Ultrasensitive Detection of Aggregated α-Synuclein in Glial Cells, Human Cerebrospinal Fluid, and Brain Tissue Using the RT-QuIC Assay: New High-Throughput Neuroimmune Biomarker Assay for Parkinsonian Disorders
Ultrasensitive Detection of Aggregated α-Synuclein in Glial Cells, Human Cerebrospinal Fluid, and Brain Tissue Using the RT-QuIC Assay: New High-Throughput Neuroimmune Biomarker Assay for Parkinsonian Disorders
复制标题
DOI：
10.1007/s11481-019-09835-4
复制
发表时间：
2019-09-01
影响因子：
6.2
通讯作者：
Kanthasamy, Anumantha G.
中科院分区：
文献类型：
--
作者：
Manne, Sireesha;Kondru, Naveen;Kanthasamy, Anumantha G.
成人发作的神经退行性疾病，如帕金森病（PD）和路易体痴呆（DLB），其共有聚集的α-突触核蛋白（α Syn（agg））的积累作为其标志性分子病理学，统称为α-突触核蛋白病。诊断α-突触核蛋白病需要在死后检测不同脑区的α-Syn（agg）。最近在活体患者中测量α Syn（agg）的努力包括定量不同生物流体中的α Syn（agg）作为PD的生物标志物。我们采用实时振荡诱导转换（RT-QuIC）测定来检测非常低水平的α-Syn（agg）。我们首先通过使用单体重组人野生型α-Syn作为底物和α-Syn（agg）作为种子，优化RT-QuIC的灵敏度、特异性和再现性。接下来，我们将小鼠小胶质细胞暴露于α-Syn预形成的原纤维（α-Syn（PFF））24小时。RT-QuIC分析显示，α-Syn（PFF）在30 min内被小鼠小胶质细胞快速摄取，并在24 h内被清除。然后，我们评估了用于检测人PD、DLB和阿尔茨海默病（AD）死后脑匀浆（BH）沿着PD和进行性核上性麻痹（PSP）脑脊液（CSF）样品中的α Syn（agg）的α Syn RT-QuIC测定，然后测定α Syn（agg）的蛋白质聚集率（PAR）。与年龄匹配的健康对照和AD相比，PD和DLB BH样品不仅显示出显著更高的α Syn（agg）PAR，而且RT-QuIC也具有高度可重复性，灵敏度为94%，特异性为100%。同样，与年龄匹配的健康对照相比，PD CSF样本的α Syn（agg）PAR显著更高，灵敏度和特异性均为100%。总体而言，RT-QuIC检测试剂盒可准确检测α-Syn（agg）接种活性，为α-突触核蛋白病和其他蛋白质错误折叠疾病的生前诊断提供了潜在工具。
Adult-onset neurodegenerative disorders, like Parkinson's disease (PD) and dementia with Lewy bodies (DLB), that share the accumulation of aggregated alpha-synuclein (alpha Syn(agg)) as their hallmark molecular pathology are collectively known as alpha-synucleinopathies. Diagnosing alpha-synucleinopathies requires the post-mortem detection of alpha Syn(agg) in various brain regions. Recent efforts to measure alpha Syn(agg) in living patients include quantifying alpha Syn(agg) in different biofluids as a biomarker for PD. We adopted the real-time quaking-induced conversion (RT-QuIC) assay to detect very low levels of alpha Syn(agg). We first optimized RT-QuIC for sensitivity, specificity, and reproducibility by using monomeric recombinant human wild-type alpha Syn as a substrate and alpha Syn(agg) as the seed. Next, we exposed mouse microglia to alpha Syn pre-formed fibrils (alpha Syn(PFF)) for 24 h. RT-QuIC assay revealed that the alpha Syn(PFF) is taken up rapidly by mouse microglia, within 30 min, and cleared within 24 h. We then evaluated the alpha Syn RT-QuIC assay for detecting alpha Syn(agg) in human PD, DLB, and Alzheimer's disease (AD) post-mortem brain homogenates (BH) along with PD and progressive supranuclear palsy (PSP) cerebrospinal fluid (CSF) samples and then determined protein aggregation rate (PAR) for alpha Syn(agg). The PD and DLB BH samples not only showed significantly higher alpha Syn(agg) PAR compared to age-matched healthy controls and AD, but RT-QuIC was also highly reproducible with 94% sensitivity and 100% specificity. Similarly, PD CSF samples demonstrated significantly higher alpha Syn(agg) PAR compared to age-matched healthy controls, with 100% sensitivity and specificity. Overall, the RT-QuIC assay accurately detects alpha Syn(agg) seeding activity, offering a potential tool for antemortem diagnosis of alpha-synucleinopathies and other protein-misfolding disorders.