Jan Xu

Matrix metalloproteinase-9 degrades amyloid-β fibrils in vitro and compact plaques in situ

The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-β fibrils (fAβ), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble Aβ (sAβ) could degrade fAβ as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fAβ and that this ability is not shared by other sAβ-degrading enzymes examined, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin. fAβ was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fAβ breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fAβ were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of Aβ that are cleaved during its degradation. Only MMP-9 digests contained fragments (Aβ1-20 and Aβ1-30) from fAβ1-42 substrate; the corresponding cleavage sites are thought to be important for β-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 fAβ and may contribute to ongoing clearance of plaques from amyloid-laden brains.

Matrix Metalloproteinases Expressed by Astrocytes Mediate Extracellular Amyloid-β Peptide Catabolism

It has been postulated that the development of amyloid plaques in Alzheimers disease (AD) may result from an imbalance between the generation and clearance of the amyloid-β peptide (Aβ). Although familial AD appears to be caused by Aβ 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 Aβ. Furthermore, astrocytes have recently been implicated as a potential cellular mediator of Aβ 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 Aβ 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 Aβ, lowering levels and producing several fragments after incubation with synthetic human Aβ1–40 and Aβ1–42. 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 Aβ were found in the brains of mmp-2 and -9 KO mice compared with wild-type controls. Furthermore, pharmacological inhibition of the MMPs with N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM 6001) increased brain interstitial fluid Aβ levels and elimination of half-life in APPsw mice. These results suggest that MMP-2 and -9 may contribute to extracellular brain Aβ clearance by promoting Aβ catabolism.

Involvement of de novo ceramide biosynthesis in tumor necrosis factor- α/cycloheximide-induced cerebral endothelial cell death

Cytokines, including tumor necrosis factor-α (TNF-α), 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-α 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 C2 ceramide, but not the biologically inactive C2 dihydroceramide, also induced DNA laddering and BCEC death in a concentration- and time-dependent manner. TNF-α/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-α/CHX-induced cell death. However, addition of fumonisin B1, a selective ceramide synthase inhibitor, attenuated TNF-α/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 novosynthesis is an alternative mechanism to sphingomyelin hydrolysis in the BCEC death process initiated by TNF-α/CHX.

Methylprednisolone inhibition of TNF-α expression and NF-kB activation after spinal cord injury in rats

Post-traumatic inflammatory reaction has been implicated in the secondary injury after SCI. TNF-alpha is a key inflammatory mediator, which plays a pathogenetic role in cell death in inflammatory disorders and traumatic brain injury. TNF-alpha exerts its effector actions, at least partially, through the activation of a pro-inflammatory transcription factor, NF-kB, which in turn upregulates such genes as iNOS, cytokines, adhesive molecules, and others. Consistent with a post-traumatic inflammatory reaction after SCI, we noted an increase in TNF-alpha expression by Western blotting (4.5-fold increase at 1 day after SCI, P<0.01) and immunohistochemistry in a rat SCI model. Post-traumatic TNF-alpha expression was accompanied by an increase in NF-kB binding activity in nuclear proteins isolated from the injured cord (3.9-fold increase, P<0.01). MP is the only drug proven effective in improving neurological function in patients with acute SCI. The mechanism of action of MP is not fully understood, but is thought to be related to its antioxidant effects. MP is also a potent anti-inflammatory agent, which has been recently shown to inhibit NF-kB binding activity. MP (30 mg/kg, i.v.) given immediately after SCI reduced TNF-alpha expression by 55% (P<0.01) and NF-kB binding activity. These findings suggest that post-traumatic inflammatory activity that is mediated by the TNF-alpha-NF-kB cascade can be suppressed by MP.

Amyloid-β peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway

Amyloid-β peptide (Aβ) accumulation in senile plaques, a pathological hallmark of Alzheimers 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.

iNOS and nitrotyrosine expression after spinal cord injury

Secondary tissue damage after spinal cord injury (SCI) may be due to inflammatory mediators. After SCI, the nuclear factor-kappaB (NF-kappaB) transcription factor can activate many pro-inflammatory genes, one of which is inducible nitric oxide synthase (iNOS). iNOS catalyzes the synthesis of nitric oxide (NO), a key inflammatory mediator, which in turn reacts with superoxide to generate peroxynitrite. Peroxynitrite is a strong oxidant that can damage cellular enzymes, membranes, and subcellular organelles through the nitration of tyrosine residues on proteins. The presence of nitrotyrosine (NT) is an indirect chemical indicator of toxic NO and peroxynitrite-induced cellular damage. Using a New York University (NYU) impactor to induce SCI in adult rats, we examined the temporal and cellular expression of iNOS and NT. We observed a progressive increase in iNOS expression in the injured cord starting at day 1 with maximal expression occurring at day 7, as determined by Western blot analysis. iNOS expression corresponded temporally to an increase in iNOS enzyme activity after SCI. In parallel with the progressive increase in iNOS activity, NT expression also increased with time after SCI. The iNOS and NT immunoreactivity was localized in neurons, astrocytes, endothelial cells and ependymal cells at the epicenter and adjacent to the region of spinal cord impact and injury. Results from the present study suggest that increased iNOS and peroxynitrite anion, as reflected by the progressive accumulation of NT in the injured impacted spinal cord, may contribute to the secondary injury process after SCI.

Oxygen-Glucose Deprivation Induces Inducible Nitric Oxide Synthase and Nitrotyrosine Expression in Cerebral Endothelial Cells

BACKGROUND AND PURPOSE The cerebral endothelial cells (ECs) are a primary target of hypoxic or ischemic brain insults. EC damage may contribute to postischemic secondary injury. Massive production of NO after inducible NO synthase (iNOS) expression has been implicated in cell death. This study aimed to characterize bovine cerebral EC death in relation to iNOS expression after oxygen-glucose deprivation (OGD) in vitro. METHODS OGD in bovine cerebral ECs in culture was induced by deleting glucose in the medium and by incubating the cells in a temperature-controlled anaerobic chamber. The extent of cell death was assessed by trypan blue exclusion, MTT assay, and LDH release. ELISA, gel electrophoresis, and staining by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling were used to examine DNA fragmentation. The expression of iNOS mRNA and protein was detected by reverse transcription-polymerase chain reaction and Western blotting, respectively. Nitrotyrosine expression was confirmed with Western blot analysis and immunostaining. RESULTS Bovine cerebral EC death was dependent on the duration of OGD and showed selected biochemical, morphological, and pharmacological features suggestive of apoptosis. OGD also induced the expression of iNOS mRNA and protein in bovine cerebral ECs. Increased expression of nitrotyrosine, the product formed by peroxynitrite reaction with proteins, was also detected after OGD. The involvement of iNOS in EC death was suggested by partial reduction of cell death by NO synthase inhibitors, including L-N(G)-(1-iminoethyl)ornithine and nitro-L-arginine, and an NO scavenger, the Fe(2+)-N-methyl-D-glucamine dithiocarbamate complex. CONCLUSIONS OGD-induced bovine cerebral EC death involves an apoptotic process. Induction of iNOS with subsequent peroxynitrite formation may contribute to bovine cerebral EC death caused by OGD.

Induction of secretory phospholipase A2 in reactive astrocytes in response to transient focal cerebral ischemia in the rat brain

Although mRNA expression of group IIA secretory phospholipase A2 (sPLA2‐IIA) has been implicated in responses to injury in the CNS, information on protein expression remains unclear. In this study, we investigated temporal and spatial expression of sPLA2‐IIA mRNA and immunoreactivity in transient focal cerebral ischemia induced in rats by occlusion of the middle cerebral artery. Northern blot analysis showed a biphasic increase in sPLA2‐IIA mRNA expression following 60‐min of ischemia–reperfusion: an early phase at 30 min and a second increase at a late phase ranging from 12 h to 14 days. In situ hybridization localized the early‐phase increase in sPLA2‐IIA mRNA to the affected ischemic cortex and the late‐phase increase to the penumbral area. Besides sPLA2‐IIA mRNA, glial fibrillary acidic protein (GFAP) and cyclo‐oxygenase‐2 mRNAs, but not cytosolic PLA2, also showed an increase in the penumbral area at 3 days after ischemia–reperfusion. Immunohistochemistry of sPLA2‐IIA indicated positive cells in the penumbral area similar to the GFAP‐positive astrocytes but different from the isolectin B4‐positive microglial cells. Confocal microscopy further confirmed immunoreactivity of sPLA2‐IIA in reactive astrocytes but not in microglial cells. Taken together, these results demonstrate for the first time an up‐regulation of the inflammatory sPLA2‐IIA in reactive astrocytes in response to cerebral ischemia–reperfusion.

Protein Phosphatase 2A Regulates bim Expression via the Akt/FKHRL1 Signaling Pathway in Amyloid-β Peptide-Induced Cerebrovascular Endothelial Cell Death

Amyloid-β peptide (Aβ)-induced death in cerebral endothelial cells (CECs) is preceded by mitochondrial dysfunction and signaling events characteristic of apoptosis. Mitochondria-dependent apoptosis engages Bcl-2 family proteins, especially the BH3-only homologues, which play a key role in initiating the apoptotic cascade. Here, we report that the expression of bim, but not other BH3-only members, was selectively increased in cerebral microvessels isolated from 18-month-old APPsw (Tg2576) mice, a model of cerebral amyloid angiopathy (CAA), suggesting a pivotal role for Bim in Aβ-induced cerebrovascular degeneration in vivo. A similar expression profile was observed in Aβ-treated CECs. Furthermore, Aβ induction of bim expression involved a pro-apoptotic transcription factor, FKHRL1. FKHRL1 bound to a consensus sequence in the bim promoter region and was activated by Aβ before bim expression. FKHRL1 activity was negatively regulated by phosphorylation catalyzed by Akt, an anti-apoptotic kinase. Akt upregulation by adenoviral gene transfer inhibited Aβ-induced FKHRL1 activation and bim induction. In addition, Aβ increased the activity of protein phosphatase 2A (PP2A), a ceramide-activated protein phosphatase. Suppression of PP2A activity by RNA interference or a specific inhibitor, okadaic acid, effectively suppressed Aβ-induced Akt inactivation and FKHRL1 activation, leading to an attenuation of bim expression and cell death in CECs. Coimmunoprecipitation experiments revealed that Aβ enhanced the binding of the PP2A regulatory subunit PP2ACαβ to Akt. These results implicate PP2A as an early regulator of Aβ-induced bim expression and CEC apoptosis via the Akt/FKHRL1 signaling pathway. We raise the possibility that this pathway may play a role in cerebrovascular degeneration in CAA.

Pyrrolidine dithiocarbamate induces bovine cerebral endothelial cell death by increasing the intracellular zinc level.

Abstract: The antioxidant and metal‐chelating effects of pyrrolidine dithiocarbamate (PDTC) have been extensively studied. PDTC prevents cell death induced by various insults. However, PDTC itself may cause cell death in selected experimental paradigms. PDTC induced bovine cerebral endothelial cell death. However, in serumdepleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. The metal chelators bathocuproine disulfonic acid, o‐phenanthroline, bathophenanthroline disulfonic acid, and N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN) prevented the cell death induced by PDTC. In a serum‐deprived condition, addition of exogenous metals, copper or zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as cooper or zinc in serum may mediate the cytotoxic effect of PDTC. The potency of zinc for PDTC‐induced endothelial cell death was greater than that of copper. Zn‐EDTA did not block PDTC‐induced cell death, whereas Ca‐EDTA and Cu‐EDTA were able to prevent this PDTC effect. PDTC increased the intracellular fluorescence of the zinc probe dye N‐(6‐methoxy‐8‐quinolyl)‐p‐toluenesulfonamide, which was quenched by TPEN or various EDTA preparations but not by Zn‐EDTA. Results suggest that an increase in intracellular zinc concentration is required in PDTC‐induced cerebral endothelial cell death.