Bone Morphogenetic Proteins BMP-6 andBMP-7 Have Differential Effects on Survivaland Neurite Outgrowth of CerebellarGranule Cell Neurons

Takeshi Yabe, Ivy Samuels, and Joan P. Schwartz*Neurotrophic Factors Section, National Institute of Neurological Disorders and Stroke,National Institutes of Health, Bethesda, Maryland

The bone morphogenetic proteins (BMPs) play an induc-tive role in the generation of cerebellar granule cellsembryonically. Therefore, we chose to look at their ef-fects on cerebellar granule cell survival and differentia-tion postnatally. The cells express mRNA for both BMP-6and BMP-7, as well as for the receptors BMPRIA andBMPRII, demonstrating that the postnatal cells have theability to form the heterodimer receptors needed to re-spond to BMPs. BMP-7 promotes cell survival, with amaximal effect at 10 ng/ml, whereas tenfold more BMP-6is needed: Both were active over the course of 8 days inculture. In addition, both BMPs were able to protect theneurons against death from induced apoptosis (exposureto serum-free, low-potassium medium) or exposure toglutamate. However, only BMP-6 could stimulate neuriteoutgrowth, measured with a neurofilament ELISA, aneffect that was seen over the first 6 days in culture. Theseresults, taken together with others in the literature, sug-gest that the BMPs have strong neurotrophic effects thatare both neuron specific and BMP specific.Published 2002 Wiley-Liss, Inc.†

Key words: cerebellar granule cell neurons; bone mor-phogenetic protein-6; bone morphogenetic protein-7;survival; neurite outgrowth

Bone morphogenetic proteins (BMPs), members of asubclass of proteins in the transforming growth factor-�family, have a number of effects in both the developingand the adult nervous systems. Both the BMPs and theirreceptors, BMPR-I and BMPR-II, are expressedthroughout the brain, embryonically and in adults (forreview see Mehler et al., 1997; Ebendal et al., 1998).BMP-6 and BMP-7 (also known as osteogenic protein-1,OP-1) are expressed by both neurons and astrocytes(Schluesener and Meyermann, 1994; Tomizawa et al.,1995; Jordan et al., 1997; Soderstrom and Ebendal, 1999).The BMPs affect neurogenesis and stimulate differentia-tion of specific neuronal subtypes, such as adrenergic neu-rons, during embryogenesis (Liem et al., 1995; Reissmanet al., 1996; Shah et al., 1996; Furuta et al., 1997). More

recently, they have been shown to affect survival and/ordifferentiation of postnatal dopaminergic neurons ( Jordanet al., 1997), serotonergic neurons (Galter et al., 1999),and sympathetic neurons (Liem et al., 1995; Guo et al.,1998) as well as neurons in the olfactory system (Shou etal., 2000), the eye (Trousse et al., 2001), the cortex (Le-Roux et al., 1999), and the hippocampus (Withers et al.,2000). Effects range from increased survival to stimulationof differentiation, including increases in neurotransmittersynthetic enzymes and uptake systems as well as processoutgrowth.

A study by Alder et al. (1999) demonstrated thatBMP-6 and BMP-7 play an inductive role in the gener-ation of cerebellar granule cells (CGCs); pretreatment inculture with BMP-6 or BMP-7 allows cells in the E8.5neural tube to differentiate into CGCs when transplantedin vivo. BMP-6 and BMP-7 are both found in the cere-bellum, and BMP-6 in particular is expressed in radial glia.Because radial glia play a major role postnatally in themigration and differentiation of CGCs, we have chosen toanalyze the effects of these two BMPs on CGCs culturedfrom postnatal day 8 rat brain.

MATERIALS AND METHODS

Primary CGC Culture

CGCs were prepared from 8-day-old Sprague Dawley ratpups (Charles River, Wilmington, MA) as previously described(Taniwaki et al., 1995). The rats were used under a protocolapproved by the NINDS Animal Care and Use Committee.Cells were plated in serum-containing medium [Eagle’s basalmedium (BME) with 25 mM KCl, 2 mM glutamine, 100 �g/mlgentamycin, and 10% heat-inactivated fetal bovine serum (FBS)]

The first two authors contributed equally to this paper.

*Correspondence to: Dr. Joan P. Schwartz, NTFS, NINDS, NIH, Build-ing 36, Room 4A31, Bethesda, MD 20892-4126.E-mail: jps@helix.nih.gov

Received 28 November 2001; Revised 18 January 2002; Accepted 18January 2002

Published online 15 March 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jnr.10210

Journal of Neuroscience Research 68:161–168 (2002)

Published 2002 Wiley-Liss, Inc. †This article is a US Government workand, as such, is in the public domain in the United States of America.

in poly-L-lysine-coated 10 cm dishes, eight-well chamber slides,or 96-well plates at 3.5 � 105 cells/cm2 and grown at 37°C ina humidified atmosphere. Cytosine arabinoside was added at 1day in vitro (DIV1) at a final concentration of 10 �M. TheCGC cultures (DIV7) consisted of �98% neurons (stained withanti-NSE antibody), �1% astrocytes (stained with anti-GFAPantibody), and �1% microglia (stained with anti-OX42 anti-body). All culture reagents were obtained from Life Technolo-gies (Gaithersburg, MD).

Astrocyte Cultures

Astrocytes were prepared from the cerebral cortex of alitter of postnatal day 2 (PD2) rat pups and cultured as previouslyreported (Schwartz and Wilson, 1992) in Dulbecco’s modifiedEagle’s medium (DMEM; high glucose) containing 10% FBS,1 mM pyruvate, 2 mM glutamine, and 25 �g/ml gentamycin ina humidified atmosphere of 90% air/10% CO2. When culturesbecame confluent, the flasks were shaken at 225 rpm overnight,and the medium was changed the next morning. Following thethird overnight shake, the cells were trypsinized and cultured for24 hr in 10 �M cytosine arabinoside. When flasks becameconfluent again, the cells were subcultured for experiments. Allexperiments were carried out when the cultures had becomeconfluent.

Microglial Cultures

Microglia were prepared from 8-day-old Sprague Dawleyrat pups. The cortex was dissected from one litter of rats, and themeninges were removed. A cell suspension was prepared bytrituration with a 10 ml pipette, followed by passage through19-, 22-, and 25-gauge needles. The suspension was filteredthrough a nylon mesh (60 �m). After centrifugation at 230g for10 min at room temperature, the pellet was resuspended inserum-containing medium (BME with 25 mM KCl, 2 mMglutamine, 100 �g/ml gentamycin, and 10% heat-inactivatedFBS). Two weeks later, the medium was changed, and the flaskswere shaken gently by hand for 3 min, after which the super-natants were removed and replated for use as pure (�99%)microglial cultures.

MTS Assay

Cell survival was measured with the MTS Cell Prolifer-ation Assay Kit (Promega, Madison, WI) using cells plated in96-well plates. Briefly, CGCs in 96-well plates were incubatedin a CO2 incubator for 2 hr with MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] and PMS (phenazine methosulfate; finalconcentration 333 �g/ml MTS and 25 �M PMS). In thepresence of PMS, mitochodrial dehydrogenases in live cellsmetabolize MTS into a water-soluble formazan product that canbe measured by spectrophotometry at 490 nm.

Lactate Dehydrogenase Assay

Lactate dehydrogenase (LDH), a stable cytoplasmic en-zyme present in all cells, is rapidly released into the culturemedium following damage to the plasma membrane. LDHactivity was measured with the commercially available Cytotox-icity Detection Kit (Boehringer Mannheim, Indianapolis, IN).For measurement of released LDH, culture medium was col-

lected and centrifuged to remove contaminating cells and cel-lular debris. To assay total LDH activity, 100 �l of 1% TritonX-100 were then added to the wells and the cells incubated for30 min. The percentage of LDH release was then calculated asthe LDH in the culture medium divided by total LDH (cellularplus medium LDH). For the actual assay, 100 �l of each samplewere transferred to a 96-well microtiter plate, 100 �l of LDHassay reagent added per well, and samples incubated for up to30 min at room temperature, after which the absorbance ofsamples was measured at 490 nm.

TUNEL Assay

Apoptosis was detected by TUNEL assay using the In SituCell Death Detection Kit according to the manufacturer’s in-structions (Roche). Briefly, CGCs were fixed in 4% paraformal-dehyde for 1 hr at room temperature and then incubated inpermeabilization buffer (0.1% Triton X-100 in 0.1% sodiumcitrate) for 2 min on ice. The cells were then incubated withTUNEL reaction medium at 37°C for 1 hr. After incubation,cells were stained with propidium iodide (5 �g/ml) for 5 min.DNA fragmentation was evaluated using a laser confocal micro-scope.

Neurofilamaent ELISA

The neurofilament ELISA was performed according tothe method of Doherty et al. (1984), with slight modifications(Taniwaki et al., 1995). Cultures grown in 96-well plates werefixed with 4% paraformaldehyde in phosphate-buffered saline(PBS) at 4°C for 2 hr. The fixed cells were permeabilized bytreatment for 15 min with 0.1% Triton X-100 in PBS, followedby incubation for 60 min with PBS containing 10% goat serumto block nonspecific binding. The cultures were then incubatedwith a monoclonal antineurofilament antibody overnight at 4°C(RMO-42 diluted 1:100; Lee et al., 1988). After two washeswith PBS containing 10% goat serum, cells were incubated withsecondary antibody (horseradish peroxidase-conjugated goatanti-mouse diluted 1:1,000) for 1 hr. After sequential washingwith PBS and water, the cultures were incubated with 0.2%o-phenylenediamine and 0.02% H2O2 in 50 mM citrate buffer,pH 5.0, for 30 min. Product formation was quantified byreading the optical density (OD) of an aliquot of the reactionproduct at 490 nm using a Dynatech microplate reader.

Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted from CGCs, according to themanufacturer’s instructions for Trizol reagent (Life Technolo-gies). Two micrograms of total RNA were converted to first-strand cDNA using the First-Strand cDNA Synthesis Kit (Am-ersham Pharmacia Biotech, Piscataway, NJ). The resultingcDNA was subjected to PCR analysis. The PCR amplificationmixture, in a final volume of 25 �l, consisted of 1� Taq DNApolymerase buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.5 �M ofeach specific primer, and 2.5 U Taq DNA polymerase (LifeTechnologies), according to the manufacturer’s instructions.Cycle number was chosen such that amplification of the prod-ucts was in the linear range with respect to the amount of inputcDNA. Each cycle consisted of 30 sec at 94°C for denaturation,30 sec at 60°C for annealing, and 60 sec at 72°C for extension.The PCR products were stained with ethidium bromide after

162 Yabe et al.

agarose gel electrophoresis and photographed using Polaroidfilm type 667. A sample without RNA was always negative; ratcortex RNA was used as a positive control for the products(results not shown).

The actual sequences of specific primers are as follows:BMPRIA (sense) 5�-TGACGTTAGCACCAGAGGACA-3�,(antisense) 5�-AATACAGACAGCCATAGAGAT-3�; BMP-RII (sense) 5�-GCTTCGCAGAATCAAGAACG-3�, (anti-sense) 5�-GTGGACTGAGTGGTGTTGTG-3�; BMP-6(sense) 5�-CGCGCTCCGGCCTCGCTCCGCCGCT-3�,(antisense) 5�-GAGTGGAGCGGACTTCAGCCGCCCT-3�;BMP-7 (sense) 5�-AGATCCTGTCCATCTTAGGGTTGC-3�, (antisense) 5�-AGTCCTTATAGATCCTGAATTCGGC-3�.

Statistical Analysis

Statistical analysis was carried out using either one-wayANOVA with Newman-Keul’s post hoc test or two-wayANOVA.

RESULTSCGCs Express BMP Receptor Subunit and BMPmRNAs

Before studying the putative roles of BMP-6 andBMP-7 in CGCs, we analyzed by RT-PCR whetherCGCs express BMP receptors as well as the BMPs them-selves. As shown in Figure 1, mRNA for the BMP recep-tor subunit types IA and II was detected in CGCs, indi-cating that CGCs should be able to form the active high-affinity receptor heterodimers capable of mediating BMP

signals (ten Dijke et al., 1996). Strong expression ofBMP-6 mRNA was also detected in CGCs, whereasBMP-7 mRNA expression was very low. Cultured astro-cytes also expressed both receptor subtypes as well asBMP-6 mRNA but no BMP-7 mRNA. Microglia, how-ever, though also expressing receptor type 1A and BMP-6mRNA, contained only low expression of BMPRII andno BMP-7 mRNA.

BMP-6 and BMP-7 Increase Survival of CGCsTo examine the effects of the BMPs on neuronal

survival, CGCs prepared from postnatal day 8 rats wereincubated with BMP-6 or BMP-7 over the concentrationrange of 3–100 ng/ml for 3 days, and cell viability wasdetermined using the MTS assay. As shown in Figure 2,incubation with BMP-7 significantly increased neuronalsurvival over a concentration range of 3–100 ng/ml, witha maximal effect achieved at 10 ng/ml. BMP-6 also in-creased neuronal survival but only at concentrations of 30or 100 ng/ml and with a maximal effect smaller than thatof BMP-7 (Fig. 2). This difference between the effects ofBMP-6 and those of BMP-7 was significant by two-wayANOVA. Time course analysis revealed that exposure ofcells to BMP-6 (100 ng/ml) or BMP-7 (3 ng/ml) resultedin a statistically significant difference in survival, measuredusing the MTS assay, between BMP-treated and untreatedcultures by DIV3; the difference lasted up to DIV8 (Fig.3). There was no significant difference (two-wayANOVA) between the effects of the two BMPs, butneither BMP had as large an effect at early times (DIV3–6)as the neurotrophic factor pigment epithelium-derivedfactor (PEDF), used as a positive control (Taniwaki et al.,1995). We have demonstrated previously (Taniwaki et al.,

Fig. 1. Expression of mRNA for BMP-6 and BMP-7 and their recep-tor subunits in cerebellar granule cells (CGCs), astrocytes, and micro-glia. Total RNA was extracted from CGCs (DIV2), cultured neonatalstriatal astrocytes, or cultured cortical microglia and reverse transcribed,followed by PCR for 40 cycles with specific primers as described inMaterials and Methods. The samples were run on agarose gels, and thefigure shows an ethidium bromide-stained image. Analysis for theunchanging cyclophilin mRNA demonstrated that equal amounts ofmRNA were used for each sample (results not shown). Samples with-out RNA were always negative; rat cortex RNA served as a positivecontrol for the products (results not shown). Marker DNA shown is the100 bp DNA ladder from Invitrogen (La Jolla, CA). Expected productsizes were the following: BMPRIA, 350 bp; BMPRII, 348 bp; BMP-6,364 bp; BMP-7, 350 bp.

Fig. 2. Dose-response analysis for effects of BMP-6 and BMP-7 onsurvival of CGCs. CGCs were prepared and incubated with the indi-cated concentrations of BMP-6 (circles) or BMP-7 (squares) or werenot treated. On DIV4, cell survival was analyzed using the MTS assay,and results are presented as increase in MTS absorbance of the treatedcells over control, with the control set equal to 1.0. Values are mean �SEM for n � 6, derived from one of three experiments. Effects of bothBMPs are significant by two-way ANOVA at P � 0.001.

Effects of BMP-6 and BMP-7 on Cerebellar Granule Cells 163

1995) that CGCs prepared from postnatal day 8 rat pupsare postmitotic, so the increase in cell number shown hereis a survival effect rather than an effect on mitosis. Becausecytosine arabinoside is included in the cultures, no celldivision is possible.

BMP-6 and BMP-7 Protect CGCs Against InducedApoptosis and Glutamate Toxicity

CGCs cultured in serum require high (25 mM) KClfor survival and undergo apoptosis when switched toserum-free medium containing 5 mM KCl (D’Mello etal., 1993; Yan et al., 1994; Galli et al., 1995). To analyzethe effects of BMP-6 and BMP-7 on induced apoptosis inCGCs, we performed the TUNEL assay. When DIV2cells were switched to serum-free medium containing low(5 mM) KCl, approximately 50% of the cells wereTUNEL positive 6 hr later (Fig. 4B vs. A). Neuronal deathcaused by low potassium was dramatically reduced by20 hr preincubation with 100 ng/ml BMP-6 (Fig. 4C) orBMP-7 (Fig. 4D). Comparable data were obtained in theMTS assay: The switch to low potassium lowered theMTS value from 0.223 � 0.010 (n � 15) to 0.139 �0.002 (n � 16). Addition of 100 ng/ml BMP-6 or BMP-7restored the value to 0.178 � 0.005 (n � 13) or 0.171 �0.004 (n � 15), respectively. All of the differences weresignificant by one-way ANOVA.

The BMPs were also effective in protecting DIV8CGCs against exposure to 100 �M glutamate for 1 hr.Glutamate caused a reduction in the ability of CGCs tometabolize MTS (Fig. 5A) and increased release of LDHactivity to the culture medium (Fig. 5B). Both effects wereat least partially attenuated by 24 hr pretreatment with

BMP-6 or BMP-7. The two BMPs had equivalent effectson maintaining metabolism of MTS. In contrast, BMP-6had a minimal effect on release of LDH, whereas BMP-7was as effective as the neurotrophic factor PEDF (Tani-waki et al., 1997) in preventing LDH release.

BMP-6, but not BMP-7, Induces NeuriteOutgrowth of CGCs

CGCs treated with 100 ng/ml of BMP-6 for 4 daysshowed increased neurite outgrowth and branching (Fig.6C) relative to the untreated control cells (Fig. 6A) or toBMP-7-treated cells (Fig. 6B). To quantitate effects of theBMPs on neurite outgrowth of CGCs, a neurofilamentprotein ELISA was used. Immunohistochemical studieshad demonstrated that the monoclonal antibody RMO-42, which recognizes the phosphorylated domains ofneurofilament-H and neurofilament-M (Lee et al., 1988),stained only the neurites of CGCs in culture. In theELISA, absorbance is linearly proportional to the contentof neurofilament-H (200 kDa) plus neurofilament-M (160kDa) in the cells, thus providing an indirect quantitation ofthe number of neurites (Taniwaki et al., 1995). WhenCGCs were incubated with BMP-6 from DIV1 to DIV4,the content of neurofilament protein was significantlyincreased at doses of 30 and 100 ng/ml (Fig. 7). In con-trast, BMP-7 did not affect the expression level of neuro-filament protein at any concentration from 3 to100 ng/ml. Smaller increases were seen in response toBMP-6 between DIV4 and DIV6; by DIV8, neurite for-mation of untreated CGCs had reached the same plateaulevel as that in BMP-6-treated cells (results not shown).

DISCUSSIONVery different results have been obtained for local-

ization of BMP-6 and BMP-7 to specific neural cells.Some authors reported expression of BMP-6 and/orBMP-7 only in neurons (Tomizawa et al., 1995; Martinezet al., 2001), whereas others found expression only inastrocytes and radial glia (Schluesener and Meyermann,1994; Jordan et al., 1997). Soderstrom and Ebendal (1999)showed expression in both neurons and glia. Our in vitroresults demonstrate that BMP-6 and BMP-7 mRNA areexpressed in cerebellar granule neurons, whereas bothastrocytes and microglia express BMP-6 mRNA. That allthree types of cells also contain mRNA for the tworeceptors BMPRIA and BMPRII suggests the possibilityof the BMPs mediating signalling among these cell types.

The BMPs apparently function in the cerebellumthroughout early development. Alder et al. (1999) showedthat both BMP-6 and BMP-7 are expressed in dorsalmidline cells adjacent to the rhombic lip and have induc-tive activities, stimulating the generation of CGC progen-itors in vitro. In our experiments, both BMP-6 andBMP-7 act as survival factors for postnatal CGCs. BMP-7was significantly more potent, acting at a tenfold lowerconcentration to produce a larger effect. In contrast,BMP-6 is more active on dopaminergic neurons ( Jordanet al., 1997), whereas neither affected survival of hip-pocampal, cortical, or septal cholinergic neurons (LeRoux

Fig. 3. Time course of effect of BMP-6 and BMP-7 on CGC survival.CGCs were prepared and incubated with BMP-6 (100 ng/ml; circles),BMP-7 (3 ng/ml; squares), or PEDF (7 nM; asterisks) or were nottreated. The neurotrophic factor PEDF was used as a positive control(Taniwaki et al., 1995). The drugs were added on DIV1, and cellsurvival was analyzed by MTS assay on DIV3, DIV4, DIV6, and DIV8.Values are mean � SEM for n � 6; the experiment was repeated threetimes. The DIV1 value was arbitrarily set equal to 1.0. Effects of bothBMP-6 and BMP-7 are significant relative to control cells by two-wayANOVA (P � 0.001) but not different from each other.

164 Yabe et al.

et al., 1999; Withers et al., 2000; Nonner et al., 2001).BMP-7 had almost no effect on peripheral ganglion neu-rons but potentiated the effect of either neurotrophin-3(NT-3) or GDNF (Bengtsson et al., 1998).

In addition to the survival effect, both the BMPscould protect the CGCs against either induced apoptosisor exposure to glutamate, the two mechanisms of neuronaldeath observed in the brain following various types ofinjury. Again, BMP-7 was significantly more effective inthese culture assays. BMP-7 has also been shown to beeffective in two in vivo injury models. When it wasadministered prior to a bilateral carotid artery ligation, theinfarct size was decreased, whereas, when it was given 1 hrpostligation, there was no effect on infarct size, but sur-vival of the rats was significantly increased (Perides et al.,1995). Similarly, Kawamata et al. (1998) showed that

administration of BMP-7 1–4 days following middle ce-rebral artery occlusion increased functional recovery. Ourresults suggest that at least part of these effects may derivefrom protection of the neurons against apoptotic and/orglutamate-induced death.

Although BMP-7 was more active in promotingsurvival of the CGCs under all conditions, it had abso-lutely no effect on neurite outgrowth, whereas BMP-6was very active. The only other report demonstrating thatBMP-6 stimulated dendritic growth was for rat sympa-thetic neurons, and the authors found that BMP-6 andBMP-7 were equally active (Guo et al., 1998). In contrast,other studies have demonstrated that BMP-7 increaseddendrite formation from hippocampal neurons (Withers etal., 2000), cortical neurons (LeRoux et al., 1999), andmotor neurons in spinal cord transplants to the eye

Fig. 4. BMPs protect CGCs aginst low-potassium-induced apoptosis.CGCs (DIV1) were incubated with or without 100 ng/ml BMP-6 orBMP-7 for 20 hr in serum-containing medium with 25 mM KCl. OnDIV2, the CGCs were switched to conditioned serum-containing me-dium with 25 mM KCl or to serum-free medium with 5 mM KCl

(induced apoptosis). After 6 hr in culture, the cells were fixed, stained withTUNEL (green fluorescence), and counterstained with propidium iodide(red fluorescence). A: No treatment. B: Induced apoptosis. C: BMP-6(100 ng/ml) plus induced apoptosis. D: BMP-7 (100 ng/ml) plus inducedapoptosis. Similar results were obtained in four experiments.

Effects of BMP-6 and BMP-7 on Cerebellar Granule Cells 165

(Granholm et al., 1999). BMP-7 potentiated the effect ofneurotrophic factors on peripheral ganglion neurons buthad no effect alone (Bengtsson et al., 1998).

This differential effect of BMP-6 suggests the pres-ence of a receptor that responds only to BMP-6. TheBMPs are known to stimulate activin receptors also, andactivin and BMP receptor subunits can differentially het-

erodimerize (ten Dijke et al., 1996; Ebendal et al., 1998;Luisi et al., 2001; Miyazono et al., 2001). Some of thesereceptors have been localized to both neurons and astro-cytes in the adult brain although not yet to microglia (Laiet al., 1996). Two possibilities could thus explain thedifferential effect of BMP-6 on neurite outgrowth:BMP-6 may act directly on a neuronal BMP/activin het-erodimer receptor expressed on CGCs that does not rec-ognize BMP-7, or BMP-6 could activate a receptor ex-pressed only on glia and the glia in turn release a trophicfactor capable of stimulating neurite outgrowth from theCGCs. Our results have shown expression of two of theBMP receptors in the CGCs, astrocytes, and microglia,but we have not yet examined activin receptor expression.There are currently no data demonstrating a BMP/activinreceptor that differentially recognizes BMP-6 but notBMP-7, although there are data showing different effectsin response to different combinations of the receptors (tenDijke et al., 1996). However, few of these studies have

Fig. 5. Effect of BMP-6 and BMP-7 on glutamate toxicity. CGCs(DIV7) were incubated with or without BMP-6 or BMP-7(100 ng/ml) for 24 hr. The cultures were then exposed to 100 �Mglutamate for 1 hr, and MTS absorbance (A) or LDH release (B) wasanalyzed 24 hr later. The control values were set equal to 1.0 and otherscalculated relative to that. Values are mean � SEM for n � 10–12(MTS) and n � 6 (LDH). The experiment was repeated four times.The differences between control and glutamate, and between glutamatewith or without BMP-6 or BMP-7, are significant by one-wayANOVA with Newman-Keuls post hoc test (P � 0.001 for all MTSresults; P � 0.001 for control vs. glutamate; P � 0.01 for glutamate �BMP-6 or BMP-7 for LDH results).

Fig. 6. Phase-contrast images of CGCs on DIV4 with or without BMPtreatment. CGCs were cultured from DIV0 to DIV4 with no treatment(A), 100 ng/ml BMP-7 (B), and 100 ng/ml BMP-6 (C). �400.

166 Yabe et al.

examined expression or function of different combinationsof these receptors in neural cells.

Taken together, these results suggest that bothBMP-6 and BMP-7 have strong neurotrophic effects on avariety of neurons. However, the effects are clearly neuronsubtype specific and in some instances require the copres-ence of a classical neurotrophic factor. Furthermore, theeffects of the two BMPs can be quite distinct, as exem-plified by their actions on CGCs: BMP-7 promotes sur-vival at lower doses against a variety of insults, whereasBMP-6 stimulates neurite outgrowth. Their differentialdistribution in different types of neural cells further sup-ports a specific set of responses in each brain region.

ACKNOWLEDGMENTSWe thank Ms. Delores Wilson for help with all the

assays and cell culture. The BMPs were obtained fromCreative Biomolecules.

REFERENCESAlder J, Lee KJ, Jessell TM, Hatten ME. 1999. Generation of cerebellar

granule neurons in vivo by transplantation of BMP-treated neural pro-genitor cells. Nat Neurosci 2:535–540.

Bengtsson H, Soderstrom S, Kylberg A, Charette MF, Ebendal T. 1998.Potentiating interactions between morphogenetic protein and neurotro-phic factors in developing neurons. J Neurosci Res 53:559–568.

D’Mello SR, Galli C, Ciotti T, Calissano P. 1993. Induction of apoptosisin cerebellar granule neurons by low potassium: inhibition of death byinsulin-like growth factor I and cAMP. Proc Natl Acad Sci USA 90:10989–10993.

Doherty P, Dickson JG, Flanigan TP, Walsh FS. 1984. Quantitative eval-uation of neurite outgrowth in cultures of human foetal brain and dorsalroot ganglion cells using an enzyme-linked immunoadsorbent assay forhuman neurofilament protein. J Neurochem 42:1116–1122.

Ebendal T, Bengtsson H, Soderstrom S. 1998. Bone morphogenetic pro-teins and their receptors: potential functions in the brain. J Neurosci Res51:139–146.

Furuta Y, Piston DW, Hogan BLM. 1997. Bone morphogenetic proteins(BMPs) as regulators of dorsal forebrain development. Development124:2203–2212.

Galli C, Meucci O, Scorziello A, Werge TM, Calissano P, Schettini G.1995. Apoptosis in cerebellar granule cells is blocked by high KCl,forskolin, and IGF-1 through distinct mechanisms of action: the involve-ment of intracellular calcium and RNA synthesis. J Neurosci 15:1172–1179.

Galter D, Bottner M, Krieglstein K, Schomig E, Unsicker K. 1999. Dif-ferential regulation of distinct phenotypic features of serotonergic neuronsby bone morphogenetic proteins. Eur J Neurosci 11:2444–2452.

Granholm A-Ch, Sanders LA, Ickes B, Albeck D, Hoffer BJ, Young DA,Kaplan PL. 1999. Effects of osteogenic protein-1 (OP-1) treatment onfetal spinal cord transplants to the anterior chamber of the eye. CellTransplant 8:75–85.

Guo X, Rueger D, Higgins D. 1998. Osteogenic protein-1 and relatedbone morphogenetic proteins regulate dendritic growth and the expres-sion of microtubule-associated protein-2 in rat sympathetic neurons.Neurosci Lett 245:131–134.

Jordan J, Bottner M, Schluesener HJ, Unsicker K, Krieglstein K. 1997.Bone morphogenetic proteins: neurotrophic roles for midbrain dopami-nergic neurons and implications of astroglial cells. Eur J Neurosci 9:1699–1710.

Kawamata T, Ren JM, Chan TCK, Charette M, Finklestein SP. 1998.Intracisternal osteogenic protein-1 enhances functional recovery follow-ing focal stroke. Neuroreport 9:1441–1445.

Lai M, Sirimanne E, Williams CE, Gluckman PD. 1996. Sequential patternsof inhibin subunit gene expression following hypoxic-ischemic injury inthe rat brain. Neuroscience 70:1013–1024.

Lee VM-Y, Otvos L Jr, Schmidt ML, Trojanowski JQ. 1988. Alzheimerdisease tangles share immunological similarities with multiphosphoryla-tion repeats in the two large neurofilament proteins. Proc Natl Acad SciUSA 85:7384–7388.

LeRoux P, Behar S, Higgins D, Charette M. 1999. OP-1 enhances den-dritic growth from cerebral cortical neurons in vitro. Exp Neurol 160:151–163.

Liem KF, Tremml G, Roelink H, Jessell TM. 1995. Dorsal differentiationof neural plate cells induced by BMP-mediated signals from epidermalectoderm. Cell 82:969–979.

Luisi S, Florio P, Reis FM, Petraglia F. 2001. Expression and secretion ofactivin A: possible physiological and clinical implications. Eur J Endocri-nol 145:225–236.

Martinez G, Carnazza ML, Di Giacomo C, Sorrenti V, Vanella A. 2001.Expression of bone morphogenetic protein-6 and transforming growthfactor-�1 in the rat brain after a mild and reversible ischemic damage.Brain Res 894:1–11.

Mehler MF, Mabie PC, Zhang D, Kessler JA. 1997. Bone morphogeneticproteins in the nervous system. Trends Neurosci 20:309–317.

Miyazono K, Kusanagi K, Inoue H. 2001. Divergence and convergence ofTGF-�/BMP signalling. J Cell Physiol 187:265–276.

Nonner D, Barrett E, Kaplan P, Barrett JN. 2001. Bone morphogeneticproteins (BMP6 and BMP7) enhance the protective effect of neurotro-phins on cultured septal cholinergic neurons during hypoglycemia.J Neurochem 77:691–699.

Perides G, Jensen FE, Edgecomb P, Rueger DC, Charness ME. 1995.Neuroprotective effect of human osteogenic protein-1 in a rat model ofcerebral hypoxia/ischemia. Neurosci Lett 187:21–24.

Reissman E, Ernsberger U, Francis-West PH, Rueger D, Brickell PM, RohrerH. 1996. Involvement of bone morphogenetic protein-4 and bone morpho-genetic protein-7 in the differentiation of the adrenergic phenotype in de-veloping sympathetic neurons. Development 122:2079–2088.

Fig. 7. Effect of BMP-6 (squares) and BMP-7 (circles) on neurofila-ment content. CGCs were cultured from DIV0 to DIV4 with theindicated concentrations of BMP-6, BMP-7, or an equivalent volumeof H2O. On DIV4, the cells were fixed and analyzed for neurofilamentcontent using the ELISA described in Materials and Methods. Thecontrol value was set equal to 1.0, and values represent the increase overcontrol. Values are mean � SEM for n � 4–10. The experiment wasrepeated once. Results were analyzed by two-way ANOVA; the dif-ference between BMP-6 and BMP-7 is significant at P � 0.001.

Effects of BMP-6 and BMP-7 on Cerebellar Granule Cells 167

Schluesener HJ, Meyermann R. 1994. Expression of BMP-6, a TGF-�related morphogenetic cytokine, in rat radial glial cells. Glia 12:161–164.

Schwartz JP, Wilson DJ. 1992. Preparation and characterization of type 1astrocytes cultured from adult rat cortex, cerebellum, and striatum. Glia5:75–80.

Shah NM, Groves AL, Anderson DJ. 1996. Alternative neural crest cell fates areinstructively promoted by TGF superfamily members. Cell 85:331–344.

Shou J, Murray RC, Rim PC, Calof AL. 2000. Opposing effects of bonemorphogenetic proteins on neuron production and survival in the olfac-tory receptor neuron lineage. Development 127:5403–5413.

Soderstrom S, Ebendal T. 1999. Localized expression of BMP and GDFmRNA in the rodent brain. J Neurosci Res 56:482–492.

Taniwaki T, Becerra SP, Chader GJ, Schwartz JP. 1995. Pigmentepithelium-derived factor (PEDF) is a survival factor for cerebellar granulecells in culture. J Neurochem 64:2509–2517.

Taniwaki T, Hirashima N, Becerra SP, Chader GJ, Etcheberrigaray R,Schwartz JP. 1997. Pigment epithelium-derived factor (PEDF) protects

cultured cerebellar granule cells against glutamate-induced neurotoxicity.J Neurochem 68:26–32.

ten Dijke P, Miyazono K, Helden C-H. 1996. Signaling via hetero-oligomeric complexes of type I and type II serine/threonine kinasereceptors. Curr Opin Cell Biol 8:139–145.

Tomizawa K, Matsui H, Kondo E, Miyamoto K, Tokuda M, Itano T,Nagahata S, Akagi T, Hatase O. 1995. Developmental alteration andneuron-specific expression of bone morphogenetic protein-6 (BMP-6)mRNA in rodent brain. Mol Brain Res 28:122–128.

Trousse F, Esteve P, Bovolenta P. 2001. BMP4 mediates apoptotic celldeath in the developing chick eye. J Neurosci 21:1292–1301.

Withers GS, Higgins D, Charette M, Banker G. 2000. Bone morphoge-netic protein-7 enhances dendritic growth and receptivity to innervationin cultured hippocampal neurons. Eur J Neurosci 12:106–116.

Yan GM, Ni B, Weller M, Wood KA, Paul SM. 1994. Depolarization orglutamate receptor activation blocks apoptotic cell death of culturedcerebellar granule neurons. Brain Res 656:43–51.

168 Yabe et al.