Neuroscience Discovery Group, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland.
J Biol Chem. 2012 Sep 28;287(40):33691-705. doi: 10.1074/jbc.M112.379792. Epub 2012 Jul 27.
Familial Parkinson disease (PD) can result from α-synuclein gene multiplication, implicating the reduction of neuronal α-synuclein as a therapeutic target. Moreover, α-synuclein content in human cerebrospinal fluid (CSF) represents a PD biomarker candidate. However, capture-based assays for α-synuclein quantification in CSF (such as by ELISA) have shown discrepancies and have limited suitability for high-throughput screening. Here, we describe two sensitive, in-solution, time-resolved Förster's resonance energy transfer (TR-FRET)-based immunoassays for total and oligomeric α-synuclein quantification. CSF analysis showed strong concordance for total α-synuclein content between two TR-FRET assays and, in agreement with a previously characterized 36 h protocol-based ELISA, demonstrated lower α-synuclein levels in PD donors. Critically, the assay suitability for high-throughput screening of siRNA constructs and small molecules aimed at reducing endogenous α-synuclein levels was established and validated. In a small-scale proof of concept compound screen using 384 well plates, signals ranged from <30 to >120% of the mean of vehicle-treated cells for molecules known to lower and increase cellular α-synuclein, respectively. Furthermore, a reverse genetic screen of a kinase-directed siRNA library identified seven genes that modulated α-synuclein protein levels (five whose knockdown increased and two that decreased cellular α-synuclein protein). This provides critical new biological insight into cellular pathways regulating α-synuclein steady-state expression that may help guide further drug discovery efforts. Moreover, we describe an inherent limitation in current α-synuclein oligomer detection methodology, a finding that will direct improvement of future assay design. Our one-step TR-FRET-based platform for α-synuclein quantification provides a novel platform with superior performance parameters for the rapid screening of large biomarker cohorts and of compound and genetic libraries, both of which are essential to the development of PD therapies.
家族性帕金森病（PD）可由α-突触核蛋白基因倍增引起，提示减少神经元α-突触核蛋白是一种治疗靶点。此外，人脑脊液（CSF）中的α-突触核蛋白含量代表 PD 生物标志物候选物。然而，用于 CSF 中α-突触核蛋白定量的基于捕获的测定法（例如 ELISA）已显示出差异，并且不适合高通量筛选。在这里，我们描述了两种用于总和寡聚α-突触核蛋白定量的灵敏、溶液内、时间分辨的Förster 共振能量转移（TR-FRET）免疫测定法。CSF 分析表明，两种 TR-FRET 测定法之间总α-突触核蛋白含量具有很强的一致性，并且与先前表征的基于 36 小时协议的 ELISA 一致，证明 PD 供体中的α-突触核蛋白水平较低。至关重要的是，建立并验证了该测定法用于筛选旨在降低内源性α-突触核蛋白水平的 siRNA 构建体和小分子的高通量筛选的适用性。在使用 384 孔板的小规模概念验证化合物筛选中，对于已知降低和增加细胞内α-突触核蛋白的分子，信号范围分别为载体处理细胞的平均值的<30 至>120%。此外，激酶定向 siRNA 文库的反向遗传筛选确定了七个调节α-突触核蛋白蛋白水平的基因（五个其敲低增加和两个降低细胞α-突触核蛋白蛋白）。这为调节α-突触核蛋白稳态表达的细胞途径提供了新的重要生物学见解，这可能有助于指导进一步的药物发现工作。此外，我们描述了当前α-突触核蛋白寡聚物检测方法学的固有局限性，这一发现将指导未来测定设计的改进。我们用于α-突触核蛋白定量的一步 TR-FRET 平台提供了一种新颖的平台，具有卓越的性能参数，可用于快速筛选大型生物标志物组以及化合物和遗传文库，这对于 PD 治疗的发展都是必不可少的。
