蛋白磷酸酯酶2A在胎鼠海马神经元极性形成中的作用

发布时间：2018-08-07 17:26

【摘要】： 典型的神经元有一个轴突和多个树突。对于一个神经元来说,树突往往是接受信息的部位,轴突往往是输出信息的部位,因此,神经元的极性是保证信息在神经系统内部及神经系统与其他系统之间有序流动的物质基础。海马神经元处于生长状态的轴突含有丰富的脑衰蛋白反应调节蛋白2(CRMP-2),轴突生长锥主要以非磷酸化CRMP-2活性形式存在。过度表达CRMP-2可以导致多轴突形成,反之抑制CRMP-2不利于轴突的生长与形成[1]。在体外,CRMP-2与管蛋白异二聚体相互作用而促进微管的聚集[2]。海马神经元GSK-3β可以磷酸化CRMP-2 Thr-514位点使其失活,而GSK-3β活性下调使得CRMP-2 Thr-514位点磷酸化水平下降,最终促进了神经元轴突的发生、发展和成熟[3]。蛋白质磷酸化和去磷酸化受蛋白激酶和蛋白磷酸酯酶的双重调节,而蛋白磷酸酯酶-2A(PP2A)是脑最重要的丝氨酸/苏氨酸蛋白磷酸酯酶[4,5]。我们最近的研究显示:采用不同浓度PP2A的特异性抑制剂冈田酸(okadaic acid, OA)作用于培养12 h的胎鼠原代海马神经元,神经元轴突的形成和神经元极性的建立均受到了明显的影响;本研究用10 nmol/L OA和PP2A激动剂神经鞘氨醇(D-erythro-Sphingosine)(10 nmol/L)处理培养12 h的原代海马神经元。结果显示10 nmol/L OA组神经元轴突生长受到明显的抑制,轴突长度明显短于DMSO对照组,且无轴突神经元数量明显增多而单轴突神经元则显著减少(P0.001);PP2A激动剂组不仅单轴突长度明显长于对照组(P0.001),而且统计学结果显示30%神经元出现了多轴突(P0.001),同时单轴突神经元减少(P0.01)。用免疫印迹检测PP2Ac亚基及其甲基化水平,结果显示:D-erythro-Sphingosine组PP2A甲基化水平升高,OA组PP2A甲基化水平降低,PP2Ac亚基水平无变化。当神经元培养48 h即维持阶段分别应用10 nmol/L OA和10 nmol/L的D-erythro-Sphingosine持续作用48 h。与DMSO处理组相比可见,10 nmol/L OA处理组神经元无轴突长度缩短(P0.05)或极性缺失;D-erythro-Sphingosine处理组神经元没有出现明显的多轴突形成(P0.05),单轴突长度变化无统计学意义。然后将神经元培养24 h后共转染RFP(or GFP)/pcDNA4.0、RFP(or GFP)/PP2A wild type(wt)和RFP(or GFP)/PP2Adn培养48 h,固定做免疫荧光可见转染pcDNA4.0对照组神经元极性已经建立,大多数神经元有一个轴突和多个树突;PP2Awt组单轴突延长(P0.01)同时多轴突增多(P0.001);PP2Adn组多出现短小的突起(P0.001)。这些结果提示PP2Awt促进了神经元单轴突的生长和多轴突的形成而PP2Adn则严重阻碍了轴突的形成和神经元极性的建立。上述结果说明激活PP2A可诱导海马神经元轴突的形成。为了进一步了解所形成的多轴突是否具有功能,我们观察了多轴突对FM-64的摄取与释放。结果显示:45 mM K+刺激1 min于共聚焦显微镜下观察,PP2A激活形成的多轴突可以摄取FM4-64呈红色;FM着色后再给予90 mM K+刺激可见FM着色减弱,提示激活PP2A形成的多轴突具有突触囊泡摄取和释放的重复循环功能。为了探讨PP2A影响神经元极性的可能机制,将神经元培养24 h后共转染RFP/pcDNA4.0和RFP/PP2Awt,免疫荧光检测CRMP-2 Thr514位点磷酸化水平。结果发现激活PP2A可以明显降低神经元胞体和突起CRMP-2 Thr-514位点磷酸化水平。结论:PP2A活性上调可能通过去磷酸化CRMP-2,进而导致神经元单轴突的延长和多轴突的形成,所形成的轴突具备突触囊泡摄取和释放功能。
[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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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