Supplementary data for: IRG1/itaconate inhibits microglial senescence-like transition by modulating mitochondrial dynamics through RhoA alkylation in subarachnoid hemorrhage

Aging is a potent risk factor for poor prognosis in subarachnoid
hemorrhage (SAH), yet the molecular mechanisms underlying the age-related
exacerbation of early brain injury remain incompletely understood. This
study investigates the immunometabolic regulation of the microglial
senescence-like transition following SAH, focusing on the
immune-responsive gene 1 (IRG1)/itaconate axis. We observed that the
endogenous upregulation of IRG1 and itaconate is a protective response to
hemorrhagic stress that is significantly blunted in aged mice.
Microglia-specific IRG1 deficiency exacerbated SAH-induced brain injury,
characterized by an accelerated senescence-like transition and the
secretion of senescence-associated secretory phenotype (SASP) factors.
Mechanistically, we demonstrate that IRG1 deficiency leads to excessive
mitochondrial fission and dysfunction via the hyperactivity of
Dynamin-related protein 1 (Drp1). Using click chemistry-based proteomics
and site-directed mutagenesis, we identified that itaconate exerts its
neuroprotective effects by directly alkylating the small GTPase RhoA at
the cysteine 107 (C107) residue. This specific post-translational
modification inhibits RhoA-GTP binding and downstream ROCK1 activation,
thereby suppressing Drp1-mediated mitochondrial fragmentation.
Importantly, treatment with the cell-permeable itaconate derivative
4-octyl itaconate (4-OI) rescued mitochondrial dynamics and attenuated
microglial senescence and neurological deficits, whereas the RhoA-C107S
mutation abolished these protective effects. Collectively, our findings
unveil a novel metabolic-mitochondrial checkpoint involving the
IRG1/itaconate-RhoA-Drp1 axis. Restoring this pathway represents a
promising therapeutic strategy to combat the age-related exacerbation of
neuroinflammation and improve outcomes in SAH patients.