Elsevier

Neurobiology of Disease

Volume 17, Issue 1, October 2004, Pages 99-107
Neurobiology of Disease

Neutral sphingomyelinase activation in endothelial and glial cell death induced by amyloid beta-peptide

https://doi.org/10.1016/j.nbd.2004.06.001Get rights and content

Abstract

We have explored the molecular mechanism underlying amyloid beta-peptide (Aβ)-mediated cytotoxicity in vitro. Exposure of murine cerebral endothelial cells (CECs) or C6 glioma cells to Aβ25-35 resulted in dose-dependent cell death. Ceramide is a pro-apoptotic lipid mediator. Forced elevation of cellular ceramide levels, either by application of an exogenous C2 ceramide analogue or bacterial sphingomyelinase that induces endogenous ceramide release from sphingomyelin, mimicked Aβ25–35 cytotoxicity in both CECs and C6 glioma cells. Aβ25–35-induced synthesis of ceramide was selectively mediated by activation of neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase (aSMase) or ceramide synthase. Both 3-O-Me-SM and N-acetyl-l-cysteine, the selective and nonselective pharmacological inhibitors of nSMase, respectively, suppressed nSMase activation, ceramide production, and cytotoxic action induced by Aβ25–35 in CECs. Furthermore, genetic knockdown of nSMase by an antisense strategy rendered C6 glioma cells specifically resistant to Aβ25–35 cytotoxicity without affecting their vulnerability to serum deprivation. Together, nSMase activation with subsequent ceramide production may contribute, at least partially, to Aβ25–35 cytotoxicity in cell types with cerebral endothelial and glial lineage.

Introduction

Cerebrovascular amyloid deposition is a prominent pathological feature in the brain of patients with cerebral amyloid angiopathy and in some patients with Alzheimer's disease (AD) Kalaria, 1997, Thomas et al., 1997. The major component of amyloid deposits is amyloid beta-peptide (Aβ), a 39–43 amino acid fragment derived from amyloid precursor protein (Estus et al., 1992). Aβ is a key mediator of neuronal degeneration in AD (Selkoe, 1991) and may also contribute to cerebrovascular pathology in AD brains (Thomas et al., 1996). Aβ is cytotoxic not only to neurons Pike et al., 1997, Shearman et al., 1994, Yankner et al., 1989, Yu et al., 1998, Zhang et al., 1994, but also to non-neuronal cells such as endothelial cells (Thomas et al., 1996), including those derived from brain (Huang et al., 1998), vascular smooth muscle cells Davis-Salinas et al., 1995, Kawai et al., 1993, and oligodendrocytes Lee et al., 2004, Xu et al., 2001a. Aβ cytotoxicity is accompanied by enhanced oxidative stress Behl et al., 1994, Davis, 1996, Schapira, 1996, Ca2+ influx (Suo et al., 1997), K+ efflux (Yu et al., 1998), activation of microglia to produce reactive nitrogen intermediates and tumor necrosis factor-alpha (TNF-α) (Meda et al., 1995), impairment of glucose transport (Blanc et al., 1997), and the induction of cytokine expression and secretion (Suo et al., 1998b). However, the molecular mechanism underlying Aβ cytotoxicity remains to be fully delineated.

Aβ-induced apoptosis in selected cell types may involve cell death receptor mechanism engaging p75 neurotrophin receptor and Fas ligand de la Monte et al., 1997, Yaar et al., 1997. Fas and p75 receptor activation in the apoptotic cascade is mediated by the sphingomyelin/ceramide signal processes Dobrowsky et al., 1995, Hannun, 1996. The sphingomyelin–ceramide pathway is a ubiquitous and evolutionarily conserved signaling system that links specific cell surface receptors and environmental stress to the nucleus. Ceramide is a lipid mediator of several cellular processes including apoptosis (Verheij et al., 1996). Three enzymes have been established in the ceramide biosynthetic pathways. They are plasma membrane-associated neutral sphingomyelinase (nSMase), lysosomal acidic sphingomyelinase (aSMase), and ceramide synthase (Hannun, 1996). All three have been implicated in selective cell death paradigms Hannun, 1996, Kolesnick and Kronke, 1998, Verheij et al., 1996. AD brains contain lower levels of sphingomyelin (Soderberg et al., 1992), raising the possibility of increased ceramide formation.

In the present study, we explored the link of ceramide biosynthetic pathways to Aβ25–35 cytotoxicity in cell types with cerebral endothelial and glial lineage. We demonstrate that nSMase activation with subsequent generation of ceramide contributes, at least in part, to Aβ25–35 cytotoxicity in vitro. These findings are in agreement with our recent demonstration that nSMase is involved in Aβ-mediated oligodendroglial apoptosis (Lee et al., 2004).

Section snippets

Reagents and cell culture

All the reagents were from Sigma (St. Louis, MO) unless otherwise specified. Previously, we have reported that Aβ1–40 and Aβ25–35 were of equal potency in inducing apoptosis in CECs derived from bovine and murine brains (Xu et al., 2001b) and oligodendrocytes (Xu et al., 2001a). In the present studies on Aβ activation of nSMase, Aβ25–35 was used exclusively. Murine CECs, a brain endothelial cell line stably overexpressing polyoma middle T antigen, were a generous gift of Dr. William A. Frazier,

Induction of CEC and C6 glioma cell death by Aβ25–35

Exposure of CECs to Aβ25–35 caused dose-dependent cell death based on both MTT reduction (Fig. 1A) and trypan blue exclusion (Fig. 1B) assays. Since Aβ1–40 and Aβ25–35 showed approximately equal potency in inducing CEC death (Xu et al., 2001b), effects of Aβ1–40 were not repeated here. Aβ25–35 also dose-dependently led to C6 glioma cell death based on the results of LDH release (Fig. 1C) and MTT reduction (Fig. 1D) assays. Together with our previous findings demonstrating Aβ cytotoxicity in

Discussion

A potential role of altered brain lipid composition in AD pathogenesis is emerging. Both AD brains (Soderberg et al., 1992) and mutant presenilin 2 transgenic mice mimicking early-onset familial AD (Sawamura et al., 2000) contained significantly lower levels of sphingomyelin. Sphingomyelin degradation may lead to the release of ceramide. Thus, reduced sphingomyelin content in AD brains may indirectly imply a higher ceramide level. Activation of sphingolipid turnover with chronic generation of

Acknowledgements

This work was supported by an NIH grant (NS41525) and a National Science Council in Taiwan grant (NSC92-2321-B-038-003) to Chung Y. Hsu and National Science Council in Taiwan (NSC92-2314-B-320-003) to Ding-I Yang.

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