Acute knockdown of Depdc5 leads to synaptic defects in mTOR-related epileptogenesis
Acute knockdown of Depdc5 leads to synaptic defects in mTOR-related epileptogenesis
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DOI：
10.1016/j.nbd.2020.104822
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发表时间：
2020-06-01
影响因子：
6.1
通讯作者：
Benfenati, Fabio
中科院分区：
文献类型：
--
作者：
De Fusco, Antonio;Cerullo, Maria Sabina;Benfenati, Fabio
DEPDC 5是GATOR 1复合物的一部分，作为雷帕霉素复合物1（mTORC 1）的机制靶点的关键抑制剂。DEPDC 5中导致mTOR过度激活的功能缺失突变已被确定为病灶性或非病灶性局灶性癫痫的最常见原因。然而，DEPDC 5功能丧失触发神经元和网络过度兴奋的确切机制仍不清楚。在这项研究中，我们研究了过度兴奋的细胞机制，通过比较组成型杂合Depdc 5基因敲除小鼠与不同水平的急性Depdc 5缺失（约40%和约80%的神经元敲除Depdc 5蛋白）的RNA干扰在原代皮层培养。虽然杂合的Depdc 5（+/-）神经元仅具有细微表型，但急性敲低的神经元表现出强剂量依赖性表型，其特征在于mTOR超活化、索马大小增加、树突树枝化、兴奋性突触传递和内在兴奋性。由急性敲低Depdc 5缺陷引起的强大的突触表型突出了Depdc 5敲低的时间动力学在触发表型变化中的重要性，这让人联想到患有局灶性皮质发育不良的患者中的体细胞二次打击机制。这些发现揭示了一种新的突触表型，该表型与Depdc 5敲低有因果关系，突出了Depdc 5的发育作用。有趣的是，突触缺陷似乎只影响兴奋性突触，而抑制性突触正常发育。mEPSC的频率和振幅增加，伴随着兴奋性突触密度增加和谷氨酸受体表达增加，可能产生激发/抑制失衡，触发癫痫发生。
DEP-domain containing 5 (DEPDC5) is part of the GATOR1 complex that functions as key inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1). Loss-of-function mutations in DEPDC5 leading to mTOR hyperactivation have been identified as the most common cause of either lesional or non-lesional focal epilepsy. However, the precise mechanisms by which DEPDC5 loss-of-function triggers neuronal and network hyperexcitability are still unclear. In this study, we investigated the cellular mechanisms of hyperexcitability by comparing the constitutive heterozygous Depdc5 knockout mouse versus different levels of acute Depdc5 deletion (approximate to 40% and approximate to 80% neuronal knockdown of Depdc5 protein) by RNA interference in primary cortical cultures. While heterozygous Depdc5(+/-) neurons have only a subtle phenotype, acutely knocked-down neurons exhibit a strong dose-dependent phenotype characterized by mTOR hyperactivation, increased soma size, dendritic arborization, excitatory synaptic transmission and intrinsic excitability. The robust synaptic phenotype resulting from the acute knockdown Depdc5 deficiency highlights the importance of the temporal dynamics of Depdc5 knockdown in triggering the phenotypic changes, reminiscent of the somatic second-hit mechanism in patients with focal cortical dysplasia. These findings uncover a novel synaptic phenotype that is causally linked to Depdc5 knockdown, highlighting the developmental role of Depdc5. Interestingly, the synaptic defect appears to affect only excitatory synapses, while inhibitory synapses develop normally. The increased frequency and amplitude of mEPSCs, paralleled by increased density of excitatory synapses and expression of glutamate receptors, may generate an excitation/inhibition imbalance that triggers epileptogenesis.