A new role for matrix metalloproteinase-3 in the NGF metabolic pathway: Proteolysis of mature NGF and sex-specific differences in the continuum of Alzheimer's pathology

Abstract

Matrix metalloproteinase-3 (MMP-3) has been associated with risk of Alzheimer's disease (AD). In this study we introduce a novel role for MMP-3 in degrading nerve growth factor (NGF) in vivo and examine its mRNA and protein expression across the continuum of AD pathology. We provide evidence that MMP-3 participates in the degradation of mature NGF in vitro and in vivo and that it is secreted from the rat cerebral cortex in an activity-dependent manner. We show that cortical MMP-3 is upregulated in the McGill-R-Thy1-APP transgenic rat model of AD-like amyloidosis. A similar upregulation was found in AD and MCI brains as well as in cognitively normal individuals with elevated amyloid deposition. We also observed that frontal cortex MMP-3 protein levels are higher in males. MMP-3 protein correlated with more AD neuropathology, markers of NGF metabolism, and lower cognitive scores in males but not in females. These results suggest that MMP-3 upregulation in AD might contribute to NGF dysmetabolism, and therefore to cholinergic atrophy and cognitive deficits, in a sex-specific manner. MMP-3 should be further investigated as a biomarker candidate or as a therapeutic target in AD.

Keywords: Alzheimer's disease; Basal forebrain cholinergic neurons; Matrix metalloproteinase-3; NGF metabolic pathway; Nerve growth factor; proNGF.

Figure 1.. Proteolysis of mature NGF by MMP-3 in vitro.

A: Representative Western blot showing that the coincubation of 10 or 20 nanograms of recombinant human mature NGF with 0, 3.7, 7.4, or 14.8 mM of active recombinant human MMP-3 overnight at 37°C led to progressive reductions in NGF immunoreactivity. B: Quantitative analysis of the dose-dependent degradation of mature NGF incubated with 0 to 3.7 mM MMP-3 as in A, expressed as a proportion of the quantified immunoreactivity of the untreated sample within a set and averaged between the 10 and 20 ng conditions. Bars indicate the average plus the SEM. mNGF = mature Nerve Growth Factor, MMP-3 = Matrix Metalloproteinase-3, IOD = Integrated Optic Density.

Figure 2.. MMP-3 contributes to the proteolysis of mature NGF in vivo.

A: to test the ability of MMP-3 to degrade mNGF in vivo, either MMP-3 or the selective MMP-3 inhibitor, UK356618 was stereotaxically injected into the medial hippocampus on one side, while the contralateral medial segment received an injection of vehicle solution. The injected medial segments were compared to each other and to the non-injected anterior and posterior segments on each side. B: The injection of 2.0 μl of MMP-3 in saline solution (1.0 μg/μl) into the medial hippocampus led to a near-total abrogation of mNGF immunoreactivity in this segment in comparison both to the anterior and posterior ipsilateral segments, as well as to each contralateral segment. C: The injection of 2.0 μl of 0.5 μM UK356618, prepared in DMSO, led to a ~25% increase in mNGF immunoreactivity in the injected segment when compared to the ipsilateral anterior and posterior segments, as well as to all contralateral segments. No such effect was observed for the injection of DMSO on the contralateral side. Data is displayed as scatterplots showing individual values and the mean +/−SEM. All groups comprised 6 rats, 3 males and 3 females. MMP-3 = Matrix Metalloproteinase-3, NGF = Nerve Growth Factor, IOD = Integrated Optic Density.

Figure 3.. Activity-dependent release of MMP-3 in response to cholinergic stimulation in superfused ex-vivo rat brain cortical tissue.

MMP-3 levels in ex vivo rat cerebral cortex superfusates are elevated 5 minutes after stimulation with the cholinergic agonist carbachol, decrease by roughly 25% after 10 minutes, decrease by 50–75% after 15 minutes, and return to baseline by 30 minutes. These responses are not affected by the cell-impermeant calcium chelator BAPTA (Panel A) but are abrogated by the cell-permeant calcium chelator BAPTA-AM (Panel B). Samples from a single experiment were run on the same blot and values are expressed as proportions of the maximum immunoreactivity observed; each dot represents the average of three experiments. MMP-3: Matrix Metalloproteinase-3, IOD: Integrated Optical Density.

Figure 4.. Analysis of MMP-3 expression (2^−ΔΔCq) in cerebral cortex from the McGill-R-Thy1-APP transgenic rat model of AD-like amyloidosis.

A: At 3 months of age, MMP-3 mRNA was upregulated in homozygous transgenics compared to heterozygotes and wild-types, which were not different one from another. B/C: At both the six and the 13–15 month time-points, MMP-3 mRNA in both the heterozygous and homozygous transgenics were increased relative to the wild-types. D: Confirming that these transcriptional differences have relevance for protein levels, we showed that MMP-3 protein was also elevated in heterozygous and homozygous transgenics relative to the wild-types at the 13–15 months timepoint. E: Illustrative Western Blot for MMP-3 in wildtype, hemizygous, and homozygous McGill-R-Thy1-APP rats, with GAPDH for reference. For all figures, stars indicate the significance of Bonferonni post-hoc tests vs. APP −/− in a 4x1 ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001 Data represents Cq or Integrated Optic Densities, with bars representing mean values and error bars representing the SEM. MMP-3 = Matrix Metalloproteinase-3.

Figure 5.. Analysis of MMP-3 expression in the continuum of human Alzheimer’s disease.

(A) Levels of MMP-3 mRNA, (B) proMMP-3 and (C) MMP-3 protein were assessed in dorsolateral/medial prefrontal cortex samples from individuals with no cognitive impairment and low amyloid deposition (LA-NCI; n = 33), individuals with no cognitive impairment and AD/MCI-like levels of amyloid deposition (HA-NCI; n = 21), individuals with mild cognitive impairment (MCI; n = 19) and patients with AD dementia (AD; n = 24). MMP-3 mRNA (A), proMMP-3 protein (B), and mature MMP-3 protein (C) were all increased in AD and MCI, as well as in HA-NCI individuals compared to the LA-NCI group. D: A significant main effect of sex was observed, with males having higher levels of mature MMP-3 protein across all clinical classifications, but only demonstrating post-hoc significance vs. females in LA-NCI individuals. E: An illustrative Western Blot for proMMP-3 and MMP-3 in LA-NCI, HA-NCI, MCI, and AD, with GAPDH for reference. For A-C, stars indicate the significance of Bonferonni post-hoc tests vs. LA-NCI in a 4x1 ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, while in D, they indicate post-hoc Bonferonni tests for male vs. female within clinical groups in a 4x2 ANOVA; the stars indicate equivalent significance. Panel A represents Cq values, while panels B-D represent Integrated Optic Density values, all expressed as a fold change from the LA-NCI group. All bars represent mean values with errors bars representing the SEM. MMP-3 = Matrix Metalloproteinase-3, proMMP-3: precursor to MMP-3

Figure 6.. Schematic of the NGF Metabolic Pathway in health in in the context of AD neuropathology.

A: Under normal conditions, mNGF is derived from proNGF by plasmin (3), itself derived form plasminogen by tissue plasminogen activator (tPA) (2). tPA activity is controlled by neuroserpin, its endogenous inhibitor (1). MMP-9 and, we now show, MMP-3 degrade mNGF (7); these are regulated by the tissue inhibitor of metalloproteinases 1 (TIMP1) (8). Under normal conditions sufficient mNGF dimerizes and binds p75/TrkA receptor complexes (4). The activated receptors are internalized and retrogradely transported to the cell body where they ultimately exert their trophic effect (6). B: In the context of AD pathology, increased neuroserpin/decreased tPA (1) lead to reductions in local plasmin concentrations, reducing the maturation of proNGF to mNGF (2). Decreased TIMP1 (3) and increased MMP-3/MMP-9 lead to the excessive degradation of mNGF (4). As a result, trophic support to BFCNs is compromised (5). mNGF = mature Nerve Growth Factor, proNGF = precursor to NGF, tPA = tissue plasminogen activator, BFCN = basal forebrain cholinergic neuron, MMP-3/9 = matrix metalloproteinase 3/9, TIMP1 = tissue inhibitor of metalloproteinases 1