Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214, and Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, United Kingdom.
J Neurosci. 2013 Oct 2;33(40):15767-78. doi: 10.1523/JNEUROSCI.1175-13.2013.
Shank3, which encodes a scaffolding protein at glutamatergic synapses, is a genetic risk factor for autism. In this study, we examined the impact of Shank3 deficiency on the NMDA-type glutamate receptor, a key player in cognition and mental illnesses. We found that knockdown of Shank3 with a small interfering RNA (siRNA) caused a significant reduction of NMDAR-mediated ionic or synaptic current, as well as the surface expression of NR1 subunits, in rat cortical cultures. The effect of Shank3 siRNA on NMDAR currents was blocked by an actin stabilizer, and was occluded by an actin destabilizer, suggesting the involvement of actin cytoskeleton. Since actin dynamics is regulated by the GTPase Rac1 and downstream effector p21-activated kinase (PAK), we further examined Shank3 regulation of NMDARs when Rac1 or PAK was manipulated. We found that the reducing effect of Shank3 siRNA on NMDAR currents was mimicked and occluded by specific inhibitors for Rac1 or PAK, and was blocked by constitutively active Rac1 or PAK. Immunocytochemical data showed a strong reduction of F-actin clusters after Shank3 knockdown, which was occluded by a PAK inhibitor. Inhibiting cofilin, the primary downstream target of PAK and a major actin depolymerizing factor, prevented Shank3 siRNA from reducing NMDAR currents and F-actin clusters. Together, these results suggest that Shank3 deficiency induces NMDAR hypofunction by interfering with the Rac1/PAK/cofilin/actin signaling, leading to the loss of NMDAR membrane delivery or stability. It provides a potential mechanism for the role of Shank3 in cognitive deficit in autism.
Shank3 是一种在谷氨酸能突触处起支架蛋白作用的基因，是自闭症的遗传风险因素。在这项研究中，我们研究了 Shank3 缺失对 NMDA 型谷氨酸受体的影响，该受体是认知和精神疾病的关键参与者。我们发现，用小干扰 RNA（siRNA）敲低 Shank3 会导致大鼠皮质培养物中 NMDA 受体介导的离子或突触电流以及 NR1 亚基的表面表达显著减少。Shank3 siRNA 对 NMDAR 电流的作用被肌动蛋白稳定剂阻断，并被肌动蛋白解稳定剂封闭，这表明肌动蛋白细胞骨架的参与。由于肌动蛋白动力学受 GTPase Rac1 和下游效应物 p21 激活激酶（PAK）调节，我们进一步研究了 Rac1 或 PAK 操纵时 Shank3 对 NMDAR 的调节作用。我们发现，Shank3 siRNA 对 NMDAR 电流的减少作用被 Rac1 或 PAK 的特异性抑制剂模拟和封闭，而 Rac1 或 PAK 的组成性激活则阻断了这种作用。免疫细胞化学数据显示，Shank3 敲低后 F-肌动蛋白簇强烈减少，被 PAK 抑制剂封闭。抑制 PAK 的主要下游靶标肌动蛋白解聚因子 cofilin，可防止 Shank3 siRNA 减少 NMDAR 电流和 F-肌动蛋白簇。综上所述，这些结果表明，Shank3 缺失通过干扰 Rac1/PAK/cofilin/actin 信号通路导致 NMDAR 功能低下，从而导致 NMDAR 膜传递或稳定性丧失，从而导致 NMDAR 功能低下。这为 Shank3 在自闭症认知缺陷中的作用提供了一个潜在的机制。
