The rs2516839 variation of USF1 gene is associated with 4-year mortality of nonagenarian women: The Vitality 90+ study.

Upstream transcription factor 1 (USF1) regulates the transcription of many genes related to cell and organism survival processes such as stress and immune response, regulation of cellular senesce, and carcinogenesis. In this study, our aim was to investigate the effect of USF1 single nucleotide variations (SNVs) on longevity in the Vitality 90+ study, a population-based study of nonagenarians (90 ±1 years of age) living in the area of Tampere municipality, Finland. Altogether 509 voluntary nonagenarians (115 males, 394 females) were genotyped using the 5'-nuclease assay for rs2774279G > A, rs2516839T > C, and rs2073658C > T SNVs. During the 4 years of follow-up, the total mortality rate was 64.2%. In the study, we found that the frequency of C-allele of rs2516839 among nonsurviving nonagenarians (52.5%) was higher than those who survived (41.2%; P = 0.0006, odds ratio = 1.575, 95% confidence interval [CI]: 1.215-2.041). Furthermore, carriage of this variation and its haplotypes had a significant gender by genotype interaction (P < 0.05) on mortality. Kaplan-Meier log-rank test during 4-years of follow-up showed significantly higher mortality rate in the case of CC genotype carriage than other genotype carriages in nonagenarian women (P < 0.0001). In addition, after adjusting for age in Cox regression analysis, cardiovascular disease, diabetes, infectious disease, dementia, and living place (nursing home or home), CC genotype of rs2516839T > C was found to be associated with shorter life expectancy in nonagenarian women (hazard ratio = 2.27; 95% CI, 1.34-3.85 P = 0.002). In conclusion, rs2516839 variation and related haplotypes of the USF1 gene are strongly related to all-cause mortality in Finnish nonagenarians, especially among women.

Autoimmunity and longevity: presence of antinuclear antibodies is not associated with the rate of inflammation or mortality in nonagenarians.

There are reports demonstrating elevated levels of autoantibodies in elderly people. We now analyzed whether the strong inflammatory response associated with aging is interrelated with the production of autoantibodies, antinuclear antibodies (ANA). In a cohort of 284 nonagenarians the rate of ANA positivity was 12.3%, which is significantly (p<0.001) higher than that in the middle-aged controls (2.8%). The mortality data of this cohort was collected after a 4-year follow-up. The ANA positivity at the age of 90 did not have any effect on the rate of survival, or on the levels of serum markers of inflammation.

CRP gene is involved in the regulation of human longevity: a follow-up study in Finnish nonagenarians.

Chronic low-grade inflammation is involved in the pathogenesis of many disease conditions in humans and it is frequently quantified by measuring the blood concentration of C-reactive protein (CRP). Here we show that the CRP concentration in old people (nonagenarians) is, at least partially, genetically determined, and that the high producer genotype is associated with a shorter life expectancy during follow-up. Thus, the data imply that the CRP gene may be a longevity gene in humans.

Indoleamine 2,3-dioxygenase activity in nonagenarians is markedly increased and predicts mortality.

Indoleamine 2,3-dioxygenase (IDO), an enzyme degrading tryptophan (trp) to kynurenine (kyn), suppresses T cell activity. Ageing of the immune system, immunosenescence, includes a decline in T cell function. We therefore sought to establish whether IDO activity is involved in immunosenescence and whether it predicts mortality in aged subjects. We measured kyn/trp, reflecting IDO activity, in 284 nonagenarians and 309 blood donor controls. IDO activity was significantly higher in nonagenarians compared with controls and IDO activity at study entry predicted subsequent mortality in nonagenarians. Thus, increased IDO activity might be a mechanism involved in the decline of T cell responses in immunosenescence.

The trajectory of the blood DNA methylome ageing rate is largely set before adulthood: evidence from two longitudinal studies.

The epigenetic clock, defined as the DNA methylome age (DNAmAge), is a candidate biomarker of ageing. In this study, we aimed to characterize the behaviour of this marker during the human lifespan in more detail using two follow-up cohorts (the Young Finns study, calendar age i.e. cAge range at baseline 15-24 years, 25-year-follow-up, N = 183; The Vitality 90+ study, cAge range at baseline 19-90 years, 4-year-follow-up, N = 48). We also aimed to assess the relationship between DNAmAge estimate and the blood cell distributions, as both of these measures are known to change as a function of age. The subjects' DNAmAges were determined using Horvath's calculator of epigenetic cAge. The estimate of the DNA methylome age acceleration (Δ-cAge-DNAmAge) demonstrated remarkable stability in both cohorts: the individual rank orders of the DNAmAges remained largely unchanged during the follow-ups. The blood cell distributions also demonstrated significant intra-individual correlation between the baseline and follow-up time points. Interestingly, the immunosenescence-associated features (CD8+CD28- and CD4+CD28- cell proportions and the CD4/CD8 cell ratio) were tightly associated with the estimate of the DNA methylome age. In summary, our data demonstrate that the general level of Δ-cAge-DNAmAge is fixed before adulthood and appears to be quite stationary thereafter, even in the oldest-old ages. Moreover, the blood DNAmAge estimate seems to be tightly associated with ageing-associated shifts in blood cell composition, especially with those that are the hallmarks of immunosenescence. Overall, these observations contribute to the understanding of the longitudinal aspects of the DNAmAge estimate.

Interleukin-6 -174G/C polymorphism and longevity: a follow-up study.

Increased rate of inflammation has been observed to be associated with aging. This is manifested, e.g. as increased blood levels of proinflammatory cytokines, such as interleukin-6 (IL-6). The production of IL-6 is, at least partially, genetically determined the single nucleotide polymorphism (SNP) at the promoter (-174G/C) being decisive. Consequently, some studies have demonstrated that the -174G/C genotype frequencies are different in very old persons as compared to younger ones. However, the results published this far have been conflicting. One of the main confounding factors in these kind of case/control association studies is the undetected difference in the population structure. To avoid this, we now have collected the mortality data of our cohort of 285 nonagenarians (representing mortality between 90 and 95 years of age) and correlated these to the IL-6 genotype. The frequency of -174 allele G was clearly higher in the survivors (n = 114) than in the non-survivors (n = 171).

Transcriptomic and epigenetic analyses reveal a gender difference in aging-associated inflammation: the Vitality 90+ study.

Aging is associated with a pro-inflammatory state, often referred to as inflammaging. The origin of the pro-inflammatory mediators and their role in the pathogenesis of the aging-associated diseases remain poorly understood. As aging is also associated with profound changes in the transcriptomic and epigenetic (e.g., DNA methylation) profiles of cells in the peripheral blood, we analyzed the correlation of these profiles with inflammaging using the "classical" marker interleukin-6 as an indicator. The analysis of the whole-genome peripheral blood mononuclear cell (PBMC) gene expression revealed 62 transcripts with expression levels that significantly correlated with the plasma interleukin-6 (IL-6) levels in men, whereas no correlations were observed in women. The Gene Ontology analysis of plasma IL-6-associated transcripts in men revealed processes that were linked to the inflammatory response. Additionally, an Ingenuity Pathway Analysis (IPA) pathway analysis identified Tec kinase signaling as an affected pathway and upstream regulator analysis predicted the activation of IL-10 transcript. DNA methylation was assessed using a HumanMethylation450 array. Seven genes with expression profiles that were associated with the plasma IL-6 levels in men were found to harbor CpG sites with methylation levels that were also associated with the IL-6 levels. Among these genes were IL1RN, CREB5, and FAIM3, which mapped to a network of inflammatory response genes. According to our results, inflammaging is manifested differently at the genomic level in nonagenarian men and women. Part of this difference seems to be of epigenetic origin. These differences point to the genomic regulation of inflammatory response and suggest that the gender-specific immune system dimorphism in older individuals could be accounted for, in part, by DNA methylation.

Reactions of rat sympathetic neurons to ethanol exposure are age-dependent.

Age-differences in the sensitivity of peripheral sympathetic neurons to chronic ethanol exposure and ethanol withdrawal were studied in male Wistar rats aged 4 months, 12 months, or 24 to 25 months. The superior cervical ganglia (SCG) of the young (4 months) and the 2-year-old rats responded to a 12-day or 4-week ethanol exposure with significantly increased catecholamine turnover, while the ganglia of the middle-aged rats (12 months) showed only a minor increase in the intensity of catecholamine fluorescence and tyrosine hydroxylase immunoreactivity. Extensive neuronal vacuolation was found in the 4 months ethanol-exposed SCG, probably as a reaction of a subpopulation of neurons to increased stimulation. Ethanol-induced neuronal loss was most prominent in the SCG of the oldest age group. Contrary to the marked changes in SCG functional and morphometric parameters, the pelvic sympathetic neurons in the hypogastric ganglion showed no significant changes after ethanol exposure. The pattern of ethanol-induced morphological alterations found in the present study did not provide unambiquous support for either the "accelerated aging" or the "increased vulnerability" concept regarding ethanol-aging interactions in the nervous system.

Sympathetic neurons outlive the original host after transplantation to the rat submandibular gland.

Sympathetic ganglion tissue of aged (36 months) Wistar rats was allotransplanted into the submandibular gland (SMG) of young (3 months) animals to study whether sympathetic neurons can outlive the original host. The viability of the transplants was evaluated one year postgrafting, using the formaldehyde-induced fluorescence technique (FIF) for histochemical demonstration of catecholamines, tyrosine hydroxylase (TH) immunohistochemistry, and morphometry. One year after transplantation, grafted superior cervical ganglion (SCG) cells demonstrated catecholamine fluorescence and tyrosine hydroxylase immunoreactivity. The transplants consisted of groups of sympathetic neurons dispersed in a fibrous matrix. After long postgrafting time, the sympathetic neurons of aged rats showed several signs of enhanced degeneration; increased autofluorescent lipopigment, decreased neuronal density and reduced catecholamine fluorescence. The mean diameter of the transplanted aged neurons was significantly decreased. The histograms of grouped diameter values showed a shift to smaller cells in ganglion transplants. A subpopulation of small and medium-sized grafted neurons sent out fluorescent fibers, which were located in a fibrous scar area but did not extend into submandibular host tissue. The results indicate that a long postgrafting time induced degeneration which is comparable to normal aging changes in grafted very old neurons. Thus, aged sympathetic neurons maintain plasticity to survive as transplants, and under favourable conditions these neurons outlive the original host.

Indices of neurotransmitter synthesis and release in aging sympathetic nervous system.

Activities of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and choline acetyltransferase (ChAT) were assayed in sympathetic ganglia and adrenal glands of young and aged Fischer-344 rats. The recovery of reserpine-depleted catecholamine stores and catecholamine loss following inhibition of synthesis and intraneuronal degradation also were assessed in sympathetic ganglia of young and aged rats. No age differences were observed in DBH activity of any tissues examined or in the activities of any enzymes in the coeliac-mesenteric ganglion complex. However, TH and ChAT activities were significantly higher in the superior cervical ganglia and adrenal glands of aged rats. In the hypogastric ganglion, only TH activity was higher in the old rats. Recoveries of reserpine-depleted catecholamine stores in the superior cervical and hypogastric ganglia of aged rats were slower than in young rats. Catecholamine loss following inhibition of synthesis and intraneuronal degradation was faster in the superior cervical ganglia but not in the hypogastric ganglia of old rats as compared with young rats. These findings suggest that neurotransmitter synthesis and release are enhanced with age in the superior cervical ganglion. The lack of age-related changes in the hypogastric ganglion might reflect the different cellular composition as well as the physiological role of this ganglion.

Age-related changes in the peroxyl radical scavenging capacity of human plasma.

Aging and the diseases that typically follow with increasing age, notably atherosclerosis and cancer, are often proposed to be involved in increased oxidative stress. Animal studies, on the other hand, show no clear-cut pattern of age-related changes in enzymatic antioxidant defences. In this study we have demonstrated that total peroxyl radical scavenging antioxidant capacity (TRAP) in human plasma changes with age. We also found that among the antioxidants in human plasma there exists a major fraction of so far unidentified antioxidant(s). A chemiluminescent TRAP assay was used to determine the presence of peroxyl radical scavenging antioxidants in human plasma. The material consisted of 87 healthy volunteers, aged 20-96 years, who used no regular medication, vitamins, or trace elements. In females, total antioxidant capacity increased significantly during the life span. The increase in TRAP was mainly due to unidentified antioxidants. In males, TRAP increased until age 51-74, and then significantly decreased. The decrease observed among males was also due to the sharp decline in the concentration of unidentified antioxidants.

Immunocytochemical localization of enkephalin and substance P in the dorsal tegmental nuclei in human fetal brain.

The peroxidase-antiperoxidase (PAP) technique is used to examine the localization of methionine [Met 5]-enkephalin and substance P in the dorsal tegmental nuclei of the human fetal brain. Specific antiserum to both peptides is immunocytochemically detectable in neuronal perikarya and varicose processes in the human dorsal tegmental nuclei at 17-21 weeks of gestation. However the two peptides show a differential distribution in the central versus peripheral portions of the nucleus. Enkephalin-like immunoreactivity is primarily localized in perikarya along the pripheral borders of the tegmental nuclei, whereas substance P is present primarily in varicose terminals and a few perikarya in the more central portions. These findings suggest that substance P-containing terminals in dorsal tegmental nuclei are probably derived from afferent axons and that enkephalin is present in intrinsic neurons.

Immunocytochemical localization of tyrosine hydroxylase in the human fetal nervous system.

The peroxidase-antiperoxidase (PAP) technique is used to determine the cellular localization of tyrosine hydroxylase in the human fetal nervous system. Antiserum to trypsin-treated tyrosine hydroxylase from the bovine adrenal medulla can be detected immunocytochemically in peripheral sympathetic neurons in the 8-mm (5-week) fetus, but can not be detected in the central nervous system until later stages of development. The cytological features and distribution of the neuronal perikarya and processes labeled for the enzyme are similar to those of the catecholaminergic neurons previously identified by histofluorescence. These findings indicate that specific neurons in the human fetus have at least one of the enzymes necessary for the biosynthesis of catecholamines and that cross-species reactivity exists between antiserum produced to the bovine tyrosine hydroxylase and human tissues.

Light and electron microscopic features of peripheral ganglion cells cultured from young and aged Wistar rats.

Neurons of the dorsal root ganglion (DRG) and sympathetic superior cervical ganglion (SCG) were cultured as explants from young adult (3 months old) and aged (28 months old) Wistar rats. Both aged DRG and SCG neurons showed delayed neurite outgrowth compared to young adult neurons. Young and some aged cultured neurons had an ultrastructure similar to that found in normal uncultured cells, but most of the aged cultured neurons displayed a heavy accumulation of homogenous lipid-like inclusions in addition to classic age pigments. Occasionally, large neurofilament aggregates were seen in aged DRG neurons. They were sometimes localized perinuclearly, resembling neurofibrillary tangles. The results show that even very old peripheral neurons survive in culture. As aged cultured neurons show degenerative changes not observed in young adult neurons, it is suggested that cultured peripheral neurons of different ages could provide useful means for neuronal aging studies.

Effect of sialectomy on the superior cervical ganglion sympathetic neurons in young adult and aged mice.

The dependence of superior cervical ganglion (SCG) adrenergic neurons on their target organ submandibular salivary gland containing high concentrations of nerve growth factor was studied in adult and aged male mice. The submandibular salivary glands were removed (sialectomy) either uni- or bilaterally, and the SCG were studied by fluorescence microscopy and histochemically. Catecholamine fluorescence and tyrosine hydroxylase immunoreactivity decreased after sialectomy, suggesting reduced noradrenaline production. Neuronal density was lower in the aged controls than in the young controls. In both age groups, sialectomy reduced the density of catecholamine-producing neurons. In the mouse SCG, there was remarkable heterogeneity in the size of neuronal somata. In aged control mice there was a greater number of large-size neurons than in young adult control mice. Six weeks postoperatively, no large catecholamine-producing neurons could be observed in the ganglia. Yellow autofluorescent lipopigments accumulated with age in the adrenergic neurons. Sialectomy increased the accumulation of lipopigments in both young and aged neurons. Sialectomy resulted in (a) reduced catecholamine fluorescence, (b) reduced tyrosine hydroxylase immunoreactivity, (c) reduced number of catecholamine neurons, (d) increased autofluorescent lipopigment. Ageing resulted in (a) reduced number of neurons, (b) increased ratio of large to small neurons, (c) increased autofluorescent lipopigment. Alterations after sialectomy were more detrimental in the aged ganglia than in the young adult ganglia. The discontinuation of the retrograde supply of nerve growth factor may contribute to these alterations.

Lifelong ethanol consumption enhances the age-related changes in rat sympathetic neurons.

The effects of aging and chronic ethanol administration on the histochemical and morphometric features of rat superior cervical ganglion were studied in a rat strain selected for voluntary alcohol consumption. Ethanol was administered to the experimental group ad libitum (10% v/v in drinking water) from 3 months to 28 months of age, the average ethanol intake being 6.4-5.4 g/kg per day. The sympathetic neurons of the ethanol consuming rats showed several signs of enhanced degeneration, e.g. decreased neuronal packing density, increased amount of age-pigment and decreased intensity of catecholamine histofluorescence and tyrosine hydroxylase immunoreactivity. The results may indicate a selective vulnerability of peripheral sympathetic neurons rather than a universal accelerated aging due to chronic ethanol exposure.

The effect of bleaching on the lipopigments in the human sympathetic neurons.

The effect of exposure to H2O2 on the lipopigments of the human sympathetic neurons was investigated by fluorescence and electron microscopy. In intact ganglia two subtypes of the lipopigment were seen: the yellow fluorescent pigment (emission maximum at 510 nm), which under the electron microscope had two components (vacuoles and osmiophilic matrix) and the orange fluorescent pigment (emission maximum at 535 nm), which in electron microscopy showed a third, highly osmiophilic component. Bleaching did not change the emission spectrum of the yellow pigment, while that of orange pigment shifted from 535 nm to 485 nm. Under electron microscopy the highly osmiophilic component disappeared in bleached orange pigment bodies. The characteristics of orange autofluorescent lipopigment are similar to those described for neuromelanin in the substantia nigra. The results suggest that neuromelanin accumulates in aging sympathetic neurons.

Effect of vitamin E and selenium supplement on the aging peripheral neurons of the male Sprague-Dawley rat.

The effect of life-long diets containing different concentrations of selenium and vitamin E on the age pigment accumulation in the rat superior cervical ganglion, vagal ganglion and dorsal root ganglion, was studied using microspectrofluorometry. All types of ganglia showed unchanged amounts of age pigments at low or high concentrations of selenium, whereas dietary concentration of vitamin E regulated age pigment content in the dorsal root ganglion, but not in the superior cervical and vagal ganglion. Vitamin E deficiency induced a three-fold increase in age pigment content in dorsal root ganglion at 8 months of age, whereas high vitamin E concentration was associated with a lesser amount of pigments at 18 months of age. Emission spectra of age pigment recorded from the dorsal root ganglion and vagal ganglion were different from that from the superior cervical ganglion, but were independent of dietary concentrations of selenium or vitamin E. The results suggest that exogenous antioxidants may play a more crucial role in lipid peroxidation and accumulation of age pigment in dorsal root ganglion than in autonomic ganglia.

Microspectrofluorometric quantitation of autofluorescent lipopigment in the human sympathetic ganglia.

The age-related changes in lipopigment autofluorescence were studied by microspectrofluorometry in three different types of human neurons: the sympathetic neurons of the stellate and superior mesenteric ganglion and pyramidal neurons of the frontal cortex. The age-related increase in lipopigment autofluorescence was more rapid in stellate ganglion but similar linear increases were found also in superior mesenteric ganglion and frontal cortex. There was an age-related shift in the autofluorescence from yellow to orange in the ganglia. This may be due to the accumulation of neuromelanin in noradrenergic neurons. Lipopigments were identified in sympathetic neurons at the age of 4 months and all neurons carried pigment granules after the age of 64 years. It is concluded that lipopigment autofluorescence is a useful marker for cellular ageing in both the peripheral and the central nervous system.

Histochemically demonstrable catecholamines in the sympathetic nervous system of trisomic 16 and normal fetal mice.

The formaldehyde-induced fluorescence (FIF) and micro-spectrofluorometric techniques were used to study catecholamines in the sympathetic nervous system of normal and trisomy 16 fetal mice with a gestation age of 15 days, an animal model for human trisomy 21 (Down's syndrome). FIF intensity in the stellate sympathetic ganglion of trisomic embryos did not differ from that of controls, whereas in the adrenal medulla the FIF intensity was 38% less in trisomic than in control embryos. When adrenal medullary cells from embryos with a gestational age of 15 days were maintained in culture for 7-10 days, a difference in FIF intensity between groups was still evident. The rate of noradrenaline uptake in cultured adrenal medullary cells also was significantly less in trisomic than in control fetal mice. The results suggest that the development of adrenal medulla is slowed in trisomic 16 mice and that uptake of noradrenaline by trisomic adrenal medullary cells is impaired.