M Sanchez-Hidalgo

C Alarcon De La Lastra

M P Carrascosa-Salmoral

M C Naranjo

A Gomez-Corvera

B Caballero

Dr Juan M Guerrero
email: guerrero@us.es

Age-related changes in melatonin synthesis in rat extrapineal tissues

Keywords: extrapineal melatonin, NAT, HIOMT, aging, neuroimmunomodulation

In the search of new therapeutic targets improving the quality of life of elderly, melatonin, "the chemical expression of darkness", seems to play a remarkable role in aging process possibly due to its antioxidant, immunoenhancer and antiaging properties.

The present study was designed to elucidate effects of aging in melatonin extrapineal synthesis and investigate evident age-related alterations in the action mechanisms involved. The presence of the two key enzymes involved in melatonin synthesis, arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) was analyzed in thymus, spleen, liver, kidney and heart of 3-and 12 monthold rats using real time PCR as well as its functionality by enzymatic activity assays. In addition, extrapineal melatonin content was measured by a competitive enzyme immunoassay (ELISA). The results of this study reveal that all rat tissues studied including thymus, and for the first time, spleen, liver, kidney and heart have the necessary machinery to synthesize melatonin. Moreover, we report an age-related decline in rat extrapineal melatonin synthesis with a consequent HIOMT functionality decrease in spleen, liver and heart during physiological aging. On the contrary, NAT enzymatic activity maintains unchanged without evident alterations with advancing age. Moreover, diminished melatonin concentrations were measured in these tissues cited above during aging except in the thymus, where, surprisingly, melatonin content, NAT/HIOMT expression, and enzymatic functionality assays revealed no significant alterations with age. As a conclusion, we report evident age-related changes in melatonin synthesis in some rat peripheral organs. We suggest that thymus may develop compensatory mechanisms to counteract the loss of immune activity and consequently, the loss of this potent antioxidant, during physiological aging.

Introduction

Melatonin (N-actetyl-5-methoxy-tryptamine), an indolamine derived from the amino-acid tryptophan via synthesis of serotonin, was first isolated by Lerner et al in 1958(Lerner et al., 1958). Melatonin, is produced by the pineal gland and secreted into the blood in a circadian manner. Its pineal production is triggered by darkness in the retina and the pineal gland, conferring the diurnal rhythm of melatonin, while in the peripheral system it is continuously produced in the gastrointestinal tract, especially in the stomach, and is triggered by innate immune response in the immune competent cells.

Melatonin participates in diverse physiological functions and has great functional versatility related to the regulation of circadian rhythms and seasonal behaviour [START_REF] Reiter | Melatonin: the chemical expression of darkness[END_REF], sexual development (Roy and Belsham, 2001), retinal physiology [START_REF] Lundmark | Role of melatonin in the eye and ocular dysfunctions[END_REF], osteogenesis [START_REF] Sanchez-Hidalgo | Melatonin inhibits fatty acid-induced triglyceride accumulation in ROS17/2.8 cells: implications for osteoblast differentiation and osteoporosis[END_REF], tumour inhibition, antioxidant, anti-aging [START_REF] Anisimov | Melatonin as antioxidant, geroprotector and anticarcinogen[END_REF] and immunoenhancer properties [START_REF] Guerrero | Melatonin-immune system relationships[END_REF]. More recently, it has been also implicated in bone homeostasis, cardiovascular, neurological and gastrointestinal systems [START_REF] Pandi-Perumal | Melatonin: Nature's most versatile biological signal?[END_REF].

The enzymatic machinery for melatonin biosynthesis in pinealocytes was first identified by [START_REF] Axelrod | The pineal gland: a neurochemical transducer[END_REF][START_REF] Axelrod | The pineal gland: a neurochemical transducer[END_REF]. Its biosynthesis is mediated by the sequential action of AA-NAT and HIOMT. The primary neurotransmitter, noradrenalinee, is released during the night from the postganglionic symphatic fibers of the superior cervical ganglion that terminate in the pinealocytes where it couples to adrenergic receptors. This leads to an activation of adenylate cyclase with a consequent rise in cAMP levels, promoting the novo protein synthesis and stimulating the ratelimiting enzyme in melatonin production, arylalkylamine-N-acetyltransferase (AA-NAT) [START_REF] Klein | The melatonin rhythm generating system: developmental aspects[END_REF]. As a result, AA-NAT N-acetylates serotonin to Nacetylserotonin (NAS) and, as long as NAS is generated, is quickly O-methylated by hydroxyindole-O-methyltransferase (HIOMT) to finally produce melatonin (Axelrod y Weissbach, 1960). The dramatic rise in AA-NAT (10-to 100-fold) leads to melatonin synthesis which is released during periods of darkness in a circadian manner [START_REF] Reiter | Melatonin: the chemical expression of darkness[END_REF]. In contrast, the nocturnal increase in pineal HIOMT activity is weak but significant. HIOMT gene expression is high during daytime but displays an additional two fold increase at night. Once synthesized and released into blood at night, melatonin is transferred through biological membranes and is found throughout the organism in all body tissues within a very short period. This presence does not necessarily indicate an endogenous synthesis because the high lipophilic nature permits its transfer. To date, extrapineal melatonin synthesis has been demonstrated only in retina, harderian gland, brain, gut, ovary, testes, inner ear and immune system including bone marrow, lymphocytes, macrophages and, more recently, in thymus [START_REF] Kvetnoy | Extrapineal melatonin: location and role within diffuse neuroendocrine system[END_REF][START_REF] Bubenik | Gastrointestinal melatonin: localization, function, and clinical relevance[END_REF][START_REF] Carrillo-Vico | Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance[END_REF][START_REF] Slominski | On the role of melatonin in skin physiology and pathology[END_REF]. In general, while pineal melatonin seems to act as a typical hormone reaching target cells through the bloodstream, extrapineal melatonin may play a key role as an intra-, auto-and paracrine signal molecule in those tissues where it is synthesized [START_REF] Carrillo-Vico | Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance[END_REF].

The consequences of aging are also seen in melatonin production. The secretory dynamic of pineal melatonin attenuates gradually with age, resulting in a loss of nocturnal peak of melatonin with nearly undetectable plasma levels at 20-22 months of age in rodents [START_REF] Reiter | Pineal melatonin rhythm: reduction in aging Syrian hamsters[END_REF] and at 60-70 in humans [START_REF] Touitou | Age-and sex-associated modification of plasma melatonin concentrations in man. Relationship to pathology, malignant or not, and autopsy findings[END_REF]. Recent studies have suggested that age-related decline in melatonin concentration involves a loss of its antioxidant ability against damage produced by free radicals [START_REF] Reiter | Melatonin reduces oxidant damage and promotes mitochondrial respiration: implications for aging[END_REF]. Thus, the loss of this potent antioxidant during aging may be consequential of the onset of age-related diseases. In fact, some studies in rodents have suggested that such diminished melatonin secretion may accelerate aging [START_REF] Reiter | Oxidative processes and antioxidative defense mechanisms in the aging brain[END_REF] whereas melatonin administration has an immune reconstituting effect in aging [START_REF] Tian | Melatonin rejuvenates degenerated thymus and redresses peripheral immune functions in aged mice[END_REF]. As mentioned above, pineal and serum melatonin levels decrease with advancing age, however little is known about the physiological roles of melatonin in extrapineal tissues during aging. The relevance of pineal and extrapineal melatonin in maintaining immune surveillance and defence response and the implication of reducing melatonin production by aging could support the idea that age-related decrease in extrapineal melatonin production should be very important for the developing of age-related diseases. Furthermore, the present study was designed to obtain a better understanding about melatonin produced in situ in rat peripheral organs, including the immune system, with age. That is why we studied the presence of the two key enzymes involved in melatonin synthesis, NAT and HIOMT, in thymus, spleen, liver, kidney and heart of rats of 3-and 12 month-old using real time PCR as well as its functionality by enzymatic activity assays. In addition, the extrapineal melatonin content was measured by a competitive enzyme immunoassay (ELISA).

Material and methods

Animals and tissue preparations

Experiments were performed on male Wistar rats (3-and 12 month-old) supplied by Production and Experimentation Animal Services from the University of Granada, Spain. Animals were placed singly in cages at a controlled room temperature 24-25C, humidity 70-75%, under controlled photoperiods of 12 h light: 12 h darkness (lights were turned off daily from 20:00 to 08:00h) and were maintained with a normal laboratory diet (Panlab, Barcelona, Spain) and water ad libitum. The experiments followed a protocol approved by the local animal Ethics Committee and the Local Government and all experiments were in accordance to the recommendations of the European Union regarding animal experimentation (Directive of the European Counsel 86/609/EC).

When required, 3-and 12 month-old rats were sacrificed by decapitation between 12:00 and 15:00 h, and the organs were carefully removed under sterile conditions, briefly rinsed in normal saline solution, frozen on solid CO 2 and stored at -80ºC until their use for RNA extraction, enzyme activities and melatonin determinations.

Total RNA extraction and reverse transcription

Total RNA was extracted from samples by a modification of Chomczynski and Sacchi´s method using TriPure Isolation Reagent (Roche, Mannheim, Germany) as a denaturing solution and appropriate chloroform volume. After cell lysis and RNA extraction, RNA was precipitated with isopropanol, and the pellet was washed in 75% ethanol. The RNA samples were recovered by centrifugation at 14000 g for 5 min and then dried. Each RNA pellet was dissolved in 50 or 100 μL RNase-free water and the quantified spectrophotometrically at 260 nm. In an attempt to discard DNA contamination before cDNA synthesis, the RNA samples underwent a purification process. A determined volume of reaction buffer 10x (100 mM Tris pH 7.5, 25 mM MgCl 2, 5 mM CaCl 2 ) and DNAsa I (Roche, 2U/ 10 μg ARN) were added to RNA extracted volume and incubated at 37 ºC for 30 min. The enzymatic inactivation was at 65ºC for 10 min. The samples were centifuged at 2500g at 4ºC 5 min after phenolchloroform addition and RNA was precipitated with absolute ethanol, dissolved in 50 μL RNase-free water and the quantity and quality of RNA was assessed spectrophotometrically at 260 nm and 260/280 nm, respectively. Single-strand cDNA was then synthesized using the following method: 5 μg of RNA was denatured in 19 μL RNase-free water at 85ºC for 10 min, and then rapidly chilled on ice. Then, 21 μL of a mixture formed by 1 X reverse transcription (RT) buffer, 20 mM dithiothreitol (DTT), 2´-deoxyribonucleoside-5´-triphosphates (0.5 mM of each), 40 U recombinant RNasin ribonuclease inhibitor, 0.5 μg oligo (dT) 15 primer and 200 U Moloney murine leukemia virus reverse transcriptase (M-MLV RT) were added to give a final volume of 40 μL and incubated for 60 min at 42ºC (all reagents were purchased from Promega, Madison, WI, USA). The RT reaction was terminated by placing it on ice after deactivation at 95ºC for 5 min.

Real Time RT-PCR

Real time quantitative PCR was performed by the MiniOpticon cycler system (BioRad) using the 2 X SensiMix DNA Kit (Quantace). The cDNA was amplified in a reaction containing 5 μL of RT product as template DNA (200 ng), 1 X Sensi Mix (reaction buffer, dNTPs, Taq DNA polymerase, 3 mM MgCl 2 ), 1 X SYBR Green and 0.2 μM sense and antisense primers of the gene under study or 0.1 μM for the housekeeping gene (hprt), in a final volume of 25 μL. The sequences of primers are shown in Table 1.

NAT activity assay

NAT activity was determined by the method of [START_REF] Champney | Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland[END_REF]. Each tissue sample (thymus, spleen, liver, kidney and heart) was homogenated at 4º C in an appropriate volume of 0.05 M phosphate-buffered saline buffer (PBS), pH 6.8, using a cell sonicator (Sonics and Materials Inc., Danbury CT, USA). Ten microliters of this homogenate were mixed with 10 µL PBS containing 40n Ci [1-14 C] acetylcoenzyme A and 5.6 mM tryptamine. The reaction was carried out for 20 min at 37ºC, and was stopped by the addition of 100 µL 0.2 M sodium borate buffer, pH 10, and 1ml chloroform at 4ºC. The N-acetyltrypamine produced was extracted with chloroform and its radioactivity was measured by liquid scintillation spectrometry with a beta counter.

NAT activity was expressed as pmol N-acetyltryptamine produced per mg protein per h.

Protein content was measured following the Bradford protocol [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

HIOMT activity assay

HIOMT activity was determined by the method of [START_REF] Champney | Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland[END_REF] by measuring the amount of melatonin formed from N-acetylserotonin and S-adenosyl-Lmethionine. Each sample was homogenated in 0.05 M PBS, pH 6.8. Forty microliters of this homogenate were mixed with 20 µL 0.05 M PBS, pH 7.9, containing 20n Ci S-[methyl-14 C] adenosyl-L-methionine and 3 mM N-acetylserotonin. The reaction was incubated for 30 min at 37ºC and stopped by the addition of 100 µL 0.2 M sodium borate buffer pH 10 and 1 mL chloroform at 4ºC. Synthesized melatonin was measured following extraction in 1 mL chloroform and the radioactivity by liquid scintillation spectrometry counted with a beta counter. HIOMT activity was expressed as pmol melatonin per mg protein per h. Protein content was measured following the Bradford protocol [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

Melatonin determination by ELISA

Melatonin levels were determined by a competitive enzyme immunoassay kit (Immuno Biological Laboratories, Hamburg, Germany). Tissues were weighed (60-100 mg) and homogenated in an appropriate volume of PBS in a sonicator (Sonics and Materials Inc., Danbury CT, USA) and centrifuged at 3000g for 10 min. Tissue homogenate supernatants were used for melatonin determinations. An aliquot was used to measure protein concentration so as to refer the melatonin measures to mg protein following the Bradford colorimetric method.

Melatonin was extracted from 500 µL of samples, standards, and controls (90-100% yield recovery) using C18 reversed-phase columns (IBL-Hamburg, Germany) and methanol elution. The dried extracts (after evaporating methanol) were reconstituted with water. Then, each sample was added to the corresponding well coated with captured antibody goat anti-rabbit Ig of a 96-well microtiter plate. Each microtiter plate was filled either with 50 µL blank reagent, extracted calibrators, extracted samples or extracted standard solutions (containing 0, 3, 10, 30, 100 or 300 pg /mL melatonin).

Then, 50 µL melatonin biotin and 50 µL rabbit-antiserum were added into each well, shaken carefully, sealed with adhesive foil and incubated overnight (14-20 h) at 2-8ºC.

After washing three times with 250 µL diluted assay buffer, 150 µL anti-biotin conjugate to alkaline phosphatase was added into each well and incubated for 2 h at room temperature. The reaction was developed using p-nitrophenyl phosphate (PNPP) substrate solution and optical densities were determined at 450 nm in an automatic microplate reader. The melatonin concentration was inversely proportional to the optical density measured. Melatonin standards were used to construct a calibration curve against which the unknown samples were calculated. The sensitivity of the melatonin assay was 3.0 pg/ mL. Both the intra-and inter-assay coefficients of variation (CV) were less than 10 %.

Statistical data analysis

The presented data are expressed as means ± standard error of the mean (SEM).

The data were evaluated with Graph Pad Prism (Version 2.01) software. The statistical significance of any difference in each parameter among the groups was evaluated by Student´s t-test or the Mann-Whitney U-test (for non-parametric values). P values of < 0.05 were considered statistically significant.

Results

RNA from all samples was isolated and RT and PCR amplification assays were performed. cDNA obtained from the pineal gland was used as a positive control.

Quantitative real-time PCR assays were established for HPRT (housekeeping gene), AA-NAT (rate-limiting enzyme in melatonin synthesis) and HIOMT (the final enzyme of the biosynthetic pathway) (Figs. 1 and2). For each PCR run, a negative control was systematically added, in which RNase-free water replaced cDNA. In addition, a single peak was observed for each of the products by melting curve analysis, performed routinely in all samples after amplification. Product Tm values were: HPRT 81ºC, AA-NAT 86.6ºC, HIOMT 84.5ºC. No product was detected in the absence of RT in any assay for any of the products, thus indicating that there was no genomic contamination. Data for 12 month-old samples were normalized by those obtained for 3 month-old samples.

As shown in figures 1 and 2, significant levels of AA-NAT and HIOMT transcripts were described in all tissues studied. According to AA-NAT expression, the relative quantification assay revealed significant differences in AA-NAT mRNA expression of 12 month-old rats when compared with values obtained in control group (Fig. 1). Particularly, a significant decrease in AA-NAT mRNA expression was observed in spleen and liver from middle-aged rats (P< 0.01 and P< 0.001, respectively). On the contrary, AA-NAT mRNA expression levels were significantly higher in kidney (P< 0.01) and heart (P< 0.05) during aging while no significant differences were observed between values of thymus from 3-and 12 month-old rats.

Likewise, we studied HIOMT mRNA levels in the tissues cited above; values of relative expression of HIOMT mRNA in thymus, spleen, kidney, liver and heart from 3-and 12 month-old rats are shown in figure 2. Once again, we observed age-related significant differences depending on the tissue studied. For instance, we observed a significant increase in HIOMT mRNA levels in spleen and heart of 12 month-old rats (P< 0.05) whereas an age-related reduction (P< 0.001) was observed in liver when compared with young control group. No significant differences related to HIOMT expression were detected in thymus and kidney as age increased.

To obtain a deeper insight of mechanisms involved in melatonin regulation of the immune system and other peripheral organs during aging, we analyzed the functionality of the enzymatic machinery involved in melatonin synthesis by measuring the enzymatic activity of NAT and HIOMT. Pineal homogenate was used as a positive control of the assay. Results obtained in enzymatic activity assays confirmed evident age-related changes in NAT and HIOMT activity levels (Figs. 3 and4). As regards to and 12 month-old rats. Results obtained in liver and kidney demonstrate that middleaged rats increased the NAT activity levels, showing a twofold increase when compared with control group animals, P< 0.05 and P< 0.001, respectively. However, the NAT activity levels from the rest of tissues studied remained unchanged with advancing age.

In relation to HIOMT activity, we observed a significant age-related reduction in spleen, liver and heart of 12 month-old rats, P< 0.05 (Fig. 4). However, HIOMT activity values remained constant in middle-aged rat thymus and kidney showing no age-related modifications.

Since melatonin levels produced by pineal gland have been found to decrease with age, we wished to know what would happen to melatonin produced in situ in peripheral organs including immune system. Therefore, melatonin content in extrapineal tissues of rats 12 month-old was measured by ELISA and compared with melatonin content obtained from the young control group (Fig. 5). The results, expressed as pg melatonin /µg protein, showed unexpectedly higher levels of melatonin in kidney and thymus of middle-aged rats when compared with those obtained in 3 month-old rats, P< 0.05 and P< 0.001, respectively. However, melatonin content produced by the spleen, liver and heart decreased significantly with age, P< 0.05.

Discussion

Aging is a physiological process that involves a number of biochemical reactions, with molecular changes that are manifested in the organism. Particularly, the impairment of the immune efficiency that occurs in aging [START_REF] Miller | Aging and immune function[END_REF] known as immunosenescence is associated with increased incidence of cancer, infectious and degenerative diseases. Innate, cellular and humoral immunity all exhibit increased deterioration with age. Moreover, it has reported that aging also implies a decline in the production of several hormones including melatonin, and this drop seems to play a remarkable role in contributing to the immunosenescence due to its immunoenhancer and anti-aging properties [START_REF] Srinivasan | Melatonin, immune function and aging[END_REF].

Since melatonin was first isolated five decades ago until it was identified in extrapineal tissues, twenty years later, it was considered as a hormone exclusive to the pineal gland. To date, its presence has been described in a large number of extrapineal sites including immune system, gut, reproductive organs, brain and skin [START_REF] Hardeland | Melatonin, hormone of darkness and more -occurrence, control mechanisms, actions and bioactive metabolites[END_REF]. Although the presence of melatonin in extrapineal sites is becoming more and more evident, the potential physiological actions of this endogenous melatonin have not yet been studied. On the other hand, it is well-known that reduced melatonin concentrations have been described in the pineal gland, serum and also in urine during aging (Reiter et al., 1981;[START_REF] Miguez | Changes with age in daytime and nighttime contents of melatonin, indoleamines, and catecholamines in the pineal gland: a comparative study in rat and Syrian hamster[END_REF][START_REF] Pang | Decreased serum and pineal concentrations of melatonin and N-acetylserotonin in aged male hamsters[END_REF][START_REF] Bondarenko | The comparative evaluation of the effect of the season on the formation of a nighttime melatonin peak in young sexually mature and old rats[END_REF].

However, what happens to melatonin produced in situ in peripheral organs with advancing age as well as the melatonin physiological functions implicated in these tissues remains undefined. Therefore, the present study was designed to elucidate effects of aging in melatonin extrapineal synthesis and investigate the possible agerelated alterations in the action mechanisms implicated. The results of this study demonstrate that all rat tissues studied including thymus, and for the first time, spleen, liver, kidney and heart have the necessary machinery to synthesize melatonin, which would indicate that at least a part of observed melatonin levels come from endogenous synthesis. We also detected expression and activities of the two key enzymes involved in melatonin synthesis as well as significant changes in melatonin levels with advancing age. Our data reveal, not only the presence of NAT and HIOMT mRNAs in rat thymus, spleen, liver, kidney and heart, but also age-related expression differences of both key melatonin biosynthetic enzymes using the relative quantification method. Regarding the immune system, enzymatic machinery expression involved in melatonin synthesis has been demonstrated in lymphoid organs including thymus [START_REF] Naranjo | Melatonin biosynthesis in the thymus of humans and rats[END_REF], bone marrow [START_REF] Tan | Identification of highly elevated levels of melatonin in bone marrow: its origin and significance[END_REF][START_REF] Conti | Evidence for melatonin synthesis in mouse and human bone marrow cells[END_REF], peripheral blood mononuclear leucocytes (Pozo et al., 2004) and in human cultured lymphocytes [START_REF] Carrillo-Vico | Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance[END_REF]. Our results confirmed that all extrapineal tissues studied express both necessary key enzymes for melatonin synthesis, as opposed to Stefulj et al., who showed that neither rat immune system (thymus and spleen) nor other peripheral organs (kidney, liver and heart) contain reliable amounts NAT and/or HIOMT [START_REF] Stefulj | Gene expression of the key enzymes of melatonin synthesis in extrapineal tissues of the rat[END_REF]. This discrepancy might be due to a different amplification method used (PCR conventional vs. real time PCR).

AA-NAT enzyme seems to be present in all peripheral tissues studied from 3and 12 month-old rats, except in kidney and spleen of 12 month-old rats where we clearly observed a trend to decrease with age. However, NAT enzymatic assay revealed that NAT enzyme continues to be functional in all cited tissues in spite of aging process.

Likewise, HIOMT expression study revealed the presence of this enzyme in all of them as well as a significant increase in spleen and heart during aging. On the contrary, HIOMT expression levels diminished significantly in liver of 12 month-old rats.

Unexpectedly, the diurnal rhythm of melatonin content did not follow the same pattern as the enzymes. This discrepancy in expression/activity agrees with the published literature [START_REF] Bernard | Transcriptional regulation of hydroxyindole O-methyltransferase in the chicken pineal gland: day/night changes and long-term effects of light and darkness[END_REF][START_REF] Sakamoto | Circadian expression of serotonin N-acetyltransferase mRNA in the rat retina[END_REF].

Melatonin content measurement revealed a significant decrease in spleen, liver and heart from middle-aged rats whereas melatonin levels were significantly higher in thymus and kidney. Particularly, the increase observed in melatonin content in the kidney is according to the increase in NAT activity, suggesting that there is a direct correlation between both parameters. Nevertheless, these results agree with those obtained in HIOMT enzymatic activity assay. When melatonin levels and HIOMT functionality decrease significantly in spleen, heart and liver during rat physiological aging process, NAT enzymatic activity remains unchanged (spleen and heart) or increases (liver) possibly due to a feedback mechanism by which the organism attempts to compensate the drop in this potent antioxidant. Particularly, the age-related decrease in melatonin levels observed in these tissues did not result in a corresponding reduction in NAT activity. These data suggest that HIOMT enzymatic activity determines the level of melatonin synthesis in the above mentioned tissues during aging. In accordance with Simonneaux and Ribelayga, any modification in HIOMT activity could modulate, consequently, the melatonin production phase [START_REF] Simonneaux | Generation of the melatonin endocrine message in mammals: a review of the complex regulation of melatonin synthesis by norepinephrine, peptides, and other pineal transmitters[END_REF]. In this sense, HIOMT enzyme may be the first key enzyme involved in melatonin synthesis that suffers the consequences of aging. In agreement with previous results reported by Liu and Borjigin we suggest that HIOMT should be considered the ratelimiting enzyme in the melatonin synthesis during aging [START_REF] Liu | N-acetyltransferase is not the rate-limiting enzyme of melatonin synthesis at night[END_REF].

With advancing age, the amount of free radicals increases considerably in cells, leading them to cell death. In this sense, although the melatonin physiological role in extrapineal tissues is not fully known, we should take into account its antioxidant and anti-aging properties [START_REF] Reiter | Melatonin reduces oxidant damage and promotes mitochondrial respiration: implications for aging[END_REF]. It has been demonstrated that melatonin can prevent age-related diseases [START_REF] Karasek | Melatonin, human aging, and age-related diseases[END_REF] and some studies in rodents suggest that such diminished melatonin secretion may accelerate aging [START_REF] Reiter | Oxidative processes and antioxidative defense mechanisms in the aging brain[END_REF]. Likewise, an accumulation of oxidative products in many organs has been described after pinealectomy in aging rats (Reiter et al., 1999).Consequently, if melatonin levels contribute to the antioxidant capacity, such ability would be reduced in advanced age and could have severe effects. As a result, an age-related decline in melatonin levels may contribute to an increase in the oxidative damage and the onset of age-related degenerative diseases due to an accumulation of free radicals. Endogenous melatonin may act as free radical scavenger protecting the cells against oxidative damage associated to the aging. Surprisingly, of the rat extrapineal tissues studied, we have to highlight the results obtained in thymus for thymic mRNA expression and enzyme activity studies as well as the melatonin content measured since these parameters remained unchanged during aging. This could be explained by taking into account the bidirectional communication circuit between pineal melatonin and the immune system [START_REF] Skwarlo-Sonta | Bidirectional communication between the pineal gland and the immune system[END_REF][START_REF] Markus | The immune-pineal axis: a shuttle between endocrine and paracrine melatonin sources[END_REF]. The pineal gland, via the rhythmic synthesis and release of melatonin, influences the development, differentiation and function of the immune system. This connection is supported by studies which have demonstrated a correlation between melatonin production and circadian and seasonal variations in the immune system, effects of surgical or functional pinealectomy on the immune system, as well as the presence of melatonin receptors in the immune system [START_REF] Guerrero | Melatonin-immune system relationships[END_REF]. Our results suggest that a feedback circuit may exist between the thymus and the pineal which balances the immunologic function in the body. In this sense, when the pineal gland starts to degenerate, the thymus may develop compensatory mechanisms such as maintaining NAT enzymatic functionality to counteract the loss of immune activity.

A bloodstream melatonin retention cannot completely be ruled out. Thus, we suggest that, despite action mechanisms involved in melatonin synthesis fail with advancing age, melatonin may be retained by the thymus from the bloodstream or intracellular space for transport to other organs where it exerts its effects or synthesized by the thymus, or both, counteracting the age-associated alterations. Our results agree with the high melatonin levels described in bone marrow [START_REF] Tan | Identification of highly elevated levels of melatonin in bone marrow: its origin and significance[END_REF], rat thymus [START_REF] Naranjo | Melatonin biosynthesis in the thymus of humans and rats[END_REF] and those reported by [START_REF] Jimenez-Jorge | Melatonin synthesis and melatonin-membrane receptor (MT1) expression during rat thymus development: role of the pineal gland[END_REF].

Pinealectomized rats have clearly increased thymic melatonin content evidencing an endogenous synthesis and suggesting that either pineal melatonin could be inhibiting thymic melatonin synthesis and when rat pinealecthesized melatonin is absent, the thymus could show a compensatory rise in melatonin production [START_REF] Jimenez-Jorge | Melatonin synthesis and melatonin-membrane receptor (MT1) expression during rat thymus development: role of the pineal gland[END_REF].

Taken together, our study reveals that all rat extapineal tissues studied including thymus, and for the first time, spleen, liver, kidney and heart have the necessary machinery to synthesize melatonin. Moreover, we report an age-related decline in rat extrapineal melatonin synthesis in some organs. When melatonin levels and HIOMT functionality decrease in some organs during rat physiological aging process, the thymus would try to counteract the loss of this potent antioxidant by maintaining NAT enzymatic activity unchanged via pineal gland axis. Nonetheless, further studies are required in order to obtain a better understanding about the role of melatonin in these extrapineal tissues under physiological conditions as well as the relevance of extrapineal melatonin synthesis, including the regulation on the immune system, with advancing age.

  The PCR reaction started by a 10 min activation of hot-start DNA polymerase at 95ºC followed by 45 cycles of target cDNA amplification. The template was initially denatured at 95ºC 10 s followed by 45-cycle program with 20 s annealing at 57ºC (AA-NAT) or 45 s annealing at 58-60ºC (HIOMT) and 20 s elongation at 72ºC and acquisition of SYBR Green fluorescence. In addition, the ubiquitously expressed hprt mRNA was used to monitor the quality of RNAs and the efficiency of the RT and PCR processes. During PCR the intercalate agent is separated resulting in increased fluorescence. The MiniOpticon instrument reads each well every few seconds and measures the increase in fluorescence which is a direct consequence of the target amplification during PCR. The results are reported as threshold cycle (Ct) values, the cycle at which fluorescence readings exceed the mean baseline readings. The mathematical model of 2 -ΔΔCT (Livak) was used for relative quantification of AA-NAT and HIOMT to hprt using the equation: ΔΔC T = (Ct, target -Ct, hprt) Time x -(Ct, target -Ct, hprt)Time 0; where time x is any time point and time 0 represents the 1 X expression of the target gene normalized to hprt. The mean, and SD were then determined from the triplicate samples at each time point. The value of the mean fold change at zero time should be very close to one (2 0 = 1).

Figure 1 .

 1 Figure 1. Real time PCR analysis of AA-NAT mRNA expression in extrapineal tissues

Figure 2 .

 2 Figure 2. Real time PCR analysis of HIOMT mRNA expression in extra pineal tissues

Figure 3 .

 3 Figure 3. NAT activity levels in extrapineal tissues from different-aged rats. NAT

Figure 4 .

 4 Figure 4. HIOMT activity in extrapineal tissues from different-aged rats. HIOMT

Figure 5 .

 5 Figure 5. ELISA melatonin measurement in extrapineal tissue homogenates from 3-and

Acknowledgements

This work was supported by grants of the Andalucía (P06-CTS-1604) and Spanish (PI 060091; RD06/00130001) governments. MSH gratefully acknowledges support from a Postgraduate National Program of FPU fellowship and financial sponsorship from the Spanish MEC.


Jedrzej Hoffmann

Young Erben

Andreas M Zeiher

PhD Stefanie Dimmeler
email: dimmeler@em.uni-frankfurt.de

MD Ioakim Spyridopoulos
email: ioakim.spyridopoulos@newcastle.ac.uk

Telomere length-heterogeneity among myeloid cells is a predictor for chronological ageing

Keywords: Telomere length, ageing, lymphocytes, granulocytes, monocytes

We have previously shown that mean telomere length (TL) of granulocytes reflects TL of myeloid bone marrow cells extremely well. Here we analysed the distribution of TL from peripheral blood granulocytes and lymphocytes in 61 female and 68 age-matched male healthy volunteers. We show that the age-dependent decline in TL occurs more rapid in peripheral blood lymphocytes (53 bp/year) than in granulocytes (39 bp/year; p<0.001), while women having longer telomeres than men. We also observed the best correlation for age and telomere length to be present in lymphocytes. The coefficient variation (CV) of TL distribution in granulocytes and monocytes was significantly higher in older compared to younger individuals, indicating an increase in telomere length heterogeneity of myeloid cells and their bone marrow residing precursors during ageing. In a multivariate statistical model, CV and lymphocyte TL were able to explain 50% of chronological ageing.

Introduction

Telomeres, hexameric TTAGGG DNA repeats the ends of chromosomes, are widely regarded as the internal biological clock of a living organism, and shorten by a few base pairs with every cell division. In addition, they are also crucial to maintaining chromosomal integrity (Blasco, 2005). Age-related pathologies with short telomeres include infertility, impaired wound-healing and liver cirrhosis. Individuals with shorter leukocyte telomeres have a significantly higher risk of developing coronary heart disease or of dying from cardiovascular and infectious disease (Brouilette et al., 2007;Cawthon et al., 2003). We have published recently that telomere length from granulocytes correlates extremely well with their bone marrow residing myeloid precursors (r= 0.94, p=0.001), justifying the quantification of TL from peripheral blood granulocytes as an indicator of stem cell divisions (Spyridopoulos et al., 2008). The enzyme telomerase counteracts telomere loss in proliferating cells and extends their capacity for replication. Human embryonic stem cells express the telomerase reverse transcriptase (TERT) gene and demonstrate telomerase activity. TERT has an important role in the maintenance of human ESC pluripotency, cell cycle regulation, and in vitro differentiation capacity (Saretzki et al., 2008;Yang et al., 2008).

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Materials and methods

Study population

Peripheral blood was collected from 129 healthy volunteers without any past medical history.

All individuals had to be physically fit and non-obese, walking or jogging at least 20 km / week. Active smokers or individuals on any kind of medical treatment were also excluded from the study. For further studies on specific myeloid cell subpopulations, we used additional 27 male healthy volunteers (14 young and 13 old). All studies have been performed under approval by our institutional ethics board and informed consent was obtained from all volunteers.

Isolation of peripheral blood cells

Red blood cells from EDTA blood were lysed by incubating the pelleted cells twice with ammonium chloride solution (Stem-Cell Technologies), each time in a 1:4 ratio for 10 minutes on ice. Nucleated cells were recovered by centrifugation. CD14 + monocytes were isolated from peripheral blood mononuclear cells by Ficoll density gradient centrifugation, followed by immunomagnetic bead sorting (Milteny Biotech, Gladbach, Germany) according to the manufacturer's protocol.

Measurement of telomere length (Flow-FISH)

Telomere length measurements were carried out as previously described. (Baerlocher et al., 2006;Spyridopoulos et al., 2008;Spyridopoulos et al., 2004) In brief, each sample, containing 100,000 bovine thymocytes as an internal standard and 200,000 cells from the donor, was resuspended in a hybridization mixture containing telomere-specific N-terminal FITC-conjugated (C 3 TA 2 ) 3 PNA probe, washed and counterstained with propidium iodide prior to FACS analysis. Telomere length measurement in CD15 + granulocytes was performed by Multicolor Flow-FISH following whole blood lysis.

Statistics

Data are reported as median and interquartile ranges [IQR] if not stated otherwise.

Correlations between age and telomere-related parameters were performed with the use of

Results

Characteristics of 129 healthy volunteers are shown in Table 1. Men and women were agematched with a median age of 41 and 40 years, respectively. HDL cholesterol was as expected higher in female compared to male volunteers (p=0.039) while LDL, triglycerides and HbA1c were equally distributed among both genders. Other cardiovascular risk factors such as hypertension and smoking were excluded a priori. We measured mean telomere length (TL) of peripheral granulocytes and lymphocytes by the Flow-FISH method, which also allows evaluation of telomere length distribution. Interestingly, men had significantly shorter telomere length in lymphocytes than women (median 6.6 kbp vs. 7.6 kbp; p=0.03), while granulocyte telomeres did not differ significantly (Table 1). The coefficient variation (CV) of telomere fluorescence histograms in lymphocytes and granulocytes, which represents telomere length heterogeneity of either population in a single individual, did not differ between genders. We found a strong correlation between lymphocyte telomere length and chronological age for the whole population (Spearman rank correlation -0.601 vs. -0.501 in granulocytes, Table 2). This difference remained stronger in lymphocytes when analysing male and female volunteers separately.

To further investigate TL heterogeneity, we analysed the relation between the CV of TL and age. Surprisingly, CV of granulocytes showed a strong correlation with age (Spearman rank correlation 0.452, p<0.001), while lymphocyte CV correlated rather weak with age (Spearman rank correlation 0.279, Table 2). As expected, we also found an age-dependent decrease in leukocyte TL with a more rapid decrease of TL in peripheral blood lymphocytes (53 bp/year) compared to granulocytes (39 bp/year) (Figure 1A). This difference was statistically significant (p<0.0001). The correlation between granulocyte CV and age ws strongest in male volunteers (Spearman rank correlation 0.50, p<0.0001, Figure 1B), which prompted us to conduct further experiments on myeloid cells in an additional male population, consisting of 14 healthy young volunteers (mean age 25 years) and 13 older healthy volunteers (mean age 65 years). To acquire even more specific data on CV of myeloid cells, we analysed immunomagnetically isolated CD14 + monocytes (purity over 95% as confirmed by flow cytometry) and surface-stained CD15 + granulocytes using standard Flow-FISH or Multicolor Flow-FISH, respectively. Here, we found that CV differed significantly between young and old healthy volunteers, both for monocytes (12% vs. 14%, p<0.001; Figure 1C) and granulocytes (12% vs. 14%, p<0.001; Figure 1C).

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Finally, multivariate regression analysis revealed that both, CV of granulocytes and TL of lymphocytes were independent predictors of age (data not shown). In this model, the combination of these 2 parameters explained 50% of chronological ageing in our population (p<0.001).

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Discussion

Our data show for the first time that lymphocyte TL decreases significantly faster and, most importantly, correlates better with age than granulocyte TL. The latter is most likely related to the compartment of memory lymphocytes. Since memory T cells contain much shorter telomeres (Rufer et al., 1999), their increasing number with age adds quicker to the decrease in lymphocyte TL. Granulocytes, on the other hand, shorten at the same rate as naïve T cells (Rufer et al., 1999). Our findings are also consistent with previous work from others and our own data, demonstrating a similar decline in telomere length in total lymphocytes from adult volunteers.

Furthermore, we found women to have significantly longer telomeres in the lymphocyte population compared to the male population. The difference of 365 base pairs would correspond to a life span of approximately 6 years. One possible explanation for this finding is the expression of the estrogen receptor in T lymphocytes. In fact, treatment of T cells with estrogen enhances telomerase activity in these cells (Effros et al., 2005). Guan and coworkers found that in the Japanese population, until the age of 50, telomere length decreases faster in men compared to women (Guan et al., 2007). This ratio was reversed in postmenopausal women.

Finally, we detected an age-dependent increase in telomere length distribution among myeloid peripheral blood cells. Previously it has only been described that telomere length variation in a single individual is more pronounced in lymphocytes than in granulocytes (Rufer et al., 1999), which is intuitive, given the difference in TL between naïve and memory lymphocytes. The latter are almost 2000 base pairs shorter, independent of age. The novel and therefore surprising finding in our cohort though was that TL heterogeneity of myeloid cells increased with age as well, yet this effect was more pronounced in male volunteers. To exclude that this could have been a technical issue attributed to the identification of granulocytes by their scatter properties in flow cytometry, we chose to extend our experiments with an additional study population, consisting of 14 young and 13 old healthy male volunteers. This time we used immunomagnetic bead sorting as well as Multicolor Flow-FISH to specifically analyse CD14 + monocytes and CD15 + granulocytes based on their surface antigen properties. The findings in both cell populations confirmed our initial results, in that TL heterogeneity in myeloid cells increases with age.

What is the relevance of such a finding? We have published previously that TL of granulocytes reflects TL of myeloid bone marrow precursors extremely well (Spearman rank

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7/11 correlation greater 0.95) (Spyridopoulos et al., 2008). Together, our results strongly suggest that TL heterogeneity of bone marrow residing precursors increases with Therefore individuals with the same mean TL but higher TL heterogeneity will have both some shorter and some longer telomeres. This implies that a proportion of myeloid precursors and stem cells might reach critical telomere length faster, which could potentially contribute to the exhaustion of bone marrow function (Kissel et al., 2007). Although two other studies have shown that shorter fragments of telomeres increase with ageing, these findings were raised in total leukocyte populations (Guan et al., 2007;Kimura et al., 2008), which apparently also include the very short telomeres from CD4 and CD8 memory lymphocytes. Kimura and coworkers have also shown that the association of mortality with mean leukocyte TL was much weaker than the association with shorter telomeres. Telomere length distribution of leukocytes was a better predictor of mortality. Prospective studies will have to show whether TL in specific leukocyte subpopulations or TL heterogeneity in myeloid cells will demonstrate a better correlation with markers of cardiovascular disease as compared to the commonly used leukocyte TL.

  Unlike in germ cells, replication of hematopoetic stem cells leads to telomere shortening and -in the long term -to the subsequent exhaustion of proliferative capacity. It remains unclear which factors determine telomere shortening in vivo. The goal of this study was to investigate which telomere-associated parameters in myeloid and lymphoid leukocyte subpopulations best determine chronological ageing. For this, we used the flow FISH technique which enabled us to determine TL in different cell populations as well as the heterogeneity of TL distribution in individual volunteers.

  Spearman rank coefficient. For stepwise multivariate regression analysis, we retained all variables with p-values < 0.05 from the univariate analysis. We excluded variables with missing values in a pairwise procedure. Tests were two sided and p-values < 0.05 regarded statistically significant. All statistical tests were performed using SPSS Version 15.0 for Windows (SPSS Inc., Chicago, USA). For comparison of correlating r's Steiger's and Fishers's Z-test Dr. Calvin P. Garbins software (FZT.exe) was used (www-class.unl.edu/ ACCEPTED MANUSCRIPT 4/11 psycrs/statpage/comp.html).

Figure 1

 1 Figure 1

Acknowledgements

The assistance of Carmen Schön is greatly appreciated. This work was supported by grants of the Deutsche Forschungsgesellschaft (DFG Sp-502/4-2).

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Dr Josef Marksteiner
email: josef.marksteiner@kabeg.at

Christian Humpel

in peripheral blood mononuclear cells of patients with mild cognitive impairment

Glycogen-synthase kinase-3ß is decreased

The enzyme glycogen-synthase kinase-3 (GSK-3) plays an important role in Alzheimers disease (AD) due to its role of hyper-phosphorylation of Tau [START_REF] Hanger | Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase[END_REF][START_REF] Mandelkow | Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau[END_REF]. GSK-3 occurs in 2 forms, alpha and beta [START_REF] Phiel | GSK-3alpha regulates production of Alzheimer´s disease amyloid-beta peptides[END_REF][START_REF] Bhat | GSK3beta signalling: casting a wide net in Alzheimer's disease[END_REF][START_REF] Eldar-Finkelman | Glycogen synthase kinase 3: an emerging therapeutic target[END_REF] and both forms are widely distributed and in circulating lymphocytes. GSK-3 is regulated by phosphorylation at serine 9 in GSK-3ß and serine 21 in GSK-3α . GSK-3 has been shown to co-localize with dystrophic neurites and neurofilbrillary tangles [START_REF] Yamaguchi | Preferential labeling of Alzheimer neurofibrillary tangles with antisera for tau protein kinase (TPK) I/glycogen synthase kinase-3 beta and cyclin-dependent kinase 5, a component of TPK II[END_REF]Hooper et al., 2007), and active GSK-3 is contained in neurons with pretangle changes [START_REF] Pei | Distribution of active glycogen synthase kinase 3beta (GSK-3beta) in brains staged for Alzheimer disease neurofibrillary changes[END_REF]. GSK-3 expression is up-regulated in circulating peripheral lymphocytes in both AD and MCI patients [START_REF] Hye | Glycogen synthase kinase-3 is increased in whote cells early in Alzheimer´s disease[END_REF]. The aim of the present study was to investigate GSK-3ß by quantitative ELISA in our patient samples, in order to further examine whether GSK-3ß could be established as a biological marker in peripheral blood samples.

Healthy subjects (n=12) and patients with AD (n=44), with MCI (n=57) and with depression (n=25) were assessed by the same diagnostic procedure. Psychiatrists clinically examined all subjects, performed a standardized neurological examination, reviewed medical records. Furthermore, neuropsychological assessment and magnetic resonance neuroimaging was performed as reported previously (Marksteiner et al., submitted). EDTA blood (10 ml) was collected during normal routine clinical treatments and processed within 90 minutes. Peripheral blood mononuclear cells (PBMC) were separated from whole blood on a continuous Biocoll density gradient (1.077 g/ml) after centrifugation (400xg, 30 min). PBMC were visible as a white stratum between plasma phase and Biocoll-Paque. The interphase with the PBMC was carefully removed, washed in 50 ml PBS, and the pellet was immediately frozen at -80°C until use. The pellet was dissolved in 400 µl PBS + protease inhibitor cocktail (Sigma), sonicated on ice (30 sec, 125W/cm2, 140 µm amplitude, 100%), centrifuged 10 min 13 000xg at 4°C, and the supernatant used for the ELISA. Total protein was determined by Bradford assay using bovine serum albumine as a standard. GSK-3ß was determined by a commercial enzyme-linked immunoassay (ELISA) from TiterZyme (catNo. 900-144). The ELISA detects GSK-3ß with 100% but with less than 0.01% crossreactivity to GSK-3α . The standard curve was linear up to 5 ng/ml and had a sensitivity of 74 pg/ml. Briefly, PBMC lysates were diluted 1+1 with assay puffer, then applied to the pre-coated wells, incubated for 1hr

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at room, washed and then the capture antibody was added. After incubation for 1 hr at room, wells were washed, the conjugate was added, wells incubated for 30 min at room, washed and then the substrate was added and the color measured in an ELISA reader at 450 nm for 30 min. Analysis was determined in duplicates and correlated to a standard curve. Statistical analysis was performed by one Way ANOVA with a subsequent Fisher-PLSD posthoc test where p<0.05 was significant.

The patients´ characteristics are shown in table 1. AD patients were significantly older (table 1). The MMSE score was not statistically different between control subjects, patients with MCI and with depression, but significantly lower in AD patients (table 1). GSK-3ß levels were significantly decreased in MCI patients, but not in patients with depression. The numerical reduction of GSK-3 in AD was not statistically significant (table 1).

GSK-3 has been subsequently shown to function in a wide range of cellular processes, including differentiation, growth, motility and apoptosis [START_REF] Forde | Glycogen synthase kinase 3: a key regulator of cellular fate[END_REF][START_REF] Manoukian | Role of glycogen synthase kinase-3 in cancer: regulation by Wnts and other signaling pathways[END_REF]. GSK-3 involves several different signalling pathways, such as. e.g. the Akt, Zak-1, Wnt, RCN or glucocorticoid receptor pathways [START_REF] Forde | Glycogen synthase kinase 3: a key regulator of cellular fate[END_REF][START_REF] Manoukian | Role of glycogen synthase kinase-3 in cancer: regulation by Wnts and other signaling pathways[END_REF]. Aberrant regulation of GSK-3 has been implicated in several human pathologies. including Alzheimer's disease, non-insulin-dependent diabetes mellitus or cancer [START_REF] Forde | Glycogen synthase kinase 3: a key regulator of cellular fate[END_REF]. The functional role of GSK-3 in PBMCs has to be eludicated. So far, only one paper has reported Tau expression in human blood lymphocytes at very low levels in healthy subjects [START_REF] Kvetnoy | Tauprotein expression in human blood lymphocytes: a promising marker and suitable sample for life-time diagnosis of Alzheimer's disease[END_REF]. The same group reported enhanced Tau levels in blood lymphocytes of AD patients [START_REF] Kvetnoy | Tauprotein expression in human blood lymphocytes: a promising marker and suitable sample for life-time diagnosis of Alzheimer's disease[END_REF]. While [START_REF] Hye | Glycogen synthase kinase-3 is increased in whote cells early in Alzheimer´s disease[END_REF] showed enhanced expression of GSK3-α /ß in white blood cells, the present data indicate a decrease of GSK-3ß in PBMC of MCI patients as measured by quantitative ELISA. We can not offer an explanation for these contradictory results. Some of the differences may be explained by differences in the methodological procedures. Interestingly, [START_REF] Castri | Reduced insulin-induced phosphatidylinositol-3-kinase activation in peripheral blood mononuclear leucocytes from patients with Alzheimer's disease[END_REF] reported that the enzyme phosphatidylinositol-3-kinase (PI3K) is reduced in PBMC in AD patients. This enzyme is directly linked to GSK-3, because the insulin-induced PI3K controls GSK-3 activity. Thus it may be possible that both enzymes, GSK-3 and PI3K are regulated the same way in PBMC in dementia. In conclusion, further experiments are necessary to clarify whether GSK-3 is a suitable biological marker for MCI or AD.

This study was supported by the Austrian Science Funds (L429-B05).

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Table 1 : Glycogen synthase kinase-3ß (GSK-3ß) levels in peripheral blood mononuclear cells

 1 PBMC were isolated from EDTA blood of healthy subjects, patients with mild cognitive impairment (MCI) or with Alzheimer´s disease (AD) or with depression. The

	Group	Age	n	MMSE score	GSK-3ß (ng/mg protein)
	Control	68±3	12	28.4±0.3	14.3±2.2
	MCI	71±1 ns	57	27.0±0.4 ns	9.0±0.5 *
	AD	76±1 *	44	18.6±1 ***	10.2±0.9 ns
	Depression 68±2 ns	25	28.0±0.2 ns	11.0±1.1 ns

Mini Mental score (MMSE) was significantly lower in AD patients. Valus are given as mean±SEM; n = number of subjects; Statistical analysis was performed by one way ANOVA with a subsequent Fisher PLSD posthoc test (* p<0.05, *** p<0.001; ns not significant).