Abstract
BackgroundChanges in serotonin transporter (SERT) function have been implicated in autism. SERT function is influenced by the number of transporter molecules present at the cell surface, which is regulated by various cellular mechanisms including interactions with other proteins. Thus, we searched for novel SERT-binding proteins and investigated whether the expression of one such protein was affected in subjects with autism.MethodsNovel SERT-binding proteins were examined by a pull-down system. Alterations of SERT function and membrane expression upon knockdown of the novel SERT-binding protein were studied in HEK293-hSERT cells. Endogenous interaction of SERT with the protein was evaluated in mouse brains. Alterations in the mRNA expression of SERT (SLC6A4) and the SERT-binding protein in the post-mortem brains and the lymphocytes of autism patients were compared to nonclinical controls.ResultsN-ethylmaleimide-sensitive factor (NSF) was identified as a novel SERT-binding protein. NSF was co-localized with SERT at the plasma membrane, and NSF knockdown resulted in decreased SERT expression at the cell membranes and decreased SERT uptake function. NSF was endogenously co-localized with SERT and interacted with SERT. While SLC6A4 expression was not significantly changed, NSF expression tended to be reduced in post-mortem brains, and was significantly reduced in lymphocytes of autistic subjects, which correlated with the severity of the clinical symptoms.ConclusionsThese data clearly show that NSF interacts with SERT under physiological conditions and is required for SERT membrane trafficking and uptake function. A possible role for NSF in the pathophysiology of autism through modulation of SERT trafficking, is suggested.
Highlights
Changes in serotonin transporter (SERT) function have been implicated in autism
After SDS-PAGE and silver staining of the gels, at least ten specific bands were observed in the lane containing proteins eluted from glutathione S-transferase (GST)-N-terminal domain of the rat SERT (N-SERT) beads incubated with brain lysates, and at least three bands were observed in the lane containing proteins eluted from GST-C-terminal domain of the rat SERT (C-SERT) beads incubated with brain lysates (Figure 1A)
We focused on the interaction between N-ethylmaleimide-sensitive factor (NSF) and SERT in the present study for the following reasons
Summary
Changes in serotonin transporter (SERT) function have been implicated in autism. SERT function is influenced by the number of transporter molecules present at the cell surface, which is regulated by various cellular mechanisms including interactions with other proteins. The reduction in the anterior and posterior cingulate cortices was associated with an impairment of social cognition in autistic subjects, and a significant correlation was found between repetitive and/or obsessive behavior and interests and a reduction in SERT binding in the thalamus [17]. These results suggested that SERT protein levels and/or its transport capacity were decreased in the brains of autistic patients. Azmitia and colleagues reported increased immunoreactivity to a SERT antibody of serotonin axons in the post-mortem cortices of autism patients [18]
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