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Cypin regulates dendrite patterning in hippocampal neurons by promoting microtubule assembly

Abstract

Dendrite branching has an important role in normal brain function. Here we report that overexpression of cypin, a protein that has guanine deaminase activity and is expressed in developing processes in rat hippocampal neurons, results in increased dendrite branching in primary culture. Mutant cypin proteins that lack guanine deaminase activity act in a dominant-negative manner when expressed in primary neurons. Furthermore, we knocked down cypin protein levels using a new strategy: expressing a 5′ end-mutated U1 small nuclear RNA (snRNA) to inhibit maturation of cypin mRNA. Neurons that express this mutant snRNA show little or no detectable cypin protein and fewer dendrites than normal. In addition, we found that cypin binds directly to tubulin heterodimers and promotes microtubule polymerization. Thus, our results demonstrate a new pathway by which dendrite patterning is regulated, and we also introduce a new method for decreasing endogenous protein expression in neurons.

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Figure 1: Expression of cypin protein in developing hippocampal neurons in culture.
Figure 2: Zinc binding and CRMP homology domains are required for normal guanine deaminase activity.
Figure 3: Cypin induces dendrite outgrowth and branching.
Figure 4: Mutated U1 snRNAs knock down cypin protein expression.
Figure 5: Knockdown of cypin protein expression results in decreased dendrite number.
Figure 6: Dominant-negative (DN) constructs of Rho, Rac and Cdc42 block cypin-mediated dendrite branching.
Figure 7: Cypin binds to directly to tubulin heterodimers and promotes assembly.
Figure 8: Activity regulates cypin protein, which is enriched in inhibitory interneurons.

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Acknowledgements

We thank C. Rongo and L. Covey for insightful discussion and critical reading of our manuscript. We would also like to thank C. Barbieri for performing some preliminary experiments and A. Ghosh for constructs encoding dominant negative small GTPases. B.L.F. would like to thank M. Miller for his support. This work was supported in part by Busch Biomedical Grants (to B.L.F. and S.I.G.), New Jersey Commission on Spinal Cord Research Grant 03–004 and National Science Foundation grant IBN–0234206(to B.L.F.) and a Discovery Grant from Johnson and Johnson and National Institutes of Health grant GM57286 (to S.I.G.).

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Correspondence to Bonnie L Firestein.

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Akum, B., Chen, M., Gunderson, S. et al. Cypin regulates dendrite patterning in hippocampal neurons by promoting microtubule assembly. Nat Neurosci 7, 145–152 (2004). https://doi.org/10.1038/nn1179

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