Project Grant F31DA067081

COCAINE SELF-ADMINISTRATION AND DRD2 SPLICING VARIANTS - PROJECT SUMMARY THE DOPAMINE D2 RECEPTOR (D2R), ENCODED BY THE DRD2 GENE, IS A CRITICAL MODULATOR OF REWARD, MOTIVATION, AND EMOTION, AND HAS BEEN A TARGET FOR THERAPEUTIC DEVELOPMENT. WHILE THE CANONICAL DRD2 TRANSCRIPT IS WELL CHARACTERIZED, EMERGING EVIDENCE SUGGESTS THAT ALTERNATIVE SPLICING CAN GENERATE MULTIPLE TRANSCRIPT VARIANTS AND PROTEIN ISOFORMS. THESE VARIANTS MAY INFLUENCE D2R PHARMACOLOGY AND RESPONSIVENESS TO LIGANDS; HOWEVER, THE FULL-LENGTH IDENTITY, BRAIN REGION- AND CELL TYPE-SPECIFIC DISTRIBUTION, AND FUNCTIONAL RELEVANCE OF THESE VARIANTS, PARTICULARLY IN THE CONTEXT OF COCAINE USE DISORDER (CUD), ARE POORLY UNDERSTOOD DUE TO LIMITATIONS OF SHORT-READ RNA SEQUENCING THAT OFTEN FAIL TO CAPTURE TRANSCRIPT ISOFORMS. IN CONTRAST, LONG-READ RNA SEQUENCING ENABLES THE DIRECT READOUT OF FULL-LENGTH RNA MOLECULES FOR ACCURATE IDENTIFICATION AND QUANTIFICATION OF TRANSCRIPT VARIANTS. TO ADDRESS THE CURRENT KNOWLEDGE GAP, WE EMPLOYED LONG-READ RNA SEQUENCING ON BRAIN TISSUE FROM RATS WITH A HISTORY OF COCAINE SELF-ADMINISTRATION (SA) AND SALINE CONTROLS. THIS APPROACH REVEALED MULTIPLE PREVIOUSLY UNANNOTATED DRD2 TRANSCRIPT VARIANTS IN THE VENTRAL TEGMENTAL AREA (VTA) AND NUCLEUS ACCUMBENS, TWO CRITICAL BRAIN REGIONS INVOLVED IN COCAINE REWARD. THESE VARIANTS EXHIBIT STRUCTURAL DIFFERENCES, INCLUDING PARTIAL OR COMPLETE DELETIONS OF CERTAIN EXONS ENCODING RECEPTOR DOMAINS CRITICAL FOR LIGAND BINDING AND G-PROTEIN COUPLING. NOTABLY, THEY DISPLAY REGION-SPECIFIC EXPRESSION PATTERNS THAT ARE SENSITIVE TO COCAINE EXPOSURE, WITH NO OVERLAP ACROSS BRAIN REGIONS. THIS PROPOSAL WILL DETERMINE THE ROLES OF TWO SPECIFIC DRD2 TRANSCRIPT VARIANTS UPREGULATED IN THE VTA AFTER COCAINE SA. MY CENTRAL HYPOTHESIS IS THAT DRD2 VARIANTS IN THE VTA ALTER D2R FUNCTION, DOPAMINE TRANSMISSION AND COCAINE SA. USING CELL-BASED ASSAYS AND ANIMAL MODELS OF CUD, I WILL TEST THIS HYPOTHESIS THROUGH THREE SPECIFIC AIMS: 1) EXAMINE THE CELLULAR LOCALIZATION AND SIGNALING OF EACH DRD2 TRANSCRIPT VARIANT IN CELL-BASED HETEROLOGOUS SYSTEMS; (2) INVESTIGATE IF DRD2 TRANSCRIPT VARIANTS IN THE VTA INFLUENCE COCAINE SA IN ANIMALS; AND 3) DETERMINE IF DRD2 VARIANTS IN THE VTA ALTER D2R FUNCTION AND DOPAMINE TRANSMISSION IN ANIMALS. FINDINGS FROM THIS PROJECT WILL ADVANCE OUR UNDERSTANDING OF THE COMPLEXITY OF D2R BIOLOGY, REVEAL NOVEL MOLECULAR MECHANISMS UNDERLYING CUD, AND INFORM THE DEVELOPMENT OF TARGETED THERAPIES BASED ON RECEPTOR ISOFORM SPECIFICITY.