神经甾体激素硫化孕烯醇酮改善Aβ痴呆小鼠认知行为的分子机制研究

发布时间：2018-07-11 15:25

本文选题：β-淀粉肽(Aβ) + 神经再生　；参考：《南京医科大学》2012年博士论文

【摘要】：随着人口老龄化的出现,阿尔茨海默病(Alzheimer's disease, AD)的发病率呈明显上升趋势,预测到2050年将超过9000万。由于缺乏有效的预防、诊断和治疗措施,AD已成为死亡病因的第四位。 AD是以进行性认知功能减退为关键临床特征,以大量老年斑形成、神经元纤维缠结和胆碱能神经元缺失为病理学特征的神经系统退行性疾病。β淀粉肽(β-amyloid peptide, Aβ)是老年斑的主要成分。Aβ损害胆碱能神经系统被认为是AD早期认知障碍的病理机制之一。研究证明,α7烟碱型乙酰胆碱能受体(α7nicotinic acetylcholine receptor, α7nAChR)是Ap的靶蛋白。Ap与α7nAChR结合,不仅能损害α7nAChR的功能,同时也能增加Ap的积聚和沉积,增强Ap的神经毒作用。本实验室的前期研究报道,Aβ25-35侧脑室注射能损伤海马的α7nAChR,导致认知功能障碍。此外,Aβ还能通过破坏神经细胞内钙稳态,产生大量自由基,激活细胞凋亡信号通路等引起神经细胞的大量死亡成年哺乳动物的神经干细胞可以分化为神经细胞——成年神经发生。海马齿状回(dentate gyrus, DG)的新生神经细胞与成熟颗粒细胞具有相同的形态和功能特征,能与CA3区神经元和内嗅皮层的传入纤维建立突触联系,产生突触传递功能。海马DG的神经发生已被证实与空间认知行为密切相关,即增加新生神经细胞数量能提高学习记忆功能,而阻碍成年神经发生则造成记忆功能减退。我们的前期研究结果显示,Aβ能损伤新生神经细胞的突起生长,导致成熟的新生神经细胞数量明显减少。我们还报道了Aβ通过下调信号分子1mTOR(mammalian target of rapamycin),抑制新生神经细胞的突起生长。这些结果提示,Aβ损害神经再生可能是AD患者认知行为进行性减退的重要病理机制之一。神经甾体激素硫化孕烯醇酮(pregnenolone sulfate, PREGS)的脑内水平被认为与AD的发病率、老年斑的形成呈负性相关。AD患者脑内PREGS水平明显低于同龄非认知障碍人群。我们的前期研究结果发现,PREGS体外给药能改善Aβ大鼠的认知功能,但是有关其机制尚不清楚。大量的研究报道,PREGS是N-甲基-D-天冬氨酸受体(N-methyl-D-aspartic acid receptor, NMDA-R)的正性调节剂,γ-氨基丁酸A型受体(y-aminobutyric acid type A receptor, GABAAR)的负性调节剂。PREGS能够增强α7nAChR功能,促进海马神经元的谷氨酸释放。此外,PREGS还能激活sigma-1受体(sigma-1receptor, σ1R)。 σlR激动剂PRE084通过上调P13K(phosphatidylinositol-3-kinase)—Akt信号通路,能阻止Aβ下调mTOR—p70S6k(70kDa ribosomal protein S6kinase)信号分子的磷酸化水平,保护新生神经细胞的突起生长。早期的研究已报道,PREGS通过抑制GABAAR活性,能促进神经干细胞的增殖。此外,NMDA-R的功能活性也已经被证明与新生神经元的存活、成熟和神经回路的整合密切相关。我们的前期研究已证明了a7nAChR激动剂DMXB能剂量依赖地阻止Aβ损害a7nAChR功能,保护海马的突触传递功能和可塑性,改善Aβ痴呆小鼠的认知功能。本课题将重点研究PREGS对Aβ损害神经再生、Aβ诱导神经细胞凋亡的影响及其分子机制,以系统地阐明PREGS改善Aβ痴呆小鼠认知行为的机制,为PREGS能用于AD的预防和治疗提供理论依据。研究目的 1.明确PREGS对Aβ损害神经再生的作用及其调控机制； 2.明确PREGS对Aβ诱导神经细胞凋亡的作用及其分子机制。第一部分PREGS对Aβ损害神经再生的作用及其调控机制材料与方法 1.动物模型：8月龄雄性APPswe/PS1dE9转基因(APP/PS1)小鼠的基因型鉴定。部分机制研究采用雄性ICR小鼠(25-30g)。 2. PREGS处理-新生细胞标记： (1) APP/PS1小鼠：用5-溴脱氧尿嘧啶核苷(bromodeoxyuridine, BrdU)连续12天腹腔注射标记有丝分裂细胞。从BrdU注射前7天开始给予PREGS(20mg/kg/day)的皮下注射,连续34天。分别在BrdU末次注射后24小时(1天龄)或第28天(28天龄),进行BrdU免疫组织化学染色。 (2)ICR小鼠：BrdU间隔6小时连续3次注射。BrdU给药的当天记为BrdU的第0天。在BrdU给药后的第0-1天、5-6天、10-11天、15-16天或20-21天分别给予连续2天的PREGS(3nmol)侧脑室注射。在BrdU给药后的第22天(22天龄)进行BrdU免疫组织化学染色。 3.增殖细胞核抗原(proliferating cell nuclear antigen, Ki67)免疫染色：检测干细胞增殖。 4. DCX (Doublecortin)免疫染色：测量新生神经细胞的突起长度。 5. BrdU与神经特异性核蛋白(neuron specific protein, NeuN)或胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)双标记免疫荧光染色：检测神经前体细胞的分化和新生神经元的存活和成熟。 6.Aβ免疫染色：检测老年斑。 7.酶联免疫吸附实验(enzyme linked immunosorbent assay, ELISA):检测海马脑源性生长因子(brain derived neurotrophic factor, BDNF)。 8.场电位记录：ICR小鼠给予PREGS腹腔注射60min后取脑,制作海马脑片,检测海马DG的兴奋性突触后电位(excitatory post-synaptic potentiation, EPSP)和双脉冲易化(paired-pulse facilitation, PPF)。 9. Morris水迷宫：检测空间认知功能。结果 1.与同窝或同周龄的野生型对照组小鼠相比,8月龄APP/PS1小鼠的海马区域出现大量的老年斑,并表现水迷宫登台潜伏期的明显延长。 2.与对照组小鼠相比,APP/PS1小鼠海马DG的1天龄BrdU免疫阳性(BrdU+)细胞数量增加约30%,Ki67免疫阳性(Ki67+)细胞数量显著增加,提示Aβ刺激干细胞的增殖。PREGS处理不影响APP/PS1小鼠的神经干细胞过增殖。 3.与对照组小鼠相比,APP/PS1小鼠DCX免疫阳性(DCX+)细胞突起长度显著减小。PREGS处理能保护APP/PS1小鼠新生神经元的突起生长。 4.与对照组小鼠相比,APP/PS1小鼠28天龄BrdU+细胞数量减少约50%,BrdU和NeuN免疫双阳性(BrdU+/NeuN+)细胞数量显著减少,而BrdU和GFAP免疫双阳性(BrdU+/GFAP+)细胞数量没有改变,提示Aβ损害新生神经元的存活和成熟。PREGS处理能增加APP/PS1小鼠成熟新生神经元的数量。 5. PREGS能减少APP/PS1小鼠脑Ap的沉积。 6. PREGS能改善APP/PS1小鼠海马BDNF水平的降低。 7.在ICR小鼠,BrdU注射后的第10-16天PREGS处理能引起22天龄BrdU+细胞数量增加。PREGS能引起EPSP斜率的持续性增加和双脉冲比率(paired-pulse ratio, PPR)的降低,提示突触前神经递质的释放增加。a7nAChR拮抗剂、σ1R拮抗剂或NMDA-R拮抗剂的前处理都能抑制PREGS促进突触前神经递质释放和促新生神经元存活的作用,提示PREGS通过增强向新生神经元的兴奋性传入,减少非活性化新生神经元的死亡。 8. PREGS能改善APP/PS1小鼠的水迷宫登台潜伏期延长。结论 1. PREGS通过减少Aβ沉积和提高BDNF水平,能阻止Aβ损害新生神经元突起生长和存活。 2. PREGS增加向新生神经元的传入神经兴奋,减少非活性新生神经元的死亡,促进新生神经元的存活和成熟。 3. PREGS通过保护Aβ小鼠的神经再生,可以改善Aβ痴呆小鼠的认知行为。第二部分PREGS对Aβ诱导神经细胞凋亡的作用及其分子机制材料与方法 1.动物模型：Aβ25-35(9nmol)进行侧脑室注射制备Aβ痴呆小鼠模型。 2.药物处理：从Aβ给药后第二天开始连续7天腹腔注射PREGS(20mg/kg/day)。 3. Morris水迷宫：检测空间记忆功能。 4.组织细胞检查：海马CA1区神经细胞计数。 5. TUNEL染色：检查细胞凋亡 6. Western blot:分析ERK1/2、Akt磷酸化水平和caspase-3。结果 1.与对照组小鼠相比,Aβ25-35小鼠水迷宫登台潜伏期明显延长,海马CA1区神经细胞数量减少和TUNEL阳性细胞明显增加。 2. PREGS处理能改善Aβ25-35小鼠的登台潜伏期延长,减少海马CA1区神经细胞的凋亡 3.σ1R拮抗剂NE100、a7nAChR拮抗剂MLA能阻止PREGS的抗凋亡作用。 4.与对照组相比,Aβ25-35小鼠海马的ERK2和Akt磷酸化水平减少,caspase-3增加。PREGS处理能增加Aβ25-35小鼠的ERK2和Akt磷酸化水平,降低caspase-3。 5.σ1R拮抗剂NE100能阻断PREGS对ERK2、Akt和caspase-3的调节,而a7nAChR拮抗剂MLA只能阻断PREGS对Akt和caspase-3调节。 6.ERK抑制剂U0126.PI3K抑制剂LY294002能阻断PREGS对Aβ25-35小鼠的抗凋亡作用。 7.P13K抑制剂LY294002能阻止PREGS改善Aβ25-35痴呆小鼠的认知行为。结论 1.Aβ抑制细胞保护因子ERK2和抗凋亡因子Akt的活性,激活caspase-3促进海马神经元凋亡 2. PREGS通过激活σ1R介导的PI3k-Akt和ERK信号通路,同时启动a7nAChR介导PI3k-Akt的信号通路,阻止Ap的神经毒性,以改善Aβ25-35小鼠的认知行为。
[Abstract]:With the aging of the population, the incidence of Alzheimer's disease (AD) is on the rise, predicted by more than 90 million in 2050. Due to the lack of effective prevention, diagnosis and treatment, AD has become the fourth cause of death.
AD is a neurodegenerative disease which is characterized by progressive cognitive impairment, with a large number of senile plaques, neurofibrillary tangles and cholinergic neurons missing. Beta amyloid (beta -amyloid peptide, A beta) is the main component of the senile plaques, the.A beta damage to the cholinergic nervous system is considered to be early AD cognition. One of the pathological mechanisms of the obstacle has proved that alpha 7 nicotinic acetylcholinergic receptor (alpha 7nicotinic acetylcholine receptor, alpha 7nAChR) is the binding of Ap target protein.Ap to alpha 7nAChR, not only to damage the function of alpha 7nAChR, but also to increase the accumulation and deposition of Ap, and to enhance the neurotoxicity of Ap. The previous study in this laboratory, A beta 25-3 5 lateral ventricle injections can damage the alpha 7nAChR of the hippocampus, which leads to cognitive dysfunction. In addition, A beta can also cause a large number of free radicals by destroying the calcium homeostasis in the nerve cells, activating the apoptotic signaling pathway and so on, causing a large number of death of the nerve cells.
The neural stem cells of adult mammals can differentiate into neural cells - adult neurogenesis. The newborn neural cells of the dentate gyrus (DG) have the same morphological and functional characteristics as the mature granular cells, and can establish synaptic connections with the afferent fibers of the CA3 and the olfactory cortex and produce synaptic transmission. The neurogenesis of horse DG has been proved to be closely related to spatial cognitive behavior, that is, increasing the number of new nerve cells can improve the learning and memory function, while hindering the adult neurogenesis can cause memory impairment. Our previous study showed that A beta could damage the growth of the new neural cells and lead to the number of mature new nerve cells. We also reported that A beta, through the down regulation of the signal molecule 1mTOR (mammalian target of rapamycin), inhibits the protuberance growth of newborn nerve cells. These results suggest that A beta damage to the nerve may be one of the important pathological mechanisms of progressive degeneration of cognitive behavior in AD patients.
The brain levels of pregnenolone sulfate (PREGS) are considered to be associated with the incidence of AD, and the formation of the senile plaque is negatively correlated with the PREGS level in the brain of the.AD patients. Our previous study found that PREGS in vitro administration could improve the cognitive function of A beta rats. It is not clear about its mechanism. A large number of studies have reported that PREGS is a positive regulator of the N- methyl -D- aspartic acid receptor (N-methyl-D-aspartic acid receptor, NMDA-R), and the negative regulator of the A type receptor of gamma aminobutyric acid (Y-aminobutyric acid type A) can enhance the alpha function and promote the Valley of hippocampal neurons. In addition, PREGS also activates the sigma-1 receptor (sigma-1receptor, sigma 1R). The sigma lR agonist PRE084 can prevent the phosphorylation level of A beta by the P13K (phosphatidylinositol-3-kinase) - Akt signaling pathway, and protects the protuberance growth of newborn nerve cells. The study has reported that PREGS can promote the proliferation of neural stem cells by inhibiting GABAAR activity. In addition, the functional activity of NMDA-R has also been proved to be closely related to the survival of new neurons and the integration of maturation and neural circuits. Our previous studies have shown that the a7nAChR irritable agent DMXB can prevent A beta damage to a7nAChR dose in a dose dependent manner. To protect the synaptic transmission function and plasticity of the hippocampus and improve the cognitive function of A beta dementia mice. This topic will focus on the effect of PREGS on the regeneration of A beta injury and the effect of A beta induced neuronal apoptosis and its molecular mechanism, so as to systematically elucidate the mechanism of PREGS to improve the cognitive behavior of A beta dementia mice, and to provide PREGS for the prevention and treatment of AD. Provide a theoretical basis.
research objective
1. to clarify the role and regulation mechanism of PREGS on A beta damaging nerve regeneration.
2. to clarify the effect of PREGS on A beta induced neuronal apoptosis and its molecular mechanism.
Part one is the effect of PREGS on A beta damaging nerve regeneration and its regulatory mechanism.
Materials and methods
1. animal model: genotype identification of 8 month old male APPswe/PS1dE9 transgenic (APP/PS1) mice.
Male ICR mice (25-30g) were used in some mechanism studies.
2. PREGS treatment - new cell markers:
(1) APP/PS1 mice: 5- bromodeoxyuridine (bromodeoxyuridine, BrdU) was injected into the mitotic cells by intraperitoneal injection for 12 days. PREGS (20mg/kg/day) was injected subcutaneously from 7 days before BrdU injection, for 34 days for consecutive 24 hours (1 days of age) or twenty-eighth days (28 days of age) after the final injection of BrdU, respectively. Color.
(2) ICR mice: the day of 6 hours of BrdU interval of 3 consecutive injections of.BrdU was recorded as zeroth days of BrdU. The 0-1 day after BrdU, 5-6 days, 10-11 days, 15-16 days, and 20-21 days were given for 2 days of PREGS (3nmol) intraventricular injection respectively. BrdU immunohistochemical staining was performed on the day after BrdU Administration (22 days of age).
3. proliferating cell nuclear antigen (Ki67) immunostaining: detection of stem cell proliferation.
4. DCX (Doublecortin) immunostaining: the length of neurite protrusion was measured.
5. BrdU and neural specific nucleoprotein (neuron specific protein, NeuN) or glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP) double labeled immunofluorescence staining: to detect the differentiation of neural precursor cells and the survival and maturation of newborn neurons.
6.A beta immunostaining: detection of senile plaques.
7. enzyme linked immunosorbent assay (ELISA): detection of hippocampus brain derived growth factor (brain derived neurotrophic factor, BDNF).
8. field potentials recording: ICR mice were given PREGS after 60min intraperitoneal injection to take the brain, make hippocampus brain slices, detect the excitatory postsynaptic potential of the hippocampus DG (excitatory post-synaptic potentiation, EPSP) and double pulse susceptibility (paired-pulse facilitation, PPF).
9. Morris water maze: detection of spatial cognitive function.
Result
1. a large number of senile plaques appeared in the hippocampus of 8 month old APP/PS1 mice compared with the wild type control group with the same or the same age, and the latency of the water maze was obviously prolonged.
2. compared with the control group, the number of BrdU immunoreactive (BrdU+) cells in the hippocampal DG of APP/PS1 mice increased by about 30%, and the number of Ki67 immunoreactive (Ki67+) cells increased significantly, suggesting that the proliferation of A beta stimulated stem cells did not affect the proliferation of neural stem cells in APP/PS1 mice.
3. compared with the control group, the DCX immunoreactive (DCX+) cell protuberance length of the APP/PS1 mice was significantly reduced by the.PREGS treatment to protect the growth of the new neurons in the APP/PS1 mice.
4. compared with the control group, the number of BrdU+ cells in APP/PS1 mice at 28 days of age decreased by about 50%, the number of BrdU and NeuN double positive (BrdU+/NeuN+) cells decreased significantly, while the number of BrdU and GFAP immunologic double positive (BrdU+/GFAP+) cells did not change, suggesting that A beta damage to the survival of newborn neurons and mature.PREGS treatment could increase the maturity of APP/PS1 mice. The number of newborn neurons.
5. PREGS can reduce the deposition of Ap in the brain of APP/PS1 mice.
6. PREGS could improve the level of BDNF in hippocampus of APP/PS1 mice.
7. in ICR mice, PREGS treatment on the 10-16 day after injection can cause an increase in the number of BrdU+ cells at 22 days of age to cause a continuous increase in the EPSP slope and the decrease of the double pulse ratio (paired-pulse ratio, PPR). It suggests that the release of the transmitter of the synapse increases the.A7nAChR antagonist, and the pretreatment of the sigma 1R antagonist or the NMDA-R antagonist can be used. Inhibition of PREGS promotes the release of neurotransmitters and the survival of newborn neurons, suggesting that PREGS reduces the death of inactive newborn neurons by enhancing the excitatory afferent to newborn neurons.
8. PREGS could improve the latency of water maze in APP/PS1 mice.
conclusion
1. PREGS, by reducing A beta deposition and raising BDNF level, can prevent A beta from damaging the growth and survival of new neurons.
2. PREGS increased the afferent nerve stimulation to neonatal neurons, reduced the death of inactive neonatal neurons, and promoted the survival and maturation of neonatal neurons.
3. PREGS can improve the cognitive behavior of A beta dementia mice by protecting nerve regeneration from A beta mice.
The second part is the effect of PREGS on A beta induced neuronal apoptosis and its molecular mechanism.
Materials and methods
1. animal model: A beta 25-35 (9nmol) was used to prepare A beta dementia mouse model by lateral ventricle injection.
2. drug treatment: PREGS (20mg/kg/day) was administered intraperitoneally for 7 consecutive days on the second day after administration of A beta.
3. Morris water maze: testing space memory function.
4. histiocytic examination: nerve cell count in hippocampal CA1 area.
5. TUNEL staining: examination of cell apoptosis
6. Western blot: analysis of ERK1/2, Akt phosphorylation levels and caspase-3.
Result
1. compared with the control group, the incubation period of the A maze 25-35 mice was significantly prolonged, and the number of nerve cells in the CA1 area of hippocampus decreased and the TUNEL positive cells increased significantly.
2. PREGS treatment could prolong the incubation period of A beta 25-35 mice and reduce the apoptosis of CA1 cells in hippocampus.
The 3. Sigma 1R antagonist NE100 and a7nAChR antagonist MLA can prevent the anti apoptotic effect of PREGS.
4. compared with the control group, the phosphorylation level of ERK2 and Akt in the hippocampus of A beta 25-35 mice decreased, and the caspase-3 increase.PREGS treatment could increase the level of phosphorylation of ERK2 and Akt in A beta 25-35 mice and reduce caspase-3..
The 5. Sigma 1R antagonist NE100 can block the regulation of PREGS on ERK2, Akt and Caspase-3, while a7nAChR antagonist MLA can only block the regulation of PREGS on Akt and caspase-3.
6.ERK inhibitor U0126.PI3K inhibitor LY294002 can block the anti apoptotic effect of PREGS on A beta 25-35 mice.
7.P13K inhibitor LY294002 can prevent PREGS from improving the cognitive behavior of A beta 25-35 dementia mice.
conclusion
1.A beta inhibits the activity of cytoprotective factor ERK2 and anti apoptotic factor Akt, and activates caspase-3 to promote apoptosis of hippocampal neurons.
2. PREGS activates the PI3k-Akt and ERK signaling pathway mediated by sigma 1R, and activates PI3k-Akt signaling pathway mediated by a7nAChR to prevent the neurotoxicity of Ap in order to improve the cognitive behavior of A beta 25-35 mice.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16

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