Focal cortical dysplasia, hemimegalencephaly and cortical tubers are paediatric epileptogenic malformations of cortical development (MCDs) frequently pharmacoresistant and mostly treated surgically by the resection of epileptic cortex. Availability of cortical resection samples has allowed significant mechanistic discoveries directly from human material. Causal brain somatic or germline mutations in the AKT/PI3K/DEPDC5/MTOR genes have been identified. GABAA-mediated paradoxical depolarization, related to altered chloride (Cl−) homeostasis, has been shown to participate to ictogenesis in human paediatric MCDs. However, the link between genomic alterations and neuronal hyperexcitability is unclear. Here, we studied the post-translational interactions between the mTOR pathway and the regulation of cation–chloride cotransporters (CCCs), KCC2 and NKCC1, that are largely responsible for controlling intracellular Cl− and, ultimately, GABAergic transmission. For this study, 35 children (25 MTORopathies and 10 pseudo-controls, diagnosed by histology plus genetic profiling) were operated for drug-resistant epilepsy. Postoperative cortical tissues were recorded on a multi-electrode array to map epileptic activities. CCC expression level and phosphorylation status of the WNK1/SPAK-OSR1 pathway was measured during basal conditions and after pharmacological modulation. Direct interactions between mTOR and WNK1 pathway components were investigated by immunoprecipitation. Membranous incorporation of MCD samples in Xenopus laevis oocytes enabled measurement of the Cl− conductance and equilibrium potential for GABA. Of the 25 clinical cases, half harboured a somatic mutation in the mTOR pathway, and pS6 expression was increased in all MCD samples. Spontaneous interictal discharges were recorded in 65% of the slices. CCC expression was altered in MCDs, with a reduced KCC2/NKCC1 ratio and decreased KCC2 membranous expression. CCC expression was regulated by the WNK1/SPAK-OSR1 kinases through direct phosphorylation of Thr906 on KCC2, which was reversed by WNK1 and SPAK antagonists (N-ethylmaleimide and staurosporine). The mSIN1 subunit of MTORC2 was found to interact with SPAK-OSR1 and WNK1. Interactions between these key epileptogenic pathways could be reversed by the mTOR-specific antagonist rapamycin, leading to a dephosphorylation of CCCs and recovery of the KCC2/NKCC1 ratio. The functional effect of such recovery was validated by the restoration of the depolarizing shift in the equilibrium potential for GABA by rapamycin, measured after incorporation of MCD membranes into X. laevis oocytes, in line with a re-establishment of normal Cl− reversal potential. Our study deciphers a protein interaction network through a phosphorylation cascade between MTOR and WNK1/SPAK-OSR1 leading to deregulation of chloride cotransporters, increased neuronal Cl− levels and GABAA dysfunction in malformations of cortical development, linking genomic defects and functional effects and paving the way to target epilepsy therapy.

中文翻译：

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MTOR 相关皮质发育畸形中的氯化物去调和 GABA 去极化


局灶性皮质发育不良、偏侧巨脑畸形和皮质结节是小儿皮质发育 （MCD） 的致痫性畸形，通常具有耐药性，主要通过切除癫痫皮层进行手术治疗。皮质切除样本的可用性允许直接从人体材料中发现重要的机制。已鉴定出 AKT/PI3K/DEPDC5/MTOR 基因中的致病脑体细胞或种系突变。GABAA 介导的反常去极化，与改变的氯化物 （Cl−） 稳态有关，已被证明参与人类儿科 MCD 的发作发生。然而，基因组改变与神经元过度兴奋之间的联系尚不清楚。在这里，我们研究了 mTOR 通路与阳离子-氯化物协同转运蛋白 （CCC）、KCC2 和 NKCC1 的调节之间的翻译后相互作用，它们主要负责控制细胞内 Cl− 并最终控制 GABA 能传递。在这项研究中，35 名儿童 （25 名 MTOR 病和 10 名假对照，通过组织学加基因分析诊断）接受了耐药性癫痫手术。在多电极阵列上记录术后皮质组织以绘制癫痫活动图。在基础条件下和药理学调节后测量 WNK1/SPAK-OSR1 通路的 CCC 表达水平和磷酸化状态。通过免疫沉淀研究 mTOR 和 WNK1 通路成分之间的直接相互作用。在非洲爪蟾卵母细胞中膜状掺入 MCD 样品能够测量 GABA 的 Cl− 电导和平衡电位。在 25 例临床病例中，一半在 mTOR 通路中携带体细胞突变，并且所有 MCD 样本中 pS6 表达均增加。 在 65% 的切片中记录到自发发作间期放电。CCC 在 MCD 中的表达发生改变，KCC2/NKCC1 比值降低，KCC2 膜表达降低。CCC 表达受 WNK1/SPAK-OSR1 激酶通过直接磷酸化 Thr906 在 KCC2 上调节，而被 WNK1 和 SPAK 拮抗剂 （N-乙基马来酰亚胺和星形孢菌素） 逆转。发现 MTORC2 的 mSIN1 亚基与 SPAK-OSR1 和 WNK1 相互作用。这些关键致癫痫途径之间的相互作用可被 mTOR 特异性拮抗剂雷帕霉素逆转，导致 CCC 去磷酸化和 KCC2/NKCC1 比值的恢复。这种恢复的功能效应通过雷帕霉素恢复 GABA 平衡电位的去极化变化来验证，在将 MCD 膜掺入 X. laevis 卵母细胞后测量，与正常 Cl− 逆转电位的重建一致。我们的研究通过 MTOR 和 WNK1/SPAK-OSR1 之间的磷酸化级联破译了蛋白质相互作用网络，导致氯协同转运蛋白的失调、神经元 Cl-水平增加和皮质发育畸形中的 GABAA 功能障碍，将基因组缺陷和功能效应联系起来，并为靶向癫痫治疗铺平了道路。