Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid.
Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid.
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DOI：
10.1038/s41596-022-00787-3
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发表时间：
2023-04
期刊：
影响因子：
14.8
通讯作者：
Soto, Claudio
中科院分区：
文献类型：
--
作者：
Concha-Marambio, Luis;Pritzkow, Sandra;Shahnawaz, Mohammad;Farris, Carly M. M.;Soto, Claudio
错误折叠的 α-突触核蛋白 (αSyn) 聚集是帕金森病 (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-SAA）已显示出作为突触核蛋白病生化诊断测试的前景。 αSyn-SAA 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进行繁殖。在这些测定中，生物体液中循环的 αSyn 种子通过循环过程进行扩增，该过程包括聚集体碎裂成较小的自繁殖种子，然后以重组 αSyn (rec-αSyn) 为代价进行延伸。种子的扩增允许通过淀粉样蛋白特异性荧光染料（例如硫黄素 T）进行检测。过去已经发表过几份 αSyn-SAA 报告，名称为“蛋白质错误折叠循环扩增”（αSyn-PMCA）和“实时震动诱导转换”。在这里，我们描述了 αSyn-SAA 的方案，最初报道为 αSyn-PMCA，它允许检测 PD、路易体痴呆或多系统萎缩 (MSA) 患者脑脊液样本中的 αSyn 聚集体。此外，这种 αSyn-SAA 可以区分 PD 患者和 MSA 患者的 αSyn 聚集体，甚至在后来发展为 PD 或 MSA 的纯自主神经衰竭患者的回顾性样本中也是如此。我们还描述了对原始协议的修改，以开发该检测的优化版本。优化版本缩短了检测长度，减少了所需的rec-αSyn 量，并减少了不确定结果的数量。该方案每个 96 孔板的实际操作时间约为 2 小时，并且可由受过人体样本基本实验培训的人员执行。
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.
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