【作者】 孙旭莹；

【导师】 王建枝； 刘蓉；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2012， 博士

【摘要】 兴奋性突触传递是神经元最基本的功能,NMDA受体(N-Methyl-D-aspartate receptor, NMDAR)是神经系统中最主要的兴奋性离子型受体之一,其在学习记忆,突触可塑性,神经发育等方面具有重要作用,但NMDA受体过度激活导致谷氨酸聚集于突触间隙所诱导的神经毒性作用也是许多神经退行性疾病的共同发病机制。阿尔茨海默病(Alzheimer’s disease, AD)是成人痴呆症最主要的病因,其中tau蛋白过度磷酸化和聚集是AD脑内的主要病理特征之一。兴奋性突触传递与tau病变之间的联系目前少见报道。本研究探讨了谷氨酸能兴奋性突触传递增强对tau蛋白表达和磷酸化的影响及其在AD样神经退行性变中的作用。本文第一部分探讨了短时间突触传递增强对tau蛋白磷酸化的影响和内在机制。成人脑内约有一半的谷氨酸能神经元是谷氨酸-锌能神经元,即突触兴奋时锌离子与谷氨酸一起释放至突触间隙。本研究阐明了谷氨酸-锌能神经元兴奋时突触释放的锌离子通过抑制蛋白磷酸酯酶2A (Protein phosphatase2A, PP2A)的活性导致tau蛋白过度磷酸化。从理论上解释了谷氨酸-锌能神经元在大脑中的分布区域(海马,杏仁核,内嗅皮层及颞叶皮层区域)与过度磷酸化tau蛋白聚集形成的神经元纤维缠结(Neurofibrillary tangles, NFTs)的分布相重叠的可能机制,为锌稳态失衡参与AD发病提供了新线索,也为临床干预tau相关病变提供了新靶点本文第二部分探讨了NMDA受体慢性激活时,特别是突触外NMDA受体(Extrasynaptic NMDA receptor, E-NMDAR)激活后对tau蛋白的作用。首次报道突触外NMDA受体慢性激活后可导致总tau蛋白、磷酸化及非磷酸化tau蛋白水平均显著增加,tau的mRNA水平也明显增加,突触内NMDA受体激活时tau蛋白表达则无明显变化。在敲除tau蛋白的小鼠神经元中激活E-NMDA受体,与野生型神经元相比神经元死亡减少,生存信号分子ERK活性增高,磷酸化CREB水平也明显升高,神经元活性得以保存。研究证明了突触外NMDA受体激活通过tau蛋白过度表达对神经元产生毒性作用,E-NMDA受体可能成为治疗AD等tau相关疾病的干预靶点。总而言之,本研究着重阐明了谷氨酸能突触传递增强对神经元内tau蛋白表达及磷酸化的长期和短期作用,其结果为NMDA受体过度激活在阿尔茨海默病发病中的病理作用机制提供了全新资料。第一部分突触传递释放的锌通过抑制蛋白磷酸酯酶2A导致tau蛋白过度磷酸化在阿尔茨海默病(Alzheimer’s Disease,AD)病人脑中,过度磷酸化的微管相关蛋白tau是神经元纤维缠结(Neurofibrillary tangles, NFTs)的主要成分,而神经元纤维缠结主要分布于富含锌离子的谷氨酸-锌能神经元所在的部位如海马、内嗅皮层、杏仁核等,提示谷氨酸-锌能神经元突触传递释放的锌离子可能参与了tau蛋白的磷酸化。为了验证这种假说,我们在体外培养的大鼠海马脑片及原代神经元用谷氨酸或Bic/4-AP处理来模拟突触兴奋性增强,采用锌离子螯合剂螯合突触释放的锌,探讨了突触传递释放的锌在tau蛋白磷酸化中的作用。实验结果显示,谷氨酸或Bic/4-AP处理后,tau蛋白在Ser396,Ser404, Thr231,Thr205等AD相关的位点发生过度磷酸化,而预先用细胞内或细胞外锌离子鳌合剂处理,或通过移除细胞外液中的钙离子来阻断突触锌离子的释放均可逆转由突触兴奋性增高导致的tau蛋白过度磷酸化。通过检测培养基中锌离子浓度,我们证实了在两种突触兴奋性增高的模型中锌离子的释放。外源性地给予硫酸锌处理,也导致海马脑片的tau蛋白过度磷酸化。进一步对tau蛋白过度磷酸化的机制进行探讨发现,突触兴奋性增高导致tau蛋白过度磷酸化时伴随蛋白磷酸酯酶2A(Protein phosphatase2A, PP2A)的活性下降,且用锌的鳌合剂预孵育可逆转PP2A的失活。以上实验结果表明：突触间隙释放的锌离子通过抑制蛋白磷酸酯酶2A导致tau蛋白过度磷酸化,AD脑中谷氨酸-锌能神经元锌稳态失衡可能参与了AD患者脑中PP2A失活和tau相关病变的发生。第二部分突触外NMDA受体对tau蛋白表达的影响及其在神经退行性变中的作用神经系统中,突触内外NMDA受体在调节突触可塑性、基因表达及神经元命运方面有不同的作用。突触外NMDA受体(extrasynaptic NMDA receptors, E-NMDARs)激活可导致神经元死亡和神经退行性变,但其内在机制尚未完全阐明。在本研究中,我们利用培养的原代皮质神经元模型,首次报道E-NMDAR受体激活后可导致总的,磷酸化及非磷酸化的tau蛋白水平增加,tau蛋白mRNA水平也明显增加,伴随神经元活性下降和轴突退行性变。选择性的E-NMDAR受体拮抗剂美金胺可逆转E-NMDAR受体激活所导致的tau蛋白表达增加。在敲除tau蛋白的小鼠神经元中激活E-NMDA受体,发现神经元死亡较野生型神经元减少,生存信号分子ERK活性增高,磷酸化CREB水平也明显升高。以上结果提示,E-NMDA受体激活通过tau蛋白表达增加介导了其对神经元的毒性效应,选择性干预E-NMDA受体可能成为治疗tau相关神经退行性病变如阿尔茨海默病(Alzheimer’s Disease,AD)的有效手段,而tau蛋白也可能成为神经元兴奋性毒性损伤中的一个治疗靶点。更多还原

【Abstract】 Excitatory neurotransmission is the basic function of neurons; N-Methyl-D-aspartate receptor (NMDAR) is one of the predominant excitatory ionotropic receptors in human brain, which plays important roles in learning and memory, synaptic plasticity, and neuronal development. However, excessive activation of NMDA receptors-induced glutamate accumulation in synaptic cleft also causes neuronal toxicity, which is now realized to be an upstream factor of many neurodegenerative diseases. Alzheimer’s disease is the most common disease causing adult dementia; abnormal tau phosphorylation and accumulation is the characteristic pathological changes in AD brain. Till now the relationship between excitatory neurotransmission and tauopathy has not yet been elucidated. In the present study, we explored the effect of excitatory neurotransmission on tau expression and phosphorylation, and its role in AD-like neurodegeneration.In part I, we explored the changes of tau phosphorylation in neurons with short term activation of synaptic transmission. Nearly half of the glutamatergic neurons are "gluzinergic" neurons, which release glutamate and zinc simultaneously upon excitation. Our results suggest that synaptically released zinc promotes tau hyperphosphorylation through protein phosphatase2A (PP2A) inhibition. This finding explains the possible underlying mechamism for that location of zinc containing glutamatergic neurons is largely overlapped with neurofibrillary tangles (NFTs, which is formed by hyperphosphorylated tau) in AD susceptible brain regions such as hippocampus, amygdala, entorhinal and temporal cortex.In part II, we explored the effect of chronic activation of NMD A receptors, especially extrasynaptic NMDA receptors (E-NMDAR) on tau protein expression. We reported for the first time that prolonged activation of E-NMDAR dramatically increased total, phosphorylated and dephosphorylated tau levels, with simultaneous increase of tau mRNA level, while chronic activation of synaptic NMDAR showed no effect on tau protein expression. Activation of E-NMDA receptors in tau knockout mice neurons resulted in less neuronal death, increased level of survival signaling molecules such as active extracellular signal-regulated kinase (ERK) and phosphorylated CREB. These results indicate that tau overexpression mediates the neuronal toxicity of E-NMDAR activation. E-NMDAR may be a promising therapeutic target for AD and related tauopathy.In a summary, the present study focused on the effects of short or long term activation of glutamatergic synaptic transmission on tau expression and phosphorylation, the results disclosed new clues for the role of excessive activation of NMDA receptors in AD pathogenesis.Part ⅠSynaptic released zinc promotes tau hyperphosphorylation by inhibition of PP2AHyperphosphorylated tau is the major component of neurofibrillary tangles in Alzheimer’s disease (AD), and the tangle distribution is largely overlapped with zinc containing glutamatergic neurons, suggesting that zinc released in synaptic terminals may play a role in tau phosphorylation. To explore this possibility, we treated the cultured hippocampal slices or primary neurons with glutamate or Bic/4-AP to increase the synaptic activity with or without pre-treatment of zinc chelators, and then detected the phosphorylation levels of tau. We found that glutamate or Bic/4-AP treatment caused tau hyperphosphorylation at multiple AD-related sites, including Ser396, Ser404, Thr231, and Thr205, while application of intracellular or extracellular zinc chelators, or blockade of zinc release by extracellular calcium omission almost abolished the synaptic activity-associated tau hyperphosphorylation. The zinc release and translocation of excitatory synapses in the hippocampus was detected, and zinc-induced tau hyperphosphorylation was also observed in cultured brain slices incubated with exogenously supplemented zinc. Tau hyperphosphorylation induced by synaptic activity was strongly associated with inactivation of protein phosphatase2A (PP2A), and this inactivation can be reversed by pre-treatment of zinc chelator. Together, these results suggest that synaptically released zinc promotes tau hyperphosphorylation through PP2A inhibition. Part ⅡEffect of extrasynaptic NMDA receptors on tau protein expression and its role in neurodegenerationSynaptic and extrasynaptic NMDA receptors (E-NMDAR) play distinct roles in neuronal plasticity, gene regulation, and neuronal survival. Activation of E-NMDA receptors may induce neuronal degeneration and neuronal death, however, the underlying mechanisms remain elusive.In the present study,, we reported for the first time that in cultured primary cortical neurons, activation of E-NMDA receptors dramatically increased the protein levels of total, phosphorylated and dephosphorylated tau, as well as the tau mRNA levels, which was accompanied with axonal degeneration and decreased neuronal survival. Memantine, a selective antagonist of E-NMDA receptors, reversed E-NMDAR-induced tau expression. Activation of E-NMDA receptors in tau knockout mice neurons resulted in less neuronal death, increased survival signaling molecules such as active extracellular signal-regulated kinases (ERk) and phosphorylated CREB.These results suggested that tau overexpression may mediate the toxicity of E-NMDA receptor activation; selective targeting E-NMDA receptor may be a promising therapeutic strategy for tauopathy like Alzheimer’s Disease (AD), and tau may also be a target for the development of therapy for neuronal excitoxicity.更多还原