Modulation of mitochondria-ER contacts decrease inflammasome formation and restores amyloid β-peptide phagocytosis in adult mouse microglia

Abstract Background Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease, currently devoid of a cure. AD’s clinical manifestations stem from a multitude of dysfunctional cellular processes, regulated by mitochondria-endoplasmic contact sites (MERCS), which undergo physical alterations and malfunction in AD brain. Despite ongoing research, the understanding of MERCS in AD remains in its nascent stages. We postulate that these subcellular interfaces are responsible for AD progression. Neuroinflammation contributes significantly to neurodegeneration and is primarily driven by microglia, the innate immune cells in the brain. In AD, activated microglia secrete pro-inflammatory cytokines that compromise neuronal vitality. The production of these cytokines is promoted by NLRP3 inflammasome. Although inflammasome activation has been observed at MERCS, the underlying MERCS-mediated mechanisms governing regulation of inflammasome activation remain to be elucidated. Methods Primary microglia were isolated from 3-4 months old wild-type (WT) and App NL-G-F mice (AD). MERCS ultrastructure was analyzed by transmission electron microscopy. Mitochondrial Ca 2+ level and metabolic function were assessed using Rhod-2 AM fluorescence and Seahorse extracellular flux analysis respectively. Inflammasome activation was induced by lipopolysaccharide and nigericin and evaluated by IL-1β ELISA, caspase-1 activity assay, and ASC immunocytochemistry. MERCS were genetically modulated via siRNA-mediated knockdown of MERCS-associated proteins, and ER-to-mitochondria Ca²⁺ transfer was pharmacologically inhibited using Xestospongin C and MCU-i11. Microglial Aβ phagocytosis was quantified using fluorescence-conjugated Aβ 1-42 . Results AD microglia exhibited increased MERCS number and contact length, accompanied by a reduction in mitochondria-ER proximity. These structural changes were associated with elevated mitochondrial Ca 2+ levels and enhanced respiratory activity, indicating metabolic reprogramming and functional change. Structural and functional decrease of microglial MERCS attenuated NLRP3 inflammasome activation and restored inflammasome-associated impairments in Aβ phagocytosis. Pharmacological inhibition of Ca 2+ channels at MERCS identified ER-to-mitochondria Ca 2+ transfer as a key regulatory mechanism for inflammasome activation. Conclusions Our findings identify microglial MERCS remodeling as an early event in AD and establish ER–mitochondria coupling as an upstream regulator of energy metabolism, inflammation, and Aβ clearance. Targeting MERCS may therefore represent a promising strategy to modulate neuroinflammation while preserving essential microglial functions in AD.