Srinivas Ayyadevara

Remarkable longevity and stress resistance of nematode PI3K-null mutants.

The great majority of lifespan‐augmenting mutations were discovered in the nematode Caenorhabditis elegans. In particular, genetic disruption of insulin‐like signaling extends longevity 1.5‐ to 3‐fold in the nematode, and to lesser degrees in other taxa, including fruit flies and mice. C. elegans strains bearing homozygous nonsense mutations in the age‐1 gene, which encodes the class‐I phosphatidylinositol 3‐kinase catalytic subunit (PI3KCS), produce progeny that were thought to undergo obligatory developmental arrest. We now find that, after prolonged developmental times at 15–20 °C, they mature into extremely long‐lived adults with near‐normal feeding rates and motility. They survive to a median of 145–190 days at 20 °C, with nearly 10‐fold extension of both median and maximum adult lifespan relative to N2DRM, a long‐lived wild‐type stock into which the null mutant was outcrossed. PI3K‐null adults, although a little less thermotolerant, are considerably more resistant to oxidative and electrophilic stresses than worms bearing normal or less long‐lived alleles. Their unprecedented factorial gains in survival, under both normal and toxic environments, are attributed to elimination of residual and maternally contributed PI3KCS or its products, and consequent modification of kinase signaling cascades.

Lifespan and stress resistance of Caenorhabditis elegans are increased by expression of glutathione transferases capable of metabolizing the lipid peroxidation product 4-hydroxynonenal

Caenorhabditis elegans expresses a glutathione transferase (GST) belonging to the Pi class, for which we propose the name CeGSTP2‐2. CeGSTP2‐2 (the product of the gst‐10 gene) has the ability to conjugate the lipid peroxidation product 4‐hydroxynonenal (4‐HNE). Transgenic C. elegans strains were generated in which the 5′‐flanking region and promoter of gst‐10 were placed upstream of gst‐10 and mGsta4 cDNAs, respectively. mGsta4 encodes the murine mGSTA4‐4, an enzyme with particularly high catalytic efficiency for 4‐HNE. The localization of both transgenes was similar to that of native CeGSTP2‐2. The 4‐HNE‐conjugating activity in worm lysates increased in the order: control < mGsta4 transgenic < gst‐10 transgenic; and the amount of 4‐HNE‐protein adducts decreased in the same order, indicating that the transgenic enzymes were active and effective in limiting electrophilic damage by 4‐HNE. Stress resistance and lifespan were measured in transgenic animals (five independent lines each) and were compared with two independent control lines. Resistance to paraquat, heat shock, ultraviolet irradiation and hydrogen peroxide was greater in transgenic strains. Median lifespan of mGsta4 and gst‐10 transgenic strains vs. control strains was increased by 13% and 22%, respectively. In addition to the cause–effect relationship between GST expression and lifespan observed in the transgenic lines, correlative evidence was also obtained in a series of congenic lines of C. elegans in which lifespan paralleled the 4‐HNE‐conjugating activity in whole‐animal lysates. We conclude that electrophilic damage by 4‐HNE may contribute to organismal aging.

Lifespan extension in hypomorphic daf‐2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2‐2

Electrophilic stress caused by lipid peroxidation products such as 4‐hydroxynonenal (4‐HNE) and/or related compounds may contribute to aging. The major mode of 4‐HNE metabolism involves glutathione conjugation catalyzed by specialized glutathione transferases. We have previously shown that glutathione transferase CeGSTP2‐2, the product of the Caenorhabditis elegans gst‐10 gene, has the ability to conjugate 4‐HNE, and that its overexpression extends lifespan of C. elegans. We now demonstrate that the expression level of CeGSTP2‐2 correlates highly with lifespan in a series of hypomorphic daf‐2 mutants of C. elegans. The overexpression of CeGSTP2‐2 in daf‐2 is abrogated in daf‐16; daf‐2 mutants, indicating that expression of the gst‐10 gene is modulated by insulin‐like growth factor signaling. To determine whether the relationship between CeGSTP2‐2 and lifespan is causal, we used RNAi to knock down CeGSTP2‐2. Treatment with gst‐10‐specific dsRNA decreased CeGSTP2‐2 protein in wild‐type N2 and in daf‐2 strains to an approximately equal level. The ability to conjugate 4‐HNE was similarly decreased by RNAi, suggesting that the increment of that activity in daf‐2 over N2 is due largely to the overexpression of CeGSTP2‐2. RNAi‐mediated knock‐down of CeGSTP2‐2 led to an increased susceptibility to 4‐HNE, paraquat, and heat shock, and to a shortening of lifespan by 13% in both N2 and daf‐2 strains. These results indicate that CeGSTP2‐2 significantly contributes to the maintenance of the soma, and that this function is augmented in daf‐2 mutants concordantly with other longevity assurance genes, probably via insulin‐like growth factor signaling.

Aspirin Inhibits Oxidant Stress, Reduces Age-Associated Functional Declines, and Extends Lifespan of Caenorhabditis elegans

AIMS Oxidative stress and inflammation are leading risk factors for age-associated functional declines. We assessed aspirin effects on endogenous oxidative-stress levels, lifespan, and age-related functional declines, in the nematode Caenorhabditis elegans. RESULTS Both aspirin and its salicylate moiety, at nontoxic concentrations (0.5-1 mM), attenuated endogenous levels of reactive oxygen species (p<0.001), and upregulated antioxidant genes encoding superoxide dismutases (especially sod-3, p<0.001), catalases (especially ctl-2, p<0.0001), and two glutathione-S-transferases (gst-4 and gst-10; each p<0.005). Aspirin, and to a lesser degree salicylate, improved survival of hydrogen peroxide, and in the absence of exogenous stress aspirin extended lifespan by 21%-23% (each p<10(-9)), while salicylate added 14% (p<10(-6)). Aspirin and salicylate delayed age-dependent declines in motility and pharyngeal pumping (each p<0.005), and decreased intracellular protein aggregation (p<0.0001)-all established markers of physiological aging-consistent with slowing of the aging process. Aspirin fails to improve stress resistance or lifespan in nematodes lacking DAF-16, implying that it acts through this FOXO transcription factor. INNOVATION Studies in mice and humans suggest that aspirin may protect against multiple age-associated diseases by reducing all-cause mortality. We now demonstrate that aspirin markedly slows many measures of aging in the nematode. CONCLUSIONS Aspirin treatment is associated with diminished endogenous oxidant stress and enhanced resistance to exogenous peroxide, both likely mediated by activation of antioxidant defenses. Our evidence indicates that aspirin attenuates insulin-like signaling, thus protecting against oxidative stress, postponing age-associated functional declines and extending C. elegans lifespan under benign conditions.

Prior exposure to oxidized low-density lipoprotein limits apoptosis in subsequent generations of endothelial cells by altering promoter methylation

Oxidized LDL (ox-LDL) plays a critical role in atherogenesis, including apoptosis. As hypercholesterolemia causes epigenetic changes resulting in long-term phenotypic consequences, we hypothesized that repeated and continuous exposure to ox-LDL may alter the pattern of apoptosis in human umbilical vein endothelial cells (HUVECs). We also analyzed global and promoter-specific methylation of apoptosis-related genes. As expected, ox-LDL evoked a dose-dependent increase in apoptosis in the first passage HUVECs that was completely abrogated by lectin-like ox-LDL receptor (LOX-1)-neutralizing antibody. Ox-LDL-induced apoptosis was associated with upregulation of proapoptotic LOX-1, ANXA5, BAX, and CASP3 and inhibition of antiapoptotic BCL2 and cIAP-1 genes accompanied with reciprocal changes in the methylation of promoter regions of these genes. Subsequent passages of cells displayed attenuated apoptotic response to repeat ox-LDL challenge with blunted gene expression and exaggerated methylation of LOX-1, BAX, ANXA5, and CASP3 genes (all P < 0.05 vs. first exposure to ox-LDL). Treatment of cells with LOX-1 antibody before initial ox-LDL treatment prevented both gene-specific promoter methylation and expression changes and reduction of apoptotic response to repeat ox-LDL challenge. Based on these data, we conclude that exposure of HUVECs to ox-LDL induces epigenetic changes leading to resistance to apoptosis in subsequent generations and that this effect may be related to the LOX-1-mediated increase in DNA methylation.

Positive Feedback between Transcriptional and Kinase Suppression in Nematodes with Extraordinary Longevity and Stress Resistance

Insulin/IGF-1 signaling (IIS) regulates development and metabolism, and modulates aging, of Caenorhabditis elegans. In nematodes, as in mammals, IIS is understood to operate through a kinase-phosphorylation cascade that inactivates the DAF-16/FOXO transcription factor. Situated at the center of this pathway, phosphatidylinositol 3-kinase (PI3K) phosphorylates PIP2 to form PIP3, a phospholipid required for membrane tethering and activation of many signaling molecules. Nonsense mutants of age-1, the nematode gene encoding the class-I catalytic subunit of PI3K, produce only a truncated protein lacking the kinase domain, and yet confer 10-fold greater longevity on second-generation (F2) homozygotes, and comparable gains in stress resistance. Their F1 parents, like weaker age-1 mutants, are far less robust—implying that maternally contributed trace amounts of PI3K activity or of PIP3 block the extreme age-1 phenotypes. We find that F2-mutant adults have <10% of wild-type kinase activity in vitro and <60% of normal phosphoprotein levels in vivo. Inactivation of PI3K not only disrupts PIP3-dependent kinase signaling, but surprisingly also attenuates transcripts of numerous IIS components, even upstream of PI3K, and those of signaling molecules that cross-talk with IIS. The age-1(mg44) nonsense mutation results, in F2 adults, in changes to kinase profiles and to expression levels of multiple transcripts that distinguish this mutant from F1 age-1 homozygotes, a weaker age-1 mutant, or wild-type adults. Most but not all of those changes are reversed by a second mutation to daf-16, implicating both DAF-16/ FOXO–dependent and –independent mechanisms. RNAi, silencing genes that are downregulated in long-lived worms, improves oxidative-stress resistance of wild-type adults. It is therefore plausible that attenuation of those genes in age-1(mg44)-F2 adults contributes to their exceptional survival. IIS in nematodes (and presumably in other species) thus involves transcriptional as well as kinase regulation in a positive-feedback circuit, favoring either survival or reproduction. Hyperlongevity of strong age-1(mg44) mutants may result from their inability to reset this molecular switch to the reproductive mode.

Caenorhabditis elegans PI3K mutants reveal novel genes underlying exceptional stress resistance and lifespan.

Two age‐1 nonsense mutants, truncating the class‐I phosphatidylinositol 3‐kinase catalytic subunit (PI3KCS) before its kinase domain, confer extraordinary longevity and stress‐resistance to Caenorhabditis elegans. These traits, unique to second‐generation homozygotes, are blunted at the first generation and are largely reversed by additional mutations to DAF‐16/FOXO, a transcription factor downstream of AGE‐1 in insulin‐like signaling. The strong age‐1 alleles (mg44, m333) were compared with the weaker hx546 allele on expression microarrays, testing four independent cohorts of each allele. Among 276 genes with significantly differential expression, 92% showed fewer transcripts in adults carrying strong age‐1 alleles rather than hx546. This proportion is significantly greater than the slight bias observed when contrasting age‐1 alleles to wild‐type worms. Thus, transcriptional changes peculiar to nonsense alleles primarily involve either gene silencing or failure of transcriptional activation. A subset of genes responding preferentially to age‐1‐nonsense alleles was reassessed by real‐time polymerase chain reaction, in worms bearing strong or weak age‐1 alleles; nearly all of these were significantly more responsive to the age‐1(mg44) allele than to age‐1(hx546). Additional mutation of daf‐16 reverted the majority of altered mg44‐F2 expression levels to approximately wild‐type values, although a substantial number of genes remained significantly distinct from wild‐type, implying that age‐1(mg44) modulates transcription through both DAF‐16/FOXO‐dependent and independent channels. When age‐1‐inhibited genes were targeted by RNA interference (RNAi) in wild‐type or age‐1(hx546) adults, most conferred significant oxidative‐stress protection. RNAi constructs targeting two of those genes were shown previously to extend life, and RNAi’s targeting five novel genes were found here to increase lifespan. PI3K‐null mutants may thus implicate novel mechanisms of life extension.

Quantitative trait loci define genes and pathways underlying genetic variation in longevity

Quantitative trait locus (QTL) mapping provides a means to discover and roughly position regions of the genome that harbor genes responsible for natural variation in a complex trait. QTL mapping has been utilized extensively in the pursuit of genes contributing to longevity, chiefly in two animal models, the nematode Caenorhabditis elegans and the dipteran insect Drosophila melanogaster. Research on both species has demonstrated that a relatively small set of loci accounts for most of their genetic variance in lifespan. QTL mapping complements the discovery of longevity genes by mutagenesis screens, because the two procedures are predicted to unveil overlapping but distinct types of genes. We argue that information gained from animal models, even invertebrates, can greatly facilitate the process of gene identification and testing of homologous genes in humans.

Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome-blocking complexes

Age‐dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP‐43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation‐prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc‐54p/Q40::YFP, a model of polyglutamine array diseases such as Huntingtons, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody‐coupled magnetic beads, were characterized by high‐resolution mass spectrometry. Three Q40::YFP‐associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimers disease models, nematodes with muscle or pan‐neuronal Aβ1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine‐rich, favoring hydrophobic interactions with other random‐coil domains. A particularly potent modulator of aggregation, CRAM‐1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72‒86% (P < 10−6). In worms expressing Aβ1–42, knockdown of cram‐1 reduced β‐amyloid 60% (P < 0.002) and slowed age‐dependent paralysis > 30% (P < 10−6). In wild‐type worms, cram‐1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM‐1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM‐1 (or human orthologs) with ubiquitin, and we verified that CRAM‐1 reduces degradation of a tagged‐ubiquitin reporter. We propose that CRAM‐1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.

A Small-Molecule Inhibitor of RAD51 Reduces Homologous Recombination and Sensitizes Multiple Myeloma Cells to Doxorubicin.

We previously reported high expression of RAD51 and increased homologous recombination (HR) rates in multiple myeloma (MM) cells, and showed that genomic instability and disease progression are commensurate with HR levels. Moreover, high RAD51 expression in vivo is associated with chemoresistance and poor patient survival. Doxorubicin (DOX) is one of the most widely used drug treatments in MM chemotherapy. DOX is cytotoxic because it induces DNA double-strand breaks, which can be repaired by RAD51-mediated HR; activation of this pathway thus contributes to resistance. To investigate the role of RAD51 in MM drug resistance, we assessed the ability of B02, a small-molecule inhibitor of RAD51, to enhance DOX sensitivity of MM cells. Combining low-toxicity doses of DOX and B02 resulted in significant synthetic lethality, observed as increased apoptosis and reduced viability compared to either agent alone, or to the product of their individual effects. In contrast, the combination did not produce significant synergy against normal human CD19+ B cells from peripheral blood. DOX induced RAD51 at both mRNA and protein levels, while arresting cells in S and G2. DOX treatment also increased the number of RAD51 foci, a marker of HR repair, so that the fraction of cells with ≥5 foci rose fourfold, whereas γH2AX foci rose far less, implying that most new breaks are repaired. When B02 treatment preceded DOX exposure, the induction of RAD51 foci was severely blunted, whereas, γH2AX foci rose significantly relative to basal levels or either agent alone. In MM cells carrying a chromosomally integrated reporter of HR repair, DOX increased HR events while B02 inhibition of RAD51 blocked the HR response. These studies demonstrate the crucial role of RAD51 in protecting MM cells from genotoxic agents such as DOX, and suggest that specific inhibition of RAD51 may be an effective means to block DNA repair in MM cells and thus to enhance the efficacy of chemotherapy.