cAMP-PKA通路的调节及其在眼优势可塑性中的作用

发布时间：2018-03-14 20:55

本文选题：磷酸二酯酶4D　切入点：咯利普兰　出处：《中国科学技术大学》2015年博士论文　论文类型：学位论文

【摘要】：脑神经发育可塑性的分子机制一直是脑科学研究的热点。对视皮层的研究工作开始的较早,其结构也了解的比较透彻,因此,视觉可塑性是研究大脑神经发育可塑性常用的经典模型。眼优势可塑性(ocular dominance plasticity,ODP)是指关键期内单眼剥夺(monocular deprivation,MD)引起的初级视皮层细胞对剥夺眼反应的降低,而对非剥夺眼的反应增加。环单磷酸腺苷(cyclic adenosine monophosphate,cAMP)-蛋白激酶A(protein kinase A,PKA)是重要的第二信使通路,有很多报道都已经证明cAMP-PKA通路对ODP的表达至关重要,但其上下游的调控机制并不清楚。本文主要是研究调控cAMP-PKA通路的两个重要分子,磷酸二酯酶4D(phosphodiesterase 4D,PDE4D)和 A 型激酶锚定蛋白(A-kinase anchoring proteins,AKAPs)如何影响 ODP。前人的研究已经证明cAMP参与到关键期内视觉可塑性的表达。PDE4是cAMP的主要水解蛋白,可以调控cAMP的时空分布。PDE4D是PDE4家族中的一个重要成员,有很多报道证明它通过特异性的水解cAMP从而影响突触可塑性,但是却几乎没有人研究它是否影响视觉可塑性。因此,我们首先在小鼠以及猫上研究了PDE4D在发育过程中如何参与调节视觉可塑性。在小鼠上,通过选取发育时程中不同时间点的双眼区的脑组织进行蛋白质印迹(western blot)测定,我们发现视皮层中的PDE4D的表达时程与ODP的表达时程相吻合。在发育早期PDE4D的蛋白水平较低,在关键期达到峰值,而关键期后水平下降。然而改变视觉经验,例如暗饲养(dark rearing,DR)或者MD都没有影响PDE4D在视皮层中的蛋白含量,表明PDE4D的表达是不依赖于视觉经验的。对关键期内MD的小鼠腹腔注射PDE4的选择性抑制剂咯利普兰(rolipram)之后,通过使用内源光学信号成像的方法检测到它并没有影响小鼠的眼优势(ocular dominance,OD)的偏移。同时我们也在1个月和2个月的幼猫上进行了电生理的实验,通过缓释微泵向左侧视皮层给药(rolipram)或者对照二甲基亚砜(Dimethyl Sulphoxide,DMSO),两天之后再进行右眼的MD,对于1个月的幼猫MD1天,而两个月的幼猫MD4天,记录结果显示抑制剂rolipram同样也没有影响MD造成的OD的偏移。因此,根据这些结果,我们认为PDE4D与视觉系统发育相关,而与视觉经验诱发的可塑性可能无关。其次,我们对PKA的锚定蛋白AKAPs进行了研究。之前已经有研究报道过PKA的活性对ODP的表达起到重要作用。向视皮层施加PKA的抑制剂,或者敲除PKA RII亚基,都能影响关键期内MD造成的OD的偏移。PKA有众多的靶目标,越来越多的证据表明,AKAPs对于PKA在细胞不同区域发挥其特异的作用至关重要。在本研究中,首先用胞外电生理的方法测量了 AKAP150的基因敲除鼠的关键期内的ODP。结果发现,敲除AKAP150的小鼠MD4天后,OD的偏移程度小于单纯MD的对照组,即阻断了 ODP。为了排除基因敲除可能造成的副作用,又使用了抑制肽段St-Ht31(一种穿膜肽段,干扰PKA与AKAPs的结合)以及其对照肽段St-H31P来研究PKA的区域化在ODP中的作用。实验动物主要是小鼠和猫,通过脑内埋管或者缓释微泵直接向视皮层给药St-Ht31/St-H31P,然后MD4天之后,使用电生理胞外单细胞记录的方法研究发现,施加St-Ht31的小鼠,MD诱发的OD的偏移被部分阻断,而St-Ht31P组与对照组的偏移程度一致,并且抑制肽段不影响视皮层神经元的功能特性。这表明阻断PKA与AKAPs的结合能部分抑制小鼠的ODP。但是施加St-Ht31的幼年猫,MD4天之后引起的OD偏移与St-Ht31P组和MD对照组却没有显著差异,说明它没有影响幼猫ODP的表达。这些结果表明,PKA信号通路通过与锚定蛋白的结合实现其特异性的区域化,对于小鼠ODP是至关重要。可能由于物种差异,St-Ht31未能影响猫的ODP。本文对ODP的研究有着重要的临床意义。弱视就是在关键期内,视觉神经系统发生障碍造成的一种疾病,它能严重影响人们生活的质量,我国就有几千万的弱视患者。我们对视觉可塑性的研究有助于人们了解视觉发育的基本规律,进一步阐明其可塑性和关键点,并为弱视的发病机制及临床治疗提供理论基础。
[Abstract]:The molecular mechanism of brain plasticity of neural development is always the research focus in brain science. The earlier research work of visual cortex began, its structure is also more thorough understanding, therefore, visual plasticity is the classic models used in the study on the plasticity of brain development. Ocular dominance plasticity (ocular dominance, plasticity, ODP) is the key during the period of monocular deprivation (monocular deprivation, MD) caused by the decrease of cells in primary visual cortex of the deprived eye responses, while the non deprived eye responses increased. Cyclic AMP (cyclic adenosine monophosphate, cAMP) - protein kinase A (protein kinase A, PKA) is an important second messenger pathways, there are a lot of reports the expression of cAMP-PKA have proved crucial pathway of ODP, but its downstream regulatory mechanism is not clear. This paper is to study the regulation of cAMP-PKA pathway two important points, phosphodiesterase 4D (pH Osphodiesterase 4D, PDE4D) and A kinase anchoring protein (A-kinase anchoring, proteins, AKAPs) to study effect of ODP. predecessors has been proved that cAMP involved in the expression of.PDE4 in the critical period of visual plasticity is mainly hydrolyzed protein cAMP, can regulate cAMP distribution of.PDE4D is an important member of the PDE4 family, have many reports show that it can affect synaptic plasticity by hydrolysis of cAMP specific, but almost no one to study whether it affects visual plasticity. Therefore, we first study the PDE4D in the process of development and how to participate in the regulation of visual plasticity in mice and cats. In mice, by Western blotting at different time point selection the development process of the binocular zone (Western blot) determination of brain tissue, we found expression in the visual cortex of the PDE4D expression and the time course of ODP in early development coincide. The protein level of PDE4D was low, and reached the peak in the critical period, and after the critical period decreased. However the change of visual experience, such as dark rearing (dark rearing, DR or MD) did not affect the content of protein in the visual cortex of PDE4D, indicating that PDE4D expression is not dependent on visual experience. Intraperitoneal injection in mice PDE4 on the key period of MD selective inhibitor rolipram (rolipram), through the detection method using optical intrinsic signal imaging that it does not affect mice ocular dominance (ocular dominance, OD). At the same time we also offset in 1 months and 2 months of cat electrophysiology through the experiment, sustained-release drug pump to the left visual cortex (rolipram) or control two dimethyl sulfoxide (Dimethyl Sulphoxide, DMSO), two days after the right MD, the cat MD1 day of the 1 month, and two months of cat MD4 days, record the results The offset shown is also no effect of MD inhibitor rolipram caused by OD. Therefore, according to these results, we believe that PDE4D is related to the visual system development and plasticity and visual experience may not be induced. Secondly, the PKA anchoring protein AKAPs was studied. Previous studies have reported that the activity of PKA the expression of ODP plays an important role in applying PKA to the inhibitor. The visual cortex, or knockdown of PKA RII subunit, can offset.PKA critical period effects caused by MD OD is the target of many, more and more evidence that AKAPs is vital to its specific PKA in different regions in the cell. In this study, firstly, using extracellular electrophysiological measurements of AKAP150 gene knockout mice in the critical period of the ODP. results showed that AKAP150 knockout mice after MD4 days, the control group OD less than MD offset degree, namely resistance In order to eliminate the broken ODP. knockout possible side effects, and the use of inhibitory peptide St-Ht31 (a transmembrane peptide, with the interference of PKA and AKAPs) and the control peptide St-H31P of PKA region in ODP. The effect of experimental animal is mainly through mice and cats, the brain tube or release micro pump directly to the visual cortex after administration of St-Ht31/St-H31P, then MD4 days, research methods of single cell recording using electrophysiological extracellular, applied St-Ht31 mice, MD induced OD migration was partially blocked, while the St-Ht31P group and the control group shift consistency, and inhibitory peptide section does not affect the functional properties of visual cortical neurons. This suggests that blocking the combination of PKA and AKAPs could partially inhibit mouse ODP. but St-Ht31 on the young cat, MD4 days after induced OD migration and St-Ht31P group and MD control group had no significant difference, indicating that it No effect on the expression of cat ODP. These results suggest that the PKA signaling pathway by binding to the anchor protein region realize its specificity, is essential for mouse ODP. May be due to species differences did not affect St-Ht31 have important clinical significance of cat ODP. the study of ODP is in the critical period of amblyopia. In a visual nervous system disorder caused by disease, it can seriously affect the quality of people's lives, there are tens of millions of amblyopia in China. We research on visual plasticity can help people understand the visual development of the basic law, to further elucidate the plasticity and key points, and provide a theoretical basis for the the mechanism of amblyopia and clinical treatment.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R777.44

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