5A_01_S

LONGEVITY ASSURANCE MOLECULAR PATHWAYS IN HUMAN CELLS

Efstathios S. Gonos

National Hellenic Research Foundation, Athens, Greece

e-mail: sgonos@eie.gr

Ageing and longevity are two multifactorial biological phenomena whose knowledge at molecular level is still limited. We have developed a clonal senescence induced system and we have cloned several senescence associated genes. Analysis of the function of one of the isolated genes, encoding for Clusterin/Apolipoprotein J (CLU), suggests that it is a novel survival factor. CLU is found over-expressed in vitro under a variety of stress conditions and in vivo in samples from patients suffering from various age-related diseases as well as in primary tumours which have acquired chemotherapeutic drug resistance. In addition, it has been demonstrated that inhibition of endogenous CLU expression by RNA interference induces growth retardation, higher rates of endogenous cellular death and sensitizes human cells to stress (Cancer Res 64, 1834-1842, 2004). Recent findings indicate that effective and sustained CLU depletion by siRNA induces late morphological alterations, growth arrest at the G₁/S checkpoint and activation of the mitochondrial axis of apoptosis that engages caspase-9. Moreover, CLU knock-down resulted in down regulation of the BH pro-survival (bcl-2 and bcl-X_L) proteins and activation of p53 and its downstream targets, namely p21^WAF1/CIP1 and bax.

We have also attempted an overall molecular and biochemical approach regarding proteasome function in replicative senescence and cell survival. We have observed reduced levels of proteasomal peptidase activities coupled with increased levels of oxidized and ubiquitinated proteins in senescent cells. We have found the catalytic subunits of the 20S complex and subunits of the 19S regulatory complex to be down-regulated in senescent cells. This is accompanied by a decrease in the level of both 20S and 26S complexes (J Biol Chem 278, 28026-28037, 2003). In support, partial inhibition of proteasomes in young cells by specific inhibitors induced a senescence-like phenotype. Stable over-expression of b subunits or POMP in human cell lines resulted in enhanced proteasome assembly and activities and increased cell survival following treatments with various oxidants. Moreover, stable over-expression of b ₅ subunit delayed senescence in human fibroblasts (J Biol Chem 280, 11840-11850, 2005). Finally in search of natural compounds that may activate proteasome, we have identified that the main constituent of olives, oleuropein, exerts stimulatory effects on proteasome. Importantly, continuous treatment of human fibroblasts cultures with oleuropein delays senescence by approximately 15%.

5A_02_S

Chemical Stress Response Mimetics Extend Lifespan

Gordon J. Lithgow

The Buck Institute, 8001 Redwood Blvd., Novato, CA 94945, USA e-mail: glithgow@buckinstitute.org

There are considerable mechanistic links between organismal stress resistance and aging. We have shown previously that long-lived mutants of the nematode C. elegans are resistant to thermal stress and over-accumulate small heat shock proteins (shsps) which alone can extend lifespan. A large number of genes determine normal aging rate in the nematode and mutations in these genes tend to be highly pleiotropic including effects on organismal stress resistance and stress gene expression. We undertook genetic screen for mutations that conferred increased resistance to multiple stresses and were surprised when we tagged a gene shown in other organisms to affect checkpoint functions. Checkpoints are evolutionarily conserved signal transduction pathways that arrest cell division in response to DNA damage or stalled replication forks. We demonstrated that CID-1, CHK-1 or CDC-25 checkpoint proteins also determine organismal stress resistance and lifespan. This pathway and other signaling pathways have remarkable effects on lifespan; we have observed some mutations extend lifespan 500%.

Each of these genetic modulators of aging are potential targets for chemical interventions. We will describe a series of screens of chemical compound libraries and show that chemical modulators of aging can be identified. These may open up new therapeutic avenues for age-related diseases.

5A_03_S

Age dependent collapse of protein folding homeostasis

Anat Ben-Zvi and Richard I. Morimoto

Department of Biochemistry, Molecular Biology, and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL 60208, USA

The balance between protein folding and degradation forms the cellular protein-folding homeostasis, which can compensate for inherent, off pathway misfolding. During the life span of an organism there are changes in expression of cellular folding and degradation components as well as an accumulation of damage proteins, however, the impact of these changes on the cellular folding homeostasis is not known. Using meta-stable proteins in C. elegans to probe for changes in the folding capacity, we find that there is an age dependent collapse of the cellular folding environment. This drop in folding capacity is modified genetically, by the Hsf-1 and Daf-16 pathways promoting or postponing homeostasis decline. We find that both pathways contribute independently to protein folding homeostasis but down regulation of either pathway is sufficient to disturb folding homeostasis. We suggest that the balance between misfolding loads and folding and turnover machinery determines this age dependent folding homeostasis collapse.

5A_04_S

Mild stress-induced hormesis and anti-aging effects on human cells

Suresh Rattan

Laboratory of Cellular Ageing, Department of Molecular Biology, University of Aarhus, Denmark. (Email: rattan@mb.au.dk )

The phenomenon in which adaptive responses to low doses of otherwise harmful conditions improve the functional ability of cells and organisms is known as hormesis. Several physical, chemical and biological stressors exhibit hormetic effects, including heavy metals, pesticides, antibiotics, chemotherapeutic agents, ethanol, aldehydes, chloroform, pro-oxidants, hypergravity and ionizing radiation. The key conceptual features of hormesis are the disruption of homeostasis, the modest overcompensation, and the re-establishment of homeodynmaics. A critical component of the homeodynamic property of living systems is their capacity to respond to stress. Thermoregulation, detoxification, cell proliferation/apoptosis, DNA repair, heat shock protein synthesis, protein turnover and antioxidative responses are some of the main homeostatic responses. Therefore, it is observed that if cells and organisms are exposed to brief periods of mild stress so that their stress response-induced gene expression is upregulated and the related pathways of maintenance and repair are stimulated, several anti-aging, health-improving and longevity-promoting hormetic effects occur. Extensive studies performed in our labs have shown that repeated mild heat stress has anti-aging hormetic effects on various cellular and biochemical characteristics of human skin fibroblasts undergoing aging. These effects include the maintenance of chaperone protein profiles, reduction in the accumulation of oxidatively and glycoxidatively damaged proteins, stimulation of the proteasomal activities for the degradation of abnormal proteins, improved cellular resistance to ethanol, hydrogenperoxide and ultraviolet-B rays, and enhanced levels of various antioxidant enzymes. Further research will elucidate the mechanisms and possibilities of human applications of physical and mental stress as a beneficial challenge.

5A_05_S

Mitochondrial J Haplotype and Anti-oxidant status and in the Belfast Elderly Longitudinal Free-living Ageing STudy (BELFAST)

Irene Maeve Rea¹, Owen A Ross^{2, 5}, G Pooler Archbold³, Ian R Young⁴, Martin Curran², Derek Middleton²

¹ Department of Geriatric Medicine, Queens University Belfast, ²Northern Ireland Tissue Typing and Immunogenetics Laboratory, Belfast City Hospital, ³Biochemistry Laboratory, Belfast City Hospital, ⁴Nutrition and Metabolism Group, Queens University Belfast, ⁵Mayo Clinic, Jacksonville, USA

Mitochondria are the chief source of both energy and oxidants inside the cell. This dual role can damage cells despite an array of antioxidants available to mop up endogenous oxidants including hydrogen peroxide, superoxide and hydroxyl radicals. The mitochondrial haplotype J seems related to ‘successful’ ageing in Italian, Finnish and Irish groups of nonagenarian/centenarians. In this study we questioned whether J haplotype octo/nonagenarians from the BELFAST study demonstrated enhanced anti-oxidant profile or other phenotype characteristics.

Methods: Briefly, community-living, mentally alert (Folstein >27/30) octo/nonagenarian subjects were enlisted as part of BELFAST study. DNA typing for mitochondrial haplotypes was collected and blood for serum antioxidants together with Blood pressure measurements. Serum uric acid, bilirubin, ceruloplasmin, glutathione, selenium and vitamins C, A, E and a-carotene were not significantly different between J and non J mitochondrial haplotypes but whole blood and serum gluthatione peroxidase (GSHpx) were lower in J haplotype octo/nonagenarians. Both systolic and diastolic blood pressure was significantly lower in J haplotype octo/nonagenarians.

Conclusion: BELFAST study octo/nonagenarian subjects, carrying the J haplotype, do not show enhanced antioxidant status for most of the major antioxidants, including uric acid and vitamin C, but show reduced whole blood and serum glutathione peroxidase status. Interestingly J mitochondrial haplotype octo/nonagenarians did have lower systolic and diastolic blood pressure.

5A_06_S

Influence of age and ethanol intoxication on Heme Oxygenase 1 expression in rat liver.

Anna Lisa Furfaro, Stefania Patriarca, Emanuela Balbis, Cinzia Domenicotti, Damiano Cottalasso, Umberto Maria Marinari, Maria Adelaide Pronzato, Nicola Traverso

Department of Experimental Medicine, Section of General Pathology, University of Genova, Via L.B. Alberti 2, 16132, Genova, Italy; annalisa.furfaro@virgilio.it

Many authors recognize that heme oxygenase 1 (HO-1) expression is a marker of cellular response to oxidative stress; since ageing is related to oxidative “wear and tear”, HO-1 may represent a candidate biomarker of ageing. In this study, we evaluated the hepatic expression of HO-1 mRNA in 2.5-24 month-old rats; this expression was higher at 6 months than at 2.5 months of age, but thereafter increased no further. However, while 2.5 and 6-month-old rats responded to acute ethanol intoxication by displaying increased expression of liver HO-1 mRNA, 18 month-old rats did not show any response; this phenomenon suggests that during development and ageing the transcriptional response to oxidative stress is progressively impaired. This may be due to decreased transcriptional ability to respond to stress in older animals, rather than by a reduction in oxidative stress. Grants from the University of Genova, MIUR PRIN #2004063943_001 and #2004068552_002, and FIRB 2001 #RBAU01JBH8_003.

5B_01_S

Heat shock proteins and protective effects in the nervous system

Ian R. Brown, Sheng Chen and Ari Chow

Center for the Neurobiology of Stress, University of Toronto at Scarborough, Toronto, Ontario, Canada M1C1A4

Hsp70 is a multi-gene family composed of stress-inducible members (Hsp70) and other members that are constitutively expressed (Hsc70). The heat shock proteins Hsp70 and Hsc70 exhibit similar molecular structure and biochemical functions. Constitutively expressed Hsc70 is enriched in the mammalian nervous system compared with non-neural tissues and present at high levels in neuronal cell bodies. After thermal stress, Hsc70 is translocated to synapse-enriched areas of the cerebral cortex where it associates with Hsp40 to form a complex that can refold denaturated proteins. These results suggest that the heat shock response in the nervous system involves not only the synthesis of stress-inducible Hsps but also translocation of constitutively expressed Hsc70 to synapse-enriched areas where it could participate in neuroprotective mechanism that preserve synaptic function during times of stress. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and ALS have been termed 'protein misfolding disorders'. These diseases differ widely in frequency and impact different classes of neurons. Hsps provide a line of defense against misfolded, aggregation-prone proteins and are among the most potent suppressors of neurodegeneration in animal models. Analysis of constitutively expressed heat shock proteins revealed variable levels of Hsc70 and Hsp27 in different classes of neurons in the adult brain. The differing levels of these constitutively expressed heat shock proteins in neuronal cell populations may confer a variable buffering capacity against protein misfolding disorders that correlates with the relative frequencies of the previously mentioned neurodegenerative diseases. Upregulation of Hsps could offer a therapeutic strategy to counter conformational changes in neuronal proteins that trigger pathogenic cascades resulting in neurodegenerative diseases. Differentiated neurons in both in vivo and in vitro systems have been reported to be refractory to Hsp induction by conventional heat shock. We will report on a compound that induces Hsps in differentiated neurons with the interesting feature that it induces a wider set of Hsps in differentiated human neurons compared to differentiated rodent neurons.

5B_02_S

Molecular Chaperones and Protection from Cerebral Ischemia

Rona G. Giffard, Yibing Ouyang, LiJun Xu and Midori Yenari

Department of Anesthesia, Stanford University, Stanford, CA USA

Stroke is the third leading cause of death in many countries. Data from both animal stroke models and primary cultures have demonstrated that Hsp70 overexpression can reduce cerebral ischemic injury. We have used viral vectors and DNA transfection to overexpress Hsp70 in the brain and in brain cells. We have previously reported that overexpression of Hsp70 and Hsp40 reduce ischemic injury in in vitro models of ischemia, and Hsp70 overexpression is effective in rodent models of stroke. To delineate which domains within Hsp70 are important for protection from ischemia in the brain we compared two mutants to full length wild type Hsp70. One was a point mutant in the ATPase domain- K71E, the second was a deletion mutant encoding the carboxy terminal domain, amino acids 381-640. The constructs were injected intracerebroventricularly, resulting in transfection of both neurons and astrocytes. We observed that both the ATPase deficient mutant and the carboxyterminal domain alone were similar to the wild type HSP70 in ability to significantly reduce focal ischemic injury in the rat. Additional effects of Hsp70 that could contribute to protection were studied in Hsp70 overexpressing mice subjected to focal ischemia. Here we found evidence of reduction of inflammation- reduced activation of NFkB, and decreased expression of iNOS, in the Hsp70 overexpressing mice. We also found previously that Hsp70 overexpression was associated with higher levels of the antiapoptotic protein Bcl-2. We have thus found at least three mechanisms contributing to protection of the brain from ischemia by Hsp70. These are binding of unfolded proteins, inhibition of inflammation and inhibition of apoptosis. Hsp70 is a multifaceted protein, and at least several of its effects are involved in protecting the brain from ischemia.

5B_03_S

Manipulating the heat shock response as a therapeutic strategy in ALS

Bernadett Kalmar and Linda Greensmith

Institute of Neurology, UCL, UK. l.greensmith@ion.ucl.ac.uk

Heat shock proteins (hsps) are know to play a role in neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), in which motoneurons degenerate, resulting in muscle weakness, paralysis and finally death, usually within 2-5 years of diagnosis. Evidence suggests a relationship between the level of the heat shock response (HSR) and the specific vulnerability of motoneurons to degeneration in ALS. For example, motoneurons have an unusually high threshold for the activation of the HSR which may result in abnormal protein folding and trafficking and an increased susceptibility to apoptotic insults. We have recently up-regulated the HSR in a mouse model of ALS. Treatment of SOD1^G93A mice with arimoclomol, induces the expression of hsp70 and hsp90, resulting in a delay in disease progression and a significant increase in survival. However, the effects of arimoclomol are unlikely to result only by increasing hsp levels in motoneurons since genetic manipulations that increase hsp70 in SOD1^G93A mice fail to alter disease progression. It is not only the mechanism, but the site of action of arimoclomol and hsps in SOD1^G93A mice that remains unclear. We are characterising the HSR in this model of ALS as a function of disease progression and testing the differential neuroprotective effects of a variety of manipulations that up-regulate expression of hsps. Our results show that not all agents that activate the HSR will necessarily have beneficial neuroprotective effects on motoneurons. Moreover, activation of the HSR in non-neuronal cells may have greater neuroprotective effects than targeting the HSR within motoneurons themselves. We hope that a better understanding of the role of the hsps in ALS will help to optimize targeting of the HSR as an effective therapeutic strategy for this fatal disease.

5B_04_S

Neuroprotective Effects of Extracellular Hsp70

Michael Tytell

Neurobiology & Anatomy, Wake Forest Univ. School of Med., Winston-Salem, NC 27157, tytellm@wfu.edu

When the cytoprotective function of Hsps was a relatively new concept in the 1980s, we and Hightower respectively observed that Hsp70 was passed from one cell to another and released into the extracellular fluid (Tytell & Lasek, Brain Res 324:223, 1984; Hightower & Guidon, J Cell Physiol 138:257, 1989). These were unexpected results because the Hsp70 family was thought to be exclusively intracellular proteins. In the two decades since, extracellular Hsp70 has become well-documented. Our 1984 observation that glial Hsp70 was transferred into the axon led to the novel hypothesis that, in the nervous system at least, the stress tolerance of neurons was not solely dependent on their own Hsp70, but could be supplemented by additional Hsp70 from adjacent glia. For neurons, this hypothesis has major implications because those with long axons (mm to meters in length) require newly synthesized proteins to be transported from the neuronal cell body to remain functional. If stress or injury occurs in axons at some distance from their cell bodies, hours to days are required for newly synthesized Hsp70 to reach the injury site, which is too long to be of use. Thus, supplying Hsp70 at the injury site would be more effective in maintaining neuronal and axonal function. We have tested this idea in a number of models. . In the injured sciatic nerve, local application of Hsp70 improved sensory and motor neuron survival. Similarly, in the light-damaged rat eye, Hsp70 injected into the vitreous chamber promoted survival of photoreceptors. In cultured glia and neurons, we found that glia released Hsp70 and that neurons took up Hsp70 from the medium, leading to greater resistance to apoptosis induced chemically or by lack of trophic factor. Therefore, Hsp70 has potential as a therapeutic agent in acute nervous system injury.

5C_01_S

Metallothionein-mediated immunomodulation; new roles for an old stress response

*Lynes, M.A., *Yin, X., *Unfricht, D.W., *Marusov, G., **Lyons, B.L., ^†Emeny, R.T., and ^†Lawrence, D.A.

*University of Connecticut, Storrs, CT, USA; **Jackson Laboratory, Bar Harbor, ME, USA; ^†Wadsworth Center, NYSDH, Albany, NY, USA

Metallothionein (MT) is an unusual stress response protein with a novel and intriguing structure and a set of distinct biological functions. The protein is remarkably cysteine rich, and many of its functions revolve around cysteine-associated thiols. This highly conserved protein is known to be a reservoir of essential divalent heavy metals, it sequesters toxicants and scavenges free radicals, and it can act to regulate transcription factor activity. In each of these instances, the principal site of action is thought to be in the nuclear and cytosolic compartments, and this perspective is consistent with the observation that MT does not have a signal sequence for export through the endoplasmic reticulum and golgi apparatus. Despite this signal sequence absence, there is strong evidence that MT can be released to extracellular spaces in significant amounts under stressful circumstances. We have found that MT in the extracellular environment can act as a chemotactic agent, and that this response may be mediated by interactions with G-coupled protein receptors. Our recent studies have focused on mechanisms of cadmium-mediated immunomodulation, and the effect of manipulations of MT gene dose on the progression of humoral immunity against T-dependent antigens during low dose cadmium or zinc exposure. At doses of metal that do not influence the humoral response in wild type mice, mice that carry a targeted disruption of the Mt1 and Mt2 genes show significant immunosuppression in the presence of cadmium, but not zinc. In contrast, both cadmium and zinc suppress the humoral response of mice that carry the Mt1 transgene construct that drives over-expression of metallothionein. This suppression is associated with changes in the cytokine profile produced by stimulated splenocytes. Both cadmium and zinc are inducers of MT mRNA and protein expression, but at sub-toxic concentrations of these metals only cadmium provokes a selective release of MT to the extracellular environment. We have also explored the impact of changes to the MT gene dose in mice infected with Listeria monocytogenes. Clearance of L.m. was accelerated in mice that carry both the targeted disruptions of Mt1 and Mt2, as well as in mice that carry the Mt1 transgene construct when compared to congenic wild type controls. We hypothesize that MT may represent an central regulator of immune functions that occur under stressful circumstances, and that manipulations of MT levels in the extracellular environment may provide an avenue for the management of inflammatory, infectious, autoimmune and neoplastic disease processes.

5C_02_S

Copper metabolic disorder in myocardial pathogenesis

Y. James Kang, DVM, Ph.D.

Department of Medicine and Department of Pharmacology and Toxicology, University of Louisville School of Medicine,Louisville, KY, USA

The association of copper (Cu) with cardiomyopathy has been recognized for a long time, but its clinical significance has not been explored until recently. In diabetic patients and rat model, Cu chelation therapy using a Cu chelator trientine has been shown to ameliorate cardiac pathological changes and improve heart function. In contrary, we have observed that dietary supplementation with physiologically relevant levels of Cu can reverse established hypertrophic cardiomyopathy in aortic banding mouse model even in the presence of pressure overload. Both results have indications for Cu metabolic disorders in cardiomyopathy in humans. However, it is important to distinguish the fundamental differences in Cu metabolic disorder between diabetic and pressure overload cardiomyopathy. Both diabetes and pressure overload cause cardiac oxidative stress, mitochondrial damage, hypertrophy and dysfunction. However, we have observed that Cu levels slightly increase in the heart, but significantly decrease in the liver of streptozotocin (STZ)-induced diabetic mice. In contrary, Cu concentrations decrease in the heart, but do not change in the liver of pressure overload mice. It is possible that diabetic complications lead to systemic disorder of Cu metabolism and disruption of Cu detoxification in the liver, thus increasing the risk of Cu toxicity to organs including the heart, but in pressure overload the heart is the primary organ of Cu metabolic disorder leading to cardiac Cu deficiency but maintaining liver detoxification function. In this context, Cu chelation would be beneficial to patients with diabetic cardiomyopathy, but Cu supplementation would improve the condition of pressure overload cardiomyopathy. Both cases have been demonstrated in animal studies and it is important to apply the information generated from animal studies to human cardiomyopathy patients. These studies were supported in part by US-NIH grants HL59225 and HL63760.

5C_03_S

Brain injury and oxidative stress: think zinc!

Stefano Sensi1,2

Depts. of Neurology, and Basic and Applied Medical Sciences, 1Center of Excellence on Aging / University

“G. d’Annunzio”, Chieti-Pescara, Italy, 2University of California-Irivine, Irvine., USA, ssensi@uci.edu

Zn2+ is a potent mediator of neuronal injury both “in vitro”and “in vivo”, and trans-synaptic movement of the cation from pre- to post-synaptic neurons has been shown to greatly contribute to a variety of neurological conditions including cerebral ischemia, brain trauma and epilepsy. Zn2+ can enter neurons through glutamate receptor-associated channels [NMDA and AMPA/kainate channels (Ca-A/K channels), voltage-sensitive calcium channels (VSCC), or Zn2+-sensitive membrane transporters (like the Na+/Ca2+ and Na+/Zn2+ exchangers). Mechanisms by which Zn2+ exerts its potent neurotoxic effects include both mitochondrial and extra-mitochondrial pathways. Experiments in cortical neurons have shown that mitochondria play an important role in restoring Zn2+i homeostasis but this Zn2+ uptake leads also to prolonged mitochondrial depolarization and free radicals generation. In addition to roles in acute injury, Zn2+ might play roles in the selective neurodegeneration associated with aging and Alzheimer’s disease (AD). Indeed, cumulative effects of repeated Zn2+ exposures could contribute to the oxidative damage and mitochondrial dysfunction seen in AD. Interestingly, Zn2+ homeostasis is affected by oxidative stress, as reactive oxygen species are potent triggers

for injurious cation release from Zn2+ binding proteins (metallothioneins). In this talk, we examine how Zn2+ dyshomeostasis and oxidative stress might act synergistically to promote degeneration in the context of several neurological conditions.

5C_04_S

Cytoprotective Metal Ions

George A. Perdrizet ^a and Lawrence E. Hightower ^b

^a Dept. Trauma, Hartford Hospital and Univ. Connecticut, Hartford, CT 06102

^b Dept Cell and Molecular Biology, Univ. Connecticut, Storrs, 06269

The cellular stress response and stress protein expression form the biologic basis of stress conditioning protocols. Currently most information on inducible cytoprotection has been focused on the use of hyperthermia as the inducing agent. Heat shock preconditioning has amply demonstrated the proof of concept that stress conditioning protocols have the potential to provide beneficial cytoprotection in the setting of elective invasive medical and surgical procedures. While whole-body hyperthermia is a very effective agent, a pharmacologic agent would be more attractive as a clinical method to achieve a cytoprotected state in diseased humans. Metal ions have been shown to be potent inducers of the cellular stress response, are anti-inflammatory and capable of providing clinically relevant cytoprotection. We have successfully used the chloride salts of tin and zinc to provide in vivo cytoprotection in animal models of: 1. Rodent renal artery occlusion, 2. rabbit spinal cord ischemia 3. acute pulmonary inflammation, rabbit fat embolism syndrome (intravenous administration of oleic acid), 4. acute inflammation within the rodent mesenteric blood vessels. In these models, evidence for induction of stress proteins and inhibition of acute inflammation will be presented as potential mechanisms for the observed cytoprotection. The nature and activities of tin and zinc metals will be compared and contrasted.

5C_05_S

Influence of mercury on rat renal glucocorticoid receptor association with Hsp90 and Hsp70

Jadranka Dundjerski, Jelena Brkljačić, Tatjana Perišić, and Gordana Matić

Department of Biochemistry, Institute for Biological Research »Siniša Stanković«, Belgrade, Serbia, email: jadund@ibiss.bg.ac.yu

Unliganded glucocorticoid receptor (GR) is located in cytoplasm in form of multiprotein heterocomplex with various proteins including Hsp90 and Hsp70. The structure, composition, assembly and functional significance of this complex depends on cell type, physiological demands and environmental conditions. It is known that toxic effects of mercury could be reflected on structure and function of transcriptional factors such as GR. The influence of mercury on association of rat kidney GR with heat shock proteins Hsp90 and Hsp70 and on cytosolic levels of these Hsps was investigated. The GR heterocomplexes with Hsp90 and Hsp70 were immunopurified from renal cytosol of rats administered with different doses of mercury (1, 2 and 3 mg Hg/kg b.w.). A quantitative immunoblotting procedure was applied to determine the levels of GR, Hsp90 and two Hsp70 isoforms (constitutive Hsp73 and inducible Hsp72) in the renal cytosol, as well as the amounts of these proteins within GR heterocomplexes immunoprecipitated by anti-GR antibody. Mercury was found to stimulate GR association with all examined Hsps. The most prominent effect of the metal was stimulation of Hsp72 interaction with GR. On the other hand, the metal administration led to an increase of Hsp90 level in the cytosol, while the cytosolic levels of Hsp70 isoforms remained unaltered. These findings suggest that association of Hsps, at least Hsp70, with the GR might be ascribed to changes in the affinity of their interaction rather than to changes in Hsps availability in the cytosol. Therefore, GR heterocomplex assembly seems to be a controlled process enabling chaperoning and functioning of the GR in concert with physiological demands.

5C_06_S

Differential stress proteins expression in NRK-52E cells exposed to Hg(II) or Pb(II)

^§Stacchiotti A, *Morandini F, *Bettoni F, ^§Foglio E, °Cadei M, ^§Rodella LF, °Grigolato P and *Aleo MF

^§Human Anatomy, °2^nd Pathology and *Biochemistry Units, School of Medicine, University of Brescia, Viale Europa 11, 25123-Brescia, Italy. Corresponding author: aleo@med.unibs.it

Mercury Hg(II) and lead Pb(II), are two hazardous environmental contaminants having nephrotoxic effects and whose toxic action appear to be associated to the cellular increase of reactive oxygen species (ROS). Cells respond to oxidative damage by synthesizing highly conserved stress proteins, heat shock (HSP) and glucose related proteins (GRP). These molecular chaperones protect other cellular proteins and organules from injuries induced by a variety of stressors, including heavy metals. So, the present in vitro study is undertaken to compare the expression of five stress proteins in rat proximal tubular cells exposed to subcytotoxic concentrations of Hg(II) or Pb(II) salts. Proliferating NRK-52E cells received for 24h culture media containing 20m M HgCl₂ or 60m M PbCl₂ then the presence and abundance of HSP25, HSP60, HSP70, GRP75, GRP78 were analysed by immunohistochemistry and Western blotting. Concomitantly, in order to check if stress proteins response might be really related to a renal oxidative damage, ROS and glutathione (GSH) levels were measured by flow cytometry and spettrophotometric analysis. Our data proved that in NRK-52E both HgCl₂ and PbCl₂ treatments enhance the expression of constitutive chaperones (HSP25, GRP75 and GRP78) and the cell GSH content, even if at different grade. Interestingly, only Hg(II) ions stimulate either the ROS formation or the inducible HSP70 protein. These results suggest that, in our in vitro system, Hg(II)-induced ROS production and expression of peculiar stress proteins could be a consequence of a different mechanism of action of this metal with respect to Pb(II).

5D_01_S

Molecular understanding of HPA axis involvement in stress/immune circuits

Eduardo Arzt

Laboratorio de Fisiologia y Biologia Molecular, Universtity of Buenos Aires, and CONICET, Argentina

The immune-neuroendocrine systems have an intimate cross communication making possible a satisfactory response to environmental changes and stress. The hypothalamic-pituitary-adrenal axis (HPA) has a key role in the interaction between the immune and neuroendocrine systems and in the stress response. Cytokines activate the HPA axis and induce a rise in glucocorticoid levels, which are instrumental in order to control immune-cytokine overreactions, acting on T lymphocytes and other ctyokine/target cells. The specificity of hormonal and cytokine signals on their target cells is crucial to provide specificity to their actions. The cross-talk between cytokine-induced transcription factors such as Tbet, GATA-3, NFk B, and AP-1 and steroid (i.e. glucocorticoid) receptors involve both genomic and non-genomic actions, and constitutes the mechanism for fine tuning both hormone and cytokine responses. Corticotrophin releasing hormone (CRH) is the key mediator of the HPA axis of the central nervous system response needed to adapt to stressful conditions. The final outcome of CRH/CRH1 signaling depends on the context of specific cells and ligands, cross-talk of signaling pathways and the effector actions of the pathways once they are activated. This specificity bears consequences at the CNS level where CRH activates through the same receptor (CRHR1) different signaling pathways depending on neuroanatomical context. All these molecular interactions represent a key step for understanding, at the cellular and genetic level, the specificity and ultimate response of physiological neuroendocrine-immune interactions during stress.

5D_02_S

Interleukin-1 signaling at the intersection of neuronal, immune and metabolic stress

Tamas Bartfai

The Harold L Dorris Neurological research Institute. Molecular and Integrative Neurosciences Department , The Scripps Research Institute , La Jolla Ca 92037 USA

Macrophages , adipocytes and macrophages in white adipose tissue, microglia and neurons can synthesize the proinflammatory cytokine and endogenous pyrogen ; IL-1 b in response to acute immune, mechanic, oxidative and hypoxic insult , and to excitatory neurotoxic events. IL-1 synthesis is also induced by chronic elevation of misfolded proteins or metabolic/endocrine stressors like elevated leptin levels. The IL-1 levels being highly inducible within short time serve as a common signal in neuronal immune and endocrine signaling.

The wide spread expression of IL-1type 1 receptor on neurons, pancreatic-b cells and macrophages ,T and B cells accounts for the global inflammatory effects f IL-1 .

The acute stress leading to rapid activation of the HPA axis, lowering seizure threshold and fever response by IL-1 utilizes the rapid transcription independent neuronal effects of IL-1 . The molecular steps of this signaling have been delineated in molecular detail showing that IL-1R1-MyD88-Neutral Spingomyelinase-ceramide cSrc – phosphoprotein steps mediate the early IL-1 response. After 45-60 min the classical Toll signaling pathway involving the NFKB mediated induction of COX2 and the subsequent production of the inflammatory mediator PGE2.

The chronic inflammatory stress presented by obesity and the role of IL-1 in the conversion from insulin resistance to type 2 diabetes will also be discussed.

5D_03_S

Role of lipids in the modulation of human T cell activation

Fulop T¹, Brassard P², Larbi A¹, Frisch F², Fortin C¹, Carpentier A².

¹Division of Geriatrics and the Program of Immunology, ²Division of Endocrinology, Department of Medicine, University of Sherbrooke, Sherbrooke, Qc, Canada

Studies have shown suppressive effects of PUFA on T cell functions suggesting that lipids can have potent immunomodulatory effects. It is now accepted that the membrane of T cells is heterogeneous and contains microdomains called lipid rafts (LR) playing an important role in TCR signalling. Thus, variations in lipid levels and composition may determine their effects on immune response. Each time when lipids are ingested the immune system is submitted to a lipid stress. The precise mechanism for this effect has not been fully investigated in vivo in humans. Our aim was to determine whether there are differences in T cell functions and signalling depending on the way of lipid administration and composition. Peripheral T cells were isolated from healthy subjects before and after 2-hours of an intravenous infusion of heparin + Intralipid (HI) during a euglycemic hyperinsulinemic clamp to induce a 2.5-fold elevation in plasma linoleic acid concentration. Similar experimental setting was designed after an oral meal (OM).. HI and OM reduced peripheral T cell membrane fluidity and altered lipid raft organisation. Both associated with reduced T cell proliferation upon CD3 + CD28 co-stimulation. Tyrosine phosphorylation of LAT and activation of Akt in T cells were also impaired without a reduction in T cell receptor expression. The LR polarization was also altered. A selective increase in plasma linoleic acid concentration and in intravascular lipolysis therefore have a suppressive effect on peripheral T cell CD28-dependent activation and this effect associates with changes in plasma membrane properties. The lipid composition of nutritional therapy in patients at high risk of septic complications may be crucial and may also be of relevance for type 2 diabetes. Furthermore, oral nutrition rich in lipids constituting a chronic lipid stress, at long term, could contribute to the observed immunosenescence.

5D_04_S

Neuroendocrine Response in Susceptibility to Inflammatory, Autoimmune and Infectious Diseases

Esther M. Sternberg M.D.

NIMH/NIH, Bethesda, MD, USA

mThe central nervous system (CNS) plays an important role in regulating immunity and in susceptibility and resistance to autoimmune, inflammatory and infectious diseases. Cytokines released during inflammation mediate changes in brain function, inducing sickness behaviors, sleep, fever and activation of the hormonal stress response (hypothalamic-pituitary-adrenal axis, HPA axis). In turn neural and neuroendocrine responses, including the HPA axis, sympathetic and parasympathetic responses, regulate immune responses, thus providing important extra-immune system feedback control of immunity. HPA axis activation inhibits inflammation through the generally anti-inflammatory action of the glucocorticoids. However, physiologic concentrations of glucocorticoids are immunomodulatory, shifting cytokine production from a TH1 to a TH2 response and enhancing delayed type hypersensitivity. Interruptions of the HPA axis at any level and through multiple mechanisms, whether genetic, surgical or pharmacological, render inflammatory resistant hosts susceptible to inflammatory disease. In contrast, over-activation of this axis, as in chronic stress, enhances severity of infections, through the immunosuppressive effects of the glucocorticoids. The association of a blunted HPA axis with autoimmune/inflammatory disease occurs across species, strains and diseases, and in a variety of human autoimmune/inflammatory illnesses. Tissue resistance to glucocorticoids resulting from polymorphisms, mutations or dysfunction of the glucocorticoid receptor (GR) is also associated with enhanced autoimmune/inflammatory disease expression. We have described a new mechanism for glucocorticoid resistance related to bacterial toxin repression of the GR and other nuclear hormone receptors. This suggests a potential new mechanism for shock and inflammatory sequelae of bacterial infections, and potential new approaches to developing therapies for these conditions.

References:

Sternberg EM. Neural regulation of innate immunity: a coordinate non-specific host response to pathogens. Nature Reviews Immunology 6(4):318-28, 2006.

Marques-Deak A, Cizza, G and Sternberg EM. Brain-immune interactions and disease susceptibility. Molecular Psychiatry, 2005, 10 (3), 239-250.

Webster JI, Tonelli L, Sternberg EM. Neuroendocrine Regulation of Immunity. Annual Review of Immunology. 2002. 20:125-163

The Balance Within. The Science Connecting Health and Emotions. by E.M. Sternberg, M.D., W.H. Freeman, New York. Hard cover 2000; paperback Holt, Times Imprint, 2001

5D_05_S

Adrenocortical hyperresponsiveness during postnatal development of stress system in mice

A. Yu. Shevchenko, T. V. Yakovleva, E. N. Makarova, N. M. Bazhan

Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrenteva 10, Novosibirsk, 630090 Russia

The stress system in the mouse from postnatal days 1-12 is characterized by very low basal corticosterone levels and an inability of mild stressors to induce an enhanced corticosterone release. This period known as the stress-hyporesponsive period. We previously demonstrated that in 3-week-old mice basal plasma corticosterone levels and adrenal corticosterone production were higher than both in 2-week-old mice and in adult mice. It is unknown whether stress-response is increased and what changes in regulation of adrenal steroidogenesis occur in 3-week-old mice. The aim of this study was to compare stress-response, as well as activity of adenylate cyclase and steroidogenic enzymes in adrenal cells in 3-week-old mice with that in adult 15-week-old mice. Stress-induced plasma corticosterone level in 3-week-old mice was greater and developed faster than in adult mice. Corticosterone production stimulated by ACTH, exogenous cAMP and progesterone, and ACTH- and forskolin-induced cAMP accumulation in vitro were higher in 3-week-old mice than in adult mice. Thus, our data demonstrate that in the mouse the stress-hyperresponsive period occurs during the third week after birth. This period is characterized by very high basal and stress induced plasma corticosterone levels, which seems to be related to adrenal ACTH-responsiveness. Adrenocortical hyperresponsiveness during the third week is caused by high adenylate cyclase activity and high intracellular steroidogenic enzyme activity.
This work was supported by research grant from The State Program Supporting Leading Scientific Schools in Russia No. 7071.2006.4. and Lavrentiev contest grant from The Siberian Branch of the Russian Academy of Sciences.

5D_06_S

Stress-induced corticosterone enhance brain surveillance by T cells

Gil. M. Lewitus¹, Hagit Cohen² and Michal Schwartz¹

Acute psychological stress was shown to act as an endogenous adjuvant, consequently enhancing cell-mediated immunity. Independent work carried out by our research group, has demonstrated that whilst T cell-mediated immunity, recognizes antigens residing in the CNS (autoimmune cells), and well-regulated with respect to activity and length of time can fight off abnormal behavior resulting from the stress. We examined the mechanism underlying the stress-induced T cell-mediated protective autoimmunity. We found that short exposure of mice to stressor (predator odor), especially in the hippocampus and the paraventricular nucleus, enhanced T-cell infiltration to the CNS. The peak of infiltration was 72 hr following mice exposure to the stressor. The T cells trafficking to the CNS was associated with ICAM-1 expression. This peak was preceded by a transient elevation of corticosterone. Assessment of the relationship between the stress-induced increase of corticosterone and infiltration of T cells revealed; while corticosterone induces T cells mobilization to the CNS by inducing the expression of ICAM-1, the T cells are necessary for the induction of corticosterone following exposure to acute stress. Moreover, the beneficial effect of the elevation of corticosterone after the stress is partly mediated by the T cells. These results identify a new relationship between acute stress, corticosterone, and adaptive immunity and support the argument that the immune system is needed to maintain normal brain homeostasis. They may also point the way to the development of immune-based therapies for post traumatic stress disorder.

5E_01_S

The mechanisms behind the brain’s response to stress

Marian Joëls

SILS-CNS, University of Amsterdam, The Netherlands

When an organism experiences a stressful situation, this is perceived through the brain and thus gives rise to the activation of the sympathetic nervous system and the hypothalamo-pituitary-adrenal axis. As a result brain cells, including those involved in the initial appreciation of the stressor, are exposed to elevated levels of monoamines like noradrenaline, to specific neuropeptides and to corticosteroids. We have examined the effects of these hormones on neuronal function in limbic regions, particularly the hippocampal CA1 area. In the initial phase after stress (<1 hr), rapid effects of noradrenaline, CRH and vasopressin will increase the excitability. Corticosterone, through a non-genomic pathway, plays a permissive role in the effects of the other stress hormones but can also by itself increase local excitability and facilitate the induction of long-term potentiation (LTP). Collectively, the excitatory effects of stress hormones in this phase may help to promote focused attention, vigilance and start the encoding of information about the event. At the same time, though, gene-mediated cascades are started via corticosterone binding to its nuclear receptors, which will alter transcriptional activity of responsive genes. These effects develop slowly (1-2 hrs), i.e. at a point in time that hormone concentrations are back to the pre-stress level; the effects can last for hours. The gene-mediated actions include an enhanced influx of calcium -hence more firing frequency accommodation-, reduction of noradrenergic responses and suppression of LTP induction. These actions will help to normalize the earlier aroused activity in the hippocampus, which can be regarded as a ‘central’ negative feedback. As information reaching the circuits at this time must be very salient to pass the threshold for LTP induction, earlier encoded information will be preserved.

5E_02_S

Genetic manipulations of hormonal signaling in the hippocampus

Daniela Kaufer

Department of Integrative Biology&Helen Wills Neuroscience Institute University of California, Berkeley,CA

Glucocorticoids (GCs), the adrenal steroids released during stress, compromise the ability of neurons to survive neurological injury. In contrast, estrogen protects neurons against such injuries. We designed three genetic interventions to manipulate GCs actions, which reduced their deleterious effects in rat both in vitro and in vivo. The most effective was a chimeric receptor combining the ligand-binding domain of the glucocorticoid receptor (GR) and DNA-binding domain of the estrogen receptor. Expression of this receptor reversed the outcome of the stress response by rendering GCs protective rather than destructive. Our findings elucidate principal steps in the neuronal stress response pathway, all of which are amenable to therapeutic intervention. GCs are also implicated in reducing adult hippocampal neurogenesis. There has been little evidence for the presence of type 1 GR or type 2 (mineralcorticoid) receptors in neuronal precursor cells (NPC), and therefore suggested that GCs must indirectly inhibit NPC proliferation, though the mechanism has remained obscure. We demonstrate that GR mRNA is transcribed and yields a cytoplasm-localized receptor in isolated NPC from the adult hippocampus. Treatment of NPC grown in vitro with GCs induces decreased proliferation index, and a down-regulation of Nestin, a protein marker that is down regulated as NPC stop dividing and differentiate. This response is blocked using the GR-specific antagonist indicating that the GCs response is mediated by the glucocorticoid receptor. We demonstrate decreased proliferation and a shift in cell fate choice in NPC following GCs treatment, which is blocked by the anti-GCs genetic manipulation. The apparent responsiveness of NPC to GCs suggests that neurogenesis is directly modulated via GR signalling pathways.

5E_03_S

Stress and transcription: Specific inactivation of the Glucocorticoid Receptor gene in the Dopaminergic system: New insights on drug addiction

Francois Tronche CNRS UMR7148 « Molecular Genetics, Neurophysiology and Behavior », College de France, 75005 Paris France; francois.tronche@gmail.com

A dysfunction of the stress response is suspected in the etiology of metabolic and behavioral disorders (ie anxiety, depression, drug dependence). A major component of this response involves the release of adrenal glucocorticoids (GCs) and the activation of the glucocorticoid receptor (GR), a transcription factor.

To study GR function in vivo, we develop and analyze animal models lacking GR in given brain cell populations. Molecular, physiological, electrophysiological and behavioral studies of these animals should allow us to locate the cell types involved and to better define the mechanisms underlying GR function in brain physiology and in pathological situations.

We previously showed that the selective inactivation of the glucocorticoid receptor (GR) gene in mice brains (GRNesCre) profoundly reduces motivation for cocaine. More recently, we showed that these behavioural effects are associated with a change in the impulse activity of midbrain dopamine neurones.

To determine in which cell type the function of GR is required to modulate motivation for cocaine, we generated animal models in which GR is selectively inactivated in either pre-synaptic dopamine neurons (GRDATCre) or post-synaptic cells (GRD1Cre). Characterization of these models will be presented. To address the question of the interaction of GCs and serotonergic pathway, we generated a mouse transgenic line that allow Cre recombination in all 5-HT1A neurones and obtained conditional GR inactivation. Analysis of this animal model will be presented.

5E_04_S

Glucocorticoids and pancreas development: studies in rodents and human

Bernadette Breant

Centre de Recherche des Cordeliers, UMRS 872 - Université Paris VI, INSERM U872, Paris, France

Low birth weight is strongly predictive of hypertension, cardiovascular diseases, obesity, insulin resistance and diabetes. The mechanisms by which fetal undernutrition or low birth weight, increases the risk of developing these diseases are unclear. In a rat model of undernutrition, involving an overall reduction in maternal food intake, we investigated the hypothesis of a primary defect in beta-cell development. In this model, fetuses with intrauterine growth retardation show decreased beta-cell mass, which persists into adulthood and ultimately causes glucose intolerance, thereby mimicking features of the metabolic syndrome. Maternal undernutrition caused glucocorticoid elevation, which, in turn, causes a reduction in beta-cell mass in the fetuses. Our data also suggest a key role of glucocorticoids under normal nutritional supply. By combining in-vitro studies with in-vivo investigations in mice lacking the glucocorticoid receptor (GR) in the whole organism or in specific pancreatic cell populations, we show that the GR is critical for pancreatic architecture and survival, as well as for beta-cell mass expansion during a critical developmental window. Glucocorticoids act on precursor cells before the onset of hormone gene expression and are likely to programme beta-cell differentiation by modifying the balance of specific transcription factors. To investigate whether this would also apply to human pancreatic development, we study the expression and localization of the GR and that of various transcription factors and pancreatic hormones on fetal pancreatic specimens. The results are consistent with a possible role for glucocorticoids during human pancreatic development.

5E_05_S

Interplay of stress-related hormones in the control of insect fitness

N.E. Gruntenko, N.A. Chentsova, N.V. Adonyeva, E.K. Karpova, E.V. Bogomolova and I.Yu. Rauschenbach

Institute of Cytology and Genetics SD RAS, Novosibirsk 630090, Russia; nataly@bionet.nsc.ru

In insects, biogenic amines, dopamine (DA) and octopamine (OA), and gonadotropins, juvenile hormone (JH) and 20-hydroxyecdysone (20E), are the main components of neurohormonal stress-reaction. For the progress of oogenesis in Drosophila under normal and stress conditions a proper balance between JH and 20E is of a paramount importance: imbalance (a shift of the balance in the direction of JH or 20E) leads to dramatic changes in oogenesis and fecundity (Soller et al., 1999; Gruntenko et al., 2003, 2005; Rauschenbach et al., 2004). Here we demonstrate that (I) D. virilis and D. melanogaster females possess a mechanism of reciprocal regulation of JH and 20E which is essential to maintain the JH/20E balance: (a) a rise in JH titre leads to a rise in the activity of ecdysone 20-monooxygenase (which converts ecdysone to 20E) and their 20E titre; (b) a rise in 20E titre results in a dose-dependent rise in JH levels; (c) 20E regulates JH indirectly via DA metabolic system - a rise in 20E titre increases DA content in young females and decreases it in mature females, thus leading to a rise in JH levels in both; (d) there is a feedback in the regulation of JH by DA - a rise in JH titre leads to a decrease in DA in young females and its rise in mature females. (II) Under normal conditions, reproduction is regulated by genes that control DA metabolic pathways (indirectly via JH); under unfavorable conditions, reproduction is regulated by genes that control OA metabolism. (III) Individual stress-resistance depends on expression of genes controlling the background level of DA. The study was supported by RFBR grants ## 07-04-00194, 06-04-48357.

5E_06_S

Dissecting CRHR1-mediated pathways via microarray technology

C. Graf, P. Weber, B. Pütz, F. Holsboer, W. Wurst, J.M. Deussing

Max-Planck-Institute of Psychiatry, Kraepelinstr. 2, 80804 München; cgraf@mpipsykl.mpg.de

The corticotropin releasing hormone (CRH) system is involved in endocrine, autonomic and behavioural responses to stress. The biological actions of CRH-like neuropeptides are mediated by G-protein-coupled receptors, CRH receptor 1 (CRHR1) and CRHR2. CRHR1 is widely expressed in the mammalian brain (e.g. cerebral cortex, cerebellum, amygdala, hippocampus) and in the pituitary gland. Mice deficient for CRHR1 display decreased anxiety-like behaviour and dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. Ligand binding increases the affinity of the CRHR to G-proteins. Binding of a G?s-protein will activate adenylate cyclase (AC) and protein kinase A (PKA) as well as other cyclic adenosine monophoshpate (cAMP) dependent pathways. The reported coupling of additional G-proteins to CRHR1 suggests that also other second messengers are involved in CRHR-signaling. In order to identify specific target genes of CRHR1-mediated signaling pathways we applied cDNA and oligonucleotide microarray technology using a mouse corticotroph cell line (AtT20) and pituitaries of CRHR1-deficient mice. 102 genes in vitro and more than 400 genes in vivo were found stress- and/or CRH- and CRHR1-dependently regulated. A subset of candidate genes was validated in independent material by quantitative real time PCR (qRT-PCR). These candidates are involved e.g. in cAMP, mitogen activated protein kinase (MAPK) or epidermal growth factor receptor (EGFR) signaling. In order to examine their functional role the effect of these genes on different known target genes downstream of CRHR1 signaling is analyzed using reporter assays.

Module 5 – Poster lectures:

5A_01_P

AISA ("Anti Inflammatory Senescence Actives") a plant derived approach of anti-stress

Jean-François Bisson, Chantal Menut, Marc Moutet and Patrizia d’Alessio

ETAP-54500 Vandoeuvre-les-Nancy ; Laboratoire de Chimie Bio-Moléculaire-UMR 5032 ENSCM, 34296 Montpellier ; EandC Biocitech France; AISA Therapeutics CHU Paul Brousse 12, ave Paul Vaillant-Couturier 94807 Villejuif France dalessio@vjf.inserm.fr

Psycho-social stress induces arterial vasomotor alterations such as vasospams and transitory hypertension. Moreover, stress has a profound impact on vascular endothelium, inducing a systemic inflammation. Starting from plant extracts, we have isolated through bio-guided selection, characterized and patented 4 mono-terpens, that we have named "AISA", Anti-Inflammatory Senescence Actives, targeting activated endothelium. In our in vitro platform, they inhibit the expression of adhesion molecules and the polymerization of actin fibers, following the activation by TNF-a. Inhibition is conserved at all replicative passages of vascular endothelial cells. In pre-clinical models, AISA 5203-L is well tolerated and has a good oral bio-availability. This molecule has shown convincing anti-inflammatory performance in a rat TNBS colitis model, comparable to those obtained with ibuprofen. Using AISA-5203-L in a stress model, a substantial anti-stress activity could be documented by a FOB (Functional Observation Battery) of 56 tests (patent pending).

We would like to propose that a new anti-inflammatory molecule of plant origin interfering with cell replicative senescence would be responsible of relevant anti-stress effects. This hypothesis would permit the positioning of AISA molecules as candidates therapeutic applications in multi-factorial diseases, such as hypertension, neuro-degenerative diseases, chronic pain syndrome, obesity and depression.

5A_02_P

INTERACTION OF HSP-16.2 WITH HISTONES AFTER HEAT-SHOCK IN THE NEMATODE C. ELEGANS.

Alavez S. and Lithgow GJ.

The Buck Institute, 8001 Redwood Blvd. Novato, CA 94945 USA. salavez@buckinstitute.org

Stress response is a very significant determinate of the life history of C. elegans. As the first metazoan genome to be sequenced, C. elegans is a major model for molecular genetic studies of aging because of the availability of a range of single gene mutant lines with greatly extended lifespan. Interestingly, these long-lived variants are also stress tolerant. Among others, we demonstrated that at least one chaperone gene encoding heat shock protein -16 (hsp-16) works together with reduced insulin signaling to bring about lifespan extension. However, the mechanism by which hsp-16 increases lifespan or thermotolerance in this animal model remains unclear.

We have now obtained an stable transgenic line that over-express the fusion protein HSP-16.2::GFP under the control of hsp-16.2 promoter that presents a remarkable thermotolerance and a high expression of the fusion protein after heat-shock (HS) measured by westernblot. The lifespan of these over-expressing lines is extended (36%, p<0.001) as compared to control strain, N2. This strain also presents a widespread expression of this transgene in multiple tissues. We have found a relocalization of HSP-16.2::GFP to the nuclei of several neurons of the nerve ring and some intestinal cells after HS in this strain as revealed by DAPI and GFP colocalization. On line with this observation, we have identified Histone-3 and Histone-4 through MALDI-TOF and LC/MS mass spectrometry by coimmunoprecipitation with an HSP-16.2 specific antibody and posterior separation of the proteins via 1-dimensional gel electrophoresis after HS.

These results show that histones are targets of HSP-16.2 during stress conditions (HS) and suggest a nuclear role of chaperones to regulate thermotolerance and maybe other stressing conditions like aging.

5A_03_P

Lipoic acid: How an increase in Respiration can Decrease Stress and increase Lifespan in C. elegans

Olivier Descamps, Michael Benedetti, Amanda L. Foster, Maithili Vantipalli, James N. Sampayo, Matthew S. Gill, Anders Olsen, and Gordon J. Lithgow

Aging is the most important risk factor in developed countries. Consequently, there is considerable interest in the pharmacological manipulation of aging as a way to uncover new avenues for development of therapeutics. Genetic alterations can extend the lifespan of the nematode C. elegans as much as five-fold and in principle chemical compounds could do the same. Based on the mechanistic relationship between aging and stress response, we have screened for compounds that enhance stress resistance. Lipoic acid (LA), a cofactor of the Krebs cycle enzymes pyruvate and glyceraldehyde dehydrogenases, increases respiration and lifespan in the adult nematode worm C. elegans. This seems somewhat paradoxical in the context of the rate of living theory of aging in which lifespan is inversely proportional to the rates of metabolic processes. We investigated the effect of the LA treatment on the mitochondrial physiology of C. elegans. We find that lipoic acid decreases the mitochondrial membrane potential and see some evidence for an increase in the amount of mitochondria. This in turn results in lower oxygen radical production (steady state superoxide anion levels), which may explain the lifespan extension. The LA-treated worms are not overtly hyperactive and have the same levels of ATP and fat content as untreated controls. Our working hypothesis is that stimulation of the Krebs cycle, without changing the other metabolic parameters, increases the number of mitochondria and in consequence lowers the burden of activity in each of the mitochondria, resulting in less stress and longer lifespan.

5A_04_P

Early stress response in FXTAS an inherited neurodegenerative disorder

D. Garcia-Arocena¹, C.K. Iwahashi¹, F. Tassone¹, J.B. Brower², E.M. Berry-Kravis³, R. Willemsen², P.J. Hagerman¹

¹ Dept. Biochemistry and Molecular Medicine, University of California Davis, School of Medicine, One Shields Ave, Davis, 95616, USA

² Dept. Clinical Genetics, Erasmus University, Rotterdam, The Netherlands

³ Neurology and Biochemistry, Rush University Medical Center, Chicago, Illinois, USA

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that appears to affect carriers of premutation alleles (55 to 200 CGG repeats) of the FMR1 gene. The pathogenesis of FXTAS involves the direct toxic effect of the elevated levels of expanded CGG repeat FMR1 mRNA. We have recently described neural cell models that are capable of recapitulating the formation of intranuclear inclusions, the pathologic hallmark of FXTAS. We have discovered that several proteins including lamin A/C and the stress response proteins a B-crystallin and Hsp27 genes are dysregulated in response to expression of the expanded CGG repeat. To quantify these observations we analyzed the expression of FMR1, a B-crystallin, Hsp27, and lamin A/C by real time quantitative PCR in cultured human cells derived from subjects with severe FXTAS and age-matched controls. We also studied cultured cells derived from mice with expanded CGG repeats and wt controls. Our results show that subjects with premutations have elevated levels of expression of stress response genes and cellular redistribution in cultured cells. Immunofluorescent studies of additional proteins and flow cytometry DNA analysis indicate cell cycle dysregulation in cells with expanded CGG repeats consistent with accelerated aging in both mouse and human models. We discuss the implications of these results on neural plasticity.

5A_05_P

Diurnal and age changes in stress responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis and erythrocyte antioxidant enzymes

N. D. Goncharova¹, T. N. Bogatyrenko²

¹Institute of Medical Primatology, Sochi-Adler, Veseloye 1, 354376, Russia; ²Institute of Problems of Chemical Physics, Chernogolovka, Russia

The aim of the study was to examine chronobiological characteristics of the HPA axis functioning and the antioxidant enzyme activities in stress and aging. Female Macaca mulatta of 6-8 years (young mature) and 20-27 years (old) were subjected to acute psycho-emotional stress (two hours immobilization) at 9.00 or 15.00 h. Levels of ACTH, cortisol (F), dehydroepiandrosterone sulfate (DHEAS), and testosterone (T) in peripheral blood plasma were measured before the stress and 5, 15, 30, 60, 120, 240 min and 24 h after the challenge. In parallel, activities of superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase (GR), glutathione-S-transferase, and lipid peroxidation (LPO) were measured in hemolyzed erythrocytes. Young monkeys demonstrated much higher increase of ACTH, F, T, DHEAS levels and GR activity in response to the stress imposed at 15.00 than to identical stress imposed at 9.00. However, no such circadian differences in dynamics of the hormonal secretion and GR activity after the stress were found for old animals. Young monkeys demonstrated also much higher accretion of ACTH, F, T, DHEAS levels and GR activity in comparison with old monkeys in response to the afternoon stress. The changes in GR activity with stress and aging correlated well with the changes in the F, DHEAS, and T level. SOD activity in old monkeys was lower than in young ones before the stress and increased in response to the stress that was accompanied by light decrease of LPO. In contrast to old monkeys, young ones demonstrated decrease of SOD activity and some increase of LPO with stress. There was a high level of correlation between the stress changes in SOD activity and the changes in the F level in old monkeys but there was no correlation in young monkeys. The lack of correlation between stress dynamics of F level and SOD activity may be caused by marked activation of adrenal T secretion in young monkeys. These results suggest that the HPA axis plays an important role in regulation of antioxidant enzymes defense in stress conditions and that this regulation shows age differences.

Supported by the RFBR (grant 06-04-97616-r_yug_?)

5A_06_P

Potential use of plant adaptogens in age-related disorders

Makarov V., Makarova M.N., Stoloschyck N.V.

Interregional Center "Adaptogen", Piskarevsky ave., 47/5, St-Petersburg, 195067, Russia

Wikman G., Panossian A.

Swedish Herbal Institute, Prinsgatan 12 5tr, SE-413 05 GÖTEBORG, Sweden

In traditional medicinal systems, members of a small group of higher plants were considered to possess restorative properties and were used as general tonics in the treatment of disease and in convalescence. Through a series of studies conducted in the early 1950s, Soviet scientists first established that several of these plants also possessed the capacity to enhance the “state of non-specific resistance” (SNSR) of an organism to stress. Plants that exhibit this “adaptogenic effect” are currently used to increase mental and physical work capacity and performance against a background of fatigue and stress, and also to improve the quality of life. Additionally, the efficacy of plant adaptogens in the relief of mild and moderate depression has recently been demonstrated. The aim of the present study was to investigate the effect of adaptogens, administered in the form of standardised extracts of Rhodiola rosea, Schisandra chinensis and Eleutherococcus senticosus in their fixed combinations (ADAPT-232, ADAPT-Plus and ADAPT-Extra-Plus), on the age-related deterioration of function of the innate defence, cardiovascular and central nervous systems in 2 year old rats. Drugs and placebo were administered daily over a period of 4 months, and alterations in adrenal weight, ECG parameters, motor activity, learning ability, duration of hexanal-induced sleep, formation of immobilisation stress-induced stomach ulcers, spontaneous promotion of tumours, apoptosis of spleen lymphocytes, and levels of 17- hydroxy-corticosteroids in the urine, and cholesterol, albumins and total proteins in the blood, were monitored. The results showed that repeated administration of adaptogens can diminish or prevent a range of age-related disorders including reduced liver detoxifying function, malfunction of the central nervous system (i.e. loss of memory and learning ability), development and progression of cardiac insufficiency and hypercholesterolemia, impaired protein synthesis, reduced activity of the hormonal system, increased sensitivity to stress (hypodynamia-induced damage to the stomach and adrenals), impaired apoptosis, and spontaneous promotion of tumours. It is concluded that adaptogens have potential value in the treatment of age-related disorders of the stress system in the elderly and may be effective in maintaining the health status of such individuals at the normal level.

5A_07_P

Tissue and Cellular Responses to Stresses of Hypoxia and Hyperoxia of Importance in Tissue Engineering

George K. B. Sándor, Riitta Suuronen

Regea Institute for Regenerative Medicine, Tampere University, Tampere, Finland and The Hospital for Sick Children, University of Toronto, Toronto, Canada. ( george.sandor@utoronto.ca )

Future reconstructive surgical procedures will have the possibility of using tissue engineered constructs to rebuild missing or defective body parts in humans and in veterinary medicine. Such constructs require three essential components: cells, a scaffold and signalling molecules. A great deal of attention has been paid to the harvesting of stem-cells both of fetal and autogenous adult origins which are to be expanded in tissue culture. Such expanded stem-cell derrived populations of cells are used to populate resorbable scaffolds. Much work has been done in understanding the potential sources and roles of key bioactive cell signalling molecules such as Bone Morphogenetic Protein (BMP). Cells that exist in isolation or as parts of complex organs and tissues have known responses to certain stressors. Cells and tissues that are grown ex vivo in extra corporeal bioreactors must be provided with environments that minimize cellular stress and promote maximal growth when cellular populations are to be expanded. These cellular constructs are sensitive to varying concentrations of ascorbic acid, dexamethasone and b -glycerophosphate. The reactions of cells and tissues to hypoxic and hyperoxic environments are also important. At the molecular level these reactions occur in part due to superoxide radicals while at the tissue level they are mediated by bioactive signalling proteins such as Vascular Endothelial Growth Factor (VEGF). There is evidence that both hypoxia and hyperoxic states may promote angiogenesis and further tissue growth. This presentation will review our understanding of these processes and mechanisms in the context of tissue engineering.

5A_08_P

CREB-INDUCING ACTION OF HYPERICUM PERFORATUM MAY BE RESPONSIBLE FOR ITS PREVENTIVE EFFECTS ON STRESS- AND AGING-RELATED MEMORY IMPAIRMENT.

Emil Trofimiuk, Adam Hołownia, Jan J. Braszko

Department of Clinical Pharmacology, Medical University of Bialystok, Waszyngtona 15A, 15-274 Bialystok, Poland.

Chronic stress impairs a number of aspects of cognition such as acquisition of memory, its consolidation and recall. Similar alterations were observed in aged rats. It is well known that stress causes acceleration of the brain aging. In stressed as well as in aged rats disturbances of hypothalamo-pituitary-adrenal axis and consecutive hypercortisolemia are seen. Excessive cortisolemia leads to a number of neurochemical and neuroanatomical changes in brain, especially in the hippocampus, which is particularly vulnerable because of high density of glucocorticoid receptors. As a result neurogenesis is disturbed in the hippocampal areas CA1 and CA3 and the neuronal atrophy occurs leading to the decrease of its volume and lowering total number of neurons and their ramifications.

We recently found, that dried crude herb of H. perforatum (350 mg kg^-1 for 21 days orally) reversed negative effects of chronic stress on cognition (spatial reference and working memory, recognition memory, conditioned behaviour, learning, acquisition and recall of the avoidance behaviour), but still our knowledge about the mechanisms of this improvement is poor. It is known that H. perforatum normalizes the dopaminergic and noradrenergic transmission in medial prefrontal cortex, normalizes decreased by stress 5-HT1A and 5-HT2 receptors, balance exaggerated NMDA receptor function, and also restores normal cortisolemia.

This study assessed an association of the H. perforatum improvement of age-related memory impairments with the increase of CREB and phosphorylated CREB in hippocampus. Middle-aged rats (78-weeks old) displayed clear-out decline in the acquisition of spatial working memory in the Morris water maze similar that in the young rats (8-weeks old) after 21-day restraint stress. Chronic administration of H. perforatum effectively prevented these negative effects of aging and stress (p<0.01) that could be partially explained by the concomitant increase of CREB phosphorylation in the hippocampi (p<0.01) of aged rats.

This study seems to justify the conclusion that St John’s wort in a crude form may have a considerable action relieving stress- and aging-related cognitive deficits by an activation of CREB regulated genes in hippocampus.

5A_09_P

Evaluation of Photo-stress of Skin Using Cathepsin L

Masaki Yamaguchi¹, Kazutaka Teramura¹, Yusuke Tahara¹, Teruhiko Makino², Tadamichi Shimizu², Akira Date³

¹Graduate School of Science and Engineering, University of Toyama, Japan

²Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Japan

³ Procter & Gamble Japan K.K., Japan

Objective: A noninvasive determination of skin surface proteolytic activity may be important for medical purposes. The aim of this research is to explore the relationship between photo-stress of skin and cathepsin L activity in the epidermis of human. Materials and Methods: Nine healthy male outdoor workers were enrolled (36.1 ± 12.2 yr, mean ± SD). The study protocol was approved by the Ethical Committee of the Institutional Review Board of the University of Toyama. A tape stripping technique using D-SQUAME tape (Cuderm Co., TX) was used to collect the human stratum corneum from both the posterior (solar-exposed area) and anterior (solar-protected area) regions of the forearm. The cathepsin L activity was analysed by the azocasein assay using a spectrophotometer. Unused tape stripping was analysed as a negative control. The calibration curve for cathepsin L activity was measured using a standard sample (human liver, EMD Chemicals Inc., Germany). Results and Discussion: The relative absorbance of the solar-exposed areas ranged between 0.045 and 0.107 and that of the solar-protected areas ranged between 0.064 and 0.125. The mean relative absorbance of the negative control, solar-protected and solar UV-unexposed areas were 0.002, 0.084 and 0.101, respectively. The cathepsin L activities of the solar-exposed and solar-protected areas were equivalent to 5.6 and 6.8 U/l. Thus, the cathepsin L activity of the solar-exposed area was lower than the solar-protected area (paired Student’s t-test, P < 0.05). Conclusion: Cathepsin L activity may be a biomarker of the photo-stress.

Specific downregulation of Hsp72 or Hsp27 IN CANCER cells suppresses the DNA damage Response

Julia Yaglom

Assistant Professor, Boston University Medical Center, 715 Albany St., Boston, MA, 02118, Yaglom@biochem.bumc.bu.edu