The ubiquitin E3 ligase TRIM59 in myeloid cell regulates complement C3 through degrading CCAAT/enhancer binding protein β to promote neuron survival after ischemic stroke

BACKGROUND: Ischemic stroke is a cerebrovascular disease that activates the complement system, leading to inflammation and neuronal injury. TRIM59, an E3 ubiquitin ligase, has been extensively studied in cancer, sepsis, and myocardial infarction, and is upregulated in macrophages after ischemic stroke. This study used myeloid cell Trim59 conditional knockout and macrophage-specific Trim59 overexpression mice to investigate the role of macrophage TRIM59 in regulating complement C3 release and neuronal phagocytosis following cerebral ischemia. METHODS: Myeloid cell Trim59 conditional knockout (Trim59-cKO) mice were generated and bred using the Cre-loxP system, and macrophage-specific Trim59 overexpressing (AAV-Trim59) mice were constructed via intra-bone marrow injection. Middle cerebral artery occlusion/reperfusion (MCAO/R) and photothrombotic (PT) surgery were used to mimic the cerebral ischemia in mice. RT-qPCR, ELISA and immunofluorescence staining were performed to detect the expression of complement molecules and synaptic engulfment. Neurobehavioral tests were performed during recovery phase. Western blot and co-immunoprecipitation (Co-IP) assay were utilized to analyze the effect of TRIM59 on C3 expression and its transcription factor CCAAT/enhancer binding protein β (c/EBPβ). RESULTS: mice, Trim59-cKO promoted excessive expression and release of complement components C3 and C9 in macrophages after cerebral ischemia, resulting in aggravated pathological injury and reduced neuronal survival. In contrast, macrophage-specific TRIM59 overexpression reversed the above results and alleviated acute-phase injury. Furthermore, deficiency of Trim59 led to excessive macrophage-derived C3 deposition around neurons, enhanced phagocytic activity, and aggravated synaptic loss during the acute phase after MCAO/R injury, ultimately causing delayed recovery of cerebral blood flow, impaired learning and memory function, and lower survival rates during the chronic phase after PT induced ischemic stroke. The transcription factor c/EBPβ was identified as a key mediator of macrophage TRIM59-dependent regulation of C3 expression, verified by silencing of c/EBPβ in Trim59 knockout macrophages could attenuate the neuronal C3 deposition and synaptic damage. Further research shows that TRIM59 promotes K48-linked ubiquitination and proteasomal degradation of c/EBPβ through its RING domain. CONCLUSION: Macrophage TRIM59 is critical for controlling excessive complement C3 activation, release, and phagocytosis after ischemic stroke by promoting ubiquitin-mediated degradation of its transcription factor c/EBPβ, providing a potential therapeutic target for cerebral ischemia.