Cell Biology and Gene Expression Section, Laboratory of Neurogenetics, National Institute on Aging, NIH, Bethesda, MD, USA.
Molecular Neuropathology Section, Laboratory of Neurogenetics, National Institutes on Aging, NIH, Bethesda, MD, USA.
J Parkinsons Dis. 2023;13(2):255-270. doi: 10.3233/JPD-213085.
Synucleinopathies are a group of neurodegenerative disorders that are pathologically characterized by intracellular aggregates called Lewy bodies. Lewy bodies are primarily composed of α-synuclein (asyn) protein, which is mostly phosphorylated at serine 129 (pS129) when aggregated and therefore used as a marker for pathology. Currently commercial antibodies against pS129 asyn stain aggregates well but in healthy brains cross react with other proteins, thus making it difficult to specifically detect physiological pS129 asyn.
To develop a staining procedure that detects endogenous and physiological relevant pS129 asyn with high specificity and low background.
We used the fluorescent and brightfield in situ proximity ligation assay (PLA) to specifically detect pS129 asyn in cell culture, mouse, and human brain sections.
The pS129 asyn PLA specifically stained physiological and soluble pS129 asyn in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal. However, this technique was not successful in detecting Lewy bodies in human brain tissue.
We successfully developed a novel PLA method that can, in the future, be used on in vitro and in vivo samples as a tool to explore and better understand the cellular localization and function of pS129 asyn in health and disease.
突触核蛋白病是一组神经退行性疾病，其病理学特征为细胞内聚集体，称为路易体。路易体主要由α-突触核蛋白（asyn）组成，当聚集时，asyn 蛋白主要在丝氨酸 129 位（pS129）磷酸化，因此被用作病理学标志物。目前，针对 pS129 asyn 的商业抗体可以很好地染色聚集体，但在健康大脑中与其他蛋白质发生交叉反应，因此难以特异性检测生理状态下的 pS129 asyn。
开发一种能够高度特异性和低背景检测内源性和生理相关 pS129 asyn 的染色程序。
我们使用荧光和明场原位邻近连接检测（PLA）技术特异性检测细胞培养物、小鼠和人脑切片中的 pS129 asyn。
pS129 asyn PLA 特异性染色细胞培养物、小鼠脑切片和人脑组织中的生理和可溶性 pS129 asyn，没有明显的交叉反应或背景信号。然而，该技术未能成功检测人脑组织中的路易体。
我们成功开发了一种新的 PLA 方法，该方法将来可用于体外和体内样本，作为探索和更好地理解 pS129 asyn 在健康和疾病中的细胞定位和功能的工具。
