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

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.