DEPDC5 和 NPRL3 调节神经祖细胞和神经元中的细胞大小、丝状伪足的生长以及 mTOR 的定位。

DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.

机构信息

Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, United States; Department of Neurology, University of Maryland School of Medicine, Baltimore, MD, United States.

Department of Neurology, University of Maryland School of Medicine, Baltimore, MD, United States.

出版信息

Neurobiol Dis. 2018 Jun;114:184-193. doi: 10.1016/j.nbd.2018.02.013. Epub 2018 Feb 24.

DOI:10.1016/j.nbd.2018.02.013

PMID:29481864

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6413529/

Abstract

Mutations in DEPDC5 and NPRL3 subunits of GATOR1, a modulator of mechanistic target of rapamycin (mTOR), are linked to malformations of cortical development (MCD). Brain specimens from these individuals reveal abnormal cortical lamination, altered cell morphology, and hyperphosphorylation of ribosomal S6 protein (PS6), a marker for mTOR activation. While numerous studies have examined GATOR1 subunit function in non-neuronal cell lines, few have directly assessed loss of GATOR1 subunit function in neuronal cell types. We hypothesized that DEPDC5 or NPRL3 shRNA-mediated knockdown (DEPDC5/NPRL3 KD) leads to inappropriate functional activation of mTOR and mTOR-dependent alterations in neuronal morphology. Neuronal size was determined in human specimens harboring DEPDC5 or NPRL3 mutations resected for epilepsy treatment. DEPDC5/NPRL3 KD effects on cell size, filopodial extension, subcellular mTOR complex 1 (mTORC1) localization, and mTORC1 activation during nutrient deprivation were assayed in mouse neuroblastoma cells (N2aC) and mouse subventricular zone derived neural progenitor cells (mNPCs). mTORC1-dependent effects of DEPDC5/NPRL3 KD were determined using the mTOR inhibitor rapamycin. Changes in mTOR subcellular localization and mTORC1 pathway activation following DEPDC5/NPRL3 KD were determined by examining the proximity of mTOR to the lysosomal surface during amino acid starvation. Neurons exhibiting PS6 immunoreactivity (Ser 235/236) in human specimens were 1.5× larger than neurons in post-mortem control samples. DEPDC5/NPRL3 KD caused mTORC1, but not mTORC2, hyperactivation, soma enlargement, and increased filopodia in N2aC and mNPCs compared with wildtype cells. DEPDC5/NPRL3 KD led to inappropriate mTOR localization at the lysosome along with constitutive mTOR activation following amino acid deprivation. DEPDC5/NPRL3 KD effects on morphology and functional mTOR activation were reversed by rapamycin. mTOR-dependent effects of DEPDC5/NPRL3 KD on morphology and subcellular localization of mTOR in neurons suggests that loss-of-function in GATOR1 subunits may play a role in MCD formation during fetal brain development.

摘要

GATOR1 是雷帕霉素（mTOR）的机械靶点调节剂，其 DEPDC5 和 NPRL3 亚基的突变与皮质发育畸形（MCD）有关。这些个体的脑标本显示皮质层排列异常、细胞形态改变以及核糖体 S6 蛋白（PS6）过度磷酸化，PS6 是 mTOR 激活的标志物。虽然许多研究都检查了非神经元细胞系中 GATOR1 亚基的功能，但很少有研究直接评估神经元细胞类型中 GATOR1 亚基功能丧失的情况。我们假设 DEPDC5 或 NPRL3 shRNA 介导的敲低（DEPDC5/NPRL3 KD）导致 mTOR 的不当功能激活以及神经元形态的 mTOR 依赖性改变。在因癫痫治疗而切除的携带 DEPDC5 或 NPRL3 突变的人类标本中测定神经元大小。在小鼠神经母细胞瘤细胞（N2aC）和小鼠侧脑室下区衍生的神经前体细胞（mNPC）中检测 DEPDC5/NPRL3 KD 对细胞大小、丝状伪足延伸、细胞内 mTOR 复合物 1（mTORC1）定位以及营养剥夺期间 mTORC1 激活的影响。使用 mTOR 抑制剂雷帕霉素确定 DEPDC5/NPRL3 KD 的 mTORC1 依赖性影响。通过在氨基酸饥饿时检查 mTOR 与溶酶体表面的接近程度，确定 DEPDC5/NPRL3 KD 后 mTOR 亚细胞定位和 mTORC1 途径激活的变化。在人类标本中表现出 PS6 免疫反应性（Ser 235/236）的神经元比死后对照样本中的神经元大 1.5 倍。与野生型细胞相比，DEPDC5/NPRL3 KD 导致 mTORC1 但不导致 mTORC2 过度激活、体增大和丝状伪足增加在 N2aC 和 mNPCs 中。DEPDC5/NPRL3 KD 导致 mTOR 在溶酶体中的位置不当，以及在氨基酸剥夺后 mTOR 的组成性激活。雷帕霉素逆转了 DEPDC5/NPRL3 KD 对形态和功能 mTOR 激活的影响。DEPDC5/NPRL3 KD 对神经元形态和 mTOR 细胞内定位的 mTOR 依赖性影响表明，GATOR1 亚基的功能丧失可能在胎儿大脑发育过程中的 MCD 形成中发挥作用。