Contactin associated protein-like 2 (CNTNAP2) has emerged as a prominent susceptibility gene implicated in multiple complex neurodevelopmental disorders, including autism spectrum disorders (ASD), intellectual disability (ID), and schizophrenia (SCZ). The presence of seizure comorbidity in many of these cases, as well as inhibitory neuron dysfunction in Cntnap2 knockout (KO) mice, suggests CNTNAP2 may be crucial for proper inhibitory network function. However, underlying cellular mechanisms are unclear. Here we show that cultured Cntnap2 KO mouse neurons exhibit an inhibitory neuron-specific simplification of the dendritic tree. These alterations can be replicated by acute knockdown of CNTNAP2 in mature wild-type (WT) neurons and are caused by faulty dendrite stabilization rather than outgrowth. Using structured illumination microscopy (SIM) and stimulated-emission depletion microscopy (STED), two super-resolution imaging techniques, we uncovered relationships between nanoscale CNTNAP2 protein localization and dendrite arborization patterns. Employing yeast two-hybrid screening, biochemical analysis, in situ proximity ligation assay (PLA), SIM, and phenotype rescue, we show that these effects are mediated at the membrane by the interaction of CNTNAP2’s C-terminus with calcium/calmodulin-dependent serine protein kinase (CASK), another ASD/ID risk gene. Finally, we show that adult Cntnap2 KO mice have reduced interneuron dendritic length and branching in particular cortical regions, as well as decreased CASK levels in the cortical membrane fraction. Taken together, our data reveal an interneuron-specific mechanism for dendrite stabilization that may provide a cellular mechanism for inhibitory circuit dysfunction in CNTNAP2-related disorders.
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This work was supported by the grants NS100785 and MH097216 from the NIH-NIMH to P.P and F30MH096457 to R.G. SIM imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by the NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. Structured illumination microscopy was performed on a Nikon N-SIM system, purchased through the support of NIH 1S10OD016342–01. We thank Dr. Joshua Zachary Rappoport for help with SIM imaging, Dr. Daniel Vogt for his consultation, and Xi Chao for help with figure illustrations.
R.G. led the project and performed all confocal and SIM imaging experiments, K.M. performed STED imaging experiments, R.G., A.E.M.-Z, S.Y., and T.A.R. performed in vivo experiments, R.G., N.H.P., M.P.F., and M.D.M.-de.S. performed biochemistry experiments, G.Z. assisted with data analysis. P.P. supervised the project while J.G.C. and D.J.S. advised. R.G. and P.P. wrote the manuscript.
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The authors declare that they have no conflict of interest.
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Gao, R., Piguel, N.H., Melendez-Zaidi, A.E. et al. CNTNAP2 stabilizes interneuron dendritic arbors through CASK. Mol Psychiatry 23, 1832–1850 (2018). https://doi.org/10.1038/s41380-018-0027-3
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