OTUD1 exacerbates sepsis-associated encephalopathy by promoting HK2 mitochondrial release to drive microglia pyroptosis

Abstract Background Sepsis-associated encephalopathy (SAE), a life-threatening neurological complication of systemic infection, contributes substantially to sepsis-related mortality. Accumulating evidence demonstrates that microglia-driven neuroinflammation emerges as a central pathogenic mechanism underlying SAE. Here, we identify ovarian tumor deubiquitinase 1 (OTUD1) as a critical mediator of SAE pathogenesis. We demonstrate that OTUD1 promotes hexokinase 2 (HK2) dissociation from mitochondria via selective K63-linked deubiquitination, triggering microglia pyroptosis and neuroinflammation. Our findings address a key knowledge gap by elucidating the OTUD1-HK2 axis as a novel regulatory pathway in SAE, offering potential therapeutic targets to mitigate cognitive deficits in sepsis. Methods Single-cell RNA sequencing was used to identify SAE-specific microglia subpopulations and analyze the expression of deubiquitinases within these subpopulations. OTUD1 knockout mice were generated to investigate the role of OTUD1 in SAE. Both wild-type and OTUD1 knockout mice were subjected to cecal ligation and puncture to induce SAE. In vitro, primary microglia and BV2 cells were treated with LPS and nigericin to simulate inflammatory conditions. Cognitive function of the mice was assessed through behavioral tests. Neuronal and synaptic damage were evaluated using HE and Nissl staining, as well as transmission electron microscopy. ELISA and qPCR were used to detect neuroinflammation. Western blot and immunofluorescence were employed to analyze protein expression. Molecular docking, 3D confocal microscopy, and co-immunoprecipitation were conducted to detect the interaction between OTUD1 and HK2. Finally, the correlation between OTUD1 and SAE was evaluated by analyzing clinical samples. Results Through single-cell RNA seq and subpopulation analysis, we identified an SAE-associated microglia (SAM) subpopulation with high expression of pyroptosis-related genes. Deubiquitinase expression analysis showed significantly elevated OTUD1 expression in SAM. OTUD1 deficiency attenuated neural damage and cognitive dysfunction in SAE mice in vivo. Further experiments revealed that OTUD1 regulates pyroptosis in microglia, affecting the progression of SAE. Mechanistically, OTUD1 directly binds to the C-terminal domain of HK2 through its Ala-rich domain and selectively cleaves K63-linked polyubiquitin chains on HK2 to promote the dissociation of HK2 from mitochondria, thereby activating the NLRP3 inflammasome and pyroptosis. Conclusions In SAE, OTUD1 deubiquitinates HK2, promoting its dissociation from mitochondria, which triggers microglia pyroptosis, leading to neuronal damage and cognitive impairment.