【作者】 谢敏；

【导师】 陈其才； 廖晓梅；

【作者基本信息】 华中师范大学 ， 动物学， 2014， 博士

【摘要】 泛素-蛋白酶体系统(ubiquitin-proteasome system, UPS)的主要生物学功能是降解细胞内聚集的或错误折叠的蛋白质以调节多种生物学过程,从而维持细胞的正常生理状态及功能。UPS中各个成分的功能障碍都会对生物体产生严重的负面影响,尤其是当这些异常作用发生在中枢神经系统中,不仅会引起记忆相关蛋白的表达缺失,还有可能会损伤突触可塑性和记忆功能的维持,从而造成记忆减退或神经变性。NMDA受体NR2B亚单位(N-methyl-D-aspartate receptor subtype2B, NR2B)在多种突触信号事件、蛋白质的相互作用以及人类神经退行性疾病中行使着极其重要的生物学功能。研究显示,UPS的许多底物都能够对NR2B亚单位的生物学功能产生影响,包括微管相关蛋白tau、突触蛋白、蛋白激酶以及蛋白磷酸酯酶等,因此,我们推测UPS可能通过其底物蛋白作用于NR2B亚单位,从而调节突触可塑性和学习记忆。本研究选取转录因子cAMP反应元件结合蛋白(cAMP response element binding protein, CREB)为研究对象,分别从细胞、脑片和动物整体水平探索UPS成分功能障碍对记忆功能的调控及分子机制。作为转录因子,CREB在调节神经元蛋白的表达中发挥关键作用,且该调节作用对于突触可塑性和记忆的形成与维持至关重要。除了蛋白酶体,CREB的转录活性还受到NR2B亚单位介导的下游分子途径所调控,因此,探索泛素-蛋白酶体系统成分功能障碍对CREB活性调节的分子机制是揭示影响学习记忆功能的有效途径之一。1.蛋白酶体活性抑制诱导CREB去磷酸化(细胞系水平,N2a细胞)。研究结果表明蛋白酶体活性抑制可以通过两条途径下调CREB-Serl33位点的磷酸化水平,从而下调CREB的活性。(1)蛋白酶体活性抑制会引起tau蛋白的异常磷酸化和泛素化,且这些异常的tau蛋白能够通过下调NMDA受体NR2B亚单位在Tyr1472位点的磷酸化水平而调节NR2B亚单位正常的生理功能,最终导致CREB活性位点Ser133位点的磷酸化水平显著下降；(2)抑制蛋白酶体活性诱导激酶蛋白激酶A (protein kinase A, PKA)和糖原合成酶激酶-3β (glycogen synthase kinase-3p, GSK-3p)的活性发生改变,进而导致CREB-Ser133去磷酸化水平的增加。2.去泛素化酶泛素C末端水解酶L1(ubiquitin C-terminal hydrolase L1, UCH-L1)的活性抑制参与海马脑片中CREB的去磷酸化调节(器官型海马脑片水平,C57BL/6小鼠海马脑片)。研究表明,UCH-L1会影响CREB的磷酸化水平及其转录活性,但其具体机制尚不清楚。为此,本研究用UCH-L1抑制剂LDN处理小鼠海马脑片以探索UCH-L1活性抑制对CREB活性的影响。实验结果表明,UCH-L1活性抑制导致海马脑片中CREB-Ser133的磷酸化水平显著下降。同时,UCH-L1抑制还引起tau蛋白的过度磷酸化、酪氨酸激酶Fyn活性下调、NR2B亚单位在Tyr1472位点的磷酸化水平下降以及突触蛋白PSD-95(postsynaptic density protein95)含量的增加。过度磷酸化的tau蛋白可通过干扰Fyn/NR2B/PSD-95复合体的形成,从而影响NR2B亚单位的生物活性,最终导致CREB活性的下调。除此之外,UCH-L1活性抑制引起的PKA活性下降也参与CREB的活性调节。3.蛋白酶体和UCH-L1功能障碍对小鼠空间记忆影响的分子机制(动物整体水平,APP/PS1双转基因小鼠)。APP/PS1双转基因小鼠是一种阿尔茨海默病(Alzheimer’s disease, AD)模型小鼠,其主要病理特性是存在老年斑沉积且对神经细胞具有毒性作用。结果显示,APP/PS1小鼠具有明显的空间记忆缺陷。进一步检测发现APP/PS1小鼠海马和皮层部位的CREB的活性较对照组小鼠显著降低,且调节CREB活性的分子途径发生改变。一方面脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)的水平显著下调与CREB活性下降密切相关；另一方面过度磷酸化的tau蛋白可能通过导致NR2B-Tyr1472位点磷酸化水平下降而影响NR2B亚单位的正常生理功能从而导致CREB的去磷酸化。另外,有研究显示,蛋白酶体和UCH-L1参与上述分子途径的活性调节。本研究发现,APP/PS1小鼠中蛋白酶体和UCH-L1的活性和表达水平都显著下调,表明APP/PS1小鼠空间学习记忆的缺陷可能是由泛素-蛋白酶体系统成分功能障碍导致的CREB活性下调引起的。因此,根据以上实验结果(1-3部分),本研究从proteasome and UCH-L1—abnormal tau protein—NR2B/CREB这一分子途径揭示了APP/PS1小鼠空间记忆障碍的分子机制。更多还原

【Abstract】 The majory function of ubiquitin-proteasome system (UPS) is to regulate variety of biological processes through degradating intracellular aggregation or misfolded proteins, and depend on this process the normal physiological state and functions of cells can be maintained. The components of UPS dysfunction have a seriously negative impact on the organism, especially when the occurrence of these abnormal effects in central nervous system would induce the lost expression of memory-related proteins or even impaire the maintenance of synaptic plasticity and memory function which further cause memory decline or neurodegenerative disorders. N-Methyl-D-aspartate (NMDA) receptor NR2B subunit plays extremely biological functions in synaptic signaling events, protein-protein interactions and human neurodegeneration. A growing body of evidence suggests that many substrates of UPS have an effect on NR2B biological function, including microtubule-associated protein tau, synaptic proteins, protein kinases and phosphatase and so on, thus a consideration is suggested that UPS probably regulates NR2B subunit through its substrates and further affects synaptic plasticity and learning and memory.The current research focused on cAMP response element binding protein (CREB), and the molecular mechanisms of dysfunctional UPS components on the regulation of memory function were explored from the levels of cells, hippocampal slices to animals. As a transcription factor, CREB is crucial for regulating the expression of neuronal proteins which is required for the synaptic plasticity and formation and maintenance of memory. In addition to the proteasome, the transcriptional activity of CREB can also be modulated by the downstream molecular signaling of NMDA receptor. Therefore, to explore the molecular mechanisms of dysfunctional UPS components on the regulation of CREB activity are an effective pathway to learning and memory function.1. Proteasome inhibition induces CREB dephosphorylation (Cell lines, N2a cells). The current study indicated that proteasome activity inhibition down-regulated the level of phosphorylated CREB at Ser133site via two pathways which further decreased CREB activity.(1) Inhibition of proteasome activity caused abnormal phosphorylation and ubiquitination of tau protein. Moreover, the abnormal tau protein affected the normal physiological function of NMDA receptor NR2B subunit by down-regulating the level of phosphorylated NR2B at Tyr1472site which eventually led to a significant decrease in phosphorylation of CREB at Ser133site.(2) Inhibition of proteasome activity induced the alternation of protein kinase A (PKA) and glycogen synthase kinase-3β (GSK-3(3) activities which further resulted in an increase in dephosphorylation of CREB-Ser133.2. Inhibition of deubiquitination enzyme ubiquitin C-terminal hydrolase L1(UCH-L1) is involved in CREB dephosphorylation in hippocampal slices (organotypic hippocampal slices, slices from C57BL/6mice). Researches have demonstrated that UCH-L1have an effect on phosphorylation and transcriptional activity of CREB, but the mechnisams still unclear. Therefore, this current research used UCH-L1inhibitor LDN to treat mice hippocampal slices and explored the effects of UCH-L1inhibition on CREB activity. The results indicated that inhibition of UCH-L1activity induced a significant reduction of phosphorylated CREB-Ser133. Meanwhile, the hyperphosphorylation of tau protein, down-regulation of tyrosin kinase Fyn activity, reduction of phosphorylated NR2B at Tyr1472site and increased level of synaptic protein PSD-95(postsynaptic density protein95) were observed after UCH-L1inhibition. Hypephosphorylated tau protein probably disturbed the formation of Fyn/NR2B/PSD-95complex to affect the biological activity of NR2B subunit and ultimately led to decreased CREB activity. Moreover, inhibition of UCH-L1activity induced reductive PKA activity also involved in the regulation of CREB activity.3. The molecular mechanisms of dysfunctional proteasome and UCH-L1affect the spatial memory in mice (Animal, APP/PS1double transgenic mice). APP/PS1double transgenic mouse is an Alzheimer’s disease (AD) mouse model and the mainly pathological characteristic of the mouse is the deposition of senile plaque which has cytotoxicity. The results showed that APP/PS1mice have significant spatial memory deficits. Further detection found that compared with the control group, CREB activity in hippocampus and cortex in APP/PS1mice significantly reduced. In addition, the alterations of the molecular pathways that regulated the CREB activity were observed in APP/PS1mice. Firsly, the obviously decreased level of brain-derived neurotrophic factor (BDNF) was closely related to the down-regulation of CREB activity; secondly, hyperphosphorylated tau protein had an effect on the phosphorylation of NR2B at Tyr1472site which was likely to decrease the normal physiological function of NR2B subunit and further resulting dephosphorylation of CREB. Additonally, proteasome and UCH-L1were involved in the regulation of the activity of above-mentioned molecular pathways. However, in the current study, both the expressions and activities of proteasome and UCH-L1were significantly decreased in APP/PS1mice, indicating the defective spatial learning and memory in APP/PS1might be caused by down-regulation of CREB activity which induced by dysfunctional ubiquitin-proteasome system components. Therefore, according to the above results (part1to3), our study from proteasome and UCH-L1to abnormal tau protein and to NR2B/CREB this molecular pathway revealed the mechanisms of impaired spatial memory function of APP/PS1mouse.更多还原