A self-perpetuating neuron-intrinsic GSDMD-mtDNA-AIM2 inflammasome axis drives neuronal pyroptosis and cognitive impairment after traumatic brain injury.

INTRODUCTION: Traumatic brain injury (TBI) is a leading cause of mortality and long-term disability worldwide, producing acute neurological deficits and lasting cognitive impairment. Post-injury neuroinflammation is a principal driver of secondary damage and contributes substantially to TBI-induced cognitive dysfunction (TBI-CD), yet the cell-autonomous mechanisms operating within neurons remain incompletely characterised. The AIM2 inflammasome - a cytosolic sensor of double-stranded DNA that drives pro-inflammatory cytokine release and pyroptosis - has been studied primarily in myeloid cells, and its role within neurons after TBI is unclear.

METHODS: Here, using a controlled cortical impact (CCI) mouse model, an in vitro mechanical-injury model, AAV-mediated neuron-specific AIM2 knockdown and a comprehensive set of behavioural and molecular assays, we define a neuron-intrinsic, self-perpetuating GSDMD-mtDNA-AIM2 axis that drives neuronal pyroptosis and cognitive decline after TBI.

RESULTS: CCI triggered acute (24 h) AIM2 inflammasome activation specifically in cortical and hippocampal neurons, neuronal pyroptosis and CA3 neuronal loss. AAV-mediated knockdown of AIM2 in hippocampal CA3 neurons significantly reduced neuronal loss in this region and rescued cognitive performance in the Y-maze, novel object recognition and Morris water maze at 14 and 28 days post-injury. Mechanistically, mechanical injury caused early (3-6 h) release of mitochondrial DNA (mtDNA) - but not nuclear DNA - into the neuronal cytosol, where it directly activated the AIM2 inflammasome and engaged caspase-1/GSDMD-dependent pyroptosis; ethidium bromide-mediated mtDNA depletion reversed each pyroptotic marker. Within the first 0.5-3 h post-injury, activated GSDMD N-terminal fragments (GSDMD-NT) translocated to mitochondria, disrupted mitochondrial membrane potential (ΔΨm) and promoted further mtDNA leakage; CRISPR knockout of GSDMD, but not of Bax, prevented this injury-induced mitochondrial dysfunction. Delayed pharmacological inhibition of caspase-1 (VX-765 applied 6 h after injury, after the first wave) selectively suppressed a discrete second wave of cytosolic mtDNA release at 9-12 h and attenuated late-phase LDH release, providing direct experimental evidence for a self-perpetuating second wave of mtDNA-driven AIM2 activation.

CONCLUSION: These findings define a self-perpetuating, neuron-intrinsic GSDMD-mtDNA-AIM2 inflammasome-pyroptosis axis as a driver of TBI-CD, and identify neuron-targeted AIM2 silencing as a candidate therapeutic strategy for limiting post-TBI neuroinflammation and cognitive decline.