Neural Regeneration Research ›› 2019, Vol. 14 ›› Issue (11): 1977-1985.doi: 10.4103/1673-5374.259620

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Silencing Huwe1 reduces apoptosis of cortical neurons exposed to oxygen-glucose deprivation and reperfusion

Guo-Qian He 1 , Wen-Ming Xu 2 , Hui-Juan Liao 2 , Chuan Jiang 2 , Chang-Qing Li 3 , Wei Zhang 4   

  1. 1 Department of Pediatrics, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, Sichuan Province, China
    2 Joint Laboratory of Reproductive Medicine, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, Sichuan Province, China
    3 Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
    4 Department of Medical Oncology, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, Cancer Hospital Affiliated to School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan Province, China
  • Online:2019-11-15 Published:2019-11-15
  • Contact: Wei Zhang, MD, zw493493@163.com.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81771642 (to WMX); the New Bud Research Foundation of West China Second University Hospital of China (to GQH).

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Abstract:

HECT, UBA and WWE domain-containing 1 (Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase (JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor (SP600125) or a p38MAPK inhibitor (SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38MAPK and of apoptosis-related proteins (p53, Gadd45a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018 (approval No. 2018013).

Key words: nerve regeneration, ischemic stroke, oxygen-glucose deprivation and reperfusion, ischemia/reperfusion, cortical neuron, ubiquitin proteasome system, Huwe1, apoptosis, therapeutic targets, cell culture, cell death, neural regeneration