Inserm, U913, Nantes, F-44093, France.
Nantes University, Nantes, F-44093, France.
J Parkinsons Dis. 2015;5(4):765-72. doi: 10.3233/JPD-150670.
Alpha-synuclein containing inclusions in neurons, the characteristic pathological lesions of Parkinson's disease (PD), are not limited to the central nervous system, but also affect the enteric nervous system (ENS). This suggests that the ENS offer some potential as a surrogate of central nervous system pathology and that it may represent an original source of biomarkers for PD. However, the usefulness of α-synuclein detection in gastrointestinal biopsies as a biomarker for PD is still unclear, as the different immunohistochemical methods employed to date have led to conflicting results.
Our aim is to propose an optimized immunoblotting method for the detection of endogenous α-synuclein in the healthy ENS that may be used to supplement the immunohistochemical analysis.
Primary culture of rat ENS and homogenates of human small intestine were analyzed by Western Blot using seven different α-synuclein and phospho-α-synuclein antibodies along with two methods that increase α-synuclein retention on blot membranes, namely incubation of the membranes with paraformaldehyde (PFA) or treatment of samples with the crosslinker dithiobis[succinimidylpropionate] (DSP).
A moderate improvement in the detection of endogenous enteric α-synuclein was observed following membrane fixation with PFA for only two of the seven antibodies we tested. Immunodetection of total and phosphorylated α-synuclein in the ENS was markedly improved when samples were treated with DSP, regardless of the antibody used.
Our results demonstrate that the detection of α-synuclein in the gut by Western Blot can be optimized by using methods for enhanced membrane retention of the protein along with the appropriate antibody. Such an optimized protocol opens the way to the development of novel biomarkers for PD that will enable a quantification of α-synuclein in gastrointestinal biopsies.
神经元中含有α-突触核蛋白的包涵体是帕金森病（PD）的特征性病理病变，不仅局限于中枢神经系统，还影响肠神经系统（ENS）。这表明 ENS 提供了一些作为中枢神经系统病理学替代物的潜力，并且它可能代表 PD 生物标志物的原始来源。然而，用胃肠道活检中的α-突触核蛋白检测作为 PD 的生物标志物的有用性尚不清楚，因为迄今为止使用的不同免疫组织化学方法导致了相互矛盾的结果。
我们的目的是提出一种优化的免疫印迹方法，用于检测健康 ENS 中的内源性α-突触核蛋白，该方法可用于补充免疫组织化学分析。
使用 Western Blot 分析大鼠 ENS 的原代培养物和人小肠的匀浆，共使用七种不同的α-突触核蛋白和磷酸化α-突触核蛋白抗体，以及两种增加α-突触核蛋白在印迹膜上保留的方法，即膜用多聚甲醛（PFA）孵育或用交联剂二硫代双琥珀酰亚胺基丙酸酯（DSP）处理样品。
在我们测试的七种抗体中，只有两种抗体在膜用 PFA 固定后观察到内源性肠内α-突触核蛋白的检测略有改善。无论使用哪种抗体，用 DSP 处理样品都可以显著改善 ENS 中总和磷酸化α-突触核蛋白的免疫检测。
我们的结果表明，通过使用增强蛋白在膜上保留的方法以及适当的抗体，可以优化通过 Western Blot 检测肠道中的α-突触核蛋白。这种优化的方案为开发用于 PD 的新型生物标志物开辟了道路，从而能够对胃肠道活检中的α-突触核蛋白进行定量。
