Microglial NEDD4L activates mtDNA-cGAS-STING pathway and promotes neuroinflammation after intracerebral hemorrhage through impairing mitophagy

Background: The resultant microglial activation and sustained neuroinflammatory response are key contributors to secondary injury, promoting blood-brain barrier disruption, cerebral edema, and progressive neuronal loss. Although NEDD4L has been implicated in inflammation and immunity, the functions of NEDD4L in acute brain injury and microglia-specific responses remain largely unknown in intracerebral hemorrhage( ICH). Methods: Using proteomic sequencing, single-cell RNA sequencing, Immunofluorescence Staining. flow cytometry analysis, we investigated cellular distribution and expression of NEDD4L in ICH. Genetically engineered mouse models, neurological function testing, flow cytometry analysis, engineering extracellular vesicle (EV) isolation and TUNEL staining, we investigated the role of NEDD4L in ICH. Protein interactions was used for mechanism analysis. Results: We identified a substantial upregulation of the ubiquitin ligase NEDD4L in isolated microglia following ICH, and demonstrated that its increase is mediated by STAT2-dependent transcriptional mechanisms. Microglial subpopulations with high NEDD4L expression exhibit a pronounced pro-inflammatory phenotype. Moreover, knockout of NEDD4L specifically in microglia conferred neuroprotection and attenuated innate immune responses by the cytosolic DNA-sensing pathway. Mechanistically, NEDD4L promotes the ubiquitination of Parkin, resulting in the leakage of mitochondrial DNA into the cytoplasm and subsequent activation of the cGAS–Sting inflammatory cascade. Engineered extracellular vesicles of NEDD4L-specific siRNA successfully mitigated neuroinflammation, tissue damage, and functional deficits—in both ICH and TBI mouse models. Conclusions: we unveil a novel role for NEDD4L in modulating microglia-driven neuroinflammation after acute brain injury and highlight its function in activating the mtDNA–cGAS–Sting signaling axis.