Clearance of extracellular and cell-associated amyloid β peptide through viral expression of neprilysin in primary neurons
Clearance of extracellular and cell-associated amyloid β peptide through viral expression of neprilysin in primary neurons
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DOI：
10.1093/oxfordjournals.jbchem.a003040
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发表时间：
2001-12-01
影响因子：
2.7
通讯作者：
Saido, TC
中科院分区：
文献类型：
--
作者：
Hama, E;Shirotani, K;Saido, TC
淀粉样β肽（A β）是阿尔茨海默病（AD）的致病因子，是一种在脑内不断合成和分解的生理代谢产物。我们先前证明脑啡肽酶是体内主要的A β降解酶.为了研究脑啡肽酶活性在脑中的操纵是否是调节A β水平的有效策略，我们使用辛德毕斯病毒载体在原代皮层神经元中表达脑啡肽酶，并检查对A β代谢的影响。相应的重组蛋白，在细胞体和过程中表达，表现出噻吩敏感的内肽酶活性，而在活性位点的氨基酸取代的突变neprilysin没有表现出任何这样的活动。野生型脑啡肽酶的表达，而不是突变体的表达，导致培养基中A β 40和42水平以剂量依赖性方式显著降低。此外，脑啡肽酶表达还导致细胞相关A β减少，其可能比细胞外A β更具神经毒性。这些结果表明，操纵神经元（脑中A β的主要来源）中的脑啡肽酶活性将是用于控制A β水平并因此控制脑组织中A β相关病理的相关策略。
Amyloid beta peptide (A beta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite constantly anabolized and catabolized in the brain. We previously demonstrated that neprilysin is the major A beta -degrading enzyme in vivo. To investigate whether or not manipulation of neprilysin activity in the brain would be an effective strategy for regulating A beta levels, we expressed neprilysin in primary cortical neurons using a Sindbis viral vector and examined the effect on A beta metabolism. The corresponding recombinant protein, expressed in the cell bodies and processes, exhibited thiorphan-sensitive endopeptidase activity, whereas a mutant neprilysin with an amino acid substitution in the active site did not show any such activity. Expression of the wildtype neprilysin, but not the mutant, led to significant decreases in both the A beta 40 and 42 levels in the culture media in a dose-dependent manner. Moreover, neprilysin expression also resulted in reducing cell-associated A beta, which could be more neurotoxic than extracellular A beta. These results indicate that the manipulation of neprilysin activity in neurons, the major source of A beta in the brain, would be a relevant strategy for controlling the A beta levels and thus the A beta -associated pathology in brain tissues.