Elevated matrix Metalloproteinase-9 associated with reduced cerebellar perineuronal nets in female mice with toxoplasmosis

Abstract

Brain infection by the parasite Toxoplasma gondii is thought to impair learning and memory, although the underlying mechanisms remain largely unknown. Recent studies suggest that perineuronal nets (PNNs) and their key regulator, matrix metalloproteinase-9 (MMP-9), have essential roles in synaptic plasticity associated with learning and memory. We investigated their roles in a chronic toxoplasmosis model using female mice. In mice with a high parasite burden of chronic infection, we found that MMP-9 expression was increased in the peripheral circulation and the brain. A correlation was found between the serum levels of MMP-9 and antibodies to the Toxoplasma matrix antigen MAG1, a surrogate marker for Toxoplasma tissue cysts in the brain. MMP-9 elevation was accompanied by increased expression of its endogenous regulators, TIMP-1 and NGAL. An increase in the levels of GSK-3α/β was observed, alongside a decrease in inhibitory GSK-3α/β (Ser-21/Ser-9) phosphorylation. MMP-9 expression was notably associated with the loss of PNNs but increased expression of the synaptic vesicle protein synaptophysin. There was a trend toward a negative correlation between MMP-9 and aggrecan expression, a critical PNN component. Together, these results suggest that chronic Toxoplasma infection can cause an increase in MMP-9 expression, resulting in the degradation of PNNs, which provides a possible mechanism for Toxoplasma-associated deficits in learning and memory.

Keywords: Learning and memory; MMP-9; Perineuronal nets; Synaptic plasticity; Toxoplasma gondii; Toxoplasma matrix antigen MAG1.

Fig. 1

Cyst-associated elevation of serum MMP-9 levels. Female CD-1 mice were infected intraperitoneally with 500 tachyzoites of GT1 strain and sacrificed at 5 months post-infection. (A) Distribution of MMP-9 concentration among the five groups stratified by antibody profiles. IgG indicates exposure to Toxoplasma, while MAG1 antibody serves as a serologic marker for cyst burden. Data are shown as mean ± SD, with individual values. (B) A strong positive correlation was found between MMP-9 and MAG1 antibody levels in mice with MAG1 antibodies, including the MAG1-high and MAG1-low groups. Statistically significant differences were determined by one-way ANOVA followed by Bonferroni's correction for multiple comparisons (A) or Pearson's correlation analysis (B). ****, P < 0.0001.

Fig. 2

Elevated expression of MMP-9, TIMP-1, and NGAL in the cerebellum of mice bearing a high burden of tissue cysts. The blot was probed with anti-MMP-9, anti-TIMP-1, and anti-NGAL antibodies under non-reducing (A) or reducing (B) conditions, among MAG1-high, MAG1-low, and control mice (n = 3 per group), alongside β-actin loading controls. Each data point and means obtained by densitometry analysis of blots were shown. A dagger indicates significant differences between experimental groups. *p < 0.05, **p < 0.01 (one-way ANOVA followed by Bonferroni's correction for multiple comparisons). M/T, MMP-9/TIMP-1 complex; M/N, MMP-9/NGAL complex.

Fig. 3

Cyst-associated elevation of enzymatic activity of the MMP-9 monomer. Enzymatic levels of MMP-9 and MMP-2 were analyzed using gelatin-substrate zymography among MAG1-high, MAG1-low, and control mice (n = 3 per group). Each data point and means obtained by densitometry analysis of blots were shown. *p < 0.05 (one-way ANOVA followed by Bonferroni's correction for multiple comparisons).

Fig. 4

Mice with a high burden of tissue cysts showed elevated levels of GSK-3 and reduced levels of pGSK-3. Western blot analysis of cerebellar protein extracts from MAG1-high or control mice probed with antibodies against GSK-3α/β (A) and pGSK-3αSer21/βSer9 (B). Each data point and means obtained by densitometry analysis of blots were shown. *p < 0.05, **p < 0.01 (unpaired t-test). N = 5 per group.

Fig. 5

Elevated cerebellar expression of MMP-9, particularly in the Purkinje cells, of MAG1-high mice. (A–C) Little MMP-9-positive staining was found in the granular, molecular, and Purkinje cell layers of uninfected control mice. (D–F) There was an increased cerebellar MMP-9 staining in the MAG1-high mice. Intense staining of MMP-9 was observed in the cytoplasmic membranes and at the terminal branches of Purkinje cells (green arrow, E). Numerous MMP-9-positive structures in the granular and molecular layers appeared as dot-shaped or cytoplasmic membrane staining (white arrowhead, Fig. 5E–F). Scale bars are indicated. gl: granular layer; ml: molecular layer.

Fig. 6

A diminished WFA staining was associated with MMP-9 expression in MAG1-high mice. The WFA staining showed a lattice-like pattern in the granular layer and was present in the terminal branches of Purkinje cells (red arrows), as shown in the control mice (A). The white arrows indicate the presence of pericellular nets (green) surrounding Golgi neurons located in the granular layer, in the proximal dendrites of interneurons in the molecular layer, and at the terminal branches of Purkinje cells (D–E). In contrast, MAG1-high mice had diminished WFA labeling in the cerebellar cortex (F). Double-labeling of WFA and MMP-9 (A-C, F–H) showed that MMP-9 expression was associated with diminished WFA staining (H). The blue arrows point to MMP-9 staining (red) occurring in the granular layer and the Purkinje cell bodies (I–J). Scale bar = 100 μm or 20 μm. gl: granular layer; ml: molecular layer. Protein extracts from cerebellar homogenates of MAG1-high and control mice were probed with antibodies against aggrecan (K) and brevican (L), alongside MMP-9 and β-actin. Each data point and means obtained by densitometry analysis of blots were shown. Correlations between PNN protein expression and MMP-9 were evaluated. Control (n = 5, circles); MAG1-high (n = 6, black dots). Statistically significant differences were determined by unpaired t-test or Pearson's correlation analysis. *p < 0.05, ns: not significant.

Fig. 7

Mice with a high burden of tissue cysts exhibited increased synaptophysin expression in the cerebellum, located adjacent to MMP-9. (A) Western blot analysis of cerebellar protein extracts from MAG1-high, MAG1-low, or control mice probed with antibodies against synaptophysin. Each data point and means obtained by densitometry analysis of blots were shown. Compared to the uninfected control (B), synaptophysin levels in the granular layer were elevated in MAG1-high mice (C). The white boxes inserted are at ×40 Magnification. (D–F) Confocal images show the proximity of the staining for MMP-9 and synaptophysin throughout the cytoplasm and processes of neuronal cells (white arrows). DAPI was used to identify nuclei (blue). Scale bar, 200 μm or 100 μm (inserts). *p < 0.05 (one-way ANOVA followed by Bonferroni's correction for multiple comparisons). Syn: synaptophysin.