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 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 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.