mtorc2 controls dendritic arbor development through akt-mtorc1-p70s6k pathway

mTORC2通过Akt-mTORC1-p70S6K通路调控树突分枝发育

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ID: 225527
2014
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Abstract
Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that was identified as one of the important regulators of dendritogenesis. mTOR forms two functionally separate complexes (mTORC) in mammalian cells. The complexes are defined by unique mTOR binding partners, Raptor for the mTORC1, and Rictor for the mTORC2. These two complexes control different cellular processes and their roles seem non-overlapping. mTORC1 was initially characterized as a protein translation regulator while mTORC2 was shown to affect cytoskeleton. Both processes are known to be essential for neuron development, but until now the role of particular mTORCs in dendritogenesis was not studied.
Therefore, in our study we precisely characterized mTOR involvement in dendrite development. Using RNA interference in developing rat hippocampal neurons in culture we targeted either Raptor or Rictor to separate activities of mTORC1 and mTORC2, respectively. Dendritic arbors of neurons, depleted of either one of these proteins were reduced. However, no obvious differences were observed in context of dendrite number, length, dendritic arbor complexity and dynamics of dendritic growth and retraction between cells with either Raptor or Rictor knockdown. Yet on the molecular level we were able to pin-point differences between cells lacking Rictor and Raptor. For example, Rictor, but not Raptor knockdown phenotype could be rescued by overexpression of a constitutively active mutant of Akt (myr-Akt), a kinase known to be mTORC2 effector. Besides of being mTORC2 effector, Akt is the most important positive regulator of mTORC1. Since, Rictor and Raptor knockdown have similar and not additive dendritic phenotypes, we hypothesized that mTORC1 acts downstream of mTORC2 during dendritic growth. Therefore, we tested the effects of Rictor knockdown in neurons on phosphorylation of S6 (Ser-235/236) and eIF4B (Ser-422), canonical targets for mTORC1. Indeed, downregulation of Rictor, significantly inhibited phosphorylation of these proteins. Because p70S6K is a kinase responsible for the phosphorylation of S6 and eiF4B in response to Akt-mTORC1 activation, we tested if mTORC2 controls dendritic arbor growth, at least partially, via the Akt-mTOR-p70S6K pathway. In fact, the overexpression of a p70S6KT389E mutant that mimics the phosphorylation of p70S6K1 induced by mTOR, not only restored P-S6 to control levels but also, to some extent, rescued dendritic arbors of cells transfected with shRNAs against Rictor.
Together, our research indicates that 1) both mTOR complexes are crucial for the proper dendritic arbor morphology, and 2) mTORC2 is upstream of mTORC1 in hippocampal neurons in culture.
哺乳动物雷帕霉素靶蛋白（mTOR）是一种丝氨酸/苏氨酸蛋白激酶，被确定为树突发生的重要调控因子之一。在哺乳动物细胞中，mTOR形成两个功能独立的复合物（mTORC）。这两个复合物由独特的mTOR结合伴侣蛋白所界定，即mTORC1的Raptor和mTORC2的Rictor。这两个复合物调控不同的细胞过程，且其作用似乎互不重叠。mTORC1最初被表征为蛋白质翻译调控因子，而mTORC2则被证实可影响细胞骨架。已知这两个过程对神经元发育均至关重要，但迄今为止，特定mTORC在树突发生中的作用尚未得到研究。
因此，在本研究中，我们精确表征了mTOR在树突发育中的参与情况。通过在培养的发育期大鼠海马神经元中利用RNA干扰技术分别靶向Raptor或Rictor，我们得以分别区分mTORC1和mTORC2的活性。缺失这两种蛋白质中任一种的神经元树突分支均减少。然而，在树突数量、长度、树突分支复杂性以及树突生长与回缩的动力学方面，Raptor敲减与Rictor敲减细胞之间未观察到明显差异。但在分子水平上，我们能够精确定位缺乏Rictor和Raptor的细胞之间的差异。例如，过表达Akt的组成型活性突变体（myr-Akt，一种已知的mTORC2效应激酶）可以挽救Rictor敲减表型，但不能挽救Raptor敲减表型。Akt除了作为mTORC2的效应分子外，还是mTORC1最重要的正向调控因子。由于Rictor和Raptor敲减具有相似且非累加的树突表型，我们推测在树突生长过程中mTORC1作用于mTORC2的下游。因此，我们检测了神经元中Rictor敲减对mTORC1经典靶标S6（Ser-235/236）和eIF4B（Ser-422）磷酸化的影响。事实上，下调Rictor显著抑制了这些蛋白质的磷酸化。由于p70S6K是响应Akt-mTORC1激活而负责S6和eIF4B磷酸化的激酶，我们检测了mTORC2是否至少部分通过Akt-mTOR-p70S6K途径控制树突分支生长。事实上，过表达模拟mTOR诱导的p70S6K1磷酸化的p70S6KT389E突变体，不仅使P-S6恢复至对照水平，而且在一定程度上挽救了转染靶向Rictor的shRNA细胞的树突分支。
综上所述，我们的研究表明：1）两种mTOR复合物对于正常的树突分支形态均至关重要；2）在培养的海马神经元中，mTORC2位于mTORC1的上游。
Reference Key
urbanska2014frontiersmtorc2 Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors ;Malgorzata Barbara Urbanska
Journal Vacuum
Year 2014
DOI
10.3389/conf.fnsys.2014.05.00022
URL
http://journal.frontiersin.org/Journal/10.3389/conf.fnsys.2014.05.00022/full
Keywords
neurons mtor raptor rictor neuropsychiatry biological psychiatry

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