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Nestin negatively regulates postsynaptic differentiation of the neuromuscular synapse

Nature Neuroscience volume 14, pages 324–330 (2011)Cite this article

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Abstract

Positive and negative regulation of neurotransmitter receptor aggregation on the postsynaptic membrane is a critical event during synapse formation. Acetylcholine (ACh) and agrin are two opposing signals that regulate ACh receptor (AChR) clustering during neuromuscular junction (NMJ) development. ACh induces dispersion of AChR clusters that are not stabilized by agrin via a cyclin-dependent kinase 5 Cdk5)-mediated mechanism, but regulation of Cdk5 activation is poorly understood. We found that the intermediate filament protein nestin physically interacts with Cdk5 and is required for ACh-induced association of p35, the co-activator of Cdk5, with the muscle membrane. Blockade of nestin-dependent signaling inhibited ACh-induced Cdk5 activation and the dispersion of AChR clusters in cultured myotubes. Similar to the effects of Cdk5 gene inactivation, knockdown of nestin in agrin-deficient mouse embryos ubstantially restored AChR clusters. These results suggest that nestin is required for ACh-induced, Cdk5-dependent dispersion of AChR clusters during NMJ development.

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Figure 1: Colocalization of nestin and AChR clusters and phosphorylation of nestin on ACh stimulation.
Figure 2: Nestin is required for the recruitment of p35 to the muscle membrane to activate Cdk5.
Figure 3: ACh-induced dispersion of AChR clusters was blocked when nestin was knocked down.
Figure 4: Phosphorylation of nestin by Cdk5 is critical for the dispersion of AChRs in myotubes.
Figure 5: The T316A nestin mutant preferentially binds Cdk5 and inhibits Cdk5 activation.
Figure 6: Knockdown of nestin expression in muscle.
Figure 7: Knockdown of nestin expression in vivo rescues the postsynaptic AChR clusters in Agrn−/− mice.

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  • 13 February 2011

    In the version of this article initially published online, the bottom two panels of Figure 7 were incorrectly labeled. Instead of reading Agrn, they should read Argn–/–. In addition, a grant number was missing from the Acknowledgments. The second sentence of the Acknowledgments should read “This study is supported by US National Institutes of Health grants HD034534, NS047345, NS060833 and NS044420 (K.-F.L.), Muscular Dystrophy Association fellowship MDA4230 (J.Y.), and Academy of Finland, the Research Institute of the Åbo Akademi University Foundation (J.E.E.)." These errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank L.-H. Tsai for the Cdk5 and p35 constructs and A. Nagy for communicating his unpublished results. This study is supported by US National Institutes of Health grants HD034534, NS047345, NS060833 and NS044420 (K.-F.L.), Muscular Dystrophy Association fellowship MDA4230 (J.Y.), and Academy of Finland, the Research Institute of the Åbo Akademi University (J.E.E.).

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Authors and Affiliations

  1. The Salk Institute, La Jolla, California, USA

    Jiefei Yang, Bertha Dominguez, Fred de Winter, Thomas W Gould & Kuo-Fen Lee

  2. Department of Biosciences, Abo Akademi University, Turku, Finland

    John E Eriksson

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  1. Jiefei Yang
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Contributions

J.Y. designed and performed majority of experiments, conducted data analysis and wrote the manuscript. B.D. conducted the transgenic mouse experiment. F.d.W. conducted the transgenic mouse experiment and assisted in data interpretation. T.W.G. assisted in data interpretation and wrote the manuscript. J.E.E. provided constructs and assisted in data interpretation. K.-F.L. supervised the project, designed experiments and wrote the manuscript.

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Correspondence to Kuo-Fen Lee.

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Yang, J., Dominguez, B., de Winter, F. et al. Nestin negatively regulates postsynaptic differentiation of the neuromuscular synapse. Nat Neurosci 14, 324–330 (2011). https://doi.org/10.1038/nn.2747

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