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SAP97 and CASK mediate sorting of NMDA receptors through a previously unknown secretory pathway

Nature Neuroscience volume 12, pages 1011–1019 (2009)Cite this article

Abstract

Synaptic plasticity is dependent on the differential sorting, delivery and retention of neurotransmitter receptors, but the mechanisms underlying these processes are poorly understood. We found that differential sorting of glutamate receptor subtypes began in the endoplasmic reticulum of rat hippocampal neurons. As AMPA receptors (AMPARs) were trafficked to the plasma membrane via the conventional somatic Golgi network, NMDA receptors (NMDARs) were diverted from the somatic endoplasmic reticulum into a specialized endoplasmic reticulum subcompartment that bypasses somatic Golgi, merging instead with dendritic Golgi outposts. This endoplasmic reticulum subcompartment was composed of highly mobile vesicles containing the NMDAR subunits NR1 and NR2B, the microtubule-dependent motor protein KIF17, and the postsynaptic adaptor proteins CASK and SAP97. Our data demonstrate that the retention and trafficking of NMDARs in this endoplasmic reticulum subcompartment requires both CASK and SAP97. These findings indicate that NMDARs are sorted away from AMPARs via a non-conventional secretory pathway that utilizes dendritic Golgi outposts.

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Figure 1: NMDARs exit the soma via a somatic Golgi–independent pathway and insert into dendritic Golgi outposts.
Figure 2: NMDARs traffic in a mobile, vesicular, endoplasmic reticulum subcompartment.
Figure 3: SAP97 and CASK alter NMDAR trafficking in HEK293 cells.
Figure 4: SAP97 co-distributes with NR1 and CASK in nonsynaptic, mobile puncta.
Figure 5: SAP97, NMDARs and CASK form a complex in brain.
Figure 6: Knockdown of SAP97 or CASK reroutes NMDARs to the somatic Golgi and away from dendritic Golgi outposts.
Figure 7: Knockdown of SAP97 or CASK reduces synaptic NMDARs.

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References

  1. Dumas, T.C. Developmental regulation of cognitive abilities: modified composition of a molecular switch turns on associative learning. Prog. Neurobiol 76, 189–211 (2005).

    Article  CAS  Google Scholar 

  2. Malinow, R. & Malenka, R.C. AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25, 103–126 (2002).

    Article  CAS  Google Scholar 

  3. Malenka, R.C. & Bear, M.F. LTP and LTD: an embarrassment of riches. Neuron 44, 5–21 (2004).

    Article  CAS  Google Scholar 

  4. Rao, A., Kim, E., Sheng, M. & Craig, A.M. Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture. J. Neurosci. 18, 1217–1229 (1998).

    Article  CAS  Google Scholar 

  5. Friedman, H.V., Bresler, T., Garner, C.C. & Ziv, N.E. Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment. Neuron 27, 57–69 (2000).

    Article  CAS  Google Scholar 

  6. Washbourne, P., Bennett, J.E. & McAllister, A.K. Rapid recruitment of NMDA receptor transport packets to nascent synapses. Nat. Neurosci. 5, 751–759 (2002).

    Article  CAS  Google Scholar 

  7. Montgomery, J.M., Zamorano, P.L. & Garner, C.C. MAGUKs in synapse assembly and function: an emerging view. Cell. Mol. Life Sci. 61, 911–929 (2004).

    Article  CAS  Google Scholar 

  8. Kennedy, M.J. & Ehlers, M.D. Organelles and trafficking machinery for postsynaptic plasticity. Annu. Rev. Neurosci. 29, 325–362 (2006).

    Article  CAS  Google Scholar 

  9. Greger, I.H. & Esteban, J.A. AMPA receptor biogenesis and trafficking. Curr. Opin. Neurobiol. 17, 289–297 (2007).

    Article  CAS  Google Scholar 

  10. Park, M., Penick, E.C., Edwards, J.G., Kauer, J.A. & Ehlers, M.D. Recycling endosomes supply AMPA receptors for LTP. Science 305, 1972–1975 (2004).

    Article  CAS  Google Scholar 

  11. Yudowski, G.A. et al. Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors. J. Neurosci. 27, 11112–11121 (2007).

    Article  CAS  Google Scholar 

  12. Cognet, L., Groc, L., Lounis, B. & Choquet, D. Multiple routes for glutamate receptor trafficking: surface diffusion and membrane traffic cooperate to bring receptors to synapses. Sci. STKE 2006, pe13 (2006).

    PubMed  Google Scholar 

  13. Sheng, M. & Hoogenraad, C.C. The postsynaptic architecture of excitatory synapses: a more quantitative view. Annu. Rev. Biochem. 76, 823–847 (2006).

    Article  Google Scholar 

  14. Setou, M., Nakagawa, T., Seog, D.H. & Hirokawa, N. Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport. Science 288, 1796–1802 (2000).

    Article  CAS  Google Scholar 

  15. Sans, N. et al. Synapse-associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway. J. Neurosci. 21, 7506–7516 (2001).

    Article  CAS  Google Scholar 

  16. Wyszynski, M. et al. Interaction between GRIP and liprin-alpha/SYD2 is required for AMPA receptor targeting. Neuron 34, 39–52 (2002).

    Article  CAS  Google Scholar 

  17. Guillaud, L., Setou, M. & Hirokawa, N. KIF17 dynamics and regulation of NR2B trafficking in hippocampal neurons. J. Neurosci. 23, 131–140 (2003).

    Article  CAS  Google Scholar 

  18. Lisé, M.F. et al. Involvement of myosin Vb in glutamate receptor trafficking. J. Biol. Chem. 281, 3669–3678 (2006).

    Article  Google Scholar 

  19. Setou, M. et al. Glutamate receptor-interacting protein GRIP1 directly steers kinesin to dendrites. Nature 417, 83–87 (2002).

    Article  CAS  Google Scholar 

  20. Perestenko, P.V. & Henley, J.M. Characterization of the intracellular transport of GluR1 and GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits in hippocampal neurons. J. Biol. Chem. 278, 43525–43532 (2003).

    Article  CAS  Google Scholar 

  21. Sans, N. et al. NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nat. Cell Biol. 5, 520–530 (2003).

    Article  CAS  Google Scholar 

  22. Sans, N. et al. mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression. Nat. Cell Biol. 7, 1179–1190 (2005).

    Article  Google Scholar 

  23. Bassand, P., Bernard, A., Rafiki, A., Gayet, D. & Khrestchatisky, M. Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD-95 and SAP97. Eur. J. Neurosci. 11, 2031–2043 (1999).

    Article  CAS  Google Scholar 

  24. Gardoni, F. et al. CaMKII-dependent phosphorylation regulates SAP97/NR2A interaction. J. Biol. Chem. 278, 44745–44752 (2003).

    Article  CAS  Google Scholar 

  25. Mauceri, D., Gardoni, F., Marcello, E. & Di Luca, M. Dual role of CaMKII-dependent SAP97 phosphorylation in mediating trafficking and insertion of NMDA receptor subunit NR2A. J. Neurochem. 100, 1032–1046 (2007).

    Article  CAS  Google Scholar 

  26. Pierce, J.P., Mayer, T. & McCarthy, J.B. Evidence for a satellite secretory pathway in neuronal dendritic spines. Curr. Biol. 11, 351–355 (2001).

    Article  CAS  Google Scholar 

  27. Pfeffer, S. Membrane domains in the secretory and endocytic pathways. Cell 112, 507–517 (2003).

    Article  CAS  Google Scholar 

  28. Dascher, C. & Balch, W.E. Dominant inhibitory mutants of ARF1 block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus. J. Biol. Chem. 269, 1437–1448 (1994).

    CAS  PubMed  Google Scholar 

  29. Horton, A.C. & Ehlers, M.D. Dual modes of endoplasmic reticulum-to-Golgi transport in dendrites revealed by live-cell imaging. J. Neurosci. 23, 6188–6199 (2003).

    Article  CAS  Google Scholar 

  30. Schuman, E.M., Dynes, J.L. & Steward, O. Synaptic regulation of translation of dendritic mRNAs. J. Neurosci. 26, 7143–7146 (2006).

    Article  CAS  Google Scholar 

  31. Bannai, H., Inoue, T., Nakayama, T., Hattori, M. & Mikoshiba, K. Kinesin-dependent, rapid, bi-directional transport of ER subcompartment in dendrites of hippocampal neurons. J. Cell Sci. 117, 163–175 (2004).

    Article  CAS  Google Scholar 

  32. Mironov, S.L. & Symonchuk, N. ER vesicles and mitochondria move and communicate at synapses. J. Cell Sci. 119, 4926–4934 (2006).

    Article  CAS  Google Scholar 

  33. Jo, K., Derin, R., Li, M. & Bredt, D.S. Characterization of MALS/Velis-1, -2, and -3: a family of mammalian LIN-7 homologs enriched at brain synapses in association with the postsynaptic density–95/NMDA receptor postsynaptic complex. J. Neurosci. 19, 4189–4199 (1999).

    Article  CAS  Google Scholar 

  34. Butz, S., Okamoto, M. & Sudhof, T.C. A tripartite protein complex with the potential to couple synaptic vesicle exocytosis to cell adhesion in brain. Cell 94, 773–782 (1998).

    Article  CAS  Google Scholar 

  35. Lee, S., Fan, S., Makarova, O., Straight, S. & Margolis, B. A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia. Mol. Cell. Biol. 22, 1778–1791 (2002).

    Article  CAS  Google Scholar 

  36. Leonoudakis, D., Conti, L.R., Radeke, C.M., McGuire, L.M. & Vandenberg, C.A. A multiprotein trafficking complex composed of SAP97, CASK, Veli and Mint1 is associated with inward rectifier Kir2 potassium channels. J. Biol. Chem. 279, 19051–19063 (2004).

    Article  CAS  Google Scholar 

  37. Tiffany, A.M. et al. PSD-95 and SAP97 exhibit distinct mechanisms for regulating K+ channel surface expression and clustering. J. Cell Biol. 148, 147–158 (2000).

    Article  CAS  Google Scholar 

  38. Leonard, A.S., Davare, M.A., Horne, M.C., Garner, C.C. & Hell, J.W. SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit. J. Biol. Chem. 273, 19518–19524 (1998).

    Article  CAS  Google Scholar 

  39. Nakagawa, T. et al. Quaternary structure, protein dynamics and synaptic function of SAP97 controlled by L27 domain interactions. Neuron 44, 453–467 (2004).

    Article  CAS  Google Scholar 

  40. Dotti, C.G. & Poo, M.M. Neuronal polarization: building fences for molecular segregation. Nat. Cell Biol. 5, 591–594 (2003).

    Article  CAS  Google Scholar 

  41. Dresbach, T. et al. Assembly of active zone precursor vesicles: obligatory trafficking of presynaptic cytomatrix proteins Bassoon and Piccolo via a trans-Golgi compartment. J. Biol. Chem. 281, 6038–6047 (2006).

    Article  CAS  Google Scholar 

  42. Tang, B.L. Protein trafficking mechanisms associated with neurite outgrowth and polarized sorting in neurons. J. Neurochem. 79, 923–930 (2001).

    Article  CAS  Google Scholar 

  43. Claudio, T. Stable expression of heterologous multisubunit protein complexes established by calcium phosphate– or lipid-mediated cotransfection. Methods Enzymol. 207, 391–408 (1992).

    Article  CAS  Google Scholar 

  44. Banker, G. & Goslin, K. Developments in neuronal cell culture. Nature 336, 185–186 (1988).

    Article  CAS  Google Scholar 

  45. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    Article  CAS  Google Scholar 

  46. Wu, H., Reuver, S.M., Kuhlendahl, S., Chung, W.J. & Garner, C.C. Subcellular targeting and cytoskeletal attachment of SAP97 to the epithelial lateral membrane. J. Cell Sci. 111, 2365–2376 (1998).

    CAS  PubMed  Google Scholar 

  47. Bresler, T. et al. Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly. J. Neurosci. 24, 1507–1520 (2004).

    Article  CAS  Google Scholar 

  48. Phend, K.D., Rustioni, A. & Weinberg, R.J. An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry. J. Histochem. Cytochem. 43, 283–292 (1995).

    Article  CAS  Google Scholar 

  49. Fujisawa, S. & Aoki, C. In vivo blockade of N-methyl-D-aspartate receptors induces rapid trafficking of NR2B subunits away from synapses and out of spines and terminals in adult cortex. Neuroscience 121, 51–63 (2003).

    Article  CAS  Google Scholar 

  50. Leal-Ortiz, S. et al. Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis. J. Cell Biol. 181, 831–846 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to thank S. Leal-Ortiz for assistance with the generation of lentivirus and shRNA, L.A. Needleman and J. Marks. Also, thanks to B. Margolis for the CASK constructs, J. Lippincott-Schwartz for the Galtase-GFP construct and V. Bindokas for help and advice with imaging protocols. This work was supported by the Nancy Pritzker Family and US National Institutes of Health grant DA016758 to C.C.G., a Deutsche Forschungsgemeinschaft postdoctoral fellowship to C.G.S., The Marsden Fund (Royal Society of New Zealand) to J.M.M., US National Institutes of Health grants NS043782, DA13602 and DA019695 to W.N.G., and the Albert & Ellen Grass Faculty Award to W.N.G. and J.M.

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Author notes

  1. Craig C Garner and William N Green: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Psychiatry and Behavioral Science, Stanford University, Palo Alto, California, USA

    Okunola Jeyifous, Clarissa L Waites, Christian G Specht & Craig C Garner

  2. Center for Neural Science and Department of Biology, New York University, New York, New York, USA

    Sho Fujisawa & Chiye Aoki

  3. Department of Physiology, University of Auckland, Auckland, New Zealand

    Manja Schubert, Tharani de Silva & Johanna M Montgomery

  4. Department of Neurobiology, University of Chicago, Chicago, Illinois, USA

    Eric I Lin & William N Green

  5. Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island, USA

    John Marshall

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Contributions

O.J. conducted the majority of the experiments and data analysis and co-wrote the manuscript. C.L.W. contributed to the shRNA experiments and helped in the editing of the manuscript. C.G.S. designed, constructed and aided in the testing of the shRNAs. S.F. performed the electron microscopy studies. M.S. carried out temperature block, BFA and some of the live-imaging experiments. E.L. performed flow cytometry experiments. J.M. provided advice and aided in the interpretation of data. C.A. supervised the electron microscopy studies. T.d.S. assisted with the temperature block experiments. J.M.M. supervised and designed studies and helped with the editing of the manuscript. C.C.G. and W.N.G. supervised the project and co-wrote the manuscript.

Corresponding authors

Correspondence to Craig C Garner or William N Green.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Results (PDF 936 kb)

Supplementary Video 1

Movement of a vesicle containing RFP-SAP97 and NR1-GFP. E18 hippocampal neurons were transfected at 14 DIV with RFP-SAP97 (left) and NR1-GFP (right). Time-lapse imaging was performed at 1 d post-transfection and image frames were acquired every 5 s for 2 min. The frame sequence highlights the image field displayed in Figure 4c. (MOV 305 kb)

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Jeyifous, O., Waites, C., Specht, C. et al. SAP97 and CASK mediate sorting of NMDA receptors through a previously unknown secretory pathway. Nat Neurosci 12, 1011–1019 (2009). https://doi.org/10.1038/nn.2362

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