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Cytosolic phospholipase A₂ mediates neuronal apoptosis induced by soluble oligomers of the amyloid-ß peptide

Badreddine Kriem^*, Isabelle Sponne^*, Alexandre Fifre^*, Catherine Malaplate-Armand^*, Karine Lozac’h-Pillot^*, Violette Koziel^*, Frances T. Yen-Potin, Bernard Bihain, Thierry Oster^*, Jean-Luc Olivier^* and Thierry Pillot^*,,1

^* Inserm EMI 0014, Université de Nancy I, Vandoeuvre, France; and
Laboratoire de Médecine et Thérapeutique Moléculaire, Vandoeuvre, France

¹Correspondence: Inserm EMI n°0014, Université de Nancy I, Faculté de Médecine, 9 Ave. de la Forêt de Haye, Vandoeuvre-les-Nancy 54505, France. E-mail: thierry.pillot{at}medecine.uhp-nancy.fr

SPECIFIC AIMS

We investigated whether the activation of the cytoplasmic calcium-dependent phospholipase A₂ (cPLA₂) and the arachidonic acid cascade were involved in the neuronal apoptosis induced by soluble oligomers of Aß peptide.

PRINCIPAL FINDINGS

1. Soluble oligomers of Aß peptide induced a rapid and calcium-dependent release of arachidonic acid from cortical neurons
This release of arachidonic acid is cPLA₂ dependent.

2. The release of arachidonic acid from cells upon soluble Aß treatment was specifically inhibited by cPLA₂ antisense oligonucleotides and was associated with a subcellular relocalization of cPLA₂

3. cPLA₂ antisense oligonucleotides inhibited both the decrease of cell viability and the apoptotic events induced by soluble oligomers of Aß(1–40) and Aß(1–42) peptides (Fig. 1 )

View larger version (15K): [in this window] [in a new window]   Figure 1. cPLA2 is involved in the neurotoxicity of Aß. To test whether cPLA2 is involved in the apoptotic pathway induced by Aß, cortical neurons were preincubated for 48 h with 4 µM antisense or sense cPLA2 ODNs, then treated for 24 h in the presence of 1 µM Aß. Cell viability was monitored using the MTT assay (A) and apoptotic nuclei visualized and quantified after DAPI staining (B). Both cPLA2 antisense ODNs antagonized neuronal cell death and apoptosis induced by nonfibrillar Aß, whereas sense ODNs used as negative controls had no effect. No significant differences were found between control cells and cells treated with sense or antisense ODN only.

4. The pharmacological inhibition of the arachidonic acid metabolic pathway prevented both the release of arachidonic acid from cortical neurons and the cell death induced by soluble oligomers of the Aß peptide (Fig. 2 )
A specific inhibitor of cPLA₂ rescued neurons from Aß peptide-induced cell death whereas inhibitors of secreted and calcium-independent cPLA₂ had no effect.

View larger version (14K): [in this window] [in a new window]   Figure 2. Inhibiting AA metabolic pathway prevents AA release and cell death induced by Aß. Cortical neurons were preincubated for 2 h with 25 µM MAFP, 1 µM PD98059, 5 µM SB202190, or 50 µM calphostin C, then incubated with 1 µM Aß. The release of AA was monitored as described above after 10 min of incubation (A). The cell viability was measured using the MTT assay (B) and apoptotic nuclei visualized and quantified using DAPI staining (C) after 24 h of incubation with Aß.

5. The activation of cPLA₂ and subsequent cell death induced by soluble oligomers of Aß peptide were shown to be MAP kinase dependent

6. We demonstrated that cyclooxygenase-2, which uses arachidonic acid as a substrate for eicosanoid production, participated in the apoptotic pathway induced by soluble Aß peptide

CONCLUSIONS AND SIGNIFICANCE

There is considerable evidence that phospholipid metabolism is altered in Alzheimer’s disease (AD) and that membrane defects contribute significantly to disease pathology. Increases in free fatty acids, eicosanoids, and products of lipid peroxydation are well known to occur early during progression of AD, leading to the hypothesis that phospholipases play a role in the production of second messengers involved in neurodegenerative disorders, specifically during early stages of AD development. Increased immunoreactivity of the cytosolic Ca²⁺-dependent phospholipase A₂ (cPLA₂) and of cPLA₂ transcript are observed in AD cortex as compared with that of age-match control subjects. The issue addressed in this study was to determine whether cPLA₂ signaling contributes to neuronal cell death. This work identifies a novel mechanism for soluble Aß-induced neuronal apoptosis. Our results reveal a causal relationship between Aß exposure and the activation of a cPLA₂ pathway, which is likely to involve AA and COX-2 derived metabolites. The novelty and physiological relevance of our results is that calcium-dependent cPLA₂ mediates the neuronal apoptosis induced by soluble oligomers of Aß. The beneficial effects of inhibiting the cPLA₂ metabolic pathway may therefore represent a novel therapeutic approach against Aß-induced apoptosis.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic diagram. The cytosolic phospholipase A2 mediates neuronal apoptosis induced by soluble oligomers of the amyloid-ß peptide. Due to the fusogenic properties of the soluble Aß peptide, soluble oligomers of Aß interact with the lipid phase of the plasma membrane and induce a rapid biphasic modification of the fluidity of the membrane. These physical modifications of the membrane result in activation and translocation of cPLA2 and of the arachidonic acid cascade. Inhibitors of cPLA2 and COX-2 significantly decrease apoptosis of cortical neurons induced by soluble Aß peptide. We demonstrated that neuronal apoptosis induced by soluble oligomers of Aß proceeds through an early perturbation of the cytoskeleton of cortical neurons and caspase activation. Further investigations are required to determine whether the cPLA2 pathway is involved in modification of the microtubule network.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1807fje;

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