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

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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.