LINGO-1参与介导睡眠剥夺对APP剪切的调节作用

发布时间：2018-03-23 21:56

本文选题：LINGO-1　切入点：BACE1　出处：《第二军医大学》2013年硕士论文

【摘要】：阿尔茨海默病（Alzheimer’s diseases，AD）是老年人常见的神经系统变性疾病，是痴呆最常见的病因。流行病学调查显示，44%的AD患者伴发有睡眠和（或）昼夜节律紊乱。现在也有越来越多的实验证明，睡眠障碍可能是阿尔茨海默病的重要诱因之一，kang等研究发现，脑组织液中Aβ浓度波动具有昼夜节律性，对APPswe/PS1dE9转基因小鼠慢性睡眠剥夺后，其脑组织中Aβ的沉积显著增加。目前观点认为这可能与睡眠障碍时神经元活性、氧化应激及褪黑素水平的改变相关，然而具体机制尚不明确。 LINGO-1(LRR and Ig domain-containing, Nogo Receptor interacting protein-1)是由15号染色体(15q24.3, GI:15029689)编码，特异性表达在中枢神经系统的跨膜糖蛋白，可负性调节神经元的存活、轴突再生及髓鞘化，因而与神经系统发育、神经系统损伤修复、帕金森病、特发性震颤及神经系统脱髓鞘病变密切相关。目前有观点研究认为，LINGO-1也参与调节了AD的病理过程，LINGO-1能够与APP相互结合，通过促进β-分泌酶的剪切作用调节APP的代谢，其机制仍未阐明，作者认为，LINGO-1可能促进了β分泌酶的活性或表达，也可能是抑制了α-分泌酶的作用。然而，LINGO-1的调控机制尚不明确，其表达增高见于PD患者、PD动物模型、中枢神经系统损伤模型及携带lingo1rs9652490等位基因的人群，神经元的损伤和死亡也会引起LINGO-1表达的增加，据此，Zhou等认为，LINGO-1的表达变化可能与氧化应激及基因突变相关。Trifunovski等则证实神经元活性增加可以促进LINGO-1表达。而睡眠/觉醒状态与神经元活性密切相关，有研究显示，睡眠剥夺可导致机体的默认模式网络功能持续激活。以上这些证据提示，睡眠/觉醒可能会通过影响神经元活性影响LINGO-1的表达。LINGO-1可能参与介导了睡眠剥夺对APP代谢的调节。本课题研究包括两个部分。第一部分我们通过片段睡眠剥夺模型和Western blot技术研究了睡眠/觉醒对LINGO-1表达的影响，第二部分并利用RNA干扰及免疫共沉淀等方法研究LINGO-1是否参与介导睡眠剥对APP代谢的调节，并对其机制进行探索。本研究的主要结果如下： 1片段睡眠后ICR小鼠不同脑区LINGO-1表达上调，且与片段睡眠剥夺时间的长度相关。ICR小鼠片段睡眠剥夺三天后，分别取额叶、颞叶和下丘脑。采用Western blot方法检测，与对照组相比，上述脑区LINGO-1表达均有所上调。对ICR小鼠分别给予1、3、5、7天的睡眠剥夺，然后检测不同时长睡眠剥夺后LINGO-1表达的变化趋势，发现LINGO-1的表达与片段睡眠剥夺的时长呈正相关。 2LINGO-1的表达具有昼夜节律性。用Western blot分别检测0:00、4:00、8：00、12:00、16:00和20:00颞叶LINGO-1的表达，发现4:00LINGO-1的表达明显高于其他时间点。 3片段睡眠剥夺后小鼠颞叶BACE1表达的增加。ICR小鼠片段睡眠剥夺三天后，取颞叶脑组织用Western blot检测BACE1的表达,发现BACE1的表达明显增高。 4LINGO-1参与调控BACE1的表达。用lingo-1RNA干扰慢病毒转染神经元，收集细胞裂解液，用Western blot检测BACE1的表达，发现内源性干扰LINGO-1的表达后，BACE1的表达下调。 5LINGO-1与BACE1在体内不直接结合。取ICR小鼠脑组织裂液，用免疫共沉淀技术分别用anti-BACE1antibody和anti-LINGO-1antibody从正反向验证，发现二者在体内不存在直接的结合。 6片段睡眠剥夺可导致GSK3β磷酸化水平下调。ICR小鼠片段睡眠剥夺三天后，取颞叶脑组织用Western blot检测p-GSK3β和total-GSK3β的表达，发现与对照组相比，GSK3β磷酸化水平下调。 7LINGO-1参与调控GSK3β的磷酸化水平。用lingo-1RNA干扰慢病毒转染神经元，收集细胞裂解液，用Western blot检测的表达p-GSK3β和total-GSK3β的表达，，发现内源性干扰LINGO-1的表达后，GSK3β磷酸化水平升高。 8LINGO-1与GSK-3β在体内相互结合。取ICR小鼠脑组织裂解液，用免疫共沉淀技术证实，LINGO-1与GSK3β在体内相互结合。综上所述，本实验研究发现，LINGO-1的表达受睡眠/觉醒的调控，LINGO-1可通过调节GSK3β的磷酸化水平来间接调节BACE1的表达，从而参与介导睡眠剥夺对APP代谢的调节。
[Abstract]:Alzheimer's disease (Alzheimer 's diseases, AD) is a neurodegenerative disease common in the elderly, is the most common cause of dementia. Epidemiological investigation showed that 44% of patients with AD associated with sleep and circadian rhythm disorder (or). Now there are more and more experiments showed that sleep disorders may be one of the important causes of Alzheimer's disease disease, Kang found that fluctuations in the concentration of brain tissue in A beta liquid with circadian rhythm, chronic sleep deprivation on APPswe/PS1dE9 transgenic mice after deposition in the brain tissues of A beta increased significantly. At present the view that this may be a sleep disorder related neuronal activity, oxidative stress and melatonin levels change, but specific the mechanism is not clear.
LINGO-1 (LRR and Ig domain-containing Nogo Receptor interacting protein-1) is composed of 15 chromosomes (15q24.3, GI:15029689) encoding, the specific expression of transmembrane glycoprotein in the central nervous system, can negatively regulate neuronal survival, axonal regeneration and myelination, and nervous system development and nervous system damage, repair, Parkinson's disease. Essential tremor and nervous system demyelinating lesions are closely related. It is research that LINGO-1 is also involved in the regulation of the pathological process of AD, LINGO-1 can combine with APP, APP regulated by shear promotes beta secretase enzyme metabolism, its mechanism is still unclear, the author thinks that LINGO-1 may promote beta secretase the activity or expression may be inhibited alpha secretase.
However, the regulation mechanism of LINGO-1 is not clear, its expression is increased in patients with PD, the animal model of PD, model of central nervous system injury and lingo1rs9652490 allele groups, neuronal damage and death will lead to increased expression of LINGO-1 in Zhou think that the expression of LINGO-1 may be associated with oxidative stress and gene related to the mutation of.Trifunovski are confirmed that neuronal activity can increase the expression of LINGO-1. And the sleep / wake state and neuronal activity are closely related, studies have shown that sleep deprivation can lead to the body of the default mode network function. These evidences suggest that sustained activation of the above, the sleep / wake may affect.LINGO-1 expression by neuronal activity may be involved in the effect of LINGO-1 by sleep deprivation, regulate the metabolism of APP.
This thesis consists of two parts. The first part of our influence by fragment sleep deprivation model and Western blot technology to study the sleep / wake on the expression of LINGO-1, whether the second part is the research and use of RNA interference and immunoprecipitation methods involved in LINGO-1 mediated regulation of APP metabolism, sleep deprivation, and the mechanism was explored.
The main results of this study are as follows:
1 fragments of ICR mice after sleep in different brain regions in LINGO-1 expression, and fragments of sleep deprivation time correlated with the length of.ICR fragment of mice were taken after three days of sleep deprivation, frontal lobe, temporal lobe and hypothalamus. Using Western blot detection method, compared with the control group, the expression of LINGO-1 in the brain were increased in ICR mice were given. 1,3,5,7 days of sleep deprivation, the change trend of the expression of LINGO-1 and then detected long after sleep deprivation, found the expression of LINGO-1 and the length of fragments of sleep deprivation was positively correlated.
The expression of 2LINGO-1 was circadian. Western blot was used to detect 0:00,4:00,8:00,12:00,16:00 and LINGO-1 expression in temporal lobe at 20:00, respectively. It was found that the expression of 4:00LINGO-1 was significantly higher than that of other time points.
The expression of BACE1 in temporal lobe increased after 3 sleep deprivation. After three days of sleep deprivation in.ICR mice, the expression of BACE1 was detected by Western blot in temporal lobe brain tissue, and the expression of BACE1 increased significantly.
4LINGO-1 was involved in the regulation of BACE1 expression. Lingo-1RNA interference was used to transfect neurons, and the cell lysate was collected. The expression of BACE1 was detected by Western blot. The expression of BACE1 was down regulated after the expression of endogenous interfering LINGO-1.
5LINGO-1 and BACE1 did not directly bind to the body. ICR mice brain tissue fluid was extracted by positive co immunization with anti-BACE1antibody and anti-LINGO-1antibody from the positive and negative sides. It was found that there was no direct combination between the two groups in vivo.
6 fragment sleep deprivation can lead to the phosphorylation level of GSK3 beta. After three days of sleep deprivation in.ICR mice, Western blot was used to detect the expression of p-GSK3 beta and total-GSK3 beta in temporal lobe brain tissue. It was found that the level of GSK3 beta phosphorylation was down regulated compared with the control group.
7LINGO-1 is involved in regulating the phosphorylation level of GSK3 beta. Lingo-1RNA interference with lentivirus transfected neurons, collecting cell lysate, and using Western blot to detect the expression of p-GSK3 beta and total-GSK3 beta, we found that the expression of GSK3 beta increased after the interference of LINGO-1 expression.
8LINGO-1 and GSK-3 beta were combined in the body. The brain tissue lysate of ICR mice was taken. It was confirmed by the immunoprecipitation technique that LINGO-1 and GSK3 beta were combined with each other in the body.
To sum up, we found that the expression of LINGO-1 is regulated by sleep / wakefulness. LINGO-1 can indirectly regulate the expression of BACE1 by regulating the phosphorylation level of GSK3 beta, thereby participating in the regulation of APP metabolism by sleep deprivation.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

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