小檗碱对心肌缺血再灌注损伤大鼠线粒体自噬及对PINK1/Parkin通路的影响

目的 探讨小檗碱对心肌缺血再灌注损伤大鼠线粒体自噬及PTEN诱导激酶1(PTEN induced putative kinase 1,PINK1)/帕金森病蛋白(Parkin)通路的影响.方法 建立心肌缺血再灌注损伤大鼠模型,随机分组为模型组、小檗碱低、高剂量(75、150 mg/kg)组,自噬抑制剂三甲基腺嘌呤(3-...     

Parkin基因甲基化在鼻咽癌发生发展中的作用

目的探讨Parkin基因甲基化在鼻咽癌发生发展中的作用。方法应用甲基化特异性PCR技术对5个鼻咽癌细胞株（CNE1、CNE2、TW03、HONE1、C666）和1个正常鼻咽上皮细胞株（NP69）、54例鼻咽癌组织和16例正常鼻咽上皮组织的Parkin基因启动子区甲基化状态进行检测,并分析鼻咽癌组织中Parkin基因甲基化与临床特征的关系;应用RT-PCR检测加入甲基转移酶抑制剂（5-氮-2-脱氧胞苷）后CNE1、CNE2中Parkin基因转录表达,分析去甲基化对Parkin基因转录表达的影响。结果1个正常鼻咽上皮细胞株和16例正常鼻咽上皮组织中均未检测到Parkin基因甲基化;5个鼻咽癌细胞株和54例鼻咽癌组织中Parkin基因甲基化频率分别为60.00%和62.96%;54例鼻咽癌患者中Parkin基因甲基化状态与临床因素均无关（均P＞0.05）。经过5-氮-2-脱氧胞苷处理后,Parkin基因在CNE1、CNE2中转录表达增加,分别从0提高至3.65和2.15。结论鼻咽癌中Parkin基因甲基化与转录表达密切相关,是基因表达调节的一种重要方式;可能成为鼻咽癌早期分子生物学辅助诊断的标志物和去甲基化基因治疗的靶点。     

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...     

Protective effects of Buyinqianzheng Formula on mitochondrial morphology by PINK1/Parkin pathway in SH-SY5Y cells induced by MPP+

ObjectiveBuyinqianzheng Formula (BYQZF) is clinically employed in traditional Chinese medicine to treat Parkinson’s disease (PD) by improving mitochondrial dysfunction. However, the underlying mechanisms by which BYQZF affects mitochondrial morphology remain unknown. Therefore, we observed the effects of BYQZF on mitochondria from the perspective of the PINK1/Parkin pathway.MethodsCell survival rates were assessed by Cell Counting Kit-8 assay. Expression levels of PINK1 and Parkin mRNA were examined by qRT-PCR. Protein expression levels of PINK1, PINK1-Ser228, Parkin, Parkin-Ser65, Drp1, and Drp1-Ser637 were examined by western blotting. PINK1, Parkin, and MitoTracker® Red CMXRos (MTR) were stained by triple-labeled immunofluorescence, and observed under laser confocal microscopy.ResultsCell survival rate, mitochondrial form factor, mean length and number of mitochondrial network branches, mitochondrial activity, mRNA expression levels of PINK1 and Parkin, and protein expression levels of PINK1, Parkin, and Drp1-Ser637 were reduced after 1-methyl-4-phenylpyridinium (MPP⁺) intervention. In contrast, Pearson’s correlation coefficients between PINK1 and Parkin, and between Parkin and MTR, as well as protein expression levels of PINK1-Ser228, Parkin-Ser65, and Drp1 increased significantly after MPP⁺ intervention. Treatment with BYQZF increased cell survival rate, mitochondrial form factor, mean length and number of mitochondrial network branches, mitochondrial activity, mRNA expression levels of PINK1 and Parkin, and expression of PINK1, Parkin, and Drp1-Ser637 proteins. Pearson’s correlation coefficients between PINK1 and Parkin, and between Parkin and MTR, as well as protein expression levels of PINK1-Ser228, Parkin-Ser65, and Drp1 decreased after BYQZF treatment.ConclusionThese results demonstrate that BYQZF has a protective effect on mitochondrial molecular mechanisms in the PD cell model, and the mechanism is related to the PINK1/Parkin pathway.     

Parkin蛋白在鼻咽癌中表达及临床意义

目的 研究Parkin蛋白在鼻咽癌中表达及临床意义.方法 运用Elivision TM S-P免疫组化技术的方法检测54例鼻咽癌组织和16例正常鼻咽上皮组织Parkin蛋白表达水平,分析Parkin蛋白表达与鼻咽癌患者临床病理特征关系.结果 54例鼻咽癌组织Parkin蛋白表达水平为“-”23例,“＋”12例,“＋＋”7例,“＋＋＋”12例,16例正常鼻咽上皮组织Parkin蛋白表达水平为“-”0例,“＋”0例,“＋＋”3例,“＋＋＋”13例,鼻咽癌组织Parkin蛋白表达明显降低（P＜0.05）,Parkin蛋白表达与患者年龄、性别、T分期、TNM分期、病理类型无明显关系（P＞0.05）,而与淋巴结转移有关（P＜0.05）.结论 Parkin基因在鼻咽癌的发生发展中起抑癌基因作用,Parkin蛋白表达可作为判断鼻咽癌临床预后预测指标.     

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.     

LRRK2 and parkin immunoreactivity in multiple system atrophy inclusions

Certain genetic defects in LRRK2 and parkin are pathogenic for Parkinson’s disease (PD) and both proteins deposit in the characteristic Lewy bodies. LRRK2 is thought to be involved in the early initiation of Lewy bodies. The involvement of LRRK2 and parkin in the similar cellular deposition of fibrillar α-synuclein in glial cytoplasmic inclusions (GCI) in multiple system atrophy (MSA) has not yet been assessed. To determine whether LRRK2 and parkin may be similarly associated with the abnormal deposition of α-synuclein in MSA GCI, paraffin-embedded sections from the basal ganglia of 12 patients with MSA, 4 with PD and 4 controls were immunostained for LRRK2, parkin, α-synuclein and oligodendroglial proteins using triple labelling procedures. The severity of neuronal loss was graded and the proportion of abnormally enlarged oligodendroglia containing different combinations of proteins assessed in 80–100 cells per case. Parkin immunoreactivity was observed in only a small proportion of GCI. In contrast, LRRK2 was found in most of the enlarged oligodendroglia in MSA and colocalised with the majority of α-synuclein-immunopositive GCI. Degrading myelin sheaths containing LRRK2-immunoreactivity were also observed, showing an association with one of the earliest oligodendroglial abnormalities observed in MSA. The proportion of LRRK2-immunopositive GCI was negatively associated with an increase in neuronal loss and α-synuclein-immunopositive dystrophic axons. Our results indicate that an increase in LRRK2 expression occurs early in association with myelin degradation and GCI formation, and that a reduction in LRRK2 expression in oligodendroglia is associated with increased neuronal loss in MSA.