A unique microglia subset associated with aggressive α-synucleinopathy uncovered in a rapidly progressive multiple system atrophy cerebellar type model

Multiple system atrophy (MSA) is a progressive and fatal α-synucleinopathy characterized by α-synuclein-positive (α-syn⁺) glial cytoplasmic inclusions in oligodendrocytes. The cerebellar variant (MSA-C) primarily affects olivopontocerebellar fibers, resulting in extensive demyelination and glial activation. To model this pathology, we developed a Tet-Off-based MSA-C mouse model with oligodendrocyte-specific overexpression of human A53T α-syn. Upon doxycycline withdrawal at 8 weeks of age, mice developed progressive cerebellar ataxia by 26 weeks and succumbed by 30 weeks. These mice exhibited severe demyelination and marked activation of microglia and astroglia in the brainstem and cerebellum, along with widespread propagation of α-syn oligomers and phosphorylated α-syn (p-α-syn) aggregates in oligodendrocytes, astrocytes, and neurons. Single-cell RNA sequencing of CD11b⁺ cells from the brain and spinal cord identified a distinct microglial cluster expressing Toll-like receptor 2 (Tlr2), transglutaminase 2 (Tgm2), arginase-1, macrophage scavenger receptor-1 (Msr1), inflammatory genes (such as Nfkbia, Nfkbiz, and Il1b), and chemokines (including Ccl3, Ccl4, and Ccl12). These microglia were located adjacent to p-α-syn aggregates and were distinct from previously described protective disease-associated microglia and border-associated macrophages. TLR2- and TGM2⁺Iba1⁺ microglia were particularly enriched in demyelinating lesions. Prophylactic administration of the CSF1R inhibitor BLZ945 exacerbated motor deficits and demyelination, significantly increasing this microglial population. Similarly, MSR1⁺ and CD68⁺ microglia/macrophages were observed in early pontocerebellar lesions of six human MSA-C autopsy cases. These findings suggest that this pro-inflammatory microglia subset plays a central role in disease progression and may represent a promising therapeutic target for modifying the course of MSA-C and related synucleinopathies.