HtrA1-dependent proteolysis of TGF-β controls both neuronal maturation and developmental survival

Transforming growth factor-β (TGF-β) signalling controls a number of cerebral functions and dysfunctions including synaptogenesis, amyloid-β accumulation, apoptosis and excitotoxicity. Using cultured cortical neurons prepared from either wild type or transgenic mice over-expressing a TGF-β responsive luciferase reporter gene (SBE-Luc), we demonstrated a progressive loss of TGF-β signalling during neuronal maturation and survival. Moreover, we showed that neurons exhibit increasing amounts of the serine protease HtrA1 (high temperature responsive antigen 1) and corresponding cleavage products during both in vitro neuronal maturation and brain development. In parallel of its ability to promote degradation of TGF-β1, we demonstrated that blockage of the proteolytic activity of HtrA1 leads to a restoration of TGF-β signalling, subsequent overexpression of the serpin type -1 plasminogen activator inhibitor (PAI-1) and neuronal death. Altogether, we propose that the balance between HtrA1 and TGF-β could be one of the critical events controlling both neuronal maturation and developmental survival. Keywords: HtrA1 / neuronal survival / PAI-1 / TGF-β signalling / tPA Total RNA were extracted from 3 cultures of 2 DIV Human neurons. For each stage, equal amounts of each RNA were pooled and 5µg were reverse-transcribed, labelled and hybridized on pangenomic microarrays. Each pool was hybridized in duplicate dye-swap independent experiments.

转化生长因子-β（Transforming growth factor-β, TGF-β）信号通路调控诸多脑功能与脑功能异常事件，涵盖突触发生、β淀粉样蛋白积累、细胞凋亡以及兴奋性毒性等过程。本研究使用源自野生型或过表达TGF-β应答性荧光素酶报告基因（SBE-Luc）的转基因小鼠的体外培养皮层神经元，证实神经元成熟与存活过程中TGF-β信号通路会发生进行性丧失。此外，本研究发现，在体外神经元成熟与脑发育过程中，神经元内的丝氨酸蛋白酶HtrA1（high temperature responsive antigen 1）及其相应剪切产物的含量会逐渐升高。与此同时，鉴于HtrA1可促进TGF-β1的降解，本研究证实阻断HtrA1的蛋白水解活性能够恢复TGF-β信号通路，继而引发纤溶酶原激活物抑制剂-1（plasminogen activator inhibitor-1, PAI-1）的过表达以及神经元死亡。综上，本研究提出HtrA1与TGF-β之间的平衡或许是调控神经元成熟与发育存活的关键事件之一。
关键词：HtrA1 / 神经元存活 / PAI-1 / TGF-β信号通路 / 组织型纤溶酶原激活剂（tissue-type plasminogen activator, tPA）
从3批2天体外培养（days in vitro, DIV）的人神经元培养物中提取总RNA。针对每个培养阶段，将等量的各份RNA混合后取5μg进行反转录、标记，并在全基因组微阵列上进行杂交。每个混合样本均通过独立的重复染料互换实验完成两次杂交。