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      Promotion of Both Proliferation and Neuronal Differentiation in Pluripotent P19 Cells with Stable Overexpression of the Glutamine Transporter slc38a1

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          We previously demonstrated the functional expression in newborn rat neocortical astrocytes of glutamine transporter (GlnT = slc38a1) believed to predominate in neurons over astroglia in the brain. In order to evaluate the possible role of this transporter in neurogenesis, we attempted to establish stable transfectants of GlnT in mouse embryonal carcinoma P19 cells endowed to proliferate for self-renewal and differentiate into progeny cells such as neurons and astroglia, in addition to in vitro pharmacological profiling of the green tea ingredient theanine, which is shown to be a potent inhibitor of glutamine transport mediated by GlnT in cultured neurons and astroglia.

          Methodology/Principal Findings

          The full-length coding region of rat GlnT was inserted into a vector for gene transfection along with selection by G418, followed by culture with all- trans retinoic acid under floating conditions and subsequent dispersion for spontaneous differentiation under adherent conditions. Stable overexpression of GlnT led to marked increases in the size of round spheres formed during the culture for 4 days and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction, with concomitant promotion of subsequent differentiation into cells immunoreactive for a neuronal marker protein. In these stable GlnT transfectants before differentiation, drastic upregulation was seen for mRNA expression of several proneural genes with a basic helix-loop-helix domain such as NeuroD1. Although a drastic increase was seen in NeuroD1 promoter activity in stable GlnT transfectants, theanine doubled NeuroD1 promoter activity in stable transfectants of empty vector (EV), without affecting the promoter activity already elevated in GlnT transfectants. Similarly, theanine promoted cellular proliferation and neuronal differentiation in stable EV transfectants, but failed to further stimulate the acceleration of both proliferation and neuronal differentiation found in stable GlnT transfectants.


          GlnT would promote both proliferation and neuronal differentiation through a mechanism relevant to the upregulation of particular proneural genes in undifferentiated P19 cells.

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          Most cited references 47

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          Bidirectional transport of amino acids regulates mTOR and autophagy.

          Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.
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            Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.

            Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.
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              Mash1 regulates neurogenesis in the ventral telencephalon.

              Previous studies have shown that mice mutant for the gene Mash1 display severe neuronal losses in the olfactory epithelium and ganglia of the autonomic nervous system, demonstrating a role for Mash1 in development of neuronal lineages in the peripheral nervous system. Here, we have begun to analyse Mash1 function in the central nervous system, focusing our studies on the ventral telencephalon where it is expressed at high levels during neurogenesis. Mash1 mutant mice present a severe loss of progenitors, particularly of neuronal precursors in the subventricular zone of the medial ganglionic eminence. Discrete neuronal populations of the basal ganglia and cerebral cortex are subsequently missing. An analysis of candidate effectors of Mash1 function revealed that the Notch ligands Dll1 and Dll3, and the target of Notch signaling Hes5, fail to be expressed in Mash1 mutant ventral telencephalon. In the lateral ganglionic eminence, loss of Notch signaling activity correlates with premature expression of a number of subventricular zone markers by ventricular zone cells. Therefore, Mash1 is an important regulator of neurogenesis in the ventral telencephalon, where it is required both to specify neuronal precursors and to control the timing of their production.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                24 October 2012
                : 7
                : 10
                [1 ]Laboratory of Molecular Pharmacology, Kanazawa University Graduate School, Kanazawa, Japan
                [2 ]R & D Division, Itoen Ltd., Makinohara City, Japan
                Rutgers University, United States of America
                Author notes

                Competing Interests: The authors have read the journal’s policy and have the following conflicts. Dr. Takami Kakuda is an employee of Itoen Co. Ltd., Japan. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials

                Conceived and designed the experiments: MO TK YY. Performed the experiments: MO TT NN RF YHK EH. Analyzed the data: MO TK EH YY. Contributed reagents/materials/analysis tools: MO TT NN RF YHK EH. Wrote the paper: MO TK YY.


                Current address: Department of Biomolecular Science, Fukushima Medical University School of Medicine, Fukushima, Japan


                Current address: Laboratory of Molecular Pharmacotherapeutics, Kanazawa University Graduate School, Kanazawa, Japan


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 12
                This work was supported in part by Grants-in-Aid for Scientific Research to T.T. (No. 22500330) and Y.Y. (No. 24650196) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Computational Biology
                Molecular Genetics
                Gene Expression
                Developmental Biology
                Cell Differentiation
                Gene Expression
                Molecular Cell Biology
                Cellular Types
                Gene Expression
                Cellular Neuroscience
                Molecular Neuroscience



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