Identification of methylation-regulated genes modulating microglial phagocytosis in hyperhomocysteinemia-exacerbated Alzheimer's disease

Abstract

Background: Hyperhomocysteinemia (HHcy) has been linked to development of Alzheimer's disease (AD) neuropathologically characterized by the accumulation of amyloid β (Aβ). Microglia (MG) play a crucial role in uptake of Aβ fibrils, and its dysfunction worsens AD. However, the effect of HHcy on MG Aβ phagocytosis remains unstudied.

Methods: We isolated MG from the cerebrum of HHcy mice with genetic cystathionine-β-synthase deficiency (Cbs^-/-) and performed bulk RNA-seq. We performed meta-analysis over transcriptomes of Cbs^-/- mouse MG, human and mouse AD MG, MG Aβ phagocytosis model, human AD methylome, and GWAS AD genes.

Results: HHcy and hypomethylation conditions were identified in Cbs^-/- mice. Through Cbs^-/- MG transcriptome analysis, 353 MG DEGs were identified. Phagosome formation and integrin signaling pathways were found suppressed in Cbs^-/- MG. By analyzing MG transcriptomes from 4 AD patient and 7 mouse AD datasets, 409 human and 777 mouse AD MG DEGs were identified, of which 37 were found common in both species. Through further combinatory analysis with transcriptome from MG Aβ phagocytosis model, we identified 130 functional-validated Aβ phagocytic AD MG DEGs (20 in human AD, 110 in mouse AD), which reflected a compensatory activation of Aβ phagocytosis. Interestingly, we identified 14 human Aβ phagocytic AD MG DEGs which represented impaired MG Aβ phagocytosis in human AD. Finally, through a cascade of meta-analysis of transcriptome of AD MG, functional phagocytosis, HHcy MG, and human AD brain methylome dataset, we identified 5 HHcy-suppressed phagocytic AD MG DEGs (Flt1, Calponin 3, Igf1, Cacna2d4, and Celsr) which were reported to regulate MG/MΦ migration and Aβ phagocytosis.

Conclusions: We established molecular signatures for a compensatory response of Aβ phagocytosis activation in human and mouse AD MG and impaired Aβ phagocytosis in human AD MG. Our discoveries suggested that hypomethylation may modulate HHcy-suppressed MG Aβ phagocytosis in AD.

Keywords: Alzheimer’s; Amyloid β; Hyperhomocysteinemia; Hypomethylation; Microglia; Phagocytosis.