Amyloid beta (Abeta) oligomers accumulate in brain tissue of Alzheimer disease patients and are related to pathogenesis. The precise mechanisms by which Abeta oligomers cause neurotoxicity remain unresolved. In this study, we investigated the role of ionotropic glutamate receptors on the intracellular Ca2+ overload caused by Abeta. Using rat cortical neurons in culture and entorhinal-hippocampal organotypic slices, we found that Abeta oligomers significantly induced inward currents, intracellular Ca2+ increases and apoptotic cell death through a mechanism requiring NMDA and AMPA receptor activation. The massive entry of Ca2+ through NMDA and AMPA receptors induced by Abeta oligomers caused mitochondrial dysfunction as indicated by mitochondrial Ca2+ overload, oxidative stress and mitochondrial membrane depolarization. Importantly, chronic treatment with nanomolar concentration of Abeta oligomers also induced NMDA- and AMPA receptor-dependent cell death in entorhinal cortex and hippocampal slice cultures. Together, these results indicate that overactivation of NMDA and AMPA receptor, mitochondrial Ca2+ overload and mitochondrial damage underlie the neurotoxicity induced by Abeta oligomers. Hence, drugs that modulate these events can prevent from Abeta damage to neurons in Alzheimer's disease.

SummaryNeurotoxic effects of amyloid b peptides are mediated through deregulation of intracellular Ca 2+ homeostasis and signaling, but relatively little is known about amyloid b modulation of Ca 2+ homeostasis and its pathological influence on glia. Here, we found that amyloid b oligomers caused a cytoplasmic Ca 2+ increase in cultured astrocytes, which was reduced by inhibitors of PLC and ER Ca 2+ release. Furthermore, amyloid b peptides triggered increased expression of glial fibrillary acidic protein (GFAP), as well as oxidative and ER stress, as indicated by eIF2a phosphorylation and overexpression of chaperone GRP78. These effects were decreased by ryanodine and 2APB, inhibitors of ryanodine receptors and InsP 3 receptors, respectively, in both primary cultured astrocytes and organotypic cultures of hippocampus and entorhinal cortex. Importantly, intracerebroventricular injection of amyloid b oligomers triggered overexpression of GFAP and GRP78 in astrocytes of the hippocampal dentate gyrus. These data were validated in a triple-transgenic mouse model of Alzheimer's disease (AD). Overexpression of GFAP and GRP78 in the hippocampal astrocytes correlated with the amyloid b oligomer load in 12-month-old mice, suggesting that this parameter drives astrocytic ER stress and astrogliosis in vivo. Together, these results provide evidence that amyloid b oligomers disrupt ER Ca 2+ homeostasis, which induces ER stress that leads to astrogliosis; this mechanism may be relevant to AD pathophysiology.

A role for neuropeptide receptors in glial tumorigenesis has recently been proposed. Although angiotensin receptors are known to mediate proliferative effects in many cell types, including brain astrocytes, the possible participation of these receptors in glial tumorigenesis remains unknown. In the present study, we have examined the expression of the molecularly defined angiotensin receptor subtypes AT(1a), AT(1b), and AT(2) in normal perinatal rat astrocytes and in a panel of tumor adult astrocytoma cells, using the reverse transcriptase-polymerase chain reaction (RT-PCR). Subsequently, we compared the mitogenic effect of the angiotensins A(1-8), A(2-8), A(3-8) and the heptapeptide "metabolite" A(1-7), on both normal and tumor astrocytes, measured in terms of the incorporation of tritiated thymidine. Our results indicate that AT(1a), AT(1b), and AT(2) angiotensin receptor mRNA is commonly expressed by many of these cells. Of notable exception is the astrocytoma U373 which was not found to express AT(1) or AT(2) mRNA. Chronic (24-h) incubation of cells with A(1-8) and A(1-7) lead to the induction of mitogenesis, even in the AT(1) and AT(2) mRNA negative astrocytoma cell line U373. Moreover, pharmacological analysis indicated that the observed mitogenic effects are not mediated by the AT(1) or AT(2) type receptors, but rather by a novel, specific A((1-7)) angiotensin receptor, since mitogenesis was shown to be partially blocked by the A(1-7) analogue D-Ala(7)A(1-7) and by the protease inhibitor orthophenanthroline (100 microM). Using Fura-2 spectrophotometry, we found that activation of this receptor does not alter intracellular calcium levels; however, preincubation with the protein kinase kinase inhibitor U0126 (10 microM) was found to inhibit these mitogenic effects partially. Overall, these results which demonstrate that normal and tumor astrocytes express a greater variety of angiotensin receptor subtypes than previously thought, support the idea that A(1-7) and its receptor signaling system may play an important role in shaping the astrocyte population during development. Moreover, the untimely expression of this A((1-7)) receptor may represent an important etiological component in the development of brain astrocytomas.