Cerebrospinal Fluid Contains Biologically Active Bone Morphogenetic Protein-7 B. Dattatreyamurty,* E. Roux,* C. Horbinski,† P. L. Kaplan,* L. A. Robak,‡ H. N. Beck,§ P. Lein,§ D. Higgins,† and V. Chandrasekaran† *Creative Biomolecules Inc., Hopkinton, Massachusetts 01748; †Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York 14214; ‡Canisius College, Buffalo, New York 14208; and §Department of Environmental Health Science, Bloomburg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21136 Received November 27, 2001; accepted May 8, 2001; published online August 24, 2001 Bone morphogenetic proteins (BMPs) regulate the development and function of many types of neurons. However, little is known of the actual concentrations of BMPs in the various parts of the brain. In this study, we considered the possibility that BMPs might be present in cerebrospinal fluid (CSF). Western blot analysis of normal adult bovine CSF revealed the pres- ence of dimeric and monomeric forms of BMP-7, and the concentration of this molecule was found to be 12 ng/ml in a radioimmunoassay. Since BMP-7 is known to induce dendritic growth in rat sympathetic neu- rons, this was used as a bioassay to examine the bio- logical activity of the BMP-7 present in CSF. Addition of normal bovine CSF to cultures of sympathetic neu- rons produced a dose-dependent increase in dendritic growth and the magnitude of this response approxi- mated that obtained with maximally effective concen- trations of exogenous BMP-7. Moreover, CSF-induced dendritic growth was inhibited by follistatin, a protein that can sequester BMPs, and by either of two mono- clonal antibodies that react with BMP-7. These results show that, unlike most other neurotrophic factors, BMP-7 is a constituent of normal CSF and is present at concentrations sufficient to elicit a near maximal bio- logical response. © 2001 Elsevier Science Key Words: bone morphogenetic protein; cerebrospi- nal fluid; sympathetic neuron; dendrite; Smad-1; bo- vine. INTRODUCTION Bone morphogenetic proteins (BMPs) 1 are members of the TGF- family of growth factors (29). They were initially identified due to their ability to induce bone formation (49, 50, 58). However, BMPs were subse- quently found to regulate critical aspects of develop- ment of the nervous system (9, 34, 43) including specification of cell fate (13, 54), regulation of neural cell survival, determination of neuronal shape and transmitter phenotype (11, 36, 55), segmentation of rhombomeres (15), and dorsal ventral patterning (41, 54). Mature neurons from adult sympathetic ganglia also retain the ability to respond to BMPs (35) and BMP-7 facilitates the recovery from ischemic in- jury induced by ligation of the middle cerebral artery (28, 39). Transcripts for one or more BMPs have been de- tected in many regions of the developing central ner- vous system including cerebral cortex, hippocampus, striatum, cerebellum, and spinal cord (13, 34, 43, 52) and they are present in both neurons and glia. Expres- sion of mRNAs for BMPs typically declines in the adult nervous system, but signals are still detectable in dis- crete structures of the mature brain (9, 34, 43, 53). However, actual concentrations of these proteins in adult brain tissues and fluids are unknown and so it is difficult to assess their functional significance. In this study, we have examined the expression and activity of BMP-7 in cerebrospinal fluid (CSF) from adult brain. We focused on CSF, because mRNA for BMP-7 (26, 52) and several other TGF- family members is highly expressed in the choroid plexus (3, 9, 12, 13, 34, 52, 53). Moreover, it is known that subventricular zone cells remain sensitive to BMPs in adult brain (38) and that the processes of certain neurons in the hypothalamus and spinal cord are in direct contact with CSF (23). Thus, CSF has the potential to act as a reservoir and source of trophic factors for neurons in the adult CNS. Trophic factors belonging to the neurotrophin family (NGF and BDNF) (31, 32, 44, 47) and the interleukin-6 cytokine family (IL-6, ciliary neurotrophic factor, leu- kemia inhibitory factor) (14, 17, 48) are typically ex- pressed at extremely low levels in CSF such that their biological activity is undetectable. Other trophic fac- tors such as insulin-like growth factors-I and -II are 1 Abbreviations used: CSF, cerebrospinal fluid; BMP, bone mor- phogenetic protein; RIA, radioimmunnoassay; GDF, growth/differen- tiation factor. Experimental Neurology 172, 273–281 (2001) doi:10.1006/exnr.2001.7728, available online at http://www.idealibrary.com on 273 0014-4886/01 $35.00 © 2001 Elsevier Science All rights reserved.