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Go with the Flow: Cerebrospinal Fluid Flow Regulates Neural Stem Cell Proliferation
Cell Stem Cell
Previews Go with the Flow: Cerebrospinal Fluid Flow Regulates Neural Stem Cell Proliferation Naoko Kaneko1 and Kazunobu Sawamoto1,2,* 1Department of Developmental and Regenerative Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467-8601, Japan 2Division of Neural Development and Regeneration, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2018.05.015
Adult neural stem cells in the wall of brain ventricles make direct contact with cerebrospinal fluid. In this issue of Cell Stem Cell, Petrik et al. (2018) demonstrate that these neural stem cells sense the flow of cerebrospinal fluid through a transmembrane sodium channel, ENaC, which regulates their proliferation. While most of the neurons in the brain are generated during the embryonic period, many animal species maintain neural stem cells (NSCs) after birth that can generate new neurons throughout life. The generation of new neurons by adult NSCs is influenced by environmental changes and contributes to the dynamic plasticity of neuronal circuits (in olfaction, for example) and to brain regeneration after injury. The largest germinal zone in the postnatal brain, referred to as the subependymal zone (SEZ) or the ventricular-subventricular zone (V-SVZ), is located at the lateral wall of lateral ventricles, where a single layer of multi-ciliated ependymal cells delineates the ventricle and the parenchyma (Figure 1A). In this region, the NSCs proliferate slowly, giving rise to rapidly dividing intermediate progenitors, which produce migratory immature neurons called neuroblasts (Lim and Alvarez-Buylla, 2016). The mechanisms that regulate NSC function in response to microenvironmental cues have been widely studied. Detailed morphological analyses have revealed the interesting cytoarchitecture of the NSCs: while they extend a long basal process that makes contact with the parenchymal vasculature, they also extend an apical protrusion into the ependymal layer; this apical protrusion, which ends with a primary cilium, makes direct contact with cerebrospinal fluid (CSF) in the ventricle (Lim and Alvarez-Buylla, 2016). Previous studies suggested that the flow of CSF is involved in polarizing the ependymal cells for their directional ciliary beating (Guirao et al., 2010) and in forming protein gradients that direct neuroblast migration (Sawa-
moto et al., 2006). CSF contains a variety of factors that influence the activity of NSCs (Lehtinen et al., 2011; Silva-Vargas et al., 2016), and the NSCs’ primary cilium is thought to act like an ‘‘antenna’’ to catch signals from the CSF. However, the molecular sensors detecting CSF flow in the NSCs have remained a mystery. Magdalena Go¨tz and her colleagues (Petrik et al., 2018) focused on the epithelium sodium channel (ENaC) as a possible mechanosensor of CSF flow in the NSCs, based on their previous work on the NSC transcriptome (Beckervordersandforth et al., 2010) and the ENaC’s functional properties. The ENaC is a voltageand ligand binding-independent sodium selective transmembrane channel that contributes to the sodium absorption from extracellular fluid in the kidney and other organs. Although the ENaC is usually open, providing a constant sodium influx into cells, its conductance and/or opening probability can be changed by various stimuli, including fluid shear stress (Wang et al., 2009). The authors show by immunohistochemistry that the ENaC protein is expressed in most NSCs and in some of their progeny (intermediate progenitors and neuroblasts). They found that reducing the expression or blocking the function of ENaC significantly impaired the proliferation and survival of NSCs and their progeny in vitro. In a mouse line in which the ENaC expression was genetically deleted in the NSCs and their progeny, the proliferation of these cells and the number of neuroblasts in the olfactory bulbs were significantly decreased, while the cells’ survival and migration were not obviously affected.
These observations suggested that the ENaC is required for the proliferation of NSCs and their progeny under normal basal conditions in vivo. To explore the mechanosensory mechanism, the authors performed a set of experiments in which they exposed dissected whole-mount ventricular walls to artificial CSF flow. Notably, a 4 hr exposure to flow increased the proliferation of NSCs and their progeny and activated extracellular-signal-related kinase (Erk), a key signaling molecule for cell division, in an ENaC-dependent manner. Through time-lapse imaging experiments using ion indicators, the authors demonstrated that in response to CSF flow, sodium enters the NSCs through the ENaC, and the elevated intracellular sodium causes the calcium-oscillation frequency to increase in these cells. The effect of CSF flow on the calcium event was observed in NSCs in the surface layer, but not in ependymal cells or in NSCs in the deeper layer, suggesting that NSCs sense the CSF flow in the ventricle with their apical process to induce these changes. Furthermore, chemical blockade of the calcium release-activated calcium (CRAC) channel abolished the artificial CSF-flow-induced increase in cell proliferation and Erk activation. Taken together, the authors propose that, in response to CSF flow, an ENaC-mediated increase in intracellular sodium activates calcium signaling and Erk to promote NSC/progenitor proliferation (Figure 1B). There is increasing interest in the effects of mechanical force on stem cell functions (Vining and Mooney, 2017), and several molecules involved in mechanical force sensing have been
Cell Stem Cell 22, June 1, 2018 ª 2018 Elsevier Inc. 783
Cell Stem Cell
Previews REFERENCES
Beckervordersandforth, R., Tripathi, P., Ninkovic, J., Bayam, E., Lepier, A., Stempfhuber, B., Kirchhoff, F., Hirrlinger, J., Haslinger, A., Lie, D.C., et al. (2010). In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells. Cell Stem Cell 7, 744–758. Guirao, B., Meunier, A., Mortaud, S., Aguilar, A., Corsi, J.M., Strehl, L., Hirota, Y., Desoeuvre, A., Boutin, C., Han, Y.G., et al. (2010). Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia. Nat. Cell Biol. 12, 341–350. He, L., Si, G., Huang, J., Samuel, A.D.T., and Perrimon, N. (2018). Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel. Nature 555, 103–106.
Figure 1. Neural Stem Cells Translate Cerebrospinal Fluid Flow to Cell Proliferation (A) Illustration of the cytoarchitecture of the neurogenic niche (V-SVZ), consisting of the SVZ/SEZ and a monolayer of ependymal cells (pale purple), which have motile cilia that propel the CSF. Neural stem cells (NSCs, blue) extend an apical protrusion with a primary cilium that makes direct contact with the CSF in the ependymal layer, and a basal process that associates with blood vessels (BV) in the SVZ/SEZ. The slowly dividing NSCs give rise to rapidly dividing intermediate progenitors (NPCs, yellow green), which produce migratory neuroblasts (red). The caudal side of the brain is up, and the rostral side is down. (B) The fluid flow-dependent regulatory mechanism for NSC proliferation demonstrated by Petrik et al. (2018). Fluid flow causes sodium to enter NSCs through the epithelium sodium channel (ENaC), leading to a calcium-release-activated calcium (CRAC) channel-associated increase in calcium oscillation and an elevated level of phosphorylated Erk (pErk), which promotes proliferation.
reported to regulate stem cell function. However, whether stem cells can directly sense mechanical signaling in vivo has been unclear. A recent study using fruit flies revealed that intestinal stem cells sense mechanical stimulation through a mechanosensitive cation channel, Piezo, to control their proliferation and differentiation (He et al., 2018). In Petrik et al. (2018), the authors have elegantly combined in vivo genetic experiments in adult mice with cutting-edge ex vivo experiments using acute brain slices and whole-mount ventricular walls exposed to artificial CSF flow, to demonstrate that NSCs sense the CFS flow to control their proliferation. A critical remaining issue is the biological and/or pathological significance of the monitoring of CFS flow by NSCs. This issue has not been studied,
784 Cell Stem Cell 22, June 1, 2018
because it is technically impossible to precisely control the CSF flow in the deep and closed intracranial space of the lateral ventricles in live animals. The authors suggest that NSCs may simply seek to detect the CSF flow to sense their presence at the ventricle, rather than to measure the fluid flow speed like endothelial or kidney cells do. The CSF flow changes due to physiological events such as cardiac output and the respiratory cycle and due to pathological conditions such as inflammation, intracranial bleeding, hydrocephalus, and traumatic brain injury. Considering the functions of NSCs in neuronal plasticity and regeneration, it is possible that NSCs sense CSF flow to control their activity in response to changes in physiological and pathological conditions of the brain.
Lehtinen, M.K., Zappaterra, M.W., Chen, X., Yang, Y.J., Hill, A.D., Lun, M., Maynard, T., Gonzalez, D., Kim, S., Ye, P., et al. (2011). The cerebrospinal fluid provides a proliferative niche for neural progenitor cells. Neuron 69, 893–905. Lim, D.A., and Alvarez-Buylla, A. (2016). The adult ventricular-subventricular zone (V-SVZ) and olfactory bulb (OB) neurogenesis. Cold Spring Harb. Perspect. Biol. 8, a018820. Petrik, D., Myoga, M.H., Grade, S., Gerkau, N.J., Pusch, M., Rose, C.R., Grothe, B., and Go¨tz, M. (2018). Epithelial sodium channel regulates adult neural stem cell proliferation in a flow-dependent manner. Cell Stem Cell 22, this issue, 865–878. Sawamoto, K., Wichterle, H., Gonzalez-Perez, O., Cholfin, J.A., Yamada, M., Spassky, N., Murcia, N.S., Garcia-Verdugo, J.M., Marin, O., Rubenstein, J.L., et al. (2006). New neurons follow the flow of cerebrospinal fluid in the adult brain. Science 311, 629–632. Silva-Vargas, V., Maldonado-Soto, A.R., Mizrak, D., Codega, P., and Doetsch, F. (2016). Agedependent niche signals from the choroid plexus regulate adult neural stem cells. Cell Stem Cell 19, 643–652. Vining, K.H., and Mooney, D.J. (2017). Mechanical forces direct stem cell behaviour in development and regeneration. Nat. Rev. Mol. Cell Biol. 18, 728–742. Wang, S., Meng, F., Mohan, S., Champaneri, B., and Gu, Y. (2009). Functional ENaC channels expressed in endothelial cells: a new candidate for mediating shear force. Microcirculation 16, 276–287.
Previews Go with the Flow: Cerebrospinal Fluid Flow Regulates Neural Stem Cell Proliferation Naoko Kaneko1 and Kazunobu Sawamoto1,2,* 1Department of Developmental and Regenerative Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467-8601, Japan 2Division of Neural Development and Regeneration, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2018.05.015
Adult neural stem cells in the wall of brain ventricles make direct contact with cerebrospinal fluid. In this issue of Cell Stem Cell, Petrik et al. (2018) demonstrate that these neural stem cells sense the flow of cerebrospinal fluid through a transmembrane sodium channel, ENaC, which regulates their proliferation. While most of the neurons in the brain are generated during the embryonic period, many animal species maintain neural stem cells (NSCs) after birth that can generate new neurons throughout life. The generation of new neurons by adult NSCs is influenced by environmental changes and contributes to the dynamic plasticity of neuronal circuits (in olfaction, for example) and to brain regeneration after injury. The largest germinal zone in the postnatal brain, referred to as the subependymal zone (SEZ) or the ventricular-subventricular zone (V-SVZ), is located at the lateral wall of lateral ventricles, where a single layer of multi-ciliated ependymal cells delineates the ventricle and the parenchyma (Figure 1A). In this region, the NSCs proliferate slowly, giving rise to rapidly dividing intermediate progenitors, which produce migratory immature neurons called neuroblasts (Lim and Alvarez-Buylla, 2016). The mechanisms that regulate NSC function in response to microenvironmental cues have been widely studied. Detailed morphological analyses have revealed the interesting cytoarchitecture of the NSCs: while they extend a long basal process that makes contact with the parenchymal vasculature, they also extend an apical protrusion into the ependymal layer; this apical protrusion, which ends with a primary cilium, makes direct contact with cerebrospinal fluid (CSF) in the ventricle (Lim and Alvarez-Buylla, 2016). Previous studies suggested that the flow of CSF is involved in polarizing the ependymal cells for their directional ciliary beating (Guirao et al., 2010) and in forming protein gradients that direct neuroblast migration (Sawa-
moto et al., 2006). CSF contains a variety of factors that influence the activity of NSCs (Lehtinen et al., 2011; Silva-Vargas et al., 2016), and the NSCs’ primary cilium is thought to act like an ‘‘antenna’’ to catch signals from the CSF. However, the molecular sensors detecting CSF flow in the NSCs have remained a mystery. Magdalena Go¨tz and her colleagues (Petrik et al., 2018) focused on the epithelium sodium channel (ENaC) as a possible mechanosensor of CSF flow in the NSCs, based on their previous work on the NSC transcriptome (Beckervordersandforth et al., 2010) and the ENaC’s functional properties. The ENaC is a voltageand ligand binding-independent sodium selective transmembrane channel that contributes to the sodium absorption from extracellular fluid in the kidney and other organs. Although the ENaC is usually open, providing a constant sodium influx into cells, its conductance and/or opening probability can be changed by various stimuli, including fluid shear stress (Wang et al., 2009). The authors show by immunohistochemistry that the ENaC protein is expressed in most NSCs and in some of their progeny (intermediate progenitors and neuroblasts). They found that reducing the expression or blocking the function of ENaC significantly impaired the proliferation and survival of NSCs and their progeny in vitro. In a mouse line in which the ENaC expression was genetically deleted in the NSCs and their progeny, the proliferation of these cells and the number of neuroblasts in the olfactory bulbs were significantly decreased, while the cells’ survival and migration were not obviously affected.
These observations suggested that the ENaC is required for the proliferation of NSCs and their progeny under normal basal conditions in vivo. To explore the mechanosensory mechanism, the authors performed a set of experiments in which they exposed dissected whole-mount ventricular walls to artificial CSF flow. Notably, a 4 hr exposure to flow increased the proliferation of NSCs and their progeny and activated extracellular-signal-related kinase (Erk), a key signaling molecule for cell division, in an ENaC-dependent manner. Through time-lapse imaging experiments using ion indicators, the authors demonstrated that in response to CSF flow, sodium enters the NSCs through the ENaC, and the elevated intracellular sodium causes the calcium-oscillation frequency to increase in these cells. The effect of CSF flow on the calcium event was observed in NSCs in the surface layer, but not in ependymal cells or in NSCs in the deeper layer, suggesting that NSCs sense the CSF flow in the ventricle with their apical process to induce these changes. Furthermore, chemical blockade of the calcium release-activated calcium (CRAC) channel abolished the artificial CSF-flow-induced increase in cell proliferation and Erk activation. Taken together, the authors propose that, in response to CSF flow, an ENaC-mediated increase in intracellular sodium activates calcium signaling and Erk to promote NSC/progenitor proliferation (Figure 1B). There is increasing interest in the effects of mechanical force on stem cell functions (Vining and Mooney, 2017), and several molecules involved in mechanical force sensing have been
Cell Stem Cell 22, June 1, 2018 ª 2018 Elsevier Inc. 783
Cell Stem Cell
Previews REFERENCES
Beckervordersandforth, R., Tripathi, P., Ninkovic, J., Bayam, E., Lepier, A., Stempfhuber, B., Kirchhoff, F., Hirrlinger, J., Haslinger, A., Lie, D.C., et al. (2010). In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells. Cell Stem Cell 7, 744–758. Guirao, B., Meunier, A., Mortaud, S., Aguilar, A., Corsi, J.M., Strehl, L., Hirota, Y., Desoeuvre, A., Boutin, C., Han, Y.G., et al. (2010). Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia. Nat. Cell Biol. 12, 341–350. He, L., Si, G., Huang, J., Samuel, A.D.T., and Perrimon, N. (2018). Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel. Nature 555, 103–106.
Figure 1. Neural Stem Cells Translate Cerebrospinal Fluid Flow to Cell Proliferation (A) Illustration of the cytoarchitecture of the neurogenic niche (V-SVZ), consisting of the SVZ/SEZ and a monolayer of ependymal cells (pale purple), which have motile cilia that propel the CSF. Neural stem cells (NSCs, blue) extend an apical protrusion with a primary cilium that makes direct contact with the CSF in the ependymal layer, and a basal process that associates with blood vessels (BV) in the SVZ/SEZ. The slowly dividing NSCs give rise to rapidly dividing intermediate progenitors (NPCs, yellow green), which produce migratory neuroblasts (red). The caudal side of the brain is up, and the rostral side is down. (B) The fluid flow-dependent regulatory mechanism for NSC proliferation demonstrated by Petrik et al. (2018). Fluid flow causes sodium to enter NSCs through the epithelium sodium channel (ENaC), leading to a calcium-release-activated calcium (CRAC) channel-associated increase in calcium oscillation and an elevated level of phosphorylated Erk (pErk), which promotes proliferation.
reported to regulate stem cell function. However, whether stem cells can directly sense mechanical signaling in vivo has been unclear. A recent study using fruit flies revealed that intestinal stem cells sense mechanical stimulation through a mechanosensitive cation channel, Piezo, to control their proliferation and differentiation (He et al., 2018). In Petrik et al. (2018), the authors have elegantly combined in vivo genetic experiments in adult mice with cutting-edge ex vivo experiments using acute brain slices and whole-mount ventricular walls exposed to artificial CSF flow, to demonstrate that NSCs sense the CFS flow to control their proliferation. A critical remaining issue is the biological and/or pathological significance of the monitoring of CFS flow by NSCs. This issue has not been studied,
784 Cell Stem Cell 22, June 1, 2018
because it is technically impossible to precisely control the CSF flow in the deep and closed intracranial space of the lateral ventricles in live animals. The authors suggest that NSCs may simply seek to detect the CSF flow to sense their presence at the ventricle, rather than to measure the fluid flow speed like endothelial or kidney cells do. The CSF flow changes due to physiological events such as cardiac output and the respiratory cycle and due to pathological conditions such as inflammation, intracranial bleeding, hydrocephalus, and traumatic brain injury. Considering the functions of NSCs in neuronal plasticity and regeneration, it is possible that NSCs sense CSF flow to control their activity in response to changes in physiological and pathological conditions of the brain.
Lehtinen, M.K., Zappaterra, M.W., Chen, X., Yang, Y.J., Hill, A.D., Lun, M., Maynard, T., Gonzalez, D., Kim, S., Ye, P., et al. (2011). The cerebrospinal fluid provides a proliferative niche for neural progenitor cells. Neuron 69, 893–905. Lim, D.A., and Alvarez-Buylla, A. (2016). The adult ventricular-subventricular zone (V-SVZ) and olfactory bulb (OB) neurogenesis. Cold Spring Harb. Perspect. Biol. 8, a018820. Petrik, D., Myoga, M.H., Grade, S., Gerkau, N.J., Pusch, M., Rose, C.R., Grothe, B., and Go¨tz, M. (2018). Epithelial sodium channel regulates adult neural stem cell proliferation in a flow-dependent manner. Cell Stem Cell 22, this issue, 865–878. Sawamoto, K., Wichterle, H., Gonzalez-Perez, O., Cholfin, J.A., Yamada, M., Spassky, N., Murcia, N.S., Garcia-Verdugo, J.M., Marin, O., Rubenstein, J.L., et al. (2006). New neurons follow the flow of cerebrospinal fluid in the adult brain. Science 311, 629–632. Silva-Vargas, V., Maldonado-Soto, A.R., Mizrak, D., Codega, P., and Doetsch, F. (2016). Agedependent niche signals from the choroid plexus regulate adult neural stem cells. Cell Stem Cell 19, 643–652. Vining, K.H., and Mooney, D.J. (2017). Mechanical forces direct stem cell behaviour in development and regeneration. Nat. Rev. Mol. Cell Biol. 18, 728–742. Wang, S., Meng, F., Mohan, S., Champaneri, B., and Gu, Y. (2009). Functional ENaC channels expressed in endothelial cells: a new candidate for mediating shear force. Microcirculation 16, 276–287.