Over-expression of an S-adenosylmethionine-dependent methyltransferase leads to an extended lifespan of Podospora anserina without impairments in vital functions

PaMTH1, a putative methyltransferase previously described to increase in abundance in total protein extracts during aging of Podospora anserina is demonstrated to accumulate in the mitochondrial cell fraction of senescent cultures. The protein is localized in the mitochondrial matrix and displays a methyltransferase activity utilizing flavonoids as substrates. Constitutive over-expression of PaMth1 in P. anserina results in a reduced carbonylation of proteins and an extended lifespan without impairing vital functions suggesting a protecting role of PaMTH1 against oxidative stress.     

Poly(ADP-Ribose) Polymerase Is a Substrate Recognized by Two Metacaspases of Podospora anserina

The two metacaspases MCA1 and MCA2 of the fungal aging model organism Podospora anserina (PaMCA1 and PaMCA2, respectively) have previously been demonstrated to be involved in the control of programmed cell death (PCD) and life span. In order to identify specific pathways and components which are controlled by the activity of these enzymes, we set out to characterize them further. Heterologous overexpression in Escherichia coli of the two metacaspase genes resulted in the production of proteins which aggregate and form inclusion bodies from which the active protein has been recovered via refolding in appropriate buffers. The renaturated proteins are characterized by an arginine-specific activity and are active in caspase-like self-maturation leading to the generation of characteristic small protein fragments. Both activities are dependent on the presence of calcium. Incubation of the two metacaspases with recombinant poly(ADP-ribose) polymerase (PARP), a known substrate of mammalian caspases, led to the identification of PARP as a substrate of the two P. anserina proteases. Using double mutants in which P. anserina Parp (PaParp) is overexpressed and PaMca1 is either overexpressed or deleted, we provide evidence for in vivo degradation of PaPARP by PaMCA1. These results support the idea that the substrate profiles of caspases and metacaspases are at least partially overlapping. Moreover, they link PCD and DNA maintenance in the complex network of molecular pathways involved in aging and life span control.     

A Podospora anserina longevity mutant with a temperature-sensitive phenotype for senescence.

A Podospora anserina longevity mutant was identified with a temperature-sensitive phenotype for senescence. This mutant, termed TS1, grew for over 3 m at 27 degrees C, but when shifted to 34 degrees C, it underwent senescence between 10 and 18 cm. A previously described senescence-associated plasmid, alpha senDNA, derived from the mitochondrial genome, was not detected in TS1 at 27 degrees C but was present in senescent cultures at 34 degrees C. A similar result was observed in progeny strains obtained by crossing the TS1 mutant with a wild-type strain. Other mitochondrial excision-amplification DNAs in addition to alpha senDNA were also observed in the senescent cultures. Most were derived from a specific region of the mitochondrial genome. These results provide evidence that alpha senDNA is involved in TS1 senescence and suggest that this plasmid may play a role in the formation of other mitochondrial excision-amplification plasmids.     

Does autophagy mediate age-dependent effect of dietary restriction responses in the filamentous fungus Podospora anserina?

Autophagy is a well-conserved catabolic process, involving the degradation of a cell''s own components through the lysosomal/vacuolar machinery. Autophagy is typically induced by nutrient starvation and has a role in nutrient recycling, cellular differentiation, degradation and programmed cell death. Another common response in eukaryotes is the extension of lifespan through dietary restriction (DR). We studied a link between DR and autophagy in the filamentous fungus Podospora anserina, a multicellular model organism for ageing studies and mitochondrial deterioration. While both carbon and nitrogen restriction extends lifespan in P. anserina, the size of the effect varied with the amount and type of restricted nutrient. Natural genetic variation for the DR response exists. Whereas a switch to carbon restriction up to halfway through the lifetime resulted in extreme lifespan extension for wild-type P. anserina, all autophagy-deficient strains had a shorter time window in which ageing could be delayed by DR. Under nitrogen limitation, only PaAtg1 and PaAtg8 mediate the effect of lifespan extension; the other autophagy-deficient mutants PaPspA and PaUth1 had a similar response as wild-type. Our results thus show that the ageing process impinges on the DR response and that this at least in part involves the genetic regulation of autophagy.     

Role for Sit4p-dependent mitochondrial dysfunction in mediating the shortened chronological lifespan and oxidative stress sensitivity of Isc1p-deficient cells

Saccharomyces cerevisiae cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, display a shortened lifespan and an increased sensitivity to oxidative stress. A lipidomic analysis revealed specific changes in sphingolipids that accompanied the premature ageing of Isc1p-deficient cells under severe calorie restriction conditions, including a decrease of dihydrosphingosine levels and an increase of dihydro-C(26) -ceramide and phyto-C(26) -ceramide levels, the latter raising the possibility of activation of ceramide-dependent protein phosphatases. Consequently, deletion of the SIT4 gene, which encodes for the catalytic subunit of type 2A ceramide-activated protein phosphatase in yeast, abolished the premature ageing and hydrogen peroxide sensitivity of isc1Δ cells. SIT4 deletion also abolished the respiratory defects and catalase A deficiency exhibited by isc1Δ mutants. These results are consistent with catabolic derepression associated with the loss of Sit4p. The overall results show that Isc1p is an upstream regulator of Sit4p and implicate Sit4p activation in mitochondrial dysfunction leading to the shortened chronological lifespan and oxidative stress sensitivity of isc1Δ mutants.     

Extension of replicative lifespan in WI-38 human fibroblasts by dexamethasone treatment is accompanied by suppression of p21 Waf1/Cip1/Sdi1 levels

Numerous studies have shown that supplementation of the growth medium of human fibroblasts with dexamethasone at physiologic concentrations extends replicative lifespan up to 30%. While this extension of lifespan has been used to probe various aspects of the senescent phenotype, no mechanism for the increased lifespan of human fibroblasts grown in the presence of dexamethasone has ever been identified. In the present study we present evidence that the extended lifespan of human lung fibroblasts (WI-38 cells) that occurs when these cells are maintained in culture medium supplemented with dexamethasone is accompanied by a suppression of p21(Waf1/Cip1/Sdi1) levels, which normally increase as these cells enter senescence, while p16(INK4a) levels are unaffected. These results suggest that the delay of senescence in cultures grown in the presence of dexamethasone is due to a suppression of the senescence related increase in p21(Waf1/Cip1/Sdi1). These results are consistent with models of replicative senescence in which p53 and p21(Waf1/Cip1/Sdi1) play a role in the establishment of the senescent arrest.     

Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, wound-healing, and morphogenetic responses in senescent endothelial cells

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P(1-3) receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P(1) and S1P(2) was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P(2) receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P(2) to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P(2)-mediated activation of the lipid phosphatase PTEN.     

Mitochondrial metabolism and aging in the filamentous fungus Podospora anserina

The filamentous fungus Podospora anserina has a limited lifespan. In this organism, aging is systematically associated to mitochondrial DNA instability. We recently provided evidence that the respiratory function is a key determinant of its lifespan. Loss of function of the cytochrome pathway leads to the compensatory induction of an alternative oxidase, to a decreased production of reactive oxygen species and to a striking increase in lifespan. These changes are associated to the stabilization of the mitochondrial DNA. Here we review and discuss the links between these different parameters and their implication in the control of lifespan. Since we demonstrated the central role of mitochondrial metabolism in aging, the same relationship has been evidenced in several model systems from yeast to mice, confirming the usefulness of simple organisms as P. anserina for studying lifespan regulation.     

Effect of the Tumor Promoter Phorbol 12-Myristate 13-Acetate (PMA) on Proliferation of Young and Senescent WI-38 Human Diploid Fibroblasts

The effects of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on the proliferation, protein kinase C activity (PKC), and c-fos gene expression were examined in cultures of young and senescent (90-95% lifespan completed) WI-38 human diploid fibroblasts. We observed that, following stimulation with medium containing 10% fetal bovine serum (FBS), the translocation of PKC from the cytosol to the particulate compartment was less efficient in senescent WI-38 cells than in young cells. However, when PMA was added to the medium, the intracellular distribution of PKC activity in old cells became nearly identical to that observed in young cells. The inducibility of c-fos mRNA by serum addition, which is a protein kinase C-dependent event [64], was significantly amplified in the presence of PMA. Moreover, the duration of peak c-fos expression, after stimulation by FBS and PMA, increased in senescent cells as compared to young cells. Our results reveal that the normal signal transduction pathway is altered in senescent, slowly proliferating human fibroblasts and that it can be partially restored in the presence of the tumor promoter PMA.     

Renewed DNA synthesis in senescent WI-38 cells by expression of an inducible chimeric c-fos construct.

As normal human cells approach the end of their proliferative lifespan in vitro they lose responsiveness to a variety of growth factors, to which they respond with DNA replication when they are young. Recently it has been reported that the protooncogene c-fos is not expressed in senescent cells (Seshadri and Campisi, 1990). In this study we have found that both c-jun and jun B, partners of c-fos in heterodimeric transactivating complexes, are equivalently expressed in young and senescent cells at both early (1-6 hr) and late (12 or 16 hr) time points following serum stimulation of quiescent cells. We have also investigated the effect of the enforced expression of c-fos in senescent WI-38 cells using an inducible construct carrying the murine c-fos gene under the control of the sheep metallothionein promoter. We have found that the transient transfection and subsequent activation of the conditional promoter with Zn++ stimulated DNA synthesis in a significant fraction of senescent cells which had completed 90%-95% of their proliferative lifespan. However, populations which had completed 100% of their proliferative life span and nondividing cultures which had been selected with BrdU did not respond to the expression of the c-fos gene. These results demonstrate that one of the primary events associated with senescence in human cells is the suppression of c-fos gene expression, but additional phenotypic changes must also occur in order to explain the ultimate loss of proliferative responsiveness of these cells.     

Podospora anserina does not senesce when serially passaged in liquid culture.

A procedure was developed for the prolonged growth of the ascomycete fungus Podospora anserina in liquid culture to determine the effects of such growth on the senescence phenotype. Senescence in P. anserina, which is maternally inherited and associated with the excision and amplification of specific mitochondrial plasmids, occurs when this species is grown on solid medium. In two independent experiments no evidence of senescence was observed as mycelia were serially passaged in liquid culture. Further, when separable mycelial masses, termed puff balls, from the liquid cultures were plated on solid medium, a significant increase in their average longevity was observed. The apparent immortality of P. anserina in liquid culture was not dependent upon mitochondrial DNA rearrangements, nor was it affected by the presence of a previously described senescence plasmid, alpha senDNA. Evidence was obtained indicating that growth in liquid culture exerts selective pressure to maintain the wild-type mitochondrial genome.     

Modulation of replicative senescence of diploid human cells by nuclear ERK signaling

Normal somatic cells have a limited replicative lifespan, and serial subcultivation ultimately results in senescence. Senescent cells are irreversibly growth-arrested and show impaired responses to mitogens. Activation of the ERK signaling pathway, an absolute requirement for cell proliferation, results in nuclear relocalization of active ERKs, an event impaired in senescent fibroblasts. This impairment coincides with increased activity of the nuclear ERK phosphatase MKP2. Here we show that replicative lifespan can be altered by changes in nuclear ERK activity. Ectopic expression of MKP2 results in premature senescence. In contrast, knock-down of MKP2 expression, through transduction of MKP2 sequence-specific short hairpin RNA, or expression of the phosphatase resistant ERK2(D319N) mutant, abrogates the effects of increased endogenous MKP2 levels and senescence is postponed. Nuclear targeting of ERK2(D319N) significantly augments its effects and the transduced cultures show higher than 60% increase in replicative lifespan compared with cultures transduced with wt ERK2. Long-lived cultures senesce with altered molecular characteristics and retain the ability to express c-fos, and Rb is maintained in its inactive form. Our results support that MKP2-mediated inactivation of nuclear ERK2 represents a key event in the establishment of replicative senescence. Although it is evident that senescence can be imposed through multiple mechanisms, restoration of nuclear ERK activity can bypass a critical senescence checkpoint and, thus, extend replicative lifespan.     

Aging in vitro and D-glucose uptake kinetics of diploid human fibroblasts

By use of a rapid technique, initial rates of D-glucose transport were obtained during the lifespan in vitro of a commercially available strain of human embryo lung fibroblasts (Flow 2000). The apparent Km of the D-glucose carrier did not change during senescence in vitro: x̄ = 1.8 mM (range 1.3–2.3) in phase II, x̄ = 1.8 mM (range 1.5–2.2) in phase III. Transport rates remained constant in stationary phase II cultures, which had completed between 30% and 80% of their replicative lifespan. A wide variation, however, was observed in terminally differentiated cells (phase III), which showed a two- to threefold increase in average cell size and protein content. In some senescent cultures, glucose transport calculated on a per cell basis was also two-to threefold increased, while it was strongly decreased (-75%) in others. When calculated per unit of cell water, protein, and surface area, respectively, transport rates in phase III cultures ranged from values established for stationary phase II cultures down to very low values. Detaching cells flushed off from senescent cultures did not show measurable rates of glucose transport into the inulin impermeable cell space. Present evidence argues against the idea that an impairment of D-glucose transport might precede loss of replicative potential in aging human fibroblasts. Instead our data indicate that the transport capacity of cell membrane finally decreases during postreplicative senescence in terminally differentiated cells.     

Increased organization of cytoskeleton accompanying the aging of human fibroblasts in vitro

Fibroblastic cells of human origin have a limited lifespan in culture. One of the senescence-associated phenotypic changes is an increase in the abundance of cytoplasmic filaments. Human skin fibroblasts (strain 0011), derived from an 8-week-old male fetus, were passaged according to a predetermined schedule and examined at successive population doubling levels. In young rapidly growing cultures, fluorescence microscopy with NBD-Phallacidin shows a normal organization of the actin-containing fibers, microtubules and intermediate filaments, as has been described previously. At stages close to the end of the in vitro lifespan of the cell strain, large flat fibroblasts are the predominant cell type in culture. These large senescent fibroblasts contain numerous prominent actin fibers traversing the entire long axis of the cytoplasm. The fibers are often located adjacent to each other and appear to form a sheet on the ventral side of the cytoplasm. Staining of senescent cells with anti-tubulin antibody reveals an increase in the abundance of microtubules per cell and the distribution pattern is altered through the increase in the number of organization centers. Intermediate filaments are also more abundant and display tightly packed fibrillar sheets or bundles. Electron microscopic studies have confirmed the increased organization of microfilaments into bundles in senescent cells. These results suggest that during in vitro senescence, the increase in cell size is correlated with increased organization of the cytoskeleton. The presence of a rigid cytoskeletal structure may contribute in part to the inability of the senescent cell to replicate.