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Membrane cholesterol interferes with neuronal apoptosis induced by soluble oligomers but not fibrils of amyloid-ß peptide¹

ISABELLE SPONNE^*,2, ALEXANDRE FIFRE^*,2, VIOLETTE KOZIEL^*, THIERRY OSTER, JEAN-LUC OLIVIER^* and THIERRY PILLOT^*,3

^* INSERM EMI 0014, University of Nancy I, Vandoeuvre, France; and
LIMBP, University of Metz, Metz, France

³Correspondence: INSERM EMI 0014, Université de Nancy I, 9 Avenue de la Forêt de Haye, BP 184, 54505 Vandoeuvre, France. E-mail: thierry.pillot{at}bcmn.facmed.u-nancy.fr

SPECIFIC AIMS

We investigated whether cholesterol content of plasma membrane of cortical neurons could modulate neurotoxicity induced by soluble oligomers of Aß peptide.

PRINCIPAL FINDINGS

1. Enrichment of plasma membrane with exogenous cholesterol protected cortical neurons from soluble Aß(1-40) peptide-induced neurotoxicity in a time- and dose-dependent manner

2. Neuroprotective effects of cholesterol did not involved a direct interaction between cholesterol and Aß(1-40) peptide, but rather a modulation of physical properties of the lipid bilayer due to cholesterol insertion

3. Cholesterol enrichment inhibits apoptotic events induced by soluble oligomers of Aß peptide, including cytoskeleton perturbations, fragmentation and condensation of nuclear DNA, caspase-3 activation and production of reactive oxygen species.

By contrast, cholesterol depletion rendered cells more susceptible to Aß peptide-induced neurotoxicity.

4. Enrichment of the plasma membrane by cholesterol had no effect on neurotoxicity of amyloid fibrils formed by Aß(1-40) peptide

5. Using model membrane, we demonstrated that increase of cholesterol molar ratio within liposomes inhibited their aggregation and fusion induced by Aß(1-40) peptide

6. We obtained similar data when using C-terminal fusogenic Aß(29-40) peptide to induce apoptotic cell death and liposome fusion

These data strongly suggest that cholesterol might interfere with Aß-induced cell death by modifying the structure and physical properties of plasma membrane of cortical neurons.

CONCLUSIONS AND SIGNIFICANCE

Our results demonstrate that cholesterol controls sensitivity of primary cortical neurons to soluble oligomeric Aß. Model membrane and cell studies (Fig. 1 and Fig. 2 ) lead to the same general conclusion: by modulating fluidity of membranes, cholesterol content of neuronal membranes may specifically influence insertion of soluble Aß in plasma membrane and its properties to disturb membrane structure and ultimately to induce cell death. Our data show no inhibitory effect of cholesterol toward fibrillar Aß(1-40) peptide-induced toxicity of primary cortical neurons, and support the idea that modulation of cholesterol and/or other factors for treatment of AD will depend on which Aß conformations are mainly involved during the disease (Fig. 3 ).

View larger version (84K): [in this window] [in a new window]   Figure 1. Cholesterol enrichment protects cortical neurons from soluble Aß peptide-induced cytoskeleton disruption. Cells were preincubated for 3 h in the presence of 10 µg/mL cholesterol and further exposed to 5 µM soluble Aß(1-40) for 24 h. The microtubule network was visualized using immunofluorescence with an anti ß-tubulin monoclonal antibody (A). No differences were found between cholesterol-treated and control cells. B) Microtubule perturbations induced by soluble Aß(1-40) peptide were quantified at indicated time using immunofluorescence with an anti ß-tubulin monoclonal antibody. Data are means (±SE) of 3 different experiments with 4 determinations each and are normalized to the effect of vehicle, designated as 100% (*P<0.05; **P<0.01; ***P<0.001). Differences among subgroups for each condition were performed by ANOVA followed by a Scheffe's post hoc test. #P <0.05, between cells treated with Aß alone and cells treated with Aß in the presence of cholesterol. No significant differences were found between cholesterol-treated and control cells.

View larger version (22K): [in this window] [in a new window]   Figure 2. Cholesterol enrichment inhibits apoptotic events induced by soluble Aß peptide. Cells were pre-incubated for 3 h in the presence of 10 µg/mL cholesterol and further exposed to 5 µM soluble Aß(1-40) for 24 h. Apoptotic nuclei were visualized and quantified after DAPI staining (A) and oligonucleosome production was monitored using an ELISA test (B). Activation of caspase-3 (C) and production of reactive oxygen species (D) were monitored. Data are means (±SE) of 3 different experiments with 4 determinations each and are normalized to the effect of vehicle, designated as 100%. No significant differences were found between caspase inhibitor treated and control cells.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic diagram. Cholesterol level in the plasma membrane of cortical neurons specifically modulates cell death triggered by soluble oligomers of Aß peptide. 1) Due to fusogenic properties of soluble Aß peptide (18 and 38), soluble oligomers of Aß interact with the lipid phase of plasma membrane and induce a rapid biphasic modification of the fluidity of the membrane. These physical modifications of the membrane result in an early perturbation of the cytoskeleton of cortical neurons and subsequent cell death. Cell death induced by soluble oligomers of Aß is prevented by cholesterol-dependent stiffness of the membrane. 2) By contrast, Aß fibrils may interact with an unknown receptor leading to activation of caspase 8 and a further caspase-dependent cell death.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0372fje;

² These authors contributed equally to this work.

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