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 α-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 α-synuclein
accumulation within oligodendrocytes and the consequences for 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 patients for all
patients of our sample set which contains both MSA-C and MSA-P cases. 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 validate the changes identified by snRNA-seq in tissue with immunohistochemistry and multiplexed
fluorescence in situ hybridization with RNAscope, allowing the assessment of whether cells bearing
dysregulated gene expression patterns have evidence of dysfunction. Initially focusing on oligodendrocytes, we
will determine whether cells bearing GCI exhibit dysregulated transcriptomes, whether α-synuclein mRNA is
overexpressed in these cells, and whether this affects myelin integrity. In Aim 3, we will test whether forced
overexpression of α-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 α-synuclein
expression is increased in cells with GCI, and whether de novo expression of α-synuclein mRNA fully
recapitulates the cell states associated with MSA.