Multifaceted functions of Drp1 in hypoxia/ischemia- induced mitochondrial quality imbalance: from regulatory mechanism to targeted therapeutic strategy

Hypoxic-ischemic injury is a common pathological dysfunction in clinical settings.Mitochondria are sensitive organelles that are readily damaged following ischemia and hypoxia.Dynamin-related protein 1(Drp1)regulates mitochondrial quality and cellular functions via its oligomeric changes and multiple modifications,which plays a role in mediating the induction of multiple organ damage during hypoxic-ischemic injury.However,there is active controversy and gaps in knowledge regarding the modification,protein interaction,and functions of Drp1,which both hinder and promote development of Drp1 as a novel therapeutic target.Here,we summarize recent findings on the oligomeric changes,modification types,and protein interactions of Drp1 in various hypoxic-ischemic diseases,as well as the Drp1-mediated regulation of mitochondrial quality and cell functions following ischemia and hypoxia.Additionally,potential clinical translation prospects for targeting Drp1 are discussed.This review provides new ideas and targets for proactive interventions on multiple organ damage induced by various hypoxic-ischemic diseases.

Electroacupuncture preconditioning protects against focal cerebral ischemia/reperfusion injury via suppression of dynamin-related protein 1

Electroacupuncture preconditioning at acupoint Baihui （GV20） can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 （Drp1） can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 （depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day）. Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.

PTEN-induced kinase 1-induced dynamin-related protein 1 Ser637 phosphorylation reduces mitochondrial fission and protects against intestinal ischemia reperfusion injury

BACKGROUND Intestinal ischemia reperfusion(I/R)occurs in various diseases,such as trauma and intestinal transplantation.Excessive reactive oxygen species(ROS)accumulation and subsequent apoptotic cell death in intestinal epithelia are important causes of I/R injury.PTEN-induced putative kinase 1(PINK1)and phosphorylation of dynamin-related protein 1(DRP1)are critical regulators of ROS and apoptosis.However,the correlation of PINK1 and DRP1 and their function in intestinal I/R injury have not been investigated.Thus,examining the PINK1/DRP1 pathway may help to identify a protective strategy and improve the patient prognosis.AIM To clarify the mechanism of the PINK1/DRP1 pathway in intestinal I/R injury.METHODS Male C57BL/6 mice were used to generate an intestinal I/R model via superior mesenteric artery occlusion followed by reperfusion.Chiu’s score was used to evaluate intestinal mucosa damage.The mitochondrial fission inhibitor mdivi-1 was administered by intraperitoneal injection.Caco-2 cells were incubated in vitro in hypoxia/reoxygenation conditions.Small interfering RNAs and overexpression plasmids were transfected to regulate PINK1 expression.The protein expression levels of PINK1,DRP1,p-DRP1 and cleaved caspase 3 were measured by Western blotting.Cell viability was evaluated using a Cell Counting Kit-8 assay and cell apoptosis was analyzed by TUNEL staining.Mitochondrial fission and ROS were tested by MitoTracker and MitoSOX respectively.RESULTS Intestinal I/R and Caco-2 cell hypoxia/reoxygenation decreased the expression of PINK1 and p-DRP1 Ser637.Pretreatment with mdivi-1 inhibited mitochondrial fission,ROS generation,and apoptosis and ameliorated cell injury in intestinal I/R.Upon PINK1 knockdown or overexpression in vitro,we found that p-DRP1 Ser637 expression and DRP1 recruitment to the mitochondria were associated with PINK1.Furthermore,we verified the physical combination of PINK1 and p-DRP1 Ser637.CONCLUSION PINK1 is correlated with mitochondrial fission and apoptosis by regulating DRP1 phosphorylation in intestinal I/R.These results suggest that the PINK1/DRP1 pathway is involved in intestinal I/R injury,and provide a new approach for prevention and treatment.

Mitochondrial components transferred by MSC-derived exosomes promoted bone regeneration under high salt microenvironment via DRP1/Wnt signaling

Bone homeostasis relies on the dynamic balance of osteoblast mediated bone construction and osteoclast-based bone resorption processes,which has been reported to be controlled by various mineral ions.However,there is no direct evidence of the effect and the underlying mechanism of high salt stimulation on bone metabolism.In this study,we demonstrated that high salt stimulation promoted excessive mitochondrial fission mediated by dynamin-related protein 1 in mesenchymal stem cells,which resulted in impaired mitochondrial morphology and function.Consequently,this impairment hindered the bone formation of mesenchymal stem cells,resulting in osteopenia in mice.Mechanically,the impaired property of mesenchymal stem cells which was caused by high salt was controlled by dynamin-related protein 1 mediated mitochondrial fission,which inhibited the classical Wnt signaling pathway.Furthermore,the osteogenic property of mesenchymal stem cells decreased by high salt could be restored by exosomes to transfer the mitochondrial DNA into the impaired mesenchymal stem cells.This study provides not only new strategies for promoting bone regeneration but also new insights into the effect and mechanism of exosome-mediated delivery.

Hypoxia promotes pulmonary vascular remodeling via HIF-1α to regulate mitochondrial dynamics

Background Increasing research suggests that mitochondrial defect plays a major role in pulmonary hypertension(PH) pathogenesis. Mitochondrial dynamics and quality control have a central role in the maintenance of the cell proliferation and apoptosis balance. However, the molecular mechanism underlying of this balance is still unknown. Methods To clarify the biological effects of hypoxic air exposure and hypoxia-inducible factor-1α(HIF-1α) on pulmonary arterial smooth muscle cell(PASMC) and pulmonary arterial hypertension rats, the cells were cultured in a hypoxic chamber under oxygen concentrations. Cell viability, reactive oxygen species level, cell death, mitochondrial morphology, mitochondrial membrane potential, mitochondrial function and mitochondrial biosynthesis, as well as fission-and fusion-related proteins, were measured under hypoxic conditions. In addition, rats were maintained under hypoxic conditions, and the right ventricular systolic pressure, right ventricular hypertrophy index and right ventricular weight/body weight ratio were examined and recorded. Further, we assessed the role of HIF-1α in the development and progression of PH using HIF-1α gene knockdown using small interfering RNA transfection. Mdivi-1 treatment was performed before hypoxia to inhibit dynamin-related protein 1(Drp1). Results We found that HIF-1α expression was increased during hypoxia, which was crucial for hypoxia-induced mitochondrial dysfunction and hypoxia-stimulated PASMCs proliferation and apoptosis. We also found that targeting mitochondrial fission Drp1 by mitochondrial division inhibitor Mdivi-1 was effective in PH model rats. The results showed that mitochondrial dynamics were involved in the pulmonary vascular remodeling under hypoxia in vivo and in vitro. Furthermore, HIF-1α also modulated mitochondrial dynamics in pulmonary vascular remodeling under hypoxia through directly regulating the expression of Drp1. Conclusions In conclusion, our data suggests that abnormal mitochondrial dynamics could be a marker for the early diagnosis of PH and monitoring disease progression. Further research is needed to study the signaling pathways that govern mitochondrial fission/fusion in PH.

Dynamin-related protein 1在线粒体分裂和细胞凋亡中的作用

Dynamin-related protein 1属于动力蛋白GTP酶超家族,是线粒体分裂体系的组成成分,在线粒体分裂中具有重要作用。在不同物种中,dynamin-related protein 1在与多种分子相互作用后,可以定位于线粒体并组装成高级结构,引起膜的收缩和分裂。Dynamin-related protein 1功能的消失会增强线粒体的融合和线粒体之间的连通性。Dynamin-related protein 1在细胞凋亡等多种细胞功能中也具有重要作用。

运用酵母双杂交系统筛选肌营养不良相关蛋白2(DRP2)相互作用蛋白(英文)

目的应用酵母双杂交系统筛选小鼠、大鼠、人类大脑文库中与DRP2相互作用的蛋白,分析中枢神经系统中DRP2分子复合体的组成成分。方法设计3个含有DRP2不同蛋白结构域的诱饵质粒,在完成诱饵质粒的自激活性的鉴定后,对小鼠、大鼠、人类脑MATCHMAKER cDNA文库进行筛选。结果运用DRP2蛋白N-Bait筛选大鼠脑MATCHMAKER cDNA文库,笔者获得了一个阳性文库质粒。该质粒的插入子编码一种SNAP25相互作用的蛋白质Scoilin。Scoilin又被命名为Zwint-1蛋白。同时笔者还确定了这两种相互作用蛋白质的精确结构域:DRP2蛋白以其含有两个Spectrin重复和一个WW蛋白结构域的氨基端与Scoilin(Zwint-1)全蛋白相互作用。结论应用酵母双杂交,笔者于大鼠大脑中找到了DRP2的相互作用的蛋白质Scoilin(Zwint-1),并且确定了这两种相互作用蛋白质的精确结构域。

带有caspase富集功能域的凋亡抑制因子对急性病毒性心肌炎小鼠心肌的保护作用

目的研究急性病毒性心肌炎(AVMC)小鼠的带有caspase富集功能域的凋亡抑制因子(ARC)的表达及对心肌的保护。方法将60只成功建立AVMC模型的小鼠随机分为ARC反义寡核苷酸组(A组)、ARC错义寡核苷酸组(B组)和生理盐水对照组(C组),每组20只。分期分别采集标本测定外周血心肌钙蛋白T(cTnT)、心肌钙蛋白I(cTnⅠ)、肌酸激酶(CK)、肌酸激酶同功酶(CK-MB)水平和ARC、动力相关蛋白1(Drp1)表达等数据。结果造模后第7、14、21天,3组外周血cTnT、cTnⅠ、CK、CK-MB水平差异有统计学意义(P<0.05),C组与B组相近(P>0.05),但均低于A组(P<0.05)。造模后第7天,3组ARC与Drp1蛋白表达水平差异有统计学意义(P<0.05),C组ARC表达与B组相近(P>0.05),但均高于A组(P<0.05),C组Drp1蛋白表达与B组相近(P>0.05),但均低于A组(P<0.05);造模后第14、21天3组ARC与Drp1蛋白表达水平差异无统计学意义(P>0.05)。结论小鼠发生AVMC后高表达的ARC可抑制心肌细胞凋亡,对心肌组织有保护作用。

线粒体动力学调控对肾缺血-再灌注损伤的影响

缺血-再灌注损伤(IRI)是肾移植术后早期移植肾功能障碍的主要原因之一。我国器官移植已经进入公民逝世后器官捐献时代,供者心肺复苏风险增加、低灌注时间和热缺血时间延长等可导致移植肾IRI,影响受者和移植肾的近期及远期临床结局。在IRI等应激状态下,以线粒体分裂和融合的动态调控为主要表现的线粒体动力学机制对线粒体的生物学功能具有重要影响,其损伤引发的细胞凋亡是导致急性肾损伤的关键环节,主要表现为线粒体动力学调控机制失调。本文综述了线粒体动力学调控对肾IRI影响机制的研究进展,以期为改善肾移植临床结局提供参考。

p66Shc-mediated oxidative stress is involved in gestational diabetes mellitus

BACKGROUND Gestational diabetes mellitus(GDM)is associated with a heightened level of oxidative stress,which is characterized by the overproduction of reactive oxygen species(ROS)from mitochondria.Previous studies showed that mitochondrial dysfunction is regulated by dynamin-related protein 1(Drp1)and p66Shc in GDM.AIM The aim was to investigate the expression of Drp1 and p66Shc and their possible mechanisms in the pathogenesis of GDM.METHODS A total of 30 pregnant women,15 with GDM and 15 without GDM,were enrolled.Peripheral blood mononuclear cells and placental tissue were collected.The human JEG3 trophoblast cell line was cultivated in 5.5 mmol/L or 30 mmol/L glucose and transfected with wild-type(wt)-p66Shc and p66Shc siRNA.P66Shc and Drp1 mRNA levels were detected by quantitative real-time polymerase chain reaction.The expression of p66Shc and Drp1 was assayed by immunohistochemistry and western blotting.ROS was assayed by dihydroethidium staining.RESULTS The p66Shc mRNA level was increased in the serum(P<0.01)and placentas(P<0.01)of women with GDM,and the expression of Drp1 mRNA and protein were also increased in placentas(P<0.05).In JEG3 cells treated with 30 mmol/L glucose,the mRNA and protein expression of p66Shc and Drp1 were increased at 24 h(both P<0.05),48 h(both P<0.01)and 72 h(both P<0.001).ROS expression was also increased.High levels of Drp1 and ROS expression were detected in JEG3 cells transfected with wt-p66Shc(P<0.01),and low levels were detected in JEG3 cells transfected with p66Shc siRNA(P<0.05).CONCLUSION The upregulated expression of Drp1 and p66shc may contribute to the occurrence and development of GDM.Regulation of the mitochondrial fusion-fission balance could be a novel strategy for GDM treatment.

Aldehyde dehydrogenase 2 preserves mitochondrial morphology and attenuates hypoxia/reoxygenationinduced cardiomyocyte injury

BACKGROUND:Disturbance of mitochondrial fi ssion and fusion(termed mitochondrial dynamics)is one of the leading causes of ischemia/reperfusion(I/R)-induced myocardial injury.Previous studies showed that mitochondrial aldehyde dehydrogenase 2(ALDH2)conferred cardioprotective effect against myocardial I/R injury and suppressed I/R-induced excessive mitophagy in cardiomyocytes.However,whether ALDH2 participates in the regulation of mitochondrial dynamics during myocardial I/R injury remains unknown.METHODS:In the present study,we investigated the effect of ALDH2 on mitochondrial dynamics and the underlying mechanisms using the H9c2 cells exposed to hypoxia/reoxygenation(H/R)as an in vitro model of myocardial I/R injury.RESULTS:Cardiomyocyte apoptosis was significantly increased after oxygen-glucose deprivation and reoxygenation(OGD/R),and ALDH2 activation largely decreased the cardiomyocyte apoptosis.Additionally,we found that both ALDH2 activation and overexpression significantly inhibited the increased mitochondrial fission after OGD/R.Furthermore,we found that ALDH2 dominantly suppressed dynamin-related protein 1(Drp1)phosphorylation(Ser616)and adenosine monophosphate-activated protein kinase(AMPK)phosphorylation(Thr172)but not interfered with the expression levels of mitochondrial shaping proteins.CONCLUSIONS:We demonstrate the protective effect of ALDH2 against cardiomyocyte H/R injury with a novel mechanism on mitochondrial fission/fusion.

Rescue axonal defects by targeting mitochondrial dynamics in hereditary spastic paraplegias

Impaired axonal development and degeneration underlie debilitating neurodegenerative diseases including hereditary spastic paraplegia, a large group of inherited diseases. Hereditary spastic paraplegia is caused by retrograde degeneration of the long corticospinal tract axons, leading to progressive spasticity and weakness of leg and hip muscles. There are over 70 subtypes with various underlying pathophysiological processes, such as defective vesicular trafficking, lipid metabolism, organelle shaping, axonal transport, and mitochondrial dysfunction. Although hereditary spastic paraplegia consists of various subtypes with different pathological characteristics, defects in mitochondrial morphology and function emerge as one of the common cellular themes in hereditary spastic paraplegia. Mitochondrial morphology and function are remodeled by mitochondrial dynamics regulated by several key fission and fusion mediators. However, the role of mitochondrial dynamics in axonal defects of hereditary spastic paraplegia remains largely unknown. Recently, studies reported perturbed mitochondrial morphology in hereditary spastic paraplegia neurons. Moreover, downregulation of mitochondrial fission regulator dynamin-related protein 1, both pharmacologically and genetically, could rescue axonal outgrowth defects in hereditary spastic paraplegia neurons, providing a potential therapeutic target for treating these hereditary spastic paraplegia. This mini-review will describe the regulation of mitochondrial fission/fusion, the link between mitochondrial dynamics and axonal defects, and the recent progress on the role of mitochondrial dynamics in axonal defects of hereditary spastic paraplegia.

中成药木丹颗粒对糖尿病大鼠坐骨神经干预作用的观察

目的探讨线粒体分裂相关蛋白(Drp-1)与糖尿病周围神经病变(DPN)的关系及木丹颗粒的干预作用。方法 50只大鼠随机分为糖尿病对照(DM)组、木丹颗粒(MD)组、α-硫辛酸(LA)组和木丹颗粒联合α-硫辛酸(CT)组,另设正常(NC)组。12周后检测各组坐骨神经传导速度(sNCV)、血清中总超氧化物歧化酶(T-SOD)、谷胱甘肽(GSH)、丙二醛(MDA)和过氧化氢酶(CAT)的水平及坐骨神经中Drp-1、Bax、Bcl-2mRNA相对表达量。结果 NC、DM、MD、LA、CT组Drp-1 mRNA相对表达量分别为(0.99±0.07)、(1.78±0.08)、(1.48±0.09)、(1.44±0.10)和(1.30±0.05);Bax mRNA相对表达量分别为(1.09±0.21)、(7.08±0.86)、(4.95±0.83)、(4.52±0.88)和(2.86±0.54);Bcl-2 mRNA相对表达量分别为(7.80±0.40)、(2.99±0.64)、(4.06±0.81)、(4.09±0.80)和(5.62±0.83)。与NC组比较,DM组T-SOD、GSH、CAT和sNCV降低,MDA升高(P<0.05);与DM组比较,MD、LA及CT组TSOD、GSH、CAT和sNCV升高,MDA降低(P<0.05),其中CT组变化更明显。结论 Drp-1表达升高可能参与DPN发病,木丹颗粒能干预Drp-1表达,减少细胞凋亡,并降低氧化应激水平,对糖尿病大鼠坐骨神经起到一定的保护作用。

Plant Peroxisome Multiplication:Highly Regulated and Still Enigmatic

Plant peroxisomes play a key role in numerous physiological processes and are able to adapt to environmental changes by altering their content, morphology, and abundance. Peroxisomes can multiply through elongation, constriction, and fission; this process requires the action of conserved, as well as species-specific proteins. Genetic and morphological analyses have been used with the model plant Arabidopsis thaliana to determine at the mechanistic level how plant peroxisomes increase their abundance. The five-member PEXll family promotes early steps of peroxisome multiplication with an unknown mechanism and some subfamily specificities. The dynamin-related protein （DRP）3 subfamily of dynaminrelated large guanosine triphosphatases mediates late steps of both peroxisomal and mitochondrial multiplication. New genetic and biochemical tools will be needed to identify additional, especially plant-specific, constituents of the peroxisome multiplication pathways.