Alpha-synuclein aggregates are phosphatase resistant.
Alpha-synuclein aggregates are phosphatase resistant.
复制标题
α-突触核蛋白聚集体具有磷酸酶抗性。
DOI：
10.1101/2023.11.20.567854
复制
发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Killinger,BA
中科院分区：
文献类型：
--
作者：
Choi,SG;Tittle,T;Garcia-Prada,D;Kordower,JH;Melki,R;Killinger,BA
α-突触核蛋白是一种本质上无序的蛋白质，在统称为突触核蛋白病的几种神经退行性疾病中聚集在大脑中。α-突触核蛋白（αsyn）丝氨酸129（PSER 129）的磷酸化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，并用作疾病包涵体的特异性标志物。然而，最近的观察结果挑战了这一假设，证明PSER 129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。在这里，我们测试了实验条件，在该条件下可以在大脑中检测和区分两种不同的PSER 129池：内源性PSER 129和聚集性PSER 129。结果显示，在小鼠脑中，灌注固定条件极大地影响内源性PSER 129的检测，延迟灌注固定（死后30分钟和1小时间隔）后几乎检测不到内源性PSER 129。灌注前暴露于麻醉剂（例如氯胺酮和甲苯噻嗪）不会显著影响内源性PSER 129检测或水平。在原位，非特异性磷酸酶（即，小牛碱性磷酸酶，CIAP）选择性地去磷酸化内源性PSER 129，而预制的纤维接种的聚集体和真正的疾病聚集体（路易病理和帕普兰托斯体在帕金森氏病和多系统脑萎缩，分别）是耐CIAP介导的去磷酸化。通过样品变性，聚集体的磷酸酶抗性被废除。我们的结论是，在哺乳动物的大脑中，有一个丰富的内源性PSER 129的稳定状态，PSER 129与病理学的关联，至少部分是由于αsyn聚集体的去磷酸化阻力。我们的研究结果对PSER 129在突触核蛋白病大脑中积累的机制有影响。
Alpha-synuclein is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of alpha-synuclein (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 tested experimental conditions under which two distinct PSER129 pools, endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the brain. Results showed that in the mouse brain, perfusion fixation conditions greatly influenced detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30min and 1hr post-mortem interval). Exposure to anesthetics (eg, ketamine and xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase (ie, calf alkaline phosphatase, CIAP) selectively dephosphorylated endogenous-PSER129 while preformed fibril-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. Phosphatase resistance of aggregates was abolished by sample denaturation. We conclude that in the mammalian brain, there is an abundant steady state of endogenous-PSER129, and PSER129 association with pathology is, at least partially, due to dephosphorylation resistance of αsyn aggregates. Our findings have implications for the mechanism of PSER129-accumulation in the synucleinopathy brain.