Extracellular signal-regulated kinases regulate dendritic growth in rat sympathetic neurons

Abstract

NGF activates several signaling cascades in sympathetic neurons. We examined how activation of one of these cascades, the ERK/MAP (extracellular signal-regulated kinase/mitogen-activated protein) kinase pathway, affects dendritic growth in these cells. Dendritic growth was induced by exposure to NGF and BMP-7 (bone morphogenetic protein-7). Exposure to NGF increased phosphorylation of ERK1/2. Unexpectedly, two MEK (MAP kinase kinase) inhibitors (PD 98059 and U 0126) enhanced dendritic growth, and a ligand, basic FGF, that activates the ERK pathway inhibited the growth of these processes. The enhancement of dendritic growth by PD 98059 was associated with an increase in the number of axo-dendritic synapses, and it appeared to represent a specific morphogenic effect because neither axonal growth nor cell survival was affected. In addition, increased dendritic growth was not observed after exposure to inhibitors of other signaling pathways, including the phosphatidylinositol-3-kinase inhibitor LY 294002. Dendritic growth was also increased in cells transfected with dominant-negative mutants of MEK1 and ERK2 but not with dominant-negative mutants of MEK5 and ERK5, suggesting that ERK1/2 is the primary mediator of this effect. Exposure to BMP-7 induces nuclear translocation of Smad1 (Sma- and Mad-related protein 1), and PD 98059 treatment potentiated nuclear accumulation of Smad-1 induced by BMP-7 in sympathetic neurons, suggesting a direct enhancement of BMP signaling in cells treated with an MEK inhibitor. These observations indicate that one of the signaling cascades activated by NGF can act in an antagonistic manner in sympathetic neurons and reduce the dendritic growth induced by other NGF-sensitive pathways.

Figure 1.

Effects of BMP-7 and PD98059 on the morphology of sympathetic neurons. Phase-contrast (A, C, E, G) and fluorescence (B, D, F, H–J) micrographs of neurons immunostained with an mAb to MAP2. Neurons in control cultures had only axons (A, B). Neurons exposed to BMP-7 (10 ng/ml) for 5 d extended dendrites (C, D), and this response was enhanced by treatment with PD 98059 (10 μm; E, F). PD 98059 alone had no effect on dendritic growth (G, H). I and J are low-power micrographs of cells exposed to BMP alone (I) or in the presence of PD 98059 (J). Scale bars, 25 μm.

Figure 2.

Inhibition of MEK1 enhances BMP-7-induced dendritic growth. Sympathetic neurons were exposed to various concentrations of the MEK inhibitor PD 98059 (PD) (A) with or without BMP-7 (10 ng/ml) for 5 d. Alternatively, cells were exposed to varying concentrations of BMP-7 (B, C) in the presence of PD 98059 (10 μm). The number of dendrites per cell (B) and total dendritic length (C) was assessed by immunostaining with mAb to MAP2 (n ≥ 60 cells). *p < 0.05 versus control.

Figure 3.

Effects of an MEK inhibitor on synapse formation in cultures of sympathetic neurons. A, Neurons were treated with BMP-7 (10 ng/ml) with or without PD 98059 (10 μm) for 5 d. Dendritic morphology and presynaptic specializations were analyzed by double immunostaining with antibodies to MAP2 (green) and SV2 (red) (n ≥ 30 cells per condition). SV2-positive puncta that are associated with dendrites have been shown previously to represent sites of synaptic contact (Fletcher et al., 1994). *p < 0.05 versus control. B, Fluorescence micrograph of a neuron treated with BMP-7 (10 ng/ml) and PD 98059 (10 μm) for 5 d.

Figure 4.

Effects of U 0126 on dendritic growth. Sympathetic neurons were exposed to various concentrations of the MEK inhibitor U 0126 with or without BMP-7 (10 ng/ml) for 5 d. Cellular morphology (n ≥ 60 cells) was assessed by immunostaining with mAb to MAP2. *p < 0.05 versus BMP-7 alone.

Figure 5.

Dominant-negative MEK1 potentiates BMP-7-induced dendritic growth. For transfections, sympathetic neurons were plated at threefold higher density (∼30 cells/mm²) than in previous experiments. Under these conditions, BMP-7 (10 ng/ml) still induced dendritic growth, but the magnitude of the response was reduced (A). Neurons were cotransfected with plasmids containing EGFP and wild-type MEK1 (MEK1 WT) or dominant-negative mutant (MEK1 K97M) (B) or with a constitutively active mutant (C). Two days later, cells were treated with BMP-7 (10 ng/ml). On day 5, cellular morphology was assessed by immunostaining with an mAb to MAP2 (D, bottom; n ≥ 60 per group). Fluorescence micrograph of a nontransfected neuron and a neuron cotransfected with EGFP and dominant-negative MEK (MEK1 K97M) (D). Transfected cells were identified by expression of EGFP (D, top). *p < 0.05 versus BMP-7.

Figure 6.

Dominant-negative ERK2 enhances dendritic growth. Sympathetic neurons were cotransfected with plasmids containing EGFP and one of the following constructs: dominant-negative ERK2 (AEF; Thr 183 and Tyr 185 replaced with alanine and phenylalanine) (Kato et al., 1997), dominant-negative ERK5 (AEF), or dominant-negative MEK5 (A). Two days later, cells were treated with BMP-7 (10 ng/ml). On day 5, cellular morphology was assessed by immunostaining with an mAb to MAP2. Transfected cells were identified by expression of EGFP. *p < 0.05 versus BMP-7.

Figure 7.

Inhibition of MEK1 does not alter cell survival. Sympathetic neurons were treated with BMP-7 (10 ng/ml) with or without PD 98059 (PD) (10 μm) for 5 d. Subsequently, cell survival was determined by counting the number of MAP2-positive cells.

Figure 8.

Inhibition of MEK1 activity does not affect axonal growth. A, Dissociated neurons were plated onto coverslips coated with laminin (2 μg/ml) in control medium (C2) or in the presence of PD 98059 (10 μm) and/or BMP-7 (10 ng/ml) for 12 hr. Axons were identified by immunostaining with an mAb that recognizes phosphorylated forms of NF-Hand NF-M (n ≥ 40 cells). B–D, Neurons were plated at low density onto gridded coverslips. After elimination of non-neuronal cells, neurons were grown in medium with or without PD 98059 (10μm) and/or BMP-7 (10 ng/ml) for 5 d. Representative areas of the cultures were photographed just before treatments and after 1, 3, and 5 d of treatment. The total linear length of all axonal processes with the designated area was determined using NIH Image J software. C, Phase-contrast micrograph of a representative area, taken before experimental treatment was begun. When the same area was relocated after 5 d of treatment with PD 98059, the axonal network had become much denser and new processes had appeared (D). Circle represents the defined area inside which axons were serially traced; dark lines indicate alignment reference points for repeated placement of saved circle template. B, Total axonal length (15 areas).

Figure 9.

FGF inhibits BMP-7-induced dendritic growth. Sympathetic neurons were cultured in the control media containing 10 ng/ml NGF to maintain cell survival. After elimination of non-neuronal cells, neurons were exposed to BMP-7 (5 ng/ml) with or without FGF (100 ng/ml). On day 5, cellular morphology (n ≥ 60 cells) was assessed by immunostaining using mAb to MAP2. *p < 0.05 versus BMP-7.

Figure 10.

Inhibition of MEK1 increases the nuclear accumulation of Smad-1. Sympathetic neurons were cultured under control conditions (A, B) or in the presence of a maximally effective dose of BMP-7 (100 ng/ml) (C, D) for 2 hr. In addition, cultures were treated with BMP-7 at a concentration (10 ng/ml) close to ED₅₀ in the presence (G, H) or absence (E, F) of PD 98059. PD 98059 (10 μm) was added 30 min before BMP-7 was maintained in the medium thereafter. Cells were immunostained with an antibody that reacts with Smad-1, and optical sections (1 μm) were obtained with a Bio-Rad confocal microscope. Phase-contrast (A, C, E, G) and fluorescence (B, D, F, H) micrographs.