ROS-xCT metabolic coupling defines an astrocyte subtype that drives glutamate excitotoxicity during Alzheimer's disease progression.

AIMS: This study aims to systematically identify the cellular drivers of brain microenvironmental imbalance during Alzheimer's disease (AD) progression by integrating high-resolution single-nucleus RNA sequencing (snRNA-seq) with intercellular communication analyses. In the work, the functional transitions and metabolic mechanisms were specifically investigated for distinct astrocyte subpopulations underlying glutamate dysregulation. MAIN METHODS: We analyzed ROSMAP snRNA-seq data from 427 individuals to identify astrocyte subpopulations, assess their associations with AD Braak stages, and infer intercellular communication using CellChat. For validation, primary mouse astrocytes were exposed to oligomeric tau, oligomeric Aβ₁₋₄₂, or activated microglia-conditioned medium (aMCM). Mdivi-1 was applied to aMCM-stimulated astrocytes to examine oxidative stress-related changes in glutamate-handling genes. KEY FINDINGS: During disease progression, Ast.1 exhibited a transcriptional signature consistent with impaired glutamate clearance machinery and became the predominant source of inferred astrocyte-to-neuron glutamate signaling. Mechanistically, microglia-derived neuroinflammation robustly induces severe mitochondrial oxidative stress in astrocytes, specifically upregulating the SLC7A11 (xCT) antiporter. This compensatory antioxidant defense enforces a deleterious metabolic trade-off, directly coupling intracellular redox homeostasis with continuous pathological glutamate efflux. Crucially, targeted inhibition of oxidative stress via mdivi-1 significantly suppressed xCT hyperactivation and partially restored the expression of glutamate-handling genes. SIGNIFICANCE: This study defines Ast.1 as a pivotal driver of microenvironmental collapse in AD, unveiling a novel microglia-astrocyte-neuron pathogenic cascade mediated by the ROS-xCT metabolic hub. Targeting this astrocytic vulnerability offers a promising therapeutic strategy for preventing neurodegeneration.