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CCDC50 Alleviates Cerebral Ischemia-Reperfusion Injury via Enhancing Autophagy to Inhibit STING/IRF3-Mediated
Chunyan Lei1, Juan Yang2, Peng Bai3
1Department of First Neurology, The First Affiliated Hospital of Kunming Medical University, 295 Xichang Road, Kunming City, Yunnan Province, 650032, China.
Abstract:
Ischemia-reperfusion injury (IRI) is a major reason for adverse prognosis in ischemic stroke, with multiple signaling pathways involved in this process. This study aims to explore the role of the novel autophagy receptor CCDC50 in cerebral IRI and its molecular mechanism. Cellular oxygen-glucose deprivation/reoxygenation model and middle cerebral artery occlusion (MCAO) animal model were adopted in this study. Quantitative real-time polymerase chain reaction, Western blot, immunofluorescence, and immunohistochemistry assays were applied to detect the expression and subcellular localization of key molecules including LL37, CCDC50, STING, IRF3, Rspondin3, and autophagy-related proteins. Co-immunoprecipitation was performed to explore protein-protein interactions. Enzyme-linked immunosorbent assay and flow cytometry were utilized to determine the levels of inflammatory cytokines and the M1/M2 polarization phenotypes of microglia. Additionally, 2,3,5-triphenyltetrazolium chloride staining was carried out to measure cerebral infarct volume. Results revealed that LL37 directly bound to CCDC50 and facilitated its ubiquitination and degradation. This process disrupted the interaction between CCDC50 and STING, thereby triggering the activation of the STING/IRF3 signaling pathway. Conversely, CCDC50 enhanced autophagy and restrained the STING/IRF3 pathway activation. It further drove microglial polarization toward the anti-inflammatory M2 phenotype, blocked pro-inflammatory M1 phenotype transition and inflammatory cytokine secretion, and ultimately alleviated neuronal damage. The above in vitro molecular mechanisms were also validated in the MCAO model. By enhancing autophagy, CCDC50 suppresses the STING/IRF3 pathway and facilitates M2 polarization of microglia, ultimately mitigating cerebral ischemia-reperfusion injury. In contrast, LL37 counteracts this protective effect by mediating CCDC50 degradation. This study identifies novel potential therapeutic targets for ischemic stroke.
Insights
The autophagy receptor CCDC50 protects against brain damage from ischemia-reperfusion injury (IRI) by enhancing autophagy and suppressing inflammation. The protein LL37 counteracts this effect, offering potential therapeutic targets for ischemic stroke.
Area of Science:
- Neuroscience
- Cell Biology
- Immunology
Background:
- Ischemia-reperfusion injury (IRI) significantly worsens outcomes in ischemic stroke.
- Multiple signaling pathways mediate the complex processes involved in cerebral IRI.
- The role of the autophagy receptor CCDC50 in cerebral IRI remains largely unexplored.
Purpose of the Study:
- To investigate the function of the novel autophagy receptor CCDC50 in cerebral IRI.
- To elucidate the molecular mechanisms underlying CCDC50's role in IRI.
- To identify potential therapeutic targets for mitigating stroke-related brain damage.
Main Methods:
- Established cellular oxygen-glucose deprivation/reoxygenation and middle cerebral artery occlusion (MCAO) animal models.
- Utilized quantitative real-time PCR, Western blot, immunofluorescence, and immunohistochemistry to assess protein expression and localization.
- Employed co-immunoprecipitation, ELISA, flow cytometry, and TTC staining to analyze protein interactions, inflammatory markers, microglial polarization, and infarct volume.
Main Results:
- LL37 directly binds and degrades CCDC50, disrupting CCDC50-STING interaction and activating the STING/IRF3 pathway.
- CCDC50 enhances autophagy, suppresses the STING/IRF3 pathway, and promotes M2 microglial polarization.
- CCDC50 alleviates neuronal damage by reducing inflammation and M1 microglial phenotype, an effect counteracted by LL37.
Conclusions:
- CCDC50 exerts a protective role in cerebral IRI by enhancing autophagy and suppressing the STING/IRF3 pathway.
- LL37 antagonizes CCDC50's protective effects through protein degradation, highlighting a novel regulatory axis.
- Targeting the CCDC50-LL37 interaction presents a promising therapeutic strategy for ischemic stroke.