加味丹参饮含药血清预处理对缺氧/复氧H9C2心肌细胞自噬相关基因Atg5和Beclin1 mRNA表达的影响

目的探讨加味丹参饮含药血清预处理对缺氧/复氧H9C2心肌细胞自噬及其相关基因Atg5、Beclin1 mRNA表达的影响。方法将体外培养的H9C2心肌细胞随机分为空白血清组(CG)、缺氧/复氧组(HRG)、加味丹参饮含药血清组(JDG)、JDG+自噬抑制剂(3-MA)组(JIG)、JDG+自噬激动剂(RAPA)组(JAG)。倒置显微镜观察各组心肌细胞生长及形态变化;MTT比色法测定心肌细胞存活率;透射电子显微镜观察心肌细胞自噬体形态及数量;实时荧光定量PCR检测自噬相关基因Atg5、Beclin1 mRNA表达。结果与CG比较,HRG镜下心肌细胞变性坏死程度增加,细胞存活率下降(P<0.01),电镜下自噬体增多,实时荧光定量PCR示Atg5、Beclin1 mRNA表达上调;与HRG相比,JDG及JAG镜下心肌细胞变性坏死程度减轻,细胞存活率显著升高(P<0.01),电镜下自噬体增加,Atg5、Beclin1 mRNA表达进一步上调(P<0.05)。结论加味丹参饮预处理通过上调自噬相关基因Atg5、Beclin1 mRNA表达,增强细胞自噬,保护缺氧/复氧损伤H9C2心肌细胞。     

自噬介导FAS/AD抑制Bel-7402细胞的增殖

目的探讨在FAS/AD抑制Bel-7402细胞增殖过程中自噬的作用。方法通过MTT法检测3-甲基腺嘌呤(3-MA)对细胞增殖的影响,通过免疫荧光法检测LC3及Beclin 1的表达变化,通过Western印迹检测Atg12-5和Atg7的表达,以揭示FAS/AD对Bel-7402细胞的自噬作用。结果与对照组相比,FAS/AD组的细胞的自噬现象明显,LC3及Beclin 1高表达,同时Atg12-5和Atg7也高表达,而FAS/AD/MA组的活力明显降低,自噬现象较弱,同时LC3、Beclin 1及Atg12-5和Atg7的表达降低。结论 3-MA能明显减弱FAS/AD诱导的细胞自噬作用,且自噬介导FAS/AD抑制BEL-7402细胞增殖。     

自噬相关基因Atg4B及LC3-Ⅱ在肌萎缩侧索硬化症转基因鼠海马中的表达及意义

目的观察自噬相关基因Atg4B及LC3-Ⅱ在肌萎缩侧索硬化症(ALS)转基因鼠海马组织中的表达变化,探讨细胞自噬机制在ALS发病中的作用及意义。方法选择SOD1-G93A转基因鼠进行试验,分别于发病早、中、晚期,通过RT-PCR、免疫荧光检测小鼠海马组织中Atg4B、LC3-Ⅱ在m RNA、蛋白水平的表达及变化。结果与野生型鼠相比较,转基因鼠的Atg4B在m RNA及蛋白水平表达均下调;LC3-Ⅱ在蛋白水平表达上调,在m RNA水平变化不明显。结论自噬相关基因Atg4B及LC3-Ⅱ参与了ALS发病,ALS海马组织的病变与自噬异常有关。     

Parkin介导的线粒体自噬在高糖高脂导致的心肌细胞损伤中的保护作用

目的 明确Parkin（一种E3泛素化连接酶）介导的线粒体自噬对高糖高脂导致的原代心肌细胞损伤的保护作用。 方法 以LV-lacZ（LacZ空病毒）或LV-Parkin（Parkin过表达慢病毒）转染SD大鼠原代心肌细胞48 h,再用含葡萄糖（5.5 mmol/L NG）的培养基或含棕榈酸盐（500 μmol/L HF）和葡萄糖（25 mmol/L HG）的高糖高脂培养基培养心肌细胞24 h。 实验分组:①阴性对照组（NG-LacZ）,②正常Parkin过表达组（NG-Parkin）,③高糖高脂阴性对照组（HG-HF-LacZ）,④高糖高脂Parkin过表达组（HG-HF-Parkin）。用Western blot法检测PTEN介导的假定激酶蛋白1（PINK1）、Parkin、P62（一种自噬相关蛋白）、微管相关蛋白1轻链3（LC3）蛋白表达水平。采用JC-1染色法检测活细胞内线粒体膜电位水平。免疫荧光法检测自噬体数量,TUNEL法检测细胞凋亡率。 结果 与对照组相比,高糖高脂处理的原代心肌细胞自噬相关蛋白LC3-Ⅱ,P62表达水平上调（P<0.05）,PINK1表达未发生统计学差异,Parkin表达水平下调（P<0.05）,自噬体数量增多,线粒体膜电位下降功能损伤,心肌细胞凋亡率升高（P<0.05）。而用高糖高脂处理LV-Parkin转染的心肌细胞,LC3-Ⅱ蛋白表达水平进一步升高（P<0.05）,而P62表达水平显著下降（P<0.05）,自噬体数量进一步增多,细胞内线粒体膜电位水平上升,心肌细胞凋亡率下降（P<0.05）。 结论 高糖高脂可引起SD大鼠原代心肌细胞自噬流量降低,自噬小体增多,线粒体自噬发生障碍。Parkin过表达慢病毒通过激活心肌细胞内线粒体自噬途径,提高自噬流量,改善线粒体功能,降低心肌细胞凋亡率。     

苯并(a)芘通过降低突触融合蛋白17和溶酶体关联膜蛋白2表达阻抑人脐静脉内皮细胞自噬流

目的 研究苯并(a)芘(BaP)影响人脐静脉内皮细胞(HUVEC)自噬的机制。方法 HUVEC经BaP(2.5、5、10 μmol/L)处理24 h后,分别采用间接免疫荧光法、Western blot、吖啶橙染色和单丹磺酰尸胺染色等技术方法,检测自噬体及其内容物降解、目标蛋白微管相关蛋白1轻链3(LC3)、选择性自噬接头蛋白p62、Beclin-1、自噬相关蛋白5(Atg5)、Atg7、Atg12、组织蛋白酶B(CTSB)、组织蛋白酶D(CTSD)、突触融合蛋白17(STX17)、溶酶体关联膜蛋白2(LAMP2)表达、溶酶体数量与功能,及相关上游关键调控蛋白丝氨酸-苏氨酸蛋白激酶(Akt)、细胞外调节蛋白激酶(ERK)、转录因子EB(TFEB)磷酸化水平。结果 BaP暴露组HUVEC内,检测结果显示:(1)LC3 puncta水平、LC3Ⅱ/LC3Ⅰ比率增加,自噬起始关键蛋白(Beclin-1、Atg5、Atg7、Atg12)表达升高,Akt蛋白磷酸化则明显降低;(2)p62 puncta和p62蛋白水平明显升高;(3)溶酶体数量增加,并伴随着溶酶体特征性水解酶(CTSB、CTSD)表达升高,相应地ERK、TFEB磷酸化水平增加;(4)调控自噬体与溶酶体融合的关键蛋白STX17与LAMP2降低。结论 BaP通过降低STX17与LAMP2表达水平,阻抑了HUVEC正常的自噬流。     

过表达Atg5蛋白对巨噬细胞泡沫化程度的影响

目的探讨过表达Atg5蛋白对巨噬细胞泡沫化程度的影响。方法采用Atg5慢病毒和对照组病毒感染Raw264.7巨噬细胞,建立对照细胞系(对照组)和Atg5过表达细胞系(观察组)。两组细胞分别加入50 mL/L ox-LDL诱导形成泡沫化细胞。采用Western blot分析两组细胞蛋白p62、LC3、CD36和SR-A的变化;采用高效液相法检测两组细胞内胆固醇脂(CE)、游离胆固醇(FC)和总胆固醇(TC)的变化;采用免疫荧光和电子显微镜分析两组细胞内脂滴的含量;采用ELISA检测两组细胞培养上清中炎症因子TNF-α、IL-6和IL-1β的水平。结果与对照组比较,观察组细胞自噬底物p62水平显著下调,LC3-II水平显著上调(P0.05)。与对照组比较,观察组巨噬细胞CE、FC和TC含量均显著下调(P0.05)。观察组细胞脂质水平和脂滴的数量较对照组明显下降(P0.05)。与对照组比较,观察组细胞炎症因子TNF-α、IL-6和IL-1β的水平显著下调(P0.05)。结论自噬核心蛋白Atg5过表达可显著提高巨噬细胞自噬水平,可能通过自噬降解胞内胆固醇等脂质,预防巨噬细胞泡沫化,进而降低动脉粥样硬化的发生。     

自噬核心蛋白Atg101对脂肪细胞衰老的影响

目的初步探究自噬核心蛋白Atg101对白色脂肪细胞衰老的影响。方法构建Atg101敲减的3T3-L1成熟脂肪细胞模型, 验证Atg101对自噬相关蛋白LC3和p62蛋白的影响。构建并分析人类皮下脂肪组织的RNA-seq数据库, 基于Atg101与其他基因FPKM值的Pearson相关系数(R2>0.4,P<0.050)预测共表达基因集并进行KEGG和Reactome富集分析。构建年轻小鼠(8周龄)和老龄小鼠(18个月龄)模型, 实时定量PCR(RT-qPCR)和Western印迹法检测Atg101在腹股沟皮下脂肪和内脏脂肪中的表达水平。进一步通过RNA-seq、Western印迹和RT-qPCR检测白色脂肪细胞衰老相关分泌表型(senescence-associated secretory phenotype, SASP)、细胞周期及线粒体稳态相关基因的表达差异, 分析Atg101敲减对脂肪细胞衰老的影响。结果在3T3-L1脂肪细胞敲减Atg101后自噬相关蛋白LC3-Ⅱ显著降低, p62蛋白明显上调, 提示细胞自噬受损。KEGG富集分析发现Atg101共表达基因集主要富集...     

高糖对心肌细胞损害调节新机制:烟酰胺核糖通过Sirt3- p53/PGC-1α改善线粒体自噬及线粒体合成

目的 明确烟酰胺核糖（nicotinamide riboside,NR）对成年小鼠心肌细胞在高糖条件下线粒体自噬及线粒体合成的影响及潜在机制。方法 分离成年小鼠心肌细胞后,将细胞分为:①对照组;②高糖组;③高糖+NR组;④高糖+NR+Sirt3siRNA;⑤高糖+NR+过氧化物酶体增生物活化受体γ共激活剂(Peroxisome proliferator-activated receptor γ co-activator,PGC)-1α siRNA组;⑥高糖+NR+nutlin-3组。采用TUNEL法,流式细胞术检测凋亡指数(AI),JC-1染色检测线粒体膜电位,Western blot法检测自噬相关蛋白Atg5、LC3,p62等蛋白表达水平,线粒体自噬相关蛋白Parkin,线粒体合成相关蛋白PGC-1α,Tfam蛋白表达水平,Sirt3。结果 与对照组相比,高糖干预后细胞AI增加（P<0.01）,线粒体膜电位降低（P<0.01）,PGC-1α、Tfam、Sirt3、Atg5、LC3和Parkin表达降低（P<0.01）、p62表达升高（P<0.01）。加入NR后,细胞凋亡减少（P<0.01）,线粒体膜电位增高（P<0.01）,PGC-1α、Tfam、Sirt3、Atg5、LC3和Parkin表达增高（P<0.01）、p62表达降低（P<0.01）,然而,加入Sirt3siRNA,NR的作用减弱（P<0.01）;加入PGC-1αsiRNA,NR作用减弱（P<0.01）,加入nutlin-3后,NR作用减弱（P<0.01）。结论 NR通过加入p53激动剂nutlin-3,改善线粒体自噬,NR通过提高PGC-1α表达水平改善线粒体合成,最终减轻高糖对成年小鼠心肌细胞的损伤。     

血管紧张素Ⅱ诱发自噬对血管平滑肌细胞表型转换的调控作用

目的观察血管紧张素Ⅱ(AngⅡ)对小鼠主动脉血管平滑肌细胞(VSMC)自噬的影响以及自噬对细胞表型转换的调控作用。方法原代培养小鼠VSMC,用10~(-6)mol/L AngⅡ作用VSMC不同时间,采用Western blot检测微管相关蛋白轻链-3-Ⅱ(LC3-Ⅱ)的表达以观察AngⅡ对VSMC自噬的影响,透射电镜观察对照组及AngⅡ组的自噬小体。用Western blot检测自噬抑制剂3-MA和Baf-A1干预后对AngⅡ诱导自噬及细胞表型转换的影响。使用siRNA抑制自噬相关基因Atg7的表达,qRT-PCR检测转染后Atg7的表达变化,Western blot检测转染siRNA Atg7后对LC3-Ⅱ及细胞表型蛋白标志物的影响。结果 AngⅡ以时间依赖方式促进LC3-Ⅱ表达,自噬抑制剂3-MA抑制AngⅡ促LC3-Ⅱ的表达作用,而Baf-A1则增强AngⅡ促LC3-Ⅱ的表达作用,两种自噬抑制剂均可抑制AngⅡ促VSMC表型转换作用。转染siRNA Atg7后显著抑制AngⅡ促LC3-Ⅱ的表达作用,并可抑制AngⅡ促细胞表型转换作用。结论 AngⅡ促进VSMC从收缩表型转化为合成表型可能是自噬依赖性的,抑制自噬可以抑制AngⅡ诱导的VSMC表型转换。     

白细胞介素10通过促进线粒体自噬调节心肌细胞生物氧化

目的探讨白细胞介素10(IL-10)对心肌细胞葡萄糖代谢的影响及其可能机制。方法使用棕榈酸干预复制心肌脂毒性和葡萄糖利用减少细胞模型,外源性IL-10干预后,以荧光标记的2-脱氧葡萄糖(2-NBDG)摄入法检测心肌细胞的葡萄糖摄取,检测乳酸及体外氧化磷酸分别代表葡萄糖无氧酵解及有氧氧化水平;使用免疫荧光检测线粒体自噬;使用Real-Time PCR及Western Blot检测线粒体自噬关键基因PINK1表达。结果与对照组比较,IL-10处理后,心肌细胞摄入2-NBDG量约增加1倍;细胞培养上清乳酸含量减少近80%;细胞内乳酸含量亦显著减少。IL-10促进微管相关蛋白1轻链3-绿色荧光蛋白(LC3-GFP)在心肌细胞线粒体聚集,促进线粒体自噬,并上调PTEN诱导假定激酶1 (PINK1)表达。结论 IL-10通过促进葡萄糖转入及氧化磷酸化恢复心肌细胞供能物质组成;其对氧化磷酸化的作用可能是通过上调PINK1进而促进线粒体自噬实现的。     

衰老心肌自噬减退的新机制-转录因子EB的关键作用

目的 探讨转录因子EB（TFEB）在衰老心肌自噬减退中的作用。方法 采用老年（22月龄）雄性C57BL/6小鼠为实验对象,以成年（4月龄）雄性C57BL/6小鼠为对照,分析心肌自噬水平、心肌TFEB表达水平。结果 与成年心肌相比,衰老心肌自噬水平显著降低（P<0.05）。衰老心肌中自噬体标志物Atg5、LC3和Beclin-1,溶酶体标志物LAMP1在蛋白和mRNA水平上均出现降低。与成年心肌相比,衰老心肌TFEB蛋白水平显著降低（P<0.05）,衰老心肌细胞核内的TFEB水平下降更为显著（P<0.05）,提示衰老心肌TFEB转录能力减退。给予小剂量雷帕霉素处理,可提高衰老心肌细胞核内TFEB水平,并且改善LC3及LAMP1的mRNA和蛋白水平,提高衰老心肌自噬水平。结论 本研究发现衰老导致的心肌TFEB水平降低严重影响心肌自噬能力,提示TFEB是心肌自噬增龄性减退机制中的关键调节因子。     

Chronic akt activation accentuates aging-induced cardiac hypertrophy and myocardial contractile dysfunction: role of autophagy

Aging is often accompanied with geometric and functional changes in the heart, although the underlying mechanisms remain unclear. Recent evidence has described a potential role of Akt and autophagy in aging-associated organ deterioration. This study was to examine the impact of cardiac-specific Akt activation on aging-induced cardiac geometric and functional changes and underlying mechanisms involved. Cardiac geometry, contractile and intracellular Ca²⁺ properties were evaluated using echocardiography, edge-detection and fura-2 techniques. Level of insulin signaling and autophagy was evaluated by western blot. Our results revealed cardiac hypertrophy (enlarged chamber size, wall thickness, myocyte cross-sectional area), fibrosis, decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca²⁺ release and clearance in aged (24–26 month-old) mice compared with young (3–4 month-old) mice, the effects of which were accentuated by chronic Akt activation. Aging enhanced Akt and mTOR phosphorylation while reducing that of PTEN, AMPK and ACC with a more pronounced response in Akt transgenic mice. GSK3β phosphorylation and eNOS levels were unaffected by aging or Akt overexpression. Levels of beclin-1, Atg5 and LC3-II-to-LC3-I ratio were decreased in aged hearts, the effect of which with the exception of Atg 5 was exacerbated by Akt overactivation. Levels of p62 were significantly enhanced in aged mice with a more pronounced increase in Akt mice. Neither aging nor Akt altered β-glucuronidase activity and cathepsin B although aging reduced LAMP1 level. In addition, rapamycin reduced aging-induced cardiomyocyte contractile and intracellular Ca²⁺ dysfunction while Akt activation suppressed autophagy in young but not aged cardiomyocytes. In conclusion, our data suggest that Akt may accentuate aging-induced cardiac geometric and contractile defects through a loss of autophagic regulation.     

The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy

Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway.     

Autophagy in the heart and liver during normal aging and calorie restriction

Autophagy is a highly regulated intracellular process for the degradation of cellular constituents and essential for the maintenance of a healthy cell. We evaluated the effects of age and life-long calorie restriction on autophagy in heart and liver of young (6 months) and old (26 months) Fisher 344 rats. We observed that the occurrence of autophagic vacuoles was higher in heart than liver. The occurrence of autophagic vacuoles was not affected by age in either tissue, but was increased with calorie restriction in heart but not in liver. Next, we examined the expression of proteins involved in the formation and maturation of autophagosomes (beclin-1, LC3, Atg7, Atg9) or associated with autolysosomes and lysosomes (LAMP-1; cathepsin D). In hearts of both ad libitum-fed and calorie-restricted rats, we observed an increase in expression of beclin-1 and procathepsin D, but not mature cathepsin D, and a decrease in expression of LAMP-1 because of aging. In hearts, calorie restriction stimulated the expression of Atg7 and Atg9 and the lipidation of Atg8 (elevated LC3-II/I ratios) in aged rats. In hearts of ad libitum-fed rats, expression of Atg7 and lipidation of Atg8 were unaffected by age, while the cellular levels of Atg9 were lower in aged animals. Furthermore, we observed that the age- and diet-dependent expression levels of those proteins differed between heart and liver. In conclusion, autophagy in heart and liver did not decrease with age in ad libitum-fed rats, but was enhanced by calorie restriction in the heart. Thus, calorie restriction may mediate some of its beneficial effects by stimulating autophagy in the heart, indicating the potential for cardioprotective therapies.     

Impaired autophagic function in rat islets with aging

Type 2 diabetes is characterized by a deficit in β-cell function and mass, and its incidence increases with age. Autophagy is a highly regulated intracellular process for degrading cytoplasmic components, particularly protein aggregates and damaged organelles. Impaired or deficient autophagy is believed to cause or contribute to aging and age-related disease. Autophagy may be necessary to maintain structure, mass, and function of pancreatic β-cells. In this study, we investigated the effects of age on β-cell function and autophagy in pancreatic islets of 4-month-old (young), 14-month-old (adult), and 24-month-old (old) male Wistar rats. We found that islet β-cell function decreased gradually with age. Protein expression of the autophagy markers LC3/Atg8 and Atg7 exhibited a marked decline in aged islets. The expression of Lamp-2, a good indicator of autophagic degradation rate, was significantly reduced in the islets of old rats, suggesting that autophagic degradation is decreased in the islets of aged rats. However, protein expression of beclin-1/Atg6, which plays an important role in the induction and formation of the pre-autophagosome structure by associating with a multimeric complex of autophagy regulatory proteins (Atg14, Vps34/class 3 PI3 kinase, and Vps15), was most prominent in the islets of adult rats, and was higher in 24-month-old islets than in 4-month-old islets. The levels of p62/SQSTM1 and polyubiquitin aggregates, representing the functions of autophagy and proteasomal degradation, were increased in aging islets. 8-Hydroxydeoxyguanosine, a marker of mitochondrial and nuclear DNA oxidative damage, exhibited strong immunostaining in old islets. Analysis by electron microscopy demonstrated swelling and disintegration of cristae in the mitochondria of aged islets. These results suggest that β-cell and autophagic function in islets decline simultaneously with increasing age in Wistar rats, and that impaired autophagy in the islets of older rats may cause accumulation of misfolded and aggregated proteins and reduce the removal of abnormal mitochondria in β-cells, leading to reduced β-cell function. Dysfunctional autophagy in islets during the aging process may be an important mechanism leading to the development of type 2 diabetes.     

Cardiac overexpression of metallothionein rescues cardiac contractile dysfunction and endoplasmic reticulum stress but not autophagy in sepsis

Sepsis is characterized by systematic inflammation where oxidative damage plays a key role in organ failure. This study was designed to examine the impact of the antioxidant metallothionein (MT) on lipopolysaccharide (LPS)-induced cardiac contractile and intracellular Ca²⁺ dysfunction, oxidative stress, endoplasmic reticulum (ER) stress and autophagy. Mechanical and intracellular Ca²⁺ properties were examined in hearts from FVB and cardiac-specific MT overexpression mice treated with LPS. Oxidative stress, activation of mitogen-activated protein kinase pathways (ERK, JNK and p38), ER stress, autophagy and inflammatory markers iNOS and TNFα were evaluated. Our data revealed enlarged end systolic diameter, decreased fractional shortening, myocyte peak shortening and maximal velocity of shortening/relengthening as well as prolonged duration of relengthening in LPS-treated FVB mice associated with reduced intracellular Ca²⁺ release and decay. LPS treatment promoted oxidative stress (reduced glutathione/glutathione disulfide ratio and ROS generation). Western blot analysis revealed greater iNOS and TNFα, activation of ERK, JNK and p38, upregulation of ER stress markers GRP78, Gadd153, PERK and IRE1α, as well as the autophagy markers Beclin-1, LCB3 and Atg7 in LPS-treated mouse hearts without any change in total ERK, JNK and p38. Interestingly, these LPS-induced changes in echocardiographic, cardiomyocyte mechanical and intracellular Ca²⁺ properties, ROS, stress signaling and ER stress (but not autophagy, iNOS and TNFα) were ablated by MT. Antioxidant N-acetylcysteine and the ER stress inhibitor tauroursodeoxycholic acid reversed LPS-elicited depression in cardiomyocyte contractile function. LPS activated AMPK and its downstream signaling ACC in conjunction with an elevated AMP/ATP ratio, which was unaffected by MT. Taken together, our data favor a beneficial effect of MT in the management of cardiac dysfunction in sepsis.     

Age-related changes in the function of autophagy in rat kidneys

Autophagy is a highly regulated intracellular process for the degradation of cytoplasmic components, especially protein aggregates and damaged organelles. It is essential for maintaining healthy cells. Impaired or deficient autophagy is believed to cause or contribute to aging and age-related disease. In this study, we investigated the effects of age on autophagy in the kidneys of 3-, 12-, and 24-month-old Fischer 344 rats. The results revealed that autophagy-related gene (Atg)7 was significantly downregulated in kidneys of increasing age. The protein expression level of the autophagy marker light chain 3/Atg8 exhibited a marked decline in aged kidneys. The levels of p62/SQSTM1 and polyubiquitin aggregates, representing the function of autophagy and proteasomal degradation, increased in older kidneys. The level of 8-hydroxydeoxyguanosine, a marker of mitochondrial DNA oxidative damage, was also increased in older kidneys. Analysis by transmission electron microscope demonstrated swelling and disintegration of cristae in the mitochondria of aged kidneys. These results suggest that autophagic function decreases with age in the kidneys of Fischer 344 rats, and autophagy may mediate the process of kidney aging, leading to the accumulation of damaged mitochondria.     

mi R-30b inhibits autophagy to alleviate hepatic ischemiareperfusion injury via decreasing the Atg12-Atg5 conjugate

AIM: To explore the role and potential mechanism of miR-30 b regulation of autophagy in hepatic ischemiareperfusion injury(IRI).METHODS: An animal model of hepatic IRI was generated in C57BL/6 mice. For in vitro studies, AML12 cells were immersed in mineral oil for 1 h and then cultured in complete Dulbecco's Modified Eagle's Medium(DMEM)/F12 to simulate IRI. Mice and cells were transfected with miR-30 b agomir/mimics or antagomir/inhibitor to examine the effect of miR-30 b on autophagy to promote hepatic IRI. The expression of miR-30 b was measured by real-time polymerase chain reaction. Apoptotic cells were detected by terminal uridine nickend labeling(TUNEL) staining, and cell viability was detected by methylthiazole tetrazolium assay. The expression of light chain 3, autophagy-related gene(Atg)12, Atg5, P62, and caspase-3 were detected by western blotting analysis.RESULTS: miR-30 b levels were significantly downregulated after hepatic IRI, and the numbers of autophagosomes were increased in response to IRI both in vivo and in vitro. These findings demonstrate that low levels of miR-30 b could promote hepatic IRI. Furthermore, we found that miR-30 b interacted with Atg12-Atg5 conjugate by binding to Atg12. Overexpression of miR-30 b diminished Atg12 and Atg12-Atg5 conjugate levels, which promoted autophagy in response to IR. In contrast, downregulation of miR-30 b was associated with increased Atg12-Atg5 conjugate levels and increased autophagy.CONCLUSION: miR-30 b inhibited autophagy to alleviate hepatic ischemia-reperfusion injury via decreasing the Atg12-Atg5 conjugate.     

Autophagy in the myocardium: Dying for survival?

Autophagy is an intracellular phenomenon in which a cell digests its own constituents. Autophagy is well conserved in nature from lower eukaryotes to mammals and has been attributed to disparate physiological events - including cell death, the mechanism of which is different from apoptosis. However, unlike in apoptosis, in which a family of cysteine proteases (caspases) and a number of other regulatory proteins have been identified and characterized, the mechanism of autophagic cell death remains unclear. In addition, the general mechanisms by which autophagy is initiated and modulated are just emerging, and several lines of evidence indicate that activated class I phosphatidylinositol 3-kinase and mammalian target of rapamycin (mTOR) inhibit autophagy, while class III phosphatidylinositol 3-kinase acts as a facilitator. Autophagy has been attributed to a number of cardiac disorders, such as ischemic cardiomyopathy, cardiac hypertrophy, hemochromatosis and myocardial aging. Induction of ventricular hypertrophy is associated with decreased autophagy, whereas it is enhanced during the regression of hypertrophy. Induction of acute cardiotoxicity by the anticancer drug anthracycline is also associated with massive cardiomyocyte loss due to autophagy (and apoptosis). Myocyte loss due to autophagy has also been reported during progression from compensated hypertrophy to heart failure in a pressure-overloaded model. Although the depth and dimension of the regulatory network that modulates autophagy in mammalian cells has yet to emerge, existing evidence suggests that it is an integral part of maintaining cellular metabolism, organelle homeostasis and redox equilibrium. Thus, it is a likely possibility that autophagy plays a crucial role in maintaining healthy myocytes in the myocardium.     

Assembly and dynamics of the autophagy-initiating Atg1 complex

The autophagy-related 1 (Atg1) complex of Saccharomyces cerevisiae has a central role in the initiation of autophagy following starvation and TORC1 inactivation. The complex consists of the protein kinase Atg1, the TORC1 substrate Atg13, and the trimeric Atg17–Atg31–Atg29 scaffolding subcomplex. Autophagy is triggered when Atg1 and Atg13 assemble with the trimeric scaffold. Here we show by hydrogen–deuterium exchange coupled to mass spectrometry that the mutually interacting Atg1 early autophagy targeting/tethering domain and the Atg13 central domain are highly dynamic in isolation but together form a stable complex with ∼100-nM affinity. The Atg1–Atg13 complex in turn binds as a unit to the Atg17–Atg31–Atg29 scaffold with ∼10-μM affinity via Atg13. The resulting complex consists primarily of a dimer of pentamers in solution. These results lead to a model for autophagy initiation in which Atg1 and Atg13 are tightly associated with one another and assemble transiently into the pentameric Atg1 complex during starvation.The engulfment of cytosolic contents by autophagy is an ancient mechanism for cell survival and homeostasis (1, 2). This process is conserved throughout the Eukarya. Autophagy consists of the surrounding of cellular material in a double-membrane structure known as the phagophore (1), which matures into the autophagosome and fuses with the lysosome. The small-molecule metabolites generated by lysosomal degradation replenish energy stores and biosynthetic precursors. Autophagy, or its dysfunction, has roles in neurodegenerative disease, cancer, infection, inflammation, and aging (3). Despite its central importance in human health and disease, current knowledge of autophagosome biogenesis at the structural and molecular mechanistic level is limited (4). Our laboratory and many others have therefore embarked on a protein-by-protein effort to dissect the structures and interactions responsible for the remarkable process of autophagosome biogenesis.In yeast, autophagosome biogenesis commences at a single locus known as the phagophore assembly site (PAS). The autophagosome is nucleated, at least in part, from a cluster of a small number of vesicles with radii of 15–30 nm that contain the integral membrane protein autophagy-related 9 (Atg9) (5–7). The Atg1 complex, consisting of the subunits Atg1, Atg13, Atg17, Atg29, and Atg31, is thought to have a central role in autophagy initiation at the PAS. Atg1 is a protein kinase, yet the Atg1 complex is thought to have essential roles very early in autophagy that are independent of its kinase activity (8). These probably include organizing the vesicle cluster that goes on to form the phagophore (9). The kinase activity of Atg1 is also essential, in part because it phosphorylates Atg9 (10). In human cells, the Unc51-like kinase 1 (ULK1) and ULK2 complexes are largely conserved and thought to serve similar functions (11). The subunits Atg17, Atg29, and Atg31 appear to be capable of assembling at the PAS constitutively. They thus appear to serve as a preexisting scaffold for the recruitment of Atg1 and Atg13 upon activation.The crystal structure of the Atg17–Atg31–Atg29 complex showed that it dimerizes into a structurally unique double crescent (9, 12). Dimerization occurs via the C terminus of Atg17, and is required for formation of the PAS and for autophagy (9). Autophagy initiation also requires the recruitment of Atg1 and Atg13 to the PAS downstream of Atg17–Atg31–Atg29 (13). Atg1 consists of an N-terminal protein kinase domain, a predicted flexible linker, and a C-terminal early autophagy targeting/tethering (EAT) domain. Atg13 consists of an N-terminal HORMA domain (14) and a very long predicted unstructured central and C-terminal region. The presence of extensive regions of presumed intrinsic disorder in the Atg1 and Atg13 subunits has slowed progress in understanding the structure and assembly of the complete pentameric Atg1 complex. Given the essential role of the Atg1 complex in autophagy initiation, we set out to probe its dynamics using hydrogen–deuterium exchange (HDX) coupled to mass spectrometry (MS). In HDX-MS, the intrinsic exchange rate of amide protons is used to measure protein dynamics (15–17). Highly ordered regions of proteins exchange protons slowly, whereas dynamic regions exchange them rapidly.The translocation of the Atg1 and Atg13 subunits to the PAS upon TORC1 inactivation is a critical event in early autophagy and has been the topic of intensive investigation and debate. Dephosphorylated Atg13 is thought to act as a bridge between Atg1 and Atg17, triggering the assembly of the subunits into a dimer of pentamers. Mutation of the eight identified Atg13 phosphorylation sites to Ala induces Atg1 complex formation in yeast in the absence of autophagy induction (18). Recently, a constitutive interaction between Atg1 and Atg13 was observed (19), consistent with findings in human (20–22) and Drosophila (23) cells. The newer report suggests that the Atg1–Atg13 complex is constitutive and is regulated primarily at the level of its conformation, rather than its assembly. In this work, we probe the dynamics and stability of the Atg1 EAT domain and find that much of it is mobile in the absence of Atg13. We also find that the affinity of Atg1 and Atg13 for one another is very high, on the order of ∼100 nM. By contrast, the affinity of the preassembled Atg1–Atg13 and Atg17–Atg31–Atg29 complexes for one another is two orders of magnitude weaker. These findings have implications for models of Atg1 recruitment to the PAS and so for the mechanism of autophagy initiation.