Age-related impairments in neuronal plasticity markers ... - Springer Link

Biogerontology (2008) 9:441–454 DOI 10.1007/s10522-008-9168-0

RESEARCH ARTICLE

Age-related impairments in neuronal plasticity markers and astrocytic GFAP and their reversal by late-onset short term dietary restriction Manpreet Kaur Æ Sandeep Sharma Æ Gurcharan Kaur

Received: 10 April 2008 / Accepted: 1 August 2008 / Published online: 2 September 2008 Ó Springer Science+Business Media B.V. 2008

Abstract Recent studies on the effects of dietary restriction (DR) in rodents and primates have shown that even late-onset short-term regimens can bring about comparable beneficial changes seen in animals subjected to life-long DR. We studied the effect of aging on the expression of neural cell adhesion molecule (NCAM), its polysialylated form PSANCAM and astrocytic marker glial fibrillary acidic protein (GFAP) by immunohistofluorescent staining and immunoblotting in 1, 3, 6, 18 and 24 months old male wistar rats. Maximum expression of NCAM and PSA-NCAM was observed in sub-granular zone (SGZ) or granular cell layer (GCL) of hippocampus, arcuate region and paraventricular area of hypothalamus and piriform cortex layer II from 1 and 3 months old rats, thereafter, gradual downregulation was observed in 6, 18 and 24 months old rats. Progressive increase in astrocytic GFAP expression was noticed in these regions of brain with age. We further addressed whether DR initiated in late adulthood in 24 months old rats confers beneficial effects and can reverse changes in expression of NCAM, PSA-NCAM and GFAP. These results suggest that even late-onset short term DR regimen in old rats can have beneficial effects on neuroplasticity.

M. Kaur S. Sharma G. Kaur (&) Department of Biotechnology, Guru Nanak Dev University, Amritsar 143005, Punjab, India e-mail: [email protected]

Keywords Aging Late-onset dietary restriction Astrocytes GFAP PSA-NCAM Neuroplasticity

Introduction Research during the last decade has tremendously increased our understanding of structural plasticity of brain i.e. how neuronal circuits can be modified by experience, learning and environmental enrichment. The neuroprotective potential of maintaining experimental animals under prolonged conditions of reduced caloric intake has been tested in several animal models (Bruce-Keller et al. 1999; Yu and Mattson 1999; Duan et al. 2003; Maswood et al. 2004). Dietary restriction (DR) reduced the degeneration of dopaminergic neurons in a mouse model of Parkinson’s disease (Maswood et al. 2004). In rats, degeneration of hippocampal CA3 region neurons and striatal lesions respectively, obtained through systemic and/or local injections of kainic acid or malonate were substantially reduced in animals kept for 2–3 months under conditions of limited access to food (Bruce-Keller et al. 1999; Sharma and Kaur 2005). In transgenic models, DR increased the resistance of hippocampal neurons to excitotoxic injuries in mutant presenilin-1 mice (Zhu et al. 1999). In view of the recent reports on DNA array of gene expression for the effect of aging and DR in rodents and primates, even late onset short-term regimens

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have been shown to produce a large part of the changes seen in animals subjected to lifelong DR (Lee et al. 1999; Cao et al. 2001). Transcriptional patterns suggest that DR retards aging by causing a metabolic shift towards increased protein turnover and decreased macromolecular damage (Goto et al. 2002). Although benefits of DR on the cardiovascular, immune and endocrine systems have been demonstrated, their effects on the nervous system are only now being studied. Studies of rats and mice maintained on a DR feeding regimen suggest that DR may slow age-related molecular changes in the brain including increases in levels of GFAP and oxidative damage to proteins and DNA (Cao et al. 2001; Duan et al. 2003). Age-associated impairments and cognitive decline may involve changes in ability of neuronal networks to undergo structural remodeling. A number of recent studies suggest an important role for the neural cell adhesion molecule (NCAM) in regeneration, learning and memory functions in the adult nervous system (Ronn et al. 2000; Cambon et al. 2004; Sandi 2004). NCAM expressed on the surface of neurons, oligodendrocytes and astrocytes in the prototypic immunoglobulin-like (Ig) member of a family of cell adhesion molecules (CAM’s) which induce cell adhesion by homophilic binding (Crossin and Krushel 2000). NCAM-deficient mice are impaired in spatial learning. Strekalova et al. (2006) observed abnormal processing and/or shedding of L1 and NCAM in CSF in dementia-related neurodegeneration and age, respectively, reflecting changes in adhesion molecule-related cell interactions. Polysialylated form of neural cell adhesion molecule i.e. PSA-NCAM is known to modulate cell interactions and promote plasticity in the developing nervous system. In particular, it facilitates translocation of neural and nonneural precursor cells and shields them from inappropriate interactions and optimizes neurite sprouting and branching during axon pathfinding, neuroendocrine structural plasticity and target innervation as well as reducing the stabilization of inappropriate synapses (Kaur et al. 2002; Parkash and Kaur 2005; Bonfanti 2006; Rutishauser 2008). Although in most cases PSA expression is downregulated by the end of embryonic development, it is retained within certain regions of the adult brain believed to exhibit different forms and degrees of plasticity, for example the hippocampus, hypothalamus,

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Biogerontology (2008) 9:441–454

brainstem and rostral migratory stream. PSA-NCAM expression during aging is strongly downregulated in middle-aged individuals (Seki 2003). A recent study from our lab has reported a strong upregulation of PSA-NCAM expression and reversing changes in glial fibrillary acidic protein (GFAP) levels by alternate day dietary restriction regimen in young adult rats (Sharma and Kaur 2008). Two different paradigms of DR have been widely employed because of their highly reproducible abilities to increase lifespan in rats and mice. In one paradigm, the animals receive food daily, but are limited to a specified amount, which is typically 30– 50% less than ad libitum consumption of the control group. The second paradigm involves periodic fasting in which the animals are deprived of food for a full day, every other day, and are fed ad libitum on the intervening days (Mattson 2003). Analysis of various physiological parameters in animals maintained on these two different regimens have revealed several similar changes including decreased body temperature, decreased heart rate and blood pressure, and decreased glucose and insulin levels. The morphological plasticity in the adult mammalian CNS is highly restricted, occurring mainly in the olfactory bulbs, hippocampus and hypothalamus and has been correlated with the enhanced expression of molecules involved in synaptic plasticity such as PSA-NCAM. The hippocampus is particularly susceptible to age related neurodegeneration. Recent findings suggest that hippocampal neurogenesis is diminished during aging (Leuner et al. 2007), an effect counteracted by dietary restriction and exercise manipulations that attenuate age related hippocampal dysfunction (Stranahan et al. 2007; van Praag et al. 2005; Lee et al. 2003). The present study reports age related changes in NCAM and PSA-NCAM expression in hippocampus, hypothalamus and piriform cortex brain regions and further addresses whether late-onset intermittent fasting DR regimen in middle aged and aged animals can promote NCAM and PSANCAM expression, which are known to mediate cell interactions to facilitate synaptic plasticity, memory processes and neuroregeneration. The expression of astrocytic marker GFAP was also studied to test whether late onset DR regimen can reverse age associated reactive gliosis. We are particularly interested in exploring the mechanisms of antiaging effects, if there are any, of late-onset short-term rather than lifelong DR in animals because it could be


extended to humans in a practical manner. The work is based on the idea of neurohormetic intervention of aging showing beneficial effects of adult-onset shortterm DR.

Materials and methods Chemicals The primary antibodies used for the western blot analysis and immunofluorescence were monoclonal anti-GFAP (Clone G-A-5, Sigma); anti-NCAM (Clone AF11, a gift sample from Dr. K. Ono); anti-PSA-NCAM (Clone 12E3, a gift sample from Dr. T. Seki) and monoclonal anti-a-tubulin (Clone Dm 1A, Sigma). The secondary antibodies used were anti-mouse IgG-FITC conjugated (Sigma) or antimouse IgG-peroxidase conjugated (Bangalore Genei). All other chemicals and reagents were the purest, available commercially from local suppliers. Experimental animals Wistar strain male albino rats in the age group of 1, 3, 6, 18 and 24 months were used for these experiments. Animal care and procedures were followed in accordance with the guidelines of Institutional Animal Ethical Committee. The paradigm of DR involved periodic fasting, in which 21 months old rats were deprived of food for a full day, every other day, and were fed ad libitum on the intervening day for 3 months. Food was provided or removed at 10 am every day. Water was available ad libitum to all the animals. Another group of rats of similar age was fed ad libitum and used as control. The body weights were recorded every fifteenth day in DR and age matched ad libitum fed (AL) rats. Blood glucose determination was done every month. Immunofluorescent staining of GFAP, NCAM and PSA-NCAM After completion of 3 months of dietary restriction, brains were perfused transcardially with 4% Paraformaldehyde in Phosphate buffered saline (PBS 0.1 M). Brains were kept in same fixative overnight at 4°C and then cryopreserved in 20% and 30% sucrose in phosphate buffered saline for 24 h each at 4°C (Sharma

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and Kaur 2005, 2007). Thirty lm thick coronal sections were cut using cryostat microtome and free floating sections were treated in the following manner: three 5 min washes in 0.1 M PBS, pH 7.4; 30 min in 0.3% Triton-X100 (0.1% Triton-X100 for (PSA-NCAM) in 0.1 M PBS for permeabilization. Then sections preincubated for 1 h at room temperature in a blocking solution (5% Normal goat serum in PBS with 0.3% Triton-X100 and 0.01% sodium azide). The sections were incubated with mouse anti-GFAP; anti-NCAM and anti-PSA-NCAM (1:500) in 0.1% Triton-X100 and 1% bovine serum albumin serum in PBS for 24 h at 4°C. Specificity of staining was determined by negative staining control procedures without adding primary antibody to incubation buffer. The sections were then washed for 15 min with four changes of 0.1% PBST at room temperature. The sections were incubated with anti mouse IgG-FITC (1:200) and IgMFITC (1:200 for PSA-NCAM) in 0.32% Triton-PBS for 2 h. After that sections were washed with 0.01% PBST for 15 min. Tissue sections were then coverslipped using the appropriate anti-fading mounting medium (500 mg propyl-gallate 20 ml ethanol/PBS (pH 7.4) (1:1) and 90 ml glycerol) for fluorescence detection. Data analysis The stained sections were examined using Nikon epifluorescent microscope E600. Images were captured using Cool Snap ProTM CCD camera (Media Cybernetics) and the pictures were analyzed using Image Pro-plusTM software version 4.5.1 from the Media cybernetics (Media Cybernetics, USA). The intensity of GFAP immunoreactivity was quantified in randomly selected fields in each section using the count/size command of image pro-plus software. Five consecutive sections each from 4 to 5 animals in all groups were used for data analysis. An area of interest (AOI) was selected and placed within region on each section and density (green) measurement was made for staining intensity of GFAP. This procedure was standardized in order to sample the same area of all brain regions studied. Similarly, intensity analysis was carried out for NCAM and PSA-NCAM immunoreactivity. All the slides were coded for their group assignment and code was not broken till the intensity was measured. An examiner blind to the group assignment of each animal did density measurement.

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Western blotting of GFAP and NCAM Protein sample preparation and quantitation After cervical dislocation, the animals were decapitated and the brains were removed immediately. Brains were dissected, and brain regions hippocampus, hypothalamus and cortices were dissected and 5% homogenate was prepared in 50 mM Tris–HCl (pH 7.4) containing 0.5 mM DTT (Amersham), 100 lM Na3Vo4 (Sigma), and 0.2 mM phenylmethylsulfonyl fluoride (Sigma) and centrifuged for 5 min at 10,000 rpm. Protein content in the supernatant was determined by the Bradford method. Each homogenate was then diluted in homogenization buffer so as to give a final concentration of 2 mg protein/ml (2 lg/ll). The samples were mixed 1:1 with sample buffer [0.25 M Tris–HCl (pH 6.8), 20% glycerol, 4% sodium dodecyl sulfate (SDS), 10% b-mercaptoethanol and 1 mg bromophenol blue] and stored at -20°C. Western blot hybridization An aliquot of each sample (30 lg each for GFAP and NCAM) was electrophoresed on a one dimensional 10% and 7.5% polyacrylamide gel, respectively, under standard denaturing conditions according to method of Laemmli (1970). The separated proteins were then blot transferred onto a PVDF membrane (Hybond-P, Amersham Pharmacia Biotech) using the semidry Novablot system (Amersham Pharmacia) at 25 V for 3 h. The membranes were placed in 5% skim milk solution in Tris buffer saline-Tween-20 (TBS-T) buffer (13.3 mM Tris. 0.8%, w/v, NaCl; pH 7.6) containing 0.2% Tween-20 (Sigma) for 4 h. The membranes were then hybridized with primary antibody anti-GFAP and anti-NCAM diluted 1:5000 in 5 ml of TBS-T for 2 h at room temperature. Following four 15-min washes in TBS-T, blots were incubated with the secondary antibody anti-mouse IgG; peroxidiase conjugated diluted 1:7000 in 5 ml of TBS-T for 1 h at room temperature. Membranes were again washed in TBS-T buffer (3 9 15 min). The blots were developed using enhanced chemiluminescence (ECL PlusTM) western blot detection system (Amersham Biosciences) and exposed to Hyper film ECL. The films were then developed and the antibody-labeling intensity was analyzed using

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Gel-documentation system (AlphaEaseTM, Alpha Innotech Corporation). In order to account for potential variations in protein estimation and sample loading, expression of each protein was compared to that of a-tubulin in each sample. Tubulins are abundant cytoskeletal proteins that are highly expressed in brain and a-tubulin in particular is known to show stabilized expression in the adult stage of life. Each blot was stripped in 62.5 mM Tris, 2% SDS and 100 mM 2-mercaptoethanol (pH 6.7) for 30 min at 50°C and reprobed with an anti-a-tubulin antibody (1:3000) and relative optical density (ROD) measured as described above. The values for each sample were then expressed as ROD obtained using a-tubulin. Statistical analysis Values are expressed as mean ± SEM. A one-way analysis of variance (ANOVA) was used to compare the results in different groups of rats. When ANOVA detected a difference, these sets of ad libitum fed and dietary restricted rats were subjected to post hoc comparison using the Bonferroni’s test (SigmaStat 2.03) for pairwise multiple comparisons to determine the statistical significance, which was assumed to be different when the comparison showed a significance level of P \ 0.05.

Results This every other day regimen of dietary restriction is the simplest and most often used way to obtain effective decrease of calorie intake, which has been estimated to be around 30–40% less as compared to ad libitum fed animals, with a reduction of more than 20% of body weight. In the present study old rats on DR regimen showed 18% reduction in body weight as compared to their AL counterparts (Fig. 1). It was also observed that rats placed on DR binge on food next day and they almost consume 1.4 times more food as compared to AL rats on feeding day. The overall reduction in food intake of DR was estimated to be around 25–30%. The average blood glucose was also measured after every two weeks and in AL rats, the average blood glucose level was 110 mg/dl which was reduced in DR rats to 89.53 mg/dl (Fig. 1). Other physiological parameters related to metabolic rate,


Fig. 1 Comparison of body weight gain (gm) food consumption (gm) and blood glucose (mg dl) in dietary restricted (DR) and ad libitum fed (AL) rats. These parameters were recorded every fifteenth day in 21 months old rats after initiation of alternate day feeding regimen till 24 months of age in both DR and AL rats. Values are mean ± SEM and are percentage of initial values of each parameter. * P \ 0.05 versus vehicletreated AL rats, Bonferroni’s test after ANOVA

such as body temperature and blood pressure have been consistently documented to decrease in previous studies under similar experimental conditions of dietary restriction regimen (Weindruch and Sohal 1997; Mattson et al. 2003). NCAM and PSA-NCAM expression in aging brain and their reversal by DR In the present study, we examined age-related expression of NCAM, its polysialylated form PSANCAM and astrocytic marker GFAP. Immunofluorescence staining of these proteins was studied in 1, 3, 6, 18 and 24 months old rats in hippocampus, hypothalamus and piriform cortex regions. Immunohistofluorescent study showed strong expression of NCAM in dentate gyrus region of hippocampus, arcuate region and paraventriculare area of hypothalamus and piriform cortex region of the 1 month old rats, which was maintained in 3 and 6 months old rats

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(Fig. 2, P \ 0.05). Abundant NCAM immunoreactive cells could be seen in SGZ area with well stained processes. Thereafter, a significant decrease in NCAM-ir was observed in these areas from 18 and 24 months old rats as compared to their younger counterparts (Figs. 2 and 3, P \ 0.05). Old rats on DR showed significantly enhanced NCAM-ir in dentate gyrus region of hippocampus, hypothalamus and piriform cortex as compared to 24 months old AL rats (P \ 0.05). The results of PSA-NCAM immunoreactivity in dentate gyrus region of hippocampus, hypothalamus and piriform cortex are presented in Fig. 4. In the hippocampus of 1 and 3 months old rats, PSANCAM-ir was mainly localized in the dentate gyrus region (Fig. 4). Abundant PSA-NCAM immunoreactive cells could be found in the innermost portion of the granule cell layer or subgranular zone (SGZ). Most of these cells had the typical morphology of granule neurons with a well developed apical dendritic tree. Several processes, probably corresponding to granule cell axons/mossy fibers, were found in the hilus. There was abrupt reduction in PSA-NCAM-ir in dentate gyrus region of the hippocampus in 6 month old rats as compared to 1 and 3 months old rats (Figs. 3, 4, P \ 0.05). A very weak PSA-NCAM-ir was observed in 18 and 24 months old rats, which was significantly low as compared to their younger counterparts of 1, 3 and 6 months old rats (P \ 0.05). Late-onset DR in 24 months old rats significantly upregulated the PSA-NCAM-ir in dentate gyrus region of hippocampus as compared to 24 months old AL rats (P \ 0.05). Similar changes in PSA-NCAM-ir were observed in hypothalamus and piriform cortex regions of brain in 3 months old rats as compared to 1 month old rats (P \ 0.05). A large number of PSA-NCAM positive cells with long processes were seen in hypothalamus of 3 and 6 month old rats projecting toward external zone of median eminence arcuate region (Fig. 2). However a gradual decrease in PSA-NCAM-ir was observed in hypothalamus and piriform cortex from 6 months to 18 and 24 months old rats. However, a clear difference in morphology of PSA-NCAM positive cells was evident with advancing age e.g. in 1 month old rats the PSA-NCAM positive cells were having short processes in piriform cortex region, whereas, in 3 and 6 month old rats, PSA-NCAM positive cells with large and well defined processes could be seen. There

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Fig. 2 Representative Immunohistofluorescent images of 30 lm thick coronal sections showing immunostaining for NCAM in 1, 3, 6, 18, 24 months old AL and 24 months old DR rats. Note the higher NCAM-ir in 1, 3 and 6 months old rats and a marked low NCAM-ir in 18 and 24 months old rats. Also, note the higher NCAM-ir in 24 months old DR rats as compared to 24 months old AL rats in SGZ and GCL of dentate gyrus region of hippocampus, arcuate and paraventricular region of hypothalamus and piriform cortex layer II. Scale bars = 100 lm DG— dentate gyrus, SGZ— subgranular layer, h—hilus, 3 V—third ventricle

was significant decline in PSA-NCAM-ir in 18 and 24 month old rats in hypothalamus and piriform cortex as compared to their younger counterparts (P \ 0.05). We observed a significant increase in PSA-NCAM-ir in hypothalamus and piriform cortex regions of old DR rats as compared to 24 month old rats (P \ 0.05). The results of immunohistofluorescent localization of NCAM were further confirmed by quantitative

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analysis of immunoblots. NCAM and a-tubulin labeling in the representative samples from hippocampus, hypothalamus and piriform cortex of 3 month old, 24 month old AL and 24 month old DR rats and the mean value for the percentage of NCAM-120, -140, -180 kDa isoforms to a-tubulin ROD is illustrated in Fig. 5. The quantitative analysis of immunoblots revealed significant increase in expression of NCAM-120 and -140 isoforms in


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Fig. 3 Intensity measurement data of the NCAM-ir, PSA-NCAM-ir and GFAP-ir in hippocampus, hypothalamus and piriform cortex regions from 1, 3, 6, 18, 24 months old AL and 24 months old DR rats. Mean values of NCAM-ir, PSA-NCAM-ir and GFAPir intensity levels for each of the groups expressed as percentage of 1 month old rat as control. * P \ 0.05, Bonferroni’s test after ANOVA

hippocampus, hypothalamus and cortices of 24 months old AL and DR rats as compared to young adult rats (Fig. 5, P \ 0.05). NCAM-180 expression was found to be significantly higher in hippocampus, hypothalamus and cortices of young adult rats as compared to 24 months old AL rats (P \ 0.05). Further, there was significant increase in NCAM-180 expression in hippocampus, hypothalamus and cortices of 24 months old DR rats as compared to 24 months old AL rats (P \ 0.05).

GFAP expression in aging and its attenuation by DR We studied the expression of GFAP in brain areas namely dentate gyrus region of hippocampus, piriform cortex and hypothalamus in 1, 3, 6, 18, 24 months old rats and 24 month old DR rats. Results are presented in Fig. 6. We observed increase in GFAP-ir with age in dentate gyrus region, piriform cortex and also in hypothalamus (Fig. 6). Enhanced GFAP-ir was evident in 3

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Fig. 4 Representative immunohistofluorescent images of 30 lm thick coronal sections showing immunostaining for PSANCAM 1, 3, 6, 18, 24 months old AL and 24 months old DR rats. A strong expression of PSA-NCAM-ir is evident in 1 and 3 months old rats, which decreased in 6 months old rats and a very low PSA-NCAM-ir is seen in 18 and 24 months old rats. Late-onset DR enhanced PSA-NCAM-ir in 24 month old DR rats as compared to 24 months old AL rats. DG—dentate gyrus, SGZ—subgranular layer, h—hilus, 3 V—third ventricle. Scale bars = 80 lm (A-L)

and 6 months old rats in different brain regions as compared to 1 month old rats (P \ 0.05), which further increased significantly in 18 and 24 months old rats (P \ 0.05). Aging not only increased the staining intensity and number of GFAP positive cells, but it also induced the phenomenon of reactive astrocytosis as revealed by altered morphology of astrocytes i.e. hypertrophy of cells and much thicker processes, which were more pronounced in 18 and 24 months old rats. DR

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was found to attenuate these changes as revealed by decreased GFAP-ir in these brain regions as compared to 24 months old AL rats (Fig. 6). GFAP and a-tubulin labeling in the representative samples from hippocampus, hypothalamus and piriform cortex regions of 3 month old, 24 month old AL and DR rats is shown in Fig. 7. The mean value for the percentage of GFAP to a-tubulin ROD for all the groups is also illustrated. The quantitative analysis of


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Fig. 5 Representative Western blot hybridization signals using antibodies specific for NCAM and a-tubulin from hippocampus, hypothalamus and piriform cortex from 1, 3, 6, 18, 24 months old

AL and 24 months old DR rats. Mean values of NCAM isoform levels for each of the groups expressed as percentage of a-tubulin labeling. * P \ 0.05, Bonferroni’s test after ANOVA

immunoblots revealed higher GFAP expression in hippocampus, hypothalamus and piriform cortex region of brain from 24 months old rats as compared to young adults (P \ 0.05). A significant decrease in GFAP content was observed in 24 months old DR rats as compared to 24 month old AL rats from hippocampus, hypothalamus and cortex regions of brain (P \ 0.05).

rats. Abundant NCAM immunoreactive cells could be seen in SGZ with well stained processes. There was no significant difference in NCAM-ir in 3 and 6 months old rats as compared to 1 month old rats. These groups also showed strong immunoreactive cells for NCAM with several well defined processes (Fig. 2). A gradual decline was seen thereafter in 18 and 24 months old rats for NCAM-ir, however this change was not as abrupt as in case of PSA-NCAMir. A similar pattern for NCAM-ir was observed in hypothalamus and cortex regions of these rats. In 18 and 24 months old rats, the 120 and 140 kDa isoforms of NCAM were specifically upregulated as compared to 3 month old young adults, whereas, the 180 kDa isoform expression was reduced in all the brain regions as is evident from western blotting data analysis. This indicates that the metabolism of different isoforms of NCAM is individually regulated during aging. Moreover, 180 kDa is the major isoform, which is modified by posttranslational polysialylation i.e. to PSA-NCAM. Selective upregulation of low molecular weight isoforms of NCAM and their mRNAs have been earlier demonstrated in aging muscle fibres (Anderson et al. 1993). We observed strong PSA-NCAM immunoreactivity in dentate gyrus region of hippocampus in 1 month

Discussion In order to know how the expression of NCAM, its polysialylated form PSA-NCAM and astrocytic marker GFAP is affected by aging, immunofluorescence staining of these proteins was studied in 1, 3, 6, 18 and 24 months old rats from hippocampus, hypothalamus and piriform cortex regions of brain which showed intense NCAM and PSA-NCAM expression in young-adult rats. Aging attenuates NCAM and PSA-NCAM expression A strong expression of NCAM was observed in dentate gyrus region of hippocampus in 1 month old

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Fig. 6 Representative immunohistofluorescent images of 30 lm thick coronal sections showing immunostaining for GFAP 1, 3, 6, 18, 24 months old AL and 24 months old DR rats. Note the marked increase in GFAP immunoreactivity with aging and a strong GFAP-ir in 18 and 24 months old rats. DR attenuated the GFAP-ir in 24 months old DR rats as compared to AL rats. Scale bars = 100 lm (A-L). DG—dentate gyrus, HYP—hypothalamus, PC—piriform cortex

old rats, which gradually decreased in 3 and 6 months old rats (Fig. 4). In the hippocampus of 1 and 3 months old rats, PSA-NCAM-ir was mainly localized in the dentate gyrus region (Fig. 4). Abundant PSA-NCAM immunoreactive cells could be found in the innermost portion of the GCL or SGZ. Most of these cells had the typical morphology of granule neurons with a well developed apical dendritic tree. Several processes, probably corresponding to granule

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cell axons/mossy fibers, were found in the hilus. There was abrupt reduction in PSA-NCAM-ir in dentate gyrus region of the hippocampus in 6 month old rats and a very weak PSA-NCAM-ir was observed in 18 and 24 months old rats. Three and 6 months old rats showed highest PSA-NCAM-ir in hypothalamic region. A large number of PSA-NCAM positive cells with long processes were seen in hypothalamus of young adult rats projecting toward external zone of


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Fig. 7 Representative Western blot hybridization signals using antibodies specific for GFAP and atubulin from hippocampus, hypothalamus and piriform cortex from 1, 3, 6, 18, 24 months old AL and 24 months old DR rats. Mean values of GFAP levels for each of the groups expressed as percentage of a-tubulin labeling. * P \ 0.05, Bonferroni’s test after ANOVA

median eminence arcuate region (Fig. 4). However a gradual decrease in PSA-NCAM-ir was observed in 18 and 24 months old rats. Similar pattern of changes in PSA-NCAM-ir was observed in piriform cortex layer II. Late-onset short-term DR reverses age-associated decline in NCAM and PSA-NCAM expression A significant increase in NCAM-ir was seen in dentate gyrus region of old DR rats as compared to old AL rats (Fig. 2). In the hypothalamus and piriform cortex as well as an increase in expression of NCAM-ir in old DR rats was observed as compared to old AL rats. These results were further confirmed by NCAM immunoblotting, which showed that with aging there is decrease in high molecular weight isoforms such as NCAM 180 kDa (Fig. 5). We observed that late-onset DR restored this agerelated decline in NCAM content, with old DR rats showing statistically significant increase in NCAM isoforms 180- and 140-kDa as compared to age matched AL rats in hippocampus, hypothalamus and cortex regions. We further observed a significantly enhanced PSA-NCAM-ir in dentate gyrus region of old DR rats as compared to old AL rats. In the SGZ of DR rats, increase in number of cells immunoreactive for PSA-NCAM is evident; these cells have clear processes projecting toward granule cell layer (Fig. 4). A number of immunoreactive cells positive for PSA-NCAM expression could also be seen in

hypothalamic region of both AL and DR rats, but the number of PSA-NCAM immunoreactive cells in the old AL rats was much lower as compared to old DR rats which showed high expression of PSA-NCAM-ir in this area (Fig. 4). In the cortical region, most PSANCAM immunolabelled cells appeared concentrated in layer II. Hippocampus, hypothalamus and piriform cortex regions have been shown earlier to have physiological plasticity as well as structural and neuroendocrine plasticity (Rutishauser 2008; Parkash and Kaur 2005). Duveau et al. (2007) reported that hypothermiainduced stress caused upregulation of NCAM polysialylation in hippocampus and further showed that protection against kainic acid induced cell death by heat shock pre-exposure was abolished in the absence of NCAM polysialylation. Recent study from our lab showed that DR enhances kainate induced increase in NCAM while blocking the glial activation in adult rat brain (Sharma and Kaur 2008). The present and the previous similar reports suggest that cell adhesion molecules may play an important role in promoting brain plasticity, which in turn might permit remodeling of neuronal structures required for neuroprotection. In another recent study from our lab (Sharma and Kaur 2007), we have reported that DR initiated in late adulthood (15 months old rats) conferred beneficial effects such as attenuation of oxidative stress, upregulation of HSP 70, NCAM and PSA-NCAM expression and reduced levels of GFAP. Polysialylation of NCAM is considered to be a

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permissive factor which allows activity dependent neuronal and glial remodeling to occur whenever the proper inductive stimulus intervenes (Theodosis et al. 1999; Parkash and Kaur 2007; Singh and Kaur 2007). Majority of the previous studies report the effect of long term dietary restriction regimen in relation to aging. Recent studies involving short-term dietary restriction have documented similar beneficial effects against vulnerability to excitotoxic and metabolic insults (Bruce-Keller et al. 1999; Lee et al. 2003). Moreover, a genomic profiling study of short and long term caloric restriction also showed that short term calorie restriction (4 weeks) reproduced nearly 70% of the effects of long term calorie restriction on genes that changed expression with age (Cao et al. 2001). Aging upregulated GFAP expression The expression of GFAP-ir, which is an astrocytic cell marker, was studied in different age group rats. Normal expression of GFAP was evident in dentate gyrus region of hippocampus in 1, 3 and 6 month old rats. A strong upregulation of GFAP-ir was seen in 18 and 24 month old rats (Fig. 6). A significant difference in number of GFAP-ir cells could be seen with advancing age with 18 and 24 months old rats showing maximum GFAP-ir cells. Moreover number of GFAP positive cells exhibiting features of reactive gliosis could be seen in AL rats. A gradual increase in expression of GFAP was also seen in 1, 3, 6, and 18 month old rats and highly intense staining of GFAP in 24 month old rats from both hypothalamus and piriform cortex regions. Many recent studies have reported a correlation between reactive gliosis during aging and age-related functional deficits in learning and memory and impaired synaptic plasticity (Rozovsky et al. 2005). Prolla and Mattson (2001) observed that GFAP expression is progressively increased during aging in humans as well as in rodent models. Although the functions of reactive astrocytes are not well understood but both harmful and beneficial activities have been attributed to these cells (Hansson et al. 2000; Sofroniew 2005). An inverse correlation persists between reactive astrocytes and neurite outgrowth (Rozovsky et al. 2005). The present study also showed that enhanced astrocytic gliosis was associated with reduced expression of NCAM and PSA-NCAM, markers of neurite plasticity. Another study from our lab showed

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negative correlation between gonadotropin releasing hormone (GnRH) axon sprouting during proestrous phase of estrous cycle and retraction of glial processes (Parkash and Kaur 2005). Late-onset short-term DR attenuates age-related increase in GFAP expression In the dentate gyrus region of hippocampus, several immunoreactive astrocytes could be seen, displaying properties of reactive astrocytes as revealed by altered morphology of astrocytes i.e. hypertrophy of cells and much thicker processes (Fig. 6). These reactive astrocytes were evident in both AL and DR rats; however old AL rats showed pronounced increase in number of such cells (Fig. 6). In the hypothalamic area also strong expression of GFAP-ir was observed in both AL and DR rats, with DR rats showing lower GFAP-ir, which was restricted to internal zone of median eminence region of hypothalamus. In piriform cortex, the number of immunoreactive astrocytes was clearly more in old AL rats as compared to old DR rats. We further examined the GFAP content by immunoblotting which revealed similar results as seen with immunofluorescence staining. A significant increase was observed in GFAP content with aging. Late-onset DR effectively attenuated this age-related increase in GFAP expression as seen from immunoblots from hippocampus, hypothalamus and cortex regions (Fig. 7). The present and other recent findings may suggest that age associated increase in GFAP expression is not only linked to neurodegeneration and further allows to hypothesize that the age-related increase of reactive astrogliosis may be an important factor to impair synaptic plasticity during aging. The upregulation of NCAM and PSA-NCAM expression in rats on short-term DR regimen suggest that even late onset DR has beneficial effects and may reverse memory and learning functions, since both NCAM and PSA-NCAM play important role in neuroregeneration and learning in the adult nervous system (Ronn et al. 2000; Venero et al. 2006) and NCAM deficient mice have been shown to have impaired memory (Cambon et al. 2004; Sandi 2004).We are further carrying out work to study expression of synaptic proteins and CaMK-II, which are known to be downregulated by aging. There is also need to further correlate the changes observed in NCAM,


PSA-NCAM expression with memory and learning functional tests and signaling functions at the synapses (Dalva et al. 2007). A recent study reports that administration of FGL, a peptide mimetic of neural cell adhesion molecule, during the 4 weeks of continuous stress not only prevented the deleterious effects of chronic stress on spatial memory, but also reduced the survival of the newly generated hippocampal cells in aging animals (Borcel et al. 2008). The simultaneous enhanced expression of HSP 70 (data not reported) in discrete brain regions from old DR rats support the idea of neurohormetic intervention of age-related brain function impairment by lateonset short-term DR regimen. Understanding the molecular mechanisms of beneficial effects of dietary restriction on the brain may also lead to development of novel therapeutic agents which may reproduce the beneficial effects such as NCAM or PSA-NCAM mimetics. They may serve to modulate cell–cell interactions through FGF receptor signaling pathway to reverse age and disease impaired brain functions. These findings from animal studies suggest that moderate levels of DR even in late age may benefit humans by reducing the incidence and severity of AD, PD and stroke. Effects of DR on cognition in aged animals have not been studied in detail; however future studies on cognitive status in aged animals on DR may provide some insight into mechanism/s by which DR could preserve cognitive health in aged animals. Acknowledgements This grant was funded by Indian Council of Medical Research (ICMR) under National Task Force Project—an initiative on Aging Research. Both Ms. Manpreet Kaur and Mr. Sandeep Sharma are thankful to the ICMR for research fellowship grant.

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