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Associations between Huwe1 and autophagy in rat cerebral neuron oxygen?glucose deprivation and reperfusion injury.

ABSTRACT: Autophagy and the ubiquitin proteasome system (UPS) are two major protein degradation pathways involved in brain ischemia. Autophagy can compensate for UPS impairment?induced cellular dysfunction. HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1 (Huwe1), an E3 ubiquitin ligase, serves critical roles in nervous system plasticity, regeneration and disease. However, the role of Huwe1 in autophagy in brain ischemia/reperfusion (I/R) injury remains unknown. The aim of the present study was to investigate the crosstalk between autophagy and the UPS in brain ischemia. The present study established an oxygen?glucose deprivation and reperfusion (OGD/R) model in rat primary cortex neurons in vitro. Lentiviral interference was used to silence the expression of Huwe1. An autophagy promoter (rapamycin), an autophagy inhibitor (wortmannin) and a JNK pathway inhibitor (SP600125) were also used in the current study. Cellular autophagy?related proteins, including Beclin?1, autophagy related (ATG) 7, ATG5, ATG3 and microtubule associated protein 1 light chain 3 ?, and apoptosis?related proteins, such as P53, cleaved caspase 3, Bax and Bcl2, were detected via western blotting and immunocytochemistry. Neuronal apoptosis was evaluated using a TUNEL assay. The results demonstrated that silencing Huwe1 increased the expression levels of autophagy?related proteins at 24 h after OGD/R. Treatment with a JNK inhibitor or cotreatment with Huwe1 shRNA significantly increased autophagy. Rapamycin increased apoptosis under OGD/R conditions. However, treatment with Huwe1 shRNA decreased the number of TUNEL?positive cells at 24 h after OGD/R. Cotreatment with Huwe1 shRNA and wortmannin alleviated neuronal apoptosis under OGD/R conditions compared with cotreatment with DMSO. Collectively, the present results suggested that silencing Huwe1 was accompanied by a compensatory induction of autophagy under OGD/R conditions. Furthermore, the JNK pathway may be a key mediator of the interaction between Huwe1 and autophagy in response to UPS impairment.

SUBMITTER: He GQ

PROVIDER: S-EPMC7646962 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Associations between Huwe1 and autophagy in rat cerebral neuron oxygen‑glucose deprivation and reperfusion injury.

He Guo-Qian GQ Chen Yan Y Liao Hui-Juan HJ Xu Wen-Ming WM Zhang Wei W He Guo-Lin GL

Molecular medicine reports 20201019 6

Autophagy and the ubiquitin proteasome system (UPS) are two major protein degradation pathways involved in brain ischemia. Autophagy can compensate for UPS impairment‑induced cellular dysfunction. HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1 (Huwe1), an E3 ubiquitin ligase, serves critical roles in nervous system plasticity, regeneration and disease. However, the role of Huwe1 in autophagy in brain ischemia/reperfusion (I/R) injury remains unknown. The aim of the present stu ...[more]

PMID: 33173969

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Project description: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).

Project description:Shuxuetong injection composed of leech (Hirudo nipponica Whitman) and earthworm (Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells (bEnd.3) were incubated in glucose-free Dulbecco's modified Eagle's medium containing 95% N2/5% CO2 for 6 hours, followed by high-glucose medium containing 95% O2 and 5% CO2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 concentrations (diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier permeability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1?, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspase-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-?B p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula occludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspase-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1?, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 expression, reduces nuclear factor-?B p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular signal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest that Shuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascular endothelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-?B p65 signaling pathway.

Project description:The neuroprotective role of propofol in transient global and focal cerebral ischemia reperfusion (I/R) animal model has recently been highlighted. However, no studies have conducted to explore the relationship between mitochondrial fission/fusion and I/R injury under the intervention of propofol. Moreover, neuroprotective mechanism of propofol is yet unclear. Culturing primary hippocampal cells were subjected to oxygen-glucose deprivation and re-oxygenation (OGD/R) model, as a model of cerebral I/R in vitro. Methods CCK-8 assay was used to test the effect of propofol on cell viability. We examined the effect of propofol on mitochondrial ultrastructure and mitochondrial fission evoked by OGD/R with transmission electron microscopy and immunofluorescence assay. To investigate possible neuroprotective mechanisms, the authors then examined whether propofol could inhibit calcium-overload, calcineurin (CaN) activation and the phosphorylation of dynamin-related protein 1 (Drp1) during the period of OGD/R, as well as the combination of Drp1-ser 637 and fission 1 (Fis1) protein by immunofluorescence assay, ELISA and double-labeling immunofluorescence analysis. Finally, the expression of Drp1-ser 637 and Fis1, apoptosis inducing factor (AIF) and cytochrome C (Cyt C) were detected by western blot. When added in culture media during OGD period, propofol (0.1?M-50?M) could alleviate neurons injury and protect mitochondrial ultrastructure, meanwhile inhibit mitochondrial fission. Furthermore, the concentration of intracellular free Ca2+, CaN activition and the phosphorylation of Drp1-ser637 were suppressed, as well as the translocation and combination of Drp1-ser 637 and Fis1. The authors also found that the expression of Cyt C, AIF, Drp1-ser637 and Fis1 were down-regulated. Notably, high dose of propofol (100?M-200?M) were confirmed to decrease the survival of neurons based on results of cell viability. Propofol could inhibit mitochondrial fission and mitochondrial apoptotic pathway evoked by OGD/R in rat hippocampal neurons, which may be via depressing calcium-overload.

Project description:Endothelial dysfunction, referring to a disturbance in the vascular homeostasis, has been implicated in many disease conditions including ischemic/reperfusion injury and atherosclerosis. Endothelial mitochondria have been increasingly recognized as a regulator of calcium homeostasis which has implications in the execution of diverse cellular events and energy production. The mitochondrial calcium uniporter complex through which calcium enters the mitochondria is composed of several proteins, including the pore-forming subunit MCU and its regulators MCUR1, MICU1, and MICU2. Mitochondrial calcium overload leads to opening of MPTP (mitochondrial permeability transition pore) and results in apoptotic cell death. Whereas, blockage of calcium entry into the mitochondria results in reduced ATP production thereby activates AMPK-mediated pro-survival autophagy. Here, we investigated the expression of mitochondrial calcium uniporter complex components (MCU, MCUR1, MICU1, and MICU2), induction of autophagy and apoptotic cell death in endothelial cells in response to oxygen-glucose deprivation. Human pulmonary microvascular endothelial cells (HPMVECs) were subjected to oxygen-glucose deprivation (OGD) at 3-h timepoints up to 12 h. Interestingly, except MCUR1 which was significantly downregulated, all other components of the uniporter (MCU, MICU1, and MICU2) remained unchanged. MCUR1 downregulation has been shown to activate AMPK mediated pro-survival autophagy. Similarly, MCUR1 downregulation in response to OGD resulted in AMPK phosphorylation and LC3 processing indicating the activation of pro-survival autophagy. Despite the activation of autophagy, OGD induced Caspase-mediated apoptotic cell death. Blockade of autophagy did not reduce OGD-induced apoptotic cell death whereas serum starvation conferred enough cellular and functional protection. In conclusion, the autophagic flux induced by MCUR1 downregulation in response to OGD is insufficient in protecting endothelial cells from undergoing apoptotic cell death and requires enhancement of autophagic flux by additional means such as serum starvation.

Dataset Information

Associations between Huwe1 and autophagy in rat cerebral neuron oxygen?glucose deprivation and reperfusion injury.

Publications

Associations between Huwe1 and autophagy in rat cerebral neuron oxygen‑glucose deprivation and reperfusion injury.

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