Mitochondrial quality control in amyotrophic lateral sclerosis:towards a common pathway?

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by loss of upper and lower motor neurons. Different mechanisms contribute to the disease initiation and progression, including mitochondrial dysfunction which has been proposed to be a central determinant in ALS patho-genesis. Indeed, while mitochondrial defects have been mainly described in ALS-linked SOD1 mutants, it is now well established that mitochondria become also dysfunctional in other ALS conditions. In such con-text, the mitochondrial quality control system allows to restore normal functioning of mitochondria and to prevent cell death, by both eliminating and replacing damaged mitochondrial components or by degrading the entire organelle through mitophagy. Recent evidence shows that ALS-related genes interfere with the mitochondrial quality control system. This review highlights how ineffective mitochondrial quality control may render motor neurons defenseless towards the accumulating mitochondrial damage in ALS.     

麝香保心丸调节PINK1/Parkin信号通路对糖尿病视网膜病变大鼠的影响

[目的] 探讨麝香保心丸（SBP）对糖尿病视网膜病变（DR）大鼠线粒体自噬及PTEN诱导激酶蛋白1（PINK1）/细胞质E3-泛素连接酶（Parkin）信号通路的影响。[方法] 建立DR大鼠模型,将大鼠随机分为正常组（Control组）、模型组（DR组）、麝香保心丸低剂量组（SBP-L组）、麝香保心丸高剂量组（SBP-H组）、麝香保心丸高剂量+雷帕霉素组（SBP-H+RAP组）。检测各组大鼠空腹血糖（FPG）及总胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白胆固醇（LDL-C）水平;苏木精-伊红（HE）染色观察各组大鼠DR情况;酶联免疫吸附实验（ELISA）检测各组大鼠血管内皮生长因子（VEGF）、高迁移率族蛋白1（HMGB1）水平;透射电镜观察各组大鼠细胞线粒体形态;免疫组化检测线粒体自噬相关蛋白选择性自噬接头蛋白sequestosome-1（SQSTM1/P62）、微管相关蛋白轻链3Ⅱ（LC3-Ⅱ）的表达;蛋白免疫印迹法检测各组大鼠PINK1、Parkin蛋白表达。[结果] 与Control组比较,DR组视网膜结构破坏严重,细胞排列紊乱,水肿明显,细胞间隙变宽,线粒体损伤、肿胀明显,形态异常,FPG、TC、TG、LDL-C、VEGF、HMGB1、LC3-Ⅱ、PINK1、Parkin表达水平显著升高,SQSTM1/P62表达水平显著降低（P＜0.05）;与DR组比较,SBP-L组、SBP-H组视网膜细胞结构改善,细胞排列逐渐整齐,细胞水肿减轻,细胞、线粒体形态均趋于正常,线粒体肿胀减轻,FPG、TC、TG、LDL-C、VEGF、HMGB1、LC3-Ⅱ、PINK1、Parkin表达水平显著降低,SQSTM1/P62表达水平显著升高（P＜0.05）;与SBP-H组相比,SBP-H+RAP组视网膜细胞结构破坏加重,细胞排列紊乱,水肿明显,线粒体肿胀加重,形态异常,FPG、TC、TG、LDL-C、VEGF、HMGB1、LC3-Ⅱ、PINK1、Parkin表达水平显著升高,SQSTM1/P62表达水平显著降低（P＜0.05）。[结论] 麝香保心丸通过抑制PINK1/Parkin信号通路抑制DR大鼠线粒体自噬。     

丹皮酚对大鼠心肌梗死后心脏功能的改善及其对Pink1/Parkin的影响＊

目的 检测丹皮酚（paeonol）对大鼠心梗后心肌重构和心脏功能的改善作用并探究其作用机制。方法 采用SD大鼠和人心肌细胞H9C2分别建立体内心肌梗死动物模型和体外心肌细胞缺氧细胞模型。Western blot检测Pink1、Parkin、LC3Ⅰ、LC3Ⅱ、Beclin-1和Mfn2的表达水平。本研究通过ELISA试剂盒检测心肌损伤标志物乳酸脱氢酶（LDH）和肌酸激酶同工酶（CK-MB）在血浆中的含量。细胞凋亡率采用TUNEL检测。使用超声心动图和生物机能采集系统检测大鼠心脏功能和心室重构情况。结果 丹皮酚减少了心肌损伤标志物LDH和CK-MB的释放，并减弱了H9C2细胞凋亡。丹皮酚激活了Pink1/Parkin信号通路，并因此增强了线粒体自噬。进一步的研究表明，丹皮酚显著减轻了SD大鼠心梗后心室重构情况并改善了心脏功能。结论 丹皮酚通过激活Pink1/Parkin信号通路促进线粒体自噬，进而减轻了心肌细胞损伤，改善了大鼠心脏功能和心室重构情况。     

Low frequency of Parkin, Tyrosine Hydroxylase, and GTP Cyclohydrolase I gene mutations in a Danish population of early-onset Parkinson''s Disease

Autosomal recessive Parkinson's disease (PD) with early-onset may be caused by mutations in the parkin gene (PARK2). We have ascertained 87 Danish patients with an early-onset form of PD (age at onset < or =40 years, or < or =50 years if family history is positive) in a multicenter study in order to determine the frequency of PARK2 mutations. Analysis of the GTP cyclohydrolase I gene (GCH1) and the tyrosine hydroxylase gene (TH), mutated in dopa-responsive dystonia and juvenile PD, have also been included. Ten different PARK2 mutations were identified in 10 patients. Two of the patients (2.3%) were found to have homozygous or compound heterozygous mutations, and eight of the patients (9.2%) were found to be heterozygous. A mutation has been identified in 10.4% of the sporadic cases and in 15.0% of cases with a positive family history of PD. One patient was found to be heterozygous for both a PARK2 mutation and a missense mutation (A6T) in TH of unknown significance. It cannot be excluded that both mutations contribute to the phenotype. No other putative disease causing TH or GCH1 mutations were found. In conclusion, homozygous, or compound heterozygous PARK2 mutations, and mutations in GCH1 and TH, are rare even in a population of PD patients with early-onset of the disease.     

Parkin蛋白抑制三阴性乳腺癌MDA-MB-231细胞的有氧糖酵解和增殖迁移

目的 评估Parkin蛋白对乳腺癌MDA-MB-231细胞有氧糖酵解和生长增殖的影响。方法 首先采用Western blot检测Parkin蛋白在人乳腺上皮细胞MCF-10A和乳腺癌MDA-MB-231细胞中的表达。随后构建高表达Parkin的乳腺癌MDA-MB-231细胞株为实验组,以转染空载体的MDA-MB-231细胞为对照组,分别采用葡萄糖、乳酸和ATP检测试剂盒检测两组细胞葡萄糖摄取量,乳酸和ATP生成量,并通过Western blot检测糖酵解相关蛋白GLUT1、PKM2和LDHA表达水平;运用细胞增殖实验、平板克隆及细胞划痕实验评估细胞的增殖和迁移能力;采用Western blot检测凋亡相关蛋白Bax、Bcl-2和cleaved Caspase-3的表达水平。结果 与MCF-10A细胞相比,MDA-MB-231细胞Parkin蛋白表达水平显著下降（P<0.01）。高表达Parkin可使乳腺癌MDA-MB-231细胞的葡萄糖摄取和乳酸生成下降,ATP水平上升（P<0.05）,其增殖和迁移能力下降（P<0.01）;与对照组相比,Parkin组细胞糖酵解相关蛋...     

Mechanism of mitochondrial protection by the Buyin Qianzheng formula in a Parkin overexpression cell modelOA

Objective: To identify the molecular mechanisms of the effects of the Buyin Qianzheng formula(BYQZF)on the mitochondrial dynamics in a Parkin overexpression Parkinson’s disease(PD) cell model.Methods: First, a stable Parkin overexpression cell model was constructed using plasmid transfection.Then, we examined the protective effect of BYQZF on the mitochondrial dysfunction of the Parkin overexpression PD cell model induced by neurotoxin 1-methyl-4-phenylpyridinium ion(MPPt). The m RNA expression ...     

Intercepting the Lipid-Induced Integrated Stress Response Reduces Atherosclerosis

Background

Eukaryotic cells can respond to diverse stimuli by converging at serine-51 phosphorylation on eukaryotic initiation factor 2 alpha (eIF2α) and activate the integrated stress response (ISR). This is a key step in translational control and must be tightly regulated; however, persistent eIF2α phosphorylation is observed in mouse and human atheroma.

何首乌苷对大脑中动脉栓塞再灌注模型大鼠皮层线粒体自噬的调控作用及神经保护机制

目的 研究何首乌苷（2,3,5,4′-tetrahydroxyl diphenylethylene-2-O-glucoside,THSG）在脑缺血再灌注（cerebralischemia/reperfusion,CI/R）中对大鼠皮层神经元的保护作用。方法 采用线栓法建立大鼠短暂性大脑中动脉栓塞再灌注（transient middle cerebral artery occlusion/reperfusion,tMCAO/R）模型。雄性SD大鼠随机分为假手术组、模型组和THSG低、中、高剂量（10、20、40 mg/kg）组,每组10只。采用Longa 5分法评估神经功能缺损情况; TTC染色检测大鼠脑梗死体积百分比;苏木素-伊红（HE）染色检测神经元结构; Nissl染色检测Nissl小体数量; Western blotting检测半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3,Caspase-3）、B淋巴细胞瘤-2基因（B-cell lymphoma-2,Bcl-2）、Bcl-2相关X蛋白（Bcl-2-associated X protein,Bax）、PTEN诱导的激酶1（PTEN induced putative kinase 1,PINK1）、Parkin、微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3,LC3）、p62的蛋白表达;透射电镜观察皮层神经元线粒体自噬情况;免疫荧光检测神经元Parkin、LC3的表达。结果 THSG显著降低了MCAO/R大鼠脑梗死体积百分比和神经功能缺损评分（P＜0.05、0.001）,降低了MCAO/R大鼠皮层神经元结构损伤和Nissl小体丢失的现象,下调了皮层区cleaved Caspase-3、Bax、p62蛋白表达（P＜0.05、0.01、0.001）,上调了皮层区Bcl-2、LC3-Ⅱ/LC3-Ⅰ蛋白表达（P＜0.01）,促进皮层神经元中自噬体的数量增加以及PINK1、Parkin蛋白表达（P＜0.05、0.001）。结论 THSG能显著改善MCAO/R大鼠皮层神经元损伤,其神经保护机制与促进PINK1/Parkin依赖性线粒体自噬途径有关。     

散发性帕金森病Parkin基因突变检测

目的 探讨散发性帕金森病病人Parkin基因的突变情况.方法 应用聚合酶链反应-单链构象多态性技术(PCR-SSCP), 对18例帕金森病病人和20例正常人Parkin基因的第3、4、5、6、7外显子突变情况进行检测分析.结果 4例病人有Parkin基因第3外显子点突变, 1例病人有Parkin基因第4外显子点突变,2例病人有Parkin基因第5外显子点突变.所有病例均未检测到Parkin基因外显子缺失突变.结论 散发性帕金森病病人中存在Parkin基因点突变,Parkin基因点突变可能是散发性帕金森病发病原因之一.     

ALS- and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy

TANK-binding kinase 1 (TBK1) is a multifunctional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia, including missense mutations that disrupt the abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presence of either wild-type or kinase-inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type–specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals.

TNF receptor–associated family member–associated NF-κB activator (TANK)-binding kinase 1 (TBK1) plays a critical role in several cellular pathways implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS), including selective clearance of mitochondria and regulation of inflammation. More than 90 mutations in TBK1 have been linked to ALS, including several mutations identified in patients with the co-occurring degenerative disease, fronto-temporal dementia (ALS-FTD) (1, 2). Some TBK1 mutations are classified as loss of function variants while others are missense mutations with unclear contributions to disease pathogenesis (1, 3–6). The latter category includes mutations shown to disrupt the ability of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation (7–9). Given the importance of TBK1 in mitophagy (10), and the necessity of mitochondrial quality control to the maintenance of neuronal homeostasis (11, 12), functional analysis of ALS-associated missense mutations in TBK1 is necessary to determine the impact of mutant TBK1 in the neurodegeneration characteristic of ALS.TBK1 has three primary domains, 1) a kinase domain, 2) a ubiquitin-like domain, and 3) a scaffold dimerization domain, which are followed by a flexible C terminus domain (CTD) (Fig. 1A) (13–15). Two TBK1 monomers dimerize along their scaffold dimerization domains, while kinase activity is activated via autophosphorylation of the critical serine residue 172 (S172) within the activation loop of the kinase domain (14). Due to the conformation of the TBK1 dimer, it is unlikely that the monomers within a dimer can self-activate, so multimer formation is thought to be required for trans-autophosphorylation and kinase activation (13, 14). TBK1 multimerization may be promoted by association of TBK1 via its CTD with adaptor proteins including OPTN, TANK, Sintbad, and NAK-associated protein 1 (NAP1) (7, 16, 17). ALS-linked missense mutations are distributed throughout the protein, with some mutations disrupting dimerization, kinase activity, or both and others disrupting the association of TBK1 with adaptors, potentially inhibiting TBK1 multimerization and activation (Fig. 1B) (3, 6–8).Open in a separate windowFig. 1.ALS-linked TBK1 mutations are found throughout the molecule and induce biochemical, biophysical, and cellular deficits. (A) Protein databank structure for TANK-binding kinase 1 (TBK1) (PDB 4IWO) (13). Domains are designated by color coding: kinase domain residues 1 to 308 (blue), ubiquitin-like domain residues 309 to 387 (yellow), and scaffolding dimerization domain residues 388 to 657 (red). ALS-linked mutations are indicated by arrows and labels of their respective colors. Some mutations likely disrupt the structure of TBK1, a phenomenon not represented by this model. (B) Table summarizing biochemical results for the ALS-linked mutants published by Ye et al. (8) and the engineered kinase-inactive D135N-TBK1 (gray). (C) Confocal section of a HeLa cell (outlined in white) expressing a mitochondria-localized fluorophore (blue), Parkin (green), and WT-TBK1 (magenta), fixed after treatment with CCCP for 90 min. The Inset (white box) and zoom images (Right) exhibit rounded mitochondria that have recruited Parkin and TBK1. A volume rendering is also shown (Right, bottom row). (Scale bars: zoom out, 10 μm; zoom in, 2 μm.) (D) Relative signal intensities for mitochondria, Parkin, and TBK1 are quantified across the diameter of a damaged mitochondria (white dashed line in C, zoom). (E and F) HeLa cells depleted of endogenous TBK1 expressing Parkin, OPTN, and WT- (E) or E696K- (F) TBK1, fixed after treatment with CCCP for 90 min. Inset (white box) and zoom images (Left) demonstrate multiple rings with colocalized mitophagy components. (Scale bars: zoom out, 10 μm; zoom in, 4 μm.) (G) Quantification of E and F as rings/μm² for each cell. n = 22 to 25 cells from three independent experiments. Dashed line, median; dotted lines, 25th and 75th quartiles. ****P < 0.0001 by Student’s unpaired t test. Images E and F shown here are insets; for representative images of whole fields, reference SI Appendix, Fig. S2B.TBK1 is an essential regulator of mitophagy, a stepwise pathway for clearance of damaged mitochondria (10, 18). Mitophagy is triggered by loss of mitochondrial membrane potential, leading to the stabilization of PTEN-induced putative kinase 1 (PINK1) on the outer mitochondrial membrane (OMM) (19) where it phosphorylates ubiquitin (20). Phosphorylated ubiquitin recruits the E3 ubiquitin ligase, Parkin (20, 21), which is activated by PINK1 phosphorylation and then ubiquitinates OMM proteins (20, 22–25). These modifications promote proteasomal degradation of mitofusins, preventing the damaged organelle from re-fusing with the healthy mitochondrial network and resulting in a small, rounded mitochondrion (26). Ubiquitination of OMM proteins also promotes recruitment of the mitophagy receptors OPTN, nuclear dot 52 kDa protein (NDP52), Tax1-binding protein 1 (TAX1BP1), next to BRCA gene 1 protein (NBR1), and p62/sequestosome1 (10, 27–30), though OPTN and NDP52 are sufficient and redundant in carrying out mitochondrial clearance in HeLa cells (29). Phosphorylation of OPTN at S177 by TBK1 at the OMM enhances the binding of OPTN to ubiquitin chains (18). OPTN then drives recruitment of the core autophagy machinery, including the unc-51-like autophagy activating kinase (ULK1) complex, to initiate formation of the double membraned phagophore that engulfs the damaged organelle (31–33). In this process, microtubule-associated protein 1A/1B-light chain 3 (LC3) is lipidated and subsequently incorporated into the elongating phagophore (10, 27, 34). The LC3-interacting region of OPTN facilitates efficient engulfment by the autophagosome (10), while TBK1-mediated phosphorylation of OPTN enhances the binding of the receptor to LC3 (35). A feed-forward mechanism in which initial LC3-positive membranes recruit more OPTN and NDP52 leads to accelerated mitochondrial engulfment (36). The newly formed compartment fuses with lysosomes to complete degradation of the organelle (30, 37, 38).We undertook a functional analysis of ALS-associated TBK1 missense mutations that have been characterized by biochemical and biophysical assays but confer unknown effects on the cellular pathways that involve TBK1. We determined the extent of recruitment of TBK1 mutants to depolarized, Parkin-positive mitochondria, the effect of mutant TBK1 expression on OPTN recruitment and phosphorylation, and the resulting downstream engulfment of fragmented mitochondria by LC3-positive autophagosomes. Expression of some ALS-linked mutations profoundly disrupted TBK1 recruitment and activity during mitochondrial clearance, while others only marginally affected the pathway. Neurons expressing TBK1 mutations demonstrated higher baseline levels of mitochondrial stress and an inability to manage induced oxidative damage, both of which may contribute to neurodegeneration. Our data suggest a nuanced model of TBK1 function, wherein TBK1 phosphorylates OPTN directly, while TBK1 recruitment also facilitates OPTN phosphorylation via an ULK1-dependent pathway. Furthermore, we demonstrate that ALS and ALS-FTD–associated missense mutations in TBK1 can lead to disordered or delayed mitochondrial clearance and a cellular deficiency in mitochondrial homeostasis.