小鼠慢性应激模型海马中tau蛋白磷酸化及其对线粒体轴浆运输的影响

发布时间：2018-04-13 02:10

本文选题：抑郁(Depression) + 母子剥夺(Maternal　；参考：《中国科学技术大学》2011年博士论文

【摘要】：Tau蛋白是一种微管相关蛋白,其磷酸化受到严格调控,在神经元中和轴浆运输密切相关。据研究报道,急性应激中,啮齿类动物海马中tau蛋白的磷酸化和下丘脑-垂体-肾上腺(Hypothalamo-Pituitary-Adrenal,HPA)轴的过度激活密切相关。而在抑郁病人中,HPA轴的过度激活是基本的特征之一。因此,在抑郁症中,tau蛋白的磷酸化情况以及和轴浆运输的关系需要进一步研究。我们的假设是:在抑郁模型中,HPA轴的过度激活会引起tau蛋白的过度磷酸化并进一步影响到轴浆运输。为验证该假设,我们首先建立了母子剥夺抑郁模型,对其进行了行为学测试,并检测了HPA轴活性。然后检测了海马中tau蛋白的磷酸化水平。为了研究轴浆运输的变化,我们选择了检测海马突触体中线粒体水平的变化。结合CRH(Corticotropin-Releasing Hormone,CRH)受体1(CRH Receptor1,CRHR1)和糖皮质激素受体(Glucocorticoid Receptor,GR)的拮抗剂,研究了CRH直接的中枢作用机制和HPA轴的反馈机制在tau蛋白磷酸化和线粒体运输中的作用,。为了进一步检测tau蛋白磷酸化和线粒体运输的关系,我们用实时成像技术,观察当原代神经元中tau蛋白磷酸化水平发生变化时线粒体运输的动态变化。结果发现: 1.母子剥夺抑郁模型具有典型的抑郁样行为和过度激活的HPA轴。在旷场、糖水偏好和明暗箱行为学测试中,抑郁模型具有典型的抑郁样行为。和对照组相比,抑郁模型具有显著升高的血浆cortitosterone和下丘脑CRH mRNA水平。 2.抑郁模型海马中tau蛋白的磷酸化水平显著升高。和对照组相比,抑郁鼠的海马中tau蛋白的总表达水平没有显著变化,但在在AT-8磷酸化位点显著升高,在tau-1非磷酸化位点显著降低。 3.抑郁模型海马突触体中的线粒体水平显著升高。和对照组相比,抑郁模型海马突触体中的线粒体水平显著升高。 4. CRHR1而不是GR介导了tau蛋白的磷酸化水平升高和突触体中线粒体水平的升高。 CRHR1的拮抗剂CP154526能显著降低tau蛋白的磷酸化,并能恢复突触体中线粒体的水平,而GR的拮抗剂RU486却没有这些作用。 5.在原代神经元中,当tau蛋白磷酸化水平降低时,线粒体的轴浆运输水平下调。我们用锂盐抑制Gsk3beta的活性从而降低原代神经元中tau蛋白的磷酸化水平,我们观察到线粒体的轴浆运输水平显著下降。上述结果表明,在抑郁模型中CRHR1介导了tau蛋白的过度磷酸化,同时伴随着突触体中线粒体水平的升高。这向我们揭示,在抑郁模型中,CRH的中枢作用参与了线粒体运输的调控,这可能为研究抑郁发病机制提出了一条新思路。
[Abstract]:Tau protein is a microtubule-associated protein, its phosphorylation is strictly regulated, transport in neurons and axonal transport is closely related.It has been reported that the phosphorylation of tau protein in the hippocampus of rodents is closely related to the hyperactivation of the hypothalamo-Pituitary-Adrenalar axis during acute stress.Hyperactivation of HPA axis is one of the basic characteristics in depressive patients.Therefore, the phosphorylation of tau and its relationship with axonal transport in depression need further study.Our hypothesis is that excessive activation of the HPA-axis in depression models leads to excessive phosphorylation of tau proteins and further affects axonal transport.To verify this hypothesis, we first established a model of mother-son deprivation depression, tested it by behavioral tests, and measured the activity of the HPA axis.Then the phosphorylation level of tau protein in hippocampus was detected.In order to study the changes of axonal transport, we chose to detect the changes of mitochondrial level in hippocampal synaptosomes.The direct central mechanism of CRH and the feedback mechanism of HPA axis in the phosphorylation of tau protein and mitochondrial transport were studied by combining the antagonists of 1(CRH Receptor1CRHR1 and Glucocorticoid Receptorgrass).In order to further investigate the relationship between tau protein phosphorylation and mitochondrial transport, we used real-time imaging technique to observe the dynamic changes of mitochondrial transport when the phosphorylation level of tau protein in primary neurons changed.It was found that:1.The model of mother and child deprivation depression has typical depressive behavior and overactivated HPA axis.In open-field, sugar water preference and dark box behavioral tests, the depression model had typical depressive behavior.Compared with the control group, the depression model had significantly increased plasma cortitosterone and hypothalamic CRH mRNA levels.2.The level of phosphorylation of tau protein in hippocampus of depression model was significantly increased.Compared with the control group, there was no significant change in the total expression of tau protein in the hippocampus of the depressed rats, but it was significantly increased at the AT-8 phosphorylation site and decreased significantly at the tau-1 non-phosphorylation site.3.The level of mitochondria in hippocampal synaptosomes was significantly increased in depression model.Compared with the control group, the level of mitochondria in the hippocampal synaptosomes was significantly increased in the depression model.4.CRHR1, rather than gr, mediates increased phosphorylation of tau protein and mitochondrial level in synaptosomes.CP154526, an antagonist of CRHR1, could significantly reduce the phosphorylation of tau protein and restore mitochondrial level in synaptosomes, but RU486, the gr antagonist, did not.5.In primary neurons, when the phosphorylation level of tau protein decreased, the axoplasmic transport level of mitochondria was down-regulated.We use lithium salt to inhibit the activity of Gsk3beta and thus reduce the phosphorylation of tau protein in primary neurons.We observed a significant decrease in the axonal transport level of mitochondria.These results suggest that CRHR1 mediates the excessive phosphorylation of tau protein in the depression model and is accompanied by an increase in the mitochondrial level in the synaptosome.This reveals that the central role of CRH participates in the regulation of mitochondrial transport in depressive models, which may provide a new way to study the pathogenesis of depression.
【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】：2011
【分类号】：R749.4;R-332

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