Jana Burianova

M.D. Ladislav Ouda
email: ouda@biomed.cas.cz

Oliver Profant

Josef Syka

Age-related changes in GAD levels in the central auditory system of the rat

Keywords: GAD, GABA, aging, inferior colliculus, auditory cortex, rat, western blot

published or not. The documents may come

INTRODUCTION

Auditory function in mammals is known to be significantly affected by aging, ultimately resulting in presbycusis (for review see [START_REF] Syka | Plastic changes in the central auditory system after hearing loss, restoration of function, and during learning[END_REF][START_REF] Gates | Presbycusis[END_REF][START_REF] Ohlemiller | Clinical Characterization of Age-Related Hearing Loss and Its Neural and Molecular Bases[END_REF]). In the human population, a loss of speech understanding with aging constitutes an important health and social impairment [START_REF] Frisina | Speech recognition in noise and presbycusis: relations to possible neural mechanisms[END_REF][START_REF] Popelar | Cochlear function in young and adult Fischer 344 rats[END_REF][START_REF] Gordon-Salant | Recognition of time-compressed and natural speech with selective temporal enhancements by young and elderly listeners[END_REF]. Pathological changes in presbycusis occur both in the inner ear and in the central auditory system. The peripheral component of presbycusis, which comprises mainly alterations of the inner and outer hair cells and/or stria vascularis, is relatively well understood [START_REF] Schuknecht | Cochlear pathology in presbycusis[END_REF][START_REF] Parham | Distortion product otoacoustic emissions in the C57BL/6J mouse model of age-related hearing loss[END_REF][START_REF] Spongr | Quantitative measures of hair cell loss in CBA and C57BL/6 mice throughout their life spans[END_REF][START_REF] Harding | The effect of an age-related hearing loss gene (Ahl) on noise-induced hearing loss and cochlear damage from low-frequency noise[END_REF][START_REF] Buckiova | Aging cochleas in the F344 rat: morphological and functional changes[END_REF]. The central component of presbycusis is thought to be associated with age-related alterations in the processing of the temporal parameters of complex acoustical stimuli occurring within the central auditory system.

Inhibitory systems in the brain are apparently strongly involved in the temporal processing of acoustical stimuli with the aim of sharpening responses to rapid complex sounds [START_REF] Walton | Neural correlates of behavioral gap detection in the inferior colliculus of the young CBA mouse[END_REF][START_REF] Strouse | Temporal processing in the aging auditory system[END_REF][START_REF] Krishna | Auditory temporal processing: responses to sinusoidally amplitude-modulated tones in the inferior colliculus[END_REF][START_REF] Liang | Neural representations of sinusoidal amplitude and frequency modulations in the primary auditory cortex of awake primates[END_REF][START_REF] Tremblay | Aging alters the neural representation of speech cues[END_REF][START_REF] Ostroff | Aging and the processing of sound duration in human auditory cortex[END_REF][START_REF] Popelar | Cochlear function in young and adult Fischer 344 rats[END_REF]. Since gama-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central auditory system [START_REF] Markram | Interneurons of the neocortical inhibitory system[END_REF], a decrease in GABA inhibition may significantly contribute to hearing deterioration with aging.

Recently, growing evidence has accumulated about the role of GABA-expressing neurons in inhibitory networks in different parts of the brain [START_REF] Caspary | Central auditory aging: GABA changes in the inferior colliculus[END_REF][START_REF] Gupta | Organizing principles for a diversity of GABAergic interneurons and synapses in the neocortex[END_REF][START_REF] Buzsáki | Inhibition and brain work[END_REF]. The distribution of GABA-immunoreactive (-ir) neurons and inputs in the auditory cortex has been described in detail for cats and monkeys and implies that GABAir neurons are present in the AC in relatively large numbers, approximately 20-25% of the total population of neurons [START_REF] Hendry | Numbers and proportions of GABA-immunoreactive neurons in different areas of monkey cerebral cortex[END_REF]Prieto et al., 1994a,b). In the IC, a similar portion, 20-30% of the total number of neurons, was found to be GABA-ir in cats and rats [START_REF] Oliver | Morphology of GABAergic neurons in the inferior colliculus of the cat[END_REF]Merchan et al, 2005). GABA is synthesized by the decarboxylation of glutamate, and the reaction is catalysed by the key rate-limiting enzyme glutamate decarboxylase (GAD). In the mammalian brain, two GAD isoforms of 65 000 and 67 000 molecular weight (GAD 65 and GAD 67) are present (Erlander et al., 1991). Most GABAexpressing neurons contain both isoforms, with GAD 65 being more prevalent in axonal terminals and membranes preferentially synthesizing GABA for vesicular release, while GAD 67 is distributed throughout the neuron including the soma, preferentially synthesizing nonvesicular cytoplasmic GABA (Erlander an Tobin, 1991;[START_REF] Feldblum | Different distributions of GAD65 and GAD67 mRNAs suggest that the two glutamate decarboxylases play distinctive functional roles[END_REF][START_REF] Feldblum | Comparative distribution of GAD65 and GAD67 mRNAs and proteins in the rat spinal cord supports a differential regulation of these two glutamate decarboxylases in vivo[END_REF][START_REF] Esclapez | Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms[END_REF][START_REF] Hendrickson | Differential localization of two glutamic acid decarboxylases (GAD65 and GAD67) in adult monkey visual cortex[END_REF]. The two GAD isoforms might be differently involved in the spatial and temporal processing and/or coding of information by GABA-expressing neurons (for review see [START_REF] Soghomonian | Two isoforms of glutamate decarboxylase: why?[END_REF][START_REF] Wei | Post-translational Regulation of L: -Glutamic Acid Decarboxylase in the Brain[END_REF].

Several lines of evidence suggest a decreasing function of GABA-mediated inhibition within the central auditory pathway in aged animals. For example, single unit recordings illustrate the presence of a less precise processing of complex sounds and extended excitatory areas in the IC (Palombi and Caspari 1996;[START_REF] Walton | Age-related alteration in processing of temporal sound features in the auditory midbrain of the CBA mouse[END_REF][START_REF] Walton | Age-related alterations in the neural coding of envelope periodicities[END_REF][START_REF] Simon | Age reduces response latency of mouse inferior colliculus neurons to AM sounds[END_REF]. Age-related alterations in the fine-tuned receptive fields and a relative increase in poorly tuned receptive fields could influence the processing of sounds in the central auditory system (Turner et al., 2005a,b). The results of measurements of GABA or GAD levels and GABA receptor subunit levels and binding intensity correspond with the electrophysiological reports. In Fischer 344 rats, a significant age-related decrease in the number of GABA immunoreactive cells was found in the central nucleus of the IC (CIC) combined with a decrease in the enzymatic activity of GAD and in the release of GABA [START_REF] Caspary | Immunocytochemical and neurochemical evidence for age-related loss of GABA in the inferior colliculus: implications for neural presbycusis[END_REF][START_REF] Raza | Age-related changes in brainstem auditory neurotransmitters: measures of GABA and acetylcholine function[END_REF][START_REF] Caspary | Central auditory aging: GABA changes in the inferior colliculus[END_REF]. In addition, a decline in the number of GABA-ergic synaptic terminals and synapses of GABA-ir neurons was observed in the CIC with no detectable neuronal losses [START_REF] Helfert | Age-related synaptic changes in the central nucleus of the inferior colliculus of Fischer-344 rats[END_REF]. Also GABA-A and GABA-B receptor binding intensity was reported to decline, and the protein levels of the receptor subunits were found to be altered during aging in the IC of Sprague-Dawley and Fischer 344 rats (Guttierez et al, 1994;Millbrandt et al., 1994Millbrandt et al., , 1997;;[START_REF] Caspary | Age-related changes in GABAA receptor subunit composition and function in rat auditory system[END_REF][START_REF] Schmidt | Age-related decline of functional inhibition in rat cortex[END_REF].

Decreases in the levels of GAD65 and GAD67 mRNAs and in the optical density of GAD67immunoreactive cells were observed in the auditory cortex of old Fischer 344/Brown Norway rat [START_REF] Ling | Age-related loss of the GABA synthetic enzyme glutamic acid decarboxylase in rat primary auditory cortex[END_REF]. However, it is not well known whether the age-related decline in the GABA system is activity-dependent and primarily follows the deterioration of the sensory inputs with aging or whether it rather results from changes occurring within the central auditory system with aging (for review see [START_REF] Caspary | Inhibitory neurotransmission, plasticity and aging in the mammalian central auditory system[END_REF]. In addition, no clear evidence of inter-strain differences in these changes has been reported, in contrast to the findings in calcium binding proteins that are well known to colocalize in neurons with GABA [START_REF] Kosaka | GABAergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus[END_REF][START_REF] Kawaguchi | Neurochemical features and synaptic connections of large physiologically-identified GABAergic cells in the rat frontal cortex[END_REF]. In this case, a significant strain-specificity in their age-related expression was shown for calretinin and parvalbumin [START_REF] Zettel | Age-related changes in calbindin D-28k and calretinin immunoreactivity in the inferior colliculus of CBA/CaJ and C57Bl/6 mice[END_REF][START_REF] Zettel | Early bilateral deafening prevents calretinin up-regulation in the dorsal cortex of the inferior colliculus of aged CBA/CaJ mice[END_REF][START_REF] Ouda | Changes in parvalbumin immunoreactivity with aging in the central auditory system of the rat[END_REF].

In our study we attempted to evaluate the age-related changes in both GAD isoforms in the central auditory system in Long-Evans rats, a strain with a very limited age-related loss of hearing function [START_REF] Syka | Auditory frequency and intensity discrimination in pigmented rats[END_REF][START_REF] Popelar | Age-related changes in cochlear and brainstem auditory functions in Fischer 344 rats[END_REF]. Immunohistochemistry and western blot protein analysis were used to compare GAD 65 and 67 expression in the inferior colliculus (IC) and auditory cortex (AC) in young and old rats. In addition, the same analysis was also performed on a representative sample of Fischer 344 rats, a strain with pronounced hearing deterioration with aging, with the aim of studying possible inter-strain differences.

MATERIALS AND METHODS

Animals

Thirty rats, strain Long-Evans (15 young animals 3-5 months old and 15 aged animals 30-35 months old), and twelve rats of the strain Fischer 344 (7 young animals 3-5 months old and 5 aged animals 24 months old) were used in the experiments. The Long-Evans rats were obtained from a local facility and, starting at 2 months of age, were reared and aged (aging group) in-house, under known rearing conditions and with a known health history. Fischer 344 rats were purchased at 2 months of age from Charles River Deutschland (Sulzfeld, Germany) and then reared in-house under the same conditions. No signs of middle ear infection were present in any animal during their stay in the animal facility. The care and use of the animals and all experimental procedures were performed in compliance with the guidelines of the Ethical Committee, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, and the Declaration of Helsinki.

GAD67 immunohistochemistry

Long-Evans rats, 3-5 (n=5) and 30-35 (n=5) months old, and Fischer 344 rats, 3-5 (n=5) and 24 (n=3) months old, were placed under deep anesthesia (ketamine 35 mg/kg + xylazine 6 mg/kg, i.m.) then transcardially perfused with saline followed by 4% paraformaldehyde fixative in 0.1 M phosphate buffer (pH 7.4). Brains were removed within 15 min of perfusion, postfixed 1 hour at 4° C (same fixative) and then cryoprotected with 30% sucrose in phosphate buffer overnight. Coronal sections (40 m thick) were cut with a freezing microtome.

Free-floating sections were preblocked in normal serum for 1 h and then incubated at 4° C with anti-GAD67 (mouse monoclonal, Chemicon, 1:2000) diluted in PBS containing 1% normal serum. After 24 hours, sections were incubated with a biotinylated secondary antibody (Vector, 1:200) for 45 min and then with peroxidase-labeled ABC reagent (Vector). The antibody labeling was visualized by incubating the sections for 3 minutes in 0.05% diaminobenzidine (DAB) with 0.01% hydrogen peroxide. Sections were mounted on slides, dehydrated and coverslipped.

GAD65 immunohistochemistry

Long-Evans rats, 3-5 (n=4) and 30-35 (n=4) months old, were subjected to the same procedure leading to the preparation of histological sections as in the case of rats for GAD 67 staining.

Free-floating sections were preblocked in normal serum for 1 h and then incubated at 4° C with anti-GAD65 (rabbit polyclonal, Chemicon, 1:750) diluted in PBS containing 1% normal serum. After 72 hours, sections were incubated with a biotinylated secondary antibody (Biosource, 1:200) for 1 hour and then with peroxidase-labeled ABC reagent (Vector). The antibody labeling was visualized by incubating the sections for 3 minutes in 0.05% diaminobenzidine (DAB) with 0.01% hydrogen peroxide. Sections were mounted on slides, dehydrated and coverslipped.

Western Blot Protein Analysis

Long-Evans rats, 3-5 (n=6) and 30-35 (n=6) months old, and Fischer 344 rats, 4 (n=2) and 24 (n=2) months old, were used in the analysis. Anesthetized rats (ketamine 35 mg/kg + xylazine 6 mg/kg, i.m) were decapitated, their brains quickly extracted and rinsed in ice-cold physiological solution, and the inferior coliculli (ICs) and auditory (ACs) and visual cortices (VCs) were rapidly removed bilaterally. The samples included the whole IC, the Te1+Te3 (Zilles 1995) areas of the AC and the V1+V2 areas of the VC [START_REF] Paxinos | The rat brain[END_REF].

All samples were immediately put into dry ice after extraction and stored frozen at -80°C until processed.

For the analysis, ICs, ACs and VCs were homogenized by a Potter-Elvehjem homogenizer in 0.05 M Tris-NaCl (pH 7.4) buffer with protease inhibitors (Sigma). The homogenate was centrifuged at 10 000 g for 10 min at 4°C. To ensure similar protein loading, the total protein concentration of these extracts was determined by using the Bradford method with BSA as the standard. Samples (cytosolic fraction) were incubated in boiling water for 10 min at 80°C in sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer containing 10% glycerol, 2% SDS, 0.05% bromphenol blue and 4M dithiotreitol. Samples were then subjected to Tris/Tricine/SDS-PAGE electrophoresis on a 3% bis-acrylamide polyacrylamide gel at 30 mA/gel for 150 min on a Mini-Protean II apparatus (Bio-Rad). After electrophoresis, the resolved proteins were transferred (Bio-Rad Mini Protean II transblot apparatus at 350 mA for 60 min at 4 °C in 25 mM Tris, 192 mM glycine, 20% methanol, 0,1% SDS) to a nitrocellulose membrane (Amersham, Biosciences). Membranes were incubated in 5% non-fat dry milk in 10% Tris-buffered saline with 0.05% Tween 20 (TBST) for 60 min at room temperature to block nonspecific protein binding. After being washed in TBST buffer (3 times quickly, 3 x 5 min each), the membranes were probed with GAD 67-specific (mouse monoclonal, Chemicon, 1:5000 in TBST), GAD 65-specific (rabbit polyclonal, Chemicon, 1:1200 in TBST) or actin-specific (mouse monoclonal, Chemicon, 1:10 000 in TBST) primary antisera overnight at 4°C. The membranes were washed again and incubated with goat anti-mouse IgG or anti-rabbit IgG antibody conjugated with horseradish peroxidase (Upstate, 1:7500; Chemicon, 1:3333 in TBST) for 2 h at room temperature. Before enhanced chemiluminescence (ECL), the membranes were washed as described above and stored in TBST for at least 2 h. For ECL, substrates A (Luminol solution) and B (H 2 O 2 solution) were prepared, mixed 40:1 (Amersham Biosciences) just before use, and poured on the membranes.

The specific signals were detected on autoradiographic film (Kodak MXB). Films were developed at room temperature, stopped, fixed, washed under running cold water and airdried. Scanning (Canon CanoScan 8400F) and ImageQuant software were used for quantifying the relative abundance of GAD and actin in individual samples. The amount of protein applied to the gel varied for each isoform and fraction to achieve linearity with the intensity x area (volume) of the band on the western blot. To ensure the specificity of GAD67, GAD65 and actin immunoreactive proteins, prestained molecular weight protein standards were used (Invitrogen). The levels of GAD65 and GAD67 were calculated as the ratio of the optical density of the antibody of interest to the optical density of the antibody directed against -actin.

Immunohistochemistry -stereological quantification

GAD65 and 67 immunoreactivity was examined in all sections containing the inferior colliculus, auditory and visual cortices, delineated according to anatomical atlases (Zilles, 1985;[START_REF] Paxinos | The rat brain[END_REF] and with the help of Nissl-stained sections (topographical borders of cortical areas) (Fig. 1A,B). For the IC, sections were sampled from bregma -8.0 mm to -9.2 mm. Sections containing the auditory cortex were sampled from bregma -4.2 mm to bregma -6.0 mm (Zilles, 1995), identified by measuring 2.0-2.5 mm from the rhinal fissure for Te3 and 3.5-4.0 mm for Te1. As an auxiliary criterion, the line going through the dorsal margin of the diencephalon indicated the control dorsal (upper) border of the auditory cortex.

For the visual cortex (V1, V2 -Paxinos and Watson, 1998), sections were sampled from bregma -5.0 mm to -8.0 mm. To estimate the total number of GAD65 and 67-ir neurons, an unbiased stereological method, the optical fractionator, was used [START_REF] West | Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator[END_REF] for review see Mayhew and Gundersen, 1996). The optical fractionator method is unaffected by tissue shrinkage, and therefore it was not necessary to measure this variable. Measurements were determined using bright-field microscopy (Leica DMRXA microscope) connected to a video camera, which transmitted the microscopic image to a monitor (Optronics, 1600x1200 px) coupled with Neurolucida software (MicroBrightField). Two sets of motors were connected to the microscope to move the section a known distance in the x-and y-directions.

The z-direction was measured using a microcator. A 10x objective lens was used to delineate the regions of interest and the starting position of the counting frame. We adapted the Neurolucida-generated grid superimposed on the screen with the use of a random number table to establish the random starting position of the counting frame in the area of interest.

A 100x oil-immersion objective was used and the appropriate counting frame superimposed on the screen. The counting frames used to obtain the sampling had the same size (3880 m²). The upper and lower surfaces of the sections were examined to determine if the margins of the tissue sections were depleted of neuronal nuclei, as suggested by [START_REF] Andersen | Pronounced loss of cell nuclei and anisotropic deformation of thick sections[END_REF]. The height of the optical dissector was constant at 5 m, and the first and last 5 m of the section thickness was omitted from the analysis. Approximately 200 neurons per animal were counted in each examined structure. Each neuron in the counting frame was counted when its distinct nucleus came to maximum focus. At maximum focus, the cell body was outlined by a cursor on the computer screen, and the morphological parameters and optical density were measured automatically using Neurolucida software. Only positive somas that exceeded 5 m in diameter and with a distinct nucleus were counted and used for analysis.

Variation in the background immunostaining affects the real values of the optical density of the neuronal soma. In studies of aging, there are not specific "indifferent reference structures" available in the brain for normalization, hence we calculated the normalized value of the optical density in each section as the ratio between the average optical density of the analyzed GAD-ir neurons in the section and the average optical density of a corresponding number of immunonegative cells in the same section (compare [START_REF] Ling | Age-related loss of the GABA synthetic enzyme glutamic acid decarboxylase in rat primary auditory cortex[END_REF]. In most cases the immunopositivity of the neurons was explicit; in a few disputable cases, a selected neuron was omitted from the analysis as "non-GAD-ir" unless the measured optical density of its soma was higher than double the average optical density of immunonegative cells in the same section.

Statistical analysis

The results of the statistical analysis were expressed as mean ± S.E.M. The significance of the differences between young and old groups of rats was assessed by the non-parametric two-tailed Mann-Whitney test with the use of GraphPad Prisma software (version 4.0) for both immunohistochemistry and western blots. The significance of differences in western blot protein analysis among protein levels and among the magnitudes of changes in different brain structures (IC vs. AC vs.VC) in young and in old rats was assessed by a one-way ANOVA test and Bonferroni`s multiple comparison test with the use of GraphPad Prisma software (version 4.0). P values of 0.05 or less were considered statistically significant. For each statistical test, each examined animal was represented by one number to ensure the maximal independence of the data entering the tests.

RESULTS

I. GAD65 and 67 changes with aging in the Long-Evans strain

Inferior colliculus GAD65 and 67-ir neurons were distributed in both young and old animals throughout all three subdivisions of the IC. In the central nucleus of the IC, cell bodies of variable sizes and shapes prevailed, with a large variation in the intensity of immunostaining. The majority of GAD-ir neurons in the external cortex of the IC (EIC) were large spindle and multipolar cells or smaller oval neurons, in both the 2nd and 3rd layers of the EIC (for details see [START_REF] Oliver | Morphology of GABAergic neurons in the inferior colliculus of the cat[END_REF]. Only a few small immunoreactive neurons were present in the dorsal cortex of the IC (Fig. 2A,B).

Significant age-related changes in GAD immunoreactivity were found in the CIC and EIC (Fig. 3A-D). In the CIC, the optical density of GAD65 and 67-ir neuronal somas decreased by 23% (P=0.028, U=0) and 25% (P=0.008, U=0), respectively. The number of GAD65 and 67-ir cells decreased slightly by 17% (P=0.028, U=0) and 9% (N.S.), respectively. In the EIC only in GAD67-ir somas the optical density decreased significantly by 14% (P=0.032, U=2), while the number of GAD65 and 67-ir did not differ between old and young animals. No significant age-related changes in the volume of GAD-ir somas were found in the IC with aging.

Western blot analysis demonstrated a significant age-related decline in the levels of GAD65 and GAD67 proteins in the whole IC of old rats in comparison with young animals of 51% and 49%, respectively (both P=0.002, U=0) (Fig. 4). The analyzed samples included all three subdivisions of the IC. A remarkable finding of the additional statistical analysis, in both young and old animals, the levels of GAD65 and GAD67 proteins in the IC were almost three times higher compared to the levels in both the auditory and visual cortices (both proteins P<0.001, Bonferroni`s multiple comparison test).

Auditory cortex

Independently of age, GAD65 and 67-ir cells were scattered throughout all layers, with slightly higher numbers observed in the superficial cortical layers (I -IV) than in the deeper layers (V -VI) (for description see Prietto et al., 1994a,b) The neuropil staining followed the same pattern with greater positivity in the superficial layers (II-IV) (Fig. 2C,D).

The results of immunohistochemical staining indicated a significant decrease in the optical density of GAD65 and 67-ir somas in old animals of 21% (P=0.028, U=0) and 22% (P=0.016, U=1), respectively (Fig. 3A-D). The number of GAD65-ir neurons did not change in old rats in comparison with young animals (only a slight decrease of 7%, N.S.), while the number of GAD67-ir neurons decreased more markedly by 13% (P=0.032, U=2). No layer-specificity or inter-areal differences (Te1 vs. Te3) of these changes were observed; similarly, the average volume of GAD-ir somas did not change with aging.

Western blot protein analysis revealed a significant age-related decrease in the levels of GAD65 and GAD67 of 43% and 52%, respectively (both P=0.002, U=0) (Fig. 4). The analyzed samples included the Te1+Te3 areas. Differences in GAD65 and 67 protein levels between the auditory and visual cortices were non-significant in young animals. In old rats, GAD67 protein levels in the auditory cortex were lower than in the visual cortex (P<0.05, Bonferroni`s multiple comparison test), whereas for GAD65 protein levels, the difference between the auditory and visual cortices was still non-significant. The declines found in all three examined brain regions are apparent on representative blots shown in Fig. 5.

Visual cortex

GAD65 and 67 immunoreactive neurons were present in all cortical layers of the visual cortex; their distribution and neuropil staining followed a similar pattern as in the auditory cortex.

The optical density was found to decrease by 11% for GAD65-ir somas (N.S.) and 18% for GAD67-ir somas (P=0.016, U=1,5) (Fig. 3A-D). In contrast, the numbers of GAD65 and 67-ir neurons did not change with aging (the decrease was in the range of 5%, N.S). As in the AC, no significant changes in the average volumes of immunoreactive somas or layer-specificity were found with aging.

In western blot analysis, decreases in the levels of GAD65 and GAD67 proteins were found in the visual cortex as well: a decrease of 22% for GAD65 (P=0.041, U=5) and 20% for GAD67 (P=0.028, U=2) (Fig. 4). However, the decrease was less apparent compared to both the auditory cortex and the IC (P<0.01, Bonferroni`s multiple comparison test), while a comparison between decreases in the IC and AC showed no difference. These results held true for both GAD65 and GAD67.

II. GAD65 and 67 changes with aging in the Fischer 344 strain

In principle, the age-related changes in Fischer 344 rats followed the same pattern as the age-related changes in the Long-Evans animals; however, the relatively low number of animals of this strain used in our study precluded a detailed statistical evaluation of the results. Using immunohistochemistry, only differences in GAD67 staining were studied: the optical density of GAD67-ir neuronal somas decreased by 21% (P=0.035, U=0), 13%, 14% and 15% in the CIC, EIC, AC and VC, respectively, while the number of GAD67-ir cells decreased by 6%, 10%, 10% and 6% in the same structures (Fig. 3E-F).

Both GAD65 and GAD67 proteins were examined by western blot protein analysis. In the IC, GAD65 protein levels decreased by 46% and GAD67 levels by 44% (Fig. 4). The decline of GAD65 protein levels in the AC was 34% and that of GAD67 was 49%; in the VC decreases of 19% in GAD65 protein levels and 20% in GAD67 levels were observed. In young and old Fischer 344 animals, the relative ratios between GAD65 and GAD67 protein levels in the IC, AC and VC followed a very similar pattern as that seen in young and old Long-Evans rats. However, due to the limited number of animals, the data did not show statistically significant differences (P<0.05 only when GAD65 or 67 protein levels in the IC were compared with the levels in the AC and VC cortices, Bonferroni`s test).

DISCUSSION

Our results demonstrate significant age-related changes in GAD65 and 67 immunoreactivity in the inferior colliculus and auditory cortex of the rat auditory system, which comprise decreases in the number and optical density of GAD65 and 67-ir neuronal somas and a decline in the levels of GAD65 and 67 proteins. The obtained data demonstrate a rather uniform character of the changes. First, the age-related changes in both GAD65 and 67 proteins shared very similar patterns. Second, the results obtained with immunohistochemical techniques were in good agreement with the results acquired with western blotting. Third, similar tendencies in the changes were present in both the IC and AC. In summary, the decrease in GAD expression with aging occurs similarly at both the subcortical and cortical levels of the rat central auditory system and involves both GAD isoforms.

The overall reductions in the levels of GAD65 and 67 proteins reported in the present work are in accordance with previous publications that reported an age-related decline in GABA and GAD levels in the IC and AC. In the IC, the number of GABA immunoreactive cells was described to be decreased in the central nucleus of the IC (CIC) in old rats. This decrease was associated with a decrease in enzymatic activity, the levels of GABA and the release of GABA [START_REF] Caspary | Immunocytochemical and neurochemical evidence for age-related loss of GABA in the inferior colliculus: implications for neural presbycusis[END_REF][START_REF] Raza | Age-related changes in brainstem auditory neurotransmitters: measures of GABA and acetylcholine function[END_REF], Guttierez et al, 1994;[START_REF] Caspary | Central auditory aging: GABA changes in the inferior colliculus[END_REF]. No changes in the optical density of GABA or GAD stained cells in the IC with aging have yet been reported, but such a decline, comparable to our results, was observed in the auditory cortex of old Fischer 344/Brown Norway F1 hybrid rats [START_REF] Ling | Age-related loss of the GABA synthetic enzyme glutamic acid decarboxylase in rat primary auditory cortex[END_REF]. In addition, the authors also reported a decline in GAD65 and 67 mRNA labeling in the auditory cortex, consistent with the decreased optical density of GAD67-ir neurons. No cortical layer specificity for the reported changes was found in their study, which is in agreement with our results. A decline in the optical density of neuronal somas may reflect either a decrease in the expression of the protein or changes in the volume of the cell (for example, [START_REF] Krzywkowski | Age-related changes in parvalbumin-and GABA-immunoreactive cells in the rat septum[END_REF]. Since there were no significant changes in the average volume of GAD-ir somas in the present work, the results support the diminished expression of the proteins in this case. This is in agreement with the decreased levels of GAD proteins found in our western blotting analysis.

The observed decreased number of GAD-ir (GAD65 and GAD67) neurons in the IC and AC in the present paper cannot be explained simply by a non-selective neuronal loss because in our previous experiments, we did not find any reduction in the total number of neurons in old Long-Evans and Fischer 344 rats on Nissl-stained sections [START_REF] Ouda | Changes in parvalbumin immunoreactivity with aging in the central auditory system of the rat[END_REF]. This finding is in agreement with the data of other authors, who reported no significant changes accompanying aging in the total number of neurons in the inferior colliculus, hippocampus or cortex in rats [START_REF] Helfert | Age-related synaptic changes in the central nucleus of the inferior colliculus of Fischer-344 rats[END_REF][START_REF] Poe | Age-related decline of presumptive inhibitory synapses in the sensorimotor cortex as revealed by the physical disector[END_REF][START_REF] Merrill | Conservation of neuronal number and size in the entorhinal cortex of behaviorally characterized aged rats[END_REF][START_REF] Stanley | Aging in the rat hippocampus is associated with widespread reductions in the number of glutamate decarboxylase-67 positive interneurons but not interneuron degeneration[END_REF]. In our experiments, the decrease in the optical density of GAD-immunoreactive somas rather suggests the diminished expression of the protein, beyond the level detectable by immunohistochemical methods, as the major reason for the reduced number of GAD-ir neurons.

An interesting finding in our experiments is the striking difference in the regional levels of GAD found with western blotting. The approximately three-fold higher levels of GAD65 and 67 in the IC in comparison with the auditory and visual cortices are in good agreement with previously reported ratios of both isoform levels in the midbrain and thalamus on one side and the neocortex on the other side [START_REF] Sheikh | Regional distribution and relative amounts of glutamate decarboxylase isoforms in rat and mouse brain[END_REF]. The functional implication of these differences remains to be determined. In our immunohistochemical analysis, no significant indication of striking differences between the IC and cortex was observed. Nevertheless, our immunohistochemical method was not designed to evaluate dendritic branching or GAD-ir puncta, which could contain a significant proportion of GAD. The fundamental role of inhibition in the IC is well known, including the strong representation of GABA terminals, which also implies the presence of high GAD levels in the IC [START_REF] Gerken | Central tinnitus and lateral inhibition: an auditory brainstem model[END_REF][START_REF] Pollak | Roles of inhibition for transforming binaural properties in the brainstem auditory system[END_REF][START_REF] Frisina | Subcortical neural coding mechanisms for auditory temporal processing[END_REF][START_REF] Batra | Monaural and binaural processing in the ventral nucleus of the lateral lemniscus: a major source of inhibition to the inferior colliculus[END_REF]Merchan et al., 2005).

In addition to the Long-Evans strain, a relatively small but representative sample of young and old Fischer 344 rats was examined in the present work. Long-Evans and Fischer 344 represent two rat strains with a variety of morphological, physiological and behavioral differences. For example, similarly to other inbred strains (Wistar, Sprague-Dawley and Dark-Agouti), Fischer 344 rats display large cognitive deficits in different tests of spatial memory in contrast to wild rats and Long-Evans rats [START_REF] Harker | Place and matching-to-place spatial learning affected by rat inbreeding (Dark-Agouti, Fischer 344) and albinism (Wistar, Sprague-Dawley) but not domestication (wild rat vs. Long-Evans, Fischer-Norway)[END_REF]. A faster and more pronounced deterioration of hearing function with aging was found in the Fischer 344 strain in comparison with Long-Evans rats, resulting in larger hearing threshold shifts, a diminution of distortion product otoacoustic emissions and a decrease in middle-ear compliance [START_REF] Popelar | Cochlear function in young and adult Fischer 344 rats[END_REF][START_REF] Popelar | Age-related changes in cochlear and brainstem auditory functions in Fischer 344 rats[END_REF]. In addition, the deterioration of the stria vascularis and ligamentum spirale was observed in aged Fischer 344 rats [START_REF] Buckiova | Collagen changes in the cochlea of aged Fischer 344 rats[END_REF][START_REF] Buckiova | Aging cochleas in the F344 rat: morphological and functional changes[END_REF]. Age-related sensory deficits in this strain are also present in visual function due to retinal degeneration [START_REF] Di Loreto | The influences of age, retinal topography, and gender on retinal degeneration in the Fischer 344 rat[END_REF]. In spite of this, we observed very similar age-related changes in GAD65 and 67 protein levels and in the immunohistochemical results in Fischer 344 rats and Long-Evans rats. Taking into account the similar effects of aging on GAD levels described in the Fischer 344/Brown Norway hybrid rats [START_REF] Ling | Age-related loss of the GABA synthetic enzyme glutamic acid decarboxylase in rat primary auditory cortex[END_REF], it might be suggested that age-related changes in GAD proteins in the IC and AC share a uniform tendency, which is not dependent on the rat strain. A similar situation exists in another species, in mice: hearing function in the CBA/CaJ strain of mice deteriorates very slowly as in the Long-Evans strain of rats, whereas the C57BL/6J strain is known to undergo rapid deterioration with early deafness [START_REF] Parham | Distortion product otoacoustic emissions in the C57BL/6J mouse model of age-related hearing loss[END_REF][START_REF] Spongr | Quantitative measures of hair cell loss in CBA and C57BL/6 mice throughout their life spans[END_REF]. In spite of these differences in age-related defects of auditory function, which are mostly caused by pathologies in the inner ear, significant age-related alterations of the inhibitory function in the central auditory system were reported in both strains of mice [START_REF] Walton | Age-related alteration in processing of temporal sound features in the auditory midbrain of the CBA mouse[END_REF][START_REF] Walton | Age-related alterations in the neural coding of envelope periodicities[END_REF][START_REF] Felix | Excitatory, inhibitory and facilitatory frequency response areas in the inferior colliculus of hearing impaired mice[END_REF]. Therefore, we may tentatively suggest that the age-related decline in GAD expression in the IC and AC demonstrated in our experiments does not depend primarily on peripheral deafferentation but is, at least partially, of central origin.

The present results are to some extent different from our previous findings concerning agerelated changes in parvalbumin immunoreactivity in the IC and AC of Long-Evans and Fischer 344 rats [START_REF] Ouda | Changes in parvalbumin immunoreactivity with aging in the central auditory system of the rat[END_REF]. Specifically, there was no reduction in parvalbumin immunoreactivity in old Long-Evans rats, while there was a pronounced decline in the number of parvalbumin-ir cells in the AC of old Fischer 344 rats. Since parvalbuminexpressing cells in the central nervous system form a subpopulation of GABA-expressing neurons [START_REF] Kosaka | GABAergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus[END_REF]Freund and Buzsáki, 1996;[START_REF] Kawaguchi | Neurochemical features and synaptic connections of large physiologically-identified GABAergic cells in the rat frontal cortex[END_REF], it could be expected that the changes in parvalbumin and GAD expression with aging would have a more similar character. However, different age-related changes in the immunoreactivity of parvalbumin and the expression of GABA (GAD) were reported, for example, in the rat septum, hippocampus and sensorimotor cortex (Krzywkovski et al., 1995;[START_REF] Vela | Rat hippocampal GABAergic molecular markers are differentially affected by ageing[END_REF][START_REF] Potier | Age-related alterations of GABAergic input to CA1 pyramidal neurons and its control by nicotinic acetylcholine receptors in rat hippocampus[END_REF][START_REF] Shi | Maintenance of inhibitory interneurons and boutons in sensorimotor cortex between middle and old age in Fischer 344 X Brown Norway rats[END_REF].

Age-related alteration in GABA inhibition was a feature also present in the visual cortex.

Our observation of decreased GAD65 and 67 immunoreactivity and protein levels in the visual cortex is in agreement with [START_REF] Hua | Decreased proportion of GABA neurons accompanies age-related degradation of neuronal function in cat striate cortex[END_REF], who found a reduction in the number of GABA-ir neurons in the primary visual cortex of the cat with aging while no changes in the total number of neurons were observed. In addition, a decreased function of the GABA system was reported in the primary visual cortex of aged monkeys [START_REF] Schmolesky | Degradation of stimulus selectivity of visual cortical cells in senescent rhesus monkeys[END_REF][START_REF] Leventhal | GABA and its agonists improved visual cortical function in senescent monkeys[END_REF]. Western blot protein analysis in our experiments showed a statistically less pronounced decline in GAD65 and GAD67 levels with aging in the VC compared to the AC, which may signal different GABA-related aging processes in the two cortical sensory areas. A decreased function of the GABA inhibitory system with aging was also documented in the rat hippocampus [START_REF] Stanley | Aging in the rat hippocampus is associated with widespread reductions in the number of glutamate decarboxylase-67 positive interneurons but not interneuron degeneration[END_REF][START_REF] Shi | Stereological quantification of GAD-67-immunoreactive neurons and boutons in the hippocampus of middle-aged and old Fischer 344 x Brown Norway rats[END_REF][START_REF] Ling | Age-related loss of the GABA synthetic enzyme glutamic acid decarboxylase in rat primary auditory cortex[END_REF], while no reduction in the number of GABA neurons or GAD immunoreactivity was found in the septum, sensorimotor cortex or parietal cortex (Krzywkovski et al., 1995;[START_REF] Poe | Age-related decline of presumptive inhibitory synapses in the sensorimotor cortex as revealed by the physical disector[END_REF][START_REF] Ling | Age-related loss of the GABA synthetic enzyme glutamic acid decarboxylase in rat primary auditory cortex[END_REF][START_REF] Shi | Maintenance of inhibitory interneurons and boutons in sensorimotor cortex between middle and old age in Fischer 344 X Brown Norway rats[END_REF]. Therefore, it is evident that aging is not necessarily accompanied with a homogenous decline of GABA levels in all brain regions.

The alteration of GABA-mediated inhibition in the central auditory pathway may have functional implications. The decreased inhibition may result in a broadening of the excitatory areas and thus in the poorer tuning of the neuronal receptive fields, which is in agreement with existing electrophysiological reports (Palombi and Caspari 1996[START_REF] Walton | Age-related alterations in the neural coding of envelope periodicities[END_REF], Turner et al., 2005a,b). The reduction of fine-tuned receptive fields results in poorer discrimination of the temporal parameters of sounds [START_REF] Narayan | Delayed inhibition in cortical receptive fields and the discrimination of complex stimuli[END_REF]. The reduced temporal acuity limits the gap detection thresholds and gap duration difference limen, which are belived to be essential for the successful processing of complex sounds including language (Gordon-Salant and Fitzgibbons, 1994;[START_REF] Grose | Temporal processing deficits in the pre-senescent auditory system[END_REF][START_REF] Gordon-Salant | Recognition of time-compressed and natural speech with selective temporal enhancements by young and elderly listeners[END_REF]. With aging, the gap detection thresholds significantly increase and gap duration difference limen worsens [START_REF] Rybalko | Age-related deterioration of hearing in rats[END_REF] These data support the conclusion that the age-related decline of GABA levels could contribute significantly to the deterioration of hearing function associated with aging in mammals, including presbycusis in man.

Legends to figures:

Fig. 1

 1 Fig. 1Topographical overview of the IC (A), AC and VC (B) and their subdivisions. Drawings

Fig. 2

 2 Fig. 2Illustration of GAD67 immunoreactivity in the EIC (A), CIC (B) and in the deep and

Fig. 3 Fig. 4

 34 Fig. 3Results of the quantitative evaluation of GAD65 and 67 immunoreactivity in Long-Evans and

Fig. 5

 5 Fig.5

Fig.1

Fig.3

Fig.4

Acknowledgements:

The authors wish to thank Mrs. J. Janouskova and Mr. J. Setnicka for their technical assistance. This study was supported by the Grant Agency of the Czech Republic (309/07/1336), by the Grant Agency of the Ministry of Health of the Czech Republic (NR8113-4), and by AV0Z50390512 and LC 554.


Juulia Jylhävä
email: juulia.jylhava@uta.fi

Carita Eklund

Marja Jylhä

Antti Hervonen

Terho Lehtimäki

Pekka Karhunen

Mikko Hurme

Complement factor H 402His variant confers an increased mortality risk in Finnish nonagenarians: The Vitality 90+ Study

Keywords: nonagenarians, longevity, complement factor H, inflammation, mortality

Ageing is characterized by chronic low-grade inflammation and the expected lifespan may be affected by several immunological and inflammatory mediators. In this study, we investigated whether the functional Tyr402His polymorphism (rs1061170) on complement factor H (CFH) gene, which is a key inflammatory downregulator, modulates the longevity of 491 nonagenarians in the Vitality 90+ study. Logistic regression analysis and Kaplan-Meier method with the log rank test were used to examine the effect of the CFH Tyr402His polymorphism on 4-year mortality. After follow-up, we observed that risk factor-adjusted mortality was significantly higher among the carriers of CFH 402His allele compared to non-carriers (OR 1.78, 95% CI 1.19-2.67, p=0.005) and that the survival curves of CFH 402His carriers and non-carriers deviated significantly (p=0.016).

We propose that the increased mortality is inflammation-related and mediated by aberrant complement regulation by the CFH 402His variant.

Introduction

Determination of human lifespan, especially in old age, is largely affected by the antigenic burden that the immune system encounters and has to deal with (reviewed in [START_REF] Franceschi | Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans[END_REF][START_REF] Vasto | Inflammatory networks in ageing, age-related diseases and longevity[END_REF]. Age-related low-grade inflammation is characterized by a 2 to 4 -fold increase in inflammatory mediators and this increase has often predicted a shorter life expectancy (reviewed in [START_REF] Franceschi | Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans[END_REF][START_REF] Vasto | Inflammatory networks in ageing, age-related diseases and longevity[END_REF]. In addition to physiological factors such as BMI, sex hormones, plasma lipids, smoking and chronic diseases that modulate the systemic inflammatory state, genetics has frequently been reported to contribute to the immunosenescence and ensuing mortality. The polymorphisms of the key immune mediators which affect the level or the function of the protein have gained recent interest as predictors of longevity [START_REF] Balistreri | Role of Toll-like receptor 4 in acute myocardial infarction and longevity[END_REF]reviewed in Christensen et al., 2006). Nevertheless, the results have frequently been contradictory -either due to confounding co-morbidity, case-control bias, too liberal p-values or insufficient power. Moreover, the majority of the reported associations are limited to certain populations and have not been replicated by others (reviewed in [START_REF] Christensen | The quest for genetic determinants of human longevity: challenges and insights[END_REF][START_REF] Budovsky | Longevity network: construction and implications[END_REF].

Complement is a sophistically ordered cascade functioning at the heart of the immune system by stimulating the inflammation, participating in waste and pathogen disposal as well as interlinking the components of innate and adaptive immunity. Vigorous complement activators include CRP, amyloid proteins, antibodies, oxidized lipoproteins and cell debris, all of which are abundant in old age. Complement activity is regulated by the complement factor H (CFH) which downregulates the activation cascade when bound to CRP. The Tyr402His tagging polymorphism on CFH creates a proinflammatory functional variant (402His) which has a markedly reduced CRPbinding capability and thus an inability to downregulate the complement sufficiently [START_REF] Laine | Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein[END_REF]. In brief, the CFH 402His variant loses its anti-inflammatory properties, which leads to unbalanced and excessive inflammatory reaction [START_REF] Laine | Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein[END_REF]. Therefore, we wanted to examine whether this polymorphism is associated with increased mortality during 4-year follow-up in the oldest-old population. Along these lines, since the CFH 402His variant has been consistently linked with age-related macula degeneration (AMD) [START_REF] Edwards | Complement factor H polymorphism and age-related macular degeneration[END_REF][START_REF] Thakkinstian | Systematic review and meta-analysis of the association between complement factor H Y402H polymorphisms and age-related macular degeneration[END_REF][START_REF] Skerka | Defective complement control of factor H (Y402H) and FHL-1 in age-related macular degeneration[END_REF] it is conceivable that CFH may be a candidate gene in other inflammation-related processes, such as age-associated mortality.

Methods

The study population (n=491; 380 females, 111 males) consisted of two population-based cohorts of people living in the city of Tampere, Finland, who were studied in the Vitality 90+ project at the age of 90, those born in 1907-08 were studied in 1998 (n= 212), and those born in 1909-10 were studied in 2000 (n=279). The study design was approved by the local ethics committee and all participants gave their informed consent. The physical examination and clinical parameters have been described elsewhere [START_REF] Goebeler | Medical history, cognitive status and mobility at the age of 90. A population-based study in Tampere, Finland[END_REF]. Smoking habits and plasma concentrations of CRP, IL-6 and HDL-cholesterol were determined as previously described [START_REF] Goebeler | Medical history, cognitive status and mobility at the age of 90. A population-based study in Tampere, Finland[END_REF][START_REF] Jylhä | Interleukin-1 receptor antagonist, interleukin-6, and C-reactive protein as predictors of mortality in nonagenarians: the vitality 90+ study[END_REF] and CRP and IL-6 were log-transformed prior to analyses due to their skewed distributions.

Genotyping of the CFH Tyr402His polymorphism (+1277T>C, rs1061170) was performed with Assay-By-Design from Applied Biosystems under standard conditions using the ABI Prism 7900HT Sequence Detection System (Taqman, Applied Biosystems, Foster City, CA, USA).

Results

Genotype frequencies of the CFH +1277T>C polymorphism in this cohort followed the Hardy-Weinberg equilibrium and were TT 27.3%, TC 52.3% and CC 20.4%. A significant risk of increased mortality was observed for the 402His carriers in logistic regression analysis (OR 1.78, 95% CI 1.19-2.67, p=0.005, Table 1) and Kaplan-Meier analysis with the log rank test revealed that death rate during 4-year follow-up was significantly higher among the carriers of the CFH 402His variant than among non-carriers (p=0.016; Figure 1). Risk factor adjustment in logistic regression analysis with sex, cardiovascular diseases and diabetes (p= 0.001; OR 2.06; 95% CI 1.33-3.20) or smoking, BMI, CRP, IL-6, HDL-cholesterol, sex, cardiovascular diseases and diabetes (p=0.015; OR 2.05; 95% CI 1.08-3.81) did not change the result.

Discussion

The results of this population-based study show that the CFH 402His variant confers a significant risk of mortality in old age when inflammatory mechanisms, especially those of innate immunity, are known to play a crucial role in determining the remaining lifespan. Importantly, as being a longitudinal follow-up study, the potentially biased case-control setting was circumvented.

Moreover, the fact that risk factor-adjustment did not attenuate this effect, indicates that the higher mortality rate of the CFH 402His carriers is not affected by confounding factors or co-morbidities.

However, since smoking and BMI data as well as the plasma concentrations of CRP, IL-6 and HDL-cholesterol were only available from 210 individuals, their effect on mortality could only be analyzed for this sub-cohort instead of the whole cohort (n=491).

Using this same follow-up approach and partially same cohort, we have previously shown that 1) the high CRP producing haplotypes of the CRP gene [START_REF] Hurme | CRP gene is involved in the regulation of human longevity: a followup study in Finnish nonagenarians[END_REF] and 2) the high IL-6 producing genotypes [START_REF] Hurme | Interleukin-6 -174G/C polymorphism and longevity: a follow-up study[END_REF] are less prevalent in nonagenarians surviving the 4-year follow-up time. These data are in line with the reports by [START_REF] Bonafe | A gender--dependent genetic predisposition to produce high levels of IL-6 is detrimental for longevity[END_REF][START_REF] Rea | Paraoxonase polymorphisms PON1 192 and 55 and longevity in Italian centenarians and Irish nonagenarians. A pooled analysis[END_REF], who have shown that pro-inflammatory genotypes are associated with increased mortality in the elderly populations. As hypothesized, this phenomenon is likely to be the result of the "tradeoff" from effective resistance to infectious diseases earlier in life (reviewed in [START_REF] Franceschi | Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans[END_REF]). Regarding the CFH Tyr402His polymorphism, [START_REF] Mooijaart | Complement Factor H polymorphism Y402H associates with inflammation, visual acuity, and cardiovascular mortality in the elderly population at large[END_REF] have recently demonstrated in the Leiden 85-plus Study that the 402His allele associates in a gene dose dependent fashion with increased inflammation and cardiovascular mortality but not with all-cause mortality in old population. Since our results concern all-cause mortality and are independent of gene dose, direct comparison of these results is not feasible. In addition, as the data concerning this polymorphism and longevity are so far scarce, we cannot exclude the possibility that a populationspecific character is involved in the association. Further research and functional studies are thus required in order to clarify the exact role of the CFH in age-associated inflammation.

As it has been reported that the CFH 402His variant has a reduced capacity to downregulate complement activation and control the inflammation [START_REF] Laine | Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein[END_REF], it is conceivable that the fine-tuning of systemic inflammation could be compromised among the carriers of the CFH 402His variant. We suggest that the novel finding reported here about all-cause mortality among the CFH 402His carriers is related to this phenomenon and that these individuals are predisposed to a chronic proinflammatory state which manifests as an increased risk of mortality in very old age. between the carrier and non-carrier groups: the 402His carriers have higher mortality count and decreased survival compared to the non-carriers.

ACCEPTED MANUSCRIPT

  The subjects with either His/His (CC) or His/Tyr (CT) genotype were classified as 402His carriers and the subjects with Tyr/Tyr (TT) genotype as non-carriers. The division into CFH 402His carriers and non-carriers was based on the observation that when the genotypes (Tyr/Tyr, Tyr/His and His/His) were tested separately, identical behaviour was observed for Tyr/His and His/His genotypes in Kaplan-Meier analysis. Statistical analyses were carried out with SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Logistic regression analysis and Kaplan-Meier method with the log rank test were used to examine the effect of the CFH Tyr402His polymorphism on 4-year mortality. Risk factor adjustment included cardiovascular diseases, diabetes and sex (available for 491 individuals) as well as smoking, BMI, CRP, IL-6, HDL-cholesterol and sex (available for 210 individuals).

Figure 1 .

 1 Figure 1. Survival curves of 402His carriers and non-carriers in Kaplan-Meier analysis during 4-year follow-up among Finnish nonagenarians. The mortality rates differ markedly (p=0.016)

Table 1 . Effect of CFH 402His carrier status on the mortality of Finnish nonagenarians in logistic regression analysis during the 4-year follow-up.

 1 The mortality count among 402His carriers is significantly higher than among 402His non-carriers.

		Survivors		Nonsurvivors
		n	(%)	n	(%)	P	OR	95% CI
	402His carriers	114	(31.9)	243	(68.1)	0.005	1.78	1.19-2.67
	402His non-carriers	61	(45.6)	73	(54.5)

Acknowledgements

The authors wish to thank Eija Spåre, Sinikka Repo-Koskinen and Nina Peltonen for their expert technical assistance.

Sources of Funding

The work was supported by the Competitive Research Funding of the Pirkanmaa Hospital District and The Emil Aaltonen Foundation (T.L.).


Alan R Hipkiss
email: alanandjill@lineone.net

On the enigma of carnosine's anti-ageing actions

Carnosine ( -alanyl-L-histidine) has described as a forgotten and enigmatic dipeptide. Carnosine's enigma is particularly exemplified by its apparent anti-ageing actions; it suppresses cultured human fibroblast senescence and delays ageing in senescence-accelerated mice and Drosophila, but the mechanisms reponsible remain uncertain. In addition to carnosine's well-documented anti-oxidant, anti-glycating, aldehyde-scavenging and toxic metal-ion chelating properties, its ability to influence the metabolism of altered polypeptides, whose accumulation characterises the senescent phenotype, should also be considered. When added to cultured cells, carnosine was found in a recent study to suppress phosphorylation of the translational initiation factor eIF4E resulting in decreased translation frequency of certain mRNA species. Mutations in the gene coding for eIF4E in nemtodes extend organism lifespan, hence carnosine's anti-ageing effects may be a consequence of decreased error-protein synthesis which in turn lowers formation of protein carbonyls and increases protease availability for degradation of polypeptides altered postsynthetically. Other studies have revealed carnosine-induced upregulation of stress protein expression and nitric oxide synthesis, both of which may stimulate proteasomal elimination of altered proteins. Some anti-convulsants can enhance nematode longevity and suppress the effects of a protein repair defect in mice, and as carnosine exerts anti-convulsant effects in rodents, it is speculated that the dipeptide may participate in the repair of protein isoaspartyl groups. These new observations only add to the enigma of carnosine's real in vivo functions. More experimentation is clearly required.

Carnosine and ageing.

The naturally-occurring dipeptide carnosine ( -alanyl-L-histidine) has been described as forgotten and enigmatic [START_REF] Bauer | Carnosine and homocarnosine, the forgotten, enigmatic peptides of the brain[END_REF]: the present paper is an attempt to rectify the former opinion, but, however only adds to the latter view. Amongst carnosine's enigmatic properties are its apparent anti-ageing actions; the dipeptide is one of a very few naturally-occurring compounds which suppresses cell senescence, induces rejuvenating effects (McFarland and[START_REF] Mcfarland | Retardation of senescence in cultured human diploid fibroblasts by carnosine[END_REF]1999), and protects against telomere shortening [START_REF] Shao | L-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts[END_REF] in cultured human fibroblasts. The dipeptide also extends the life-span of senescence-accelerated mice [START_REF] Yuneva | Effect of carnosine on age-induced changes in senescence-accelerated mice[END_REF] and Drosophila [START_REF] Yuneva | Effect of carnosine on Drosophila melanogaster lifespan[END_REF]. There are many possible mechanisms by which carnosine exerts these anti-ageing actions however, some of which were summarized a decade ago [START_REF] Hipkiss | Carnosine, a protective anti-ageing peptide?[END_REF]. Meanwhile there have been a number of publications suggesting further possible routes by which carnosine could suppress additional processes associated with ageing at cellular and whole organism levels.

Carnosine and the metabolism of altered proteins

The most prominent biochemical symptom of ageing and many age-related conditions is proteotoxic stress resulting from the accumulation of altered proteins which arise from biosynthetic errors and/or deleterious post-synthetic polypeptide modifications [START_REF] Rosenberger | Senescence and the accumulation of abnormal proteins[END_REF]Hipkiss, 2006a;[START_REF] Schoneich | Protein modification in aging: an update[END_REF][START_REF] Morimoto | Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging[END_REF]. While carnosine might suppress the post-synthetic generation of altered proteins, including anti-oxidant activity, toxic metal-ion chelation, anti-glycating and aldehyde/carbonyl-binding activities [START_REF] Hipkiss | Carnosine, a protective anti-ageing peptide?[END_REF], there is no unequivocal proof that any of these proposals can adequately account for all carnosine's effects on model ageing systems.

Carnosine, slowing protein synthesis and ageing

A number of recent studies have shown that ageing can be delayed by partial inhibition of protein synthesis. Studies in Caenorhabditis elegans have shown that senescence is delayed, stress resistance enhanced and life-span extended in mutants defective in the mRNA translation initiation factor eIF4E [START_REF] Pan | Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans[END_REF][START_REF] Syntichaki | eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans[END_REF]. Similarly, C. elgans lifespan was extended and stress resistance enhanced when translation was inhibited when synthesis of eleven ribosomal proteins was suppressed using inhibiting-RNAs (RNAi) [START_REF] Hansen | Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans[END_REF]. Furthermore, certain ribosomal protein defects have beneficial effects on yeast longevity [START_REF] Chiocchetti | Ribosomal proteins Rp110 and Rps6 are potent regulators of yeast replicative life span[END_REF]. While the explanation of these effects is debated [START_REF] Kaeberlein | Protein translation[END_REF], it is likely that the lowered rate of bulk protein synthesis, resulting from the lowered translation initiation frequency, also decreases biosynthesis of erroneous proteins (Hipkiss, 2007a). This lowered production of biosynthetic error-proteins could directly lower the load that the chaperone and proteolytic apparatus must deal with: the chaperone/proteolytic apparatus is responsible for the elimination of altered protein generated both post-synthetically and those formed by biosynthetic errors. It should also be noted that, compared to normal gene products, error-proteins are more readily glycated and oxidatively-damaged by ROS [START_REF] Dukan | Proteins are oxidatively carbonylated in response to reduced transcriptional and translation[END_REF][START_REF] Fredriksson | Induction of the heat shock regulon in response to increased mistranslation requires oxidative modification of malformed proteins[END_REF] than the normal gene products, thus the mutant organisms would generate fewer protein carbonyls that normally characterize the senescent state (Stadtman, 1992). Consequently the decreased level of biosynthetic error-proteins would not only decrease formation of protein carbonyls, but also increase the relative availability of chaperone and proteolytic activities for the recognition and elimination of altered proteins arising from deleterious post-synthetic modification (Hipkiss, 2007a).

Interestingly, methionine restriction (40% and 80%) has been shown to delay ageing in rats and mice [START_REF] Miller | Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-1 and insulin levels, and increases hepatocyte MIF levels and stress resistance[END_REF][START_REF] Naudi | Methionine restriction decreases endogenous oxidative molecular damage and increases mitochondrial biogenesis and uncoupling protein 4 in rat brain[END_REF]. Because methionine is the initiating amino acid in protein biosynthesis, this may again indicate that decreased translation initiation is an effective anti-ageing strategy. Therefore methionine restriction could also decrease biosynthetic formation of error-proteins, similar to the effects of the defective eIF4E initiation factor in nematodes outlined above (Hipkiss, 2008a).

A recent paper has revealed that carnosine can also exert suppressive effects on mRNA translation initiation. [START_REF] Son | Inhibitory effect of carnosine on interleukin-8 in intestinal epithelial cells through translational regulation[END_REF] showed that the dipeptide inhibits interleukin-8 mRNA translation by suppressing phosphorylation of initiation factor eIF4E in peroxide-activated intestinal epithelial cells and Caco-2 cells. Phosphorylation of eIF4E is required for effective mRNA translation, which explains the observed carnosine-mediated decreased synthesis of the pro-inflammatory cytokine. Carnosine was also shown to inhibit phosphorylation of other regulatory proteins ERK1/2 and p38 MAP kinase. Because defective eIF4E limits mRNA translation initiation and results in lifespan extension in C. elegans (as described above), it is possible that carnosine's beneficial effects on fibroblast senescence and lifespan are mediated via a similar mechanism in human cells. This should be relatively easy to test.

Carnosine and post-synthetic protein modification

Carnosine has the potential to inhibit much deleterious post-translational polypeptide modification caused by oxidative, nitroxidative and glycoxidative agents.

There is much evidence, mostly obtained from model systems, suggesting that carnosine can suppress protein modification mediated by reactive oxygen species (ROS) [START_REF] Kohen | Antioxidant activity of carnosine, homocarnosine and anserine present in muscle and brain[END_REF][START_REF] Boldyrev | Protection of proteins from oxidative stress: a new illusion or a novel strategy?[END_REF]Alhamdani et al., 2007aAlhamdani et al., & 2007b)), reactive nitrogen species (RNS) [START_REF] Calabrese | Protective effect of carnosine during nitrosactive stress in astroglial cell cultures[END_REF][START_REF] Fontana | Prevention of peroxynitratedependent damage by carnosine and related sulphonamide pseudopeptides[END_REF], glycating agents [START_REF] Hipkiss | Non-enzymic glycosylation of the dipeptide L-carnosine, a potential anti-protein-cross-linking agent[END_REF][START_REF] Vinson | Inhibition of protein glycation and advanced glycation end products by ascorbic acid and other vitamins and nutrients[END_REF][START_REF] Seidler | Carnosine prevents glycation-induced changes in electrophoretic mobility of aspirate aminotransferase[END_REF] and deleterious aldehydes such as malondialdehyde [START_REF] Hipkiss | Protective effects of carnosine against malondialdehyde-induced toxicity towards cultured rat brain endothelial cells[END_REF](Hipkiss et al. and 1998a(Hipkiss et al. and 1998b)), hydroxynonenal [START_REF] Aldini | Carnosine is a quencher of 4-hydroxy-nonenal: though what is the mechanism of reaction?[END_REF]2005), acrolein [START_REF] Carini | Acroleinsequestering ability of endogenous dipeptides: characterization of carnosine and homocarnosine/acrolein adducts by electrospray ionization tandem mass spectrometry[END_REF] and methylglyoxal (MG) [START_REF] Hipkiss | Carnosine protects proteins against methylglyoxalmediated modifications[END_REF]. Carnosine also inhibited the cross-linking of MG-modified protein with normal polypeptides [START_REF] Hipkiss | Carnosine protects proteins against methylglyoxalmediated modifications[END_REF], most likely by forming adducts with the MG-induced protein carbonyl groups [START_REF] Brownson | Carnosine reacts with a glycated protein[END_REF].

Recent studies in mice have shown that decreased intake, or formation, of protein-AGEs can make major contributions to lifespan extension in mice [START_REF] Cai | Reduced oxidant stress and extended lifespan in mice exposed to a low glycotoxin diet. Association with increased AGER1 expression[END_REF]Uribarri et al., 2007a and2007b). MG, a spontaneously-generated glycolytic by-product, induces senescence in cultured fibroblasts [START_REF] Sejersen | Dicarbonyl-induced accelerated aging in vitro in human skin fibroblasts[END_REF], and is a primary source of intracellular glycation in both yeast [START_REF] Gomes | Protein glycation in vivo: functional and structural effects on yeast enolase[END_REF] and mice [START_REF] Morcos | Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in C. elegans[END_REF]. It is likely that MG generation is suppressed by caloric restriction and every-other-day feeding (Hipkiss 2006b and2008b), both of which delay ageing. Carnosine's ability to react with MG and protect proteins against MG-induced damage may contribute to the dipeptide's apparent anti-ageing activity (Hipkiss, 2007b). The presence of carnosine-aldehyde adducts in muscle and urine [START_REF] Orioli | LC-ESI-MS/MS determination of 4-hydroxy-trans-2-nonenal Michael adducts with cysteine and histidine-containing peptides as early markers of oxidative stress inexcitable tissues[END_REF][START_REF] Orioli | HNE Michael adducts to histidine and histidine-containing peptides as biomarkers of lipidderived carbonyl stress in urines: LC-MS/MS profiling in Zucker obese rats[END_REF] provides evidence for the occurrence in vivo of at least some of carnosine's putative aldehyde-scavenging actions. It should be pointed out that while no protein-carnosine adducts have been detected in vivo, NMR data has indicated the presence of carnosine-phosphatidylcholine adducts in human leg muscle, possibly arising from oxidation of amino lipids and their subsequent reaction with the dipeptide (Schroder et al., 2004).

Carnosine and proteolysis of error-proteins

Carnosine may improve cellular ability to deal with altered proteins.

Decreased intracellular elimination of aberrant polypeptides by proteasomal activity and autophagy can contribute to altered protein accumulation, while upregulation of certain proteolytic functions is associated with increased longevity [START_REF] Min | CHIP deficiency decreases longevity with accelerated aging phenotypes accompanied by altered protein quality control[END_REF][START_REF] Yun | Proteasomal adaptation to environmental stress links resistance to proteotoxicity with longevity in Caenorhabditis elegans[END_REF][START_REF] Bonelli | Low-level caloric restriction rescues proteasome activity and Hsc70 level in liver of aged rats[END_REF][START_REF] Cuervo | Calorie restriction and aging: the ultimate "cleansing diet[END_REF]Bergami et al., 2007). There have been three reports indicating that carnosine may stimulate intracellular proteolysis. Hipkiss et al. (1998a) showed that proteolysis was stimulated in carnosine-treated cultured human fibroblasts, and [START_REF] Bharadwaj | L-Carnosine and verapamil inhibit hypoxia-induced expression of hypoxia inducible factor (HIF1-α) in H9c2 cardiomyoblasts[END_REF] found that carnosine stimulated degradation of HIF1-in cardiomyoblasts. It has also been reported that carnosine stimulates neutral, non-lysosomal, proteolysis in cell-free extracts of rat brain [START_REF] Bonner | Lysosomal and non-lysosomal protease activities of the brain in response to ethanol feeding[END_REF]. Conceivably these effects could occur by the dipeptide stimulating synthesis of nitric oxide (Nicoleti et al., 2007;[START_REF] Tomonaga | Nitric oxide involves carnosine-induced hyperactivity in chicks[END_REF], which in turn may activate proteasomal-mediated protein degradation [START_REF] Thomas | Hydrogen peroxide induces nitric oxide and proteasome activity in endothelial cells: a bell-shaped signaling response[END_REF].

Carnosine has also been shown to stimulate expression of vimentin [START_REF] Ikeda | Carnosine stimulates vimentin expression in cultured rat fibroblasts[END_REF], a cytoskeletal protein which turns out to be very readily glycated by deleterious aldehydes [START_REF] Kueper | Vimentin is the specific target in skin glycation. Structural prerequisites, functional consequences, and role in skin aging[END_REF]. Although the significance of this observation is uncertain, the possibility arises that vimentin alongside carnosine may help to control protein glycation. However it has also been shown in COS cells that the enzyme oxidized protein hydrolase (OPH) is coexpressed with vimentin: as its name states, OPH degrades oxidized polypeptides in collaboration with proteasomes (Shimizi et al., 2004). It should be possible to test whether carnosine stimulates expression of OPH in cultured fibroblasts or other cells.

Carnosine and the stress response

Stress/chaperone proteins participate in the recognition and proteolytic elimination of altered proteins and their upregulation is associated with increased organism longevity and suppression of some age-related diseases [START_REF] Morimoto | Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging[END_REF][START_REF] Calabrese | Cellular stress response: a novel target for chemoprevention and nutritional neuroprotection in aging, neurodegenerative disorders and longevity[END_REF][START_REF] Erjavec | Accelerated aging andfailure to segregate damaged proteins in Sir2 mutants can be suppressed by overproducing the protein aggregation-remodeling factor Hsp104p[END_REF]. Dietary restriction, which delays ageing in a variety of organisms and suppresses proteotoxicity, requires the action of a heatshock transcription factor [START_REF] Steinkraus | Dietary restriction suppresses proteotoxicity and enhances longevity by an hsf-1-dependent mechanism in Caenorhabditis elegans[END_REF]. Carnosine-zinc complexes (poleprezinc) stimulate expression of certain stress (heat-shock) proteins [START_REF] Odashima | Induction of a 72-kDa heat-shock protein in cultured rat gastric mucosal cells and rat gastric mucosa by zinc-L-carnosine[END_REF]2006;[START_REF] Ohkawara | Polaprezinc protects human colon cells from oxidative injury induced by hydrogen peroxide relevant to sytoprotective heat shock proteins[END_REF] which could improve cellular ability to deal with altered proteins and may again contribute to the dipeptide's apparent antiageing activity. Some studies carried out 30-40 years ago showed that hydrocortisone or cortisone (Cristofolo and Kabakjian, 1975;[START_REF] Macieira-Coelho | Action of cortisone on human fibroblasts in vitro[END_REF] have positive effects on the life-span of cultured human fibroblasts. These findings have recently been reactivated where the beneficial effects of glucocorticoids towards cultured human fibroblasts have again been demonstrated [START_REF] Kletsas | Prior chronic in vivo glucocorticoid excess leads to an anabolic phenotype and an extension of cellular life span of skin fibroblasts in vitro[END_REF]. It is possible that carnosine's effects on fibroblast lifespan (McFarland and Holliday, 1994) could be exerted via glucocorticoid upregulation because it has also been shown that intracerebroventricular carnosine administration stimulates corticosterone release in chick brain [START_REF] Tsuneyoshi | Central administration of dipeptides, beta-alanyl-BCAAs, induces hyperactivity in chicks[END_REF].

Carnosine, anti-convulsant activity and aging

Studies using the nematode Caenorhabditis elegans have shown, somewhat surprisingly, that certain anti-convulsant agents suppress ageing and extend organism lifespan [START_REF] Evason | Anticonvulsant medications extend worm life-span[END_REF]2008;[START_REF] Kornfeld | Effects of anticonvulsant drugs on life span[END_REF]. The mechanism of anticonvulsant anti-ageing action is uncertain but may include histone deacetylase inhibition and/or effects on nematode nervous system [START_REF] Leng | Synergistic neuroprotective effects of lithium and valproic acid or other histone deactylase inhibitors in neurons: roles of glycogen synthase kinase-3 inhbition[END_REF]).

Carnosine appears to possess anticonvulsant activity in rats [START_REF] Wu | Carnosine, precursor of histdine, ameliorates pentylenetetrazole-induced kindled seizures in rat[END_REF][START_REF] Kozan | Anticonvulsant effect of carnosine on penicillium-induced epileptiform activity in rats[END_REF] and mice (Zhu et al., 2007). It is interesting to note that treatment of humans with anticonvulsants gabapentin and topiramate raise levels of homocarnosine, a carnosine homologue, in cerebrospinal fluid (CSF) [START_REF] Petroff | Vigabatrin increases human brain homocarnosine and improves seizure control[END_REF]2006), and that, while carnosine is absent from human CSF, the levels of CSF homocarnosine decrease markedly with age [START_REF] Jansen | A novel, quantitative assay for homocarnosine in cerebrospinal fluid using stableisotope dilution liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Huang | Separation and determination of carnosine-related peptides using capillary electrophoresis with laser-induced fluorescence detection[END_REF].

Anticonvulsants have also been shown to suppress some of the deleterious effects of deficiency of the protein repair enzyme, protein isoaspartate methyl transferase (PIMT), in mouse brain [START_REF] Kim | Phenotypic analysis of seizure-prone mice lacking L-isoaspartate (D-aspartate) O-methyltransferase[END_REF]: PIMT deficiency causes accumulation of aberrant proteins in mouse brain and induces of epileptic seizures. It may be significant that some anticonvulsants, e.g. ethosuximide, which suppress ageing in C. elegans, structurally resemble the succinimide intermediate generated following spontaneous asparagine deamidation. During PIMT-mediated repair of protein isoaspartate residues, methylation of the succinimide intermediate occurs. It has recently been shown that, following the methylation reaction, the isoaspartyl isopeptide bond can be selectively cleaved by hydroxylamine [START_REF] Zhu | Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming[END_REF].

The related compound, N-t-butylhydroxylamine, appears to suppress senescence of cultured human fibroblasts in a manner somewhat similar to carnosine [START_REF] Atamna | N-t-butylhydroxylamine, a hydrolysis product of -phenyl-N-t-butylnitrone, is more potent in delaying senescence in human lung fibroblasts[END_REF], and it was proposed that N-t-butylhydroxylamine and carnosine may mediate their anti-ageing effects by acting similarly to each other, perhaps as protein carbonyl scavengers [START_REF] Hipkiss | On the anti-aging activities of aminoguanidine and N-tbutylhyroxylamine[END_REF]. However, following the observations of [START_REF] Zhu | Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming[END_REF], one speculates that carnosine might participate in protein repair, possibly by also cleaving the isopeptide bonds of protein isoaspartic acid residues, analogous to hydroxylamine. There is another theoretical position where carnosine could be involved in PIMT-mediated protein repair. PIMT catalyses the methylation of the succinyl intermediate forming a methyl ester which then undergoes spontaneous demethylation releasing methanol, the fate of which is likely to be formation of formaldehyde, which carnosine could scavenge.

Evidence for carnosine's potential to participate in asparagine and glutamine deamidation was shown by [START_REF] Kuroda | Investigation on the formation and the determination of -glutamyl--alanylhistidine and related isopeptide in the macromolecular fraction of beef soup[END_REF] who detected -aspartyl-carnosineand -glutamyl-carnosine-adducts, respectively, when free asparagine and free glutamine spontaneously deaminate in the presence of dipeptide. Interestingly whilst -glutamyl-carnosine has been detected in muscle preparations, -aspartyl-carnosine has not [START_REF] Kuroda | Distribution of -glutamyl--alanylhistidine isopeptide in the macromolecular fractions of commercial meat extracts and correlation with the color of the macromolecular fractions[END_REF]; perhaps this difference reflects that fact that isoaspartate residues, generated primarily by the spontaneous deamidation of asparagine residues, are repaired by PIMT, whereas there is no corresponding enzyme for the repair of -glutamyl peptide bonds formed following the spontaneous deamidation of glutamine residues.

Carnosine and other known modulators of ageing: sirtuins, TOR, resveratrol, caloric restriction and aerobic exercise

Control of ageing appears to be exerted by a variety of mechanisms mostly involving changes to energy supply and/or increased expression of agents which increase an organism's resistance to stress. It is totally unknown whether carnosine participates in any of the proposed mechanisms or pathways thought to be responsible. A large number of papers have indicated that certain sirtuins, acting as histone/protein deacetylases, exert anti-ageing activity and that they may also be required when ageing is suppressed by caloric restriction [START_REF] Leibiger | Sirt1: a metabolic master switch that modulateslifespan[END_REF][START_REF] Lin | Nicotinamide adenine dinucleotide, a metabolic regulator of transcription, longevity and disease[END_REF][START_REF] Longo | Sirtuins in aging and age-related disease[END_REF]. Although totally speculative, there are two theoretical possibilities where carnosine could be involved in protein deacetylation. Sirtuin-mediated protein deacetylation eventually generates acetyl-ADP-ribose as a product (Howizt et al., 2003). ADP-ribose and acetyl-ADPribose are powerful glycating agents which have the potential to damage proteins, DNA etc. Carnosine could act as an acetyl acceptor forming N-acetyl-carnosine, which has been isolated from various biological sources. Following loss of the acetyl group from the acetyl-ADP-ribose, the ADP-ribose could be scavenged by carnosine, thereby eliminating random glycation damage. It should be relatively easy to test whether any of these reactions occur by searching for the predicted carnosine adducts.

Alternatively it is theoretically possible that N-acetyl-carnosine could behave as an acetyl donor in protein acetylation mediated by histone/protein acetylases.

Inhibition of the target of rifamycin (TOR) appears to extend life-span by stimulating respiration and upregulating mitogenesis (Bonawitz et al., 2007); these changes also occur when ageing is suppressed by dietary restriction and increased aerobic exercise. Again there is no evidence as to whether carnosine is directly involved in TOR signalling or sirtuin activity. It is possible that carnosine might be indirectly involved however; any resultant increase in mitochondrial activity and mitogenesis could decrease glycolysis together with generation of the toxic glycating agent, methylglyoxal. Carnosine's ability to scavenge methylglyoxal could therefore be additionally beneficial by augmenting elimination of the deleterious aldehyde.

Resveratrol is thought to behave as an anti-ageing agent by mimicking the effects of caloric restriction, i.e. inhibiting TOR and activating sirtuin activity [START_REF] Orallo | Trans-resveratrol: a magical elixir of eternal youth?[END_REF][START_REF] Knutson | resversatrol and novel potent activators of SIRT1: effects on aging and age-related diseases[END_REF]. Resveratrol and carnosine seem to have a number of properties in common (they are anti-oxidants, and possess antiinflammatory, anti-carcinogenic and platelet anti-aggregatory activity). Therefore one has to consider, admitted very speculatively, the possibility that carnosine could also activate SIRT1 in a manner similar to that proposed for resveratrol [START_REF] Borra | Mechanism of human SIRT1 activation by resveratrol[END_REF]; at least both molecules are approximately the same size.

Future research

From the above suggestions it is clear that much more research is required to verify or eliminate the various mechanisms proposed to explain the anti-ageing effects of carnosine. Amongst the more obvious areas of investigation would be to search for some of the various carnosine adducts that have been predicted following the dipeptide's putative reaction with toxic aldehydes and carbonyl compounds. It should also be relatively simply to determine if carnosine stimulates proteolysis (proteasomal or autophagic) of altered proteins, and to test whether carnosine binds to SIRT1 and/or other sirtuins and stimulates their activity.

Conclusions.

Carnosine has been described as an enigmatic peptide [START_REF] Bauer | Carnosine and homocarnosine, the forgotten, enigmatic peptides of the brain[END_REF] and many recent observations certainly confirm this opinion in relation to its effects on ageing.

Carnosine seemingly has the potential to intervene in most of the processes that are thought to contribute to the aged phenotype, as well as mimic, to some degree, the effects those manipulations (possibly including the effects of resveratrol) which promote longevity. That carnosine can also interfere with mRNA translation by inhibiting phosphorylation of certain initiation factors, as well as upregulate synthesis of protective stress/chaperone proteins, reveal additional potential mechanisms of action. The unexpected role of anticonvulsants as anti-ageing agent, at least in nematodes, and the association of carnosine/homocarnosine with anticonvulsant activity in rodents and humans are further testament to the enigmatic pluripotency of this almost non-toxic peptide. Possible beneficial effects of the dipeptide on agerelated pathologies such as diabetes [START_REF] Hipkiss | Glycation, ageing and carnosine: are carnivorous diets beneficial?[END_REF](Hipkiss, & 2006c), Alzheimer's disease (Hipkiss, 2007c), Parkinsons's disease [START_REF] Boldyrev | Carnosine increases efficiency of DOPA therapy of Parkinson's disease: a pilot study[END_REF] and stroke (Dobrota et al., 2005) have therefore been proposed.

Finally perhaps the enigma of carnosine's "true" function(s) is merely a reflection of the fact that relatively little experimental work has been performed in some of the areas under discussion; much more research is required to determine whether any of these proposals are justified.

Acknowledgement

The author thanks an anonymous reviewer for helpful suggestions.