Age-dependent decline of nogo-a protein in the mouse cerebrum.

Nogo-A, a myelin-associated neurite growth inhibitory protein, is implicated in synaptic plasticity. It binds to its receptor namely the Nogo-66 receptor1 (NgR1) and regulates filamentous (F) actin dynamics via small GTPases of the Rho family, RhoA kinase (ROCK), LimK and cofilin. These proteins are associated with the structural plasticity, one of the components of synaptic plasticity, which is known to decline with normal aging. So, the level of Nogo-A and its receptor NgR1 are likely to vary during normal brain aging. However, it is not clearly understood how the levels of Nogo-A and its receptor NgR1 change in the cerebrum during aging. Several studies show an age- and gender-dependent decline in synaptic plasticity. Therefore, the present study was planned to analyze the relative changes in the mRNA and protein levels of Nogo-A and NgR1 in both male and female mice cerebrum during normal aging. Western blot analysis has shown decrease in Nogo-A protein level during aging in both male and female mice cerebrum. This was further confirmed by immunofluorescence analysis. RT-PCR analysis of Nogo-A mRNA showed no significant difference in the above-mentioned groups. This was also supported by in situ hybridization. NgR1 protein and its mRNA expression levels showed no significant alteration with aging in the cerebrum of both male and female mice. Taken together, we speculate that the downregulation of Nogo-A protein might have a role in the altered synaptic plasticity during aging.

Role of CREB signaling in aging brain.

During aging, brain undergoes several changes which influence its function through alteration in the expression of genes. Some of these genes are regulated by estrogen which requires a host of coregulator proteins including CREB. In brain, CREB is expressed in different regions and regulates a wide range of functions such as cellular growth, proliferation and memory in response to a variety of intracellular signaling events including synaptic efficacy and long-lasting changes in synaptic plasticity. In response to signals at the cell surface, CREB is phosphorylated in the nucleus by various protein kinases via secondary messengers such as cAMP and/or Ca+2 for regulating specific genes. Alterations in CREB signaling lead to cognitive deficits as observed in normal aging and neurodegenerative diseases. In brain, the expression of CREB changes with age, but its variation with sex is not known. So, in this review paper, we summarize recent findings indicating age and sex dependent expression of CREB and its interaction with estrogen receptor (ER)ß, and the role of CREB signaling in brain aging and diseases. Such understanding of CREB signaling through ER may help to design therapeutic strategies for age related cognitive deficits and neurodegenerative disorders.

Expression of Trk A and Src and their interaction with ERß ligand binding domain show age and sex dependent alteration in mouse brain.

Following the binding of estrogen to estrogen receptor (ER)ß ligand binding domain (LBD) and its interaction with the target genes, a host of nuclear proteins is recruited to regulate the expression of specific genes(s). It is not known which proteins interact with ERßLBD and whether they vary with age and sex in the brain. Therefore, using pull down assay, immunoprecipitation and immunoblotting, we report that cell signaling molecules Trk A and Src interacted with ERßLBD, and showed alteration in the level of interaction and expression in the brain of AKR strain young (6 weeks), adult (25 weeks) and old (70 weeks) mice of both sexes. Trk A showed decreasing interaction with age, and lower expression in adult as compared to young and old males, whereas female mice exhibited decline in both interaction and expression as a function of age. On the other hand, Src interaction with ERßLBD decreased, but its expression increased with age in males, whereas the interaction and expression was lower in adult but higher in old as compared to young females. These findings suggest the implication of Trk A and Src in ERß mediated brain functions and related disorders during aging.

Gonadal steroids do not affect apolipoprotein E expression in aging mouse cerebral cortex.

The allelic variant of apolipoprotein (Apo) E4 is a known risk factor for the development of most common late onset form of Alzheimer's disease (AD). As aging is associated with reduced circulating level of gonadal steroid hormones, hormone replacement therapies have been used for the possible treatment of AD. Both estrogen and testosterone have beneficial effects on brain due to interaction with apoE, but the underlying mechanism is still not clear. In this article, we report the effects of gonadectomy and hormone supplementation on apoE protein level in male and female mouse cerebral cortex during normal aging. We could not get any effect of gonadectomy and estradiol or testosterone treatment in adult and old mice of either sex. This suggests that during normal aging apoE protein level is not affected due to steroid hormone withdrawal or supplementation in the mouse cerebral cortex.

Age-related binding of proximal region of ApoE promoter to nuclear proteins of mouse cerebral cortex.

Mouse ApolipoproteinE (ApoE) gene has maximum promoter activity in the proximal region (-212 to +54) which includes different regulatory elements. These elements bind to specific protein factors and influence the expression of genes which are involved in key brain functions that decline with age. As there is no information on the binding of apoE promoter to nuclear proteins as a function of age, we have analyzed the binding of USF, AP1 and one negative element sequence present in ApoE proximal promoter to nuclear proteins of the cerebral cortex of mice of different ages. The findings show the formation of one complex with USF and two complexes with AP1 and negative element. The intensity of these complexes varies with age, indicating differential binding of protein factors to specific elements of apoE promoter, which reflect age-related regulation of apoE -mediated brain functions.

NMR analysis reveals 17ß-estradiol induced conformational change in ERß ligand binding domain expressed in E. coli.

Nuclear magnetic resonance (NMR) spectroscopy is a useful biophysical technique to study the ligand-protein interaction. In this report, we have used bacterially produced ERß and its domains for studying the functional analysis of ligand-protein interaction. Briefly, ERß and its transactivation domain (TAD) and ligand binding domain (LBD) were subcloned and overexpressed using a prokaryotic expression system. The recombinant proteins were purified using Ni(+2)-IDA affinity chromatography and analyzed by NMR. Purified ERß and TAD show similar conformation in the absence or presence of 17ß-estradiol. However, LBD shows altered conformation in the presence of 17ß-estradiol. These findings suggest that ERß produced in bacteria exhibits a conformation such that its LBD remains masked and consequently it binds less to 17ß-estradiol. Such study may help to develop the therapeutic approaches for controlling the estradiol-mediated gene expression in hormone dependent diseases.

Role of nutraceuticals in cognition during aging and related disorders.

Cognitive abilities are compromised with advancing age posing a great risk for the development of dementia and other related brain disorders. Genetic susceptibility as well as environmental exposures determine the fate of cognitive aging and its transition to pathological states. Emerging epidemiological and observational studies have revealed the importance of lifestyle factors including dietary patterns and nutritional intake in the maintenance of cognitive health and reducing the risk of neurodegenerative disorders. In this context, nutraceutical interventions have gained considerable attention in preventing age-related cognitive deficits and counteracting pathological processes. Nutraceuticals include dietary plants and derivatives, food supplements and processed foods with nutritional and pharmaceutical values. The present review highlights the importance of nutraceuticals in attenuating cognitive aging and its progression to dementia, with specific emphasis on chemical constituents, neurocognitive properties and mechanism of action.

Interaction of Estrogen Receptor Associated Protein (ERAP) 140 with ER beta decreases but its expression increases in aging mouse cerebral cortex.

Following binding to cognate ligand, estrogen receptor (ER) beta interacts with specific responsive elements of the target genes and recruits a host of nuclear proteins for hormone dependent gene regulation. However, it is poorly known which proteins interact with ER beta in mouse brain and whether their interaction and expression change with age. In this report, we have used his-tag mouse ER beta for interaction with nuclear proteins of cerebral cortex of young (6 +/- 1 weeks), adult (25 +/- 2 weeks), and old (70 +/- 5 weeks) female mice. We have identified estrogen receptor-associated protein (ERAP) 140 as one of the interacting proteins and studied its interaction by pull down immunoblotting, far-Western blotting and immunoprecipitation, and expression by western blotting. The data show that ERAP 140 interacts with ER beta and its interaction decreases but its expression increases with age in mouse cerebral cortex, suggesting its role in estrogen-mediated brain functions during aging.

GST-tagged mouse estrogen receptor alpha-transactivation domain fusion protein is specifically degraded during its over-expression in E. coli and purification.

Escherichia coli BL21 (DE3) is commonly used for the overproduction of fusion proteins. Using this system, we recently reported the overproduction of histidine-tagged mouse estrogen receptor (ER) alpha-ligand binding domain as an intact 30 kD protein and its inhibitory effect on the growth of bacteria. However, when GST-tagged mouse ERalpha transactivation domain (TAD) was overproduced using this system, it showed no effect on the growth of bacteria but was specifically degraded during its expression and purification. Here we report the expression of 47 kD GST-tagged mouse ERalpha-TAD protein, which was degraded partially and specifically into 46 and 43 kD fragments. This fusion protein was further degraded into 37, 31, 29 and 26 kD fragments during its purification by affinity chromatography. Such specific degradation of GST-tagged mouse ERalpha-TAD during its overproduction in E. coli and purification indicates the induction of specific protease and suggests the modification of expression system.

Quantitative metabolic biomarker analysis of mild cognitive impairment in eastern U.P. and Bihar population.

Mild cognitive impairment (MCI) is a transition phase between healthy individuals and Alzheimer's disease (AD). Therefore, diagnosis of MCI at early stage will help to delay or prevent its progression to disease. In the present study, we aim to identify the metabolic biomarkers, which can help in the diagnosis of MCI. We have screened 2000 elderly individuals from north India, out of which 200 were identified as MCI. We continued our study on 10 MCI individuals who regularly participated in the follow-up. The age and gender matched 10 healthy individuals were taken as control. These control and MCI individuals were subjected to neuropsychological examination such as Hindi mental state examination (HMSE) and Montreal cognitive assessment (MOCA) followed by 1H Nuclear Magnetic Resonance (NMR) analysis. Remarkable changes were noted between control and MCI individuals at metabolic level. In silico study showed the involvement of eight metabolites in MCI. We found higher level of lactate, N-acetyl aspartate, histidine and lower level of formate, choline, alanine, creatinine and glucose in blood plasma of MCI individuals compared to control. Further, In silico study showed that choline might be directly associated with MCI or AD. Such In silico study with quantitative metabolite analysis of plasma could be used as diagnostic biomarkers for the identification of MCI.

Interaction of estrogen receptor-alpha ligand binding domain with nuclear proteins of aging mouse brain.

After the interaction of estrogen with the ligand binding domain (LBD) of mouse estrogen receptor-alpha (mERalpha) and hormone-responsive elements of target genes, many nuclear proteins are recruited to regulate the expression of specific genes. Because it is not known which brain proteins interact with LBD or whether these proteins vary with age and sex, we used pull-down assay and far Western blotting to detect five nuclear proteins of 160, 140, 87, 60, and 46 kD in the mouse brain. These interacting proteins were identified as PELP1, RIP140, PGC1alpha, BAF60, and ADA3, respectively. The level of PELP1, RIP140, PGC1alpha, and BAF60 decreased drastically in old compared with adult male mice, whereas the ADA3 level showed no significant change. PELP1, PGC1alpha, and BAF60 levels were lower in old male compared with female mice. Thus we report the identification and interaction of five nuclear proteins with mERalpha-LBD, indicating their role in estrogen signaling and brain functions during aging.

Interaction of estrogen receptor-alpha transactivation domain with nuclear proteins of mouse brain: p68 RNA helicase shows age- and sex-specific change.

Estrogen receptor (ER)-alpha interacts with nuclear proteins to mediate its multiple functions in the brain. However, it is not known which proteins interact with the ERalpha-transactivation domain (TAD) in mouse brain and whether they change with age and sex. Therefore, we have used affinity-purified GST-tagged mouse ERalpha-TAD fusion protein for interaction with nuclear proteins from the mouse brain. The pull-down assay and far-Western blotting detected four nuclear proteins of 100, 80, 68, and 50 kD. We have recently identified the 80-kD protein as MTA1 and demonstrated its decrease in old age. Here we report alteration in the interaction and expression of the 68-kD protein of adult and old mice of both sexes. This protein was identified as p68 RNA helicase through NCBI database search, immunoprecipitation, and immunoblotting. Further analysis showed that the extent of its interaction was relatively lower in old mice of both sexes and in male mice of both ages compared with their counterparts. However, the expression of p68 was significantly lower in old males compared with adult males, although other groups did not show significant changes. Such age- and sex-specific interaction of p68 suggests its implication in ERalpha-mediated brain functions during aging.

Interaction of estrogen receptor alpha transactivation domain with MTA1 decreases in old mouse brain.

We have reported earlier that estrogen receptor (ER) alpha-transactivation domain (TAD) interacted with four nuclear proteins of 100 kD, 80 kD, 68 kD, and 50 kD of mouse brain and identified 68 kD as p68 RNA helicase and 50 kD as beta-tubulin. In this paper, we describe the identification of 80 kD nuclear protein as metastasis associated protein 1 (MTA1) and its interaction and expression in the brain of aging mice. Far-Western blotting and immunoprecipitation data revealed lower interaction of MTA1 in old than adult mice of both sexes. Furthermore, adult male showed lower expression of protein as compared to adult female. Altogether these findings suggest that age-dependent decrease in the expression of MTA1 and its interaction with ERalpha-TAD may influence the estrogen-mediated signaling pathway during aging of mouse brain.

Role of steroid hormone coregulators in health and disease.

The involvement of coactivators and corepressors, collectively termed as coregulators, increases the complexity of regulation of steroid hormone action. Following the interaction of the steroid hormone-receptor complex with the specific nucleotide sequences of target genes, the coregulators are recruited for activation or suppression of specific genes. The coregulators regulate a number of hormonal events during pregnancy, sex differentiation, development, reproduction and sexual behavior. They also exert equally important functions in non-reproductive tissues like heart, kidney, pancreas, bone and brain. The mutation and/or aberrant expression of these coregulators affect the normal function of steroid hormones and result in physiological abnormalities leading to the development of diseases. Therefore, understanding the role of coregulators in steroid hormone action is important and would help in developing the therapeutic strategy for the treatment of steroid-related diseases. In this review article, we describe the coregulators and their implication in health and pathogenesis of diseases. Furthermore, the possible therapeutic approach has been discussed for the treatment of steroid-related diseases, which will be of future interest in the field of medical sciences.

Correction to: Age-related expression of Neurexin1 and Neuroligin3 is correlated with presynaptic density in the cerebral cortex and hippocampus of male mice.

The corresponding author noticed in his published paper that the images (30 weeks, CC, 10, 30 and 50 weeks DG) of fig. 3b are inadvertently duplicated with the images of fig. 3a. Now, these images have been replaced in the corrected panel (Fig. 3b) below.

PS2 protein expression is upregulated by sex steroids in the cerebral cortex of aging mice.

Mutations in presenilin (PS) genes cause majority of early onset Alzheimer's disease (AD), an age related neurodegenerative disorder. PS proteins undergo proteolytic cleavage to produce biologically active fragments, which constitute the catalytic core of the gamma-secretase enzyme. This enzyme cleaves beta-amyloid precursor protein (betaAPP) to generate Abeta peptides, which are influenced by sex steroids. Recently we have reported the downregulation of PS1 expression by sex steroids in the brain of adult mice. Here we have examined the effect of gonadectomy and subsequent administration of gonadal hormones 17beta-estradiol and testosterone on the level of PS2 C-terminal fragment (CTF) in the cerebral cortex of adult and old AKR strain mice of both sexes. PS2 expression was downregulated following gonadectomy, but upregulated by supplementation of gonadal steroids in both age groups and sexes. Thus these results demonstrate up-regulation of PS2 protein expression by sex steroids, which in turn may influence PS2 associated brain functions.

Histone Deacetylase 2 Inhibition Attenuates Downregulation of Hippocampal Plasticity Gene Expression during Aging.

The brain undergoes several anatomical, biochemical, and molecular changes during aging, which subsequently result in downregulation of synaptic plasticity genes and decline of memory. However, the regulation of these genes during aging is not clearly understood. Previously, we reported that the expression of histone deacetylase (HDAC)2 was upregulated in the hippocampus of old mice and negatively correlated with the decline in recognition memory. As HDAC2 regulates key synaptic plasticity neuronal immediate early genes (IEGs), we have examined their expression and epigenetic regulation. We noted that the expression of neuronal IEGs decreased both at mRNA and protein level in the hippocampus of old mice. To explore the underlying regulation, we analyzed the binding of HDAC2 and level of histone acetylation at the promoter of neuronal IEGs. While the binding of HDAC2 was higher, H3K9 and H3K14 acetylation level was lower at the promoter of these genes in old as compared to young and adult mice. Further, we inhibited HDAC2 non-specifically by sodium butyrate and specifically by antisense oligonucleotide to recover epigenetic modification, expression of neuronal IEGs, and memory in old mice. Inhibition of HDAC2 increased histone H3K9 and H3K14 acetylation level at the promoter of neuronal IEGs, their expression, and recognition memory in old mice as compared to control. Thus, inhibition of HDAC2 can be used as a therapeutic target to recover decline in memory due to aging and associated neurological disorders.

Transcription of estrogen receptor alpha and beta in mouse cerebral cortex: effect of age, sex, 17beta-estradiol and testosterone.

Estrogen actions are mainly mediated by estrogen receptor (ER)alpha and ERbeta which in turn are regulated by several factors including age, sex and gonadal steroid hormones 17beta-estradiol and testosterone. In the present study, we have used nuclear run-off assay to examine the effect of these factors on the rate of transcription of ERalpha and ERbeta of mouse cerebral cortex. The run-off assay result was further corroborated with the measurement of steady state level of ERalpha and ERbeta mRNA by semiquantitative RT-PCR method. Our results reveal that ERalpha transcription rate decreases in old mice of both sexes, whereas ERbeta transcription rate decreases only in old females when compared to their adult counterparts. 17beta-Estradiol supplementation reduces the transcription rate of ERalpha and ERbeta in all groups except in adult male while testosterone treatment down regulates the transcription rate of ERalpha and ERbeta in all groups. The semiquantitative RT-PCR analysis reveals that the level of ERalpha mRNA decreases in old male but shows no effect in old female as compared to adult counterpart. In contrast, ERbeta transcript level decreases in old mice of both sexes. Furthermore, ERalpha mRNA level is higher in adult female than in adult male but no sex-dependent difference is seen in ERbeta mRNA level. Supplementation of 17beta-estradiol shows no significant alteration but testosterone reduces the ERalpha level in male mice, while 17beta-estradiol and testosterone down regulate the ERalpha level in female mice of both ages. In case of ERbeta, 17beta-estradiol decreases the transcript level in all groups except adult male while testosterone treatment results in the down regulation of transcript level in all groups. Thus these findings suggest differential effects of age, sex, 17beta-estradiol and testosterone supplementation on the transcription of mouse ER genes which may account for differences in the protein levels of ERalpha and ERbeta and their functions in the brain.

Epigenetic Regulation of Memory-Therapeutic Potential for Disorders.

BACKGROUND: Memory is a vital function which declines in different physiological and pathological conditions such as aging and neurodegenerative diseases. Research in the past has reported that memory formation and consolidation require the precise expression of synaptic plasticity genes. However, little is known about the regulation of these genes. Epigenetic modification is now a well established mechanism that regulates synaptic plasticity genes and neuronal functions including memory. Therefore, we have reviewed the epigenetic regulation of memory and its therapeutic potential for memory dysfunction during aging and neurological disorders. METHOD: Research reports and online contents relevant to epigenetic regulation of memory during physiological and pathological conditions have been compiled and discussed. RESULTS: Epigenetic modifications include mainly DNA methylation and hydroxymethylation, histone acetylation and methylation which involve chromatin modifying enzymes. These epigenetic marks change during memory formation and impairment due to dementia, aging and neurodegeneration. As the epigenetic modifications are reversible, they can be modulated by enzyme inhibitors leading to the recovery of memory. CONCLUSION: Epigenetic modifications could be exploited as a potential therapeutic target to recover memory disorders during aging and pathological conditions.

Anxiety like behavior due to perinatal exposure to Bisphenol-A is associated with decrease in excitatory to inhibitory synaptic density of male mouse brain.

Bisphenol-A (BPA) is a synthetic endocrine disruptor which causes anxiety like behavior in rodents, though the underlying mechanism is not clearly understood. As excitatory-inhibitory synaptic proteins are the key regulators of anxiety, we have examined the effect of perinatal exposure to BPA on this behavior and the expression of excitatory (PSD95), inhibitory (gephyrin) and presynaptic density marker (synaptophysin) proteins in cerebral cortex and hippocampus of 3 and 8 weeks postnatal male mice. In open field (OF) test, BPA exposure reduced the time spent, number of entries and distance travelled in the central zone as compared to control in 8 weeks mice. On the other hand, elevated plus maze (EPM) results showed decrease in time spent and number of entries to the open arms. Immunoblotting and immunofluorescence analysis showed significant downregulation of PSD95 and synaptophysin, but upregulation of gephyrin, leading to reduction in excitatory to inhibitory protein ratio and synaptic density in postnatal 3 and 8 weeks mice. Thus, our findings show that the anxiety like behavior due to perinatal exposure to BPA is associated with decrease in excitatory to inhibitory synaptic density in postnatal male mice.