Role of pro-brain-derived neurotrophic factor (proBDNF) to mature BDNF conversion in activity-dependent competition at developing neuromuscular synapses

Abstract

Formation of specific neuronal connections often involves competition between adjacent axons, leading to stabilization of the active terminal, while retraction of the less active ones. The underlying molecular mechanisms remain unknown. We show that activity-dependent conversion of pro-brain-derived neurotrophic factor (proBDNF) to mature (m)BDNF mediates synaptic competition. Stimulation of motoneurons triggers proteolytic conversion of proBDNF to mBDNF at nerve terminals. In Xenopus nerve-muscle cocultures, in which two motoneurons innervate one myocyte, proBDNF-p75(NTR) signaling promotes retraction of the less active terminal, whereas mBDNF-tyrosine-related kinase B (TrkB) p75NTR (p75 neurotrophin receptor) facilitates stabilization of the active one. Thus, proBDNF and mBDNF may serve as potential "punishment" and "reward" signals for inactive and active terminals, respectively, and activity-dependent conversion of proBDNF to mBDNF may regulate synapse elimination.

Fig. 1.

Activity-dependent synaptic competition in culture. (A) Schematic diagram (Left) and confocal image (Right) showing a triplet in which a spherical myocyte (M) (indicated by white dotted lines) is innervated by two spinal neurons (N), one labeled with FITC (green) and one labeled in rhodamine (red), in nerve–muscle coculture. (B) Time course of synaptic competition. A higher magnification of A is shown. Stimulation of the red neuron, by photo-uncaging of MNI-glutamate in the soma area using a two-photon laser, caused the unstimulated (green) axon terminal to retract from the synaptic target (indicated by a yellow arrow, upper row). In contrast, the axon terminal (red) from the stimulated neuron did not retract, but elongated a little (white arrows, middle row). The phase and two-color fluorescence images of the triplet at multiple time points (lower row). (Scale bar: 10 μm.) The phase (Bottom) and two-color fluorescence images (Top and Middle) of the triplet at “0” and “120” min are shown. (C) Quantification of axonal retraction and elongation measured 120 min after stimulation of one of the neurons in the triplets. *P < 0.01.

Fig. 2.

Activity-dependent cleavage of proBDNF detected by fluorogenic probes. (A) Schematic diagram depicting the design of a fluorogenic indicator of proBDNF cleavage. A peptide containing the proBDNF cleavage site was placed between a fluorophore and a quencher. Upon proteolytic cleavage, the quencher was dissociated from the fluorophore, leading to emission of fluorescence. (B) Sample images of fluorogenic probes in phase (Left) and fluorescence (Right) before and after neuronal stimulation. Polystyrene beads soaked with fluorogenic probes were positioned on (yellow arrows) or near (white arrows) an axonal process (indicated by white dotted lines), respectively, by micromanipulation. A neuronal cell body was stimulated by photolysis of caged-glutamate (MNI-glutamate) with a UV laser. Note that after stimulation, only beads on, but not near, the axonal processes showed an increase in fluorescence. (C) Time course of cleavage-dependent increase in fluorescence intensity upon stimulation of spinal neurons. Fluorescence intensities measured on contacted beads or free beads were normalized to that at “0” time point and presented as ratios (F/F₀) over time. (D) Quantification of relative fluorescence intensities in beads 30 min after stimulation of neurons under various conditions. Concentrations of protease inhibitors (In.): general protease inhibitor mixture, 50 μM; pan MMP inhibitor, 60 μM; tPA inhibitor, 10 μM; furin inhibitor, 40 μM; MMP2 inhibitor, 60 μM; MMP3 inhibitor, 50 nM; MMP8 inhibitor, 20 nM; MMP9 inhibitor, 50 μM; MMP13 inhibitor, 80 nM. B, beads; M, muscle cell; N, neuron.

Fig. 3.

Synaptic stabilization mediated by mBDNF/TrkB. (A) Down-regulation of TrkB by TrkB-morpholino (TrkB-morp.) led to retraction of both stimulated and unstimulated axon terminals. In a triplet system, stimulation of a neuron expressing TrkB morpholino (green) resulted in retraction of the red axon as well as the green axon. (Scale bar: 10 μm.) (B) Treatment with mBDNF prevented activity-dependent synaptic retraction. The culture was treated with mBDNF before stimulating the soma of a red neuron. The terminals from both red and green neurons remained in the synaptic site without any retraction. (Scale bar: 20 μm.) (C) Quantification of axonal retraction and elongation measured 120 min after stimulation of one of the neurons in the triplets.

Fig. 4.

Synaptic retraction mediated by proBDNF/p75^NTR. (A) Down-regulation of p75^NTR by siRNA prevents activity-dependent synaptic retraction. Fluorescence images show a double-innervated myocyte (white dotted lines) by a neuron expressing p75^NTR siRNAs (green) and a rhodamine-labeled control neuron (red). The soma of the red neuron (outside the field) was stimulated by photo-uncaging and the axon terminals from both neurons were monitored by time-lapse microscopy. (Scale bar: 20 μm.) Even after 120 min, the axon terminals (white arrow) of both red and green neurons remained unchanged at the synaptic site. (B) Time-lapse images showing retraction of both stimulated (red) and unstimulated (green) axons in the presence of a mixture of protease inhibitors (50 μM). (Scale bar: 20 μm.) (C) Quantification of axonal retraction and elongation measured 120 min after stimulation of one of the neurons in the triplets.