Porphyromonas gingivalis outer membrane vesicles drive neuroinflammation via TGM2-mediated mitochondria-associated endoplasmic reticulum membranes.

BACKGROUND: Porphyromonas gingivalis (P.g), a key periodontal pathogen, is implicated in Alzheimer's disease (AD). Given that mitochondrial dysfunction is a common event happened in neurodegenerative diseases, mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) play a potential role in AD pathology. This study aimed to investigate the impact of P.g derived outer membrane vesicles (P.g-OMVs) on neuroinflammation in the onset and progression of AD, and to elucidate the underlying mechanism. METHODS: Eight-week-old male C57BL/6 mice received bilateral gingival injections of P.g-OMVs (4 × 10⁸ particles) 3 times weekly for 8 weeks to assess cognitive impairment and neuroinflammation. RNA-seq identified differentially expressed genes (DEGs) and enriched pathway in brain tissues. In vitro, HT22 cells were treated with P.g-OMVs (5 µg/mL), and the expressions of inflammatory cytokines, transglutaminase 2 (TGM2) and ferroptosis markers were quantified. MAM formation and mitochondrial function regulated by TGM2, including mitochondrial Ca2+ accumulation and membrane potential, were assessed, which further elucidated after knocking down TGM2 by siRNA. RESULTS: P.g-OMVs induced significant cognitive impairment and​ neurodegeneration in mice, evidenced by escape latency in the MWM (p < 0.001) and accumulation of amyloid β plaques and hyperphosphorylated tau. RNA-seq highlighted DEGs enriched in mitochondrial pathways, notably targeting TGM2. In vitro, P.g-OMVs triggered inflammation evidenced by increased pro-inflammatory cytokines (IL-1β, IL-17, etc.) and reduced levels of IL-10 (p < 0.05), and led to the upregulation of TGM2 (p < 0.05). Crucially, co-localization of mitochondria and ER suggested that P.g-OMVs promoted MAM formation in neurons, accompanied by ​aberrant mitochondrial Ca2+ levels​ and membrane potential. Additionally, altered expressions of ferroptotic markers were observed, including increased level of ACSL4 (p < 0.01) and decreased levels of GPX4 and xCT (p < 0.01). TGM2 knockdown reversed the inflammation, ferroptosis, mitochondrial dysfunction and MAM formation. CONCLUSIONS: Our findings demonstrate that P.g-OMVs drive​ neuroinflammation and neuronal ferroptosis through a MAMs-associated mechanism. TGM2 acts as a key mediator, promoting MAM formation and subsequent mitochondrial dysfunction. These findings highlight TGM2 represents a potential therapeutic target for neuroinflammatory conditions linked to oral pathogens.