Rheb Regulates Mouse Cortical Development Through MTOR/S6 Pathway

The cerebral cortex, an extensive sheet of neural tissue at the superficial part of the cortex, is involved in a variety of higher cognitive, emotional, sensory and motor functions. The emergence of a six-layered neocortex, the phylogenetically most recent part of the cerebral cortex, and its extensive projections to subcortical regions are key features of mammalian evolution. The malformation of neocortical organization and circuitry might lead to cognitive impairments and increase susceptibility to major psychiatric and neurological disorders. Mammalian cortical development requires several subtle processes, including progenitor cellproliferation, neuronal migration,axonal pathfinding, dendritogenesis and synaptogenesis. The cerebral cortex in mammalian is formed in an “inside-out” manner, which means that the early born neurons are locatedin the deeper layers of cortical plate, while the late-born neurons have to pass the formed layer structures to occupy the superficial layers.Rheb is first identified as a GTPase whose expression is induced in rat brain by NMDA-dependent synaptic activity. Since then, Rheb is found to be highly conserved during evolution from yeast to human and plays essential roles in cell cycle, cell growth and amino acid uptake. Rheb, only 21 kDa with a length of 184 amino acids, belongs to Ras superfamily GTPase and acts as a molecular switch in varieties of cellular functions. The N-terminal 169 amino acids make up the GTPase domain and the 15 remaining C-terminal residues make up a high variable region ending in a CAAX motif, which can be farnesylated at the cysteine to make Rheb anchor on membrane. Rheb is active by binding to GTP, while itisinactive when it binds to GDP. Rheb is regulated by PI3 K pathway including PKB(also known as AKT) and TSC1/2 and it is the upstream of mTOR complex 1(mTORC1) which phosphorylates 4E-BP1 and S6, causing the activation of protein synthesis. Furthermore, Rheb also plays vital roles in central nerve system by promoting the axonal elongation and outgrowth. Deletion of Rheb in neural progenitor cells by Nes-cre system causes the reduction of phosphor-S6 and cortical thickness. However, Rheb transgenic mice, including knock-in mutant, exhibit increased myelination associated with an increase in mature oligodendrocytes and in cortical thickness. Additionally, Rheb improves the degenerative diseases including Alzheimer’s disease(AD), Parkinson’s disease(PD) and Huntington’s disease(HTT), through promoting the secretion of brain-derived neurotrophic factor(BDNF) or glial cell line-derived neurotrophic factor(GDNF), indicating the importance of Rheb in central nerve system. However, the function of Rheb in embryonic corticogenesis is still largely unknown.In the present study, Rheb overexpression plasmid(Rhebwt), RNAi(Rhebsh1, Rhebsh2) and Rheb mutant plasmids(RhebQ64L,RhebC181 S,RhebD60K) were constructed. Through primary neural culture and in utero electroporation combined with immunohistochemistry and microscope image, we analyzed the function and mechanism of Rheb in mouse cortical development. Here, following arethe main findings:1. Rheb overexpression plasmid and its RNAi and mutants were constructed successfully.2. The expression of Rheb affected the neuronal morphology and mouse cortical development. Overexpression of Rheb in primary cultures of neurons showed that Rheb promoted axonaloutgrowth. In vivo experiments showed that Rheb has an effect on mouse cortical development including neuronal migration and soma size, length of leading process and axon.3. The activity of Rheb played essential roles in neuronal morphology and mouse cortical development. The activity of Rheb64,Rheb181 and Rheb60 decreased successively and the soma size and axon length showed the same trend with its activity in vitro. Furthermore, the similar decreasing tendency was observed in neuronal migration, neuronal soma size, length of leading process and axon length in vivo, indicating that Rheb activity was vital for neuronal morphology and mouse cortical development.4. Rheb regulated neuronal morphology and mouse cortical development through mTOR/S6 pathway in a rapamycin-dependent manner. Overexpression of Rheb in neurons increased phosphorylation of S6,while the phosphorylation of S6 decreased significantlywhen the neurons weretreated with rapamycin.Taken together, Rheb regulates neuronal morphology and mouse cortical development through mTOR/S6.