| Alzheimer’s disease (AD), a neurodegenerative disease, distinguished by progressive memory/cognitive impairment and personality changes represents the most common type of dementia. AD is associated with accumulation of excess amounts of Beta-amyloid protein (Aβ) in the form of extracellular senile plaques, with disruption of neuronal interactions and nerve cell death. But our understanding of its neurotoxic mechanisms is limited. In AD model or cell culture, Aβ binding receptor is a key to its neurotoxicity.Selective neuronal loss, senile plaques and neurofibrillary tangles represent the most salient pathological changes of AD. Senile plaques are related with β-amyloid (Aβ) deposits. Accumulation of β-amyloid (Aβ) in brain plays an important role in the pathogenesis of Alzheimer’s disease. Growing evidence indicates that overproduction of Aβ and consequent appearance of amyloid plaques dramatically diminish the survival of newborn neurons.Adult neurogenesis in the hippocampus dentate gyrus (DG) exists in most mammalian species including humans occurs via proliferation of progenitor cells and migration, differentiation, and the resulting daughter cells develop into neurons. The newborn granule neurons integrate in the synaptic circuitry of the hippocampus CA3 field to contribute to the electrical activation and make a functional hippocampus. However, the newborn neurons have higher excitability leads to a facilitated production of long-term potentiation (LTP), which is different from those of mature granule cells. Administration of Aβ makes changes in LTP in the hippocampus and consequently impairs cognition and memory in rodents.There is evidence that depletion of ovarian hormones at menopause increases the risk of AD in postmenopausal women, and estrogen treatment improves verbal memory and decreases Aβ accumulation in 3xTg-AD. In addition, the activation of estrogen receptor (ER) increases hippocampal neurogenesis. Elevation or decrease of the E2 concentration alters rapidly the density and morphology of spines in CA1 neurons, which is observed in female rats with ovariectomy. In male meadow voles, the treatment with estradiol (E2) is able to enhance the survival of young neurons. At the synaptic level, elevated serum E2 increases the magnitude of LTP at Schaffer collateral-CAl synapses. Although increasing evidence shows that E2 has neuroprotective effect on the neuropathology and behavior in AD-mice, the mechanistic link has yet to be demonstrated.Studies showed that MAPKs signaling pathway was associated with abnormal deposition and neurotoxicity of Aβ, which may activated MAPKs pathway and triggered apoptosis. Akt activation can phosphorylate a series of substrate proteins and regulate apoptosis signal. E2 treatment can improve brain injury by activating the AKT pathway. Protective effects of Estradiol on dementia may involve ynaptic growth, brain derived neurotrophic factor, regulation of MAPK and Akt signaling pathway protein expressiond and reduction of Aβ. Antioxidation and anti inflammation may also be involved in the process of neurotoxicity. However, It is still necessary to further study how E2 regulates the signal pathway and Plays a protective role.Part 1:Protective effects of Estradiol on newborn neurons in hippocampus of AP25-35 miceObject:In this study, male mice inserted with AP25.35 into lateral ventricular were used as a model for AD pathology. E2 was given after the last BrdU-injection in vehicle treated control mice and AP25-35-mice. This study explored the beneficial effect of E2 in AD using Morris water maze task, histological examination and electrophysiological recording technique in AD model mice created by intracerebroventricular (i.c.v.) injection of Aβ25.35.1. To explore the effects of estradiol on cognition and memory function of AD model mice induced by Aβ25-35.2. To investigate the effect of Aβ25-35 on the survival of newborn neurons in DG, EPSPs and the induction of LTP.3. To investigate the effect of E2 on newborn neurons, EPSPs and the induction of LTP in Aβ25-35-mice.Methods:1. Preparetion of AD model. Mice were intracerebroventriculy injected with the "aged" Aβ25-35 (3 nmol/3 μl/mouse).2. All mice were decapitated under deep anesthesia with ethyl ether. The brains were rapidly removed and coronal brain slices (400 μm) were cut using a vibrating microtome in ice-cold cutting solution.3. Long-term intervation with E2. Mice were intracerebroventriculy injected with the "aged" Aβ25-35 (3nmol/3μl/mouse), while the control mice with saline (3μl/mouse). Mice were subcutaneously (s.c.) injected with E2 on day 6-28 after the last BrdU-injection (10 μg/day). Contral groups were randomly divided into three groups according to the day 14 (n=8),21 day (n=8) and 28 day (n=14) after the last BrdU-injection; AD model groups were randomly divided into three groups according to the day 14 (n=8),21 day (n=8) and 28 day (n=14) after the last BrdU-injection; Contral groups treated with E2 (E2 treatment,10μg/day, n=8); AD model groups treated with E2 (E2 treatment,10μg/day, n= 14).4. Short-term intervation with E2:Aβ25-35 was infused on day 25 after BrdU-injection, and then treated with E2 for three days. Contral groups (n=8); AD model groups (n=14); Contral groups treated with E2 (E2 treatment for three days, n=8); AD model groups treated with E2 (E2 treatment for three days, n=14).5. AD mice were used to detect the spatial cognitive ability and memory function by Morris water maze. Contral groups (n=8); AD model groups (n=8); Contral groups treated with E2 (E2 treatment,10μg/day, n=8); AD model groups treated with E2 (E2 treatment,1,5,10,15,20μg/day, n=40).6. BrdU immunostaining:Five-bromo-2-deoxyuridine (BrdU), the thymidine analog that incorporates into the DNA of dividing cells during S phase, was used for mitotic labeling. Briefly. Mice received thrice intraperitoneal (i.p.) injection of BrdU (50 mg/kg) with interval of 6 h.7. DCX immunostaining:As a microtubule-associated protein, DCX is expressed specifically in newly generated neurons, reaching a peak during the 2nd week, we examined the effect of Aβ25-35 on the neurite growth of newborn neurons by DCX-staining.8. Field potential recording:EPSP was recorded from molecular layer of DG with a 5 MΩ resistance glass microelectrode. LTP was induced by conditioning stimuli (CS). If the EPSP slopes were larger 20% than baseline, LTP was determined.Results:1. E2 improved the spatial cognitive ability and learning memory function (P<0.01) in AD mice. Dose of lOug/day E2 showed a significant dose dependent relationship.2. In comparison with control mice, the numbers of 21-day-old and 28-day-old Bride+cells in Aβ25-35-mice were significantly decreased compared to vehicle treated control mice (P<0.01), while the number of 14-day-old BrdU+cells was not altered in Aβ25-35-mice (P>0.05).3. The density of DCX+dendrites per DCX+ cell in Aβ25-35-mice was obviously increased compared to vehicle treated control mice (P<0.01).4. Input/output (I/O) curve showed that the slope of EPSP in DG regions of Ap25-35-mice decreased compared to that of vehicle treated control mice (P<0.05). The high-frequency stimuli evoked a stable potentiation of EPSP slope in control DG, but failed to induce LTP in AP25-35-DG.5. The administration with E2 slightly increased the number of 28-day-old BrdU+ cells in control mice (P>0.05), whereas it significantly attenuated the loss of 28-day-old BrdU+cells in Aβ25-35-mice (P<0.01). E2-treatment reversed Aβ25-35-induced synaptic dysfunction (P<0.05), and rescued LTP-induction in Aβ25-35-DG6. Although short-term E2-treatment slightly increased the number of 28-day-old BrdU+cells in control mice and Aβ25-35-mice, there was no statistical difference between vehicle-and E2-treated control mice or Aβ25-35-mice (P>0.05). Short-term E2-treatment enhanced the basal transmission of perforant path-granular synapses (P<0.05), but failed to induce LTP in Aβ25-35-mice.Conclusion:1. E2 improved the spatial cognitive ability and learning memory function in AD mice.2. AP25-35 impairs survival and neurite growth of newborn neurons in DG.3. Aβ25-35 suppresses LTP-induction in DG.4. Long-term administration with E2 prevents Aβ25-35-impaired survival of newborn neurons and LTP-induction. But Short-term administration with E2 failed to rescue the survival of the newborn neurons accompanied by LTP-induction in DG.5. The effect of E2 in Aβ25-35-impaired LTP induction depends on its neuroprotection improvement.Part 2:Estradiol inhibits Aβ25-35-induced apoptosis in HT22 cells through regulation of MAPKs and AKT pathwaysObject:In this study, HT22 cells Rat hippocampal neurons treated with Aβ25-35 were used as a model for AD pathology.1. To investigate the effect and molecular mechanism of Aβ25-35-induced neurotoxicity in vitro.2. To investigate the effect and molecular mechanism E2 on AP25-35-induced neurotoxicity in vitro.Methods:1. Cell culture. Proliferation growth medium composed of 10% fetal bovine serum and 1%pairs of anti was added to HT22 cells at 37℃C in 5% CO2, which were used in the following experiments after the cells stability index increased.2. In vitro model. HT22 cells were inoculated into 96-well microplates in the form of 5000 cells/well and incubated at 37℃ in 5% CO2 for 24 hours. HT22 cells were randomly divided into four groups:controls, Aβ25-35, estradiol (E2) and E2+Ap25-35-HT22 cells in Aβ25-35 group were treated with Aβ25-35(5,10,20,40μM) for 24 h. HT22 cells in E2 group were treated with E2 (0.01,0.1, 1.0μM) for 24 h. HT22 cells in E2+AP25-35 group were pretreated with E2 (1.0μM) for one hour prior to treatment with Aβ25-35 (10μM) for 24.3. Cell viability. Cell viability was assessed using a MTT assay after the end of drug treatment.4. Cell morphology. The morphology of HT22 cells was observed by phase contrast microscope.5. TUNEL-DAPI double staining was used to detect apoptosis.6. Western blotting. The expression of MAPKs and AKT signaling pathway was detected by blotting Western.Results:1. MTT results revealed that Aβ25-35 showed dose-dependent inhibition of HT22 cell growth in vitro (P<0.01). E2 was no significant cytotoxicity within 24 hour(P>0.05). However, this inhibition was significantly diminished by pretreatment with E2(P<0.01).2. TUNEL-DAPI double staining results showed that Aβ25-35 could significantly induced HT22 cells apoptosis (P<0.01). However, HT22 cells apoptosis was significantly inhibited by pretreatment with E2 (P<0.01).3. Aβ25-35 could significantly affect the expression of MAPKs pathway and PI3K/AKT pathway. The intervention of AP25-35 showed activation of phosphorylation of JNK and p38 (P<0.01), inactivation of phosphorylation of ERK and Akt (P<0.01). However, E2 significantly inhibited AP25-35-induced activation of phosphorylation of JNK and p38 (P<0.01), inactivation of phosphorylation of ERK and Akt(P<0.01).Conclusion:1. Aβ25-35 induced HT22 cell apoptosis by affecting the expression of MAPKs pathway and PI3K/AKT pathway protein and showed obvious cytotoxicity in vitro.2. E2 significantly inhibited Aβ25-35-induced apoptosis by regulating the expression of MAPKs pathway and PI3K/AKT pathway protein and weakened the inhibitory effect of AP25-35 on HT22 cells. |
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