Yang F et al. (2009), Pro-BDNF-induced synaptic depression and retrac...

XB-ART-39972

J Cell Biol 2009 May 18;1854:727-41. doi: 10.1083/jcb.200811147.

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Pro-BDNF-induced synaptic depression and retraction at developing neuromuscular synapses.

Yang F , Je HS , Ji Y , Nagappan G , Hempstead B , Lu B .

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Postsynaptic cells generate positive and negative signals that retrogradely modulate presynaptic function. At developing neuromuscular synapses, prolonged stimulation of muscle cells induces sustained synaptic depression. We provide evidence that pro-brain-derived neurotrophic factor (BDNF) is a negative retrograde signal that can be converted into a positive signal by metalloproteases at the synaptic junctions. Application of pro-BDNF induces a dramatic decrease in synaptic efficacy followed by a retraction of presynaptic terminals, and these effects are mediated by presynaptic pan-neurotrophin receptor p75 (p75(NTR)), the pro-BDNF receptor. A brief stimulation of myocytes expressing cleavable or uncleavable pro-BDNF elicits synaptic potentiation or depression, respectively. Extracellular application of metalloprotease inhibitors, which inhibits the cleavage of endogenous pro-BDNF, facilitates the muscle stimulation-induced synaptic depression. Inhibition of presynaptic p75(NTR) or postsynaptic BDNF expression also blocks the activity-dependent synaptic depression and retraction. These results support a model in which postsynaptic secretion of a single molecule, pro-BDNF, may stabilize or eliminate presynaptic terminals depending on its proteolytic conversion at the synapses.

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Species referenced: Xenopus
Genes referenced: bdnf mmp3 ngfr
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	Figure 2. Pro-BDNF–mediated synaptic depression is mediated by presynaptic p75NTR. (A) Down-regulation of p75NTR by siRNA. p75NTR-specific siRNA and scrambled (Scrm.) analogues were introduced into the developing Xenopus by embryo injection. Neural tubes were dissected, and proteins were extracted. Western blots were performed using anti-p75NTR–specific antibody together with an antitubulin antibody for loading controls. Note a reduction of p75NTR expression in p75NTR siRNA–injected but not in uninjected (control [Ctl.]) or scrambled siRNA–injected embryos (left). Quantitative analysis of p75NTR expression in four independent experiments (right) is shown. The number in the bar indicates the number of Western blots performed. (B) Attenuation of pro-BDNF effect on SSCs by presynaptic expression of siRNA and its rescue by coexpression with rat p75NTR. p75NTR siRNA was expressed in presynaptic neurons (N+), postsynaptic myocytes (M+), or not at all (N−). Scrambled p75NTR siRNA was used as a control. In the last set of experiments, coexpression of p75NTR siRNA with rat p75NTR mRNA reversed the effect of p75NTR siRNA. (C) Effect of p75NTR siRNA on pro-BDNF–induced depression of ESCs. Amplitude (left) and delay of onset (right) of ESCs at control synapses (N−) and synapses expressing p75NTR siRNA (N+) are presented. The numbers in the bars indicate the number of synapses recorded. (A–C) Error bars represent SEM. *, P < 0.05.
	Figure 3. Pro-BDNF–induced synaptic retraction mediated by presynaptic p75NTR. (A) Time-lapse images showing the retraction of a nerve terminal from its target myocyte (M) after pro-BDNF treatment. The spinal neuron (N) is labeled with GFP (green), and AChRs on the postsynaptic membrane are labeled with rhodamine-conjugated α-BTX (red). Pro-BDNF was applied at time 0. Enlarged images of an area within the white box are shown at the left and were captured at the indicated times (shown in minutes) after pro-BDNF application. The GFP-labeled axonal terminal (yellow arrows) clearly occupied the synaptic site (colocalized with AChRs; yellow) before treatment but progressively withdrew from the junction after pro-BDNF treatment. Note that the cell body of the neuron remained healthy even after 2 h. (B) Time-lapse images showing that pro-BDNF failed to induce retraction of an axon of a neuron expressing p75NTR siRNA. Note that upon application of pro-BDNF, the terminals either did not retract (white arrows) or continued to extend further (white arrowheads). The inset is magnified to show more detailed images of the synapses. (C) Time course of synaptic retraction under different conditions. The number of experiments performed in each condition is indicated in parentheses. Error bars represent SEM. Scrm., scrambled. Bars, 10 µm.
	Figure 4. Simultaneous recordings of pro-BDNF–induced synaptic depression and imaging of terminal retraction. (top) Time-lapse images showing progressive retraction of a nerve terminal from a myocyte after pro-BDNF treatment. The dotted circle indicates the myocytes recorded; the dotted line indicates the recording pipette; the arrows indicate the axon terminal. (bottom) Simultaneous recording of SSC recording. The recording shows a rapid and persistent decrease in SSC frequency after pro-BDNF application. Note that synaptic depression precedes axonal retraction from its target myocyte. n = 4 independent experiments. Bar, 10 µm.
	Figure 5. Synaptic modulation elicited by stimulation of myocytes expressing cleavable and cleavage-resistant pro-BDNF. (A) Superimposed differential interference contrast and GFP fluorescence image showing a motor neuron (N) innervating a myocyte (M) expressing cleavage-resistant pro-BDNF (green). The dashed lines indicate the recording pipette. (B and C) Examples (left) and summary (right) of SSC recordings before and after stimulation of postsynaptic myocytes expressing cleavable (Clev.; B) and cleavage-resistant (uncleavable [Unclev.]; C) AA–pro-BDNF. Repetitive depolarization was applied to the postsynaptic myocytes through a whole-cell recording pipette (1 Hz for 30 s) under the current-clamp condition (Stim.). Note that at synapses expressing cleavable pro-BDNF, SSC frequency increased after postsynaptic depolarization. In contrast, at synapses expressing cleavage-resistant pro-BDNF, SSC frequency decreased after postsynaptic depolarization. The numbers in parentheses indicate the number of synapses recorded. (D) Quantitative analysis of amplitude (left), rise time (middle), and decay time (right) of SSCs before and after stimulation (Stim.) of postsynaptic myocytes expressing cleavable and cleavage-resistant pro-BDNF. The numbers in the bars indicate the number of synapses recorded. *, P < 0.05. (B–D) Error bars represent SEM. Bar, 10 µm.
	Figure 6. Synaptic depression induced by postsynaptic secretion of endogenous pro-BDNF, which is processed by MMP3 and MMP9. (A, top) A schematic diagram showing a synapse in which a spherical myocyte (M) is innervated by single spinal neurons (N). The dashed lines indicate the recording pipette. (bottom) The experimental protocol is shown. After baseline recording (10 min), protease inhibitors were applied for 10 min. Repetitive depolarization was applied to the postsynaptic myocytes through a whole-cell recording pipette (1 Hz for 30 s) under current-clamp condition, and synaptic efficacy was monitored. (B) Quantitative analysis of depolarization-induced depression caused by treatment with protease inhibitors (In.). Note that treatment with protease inhibitors alone or stimulation alone did not alter SSC frequency, but stimulation in the presence of protease inhibitors markedly reduced synaptic efficacy. (C) Specific MMPs involved in depolarization-induced synaptic depression. 50 nM MMP3 inhibitor, 50 µM MMP9 inhibitor, or both were applied to the cultures for at least 10 min. Synaptic depression was induced by postsynaptic stimulation (stim.) as described in Fig. 4. Note that postsynaptic stimulation in the presence of both MMP3 and MMP9 inhibitors but not either one alone triggered synaptic depression. (B and C) The numbers in the bars indicate the number of synapses recorded. Error bars represent SEM. *, P < 0.05.
	Figure 7. Synaptic depression induced by postsynaptic secretion of endogenous pro-BDNF, which activates presynaptic p75NTR. (A) Down-regulation of pro-BDNF by BDNF morpholino. Xenopus BDNF–specific morpholino (Morph.) and scrambled (Scrm.) analogues were introduced by embryo injection. Neural tubes were dissected, and proteins were extracted. Western blots were performed using anti-BDNF antibodies. The blot was reprobed with an antitubulin antibody for loading controls. Note a reduction of pro-BDNF expression in BDNF morpholino–injected but not in uninjected (control [Ctl.]) or scrambled morpholino–injected embryos (left). Quantitative analysis of pro-BDNF expression in four independent experiments (right) is shown. The numbers in the bars indicate the number of Western blots performed. (B) Effect of presynaptic (N+) or postsynaptic (M+) expression of BDNF morpholino on depolarization-induced depression. (C) Effect of presynaptic or postsynaptic expression of p75NTR siRNA on depolarization-induced depression. (B and C) The numbers in the bars indicate the number of synapses recorded. (A–C) Error bars represent SEM. *, P < 0.05. In., inhibitor; stim., stimulation.
	Figure 8. Synaptic retraction induced by postsynaptic secretion of endogenous pro-BDNF. Nerve terminals were labeled by rhodamine-dextran (red). The expression of BDNF morpholino (morph.) is indicated by the green fluorescence. Repetitive depolarization was applied to the postsynaptic myocytes (M) through a whole-cell recording pipette (2 Hz for 2 min) under current-clamp condition, and the axon terminal was visualized by time-lapse microscopy. (A) Synaptic retraction induced by repetitive postsynaptic stimulation. The axon was filled with rhodamine-dextran to visualize the axon terminal. After 30 min, the axon terminal from a stimulated myocyte retracted (indicated by yellow arrows). (B and C) Effect of presynaptic (B) or postsynaptic (C) expression of BDNF morpholino on depolarization-induced synaptic retraction. Postsynaptic expression of BDNF morpholino in myocytes blocked synaptic retracted induced by repetitive postsynaptic stimulation (indicated by white arrows). However, presynaptic expression of BDNF morpholino in spinal neurons could not block the depolarization-induced synaptic retraction (indicated by yellow arrows). The insets are magnified to show more detailed images of the synapses. (A–C) The dotted circles indicate myocytes recorded with a patch electrode. Bars, 10 µm.

References [+] :

Balice-Gordon, Gradual loss of synaptic cartels precedes axon withdrawal at developing neuromuscular junctions. 1993, Pubmed