Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Korea.
Department of Biochemistry, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
Protein Pept Lett. 2021;28(1):115-120. doi: 10.2174/0929866527666200420105352.
The accumulation of aggregated α-synuclein (αSyn) is known as one of the critical reasons to exhibit their variable molecular pathologies and phenotypes in synucleinopathies. Recent studies suggested that the real-time quaking-induced conversion (RT-QuIC) assay is one of the potential methods to detect these αSyn aggregates and could detect the aggregated αSyn in the brain tissue and cerebrospinal fluid (CSF) using the propensity of the prion-like oligomerization.
We tried to optimize the αSyn RT-QuIC assay based on the aggregation of αSyn in brain samples of synucleinopathies by comparing the conditions of the recently reported αSyn RTQuIC assays.
This study applied a highly sensitive RT-QuIC assay using recombinant αSyn (rαSyn) to detect aggregated αSyn in the brain tissue from dementia with Lewy bodies (DLB).
This study compared αSyn RT-QuIC assays under conditions such as beads, rαSyn as a substrate, reaction buffers, and fluorescence detectors. We observed that the addition of beads and the use of 6x His-tagged rαSyn as a substrate help to obtain higher positive responses from αSyn RT-QuIC assay seeding with brain homogenate (BH) of DLB and phosphate buffer-based reaction showed higher positive responses than HEPES buffer-based reaction on both fluorescent microplate readers. We also observed that the DLB BHs gave positive responses within 15-25h, which is faster high positive responses than recently reported assays.
This established αSyn RT-QuIC assay will be able to apply to the early clinical diagnosis of αSyn aggregates-related diseases in various biofluids such as CSF.
聚集的α-突触核蛋白（αSyn）的积累被认为是在突触核蛋白病中表现出其可变分子病理学和表型的关键原因之一。最近的研究表明，实时震动诱导转化（RT-QuIC）测定是检测这些αSyn 聚集物的潜在方法之一，并且可以使用类朊病毒寡聚化的倾向来检测脑组织和脑脊液（CSF）中的聚集αSyn。
我们通过比较最近报道的αSyn RT-QuIC 测定条件，尝试基于突触核蛋白病中脑样本中αSyn 的聚集来优化 αSyn RT-QuIC 测定。
本研究应用了一种使用重组αSyn（rαSyn）的高度敏感的 RT-QuIC 测定法，以检测Lewy 体痴呆（DLB）脑组织中的聚集αSyn。
本研究比较了 RT-QuIC 测定条件，例如珠子、作为底物的 rαSyn、反应缓冲液和荧光探测器。我们观察到，添加珠子和使用 6x His 标记的 rαSyn 作为底物有助于从 DLB 脑匀浆（BH）接种的 αSyn RT-QuIC 测定中获得更高的阳性反应，基于磷酸盐缓冲液的反应比基于 HEPES 缓冲液的反应显示出更高的阳性反应在荧光微孔板读数器上。我们还观察到，DLB BH 在 15-25 小时内给出阳性反应，比最近报道的测定更快。
本建立的 αSyn RT-QuIC 测定法将能够应用于各种生物液（如 CSF）中与αSyn 聚集物相关疾病的早期临床诊断。
