Journal: bioRxiv

Article Title: Matrix Metalloproteinase-9 controls structural synaptic plasticity via BDNF-dependent signaling

doi: 10.1101/2023.12.08.569797

Figure Lengend Snippet: Spine-head enlargement during sLTP depends on MMP-9 activity. A Experimental arrangement: GFP-expressing neuron in CA1 subfield of hippocampus. Individual spine in secondary dendrite was stimulated using sLTP protocol leading to the spine enlargement. B Time-lapse z-integrated (z-stack) images of spines stimulated at time 0 min by sLTP protocol. Cross indicate a spot of two-photon uncaging, scale bar: 1 μm. C Averaged time courses of changes in spine volume (measured as ΔF/F 0 ) for spines stimulated by uncaging. Data are means ± SEM, DMSO (n = 50 spines; 27 cells, 15 animals), Inhibitor I (n = 46 spines; 21 cells, 10 animals). Grey box indicates duration of sLTP protocol. D Statistical analysis of (C); Averaged spine volume changes for three phases: 1-3 min. (transient), 9-11 and 15-20 min. (sustained). Gray dots represent individual values for spines, bars are means ± SEM (black – DMSO, red – Inhibitor I). Repeated measures ANOVA (Time (F (1.175, 110.5) = 33.60, p < 0.0001); Inhibitor (F (1, 94) = 18.63, p < 0.0001); TimelInhibitor (F (2, 188) = 0.6494, p = 0.5235)) followed by Šidák’s multiple comparison test (DMSO vs Inhibitor I at 1-3 min (p = 0.0350, 95% C.I. = [0.05124 to 1.863], 9-11 min (p = 0.0002, 95% C.I. = [0.3747 to 1.358]) and at 15-20 min (p = 0.0002, 95% C.I. = [0.2553 to 0.9569])). E as in (C), but for WT (n = 44 spines; 13 cells, 7 animals) and MMP-9 KO animals (n = 51 spines; 14 cells, 7 animals). F Statistical analysis of (E), marks as in (D). Bars are means ± SEM (black – WT, blue – MMP-9 KO). Repeated measures ANOVA (Time (F (1.463, 136.1) = 12.56, p < 0.0001); MMP-9 KO (F (1, 93) = 8.444, p = 0.0046); TimelMMP-9 KO (F (2, 186) = 3.253, p = 0.0409) followed by Šidák’s multiple comparison test (WT vs MMP-9 KO at 1-3 min (p = 0.0196, 95% C.I. = [0.08769 to 1.311]).

Article Snippet: pCAG_GFP, pCAG_mCherry, pCAG_MMP-9 – full length mouse (MMP-9) from pcDNA3.1_MMP-9-HA (Addgene #121172) cloned into pCAG vector, pCAG_MMP- 9a1205c - full length mouse MMP-9 with point mutation in single nucleotide 1205 from adenine to cytosine resulting in codon change E402A which results in lack of MMP-9 enzymatic activity.

Techniques: Activity Assay, Expressing, Comparison

Journal: bioRxiv

Article Title: Matrix Metalloproteinase-9 controls structural synaptic plasticity via BDNF-dependent signaling

doi: 10.1101/2023.12.08.569797

Figure Lengend Snippet: MMP-9 release during sLTP. A MMP9-SEP fluorescence is quenched by low (< 6.0) pH inside vesicles. Stimulation of the spine promotes exocytosis. Once the vesicle fuses with the cell membrane, pore opens and the acidic environment in the vesicle’s lumen is neutralized (pH 7.4) allowing for observation of fluorescence of SEP. B Time-lapse t-integrated (t-stack) images of spines stimulated by sLTP protocol – average of 10 consecutive frames collected after indicated uncaging pulses. Separate channels of MMP9- SEP and mCherry are shown. Crosses indicate a spot of two-photon uncaging, scale bar: 1 μm. C Averaged time courses of changes in volume (measured as ΔR/R 0 for mCherry channel) for spines stimulated by uncaging. Data are means ± SEM (n = 13 spines, 3 cells, 1 animal), timing of glutamate pulses during sLTP protocol are indicated by black bars. D Averaged time courses of changes in SEP fluorescence (measured as ΔF/F 0 ) for spines stimulated by uncaging. Data are means ± SEM (Control n = 13 spines, 3 cells, 1 animal; + AP5 n = 13 spines, 3 cells, 2 animals; + TeTx n = 12 spines, 1 cell, 1 animal), timing of glutamate uncaging laser pulses during sLTP protocol are indicated by black bars. E Heatmap representing averaged fluorescence of MMP9-SEP in stimulated spines (n=13). Each colored rectangle represents relative fluorescence intensity of SEP. Imaging data were divided into blocks (columns) of 16 frames matching uncaging frequency, so that uncaging pulse always occurs in a second frame of the block. sLTP protocol starts after recording baseline of 7 blocks (∼ 14 s) and lasts for 60 seconds (30 pulses, 0.5 Hz). White dots represent uncaging laser pulse which occurs always during the second frame in a block during sLTP- evoking protocol. F same as (E) but for MMP9-Gamillus. G Averaged time courses of changes in MMP9-SEP and corresponding mCherry fluorescence (measured as ΔF/F 0 ) for spines stimulated by uncaging. The plot represents superimposed and averaged blocks of 16 frames (2.048 s) for normalized fluorescence from 2 nd to 18 th uncaging pulse. Data are means ± SEM (n = 13 spines, 3 cells, 1 animal), timing of glutamate uncaging laser pulses during sLTP protocol are indicated by dashed vertical line at time = 0 s. H Peak of the uncaging-triggered change in MMP9-SEP and mCherry fluorescence (calculated as a mean of ΔF/F 0 for the three subsequent frames after uncaging pulse: 0.128- 0.384 s). Dots with lines represent values for individual spines for MMP9-SEP and mCherry fluorescence paired together. Bars are means. Paired t-test (t=3.149, df=12, p = 0.0084;). I Same as in (G) but for MMP9-Gamillus (n = 13 spines, 5 cells, 2 animals). J same as in (H) but for MMP9-Gamillus. Paired t-test (t=1.024, df=12, p = 0.3262).

Article Snippet: pCAG_GFP, pCAG_mCherry, pCAG_MMP-9 – full length mouse (MMP-9) from pcDNA3.1_MMP-9-HA (Addgene #121172) cloned into pCAG vector, pCAG_MMP- 9a1205c - full length mouse MMP-9 with point mutation in single nucleotide 1205 from adenine to cytosine resulting in codon change E402A which results in lack of MMP-9 enzymatic activity.

Techniques: Fluorescence, Membrane, Control, Imaging, Blocking Assay

Journal: bioRxiv

Article Title: Matrix Metalloproteinase-9 controls structural synaptic plasticity via BDNF-dependent signaling

doi: 10.1101/2023.12.08.569797

Figure Lengend Snippet: TrkB activation depends on MMP-9 activity. A TrkB FRET sensor schematic. B Two-photon FLIM images of TrkB activation averaged at indicated time points. Warmer colors represent shorter lifetimes, increased binding fraction and higher TrkB activity. White cross indicate uncaging spot, scale bar: 1 μm. C Averaged time courses of TrkB activation (measured as a change of the sensor binding fraction) for spines stimulated by uncaging. Data are means ± SEM. Grey box indicates duration of sLTP protocol. D Statistical analysis of (C). Averaged TrkB activation (measured as a Δ Binding Fraction) for transient phase (1-3 min after sLTP induction) and sustained phase (9-11 min. after sLTP induction) in spines incubated with DMSO (n = 70 spines, 29 cells, 16 animals) or Inhibitor I (n = 49 spines, 21 cells, 10 animals). Gray dots represent individual values for spines, bars (black – DMSO, red – Inhibitor I) are means ± SEM. Repeated measures ANOVA (Time (F (1, 117) = 11.46, p = 0.0010); Inhibitor (F (1, 117) = 10.80, p = 0.0013); Time=Inhibitor (F (1, 117) = 0.1103, p = 0.7403)) followed by Šidák’s multiple comparison test (DMSO vs Inhibitor I at 1-3 min (p = 0.0113, 95% C.I. = [0.005425 to 0.05049]) and at 9-11 min (p = 0.0045, 95% C.I. = [0.008394 to 0.05346])). E Same as in (C). Averaged time courses of TrkB activation observed in spines obtained from WT, and MMP-9 KO mice. F same as in (D). WT (black bars, n = 66 spines; 22 cells, 10 animals), MMP-9 KO (blue bars, n = 73 spines; 25 cells, 11 animals). Repeated measures ANOVA (Time F (1, 137) = 5.084, p = 0.0257; MMP-9 KO F (1, 137) = 7.982, p = 0.0054; Time=MMP-9 KO F (1, 137) = 0.1253, p = 0.7238)) followed by Šidák’s multiple comparison test (WT vs MMP-9 KO at 1-3 min (p = 0.0351, 95% C.I.=[0.001241 to 0.04204] and at 9-11 min (p = 0.0149, 95% C.I. = [0.004051 to 0.04485])). G Representative immunoblot of digestion reaction of proBDNF incubated with active MMP- 9, inactive MMP-9 (E402A), or only in the reaction buffer. Arrows indicate proBDNF (∼26 kDa) and mBDNF (∼14 kDa). H Quantification of three separate Western-blots of proBDNF digestion. Gray dots represent individual values of mBDNF band intensity in separate experiments and Western-blots for each experimental condition. Bars are means ± SEM. One-way ANOVA (F (2, 6) = 20.38, p = 0.0021) followed by Tukey’s multiple comparisons test (Buffer vs MMP-9 p = 0.0034, 95% C.I. = [-213821761 to -62305372] and E402A vs MMP-9 p = 0.0038, 95% C.I. = [59136932 to 210653321]).

Article Snippet: pCAG_GFP, pCAG_mCherry, pCAG_MMP-9 – full length mouse (MMP-9) from pcDNA3.1_MMP-9-HA (Addgene #121172) cloned into pCAG vector, pCAG_MMP- 9a1205c - full length mouse MMP-9 with point mutation in single nucleotide 1205 from adenine to cytosine resulting in codon change E402A which results in lack of MMP-9 enzymatic activity.

Techniques: Activation Assay, Activity Assay, Binding Assay, Incubation, Comparison, Western Blot