Current Aging Science (v.6, #1)

Ovarian Aging: Melatonin Regulation of the Cytometric and Endocrine Evolutive Pattern by Blanca E. Fernandez, Elena Diaz, Carmen Fernandez, Paula Nunez, Beatriz Diaz (1-7).
We have investigated the effect of melatonin administration on the cytometric and endocrine functions of theovary during aging. Young cyclic (3 months old), middle-aged pre-acyclic (13 months old), and old acyclic (22 monthsold) female Wistar rats were used for two months, in both control and melatonin-treated groups. Cell cycle by flow cytometry:the percentage of ovarian cells in the G0-G1 phase was the highest in both control and melatonin-treated rats.However, melatonin treatment significantly reduced (P< 0.05) the percentage of cells in the G0-G1 phase compared tocontrol rats. This reduction of cells in the G0-G1 phase is derived to the S phase in cyclic and acyclic rats. The possibilitythat a tumoral process leads to a proliferative effect observed in young and acyclic melatonin-treated rats was ruled outbecause no significant differences were found for p53 and Ki67 expression levels between control and melatonin groups.Density of oocytes: the oocyte number per ovary unit volume was not affected by melatonin treatment in the three ageranges studied. Melatonin treatment in middle-aged (pre-acyclic) rats resulted in significantly higher (P< 0.05) ovarianvolume; higher oocyte volume, without significant differences, and oocytes in circular form were significantly (P< 0.05)higher than in control rats. Melatonin treatment during pre-acyclic age range could resynchronize the estrous cycle periodicity.Melatonin treatment was able to maintain the same levels of estradiol in the pre-acyclic age groups studied as thoseobserved in the young cyclic rats. The present results indicate that melatonin administration to middle-aged female ratsproduces beneficial effects that extend the reproductive function of the ovary.

A very small tripeptide amide L-pyroglutamyl-L-histidyl-L-prolineamide (L-PHP, Thyrotropin-ReleasingHormone, TRH), was first identified in the brain hypothalamus area. Further studies found that L-PHP was expressed inpancreas. The biological role of pancreatic L-PHP is still not clear. Growing evidence indicates that L-PHP expression inthe pancreas may play a pivotal role for pancreatic development in the early prenatal period. However, the role of L-PHPin adult pancreas still needs to be explored. L-PHP activation of pancreatic β cell Ca2+ flow and stimulation of β-cell insulinsynthesis and release suggest that L-PHP involved in glucose metabolism may directly act on the β cell separatefrom any effects via the central nervous system (CNS). Knockout L-PHP animal models have shown that loss of L-PHPexpression causes hyperglycemia, which cannot be reversed by administration of thyroid hormone, suggesting that the absenceof L-PHP itself is the cause. L-PHP receptor type-1 has been identified in pancreas which provides a possibility forL-PHP autocrine and paracrine regulation in pancreatic function. During pancreatic damage in adult pancreas, L-PHP mayprotect beta cell from apoptosis and initiate its regeneration through signal pathways of growth hormone in β cells. L-PHPhas recently been discovered to affect a broad array of gene expression in the pancreas including growth factor genes.Signal pathways linked between L-PHP and EGF receptor phosphorylation suggest that L-PHP may be an important factorfor adult β - cell regeneration, which could involve adult stem cell differentiation. These effects suggest that L-PHPmay benefit pancreatic β cells and diabetic therapy in clinic.

According to our conception, the aging process is caused by cell proliferation restriction-induced accumulationof various macromolecular defects (mainly DNA damage) in cells of a mature organism or in a cell population. In the caseof cell cultures, the proliferation restriction is related to so-called contact inhibition and to the Hayflick's limit, while inthe case of multicellular organisms, it is related to the appearance, in the process of differentiation, of organs and tissuesconsisting of postmitotic and very slowly dividing cells. It is assumed that the proliferation of intact cells prevents accumulationof various errors in a cell population. However, the continuous propagation of all the cells in a multicellular organismis absolutely incompatible with its normal functioning. Thus, the program of development, when it generatespostmitotic or slowly dividing cells, automatically leads also to the onset of the aging process (mortality increase withage). Therefore, any additional special program for aging simply becomes unnecessary. This, however, doesn't reject, forsome organisms, the reasonability of programmed death, which makes possible the elimination of harmful, from the speciespoint of view, individuals. It is also very important to emphasize that increase or decrease of an organism's lifespanunder the effects of various external factors is not always necessarily related to modification of the aging process, thoughthe experimental results in the field are usually interpreted in just this way. I called the experimental-gerontological modelssimilar to the Hayflick?s model "correlative", since they are based on some correlations only and not related necessarilyto the gist of the aging phenomenon. So, for the Hayflick's model, it is the relationship between population doublinglevel and donor age, between population doubling potential and species lifespan, between some cell changes in vivo and invitro, and so forth. If the rationale of the "Hayflick phenomenon" is used, we can't explain why we age. Nevertheless,many authors virtually put a sign of equality between aging in vitro and aging in vivo, which generates conclusions thatare of quite doubtful accuracy. A classic illustration of this is the telomere concept of aging. Originally, the principle ofshortening end-segments of DNA (telomeres) during each cell division was formulated at the beginning of seventies bythe Russian scientist Aleksey Olovnikov and used by him to explain the limited "proliferative" lifespan in vitro of normalcells. Subsequently, the existence of this phenomenon was confirmed by the results of many research reports, the culminationof which was a publication in which the authors demonstrated the possibility of increasing the proliferative potentialof normal cells by introducing the enzyme telomerase to them, thus restoring the lost telomere segments. At the moment itlooks like the telomere shortening contributes to aging in vitro only, but not to aging in vivo because an organism neverrealizes the full proliferative potential of its cells. Besides, the most "responsive to aging" are the organs and tissues consistingof postmitotic cells, for which the concept of proliferative potential loses any meaning in practical terms. We developedanother "correlative" model?a model for testing of geroprotectors and geropromoters ? the "cell kinetics model."It is based on the well-known correlation between the "age" of cultured cells (age of their donor) and their saturation density.The model allowed us to perform preliminary testing of a lot of different compounds and factors that are interestingfrom a gerontological point of view, but it revealed no information about the real mechanisms of aging. However, the secondmodel we use in our studies ? the "stationary phase aging" model ? obviously, is a "gist" model. It is based on the assumptionthat in the cells of stationary cultures various intracellular changes similar to those of an aging organism can beobserved. The proliferation restriction in the case is provided, as a rule, just by contact inhibition. Many experimental resultsconfirming this assumption were obtained. "Age-related" changes that are well known from organismal studies wereshown to really occur in our experimental stationary cell culture model. Besides, such experiments can be carried out onnearly any type of cells from various biological species. Thus, the evolutionary approach to analysis of the data is provided.Moreover, the changes in the stationary cell cultures become detectable very soon ? as a rule, in 2 to 3 weeks afterbeginning the experiment. All this allows us to suppose that the "stationary phase aging" model should provide a very effectiveapproach to testing of different substances and their cocktails on their activities in terms of accelerating or retardingaging ? of course, if their effect is realized on the cell level only.

Neuroimmunomodulation and Aging: A Role for Transferrin and the Hypothalamus/Thymus Axis by Vladimir A. Lesnikov, Marina P. Lesnikova, H. Joachim Deeg (21-28).
Advanced age is associated with an increased incidence of immune and degenerative disorders, mediated bymetabolic changes, dysregulation of proinflammatory signals, and apoptosis. Concurrently, there is a progressive declinein self-recognition. Investigations on biologic functions of transferrin (Tf) other than iron transport showed that Tf has aprofound cytoprotective (anti-apoptotic) effect on lympho-hematopoietic cells and the thymus, and interferes with stressinducedsignals. Tf protects hepatocytes against Fas-induced cell death by reducing BID cleavage, inhibiting caspase-3and -9 activation and up-regulating survival signals such as Bcl-xL. The involvement in the regulation of alloreactivityand apoptosis suggests that Tf participates in the maintenance of ?self-identity? mechanisms, which are tightly linked tothe capacity of the immune system to recognize and react against any noxious agent. Some of the disorders associatedwith aging are thought to be related to thymic involution, reflecting alterations in the interplay of neural, endocrine andimmune factors. We established a murine model of thymic involution induced by stereotactically placed electrolytic lesionsin the anterior hypothalamic area. The events observed in this model mimic those observed during senescence includingthymus involution, i.e. enhanced glucocorticoid reaction to distress, and obesity. The described properties of Tfcan be exploited to modify immune responses and provide cytoprotection against pro-apoptotic and cytotoxic signalswhen neuroimmunomodulatory mechanisms are impaired, as is the case with aging.

Why Is Homocysteine Toxic for the Nervous and Immune Systems? by Alexander Boldyrev, Ekaterina Bryushkova, Anna Mashkina, Elizaveta Vladychenskaya (29-36).
Hyperhomocysteinemia is a risk factor for a number of neurodegenerative and cardiovascular diseases. Wehave shown that homocysteine induces excitotoxic effects in cells expressing glutamate receptors of the NMDA class.These receptors were found not only in neurons but also in immune-competent cells, neutrophils, red blood cells, cardiomyocytes,and osteoblasts. Activation of these cells by homocysteine results in an increase in cytoplasmic calcium ions,accumulation of reactive oxygen species, and activation of MAP kinase. An overload of immune-competent cells activatesboth necrotic and apoptotic cell death, whereas the neuropeptide carnosine (an antioxidant and immune modulator) protectscells against both processes. In a model of prenatal hyperhomocysteinemia in rats, we have found that carnosine protectsanimals against homocysteine toxicity with no change of the blood homocysteine levels. The efficiency of carnosinehas also been demonstrated in clinical trials of chronic brain ischemia and Parkinson?s disease.

Metallothionein I+II Expression as an Early Sign of Chronic Relapsing Experimental Autoimmune Encephalomyelitis in Rats by Hrvoje Jakovac, Marin Tota, Damir Grebic, Tanja Grubic-Kezele, Vesna Barac-Latas, Ines-Mrakovcic-Sutic, Cedomila Milin, Biserka Radosevic-Stasic (37-44).
Metallothioneins (MTs) are small, cysteine-rich proteins which have been implicated in various forms of stressproviding cytoprotective action against oxidative injury, DNA damage and apoptosis. Owing to their high affinity forphysiological metals, such as zinc and copper MTs are also critical components of regulatory proteins involved in cellgrowth and multiplication, as well as in the maintenance of immune homeostasis.To elucidate the role of MTs in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MTI+II proteins and the content of free Zn ions in the brain, spinal cord and in the liver early in the course of chronic relapsingexperimental autoimmune encephalomyelitis (CR-EAE) pathogenesis, i.e. before the onset of any clinical symptoms.Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenatein CFA. Control animals were treated with CFA alone. The data, obtained by immuno-histochemistry and insitu fluorescent labeling of free zinc ions, have shown that in the presymptomatic phase of CR-EAE (on the seventh postimmunizationday) MTs I+II were markedly upregulated in the cells that form blood-brain and blood-cerebrospinal fluidbarriers, as well as in the cerebellar parenchyma and hippocampal dentate gyri. Furthermore, we found that the liver alsobecomes a site of extensive MTs I+II synthesis shortly after immunization. Simultaneously, tissue content of free zincions increased at the sites of MTs induction, reflecting their antioxidative activity.The data, described in this paper point to regulatory and neuroprotective role of MTs in the pathogenesis of CR-EAE.

Reactive Astrocytes Are Key Players in Nigrostriatal Dopaminergic Neurorepair in the Mptp Mouse Model of Parkinson?s Disease: Focus on Endogenous Neurorestoration by Francesca L.&#8217; Episcopo, Cataldo Tirolo, Nuccio Testa, Salvo Caniglia, Maria Concetta Morale, Bianca Marchetti (45-55).
Parkinsons?disease (PD), a common neurodegenerative disorder, is characterized by progressive loss of dopaminergic(DAergic) neurons in the subtantia nigra pars compacta (SNpc) and gliosis. The cause and mechanisms underlyingthe demise of nigrostriatal DAergic neurons are not completely clarified, but interactions between genes and environmentalfactors are recognized to play a critical role in modulating the vulnerability to PD. Current evidence points to reactiveglia as a pivotal factor in PD, but whether astroglia activation may protect or exacerbate DAergic neuron loss is presentlythe subject of much debate. Astrocytes and microglia are the key players in neuroinflammatory responses, by secretingan array of pro- and anti-inflammatory cytokines, anti-oxidant and neurotrophic factors. Here, the contribution of astrocytesand their ability to influence DAergic neurodegeneration, neuroprotection and neurorepair will be discussed. Inparticular, the dynamic interplay between astrocyte-derived factors and neurogenic signals in MPTP-induced plasticity ofnigrostriatal DAergic neurons will be summarized together with recent findings showing that reactive astrocytes may contributeto promote DAergic neurogenesis from midbrain adult neural stem/precursor cells (NPCs). Within a host of astrocyte-derived factors, we unveiled Wingless-type MMTV integration site (Wnt)/?-catenin signalling was unveiled, as astrong candidate in MPTP-induced DAergic neuroplasticty/neurorepair. Understanding the intrinsic plasticity of nigrostriatalDAergic neurons and decifering the signals facilitating the crosstalk between astrocytes and midbrain neuroprogenitorsmay have implications for the role of stem cells technology in PD and for identifying potential therapeutic targets topromote endogenous neurorepair.

Physiologic autoantibodies, that is, those with an active physiologic role, are an important part of the normalhuman immune system and are essential in maintaining homeostasis. Evidence suggests that the body uses autoantibodiesto prevent disease and to self-treat diseases once started. This suggests a potential therapeutic role for autoantibodies, or,even better, a way to use them to prevent disease. Their capacity to remove aged, damaged cells is well established.Immunoglobulin (Ig) G autoantibodies bind to senescent cell antigen (SCA), which is an altered band 3 anion exchangerprotein found mainly on aged cells. Once bound, IgG triggers the removal of the senescent cells by macrophages.</p>;Band 3 is altered primarily by oxidation, which in turn generates SCA. These studies demonstrated that oxidation cangenerate neoantigens that the immune system will recognize. Band 3 isoforms are ubiquitous: they have been found in allmammalian cells and species so far examined.The innate immune response to band 3 membrane proteins, and their regulation of cellular lifespan and therapeutic potentialwill be presented. Examples of other potential innate and physiologic autoantibodies include neuroprotective antibodiesto amyloidgenic toxic peptides and antibodies to oxidized LDL (OxLDL), which modify the natural progression ofatherosclerosis.

Marine Therapy and Its Healing Properties by Juan Alberola, Francisco Coll (63-75).
This study demonstrates the effects of Quinton?s isotonic and hypertonic solution on mononuclear cells of peripheralblood. This involved assessing cell viability, morphology, number and size of aggregated cells; possible effectson cellular proliferation; effects on cellular proliferation in different lymphocyte populations; effect on hemoglobin releasedinto the medium.

It has been suggested that arterial stiffness is one of the most important risk factors for the development of acardiac infarction or stroke. As cardiovascular disease remains the number one killer of individuals before the age of 75,the early detection of cardiovascular disease and its prevention remains paramount in order to sustain a healthy longevity.This article looks at the latest noninvasive technology that can measure arterial stiffness quickly and easily and also highlightsa small open trial in which supplements were used to determine their efficacy in helping to reverse/improve arterialstiffness.

Aging, Cancer, and Longevity: The Uncertain Road by Novera H. Spector, Katica Jovanova-Nesic, Alida M. Gertz (86-91).
First, the latest scientific and clinical reports will be evaluated to separate the wheat from the chaff, that is,good data versus merely anecdotal evidence. Thus, the famous (infamous) Stromboli Cocktail will be brought up to date.Second, longevity statistics will be reviewed: Why do the most scientifically advanced countries have such low (comparatively)life expectancies? Scientific knowledge expands exponentially each decade, whereas there have been no significantadvances in our knowledge, government, economics, politics, anti-corruption, and so forth since the dawn of history.What can we expect in the future? Will the human species outlive the cockroach? Can we expect to get closer to that theoreticalasymptote of 120 years of human life? Will this ceiling ever be lifted? Finally, we offer two vital challenges to scientistsof today.

Thyrotropin-releasing hormone (TRH) aroused our interest when we were engaged in related experiments, sowe decided to study its effects on organs, tissues, and aging-related metabolic and hormonal markers when administeredin acute or chronic (oral) doses at various time points in its cyclic circadian pattern. We also wanted to determine what effects,if any, it had on aging processes in two essential systems, namely gonadal-reproductive and kidney-urinary. Our resultsshow positive changes as a result of short-term acute and long-term chronic oral administration of TRH to old micethat included rapid correction to more juvenile levels of most typical aging-related hormonal and metabolic measurements.Remarkably, testes function was maintained by means of a 4-month oral treatment with TRH in aging mice. As we suspectedupon seeing a significant increase in testes weight, TRH resulted in maintenance or even reconstitution of testesstructure and function when administered in the drinking water. This was demonstrated by the active formation and proliferationof mature spermatogonia and the intensive spermatogenesis in the follicles. The same TRH treatment led to protectionfor the kidneys from amyloid and hyalin infiltration of tubuli and glomeruli, which typically occurs in aging mice. Infact, we observed massive deposits of amyloid and hyalin material infiltrating the shrunken glomeruli and negatively affectingfiltration capacity of the untreated mice, whereas this was barely present in the TRH-treated mice. Advanced hyalindegeneration could also be observed in the tubular vessels of the untreated control mice.These experiments with TRH supplementation show clear aging-delaying and apparently even aging-reversing effects ofthe neuropeptide, whether it was administered parenterally or orally. TRH, like melatonin, is an anti-aging agent with abroad spectrum of activities that, because of their actions, suggest that TRH has a fundamental role in the regulation ofmetabolic and hormonal functions.

Is there a Possible Single Mediator in Modulating Neuroendocrine?thymus Interaction in Ageing? by Eugenio Mocchegiani, Marco Malavolta, Laura Costarelli, Robertina Giacconi, Francesco Piacenza, Fabrizia Lattanzio, Andrea Basso (99-107).
The restoration of the thymic functions and the thymic re-growth may be achieved in old mice by some endocrinological(melatonin) or nutritional interventions (arginine or zinc), suggesting that the thymic involution in old age is aphenomenon secondary to age-related alterations occurring in neuroendocrine?thymus interactions. The targets for thethymic restoration may be hormone receptors and cytokines, strictly related to the presence of two nutritional factors, suchas arginine and zinc, which are in turn essential for the efficiency of neuroendocrine?immune network both in ontogenyand ageing. The effect of melatonin is largely due to the presence of its specific receptors on cell membrane of thymocytesand Thymic Epithelial Cells (TECs). TECs synthesize thymulin peptide that is required for T-cell differentiation andmaturation within the thymus gland. In this context, the role of zinc is pivotal because it is involved, through ?zinc fingermotifs?, in the gene expression of melatonin receptors, in cell proliferation, apoptosis and thymulin reactivation. Zinc isalso required for the biological action of arginine, via Nitric Oxide pathway. Therefore, the beneficial effect of melatoninor arginine on neuroendocrine-thymus interaction in ageing can also occur via a better zinc pool redistribution within thebody where the capability of the zinc-binding proteins Metallothioneins (MT) in zinc release has a key role. These findingssuggest that zinc, via MT buffering, can be a single mediator in modulating neuroendocrine-thymus interaction inageing.

The majority of chronic diseases, most notably those accompanying aging, result from progressive deteriorationof central neuroimmunoendocrine control, often referred to as immunological surveillance. This is as true of cancer as it isof the development of cardiovascular, autoimmune, and neurodegenerative disease, in all of these immunological surveillancebreak downs, leading to an unraveling of the neuroimmunoendocrine process that inhibits proliferation of preneoplasticand neoplastic cells already existing in the body. The onset of cancer is anticipated by changes in the hormonalimmunecoordination resulting in chronic quantitative alterations in the synthesis and release of hormones and the loss ofthe natural synchronicity of that release, which occurs according to circadian rhythms in the healthy organism, principallyunder the control of the pineal network.Periodic circadian hormonal release is the source of immune system regulation, thus altering hormone rhythms impairs theimmune system?s ability to maintain control over emerging tumor cells, not necessarily to eliminate them, but to inhibitproliferation. Malignancy, then, is the result of suppression of or interference with the regular release of hormones thatmaintain strict regulation of the thymo-lymphatic immune system?s maturation and activity. This understanding meansthat we can act to prevent cancer by means of efficiently monitoring and maintenance of physiological hormonal values.For the cyclic synthesis of malignancies that are metastasized, a means of xenogeneic bone marrow transplantation is proposedas an alternative therapeutic approach.

There is virtually no correlation between what are generally accepted to be the symptoms of deficient androgen inmen and levels of androgens as measured in the laboratory. Now that androgen deficiency is being shown to play a part inconditions as diverse as metabolic syndrome, diabetes, and coronary heart disease, a hypothesis is needed to explain this apparentdiscrepancy between measured androgen levels and our understanding of the symptoms of androgen deficiency.When the possible mechanisms for androgen actions are considered, one explanation emerges that androgen may actmuch like insulin in persons with type 2 diabetes mellitus: the degree of androgen resistance may be variable dependingon the organs or systems considered. Therefore, the symptoms can result from altered or damaged synthesis of androgensynthesis or regulation, elevated androgen binding, a reduction in tissue response, or decreased as a result of polymorphismand aging. Genomic transcription and translation may also be affected.As with diabetes, in adult male androgen deficiency, it is suggested that the definition of androgen deficiency should bebased on individual physiology, with the requirements of the individual at a particular stage of life setting the baselineagainst which any deficiency of androgens or androgen metabolites, either absolute or relative, is determined. This approachwill affect the terminology, etiology, diagnosis, and treatment of androgen deficiency.

Age-Related Decline in Melatonin and Its MT1 Receptor Are Associated with Decreased Sensitivity to Melatonin and Enhanced Mammary Tumor Growth by Steven M. Hill, Chi Cheng, Lin Yuan, Lulu Mao, Rolf Jockers, Bob Dauchy, David E. Blask (125-133).
The pineal hormone melatonin (MLT) has potent anti-breast cancer activity, its actions are heavily mediatedvia the MT1 receptor and subsequent modulation of downstream signaling pathways including cAMP/PKA, Erk/MAPK,p38, and Ca2+/calmodulin. Also, via the MT1 pathway, MLT can repress the transcriptional activity of some mitogenicnuclear receptors including ER?, GR, and ROR?, while potentiating the activity of other receptors (RAR? and RXR?)involved in differentiation, anti-proliferation, and apoptosis. A review of the literature supports the view that MLT, via itsMT1 receptor, can suppress all phases of breast cancer including initiation, promotion, and progression. During the fifthand sixth decades of life, the production of MLT diminishes, concurrently with an increase in the incidence of breast cancer.Inasmuch as MLT has been demonstrated to have anti-cancer activity, we hypothesized that there may be a causallink between the reduction in MLT production in the pineal gland and the incidence of breast cancer which increases withage. We designed this study to establish whether a truly inverse relationship exists between tissue-isolated mammary tumorgrowth in young (2 months), adult (12 months), and old (20 months) female Buffalo rats and the decrease in bothMLT and the MT1 receptor with age, such that a causal link could be found. Serum MLT levels were measured in boththe light and dark phases. A significant 29% decrease in serum MLT levels, measured at the nocturnal peak, was foundin the adult and senescent rats (75% decrease) in comparison to that in young rats. In young rats, the nocturnal pinealgland MLT content exceeded daytime levels by 19-fold compared to a sevenfold increase in old mice. Also, the MT1 receptorwas found to be significantly lower in the nighttime and early morning in the senescent rat uterus as comparedto uteri from young and adult rats. Analysis of the rate of growth in transplanted, tissue-isolated, mammary tumors inducedby N-nitroso-n-methyl-urea (NMU) showed a significant increase in the senescent rats, but not in the young or adultrats Additionally, diminished response to the inhibitory action on tumor growth of exogenous MLT was noted in senescentrats such that tumor growth was suppressed by only 33% compared to 48% and 66% in adult and young rats, respectively.The diminution of the response of tumors to exogenous MLT was found to correlate with reduced MT1 receptorexpression in senescent compared to young and adult rats. These data suggest that the observed age-associated enhancedgrowth of tumors is related to the much reduced levels of MLT and its receptor in aged animals which reduce the sensitivityof tumors to inhibition by exogenous MLT.