IFI204 drives gasdermin D–mediated mitochondrial permeabilization to amplify neuronal pyroptosis in ischemic stroke

Gasdermin D (GSDMD)-mediated pore formation on mitochondrial membranes is known to exacerbate pyroptosis. The cytosolic DNA sensor interferon activated gene 204 (IFI204) can activate the inflammasome to induce pyroptosis. However, whether and how IFI204 regulates mitochondrial membrane permeabilization to drive pathological outcomes in ischemic stroke remains unclear. Here, using a mouse model of middle cerebral artery occlusion (MCAO), we demonstrate that IFI204 was predominantly expressed in neurons and increased to peak at 24 h after ischemic injury. Neuron-specific deletion of IFI204 alleviated cerebral infarction, reduced neuronal degeneration, and restored long-term sensorimotor coordination and cognitive function. These protective effects correlated with attenuated neuronal pyroptosis and mitochondrial dysfunction, as evidenced by decreased levels of GSDMD N-terminal fragment (GSDMD-N) and reduced mitochondrial colocalization. Conversely, adeno-associated virus-mediated re-expression of IFI204 in knockout mice restores these pathological features. In vitro, IFI204 is both necessary and sufficient to trigger this cascade. Transcriptomic profiling revealed a significant downregulation of the stimulator of interferon genes (STING) within the NOD-like receptor signaling pathway in IFI204-deficient neurons. Mechanistically, glutathione S-transferase (GST) pull-down assays confirmed a direct interaction between the pyrin domain (PYD) of IFI204 and STING. This interaction triggers caspase-1 activation and GSDMD cleavage, generating GSDMD-N, which subsequently forms pores specifically on mitochondrial membranes. These pyroptotic pores disrupted mitochondrial integrity, exacerbating dysfunction, and facilitating the cytosolic release of mitochondrial DNA (mtDNA), cytochrome c, and aconitase 2. Notably, the released mtDNA further activated IFI204, establishing a pathogenic feed-forward cycle that exacerbates mitochondrial damage and inflammatory neuronal death. Genetic ablation of STING partially abrogated the pyroptosis-promoting effect of IFI204. Collectively, these findings demonstrate that IFI204-driven cytosolic mtDNA sensing underlies a neuronal inflammatory mechanism responsible for pyroptosis and mitochondrial damage in ischemic stroke.