Gene delivery of Homer1c rescues spatial learning in a rodent model of cognitive aging

Homer1c has been shown to play a role in learning and memory. Overexpression of Homer1c in the hippocampus can improve memory in normal rats and can also rescue spatial learning deficits in Homer1 knockout mice. In a previous study, we found that Homer1c mRNA is upregulated after a spatial learning paradigm in aged rats that successfully learn the task, when compared to aged rats that are learning-impaired (AI). This study was designed to validate the role of Homer1c in successful cognitive aging. In this article, we report that gene delivery of Homer1c into the hippocampus of aged learning-impaired rats significantly improves individual performance on an object location memory task. The learning ability of these rats on the Morris Water Maze was also superior to that of AI control rats. In summary, using 2 independent spatial memory tasks, we demonstrate that Homer1c is sufficient to improve the spatial learning deficits in a rodent model of cognitive aging. These results point to Homer1c as a potential therapeutic target for improving age-related cognitive impairment.     

Preserved sigma1 (sigma1) receptor expression and behavioral efficacy in the aged C57BL/6 mouse

The sigma(1) (sigma(1)) receptor represents a unique intracellular neuronal protein modulating several neurotransmitter responses with relevant effects on cognitive functions. We examined here its expression and behavioral efficacy during aging. The sigma(1) receptor expression was examined in young (2 months old) and aged (24 months old) C57BL/6 mouse brain using comparative RT-PCR and immunohistochemistry. The promnesic effect of PRE-084, a selective sigma(1) agonist, was assessed using a water-maze procedure. The sigma(1) mRNA expression was not affected during aging in the olfactory bulb, hippocampus, hypothalamus, cortex or cerebellum. The sigma(1) immunolabeling was intense in the olfactory bulb, hippocampus, hypothalamus and midbrain of the young mouse and the distribution appeared unchanged in the aged. The subcellular localization was similar in aged and younger animals, the protein being present on nuclear, mitochondrial, endoplasmic reticular and plasmic membranes. At the behavioral level, aged C57BL/6 mice showed deficits in the invisible platform learning, but not when the platform was visible. Animals subjected to a transfer test under repeated treatment with saline or PRE-084 significantly learned the new platform location. This study shows that sigma(1) receptor expression is preserved in aged animals and demonstrates the efficacy of a selective sigma(1) agonist against age-related memory deficits. Targeting this unique receptor may offer an original drug strategy during aging.     

Prominent hippocampal CA3 gene expression profile in neurocognitive aging

Research in aging laboratory animals has characterized physiological and cellular alterations in medial temporal lobe structures, particularly the hippocampus, that are central to age-related memory deficits. The current study compares molecular alterations across hippocampal subregions in a rat model that closely mirrors individual differences in neurocognitive features of aging humans, including both impaired memory and preserved function. Using mRNA profiling of the CA1, CA3 and dentate gyrus subregions, we have distinguished between genes and pathways related to chronological age and those associated with impaired or preserved cognitive outcomes in healthy aged Long-Evans rats. The CA3 profile exhibited the most prominent gene expression differences related to cognitive status and of the three subregions, best distinguished preserved from impaired function among the aged animals. Within this profile differential expression of synaptic plasticity and neurodegenerative disease-related genes suggests recruitment of adaptive mechanisms to maintain function and structural integrity in aged unimpaired rats that does not occur in aged impaired animals.     

Memantine reduces oxidative damage and enhances long-term recognition memory in aged rats

Many neurodegenerative diseases, including Alzheimer's (AD), Parkinson's (PD) and Huntington's diseases (HD), are caused by different mechanisms but may share a common pathway to neuronal injury as a result of the overstimulation of glutamate receptors. It has been suggested that this pathway can be involved in generation of cognitive deficits associated with normal aging. Previous studies performed in our laboratory have demonstrated that aged rats presented recognition memory deficits. The aim of the present study was to evaluate the effect of memantine, a low-affinity N-methyl-D-aspartate (NMDA) receptor antagonist, on age-induced recognition memory deficits. Additionally, parameters of oxidative damage in cerebral regions related to memory formation were evaluated. In order to do that, male Wistar rats (24 months old) received daily injections of saline solution or memantine (20 mg/kg i.p.) during 21 days. The animals were submitted to a novel object recognition task 1 week after the last injection. Memantine-treated rats showed normal recognition memory while the saline group showed long-term recognition memory deficits. The results show that memantine is able to reverse age-induced recognition memory deficits. We also demonstrated that memantine reduced the oxidative damage to proteins in cortex and hippocampus, two important brain regions involved in memory formation. Thus, the present findings suggest that, at least in part, age-induced cognitive deficits are related to oxidative damage promoted by NMDA receptor overactivation.     

Female CREBalphadelta- deficient mice show earlier age-related cognitive deficits than males

Age-related changes in the hippocampus increase vulnerability to impaired learning and memory. Our goal is to understand how a genetic vulnerability to cognitive impairment can be modified by aging and sex. Mice with a mutation in the cAMP response element binding (CREB) protein gene (CREB(alphadelta-) deficient mice) have a mild cognitive impairment and show test condition-dependent learning and memory deficits. We tested three ages of CREB(alphadelta-) deficient and wild-type (WT) mice in two Morris water maze (MWM) protocols: four trials per day with a 3-5 min inter-trial interval (ITI) (MWM4) and two trials per day with a 1 min ITI (MWM2). All CREB(alphadelta-) deficient mice performed well in the easier MWM4, except for the aged females that performed poorly. In the harder MWM2, young male and female and middle-aged male CREB(alphadelta-) deficient mice performed well, but aged male and all middle-aged and aged female CREB(alphadelta-) deficient mice were impaired. These results show that mice with a genetic vulnerability to impaired learning and memory exhibit increased vulnerability with age that is most apparent among females. Thus, a genetic predisposition to cognitive impairment may render females more vulnerable than males to such deficits with age.     

Senescence of chondrocytes in aging articular cartilage: GADD45β mediates p21 expression in association with C/EBPβ in senescence-accelerated mice

Growth arrest and DNA damage-inducible protein 45β (GADD45β) is expressed in normal and early osteoarthritic articular cartilage. We recently reported that GADD45β enhances CCAAT/enhancer binding protein β (C/EBPβ) activation in vitro. This study was undertaken in order to determine whether GADD45β is expressed with C/EBPβ in aging articular cartilage. We also investigated whether the synergistic expression of GADD45β and C/EBPβ may be involved in the mechanism of chondrocyte senescence. Senescence-accelerated mice (SAMP1) were used as a model of aging. GADD45β, C/EBPβ, and p21 were analyzed by immunohistochemistry. A luciferase reporter assay using ATDC5 cells was performed in order to examine p21 as a target gene of the GADD45β/C/EBPβ cascade. GADD45β exhibited increased expression in the aging articular cartilage of SAMP1 mice compared to that in control mice. The co-localization of GADD45β and C/EBPβ was confirmed by double immunostaining. The synergistic mechanisms of GADD45β and C/EBPβ on the gene regulation of p21, a molecule related to cellular senescence, were verified by a p21-luciferase reporter assay. Co-expression of C/EBPβ and p21 was confirmed. These observations suggest that the synergism between GADD45β and C/EBPβ may play an important role in cellular senescence in the aging articular cartilage.     

Neuronal and inducible nitric oxide synthase expressions and activities in the hippocampi and cortices of young adult,aged cognitively unimpaired,and impaired Long-Evans rats

Nitric oxide (NO) is a neurosignaling molecule that appears to play a significant role in learning and memory. This molecule has also been implicated in neurotoxicity due to its oxidative properties. Previous experiments from our laboratories have demonstrated elevated hippocampal and cortical neuronal nitric oxide synthase (NOS) mRNA levels in aged cognitively unimpaired and impaired Long-Evans rats, which could represent either increased neuronal NOS activity thereby leading to NO-mediated neurotoxicity, or a compensatory response by aged neurones to maintain physiological nitric oxide output. The current study measured the protein expression and activity levels of neuronal and inducible NOS in young adult (6 months) and aged (24-26 months) Long-Evans rats by means of western blotting and NOS activity assay. Aged animals were assigned as either cognitively unimpaired or aged with moderate cognitive impairments based on their performances in the Morris water maze behavioural task. Our results showed that hippocampal and cortical neuronal NOS expressions were significantly decreased in aged animals. These aged animals also exhibited increased hippocampal and cortical inducible NOS expressions. Between the two aged animal groups, cognitively impaired rats showed significantly lower hippocampal and cortical neuronal but higher hippocampal inducible NOS expressions. Young adult rats exhibited significantly higher hippocampal and cortical NOS activities than the aged animals. Aged animals with cognitive deficits showed significantly lower hippocampal NOS activity than cognitively unimpaired aged rats.Our data indicate that aging is associated with a decline in neuronal but elevated inducible NOS functioning in brain areas involved in learning and memory. These phenomena could contribute to the cognitive deficits observed in a sub-population of aged animals.     

CREB-binding protein levels in the rat hippocampus fail to predict chronological or cognitive aging

Normal cognitive aging is associated with deficits in memory processes dependent on the hippocampus, along with large-scale changes in the hippocampal expression of many genes. Histone acetylation can broadly influence gene expression and has been recently linked to learning and memory. We hypothesized that CREB-binding protein (CBP), a key histone acetyltransferase, may contribute to memory decline in normal aging. Here, we quantified CBP protein levels in the hippocampus of young, aged unimpaired, and aged impaired rats, classified on the basis of spatial memory capacity documented in the Morris water maze. First, CBP-immunofluorescence was quantified across the principal cell layers of the hippocampus using both low and high resolution laser scanning imaging approaches. Second, digital images of CBP immunostaining were analyzed by a multipurpose classifier algorithm with validated sensitivity across many types of input materials. Finally, CBP protein levels in the principal subfields of the hippocampus were quantified by quantitative Western blotting. CBP levels were equivalent as a function of age and cognitive status in all analyses. The sensitivity of the techniques used was substantial, sufficient to reveal differences across the principal cell fields of the hippocampus, and to correctly classify images from young and aged animals independent of CBP immunoreactivity. The results are discussed in the context of recent evidence suggesting that CBP decreases may be most relevant in conditions of aging that, unlike normal cognitive aging, involve significant neuron loss.     

Evidence for early cytotoxic aggregates in transgenic mice for human transthyretin Leu55Pro

Familial amyloidotic polyneuropathy (FAP) is a lethal autosomal dominant disorder characterized by systemic extracellular deposition of transthyretin (TTR) amyloid fibrils. Several groups have generated transgenic mice carrying human TTR Val30Met, the most common mutation in FAP. To study amyloidogenicity and cytotoxicity of different TTRs, we produced transgenic mice expressing human TTR Leu55Pro, one of the most aggressive FAP-related mutations. TTR deposition and presence of amyloid fibrils was investigated and compared to animals carrying the human TTR Val30Met gene kept under the same conditions. Deposition in a C57BL/6J background (TTR-Leu55Pro mice) and in a TTR-null background [TTR-Leu55Pro X TTR-knockout (KO) mice] was compared. Animals in a C57BL/6J background presented early (1 to 3 months) nonfibrillar TTR deposition but amyloid was absent. In a TTR-null background, presence of amyloid fibrils was detected starting at 4 to 8 months with a particular involvement of the gastrointestinal tract and skin. This data suggested that TTR homotetramers are more prone to fibril formation than TTR murine wild-type/human mutant heterotetramers. The nature of the deposited material was further investigated by immunocytochemistry. Both amorphous aggregates and small TTR fibrils were present in TTR-Leu55Pro X TTR-KO transgenics. We observed that these TTR deposits mimic the toxic effect of TTR deposits in FAP: animals with TTR deposition, present approximately twofold increased levels of nitrotyrosine in sites related to deposition. The TTR-Leu55Pro X TTR-KO mice here described are an important tool for the dual purpose of investigating factors involved in amyloidogenesis and in cytotoxicity of deposited TTR.     

Differential response of AMPA and NMDA glutamate receptors of Purkinje cells to aging of the chicken cerebellum

Aging can lead to cognitive, affective, learning, memory and motor deficits. Since the cerebellum and glutamatergic neurotransmission are involved in several of those functions, the present work aimed at studying the expression of AMPA and NMDA glutamate receptor subunits in the chick cerebellum during aging. Young (30 days old) and aged (ca. 4 years old) chickens (Gallus gallus) were used in order to evaluate the expression of GluR1, GluR2/3 and NR1 subunits. The cerebella of young and aged chickens were subjected to immunohistochemical and immunoblotting techniques. Numbers of GluR1, GluR2/3 and NR1-positive cells and optical density of the immunoblotting data were analyzed and submitted to statistical analysis using ANOVA and the Bonferroni post hoc test. Mean density of Purkinje cells stained for Giemsa, GluR1, GluR2/3 and NR1 in the cerebellum all showed a statistically significant decrease in aged animals when compared to the young animals (Giemsa, P < 0.01; GluRs and NR1, P < 0.03). However, the ratio of GluR1 and GluR2/3-positive Purkinje cells in relation the total number of Purkinje cells found in each time point decreased with aging (ca. 10%), whereas the ratio of NR1-positive cells increased (ca. 9%). The immunoblotting data showed a significant decrease of GluR1 (ca. 66%) and GluR2/3 (ca. 55%) protein expression with aging, but did not reveal changes for NR1. Our data suggest that aging can lead to differential changes in the pattern of expression of glutamate receptor subunits, which can underlie at least part of the cognitive and motor disorders found in aged animals.     

Allopregnanolone restores hippocampal-dependent learning and memory and neural progenitor survival in aging 3xTgAD and nonTg mice

We previously demonstrated that allopregnanolone (APα) increased proliferation of neural progenitor cells and reversed neurogenic and cognitive deficits prior to Alzheimer's disease (AD) pathology (Wang, J.M., Johnston, P.B., Ball, B.G., Brinton, R.D., 2005. The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell-cycle gene and protein expression. J. Neurosci. 25, 4706-4718; Wang, J.M., Singh, C., Liu, L., Irwin, R.W., Chen, S., Chung, E.J., Thompson, R.F., Brinton, R.D., 2010. Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer's disease. Proc. Natl. Acad. Sci. U. S. A. 107, 6498-6503). Herein, we determined efficacy of APα to restore neural progenitor cell survival and associative learning and memory subsequent to AD pathology in male 3xTgAD mice and their nontransgenic (nonTg) counterparts. APα significantly increased survival of bromodeoxyuridine positive (BrdU+) cells and hippocampal-dependent associative learning and memory in 3xTgAD mice in the presence of intraneuronal amyloid beta (Aβ) whereas APα was ineffective subsequent to development of extraneuronal Aβ plaques. Restoration of hippocampal-dependent associative learning was maximal by the first day and sustained throughout behavioral training. Learning and memory function in APα-treated 3xTgAD mice was 100% greater than vehicle-treated and comparable to maximal normal nonTg performance. In aged 15-month-old nonTg mice, APα significantly increased survival of bromodeoxyuridine-positive cells and hippocampal-dependent associative learning and memory. Results provide preclinical evidence that APα promoted survival of newly generated cells and restored cognitive performance in the preplaque phase of AD pathology and in late-stage normal aging.     

Reduced serine racemase expression contributes to age-related deficits in hippocampal cognitive function

To gain insight into the contribution of d-serine to impaired cognitive aging, we compared the metabolic pathway and content of the amino acid as well as d-serine-dependent synaptic transmission and plasticity in the hippocampus of young and old rats of the Wistar and Lou/C/Jall strains. Wistar rats display cognitive impairments with aging that are not found in the latter strain, which is therefore considered a model of healthy aging. Both mRNA and protein levels of serine racemase, the d-serine synthesizing enzyme, were decreased in the hippocampus but not in the cerebral cortex or cerebellum of aged Wistar rats, whereas the expression of d-amino acid oxidase, which degrades the amino acid, was not affected. Consequently, hippocampal levels of endogenous d-serine were significantly lower. In contrast, serine racemase expression and d-serine levels were not altered in the hippocampus of aged Lou/C/Jall rats. Ex vivo electrophysiological recordings in hippocampal slices showed a marked reduction in N-methyl-d-aspartate-receptor (NMDA-R)-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) in the CA1 area of aged Wistar rats, which were restored by exogenous d-serine. In contrast, NMDA-R activation, LTP induction and responses to d-serine were not altered in aged Lou/C/Jall rats.These results further strengthen the notion that the serine racemase-dependent pathway is a prime target of hippocampus-dependent cognitive deficits with aging. Understanding the processes that specifically affect serine racemase during aging could thus provide key insights into the treatment of memory deficits in the elderly.     

Deficits in the expression of the NR2B subunit in the hippocampus of aged Fisher 344 rats

The NMDA receptor (NMDAR) has been implicated in the induction of LTP at hippocampal synapses, and has been proposed to play a significant role in the involvement of the hippocampus with learning and memory. Aged rats are known to have deficits in LTP, learning and memory. We tested the hypothesis that aged rats might have deficits in expression of NMDAR subunits. Aged rats have significantly lower levels of NR2B mRNA and protein compared to young animals. This complements a recent report which showed improved learning and memory in mice which overexpress NR2B. No changes were seen in either the mRNA or the protein levels of the NMDAR subunit NR2A, nor in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate receptor (AMPAR) subunit GluR2. Our data support the hypothesis that age related alterations in the expression of the NMDAR NR2B subunit might underlie deficits in LTP and learning and memory in aged animals.     

Protein kinase C activity is associated with prefrontal cortical decline in aging

Aging is associated with deficiencies in the prefrontal cortex, including working memory impairment and compromised integrity of neuronal dendrites. Although protein kinase C (PKC) is implicated in structural plasticity, and overactivation of PKC results in working memory impairments in young animals, the role of PKC in prefrontal cortical impairments in the aged has not been examined. This study provides the first evidence that PKC activity is associated with prefrontal cortical dysfunction in aging. Pharmacological inhibition of PKC with chelerythrine rescued working memory impairments in aged rats and enhanced working memory in aged rhesus monkeys. Improvement correlated with age, with older monkeys demonstrating a greater degree of improvement following PKC inhibition. Furthermore, PKC activity within the prefrontal cortex was inversely correlated with the length of basal dendrites of prefrontal cortical neurons, as well as with working memory performance in aged rats. Together these findings indicate that PKC is dysregulated in aged animals and that PKC inhibitors may be useful in the treatment of cognitive deficits in the elderly.     

Rescue of cognitive aging by long-lasting environmental enrichment exposure initiated before median lifespan

The rescue of cognitive function through environmental enrichment (EE) during aging has been extensively documented. However, the age at onset, the duration of EE, and the cerebral mechanisms required to obtain the greatest benefits still remain to be determined. We have recently shown that EE applied for 3 mo after the median lifespan, i.e., the age at which 50% of the population is still alive (from 17 to 20 mo in NMRI mice), failed to prevent cognitive deficits in senescent animals. In the present study, mice were exposed to EE prior to the median lifespan, and for a longer total duration (from 14 to 20 mo), before the assessment of memory performance and the electrophysiological properties of hippocampal neuronal networks. The EE prevented memory deficits and reduced anxiety as the animal aged. Moreover, EE attenuated the age-related impairment of basal glutamatergic neurotransmission in CA1 hippocampal slices, and reversed the decrease in isolated N-methyl-D-Aspartate receptor (NMDA-R)-dependent synaptic potentials. Surprisingly, EE did not prevent the age-related alteration of theta-burst-induced long-term potentiation (LTP). This study therefore suggests that EE needs to be initiated before the age corresponding to the median lifespan and/or required long duration (> 3 mo) to have an effect on cognitive aging. In addition, we show that EE probably acts through theta-burst-independent mechanisms of synaptic plasticity.     

Hippocampal volume is preserved and fails to predict recognition memory impairment in aged rhesus monkeys (Macaca mulatta)

Aged monkeys exhibit deficits in memory mediated by the medial temporal lobe system, similar to the effects of normal aging in humans. The contribution of structural deterioration to age-associated memory loss was explored using magnetic resonance imaging techniques. We quantified hippocampal, cerebral and ventricular volumes in young (n = 6, 9-12 years) and aged (n = 6, 24-29 years) rhesus monkeys. Eleven subjects were tested on a recognition memory task, delayed non-matching-to-sample (DNMS). Compared to young animals, aged monkeys exhibited robust learning deficits and significant memory impairments when challenged with longer retention intervals. Hippocampal volume was statistically equivalent across age groups, differing by less than 6%, and there was no correlation between this measure and DNMS performance. Variability in cerebral volume was greater in the aged compared to young monkeys and this parameter was marginally correlated with DNMS performance with a 10-min delay. These findings confirm and extend the conclusion of recent post-mortem histological analyses demonstrating that normal cognitive aging occurs independently of gross structural deterioration in the primate hippocampus.     

The effects of aging on N-methyl-d-aspartate receptor subunits in the synaptic membrane and relationships to long-term spatial memory

There are declines in the protein expression of the NR2B (mouse ε2) and NR1 (mouse ζ1) subunits of the N-methyl-d-aspartate (NMDA) receptor in the cerebral cortex and hippocampus during aging in C57BL/6 mice. This study was designed to determine if there is a greater effect of aging on subunit expression and a stronger relationship between long-term spatial memory and subunit expression within the synaptic membrane than in the cell as a whole. Male, C57BL/6JNIA mice (4, 11 and 26 months old) were tested for long-term spatial memory in the Morris water maze. Frontal cortex, including prefrontal regions, and hippocampus were homogenized and fractionated into light and synaptosomal membrane fractions. Western blots were used to analyze protein expression of NR2B and NR1 subunits of the NMDA receptor. Old mice performed significantly worse than other ages in the spatial task. In the frontal cortex, the protein levels of the NR2B subunit showed a greater decline with aging in the synaptic membrane fraction than in the whole homogenate, while in the hippocampus a similar age-related decline was observed in both fractions. There were no significant effects of aging on the expression of the NR1 subunit. Within the middle-aged mouse group, higher expression of both NR2B and NR1 subunits in the synaptic membrane of the hippocampus was associated with better memory. In the aged mice, however, higher expression of both subunits was associated with poorer memory. These results indicate that aging could be altering the localization of the NR2B subunit to the synaptic membrane within the frontal cortex. The correlational results suggest that NMDA receptor functions, receptor subunit composition, and/or the environment in which the receptor interacted in the hippocampus were not the same in the old animals as in younger mice and this may have contributed to memory declines during aging.     

Investigation of a functional quinine oxidoreductase (NQO2) polymorphism and cognitive decline

NAD(P)H dehydrogenase quinone 2 (NQO2) is a quinone reductase whose functions include the reduction of both oxidative stress during the redox cycle and neurotoxicity caused by the metabolism of catecholamines. We have investigated a functional non-synonymous exon 3 single nucleotide polymorphism (rs1143684) within the NQO2 gene for association with cognitive decline using a cohort of 722 community-dwelling older individuals aged 50 years and over. The volunteers had completed tests that measured fluid intelligence, processing speed, immediate/delayed verbal recall and semantic memory. We observed a nominal significant association between this polymorphism and the trajectory of delayed memory recall over time (p=0.029). No other associations were seen with the decline of other cognitive abilities.     

Aging impairs hippocampus-dependent long-term memory for object location in mice

The decline in cognitive function that accompanies normal aging has a negative impact on the quality of life of the elderly and their families. Studies in humans and rodents show that spatial navigation and other hippocampus-dependent functions are particularly vulnerable to the deleterious effects of aging. However, reduced motor activity and alterations in the stress response that accompany normal aging can hinder the ability to study certain cognitive behaviors in aged animals. In an attempt to circumvent these potential confounds, we used a hippocampus-dependent object-place recognition task to show that long-term spatial memory is impaired in aged mice. Aged animals performed similarly to young adult mice on an object recognition task that does not rely on hippocampal function.