Autophagy in aging and neurodegenerative diseases: implications for pathogenesis and therapy

Neurodegenerative diseases, such as Alzheimer's disease Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, share a common cellular and molecular pathogenetic mechanism involving aberrant misfolded protein or peptide aggregation and deposition. Autophagy represents a major route for degradation of aggregated cellular proteins and dysfunctional organelles. Emerging studies have demonstrated that up-regulation of autophagy can lead to decreased levels of these toxic aggregate-prone proteins, and is beneficial in the context of aging and various models of neurodegenerative diseases. Understanding the signaling pathways involved in the regulation of autophagy is crucial to the development of strategies for therapy. This review will discuss the cellular and molecular mechanisms of autophagy and its important role in the pathogenesis of aging and neurodegenerative diseases, and the ongoing drug discovery strategies for therapeutic modulation.     

微管相关蛋白1轻链3及Beclin-1在硅沉着病大鼠肺泡巨噬细胞中的表达

目的观察自噬相关蛋白——微管相关蛋白1轻链3(LC-3)、Beclin-1在硅沉着病大鼠肺泡巨噬细胞中的表达,从细胞自噬角度探讨硅沉着病形成的分子机制。方法 50只成年雄性SD大鼠随机分为2组(n=25):对照组和硅沉着病模型组,每组再分5个时相组。采用非暴露气管灌注二氧化硅(SiO2)粉尘混悬液法(50g/L)建立大鼠硅沉着病模型。分别于造模后1、3、7、14及28 d分批处死5只大鼠,进行原位支气管肺泡灌洗,获取肺泡巨噬细胞,进行培养纯化和富集后用于后续研究。HE染色及透射电子显微镜观察肺泡巨噬细胞形态学改变;免疫细胞化学法检测LC-3、Beclin-1的表达及分布;免疫印迹法检测LC-3、Beclin-1的蛋白表达量。结果与对照组相比模型组肺泡巨噬细胞体积较大,胞质丰富,部分细胞内可见硅沉着吞噬颗粒,电镜下可见自噬体形成;模型组LC-3、Beclin-1在各时间点的表达较对照组均增多(P0.05),1 d即开始增多,随着时间的延长表达逐渐增多,至14 d时达高峰(P0.05),28d时回落,但仍高于对照组的表达。结论在硅沉着病大鼠肺泡巨噬细胞中有自噬的激活,肺泡巨噬细胞自噬参与了大鼠硅沉着病的病理进程。     

微管末端结合蛋白1在Siha细胞自噬时的表达和意义

目的 探讨宫颈癌Siha细胞自噬过程中微管末端结合蛋白1（EB1）基因的表达变化。 方法 Hank平衡盐溶液（HBSS）和秋水仙素处理体外培养的宫颈癌Siha细胞,分别采用实时定量PCR和Western blotting检测EB1基因、LC3和p62的表达,荧光显微镜检测特异性绿色荧光蛋白（GFP)-LC3以证实自噬体形成。结果 宫颈癌Siha细胞随HBSS作用时间延长,LC3 mRNA、EB1 mRNA、LC3-Ⅱ蛋白和EB1蛋白的表达均呈时间依赖性增加,差异具有统计学意义(P<0.05)。EB1 mRNA和LC3 mRNA表达呈正相关,具有统计学意义(P<0.05)。p62 mRNA和蛋白在HBSS作用后的表达呈时间依赖性降低,差异具有统计学意义(P<0.05),EB1 mRNA和p62 mRNA表达呈负相关,具有统计学意义(P<0.05)。GFP-LC3结果显示,在HBSS作用12 h后,Siha细胞胞质中GFP-LC3强度和数目明显增加,含有GFP信号的细胞数量也明显增加。秋水仙素处理后,LC3、p62和EB1 mRNA及蛋白均未发生显著改变,差异无统计学意义(P>0.05),但此时几乎检测不到含GFP-LC3荧光信号的细胞。 结论 EB1在饥饿状态Siha细胞中高表达,此时伴随LC3表达增加、p62表达降低以及自噬体形成增加,而用秋水仙素破坏EB1赖以作用的微管时,上述现象消失,充分表明EB1参与细胞自噬的可能。     

DHRSX,A Novel Non-Classical Secretory Protein Associated With Starvation Induced Autophagy

Dehydrogenase/reductase (SDR family) X-linked (DHRSX) is a novel human gene without any substantial functional annotation and was initially cloned and identified in our laboratory. In this study, we present evidence that it encodes a non-classical secretory protein and promotes starvation induced autophagy. Using the Baf.A1 assay and N-terminal sequencing, we showed that DHRSX is secreted in a non-classical form. We expressed and purified a recombinant human GST-DHRSX fusion protein. Functional studies revealed that HeLa and U2OS cells overexpressing DHRSX or treated with the GST-DHRSX fusion protein exhibited higher levels of starvation-induced autophagy, resulting in increased endogenous LC3-II levels, a punctate GFP-LC3 distribution, and structures associated with autophagy, with a lower accumulation of autophagy substrates such as p62 and polyQ80. Accordingly, knockdown of endogenous DHRSX through specific siRNAs reduced LC3-II levels obviously in U2OS cells induced by starvation. Collectively, these results demonstrate that DHRSX is a novel non-classical secretory protein involved in the positive regulation of starvation induced autophagy and provide a new avenue for research on this protein family and autophagy regulation.     

Accelerating the clearance of mutant huntingtin protein aggregates through autophagy induction by europium hydroxide nanorods

Autophagy is one of the well-known pathways to accelerate the clearance of protein aggregates, which contributes to the therapy of neurodegenerative diseases. Although there are numerous reports that demonstrate the induction of autophagy with small molecules including rapamycin, trehalose and lithium, however, there are few reports mentioning the clearance of aggregate-prone proteins through autophagy induction by nanoparticles. In the present article, we have demonstrated that europium hydroxide [Eu^III(OH)₃] nanorods can reduce huntingtin protein aggregation (EGFP-tagged huntingtin protein with 74 polyQ repeats), responsible for neurodegenerative diseases. Again, we have found that these nanorods induce authentic autophagy flux in different cell lines (Neuro 2a, PC12 and HeLa cells) through the expression of higher levels of characteristic autophagy marker protein LC3-II and degradation of selective autophagy substrate/cargo receptor p62/SQSTM1. Furthermore, depression of protein aggregation clearance through the autophagy blockade has also been observed by using specific inhibitors (wortmannin and chloroquine), indicating that autophagy is involved in the degradation of huntingtin protein aggregation. Since [Eu^III(OH)₃] nanorods can enhance the degradation of huntingtin protein aggregation via autophagy induction, we strongly believe that these nanorods would be useful for the development of therapeutic treatment strategies for various neurodegenerative diseases in near future using nanomedicine approach.     

Hinokitiol induces cell death and inhibits epidermal growth factor-induced cell migration and signaling pathways in human cervical adenocarcinoma

ObjectiveThe aim of this study was to examine the antitumor activity of hinokitiol for its clinical application in the treatment of human cervical carcinoma.Materials and methodsCervical carcinoma HeLa cells were treated by different concentrations of hinokitiol. Flow cytometry was used to analyze cell cycle. Senescence-associated β-galactosidase (SA-β-gal) assay was used to identify senescent cells. The effects of hinokitiol on EGF-induced cell migration were determined by wound healing and transwell migration assays. Western blot was used to detect proteins involved in cell cycle progression, apoptosis, autophagy, and EGF-induced signaling pathways.ResultsHinokitiol suppressed cell viability in a dose-dependent manner. Flow cytometric analysis indicated that hinokitiol treatment resulted in cell cycle arrest at G1 phase, with reduced number of cells in the G2/M phase. Western blot analysis further demonstrated that hinokitiol treatment increased the levels of p53 and p21, and concomitantly reduced the expression of cell cycle regulatory proteins, including cyclin D and cyclin E. SA-β-gal assay showed that hinokitiol treatment significantly induced β-galactosidase activity. In addition, treatment with hinokitiol increased the accumulation of the autophagy regulators, beclin 1 and microtubule-associated protein 1 light chain 3 (LC3-II), in a dose-dependent manner; however, it did not induce caspase-3 activation and poly ADP ribose polymerase (PARP) cleavage. In addition, epidermal growth factor-induced cell migration and c-Jun N-terminal kinase (JNK) and focal adhesion kinase (FAK) phosphorylation were significantly inhibited by hinokitiol.ConclusionOur findings revealed that hinokitiol might serve as a potential therapeutic agent for cervical carcinoma therapy.     

ARHI与妇科恶性肿瘤的关系

自噬是细胞通过自我消化实现大分子物质的循环再利用,维持细胞自身稳定的生物学过程。自噬活性的变化、自噬性细胞死亡与恶性肿瘤的发生、发展有关。自噬相关基因ARHI通过调节自噬活性,在肿瘤的发生、发展中发挥作用。ARHI调节自噬信号通路中某些关键激酶可能为肿瘤的治疗提供新的药物作用靶点。     

雷公藤甲素通过诱导细胞自噬促进结肠癌CT26细胞死亡

目的探讨雷公藤甲素诱导结肠癌CT26细胞自噬与细胞死亡之间的关系。方法体外培养小鼠结肠癌CT26细胞,应用免疫荧光、荧光双标自噬腺病毒载体(Ad-mRFP-GFP-LC3)技术,在激光扫描共焦显微镜下观察LC3的表达轮廓及水平,并结合免疫印迹法对LC3蛋白、P62蛋白做半定量分析;进而用单溶液细胞增殖检测(MTS)试剂测定CT26细胞增殖抑制率;用Annexin V-FITC/PI双染、流式细胞术检测细胞凋亡/死亡率。结果雷公藤甲素能诱导CT26细胞出现明确的自噬流;激活细胞自噬可提高CT26细胞的晚期凋亡率。与雷公藤甲素组比较,雷帕霉素与其联用能显著增强CT26细胞死亡。结论自噬可能介导了雷公藤甲素诱导CT26细胞死亡的过程。     

流体剪切力调节血管内皮细胞自噬的研究进展

细胞自噬作为细胞的一种防御和应激调控机制,参与维持细胞的代谢平衡。在血管内皮细胞中,自噬是影响内皮细胞功能的一个重要因素。流体剪切力也是影响血管内皮细胞形态和功能的重要因素之一。而血管内皮细胞生理功能的维持在整个心血管系统的正常运转中起着重要的作用。我们主要对血管内皮细胞的功能,细胞自噬和流体剪切力对血管内皮细胞功能的调节作用,以及流体剪切力对血管内皮细胞自噬的调节作用等方面进行综述。