Proteomic profile of nuclei containing p62-positive inclusions in a patient with neuronal intranuclear inclusion disease
Proteomic profile of nuclei containing p62-positive inclusions in a patient with neuronal intranuclear inclusion disease
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DOI：
10.1016/j.nbd.2023.105989
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发表时间：
2023-01
影响因子：
6.1
通讯作者：
M. Kurihara;T. Mano;Fumihiro Eto;I. Yao;Kenichiro Sato;G. Ohtomo;T. Bannai;Shota Shibata;H. Ishiura;M. Ikemura;T. Matsubara;M. Morishima;Yuko Saito;Shigeo Murayama;Tatsushi Toda;M. Setou;A. Iwata
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文献类型：
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作者：
M. Kurihara;T. Mano;Fumihiro Eto;I. Yao;Kenichiro Sato;G. Ohtomo;T. Bannai;Shota Shibata;H. Ishiura;M. Ikemura;T. Matsubara;M. Morishima;Yuko Saito;Shigeo Murayama;Tatsushi Toda;M. Setou;A. Iwata
神经元核内包涵体病(NIID)是一种以神经元、神经胶质细胞和其他体细胞中嗜酸性透明质核内包涵体为特征的神经退行性疾病。虽然在大多数东亚NIID患者中已发现NOTCH2NLC中CGG重复扩增，但NIID的病理生理机制尚不清楚。泛素和p62阳性的核内包涵体是NIID的病理标志。靶向免疫染色研究已经确定了这些包涵体中存在的其他几种蛋白质。然而，含有这些包涵体的核内的整体分子变化尚不清楚。在此，我们分析了一例NOTCH2NLC有CGG重复扩增的NIID患者p62阳性包涵体细胞核的蛋白质组学特征，以发现与NIID病理生理相关的候选蛋白质。我们使用荧光激活的细胞分选和LC-MS/MS(LC-MS/MS)来定量检测在含有p62阳性包涵体的细胞核中鉴定的每一种蛋白质。通过免疫荧光在三名遗传确诊的NIID患者的尸检脑样本中证实了增加的蛋白质的分布。总体而言，共鉴定了526种蛋白质，其中243种蛋白质被一致地用MS进行了定量，持续观察到有p62阳性包涵体的细胞核中的20种蛋白质比没有包涵体的核中的蛋白质增加了1.4倍。对15种在LC-MS/MS分析中具有中等或高度置信度的蛋白质进行了进一步的评估。基因本体丰富分析显示，包括聚(A)RNA结合、核小体DNA结合和蛋白质结合在内的几个术语的丰富。免疫荧光研究证实，蛋白质组学分析鉴定的RNA结合蛋白增加的荧光强度，即hnRNP A2/B1、hnRNP A3和hnRNP C1/C2，在有p62阳性包涵体的细胞核中比在无包涵体的核中更高，这并不局限于核内包涵体。我们在含有p62阳性包涵体的细胞核中发现了几种增加的蛋白质。虽然需要更大规模的研究来验证我们的结果，但这些蛋白质组学数据可能会为理解NIID的病理生理学奠定基础。
Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease characterized by eosinophilic hyaline intranuclear inclusions in the neurons, glial cells, and other somatic cells. Although CGG repeat expansions inNOTCH2NLChave been identified in most East Asian patients with NIID, the pathophysiology of NIID remains unclear. Ubiquitin- and p62-positive intranuclear inclusions are the pathological hallmark of NIID. Targeted immunostaining studies have identified several other proteins present in these inclusions. However, the global molecular changes within nuclei with these inclusions remained unclear. Herein, we analyzed the proteomic profile of nuclei with p62-positive inclusions in a NIID patient with CGG repeat expansion inNOTCH2NLCto discover candidate proteins involved in the NIID pathophysiology. We used fluorescence-activated cell sorting and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify each protein identified in the nuclei with p62-positive inclusions. The distribution of increased proteins was confirmed via immunofluorescence in autopsy brain samples from three patients with genetically confirmed NIID. Overall, 526 proteins were identified, of which 243 were consistently quantified using MS. A 1.4-fold increase was consistently observed for 20 proteins in nuclei with p62-positive inclusions compared to those without. Fifteen proteins identified with medium or high confidence in the LC-MS/MS analysis were further evaluated. Gene ontology enrichment analysis showed enrichment of several terms, including poly(A) RNA binding, nucleosomal DNA binding, and protein binding. Immunofluorescence studies confirmed that the fluorescent intensities of increased RNA-binding proteins identified by proteomic analysis, namely hnRNP A2/B1, hnRNP A3, and hnRNP C1/C2, were higher in the nuclei with p62-positive inclusions than in those without, which were not confined to the intranuclear inclusions. We identified several increased proteins in nuclei with p62-positive inclusions. Although larger studies are needed to validate our results, these proteomic data may form the basis for understanding the pathophysiology of NIID.