Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama.
J Comp Neurol. 2018 Aug 15;526(12):1978-1990. doi: 10.1002/cne.24468.
α-Synuclein (α-syn) is an abundant presynaptic protein that is the primary constituent of inclusions that define Lewy body diseases (LBDs). In these inclusions, α-syn is phosphorylated at the serine-129 residue. Antibodies directed to this phosphorylation site are used to measure inclusion abundance and stage disease progression in preclinical models as well as in postmortem tissues in LBDs. While it is critical to reliably identify inclusions, phospho-specific antibodies often cross-react with nonspecific antigens. Four commercially available monoclonal antibodies, two from rabbits (clones EP1536Y and MJF-R13) and two from mice (81a and pSyn#64), have been the most widely used in detecting pS129-α-syn inclusions. Here, we systematically evaluated these antibodies in brain sections and protein lysates from rats and mice. All antibodies detected pS129-α-syn inclusions in the brain that were induced by preformed α-syn fibrils in wild-type rats and mice. Antibody titrations revealed that clones EP1536Y and 81a comparably labeled inclusions in both the perikarya and neuronal processes in contrast to clones MJF-R13 and pSyn#64 that incompletely labeled inclusions at various antibody concentrations. Except for EP1536Y, the clones produced nonspecific diffuse neuropil labeling in α-syn knockout mice as well as mice and rats injected with monomeric α-syn, with some nonspecific staining titrating with pS129-α-syn inclusions. By immunoblot, all the clones cross-reacted with proteins other than α-syn, warranting caution in interpretations of specificity. Clone EP1536Y uniquely and robustly detected endogenous pS129-α-syn in highly soluble protein fractions from the mouse brain. In summary, EP1536Y had the highest sensitivity and specificity for detecting pS129-α-syn.
α-突触核蛋白（α-syn）是一种丰富的突触前蛋白，是定义路易体病（LBD）的包涵体的主要成分。在这些包涵体中，α-syn 在丝氨酸-129 残基处发生磷酸化。针对该磷酸化位点的抗体被用于测量临床前模型和 LBD 死后组织中的包涵体丰度和疾病进展阶段。虽然可靠地识别包涵体至关重要，但磷酸化特异性抗体通常与非特异性抗原发生交叉反应。四种市售的单克隆抗体，两种来自兔（克隆 EP1536Y 和 MJF-R13），两种来自小鼠（81a 和 pSyn#64），已被最广泛用于检测 pS129-α-syn 包涵体。在这里，我们系统地评估了这些抗体在大鼠和小鼠脑组织切片和蛋白裂解物中的应用。所有抗体均检测到野生型大鼠和小鼠中预先形成的 α-syn 纤维诱导的 pS129-α-syn 包涵体。抗体滴定表明，与不完全标记各种抗体浓度下包涵体的克隆 MJF-R13 和 pSyn#64 相比，克隆 EP1536Y 和 81a 可比地标记了胞体和神经元突起中的包涵体。除了 EP1536Y 之外，这些克隆在α-syn 敲除小鼠以及注射单体α-syn 的小鼠和大鼠中产生了非特异性弥漫性神经胶质标记，一些非特异性染色与 pS129-α-syn 包涵体滴定。通过免疫印迹，所有克隆均与除α-syn 之外的蛋白质发生交叉反应，这需要谨慎解释特异性。克隆 EP1536Y 独特且强烈地检测到来自小鼠大脑的高度可溶性蛋白级分中的内源性 pS129-α-syn。综上所述，EP1536Y 对检测 pS129-α-syn 具有最高的敏感性和特异性。
