Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.
Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.
Mol Psychiatry. 2018 Sep;23(9):1832-1850. doi: 10.1038/s41380-018-0027-3. Epub 2018 Apr 9.
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.
接触蛋白相关蛋白 2（CNTNAP2）已成为一个突出的易感基因，与多种复杂的神经发育障碍有关，包括自闭症谱系障碍（ASD）、智力障碍（ID）和精神分裂症（SCZ）。许多情况下存在癫痫共病，以及 Cntnap2 敲除（KO）小鼠抑制性神经元功能障碍，表明 CNTNAP2 对于适当的抑制性网络功能可能至关重要。然而，潜在的细胞机制尚不清楚。在这里，我们展示了培养的 Cntnap2 KO 小鼠神经元表现出抑制性神经元特有的树突分支简化。这些改变可以通过在成熟的野生型（WT）神经元中急性敲低 CNTNAP2 来复制，并且是由错误的树突稳定而不是生长引起的。使用结构照明显微镜（SIM）和受激发射损耗显微镜（STED），两种超分辨率成像技术，我们发现了纳米级 CNTNAP2 蛋白定位与树突分支模式之间的关系。通过酵母双杂交筛选、生化分析、原位邻近连接测定（PLA）、SIM 和表型挽救，我们表明这些影响是通过 CNTNAP2 的 C 末端与钙/钙调蛋白依赖性丝氨酸蛋白激酶（CASK）之间的膜相互作用介导的，CASK 是另一个 ASD/ID 风险基因。最后，我们发现成年 Cntnap2 KO 小鼠的特定皮层区域的中间神经元树突长度和分支减少，以及皮层膜部分的 CASK 水平降低。总之，我们的数据揭示了一种神经元特异性的树突稳定机制，它可能为 CNTNAP2 相关疾病中抑制性回路功能障碍提供了一种细胞机制。
