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Recent data have revealed that soluble oligomeric forms of amyloid peptide (Abeta) may be the proximate effectors of the neuronal injury and death occurring in Alzheimer's disease (AD). However, the molecular mechanisms associated with the neuronal cell death induced by the nonfibrillar Abeta remain to be elucidated. In this study, we investigated the role of the cytosolic Ca2+-dependent phospholipase A2 (cPLA2), and its associated metabolic pathway, i.e., the arachidonic acid (AA) cascade, in the apoptotic cell death induced by soluble oligomers of Abeta. The treatment of rat cortical neurons with low concentrations of soluble Abeta(1-40) or Abeta(1-42) peptide resulted in an early calcium-dependent release of AA associated with a transient relocalization of cPLA2. Both cPLA2 antisense oligonucleotides and a selective inhibitor of cPLA2 activity abolished the release of AA from neurons and also protected cells against apoptosis induced by Abeta. Furthermore, inhibitors of the PKC, p38, and MEK/ERK pathways that are involved in cPLA2 phosphorylation and activation reduced Abeta-induced cell death. Finally, we demonstrate that inhibitors of cyclooxygenase-2 reduced the Abeta-induced cell death by 55%. Our studies suggest a novel neuronal response of soluble oligomers of Abeta, which occurs through a cPLA2 signaling cascade and an AA-dependent death pathway. This may prove to be crucial in AD processes and could provide important targets for drug development.

In the present study, we have determined the nature and the kinetics of the cellular events triggered by the exposure of cells to non-fibrillar amyloid-␤ peptide (A␤). When cortical neurons were treated with low concentrations of soluble A␤ (1-40), an early reactive oxygen species (ROS)-dependent cytoskeleton disruption precedes caspase activation. Indeed, caspase activation and neuronal cell death were prevented by the microtubulestabilizing drug taxol. A perturbation of the microtubule network was noticeable after being exposed to A␤ for 1 h, as revealed by electron microscopy and immunocytochemistry. Microtubule disruption and neuronal cell death induced by A␤ were inhibited in the presence of antioxidant molecules, such as probucol. These data highlight the critical role of ROS production in A␤-mediated cytoskeleton disruption and neuronal cell death. Finally, using FRAP (fluorescence recovery after photo bleaching) analysis, we observed a time-dependent biphasic modification of plasma membrane fluidity, as early as microtubule disorganization. Interestingly, molecules that inhibited neurotubule perturbation and cell death did not affect the membrane destabilizing properties of A␤, suggesting that the lipid phase of the plasma membrane might represent the earliest target for A␤. Altogether our results convey the idea that upon interaction with the plasma membrane, the non-fibrillar A␤ induces a rapid ROS-dependent disorganization of the cytoskeleton, which results in apoptosis.A common feature of Alzheimer's disease (AD), 1 the most common form of dementia, is the accumulation and the aggregation of the amyloid-␤ peptide (A␤), a 39-to 43-amino acid peptide derived from the proteolytic cleavage of the amyloid precursor protein (1, 2). Although A␤ represents a key factor in AD (3), the nature of the toxic form of A␤ early involved in AD pathology remains unclear. Whether it is the fibrillar or the non-fibrillar peptides that are the more deleterious remains a controversial issue (4). The amyloid cascade hypothesis causally links AD clinico-pathological process and neuronal cell death to the aggregation and deposition of A␤ (5-7). However, this hypothesis has been challenged by recent evidences indicating that the non-fibrillar A␤ also plays a major role in AD (8, 9). A recent elegant study has demonstrated that the fibrils from AD brain are composed of amyloid peptide moieties arranged at right angles to the backbone of the amyloid P protein wrapped in glycosaminoglycans (10). Thus, the fibrils are not simply made of chains of self-aggregated A␤ and do not comprise long chains of multimeric A␤, similar to those used to evaluate the neurotoxicity of the fibrillar A␤ in vitro and in vivo. Moreover, the synaptic loss in AD brain has been correlated with the soluble pool of A␤ peptides rather than the fibrillar one, implying that the non-fibrillar A␤ may be a crucial pathological factor in AD (11-13). Several studies, based on the use of transgenic mice, have demonstrated that neurodegeneration and specific spatia...

A growing body of evidence supports the notion that soluble oligomeric forms of the amyloid ␤-peptide (A␤) may be the proximate effectors of neuronal injuries and death in the early stages of Alzheimer disease. However, the molecular mechanisms associated with neuronal apoptosis induced by soluble A␤ remain to be elucidated. We recently demonstrated the involvement of an early reactive oxygen species-dependent perturbation of the microtubule network (Sponne, I., Fifre, A., Drouet, B., Klein, C., Koziel, V., Pincon-Raymond, M., Olivier, J.-L., Chambaz, J., and Pillot, T. Microtubules are polymers of ␣-and ␤-tubulin dimers that mediate many functions in neurons, including organelle transport and cell shape establishment and maintenance as well as axonal elongation and growth cone steering in neurons. The polymerization, stabilization, and dynamic properties of microtubules are influenced by interactions with microtubule-associated proteins (MAPs).3 Members of this protein family are classified by size: high molecular mass proteins (MAP1A, MAP1B, MAP2a, and MAP2b) and intermediate molecular mass proteins (MAP2c, MAP2d, and tau) (1-3). Numerous studies have shown that neuronal apoptotic cell death involves alterations of the microtubule network consequent to calpain and effector caspase activation (4, 5). These calcium-dependent proteases are responsible for the degradation and turnover of a broad repertoire of MAP substrates, some of which they share, such as ␣II-spectrin and tau, and some of which are specific, as is the case of calpain-degraded MAP1B and MAP2.Intraneuronal neurofibrillary tangles (NFTs) are one of the histopathological hallmarks in brains of patients diagnosed with Alzheimer disease (AD), a progressive dementia that manifests primarily as a profound inability to form new memories. These NFTs are composed of hyperphosphorylated tau organized into paired helical filaments (PHFs) (6). In addition, AD is also associated with the presence of extracellular senile plaques (7) formed as a consequence of the gradual accumulation and aggregation of the amyloid ␤-peptide (A␤) into fibrils (8). Despite evidence that A␤ represents a key factor in AD (9), the nature of the toxic form of A␤ involved early in AD pathology remains to be determined. The issue of which pool (soluble or aggregated) of A␤ in brain is more deleterious in the early stages of AD is still controversial (10). However, clinicopathological hallmarks of AD correlate far better with the soluble pool of A␤ (11,12). Moreover, several studies in transgenic mice have indicated that specific cognitive deficits, neurodegeneration, and synaptic loss might occur before any histologically detectable formation of senile plaques (13,14). So, in reports from our group (15-18) and others (19,20), attention has been paid to the soluble oligomeric forms of A␤ as the principal mediators of neurodegeneration in the early stages of AD development (10, 19 -23).Increasing evidence suggests that the selective neuronal cell death in AD involves activation of caspa...

A growing body of evidence supports the notion that soluble oligomers of amyloid-b (Ab) peptide interact with the neuronal plasma membrane, leading to cell injury and inducing deathsignalling pathways that could account for the increased neurodegeneration occurring in Alzheimer's disease (AD). Docosahexaenoic acid (DHA, C22:6, n-3) is an essential polyunsaturated fatty acid in the CNS and has been shown in several epidemiological and in vivo studies to have protective effects against AD and cognitive alterations. However, the molecular mechanisms involved remain unknown. We hypothesized that DHA enrichment of plasma membranes could protect neurones from apoptosis induced by soluble Ab oligomers. DHA pre-treatment was observed to significantly increase neuronal survival upon Ab treatment by preventing cytoskeleton perturbations, caspase activation and apoptosis, as well as by promoting extracellular signal-related kinase (ERK)-related survival pathways. These data suggest that DHA enrichment probably induces changes in neuronal membrane properties with functional outcomes, thereby increasing protection from soluble Ab oligomers. Such neuroprotective effects could be of major interest in the prevention of AD and other neurodegenerative diseases.

Neuronal cell death in Alzheimer's disease (AD) is partly induced by the interaction of the amyloid-beta peptide (Abeta) with the plasma membrane of target cells. Accordingly, recent studies have suggested that cholesterol, an important component of membranes that controls their physical properties and functions, plays a critical role in neurodegenerative diseases. We report here that the enrichment of the neuronal plasma membrane with cholesterol protects cortical neurons from apoptosis induced by soluble oligomers of the Abeta(1-40) peptide. Conversely, cholesterol depletion using cyclodextrin renders cells more vulnerable to the cytotoxic effects of the Abeta-soluble oligomers. Increasing the cholesterol content of small unilamellar liposomes also decreases Abeta-dependent liposome fusion. We clearly demonstrate that cholesterol protection is specific to the soluble conformation of Abeta, because we observed no protective effects on cortical neurons treated by amyloid fibrils of the Abeta(1-40) peptide. This may provide a new opportunity for the development of an effective AD therapy as well as elucidate the impact of the cholesterol level during AD development.

We recently demonstrated that the 118 -135 putative transmembrane domain of prion protein (PrP) exhibited membrane fusogenic properties and induced apoptotic neuronal cell death of rat cortical neurons, independently of its aggregation state. The aim of the present study was to analyze the in vivo neurotoxicity of the prion fragment P118 -135 and to evaluate the potential role of the physiological isoform of PrP in the P118 -135-induced cell death. Here, we demonstrate that the nonfibrillar P118 -135 is cytotoxic to retinal neurons in vivo as monitored by intravitreal inoculation and recording of the electrical activity of retina and tissue examination. Moreover, knock-out PrP gene mice exhibit similar sensitivity to the nonfibrillar P118 -135-induced cell death and electrical perturbations, strongly suggesting that cell death occurs independently of PrP expression. Interestingly, a variant nonfusogenic P118 -135 peptide (termed P118 -135) had no effects on in vivo neuronal viability, suggesting that the P118 -135-induced cell death is mediated by its membrane destabilizing properties. These data have further been confirmed in vitro. We show that the fusogenic peptide P118 -135 induces death of cultured neurons from both wild-type and knock-out PrP gene mice via an apoptotic-mediated pathway, involving early caspase activation and DNA fragmentation. Altogether these results emphasize the neurotoxicity of the fusogenic nonfibrillar PrP transmembrane domain and indicate that fibril formation and PrP expression are not obligatory requirements for neuronal cell death. The use of synthetic prion peptides could provide insights into the understanding of neuronal loss mechanisms that take place during the development of the various types of spongiform encephalopathies.

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