Identification of autophagy as a longevity-assurance mechanism in the aging model Podospora anserina

The filamentous ascomycete Podospora anserina is a well-established aging model in which a variety of different pathways, including those involved in the control of respiration, ROS generation and scavenging, DNA maintenance, proteostasis, mitochondrial dynamics, and programmed cell death have previously been demonstrated to affect aging and life span. Here we address a potential role of autophagy. We provide data demonstrating high basal autophagy levels even in strains cultivated under noninduced conditions. By monitoring an N-terminal fusion of EGFP to the fungal LC3 homolog PaATG8 over the lifetime of the fungus on medium with and without nitrogen supplementation, respectively, we identified a significant increase of GFP puncta in older and in nitrogen-starved cultures suggesting an induction of autophagy during aging. This conclusion is supported by the demonstration of an age-related and autophagy-dependent degradation of a PaSOD1-GFP reporter protein. The deletion of Paatg1, which leads to the lack of the PaATG1 serine/threonine kinase active in early stages of autophagy induction, impairs ascospore germination and development and shortens life span. Under nitrogen-depleted conditions, life span of the wild type is increased almost 4-fold. In contrast, this effect is annihilated in the Paatg1 deletion strain, suggesting that the ability to induce autophagy is beneficial for this fungus. Collectively, our data identify autophagy as a longevity-assurance mechanism in P. anserina and as another surveillance pathway in the complex network of pathways affecting aging and development. These findings provide perspectives for the elucidation of the mechanisms involved in the regulation of individual pathways and their interactions.     

Identification and Characterization of MicroRNAs by High Through-Put Sequencing in Mesenchymal Stem Cells and Bone Tissue from Mice of Age-Related Osteoporosis

The functional deficiencies of bone marrow-derived mesenchymal stem cells (MSCs) may contribute to the aging process and age-related diseases, such as osteoporosis. Although it has been reported that microRNAs (miRNAs) played an important role in mechanisms of gene regulation of aging, and their expression profiles in MSCs osteogenic differentiation were established in recent years, but it is still elusive for the dynamic patterns of miRNAs in aging process. Importantly, the miRNAs in aged bone tissue had not been yet reported so far. Here, we combined high through-put sequencing with computational techniques to detect miRNAs dynamics in MSCs and bone tissue of age-related osteoporosis. Among the detected miRNAs, 59 identified miRNAs in MSCs and 159 in bone showed significantly differential expressions. And more importantly, there existed 8 up-regulated and 30 down-regulated miRNAs in both MSCs and bone during the aging process, with the majority having a trend of down-regulation. Furthermore, after target prediction and KEGG pathway analysis, we found that their targeted genes were significantly enriched in pathways in cancer, which are complex genetic networks, comprise of a number of age-related pathways. These results strongly suggest that these analyzed miRNAs may be negatively involved in age-related osteoporosis, given that most of them showed a decreased expression, which could lay a good foundation for further functional analysis of these miRNAs in age-related osteoporosis.     

Time course of gene expression profiling in the liver of experimental mice infected with Echinococcus multilocularis

Background

Alveolar echinococcosis (AE) is a severe chronic parasitic disease which behaves like a slow-growing liver cancer. Clinical observations suggest that the parasite, Echinococcus multilocularis (E. multilocularis) influences liver homeostasis and hepatic cell metabolism. However, this has never been analyzed during the time course of infection in the common model of secondary echinococcosis in experimental mice.

Methodology/Principal Findings

Gene expression profiles were assessed using DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis metacestode in the liver of susceptible mice. Data were collected at different time points to monitor the dynamic behavior of gene expression. 557 differentially expressed genes were identified at one or more time points, including 351 up-regulated and 228 down-regulated genes. Time-course analysis indicated, at the initial stage of E. multilocularis infection (month 1–2), that most of up-regulated pathways were related to immune processes and cell trafficking such as chemokine-, mitogen-activated protein kinase (MAPK) signaling, and down-regulated pathways were related to xenobiotic metabolism; at the middle stage (month 3), MAPK signaling pathway was maintained and peroxisome proliferator-activated receptor (PPAR) signaling pathway emerged; at the late stage (month 6), most of up-regulated pathways were related to PPAR signaling pathway, complement and coagulation cascades, while down-regulated pathways were related to metabolism of xenobiotics by cytochrome P450. Quantitative RT-PCR analysis of a random selection of 19 genes confirmed the reliability of the microarray data. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was increased in the liver of E. multilocularis infected mice from 2 months to 6 months.

Conclusions

E. multilocularis metacestode definitely exerts a deep influence on liver homeostasis, by modifying a number of gene expression and metabolic pathways. It especially promotes hepatic cell proliferation, as evidenced by the increased PCNA constantly found in all the experimental time-points we studied and by an increased gene expression of key metabolic pathways.     

Stress-dependent opposing roles for mitophagy in aging of the ascomycete Podospora anserina

Mitochondrial dysfunction is causatively linked to organismal aging and the development of degenerative diseases. Here we describe stress-dependent opposing roles of mitophagy, the selective autophagic degradation of mitochondria, in aging and life-span control. We report that the ablation of the mitochondrial superoxide dismutase which is involved in reactive oxygen species (ROS) balancing, does not affect life span of the fungal aging model Podospora anserina, although superoxide levels are strongly increased and complex I-dependent respiration is impaired. This unexpected phenotype depends on functional autophagy, particularly mitophagy, which is upregulated during aging of this mutant. It identifies mitophagy as a prosurvival response involved in the control of mitohormesis, the well-known beneficial effect of mild mitochondrial oxidative stress. In contrast, excessive superoxide stress turns mitophagy to a prodeath pathway and leads to accelerated aging. Overall our data uncover mitophagy as a dynamic pathway that specifically responds to different levels of mitochondrial oxidative stress and thereby affects organismal aging.     

p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene

p73, a member of the p53 tumor suppressor family, is involved in neurogenesis, sensory pathways, immunity, inflammation, and tumorigenesis. How p73 is able to participate in such a broad spectrum of different biological processes is still largely unknown. Here, we report a novel role of p73 in regulating lipid metabolism by direct transactivation of the promoter of autophagy-related protein 5 (ATG5), a gene whose product is required for autophagosome formation. Following nutrient deprivation, the livers of p73-deficient mice demonstrate a massive accumulation of lipid droplets, together with a low level of autophagy, suggesting that triglyceride hydrolysis into fatty acids is blocked owing to deficient autophagy (macrolipophagy). Compared with wild-type mice, mice functionally deficient in all the p73 isoforms exhibit decreased ATG5 expression and lower levels of autophagy in multiple organs. We further show that the TAp73α is the critical p73 isoform responsible for inducing ATG5 expression in a p53-independent manner and demonstrate that ATG5 gene transfer can correct autophagy and macrolipophagy defects in p73-deficient hepatocytes. These data strongly suggest that the p73–ATG5 axis represents a novel, key pathway for regulating lipid metabolism through autophagy. The identification of p73 as a major regulator of autophagy suggests that it may have an important role in preventing or delaying disease and aging by maintaining a homeostatic control.     

Unmasking a temperature-dependent effect of the P. anserina i-AAA protease on aging and development

Different molecular pathways involved in maintaining mitochondrial function are of fundamental importance to control cellular homeostasis. Mitochondrial i-AAA protease is part of such a surveillance system, and PaIAP is the putative ortholog in the fungal aging model Podospora anserina. Here, we investigate the role of PaIAP in aging and development. Deletion of the gene encoding PaIAP resulted in a specific phenotype. When incubated at 27°C, spore germination and fruiting body formation are not different from that of the corresponding wild-type strain. Unexpectedly, the lifespan of the deletion strain is strongly increased. In contrast, cultivation at an elevated temperature of 37°C leads to impairments in spore germination and fruiting body formation and to a reduced lifespan. The higher PaIAP abundance in wild-type strains of the fungus grown at elevated temperature and the phenotype of the deletion strain unmasks a temperature-related role of the protein. The protease appears to be part of a molecular system that has evolved to allow survival under changing temperatures, as they characteristically occur in nature.Key words: Podospora anserina, aging, mitochondria, protein quality control, temperature, i-AAA protease     

不同温度条件下鲁氏耶尔森氏菌的链特异性转录组分析

为鉴定鱼源鲁氏耶尔森氏菌(Yersinia ruckeri) SC09菌株水生环境中不同温度的转录组水平上的差异, 研究采用链特异性转录组测序(Strand-specific RNA-seq)技术对菌体生理温度(28℃)和实验培养温度(37℃)下进行链特异性测序, 原始数据质控后, 筛选得到差异表达基因, 通过KEGG (Kyoto Encyclopedia of Genes and Genomes)数据库对差异表达基因进行富集分析, 并利用Rockhopper软件筛选出的重要原核生物基因簇进行验证。结果显示, 共筛选获得173个显著差异表达基因(P<0.05), 其中包括58个上调基因, 主要富集到一些特殊的碳水化合物代谢相关的通路中; 以及115个下调基因, 主要富集到双组份信号系统中与三羧酸循环相关的代谢通路上, 同时部分基因富集到编码鞭毛素相关的基因簇中。结果表明, 相对于37℃的实验室培养温度, 在水生环境的生理温度条件下(28℃) SC09菌株拥有较高的运动性和较强的葡萄糖代谢, 但相对的SC09菌株代谢一些特殊糖类的能力减弱。     

Autophagy compensates impaired energy metabolism in CLPXP‐deficient Podospora anserina strains and extends healthspan

The degradation of nonfunctional mitochondrial proteins is of fundamental relevance for maintenance of cellular homeostasis. The heteromeric CLPXP protein complex in the mitochondrial matrix is part of this process. In the fungal aging model Podospora anserina, ablation of CLPXP leads to an increase in healthy lifespan. Here, we report that this counterintuitive increase depends on a functional autophagy machinery. In PaClpXP mutants, autophagy is involved in energy conservation and the compensation of impairments in respiration. Strikingly, despite the impact on mitochondrial function, it is not mitophagy but general autophagy that is constitutively induced and required for longevity. In contrast, in another long‐lived mutant ablated for the mitochondrial PaIAP protease, autophagy is neither induced nor required for lifespan extension. Our data provide novel mechanistic insights into the capacity of different forms of autophagy to compensate impairments of specific components of the complex mitochondrial quality control network and about the biological role of mitochondrial CLPXP in the control of cellular energy metabolism.     

基于转录组分析铜绿假单胞菌DN1降解荧蒽特性

[背景]铜绿假单胞菌DN1是一株从石油污染土壤中分离筛选到的具有广谱降解功能的菌株。[目的]深入了解荧蒽胁迫条件下铜绿假单胞菌DN1降解污染物过程中重要的降解相关基因信息。[方法]通过高通量测序技术对铜绿假单胞菌DN1进行转录组测序,对其所有的转录本进行KEGG (kyoto encyclopedia of genes and genomes)分类和Pathway注释、GO (gene ontology)分类和富集分析。[结果]转录组测序显示:与对照组相比,荧蒽诱导组检测到6 189个基因,其中1 919个基因上调表达,1 603个基因下调表达。KEGG注释分析显示差异上调表达基因匹配到了112个KEGG代谢途径,注释到"代谢途径"的1 408个基因(约占总差异基因的73.4%)中有317个基因参与了碳氢化合物代谢及含有苯环结构的异源生物质的生物降解,占"代谢途径"的16.53%,暗示了菌株DN1降解荧蒽可能与这些途径有密切关系。另外,主要代谢途径中的差异表达基因主要集中在ABC转运系统、氨基酸生物合成、双组分系统及碳代谢,这些途径大多数参与了底物的识别转运、信号转导及基因表达调控。[结论]进一步拓展了铜绿假单胞菌DN1在荧蒽胁迫条件下的代谢途径和逆境反应,也为微生物修复环境污染物研究夯实了理论基础。     

雷帕霉素靶蛋白与自噬在衰老中的交互作用

衰老是一个非常复杂的过程,与细胞和组织中累积的各种大分子（DNA、蛋白质和脂质）损伤密不可分,并且是由细胞中不同的信号通道共同调控的结果,而雷帕霉素靶标途径就是其中的一种。该途径整合了各种来自细胞内外的信号以调控细胞的生长、增殖和代谢。越来越多证据表明,雷帕霉素靶蛋白（target of rapamycin,TOR）控制着细胞和组织老化的速度,影响着整个机体衰老过程。另外TOR参与调控自噬的发生,而自噬能使生物大分子和细胞器降解并回收重复利用。多种生物模型研究发现,衰老其实是与自噬的不足有关联。本文对TOR和自噬在衰老过程中的作用和相互关系进行综述,为发展与老年疾病相关的新型治疗方法提供思路。