMES.

DEVELOPMENTAL PHYSIOLOGY OF CILIATED EPITHELIA

DYNAMICS OF ACTIVITY-INDUCED TRANSCRIPTION IN SINGLE DENTATE GRANULE CELLS

PHYSIOLOGY AND FUNCTION OF BASAL GANGLIA SUBCIRCUITS IN SEQUENCE LEARNING

REGULATION OF HEPATIC GLUCONEOGENESIS BY THE CREB:TORC2 PATHWAY

SPATIOTEMPORAL PATTERNS OF NEURAL ACTIVITY AND THEIR ROLE IN PERCEPTION

THE CONTRIBUTION OF OLD AGE-ASSOCIATED PROTEOTOXICITY TO INFLAMMATION AND DEMENTIA

THE ROLE OF LONG-LIVED PROTEINS IN THE SURVIVAL OF NERVE CELLS

NEURAL CONTROL OF SKILLED MOVEMENTS: AN ETHOLOGICAL DISSECTION OF GENETICALLY TRACTABLE MAMMALIAN MOTOR CIRCUITS

EPIGENETIC ALTERATIONS OF THE DEVELOPING BRAIN IN ANIMAL MODELS OF SCHIZOPHRENIA

REGULATION OF NF-KAPPA B

TOOLS AND TECHNOLOGIES TO ENABLE GENOME-WIDE SCREENS IN ARABIDOPSIS

ESTABLISHMENT AND MODULATION OF DNA METHYLATION PATTERNS IN ARABIDOPSIS

TRANSCRIPTOMIC SINGLE-CELL PROFILING IN BREATHING-SPECIFIC PARABRACHIAL MU-OPIOID RECEPTOR NEURONS - ABSTRACT OPIOIDS ARE THE MOST COMMONLY USED AND MOST EFFECTIVE ANALGESICS, AND ARE THE FIRST LINE OF DEFENSE AGAINST ACUTE AND SEVERE PAIN. HOWEVER, THIS DRAMATIC ABILITY TO MITIGATE PAIN COMES WITH MANY SIDE EFFECTS. THESE INCLUDE CONSTIPATION, NAUSEA, SEDATION, DIZZINESS, RESPIRATORY DEPRESSION, DEPENDENCE, AND ADDICTION. AMONG THESE, RESPIRATORY DEPRESSION IS THE MAJOR DRIVER OF DEATH BY OPIOID OVERDOSE. ACCORDING TO THE CENTER FOR DISEASE CONTROL (CDC), NEARLY 50,000 PEOPLE DIED IN 2019 BY OPIOID-INDUCED RESPIRATORY DEPRESSION (OIRD) IN THE UNITED STATES, AND THE DEATH RATE IS RISING RAPIDLY DUE TO INCREASED MISUSE AND ADDICTION TO BOTH PRESCRIPTION AND ILLICIT OPIOIDS. THUS, THE US IS CURRENTLY EXPERIENCING A SERIOUS NATIONAL PUBLIC HEALTH CRISIS THAT IS ALSO TAKING A TOLL ON SOCIAL ECONOMIC WELFARE. DESPITE THESE DIRE NUMBERS, RESEARCH ELUCIDATING THE NEURAL MECHANISMS OF OIRD, WHICH COULD IDENTIFY THERAPEUTIC TARGETS, HAS NOT BEEN RIGOROUSLY INVESTIGATED. ANIMAL STUDIES HAVE SHOWN THAT OIRD AND OPIOID ANALGESIA ARE BOTH MEDIATED BY THE Μ-OPIOID RECEPTOR (MOR), HOWEVER THE NEURAL CIRCUITS AND BRAIN REGIONS RESPONSIBLE FOR OIRD AND OPIOID ANALGESIA ARE NOT FULLY UNDERSTOOD. THE PROPOSED RESEARCH AIMS TO DISSECT THE NEURAL CIRCUITS THAT SELECTIVELY MEDIATE OIRD OR OPIOID ANALGESIA USING CUTTING-EDGE MOLECULAR, PHYSIOLOGICAL, BEHAVIORAL, AND IMAGING TECHNIQUES. PROJECTION-SPECIFIC SINGLE-CELL TRANSCRIPTOMIC ANALYSIS WILL THEN BE USED TO IDENTIFY FUNCTIONAL MARKERS EXPRESSED IN THE MOR- EXPRESSING NEURONS THAT SPECIFICALLY MEDIATE OIRD, NOT OPIOID ANALGESIA. SUCCESSFUL COMPLETION OF THE PROPOSED RESEARCH WILL IDENTIFY NOVEL THERAPEUTIC TARGETS THAT SELECTIVELY RESCUE OIRD WITHOUT ALTERING ANALGESIC EFFECTS OF OPIOIDS.

INTERSECTIONAL GENETIC DISSECTION OF SPINAL CIRCUITS PROCESSING PAIN AND ITCH

INVESTIGATING THE EFFECTS OF STRUCTURAL VARIANTS ON 3D GENOME ORGANIZATION AND GENE REGULATION IN CANCER GENOMES

ENGINEERING HUMAN ISLET-LIKE ORGANOIDS FOR TRANSPLANTATION

STRUCTURAL BASIS FOR ACTIVITY OF AND RESISTANCE TO HIV INTEGRASE INHIBITORS

STRUCTURAL BASIS FOR ACTIVITY OF AND RESISTANCE TO HIV INTEGRASE INHIBITORS - ABSTRACT THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1, HEREAFTER REFERRED TO AS HIV) CURRENTLY INFECTS ~40 MILLION PEOPLE WORLDWIDE, AND THE NUMBER OF INFECTED INDIVIDUALS CONTINUES TO RISE. IN THE ABSENCE OF A CURE, ANTIRETROVIRAL THERAPY REPRESENTS THE PRIMARY TREATMENT OPTION, BECAUSE IT SLOWS DISEASE PROGRESSION AND REDUCES NEW INFECTIONS. INTEGRASE (IN) STRAND TRANSFER INHIBITORS (INSTIS) ARE A CLASS OF ANTIRETROVIRAL THERAPEUTICS THAT BLOCK INTEGRATION OF VIRAL DNA INTO HOST CHROMOSOMES, A PROCESS THAT IS MEDIATED BY THE VIRAL IN ENZYME, WHICH ASSEMBLES INTO OLIGOMERIC NUCLEOPROTEIN COMPLEXES ON THE ENDS OF VIRAL DNA, TERMED “INTASOMES”. INSTIS SELECTIVELY TARGET INTASOMES AND REPRESENT FIRST-LINE THERAPIES IN THE CLINIC. HOWEVER, THE EMERGENCE OF IN VARIANTS RESISTANT TO INSTIS IS BECOMING A GREATER CLINICAL PROBLEM. STRUCTURAL BIOLOGY APPROACHES CAN SHED LIGHT ON THE MECHANISMS UNDERLYING DRUG ACTION AND RESISTANCE, PROVIDING USEFUL INFORMATION FOR RATIONALLY IMPROVING THE CURRENT INSTIS. WHEN COMPLEMENTED WITH ANCILLARY TECHNIQUES, SUCH AS BIOCHEMICAL ACTIVITY ASSAYS, BIOPHYSICAL THERMODYNAMIC AND KINETIC MEASUREMENTS, CELLULAR VIROLOGY, AND DIVERSE COMPUTATIONAL APPROACHES INCLUDING FREE ENERGY CALCULATIONS, THE STRUCTURES PRECISELY DETAIL MECHANISMS OF RESISTANCE AGAINST SPECIFIC INSTIS AND PROVIDE GUIDANCE FOR DESIGNING AND DEVELOPING NOVEL 3RD GENERATION INSTIS TO FIGHT INFECTIONS. IN THIS PROPOSAL, APPROACHES CENTERED AROUND USING REVOLUTIONARY ADVANCES IN CRYO-ELECTRON MICROSCOPY FOR STRUCTURAL STUDIES WILL SHOW HOW INSTIS INTERACT WITH THEIR NATURAL DRUG TARGET, THE HIV INTASOME (BOTH WT AND MUTANT), AND ELUCIDATE THE MECHANISMS BY WHICH RESISTANCE TO THESE DRUGS EMERGES. THERE ARE THREE SPECIFIC AIMS THAT WILL: (1) EXTEND AND BUILD UPON CURRENT EFFORTS TO PROVIDE A MECHANISTIC UNDERSTANDING OF BOTH WHY AND HOW SELECT VIRAL RESISTANT VARIANTS (VRVS) ARISE IN RESPONSE TO THE CLINICALLY USED DRUG DOLUTEGRAVIR (DTG) OR THE MOST POTENT DEVELOPMENTAL IN-HOUSE COMPOUND THAT 4D, WHICH IS CURRENTLY UNDER PRE-CLINICAL EVALUATION; (2) BROADLY IDENTIFY AND ANALYZE NOVEL MECHANISMS AND PATHWAYS OF DRUG RESISTANCE THAT ARISE IN RESPONSE TO TREATMENT WITH 2ND GENERATION DRUGS, HIGHLIGHTING BOTH PRIMARY AND COMPENSATORY MUTATIONS, AND PROVIDING STRATEGIES TO PREDICT FUTURE VARIANTS; (3) SELECT FOR RESIDUAL RESISTANT VARIANTS ARISING IN RESPONSE TO TREATMENT WITH NOVEL 3RD GENERATION INSTIS THAT WERE SYNTHESIZED BASED ON THE CONCEPT OF SUBSTRATE MIMICRY, MANY OF WHICH EFFECTIVELY INHIBIT VIRAL RESISTANT VARIANTS THAT ARISE IN RESPONSE TO TREATMENT WITH 2ND GENERATION CLINICALLY USED INSTI DRUGS, AND EXPLAIN MECHANISMS UNDERLYING THE SUPERIOR POTENCY OF NOVEL COMPOUNDS. THIS WORK WILL IMPROVE OUR UNDERSTANDING OF AN IMPORTANT CLASS OF DRUGS USED TO TREAT PEOPLE LIVING WITH HIV, IDENTIFY MECHANISMS, PATHWAYS, AND PATTERNS OF CLINICALLY RELEVANT RESISTANCE TO INSTIS, AND PROVIDE SPECIFIC GUIDELINES FOR THEIR RATIONAL IMPROVEMENT.

PREVENTING THE TRANSMISSION OF MITOCHONDRIAL DISEASES THROUGH HETEROPLASMIC SHIFT

THE ROLE OF DNA METHYLATION DYNAMICS AND PATTERNING IN POSTMITOTIC NEURONAL-MATURATION

EPIGENOMIC MAPPING APPROACHES FOR CELL-TYPE CLASSIFICATION IN THE BRAIN

LENTIVIRAL VECTORS FOR GENE TRANSFER

CROSS-TALK BETWEEN THE CIRCADIAN CLOCK AND THE CAMP SIGNALING PATHWAY

MOLECULAR SIGNAL INTEGRATION FOR ROOT GROWTH CONTROL

ROLE OF LKB1 AND AMPK IN METFORMIN AND TZD CONTROL OF GLUCOSE METABOLISM IN LIVER

THE CONTRIBUTION OF KIDNEY DISEASE TO AGE-ASSOCIATED COGNITIVE DECLINE

C. ELEGANS AS A MODEL FOR TELOMERE MAINTENANCE IN CANCER

INITIAL EVENTS IN PHOTORECEPTOR SIGNALING

GENETIC CONTROL OF MOTOR NEURON DEVELOPMENT AND FUNCTION

UNDERSTANDING THE ROLE OF AUTOPHAGY-REGULATED CELL DEATH IN THE ESCAPE FROM REPLICATIVE CRISIS

DEVELOPMENT OF MARMOSET MODELS OF NEURODEGENERATIVE DISEASE USING EMBRYONIC STEM CELL-BASED GENE-EDITING APPROACHES

MULTISCALE MODELING AND LARGE-SCALE RECORDINGS OF TRAUMA-INDUCED EPILEPTOGENESIS

ENVIRONMENTAL CONTRIBUTION TO NEURONAL-METHYLOME DYNAMICS IN ANIMAL MODELS OF AUTISM SPECTRUM DISORDERS

DEVELOPING A NONINVASIVE METHOD TO MANIPULATE SPECIFIC CELL TYPES WITHIN THE MAMMALIAN BRAIN

OPTIMIZED CIRCADIAN RHYTHMS FOR THE PREVENTION OF ALZHEIMER'S DISEASE

A NUCLEUS-TO-MITOCHONDRIA NUCLEIC ACID-SENSING PATHWAY PREVENTS BYPASS OF AGE-ASSOCIATED PROLIFERATIVE BOUNDARIES - PROJECT SUMMARY A NUCLEUS-TO-MITOCHONDRIA NUCLEIC ACID-SENSING PATHWAY PREVENTS BYPASS OF AGE-ASSOCIATED PROLIFERATIVE BOUNDARIES DEVELOPMENT OF IMMORTALITY AS FUNCTION OF AGE IS DEPENDENT ON THE ABILITY OF CELLS TO ESCAPE FROM AT LEAST TWO DISTINCT PROLIFERATIVE BARRIERS, REPLICATIVE SENESCENCE AND CRISIS. BOTH SERVE AS CRITICAL TUMOR-SUPPRESSORS, BUT THE PATHWAYS GOVERNING THEM ARE DISTINCT. REPLICATIVE SENESCENCE IS TRIGGERED BY SHORT FUNCTIONAL TELOMERES, DEPENDENT ON THE P53/PRB TUMOR SUPPRESSOR PATHWAYS AND CHARACTERIZED BY PERMANENT CELL CYCLE ARREST AND CONTINUED METABOLISM. WHEN P53/PRB PATHWAYS ARE DYSFUNCTIONAL, SENESCENCE ENTRY IS COMPROMISED, AND CELLS CONTINUE TO PROLIFERATE UNTIL THEIR TELOMERES BECOME DYSFUNCTIONAL AND CHROMOSOME FUSIONS ARISE. THIS TRIGGERS REPLICATIVE CRISIS, A P53/PRB-INDEPENDENT STATE, WHERE THE VAST MAJORITY OF CELLS RAPIDLY SUCCUMB TO CELL DEATH. HOWEVER, RARE CELLS CAN EVEN OVERCOME THIS BARRIER AND BECOME NEOPLASTIC, POINTING TO REPLICATIVE CRISIS AS ONE OF THE FINAL BARRIERS AGAINST AGE-ASSOCIATED TUMOR CELL INITIATION. RECENTLY, IT WAS DISCOVERED THAT CELL DEATH IN CRISIS IS GOVERNED BY MACROAUTOPHAGY THROUGH A PATHWAY IN WHICH CYTOPLASMIC DNA SPECIES FROM FUSED AND BROKEN CHROMOSOMES ACTIVATE THE CGAS-STING CYTOPLASMIC DNA-SENSING RESPONSE THAT NORMALLY DETECTS VIRAL DNA. SUPPRESSION OF AUTOPHAGY ALLOWED CELLS TO BYPASS CRISIS AND CONTINUE TO PROLIFERATE, WHILE ACCUMULATING GENOME INSTABILITY. THIS DISCOVERY REPRESENTED THE FIRST CRISIS-BYPASS SYSTEM, WHICH ALLOWED THE DESIGN OF A CRISPR SUPPRESSION SCREEN AIMED AT IDENTIFYING FACTORS REQUIRED TO PROTECT CELLS AGAINST AGE-ASSOCIATED CANCER INITIATION. ANOTHER NUCLEIC ACID SENSOR, ZBP1 EMERGED AS CRITICAL FOR THE CRISIS PROGRAM, WHICH WAS CONFIRMED BY ZBP1 SUPPRESSION ALLOWING CELLS TO PROLIFERATE BEYOND CRISIS. HERE, IN THREE SYNERGISTIC AIMS IT IS PROPOSED TO DECIPHER THE MECHANISM UNDERLYING THE ZBP1-DEPENDENT INHIBITION OF CANCER INITIATION. AIM1 WILL DETERMINE THE INTERACTIONS BETWEEN DYSFUNCTIONAL TELOMERES, TELOMERIC (TERRA) TRANSCRIPTS AND ZBP1 AND DEFINE THE MECHANISMS OF ZBP1-MEDIATED INNATE IMMUNE SIGNALING ON MITOCHONDRIA DURING CRISIS. AIM2 IS DESIGNED TO INVESTIGATE THE MECHANISM OF THE MITOCHONDRIAL LOCALIZATION OF THE CRISIS-SPECIFIC ISOFORM OF ZBP1 AND ITS RELEVANT INTERACTING PARTNERS. FINALLY, THE ABILITY TO ALLOW CELLS TO PROLIFERATE BEYOND CRISIS REVEALED THE EXISTENCE OF A THIRD PREVIOUSLY UNKNOWN PROLIFERATIVE BARRIER AGAINST CANCER INITIATION (CALLED M3), WHICH WILL BE EXTENSIVELY CHARACTERIZED IN AIM 3. SUCCESSFUL COMPLETION OF THESE AIMS WILL SHED NEW LIGHT ON CROSSTALK BETWEEN TELOMERES, MITOCHONDRIA AND INFLAMMATION (THREE ESTABLISHED HALLMARKS OF AGING), THE ROLE OF A TELOMERE-TO-MITOCHONDRIA INNATE IMMUNE SIGNALING PATHWAY IN THE PREVENTION OF AGE-ASSOCIATED CANCER AND ESTABLISH BIOMARKERS AND NEW APPROACHES TO UNDERSTAND THE RELEVANCE OF THE NEW M3 PROLIFERATIVE BARRIER AS TUMOR-SUPPRESSOR.

CHARACTERIZATION OF SPINAL CIRCUITS UNDERLYING MOTOR SYNERGY FUNCTION - ABSTRACT: THE CNS PERFORMS EXTREMELY COMPLEX COMPUTATIONS WITH REMARKABLE EFFICIENCY. THIS IS EXEMPLIFIED BY THE ABILITY TO SEAMLESSLY EXECUTE MOTOR BEHAVIORS THAT NECESSITATE THE COORDINATION OF MULTIPLE MUSCLE GROUPS CONTROLLING JOINTS WITH MANY DEGREES OF FREEDOM. IT IS THOUGHT THAT ONE STRATEGY TO SIMPLIFY MOTOR COMPUTATIONS IS TO ADOPT A CIRCUIT ORGANIZATION THAT LINKS COMBINATIONS OF MOTOR POOLS INTO FUNCTIONAL UNITS CALLED “SYNERGIES” OR “PRIMITIVES”. THUS, THE CIRCUIT ELEMENTS THAT UNDERLIE MOTOR SYNERGIES ARE THOUGHT TO REPRESENT THE BASIC BUILDING BLOCKS FOR ORCHESTRATING THE NEURAL CONTROL OF ROUTINE MOTOR BEHAVIORS. ELEGANT STIMULATION AND RECORDING EXPERIMENTS FROM LABS WORKING WITH AMPHIBIANS, RODENTS, AND PRIMATES HAVE FOUND EVIDENCE FOR MOTOR SYNERGY CIRCUITS WITHIN THE SPINAL CORD. THE MAJOR QUESTIONS ADDRESSED IN THIS GRANT ARE: (A) WHAT IS THE UNDERLYING CELLULAR AND CONNECTIVITY ORGANIZATION OF LUMBAR SPINAL MOTOR SYNERGY CIRCUITS, (B) WHAT NEURONAL SUBTYPES COMPRISE THESE CIRCUITS, AND (C) WHAT INTRINSIC AND EXTRINSIC FACTORS SHAPE THE FORMATION OF THESE CIRCUITS? THE LABORATORY HAS USED TRANS-SYNAPTIC NEURONAL TRACING, OPTOGENETICS, AND MOLECULAR SCREENS TO IDENTIFY A HETEROGENOUS (SATB1+, SATB2+, TCFAP2B+, TCF4+) POPULATION OF INTERCONNECTED EXCITATORY AND INHIBITORY PRE- MOTOR INTERNEURONS WITHIN LAMINA V OF THE LUMBAR SPINAL CORD. BASED ON THEIR PROPERTIES THESE LAMINA V CELLS ARE GENERICALLY REFERRED TO AS MOTOR SYNERGY ENCODERS (MSE). THE HYPOTHESIZE IS THAT THE MSE CELL NETWORK COMPRISES A MAJOR COMPUTATIONAL NODE FOR MOTOR CONTROL WITHIN THE SPINAL CORD. THESE CELLS RECEIVE INPUTS FROM THE CORTEX AND SENSORY NEURONS SUCH AS THOSE THAT RELAY PROPRIOCEPTIVE INFORMATION. THUS, MSE NEURONS ARE WELL POSITIONED TO MEDIATE COORDINATED MUSCLE ACTIVATION PATTERNS ARISING FROM COMMAND CENTERS FOR VOLITIONAL MOVEMENT AS WELL AS REFLEX PATHWAYS ACTIVATED BY SENSORY FEEDBACK LOCALLY WITHIN THE SPINAL CORD. THE AIMS OF THIS GRANT ARE DESIGNED TO UNRAVEL THE WIRING AND CELLULAR CONSTITUENTS WITHIN MOTOR SYNERGY CIRCUITS, AND TO EXAMINE HOW THESE CIRCUITS FORM DURING EMBRYONIC DEVELOPMENT AND EARLY POSTNATAL LIFE. AIM 1 WILL CREATE A CELLULAR ATLAS AND CONNECTIVITY MAP OF MSE NEURONS. THIS WILL DEFINE WHETHER THE MOLECULAR HETEROGENEITY OF MSE NEURONS CORRESPONDS TO SEPARATE MOTOR POOL CIRCUIT-MODULES OR PHYSIOLOGICALLY-DIFFERENT CLASSES OF NEURONS USED FOR CONTROLLING ALL MOTOR POOLS. AIM 2 WILL DEFINE THE PATTERN OF PROPRIOSPINAL FEEDBACK FROM MUSCLES ONTO MSE NEURONS. HERE THE GOAL IS TO ESTABLISH WHETHER THE MSE CIRCUIT IS BASED ON SIMPLE LABELED LINE PATHWAYS OR HAS A MORE COMPLEX INPUT-OUTPUT RELATIONSHIP. AIM 3 WILL USE TRANSCRIPTION FACTOR KNOCKOUTS TO DETERMINE WHETHER HARDWIRED INTRINSIC GENETIC PROGRAMS ESTABLISH THE MSE CIRCUITRY. AIM 4 WILL TEST WHETHER THE FUNCTIONAL MSE NETWORK ARISES FROM ACTIVITY DEPENDENT FEEDBACK FROM PROPRIOCEPTIVE SENSORY NEURONS. TAKEN TOGETHER, THESE AIMS WILL PROVIDE A DETAILED MOLECULAR-CELLULAR UNDERSTANDING OF A CRITICAL NODE WITHIN THE LOCAL SPINAL SYSTEM FOR COMPUTING AND COORDINATING MOTOR ACTIVATION PATTERNS. THESE FINDINGS MAY HELP TARGET MOTOR CIRCUITS USING GENETICS AND/OR NEURAL ACTIVITY TO FACILITATE RECOVERY FROM SPINAL CORD INJURY.

UNDERSTANDING HOW METABOLIC HETEROGENEITY IN CANCER AFFECTS THE TUMOR MICROENVIRONMENT AND ANTI-TUMOR IMMUNITY

EYE DEVELOPMENT: GENETIC STUDIES

EARLY EVENTS IN CORTICAL DEVELOPMENT

TAS::89 0222::TAS NEW; TITLE: NEXT GENERATION INTERACTOMES FOR PLANT SYSTEMS BIOLOGY AND BIOMASS FEEDSTOCKS RESEARCH; PI: JOSEPH ECKER

CONTRIBUTION OF THE PARABRACHIAL CGRP-EXPRESSING NEURONS TO THE PATHOPHYSIOLOGY OF PANIC DISORDER

CYCLIN T AND HIV1 TAT TRANSACTIVATION

IDENTIFICATION OF OLD-AGE-ASSOCIATED ALZHEIMER'S DISEASE DRUG TARGETS

ULTRA SENSITIVE SINGLE MOLECULE SPECTROSCOPY WITH PLASMONIC ANTENNAS

DEFINING THE ANATOMICAL, MOLECULAR AND FUNCTIONAL LOGIC OF INTERNAL COPY CIRCUITS INVOLVED IN DEXTEROUS FORELIMB BEHAVIORS

NOVEL APPROACHES TO STUDY MICROGLIA PHYSIOLOGY AND PATHOLOGY IN THE INTACT BRAIN

DECIPHER MEMBRANE PATTERNS IN SITU WITH SUPER-RESOLUTION AND DYNAMIC MICROSCOPY

USING GENOMIC PERTURBATIONS TO UNDERSTAND TRAIT-ASSOCIATED HUMAN GENETIC VARIATION - PROJECT SUMMARY/ABSTRACT GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE IDENTIFIED THOUSANDS OF GENETIC VARIANTS ASSOCIATED WITH METABOLIC, CARDIOVASCULAR, AUTOIMMUNE AND OTHER DISEASES. THESE VARIANTS HAVE THE POTENTIAL TO REVEAL MOLECULAR MECHANISMS THAT UNDERPIN HUMAN DISEASES, BUT THEIR INTERPRETATION IS EXTREMELY CHALLENGING, BECAUSE MOST ARE WITHIN NON-CODING GENOMIC REGIONS WITH UNKNOWN FUNCTION. THE LONG-TERM GOAL OF THE PROPOSED RESEARCH IS TO ELUCIDATE THE MOLECULAR BASIS OF COMPLEX DISEASES BY ASSEMBLING COMPREHENSIVE CATALOGS OF REGULATORY SEQUENCES AND ILLUMINATING HOW NON-CODING GENETIC VARIANTS AFFECT GENE REGULATION. THIS PROPOSAL WILL LEVERAGE THE POWER OF HIGH-THROUGHPUT GENOMIC PERTURBATIONS AND COMPUTATIONAL ANALYSES TO DISCOVER REGULATORY SEQUENCES, INTERPRET NON-CODING GENETIC VARIANTS, AND CONNECT DISEASE-ASSOCIATED VARIANTS TO THE GENES THEY REGULATE. RESEARCH FOCUS 1 WILL SYSTEMATICALLY DISCOVER NOVEL REGULATORY SEQUENCES USING CRISPR-DIRECTED TILING DELETION SCREENS, WHICH CAN DISCOVER REGULATORY SEQUENCES THAT ARE INVISIBLE TO OTHER APPROACHES. THESE SCREENS WILL BE PERFORMED IN PRIMARY T CELLS AND APPLIED TO MEGABASE-SCALE REGIONS SURROUNDING T CELL DIFFERENTIATION GENES, WHICH ARE RICH IN UNCHARACTERIZED GWAS HITS. TO DETERMINE HOW FREQUENTLY GWAS HITS AFFECT NOVEL REGULATORY SEQUENCES LACKING CANONICAL ENHANCER MARKS, FINE-MAPPED GWAS VARIANTS WILL BE INTERSECTED WITH REGULATORY SEQUENCES DISCOVERED BY THE SCREENS. THE FUNCTION OF NOVEL REGULATORY SEQUENCES WILL BE DETERMINED WITH DELETIONS FOLLOWED BY EXPERIMENTS TO MEASURE 3D CHROMATIN CONTACTS, GENE EXPRESSION, AND CELLULAR PROLIFERATION. RESEARCH FOCUS 2 WILL UTILIZE SINGLE-CELL GENOME PERTURBATIONS TO CONNECT THOUSANDS OF VARIANTS ASSOCIATED WITH HUMAN DISEASES TO THE GENES THEY REGULATE ACROSS MULTIPLE CELL TYPES. SEQUENCES CONTAINING POTENTIALLY CAUSAL GWAS VARIANTS WILL BE TARGETED WITH CRISPR INTERFERENCE AND GENE EXPRESSION WILL BE MEASURED WITH SINGLE-CELL RNA-SEQ IN A MIXTURE OF DISEASE-RELEVANT IMMUNE CELLS. USING THE SINGLE-CELL DATA, PERTURBED SEQUENCES WILL BE CONNECTED TO CHANGES IN GENE EXPRESSION IN SPECIFIC CELL TYPES. VARIANTS PREDICTED TO REGULATE GENE EXPRESSION WILL BE VALIDATED BY MODIFYING ALLELES WITH GENOME EDITING. THE EXPECTED OUTCOMES OF THIS PROJECT ARE (I) SYSTEMATIC CATALOGS OF REGULATORY SEQUENCES FOR GENES INVOLVED IN T CELL DIFFERENTIATION, (II) MOLECULAR CHARACTERIZATION OF NOVEL UNMARKED REGULATORY SEQUENCES THAT CONTAIN GWAS HITS AND (III) CONNECTIONS BETWEEN SEQUENCES CONTAINING GWAS HITS AND GENES THAT THEY REGULATE IN SPECIFIC CELL TYPES. THIS PROPOSAL WILL ESTABLISH GENOMIC PERTURBATIONS AS A NEW STRATEGY TO INTERPRET NON-CODING VARIANTS, UNCOVER IMPORTANT NEW REGULATORY BIOLOGY, AND ACCELERATE MECHANISTIC UNDERSTANDING OF DISEASE-ASSOCIATED VARIANTS.

TAM RECEPTOR SIGNALING IN NEURODEGENERATIVE DISEASE

REGULATION OF BETA CELL GENES BY GLUCOSE AND INCRETINS

NEURONAL SENESCENCE AND INFLAMMATION IN ALZHEIMER'S DISEASE - PROJECT SUMMARY AGE IS THE SINGLE STRONGEST PREDICTIVE FACTOR FOR ALZHEIMER'S DISEASE (AD). THE VAST MAJORITY OF AD CASES ARISE SPORADICALLY IN THE SEVENTH DECADE OF LIFE OR BEYOND, WITH NO KNOWN ETIOLOGY ASIDE FROM ADVANCED AGE. TO UNDERSTAND CELLULAR AND MOLECULAR CHANGES AT THE ROOT OF AD, STUDIES NEED EXPERIMENTAL METHODS THAT DISCRIM- INATE HEALTHY FROM PATHOLOGICAL AGING. UNTIL NOW, OPTIONS FOR IN VITRO STUDY OF AGED HUMAN NEURONS, THE CELL TYPE DEVASTATED BY AD, HAVE BEEN LACKING. THIS PROPOSAL WILL USE INDUCED NEURONS (INS), A METHOD THAT GENERATES NEURONS FROM FIBROBLASTS BY DIRECT TRANSDIFFERENTIATION, TO STUDY THE CELLULAR PROCESSES IN NEURONAL AGING AND AD. UNLIKE INDUCED PLURIPOTENT STEM CELL (IPSC)-DERIVED NEURONS, WHICH PASS THROUGH A STEM CELL INTERMEDIATE PHASE AND BECOME PHYSIOLOGICALLY REJUVENATED, INS RETAIN AGE-RELATED FEATURES OF THEIR SOURCE FIBROBLASTS. GENE EXPRESSION, EPIGENETIC MARKS, AND CELL METABOLISM IN INS CORRESPOND TO THE AGE OF THE DONOR. USING THE IN SYSTEM, THIS PROPOSAL WILL EVALUATE THE INTERACTION BETWEEN TWO AGE-CORRELATED FACTORS ASSOCIATED WITH THE AD BRAIN: CELLULAR SENESCENCE AND INFLAMMATION. PRELIMINARY DATA DEMONSTRATE THAT INS FROM AD PATIENTS ARE MORE LIKELY TO SHOW PROTEIN MARKERS OF SENESCENCE AND UPREGULATION OF SENESCENCE-ASSOCIATED GENES THAN INS FROM HEALTHY AGED CONTROLS (CTL). IN AIM 1, SENESCENT AND NON-SENESCENT CELLS FROM AD AND CTL IINES WILL BE EXAMINED BY RNA-SEQ AND ATAC-SEQ. THESE ANALYSES WILL IDENTIFY PRE-EXISTING DIFFERENCES IN NON-SENESCENT AD NEURONS THAT DIFFERENTIATE THEM FROM CTL AS WELL AS SPECIFIC COMPONENTS OF CELLULAR SENESCENCE THAT ARE UNIQUE TO NEURONS, A CELL TYPE IN WHICH THE EXISTENCE OF SENESCENCE REMAINS CONTROVERSIAL. IN OTHER TISSUES, SENESCENT CELLS RELEASE INFLAMMATORY FACTORS (COLLECTIVELY TERMED THE SENESCENCE ASSOCIATED SECRETORY PHENO- TYPE OR SASP) THAT DISRUPT THE LOCAL ENVIRONMENT, CONTRIBUTING TO AGE-RELATED TISSUE DYSFUNCTION. IN AIM 2, THE ABILITY OF SECRETED FACTORS FROM AD AND CTL INS TO ACTIVATE HUMAN ASTROCYTES AND MICROGLIA WILL BE ASSESSED. THESE EXPERIMENTS WILL GENERATE A LIST OF NEURONAL COMPONENTS OF THE SASP AND IDENTIFY WHICH ARE CONTRIBUTORS TO THE CHRONIC INFLAMMATION THAT ARISES WITH AD. FINALLY, AIM 3 WILL EXAMINE CELLULAR PROCESSES HYPOTHESIZED TO BE UPSTREAM OF SENESCENCE. PRIOR WORK AND PRELIMINARY DATA SHOW THAT AD AND CTL INS DIFFER IN THEIR CELLULAR METABOLISM AND INTRACELLULAR NUCLEOTIDE POOLS. THE NUCLEOTIDE CONTENT OF AD AND CTL INS WILL BE COMPARED, AND SMALL MOLECULES WILL BE USED TO MANIPULATE COMPONENTS OF NUCLEOTIDE SYNTHESIS OR SALVAGE AND TEST THE RESULTING IMPACT ON SENESCENCE. THESE STUDIES WILL REVEAL WHETHER INHERENT DIFFERENCES IN NUCLEOTIDE METAB- OLISM LEAD TO INCREASED RATES OF CELLULAR SENESCENCE AND CONTRIBUTE TO THE INFLAMMATORY RESPONSE. AS A WHOLE, THESE STUDIES WILL PROVIDE NOVEL INSIGHTS INTO HOW SENESCENCE AND INFLAMMATION INTERACT WITHIN THE AGED NEURONAL ENVIRONMENT THAT IS PERMISSIVE TO AD.

TOOLS FOR MAPPING MAMMALIAN NERVOUS SYSTEM CONNECTIONS WITH MODIFIED RABIES VIRUS

CIRCUITRY UNDERLYING RESPONSE SUMMATION IN MOUSE AND PRIMATE: THEORY AND EXPERIMENT

ASSESSING CELLULAR AGING IN OLD AND REJUVENATED NEURONS FROM ALZHEIMER PATIENTS - SUMMARY ALZHEIMER’S DISEASE (AD) AFFECTS OVER 50 MILLION PEOPLE WORLDWIDE. THE VAST MAJORITY OF PATIENTS, AD DEVELOPS SPORADICALLY IN THE ABSENCE OF ANY KNOWN ETIOLOGY OTHER THAN ADVANCED AGE. DESPITE THE FACT THAT AD HAS BEEN STUDIED FOR OVER A CENTURY, ITS PATHOGENESIS IS STILL NOT COMPLETELY UNDERSTOOD, AND DRUGS THAT COULD STOP OR REVERSE THE DISEASE PROGRESSION ARE NOT YET AVAILABLE. SIMILAR TO THE AGE-DEPENDENT INCIDENCE OF MANY CANCERS, AD ONSET IS BELIEVED TO BE CAUSED BY MULTIPLE HITS OF ENVIRONMENTAL, GENOMIC, AND AGING-RELATED FACTORS. TO BETTER UNDERSTAND THE CELLULAR AND MOLECULAR INTERACTIONS BETWEEN HUMAN AGING AND AD PATHOGENESIS, INDUCED NEURONS (INS) DIRECTLY CONVERTED FROM AD PATIENT FIBROBLASTS OFFER UNIQUE POSSIBILITIES TO MODEL AND STUDY THE DISEASE IN A HUMAN AGE-EQUIVALENT NEURONAL MODEL. THE TEAMS AROUND DR. GAGE AND DR. MERTENS HAVE RECENTLY SHOWN THAT DIRECT CONVERSION OF HUMAN AD PATIENT-DERIVED FIBROBLASTS INTO INDUCED NEURONS (INS) PRESERVES SIGNATURES OF CELL AGING AND SPORADIC AD AND ALLOWS FOR THE DETECTION OF CELLULAR PATHOLOGIES AND DISEASE DRIVERS. NEURONAL HYPO-MATURITY REPRESENTS A FUNDAMENTAL AD-RELATED CELLULAR STATE IN INS, AND THE CANCER-ASSOCIATED PYRUVATE KINASE M2 (PKM2) SPLICE VARIANT EMERGED AS A KEY PLAYER THAT COMPROMISES MATURE NEURONAL METABOLISM AND NEURONAL IDENTITY. PKM2 PROMOTES NEURONAL VULNERABILITY AND DE-DIFFERENTIATION VIA METABOLIC CHANGES IN THE CYTOPLASM, AND VIA EPIGENETIC PROCESSES IN THE NUCLEUS, BUT THE RELATIVE CONTRIBUTION OF THE TWO MECHANISMS REMAINS ELUSIVE AND MIGHT DIFFER SUBSTANTIALLY BETWEEN CANCER CELLS AND POST-MITOTIC NEURONS. TO UNDERSTAND PKM2 IN AD, AND TO DEVELOP PKM-DIRECTED THERAPEUTICS, MORE KNOWLEDGE REGARDING (1) THE RELATIONSHIP BETWEEN NEURONAL PKM2 AND HALLMARKS OF AD IN THE HUMAN BRAIN, (2) THE FUNDAMENTAL CELL BIOLOGICAL FUNCTIONS OF PKM2 IN AGED HUMAN NEURONS, AND (3) THE CROSSTALK BETWEEN PKM-COMPROMISED NEURONS AND THEIR GLIAL ENVIRONMENT IS NEEDED. THIS PROJECT WILL CHALLENGE THE IMPORTANCE OF SHARED PATHOGENIC PATHWAYS BETWEEN CANCER AND AD, AND ASSESS AGE-DEPENDENTLY COMPROMISED NEURONS IN THE CONTEXT OF AD. FIRST, THE TEAM WILL STUDY THE RELATIONSHIP BETWEEN CANCER-RELATED PKM2-POSITIVE NEURONS AND AD PATHOLOGY IN THE POST-MORTEM HUMAN BRAIN. SECOND, THE MECHANISTIC IMPACT OF PKM2 IMBALANCE ON THE METABOLIC STATE AND NEURONAL FATE STABILITY OF PATIENT-SPECIFIC INS WILL BE ASSESSED BY TRANSGENE- AND GENOME EDITING-BASED APPROACHES. THIRD, PHARMACOLOGICAL COMPOUNDS FROM THE CANCER FIELD AS A BASIS FOR DEVELOPING PKM2-TARGETED THERAPEUTICS FOR AD WILL BE LEVERAGED. FOURTH, USING A NOVEL HUMAN IN-BASED THREE-DIMENSIONAL MULTICELLULAR MODEL, THE TEAM WILL STUDY NEURON-ASTROCYTE COUPLING IN THE CONTEXT OF METABOLICALLY CHALLENGED INS AND THEIR METABOLIC CROSSTALK WITH HUMAN ASTROCYTES. THE ULTIMATE GOALS OF THESE FOUR AIMS ARE TO GAIN INSIGHT INTO THE ROLES OF THE WELL- ESTABLISHED CANCER PROTEIN PKM2 IN AGE-RELATED NEURODEGENERATION AND TO EXPLOIT THIS KNOWLEDGE TO DEVELOP THERAPEUTIC STRATEGIES AGAINST AD.

SENSORY AND SUPRASPINAL CONTROL OF LOCOMOTION

MOLECULAR MECHANISMS AND THERAPEUTIC APPROACHES TO MALIGNANT GLIOMAS

TARGETED DEGRADATION OF EXTRACELLULAR PROTEINS TO ENHANCE BRAIN PLASTICITY - PROJECT SUMMARY THE YOUNG BRAIN DISPLAYS REMARKABLE PLASTICITY. THIS INCLUDES AN ABILITY TO REMODEL NEURONAL SYNAPTIC CONNECTIONS TO LEARN NEW TASKS IN HEALTH, AS WELL AS BEING ABLE TO REPAIR CONNECTIONS AND RESTORE FUNCTION AFTER INJURY. IN THE ADULT BRAIN THERE ARE ACTIVE MECHANISMS IN PLACE THAT MAINTAIN NEURONAL CIRCUIT CONNECTIVITY AND SYNAPTIC STABILITY, WHICH IS NECESSARY FOR TYPICAL BRAIN FUNCTION, BUT ACTS AS A BARRIER TO TARGETED SYNAPTIC REMODELING IN SITUATIONS WHERE THIS IS BENEFICIAL, FOR EXAMPLE TO ENHANCE LEARNING, OR TO REPAIR NEURONS AFTER INJURY. REMOVAL OF FACTORS FROM THE ADULT BRAIN THAT LIMIT PLASTICITY IS SUFFICIENT TO ENABLE ENHANCED REMODELING AND REPAIR. HOWEVER, THESE MANIPULATIONS LEAD TO A PERMANENT REOPENING OF PLASTICITY STATES, WHICH IS DETRIMENTAL TO LONG-TERM BRAIN FUNCTION BY NON-SPECIFICALLY DESTABILIZING SYNAPTIC CONNECTIONS. THEREFORE, NEW APPROACHES ARE NEEDED TO TRANSIENTLY ENHANCE NEURONAL PLASTICITY STATE TO ENABLE CONTROLLED SYNAPTIC REMODELING AND REPAIR. THERE IS COMPELLING EVIDENCE THAT MANIPULATING PROTEINS IN THE EXTRACELLULAR SPACE SURROUNDING NEURONS IS SUFFICIENT TO ENHANCE PLASTICITY AND SYNAPTIC REMODELING. FOR EXAMPLE, PROTEINS SECRETED BY NON-NEURONAL GLIAL CELLS, SPECIFICALLY ASTROCYTES, ARE SUFFICIENT TO INDUCE SYNAPSE MATURATION AND STABILIZATION, AND PERMANENT REMOVAL OF THESE FACTORS FROM ADULT ASTROCYTES ENABLES ENHANCED PLASTICITY AND REPAIR. THIS DEMONSTRATES A ROLE FOR SPECIFIC SECRETED PROTEINS IN REPRESSING PLASTICITY IN THE ADULT BRAIN, SUGGESTING THEIR TARGETED REMOVAL MAY BE BENEFICIAL. THEREFORE, THE FIRST GOAL OF THIS PROPOSAL IS TO ASK IF ACUTE DEGRADATION OF SPECIFIC EXTRACELLULAR PROTEINS IS ABLE TO REOPEN BRIEF, CONTROLLED, WINDOWS OF PLASTICITY TO ENABLE ENHANCED LEARNING OR TO PROMOTE SYNAPTIC REPAIR AFTER INJURY. THIS WILL BE ACHIEVED BY DEVELOPING A GENETICALLY ENCODED SYSTEM FOR TARGETED DEGRADATION OF EXTRACELLULAR PROTEINS (TDEP). TDEP WILL USE NANOBODIES THAT BIND THE PROTEIN OF INTEREST, COUPLED TO A DEGRADATION-TARGETING SEQUENCE FOR UPTAKE AND REMOVAL BY ENDOGENOUS BRAIN CELLS. AS PROOF-OF-CONCEPT TDEP WILL BE DEVELOPED TO DEGRADE KNOWN ASTROCYTE-SECRETED PROTEINS THAT STABILIZE SYNAPSES, AND DETERMINE WHETHER ACUTE PROTEIN DEGRADATION IS SUFFICIENT TO REOPEN TRANSIENT WINDOWS OF SYNAPTIC PLASTICITY, ASSAYED USING VISUAL SYSTEM PLASTICITY, INJURY MODELS AND LEARNING AND MEMORY PARADIGMS. THE SECOND GOAL IS TO IDENTIFY THE COMPLETE REPERTOIRE OF EXTRACELLULAR PROTEINS THAT CONTRIBUTE TO REPRESSING PLASTICITY IN ADULTHOOD, AND THEIR CELLULAR SOURCE, TO ENABLE THEIR PRECISE TARGETING FOR DEGRADATION AND PLASTICITY ENHANCEMENT. THIS WILL BE ACHIEVED BY LABELING NEWLY-SYNTHESIZED PROTEINS SECRETED FROM SPECIFIC BRAIN CELLS UNDER DIFFERENT PLASTICITY CONDITIONS, USING PROXIMITY LABELING OF PROTEINS WITH BIOTIN BY THE ENZYME TURBOID, TARGETED TO DIFFERENT SUBCELLULAR COMPARTMENTS FROM WHICH EXTRACELLULAR PROTEINS ORIGINATE. THIS WILL GENERATE AN ATLAS OF THE CELLULAR ORIGIN OF EXTRACELLULAR PROTEINS, AND WILL BE USED TO IDENTIFY CANDIDATES FOR TDEP TARGETING FOR PLASTICITY ENHANCEMENT. THE OUTCOME WILL BE A TOOLKIT OF GENETIC REAGENTS THAT ENABLE PRECISE CONTROL OF THE NEURONAL ENVIRONMENT TO PROMOTE BRAIN HEALTH AND REPAIR, WITH LASTING IMPACT ON MULTIPLE AREAS OF NEUROSCIENCE WHERE ENHANCING BRAIN PLASTICITY WOULD IMPROVE FUNCTION.

SONOGENETIC CONTROL OF NEURONS IN A LARGE VOLUME OF THE RODENT BRAIN

CLOUDSLEAP: MAXIMIZING ACCESSIBILITY TO DEEP LEARNING-BASED MOTION CAPTURE - CLOUDSLEAP – PROJECT SUMMARY/ABSTRACT UNDERSTANDING HOW THE BRAIN PRODUCES COMPLEX BEHAVIOR IS A CENTRAL GOAL OF NEUROSCIENCE, BUT QUANTIFYING BEHAVIOR IS TECHNICALLY CHALLENGING, PARTICULARLY IN UNRESTRAINED AND NATURALISTIC SETTINGS. TOOLS THAT ARE ABLE TO OVERCOME THESE LIMITATIONS LEVERAGE DEEP LEARNING TO ACHIEVE ROBUST MARKERLESS MOTION CAPTURE, ENABLING CHARACTERIZATION OF BEHAVIOR THROUGH PRECISE POSITIONAL TRACKING OF BODY PARTS FROM STANDARD VIDEOS OF BEHAVIOR. UNFORTUNATELY, LIKE MOST DEEP LEARNING SYSTEMS, EXISTING POSE TRACKING SOFTWARE REQUIRES TECHNICAL EXPERTISE TO MANAGE THE COMPLEX SOFTWARE DEPENDENCIES REQUIRED FOR DEEP LEARNING, AND INVESTMENT IN EXPENSIVE COMPUTATIONAL HARDWARE (GPUS), BOTH OF WHICH CURTAIL EQUITABLE ACCESS TO THIS TECHNOLOGY. THIS PROJECT PROPOSES CLOUDSLEAP, A PLATFORM THAT BUILDS ON THE WIDELY USED MULTI-ANIMAL POSE TRACKING SOFTWARE SLEAP TO ADDRESS THESE BARRIERS BY PROVIDING THE INFRASTRUCTURE NECESSARY TO RUN THE ENTIRE POSE TRACKING WORKFLOW THROUGH CLOUD-BASED SYSTEMS. THIS PLATFORM ENABLES ANNOTATION, VISUALIZATION AND SHARING POSE TRACKING DATASETS DIRECTLY FROM THE BROWSER, ELIMINATING THE NEED FOR INSTALLATION AND MANAGEMENT OF DESKTOP-BASED SOFTWARE. CLOUDSLEAP WILL SUPPORT DATA FORMATS FROM ALL CURRENTLY EXISTING TOOLS FOR POSE TRACKING, AND WILL BE INTEGRATED WITH EXISTING DATA STANDARDS AND REPOSITORIES SUCH AS NEURODATAWITHOUTBORDERS AND DANDI TO ENSURE THAT ALL OUTPUTS OF CLOUDSLEAP ARE STANDARDIZED AND FAIR-COMPLIANT. USERS WILL BE ABLE TO USE CLOUDSLEAP TO TRAIN POSE TRACKING MODELS ON THEIR OWN DATA THROUGH A CLOUD-BASED JOB ORCHESTRATION SYSTEM, ELIMINATING THE COMPLEXITY OF DEEP LEARNING LIBRARY DEPENDENCIES. LEVERAGING THE HIGHLY EFFICIENT MODEL CONFIGURATIONS PROVIDED BY SLEAP, CLOUDSLEAP WILL PROVIDE USERS WITH FREE COMPUTATIONAL RESOURCES ON THE CLOUD TO TRAIN POSE MODELS. THIS CAPABILITY EFFECTIVELY ELIMINATES THE NEED FOR INVESTMENT IN LOCAL GPU HARDWARE, THEREBY REMOVING THE SINGLE BIGGEST BARRIER TO ENTRY FOR RESEARCHERS FROM UNDER-RESOURCED INSTITUTIONS. THE ENTIRE PLATFORM WILL BE DEVELOPED AS OPEN-SOURCE SOFTWARE ON PUBLIC REPOSITORIES FROM THE START, AND ALL DATA USED FOR TESTING AND DEVELOPMENT WILL BE FREELY AVAILABLE. A CORE GOAL FOR THIS PROJECT IS TO ENSURE THAT CLOUDSLEAP MAXIMIZES ACCESSIBILITY TO BEHAVIOR QUANTIFICATION TECHNOLOGY TO THE WIDEST RANGE OF PRACTITIONERS. TO THIS END, THE FIRST AIM OF THIS PROPOSAL WILL BE TO ESTABLISH A BROAD-BASED COMMUNITY OF BETA TESTERS THAT REPRESENTS THE DIVERSITY OF INSTITUTIONS IN THE BRAIN INITIATIVE AND WIDER NEUROSCIENCE COMMUNITY. BETA TESTERS WILL BE INVITED TO COLLABORATE THROUGHOUT DEVELOPMENT VIA REGULAR VIRTUAL TOWN HALL MEETINGS, IN-PERSON EVENTS, DIRECT COMMUNICATION CHANNELS AND OPEN DISCUSSION FORUMS. THESE EFFORTS WILL ADDITIONALLY PRODUCE DOCUMENTATION AND DIDACTIC MATERIALS THAT WILL BE USED FOR TRAINING AND EDUCATION ACTIVITIES. BY ENSURING THAT DIVERSE PERSPECTIVES ARE INCLUDED FROM THE VERY ONSET OF THE PROJECT, CLOUDSLEAP WILL ENABLE TRULY EQUITABLE ACCESS AND DISSEMINATION OF A CRUCIAL PART OF THE MODERN NEUROSCIENCE TOOLKIT.

HORMONAL REGULATION OF MAMMALIAN GENE EXPRESSION - NUCLEAR RECEPTORS (NRS) INFLUENCE DEVELOPMENT AND PHYSIOLOGY BY CONTROLLING PATTERNS OF GENE EXPRESSION VIA LIGAND-DIRECTED CHROMATIN MODIFICATIONS. THE RESULTANT INDUCED EPIGENOMIC STATE MOBILIZES NETWORKS OF GENES TO ENGENDER UNIQUE CELL FUNCTIONS AND PHYSIOLOGY. THIS PROPOSAL EXTENDS ON PREVIOUS STUDIES THAT ELUCIDATED THE ROLES OF THE NRS PPARD AND ERRA/G IN CONTEXT-SPECIFIC TRANSCRIPTIONAL ACTIVITIES THAT REPROGRAM SKELETAL MUSCLE METABOLISM AND PHYSIOLOGY TO INCREASE ENDURANCE PERFORMANCE. A CENTRAL FOCUS OF EXERCISE PHYSIOLOGY HAS BEEN THE FUNCTIONAL ASSOCIATION OF INDUCED TRANSCRIPTIONAL PROGRAMS WITH CHANGES IN HISTONE ACETYLATION, INCLUDING UNDERSTANDING HOW THE ACTIVATION OF PPARD AND ERRA/G AND THEIR COACTIVATOR PGC1A DRIVE OXIDATIVE METABOLIC PROGRAMS THAT LEAD TO INCREASED ENDURANCE PERFORMANCE. IN CONTRAST, LITTLE IS KNOWN ABOUT THE POTENTIAL CONTRIBUTIONS OF REPRESSIVE PROGRAMS AND THE EXPECTED HISTONE DEACETYLATION. BCL6 WAS IDENTIFIED AS A NOVEL, EXERCISE-RESPONSIVE TRANSCRIPTIONAL REPRESSOR IN BOTH HUMANS AND MICE, HOWEVER ITS IMPORTANCE IN MUSCLE PHYSIOLOGY HAS NOT BEEN EXPLORED. INTERESTINGLY, AN INTERACTION BETWEEN BCL6 AND PPARD, THE MASTER REGULATOR OF FATTY ACID SUBSTRATE UTILIZATION, HAS BEEN SHOWN IN OTHER CELL TYPES SUGGESTING THAT BCL6 MAY HAVE DUAL ROLES IN SKELETAL MUSCLE; EFFECTING TRANSCRIPTIONAL OUTCOMES THROUGH ITS DIRECT CHROMATIN BINDING AS WELL AS INDIRECTLY BY MODULATING THE PPARD TRANSCRIPTIONAL PROGRAM. THIS PROPOSAL UTILIZES PHARMACOLOGIC STRATEGIES AND MUSCLE-SPECIFIC GENETIC KNOCKOUT MOUSE MODELS IN COMBINATION WITH EPIGENETIC AND TRANSCRIPTIONAL MAPPING TECHNIQUES TO DELINEATE BCL6’S FUNCTION IN EXERCISE PHYSIOLOGY. IN AIM 1, THE BCL6 TRANSCRIPTIONAL COMPLEX WILL BE CHARACTERIZED USING STATE-OF-THE-ART APEX2 PROXIMITY LABELING TECHNIQUES IN WT AND PPARD KO PRIMARY SKELETAL MUSCLE MYOTUBES WITH AND WITHOUT PHARMACOLOGIC AGENTS TARGETING BCL6. CHANGES IN THE BCL6 COMPLEX IN RESPONSE TO LIGANDS WILL BE CORRELATED WITH ALTERED EPIGENOMIC AND GENE EXPRESSION STATES TO ASSOCIATE SPECIFIC COMPLEXES WITH FUNCTIONAL OUTCOMES. IN AIM 2, THE CONTRIBUTIONS OF BCL6 TO EXERCISE-INDUCED PHYSIOLOGIC AND TRANSCRIPTIONAL CHANGES WILL BE DETERMINED USING GENETIC (MUSCLE-SPECIFIC BCL6 KNOCKOUT MICE) AND PHARMACOLOGIC (BCL6 INHIBITOR) LOSS-OF-FUNCTION MOUSE MODELS, WHILE RELATED STUDIES IN MUSCLE-SPECIFIC PPARD KNOCKOUT MICE WILL DISTINGUISH THE PPARD DEPENDENT AND INDEPENDENT EFFECTS. LASTLY, AIM 3 WILL EXPLOIT THE ADVANCES OF THE CUT&RUN TECHNOLOGY TO MAP THE BCL6 CHROMATIN BINDING SITES IN SEDENTARY AND EXERCISED SKELETAL MUSCLE. COMBINED WITH THE CHANGES INDUCED IN PPARD AND ERRG BINDING, IN ADDITION TO ALTERATIONS IN HISTONE REGULATORY MARKS, THESE STUDIES WILL MOLECULARLY DISSECT THE COMPETING REPRESSIVE AND ACTIVATING TRANSCRIPTIONAL PROGRAMS IN MUSCLE. MOREOVER, ASSOCIATING THESE FINDINGS WITH THE TRANSCRIPTIONAL COMPLEXES IDENTIFIED IN AIM 1 AND THE TRANSCRIPTOMICS DATA FROM AIM 2 WILL PROVIDE A COMPREHENSIVE VIEW OF BCL6-DRIVEN TRANSCRIPTIONAL REPRESSION AND ITS CONTRIBUTION TO TRANSCRIPTIONAL REGULATION OF SKELETAL MUSCLE IN BOTH THE RESTING AND EXERCISE STATE.

EVOLUTION OF SEXUALLY DIMORPHIC GERM CELLS IN VOLVOX CARTERI

PHARMACOLOGICAL TARGETING OF CIRCADIAN CLOCK COMPONENTS TO TREAT GLIOBLASTOMA

FUNCTIONAL ANALYSIS OF INHIBITORY SPINAL CORD INTERNEURONS

MITOCHONDRIA-TO-NUCLEUS SIGNALING IN COLORECTAL CANCER

DIURNAL RHYTHM IN NUTRIENT METABOLISM FOR METABOLIC HOMEOSTASIS

EPIGENETIC REGULATION OF EMBRYONIC STEM CELLS BY ATP-DEPENDENT BAF CHROMATIN REMODELING COMPLEXES

DISSECTING MOLECULAR ELEMENTS OF THREAT BEHAVIOR

ASSESSING CELLULAR AGING IN OLD AND REJUVENATED NEURONS FROM ALZHEIMER PATIENTS

MECHANISMS OF FORMATION OF 3D GENOME STRUCTURES

BREAKING BARRIERS IN STRUCTURAL BIOLOGY: NOVEL CRYOEM METHODS AND APPLICATIONS

NEURAL MECHANISMS OF SHAPE PERCEPTION

FUNCTIONAL ANATOMY OF NEUROIMMUNE INTERACTIONS

GENETIC REGULATION OF THE RESPONSE TO DIETARY RESTRICTION

NEURAL MECHANISMS OF VISUAL SENSITIVITY

RECEPTOR DECOY INHIBITOR OF ANTHRAX TOXIN

ASTROCYTE REGULATION OF SYNAPSE MATURATION

EXPLORING NEURAL CIRCUIT MECHANISMS OF SOCIAL CONTACT AND SOCIAL ISOLATION

FUNCTIONAL ANALYSIS OF ADULT HIPPOCAMPAL NEUROGENESIS

PROBING PERCEPTION AND ATTENTION USING PRIMATE OPTOGENETICS

TRANSCRIPTIONAL FACTORS IN FOREBRAIN PATTERNING

FUNCTIONAL DISSECTION OF CEREBELLAR OUTPUT CIRCUITS THAT ORCHESTRATE LIMB MOTOR CONTROL - PROJECT SUMMARY THE CEREBELLUM IS ESSENTIAL FOR COORDINATING MOTOR BEHAVIOR THROUGH RAPID ADJUSTMENTS OF ONGOING MOVEMENTS. TO REFINE MOVEMENT, THE CEREBELLUM PROCESSES MOTOR AND SENSORY INFORMATION, AND TRANSMITS OUTPUT THAT ULTIMATELY MODULATES MOTOR NEURON ACTIVITY TO ENSURE SUCCESSFUL EXECUTION. THE PATH THROUGH WHICH THE CEREBELLUM CAN INFLUENCE LIMB MOVEMENT IS THROUGH OUTPUT CIRCUITS IN THE CEREBELLAR NUCLEI (CN). YET LITTLE IS KNOWN ABOUT HOW CN CIRCUITS ARE ORGANIZED AND WHETHER DISCRETE PATHWAYS ARE DEDICATED TO SPECIFIC MOTOR FUNCTIONS FOR LIMB CONTROL. THE RECENT IDENTIFICATION OF A MOLECULARLY DISTINCT SUBSET OF CN NEURONS THAT PROJECT TO THE CEREBRAL CORTEX VIA THE THALAMUS AND CAN AFFECT FORELIMB MOVEMENT BEGINS TO REVEAL A BROADER NEURAL SUBTYPE LOGIC TO CEREBELLAR OUTPUT. FURTHER EXPLORATION INDICATES THAT THERE ARE ALSO CN NEURONS THAT DIRECTLY INNERVATE THE CERVICAL SPINAL CORD. THESE DESCENDING PROJECTIONS COULD PROVIDE A MORE DIRECT ROUTE FOR INFLUENCING MOTOR OUTPUT, BUT THE SPECIFIC ROLES THEY HAVE IN MOVEMENT EXECUTION AND REFINEMENT REMAIN POORLY UNDERSTOOD. BASED ON PRELIMINARY EVIDENCE, THE MAJOR HYPOTHESIS OF THIS PROPOSAL IS THAT ANATOMICALLY AND MOLECULARLY DISTINCT SUBSETS OF CN NEURONS HAVE DISCRETE CONTRIBUTIONS TO FORELIMB MOTOR CONTROL. SPECIFICALLY, CEREBELLAR-SPINAL CIRCUITS PLAY A CRITICAL ROLE IN RAPID ONLINE CORRECTION, AND THEIR FUNCTIONAL OUTPUT IS NEEDED TO PREVENT FORELIMB ATAXIA. THREE AIMS WILL EXPLORE HOW SPINAL-PROJECTING CN CIRCUITS DIFFER FROM THE MORE HEAVILY STUDIED CN CIRCUITS THAT PROJECT TO THE THALAMUS. AIM 1 DEFINES SPECIFIC SUBTYPES OF NEURONS WITHIN THE CEREBELLAR NUCLEI BY DELINEATING THEIR INPUT AND OUTPUT CONNECTIVITY AND MOLECULAR IDENTITIES. THE DISTINCTIONS IN EFFERENT AND AFFERENT CONNECTIVITY OF SPINAL- AND THALAMUS-PROJECTING CN NEURONS WILL BE DEFINED USING COMBINATORIAL GENETIC AND VIRAL CIRCUIT TRACING TOOLS IN MICE. IN ADDITION, MOLECULAR DISTINCTIONS BETWEEN THESE TWO CLASSES OF CEREBELLAR OUTPUT NEURONS WILL BE IDENTIFIED USING SINGLE NUCLEI RNA-SEQUENCING AND MULTIPLEXED IN SITU HYBRIDIZATION. AIM 2 SETS OUT TO ESTABLISH THE FUNCTIONAL CONNECTIVITY BETWEEN THESE DISTINCT CN SUBPOPULATIONS AND FORELIMB MUSCLES AND EXAMINES HOW SPINAL- AND THALAMUS-PROJECTING CEREBELLAR OUTPUT PATHWAYS INFLUENCE GOAL-DIRECTED FORELIMB MOVEMENTS. SELECTIVE OPTOGENETIC PERTURBATION, ELECTROMYOGRAPHIC (EMG) RECORDING, AND HIGH-RESOLUTION KINEMATIC ANALYSIS WILL BE USED TO DETERMINE HOW SPINAL- AND THALAMUS-PROJECTING CN NEURONS DIFFERENTIALLY AFFECT MUSCLE ACTIVITY AND ONLINE REFINEMENT OF BEHAVIOR. AIM 3 EXPLORES HOW THE ACTIVITY OF DISCRETE CN OUTPUT PATHWAYS CORRELATES WITH FORELIMB ONLINE CORRECTION AND ENDPOINT PRECISION. THIS GOAL WILL BE ACCOMPLISHED BY RECORDING FROM SPINAL- AND THALAMUS- PROJECTING CN POPULATIONS AND FORELIMB MUSCLES DURING DEXTEROUS REACHING BEHAVIORS, AND BY APPLYING GENERALIZED LINEAR MODELS TO DETERMINE IF NEURAL ACTIVITY PREDICTS EMG AND KINEMATIC MOVEMENT FEATURES. BY DEFINING THE ORGANIZATION OF TWO MAJOR CEREBELLAR OUTPUT PATHWAYS AND IDENTIFYING THE WAYS IN WHICH THEY INFLUENCE DEXTEROUS MOVEMENTS, THIS WORK WILL PROVIDE INSIGHT INTO HOW DIVERSE CIRCUITS DIFFERENTIALLY PARTICIPATE IN MOTOR CONTROL, AND CLARIFY HOW INJURY AND DISEASE OF CEREBELLAR CIRCUITS CAN LEAD TO MOTOR IMPAIRMENTS IN HUMANS.

GENETIC ACCESS TO CORTICAL CELL TYPES WITH EPIGENETIC ASSAYS AND HIGH-THROUGHPUT, LOW-COST ENHANCER SCREENING

OPTIMIZATION OF CHRONONUTRITION TO REDUCE THE RISK OF DISEASE IN SHIFT WORKERS - PROJECT SUMMARY. GREATER THAN 20% OF THE ACTIVE US WORKFORCE ARE SHIFT WORKERS WHO, BECAUSE OF THEIR WORK SCHEDULES, HAVE DISRUPTED EATING PATTERNS, SLEEP/WAKE CYCLES, AND LIGHT EXPOSURE, RESULTING IN CIRCADIAN RHYTHM DISRUPTION. UNFORTUNATELY, THE ONLY ASPECT THAT CAN BE MODIFIED IS THE TIMING OF DIETARY INTAKE. SHIFT WORK-RELATED LATE NIGHT AND ERRATIC EATING PATTERNS INCREASE THE RISK OF OBESITY AND CARDIOMETABOLIC DISEASES, AND ACCELERATE DISEASE PROGRESSION AND EMERGENCE OF COMPLICATIONS. EVEN A FEW YEARS OF SHIFT WORK RESULTS IN DETRIMENTAL EFFECTS ON OVERALL CARDIOMETABOLIC HEALTH THAT PERSIST LONG AFTER THE SHIFT WORK CEASES. HENCE, SHIFT WORKERS BEAR A DISPROPORTIONATELY HEAVY BURDEN IN TERMS OF OBESITY AND CARDIOMETABOLIC DISORDERS. EXACERBATING THIS PROBLEM IS THE FACT THAT THE VAST MAJORITY (>99.98%) OF STUDIES EXAMINING BEHAVIORAL AND PHARMACOLOGICAL INTERVENTIONS ACTIVELY EXCLUDE SHIFT WORKERS. MAJOR CHALLENGES TO INCORPORATING SHIFT WORKERS INTO CLINICAL RESEARCH ARE THEIR VARIABLE SCHEDULES AND DAYTIME SLEEPING HABITS, MAKING FREQUENT IN-PERSON STUDY VISITS OR GROUP-BASED INTERVENTIONS (SUCH AS DIET AND NUTRITION CLASSES) IMPRACTICAL. TO OVERCOME THIS SIGNIFICANT BARRIER TO CARING FOR SHIFT WORKERS, AN EFFECTIVE INTERVENTION SHOULD ADDRESS AND IMPROVE EATING PATTERNS THROUGH LIFESTYLE INTERVENTIONS THAT ARE PRAGMATIC FOR REAL-WORLD APPLICATIONS THAT CAN BE APPLIED REMOTELY. THIS CAN BE ACHIEVED BY USING CONTEMPORARY METHODS FOR EFFECTIVE PATIENT EDUCATION, ENGAGEMENT, MONITORING, RETENTION, AND THE PROMOTION OF INTERVENTION ADHERENCE. THE PROPOSED CLINICAL STUDY MAKES INNOVATIVE USE OF DIGITAL AND MOBILE TECHNOLOGIES TO STUDY THE EFFECT OF TIME-RESTRICTED EATING (TRE) AND TRE WITH A LOW-GLYCEMIC SNACK (TRE-LGS) DURING NIGHT SHIFTS AS DIETARY INTERVENTIONS TO IMPROVE THE HEALTH OF SHIFT WORKERS, SPECIFICALLY NURSES AND NURSING ASSISTANTS WORKING NIGHT SHIFTS. TRE IS A BEHAVIORAL EATING PATTERN INTERVENTION THAT COMBINES THE IN-DEPTH SCIENTIFIC KNOWLEDGE OF THE BENEFICIAL EFFECTS OF FASTING WITH THE IMPACT OF MEAL TIMING ON BIOLOGICAL RHYTHMS. A WELL-VALIDATED SMARTPHONE APP, MYCIRCADIANCLOCK, HAS BEEN USED TO MONITOR AND FACILITATE A TRE EATING PATTERN INTERVENTION IN PRIOR PUBLISHED CLINICAL TRIALS AND WILL BE USED IN THIS STUDY. WEARABLE SENSORS WILL BE USED FOR LONGITUDINAL AND PERIODIC MEASUREMENTS OF ACTIVITY AND SLEEP PATTERNS, INTERSTITIAL BLOOD GLUCOSE, BLOOD PRESSURE, AND BODY WEIGHT. A COMBINATION OF LIVE VIDEO CONSULTATION AND DIGITAL EDUCATIONAL MATERIALS WILL BE USED TO GUIDE PARTICIPANTS THROUGH THE INTERVENTION. THE PROPOSED STUDY WILL RECRUIT OVERWEIGHT AND OBESE ADULT NIGHT SHIFT WORKERS WHO WILL BE RANDOMIZED INTO ONE OF THREE ARMS: 1) STANDARD OF CARE (SOC) WHO WILL BE ADVISED TO FOLLOW A MEDITERRANEAN DIET, 2) SOC + A PERSONALIZED 10 HOURS OF TRE INTERVENTION, OF 3) TRE WITH A PROVIDED LOW GLYCEMIC SNACK DURING NIGHT SHIFTS. ALL PARTICIPANTS WILL UNDERGO 6-MONTHS OF GUIDED INTERVENTION AND 6 MONTHS OF SELF-GUIDED INTERVENTION. ALL GROUPS WILL BE EXPOSED TO THE SAME DIGITAL PLATFORM FOR THE DELIVERY AND MONITORING OF INTERVENTION. IT IS HYPOTHESIZED THAT TRE AND TRE+LGS WILL BE EFFECTIVE AND VIABLE APPROACHES TO PROMOTE WEIGHT LOSS AND IMPROVE METABOLIC HEALTH IN SHIFT WORKERS.

OPTOGENETIC DISSECTION OF BRAIN NETWORK DEFICITS IN ALZHEIMER'S DISEASE

THE INVISIBLE PHOSPHOPROTEOME: NEW TOOLS TO STUDY HISTIDINE PHOSPHORYLATION

UNDERSTANDING DNA BREAK REPAIR PATHWAY CHOICE REGULATION BY THE CNHEJ INHIBITOR CYREN

FUNCTION AND METABOLISM OF AGING LIPIDS IN THE BRAIN - PROJECT SUMMARY/ABSTRACT LIPIDS MAKE UP HALF OF THE BRAIN’S WEIGHT AND ARE REQUIRED FOR THE PROPER DEVELOPMENT, STRUCTURE, AND FUNCTION OF HEALTHY BRAINS. AS WE AGE, THE RISK OF COGNITIVE DECLINE AND NEURODEGENERATIVE DISEASES INCREASES. THIS PROPOSAL SEEKS TO UNDERSTAND WHETHER AGING CHANGES THE LIPID COMPOSITION OF THE BRAIN AND WHETHER THESE CHANGES CONTRIBUTE TO AGING AND/OR PREDISPOSE US TO DISEASE. UNFORTUNATELY, THE FIELD CURRENTLY LACKS A DEEP UNDERSTANDING OF LIPID COMPOSITION AND DYNAMICS IN THE AGING BRAIN, PREVENTING PROGRESS TOWARD THIS LARGER GOAL. USING A UNIQUE APPROACH TO LIPIDOMICS THAT INTEGRATES CHEMISTRY TO EXAMINE THE BRAIN LIPIDOME OF AGING MICE LED TO THE DISCOVERY OF THE UNANNOTATED 3-SULFO GALACTOSYL DIACYLGLYCEROL (SGDG) LIPIDS AS THE MOST SIGNIFICANTLY DECREASED LIPIDS IN THE AGING BRAIN. FURTHERMORE, SGDGS ARE A NOVEL CLASS OF ANTI-INFLAMMATORY LIPIDS, SUGGESTING THAT DECREASED SGDG LEVELS WITH AGING MAY PROMOTE BRAIN INFLAMMATION. THE DISCOVERY OF SGDGS AS AGE-REGULATED LIPIDS REPRESENT A NEW FRONTIER AT THE INTERFACE OF AGING RESEARCH AND LIPID BIOLOGY. THIS PROPOSAL SEEKS TO ANSWER THE MOST PRESSING QUESTIONS ABOUT SGDGS AND THEIR ROLE IN AGING AND DISEASE. SPECIFICALLY, THIS PROPOSAL ASKS HOW SGDG LEVELS ARE CONTROLLED DURING AGING AND NEURODEGENERATIVE DISEASE (AIM 1); DETERMINE WHETHER SGDGS ARE BIOACTIVE IN VIVO AND WHETHER THEY REVERSE INFLAMMATION OR ANY OTHER AGE- OR DISEASE-ASSOCIATED PHENOTYPES (AIM 2); AND MEASURE SGDG DISTRIBUTION IN THE BRAIN AND MYELIN OF MOUSE MODELS OF AGING AND NEUROGENERATIVE DISEASE (AIM 3). TO ACCOMPLISH THESE GOALS, THE APPROACH RELIES HEAVILY ON CHEMISTRY TO SYNTHESIZE OF SGDGS AND SGDG ANALOGS AND USE THESE UNIQUE REAGENTS FOR CELL BIOLOGY, IN VIVO PHARMACOLOGY AND BIOLOGY, PROTEOMICS, GENOMICS, AND IMAGING METHODS TO ELUCIDATE SGDG’S ROLE IN AGING AND NEURODEGENERATIVE DISEASE. THESE STUDIES WILL EXPLAIN HOW AGE-RELATED DIFFERENCES IN LIPIDS AND LIPID METABOLISM CAN CONTRIBUTE TO AGING AND NEURODEGENERATIVE DISEASE. FURTHERMORE, SINCE SGDGS ARE ALSO FOUND IN HUMAN AND PRIMATE BRAINS, THIS INFORMATION MAY CONTRIBUTE TO THE LONGER-TERM GOAL OF KNOWING WHICH AGE-REGULATED CHANGES IN LIPIDS AND LIPID METABOLISM PREDISPOSE HUMANS TO COGNITIVE DECLINE AND NEURODEGENERATION.

SENSITIVE AND TREATABLE PERIODS OF BRAIN-REDOX IMBALANCE IN SCHIZOPHRENIA

ADOLESCENT ALCOHOL EXPOSURE AND ALTERED ADULT HPA AXIS ACTIVITY

REGULATION OF FOLLISTATIN EXPRESSION BY ACTIVIN

IONIC CELL SIGNALING IN SMALL SPACES

MITOCHONDRIAL DNA STRESS ACTIVATION OF INTERFERON SIGNALING AND LUPUS PATHOLOGY

GENETIC ANALYSIS OF THE NEUROTROPHIN RECEPTOR P75 IN NEURAL DEVELOPMENT

TAM RECEPTOR CONTROL OF MICROGLIAL FUNCTION AND NERVOUS SYSTEM HOMEOSTASIS

MODELING ALZHEIMER'S DISEASE RELATED DEMENTIAS IN THE MARMOSET

MONITORING PRESYNAPTIC RELEASE OF NEUROPEPTIDES IN AWAKE BEHAVING ANIMALS - ABSTRACT NEUROMODULATORS, SUCH AS NEUROPEPTIDES AND BIOGENIC AMINES ARE PRODUCED AND RELEASED BY NEURONS TO COMMUNICATE WITH EACH OTHER. THEY ACT AS NEUROTRANSMITTERS, AS WELL AS NEUROMODULATORS, TO PROFOUNDLY INFLUENCE THE FUNCTION OF NEURAL CIRCUITS, THEREBY REGULATING CRITICAL BRAIN FUNCTIONS, INCLUDING AROUSAL, FEEDING, METABOLISM, SOCIAL BEHAVIOR, FEAR/ANXIETY, LEARNING/MEMORY, REWARD, AND SEXUAL BEHAVIORS. DYSFUNCTION IN NEUROMODULATOR SIGNALING IS ASSOCIATED WITH MANY NEUROLOGICAL AND NEUROPSYCHIATRIC DISORDERS. THEREFORE, UNDERSTANDING THE MECHANISM OF NEUROMODULATOR FUNCTIONS IN THE BRAIN IS IMPORTANT FOR UNDERSTANDING BRAIN DISORDERS. AMONG THESE, NEUROPEPTIDES ARE BY FAR THE MOST DIVERSE CLASS OF NEUROMODULATORS IN THE BRAIN, AND THEY ARE INVOLVED IN A RANGE OF PHYSIOLOGICAL, EMOTIONAL, AND COGNITIVE PROCESSES IN THE BRAIN. ALTHOUGH MORE THAN 100 NEUROPEPTIDES AND THEIR DOWNSTREAM RECEPTORS HAVE BEEN DISCOVERED, CIRCUIT-BASED MECHANISMS OF NEUROPEPTIDE FUNCTIONS HAVE NOT BEEN ADEQUATELY EXPLORED. THIS IS PRIMARILY BECAUSE THE FIELD LACKS TOOLS FOR MONITORING THE RELEASE OF NEUROPEPTIDES IN A TEMPORALLY PRECISE MANNER AS ANIMALS PERFORM BEHAVIORS. TO FILL THIS VOID, THE RESEARCH PROPOSED HERE WILL DEVELOP A NOVEL QUANTITATIVE TECHNIQUE FOR DETECTING NEUROPEPTIDE RELEASE FROM THE PRESYNAPTIC TERMINALS IN AWAKE BEHAVING MICE. THIS PROPOSAL WILL DEVELOP A SENSOR THAT DETECTS THE RELEASE OF DENSE-CORE VESICLES THAT SPECIFICALLY PACKAGES NEUROPEPTIDES IN THE AXONAL TERMINALS. THIS INNOVATIVE APPROACH WILL ALLOW US TO MONITOR THE PRESYNAPTIC RELEASE OF ESSENTIALLY ALL NEUROPEPTIDES, THEREBY ADDRESSING FUNDAMENTAL QUESTIONS ABOUT THE NEUROPEPTIDERGIC REGULATION OF BRAIN FUNCTIONS.

NEURAL BASIS OF GESTURAL COMMUNICATION: EVIDENCE FROM SIGN LANGUAGE

MECHANISMS CONTROLLING HORMONE-MEDIATED PLANT GROWTH IN RESPONSE TO THE ENVIRONMENT

NEURAL CIRCUITS AND FUNCTION IN PRIMARY VISUAL CORTEX

COORDINATION OF VOLUNTARY EYE MOVEMENTS

VIRAL ONCOPROTEINS: REVEALING NOVEL STRUCTURAL MOTIFS TO TARGET TUMOR SUPPRESSORS

PATTERNING OF CILIATED EPITHELIA BY MECHANICAL STRAIN

THE ROLE OF HISTONE CHAPERONE ASF1 IN ALTERNATIVE LENGTHENING OF TELOMERES

AMPK AND AMPK-RELATED KINASES IN LUNG CANCER DEVELOPMENT AND TREATMENT

TELOMERE REPLICATION AND SENESCENCE IN YEAST

NON-ESSENTIAL AMINO ACIDS AND SPHINGOLIPID DIVERSITY IN CANCER PROGRESSION

DEREGULATION OF CTCF IN EPIGENETIC GENE SILENCING IN HUMAN CANCERS

TELOMERE REPLICATION AND SENESCENCE IN YEAST

TAM RECEPTOR REGULATION OF INNATE IMMUNITY

STRESS AND CRF SIGNALING IN ALZHEIMER?S DISEASE PATHOGENESIS

NEURAL MECHANISMS OF FEATURE INTEGRATION

MOTION AND FORM PROCESSING IN EXTRASTRIATE VISUAL CORTEX

PAN-LIPOXYGENASE INHIBITORS FOR CNS DISEASE

MODELING ALZHEIMER'S DISEASE RELATED DEMENTIAS IN THE MARMOSET - ABSTRACT THE LONG-TERM GOAL OF THIS PROJECT IS TO MODEL ALZHEIMER'S DISEASE RELATED DEMENTIAS (ADRD) IN LONGER-LIVING MAMMALS. THESE MULTI-DIMENSIONAL MAMMALIAN ADRD MODELS, INCLUDING FRONTOTEMPORAL DEGENERATION (FTD), LEWY BODY DEMENTIA (LBD), VASCULAR CONTRIBUTIONS TO COGNITIVE IMPAIRMENT AND DEMENTIA (VCID), AND MIXED ETIOLOGY DEMENTIAS, ARE AIMED AT INFORMING HUMAN ADRD ACROSS THE DISEASE-RELEVANT STAGES AND SERVE AS TOOLS TO INTERROGATE DISEASE MECHANISMS AND IDENTIFY THERAPEUTIC TARGETS. ADRD DEVELOPS AS A RESULT OF A COMPLEX SERIES OF EVENTS THAT TAKE PLACE IN THE BRAIN OVER A LONG PERIOD OF TIME—THE PRODROMAL PHASE CAN LAST 10-20 YEARS IN HUMANS—BEFORE DEMENTIA IS CLINICALLY DIAGNOSED. UNDERSTANDING THIS EARLY STAGE WILL BE CRITICALLY IMPORTANT FOR DEVELOPING DIAGNOSTIC TOOLS (E.G., NEUROIMAGING) AND FOR SCREENING COMPOUNDS TO PREVENT OR MODIFY DISEASE PROGRESSION IN HUMANS. A VARIETY OF ADRD MOUSE MODELS HAVE BEEN DEVELOPED TO ADVANCE RESEARCH INTO THE UNDERLYING MOLECULAR AND CELLULAR MECHANISMS, TO IDENTIFY THERAPEUTIC TARGETS, AND TO TEST THERAPEUTIC CANDIDATES. WHILE EXISTING ADRD MOUSE MODELS HAVE ENABLED PROGRESS TOWARD EACH OF THESE OBJECTIVES, THOSE AVAILABLE TO DATE DO NOT RECAPITULATE THE FULL SPECTRUM AND COMPLEXITY OF THE MOLECULAR, CELLULAR, BEHAVIORAL AND COGNITIVE PATHOLOGY OBSERVED IN TYPICAL DEMENTIAS. SEVERAL LINES OF EVIDENCE SUGGEST THAT MARMOSETS HAVE EMERGED AS A NON-HUMAN PRIMATE MODEL FOR BOTH BASIC AND TRANSLATIONAL NEUROSCIENCE TO BRIDGE THE GAP BETWEEN MICE AND HUMANS. FIRST, MARMOSETS AND HUMANS HAVE VERY SIMILAR COMPLEXITY IN BRAIN STRUCTURES, COGNITIVE/SOCIAL BEHAVIORAL REPERTOIRES, METABOLISMS AND IMMUNE FUNCTIONS. SECOND, AS COMPARED TO OTHER PRIMATES, IT IS HIGHLY ECONOMICAL AND SCALABLE FOR UNDERSTANDING BRAIN FUNCTIONS AND PRECLINICAL TESTS BECAUSE THEY HAVE A SHORT LIFESPAN, SMALL BODY SIZE, AND HIGH REPRODUCTIVE POWER. FINALLY, A SET OF GENE EDITING TOOLS IS AVAILABLE TO GENERATE VARIOUS TYPES OF GENETICALLY MODIFIED MARMOSETS. SEVERAL LINES OF EVIDENCE LED US TO ELUCIDATE THE ROLE OF THE TRIGGERING RECEPTOR EXPRESSED ON MYELOID CELLS 2 (TREM2) PATHWAYS IN ADRD. FIRST, HOMOZYGOUS TREM2 NULL MUTATIONS HAVE BEEN LINKED TO A RECESSIVE EARLY-ONSET DEMENTIA SYNDROME CALLED NASU–HAKOLA DISEASE. SEVERAL TREM2 VARIANTS ARE PATHOLOGIC TO FTD AND A MAJOR RISK FACTOR/MODIFIER FOR AD, PARKINSON'S DISEASE, AND AMYOTROPHIC LATERAL SCLEROSIS (SEE HTTPS://WWW.ALZFORUM.ORG/MUTATIONS/TREM2). TREM2 MUTATION PRECIPITATES CHANGES IN VASCULAR CHANGES AND BLOOD BRAIN BARRIER DAMAGES AND MAY CONTRIBUTE TO VCID. SECOND, MICROGLIA HAVE EMERGED AS A KEY CELL TYPE IN THE MAINTENANCE OF CENTRAL NERVOUS SYSTEM HOMEOSTASIS, FOR WHICH TREM2 MEDIATES MULTIPLE CRITICAL SIGNALING PATHWAYS. THIRD, AGING IS A MAJOR RISK FACTOR FOR ADRD. DECREASED TREM2 EXPRESSION DURING AGING MAY ACCELERATE ADRD DEVELOPMENT. TAKEN TOGETHER , TREM2 IS POISED TO INFLUENCE NEURONAL AND VASCULAR SYSTEMS ASSOCIATED WITH THE DEVELOPMENT OF MULTIPLE FORMS OF ADRD. TO ELUCIDATE THE ROLE OF TREM2 IN ADRD, DIFFERENT GENETICALLY MODIFIED MARMOSETS WILL BE GENERATED AND CHARACTERIZED.

IMAGING LOCAL AND GLOBAL CHROMATIN STRUCTURE AS A 3D CONTINUUM WITHIN THE NUCLEUS

CAREER: STRUCTURAL INTERPLAY BETWEEN CHROMATIN REMODELING AND LENTIVIRAL INTEGRATION

EARLY EVENTS IN CORTICAL DEVELOPMENT

CELLULAR FACTORS IN GAMMARETROVIRUS REPLICATION

THE ROLE OF THE TUMOR SUPPRESSOR ARID1A IN R LOOP HOMEOSTASIS AND TUMOR IMMUNITY - PROJECT SUMMARY CANCER IMMUNOTHERAPY IS THE PRACTICE OF HARNESSING THE ACTIVITY OF THE IMMUNE SYSTEM TO COMBAT CANCER. IMMUNE CHECKPOINT BLOCKADE (ICB) IS A TYPE OF CANCER IMMUNOTHERAPY THAT UNLEASHES THE ACTIVITY OF CYTOTOXIC T CELLS BY BLOCKING THE INTERACTION OF INHIBITORY RECEPTORS ON T CELLS WITH LIGANDS EXPRESSED ON TUMOR CELLS. ICB HAS BEEN AN EFFECTIVE TREATMENT FOR MANY, RESULTING IN DURABLE, EVEN CURATIVE ANTI-TUMOR IMMUNE RESPONSES. HOWEVER, IN MOST CANCER TYPES, LESS THAN 50% OF PATIENTS RESPOND, SPURRING EFFORTS TO UNDERSTAND GENETIC AND MOLECULAR SIGNATURES ASSOCIATED WITH ICB RESPONSE, AS WELL AS THERAPIES THAT CAN BE USED IN COMBINATION TO IMPROVE ICB EFFICACY. RETROSPECTIVE STUDIES HAVE SHOWN THAT MUTATIONS IN THE ARID1A GENE ARE ENRICHED AMONG PATIENTS THAT RESPOND TO ICB IN PAN-CANCER AND CANCER TYPE SPECIFIC TRIALS. ARID1A IS A SUBUNIT OF THE SWI/SNF CHROMATIN REMODELING COMPLEX, WHICH UTILIZES ENERGY DERIVED FROM ATP HYDROLYSIS TO MOVE NUCLEOSOMES ALONG DNA. GENES ENCODING SUBUNITS OF THE SWI/SNF COMPLEX ARE FREQUENTLY MUTATED IN HUMAN CANCER, WITH ARID1A BEING THE MOST FREQUENTLY MUTATED SUBUNIT AND THE THIRD MOST COMMONLY MUTATED TUMOR SUPPRESSOR BEHIND TP53 AND CDKN2A. ARID1A MUTATION COULD THUS SERVE AS AN IMPORTANT BIOMARKER FOR CANCER IMMUNOTHERAPY, POTENTIALLY AFFECTING TREATMENT OF HUNDREDS OF THOUSANDS OF CANCER PATIENTS. IT IS NOT KNOWN WHETHER ARID1A MUTATION IS CAUSAL OR HOW ARID1A MUTATION SENSITIZES TUMORS TO ICB TREATMENT. INDEED, ARID1A MUTATION IS COMMONLY FOUND IN MICROSATELLITE UNSTABLE (MSI) OR TUMOR MUTATION BURDEN-HIGH (TMB-HIGH) TUMORS, WHICH ARE THEMSELVES INDEPENDENT PREDICTORS OF ICB RESPONSIVENESS. HOWEVER, ARID1A MUTATION IS BENEFICIAL INDEPENDENT OF MSI AND TMB STATUS AND ADDITIVE FOR PATIENTS WITH MSI OR TMB-HIGH TUMORS. IT WAS FOUND THAT ARID1A MUTANT TUMORS GROW MORE SLOWLY THAN ISOGENIC WILD-TYPE LINES IN MURINE MODELS, WITH INCREASED IMMUNE INFILTRATION AND T CELL ACTIVATION. FURTHER, ARID1A MUTANT CANCER CELLS UPREGULATE A SUBSET OF GENES IN THE TYPE I INTERFERON RESPONSE, INCLUDING CHEMOKINES AND ANTIGEN PRESENTATION AND PROCESSING GENES. MECHANISTICALLY, GENETIC OR PHARMACOLOGIC INHIBITION OF ARID1A RESULTS IN INCREASED R LOOPS, AS WELL AS CYTOPLASMIC ACCUMULATION OF RNA:DNA HYBRIDS AND SSDNA. THIS PROPOSAL WILL UTILIZE GENETIC DELETION AND SMALL MOLECULE INHIBITORS OF ARID1A TO DETERMINE 1) HOW ARID1A DEFICIENCY CAUSES CYTOSOLIC NUCLEIC ACID RELEASE, 2) THE PATHWAY BY WHICH INFLAMMATORY GENES ARE ACTIVATED IN ARID1A DEFICIENT CELLS, 3) THE ROLE OF R LOOP DRIVEN INFLAMMATORY RESPONSES IN ARID1A MUTANT ANTI-TUMOR IMMUNITY AND ICB RESPONSE. THIS WILL BE ACCOMPLISHED USING ARID1A MUTANT HUMAN CANCER CELL LINES AND MOUSE MODELS OF ARID1A MUTANT CANCER. FINALLY, STUDIES PIONEERING THE IN VIVO USE OF SMALL MOLECULE INHIBITORS OF ARID1A IN COMBINATION WITH ICB WILL BE PERFORMED. THESE STUDIES WILL REVEAL THE MOLECULAR MECHANISM BY WHICH ARID1A MUTANT CANCERS RESPOND TO ICB WITH THE POTENTIAL TO IMPROVE PATIENT SELECTION FOR ICB AND TO AUGMENT ICB EFFICACY WITH ARID1A INHIBITORS.

INDUCIBLE MOUSE MODELS OF MITOCHONDRIAL ROS SIGNALING AND ENVIRONMENT STRESS

TRACKING THE MECHANISMS OF ADAPTATION TO AUTOPHAGY INHIBITION - SUMMARY ACQUIRED RESISTANCE TO ANTI-CANCER THERAPEUTICS HAS PROVEN TO BE ONE OF THE LARGEST HURDLES IN CANCER CELL BIOLOGY BECAUSE CANCER CELLS HAVE THE REMARKABLE ABILITY TO ADAPT TO DIVERSE CONDITIONS. FOR EXAMPLE, WHEN ESSENTIAL METABOLIC PROCESSES ARE BLOCKED, SOME CANCER CELLS DIE, BUT SUBSETS OF CELLS CAN SURVIVE AND ACQUIRE RESISTANCE. THE ORGANELLE RECYCLING PROCESS, AUTOPHAGY, PROVIDES AN EXCELLENT PARADIGM TO STUDY METABOLIC ADAPTATIONS IN CANCER. MANY CANCER CELLS ARE ADDICTED TO AUTOPHAGY TO MAINTAIN HOMEOSTASIS AND REGENERATE NUTRIENTS, BUT PREVIOUS WORK HIGHLIGHTED THE ABILITY OF RARE CELLS TO RAPIDLY ADAPT AND ACQUIRE NEW DEPENDENCIES ON ALTERNATE METABOLIC PATHWAYS. RAPID AND TRANSIENT ADAPTATIONS TO STRESS THAT MANIFEST IN THE METABOLOME, EPIGENOME AND TRANSCRIPTOME HAVE BEEN UNDERSTUDIED. THIS PROPOSAL SUGGESTS THAT RESISTANCE MECHANISMS ARE MORE COMPLEX THAN JUST PRE-EXISTING GENETIC DIFFERENCES BETWEEN HETEROGENEOUS TUMOR CELLS, BUT INSTEAD INCLUDE RAPID SIGNALING EVENTS, BROAD STRESS AND METABOLIC RESPONSES, EPIGENETIC CHANGES, AND THE ACQUISITION OF NEW GENETIC MUTATIONS. HOW AND WHEN EACH OF THESE FACTORS CONTRIBUTE TO RESISTANCE REMAINS UNKNOWN. MANY STUDIES ANALYZE ADAPTED POPULATIONS AFTER THEY HAVE UNDERGONE SELECTION. THE APPROACH TAKEN HERE IS DIFFERENT: THESE STUDIES AIM TO OBSERVE THE PROCESS OF SELECTION AND ADAPTATION IN ACTION. THE PROPOSED PROJECTS WILL DEVELOP A SET OF NOVEL TOOLS AND MODEL SYSTEMS TO TRACK THE DYNAMIC INTERACTIONS OF RAPID SIGNALING, STRESS AND METABOLIC RESPONSES, ALONG WITH TRANSCRIPTIONAL CHANGES, EPIGENETIC CHANGES, AND GENETIC ALTERATIONS – ALL WITH TEMPORAL PRECISION. DESPITE DECADES OF STUDIES ON THERAPEUTIC RESISTANCE, FUNDAMENTAL QUESTIONS REMAIN. FOR EXAMPLE, IT IS CRITICAL TO DETERMINE WHETHER: A) CANCER CELLS UNDERGO A CHANGE IN STATE AND ADAPT IN RESPONSE TO A TREATMENT, OR B) A TREATMENT SIMPLY SELECTS FOR A PRE-EXISTING STATE THAT IS HETEROGENOUS AND ALREADY RESISTANT. IT IS CRITICAL TO DIFFERENTIATE THE DYNAMICS BETWEEN THESE TWO MODELS TO DETERMINE WHETHER A GIVEN RESISTANCE MECHANISM SHOULD BE TARGETED AS A COMBINATION THERAPY (A CONSEQUENCE OF MODEL A), OR INSTEAD USED AS A BIOMARKER FOR PATIENT SELECTION (A CONSEQUENCE OF MODEL B). SOME PATIENTS RESPOND REMARKABLY WELL TO AUTOPHAGY INHIBITION AND THE FIELD IS DESPERATE FOR BOTH BIOMARKERS ASSOCIATED WITH THESE PATIENTS TO IMPROVE PATIENT SELECTION, AND FOR WAYS TO PREVENT THERAPY RESISTANCE. TO THIS END, THESE STUDIES WILL FACILITATE THE DEVELOPMENT OF BETTER AUTOPHAGY-TARGETING CANCER THERAPEUTICS. MOREOVER, UNDERSTANDING THE TEMPORALLY DYNAMIC CONTRIBUTIONS OF DIFFERENT KINDS OF ADAPTATIONS WILL GENERATE NEW MODELS OF CANCER CELL DRUG RESISTANCE, BEYOND THOSE THAT MODEL AUTOPHAGY MODULATION.

TRANSGENIC MICE AND BIOINFORMATIC TOOLS TO TRACK ASTROCYTE DIVERSIFICATION INSITU

GENETIC ANALYSIS OF ERBB2 IN MAMMALIAN DEVELOPMENT

ELUCIDATING CELLULAR ACTIVITY PATTERNS UNDERLYING SPINAL CORD FUNCTION

PROTO-ONCOGENES IN AXON GUIDANCE

NEURONAL CORRELATES OF VISUAL MOTION PROCESSING

REGULATION OF NF-KAPPA B

A NOVEL FAMILY OF NEUROPROTECTIVE COMPOUNDS FOR STROKE

MECHANISMS OF NUCLEOPORIN-MEDIATED GENE REGULATION

ARABIDOPSIS 2010: 1,001 GENOMES PROJECT

DISSECTING LOCAL (N) AND LONG RANGE (Z) CIRCUIT MODULES WITHIN THE SPINAL CORD - PROJECT SUMMARY/ABSTRACT: SPINAL CORD CIRCUITS ARE THE FINAL STEP IN MOTOR COMPUTATIONS AND ARE A SITE WHERE DESCENDING SIGNALS ARE INTEGRATED WITH SENSORY CUES TO DRIVE MOTOR BEHAVIOR. ELEVEN CARDINAL SPINAL INTERNEURON TYPES HAVE BEEN IDENTIFIED WITH DISTINCT DEVELOPMENTAL ORIGINS, NEUROTRANSMITTER PHENOTYPES, AND IPSI- VERSUS CONTRA-LATERAL CONNECTIONS. ELEGANT GENETIC APPROACHES HAVE TARGETED THESE CARDINAL INTERNEURONS REVEALING ROLES IN MOTOR CONTROL, BUT UNDERSTANDING OF HOW SPINAL NEURAL NETWORKS COMPRISED OF HETEROGENOUS CELLS FROM MULTIPLE CARDINAL DOMAINS FUNCTION TOGETHER TO CONTROL FEATURES SUCH AS PRECISION, COORDINATION, AND AUTOMATICITY OF MOTOR BEHAVIORS REMAINS UNCLEAR. THIS GRANT FOCUSES ON A RECENT DISCOVERY (OSSEWARD ET AL., SCIENCE 2021) THAT EACH CARDINAL INTERNEURON POPULATION IS PROFOUNDLY DIVIDED INTO GENETICALLY-DISTINCT LOCAL AND LONG-RANGE NEURONS (1). THE GOAL OF THIS GRANT IS TO DETERMINE IF THESE LOCAL/LONG RANGE DIVISIONS OF CARDINAL NEURONS CORRESPOND TO DISTINCT CIRCUIT MODULES FOR MOTOR CONTROL. TRANSCRIPTOMIC PROFILING REVEALED A CODE SPANNING EACH CARDINAL INTERNEURON DOMAIN IN WHICH MEDIALLY-LOCATED NEURONS WITH SHORT RANGE CONNECTIONS ARE MARKED BY NEUROD2 (CALLED N-TYPE) AND LATERALLY-LOCATED CELLS WITH LONG RANGE PROJECTIONS EXPRESS ZFHX3 (CALLED Z-TYPE). HERE THIS PROPOSAL WILL TEST WHETHER THESE N/Z-MARKERS DEFINE FUNCTIONAL NEURAL UNITS FOR CONTROLLING COHERENT FEATURES OF MOTOR BEHAVIOR BY COMPARING THE CONNECTIVITY, FUNCTION, AND NEURAL-ACTIVITY OF N-TYPE AND Z-TYPE CELLS WITHIN TWO DISTINCT CARDINAL INTERNEURON POPULATIONS: INHIBITORY V1 AND EXCITATORY V2A NEURONS. THIS GRANT HYPOTHESIZES THAT LONG RANGE Z-TYPE NEURONS HAVE CRITICAL FUNCTIONS RELATED TO “BROADCASTING” MOTOR COMMANDS ACROSS MULTIPLE SPINAL SEGMENTS TO FACILITATE DEXTERITY AND INTERLIMB COORDINATION, WHEREAS N-TYPE NEURONS PROCESS LOCAL INFORMATION FOR RAPID REFLEXES AND L/R LIMB CONTROL. ALTHOUGH INHIBITORY-V1 AND EXCITATORY-V2A INTERNEURONS ARE PHYSIOLOGICALLY DIFFERENT, THIS GRANT EXPLORES THE POSSIBILITY THAT THE N- VERSUS Z-CELL TYPES FROM EACH CARDINAL CLASS MAY HAVE RELATED FUNCTIONS THAT TRANSCEND EVEN THEIR NEUROTRANSMITTER STATUS. BY DEFINING THE FOUNDATIONAL ORGANIZATIONAL FEATURES OF MOTOR CIRCUITS, THESE STUDIES MAY REVEAL A HIGHER LOGIC FOR HOW CIRCUIT MODULES ARE CONSTRUCTED WITH DIFFERENT CARDINAL NEURON TYPES. TO EXPLORE THE INNOVATIVE HYPOTHESIS THAT FUNCTIONAL UNITS SPAN CARDINAL CELL TYPES, A NOVEL SET OF MOUSE INTERSECTIONAL GENETIC TOOLS FOR LABELING/TARGETING THE N/Z DIVISIONS OF THE V1 AND V2A NEURONS WAS GENERATED. A NEW METHOD FOR MULTI-ELECTRODE IN VIVO NEURONAL RECORDING OF SPINAL NEURONS IN BEHAVING ANIMALS WAS ALSO ESTABLISHED. AIM 1 (OUTPUTS) AND AIM 2 (INPUTS) WILL DEFINE V1- AND V2A-N/Z CONNECTIONS TO UNDERSTAND THE ANATOMICAL SIMILARITIES AND DIFFERENCES OF N/Z-NEURONS WITHIN TWO DISTINCT CARDINAL POPULATIONS. AIM 3 WILL PERTURB THE FUNCTION OF V1- AND V2A-N/Z SUBTYPES TO UNDERSTAND THEIR ROLE IN MOTOR BEHAVIOR. AIM 4 WILL RECORD THE NEURAL ACTIVITY OF V1- AND V2A-N/Z SUBTYPES DURING MOTOR BEHAVIOR TO DETERMINE WHETHER THEIR ACTIVITY IS LINKED TO SPECIFIC MOTOR FUNCTIONS. THESE STUDIES AIM TO REVEAL ENTIRELY NEW ORGANIZATIONAL FEATURES OF SPINAL MOTOR CIRCUITRY.

INTRINSIC AND SYNAPTIC MECHANISMS OF EPILEPTOGENESIS TRIGGERED BY CORTICAL TRAUMA

MECHANISMS OF GENETIC INSTABILITY AND TUMOR SUPPRESSION

IMAGING LOCAL AND GLOBAL CHROMATIN STRUCTURE AS A 3D CONTINUUM WITHIN THE NUCLEUS

NEURAL MECHANISMS UNDERLYING ADAPTIVE OPTIMIZATION OF VISUAL SENSITIVITY

REGULATION OF TELOMERE HOMEOSTASIS BY REPLICATION FORK PROGRESSION

TRAINING PROGRAM IN VISUAL NEUROSCIENCE

SPONTANEOUS REPLICATION FORK COLLAPSE REGULATES TELOMERE LENGTH HOMEOSTASIS IN WILD TYPE YEAST - PROJECT SUMMARY/ABSTRACT: TELOMERES PRESENT UNIQUE CHALLENGES FOR GENOMES WITH LINEAR CHROMOSOMES, INCLUDING THE INABILITY OF THE SEMI-CONSERVATIVE DNA REPLICATION MACHINERY TO FULLY DUPLICATE THE ENDS OF LINEAR MOLECULES. THIS IS SOLVED IN VIRTUALLY ALL EUKARYOTES BY THE ENZYME TELOMERASE, THROUGH THE ADDITION OF TELOMERIC REPEATS ONTO CHROMOSOME ENDS. IT IS WIDELY ASSUMED THAT THE PRIMARY SITE OF ACTION FOR TELOMERASE IS THE SINGLE-STRANDED G-RICH OVERHANG AT THE ENDS OF CHROMOSOMES, FORMED AFTER DNA REPLICATION IS COMPLETE. A NEWLY DEVELOPED ASSAY THAT MONITORS SPONTANEOUS MONITOR FORK COLLAPSE AT AN INTERSTITIAL TELOMERIC TRACT HAS DEMONSTRATED THERE IS A SECOND SUBSTRATE FOR TELOMERASE IN WILD TYPE YEAST, WHICH IS A COLLAPSED FORK GENERATED DURING REPLICATION OF DUPLEX TELOMERIC DNA. NEWLY COLLAPSED FORKS ARE EXTENSIVELY ELONGATED BY TELOMERASE IN A SINGLE CELL DIVISION, INDICATING THAT A MAJOR SOURCE OF NEWLY SYNTHESIZED TELOMERIC REPEATS IN WILD TYPE CELLS OCCURS AT COLLAPSED FORKS. FURTHERMORE, THE ABILITY OF TELOMERASE TO ELONGATE NEWLY COLLAPSED FORKS IS DEPENDENT ON FORK REMODELING PROTEINS. IN PARALLEL, A RE-EXAMINATION OF THE ROLE OF A TELOMERE-DEDICATED RPA-LIKE COMPLEX (T- RPA) IN BUDDING YEAST ARGUES THAT THIS COMPLEX FACILITATES LAGGING STRAND SYNTHESIS DURING DUPLEX DNA REPLICATION, RATHER THAN PROTECTING TELOMERES IN FROM UNREGULATED RESECTION. ADDITIONAL DATA ARGUES THAT THIS COMPLEX COLLABORATES WITH THE CANONICAL RPA COMPLEX TO STABILIZE REPLICATION FORKS DURING DUPLEX TELOMERIC DNA REPLICATION. COLLECTIVELY, THESE OBSERVATIONS PROVIDE A SUBSTANTIAL CHALLENGE TO CURRENT MODELS FOR HOW TELOMERE HOMEOSTASIS IS MAINTAINED IN WILD TYPE YEAST. THIS APPLICATION TESTS THE MODEL THAT THE ACTIVITY OF TELOMERASE IN RESPONSE TO SPONTANEOUS FORK COLLAPSE IS A MAJOR DETERMINANT OF TELOMERE LENGTH REGULATION. AIM 1 WILL TEST THE HYPOTHESIS THAT TELOMERASE ACTIVITY AT NEWLY COLLAPSED FORKS PROCEEDS THROUGH A REGULATORY PATHWAY DISTINCT FROM HOW TELOMERASE ENGAGES FULLY REPLICATED CHROMOSOME TERMINI. AIM 2 WILL TEST THE HYPOTHESIS THAT TWO RPA COMPLEXES, ONE DEDICATED TO THE LEADING STRAND (RPA) AND THE OTHER (T-RPA) BOUND TO THE LAGGING STRAND, COLLABORATE TO PROMOTE STABILIZATION OF THE FORK DURING REPLICATION OF DUPLEX TELOMERIC DNA. THE THIRD AIM WILL EXAMINE A NEW ROLE FOR THE CANONICAL RPA COMPLEX IN REGULATING TELOMERASE, THROUGH SURFACES THAT ARE HIGHLY CONSERVED FROM YEAST TO HUMANS.