Yeast chronological lifespan and proteotoxic stress: is autophagy good or bad?

Autophagy, a highly conserved proteolytic mechanism of quality control, is essential for the maintenance of metabolic and cellular homoeostasis and for an efficient cellular response to stress. Autophagy declines with aging and is believed to contribute to different aspects of the aging phenotype. The nutrient-sensing pathways PKA (protein kinase A), Sch9 and TOR (target of rapamycin), involved in the regulation of yeast lifespan, also converge on a common targeted process: autophagy. The molecular mechanisms underlying the regulation of autophagy and aging by these signalling pathways in yeast, with special attention to the TOR pathway, are discussed in the present paper. The question of whether or not autophagy could contribute to yeast cell death occurring during CLS (chronological lifespan) is discussed in the light of our findings obtained after autophagy activation promoted by proteotoxic stress. Autophagy progressively increases in cells expressing the aggregation-prone protein α-synuclein and seems to participate in the early cell death and shortening of CLS under these conditions, highlighting that autophagic activity should be maintained below physiological levels to exert its promising anti-aging effects.     

Enhancement of mitochondrial function correlates with the extension of lifespan by caloric restriction and caloric restriction mimetics in yeast

Caloric restriction mimetics (CRMs) have been developed to mimic the effects of caloric restriction (CR). However, research reports for the effects of CRMs are often times inconsistent across different research groups. Therefore, in this study, we compared seven identified CRMs which extend the lifespans of various organisms including caffeine, curcumin, dapsone, metformin, rapamycin, resveratrol, and spermidine to CR for mitochondrial function in a single model, Saccharomyces cerevisiae. In this organism, rapamycin extended chronological lifespan (CLS), but other CRMs failed to extend CLS. Rapamycin enhanced mitochondrial function like CR did, but other CRMs did not. Both CR and rapamycin worked on mitochondrial function, but they worked at different windows of time during the chronological aging process.     

Regulation of yeast chronological life span by TORC1 via adaptive mitochondrial ROS signaling

Here we show that yeast strains with reduced target of rapamycin (TOR) signaling have greater overall mitochondrial electron transport chain activity during growth that is efficiently coupled to ATP production. This metabolic alteration increases mitochondrial membrane potential and reactive oxygen species (ROS) production, which we propose supplies an adaptive signal during growth that extends chronological life span (CLS). In strong support of this concept, uncoupling respiration during growth or increasing expression of mitochondrial manganese superoxide dismutase significantly curtails CLS extension in tor1Δ strains, and treatment of wild-type strains with either rapamycin (to inhibit TORC1) or menadione (to generate mitochondrial ROS) during growth is sufficient to extend CLS. Finally, extension of CLS by reduced TORC1/Sch9p-mitochondrial signaling occurs independently of Rim15p and is not a function of changes in media acidification/composition. Considering the conservation of TOR-pathway effects on life span, mitochondrial ROS signaling may be an important mechanism of longevity regulation in higher organisms.     

A genomic analysis of chronological longevity factors in budding yeast

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.     

SNCA (α-synuclein)-induced toxicity in yeast cells is dependent on Sir2-mediated mitophagy

SNCA (α-synuclein) misfolding and aggregation is strongly associated with both idiopathic and familial forms of Parkinson disease (PD). Evidence suggests that SNCA has an impact on cell clearance routes and protein quality control systems such as the ubiquitin-proteasome system (UPS) and autophagy. Recent advances in the key role of the autosomal recessive PARK2/PARKIN and PINK1 genes in mitophagy, highlighted this process as a prominent new pathogenic mechanism. Nevertheless, the role of autophagy/mitophagy in the pathogenesis of sporadic and autosomal dominant familial forms of PD is still enigmatic. The yeast Saccharomyces cerevisiae is a powerful “empty room” model that has been exploited to clarify different molecular aspects associated with SNCA toxicity, which combines the advantage of being an established system for aging research. The contribution of autophagy/mitophagy for the toxicity induced by the heterologous expression of the human wild-type SNCA gene and the clinical A53T mutant during yeast chronological life span (CLS) was explored. A reduced CLS together with an increase of autophagy and mitophagy activities were observed in cells expressing both forms of SNCA. Impairment of mitophagy by deletion of ATG11 or ATG32 resulted in a CLS extension, further implicating mitophagy in the SNCA toxicity. Deletion of SIR2, essential for SNCA toxicity, abolished autophagy and mitophagy, thereby rescuing cells. These data show that Sir2 functions as a regulator of autophagy, like its mammalian homolog, SIRT1, but also of mitophagy. Our work highlights that increased mitophagy activity, mediated by the regulation of ATG32 by Sir2, is an important phenomenon linked to SNCA-induced toxicity during aging.     

SKO1 deficiency extends chronological lifespan in Saccharomyces cerevisiae

ABSTRACT

Sko1 plays a key role in the control of gene expression by osmotic and oxidative stress in yeast. We demonstrate that the decrease in chronological lifespan (CLS) of hog1Δ cells was suppressed by SKO1 deletion. sko1Δ single mutant cells were shown to have a longer CLS, thus implicating Sko1 in the regulation of their CLS.     

A genomic analysis of chronological longevity factors in budding yeast

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.Key words: aging, genomic, screen, lifespan, yeast, C. elegans, pH, chronological, replicative     

Calorie restriction extends the chronological lifespan of Saccharomyces cerevisiae independently of the Sirtuins

Calorie restriction (CR) extends the mean and maximum lifespan of a wide variety of organisms ranging from yeast to mammals, although the molecular mechanisms of action remain unclear. For the budding yeast Saccharomyces cerevisiae reducing glucose in the growth medium extends both the replicative and chronological lifespans (CLS). The conserved NAD(+)-dependent histone deacetylase, Sir2p, promotes replicative longevity in S. cerevisiae by suppressing recombination within the ribosomal DNA locus and has been proposed to mediate the effects of CR on aging. In this study, we investigated the functional relationships of the yeast Sirtuins (Sir2p, Hst1p, Hst2p, Hst3p and Hst4p) with CLS and CR. SIR2, HST2, and HST4 were not major regulators of CLS and were not required for the lifespan extension caused by shifting the glucose concentration from 2 to 0.5% (CR). Deleting HST1 or HST3 moderately shortened CLS, but did not prevent CR from extending lifespan. CR therefore works through a Sirtuin-independent mechanism in the chronological aging system. We also show that low temperature or high osmolarity additively extends CLS when combined with CR, suggesting that these stresses and CR act through separate pathways. The CR effect on CLS was not specific to glucose. Restricting other simple sugars such as galactose or fructose also extended lifespan. Importantly, growth on nonfermentable carbon sources that force yeast to exclusively utilize respiration extended lifespan at nonrestricted concentrations and provided no additional benefit when restricted, suggesting that elevated respiration capacity is an important determinant of chronological longevity.     

New rapid screening method for anti-aging compounds using budding yeast and identification of beauveriolide I as a potent active compound

The chronological lifespan (CLS) of budding yeast is a model for the aging of post-mitotic cells in higher eukaryotes. We report here the development of a new method to assess yeast CLS. The new assay is simple, convenient and labor-saving. We applied this new method to screen natural compounds isolated from mushrooms and discovered beauveriolide I as a potent anti-aging agent.     

Mitochondrial respiratory thresholds regulate yeast chronological life span and its extension by caloric restriction

We have explored the role of mitochondrial function in aging by genetically and pharmacologically modifying yeast cellular respiration production during the exponential and/or stationary growth phases and determining how this affects chronological life span (CLS). Our results demonstrate that respiration is essential during both growth phases for standard CLS, but that yeast have a large respiratory capacity, and only deficiencies below a threshold (~40% of wild-type) significantly curtail CLS. Extension of CLS by caloric restriction also required respiration above a similar threshold during exponential growth and completely alleviated the need for respiration in the stationary phase. Finally, we show that supplementation of media with 1% trehalose, a storage carbohydrate, restores wild-type CLS to respiratory-null cells. We conclude that mitochondrial respiratory thresholds regulate yeast CLS and its extension by caloric restriction by increasing stress resistance, an important component of which is the optimal accumulation and mobilization of nutrient stores.     

酵母Sirtuins在细胞寿命中的作用

酿酒酵母(以下简略为酵母)作为寿命分析模型广泛应用于寿命研究领域。酵母寿命分析方法有两种,分别是复制型酵母寿命分析法和时序型酵母寿命分析法。目前,通过酵母寿命分析模型已识别出包括SIR2在内的多个寿命调节基因。SIR2是目前较好的被确立起来的寿命调节基因,具有NAD依赖型脱乙酰化酶的活性,从原核生物到真核生物都有良好的保守性。Sirtuins (Sir2蛋白家族的总称)在细胞内具有功能上的多样性,其中包括对于压力耐受的调节、基因转录的调节、代谢通路的调节以及寿命调节作用等。Sir2是Sirtuins家族最早发现的成员,其功能是参与异染色质结构域转录的沉默调节,同时还参与复制型酵母寿命的调节。已证明,SIR2的缺失会缩短酵母的寿命,基因表达的增高会延长寿命。Sir2的高等真核生物的同源蛋白也被证实参与衰老相关疾病的调节。本文中,我们将阐述Sir2以及Sir2的酵母同源蛋白Hst1-Hst4的功能,以及由它们调节的酵母寿命。     

Small Molecule Enhancers of Rapamycin-Induced TOR Inhibition Promote Autophagy,Reduce Toxicity in Huntington’s Disease Models and Enhance Killing of Mycobacteria by Macrophages

Upregulation of autophagy may have therapeutic benefit in a range of diseases that include neurodegenerative conditions caused by intracytosolic aggregate-prone proteins, such as Huntington’s disease, and certain infectious diseases, such as tuberculosis. The best-characterized drug that enhances autophagy is rapamycin, an inhibitor of the TOR (target of rapamycin) proteins, which are widely conserved from yeast to man. Unfortunately, the side effects of rapamycin, especially immunosuppression, preclude its use in treating certain diseases including tuberculosis, which accounts for approximately 2 million deaths worldwide each year, spurring interest in finding novel drugs that selectively enhance autophagy. We have recently reported a novel two-step screening process for the discovery of such compounds. We first identified compounds that enhance the growth-inhibitory effects of rapamycin in the budding yeast Saccharomyces cerevisiae, which we termed small molecule enhancers of rapamycin (SMERs). Next we showed that three SMERs induced autophagy independently, or downstream of mTOR, in mammalian cells, and furthermore enhanced the clearance of a mutant huntingtin fragment in cell disease models. These SMERs also protected against mutant huntingtin fragment toxicity in Drosophila. We have subsequently tested two of the SMERs in models of tuberculosis and both enhance the killing of mycobacteria by primary human macrophages.

Addendum to:

Small Molecules Enhance Autophagy and Reduce Toxicity in Huntington's Disease Models

S. Sarkar, E.O. Perlstein, S. Imarisio, S. Pineau, A. Cordenier, R.L. Maglathlin, J.A. Webster, T.A. Lewis, C.J. O'Kane, S.L. Schreiber and D.C. Rubinsztein

Nat Chem Biol 2007; 3:331-8     

High plasma concentrations of acyl-coenzyme A binding protein (ACBP) predispose to cardiovascular disease: Evidence for a phylogenetically conserved proaging function of ACBP

Autophagy defects accelerate aging, while stimulation of autophagy decelerates aging. Acyl-coenzyme A binding protein (ACBP), which is encoded by a diazepam-binding inhibitor (DBI), acts as an extracellular feedback regulator of autophagy. As shown here, knockout of the gene coding for the yeast orthologue of ACBP/DBI (ACB1) improves chronological aging, and this effect is reversed by knockout of essential autophagy genes (ATG5, ATG7) but less so by knockout of an essential mitophagy gene (ATG32). In humans, ACBP/DBI levels independently correlate with body mass index (BMI) as well as with chronological age. In still-healthy individuals, we find that high ACBP/DBI levels correlate with future cardiovascular events (such as heart surgery, myocardial infarction, and stroke), an association that is independent of BMI and chronological age, suggesting that ACBP/DBI is indeed a biomarker of “biological” aging. Concurringly, ACBP/DBI plasma concentrations correlate with established cardiovascular risk factors (fasting glucose levels, systolic blood pressure, total free cholesterol, triglycerides), but are inversely correlated with atheroprotective high-density lipoprotein (HDL). In mice, neutralization of ACBP/DBI through a monoclonal antibody attenuates anthracycline-induced cardiotoxicity, which is a model of accelerated heart aging. In conclusion, plasma elevation of ACBP/DBI constitutes a novel biomarker of chronological aging and facets of biological aging with a prognostic value in cardiovascular disease.     

The migration of mitochondrial DNA fragments to the nucleus affects the chronological aging process of Saccharomyces cerevisiae

Migration of fragmented mitochondrial DNA (mtDNA) to the nucleus has been shown to occur in multiple species including yeast, plants, and mammals. Several human diseases, including Pallister–Hall syndrome and mucolipidosis, can be initiated by mtDNA insertion mutagenesis of nuclear DNA. In yeast, we demonstrated that the rate of mtDNA fragments translocating to the nucleus increases during chronological aging. The yeast chronological lifespan (CLS) is determined by the survival of nondividing cell populations. Whereas yeast strains with elevated migration rates of mtDNA fragments to the nucleus showed accelerated chronological aging, strains with decreased mtDNA transfer rates to the nucleus exhibited an extended CLS. Although one of the most popular theories of aging is the free radical theory, migration of mtDNA fragments to the nucleus may also contribute to the chronological aging process by possibly increasing nuclear genomic instability in cells with advanced age.     

Lifespan Extension by Methionine Restriction Requires Autophagy-Dependent Vacuolar Acidification

Reduced supply of the amino acid methionine increases longevity across species through an as yet elusive mechanism. Here, we report that methionine restriction (MetR) extends yeast chronological lifespan in an autophagy-dependent manner. Single deletion of several genes essential for autophagy (ATG5, ATG7 or ATG8) fully abolished the longevity-enhancing capacity of MetR. While pharmacological or genetic inhibition of TOR1 increased lifespan in methionine-prototroph yeast, TOR1 suppression failed to extend the longevity of methionine-restricted yeast cells. Notably, vacuole-acidity was specifically enhanced by MetR, a phenotype that essentially required autophagy. Overexpression of vacuolar ATPase components (Vma1p or Vph2p) suffices to increase chronological lifespan of methionine-prototrophic yeast. In contrast, lifespan extension upon MetR was prevented by inhibition of vacuolar acidity upon disruption of the vacuolar ATPase. In conclusion, autophagy promotes lifespan extension upon MetR and requires the subsequent stimulation of vacuolar acidification, while it is epistatic to the equally autophagy-dependent anti-aging pathway triggered by TOR1 inhibition or deletion.     

Small molecule enhancers of rapamycin-induced TOR inhibition promote autophagy, reduce toxicity in Huntington's disease models and enhance killing of mycobacteria by macrophages

Upregulation of autophagy may have therapeutic benefit in a range of diseases that includes neurodegenerative conditions caused by intracytosolic aggregate-prone proteins, such as Huntington's disease, and certain infectious diseases, such as tuberculosis. The best-characterized drug that enhances autophagy is rapamycin, an inhibitor of the TOR (target of rapamycin) proteins, which are widely conserved from yeast to man. Unfortunately, the side effects of rapamycin, especially immunosuppression, preclude its use in treating certain diseases including tuberculosis, which accounts for approximately 2 million deaths worldwide each year, spurring interest in finding novel drugs that selectively enhance autophagy. We have recently reported a novel two-step screening process for the discovery of such compounds. We first identified compounds that enhance the growth-inhibitory effects of rapamycin in the budding yeast Saccharomyces cerevisiae, which we termed small molecule enhancers of rapamycin (SMERs). Next we showed that three SMERs induced autophagy independently, or downstream of mTOR, in mammalian cells, and furthermore enhanced the clearance of a mutant huntingtin fragment in Huntington's disease cell models. These SMERs also protected against mutant huntingtin fragment toxicity in Drosophila. We have subsequently tested two of the SMERs in models of tuberculosis and both enhance the killing of mycobacteria by primary human macrophages.     

The effect of rapamycin on biodiesel-producing protist Euglena gracilis

Rapamycin induces autophagy with lipid remodeling in yeast and mammalian cells. To investigate the lipid biosynthesis of Euglena gracilis, rapamycin was supplemented in comparison with two model algae, Chlamydomonas reinhardtii and Cyanidioschyzon merolae. In Euglena, rapamycin induced the reduction of chlorophylls and the accumulation of neutral lipids without deterring its cell proliferation. Its lipidomic profile revealed that the fatty acid composition did not alter by supplementing rapamycin. In Chlamydomonas, however, rapamycin induced serious growth inhibition as reported elsewhere. With a lower concentration of rapamycin, the alga accumulated neutral lipids without reducing chlorophylls. In Cyanidioschyzon, rapamycin did not increase neutral lipids but reduced its chlorophyll content. We also tested fatty acid elongase inhibitors such as pyroxasulfone or flufenacet in Euglena with no significant change in its neutral lipid contents. In summary, controlled supplementation of rapamycin can increase the yield of neutral lipids while the scheme is not always applicable for other algal species.