Enhanced Phospholipase A2 group 3 expression by oxidative stress decreases the insulin-degrading enzyme. Mus musculus

Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa-/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa-/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa-/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD. Overall design: Gene expression in cerebral cortex and cerebellum of mice were determined using Agilent chips. To ensure higher quality results in gene expression data, we conducted microarrays on 4 mice per group. Young mice were 2 months old and the other aged mice were 29 months old at the time of use. Data were standardized using global normalization and pro-cessed by R-program. An absolute fold change threshold of greater than 1.5 was required to be considered for further analyses. Expression values were in log2 scale.

氧化应激在神经退行性疾病中广泛存在，大脑特定区域的氧化损伤与选择性神经元变性紧密关联。本团队此前研究发现，阿尔茨海默病（Alzheimer disease, AD）模型小鼠的大脑皮质中胰岛素降解酶（insulin-degrading enzyme, IDE）水平降低，且与α-生育酚转移蛋白敲除（alpha-tocopherol transfer protein knockout, Ttpa-/-）小鼠杂交后，AD的表型特征会显著加速出现。为进一步探究慢性氧化应激在AD病理生理进程中的作用，我们以年轻野生型小鼠与老年Ttpa-/-小鼠为实验对象，开展了DNA微阵列分析。在表达发生显著改变的基因中，磷脂酶A2家族3型（Phospholipase A2 group 3, Pla2g3）进入研究视野；通过计算机模拟（in silico）分析与老年Ttpa-/-小鼠实验验证，该基因呈现出慢性氧化应激诱导的大脑特异性上调表达谱。免疫组织化学实验进一步证实，与对照脑组织相比，人类AD患者脑组织内星形胶质细胞来源的Pla2g3表达水平显著升高。此外，向HEK293细胞中转染人源Pla2g3后，内源性IDE的表达呈现剂量依赖性下调趋势。本研究结果揭示了Pla2g3在降低IDE水平过程中的关键调控作用，并提示大脑特异性高表达Pla2g3参与了AD的起始和/或进展过程。实验整体设计：本研究通过安捷伦（Agilent）芯片检测小鼠大脑皮层与小脑的基因表达水平。为保障基因表达数据的可靠性，每组设置4只小鼠进行微阵列实验。实验所用年轻小鼠为2月龄，老年小鼠为29月龄。数据通过全局标准化方法完成归一化处理，并采用R语言程序进行后续分析。后续筛选仅纳入绝对倍数变化大于1.5的基因，基因表达值以log2标度进行呈现。