Alpha-synuclein aggregates are phosphatase resistant.

IF 6.2 2区 医学 Q1 NEUROSCIENCES Acta Neuropathologica Communications Pub Date : 2024-05-31 DOI:10.1186/s40478-024-01785-0
S G Choi, T Tittle, D Garcia-Prada, J H Kordower, R Melki, B A Killinger
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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.

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α-突触核蛋白聚集体对磷酸酶具有抗性。
α-突触核蛋白(αsyn)是一种内在紊乱的蛋白质,在几种统称为突触核蛋白病的神经退行性疾病中会在大脑中聚集。αsyn在丝氨酸129(PSER129)处的磷酸化在健康人脑中被认为是罕见的,但在病理αsyn聚集体中却富集,并被用作疾病内含物的特异性标记。然而,最近的观察结果挑战了这一假设,证明 PSER129 是神经元活动的结果,可在未患病的哺乳动物大脑中轻易检测到。在这里,我们研究了在哺乳动物大脑中检测和区分两种不同的 PSER129 池(即内源性 PSER129 和聚集性 PSER129)的实验条件。结果表明,在野生型(WT)小鼠脑中,灌注固定条件在很大程度上影响了内源性-PSER129的检测,延迟灌注固定(30分钟和1小时尸检间隔)后几乎检测不到内源性-PSER129。灌注前使用麻醉剂(如氯胺酮或甲苯噻嗪)不会显著影响内源性-PSER129的检测或水平。原位非特异性磷酸酶小牛碱性磷酸酶(CIAP)可选择性地使内源性-PSER129去磷酸化,而αsyn预成纤维蛋白(PFF)种子聚集体和真正的疾病聚集体(分别为帕金森病和多系统萎缩脑中的路易病理和帕普-兰托斯体)对CIAP介导的去磷酸化具有抗性。样品变性可消除聚集体的磷酸酶抗性,抗 CIAP 的 PSER129 与抗蛋白酶 K(PK)的 αsyn(即聚集体的标志物)密切相关。通过 CIAP 预处理,可以在积聚非聚集 PSER129 的小鼠模型中高度特异性地检测出种子αsyn 聚集。我们的结论是,αsyn 聚集体不受磷酸酶的影响,CIAP 预处理提高了聚集-PSER129 的检测特异性,尤其是在保存完好的生物样本(如灌注固定或急冻的哺乳动物组织)中,因为在这些样本中内源性-PSER129 的干扰概率很高。我们的发现对PSER129在突触核蛋白病大脑中的聚集机制具有重要意义,并提供了一种简单的实验方法来区分内源性和聚集性PSER129。
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来源期刊
Acta Neuropathologica Communications
Acta Neuropathologica Communications Medicine-Pathology and Forensic Medicine
CiteScore
11.20
自引率
2.80%
发文量
162
审稿时长
8 weeks
期刊介绍: "Acta Neuropathologica Communications (ANC)" is a peer-reviewed journal that specializes in the rapid publication of research articles focused on the mechanisms underlying neurological diseases. The journal emphasizes the use of molecular, cellular, and morphological techniques applied to experimental or human tissues to investigate the pathogenesis of neurological disorders. ANC is committed to a fast-track publication process, aiming to publish accepted manuscripts within two months of submission. This expedited timeline is designed to ensure that the latest findings in neuroscience and pathology are disseminated quickly to the scientific community, fostering rapid advancements in the field of neurology and neuroscience. The journal's focus on cutting-edge research and its swift publication schedule make it a valuable resource for researchers, clinicians, and other professionals interested in the study and treatment of neurological conditions.
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