Dynamic analysis of 4E-BP1 phosphorylation in neurons with Tsc2 or Depdc5 knockout.
Dynamic analysis of 4E-BP1 phosphorylation in neurons with Tsc2 or Depdc5 knockout.
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DOI：
10.1016/j.expneurol.2020.113432
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发表时间：
2020-12
影响因子：
5.3
通讯作者：
Crino PB
中科院分区：
文献类型：
--
作者：
Iffland PH 2nd;Barnes AE;Baybis M;Crino PB
TSC 1或TSC 2突变导致严重的硬化症复合体（TSC），并导致雷帕霉素（mTOR）的机制性靶点过度活化，这通过核糖体S6蛋白和4-延伸因子结合蛋白（4 E-BP 1）的过度磷酸化来证明。氨基酸（AA）水平调节非神经细胞中mTOR依赖性S6和4 E-BP 1磷酸化，但尚未在神经元中研究。在Tsc 2和Depdc 5（与癫痫相关的不同mTOR调节基因）CRISPR编辑的Neuro 2a（N2 a）细胞和分化的神经元中分析AA水平对mTOR信号传导和S6和4 E-BP 1磷酸化的影响。Tsc 2或Depdc 5敲除（KO）导致S6和4 E-BP 1过度磷酸化和细胞索马体增大，但Tsc 2 KO N2 a细胞在无AA（AAF）培养基中孵育后显示S6磷酸化（Ser 240/244）和细胞索马体大小减少，而Depdc 5 KO细胞则没有。使用与4 E-BP 1偶联的CFP/YFP FRET生物传感器，我们测定了Tsc 2或Depdc 5 KO后活N2 a细胞和分化神经元中的4 E-BP 1磷酸化。AAF条件降低Tsc 2 KO N2 a细胞中的4 E-BP 1磷酸化，但对Depdc 5 KO细胞没有影响。在Tsc 2或Depdc 5 KO后，雷帕霉素阻断S6蛋白磷酸化，但对4 E-BP 1磷酸化没有影响。共聚焦成像证明，在Tsc 2 KO而不是Depdc 5 KO细胞中，AAF介质促进mTOR从溶酶体中移出，功能性灭活mTOR，证明AA水平调节溶酶体mTOR定位，并部分解释Tsc 2与Depdc 5 KO后AAF条件的差异效应。AA水平和雷帕霉素差异调节Tsc 2 KO与Depdc 5 KO后神经元中S6和4 E-BP 1磷酸化和mTOR溶酶体定位。mTOR相关癫痫中的神经元mTOR信号传导可能对mTOR抑制剂和细胞氨基酸水平具有不同的反应。
TSC1 or TSC2 mutations cause Tuberous Sclerosis Complex (TSC), and lead to mechanistic target of rapamycin (mTOR) hyperactivation evidenced by hyperphosphorylation of ribosomal S6 protein and 4-elongation factor binding protein (4E-BP1). Amino acid (AA) levels modulate mTOR-dependent S6 and 4E-BP1 phosphorylation in non-neural cells, but this has not been investigated in neurons. The effects of AA levels on mTOR signaling and S6 and 4E-BP1 phosphorylation were analyzed in Tsc2 and Depdc5 (a distinct mTOR regulatory gene associated with epilepsy) CRISPR-edited Neuro2a (N2a) cells and differentiated neurons. Tsc2 or Depdc5 knockout (KO) led to S6 and 4E-BP1 hyperphosphorylation and cell soma enlargement, but while Tsc2 KO N2a cells exhibited reduced S6 phosphorylation (Ser240/244) and cell soma size after incubation in AA free (AAF) media, Depdc5 KO cells did not. Using a CFP/YFP FRET-biosensor coupled to 4E-BP1, we assayed 4E-BP1 phosphorylation in living N2a cells and differentiated neurons following Tsc2 or Depdc5 KO. AAF conditions reduced 4E-BP1 phosphorylation in Tsc2 KO N2a cells but had no effect in Depdc5 KO cells. Rapamycin blocked S6 protein phosphorylation but had no effect on 4E-BP1 phosphorylation, following either Tsc2 or Depdc5 KO. Confocal imaging demonstrated that AAF media promoted movement of mTOR off the lysosome, functionally inactivating mTOR, in Tsc2 KO but not Depdc5 KO cells, demonstrating that AA levels modulate lysosomal mTOR localization and account, in part, for differential effects of AAF conditions following Tsc2 versus Depdc5 KO. AA levels and rapamycin differentially modulate S6 and 4E-BP1 phosphorylation and mTOR lysosomal localization in neurons following Tsc2 KO versus Depdc5 KO. Neuronal mTOR signaling in mTOR associated epilepsies may have distinct responses to mTOR inhibitors and to levels of cellular amino acids.
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影响因子：
6.1
作者：
Iffland PH 2nd;Baybis M;Barnes AE;Leventer RJ;Lockhart PJ;Crino PB
通讯作者：
Crino PB
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
DOI：
10.1038/s41436-018-0060-2
发表时间：
2019-03
期刊：
Genetics in medicine : official journal of the American College of Medical Genetics
影响因子：
--
作者：
Baldassari S;Picard F;Verbeek NE;van Kempen M;Brilstra EH;Lesca G;Conti V;Guerrini R;Bisulli F;Licchetta L;Pippucci T;Tinuper P;Hirsch E;de Saint Martin A;Chelly J;Rudolf G;Chipaux M;Ferrand-Sorbets S;Dorfmüller G;Sisodiya S;Balestrini S;Schoeler N;Hernandez-Hernandez L;Krithika S;Oegema R;Hagebeuk E;Gunning B;Deckers C;Berghuis B;Wegner I;Niks E;Jansen FE;Braun K;de Jong D;Rubboli G;Talvik I;Sander V;Uldall P;Jacquemont ML;Nava C;Leguern E;Julia S;Gambardella A;d'Orsi G;Crichiutti G;Faivre L;Darmency V;Benova B;Krsek P;Biraben A;Lebre AS;Jennesson M;Sattar S;Marchal C;Nordli DR Jr;Lindstrom K;Striano P;Lomax LB;Kiss C;Bartolomei F;Lepine AF;Schoonjans AS;Stouffs K;Jansen A;Panagiotakaki E;Ricard-Mousnier B;Thevenon J;de Bellescize J;Catenoix H;Dorn T;Zenker M;Müller-Schlüter K;Brandt C;Krey I;Polster T;Wolff M;Balci M;Rostasy K;Achaz G;Zacher P;Becher T;Cloppenborg T;Yuskaitis CJ;Weckhuysen S;Poduri A;Lemke JR;Møller RS;Baulac S
通讯作者：
Baulac S
影响因子：
64.5
作者：
Demetriades C;Doumpas N;Teleman AA
通讯作者：
Teleman AA
DOI：
10.1073/pnas.1605740113
发表时间：
2016-10-04
影响因子：
11.1
作者：
Lin, Tiffany V.;Hsieh, Lawrence;Bordey, Angelique
通讯作者：
Bordey, Angelique