Regulation of neurite growth by tumour necrosis superfamily member RANKL

Abstract

RANKL (receptor-activator of NF-κB ligand, TNFSF11) is a member of the TNF superfamily that regulates bone remodelling and the development of the thymus, lymph nodes and mammary glands. While RANKL and its membrane bound receptor RANK (TNFRSF11A) are expressed in the adult central nervous system and have been implicated in thermoregulation, the potential function of RANK signalling in the developing nervous system remains unexplored. Here, we show that RANK is expressed by sympathetic and sensory neurons of the developing mouse peripheral nervous system and that activating RANK signalling in these neurons during perinatal development by either treating cultured neurons with soluble RANKL or overexpressing RANK in the neurons inhibited neurotrophin-promoted neurite growth without affecting neurotrophin-promoted neuronal survival. RANKL is expressed in tissues innervated by these neurons, and studies in compartment cultures demonstrated that RANKL is capable of acting directly on neurites to inhibit growth locally. Enhancing RANK signalling in cultured neurons resulted in NF-κB activation and phosphorylation of the p65 NF-κB subunit on serine 536. Transfecting neurons with a series of mutated signalling proteins showed that NF-κB activation and p65 phosphorylation occurred by an IKKβ-dependent mechanism and that blockade of this signalling pathway prevented neurite growth inhibition by RANKL. These findings reveal that RANKL is a novel negative regulator of neurite growth from developing PNS neurons and that it exerts its effects by IKKβ-dependent activation of NF-κB.

Figure 1.

RANK is expressed in developing SCG neurons and its activation inhibits NGF-promoted neurite growth. (a) Levels of RANK mRNA relative to the reference mRNAs, GAPDH and SDHA, in RNA extracted from SCG at ages ranging from E14 to P5. The mean ± s.e.m. of data obtained from four separate sets of ganglia at each age are shown. (b) Photomicrographs of a P0 SCG neuron immunostained for RANK and β-III tubulin after 24 h incubation in medium containing 10 ng ml^–1 NGF. Scale bar = 10 μm. A non-neuronal cell whose nucleus, along with the neuronal nucleus, is stained with the nuclear marker DAPI was unlabelled by the anti-RANK antibody. (c) Representative photomicrographs of P0 SCG neurons labelled with calcein AM after 24 h incubation in medium containing either 10 ng ml⁻¹ NGF or NGF plus 10 ng ml^–1 recombinant RANKL. (d) Confocal optical section through a neuron cell body. Scale bar = 10 μm. (e) Representative camera lucida drawings of neurons illustrating the range of morphologies of P0 SCG neurons after 24 h incubation in medium containing either NGF or NGF plus RANKL. The neurons illustrated correspond to percentiles 25, 50, 75 and 100 of the sampled populations in terms of total neurite length. Scale bar = 20 μm. (f,g) Sholl profiles, total length and number of branching points in the neurite arbours of P0 SCG neurons incubated for 24 h in medium containing 10 ng ml⁻¹ NGF alone, NGF plus 10 ng ml^–1 RANKL or NGF plus RANKL plus either (f) 10 ng ml⁻¹ OPG or (g) 10 ng ml^–1 RANKfc. To facilitate comparison of separate experiments, the length and branching point data are expressed as a percentage of the NGF values. Mean ± s.e.m. are shown, **p < 0.001, statistical comparison with NGF data. The non-normalized neurite length and branching datasets for all experiments carried out are provided in electronic supplementary material, spreadsheet S1.

Figure 2.

Recombinant RANKL and RANK overexpression impair NGF-promoted neurite growth from SCG neurons over an extended period of development. (a) Sholl profiles of SCG neurons isolated from (i) P0, (ii) P5 and (iii) P10 mice after 24 h incubation with either 10 ng ml⁻¹ NGF (unfilled circles) or NGF plus 10 ng ml⁻¹ RANKL (filled circles). (b) Sholl profiles of SCG neurons isolated from (i) P0, (ii) P5 and (iii) P10 mice that were incubated for 24 h in medium containing 10 ng ml⁻¹ NGF after transfection with a YFP expression plasmid together with either an empty control plasmid (control, unfilled circles) or a YFP expression plasmid together a plasmid expressing RANK (filled circles). Means ± s.e.m. of typical experiments are shown at each age. The neurite length and branching datasets for all experiments carried out are provided in electronic supplementary material, spreadsheet S2.

Figure 3.

RANKL is expressed in sympathetic targets and acts directly on sympathetic axons to suppress NGF-promoted growth. (a) Levels of RANKL mRNA relative to GAPDH and SDHA mRNAs in RNA extracted from submandibular gland at ages ranging from P0 to P10. The mean ± s.e.m. of data obtained from four separate sets of glands at each age are shown. (b) Immunohistochemical localization of RANKL in a section through the submandibular gland at P0. A section incubated with secondary antibody alone (control) is also shown. Scale bar = 50 μm. (c) Levels of RANKL mRNA relative to GAPDH and SDHA mRNAs in RNA extracted from pineal gland at ages ranging from P0 to P10. The mean ± s.e.m. of data obtained from four separate sets of glands at each age are shown. (d) Representative images of calcein-AM-labelled P0 SCG neurons cultured for 24 h in a two-compartment microfluidic device containing 10 ng ml⁻¹ NGF in both compartments (left) or NGF in both compartments plus 10 ng ml⁻¹ RANKL in the axon compartment (right). Scale bar = 50 μm. (e) Bar chart of mean axon length of neurons projecting axons into the axon compartment in cultures containing only NGF in both compartments or NGF in both compartments plus RANKL in the axon compartment. The results are expressed as a percentage of axon length in cultures containing NGF alone in both compartments (mean ± s.e.m. are shown, **p < 0.001, statistical comparison with cultures containing NGF alone in both compartments). The non-normalized datasets of all experiments carried out are provided in electronic supplementary material, spreadsheet S3.

Figure 4.

Recombinant RANKL and RANK overexpression activate NF-κB in SCG neurons. (a) NF-κB reporter signal in P0 SCG neurons incubated for 4 and 24 h in medium containing 10 ng ml⁻¹ NGF alone (unfilled bars) and 10 ng ml^–1 NGF plus 10 ng ml^–1 RANKL (filled bars). (b) NF-κB reporter signal in P0 SCG neurons transfected with either a RANK (filled bars) expression plasmid or a control (unfilled bars) empty plasmid and incubated for 4 and 24 h in medium containing 10 ng ml^–1 NGF. The data are normalized to the respective control signal. Mean ± s.e.m. are shown, **p < 0.001, statistical comparison with the respective controls. The non-normalized datasets of these experiments are provided in electronic supplementary material, spreadsheet S4.

Figure 5.

Neurite growth inhibition associated with RANK overexpression requires IKKβ- (but not IKKα-) mediated NF-κB activation. (a–c) Bar charts of neurite length and branch point number, and Sholl profiles, of P0 SCG neurons after 24 h incubation in medium containing 10 ng ml⁻¹ NGF following co-transfection with a YFP plasmid together with plasmids expressing: (a) RANK, dominant-negative IKKα (dnIKKα) and RANK plus dnIKKα; (b) RANK, dominant-negative IKKβ (dnIKKβ) and RANK plus dnIKKβ; and (c) RANK, S32A/S36A IκBα and RANK plus IκBα. To facilitate comparison of data from all experiments, the length and branch point data are expressed as a percentage of control-transfected values. Means ± s.e.m. are shown, **p < 0.001, statistical comparison with the respective controls. The non-normalized neurite length and branching datasets for all experimental results are provided in electronic supplementary material, spreadsheet S5.

Figure 6.

Phosphorylation of the p65 NF-κB subunit at ser536 is required for RANK-mediated inhibition of neurite growth. (a) Representative Western blot of phospho-S536-p65 and total p65, and graph of time course of p65 phosphorylation in cultured SCG neurons following stimulation with 10 ng ml⁻¹ RANKL after 12 h pre-incubation with 10 ng ml⁻¹ NGF. Each data point represents the mean ± s.e.m. of six separate Western blot assays. (b) Sholl profiles and bar charts of (c) total neurite length and (d) branch point number of the neurite arbours of P0 SCG neurons after 24 h incubation in medium containing 10 ng ml⁻¹ NGF following co-transfection with a YFP plasmid together with either an empty control plasmid or plasmids expressing RANK, S536A-p65 or RANK plus S536A-p65. The length and branch point data are expressed as a percentage of data from control-transfected neurons. Means ± s.e.m. of typical experiments are shown (50–90 neurons per condition, **p < 0.001, statistical comparison with the respective controls). Sholl plots from representative experiments are shown. The non-normalized neurite length and branching datasets for all experiments carried out are provided in electronic supplementary material, spreadsheet S6.

Figure 7.

RANK is expressed in developing trigeminal and nodose neurons, and its activation inhibits neurotrophin-promoted neurite growth. (a,d) Relative levels of RANK mRNAs in total RNA extracted from (a) nodose and (d) trigeminal ganglia at the ages indicated. The levels of these mRNAs were normalized to the level of the reference GAPDH and SDHA mRNAs at each age. The mean ± s.e.m. of data obtained from at least three separate sets of dissected ganglia at each age are shown. (b,c) Sholl profiles of the neurite arbours of P1 nodose neurons incubated for 24 h with 10 ng ml⁻¹ BDNF and additionally (b) treated with 10 ng ml⁻¹ RANKL or (c) transfected with either an empty control plasmid or a RANK expression plasmid. (e,f) Sholl profiles of the neurite arbours of P1 trigeminal neurons incubated for 24 h with 10 ng ml⁻¹ NGF and additionally (e) treated with 10 ng ml⁻¹ RANKL or (f) transfected with either an empty control plasmid or a RANK expression plasmid. Sholl plots from representative experiments are shown. The datasets of all experiments carried out are provided in electronic supplementary material, spreadsheet S7. (b,e) Unfilled circles, control; filled circles, RANK. (c,f) Unfilled circles, control; filled circles, RANKL.