The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-␤ fibrils (fA␤), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble A␤ (sA␤) could degrade fA␤ as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fA␤ and that this ability is not shared by other sA␤-degrading enzymes examined, including endothelinconverting enzyme, insulin-degrading enzyme, and neprilysin. fA␤ was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fA␤ breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fA␤ were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of A␤ that are cleaved during its degradation. Only MMP-9 digests contained fragments (A␤ 1-20 and A␤ 1-30 ) from fA␤ 1-42 substrate; the corresponding cleavage sites are thought to be important for ␤-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 fA␤ and may contribute to ongoing clearance of plaques from amyloid-laden brains.

It has been postulated that the development of amyloid plaques in Alzheimer's disease (AD) may result from an imbalance between the generation and clearance of the amyloid-␤ peptide (A␤). Although familial AD appears to be caused by A␤ overproduction, sporadic AD (the most prevalent form) may result from impairment in clearance. Recent evidence suggests that several proteases may contribute to the degradation of A␤. Furthermore, astrocytes have recently been implicated as a potential cellular mediator of A␤ degradation. In this study, we examined the possibility that matrix metalloproteinases (MMPs), proteases known to be expressed and secreted by astrocytes, could play a role in extracellular A␤ degradation. We found that astrocytes surrounding amyloid plaques showed enhanced expression of MMP-2 and MMP-9 in aged amyloid precursor protein (APP)/presenilin 1 mice. Moreover, astrocyte-conditioned medium (ACM) degraded A␤, lowering levels and producing several fragments after incubation with synthetic human A␤ 1-40 and A␤ 1-42 . This activity was attenuated with specific inhibitors of MMP-2 and -9, as well as in ACM derived from mmp-2 or -9 knock-out (KO) mice. In vivo, significant increases in the steady-state levels of A␤ were found in the brains of mmp-2 and -9 KO mice compared with wild-type controls.

The accumulation of aggregated amyloid-β (Aβ) in amyloid plaques is a neuropathological hallmark of Alzheimer's disease (AD). Reactive astrocytes are intimately associated with amyloid plaques; however, their role in AD pathogenesis is unclear. We deleted the genes encoding two intermediate filament proteins required for astrocyte activation-glial fibrillary acid protein (Gfap) and vimentin (Vim)-in transgenic mice expressing mutant human amyloid precursor protein and presenilin-1 (APP/PS1). The gene deletions increased amyloid plaque load: APP/PS1 Gfap(-/-)Vim(-/-) mice had twice the plaque load of APP/PS1 Gfap(+/+)Vim(+/+) mice at 8 and 12 mo of age. APP expression and soluble and interstitial fluid Aβ levels were unchanged, suggesting that the deletions had no effect on APP processing or Aβ generation. Astrocyte morphology was markedly altered by the deletions: wild-type astrocytes had hypertrophied processes that surrounded and infiltrated plaques, whereas Gfap(-/-)Vim(-/-) astrocytes had little process hypertrophy and lacked contact with adjacent plaques. Moreover, Gfap and Vim gene deletion resulted in a marked increase in dystrophic neurites (2- to 3-fold higher than APP/PS1 Gfap(+/+)Vim(+/+) mice), even after normalization for amyloid load. These results suggest that astrocyte activation limits plaque growth and attenuates plaque-related dystrophic neurites. These activities may require intimate contact between astrocyte and plaque.

Amyloid plaques are primarily composed of extracellular aggregates of amyloid-␤ (A␤) peptide and are a pathological signature of Alzheimer's disease. However, the factors that influence the dynamics of amyloid plaque formation and growth in vivo are largely unknown. Using serial intravital multiphoton microscopy through a thinned-skull cranial window in APP/PS1 transgenic mice, we found that amyloid plaques appear and grow over a period of weeks before reaching a mature size. Growth was more prominent early after initial plaque formation: plaques grew faster in 6-month-old compared with 10-month-old mice. Plaque growth rate was also size-related, as smaller plaques exhibited more rapid growth relative to larger plaques. Alterations in interstitial A␤ concentrations were associated with changes in plaque growth. Parallel studies using multiphoton microscopy and in vivo microdialysis revealed that pharmacological reduction of soluble extracellular A␤ by as little as 20 -25% was associated with a dramatic decrease in plaque formation and growth. Furthermore, this small reduction in A␤ synthesis was sufficient to reduce amyloid plaque load in 6-month-old but not 10-month-old mice, suggesting that treatment early in disease pathogenesis may be more effective than later treatment. In contrast to thinned-skull windows, no significant plaque growth was observed under open-skull windows, which demonstrated extensive microglial and astrocytic activation. Together, these findings indicate that individual amyloid plaque growth in vivo occurs over a period of weeks and may be influenced by interstitial A␤ concentration as well as reactive gliosis.

In sporadic Alzheimer's disease (AD), impaired A␤ removal contributes to elevated extracellular A␤ levels that drive amyloid plaque pathogenesis. Extracellular proteolysis, export across the blood-brain barrier, and cellular uptake facilitate physiologic A␤ clearance. Astrocytes can take up and degrade A␤, but it remains unclear whether this function is insufficient in AD or can be enhanced to accelerate A␤ removal. Additionally, age-related dysfunction of lysosomes, the major degradative organelles wherein A␤ localizes after uptake, has been implicated in amyloid plaque pathogenesis. We tested the hypothesis that enhancing lysosomal function in astrocytes with transcription factor EB (TFEB), a master regulator of lysosome biogenesis, would promote A␤ uptake and catabolism and attenuate plaque pathogenesis. Exogenous TFEB localized to the nucleus with transcriptional induction of lysosomal biogenesis and function in vitro. This resulted in significantly accelerated uptake of exogenously applied A␤42, with increased localization to and degradation within lysosomes in C17.2 cells and primary astrocytes, indicating that TFEB is sufficient to coordinately enhance uptake, trafficking, and degradation of A␤. Stereotactic injection of adeno-associated viral particles carrying TFEB driven by a glial fibrillary acidic protein promoter was used to achieve astrocyte-specific expression in the hippocampus of APP/PS1 transgenic mice. Exogenous TFEB localized to astrocyte nuclei and enhanced lysosome function, resulting in reduced A␤ levels and shortened half-life in the brain interstitial fluid and reduced amyloid plaque load in the hippocampus compared with control virus-injected mice. Therefore, activation of TFEB in astrocytes is an effective strategy to restore adequate A␤ removal and counter amyloid plaque pathogenesis in AD.