应激通过谷氨酸系统诱导老年痴呆相关的tau蛋白磷酸化及其Munc18的介导作用研究

Alzheimer's disease (AD) is a chronic neurodegenerative disorder marked by a progressive loss of memory and cognitive function. The neuropathological features are beta-amyloid plaques and phosphorylated tau-laden neurofibrillary tangles. The etiology of the sporadic cases remains unclear and likely involves complex interactions between various genetic and environmental factors. Stressful lifestyle or psychological distress for AD was noticed recently, because life stressors are wildly distributed in modern society. However,the molecular mechanisms linking stress to AD pathogenesis remain to be elucidated. Recent studies have implicated corticotropin releasing factor (CRF) as a more likely causal factor in tau phosphorylation, whereas glucocorticoid receptor-dependent pathway did not play a pivital role in tau phosphorylation. Meanwhile, our study indicated glutamate neurotransmission taking a role in stress induced tau phosphorylation. Moreover, we found Munc18 a CDK5 activator translocation from nuclear to cytoplasm after glutamate stimulation or stress stimulation. And preliminary experiments also implied that Munc18 could bind with Mecp2 a transcript factor. These results inferred that Munc18 might take part in tau phosphorylation under stress. It was known that brain regions vulnerable to early injury in AD, appear to overlap a set of cortices which are important for memory and cognition, labeled the ''Default Mode Network'' . Regions within the default network show functional connectivity that converges on the posterior cingulate extending into the precuneus. Therefore, in this study, we will aim at the posterior cingulate, studying the mechanism of glutamate mediating stress induced tau phosphorylation, and exploring the machinery of interactions among the glutamate, CRF and beta-amyloid to confirm which one is more important in stress induced AD related brain injury and occupies in upstream of the mechanisms. Studies will also resolve how the Munc18 affect the tau phosphorylation under glutamate in stress induced tau phosphorylation, by its activation of CDK5 and/or regulation of gene transcription. Finally, we will explore the interfering of glutamate receptors and Munc18 in posterior cingulate to evaluate stress caused the tau phosphorylation, amyloid deposition in DMN and in other brain regions, as well as the effects on stress induced memory impairment, anxiety and depression. These works would be helpful for uncovering the pathological mechanism of AD, and for developing the treatment of AD at the point of psychological stress.

Alzheimer 病（AD）是以学习记忆和认知功能损害为特征的神经退行性疾病。AD相关的危险因素很多，神经精神应激因素可促进AD病理发展。我们前期研究显示谷氨酸系统参与应激诱导的AD相关的tau蛋白磷酸化，并观察到谷氨酸可诱导胞浆中CDK5激活蛋白Munc18分布增加，推测其参与介导作用。鉴于默认神经网络（DMN）参与记忆和认知活动，其中后扣带回及周围皮质涉及应激机制，同时为AD最易损脑区之一。本项目拟以后扣带回为主，研究应激条件下谷氨酸参与tau蛋白磷酸化机制，及其中与促肾上腺皮质素释放因子CRF和beta-amyloid的相互关系；研究Munc18在下游介导tau蛋白磷酸化和基因表达调控改变,及其与学习记忆能力改变的关系；针对后扣带皮层的谷氨酸信号传递系统和Munc18进行干预。这些研究将不仅对于认识AD发病机制提供新的视角，也将对从应激角度选择合适的防治措施提供资料。

项目主要内容为应激诱导痴呆相关tau磷酸化中谷氨酸递质系统的作用及机制。1. 慢性束缚应激（CRS）可导致预设网络脑区（default network）tau磷酸化和剪切增加，该网络重要结构后扣带回tau异常变化并不比前额叶皮层更显著。2. CRS可导致谷氨酸转运体（EAAT1、EAAT2）表达下降，提示应激使谷氨酸不能有效清除。而细胞模型上，一些NMDA受体拮抗剂可抑制tau磷酸化。非竞争性NMDA受体拮抗剂美金刚（MEM）干预CRS，可减轻动物焦虑行为，该干预抑制16天CRS诱导的tau磷酸化，机制涉及恢复tau相关激酶和磷酸酶功能、及谷氨酸受体表达异常。但长期应用MEM于应激（28天）治疗可使得tau磷酸化反而增加，机制涉及激酶、磷酸酶和伴侣分子异常，上游机制涉及NR2B表达异常。结果表明谷氨酸系统参与应激导致的tau异常，并为MEM临床用药提供参考。3.老年动物中，应激诱导tau磷酸化更明显，并易导致tau的沉积；tau异常沉积机制与老年动物应激后多种伴侣分子变化异常有关，尤其是HSP42/HDJ2与PIN1。培养细胞上敲低HDJ2与PIN1可使细胞内tau水平增加。本研究首次提出老化背景下应激与AD的关系。CRS诱导tau异常伴随谷氨酸受体调节激酶Fyn表达和磷酸化异常，提示其激活。Fyn抑制剂迷迭香酸（RA）可抑制CRS诱导tau磷酸化及其聚集；RA逆转CRS诱导的伴侣分子异常是减少tau异常聚集原因。结果提示Fyn是干预慢性应激诱导tau异常变化的靶标。5. 实验研究了tau磷酸化与tau D421位点剪切tau的关系，AT8位点模拟磷酸化可抑制剪切；加入PHF1位点模拟磷酸化，逆转AT8磷酸化效应。另，刚果红可抑制Tau剪切。结果首次揭示AT8磷酸化调控D421位点剪切，为解释AD tau病理提供新的解释。5. Munc18在CRS或神经兴奋刺激下出核增加。分子生物学鉴定Munc18出核入核序列。过表达Munc18在胞浆中分布可导致tau磷酸化增加，提示Munc18穿梭涉及对tau的调节。敲低Munc18导致细胞核内分布减少，使多种基因表达改变，包括BDNF；并导致动物出现焦虑表型，该现象与CRS动物症状类似，提示应激后Munc18细胞核内水平下降参与应激导致的行为异常。研究结果对认识应激导致脑功能异常提供了新资料。