CKMT1 Counteracts VDAC1 Oligomerization‐Induced Neuronal Pyroptosis Through Steric Hindrance in Ischemic Stroke

Ischemic stroke is one of the leading causes of death and disability worldwide; yet effective therapeutic options remain limited. Cytosolic leakage of mitochondrial DNA (mtDNA) is a critical driver of ischemic brain injury. Voltage-dependent anion channel 1 (VDAC1), embedded in the mitochondrial outer membrane, promotes mtDNA leakage through oligomerization. However, the regulatory mechanisms underlying VDAC1 oligomerization in ischemic stroke remain poorly understood. This study demonstrates that ischemic stroke promotes VDAC1 oligomerization, leading to mtDNA leakage, activating the absent in melanoma 2 (AIM2) inflammasome, and subsequent neuronal pyroptosis. Pharmacological inhibition of VDAC1 oligomerization using VBIT-4 effectively disrupts this cascade. Creatine kinase mitochondrial 1 (CKMT1) is identified as a novel endogenous inhibitor of VDAC1 oligomerization. CKMT1 inhibits VDAC1 oligomerization through a dual-region synergistic mechanism. Specifically, the N-terminal domain of CKMT1 (residues R75 and E121) forms a stable interface with the C-terminal domain of VDAC1 (residues R218 and E189), whereas the CKMT1 signal peptide (residues 1-39) sterically hinders VDAC1 dimerization, thereby blocking oligomerization. Moreover, neuronal overexpression of CKMT1 significantly attenuates neuronal apoptosis, whereas CKMT1 knockdown aggravates neuronal injury. Collectively, these findings demonstrate that CKMT1 protects against cerebral ischemic injury by suppressing the VDAC1-mtDNA-AIM2 axis and identify CKMT1 as a novel therapeutic target for ischemic stroke.