Hani Atamna

Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging

Heme, a major functional form of iron in the cell, is synthesized in the mitochondria by ferrochelatase inserting ferrous iron into protoporphyrin IX. Heme deficiency was induced with N-methylprotoporphyrin IX, a selective inhibitor of ferrochelatase, in two human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astrocytoma), as well as in rat primary hippocampal neurons. Heme deficiency in brain cells decreases mitochondrial complex IV, activates nitric oxide synthase, alters amyloid precursor protein, and corrupts iron and zinc homeostasis. The metabolic consequences resulting from heme deficiency seem similar to dysfunctional neurons in patients with Alzheimers disease. Heme-deficient SHSY5Y or U373 cells die when induced to differentiate or to proliferate, respectively. The role of heme in these observations could result from its interaction with heme regulatory motifs in specific proteins or secondary to the compromised mitochondria. Common causes of heme deficiency include aging, deficiency of iron and vitamin B6, and exposure to toxic metals such as aluminum. Iron and B6 deficiencies are especially important because they are widespread, but they are also preventable with supplementation. Thus, heme deficiency or dysregulation may be an important and preventable component of the neurodegenerative process.

N-t-Butyl Hydroxylamine, a Hydrolysis Product of α-Phenyl-N-t-butyl Nitrone, Is More Potent in Delaying Senescence in Human Lung Fibroblasts

α-Phenyl-N-t-butyl nitrone (PBN), a spin trap, scavenges hydroxyl radicals, protects tissues from oxidative injury, and delays senescence of both normal human lung fibroblasts (IMR90) and senescence-accelerated mice.N-t-butyl hydroxylamine and benzaldehyde are the breakdown products of PBN. N-t-Butyl hydroxylamine delays senescence of IMR90 cells at concentrations as low as 10 μm compared with 200 μm PBN to produce a similar effect, suggesting thatN-t-butyl hydroxylamine is the active form of PBN. N-Benzyl hydroxylamine and N-methyl hydroxylamine compounds unrelated to PBN were also effective in delaying senescence, suggesting the active functional group is theN-hydroxylamine. All the N-hydroxylamines tested significantly decreased the endogenous production of oxidants, as measured by the oxidation of 2′,7′-dichlorodihydrofluorescin and the increase in the GSH/GSSG ratio. The acceleration of senescence induced by hydrogen peroxide is reversed by the N-hydroxylamines. DNA damage, as determined by the level of apurinic/apyrimidinic sites, also decreased significantly following treatment withN-hydroxylamines. The N-hydroxylamines appear to be effective through mitochondria; they delay age-dependent changes in mitochondria as measured by accumulation of rhodamine-123, they prevent reduction of cytochrome CFeIII by superoxide radical, and they reverse an age-dependent decay of mitochondrial aconitase, suggesting they react with the superoxide radical.

Deep Sequencing of Serum Small RNAs Identifies Patterns of 5' tRNA Half and YRNA Fragment Expression Associated with Breast Cancer

Small noncoding RNAs circulating in the blood may serve as signaling molecules because of their ability to carry out a variety of cellular functions. We have previously described tRNA- and YRNA-derived small RNAs circulating as components of larger complexes in the blood of humans and mice; the characteristics of these small RNAs imply specific processing, secretion, and physiological regulation. In this study, we have asked if changes in the serum abundance of these tRNA and YRNA fragments are associated with a diagnosis of cancer. We used deep sequencing and informatics analysis to catalog small RNAs in the sera of breast cancer cases and normal controls. 5′ tRNA halves and YRNA fragments are abundant in both groups, but we found that a breast cancer diagnosis is associated with changes in levels of specific subtypes. This prompted us to look at existing sequence datasets of serum small RNAs from 42 breast cancer cases, taken at the time of diagnosis. We find significant changes in the levels of specific 5′ tRNA halves and YRNA fragments associated with clinicopathologic characteristics of the cancer. Although these findings do not establish causality, they suggest that circulating 5′ tRNA halves and YRNA fragments with known cellular functions may participate in breast cancer syndromes and have potential as circulating biomarkers. Larger studies with multiple types of cancer are needed to adequately evaluate their potential use for the development of noninvasive cancer screening.

N-t-Butyl hydroxylamine is an antioxidant that reverses age-related changes in mitochondria in vivo and in vitro

N‐t‐butyl hydroxylamine (NtBHA) delays senescence‐dependent changes in human lung fibroblasts (IMR90) (Atamna et al., J. Biol Chem. 275, 6741–6748). The current study examines the effect of NtBHA on mitochondria in old and young rats and human primary fibroblasts (IMR90). In NtBHA‐treated rats, the age‐dependent decline in food consumption and ambulatory activity was reversed without affecting body weight. The respiratory control ratio of mitochondria from liver of old rats improved after feeding NtBHA. These findings suggest that NtBHA improved mitochondrial function in vivo. The age‐dependent increase in proteins with thiol‐mixed disulfides was significantly lower in old rats treated with NtBHA. NtBHA was effective only in old rats;no significant effect was observed in young rats. In IMR90 cells, NtBHA delayed senescence‐associated changes in mitochondria and cellular senescence induced by maintaining the cells under suboptimal levels of growth factors. Proteasomal activity was also higher in cells treated with NtBHA than in untreated cells. NtBHA accumulates in cells 10‐ to15‐fold the extracellular concentration and is maintained by mitochondrial NADH. NtBHA is an antioxidant that is recycled by mitochondrial electron transport chain and prevents radical‐induced toxicity to mitochondria.—Atamna, H., Robinson, C., Ingersoll, R., Elliott, H., Ames, B. N. N‐t‐Butyl hydroxylamine is an antioxidant that reverses age‐related changes in mitochondria in vivo and in vitro. FASEB J. 15, 2196–2204 (2001)

Iron Accumulation During Cellular Senescence in Human Fibroblasts In Vitro

Iron accumulates as a function of age in several tissues in vivo and is associated with the pathology of numerous age-related diseases. The molecular basis of this change may be due to a loss of iron homeostasis at the cellular level. Therefore, changes in iron content in primary human fibroblast cells (IMR-90) were studied in vitro as a model of cellular senescence. Total iron content increased exponentially during cellular senescence, resulting in 10-fold higher levels of iron compared with young cells. Low-dose hydrogen peroxide (H2O2) induced early senescence in IMR-90s and concomitantly accelerated iron accumulation. Furthermore, senescence-related and H2O2-stimulated iron accumulation was attenuated by N-tert-butylhydroxylamine (NtBHA), a mitochondrial antioxidant that delays senescence in vitro. However, SV40-transformed, immortalized IMR-90s showed no time-dependent changes in metal content in culture or when treated with H2O2 and/or NtBHA. These data indicate that iron accumulation occurs during normal cellular senescence in vitro. This accumulation of iron may contribute to the increased oxidative stress and cellular dysfunction seen in senescent cells.

Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue.

Methylene blue (MB) delays cellular senescence, induces complex-IV, and activates Keap1/Nrf2; however, the molecular link of these effects to MB is unclear. Since MB is redox-active, we investigated its effect on the NAD/NADH ratio in IMR90 cells. The transient increase in NAD/NADH observed in MB-treated cells triggered an investigation of the energy regulator AMPK. MB induced AMPK phosphorylation in a transient pattern, which was followed by the induction of PGC1α and SURF1: both are inducers of mitochondrial and complex-IV biogenesis. Subsequently MB-treated cells exhibited >100% increase in complex-IV activity and a 28% decline in cellular oxidants. The telomeres erosion rate was also significantly lower in MB-treated cells. A previous research suggested that the pattern of AMPK activation (i.e., chronic or transient) determines the AMPK effect on cell senescence. We identified that the anti-senescence activity of MB (transient activator) was 8-times higher than that of AICAR (chronic activator). Since MB lacked an effect on cell cycle, an MB-dependent change to cell cycle is unlikely to contribute to the anti-senescence activity. The current findings in conjunction with the activation of Keap1/Nrf2 suggest a synchronized activation of the energy and cellular defense pathways as a possible key factor in MBs potent anti-senescence activity.

mRNA-Seq reveals complex patterns of gene regulation and expression in the mouse skeletal muscle transcriptome associated with calorie restriction

Sarcopenia is an age-associated loss of skeletal muscle mass and strength that increases the risk of disability. Calorie restriction (CR), the consumption of fewer calories while maintaining adequate nutrition, mitigates sarcopenia and many other age-related diseases. To identify potential mechanisms by which CR preserves skeletal muscle integrity during aging, we used mRNA-Seq for deep characterization of gene regulation and mRNA abundance in skeletal muscle of old mice compared with old mice subjected to CR. mRNA-Seq revealed complex CR-associated changes in expression of mRNA isoforms, many of which occur without a change in total message abundance and thus would not be detected by methods other than mRNA-Seq. Functional annotation of differentially expressed genes reveals CR-associated upregulation of pathways involved in energy metabolism and lipid biosynthesis, and downregulation of pathways mediating protein breakdown and oxidative stress, consistent with earlier microarray-based studies. CR-associated changes not noted in previous studies involved downregulation of genes controlling actin cytoskeletal structures and muscle development. These CR-associated changes reflect generally healthier muscle, consistent with CRs mitigation of sarcopenia. mRNA-Seq generates a rich picture of the changes in gene expression associated with CR, and may facilitate identification of genes that are primary mediators of CRs effects.

Delaying Aging with Mitochondrial Micronutrients and Antioxidants

MITOCHONDRIAL DECAY IN AGING. Mitochondria decay with age due to the oxidation of mtDNA, proteins, and lipids. Some of this decay can be reversed in old rats by feeding them normal mitochondrial metabolites at high levels. Aging appears to be in good part due to the oxidants produced as by-products of normal metabolism by mitochondria [1-5]. In old rats mitochondrial membrane potential, cardiolipin levels, respiratory control ratio, and overall cellular O2 consumption are lower than in young rats and the level of oxidants (per unit O2) and mutagenic aldehydes from lipid peroxidation is higher [3]. Ambulatory activity declines markedly in old rats. Spatial memory as measured by the Morris water maze, also declines with age. Feeding old rats the normal mitochondrial metabolites acetyl carnitine and lipoic acid for a few weeks, restores mitochondrial function, lowers oxidants to the level of a young rat, and increases ambulatory activity and spatial memory [6-11]. Thus, these two metabolites can be considered necessary for health in old age and are therefore “conditional micronutrients”. This restoration suggests a plausible mechanism [11]: with age increased oxidative damage to proteins, such as acyl carnitine transferase (whose activity declines with age), and lipid membranes, particularly in mitochondria, causes a deformation of structure of key enzymes, with a consequent lessening of affinity (Km) for the enzyme substrate; an increased level of the substrate restores the velocity of the reaction, and thus restores function. Hepatocytes were isolated from young (3-5 months) and old (24-28 months) rats and incubated with various concentrations of tert-butylhydroperoxide (t-BuOOH) [10]. The t-BuOOH concentration that killed 50% of cells (LC50) in 2 hr declined nearly two-fold from 721 ± 32 µM in cells from young rats to 391 ± 31 µM in cells from old rats. This increased sensitivity of hepatocytes from old rats may be due, in part, to changes in glutathione (GSH) levels, because total cellular and mitochondrial GSH were 37.7% and 58.3% lower, respectively, compared to cells from young rats. Cells from old animals were incubated with either (R)- or (S)-lipoic acid (100 µM) for 30 min prior to the addition of 300 µM t-BuOOH. The physiologically relevant (R)-form, a coenzyme in mitochondria, as opposed to the (S)-form significantly protected hepatocytes against t-BuOOH toxicity. Dietary supplementation of (R)-lipoic acid [0.5% (wt/wt)] for 2 weeks also completely reversed the age-related decline in hepatocellular GSH levels and the increased vulnerability to t-BuOOH as well. An identical supplemental diet fed to young rats did not enhance the resistance to t-BuOOH, indicating that antioxidant protection was already optimal in young rats. Thus, this study shows that cells from old animals are more susceptible to oxidant insult and (R)-lipoic acid, after reduction to an antioxidant in the mitochondria, effectively reverses this age-related increase in oxidant vulnerability.

Organ reserve, excess metabolic capacity, and aging

Abstract“Organ reserve” refers to the ability of an organ to successfully return to its original physiological state following repeated episodes of stress. Clinical evidence shows that organ reserve correlates with the ability of older adults to cope with an added workload or stress, suggesting a role in the process of aging. Although organ reserve is well documented clinically, it is not clearly defined at the molecular level. Interestingly, several metabolic pathways exhibit excess metabolic capacities (e.g., bioenergetics pathway, antioxidants system, plasticity). These pathways comprise molecular components that have an excess of quantity and/or activity than that required for basic physiological demand in vivo (e.g., mitochondrial complex IV or glycolytic enzymes). We propose that the excess in mtDNA copy number and tandem DNA repeats of telomeres are additional examples of intrinsically embedded structural components that could comprise excess capacity. These excess capacities may grant intermediary metabolism the ability to instantly cope with, or manage, added workload or stress. Therefore, excess metabolic capacities could be viewed as an innate mechanism of adaptability that substantiates organ reserve and contributes to the cellular defense systems. If metabolic excess capacities or organ reserves are impaired or exhausted, the ability of the cell to cope with stress is reduced. Under these circumstances cell senescence, transformation, or death occurs. In this review, we discuss excess metabolic and structural capacities as integrated metabolic pathways in relation to organ reserve and cellular aging.n

Caloric restriction impacts plasma microRNAs in rhesus monkeys

Caloric restriction (CR) is one of the most robust interventions shown to delay aging in diverse species, including rhesus monkeys (Macaca mulatta). Identification of factors involved in CR brings a promise of translatability to human health and aging. Here, we show that CR induced a profound change in abundance of circulating microRNAs (miRNAs) linked to growth and insulin signaling pathway, suggesting that miRNAs are involved in CRs mechanisms of action in primates. Deep sequencing of plasma RNA extracts enriched for short species revealed a total of 243 unique species of miRNAs including 47 novel species. Approximately 70% of the plasma miRNAs detected were conserved between rhesus monkeys and humans. CR induced or repressed 24 known and 10 novel miRNA species. Regression analysis revealed correlations between bodyweight, adiposity, and insulin sensitivity for 10 of the CR‐regulated known miRNAs. Sequence alignment and target identification for these 10 miRNAs identify a role in signaling downstream of the insulin receptor. The highly abundant miR‐125a‐5p correlated positively with adiposity and negatively with insulin sensitivity and was negatively regulated by CR. Putative target pathways of CR‐associated miRNAs were highly enriched for growth and insulin signaling that have previously been implicated in delayed aging. Clustering analysis further pointed to CR‐induced miRNA regulation of ribosomal, mitochondrial, and spliceosomal pathways. These data are consistent with a model where CR recruits miRNA‐based homeostatic mechanisms to coordinate a program of delayed aging.