【作者】 张贵丽；

【导师】 冯加纯；

【作者基本信息】 吉林大学 ， 神经病学， 2011， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease, AD)是一种病因不明的神经系统退行性疾病。病理学特征为神经细胞外老年斑(senile plaque, SP)沉积、神经细胞内神经原纤维缠结(neurofibrillary tangles, NFTs)和神经细胞缺失。β淀粉样蛋白(β-amyloid protein,Aβ)是老年斑的主要成分,Aβ是各种原因诱发AD的中心环节,但是Aβ发挥神经毒性作用的具体机制,迄今为止尚不十分清楚,目前临床应用治疗AD的药物多有明显的副作用,而且疗效不够确切,还须进一步实验研究。长时程增强(long-term potentiation, LTP)是突触功能可塑性的主要形式,可作为研究学习记忆的细胞水平的模型,它反映了突触可塑性变化中的神经电生理活动方面的改变。在AD发病过程中,Aβ的神经毒性作用可以通过影响突触后结构成分特别是影响Postsynaptic density-95 (PSD-95)而导致LTP的抑制及突触功能障碍。因此,PSD-95蛋白的表达水平变化为大鼠海马的突触可塑性提供了另一个检测指标,与学习和记忆能力变化密切相关。近来Wnt信号通路在调节神经发育、突触发生、突触可塑性等方面的作用越来越受到重视,大量研究提示Wnt信号通路的活化能够增加PSD-95蛋白的表达,增强LTP及学习记忆能力。虽然大量研究证据都将Wnt信号通路与AD的发病机制联系在一起,但是过去的报道只是提供了一些提示性的结果,并不足以支持Wnt-5a配体对Aβ的神经毒性具有保护作用,且发挥作用机制尚不明确。推测Wnt-5a配体可能为拮抗Aβ的神经毒性作用开辟新的治疗药物,但是还需要进一步的实验证实其神经保护性作用,并深入研究其发挥作用的机制。本文中提供一些证据证实Wnt-5a配体能够拮抗Aβ导致突触可塑性损害,并且在此基础上还首次评价了Wnt-5a对Aβ引发的认知功能损害的保护作用,进一步的研究了Wnt信号通路在AD发病机制中的作用。更多还原

【Abstract】 Background:Alzheimer’s disease(AD) is the most prevalent neurodegenerative disease leading to dementia. It has been reported widely that Aβoligomers block hippocampal long-term potentiation (LTP) and impair rodent spatial memory. However, the mechanisms underlying Aβ-neurotoxicity are unknown so far. At present, it is necessary to find the new clinical therapeutical drugs for AD patients. Recently, it is found that Wnt signaling pathway plays a role in multiple modulation of synaptic differentiation and synaptic plasticity during neuronal development. A lot of studyies suggest that activation of Wnt signaling pathway is able to increase the expression of PSD-95 protein, upregulate LTP and enhance learning and memory funtion. Although various evidence has associated Wnt signaling with AD pathogenesis, previous experiments just provide some suggestive results which are not adequate for supporting a major protective role of Wnt-5a ligands in Aβneurotoxicity and the mechanism of protevtive effect is not definitive. Wnt-5a ligands may protect against Aβneurotoxicity and develop a new therapeutical strategy for the AD patient in the future, but it is nessesary to carry out the further experiment to investigate the neuroprotective role and the mechanism of the effect.Objective:The present study observes the roles of Wnt-5a ligands which play in the Aβ25-35-induced depression of synaptic plasticity, moreover, we examine whether the Wnt antagonist sFRP affects Wnt-5a ligands’effect on LTP; We further investigate the effect of Wnt-5a ligands on the cognitive functin of AD model rats; We discuss the postsynaptic mechanism through which Wnt signaling pathway play the protective effect on the AD pathogenesis.Methods:Making AD model with intracerebroventricular (i.c.v) injection of Aβ25-35, we examine the effects of intracerebroventricular (i.c.v.) injection of Wnt-5a ligands on the Aβ25-35-induced suppression of LTP in the rat hippocampal CA1 region in vivo with field potential recording technique and clarify whether Wnt antagonist sFRP affects the role of Wnt-5a. Morris water maze is used to examine the spatial learing and menory, annalyzing the change of PSD-95 proteins expression in the CA1 region of rat hippocampus by means of immunofluorescence and Western-Blot methods.Results:(1) LTP in vivo was successfully induced in the control group after applying high-frequency stimulus (HFS). I.c.v. injection of 10 nmol Aβ25-35 had no effect on baseline synaptic transmission, but it significantly depressed (P<0.01) the induction of LTP after HFS as compared to control group; Application of 10 pmol Wnt-5a alone enhanced baseline synaptic transmission without significant effect (P>0.05) on HFS-induced LTP compared with control; However, co-injection of Wnt-5a and Aβ25-35 reversed the depression of LTP by Aβ25-35 alone in a dose-dependent manner. After i.c.v. co-injection of 2 pmol Wnt-5a with 10nmol AP25-35, the LTP post HFS had no significance compared with Aβ25-35 alone. However, i.c.v. co-injection of 10 pmol (P<0.05) or 50 pmol (P<0.01) with 10nmol Aβ25-35 is sigficantly higher than Aβ25-35; Separate application or co-application of Wnt-5a and Aβ25-35 showed no significant effect (P>0.05) on paired pulse-evoked facilitation. Pretreatment with 100 pmol sFRP obviously attenuated the protection of Wnt-5a against Aβ25-35-induced LTP impairment (P<0.05).(2) Under morris water maze, compared with the control group, AD group significantly increased (P<0.01) the mean escape latency, path length and reduced the number (P<0.01) that the rats crossed over the position where the platform located, percentage of time (P<0.05) spent in the target quadrant area relative to the total time in the pool; Compared with the control group, there was no significant change (P>0.05) in the mean escape latency, path length, the percentage of time spent in the target quadrant area relative to the total time and the number of crossing over the target position in Wnt-5a alone group. Compared with the AD group, co-application of 2 pmol Wnt-5a and Aβ25-35 had no significance (P>0.05) in mean escape latency, path length, the percentage of time spent in the target quadrant area relative to the total time and the number of crossing over the target position. I.c.v. co-injection of 10 pmol or 50 pmol Wnt-5a with 10nmol Aβ25-35 significantly reduced (P<0.05) the mean escape latency, path length and increased (P<0.05) the percentage of time spent in the target quadrant area relative to the total time, the number of crossing over the target position. The improved level of the high dose is better than the low one (P<0.05).(3) According to immunofluorescence and Western blot analysis, PSD-95 protein expression in the CA1 region of the rat hippocampus was significantly decreased in Aβ25-35 group (P<0.01, compared with control group). I.c.v. co-injection of 10 pmol (P<0.05) or 50 pmol (P<0.01) Wnt-5a with 10nmol Aβ25-35 signifigant reversed decresed PSD-95 protein level triggerd by Aβ25-35. Moreover, the increased level of the high dose is more than the low one (P<0.05). On the other hand, there is no difference between Wnt-5a alone group and control group (P>0.05).Conclusion:(1) Application of Wnt-5a ligands activate the Wnt signaling pathway to occlude the Aβ25-35 induced LTP depression;(2) Wnt-5a ligands improve the impairment of spatial learning and memory of AD model rats with intracerebroventricular (i.c.v) injection of Aβ25-35;(3) It is clarified that Wnt-5a ligands play protective roles in the synaptic plasticity and cognitive function of AD through the postsynaptic mechanism especially the moducation of PSD-95 protein.更多还原