Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Disrupted-in-schizophrenia 1 regulates transport of ITPR1 mRNA for synaptic plasticity

Nature Neuroscience volume 18, pages 698–707 (2015)Cite this article

Subjects

Abstract

Disrupted-in-schizophrenia 1 (DISC1) is a susceptibility gene for major psychiatric disorders, including schizophrenia. DISC1 has been implicated in neurodevelopment in relation to scaffolding signal complexes. Here we used proteomic analysis to screen for DISC1 interactors and identified several RNA-binding proteins, such as hematopoietic zinc finger (HZF), that act as components of RNA-transporting granules. HZF participates in the mRNA localization of inositol-1,4,5-trisphosphate receptor type 1 (ITPR1), which plays a key role in synaptic plasticity. DISC1 colocalizes with HZF and ITPR1 mRNA in hippocampal dendrites and directly associates with neuronal mRNAs, including ITPR1 mRNA. The binding potential of DISC1 for ITPR1 mRNA is facilitated by HZF. Studies of Disc1-knockout mice have revealed that DISC1 regulates the dendritic transport of Itpr1 mRNA by directly interacting with its mRNA. The DISC1-mediated mRNA regulation is involved in synaptic plasticity. We show that DISC1 binds ITPR1 mRNA with HZF, thereby regulating its dendritic transport for synaptic plasticity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Identification of RNA-binding proteins as novel DISC1 interactors.
Figure 2: Movement of DISC1-GFP and ITPR1 3′ UTR in hippocampal dendrites.
Figure 3: Inhibition of dendritic localization of ITPR1 3′ UTR by the loss of DISC1.
Figure 4: Interaction of DISC1 with ITPR1 3′ UTR RNA.
Figure 5: Requirement of interaction between DISC1 and RNA for dendritic transport of ITPR1 3′ UTR.
Figure 6: Requirement of interaction between DISC1 and HZF for dendritic transport of ITPR1 3′ UTR.
Figure 7: Involvement of DISC1-mediated mRNA regulation in synaptic plasticity.

Similar content being viewed by others

References

  1. Blackwood, D.H. et al. Schizophrenia and affective disorders—cosegregation with a translocation at chromosome 1q42 that directly disrupts brain-expressed genes: clinical and P300 findings in a family. Am. J. Hum. Genet. 69, 428–433 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Millar, J.K. et al. Disruption of two novel genes by a translocation co-segregating with schizophrenia. Hum. Mol. Genet. 9, 1415–1423 (2000).

    CAS  PubMed  Google Scholar 

  3. Brandon, N.J. & Sawa, A. Linking neurodevelopmental and synaptic theories of mental illness through DISC1. Nat. Rev. Neurosci. 12, 707–722 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Chubb, J.E., Bradshaw, N.J., Soares, D.C., Porteous, D.J. & Millar, J.K. The DISC locus in psychiatric illness. Mol. Psychiatry 13, 36–64 (2008).

    CAS  PubMed  Google Scholar 

  5. Taya, S. et al. DISC1 regulates the transport of the NUDEL/LIS1/14-3-3ɛ complex through kinesin-1. J. Neurosci. 27, 15–26 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Shinoda, T. et al. DISC1 regulates neurotrophin-induced axon elongation via interaction with Grb2. J. Neurosci. 27, 4–14 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Brandon, N.J. et al. Understanding the role of DISC1 in psychiatric disease and during normal development. J. Neurosci. 29, 12768–12775 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Kuroda, K. et al. Behavioral alterations associated with targeted disruption of exons 2 and 3 of the Disc1 gene in the mouse. Hum. Mol. Genet. 20, 4666–4683 (2011).

    CAS  PubMed  Google Scholar 

  9. Kvajo, M., McKellar, H. & Gogos, J.A. Avoiding mouse traps in schizophrenia genetics: lessons and promises from current and emerging mouse models. Neuroscience 211, 136–164 (2012).

    CAS  PubMed  Google Scholar 

  10. Nakata, K. et al. DISC1 splice variants are upregulated in schizophrenia and associated with risk polymorphisms. Proc. Natl. Acad. Sci. USA 106, 15873–15878 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Ishizuka, K. et al. Evidence that many of the DISC1 isoforms in C57BL/6J mice are also expressed in 129S6/SvEv mice. Mol. Psychiatry 12, 897–899 (2007).

    CAS  PubMed  Google Scholar 

  12. Duan, X. et al. Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain. Cell 130, 1146–1158 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Mao, Y. et al. Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell 136, 1017–1031 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Kim, J.Y. et al. DISC1 regulates new neuron development in the adult brain via modulation of AKT-mTOR signaling through KIAA1212. Neuron 63, 761–773 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Koike, H., Arguello, P.A., Kvajo, M., Karayiorgou, M. & Gogos, J.A. Disc1 is mutated in the 129S6/SvEv strain and modulates working memory in mice. Proc. Natl. Acad. Sci. USA 103, 3693–3697 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Clapcote, S.J. & Roder, J.C. Deletion polymorphism of Disc1 is common to all 129 mouse substrains: implications for gene-targeting studies of brain function. Genetics 173, 2407–2410 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Kamiya, A. et al. A schizophrenia-associated mutation of DISC1 perturbs cerebral cortex development. Nat. Cell Biol. 7, 1167–1178 (2005).

    PubMed  Google Scholar 

  18. Kubo, K. et al. Migration defects by DISC1 knockdown in C57BL/6, 129X1/SvJ, and ICR strains via in utero gene transfer and virus-mediated RNAi. Biochem. Biophys. Res. Commun. 400, 631–637 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Niwa, M. et al. Knockdown of DISC1 by in utero gene transfer disturbs postnatal dopaminergic maturation in the frontal cortex and leads to adult behavioral deficits. Neuron 65, 480–489 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Bannai, H. et al. An RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1) is a component of mRNA granule transported with inositol 1,4,5-trisphosphate receptor type 1 mRNA in neuronal dendrites. J. Biol. Chem. 279, 53427–53434 (2004).

    CAS  PubMed  Google Scholar 

  21. Kanai, Y., Dohmae, N. & Hirokawa, N. Kinesin transports RNA: isolation and characterization of an RNA-transporting granule. Neuron 43, 513–525 (2004).

    CAS  PubMed  Google Scholar 

  22. Kiebler, M.A. & Bassell, G.J. Neuronal RNA granules: movers and makers. Neuron 51, 685–690 (2006).

    CAS  PubMed  Google Scholar 

  23. Anderson, P. & Kedersha, N. RNA granules. J. Cell Biol. 172, 803–808 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Iijima, T. et al. Hzf protein regulates dendritic localization and BDNF-induced translation of type 1 inositol 1,4,5-trisphosphate receptor mRNA. Proc. Natl. Acad. Sci. USA 102, 17190–17195 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Knowles, R.B. et al. Translocation of RNA granules in living neurons. J. Neurosci. 16, 7812–7820 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Köhrmann, M. et al. Microtubule-dependent recruitment of Staufen-green fluorescent protein into large RNA-containing granules and subsequent dendritic transport in living hippocampal neurons. Mol. Biol. Cell 10, 2945–2953 (1999).

    PubMed  PubMed Central  Google Scholar 

  27. Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).

    CAS  PubMed  Google Scholar 

  28. Rook, M.S., Lu, M. & Kosik, K.S. CaMKIIα 3′ untranslated region-directed mRNA translocation in living neurons: visualization by GFP linkage. J. Neurosci. 20, 6385–6393 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Hirokawa, N. & Takemura, R. Molecular motors and mechanisms of directional transport in neurons. Nat. Rev. Neurosci. 6, 201–214 (2005).

    CAS  PubMed  Google Scholar 

  30. Presley, J.F. et al. ER-to-Golgi transport visualized in living cells. Nature 389, 81–85 (1997).

    CAS  PubMed  Google Scholar 

  31. King, S.J. & Schroer, T.A. Dynactin increases the processivity of the cytoplasmic dynein motor. Nat. Cell Biol. 2, 20–24 (2000).

    CAS  PubMed  Google Scholar 

  32. Dictenberg, J.B., Swanger, S.A., Antar, L.N., Singer, R.H. & Bassell, G.J. A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome. Dev. Cell 14, 926–939 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Nakata, T. & Hirokawa, N. Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head. J. Cell Biol. 162, 1045–1055 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Bayer, T.S., Booth, L.N., Knudsen, S.M. & Ellington, A.D. Arginine-rich motifs present multiple interfaces for specific binding by RNA. RNA 11, 1848–1857 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Bramham, C.R. & Wells, D.G. Dendritic mRNA: transport, translation and function. Nat. Rev. Neurosci. 8, 776–789 (2007).

    CAS  PubMed  Google Scholar 

  36. Miller, S. et al. Disruption of dendritic translation of CaMKIIα impairs stabilization of synaptic plasticity and memory consolidation. Neuron 36, 507–519 (2002).

    CAS  PubMed  Google Scholar 

  37. Jones, S.W. et al. Characterisation of cell-penetrating peptide-mediated peptide delivery. Br. J. Pharmacol. 145, 1093–1102 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Eberhart, D.E., Malter, H.E., Feng, Y. & Warren, S.T. The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals. Hum. Mol. Genet. 5, 1083–1091 (1996).

    CAS  PubMed  Google Scholar 

  39. Hirokawa, N., Niwa, S. & Tanaka, Y. Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease. Neuron 68, 610–638 (2010).

    CAS  PubMed  Google Scholar 

  40. Bardo, S., Cavazzini, M.G. & Emptage, N. The role of the endoplasmic reticulum Ca2+ store in the plasticity of central neurons. Trends Pharmacol. Sci. 27, 78–84 (2006).

    CAS  PubMed  Google Scholar 

  41. Iijima, T. et al. Impaired motor functions in mice lacking the RNA-binding protein Hzf. Neurosci. Res. 58, 183–189 (2007).

    CAS  PubMed  Google Scholar 

  42. Rudy, B. & McBain, C.J. Kv3 channels: voltage-gated K+ channels designed for high-frequency repetitive firing. Trends Neurosci. 24, 517–526 (2001).

    CAS  PubMed  Google Scholar 

  43. Strumbos, J.G., Brown, M.R., Kronengold, J., Polley, D.B. & Kaczmarek, L.K. Fragile X mental retardation protein is required for rapid experience-dependent regulation of the potassium channel Kv3.1b. J. Neurosci. 30, 10263–10271 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Moosmang, S. et al. Role of hippocampal Cav1.2 Ca2+ channels in NMDA receptor-independent synaptic plasticity and spatial memory. J. Neurosci. 25, 9883–9892 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Hayashi-Takagi, A. et al. Disrupted-in-Schizophrenia 1 (DISC1) regulates spines of the glutamate synapse via Rac1. Nat. Neurosci. 13, 327–332 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Maher, B.J. & LoTurco, J.J. Disrupted-in-schizophrenia (DISC1) functions presynaptically at glutamatergic synapses. PLoS ONE 7, e34053 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. McBride, J.L. et al. Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc. Natl. Acad. Sci. USA 105, 5868–5873 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Baek, S.T. et al. Off-target effect of doublecortin family shRNA on neuronal migration associated with endogenous microRNA dysregulation. Neuron 82, 1255–1262 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Kang, E. et al. Interaction between FEZ1 and DISC1 in regulation of neuronal development and risk for schizophrenia. Neuron 72, 559–571 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhou, M. et al. mTOR inhibition ameliorates cognitive and affective deficits caused by Disc1 knockdown in adult-born dentate granule neurons. Neuron 77, 647–654 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Watanabe, N. & Mitchison, T.J. Single-molecule speckle analysis of actin filament turnover in lamellipodia. Science 295, 1083–1086 (2002).

    CAS  PubMed  Google Scholar 

  52. Yeap, B.B. et al. Novel binding of HuR and poly(C)-binding protein to a conserved UC-rich motif within the 3′-untranslated region of the androgen receptor messenger RNA. J. Biol. Chem. 277, 27183–27192 (2002).

    CAS  PubMed  Google Scholar 

  53. Li, Y., Jiang, Z., Chen, H. & Ma, W.J. A modified quantitative EMSA and its application in the study of RNA–protein interactions. J. Biochem. Biophys. Methods 60, 85–96 (2004).

    CAS  PubMed  Google Scholar 

  54. Sugimoto, M., Gromley, A. & Sherr, C.J. Hzf, a p53-responsive gene, regulates maintenance of the G2 phase checkpoint induced by DNA damage. Mol. Cell. Biol. 26, 502–512 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Funahashi, Y. et al. ERK2-mediated phosphorylation of Par3 regulates neuronal polarization. J. Neurosci. 33, 13270–13285 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Arimura, N. et al. Anterograde transport of TrkB in axons is mediated by direct interaction with Slp1 and Rab27. Dev. Cell 16, 675–686 (2009).

    CAS  PubMed  Google Scholar 

  57. Nguyen, P.V. & Kandel, E.R. A macromolecular synthesis-dependent late phase of long-term potentiation requiring cAMP in the medial perforant pathway of rat hippocampal slices. J. Neurosci. 16, 3189–3198 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Huang, Y.Y. et al. Genetic evidence for a protein-kinase-A-mediated presynaptic component in NMDA-receptor-dependent forms of long-term synaptic potentiation. Proc. Natl. Acad. Sci. USA 102, 9365–9370 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge T. Takumi (Hiroshima University, Higashihiroshima, Japan) for providing rat Staufen cDNA, S. Okabe (University of Tokyo, Tokyo, Japan) for providing the pAct expression vector, H. Song (Johns Hopkins University school of Medicine, Baltimore, Maryland) for providing the Disc1 shRNA vector and K. Kosik (University of California, Santa Barbara, Santa Barbara, California) for providing the RSV-MS2-CAMK2A 3′ UTR construct. We thank T. Fujiwara (Kobe University) and A. Sawa (Johns Hopkins University) for helpful discussions. We also thank Y. Funahashi, Y. Fujino, H. Yano and T. Watanabe for providing materials; K. Kobayash and S. Suzuki for technical assistance; and T. Ishii for secretarial assistance. This work was supported in part by a Grant-in-Aid for the Strategic Research Program for Brain Science (SRPBS; Theme G) (26-J-JG38), a Grant-in-Aid for Scientific Research (S) (20227006), an Academic Frontier Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and a Grant-in-Aid for Creative Scientific Research from the Japan Society for the Promotion of Science, Japan Science and Technology Agency, CREST (26-J-Jc08).

Author information

Authors and Affiliations

  1. Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan

    Daisuke Tsuboi, Keisuke Kuroda, Takashi Namba, Yukihiko Iizuka, Shinichiro Taya, Tomoyasu Shinoda, Takao Hikita, Shinsuke Muraoka, Ai Nimura & Kozo Kaibuchi

  2. JST, CREST, Tokyo, Japan

    Daisuke Tsuboi, Keisuke Kuroda, Takashi Namba, Yukihiko Iizuka, Shinichiro Taya, Michiro Iizuka & Kozo Kaibuchi

  3. Department of Physiology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan

    Motoki Tanaka & Masahiro Sokabe

  4. Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, Kodaira, Tokyo, Japan

    Shinichiro Taya

  5. Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan

    Tomoyasu Shinoda

  6. Department of Developmental and Regenerative Biology, Graduate School of Medical Science, Nagoya City University, Nagoya, Aichi, Japan

    Takao Hikita

  7. Department of Neural Regeneration and Cell Communication, Mie University Graduate School of Medicine, Tsu, Mie, Japan

    Michiro Iizuka & Akira Mizoguchi

  8. Department of Neuronal Cell Biology, Okazaki Institute for Integrative Bioscience and National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, Aichi, Japan

    Nobuyuki Shiina

  9. Department of Physiology, Keio University School of Medicine, Shinjuku, Tokyo, Japan

    Hideyuki Okano

  10. Brain Science Institute, RIKEN, Wako, Saitama, Japan

    Katsuhiko Mikoshiba

Authors
  1. Daisuke Tsuboi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Keisuke Kuroda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Motoki Tanaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takashi Namba
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yukihiko Iizuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shinichiro Taya
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tomoyasu Shinoda
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Takao Hikita
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shinsuke Muraoka
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Michiro Iizuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ai Nimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Akira Mizoguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Nobuyuki Shiina
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Masahiro Sokabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Hideyuki Okano
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Katsuhiko Mikoshiba
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Kozo Kaibuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.T. carried out most of the cell biology experiments; K. Kuroda and S.T. prepared the gene-knockout mouse and antibodies to DISC1; T.N. carried out in vivo experiments with in utero electroporation; Y.I., T.S. and T.H. carried out biochemical experiments; M.I. and A.M. carried out immunoelectron microscopy analysis; Y.I., S.M. and A.N. carried out affinity chromatography; M.T. and M.S. carried out an electrophysiological study; N.S., H.O. and K.M. made materials and analyzed results; and K. Kaibuchi guided the research and wrote the paper.

Corresponding author

Correspondence to Kozo Kaibuchi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Quantitative binding and colocalization of DISC1 to RNA-binding proteins.

(a) Effect of RNase A treatment on interactions of DISC1 with RNA-binding proteins. The brain cytosol fraction was treated with or without RNase A prior to loading for DISC1-affinity column chromatography. Immunoblotting analyses were performed on the eluates with the indicated antibodies. The bars represent the quantified western blot signals normalized to input. The mean values ± s.e.m. from triplicate western blot experiments (n = 3) are shown. For statistical analysis we used the Mann-Whitney U-test (RNase A (–) versus RNase A (+) in KIF5, HnRNPU, SYNCRIP, HZF, PURα and RACK1). The asterisks indicate statistical significance (P < 0.05). (b) Quantification of colocalization of DISC1 with endogenous components of RNA granules. The bar graphs show the average Pearson’s correlation coefficient for each antigen pair as indicated in Fig. 1f, Supplementary Fig. 2d–f and Supplementary Fig. 2i. The mean values (mean ± s.e.m.) were calculated in five different neurons (n = 5). For statistical analysis we used ANOVA with Bonferroni post hoc test (DISC1-HZF, DISC1-SYNCRIP, DISC1-AGO2 and DISC1-FMRP versus DISC1-eIF4G, P < 0.05, F(4, 20) = 23.81). The asterisks indicate statistical significance (P < 0.05).

Supplementary Figure 2 Subcellular localization of DISC1 in dendrites.

(a,b) DISC1 and MAP2 staining in hippocampal CA1 pyramidal neurons of a mouse brain slice (a) and in cultured hippocampal neurons (b). Scale bar, 10 μm (5 mm in magnified images). (c–i) Staining is shown for DISC1 and GFP-rStaufen (c), SYNCRIP (d), FRMP (e), eIF4G (f), Venus–ITPR1 3' UTR (g), HZF (h) or Ago2/EIF2C2 (i). Scale bars, 20 μm. Higher-magnification images of the boxed areas are shown in the lower right panels, in which asterisks and arrowheads indicate larger and smaller granules in dendrites, respectively. Scale bars in the magnified images represent 5 μm. (j,k) Immunoelectron microscopic localization of DISC1 in CA1 pyramidal neurons of an adult mouse brain. DISC1 immunoreactivity (IR) was detected at neighboring sites of the spine and along microtubules. The arrowheads and arrows represent postsynaptic density and IR of DISC1, respectively. D, dendritic shaft; Pre, presynapse; M, mitochondria. Scale bars represent 500 nm (j) and 1 μm (k).

Supplementary Figure 3 Depletion of DISC1, HZF or SYNCRIP in hippocampal neurons.

(a) DISC1 expression in Disc1−/− mice. Hippocampal neurons were dissociated from hippocampus of wild-type and Disc1−/− mice. After 2 d in culture, total protein amounts in the cell lysates were measured by BCA assay followed by immunoblotting with DISC1c-Ab. The asterisks indicate the degradation products of mouse DISC1 recombinant protein (GST-mDISC1). (b,c) Specific knockdown of HZF (b) and SYNCRIP (c) expression by RNAi. Hippocampal neurons were transfected with siRNAs against HZF or SYNCRIP. The graphs represent the densitometry analysis of western blots after normalization to β-tubulin expression. The knockdown efficiencies were calculated in three independent experiments (n = 3). *P < 0.05, Mann-Whitney U-test. Bars show mean ± s.e.m. (d) Effects of HZF and SYNCRIP knockdown on the dendritic localization of ITPR1 3ʹ UTR. Hippocampal neurons were transfected with the indicated siRNAs and Venus–ITPR1 3ʹ UTR plasmids at DIV 12. The transfected neurons were fixed at DIV 14 and immunostained with antibodies to Venus (green) and MAP2 (red). The blue bars indicate the distance between the soma and the farthest transported granules containing ITPR1 3ʹ UTR. The magnified images of these granules are shown in the boxed areas. Scale bars, 15 μm.

Supplementary Figure 4 Effect of overexpression of DISC1 or KIF5A-headless on dendritic localization of ITPR1 3ʹ UTR.

Hippocampal neurons were transfected with Myc-GST, Myc-hDISC1-FL or Myc-KIF5A-headless (HL) and Venus–ITPR1 3ʹ UTR plasmids at DIV 12. The transfected neurons were fixed at DIV 14 and immunostained with antibodies to Venus (green) and MAP2 (red). Blue bars indicate the distance between the soma and the farthest transported granules containing ITPR1 3ʹ UTR. Magnified images of the granules are shown in the boxed areas. Scale bars, 15 μm.

Supplementary Figure 5 Electrophoretic mobility shift assay for the interaction between DISC1 and ITPR1-3'UTR RNA.

(a) RNA EMSA assay was performed by incubating biotin-labeled ITPR1 3' UTR RNA with purified GST-hDISC1-FL, GST-hDISC1-CH or GST recombinant protein. (b,c) Dissociation constant of DISC1 for ITPR1 3' UTR RNA. The relative amount of the bound RNA was plotted against the protein concentration. The dissociation constant was calculated as half-maximum binding.

Supplementary Figure 6 Identification of DISC1-bound mRNAs.

(a) Enrichment of mRNAs in DISC1 immunoprecipitate. Mouse brain lysate was subjected to an immunoprecipitation assay with anti-DISC1. The co-immunoprecipitated mRNAs were purified from the DISC1 immunoprecipitate and analyzed by quantitative RT-PCR. The mRNA enrichment was determined by normalizing the DISC1 signal (DISC1-Ct value) against the IgG signal (control-Ct value) in three independent experiments (n = 3). The red bars indicate the genes for which there was a statistically significant difference in the mRNA enrichment compared to the control PGAM1. (ANOVA, Dunnett's test (the indicated genes versus PGAM1), P < 0.01, F(21, 44) = 15.39). **P < 0.01. Bars show mean ± s.e.m. (b) Direct interactions of DISC1 with a subset of neuronal mRNAs. Recombinant GST or GST-hDISC1-N1 protein was incubated with biotin-labeled transcripts for ITPR1 (IP3R1), CACNA1C, CACNA2D1, KCNC1, KCNC4, SCN2A and KALRN. After UV cross-linking and digestion of the unbound RNA, RNA-protein complexes were detected using immunoblot analysis with streptavidin-HRP.

Supplementary Figure 7 Interactions of DISC1 mutants with DISC1 interactors and RNA.

(a) MBP pulldown assays were performed with COS7 cell lysates expressing the indicated Myc fusion proteins. The cell lysates were mixed with 20 pmol of MBP, MBP-hDISC1-FL, MBP-hDISC1-ΔARM, GST, GST-hDISC1-ΔKBR or GST-hDISC1-N1-ΔARM recombinant protein and then pulled down by amylose or glutathione magnetic beads. The pulldown samples were subjected to SDS-PAGE and immunoblot analyses with anti-Myc. The asterisks mark the full length of Myc-HZF, -KIF5-HL, -GSK3β, -LIS1 and -PDE4B1. An open circle marks a nonspecific band. Aliquots of original samples (0.5% input) and eluates (10%) were subjected to SDS-PAGE. (b) Recombinant GST or GST-hDISC1-ΔKBR protein was incubated with biotin-labeled ITPR1 3ʹ UTR and unlabeled 3ʹ UTR (100-fold molar excess relative to the biotin-labeled RNA). After UV cross-linking and digestion of the unbound RNA, RNA-protein complexes were detected using immunoblotting with streptavidin-HRP. The asterisk indicates the degradation product of GST-hDISC1 recombinant protein.

Supplementary Figure 8 Rescue of transport defect of ITPR1 3' UTR in Disc1−/− mice with hDISC1-FL and hDISC1-ΔKBR.

(a) Localization of ITPR1 (IP3R1) 3' UTR in Disc1−/− neurons expressing the indicated Myc fusion proteins. Cultured neurons from Disc1−/− mice were transfected with the indicated plasmids at DIV 12. The transfected neurons were fixed at DIV 14 and immunostained with antibodies to Venus (green) and MAP2 (red). The blue bars indicate the distance between the soma and the farthest transported granules containing ITPR1 3' UTR. Magnified images of these granules are shown in the boxed areas. Scale bars, 15 μm. (b) Relative frequency of the transport distance of ITPR1 3' UTR–containing granules. The transport distances of ITPR1 3’ UTR granules were categorized into five groups. Data in b are from three independent experiments for each treatment, with at least 25 neurons per experiment (n = 75). Significant differences between transport distances were determined by one-way ANOVA with Bonferroni post hoc test (Disc1−/−–Myc-GST versus Disc1−/−–hDISC1-FL, P < 0.01, F(2, 222) = 52.56). Bars show mean ± s.e.m.

Supplementary Figure 9 Determination of the binding region between DISC1 and HZF.

(a,b) GST pulldown assays were performed with the recombinant GST fusion proteins and COS7 cell lysates expressing FLAG-HZF (a) or the indicated Myc fusion proteins (b). Proteins that were bound to the immobilized beads with GST fusion proteins were eluted and analyzed using immunoblot analyses with anti-FLAG and anti-Myc. The asterisks mark the full length of Myc fusion proteins in b. Aliquots of original samples (1% input) and eluates (10%) in a and b were subjected to SDS-PAGE.

Supplementary Figure 10 Impairment of dendritic localization of ITPR1 3' UTR by dominant-negative forms of DISC1.

(a) Inhibitory effect of hDISC1-N1-ΔARM on interaction between DISC1 and HZF. His pulldown assays were performed in the COS7 cell lysates expressing GFP-hDISC1-FL and the indicated Myc fusion proteins. 20 pmol of the purified recombinant His-HZF or His-GST protein was mixed with the cell lysates. Proteins that were bound to the immobilized beads with His fusion proteins were eluted and analyzed using immunoblot analyses with anti-GFP, anti-Myc and anti-His. Aliquots of original samples (1% input) and eluates (10%) in a were subjected to SDS-PAGE. (b) Dendritic localization of Venus–ITPR1 (IP3R1) 3' UTR in the neurons expressing hDISC1-N1-ΔARM. Cultured hippocampal neurons were transfected with the indicated plasmids at DIV 12. The transfected neurons were immunostained with antibodies to Venus (green) and MAP2 (red) at DIV 14. The blue bars indicate the distance between the soma and the farthest transported granules containing ITPR1 3' UTR. Magnified images of these granules are shown in the boxed areas. Scale bars, 15 μm.

Supplementary Figure 11 Impairment of dendritic localization of ITPR1 3' UTR by dominant-negative form of HZF.

(a) Inhibitory effect of HZF-ΔZn3 on interaction between DISC1 and HZF. COS7 cell lysates expressing the indicated Myc fusion proteins and FLAG-HZF were used in a GST pulldown assay. The cell lysates were mixed with 20 pmol of GST or GST-hDISC1-FL and pulled down by glutathione Sepharose beads. The pulldown samples were subjected to SDS-PAGE and immunoblot analysis with anti-Myc or anti-FLAG. Aliquots of original samples (1% input) and eluates (10%) were subjected to SDS-PAGE. (b) Dendritic localization of Venus–ITPR1 (IP3R1) 3' UTR in the neurons expressing HZF-ΔZn3. Cultured hippocampal neurons were transfected with the indicated plasmids at DIV 12. The transfected neurons were immunostained with antibodies to Venus (green) and MAP2 (red). The blue bars indicate the distance between the soma and the farthest transported granules containing ITPR1 3' UTR. Magnified images of these granules are shown in the boxed areas. Scale bars, 15 μm.

Supplementary Figure 12 Impairments of mRNA transport and long-term potentiation in Disc1−/− mice.

(a) Dendritic localization of ITPR1 3' UTR in organotypic slices from wild-type and Disc1−/− mice. Hippocampal slices (slice thickness, 250 μm) were prepared from wild-type and Disc1−/− mice at 6 postnatal days. After 3 d in culture, the plasmids were introduced into the slices by biolistic transfection. The transport distances of Venus–ITPR1 3' UTR were measured in the DNA-transfected neurons of slices at 2 d after transfection. The blue bars indicate the distance between the soma and the farthest transported granules containing ITPR1 3' UTR. Scale bars, 15 μm. (b) Relative frequency of the transport distance of ITPR1 3' UTR–containing granules. The transport distances of ITPR1 3' UTR granules were categorized into five groups. The data in b are from three independent experiments for each treatment and at least ten neurons per experiment (n = 30). Significant differences between the transport distances were determined by one-way ANOVA with Bonferroni post hoc test (wild-type versus Disc1−/−, P < 0.01, F(1, 58) = 33.81). Bars show mean ± s.e.m. (c,d) The late phase of LTP (L-LTP) in the dentate gyrus of hippocampal slices from wild-type and Disc1−/− mice (C57BL/6J, 3 weeks in age). (c) Ten trains of tetanic stimulation (100 Hz for 100 pulses with 60-s intervals; indicated by arrow) elicited L-LTP that persisted for at least 3 h in wild-type mice (137.3% ± 8.2% of baseline 180 min after the tetanic stimulation, n = 4). In Disc1−/− mice, a potentiation of comparable magnitude was observed after the tetanic stimulation but declined to near-baseline level by 3 h (100.0% ± 6.3% of baseline 180 min after the tetanic stimulation, n = 4). The data are expressed as the mean ± s.d.(d) LTP amplitude was averaged at 30 min and 180 min after high-frequency stimulation in c and was expressed as a percentage of the baseline value. For statistical analysis we used the Mann-Whitney U-test (LTP amplitude: wild-type versus Disc1−/− at 30 min, n = 4; wild-type versus Disc1−/− at 180 min, n = 4). *P < 0.05. Bars show mean ± s.d.

Supplementary Figure 13 Novel function of DISC1 and off-target effect of Disc1-shRNA during neurodevelopment.

(a) A model for DISC1 functions in mature neurons. We propose that DISC1 binds ITPR1 mRNA with HZF, thereby regulating its dendritic transport for synaptic plasticity. DISC1 directly associates with a subset of mRNAs that encode synaptic proteins, including ITPR1. DISC1 forms a complex with HZF and ITPR1 mRNA, resulting in stable RNA granules. Because DISC1 can link kinesin-1 to HZF and ITPR1 mRNA, DISC1 may be involved in the conversion from static RNA granules to motile ones (namely, RNA-transporting granules). The localized mRNAs would be translated at activated spines, and then newly synthesized proteins would contribute to synaptic plasticity. (b) Phenotypic discrepancy between Disc1 knockout and knockdown. shRNA-C1 (control) or shRNA-D1 (shRNA against Disc1) knockdown vectors (provided by Dr. Song) were co-electroporated with Tα-LPL-pmKO and Tα-Cre into cerebral cortices at E15 and fixed at P1. Coronal sections were prepared and immunostained with anti-Kusabira Orange (red). Nuclei were stained with Hoechst 33342 (blue). WM, white matter. Scale bars, 50 μm.

Supplementary Figure 14 Full-length images of blots and gels presented in Figures 1–6.

(a) Figure 1b. (b) Figure 1c. (c) Figure 1d. (d) Figure 4f. (e) Figure 6a. Sidelines indicate the membranes used in the immunoblotting with the indicated antibodies. Rectangles indicate the regions used in the figures. Dup., duplication sample.

Supplementary Figure 15 Full-length images of blots and gels presented in Figures 6 and 7.

(a) Figure 6b. (b) Figure 7b. Sidelines indicate the membranes used in the immunoblotting with the indicated antibodies. Rectangles indicate the regions used in the figures.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Tables 1 and 2 (PDF 11100 kb)

Supplementary Methods Checklist (PDF 196 kb)

Time-lapse analysis of DISC1-GFP movement in dendrites.

Rat hippocampal neurons were transfected with DISC1-GFP at DIV 5 and observed 1 d after the transfection. The images were taken every 1 s for 30 s. The arrows in this video show examples of anterograde transport of DISC1-GFP granules. (AVI 44 kb)

Time-lapse analysis of Venus–ITPR1 3' UTR movement in dendrites.

The images were taken every 1 s for 30 s under the conditions described above (Supplementary Video 1). The arrows in this video show examples of the anterograde transport of Venus–ITPR1 3' UTR granules. (AVI 57 kb)

Cotransport of DISC1-mCherry with Venus–ITPR1 3' UTR.

The images were taken every 1 s for 15 s under the conditions described above (Supplementary Video 1). Granules containing both DISC1-mCherry and Venus–ITPR1 3' UTR moved in an anterograde fashion along a dendrite. (AVI 241 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tsuboi, D., Kuroda, K., Tanaka, M. et al. Disrupted-in-schizophrenia 1 regulates transport of ITPR1 mRNA for synaptic plasticity. Nat Neurosci 18, 698–707 (2015). https://doi.org/10.1038/nn.3984

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.3984

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing