Assocation Between Leber's Hereditary Optic Neuropathy and MT-ND1 3460G>A Mutation-Induced Alterations in Mitochondrial Function,Apoptosis, and Mitophagy

PurposeTo investigate the molecular mechanism underlying the Leber''s hereditary optic neuropathy (LHON)-linked MT-ND1 3460G>A mutation.MethodsCybrid cell models were generated by fusing mitochondrial DNA-less ρ⁰ cells with enucleated cells from a patient carrying the m.3460G>A mutation and a control subject. The impact of m.3460G>A mutations on oxidative phosphorylation was evaluated using Blue Native gel electrophoresis, and measurements of oxygen consumption were made with an extracellular flux analyzer. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Assays for apoptosis and mitophagy were undertaken via immunofluorescence analysis.ResultsNineteen Chinese Han pedigrees bearing the m.3460G>A mutation exhibited variable penetrance and expression of LHON. The m.3460G>A mutation altered the structure and function of MT-ND1, as evidenced by reduced MT-ND1 levels in mutant cybrids bearing the mutation. The instability of mutated MT-ND1 manifested as defects in the assembly and activity of complex I, respiratory deficiency, diminished mitochondrial adenosine triphosphate production, and decreased membrane potential, in addition to increased production of mitochondrial ROS in the mutant cybrids carrying the m.3460G>A mutation. The m.3460G>A mutation mediated apoptosis, as evidenced by the elevated release of cytochrome c into the cytosol and increasing levels of the apoptotic-associated proteins BAK, BAX, and PARP, as well as cleaved caspases 3, 7, and 9, in the mutant cybrids. The cybrids bearing the m.3460G>A mutation exhibited reduced levels of autophagy protein light chain 3, accumulation of autophagic substrate P62, and impaired PTEN-induced kinase 1/parkin-dependent mitophagy.ConclusionsOur findings highlight the critical role of m.3460G>A mutation in the pathogenesis of LHON, manifested by mitochondrial dysfunction and alterations in apoptosis and mitophagy.     

Nonstructural Protein NS1 of Influenza Virus Disrupts Mitochondrial Dynamics and Enhances Mitophagy via ULK1 and BNIP3

Nonstructural protein 1 (NS1) of influenza virus (IFV) is essential for evading interferon (IFN)-mediated antiviral responses, thereby contributing to the pathogenesis of influenza. Mitophagy is a type of autophagy that selectively removes damaged mitochondria. The role of NS1 in IFV-mediated mitophagy is currently unknown. Herein, we showed that overexpression of NS1 protein led to enhancement of mitophagy. Mitophagy induction via carbonyl cyanide 3-chlorophenylhydrazone treatment in IFV-infected A549 cells led to increased viral replication efficiency, whereas the knockdown of PTEN-induced kinase 1 (PINK1) led to the opposite effect on viral replication. Overexpression of NS1 protein led to changes in mitochondrial dynamics, including depolarization of mitochondrial membrane potential. In contrast, infection with NS1-deficient virus resulted in impaired mitochondrial fragmentation, subsequent mitolysosomal formation, and mitophagy induction, suggesting an important role of NS1 in mitophagy. Meanwhile, NS1 protein increased the phosphorylation of Unc-51-like autophagy activating kinase 1 (ULK1) and the mitochondrial expression of BCL2- interacting protein 3 (BNIP3), both of which were found to be important for IFV-mediated mitophagy. Overall, these data highlight the importance of IFV NS1, ULK1, and BNIP3 during mitophagy activation.     

Mechanisms and roles of mitophagy in neurodegenerative diseases

Mitochondria are double‐membrane‐encircled organelles existing in most eukaryotic cells and playing important roles in energy production, metabolism, Ca²⁺ buffering, and cell signaling. Mitophagy is the selective degradation of mitochondria by autophagy. Mitophagy can effectively remove damaged or stressed mitochondria, which is essential for cellular health. Thanks to the implementation of genetics, cell biology, and proteomics approaches, we are beginning to understand the mechanisms of mitophagy, including the roles of ubiquitin‐dependent and receptor‐dependent signals on damaged mitochondria in triggering mitophagy. Mitochondrial dysfunction and defective mitophagy have been broadly associated with neurodegenerative diseases. This review is aimed at summarizing the mechanisms of mitophagy in higher organisms and the roles of mitophagy in the pathogenesis of neurodegenerative diseases. Although many studies have been devoted to elucidating the mitophagy process, a deeper understanding of the mechanisms leading to mitophagy defects in neurodegenerative diseases is required for the development of new therapeutic interventions, taking into account the multifactorial nature of diseases and the phenotypic heterogeneity of patients.     

Mitochondrial transport serves as a mitochondrial quality control strategy in axons: Implications for central nervous system disorders

Axonal mitochondrial quality is essential for neuronal health and functions. Compromised mitochondrial quality, reflected by loss of membrane potential, collapse of ATP production, abnormal morphology, burst of reactive oxygen species generation, and impaired Ca²⁺ buffering capacity, can alter mitochondrial transport. Mitochondrial transport in turn maintains axonal mitochondrial homeostasis in several ways. Newly generated mitochondria are anterogradely transported along with axon from soma to replenish axonal mitochondrial pool, while damaged mitochondria undergo retrograde transport for repair or degradation. Besides, mitochondria are also arrested in axon to quarantine damages locally. Accumulating evidence suggests abnormal mitochondrial transport leads to mitochondrial dysfunction and axon degeneration in a variety of neurological and psychiatric disorders. Further investigations into the details of this process would help to extend our understanding of various neurological diseases and shed light on the corresponding therapies.     

异丙酚通过诱导幼鼠海马神经元线粒体自噬减轻其肝缺血再灌注后脑损伤

目的:探究异丙酚对肝缺血再灌注后幼鼠海马神经元线粒体自噬的影响。方法: C57BL/6两周龄的小鼠40只被随机分成4组（n=10）:假手术组(S组),肝缺血再灌注组（IR组）,异丙酚组（P组）,异丙酚+自噬抑制剂3-MA组（3-MA组）,除S组仅进行开关腹操作外,其余各组均行缺血60 min,再灌注6h建模。取血组织标本及海马组织标本。ELISA法检测血清中脑损伤标志物神经元特异性烯醇化酶（NSE）和S100β浓度及炎症因子白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）的浓度;透射电镜法观察自噬小体和线粒体超微结构;Western blot法检测自噬相关蛋白LC3Ⅱ、Nix及凋亡蛋白Caspase-3的表达水平。结果:（1）脑损伤标志物和炎症因子水平, 3-MA组最高,其次为IR组,P组居中,S组最低（P＜0.05）;（2）自噬小体,P组最多,且线粒体结构较完整,依次为IR和S组,3-MA组自噬最少,线粒体肿胀,分裂最多;（3）LC3Ⅱ、Nix蛋白表达水平同上述线粒体自噬水平一致,Caspase-3与此相反（P＜0.05）。结论:异丙酚可促进肝缺血再灌注后幼鼠海马神经元的线粒体自噬,减轻炎症反应,减少细胞凋亡。     

Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation

Mitophagy is a cellular quality control pathway in which the E3 ubiquitin ligase parkin targets damaged mitochondria for degradation by autophagosomes. We examined the role of optineurin in mitophagy, as mutations in optineurin are causative for amyotrophic lateral sclerosis (ALS) and glaucoma, diseases in which mitochondrial dysfunction has been implicated. Using live cell imaging, we demonstrate the parkin-dependent recruitment of optineurin to mitochondria damaged by depolarization or reactive oxygen species. Parkin’s E3 ubiquitin ligase activity is required to ubiquitinate outer mitochondrial membrane proteins, allowing optineurin to stably associate with ubiquitinated mitochondria via its ubiquitin binding domain; in the absence of parkin, optineurin transiently localizes to damaged mitochondrial tips. Following optineurin recruitment, the omegasome protein double FYVE-containing protein 1 (DFCP1) transiently localizes to damaged mitochondria to initialize autophagosome formation and the recruitment of microtubule-associated protein light chain 3 (LC3). Optineurin then induces autophagosome formation around damaged mitochondria via its LC3 interaction region (LIR) domain. Depletion of endogenous optineurin inhibits LC3 recruitment to mitochondria and inhibits mitochondrial degradation. These defects are rescued by expression of siRNA-resistant wild-type optineurin, but not by an ALS-associated mutant in the ubiquitin binding domain (E478G), or by optineurin with a mutation in the LIR domain. Optineurin and p62/SQSTM1 are independently recruited to separate domains on damaged mitochondria, and p62 is not required for the recruitment of either optineurin or LC3 to damaged mitochondria. Thus, our study establishes an important role for optineurin as an autophagy receptor in parkin-mediated mitophagy and demonstrates that defects in a single pathway can lead to neurodegenerative diseases with distinct pathologies.Damaged mitochondria are selectively turned over in eukaryotic cells via mitophagy, a process in which double-membraned autophagosomes sequester and ultimately degrade mitochondria via lysosomal fusion (1, 2). This process is regulated by phosphatase and tensin homolog-induced putative kinase protein 1 (PINK1) and parkin, two proteins linked to hereditary forms of Parkinson’s disease (3, 4). PINK1 is stabilized on the outer membrane of damaged mitochondria and recruits the E3 ubiquitin ligase parkin, which ubiquitinates proteins on the outer mitochondrial membrane (OMM) (5–13). Parkin-mediated ubiquitination of damaged mitochondria is followed by autophagosome formation and engulfment of mitochondria (1, 2). However, the proteins involved in dynamically recruiting autophagic machinery to ubiquitinated damaged mitochondria still remain elusive.Optineurin is an autophagy receptor, characterized by its ability to bind ubiquitin via its ubiquitin binding in ABIN (A20 binding and inhibitor of NF-κB) and NEMO (NF-κB essential modulator) (UBAN) domain (14) and the autophagosome-associated protein LC3 (microtubule-associated protein light chain 3) via its LC3 interacting region (LIR) domain (15). Optineurin regulates autophagosome maturation (16) and autophagic degradation of Salmonella and protein aggregates (15, 17). However, optineurin’s role in mitophagy has not been previously studied. Mutations in optineurin lead to primary open-angle glaucoma (18) and amyotrophic lateral sclerosis (ALS) (19), two neurodegenerative diseases in which mitochondrial defects have been observed (20, 21). Thus, optineurin may play a role in regulating the autophagic turnover of damaged mitochondria during mitophagy.Here, we use confocal live cell imaging to show that parkin is both necessary and sufficient to stabilize optineurin on the surface of damaged mitochondria. In the absence of parkin, optineurin puncta transiently localize to damaged mitochondria but do not remain stably associated. In cells expressing parkin, optineurin is recruited to mitochondria following parkin recruitment, and this recruitment is stabilized via the UBAN domain. Following optineurin recruitment, double FYVE-containing protein 1 (DFCP1) puncta transiently localize to parkin/optineurin-labeled damaged mitochondria to mark the initial site of autophagosome formation (22). This is followed by LC3 recruitment and subsequent autophagosome formation around optineurin-labeled damaged mitochondria. Importantly, we find that depletion of optineurin inhibits autophagosome recruitment to damaged mitochondria, leading to increased levels of the mitochondrial matrix protein Hsp60 and mtDNA content within cells. This defect in mitochondrial degradation is rescued by wild-type optineurin but not by the E478G UBAN mutant in optineurin causative for ALS (19) or by an optineurin LIR mutant unable to bind LC3 (15). Optineurin and p62, previously implicated in mitophagy (23–26), are independently recruited to distinct domains on damaged mitochondria. In contrast to our observations with optineurin, depletion of p62 did not inhibit LC3 recruitment or efficient degradation of damaged mitochondria. Thus, our study shows an important role for the autophagy receptor optineurin in parkin-mediated mitophagy and provides support for the hypothesis that defective mitochondrial quality control may contribute to ALS pathogenesis.     

中医药靶向调控线粒体质量控制系统治疗心肌缺血再灌注损伤研究进展

心肌缺血再灌注损伤（myocardial ischemia reperfusion injury,MIRI）是心梗后血流恢复再灌注诱发的一种疾病,以心肌细胞坏死、凋亡、线粒体功能障碍为主要病理特征,可能出现心肌顿抑、再灌注性心律失常和再发心绞痛等症状。线粒体是细胞代谢的基石,而线粒体质量控制系统（mitochondrial quality control,MQC）是线粒体内环境稳态的根基,也是反映线粒体质量的一个网络系统,对于监测和维持线粒体内环境稳态至关重要。而MQC紊乱可能导致MIRI。近年来,中医药疗法可通过多途径、多靶点、多层次显著改善MIRI。通过对中医药基于MQC治疗MIRI的研究进展进行梳理,为中医药治疗MIRI相关基础研究的转化以及拓展临床思路提供借鉴。     

The emerging multifaceted role of PINK1 in cancer biology

For its various important functions in cells, phosphatase and tensin homolog‐induced kinase 1 (PINK1) has drawn considerable attention for the role it plays in early‐onset Parkinson''s disease. In recent years, emerging evidence has supported the hypothesis that PINK1 plays a part in regulating many physiological and pathophysiological processes in cancer cells, including cytoplasmic homeostasis, cell survival, and cell death. According to the findings of these studies, PINK1 can function as a tumor promoter or suppressor, showing a duality that is dependent on the context. In this study we review the mechanistic characters relating to PINK1 based on available published data from peer‐reviewed articles, The Cancer Genome Atlas data mining, and cell‐based assays. This mini review focuses on some of the interplays between PINK1 and the context and recent developments in the field, including its growing involvement in mitophagy and its nonmitophagy organelles‐related function. This review aims to help readers better grasp how PINK1 is functioning in cell physiological and pathophysiological processes, especially in cancer biology.     

Mitochondrial biogenesis and dynamics in the developing and diseased heart

The mitochondrion is a complex organelle that serves essential roles in energy transduction, ATP production, and a myriad of cellular signaling events. A finely tuned regulatory network orchestrates the biogenesis, maintenance, and turnover of mitochondria. The high-capacity mitochondrial system in the heart is regulated in a dynamic way to generate and consume enormous amounts of ATP in order to support the constant pumping function in the context of changing energy demands. This review describes the regulatory circuitry and downstream events involved in mitochondrial biogenesis and its coordination with mitochondrial dynamics in developing and diseased hearts.     

电针百会、神庭调控BNIP3L介导的线粒体自噬和减轻脑缺血再灌注损伤的机制研究CSCD

目的:探讨电针百会、神庭调节线粒体自噬减轻脑缺血再灌注损伤的作用机制。方法:采用随机数字表法将60只SD大鼠随机分为假手术组15只和手术组45只。假手术组只分离、暴露血管,不结扎,不插入尼龙线。手术组大鼠采用大脑中动脉线栓阻塞法(MCAO)制备大鼠局灶性脑缺血-再灌注(I/R)模型,术后2 h采用Zea Longa法进行神经功能评分,最终纳入30只手术组大鼠。进一步将纳入的手术组大鼠随机分为模型组、电针组和电针+3-MA组,每组10只。造模分组成功后,各组给予电针等相应方式干预7 d,采用Zea Longa法于第1天、第3天、第5天、第7天再次进行神经功能评估。干预7 d后获取各组大鼠左侧大脑皮层组织,苏木精-伊红(HE)染色后观察脑组织病理学变化,Western blot法检测LC3-Ⅱ/LC3-Ⅰ蛋白表达水平,组织免疫荧光技术检测线粒体自噬相关蛋白表达共定位情况(BNIP3L和SQSTM1标记),TUNEL法检测各组大鼠神经细胞凋亡水平。结果:(1)造模2 h后手术组大鼠神经功能评分均显著升高,假手术组大鼠未表现出神经功能缺损症状,评分均为0分。干预后第7天模型组及电针+3-MA组大鼠神经功能评分仍显著升高,与假手术组比较,差异有统计学意义(P<0.05)。而电针组大鼠在电针干预后神经功能评分显著降低,与模型组及电针+3-MA组比较,差异均有统计学意义(P<0.05)。(2)HE染色结果可见,假手术组神经元胞体较大,多角形(多个突起),核着色浅,胞质可见特征性结构尼氏体;模型组神经元皱缩,胞质结构不清,胞浆嗜依红染色增强,尼氏体边聚或消失,核固缩,有的胞体变形缩小,呈三角形,细胞周围间隙增宽;电针组可以在一定程度上减轻缺血再灌注所致的病理损伤。(3)Western blot结果表明,MCAO后第7天模型组自噬水平(LC3-Ⅱ/LC3-Ⅰ)下降,与假手术组比较,差异有统计学意义(P<0.05);电针组可以激活自噬水平,与模型组比较,差异有统计学意义(P<0.05)。(4)模型组中皮质梗死区域线粒体自噬水平缺失,表现为红色荧光和绿色荧光强度减弱,而电针干预可以激活梗死区域神经细胞的线粒体自噬,表现为红色荧光和绿色荧光的表达增高及共定位增强(橘黄色)。(5)TUNEL检测结果表明,模型组大鼠凋亡率升高,与假手术组比较,差异有统计学意义(P<0.05);电针组的凋亡率降低,与模型组比较,差异有统计学意义(P<0.05)。电针+3-MA组结果表明,自噬抑制剂3-MA可以逆转电针对MCAO模型大鼠的神经保护作用。结论:电针百会、神庭可以减轻脑缺血再灌注模型大鼠神经功能损伤,其具体机制和调控BNIP3L介导的线粒体自噬、减轻脑缺血再灌注损伤和细胞凋亡有关。