The Study Of The Relationship Between Hippocampal ERs Gene Expression And Brain Degeneration And Learning And Memory Function

Along with the raising of the living standard, the average life expectancy of women has been increased dramatically. The time of menopausal has remained essentially constant, however, more women will live a larger fraction of their lives in a postmenopausal hypoestrogenic state than ever before.Estrogen plays important roles in regulating physiologic processes in the body. The cessation of menstrual cyclicity and the resulting hypoestrogenicity broadly impact women's health. The morbidities of some disease of the central nervous system (CNS) have increased dramatically in postmenopausal women, including Alzheimer's disease. Some evidences have demonstrated that estrogen acts as a potent neuroprotective and neurotrophic factor in CNS. It influences memory and cognition function, decreases the risk and delays the onset of some neurological diseases, and attenuates the extent of cell death caused by brain injuries. Clearly, it is critical for us to understand the circumstances under which estrogen exerts neuroprotective and neurotrophic action and the cellular and molecular mechanisms underling it. The functions of learning and memory of hippocampus and the neurotrophic andneuroprotective profits of estrogen have been studied, so we hypothesized that estrogen deficiency has a close relationship with the decline of the learning and memory functions. It has been found that estrogen replacement therapy ameliorated the learning and memory-related functions on various aspects, such as modulating monoamine contents, improving the BDNF expression and the anti-apoptotic protein Bcl-2 expression, stabilizing MMP and potentiating neuronal synaptic plasticity.At present, the mechanism of learning and memory and the relationship between dynamic changes of ERs gene expression and learning and memory in CNS are not well understood yet. The objective of this study is to explore the relationship between estrogen receptors expression and learning and memory function by means of examining the mRNA of the two subtypes of estrogen receptors. Real-time quantitative reverse transcription -polymerase chain reaction and in situ hybridization histochemistry were employed to detect the expression of ERalpha and ERbeta. The distribution and dynamic changes of ERalpha and ERbeta in hippocampus and supaoptic nucleus were observed in the aging process of senescence-accelerated mouse (SAM). ERalpha and ERbeta gene expression were also observed in ovariectomized and orchiectomized mice and in mice who acquired learning and memory by shuttle box test.1. Dynamic changes of hippocampal ERs gene expression in the aging process of senescence-accelerated mouse (SAM)Senescence-accelerated mouse/prone8 (SAMP8), a substrain of SAM, is special for its early onset of learning and memory deterioration with typically pathological changes in brain aging. Senescence-accelerated mouse/resistance 1 (SAMRl), is another substrain of SAM, whose physiological characteristics matches those of normal mice. We used SAMP8 and SAMRl as models to descript the characters of aging. At present, the mechanism of learning and memory and the relationship between ERs gene expression and learning and memory are not well understood yet. In this study, the dynamic changes of hippocampal ERalpha and ERbetamRNA in the aging process of SAM were measured quantitatively.1.1 Dynamic changes of hippocampal ERalpha gene expressionThe results showed that in their own aging process, hippocampal ERalpha gene expressed constantly, no significant difference was observed in both SAMP8 and SAMR1.1.2 Dynamic changes of hippocampal ERbeta gene expressionThe results showed that in SAMP8, the expression level of hippocamal ERbeta gene was fairly high at the 3rd and 6th month, and began to drop at the 9th month. In SAMR1, the expression level is higher from 3 to 12 months than in 15 months. It is notable that the expression level is higher in female than in male in both SAMP8 and SAMR1.1.3 Dynamic changes of hippocampal IDE gene expressionThe deposition of amyloid beta-protein (A beta ) is an early and essential feature in the pathogenesis of Alzheimer's Disease. Degradation and clearance of intracellularly deposited A beta is critical for AD treatment. Insulin-degrading enzyme (IDE, insulysin) is a 110-kDa neutral metallopeptidase that can degrade a number of peptides including A beta. The gene encoding IDE locates closely to genes encoding late-onset Alzheimer's disease (LOAD), which make IDE a functional and positional candidate for this disorder. IDE exert a principal role in the degradation and clearance of naturally secreted A beta by neurons and microglia. IDE hydrolyzes A beta peptides into products that are neither neurotoxicity nor depositing plaque formation, which implicated in the removal of extracellular A beta. Comparison of IDE activity from AD brain cytosolic fractions and age-matched controls revealed a significant decrease in A beta degrading activity in the first group, supporting the hypothesis that a reduced IDE activity may contribute to A beta accumulation in the brain.Our result showed that the expression level of hippocampal IDE mRNA was fairly high at the 3rd and 6th month, and began to drop at the 9th month in female SAMP8, the tendency in male SAMP8 was similar to female SAMP8. It maintained highlevel at the 3rd to the 12th month, and then dropped at the 15th month. In female SAMR1, the tendency in male SAMR1 was similar to female SAMR1.1.4 The gender difference of ERalpha, ERbeta and IDE gene expression in the aging process of senescence-accelerated mouse(SAM)ERalpha: There were no gender differences of ERalpha gene expression in bothSAMP8andSAMRl.ERbeta: The expression of ERbeta gene decreased gradually with age in both maleand female SAMP8. But the levels of ERbeta gene expression in female SAMP8was much lower and decreased faster than that of male SAMP8.The tendencies of ERbeta gene expression of male and female SAMR1 weresimilar. But in month of 3,6,9,12, ERbeta gene expression in female was muchhigher than that of male in month 15, ERbeta expression of both genders came tothe same level.IDE: In both SAMP8 and SAMR1, there were no gender differences in IDE geneexpression.1.5. The substrain difference of ERalpha, ERbeta and IDE gene expression in SAM hippocampal.ERalpha: SAMP8 and SAMR1 showed ERalpha expression difference in neither the regularities in the aging process nor the expression levels in each individual month.ERbeta: ERbeta gene expression levels of the two substrains were almost equal in 3,6 months-old mice, while in 9,12,15 months-old mice, it was significantly lower in SAMP8 than in SAMR1. As to the whole aging process, in SAMP8, the quantity of ERbeta mRNA was fairly high at the 3rd and 6th month, and began to drop at the 9th month. In SAMR1, it maintained high level at the 3rd to the 12th month, and then dropped at the 15th month. In brief, the occurrence of significant decrease of ERbeta mRNA was earlier in SAMP8 than in SAMR1, which seems to be relatedto the different learning and memory function of each substrain. The above phenomena were obtained in both male and female mice.IDE: IDE gene expression levels of the two substrains were nearly equal in 3,6,9 months-old mice, while in 12,15 months-old mice, it was significantly lower in SAMP8 than in SAMR1. As to the whole aging process, in SAMP8, the quantity of IDE mRNA was fairly high at the 3rd and 6th month, and began to drop at the 9th month. In SAMR1, it maintained high level at the 3rd to the 12th month, and then dropped at the 15th month. In brief, the occurrence of significant decrease of IDE mRNA was earlier in SAMP8 than in SAMR1. The phenomena were obtained in both male and female mice.1.6 The expression difference between hippocampal ERalpha and ERbeta in the aging process of senescence-accelerated mouse(SAM)In the aging process, hippocampal ERalpha gene expressed constantly, no substrain difference and no gender difference were observed. However, ERbeta gene expression decreased gradually along with aging. With regard to the 3 to 9 months-old SAMP8 and 3 to 12 months-old SAMR1, the expression of ERbeta gene were significantly higher than that of ERalpha, in each individual month. The gap between the expression level of the genes become narrow as mice got older, and it even disappeared in the 15th month. These results suggest that ERalpha and ERbeta gene differ greatly in regulating the function of learning and memory in the aging process of SAM. ERbeta gene is more important than ERalpha gene on learning and memory.2. The effect of learning and memory behavior on hippocampal ERs gene expressionShuttle Box Test was employed to obtain mice whose conditional avoidance response (CAR) were 20%, 50% and 90% respectively. Serum levels of estrogen testosterone were measured and expressions of ERs gene were also analyzed.In the female mice, the E2 content in serum was raised along with the increase ofCAR. There is also a tendency of increase in serum T, but no marked differencewas observed. ERalpha mRNA was decreased during the memory retention whileERbeta mRNA was increased at the same time.In male mice, E2 content in serum were also raised, T content in serum reachedpeak when CAR was 50%, and dropped sharply when CAR was 90%. ERalphamRNA was decreased during the memory retention while ERbeta mRNA wasincreased at the same time.We also observed that the expression of ERbeta gene was much higher than that ofERalpha gene. The levels of ERalpha mRNA were equal in male and female mice.ERbeta mRNA in female mice was significantly higher than in male mice.3. The effects of 17 0 -estradiol and testosterone propionate on ERs gene expression in ovariectomized miceThe experimental result showed that serum E2 content of OVX (ovariectomized) group was markedly decreased compared with SHAM E2 content. Serum T content of OVX group remained unchanged but was significantly elevated after TP (testosterone propionate) replacement.Compared with SHAM group, ERalpha gene expression in OVX group was greatly increased while ERbeta gene expression in this group was greatly decreased. 17 ï¿¡ -E2 replacement significantly lowered the ERalpha gene expression and elevated ERbeta gene expression. The expression of ERalpha and ERbeta gene changed neglectablely after TP replacement. ERbeta gene expressed highly than ERalpha gene did in each individual group.4. The effects of 17 ï¿¡ -estradiol and testosterone propionate on ERs gene expression in orchidectomized miceThe experimental result showed that serum E2 and T content of ORX (orchidectomized) group were markedly decreased compared with SHAM. And they were significantly elevated after 17 13-E2 or TP(testosterone propionate)replacement.Compared with SHAM group, ERalpha gene expression in ORX group was greatly increased while ERbeta gene expression in this group was greatly decreased. 17 3 -E2 or TP replacement significantly lowered the ERalpha gene expression and elevated ERbeta gene expression. ERbeta gene expressed highly than ERalpha gene did in each individual group.5. Dynamic changes of ERs gene expression of supraoptic nucleus in SAMThe experimental result showed that in SAMP8 and SAMR1, ERalpha gene expressed constantly in different month (3,6,9,12,15 month), no significant differences were observed. However, as to the whole aging process, in SAMP8, the quantity of ERbeta mRNA increased from the 3rd month, peaked at the 6th month, and then began to drop gradually till the 15th month. In SAMR1, it maintained high level at the 3rd to the 12th month, and then dropped greatly at the 15th month. ERbeta gene expression level was lower at the 12th and 15th month in SAMP8 than in SAMR1. ERbeta gene expression decreased gradually while ERalpha gene expressed constantly during the cause of aging, and in each individual month, ERbeta mRNA was much more than ERalpha mRNA in both SAMP8 and SAMR1. The above results indicated that ERbeta gene expression play a more important role in regulating the function of supraoptic nucleus in aging process.6. ConclusionThe experimental results suggest that the progressive decline of the learning and memory related function is closely related to the dynamic changes of central ERs gene and IDE gene expression. In the aging process of SAMP8 and SAMR1, hippocampal ERalpha gene expressed constantly, no substrain differences and no gender differences were observed. However the expression levels of hippocampal ERbeta gene presented all a tendency of decrease, and the tendency appeared earlier in SAMP8 than in SAMR1. In the same sustrain, the tendency wasconsistent between female and male. As to the whole aging process, the expression level of ERbeta gene was higher in female than in male. Compared with ERbeta, the phenomena of IDE gene expression were similar, but no gender difference existed. Gene expression of ERs and IDE in the hippocampus and the supraoptic nucleus were consistent. There was also a close relationship between the retention of conditional avoidance response (CAR) and serum E2, T content and the quantity of ERs mRNA. With the increasing of memory retention, serum E2 and T content raised, and the expression levels of ERalpha gene decreased gradually, moreover, the expression levels of ERbeta gene increased gradually. In OVX and ORX mice, the serum E2 and T content decreased, the expression level of ERbeta gene also decreased, but the expression level of ERalpha gene increased markedly. After 17 0 -E2 and TP replacement, compared with OVX or ORX groups, the serum E2 and T content increased, the expression level of ERbeta gene also increased, but the expression levels of ERalpha gene decreased markedly.The results indicate that ERbeta gene expression in the central nervous system present a closer relationship to learning and memory function while ERalpha shows nearly no influence on it. Estrogen inhibits the expression of ERalpha and promotes that of ERbeta in central nervous system. Female mice are more subject to estrogen influence than male mice. The beta subtype of ERs exerts a more important role in the learning and memory acquirement mediated by ERs.