Myelination has been evolved in the vertebrate system as an essential mechanism to facilitate axonal signal transduction and provide trophic support for neurons. Formation and maintenance of myelin sheaths are tightly regulated throughout the development by diverse cell metabolic pathways. Consequently, the impairment of myelin homeostasis, for instance by an altered metabolism of myelinating glial cells, can lead to severe axonal degeneration. Inherited peripheral neuropathies such as the most commonly inherited Charcot-Marie-Tooth (CMT) diseases are a heterogeneous group of disorders, caused by mutations in more than 80 different genes. The demyelinating CMT-type 4B results from loss-of-function or missense mutations in myotubularin-related protein 2 (MTMR2), MTMR5 or MTMR13. These proteins belong to the family of MTMR phosphatidylinositol (PI) phosphatases that specifically hydrolyze the endomembrane signaling lipids PI(3)P and PI(3,5)P 2 at the D-3 position. How dysregulated PI(3)-phosphate conversion in myelinating Schwann cells can lead to such a severely affected myelin homeostasis, as observed in the absence of CMT-associated MTMRs, is still elusive. Here, we show that the small GTPase Rab35, a critical regulator of endomembrane trafficking, interacts with CMT-associated MTMR lipid phosphatases. Rab35 binds and recruits the pseudophosphatases MTMR13 and MTMR5, and via these, also the active phosphatase MTMR2. In agreement with the critical involvement of these proteins in the myelin homeostasis of the peripheral nervous system (PNS), Schwann cell-specific Rab35 ablation results in severe peripheral demyelination in mice. Sciatic nerves of these animals are characterized by aberrantly myelinated fibers with Tomacula and myelin outfoldings. This progressive focal hypermyelination is accompanied by abnormally elevated activity of mTORC1, a central cell signaling hub that needs to be crucially balanced for proper myelination. Inhibiting mTORC1 activity by pharmaceutical treatment of these mice using Rapamycin leads to a partial amelioration of the nerve morphology. Moreover, reduced myelin segment formation and myelin abnormalities in Rab35-depleted Schwann cells ex vivo, in Schwann cell and dorsal root ganglion neuron co-cultures, are rescued by Rapamycin application. These findings strongly indicate that mTORC1 hyperactivity contributes to the observed impairment of myelin homeostasis in conditional Rab35 knockout (KO) animals. Furthermore, the absence of Rab35 or the active phosphatase MTMR2, or both, result in elevated mTORC1 activity in different cultured cell types, including primary cells of the nervous system. This hyperactivity is displayed independently of receptor tyrosine kinase (RTK) and AKT activation. Notably, physiological mTORC1 activity can be restored in Rab35-depleted cells by overexpression of MTMR2, suggesting a sequential function of the small GTPase and active MTMR complexes in repressing mTORC1. In accordance with the lipid phosphatase activity of MTMR2, we further show that dysregulated PI(3)-phosphate levels in the absence of Rab35 are causative for the observed mTORC1 hyperactivation, and likely result from an impaired recruitment of MTMR complexes. Rab35-depleted cells display an accumulation of PI(3)P and likely its product, PI(3,5)P 2 . Importantly, pharmacological interference with VPS34-mediated PI(3)P synthesis restores physiological levels of mTORC1 activity. We observe this mechanism in non-myelinating cells as well as in Schwann cells. Elevated mTORC1 activity and PI(3)P accumulation are accompanied by increased levels of the myelin protein P0 in differentiated Rab35 KO Schwann cells in culture. Importantly, P0-protein levels are reduced and comparable to WT levels upon pharmacological inhibition of mTORC1, VPS34, and especially the PI(3,5)P 2 -synthesizing enzyme PIKfyve. This suggests that PI(3,5)P 2 is the critical lipid for mTORC1 activation in PNS myelinating glial cells and thus, provides a possible explanation for the crucial effect of the loss of MTMR proteins on myelin homeostasis. Moreover, our data indicate a similar role for Rab35 in myelinating glial cells of the central nervous system (CNS). Taken together, we could identify Rab35 as a novel critical regulator of both, myelin homeostasis and mTORC1 activity. We propose a mechanism in which Rab35 represses mTORC1 activity by the recruitment of PI(3)-phosphate-hydrolyzing MTMR complexes to lysosomal sites. These findings may have implications for a potential therapeutic treatment of CMT4B-patients by pharmaceutical mTORC1-inhibition, for instance with Rapamycin.
