Finite Replicative Lifespan Research Articles

S-07-1: TARGETING SENESCENT CELLS AS A NOVEL THERAPEUTIC STRATEGY FOR AGE-ASSOCIATED DISEASE

Epidemiological studies have shown that age is the dominant risk factor for lifestyle-related diseases. The incidence and the prevalence of diabetes, heart failure, coronary heart disease and hypertension increase with advancing age. However, the molecular mechanisms underlying the increased risk of such diseases that is conferred by aging remain unclear. Cellular senescence is originally described as the finite replicative lifespan of human somatic cells in culture. Cellular senescence is accompanied by a specific set of phenotypic changes in morphology and gene expression including negative regulators of the cell cycle such as p53. Primary cultured cells from patients with premature aging syndromes are known to have a shorter lifespan than cells from age-matched healthy persons. It is also reported that the number of senescent cells increases in various tissues with advancing age. Interestingly, such accumulation of senescent cells in aged animals is attenuated by caloric restriction that regulates the lifespan regulatory system and delays age-associate phenotypes. I therefore hypothesize that cellular senescence in vivo contributes to the pathogenesis of age-associated disease and have shown a critical role of cellular senescence in age-related pathologies. However, a direct inhibition of cellular aging signaling would lead to the increased incidence of cancer, so we need to develop anti-senescent therapy without cancer development. Here I will show our recent data on a novel strategy of anti-senescent therapy for age-associated disease by targeting cellular senescence (Seno-antigens, Seno-metabolites, SASP), which would not promote tumorigenesis.

Protein aggregation triggers a declining libido in elder yeasts that still have a lust for life.

Many organisms have to face a physiological decline that is associated with age. Humans and even budding yeast accumulate scars and cellular damages. A single yeast cell can only produce a limited number of daughter cells and thus has a finite replicative lifespan. Many studies have now identified molecular ageing factors and defects in organelle functions linked to the ageing process. However, at the cellular level, the most striking phenotype of yeast elders is their loss of mating ability. This sterility in old cells has been linked to a loss of response to mating pheromone, the peptide that haploid yeast cells send to opposite mating type cells in order to signal their presence and readiness to mate. Our results (Schlissel et al., 2017) demonstrate that old cells are unable to respond to mating pheromone due to age-induced aggregation of the protein Whi3. We recently discovered that Whi3 changes conformation and coalesces when cells experience and memorise a deceptive mating attempt. Together, these results prompt the question of how proteins physiologically aggregating behave during ageing, induce age associated phenotypes and influence the ageing process itself.

Kinetics of Formation and Asymmetrical Distribution of Hsp104-Bound Protein Aggregates in Yeast

Budding yeast cells have a finite replicative life span; that is, a mother cell produces only a limited number of daughter cells before it slows division and dies. Despite the gradual aging of the mother cell, all daughters are born rejuvenated and enjoy a full replicative lifespan. It has been proposed that entry of mother cells into senescence is driven by the progressive accumulation and retention of damaged material, including protein aggregates. This additionally allows the daughter cells to be born damage free. However, the mechanism underlying such asymmetrical segregation of protein aggregates by mother and daughter cells remains controversial, in part because of the difficulties inherent in tracking the dynamics and fate of protein aggregates in vivo. To overcome such limitations, we have developed single-cell real-time imaging methodology to track the formation of heat-induced protein aggregates in otherwise unperturbed dividing cells. By combining the imaging data with a simple computational model of protein aggregation, we show that the establishment of asymmetrical partitioning of protein aggregates upon division is driven by the large bud-specific dilution rate associated with polarized growth and the absence of significant mother/bud exchange of protein aggregates during the budded phase of the cell cycle. To our knowledge, this study sheds new light on the mechanism of establishment of a segregation bias, which can be accounted for by simple physical arguments.

Isolation and Primary Culture of Murine Podocytes with Proven Origin.

Genetic studies on hereditary kidney diseases and in vivo experimental model studies have revealed a critical role for the podocyte in glomerular function and disease. Primary podocyte cultures as well as immortalized podocyte cell lines have been used extensively to study podocyte function. Although, primary cells often more closely resemble the in vivo cells, they may have only a finite replicative life span before they reach senescence. Therefore, the success of studies using primary cell cultures depends on standardized isolation and culture protocols that allow reproducible generation of stable primary cultures.This chapter describes the isolation of primary podocytes with a proven origin using the novel technology of cell-specific genetic tagging. Podocytes are isolated from glomeruli from a podocyte-specific transgenic reporter mouse. The podocyte-specific reporter gene beta-galactosidase is used to identify and specifically isolate the labeled podocytes from other glomerular cells by FACS.

Yeast replicative aging: a paradigm for defining conserved longevity interventions

The finite replicative life span of budding yeast mother cells was demonstrated as early as 1959, but the idea that budding yeast could be used to model aging of multicellular eukaryotes did not enter the scientific mainstream until relatively recently. Despite continued skepticism by some, there are now abundant data that several interventions capable of extending yeast replicative life span have a similar effect in multicellular eukaryotes including nematode worms, fruit flies, and rodents. In particular, dietary restriction, mTOR signaling, and sirtuins are among the most studied longevity interventions in the field. Here, we describe key conserved longevity pathways in yeast and discuss relationships that may help explain how such broad conservation of aging processes could have evolved.

Necdin modulates proliferative cell survival of human cells in response to radiation-induced genotoxic stress

BackgroundThe finite replicative lifespan of cells, termed cellular senescence, has been proposed as a protective mechanism against the proliferation of oncogenically damaged cells, that fuel cancer. This concept is further supported by the induction of premature senescence, a process which is activated when an oncogene is expressed in normal primary cells as well as following intense genotoxic stresses. Thus, deregulation of genes that control this process, like the tumor suppressor p53, may contribute to promoting cancer by allowing cells to bypass senescence. A better understanding of the genes that contribute to the establishment of senescence is therefore warranted. Necdin interacts with p53 and is also a p53 target gene, although the importance of Necdin in the p53 response is not clearly understood.MethodsIn this study, we first investigated Necdin protein expression during replicative senescence and premature senescence induced by gamma irradiation and by the overexpression of oncogenic RasV12. Gain and loss of function experiments were used to evaluate the contribution of Necdin during the senescence process.ResultsNecdin expression declined during replicative aging of IMR90 primary human fibroblasts or following induction of premature senescence. Decrease in Necdin expression seemed to be a consequence of the establishment of senescence since the depletion of Necdin in human cells did not induce a senescence-like growth arrest nor a flat morphology or SA-β-galactosidase activity normally associated with senescence. Similarly, overexpression of Necdin did not affect the life span of IMR90 cells. However, we demonstrate that in normal human cells, Necdin expression mimicked the effect of p53 inactivation by increasing radioresistance.ConclusionThis result suggests that Necdin potentially attenuate p53 signaling in response to genotoxic stress in human cells and supports similar results describing an inhibitory function of Necdin over p53-dependent growth arrest in mice.

Motility and Segregation of Hsp104-Associated Protein Aggregates in Budding Yeast

During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother and that Bni1p formin regulates this transport. Here, we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking, and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data do not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.

Telomere dynamics in dyskeratosis congenita: the long and the short of iPS

Seminal experiments by Hayflick in the 1960s demonstrated that normal human diploid cells have a finite replicative life span in culture 1. The Hayflick “limit” is explained at least in part by the decay in telomeres, repeat sequences that cap the ends of chromosomes 2. Telomeric repeats are only incompletely extended by DNA polymerase and thus erode with each cell division. When telomeres become critically short, cells stop dividing and senesce. The telomere length in somatic cells is an “endowment” inherited from the stem cells from which they derive. Stem cells contain telomerase, a multisubunit ribonucleoprotein polymerase that adds repeats to telomere ends and thereby counteracts telomere attrition and replicative senescence, allowing stem cells to self-renew and maintain adult tissues throughout life. Unlike most telomerase components, the telomerase reverse transcriptase (TERT) is absent in somatic cells, and therefore the gatekeeper of telomerase function. TERT is expressed in highly self-renewing or immortalized cells such as embryonic cells, germ cells, cancer cells, and some adult stem cells, and forced expression of TERT in somatic cells overcomes replicative senescence and contributes to malignant transformation. Reprogramming of somatic cells to an embryonic state by nuclear transfer or factor-mediated direct reprogramming activates TERT, and is associated with the acquisition of self-renewal capacity 3, 4.

Abstract 1231: The identification of novel senescence-associated miRNAs

Abstract Cellular senescence was first described by Len Hayflick as the finite replicative lifespan of human fibroblasts in culture. Since then, it has emerged that senescence can be induced via multiple mechanisms, including radiochemotherapy and oncogenic stimuli, and that senescence rather than apoptosis occurs when damage to the cellular genome threatens to overwhelm the DNA repair machinery. Moreover, stress-induced premature senescence may have serious implications for the success of conventional chemotherapeutic regimes, as senescent cells adopt an anti-apoptotic and mitogenic secretory phenotype that can influence the growth of both normal and malignant neighbouring cells. Induction of p21 expression (via a stably integrated, IPTG-inducible transgene) in HT1080 fibrosarcoma cells causes a senescent-like phenotype characterised by an inability to divide or form colonies, activation of senescence-associated β-galactosidase expression, increased granularity and a large, flattened morphology. Indeed, along with p53, p21 has been shown to act as a potent regulator of accelerated senescence in tumour cells and a key mediator of the senescence-associated secretory phenotype. Here we describe a microRNA (miRNA) profiling study to identify novel miRNAs that are differentially expressed upon p21-mediated senescence. Total RNA from p21-induced (senescent) and p21-uninduced (cycling) HT1080 cells was isolated in duplicate using the mirVana miRNA isolation kit (Ambion) and miRNAs were reverse transcribed using the Megaplex RT primer human pool set (Applied Biosystems). miRNA profiling was performed on TaqMan Human MicroRNA Array Sets (Applied Biosystems) which enable the simultaneous relative quantification of the expression 667 human miRNAs. Differentially expressed miRNAs were identified using the ααCT method with RealTime Statminer data analysis software (Integromics) using 4 endogenous controls for data normalisation. Twenty statistically significant (p<0.05) differentially expressed miRNAs were identified following the induction of senescence. Four miRNAs were expressed only in cycling cells (hsa-miR-100*, hsa-let-7b, hsa-miR-30d, hsa-miR-422a) while expression was lost in senescent cells, and 3 miRNAs expressed only after the induction of p21 expression (hsa-miR-188-5p, hsa-miR-345, hsa-miR-425). Expression levels of miRNAs hsa-miR-100*, hsa-let-7b were examined in individual Taqman assays (Applied Biosystems) and higher expression of hsa-miR-100* and hsa-let-7b in cycling cells was confirmed. All of the differentially expressed miRNAs have previously been related to cancer in the literature, but there are no findings yet about their role in cellular senescence. Although their potential to act as regulators of senescence requires further validation, we believe these miRNAs represent novel biomarkers of p21-mediated senescence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1231. doi:10.1158/1538-7445.AM2011-1231

Abstract 3022: The identification of novel senescence-associated miRNAs

Abstract Cellular senescence was first described by Len Hayflick as the finite replicative lifespan of human fibroblasts in culture. Since then, it has emerged that senescence can be induced via multiple mechanisms, including radiochemotherapy and oncogenic stimuli, and that senescence rather than apoptosis occurs when damage to the cellular genome threatens to overwhelm the DNA repair machinery. Moreover, stress-induced premature senescence may have serious implications for the success of conventional chemotherapeutic regimes, as senescent cells adopt an anti-apoptotic and mitogenic secretory phenotype that can influence the growth of both normal and malignant neighbouring cells. Induction of p21 expression (via a stably integrated, IPTG-inducible transgene) in HT1080 fibrosarcoma cells causes a senescent-like phenotype characterised by an inability to divide or form colonies, activation of senescence-associated β-galactosidase expression, increased granularity and a large, flattened morphology. Indeed, along with p53, p21 has been shown to act as a potent regulator of accelerated senescence in tumour cells and a key mediator of the senescence-associated secretory phenotype. Here we describe a microRNA (miRNA) profiling study to identify novel miRNAs that are differentially expressed upon p21-mediated senescence. Total RNA from p21-induced (senescent) and p21-uninduced (cycling) HT1080 cells was isolated in duplicate using the mirVana miRNA isolation kit (Ambion) and miRNAs were reverse transcribed using the Megaplex RT primer human pool set (Applied Biosystems). miRNA profiling was performed on TaqMan Human MicroRNA Array Sets (Applied Biosystems) which enable the simultaneous relative quantification of the expression 667 human miRNAs. Differentially expressed miRNAs were identified using the ααCT method with RealTime Statminer data analysis software (Integromics) using 4 endogenous controls for data normalisation. Twenty statistically significant (p<0.05) differentially expressed miRNAs were identified following the induction of senescence. Four miRNAs were expressed only in cycling cells (hsa-miR-100*, hsa-let-7b, hsa-miR-30d, hsa-miR-422a) while expression was lost in senescent cells, and 3 miRNAs expressed only after the induction of p21 expression (hsa-miR-188-5p, hsa-miR-345, hsa-miR-425). All of the exclusively expressed miRNAs have previously been related to cancer in the literature, but there are no findings yet about their role in cellular senescence. Although their potential to act as regulators of senescence requires further validation, we believe these miRNAs represent novel biomarkers of p21-mediated senescence. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3022.

Modeling growth and telomere dynamics in Saccharomyces cerevisiae

A general branching process is proposed to model a population of cells of the yeast Saccharomyces cerevisiae following loss of telomerase. Previously published experimental data indicate that a population of telomerase-deficient cells regain exponential growth after a period of slowing due to critical telomere shortening. The explanation for this phenomenon is that some cells engage telomerase-independent pathways to maintain telomeres that allow them to become "survivors." Our model takes into account random variation in individual cell cycle times, telomere length, finite replicative lifespan of mother cells, and survivorship. We identify and estimate crucial parameters such as the probability of an individual cell becoming a survivor, and compare our model predictions to experimental data.

The ACTCellerate Initiative: Large-Scale Combinatorial Cloning of Novel Human Embryonic Stem Cell Derivatives

Human embryonic stem cells offer a scalable and renewable source of all somatic cell types. Human embryonic progenitor (hEP) cells are partially differentiated endodermal, mesodermal and ectodermal cell types that have not undergone terminal differentiation and express an embryonic pattern of gene expression. Here, we describe a large-scale and reproducible method of isolating a diverse library of clonally purified hEP cell lines, many of which are capable of extended propagation in vitro. Initial microarray and non-negative matrix factorization gene-expression profiling suggests that the library consists of at least 140 distinct clones and contains many previously uncharacterized cell types derived from all germ layers that display diverse embryo- and site-specific homeobox gene expression. Despite the expression of many oncofetal genes, none of the hEP cell lines tested led to tumor formation when transplanted into immunocompromised mice. All hEP lines studied appear to have a finite replicative lifespan but have longer telomeres than most fetal- or adult-derived cells, thereby facilitating their use in the manufacture of purified lineages for research and human therapy.

Abstract 1123: Critical Role Of Adipose Senescence In Insulin Resistance And Vascular Dysfunction

Cellular senescence is originally described as the finite replicative lifespan of human somatic cells in culture. As a consequence of semi-conservative DNA replication, the extreme terminals of the chromosomes are not duplicated completely, resulting in successive shortening of telomeres with each cell division. Telomerase is a ribonucleoprotein that adds telomeres to the ends of chromosomes. Critically short telomeres are thought to trigger DNA damage response, thereby inducing cellular senescence. Accumulating evidence has suggested that senescent cells promote aging phenotypes or age-related pathologies. Here we show that adipose senescence is critically involved in the regulation of insulin resistance that underlies age-associated cardiovascular disease. The later generation of telomerase-deficient mice with short telomeres exhibited insulin resistance and vascular dysfunction when fed on a high-calorie diet. Adipose tissue of these mice revealed senescence-like phenotypes such as an increase in neutral β galactosidase activity and upregulation of p53 and pro-inflammatory cytokines. Serum levels of pro-inflammatory cytokines were markedly elevated in telomerase-deficient mice and treatment of these mice with a neutralizing antibody against TNF-α significantly improved insulin and glucose intolerance. Removal of senescent adipose tissue reduced serum levels of pro-inflammatory cytokines and thereby improved insulin resistance in telomerase-deficient mice. Conversely, implantation of senescent adipose tissue to wild-type mice impaired insulin sensitivity and glucose tolerance in recipients. Introduction of telomere dysfunction to young adipose tissue markedly upregulated p53 expression and increased the production of pro-inflammatory cytokines. Inhibition of p53 activity significantly improved senescence-like phenotypes of adipose tissue, insulin resistance, and vascular dysfunction in telomerase-deficient mice. These results disclose a novel mechanism of insulin resistance and suggest that adipose senescence is a potential therapeutic target for the treatment of diabetes and diabetic vasculopathy.

TAK1 represses transcription of the human telomerase reverse transcriptase gene

In human cells, telomerase activity is tightly regulated by the expression of its catalytic subunit, namely, the human telomerase reverse transcriptase (hTERT). However, the molecular mechanisms involved in the regulation of hTERT expression have not been completely clarified. We have previously reported that transforming growth factor beta (TGF-beta) represses the expression of the hTERT gene. In the present study, we demonstrated that TGF-beta-activated kinase 1 (TAK1), originally identified as a mitogen-activated kinase kinase kinase, represses the hTERT core promoter activity in an E-box-independent manner, and it also represses the transcription of the hTERT gene in human lung adenocarcinoma cell line, A549 cells. This TAK1-induced repression was found to be caused by the recruitment of histone deacetylase to Sp1 at the hTERT promoter and a consequent reduction in the amount of acetylated histone H4 at the hTERT promoter. Finally, we demonstrated that TAK1 induces cellular senescence programs in normal human diploid cells. Thus, we assume that TAK1 triggers the repression mechanisms of the hTERT gene as a result of evoking cellular senescence programs. Considered together, TAK1 is thought to play a causative role in the determination of a finite replicative lifespan of normal and cancer cells.

Intestinal crypt properties fit a model that incorporates replicative ageing and deep and proximate stem cells

A model of intestinal crypt organization is suggested based on the assumption that stem cells have a finite replicative life span. The model assumes the existence in a crypt of a quiescent ('deep') stem cell and a few more actively cycling ('proximate') stem cells. Monte Carlo computer simulation of published intestinal crypt mutagenesis data is used to test the model. The results of the simulation indicate that stabilization of the crypt mutant phenotype following treatment with external mutagen is consistent with a stem cell replicative life span of about 40 divisions for mouse colon and 90-100 divisions for mouse small intestine, corresponding to a deep stem cell cycle time of about 3.9 and 8.5 weeks for colon and small intestine, respectively. Simulation of the data obtained for human colorectal crypts suggests that the proximate stem cell cycle time is about 80 h, assuming a replicative life span of 50-150 divisions, and that the deep stem cell divides approximately every 30 weeks.

Bmi-1 Is a Novel Molecular Marker of Nasopharyngeal Carcinoma Progression and Immortalizes Primary Human Nasopharyngeal Epithelial Cells

The Bmi-1 oncoprotein regulates proliferation and oncogenesis in human cells. Its overexpression leads to senescence bypass in human fibroblasts and immortalization of human mammary epithelial cells. In this study, we report that compared with normal nasopharyngeal epithelial cells (NPEC), Bmi-1 is overexpressed in nasopharyngeal carcinoma cell lines. Importantly, Bmi-1 was also found to be overexpressed in 29 of 75 nasopharyngeal carcinoma tumors (38.7%) by immunohistochemical analysis. In contrast to nasopharyngeal carcinoma, there was no detectable expression of Bmi-1 in noncancerous nasopharyngeal epithelium. Moreover, high Bmi-1 expression positively correlated with poor prognosis of nasopharyngeal carcinoma patients. We also report that the overexpression of Bmi-1 leads to bypass of senescence and immortalization of NPECs, which normally express p16(INK4a) and exhibit finite replicative life span. Overexpression of Bmi-1 in NPECs led to the induction of human telomerase reverse transcriptase activity and reduction of p16(INK4a) expression. Mutational analysis of Bmi-1 showed that both RING finger and helix-turn-helix domains of it are required for immortalization of NPECs. Our findings suggest that Bmi-1 plays an important role in the development and progression of nasopharyngeal carcinoma, and that Bmi-1 is a valuable marker for assessing the prognosis of nasopharyngeal carcinoma patients. Furthermore, this study provides the first cellular proto-oncogene immortalized nasopharyngeal epithelial cell line, which may serve as a cell model system for studying the mechanisms involved in the tumorigenesis of nasopharyngeal carcinoma.

Functional impairment of telomerase in sublines derived from human lung adenocarcinoma exposed to mild oxidative stress

We investigated the effects of a sublethal concentration of H 2O 2 on cancer cells by using sublines derived from human lung adenocarcinoma cell line A549 cells exposed to 200 μM H 2O 2. These sublines (AST cells) showed an elongated morphology distinct from the rounded morphology of A549 cells. Notably, AST cells demonstrated telomere shortening despite displaying telomerase activity and expressing human telomerase reverse transcriptase (hTERT). This functional impairment of telomerase occurred due to perturbed subcellular localization of hTERT in AST cells. Endogenous as well as ectopically expressed hTERT was localized in the nuclei of A549 cells; however, in AST cells, the localization was mainly in the cytoplasm. Furthermore, these AST cells demonstrated decreased tumorigenic features both in vitro and in vivo. These results suggest that depletion of hTERT from nuclei not only endows cancer cells with a finite replicative life span accompanied by telomere shortening, but also decreases the tumorigenicity of cancer cells.

Prohibitins and Ras2 protein cooperate in the maintenance of mitochondrial function during yeast aging.

The yeast Saccharomyces cerevisiae has a finite replicative life span. Yeasts possess two prohibitins, Phb1p and Phb2p, in similarity to mammalian cells. These proteins are located in the inner mitochondrial membrane, where they are involved in the processing of newly-synthesized membrane proteins. We demonstrate that the elimination of one or both of the prohibitin genes in yeast markedly diminished the replicative life span of cells that lack fully-functional mitochondria, while having no effect on cells with functioning mitochondria. This deleterious effect was suppressed by the deletion of the RAS2 gene. The expression of PHB1 and PHB2 declined gradually up to 5-fold during the life span. Cells in which PHB1 was deleted in conjunction with the absence of a mitochondrial genome displayed remarkable changes in mitochondrial morphology, distribution, and inheritance. This loss of mitochondrial integrity was not seen in cells devoid of PHB1 but possessing an intact mitochondrial genome. In a subset of the cells, the changes in mitochondrial integrity were associated with increased production of reactive oxygen species, which co-localized with the altered mitochondria. The mitochondrial deficits described above were all suppressed by deletion of RAS2. Our data, together with published information, are interpreted to provide a unified view of the role of the prohibitins in yeast aging. This model posits that the key initiating event is a decline in mitochondrial function, which leads to progressive oxidative damage that is exacerbated in the absence of the prohibitins. This aggravation of the initial damage is ameliorated by the suppression of the production of mitochondrial proteins in the absence of Ras2p signaling of mitochondrial biogenesis.

Prohibitins and Ras2 protein cooperate in the maintenance of mitochondrial function during yeast aging.

The yeast Saccharomyces cerevisiae has a finite replicative life span. Yeasts possess two prohibitins, Phb1p and Phb2p, in similarity to mammalian cells. These proteins are located in the inner mitochondrial membrane, where they are involved in the processing of newly-synthesized membrane proteins. We demonstrate that the elimination of one or both of the prohibitin genes in yeast markedly diminished the replicative life span of cells that lack fully-functional mitochondria, while having no effect on cells with functioning mitochondria. This deleterious effect was suppressed by the deletion of the RAS2 gene. The expression of PHB1 and PHB2 declined gradually up to 5-fold during the life span. Cells in which PHB1 was deleted in conjunction with the absence of a mitochondrial genome displayed remarkable changes in mitochondrial morphology, distribution, and inheritance. This loss of mitochondrial integrity was not seen in cells devoid of PHB1 but possessing an intact mitochondrial genome. In a subset of the cells, the changes in mitochondrial integrity were associated with increased production of reactive oxygen species, which co-localized with the altered mitochondria. The mitochondrial deficits described above were all suppressed by deletion of RAS2. Our data, together with published information, are interpreted to provide a unified view of the role of the prohibitins in yeast aging. This model posits that the key initiating event is a decline in mitochondrial function, which leads to progressive oxidative damage that is exacerbated in the absence of the prohibitins. This aggravation of the initial damage is ameliorated by the suppression of the production of mitochondrial proteins in the absence of Ras2p signaling of mitochondrial biogenesis.

The impact of brewing yeast cell age on fermentation performance, attenuation and flocculation.

Individual cells of the yeast Saccharomyces cerevisiae exhibit a finite replicative lifespan, which is widely believed to be a function of the number of divisions undertaken. As a consequence of ageing, yeast cells undergo constant modifications in terms of physiology, morphology and gene expression. Such characteristics play an important role in the performance of yeast during alcoholic beverage production, influencing sugar uptake, alcohol and flavour production and also the flocculation properties of the yeast strain. However, although yeast fermentation performance is strongly influenced by the condition of the yeast culture employed, until recently cell age has not been considered to be important to the process. In order to ascertain the effect of replicative cell age on fermentation performance, age synchronised populations of a lager strain were prepared using sedimentation through sucrose gradients. Each age fraction was analysed for the ability to utilise fermentable sugars and the capacity to flocculate. In addition cell wall properties associated with flocculation were determined for cells within each age fraction. Aged cells were observed to ferment more efficiently and at a higher rate than mixed aged or virgin cell cultures. Additionally, the flocculation potential and cell surface hydrophobicity of cells was observed to increase in conjunction with cell age. The mechanism of ageing and senescence in brewing yeast is a complex process, however here we demonstrate the impact of yeast cell ageing on fermentation performance.