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CDKL5 ensures excitatory synapse stability by reinforcing NGL-1–PSD95 interaction in the postsynaptic compartment and is impaired in patient iPSC-derived neurons

Nature Cell Biology volume 14, pages 911–923 (2012)Cite this article

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Abstract

Mutations of the cyclin-dependent kinase-like 5 (CDKL5) and netrin-G1 (NTNG1) genes cause a severe neurodevelopmental disorder with clinical features that are closely related to Rett syndrome, including intellectual disability, early-onset intractable epilepsy and autism. We report here that CDKL5 is localized at excitatory synapses and contributes to correct dendritic spine structure and synapse activity. To exert this role, CDKL5 binds and phosphorylates the cell adhesion molecule NGL-1. This phosphorylation event ensures a stable association between NGL-1 and PSD95. Accordingly, phospho-mutant NGL-1 is unable to induce synaptic contacts whereas its phospho-mimetic form binds PSD95 more efficiently and partially rescues the CDKL5-specific spine defects. Interestingly, similarly to rodent neurons, iPSC-derived neurons from patients with CDKL5 mutations exhibit aberrant dendritic spines, thus suggesting a common function of CDKL5 in mice and humans.

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Figure 1: CDKL5 localizes in dendrites and gathers at excitatory synapses both in vitro and in vivo.
Figure 2: CDKL5 knockdown alters spine morphology and synaptic activity.
Figure 3: CDKL5 knockdown impairs spine structure and excitatory synapse density in vivo.
Figure 4: CDKL5 and NGL-1 interact in vitro and in vivo.
Figure 5: CDKL5 mediates phosphorylation in NGL-1 at Ser 631.
Figure 6: CDKL5-dependent phosphorylation of NGL-1 is necessary for NGL-1–PSD95 binding and correct spine morphogenesis.
Figure 7: Assessment of pluripotency-associated markers in iPSCs and forebrain identity of iPSC-derived neurons.
Figure 8: Patient-specific iPSC-derived neurons exhibit a normal differentiation rate, but aberrant spine structures.

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Acknowledgements

We gratefully acknowledge T. Bienvenu for providing primary fibroblasts from CDKL5 patients, H. Van Esch and H. Archer for providing DNA samples from patients, S. Freier, A. Walther, A. Grimme, U. Fischer and B. Moser for their excellent technical assistance, and L. Musante for helpful discussions. L. Pecciarini is acknowledged for karyotype analysis of the iPSC lines. This study was supported by the Telethon Foundation, Ministry of Health, IIT-SEED project, EuroRETT network to V.B. and the EU-FP7 project GENCODYS (241995) to V.M.K.

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Author notes

  1. Federica Ungaro and Melanie Hambrock: These authors contributed equally to this work

  2. Equally contributing senior authors

  3. Correspondence should be addressed to V.M.K. or V.B. (e-mail:kalscheu@molgen.mpg.de orbroccoli.vania@hsr.it)

Authors and Affiliations

  1. Division of Neuroscience, Stem Cell and Neurogenesis Unit, San Raffaele Scientific Institute, 20132 Milan, Via Olgettina 58, Italy

    Sara Ricciardi, Federica Ungaro, Gilda Stefanelli, Alessandro Sessa & Vania Broccoli

  2. Department of Human Molecular Genetics, Max Planck Institute for Molecular Genetics, Ihnestr. 73, D-14195 Berlin, Germany

    Melanie Hambrock, Nils Rademacher & Vera M. Kalscheuer

  3. Department of Human Physiology, University of Milan Medical School, Milan 20133, Italy

    Dario Brambilla

  4. Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milan 20132, Italy

    Cinzia Magagnotti & Angela Bachi

  5. Department of Structural and Functional Biology, Laboratory of Genetic and Epigenetic Control of Gene Expression, University of Insubria, 21052 Busto Arsizio, VA, Italy

    Elisa Giarda & Charlotte Kilstrup-Nielsen

  6. Consiglio Nazionale delle Ricerche Neuroscience Institute, Milan 20129, Italy

    Chiara Verpelli & Carlo Sala

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Contributions

V.M.K. and N.R. initiated this study. S.R., M.H., N.R, V.M.K. and V.B. contributed to its conceptualization and V.M.K and V.B. directed it throughout; S.R. performed and analysed neuronal experiments. F.U. generated iPSCs and characterized iPSC-derived neurons. M.H. performed and analysed immunoprecipitation assays and Phos-tag assays. M.H. and N.R. performed mutation search. G.S. was involved in mutagenesis experiments. D.B. performed electrophysiological recordings. A.S. performed electroporation experiments. C.M. and A.B. performed 2D gel analysis. E.G. produced the CDKL5-specific antibody. C.V. and C.S. assisted with the preparation of primary neurons. C.K-N. assisted with the in vitro phosphorylation assays. V.M.K., V.B., S.R., M.H. and N.R. interpreted the results. V.B., V.M.K. and S.R. wrote the manuscript.

Corresponding authors

Correspondence to Vera M. Kalscheuer or Vania Broccoli.

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Ricciardi, S., Ungaro, F., Hambrock, M. et al. CDKL5 ensures excitatory synapse stability by reinforcing NGL-1–PSD95 interaction in the postsynaptic compartment and is impaired in patient iPSC-derived neurons. Nat Cell Biol 14, 911–923 (2012). https://doi.org/10.1038/ncb2566

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