The 2^nd Ukraine-Israel Symposium on the Biology of Aging

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Kiev, Ukraine – June 18, 2008

PROGRAM

The 2^nd Ukraine-Israel Symposium
on the Biology of Aging

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Kiev, Ukraine – June 18, 2008

ORGANIZING COMMITTEE

Co-Chairs:

Khachik Muradian, Institute of Gerontology AMS Ukraine

Vadim Fraifeld, Ben-Gurion University of the Negev

ACKNOWLEDGEMENTS

The Organizing Committee wishes to acknowledge the following contributors whose generous support has made this Symposium possible:

Institute of Gerontology AMS Ukraine

Ben-Gurion University of the Negev

Institute of Biology of Aging (Moscow)

The 2^nd Ukraine-Israel Symposium on the Biology of Aging

Kiev – June 18, 2008

Program

9.30 – 9.40 Welcome and Opening

9.40 – 10.05 New Aspects of Neurodegeneration and Neuroinflammation: Involvement

of the Cholinergic System

Talma Brenner

10.05 – 10.30 Emotiogenic Hypothalamic Zones and Organism’s Vital Capacity in Aging

Vladislav Bezrukov

10.30 – 10.55 How to Protect the Elderly from West Nile Virus?

Bracha Rager

10.55 – 11.15 Coffee break

11.15 – 11.40 The Importance of Molecules of the IL-1 Family in Carcinogenesis and Tumor Invasiveness

Ron Apte

11.40 – 12.05 Stem Cell Technologies in Gerontological Research

Gennady Butenko

12.05 – 12.30 The Immune System in Heterochronic Parabiosis: Pilot Study

Irina Pishel

12.30 – 12.55 Visualization of Molecular Processes in Immunocytes

Alex Braiman

12.55 – 14.30 Lunch

14.30 – 14.55 Yet Another Hypothesis of Aging: Networks in Focus

Vadim Fraifeld

14.55 – 15.20 Gene Therapy of Atherosclerosis

Svetlana Novikova

15.20 – 15.45 MicroRNAs: Relevance to Aging and Age-related Diseases

Marina Wolfson

15.45 – 16.10 Early-life Etiology of Age-related Diseases: Epidemiological Study

Alexander Vaiserman

16.10-16.30 Coffee break

16.30 – 16.55 Mitochondria and Longevity: A New Wind of the Old Story

Vadim Fraifeld, Khachik Muradian

16.55 – 17.20 We’ve Hit the Bottom. There is No Place to Go But Up

Albert Timchenko

17.20 – 17.45 Kinetic Isotope Effect - A New Prospect for Slower Aging?

Nikolay Pestov

17.45 – 18.00 Gene Therapy of Brain Injury in Rats of Different Ages

Sergey Mikhalsk

18.00 – 18.10 Closing Remarks

ABSTRACTS

The Importance of Molecules of the IL-1 Family in Carcinogenesis and Tumor Invasiveness

Ron N. Apte

The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences,
Ben-Gurion University of the Negev, Beer-Sheva, Israel

Interleukin-1 (IL-1) is a pleiotropic pro-inflammatory and immunostimulatory cytokine with diverse effects on malignant processes. The IL-1 family consists of two agonistic proteins, namely IL-1a and IL-1β, and one antagonistic protein, the IL-1 receptor antagonist (IL-1Ra), which binds to IL-1 receptors without transmitting an activation signal and thus represents a physiological inhibitor of pre-formed IL-1. In their recombinant form, IL-1a and IL-1β exert the same biological activities and bind to the same receptors. However, in the physiological milieu, IL-1a and IL-1β differ dramatically in the sub-cellular compartments in which they are active; IL-1a is mainly active as a cell-associated cytokine (cytosolic and membrane-associated forms), while IL-1β is active only in its mature secreted form. As a ubiquitous mediator at the site of tumor development, IL-1 can be produced by microenvironmental cellular elements as well as by the malignant cells. Previously, we have shown, using fibrosarcoma cells transfected with the active forms of IL-1, that IL-1a expression on the membrane of tumor cells increases their immunogenicity and leads to tumor eradication, while tumor cells which actively secrete IL-1β are of more malignant. To distinguish between tumor cell and host-derived IL-1, we used knockout (KO) mice that lack functional genes of members of the IL-1 family, i.e., IL-1a, IL-1β, IL-1a and IL-1β (double KO) and IL-1Ra KO mice as well as 3-methylcholanthrene (3-MCA)-induced tumors in control and IL-1 KO mice. The results indicate that microenvironmental IL-1β is essential for determining the malignant phenotype of transplantable tumors as well as for the induction of tumors following chemical carcinogenesis. We have also used fibrosarcoma cell lines obtained from control and the various IL-1 KO mice and the same set of recipient mice, to assess whether IL-1 of tumor cell- or host-origin are essential for invasiveness. The results indicate that IL-1β of both the malignant cell- and the host-origin synergize in controlling invasiveness and metastasis of the tumor. Also, different forms of IL-1 that are expressed in the microenvironment of the host, where 3-MCA tumors develop, determine features of the malignant cells, such as invasiveness and immunogenicity. Altogether, the results point to differential effects of IL-1β and IL-1a in malignant processes and points to the therapeutic feasibility of the IL-1Ra, which neutralizes soluble IL-1 (mainly IL-1β), in tumor therapy, apart from its use in treatment of autoimmune diseases, such as Rheumatoid arthritis.

Emotiogenic Hypothalamic Zones and Organism’s Vital Capacity in Aging

Vladislav V. Bezrukov, Tatyana A. Dubiley, and Yuri E. Rushkevich

Institute of Gerontology AMS Ukraine, Kiev

As Vladimir Frolkis had suggested, the decline of organism’s adaptive capacity and viability could be caused by the shift of the balance between anti-aging and aging processes in favor of the latter. The role of the integrative centers of the brain in these events is widely recognized. The hypothalamic emotiogenic zones – lateral hypothalamic area (LHA) and ventromedial hypothalamic nucleus (VMN) are believed to belong to the vitally important centers of homeostasis maintenance. The results of our experiments demonstrated that LHA and VMN functions decline in aging. Chronic LHA stimulation improved functions of this structure in old rats, corrected the mechanisms of neuro-humoral regulation and retarded the aging rate. Therefore, LHA can be considered as a morpho-functional basis of the central anti-aging mechanism. On the other hand, chronic VMA stimulation led to the decrease in survivorship and life span of old rats. The effect was probably the result of metabolic and functional impairments. It is assumed that the emotiogenic hypothalamic areas, as a part of emotional reinforcement system, determine organism’s viability. Under favorable external circumstances and/or successful actions accompanied by the formation of positive emotional states, the LHA could induce anti-aging mechanisms, thus increasing the individual’s viability. Under unfavorable conditions and in the absence of possibilities for a solution of actual tasks, the VMN activation reduces the viability of an “unfit” organism and thus eliminates the less adaptable individuals.

Visualization of Molecular Processes in Immunocytes

Alex Braiman

The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

T lymphocytes are an integral and indispensable part of the immune system, which participate in a vast majority of immunological responses. Compromised function of T lymphocytes results in severe, often fatal, immunodeficiencies or auto-immune diseases. T cell activation begins when the T-cell antigen receptor (TCR) is stimulated by an antigen. Initially, the composition of the TCR-proximal signaling complexes and molecular interactions underlying its formation were studied primarily using biochemical methods. Although many characteristics of these complexes have been unraveled up to date, the precise details of their structure and functionality remain poorly understood. The main difficulty for solving this puzzle is a highly dynamic and heterogeneous nature of molecular interactions within the complexes. Recent developments in optical microscopy and imaging techniques allowed direct visualization of signaling complexes in live lymphocytes. Labeling of various cellular proteins with fluorescent markers allowed tracking their location and interactions within the cell in the course of T cell activation.

New Aspects of Neurodegeneration and Neuroinflammation: Involvement of the Cholinergic System

Talma Brenner

Department of Neurology, Hadassah Hebrew University Medical Center, Jerusalem, Israel

The maintenance of a balanced cholinergic homeostasis is crucial for the function of the central nervous system (CNS), peripheral nervous system and the neuromuscular junction. However, it appears that the cholinergic system is not restricted to neurons and synapses but may also involve immune reactions. Immune cells possess a complete cholinergic system consisting of acetylcholine (ACh) muscarinic and nicotinic receptors, choline acetyltransferase and acetylcholinesterase (AChE). Since the nervous system is a major producer of ACh the immune cholinergic system can mediate neuro-immune interactions, or it may serve as an internal regulator of immune responses. Alzheimer’s disease (AD) is almost invariably associated with a disruption of cholinergic balance, as well as with marked activation of an innate immune response near the amyloid plaques. We investigated the anti-inflammatory effects of AChE inhibitors (AChEI) at the cellular and molecular levels during neuroinflammation. In experimental autoimmune encephalomyelitis (EAE), the inflammatory demyelinating model used for the study of multiple sclerosis (MS), AChEI reduced the clinical severity of the disease, CNS inflammation and axonal damage. The reactivity of encephalitogenic T-cells was also suppressed. This was performed by increasing the ACh concentration near immune cells and making it available for interaction with the alpha-7 nicotinic ACh receptor, expressed on these cells. This outcome is additional to the effect of AChEI on neurons and synapses. Our findings point to a novel role for AChEI which may be relevant in CNS inflammatory diseases and emphasize the importance of the cholinergic balance in neurological disorders such as AD and myasthenia gravis, in which these agents are used.

Yet Another Hypothesis of Aging: Networks in Focus

Arie Budovsky and Vadim Fraifeld

The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Hundreds of genes have been identified to be involved in the control of lifespan in model organisms and in human age-related diseases (ARDs). These studies highlighted that aging, longevity, and ARDs are associated with multiple genetic factors. Yet, the trend to focus on individual genes and/or their products continues to dominate, reflecting in part a paradigm in biomedical research – searching for the specific targets that offer the potential for the development of highly specific drugs. Despite enormous efforts and accumulated knowledge, our capabilities for tackling aging and ARDs, and ultimately to promote longevity are still very modest. What is lacking -- essential knowledge of key players or efficient analytic tools, or both? Here we discuss how the existing data may be integrated and analyzed using a network-based approach, with a particular focus on the role of protein-protein interaction (PPIs) networks in linking the human aging, longevity and ARDs. A gradual decrease in the PPI network robustness is proposed to be one of the common pathways for both aging and ARDs.

Merging Aging with Rejuvenation: A Tale of Dedifferentiation

Meytal Damri^1,2, Vadim Fraifeld², Marina Wolfson², and Gideon Grafi¹

¹The Jakob Blaustein Institutes for Desert Research and ²The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of
the Negev, Beer-Sheva, Israel

Dedifferentiation signifies that differentiated cells retain developmental potentialities and are capable to acquire stem cell-like state. This process precedes switch in cell fate including transdifferentiation, reentry into the cell cycle and even a commitment for cell death. In plants, dedifferentiation characterizes the transition of differentiated leaf cells to protoplasts (plant cells devoid of cell walls) and is accompanied by global chromatin decondensation and extensive changes in gene expression profile. We aim to investigate the hypothesis that senescing cells acquire dedifferentiated, stem cell-like state before commitment for death is established. We selected Arabidopsis and tobacco plants for this study and induced leaf senescence by exposing plants to dark. Because chromatin decondensation is a feature characteristic of animal stem cells as well as dedifferentiating cells, we currently study various aspects of chromatin reorganization accompanying senescence. Our initial study showed that senescing cells display extensive posttranslational modifications of core histone proteins, which are accompanied by substantial changes in gene expression profile. Also, transcriptome profiling showed common features between senescing and dedifferentiating cells.

Effect of Cationic Liposome-mediated APOE3 Gene Therapy on Structure of Hippocampus and Attenuation of Cognitive Impairment after Traumatic Brain Injury in Rats

S.A. Mikhalsky¹, V.V. Biloshytskyy², T.Yu. Kvitnitskaya-Ryzhova¹, L.A. Tsyba², and N.Ya. Gridina²

¹Institute of Gerontology AMS Ukraine, ²Institute of Neurosurgery AMS Ukraine, Kyiv

Apolipoprotein E (APOE, gene; apoE, protein) has been shown to support effective repair and remodeling after neuronal injury, suggesting application of apoE in treatment of traumatic brain injury (TBI). In this study, we tested the hypothesis that cationic liposome-mediated APOE3 gene transfer in vivo can attenuate cognitive dysfunction associated with TBI.

Material and methods: Severe diffuse TBI was performed in adult male Wistar rats using the weight drop impact acceleration model (1.5 m/450 g). The mixture of DOTAP liposomes and 25 µg of recombinant plasmid pCMV- APOE3 cDNA was infused intraventicularly after TBI using ALZET osmotic pump. Animals (n = 20) were randomized into one of four groups: (1) sham-injured and vehicle–treated, (2) brain-injured and vehicle-treated, (3) sham-injured and APOE3-transfected, (4) brain-injured and APOE3-transfected.

Results: RT-PCR confirmed the increased expression of APOE3 mRNA in the brain tissue at day 10 after infusion. After TBI, CA1 hippocampus exhibited delayed neuronal loss by 10 days. Such a loss was attenuated in APOE3-transfected animals (24% rate of loss as compared to 40% in vehicle-treated rats, p<0.05). At the same time, linear density of gliocytes and microgliocytes increased by 23% and 370%, respectively. The structure of axons and myelin membranes became normal, lipofuscin in neurons and astrocytes decreased. Cognitive function was assessed using the Morris Water Maze at day 7-10 after TBI. Brain-injured, vehicle-treated rats demonstrated a profound cognitive deficit (p<0.01) as compared to sham (non-injured) animals. APOE3 gene transfer caused a significant improvement in the learning ability and memory retention, which were different (p<0.05) from injured, non-treated group.

Conclusion: Cationic liposome-mediated APOE3 gene transfer had a marked positive effect on the structure and ultrastructure of the hippocampus, including a considerable reduction of the TBI-induced neuronal death, axon damage, gliosis and microglial reaction. The APOE3 gene transfer may have a therapeutic potential in treatment of TBI-induced cognitive dysfunction.

Mitochondria and Longevity: A New Wind of the Old Story

Khachik Muradian¹, Gilad Lehmann², and Vadim Fraifeld²

¹Institute of Gerontology AMS Ukraine, Kiev and ²The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of
the Negev, Beer-Sheva, Israel

In animal cells, mitochondria are semiautonomous organelles of virtually “hostile” (bacterial) origin, with their own code and genome (mtDNA). The semiautonomy and restricted resources could result in occasional “conflicts of interests” with other cellular components, in which mitochondria have greater chances to be “the weakest link,” thus limiting longevity. Two principal questions are addressed: (1) to what extent the mammalian maximum life span (MLS) is associated with mtDNA base composition? (2) Does mtDNA base composition correlate with other important mitochondria-associated variables – resting metabolic rate (RMR) and body temperature – and whether they complement each other in determination of MLS? Analysis of 140 mammalian species revealed significant correlations between MLS and the content of the four mtDNA nucleotides. Guanine (G) dominates over other bases in correlative links with MLS. An increase in G content and the subsequent accumulation of thermodynamically more stable GC pairs would result in a higher stability of mtDNA ensuring thus a higher resistance of mtDNA against denaturizing factors, for example, temperature fluctuations. This could be particularly relevant for endothermic organisms. Indeed, we found a significant positive correlation between the GC content and typical body temperature in mammals (r = 0.35; p < 0.002), which was even more evident when birds were also included in the analysis (r = 0.6; p < 10^-9). The most remarkable finding of this study is that multivariate stepwise analysis selected only the GC content and RMR, which together explained 77% of variation in MLS (p < 10^-25). To the authors’ knowledge, it is the highest coefficient of MLS determination that has ever been reported for a comparable sample size. Taking into account substantial errors in estimation of MLS and RMR, it could mean that the GC and RMR explain most of the MLS biological variation. Other putative players in MLS determination should have relatively small contribution or their effects should be realized via the same channels. Although further research is clearly warranted, the extraordinary high correlation of mtDNA GC and RMR with MLS suggests a “direct hitting” of the core longevity targets, inferring mitochondria as a primary object for longevity-promoting interventions.

Gene Therapy of Atherosclerosis

S.N. Novikova¹, O.K. Toporova², Yu.M. Gilchuk², and V.A. Kordjum²

¹Institute of Gerontology AMS Ukraine, Kiev;

²Institute of Molecular Biology and Genetics NAS Ukraine

Approaches to gene therapy of atherosclerosis have been developed by introduction of human apoA1 gene (the main protein component of the lipoprotein high density ligand) into the liver cells. Plasmid vector for the expression of full-sized human apoA1gene in mammalian cells in vitro and in vivo was constructed. The construct contains an expression cassette for the human apoA1 gene flanked by inverted terminal repeats of human adeno-associated virus. For the gene transfer, the polycationic transfection reagents were used, ensuring high degree of transfection without toxic effect, both in vitro and in vivo. Animal studies (rats and rabbits) demonstrated that human protein ApoA1 could be detected in blood plasma 3 months following apoA1 gene injection. The presence of human apoA1 gene fragments was confirmed by PCR-analysis and DNA hybridization. The apoA1 gene transfection prevented hypercholesterinemia caused by cholesterol loading and enhanced the process of spontaneous regression after cessation of starvation. The results of morphological examination further confirmed the therapeutic effect of apoA1 gene introduction. In conclusion, the data obtained on the model of experimental atherosclerosis suggest the therapeutic efficacy of apoA1 gene transfection.

Kinetic Isotope Effect -- A New Prospect For Slower Aging?

Nikolay B. Pestov¹ and Mikhail S. Shchepinov²

¹Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia, ²Retrotope Inc., Oxford, UK

A well-known mechanism of aging is oxidation of proteins, nucleic acids, lipids, and other components in living cells by reactive oxygen species (ROS). A new hypothesis is put forward that the pace of this destructive oxidation can be decelerated using kinetic isotope effect. Naturally occurring heavy isotopes such as ²H (deuterium) and ¹³C (carbon-13) can be incorporated into building blocks such as amino acids, fatty acids and nucleotides. Controlled consumption of such “heavy food” can be used to achieve desirable degree of substitution and to make cellular biopolymers partially protected against ROS. At least one exciting feature of this approach to enhanced longevity is that it does not employ xenobiotics or any risky interventions. At present, the high cost of stable isotopes is the major obstacle to their widespread use.

Supported by the Institute of Biology of Aging (Moscow)

The Immune System in Heterochronic Parabiosis: Pilot Study

I. Pishel, T. Dubiley, A. Rodnichenko, N. Utko, Yu. Leonov, S. Migovan, M. Azarskova, V. Kyryk, T. Badova, P. Klimenko, Kh. Muradian, and

G. Butenko

Institute of Gerontology AMS Ukraine, Kiev

Age-related decline in immune functions is well documented. This decline is attributed to numerous factors including decreased thymus function, a dramatic decrease in the proportion of naive T cells with a concomitant increase in T cells with memory phenotypes, an altered cytokine production by APC, and alterations in lymphocyte intracellular signal transduction pathways. To ameliorate the age-associated changes, heterochronic parabiotic pairs with common blood circulation were constructed. Surprisingly, the young heterochronic partner exhibited a declined primary immune response, whereas no changes were observed in the old partner. To further clarify the problem, the parabiotic pairs with different terms of coexistence (3, 6 and 12 weeks) were explored. After 3 weeks of parabiosis, CD4/CD8 ratio in old partners was increased to the “young” level, without any significant changes in other parameters. This rejuvenation effect was temporary and disappeared later. At the same time, in young heterochronic partners, CD8+44+ cell number in the spleen was increased to the level of old mice. Moreover, the elevation remained stable in the later terms of parabiosis. After 12 weeks of parabiosis, the spleen CD4+44+ cell number, macrophage phagocytosis, and T-cell proliferation in vitro were decreased to the level of old mice. These results could indicate a progressive decline in immune functions in young heterochronic partners. The mechanisms of the age-related changes are not clear. Wagers et al. (2002) demonstrated substantial chimerism of hematopoietic but not non-hematopoietic cells in young parabionts, suggesting that factors responsible for the age-related immune decline may circulate in the serum, or may be induced by interaction between young circulated lymphocytes and stromal or antigen-presented cells from the old partners. Both possibilities will soon be examined in our labs.

Supported by the Institute of Biology of Aging (Moscow)

How to Protect the Elderly from West Nile Virus Infection?

Bracha Rager

The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

West Nile virus (WNV), the etiologic agent of West Nile fever (WNF), was first isolated in the West Nile District of Uganda in 1937, and is endemic in Europe, Africa, West Asia, and the Middle East. From 1994 it has re-emerged in Europe and Israel. Its first unexpected appearance in the United States was in 1999 and it has continued to spread each year since then. During the 1999–2003 outbreaks of WNV in the USA, more than 10,000 cases were reported, including 533 deaths. WNV is a single stranded plus-RNA virus classified within the Flaviviridae family. It is maintained in nature in a mosquito-bird-mosquito cycle, while avians, humans, horses and other mammals serve as incidental hosts. Recently, it has been shown that WNV can also be transmitted through blood transfusion, organ transplantation, laboratory-acquired infection and breast feeding. Its clinical symptoms range from asymptomatic infection, including fever and headache, to serious cases with myocarditis, meningitis, encephalitis, and a polio-like paralytic syndrome . The level of exposure resulting in infection and disease is not established yet. Meanwhile, according to epidemiologic studies, ~80% of WNV infection remains subclinical, and ~20% progresses to a febrile illness. More than 30% of cases in the US lead to meningitis and/or encephalitis. After this severe neurological disease, patients usually need a prolonged rehabilitation. In the immunocompromised and the elderly, this infection can be life threatening. Despite its wide geographic distribution and frequency, little is known about the pathogenesis of WNV, especially regarding encephalitis. Impairment of neuronal function and neuronal cell death are believed to be the end pathophysiological result of WNV. Experiments of WNV encephalitis performed in mice have shown neuronal degeneration accompanied by microgliosis and perivascular inflammation. Inflammatory reactions occur in the brain in case of various CNS diseases, including neurodegenerative diseases, Alhzheimer's disease, Parkinson’s disease, stroke, head trauma, and infection. To understand better the clinical pattern of WNV infection, we investigated the influence of WNV on acetylcholinesterase (AChE) enzyme activity. We also examined in the present study the prophylactic and therapeutic efficacy of pooled human plasma or IVIG-IL, from healthy Israeli blood donors. It was found that the full recovery of mice after infection with a lethal dose of WNV was dependent on the dose and time of the IVIG administration. Full protection was achieved when WNV infected mice were treated with pooled plasma or IVIG-IL. These studies indicate that antibodies play a major role in protection and recovery from WNV infection and that IVIG can be used as first line therapy of WNV infection.

Vascular Responses to Acetylcholine and Nitrovasodilators

in Young and Old Rats

Nina V. Sykalo

State Institute “Institute of Gerontology AMS Ukraine”, Kyiv

Disregulation of vessel tone and reactivity play an important role in development of age-related cardiovascular pathology. To certain extent these events are attributed to the endothelium-dependent factors. Endothelial cells secret numerous vasoactive agents including endothelium-derived relaxing factor – NO (ERF-NO). The aim of our work was to study the role of endothelium in alterations of the vessels’ smooth muscles reactivity in aging The effects of endothelium-dependent (acetylcholine, Ach) and endothelium-independent relaxants (NO solution and NO donors – sodium nitroprusside and nitroglycerine) on isolated segments of thoracic aorta of adult (7–8 mo) and old (24–26 mo) Wistar rats were investigated. A significant age-related decrease in dose-dependent vasodilating effect of Ach (10^-10–10^-5 mol/l) was revealed. L-arginine (10^-6 mol/l), a precursor of ERF-NO synthesis provoked a twofold increase in Ach-induced dilation in old rats. No significant differences were found in the vasodilating effects of NO solution (10^-7–10^-4 mol/l), sodium nitroprusside (10^-10–10^-6 mol/l) and nitroglycerine (10^-9–10^-5 mol/l) in adult and old animals. It may be concluded that the
NO-mediated mechanism of relaxation of vascular smooth muscles is not affected by age, whereas the age-related decline in vasodilating reactions could be linked with damage of the endothelium and/or a decreased release of NO.

Atmospheric Oxygen Content Modulates Life Span: Experiments on D. melanogaster

Albert Timchenko, Natalie Utko, and Khachik Muradian

Institute of Gerontology AMS Ukraine, Kiev

There is a growing consensus underpinned by numerous evolutionary and experimental findings, which suggest that aging rate is inversely proportional to the intensity of metabolic processes (according to A.T., ‘The first law of gerontology’). This statement, however, is not all encompassing, i.e., there are many exceptions. Moreover, the relationship is poorly studied in direct experiments, e.g., in attempts to extend life span by modulation of the gaseous exchange rate. The present study is one of few in a series of ‘trial and error’ experiments undertaken in order to evaluate the possibility for modulating the life span and stress-resistance due to elevated or declined content of atmospheric oxygen, carbon dioxide and other gases - a problem actual not only in gerontology, but also in medicine, in space explorations, submarines and other relevant areas with usage of ‘artificial’ or modified atmosphere. Different degree of hypo- and hyperoxia or hypercapnia was modeled by injection of 100% nitrogen, oxygen or carbon dioxide into the hermetic syringes with experimental animals. Control flies lived in standard conditions out of syringes or in syringes with openings ensuring exchange of atmospheric air. The renewal of fresh food and registration of died animals was carried out on the ‘every-other-day’ basis. The oxygen and carbon dioxide contents in the syringes were controlled by a gaseous analyzer immediately before and after each food renewal. Two practically identical series of experiments were performed during the winter-spring and spring-summer seasons. Surprisingly, the life span of both control and experimental flies was significantly shorter in the winter-spring experiments. Nevertheless, in the both series we observed a dose-dependent decrease of the mean life span at the hyperoxia and extended life span at the hypoxia or hypercapnia. All in all, longevity inversely correlated with the oxygen consumption, i.e., animals exposed to hyperoxia demonstrated higher rates of oxygen consumption and shorter life span and vice versa. In assessment of the stress resistance, the heat shock (38 ^oC, 30 min) survival was increased in hypercapnia- and decreased in hyperoxia-treated flies, whereas the survival after exposure to 15% hydrogen peroxide was several folds higher at the both hypercapnia and hyperoxia. No significant effects of hyperoxia or hypercapnia on survival were observed in the alkaline feeding medium (pH = 11.8).

ORION – A New Anti-aging and Anti-stress Remedy?

N. Utko¹, V. Bezrukov¹, O. Zhukovsky³, V. Razumenko², V. Polyakov,

A. Orlovsky², and Kh. Muradian¹

¹Institute of Gerontology AMS Ukraine; ²Institute of Oncology AMS Ukraine;

³International Charitable Funding “Kievskaya Rus”, Ukraine

Orion is a derivative of taurocholic acid, which is well-known by modulating effects on numerous functional systems including digestion, excretion, detoxification, apoptosis, etc. Of particular interest is the impressive anti-cancer effect of the drug, shown lately by us on mouse and human cells (unpublished data). Moreover, clinical investigation of Orion has been initiated in the Institute of Oncology AMS Ukraine since 2007. Although the relation between cancer and aging is complex and there is no telling what the end game will be like, the authors cherish an idea that Orion could also possess anti-aging and longevity-promoting effects. In an attempt to shed light on the problem, pilot experiments were undertaken using Drosophila melanogaster as a test-object. After preliminary experiments aimed to establish the working range of the drug concentrations (0.001–2%), Orion was added to the feeding medium of animals starting from different ages (from oviposition to the oldest-old imagoes). The stress resistance of flies was assessed by a test-battery, including exposures to the heat-shock, 15% hydrogen peroxide, 100% oxygen, acidic (pH = 1.6) or alkaline (pH = 11.8) feeding mediums, starvation, etc. The results obtained infer substantial life span-extending and stress resistance-ensuring potential of Orion. The longevity-promoting effect was especially impressive when the drug was applied in the advanced ages (over 50–60 days). In such experiments, the so-called remaining mean life span could be extended by several folds. The beneficial effect of Orion was also apparent when considering the longest-lived flies (maximum life span). Experimental animals could often reach 110–120 days of age (the longest championing up to 127 days), whereas the control flies lived no more than 85–90 days. Orion significantly increased survival upon the heat shock and pH-related stresses, which apparently could be associated with an enhanced stability of macromolecules against denaturizing factors. However, only moderate improvement was observed in response to ROS-associated treatments (peroxide or 100% oxygen), thus implying a specific pattern of both quantitative and qualitative amelioration of the aging process. Further research is definitely warranted to clarify the real longevity-promoting and stress resistance-ensuring potential of Orion.

Early-life Etiology of Age-related Diseases: Epidemiological Study

Alexander M. Vaiserman

Laboratory of Mathematical Modeling of Aging Processes, Institute of Gerontology AMS Ukraine, Kiev

Recently, a cluster of new hypotheses of aging has been suggested, which explicitly predicts the importance of early-life events in adult health and life span modulations. It has been widely assumed that these long-lasting consequences of early-life exposures may depend on the same mechanisms as those underlying ‘cellular memory’, i.e., epigenetic inheritance systems. There is a growing body of evidence that environmentally-induced perturbations in the epigenetic processes (which involve alterations of gene expression without a change in DNA sequence) can determine different aspects of aging as well as etiology and pathogenesis of age-related diseases. To examine the relationship between the early-life impacts and late-life health, we used the month of birth as an indicator for environmental influences during pre- and early postnatal development. We found highly significant differences (p < 0.001) between month-of-birth patterns in type 1 diabetes patients before age 30 (n = 20,117) and type 2 diabetics after age 40 (n = 103,028), and individuals from the general Ukraine population. These seasonal patterns of birth were remarkably similar for the both diseases, with a peak in April 29 and a trough on December 29 for the type 1 diabetics, and with a peak on May 5 and a trough on December 19 for the type 2 diabetics. A significant (p < 0.05) but opposite month-of-birth pattern was observed for stomach cancer patients: in males (n = 36,826), peak on January 23, trough on May 17; in females (n = 27,012), peak on February 7, trough on June 11. Altogether, our results are in line with the hypotheses postulating the role of early-life events in the development of age-related diseases.

MicroRNAs: Relevance to Aging and Age-related Diseases

M. Wolfson, R. Tacutu, A. Budovsky, N. Aizenberg, and V. Fraifeld

The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Gene expression is under a tight epigenetic control which includes DNA methylation, histone modification, and a recently discovered mechanism of RNA interference (RNAi). Not surprisingly, dysregulations in the epigenetic control appear to have a profound impact on both aging and age-related diseases (ARDs) including atherosclerosis, cancer, neurodegenerative diseases, and diabetes type 2. Here we summarize in brief what is known about RNAi in aging and ARDs, with a special focus on the RNAi key players -- microRNAs (miRNAs). MicroRNAs represent a novel extremely complex layer of regulation of cell activities, from proliferation and differentiation to drug resistance and apoptosis. This rapidly developing research field is only beginning to emerge and most data have been published during the last two years. The studies on C. elegance and mice indicated that miRNA abundance could be age-related. However, the aging-associated patterns of miRNA expression may differ across species and strains, and may also be gender- and tissue-specific. Analysis of micRNA profiles of ARDs revealed both specific and common RNAs. Along with reviewing the existent data in the field, we present the results of our pilot analysis of the miRNA-mediated links between aging, longevity and ARDs using a network-based approach. For example, among the target proteins of ARD-associated miRNAs, were those that were also found in the Human Longevity Network (HLN). This was particularly noted for miR-21 and let-7, miRNAs with broad spectrum of activities, shown to be involved in more than one ARD. The list of potential miRNA targets could be substantially broadened if not only the experimentally identified target proteins but also the predicted ones would be taken into account. The potential to influence miRNA activity by antisense agents or other compounds make miRNAs promising targets for therapeutic interventions.