27-Hydroxycholesterol Modulates Microglial α-Synuclein Handling and Is Associated with AKT1/JAK-STAT/TNF Signaling in Parkinson's Disease Models.

Abstract

27-Hydroxycholesterol (27-OHC) is an oxysterol implicated in neuroinflammation, but its relationship to microglial α-synuclein (α-syn) handling and intracellular signaling in Parkinson's disease (PD) models remains incompletely defined. We combined BV2-cell assays, an rAAV-A53T mouse model, untargeted lipidomics and proteomics, network analyses, molecular docking, pathway immunoblotting, pharmacological AKT inhibition, and a cellular thermal shift assay (CETSA). In BV2 cells, 0.5-2.5 μM 27-OHC did not reduce viability, whereas concentrations ≥ 5 μM were cytotoxic. At 2.5 μM, 27-OHC increased cell-associated α-syn signal after exposure to recombinant α-syn PFFs. Brain lipidomics in the PD model showed accumulation of the broad sterol-lipid class, but did not provide absolute brain 27-OHC concentrations. Network analyses nominated AKT1, IL6, STAT3, and MMP9 as hub genes. In three independent BV2 experiments, PFF and 27-OHC exposure were associated with altered phosphorylation of AKT, STAT3, and NF-κB p65; a PFF × 27-OHC interaction was detected for STAT3 phosphorylation. MK-2206 lowered the cell-associated α-syn/GAPDH signal in the combined-treatment group, although the comparison did not remain significant after adjustment across three pairwise tests. CETSA showed greater soluble AKT1 retention after 27-OHC exposure and a higher thermal-curve area under the curve (paired p = 0.023). These findings support an association between 27-OHC exposure, microglial α-syn handling, and AKT-related signaling in BV2 cells. They do not establish direct 27-OHC-AKT1 binding or an in vivo causal effect of brain 27-OHC on microglia.

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