Abstract
The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-beta fibrils (fAbeta), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble Abeta (sAbeta) could degrade fAbeta as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fAbeta and that this ability is not shared by other sAbeta-degrading enzymes examined, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin. fAbeta was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fAbeta breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fAbeta were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of Abeta that are cleaved during its degradation. Only MMP-9 digests contained fragments (Abeta(1-20) and Abeta(1-30)) from fAbeta(1-42) substrate; the corresponding cleavage sites are thought to be important for beta-pleated sheet formation. To determine whether MMP-9 can degrade plaques formed in vivo, fresh brain slices from aged APP/PS1 mice were incubated with proteases. MMP-9 digestion resulted in a decrease in thioflavin-S (ThS) staining. Consistent with a role for endogenous MMP-9 in this process in vivo, MMP-9 immunoreactivity was detected in astrocytes surrounding amyloid plaques in the brains of aged APP/PS1 and APPsw mice, and increased MMP activity was selectively observed in compact ThS-positive plaques. These findings suggest that MMP-9 can degrade fAbeta and may contribute to ongoing clearance of plaques from amyloid-laden brains.
Highlights
(AD)3 is the senile plaque, extracellular deposits found throughout the brains of AD patients, composed primarily of the amyloid- peptide (A)
The 42-amino acid peptide (A1–42), the predominant peptide length found in senile plaques, has a remarkable propensity to aggregate at high concentrations to form a -pleated sheet structure [2, 3]
Given the purported irreversibility of A fibril formation and the resistance to degradation, one might expect that senile plaques would continue to grow throughout disease progression; careful observational studies indicate that plaque size remains relatively constant over a wide range of disease durations [4]
Summary
(AD) is the senile plaque, extracellular deposits found throughout the brains of AD patients, composed primarily of the amyloid- peptide (A). In vivo imaging in the APPsw (Tg2576) transgenic mouse model of Alzheimer’s disease (using multiphoton microscopy) demonstrated that plaques remain constant in size over a period of many months [5] These observations have led some to believe that plaques, once formed, are in dynamic equilibrium with their environment, balancing formation with degradation [6]. Experimental evidence that these proteases degrade sA in vivo is available only for a few: gene deletion of NEP [14], ECE [15], or IDE [16] in mice resulted in increased steady-state levels of A in brain, suggesting a role in regulating endogenous basal levels of A in vivo All of these proteases demonstrate the ability to degrade sA, none has been convincingly shown to degrade A fibrils or compact plaques. We further examined the expression and activity of fA-degrading proteases in the brains of transgenic mouse models of AD
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