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Timothy syndrome is associated with activity-dependent dendritic retraction in rodent and human neurons

Nature Neuroscience volume 16, pages 201–209 (2013)Cite this article

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Abstract

L-type voltage gated calcium channels have an important role in neuronal development by promoting dendritic growth and arborization. A point mutation in the gene encoding CaV1.2 causes Timothy syndrome, a neurodevelopmental disorder associated with autism spectrum disorders (ASDs). We report that channels with the Timothy syndrome alteration cause activity-dependent dendrite retraction in rat and mouse neurons and in induced pluripotent stem cell (iPSC)-derived neurons from individuals with Timothy syndrome. Dendrite retraction was independent of calcium permeation through the mutant channel, was associated with ectopic activation of RhoA and was inhibited by overexpression of the channel-associated GTPase Gem. These results suggest that CaV1.2 can activate RhoA signaling independently of Ca2+ and provide insights into the cellular basis of Timothy syndrome and other ASDs.

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Figure 1: TS-CaV1.2 causes activity-dependent dendritic retraction.
Figure 2: Decrease in dendritic complexity in the basal dendrites of layer 2/3 pyramidal cortical neurons from Cacna1c+/tm2Itl mice.
Figure 3: Human iPSCs-derived neurons affected by Timothy syndrome show activity-dependent dendritic retraction.
Figure 4: TS-CaV1.2–mediated dendritic retraction is independent of Ca2+ flux through the channel.
Figure 5: Reducing Gem expression in cortical neurons prevents activity-induced dendritic arborization.
Figure 6: Overexpression of Gem prevents dendritic retraction in TS-CaV1.2–expressing neurons and its effects are dependent on association with the CaVβ subunit.
Figure 7: TS-CaV1.2 causes dendrite retraction by activating RhoA.

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References

  1. Dolmetsch, R. Excitation-transcription coupling: signaling by ion channels to the nucleus. Sci. STKE 2003, PE4 (2003).

    Article  PubMed  Google Scholar 

  2. Tonelli, A. et al. Early onset, non fluctuating spinocerebellar ataxia and a novel missense mutation in CACNA1A gene. J. Neurol. Sci. 241, 13–17 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Wang, K. et al. Common genetic variants on 5p14.1 associate with autism spectrum disorders. Nature 459, 528–533 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Nyegaard, M. et al. CACNA1C (rs1006737) is associated with schizophrenia. Mol. Psychiatry 15, 119–121 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Tottene, A. et al. Familial hemiplegic migraine mutations increase Ca(2+) influx through single human CaV2.1 channels and decrease maximal CaV2.1 current density in neurons. Proc. Natl. Acad. Sci. USA 99, 13284–13289 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Splawski, I. et al. CACNA1H mutations in autism spectrum disorders. J. Biol. Chem. 281, 22085–22091 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Barrett, C.F. & Tsien, R.W. The Timothy syndrome mutation differentially affects voltage- and calcium-dependent inactivation of CaV1.2 L-type calcium channels. Proc. Natl. Acad. Sci. USA 105, 2157–2162 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hoda, J.C., Zaghetto, F., Singh, A., Koschak, A. & Striessnig, J. Effects of congenital stationary night blindness type 2 mutations R508Q and L1364H on Cav1.4 L-type Ca2+ channel function and expression. J. Neurochem. 96, 1648–1658 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Catterall, W.A. Structure and regulation of voltage-gated Ca2+ channels. Annu. Rev. Cell Dev. Biol. 16, 521–555 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Richards, M.W., Butcher, A.J. & Dolphin, A.C. Ca2+ channel beta-subunits: structural insights AID our understanding. Trends Pharmacol. Sci. 25, 626–632 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Pravettoni, E. et al. Different localizations and functions of L-type and N-type calcium channels during development of hippocampal neurons. Dev. Biol. 227, 581–594 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Hell, J.W. et al. Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits. J. Cell Biol. 123, 949–962 (1993).

    Article  CAS  PubMed  Google Scholar 

  13. Splawski, I. et al. Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 119, 19–31 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. McAllister, A.K., Katz, L.C. & Lo, D.C. Neurotrophin regulation of cortical dendritic growth requires activity. Neuron 17, 1057–1064 (1996).

    Article  CAS  PubMed  Google Scholar 

  15. Redmond, L., Kashani, A.H. & Ghosh, A. Calcium regulation of dendritic growth via CaM kinase IV and CREB-mediated transcription. Neuron 34, 999–1010 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Wong, R.O. & Ghosh, A. Activity-dependent regulation of dendritic growth and patterning. Nat. Rev. Neurosci. 3, 803–812 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Aizawa, H. et al. Dendrite development regulated by CREST, a calcium-regulated transcriptional activator. Science 303, 197–202 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Lohmann, C. & Wong, R.O. Regulation of dendritic growth and plasticity by local and global calcium dynamics. Cell Calcium 37, 403–409 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Fink, C.C. et al. Selective regulation of neurite extension and synapse formation by the beta but not the alpha isoform of CaMKII. Neuron 39, 283–297 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Lohmann, C., Myhr, K.L. & Wong, R.O. Transmitter-evoked local calcium release stabilizes developing dendrites. Nature 418, 177–181 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Oyama, F. et al. Gem GTPase and tau: morphological changes induced by gem GTPase in cho cells are antagonized by tau. J. Biol. Chem. 279, 27272–27277 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Mahalakshmi, R.N. et al. Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport. Traffic 8, 1164–1178 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Finlin, B.S. et al. Regulation of L-type Ca2+ channel activity and insulin secretion by the Rem2 GTPase. J. Biol. Chem. 280, 41864–41871 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Beguin, P. et al. Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem. Nature 411, 701–706 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Beguin, P. et al. RGK small GTP-binding proteins interact with the nucleotide kinase domain of Ca2+-channel beta-subunits via an uncommon effector binding domain. J. Biol. Chem. 282, 11509–11520 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Beguin, P. et al. 14–3-3 and calmodulin control subcellular distribution of Kir/Gem and its regulation of cell shape and calcium channel activity. J. Cell Sci. 118, 1923–1934 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Finlin, B.S. et al. Analysis of the complex between Ca2+ channel beta-subunit and the Rem GTPase. J. Biol. Chem. 281, 23557–23566 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Aresta, S., de Tand-Heim, M.F., Beranger, F. & de Gunzburg, J. A novel Rho GTPase-activating-protein interacts with Gem, a member of the Ras superfamily of GTPases. Biochem. J. 367, 57–65 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ward, Y. et al. The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway. J. Cell Biol. 157, 291–302 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bader, P.L. et al. Mouse model of Timothy syndrome recapitulates triad of autistic traits. Proc. Natl. Acad. Sci. USA 108, 15432–15437 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Dolmetsch, R. & Geschwind, D.H. The human brain in a dish: the promise of iPSC-derived neurons. Cell 145, 831–834 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Yazawa, M. et al. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471, 230–234 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pasca, S.P. et al. Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome. Nat. Med. 17, 1657–1662 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Splawski, I. et al. Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations. Proc. Natl. Acad. Sci. USA 102, 8089–8096 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Dolmetsch, R.E., Pajvani, U., Fife, K., Spotts, J.M. & Greenberg, M.E. Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. Science 294, 333–339 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Yang, J., Ellinor, P.T., Sather, W.A., Zhang, J.F. & Tsien, R.W. Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels. Nature 366, 158–161 (1993).

    Article  CAS  PubMed  Google Scholar 

  38. Parent, L. & Gopalakrishnan, M. Glutamate substitution in repeat IV alters divalent and monovalent cation permeation in the heart Ca2+ channel. Biophys. J. 69, 1801–1813 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lee, T., Winter, C., Marticke, S.S., Lee, A. & Luo, L. Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis. Neuron 25, 307–316 (2000).

    Article  CAS  PubMed  Google Scholar 

  40. Swetman, C.A. et al. Extension, retraction and contraction in the formation of a dendritic cell dendrite: distinct roles for Rho GTPases. Eur. J. Immunol. 32, 2074–2083 (2002).

    Article  CAS  PubMed  Google Scholar 

  41. Kranenburg, O. et al. Activation of RhoA by lysophosphatidic acid and Galpha12/13 subunits in neuronal cells: induction of neurite retraction. Mol. Biol. Cell 10, 1851–1857 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Totsukawa, G. et al. Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J. Cell Biol. 150, 797–806 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Casanova, M.F. et al. Minicolumnar abnormalities in autism. Acta Neuropathol. 112, 287–303 (2006).

    Article  PubMed  Google Scholar 

  44. Belmonte, M.K. et al. Autism and abnormal development of brain connectivity. J. Neurosci. 24, 9228–9231 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Geschwind, D.H. & Levitt, P. Autism spectrum disorders: developmental disconnection syndromes. Curr. Opin. Neurobiol. 17, 103–111 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Belmonte, M.K. & Bourgeron, T. Fragile X syndrome and autism at the intersection of genetic and neural networks. Nat. Neurosci. 9, 1221–1225 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Kaufmann, W.E. & Moser, H.W. Dendritic anomalies in disorders associated with mental retardation. Cereb. Cortex 10, 981–991 (2000).

    Article  CAS  PubMed  Google Scholar 

  48. Cohen-Kutner, M., Nachmanni, D. & Atlas, D. CaV2.1 (P/Q channel) interaction with synaptic proteins is essential for depolarization-evoked release. Channels (Austin) 4, 266–277 (2010).

    Article  CAS  Google Scholar 

  49. Wiser, O. et al. The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery. Proc. Natl. Acad. Sci. USA 96, 248–253 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Tanabe, T., Beam, K.G., Adams, B.A., Niidome, T. & Numa, S. Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling. Nature 346, 567–569 (1990).

    Article  CAS  PubMed  Google Scholar 

  51. Green, E.M., Barrett, C.F., Bultynck, G., Shamah, S.M. & Dolmetsch, R.E. The tumor suppressor eIF3e mediates calcium-dependent internalization of the L-type calcium channel CaV1.2. Neuron 55, 615–632 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Gomez-Ospina, N., Tsuruta, F., Barreto-Chang, O., Hu, L. & Dolmetsch, R. The C terminus of the L-type voltage-gated calcium channel Ca(V)1.2 encodes a transcription factor. Cell 127, 591–606 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Yuan, B., Latek, R., Hossbach, M., Tuschl, T. & Lewitter, F. siRNA Selection Server: an automated siRNA oligonucleotide prediction server. Nucl. Acids Res. 32, W130–W134 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Paradis, S. et al. An RNAi-based approach identifies molecules required for glutamatergic and GABAergic synapse development. Neuron 53, 217–232 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Xia, Z., Dudek, H., Miranti, C.K. & Greenberg, M.E. Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism. J. Neurosci. 16, 5425–5436 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Kaech, S. & Banker, G. Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).

    Article  CAS  PubMed  Google Scholar 

  58. Meijering, E. et al. Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry A 58, 167–176 (2004).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank K. Timothy and the individuals with Timothy syndrome who participated in this study, J. Bernstein and J. Hallmayer for recruiting the subjects for this study, and E. Nigh for critical reading of the manuscript. J.F.K. was supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award (F31 NS055549-03) from the National Institute of Neurological Disorders and Stroke. Financial support was provided by a US National Institutes of Health Director's Pioneer Award and a Simons Foundation Grant to R.E.D.; the International Brain Research Organization Outstanding Research Fellowship and the Tashia and John Morgridge Endowed Fellowship to S.P.P.; a Japan Society of the Promotion for Science Postdoctoral Fellowship for Research Abroad and American Heart Association Western States to M.Y.; and a California Institute for Regenerative Medicine Postdoctoral Fellowship to O.S. We are grateful for funding from B. and F. Horowitz, M. McCafferey, B. and J. Packard, P. Kwan and K. Wang.

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  1. Jocelyn F Krey

    Present address: Present address: Oregon Hearing Research Center, Oregon Health and Sciences University, Portland, Oregon, USA.,

Authors and Affiliations

  1. Department of Neurobiology, Stanford University School of Medicine, Stanford, California, USA

    Jocelyn F Krey, Sergiu P Paşca, Aleksandr Shcheglovitov, Masayuki Yazawa, Rachel Schwemberger & Ricardo E Dolmetsch

  2. Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York, USA,

    Randall Rasmusson

  3. Allen Institute for Brain Science, Seattle, Washington, USA

    Ricardo E Dolmetsch

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Contributions

R.E.D. and J.F.K. designed the experiments and wrote the manuscript; J.F.K. performed all of the cellular assays in rodent cells, including the calcium imaging and immunocytochemistry studies, all of the mice breeding and the in vivo studies of dendritic arborization; S.P.P. differentiated iPSCs into neurons and performed the dendritic arborization experiments in human cells; A.S. performed and analyzed the electrophysiology experiments; M.Y. generated and characterized the iPSCs; R.S. contributed to the analysis of dendrites in iPSC-derived neurons; R.R. generated the Timothy syndrome mice.

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Correspondence to Ricardo E Dolmetsch.

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Krey, J., Paşca, S., Shcheglovitov, A. et al. Timothy syndrome is associated with activity-dependent dendritic retraction in rodent and human neurons. Nat Neurosci 16, 201–209 (2013). https://doi.org/10.1038/nn.3307

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