| Alzheimer’s disease is a common neurodegenerative disease of the brain. Alzheimer’s disease is mainly characteristic with memory loss, cognitive impairment, and abnormal behavior. The hippocampus always is the first damaged area in the brain of patient with Alzheimer’s disease. The main pathology changes in the damaged brain are widespread senile plaques and neurofibrillary tangles. Aberrant splicing of amyloid precursor protein produced deposition of soluble β-amyloid in the brain, and Aβ caused the formation of senile plaques, the toxic effect of Aβ is considered directly related to the occurrence and development of AD. Hyperphosphorylation of Tau protein causes fiber degeneration and the formation of double helix neurofilament and neurofibrillary tangles, which causes the function losses of nerve. The report of International AD Federation in 2009 shows, the numbers of AD patients have reached 35000000 all around the world. There were more than 6000000 of AD patients in China. But the exact etiology and pathogenesis of AD is still unclear until now, and a break of the treatment to AD was needed.The distribution of sex hormone in the brain and its interaction with AD had got much attention in the treatment for AD. Studies showed that androgen can reduce the neurodegenerative brain diseases, and its protective mechanisms include: ⑴ lower the deposition of Aβ; ⑵ protect neurons through classic and fast non-classic androgen pathway to inhibiting apoptosis of neural cells. The downstream metabolites of androgen, which can combine with the special receptor complexes, then move into the cell, and connect nuclear receptors, combine with chromatin, affects the synthesis of RNA and DNA at last.The researches in age related loss of androgen points out that, the increased Aβ levels in brain was closely related to the pathogenesis of AD. The regulation mechanism of Androgen to Aβ is unclear, but it may play a role through three aspects, the effects dependent on androgen receptor; testosterone converse to estradiol through aromatization, and then affect AD through the estrogen pathway; Affect the hypothalamic pituitary gonadal axis. Using of androgen metabolite dihydrotestosterone, that can exclude the effects of estrogen pathway, is more conducive to androgen action mechanism research.The high expression of AR is mainly located in the areas that are responsible for the learning and memory in the brain. Among those areas, the hippocampus plays a crucial role in spatial memory. It is reported that androgen levels is closely related to cognitive function androgen, but how androgens to control the cognitive function is not very clear until now.SAM mice(senescence accelerated mouse, SAM) are a kind of inbred aging model with uniform genetic background and stable aging pathological features. According to the aging speed, SAM can be divided into two strains, SAMR and SAMP. SAMR strains are resistant to senescence, those physiological index and average survival period were similar to normal animal, and often be used as homologous SAMP normal control group. SAMP is a fast aging strain. Among the SAM strains, senescence-accelerated-prone mouse8(SAMP8) is characterized by an age-related spontaneous deterioration in learning and memory abilities. This deterioration is often preceded or accompanied by cortical and hippocampal changes in gene expression and other various pathological features that are similar to Alzheimer’s disease(AD). Numerous studies have demonstrated that SAMP8 is a good, naturally derived animal model for the investigation of fundamental AD mechanisms.Current research suggests that there is a transition state or stage in the growth of SAMP8 mice, which is similar to characteristic changes in mild cognitive impairment(MCI) stage of patients in human. MCI is currently described as a transitional condition with cognitive changes between normalaging and Alzheimer’s dementia. Our previous results demonstrate a fluctuating trend in dendritic spine density and Aβ deposits at approximately 5 months of age, along with signs of aging including of declining memory ability and storage capacity of spatial learning. AD like pathological changes also appeared in the related brain regions, this suggesting that the rat is going into the aging period, so we called this period as mild cognitive impairment, or the early stage of cognitive impairment. Our previous study also confirmed SAMP8 mice in 7 months have appeared the characteristic of AD like symptoms and pathological changes in some damaged lesions of brain, along with the decreased learning and memory ability. These signs of aging suggested that animals had entered a rapid aging period with cognitive disorder and dementia.There was a close relationship between synaptic plasticity and sex hormones. Studies have reported the density of synapses in the hippocampal in the adult female rat is sensitive to estradiol manipulation and fluctuates naturally as the levels of ovarian steroids. In adult female rats, in vivo estradiol level is associated with hippocampal pyramidal cell synapse density, quantity and density of dendritic spines. The density of dendritic spines of pyramidal neurons in the brain regions related to learning and memory, such as the hippocampus, is associated with estrogen level. So the regulation mechanism of androgen to synaptic plasticity is also been concerned. Similarly, the density of dendritic spines of pyramidal neurons in the hippocampus is also modulated in vivo by the depletion and replacement of androgens.The normal morphology and function of dendritic spines is the basis of synaptic growth, and is an important mechanism of learning and memory of brain. Numerous studies have demonstrated that the number of synapses decreased in the early cognitive impairment stage of AD, the specific synaptic degeneration related to memory also can be seen at the same time. Evidences show that synaptic degeneration will occur firstly in the dendritic spines. Brain development regulatory protein(Drebrin) is an F-actin-binding proteinlocalized to postsynaptic dendritic spines at excitatory synapses, where it plays a role in synaptic plasticity by regulating spine morphogenesis, organizing post-synaptic densities, and targeting receptors. Drebrin was shown to be critical for the postsynaptic targeting of the protein PSD-95, which is also involved in excitatory postsynaptic plasticity and reduced in the MCI hippocampus. Studies to animal models of AD displayed Aβ accumulation will damage the regulation of postsynaptic actin, and then reduce the expression of Drebrin in postsynaptic area, and this reduction is correlated with the severity of cognitive impairment. So Drebrin may play an important role in regulation to outgrowth of neurite.Postsynaptic densities-95(PSD-95) is a main component of the postsynaptic density protein, associated with a variety of synaptic function, such as ion channels, synaptic activity and intracellular signal transduction pathway. PSD-95 is a very important scaffold protein at the postsynaptic membrane involved in synaptic function change, will increase with the enhancement of synaptic transmission function.Cyclic AMP response element binding protein(CREB) is a constitutively expressed nuclear transcription factor that regulates the expression of genes involved in neuronal survival and function. CREB is essential for the formation and retention of memory in several species. CREB-mediated gene expression is increased in the hippocampus during LTP. Spatial learning deficits in rats are observed after intra-hippocampal infusion of CREB antisense oligos. CREB is also an important nuclear target that couples neurotrophin-mediated signaling to neuronal survival. CREB undergoes phosphorylation at serine 133 in response to multiple signaling pathways. The phosphorylated form of CREB binds to the co-activators, CREB binding protein(CBP) and p300 resulting in the facilitation of target gene expression. In AD post-mortem brain, there is a decrease in the levels of CREB-regulated BDNF. In cultured neurons, Aβ interferes with CREB activation by c AMP and BDNF. Our previous study suggested that the CREB is an intermediate or final adjustment signal molecule in the nervous system degenerative diseases,such as Alzheimer’s disease(AD). With the occurrence and development of Aβ the upregulation of CREB protein expression decreased, treatment with testosterone will down-regulate the deposition of Aβ and increase CREB protein expression as well. So the molecules associated with CREB signaling pathway were thought as the probably target of intervention to nervous system disease.Extracellular regulated protein kinase 1/2(ERK1/2) is closely related to the proliferation and differentiation of cell, also related to the transcription and expression of gene. The mitogen-activated protein kinase(MAPK) signaling pathways play pivotal roles in cellular processes such as proliferation, apoptosis, gene regulation, differentiation, and cell motility. The typical mammalian MAPK pathways ERK1/2, JNK, p38 MAPK, and ERK5 operate through a concatenation of three successive phosphorylation events mediated by a MAPK kinase, a MAPK kinase, and a MAPK. It is reported that ERK-MAPK pathway is associated with neuron protection of androgen. In microglial cells, MAPK pathways JNK and P38 plays a major role in phagocytosis of Aβ as an inflammatory response. There are some reports that excessive activation of ERK can also increase the deposition of Aβ. In the situation of peroxide injury, inhibit the inflammatory reaction of ERK and glial cells can reduce the aggregation of Aβ and halt the progress of AD. Recently, some research focused to cancer suggests the existence of ERK-CREB signaling loop. But there few reports were seen in other field, especially in nervous system. We thought that there is a close relationship between androgen and ERK in the central nervous system. Investigate regulations of DHT to the expression of ERK and CREB will help us to understand the mechanisms and principles of protective effect of DHT to hippocampus in SAMP8 mouse.Our previous studies found that testosterone and dihydrotestosterone can improve the spatial memory ability of the castrated mice and rats. Some scholars thought that identify and treat AD early before the stage of Aβ accumulation will improve the prognosis. In our study, we used DHT, anon-aromatizable androgen, to explore the protective mechanisms and neurological basis of DHT specifically. This study will observe the effects of castration on spatial memory ability in early adult SAMP8 male mice, and explore the protective effects of dihydrotestosterone on cognitive impairment and spatial memory ability in early SAMP8 male mice.To sum up, this research will observe the effects of physiological dose of DHT on hippocampal synaptic plasticity, especially its influence on early cognitive impairment in castrated male SAMP8 mice. At the same time, we will observe the expression of ERK1/2 and downstream protein CREB in prefrontal cortex and hippocampus. On the other hand, we will observe the ability of spatial learning and memory by Morris water maze test among different groups of mice to investigate the protection of androgen in MCI mice due to AD.Part one Effects of dihydrotestosterone to spatial learning memory on early stage of cognitive impairment in male MCI SAMP8 mice due to ADObjective: To explore whether dihydrotestosterone(DHT) could improve spatial learning and memory in castrated SAMP8 mice at MCI stage due to AD.Methods: Twenty-four male SAMP8 mice were divided into three groups(n=8): sham gonadally-intact and Vehicle SAMP8 group(P8 group), Castrated SAMP8 group(Castrated group), DHT treated Castrated SAMP8 group(DHT group). And eight male SAMR1 mice were used as control group(R1 group). Surgery : Surgeries were conducted using aseptic procedures. Under Chloral hydrate anesthesia(0.5ml/100g), sixteen P8 mice were bilaterally castrated and eight mice received sham-castrations. For castration, both tests were extracted through a small incision made at the posterior tip of the scrotum. Sham operations involved incisions into the skin and muscle layers of the scrotum without removing the testes. One week or more was allowed for recovery from surgery prior to any behavioral testing.The MWM test was administered to analyze the effect of DHT on spatiallearning and memory in mice.MWM test:Each animal received 4 trials per day for 5 consecutive days. For each trial, the time for the mice to find the hidden platform was recorded. On day 6, the platform was removed, and the mice were allowed to swim freely for 120 s as in the probe trial. The time spent in the target quadrant and the numbers of platform crossings were measured.Results:1 Acquisition trial performance: Effects of DHT on escape latency performance in the MWM test by five groups of mice.All mice showed different decreases in escape latency over the five days of acquisition trials on the MWM.There were not significant differences among the five groups from the first day to the second day. During the third day to fifth day of acquisition trials, the escape latency of R1 group decreased significantly when compared with other three groups(P < 0.05). During the forth and fifth day, the escape latency of P8 group decreased significantly when compared with Castrated group(P < 0.05). The significant difference also be seen in DHT group when compared with the Castrated group in the last day(P < 0.05).2 Probe trails: Effects of DHT on the amount of time spent in the target quadrant and in the MWM test by five groups of mice.During the probe trails, there were significant differences among groups in the amount of time spent in the target quadrant(P < 0.05). The R1 group spent a significantly longer percentage of time in the target quadrant than the other groups(P < 0.05). And there were significant differences were seen when DHT group and P8 group compared with the Castrated group(P < 0.05).Compared to the R1 group, the times of crossing platform was significantly reduced in the other three groups. There is a significant difference in the P8 group, Castrated group, DHT group when compared with R1 group(P < 0.05). The times of crossing platform in DHT group and P8 group is higher than Castrated group, an there were significant differences(P < 0.05).Conclusions: 1 Castration impaired the spatial learning and memory in 5 months old SAMP8 mice at MCI stage due to AD. 2 DHT injection improved cognitive performance of Castrated mice at MCI stage due to AD.Part Two Effects of dihydrotestosterone on expression of synaptic plasticity related proteins SYN, PSD-95 and Drebrin in MCI SAMP8 male mice due to ADObjective: Observe the effects of dihydrotestosterone on synaptic plasticity in morphology and associated protein SYN, PSD-95, Drebrin protein. Investigate whether the early using of dihydrotestosterone in MCI SAMP8 male mice due to AD can affect hippocampal synaptic plasticity.Methods: Fifteen male SAMP8 mice were divided into three groups(n=5): sham gonadally-intact and Vehicle SAMP8 group(P8 group), Castrated SAMP8 group(Castrated group), DHT treated Castrated SAMP8 group(DHT group). And five male SAMR1 mice were used as control group(R1 group). We used Immunohistochemical staining, Quantitative real-time PCR analysis and Western blotting analysis to observe the expression of SYN, PSD-95 and Drebrin in the hippocampal.Results:1 Immunohistochemical stainingSYN detected by Immunohistochemical staining at all the four groups were, P8 group 0.887±0.089, Castrated group 0.553±0.034, DHT group 0.843±0.107 and R1 group 1.076±0.086. PSD-95 detected by Immunohistochemical staining at all the four groups were, P8 group 0.983±0.116, Castrated group 0.636±0.046, DHT group 0.935±0.073, R1 group 1.306±0.085. Drebrin detected by Immunohistochemical staining at all the four groups were, P8 group 0.673±0.058, Castrated group 0.489±0.044, DHT group 0.729±0.075, R1 group 0.987±0.112. The expressions of SYN, PSD-95 and Drebin in R1 group were higher than the other groups(P < 0.05). And there were significant differences were seen when DHT group and P8 group when compared with the Castrated group(P < 0.05).2 Quantitative real-time PCRThe expression of Syn detected by Quantitative real-time PCR at all the four groups were, P8 group 1.231±0.111, Castrated group 0.853±0.079, DHT group1.194±0.064 and R1 group 1.426±0.110. The expression of Psd95 detected by Quantitative real-time PCR at all the four groups were, P8 group 0.894±0.052, Castrated group 0.421±0.058, DHT group 0.861±0.027, R1 group 1.175±0.075. The expression of Drebin detected by Quantitative real-time PCR at all the four groups were, P8 group 0.443±0.041, Castrated group 0.329±0.019, DHT group 0.422±0.018, R1 group 0.528±0.043. The expressions of Syn, Psd95 and Drebin in R1 group were higher than the other groups(P < 0.05). And there were significant differences were seen when DHT group and P8 group when compared with the Castrated group(P < 0.05).3 Western blotting analysisThe expression of SYN protein detected by Western blotting analysis when normalized to GAPDH at all the four groups were, P8 group 0.891±0.126, Castrated group 0.453±0.074, DHT group 0.947±0.117, R1 group 1.133±0.117. The expression of PSD-95 protein detected by Western blotting analysis when normalized to GAPDH at all the four groups were, P8 group 0.205±0.025, Castrated group 0.109±0.007, DHT group 0.193±0.018, R1 group 0.259±0.018. The expression of Drebrin protein detected by Western blotting analysis when normalized to GAPDH at all the four groups were, P8 group 0.613±0.092, Castrated group 0.189±0.045, DHT group 0.632±0.058, R1 group 0.773±0.055. The expressions SYN, PSD-95 and Drebrin in R1 group were higher than the other groups(P < 0.05). And there were significant differences were seen when DHT group and P8 group when compared with the Castrated group(P < 0.05).Conclusion: 1 The decline of androgen due to castration decreased the synaptic plasticity and the expression of synapse associated protein SYN, PSD-95 and Drebrin. 2 Treated the Castrated mice with DHT can improve the synaptic plasticity and the expression of synapse associated protein SYN, PSD-95 and Drebrin.Part Three Effects of dihydrotestosterone on expression of proteins ERK1, ERK2 and CREB in hippocampus of MCI SAMP8 male mice due to ADObjective: Observe the effects of dihydrotestosterone on expressions of proteins ERK1, ERK2 and CREB. Investigate whether the early using of dihydrotestosterone in MCI SAMP8 male mice due to AD can affect signal transduction of ERK1/2 and CREB.Methods: Fifteen male SAMP8 mice were divided into three groups(n=5): sham gonadally-intact and Vehicle SAMP8 group(P8 group), Castrated SAMP8 group(Castrated group), DHT treated Castrated SAMP8 group(DHT group). And five male SAMR1 mice were used as control group(R1 group). We used Immunohistochemical staining, Quantitative real-time PCR analysis and Western blotting analysis to observe the expression of ERK1/2 and CREB in the hippocampal.Results:1 Immunohistochemical(IHC)stainingERK1 detected by IHC staining at all the four groups were, P8 group 0.240±0.046,Castrated group 0.214±0.025,DHT group 0.226±0.035,R1 group 0.254±0.028. ERK2 detected by IHC staining at all the four groups were, P8 group0.415±0.032, Castrated group 0.255±0.018, DHT group 0.388±0.025,R1 group 0.646±0.037. CREB detected by IHC staining at all the four groups were, P8 group 1.472±0.153,Castrated group 1.219±0.094,DHT group 1.535±0.132,R1 group 1.957±0.127.The expressions of ERK1, ERK2 and CREB in R1 group were higher than the other groups(P < 0.05). And there were significant differences of ERK2 and CREB were seen when DHT group and P8 group when compared with the Castrated group(P < 0.05).2 Quantitative real-time PCRThe expression of Erk1 detected by Quantitative real-time PCR at all the four groups were, P8 group 0.508±0.031,Castrated group 0.475±0.014, DHT group 0.491±0.052,R1 group 0.673±0.056. The expression of Erk2 detectedby Quantitative real-time PCR at all the four groups were, P8 group 0.944±0.068,Castrated group 0.533±0.026, DHT group 0.875±0.085,R1 group 1.173±0.109. The expression of Creb detected by Quantitative real-time PCR at all the four groups were, P8 group 1.246±0.060, Castrated group0.598±0.075, DHT group 1.177±0.100,R1 group 1.598±0.064.The expressions of Erk1, Erk2 and Creb in R1 group were higher than the other groups(P < 0.05). And there were significant differences of Erk2 and Creb were seen when DHT group and P8 group when compared with the Castrated group(P < 0.05).3 Western blotting analysisThe expression of ERK1 protein detected by Western blotting analysis when normalized to GAPDH at all the four groups were, P8 group 0.613±0.054,Castrated group 0.552±0.027,DHT group 0.587±0.034,R1 group 0.750±0.051. The expression of ERK2 protein detected by Western blotting analysis when normalized to GAPDH at all the four groups were, P8 group0.835±0.043,Castrated group 0.561±0.033,DHT group 0.805±0.065,R1 group 1.059±0.050.The expression of CREB protein detected by Western blotting analysis when normalized to GAPDH at all the four groups were, P8 group 1.113±0.152,Castrated group 0.589±0.075,DHT group 1.032±0.118,R1 group 1.376±0.107. The expression of p-CREB protein detected by Western blotting analysis when normalized to GAPDH at all the four groups were, P8 group 0.567±0.027,Castrated group 0.353±0.026,DHT group 0.540±0.037,R1 group 0.750±0.055.The expressions of ERK1, ERK2, CREB and p-CREB in R1 group were higher than the other groups(P < 0.05). And there were significant differences of ERK2, CREB and p-CREB were seen when DHT group and P8 group when compared with the Castrated group(P < 0.05).Conclusion: 1 The decline of androgen due to castration decreased the synaptic plasticity and the expression of synapse associated protein ERK2 andCREB. 2 Treated the Castrated mice with DHT can improve the synaptic plasticity and the expression of synapse associated protein ERK2 and CREB. |
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