MIOS/GATOR2 Mediated Amino Acid Signaling Pathway Regulates Brain Myelination

Myelin is a compact and insulating multiple layers of membrane around axons in the nervous system,which facilitates nerve impulses to conduct in "jumping" manner.In addition,myelin provides metabolic support to the axons and protects neurons from damage.Cell growth,division and differentiation depend on the support of nutrients and intracellular signaling conduction,especially the mTORC1 pathway activated by growth factors and amino acids.Previous studies have identified RHEB-mTORC1 signaling is essential for oligodendrocyte differentiation and myelin development in the CNS.Amino acids have been found to be involved in the process of intracellular signal transduction.However,the physiological functions of their signaling pathway in mammalian tissue and cell development have not been reported yet.Therefore,this thesis will mainly investigate the role of amino acid sensing/signaling pathways in the development of myelin in the mammalian CNS,explore the potential mechanism of amino acid sensing/signaling pathway in regulating myelin development.To investigate the function of amino acid sensing/signaling pathway in development central nervous system,we generated Mios(encoding a component of GATOR2,a key protein complex in amino acid sensing/signaling pathway)knockout mouse in neural stem cells by Cre-loxp technology.Previous studies demonstrated that Mios knockout reduced mTORC1 activity regulated by amino acids in Drosophila and in vitro cells.However,in our study we only found mTORC1 activity reduction in oligodendrocytes Mios knockout,but not in other nerve cells(such as neurons).We also demonstrated that it was blocked amino acids signaling pathway caused mTORC1 activity reduction in Mios-deleted oligodendrocytes through in vitro experiments,and further studies showed amino acids regulating mTORC1 activity was cell-specific.In order to clarify the function of amino acid sensing/signaling pathway in cell development,we also detected nerve cells generation in Mios neural stem cells knockout mice.We found that the number of oligodendrocytes was significantly reduced(including OPCs and mature oligodendrocytes);however,the number of neurons and astrocytes were not altered in Mios knockout compared with control mice.These results suggest that the amino acid sensing/signaling pathway mediated by MIOS/GATOR2 also has cell specificity in regulating nerve cells development.Further experiments in cell biology found that amino acid sensing/signaling pathway may regulate oligodendrocyte development through controlling the cell cycle progression in OPCs.Oligodendrocytes develop and wrap axons to form myelin in the CNS,and their dysplasia may lead to hypomyelination.To determine whether MIOS deletion also causes myelin disorder in the CNS,we further examined myelin development in Mios knockout mice.Combined with myelin-specific staining(Black Gold and Luxol Fast Blue),Western Blot and qPCR detection for myelin protein expressions,and electron microscopy observing wrapped myelin in the optic nerve and corpus callosum,we found severe hypomyelination in Mios knockout mice.Further examination in different aged Mios knockout mice showed consistent myelin defects,suggesting that hypomyelination in Mios knockout mice are persistent defects.To determine whether the defects in oligodendrocyte and myelin development in Mios knockout could be rescued by mTORC1 activity recovering,we cross Rheb(S16H)(Activating mTORC1 continuously)transgene mice with Mios knockout mice to increase mTORC1 activity.We found that Rheb(S16H)overexpression significantly up-regulates mTORC1 in oligodendrocytes,but that failed to rescue the oligodendrocytes number reduction and myelin reduced caused by Mios knockout,indicating that MIOS/GATOR2 mediated amino acid sensing/signaling pathway is an essential pathway in CNS myelin development,and amino acids signaling might synergistically regulate mTORC1 and oligodendrocyte myelination with RHEB mediated growth factor signaling.In conclusion,this thesis establishes an amino acid sensing/signaling pathway blocking mouse model,that is Mios knockout mice.The mouse line exhibited oligodendrocyte developmental deficiency and hypomyelination,demonstrating that amino acids act as signaling molecules in cells,which regulates myelination in the mammalian CNS.We also found that only oligodendrocytes in the brain had impairment in AAs-activating mTORC1 and cytogenesis after Mios deleted,suggesting that amino acid sensing/signaling pathway is cell-specific in intracellular mTORC1 activation and cell development regulation.This study provides a good starting point in functional exploring of amino acid sensing/signaling pathway in mammalian CNS development.It provides new insights into the mechanisms of oligodendrocyte development and myelination.In addition,our findings have guiding significance in exploring the pathogenesis of demyelinating diseases(such as multiple sclerosis,MS)and therapy of neurodegenerative diseases(such as Alzheimer’s disease,AD).