DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.
DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.
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DOI：
10.1016/j.nbd.2018.02.013
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发表时间：
2018-06
影响因子：
6.1
通讯作者：
Crino PB
中科院分区：
文献类型：
--
作者：
Iffland PH 2nd;Baybis M;Barnes AE;Leventer RJ;Lockhart PJ;Crino PB
GATOR 1的DEPDC 5和NPRL 3亚基（雷帕霉素机制靶点（mTOR）的调节剂）突变与皮质发育畸形（MCD）有关。来自这些个体的脑标本显示异常的皮质层压，改变的细胞形态，以及核糖体S6蛋白（PS 6）的过度磷酸化，这是mTOR激活的标志物。虽然许多研究已经检查了GATOR 1亚基在非神经元细胞系中的功能，但很少有直接评估神经元细胞类型中GATOR 1亚基功能的丧失。我们假设DEPDC 5或NPRL 3 shRNA介导的敲低（DEPDC 5/NPRL 3 KD）导致mTOR的不适当功能激活和mTOR依赖性神经元形态学改变。神经元的大小，确定在人类标本窝藏DEPDC 5或NPRL 3突变切除癫痫治疗。在小鼠神经母细胞瘤细胞（N2 aC）和小鼠脑室下区衍生的神经祖细胞（mNPC）中测定DEPDC 5/NPRL 3 KD对细胞大小、丝状伪足延伸、亚细胞mTOR复合物1（mTORC 1）定位和营养剥夺期间mTORC 1活化的影响。使用mTOR抑制剂雷帕霉素测定DEPDC 5/NPRL 3 KD的mTORC 1依赖性作用。通过检查氨基酸饥饿期间mTOR与溶酶体表面的接近度，确定DEPDC 5/NPRL 3 KD后mTOR亚细胞定位和mTORC 1通路活化的变化。人类样本中表现出PS 6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，DEPDC 5/NPRL 3 KD导致N2 aC和mNPC中mTORC 1而非mTORC 2过度活化、索马增大和丝状伪足增加。DEPDC 5/NPRL 3 KD导致mTOR不适当地定位于溶酶体沿着氨基酸剥夺后的组成性mTOR活化。雷帕霉素逆转了DEPDC 5/NPRL 3 KD对形态和功能性mTOR活化的影响。DEPDC 5/NPRL 3 KD对mTOR在神经元中的形态学和亚细胞定位的mTOR依赖性作用表明，GATOR 1亚基的功能丧失可能在胎儿脑发育期间的MCD形成中起作用。
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 GATOR 1 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.
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影响因子：
11.2
作者：
Desikan, Rahul S.;Barkovich, A. James
通讯作者：
Barkovich, A. James
影响因子：
8.8
作者：
Chantranupong L;Wolfson RL;Orozco JM;Saxton RA;Scaria SM;Bar-Peled L;Spooner E;Isasa M;Gygi SP;Sabatini DM
通讯作者：
Sabatini DM
影响因子：
5.3
作者：
Asada, Naoyuki;Sanada, Kamon;Fukada, Yoshitaka
通讯作者：
Fukada, Yoshitaka
影响因子：
6.1
作者：
Srivastava, Isha N.;Shperdheja, Jona;Crino, Peter B.
通讯作者：
Crino, Peter B.
影响因子：
15.9
作者：
Orlova, Ksenia A.;Parker, Whitney E.;Crino, Peter B.
通讯作者：
Crino, Peter B.