Microglial hyperactivation contributes to Parkinson's disease (PD) progression, yet the upstream microenvironmental cues that sustain this state remain incompletely understood. While α-synuclein (α-Syn) aggregation is a primary trigger, aging and PD are also associated with microvascular and perfusion abnormalities. However, how vascular-associated hypoxic stress interacts with protein toxicity in microglial fate determination remains unclear. We integrated human single-nucleus RNA sequencing (snRNA-seq) data, a chronic progressive transgenic mouse model (9-month-old A53T), and an in vitro "double-hit" model. Neuropathological and immunofluorescence analyses were employed to assess the neurovascular unit and microglial phenotypes. The snRNA-seq analysis of human PD brains revealed a Disease-Associated Microglia (DAM) subset characterized by enrichment of hypoxia and glycolysis pathways, with HIF1A acting as a central node. In vivo, 9-month-old A53T mice exhibited motor deficits and dopaminergic degeneration, accompanied by reduced CD31+ microvascular coverage in the substantia nigra. This reduction in CD31+ vascular coverage was associated with microglial HIF1A accumulation and increased IBA1-defined soma area. In vitro, physical hypoxia amplified α-Syn preformed fibril (PFF)-induced microglial reactivity, intracellular accumulation of phosphorylated α-Syn (p-αSyn). Our study supports a "double-hit" model in which hypoxia-associated stress may amplify α-Syn-induced microglial dysfunction through HIF1A-linked metabolic remodeling and impaired autophagy-related protein handling. Targeting neurovascular-immune interactions may offer therapeutic opportunities for advanced PD.
