Bone morphogenetic protein-4 inhibits adult neurogenesis and is regulated by fractone-associated heparan sulfates in the subventricular zone Frederic Mercier *, Vanessa Douet Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii, Biomed T401, 1960 East-West Road, Honolulu, HI 96822, USA 1. Introduction The cerebrospinal fluid (CSF) circulating in the brain ventricles carries ions and numerous signaling molecules including growth factors, cytokines, chemokines and neurohormones. These signaling molecules are produced by the choroid plexus (Chodobski et al., 1997; Redzic et al., 2005; Stopa et al., 2001; Zappaterra and Lehtinen, 2012), circumventricular organs (Frautschy et al., 1991), and by the ependymal lining (ventricular zone or VZ) and subventricular zone (SVZ) (Bain et al., 2013) that together form the ventricle walls. How do the CSF-borne signaling molecules penetrate from the ventricle to the VZ/SVZ? The ependyma does not form a barrier for the passage of growth factors and other molecules injected in the ventricle (Brightman, 1965; Anderson et al., 1995). Biological molecules reach the SVZ by two routes: trans-ependymal diffusion and direct diffusion in the intercellular space between ependymocytes (Brightman, 1965, 2002). Opened tight junctions (fascia adherens) are found in the luminal side of adjacent ependymocytes, lying in direct continuity with the ventricular lumen, allowing for the passage of the CSF and its signaling molecules. The regular ‘‘proof’’ Journal of Chemical Neuroanatomy 57–58 (2014) 54–61 A R T I C L E I N F O Article history: Received 1 December 2013 Received in revised form 12 March 2014 Accepted 12 March 2014 Available online 27 March 2014 Keywords: Bone morphogenetic protein-4 Cell proliferation Extracellular matrix Heparan sulfate proteoglycans Adult neurogenesis Subventricular zone A B S T R A C T Fractones are extracellular matrix structures that display a fractal ultrastructure and that are visualized as puncta after immunolabeling for laminin or heparan sulfate proteoglycans. In the adult brain, fractones are found throughout the subventricular zone (SVZ). The role of fractones is just emerging. We have recently shown that fractones sequester fibroblast growth factor-2 and bone morphogenetic protein-7 from the brain ventricles to regulate cell proliferation in the SVZ of the lateral ventricle, the primary neural stem cell niche and neurogenic zone in adulthood. Here, we have examined in vivo the effect of bone morphogenetic protein-4 (BMP-4) on cell proliferation in the SVZ and we have determined whether BMP-4 interacts with fractones to promote this effect. To examine BMP-4 effect on cell proliferation, BMP-4 was intracerebroventricularly injected, and bromodeoxyuridine immunolabeling was performed on frozen sections of the adult mouse brain. To identify the location of BMP-4 binding, biotinylated-BMP-4 was injected, and its binding localized post-mortem with streptavidin, Texas red conjugate. Injection of heparitinase-1 was used to desulfate fractones and determine whether the binding and the effect of BMP-4 on cell proliferation are heparan sulfate-dependent. BMP-4 inhibited cell proliferation in the SVZ neurogenic zone. Biotinylated-BMP-4 bound to fractones and some adjacent blood vessels. Co-injection of heparitinase-1 and biotinylated-BMP-4 resulted in the absence of signal for biotinylated-BMP-4, indicating that the binding was heparan sulfate dependent. Moreover, preventing the binding of BMP-4 to fractones by heparitinase-1 reinforced the inhibitory effect of BMP-4 on cell proliferation in the SVZ. These results show that BMP-4 inhibits cell proliferation in the SVZ neurogenic zone and that the binding of BMP-4 to fractone-associated heparan sulfates moderates this inhibitory effect. Together with our previous results, these data support the view that fractones capture growth factors and modulate their activity in the neural tissues lining the ventricles. ß 2014 Elsevier B.V. All rights reserved. Abbreviations: BMP-4, bone morphogenetic protein-4; BrdU, bromodeoxyuridine; CSF, cerebrospinal fluid; ECM, extracellular matrix; FGF-2, fibroblast growth factor- 2; HSPG, heparan sulfate proteoglycans; ICV, intracerebroventricular; IgM, immunoglobulin M; LRP-2, low-density lipoprotein receptor-related protein-2; NS-HSPG, N-sulfated heparan sulfate proteoglycans; NSPC, neural stem and progenitor cells; SVZ, subventricular zone; VZ, ventricular zone. * Corresponding author. Tel.: +1 808 956 7414; fax: +1 808 692 1980. E-mail addresses: fmercier@hawaii.edu, fmercier@pbrc.hawaii.edu (F. Mercier), douet@hawaii.edu (V. Douet). Contents lists available at ScienceDirect Journal of Chemical Neuroanatomy jo ur n al ho mep ag e: www .elsevier .c om /lo cate/jc h emn eu http://dx.doi.org/10.1016/j.jchemneu.2014.03.005 0891-0618/ß 2014 Elsevier B.V. All rights reserved.