Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology Shanghai 200237, China.
Am J Transl Res. 2013 Sep 25;5(6):608-21. eCollection 2013.
We investigated the subcellular distribution of NEP protein and activity in brains of human individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD dementia, as well as double transgenic mice and human neuronal cell line treated with Aβ and 4-hydroxy-2-nonenal (HNE). Total cortical neuronal-related NEP was significantly increased in MCI compared to NCI brains. NeuN was decreased in both MCI and AD, consistent with neuronal loss occurring in MCI and AD. Negative relationship between NEP protein and NeuN in MCI brains, and positive correlation between NEP and pan-cadherin in NCI and MCI brains, suggesting the increased NEP expression in NCI and MCI might be due to membrane associated NEP in non-neuronal cells. In subcellular extracts, NEP protein decreased in cytoplasmic fractions in MCI and AD, but increased in membrane fractions, with a significant increase in the membrane/cytoplasmic ratio of NEP protein in AD brains. By contrast, NEP activity was decreased in AD. Similar results were observed in AD-mimic transgenic mice. Studies of SH-SY5Y neuroblastoma showed an up-regulation of NEP protein in the cytoplasmic compartment induced by HNE and Aβ; however, NEP activity decreased in cytoplasmic fractions. Activity of NEP in membrane fractions increased at 48 hours and then significantly decreased after treatment with HNE and Aβ. The cytoplasmic/membrane ratio of NEP protein increased at 24 hours and then decreased in both HNE and Aβ treated cells. Both HNE and Aβ up-regulate NEP expression, but NEP enzyme activity did not show the same increase, possibly indicating immature cytoplasmic NEP is less active than membrane associated NEP. These observations indicate that modulation of NEP protein levels and its subcellular location influence the net proteolytic activity and this complex association might participate in deficiency of Aβ degradation that is associated with amyloid deposition in AD.
我们研究了无认知障碍（NCI）、轻度认知障碍（MCI）和 AD 痴呆患者大脑中 NEP 蛋白和活性的亚细胞分布，以及用 Aβ 和 4-羟基-2-壬烯醛（HNE）处理的双转基因小鼠和人神经元细胞系。与 NCI 大脑相比，MCI 大脑中总皮质神经元相关的 NEP 显著增加。MCI 和 AD 中的 NeuN 减少，与 MCI 和 AD 中发生的神经元丢失一致。MCI 大脑中 NEP 蛋白与 NeuN 呈负相关，NCI 和 MCI 大脑中 NEP 与泛钙粘蛋白呈正相关，提示 NCI 和 MCI 中 NEP 表达增加可能是由于非神经元细胞中膜相关的 NEP。在亚细胞提取物中，MCI 和 AD 中细胞质部分的 NEP 蛋白减少，而膜部分增加，AD 大脑中 NEP 蛋白的膜/细胞质比值显著增加。相比之下，AD 中 NEP 活性降低。在 AD 模拟转基因小鼠中也观察到了类似的结果。对 SH-SY5Y 神经母细胞瘤的研究表明，HNE 和 Aβ 诱导细胞质部分 NEP 蛋白上调；然而，细胞质部分的 NEP 活性降低。HNE 和 Aβ 处理后，膜部分的 NEP 活性在 48 小时增加，然后显著降低。NEP 蛋白的细胞质/膜比值在 24 小时增加，然后在 HNE 和 Aβ 处理的细胞中降低。HNE 和 Aβ 均上调 NEP 表达，但 NEP 酶活性没有相同的增加，这可能表明不成熟的细胞质 NEP 比膜相关的 NEP 活性更低。这些观察表明，NEP 蛋白水平及其亚细胞位置的调节影响净蛋白水解活性，这种复杂的关联可能参与与 AD 中淀粉样沉积相关的 Aβ 降解不足。
