BDNF signaling requires Matrix Metalloproteinase-9 during structural synaptic plasticity

  1. Diana Legutko
  2. Bożena Kuźniewska
  3. Katarzyna Kalita
  4. Ryohei Yasuda
  5. Leszek Kaczmarek
  6. Piotr Michaluk

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Abstract

Synaptic plasticity underlies learning and memory processes as well as contributes, in its aberrant form, to neuropsychiatric disorders. One of its major forms is structural long-term potentiation (sLTP), an activity-dependent growth of dendritic spines that harbor excitatory synapses. The process depends on the release of brain-derived neurotrophic factor (BDNF), and activation of its receptor, TrkB. Matrix metalloproteinase-9 (MMP-9), an extracellular protease is essential for many forms of neuronal plasticity engaged in physiological as well as pathological processes. Here, we utilized two-photon microscopy and two-photon glutamate uncaging to demonstrate that MMP-9 activity is essential for sLTP and is rapidly (∼seconds) released from dendritic spines in response to synaptic stimulation. Moreover, we show that either chemical or genetic inhibition of MMP-9 impairs TrkB activation, as measured by fluorescence lifetime imaging microscopy of FRET sensor. Furthermore, we provide evidence for a cell-free cleavage of proBDNF into mature BDNF by MMP-9. Our findings point to the autocrine mechanism of action of MMP-9 through BDNF maturation and TrkB activation during sLTP.

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    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In "BDNF signaling requires matrix metalloproteinase-9 during structural synaptic plasticity", Legutko et al. used two-photon microscopy and glutamate uncaging to show that rapid release (seconds) of MMP-9 from dendritic spines following synaptic stimulation as well as MMP-9 dependent activation of TrkB. The authors also show and MMP-dependent increase on dendritic spine volume. These data support the possibility that MMP-9 rapidly activates BDNF to promote the spine maturation required for LTP. All is all the manuscript is well written, and the data is convincing and important.

    Answer: We thank the reviewer for that comment.

    Questions/Concerns:

    • The authors show cell free cleavage of BDNF by recombinant MMP-9. It would be more convincing to show that MMP-9 cleaved BDNF using concentrated supernatants following synaptic stimulation in control versus inhibitor treated slices. Answer: In the present study we focus on a single-spine approach; thus, we did not include general stimulation techniques and biochemical analyses. To our knowledge, there is no method to show BDNF cleavage by MMP-9 directly at a single synapse. We agree with the reviewer that the general stimulation is important; however, at the synapse, there is potentially a whole array of proteases such as plasmin, tissue plasminogen activator (tPA) that might not only create catalytic cascade and proteolytically activate MMP-9 but also directly cleave proBDNF. When stimulating neurons and analysing supernatants, it is therefore impossible to determine if MMP-9 directly digests proBDNF to mBDNF or, alternatively, whether it is just a part of a proteolytic cascade leading to BDNF maturation. Therefore, our result where we use recombinant proteins provide an important piece of evidence that MMP-9 can indeed cleave proBDNF directly. Of note, experiments using brain extracts have been published previously, for example in a paper of Mizoguchi et al., J.Neurosci. (2011); DOI:10.1523/JNEUROSCI.3118-11.2011, where the authors showed increased cleavage of BDNF after pentylenetetrazole kindling and the kindling induced proBDNF cleavage was decreased in MMP-9 KO mice.

    • The concentration of the MMP-9/13 inhibitor used was quite high and would also inhibit MMP-1, -3 and -7. This concern is, however, abrogated by the use of the MMP-9 KO. But it might be important to mention that the inhibitor is not MMP-9 specific at higher concentrations. Answer: To comply with this remark, we have stressed the notion in the Discussion of the revised ms.:

    "There are over twenty MMPs with overlapping substrate specificity (Fields, 2015; Cieplak & Strongin, 2017) and there are no fully specific, commercially available inhibitors for MMP-9. Since Inhibitor I might affect also other MMPs, to further test the involvement of the protease in sLTP, we have used hippocampal slice cultures prepared from MMP-9 KO mice and their WT littermates (Fig. 1E, 1F)."

    • In figure 1C vs E, as well as Fig 3C vs E, it appears that the DMSO to inhibitor (1C and 3C) change is larger than the WT vs MMP-9 KO (1E and 3E). Is this possibly because DMSO has a potentiating effect and/or because the inhibitor is getting other MMPs or the MMP-9 KO has compensatory increases in other MMPs? __Answer: __At the concentrations used in the study (not exceeding 0.08%), we do not consider DMSO having any potentiating effect. As we discuss in the manuscript, the difference between DMSO control and MMP-9 WT is most likely due to differences between genetic lines of the mice. This is also a reason why each set of experiments has its own control. Of note, in the paper preceding this study, Harvard et al., Nature (2016); doi:10.1038/nature19766, spine volume change induced by uncaging, vary between 200 and 300% depending on mice strain used in the experiment.

    • The idea that MMP-9 and pro-BDNF are in the same vesicular stores is an interesting and very plausible one. Perhaps the authors could discuss what is known about the types of vesicles thought to harbor these two proteins. Answer: To follow on this remark, we added information about the vesicles containing BDNF and MMP-9 in the Discussion:

    "Given that the release kinetics of BDNF and MMP-9 are similar, one could speculate that the effect of MMP-9 inhibition on early TrkB activation can be achieved because both, MMP-9 and BDNF are co-localized and co-released from the same release vesicles. BDNF is widely considered to be stored and released from Large Dense-Core Vesicles (Dieni et al, 2012; Kojima et al, 2020), and MMP-9 release although not studied in neurons but in cell lines, also points to the same type of vesicles (Stephens et al, 2019)."

    • It might be useful to add to the discussion pathological conditions such as major depression and post-stroke plasticity in which MMP-9 dependent BDNF activation could be important. Answer: We thank the reviewer for that suggestion. We have added the information about MMP-9 and BDNF link in the brain pathologies in the Discussion:

    "Additionally, our data may provide a functional link between the involvement of MMP-9 and BDNF in various brain pathologies, in which such a link has previously been implicated, for example in addiction (Cheng et al, 2019), schizophrenia (Pan et al, 2022; Yamamori et al, 2013), ischemic stroke (Li et al, 2022) or even following cochlear implantation (Matusiak et al, 2023)."

    Reviewer #1 (Significance (Required)):

    The results are significant to understanding synaptic plasticity in health and disease.

    __Answer: __We thank the reviewer for that comment stressing the importance of our study.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The study addresses the molecular mechanisms of activity-dependent morphological plasticity of dendritic spines, focusing on the role of MMP-9 and BDNF-TrkB in the signalling and biochemical activities that lead to and maintain spine enlargement ('structural LTP', sLTP) induced experimentally.

    It is based on a combination of 2-photon imaging of spine morphology, 2-photon imaging of MMP-9-SEP fluorescence, 2-photon FLIM of a biosensor for TrkB activity and 2-photon glutamate uncaging in organotypic hippocampal brain slices. In addition, it includes an assay of protein digestion based on Western immuno blots.

    As results, the study reports that diminishing MMP-9 activity (pharmacologically or genetically) in the slices reduces sLTP, that repetitive glutamate uncaging evokes the release of MMP-9 from the spines that undergo sLTP, and that this effect can be blocked by pharmacological blockade of NMDA or exocytosis, that repetitive glutamate uncaging on a spine increases TrkB activation in the spine, and that this effect is diminished in slices from MMP-9 KO animals or treated by an MMP-9 blocker, and that MMP-9 can cleave proto-BDNF into mature BDNF in a cell-free medium.

    The experiments are technically challenging but they are well conceived, designed and executed. The conclusions are well supported by the results, which are clearly discussed in light of the substantial and somewhat contradictory literature.

    Reviewer #2 (Significance (Required)):

    The study provides a finer view of the dynamic role of MMP-9 in activity-dependent spine plasticity, reinforcing and expanding existing knowledge on this timely topic.

    The study is well executed and the conclusions are warranted. The study is an experimental tour de force, even if the biological results and insights are rather incremental and don't force us to revise our main assumptions or expectations.

    Answer: We thank the reviewer for that comment and the appreciation of our work.

    I only have a few questions and suggestions:

    • 2: Do TeTx and AP5 treatments also block spine enlargement? The MMP9-SEP and mCherry signals in the spines are going up, what about their ratio F/R? __Answer: __Yes, we do have results showing that TeTx and AP5 block spine enlargement, however we did not present them in the original manuscript. The AP5 application on spine enlargement was previously demonstrated for example by Tanaka and co-workers (2008); DOI: 10.1126/science.1152864, and the effect of TeTx on LTP and insertion of AMPA receptors has also been demonstrated multiple times for example by Penn et al., Nature (2017); doi:10.1038/nature23658. To comply with the reviewer's request we have included the data in the revised version of the manuscript (Figure 2C).

    As far as the F/R ratio is concerned we shall stress that the aim of our experiments was to show the release of MMP-9 into extracellular space upon uncaging. We have initially tried to analyse the ratio of F/R, however the green signal that comes from MMP9-SEP does not accumulate at the spine, apparently being rapidly diffused. Therefore, the overall red signal for mCherry increases much faster (mCherry fills the cytoplasm in the spine) than the MMP9-SEP; therefore, the F/R ratio is decreasing over time. Figure 2G shows that increases in MMP9-SEP fluorescence are only transient (around 0.5 s) after uncaging pulses.

    • 3B shows increased TrkB activation after glutamate uncaging, but is it possible to see the spine enlargement in the FRET-FLIM signal/images? Answer: Yes, it is possible to observe spine enlargement during FRET-FLIM experiments by counting photons from the red channel (RFP) as well as from the green one (GFP), however due to technical difficulties spine volume change was measured in separate experiments.

    • Fix: mW and Chameleon in the Methods section - corrected

    • Consider streamlining the Discussion a bit - we have reviewed the discussion

    • Consider adding a schematic to summarise the new and existing findings Answer: We thank the reviewer for the suggestion, we have added a schematic summarising the paper as a separate figure (Fig.4).

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In this short report, Legutko et al address the role of MMP9 in BDNF signaling in the context of structural long-term potentiation (sLTP). In particular, they assess whether MMP9 is secreted fast enough to mediate the cleavage of proBDNF in mBDNF during sLTP. The study uses 2-photon imaging of hippocampal organotypic slices, glutamate uncaging and FRET-based sensors of TrkB activity. The authors demonstrate that MMP9 is secreted within seconds upon 2-photon glutamate uncaging and that MMP9 secretion precedes spine enlargement. They also show that MMP9 can cleave proBDNF in vitro. However, the role of MMP9 in sLTP and associated TrkB signaling remains speculative at the end of the manuscript.

    Major comments

    • The title of the first result section "spine head enlargement during structural plasticity depends on MMP9 activity" is an overstatement. The authors provide evidence that MMP inhibition and MMP9 KO decrease spine enlargement during the early phase of sLTP. However, after the first few minutes, spines still display long-term enlargement, and no difference between WT and MMP9 KO mice can be detected. These data suggest that MMP9 is only involved in the initial phase of sLTP, and that other MMPs are involved in sLTP.

    __Answer: __We thank the reviewer for that comment. We have change the wording in the revised manuscript to accommodate the suggestion.

    • The authors cannot conclude that "spine head enlargement during sLTP depends on MMP9 activity".

    __Answer: __We thank the reviewer for that comment. We have changed the title and wording in the revised manuscript to accommodate the suggestion.

    • The authors should apply Inhibitor I on MMP9 KO slices to determine if MMPs other than MMP9 are involved in spine enlargement.

    __Answer: __We thank the reviewer for the suggestion, and indeed we agree that other MMPs might be involved in spine enlargement induced by glutamate uncaging. Furthermore, applying Inhibitor I will not resolve the question which MMPs or other proteases are involved in the spine enlargement. Applying Inhibitor I on MMP-9 KO slices would only eliminate one of the proteases. To deal with this difficult issue, we have used slices from MMP-9 KO mice and showed the influence of MMP-9 on the transient phase of spine enlargement induced by glutamate uncaging.

    • If Inhibitor I still impacts sLTP in MMP9 KO slice, it would greatly benefit this study to determine which MMPs are involved (for example by analyzing the expression patterns of MMPs in their neurons and selectively inactivating those expressed with shRNAs).

    Answer: The proposed experiment is an excellent suggestion for a future project however it is not an easy experiment to perform. MMPs expression pattern could be assessed by single cell RNA sequencing to distinguish it from for example astrocytic expression, however it often fails to detect mRNAs which are expressed at low level. For example mRNA coding MMP-9 belongs to this group as its mRNA is kept at very low level, see, e.e.g, Konopacki et al., Neuroscience (2007); https://doi.org/10.1016/j.neuroscience.2007.08.026, Dziembowska et al. J.Neurosci (2012); https://doi.org/10.1523/JNEUROSCI.6028-11.2012. There is also quite low correlation between mRNA levels and protein levels at a global scale, see e.g., Reimegård et al., Comm. Biol. (2021); https://doi.org/10.1038/s42003-021-02142-w, therefore predictive power of mRNA sequencing for the importance of a particular protein might not be sufficiently informative. Moreover, the situation is even more complex in neurons which are strongly compartmentalized, and where local translation plays a significant role. We have previously studied this particular aspect for MMP-9, Dziembowska et al. J.Neurosci. (2012); DOI:10.1523/JNEUROSCI.6028-11.2012..

    • The title of the third/last result section "TrkB signaling depends on MMP9 activity" is also an overstatement. In Figure 3, the authors show that the pharmacological inhibition of MMPs slightly inhibits TrkB signaling in the early phase of sLTP, and almost abolishes TrkB signaling in the second phase (> 3 min after uncaging). However, the data suggesting a specific role for MMP9 in TrkB signaling are not convincing (Figure 3E-F). The activation of trkB during sLTP is weak even in WT, the peak of trkB activation upon glutamate uncaging in not disrupted in MMP9 KO mice, and the data are noisy. It is a major concern that the authors cannot convincingly show that TrkB signaling is altered in MMP9-deficient neurons. Answer: To the best of our knowledge, using FRET-FLIM sensors is the best and state-of-the-art method to track biochemical changes (such as receptor activation) in real time using live preparations. The method is very sensitive and published previously by one of the authors of the current study where TrkB sensor is activated in the same magnitude (Harward et al., Nature, 2016; doi: 10.1038/nature19766). Moreover similar magnitude of sensor activation was reported previously in single dendritic spines for other sensors using FLIM-FRET method: Rho GTPases (Hedrick et al., Nature 2016; doi: 10.1038/nature19784), IGF1R (Tu et al., Sci Advanc. 2023; doi: 10.1126/sciadv.adg0666) or CaMKII (Chang et al., Nat. Commun. 2019; https://doi.org/10.1038/s41467-019-10694-z). The noise is to be expected, as we are imaging small compartments in a short time where collecting enough number of photons is challenging. Similarly to previous studies using FRET-FLIM sensors, we bin experimental points to reduce noise for statistical analysis. Notably, the biological effect we observe, namely sensor activation, is well above the experimental noise that in inevitable in this experimental approach. For statistical analyses we have used repeated measures ANOVA, which is very sensitive to noise and signal fluctuation. The differences we measure are statistically significant.
    • The authors discuss that the problem might stem from mouse genetic backgrounds. However, if the MMP9 KO mouse model is not appropriate to answer the question, the authors should use another one (i.e. MMP9 knockdown using sh/siRNAs).

    Answer: We believe that the effect of MMP-9 KO in this experiment is evident, as supported by Fig. 3 E,F and statistical analysis. Furthermore, the experiment with the inhibitor further supports our reasoning.

    • In addition to the graphs, the authors should mention in the text the percentage of inhibition compared to WT). This would make the results easier to read.

    Answer: To comply with this request the appropriate information has been added to the revised manuscript.

    • The change in TrkB activation following glutamate uncaging is low (max 5-7 % at the peak, compared to 200% for spine volume). This raises the question of the physiological relevance of TrkB activation in this model. The authors should include experiments with a trkB inhibitor to assess whether it prevents sLTP in WT and MMP9 KO mice. They should also discuss other potential targets of MMP9. This would strengthen the rationale of the experiments. Answer: Previously published results using the same TrkB sensor (Harward et al., Nature, 2016; doi: 10.1038/nature19766), show exactly the same change in binding fraction calculated from a change in GFP fluorescence lifetime. These data are also in agreement with well-established standard in the field, see, e.g., Rho GTPases (Hedrick et al., Nature 2016; doi: 10.1038/nature19784), IGF1R (Tu et al., Sci Advanc. 2023; doi: 10.1126/sciadv.adg0666) or CaMKII (Chang et al., Nat. Commun. 2019; https://doi.org/10.1038/s41467-019-10694-z). In response to the comment we have addressed this issue in the Discussion in the revised ms.

    Minor comments

    • In the introduction, the authors should provide more context. Could the authors develop the "long standing debate on which enzymes process proBDNF to mBDNF"? Answer: We have removed the sentence as we realized it was too confusing and the paper does not compare between different proteases which may process proBDNF to mBDNF.

    • In the result section:

    • First paragraph, the last sentence should be moved from the end of the paragraph to before "During sLTP induction...".

    Answer: Following the reviewer suggestion, we have moved the sentence.

    • Several paragraphs in the result section lack a proper conclusion/interpretation, which makes it difficult to read. Examples: after (Fig. 2E), after (Fig. 2F). The authors should explicit what their results mean.

    Answer: We have changed the paragraphs and tried to explain the results better.

    • Clarify when and for how long the MMP inhibitor was applied. Answer: The inhibitor was applied 30 min. before stimulation. We have added the information in the Methods section.

    • In figure 1, The authors observe a specific alteration of the early, transient, sustained increase in spine head volume in MMP9 KO mice. The later phase of sLTP is not impacted, which means that sLTP is induced and maintained in the KO. Could the authors discuss the role/importance of this transient peak in spine head volume? Answer: In response to this comment, we have discussed this issue in the revised ms. as follows:

    " The transient spine expansion might be important for the remodeling of the synapse (Lang et al, 2004) and is associated with NMDAR-dependent formation of "memory gel" created by enlargement pool of actin (Honkura et al, 2008; Kasai et al, 2010; Bonilla-Quintana & Rangamani, 2024). It has also been reported that TrkB activity can influence actin dynamics (Woo et al, 2019; Hedrick et al, 2016), in some instances in concert with integrin 1 (Wang et al, 2016), which is also activated by MMP-9 (Wang et al, 2008; Michaluk et al, 2009, 2011) and further supports our observations."

    Reviewer #3 (Significance (Required)):

    The manuscript aims to bring conceptual advance in our understanding of structural synaptic plasticity by investigating the role and timing of MMP9 secretion in TrkB signaling. Previous work from the Yasuda lab and others have shown that trkB is activated early on by BDNF during sLTP. However, how, when and where BDNF is cleaved from proBDNF in mBDNF is poorly understood. The authors demonstrate that the pharmacological inhibition of metalloproteases attenuates structural long-term plasticity (sLTP) and that MMP9 is secreted early enough to cleave proBDNF. They also show that MMP9 can cleave proBDNF in BDNF in vitro. Whether MMP9 specifically cleaves BDNF during sLTP and whether this cleavage is physiologically relevant for sLTP remain an open question.

    This report will be of interest to neurobiologists interested in the molecular mechanisms of synaptic plasticity.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    In this short report, Legutko et al address the role of MMP9 in BDNF signaling in the context of structural long-term potentiation (sLTP). In particular, they assess whether MMP9 is secreted fast enough to mediate the cleavage of proBDNF in mBDNF during sLTP. The study uses 2-photon imaging of hippocampal organotypic slices, glutamate uncaging and FRET-based sensors of TrkB activity. The authors demonstrate that MMP9 is secreted within seconds upon 2-photon glutamate uncaging and that MMP9 secretion precedes spine enlargement. They also show that MMP9 can cleave proBDNF in vitro. However, the role of MMP9 in sLTP and associated TrkB signaling remains speculative at the end of the manuscript.

    Major comments

    1. The title of the first result section "spine head enlargement during sLTP depends on MMP9 activity" is an overstatement. The authors provide evidence that MMP inhibition and MMP9 KO decrease spine enlargement during the early phase of sLTP. However, after the first few minutes, spines still display long-term enlargement, and no difference between WT and MMP9 KO mice can be detected. These data suggest that MMP9 is only involved in the initial phase of sLTP, and that other MMPs are involved in sLTP.
      • The authors cannot conclude that "spine head enlargement during sLTP depends on MMP9 activity".
      • The authors should apply Inhibitor I on MMP9 KO slices to determine if MMPs other than MMP9 are involved in spine enlargement.
      • If Inhibitor I still impacts sLTP in MMP9 KO slice, it would greatly benefit this study to determine which MMPs are involved (for example by analyzing the expression patterns of MMPs in their neurons and selectively inactivating those expressed with shRNAs).
    2. The title of the third/last result section "TrkB signaling depends on MMP9 activity" is also an overstatement. In Figure 3, the authors show that the pharmacological inhibition of MMPs slightly inhibits TrkB signaling in the early phase of sLTP, and almost abolishes TrkB signaling in the second phase (> 3 min after uncaging). However, the data suggesting a specific role for MMP9 in TrkB signaling are not convincing (Figure 3E-F). The activation of trkB during sLTP is weak even in WT, the peak of trkB activation upon glutamate uncaging in not disrupted in MMP9 KO mice, and the data are noisy. It is a major concern that the authors cannot convincingly show that TrkB signaling is altered in MMP9-deficient neurons.
      • The authors discuss that the problem might stem from mouse genetic backgrounds. However, if the MMP9 KO mouse model is not appropriate to answer the question, the authors should use another one (i.e. MMP9 knockdown using sh/siRNAs).
      • In addition to the graphs, the authors should mention in the text the percentage of inhibition compared to WT). This would make the results easier to read.
    3. The change in TrkB activation following glutamate uncaging is low (max 5-7 % at the peak, compared to 200% for spine volume). This raises the question of the physiological relevance of TrkB activation in this model. The authors should include experiments with a trkB inhibitor to assess whether it prevents sLTP in WT and MMP9 KO mice. They should also discuss other potential targets of MMP9. This would strengthen the rationale of the experiments.

    Minor comments

    1. In the introduction, the authors should provide more context. Could the authors develop the "long standing debate on which enzymes process proBDNF to mBDNF"?
    2. In the result section:
      • First paragraph, the last sentence should be moved from the end of the paragraph to before "During sLTP induction...".
      • Several paragraphs in the result section lack a proper conclusion/interpretation, which makes it difficult to read. Examples: after (Fig. 2E), after (Fig. 2F). The authors should explicit what their results mean.
    3. Clarify when and for how long the MMP inhibitor was applied.
    4. In figure 1, The authors observe a specific alteration of the early, transient, sustained increase in spine head volume in MMP9 KO mice. The later phase of sLTP is not impacted, which means that sLTP is induced and maintained in the KO. Could the authors discuss the role/importance of this transient peak in spine head volume?

    Significance

    The manuscript aims to bring conceptual advance in our understanding of structural synaptic plasticity by investigating the role and timing of MMP9 secretion in TrkB signaling. Previous work from the Yasuda lab and others have shown that trkB is activated early on by BDNF during sLTP. However, how, when and where BDNF is cleaved from proBDNF in mBDNF is poorly understood. The authors demonstrate that the pharmacological inhibition of metalloproteases attenuates structural long-term plasticity (sLTP) and that MMP9 is secreted early enough to cleave proBDNF. They also show that MMP9 can cleave proBDNF in BDNF in vitro. Whether MMP9 specifically cleaves BDNF during sLTP and whether this cleavage is physiologically relevant for sLTP remain an open question.

    This report will be of interest to neurobiologists interested in the molecular mechanisms of synaptic plasticity.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The study addresses the molecular mechanisms of activity-dependent morphological plasticity of dendritic spines, focusing on the role of MMP-9 and BDNF-TrkB in the signalling and biochemical activities that lead to and maintain spine enlargement ('structural LTP', sLTP) induced experimentally.

    It is based on a combination of 2-photon imaging of spine morphology, 2-photon imaging of MMP-9-SEP fluorescence, 2-photon FLIM of a biosensor for TrkB activity and 2-photon glutamate uncaging in organotypic hippocampal brain slices. In addition, it includes an assay of protein digestion based on Western immuno blots.

    As results, the study reports that diminishing MMP-9 activity (pharmacologically or genetically) in the slices reduces sLTP, that repetitive glutamate uncaging evokes the release of MMP-9 from the spines that undergo sLTP, and that this effect can be blocked by pharmacological blockade of NMDA or exocytosis, that repetitive glutamate uncaging on a spine increases TrkB activation in the spine, and that this effect is diminished in slices from MMP-9 KO animals or treated by an MMP-9 blocker, and that MMP-9 can cleave proto-BDNF into mature BDNF in a cell-free medium.

    The experiments are technically challenging but they are well conceived, designed and executed. The conclusions are well supported by the results, which are clearly discussed in light of the substantial and somewhat contradictory literature.

    Significance

    The study provides a finer view of the dynamic role of MMP-9 in activity-dependent spine plasticity, reinforcing and expanding existing knowledge on this timely topic.

    The study is well executed and the conclusions are warranted. The study is an experimental tour de force, even if the biological results and insights are rather incremental and don't force us to revise our main assumptions or expectations.

    I only have a few questions and suggestions:

    • Fig. 2: Do TeTx and AP5 treatments also block spine enlargement? The MMP9-SEP and mCherry signals in the spines are going up, what about their ratio F/R?
    • Fig. 3B shows increased TrkB activation after glutamate uncaging, but is it possible to see the spine enlargement in the FRET-FLIM signal/images?
    • Fix: mW and Chameleon in the Methods section
    • Consider streamlining the Discussion a bit
    • Consider adding a schematic to summarise the new and existing findings
  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In "BDNF signaling requires matrix metalloproteinase-9 during structural synaptic plasticity", Legutko et al. used two-photon microscopy and glutamate uncaging to show that rapid release (seconds) of MMP-9 from dendritic spines following synaptic stimulation as well as MMP-9 dependent activation of TrkB. The authors also show and MMP-dependent increase on dendritic spine volume. These data support the possibility that MMP-9 rapidly activates BDNF to promote the spine maturation required for LTP. All is all the manuscript is well written, and the data is convincing and important.

    Questions/Concerns:

    1. The authors show cell free cleavage of BDNF by recombinant MMP-9. It would be more convincing to show that MMP-9 cleaved BDNF using concentrated supernatants following synaptic stimulation in control versus inhibitor treated slices.
    2. The concentration of the MMP-9/13 inhibitor used was quite high and would also inhibit MMP-1, -3 and -7. This concern is, however, abrogated by the use of the MMP-9 KO. But it might be important to mention that the inhibitor is not MMP-9 specific at higher concentrations.
      1. In figure 1C vs E, as well as Fig 3C vs E, it appears that the DMSO to inhibitor (1C and 3C) change is larger than the WT vs MMP-9 KO (1E and 3E). Is this possibly because DMSO has a potentiating effect and/or because the inhibitor is getting other MMPs or the MMP-9 KO has compensatory increases in other MMPs?
    4. The idea that MMP-9 and pro-BDNF are in the same vesicular stores is an an interesting and very plausible one. Perhaps the authors could discuss what is known about the types of vesicles thought to harbor these two proteins.
    5. It might be useful to add to the discussion pathological conditions such as major depression and post-stroke plasticity in which MMP-9 dependent BDNF activation could be important.

    Significance

    The results are significant to understanding synaptic plasticity in health and disease.

  5. Version published to 10.1101/2023.12.08.569797v2 on bioRxiv
  6. Version published to 10.1101/2023.12.08.569797v1 on bioRxiv