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01.12.2014 | Research | Ausgabe 1/2014 Open Access

Molecular Autism 1/2014

N-ethylmaleimide-sensitive factor interacts with the serotonin transporter and modulates its trafficking: implications for pathophysiology in autism

Zeitschrift:
Molecular Autism > Ausgabe 1/2014
Autoren:
Keiko Iwata, Hideo Matsuzaki, Taro Tachibana, Koji Ohno, Saori Yoshimura, Hironori Takamura, Kohei Yamada, Shinsuke Matsuzaki, Kazuhiko Nakamura, Kenji J Tsuchiya, Kaori Matsumoto, Masatsugu Tsujii, Toshirou Sugiyama, Taiichi Katayama, Norio Mori
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​2040-2392-5-33) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

HM and TK co-designed the study. KI and HM collected blood samples; collected, analyzed and interpreted the data and prepared the manuscript. TT and SY produced the SERT antibody. KO, HT, KY and SM collected, analyzed and interpreted the data. KN recruited participants, collected blood samples and obtained post-mortem brain samples. KJT and KM collected blood samples and undertook clinical evaluations. MT recruited participants. TS recruited participants and diagnosed ASD. TK collected, analyzed and interpreted the data and prepared the manuscript. NM analyzed and interpreted the data, and prepared the manuscript. All authors read and approved the final manuscript.

Abstract

Background

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. Thus, we searched for novel SERT-binding proteins and investigated whether the expression of one such protein was affected in subjects with autism.

Methods

Novel 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.

Results

N-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.

Conclusions

These 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.
Zusatzmaterial
Additional file 1: Figure S1: N-tail-specific binding of syntaxin-1A to SERT was confirmed by Western blot analysis. (PDF 21 KB)
13229_2014_126_MOESM1_ESM.pdf
Additional file 2: Figure S2: SERT is transported to the plasma membrane in HEK293-hSERT cells. (A, B) Double immunocytochemical staining for SERT (green) and the membrane maker cadherin (red) in HEK293-hSERT cells. (C) SERT was mainly co-localized with the membrane maker (cadherin) (merged). Scale bar: 10 μm. Results are representative of three independent experiments. (PDF 71 KB)
13229_2014_126_MOESM2_ESM.pdf
Additional file 3: Figure S3: Transfection efficacy of siRNA in HEK293-hSERT cells. We determined the proportion of siRNA-transfected HEK293-hSERT cells using a commercially available fluor-oligo kit (TYE 563 DS, Integrated DNA Technologies). The proportion of siRNA-transfected cells was 90%. Upper panels show untreated cells and lower panels show red fluorescent oligo-transfected cells. Left panels show phase-contrast images and right panels show the images obtained by fluorescence microscopy (excitation: 546 nm, emission: 590 nm). Scale bar: 50 μm. Results are representative of three independent experiments. (PDF 150 KB)
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Additional file 4: Figure S4: CBB staining of membranes from biotinylated fractions. Biotinylation experiments in HEK293-hSERT cells transfected with siRNA-2 targeting a specific NSF sequence or negative control. Transfected cells were incubated with sulfo-NHS-SS-biotin. After Western blot analysis, the membrane was stained with CBB as a protein-loading control. (PDF 34 KB)
13229_2014_126_MOESM4_ESM.pdf
Additional file 5: Figure S5: Confirmation of tcTPC efficacy. (A) Western blotting of total proteins from non-tcTPC- or tcTPC-treated mouse brains (lanes 1 and 2, respectively) using anti-SERT antibodies. Results are representative of three independent experiments. It was confirmed that SERT-containing cross-linked complexes were retained by the tcTPC method (lane 2). (B) Proteins from non-tcTPC- or tcTPC-treated mouse brains were immunoprecipitated with rat immunoglobulin G (IgG) as a negative control (lanes 1 and 5) and SERT antibodies (lanes 2 to 4 and 6 to 8), and the resulting Western blot was probed for SERT. In immunoprecipitated samples using tcTPC-treated mouse brains, SERT-containing cross-linked complexes were identified (lanes 6 to 8) in a dose-dependent manner. Results are representative of three independent experiments. (PDF 79 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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Authors’ original file for figure 5
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Authors’ original file for figure 6
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Authors’ original file for figure 7
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Authors’ original file for figure 8
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