New label‐free automated survival assays reveal unexpected stress resistance patterns during C. elegans aging

Caenorhabditis elegans is an excellent model for high‐throughput experimental approaches but lacks an automated means to pinpoint time of death during survival assays over a short time frame, that is, easy to implement, highly scalable, robust, and versatile. Here, we describe an automated, label‐free, high‐throughput method using death‐associated fluorescence to monitor nematode population survival (dubbed LFASS for label‐free automated survival scoring), which we apply to severe stress and infection resistance assays. We demonstrate its use to define correlations between age, longevity, and severe stress resistance, and its applicability to parasitic nematodes. The use of LFASS to assess the effects of aging on susceptibility to severe stress revealed an unexpected increase in stress resistance with advancing age, which was largely autophagy‐dependent. Correlation analysis further revealed that while severe thermal stress resistance positively correlates with lifespan, severe oxidative stress resistance does not. This supports the view that temperature‐sensitive protein‐handling processes more than redox homeostasis underpin aging in C. elegans. That the ages of peak resistance to infection, severe oxidative stress, heat shock, and milder stressors differ markedly suggests that stress resistance and health span do not show a simple correspondence in C. elegans.     

Regulatory Coordination between Two Major Intracellular Homeostatic Systems: HEAT SHOCK RESPONSE AND AUTOPHAGY*

The eukaryotic cell depends on multitiered homeostatic systems ensuring maintenance of proteostasis, organellar integrity, function and turnover, and overall cellular viability. At the two opposite ends of the homeostatic system spectrum are heat shock response and autophagy. Here, we tested whether there are interactions between these homeostatic systems, one universally operational in all prokaryotic and eukaryotic cells, and the other one (autophagy) is limited to eukaryotes. We found that heat shock response regulates autophagy. The interaction between the two systems was demonstrated by testing the role of HSF-1, the central regulator of heat shock gene expression. Knockdown of HSF-1 increased the LC3 lipidation associated with formation of autophagosomal organelles, whereas depletion of HSF-1 potentiated both starvation- and rapamycin-induced autophagy. HSP70 expression but not expression of its ATPase mutant inhibited starvation or rapamycin-induced autophagy. We also show that exercise induces autophagy in humans. As predicted by our in vitro studies, glutamine supplementation as a conditioning stimulus prior to exercise significantly increased HSP70 protein expression and prevented the expected exercise induction of autophagy. Our data demonstrate for the first time that heat shock response, from the top of its regulatory cascade (HSF-1) down to the execution stages delivered by HSP70, controls autophagy thus connecting and coordinating the two extreme ends of the homeostatic systems in the eukaryotic cell.     

Glutamine's protection against cellular injury is dependent on heat shock factor-1

Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. This study tested the hypothesis that GLN-mediated activation of the HSP pathway via heat shock factor-1 (HSF-1) is responsible for cellular protection. Wild-type HSF-1 (HSF-1^+/+) and knockout (HSF-1^–/–) mouse fibroblasts were used in all experiments. Cells were treated with GLN concentrations ranging from 0 to 16 mM and exposed to heat stress injury in a concurrent treatment model. Cell viability was assayed with phenazine methosulfate plus tetrazolium salt, HSP-70, HSP-25, and nuclear HSF-1 expression via Western blot analysis, and HSF-1/heat shock element (HSE) binding via EMSA. GLN significantly attenuated heat-stress induced cell death in HSF-1^+/+ cells in a dose-dependent manner; however, the survival benefit of GLN was lost in HSF-1^–/– cells. GLN led to a dose-dependent increase in HSP-70 and HSP-25 expression after heat stress. No inducible HSP expression was observed in HSF-1^–/– cells. GLN increased unphosphorylated HSF-1 in the nucleus before heat stress. This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation. knockout cells; amino acid; heat stress mechanism     

Micronucleus‐Specific Bacterium Holospora elegans Irreversibly Enhances Stress Gene Expression of the Host Paramecium caudatum

ABSTRACT. The bacterium Holospora is an endonuclear symbiont of the ciliate Paramecium. Previously, we reported that paramecia bearing the macronuclear‐specific symbiont Holospora obtusa survived better than symbiont‐free paramecia, even under high temperatures unsuitable for growth. The paramecia with symbionts expressed high levels of hsp70 mRNAs even at 25 °C, a usual growth temperature. We report herein that paramecia bearing the micronuclear‐specific symbiont Holospora elegans also acquire the heat‐shock resistance. Even after the removal of the bacteria from the hosts by treatment with penicillin, the resulting aposymbiotic paramecia nevertheless maintained their heat shock‐resistant nature for over 1 yr. Like symbiotic paramecia, these aposymbiotic paramecia also expressed high levels of both hsp60 and hsp70 mRNAs even at 25 °C. Moreover, analysis by fluorescent in situ hybridization with a probe specific for Holospora 16S rRNA revealed that the 16S rRNA of H. elegans was expressed around the nucleoli of the macronucleus in the aposymbiotic cells. This result suggests the possible transfer of Holospora genomic DNA from the micronucleus into the macronucleus in symbiotic paramecia. Perhaps this exogenous DNA could trigger the aposymbiotic paramecia to induce a stress response, inducing higher expression of Hsp60 and Hsp70, and thus conferring heat‐shock resistance.     

The selective autophagy receptor SQSTM1/p62 improves lifespan and proteostasis in an evolutionarily conserved manner

ABSTRACT

The degradation of specific cargos such as ubiquitinated protein aggregates and dysfunctional mitochondria via macroautophagy/autophagy is facilitated by SQSTM1/p62, the first described selective autophagy receptor in metazoans. While the general process of autophagy plays crucial roles during aging, it remains unclear whether and how selective autophagy mediates effects on longevity and health. Two recent studies in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster observed gene expression changes of the respective SQSTM1 orthologs in response to environmental stressors or age and showed that overexpression of SQSTM1 is sufficient to extend lifespan and improve proteostasis and mitochondrial function in an autophagy-dependent manner in these model organisms. These findings show that increased expression of the selective autophagy receptor SQSTM1 is sufficient to induce aggrephagy in C. elegans, and mitophagy in Drosophila, and demonstrate an evolutionarily conserved role for SQSTM1 in lifespan determination.     

微RNA Cre-miR914参与调控衣藻对高温胁迫的适应过程

高温胁迫是生物所面临的常见环境胁迫,因此在长期进化中生物逐渐进化出了对高温胁迫的高效适应能力.目前,有关藻类对高温胁迫的适应机制研究主要集中在生理调控及其相关的编码基因调控方面,而有关非编码基因对高温适应的调控尚无报道.在前期研究中,我们通过对衣藻细胞的定量PCR筛选和生物信息学分析发现,在多种胁迫处理后Cre-miR914表达下调且其靶基因有可能是RPL18,但对它们的作用却不清楚.本研究中利用生物信息学结合降解组测序确定了Cre-miR914的靶基因是RPL18,接着利用定量PCR验证Cre-miR914及其靶基因的表达情况,发现Cre-miR914表达在高温处理后明显下调,而RPL18表达明显上调,同时通过构建Cre-miR914过表达株和靶基因RPL18过表达株,结合高温胁迫处理和抗性表型研究,发现Cre-miR914过表达明显降低衣藻对抗高温胁迫能力,而靶基因RPL18过表达提高了衣藻对抗高温胁迫能力.本研究发现了一个microRNA参与调控藻类高温适应过程的分子机制,即衣藻通过调控Cre-miR914及其靶基因RPL18表达参与了的高温胁迫适应过程.     

A deuterohemin peptide extends lifespan and increases stress resistance in Caenorhabditis elegans

Abstract

This group has invented a novel deuterohemin containing peptide deuterohemin-AlaHisThrValGluLys (DhHP-6), which has various biological activities including protection of murine ischemia reperfusion injury, improving cell survival and preventing apoptosis. It was hypothesized that DhHP-6 is beneficial on the lifespan of Caenorhabditis elegans (C. elegans) and increases their resistance to heat and oxidative stress. C. elegans were treated with different concentrations of DhHP-6. Survival time and sensitivity to heat and paraquat were investigated. The data demonstrated that the mean survival time of C. elegans was significantly increased (p < 0.05) in the DhHP-6 treated group compared with the control group. The maximum lifespan was not affected by DhHP-6 treatment. DhHP-6 improved the survival rate of C. elegans in the acute heat stress (35°C) and rescued the C. elegans' sensitivity to paraquat in acute oxidative stress. Superoxide dismutase 3 (SOD-3) protein was up-regulated by DhHP-6 treatment. It was further demonstrated that stress resistance genes such as hsp-16.1, hsp-16.49 and sir-2.1 were regulated by DhHP-6. DAF-16 and SIR-2.1 genes are essential for the beneficial effect of DhHP-6. Therefore, the investigation into the beneficial effect of DhHP-6 on C. elegans' lifespan has the potential to develop novel drugs to prevent ageing.