ABSTRACT

Research on Parkinson’s disease (PD) has been focused on the development of PD diagnostic tools as much as the development of PD therapeutics. Several genetic culprits of PD, including DJ-1, Leucine-rich repeat kinase 2 (LRRK2), and α-synuclein (α-syn), have been investigated as markers of PD in human biofluids. Unfortunately, the approaches to develop PD diagnostic tools are impractical, and there is a considerable demand for an appropriate marker of PD. The measurement of α-syn in biofluids has recently been made more accurate by examining monomers and aggregates separately using enzyme-linked immunosorbent assay (ELISA). Previously, we reported on the development of two types of sandwich ELISA for total α-syn and MJFR-14-6-4-2 antibody-specific α-syn fibrillar oligomers. The pathogenic LRRK2 G2019S mutation is related to increased α-syn secretion in the extracellular space. We tested our established ELISA using differentiated SH-SH5Y cells transfected with LRRK2 G2019S. The secretory levels of fibrillar oligomeric α-syn divided by total α-syn were significantly increased in LRRK2 G2019S-expressing cells. Additionally, substantia nigra lysates or concentrated urine from PD patients and non-PD subjects were analyzed. We observed ambiguous changes in the levels of total or fibrillar oligomeric α-syn and their ratio between PD and non-PD. Despite the insignificant increase in the relative levels of fibrillar oligomeric α-syn to total α-syn in PD, the duration of disease progression after diagnosis significantly corresponded to the relative levels of fibrillar oligomeric α-syn to total α-syn in the urine. These results might provide greater understanding for the next stage of development of α-syn ELISAs.

摘要

帕金森氏病（PD）的研究一直侧重于PD诊断工具的开发以及PD治疗剂的开发。已经研究了PD的几种遗传元凶，包括DJ-1，富含亮氨酸的重复激酶2（LRRK2）和α-突触核蛋白（α-syn），作为人类生物流体中PD的标记。不幸的是，开发PD诊断工具的方法是不切实际的，并且对PD的合适标记物有相当大的需求。最近，通过使用酶联免疫吸附测定（ELISA）分别检查单体和聚集体，已使生物流体中α-syn的测量更加准确。以前，我们报道了两种针对总α-syn和MJFR-14-6-4-2抗体特异性α-syn纤维状寡聚物的夹心ELISA的开发。致病性LRRK2 G2019S突变与细胞外空间α-syn分泌增加有关。我们使用转染了LRRK2 G2019S的分化SH-SH5Y细胞测试了我们建立的ELISA。在LRRK2 G2019S表达细胞中，原纤维寡聚α-syn的分泌水平除以总α-syn的水平显着增加。另外，分析了来自PD患者和非PD患者的黑质溶解物或浓缩尿液。我们观察到总或原纤维寡聚α-syn的水平及其在PD和非PD之间的比率存在歧义变化。尽管PD中原纤维寡聚α-syn相对于总α-syn的相对水平无显着增加，但诊断后疾病进展的持续时间明显对应于尿液中原纤维寡聚α-syn与总α-syn的相对水平。