Institut de Neurobiologie de la Méditerranée, Institut National de la Santé et de la Recherche Médicale, Aix-Marseille University, Marseille, France.
Epilepsia. 2024 Jul;65(7):2111-2126. doi: 10.1111/epi.18000. Epub 2024 May 8.
Genetic variations in proteins of the mechanistic target of rapamycin (mTOR) pathway cause a spectrum of neurodevelopmental disorders often associated with brain malformations and with intractable epilepsy. The mTORopathies are characterized by hyperactive mTOR pathway and comprise tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) type II. How hyperactive mTOR translates into abnormal neuronal activity and hypersynchronous network remains to be better understood. Previously, the role of upregulated GluN2C-containing glutamate-gated N-methyl-D-aspartate receptors (NMDARs) has been demonstrated for germline defects in the TSC genes. Here, we questioned whether this mechanism would expand to other mTORopathies in the different context of a somatic genetic variation of the MTOR protein recurrently found in FCD type II.
We used a rat model of FCD created by in utero electroporation of neural progenitors of dorsal telencephalon with expression vectors encoding either the wild-type or the pathogenic MTOR variant (p.S2215F). In this mosaic configuration, patch-clamp whole-cell recordings of the electroporated, spiny stellate neurons and extracellular recordings of the electroporated areas were performed in neocortical slices. Selective inhibitors were used to target mTOR activity and GluN2C-mediated currents.
Neurons expressing the mutant protein displayed an excessive activation of GluN2C NMDAR-mediated spontaneous excitatory postsynaptic currents. GluN2C-dependent increase in spontaneous spiking activity was detected in the area of electroporated neurons in the mutant condition and was restricted to a critical time window between postnatal days P9 and P20.
Somatic MTOR pathogenic variant recurrently found in FCD type II resulted in overactivation of GluN2C-mediated neuronal NMDARs in neocortices of rat pups. The related and time-restricted local hyperexcitability was sensitive to subunit GluN2C-specific blockade. Our study suggests that GluN2C-related pathomechanisms might be shared in common by mTOR-related brain disorders.
机械靶标雷帕霉素（mTOR）途径蛋白的遗传变异导致一系列神经发育障碍，这些障碍通常与脑畸形和难治性癫痫有关。mTOR 病的特征是 mTOR 途径过度活跃，包括结节性硬化症（TSC）和局灶性皮质发育不良（FCD）II 型。过度活跃的 mTOR 如何转化为异常神经元活动和超同步网络仍有待更好地理解。以前，已经证明 TSC 基因种系缺陷中上调的含有 GluN2C 的谷氨酸门控 N-甲基-D-天冬氨酸受体（NMDAR）的作用。在这里，我们质疑这种机制是否会扩展到 FCD 类型 II 中反复发现的 MTOR 蛋白体细胞遗传变异的不同情况下的其他 mTOR 病。
我们使用通过在体电穿孔向背侧端脑的神经祖细胞表达载体转染野生型或致病 MTOR 变体（p.S2215F）创建的 FCD 大鼠模型。在这种镶嵌配置中，对电穿孔的棘状星状神经元进行全细胞膜片钳记录，对电穿孔区域进行细胞外记录。选择性抑制剂用于靶向 mTOR 活性和 GluN2C 介导的电流。
表达突变蛋白的神经元显示出 GluN2C 介导的自发性兴奋性突触后电流的过度激活。在突变条件下，在电穿孔神经元的区域中检测到 GluN2C 依赖性自发性尖峰活动增加，并且仅局限于出生后第 9 天至第 20 天之间的关键时间窗口。
在 FCD 类型 II 中反复发现的体细胞 MTOR 致病变体导致新生大鼠皮质中 GluN2C 介导的神经元 NMDAR 的过度激活。相关的和时间限制的局部过度兴奋性对亚基 GluN2C 特异性阻断敏感。我们的研究表明，GluN2C 相关的病理机制可能在 mTOR 相关的脑疾病中共同存在。
