Matrix metalloproteinase-7 disrupts dendritic spines in hippocampal neurons through NMDA receptor activation

Abstract

Dendritic spines are protrusions from the dendritic shaft that host most excitatory synapses in the brain. Although they first emerge during neuronal maturation, dendritic spines remain plastic through adulthood, and recent advances in the molecular mechanisms governing spine morphology have shown them to be exquisitely sensitive to changes in the micro-environment. Among the many factors affecting spine morphology are components and regulators of the extracellular matrix (ECM). Modification of the ECM is critical to the repair of injuries throughout the body, including the CNS. Matrix metalloproteinase (MMP)-7/matrilysin is a key regulator of the ECM during pathogen infection, after nerve crush and in encephalitogenic disorders. We have investigated the effects of MMP-7 on dendritic spines in hippocampal neuron cultures and found that it induces the transformation of mature, short mushroom-shaped spines into long, thin filopodia reminiscent of immature spines. These changes were accompanied by a dramatic redistribution of F-actin from spine heads into thick, rope-like structures in the dendritic shaft. Strikingly, MMP-7 effects on dendritic spines were similar to those of NMDA treatment, and both could be blocked by channel-specific antagonists. These findings are the first direct evidence that MMPs can influence the morphology of mature dendritic spines, and hence synaptic stability.

Fig. 1

MMP-7 induces F-actin reorganization in dendrites of cultured hippocampal neurons. (a–d) Confocal images of control and MMP-7 treated hippocampal neurons at 15 DIV. F-actin polymerization was visualized by rhodamine-coupled phalloidin (red), dendrites by immunostaining for MAP2 (green; a, b), and axons by immunostaining for NF-200 (green; c, d). Scale bars: 10 μm (upper); 5 μm (lower).

Fig. 2

MMP-7 treatment induces F-actin rearrangement and reduces the number of PSD-95-positive postsynaptic sites and spiny synapses in cultured hippocampal neurons. (a–d) Confocal images of control and MMP-7 treated hippocampal neurons at 15 DIV. F-actin polymerization was visualized by rhodamine-coupled phalloidin (red), (a, b) presynaptic terminals by immunostaining for synaptophysin (green) and (c, d) postsynaptic sites by immuno-staining for PSD-95 (green). Scale bars: 10 μm (upper); 5 μm (lower). (e) Quantitative analysis of the number of F-actin, synaptophysin and actin/synaptophysin double-positive clusters per 10 μm of dendrite. (f) Quantitative analysis of the number of F-actin, PSD-95 and actin/PSD-95 double-positive clusters per 10 μm of dendrite. Vertical bars indicate SD; n = 10 neurons per group; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

Quantitative evaluation of the F-actin labeling pattern in dendrites. (a) A dendritic fragment showing characteristic labeling for F-actin in baseline conditions. Image is superimposed with a sampling segment (dotted line) for labeling intensity reference. (b) The labeling intensity profile along the dendritic fragment in (a) shows average pixel values across the area within dotted lines. (c) Autocorrelation function (ACF) of the labeling profile shown in (b). Gray shaded area shows 95% confidence limits. The initial deflection indicates the presence of distinct, 1–2-μm wide spots/clusters of labeling. In comparison, random noise labeling would correspond to insignificant deviation of ACF from zero, whereas homogeneous labeling would tend to show ACF values close to unity. (d) Dendritic fragments showing characteristic labeling for F-actin in control and MMP-7 treated cultures. Scale bars: 5 μm (e). Average autocorrelation functions reflecting the labeling pattern of F-actin in dendrites in control cultures (Control, closed circles), and cultures incubated with MMP-7 (MMP-7, open squares); vertical bars, SEM (n = 20 dendrite fragments in each condition, see Fig. 1). The data indicate that the ~2-μm F-actin clusters, present in control conditions, are transformed into a more homogeneous type of labeling (statistical difference at lag distances above 1 μm is at least at p < 0.05; error bars, 95% confidence intervals).

Fig. 4

MMP-7 induces changes in dendritic spine morphology in 15-DIV hippocampal neuron cultures. (a, b) The confocal images of GFP-labeled hippocampal neurons from control and MMP-7 treated cultures. Scale bars: 10 μm (upper); 5 μm (lower). (c, d) Live images of GFP-labeled dendritic spines in 15-DIV hippocampal neurons before (0 min) and after (50 min) treatment with (c) control, or (d) MMP-7. MMP-7 treatment induced changes in dendritic spine morphology and promoted elongation of existing dendritic spines (arrows). Scale bars: 5 μm. (e) Quantification of dendritic protrusion length. (f) Quantification of dendritic protrusion density.

Fig. 5

MMP-7 effects are time and concentration dependent in 15-DIV hippocampal neuron cultures. (a) Phalloidin-labeled dendrites were calculated for rope-like or spiny F-actin labeling after treatment with a series of MMP-7 concentrations for 1 h. The percentage of neurons showing rope-like dendrite labeling increased as more MMP-7 was added. (b) The percentage of neurons with rope-like F-actin labeling increased with time, in cultures treated with 100 mU/mL of MMP-7. Vertical bars indicate SD; n = 1000 neurons per group; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6

MMP-7 mimics NMDA effects and blockade of NMDA channels prevents MMP-7 mediated F-actin rearrangement. Confocal images of 15-DIV hippocampal neurons in (a) control cultures and cultures treated with (b) MMP-7, (c) NMDA, (d) MK-801, (e) MK-801 + MMP-7, and (f) MK-801 + NMDA. F-actin polymerization was visualized by rhodamine-coupled phalloidin (red), presynaptic terminals by immunostaining for synaptophysin (green). Scale bars: 10 μm (upper); 5 μm (lower). (g) Quantitative analysis of the number of F-actin, synaptophysin and actin/synaptophysin double-positive clusters per 10 μm of dendrite. Vertical bars indicate SD; n = 10 neurons per group; *p < 0.05, **p < 0.01, ***p < 0.001. (h) Average autocorrelation functions reflecting the labeling pattern of F-actin along dendrites of cultured neurons in control and experimental conditions, as indicated; error bars, 95% confidence intervals.

Fig. 7

MMP-7 effects in cultures grown on fibronectin or poly-dl-ornithine alone. MMP-7 effects were compared in cultures grown on fibronectin or poly-dl-ornithine. Confocal images of 15-DIV hippocampal neurons in cultures grown on (a, b) fibronectin, and (d, e) poly-dl-ornithine alone. F-actin polymerization was visualized by rhodamine-coupled phalloidin (red), presynaptic terminals by immunostaining for synaptophysin (green). Scale bars: 10 μm (upper); 5 μm (lower). (c, f) Quantitative analysis of the number of F-actin, synaptophysin and actin/synaptophysin double-positive clusters per 10 μm of dendrite in cultures grown on (c) fibronectin, or (f) poly-dl-ornithine alone. Vertical bars indicate SD; n = 10 neurons per group; *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 8

MMP-7-induced F-actin reorganization is reversible. Hippocampal neuron cultures (15 DIV) were treated for 1 h with control or MMP-7 containing medium (1 + 0), which was then replaced with normal or MK-801 containing medium for 2 h (1 + 2). (a–c) Quantitative analysis of the number of F-actin, synaptophysin and actin/ synaptophysin double-positive clusters per 10 μm of dendrite. (d) Quantitative analysis of neuronal survival.