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.

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.

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.

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.

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.

Binding of transcription factors to Presenilin 1 and 2 promoter cis-acting elements varies during the development of mouse cerebral cortex.

Previously, we reported differential expression of Presenilin (PS)1 and 2 and epigenetic modifications of their gene promoter in the cerebral cortex of mice during development. We identified the crucial role of DNA methylation and H3K9/14 acetylation in stage specific PS expression during brain development. Interestingly, we noted differential DNA methylation in putative binding sites of transcription factors considered pivotal for brain development. This prompted us to study the binding of transcription factors to cis-acting elements of PS1 and PS2 promoter in the cerebral cortex of mice during development. In-silico analysis revealed various cis-acting elements of PS1 and PS2 promoter and their putative transcription factors. We selected those cis-acting elements that were proven by wet lab experiments to interact with the transcription factors crucial for brain development. Electrophoretic mobility shift assay revealed that the binding of nuclear proteins to PS1 promoter cis-acting elements like HSF-1, Cdx1, Ets-1 and Sp1 significantly increased at embryonic day (E) 12.5, postnatal day (P) 45 and 20 weeks (w) as compared to P0. The binding pattern of these factors correlated well with the PS1 expression profile, indicating their cumulative influence on PS1 gene transcription. For PS2 promoter, the binding of Nkx2.2 and HFH-2 was high at prenatal stages (E12.5 and E18.5) while that of Cdx1 and NF-κB was maximum at postnatal stages (P45 and 20w). Taken together, our study shows that the binding of HSF-1, Cdx1, Ets-1 and Sp1 to PS1 promoter and that of Nkx2.2, HFH-2, Cdx1 and NF-κB to PS2 promoter regulate their differential expression during brain development.

Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in the female mouse brain.

Adverse early life experience is prominent risk factors for numerous psychiatric illnesses, including mood and anxiety disorders. It imposes serious long-term costs on the individual as well as health and social systems. Hence, developing therapies that prevent the long-term consequences of early life stress is of utmost importance, and necessitates a better understanding of the mechanisms by which early life stress triggers long-lasting alterations in gene expression and behavior. Post-weaning isolation rearing of rodents models the behavioral consequences of adverse early life experiences in humans and it is reported to cause anxiety like behavior which is more common in case of females. Therefore, in the present study, we have studied the impact of social isolation of young female mice for 8weeks on the anxiety like behavior and the underlying molecular mechanism. Elevated plus maze and open field test revealed that social isolation caused anxiety like behavior. BDNF, a well-known molecule implicated in the anxiety like behavior, was up-regulated both at the message and protein level in cerebral cortex by social isolation. CREB-1 and CBP, which play a crucial role in BDNF transcription, were up-regulated at mRNA level in cerebral cortex by social isolation. HDAC-2, which negatively regulates BDNF expression, was down-regulated at mRNA and protein level in cerebral cortex by social isolation. Furthermore, BDNF acts in concert with Limk-1, miRNA-132 and miRNA-134 for the regulation of structural and morphological plasticity. Social isolation resulted in up-regulation of Limk-1 mRNA and miRNA-132 expression in the cerebral cortex. MiRNA-134, which inhibits the translation of Limk-1, was decreased in cerebral cortex by social isolation. Taken together, our study suggests that social isolation mediated anxiety like behavior is associated with up-regulation of BDNF expression and concomitant increase in the expression of CBP, CREB-1, Limk-1 and miRNA-132, and decrease in the expression of HDAC-2 and miRNA-134 in the cerebral cortex.

Presenilin 2 overexpression is associated with apoptosis in Neuro2a cells.

Presenilin 1 (PS1) and PS2 are evolutionarily conserved transmembrane proteins of the aspartyl protease family. Initially, they were reported to be associated with the early onset of familial, early-onset Alzheimer's disease. PS1 has been implicated in several crucial brain functions including developmental processes, synaptic plasticity, and processing of various molecules, while PS2 has been poorly studied and is considered to be a compensatory partner of PS1. Certain controversial reports have suggested that PS2 has a role in apoptosis, though the underlying mechanism is not clear. To ascertain the role of PS2 in apoptosis, mouse neuroblastoma cells (Neuro2a) were transfected with a cDNA construct encoding full length mouse PS2 and analyzed for viability, expression of PS1, PS2, Bax and p53, Bax protein, and status of chromatin condensation. Our results showed reduced viability, condensed chromatin and higher expression of Bax at mRNA and protein levels, but no change in the expression of p53 and PS1 in PS2-overexpressing Neuro2a cells. Thus, it is evident that PS2, independent of PS1, is associated with apoptosis via a Bax-mediated pathway. These findings might help in the understanding of the involvement of PS2 in apoptosis and its associated brain disorders.

Analysis of Interaction of Estradiol with Estrogen Receptor by NMR Spectroscopy.

Following binding to estradiol, estrogen receptors (ER) α and ERß recruit a number of interacting proteins and mediate a plethora of functions. The binding of estrogen with the receptors shows changes in the resonance structure and movement of protons. We cloned ERß and its trans-activation domain (TAD) and ligand-binding domain (LBD), expressed them in prokaryotic expression vectors, purified them, and studied their interaction with estradiol. In this chapter, a detailed method of preparation of recombinant proteins, SDS-PAGE, silver staining, and NMR are described. Such methods are useful to check the biological activity of bacterially expressed proteins and are applicable to basic and applied research.

Bacopa monniera (CDRI-08) Upregulates the Expression of Neuronal and Glial Plasticity Markers in the Brain of Scopolamine Induced Amnesic Mice.

Preclinical studies on animal models have discerned the antiamnesic and memory-enhancing potential of Bacopa monniera (Brahmi) crude extract and standardized extracts. These studies primarily focus on behavioral consequences. However, lack of information on molecular underpinnings has limited the clinical trials of the potent herb in human subjects. In recent years, researchers highlight plasticity markers as molecular correlates of amnesia and being crucial to design therapeutic targets. In the present report, we have investigated the effect of a special extract of B. monniera (CDRI-08) on the expression of key neuronal (BDNF and Arc) and glial (GFAP) plasticity markers in the cerebrum of scopolamine induced amnesic mice. Pre- and postadministration of CDRI-08 ameliorated amnesic effect of scopolamine by decreasing acetyl cholinesterase activity and drastically upregulating the mRNA and protein expression of BDNF, Arc, and GFAP in mouse cerebrum. Interestingly, the plant extract per se elevated BDNF and Arc expression as compared to control but GFAP was unaltered. In conclusion, our findings provide the first molecular evidence for antiamnesic potential of CDRI-08 via enhancement of both neuronal and glial plasticity markers. Further investigations on detailed molecular pathways would encourage therapeutic application of the extract in memory disorders.

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

Neurexin1 (Nrxn1) and Neuroligin3 (Nlgn3) are cell adhesion proteins, which play an important role in synaptic plasticity that declines with advancing age. However, the expression of these proteins during aging has not been analyzed. In the present study, we have examined the age-related changes in the expression of these proteins in cerebral cortex and hippocampus of 10-, 30-, 50-, and 80-week-old male mice. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis indicated that messenger RNA (mRNA) level of Nrxn1 and Nlgn3 significantly increased from 10 to 30 weeks and then decreased at 50 weeks in both the regions. However, in 80-week-old mice, Nrxn1 and Nlgn3 were further downregulated in cerebral cortex while Nrxn1 was downregulated and Nlgn3 was upregulated in hippocampus. These findings were corroborated by immunoblotting and immunofluorescence results. When the expression of Nrxn1 and Nlgn3 was correlated with presynaptic density marker synaptophysin, it was found that synaptophysin protein expression in cerebral cortex was high at 10 weeks and decreased gradually up to 80 weeks, whereas in hippocampus, it decreased until 50 weeks and then increased remarkably at 80 weeks. Furthermore, Pearson's correlation analysis showed that synaptophysin had a strong relation with Nrxn1 and Nlgn3 in cerebral cortex and with Nlgn3 in hippocampus. Thus, these findings showed that Nrxn1 and Nlgn3 are differentially expressed in cerebral cortex and hippocampus which might be responsible for alterations in synaptic plasticity during aging.

Neuropsin Expression Correlates with Dendritic Marker MAP2c Level in Different Brain Regions of Aging Mice.

Neuropsin (NP) is a serine protease, implicated in synaptic plasticity and memory acquisition through cleavage of synaptic adhesion molecule, L1CAM. However, NP has not been explored during brain aging that entails drastic deterioration of plasticity and memory with selective regional vulnerability. Therefore, we have analysed the expression of NP and correlated with its function via analysis of endogenous cleavage of L1CAM and level of dendritic marker MAP2c in different regions of the aging mouse brain. While NP expression gradually decreased in the cerebral cortex during aging, it showed a sharp rise in both olfactory bulb and hippocampus in adult and thereafter declined in old age. NP expression was moderate in young medulla, but undetectable in midbrain and cerebellum. It was positively correlated with L1CAM cleavage and MAP2c level in different brain regions during aging. Taken together, our study shows age-dependent regional variation in NP expression and its positive correlation with MAP2c level, suggesting the involvement of NP in MAP2c mediated alterations in dendritic morphology during aging.

Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development.

In our previous report, we showed that Presenilin (PS)1 and 2 have differential expression profile from early embryonic stages till adulthood in mouse cerebral cortex, suggesting that both of these proteins are crucial for brain development. Genetic manipulation studies have also shown the involvement of PS1 in brain development, but PS2 remains largely unexplored. In order to understand how PS1 and 2 mediate developmental functions, we have investigated the interaction of PS1 and 2 with proteins of mouse cerebral cortex during development. Co-immunoprecipitation (Co-IP) combined with MALDI-MS/MS analysis revealed 12 interacting partners of PS1 and 11 partners of PS2. The interacting proteins were different for PS1 and 2, and involved in cell division, glycolysis, cell adhesion and protein trafficking. Densitometric analysis of protein bands visualized after SDS-PAGE separation of Co-IP proteins revealed variation in their amount and degree of interaction during different developmental stages of mice. Further, immunoblot based validation of PS1 interacting protein Notch-1 showed maximum interaction at embryonic day (E) 12.5, decline at E18.5, upregulation from postnatal day 0 (P0) to P20 and thereafter reduction at P45 and 20 weeks. In-silico analysis of PS and its interacting proteins indicated conformation based interaction through common type of secondary structures having alpha helical, extended beta strand and random coil, and CK2, PKC phosphorylation and myristoylation motifs. Taken together, our study showed that PS1 and PS2 interact to varying extent with different proteins of mouse cerebral cortex and suggests their interaction based on specific conformation and involvement in diverse functions essential for the brain development.

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.

Presenilin 1 and 2 are expressed differentially in the cerebral cortex of mice during development.

Presenilin (PS) 1 and PS2 are multi-pass transmembrane proteins involved in vital brain functions. Studies using transgenic or conditional knockout models show that PS1 is implicated in crucial brain developmental processes. Conversely, PS2 knockout mice do not exhibit any abnormality in the brain morphology, suggesting that PS2 may not be involved in brain development. However, there is no holistic information available for endogenous expression of PS during brain development. Therefore, we have examined the distribution and expression profile of PS1 and PS2 mRNA and protein in the cerebral cortex of prenatal, neonatal and postnatal mice. The results revealed that the distribution and expression profile of PS1 and PS2 mRNA varied significantly in the cerebral cortex during development. In prenatal stages, both PS1 and PS2 mRNA showed high expression at embryonic day (E) 12.5 and downregulation at E18.5. Postnatally, PS1 mRNA showed upregulation from postnatal day 0 (P0) to P45 and thereafter reduction at 20 weeks, but PS2 mRNA showed no significant alteration. However, they did not exhibit any significant regional variation except at E18.5, when PS2 showed reduction in temporal and medial temporal lobes as compared to frontal and parietal lobes. Furthermore, PS1 showed significant change in protein expression similar to its mRNA profile. However, PS2 protein expression did not correspond to its mRNA; it was highest at E12.5, downregulated up to P20 and then upregulated at P45 and 20 weeks. Taken together, our study demonstrates for the first time that the distribution and expression profile of PS2 is different from PS1 in the mouse cerebral cortex during development.

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.

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.

Amyloid precursor protein (APP) mRNA level is higher in the old mouse cerebral cortex and is regulated by sex steroids.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid beta peptides, which are produced by the proteolytic cleavage of amyloid precursor protein (APP). As the incidence of AD is higher in females and sex steroids are implicated in this disease, we have examined the effect of sex steroids (testosterone and 17ß-estradiol) on the expression of APP mRNA and protein in the cerebral cortex of adult and old mice of both sexes. Northern blot analysis detected APP mRNA as a single 3.5-kb band and its level is increased in old as compared to adult. Following gonadectomy, its level was upregulated in female mice but downregulated in male mice. Supplementation with testosterone or estradiol decreased its levels in female mice of both ages. Testosterone supplementation increased the mRNA levels in both adult and old male mice. Estrogen supplementation decreased its level in adult but increased in old male mice. Western blot analysis detected APP specific bands ranging from 95 to 125 kD. The level of 95 kDa band representing APP695 protein showed difference in levels with age or hormone treatment. These results provide evidence for increase in APP mRNA level in the cerebral cortex of old mice and its regulation by sex steroids during aging.