Project Grant R01NS131658

SINGLE CELL TRANSCRIPTOMIC PROFILING OF MULTIPLE SYSTEM ATROPHY BRAIN - PROJECT SUMMARY/ABSTRACT MULTIPLE SYSTEM ATROPHY (MSA) IS A RARE PROGRESSIVE NEURODEGENERATIVE DISEASE CHARACTERIZED BY SELECTIVE ACCUMULATION OF A-SYNUCLEIN IN GLIAL CYTOPLASMIC INCLUSIONS (GCIS) WITHIN OLIGODENDROCYTES. CLINICALLY, MSA PATIENTS PRESENT WITH VARIOUS COMBINATIONS OF PARKINSONISM, CEREBELLAR DYSFUNCTION, AND DYSAUTONOMIA. MSA IS SUBCLASSIFIED BASED ON PREDOMINANCE OF SYMPTOMOLOGY, WHICH IS ASSOCIATED WITH THE PRIMARY SITE OF NEURODEGENERATION: MSA-P FOR PARKINSONISM AND STRIATONIGRAL DEGENERATION OR MSA-C FOR CEREBELLAR FEATURES AND OLIVOPONTOCEREBELLAR ATROPHY, THOUGH MOST CASES INVOLVE BOTH SYSTEMS. THE CAUSES OF A-SYNUCLEIN ACCUMULATION WITHIN OLIGODENDROCYTES AND ITS CONSEQUENCES TO OLIGODENDROCYTE PHYSIOLOGY IN MSA ARE LARGELY UNKNOWN. LIKEWISE, HOW OLIGODENDROCYTE DYSFUNCTION CAUSES NEURONAL DEATH REMAINS OBSCURE. IN THIS PROPOSAL, WE WILL USE SINGLE NUCLEUS RNA SEQUENCING (SNRNA-SEQ) TO GENERATE TRANSCRIPTOMES OF SINGLE CELLS FROM POSTMORTEM BRAIN TISSUE FROM MSA PATIENTS TO DELINEATE THE CELL TYPE SPECIFIC TRANSCRIPTIONAL CHANGES ASSOCIATED WITH MSA. WE WILL PROBE STRIATAL, CEREBELLAR, AND CORTICAL TISSUE SETS FROM THE SAME MSA-P OR MSA- C PATIENTS. THIS ALLOWS US TO CAPTURE THE CHANGES THAT OCCUR IN THE PRIMARY SITE OF PATHOLOGY FOR EACH MSA SUBTYPE, STRIATUM FOR MSA-P AND CEREBELLUM FOR MSA-C, ALONG WITH THE SECONDARY SITES, AND A MINIMALLY AFFECTED BRAIN REGION (CORTEX). IN AIM 1, WE WILL COLLECT ADDITIONAL MSA CASES AND GENERATE SNRNA-SEQ PROFILES FROM ALL TISSUE SETS. THESE DATA WILL BE INTEGRATED AND CLUSTERED TO IDENTIFY MAJOR CELL TYPES AND SUBTYPES FROM WHICH INFORMATIC ANALYSIS OF DIFFERENTIALLY-EXPRESSED GENES WILL BE USED TO IDENTIFY REGULATORY NETWORKS AND INFER CHANGE IN FUNCTION. IN AIM 2, WE WILL TEST WHETHER CELLS WITH ALTERED TRANSCRIPTIONAL PROFILES IDENTIFIED BY SNRNA- SEQ CORRESPOND TO THE CELLS WITH PATHOLOGICAL FEATURES OF MSA OR TO CELLS EXHIBITING DYSFUNCTION USING IMMUNOHISTOCHEMISTRY AND MULTIPLEXED FLUORESCENCE IN SITU HYBRIDIZATION WITH RNASCOPE. INITIALLY FOCUSING ON OLIGODENDROCYTES, WE WILL DETERMINE WHETHER CELLS BEARING GCI EXHIBIT DYSREGULATED TRANSCRIPTOMES, WHETHER A- SYNUCLEIN MRNA IS OVEREXPRESSED IN THESE CELLS, AND WHETHER THIS AFFECTS MYELIN INTEGRITY. IN AIM 3, WE WILL TEST WHETHER FORCED OVEREXPRESSION OF A-SYNUCLEIN IN OLIGODENDROCYTES IS SUFFICIENT TO RECAPITULATE THE SNRNA-SEQ PROFILES OBTAINED FROM MSA TISSUES USING A NONHUMAN PRIMATE MSA MODEL. UPON COMPLETION, THIS PROPOSAL WILL GENERATE AN ATLAS OF THE MSA-DEPENDENT TRANSCRIPTIONAL CHANGES OF NEARLY ALL CELL TYPES IN THE STRIATUM, CEREBELLUM, AND CORTEX, IDENTIFY KEY ALTERATIONS IN GENE EXPRESSION AND THE PATHWAYS AFFECTED, DETERMINE WHETHER A-SYNUCLEIN EXPRESSION IS INCREASED IN CELLS WITH GCI, AND WHETHER DE NOVO EXPRESSION OF A-SYNUCLEIN MRNA FULLY RECAPITULATES THE CELL STATES ASSOCIATED WITH MSA.