【作者】 杨细飞；

【导师】 王建枝；

【作者基本信息】 华中科技大学 ， 病理生理学， 2007， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease, AD)以β-淀粉样肽(β-amyloid,Aβ)大量生成和tau蛋白过度磷酸化为主要特征。Aβ与异常磷酸化的tau蛋白的联系及其分子机制尚不清楚,仍然没有有效的措施能阻止AD的发展。在目前研究中,我们发现患有家族性阿尔茨海默病早老素1-L235P(在密码子235位亮氨酸变成脯氨酸)突变的老年(16～17月龄)转基因鼠Aβ42的生成和在细胞内的沉积显著增多。同时,我们检测了tau蛋白的磷酸化修饰,发现老年转基因鼠丝氨酸396,404和198/199/202位点tau磷酸化程度比同月龄正常鼠显著升高。我们也观察到老年转基因鼠丙二醛(malondialdehyde,MDA)水平比同月龄正常鼠升高,而超氧化物歧化酶(superoxide dismutase,SOD)水平则降低,表明转基因鼠氧化应激水平增高。我们进一步检测了Akt-GSK-3β信号途径的修饰,发现老年转基因鼠Akt、GSK-3β的磷酸化程度降低,提示Akt-GSK-3β信号途径发生抑制。外源性给予辅酶Q10(一个强有力的抗氧化剂和自由基清除剂),不仅能部分降低MDA的水平,增加SOD的活性,而且能减少Aβ42的生成,降低tau蛋白过度磷酸化,同时伴有Akt-GSK-3β信号途径的恢复。这些结果表明早老素-1突变选择性地增加Aβ42的生成和在细胞内的沉积,并使tau蛋白过度磷酸化。后者可能是细胞内过多的Aβ诱导的氧化应激的下游事件,其机制可能涉及氧化应激诱导的Akt-GSK-3β信号途径的抑制。辅酶Q10通过抗氧化特性发挥其神经保护作用。这些结果也提示早老素-1突变的致病角色是淀粉样肽级联反应和tau病理的上游事件,而辅酶Q10可能是一个潜在有用的治疗阿尔茨海默病的药物。小结我们的研究发现:1)在携带L235P突变的早老素-1的老年转基因鼠的海马和皮层发生了AD样病理改变:β淀粉样变和tau蛋白过度磷酸化。2)该老年转基因鼠的相应脑区表现出了增强的氧化应激反应和Akt-GSK-3β信号途径的抑制。3)外源性补充辅酶Q10不仅降低了氧化应激反应水平,而且能减少Aβ42的生成,降低tau蛋白的过度磷酸化,同时伴有Akt-GSK-3β信号途径的恢复。这些结果表明突变的早老素-1是淀粉样肽级联反应和tau蛋白病理改变的上游事件,氧化应激可能在介导突变的早老素-1的致病作用中发挥重要作用,而辅酶Q10可能是一个潜在有用的治疗阿尔茨海默病的药物。第二部分褪黑素改善花萼海绵诱癌素诱导的轴突运输障碍和大鼠空间记忆损伤最近,我们发现褪黑素能保护SH-SY5Y成神经瘤细胞免于花萼海绵诱癌素(calyculin A,CA)引起的神经细丝损伤和神经毒性作用。在我们现在的研究中,通过双侧海马注射CA—一个有效的特异性蛋白磷酸酯酶2A(protein phosphatase -2A,PP-2A)和蛋白磷酸酯酶1(protein phosphatase-1,PP-1)的抑制剂,建立大鼠空间记忆损伤模型,在整体上研究了褪黑素对大鼠空间记忆的保护作用。结果表明在CA注射前9天腹腔内给予褪黑素,不仅显著改善了CA引起的记忆损伤,而且对CA诱导的神经细胞骨架蛋白tau和神经细丝的过度磷酸化也有保护作用。为进一步揭示褪黑素上述保护作用的潜在机制,用pEGFP标记N2a细胞内中分子量神经细丝,通过全内反射显微成像系统研究轴突运输,我们在轴突样突起中发现褪黑素能部分地逆转CA诱导的轴突运输障碍。免疫印迹结果显示褪黑素还能抑制CA诱导的骨架蛋白tau和神经细丝的过度磷酸化。此外,我们建立了轴突样突起生长的细胞模型,用以揭示褪黑素对磷酸酯酶活性处于抑制状态时细胞形态学改变的影响,并借助体视学系统分析细胞突起的长度变化。我们发现CA处理抑制细胞突起的生长,长时间使用CA处理引起细胞突起缩短,并可见早期神经退行性轴突曲张样改变,褪黑素明显阻止这些病理改变。这些结果表明褪黑素不仅能改善因PP-2A和PP-1活性抑制所致的轴突转运障碍,也能抑制神经细胞退行性变性,这些效应可能构成了褪黑素对磷酸酯酶活性抑制所引起的记忆缺失的保护作用的基础。小结我们的研究发现:褪黑素不仅部分改善了花萼海绵诱癌素诱导的神经细丝的运输障碍及细胞退化样的改变,降低了骨架蛋白tau和神经细丝的过度磷酸化,而且改善了花萼海绵诱癌素诱导的空间记忆保留障碍。这些结果提示骨架蛋白的过度磷酸化可能在介导轴突运输障碍,轴突病变及空间记忆损伤等AD病理改变中起关键作用,褪黑素通过降低骨架蛋白的过度磷酸化部分逆转上述病理改变,提示褪黑素可能是有效治疗AD的药物。更多还原

【Abstract】 Alzheimer’s disease is characterized byβ-amyloid (Aβ) overproduction and tau hyperphosphorylation. The underlying mechanisms linking Aβand aberrant tau phosphorylation are poorly understood, and therefore there are no effective therapeutic measures for arresting the AD pathology. In our present study, we found that familial Alzheimer disease mutation with presenilin 1-L235P(leucine-to-proline mutation at codon 235 ) transgenic mice (PS-1 Tg) enhanced the generation of Aβ42, and significantly increased intracellular Aβ42 deposition in the brain of the aged Tg mice (16-17 months old). We also determined the alterations of phosphorylated tau and found that phosphorylation of tau at pSer396, Tau-1 (Ser198/199/202) and pSer404 epitopes was significantly increased in the aged Tg mice compared with the age-matched wild type mice (WT). Meanwhile, we detected an increased oxidative stress reaction as evidenced by elevated level of malondialdehyde (MDA) and decreased activity of superoxide dismutase (SOD) in the aged Tg mice, compared to the WT mice. We further detected the alterations in Akt-GSK-3βsignaling pathway and found a decreased phosphorylation of Akt, glycogen synthase kinase (GSK)-3βin the aged Tg mice. Exogenous supplementation of coenzyme Q10 (CoQ10), a powerful antioxidant and free radical scavenger, not only partially decreased MDA level and up-regulated the activity of SOD, but also arrested Aβ42 level and tau hyperphosphorylation, concurrently accompanied with a restoration of Akt-GSK-3βsignaling. These results suggest that the mutated PS-1 increases Aβ42 production and intracellular deposition selectively and tau hyperphosphorylation. The latter may be a consequence of intracellular overproduced Aβinduced-oxidative stress and the mechanisms may involve depression of oxidative stress-induced Akt-GSK-3βsignaling events. The neuroprotective effects of CoQ10 may act through its antioxidant property. Our data also indicate that the pathogenic role of the mutated PS-1 is upstream of the amyloid cascade and taupathy, and CoQ10 might be potentially useful for therapy of Alzheimer’s disease.Part twoMelatonin Meliorates Calyculin A-induced Axonal Transport Defects and Spatial Memory Retention Impairment in RatsWe have recently found that melatonin protects SH-SY5Y neuroblastoma cells from calyculin A(CA)-induced neurofilament impairment and neurotoxicity. In the present study, we further investigated the in vivo effect of melatonin on spatial memory retention in rats by bilateral injection of CA, a potent and specific inhibitor of protein phosphatase-2A (PP-2A) and protein phosphatase-1 (PP-1) into hippocampus. We found that the supplementation of melatonin by intraperitoneal injection for 9 days before injecting CA not only significantly improves CA-induced memory retention deficits of the rats in the Morris Water-Maze Test, but also decreases hyperphosphorylation of neuronal cytoskeletal protein tau and neurofilaments. To further explore the underlying mechanisms of the protective effect of melatonin on spatial memory deficit in rats induced by CA, we analyzed the axonal transport by a time-lapse recording of pEGFP-labeled neurofilament-M (pEGFP-NF-M) subunit in live neuroblastoma N2a cells and found that melatonin partially reversed CA-induced impairment of transport of pEGFP- NF-M in the axon-like processes of the cells. In the meantime, we found by western blot that melatonin also decreased CA-induced hyperphosphorylation of cytoskeletal proteins. In addition, we explored the effect of melatonin on the morphological alteration of the cells during inhibition of the phosphatases by establishing a cell model showing steady outgrowth of axon-like cell processes and employed a stereological system to analyze the retraction of the processes. We found CA-treatment inhibited outgrowth of the cell processes and prolonged treatment with CA caused retraction of the processes and meanwhile, the early neurodegenerative varicosities were also obvious in the CA-treated cells, and the administration of melatonin significantly arrested these pathological alterations. These data suggest that melatonin not only meliorated impaired intracellular transport induced by suppression of PP-2A and PP-1, but also cell degeneration, which may underlie the basis of the effects of protection of melatonin on memory deficits induced by suppression of the phosphatases.更多还原