Abstracts

Gary R. Andrews

The prospects for individual and population ageing as we enter a new century pose some of the greatest social, economic and humanitarian challenges humankind as a whole has ever faced. The basic biological mechanisms that control human ageing remain ill understood but it is clear that for many individuals exhibiting predisposition to risk factors for certain chronic diseases, such as coronary heart disease, diabetes, osteoporosis, certain cancers and Alzheimer's disease, such predisposition is mediated through genetic processes that operate at a most fundamental biomolecular level interacting with nongenetic attributes. The prospect of improved understanding of the fundamental processes underlying the pathogenesis of common age-related diseases that may lead to identification of interventions that are effective in preventing, delaying or ameliorating the diseases and their consequences is compelling. It is this prospect that provides the prime justification for giving high priority to research on ageing vulnerability in a comprehensive research agenda on ageing for the 21st century.

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©2001 The Novartis Foundation

Julie K. Andersen

A growing body of evidence has implicated oxidative stress as an important factor in the neuropathology associated with Parkinson's disease. Dopaminergic nigrostriatal neurons, the predominant cells lost in Parkinson's, are believed to be highly prone to oxidative damage due to the propensity for dopamine to auto-oxidize and thereby produce elevated levels of hydrogen peroxide and catecholamine quinones. Hydrogen peroxide formed during this process can either be converted by iron to form highly reactive hydroxyl radicals or removed through reduction by glutathione. Glutathione can also conjugate with quinones formed during dopamine oxidation preventing them from facilitating the release of iron from the iron-storage molecule ferritin. Alterations in both iron and glutathione levels in the substantia nigra have been correlated with the neuronal degeneration accompanying Parkinson's disease but a direct causative role for either has yet to be definitively proved. We will discuss the use of genetically engineered cell and mouse lines generated in our laboratory as models to examine the role that alterations in iron and glutathione levels may play in neurodegeneration of dopaminergic neurons of the substantia nigra associated with Parkinson's disease, and how these two parameters may interact with one another to bring this about.

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©2001 The Novartis Foundation

Ashley I. Bush, Lee E. Goldstein

Abnormalities of protein aggregation and deposition may play an important role in the pathophysiology of a diverse set of chronically progressive degenerative disorders including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease and age-related cataracts. We propose that aberrant metalloprotein reactions may be a common denominator in these diseases. In these instances, an abnormal reaction between a protein and redox active metal ions (especially copper or iron) promote the generation of reactive oxygen species, and possibly, protein radicalization. These products then lead to chemical modification of the protein, alterations in protein structure and solubility, and oxidative damage to surrounding tissue. In this review, we explore these ideas by focusing on two common diseases of ageing, Alzheimer's disease and age-related cataracts. Understanding the metalloprotein biochemistry in both diseases may lead to a better understanding of the underlying pathophysiology in both disorders and suggests novel targets for therapeutic agents.

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©2001 The Novartis Foundation

R. N. Kalaria, C. G. Ballard, P. G. Ince, R. A. Kenny, I. G. McKeith, C. M. Morris, J. T. O'Brien, E. K. Perry, R. H. Perry and J. A. Edwardson

Age is the single most important risk factor for progressive dementia in populations worldwide. In developed countries the prevalence of dementia is estimated to be 3-5% at age 65 years and expected to double every decade thereafter. Although there is ageing-related attrition of neural tissue accompanied by profound changes in brain glia, marked neuronal loss and severe cognitive impairment are associated with pathological changes. Accelerated somatic ageing of the vasculature comprising endothelial and smooth muscle cells and slowed glial replacement are also likely pre-dispose to degenerative processes. Approximately 90% of patients with late-onset dementia have neuropathological features of Alzheimer's Disease (AD), dementia with Lewy bodies (DLB), or vascular dementia (VaD), alone or in combination. Both AD and DLB reveal extensive amyloid b deposition within senile plaques. Neurofibrillary tangles evident as tau pathology are much reduced in DLB where symptoms may be more related to cholinergic transmitter abnormalities than structural pathology. Depletion of brain acetylcholine is also encountered in VaD, which like AD and DLB may respond to cholinergic therapy. Cerebrovascular pathology, ischaemic brain damage and neurovascular instability resulting in cerebral hypoperfusion appears fundamental in the pathogenesis of late-onset dementia. The apolipoprotein E e4 allele, a major genetic susceptibility factor for AD also associated with cardiovascular pathology, may contribute to neurodegenerative changes through vascular mechanisms. The interrelationships of these multiple substrates of late-onset dementia have major implications for neuroprotective and disease slowing therapies. Measures that improve cardiovascular function and increase brain perfusion would be useful to attenuate cognitive decline.

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©2001 The Novartis Foundation

C. S. Potten, K. Martin and T. B. Kirkwood

Most organs of the body comprise populations of cells that are committed to specialized functions and that are renewed from small numbers of uncommitted progenitor or 'stem' cells. Stem cells are of central importance in the study of ageing because any senescent decline in the number or functional competence of stem cells will impair the capacity for renewal and turnover of committed cells, with potentially serious consequences for tissue homeostasis. The intestinal epithelium represents an excellent model system for the study of stem cell. Its spatial and hierarchical organisation allows the study of the function or characteristic of a given cell according to its position within the crypt. Hence, the stem cells which are located at the 4th-5th cell position from the bottom can be studied together with their daughter cells divide and differentiate while migrating along the crypt-villus axis. The ability of the stem cells to undergo apoptosis and the capacity to regenerate the epithelium following injury were investigated in mice from different ages. Stem cells from older animals showed an increased apoptotic response following exposure to low doses of ionising radiation. The regenerative capacity was estimated by measuring the crypt survival levels and the growth rate of surviving crypts after high doses of irradiation. Surviving crypts in the older mice, suggesting an impairment in the damage recognition/response mechanisms were both fewer and smaller than in young mice. The growth rate of surviving crypts was determined by measuring the crypt area and the number of cells/crypt at various times after 14 Gy irradiation. There was a growth delay of between half and one day in the older mice, and they subsequently grew more slowly. The number of cells susceptible to regenerate a crypt was also estimated. Surprisingly, they appear to be more numerous in the older mice. These studies indicate important age-related alterations in the capacity of the stem cells to regenerate the crypts after radiation-induced damage. The molecular bases of these changes are currently being investigated. Preliminary data showed alteration in the level of p53 and p21 expression, suggesting an age-related defect in the capacity to recognize damage and initiate apoptosis or repair..

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©2001 The Novartis Foundation

Amiela Globerson

The question of whether haematopoietic stem cells age has raised considerable controversy, and has been re-opened recently, as a result of the growing interest in stem cells for transplantation and gene therapy. Studies have focused on the generation of different blood cell elements and the capacity for self-renewal; properties that characterize stem cells. Taken together, it appears that basal haematopoiesis is maintained throughout life, yet, the capacity to cope with haematological stress is decreased in advanced age. In principle, stem cells derived from aged donors can be used for autologous transplantation, when needed to recover basic haematopoiesis. However, patterns of T cell development are altered in ageing, and intervention to augment T cell response still needs to be considered. Current methods for expansion and maintenance of stem cells in vitro enable examination of stem cell potential for long-term expansion and function. A critical evaluation of the possible risks of replicative senescence and developmental changes in stem cells has become feasible. Ageing effects may relate to cell replication, cell migration and lymphoid differentiation. Understanding of the mechanisms underlying these processes will enable the fidelity of stem cell expansion and maintenance of their potential for long-term function.

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©2001 The Novartis Foundation

James K. Leung and Olivia M. Pereira-Smith*

The limited proliferative potential of normal cells in culture, cell replicative senescence, is an accepted model for ageing at the cellular level. Tumour-derived, or viral- or carcinogen-transformed cells have escaped senescence and proliferate without control (immortal). We and others have found that fusion of normal with immortal human cells yields hybrids that have regained growth control and cease division. This demonstrates that the phenotype of replicative senescence is dominant and that cells immortalize because of defects in senescence-related genes. We exploited the recessive nature of immortality and by fusing different immortal cell lines with each other identified four complementation groups for indefinite division. Immortal parental cell lines with similar senescence gene defects when fused yielded hybrids with unlimited division potential and were assigned to the same complementation group. Fusion of immortal cell lines with different gene defects resulted in complementation in the hybrids, which had limited division capability. These parental cell lines were assigned to different complementation groups. Using microcell-mediated chromosome transfer, we then demonstrated that introduction of a normal human chromosome 4 induced senescence only in immortal cell lines assigned to complementation group B. We have now cloned the gene on chromosome 4, MORF4 (mortality factor on chromosome 4). It is a member of a family of seven genes and only MORF4 and the MORF-related genes MRG15 and MRGX are expressed. The predicted protein motifs strongly suggest this is a novel family of transcription factors. We have identified interacting proteins, some of which are also novel. These genes have the potential to modulate expression of a large number of genes by chromatin remodelling. They, therefore, also have the potential to affect tissue function due to changes in expression activity during ageing.

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©2001 The Novartis Foundation

Jerry W. Shay and Woodring E. Wright

Telomeres are repetitive DNA sequences at the ends of linear chromosomes. Telomerase, a cellular reverse transcriptase, helps stabilize telomere length in human stem, reproductive and cancer cells by adding TTAGGG repeats onto the telomeres. Each time a telomerase-negative cell divides some telomeric sequences are lost. When telomeres are short, cells enter an irreversible growth arrest state called replicative senescence. In most instances cells become senescent before they can become cancerous, thus the growth arrest induced by short telomeres may be a potent anti-cancer mechanism. Since most cancers express telomerase, maintenance of telomere stability is likely to be required for the long-term viability of tumours. Inhibition of telomerase results in gradual erosion of telomeres followed by cessation of proliferation or apoptosis, and thus may be a promising target for cancer therapy. Introduction of the telomerase catalytic protein component into telomerase-silent cells is sufficient to restore telomerase activity and extend cellular life span. However, cells with introduced telomerase are not cancer cells since they have not accumulated the other changes needed to become cancerous. This indicates that telomerase-induced telomere length manipulations may have utility for tissue engineering and for dissecting the molecular mechanisms underlying genetic diseases including cancer.

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©2001 The Novartis Foundation

Rita B. Effros

Immune system alterations during are complex and pleiotropic, suggestive of remodelling or altered regulation, rather than simple immune deficiency. The most dramatic changes with age occur within the T cell compartment, the arm of the immune system that protects against pathogens and tumours, consistent with the increased incidence and severity of infection and cancer in the elderly. Indeed, autopsy studies confirm infection as the major cause of death in the very old. Increased serum levels of inflammatory mediators are another hallmark of ageing, suggestive of either regulatory defects or an ongoing attack on sub-clinical neoplastic disease or infection. Qualitative changes in antibody production, including those secreted by the gut mucosal immune compartment, affect responses to foreign antigens as well as to prophylactic vaccines. Innate immunity, the first line of defence that precedes the antigen-specific T and B cell responses, also undergoes changes with age. Some of the immune effects associated with ageing are secondary to overall organismic changes, such as alterations in the viscosity of cell membranes and proteolytic cellular machinery. Evidence suggesting that immune system changes may be involved in some major age-related pathologies, such as atherosclerosis and Alzheimer's disease, will be discussed.

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©2001 The Novartis Foundation

Lis Mosekilde

The human skeleton is formed and modelled during childhood and youth through the influence of hormones and daily mechanical usage. Around the age of 20-25 years, the skeleton achieves its maximum mass and strength. Thereafter, and throughout adult life, bone is lost at an almost constant rate due to the dynamic bone turnover process: the remodelling process. During this process, small packets of bone are renewed by teams of bone cells coupled together in time and space. In an adult human skeleton there will be 1-2 million active remodelling sites at any time point. The vast number of turnover units combined with a slightly negative balance at the completion of each process leads to the age-related loss of bone mass mentioned above and, concomitantly, to loss of structural continuity and strength. The magnitude of this loss will be determined by hormonal factors, nutrition and mechanical usage. As a consequence of the remodelling process, the bone tissue of the skeleton will always be younger than the age of the individual. However, as a consequence of the remodelling process, osteopenia and osteoporotic fractures will also occur. In this article, the remodelling-induced changes in the human spine will be used as an example of ageing bone.

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©2001 The Novartis Foundation

Edward G. Lakatta, Steven J. Sollott and Salvatore Pepe

Excitation of cardiac cells is accompanied by Ca2+ influx which triggers a transient increase in cytosolic [Ca2+], (Cai), and contraction. While the amplitudes of the Cai transient and contraction increase with the extent of cell Ca2+ loading, excess Ca2+ loading leads to dysregulation of Ca2+ homeostasis, impaired contraction, arrhythmia and cell death. The cell Ca2+ load is determined by membrane structure and permeability characteristics, the intensity of stimuli that modulate Ca2+ influx or efflux via regulatory function of proteins within membranes, and reactive oxygen species (ROS), which affect both membrane structure and function. Cardiocytes of senescent hearts exhibit a reduced threshold for pathologic manifestations of excess Ca2+ loading during stimulation (physiologic or pharmacologic) that increases Ca2+ influx, e.g. in response to neurotransmitters, post-ischaemic reperfusion, or oxidative stress. Cell 'remodelling' is one cause of the relative Ca2+ intolerance of cardiocytes in the senescent heart; cells increase in size and changes occur in the amounts of proteins that regulate Ca2+ handling due, in part, to altered gene expression; another cause is a change in the composition of membranes in which Ca2+ regulatory proteins reside, e.g. an increase in membrane w6:w3 polyunsaturated fatty acids (PUFA); a third cause is an enhanced likelihood for intracellular generation of ROS. Each class of these determinants changes with ageing and reduces the threshold for Ca2+ overload to occur with the older heart. The risk of excess Ca2+ loading within senescent heart can potentially be reduced by gene therapy to restore Ca2+ regulatory proteins, by diet to reverse the membrane w6:w3 PUFA imbalance, or by antioxidants.

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©2001 The Novartis Foundation

Anthony Cerami and Peter Ulrich

The Kenneth S. Warren Laboratories, 765 Old Saw Mill River Road, Tarrytown, NY 10591, USA

Recent studies have revealed that reducing sugars, such as glucose, react with proteins through non-enzymatic glycosylation to form irreversible, covalently cross-linked proteins known as advanced glycation endproducts (AGEs). Furthermore, it has been demonstrated that this naturally occurring process, accelerated in diabetics due to hyperglycaemia, impairs biological functions leading to cardiovascular disorders, as well as diabetic and age-related complications. Pharmaceutical intervention to prevent or reverse these complications have focused on inhibiting the formation of AGEs by compounds such as dimethyl-3-phenacylthiazolium chloride or breaking the glucose derived cross-links by selective cleavage. Intervention targeted at AGE cross-links in vivo offers a way to interfere with age-related changes of tissues.

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©2001 The Novartis Foundation

The anti-ageing action of dietary restriction

Holly Van Remmen, Zhongmao Guo and Arlan Richardson*

Department of Physiology, The University of Texas Health Science Center at San Antonio, and South Texas Veterans Health Care System, San Antonio, TX 78284-7756, USA

Over 60 years ago, McCay's laboratory showed that dietary or calorie-restriction dramatically increased the lifespan of rats. Since then, numerous laboratories with a variety of strains of rats and mice have confirmed this initial observation and have shown that reducing calorie intake (without malnutrition) significantly increases both the mean and maximum survival of rodents. Currently, dietary restriction is the only experimental manipulation that has been shown to retard ageing of mammals. Although mechanism whereby dietary restriction retards ageing is currently unknown, much of the emerging data suggest that the calorie-restricted rodents live longer and age more slowly because they are more resistant to stress and have an enhanced ability to protect cells against damaging agents.

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©2001 The Novartis Foundation

D. A. Cottrell, E. L. Blakely, M. A. Johnson, G. M. Borthwick, P. I. Ince* and D. M. Turnbull

Department of Neurology, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH and *MRC Neurochemical Pathology Unit, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE, UK

The chronological accumulation of mitochondrial DNA mutations has been proposed as a potential mechanism in the physiological processes of ageing and age-related disease. We discuss the evidence behind this theory and relate some of the ageing mitochondrial changes to mitochondrial DNA disorders. In particular, we describe the aggregation of cytochrome c oxidase-deficient cells in both skeletal muscle and the CNS in normal ageing as seen in the mitochondrial DNA disorders. These mitochondrial enzyme-deficient cells have been shown in both ageing and mtDNA disorder muscle to contain high levels of a single mutated mtDNA. Whether these mutations are a primary or secondary event in the physiology of ageing remains to be determined.

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©2001 The Novartis Foundation

Douglas C. Wallace

A variety of degenerative diseases have now been shown to be caused by mutations in mitochondrial genes encoded by the mitochondrial DNA (mtDNA) or the nuclear DNA (nDNA). The mitochondria generate most of the cellular energy by oxidative phosphorylation (OXPHOS), and produce most of the toxic reactive oxygen species (ROS) as a by-product. Genetic defects that inhibit OXPHOS also cause the redirection of OXPHOS electrons into ROS production, thus increasing oxidative stress. A decline in mitochondrial energy production and an increase in oxidative stress can impinge on the mitochondrial permeability transition pore (mtPTP) to initiate programmed cell death (apoptosis). The interaction of these three factors appear to play a major role on the pathophysiology of degenerative diseases. Inherited diseases can result from mtDNA base substitution and rearrangement mutations and can affect the CNS, heart and skeletal muscle, and renal, endocrine and haematological systems. In addition, somatic mtDNA mutations accumulate with age in post-mitotic tissues in association with the age-related decline in mitochondrial function and are thought to be an important factor in ageing and senescence. The importance of mitochondrial defects in degenerative diseases and ageing has been demonstrated using mouse models of mitochondrial disease. A mtDNA mutation imparting chloramphenical resistance (CAPR) to mitochondrial protein synthesis has been transferred into mice and resulted in growth retardation and cardiomyopathy. A nDNA mutation which inactivates the heart-muscle isoform of the adenine nucleotide translocator (Ant1) results in mitochondrial myopathy and cardiomyopathy; induction of ROS production; the compensatory up-regulation of energy, antioxidant, and apoptosis gene expression; and an increase in the mtDNA somatic mutation rate. Finally, a nDNA mutation which inactivates the mitochondrial Mn superoxide dismutase (MnSOD) results in death in about 8 days due to dilated cardiomyopathy, which can be ameliorated by treatment with catalytic anti-oxidants. A partial MnSOD deficiency chronically increases oxidative stress, decreases OXPHOS function, and stimulates apoptosis. Thus, the decline of mitochondrial energy production resulting in increased oxidative stress and apoptosis does play a significant role in degenerative diseases and ageing.

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©2001 The Novartis Foundation