發(fā)布時(shí)間：2018-01-01 17:37

  本文關(guān)鍵詞：Dynamin1磷酸化介導(dǎo)的TrkB內(nèi)吞在阿爾茲海默癥中的保護(hù)作用及其機(jī)制研究　出處：《山東大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： GSK3β Dyn1 BDNF TrkB 阿茲海默癥 學(xué)習(xí)與記憶

【摘要】：一.研究目的和背景 腦源性神經(jīng)營養(yǎng)因子(BDNF)對(duì)神經(jīng)元的生長和功能起著非常重要的作用。BDNF與TrkB受體結(jié)合以后,會(huì)引起TrkB受體的二聚化,激活其酪氨酸激酶活性,進(jìn)而激活其下游的信號(hào)傳導(dǎo)通路,磷脂酶Cγ(PLCγ)、磷脂酰肌醇-3-激酶(P13K)和細(xì)胞外信號(hào)調(diào)節(jié)激酶(Erk),實(shí)現(xiàn)其生物學(xué)效應(yīng)。BDNF會(huì)誘使TrkB受體內(nèi)吞,TrkB受體的內(nèi)吞具有很重要的作用。雖然有報(bào)導(dǎo)BDNF誘導(dǎo)的TrkB內(nèi)吞是一種網(wǎng)格蛋白以及dynamin介導(dǎo)的內(nèi)吞(CME),但是具體的TrkB內(nèi)吞分子調(diào)控機(jī)制并不是很清楚。 糖原合成酶激酶30(GSK3β)在神經(jīng)元中是一種非常重要的激酶,它能夠影響神經(jīng)元的生長以及存活。近些年的研究表明,在阿爾海默癥(AD)中,GSK3β的活性異常高,這可能是導(dǎo)致AD的關(guān)鍵因素。在先前的研究中,GSK3β能夠通過磷酸化dynaminl (Dynl)的第774位絲氨酸來調(diào)控一種活性依賴的內(nèi)吞(activity-dependent bulk endocytosis)。所以,GSK3β能否通過磷酸化dynaminl來調(diào)控BDNF依賴的TrkB內(nèi)吞,以及其在AD疾病中起著什么樣的影響都具有非常大的研究價(jià)值。 二.實(shí)驗(yàn)方法 1.各種表達(dá)質(zhì)粒的構(gòu)建。 2.原代海馬神經(jīng)元的培養(yǎng)及其轉(zhuǎn)染。 3.免疫熒光定量檢測TrkB受體內(nèi)吞。 4.可裂解生物素法檢測TrkB受體內(nèi)吞。 5.TUNNEL檢測神經(jīng)元凋亡。 6.新奇事物認(rèn)知實(shí)驗(yàn)和水迷宮實(shí)驗(yàn)。 三.實(shí)驗(yàn)結(jié)果 1.GSK3β活性調(diào)控BDNF依賴的TrkB內(nèi)吞 通過可裂解生物素和免疫熒光定量兩種方法,我們發(fā)現(xiàn)過表達(dá)GSK3β的激活形式GSK3B S9A會(huì)導(dǎo)致TrkB內(nèi)吞減少,而過表達(dá)GSK3β的失活形式GSK3B R96A會(huì)促進(jìn)其內(nèi)吞。 2.GSK3β通過磷酸化Dynl的第774位的絲氨酸,實(shí)現(xiàn)對(duì)TrkB內(nèi)吞的調(diào)控。 通過小干擾RNA的方法干擾掉Dynl后,GSK3β不再調(diào)控BDNF依賴的TrkB內(nèi)吞,證明GSK3β調(diào)控TrkB內(nèi)吞是依賴Dynl的。通過查閱前人文獻(xiàn),發(fā)現(xiàn)GSK3β能夠磷酸化Dynl的第774位絲氨酸。我們過表達(dá)Dynl的第774位絲氨酸模擬磷酸化形式的突變體Dyn1S774D后發(fā)現(xiàn),TrkB內(nèi)吞受到了抑制,并且GSK3β活性不再調(diào)控BDNF依賴的TrkB內(nèi)吞。 3.TAT-Dyn1SpS對(duì)于阻斷GSK3β對(duì)Dynl的磷酸化作用 為了研究GSK3β調(diào)控TrkB內(nèi)吞的意義,我們采用一個(gè)多肽TAT-Dyn1SpS,特異性阻斷了GSK3β對(duì)Dynl的磷酸化作用,并檢測了其對(duì)TrkB內(nèi)吞的影響,以及BDNF下游信號(hào)通路的影響。我們發(fā)現(xiàn),阻斷GSK3β對(duì)Dynl的磷酸化可以促進(jìn)TrkB內(nèi)吞,并且使得下游的Akt信號(hào)通路增強(qiáng)。 4.A β通過GSK3β抑制TrkB內(nèi)吞 通過免疫熒光定量分析,我們發(fā)現(xiàn)Aβ能夠抑制TrkB內(nèi)吞,在Aβ刺激的同時(shí)加入GSK3β的抑制劑AR-A014418,或者TAT-Dyn1SpS,能夠使得抑制的TrkB內(nèi)吞得到部分地恢復(fù)。證明Aβ能夠通過GSK3β來抑制TrkB內(nèi)吞。 5.TAT-Dyn1SpS能夠恢復(fù)Aβ?lián)p傷的BDNF下游信號(hào) 通過Aβ和TAT-Dyn1SpS同時(shí)刺激神經(jīng)元,我們發(fā)現(xiàn)阻斷GSK3β對(duì)Dynl的磷酸化能夠使得Aβ造成的BDNF下游信號(hào)Akt的損失得到一定的恢復(fù)。 6.TAT-Dyn1SpS能夠增強(qiáng)BDNF對(duì)Aβ誘導(dǎo)的細(xì)胞凋亡的保護(hù)作用。 有文獻(xiàn)報(bào)導(dǎo)BDNF對(duì)Aβ誘導(dǎo)的細(xì)胞凋亡的保護(hù)作用,我們通過可裂解的Caspase-3的檢測和TUNEL分析,發(fā)現(xiàn)阻斷GSK3β對(duì)Dyn1的磷酸化能夠增強(qiáng)BDNF對(duì)Aβ誘導(dǎo)的細(xì)胞凋亡的保護(hù)作用。 7. TAT-Dyn1SpS可改善AD小鼠的學(xué)習(xí)記憶能力。 在行為學(xué)的測試中,向AD小鼠的海馬腦區(qū)注入TAT-Dyn1SpS與BDNF,通過新奇事物認(rèn)知測試和水迷宮測試,發(fā)現(xiàn)TAT-Dyn1SpS能夠增強(qiáng)BDNF對(duì)AD小鼠學(xué)習(xí)和記憶的影響。 四.結(jié)論 通過一系列的分子細(xì)胞學(xué)和行為學(xué)方法,我們證實(shí)了GSK3β能夠通過磷酸化Dynl的774位絲氨酸調(diào)控TrkB內(nèi)吞,并闡述了其生物學(xué)意義。同時(shí),我們發(fā)現(xiàn)在AD中,TrkB的內(nèi)吞受損。通過設(shè)計(jì)多肽干擾GSK3β對(duì)Dynl的作用,能夠增強(qiáng)BDNF對(duì)AD的保護(hù)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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R749.16

【共引文獻(xiàn)】

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