【摘要】： 典型的神經(jīng)元有一個(gè)軸突和多個(gè)樹突。對于一個(gè)神經(jīng)元來說,樹突往往是接受信息的部位,軸突往往是輸出信息的部位,因此,神經(jīng)元的極性是保證信息在神經(jīng)系統(tǒng)內(nèi)部及神經(jīng)系統(tǒng)與其他系統(tǒng)之間有序流動(dòng)的物質(zhì)基礎(chǔ)。海馬神經(jīng)元處于生長狀態(tài)的軸突含有豐富的腦衰蛋白反應(yīng)調(diào)節(jié)蛋白2(CRMP-2),軸突生長錐主要以非磷酸化CRMP-2活性形式存在。過度表達(dá)CRMP-2可以導(dǎo)致多軸突形成,反之抑制CRMP-2不利于軸突的生長與形成[1]。在體外,CRMP-2與管蛋白異二聚體相互作用而促進(jìn)微管的聚集[2]。海馬神經(jīng)元GSK-3β可以磷酸化CRMP-2 Thr-514位點(diǎn)使其失活,而GSK-3β活性下調(diào)使得CRMP-2 Thr-514位點(diǎn)磷酸化水平下降,最終促進(jìn)了神經(jīng)元軸突的發(fā)生、發(fā)展和成熟[3]。蛋白質(zhì)磷酸化和去磷酸化受蛋白激酶和蛋白磷酸酯酶的雙重調(diào)節(jié),而蛋白磷酸酯酶-2A(PP2A)是腦最重要的絲氨酸/蘇氨酸蛋白磷酸酯酶[4,5]。我們最近的研究顯示:采用不同濃度PP2A的特異性抑制劑岡田酸(okadaic acid, OA)作用于培養(yǎng)12 h的胎鼠原代海馬神經(jīng)元,神經(jīng)元軸突的形成和神經(jīng)元極性的建立均受到了明顯的影響;本研究用10 nmol/L OA和PP2A激動(dòng)劑神經(jīng)鞘氨醇(D-erythro-Sphingosine)(10 nmol/L)處理培養(yǎng)12 h的原代海馬神經(jīng)元。結(jié)果顯示10 nmol/L OA組神經(jīng)元軸突生長受到明顯的抑制,軸突長度明顯短于DMSO對照組,且無軸突神經(jīng)元數(shù)量明顯增多而單軸突神經(jīng)元?jiǎng)t顯著減少(P0.001);PP2A激動(dòng)劑組不僅單軸突長度明顯長于對照組(P0.001),而且統(tǒng)計(jì)學(xué)結(jié)果顯示30%神經(jīng)元出現(xiàn)了多軸突(P0.001),同時(shí)單軸突神經(jīng)元減少(P0.01)。用免疫印跡檢測PP2Ac亞基及其甲基化水平,結(jié)果顯示:D-erythro-Sphingosine組PP2A甲基化水平升高,OA組PP2A甲基化水平降低,PP2Ac亞基水平無變化。當(dāng)神經(jīng)元培養(yǎng)48 h即維持階段分別應(yīng)用10 nmol/L OA和10 nmol/L的D-erythro-Sphingosine持續(xù)作用48 h。與DMSO處理組相比可見,10 nmol/L OA處理組神經(jīng)元無軸突長度縮短(P0.05)或極性缺失;D-erythro-Sphingosine處理組神經(jīng)元沒有出現(xiàn)明顯的多軸突形成(P0.05),單軸突長度變化無統(tǒng)計(jì)學(xué)意義。然后將神經(jīng)元培養(yǎng)24 h后共轉(zhuǎn)染RFP(or GFP)/pcDNA4.0、RFP(or GFP)/PP2A wild type(wt)和RFP(or GFP)/PP2Adn培養(yǎng)48 h,固定做免疫熒光可見轉(zhuǎn)染pcDNA4.0對照組神經(jīng)元極性已經(jīng)建立,大多數(shù)神經(jīng)元有一個(gè)軸突和多個(gè)樹突;PP2Awt組單軸突延長(P0.01)同時(shí)多軸突增多(P0.001);PP2Adn組多出現(xiàn)短小的突起(P0.001)。這些結(jié)果提示PP2Awt促進(jìn)了神經(jīng)元單軸突的生長和多軸突的形成而PP2Adn則嚴(yán)重阻礙了軸突的形成和神經(jīng)元極性的建立。上述結(jié)果說明激活PP2A可誘導(dǎo)海馬神經(jīng)元軸突的形成。為了進(jìn)一步了解所形成的多軸突是否具有功能,我們觀察了多軸突對FM-64的攝取與釋放。結(jié)果顯示:45 mM K+刺激1 min于共聚焦顯微鏡下觀察,PP2A激活形成的多軸突可以攝取FM4-64呈紅色;FM著色后再給予90 mM K+刺激可見FM著色減弱,提示激活PP2A形成的多軸突具有突觸囊泡攝取和釋放的重復(fù)循環(huán)功能。為了探討PP2A影響神經(jīng)元極性的可能機(jī)制,將神經(jīng)元培養(yǎng)24 h后共轉(zhuǎn)染RFP/pcDNA4.0和RFP/PP2Awt,免疫熒光檢測CRMP-2 Thr514位點(diǎn)磷酸化水平。結(jié)果發(fā)現(xiàn)激活PP2A可以明顯降低神經(jīng)元胞體和突起CRMP-2 Thr-514位點(diǎn)磷酸化水平。結(jié)論:PP2A活性上調(diào)可能通過去磷酸化CRMP-2,進(jìn)而導(dǎo)致神經(jīng)元單軸突的延長和多軸突的形成,所形成的軸突具備突觸囊泡攝取和釋放功能。
[Abstract]:A typical neuron has an axon and a number of dendrites. For a neuron, the dendrite is often the location of information, and the axon is often the part of the output information. Therefore, the polarity of the neuron is the material basis for ensuring the orderly flow of information within the nervous system and between the nervous system and the other systems. The axons in the growth state contain rich brain failure protein response regulation protein 2 (CRMP-2), and the axon growth cone is mainly in the form of non phosphorylated CRMP-2 activity. Overexpression of CRMP-2 can lead to the formation of multiple axons. On the contrary, the inhibition of CRMP-2 is not conducive to the growth of axon and the formation of [1]. in vitro, and the interaction of CRMP-2 and tubulin different polymer promotes the formation of [1].. The accumulation of GSK-3 beta in the [2]. hippocampal neurons of microtubules can deactivate the CRMP-2 Thr-514 site, while the decrease of GSK-3 beta activity reduces the phosphorylation level of the CRMP-2 Thr-514 site, and ultimately promotes the occurrence of neuron axons. The development and maturation of [3]. protein phosphorylation and dephosphorylation are dual modulation of protein kinase and protein phosphatase. Protein phosphatase -2A (PP2A) is the most important serine / threonine phosphatase [4,5]. in the brain. Our recent study showed that the use of okadaic acid (OA), a specific inhibitor of PP2A, acted on the primary hippocampal neurons, the formation of neuron axons and the establishment of neuronal polarity in 12 h. In this study, the primary hippocampal neurons were cultured with 10 nmol/L OA and PP2A agonist neuringosine (D-erythro-Sphingosine) (10 nmol/L). The results showed that the axon growth of the 10 nmol/L OA group was significantly inhibited, the axon length was significantly shorter than that in the DMSO control group, and the number of non axon neurons was significantly increased. The multiple and single axon neurons were significantly reduced (P0.001); the PP2A agonist group was not only longer than the control group (P0.001), but the statistical results showed that the 30% neurons had multiple axons (P0.001), and the single axon neurons decreased (P0.01). The immunological trace was used to detect the PP2Ac subunit and its methylation level, and the results showed that D-erythro-Sph The level of PP2A methylation in ingosine group increased, the level of PP2A methylation in OA group decreased and the level of PP2Ac subunit was not changed. When the neuron culture was 48 h, the duration of the 10 nmol/L OA and 10 nmol/L D-erythro-Sphingosine continued 48 h. compared with the DMSO treatment group. There was no obvious multiple axon formation (P0.05) in the D-erythro-Sphingosine treatment group, and there was no significant difference in the length of the single axon. Then the neurons were cultured for 24 h and then transfected with RFP (or GFP) /pcDNA4.0, RFP (or GFP) /PP2A wild, and cultured for immunofluorescence. The polarity of neuron in the 0 control group was established, most of the neurons had an axon and multiple dendrites; in group PP2Awt, single axon lengthening (P0.01) and multiple axons increased (P0.001), and in group PP2Adn there was a short protuberance (P0.001). These results suggest that PP2Awt promotes the growth of single axon and the formation of multiple axons, while PP2Adn is seriously hindered by PP2Adn. The formation of axon and the establishment of neuronal polarity. These results show that activation of PP2A can induce the formation of axon in the hippocampal neurons. In order to further understand whether the formation of the axon has function, we observed the uptake and release of FM-64 by the multi axon. The results showed that 45 mM K+ stimulation was observed under confocal microscopy, PP2A activation was observed. The formation of multiple axons can take FM4-64 in red; after FM coloring, 90 mM K+ stimulation can be seen that FM coloring is weakened, suggesting that the multiple axons formed by the activation of PP2A have the repetitive cycle function of synaptic vesicle uptake and release. In order to explore the possible mechanism of PP2A influence on the polarity of neurons, a co transfection of RFP/pcDNA4.0 and RFP/PP2Awt after the culture of 24 h is carried out. The phosphorylation level of CRMP-2 Thr514 loci was detected by immunofluorescence. The results showed that activation of PP2A could significantly reduce the phosphorylation level of the cell body and the CRMP-2 Thr-514 site of the neurite. Conclusion: the up regulation of PP2A activity may be through dephosphorylation of CRMP-2, which leads to the extension of the single axon and the formation of the multiple axons, and the axons formed by the synapses are synapses. Vesicle uptake and release function.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R363

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