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. 2014 Mar 19;34(12):4371-81.
doi: 10.1523/JNEUROSCI.4943-13.2014.

Retrograde BMP signaling modulates rapid activity-dependent synaptic growth via presynaptic LIM kinase regulation of cofilin

Zachary D Piccioli  1 , J Troy Littleton
Affiliations

Retrograde BMP signaling modulates rapid activity-dependent synaptic growth via presynaptic LIM kinase regulation of cofilin

Zachary D Piccioli et al. J Neurosci. .

Abstract

The Drosophila neuromuscular junction (NMJ) is capable of rapidly budding new presynaptic varicosities over the course of minutes in response to elevated neuronal activity. Using live imaging of synaptic growth, we characterized this dynamic process and demonstrated that rapid bouton budding requires retrograde bone morphogenic protein (BMP) signaling and local alteration in the presynaptic actin cytoskeleton. BMP acts during development to provide competence for rapid synaptic growth by regulating the levels of the Rho-type guanine nucleotide exchange factor Trio, a transcriptional output of BMP-Smad signaling. In a parallel pathway, we find that the BMP type II receptor Wit signals through the effector protein LIM domain kinase 1 (Limk) to regulate bouton budding. Limk interfaces with structural plasticity by controlling the activity of the actin depolymerizing protein Cofilin. Expression of constitutively active or inactive Cofilin in motor neurons demonstrates that increased Cofilin activity promotes rapid bouton formation in response to elevated synaptic activity. Correspondingly, the overexpression of Limk, which inhibits Cofilin, inhibits bouton budding. Live imaging of the presynaptic F-actin cytoskeleton reveals that activity-dependent bouton addition is accompanied by the formation of new F-actin puncta at sites of synaptic growth. Pharmacological disruption of actin turnover inhibits bouton budding, indicating that local changes in the actin cytoskeleton at pre-existing boutons precede new budding events. We propose that developmental BMP signaling potentiates NMJs for rapid activity-dependent structural plasticity that is achieved by muscle release of retrograde signals that regulate local presynaptic actin cytoskeletal dynamics.

Keywords: BMP; Drosophila; actin; neuromuscular junction; synapse formation; synaptic plasticity.

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Figures

Figure 1.
Figure 1.
Rapid synaptic growth occurs spontaneously and in response to elevated activity. A, Live confocal imaging of a dissected larval NMJ presynaptically expressing mRFP-Syx13. The arrow indicates the site of a new bouton spontaneously budding and stabilizing (arrow) over the course of a minute. A second bouton can also be seen to emerge from the same budding site, but later collapses. Scale bar, 5 μm. B, Ghost boutons can be detected in fixed tissue by staining for the presynaptic neuronal membrane (anti-HRP, red) and the postsynaptic scaffold protein DLG (green), appearing as presynaptic varicosities that lack DLG staining. Scale bar, 11 μm. Arrowheads indicate ghost boutons. C, Histogram of ghost bouton frequency observed at unstimulated NMJs. N = 68 NMJs, 12 animals. D, Putative ghost boutons identified by morphology in a fixed preparation can display faint DLG accumulation (arrowheads), suggesting some newly formed varicosities are likely to be undergoing synaptic maturation. Scale bar, 5 μm.
Figure 2.
Figure 2.
Ghost bouton budding induced by high K+ stimulation is a rapid local signaling event. A, Live imaging of bouton budding (arrowheads) in response to 2 min incubations in high K+ spaced 10 min apart. New varicosity formation was visualized by presynaptic expression of membrane-tethered CD8-GFP. Scale bar, 7 μm. B, Quantification of ghost boutons in relation to existing bouton number at the NMJ following high K+ stimulation. N = 123 NMJs, 18 animals. C, New bouton budding (arrowheads) in response to high K+ stimulation is strongly dependent upon external Ca2+ but is not changed when axons are severed from motor neuron cell bodies. Scale bar, 14 μm. D, Quantification of ghost bouton budding detected by live imaging of animals presynaptically expressing membrane-tethered CD8-GFP at the indicated conditions. N (NMJs, animals): control = 38, 7; mock treated = 26, 4; 0 mm Ca2+ = 25, 4; 0.5 mm EGTA = 13, 4; axon cut = 21, 4. Error bars indicate SEM.
Figure 3.
Figure 3.
Ghost bouton budding requires normal synaptic transmission and local retrograde BMP signaling. A, Wandering third instar animals were fixed in formaldehyde after high K+ stimulation and were stained with anti-HRP and anti-DLG to identify ghost boutons. Loss of Syt1 and postsynaptic knockdown of GluRIIA and GluRIIB reduce activity-dependent budding. Likewise, loss of the postsynaptic Ca2+ sensor Syt4, or postsynaptic knockdown of Gbb with the muscle driver 24B-Gal4, reduces ghost bouton budding. Knockdown of Gbb with the muscle 6-specific H94-Gal4 preferentially reduces budding at muscle 6. Scale bar, 12 μm. Arrowheads indicate ghost boutons. B, Quantification of ghost boutons per NMJ in the indicated genetic backgrounds. N (NMJs, animals): wild-type = 57, 11; syt1AD4/N13 = 45, 6; 24B, gluRIIARNAi = 31, 4; 24B, gluRIIBRNAi = 29, 4; 24B, gbbRNAi = 20, 3; syt4BA1 = 65, 11; 24B, syt4RNAi = 3.548 ± 2.694, n = 31, 3; witA12/B11 = 37, 6. C, Quantification of baseline bouton number in the indicated genetic backgrounds. N, same as in B. D, The average number of ghost boutons that bud onto muscle 6 or muscle 7 is quantified for Gbb knockdown by the muscle 6-specific driver H94-Gal4. N (NMJs, animals): wild-type = 52, 7; H94, gbbRNAi = 40, 6. **p < 0.01; ***p < 0.001; ANOVA. Error bars indicate SEM.
Figure 4.
Figure 4.
Ghost bouton budding requires Smad signaling and is modulated by Limk activity. A, Wandering third instar larvae were fixed in formaldehyde after high K+ stimulation, and stained with anti-HRP and anti-DLG to identify ghost boutons. The overexpression of the inhibitory Smad dad causes synaptic undergrowth and a reduction in ghost bouton budding frequency. Trio protein levels correlate with ghost bouton budding frequency. Overexpression of full-length UAS-wit causes a reduction in bouton budding, while the overexpression of truncated UAS-witdCT does not. Motor neuron rescue with full-length UAS-wit did not completely rescue ghost bouton budding frequency, while rescue with UAS-witdCT rescued ghost bouton budding to a significantly greater extent. Scale bar, 12 μm. Arrowheads indicate ghost boutons. B, c164, UAS-trio animals were stimulated, fixed, and stained with anti-Trio antibody. Scale bar, 12 μm. Arrowheads indicate ghost boutons identified by morphology. C, Normalized fluorescence intensity of Trio antisera staining within ghost boutons was normalized to average fluorescence intensity of all other normal boutons at the same NMJ. N (ghost boutons, NMJs) = 77, 9. Solid line indicates mean; dashed line indicates the average normal bouton fluorescence intensity. D, Quantification of ghost boutons per NMJ in the indicated genetic background. N (NMJs, animals): wild-type = 57, 11; c164, dad = 70, 8; trioS137203 = 37, 5; c164, trio = 27, 4; witA12/B11 = 37, 6; wit rescue = 45, 7; witdCT rescue = 56, 7; c164, wit = 55, 8; c164 witdCT = 55, 8. E, Quantification of baseline bouton number. N, same as in D. F, Wit and Tsr show genetic interactions for defective ghost bouton budding. Quantification of ghost boutons per NMJ in the indicated genetic background is shown. N (NMJs, animals): wild-type = 57, 11; tsr1/+ = 24, 3; witB11/+ = 45, 6; tsr1/+;witB11/+ = 37, 5. *p < 0.05; **p < 0.01; ***p < 0.001; ANOVA. Error bars indicate SEM.
Figure 5.
Figure 5.
Ghost bouton budding is regulated by Limk and Cofilin activity. A, Presynaptic overexpression of limk strongly reduces activity-dependent bouton budding. Presynaptic overexpression of constitutively inactive tsrS3E reduces ghost bouton budding, while presynaptic overexpression of constitutively active tsrS3A increases ghost bouton budding above wild-type levels. Scale bar, 12 μm. Arrowheads indicate ghost boutons. B, Quantification of ghost bouton budding frequency in the indicated genetic background. N (NMJs, animals): wild-type = 57, 11; c164, limk = 40, 5; limkP1/Y = 36, 5; c164, tsrS3E = 28, 4; c164, tsrS3A = 36, 6. C, Quantification of baseline bouton number in the indicated genetic background. D, Live confocal imaging of GMA-GFP at NMJs driven by c164-Gal4. F-actin labeled by GMA appears as dynamic puncta with relatively even size and spacing in wild-type animals. GMA labeling in axons and extended interbouton regions is stable and uniform at wild-type NMJs (double arrow), but is interrupted by puncta and appears less uniform in tsrS3E and tsrS3E NMJs (double arrowheads). Boutons lacking discernable F-actin puncta occurred rarely at wild-type NMJs and more frequently at tsrS3E NMJs (arrows). Large and bright GMA labeling was observed in some boutons in tsrS3E and tsrS3A NMJs that was not observed in wild-type (arrowheads). Scale bar, 12 μm. *p < 0.05; **p < 0.01; ***p < 0.001; ANOVA. Error bars indicate SEM.
Figure 6.
Figure 6.
Ghost bouton budding is accompanied by local rearrangements of the presynaptic actin cytoskeleton. A, Animals presynaptically expressing the membrane marker CD8-RFP and the F-actin marker GMA-GFP were imaged before and after high K+ stimulation. New F-actin puncta (arrowheads) are observed at sites of budding (arrows) where newly formed ghost boutons (double arrowheads) attach to the main axonal arbor. Scale bar, 6 μm. B, Application of 10 μm latrunculin A to the bath solution rapidly disperses F-actin puncta, while application of 10 μm jasplakinolide causes formation and stabilization of F-actin puncta. Scale bar, 6 μm. C, Wild-type animals pretreated with latrunculin A or jasplakinolide for 15 min before high K+ stimulation display a reduction in ghost bouton budding frequency. N (NMJs, animals): no drug = 30, 4; latrunculin A = 35, 5; jasplakinolide = 30, 4. *p < 0.05; ***p < 0.001; ANOVA. Error bars indicate SEM.
Figure 7.
Figure 7.
Model for ghost bouton formation through parallel signaling pathways involving Gbb and Wit. BMP signaling through Wit is predicted to both promote and inhibit changes to the actin cytoskeleton that regulate ghost bouton formation. Gbb signaling developmentally through the phosphorylation and nuclear translocation of Mad potentiates synaptic terminals for activity-induced bouton budding by promoting transcription of the Rho GEF trio. Trio activity may also be regulated locally and acutely by synaptic activity. Wit also signals locally through Limk to inhibit Cofilin (Tsr), thereby suppressing ghost bouton formation.
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