AGING NEURON MITOCHONDRIAL FUNCTION AND REJUVENATION WITH ESTROGEN OR MITOSIS

 

G. Brewer, J. Nash, T. Jones, J. Reichensperger, M. Parihar

 

PO Box 19626, Springfield, IL 62794-9626

 

The mitochondrial oxyradical theory of aging is gaining increased support from studies of brain, heart and muscle tissue.  Is this aging caused by systemic hormones or intrinsic to  aging mitochondria in neurons, for example.  The intrinsic hypothesis is supported by our findings with isolated neurons of increased susceptibility with age to glutamate and A-beta toxicity, two endogenous toxins important to age-related neurodegenerative disease (Brewer, 1998).  For neuron-specific function, we have established culture techniques that allow us to compare neurons isolated from embryonic, middle-age (12 month) and old (24 month) rat hippocampus (Brewer, 1997).  For use with these cultured neurons, we monitored cytochrome oxidase activity (COX), cardiolipin specific staining of mitochondria with nonyl-acrydine orange (NAO), as well as labeling with mitotracker-red and immunostain for cytochrome C, fluorescent NADH, glutathione levels and respiration. Cytochrome C immunostain and mitotracker red stained equal numbers of mitochondria per cell for middle-age and old neurons, compared to higher numbers per embryonic neuron.  In contrast to mitochondrial numbers, COX activity/cell declined sharply with age of the neurons: levels of old neurons were reduced by 30% of those of middle-age neurons.   Similarly, NAO stain for mitochondria was 40% lower for old neurons compared to middle-age neurons.  By addition of FGF2 and low density passage, old neurons divide in culture (Brewer, 1999).  COX activity in these dividing old neurons was equivalent to that of middle-age neurons.  Age-related reductions in glutathione levels were exacerbated by exposure to glutamate.  NADH levels and respiration in old neurons were normal, but showed dramatic deficits in response to glutamate.  In addition to restoring youthful calcium dynamics, treatment of old neurons with 17-beta-estradiol restored NAO staining levels/cell to those of middle-age neurons and provided full neuroprotection from glutamate and A-beta toxicity.  These results demonstrate that mitochondrial function is impaired in old neurons, which correlates with increased susceptibility to toxic stress, but function can be restored by treatment with FGF2 or estradiol.

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ACE inhibition intervention: Implications for improving age-related declines in physical performance and longevity.

 

CS Carter, M Cesari, M Pahor

 

Wake Forest Univeristy School of Medicine, J. Paul Sticht Center on Aging, 1 Medical Center Blvd. Winston-Salem NC, 27157

 

Age-related disregulation of  the renin-angiotensin system (RAS) leads to variety of pathologies including end organ damage to kidney, heart, blood vessels, and more recently has been suggested to exacerbate declining cognitive and physical performance. Reversing or attenuating these effects could have a significant impact on maintaining independence and extending longevity in the elderly.  In fact, both pharmacological and genetic studies have demonstrated that lower circulating levels of angiotensin converting enzyme (ACE) results in enhanced responsiveness to exercise, increased muscle strength, and lower adiposity. The relationship between low physical performance in healthy older adults, shifting body composition (increases in fat and decreases in fat-free mass) and longevity has been documented and more recently this same assessment has been made in aging rodents.  Therefore, interventions that retard declining performance such as ACE inhibition (ACEi) may also have validity for studies of longevity.  We have conducted studies to assess the effects of ACEi on body composition and physical performance in aged rats. Animals were randomized to daily injections of 40 or 80 mg/kg of enalapril or saline at baseline (24 months of age) and were followed for 6 months.  ACEi attenuated declining physical performance as measured by both grip strength and inclined plane tasks, and was most likely explained by a significant reduction in total fat mass, as no differences in lean mass were observed between groups (as measured using DXA). A possible interpretation is that beyond its powerful hemodynamic effects, ACEi regulates many aspects of metabolic functioning, decreases oxidative stress in many tissues, and may act ubiquitously to reduce age and disease related chronic inflammatory states.  Therefore, our understanding of these results could be enhanced by a more thorough and mechanistic characterization of the effects of long-term ACEi on lifespan and declining performance.  

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ESSENTIAL ROLE OF LIMITING TELOMERES IN THE PATHOGENESIS OF WERNER SYNDROME

AbstractAuthors: S. Chang*, A. Multani, N. Cabrera, M. Naylor, P. Laud, D. Lombard, S. Pathak, R. de Pinho

MD Anderson Cancer Center, Houston, TX and Dana Farber Cancer Center, Boston, MA

Mutational inactivation of the Wrn helicase gene causes Werner Syndrome (WS), an autosomal recessive disease characterized by premature aging, elevated genomic instability and increased cancer incidence. The capacity of enforced telomerase expression to rescue premature senescence of cultured human WS cells and the lack of a phenotype in Wrn deficient mice with long telomeres have implicated telomere attrition in WS pathogenesis. Here, we show that the varied and complex in vivo and cellular phenotypes of WS are precipitated upon exhaustion of telomere reserves in mice. In mice doubly null for Wrn and the telomerase RNA component (Terc), telomere dysfunction elicits a classical WS premature aging syndrome typified by premature death, hair graying, alopecia, osteoporosis, type II diabetes, and cataracts. This model also exhibited accelerated replicative senescence and accumulation of DNA-damage foci in cultured cells as well as increased chromosomal instability and cancer, particularly non-epithelial malignancies typical of WS. This faithful mouse model of WS establishes that the complex and pleiotropic in vivo sequelae of Wrn deficiency are provoked largely by critical telomere shortening.

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THE EFFECTS OF 6-MONTHS OF CALORIE RESTRICTION ON BIOMARKERS OF AGING IN NON-OBESE HUMANS.

 

Leonie K Heilbronn*, Eric Ravussin and the Pennington CALERIE group.

 

Pennington Biomdeical Research Center, 6400 Perkins Rd, Baton Rouge, LA

 

Caloric restriction (CR) extends life span and retards age-related diseases in rats, mice, flies, worms and yeast. Several biomarkers of aging (fasting glucose, insulin, DHEAS and core temperature) have been identified in human and monkey studies of longevity. Whether prolonged CR improves biomarkers of aging in humans is unknown. Forty-eight healthy, nonsmoking, male (25-50y) and female (25-45y), overweight participants (25 < BMI < 30) were randomized into one of four groups; group 1 = 25% CR, group 2 = 12.5% CR + 12.5% increase in physical activity, group 3 = low calorie diet until 15% weight reduction, group 4 = Control. Subjects were fed by the metabolic kitchen from Weeks 1-12 whilst they were taught a calorie counting system to allow them to prepare their own meals (Weeks 12-22). Subjects returned to infeeding from Weeks 22-24. Fasting glucose, insulin, DHEAS and 24h core temperature were assessed at baseline, weeks 12 and 24. At the time of writing, only 30 subjects had completed the study and so CR groups (Groups 1-3) were combined for analysis. Average weight loss was 10% in the CR groups. Weight was not changed significantly in the controls. Fasting insulin was reduced 24% in CR groups as compared to a 5% increase in the controls (p<0.05). Glucose was reduced 1% in CR groups as compared to a 4% increase in controls (p<0.03). DHEAS and daytime core temperature were not significantly changed after 6 months. However, nighttime temperature (recorded from 10pm to 6am) was reduced by 0.2ºC in CR groups as compared to a 0.3ºC increase in controls (p=0.06). This study suggests that 6-mo. of calorie restriction in non-obese humans was sufficient to observe improvements in 3 out of 4 known biomarkers of aging. Whether these changes are sustained over longer periods of time is unknown.

 

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THE PRESENCE OF AN OPTIMAL DOSE RANEGE MAY EXPLAIN DISCREPANCIES IN THE EFFECTS OF (-)DEPRENYL (D) ON SURVIVALS OF ANIMALS IN PAST REPORTS

 

K.Kitani*1, S.Kanai2, K.Miyasaka2, M.C.Carrillo3 ,GO Ivy4

 

Tokyo Metropolitan Institute of Gerontology

 

THE PRESENCE OF AN OPTIMAL DOSE RANGE MAY EXPLAIN DISCREPANCIES IN THE EFFECTS OF (-)DEPRENYL (D) ON SURVIVALS OF ANIMALS IN PAST REPORTS

 

K.Kitani*1, S.Kanai2, K.Miyasaka2, M.C.Carrillo3 ,GO Ivy4

*1National Institute for Longevity Sciences, 36-3, Gengo, Moriokacho, Obu-shi; 2Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; 3Univ.Rosario,Rosario, Argentina; 4Univ.Toronto, Scarborough,Canada

 

To explain discrepancies in the effect of D on life spans of animals in past reports, we examined the effect of D at a dose of 0.25 mg/kg/inj s.c. 3x a week starting from 18 months of age on life spans of F344Du/Crj rats of both sexes.  Average life spans  (days) were significantly increased in both male (895.2±109.7,mean ±S.D., n=30; 967.8±88.6, n=30, control vs. D treated,+8.1%, P<0.01) and female (924.7±132.2, n=38; 987.1±133.4, n=39,+6.7%, P<0.05) rats that received D injections compared to control animals given saline.  Increases in life expectancy from 24 months of age were 44% in males and 32% in females.  We previously reported that a dose of 0.5mg/kg/inj significantly increased the life span of male F344 rats (1). However, a dose of 1.0 mg/kg/inj somewhat shortened the life span of animals, although not statistically significantly (2). Together,our data indicate that a proper dose range of D can significantly increase the life spans of rats of both sexes, however, a greater dose becomes less effective and may adversely affect the life span of animals.  The presence of this effective dose range of D may explain discrepancies in the effect of D on life spans of animals previously reported.  Furthermore, the fact that the effective dose range observed for the life prolonging effect of D parallels that for increasing activities of SOD and catalase strengthens our contention that these two effects are causally related.  Our new observation that the same dosage of D (0.25mg/kg) increases life spans of both male and female rats further supports our contention, since we previously reported that the optimally effective dose of D to increase antioxidant enzyme activities becomes closer for male and female rats as they get older, while at young ages, the optimal dose is 10 times greater in male rats (3).  Ref. 1) Life Sci. 52:281-288, 1993. 2) Life Sci. 67:577-585, 2000.3)Life Sci. 52:1925-1934,1993

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AGE-ASSOCIATED CELLULAR STRESS IN HSP-40 MOLECULAR CHAPERONE MUTANT MICE

 

S Knoblaugh, J Morton, G Moore-Sanders, A MacAuley, W Ladiges*

 

Department of Comparative  Medicine, University of Washington, Seattle, WA 98195

 

Cellular stress associated with a malfunctioning endoplasmic reticulum (ER) is causally related to the inability of molecular chaperones to maintain structural integrity of proteins. Proteins which are structurally damaged under conditions of oxidative stress or aggregation during the aging process place an increased burden on the processing capacity of the ER. The result is an exaggerated and extended ER stress response, which triggers transcriptional and translational pathways of cell death and decreased protein synthesis. ER dysfunction has been implicated in a number of age-related diseases including Parkinsons, Alzheimers, cardiovascular conditions, and diabetes. The HSP-40 molecular chaperone family member, p58ipk, functions in the later stages of the ER stress response as an off switch to prevent excessive cell death and promote protein synthesis.  Mutant mice deficient in p58ipk have evidence of an exaggerated ER stress response in several cell types including pancreatic beta cells, hepatocytes, and plasma cells. Gene expression, Western immunoblots and immunohistochemistry in cells from p58ipk mutant mice confirm the upregulation of ER stress response genes and an enhanced cell death pattern reflective of an age-dependent onset of diabetes and immune dysfunction, and an average life span of 18 months.  We conclude that ER resident proteins, including HSP-40/p58ipk, are targets for investigating specific aging and age-related disease questions. The p58ipk mutant mouse model will be of interest in intervention studies to determine capabilities of decreased disease and increased life span mediated by a functional ER.

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PHENOTYPIC, FUNCTIONAL AND GENETIC PROFILES OF THE AGING T- LYMPHOCYTES IN PRIMATES AND THEIR ALTERATION BY CALORIC RESTRICTION

 

Janko Nikolich-Zugich, Jessica Warner, Bree Fisher, Dragana Nikolich-Zugich

and Ilhem Messaoudi

 

Vaccine and Gene Therapy Institute and the Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR, USA

  

Numerous T-cell functions are diminished or dysregulated in old age, including T-cell population dysbalance (altered na ve:memory and CD4/CD8 T-cell ratios), diminished T-cell responsiveness to a variety of signals and altered cytokine networks. Yet, despite intense research, numerous gaps in the understanding of T-cell senescence still exist. Moreover, much of our current knowledge on T-cell biology and aging comes from inbred, specific pathogen-free rodents, and it is unclear which observations translate to human immune aging.

 

We begun to develop a primate model of human immune senescence using Rhesus macaques (RM). We evaluated T-cell surface phenotype, in vivo and in vitro turnover, the complexity of T-cell receptor (TCR) repertoire and correlated them to functional studies of isolated cell subsets and gene expression profile analysis. We also evaluated the ability of an experimental manipulation - caloric restriction, the only intervention that consistently and reproducibly increases longevity and quality of life in a variety of animal models - to impact upon these parameters.

 

We showed that sharp differences exist in the phenotypic and functional T-cell aging between the CD8 and CD4 T-cell subsets in: (i) cell cycle programs (as assessed by both in vitro proliferation and in vivo turnover measurement); (ii) CD28 regulation upon cell cycle entry; (iii) accumulation of immediate effector cells amongst the CD28- cells, believed to be close to or at replicative senescence. These results underscore poor reliability of CD28 as a marker for senescence, and further suggest that some of the T-cell aging phenomenology in RM can be ascribed to accentuation over time of the inherent differences in activation programs in CD8 and CD4 T-cells. We also found age-related differences in T-cell turnover, and modest, but consistent, gene expression changes in certain resting T-cell subsets. Caloric restriction was able to modulate most, if not all, of these parameters, resulting in  young-like  characteristics of examined T-cells. We conclude that the results so far are consistent with resource conservation induced by caloric restriction.

 

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cAMP forms a neurochemical correlate of tinnitus

 

A, Shaikh; P, Finlayson

 

Department of Otolaryngology, Wayne State University, Detroit, MI 48201 

 

Cochlear damage that commonly occurs in elderly is either due to ototoxicity of anticancer drugs (cisplatin) or a process of aging. Such hearing loss is often accompanied by tinnitus. It was suggested that increased spontaneous neural activity (SA) may be the underlying mechanism for tinnitus. Neurochemical consequences of cochlear ablation (CA) have also been widely explored. A significant synaptic plasticity has been reported following cochlear insults. Yet a direct relationship of increased SA with plastic neurochemical alterations remains to be recognized. In central auditory neurons, the signals emerging after CA are transduced by range of mechanisms including extracellular signal-regulated kinase (ERK) pathway. One of the functional roles of this pathway is to enhance the inhibition of phosphodisterase E 4 (PDE4), thereby elevating intracellular cAMP concentrations ([cAMP]i). We sought to determine if increasing [cAMP]i affects SA, and if so what possible mechanisms may be involved and the possible physiological/pathological ramifications. We also investigated such elevations in SA could mimic the neural code for the pure-tone. Forskolin (an agent that systematically increases [cAMP]i) specifically increases tone-evoked responses and SA of the SOC neurons in dose-dependent manner. Interestingly, the increased SA following application of 50 µM forskolin mimics the neural code that is normally generated by the tonal stimulus. We also report that effects of forskolin are predominantly due to PKA independent pathway that involves hyperpolarization activated inward conductances. These results provide for the first time, direct evidence that systematic increase in the [cAMP]i, in the auditory brainstem neurons, by application of forskolin elevates the SA, which mimics the neural code for the pure-tone. Here we suggest, increased levels of [cAMP]i that could follow cochlear insults, forms the basis of increased SA and therefore could be the “neurochemical correlate” of tinnitus.

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HIPPOCAMPAL GH AND IGF-1 EXPRESSION IN GH-DEFICIENT MICE

 

Liou Y. Sun*, Khalid Al-Regaiey, Michal M. Masternak, Jian Wang and Andrzej Bartke

 

Geriatrics Research, Department of Medicine and Department of Physiology

Southern Illinois University, Springfield, IL 62794, USA

 

Liou Y. Sun*, Khalid Al-Regaiey, Michal M. Masternak, Jian Wang and Andrzej Bartke

Geriatrics Research, Department of Medicine and Department of Physiology

Southern Illinois University, Springfield, IL 62794, USA

 

Beneficial effects of GH and IGF-1 on the development and function of the central nervous system are well documented. In spite of primary deficiency of GH and secondary IGF-1 deficiency, Ames dwarf mice live considerably longer than normal animals, exhibit apparently normal cognitive functions and maintain them into advanced age.  In an attempt to reconcile these findings, we have examined local expression of GH and IGF-1 in the hippocampus, brain region involved in learning and memory, of these mice.  RNA and protein was extracted from the hippocampus of dwarf and normal mice and assayed for the GH and IGF-1 transcripts and their encoded proteins. With the real-time RT-PCR analyses, hippocampus of Ames dwarf mice was found to express normal levels of GH and IGF-1 messenger RNA indicating ectopic GH expression and the integrity of transcription capability in dwarf mice. The identities of the PCR products were confirmed by sequencing. Hippocampal levels of GH and IGF-1 were evaluated by western blotting using the antibodies specific for the respective proteins. Both GH and IGF-1 protein levels are significantly increased in the hippocampus of Ames dwarf compared with normal mice suggesting a compensatory mechanism of peripheral hormonal deficiency. In contrast, IGF-1 expression in the liver of Ames dwarf mice is profoundly suppressed, consistent with congenital GH deficiency and lack of detectable GH and IGF-1 in peripheral circulation. In addition, Increased phosphorylation of Akt and CREB were also detected in the hippocampus of Ames dwarf mice. Our results suggest that increase in hippocampal GH and IGF-1 protein expression and subsequent activation of PI3K/Akt-CREB signal transduction cascade might contribute to the maintenance of cognitive function and is likely to be responsible for the integrity of neuronal structure, and maintenance of youthful levels of cognitive function in these long-lived mice during aging.

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PASSIVE AMYLOID IMMUNOTHERAPY CLEARS AMYLOID AND TRANSIENTLY ACTIVATES MICROGLIA IN A TRANSGENIC MOUSE MODEL OF AMYLOID DEPOSITION

 

Donna M. Wilcock1, Amyn Rojiani2, Arnon Rosenthal3, Jennifer Alamed1, David Wilson1, Nedda Wilson1, Melissa J. Freeman1, Marcia N. Gordon1, Dave Morgan1.

 

AbstractLocation: 1,2: Alzheimer’s Research Laboratory, 1Department of Pharmacology, 2Departments of Interdisciplinary Oncology and Pathology, University of South Florida, 12901 Bruce B Downs Blvd, Tampa, FL 33612, USA.

3: Rinat Neuroscience Corp. 3155 Porter Drive, Palo Alto, California, 94304, USA.

 

The role of microglia in the removal of amyloid deposits following systemically administered anti-Aß antibodies remains unclear. In the current study we injected Tg2576 APP transgenic mice weekly with anti-Aß antibody for a period of one, two or three months such that all mice were 22 months at the end of the study. In mice immunized for three months we found an improvement in alternation performance in the Y maze. Histologically, we were able to detect mouse IgG bound to congophilic amyloid deposits in those mice treated with anti-Aß antibody but not in those treated with control antibody. We found that Fcgamma receptor expression on microglia was increased following one month of treatment while CD45 was increased following two months of treatment. Associated with these microglial changes was a reduction in both diffuse and compact amyloid deposits following two months of treatment.  Interestingly, the microglia markers were reduced to control levels following three months of treatment while amyloid levels remained reduced.  Serum Aß levels and anti Aß antibody levels were elevated to similar levels at all three survival times in mice given anti-Aß injections rather than control antibody injections. These data show that antibody is able to enter the brain and bind to the amyloid deposits, likely opsonizing the Aß and resulting in Fcgamma receptor mediated phagocytosis. Together with our earlier work, our data argue that all proposed mechanisms of anti-Aß antibody mediated amyloid removal can be simultaneously active.

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Khalid Al-Regaiey, Michal M. Masternak, Michael Bonkowski, Liou Sun, Andrzej Bartke

Southern Illinois University School of Medicine, Springfield, IL 62794

Reduced IGF-1/insulin signaling and caloric restriction (CR) are known to extend life span and delay age related diseases. To address the interaction of these two interventions, we subjected normal (N) and growth hormone receptor knockout (KO) mice to CR starting at weaning for 20 months.  Molecules involved in glucose homeostasis were investigated.  CR resulted in decreased plasma glucose levels in both phenotypes (N-CR and KO-CR).  Circulating IGF-1 was reduced in N-CR and was undetectable in KO animals.  Insulin was also reduced in N-CR to levels comparable to those in KO animals. Corticosterone and adiponectin levels were higher in KO than in N animals while leptin was reduced by CR in both phenotypes. We also analyzed hepatic gene and protein expressions of molecules involved in insulin signaling using real-time PCR and western blotting, respectively. Diet and phenotype did not affect Akt gene and total protein expression while KO mice exhibited reduced Akt activation with no diet effect. Phosphorylation of protein kinase C &#947;/&#950; was not affected by either diet or phenotype. KO mice exhibited increased CREB activation. Gene expression and protein levels of Foxo1 and PGC-1 were increased in KO mice with little diet effects. Genes that are known to be regulated by CREB, Foxo1, and/or PGC-1including pepck, g6pase, igfbp-1, and MnSOD had increased expression in KO mice. Genes encoding proteins that are involved in lipolysis, including hormone-sensitive lipase (HSL) and lipo-protein lipase (LPL) were upregulated in KO mice. We conclude that CR and growth hormone resistance probably affect longevity of mice by different mechanisms and that gluconeogenesis pathway as well as fatty acids mobilization are more active in KO mice. These data also suggest that liver of KO mice is more protected against oxidative stress as indicated by increased Foxo proteins and MnSOD mRNA.

Supported by NIA.
 

44.  AGING RETARDATION BY CALORIE RESTRICTION: ROLE OF REGULATORS OF ENERGY METABOLISM.

 

R. Anderson (1), J.Barger (1), T. Pugh (1), S. Park (2), T. Prolla (2) and R. Weindruch (1).

 

(1)Department of Medicine, University of Wisconsin and GRECC, VA Hospital, Madison WI 53705 USA. (2) Department of Genetics and Medical Genetics, University of Wisconsin, Madison WI 53706 USA.

 

University of Wisconsin Madison, GRECC, VA Hospital, Madison WI 53705, USA.

 

     Caloric restriction (CR) extends lifespan in a broad spectrum of organisms and retards the progression of a wide range of age-associated biological changes; however, the underlying mechanisms are unclear.  In mice, there is an inverse linear relationship between calorie intake and lifespan extension suggesting that factors central to energy metabolism may underlie aging retardation by CR.  One hypothesis is that CR triggers an active response involving a reprogramming of energy metabolism that reduces the rate of aging and the development of several age-related diseases.  Transcription profiling of tissues from control and CR animals points to mitochondrial function as a central feature in this process.  We have used a combination of techniques to identify factors that may be involved in the mechanism of lifespan extension by CR.  We then investigated how these potential effectors of CR are regulated and examined their relationship to elements of known longevity pathways, including members of the FOXO and Sirtuin families.

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45.  REGULATION OF THE RENAL VITAMIN D 1ALPHA-HYDROXYLASE CYTOCHROME P450(CYP27B1) BY DIETARY PHOSPHORUS CHANGES WITH AGE

 

H.J. Armbrecht*, M.A. Boltz

 

Geriatric Center, St. Louis VA Medical Center, St. Louis, MO  63125

 

Young rats adapt to a low phosphorus diet by increasing plasma levels of 1,25-dihydroxyvitamin D (1,25D), the biologically active metabolite of vitamin D, which then increases intestinal phosphate absorption.  Previous studies have shown that the capacity of rats to adapt to low dietary phosphorus declines with age.  The purpose of this study was to determine whether this decreased adaptation was due to decreased expression of the renal 1alpha-hydroxylase (1-OHase), which makes 1,25D.  The 1-OHase consists of ferredoxin reductase, ferredoxin, and a terminal cytochrome P450 – CYP27B1.  Young (2 months) and adult (12 months) F344 rats were placed on a low phosphorus (0.1%) or high phosphorus (1.0%) diet for 2 weeks.  Plasma 1,25D was markedly increased by the low phosphorus diet in young animals but not in adults.  To determine whether this difference was due to decreased 1-OHase expression, mRNA levels of CYP27B1 were measured by ribonuclease protection assay.  In young animals, the low phosphorus diet increased renal CYP27B1 mRNA levels 8-fold compared to the high phosphorus diet, but there was no significant effect in adults.  The effect of low dietary phosphorus on plasma calcium, phosphate, and parathyroid hormone levels was similar in both age groups.  These results suggest that the decreased CYP27B1 expression in the adult in response to dietary phosphorus depravation is not due to changes in the major regulators of 1-OHase activity.    

 

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46.  SENEX: INTEGRATING GERIATRICS AND MOLECULAR GERONTOLOGY

 

S Ball, A Nhalil and V Mah

 

Department of Veterans Affairs, Greater Los Angeles Healthcare, 11301 Wilshire Boulevard, GRECC 11G, Los Angeles CA 90073

 

Senex is a computer application 15 years in development integrating geriatrics and molecular gerontology (from bench to bedside). It includes information (and tools for interpretation of data) that span internal medicine, molecular pathology (biochemistry, molecular biology, pathology), laboratory medicine, radiology, pharmacology, anatomy, and statistics.  Clinical and molecular information is needed from laboratory bench to patient bedside, during contact between physician and patient, educator and student, scientist and technician.  The information needs to be structured so that specific and real-time retrieval is achievable. The information needs to be presented in a manner that facilitates a conceptual understanding of the details presented.  Additionally, an information system should display a certain degree of intelligence, including flexibility in accepting input from the user, the capacity to reason with structured information, and the esthetic display of context-specific information. Implementation of such an information system should allow use to become better physicians, scientists, educators, students and/or informed citizens. Senex functions independently as a stand-alone application (i.e. not dependent upon internet access). Thus it is fast, reliable, and mobile. Senex also provides direct links into the world-wide-web, to molecular and clinical databases and to the original literature when access to the internet is available.  Senex is a large application with: 1514 organisms, 2417 anatomic structures, 222 cells, 131 cellular compartments, 13,138 molecules, 9607 proteins, 628 genes, 795 motifs,  67 molecular pathways, 1580 diseases,  540 clinical laboratory tests, 18,680 database links (6140 Swiss Prot,  6280 OMIM, 870 Prosite, 2000 PIR, 3390 Locus Link).  Senex allows a user to add proprietary information on top of the core Senex knowledge base, thus customizing the application for a user’s own information needs.  Senex runs on Macintosh and Windows computers. The Macintosh version has a microarray data analysis and data mining module. Reference: www.senex-medical-software.com

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47.  THE AGE-RELATED ACCUMULATION OF DOLICHOL IN TISSUES SATISFIES ALL CRITERIA TO BE QUALIFIED A BIOMARKER OF AGING

 

E. Bergamini, G. Cavallini, A. Donati, Z. Gori, A. Manfrini, I. Parentini

 

Centro di Ricerca di Biologia e Patologia dell’Invecchiamento dell’Universita’ di Pisa, Dipartimento di Patologia sperimentale, Via Roma 55, 56126 PISA (Italy); Tel 0039-050-2218584  Fax 0039-050-2218581   e-mail ebergami@med.unipi.it

 

It was stated that the identification of specific biomarkers of aging would be an important milestone in gerontological research, and criteria for defining biomarkers were suggested (Mooradian, 1990). In this communication evidence on male and female Sprague Dawley and Lewis rats fed ad-libitum or on regimens of food restriction and on human tissues are reviewed, showing that accumulation of dolichol in tissues of older animals meet these criteria: levels of dolichol exhibit a quantitative correlation with age in all tissues; the biological parameter is not altered by several age-dependent diseases in the same direction as that of aging; data with transplanted organs show that the age-related accumulation is not secondary to metabolic changes of aging; factors that modulate the aging rate like caloric restriction and physical exercise appropriately alter the accumulation of dolichol; this putative biomarker of aging appears to be applicable to different tissues across mammalian species including human; biomarker is applicable to the hypothalamic digoxin-mediated model for trisomy 21 (Kurup & Kurup, 2003). Reliable changes in tissue dolichol levels are seen in relatively short intervals of time compared to life-time, and levels can be tested on a small amount of tissue without causing death of the animal, which are desirable features of a biomarker of aging. The age-associated alteration in dolichol levels is likely to reflect an age-dependent derangement of free radical metabolism in membranes. Useful applications are shown, which include a study of the effect of the donor-recipient age-mismatch on the biological age of the graft tissues, and the detection of gender-related differences in aging.

Research was supported in part by a grant of MIUR (Ministero Istruzione, Universita’ e Ricerca)

Mooradian, A.D. (1990) J Gerontol 45(8) B183-186.

Kurup R.K., Kurup, P.A. (2004) Pediatr Pathol Mol Med 22(5) 411-422.

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R. Bowen, C. Atwood*

Voyager Pharmaceutical Corporation, Raleigh, North Carolina, USA
* Department of Pathology, Case Western Reserve University, Cleveland, Ohio and School of Medicine, Section of Geriatrics and Gerontology, Department of Medicine, University of Wisconsin, Madison, Wisconsin, USA

We put forth a new theory based on a novel definition of aging - any change in an organism over time. This definition includes not only the changes associated with the loss of function (i.e. senescence), but also the changes associated with the gain of function (growth and development). Using this definition, the rate of aging would be synonymous with the rate of change. The rate of change/aging is most rapid during the fetal period when organisms develop from a single cell at conception to a multicellular organism at birth. Therefore, “fetal aging” would be determined by factors regulating the rate of mitogenesis, differentiation, and cell death. We suggest these factors also are responsible for regulating aging throughout life. Thus, whatever controls mitogenesis, differentiation and cell death must also control aging. Since life-extending modalities consistently affect reproduction, and reproductive hormones are known to regulate mitogenesis and differentiation, we propose that aging is primarily regulated by the hormones that control reproduction (hence, the Reproductive-Cell Cycle Theory of Aging). In mammals, reproduction is controlled by the hypothalamic-pituitary-gonadal (HPG) axis hormones. Longevity inducing interventions, including caloric restriction, decrease fertility by suppressing HPG axis hormones and HPG hormones are known to affect signaling through the well-documented longevity regulating GH/IGF-1/PI3K/Akt/Forkhead pathway. This is exemplified by genetic alterations in C. elegans where homologues of the HPG axis pathways, as well as the daf-2 and daf-9 pathways, all converge on daf-16, the homologue of human Forkhead that functions in the regulation of cell cycle events. In summary, we propose that the hormones that regulate reproduction act in an antagonistic pleiotrophic manner to control aging via cell cycle signaling; promoting growth and development early in life in order to achieve reproduction, but later in life, in a futile attempt to maintain reproduction, become dysregulated

P. Wills¹, H. Brown-Borg²,and M.A. Chacon¹(P)   

¹Irazu Biodiscovery, LLC, Baltimore, MD 21224; ²Department of Pharmacology, Physiology and Therapeutics, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58203.

Seventy years of scientific investigation has demonstrated that caloric restriction (CR) without malnutrition is the only experimental intervention that can extend life span, and decrease the incidence and delay the onset of pathologies associated with aging.  Nevertheless, the precise mechanism by which caloric restriction induces its life-extending effect remains poorly understood.  One consequence of caloric restriction observed in experimental rats, which has not been previously investigated, is an elevation in serum levels of free fatty acids (FFA). In our studies, we found that calorie restricted rats, mice, and dwarf mice all have elevated free fatty acids.  In addition to their well-characterized role as an energy substrate, recent scientific studies have demonstrated that these molecules possess signaling properties.  Therefore, we tested the hypothesis that free fatty acids may induce (mimic) some of the protective effects seen in calorie restricted animals. Using an in vitro, cell-based ROS assay, we demonstrated that supplementation of serum from ad lib animals with free fatty acids to levels comparable to those found in the serum of caloric restricted animals conferred protection from the cytotoxic effects of H2O2.  We contend that the up-regulation of FFA and their CoA derivatives, through their effects on ion channels, oxygen utilization, and gene expression, offer a plausible mechanism for the protective phenotype associated with CR.

W. Chai, J.W. Shay, and W.E. Wright

Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9039

Replicative senescence involves progressive telomere shortening but the exact molecular mechanisms triggering the growth arrest are poorly understood. Recently, using a telomere-oligonucleotide ligation assay (T-OLA), it was reported1 that the 3’ G-rich overhang was eroded at senescence, leading to the hypothesis that loss of the 3’ G-rich overhang is the molecular signal that triggers senescence. To better understand the molecular mechanisms triggering replicative senescence, we have developed a quantitative assay to measure the length of the G-rich 3’ telomeric overhangs from cultured cells. By using this assay and the conventional non-denaturing hybridization assay for measuring G-rich overhangs, we show that normal human fibroblasts maintain their 3’ G-rich overhangs at senescence. Furthermore, cells maintain their G-rich overhangs when they bypass senescence after the inactivation of p53 and retinoblastoma proteins by human papillomavirus type 16 (HPV) oncoproteins E6 and E7 (E6/E7). Interestingly, cells expressing just HPV E7 exhibit longer overhangs when they growth arrest. We also found that expression of simian virus 40 large T-antigen induces some telomeric overhang shortening. These results demonstrate that a significant reduction in overhang length is not the molecular signal that triggers senescence.

Reference:
1. Stewart, S.A., I. Ben-Porath, V.J. Carey, B.F. O'Connor, W.C. Hahn, and R.A. Weinberg. 2003. Erosion of the telomeric single-strand overhang at replicative senescence. Nat Genet 33: 492-6.

49.  APOPTOTIC DNA FRAGMENTATION IN THE BRAINS OF YOUNG AND AGED eNOS-, iNOS- and nNOS-KNOCKOUT MICE: MEASUREMENT BY NEW ULTRASENSITIVE CE-LIF TECHNIQUE

 

VW Chow*, RR Fiscus, SB Chew

 

The Department of Physiology, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong.

 

Previously, we showed that elevation of cGMP levels in PC12 cells exposed to atrial and brain natriuretic peptides (ANP & BNP) inhibits onset of apoptosis and promotes survival [1]. We further showed that 24h-preincubation of NG108-15 cells with ANP protects against pro-apoptotic effects of nitric oxide (NO) donor (high concentrations) [2]. At lower physiological concentrations, NO, via cGMP elevations, inhibits apoptosis and improves neural survival [3]. Depending on the source of NO, whether from nNOS, iNOS or eNOS, and depending on age, NO may promote or protect against apoptosis. The present study determines if aging-induced-apoptosis in brain is affected by genetic deletion of nNOS, iNOS and eNOS using nNOS-, iNOS-, eNOS-knockout mice. Young (2-4-month-old) and aged (12-22-month-old) nNOS-/-, iNOS-/-, eNOS-/- knockout and control mice (C57BL/6J & B6129SF2/J) were used to determine levels of apoptosis in half-brain and different brain regions by a new ultrasensitive technique using capillary electrophoresis with laser-induced fluorescent detector (CE-LIF) to accurately quantify apoptotic-DNA fragmentation. Using half-brain, amounts of apoptotic-DNA-fragments were 11-fold higher in aged than in young B6129SF2/J (nNOS-/-control) mice (p<0.0001). Apoptotic-DNA-fragments in young nNOS-/- were 23-fold higher than in young B6129SF2/J (p<0.0001). Apoptotic levels in aged B6129SF2/J were 2.5-fold higher than in aged nNOS-/- (p<0.001). These data suggest that nNOS contributes to aging-dependent-increase in brain apoptosis of B6129SF2/J, but provide neuroprotective effect in young adults. Neuroprotective effects of nNOS were especially large in young hippocampus. Apoptotic-DNA-fragments in young iNOS-/- were 30-fold higher than in young C57 controls (p<0.001). Aged iNOS-/- showed 6.5-fold lower level of apoptosis compared to young adult (p<0.001). iNOS had anti-apoptotic effect in young C57 but not aged C57. eNOS showed significant neuroprotective effect in medulla oblongata of both young and aged C57. All NOS isoforms provided neuroprotective function in young adult brain, but this neuroprotection appeared change during aging.

1.    Fiscus RR, Tu AW, Chew SB. Neuroreport 2001;12:185-189.

2.    Cheng Chew SB, Leung PY, Fiscus RR. Histochem Cell Biol. 2003;120:163-171.

3.    Fiscus RR. Neurosignals 2002;11:175-190.

Support: Competitive Earmarked Grant from RGC of Hong Kong to RRF. (#CUHK 4169/02M)

 

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50.  GENE EXPRESSION CHANGES IN NEURODEGENERATIVE MICE AND CULTURED CELLS
 

B.A. Citron*^1,2, A. Surguchov^3,4, I. Surgucheva^3,4, and B. W. Festoff<sup>3,4,5

1</sup>Lab of Molecular Biology, VA Medical Center, Bay Pines, FL, ²Dept. of Biochemistry & Molecular Biology, USF College of Medicine, Tampa, FL, ³Neurobiology Research Lab, VA Medical Center, Kansas City, MO, Depts. of ⁴Neurology and ⁵Pharmacology, University of Kansas Medical School, Kansas City, KS

A variety of genes have been implicated in late onset neurodegenerative disorders. Several reports linked synucleins to motor neuron diseases including ALS. Present in aggregates, α-synuclein is important for the long term survival of neurons. The role of γ-synuclein is less clear but recent studies indicate that it regulates other neurodegenerative factors. Synucleins modulate certain proteases, however their own proteolytic degradation may be essential in the process of formation of inclusion bodies. We have been studying cultured neurons and wobbler mice, which undergo an age dependent loss of motor neurons in the cervical region of the spinal cord and model sporadic ALS. We have measured activation of transcription factors (with electrophoretic mobility shift assays) in wobbler spinal cords and cultured neurons exposed to insults. Specifically, we found that activated transcription factors SP1 and NF-kB, but not AP1 are increased in wobbler mice and in motor neurons exposed to apoptosis inducing insults (thrombin or mechanical injury). We have also examined mRNAs levels with Affymetrix gene arrays. Genes responsible for CNS maintenance (e.g. myelin-associated oligodendrocytic basic protein) are downregulated in the wobbler spinal cord. Ubiquitin C-terminal hydrolase is also expressed at lower levels in the wobbler cord and this could affect the ubiquitination of proteins that aggregate in the degenerating CNS. α-synuclein is upregulated in the wobbler compared to the wild-type littermate spinal cord. Directed elevation of α-synuclein, γ-synuclein or both, in transfected neurons resulted in a marked dysregulation of gene expression in several functional categories. For example, of the genes that are significantly expressed in these cells, more genes involved in cell adhesion are upregulated compared to genes in several other categories. The over and underrepresented functional classes in gene groups upregulated and downregulated by α- and/or γ-synuclein introduction have been determined to obtain a picture of synuclein regulatory influences.

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51.  The SIRT1 NAD+-dependent deacetylase regulates Bax-mediated apoptosis

Haim Y. Cohen*, Christine Miller, Kevin Bitterman and David A. Sinclair.

Department of Pathology, Harvard Medical School. Boston MA USA 02115.

Decreased apoptosis and increased cell survival is observed in organisms whose lifespan has been extended by calorie restriction or genetic manipulation. A crucial but poorly understood step in the stress-induced apoptotic pathway is activation of the pro-apoptotic factor Bax.  In yeast, lifespan extension by calorie restriction requires Sir2, a stress-responsive, NAD+-dependent deacetylase. Here we show that the human Sir2 homolog, SIRT1, controls cell survival in response to stress by inhibiting Bax-mediated apoptosis. SIRT1 deacetylates specific lysines in the Ku70 C-terminus, which increases the Ku70-Bax interaction and prevents Bax from relocalizing to mitochondria. We show that this pathway can be manipulated using small molecules and discuss the implications of this finding to the general decline in physiological function with age.

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52.  Modulation of IGF members expression in Brca1-knockout Mice

X Coumoul*, L Cao and C Deng

National Institute of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 9000 Rockville Pike, Bethesda MD 20892, USA

Breast cancer affects 200.000 women in the United States which results in 40.000 deaths every year. Heterozygous carriers of Breast Cancer Associated Protein 1 (Brca1) mutations are at great risk to develop breast or ovarian cancers through their lifetime. Our mice models of Brca1 deletions have shown that indeed lack of this gene plays a role in mammary tumorigenesis but also interestingly in aging process. Here, we shown that deletion of Brca1 modulates expression of several Insulin-like Growth Factor (IGF) members in mice liver. IGF members expression has previously been linked to tumorigenesis and aging process. Interestinlgy, IGF1 serum levels but also mRNA liver levels are increased as shown by microarrays and RT-PCR analysis. Other members like IGF1-Receptor, Insulin Receptor Substrate 1 or IGF-Binding Protein 1 and 2 also appears to be modulated. We also use a human cell culture system UBR60, allowing modification of Brca1 expression, to shown that those results are reproducible with a different model. Those results allow us to hypothesize that modulation of IGF members expression by Brca1 could be one of the mechanism that links lack of this protein to aging and tumorigenesis process.

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53.  THE UNFORTUNATE INFLUENCE OF THE WEATHER ON THE RATE OF AGING

 

A. de Grey

 

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK

 

Much research interest, and recently even commercial interest, has been predicated on the assumption that reasonably closely-related species -- humans and mice, for example -- should in principle respond to aging-retarding interventions with an increase in life expectancy roughly proportional to their control lifespan (life expectancy without the intervention). Here it is argued that the best-studied life-extending manipulations of mice are examples of a category that is highly unlikely to follow this rule, and more likely to exhibit only a similar absolute increase in lifespan from one species to the next, independent of the species' control lifespan. That category -- deprivation of dietary calories or of the organism's ability to metabolise or sense them -- is widely recognised to extend lifespan as an evolutionary adaptation to transient starvation in the wild, a situation which alters the organism's optimal partitioning of resources between maintenance and reproduction. What has been generally overlooked is that the extent of the evolutionary pressure to maintain adaptability to a given duration of starvation varies with the frequency of that duration, something which is -- certainly for terrestrial animals, and less directly for others -- determined principally by the weather. The pattern of starvation that the weather imposes is suggested here to be of a sort that will tend to cause all terrestrial animals, even those as far apart phylogenetically as nematodes and mice, to possess the ability to live a similar maximum absolute (rather than proportional) amount longer when food is short than when it is plentiful. This generalisation seems to be strikingly in line with available data, leading to the biomedically and commercially sobering conclusion that human interventions which manipulate caloric intake or its sensing are unlikely ever to confer more than two or three years' increase in lifespan at the most.

 

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54.  CRP LEVELS ARE STRONGLY ASSOCIATED WITH AGING 

 

SY. Ding, X.T. Tigno, and B.C. Hansen

 

Obesity, Diabetes, and Aging Animal Resource Center, Department of Physiology, School of Medicine, University of Maryland at Baltimore, 10 S. Pine St, Baltimore MD 21201

 

Epidemiological studies have consistently shown that the plasma level of C-Reactive Protein (CRP), a marker of inflammation, is a strong, independent predictor of future cardiovascular events, both in patients with a history of coronary heart disease (CHD) and in apparently healthy subjects. Inflammatory markers have also lately been implicated in aging and in type 2 diabetes. The plasma concentrations of CRP were determined by ELISA in a cohort of 33 diabetic and non-diabetic rhesus monkeys: 13 young normal (Y) with a mean age of 9.1±2.9 yr, 11 old normal (O, 25.9±1.5 yr), 8 old type 2 diabetic monkeys (DM, 26.2±2.3 yr) and 4 calorie-restricted (for more than 20 years) monkeys (CR, 30.1± 2.8 yr). Results show that CRP levels were positively correlated to age (r=0.46, p<0.01) and body weight (r=0.33, p<0.02). The mean levels of CRP were found to be highest among the O (246±16µg/L), a value significantly greater than that among the Y (184±14µg/L). However, mean CRP values did not differ significantly between O, DM (221.4±18µg/L) and CR (214±26µg/L), all of whom were elderly. The data suggests that the proportion of the variation in CRP that can be accounted for by variation in age is 0.21, and that age is a stronger predictor of CRP than weight (R2= 0.18) or metabolic status, as reflected by circulating levels of CRP in the non-human primate.

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56.  DNA EPISOMES AND REPLICATIVE SENESCENCE IN S. CEREVISIAE

 

Alaric A. Falcón*, Diego M. Ayo, Natalie Rios, and John P. Aris

 

Department of Anatomy and Cell Biology, Health Science Center, University of Florida, Gainesville, FL  32610-0235

  

We have shown that plasmids containing an autonomously replicating sequence (ARS; yeast DNA replication origin) reduce yeast replicative life span, most likely due to their accumulation during replicative aging (Plasmid accumulation reduces life span in Saccharomyces cerevisiae(Falcón and Aris, 2003).  This raises the question:  how is plasmid accumulation mechanistically linked to replicative senescence in yeast?  One possibility is that accumulated DNA exerts an effect on other cellular DNA, perhaps by influencing DNA replication and/or inheritance.  To explore this, we have studied “interactions” between different types of episomes in yeast:  recombinant plasmids, extrachromosomal rDNA circle (ERCs), and the naturally-occurring 2 micron plasmid.  A short-lived sir2∆ strain that contains elevated ERC levels transmits recombinant ARS1-plasmids to daughter cells more often than a SIR2 control strain.  However, an ARS1-CEN4-plasmid is transmitted to daughter cells equally well in sir2∆ and SIR2 strains.  Levels of ARS1-plasmids are reduced in old sir2∆ cells, compared to old SIR2 cells.  Unexpectedly, an ARS1-plasmid, but not an ARS1-CEN4-plasmid, significantly increased the frequency of loss of the 2 micron plasmid (i.e., curing of a cir+ strain to cir0).  Consistent with this, levels of the 2 micron plasmid are reduced in old cells compared to young cells.  Thus, accumulation of different types of nuclear episomal DNAs can have significant and unexpected effects on other nuclear DNAs.  These findings support a relationship between episomal DNA accumulation, effects on cellular DNA metabolism, and cell senescence in S. cerevisiae. 

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57.  INHIBITOR OF APOPTOSIS PROTEIN-2 (IAP-2) EXPRESSION LEVELS ARE DOWN-REGULATED BY CYCLIC GMP DEPLETION AND UP-REGULATED BY CYCLIC GMP ELEVATIONS INDUCED BY NATRIURETIC PEPTIDES OR  ANALOGS OF CYCLIC GMP IN NG108-15 CELLS:  MOLECLULAR MECHANISM OF CYCLIC GMP/PROTEIN KINASE G (PKG)-MEDIATED NEUROPROTECTION

 

R.R. Fiscus* and J.P. Yuen

 

Dept. Physiology (Fac. Medicine), Epithelial Cell Biology Res. Ctr. and Ctr. for Gerontology & Geriatrics, The Chinese University of Hong Kong, Shatin, NT, Hong Kong

 

Previously, we showed that cGMP elevations protect hippocampal neurons against glutamate toxicity [1], inhibit apoptosis and prolong cell survival in stressed PC12 cells [2] and protect NG108-15 cells against the pro-apoptotic effects of nitric oxide (NO, used at high/neurotoxic concentrations mimicking pathogenesis of Alzheimer’s and Parkinson’s disease) [3].  Activation of cGMP/PKG signaling pathway in neurons may represent an important physiological mechanism protecting against neurotoxicity and neurodegeneration (reviewed [4]).  Even basal levels of cGMP appear sufficient (and necessary) to prevent spontaneous development of apoptosis in some neural cells (e.g. NG108-15 cells), because depletion of cGMP by ODQ triggers apoptosis [4].  Recently, cAMP was shown to inhibit apoptosis in intestinal epithelial cells via protein kinase A (PKA)-mediated phosphorylation of CREB and increased IAP-2 expression [5].  Because PKG phosphorylates CREB at the same site as PKA [4], we hypothesized that cGMP/PKG-mediated neuroprotection may involve IAP-2 expression.  The present study found that ODQ (10-100 micromolar, 24 h), which lowered basal cGMP levels, greatly reduced protein content of IAP-2 (Western blot) in a concentration-dependent manner in NG108-15 cells.  Elevation of cGMP levels with either atrial or brain natriuretic peptides (ANP or BNP, 10-100 nM) or with 8-pCPT-cGMP (1-100 micromolar, a cell-permeable direct activator of PKG), all of which by-pass the ODQ block, completely reversed the ODQ-induced down-regulation of IAP-2.  Indeed, ANP and BNP up-regulated IAP-2 levels above normal control.  The results suggest that up-regulation of IAP-2 expression in neural cells may represent one of the molecular mechanisms that mediates cGMP/PKG-induced protection against apoptosis.  1. Barger et al (1995) J Neurochem 64:2087-2096,  2. Fiscus et al (2001) NeuroReport 12:185-189,  3. Chew et al (2003), Histochem Cell Biol 120:163-171,  4. Fiscus (2002) NeuroSignals 11:175-190,  5. Nishihara et al (2003) PNAS 100:8921-8926.  (Support: Grant # CUHK4169/02M from Research Grants Council of Hong Kong to RRF)

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58.  nINHIBITION OF CASPASE 1 IMPROVES CONTEXTUAL FEAR CONDITIONING IN AGED RATS

C. Gemma (P)*, M. Fister, C. Hudson, P.C. Bickford

James A Haley VAH, Tampa Florida and Center for Aging and Brain Repair, USF, 12901 Bruce B. Downs BLVD, Tampa, FL 33612

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59.  EXPRESSION OF HUMAN TELOMERASE (hTERT) DOES NOT PREVENT STRESS-INDUCED SENESCENCE IN NORMAL HUMAN FIBROBLASTS BUT PROTECTS THE CELLS FROM STRESS-INDUCED APOPTOSIS AND NECROSIS

Vera Gorbunova¹ (P), Andrei Seluanov¹, and Olivia M. Pereira-Smith²

¹Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030

²Department of Cellular and Structural Biology, Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX 78229-3900

Cells subjected to sub-lethal doses of stress such as irradiation or oxidative damage enter a state that closely resembles replicative senescence.  What triggers stress-induced premature senescence (SIPS) and how similar this mechanism is to replicative senescence are not well understood.  It has been suggested that stress-induced senescence is caused by rapid telomere shortening resulting from DNA damage.  In order to test this hypothesis directly, we examined whether overexpression of the catalytic subunit of human telomerase (hTERT) can protect cells from SIPS.  We therefore analyzed the response of four different lines of normal human fibroblasts with and without hTERT to stress induced by UV,  -irradiation, and H2O2.  SIPS was induced with the same efficiency in normal and hTERT-immortalized cells.  This suggests that SIPS is not triggered by telomere shortening and that nonspecific DNA damage serves as a signal for induction of SIPS.  Although telomerase did not protect cells from SIPS, fibroblasts expressing hTERT were more resistant to stress-induced apoptosis and necrosis.  We hypothesize that healing of DNA breaks by telomerase inhibits the induction of cell death, but because healing does not provide legitimate DNA repair, it does not protect cells from SIPS.

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60.  ROCKFISH: RETARDING AGING WITH "NEGLIGIBLE SENESCENCE"

 

J. Guerin

 

Portland, OR

 

Field observations have suggested for quite some time that certain fish, turtles and invertebrates have extremely long maximum lifespan potential.  Age validation techniques have since confirmed these observations.  Negligible senescence is defined in part as no observable age-related increase in mortality rate or decrease in reproduction rate after maturity, and no observable age-related decline in physiological capacity or disease resistance.  Recent data compiled on rockfish (genus Sebastes) have documented ages exceeding 200 years.  However, many rockfish species are reported under 30 years maximum observed lifespan, raising the intriguing possibility of intra-species lifespan comparison.  The Centenarian Species and Rockfish Project has 14 total pilot studies.  The three studies reported in this poster are: “Rockfish liver microarrays using existing 16,000+ zebrafish gene chips”, a collaboration between Glenn S. Gerhard, Dartmouth Medical Center, and Renee Malek, TIGR (The Institute for Genomic Research); “Heat Shock Protein comparison between younger and older rockfish”, Marcelle Morrison-Bogorad, Associate Director, Neuroscience and Neuropsychology of Aging Program, NIA; and “Electron transport abnormalities and mitochondrial DNA mutations in rockfish heart tissue”, Judd Aiken, University of Wisconsin-Madison.

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61.  ANALYSIS OF LONG LIVED C. ELEGANS DAF-2 MUTANTS USING SERIAL ANALYSIS OF GENE EXPRESSION

J Halaschek-Wiener*, S McKay, S Jones, M Marra, D Riddle, A Brooks-Wilson

Genome Sciences Centre, BC Cancer Agency, 600 West 10th Avenue, Vancouver, BC V5Z 4E6, Canada

Serial Analysis of Gene Expression (SAGE) was used to identify longevity-associated genes in a long-lived daf-2 C. elegans mutant. The daf-2 gene encodes an insulin/IGF-1 receptor-like protein and mutations therein lead to a 100% increase in mean life span. SAGE is a method to efficiently count large numbers of mRNA transcripts by sequencing short tags. We prepared adult C. elegans SAGE libraries of days 1, 6 and 10 for daf-2 and days 1 and 6 for control worms. Analyses of gene expression profiles within daf-2 libraries and between daf-2 versus control SAGE libraries (day 6 vs. 6 and day 10 vs. 6) identified not only single genes but also whole gene families that were differentially regulated. Furthermore, daf-2 mutants at day 6 show a strong hypometabolic phenotype when compared to equally aged control worms that diminishes at advanced age. Identified gene families regulate important metabolic processes including stress response, lipid-, DNA/RNA-, protein, and energy metabolisms as well as intracellular signaling and cell structure. Identical expression patterns of various members of several gene families emphasize the importance of these types of genes in longevity-related processes. Our results suggest that long-lived daf-2 mutants are severely hypometabolic in mid-life and we present evidence that specific gene families are involved in these metabolic changes.

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62.  SERUM IGF-I AND LIFESPAN IN CROSSES BETWEEN WILD AND LABORATORY STOCKS OF MICE

J.M. Harper (P)¹, S.N. Austad¹, R.A. Miller¹, R.C. Dysko²

¹Department of Pathology and Geriatrics Center, University of Michigan School of Medicine, ²Unit for Laboratory Animal Medicine, University of Michigan

 

Using F2 hybrid stocks constructed as a cross between two stocks of wild-derived mice (Id and Ma) and the C57BL/6J inbred strain, we have assessed the ability of a suite of life-history and physiological traits to act as predictors of lifespan, as a prelude to mapping genes that may regulate lifespan in mice.  The Id parental strain, the Ma parental strain, and the (Id x B6)F2 hybrid stock are all significantly longer-lived than a laboratory-derived control stock (DC), generated as the offspring of (BALB/cJ x C57BL/6J)F1 females and (C3H/HeJ x DBA/2J)F1 males (log-rank test p £ 0.02). The lifespan of the (Ma x B6)F2 hybrid stock is indistinguishable from the control (log-rank, p = 0.84).  Because serum IGF-I levels measured in 6-month old mice are lower in both the Id and Ma stocks than in the DC control (p < 0.001), we tested to see if IGF-I levels could predict life expectancy in mice of the two segregating F2 stocks.  We found in each case that low IGF-I levels at 6 months of age were associated with longer life span (r2 £ 0.19, p = 0.02).  IGF-I levels did not predict life span in either of the wild-derived parental stocks Ma and Id, nor in the laboratory control DC mice (p > 0.2 for all).  These data suggest that endogenous levels of early life IGF-I may play a key role in the regulation of lifespan, and that there are polymorphisms between at least the Id and C57BL/6J stocks of mice that influence lifespan.  The QTL mapping of the loci responsible for these effects will provide valuable insight into the hormonal and genetic control of mammalian aging.

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63.  ANALYSIS OF THE INFLUENCE OF MITOCHONDRIAL DNA POINT MUTATIONS ON LONGEVITY IN SACCHAROMYCES CEREVISIAE

 

P. Kirchman

 

Harriet L. Wilkes Honors College, Florida Atlantic University, 5353 Parkside Dr., Jupiter, FL 33458

 

Evidence from several species points to mitochondrial DNA (mtDNA) as one determinant of longevity.  Interpretation of these studies is hampered by nuclear DNA heterogeneity of the test subjects and, in some cases, by the variable environments in which subjects live(d).  To eliminate variability in both the nuclear genome and the environment, the budding yeast, Saccharomyces cerevisiae, was used to examine the influence of mtDNA variation on longevity.    Mitochondrial DNA’s containing point mutations in the COX1, COX3, or CYTB gene were transferred to a yeast strain that lacks mtDNA (rho0).  The created strains vary only in the point mutations on the mtDNA.  These strains were analyzed for variation in longevity relative to a control strain containing the non-mutated mtDNA.  No statistically significant variation in longevity has been found between any of the strains tested.  To influence longevity, variation in mitochondrial DNA may need to be more extensive than the single nucleotide mutations tested.  Analysis of additional mitochondrial mutant strains is ongoing and additional results will be reported.

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64.  MICROARRAY ANALYSIS OF GENE EXPRESSION CHANGES IN INTERLEUKIN-4-STIMULATED HUMAN VASCULAR ENDOTHELIAL CELLS

 

YW Lee*, M Toborek

 

Department of Surgery/Division of Neurosurgery, University of Kentucky College of Medicine, Lexington, Kentucky 40536

 

Oxidative stress-mediated inflammatory reactions within vascular endothelium have been implicated in the development of age-related human diseases including atherosclerosis. We have reported that interleukin-4 (IL-4) can induce the pro-oxidative and pro-inflammatory pathways in human vascular endothelial cells. The cellular and molecular regulatory mechanisms underlying this process, however, are not fully understood. In the present study, we performed GeneChip microarray analysis to investigate global gene expression patterns in human vascular endothelial cells after treatment with IL-4 using the Affymetrix GeneChip® Human Genome U133A Arrays, which contain more than 22,000 human genes. Our results showed that mRNA levels of a total of 106 genes were significantly up-regulated and 41 genes significantly down-regulated with more than a twofold change. Majority of these genes are critically involved in the regulation of inflammatory responses, apoptosis, signal transduction, transcription factors, metabolism; functions of the remaining genes are unknown. The changes in gene expression of selected genes related to inflammatory reactions such as VCAM-1, E-selectin, MCP-1 and IL-6 were verified by quantitative real-time RT-PCR and ELISA, respectively. IL-4 treatment also significantly increased the adherence of inflammatory cells to endothelial cell monolayers in a dose-dependent manner. These results may help determine the molecular mechanisms of action of IL-4 in human vascular endothelium. In addition, a better understanding of IL-4-induced vascular injury at the level of gene expression could lead to the identification of new therapeutic strategies for atherosclerosis. (This work was supported by the American Heart Association, Ohio Valley Affiliate and University of Kentucky Microarray Facility  Program)

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65.  GENDER DIFFERENCES IN NEURONAL CELL DEATH AFTER OGD INJURY AND NMDA-MEDIATED EXCITOTOXICITY IN ORGANOTYPIC HIPPOCAMPAL CULTURES

 

Hong Li*, MD, Katrin Andreasson, MD, Louise McCullough, MD, PhD

 

Department of Neurology, Johns Hopkins University, Baltimore, Maryland, 21287

 

Background and Objective: Increasing evidence has demonstrated striking sex differences in outcomes after acute neurological injury. Females are less vulnerable to acute insults associated with experimental cerebral ischemia. It is believed that the greater neuroprotection seen in females is due to circulating estrogens. However, this endogenous female neuroprotection may not be due solely to hormonal influences. The objective of this study was to determine if there are gender differences in neuronal cell death in organotypic hippocampal cultures after oxygen glucose deprivation (OGD) injury and M-Methyl-d-Aspartic Acid (NMDA)-mediated excitotoxicity. Methods: Organotypic hippocampal slice cultures were prepared from individual postnatal day 8 Sprague–Dawley rat pups. Gender genotyping was accomplished by PCR analysis from pup tail DNA. The cultures were exposed to 10 mM NMDA for 1 h or subjected to OGD for 45 min, in presence or absence of 30 mM 7NI (7 Nitroindazole; a selective nNOS inhibitor), or 10nm 17b-estradiol (E2) for 7 days prior to OGD. Neuronal death was quantified with propidium iodide (PI). Data were analyzed by one-way ANOVA followed by Newman–Keuls post hoc test. Results: Baseline neuronal cell death in control, 7NI, and E2 treated groups was equivalent in males and females. Cell death markedly increased after stimulation with NMDA or OGD (P < 0.001) in both groups, but there was a significant increase in cell death in male cultures (P < 0.001 ~ 0.05) after both OGD and NMDA treatment. Treatment with7NI and E2 reduced neuronal damage induced by NMDA and OGD (P < 0.001 ~ 0.05). Interestingly, this effect was more notable in males than in females after OGD injury (P <0.01). Conclusions: There is a consistent gender difference after OGD and NMDA toxicity, demonstrating neuroprotection in female cells. Dimorphisms in cell survival may underlie enhanced neuronal survival (or decreased apoptosis) in female brain.

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66.  ROLE OF MITOCHONDRIAL CASPASE-2 ACTIVITY IN OXIDATIVE STRESS-INDUCED APOPTOSIS

 

Marisa Lopez-Cruzan, Victoria Centonze, and Brian Herman

 

Cellular and Structural Biology Department, University of Texas Health Science Center at San San Antonio. 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900

 

The role of mitochondrial caspase-2 in oxidative stress-induced apoptosis was examined by first determining if mitochondria contain oxidative stress-inducible capase-2 activity and second, what the effect of loss of mitochondrial caspase-2 would be on mitochondrial-specific oxidative stress-induced apoptosis. Oxidative stress-induced mitochondrial-specific caspase-2 activation and apoptosis were assayed in murine NIH-3T3 fibroblasts or fibroblasts obtained from caspase-2 knockout mice using Florescence Resonance Energy Transfer (FRET)/optical microscopy (mitochondrial caspase-2 activity) and uptake of propidium iodide (apoptosis). Mitochondrially targeted FRET caspase-2 fusion protein containing the preferential caspase-2 substrate flanked by CFP and YFP, as donor and acceptor fluorophores respectively, were used as an in-situ sensor of mitochondrial caspase-2 activity. FRET efficiency, measured using acceptor photobleaching, demonstrated a time-dependent loss of FRET after exposure of cells to tert-butyl hydroperoxide, indicative of mitochondrial specific caspase-2 activation. Inhibition of complex I or III of the mitochondrial electron transport chain is known to induce reactive oxygen species (ROS) within the mitochondria. Treatment with the complex I inhibitor rotenone, the complex III antimycin A or staurosporine (STS), a non-ROS inducer of apoptosis, produced a significant amount of cell death in fibroblasts isolated from wild type mice. However, cells isolated from caspase-2 deficient mice were highly resistant to rotenone and antimycin A, but not STS-induced apoptosis. These results indicate that mitochondria contain oxidative stress-inducible caspase-2 and that caspase-2 activation is required for mitochondrial oxidative stress-induced apoptosis.

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67.  PATTERN OF GENE EXPRESSION IN LIVER OF YOUNG AND AGED RATS AFTER SHORT-TERM DIET RESTRICTION OR 2-DEOXY-D-GLUCOSE INJECTION: cDNA

 

Jacek Mamczarz*, Min Zhu, Jonna Bowker, Kara Duffy, Donald Ingram

 

Lab. Exp. Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224

 

Diet restriction (DR) in rodents increases lifespan, reduces age-related disease and pathology, increases stress responses, and maintains better function later into life compared to conventional ad libitum (AL) feeding. We have been investigating DR regimen and also DR mimetics that stimulate stress response pathways that are activated by DR. By inhibiting glycolysis, feeding or injection of 2-deoxy-D-glucose (2DG) has been proposed as a DR mimetic. In the current study we investigated the pattern of gene expression using cDNA microarray in liver of young (4 mo) and aged (26 mo) male Fischer-344 rats subjected to 3 weeks of 40% DR or 2DG injections once a day. 40% DR reduced initial body weight (BW) around 15% in young and 14% in old rats, respectively. 2DG in the dose of 125 mg/kg attenuated food intake and developmental gains in BW in young rats, while in aged rats 2DG decreased BW and food intake below initial levels. 2DG at a dose of 250 mg/kg also decreased BW and food intake in both young and aged rats; however, in aged rats the decrease was at the level of DR.  In general, DR and 2DG treatment up-regulated more genes in aged rats than recorded in young rats (around twice in DR, 6 times in 2DG-125, and 3.5 times more in 2DG-250). Down-regulation of gene expression was similar in young and old DR rats, twice higher in young 2DG-125, and twice lower in young 2DG-250, respectively, compared to aged rats. DR and 2DG-125 shared much more common patterns of gene expression in aged rats than in young, but for 2DG-250 there was a generally common pattern shared in both age groups. Analysis of gene expression patterns can be used to assess potential effectiveness of DR mimetics.

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