James C. Jiang

An intervention resembling caloric restriction prolongs life span and retards aging in yeast

The yeast Saccharomyces cerevisiae has a finite life span that is measured by the number of daughter cells an individual produces. The 20 genes known to determine yeast life span appear to function in more than one pathway, implicating a variety of physiological processes in yeast longevity. Less attention has been focused on environmental effects on yeast aging. We have examined the role that nutritional status plays in determining yeast life span. Reduction of the glucose concentration in the medium led to an increase in life span and to a delay in appearance of an aging phenotype. The increase in life span was the more extensive the lower the glucose levels. Life extension was also elicited by decreasing the amino acids content of the medium. This suggests that it is the decline in calories and not a particular nutrient that is responsible, in striking similarity to the effect on aging of caloric restriction in mammals. The caloric restriction effect did not require the induction of the retrograde response pathway, which signals the functional status of the mitochondrion and determines longevity. Furthermore, deletion of RTG3, a downstream mediator in this pathway, and caloric restriction had an additive effect, resulting in the largest increase (123%) in longevity described thus far in yeast. Thus, retrograde response and caloric restriction operate along distinct pathways in determining yeast longevity. These pathways may be exclusive, at least in part. This provides evidence for multiple mechanisms of metabolic control in yeast aging. Inasmuch as caloric restriction lowers blood glucose levels, this study raises the possibility that reduced glucose alters aging at the cellular level in mammals.

Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas

In this study, quantitative isobologram studies showed that treatment with gemcitabine and doxorubicin, known inducers of ceramide generation, in combination, supra-additively inhibited the growth of human UM-SCC-22A cells in situ. Then, possible involvement of the human homologue of yeast longevity assurance gene 1 (LASS1)/C18-ceramide in chemotherapy-induced cell death in these cells was examined. Gemcitabine/doxorubicin combination treatment resulted in the elevation of mRNA and protein levels of LASS1 and not LASS2-6, which was consistent with a 3.5-fold increase in the endogenous (dihydro)ceramide synthase activity of LASS1 for the generation of C18-ceramide. Importantly, the overexpression of LASS1 (both human and mouse homologues) enhanced the growth-inhibitory effects of gemcitabine/doxorubicin with a concomitant induction of caspase-3 activation. In reciprocal experiments, partial inhibition of human LASS1 expression using small interfering RNA (siRNA) prevented cell death by about 50% in response to gemcitabine/doxorubicin. In addition, LASS1, and not LASS5, siRNA modulated the activation of caspase-3 and caspase-9, but not caspase-8, in response to this combination. Treatment with gemcitabine/doxorubicin in combination also resulted in a significant suppression of the head and neck squamous cell carcinoma (HNSCC) tumor growth in severe combined immunodeficiency mice bearing the UM-SCC-22A xenografts. More interestingly, analysis of endogenous ceramide levels in these tumors by liquid chromatography/mass spectroscopy showed that only the levels of C18-ceramide, the main product of LASS1, were elevated significantly (about 7-fold) in response to gemcitabine/doxorubicin when compared with controls. In conclusion, these data suggest an important role for LASS1/C18-ceramide in gemcitabine/doxorubicin-induced cell death via the activation of caspase-9/3 in HNSCC. [Mol Cancer Ther 2007;6(2):712–22]

Human Homologues of LAG1 Reconstitute Acyl-CoA-dependent Ceramide Synthesis in Yeast

Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum. lag1Δ lac1Δ double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background. LAG1 and LAC1 are members of a large eukaryotic gene family that shares the Lag1 motif, and some members of this family additionally contain a DNA-binding HOX homeodomain. Here we show that several human LAG1 homologues can rescue the viability of lag1Δ lac1Δ yeast cells and restore acyl-CoA-dependent ceramide and sphingolipid biosynthesis. When tested in a microsomal assay, Lac1p and Lag1p had a strong preference for C26:0-CoA over C24:0-CoA, C20-CoA, and C16-CoA, whereas some human homologues preferred C24:0-CoA and CoA derivatives with shorter fatty acids. This suggests that LAG1 proteins are related to substrate recognition and to the catalytic activity of ceramide synthase enzymes. CLN8, another human LAG1 homologue implicated in ceroid lipofuscinosis, could not restore viability to lag1Δ lac1Δ yeast mutants.

Necessary Role for the Lag1p Motif in (Dihydro)ceramide Synthase Activity

Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro)ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro)ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro)ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B1, an in vitro inhibitor of (dihydro)ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues.

Distinct roles of processes modulated by histone deacetylases Rpd3p, Hda1p, and Sir2p in life extension by caloric restriction in yeast

Caloric restriction has been demonstrated to extend life span and postpone aging in a variety of species. The recent extension of the caloric restriction paradigm to yeast places the emphasis of the search for the longevity effectors at the cellular level. To narrow the range of potential effectors of the caloric restriction response, we have examined the effects of the histone deacetylases Rpd3p, Hda1p, and Sir2p, which have distinguishable but partially overlapping influences on global patterns of gene expression, on the life extension afforded by caloric restriction. Deletion of the RPD3 gene extended life span, and there was no additive effect of caloric restriction. Deletion of HDA1 had no effect of its own on longevity but acted synergistically with caloric restriction to increase life span. SIR2 deletion shortened life span but did not prevent extension of life span by caloric restriction. The results suggest that Rpd3p affects both processes that play an obligate and those that play a synergistic role in life extension by caloric restriction, while Hda1p and Sir2p affect processes that are not the obligate longevity effectors of caloric restriction but instead synergize with them, although in opposite directions. From the known patterns of gene expression elicited by rpd3delta, hda1delta, and sir2delta, we propose that the major longevity effectors of caloric restriction in yeast involve carbohydrate/energy metabolism and mitochondrial function.

Loss of mitochondrial membrane potential triggers the retrograde response extending yeast replicative lifespan.

In the budding yeast Saccharomyces cerevisiae, loss of mitochondrial DNA (rho0) can induce the retrograde response under appropriate conditions, resulting in increased replicative lifespan (RLS). Although the retrograde pathway has been extensively elaborated, the nature of the mitochondrial signal triggering this response has not been clear. Mitochondrial membrane potential (MMP) was severely reduced in rho0 compared to rho+ cells, and RLS was concomitantly extended. To examine the role of MMP in the retrograde response, MMP was increased in the rho0 strain by introducing a mutation in the ATP1 gene, and it was decreased in rho+ cells by deletion of COX4. The ATP1-111 mutation in rho0 cells partially restored the MMP and reduced mean RLS to that of rho+ cells. COX4 deletion decreased MMP in rho+ cells to a value intermediate between rho+ and rho0 cells and similarly increased RLS. The increase in expression of CIT2, the diagnostic gene for the retrograde response, seen in rho0 cells, was substantially suppressed in the presence of the ATP1-111 mutation. In contrast, CIT2 expression increased in rho+ cells on deletion of COX4. Activation of the retrograde response results in the translocation of the transcription factor Rtg3 from the cytoplasm to the nucleus. Rtg3–GFP translocation to the nucleus was directly observed in rho0 and rho+ cox4Δ cells, but it was blunted in rho0 cells with the ATP1-111 mutation. We conclude that a decrease in MMP is the signal that initiates the retrograde response and leads to increased RLS.

HRAS1 and LASS1 with APOE are associated with human longevity and healthy aging.

The search for longevity‐determining genes in human has largely neglected the operation of genetic interactions. We have identified a novel combination of common variants of three genes that has a marked association with human lifespan and healthy aging. Subjects were recruited and stratified according to their genetically inferred ethnic affiliation to account for population structure. Haplotype analysis was performed in three candidate genes, and the haplotype combinations were tested for association with exceptional longevity. An HRAS1 haplotype enhanced the effect of an APOE haplotype on exceptional survival, and a LASS1 haplotype further augmented its magnitude. These results were replicated in a second population. A profile of healthy aging was developed using a deficit accumulation index, which showed that this combination of gene variants is associated with healthy aging. The variation in LASS1 is functional, causing enhanced expression of the gene, and it contributes to healthy aging and greater survival in the tenth decade of life. Thus, rare gene variants need not be invoked to explain complex traits such as aging; instead rare congruence of common gene variants readily fulfills this role. The interaction between the three genes described here suggests new models for cellular and molecular mechanisms underlying exceptional survival and healthy aging that involve lipotoxicity.

Natural genetic variation in yeast longevity

The genetics of aging in the yeast Saccharomyces cerevisiae has involved the manipulation of individual genes in laboratory strains. We have instituted a quantitative genetic analysis of the yeast replicative lifespan by sampling the natural genetic variation in a wild yeast isolate. Haploid segregants from a cross between a common laboratory strain (S288c) and a clinically derived strain (YJM145) were subjected to quantitative trait locus (QTL) analysis, using 3048 molecular markers across the genome. Five significant, replicative lifespan QTL were identified. Among them, QTL 1 on chromosome IV has the largest effect and contains SIR2, whose product differs by five amino acids in the parental strains. Reciprocal gene swap experiments showed that this gene is responsible for the majority of the effect of this QTL on lifespan. The QTL with the second-largest effect on longevity was QTL 5 on chromosome XII, and the bulk of the underlying genomic sequence contains multiple copies (100-150) of the rDNA. Substitution of the rDNA clusters of the parental strains indicated that they play a predominant role in the effect of this QTL on longevity. This effect does not appear to simply be a function of extrachromosomal ribosomal DNA circle production. The results support an interaction between SIR2 and the rDNA locus, which does not completely explain the effect of these loci on longevity. This study provides a glimpse of the complex genetic architecture of replicative lifespan in yeast and of the potential role of genetic variation hitherto unsampled in the laboratory.

Suppressor analysis points to the subtle role of the LAG1 ceramide synthase gene in determining yeast longevity

Individual yeast cells display a finite replicative capacity. LAG1 was identified as a gene that is differentially expressed during the yeast replicative life span and was shown to play a role in determining yeast longevity. This gene is not essential, but simultaneous deletion of LAG1 and its close homologue LAC1 is lethal. Lag1p and Lac1p have been found to be an essential component of ceramide synthase. In this study, multicopy suppressors of the lethality of a lag1delta lac1delta double mutant were isolated to help clarify the role of LAG1 in yeast longevity. The two multicopy suppressors YBR183w (YPC1) and YPL087w (YDC1) encode ceramidases unrelated to Lag1p and Lac1p, which were previously found to support the reverse reaction of ceramide synthesis. Multiple copies of YPC1 were much more efficient than YDC1 in rescuing cell growth. They were also much more effective in rescuing the life span of a lag1delta lac1delta double mutant, sustaining a life span approaching that obtained by the restoration of LAG1 expression. Neither deletion of LAC1 nor overexpression of YPC1 had a detectable effect on wild-type life span. However, the overexpression of LAG1 had a bimodal effect on longevity, with moderate expression resulting in increased longevity and with higher expression curtailing life span. These results suggest that subtle changes in ceramide/sphingolipid metabolism are important in determining yeast longevity. They also indicate that Lag1p plays a special role in this relationship. Homologues of Lag1p have been identified in higher eukaryotes, including human, raising the possibility that ceramide and other sphingolipid metabolites play a wider role in biological aging.

Gene regulatory changes in yeast during life extension by nutrient limitation

Genetic analyses aimed at identification of the pathways and downstream effectors of calorie restriction (CR) in the yeast Saccharomyces cerevisiae suggest the importance of central metabolism for the extension of replicative life span by CR. However, the limited gene expression studies to date are not informative, because they have been conducted using cells grown in batch culture which markedly departs from the conditions under which yeasts are grown during life span determinations. In this study, we have examined the gene expression changes that occur during either glucose limitation or elimination of nonessential-amino acids, both of which enhance yeast longevity, culturing cells in a chemostat at equilibrium, which closely mimics conditions they encounter during life span determinations. Expression of 59 genes was examined quantitatively by real-time, reverse transcriptase polymerase chain reaction (qRT-PCR), and the physiological state of the cultures was monitored. Extensive gene expression changes were detected, some of which were common to both CR regimes. The most striking of these was the induction of tricarboxylic acid (TCA) cycle and retrograde response target genes, which appears to be at least partially due to the up-regulation of the HAP4 gene. These gene regulatory events portend an increase in the generation of biosynthetic intermediates necessary for the production of daughter cells, which is the measure of yeast replicative life span.