Comprehensive search of Treatment-resistant depression associated protein by time-controlled transcardiac perfusion cross-linking technique

时间控制经心灌注交联技术综合检索难治性抑郁症相关蛋白

• 批准号: 22591259
• 负责人: OHSHIRO Masaya
• 金额: $ 2.83万
• 依托单位: Hamamatsu University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2010
• 资助国家: 日本
• 起止时间: 2010 至 2012
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22591259/
• 关键词: うつ病; セロトニン・トランスポーター; 治療抵抗性; 中枢神経系; In vivo cross-link 法; In vivo cross-link法

Change in serotonin transporter (SERT) function may be implicated in treatment-resistant depression. Anti-depressant, serotonin selective reuptake inhibitor (SSRI) is acting on SERT present at the cell surface, which is regulated by various cellular mechanisms including interactions with other proteins. In this study, we have searched for novel SERT -binding proteins by time-controlled transcardiac perfusion cross-linking and a pull-down system, and investigated whether the expression of one such protein inhibited the effect of SSRI on serotonergic neuron.N-ethylmaleimide-sensitive factor (NSF) was identified by a pull-down system as a novel SERT -binding protein. Alterations of SERT function and membrane expression upon knockdown of the novel SERT -binding protein were studied in HEK293-hSERT cells. NSF co-localized with SERT at the plasma membrane, and NSF knockdown resulted in decreased SERT expression at the cell membranes and its uptake function. NSF endogenously co-localized with SERT and interacted with SERT. Time-controlled transcardiac perfusion cross-linking technique has screened 85 candidate proteins. Then, 14 proteins have identified from the candidates by shot-gun analysis score. Endogenous interaction of SERT with 14 proteins, NSF and Syntaxin 1A wereevaluated in mouse primary cultured raphe neurons. The association between proteinsand treatment-resistant depression have not evaluated within this term of grant-in-aid.

5-羟色胺转运体（SERT）功能的改变可能与难治性抑郁症有关。5-羟色胺选择性再摄取抑制剂（SSRI）是一种抗肿瘤药物，作用于细胞表面的SERT，SERT受多种细胞机制的调节，包括与其他蛋白质的相互作用。在本研究中，我们通过时间控制的经心灌流交联和下拉系统寻找新的SERT结合蛋白，并研究其中一种蛋白的表达是否抑制SSRI对多巴胺能神经元的作用，通过下拉系统鉴定N-乙基马来酰亚胺敏感因子（NSF）是一种新的SERT结合蛋白。在HEK 293-hSERT细胞中研究了敲低新型SERT结合蛋白后SERT功能和膜表达的改变。NSF与SERT共定位于质膜，并且NSF敲低导致SERT在细胞膜上的表达及其摄取功能降低。NSF与SERT内源性共定位，并与SERT相互作用。时间控制的经心灌注交联技术筛选了85个候选蛋白。然后，通过鸟枪分析评分，从候选蛋白中鉴定出14个蛋白质。在原代培养的小鼠中缝核神经元中，研究了SERT与14种蛋白质、NSF和Syntaxin 1A的内源性相互作用。蛋白质与难治性抑郁症之间的关系尚未在本项资助中进行评估。

项目成果

急性ストレスモデルにおける局所脳血流・脳糖代謝解析

急性应激模型局部脑血流及脑葡萄糖代谢分析

• 发表时间: 2012
• 作者: 亀野 陽亮;松崎 秀夫;高橋 健二;岩田 圭子;鈴木 勝昭;間賀田 泰寛;森 則夫
• 通讯作者: 森 則夫

浜松医科大学・精神医学講座

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