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.     

Effect of age and cognitive status on basal level AP-1 activity in rat hippocampus.

Activator protein-1 (AP-1) was examined at multiple levels (mRNA, DNA binding, composition) in hippocampus of young and aged rats that were behaviorally characterized for spatial memory. GFAP mRNA was measured as a gene product known to increase with aging and to be regulated by AP-1. The activity of Jun-amino terminal-kinase (JNK) was also assessed. Levels of c-jun and c-fos mRNAs were unchanged with aging or spatial learning ability. Abundance of GFAP mRNA was significantly increased in aged hippocampus but did not correlate with spatial learning. Total AP-1 binding activity was unaltered with age or cognitive ability. In hippocampus of young, aged unimpaired and aged impaired rats, AP-1 consists mainly of c-Jun, phosphorylated c-Jun (p-c-Jun), JunD, and smaller amounts of c-Fos. JNK is constitutively active in young and aged hippocampus. We conclude that the basal expression of c-fos and c-jun mRNA, overall AP-1 binding activity and AP-1 composition are not influenced by aging or cognitive ability.     

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.     

Malocclusion induces chronic stress

We examined the effect of occlusal disharmony in senescence-accelerated (SAMP8) mice on plasma corticosterone levels, spatial learning in the water maze, fos induction, hippocampal neuron number, expression of glucocorticoid receptors (GR) and glucocorticoid receptor messenger ribonucleic acid (GRmRNA) in hippocampus and inhibitor of glucocorticoid (metyrapone). Bite-raised aged mice had significantly greater plasma corticosterone levels than age-matched control mice as well as impaired spatial memory and decreased Fos induction and a number of neurons in hippocampus. GR and GRmRNA expressions were significantly decreased in aged bite-raised mice compared with age-matched control mice. Pretreatment with metyrapone inhibited not only the bite-raised induced increase in plasma corticosterone levels, but also the reduction in the number of hippocampal neurons and impaired spatial learning. These datas suggest that the bite-raised condition may enhance the aging process in hippocampus, thereby leading to impairment of spatial memory by stress.     

二氢麦角碱升高血管性痴呆小鼠海马cAMP和腺苷环化酶

目的观测血管性痴呆小鼠海马组织环磷酸腺苷(cAMP)和腺苷环化酶(AC)水平及二氢麦角碱对其的影响,探讨血管性痴呆发病的分子生物学机制。方法通过双侧颈总动脉线结、连续3次缺血-再灌注,制作血管性痴呆动物模型,并设立假手术组、二氢麦角碱组;术后29 d、30 d分别测试学习和记忆成绩;应用放射免疫法检测小鼠海马组织cAMP水平,应用原位杂交技术检测AC水平。结果与假手术组比较,模型组学习和记忆成绩均降低(P<0.05),且海马组织cAMP水平也降低(P<0.05),海马CA1区AC mRNA阳性神经元比例明显降低(P<0.05);而与模型组比较,二氢麦角碱组学习和记忆成绩均改善(P<0.05),且海马组织cAMP水平也升高(P<0.05),海马CA1区AC mRNA阳性神经元比例明显增加(P<0.05)。结论海马组织cAMP和AC水平降低可能参与了血管性痴呆的分子生物学发病机制;二氢麦角碱可以升高其cAMP和AC水平而改善临床症状。     

TrkB but not trkC receptors are necessary for postnatal maintenance of hippocampal spines

Dendritic spines are major sites of excitatory synaptic transmission and changes in their densities have been linked to alterations in learning and memory. The neurotrophins brain-derived neurotrophic factor and neurotrophin-3 and their receptors, trkB and trkC, are thought to be involved in learning, memory and long-term potentiation (LTP). LTP is known to induce trkB and trkC gene expression as well as spinogenesis in the hippocampus. In the aging hippocampus, declines in trkB and trkC mRNA levels may underlie, at least in part, impairments in spatial memory and reductions in spine densities. To determine the significance of trkB and trkC for the maintenance of dendritic spines, we have analyzed Golgi-impregnated hippocampi of adult and aged mice heterozygous for trkB, trkC, or both along with respective wildtype littermates. Deletion of one allele of trkB, but not trkC, significantly reduces spine densities of CA1 pyramidal neurons in both adult and aged mice, as compared to age-matched controls. This indicates that trkB, but not trkC, receptors are necessary for the maintenance of hippocampal spines during postnatal life.     

Comparison of hippocampal synaptosome proteins in young-adult and aged rats

The hippocampus is important in learning and memory functions but its ability to aid in these functions declines during aging. In this study, we examined hippocampal proteins whose expressions changed in the aging process. A comparison of synaptosome proteins of hippocampus prepared from young-adult (9-week-old) rats with those from aged (30-month-old) rats by two-dimensional fluorescence difference gel electrophoresis revealed 24 spots that were expressed differently among about 1000 spots detected in both young-adult and aged rat samples. Nineteen of these 24 spots were identified by peptide mass fingerprinting. These proteins included chaperone proteins and proteins related to the cytoskeleton, neurotransmission, signal transduction and energy supply. The cytoskeleton-related proteins included actin and T-complex 1, which is thought to play a role in actin folding. Actin was up-regulated but T-complex 1 was down-regulated in aged rat synapses. These results suggest that age-dependent changes of actin filament formation are related to neuronal dysfunction associated with aging.     

Correlation of increased hippocampal Sumo3 with spatial learning ability in old C57BL/6 mice

Age-related impairment of learning and memory is a common phenomenon in humans and animals, yet the underlying mechanism remains unclear. We hypothesize that a small ubiquitin-related modifier (Sumo) might correlate with age-related loss of learning and memory. To test this hypothesis, the present study evaluated age-related spatial learning and memory in C57BL/6 mice (25 aged 7 months and 21 aged 25 months) using a radial six-arm water maze (RAWM). After the behavioral test, the protein expression of Sumo3 was determined in different brain regions using Western blotting. The results showed that the 25-month-old mice had longer latency and a higher number of errors in both learning and memory phases in the RAWM task than the 7-month-old mice. Compared to the latter, the former's level of Sumo3 protein was significantly increased in the dorsal and ventral hippocampus. For the 25-month-old mice, the number of errors and the latency in the learning phase negatively correlated with the Sumo3 level in the dorsal hippocampus. These results suggest that increased Sumo3 in the hippocampus may be correlated with spatial learning ability in old C57BL/6 mice.     

Influence of diet restriction on NMDA receptor subunits and learning during aging.

This study was designed to determine if changes related to aging and diet in the mRNA expression of subunits of the NMDA receptor were associated with changes in binding to NMDA receptors and learning ability in C57Bl/6 mice. Three age groups (3, 15, and 26-27 months old) and 2 diet groups (ad libitum-fed and diet restricted) were used. The old ad libitum-fed mice had significantly poorer performance in a spatial reference memory task than all other groups. Diet restriction slightly spared glutamate binding to NMDA sites and improved zeta1, but not epsilon2, mRNA expression. Significant correlations were found between NMDA-displaceable [(3)H]glutamate binding and both learning ability and epsilon2 and epsilon1 mRNA density in several brain regions. Learning ability in the old mice also correlated with the ratios of mRNA expression for epsilon1 and epsilon2 and/or zeta1 subunits in the parietal cortex and CA1 region of the hippocampus. This suggests that it is the relationship between subunit expression levels that is important for maintaining memory functions in older animals.     

A novel missense mutant inactivates GTP cyclohydrolase I in dopa-responsive dystonia

To clarify the biological function of neurotrophin-3 (NT-3) at the postnatal stage, we created a line of transgenic mice overexpressing NT-3 under the control of the mouse nerve growth factor gene promoter. Transgenic mice showed high-level NT-3 expression in the hippocampus and several tissues. We performed behavioral tests in young-adult (7-months-old) and aged (25-months-old) mice. Although aged non-transgenic mice exhibited spatial learning impairments in the Morris water maze, overexpression of NT-3 protected against these age-dependent spatial learning impairments in mice.     

Long-term administration of green tea catechins prevents age-related spatial learning and memory decline in C57BL/6 J mice by regulating hippocampal cyclic amp-response element binding protein signaling cascade

Flavonoid-rich foods have been shown to be effective at reversing age-related deficits in learning and memory in both animals and humans. However, little investigation of the preventative effects of flavonoids on the naturally aged animals was reported. In our study, 14-month-old female C57BL/6 J mice were orally administered 0.025%, 0.05% and 0.1% green tea catechins (GTC, w/v) in drinking water for 6 months; we found that a supplementation with 0.05% or 0.1% GTC prevented age-related spatial learning and memory decline of mice in the Morris water maze. Better performance of GTC-treated mice was associated with increased levels of cAMP-response element binding protein (CREB) phosphorylation in the hippocampus. The expressions of brain-derived neurotrophic factor (BDNF) and Bcl-2, two target genes of CREB which can exhibit long-term regulatory roles in synaptic plasticity and synaptic structure, were also increased. We also found that long-term 0.05% or 0.1% GTC administration prevented age-related reductions of two representative post-synaptic density proteins PSD95 and Ca²⁺/calmodulin-dependent protein kinase II, suggesting that synaptic structural changes may be involved. These results demonstrated that long-term 0.05% or 0.1% green tea catechin administration may prevent age-related spatial learning and memory decline of female C57BL/6 J mice by regulating hippocampal CREB signaling cascade.     

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.     

Spatial memory training modifies the expression of brain-derived neurotrophic factor tyrosine kinase receptors in young and aged rats

Aging leads to alterations in the function of the hippocampus, a brain structure largely involved in learning processes. This study aimed at examining the basal levels and the impact of a learning-associated task on brain-derived neurotrophic factor (BDNF), on BDNF full-length catalytic receptor (TrkB.FL) and on the truncated forms (TrkB.T1 and TrkB.T2) receptor expression (mRNA and protein) in the hippocampus of young (2-month-old) and aged (24-month-old) Wistar rats. Spatial memory was evaluated using a water-maze procedure involving visible and invisible platform location learning. Aged rats showed higher latencies during the first two training days but rapidly exhibited learning performances similar to patterns observed with young rats. Real-time PCR measurements showed that aged rats had significantly higher levels of trkB.FL mRNAs than young rats under basal conditions. In situ hybridization analysis indicated that the highest level of trkB.FL mRNA (mRNA encoding for TrkB.FL receptor) was noted in the dentate gyrus, and in the CA2 and CA3 hippocampal layers. In contrast, there was no marked difference in trkB.T1 signal in any hippocampal region. Training induced a significant reduction in trkB.FL mRNA levels solely in aged rats. In contrast, in young and aged rats, trkB.T2 mRNA levels were significantly increased after training. Measurements of proteins revealed that learning significantly increased TrkB.FL content in aged rats. Untrained aged rats presented higher levels of BDNF and brain-derived neurotrophic factor precursor (proBDNF) proteins than young rats. Training strongly increased precursor BDNF metabolism in young and aged rats, resulting in increased levels of proBDNF in the two groups but in old rats the mature BDNF level did not change. This study shows that Wistar rats present age-related differences in the levels of BDNF and TrkB isoforms and that spatial learning differentially modifies some of these parameters in the hippocampus.     

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.     

Estrogen replacement improves spatial reference memory and increases hippocampal synaptophysin in aged female mice

Estrogen deficiency during menopause is often associated with memory dysfunction. However, inconsistencies regarding the ability of estrogen to improve memory in menopausal women highlight the need to evaluate, in a controlled animal model, the potential for estrogen to alleviate age-related mnemonic decline. The current study tested whether estrogen could ameliorate spatial reference memory decline in aged female mice. At the conclusion of testing, levels of the presynaptic protein synaptophysin, and activities of the synthetic enzymes for acetylcholine and GABA, were measured in the hippocampus and neocortex. Aged (27-28-month-old) female C57BL/6 mice were given daily subcutaneous injections of 1 microg or 5 microg of beta-estradiol-3-benzoate dissolved in sesame oil. Control mice received daily injections of sesame oil or no injections. Estradiol treatment began 5 days prior to behavioral testing and continued throughout testing. Spatial and non-spatial memory were assessed in the Morris water maze. The 5 microg dose of estradiol significantly improved spatial learning and memory in aged females. The performance of 5 microg females improved significantly more rapidly than that of control females; estradiol-treated females performed at asymptotic levels by session 2. Furthermore, 5 microg females exhibited a more robust spatial bias than controls during probe trials. In contrast, 1 microg of estradiol did not improve spatial task performance. Neither dose affected performance of the non-spatial task. In the hippocampus, synaptophysin was increased in 5 microg females relative to controls. Estrogen did not affect enzyme activities in either brain region.This study is the first to examine the effects of estrogen replacement on spatial reference memory and synaptophysin expression in aged post-estropausal female rodents. The results suggest that: (1) estrogen can profoundly improve spatial reference memory in aged females, and (2) this improvement may be related to increased hippocampal synaptic plasticity, but not modulation of the synthetic enzymes for acetylcholine and GABA.     

Changes in the expression of the NR2B subunit during aging in macaque monkeys

Humans, non-human primates and rodents show declines in spatial memory abilities with increased age. Some of these declines in mice are related to changes in the expression of the epsilon2 (epsilon2) (NR2B) subunit of the N-methyl-D-aspartate receptor. The purpose of this study was to determine whether primates show changes during aging in the mRNA expression of the NR2B subunit. In situ hybridization was performed on tissue sections from three different ages of Rhesus monkeys (Macaca mulatta; 6-8, 10-12, and 24-26 years). There was a significant decrease in the mRNA expression of the NR2B subunit overall in the prefrontal cortex and in the caudate nucleus between young and old monkeys. There were no significant changes in NR2B mRNA expression in the hippocampus or the parahippocampal gyrus. The results in the prefrontal cortex, caudate and hippocampus were similar to those seen previously in C57BL/6 mice during aging, which suggests that mice may be useful as a model for primates to further examine the age-related changes in the expression of the NR2B subunit of the NMDA receptor in several important regions of the brain.     

Alteration of CREB phosphorylation and spatial memory deficits in aged 129T2/Sv mice

Phosphorylation of cAMP-response element binding protein (CREB) is required for hippocampus-dependent long-term memory formation. The present study was designed to determine whether spatial memory deficits in aged mice were associated with alteration of hippocampal CREB phosphorylation. We examined the temporal pattern of CREB activation in 5–6 and 23–24-month-old 129T2/Sv mice trained on a spatial reference memory task in the water maze. Phosphorylated CREB (pCREB), total CREB (t-CREB) and c-Fos immunoreactivity (ir) were measured at four time points after the end of training. In young mice, pCREB-ir was significantly increased 15 and 60 min after training in the CA1 region and dentate gyrus. In aged mice sacrificed 15 min after training, pCREB-ir in these structures was reduced whereas t-CREB-ir remained unchanged compared to respective young-adults. An age-related reduction of c-Fos-ir also occurred selectively in hippocampal CA1 region. Since reduced pCREB-ir in CA1 from the 15 min-aged group strongly correlated with individual learning performance, we suggest that altered CREB phosphorylation in CA1 may account for spatial memory impairments during normal aging.