Pii: s0531-5565(00)00177-7

O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 Experimental Gerontology 35 (2000) 901–916 Experimental gerontology in Belgium: from model O. Toussainta,*, P.V. Baretb, J.-P. Brionc, P. Crasd, F. Collettee, P.P. De Deynf, V. Geeneng, P. Kienlen-Campardh, C. Labeuri, J.-J. Legrosj, J. Ne`vek, J.-N. Octaveh, G.E. Pie´rardl, E. Salmonm, P. van den Bosch de Aguilarn, M. Van der Lindene, F.V. Leuveno, aUnit of Cellular Biochemistry, University of Namur, Rue de Bruxelles, 61, B-5000 Namur, Belgium bUnit of Genetics, Catholic University of Louvain, Croix du Sud 2, box 14, B-1348 Louvain-la-Neuve, Belgium cLaboratory of Histology and Neuropathology, School of Medicine, Free University of Brussels, 808 Route de Lennik, B-1070, Bldg C-10, Brussels, Belgium dDepartment of Neurology, Laboratory of Neurobiology, University of Antwerp, Born-Bunge Found., University Hospital of Antwerp, Antwerp, Belgium eUnit of Neuropsychology, Bd du rectorat 3, B33, B-4000 Lie`ge, Belgium fLaboratory of Neurochemistry and Behavior, Born-Bunge Found. and Middelheim Hospital, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium gDepartment of Medicine, Institute of Pathology, University of Lie`ge, CHU-B23, B-4000 Lie`ge, hLaboratory of Pharmacology, Catholic University of Louvain, FARL 5410, Av. Hippocrate 54, iDepartment of Biochemistry, University of Gent, Hospitaalstraat 13, B-9000 Gent, Belgium jInterdisciplinary Research Group on Andropause. CHU-Sart-Tilman, B-4000, Lie`ge, Belgium kInstitute of Pharmacy, Free University of Brussels, Campus Plaine 205/5, B-1050 Brussels, Belgium lUnit of Dermatopathology, CHU Sart Tilman, B-4000 Lie`ge, Belgium mCyclotron Research Centre, ULg, B30, Sart Tilman, B-4000 Lie`ge, Belgium nLaboratory of Cellular Biology, Catholic University of Louvain, Place Croix du Sud, 5, oExperimental Genetics Group, Center for Human Genetics, Flanders Institute for Biotechnology-VIB, K.U. Leuven, Campus Gasthuisberg O&N 06, B-3000 Leuven, Belgium pDepartment of Biology, University of Gent, Ledeganckstraat 35, B-9000 Gent, Belgium Received 28 June 2000; received in revised form 12 July 2000; accepted 12 July 2000 Keywords: Aging; Drosophila melanogaster; Caenorhabditis elegans; Oxidative stress; Cellular senescence;Brain ageing; Alzheimer disease; Skin; Immunosenescence; Andropause * Corresponding author. Tel.: ϩ32-81-72-41-32; fax: ϩ32-81-72-41-35.
E-mail address: [email protected] (O. Toussaint).
0531-5565/00/$ - see front matter ᭧ 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 5 3 1 - 5 5 6 5 ( 0 0 ) 0 0 1 7 7 - 7 O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 1. Introduction
Unraveling the fundamental mechanisms of ageing is a pre-requisite for developing appropriate means of increasing mobility, activity, creativity and independence of theelderly. The physiological changes that occur with ageing originate in the molecularbiology of cells, contributing not only to normal human ageing, but also to age-relatedpathologies. An urgent priority, if we are to intervene in age-related pathologies, must beto understand how different cell types are altered during ageing and how these interactionshave pathobiological consequences.
In this review, we shall develop the belgian research on the molecular and cellular mechanisms of normal ageing and appearance of age-related diseases.
Belgium spends a very reduced fraction of GNP to fund non-commercially oriented research. An increased priority has been progressively attributed to applied research overthe last five years. Nevertheless, more than 15 laboratories work on experimental geron-tology and cover as different subjects as: • model systems based on lower organisms (Drosophila melanogaster, Caenorhabditis • oxidative stress-induced premature cellular senescence; • normal brain ageing, age-related neurodegenerative diseases (cells in vitro, transgenic • skin ageing using cells in vitro and non-invasive methods in humans; The scientific activities of these laboratories will be reviewed in this order.
2. Model systems
The main research topics of this research are focused on the evolutionary aspects of lifespan and the effects of mild stress on longevity. An evolutionary approach of lifespanrequires a better knowledge of the genetic basis of life history traits such as fecundity andlongevity. Experimental data and analytical approaches show that both heritability of Abbreviations: AD, Alzheimer disease; AHA, a-hydroxyacid; Apo, Apolipoprotein; APP, Amyloid precursor protein; Ab, Amyloid peptide; CHO, Chinese hamster ovary; CNS, Central nervous system; CSF, Cerebrospinalfluid; FSH, Follicle Stimulating Hormone; GnRH, gonadotropin-releasing hormone; HDFs, Human diploidfibroblasts; HRT, Hormone replacement therapy; IGF, Insulin growth factor; IL, Interleukin; LCAT, Lecithincholesterol acyl transferase; LH, luteinzing hormone; LHA, b-lipohydroxyacid; MALDI, Matrix-assisted laserdesorption ionisation; NFT, Neurofibrillary tangles; NGF, Nerve Growth Factor; PLTP, Phospholipid transferprotein; PMN, Polymorphonuclear cells; PNS, Peripheral nervous system; PS, Presenilins; Rb, Retinoblastomaprotein; ROS, Reactive oxygen species; SA b-gal, Senescence-associated b-galactosidase activity; SIPS, Stress-induced premature cellular senescence; T, Testosterone; t-BHP,tert-butylhydroperoxide; TNF-a, Tumor NecrosisFactor O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 lifespan and genetic correlation between lifespan and fecundity are null or very low. Onthe one hand, no consistent correlation is observed between early fitness and longevity inlaboratory assessments of freshly caught populations of Drosophila melanogaster (Drayeand Lints, 1996). This is in contradiction with the hypothesis of antagonistic pleiotropy.
On the other hand, an analytical reinterpretation of previous experiments indicates that theapparent success of selection by reproduction at early age may be explained by environ-mental variations (Baret and Lints, 1993). Further studies will focus on the interactionsbetween environmental (temperature) and genetic effects on lifespan.
Recent experimental work addresses the effect of stress on longevity. Populations of D. melanogaster were exposed to two types of stress: hypergravity and hyperoxygenation. Inboth cases, a lifelong exposure to stress induced a decrease in mean lifespan (Baret et al.,1994). Nevertheless, when hypergravity was applied during the first two weeks of life,positive effects were observed on male lifespan. The positive effect of a mild stress due tohypergravity was more important than similar effects obtained by heat shock stress (LeBourg et al., 2000). Further studies on positive effect on lifespan will focus on the anti-oxidant mechanisms with specific measurements of the antioxidant enzymes superoxidedismutase and catalase activities on individual flies exposed to different levels of hyper-gravity.
Research is performed on the genes and biochemical processes that govern longevity and ageing in the nematode Caenorhabditis elegans to understand the fundamentalmechanisms that determine ageing in humans. Several genes have been identifiedwhich, when mutated, extend the life span of the adult worms substantially. A subset ofthese genes has homologues in the mammalian insulin and IGF signaling pathways.
Another group of genes, the clock genes, regulate the pace of many temporal processesand metabolic activity.
All known longevity phenotypes confer resistance to multiple environmental stresses including exposure to oxidative stress, high temperature and UV. This is in agreement withthe generally accepted idea that the balance between oxidative damage due to ROS, andthe potential to resist such damage determines the pace of the age-related metabolic andphysiological alterations (Remacle et al., 1995). However, the precise interactionsbetween the stochastic and genetic driving forces are unknown. All known mutationsaltering life span in C. elegans define a novel mean life span, but do not alter the survivalprofiles appreciably and may not fundamentally alter the proper ageing process (Vanfle-teren et al., 1998).
A panel of physiological and biochemical parameters describing the metabolic state in ageing worms is currently monitored (Braeckman et al., 1999, 2000; Vanfleteren andBraeckman, 1999). As a complementary approach, the antioxidant capacity of longevitymutants is evaluated. The mechanism of extension of life span by caloric restriction isbeing established: the alterations of metabolism and stress resistance are characterized. Itis planned to identify genes that are differentially expressed in this medium. The URL ofthe laboratory is: http://allserv.rug.ac.be/~jvfleter O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 3. Oxidative stress-induced premature cellular senescence (SIPS)
A complex model of cellular ageing has been developed based on thermodynamics of open systems which predicts that instabilities like oxidative stress, at subcytotoxic levels,can accelerate the process of cellular ageing (Toussaint et al., 1991; Toussaint and Schnei-der, 1998). Replicative senescence of human diploid fibroblasts (HDFs) is a relevantmodel for studying cellular ageing. Many biomarkers of in vivo ageing appear whenthe in vitro proliferative potential of HDFs becomes exhausted. Subcytotoxic stressunder tert-butylhydroperoxide (t-BHP) or ethanol increases sharply the proportion ofHDFs positive for the senescence-associated b-galactosidase activity (SA b-gal). Theability to duplicate DNA is drastically reduced. For at least 3 weeks after the stress,HDFs present a p53-independent increase in their level of cyclin-dependent kinase inhi-bitors p21waf-1/Cip1, p16Ink-4a, and p14/15Ink-4c responsible for hypophosphorylation of theretinoblastoma protein (Rb). The level of mRNA of at least 8 genes is similarly changedin both senescent HDFs and HDFs in SIPS. The common 4977 bp deletion of mitochon-drial DNA is detected in senescent HDFs and HDFs in SIPS. A novel mitochondrial DNAdeletion was discovered in SIPS (Dumont et al., 2000a,b; Toussaint et al., 1992) (for areview, see Brack et al., 2000).
A sophisticated method was developed for quantifying the mt DNA deletions with quantitative PCR allowing to calculate the ratio of deletions to the number of total mtDNA copies present in the sample; and to quantify the formation of heteroduplexesbetween the amplified target DNA and internal standard (Frippiat et al., 2000).
The signaling pathways of cytokines such as IL-1a and TNF-a generate transient increases in reactive oxygen species. After five stimulations with these cytokines followedby 3 days of recovery, there is a significant shift from the ‘young’ HDFs morphotypes tothe ‘older’ morphotypes, and a significant increase in the proportion of HDFs positive forthe SA b-gal activity. The antioxidants vitamin E and N-acetylcysteine protect againstthese changes (Dumont et al., 2000c; Toussaint et al., 1996, 1998). These were some of thefirst results suggesting a direct pro-ageing effect of pro-inflammatory cytokines. In vivo,Blasko et al. have found that TNFa plus IFNg induce the production of Alzheimer beta-amyloid peptides and decrease the secretion of APPs (Blasko et al., 1999).
When neuroblastoma cells differentiated with NGF and HDFs are exposed to t-BHP and ethanol in conditions of partial uncoupling of the mitochondrial respiration, or decreasedconcentrations of substrates of the energy metabolism, a synergistic effect of cell deathexists between the decrease in the cellular capacities of regeneration of ATP and theconcentration of stressor. Pharmacological molecules which stimulate the mitochondrialenergy metabolism are able to decrease the oxidative stress-induced cell death. A decreasein the capacities of the cells to regenerate ATP favors SIPS (Toussaint et al., 2000a, 1994,1995a,b,c; Toussaint and Remacle, 1996).
Proteome and transcriptome analyses are performed to identify proteins and mRNAs involved in SIPS and replicative senescence of HDFs. For instance, the proteomic studiesbrought out 68 proteins which undergo expression changes out of 2100 analyzed in each2D gel (unpublished data). Most of these proteins were identified using nano-electrosprayand MALDI mass spectrometry. The roles of the genes and proteins found in these screen-ing studies are being analyzed. One gene was found to protect the cells against both O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 cytotoxicity and SIPS induced by t-BHP, H2O2, UV, and ethanol and will be tested aslongevity candidate. All the experimental models of SIPS developed also allow us to testnew compounds against senescence or SIPS (Toussaint et al., 2000b). Web site of thelaboratory: http://www.fundp.ac.be/urbc 4. Normal brain ageing and age-related neurodegenerative diseases
4.1. In vitro models and ageing of rat brain Ageing of the nervous system has been investigated both in peripheral (PNS) and central nervous system (CNS), using ageing rats as an experimental model. In PNS, itwas found that ageing is chronologically determined. A first phase affects the mitochon-dria and the second one alters the cytoskeleton. Loss and/or degradation of the targetorgans can induce retrograde degeneration.
However, a population of the oldest nerve cells never loses its ability to regenerate fibers. Basically, the same results are obtained in different areas in the CNS (Will et al.,1998). On the basis of these results which reflect normal ageing, investigations areconducted towards two objectives: determination of the factors which can improve regen-eration in the nervous system and identification of the factors which can enhance thealterations. The first approach concerns studies of neurites outgrowth and cell migration,using biomaterial substrates and defined culture medium, in order to create new neuronalnetworks (Detrait et al., 1998). The second approach concerns the action of aluminumwhich has been suspected to intervene in Alzheimer disease (AD) since Al acts on astro-cytes and the glutamate metabolism, increasing the toxic action of glutamate on theneurons. By this way aluminum could favor the onset of AD (Struys-Ponsar et al., 2000).
4.2. Analysis of human amyloid precursor protein expression and processing in cellularmodels The amyloid peptide (Ab) is the major constituent of the amyloid core of senile plaques found in the cerebral cortex of patients with AD. Ab is derived from a much largerprecursor: the amyloid precursor protein or APP. The aim of this work is to analyze, inappropriate cellular models, the expression and processing of human APP and to correlateit with cellular functions. The processing of human APP is studied in cell lines, as well asin primary cultures of neurons.
In the mammalian CHO cell line expressing the full length human APP (APP 695), APP is expressed as a membrane bound protein and is processed through both amyloidogenicand non-amyloidognenic pathways. This processing relies on secretase activities. Thecleavage of APP by the a-secretase allows the release of soluble APP (saAPP). Theamyloidogenic pathway involves b- and g-secretase activities required for Ab production(Octave et al., 2000). Insect Sf9 cells, infected by baculoviruses encoding human APP,provide an interesting expression system since these cells do not produce endogenousAPP. Interestingly, these cells do not produce Ab. These models will hopefully allow us tofurther characterize the mechanisms involved in APP trafficking and processing (Essal-mani et al., 1996).
O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 Recombinant adenoviruses were constructed to express human APP into primary cultures of rat cortical neurons. Human APP expression in neurons is followed bysaAPP and Ab production. This human APP expression exerts strong apoptotic effectsin neurons (Macq et al., 1998). The mechanisms responsible for APP-induced neuronalapoptosis are currently investigated, considering a recent hypothesis suggesting that intra-neuronal accumulation of Ab could exert neurotoxic effects by itself.
4.3. The neurobiology of AD and related neurodegenerative diseases This research program is focused on the structural and molecular analysis of neurofi- brillary tangles, a characteristic cellular lesion of AD and other age-associated neurode-generative diseases, collectively grouped under the term of “tauopathies”. Mutated formsof tau have also been recently identified in familial forms of fronto-temporal dementias(Brion, 1998).
This group has demonstrated that neurofibrillary tangles are composed of the micro- tubule-associated protein tau in AD. The modifications affecting tau proteins in AD, mostnotably hyperphosphorylation, as well as the cell biology of this protein, have been studied(Dayanandan et al., 1999). Major goals of the present research are: (1) to reproduceneurofibrillary tangles and the modifications affecting tau proteins in AD in an in vivomodel, using transfected neuronal cultures, transgenic animals; and (2) to understand howother key-proteins implied in the pathogenesis of AD (presenilins, amyloid peptide precur-sor) affect the metabolism of tau proteins.
The hyperphosphorylation of tau protein characteristic of AD can be partially repro- duced in transfected cells and in neuronal cultures by manipulating the enzymatic activ-ities of selected protein kinases and phosphatases. The modulation of the biologicalactivity of tau on the microtubule networks has also been studied by controlling theexpression of selected protein kinases, and mutated forms of tau and presenilins proteinsin cultured cells (Leroy et al., 2000). The accumulation of phosphorylated forms of tau inthe somatodendritic compartment of neurons, characteristic of the early stades precedingNFT formation, has also been achieved in transgenic models (Brion et al., 1999).
4.4. Transgenic mice: models for Alzheimer’s disease Another group has generated different strains of transgenic mice that overexpress wild- type or mutant APP, human wild-type or mutant Presenilins (PS1, PS2), human ApoE4 orhuman protein tau. All constructs were based on the mouse thy1 gene promoter to expressthe transgenes specifically in neurons. The most remarkable observation was that all APPtransgenic mouse strains displayed a similar behavioral phenotype as the original APP/RKmice (Moechars et al., 1996). The major symptoms include: disturbed behavior of reducedexploration, neophobia, increased aggression, excitotoxicity with premature death andhypersensitivity to kainic acid, but hypo-sensitivity to NMDA with reduced cognition(Moechars et al., 1999). The combined observations in the APP transgenic mouse strainsdemonstrate the marked dissociation in time of the early cognitive and behavioral deficitsobserved in all APP strains from the late and selective development of amyloid plaques inold APP/London mice. Whereas, the occurrence of plaque and vascular amyloid isexplained by the higher levels of amyloid peptides, the cognitive and behavioral O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 phenotypic traits appeared to be linked to a combination of metabolites, i.e. Ab40, b-C-stubs and secreted APP. To define their respective contributions, other more complextransgenic mouse strains are being generated.
Double transgenic mice, i.e. APP/London × PS1[A246E], develop amyloid plaques when only 6–9 months old, concomitant with increased Ab42 levels. The other APP-metabolites are relatively unchanged, which is concordant with the observation that theearly behavioral traits in the APP/Lo × PS1 double tg mice are not essentially differentfrom the single APP/London tg mice. Single PS1 tg mice that overexpress either the wild-type human PS1 or the EOFAD mutant PS1[A246E], have essentially no pathology orphenotypic abnormalities. Mice deficient in PS1 are not viable, but primary cultures ofembryonal neurons grow and differentiate normally, and were used to demonstrate thatproduction of the amyloid peptides is reduced dramatically in the absence of PS-1 (DeStrooper et al., 1998).
To implement and understand the problem of tau-pathology in AD, transgenic mice are generated that overexpress human protein tau in neurons, causing motoric impairment dueto prominent axonopathy in brain and spinal cord. Axonal dilations with accumulation ofneurofilaments, mitochondria and vesicles are prominent suggesting that defective axonaltransport causes axonal degeneration. This effect was gene-dosage related. Increasing theconcentration of the four-repeat tau protein isoform is sufficient to cause neuronal injurywithout additional requirement of intraneuronal neurofibrillary tangles (Spittaels et al.,1999).
ApoE4 is an important genetic risk-factor for AD, but besides the epidemiological evidence, its molecular contribution to the neurodegenerative pathogenesis is unknown.
Rodents do not express any ApoE in neurons, only in astrocytes. In human brain, neuronsthat express ApoE might be most vulnerable for developing neurofibrillary pathology.
Transgenic mice with neuronal expression of human ApoE4 progressively exhibit motorproblems that correlated with neuronal hyperphosphorylation of protein tau. Increasedprotein tau phosphorylation was dependent on the level of neuronal expression of humanApoE4 and on the age of the mice. Neurons in brain and spinal cord react positively withmonoclonal antibodies which specify AD related epitopes. In addition, ApoE4 transgenicmice developed axonopathy, severe motor impairment and neurogenic muscle atrophy(Tesseur et al., 2000). Numerous inclusions stained positive for ubiquitin, neurofilamentsand synaptophysin in the white matter tracts of the CNS, indicating impairment of axonaltransport. In sharp contrast, none of these symptoms were detected in transgenic mouselines that over-express human ApoE4 in astrocytes at similar levels.
Experiments in “multiple” transgenic mice are ongoing to determine which of the APP metabolites is causing the early signs of the “amyloid”-related phenotype, in relationshipwith the neuronal “ApoE4–tau” connection. This will determine the importance of APPmetabolites and of the intraneuronal tangles, the other diagnostic lesion for AD.
4.5. Neurological disorders associated with ageing: increasing pathophysiologicalinsight, optimizing molecular diagnostic and therapeutic tools A series of mice models for mental retardation and dementia have been studied. Among these rank a series of genetically manipulated animals among which a mice model O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 expressing human 751-amino acid b-APP (D’Hooge et al., 1996). Behavioral observationsdemonstrated an age-dependent spatial learning deficit in these animals in the absence ofhistopathological amyloid-related alterations.
Neurochemical parameters are investigated both in brain tissue and cerebrospinal fluid in a variety of dementia syndromes. These research projects aim at furthering pathophy-siological knowledge of these neurodegenerative diseases and focus on potential para-meters that would allow evaluation of therapeutic efficacy. Illustrative in this regard aredata generated on superoxide dismutase and interleukins in cerebrospinal fluid of patientspresenting with dementia (De Deyn et al., 1998; Engelborghs et al., 1999). New neurop-sychopharmacological treatment options are investigated in large clinical trials contribut-ing to the development of rational pharmacological approaches in age-related dementias(De Deyn et al., 1997, 1999).
4.6. Lipoproteins and amyloid b in AD These studies are focused on the analysis of lipoproteins and aspects of lipoprotein metabolism in human cerebrospinal fluid (CSF) in normal individuals and patients suffer-ing from AD. The presence of high density like lipoproteins of complex composition wasdemonstrated in this body fluid. Differences in the lipoprotein distribution between ADpatients and normals were observed especially in the amounts of apolipoprotein A-Ienriched fractions. Minor lipoprotein subclasses such as small precursor pre-b particleswere identified, suggesting an active lipoprotein synthesis by the brain cells. Moreover, itwas demonstrated that in human CSF lipid transfer proteins such as phospholipid transferprotein (PLTP) and enzymes involved in the esterification of cholesterol such as lecithincholesterol acyl transferase (LCAT) are present and enzymatically active (Demeester etal., 2000). Interestingly, the LCAT activity in CSF from AD patients is significantly lowerthan in normal individuals suggesting that the increased amounts of amyloid b peptides inCSF from AD patients directly interfere in this process. It is known that lipoproteins areactively taken up by neuronal cells by receptor mediated processes when cells are in needof cholesterol. The CSF lipoproteins are also involved in taking up excess cholesterol fromastrocytes thereby preventing excessive build up of cholesterol pools in astrocytes. Thischolesterol efflux can be directly correlated with the apolipoprotein content of the CSFtested. Taken together these studies suggest that an active lipoprotein metabolism isoccurring in the human CSF and the preliminary data suggest that this metabolism isdisturbed in patients suffering from AD.
It was also shown that amyloid b peptides are lipophilic peptides that associate with lipid membranes both of liposomes and neuronal cells. Structural studies of the lipidbound amyloid b peptides (C-terminal fragments e.g. fragments 29–40 and 29–42)suggest that at certain peptide:lipid ratio’s the amyloid b peptides assemble into aggre-gated b sheet structures. These aggregates could explain the cytotoxic effects of thesepeptides. Indeed studies on cytotoxic mechanisms in neuronal cells suggest that thesefragments are highly toxic for neuronal cells and that these processes involve apoptoticcell death evidenced by caspase 3 measurements, DNA laddering, PI staining and Facsanalysis (Decout et al., 1998; Pillot et al., 1999a,b). Another belgian laboratory has alsocontributed to the diagnosis of dementia by analysis of the paired helical filament subunit O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 protein tau and amyloid-beta in cerebrospinal fluid (Vandermeeren et al., 1993). In addi-tion, this laboratory is also involved in a surveillance program on the incidence of Creutz-feldt–Jakob’s disease in Belgium (Pals et al., 1999; Van Everbroeck et al., 1999) and itscurrent interest involves gene-environment interaction in Parkinson disease.
4.7. Brain activity measured with positron emission tomography in ageing and AD New programmes were recently developed to perform voxel-based analysis of regional brain activity measured with functional neuroimaging. Cerebral glucose uptake in normalageing was confirmed to be essentially decreased in several frontal regions, whencompared to young subjects (Garraux et al., 1999). The relative frontal hypometabolismof elderly healthy people is probably related to decreased synaptic activity due to progres-sive loss of interregional connections. Such a frontal metabolic impairment is consistentwith several neuropsychological characteristics of normal ageing. In AD an age-relateddegenerative dementia, brain activity is predominantly decreased in posterior cingulateand in temporo-parietal associative cortices. Metabolic impairment in posterior cingulatecortex is positively related to age (Salmon et al., 2000). This means that elderly patientsalready become demented with a moderate decrease of activity in multimodal associativeposterior cortices.
Elderly people present impaired performance on tasks designed to explore some execu- tive functions, especially working memory updating, planning, inhibition and abstractionof logical rules. Processing speed explained some of these age-related differences. Work-ing memory and executive deficits take place in the early stages of AD, but the preserva-tion of more automatic working memory processes suggests a possible trajectory ofcognitive impairment in AD. The early executive deficits may be due to a breakdownin connections between the main cortical areas. However, as the AD progresses, theneuropathological changes may also affect specific cortical areas, leading to deficits inmore automatic cognitive processes, as confirmed using positron emission tomography.
According to the cognitive task administered, deficits of AD patients can be explainedeither by a disconnection process or by a less efficient functioning of specific cerebralareas (Andre`s and Van der Linden, 2000; Collette et al., 2000).
5. Skin ageing using cells in vitro and non-invasive methods in humans
The prevalence of many skin ailments and diseases increases with age. This laboratory has expertise in evaluating the histological clues of ageing and in assessing the changesusing in vivo non-invasive bioengineering methods. The state-of-art in testing tensileproperties of skin during ageing was reviewed in cooperation with the European ExpertGroup on Efficacy Measurement of Cosmetics and Other Topical Products (Pie´rard et al.,1999a). From an engineering point of view, the skin and subcutaneous tissue represent anintegrated load-transmitting structure. It is subjected to intrinsic and environmental influ-ences. With ageing the intimate structures of the dermis loose their balanced tensilefunctions and respond less adequately to the casual mechanical demands. Such an inves-tigative approach was used to show evidence for the long-term beneficial effects ofhormone replacement therapy (HRT) on the skin of menopausal women. A prospective O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 longitudinal comparative trial showed a positive effect of HRT on facial skin elasticity, atleast in a subgroup of women (Pie´rard-Franchimont et al., 1999a). The efficacy of topicaltretinoin, a-hydroxyacid (AHA) and b-lipohydroxyacid (LHA) was compared usingimmunohistochemistry (Pie´rard et al., 1999b). Keratinocytes and dermal dendrocyteswere boosted by tretinoin and LHA so that their numbers and phenotypic charactersresembled those of younger skin.
The increased incidence of skin cancers in Wallonia was also scrutinized with the help of the Mosan Study Group on Pigmented Neoplasms (Pie´rard-Franchimont et al., 1999b).
It appears that the numbers of skin cancers reported by the Belgian National Registry areunderscored and unreliable.
6. Ageing and immunity
6.1. The influence of selected trace elements status on the immune system The influence on the immune system of selected trace elements status and supplementa- tion was studied in elderly subjects and in pathologic conditions showing how particularmicronutrients can modulate immune functions. In a first study, the effects on lymphocyteproliferation responses of a 6-months supplementation with either 100 mg selenium/day ora placebo were investigated in 22 elderly institutionalized subjects. Responses to mitogenstended to be lower in elderly subjects than in younger healthy adults. The proliferativeresponse to pokeweed mitogen increased significantly during selenium supplementationand reached the upper limit of the usual range for adults at the end of the trial. This trialwas the first time that demonstrated immunostimulatory effects for selenium in humans(Peretz et al., 1991a).
The relationships between immune response and selenium status was further investi- gated in 5 patients receiving home parenteral nutrition for short-bowel syndrome. With anelegant protocol including a depletion period with 20 mg selenium/day followed by adepletion period with 200 mg, it was possible to observe a significant improvement inthe lymphocyte response to 3 mitogens due to the increase in selenium daily intake.
Similarly, the response to 2 out of 3 antigens tested was also enhanced (Peretz et al.,1991b). Finally, the potential influence of zinc on the phagocytosis of polymorphonuclearcells was examined. The effects of a 60-day treatment with 45 mg zinc/day or a placebowere studied in 22 patients with inflammatory rheumatic diseases. While the phagocyticactivity of PMNs was significantly impaired in these patients before the trial, zinc supple-mentation increased the percentage of phagocytic PMNs and the mean phagocytic activity,particularly in subjects with initial low phagocytosis. This demonstrated that zinc is able tocorrect impaired phagocytosis in patients with inflammation (Peretz et al., 1994).
Before reacting against non-self infectious agents, the immune system has to tolerate the host molecular structure (self). The induction of self-tolerance is a multistep process whichbegins with the potent deletion of self-reactive T cells in the thymus (central tolerance),and which also involves inactivating mechanisms outside the thymus (peripheral O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 tolerance). Our knowledge of thymic physiology has considerably progressed during thelast few years. The thymus is the primary lymphoid organ implicated in the generation ofself-tolerant and competent T cells. From 100 T-cell progenitors that migrate in thethymus where they randomly rearrange gene segments coding for the variable part ofT-cell antigen receptor (TCR), only a maximum of 10 T cells will leave the thymus in astate of self-tolerance and competence against non-self antigens. The thymic repertoire ofneuroendocrine-related precursors, and particularly the thymic insulin-like growth factor(IGF) axis, transposes at the molecular level the dual role of the thymus in the negativeselection of self-reactive T-cells and in the development of peripheral T lymphocytes(Martens et al., 1996). Contrary to the previous assumption, it is more and more estab-lished that the thymus is responsible for the generation and maintenance of T lymphocytesthroughout life (Kecha et al., 2000), even during the course of HIV infection and afterintense chemotherapy (Jenkins et al., 1998; Mackall et al., 1995).
Clinically, immunosenescence is characterized by an increase of autoimmune responses, as well as by an increase of sensitivity to infectious agents. Both of thesephenomena result from a decrease in the homeostasis control of the immune systemand could be explained by a progressive defect of thymic normal function in shapingthe peripheral repertoire of self-tolerant and competent T cells.
While vaccinations must be promoted to palliate this progressive immunosenescence, an important benefit for the elderly may be expected from the development of therapiesoriented against thymic ageing.
In distinction to the course of reproductive ageing in women, men do not experience a rapid decline of Leydig cell function or irreversible arrest of reproductive capacity in oldage. Hence, strictu sensu, the andropause does not exist. Nevertheless, both spermatogen-esis and fertility as well as Leydig cell function do decline with age. The origin of thisdecline of Leydig cell function resides on the one hand in the testes, and is essentiallycharacterized by a decreased number of Leydig (and Sertoli) cells and on the other hand inthe hypothalamo-pituitary complex characterized by a decreased luteinizing hormone(LH) pulse amplitude. As the responsiveness of the gonadotrophs to gonadotropin-releas-ing hormone (GnRH) remains unimpaired, one may assume that the amount of GnRHreleased at each pulse is also reduced, possibly as the consequence of a reduction of thecellular mass of GnRH neurones (Vermeulen and Kaufman, 1995).
The progressive decline of gonadal function with, in particular, a decline of total and free testosterone (T) plasma levels results in a significant proportion of elderly men over60 years age presenting with subnormal T levels compared with the levels in young adults.
A great interindividual variation in T levels is observed in elderly men, a variabilityexplained in part by physiological variables and differences in life style, while associatedacute or chronic diseases may accentuate the age-related decline of T levels. The progres-sive decrease of plasma T levels has been shown to result from both primary testicularchanges and altered neuroendocrine regulation of Leydig cell function. At present, little isknown about the clinical relevance of the relative hypoandrogenism of elderly men andthere is an urgent need for more longitudinal studies, which may clarify a possible role of O. Toussaint et al. / Experimental Gerontology 35 (2000) 901–916 decreased T levels in the modulation of the clinical consequences of ageing in men. Inview of the lack of relevant controlled clinical trials having careful assessment of the risksand benefits of androgen replacement therapy in elderly men, this treatment should bereserved for selected patients with clinically and biochemically manifested hypogonad-ism, after careful screening for contraindications (Kaufman and Vermeulen, 1997).
Vermeulen et al. have also published a critical evaluation of simple methods for theestimation of free testosterone in serum, which is of use in older individuals (Vermeulenet al., 1999).
An increased response of LH and FSH in normal male aged 55 ^ 0.9 years compared to normal young adult, and a significant decrease of urinary 6 sulfatoxy melatonin excretionwere demonstrated (Legros and Delmotte, 1997) around a similar age.
An ‘Andropause Interdisciplinary Research Centre’ was recently organized at the University of Lie`ge. Its major projects are the study of: the relationship between endo-genous androgenic function and anxio-depressive states; sleep quality and endogenousmelatonin function; osteoporosis in middle aged man; dental health problems; sexualdysfunction in the elderly; cutaneous quality during early ageing. A close relationshiphas been established with the Menopause Clinic through the opening of a ‘Centre de lame´nopause et de l’andropause’ as an outpatient clinic of the hospital ‘Centre Hospitalierde Lie`ge’.
Acknowledgements
O. Toussaint is a Research Associate and F. Collette a post-doctoral fellow of the National Fund for Scientific Research (FNRS), Belgium.
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