Aging mechanisms

Aging (senescence) has long been a difficult issue to be experimentally analyzed because of stochastic processes, which contrast with the programmed events during early development. However, we have recently started to learn the molecular mechanisms that control aging. Studies of the mutant mouse, klotho, showing premature aging, raise a possibility that mammals have an “anti-aging hormone.” A decrease of cell proliferation ability caused by the telomeres is also tightly linked to senescence. Frontier experimental studies of aging at the molecular level are leading to fascinating hypotheses that aging is the price we had to pay for the evolution of the sexual reproduction system that produces a variety of genetic information and complex body structures.

Nucleolar localization of the Werner syndrome protein in human cells

Werner Syndrome (WS) is a human genetic disorder with many features of premature aging. The gene defective in WS (WRN) has been cloned and encodes a protein homologous to several helicases, including Escherichia coli RecQ, the human Bloom syndrome protein (BLM), and Saccharomyces cerevisiae Sgs1p. To better define the function of WRN protein we have determined its subcellular localization. Indirect immunofluorescence using polyclonal anti-human WRN shows a predominant nucleolar localization. Studies of WRN mutant cells lines confirmed the specificity of antibody recognition. No difference was seen in the subcellular localization of the WRN protein in a variety of normal and transformed human cell lines, including both carcinomas and sarcomas. The nucleolar localization of human WRN protein was supported by the finding that upon biochemical subcellular fractionation, WRN protein is present in an increased concentration in a subnuclear fraction enriched for nucleolar proteins. We have also determined the subcellular localization of the mouse WRN homologue (mWRN). In contrast to human WRN protein, mWRN protein is present diffusely throughout the nucleus. Understanding the function of WRN in these organisms of vastly differing lifespan may yield new insights into the mechanisms of lifespan determination.

The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.

A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

Werner syndrome (WS) is a premature aging disorder where the affected individuals appear much older than their chronological age. The single gene that is defective in WS encodes a protein (WRN) that has ATPase, helicase and 3′→5′ exonuclease activities. Our laboratory has recently uncovered a physical and functional interaction between WRN and the Ku heterodimer complex that functions in double-strand break repair and V(D)J recombination. Importantly, Ku specifically stimulates the exonuclease activity of WRN. We now report that Ku enables the Werner exonuclease to digest through regions of DNA containing 8-oxoadenine and 8-oxoguanine modifications, lesions that have previously been shown to block the exonuclease activity of WRN alone. These results indicate that Ku significantly alters the exonuclease function of WRN and suggest that the two proteins function concomitantly in a DNA damage processing pathway. In support of this notion we also observed co-localization of WRN and Ku, particularly after DNA damaging treatments.

On signal sequence polymorphisms and diseases of distribution.

We report a previously unappreciated property of the signals that target organelle-specific proteins to their subcellular sites of action. Such targeting sequences are shown to be polymorphic. We discovered this polymorphism when we cloned the mitochondrial manganese-containing superoxide dismutase from cell lines of normal individuals and patients with genetic diseases of premature aging and compared their sequences to each other and to those previously reported. The polymorphism consists of a single nucleotide change in the region of the DNA that encodes the signal sequence such that either an alanine or valine is present. Subsequently, eight cell lines were analyzed and all three possible combinations of the two signal sequences were observed. Such signal sequence polymorphisms could result in diseases of distribution, where essential proteins are not properly targeted, thereby leading to absolute or relative deficiencies of critical enzymes within specific cellular compartments. Progeria and related syndromes may be diseases of distribution.

Oxidative damage during aging targets mitochondrial aconitase

The mechanisms that cause aging are not well understood. The oxidative stress hypothesis proposes that the changes associated with aging are a consequence of random oxidative damage to biomolecules. We hypothesized that oxidation of specific proteins is critical in controlling the rate of the aging process. Utilizing an immunochemical probe for oxidatively modified proteins, we show that mitochondrial aconitase, an enzyme in the citric acid cycle, is a specific target during aging of the housefly. The oxidative damage detected immunochemically was paralleled by a loss of catalytic activity of aconitase, an enzyme activity that is critical in energy metabolism. Experimental manipulations which decrease aconitase activity should therefore cause a decrease in life-span. This expected decrease was observed when flies were exposed to hyperoxia, which oxidizes aconitase, and when they were given fluoroacetate, an inhibitor of aconitase. The identification of a specific target of oxidative damage during aging allows for the assessment of the physiological age of a specific individual and provides a method for the evaluation of treatments designed to affect the aging process.

Mitochondrial adenine nucleotide translocase is modified oxidatively during aging

The purpose of this study was to test the hypothesis that elevation in protein oxidative damage during the aging process is a targeted rather than a stochastic phenomenon. Oxidative damage to proteins in mitochondrial membranes in the flight muscles of the housefly, manifested as carbonyl modifications, was detected immunochemically with anti-dinitrophenyl antibodies. Adenine nucleotide translocase (ANT) was found to be the only protein in the mitochondrial membranes exhibiting a detectable age-associated increase in carbonyls. The age-related elevation in ANT carbonyl content was correlated with a corresponding loss in its functional activity. Senescent flies that had lost the ability to fly exhibited a relatively higher degree of ANT oxidation and a greater loss of functional activity than their cohorts of the same age that were still able to fly. Exposure of flies to 100% oxygen resulted in an increase in the level of ANT carbonyl content and a loss in its activity. In vitro treatment of mitochondria with a system that generated hydroxyl free radicals caused an increase in ANT carbonyl level and a decrease in ANT exchange activity. ANT was also the only mitochondrial membrane protein exhibiting adducts of the lipid peroxidation product 4-hydroxynonenal. Results of this study indicate that proteins in mitochondrial membranes are modified selectively during aging.

Potentially predictive and manipulable blood serum correlates of aging in the healthy human male: Progressive decreases in bioavailable testosterone, dehydroepiandrosterone sulfate, and the ratio of insulin-like growth factor 1 to growth hormone

A cross-sectional survey was made in 56 exceptionally healthy males, ranging in age from 20 to 84 years. Measurements were made of selected steroidal components and peptidic hormones in blood serum, and cognitive and physical tests were performed. Of those blood serum variables that gave highly significant negative correlations with age (r > −0.6), bioavailable testosterone (BT), dehydroepiandrosterone sulfate (DHEAS), and the ratio of insulin-like growth factor 1 (IGF-1) to growth hormone (GH) showed a stepwise pattern of age-related changes most closely resembling those of the age steps themselves. Of these, BT correlated best with significantly age-correlated cognitive and physical measures. Because DHEAS correlated well with BT and considerably less well than BT with the cognitive and physical measures, it seems likely that BT and/or substances to which BT gives rise in tissues play a more direct role in whatever processes are rate-limiting in the functions measured and that DHEAS relates more indirectly to these functions. The high correlation of IGF-1/GH with age, its relatively low correlation with BT, and the patterns of correlations of IGF-1/GH and BT with significantly age-correlated cognitive and physical measures suggest that the GH–IGF-1 axis and BT play independent roles in affecting these functions. Serial determinations made after oral ingestion of pregnenolone and data from the literature suggest there is interdependence of steroid metabolic systems with those operational in control of interrelations in the GH–IGF-1 axis. Longitudinal concurrent measurements of serum levels of BT, DHEAS, and IGF-1/GH together with detailed studies of their correlations with age-correlated functional measures may be useful in detecting early age-related dysregulations and may be helpful in devising ameliorative approaches.

Long-term suppression of Leydig cell steroidogenesis prevents Leydig cell aging

Male aging is accompanied by reduced testosterone production by the Leydig cells, the testosterone-producing cells of the testis. The mechanism by which this occurs is unknown. Based on the observations that reactive oxygen is capable of damaging components of the steroidogenic pathway and that reactive oxygen is produced during steroidogenesis itself, we hypothesized that long-term suppression of steroidogenesis might inhibit or prevent age-related deficits in Leydig cell testosterone production. To test this, we administered contraceptive doses of testosterone to groups of young (3 months old) and middle-aged (13 months old) Brown Norway rats via Silastic implants to suppress endogenous Leydig cell testosterone production. After 8 months, the implants were removed, which rapidly (days) restores the ability of the previously suppressed Leydig cells to produce testosterone. Two months after removing the implants, when the rats of the two groups were 13 and 23 months of age, respectively, the Leydig cells in both cases were found to produce testosterone at the high levels of young Leydig cells, whereas significantly lower levels were produced by the 23-month-old controls. Thus, by placing the Leydig cells in a state of steroidogenic “hibernation,” the reductions in Leydig cell testosterone production that invariably accompany aging did not occur. If hormonal contraception in the human functions the same way, the adverse consequences of reduced testosterone in later life (osteoporosis, reduced muscle mass, reduced libido, mood swings, etc.) might be delayed or prevented.

Overexpression of the Matrix Metalloproteinase Matrilysin Results in Premature Mammary Gland Differentiation and Male Infertility

To examine the role of matrilysin (MAT), an epithelial cell-specific matrix metalloproteinase, in the normal development and function of reproductive tissues, we generated transgenic animals that overexpress MAT in several reproductive organs. Three distinct forms of human MAT (wild-type, active, and inactive) were placed under the control of the murine mammary tumor virus promoter/enhancer. Although wild-type, active, and inactive forms of the human MAT protein could be produced in an in vitro culture system, mutations of the MAT cDNA significantly decreased the efficiency with which the MAT protein was produced in vivo. Therefore, animals carrying the wild-type MAT transgene that expressed high levels of human MAT in vivo were further examined. Mammary glands from female transgenic animals were morphologically normal throughout mammary development, but displayed an increased ability to produce β-casein protein in virgin animals. In addition, beginning at approximately 8 mo of age, the testes of male transgenic animals became disorganized with apparent disintegration of interstitial tissue that normally surrounds the seminiferous tubules. The disruption of testis morphology was concurrent with the onset of infertility. These results suggest that overexpression of the matrix-degrading enzyme MAT alters the integrity of the extracellular matrix and thereby induces cellular differentiation and cellular destruction in a tissue-specific manner.

Checkpoint Defects Leading to Premature Mitosis Also Cause Endoreplication of DNA in Aspergillus nidulans

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMXcdc2 in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsBrad3 and uvsDrad26 have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIMEcyclinB, but ectopic expression of active nondegradable NIMEcyclinB does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMXcdc2 and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.

Dissociation of Oct-1 from the Nuclear Peripheral Structure Induces the Cellular Aging-associated Collagenase Gene Expression

The cellular aging-associated transcriptional repressor that we previously named as Orpheus was identical to Oct-1, a member of the POU domain family. Oct-1 represses the collagenase gene, one of the cellular aging-associated genes, by interacting with an AT-rich cis-element in the upstream of the gene in preimmortalized cells at earlier population-doubling levels and in immortalized cells. In these stages of cells, considerable fractions of the Oct-1 protein were prominently localized in the nuclear periphery and colocalized with lamin B. During the cellular aging process, however, this subspecies of Oct-1 disappeared from the nuclear periphery. The cells lacking the nuclear peripheral Oct-1 protein exhibited strong collagenase expression and carried typical senescent morphologies. Concomitantly, the binding activity and the amount of nuclear Oct-1 protein were reduced in the aging process and resumed after immortalization. However, the whole cellular amounts of Oct-1 protein were not significantly changed during either process. Thus, the cellular aging-associated genes including the collagenase gene seemed to be derepressed by the dissociation of Oct-1 protein from the nuclear peripheral structure. Oct-1 may form a transcriptional repressive apparatus by anchoring nuclear matrix attachment regions onto the nuclear lamina in the nuclear periphery.

The biological clock of very premature primate infants is responsive to light

Each year more than 250,000 infants in the United States are exposed to artificial lighting in hospital nurseries with little consideration given to environmental lighting cycles. Essential in determining whether environmental lighting cycles need to be considered in hospital nurseries is identifying when the infant’s endogenous circadian clock becomes responsive to light. Using a non-human primate model of the developing human, we examined when the circadian clock, located in the hypothalamic suprachiasmatic nuclei (SCN), becomes responsive to light. Preterm infant baboons of different ages were exposed to light (5,000 lux) at night, and then changes in SCN metabolic activity and gene expression were assessed. After exposure to bright light at night, robust increases in SCN metabolic activity and gene expression were seen at ages that were equivalent to human infants at 24 weeks after conception. These data provide direct evidence that the biological clock of very premature primate infants is responsive to light.

Aging accentuates and bone marrow transplantation ameliorates metabolic defects in Fabry disease mice

Fabry disease is an X-linked metabolic disorder caused by a deficiency of α-galactosidase A (α-Gal A). The enzyme defect leads to the systemic accumulation of glycosphingolipids with α-galactosyl moieties consisting predominantly of globotriaosylceramide (Gb3). In patients with this disorder, glycolipid deposition in endothelial cells leads to renal failure and cardiac and cerebrovascular disease. Recently, we generated α-Gal A gene knockout mouse lines and described the phenotype of 10-week-old mice. In the present study, we characterize the progression of the disease with aging and explore the effects of bone marrow transplantation (BMT) on the phenotype. Histopathological analysis of α-Gal A −/0 mice revealed subclinical lesions in the Kupffer cells in the liver and macrophages in the skin with no gross lesions in the endothelial cells. Gb3 accumulation and pathological lesions in the affected organs increased with age. Treatment with BMT from the wild-type mice resulted in the clearance of accumulated Gb3 in the liver, spleen, and heart with concomitant elevation of α-Gal A activity. These findings suggest that BMT may have a potential role in the management of patients with Fabry disease.

Adaptive evolution of a candidate gene for aging in Drosophila

Examination of the phenotypic effects of specific mutations has been extensively used to identify candidate genes affecting traits of interest. However, such analyses do not reveal anything about the evolutionary forces acting at these loci, or whether standing allelic variation contributes to phenotypic variance in natural populations. The Drosophila gene methuselah (mth) has been proposed as having major effects on organismal stress response and longevity phenotype. Here, we examine patterns of polymorphism and divergence at mth in population level samples of Drosophila melanogaster, D. simulans, and D. yakuba. Mth has experienced an unusually high level of adaptive amino acid divergence concentrated in the intra- and extracellular loop domains of the receptor protein, suggesting the historical action of positive selection on those regions of the molecule that modulate signal transduction. Further analysis of single nucleotide polymorphisms (SNPs) in D. melanogaster provided evidence for contemporary and spatially variable selection at the mth locus. In ten surveyed populations, the most common mth haplotype exhibited a 40% cline in frequency that coincided with population level differences in multiple life-history traits including lifespan. This clinal pattern was not associated with any particular SNP in the coding region, indicating that selection is operating at a closely linked site that may be involved in gene expression. Together, these consistently nonneutral patterns of inter- and intraspecific variation suggest adaptive evolution of a signal transduction pathway that may modulate lifespan in nature.