Dynamin1磷酸化介导的TrkB内吞在阿尔兹海默症中的保护作用及其机制研究

发布时间：2018-01-01 17:37

  本文关键词：Dynamin1磷酸化介导的TrkB内吞在阿尔兹海默症中的保护作用及其机制研究　出处：《山东大学》2014年硕士论文　论文类型：学位论文

  更多相关文章： GSK3β Dyn1 BDNF TrkB 阿兹海默症 学习与记忆

【摘要】：一.研究目的和背景 脑源性神经营养因子(BDNF)对神经元的生长和功能起着非常重要的作用。BDNF与TrkB受体结合以后,会引起TrkB受体的二聚化,激活其酪氨酸激酶活性,进而激活其下游的信号传导通路,磷脂酶Cγ(PLCγ)、磷脂酰肌醇-3-激酶(P13K)和细胞外信号调节激酶(Erk),实现其生物学效应。BDNF会诱使TrkB受体内吞,TrkB受体的内吞具有很重要的作用。虽然有报导BDNF诱导的TrkB内吞是一种网格蛋白以及dynamin介导的内吞(CME),但是具体的TrkB内吞分子调控机制并不是很清楚。 糖原合成酶激酶30(GSK3β)在神经元中是一种非常重要的激酶,它能够影响神经元的生长以及存活。近些年的研究表明,在阿尔海默症(AD)中,GSK3β的活性异常高,这可能是导致AD的关键因素。在先前的研究中,GSK3β能够通过磷酸化dynaminl (Dynl)的第774位丝氨酸来调控一种活性依赖的内吞(activity-dependent bulk endocytosis)。所以,GSK3β能否通过磷酸化dynaminl来调控BDNF依赖的TrkB内吞,以及其在AD疾病中起着什么样的影响都具有非常大的研究价值。 二.实验方法 1.各种表达质粒的构建。 2.原代海马神经元的培养及其转染。 3.免疫荧光定量检测TrkB受体内吞。 4.可裂解生物素法检测TrkB受体内吞。 5.TUNNEL检测神经元凋亡。 6.新奇事物认知实验和水迷宫实验。 三.实验结果 1.GSK3β活性调控BDNF依赖的TrkB内吞 通过可裂解生物素和免疫荧光定量两种方法,我们发现过表达GSK3β的激活形式GSK3B S9A会导致TrkB内吞减少,而过表达GSK3β的失活形式GSK3B R96A会促进其内吞。 2.GSK3β通过磷酸化Dynl的第774位的丝氨酸,实现对TrkB内吞的调控。 通过小干扰RNA的方法干扰掉Dynl后,GSK3β不再调控BDNF依赖的TrkB内吞,证明GSK3β调控TrkB内吞是依赖Dynl的。通过查阅前人文献,发现GSK3β能够磷酸化Dynl的第774位丝氨酸。我们过表达Dynl的第774位丝氨酸模拟磷酸化形式的突变体Dyn1S774D后发现,TrkB内吞受到了抑制,并且GSK3β活性不再调控BDNF依赖的TrkB内吞。 3.TAT-Dyn1SpS对于阻断GSK3β对Dynl的磷酸化作用 为了研究GSK3β调控TrkB内吞的意义,我们采用一个多肽TAT-Dyn1SpS,特异性阻断了GSK3β对Dynl的磷酸化作用,并检测了其对TrkB内吞的影响,以及BDNF下游信号通路的影响。我们发现,阻断GSK3β对Dynl的磷酸化可以促进TrkB内吞,并且使得下游的Akt信号通路增强。 4.A β通过GSK3β抑制TrkB内吞 通过免疫荧光定量分析,我们发现Aβ能够抑制TrkB内吞,在Aβ刺激的同时加入GSK3β的抑制剂AR-A014418,或者TAT-Dyn1SpS,能够使得抑制的TrkB内吞得到部分地恢复。证明Aβ能够通过GSK3β来抑制TrkB内吞。 5.TAT-Dyn1SpS能够恢复Aβ损伤的BDNF下游信号 通过Aβ和TAT-Dyn1SpS同时刺激神经元,我们发现阻断GSK3β对Dynl的磷酸化能够使得Aβ造成的BDNF下游信号Akt的损失得到一定的恢复。 6.TAT-Dyn1SpS能够增强BDNF对Aβ诱导的细胞凋亡的保护作用。 有文献报导BDNF对Aβ诱导的细胞凋亡的保护作用,我们通过可裂解的Caspase-3的检测和TUNEL分析,发现阻断GSK3β对Dyn1的磷酸化能够增强BDNF对Aβ诱导的细胞凋亡的保护作用。 7. TAT-Dyn1SpS可改善AD小鼠的学习记忆能力。 在行为学的测试中,向AD小鼠的海马脑区注入TAT-Dyn1SpS与BDNF,通过新奇事物认知测试和水迷宫测试,发现TAT-Dyn1SpS能够增强BDNF对AD小鼠学习和记忆的影响。 四.结论 通过一系列的分子细胞学和行为学方法,我们证实了GSK3β能够通过磷酸化Dynl的774位丝氨酸调控TrkB内吞,并阐述了其生物学意义。同时,我们发现在AD中,TrkB的内吞受损。通过设计多肽干扰GSK3β对Dynl的作用,能够增强BDNF对AD的保护作用。
[Abstract]:1. The purpose and background of the study
Brain derived neurotrophic factor (BDNF) on the growth and function of neurons plays a very important role in combination with.BDNF and TrkB receptor, TrkB receptor leads to dimerization, activation of its tyrosine kinase activity, signal transduction and activation of its downstream, phospholipase C gamma (PLC gamma), phosphatidylinositol kinase (-3- P13K) and extracellular signal regulated kinase (Erk), the biological effects of.BDNF could induce TrkB in vivo by swallowing, endocytosis of TrkB receptor plays a very important role. Although there are reports of BDNF induced TrkB endocytosis is a clathrin mediated endocytosis and dynamin (CME), but the specific molecular swallow the TrkB control mechanism is not very clear.
Glycogen synthase kinase 30 (GSK3) in neurons is an important kinase, it can affect the growth and survival of neurons. Recent studies show that, in Alzheimer's disease (AD), GSK3 beta activity is unusually high, which may be a key factor in AD. In the previous study, GSK3 beta can phosphorylate dynaminl (Dynl) 774th serine to regulate endocytic activity dependent (activity-dependent bulk endocytosis). So, whether through GSK3 beta phosphorylation of dynaminl regulates BDNF dependent endocytosis of TrkB, and the AD disease plays have great research value of what effect kind of.
Two. Experimental method
1. construction of various expression plasmids.
2. primary cultured hippocampal neurons and its transfection.
3. immunofluorescence quantitative detection of TrkB was endocytic in vivo.
4. lysate biotin assay was used to detect TrkB endocytosis.
5.TUNNEL was used to detect neuronal apoptosis.
6. novelty cognition experiment and water maze experiment.
Three. Experimental results
1.GSK3 beta activity regulates BDNF dependent TrkB endocytosis
Through the two methods of cleavage biotin and immunofluorescence quantification, we found that over expression of GSK3 GSK3B activation S9A TrkB resulted in decreased TrkB endocytosis, while overexpression of GSK3 beta inactivation form GSK3B R96A promoted endocytosis.
2.GSK3 beta regulates the endocytosis of TrkB by phosphorylated 774th - bit serine of Dynl.
Through the method of small interfering RNA interference Dynl off, GSK3 is no longer dependent regulation of BDNF beta TrkB endocytosis, GSK3 beta regulation of TrkB endocytosis is dependent on Dynl. By referring to the previous literature, found that GSK3 beta can phosphorylate Dynl at serine 774th. Our overexpression of Dynl serine 774th phosphorylation in the form of simulation the mutant Dyn1S774D showed that TrkB endocytosis was inhibited, and the GSK3 activity no longer regulate BDNF dependent TrkB endocytosis.
The effect of 3.TAT-Dyn1SpS on blocking the phosphorylation of GSK3 beta to Dynl
In order to study the regulation of GSK3 beta TrkB endocytosis significance, we use a specific blocking peptide TAT-Dyn1SpS, the phosphorylation of GSK3 beta on Dynl, and detected its effects on TrkB endocytosis, and BDNF signaling pathways. We found that blocking the phosphorylation of GSK3 beta on Dynl can promote TrkB endocytosis, and the downstream Akt signaling pathway.
4.A beta inhibits endocytosis through GSK3 beta in TrkB
Through immunofluorescence quantitative analysis, we found that A beta can inhibit TrkB endocytosis, and A beta stimulation at the same time, adding GSK3 beta inhibitor AR-A014418 or TAT-Dyn1SpS can make the inhibition of TrkB endocytosis partially recover. It is proved that A beta can inhibit TrkB endocytosis through GSK3 beta.
5.TAT-Dyn1SpS can restore the downstream BDNF signal of A beta damage
By stimulating neurons at the same time by A and TAT-Dyn1SpS, we found that blocking the phosphorylation of Dynl by GSK3 beta can restore the loss of Akt downstream of BDNF signal induced by A beta.
6.TAT-Dyn1SpS can enhance the protective effect of BDNF on A beta induced apoptosis.
The protective effect of BDNF on A beta induced apoptosis is reported. Through the detection of lysable Caspase-3 and TUNEL analysis, it is found that blocking GSK3 Dyn1 to phosphorylate Dyn1 can enhance BDNF's protective effect on apoptosis induced by A beta.
7. TAT-Dyn1SpS can improve the learning and memory ability of AD mice.
In behavioral tests, TAT-Dyn1SpS and BDNF were injected into the hippocampus of AD mice. By novelty cognition test and water maze test, it was found that TAT-Dyn1SpS could enhance the effect of BDNF on learning and memory in AD mice.
Four. Conclusion
Method by molecular cytology and a series of acts, we demonstrate that GSK3 can phosphorylate Dynl beta 774 serine TrkB regulation of endocytosis, and expounds its biological significance. At the same time, we found that in AD, endocytosis of TrkB damage. Through the design of the Dynl GSK3 beta peptide interference, can enhance protective effect of BDNF on AD.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R749.16

【共引文献】

相关期刊论文 前10条

1 肖玲;舒畅;唐记华;王晓萍;王高华;;不同时程的慢性不可预计温和应激对大鼠海马神经可塑性相关蛋白水平的影响[J];神经损伤与功能重建;2013年06期

2 冯娅妮;孙艳红;马虹;;丙泊酚预处理对SK-N-SH神经母细胞缺氧/复氧损伤的保护作用[J];广东医学;2013年22期

3 林志雄;颜海锋;林举达;邹晓波;;纤维蛋白溶解酶原激活物抑制物-1基因多态性与抑郁发作及冠心病的关系[J];广东医学;2013年23期

4 Wei Xu;Chengqi Xin;Qiang Lin;Feng Ding;Wei Gong;Yuanyuan Zhou;Jun Yu;Peng Cui;Songnian Hu;;Adolescent Mouse Takes on An Active Transcriptomic Expression During Postnatal Cerebral Development[J];Genomics,Proteomics & Bioinformatics;2014年03期

5 俞捷;杨雪峰;杨孟雪;罗娅;封校亚;杨雪松;许洁;;壬基酚致雄性仔鼠脑组织差异基因的表达[J];环境卫生学杂志;2014年04期

6 李静静;叶长林;;运动诱导的脑源性神经生长因子在能量平衡调节中的作用[J];当代体育科技;2014年24期

7 刘莹;郝跃文;赵海涛;宦怡;孙立军;;bFGF和BDNF缓释微粒联合应用对缺血心肌血管生成作用的研究[J];功能与分子医学影像学(电子版);2014年03期

8 Qiang Li;Dianxiu Wu;Rui Li;Xiaojuan Zhu;Shusen Cui;;Valproic acid protects neurons and promotes neuronal regeneration after brachial plexus avulsion[J];Neural Regeneration Research;2013年30期

9 范存刚;王栋梁;张庆俊;周景儒;;人脐血和外周血单个核细胞中神经营养因子表达水平研究[J];山东医药;2013年33期

10 包丽娟;刘宁;徐松;董静;;神经生长因子制剂治疗DPN疗效的Meta分析[J];山东医药;2013年42期

相关博士学位论文 前10条

1 李莲莲;感觉神经节对脑运动神经元轴突生长的影响及其分子机制[D];兰州大学;2013年

2 李莉;脑红蛋白对神经元突起生长的作用及其潜在机制[D];华中科技大学;2013年

3 周柳青;大鼠单侧迷路切除术后BDNF-TrkB信号在小脑前庭通路中变化的研究[D];华中科技大学;2013年

4 谭涛;低频rTMS增强Aβ模型大鼠学习记忆功能的研究[D];天津医科大学;2013年

5 张楠;Noggin和BDNF基因修饰的BMSCs、羟基红花黄色素A、重复经颅磁刺激对血管性痴呆大鼠作用机制的研究[D];天津医科大学;2011年

6 黄菊香;上下游反馈整合比较PTHLH在非肿瘤肝炎及肝硬化组织和肝癌的分子网络及分析[D];北京邮电大学;2013年

7 张雪咏;Math5对大鼠视网膜Müller细胞源性干细胞向神经节细胞转化的调控作用[D];中南大学;2013年

8 王兴华;氧化铈纳米粒子的制备及其对RGC-5细胞凋亡的调控[D];华中科技大学;2013年

9 黄琴;MRI引导聚焦超声联合载基因微泡靶向开放血脑屏障促进外源基因体内转染[D];重庆医科大学;2013年

10 Elkhalifa Mahjoub Elkhalifa Ahmed;植入羊膜上皮细胞的化学去细胞肌肉组织工程支架对大鼠脊髓损伤的修复机制[D];吉林大学;2013年

，

本文编号：1365537