R&D Unit, Amprion Inc., San Diego, CA, USA.
Mitchell Center for Alzheimer's Disease and Related Brain Disorders, University of Texas McGovern Medical School, Houston, TX, USA.
Nat Protoc. 2023 Apr;18(4):1179-1196. doi: 10.1038/s41596-022-00787-3. Epub 2023 Jan 18.
Misfolded alpha-synuclein (αSyn) aggregates are a hallmark event in Parkinson's disease (PD) and other synucleinopathies. Recently, αSyn seed amplification assays (αSyn-SAAs) have shown promise as a test for biochemical diagnosis of synucleinopathies. αSyn-SAAs use the intrinsic self-replicative nature of misfolded αSyn aggregates (seeds) to multiply them in vitro. In these assays, αSyn seeds circulating in biological fluids are amplified by a cyclical process that includes aggregate fragmentation into smaller self-propagating seeds, followed by elongation at the expense of recombinant αSyn (rec-αSyn). Amplification of the seeds allows detection by fluorescent dyes specific for amyloids, such as thioflavin T. Several αSyn-SAA reports have been published in the past under the names 'protein misfolding cyclic amplification' (αSyn-PMCA) and 'real-time quaking-induced conversion'. Here, we describe a protocol for αSyn-SAA, originally reported as αSyn-PMCA, which allows detection of αSyn aggregates in cerebrospinal fluid samples from patients affected by PD, dementia with Lewy bodies or multiple-system atrophy (MSA). Moreover, this αSyn-SAA can differentiate αSyn aggregates from patients with PD versus those from patients with MSA, even in retrospective samples from patients with pure autonomic failure who later developed PD or MSA. We also describe modifications to the original protocol introduced to develop an optimized version of the assay. The optimized version shortens the assay length, decreases the amount of rec-αSyn required and reduces the number of inconclusive results. The protocol has a hands-on time of ~2 h per 96-well plate and can be performed by personnel trained to perform basic experiments with specimens of human origin.
错误折叠的α-突触核蛋白（αSyn）聚集体是帕金森病（PD）和其他突触核蛋白病的标志性事件。最近，αSyn 种子扩增测定（αSyn-SAAs）已显示出作为突触核蛋白病生物化学诊断测试的潜力。αSyn-SAAs 利用错误折叠的αSyn 聚集体（种子）的内在自我复制性质在体外扩增它们。在这些测定中，生物体液中循环的αSyn 种子通过一个周期性过程进行扩增，该过程包括将聚集体片段化为更小的自我传播种子，然后以重组αSyn（rec-αSyn）为代价伸长。种子的扩增允许通过针对淀粉样蛋白的荧光染料（例如硫黄素 T）进行检测。过去，已有几篇关于αSyn-SAA 的报告以“蛋白质错误折叠循环扩增”（αSyn-PMCA）和“实时震动诱导转化”的名称发表。在这里，我们描述了一种αSyn-SAA 协议，该协议最初以αSyn-PMCA 的名义报告，该协议允许检测来自受 PD、路易体痴呆或多系统萎缩（MSA）影响的患者的脑脊液样本中的αSyn 聚集体。此外，即使在来自仅自主衰竭患者的回顾性样本中，该αSyn-SAA 也可以区分 PD 患者与 MSA 患者的αSyn 聚集体，这些患者后来发展为 PD 或 MSA。我们还描述了对原始协议的修改，这些修改引入了优化测定版本。优化版本缩短了测定长度，减少了所需 rec-αSyn 的量，并减少了不确定结果的数量。该协议每 96 孔板的实际操作时间约为 2 小时，可以由经过培训以使用源自人体标本进行基本实验的人员执行。
