Christoph Meissner
email: christoph.meissner@uk-sh.de

Petra Bruse

Salaheldien Ali Mohamed

Anja Schulz

Hanne Warnk

Thilo Storm

Manfred Oehmichen

The 4977 bp deletion of mitochondrial DNA in human skeletal muscle, heart and different areas of the brain: a useful biomarker or more?

Keywords: human skeletal muscle, human heart, human brain, mitochondrial DNA, 4977 bp deletion, ageing

It has been suggested that deletions of mitochondrial DNA (mtDNA) are important players with regard to the ageing process. Since the early 1990s, the 4977 bp deletion has been studied in various tissues, especially in postmitotic tissues with high energy demand. Unfortunately, some of these studies included less than 10 subjects, so the aim of our study was to quantify reliable the deletion amount in nine different regions of human brain, heart and skeletal muscle in a cohort of 92 individuals. The basal ganglia contain the highest deletion amounts with values up to 2.93% and differences in deletion levels between early adolescence and older ages were up to three orders of magnitude. Values in frontal lobe were on average an order of magnitude lower, but lowest in cerebellar tissue where the amount was on average only 5 x 10 -3 of the basal ganglia. The deletion started to accumulate in iliopsoas muscle early in the fourth decade of life with values between 0.00019% and 0.0035% and was highest in a 102 year-old woman with 0.14%. In comparison to skeletal muscle, the overall abundance in heart muscle of the left ventricle was only one third. The best linear logarithmic correlation between amount of the deletion and age was found in substanta nigra with r=0.87 (p<0.0005) followed by anterior wall of the left ventricle (r=0.82; p<0.0005). With regard to mitochondrial DNA damage, we propose to use the 4977 bp deletion as an ideal biomarker to discriminate between physiological ageing and accelerated ageing. The biological meaning of mitochondrial deletions in the process of ageing is under discussion, but there is experimental evidence that large-scale deletions impair the oxidative phosphorylation in single cells and sensitize these cells to undergo apoptosis.

Introduction

It has been postulated that alterations of mitochondrial DNA (mtDNA) are involved in the progression of the ageing process. MtDNA is a double stranded DNA ring, packed into a nucleo-protein complex, called the nucleoid [START_REF] Chen | The organization and inheritance of the mitochondrial genome[END_REF][START_REF] Holt | Mammalian mitochondrial nucleoids: organizing an independently minded genome[END_REF], lacks any introns and encodes for 13 proteins necessary for the assembly of the respiratory chain, 22 tRNAs and two ribosomal RNAs [START_REF] Graeber | Neurodegeneration and aging: role of the second genome[END_REF]. The general thinking for the past three decades has been that the ageing phenotype is a consequence of oxidative damage of proteins, lipids and DNA inflicted by free radicals which in turn are generated as normal byproducts of the oxidative phosphorylation (OXPHOS) (Miquel et al., 1980;[START_REF] Harman | The aging process[END_REF][START_REF] Ames | Endogenous oxidative DNA damage, aging, and cancer[END_REF]). The mtDNA is at higher risk, because it is located in close proximity of the respiratory chain, and displays reduced repair capacity relative to nuclear DNA. Oxidative damage of mtDNA can be detected on the molecular level as base adducts like 8hydroxy-2'-deoxyguanosine (8-OHdG). These modified bases are especially prone to mispairing of repetitive elements and are correlated with deletions [START_REF] Lezza | Mitochondrial DNA 4977 bp deletion and OH8dG levels correlate in the brain of aged subjects but not Alzheimer´s disease patients[END_REF]. This observation has led to the proposal that deletions are induced via mispairing and slippage during replication [START_REF] Shoffner | Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip-replication model and metabolic therapy[END_REF]. Nowadays it is quite clear that large-scale deletions may be attributed to polymerase slippage, homologous recombination, double strand breaks, inefficient repair mechanisms, or mutations in the helicase twinkle [START_REF] Zeviani | Nuclear genes in mitochondrial disorders[END_REF], Phadnis et al., 2005;[START_REF] Srivastava | Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans[END_REF]. Furthermore, deletion events may be inflicted by tissue specific nuclear genes, interactions between the mitochondrium and the nucleus or endonucleases that could shift mtDNA haplotypes [START_REF] Ballard | The mitochondrial genome: mutation, selection and recombination[END_REF][START_REF] Srivastava | Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans[END_REF].

The vast majority of large-scale deletions is located between the origins of replication O H and O L at np 5760 and np 190 of mtDNA [START_REF] Bank | Multiple deletions of mtDNA remove the light strand origin of replication[END_REF]. There is growing experimental evidence, especially from single cell studies that the overwhelming majority of large scale deletions is generated by clonal expansion of a single stochastic mutational event in childhood or early adolescence [START_REF] Elson | Random intracellular drift explains the clonal expansion of mitochondrial DNA mutations with age[END_REF][START_REF] Bodyak | Quantification and sequencing of somatic deleted mtDNA in single cells: evidence for partially duplicated mtDNA in aged human tissues[END_REF][START_REF] Storm | Mitotic brain cells are just as prone to mitochondrial deletions as neurons: a large-scale single-cell PCR study of the human caudate nucleus[END_REF][START_REF] Khrapko | Clonal expansions of mitochondrial genomes: implications for in vivo mutational spectra[END_REF][START_REF] Gokey | Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys[END_REF]. It seems reasonable that large-scale deletions may be involved in an age dependent decline of cell or tissue function, because essential parts of coding sequences are removed. The loss of the corresponding gene products may lead to faulty assembly of the respiratory chain. The resulting impaired function of OXPHOS, especially in postmitotic tissues with high energy demand can explain the decline of cognitive function and memory, cardiovascular pathologies or loss of muscular strength.

Additionally, mitochondria play a key role in the initiation of the apoptotic cascade, which may also play a role in the pathology of the ageing phenotype [START_REF] Kujoth | The role of mitochondrial DNA mutations in mammalian aging[END_REF].

The 4977 bp deletion of mtDNA has attracted the attention of the ageing community since its age dependency in several tissues has been reported in the early 1990s [START_REF] Cortopassi | A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues[END_REF]. Since then quantitative data for nearly all tissues (Corral-Debrinski et al., 1992a;[START_REF] Lee | Differential accumulations of 4,977 bp deletion in mitochondrial DNA of various tissues in human ageing[END_REF]Papiha et al., 1998;[START_REF] Zhang | Mitochondrial DNA deletions in human cardiac tissue show a gross mosaic distribution[END_REF]Meissner et al., 2000a;[START_REF] Koch | In human keratinocytes the common deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation[END_REF] or for specific cell types [START_REF] Kao | Mitochondrial deoxyribonucleic acid 4977-bp deletion is associated with diminished fertility and motility of human sperm[END_REF][START_REF] Steuerwald | Quantification of mtDNA in single oocytes, polar bodies and subcellular components by real-time rapid cycle fluorescence monitored PCR[END_REF][START_REF] Storm | Mitotic brain cells are just as prone to mitochondrial deletions as neurons: a large-scale single-cell PCR study of the human caudate nucleus[END_REF]Mohamed et al., 2004) have been collected. This large pool of data makes the 4977 bp deletion an ideal biomarker for age-related changes in a tissue or a special compartment thereof. Interestingly, various areas of the human brain have been found to harbour substantially different amounts of the 4977 bp deletion (Corral-Debrinski et al., 1992a;[START_REF] Soong | Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain[END_REF].

Though there are a number of studies on the 4977 bp deletion in human skeletal muscle, brain and heart, some important data are still missing. Until now there is no study comparing the amount of the 4977 bp deletion in human heart, skeletal muscle and different areas of the brain with the same method or in the same individuals.

Another important point to address is the detected differences in the amount of the deletion. Two studies published more than ten years ago demonstrated a difference of roughly two orders of magnitude in general in different brain regions of 6 and 7 individuals of various ages [START_REF] Soong | Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain[END_REF]Corral Debrinski et al., 1992a). In view of recent single cell or muscle fibre studies [START_REF] Bodyak | Quantification and sequencing of somatic deleted mtDNA in single cells: evidence for partially duplicated mtDNA in aged human tissues[END_REF][START_REF] Storm | Mitotic brain cells are just as prone to mitochondrial deletions as neurons: a large-scale single-cell PCR study of the human caudate nucleus[END_REF][START_REF] Khrapko | Clonal expansions of mitochondrial genomes: implications for in vivo mutational spectra[END_REF][START_REF] Gokey | Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys[END_REF], where large deletion amounts in single cells have been observed, the sample of less than 10 individuals seems not to be sufficient to obtain reliable data for a region of the brain. So the objective of our study was to collect reliable quantitative data on the common deletion in nine different areas of postmitotic tissues with high energy demand in a cohort (n=92) of individuals.

Material and methods

2.1 Sample material 100 mg tissue was collected at autopsy from substantia nigra, caudate nucleus, putamen, frontal cortex, cerebellum, anterior and posterior wall of the left ventricle of the heart, and iliopsas muscle of a total of 92 individuals. Tissue samples were immediately frozen in liquid nitrogen and kept at -70°C until DNA was extracted. To exclude mitochondrial or degenerative diseases a complete microscopic examination of each tissue sample was performed. Only individuals dying of acute or peracute causes (e.g. stabbing, suffocation, drowning) were included in the study. Fifty subjects were sampled for each of the nine tissues. Care was taken not to exceed an interval of 48 h between death and autopsy.

DNA extraction and quantitation

Total DNA was extracted from 100 mg tissue using the Super QUICK-GENE SQG1 Kit (Immucor, Roedermark, Germany) following the instructions of the manufacturer.

Quantitation of PCR products

Fragments were detected and quantified by the ABI Prism TM 310 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany) according to the method described elsewere (Mohamed et al., 2004). Briefly, a 1-µl aliquot of each PCR reaction and 0.5 µl of the internal size standard (GS350, Applied Biosystems, Weiterstadt, Germany) labelled with the fluorescent dye ROX (Applied Biosystems, Weiterstadt, Germany)

were denaturated and separated by capillary electrophoresis (CE) using the ABI Prism TM 310 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany). When the run was finished, specific fragments were displayed as peaks in an electropherogram using the Gene Scan Analysis Software 3.1. This software determines automatically fragment length and amount of a specific PCR product by calculating the area under the peak. The calculated value of the peak area corresponds directly to the amount of the product. To ensure that the signals derived from wild type molecules were in the exponential phase, the amount of wild type mtDNA was determined applying 14-18 cycles. When the quantitative results of the ABI Prism TM 310 Genetic Analyzer clearly indicate that the PCR-reaction of the sample was in the plateau phase, cycle number was reduced and the sample was quantified three times using the optimal number of cycles. The same approach was performed for deleted mtDNA, but numbers varied between 28-32 cycles. The frequency of the common deletion was determined by the ratio of the amount of the 123 bp deletion-specific fragment and the 113 bp fragment of intact mtDNA.

Statistics

Statistical analyses were performed using the SPSS statistical Package 14.0 (SPSS GmbH Software, Munich, Germany). In case of samples where the 4977 bp deletion was not detectable, a set of arbitrary abundance values between 0.00001% and 0.00009% was generated by the random number generation system of Microsoft Excel 11.0. The mean correlation coefficient r was obtained by taking the average of r values from the regression analyses based on 10 independent sets of randomly generated values below the detection limit of 0.00009%.

Results

Applying a quantitative PCR approach we calculated the amount of the 4977 bp deletion in nine different areas of postmitotic tissues of 92 different individuals (table 1). All of the tissues revealed a positive correlation between deletion amount and advancing age of the subjects, but there was a strong variability even between individuals of same age decade. In human brain the deletion was even detectable in individuals as young as 10 or 12 years (table 1). The different areas of the brain displayed striking differences in the level of the common deletion. The basal ganglia exhibit the highest deletion amounts with values up to 2.93% in substantia nigra of a 68-year-old individual and up to 2.14% in caudate nucleus of a 76-year-old person.

We observed differences in deletion levels between early adolescence and older ages of up to 2.6 x10 3 in caudate nucleus. The frequency of the common deletion was nearly the same in substantia nigra, putamen and caudate nucleus, but values in frontal lobe brain were on average an order of magnitude lower. The lowest values of the deletion were observed in cerebellar tissue were the amount was on average only 5 x 10 -3 of the basal ganglia. In a single case the common deletion was not detectable in cerebellum, in spite of an amount of 1.12% in substantia nigra of the same 93-year-old individual. In frontal lobe the deletion was detectable at the age of 12 with an amount of 0.0016% and reached values between 0.039% and 0.18% by the end of the sixth decade and 102 years, respectively (table 1). In skeletal muscle, the amount of the 4977 bp deletion was roughly half of that detected in frontal lobe.

In general, the deletion started to appear in iliopsoas muscle in the early thirties with values between 0.00019% and 0.0035%, but interestingly there were three individuals aged 0.5,1 and 8 years with a relatively high level of up to 0.00159%. The

Discussion

This study demonstrates, that there are striking differences in the amount of the common deletion. The abundance of the 4977 deletion is highest in the basal ganglia with values of more than three orders of magnitude higher in comparison to cerebellar tissue. It has been speculated, that the higher amount of the 4977 bp deletion may be due to tissue properties. The basal ganglia are regions of high iron levels, because these regions have low or no transferrin-binding sites, whereas grey matter regions with low levels of iron have a high density of transferrin-binding sites [START_REF] Griffiths | Iron in the basal ganglia in Parkinson' s disease[END_REF]. Additionally, in corpus striatum and substantia nigra brain dopamine levels are high in comparison to other regions. Auto-oxidation and deamination of dopamine by monoamine oxidase B, which level increases in an age dependent manner [START_REF] Goldstein | The role of the regulatory enzymes of catecholamine synthesis in Parkinson' s disease[END_REF], leads to the generation of H 2 O 2 , which can react with iron via the Fenton reaction forming the highly reactive hydroxyl radical [START_REF] Soong | Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain[END_REF]. Interestingly, when the number of mtDNA molecules carrying the deletion approached more than 1.5% of all mtDNAs in the basal ganglia, a plateau in the logarithmic scale was observed. It is suggested, that in case the amount of rearranged mtDNAs within the cell reaches a certain threshold, the oxidative stress becomes too high and the cell and its mutant mtDNAs are eliminated via the opening of the mitochondrial transition pore and subsequent apoptosis. A very interesting observation is the low abundance of the common deletion in the grey matter of the cerebellum. In alignment with our findings, earlier studies from other groups showed that the mtDNA of cerebellar cells has about 0.5% to 1% of the deletion amount of the basal ganglia [START_REF] Soong | Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain[END_REF]Corral-Debrinski et al., 1992a;Meissner et al., 2000b). Therefore, the cerebellum seems to be protected by tissue properties like anti-oxidant enzyme protection or status of repair systems [START_REF] Gershon | The mitochondrial theory of aging: is the culprit a faulty disposal system rather than indigenous mitochondrial alterations?[END_REF]. The remarkable quantitative differences in the deletion between the tissues seen here, indicate that the accumulation is not universal, but tissue specific. Because tissues exhibiting the lowest deletion load (cerebellum) and tissues with the highest deletion load (basal ganglia) are made up of neurons and glial cellls, tissue properties like dopaminergic connectivity must be more important than cell properties. It should be stressed, that mitochondrial deletions do not accumulate in postmitotic neurons only, but also (and at a similar rate) in dividing cells like astrocytes [START_REF] Storm | Mitotic brain cells are just as prone to mitochondrial deletions as neurons: a large-scale single-cell PCR study of the human caudate nucleus[END_REF]. In the same manner, deletion loads between iliopsoas muscle and heart varied up to threefold on average, despite both skeletal muscle and heart muscle consisting of striate myocytes. The differences are even more striking, when all mitochondrial deletions are analysed. In comparison to skeletal muscle, heart muscle tissue exhibits a deletion pattern that is uniform across individuals, indicating that tissue properties play the key role in accumulation of mitochondrial deletions [START_REF] Meissner | Tissue-specific deletion patterns of the mitochondrial genome with advancing age[END_REF].

All of the nine areas of postmitotic tissues exhibit an increase of the 4977 bp deletion in an age-dependent manner, which is in accordance with other studies [START_REF] Soong | Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain[END_REF]Corral-Debrinski et al., 1992a;[START_REF] Zhang | Differential occurrence of mutations in mitochondrial DNA of human skeletal muscle during aging[END_REF][START_REF] Liu | Mutations in mitochondrial DNA accumulate differentially in three different human tissues during ageing[END_REF]. Our data give the best correlation between age and ln mtDNA% for the substantia nigra (Fig. 1A), while the correlation for the right ventricle is much weaker (Fig. 1I). This may be due to tissue sampling artefacts, because samples of the right ventricle sometimes include the cell layers beneath the endocardium, which are especially prone to ischemic/hypoxic conditions, and therefore would explain increased amounts of the deletion [START_REF] Merril | Mitochondrial DNA deletions and chloramphenicol treatment stimulate the autophagic transcript ATG12[END_REF]. The relatively high linear correlation of ln(dmtDNA %)

with age in all the investigated brain tissues indicates that there is a basal rate in the accumulation of deletions which may be modulated by internal or external stressors.

The 4977 bp deletion and other deletion events are induced by external factors like UV radiation [START_REF] Berneburg | Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin[END_REF], ionising radiation [START_REF] Prithivirajsingh | Accumulation of the common mitochondrial DNA deletion induced by ionizing radiation[END_REF] deuterium oxide [START_REF] Berneburg | Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion[END_REF] or by pathologic conditions like chronic hypoxia/reperfusion [START_REF] Merril | Mitochondrial DNA deletions and chloramphenicol treatment stimulate the autophagic transcript ATG12[END_REF][START_REF] Bagenholm | Free radicals are formed in the brain of fetal sheep during reperfusion after cerebral ischema[END_REF], ischemia (Corral-Debrinski et al., 1992b) or end-stage renal disease [START_REF] Lim | Large-scale mitochondrial DNA deletions in skeletal muscle of patients with end-stage renal disease[END_REF]. All of these factors are associated with an increased generation of free radicals. Mitochondrial deletions are somatically acquired during human lifetime in a tissue. The first mutagenic event appears to be stochastic oxidative damage of single base pairs of mtDNA [START_REF] Lezza | Mitochondrial DNA 4977 bp deletion and OH8dG levels correlate in the brain of aged subjects but not Alzheimer´s disease patients[END_REF], inflicted by free radicals or other oxidative stressors. It is important to mention that damage of mtDNA is largely confined to the mitochondria themselves, because free radicals cannot diffuse very far. On the contrary, the amount of free radicals has been overestimated for a long time and is probably not more than 0.2% of mitochondrially consumed oxygen [START_REF] St-Pierre | Topology of superoxide production from different sites in the mitochondrial electron transport chain[END_REF].

Additionally, there is a lot of experimental evidence that the base excision repair (BER) pathway in mitochondria is efficient and oxidative lesions like 8-OHdG are removed from the genome [START_REF] Stevnsner | Mitochondrial repair of 8-oxoguanine and changes with aging[END_REF]. Therefore local repair mechanisms may be sufficient to hold most of the oxidative mtDNA damage in check, of which there might be not as much as had been assumed in the past. In case of oxidative damage of single base pairs, this mutagenic lesion favours the generation of large scale deletions, which are clonally expanded over the passage of time in a single cell as has been shown in an elegant experimental study [START_REF] Khrapko | Clonal expansions of mitochondrial genomes: implications for in vivo mutational spectra[END_REF], that confirmed a previous computer simulation approach [START_REF] Elson | Random intracellular drift explains the clonal expansion of mitochondrial DNA mutations with age[END_REF].

But there must be a selective force at work against mitochondrial deletions. The half life of a neuronal mitochondrion lies between 18 and 24 days [START_REF] Korr | Mitochondrial DNA synthesis studied autoradiographically in various cell types in vivo[END_REF]. This means that mitochondria in the brain of an 80-year-old individual have undergone anywhere between 1200 to 1600 duplications. Given that the half life of mitochondria is the same as the half life of mtDNA [START_REF] Gross | Apparent turnover of mitochondrial deoxyribonucleic acid and mitochondrial phospholipids in the tissues of the rat[END_REF] and that turnover of mitochondrial genomes alone would determine the accumulation rate, one would calculate a 10 18 -fold increase of a single initial mtDNA deletion during the human lifespan of eighty years in a tissue [START_REF] Simonetti | Accumulation of deletions in human mitochondrial DNA during normal aging: analysis by quantitative PCR[END_REF]. Our study clearly demonstrates that the common deletion in normal human caudate nucleus or iliopsoas muscle increases from young adulthood to old age merely by a factor of 10 3 , indicating a selective force against the accumulation of mitochondrial deletions, one of which may be apoptosis.

It has been shown in several studies that mitochondrial deletions can serve as an excellent biomarker of photoageing in human skin [START_REF] Koch | In human keratinocytes the common deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation[END_REF][START_REF] Berneburg | Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin[END_REF][START_REF] Eshaghian | Mitochondrial DNA deletions serve as biomarkers of aging in the skin, but are typically absent in nonmelanoma skin cancers[END_REF]. The results of the present study lead us to suggest in agreement with others [START_REF] Ross | Mitochondrial DNA damage in lymphocytes: a role in immunosenescence?[END_REF] to use the 4977 bp deletion as a biomarker of biological ageing in general, because age seems to reveal the strongest correlation with its abundance. In case of human oocytes it has been confirmed that age exhibits the only significant correlation with the presence of the 4977 bp deletion, while other parameters like smoking or early follicular phase FSH did not reveal any correlation [START_REF] Chan | Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes[END_REF]. In recent years, a number of carefully conducted studies reported, that the level of the 4977 bp deletion is not increased in different tumour tissues [START_REF] Dani | Less DeltamtDNA4977 than normal in various types of tumors suggests that cancer cells are essentially free of this mutation[END_REF][START_REF] Dai | Mitochondrial DNA 4977 BP deletion mutations in lung carcinoma[END_REF][START_REF] Ye | Quantitative analysis of mitochondrial DNA 4977-bp deletion in sporadic breast cancer and benign breast diseases[END_REF] and in conditions associated with chronic inflammation like ulcerative colitis [START_REF] Fukushima | Paradoxical decrease of mitochondrial DNA deletions in epithelial cells of active ulcerative colitis patients[END_REF]. Though our data display a great variability even in individuals of the same age group, the large pool of data may help to discriminate between physiological ageing and pathological conditions associated with accelerated ageing. Interestingly an increase in deletion amounts is observed in association with smoking [START_REF] Ballinger | Mitochondrial genome damage associated with cigarette smoking[END_REF], ethanol consumption [START_REF] Mansouri | Multiple hepatic mitochondrial DNA deletions suggest premature oxidative aging in alcoholic patients[END_REF] or sunlight exposure [START_REF] Ray | The spectrum of mitochondrial DNA deletions is a ubiquitous marker of ultraviolet radiation exposure in human skin[END_REF], all of which seem to accelerate the ageing process. There are at least three aspects that render the deletion an excellent biomarker. First, the high sensitivity of any PCR based assay reveals this deletion even in minute amounts in lymphocytes or other blood cells (Mohamed et al., 2004). The detection limit of 0.00009% as was observed in our study, nearly equals the detection of a single deleted molecule in one million molecules and is sensitive enough to show early signs of biological ageing.

Second, the common deletion is observable in all tissues, so a comparison of different tissues in the same individual is easy to achieve. Third, there seems to be a basal rate for the accumulation of the deletion with a high correlation to age only, which is not modulated by chronic inflammation, tumour development or progression.

As outlined above the abundance of the 4977 bp deletion is only determined by conditions associated with a increased generation of free radicals. In spite of the use of the common deletion as an ideal biomarker, with regard to damage of mtDNA the measurement of a panel of different deletions may be more useful to monitor biological ageing.

The role of the common deletion and other deletion events regarding the ageing process has been elucidated recently in single cell studies and cybrid experiments. It is obvious, that large scale deletions remove coding sequences of proteins and tRNAs, that are necessary for correct assembly of the ETC. This should impair the respiratory capacity of the affected mitochondrion where the ratio of deleted to wild type mitochondrial DNA molecules determines severity. The 4977 bp deletion encompasses the genes of four subunits of NADH dehydrogenase (Complex I), subunit 3 of cytochrome oxidase (COX; Complex IV) and for subunits 6 and 8 of ATPase (Complex V). The loss of these proteins is associated with severe mitochondrial diseases like Pearson's syndrome, Kearns-Sayre syndrome or chronic progressive external ophthalmoplegia (Peng et al., 2006). In these diseases the deletion amount exceeds usually 50%-55% in a given tissue and therefore is more than one to three orders of magnitude higher than in normal ageing tissues.

Therefore it is difficult to imagine that the 4977 bp deletion may account for the age related changes in a tissue. On the cellular level there is a lot of evidence that mtDNA deletions do have functional consequences, as is reflected by the correlations demonstrated between deletions and biochemical parameters. The fate of single cells carrying high amounts of the common deletion has been shown recently. In a lot of studies it was shown that single COX-negative fibres of skeletal muscle or COXnegative neurons contain a heterogeneous array of different deletions [START_REF] Kopsidas | Stochastic mitochondrial DNA changes: bioenergy decline in type I skeletal muscle fibres correlates with a decline in the amount of amplifiable fulllength mtDNA[END_REF][START_REF] Gokey | Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys[END_REF][START_REF] Bua | Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibres[END_REF][START_REF] Bender | High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease[END_REF][START_REF] Kraytsberg | Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons[END_REF]. In all studies a strong correlation between deletions, ETC abnormalities and even sarcopenia or Parkinsonism was observed. Thus, on the single-cell level there appears to be a causal link between the age-related rise in mitochondrial deletions and the reported reductions in respiratory chain enzyme activities. From this data it seems evident that the amount of intact mtDNA correlates with unimpaired function of the respiratory chain. However, even in single cells the impact of mitochondrial deletions is still being questioned, because in human heart and skeletal muscle the observed abundance of all deletion species per cell is in the order of 100 mtDNA molecules or less [START_REF] Kajander | Human mtDNA sublimons resemble rearranged mitochondrial genoms found in pathological states[END_REF], which should not impair the metabolism of the cell. Additionally, it is yet to be proven, whether mtDNA or ETC abnormalities considerably compromise metabolic function in muscle bundles or even in complete muscles, because in muscle biopsies of aged human individuals no change in ETC activity was observed [START_REF] Rasmussen | Experimental evidence against the mitochondrial theory of aging. A study of isolated human skeletal muscle mitochondria[END_REF][START_REF] Chabi | Random mtDNA deletions and functional consequence in aged human skeletal muscle[END_REF]. Conversely, one has to keep in mind that skeletal muscle is organized in a segmental manner and the force of the muscle bundle depends on the weakest segment where COX-negative fibres are present in a significant number of cells [START_REF] Bua | Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibres[END_REF]. It has been estimated, that 6% -31% of fibres with ETC abnormalities may account for fibre breakage and, ultimately, fibre loss [START_REF] Bua | Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibres[END_REF]. As mentioned above mtDNA deletions eliminate tRNAs essential for mitochondrial translation. Translation should not be possible in mitochondria containing only deleted mitochondrial genomes (Mita et al., 1989). In a heteroplasmic state, the amount of wild type mtDNA determines the amount of synthesized proteins. The inhibition of protein synthesis could induce the autophagic transcipt ATG12, which is involved in an early step of the mammalian autophagy pathway, probably as a cytoprotective mechanism (Prigione and [START_REF] Merril | Mitochondrial DNA deletions and chloramphenicol treatment stimulate the autophagic transcript ATG12[END_REF].

There are observations leading to the conclusion that mitochondrial deletions play a role in apoptosis of single cells. Cybrids with amounts of the 4977 bp deletion ranging between 16% and 80% of all mitochondrial genomes exhibit a significantly enhanced ROS production. Treatment of these cells with H 2 O 2 induced the morphological signs of apoptosis like membrane blebbing or nuclear condensation (Peng et al., 2006).

Other cybrid experiments indicated that the generation of mitochondrial ROS is increased depending on the deletion amount, but displayed no threshold effect because elevated mitochondrial ROS production was even detectable in cells with a relatively low amount of 16% of the common deletion (Peng et al., 2006[START_REF] Indo | Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage[END_REF]. UV-induced programmed cell death was increased in cybrids harbouring amounts of the 4977 bp deletion up to 80% leading to the suggestion that large scale deletion sensitize a cell to undergo apoptosis (Schoeler et al., 2006;[START_REF] Liu | Quantitative effect of 4977 bp deletion of mitochondrial DNA on the susceptibility of human cells to UV-induced apoptosis[END_REF].

One has to keep in mind that not the common deletion alone, but up to 200 different types of deleted mitochondrial genomes have been detected in bulk tissues of skeletal muscle, human heart and brain [START_REF] Hayakawa | Age-related extensive fragmentation of mitochondrial DNA into minicircles[END_REF]Kowalenko et al., 1997;[START_REF] Meissner | Tissue-specific deletion patterns of the mitochondrial genome with advancing age[END_REF]. Therefore it seems conceivable that any enhancement of oxidative stress by external or internal conditions will initiate the apoptotic cascade in deletion bearing cells. An ongoing apoptosis may exhaust stem cell renewal capacities, leading to the ageing phenotype with features such as hair greying, cardiac enlargement or sarcopenia [START_REF] Kujoth | Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging[END_REF][START_REF] Kujoth | The role of mitochondrial DNA mutations in mammalian aging[END_REF].

Therefore the total of all mitochondrial deletions can promote a deterioration of tissue function through the loss of irreplaceable cells by programmed cell death [START_REF] Meissner | Mutations of mitochondrial DNA -cause or consequence of the ageing process?[END_REF].

Conclusion

The age-related increase of the common deletion is not universal, but clearly tissue-

Fluorometric

  quantification of the extracts was performed by applying the PicoGreen dsDNA Quantitation Kit according to the recommended assay protocol (Molecular Probes, Willow Creek, OR, USA) and a fluorometer (Bio-Rad, Munich, Germany). The PicoGreen quantitation reagent is a sensitive fluorescent stain for quantifying double-stranded DNA without interference of extracted RNA. 2.3 PCR analysis PCR was performed in 25 µl reaction mixture containing 50 mM KCl, 1.5 mM MgCl 2 and 10 mM Tris-HCl (pH 8.4), 200 µM of each of the dNTP's including dUTP, 0.5 µM each of the primers, 0.5 U AmpErase UNG, 1 U of Platinum TM Taq DNA polymerase (Life Technologies, Gaithersburg, MD, USA), and 5 ng of total DNA in the GeneAmp 2400, thermal cycler (Applied Biosystems, Weiterstadt, Germany). Cycling conditions were as follows: Beginning with one initial step with AmpErase UNG incubation at 50° C for 2 min, 14-18 or 28-32 cycles at 95°C for 15 s, 60°C for 30 sec and 72°C for 15 s were performed. Forward primers were labeled with 6-FAM by the manufacturer (Applied Biosystems, Weiterstadt, Germany). A fluorescent labelled 113 bp fragment of the undeleted mtDNA resulting from amplification with primers int1F 5-3' was used to amplify a fluorescent labeled 123 bp fragment specific for mtDNA carrying the 4977 deletion.

  highest abundance was detected in a 102-year-old woman with 0.14%. This means roughly a 1000-fold increase from the early thirties up to very old age. In human heart the 4977 bp deletion was detected around thirty years of age, too, but it was even observed in the anterior wall of the left ventricle and the right ventricle of a one year old child and even in the right ventricle of a half year old child with an abundance of 0.00475%. The highest amount (0.0929%) was observed in the posterior wall of a 31year old individual. At around age 85 the abundance of the 4977 bp deletion varied between 0.0109% and 0.0282% (table1). Interestingly the total amounts in the anterior and posterior wall of the left ventricle were very similar in the majority of the subjects, but varied up to fivefold in some individuals. In comparison to skeletal muscle, total deletion abundance in heart muscle of the left ventricle was only one third. The amount of the 4977 bp deletion displayed the greatest diversity with regard to age when the right ventricle was analysed. The lowest detectable value was 0.00015% in a normal adult 31 years old, the highest 0.17% in an 8-year-old individual. Comparing the nine different tissues examined, our data clearly show the overall abundance of the common deletion varies with tissue type, in the following descending order: substantia nigra > caudate nucleus > putamen > right ventricle > frontal lobe brain > skeletal muscle > left ventricle (anterior and posterior wall) > cerebellum. The best linear logarithmic correlation between amount of the deletion and age was achieved in substanta nigra with r= 0.87 (p<0.0005), followed by anterior wall of the left ventricle (r=0.82; p<0.0005), caudate nucleus (r=0.80; p<0.0005), putamen (r=0.79; p<0.0005), frontal lobe brain (r=0.79; p<0.0005), iliopsoas muscle, (r=0.70; p<0.0005), posterior wall of the left ventricle (r=0.67; p>0.0005), cerebellum (r= 0.66; p<0.0005) and right ventricle (r=0.57; p<0.0005). The data clearly indicate an agedependent accumulation of the 4977 bp deletion in the nine different regions of postmitotic tissues (Fig. 1A -Fig. 1I).

  specific. It has been demonstrated in our study and others that different organs or even different areas of the same organ accumulate the 4977 bp deletion at different rates, with differences up to more than three orders of magnitude. This makes this deletion an ideal biomarker to determine age related changes in a given tissue. With regard to a causal link between the amount of the common deletion and a decline of tissue function with advancing age, it is not conceivable that one single mitochondrial deletion can be the sole cause of the ageing phenotype. On the other hand, recent single cell studies and cybrid experiments have shown conclusively that the common deletion predisposes for an increased sensitivity to oxidative stress and therefore triggers apoptosis. It is well known that up to 200 different types of deleted mitochondrial genomes are detectable in postmitotic tissues. The clonal amplification of any deletion in a single cell will heighten the risk of this cell to undergo programmed cell death. An increased apoptosis in itself may lead to an exhaustion of stem cell renewal capacity that may then contribute to the ageing phenotype with a deterioration of function in tissues with high energy demand.

Table 1 .

 1 associated with chronic hypoxia and brain mitochondrial DNA deletions. Arch. Amount of the 4977 bp deletion (in %) in different regions of postmitotic tissues

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	Miquel, J., Economos, A.C., Fleming, J., Johnson, J.E. Jr., 1980. Mitochondrial role in
	cell aging. Exp. Gerontol. 15, 575-591
	Mita, S., Schmidt, B., Schon, E.A., DiMauro, S., Bonilla, E., 1989. Detection of
	"deleted" mitochondrial genomes in cytochrome-c oxidase-deficient muscle fibers of
	a patient with Kearns-Sayre syndrome. Proc. Natl. Acad. Sci USA 86, 9509-9513
	Mohamed, S.A., Wesch, D., Blumenthal, A., Bruse, P., Windler, K., Ernst, M.,
	Kabelitz, D., Oehmichen, M., Meissner, C., 2004. Detection of the 4977 bp deletion of
	mitochondrial DNA in different human blood cells. Exp Gerontol. 39, 181-188
	Papiha, S.S., Rathod, H., Briceno, I., Pooley, J., Datta, H.K., 1998. Age related
	somatic mitochondrial DNA deletions in bone. J. Clin. Pathol. 51, 117-120
	Peng, T.I., Yu, P.R., Chen, J.Y., Wang, H.L., Wu, H.Y., Wei, Y.H., Jou, M.J., 2006.
	Visualizing common deletion of mitochondrial DNA-augmented mitochondrial reactive
	oxygen species generation and apoptosis upon oxidative stress. Biochim. Biophys.
	Acta 1762, 241-255
	Phadnis, N., Sia, R.A., Sia, E.A., 2005. Analysis of repeat-mediated deletions in the
	mitochondrial genome of Saccharomyces cerevisiae. Genetics 171, 1549-1559


Dr. Dr Wolfgang Wagner
email: wolfgang_wagner@med.uni-heidelberg.de

Patrick Horn

Simone Bork

Anthony D Ho

AGING OF HEMATOPOIETIC STEM CELLS IS REGULATED BY THE STEM CELL NICHE

Keywords: hematopoietic stem cells, mesenchymal stem cell, microenvironment, differentiation, rejuvenation, senescence

Adult stem cells provide the basis for regeneration of aging tissue. Their dual ability for selfrenewal and multilineage differentiation is controlled by direct interaction with a specific microenvironment -the so called "stem cell niche". Hematopoietic stem cells (HSC) reside in the bone marrow. It is still under debate if HSC can rejuvenate infinitively or if they do not possess "true" self-renewal and undergo replicative senescence such as any other somatic cell. Furthermore, the question arises to what extent age-related changes in HSC are due to intrinsic factors or regulated by external stimuli. There is growing evidence, that the stem cell niche is most important for the regulation of cellular aging in adult stem cells. It is the stem cell niche that (i) maintains HSC in a quiescent state that reduces DNA damage as well as replicative senescence, (ii) protects from radicals and toxic compounds, (iii) regulates cell intrinsic signal cascades and (iv) modulates gene expression and epigenetic modifications in HSC. Thus, the interplay with the stem cell niche controls HSC function including the aging process of the hematopoiesis.

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Stem cells and aging

Aging is a complex process that involves every cell and organ in the body and that leads to the deterioration of many body functions over the lifespan of an individual. The reduced capacity to regenerate injured tissues or organs and an increased propensity to infections and cancers are probably the most prominent features of aging. Adult stem cells such as hematopoietic stem cells (HSC) or mesenchymal stem cells (MSC) assure lifelong regeneration of adult tissues (e.g. all different types of blood cells or bone, fat and cartilage respectively). If the rejuvenating effect of stem cells was perfect, senescent cells could be replaced indefinitely. In the context of time and aging, stem-cell research is important for two reasons. On the one hand, stem cells are a good model to study aging as they provide the basis for regeneration of aging tissue. On the other hand, finding ways to reactivate stem cells and control their target destination might create unforeseen opportunities for treatment of degenerative diseases. Hence, all aging phenomena can be interpreted as signs of aging at the level of somatic stem cells -a human body is as old as its stem cells [START_REF] Ho | Stem cells and ageing. The potential of stem cells to overcome age-related deteriorations of the body in regenerative medicine[END_REF].

All normal somatic cells in mammals stop proliferation after a certain number of cell divisions in vitro before they enter a state called senescence. Senescent cells are viable and metabolically active but no longer have the ability to proliferate and this is in contrast to reversible forms of cell cycle arrest such as quiescence. Since the first description of replicative senescence, it has been speculated that this process might also govern aging of the whole organism. Two fundamental ways have been hypothesized how this process may be governed: Aging might either be the result of a purposeful program driven by genes or rather be evoked by stochastic or random, accidental events [START_REF] Hayflick | Biological aging is no longer an unsolved problem[END_REF]. Progressive shortening of the telomeres or modified telomeric structures is thought to be a main trigger for replicative senescence. Telomerase activity in normal human cells is generally undetectable leading to successive erosion of telomeres with each cell division. In contrast germline cells, embryonic stem cells (ESC) and adult stem cells maintain their telomeres by activated telomerase and this counteracts replicative senescence. However, this mechanism 2003). There is also evidence that senescence involves DNA damage and oxidative stress [START_REF] Kiyono | Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells[END_REF][START_REF] Ho | Stem cells and ageing. The potential of stem cells to overcome age-related deteriorations of the body in regenerative medicine[END_REF]. Clearly, cellular senescence is a complex process and the sequence of its molecular events is thus far unknown.

In general, adult stem cells have a slow turnover and reside in specialized niches, protected from the environment and only a few are activated at a time [START_REF] Wilson | Bone-marrow haematopoietic-stem-cell niches[END_REF].

Most of the long-term self-renewing HSC remain in G0 phase in contrast to transient amplifying progenitors cells [START_REF] Cheshier | In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells[END_REF]. The stem cell niche governs asymmetric cell division of two daughter cells that may either self-renew to replenish the stem cell pool or to differentiate to more committed cells [START_REF] Ho | The beaty of asymmetry -asymmetric divisions and self-renewal in the hematopoietic system[END_REF]. In addition the stem cell niche might also play a crucial role in regulation of aging. In this review we will discuss how aging of HSC might be influenced and regulated by the microenvironment.

Self-renewal and differentiation -the hallmark of stem cells

Modern stem cell research began in 1963 when Siminovitch et al. proved the existence of hematopoietic stem cells (HSC) in the bone marrow (BM) of mice [START_REF] Siminovich | The distribution of colony-forming cells among spleen colonies[END_REF]. In series of experiments they showed that cells from the bone marrow could reconstitute hematopoiesis and hence rescue lethally irradiated recipient animals. Furthermore, by serial transplantations, they demonstrated self-renewal ability of the original bone marrow cells.

Based on these experiments HSC were defined by their capability for self-renewal to maintain the stem cell pool as well as multilineage differentiation. This dual function is still considered as the hallmark for adult stem cells. The question remains however, if selfrenewal of stem cell function does also imply rejuvenation or if stem cells undergo the process of aging such as any other somatic cell.

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Hematopoietic stem cells -loss or accumulation with aging?

Analysis of aging in HSC is limited by two hurdles: the available methods for their isolation as well as by the problem to precisely define their functional characteristics. The cell surface glycoprotein CD34 is used as a surrogate marker for human HSC. The majority of CD34 + cells however comprise more committed progenitor cells and HSC can be further enriched in the CD34 + /CD38 -cell fraction. In the murine system a variety of further markers have been described such as c-Kit + /Thy1.1 lo /Sca-1 + /Lin -(KTSL) or a combination of signaling lymphocyte activation molecules (SLAM) receptors (CD150 + /CD244 -/CD48 -or CD150 + /CD48 - /CD41 -) [START_REF] Kiel | SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells[END_REF]. Despite these efforts it is so far not possible to precisely define a pure population of primitive HSC with repopulation ability. Demonstration of intrinsic changes, cellular-senescence or numeric changes in HSC during the process of aging is therefore limited by cell fractions enriched in HSC and this might be a reason for contradictory observations between different studies (Dykstra and de Haan, 2008).

Furthermore, analysis of the aging process in stem cells is limited by the available methods for functional characterization. Various in vitro assays provide an estimate for the number and maintenance of primitive hematopoietic progenitor cells (HPC) such as colony-forming unit (CFU) assay, cobblestone area forming cell (CAFC) assay, long-term culture-initiating cell (LTC-IC) assay or multilineage initiating cell (ML-IC) assay (de Wynter and Ploemacher, 2001). Murine in vivo transplantation models such as competitive repopulation unit (CRU) assay or SCID-repopulating cell (SRC) assay represent a more authentic test system for engraftment and differentiation potential. On the other hand, transplantation assays are very complex, the immune system of these mice is altered and there are significant differences between different mouse strains. Finally, mice may not be an adequate model organism for the human stem cell niche. Each of these assays has its advantages and disadvantages and the results of two different assays do not necessarily correlate with each other.

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Despite these limitations recent data indicated that the numbers of HSC increase with age.

This was unexpected, as it has been anticipated that aging may be caused by the depletion of stem and progenitor cells and therefore reduced regeneration potential. Competitive repopulation assays in murine transplantation models demonstrated that older bone marrow contains a higher number of repopulating HSC (2-3 fold higher) [START_REF] Morrison | The aging of hematopoietic stem cells[END_REF]. This is in line with similar in vitro experiments using the cobblestone area forming cell (CAFC) assay as a surrogate assay for HSC that demonstrated that the number of HSC increases about four-fold with age (de Haan and Van Zant, 1999;Dykstra and de Haan, 2008). On the other hand, there is evidence that the increase in HSC number comprises mainly progenitor cells whereas a loss of more primitive HSC occurs [START_REF] Dykstra | Hematopoietic stem cell aging and self-renewal[END_REF]). Various studies have indicated that even though similar HSC concentrations could be found in young and old bone marrow, it is the functional ability per cell in the repopulation model that shows a significant reduction with increasing donor age [START_REF] Chen | Senescence and functional failure in hematopoietic stem cells[END_REF]). It has been described that HSC from older mice have a significantly lower cycling activity than those isolated from young mice (de Haan et al., 1997). Furthermore, age-related changes in HSC number in murine models are highly strain-dependent (Dykstra and de Haan, 2008). Whereas the impact of aging on the quantity and quality of murine HSC has been recently studied, information on are-related changes in human HSC is scarce. Studying this is also a challenge because these cells can maintain their function much longer than the average human lifespan. It needs to be demonstrated if the knowledge obtained from murine transplantation models can be extrapolated to the human system.

Aging of the stem cell niche

Various intrinsic programs and pathways facilitate the unique characteristics of stem cells.

These programs regulate maintenance, proliferation and differentiation of HSC but they have to be tightly controlled by the microenvironment according to the physiological needs of the organism. 1978 Schofield proposed a hypothesis in which stem cells are seen in association with other cells which determine their behavior -the so called "stem cell niche" (Schofield,

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1978). For hematopoietic stem cells two types of niches have been identified in the bone marrow: the endosteal niche and the vascular niche. There is evidence that the most primitive HSC are located close to the endosteum of the bone in proximity to stromal cells and osteoblasts. Presumably the reciprocal communication and adhesion between these cell types regulate stem cell function [START_REF] Zhang | Identification of the haematopoietic stem cell niche and control of the niche size[END_REF][START_REF] Jones | No place like home: anatomy and function of the stem cell niche[END_REF]. HSC are also found in the vicinity of sinusoidal endothelial cells, which are referred to as the vascular niche. It has been suggested that the vascular niche forms a milieu that supports proliferation, differentiation and trans-endothelial migration of HSC, whereas the endosteal niche supports quiescence and self-renewal [START_REF] Sugiyama | Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches[END_REF][START_REF] Geiger | Stem cells, aging, niche, adhesion and Cdc42: a model for changes in cell-cell interactions and hematopoietic stem cell aging[END_REF].

The precise mechanism of this interaction of HSC with their local environment is largely unknown. However, it is certain, that the composition of cell types and extracellular matrix components in the bone marrow varies extensively during lifetime. These changes are reflected by decreased bone formation and loss of bone mass and they are more pronounced in patients with osteoporosis. This is in conjunction of enhanced adipogenesis and down-regulation of osteoblastogenesis [START_REF] Justesen | Adipocyte tissue volume in bone marrow is increased with aging and in patients with osteoporosis[END_REF]. Furthermore, the composition of the mature hematopoietic cells changes during lifetime. Especially the immune system is affected by aging: the lymphoid differentiation potential in bone marrow of elderly donors is significantly reduced [START_REF] Linton | Age-related changes in lymphocyte development and function[END_REF] somewhere between these (figure 1).

1) HSC can rejuvenate infinitively

"True" self-renewal of stem cells indicates that the daughter cell possesses the same molecular features and functional abilities as the mother cell and this should also include their rejuvenation. This would enable infinite maintenance of the stem cell pool. If this was the case, adult stem cells could also provide an infinitive basis for tissue regeneration. In this context, the continuous loss or regenerative potential of aging tissue might be attributed to a reduction in the number of primitive stem cells. Prove of this thesis is hampered by the crude functional measurement of HSC function that would need to discriminate between true stem cells and progenitor cells. Furthermore, demonstration that the daughter cells share precisely the same molecular features as their parent cells is not possible due to the fact that the parent cell is no more existent after cell division (Dykstra and de Haan, 2008). Thus, the theory of infinitive rejuvenation of HSC as it is observed in germline stem cells or ESC can hardly be verified.

2) HSC undergo cellular aging

In contrast to the above mentioned thesis HSC could undergo replicative senescence such as all mammalian cells. In this case there is no "true" self-renewal as the daughter cells may one became more specified [START_REF] Giebel | Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division[END_REF]. We have exploited differences in division kinetics to separate the CD34 + /CD38 -cells into a SDF and a committed, fast-dividing fraction (FDF). Several molecular markers associated with primitive stem cell function are higher expressed in the SDF compared to the FDF [START_REF] Wagner | Molecular evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis[END_REF]. The underlying reason for quiescence or slow cell division might either be protection from mutations during DNA replication or prevention of replicative exhaustion of stem cells.

3) Cellular aging is regulated by the microenvironment

A number of studies have demonstrated that hematopoiesis is affected by the aging of the In telomerase knockout mice B-lymphopoiesis is impaired whereas myelopoiesis is increased. Furthermore telomere dysfunction and aging limited the engraftment of transplanted wild-type HSC [START_REF] Ju | Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment[END_REF]. These results provide evidence, that the supportive function or the size of the stem cell niche is modified during the progress of aging.

4) The microenvironment keeps HSC in a quiescent state to prevent replicative senescence

Whereas HSC are kept in a quiescent state under normal conditions they have to be activated to enhance hematopoiesis according to the physiological needs, especially after chemotherapy. Cell division, self-renewal and differentiation are tightly controlled by the stem cell niche [START_REF] Ho | The beaty of asymmetry -asymmetric divisions and self-renewal in the hematopoietic system[END_REF]. Keeping HSC in a quiescent state might reduce the number of mutations that could ultimately lead to the development of leukemia. On the other hand this might delay exhaustion of primitive HSC by replicative senescence. If HSC undergo replicative senescence, then aging is modulated indirectly by external stimuli of the stem cell niche that regulate their proliferation.

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Stromal cells as a model system for the stem cell niche

In 1973 Dexter and coworkers described that by co-cultivation of hematopoietic bone marrow cells with thymus cells it was possible to maintain different stages of granulocytic cells for at least ten weeks [START_REF] Dexter | Stimulation of differentiation and proliferation of haemopoietic cells in vitro[END_REF]. These early experiments demonstrated that feeder layer cells may provide a supportive cellular environment to maintain primitive function of hematopoietic cells. Although numerous studies have demonstrated the vital role of stromal feeder layers for maintenance of multi-potency of HPC in vitro, little is known about the precise cellular and molecular mechanisms of this interaction. HPC are characterized by rapid migratory activity and constantly changing morphology. They actively migrate towards stromal cells and extend magnupodia in the direction of the latter [START_REF] Wagner | Hematopoietic progenitor cells and cellular microenvironment: behavioral and molecular changes upon interaction[END_REF]. The essential role of direct cell-cell contact for the regulation of self-renewal and differentiation of adult stem cells has been shown in various organisms and cell systems. Specific junctional complexes may play a similar role in the hematopoietic system. Various adhesion proteins have been implicated in this interaction such as N-cadherin, integrin beta-1 (CD29), VCAM1, NCAM1, notch and CD44 (Wagner et al., 2007a;Wagner et al., 2007b) and further studies will be necessary to determine the relative significance of these cell adhesion proteins. This represents an artificial model system that does not take into account the complex composition of cellular and extracellular components of the stem cell niche. However, it may help to further elucidate the molecular mechanisms involved in the regulation of stem cell function including its influence on cellular aging. Supporting this concept, it has been demonstrated that stromal cells from younger bone marrow have a higher hematopoiesis supportive activity compared to those from old bone marrow samples [START_REF] Mauch | Decline in bone marrow proliferative capacity as a function of age[END_REF].

Furthermore, we observed a lower proliferation rate on hematopoiesis supportive feeder layer cells in comparison to non-supportive MSC from adipose tissue and these results indicate, that supportive stromal cells keep HPC in a quiescent state (Wagner et al., 2007a).

Finding a method for culture expansion of primitive HSC would be of high interest for stem cell biology and for clinical application. So far ex vivo expansion of HSC resulted prevailing in

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expansion of mature progenitors rather than immature HSC and this might partly be due to senescence of HSC under in vitro conditions. A better understanding of the interaction between HSC and the stem cell niche and of the mechanisms involved in regulation of stem cell aging might pave the way for reliable expansion of HSC.

Stem cell aging is regulated by the microenvironment

As mentioned above, there is evidence that the microenvironment influences aging of HSC.

The precise molecular mechanisms are still unknown. Various effectors for aging have recently been described and some of these might also subjected to external control by the stem cell niche (figure 2). The following examples show that age related changes of hematopoiesis are a result of intrinsic alterations in the HSC themselves as well as of the hematopoietic microenvironment that regulates and maintains stem cell function.

Progressive loss of telomeres

The successive shortening of telomere length with every cell division is one of the best described mechanisms involved in cellular aging. Telomeres are elongated by the enzyme telomerase. Absence of telomerase results in persistent telomere shortening and finally loss of telomere function, which is associated with replicative senescence or apoptosis. Stem cells normally express telomerase in contrast to most somatic cells. However, in HSC telomerase activity is low and thus telomere shortening still occurs during aging and limits their proliferative lifespan (Allsopp et al., 2003a). On the other hand, in a serial mouse transplantation model with telomerase over-expression HSC of both transgenic and nontransgenic animals could be serially transplanted no more than four times (Allsopp et al., 2003b). These results indicate that other mechanisms can limit stem cell function as well.

Furthermore, telomere dysfunction impairs also the stem cell niche. Using telomerase knockout mice (Terc -/-) Ju and coworkers demonstrated that telomerase dysfunction induces alterations in the bone marrow microenvironment by diminishing the stromal cell compartment and reducing the capacity of bone marrow stromal cells in their hematopoiesis

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supportive activity [START_REF] Ju | Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment[END_REF]. The Terc -/-dysfunctional environment limited the engraftment of transplanted wild-type HSC. The reduced hematopoieisis supportive activity was age dependent and correlated with progressive telomere shortening in bone marrow stromal cells. Furthermore, telomere dysfunction altered the expression of various cytokines in the plasma of aging Terc -/-mice [START_REF] Ju | Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment[END_REF]. These results provide clear evidence, that telomere shortening is neither the unique nor an exclusively intrinsic mechanism for cellular aging in HSC. In contrast the loss of telomeres induces age dependent alterations in the stem cell environment that can impair the function and engraftment of HSC.

Reactive oxygen species and other toxic compounds

Aging of HSC might also be attributed to a continuous accumulation of cellular defects for example by damaging compounds such as reactive oxygen species (ROS). ROS arise as byproducts of numerous metabolic processes and it causes direct damage to proteins, membranes and DNA. The oxidative stress mediates activation of several stress induced proteins as ERK, p38 MAPK, or p53. Glycation of proteins provides another harmful mechanism that leads to cross-linked proteins [START_REF] Finkel | Oxidants, oxidative stress and the biology of ageing[END_REF]. Over time, these proteins as well as oxidant-damaged DNA accumulate and eventually disrupt cellular function. In this context it is intriguing to note, that HSC in their stem cell niche are predominantly located at the lowest end of an oxygen gradient in the bone marrow. There is experimental evidence that that regionally defined hypoxia plays a fundamental role in regulating stem cell function [START_REF] Fehrer | Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan[END_REF][START_REF] Parmar | Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia[END_REF]. The protection of the stem cell niche from harmful compounds might play a central role in prevention of HSC aging.

Regulation of gene expression

Recent studies comparing gene expression profile of purified HSC from young and old mice showed up-regulation of genes involved in inflammatory and stress within age [START_REF] Chambers | Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation[END_REF]. Whereas the functional role of these genes has been demonstrated it is yet unknown how they are regulated in the process of aging and to which degree the

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microenvironment is involved in this mechanism. Co-cultivation of HSC with stromal cells provides a good model to get a first idea about the influence of the stem cell niche on the gene expression profile of HSC. We have previously described that a multitude of genes was differentially expressed in CD34 + /CD38 -HPC upon interaction with stromal cells. Among the genes up-regulated were many involved in regulation of cell division, reorganization of the cytoskeleton and maintenance of genetic stability [START_REF] Wagner | Hematopoietic progenitor cells and cellular microenvironment: behavioral and molecular changes upon interaction[END_REF]. Therefore, gene expression of HSC is not a purely intrinsic process but it is regulated by the interplay of different cell types of the stem cell niche.

Regarding the gene expression involved in aging of stem cells we have recently observed continuous changes in the expression of several miRNAs in the process of replicative senescence in MSC (manuscript in revision). Mature miRNAs constitute a group of endogenous small, non coding RNAs of approximately 22 nucleotides in length that are expressed in a tissue-and cell-type specific manner and play essential roles in development.

These single miRNA molecules, cleaved from 70-100 nucleotide hairpin pre-miRNA precursors exert a post-transcriptional effect on gene expression [START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF]) and some of these might be involved in the process of aging. For the nematode worm Caenorhabditis elegans it was demonstrated that over-expression of lin-4 led to extended life span, and an overall age-related decline in miRNA expression was described [START_REF] Boehm | A developmental timing microRNA and its target regulate life span in C. elegans[END_REF][START_REF] Ibanez-Ventoso | Modulated microRNA expression during adult lifespan in Caenorhabditis elegans[END_REF]. For the MSCs we assume that the up-regulation of specific miRNAs has functional implications for cellular aging and this will be addressed in the future.

Regulation of epigenetic modifications

Epigenetic mechanisms such as DNA methylation or histone acetylation regulate gene transcription at the chromatin level. As these modifications can be inherited by the daughter cells they have been demonstrated to play key roles in altering the long-term fate of HSC and hence, they might also be involved in the regulation or stem cell aging [START_REF] Ruau | Pluripotency Associated Genes are Reactivated by Chromatin Modifying Agents in Neurosphere Cells[END_REF].

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Gene expression analysis of HSC from old mice compared with those from young mice revealed that genes involved in chromatin remodelling and transcriptional silencing were found to be down-regulated. Furthermore, inapposite expression of genes normally regulated by epigenetic mechanisms was observed [START_REF] Chambers | Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation[END_REF]Rossi et al., 2007a).

Culture-induced decline of human HSC can be diminished by treatment with DNA methylation or histone acetylation interfering drugs whereas self-renewal is increased [START_REF] Young | Inhibitors of histone deacetylases promote hematopoietic stem cell self-renewal[END_REF][START_REF] Bug | Valproic acid stimulates proliferation and self-renewal of hematopoietic stem cells[END_REF]. Epigenetic modifications in the process of aging might either be due to a loss of epigenetic regulation that may drive functional attenuation of HSC or to a purposeful mechanism where changes can be transmitted to progeny. We have previously demonstrated, that various genes including DNA (cytosine-5-)-methyltransferase 1 (dnmt1)

were up-regulated upon contact between CD34 + /CD38 -HPC with stromal cells and this indicates that intrinsic epigenetic modifications can be modulated by cell-cell interaction with the microenvironment [START_REF] Wagner | Hematopoietic progenitor cells and cellular microenvironment: behavioral and molecular changes upon interaction[END_REF].

DNA damage accumulation during aging

Accumulation of endogenous DNA mutations is another key mechanisms that contributes to aging of HSC [START_REF] Kenyon | The role of DNA damage repair in aging of adult stem cells[END_REF]. Mouse experiments have revealed that aged HSC show a higher extend of DNA damage (Rossi et al., 2007a). Additionally genes involved in DNA repair and maintenance were found to be down-regulated in HSC from old mice compared with those from young mice [START_REF] Chambers | Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation[END_REF]Rossi et al., 2007a).

Furthermore, mice that were deficient for genes involved in DNA repair or maintenance show premature hematopoietic exhaustion (Rossi et al., 2007a). These data indicate that DNA repair and genomic maintenance play a key role in stem cell aging. This effect might be attenuated in HSC as a result of their quiescent (G0) cell cycle status, which results in an attenuation of checkpoint control and DNA damage in the context of DNA replication (Rossi et al., 2007b). As mentioned above, maintenance in a quiescent state and proliferation are governed by the stem cell niche and this seems to be another important mechanism for external modulation of cellular aging.

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Conclusion

Cellular aging and senescence does not necessarily represent an inevitable fate for all cells, because cellular senescence is not observed in primitive organisms such as sponges or corrals as well as in our germline cells. Aging in different species correlates with the generation time and it seems to provide an evolutional advantage for the whole species. It is intriguing to note, that this process is restricted to higher organisms with a mesoderm and highly specialized stem cell niches. External control by the microenvironment might be more robust with respect to mutations -hereby it is not a unique stem cell that might gather a

HSC HSC

A

  C C E P T E D M A N U S C R I P T is unlikely to be the only cause of this phenomenon (O'Hare et al., 2001;Di Donna et al.,

A

  C C E P T E D M A N U S C R I P T the stem cell niche undergoes extensive changes during the lifetime of the organism and these might play an important role for external control of stem cell aging. Potential mechanisms involved in aging of hematopoietic stem cells Central questions in adult stem cells and their function in the context of aging are yet unravelled: In theory, HSC might either have the unlimited potential for self-renewal including rejuvenation or they might undergo cellular aging such as other somatic cells. Furthermore, the question arises to what extent age-related changes in these cells are due to intrinsic factors or regulated by stimuli from the microenvironment -the stem cell niche. Four possible scenarios will be described in the following paragraph and the truth lies most likely

  have the same multilineage differentiation potential but after a limited number of cell cycles the daughter cells enter a senescent state and stop proliferation. In addition to the above mentioned limitations in HSC research analysis of replicative senescence is further hampered by the absence of reliable in vitro expansion protocols for primitive HSC. For mesenchymal stem cells (MSC) culture expansion protocols are established and our group has recently demonstrated that replicative senescence in these cells is a continuous and organized process (manuscript in revision). However, MSC are only poorly defined on a molecular level and verification of their stem cell function is not unequivocally demonstrated(Wagner and Ho, 2007). Exhaustion of the HSC pool does not normally occur during lifetime.Serial transplantation experiments in the murine model demonstrated that the declining repopulation ability and exhaustion of HSC can be protracted for more than five rounds of transplantation[START_REF] Harrison | Loss of stem cell repopulating ability upon transplantation. Effects of donor age, cell number, and transplantation procedure[END_REF]. Despite a remarkable proliferative capacity these results indicate cellular aging of HSC. Mathematical models for HSC differentiation demonstrate that the Hayflick hypothesis is compatible with lifelong hematopoiesis[START_REF] Dingli | Allometric scaling of the active hematopoietic stem cell pool across mammals[END_REF]. Furthermore, replicative senescence in HSC is supported indirectly by the fact that most of them remain in a quiescent state in the bone marrow. Primitive HSC can even be enriched in the slow dividing fraction (SDF) of CD34 + /CD38 -cells. In our previous work we have demonstrated that asymmetric cell division of primitive HSC correlates with asymmetric cell division kinetics[START_REF] Wagner | Molecular evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis[END_REF][START_REF] Giebel | Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division[END_REF]. One daughter cell remains quiescent or divides very slowly while the other multiplies exponentially to yield committed progenitors and lineage specific colonies. If the two daughter cells of slow dividing cells were separated by micromanipulation and analyzed individually, it has been demonstrated that one daughter cell inherited the developmental capacity of the mother cell, whereas the otherA C C E P T E D M A N U S C R I P TAging of HSC is regulated by the niche 10

  hematopoietic microenvironment. HSC have a reduced ability to home to their niches upon transplantation into old versus young recipients (Rossi et al., 2005;Liang et al., 2005). Furthermore, adhesive interaction of HSC with their niche is modulated by age-related alterations as aged mice show an increased mobilization of HSC in response to granulocytecolony-stimulating factor (G-CSF) (Xing et al., 2006). Recently Ju et al. have demonstrated that telomere dysfunction exerts especially effects on the hematopoietic microenvironment.

Fig 1 .

 1 Fig 1.Potential mechanisms involved in aging of hematopoietic stem cells. Self-renewal of HSC might be associated with rejuvenation to circumvent cellular aging in adult stem cells (A). Alternatively HSC could be bound to replicative senescence with a limited number of cell divisions such as any other somatic cell (B). Aging of HSC might also be controlled by the external stimuli from the microenvironment that regulate rejuvenation of HSC (C). Last but not least, anchorage of HSC to specific niches in the bone marrow seems to keep them in a quiescent state and hence, interaction with the microenvironment might counteract replicative senescence of HSC (D).

Fig 2 .

 2 Fig 2. Potential mechanisms for regulation of aging by the stem cell niche. Self-renewal and differentiation of stem cells are tightly controlled by the stem cell niche in the bone marrow. This figure summarizes potential mechanisms by which this interaction might also regulate the process of aging in HSC.

Acknowledgements

This work was supported by the German Ministry of Education and Research (BMBF) within the National Genome Research Network NGFN-2 (EP-S19T01) and within the supporting program "cell based regenerative medicine" (START-MSC), the German Research Foundation DFG (HO 914/7-1), the Joachim Siebeneicher-Stiftung, the Netzwerk für Alternsforschung (NAR) and the Academy of Sciences and Humanities, Heidelberg (WIN-Kolleg).

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