Abstract
Throughout life, neural stem cells proliferate and differentiate in restricted zones of the brain, termed niches, to produce new neurons and glial cells. In these niches, growth factors and extracellular matrix (ECM) molecules determine the fate of neural stem and progenitor cells (NSPC). However, the precise compounds and the mechanisms that regulate growth factors and other signaling molecules in the niches are unknown. Based on the evidence that NSPCs proliferate next to blood vessels in the dentate gyrus, the concept of a vascular niche for neurogenesis has been initially proposed. In the subventricular zone of the lateral ventricle, the most neurogenic zone in adulthood, we have found that NSPC directly contact a novel type of ECM structure that we have named fractones. Fractones contain heparan sulfate proteoglycans (HSPG) that collect and concentrate the neurogenic growth factor FGF2 at the NSPC surface and likely direct its signaling via tyrosine kinase receptors. Our preliminary results indicate that FGF2 binding to fractone-HSPG is essential for activating FGF2 at the NSPC surface. Moreover, we have found fractones express diverse HSPG at the surface of proliferating NSPC during development, even before the brain vasculature emerges. Therefore, fractones hold considerable promise for promoting growth factors at the stem cell surface to ultimately regulate neurogenesis during development and adulthood.
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References
Altman, J. (1963). Autoradiographic investigation of cell proliferation in the brain of cats and rats. Anat. Rec. 145, 573–591.
Altman, J. (1969). Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J. Comp. Neurol. 137, 433–458.
Altman, J., Das, G.D. (1966). Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats, with special references to postnatal neurogenesis in some brain regions. J. Comp. Neurol. 126, 337–390.
Alvarez-Buylla, A., Nottebohm, F. (1988). Migration of young neurons in adult avian brain. Nature 335, 353–354.
Aviezer, D., Hecht, D., Safran, M., Elsinger, M. David, G., Yayon, A. (1994). Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis. Cell 79, 1005–1013.
Bernfield, M., Kokenyesi, R., Kato, M., Hinkes, M.T., Spring, J., Gallo, R.L. (1992). Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annu. Rev. Cell Biol. 8, 365–93.
Bernfield, M., Banerjee, S.D., Koda, J.E., Rapraeger, A.C. (1994). Remodelling of basement membranes as a mechanism of morphogenetic tissue interactions. In: Trelstadt, R.L. (ed) The role of extracellular matrix in development. New York, Liss, A.R.
Bernfield, M., Gotte, M., Park, P., Reizes, O., Fitzgerald, M., Lincecum, J., Zako, M. (1999). Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68, 729–777.
Blanpain, C., Lowry, W., Georghegan, A., Polak, L., Fuchs, E. (2004). Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118, 635–648.
Brickman, Y.G., Ford, M.D., Small, D.H., Bartlett, P.F., Nurcombe, V. (1995). Heparan sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells. J. Biol. Chem. 270, 24941–24948.
Brickman, Y., Ford, M., Gallagher, J., Nurcombe, V., Bartlett, P., Turnbull, J. (1998). Structural modification of fibroblast growth factor-binding heparan sulfate at a determinative stage of neural development. J. Biol. Chem. 8:5, 4350–4359.
Brightman, M.W. (1965). The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. J. Cell Biol. 26, 99–123.
Brightman, M.W. (2002). The brain’s interstitial clefts and their glial walls. J. Neurocytol. 31, 595–603.
Chadashvili, T., Peterson, D. (2006). Cytoarchitecture of fibroblast growth factor receptor 2 (FGFR-2) immunoreactivity in astrocytes of neurogenic and non-neurogenic regions of the young adult and aged rat brain. J. Comp. Neurol. 498:1, 1–15.
Chang, Z., Meyer, K., Rapraeger, A.C., Friedl, A. (2000). Differential ability of heparan sulfate proteoglycans to assemble the fibroblast growth factor receptor complex in situ. FASEB J. 14, 137–144.
Craig, CG, Tropepe, V, Morshead, CM, Reynolds, BA, Weiss, S, van der Kooy, D. (1996). In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain. J. Neurosci. 16, 2649–2658.
Doetsch, F., Alvarez-Buylla, A. (1996). Network of tangential pathways for neuronal migration in the adult mammalian brain. Proc. Natl. Acad. Sci. USA 93, 14895–14900.
Doetsch, F., Garcia-Verdugo, J.M., Alvarez-Buylla, A. (1997). Cellular composition and three-dimensional organization in the subventricular germinal zone in the adult mammalian brain. J. Neurosci. 17, 5046–5061.
Falk, A., Frisen, J. (2002). Amphiregulin is a mitogen for adult neural stem cells. J. Neurosci. Res. 69, 757–762.
Fitzgerald, M., Hayward, I.P., Thomas, A.C., Campbell, G.R., Campbell, J.H. (1999). Matrix metalloproteinase can facilitate the heparanase-induced promotion of phenotype change in vascular smooth muscle cells. Atherosclerosis 145:1, 97–106.
Gallagher, J.T. (2001). Heparan sulfate: growth control with a restricted sequence menu. J. Clin. Invest. 108:3, 357–361
Goodger, S., Robinson, C., Murphy, K., Gasiunas, N., Harmer, N., Blundell, T., Pye, D., Gallagher, J. (2008). Evidence that heparin saccharides promote FGF2 mitogenesis through two distinct mechanisms. J. Biol. Chem. 283, 13001–13008.
Gordon, M.Y., Riley, G.P., Watt, S.M., Greaves, M.F. (1987). Compartmentalization of a haematopoeitic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment. Nature 326, 403–405.
Grobe, K., Ledin, J., Ringvall, M., Holmborn, K., Forsberg, E., Esko, J.D., Kjellén, L. (2002). Heparan sulfate and development: differential roles of the N-acetylglucosamine N-deacetylase/N-sulfotransferase isozymes. Biochim. Biophys. Acta 1573:3, 209–215.
Guimond, S., Maccarana, M., Olwin, B., Lindahl, U., Rapraeger, A. (1993). Activating and inhibitory heparin sequences for FGF-2 (basic FGF) distinct requirements for FGF-1, FGF-2, and FGF-4. J. Biol. Chem. 268, 23906–23914.
Halfter, W. (1998). Disruption of the retinal basal lamina during early embryonic development leads to a retraction of vitral endfeet, an increase number of ganglion cells, and aberrant axonal outgrowth. J. Comp. Neurol. 397, 99–104.
Hayamizu, T.F., Chan, P.T., Johanson, C.E. (2001). FGF-2 immunoreactivity in adult rat ependyma and choroid plexus: responses to global forebrain ischemia and intraventricular FGF-2. Neurol. Res. 23, 353–358.
Hienola, A., Pekkanen, M., Raulo, E., (2004). HB-GAM inhibits proliferation and enhances differentiation of neural stem cells. Mol. Cell. Neurosci. 26, 75–88.
Hienola, A., Tumova, S., Kulesskiy, E., Rauvala, H. (2006). N-syndecan deficiency impairs neural migration in the brain. J. Cell Biol. 174, 569–580.
Iozzo, R.V. (2005). Basement membrane proteoglycans: from cellar to ceiling. Nat. Rev. Mol. Cell. Biol. 6, 646–656.
Jasuja, R., Allen, B., Pappano, W., Rapraeger, A., Greenspan, D. (2004). Cell-surface heparen sulfate proteoglycans chordin antagonism of bone morphogenetic protein signaling and are necessary for cellular uptake of chordin. J. Biol. Chem. 279, 51289–51297.
Jayson, G.C., Lyon, M., Paraskeva, C., Turnbull, J.E., Deakin, J.A., Gallagher, J.T. (1998). Heparan sulfate undergoes specific structural changes during the progression from human colon adenoma to carcinoma in vitro. J. Biol. Chem. 273:1, 51–57.
Kaji, T., Yamamoto, C., Oh-i, M., Fujiwara, Y., Yamazaki, Y., Morita, T., Plaas, A.H., Wight, T.N. (2006). The vascular endothelial growth factor VEGF165 induces perlecan synthesis via VEGF receptor-2 in cultured human brain microvascular endothelial cells. Biochim. Biophys. Acta 1760:9, 1465–1474.
Kerever, A., Schnack, J., Vellinga, D., Ichikawa, N., Moon, C., Arikawa-Hirasawa, E., Efird, J.T., Mercier, F. (2007). Novel extracellular matrix structures in the neural stem cell niche capture the neurogenic factor FGF-2 from the extracellular milieu. Stem Cells 25, 2146–2157.
Kim, C.W., Goldberger, O.A., Gallo, R.L., Bernfield, M. (1994). Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-,tissue-, and development-specific patterns. Mol. Biol. Cell 5, 797–805.
Kim, S.J., Son, T.G., Kim, K., Park, H.R., Mattson, M.P., Lee, J. (2007). Interferon-gamma promotes differentiation of neural progenitor cells via the JNK pathway. Neurochem. Res. 32, 1399–1406.
Klein, G., Conzelmann, S., Beck, S., Timpl, R., Müller, C.A. (1995). Perlecan in human bone marrow: a growth-factor-presenting, but anti-adhesive, extracellular matrix component for hematopoietic cells. Matrix Biol. 14, 457–465.
Knox, S., Merry, C., Stringer, S., Melrose, J., Whitelock, J. (2002). Not all perlecans are created equal. J. Biol. Chem. 277, 14657–14665.
Kobayashi, M., Shimada, K., Ozawa, T. (1992). Human platelet-derived transforming growth factor-beta stimulates synthesis of glycosaminoglycans in cultured porcine aortic endothelial cells. Gerontology 38, 36–42.
Kuhn, H.G., Winkler, J., Kempermann, G. Thal, L.J., Gage, F.H. (1997). Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J. Neurosci. 17, 5820–5827.
Lamanna, W.C., Baldwin, R.J., Padva, M., Kalus, I., Ten Dam, G., van Kuppevelt, T.H., Gallagher, J.T., von Figura, K., Dierks, T., Merry, C.L. (2006). Heparan sulfate 6-O-endosulfatases: discrete in vivo activities and functional co-operativity. Biochem. J. 400, 63–73.
Langsdorf, A., Do, A.T., Kusche-Gullberg, M., Emerson, C.P. Jr, Ai, X. (2007). Sulfs are regulators of growth factor signaling for satellite cell differentiation and muscle regeneration. Dev. Biol. 311:2, 464–477.
Lim, D., Tramontin, A., Trevejo, J., Herrera, D., Garcia-Verdugi, J., Alvarez-Buylla, A. (2000). Noggin antagonizes BMP signaling to create a niche for adult neurogenesis. Neuron 28, 713–726.
Lindahl, U., Kusche-Gullberg, M., Kjellen, L. (1998). Regulated diversity of heparan sulfate. J. Biol. Chem. 273:29, 24979–24982.
Litwack, E.D., Ivins, J.K., Kumbasor, A., Paine-Saunolers, S., Stipp, C.S., Lanoler, A.D. (1998). Expression of the heparan sulfate proteoglycon glypicon-1 in the developing rodent. Dev. Dyn. 211, 72–87.
Lois, C., Alvarez-Buylla, A. (1994). Long distance neuronal migration in the adult mammalian brain. Science 264, 1145–1148.
Lortat-Jacob, H., Grimaud, J.A. (1991). Interferon-gamma C-terminal function: networking hypothesis. Heparan sulfate and heparin, new targets for IFNGamma, protect, relax the cytokine and regulate its activity. Cell Mol. Biol. 37:3, 253–260.
Mandelbrot, B.B. (ed) (1983). The fractal geometry of nature. Freeman, San Francisco
Martens, D.J., Seaber, R.M., van der Kooy, D. (2002). In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord. Eur. J. Neurosci. 16, 1045–1057.
Mercier, F. (2004). Astroglia as a modulation interface between meninges and neurons. In: Hatton, G.I., Parpura, V. (eds) Glial/neuronal signaling. Amsterdam, Kluwer Pub, 125–162.
Mercier, F., Hatton, G.I. (2000). Immunocytochemical basis for a meningeo-glial network. J. Comp. Neurol. 420, 445–465.
Mercier, F., Hatton, G.I. (2001). Connexin 26 and bFGF are primarily expressed in subpial and subependymal layers in adult brain parenchyma: roles in stem cell proliferation and morphological plasticity? J. Comp. Neurol. 431, 88–104.
Mercier, F., Hatton, G.I. (2004). Meninges and perivasculature as mediators of CNS plasticity. In: Bittar, E.E., Hertz, L. (eds.) Non-neuronal cells in the nervous system: function and dysfunction. Elsevier Bioscience, Amsterdam, Adv. Mol. Cell. Biol. 31, 215–253.
Mercier, F., Kitasako, J.T., Hatton, G.I. (2002). Anatomy of the brain neurogenic zones revisited: fractones and the fibroblast/macrophage network. J. Comp. Neurol. 451, 170–188.
Mercier, F., Kitasako, J.T., Hatton, G.I. (2003). Fractones and other basal laminae in the hypothalamus. J. Comp. Neurol. 455, 324–340.
Mohammadi, M., Olsen, S.K., Ibrahimi, O.A. (2005). Structural basis for fibroblast receptor activation. Cyt. Growth Fact. Rev. 16, 107–137.
Monje, M.L., Toda, H., Palmer, T.D. (2003). Inflammatory blockade restores adult hippocampal neurogenesis. Science, 302, 1760–1765.
Mulloy, B., Forster, M.J., Jones, C., Drake, A.F., Johnson, E.A., Davies, D.B. (1994). The effect of variation of substitution on the solution conformation of heparin: a spectroscopic and molecular modelling study. Carbohydr. Res. 255, 1–26.
Nakato, H., Kimata, K. (2002). Heparan sulfate fine structure and specificity of proteoglycan functions. Biochim. Biophys. Acta 1573:3, 312–318.
Narita, K., Chien, J., Mullany, S.A., Staub, J., Qian, X., Lingle, W.L., Shridhar, V. (2007). Loss of HSulf-1 expression enhances autocrine signaling mediated by amphiregulin in breast cancer. J Biol. Chem. 282:19, 14413–14420.
Nawroth, R., Zante, A., Cervantes, S., McManus, M., Helbrok, M., Rosen, S. (2007). Extracellular sulfatases, elements of the WNT signaling pathway, positively regulate growth and tumorigenicity of human pancreatic cancer cells. PLoS One 2:4, 1–11.
Ornitz, D.M., Yayon, A., Flanagan, J.G., Svahn, C.M., Levi, E., Leder, P. (1992). Heparin is required for cell-free binding of basic fibroblastic growth factor to a soluble receptor and for mitogenesis in whole cells. Mol. Cell. Biol. 12, 240–247.
Paine-Saunders, S., Vivano, B., Economides, A., Saunders, S. (2002). Heparan sulfate proteoglycans retain noggins at the cell surface: a potential mechanism for shaping bone morphogenic protein gradients. J. Biol. Chem. 277, 2089–2096.
Palmer, T.D., Ray, J., Gage, F.H. (1995). FGF-2 responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol. Cell. Neurosci. 6, 474–486.
Palmer, T.D., Willhoite, A.R., Gage, F.H. (2000). Vascular niche for adult hippocampal neurogenesis. J. Comp. Neurol. 425, 479–494.
Pencea, V., Bingaman, K.D., Wiegand, S.J., Luskin, M.B. (2001). Infusion of brain derived neurotrophic growth factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J. Neurosci. 21, 6706–6717.
Proia, P., Schiera, G., Mineo, M., Ingrassia, A.M., Santoro, G., Savettieri, G., Di Liegro, I. (2008). Astrocytes shed extracellular vesicles that contain fibroblast growth factor-2 and vascular endothelial growth factor. Int. J. Mol. Med. 21, 63–67.
Properzi, F., Lin, R., Kwok, J., Naidu, M., Van Kuppevelt, T., Dam, G., Camargo, L., Raha-Chowdhury, R., Furukawa, Y., Mikami, T., Sugahara, K. (2008). Heparan sulphate proteoglycan in glia and in the normal and injured CNS: expression of sulphotransferases and changes in sulphation. Eur. J. Neurosci. 27, 593–604.
Rapraeger, A. (1995). In the clutches of proteoglycans: how does heparan sulfate regulate FGF binding. Chem. Biol. 2, 137–144.
Reiland, J., Rapraeger, A.C. (1993). Heparan sulfate protoglycan and FGF receptor target basic FGF to different intracellular destinations. J. Cell Sci. 105, 1085–1093.
Rider, C.C. (2006). Heparin/heparan sulphate binding in the TGF-beta cytokine super family. Biochem. Soc. Trans. 34, 458–460.
Roberts, R., Gallagher, J., Spooncer, E., Allen, T.D., Bloomfield, F., Dexter, T.M. (1988). Heparan sulfate bond growth factors: a mechanism for stromal cell mediated haemopoiesis. Nature 332, 376–378.
Sakaguchi, K., Lorenzi, M.V., Bottaro, D.P., Miki, T. (1999). The acidic domain and first immunoglobulin-like loop of fibroblast growth factor receptor 2 modulate downstream signaling through glycosaminoglycan modification. Mol. Cell. Biol. 19, 6754–6764.
Saksela, O., Moscatelli, D., Sommer, A., Rifkin, D.B. (1988). Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J. Cell Biol. 107:2, 743–51.
Sawamoto, K., Wichterle, H., Gonzalez-Perez, O., Choifin, J.A., Yamada, M., Spassky, N., Murcia, N.S., Garcia-Verdugo, J.M., Marin, O., Rubenstein, J.L.M., Tessier-Lavigne, M., Okano, H., Alvarez-Buylla, A. (2006). New neurons follow the flow of cerebrospinal fluid in the adult brain. Science 311, 626–632.
Seaberg, R.M., van der Kooy, D. (2002). Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. J. Neurosci. 22, 1784–1793.
Seki, T., Array, Y. (1993). Highly polysialylated neural cell adhesion molecule (NCAM-H) is expressed by newly generated granule cells in the dentate gyrus of the adult rat. J. Neurosci. 13, 2351–2358.
Seri, B., Herrera, D.G., Gritti, A., Ferron, S., Collado, L., Vescovi, A., Garcia-Verdugo, J.M., Alvarez-Buylla, A. (2006). Composition and organization of the SCZ: a large germinal layer containing neural stem cells in the adult mammalian brain. Cereb. Cortex 16, 103–111.
Sharma, B., Iozzo, R. (1998). Transcriptional silencing of perlecan gene expression by interferon. J. Biol. Chem. 273, 4642–4646.
Smits, N.C., Robbesom, A.A., Versteeg, E.M., van de Westerlo, E.M., Dekhuijzen, P.N., van Kuppevelt, T.H. (2004). Heterogeneity of heparan sulfates in human lung. Am. J. Respir. Cell Mol. Biol. 30, 166–173.
Stopa, E.G., Berzin, T.M., Kim, S., Song, P., Kuo-Leblanc, V., Rodriguez-Wolf, M., Baird, A., Johanson, C.E. (2001). Choroid plexus growth factors: what are the implications for CSF dynamics in Alzheimer’s disease? Exp. Neurol. 167, 40–47.
Stringer, S., Gallagher, J. (1997). Heparan sulfate. Int. J. Biochem. Cell Biol. 29:5, 709–714.
Unger, E., Pettersson, I., Eriksson, U.J., Lindahl, U., Kjellén, L. (1991). Decreased activity of the heparan sulfate-modifying enzyme glucosaminyl N-deacetylase in hepatocytes from streptozotocin-diabetic rats. J. Biol. Chem. 266:14, 43–961.
Venkataraman, G., Sasisekharan, V., Herr, A.B., Ornitz, D.M., Waksman, G., Cooney, C.L., Langer, R., Sasisekharan, R. (1996). Preferential self-association of basic fibroblastic growth factor is stabilized by heparin during receptor dimerization and activation. Proc. Natl. Acad. Sci. USA 93, 845–890.
Vlodavsky, I., Korner, G., Ishai-Michaeli, R., Bashkin, P., Bar-Shavit, R., Fuks, Z. (1990). Extracellular matrix-resident growth factors and enzymes: possible involvement in tumor metastasis and angiogenesis. Cancer Metastasis Rev. 32:3, 313–318
Walker, A., Turnbul, J., Gallagher, J. (1994). Specific heparan sulfate saccharides mediate the activity of basic fibroblast growth factor. J. Biol. Chem. 269:2, 931–935.
Weiss, S., Dunne, C., Hewson, J. (1996). Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J. Neurosci. 16, 7599–7609.
Westling, C., Lindahl, U. (2002). Location of N-unsubstituted glucosamine residues in heparan sulfate. J. Biol. Chem. 277:51, 49247–49255.
Wexler, E.M., Geschwind, D.H., Palmer, T.D. (2007). Lithium regulates adult hippocampal progenitor development through canonical wnt pathway activation. Mol. Psychiatr. 13:285–292.
Whitelock, J., Murdoch, A., Iozzo, R., Underwood, A. (1996). The degradation of human endothelial cell-derived perlecan and release of bound basis fibroblast growth factor by stromelysin collagenase, plasmin, and heparanases. J. Biol. Chem. 271:17, 10079–10086.
Wieseler-Frank, J., Jekich, B.M., Mahoney, J.H., Bland, S.T., Maier, S.F., Watkins, L.R. (2007). A novel immune to CNS communication pathway: cells of the meninges surrounding the spinal cord CSF space produce proinflammatory cytokines in response to an inflammatory cytokine. Brain Behav. Immun. 21, 711–718.
Wurmser, H.M, Palmer, T.D., Gage, F.H. (2004). Neuroscience-Cellular interactions in the stem cell niches. Science 304, 1253–1255.
Yang, W.D., Gomes, R.R. Jr, Alicknavitch, M., Farach-Carson, M.C., Carson, D.D. (2005). Perlecan domain I promotes fibroblast growth factor 2 delivery in collagen I fibril scaffolds. Tissue Eng. 11:1–2, 76–89.
Yayon, A., Klagsbrun, M., Esko, J.D., Leder, P., Ornitz, D.P. (1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64, 841–848.
Yurchenko, P.D., Schyttny, J.C. (1990). Molecular architecture of basement membranes. FASEB J. 4, 1577–1590.
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Mercier, F., Schnack, J., Chaumet, M.S.G. (2011). Fractones: Home and Conductors of the Neural Stem Cell Niche. In: Seki, T., Sawamoto, K., Parent, J.M., Alvarez-Buylla, A. (eds) Neurogenesis in the Adult Brain I. Springer, Tokyo. https://doi.org/10.1007/978-4-431-53933-9_4
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