PDF(Pubmed)
Abstract：
Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson\'s disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.
摘要：
α-突触核蛋白（αsyn）是一种内在无序的蛋白质，在几种神经退行性疾病中聚集在大脑中，统称为突触核蛋白病。在丝氨酸129处的αsyn磷酸化（PSER129）在健康人脑中被认为是罕见的，但在病理性αsyn聚集体中富集，并用作疾病内含物的特定标记。然而,最近的观察通过证明PSER129是由神经元活动引起的，并且可以在未患病的哺乳动物大脑中容易地检测到，从而挑战了这一假设。这里,我们调查了两个不同的PSER129池的实验条件，即内源性PSER129和聚集的PSER129可以在哺乳动物大脑中检测和分化。结果表明,在野生型(WT)小鼠脑中,灌注固定条件极大地影响了内源性PSER129的检测,延迟灌注固定后(死后30分钟和1小时),内源性PSER129几乎检测不到.暴露于麻醉剂(例如，灌注前的氯胺酮或赛拉嗪）不会显着影响内源性PSER129的检测或水平。在原地,非特异性磷酸酶小牛碱性磷酸酶（CIAP）选择性去磷酸化内源性PSER129，而αsyn预形成原纤维（PFF）种子聚集体和真正的疾病聚集体（帕金森病和多系统萎缩脑中的路易病理学和Papp-Lantos体，分别）是对CIAP介导的去磷酸化的抗性。样品变性消除了聚集体的磷酸酶抗性，和CIAP抗性PSER129与蛋白酶K（PK）抗性αsyn密切相关（即，聚集的标记)。CIAP预处理允许在积累非聚集的PSER129的小鼠模型中高度特异性地检测接种的αsyn聚集体。我们得出的结论是，αsyn聚集体对磷酸酶是不可渗透的，和CIAP预处理增加了聚集的PSER129的检测特异性，特别是在保存完好的生物样品中(例如，灌注固定或快速冷冻的哺乳动物组织），其中很有可能受到内源性PSER129的干扰。我们的发现对突触核蛋白病脑中PSER129积累的机制具有重要意义，并提供了一种简单的实验方法来区分内源性和聚集的PSER129。
