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.

Amyloid-β peptide (Aβ) accumulation in senile plaques, a pathological hallmark of Alzheimer's disease (AD), has been implicated in neuronal degeneration. We have recently demonstrated that Aβ induced oligodendrocyte (OLG) apoptosis, suggesting a role in white matter pathology in AD. Here, we explore the molecular mechanisms involved in Aβ-induced OLG death, examining the potential role of ceramide, a known apoptogenic mediator. Both Aβ and ceramide induced OLG death. In addition, Aβ activated neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase, resulting in increased ceramide generation. Blocking ceramide degradation with N-oleoyl-ethanolamine exacerbated Aβ cytotoxicity; and addition of bacterial sphingomyelinase (mimicking cellular nSMase activity) induced OLG death. Furthermore, nSMase inhibition by 3-O-methyl-sphingomyelin or by gene knockdown using antisense oligonucleotides attenuated Aβ-induced OLG death. Glutathione (GSH) precursors inhibited Aβ activation of nSMase and prevented OLG death, whereas GSH depletors increased nSMase activity and Aβ-induced death. These results suggest that Aβ induces OLG death by activating the nSMase–ceramide cascade via an oxidative mechanism.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive dementia. Amyloid-beta peptide (Abeta), a 39-43 amino acid peptide derived from beta-amyloid precursor protein, forms insoluble fibrillar aggregates that have been linked to neuronal and vascular degeneration in AD and cerebral amyloid angiopathy. Here we demonstrate that Abeta 1-40 and a truncated fragment, Abeta 25-35, induced death of oligodendrocytes (OLGs) in vitro in a dose-dependent manner with similar potencies. Abeta-induced OLG death was accompanied by nuclear DNA fragmentation, mitochondrial dysfunction, and cytoskeletal disintegration. Abeta activation of redox-sensitive transcription factors NF-kappaB and AP-1 and antioxidant prevention of Abeta-mediated OLG death suggest that oxidative injury contributes to Abeta cytotoxicity in OLGs. Recent demonstration of Abeta deposition and white matter abnormalities in AD implies a potential pathophysiological role for Abeta-mediated cytotoxicity of OLGs in this neurodegenerative disease.

Cytokines, including tumor necrosis factor-␣ (TNF-␣), may elicit cytotoxic response through the sphingomyelin-ceramide signal transduction pathway by activation of sphingomyelinases and the subsequent release of ceramide: the universal lipid second messenger. Treatment of bovine cerebral endothelial cells (BCECs) with TNF-␣ for 16 h followed by cycloheximide (CHX) for 6 h resulted in an increase in ceramide accumulation, DNA fragmentation, and cell death. Application of a cell permeable ceramide analogue C 2 ceramide, but not the biologically inactive C 2 dihydroceramide, also induced DNA laddering and BCEC death in a concentration-and time-dependent manner. TNF-␣/CHX-mediated ceramide production apparently is not a result of sphingomyelin hydrolysis because sphingomyelin content does not decrease in this death paradigm. In addition, an acidic sphingomyelinase inhibitor, desipramine, had no effect on TNF-␣/CHX-induced cell death. However, addition of fumonisin B1, a selective ceramide synthase inhibitor, attenuated TNF-␣/CHX-induced intracellular ceramide elevation and BCEC death. Together, these findings suggest that ceramide plays at least a partial role in this paradigm of BCEC death. Our results show, for the first time, that ceramide derived from de novo synthesis is an alternative mechanism to sphingomyelin hydrolysis in the BCEC death process initiated by TNF-␣/CHX.