Role of matrix metalloproteinase-9 in neurodevelopmental deficits and experience-dependent plasticity in Xenopus laevis

  1. Sayali V Gore
  2. Eric J James
  3. Lin-chien Huang
  4. Jenn J Park
  5. Andrea Berghella
  6. Adrian C Thompson
  7. Hollis T Cline
  8. Carlos D Aizenman

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Abstract

Matrix metalloproteinase-9 (MMP-9) is a secreted endopeptidase targeting extracellular matrix proteins, creating permissive environments for neuronal development and plasticity. Developmental dysregulation of MMP-9 may also lead to neurodevelopmental disorders (ND). Here, we test the hypothesis that chronically elevated MMP-9 activity during early neurodevelopment is responsible for neural circuit hyperconnectivity observed in Xenopus tadpoles after early exposure to valproic acid (VPA), a known teratogen associated with ND in humans. In Xenopus tadpoles, VPA exposure results in excess local synaptic connectivity, disrupted social behavior and increased seizure susceptibility. We found that overexpressing MMP-9 in the brain copies effects of VPA on synaptic connectivity, and blocking MMP-9 activity pharmacologically or genetically reverses effects of VPA on physiology and behavior. We further show that during normal neurodevelopment MMP-9 levels are tightly regulated by neuronal activity and required for structural plasticity. These studies show a critical role for MMP-9 in both normal and abnormal development.

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  1. Version published to 10.7554/elife.62147 on eLife
  2. eLife

    Reviewer #3:

    The Aizenman lab has previously demonstrated the utility of Xenopus tectum as a model to examine neuronal, circuit and behavioral manifestations of VPA treatment, a teratogen associated with autism spectrum disorder in humans. In Gore et al., they demonstrate that the deficits induced by VPA treatment, including enhanced spontaneous and evoked neuronal activity, are blocked by pharmacological or morpholino based inhibition of MMP9. Inhibition of MMP9 also reverses the effects of VPA treatment on seizure susceptibility and the startle habituation response. Over-expression of MMP9 pheno-copies the effect of VPA, and inhibition of MMP9 in single tectal neuronal blocks the expression of experience-dependent structural plasticity. The results are convincing and add mechanistic insight into circuit and behavioral dysfunction induced by VPA signaling, as well as an expansion of the repertoire of plasticity mediated by MMP9 signaling.

    Minor points:

    -The time course for the introduction of VPA and MMP9 inhibitors should be reiterated in the results section.

    -Fig 1 Please report the number (or %) of tectal neurons in which MMP9 was over-expressed following whole-brain electroporation.

    -Does MMP9 transfection change the E/I ratio, as previously reported for VPA?

    -Does VPA or MMP9 inhibition change the initial large amplitude/short latency evoked response?

    Figure 2: please report statistics for total number of barrages or barrage distribution across experimental groups (latter also for Fig 3).

    Figs 3 and 5: The presentation of the immunoblots should clarify if raw or normalized (to Ponceau Blue) data were quantified.

    Fig 4: Please report a post hoc comparison following the repeated measures ANOVA

    Fig 5: Total growth and growth rates could also be included in the results section.

    Minor comments: -The discussion considers a broad range of potential targets of MMP9, including cell surface receptors, growth factors, adhesive proteins, and extracellular matrix components, many of these are left out of the abstract and introduction.

    -The statement of page 6 "Increased synaptic transmission observed in MMP9 over-expression tectal neurons is consistent with dysfunctional synaptic pruning" appears at odds with a body of literature in mouse hippocampus, included many papers cited in the discussion, demonstrating the role of MMP9 in spine elongation, synaptic potentiation and synapse maturation.

  3. eLife

    Reviewer #2:

    In the manuscript by Gore et al., the authors show evidence that MMP9 is a key regulator of synaptic and neuronal plasticity in Xenopus tadpoles. Importantly, they demonstrate a role for MMP9s in valproic acid-induced disruptions in development of synaptic connectivity, a finding that may have particular relevance to autism spectrum disorder (ASD), as prenatal exposure to VPA leads to a higher risk for the disorder. Specifically, the authors show that hyper-connectivity induced by VPA is mimicked by overexpression of MMP9 and reduced by MMP9 knockdown and pharmacological inhibition, suggesting a causal link. The experiments appear to be well executed, analyzed appropriately, and are beautifully presented. I have only a few suggestions for improvement of the manuscript and list a few points of clarification that the authors should address.

    1. The authors refer to microarray data as the rationale for pursuing the role for MMP9 in VPA-induced hyperconnectivity. How many other MMPs or proteases with documented roles in development are similarly upregulated? The authors should say how other possible candidate genes did or did not change, perhaps presenting the list with data in a table (at least other MMPs and proteases). If others have changed, the authors should discuss their data in that context.

    2. Please cite the microarray study(ies?).

    3. In a related issue, the authors should comment on the specificity of the SB-3CT, particularly with regard to other MMPs or proteases that may/may not have been found to be upregulated in the microarray experiment.

    4. Results, first paragraph: although it is in the methods, please state briefly the timing of the VPA exposure and the age/stage at which the experiments were performed. Within the methods, please give an approximate age in days after hatching for the non-tadpole experts.

    5. The finding that a small number of MMP9 overexpressing cells is fascinating. Have the authors stained the tissue for MMP9 after VPA?

    6. Do the authors have data on the intrinsic cell properties (input resistance, capacitance, etc.)? If so, they should include that data either in Supplemental information or in the text. These factors could absolutely influence hyperconnectivity or measurements of the synaptic properties, so at least the authors should discuss their findings in the context of the findings of James, et al.

    Minor Comments:

    1. Page 15: 'basaly low' may be better worded as 'low at baseline'.

    2. The color-coding is very useful and facilitates communicating the results. The yellow on Figure 5, however, is really too light. Consider another color.

  4. eLife

    Reviewer #1:

    This study is based on previous work that exposure to valproic acid (VPA), which is used to model autism spectrum disorders, produces excess local synaptic connectivity, increased seizure susceptibility, abnormal social behavior, and increased MMP-9 mRNA expression in Xenopus tadpoles. VPA is an interesting compound that is also used as an antimanic and mood stabilizing agent in the treatment of bipolar disorder, although the therapeutic targets of VPA for its treatment of mania or as a model of neurodevelopmental disorders have remained elusive. The authors validate that VPA exposed tadpoles have increased MMP9 mRNA expression and then test whether the increased levels of MMP9 mediate the effects of VPA in the tadpole model. The authors report that overexpression of MMP-9 increases spontaneous synaptic activity and network connectivity, whereas pharmacological and genetic inhibition with antisense oligos rescues the VPA induced effects, and then tie the findings to experience dependent synaptic reorganization.

    1. What is the exact nature of "increased connectivity"? Is there an increase in synapse numbers or solely an increase in dendritic complexity coupled with a functional plasticity? The authors should document properties of mEPSCs and mIPSCs recording in TTX to isolate synaptic properties. Coupling this "mini" analysis to quantification of synapse numbers will address whether the changes are solely due to structural plasticity or also due to a functional potentiation of transmission. These experiments should at least be conducted in MMP-9 overexpression, VPA treatment and VPA treatment+MMP-9 loss-of-function cases to validate the basic premise that there is an increased connectivity.

    2. It is unclear why the authors focused on MMP-9 compared to other genes dysregulated by VPA. This point should be further discussed.

    3. How does VPA alter MMP-9 levels? Is this through an HDAC dependent mechanism? Granted VPA has been proposed to work through a variety of mechanisms including HDAC inhibition.

    4. Does SB-3CT rescue the expression levels of MMP-9?

    5. How is increased MMP-9 produces the synaptic and behavioral effects? What is the downstream target (specific receptor?) that would produce the broad changes in synaptic and behavioral phenotypes? Or is this a rather non-specific effect of extracellular matrix? Based on years of data on MMP-9 function its impact on "structural plasticity" in general terms is not surprising but further mechanistic details and specific targets would help move this field forward.

  5. Version published to 10.1101/2020.05.29.123661 on bioRxiv