San Diego Biomedical Research Institute

PURE AND AUTHENTIC HIV-1 ENV IMMUNOGENS

STRUCTURAL STUDIES OF THE AUTOPHAGIC UBIQUITIN-LIKE PROTEINS

GENOME PLASTICITY DURING ES CELL DIFFERENTIATION TO NEURAL LINEAGES

ONCOGENIC PATHWAY-INDUCED FRAGILE SITES: A NEW PARADIGM FOR UNDERSTANDING GENOME INSTABILITY IN CANCER - ABSTRACT ONE OF THE GRAND CHALLENGES IN CANCER RESEARCH IS THE VAST HETEROGENEITY IN RESPONSIVENESS TO TREATMENT FOR DIFFERENT CANCER TYPES. WE HAVE MADE GREAT STRIDES IN TREATING SOME CANCERS, WHILE THE PROGNOSIS FOR OTHERS REMAINS DISMAL. LARGE SCALE WHOLE GENOME SEQUENCE (WGS) ANALYSES HAVE IDENTIFIED “BREAKPOINT SIGNATURES” OF DIFFERENT CANCER TYPES THAT PRESUMABLY REFLECT HETEROGENEITY IN THEIR UNDERLYING DISRUPTED PATHWAYS BUT, DRUG- GABLE TARGETS HAVE EMERGED ONLY IN CASES WHERE THE BREAKS CREATE ONCOGENIC FUSION PROTEINS. THE ABILITY TO PREDICT DISRUPTED PATHWAYS USING WGS WOULD BE HIGHLY IMPACTFUL FOR CANCER DIAGNOSIS AND TREATMENT. CHROMO- SOME FRAGILE SITES (FSS) MANIFEST AS GAPS AND BREAKS IN METAPHASE CHROMOSOMES WHEN CULTURED CELLS EXPERI- ENCE REPLICATION STRESS. HOWEVER, MANY FSS ARE NOT CANCER-TYPE SPECIFIC AND MANY BREAKPOINT HOTSPOTS IN CANCER ARE NOT KNOWN FSS, WHICH HAS SEVERELY LIMITED THE IMPACT OF THE FS FIELD. SINCE CULTURED CELLS OFFER THE POTENTIAL TO PROSPECTIVELY DISSECT MECHANISMS INITIATING CHROMOSOME BREAKS AND TO TRACK THEIR EXPANSION INTO COMPLEX STRUCTURAL VARIATION, THERE IS A CRITICAL NEED TO MAKE THESE IN VITRO SYSTEMS MORE CANCER RELEVANT. OUR LONGTERM GOAL IS TO ESTABLISH A SUSTAINED RESEARCH STRATEGY THAT CAN PREDICT WHICH KNOWN PATHWAYS ARE DISRUPTED IN A GIVEN CANCER TYPE FROM THEIR BREAKPOINT PATTERNS. THE OVERALL OBJECTIVE OF THIS PROPOSAL IS TO REVEAL MECHANISMS BY WHICH PERTURBATION OF CANCER-RELEVANT CELLULAR PATHWAYS PRODUCE PATHWAY-SPECIFIC PATTERNS OF FSS IN CELL CULTURE AND DETERMINE WHETHER THOSE PATTERNS CAN BE IDENTIFIED IN CANCERS. OUR CENTRAL HYPOTHESIS IS THAT THE POWER OF IN VITRO SYSTEMS TO DISSECT MECHANISMS LEADING TO CANCER BREAKPOINT SIGNATURES WILL BECOME EVIDENT ONLY WHEN SHIFTED AWAY FROM CHEMICALLY-INDUCED FSS TOWARDS FSS INDUCED BY PERTURBING KNOWN CANCER-RELEVANT PATHWAYS. WE HAVE SHOWN THAT OVEREXPRESSION OF DIFFERENT ONCOGENES LEADS TO ONCOGENE-SPECIFIC SPECTRA OF FSS AND PRE- LIMINARY DATA SUGGEST SOME OF THE MECHANISMS BY WHICH THIS OCCURS. OUR RATIONALE IS THAT UNDERSTANDING CANCER- RELEVANT MECHANISMS THAT SPECIFY FSS WILL FILL THE GAP IN LINKING IN VITRO FSS TO CANCER BREAKPOINTS. THIS WOULD BE A MAJOR STEP TOWARD A STRATEGY TO PREDICT DISRUPTED PATHWAYS FROM CANCER WGS DATA, THEREBY SUGGESTING TREAT- MENTS FOR PREVIOUSLY INTRACTABLE CANCERS. AIM1 WILL USE HIGH THROUGHPUT/RESOLUTION REPLICATION AND OLIGOPAINTS ASSAYS TO IDENTIFY DOWNSTREAM MECHANISMS DISTINGUISHING WHICH OF MANY SITES OF ONCOGENE-INDUCED REPLICATION DELAY MANIFEST AS FSS. AIM2 WILL ELUCIDATE UPSTREAM MECHANISMS CAUSING REPLICATION DELAYS AND FS AT SPECIFIC SITES. AIM3 WILL MAP FSS AT UNPREDEDENTED RESOLUTION AND MINE TUMOR SEQUENCING DATABASES FOR SIGNATURES THAT MATCH THOSE OF ONCOGENE-SPECIFIC FSS. THIS CONTRIBUTION WILL BE SIGNIFICANT BECAUSE THE ABILITY TO IDENTIFY AFFECTED PATHWAYS SOLELY FROM BREAKPOINT SIGNATURES WOULD EXPOSE TUMOR-SPECIFIC VULNERABILITIES FOR PRECISION CANCER MEDICINE. THE PROPOSED RESEARCH IS INNOVATIVE BECAUSE IT WILL REJUVENATE THE IMPACT OF FS RESEARCH, CLOSING THE GAP BETWEEN IN VITRO SYSTEMS AND IN VIVO CANCERS WHILE LEVERAGING NOVEL TECHNOLOGIES TO MANIPULATE CELLS, MAP THEIR SIGNATURES OF GENOME INSTABILITY AND MATCH THEM TO CANCER BREAKPOINT SIGNATURES.

HIV ANTIBODIES AND NK CELL ADCC: NANOMETER-SCALE TRACKING OF IMMUNE SYNAPSE DYNAMICS. - SUMMARY STRUCTURAL STUDIES HAVE ESTABLISHED THAT NATURALLY ELICITED ANTIBODIES CAN BIND TO HIV ENVELOPE (ENV) OVER A WIDE RANGE OF EPITOPES AND ANGLES MEDIATED BY THEIR FAB ARM (I.E. “IMMUNE COMPLEX GEOMETRY”). HOW THIS AFFECTS ANTIBODY DEPENDENT CELLULAR CYTOTOXICITY (ADCC) ACTIVITY, ESPECIALLY BY NATURAL KILLER (NK) CELLS IS UNKNOWN. OUR KNOWLEDGE IS LIMITED BY A POOR UNDERSTANDING OF HOW ANTIBODY IMMUNE COMPLEXES ORCHESTRATE ANTIBODY RECEPTOR BASED SIGNALING (SPECIFICALLY FOR FC GAMMA RIIIA, FCRIIIA). THE LONG-TERM GOAL IS TO ACQUIRE A MORE DETAILED UNDERSTANDING OF HOW ANTIBODIES RECRUIT FC MEDIATED CELLULAR ACTIVITY AND TO DEVELOP NOVEL STRATEGIES TO ENGINEER ANTIBODIES, DRUGS, AND VACCINES THAT CAN RECRUIT SPECIFIC EFFECTOR FUNCTIONS WITH MAXIMAL POTENCY IN VIVO. THE OBJECTIVE OF THIS PROPOSAL IS TO DETERMINE HOW IMMUNE COMPLEX GEOMETRY IMPACTS NK CELL ADCC AGAINST HIV AND IGG RECEPTOR SPATIOTEMPORAL DYNAMICS WITHIN THE NK CELL IMMUNE SYNAPSE (NKIS). OUR CENTRAL HYPOTHESIS IS THAT ANTIBODY GEOMETRY WILL MODULATE FCRIIIA INTERACTION AND ADCC ACTIVITY IN RELATION TO IMMUNE COMPLEX GEOMETRY. THE RATIONALE FOR THIS WORK IS THAT CUTTING-EDGE MICROSCOPY OBSERVATIONS WILL PROVIDE NEW INSIGHT INTO ADCC FUNCTION WITH IMMEDIATE IMPACTS ON HIV ANTIBODY THERAPEUTIC DESIGN WITH APPLICATIONS TO A BROADER RANGE OF HUMAN DISEASES. OUR CENTRAL HYPOTHESIS WILL BE TESTED IN THREE SPECIFIC AIMS: 1) DETERMINE HOW IMMUNE COMPLEX GEOMETRY INFLUENCES FCGRIIIA INTERACTION DURING ADCC; 2) PERFORM SINGLE MOLECULE TRACKING OF FCRIIIA WITHIN THE NKIS DURING ADCC; 3) DETERMINE NANOMETER-SCALE LOCALIZATION OF FCRIIIA AND SIGNALING KINASES WITHIN THE NKIS DURING ADCC. WE WILL PURSUE THESE AIMS USING THE INNOVATIVE TECHNIQUE OF MINFLUX NANOSCOPY, A SUPER-RESOLUTION FLUORESCENCE MICROSCOPY TECHNIQUE THAT IS CAPABLE OF 1- TO 3-NM SPATIAL RESOLUTIONS IN BOTH 2- AND 3-DIMENSIONS AS WELL AS SUB-MILLISECOND TRACKING OF SINGLE MOLECULES IN LIVE CELLS. CAREFULLY MEASURED IN VITRO ADCC ACTIVITY AND FÖRSTER RESONANCE ENERGY TRANSFER (FRET) MEASUREMENTS WILL ALSO COMPLEMENT OUR MINFLUX OBSERVATIONS AND BROADEN THE INTERPRETATION OF OUR RESULTS. THESE STUDIES ARE SIGNIFICANT BECAUSE THEY WILL ESTABLISH A MOLECULAR BASIS FOR ANTIBODY EFFECTOR FUNCTION, ESPECIALLY IN RELATION TO NK CELL ADCC, THAT COULD IMPROVE THERAPEUTICS FOR HIV. THE TECHNIQUES ESTABLISHED IN THIS PROPOSAL WILL ALSO BE USEFUL FOR INTERROGATING ANTIBODY ADCC FUNCTION FOR OTHER VIRAL PATHOGENS. THE EXPECTED OUTCOME OF OUR STUDIES IS THE CHARACTERIZATION OF BIOPHYSICAL PRINCIPLES THAT ALTER NK CELL ADCC ACTIVITY AS WELL AS THE MOLECULAR MECHANICS THAT FORM THE BASIS FOR SUCH ACTIVITY. THESE FINDINGS WILL HAVE AN IMPORTANT IMPACT ON HUMAN HEALTH BY OFFERING A RATIONAL BASIS FOR DESIGNING IMPROVED ANTIBODY THERAPEUTICS FOR HIV, AS WELL AS OTHER VIRUSES AND DISEASES, AND WILL INCREASE OUR BASIC UNDERSTANDING OF NK CELL ADCC.

SYNERGISTIC EFFECTS OF SILICA EXPOSURE, VIRUS INFECTION AND GENETIC PREDISPOSITION IN SYSTEMIC AUTOIMMUNITY

METHAMPHETAMINE AND HIV INTERACTIONS IN THE REGULATION OF GLIAL ACTIVATION

RESCUE OF BROADLY NEUTRALIZING MABS USING NATIVE TRIMER

THE PROTECTIVE ROLE OF MICROGLIA IN PREVENTING HYPOXIC DISRUPTION OF BLOOD-BRAIN BARRIER INTEGRITY AND VCID - PROJECT SUMMARY/ABSTRACT DEMENTIA IS A MAJOR HEALTH PROBLEM IN THE UNITED STATES. VASCULAR CONTRIBUTIONS TO COGNITIVE IMPAIRMENT AND DEMENTIA (VCID) IS THE SECOND LEADING CAUSE OF DEMENTIA BEHIND ALZHEIMER’S DISEASE (AD) BUT DESPITE THE MASSIVE IMPACT OF VCID IN THE EXPANDING ELDERLY POPULATION, ITS PATHOGENESIS IS STILL ONLY POORLY UNDERSTOOD. THE CONSENSUS IS THAT IN THE AGING BRAIN, PARTICULARLY ON A BACKGROUND OF HYPERTENSION, BLOOD VESSELS UNDERGO DEGENERATIVE CHANGES, RESULTING IN LOSS OF BLOOD-BRAIN BARRIER (BBB) INTEGRITY AND INCREASED VASCULAR RESISTANCE, WHICH TOGETHER, LEAD TO CEREBRAL HYPOPERFUSION, NEURONAL DAMAGE AND COGNITIVE DECLINE. RECENTLY, WE DESCRIBED A NOVEL ROLE FOR MICROGLIA IN THE MAINTENANCE OF VASCULAR INTEGRITY. WE DEMONSTRATED THAT CHRONIC MILD HYPOXIA (CMH; 8% O2) INDUCES TRANSIENT VASCULAR LEAK IN SPINAL CORD BLOOD VESSELS IN YOUNG (10 WEEKS OLD) MICE, THAT IS ASSOCIATED WITH MICROGLIAL ACTIVATION AND CLUSTERING AROUND LEAKY BLOOD VESSELS. INTERESTINGLY, MICROGLIAL DEPLETION PROFOUNDLY INCREASED VASCULAR LEAK AND THIS WAS ASSOCIATED WITH ASTROCYTE-VASCULAR UNCOUPLING AND LOSS OF VASCULAR TIGHT JUNCTION PROTEINS, SUGGESTING THAT MICROGLIA PLAY AN IMPORTANT PROTECTIVE ROLE IN MAINTAINING VASCULAR INTEGRITY IN THE SPINAL CORD. WE HAVE SINCE FOUND THAT CMH ALSO TRIGGERS VASCULAR LEAK IN THE BRAIN AND THAT MICROGLIAL DEPLETION EXACERBATES THIS LEAK. STRIKINGLY, IN AGED (20 MONTHS OLD) MICE, THE EXTENT OF HYPOXIC-INDUCED CEREBROVASCULAR DISRUPTION IS GREATLY ENHANCED, AS SHOWN BY INCREASED VASCULAR LEAK AND THE EMERGENCE OF MICROHEMORRHAGES, THOUGH THE IMPACT OF MICROGLIAL DEPLETION IN AGED MICE HAS YET TO BE ADDRESSED. TOGETHER, OUR DATA SUGGESTS THAT MICROGLIA PLAY AN IMPORTANT VASCULOPROTECTIVE ROLE IN YOUNG MICE, BUT THIS MECHANISM MAY BE LESS EFFECTIVE IN THE AGED BRAIN. TAKEN WITH THE OBSERVATION THAT AGING INDUCES THE APPEARANCE OF A “PRIMED”, PRO-INFLAMMATORY, DESTRUCTIVE MICROGLIAL PHENOTYPE, WE HYPOTHESIZE THAT: (I) MILD HYPOXIA TRIGGERS VASCULAR LEAK AND MICROHEMORRHAGE IN THE BRAIN, RESULTING IN NEURONAL DAMAGE AND COGNITIVE DECLINE, (II) VASCULAR DISRUPTION IS WORSE IN THE AGED AND THE HYPERTENSIVE, (III) MICROGLIA PLAY AN IMPORTANT VASCULOPROTECTIVE ROLE IN STABILIZING THE BBB, BUT THIS DECLINES WITH AGE, AND (IV) REPOPULATING THE AGED BRAIN WITH YOUNG MICROGLIA OR ATTENUATION OF MICROGLIAL ACTIVATION STATE, COULD STABILIZE THE BBB AND REDUCE COGNITIVE IMPAIRMENT. TO INVESTIGATE THESE HYPOTHESES, WE PROPOSE THREE SPECIFIC AIMS: (1) CHARACTERIZE HYPOXIA-INDUCED VASCULAR LEAK IN THE BRAIN AND DEFINE HOW THIS IS INFLUENCED BY AGE, GENDER, SEVERITY OF HYPOXIA, HYPERTENSION AND BRAIN REGION, (2) DEFINE THE CONTRIBUTION OF MICROGLIA IN PREVENTING HYPOXIA-INDUCED CEREBROVASCULAR LEAK IN YOUNG AND AGED MICE, AND (3) DEMONSTRATE THAT HYPOXIA-INDUCED BBB DISRUPTION AND COGNITIVE IMPAIRMENT ARE REDUCED BY REPOPULATING THE AGED BRAIN WITH “YOUNG” MICROGLIA OR BY ATTENUATING MICROGLIAL ACTIVATION STATE. THESE STUDIES WILL PROVIDE IMPORTANT INSIGHT INTO THE LINK BETWEEN HYPOXIC EXPOSURE, BBB DISRUPTION, NEURONAL DAMAGE AND COGNITIVE DECLINE, AND INFORM ON THE THERAPEUTIC POTENTIAL OF MANIPULATING MICROGLIAL BEHAVIOR IN THE AGED BRAIN TO RESTORE VASCULOPROTECTIVE FUNCTION.

DOPAMINE SYSTEM AS REPORTER OF HIV STATUS AND INFLAMMATION IN METH ABUSERS

MAPPING THE 3D ARCHITECTURE OF NATIVE HUMAN REPLISOMES

A VASCULO-PROTECTIVE ROLE FOR BETA4 INTEGRIN IN NEUROINFLAMMATION

A NOVEL PLASMINOGEN RECEPTOR IN BREAST CANCER

BI-DIRECTIONAL INTERACTIONS WITH TUMOR MICROENVIRONMENT ENHANCE BRCA1-IRIS OVEREXPRESSING TNBC TUMOR CELLS AGGRESSIVENESS

ROLE OF A NOVEL INTERFERON RESPONSIVE T CELL SUBSET IN ALLERGY AND ASTHMA - DISEASE ASSOCIATED WITH ALLERGIES SUCH AS ASTHMA ARE A RISING HEALTH PROBLEM WITH NO CURRENT CURATIVE SOLUTIONS. CD4+ HELPER T CELLS (TH) THAT RESPOND TO COMMON ALLERGENS PLAY AN IMPORTANT ROLE IN DRIVING AIRWAY INFLAMMATION IN ASTHMA. TO BETTER UNDERSTAND THE DIVERSITY OF T CELL SUBSETS IN ALLERGY AND ASTHMA, WE ANALYZED THE SINGLE-CELL TRANSCRIPTOME OF ~50,000 HOUSE DUST MITE (HDM) ALLERGEN-REACTIVE TH CELLS FROM ASTHMATICS AND NON- ASTHMATICS, WITH AND WITHOUT HDM ALLERGY. FROM OUR ANALYSIS, BESIDES CANONICAL CLUSTERS OF CELLS SUCH AS TH2, TH17, AND TH1, WE IDENTIFIED A NOVEL SUBSET OF ALLERGEN-REACTIVE TH CELLS CHARACTERIZED BY AN IFN RESPONSIVE GENE SIGNATURE THAT WE CALLED THIFNR CELLS (SEUMOIS ET AL. SCIENCE IMMUNOLOGY, 2020). PROPORTIONS OF THIFNR CELLS WERE SIGNIFICANTLY INCREASED IN NONALLERGIC INDIVIDUALS COMPARED TO ALLERGIC PATIENTS, SUGGESTING AN ALLERGEN-SPECIFIC HOST SPECIFIC RESPONSE EVEN IN NON-ALLERGIC INDIVIDUALS. MOREOVER, THE EXCLUSIVE PRESENCE OF THE ALLERGEN-REACTIVE TH2 CELLS IN THE ALLERGIC PATIENTS SUGGESTS A PROTECTIVE ROLE (ANTI-TH2 RESPONSE) OF THE THIFNR CELLS IN THE NON-ALLERGIC PATIENTS WITH EXPOSURE TO ALLERGEN. THIS POTENTIAL PROTECTIVE ROLE WAS REINFORCED BY OUR IN VITRO STUDIES SHOWING THAT TNF-RELATED APOPTOSIS- INDUCING LIGAND (TRAIL) PRODUCED BY THIFNR CELLS DIRECTLY INHIBITS T CELL ACTIVATION TRIGGERED BY TCR ENGAGEMENT. IN FOLLOW-UP STUDIES, WE FOUND THIFNR CELLS AMONG VIRAL-REACTIVE TH CELLS DIRECTED TOWARDS FLU OR SARS-COV2, SUGGESTING A BROADER ROLE OF THOSE CELLS IN IMMUNE RESPONSES. ALSO, WE FOUND THIFNR CELLS AS A STABLE TH SUBSET IN A LARGE COHORT OF HEALTHY INDIVIDUALS. BECAUSE OF THE RECENT DISCOVERY OF THIFNR CELLS, VERY LITTLE IS KNOWN ABOUT THEIR ORIGINS, DIFFERENTIATION, PHENOTYPE, AND FUNCTION. WE HYPOTHESIZE THAT THESE THIFNR HDM-REACTIVE T CELLS COULD PLAY A ROLE THROUGH TRAIL ENGAGEMENT IN DAMPENING TH2 INFLAMMATION IN ALLERGY AND ASTHMA. IN AIM 1, WE WILL UTILIZE INTERFERON-STIMULATED RESPONSE ELEMENT (ISRE) REPORTER MICE AND T CELL-SPECIFIC INTERFERON RECEPTOR 1 (IFNAR1) KNOCKOUT MICE TO DETERMINE THE IMPORTANCE OF THIFNR CELLS IN CONTROLLING ALLERGIC AIRWAY INFLAMMATION IN ASTHMA MODELS. IN AIM 2, WE WILL PERFORM SINGLE-CELL ATAC-SEQ PROFILING TO IDENTIFY TRANSCRIPTION FACTORS THAT MAY BE INVOLVED ESTABLISHING AND MAINTAINING THE EPIGENETIC STATE OF THIFNR CELLS. FINALLY, WE WILL TEST FUNCTIONALLY THOSE TF BY USING SHRNA KNOCKDOWN EXPERIMENTS. OVERALL, STUDIES IN THIS PROGRAM WILL IMPROVE OUR UNDERSTANDING OF HOW THIFNR CELLS ARE GENERATED IN VIVO AND HOW THEY INTERACT WITH OTHER CD4+ T CELLS SUBSETS LIKE TH2 CELLS TO CURTAIL ALLERGIC AIRWAY INFLAMMATION IN ASTHMA MODELS.

MOLECULAR EFFECTS OF CANNABINOIDS ON THE BLOOD BRAIN BARRIER IN HIV-INFECTED BRAIN - MOLECULAR EFFECTS OF CANNABINOIDS ON THE BLOOD BRAIN BARRIER IN HIV-INFECTED BRAIN INFECTION WITH THE HUMAN IMMUNODEFICIENCY VIRUS (HIV) CAUSES SIGNIFICANT DISEASE MORBIDITY IN THE BRAIN, LARGELY DUE TO HIV-ASSOCIATED BLOOD-BRAIN BARRIER (BBB) DISRUPTION, INFLAMMATION, AND PERSISTENCE OF HIV-INFECTED CNS TARGET CELLS SUCH AS MICROGLIA AND MACROPHAGES, IN SPITE OF ANTIRETROVIRAL THERAPIES (ART). CANNABIS IS A COMMONLY USED DRUG BY PEOPLE WITH HIV (PWH). RECENTLY, WE MADE THE IMPORTANT OBSERVATION THAT THE IMPACT OF CANNABIS ON BBB INTEGRITY MARKERS IS CONTEXT-DEPENDENT, WITH SIGNS OF BBB DISRUPTION IN NON-HIV SUBJECTS, BUT PARADOXICALLY IMPROVING BBB INTEGRITY IN PWH. THIS OBSERVATION MAKES IT A HIGH PRIORITY TO UNDERSTAND THE MECHANISTIC BASIS OF THIS DICHOTAMOUS INFLUENCE OF CANNABIS ON THE BBB. CANNABIS AND HIV INTERACT IN MULTIFACTORIAL WAYS THAT ARE STILL POORLY UNDERSTOOD. IMPORTANTLY, HIV BRAIN TARGET CELLS MICROGLIA/MACROPHAGES EXPRESS THE CANNABINOID RECEPTOR 2 (CB2R), WHILE ENDOTHELIAL CELLS OF THE BLOOD BRAIN BARRIER (BBB) EXPRESS BOTH CB1R AND CB2R AND OTHER CANNABINOID RECEPTORS SUCH AS GPR55. IT IS ALSO UNKNOWN HOW CANNABIS INFLUENCES HIV-ASSOCIATED BRAIN INFLAMMATION OR THE ACTIVE/LATENT STATUS OF HIV IN INFECTED MICROGLIA. BY PROTECTING VASCULAR INTEGRITY, IT FOLLOWS THAT CANNABIS WILL NOT ONLY AFFECT THE SELECT TRANSPORT OF SUBSTRATES INTO THE CNS, IT WILL ALSO DIRECTLY INFLUENCE THE INFILTRATION OF INFLAMMATORY CELLS AND THE PASSAGE OF ART OR OTHER TREATMENTS TO THE BRAIN. THE GOAL OF THIS APPLICATION IS TO TEST THE HYPOTHESIS THAT CANNABINOIDS POSITIVELY IMPACT BBB INTEGRITY IN THE HIV-INFECTED BRAIN ENVIRONMENT, LEADING TO REDUCED INFLAMMATORY CELL INFILTRATION AND ULTIMATELY PROTECTION FROM HIV-ASSOCIATED NEUROCOGNITIVE DISORDERS. OUR ANALYSIS OF TWO HUMAN BRAIN MICROVASCULAR ENDOTHELIAL CELL (HBMEC) LINES (HCMEC/D3 AND HBMEC/CI18) WITH OPPOSING VASCULAR INTEGRITY RESPONSES TO CANNABINOIDS IN THE CONTEXT OF HIV SUGGEST A MOLECULAR MECHANISM THAT CAN EXPLAIN THE CONTEXT-DEPENDENT EFFECTS OF CANNABIS IN HUMAN BBB PROPERTIES. BASED ON THIS PRELIMINARY DATA, OUR HYPOTHESIS POSITS THAT BENEFICIAL EFFECTS OF CANNABINOIDS IN THE CONTEXT OF HIV OCCUR VIA CB2R, WHILE CANNABINOID SIGNALING VIA GPR55 MAY BE DAMAGING. IN THE PROPOSED STUDIES WE WILL MODEL THE HUMAN BBB IN VITRO USING MULTICELLULAR SYSTEMS CONTAINING THE TWO HBMEC LINES IN PARALLEL, ALONG WITH PERICYTES, ASTROCYTES AND MICROGLIA, TO REPLICATE HIV-INDUCED VASCULAR PHENOTYPES CHARACTERIZED BY LOSS OF TIGHT JUNCTION PROTEINS AND INCREASED PERMEABILITY TO FLUORESCENT LABELLED- DEXTRAN. IMPLICATIONS TO INFECTION IN THE BRAIN WILL BE VALIDATED IN VIVO USING THE ECOHIV MOUSE MODEL, WHICH DEVELOPS BBB DISORDERS. SUCCESSFUL COMPLETION OF THESE EXPERIMENTS WILL DEFINE THE MOLECULAR MECHANISMS THAT UNDERLIE THE DICHOTOMOUS INFLUENCE OF CANNABIS ON THE BBB IN THE CONTEXT OF HIV, FORMING THE BASIS OF NEW APPROACHES AIMED AT OPTIMIZING BBB INTEGRITY.

ANTI-INFLAMMATORY SIGNALS AND NEURODEGENERATION - ABSTRACT NEUROIMMUNE SIGNALS REGULATE NEURONAL FUNCTION AND SURVIVAL. WE HAVE OBTAINED MULTIPLE PIECES OF EVIDENCE INDICATING THAT ACTIVATION OF THE HETERODIMERIC INTERLEUKIN-13 RECEPTOR ALPHA 1/INTERLEUKIN-4 RECEPTOR ALPHA (IL- 13RΑ1/IL-4RΑ) AFFECTS THE VIABILITY OF MIDBRAIN DOPAMINERGIC (DA) NEURONS. WE FOUND THAT IN THE BRAIN, IL- 13RΑ1/IL-4RΑ IS PREFERENTIALLY EXPRESSED ON THE NEURONS OF THE SUBSTANTIA NIGRA PARS COMPACTA (SNC) THAT ARE LOST IN PARKINSON’S DISEASE (PD). WE ALSO SHOWED THAT INTERLEUKIN 13 (IL-13), PRODUCED DURING NEUROINFLAMMATION BY MICROGLIA AND NEURONS, CAN MODULATE THE ACTIVITY OF DOPAMINERGIC CELLS AND INCREASE THEIR SUSCEPTIBILITY TO OXIDATIVE DAMAGE. THUS, HAVING ESTABLISHED THAT ACTIVATION OF IL-13RΑ1 SIGNALING CAN AFFECT THE SURVIVAL OF DOPAMINERGIC NEURONS DURING NEUROINFLAMMATION, IN THE PRESENT APPLICATION WE WISH TO DETERMINE THE MOLECULAR AND CELLULAR MECHANISMS BY WHICH THIS OCCURS. SPECIFICALLY, WE WISH TO TEST THE HYPOTHESIS THAT IL-13 AND NEURONAL IL-13RΑ1 CAUSE DAMAGE BY STIMULATING A REGULATED CELL DEATH PATHWAY CALLED FERROPTOSIS. WE ALSO WISH TO DETERMINE TO WHAT EXTENT IL-13 AND IL-13RΑ1 CONTRIBUTE TO NEURODEGENERATION IN A MOUSE MODEL OF ALPHA- SYNUCLEINOPATHY (Α-SYN), A HALLMARK TRAIT OF PD THAT IS ASSOCIATED WITH NEUROINFLAMMATION AND OXIDATIVE DAMAGE. THIS WILL HELP US DETERMINE WHETHER TARGETING IL-13RΑ1 SIGNALING MIGHT BE A VIABLE APPROACH TO SLOW NEURODEGENERATION IN HUMANS AFFECTED BY AN Α-SYNUCLEINOPATHY SUCH AS PD. THE ABILITY OF RUXOLITINIB, AN FDA- APPROVED DRUG THAT INHIBITS IL-13RΑ1 SIGNALING, AND THAT OF THE NOVEL FERROPTOSIS INHIBITOR CMS121 TO REDUCE IL- 13-MEDIATED DAMAGE IN VIVO WILL ALSO BE TESTED. FINALLY, WE PROPOSE IN VIVO EXPERIMENTS TO TEST THE HYPOTHESIS THAT A RARE GENETIC VARIANT OF IL-13 FOUND IN INDIVIDUALS DIAGNOSED WITH EARLY-ONSET PD CAN CONTRIBUTE TO MORE RAPID LOSS OF DOPAMINERGIC NEURONS IN A MOUSE WITH THE HOMOLOGUE OF THIS MUTATION. IF SUCCESSFUL, THESE EXPERIMENTS WILL PROVIDE STRONG SUPPORT FOR THE HYPOTHESIS THAT IL-13 AND IL-13RΑ1 ARE NOVEL TARGETS FOR PREVENTING PD OR SLOWING ITS PROGRESSION, AT LEAST IN A SUB-SET OF PD PATIENTS.

IDENTIFYING RESPONSE DEFINING MECHANISMS FOR BIOLOGICAL THERAPIES IN SEVEREASTHMA (INSIGHTS ); ASSESSING THE ROLE OF CD4-CTLS - PROJECT SUMMARY SEVERE ASTHMA, CHARACTERIZED BY PERSISTENT AIRWAY INFLAMMATION AND CORTICOSTEROID RESISTANCE, AFFLICTS MILLIONS OF AMERICANS WITH LIMITED THERAPEUTIC OPTIONS. CLINICIANS CURRENTLY RELY ON TYPE 2 IMMUNE BIOMARKERS FOR PATIENT CLASSIFICATION (T2HIGH VS. T2LOW) AND TREATMENT DECISIONS. HOWEVER, REAL-WORLD STUDIES REVEALED THE OVER- REPRESENTATION OF THE T2HIGH ENDOTYPE IN SEVERE ASTHMA AND THOSE TREATED WITH ANTI-T2 BIOLOGIC THERAPIES EXHIBIT CONTRASTING RESPONSES, CHALLENGING THE EFFICACY OF THIS T2 DICHOTOMY AND HIGHLIGHTING THE NEED TO EXPLORE ALTERNATIVE PATHOPHYSIOLOGICAL DRIVERS. OUR SINGLE-CELL TRANSCRIPTOME ANALYSIS OF T CELLS FROM INFLAMED AIRWAYS IN SEVERE ASTHMATICS IDENTIFIED A NOVEL SUBSET OF PATHOGENIC AIRWAY TISSUE RESIDENT CYTOTOXIC CD4+ T CELLS (CD4-CTLS). DESPITE BIOLOGIC TREATMENT, CD4- CTLS REMAINED ACTIVATED, PRODUCING INFLAMMATORY MOLECULES. CELL PROPORTIONS ANALYSIS LINKED CD4-CTLS TO ASTHMA SEVERITY AND LUNG FUNCTION IMPAIRMENT, PARTICULARLY IN MALES WITH LATE-ONSET ASTHMA. THE STUDY STRONGLY SUPPORTS CD4-CTLS' SIGNIFICANT CONTRIBUTION TO AIRWAY INFLAMMATION, POTENTIALLY HINDERING T2-BIOLOGIC RESPONSE. LED BY A MULTIDISCIPLINARY TEAM OF EXPERTS IN CLINICAL ASTHMA, AND IMMUNO-GENOMICS, DRS. KURUKULAARATCHY AND SEUMOIS, OUR INSIGHTS STUDY AIMS TO ASSESS THE IMPACT OF CD4-CTLS ON SEVERE ASTHMA PATHOGENESIS AND T2- BIOLOGICS RESPONSE AT A LARGER SCALE. AIM 1: WE WILL ANALYZE AIRWAY CD4-CTLS FROM BLOOD AND SPUTUM SAMPLES COLLECTED FROM 160 SEVERE ASTHMATIC PATIENTS BEFORE, AND 12- AND 24- MONTHS AFTER INITIATING T2-BIOLOGIC THERAPY. WE WILL EVALUATE THE IMPACT OF CD4- CTLS ON T2-BIOLOGICS RESPONSE THROUGH EXTENSIVE CLINICAL ANALYSIS. SAMPLES WILL BE COLLECTED BY THREE CLINICAL CENTERS [UNIVERSITY OF SOUTHAMPTON, UK; UNIVERSITY OF MICHIGAN, ANN ARBOR, US, AND UNIVERSITY OF CALIFORNIA, SAN DIEGO, US] ESTABLISHING THE CLINICAL SIGNIFICANCE OF CD4-CTL AS A NEW PLAYER IN ASTHMA PATHOGENESIS. AIM 2: WITH THE PLANNED COLLECTION OF SAMPLES, WE WILL GENERATE UP TO 1 MILLION SINGLE-CD4-CTL CELL TRANSCRIPTOMES ALLOWING US TO COMPREHENSIVELY DECIPHER THE PATHOGENIC CELLULAR AND MOLECULAR FEATURES OF CD4- CTLS IN ASTHMA SEVERITY AND RESPONSE TO T2-BIOLOGICS, AS WELL AS INVESTIGATE THEIR FUNCTION THROUGH TCR- REACTIVITY AND INTERACTIONS WITH STRUCTURAL CELLS ANALYSIS. IN SUMMARY, THIS UNIQUE TRANSLATIONAL PROPOSAL AIMS TO UNVEIL MECHANISMS CAUSING A LACK OR LOSS OF RESPONSE TO T2-BIOLOGICS IN SEVERE ASTHMA, FOCUSING ON CD4-CTLS. FINDINGS COULD ESTABLISH CD4-CTLS AS A BETTER PREDICTOR FOR PATIENT STRATIFICATION, LEADING TO NOVEL THERAPIES FOR UNMET PATIENT NEEDS.

THE PROTECTIVE ROLE OF MICROGLIA IN PREVENTING HYPOXIC DISRUPTION OF BLOOD-BRAIN BARRIER INTEGRITY AND VCID - PROJECT SUMMARY/ABSTRACT DEMENTIA IS A MAJOR HEALTH PROBLEM IN THE UNITED STATES. VASCULAR CONTRIBUTIONS TO COGNITIVE IMPAIRMENT AND DEMENTIA (VCID) IS THE SECOND LEADING CAUSE OF DEMENTIA BEHIND ALZHEIMER’S DISEASE (AD) BUT DESPITE THE MASSIVE IMPACT OF VCID IN THE EXPANDING ELDERLY POPULATION, ITS PATHOGENESIS IS STILL ONLY POORLY UNDERSTOOD. THE CONSENSUS IS THAT IN THE AGING BRAIN, PARTICULARLY ON A BACKGROUND OF HYPERTENSION, BLOOD VESSELS UNDERGO DEGENERATIVE CHANGES, RESULTING IN LOSS OF BLOOD-BRAIN BARRIER (BBB) INTEGRITY AND INCREASED VASCULAR RESISTANCE, WHICH TOGETHER, LEAD TO CEREBRAL HYPOPERFUSION, NEURONAL DAMAGE AND COGNITIVE DECLINE. RECENTLY, WE DESCRIBED A NOVEL ROLE FOR MICROGLIA IN THE MAINTENANCE OF VASCULAR INTEGRITY. WE DEMONSTRATED THAT CHRONIC MILD HYPOXIA (CMH; 8% O2) INDUCES TRANSIENT VASCULAR LEAK IN SPINAL CORD BLOOD VESSELS IN YOUNG (10 WEEKS OLD) MICE, THAT IS ASSOCIATED WITH MICROGLIAL ACTIVATION AND CLUSTERING AROUND LEAKY BLOOD VESSELS. INTERESTINGLY, MICROGLIAL DEPLETION PROFOUNDLY INCREASED VASCULAR LEAK AND THIS WAS ASSOCIATED WITH ASTROCYTE-VASCULAR UNCOUPLING AND LOSS OF VASCULAR TIGHT JUNCTION PROTEINS, SUGGESTING THAT MICROGLIA PLAY AN IMPORTANT PROTECTIVE ROLE IN MAINTAINING VASCULAR INTEGRITY IN THE SPINAL CORD. WE HAVE SINCE FOUND THAT CMH ALSO TRIGGERS VASCULAR LEAK IN THE BRAIN AND THAT MICROGLIAL DEPLETION EXACERBATES THIS LEAK. STRIKINGLY, IN AGED (20 MONTHS OLD) MICE, THE EXTENT OF HYPOXIC-INDUCED CEREBROVASCULAR DISRUPTION IS GREATLY ENHANCED, AS SHOWN BY INCREASED VASCULAR LEAK AND THE EMERGENCE OF MICROHEMORRHAGES, THOUGH THE IMPACT OF MICROGLIAL DEPLETION IN AGED MICE HAS YET TO BE ADDRESSED. TOGETHER, OUR DATA SUGGESTS THAT MICROGLIA PLAY AN IMPORTANT VASCULOPROTECTIVE ROLE IN YOUNG MICE, BUT THIS MECHANISM MAY BE LESS EFFECTIVE IN THE AGED BRAIN. TAKEN WITH THE OBSERVATION THAT AGING INDUCES THE APPEARANCE OF A “PRIMED”, PRO-INFLAMMATORY, DESTRUCTIVE MICROGLIAL PHENOTYPE, WE HYPOTHESIZE THAT: (I) MILD HYPOXIA TRIGGERS VASCULAR LEAK AND MICROHEMORRHAGE IN THE BRAIN, RESULTING IN NEURONAL DAMAGE AND COGNITIVE DECLINE, (II) VASCULAR DISRUPTION IS WORSE IN THE AGED AND THE HYPERTENSIVE, (III) MICROGLIA PLAY AN IMPORTANT VASCULOPROTECTIVE ROLE IN STABILIZING THE BBB, BUT THIS DECLINES WITH AGE, AND (IV) REPOPULATING THE AGED BRAIN WITH YOUNG MICROGLIA OR ATTENUATION OF MICROGLIAL ACTIVATION STATE, COULD STABILIZE THE BBB AND REDUCE COGNITIVE IMPAIRMENT. TO INVESTIGATE THESE HYPOTHESES, WE PROPOSE THREE SPECIFIC AIMS: (1) CHARACTERIZE HYPOXIA-INDUCED VASCULAR LEAK IN THE BRAIN AND DEFINE HOW THIS IS INFLUENCED BY AGE, GENDER, SEVERITY OF HYPOXIA, HYPERTENSION AND BRAIN REGION, (2) DEFINE THE CONTRIBUTION OF MICROGLIA IN PREVENTING HYPOXIA-INDUCED CEREBROVASCULAR LEAK IN YOUNG AND AGED MICE, AND (3) DEMONSTRATE THAT HYPOXIA-INDUCED BBB DISRUPTION AND COGNITIVE IMPAIRMENT ARE REDUCED BY REPOPULATING THE AGED BRAIN WITH “YOUNG” MICROGLIA OR BY ATTENUATING MICROGLIAL ACTIVATION STATE. THESE STUDIES WILL PROVIDE IMPORTANT INSIGHT INTO THE LINK BETWEEN HYPOXIC EXPOSURE, BBB DISRUPTION, NEURONAL DAMAGE AND COGNITIVE DECLINE, AND INFORM ON THE THERAPEUTIC POTENTIAL OF MANIPULATING MICROGLIAL BEHAVIOR IN THE AGED BRAIN TO RESTORE VASCULOPROTECTIVE FUNCTION.

DEVELOPMENT OF ASSAYS FOR HTS TO IDENTIFY INHIBITORS OF A NEW PPI INVOLVED IN CANCER METASTASIS

EVALUATING THE THERAPEUTIC POTENTIAL OF HYPOXIA MIMETICS IN INFLAMMATORY DEMYELINATING DISEASE - MULTIPLE SCLEROSIS (MS) IS A CHRONIC INFLAMMATORY DISEASE THAT RESULTS IN DEMYELINATION AND DEGENERATION OF AXONS IN THE CENTRAL NERVOUS SYSTEM (CNS). DISRUPTION OF THE BLOOD-BRAIN BARRIER (BBB) OCCURS AT AN EARLY STAGE OF MS AND IN THE ANIMAL MODEL EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE) AND IS CENTRAL TO THE INITIATION AND MAINTENANCE OF MS PATHOGENESIS BY PERMITTING LEUKOCYTE INFILTRATION INTO THE CNS. RECENTLY, WE MADE THE IMPORTANT DISCOVERY THAT WHEN APPLIED TO PRE-EXISTING EAE DISEASE, CHRONIC MILD HYPOXIA (CMH; 10% O2) MARKEDLY ACCELERATES NEUROLOGICAL RECOVERY, LEADING TO LONG-TERM STABLE REDUCTIONS IN DISABILITY SCORE. HISTOLOGICALLY, THIS PROTECTION CORRELATED WITH REDUCED LEVELS OF VASCULAR DISRUPTION AND LEUKOCYTE ACCUMULATION AND FASTER REMYELINATION. MECHANISTICALLY, CMH PROMOTED A VIGOROUS ANGIOGENIC RESPONSE AND INCREASED ENDOTHELIAL EXPRESSION OF TIGHT JUNCTION PROTEINS WHILE REDUCING VCAM-1 EXPRESSION, KEY INDICATORS OF ENHANCED VASCULAR INTEGRITY. IN ADDITION, CMH GREATLY ENHANCED APOPTOTIC REMOVAL OF INFILTRATED MONOCYTES DURING DISEASE REMISSION. HAVING RECENTLY FOUND THAT HYPOXIA MIMETICS (HMS), DRUGS THAT STIMULATE HYPOXIA SIGNALING PATHWAYS, ALSO ACCELERATE NEUROLOGICAL RECOVERY FROM EAE, WE NOW PLAN TO EXTEND THESE STUDIES IN TWO WAYS. FIRST, BY DEFINING A HM TREATMENT PROTOCOL THAT OPTIMIZES RECOVERY FROM EAE. SECOND, DEFINE MECHANISTICALLY HOW HYPOXIA INDUCIBLE FACTOR (HIF)-MEDIATED VASCULAR REMODELING AND APOPTOSIS OF INFILTRATED MONOCYTES CONTRIBUTE TO THIS PROTECTION. AS MS ONSET TYPICALLY OCCURS IN YOUNG TO MIDDLE-AGED PATIENTS, WE WILL STUDY THESE EVENTS BOTH IN YOUNG (10 WEEKS) AND MATURE (8 MONTHS) MICE. THE GOAL OF THIS PROPOSAL IS TO TEST THE HYPOTHESIS THAT HMS PROMOTE RECOVERY FROM INFLAMMATORY DEMYELINATING DISEASE BY ENHANCING VASCULAR INTEGRITY AND SUPPRESSING NEUROINFLAMMATION VIA HIF-MEDIATED SIGNALING. OUR HYPOTHESIS WILL BE TESTED IN THREE SPECIFIC AIMS: (1) DEFINE A HM PROTOCOL THAT OPTIMIZES RECOVERY FROM EAE, (2) EVALUATE THE CONTRIBUTION OF VASCULAR REMODELING TO THE HYPOXIA PROTECTIVE RESPONSE IN EAE, AND (3) EVALUATE THE CONTRIBUTION OF MONOCYTE APOPTOSIS TO THE HYPOXIA PROTECTIVE RESPONSE IN EAE. BUILDING ON OUR FUNDAMENTAL OBSERVATION THAT CMH ACCELERATES RECOVERY FROM EAE AND THAT THE HM DRUG FG-4592 EXERTS SIMILAR PROTECTION, BOTH IN THE RELAPSING-REMITTING AND CHRONIC PROGRESSIVE EAE MODELS, WE NOW PLAN TO EVALUATE THE TRANSLATIONAL POTENTIAL OF THESE FINDINGS BY (I) DEFINING AN OPTIMAL FG-4592 TREATMENT PROTOCOL AND (II) DEFINE MECHANISTICALLY HOW HIF-MEDIATED VASCULAR REMODELING AND APOPTOSIS OF INFILTRATED MONOCYTES CONTRIBUTE TO THIS PROTECTION. SUCCESSFUL COMPLETION OF THESE STUDIES WILL FURTHER OUR GOAL OF DEVELOPING THIS APPROACH AS A NOVEL TREATMENT FOR MS.

(PQD6)THE ROLE OF IMMUNE HOMEOSTASIS IN PROTECTION FROM CANCER CACHEXIA

METHAMPHETAMINE, HIV INTEGRATION AND LATENCY IN THE BRAIN - METHAMPHETAMINE, HIV INTEGRATION AND LATENCY IN THE BRAIN METHAMPHETAMINE (METH), A STIMULANT DRUG USED BY PEOPLE WITH HIV (PWH PATHWAYS IN ADDICTION, AGGRAVATING EFFECTS OF HIV IN THE BRAIN, WHERE RESERVOIR CELLS BEARING HIV INTEGRATED PROVIRUS CHALLENGE CURE STRATEGIES AND CONTRIBUTE TO PERPETUATING NEUROLOGICAL CONSEQUENCES, DESPITE ), INFLUENCES INFLAMMATION AND ANTIRETROVIRAL TREATMENTS (ART). NEUROTRANSMITTERS INVOLVED IN ADDICTION SUCH AS DOPAMINE (DA) MODULATE HIV TARGETS THAT EXPRESS DA RECEPTORS, INCLUDING THE MICROGLIA DIVERSE POPULATION. CHROMATIN ORGANIZATION IN HIV-1 INTEGRATION REPRESENTS AN IMPORTANT PREREQUISITE FOR UNDERSTANDING INFECTION AND LATENCY. HOWEVER, THERE IS A CRITICAL GAP IN UNDERSTANDING RELATIONSHIPS BETWEEN CHROMATIN ORGANIZATION AND PROVIRAL INTEGRATION ASSOCIATED WITH HIV-1 PERSISTENCE IN MICROGLIA CELLULAR RESERVOIRS, AND PARTICULARLY IN THE BEST EXPERIMENTAL MODEL, THE SIV- RHESUS MACAQUE. MOREOVER, IT IS UNKNOWN HOW METH MODIFIES THESE RELATIONSHIPS. WE HYPOTHESIZE THAT METH IMPACTS CHROMATIN ENHANCING VIRAL INTEGRATION SUSCEPTIBILITIES IN HIV/SIV BRAIN TARGET CELLS, AND THAT EFFECTS VARY BY BRAIN REGION THAT DIFFER IN DOPAMINERGIC PROJECTIONS. CRITICAL TO THIS GOAL IS THE PROGRESSION OF MODELS REFLECTED IN PHASES, FIRST IN VITRO USING HUMAN MICROGLIA CELL LINES AND IPSC-DERIVED, WHERE THE CHARACTERISTICS OF HIV INTEGRATION HAVE BEEN DEFINED, AND EFFECTS OF METH, OR DA, CAN BE CONTROLLED. THIS IS FOLLOWED BY MODELS EX VIVO AND IN VIVO IN THE SIV-RHESUS MACAQUES, TO REPLICATE HIV, CHRONIC METH AND ART-SUPPRESSION. IN THE R61 PHASE, WE WILL CONCENTRATE ON WHETHER CHROMATIN ACCESSIBILITY AND THE ARCHITECTURAL PROTEIN CTCF, WHICH INTERACTS WITH LEDGF/P75 TO TETHER THE HIV-1 INTEGRASE, MEDIATE INSERTIONS INTO GENOMIC BOUNDARIES OF TOPOLOGICALLY ASSOCIATED DOMAINS (TADS), MODIFIED BY METH, OR DA. WE WILL ASSESS THE IMPACT OF INTEGRATION PATTERNS ON THE INTEGRITY OF PROVIRAL GENOMES TO GAIN INSIGHTS ABOUT THE REAL SIZE OF THE FUNCTIONAL VIRAL RESERVOIR. AIM 1) DELINEATES RELATIONSHIPS BETWEEN EPIGENOMIC CHANGES IN MICROGLIA UPON METH AND PROVIRAL GENOMES INSERTED INTO THE PRE- ESTABLISHED REGIONS WITH MARKED PRESENCE OF CTCF AND H3K36ME3 (TAD BOUNDARIES), AIM 2) CONNECTS EPIGENETIC PROFILES AND HIV-1 INSERTION PATTERNS WITH THE CELLULAR AND VIRAL TRANSCRIPTION PROFILES, WHILE CONFIRMING THESE RULES IN THE SIV SYSTEM, AND AIM 3) DETERMINES THE CONTRIBUTION OF DA. GO-NO-GO: IF EPIGENOMIC CHANGES CAUSED BY METH OR DA ARE LINKED TO INTEGRATION SITES, WITH SIMILAR RULES IN ALL MODELS, THE R33 PHASE WILL MOVE TO THE IN VIVO SIV MODEL, TO TEST EFFECTS OF METH CHRONIC USE ON EPIGENOMIC VULNERABILITIES TO SIV INTEGRATION IN MICROGLIA, IN RELATION TO SUBSET HETEROGENEITY IN MESOLIMBIC AREAS WHERE DA PROJECTIONS ARE ABUNDANT COMPARED TO CONTROL REGIONS OF THE BRAIN. AIM 4) TESTS WHETHER SIV INTEGRATION SITE AVAILABILITY FOLLOWS RULES DICTATED BY CHROMATIN STATES, ACCESSIBILITY AND CTCF BINDING THAT CAN BE USED TO PREDICT SUSCEPTIBILITIES IN THE CONTEXT OF METH. AIM 5) TESTS WHETHER INTEGRATION SUSCEPTIBILITIES AND PATTERNS VARY IN BRAIN AREAS THAT DIFFER IN DOPAMINERGIC PROJECTIONS, AND HETEROGENEOUS MICROGLIA POPULATIONS. THIS PROJECT ENABLES NOVEL STUDIES ON MICROGLIA CHROMATIN AND SIV INTEGRATION DYNAMICS IN BRAIN AREAS THAT DIFFER BY FUNCTION, CELLULAR DENSITY, AND NEUROTRANSMITTERS.

STABILIZING HIV-1 TRIMERS BY LINKING GP120 SUBUNITS

METHAMPHETAMINE, HIV INTEGRATION AND LATENCY IN THE BRAIN - METHAMPHETAMINE, HIV INTEGRATION AND LATENCY IN THE BRAIN METHAMPHETAMINE (METH), A STIMULANT DRUG USED BY PEOPLE WITH HIV (PWH PATHWAYS IN ADDICTION, AGGRAVATING EFFECTS OF HIV IN THE BRAIN, WHERE RESERVOIR CELLS BEARING HIV INTEGRATED PROVIRUS CHALLENGE CURE STRATEGIES AND CONTRIBUTE TO PERPETUATING NEUROLOGICAL CONSEQUENCES, DESPITE ), INFLUENCES INFLAMMATION AND ANTIRETROVIRAL TREATMENTS (ART). NEUROTRANSMITTERS INVOLVED IN ADDICTION SUCH AS DOPAMINE (DA) MODULATE HIV TARGETS THAT EXPRESS DA RECEPTORS, INCLUDING THE MICROGLIA DIVERSE POPULATION. CHROMATIN ORGANIZATION IN HIV-1 INTEGRATION REPRESENTS AN IMPORTANT PREREQUISITE FOR UNDERSTANDING INFECTION AND LATENCY. HOWEVER, THERE IS A CRITICAL GAP IN UNDERSTANDING RELATIONSHIPS BETWEEN CHROMATIN ORGANIZATION AND PROVIRAL INTEGRATION ASSOCIATED WITH HIV-1 PERSISTENCE IN MICROGLIA CELLULAR RESERVOIRS, AND PARTICULARLY IN THE BEST EXPERIMENTAL MODEL, THE SIV- RHESUS MACAQUE. MOREOVER, IT IS UNKNOWN HOW METH MODIFIES THESE RELATIONSHIPS. WE HYPOTHESIZE THAT METH IMPACTS CHROMATIN ENHANCING VIRAL INTEGRATION SUSCEPTIBILITIES IN HIV/SIV BRAIN TARGET CELLS, AND THAT EFFECTS VARY BY BRAIN REGION THAT DIFFER IN DOPAMINERGIC PROJECTIONS. CRITICAL TO THIS GOAL IS THE PROGRESSION OF MODELS REFLECTED IN PHASES, FIRST IN VITRO USING HUMAN MICROGLIA CELL LINES AND IPSC-DERIVED, WHERE THE CHARACTERISTICS OF HIV INTEGRATION HAVE BEEN DEFINED, AND EFFECTS OF METH, OR DA, CAN BE CONTROLLED. THIS IS FOLLOWED BY MODELS EX VIVO AND IN VIVO IN THE SIV-RHESUS MACAQUES, TO REPLICATE HIV, CHRONIC METH AND ART-SUPPRESSION. IN THE R61 PHASE, WE WILL CONCENTRATE ON WHETHER CHROMATIN ACCESSIBILITY AND THE ARCHITECTURAL PROTEIN CTCF, WHICH INTERACTS WITH LEDGF/P75 TO TETHER THE HIV-1 INTEGRASE, MEDIATE INSERTIONS INTO GENOMIC BOUNDARIES OF TOPOLOGICALLY ASSOCIATED DOMAINS (TADS), MODIFIED BY METH, OR DA. WE WILL ASSESS THE IMPACT OF INTEGRATION PATTERNS ON THE INTEGRITY OF PROVIRAL GENOMES TO GAIN INSIGHTS ABOUT THE REAL SIZE OF THE FUNCTIONAL VIRAL RESERVOIR. AIM 1) DELINEATES RELATIONSHIPS BETWEEN EPIGENOMIC CHANGES IN MICROGLIA UPON METH AND PROVIRAL GENOMES INSERTED INTO THE PRE- ESTABLISHED REGIONS WITH MARKED PRESENCE OF CTCF AND H3K36ME3 (TAD BOUNDARIES), AIM 2) CONNECTS EPIGENETIC PROFILES AND HIV-1 INSERTION PATTERNS WITH THE CELLULAR AND VIRAL TRANSCRIPTION PROFILES, WHILE CONFIRMING THESE RULES IN THE SIV SYSTEM, AND AIM 3) DETERMINES THE CONTRIBUTION OF DA. GO-NO-GO: IF EPIGENOMIC CHANGES CAUSED BY METH OR DA ARE LINKED TO INTEGRATION SITES, WITH SIMILAR RULES IN ALL MODELS, THE R33 PHASE WILL MOVE TO THE IN VIVO SIV MODEL, TO TEST EFFECTS OF METH CHRONIC USE ON EPIGENOMIC VULNERABILITIES TO SIV INTEGRATION IN MICROGLIA, IN RELATION TO SUBSET HETEROGENEITY IN MESOLIMBIC AREAS WHERE DA PROJECTIONS ARE ABUNDANT COMPARED TO CONTROL REGIONS OF THE BRAIN. AIM 4) TESTS WHETHER SIV INTEGRATION SITE AVAILABILITY FOLLOWS RULES DICTATED BY CHROMATIN STATES, ACCESSIBILITY AND CTCF BINDING THAT CAN BE USED TO PREDICT SUSCEPTIBILITIES IN THE CONTEXT OF METH. AIM 5) TESTS WHETHER INTEGRATION SUSCEPTIBILITIES AND PATTERNS VARY IN BRAIN AREAS THAT DIFFER IN DOPAMINERGIC PROJECTIONS, AND HETEROGENEOUS MICROGLIA POPULATIONS. THIS PROJECT ENABLES NOVEL STUDIES ON MICROGLIA CHROMATIN AND SIV INTEGRATION DYNAMICS IN BRAIN AREAS THAT DIFFER BY FUNCTION, CELLULAR DENSITY, AND NEUROTRANSMITTERS.

DO ALLERGENS CONTRIBUTE TO NEURODEGENERATION? - ABSTRACT PARKINSON’S DISEASE (PD) IS THE SECOND MOST COMMON NEURODEGENERATIVE DISORDER AND AFFECTS APPROXIMATELY 1% OF THE POPULATION OVER THE AGE OF 60, WITH ABOUT 50,000 NEW CASES REPORTED ANNUALLY IN THE U.S. ALONE. GENETIC STUDIES HAVE IDENTIFIED AT LEAST 9 RARE, MONOGENIC CAUSES OF PD AND OVER 13 GENETIC LOCI. YET, MOST PD CASES REMAIN OF UNKNOWN ORIGIN AND ARE BELIEVED TO RESULT FROM A COMBINATION OF ENVIRONMENTAL AND GENETIC FACTORS. THE IDENTIFICATION OF THESE COMBINATIONS REMAINS A MAJOR CHALLENGE. HERE WE PROPOSE TO TEST THE HYPOTHESIS THAT ONE COMBINATION IS REPRESENTED BY ALLERGIC REACTION (ENVIRONMENTAL) AND THE RECEPTOR A1 FOR THE INTERLEUKIN 13 (IL-13RA1) (GENETIC). OUR HYPOTHESIS IS SUPPORTED BY A HUMAN CLINICAL STUDY PROVIDING A STRONG LINK BETWEEN HEIGHTENED ALLERGIC RESPONSE AND SUSCEPTIBILITY TO PD, AS WELL AS BY OUR RECENT FINDING THAT PIVOTAL REGULATORS OF ALLERGIC REACTIONS, IL-13 AND IL-13RA1, ARE LINKED TO HUMAN PD AND CONTRIBUTE TO NEURONAL LOSS IN MOUSE MODELS OF PD. IF SUCCESSFUL, WE WILL IDENTIFY ALLERGIC REACTIONS AND THE IL-13 SYSTEM AS NOVEL PATHOGENIC COMPONENTS OF PD AND WILL UTILIZE THEM AS NOVEL THERAPEUTIC TARGETS TO PREVENT PD OR REDUCE ITS PROGRESSION. ANTI-ALLERGIC THERAPIES, INCLUDING SPECIFIC IL-13 NEUTRALIZING ANTIBODIES, ARE ALREADY AVAILABLE OR ARE UNDER DEVELOPMENT, WHICH WILL REDUCE THE TIME DELAY BETWEEN BASIC RESEARCH, DEVELOPMENT OF THERAPEUTIC AGENTS, AND THEIR CLINICAL USE. IN ADDITION, THE ANIMAL MODEL WILL PROVIDE A NEW EXPERIMENTAL TOOL FOR THE SCIENTIFIC COMMUNITY TO FURTHER UNDERSTAND THE ROLE OF INFLAMMATION AND ALLERGY IN PD AND NEURODEGENERATIVE DISEASES.

ANTI-INFLAMMATORY SIGNALS AND NEURODEGENERATION - ABSTRACT NEUROIMMUNE SIGNALS REGULATE NEURONAL FUNCTION AND SURVIVAL. WE HAVE STRONG EVIDENCE INDICATING THAT ACTIVATION OF THE HETERODIMERIC INTERLEUKIN-13 RECEPTOR ALPHA 1/INTERLEUKIN-4 RECEPTOR ALPHA (IL-13RA1/IL-4RA) COMPLEX IN MIDBRAIN DOPAMINERGIC (DA) NEURONS AFFECTS THEIR VIABILITY. IN THE BRAIN, IL-13RA1/IL-4RA IS UNIQUELY EXPRESSED ON THE NEURONS OF THE SUBSTANTIA NIGRA PARS COMPACTA (SNC) THAT ARE LOST IN PARKINSON’S DISEASE (PD). WE ALSO SHOWED THAT INTERLEUKIN 13 (IL-13), PRODUCED DURING NEUROINFLAMMATION BY MICROGLIA AND NEURONS, CAN MODULATE THE ACTIVITY OF DOPAMINERGIC CELLS AND INCREASE THEIR SUSCEPTIBILITY TO OXIDATIVE DAMAGE. TO DATE, THERE IS A GAP IN UNDERSTANDING HOW NEUROINFLAMMATION CONTRIBUTES TO THE SELECTIVE LOSS OF DA NEURONS IN PD. HAVING ESTABLISHED THAT ACTIVATION OF IL-13RA1 SIGNALING CAN AFFECT THE SURVIVAL OF DOPAMINERGIC NEURONS DURING NEUROINFLAMMATION, IN THE PRESENT APPLICATION WE WISH TO ADDRESS THIS GAP. SPECIFICALLY, WE WISH TO TEST THE HYPOTHESIS THAT IL-13 AND NEURONAL IL-13RA1 CAUSE DAMAGE BY STIMULATING A REGULATED CELL DEATH PATHWAY CALLED FERROPTOSIS. WE ALSO WISH TO DETERMINE TO WHAT EXTENT IL-13 AND IL-13RA1 CONTRIBUTE TO NEURODEGENERATION IN A MOUSE MODEL OF ALPHA- SYNUCLEINOPATHY (A-SYN), A HALLMARK TRAIT OF PD THAT IS ASSOCIATED WITH NEUROINFLAMMATION AND OXIDATIVE DAMAGE. THIS WILL HELP US DETERMINE WHETHER TARGETING IL-13RA1 SIGNALING MIGHT BE A VIABLE APPROACH TO SLOW NEURODEGENERATION IN HUMANS AFFECTED BY A-SYNUCLEINOPATHY SUCH AS PD. THE ABILITY OF RUXOLITINIB, AN FDA- APPROVED DRUG THAT INHIBITS IL-13RA1 SIGNALING AND THAT OF THE NOVEL FERROPTOSIS INHIBITOR CMS121, TO REDUCE IL- 13-MEDIATED DAMAGE IN VIVO WILL ALSO BE TESTED. FINALLY, WE PROPOSE IN VIVO EXPERIMENTS UN A NOVEL MOUSE MODEL TO TEST THE HYPOTHESIS THAT A RARE GENETIC VARIANT OF IL-13 FOUND IN INDIVIDUALS DIAGNOSED WITH EARLY-ONSET PD CAN CONTRIBUTE TO MORE RAPID LOSS OF DOPAMINERGIC NEURONS IN A MOUSE WITH THE HOMOLOGUE OF THIS MUTATION. IF SUCCESSFUL, THESE EXPERIMENTS WILL PROVIDE STRONG SUPPORT FOR THE HYPOTHESIS THAT IL-13 AND IL-13RA1 ARE NOVEL TARGETS FOR PREVENTING PD OR SLOWING ITS PROGRESSION, AT LEAST IN A SUB-SET OF PD PATIENTS.

DISCOVERY OF NONCANONICAL AUTOPHAGY MODULATORS FOR ALZHEIMER'S AND RELATED DEMENTIAS - ABSTRACT DEMENTIAS SUCH AS ALZHEIMER’S DISEASE (AD), FRONTOTEMPORAL DEGENERATION (FTD), AND LEWY BODY DEMENTIA (LBD) ARE PROGRESSIVE NEURODEGENERATIVE DISORDERS CHARACTERIZED BY COGNITIVE DECLINE AND PATHOLOGICAL PROTEIN AGGREGATES, INCLUDING TAU, TDP-43, AND Α-SYNUCLEIN. THESE AGGREGATES RESULT FROM A BREAKDOWN IN PROTEOSTASIS AND IMPAIRMENTS IN MACROAUTOPHAGY, THE LYSOSOMAL PATHWAY CRUCIAL FOR CLEARING MISFOLDED PROTEINS AND DAMAGED ORGANELLES. ENHANCING AUTOPHAGY REDUCES AGGREGATE ACCUMULATION AND IMPROVES NEURONAL VIABILITY IN PRECLINICAL MODELS. HOWEVER, CURRENT STRATEGIES—SUCH AS MTOR INHIBITION OR TRANSCRIPTION FACTOR UPREGULATION—ARE LIMITED BY POOR SPECIFICITY, TOXICITY, OR INCOMPLETE ENGAGEMENT OF THE AUTOPHAGY PATHWAY. TO ADDRESS THESE LIMITATIONS, THIS PROJECT WILL ESTABLISH TWO COMPLEMENTARY INTRACELLULAR FUNCTIONAL SCREENING PLATFORMS DESIGNED TO DISCOVER NOVEL AUTOPHAGY-ACTIVATING MOLECULES THAT RESTORE PROTEOSTASIS IN NEURONAL MODELS OF DEMENTIA. THESE PLATFORMS EMPLOY GENETICALLY ENCODED LIBRARIES CAPABLE OF ENGAGING DYNAMIC AND PREVIOUSLY UNREACHED INTRACELLULAR TARGETS, ENABLING VARIED MECHANISMS OF ACTION. IN AIM 1, WE WILL DISCOVER MODULATORS FROM A SYNTHETIC LIBRARY USING AN AUTOPHAGY FLUX REPORTER IN NEURONAL CELLS. IN AIM 2, WE WILL IMPLEMENT A SECOND, ORTHOGONAL SCREENING PLATFORM BASED ON A DISTINCT PEPTIDE SCAFFOLD, ENABLING EXPLORATION OF COMPLEMENTARY CHEMICAL AND FUNCTIONAL SPACES. HITS FROM BOTH APPROACHES WILL BE VALIDATED THROUGH BIOCHEMICAL AND FUNCTIONAL ASSAYS AND TESTED IN HUMAN NEURONAL MODELS TO CONFIRM THEIR CAPACITY TO PROMOTE AGGREGATE CLEARANCE. THE OUTCOMES WILL BE NOVEL MODULATORS OF AUTOPHAGY AND A BROADLY APPLICABLE SCREENING PIPELINE TO ADVANCE THERAPEUTIC DISCOVERY FOR DEMENTIA AND OTHER PROTEINOPATHY-DRIVEN NEURODEGENERATIVE DISEASES.

AUTOIMMUNITY, CALORIE RESTRICTION, AND CORE BODY TEMPERATURE - PROJECT SUMMARY CALORIE RESTRICTION (CR) IS A BALANCED REDUCTION OF CALORIC INTAKE THAT PROMOTES HEALTHY AGING AND LONGEVITY WITHOUT CAUSING MALNUTRITION. IMPORTANTLY, IN EARLIER STUDIES, CR WAS ALSO FOUND TO REDUCE AUTOIMMUNITY IN SEVERAL MOUSE MODELS OF LUPUS. A STRIKING EFFECT OF CR IN MICE IS THE REDUCTION OF THE CORE BODY TEMPERATURE (TB) DURING SLEEP, WHICH IS LOWERED BY UP TO 10°C COMPARED TO MICE FED AD LIBITUM (AL). HERE, WE HYPOTHESIZE THAT THIS AMPLIFIED TB REDUCTION IS CRUCIAL FOR THE BENEFICIAL EFFECTS OF CR. CONSISTENT WITH THIS HYPOTHESIS, OUR PRELIMINARY RESULTS SUGGESTED THAT THE CR-ASSOCIATED INHIBITION OF LUPUS-LIKE AUTOIMMUNITY, ESPECIALLY KIDNEY DISEASE, IS LOST IF MICE ARE HOUSED UNDER CONDITIONS OF THERMONEUTRALITY (TN, ACHIEVED AT THE AMBIENT TEMPERATURE OF ~30°C), WHICH DOES NOT ALLOW BODY HEAT DISSIPATION AND HENCE PREVENTS TB REDUCTIONS IN CR MICE. THUS, STUDIES ARE PROPOSED TO INVESTIGATE THE EFFECTS OF CR AND TN ON CIRCADIAN TB PROFILES IN LUPUS MICE AT DIFFERENT DISEASE STAGES, ASSESS THE LONG-TERM IMPACT OF CR AND TN ON LUPUS DISEASE, AND DETERMINE THE EFFECTS OF CR AND TN ON GENE EXPRESSION SIGNATURES IN LUPUS KIDNEYS AND SPLEEN USING SINGLE CELL AND SPATIAL TRANSCRIPTOMICS. THE PROPOSED STUDIES ARE HIGHLY SIGNIFICANT, PARTICULARLY FOR TRANSLATIONAL APPLICATIONS OF CR IN HUMANS, SINCE HUMANS USE BEHAVIORAL THERMOREGULATION (CLOTHES, DUVETS, AIR CONDITIONING) TO LIVE AT TN.

CALORIE RESTRICTION, BODY TEMPERATURE AND ALZHEIMERS DISEASE - CALORIE RESTRICTION (CR) HAS ANTI-AGING EFFECTS, PROLONGS LIFESPAN AND REDUCES THE INCIDENCE OF AGE ASSOCIATED DISEASES. EXPERIMENTAL EVIDENCE IN ANIMAL MODELS INDICATES THAT CR AMELIORATES THE NEUROPATHOLOGIC AND BEHAVIORAL ABNORMALITIES SEEN IN TRANSGENIC MODELS OF ALZHEIMER’S DISEASE (AD). OUR PREVIOUS WORK DEMONSTRATED THAT ONE MECHANISM BY WHICH CR PROMOTES LONGEVITY IS BY REDUCING CORE BODY-TEMPERATURE (TB). SINCE VIRTUALLY ALL PROTEINS ARE SENSITIVE TO HEAT DENATURATION, NATURE HAS CREATED CELLULAR SYSTEMS TO PROTECT CELLS FROM EVEN SMALL QUANTITIES OF ABNORMALLY FOLDED TOXIC PROTEINS AS FAR BACK IN EVOLUTION AS BACTERIA. WE NOW HYPOTHESIZE THAT THE BENEFICIAL EFFECTS THAT CR HAS ON THE AD MODELS ARE AT LEAST IN PART DUE TO ITS ABILITY TO REDUCE TB AND THE SUBSEQUENT PROTEOTOXIC STRESS. HERE WE PROPOSE EXPERIMENTS TO TEST OUR HYPOTHESIS REGARDING THE ROLE OF LOWERING BODY TEMPERATURE AS BEING A MAJOR FACTOR IN BOTH LIFE EXTENSION AND THE SALUTARY EFFECT OF CR IN THE WELL VALIDATED TG2576 TRANSGENIC MODEL OF HUMAN AD.

DIFFERENTIAL ROLE OF TASL AND SLC15A4 IN TLR RESPONSES TO NUCLEIC ACIDS AND LUPUS DEVELOPMENT - PROJECT SUMMARY SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) IS CHARACTERIZED BY HYPERACTIVATION OF T CELLS, B CELLS AND DENDRITIC CELLS (DCS), PRODUCTION OF AUTOANTIBODIES TO NUCLEIC ACID-ASSOCIATED AND OTHER SELF-MOLECULES, AND IMMUNE COMPLEX-MEDIATED INFLAMMATORY DAMAGE IN MULTIPLE ORGANS. MECHANISTIC ASSESSMENTS IN BOTH MOUSE MODELS AND HUMANS HAVE IMPLICATED INNATE IMMUNE PATHWAYS OF NUCLEIC ACID SENSING IN LUPUS PATHOGENESIS, PARTICULARLY TOLL-LIKE RECEPTOR (TLR) ACTIVATION IN B CELLS AND PLASMACYTOID DENDRITIC CELLS (PDCS), PRODUCTION OF TYPE I INTERFERONS, AND EXPRESSION OF AN INTERFERON-INDUCIBLE GENE SIGNATURE OFTEN CORRELATING WITH DISEASE ACTIVITY. ADDITIONAL STUDIES INCLUDING BY OUR LABORATORY HAVE SHOWN THAT PATHOGENIC TLR RESPONSES IN SYSTEMIC AUTOIMMUNITY REQUIRE THE PARTICIPATION OF THE PEPTIDE/HISTIDINE TRANSPORTER SLC15A4, SUGGESTING THAT INHIBITION OF THIS TRANSPORTER MAY BE AN EFFECTIVE THERAPEUTIC APPROACH. VERY RECENT STUDIES, HOWEVER, SUGGESTED THAT SLC15A4 DOES NOT ACT AS A TRANSPORTER BUT RATHER AS BINDING PARTNER OF TASL, WHICH IN TURN FACILITATES TLR SIGNALING. THUS, THE FOCUS OF THIS PROPOSAL IS TO DEFINE MECHANISTICALLY HOW SLC15A4 AND TASL CONTRIBUTE TO TLR ACTIVATION. FOR THIS WE WILL USE A NOVEL TASL- DEFICIENT MOUSE MODEL THAT WILL ALLOW TO SEPARATE THE SLC15A4 ROLE AS A TRANSPORTER VS. ITS ROLE AS A PLATFORM THAT FACILITATES RECRUITMENT OF TASL. CRUCIALLY, THESE STUDIES WILL CLARIFY IF PHARMACOLOGIC INHIBITION OF THE SLC15A4 TRANSPORTER ACTIVITY IS AN APPROPRIATE STRATEGY, OR IF INSTEAD TARGETING TASL WOULD BE A BETTER APPROACH TO REDUCE INFLAMMATORY RESPONSES IN LUPUS AND OTHER AUTOIMMUNE CONDITIONS.

SIRT-1-MEDIATED REGULATION OF NEUROAIDS

KETOGENIC PROTECTION FROM ISCHEMIC STROKE: EVALUATING THE ROLE OF BLOOD-BRAIN BARRIER INTEGRITY

ENDOTHELIAL PROTEIN C RECEPTOR IN BREAST CANCER PROGRESSION

DEFINING THE ROLE OF ANTIBODY GLYCOSYLATION IN REGULATING NK CELL MEDIATED ADCC DURING HIV INFECTION - PROJECT SUMMARY/ABSTRACT MULTIPLE STUDIES HAVE DEMONSTRATED THAT NATURAL KILLER (NK) CELL ACTIVITY, INCLUDING ANTIBODY DEPENDENT CELLULAR CYTOTOXICITY (ADCC), IS CORRELATED WITH DELAYED HIV DISEASE PROGRESSION. FURTHERMORE, PROTEIN ENGINEERING HAS REVEALED THAT GLYCOSYLATION OF THE ANTIBODY FC REGION, PARTICULARLY FOR IGG1, AFFECTS FC GAMMA RECEPTOR IIIA (CD16) BINDING AFFINITY AND ADCC ACTIVITY. HOWEVER, THE MECHANISM BY WHICH FC GLYCOSYLATION ALTERS ADCC ACTIVITY REMAINS INCOMPLETELY UNDERSTOOD, SPECIFICALLY REGARDING THE SPATIOTEMPORAL LOCALIZATION OF CD16 AND ASSOCIATED SIGNALING MOLECULES. THE LONG-TERM GOAL OF THIS PROPOSAL IS TO OBTAIN A MORE COMPLETE UNDERSTANDING OF HOW ANTIBODY GLYCOBIOLOGY INTERPLAYS WITH THE INNATE IMMUNE RESPONSE TO GENERATE GUIDING PRINCIPLES FOR MORE EFFECTIVE VACCINES AND THERAPEUTICS. THE OBJECTIVE OF THIS PROPOSAL IS TO DETERMINE HOW ANTIBODY GLYCOSYLATION INFLUENCES CD16 MEDIATED NK CELL ACTIVATION AT SINGLE CELL RESOLUTION IN HIV INFECTED INDIVIDUALS AND TO DEFINE THE SPACIOTEMPORAL DYNAMICS OF CD16-BASED SIGNALING. THE RATIONALE FOR THIS WORK IS THAT THE COMBINATION OF CUTTING- EDGE MICROSCOPY OBSERVATIONS AND TEMPORAL PROTEIN ANALYSIS WILL PROVIDE NEW INSIGHTS INTO ADCC FUNCTION WITH IMMEDIATE IMPACTS ON ANTIBODY THERAPEUTIC DESIGN. OUR CENTRAL HYPOTHESIS IS THAT SPECIFIC ANTIBODY GLYCOSYLATION PROFILES WILL DIFFERENTIALLY MODULATE CD16 INTERACTIONS AND ADCC ACTIVITY THROUGH CHANGES IN CD16 SIGNALING. OUR CENTRAL HYPOTHESIS WILL BE TESTED IN TWO SPECIFIC AIMS: 1) DETERMINE HOW NARROW GLYCOSYLATION PROFILES OF MONOCLONAL ANTIBODIES (MABS) DIRECTED TO KEY SITES OF VULNERABILITY ON THE HIV ENVELOPE (ENV) AFFECT PHOSPHORYLATION STATES OF SIGNALING PROTEINS DURING NK CELL ADCC, COMPARING NK CELLS FROM HEALTHY AND HIV+ DONORS; 2) DETERMINE HOW ANTIBODY GLYCOSYLATION ALTERS THE SUBCELLULAR LOCALIZATION OF CD16 AND SIGNALING PROTEINS WITHIN THE NK CELL IMMUNOLOGICAL SYNAPSE (NKIS) THROUGHOUT EACH STAGE OF ADCC. WE WILL PURSUE THESE AIMS WITH INNOVATIVE TECHNIQUES IN MULTI-COLOR QUANTITATIVE FLUORESCENT MICROSCOPY AND SPECTRAL FLOW CYTOMETRY. DETAILED SINGLE CELL STUDIES USING MINFLUX NANOSCOPY, A SUPER-RESOLUTION FLUORESCENT MICROSCOPY TECHNIQUE CAPABLE OF 2 NM OR BETTER SPATIAL RESOLUTION IN 3D, WILL COMPLEMENT OUR PROTEOMIC ANALYSIS. THE EXPECTED OUTCOME OF OUR STUDIES IS THAT MARRYING OUR QUANTITATIVE PROTEOMIC ANALYSIS WITH MORE DETAILED MECHANISTIC STUDIES OF ADCC WILL RATIONALIZE PREVIOUSLY OBSERVED CHANGES IN CELLULAR ACTIVITY ASSOCIATED WITH DIFFERENTIAL ANTIBODY GLYCOSYLATION. OUR RESULTS WILL IMPACT HUMAN HEALTH BY PROVIDING A MOLECULAR RATIONAL FOR THE EFFECT OF ANTIBODY GLYCOSYLATION ON ADCC ACTIVITY, THUS IMPROVING ANTIBODY AND CELLULAR THERAPEUTIC DEVELOPMENT FOR HIV AS WELL AS A BROAD RANGE OF OTHER DISEASES.