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Retrograde semaphorin–plexin signalling drives homeostatic synaptic plasticity

Abstract

Homeostatic signalling systems ensure stable but flexible neural activity and animal behaviour1,2,3,4. Presynaptic homeostatic plasticity is a conserved form of neuronal homeostatic signalling that is observed in organisms ranging from Drosophila to human1,5. Defining the underlying molecular mechanisms of neuronal homeostatic signalling will be essential in order to establish clear connections to the causes and progression of neurological disease. During neural development, semaphorin–plexin signalling instructs axon guidance and neuronal morphogenesis6,7,8,9,10. However, semaphorins and plexins are also expressed in the adult brain11,12,13,14,15,16. Here we show that semaphorin 2b (Sema2b) is a target-derived signal that acts upon presynaptic plexin B (PlexB) receptors to mediate the retrograde, homeostatic control of presynaptic neurotransmitter release at the neuromuscular junction in Drosophila. Further, we show that Sema2b–PlexB signalling regulates presynaptic homeostatic plasticity through the cytoplasmic protein Mical and the oxoreductase-dependent control of presynaptic actin. We propose that semaphorin–plexin signalling is an essential platform for the stabilization of synaptic transmission throughout the developing and mature nervous system. These findings may be relevant to the aetiology and treatment of diverse neurological and psychiatric diseases that are characterized by altered or inappropriate neural function and behaviour.

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Figure 1: sema2b and PlexB are necessary for PHP.
Figure 2: Sema2b and PlexB function as a retrograde trans-synaptic signal.
Figure 3: Altered NMJ growth with normal active-zone number and integrity.
Figure 4: Sema2b, PlexB and Mical control the homeostatic potentiation of the RRP.

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Acknowledgements

We thank S. Meltzer for consultation throughout and members of the Davis laboratory for comments on an earlier version of this manuscript. Supported by NIH Grant number R01NS39313 and R35NS097212 to G.W.D.

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Authors and Affiliations

Authors

Contributions

B.O.O. conducted all experiments and analyses, including genetics, electrophysiology and light microscopy experiments and wrote the text. R.D.F. performed electron microscopy. G.W.D. helped to analyse electron micrographs and wrote the text.

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Correspondence to Graeme W. Davis.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks H. J. Bellen, A. Chedotal and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Mutations in additional semaphorin and Plexin gene family members do not alter the rapid induction of PHP.

a, Gene diagrams indicating the mutations used in this study. Colours match scale bars. b, Quantification of the percentage change in mEPSPs (solid bars) and quantal content (open bars) in the presence of PhTx for the following genotypes: wild-type (w1118), sema1aK13702, sema1bEY21782, sema2aEY08184, sema5cMI10577 and PlexAEY1654. Each genotype was either previously described as, or predicted to be, a strong loss-of-function mutant: sema1aK13702 (ref. 17), sema1bEY21782(ref. 30), sema2aEY08184 (ref. 31), sema5cMI10577 (ref. 32) and PlexAEY1654 (ref. 30). All recordings were made in 0.3 mM [Ca2+]e. Data are mean ±s.e.m. *P < 0.05; **P < 0.01; two-tailed Student’s t-test.

Extended Data Figure 2 Evidence of sema2b gene expression in larval muscle.

a, Left, representative images of a sema2b-promoter–Gal4 fusion driving UAS-cd8-GFP at low magnification; images show multiple NMJs in the peripheral musculature, all expressing GFP. Muscles 4, 6 and 7 are labelled. Segmental boundaries are indicated by the horizontal lines and the middle segment is indicated as abdominal segment 3 (A3). Right, a higher magnification image taken of muscle 6/7, the muscles in which all recordings were made in this study, revealing expression of sema2b-promoter–Gal4. The NMJ is labelled with anti-Dlg (pink). Muscle identity is indicated. b, Images were taken at an identical exposure to those in a showing that there is no background GFP immunofluorescence in the absence of the UAS-cd8-GFP reporter as a control. Scale bars, 200 μm (left) and 10 μm (right). c, Data displayed as in a. Expression of sema2b-HA is controlled by endogenous promoter sequences. Scale bars, 200 μm (left) and 10 μm (right). d, Structured illumination, super-resolution microscopy was used to image Sema2b-HA expressed as in c. A single optical section (single plane) is shown revealing close proximity between Sema2b (green; anti-HA) and the presynaptic membrane (purple) labelled with anti-HRP. Scale bar, 2 μm.

Extended Data Figure 3 Effects of exogenous application of Sema2b protein on baseline transmission.

a, Raw data and analysis of additional control genotypes for the electrophysiological analysis of the effects of application of exogenous Sema2b protein (0.3 mM [Ca2+]e). Wild type (wt; n = 5), wt + sham (n = 12), wt + Sema2b (n = 23), PlexB + sham (n = 7), PlexB + Sema2B (n = 6). b, A silver-stained protein gel of supernatant collected from S2 cells transfected with both Actin-Gal4 and UAS:Sema2b-AP, or the Actin-Gal4 plasmid alone (sham). The red box highlights that the Sema2b-AP ligand is present at the correct size when both plasmids were transfected together, but absent when the Actin-Gal4 plasmid is transfected alone (sham). Bottom, BSA standards. c, Representative traces (0.3 mM [Ca2+]e). d, Raw data (0.2 mM [Ca2+]e) for indicated genotypes without (filled bars) and with (open bars) application of exogenous Sema2b protein (100 nM protein). Application of Sema2b protein causes a 40% increase in quantal content in controls (n = 6) and this effect is blocked in larvae that overexpress a PlexB dominant-negative transgene in motor neurons (OE MN PlexB DN) (control, n = 6; OE MN PlexB DN, n = 14). e, Effects of applying Sema2b protein to the rim103 mutant (compare rim baseline to rim with Sema2b). Experiments were performed in 0.4 mM [Ca2+]e to achieve comparable levels of absolute baseline vesicle release to the experiments shown in a. Sema2b protein has no effect on mEPSP amplitudes, but potentiates both the average EPSP and quantal content in rim103, demonstrating a significant (P < 0.05) potentiation of release. Sema2b protein rescues the blockade of PHP observed in the rim103-null mutant. Application of PhTx reduces mEPSP amplitudes in rim103 (P < 0.01) and there no significant (n.s.) increase in quantal content resulting in average EPSP amplitudes that are smaller than baseline. When Sema2b is co-applied with PhTx (rim + PhTx + Sema2b), the homeostatic potentiation of quantal content is significantly potentiated (P < 0.01) consistent with a rescue of PHP. (rim103 baseline, n = 6; rim103 + Sema2b, n = 13; rim103 + PhTx, n = 7; rim103 + PhTx + Sema2b n = 10). Data are mean ±s.e.m. *P < 0.05; **P < 0.01; two-tailed Student’s t-test.

Extended Data Figure 4 Genetic interactions.

a, Averaged mEPSP and quantal content in the absence and presence of PhTx for the indicated genotypes. Both genotypes (A and B) expressed the membrane-tethered UAS-sema2b-GFP (UAS-sema2bTM-GFP) in muscle (BG57-GAL4). Expression of membrane-tethered UAS-Sema2b-GFP has no deleterious effects on neurotransmission or the expression of PHP in control larvae with a heterozygous mutation in the sema2b gene (sema2b/+) (n = 8 without PhTx and n = 8 with PhTx; genotype A). Muscle expression of membrane-tethered UAS-sema2b-GFP in the sema2b homozygous mutant background failed to rescue PHP (n = 8, n = 9; genotype B). This is in contrast to the observation that expression of wild-type UAS-sema2b in muscle fully restores PHP in the sema2b mutant background (Fig. 1). We conclude that a membrane-tethered Sema2b protein is unable to signal to the presynaptic terminal without being secreted from the postsynaptic membranes. These data are consistent with Sema2b being a secreted ligand, originating in muscle, for the induction and expression of PHP. bd, Averaged mEPSPs and quantal content in the absence and presence of PhTx for the indicated genotypes. Heterozygous mutations in rim/+ (n = 8, n = 8; without and with PhTx, respectively), PlexB/+ (n = 9, n = 9) and micalK584/+ (n = 8, n = 8; note micalK584 shortened to micalK5 in the figure) show normal PHP following PhTx-dependent inhibition of mEPSP amplitudes. Double-heterozygous combinations of rim/+ with micalK584/+ (n = 9, n = 11) or PlexB/+ with micalK584/+ (n = 8, n = 13) results in complete blockade of PHP. These genetic interactions indicate that mical, rim and PlexB all participate in a common process that is directly required for PHP. Data are mean ±s.e.m. *P < 0.05; **P < 0.01; two-tailed Student’s t-test.

Extended Data Figure 5 Synaptic localization of Mical and Act5C.

a, Transgenic expression of UAS-Act5CM44L (middle; green in merge on the right) localizes throughout the presynaptic terminal marked with anti-HRP (left; magenta). Scale bar, 5 μm. b, The transgenic expression of UAS-mical-mCherry (green), used to rescue PHP in the mical mutant, localizes to the presynaptic boutons of motor neurons labelled with anti-HRP (magenta). Scale bar, 10 μm. c, Image of a Mical protein trap (see Methods) showing endogenous localization of Mical protein in the postsynaptic muscle and enrichment at the NMJ, which is labelled with anti-HRP (magenta). Projections in the z plane (yz or xz planes) indicate the presence of Mical protein within the presynaptic bouton of the protein trap. d, To selectively image presynaptic Mical–GFP in the protein-trap background, UAS-mical-RNAi was selectively expressed in muscle (BG57-GAL4) in the protein-trap background, greatly reducing muscle Mical–GFP and revealing strong presynaptic Mical–GFP originating from the protein trap at the endogenous gene locus. The NMJ is defined by anti-HRP (magenta, left) and by anti-Dlg (magenta, right).

Extended Data Figure 6 Representative data showing that homeostatic expansion of the RRP fails in sema2b and mical mutants and following expression of mutant UAS-Act5C.

Representative traces and graphs indicating cumulative EPSCs and back extrapolation from steady state (red line) for the indicated genotypes. a, Data are shown for wild type. b, Data are shown for sema2b. c, Data are shown for micalKG. d, Data are shown for larvae expressing UAS-DN-Act5C. We note that sema2b mutants have a baseline synaptic transmission defect, with a smaller baseline initial EPSC and correspondingly smaller RRP compared to both wild-type larvae and PlexB mutants (Fig. 4). Because there is no change in the number of active-zone-associated vesicles in sema2b mutants (Fig. 3), this defect must reflect a change in the allocation of vesicles to the RRP at baseline that parallels the failure to homeostatically potentiate the RRP during PHP. However, because PlexB mutants also block PHP without a change in baseline RRP, it seems unlikely that there is a causal link between reduced baseline RRP and failed PHP in the sema2b mutant.

Extended Data Figure 7 A schematic of retrograde, trans-synaptic Sema2b–PlexB signalling.

Signalling is schematized in the context of other mechanisms that have been recently demonstrated to be necessary for PHP. Sema2b–PlexB signalling (red) is a coherent trans-synaptic, retrograde signalling system that is conveyed, via Mical, to modify presynaptic actin and potentiate the readily releasable synaptic vesicle pool. Other genes have been shown to be necessary for PHP, but none can be connected into a coherent, trans-synaptic signalling cascade. In brown, presynaptic Deg/ENaC channels are inserted into the presynaptic plasma membrane, causing sodium leak and potentiation of presynaptic calcium influx through presynaptic calcium channels (CaV2.1)33. In blue, two components residing in the synaptic extracellular matrix have been implicated in PHP. The α2δ3 auxiliary subunit of the presynaptic calcium channel is necessary for PHP34. The matrix-derived signalling protein Endostatin, a cleavage product of the collagen homologue Multiplexin, is also necessary for PHP35. In orange, the innate immune receptor, peptidoglycan-recognition protein (PGRP), is essential for PHP29. Signalling downstream of PGRP is hypothesized to reach the neuronal nucleus and could thereby mediate the long-term maintenance of PHP. A major task for the future will be to define how these diverse signalling mechanisms participate in a coordinated response that rapidly, accurately and persistently regulates presynaptic neurotransmitter release following disruption of postsynaptic glutamate receptor function. P, phosphorylation; TF, transcription factor; Ub, ubiquitination.

Extended Data Table 1 Axon guidance defects for type 1s and type 1b motor neurons

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Supplementary Information

This file contains figure 1 which shows a silver stain gel shown in full, referring to Figure 2e-h and extended data figure 3b. It also contains table 1 which shows primary data inclusive of genotypes, experimental conditions, and figure/panel to which the data refer. (PDF 834 kb)

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Orr, B., Fetter, R. & Davis, G. Retrograde semaphorin–plexin signalling drives homeostatic synaptic plasticity. Nature 550, 109–113 (2017). https://doi.org/10.1038/nature24017

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