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. 2001:2:12.
doi: 10.1186/1471-2202-2-12. Epub 2001 Sep 11.

Bone morphogenetic protein-5 (BMP-5) promotes dendritic growth in cultured sympathetic neurons

H N Beck  1 , K Drahushuk, D B Jacoby, D Higgins, P J Lein
Affiliations

Bone morphogenetic protein-5 (BMP-5) promotes dendritic growth in cultured sympathetic neurons

H N Beck et al. BMC Neurosci. 2001.

Abstract

Background: BMP-5 is expressed in the nervous system throughout development and into adulthood. However its effects on neural tissues are not well defined. BMP-5 is a member of the 60A subgroup of BMPs, other members of which have been shown to stimulate dendritic growth in central and peripheral neurons. We therefore examined the possibility that BMP-5 similarly enhances dendritic growth in cultured sympathetic neurons.

Results: Sympathetic neurons cultured in the absence of serum or glial cells do not form dendrites; however, addition of BMP-5 causes these neurons to extend multiple dendritic processes, which is preceded by an increase in phosphorylation of the Smad-1 transcription factor. The dendrite-promoting activity of BMP-5 is significantly inhibited by the BMP antagonists noggin and follistatin and by a BMPR-IA-Fc chimeric protein. RT-PCR and immunocytochemical analyses indicate that BMP-5 mRNA and protein are expressed in the superior cervical ganglia (SCG) during times of initial growth and rapid expansion of the dendritic arbor.

Conclusions: These data suggest a role for BMP-5 in regulating dendritic growth in sympathetic neurons. The signaling pathway that mediates the dendrite-promoting activity of BMP-5 may involve binding to BMPR-IA and activation of Smad-1, and relative levels of BMP antagonists such as noggin and follistatin may modulate BMP-5 signaling. Since BMP-5 is expressed at relatively high levels not only in the developing but also the adult nervous system, these findings suggest the possibility that BMP-5 regulates dendritic morphology not only in the developing, but also the adult nervous system.

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Figures

Figure 1
Figure 1
BMP-5 promotes dendritic growth in cultured sympathetic neurons. Non-neuronal cells were eliminated from SCG cultures by treatment with anti-mitotic agent during the 2nd and 3rd days in vitro. Beginning on day 5, sympathetic neurons were treated with either control medium (A), medium supplemented with 50 ng/ml BMP-7 (B), or medium containing 300 ng/ml BMP-5 (C). Six days later, cultures were immunostained with mAb against MAP2, a protein found primarily in dendrites and neuronal somata. Neurons grown under control conditions lack dendrites (A). In contrast, neurons exposed to BMP-7 (B) or BMP-5 (C) typically have several tapered dendrites. Bar, 50 μm.
Figure 2
Figure 2
BMP-5 is less potent than BMP-7 but equally efficacious in promoting dendritic growth in cultured sympathetic neurons. Beginning on the fifth day in vitro after elimination of glial cells, SCG neurons were exposed to varying concentrations of BMP-5 or BMP-7. After 6 days of exposure to BMPs, cultures were immunostained with a mAb to the dendritic marker MAP2. Dendritic growth was quantified with respect to the percent of cells with dendrites (A), number of dendrites per neuron (B) and total dendritic length (C). Data in panels B and C are presented as the mean ± S.E.M. (n = 60 per experimental condition). * p < 0.05.
Figure 3
Figure 3
The time course of dendritic growth is comparable between maximally effective concentrations of BMP-5 and BMP-7. Beginning on the fifth day in vitro, SCG neurons grown in the absence of glial cells were treated with control medium or medium supplemented with maximally effective concentrations of BMP-5 (300 ng/ml) or BMP-7 (50 ng/ml). After varying exposure times, cultures were fixed and immunostained with mAb to the dendritic marker MAP2. Dendritic growth was quantified with respect to the percent of cells with dendrites (A) and the number of dendrites per neuron (B). Data in panel B are expressed as the mean ± S.E.M. (n = 60 per experimental condition). There were no significant differences between BMP-7 and BMP-5 at any time point at p < 0.05.
Figure 4
Figure 4
BMP-5 induces phosphorylation of Smad-1 in cultured sympathetic neurons. (A) Blots of cell lysates from SCG cultures consisting solely of neuronal cells were probed using Ab that specifically recognizes the phosphorylated form of Smad-1. Under control conditions (time = 0 min), phosphorylated Smad-1 is not detected. Treatment with BMP-5 (100 ng/ml) causes a time-dependent increase in the band density of phosphorylated Smad-1. (B) Densitometric analyses of Western blots of cell lysates from purified neuronal cell cultures at varying times after exposure to BMP-5. Equal amounts of protein were loaded into all wells and each blot was probed initially for p-Smad, then stripped and successively probed for total Smad and tubulin. Data are presented as the mean ± S.E.M. (n = 2 per experimental condition).
Figure 5
Figure 5
Soluble BMP antagonists inhibit BMP-5-induced dendritic growth. Beginning on the fifth day in vitro, SCG neurons were exposed to BMP-5 (100 ng/ml) in the absence or presence of varying concentrations of follistatin (A), noggin (B) or the BMP-RIA-Fc chimera (C). On the tenth day in vitro, cultures were immunostained with mAb to nonphosphorylated forms of the M and H neurofilaments to visualize dendrites. Dendritic growth was quantified with respect to the number of dendrites per neuron expressed as the mean ± S.E.M. (n = 50 per experimental condition). * Indicates a significant difference from negative control values (- BMP-5/- antagonist) indicated by the dashed line in each bar graph at p < 0.01 and ** from positive control values (+ BMP-5/- antagonist) at p < 0.01.
Figure 6
Figure 6
The specificity of the BMP-5 Ab used in these studies as assessed by Western blot analysis. BMP-5 Ab (0.5 μg/ml) reacts with purified recombinant BMP-5 (50 ng), but not with equal amounts of purified recombinant BMP-6 or -7. Preincubation of BMP-5 Ab with specific blocking peptide (2.5 μg/ml) significantly reduces binding to BMP-5 protein. Data represented in this figure were obtained from the same immunoblot.
Figure 7
Figure 7
BMP-5 immunoreactivity is detected in co-cultures of SCG neurons and glia. Phase contrast (A,C,E) and fluorescence (B,D,F) micrographs of SCG cultures immunostained for BMP-5 after 10 days in vitro. Both glial cells and neurons express significant BMP-5 immuno-reactivity, and in neurons, the processes as well as the soma are brightly stained (A,B). Preincubation of BMP-5 Ab with blocking peptide prior to reaction with SCG cultures (C,D) reduces the intensity of immunostaining to levels comparable to the background fluorescence observed in SCG cultures reacted only with secondary Ab (E,F).
Figure 8
Figure 8
BMP-5 mRNA is expressed in SCG at times corresponding to maximal dendritic growth in SCG neurons. BMP-5 mRNA was detected by RT-PCR in total RNA extracted from rats SCG at E20, PN3 and PN7, which correspond to times of initial dendrite extension (E20) and maximal expansion of the dendritic arbor (PN3 and PN7). In contrast, PCR product was not detected in equal amounts of total RNA isolated from adult SCG.
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