Research ArticleFRAGILE X SYNDROME

Neuronal activity drives FMRP- and HSPG-dependent matrix metalloproteinase function required for rapid synaptogenesis

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Sci. Signal.  07 Nov 2017:
Vol. 10, Issue 504, eaan3181
DOI: 10.1126/scisignal.aan3181
  • Fig. 1 Mmp1 mediates rapid, activity-dependent synaptic bouton formation.

    (A) NMJs colabeled for HRP and DLG after dTRPA1 stimulation in the indicated genotypes. White asterisks mark ghost boutons. Scale bar, 5 μm. Higher-magnification images of synaptic boutons are shown below. Scale bar, 2 μm. (B) Quantified ghost bouton number per terminal after dTRPA1 stimulation: vglut-Gal4/+ [number of NMJs (n) = 28; 1.54 ± 0.30], vglut-Gal4>UAS-dTRPA1 (n = 20; 7.4 ± 0.97), vglut-Gal4, mmp1Q112*/mmp1Q112* (n = 24; 1.54 ± 0.34), and vglut-Gal4>UAS-dTRPA1, mmp1Q112*/mmp1Q112* (n = 24; 1.38 ± 0.33). Data are means ± SEM from three replicates. ***P < 0.001 by nonparametric analysis of variance (ANOVA) (Kruskal-Wallis) and Dunn’s multiple comparison posttest. Nonsignificant (P > 0.05) comparisons are not represented for (i) vglut-Gal4/+ versus vglut-Gal4, mmp1Q112*/mmp1Q112*; (ii) vglut-Gal4/+ versus vglut-Gal4>UAS-dTRPA1, mmp1Q112*/mmp1Q112*; and (iii) vglut-Gal4, mmp1Q112*/mmp1Q112* versus vglut-Gal4>UAS-dTRPA1, mmp1Q112*/mmp1Q112*. Temperature controls are shown in fig. S1.

  • Fig. 2 Synaptic Mmp1 is rapidly increased after acute neuronal stimulation.

    (A) Images of NMJs after the denoted stimulation colabeled with HRP and Mmp1 in the indicated genotypes. Heat map shows Mmp1 alone (scale below). White outline, HRP bouton. Scale bars, 2 μm. (B) Quantification of synaptic Mmp1 intensity normalized to matched controls in (i) vglut-Gal4/+ (n = 10; 1.0 ± 0.07) versus vglut-Gal4>UAS-dTRPA1 (n = 13; 1.42 ± 0.1) and (ii) w1118 control (unstimulated, n = 56; 1.0 ± 0.03) versus w1118 stimulated (high [K+], n = 57; 1.54 ± 0.07). **P < 0.01, ***P < 0.001 by unpaired t test with Welch correction (dTRPA1) or Mann-Whitney U test (high [K+]). Data are means ± SEM from three replicates for each experiment. Additional controls are shown in fig. S3.

  • Fig. 3 Mmp1 and Dlp colocalization in synaptic subdomains is increased by acute activity.

    (A) Images of Dlp::GFP NMJs labeled with HRP, GFP, and Mmp1 under basal conditions or after high [K+] stimulation. Dlp::GFP and Mmp1 signals are shown as a heat map with HRP synaptic outlines in white. Arrows indicate overlapping Dlp::GFP and Mmp1 signals. Scale bar, 5 μm. Higher-magnification images of single optical sections are shown below. Scale bar, 1 μm. Asterisks denote overlapping Dlp::GFP and Mmp1, shown pseudocolored in white in the rightmost images. (B) Quantification of both Dlp::GFP and Mmp1 fluorescence intensities normalized to unstimulated controls. (i) “Dlp::GFP”: control (n = 20; 1.0 ± 0.06) versus stimulated (n = 19; 1.48 ± 0.09). (ii) “Mmp1”: control (n = 20; 1.0 ± 0.05) versus stimulated (n = 19; 1.56 ± 0.14). ***P < 0.001 by unpaired t test with Welch correction. (C) Quantification of MCCs. (i) MA (Mmp1/Dlp::GFP) in basal control (n = 27; 0.33 ± 0.03) and after stimulation (n = 27; 0.72 ± 0.03) and (ii) MB (Dlp::GFP/Mmp1) in basal control (n = 27; 0.37 ± 0.03) and after stimulation (n = 27; 0.59 ± 0.03). ***P < 0.001 by unpaired t test (MA) or Mann-Whitney U test (MB). Data are means ± SEM from three replicates. Additional controls are shown in figs. S5 and S6.

  • Fig. 4 Synaptic Dlp positively and bidirectionally regulates secreted Mmp1 abundance.

    (A and B) Images of NMJs (A) and quantification of Mmp1 fluorescence intensity (B) from the denoted dlp reduction conditions compared to matched controls colabeled with HRP and Mmp1. Mmp1 signal intensity is shown as a heat map with HRP synaptic outlines in white. Scale bars, 2 μm. Fluorescence intensity was normalized to that from matched genetic controls: (i) w1118 (n = 34; 1.0 ± 0.04) versus dlpA187/+ (n = 37; 0.67 ± 0.04), and (ii) elav-Gal4,24B-Gal4/+ control (n = 28; 1.0 ± 0.07) versus elav-Gal4,24B-Gal4>UAS-dlpRNAi knockdown (n = 32; 0.77 ± 0.05). *P < 0.05, ***P < 0.001 by Mann-Whitney U test. (C and D) As in (A) and (B), but from the denoted dlp overexpression conditions. Scale bar, 2 μm. 24B-Gal4/+ (n = 51; 1.0 ± 0.03), 24B-Gal4>UAS-dlpWT (n = 42; 1.55 ± 0.09), and 24B-Gal4>UAS-dlp−HS (n = 34, 1.13 ± 0.06). ***P < 0.001 by nonparametric ANOVA (Kruskal-Wallis) with Dunn’s multiple comparison posttest. The nonsignificant (P > 0.05) comparison for 24B-Gal4/+ versus 24B-Gal4>UAS-dlp−HS is not shown. Data are means ± SEM from at least three independent replicates for each experiment. Abundance of Dlp in each of the genetic manipulations is shown in fig. S7.

  • Fig. 5 Dlp mediates activity-dependent regulation of synaptic Mmp1.

    (A and B) Images of NMJs (A) and quantification of Mmp1 fluorescence intensity (B) from the indicated dlp reduction conditions treated with or without high [K+] and colabeled for HRP and Mmp1. Mmp1 signal intensity is shown as a heat map with HRP synaptic outlines in white. Scale bar, 2 μm. Quantified Mmp1 fluorescence intensity from stimulated conditions (bar graphs) was normalized to unstimulated controls (red lines). (i) w1118 control (n = 17; 1.0 ± 0.08) versus w1118 stimulated (n = 19; 1.34 ± 0.06); (ii) dlpA187/+ control (n = 19; 1.0 ± 0.03) versus dlpA187/+ stimulated (n = 21; 0.85 ± 0.06); (iii) elav-Gal4,24B-Gal4/+ control (n = 26; 1.0 ± 0.04) versus elav-Gal4,24B-Gal4/+ stimulated (n = 24; 1.34 ± 0.06); and (iv) elav-Gal4,24B-Gal4>UAS-dlpRNAi control (n = 25; 1.0 ± 0.06) versus elav-Gal4,24B-Gal4>UAS-dlpRNAi stimulated (n = 18; 0.88 ± 0.06). (C and D) As in (A) and (B), but from the dlp overexpression conditions. Scale bar, 2 μm. (i) 24B-Gal4/+ control (n = 21; 1.0 ± 0.06) versus 24B-Gal4/+ stimulated (n = 18; 1.39 ± 0.07); (ii) 24B-Gal4>UAS-dlpWT control (n = 21; 1.0 ± 0.03) versus 24B-Gal4>UAS-dlpWT stimulated (n = 16; 1.64 ± 0.16); and (iii) 24B-Gal4>UAS-dlp−HS control (n = 23; 1.0 ± 0.05) versus 24B-Gal4>UAS-dlp−HS stimulated (n = 13; 1.19 ± 0.08). (B and D) For stimulated versus unstimulated pairwise comparisons (red asterisks), significance was determined by Mann-Whitney U tests, and significance across genotypes (black) was determined by unpaired t tests (B) or nonparametric ANOVA (Kruskal-Wallis; D) with Dunn’s multiple comparison posttest, as indicated by *P < 0.05, **P < 0.01, and ***P < 0.001. Nonsignificant (P > 0.05) comparisons for (i) stimulated versus unstimulated elav-Gal4,24B-Gal4>UAS-dlpRNAi (B), (ii) stimulated 24B-Gal4/+ versus stimulated 24B-Gal4>UAS-dlpWT (D), and (iii) stimulated 24B-Gal4/+ versus stimulated 24B-Gal4>UAS-dlp−HS (D) are not shown. Data are means ± SEM from three independent replicates.

  • Fig. 6 Dlp positively and bidirectionally regulates proteolytic activity at the synapse.

    (A) Images of NMJs from the indicated dlp reduction and overexpression conditions colabeled for HRP (red) and in situ zymography activity (green) compared to matched genetic controls. Gelatinase activity is shown as a heat map with HRP synaptic outlines in white. Scale bar, 2 μm. (B) Quantified in situ zymography fluorescence intensity normalized to controls for the dlp reduction: w1118 control (n = 35; 1.0 ± 0.06) and dlpA187/+ heterozygote (n = 33; 0.66 ± 0.04). Significance was determined by unpaired t test with Welch correction, as indicated by ***P < 0.0001. (C) Quantified in situ zymography fluorescence intensity normalized to controls for the dlp overexpression conditions: 24B-Gal4/+ control (n = 35; 1.0 ± 0.05), 24B-Gal4>UAS-dlpWT (n = 18; 1.5 ± 0.09), and 24B-Gal4>UAS-dlp−HS (n = 20; 1.6 ± 0.18). **P < 0.01 by nonparametric ANOVA (Kruskal-Wallis) and Dunn’s multiple comparison posttest. Data are means ± SEM from three independent replicates.

  • Fig. 7 FMRP regulation of the activity-dependent Mmp1 enhancement requires Dlp.

    (A and B) Images of NMJs (A) and quantification of Mmp1 fluorescence intensity (B) from the denoted genotypes colabeled with HRP and Mmp1. Mmp1 intensity is shown as a heat map; white outlines mark synaptic HRP. Scale bar, 2 μm. Quantification of Mmp1 fluorescence intensity was normalized to w1118 control (n = 49; 1.0 ± 0.03); dfmr150M/50M (n = 35; 1.57 ± 0.12); and dlpA187/+, dfmr150M/50M (n = 22; 1.07 ± 0.07). (C and D) As in (A) and (B), but treated with or without high [K+]. Scale bar, 2 μm. Red line, unstimulated control. (i) w1118 control (unstimulated, n = 13; 1.0 ± 0.04) versus w1118 stimulated (n = 15; 1.51 ± 0.1); (ii) dfmr150M/50M control (unstimulated, n = 15; 1.0 ± 0.07) versus dfmr150M/50M stimulated (n = 13; 1.02 ± 0.11); and (iii) dlpA187/+, dfmr150M/50M control (unstimulated, n = 14; 1.0 ± 0.04) versus dlpA187/+, dfmr150M/50M stimulated (n = 17; 1.55 ± 0.16). For stimulated versus unstimulated pairwise comparisons, significance was determined by Mann-Whitney U tests, as indicated by **P < 0.01 and ***P < 0.001 (red asterisks). In (B) and across stimulated genotypes in (D), significance was determined by nonparametric ANOVA (Kruskal-Wallis) with Dunn’s multiple comparison posttest, as indicated by *P < 0.05, **P < 0.01, and ***P < 0.001 (black asterisks). Nonsignificant (P > 0.05) comparisons for (i) w1118 versus dlpA187/+, dfmr150M/50M (B), (ii) stimulated versus unstimulated dfmr150M/50M (D), and (iii) stimulated w1118 versus stimulated dlpA187/+, dfmr150M/50M (D) are not shown. Data are means ± SEM from at least three independent replicates.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/504/eaan3181/DC1

    Fig. S1. Temperature controls for dTRPA1 activity-induced synaptic bouton formation.

    Fig. S2. Mmp2 is not required for activity-dependent synaptic bouton formation.

    Fig. S3. Mmp1 is rapidly and specifically increased after dTRPA1 neuronal stimulation.

    Fig. S4. Synaptic Mmp2 is rapidly reduced after acute neuronal stimulation.

    Fig. S5. Synaptic Dlp is rapidly increased after acute neuronal stimulation.

    Fig. S6. Synaptic Mmp1 and Dlp imaging controls at the NMJ terminal.

    Fig. S7. Synaptic Dlp changes with bidirectional dlp genetic manipulations.

    Fig. S8. Synaptic Mmp2 changes with bidirectional dlp genetic manipulations.

    Fig. S9. Activity-dependent synaptic Dlp increase occurs in the absence of Mmp1.

    Fig. S10. Synaptic Dlp in FXS disease model is restored by single-copy dlp coremoval.

  • Supplementary Materials for:

    Neuronal activity drives FMRP- and HSPG-dependent matrix metalloproteinase function required for rapid synaptogenesis

    Mary L. Dear, Jarrod Shilts, Kendal Broadie*

    *Corresponding author. Email: kendal.broadie{at}vanderbilt.edu

    This PDF file includes:

    • Fig. S1. Temperature controls for dTRPA1 activity-induced synaptic bouton formation.
    • Fig. S2. Mmp2 is not required for activity-dependent synaptic bouton formation.
    • Fig. S3. Mmp1 is rapidly and specifically increased after dTRPA1 neuronal stimulation.
    • Fig. S4. Synaptic Mmp2 is rapidly reduced after acute neuronal stimulation.
    • Fig. S5. Synaptic Dlp is rapidly increased after acute neuronal stimulation.
    • Fig. S6. Synaptic Mmp1 and Dlp imaging controls at the NMJ terminal.
    • Fig. S7. Synaptic Dlp changes with bidirectional dlp genetic manipulations.
    • Fig. S8. Synaptic Mmp2 changes with bidirectional dlp genetic manipulations.
    • Fig. S9. Activity-dependent synaptic Dlp increase occurs in the absence of Mmp1.
    • Fig. S10. Synaptic Dlp in FXS disease model is restored by single-copy dlp coremoval.

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    Citation: M. L. Dear, J. Shilts, K. Broadie, Neuronal activity drives FMRP- and HSPG-dependent matrix metalloproteinase function required for rapid synaptogenesis. Sci. Signal. 10, eaan3181 (2017).

    © 2017 American Association for the Advancement of Science

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