Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia.
Department of Pharmacology & Therapeutics, The University of Melbourne, Melbourne, VIC, 3010, Australia.
Mol Neurobiol. 2019 Jan;56(1):13-28. doi: 10.1007/s12035-018-1070-4. Epub 2018 Apr 19.
The amyloid precursor protein (APP) undergoes extensive metabolism, and its transport and proteolytic processing can be modulated by its ability to form a homodimer. We have investigated the functional consequences of stabilised APP dimer expression in cells by studying the engineered dimerisation of the APP (residue 17 in Aβ sequence) construct, which is associated with a 30% increase in APP dimer expression, on APP's neurite outgrowth promoting activity. Overexpression of APP in SH-SY5Y cells decreased neurite outgrowth upon retinoic acid differentiation as compared to overexpressing APP cells. The APP phenotype was rescued by replacing the APP media with conditioned media from APP cells, indicating that the APP mutant is impairing the secretion of a neuritogenic promoting factor. APP had altered transport and was localised in the endoplasmic reticulum. Defining the molecular basis of the APP phenotype showed that RhoA GTPase activity, a negative regulator of neurite outgrowth, was increased in APP cells. RhoA activity was decreased after APP conditioned media rescue. Moreover, treatment with the RhoA inhibitor, Y27632, restored a wild-type morphology to the APP cells. Small RNAseq analysis of APP and APP cells identified several differentially expressed miRNAs relating to neurite outgrowth. Of these, miR-34a showed the greatest decrease in expression. Lentiviral-mediated overexpression of miR-34a rescued neurite outgrowth in APP cells to APP levels and changed RhoA activation. This study has identified a novel link between APP dimerisation and its neuritogenic activity which is mediated by miR-34a expression.
淀粉样前体蛋白 (APP) 经历广泛的代谢，其运输和蛋白水解加工可以通过其形成同源二聚体的能力来调节。我们通过研究 APP（Aβ 序列中的残基 17）构建体的工程化二聚化来研究稳定的 APP 二聚体表达对 APP 的神经突生长促进活性的功能后果，该构建体与 APP 二聚体表达增加 30%相关。与过表达 APP 细胞相比，在 SH-SY5Y 细胞中过表达 APP 会在维甲酸分化时降低神经突生长。用来自 APP 细胞的条件培养基替代 APP 培养基可挽救 APP 表型，表明 APP 突变体损害了神经营养促进因子的分泌。APP 的运输发生改变，并定位于内质网。定义 APP 表型的分子基础表明，神经突生长的负调节剂 RhoA GTPase 活性在 APP 细胞中增加。APP 条件培养基挽救后 RhoA 活性降低。此外，用 RhoA 抑制剂 Y27632 处理可使 APP 细胞恢复为野生型形态。APP 和 APP 细胞的小 RNAseq 分析确定了与神经突生长相关的几种差异表达 miRNA。其中，miR-34a 的表达下降最大。miR-34a 的慢病毒介导过表达可使 APP 细胞的神经突生长恢复至 APP 水平并改变 RhoA 激活。这项研究确定了 APP 二聚化与其神经营养活性之间的新联系，该联系由 miR-34a 的表达介导。
