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

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

Tumor necrosis factor (TNF) interferes with insulin signaling through the p55 TNF receptor death domain

Tumor necrosis factor (TNF) contributes to insulin resistance by binding to the 55kDa TNF receptor (TNF-R55), resulting in serine phosphorylation of proteins such as insulin receptor (IR) substrate (IRS)-1, followed by reduced tyrosine phosphorylation of IRS-1 through the IR and, thereby, diminished IR signal transduction. Through independent receptor domains, TNF-R55 activates a neutral (N-SMase) and an acid sphingomyelinase (A-SMase), that both generate the sphingolipid ceramide. Multiple candidate kinases have been identified that serine-phosphorylate IRS-1 in response to TNF or ceramide. However, due to the fact that the receptor domain of TNF-R55 mediating inhibition of the IR has not been mapped, it is currently unknown whether TNF exerts these effects with participation of N-SMase or A-SMase. Here, we identify the death domain of TNF-R55 as responsible for the inhibitory effects of TNF on tyrosine phosphorylation of IRS-1, implicating ceramide generated by A-SMase as a downstream mediator of inhibition of IR signaling.     

Induction of apoptosis in hepatocellular carcinoma Smmc-7721 cells by vitamin K2 is associated with p53 and independent of the intrinsic apoptotic pathway

Obesity increases the risk for hepatic steatosis. Recent studies have demonstrated that high fat diet (HFD) may affect sphingolipid formation in skeletal muscles, heart, and other tissues. In this work we sought to investigate whether HFD feeding provokes changes in content and fatty acids (FAs) composition of sphingomyelin and ceramide at the level of liver and hepatic nuclei. Furthermore, we investigated whether the ceramide formation is related to the activity of either neutral sphingomyelinase (N-SMase) or acidic sphingomyelinase (A-SMase). Three weeks of HFD provision induced pronounced ceramide and sphingomyelin accumulation in both liver and hepatic nuclei, accompanied by increased activity of N-SMase but not A-SMase. Furthermore, a shift toward greater FAs saturation status in these sphingolipids was also observed. These findings support the conclusion that HFD has a major impact on sphingolipid metabolism not only in the liver, but also in hepatic nuclei.     

Multiple signaling events in amyloid beta-induced,oxidative stress-dependent neuronal apoptosis

Current evidence suggests that amyloid beta peptides (Abeta) may play a major role in the pathogenesis of Alzheimer's disease by eliciting oxidative stress and neuronal apoptosis. In this study we have used differentiated SK-N-BE neurons to investigate molecular mechanisms and regulatory pathways underlying apoptotic neuronal cell death elicited by Abeta(1-40) and Abeta(1-42) peptides as well as the relationships between apoptosis and oxidative stress. Abeta peptides, used at concentrations able to induce oxidative stress, elicit a classic type of neuronal apoptosis involving mitochondrial regulatory proteins and pathways (i.e. affecting Bax and Bcl-2 protein levels as well as release of cytochrome c in the cytosol), poly-ADP rybose polymerase cleavage and activation of caspase 3. This pattern of neuronal apoptosis, that is significantly prevented by alpha-tocopherol and N-acetylcysteine and completely abolished by specific inhibitors of stress-activated protein kinases (SAPK) such as JNKs and p38(MAPK), involved early elevation of p53 protein levels. Pretreatment of neurons with alpha-pifithrin, a specific p53 inhibitor, resulted in a 50-60% prevention of Abeta induced apoptosis. These results suggest that oxidative stress - mediated neuronal apoptosis induced by amyloid beta operates by eliciting a SAPK-dependent multiple regulation of pro-apoptotic mitochondrial pathways involving both p53 and bcl-2.     

Neutral sphingomyelinase: Localization in rat liver nuclei and involvement in regeneration/proliferation

We have studied the localization of neutral sphingomyelinase (N-SMase) in rat liver nuclei. The levels of neutral sphingomyelinase in regenerating liver nuclei were also assessed.We found that rat liver nuclei contain a sphingomyelinase having a pH optima of 7.2 and a kDa of 92. In intact nuclei, neutral sphingomyelinase was associated predominantly with the nuclear envelope. In regenerating/proliferating rat liver (during DNA synthesis), neutral sphingomyelinase was translocated from the nuclear envelope to the nuclear matrix. The levels of sphingomyelin in whole nuclei decreased in reverse proportion to an increase in the levels of neutral sphingomyelinase. By contrast, there was a corresponding increase in the levels of ceramide and sphingosine during cell regeneration/proliferation. Thus, endogenous nuclear neutral sphingomyelinase may play a role in the regulation of sphingomyelin levels and in relevant signal transduction reactions involving cell regeneration/proliferation. The potential significance of ceramide generation may be aimed at programmed cell death to allow the regeneration of liver mediated via target proteins such as, ceramide activated protein kinases/phospholipases or other unknown mechanisms.Abbreviations N-SMase neutral sphingomyelinase - A-SMase acid sphingomyelinase     

Amyloid induced suicidal erythrocyte death.

Amyloid peptides are known to induce apoptosis in a wide variety of cells. Erythrocytes may similarly undergo suicidal death or eryptosis, which is characterized by scrambling of the cell membrane with subsequent exposure of phosphatidylserine (PS) at the cell surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity and by activation of acid sphingomyelinase with subsequent formation of ceramide. Triggers of eryptosis include energy depletion and isosmotic cell shrinkage (replacement of extracellular Cl(-) by impermeable gluconate for 24 h). The present study explored whether amyloid peptide Abeta (1-42) could trigger eryptosis and to possibly identify underlying mechanisms. Erythrocytes from healthy volunteers were exposed to amyloid and PS-exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca(2+) activity (Fluo3 fluorescence) and ceramide formation (anti-ceramide antibody) were determined by FACS analysis. Exposure of erythrocytes to the amyloid peptide Abeta (1-42) (> or = 0.5 microM) for 24 h significantly triggered annexin V binding, an effect mimicked to a lesser extent by the amyloid peptide Abeta (1-40) (1 microM). Abeta (1-42) (> or = 1.0 microM) further significantly decreased forward scatter of erythrocytes. The effect of Abeta (1-42) (> or = 0.5 microM) on erythrocyte annexin V binding was paralleled by formation of ceramide but not by significant increase of cytosolic Ca(2+) activity. The presence of Abeta (1-42) further significantly enhanced the eryptosis following Cl(-) depletion but not of glucose depletion for 24 hours. The present observations disclose a novel action of Abeta (1-42), which may well contribute to the pathophysiological effects of amyloid peptides, such as vascular complications in Alzheimer's disease.     

p53-Independent ceramide formation in human glioma cells during gamma-radiation-induced apoptosis

Although the p53 tumor-suppressor gene product plays a critical role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents, human glioma cells with functional p53 were more resistant to gamma-radiation than those with mutant p53. U-87 MG cells with wild-type p53 were resistant to gamma-radiation. U87-W E6 cells that lost functional p53, by the expression of type 16 human papillomavirus E6 oncoprotein, became susceptible to radiation-induced apoptosis. The formation of ceramide by acid sphingomyelinase (A-SMase), but not by neutral sphingomyelinase, was associated with p53-independent apoptosis. SR33557 (2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxybphenethyl)amino]propyloxy)benzene-sulfonyl) indolizine, an inhibitor of A-SMase, suppressed radiation-induced apoptotic cell death. In contrast, radiation-induced A-SMase activation was blocked in glioma cells with endogenous functional p53. The expression of acid ceramidase was induced by gamma-radiation, and was more evident in cells with functional p53. N-oleoylethanolamine, which is known to inhibit ceramidase activity, unexpectedly downregulated acid ceramidase and accelerated radiation-induced apoptosis in U87-W E6 cells. Moreover, cells with functional p53 could be sensitized to gamma-radiation by N-oleoylethanolamine, which suppressed radiation-induced acid ceramidase expression and then enhanced ceramide formation. Sensitization to gamma-radiation was also observed in U87-MG cells depleted of functional p53 by retroviral expression of small interfering RNA. These results indicate that ceramide may function as a mediator of p53-independent apoptosis in human glioma cells in response to gamma-radiation, and suggest that p53-dependent expression of acid ceramidase and blockage of A-SMase activation play pivotal roles in protection from gamma-radiation of cells with endogenous functional p53.     

Crocin prevents the death of PC-12 cells through sphingomyelinase-ceramide signaling by increasing glutathione synthesis

Crocin is a pharmacologically active component of Crocus sativus L. (saffron) that has been used in traditional Chinese medicine. In a previous study, we demonstrated that crocin inhibits apoptosis in PC-12 cells by affecting the function of tumor necrosis factor-alpha. In this study, we found that depriving cultured PC-12 cells of serum/glucose causes a rapid increase in cellular ceramide levels, followed by an increase in the phosphorylation of c-jun kinase (JNK). The accumulation of ceramide was found to depend on the activation of magnesium-dependent neutral sphingomyelinase (N-SMase), but not on de novo synthesis. The serum/glucose-deprived PC-12 cells also decreased the cellular levels of glutathione (GSH), which is the potent inhibitor of N-SMase. Treating the PC-12 cells with crocin prevented N-SMase activation, ceramide production, and JNK phosphorylation. We also found that the chemical can enhance the activities of GSH reductase and gamma-glutamylcysteinyl synthase (gamma-GCS), contributing to a stable GSH supply that blocks the activation of N-SMase. Thus our data suggest that crocin combats the serum/glucose deprivation-induced ceramide formation in PC-12 cells by increasing GSH levels and prevents the activation of JNK pathway, which is reported to have a role of the signaling cascade downstream ceramide for neuronal cell death.     

Selective cytotoxicity of intracellular amyloid beta peptide1-42 through p53 and Bax in cultured primary human neurons

Extracellular amyloid beta peptides (Abetas) have long been thought to be a primary cause of Alzheimer's disease (AD). Now, detection of intracellular neuronal Abeta1--42 accumulation before extracellular Abeta deposits questions the relevance of intracellular peptides in AD. In the present study, we directly address whether intracellular Abeta is toxic to human neurons. Microinjections of Abeta1--42 peptide or a cDNA-expressing cytosolic Abeta1--42 rapidly induces cell death of primary human neurons. In contrast, Abeta1--40, Abeta40--1, or Abeta42--1 peptides, and cDNAs expressing cytosolic Abeta1--40 or secreted Abeta1--42 and Abeta1--40, are not toxic. As little as a 1-pM concentration or 1500 molecules/cell of Abeta1--42 peptides is neurotoxic. The nonfibrillized and fibrillized Abeta1--42 peptides are equally toxic. In contrast, Abeta1--42 peptides are not toxic to human primary astrocytes, neuronal, and nonneuronal cell lines. Inhibition of de novo protein synthesis protects against Abeta1--42 toxicity, indicating that programmed cell death is involved. Bcl-2, Bax-neutralizing antibodies, cDNA expression of a p53R273H dominant negative mutant, and caspase inhibitors prevent Abeta1--42-mediated human neuronal cell death. Taken together, our data directly demonstrate that intracellular Abeta1--42 is selectively cytotoxic to human neurons through the p53--Bax cell death pathway.     

The role of NADPH oxidase,neuronal nitric oxide synthase and poly(ADP ribose) polymerase in oxidative neuronal death induced in cortical cultures by brain-derived neurotrophic factor and neurotrophin-4/5

Certain neurotrophins promote or induce oxidative neuronal death in cortical cultures. However, the effector mechanisms mediating this phenomenon have not been delineated. In this study, we investigated the possibility that NADPH oxidase and nitric oxide synthase (NOS) function as such effectors. Western blot analysis showed that treatment with brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-4/5 increased the levels of NADPH oxidase subunits. Moreover, neurotrophin treatment resulted in membrane translocation of p67phox, a characteristic feature of NADPH oxidase activation. Administration of the specific NADPH oxidase inhibitor, 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF), attenuated increases in oxygen free radicals thereby suggesting that NADPH oxidase contributes to the oxidative stress induced by neurotrophins. Furthermore, neuronal death induced by BDNF or NT-4/5 was significantly attenuated by AEBSF. Treatment with BDNF has previously been shown to induce neuronal NOS (nNOS). Our data indicated that inhibitors of nNOS attenuated neuronal death induced by BDNF or NT-4/5, consistent with an active role of nNOS in the mediation of neurotrophin neurotoxicity. As in other models of oxidative cell death, BDNF-induced neuronal death was accompanied by poly(ADP ribose) polymerase (PARP) activation. AEBSF or N-nitro-l-arginine (NNA) reduced BDNF-mediated PARP activation. PARP and poly(ADP ribose) glycohydrolase (PARG) are actively involved in mediating neurotrophin neurotoxicity since inhibitors of PARP and PARG significantly reduced levels of cell death. These results suggest that NADPH oxidase and nNOS contribute to increased oxidative stress, subsequent activation of PARP/PARG, and neuronal death induced by prolonged neurotrophin exposure.     

Ceramide-1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages

It was reported previously that ceramide-1-phosphate (Cer-1-P) is mitogenic for fibroblasts (Gómez-Mu?oz, A., P. A. Duffy, A. Martin, L. O'Brien, H-S. Byun, R. Bittman, and D. N. Brindley. 1995. Mol. Pharmacol. 47: 883-889; Gómez-Mu?oz, A., L. M. Frago, L. Alvarez, and I. Varela-Nieto. 1997. Biochem. J. 325: 435-440). We now show that Cer-1-P prevents cell death in bone-marrow-derived macrophages (BMDMs) after withdrawal of macrophage colony-stimulating factor (M-CSF). Removal of M-CSF is known to induce apoptosis in these cells. Cer-1-P blocked activation of the caspase-9/caspase-3 pathway and prevented DNA fragmentation, indicating that the enhancement of cell survival was due to inhibition of apoptosis. M-CSF deprivation resulted in activation of acid sphingomyelinase (A-SMase), increased ceramide levels, and a decrease in intracellular Cer-1-P. Exogenously added Cer-1-P inhibited A-SMase in intact BMDMs at concentrations that also prevented apoptosis. Cer-1-P also inhibited A-SMase in cell homogenates, suggesting a possible direct physical interaction of Cer-1-P with the enzyme. In conclusion, these data demonstrate that Cer-1-P blocks apoptosis in BMDMs through inhibition of A-SMase, thereby reducing ceramide generation. This adds a new dimension to the understanding of the metabolic interrelationship of ceramides and Cer-1-P, and shows how altering the balance of intracellular levels of these mediators can affect cell survival.     

Effects of gentamicin on sphingomyelinase activity in cultured human renal proximal tubular cells

We have previously shown that cultured human proximal tubular cells (PT) incubated with gentamicin contain numerous "myeloid bodies." This morphological change was accompanied by the storage of phosphatidylcholine and sphingomyelin. In order to delineate the biochemical mechanisms responsible for the accumulation of sphingomyelin in cells incubated with gentamicin, we pursued detailed studies on the activity of sphingomyelinase. Characterization studies on sphingomyelinase revealed that this enzyme has a bimodal pH optima in PT cells. Optimum activity was observed at pH 5.6 (designated as acid sphingomyelinase, A-SMase) and at pH 7.4 (designated as neutral sphingomyelinase, N-SMase). The activity of both the enzymes increased proportionately in control cells as a function of days of incubation. The activity of A-SMase was 16% lower in cells incubated with gentamicin as compared to control. The most striking observation was a gradual decline in the activity of N-SMase in cells incubated with gentamicin. Thus, following 21 days of incubation of cells with 0.3 mM gentamicin, the N-SMase was 2.7-fold lower than control cells. Mg2+ stimulated and Triton X-100 inhibited the activity of N-SMase. Whereas Mg2+ had no effects, Triton X-100 stimulated the activity of the A-SMase in PT cells. Moreover, A-SMase was relatively more heat-resistant than the N-SMase. The Km values for sphingomyelin using A-SMase in control cells and cells incubated with gentamicin were 0.07 X and 0.016 X 10(-7) M, respectively, whereas the Km values for sphingomyelin using N-SMase in control cells and cells incubated with gentamicin were 1.8 X and 1.5 X 10(-7) M, respectively. These findings suggest that gentamicin exerts a competitive inhibition of the A-SMase in PT cells. In contrast, gentamicin exerts a noncompetitive inhibition of the N-SMase in PT cells. Subcellular fractionation studies revealed that A-SMase was exclusively localized in the "lysosome-rich" fraction, whereas most, if not all, the N-SMase was localized in the microsomal fraction and "plasma-membrane"-rich fraction in cultured PT cells. Cells incubated with gentamicin for 21 days contained 25% lower activity of A-SMase associated with the lysosomal fraction as compared to control. In contrast, N-SMase activity in the microsomal and plasma membrane fraction was one-half as compared to control. We conclude that gentamicin-mediated decrease in sphingomyelinase activity may be responsible for the storage of sphingomyelin in cultured human PT cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neutral sphingomyelinase: past, present and future

Sphingomyelin and its metabolic products are now known to have second messenger functions in a variety of cellular signaling pathways. At the epicenter of the sphingomyelin--cell signaling pathway is a family of phospholipases called sphingomyelinases. These enzymes cleave sphingomyelin to produce ceramide and phosphocholine. Ceramide in turn serves as a lipid second messenger that induces a variety of cell regulatory phenomenon such as programmed cell death (apoptosis), cell differentiation, cell proliferation, and sterol homeostasis. Neutral sphingomyelinase (N-SMase) is a Mg2+ sensitive enzyme that can be activated by a host of physiologically relevant and structurally diverse molecules like tumor necrosis factor-alpha (TNF-alpha), oxidized human low density lipoproteins (Ox-LDL), and several growth factors. Large amounts of ceramide accumulate in human fatty streaks and plaques along with Ox-LDL, growth factors, and proinflammatory cytokines in human atherosclerosis. A further role of ceramide and N-SMase in atherosclerosis was uncovered by the finding that Ox-LDL and TNF-alpha stimulated N-SMase activity. In turn, ceramide and/or a homolog serves as an important stress signaling molecule in signal transduction, which leads to apoptosis. Interestingly, an antibody against N-SMase can abrogate Ox-LDL and TNF-alpha induced apoptosis, and therefore may be useful for additional studies of apoptosis in experimental animals. Overexpression of recombinant human N-SMase in human aortic smooth muscle cells markedly stimulate apoptosis, presumably via the multioligomerization of the 'death domain'. Since plaque stability is an integral aspect of atherosclerosis management, activation of N-SMase and subsequent apoptosis may be vital events in the onset of plaque rupture, stroke and heart failure. In contrast to these observations in human hepatocytes, TNF-alpha mediated N-SMase activation did not induce apoptosis. Rather it stimulated the maturation of sterol regulatory element (SRE) binding protein (SREBP-1). Moreover, a cell permeable ceramide was found to reconstitute the phenomenon above in a sterol-independent fashion. These findings provide alternate avenues for therapy of patients with hypercholesterolemia and atherosclerosis. The findings reported here suggests that N-SMase plays important cell regulatory roles and provide an exciting opportunity to further these findings to understand the pathophysiology of human disease states.     

Nerve growth factor-induced p75-mediated death of cultured hippocampal neurons is age-dependent and transduced through ceramide generated by neutral sphingomyelinase.

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75(exonIII-/-) mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase(-/-) mice were as susceptible to NGF-induced death as wild type neurons. The acid ceramidase inhibitor, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.     

Calcium signals induced by amyloid beta peptide and their consequences in neurons and astrocytes in culture

In Alzheimer's disease, amyloid beta (Abeta) peptide is deposited in neuritic plaques in the brain. The Abeta peptide 1-42 or the fragment 25-35 are neurotoxic. We here review our recent explorations of the mechanisms of Abeta toxicity in hippocampal cultures. Abeta had no effect on intracellular calcium in neurons but caused striking changes in nearby astrocytes. The [Ca(2+)](c) signals started approximately 5-15 min after Abeta application and consisted of sporadic [Ca(2+)](c) pulses. These were entirely dependent on extracellular Ca(2+), independent of ER Ca(2+) stores and resulted from Ca(2+) influx, probably through Abeta-induced membrane channels. The Ca(2+) signals were closely associated with transient, episodic acidification which may reflect displacement of protons from binding sites or Ca(2+)/2H(+) exchange. Abeta caused an increased rate of generation of reactive oxygen species (ROS), also seen in astrocytes and not in neurons. The increased ROS generation was blocked by inhibitors of the NADPH oxidase, strongly suggesting that this enzyme, normally associated with immune cells, is expressed in astrocytes. ROS generation was also Ca(2+)-dependent, suggesting that Abeta activation of the enzyme may be secondary to the increase in [Ca(2+)](c). Abeta caused delayed neuronal death despite the fact that all responses were seen only in astrocytes. Neurons could not be protected by glutamate receptor antagonists, but were rescued by inhibition of the NADPH oxidase, by antioxidants and by increasing glutathione. These data suggest that Abeta causes Ca(2+)-dependent oxidative stress by activating an astrocytic NADPH oxidase, and that neuronal death follows through a failure of antioxidant support.     

The role of an astrocytic NADPH oxidase in the neurotoxicity of amyloid beta peptides

Amyloid beta peptide (Abeta) accumulates in the CNS in Alzheimer's disease. Both the full peptide (1-42) or the 25-35 fragment are toxic to neurons in culture. We have used fluorescence imaging technology to explore the mechanism of neurotoxicity in mixed asytrocyte/neuronal cultures prepared from rat or mouse cortex or hippocampus, and have found that Abeta acts preferentially on astrocytes but causes neuronal death. Abeta causes sporadic transient increases in [Ca2+]c in astrocytes, associated with a calcium dependent increased generation of reactive oxygen species (ROS) and glutathione depletion. This caused a slow dissipation of mitochondrial potential on which abrupt calcium dependent transient depolarizations were superimposed. The mitochondrial depolarization was reversed by mitochondrial substrates glutamate, pyruvate or methyl succinate, and by NADPH oxidase (NOX) inhibitors, suggesting that it reflects oxidative damage to metabolic pathways upstream of mitochondrial complex I. The Abeta induced increase in ROS and the mitochondrial depolarization were absent in cells cultured from transgenic mice lacking the NOX component, gp91phox. Neuronal death after 24 h of Abeta exposure was dramatically reduced both by NOX inhibitors and in gp91phox knockout mice. Thus, by raising [Ca2+]c in astrocytes, Abeta activates NOX, generating oxidative stress that is transmitted to neurons, causing neuronal death.     

Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3

Sodium nitroprusside (SNP), a NO donor, has been recognized as an inducer of apoptosis in various cell lines. Here, we demonstrated the intracellular formation of ceramide, a lipid signal mediator, in SNP-induced apoptosis in human leukemia HL-60 cells and investigated the mechanisms of ceramide generation. The levels of intracellular ceramide increased to, at most, 160% of the control level in a time- and dose-dependent manner when the cells were treated with 1 mM SNP. SNP also decreased the sphingomyelin level to approximately 70% of the control level and increased magnesium-dependent neutral sphingomyelinase (N-SMase) activity to 160% of the control activity 2 h after treatment. Neither acid SMase nor magnesium-independent N-SMase was affected by SNP. Caspases are thought to be key enzymes in apoptotic cell death. Acetyl-Asp-Glu-Val-Asp-aldehyde, a synthetic tetrapeptide inhibitor of caspases, inhibited magnesiumdependent N-SMase, ceramide generation, and apoptosis. Moreover, recombinant purified caspase-3 increased magnesium-dependent N-SMase in a cell-free system. These results suggest that the findings that SNP increased ceramide generation and magnesium-dependent N-SMase activity via caspase-3 are interesting to future study to determine the relation between caspases and sphingolipid metabolites in NO-mediated signaling.     

Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer's disease

The proteinase-activated receptors (PARs) are a novel family of G protein-coupled receptors, and their effects in neurodegenerative diseases remain uncertain. Alzheimer's disease (AD) is a neurodegenerative disorder defined by misfolded protein accumulation with concurrent neuroinflammation and neuronal death. We report suppression of proteinase-activated receptor-2 (PAR2) expression in neurons of brains from AD patients, whereas PAR2 expression was increased in proximate glial cells, together with up-regulation of proinflammatory cytokines and chemokines and reduced IL-4 expression (p < 0.05). Glial PAR2 activation increased expression of formyl peptide receptor-2 (p < 0.01), a cognate receptor for a fibrillar 42-aa form of beta-amyloid (Abeta(1-42)), enhanced microglia-mediated proinflammatory responses, and suppressed astrocytic IL-4 expression, resulting in neuronal death (p < 0.05). Conversely, neuronal PAR2 activation protected human neurons against the toxic effects of Abeta(1-42) (p < 0.05), a key component of AD neuropathogenesis. Amyloid precursor protein-transgenic mice, displayed glial fibrillary acidic protein and IL-4 induction (p < 0.05) in the absence of proinflammatory gene up-regulation and neuronal injury, whereas PAR2 was up-regulated at this early stage of disease progression. PAR2-deficient mice, after hippocampal Abeta(1-42) implantation, exhibited enhanced IL-4 induction and less neuroinflammation (p < 0.05), together with improved neurobehavioral outcomes (p < 0.05). Thus, PAR2 exerted protective properties in neurons, but its activation in glia was pathogenic with secretion of neurotoxic factors and suppression of astrocytic anti-inflammatory mechanisms contributing to Abeta(1-42)-mediated neurodegeneration.