Lieber Institute Inc

REGULATION OF NEURAL ACTIVITY IN FEAR CIRCUITS BY PROMOTER IV DERIVED BDNF

2/3 UNDERSTANDING PTSD THROUGH POSTMORTEM TARGETED BRAIN MULTI-OMICS

CELLULAR AND MOLECULAR ANALYSIS OF THE SCHIZOPHRENIA AND AUTISM SPECTRUM DISORDER GENE TRANSCRIPTION FACTOR 4 (TCF4)

MATERNAL SARS-COV-2 INFECTION, PLACENTA BIOLOGY, AND NEURODEVELOPMENTAL OUTCOMES IN THE OFFSPRING - PROJECT SUMMARY THE EXTENT TO WHICH SARS-COV-2 INFECTION IN PREGNANCY AFFECTS THE BIOLOGY OF THE MATERNAL-PLACENTAL-FETAL TRIAD AND NEURODEVELOPMENTAL TRAJECTORY IN OFFSPRING IS NOT KNOWN. EARLY STUDIES INDICATE THAT SARS-COV-2 CAN INDUCE ALTERATIONS IN THE PLACENTA WHICH CAN INCREASE RISK FOR ADVERSE PERINATAL OUTCOMES AND, BASED ON KNOWLEDGE FROM OTHER INFECTIOUS AND INFLAMMATORY DISORDERS IN PREGNANCY AND PRELIMINARY DATA ON THE CURRENT PANDEMIC, INCREASE RISK FOR NEURODEVELOPMENTAL DISORDERS (NDDS) IN OFFSPRING. THE LONG-TERM GOAL OF THIS PROPOSAL IS TO TIMELY IDENTIFY THE IMPACT OF THE ONGOING PANDEMIC ON THE NEURODEVELOPMENT OF INFANTS BORN FOLLOWING MATERNAL SARS-COV-2 INFECTION. THE OBJECTIVE IS TO ANALYZE THE RELATIONSHIPS BETWEEN MATERNAL SARS-COV-2 INFECTION, PLACENTA BIOLOGY, AND NEURODEVELOPMENTAL OUTCOMES, IN INTERACTION WITH GENOMIC RISK (GRSS) FOR NDDS AND SEX. THE CENTRAL HYPOTHESIS IS THAT MATERNAL SARS-COV-2 INFECTION AND INFECTION-RELATED PREGNANCY COMPLICATIONS AFFECT EARLY PATHS OF BRAIN DEVELOPMENT, PARTICULARLY IN THOSE WITH HIGH GRSS FOR NDDS AND IN MALES, AND THAT THESE EFFECTS ARE MEDIATED BY ALTERATIONS IN PLACENTA MORPHOLOGY AND MOLECULAR BIOLOGY. THE RATIONALE UNDERLYING THE PROPOSAL IS THAT COMPLETION WILL DEFINE CRITICAL TARGETS FOR IDENTIFICATION AND POTENTIALLY PREVENTION OF NDDS IN THE OFFSPRING OF INFECTED MOTHERS. THE CENTRAL HYPOTHESIS WILL BE TESTED BY PURSUING THE FOLLOWING SPECIFIC AIMS: 1) DETERMINE PREGNANCY, PLACENTAL, AND NEWBORN OUTCOMES FOLLOWING ANTENATAL SARS-COV-2 INFECTION; 2) INVESTIGATE THE RELATIONSHIP BETWEEN SARS-COV-2 MATERNAL INFECTION, GRSS FOR NDDS, AND PLACENTA MOLECULAR BIOLOGY; 3) EVALUATE THE RELATIONSHIP BETWEEN MATERNAL SARS-COV-2 INFECTION IN PREGNANCY, OFFSPRING GENOMIC RISK FACTORS FOR NDDS, AND NEURODEVELOPMENTAL OUTCOMES. WE WILL PURSUE THE AIMS USING AN INNOVATIVE COMBINATION OF PLACENTA TISSUE AND MATERNAL AND FETAL BLOOD ANALYSIS, AND COMPREHENSIVE PSYCHOMETRIC TESTING OF EARLY CHILD NEURODEVELOPMENT. WE WILL COMPARE WOMAN-PLACENTA-INFANT TRIADS EXPOSED TO SARS-COV-2 INFECTION DURING PREGNANCY TO UNVACCINATED AND VACCINATED CONTROLS. WE WILL STUDY HOW, IN A LARGE SAMPLE OF MULTIPLE ETHNICITIES, MATERNAL INFECTION AND STRESS, GRSS FOR NDDS, AND SEX, ADVERSELY AFFECT NEURODEVELOPMENTAL OUTCOMES OF THE OFFSPRING THOUGH PROCESSES MEDIATED BY ALTERATION IN PLACENTA GENE/PROTEIN EXPRESSION AND MATERNAL IMMUNE ACTIVATION. THE PROPOSED RESEARCH IS SIGNIFICANT, BECAUSE IT WILL CHARACTERIZE THE RELATIONSHIP BETWEEN MATERNAL SARS-COV-2 INFECTION AND NEURODEVELOPMENTAL OUTCOME OF THE OFFSPRING, AND ALSO IDENTIFY FACTORS, MOLECULES AND GENOMIC PREDICTORS THAT MODULATE, MEDIATE OR – AS BIOMARKERS – REVEAL SUCH RELATIONSHIP. THE PROXIMATE EXPECTED OUTCOME OF THIS WORK WILL BE AN UNDERSTANDING OF THE MECHANISMS THROUGH WHICH MATERNAL INFECTION, GENOMIC RISK AND SEX, PLACENTA BIOLOGY, AND POSTNATAL FACTORS MAY CONTRIBUTE TO DEFINE RISK FOR NDDS. THE RESULTS WILL HAVE AN IMMEDIATE POSITIVE IMPACT TO INFORM TARGETED INTERVENTIONS AND GUIDELINES FOR SARS-COV-2 EXPOSED WOMEN AND THEIR INFANTS, IMPACTING HOW CLINICIANS EVALUATE AND CARE FOR THESE CASES, AND TO IDENTIFY POTENTIAL BIOMARKERS OF RISK FOR NDDS IN OFFSPRING OF MOTHERS WITH INFECTION DURING PREGNANCY.

DRUG DISCOVERY OF COMT INHIBITORS TO TREAT COGNITIVE DEFICITS IN SCHIZOPHRENIA

BSMN REFERENCE TISSUE PROJECT (LIEBER INSTITUTE FOR BRAIN DEVELOPMENT) 1U01MH106893-01 - 1/3-SCHIZOPHRENIA GENETICS AND BRAIN SOMATIC MOSAICISM

LAMINAR DISSECTION OF CORTICAL HUMAN BRAIN GENE EXPRESSION IN NEUROPSYCHIATRIC DISORDERS - PROJECT SUMMARY/ABSTRACT THE DLPFC IS A TRUE SIX LAYERED NEOCORTEX, AND NEURONS IN DIFFERENT CORTICAL LAYERS SHOW DISTINCT EXPRESSION PATTERNS, MORPHOLOGY, PHYSIOLOGY AND PATTERNS OF CONNECTIVITY. CONVERGING EVIDENCE SUGGESTS THAT IMPAIRMENTS IN THE FORMATION OR MAINTENANCE OF SYNAPSES MAY BE INVOLVED IN SCHIZOPHRENIA AND OTHER NEUROPSYCHIATRIC DISORDERS. STUDIES IN THE POSTMORTEM BRAINS OF SUBJECTS HAVE POINTED TO SPECIFIC CELL TYPES AND REVEALED DIFFERENCES IN NEURONAL AND SYNAPTIC STRUCTURE THAT ARE LOCALIZED TO SPECIFIC LAYERS, SUGGESTING THAT GENETIC RISK FOR SCHIZOPHRENIA MAY MANIFEST WITH LAMINAR SPECIFICITY. GIVEN THE CLOSE RELATIONSHIP BETWEEN BRAIN STRUCTURE AND FUNCTION, PRECISELY ASSIGNING GENE EXPRESSION TO THE SPATIAL COORDINATES OF INDIVIDUAL CELL POPULATIONS WITHIN THIS CORTICAL CYTOARCHITECTURE WOULD SIGNIFICANTLY ADVANCE OUR UNDERSTANDING OF HOW DYSREGULATION IN THESE AREAS CONTRIBUTES TO DEBILITATING NEUROPSYCHIATRIC DISORDERS. IN THIS APPLICATION, WE PROPOSE TO GENERATE DETAILED SPATIAL TRANSCRIPTOMICS MAPS OF THE HUMAN DLPFC IN PATIENTS WITH SCHIZOPHRENIA (SCZD), BIPOLAR (BPD), MAJOR DEPRESSIVE (MDD) AND AUTISM SPECTRUM (ASD) DISORDERS, AND CONTRAST THESE LAMINAR EXPRESSION PATTERNS TO THOSE DERIVED FROM MATCHED NEUROTYPICAL CONTROLS (CONT). WE WILL USE THE 10X GENOMICS VISIUM PLATFORM, WHICH COMBINES TRANSCRIPTOME-WIDE RNA SEQUENCING WITH DETAILED HIGH-RESOLUTION HISTOLOGY AND IMMUNOFLUORESCENCE IMAGING, TO GENERATE THESE SPATIAL TRANSCRIPTOMICS PROFILES. WE WILL COMBINE THESE TOPOGRAPHIC AND CELL TYPE-SPECIFIC MAPS TO IMPLICATE LAYER- AND CELL TYPE-SPECIFIC POPULATIONS IN PSYCHIATRIC DISORDER GENETIC RISK AND ILLNESS STATE THAT WILL BE VALIDATED USING COMPLEMENTARY QUANTITATIVE IN SITU HYBRIDIZATION TECHNIQUES. THESE LAYER-SPECIFIC AND CELL TYPE-SPECIFIC EXPRESSION PROFILES CAN REFINE THE MOLECULAR CAUSES AND CONSEQUENCES OF DEBILITATING NEUROPSYCHIATRIC DISORDERS THAT CAN BE TARGETED FOR PREVENTION AND TREATMENT.

REGISTRATION OF SPATIAL GENE EXPRESSION IN KEY NODES OF REWARD-RELATED CIRCUITRY IN THE HUMAN BRAIN - PROJECT SUMMARY DRUG ADDICTION IS HIGHLY COMORBID WITH PSYCHIATRIC DISORDERS, PARTICULARLY POST-TRAUMATIC STRESS DISORDER (PTSD) AND MAJOR DEPRESSIVE DISORDER (MDD). THESE CONDITIONS SHARE UNDERLYING GENETIC RISK AND ARE EXACERBATED BY SIMILAR ENVIRONMENTAL FACTORS, INCLUDING EXPOSURE TO STRESS AND TRAUMATIC EVENTS. THE DORSAL ANTERIOR CINGULATE CORTEX (DACC), NUCLEUS ACCUMBENS (NAC) AND AMYGDALA CONSTITUTE KEY NODES OF THE BRAIN’S REWARD CIRCUITRY, AND PERTURBATIONS IN REWARD SIGNALING ARE HIGHLY IMPLICATED IN ADDICTION, MDD AND PTSD. THE HUMAN DACC, NAC AND AMYGDALA HAVE UNIQUE NEUROANATOMICAL FEATURES, WHICH CORRESPOND TO DISTINCT BIOLOGICAL FUNCTIONS. GIVEN THE CLOSE RELATIONSHIP BETWEEN BRAIN STRUCTURE AND FUNCTION, PRECISELY ASSIGNING GENE EXPRESSION TO THE SPATIAL COORDINATES OF INDIVIDUAL CELL POPULATIONS WITHIN THE CYTOARCHITECTURE CAN SIGNIFICANTLY ADVANCE OUR UNDERSTANDING OF HOW DYSREGULATION IN THESE AREAS CONTRIBUTES TO ADDICTION AND COMORBID NEUROPSYCHIATRIC DISORDERS. TOWARDS THIS GOAL, WE PROPOSE TO GENERATE DETAILED SPATIAL TRANSCRIPTOMIC MAPS, WHICH WILL BE COMBINED WITH SINGLE-NUCLEUS RNA SEQUENCING (SNRNA-SEQ) TO REGISTER MOLECULARLY-DEFINED CELL TYPES TO THEIR SPATIAL COORDINATES, FACILITATING PREDICTION OF THE ANATOMICAL LOCATIONS OF DISTINCT NEURONAL CLASSES OF CELLS WITHIN THE DACC, NAC AND AMYGDALA. THESE MOLECULARLY- AND SPATIALLY-DEFINED POPULATIONS OF CELLS WILL BE ASSOCIATED WITH GENE EXPRESSION CHANGES LINKED TO SUBSTANCE USE AND COMORBID NEUROPSYCHIATRIC DISORDERS. WE HYPOTHESIZE THAT THESE REGIONS HAVE A PRECISE MOLECULAR ARCHITECTURE THAT REVEALS 1) TOPOGRAPHICALLY ORGANIZED AND MOLECULARLY-DEFINED CELL TYPES WITHIN LAYERS OF THE DACC, AND ACROSS SUB-REGIONS OF THE NAC AND AMYGDALA; 2) SPATIAL-ENRICHMENT OF GENES ASSOCIATED WITH ADDICTION AND COMORBID NEUROPSYCHIATRIC DISORDERS. BY GENERATING THE FIRST TRANSCRIPTOME-SCALE SPATIAL MAPS OF THE HUMAN DACC, NAC AND AMYGDALA, CRITICAL INFORMATION ABOUT THE MOLECULAR LANDSCAPE OF THESE REGIONS WITHIN THE ARCHITECTURE OF THE HUMAN BRAIN WILL BE GENERATED. OUR SPATIAL REGISTRATION APPROACH WILL FACILITATE REFINED ANNOTATION OF CELL TYPES IN THE HUMAN BRAIN, AND CONTRIBUTE TO UNDERSTANDING ADDICTION AND COMORBID NEUROPSYCHIATRIC DISORDERS BY IDENTIFYING CLINICAL ASSOCIATIONS WITH MOLECULARLY- AND SPATIALLY-DEFINED CELL POPULATIONS THAT CAN BE TARGETED FOR PREVENTION AND TREATMENT.

FUNCTIONAL METHYLOMICS APPROACHES FOR SCHIZOPHRENIA IN THE FRONTAL CORTEX AND HIPPOCAMPUS

SMALL-MOLECULE PROBES FOR AUGMENTING D5 RECEPTOR SIGNALING - PROJECT SUMMARY COGNITIVE IMPAIRMENT IS A CORE FEATURE OF MANY NEUROPSYCHIATRIC DISORDERS THAT DIRECTLY CORRELATES WITH FUNCTIONAL OUTCOME FOR PATIENTS. CURRENT TREATMENT OPTIONS ARE INADEQUATE AND THERE IS A NEED FOR NOVEL THERAPEUTIC STRATEGIES. THE DOPAMINE D1-LIKE RECEPTOR FAMILY (D1 AND D5 RECEPTORS) HAS BEEN PROPOSED AS A POTENTIAL TARGET FOR IMPROVING COGNITIVE FUNCTION. CLINICAL STUDIES WITH D1/D5 FULL AGONISTS HAVE SHOWN PROMISING EFFICACY, BUT THESE COMPOUNDS SUFFER FROM SIGNIFICANT DRAWBACKS RELATED TO ADVERSE EFFECT PROFILES. THE LACK OF SELECTIVE LIGANDS AS WELL AS CONSIDERABLE SPATIAL OVERLAP BETWEEN THE D1 AND D5 RECEPTORS HAVE MADE IT DIFFICULT TO DETERMINE WHICH RECEPTOR IS RESPONSIBLE FOR SPECIFIC EFFICACY AND ADVERSE EFFECT FEATURES OF THE NONSELECTIVE AGONISTS. THERE HAVE BEEN EFFORTS TO DEVELOP SELECTIVE TOOLS FOR D1 RECEPTORS BUT, TO OUR KNOWLEDGE, THERE HAVE BEEN NO FORMAL EFFORTS TO IDENTIFY D5-SELECTIVE COMPOUNDS. RECENT RESEARCH FROM OUR GROUP AND OTHERS HAS SHOWN THAT THE D5 RECEPTOR PLAYS A CRITICAL ROLE IN COGNITIVE FUNCTION, PARTICULARLY IN COGNITIVE DOMAINS SIGNIFICANTLY DISRUPTED IN NEUROPSYCHIATRIC DISORDERS. ADDITIONALLY, MULTIPLE D5-SELECTIVE SIGNALING PATHWAYS RELEVANT FOR NEURAL FUNCTION HAVE BEEN IDENTIFIED. UNFORTUNATELY, FURTHER EXPERIMENTATION TO DETERMINE THE ROLE OF THE D5 RECEPTOR IN NEUROBIOLOGY HAS BEEN HINDERED BY THE LACK OF SELECTIVE LIGANDS. IN THIS APPLICATION, WE PROPOSE TO DESIGN AND SYNTHESIZE D5-SELECTIVE CHEMICAL PROBES. DUE TO THE HIGH DEGREE OF HOMOLOGY BETWEEN THE D5 AND D1 ORTHOSTERIC BINDING SITES, WE WILL FOCUS ON ALLOSTERIC MODULATORS. ONCE SELECTIVITY FOR D5 RECEPTORS OVER D1 RECEPTORS HAS BEEN ESTABLISHED USING CAMP INDUCTION ASSAYS, POTENCY ON CELL-BASED ASSAYS OF D5-SPECIFIC SIGNALING CASCADES WILL BE DETERMINED. COMPOUNDS SHOWN TO BE POTENT AND SELECTIVE WILL THEN UNDERGO IN VITRO SCREENING FOR METABOLIC STABILITY AND COUNTERSCREENING AGAINST A BROAD PANEL OF RECEPTORS AND ION CHANNELS. COMPOUNDS THAT ARE FOUND TO BE METABOLICALLY STABLE WITH MINIMAL OFF-TARGET BINDING, WILL BE TESTED IN VIVO TO DETERMINE IF THEY HAVE SUFFICIENT PLASMA AND BRAIN EXPOSURE TO ENGAGE THE D5 RECEPTOR AND ADVANCE TO PHARMACODYNAMIC ASSAYS. FINALLY, COMPOUNDS WILL BE TESTED TO DETERMINE THEIR ABILITY TO INDUCE BDNF EXPRESSION IN THE PREFRONTAL CORTEX, A KNOWN EFFECT OF SIGNALING THROUGH THE D5 RECEPTOR. ONCE OPTIMIZED AND CHARACTERIZED, THESE D5-SELECTIVE PROBES WILL ALLOW US TO FURTHER CHARACTERIZE THE ROLE OF THE D5 RECEPTOR IN NEURAL FUNCTION AND DETERMINE IF IT IS A VIABLE THERAPEUTIC TARGET.

REGULATION OF GENE EXPRESSION IN THE HUMAN HABENULA IN COMORBID OPIOID ADDICTION AND DEPRESSION - PROJECT SUMMARY/ABSTRACT DRUG ADDICTION IS HIGHLY COMORBID WITH NEUROPSYCHIATRIC DISORDERS, ESPECIALLY MAJOR DEPRESSIVE DISORDER (MDD). BOTH CONDITIONS ARE MODERATELY HERITABLE AND EXACERBATED BY ENVIRONMENTAL FACTORS, PARTICULARLY STRESSFUL LIFE EVENTS, SUGGESTING COMBINED GENETIC AND EPIGENETIC LIABILITY. FURTHERMORE, BOTH ILLNESSES FEATURE DYSREGULATION OF DOPAMINERGIC (DA) AND SEROTONERGIC (5HT) SYSTEMS MODULATING MOTIVATION AND COGNITIVE FUNCTION. THE HABENULA (HB) IS ONE OF THE FEW BRAIN REGIONS CONTROLLING BOTH DA AND 5HT SYSTEMS AND IS A HIGHLY ORGANIZED CENTRAL NODE FOR REWARD CIRCUITS GOVERNING MOTIVATED BEHAVIOR AND AFFECTIVE STATES. HB DYSFUNCTION IS ASSOCIATED WITH IMPAIRED MOOD REGULATION AND SUBSTANCE USE DISORDER (SUD), BUT THE GENETIC AND EPIGENETIC MECHANISMS MEDIATING THIS DYSFUNCTION ARE UNKNOWN. GIVEN THE CLOSE RELATIONSHIP BETWEEN BRAIN STRUCTURE AND FUNCTION, ASSIGNING GENE EXPRESSION TO FUNCTIONALLY DISTINCT ANATOMICAL SUBDIVISIONS AND CELL POPULATIONS WITHIN THE HUMAN HB WOULD SIGNIFICANTLY ADVANCE OUR UNDERSTANDING OF HOW HB DYSREGULATION CONTRIBUTES TO NEUROPSYCHIATRIC DISORDERS AND SUD. TOWARDS THIS END, WE PROPOSE TO EMPLOY 10X GENOMICS MULTI-OME AND VISIUM TECHNOLOGIES TO GENERATE CORRESPONDING SINGLE CELL AND SPATIAL MOLECULAR REFERENCE MAPS OF HUMAN HB TO IDENTIFY GENETICALLY-DEFINED AND TOPOGRAPHICALLY-ORGANIZED CELL TYPES ACROSS MEDIAL AND LATERAL SUBDIVISIONS OF THIS BRAIN REGION. WE WILL ALSO GENERATE TRANSCRIPTOMIC DATA FROM HOMOGENATE HUMAN HB IN PATIENTS WITH MDD AND COMORBID MDD/OPIOID USE DISORDER (OUD), AND CONTRAST THESE GENE EXPRESSION PATTERNS TO THOSE DERIVED FROM MATCHED NEUROTYPICAL CONTROLS. WE WILL INTEGRATE COMPLIMENTARY TOPOGRAPHIC AND CELL TYPE- SPECIFIC REFERENCE MAPS WITH HOMOGENATE DATA FROM PATIENTS WITH DEPRESSION AND ADDICTION TO IMPLICATE SPECIFIC HB CELL POPULATIONS AND SUBREGIONS IN ILLNESS STATE AND GENETIC RISK FOR THESE HIGHLY COMORBID CONDITIONS. BY GENERATING THE FIRST MOLECULAR NEUROANATOMICAL ATLAS OF THE HUMAN HABENULA, WE WILL FACILITATE REFINED ANNOTATION OF CELL TYPES WITH BRAIN ARCHITECTURE IN A KEY INTEGRATION HUB OF BRAIN REWARD CIRCUITRY THAT CAN BE TARGETED FOR PREVENTION AND TREATMENT OF DEBILITATING NEUROPSYCHIATRIC AND SUBSTANCE USE DISORDERS.

1/2 LARGE-SCALE, SINGLE-CELL CHARACTERIZATION OF MOLECULAR AND CELLULAR NETWORKS OF MOOD REGULATION CIRCUITRY IN MAJOR DEPRESSIVE DISORDER - WHILE THERE IS STRONG EVIDENCE SUPPORTING THE ROLE OF THE ANTERIOR CINGULATE CORTEX, BASOLATERAL AMYGDALA, AND THE HIPPOCAMPUS (ACC, BLA, HIPP) AS A KEY NEURAL NETWORK REGULATING MOOD, AND THEREFORE CENTRAL TO THE PATHOPHYSIOLOGY OF MAJOR DEPRESSIVE DISORDER (MDD), MUCH REMAINS UNKNOWN, INCLUDING WHICH GENE PATHWAYS AND WHICH SPECIFIC CELL TYPES PLAY A PRIMARY CAUSAL ROLE MEDIATING ALTERATIONS IN THIS CIRCUIT, AND WHAT CELL-TYPE CONNECTIONS, WITHIN AND BETWEEN THESE REGIONS, ARE PARTICULARLY ALTERED IN DEPRESSIVE STATES. THE OVERALL OBJECTIVE OF THIS APPLICATION IS TO GENERATE SINGLE-CELL TRANSCRIPTOMIC PROFILES TO STUDY MOLECULAR CHANGES, INCLUDING THOSE SPECIFIC TO GENETIC ANCESTRY AND SEX, ASSOCIATED WITH MDD IN THE MOOD REGULATION CIRCUIT. WHILE DISEASE BURDEN IS GREATER IN AFRICAN AMERICANS, THE IMPACT OF GENETIC ANCESTRY REMAINS UNKNOWN AS MOST GENOMIC STUDIES IN MDD SO FAR HAVE BEEN LIMITED TO SUBJECTS OF EUROPEAN DESCENT. IN ADDITION, PREVIOUS STUDIES REVEALED THAT TRANSCRIPTOMIC CHANGES ASSOCIATED WITH MDD ARE SEX-SPECIFIC, AND GENE NETWORKS ARE DIFFERENTIALLY DYSREGULATED BETWEEN SEXES. THE APPLICANTS’ RECENT SINGLE-CELL BRAIN STUDY REVEALED CELL-SPECIFIC CONTRIBUTIONS TO TRANSCRIPTOMIC CHANGES ASSOCIATED WITH MDD. THE PROPOSED PROJECT IS A LARGE-SCALE, SYSTEMATIC INVESTIGATION IN THE ACC, BLA, AND HIPP TO INTERROGATE THE TRANSCRIPTOME AT SINGLE-NUCLEUS RESOLUTION IN AN UNPRECEDENTLY LARGE AND REPRESENTATIVE SAMPLE OF MDD. THE SPECIFIC AIMS ARE TO: 1) IDENTIFY, AT THE SINGLE-CELL LEVEL TRANSCRIPTOMIC CHANGES ASSOCIATED WITH MDD IN 800 SUBJECTS ACROSS THREE LINKED BRAIN REGIONS: ACC, BLA, AND HIPP; 1B) STUDY THE IMPACT OF GENETIC ANCESTRY AND SEX; 2) DEFINE CELL NETWORKS ASSOCIATED WITH MOOD REGULATION USING MACHINE LEARNING APPROACHES; AND 3) IDENTIFY CELL-SPECIFIC EXPRESSION QUANTITATIVE TRAIT LOCI (EQTLS) COLOCALIZING WITH GENOME-WIDE SIGNIFICANT SNPS IDENTIFIED IN MDD GWAS ANALYSES. A LARGE COHORT (N=800) OF HUMAN POST-MORTEM SAMPLES OBTAINED FROM SUBJECTS WITH MDD WILL BE COMPARED TO PSYCHIATRICALLY-HEALTHY CONTROLS. THE SAMPLE (~20% AFRICAN AMERICAN AND ~30% FEMALE) WILL ALLOW FOR STUDYING THE IMPACT OF GENETIC ANCESTRY AND SEX. DROPLET-BASED SINGLE-NUCLEUS RNA SEQUENCING WILL BE APPLIED TO GENERATE TRANSCRIPTOMIC PROFILES. DEEP LEARNING APPROACHES WILL BE USED TO IDENTIFY AND ANNOTATE THE CELL TYPES AND GENE NETWORKS ASSOCIATED MDD. THE LATEST GWAS DATA IN MDD WILL BE LEVERAGED TO FINE MAP GENETIC LOCI WITH CELLULAR AND REGIONAL RESOLUTION. THE PROPOSED RESEARCH IS INNOVATIVE BECAUSE IT IS THE FIRST LARGE-SCALE INVESTIGATION OF THE ACC-BLA-HIPP CIRCUIT IN HUMANS AND WILL REPRESENT THE LARGEST SINGLE-CELL TRANSCRIPTIONAL RESOURCE OF THE HUMAN BRAIN. IT WILL IDENTIFY GENE AND CELLULAR NETWORKS ASSOCIATED WITH SEX OR GENETIC ANCESTRY, AND WILL ALSO GENERATE A VAST AMOUNT OF TRANSCRIPTOMIC DATA ON NEUROTYPICAL BRAINS. THIS RESEARCH IS SIGNIFICANT BECAUSE IT WILL GREATLY ADVANCE OUR UNDERSTANDING OF THE CELLULAR AND MOLECULAR PATHWAYS INVOLVED IN MOOD REGULATION AND MDD. THROUGH A BETTER UNDERSTANDING OF THE MECHANISMS OF DEPRESSIVE ILLNESS, WE MAY BE ONE STEP CLOSER TO DEVELOPING NOVEL TREATMENT STRATEGIES AND PERSONALIZE INTERVENTIONS.

UNDERSTANDING SCHIZOPHRENIA RISK MECHANISMS VIA MULTIMODAL ANALYSIS OF PATIENT-DERIVED NEURONS AND 3D CORTICAL ORGANOIDS AND CORRELATION WITH CLINICAL AND COGNITIVE PHENOTYPES - PROJECT SUMMARY: SCHIZOPHRENIA (SCZ) IS A COMPLEX POLYGENIC DISORDER, ENCOMPASSING A SPECTRUM OF POSITIVE AND NEGATIVE CLINICAL SYMPTOMS AND COGNITIVE DEFICITS THAT VARY CONSIDERABLY BETWEEN INDIVIDUALS. UNFORTUNATELY, PROGRESS IN UNDERSTANDING THE ETIOLOGY AND PATHOPHYSIOLOGY IS HINDERED BY THE LACK OF APPROPRIATE MODELS THAT ADEQUATELY CAPTURE BOTH THE COMPLEX AND HETEROGENEOUS NATURE OF THE GENETIC RISK AND THE DIVERSITY OF THE PHENOTYPIC MANIFESTATIONS. THE ADVENT OF INDUCED PLURIPOTENT STEM CELL (HIPSC) TECHNOLOGY PROVIDES AN IMPORTANT NEW EXPERIMENTAL PLATFORM TO STUDY THE CELLULAR AND CIRCUIT FUNCTION OF HUMAN CELLS THAT CONTAIN AN INDIVIDUAL’S FULL COMPLEMENT OF GENOMIC RISK ALLELES. THUS, THE DOOR IS NOW OPENED TO DEVELOPING IMPROVED IN VITRO MODELS THAT CAN ENHANCE UNDERSTANDING IN AN INTEGRATED CONTEXT, RELATING PATIENT GENOMES AND CELLULAR VARIABLES TO THEIR CLINICAL AND COGNITIVE PHENOTYPES. IN THIS PROPOSAL, WE DESCRIBE OUR ESTABLISHED, SYSTEMATIC, AND QUANTITATIVE HIPSC PIPELINE THAT MODELS THE CUMULATIVE CELLULAR EFFECTS OF COMMON SCZ GENETIC RISK VARIANTS BY SELECTING PATIENT AND CONTROL LINES (CON) BASED ON THEIR SCZ POLYGENIC RISK SCORE (PRS). HIPSC LINES WERE GENERATED FROM FIBROBLAST DONORS IN OUR NIMH CLINICAL SIBLING STUDY, FOR WHICH EXTENSIVE CLINICAL AND COGNITIVE DATA ARE AVAILABLE. ON OUR 2D PLATFORM, WE PERFORMED UNBIASED PHENOTYPIC DISCOVERY ON CORTICAL NEURONS DERIVED FROM 13 HIGH PRS SCZ HIPSCS AND 15 LOW PRS CON HIPSCS USING AN ARRAY OF PHYSIOLOGICAL ASSAYS, DONE BLIND TO PATIENT INFORMATION. FROM THESE EXPERIMENTS WE HAVE PUBLISHED A REPORT WITH MULTIPLE PRELIMINARY ASSOCIATIONS BETWEEN ELECTROPHYSIOLOGICAL VARIABLES AND PATIENT CLINICAL AND COGNITIVE PHENOTYPES. HERE, WE PROPOSE TO REPLICATE AND EXPAND ON THESE FINDINGS USING AN ENTIRELY NEW SET OF HIPSCS (17 SCZ, 15 CON), AGAIN CHOSEN BASED ON PRS. IN AIM 1, USING OUR 2D PLATFORM, WE PROPOSE TO REPLICATE, EXPAND, AND EXAMINE IN DEPTH CASE/CONTROL PHENOTYPES AND ASSOCIATIONS BETWEEN CELLULAR MEASURES AND CLINICAL AND COGNITIVE FEATURES OBSERVED IN SCZ PATIENTS. IN AIM 2, WE PROPOSE TO USE 3D CORTICAL ORGANOIDS TO CONFIRM OUR PRIOR VOLTAGE GATED SODIUM CHANNEL (VGSC) AND GABAERGIC PHENOTYPES AND TO DISCOVER NOVEL MOLECULAR AND CELLULAR ALTERATIONS IN EARLY CORTICAL DEVELOPMENT BY PERFORMING A MULTI-MODAL SET OF ASSAYS THAT INCLUDES SINGLE CELL ELECTROPHYSIOLOGY, BULK AND SINGLE CELL RNA SEQUENCING, PROTEOMICS, AND METABOLOMICS ON OUR ENTIRE SET OF SCZ AND CON HIPSCS (N=60). TOGETHER, THE WORK IN THESE AIMS IS DESIGNED TO IDENTIFY CELLULAR PHENOTYPES RELATED TO THE ETIOLOGY, PATHOPHYSIOLOGY, AND SYMPTOMATOLOGY OF SCHIZOPHRENIA, WITH THE GOAL OF IDENTIFYING THERAPEUTIC TARGETS AND ENABLING PRECISION PSYCHIATRY.

IMPACT OF NEUROMELANIN AND TAU ACCUMULATION DURING AGING AND DISEASE ON LOCAL GENE EXPRESSION IN THE HUMAN LOCUS COERULEUS USING SPATIALLY-RESOLVED TRANSCRIPTOMICS WITH PROTEIN DETECTION - PROJECT SUMMARY THE LOCUS COERULEUS (LC) IS A SMALL BILATERAL NUCLEUS LOCATED IN THE DORSAL PONS OF THE BRAINSTEM, AND SERVES AS THE BRAIN’S PRIMARY SITE FOR PRODUCING THE NEUROMODULATOR NOREPINEPHRINE (NE). NE-PRODUCING NEURONS IN THE LC PROJECT TO MANY REGIONS OF THE CENTRAL NERVOUS SYSTEM TO MODULATE HIGHLY DIVERGENT FUNCTIONS INCLUDING COGNITION, AROUSAL, AND MOOD. THE LC IS ONE OF THE EARLIEST SITES OF DEGENERATION IN ALZHEIMER’S DISEASE (AD), AND PROFOUND LOSS OF LC-NE NEURONS ACCELERATES WITH DISEASE PROGRESSION. SEVERAL UNIQUE ANATOMICAL, MORPHOLOGICAL AND NEUROCHEMICAL PROPERTIES LIKELY CONTRIBUTE TO VULNERABILITY OF LC-NE NEURONS. FIRST, LC-NE NEURONS SYNTHESIZE NEUROMELANIN (NM), A PROPERTY SHARED BY ONLY A FEW CELL TYPES IN THE HUMAN BRAIN. NM ACCUMULATES WITH AGING, AND ONE HYPOTHESIS IS THAT IT INITIALLY PROTECTS LC-NE NEURONS BY SEQUESTERING OR CHELATING NEUROTOXIC SUBSTANCES. HOWEVER, IN LATE LIFE AND DISEASE STATES, NM DEPOSITION MAY BE DETRIMENTAL WHEN IT OVERWHELMS CELLULAR MACHINERY, AND ACCUMULATED TOXINS ARE RELEASED FROM DEGENERATING CELLS. SECOND, ACCUMULATION OF PHOSPHORYLATED TAU (PTAU), A PRIMARY AD NEUROPATHOLOGY, IS DETECTED IN THE LC PRIOR TO ANY OTHER BRAIN REGION. IN THIS APPLICATION, WE GENERATE TRANSCRIPTOMIC-SCALE SPATIAL MAPS THAT INCORPORATE LOCALIZATION OF NM AND PTAU IN THE HUMAN LC IN BRAIN DONORS DIAGNOSED WITH AD AND CONTRAST THESE EXPRESSION PATTERNS TO BOTH MIDDLE-AGED AND AGE-MATCHED ELDERLY NEUROTYPICAL DONORS. WE WILL USE THE 10X GENOMICS VISIUM SPATIAL GENE EXPRESSION AND THE VISIUM SPATIAL PROTEOGENOMICS PLATFORMS, WHICH COMBINE TRANSCRIPTOME-WIDE RNA SEQUENCING WITH DETAILED HIGH-RESOLUTION HISTOLOGY AND IMMUNOFLUORESCENCE IMAGING TO SEGMENT AND QUANTIFY NM AND PTAU, RESPECTIVELY. WE WILL COMPUTATIONALLY ALIGN ADJACENT TISSUE SECTIONS FROM THE SAME DONOR ACROSS PLATFORMS TO DETERMINE HOW JOINT ACCUMULATION OF NM AND PTAU DURING AGING AND DISEASE IMPACTS THE LOCAL TISSUE MICROENVIRONMENT IN THE HUMAN LC. RESULTS WILL BE VALIDATED AND EXTENDED USING COMPLEMENTARY IN SITU HYBRIDIZATION PLATFORMS WITH CELLULAR RESOLUTION AND SIMULTANEOUS PROTEIN DETECTION. ACCUMULATION OF NM AND PTAU REPRESENT TWO KEY BIOLOGICAL HYPOTHESES UNDERLYING THE VULNERABILITY AND RESILIENCY OF LC-NE NEURONS TO NEURODEGENERATION IN THE HUMAN BRAIN. HENCE, GENERATION OF THESE DATA ARE IMPORTANT BECAUSE UNDERSTANDING THE MOLECULAR SEQUELAE DOWNSTREAM OF THIS ACCUMULATION IS CRITICAL TO DEVELOP STRATEGIES TO TARGET CELLS IN THE LC FOR DISEASE TREATMENT OR PREVENTION.

INTEGRATIVE APPROACHES TO IDENTIFICATION AND INTERPRETATION OF GENES UNDERLYING PSYCHIATRIC DISORDERS

INTEGRATIVE CELLULAR DECONVOLUTION OF HUMAN BRAIN RNA SEQUENCING DATA

VALIDATION OF ELECTROPHYSIOLOGICAL BIOMARKERS ASSOCIATED WITH PERFORMANCE IN A PRECLINICAL ASSAY OF SUSTAINED ATTENTION - PROJECT SUMMARY SUSTAINED ATTENTION, THE ABILITY TO FOCUS ON AN ACTIVITY OR STIMULUS OVER TIME, IS IMPAIRED IN MANY BRAIN DISORDERS. TARGETING ATTENTIONAL DEFICITS FOR TREATMENT IS CRITICAL BECAUSE THESE SYMPTOMS ARE NEGATIVELY CORRELATED WITH FUNCTIONAL OUTCOME AND QUALITY OF LIFE. CONTINUOUS PERFORMANCE TESTS (CPTS) HAVE BEEN DESIGNED TO MEASURE SUSTAINED ATTENTION IN MULTIPLE SPECIES. SIMILAR NEURAL CIRCUITS ARE ENGAGED IN BOTH HUMANS AND MODEL ORGANISMS DURING CPT PERFORMANCE, SUPPORTING THEIR USE IN TRANSLATIONAL STUDIES THAT SCREEN FOR NOVEL THERAPEUTICS. THE PRELIMBIC CORTEX (PRL), IS INVOLVED IN BOTH CONFLICT DETECTION AND ALLOCATION OF ATTENTION TO CUES BEFORE ORIENTATION, IMPORTANT COMPONENTS IN GO/NO-GO TASKS LIKE CPTS. ELECTROENCEPHALOGRAM (EEG) STUDIES IN HUMANS SHOW THAT NEURAL ACTIVITY IN THE DORSAL ANTERIOR CINGULATE CORTEX (DACC), WHICH SHOWS FUNCTIONAL AND ANATOMICAL ANALOGY TO THE RODENT PRL, IS CORRELATED WITH TASK ENGAGEMENT AND PERFORMANCE IN THE CPT. IN THIS APPLICATION, WE OPTIMIZE THE RCPT TO DETERMINE THE EFFECT OF TARGET PERCENTAGE ON THE TIME-ON-TASK VIGILANCE DECREMENT. WE THEN EVALUATE THE OPTIMIZED CPT PARADIGM BY ASSESSING THE IMPACT OF AMPHETAMINE, A KNOWN ENHANCER OF PERFORMANCE, ON THE ELECTROPHYSIOLOGICAL CORRELATES OF BEHAVIOR. FINALLY, WE MECHANISTICALLY TEST THE ROLE OF NEURAL ACTIVITY IN THE PRL-LC CIRCUIT IN CONTROLLING BEHAVIORAL PERFORMANCE AND ELECTROPHYSIOLOGICAL CORRELATES IN THE OPTIMIZED RCPT.

CELLULAR RESOLUTION SPATIO-MOLECULAR EXPRESSION ACROSS THE ANTERIOR-POSTERIOR AXIS OF THE NUCLEUS ACCUMBENS IN THE HUMAN BRAIN - PROJECT SUMMARY THE NUCLEUS ACCUMBENS (NAC) IS A KEY NODE OF THE BRAIN’S REWARD CIRCUITRY, AND PERTURBATIONS IN NAC-MEDIATED REWARD SIGNALING ARE HIGHLY IMPLICATED IN SUBSTANCE USE DISORDERS. THE HUMAN NAC HAS UNIQUE NEUROANATOMICAL FEATURES, WHICH CORRESPOND TO DISTINCT BIOLOGICAL FUNCTIONS. GIVEN THE CLOSE RELATIONSHIP BETWEEN BRAIN STRUCTURE AND FUNCTION, PRECISELY ASSIGNING SPATIAL COORDINATES TO TRANSCRIPTIONALLY-DISTINCT CELL POPULATIONS WITHIN THE INTACT CYTOARCHITECTURAL STRUCTURE IS IMPORTANT TO ADVANCE OUR UNDERSTANDING OF HOW NAC CELL TYPES MEDIATE REWARD SIGNALING AND CONTROL MOTIVATED BEHAVIOR. TOWARDS THIS GOAL, WE RECENTLY GENERATED SPATIAL TRANSCRIPTOMIC MAPS COMBINED WITH PAIRED SINGLE-NUCLEUS RNA SEQUENCING (SNRNA-SEQ) TO REGISTER MOLECULARLY-DEFINED CELL TYPES TO PRECISE SPATIAL COORDINATES ACROSS THE DORSAL-VENTRAL AND MEDIAL-LATERAL AXES OF THE HUMAN NAC. RESULTS FROM THOSE DATA SUGGEST EXISTENCE OF TOPOGRAPHICALLY ORGANIZED AND MOLECULARLY-DEFINED CELL TYPES ACROSS NAC SUB- REGIONS IN TWO-DIMENSIONAL SPACE. IN ADDITION TO TOPOGRAPHICAL EXPRESSION IN TWO-DIMENSIONAL SPACE, THE DATA ALSO SUGGEST POTENTIAL CHANGES IN THE DENSITY AND LOCALIZATION OF THESE TRANSCRIPTIONALLY-DISTINCT SUBTYPES ACROSS THE ANTERIOR-POSTERIOR AXIS. THIS APPLICATION EXTENDS OUR ONGOING STUDIES TO GENERATE A THREE-DIMENSIONAL CELLULAR RESOLUTION SPATIO-MOLECULAR MAP ACROSS THE NEUROANATOMICAL EXTENT OF THE HUMAN NAC, WHICH WILL BE USED TO COMPUTATIONALLY ASSIGN MOLECULAR IDENTITIES TO NAC CELL TYPES ACROSS DORSAL-VENTRAL, MEDIAL-LATERAL AND ANTERIOR- POSTERIOR AXES. THESE REFINED ANNOTATIONS WILL BE USED TO REGISTER GENE EXPRESSION CHANGES LINKED TO BEHAVIORAL PHENOTYPES AND GENETIC RISK ASSOCIATED WITH SUBSTANCE USE TO SPATIALLY-DEFINED NAC CELL POPULATIONS. THESE DATA ARE CRITICAL FOR UNDERSTANDING NAC FUNCTION AND IDENTIFYING CLINICAL ASSOCIATIONS WITH MOLECULARLY- AND SPATIALLY- DEFINED CELL POPULATIONS THAT CAN BE TARGETED FOR PREVENTION AND TREATMENT.

UNDERSTANDING SCHIZOPHRENIA RISK MECHANISMS VIA MULTIMODAL ANALYSIS OF PATIENT-DERIVED NEURONS AND 3D CORTICAL ORGANOIDS AND CORRELATION WITH CLINICAL AND COGNITIVE PHENOTYPES - PROJECT SUMMARY: SCHIZOPHRENIA (SCZ) IS A COMPLEX POLYGENIC DISORDER, ENCOMPASSING A SPECTRUM OF POSITIVE AND NEGATIVE CLINICAL SYMPTOMS AND COGNITIVE DEFICITS THAT VARY CONSIDERABLY BETWEEN INDIVIDUALS. UNFORTUNATELY, PROGRESS IN UNDERSTANDING THE ETIOLOGY AND PATHOPHYSIOLOGY IS HINDERED BY THE LACK OF APPROPRIATE MODELS THAT ADEQUATELY CAPTURE BOTH THE COMPLEX AND HETEROGENEOUS NATURE OF THE GENETIC RISK AND THE DIVERSITY OF THE PHENOTYPIC MANIFESTATIONS. THE ADVENT OF INDUCED PLURIPOTENT STEM CELL (HIPSC) TECHNOLOGY PROVIDES AN IMPORTANT NEW EXPERIMENTAL PLATFORM TO STUDY THE CELLULAR AND CIRCUIT FUNCTION OF HUMAN CELLS THAT CONTAIN AN INDIVIDUAL’S FULL COMPLEMENT OF GENOMIC RISK ALLELES. THUS, THE DOOR IS NOW OPENED TO DEVELOPING IMPROVED IN VITRO MODELS THAT CAN ENHANCE UNDERSTANDING IN AN INTEGRATED CONTEXT, RELATING PATIENT GENOMES AND CELLULAR VARIABLES TO THEIR CLINICAL AND COGNITIVE PHENOTYPES. IN THIS PROPOSAL, WE DESCRIBE OUR ESTABLISHED, SYSTEMATIC, AND QUANTITATIVE HIPSC PIPELINE THAT MODELS THE CUMULATIVE CELLULAR EFFECTS OF COMMON SCZ GENETIC RISK VARIANTS BY SELECTING PATIENT AND CONTROL LINES (CON) BASED ON THEIR SCZ POLYGENIC RISK SCORE (PRS). HIPSC LINES WERE GENERATED FROM FIBROBLAST DONORS IN OUR NIMH CLINICAL SIBLING STUDY, FOR WHICH EXTENSIVE CLINICAL AND COGNITIVE DATA ARE AVAILABLE. ON OUR 2D PLATFORM, WE PERFORMED UNBIASED PHENOTYPIC DISCOVERY ON CORTICAL NEURONS DERIVED FROM 13 HIGH PRS SCZ HIPSCS AND 15 LOW PRS CON HIPSCS USING AN ARRAY OF PHYSIOLOGICAL ASSAYS, DONE BLIND TO PATIENT INFORMATION. FROM THESE EXPERIMENTS WE HAVE PUBLISHED A REPORT WITH MULTIPLE PRELIMINARY ASSOCIATIONS BETWEEN ELECTROPHYSIOLOGICAL VARIABLES AND PATIENT CLINICAL AND COGNITIVE PHENOTYPES. HERE, WE PROPOSE TO REPLICATE AND EXPAND ON THESE FINDINGS USING AN ENTIRELY NEW SET OF HIPSCS (17 SCZ, 15 CON), AGAIN CHOSEN BASED ON PRS. IN AIM 1, USING OUR 2D PLATFORM, WE PROPOSE TO REPLICATE, EXPAND, AND EXAMINE IN DEPTH CASE/CONTROL PHENOTYPES AND ASSOCIATIONS BETWEEN CELLULAR MEASURES AND CLINICAL AND COGNITIVE FEATURES OBSERVED IN SCZ PATIENTS. IN AIM 2, WE PROPOSE TO USE 3D CORTICAL ORGANOIDS TO CONFIRM OUR PRIOR VOLTAGE GATED SODIUM CHANNEL (VGSC) AND GABAERGIC PHENOTYPES AND TO DISCOVER NOVEL MOLECULAR AND CELLULAR ALTERATIONS IN EARLY CORTICAL DEVELOPMENT BY PERFORMING A MULTI-MODAL SET OF ASSAYS THAT INCLUDES SINGLE CELL ELECTROPHYSIOLOGY, BULK AND SINGLE CELL RNA SEQUENCING, PROTEOMICS, AND METABOLOMICS ON OUR ENTIRE SET OF SCZ AND CON HIPSCS (N=60). TOGETHER, THE WORK IN THESE AIMS IS DESIGNED TO IDENTIFY CELLULAR PHENOTYPES RELATED TO THE ETIOLOGY, PATHOPHYSIOLOGY, AND SYMPTOMATOLOGY OF SCHIZOPHRENIA, WITH THE GOAL OF IDENTIFYING THERAPEUTIC TARGETS AND ENABLING PRECISION PSYCHIATRY.

SPATIAL REGISTRATION OF GENE EXPRESSION IN THE HUMAN BRAIN

KCNH2-3.1 POTASSIUM CHANNEL AND SCHIZOPHRENIA.

HIGH THROUGHPUT SCREENING TO DISCOVER INHIBITORS FOR IP6K1 AS A POTENTIAL TREATMENT FOR PSYCHIATRIC DISORDERS

A SYSTEMS APPROACH TO THE GENETIC STUDY OF ALCOHOL DEPENDENCE

SYSTEMATIC UNDERSTANDING OF RNA-CENTRIC MECHANISMS OF GENE REGULATION IN CHROMATIN - A MAJOR FOCUS OF MODERN RNA RESEARCH IS TO UNDERSTAND ITS REGULATORY ROLES BEYOND ITS CODING POTENTIAL. IN CHROMATIN, DIVERSE TYPES OF RNAS ARE INCREASINGLY RECOGNIZED AS REGULATORS OF GENE EXPRESSION PROGRAMS. FOR EXAMPLE, MANY SHORT OR LONG NONCODING RNAS (NCRNAS) ARE INVOLVED WITH HISTONE MODIFICATIONS THAT ARE IMPORTANT FOR EPIGENETIC GENE REGULATION. EVEN NASCENT RNAS, CONVENTIONALLY VIEWED AS INTERMEDIATES BETWEEN GENES AND PROTEINS, ARE ACKNOWLEDGED FOR THEIR REGULATORY POTENTIAL AS THEY CAN AFFECT CHROMATIN-ASSOCIATED PROTEINS (CAPS)’ ACTIVITIES. HOWEVER, UNDERSTANDING THE UNDERLYING MOLECULAR MECHANISMS OF REGULATORY RNAS REMAINS CHALLENGING. THIS DIFFICULTY ARISES FROM RNA'S INHERENT BIOCHEMICAL FLEXIBILITY, WHERE NOT ONLY ITS PRIMARY SEQUENCE BUT ALSO ITS SECONDARY AND TERTIARY STRUCTURES INFLUENCE FUNCTION. ADDITIONALLY, REGULATORY RNAS OFTEN EXHIBIT EVOLUTIONARY DIVERGENCES IN A SEQUENCE LEVEL, COMPLICATING THE INVESTIGATION. MY LABORATORY IS INTERESTED IN RNA-MEDIATED GENE REGULATION IN CHROMATIN. WE USE INTERDISCIPLINARY APPROACHES SPANNING GENETICS, COMPUTATIONAL GENOMICS, AND HIGH-THROUGHPUT BIOCHEMICAL ASSAYS TO UNDERSTAND THE MECHANISMS OF REGULATORY RNAS IN CHROMATIN. OUR CURRENT PROJECTS AIM TO DEVELOP A SYSTEMATIC APPROACH TO DETECTING REGULATORY RNAS AND THEIR FUNCTIONAL RNA MOTIFS, BASED ON RNA-PROTEIN INTERACTIONS. SPECIFICALLY, WE WILL FIRST TEST WHETHER RNA REGULATES CAP’S GENOMIC OCCUPANCIES. SECOND, WE WILL USE RNA-EDITING-BASED APPROACH TO DETECT RNAS INTERACTING WITH A CAP. THIRD, WE WILL DEVELOP A HIGH-THROUGHPUT ASSAY AND COMPUTATIONAL METHODS TO DETECT FUNCTIONAL RNA ELEMENTS BINDING TO A CAP, BASED ON RNA-CENTRIC DEEP MUTATIONAL APPROACH. LASTLY, WE WILL CHECK WHETHER FUNCTIONAL RNA ELEMENTS ARE CONSERVED BY CONSIDERING BOTH SEQUENCE AND SECONDARY STRUCTURE. WE WILL APPLY OUR APPROACH TO A HISTONE MODIFIER, LSD1/KDM1A, AND ITS INTERACTION WITH RNA, INCLUDING A LONG NCRNA, HOTAIR. OUR RESULTS WILL PROVIDE A DEEP UNDERSTANDING OF LSD1-RNA INTERACTIONS, AS WELL AS PRESENT A GENERALIZABLE APPROACH TO STUDY HOW RNA MEDIATES REGULATORY ROLES IN CHROMATIN. IN ADDITION, AS LSD1 IS A DRUG TARGET FOR MANY DISEASES SUCH AS CANCER AND PSYCHIATRIC DISORDERS, OUR RESULTS WILL OFFER INSIGHTS INTO THE DEVELOPMENT OF RNA-BASED THERAPEUTIC STRATEGIES. IN PARALLEL TO SCIENTIFIC CONTRIBUTIONS IN THE FIELD OF RNA GENOMICS, WE WILL DEVOTE OURSELVES TO MOTIVATING AND TRAINING NEXT-GENERATION SCIENTISTS TO DEVELOP INTERDISCIPLINARY SKILLS AND MINDS TO TACKLE IMPORTANT QUESTIONS IN BIOLOGY. AS BIOLOGY UNDERGOES A TRANSFORMATIVE INTEGRATION WITH DATA SCIENCE AND ARTIFICIAL INTELLIGENCE METHODOLOGIES, IT IS VITAL TO USE BOTH EXPERIMENTAL AND COMPUTATIONAL TECHNIQUES AND INTERPRET THEM APPROPRIATELY. THROUGHOUT THE PROJECT, WE WILL PROVIDE INTERDISCIPLINARY TRAINING OPPORTUNITIES TO TRAINEES.

FUNCTIONAL ANALYSIS OF THE SCHIZOPHRENIA AND AUTISM SPECTRUM DISORDER GENE TCF4 I

DISSECTING MODULAR INTERACTION OF RNA WITH PROTEIN IN CHROMATIN - PROJECT SUMMARY / ABSTRACT THE FOREFRONT OF CURRENT RNA RESEARCH CENTERS ON UNDERSTANDING THE AMAZING COMPLEXITY OF THIS MOLECULE. THERE IS AN EXPANDING APPRECIATION THAT THE ROLES OF RNA ARE MULTIFACETED AND EXTEND FAR BEYOND ITS CODING POTENTIAL. RNA HAS THE CAPACITY FOR DIVERSE STRUCTURES, NUMEROUS MODIFICATIONS, AND COMBINATORIAL INTERACTIONS, AND THUS RNA IS A FOUNDATIONAL COMPONENT FOR MANY COMPLEX REGULATORY NETWORKS. IT IS CRUCIAL TO UNDERSTAND THESE NETWORKS FROM THE LEVEL OF INDIVIDUAL MOLECULAR RNA INTERACTIONS UP THROUGH THEIR EFFECTS ON DISEASE AND DEVELOPMENT. IN PARTICULAR, MANY CHROMATIN-ASSOCIATED PROTEINS (CAPS) ADDITIONALLY INTERACT WITH RNA, AND MAY FUNCTION IN REGULATING GENE EXPRESSION BY WAY OF THESE RNA INTERACTIONS. PREVIOUS STUDIES, LIMITED TO IDENTIFYING THE RNAS BOUND BY INDIVIDUAL CAPS, DID NOT CONSIDER COMBINATORIAL RNA BINDING. HERE, I REVEAL THAT THE CAP- RNA NETWORK CONTAINS CLUSTERS OF RNAS THAT BIND MULTIPLE PROTEINS. NOTABLY, POLYCOMB GROUP AND TRITHORAX GROUP CAPS (EZH2 AND WDR5, RESPECTIVELY) SEEM TO INTERACT WITH SIMILAR RNAS DESPITE THEIR OPPOSING ROLES IN CHROMATIN MODIFICATION. THIS SUGGESTS THAT EZH2- AND WDR5- RNA INTERACTIONS MAY AFFECT OR COUNTER ONE ANOTHER, AND THAT THIS COULD MANIFEST IN ALTERED CHROMATIN STATES. I AIM TO ELUCIDATE HOW DIFFERENT CAP-RNA INTERACTIONS AFFECT ONE ANOTHER AND TO CHARACTERIZE THE RELATIONSHIP AMONG RNA-BINDING OF MULTIPLE CAPS. I HYPOTHESIZE THAT RNA CONTAINS MULTIPLE ELEMENTS, OR MODULES, THAT INTERACT WITH SPECIFIC PROTEINS, AND THAT THE FUNCTIONAL READOUT OF COMBINATORIAL INTERACTIONS IN A NETWORK CAN BE UNDERSTOOD BY MODULAR DESIGN PRINCIPLES OF RNA. I PROPOSE THE FOLLOWING AIMS TO INVESTIGATE THIS IDEA, PARTICULARLY FOCUSING ON EZH2- AND WDR5- RNA INTERACTIONS: (1) DEVELOPMENT OF A MASSIVELY-PARALLEL RNA ASSAY (MPRNA) TO IDENTIFY RNA ELEMENTS THAT BIND TO A PARTICULAR PROTEIN, (2) INVESTIGATING THE MODULAR PRINCIPLE USING COMBINATORIAL SYNTHESIS OF MULTIPLE RNA ELEMENTS, (3) UNCOVERING THE UNDERLYING MECHANISM OF RNA ELEMENT ORGANIZATION AND PROTEIN INTERACTION. THIS APPROACH WILL ESTABLISH AN INTERDISCIPLINARY FRAMEWORK FOR STUDYING RNA-CENTRIC NETWORKS, USING BOTH EXPERIMENTAL AND COMPUTATIONAL METHODS. IT WILL REVEAL INTERACTION PRINCIPLES AND MECHANISMS OF PROTEIN-RNA INTERACTIONS, AND EXAMINE FUNCTIONAL CONSEQUENCES OF THESE INTERACTIONS. IT WILL INCREASE OUR UNDERSTANDING OF RNA-BASED REGULATORY NETWORKS, PARTICULARLY IN THE CONTEXT OF CHROMATIN AND CAP-MEDIATED GENE REGULATION. IT MAY EVEN PROVIDE INSIGHT INTO THE CONTROL OF POLYCOMB- AND TRITHORAX- GROUP COMPLEXES AT SITES OF BIVALENT CHROMATIN.

NEUROPHYSIOLOGICAL BIOMARKERS IN PRECLINICAL ASSAYS OF SUSTAINED ATTENTION - PROJECT SUMMARY SUSTAINED ATTENTION, THE ABILITY TO FOCUS ON AN ACTIVITY OR STIMULUS OVER TIME, IS IMPAIRED IN MANY BRAIN DISORDERS. CONTINUOUS PERFORMANCE TESTS (CPTS) HAVE BEEN DESIGNED TO MEASURE SUSTAINED ATTENTION IN MULTIPLE SPECIES. SIMILAR NEURAL CIRCUITS ARE ENGAGED IN BOTH HUMANS AND MODEL ORGANISMS DURING CPT PERFORMANCE, SUPPORTING THEIR USE IN TRANSLATIONAL STUDIES THAT SCREEN FOR NOVEL THERAPEUTICS. THE HUMAN DORSAL ANTERIOR CINGULATE CORTEX (DACC), WHICH PLAYS CRITICAL ROLES IN ATTENTIONAL PROCESSES, SHOWS FUNCTIONAL AND ANATOMICAL SIMILARITY TO THE MOUSE PRELIMBIC REGION (PRL). THESE HOMOLOGOUS REGIONS ARE INVOLVED IN BOTH CONFLICT DETECTION AND ALLOCATION OF ATTENTION TO CUES BEFORE ORIENTATION, IMPORTANT COMPONENTS IN GO/NO-GO TASKS LIKE CPTS. ELECTROENCEPHALOGRAM (EEG) STUDIES SHOW THAT NEURAL ACTIVITY IN THE DACC IS CORRELATED WITH TASK ENGAGEMENT AND PERFORMANCE IN THE CPT. THE NEUROMODULATOR DOPAMINE IS HYPOTHESIZED TO PLAY A CRITICAL ROLE IN REGULATING ATTENTION, A NOTION SUPPORTED BY THE FACT THAT DOPAMINE D1 RECEPTOR AGONISTS AND ANTAGONISTS IMPROVE AND IMPAIR CPT PERFORMANCE, RESPECTIVELY. IN THIS APPLICATION WE PROPOSE TO OPTIMIZE A MOUSE TOUCHSCREEN-BASED CPT TO INCLUDE MEASUREMENTS OF THE EFFECTS OF STIMULUS DEGRADATION AND SESSION LENGTH (TIME ON TASK) ON ACCURACY, REACTION TIMES, AND OTHER TASK PARAMETERS. IN THIS OPTIMIZED PARADIGM WE WILL IDENTIFY ELECTROPHYSIOLOGICAL CORRELATES OF TASK PERFORMANCE BY ANALYZING SPECTRAL MEASURES OF POWER AND COUPLING FROM THE EEG AND PRL LOCAL FIELD POTENTIAL. WE WILL EVALUATE THE OPTIMIZED CPT PARADIGM BY ASSESSING BEHAVIORAL PERFORMANCE AND EEG/LFP CORRELATES IN RESPONSE TO PHARMACOLOGICAL ENHANCERS THAT ARE DOCUMENTED TO IMPROVE ATTENTION. FINALLY, WE WILL MECHANISTICALLY TEST THE ROLE OF DOPAMINERGIC INPUT TO THE DACC/PRL FROM THE VENTRAL TEGMENTAL AREA ON BEHAVIORAL PERFORMANCE AND EEG/LFP CORRELATES IN THE CPT.

ESTABLISHING COMPREHENSIVE AND QUANTITATIVE MAPS OF DNA METHYLATION IN THE DEVELO

EFFECTS OF GENES AND CHILDHOOD ENVIRONMENT ON BRAIN MECHANISMS OF SCHIZOPHRENIA R

THE ROLE OF DISC 1 IN SYNAPSE FORMATION & FUNCTION IN THE DEVELOPING NEOCORTEX

AN EXPANDED FRAMEWORK FOR RNA QUALITY CORRECTION IN EXPRESSION ANALYSES IN THE HUMAN BRAIN

EPIGENOMIC CONTRIBUTION TO THE ANTIDEPRESSANT RESPONSE

PROFILING THE HUMAN DENTATE GYRUS ACROSS THE LIFESPAN WITH SPATIALLY-RESOLVED TRANSCRIPTOMICS - PROJECT SUMMARY THE HIPPOCAMPUS PLAYS A WELL-ESTABLISHED ROLE IN LEARNING AND MEMORY ACROSS THE LIFESPAN, WITH DEVELOPMENTAL AND PLASTICITY-RELATED GENE EXPRESSION CHANGES OBSERVED FROM INFANCY THROUGH OLD AGE. DUE TO ITS STRUCTURE AND CIRCUITRY, THE REGION HAS BEEN LINKED TO A NUMBER OF CRITICAL BEHAVIORAL FUNCTIONS. HIPPOCAMPAL NEURONS ARE ORGANIZED IN DENSELY PACKED LAYERS ACCORDING TO DENDRITE-AXON POLARITY, AND SYNAPTIC CONNECTIONS WITHIN THE HIPPOCAMPUS ARE MAJOR SITES OF STRUCTURAL AND FUNCTIONAL PLASTICITY ACROSS THE LIFESPAN THAT REGULATE CRITICAL FUNCTIONS RELATED TO LEARNING, MEMORY, MOOD AND STRESS REGULATION. MANY IMPORTANT PLASTICITY-RELATED TRANSCRIPTS ARE LOCALIZED TO THE DENDRITIC COMPARTMENT, AND THEIR TRANSCRIPTION, TRANSPORT OUT OF THE NUCLEUS, AND TRANSLATION WITHIN THE DENDRITIC COMPARTMENT IS TIGHTLY REGULATED, BOTH DEVELOPMENTALLY AND BY NEURONAL ACTIVITY. ADDITIONALLY, DESPITE EXTENSIVE CHARACTERIZATION OF THE FUNCTIONAL IMPORTANCE OF POSTNATAL NEUROGENESIS IN RODENT DENTATE GYRUS (DG), INDISPUTABLE EVIDENCE OF ADULT NEUROGENESIS IN THE HUMAN DG (HDG) REMAINS ELUSIVE AND ITS PERSISTENCE THROUGHOUT THE LIFESPAN REMAINS CONTROVERSIAL. RECENTLY, CELL-TYPE SPECIFIC MOLECULAR PROFILES OF THE HUMAN HIPPOCAMPUS IN ADULTS AND ACROSS THE LIFESPAN HAVE BEEN DESCRIBED, USING SINGLE- NUCLEUS RNA SEQUENCING (SNRNA-SEQ); HOWEVER, THESE RESOURCES LACK SPATIAL RESOLUTION WITHIN THE HIPPOCAMPUS AND LOSE TRANSCRIPTOMIC INFORMATION FROM THE CYTOSOLIC COMPARTMENT. GIVEN THE TIGHT CORRELATION BETWEEN SPATIAL STRUCTURE AND FUNCTION IN HPC, AND THE PARTICULAR IMPORTANCE OF TRANSCRIPTS LOCALIZED TO THE SYNAPTIC COMPARTMENT, MOLECULAR PROFILING TECHNOLOGIES WITH THE CAPACITY TO ADDRESS THESE GAPS IN OUR KNOWLEDGE ARE NEEDED. THUS, WE PROPOSE THE GENERATION OF SPATIALLY-RESOLVED, TRANSCRIPTOME-WIDE GENE EXPRESSION PROFILES IN HDG ACROSS THE LIFESPAN, FROM NEUROTYPICAL DONORS IN FOUR AGE GROUPS ACROSS THE HUMAN LIFESPAN (INFANT, TEEN, MIDDLE-AGE, AND ELDERLY). DATA WILL BE COMPILED INTO A USER-FRIENDLY BROWSER AS A COMMUNITY RESOURCE. EXPERT NEUROBIOLOGISTS WILL PERFORM MANUAL ANNOTATIONS OF CANONICAL SUBFIELDS, WHILE BIOSTATISTICIANS WILL APPLY UNSUPERVISED CLUSTERING ALGORITHMS TO IDENTIFY NOVEL SPATIAL DOMAINS. WE WILL THEN COMPARE GENE EXPRESSION ACROSS THE LIFESPAN TO IDENTIFY DEVELOPMENTALLY- AND SPATIALLY-REGULATED GENE EXPRESSION. WE WILL VALIDATE THE CELLULAR EXPRESSION PATTERNS OF SPECIFIC NOVEL GENE MARKERS BY PERFORMING SMFISH IN OUR DONOR COHORT. THIS APPROACH WILL FACILITATE REFINED ANNOTATION OF SPATIAL GENE EXPRESSION PATTERNS IN THE HUMAN HIPPOCAMPUS, AND CONTRIBUTE TO UNDERSTANDING NEURODEVELOPMENTAL AND NEURODEGENERATIVE DISORDERS BY IDENTIFYING CLINICAL ASSOCIATIONS WITH SPATIALLY-DEFINED CELL POPULATIONS THAT CAN BE TARGETED FOR PREVENTION AND TREATMENT.

DECOMPOSING CELL TYPE-SPECIFIC MARKS IN POST-MORTEM HUMAN BRAIN STUDIES

INTEGRATED SINGLE CELL AND SPATIAL MAPPING OF HABENULA CIRCUITRY TO IDENTIFY PROJECTION-SPECIFIC MOLECULAR PATHWAYS ASSOCIATED WITH OPIOID EXPOSURE. - PROJECT SUMMARY THE NATIONAL OPIOID EPIDEMIC IS A SIGNIFICANT PUBLIC HEALTH CRISIS REQUIRING ACCELERATED EFFORTS TO TRANSLATE MECHANISTIC STUDIES INTO THERAPEUTIC DEVELOPMENTS. SUBSTANCE USE DISORDERS (SUDS) FEATURE DYSREGULATION OF BRAIN REWARD CIRCUITS, ESPECIALLY DOPAMINERGIC SYSTEMS GOVERNING MOTIVATED BEHAVIOR. THE HABENULA (HB) IS A KEY REWARD CIRCUITRY HUB THAT SENDS DIRECT PROJECTIONS TO THE VENTRAL TEGMENTAL AREA (VTA), AS WELL AS OTHER AMINERGIC CENTERS, TO MODULATE REWARD SEEKING. HB CIRCUIT DYSFUNCTION IS ASSOCIATED WITH ADDICTION, BUT THE MOLECULAR MECHANISMS MEDIATING THIS DYSFUNCTION, ESPECIALLY IN THE CONTEXT OF OPIOID USE DISORDER (OUD) ARE UNKNOWN. HERE WE PROPOSE TO USE INTEGRATED SINGLE CELL AND SPATIAL TRANSCRIPTOMIC APPROACHES TO MAP MOLECULAR CHANGES ASSOCIATED WITH CHRONIC OPIOID SELF-ADMINISTRATION IN SPECIFIC CELL POPULATIONS ACROSS THE RODENT HB (AIM 1). USING RETROGRADE LABELING AND NUCLEAR TAGGING, WE WILL ALSO GENERATE TRANSCRIPTOMIC SIGNATURES FOR HB NEURONS PROJECTING TO THE VTA (HB-VTA PROJECTIONS) AND EVALUATE THEIR SUSCEPTIBILITY TO MOLECULAR CHANGES ASSOCIATED WITH CHRONIC OPIOID EXPOSURE (AIM 2). FINALLY, WE WILL INTEGRATE THESE COMPLIMENTARY RODENT DATASETS WITH MOLECULAR DATA WE ARE GENERATING IN THE POST-MORTEM HUMAN HB IN THE CONTEXT OF OUD AS PART OF AN ONGOING NIDA-FUNDED AWARD (1R01DA55823) TO IDENTIFY SPECIFIC HB CELL POPULATIONS AND PROJECTION NEURONS ENRICHED IN GENES ASSOCIATED WITH OPIOID ADDICTION IN THE HUMAN BRAIN (AIM 3). THE PROPOSED STUDY WILL GENERATE THE FIRST SPATIALLY-RESOLVED ATLAS OF OPIOID-INDUCED GENE EXPRESSION CHANGES IN THE RODENT HB AT CELLULAR AND CIRCUIT RESOLUTION. INTEGRATION OF THIS MOLECULAR MAP WITH CLINICAL GENE SETS IN THE CONTEXT OF OUD WILL HELP TRANSLATE INSIGHTS FROM RODENT MODELS INTO CELL TYPE- AND CIRCUIT-BASED THERAPEUTIC APPROACHES FOR THE TREATMENT OF ADDICTION.

CHARACTERIZING THE DEVELOPING HUMAN BRAIN TRANSCRIPTOME AT SINGLE-BASE RESOLUTION

1/2 LARGE-SCALE, SINGLE-CELL CHARACTERIZATION OF MOLECULAR AND CELLULAR NETWORKS OF MOOD REGULATION CIRCUITRY IN MAJOR DEPRESSIVE DISORDER - SUMMARY WHILE THERE IS STRONG EVIDENCE SUPPORTING THE ROLE OF THE ANTERIOR CINGULATE CORTEX, BASOLATERAL AMYGDALA, AND THE HIPPOCAMPUS (ACC, BLA, HIPP) AS A KEY NEURAL NETWORK REGULATING MOOD, AND THEREFORE CENTRAL TO THE PATHOPHYSIOLOGY OF MAJOR DEPRESSIVE DISORDER (MDD), MUCH REMAINS UNKNOWN, INCLUDING WHICH GENE PATHWAYS AND WHICH SPECIFIC CELL TYPES PLAY A PRIMARY CAUSAL ROLE MEDIATING ALTERATIONS IN THIS CIRCUIT, AND WHAT CELL-TYPE CONNECTIONS, WITHIN AND BETWEEN THESE REGIONS, ARE PARTICULARLY ALTERED IN DEPRESSIVE STATES. THE OVERALL OBJECTIVE OF THIS APPLICATION IS TO GENERATE SINGLE-CELL TRANSCRIPTOMIC PROFILES TO STUDY MOLECULAR CHANGES, INCLUDING THOSE SPECIFIC TO GENETIC ANCESTRY AND SEX, ASSOCIATED WITH MDD IN THE MOOD REGULATION CIRCUIT. WHILE DISEASE BURDEN IS GREATER IN AFRICAN AMERICANS, THE IMPACT OF GENETIC ANCESTRY REMAINS UNKNOWN AS MOST GENOMIC STUDIES IN MDD SO FAR HAVE BEEN LIMITED TO SUBJECTS OF EUROPEAN DESCENT. IN ADDITION, PREVIOUS STUDIES REVEALED THAT TRANSCRIPTOMIC CHANGES ASSOCIATED WITH MDD ARE SEX-SPECIFIC, AND GENE NETWORKS ARE DIFFERENTIALLY DYSREGULATED BETWEEN SEXES. THE APPLICANTS’ RECENT SINGLE-CELL BRAIN STUDY REVEALED CELL-SPECIFIC CONTRIBUTIONS TO TRANSCRIPTOMIC CHANGES ASSOCIATED WITH MDD. THE PROPOSED PROJECT IS A LARGE-SCALE, SYSTEMATIC INVESTIGATION IN THE ACC, BLA, AND HIPP TO INTERROGATE THE TRANSCRIPTOME AT SINGLE-NUCLEUS RESOLUTION IN AN UNPRECEDENTLY LARGE AND REPRESENTATIVE SAMPLE OF MDD. THE SPECIFIC AIMS ARE TO: 1) IDENTIFY, AT THE SINGLE-CELL LEVEL TRANSCRIPTOMIC CHANGES ASSOCIATED WITH MDD IN 800 SUBJECTS ACROSS THREE LINKED BRAIN REGIONS: ACC, BLA, AND HIPP; 1B) STUDY THE IMPACT OF GENETIC ANCESTRY AND SEX; 2) DEFINE CELL NETWORKS ASSOCIATED WITH MOOD REGULATION USING MACHINE LEARNING APPROACHES; AND 3) IDENTIFY CELL-SPECIFIC EXPRESSION QUANTITATIVE TRAIT LOCI (EQTLS) COLOCALIZING WITH GENOME-WIDE SIGNIFICANT SNPS IDENTIFIED IN MDD GWAS ANALYSES. A LARGE COHORT (N=800) OF HUMAN POST-MORTEM SAMPLES OBTAINED FROM SUBJECTS WITH MDD WILL BE COMPARED TO PSYCHIATRICALLY-HEALTHY CONTROLS. THE SAMPLE (~20% AFRICAN AMERICAN AND ~30% FEMALE) WILL ALLOW FOR STUDYING THE IMPACT OF GENETIC ANCESTRY AND SEX. DROPLET-BASED SINGLE-NUCLEUS RNA SEQUENCING WILL BE APPLIED TO GENERATE TRANSCRIPTOMIC PROFILES. DEEP LEARNING APPROACHES WILL BE USED TO IDENTIFY AND ANNOTATE THE CELL TYPES AND GENE NETWORKS ASSOCIATED MDD. THE LATEST GWAS DATA IN MDD WILL BE LEVERAGED TO FINE MAP GENETIC LOCI WITH CELLULAR AND REGIONAL RESOLUTION. THE PROPOSED RESEARCH IS INNOVATIVE BECAUSE IT IS THE FIRST LARGE-SCALE INVESTIGATION OF THE ACC-BLA-HIPP CIRCUIT IN HUMANS AND WILL REPRESENT THE LARGEST SINGLE-CELL TRANSCRIPTIONAL RESOURCE OF THE HUMAN BRAIN. IT WILL IDENTIFY GENE AND CELLULAR NETWORKS ASSOCIATED WITH SEX OR GENETIC ANCESTRY, AND WILL ALSO GENERATE A VAST AMOUNT OF TRANSCRIPTOMIC DATA ON NEUROTYPICAL BRAINS. THIS RESEARCH IS SIGNIFICANT BECAUSE IT WILL GREATLY ADVANCE OUR UNDERSTANDING OF THE CELLULAR AND MOLECULAR PATHWAYS INVOLVED IN MOOD REGULATION AND MDD. THROUGH A BETTER UNDERSTANDING OF THE MECHANISMS OF DEPRESSIVE ILLNESS, WE MAY BE ONE STEP CLOSER TO DEVELOPING NOVEL TREATMENT STRATEGIES AND PERSONALIZE INTERVENTIONS.

PLACENTAL TRANSCRIPTOMICS AND RISK FOR SCHIZOPHRENIA - OUR MAIN OBJECTIVE IS TO FIND THE FACTORS MEDIATING THE RELATIONSHIP BETWEEN GENETIC RISK FACTORS, COMPLICATIONS IN EARLY LIFE (ELCS, I.E. PRE-, AND PERINATAL) AND ALTERED NEURO- DEVELOPMENT (ND) IN HUMANS, SO THAT, IN HIGH-RISK INDIVIDUALS, DISORDERS OF ND SUCH AS SCHIZOPHRENIA (SZ), CAN BE PREVENTED OR RESCUED IN EARLY STAGES. EPIDEMIOLOGICAL FINDINGS SHOW THAT ELCS INCREASE THE RISK FOR ND DISORDERS INCLUDING SZ; PRECLINICAL MODELS SHOW THAT ELCS ALTER ND, AN EFFECT PARTIALLY MEDIATED BY GENE EXPRESSION IN PLACENTA, WITH MALES MORE VULNERABLE THAN FEMALES. HOWEVER, THE MECHANISM LINKING ELCS WITH SZ ARE STILL ELUSIVE. OUR RECENT HUMAN WORK FOUND THAT GENETIC FACTORS CONVERGE WITH PLACENTA BIOLOGY IN AFFECTING RISK FOR SZ. HERE, WE FOCUS ON DETECTING THE MOLECULAR MEDIATORS INVOLVED: THAT IS, THE MOLECULES THAT, BY REGULATING PLACENTA'S PHYSIOLOGY, REGULATE ND, DRIVE SUSCEPTIBILITY TO SZ, AND MAY MEDIATE THE INTERACTION BETWEEN GENETIC FACTORS AND ELCS ON THE RISK FOR THE DISORDER. WE PROPOSE A MULTI-STEP STRATEGY, WHICH LEVERAGES OUR STRENGTHS IN ANALYZING TRANSCRIPTOMIC AND GENETIC DATA, USING WELL-ESTABLISHED AND INNOVATIVE METHODOLOGIES TO EXPLORE A NOVEL AREA OF RESEARCH: THE RELATIONSHIP BETWEEN GENOMIC RISK FACTORS, PLACENTA AND SZ. WE WILL FIRST INVESTIGATE THE TRANSCRIPTOMIC CORRELATES OF GENETIC RISK FACTORS FOR SZ IN THE PLACENTA, AND WE WILL ANALYZE WHETHER GENE ISOFORMS ASSOCIATED WITH RISK FOR SZ ARE CO-EXPRESSED IN PLACENTA, AS PART OF SPECIFIC BIOLOGICAL PROCESSES. USING GENOTYPE-BASED PREDICTION OF GENE EXPRESSION IN LARGE COHORTS, WE WILL IDENTIFY THE SPECIFIC PLACENTAL ISOFORMS ASSOCIATED WITH SZ CASE-CONTROL STATUS. WE WILL THEN GENERATE PLACENTAL GENOMIC PREDICTORS OF SZ RISK, AND WE WILL TEST THEIR RELATIONSHIP WITH ND OUTCOMES. TO INVESTIGATE SEX-SPECIFIC PLACENTAL PROCESSES RELEVANT FOR SZ, WE WILL STRATIFY EACH ANALYSIS BY SEX. WE EXPECT OUR DATA TO CONVERGE IN IDENTIFYING PLACENTAL MOLECULES AND PATHWAYS THAT ARE RELEVANT FOR SZ. THE DETECTION OF SUCH MOLECULES MAY BE TRANSLATED INTO THE DEVELOPMENT OF NOVEL PRENATAL PREVENTION OF SZ, AIMED AT PRESERVING / OPTIMIZING THE IMPORTANT PLACENTAL SUPPORT TO ND, AND THE DEVELOPMENT OF NOVEL POSTNATAL PREVENTION, BASED ON THE IDENTIFICATION OF HIGH-RISK INDIVIDUALS – THROUGH PLACENTAL AND GENETIC BIOMARKERS.

MODELING SCHIZOPHRENIA WITH PATIENT-SPECIFIC MUTATIONS IN GRIN2A AND SP4 - PROJECT SUMMARY: SCHIZOPHRENIA (SCZ) IS A COMMON AND DEBILITATING MENTAL ILLNESS CHARACTERIZED BY POSITIVE SYMPTOMS, NEGATIVE SYMPTOMS, AND IMPAIRED COGNITION, WITH A LIFETIME PREVALENCE APPROACHING 1% IN THE UNITED STATES. UNFORTUNATELY, PROGRESS IN UNDERSTANDING THE ETIOLOGY AND PATHOPHYSIOLOGY IS HINDERED BY THE LACK OF APPROPRIATE MODELS THAT ADEQUATELY CAPTURE BOTH THE COMPLEX AND HETEROGENEOUS NATURE OF THE GENETIC RISK AND THE DIVERSITY OF THE PHENOTYPIC MANIFESTATIONS. THE ADVENT OF INDUCED PLURIPOTENT STEM CELL (HIPSCS) TECHNOLOGY PROVIDES AN IMPORTANT NEW PLATFORM TO STUDY THE CELLULAR AND CIRCUIT BEHAVIOR OF HUMAN CELLS THAT CONTAIN AN INDIVIDUAL’S FULL COMPLEMENT OF PRIMARY SEQUENCE RISK ALLELES. THUS, THE DOOR IS NOW OPENED TO DEVELOPING IMPROVED IN VITRO MODELS THAT CAN ENHANCE OUR UNDERSTANDING OF ETIOLOGY AND PATHOPHYSIOLOGY IN AN INTEGRATED CONTEXT, RELATING PATIENT GENOMES TO THEIR CELLULAR PHENOTYPES. HERE, WE PROPOSE TO GENERATE NOVEL HIPSC LINES HARBORING PATIENT-SPECIFIC MUTATIONS IN TWO HIGHLY PENETRANT SCZ RISK GENES, GRIN2A AND SP4. GRIN2A ENCODES A SUBUNIT OF THE N-METHYL-D-ASPARTATE RECEPTOR (NMDAR) WHICH ARE A SUBCLASS OF IONOTROPIC GLUTAMATE RECEPTORS THAT PLAY A PIVOTAL ROLE IN THE DEVELOPMENT, PLASTICITY, AND PATHOPHYSIOLOGY IN THE CENTRAL NERVOUS SYSTEM. NMDAR HYPOFUNCTION IS A LEADING CAUSAL HYPOTHESIS FOR SCZ BECAUSE SUBANESTHETIC DOSES OF THE NMDAR ANTAGONISTS PRODUCES ACUTE PSYCHOSIS IN HEALTHY SUBJECTS. IN ADDITION, AUTOIMMUNE ENCEPHALITIS DUE AUTO-IMMUNOREACTIVITY TO NMDARS LEADS TO SEVERE PSYCHOSIS. SP4 BELONGS TO THE SP1 FAMILY OF TRANSCRIPTION FACTORS THAT RECOGNIZES DNA SEQUENCES TERMED GC-BOXES THAT ARE OFTEN FOUND UPSTREAM OF TRANSCRIPTION START SITES (TSS). SP4 HAS BEEN SHOWN TO FUNCTIONALLY REGULATE THE EXPRESSION OF NMDARS THROUGH DIRECT INTERACTIONS WITH GC-BOXES UPSTREAM OF THE TSS OF GRIN1, GRIN2A, AND GRIN2B. IN ADDITION, SP4 HYPOMORPHIC MICE HAVE REDUCED HIPPOCAMPAL LONG-TERM POTENTIATION, REDUCED NMDAR EXPRESSION, AND BEHAVIORAL ABNORMALITIES RELATED TO SCZ. IMPORTANTLY, BOTH GRIN2A AND SP4 ARE HIGH PRIORITY SCZ RISK GENES, BEING ASSOCIATED WITH SCZ THROUGH GENOME-WIDE ASSOCIATION STUDIES AND RARE VARIANT ANALYSIS. WE PROPOSE TO USE CRIPSR EDITING TO GENERATE PATIENT-SPECIFIC MUTATIONS IN GRIN2A AND SP4 WITH CONSIDERATION OF THE GENETIC BACKGROUND OF THE HIPSC LINES. BOTH GENES WILL BE INDIVIDUALLY EDITED IN THE SAME SIX CONTROL HIPSCS LINES WITH ELEVATED SCZ POLYGENIC RISK SCORES. WE WILL PERFORM QUALITY CONTROL EXPERIMENTS TO CONFIRM MUTATIONS ARE CORRECT AND NO OFF-TARGET MUTATIONS ARE PRESENT. VALIDATION OF FUNCTIONAL MUTATIONS WILL BE CONFIRMED WITH QPCR, WESTERN BLOT AND ELECTROPHYSIOLOGY. ONCE VALIDATED, WE WILL QUANTIFY THE EFFECT OF THESE MUTATIONS ON SPONTANEOUS NETWORK ACTIVITY USING MULTI-ELECTRODE ARRAYS AND THEIR EFFECT ON THE TRANSCRIPTOME WITH RNA SEQUENCING. THE OVERALL GOAL OF THIS PROPOSAL IS TO DEVELOP NOVEL CELLULAR MODELS OF SCZ THAT CAN INFORM US ABOUT PATHOPHYSIOLOGY AND HELP IDENTIFY CONVERGENT MOLECULAR MECHANISM IN SCZ WITH THE GOAL OF IDENTIFYING THERAPEUTIC TARGETS FOR THIS DEBILITATING DISORDER.

TEMPORAL COHERENCE OF SCHIZOPHRENIA RISK GENES IN A CRITICAL BRAIN CIRCUIT: IT'S ABOUT TIME

MOLECULAR, CELLULAR AND PHYSIOLOGICAL CORRELATES OF SUSTAINED ATTENTION IN THE LOCUS COERULEUS TO ANTERIOR CINGULATE CORTEX CIRCUIT - PROJECT SUMMARY SUSTAINED ATTENTION DEFICITS ARE A PROMINENT COGNITIVE SYMPTOM IN NEUROPSYCHIATRIC DISORDERS SUCH AS SCHIZOPHRENIA, ATTENTION-DEFICIT HYPERACTIVITY DISORDER, AND MAJOR DEPRESSIVE DISORDER AS WELL AS IN AGE-RELATED NEURODEGENERATIVE DISORDERS, INCLUDING ALZHEIMER’S DISEASE. IN HUMANS, CONTINUOUS PERFORMANCE TESTS (CPTS) ARE COMMONLY USED TO ASSESS SUSTAINED ATTENTION IN PATIENTS WITH THESE NEUROPSYCHIATRIC DISEASES. THE ANTERIOR CINGULATE CORTEX (ACC) IS ESSENTIAL FOR NORMAL CPT PERFORMANCE, AND PATIENT POPULATIONS OFTEN EXHIBIT ABERRANT ACC FUNCTION DURING THE CPT. THESE RESULTS ARE IN LINE WITH AN ESTABLISHED ROLE FOR THE ACC IN ATTENTION-GUIDED BEHAVIOR, AND POINT TO A ROLE FOR THE ACC IN THE PATHOPHYSIOLOGY OF ATTENTIONAL DEFICITS IN COMPLEX BRAIN DISORDERS. HOWEVER, THE CELLULAR AND MOLECULAR MECHANISMS THAT UNDERLIE THE ROLE OF THE ACC TO REGULATE SUSTAINED ATTENTION REMAIN UNCLEAR. THIS LACK OF KNOWLEDGE IS IMPORTANT BECAUSE IDENTIFYING THESE MECHANISMS IS CRITICAL FOR DEVELOPING TARGETED TREATMENTS FOR ATTENTION DEFICITS. THIS APPLICATION INVESTIGATES LINKS BETWEEN EXPRESSION OF NOVEL MOLECULAR AND CELLULAR TARGETS, NEURAL ACTIVITY PATTERNS IN ACC CIRCUITS AND ATTENTIONAL PERFORMANCE. OUR PRELIMINARY DATA SUGGEST THAT PROJECTION-SPECIFIC PATHWAYS BETWEEN THE LOCUS COERULEUS (LC) AND THE ACC REGULATE DISTINCT ASPECTS OF ATTENTION-GUIDED BEHAVIOR DURING THE CPT. SPECIFICALLY, WE IDENTIFIED THE GENE ENCODING APOLIPOPROTEIN E (APOE), WHICH HAS BEEN IMPLICATED IN ATTENTION AND DISORDERS ASSOCIATED WITH DISORDERS FEATURING DEFICITS IN ATTENTION, AS A POTENTIAL MOLECULAR PLAYER UNDERLYING REGULATION OF SUSTAINED ATTENTION IN THE LC-ACC CIRCUIT. WE LEVERAGE GENETIC AND CIRCUIT-SPECIFIC TOOLS TO DISSECT THE MOLECULAR, CELLULAR AND CIRCUIT UNDERPINNINGS OF SUSTAINED ATTENTION DURING A TOUCHSCREEN-BASED RODENT ANALOG OF THE HUMAN CPT (RCPT) IN MICE. SPECIFICALLY, WE 1) TEST CAUSAL RELATIONSHIPS BETWEEN APOE GENE EXPRESSION, PHYSIOLOGICAL FUNCTION IN THE LC-ACC CIRCUIT AND ATTENTIONAL PERFORMANCE, AND 2) IDENTIFY CELL TYPES AND CIRCUIT- SPECIFIC MOLECULAR TARGETS IN THE RODENT AND HUMAN ACC THAT ARE CRITICAL FOR SUSTAINED ATTENTION. TO ACHIEVE THESE AIMS WE INTEGRATE A VARIETY OF MOLECULAR AND SYSTEMS LEVEL APPROACHES INCLUDING IN VIVO ELECTROPHYSIOLOGY, SINGLE-CELL RNA-SEQUENCING, AND CRISPR-DCAS9 MEDIATED EPIGENOME EDITING COUPLED WITH QUANTIFICATION OF ATTENTION-GUIDED BEHAVIOR. FOR THE SEQUENCING STUDIES, WE CAPITALIZE ON THE POWER OF MOLECULAR GENETIC TOOLS TO TARGET AND MANIPULATE CELL-SPECIFIC POPULATIONS WITHIN THE LC-ACC CIRCUIT IN THE MOUSE AND USE THESE DATA TO GENETICALLY IDENTIFY CIRCUIT-SPECIFIC CELL TYPES IN DATA FROM POSTMORTEM HUMAN BRAIN TISSUE. THIS CROSS-SPECIES ANALYSIS SUPPORTS OUR LONG-TERM GOAL OF IDENTIFYING AND PRIORITIZING THERAPEUTIC TARGETS FOR DISORDERS THAT FEATURE ATTENTIONAL DEFICITS. THE PROPOSED RESEARCH IS SIGNIFICANT BECAUSE THE RESULTS WILL SIGNIFICANTLY ADVANCE OUR UNDERSTANDING OF THE CIRCUIT AND MOLECULAR MECHANISMS UNDERLYING SUSTAINED ATTENTION, AS WELL AS PROVIDE POTENTIAL AVENUES FOR ANATOMICALLY AND GENETICALLY-LOCALIZED THERAPEUTIC TARGETING IN DISORDERS FEATURING DYSREGULATION OF ATTENTION.

COMPREHENSIVE COMPUTATIONAL ANALYSIS OF GENETIC AND REGULATORY DIFFERENCES BETWEEN INDIVIDUALS WITH AFRICAN AND EUROPEAN ANCESTRIES ACROSS FOUR BRAIN REGIONS - PROJECT SUMMARY / ABSTRACT HEALTH DISPARITIES HAVE ENDURED FOR CENTURIES. THE DISPARITY IN RESEARCH FOR BIOLOGICAL VARIATIONS IS EVEN GREATER, WHERE THE EXPLOSION OF GENETIC AND ENVIRONMENTAL DATA HAS FOCUSED ALMOST EXCLUSIVELY ON THOSE OF EUROPEAN ANCESTRY (EA). IN NEUROSCIENCE AND GENOMICS, INDIVIDUALS WITH RECENT AFRICAN ANCESTRY (AA) ACCOUNT FOR LESS THAN 5% OF LARGE-SCALE GENOMIC STUDIES BUT ARE 20% MORE LIKELY TO EXPERIENCE A MAJOR MENTAL HEALTH CRISIS. TO COMPOUND THIS ISSUE FURTHER, STUDIES HAVE LINKED GENETIC DIFFERENCES BETWEEN AA AND EA FOR DIVERGENT RESPONSES TO ANTIPSYCHOTICS. AS A RESULT, LARGE-SCALE STUDIES FOR NEUROPSYCHIATRIC DISORDERS HAVE LIMITED DIAGNOSTIC ACCURACY FOR NON-EUROPEAN ANCESTRY INDIVIDUALS, HINDERING THE DEVELOPMENT OF EFFECTIVE EQUITABLE NEUROTHERAPEUTICS AND POTENTIALLY INCREASING HEALTH DISPARITIES. DESPITE THE CLEAR URGENT NEED, THERE ARE NO LARGE-SCALE STUDIES EXAMINING GENETIC OR REGULATORY DIFFERENCES BETWEEN AA AND EA IN THE HUMAN BRAIN. THIS PROPOSAL SEEKS TO ADDRESS THIS GAP IN RESEARCH BY COMPARING AA WITH WELL-STUDIED EA INDIVIDUALS. THE INITIAL GOAL OF THIS PROPOSAL IS TO IDENTIFY GENETIC AND REGULATORY DIFFERENCE BETWEEN AA (N=784) AND EA (N=678) IN POSTMORTEM CAUDATE NUCLEUS (N=420), DENTATE GYRUS (N=161), DORSOLATERAL PREFRONTAL CORTEX (N=434), AND HIPPOCAMPUS (N=447), COMPILED FROM ONE OF THE LARGEST POSTMORTEM BRAIN COLLECTIONS OF PSYCHIATRIC DISORDERS. THIS DIVERSE LARGE-SCALE POSTMORTEM BRAIN TISSUE WILL ALLOW US TO TEST THE HYPOTHESES OF GENETIC VARIATIONS INFLUENCE ON BRAIN REGION-SPECIFIC TRANSCRIPTIONAL AND EPIGENETIC CHANGES FOR ANCESTRY ACROSS PRENATAL AND POSTNATAL LIFE. THIS MOSAIC (MAXIMIZING OPPORTUNITIES FOR SCIENTIFIC AND ACADEMIC INDEPENDENT CAREERS) POSTDOCTORAL CAREER TRANSITION AWARD TO PROMOTE DIVERSITY WILL BE SUPPORTED BY EXCELLENT CAREER DEVELOPMENT RESOURCES AT THE LIEBER INSTITUTE FOR BRAIN DEVELOPMENT AND JOHNS HOPKINS UNIVERSITY, AND TRAINING FROM A MENTORING TEAM OF GLOBALLY RECOGNIZED EXPERTS IN THE FIELDS OF HUMAN GENETICS AND ADVANCED STATISTICAL METHODS. IT WILL PROVIDE A FIRST OF ITS KIND EXAMINATION OF ANCESTRY IN NEUROSCIENCE, WHICH WILL ADVANCE OUR UNDERSTANDING OF GENETIC VARIATION IN THE HUMAN BRAIN.

IDENTIFYING A FUNCTIONAL ROLE FOR TRANSCRIPTIONALLY DISTINCT, BLA-ACTIVATED LS ENSEMBLES IN SOCIAL BEHAVIORS - PROJECT SUMMARY SOCIAL RECOGNITION IS THE ABILITY TO DISTINGUISH BETWEEN NOVEL AND FAMILIAR CONSPECIFICS. THIS SOCIAL BEHAVIOR IS ESSENTIAL FOR ORGANISMAL SURVIVAL AS AN ORGANISM MIST BE ABLE TO DIRECT THEIR BEHAVIOR BASED ON A SOCIAL STIMULUS OR PRESENTATION. RECENT EVIDENCE FROM OUR LAB HAS IDENTIFIED THAT NEURONS PROJECTING FROM THE BASOLATERAL AMYGDALA (BLA) TO THE LATERAL SEPTUM (LS) ARE CRITICALLY INVOLVED IN SOCIAL RECOGNITION. HOWEVER, THE MECHANISMS GOVERNING ACTIVATION OF LS NEURONS BY BLA INPUT ARE UNDERSTUDIED. MY LONG-TERM GOAL IS TO BETTER UNDERSTAND THE NEURAL SUBSTRATES REGULATING SOCIAL BEHAVIORS AT THE CIRCUIT, CELLULAR, AND MOLECULAR LEVELS. THE OBJECTIVE OF THIS PROJECT – TO STUDY THE ROLE OF BLA-ACTIVATED LS NEURONAL ENSEMBLES IN REGULATING SOCIAL RECOGNITION, AND TO CHARACTERIZE BOTH THE SPATIAL TOPOGRAPHY AND TRANSCRIPTIONAL HETEROGENEITY OF THESE ENSEMBLES, IS THE FIRST STEP TOWARD THIS LONG-TERM GOAL. BASED ON PREVIOUS FINDINGS IMPLICATING THE BLA-LS CIRCUIT IN REGULATING SOCIAL RECOGNITION, MY CENTRAL HYPOTHESIS IS THAT ACTIVATING LS-PROJECTING BLA NEURONS RECRUITS A TRANSCRIPTIONALLY DISTINCT ENSEMBLE OF LS NEURONS THAT ARE REQUIRED FOR SOCIAL RECOGNITION. THE RATIONALE FOR THE PROPOSED RESEARCH IS THAT IDENTIFYING A FUNCTIONAL ROLE FOR A SPECIFIC, MOLECULARLY DEFINED LS NEURONAL SUBPOPULATION IN SOCIAL RECOGNITION WILL PROVIDE FURTHER UNDERSTANDING OF THE NEURAL SUBSTRATES REGULATING HARDCODED SOCIAL BEHAVIORS ESSENTIAL FOR ORGANISMAL SURVIVAL. THE CENTRAL HYPOTHESIS OF THIS PROPOSAL WILL BE TESTED BY PURSUING TWO SPECIFIC AIMS: 1) ELUCIDATE MOLECULAR SIGNATURES OF BLA-ACTIVATED LS ENSEMBLES ACROSS THE SPATIAL TOPOGRAPHY OF THE LS, AND 2) TEST THE NECESSITY AND SUFFICIENCY OF BLA-ACTIVATED LS NEURONAL ENSEMBLES IN SOCIAL RECOGNITION. THIS APPROACH IS INNOVATIVE BECAUSE IT PROPOSES TO USE CIRCUIT-SPECIFIC, ACTIVITY- DEPENDENT TAGGING OF NEURONAL ENSEMBLES TO BOTH FUNCTIONALLY INTERROGATE THE POPULATION AND COMPREHENSIVELY CHARACTERIZE ITS MOLECULAR SIGNATURE. THE PROPOSED RESEARCH IS SIGNIFICANT BECAUSE IT WILL SHED LIGHT ON THE NEUROBIOLOGICAL UNDERPINNINGS THAT GOVERN THE DEVELOPMENT OF SOCIAL BEHAVIORS, AND COULD POTENTIALLY IDENTIFY NOVEL THERAPEUTIC TARGETS FOR THE TREATMENT OF NEURODEVELOPMENTAL DISORDERS WITH SOCIAL DEFICITS.

INVESTIGATING THE EFFECTS OF TCF4 MUTATIONS DURING OLIGODENDROCYTE DEVELOPMENT AND MATURATION IN A HUMAN-DERIVED MODEL OF AUTISM SPECTRUM DISORDER - PROJECT SUMMARY/ABSTRACT PITT-HOPKINS SYNDROME (PTHS) IS A RARE FORM OF AUTISM SPECTRUM DISORDER (ASD) CHARACTERIZED BY DEVELOPMENTAL DELAY, BREATHING ABNORMALITIES, SEIZURE, LACK OF SPEECH, AND ABNORMAL FACIES. PTHS IS CAUSED BY DE NOVO MUTATIONS IN TRANSCRIPTION FACTOR 4 (TCF4), A KEY TRANSCRIPTION FACTOR THAT ORCHESTRATES MULTIPLE NEURODEVELOPMENTAL PROGRAMS. PREVIOUS STUDIES FROM OUR LABORATORY USING PTHS MOUSE MODELS SHOWED DYSREGULATION IN THE DENSITY OF OLIGODENDROCYTE PROGENITOR CELLS (OPCS) AND MATURITY OF FUNCTIONAL OLIGODENDROCYTES (OLS), WHICH RESULTED IN MYELINATION DEFICITS. HOWEVER, THESE DEFICIENCIES HAVE NOT BEEN STUDIED IN THE HUMAN SYSTEM. THE CURRENT PROPOSAL CENTERS ON EXPLORING THE EFFECTS OF TCF4 MUTATIONS IN HUMAN OLS GENERATED FROM PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS (IPSCS). OUR LABORATORY HAS GENERATED A COLLECTION OF IPSCS THAT HARBOR VARIOUS DISEASE-CAUSING MUTATIONS IN TCF4 THAT RESULT IN THE EXPRESSION OF PUTATIVE DOMINANT-NEGATIVE PROTEINS OR HAPLOINSUFFICIENCY. TO DETERMINE IF PATIENT-SPECIFIC MUTATIONS IN TCF4 RESULT IN DYSREGULATION OF THE OL LINEAGE, WE DIFFERENTIATED IPSC USING A TWO-DIMENSIONAL AND A THREE-DIMENSIONAL OL GENERATING PROTOCOL. THE GOALS FOR THIS PROPOSAL FOCUS ON (1) CHARACTERIZING THE EFFECTS OF MUTATIONS IN TCF4 DURING OLIGODENDROGENESIS AND OL MATURATION IN A PATIENT-DERIVED IPSC MODEL, (2) IDENTIFYING TCF4’S INTERACTOME DURING OL SPECIFICATION, AND (3) GENERATING A PLATFORM FOR PRO-MYELINATING DRUG SCREENING. THE PROPOSED APPROACH IS INNOVATIVE AND SIGNIFICANT, AS IT AIMS TO SHED LIGHT ON THE PROCESS OF MYELINATION AND THE GENETIC REGULATORS THAT GUIDE OL MATURATION AND SURVIVAL. MOREOVER, THIS PROPOSAL WILL ALLOW FOR THE DEVELOP OF 2-D AND 3-D HUMAN-DERIVED PLATFORMS WITH THE CAPACITY TO RAPIDLY SCREEN PROMISING PRO-MYELINATING DRUGS THAT COULD IMPROVE PATIENT QUALITY OF LIFE. THIS FELLOWSHIP WILL HELP THE APPLICANT DEVELOP THE EXPERTISE NECESSARY TO CONDUCT BASIC BIOLOGICAL, FUNCTIONAL AND TRANSLATIONAL RESEARCH AS AN INDEPENDENT INVESTIGATOR IN THE AREA OF NEUROPSYCHIATRIC AND NEURODEVELOPMENTAL DISEASES.

REGULATION OF FEAR EXPRESSION BY ACTIVITY-DEPENDENT BDNF IN DIRECT HIPPOCAMPAL-TO-PRELIMBIC PROJECTIONS

INVESTIGATING TCF4 AND SYNAPTIC DYSFUNCTION IN PITT-HOPKINS SYNDROME - PROJECT SUMMARY AUTISM SPECTRUM DISORDERS (ASDS) PRESENT A COMPLEX ARRAY OF NEURODEVELOPMENTAL CHALLENGES, AFFECTING APPROXIMATELY 1 IN 100 INDIVIDUALS GLOBALLY. DESPITE SUBSTANTIAL EFFORTS TO UNDERSTAND THE GENETIC UNDERPINNINGS OF ASD, THERAPEUTIC TREATMENTS REMAIN ELUSIVE. TRANSCRIPTION FACTOR 4 (TCF4) LOSS-OF-FUNCTION (LOF) MUTATIONS ARE IMPLICATED IN PITT-HOPKINS SYNDROME (PTHS), A SYNDROMIC FORM OF ASD. WHILE PTHS PATHOPHYSIOLOGY HAS BEEN EXTENSIVELY STUDIED IN MURINE MODELS, UNDERSTANDING TCF4 FUNCTION WITHIN A HUMAN CONTEXT IS IMPERATIVE. THIS PROPOSAL AIMS TO UTILIZE PATIENT-DERIVED INDUCED PLURIPOTENT CELLS (HIPSCS) TO MODEL SYNAPTIC TRANSMISSION, FOCUSING ON PRESYNAPTIC REGULATORY ROLES OF TCF4 AND THE THERAPEUTIC POTENTIAL OF RIMBP2 RE-EXPRESSION. OUR RECENTLY PUBLISHED DATA INDICATE SIGNIFICANT DEFICITS IN SPONTANEOUS EXCITATORY POSTSYNAPTIC CURRENTS (SEPSCS) AND NETWORK ACTIVITY IN PTHS HIPSC-DERIVED NEURONS, POTENTIALLY STEMMING FROM PRESYNAPTIC ABNORMALITIES. FROM OUR DIFFERENTIAL EXPRESSION GENE ANALYSIS, RIMBP2, A PRESYNAPTIC ORGANIZER, EMERGED AS THE MOST SIGNIFICANTLY DOWNREGULATED GENE, WITH ITS RE-EXPRESSION RESCUING DEFICITS IN NETWORK SYNCHRONICITY AND SEPSC FREQUENCY. IN THIS STUDY, I WILL EXPLORE THE IMPACT OF TCF4 LOF ON PRESYNAPTIC RELEASE, SUBCELLULAR PROTEIN LOCALIZATION, AND ULTRASTRUCTURE, ALONG WITH MECHANISM BY WHICH RIMBP2 RESCUES DEFICITS. IN AIM 1, I WILL EMPLOY LIVE-CELL IMAGING WITH THE GLUTAMATE SENSOR IGLUSNFR3 TO QUANTIFY PRESYNAPTIC RELEASE DEFICITS AND CHANGES IN EVOKED NEUROTRANSMITTER RELEASE, RELEASE PROBABILITY, AND COUPLING DISTANCE BETWEEN RELEASE READY VESICLES AND CALCIUM CHANNELS. ADDITIONALLY, WITH TIME-RESOLVED ELECTRON MICROSCOPY, I WILL EXAMINE DEFICITS IN SYNAPTIC ULTRASTRUCTURE. IN AIM 2, I WILL CHARACTERIZE DEFICITS INVOLVING CALCIUM CHANNEL CONDUCTANCE, EXPRESSION, AND LOCALIZATION. FIRST, I WILL MEASURE PRESYNAPTIC CALCIUM INFLUX WITH THE CALCIUM SENSOR SYNG-CAMP6F. THEN, USING QUANTITATIVE MICROSCOPY, I WILL ASSESS THE EXPRESSION AND LOCALIZATION OF CALCIUM CHANNELS ALONG WITH RELATED PROTEINS IN THE ACTIVE ZONE. FINALLY, I WILL EMPLOY LIVE-GOLD-LABELING ELECTRON MICROSCOPY TO MEASURE THE COUPLING DISTANCE BETWEEN CALCIUM CHANNELS AND DOCKED VESICLES AT THE NANOMETER SCALE. THE SIGNIFICANCE OF THIS RESEARCH LIES IN ELUCIDATING THE SYNAPTIC DEFICITS ASSOCIATED WITH TCF4 LOF, PROVIDING A FOUNDATION FOR TARGETED THERAPEUTICS. THE ANTICIPATED RESULTS COULD IDENTIFY CANDIDATE GENES AND INFORM NOVEL APPROACHES TO RESTORE PRESYNAPTIC RELEASE DEFICITS LINKED TO TCF4 DYSFUNCTION IN PTHS. THE STUDY'S INNOVATIVE METHODOLOGY, INCORPORATING ADVANCED IMAGING TECHNIQUES AND HUMAN-DERIVED MODELS, CONTRIBUTES A UNIQUE PERSPECTIVE TO UNDERSTANDING THE SYNAPTIC ARCHITECTURE OF NEURONS AFFECTED BY TCF4 MUTATIONS. WITH THE SUPPORT OF MY PROPOSED MENTORSHIP TEAM AND THE OUTSTANDING TRAINING ENVIRONMENT, THE RESEARCH AND TRAINING ACTIVITIES PROPOSED HERE WILL IDEALLY POSITION ME FOR A FUTURE CAREER IN ACADEMIC NEUROSCIENCE RESEARCH.

HIGH THROUGHPUT SCREENING FOR SPECIFIC INHIBITORS AND MODULATORS OF A NOVEL POTAS