Beta-Amyloid Toxicity in Embryonic Rat Astrocytes

Abstract

The senile plaques of Alzheimer's disease contain a high concentration of beta-amyloid (betaA) protein, which may affect the glial population in the septal nucleus, an area of increased risk in AD. BetaA toxicity was measured in septal glia, via a dose-response experiment, by quantifying the effects of three different doses (0.1, 1, and 10 microM) of betaA on cell survival. Astrocytes from embryonic day-16 rats were grown in serum-free media in a single layer culture. Cells were treated on day in vitro (DIV)1 and survival was determined on DIV3 to ascertain which concentration was most toxic. In a separate set of experiments, an attempt was made to protect glial cells from the degenerative effects of betaA, with treatments of growth factors and estrogen. BetaA (10 microM) treatment was administered on DIV1, on DIV2 the cells were treated with estrogen (EST, 10 nM), insulin-like growth factors (IGF1 and IGF2, each 10 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml) or nerve growth factor (NGF, 100 ng/ml), and on DIV3 the cells were visualized and quantified by fluorescence microscopy with DAPI (4,6-diamidino-2-phenylindole). In addition to dose-response and glial protection, experiments were also conducted to determine whether toxic effects were due to apoptosis. Our results suggest that the survival of glial populations is significantly affected in all three concentrations (0.1, 1.0, and 10 microM) of betaA. Glial protection was evident in the presence of NGF, for it showed the significantly highest survival rate relative to the betaA treatment alone. Furthermore, toxic effects of betaA appear to be due primarily to apoptosis. Significant reversal of betaA-induced apoptosis was seen with bFGF and IGF1.

Beta-amyloid toxicity and reversal in embryonic rat septal neurons

“…Some of these effects may be indirectly mediated by the glial cells as in another study [3] we have shown that Aβ decreases survival of glial cells at 0.1, 1.0, and 10 µM concentrations under the same circumstances. The glial cell increase in apoptosis due to Aβ was reversed significantly by bFGF and IGF1.…”

“…Astrocytes from the cerebral cortex were microdissected and cultured as described previously [3] and plated in 100 mm culture dishes (Corning; Corning, NY). Seven days before neuronal dissection, glial cells were transferred to 12 well cell culture cluster dishes.…”

“…Seven days before neuronal dissection, glial cells were transferred to 12 well cell culture cluster dishes. Twenty-four hours before neuronal dissection, medium was replaced with 1 ml N2.1 defined medium [3].…”

“…Neuronal Plane-Sprague-Dawley rat brains were dissected on E16 and were microdissected and cultured as described previously [3]. Neurons were plated on silane treated Poly-L-Lysine coated glass coverslips before treatment [17].…”

Concomitant astroglial atrophy and astrogliosis in a triple transgenic animal model of Alzheimer's disease

“…Several studies have shown that both aggregated Ab protein and the intact cores of Ab plaques isolated from human AD brain tissue, as well as Ab fragments can stimulate astrogliosis, which is also observed in human AD brains (Nagele et al, 2004). Experiments in vitro, have demonstrated that Ab can trigger astroglial Ca 21 signaling, mitochondrial depolarization, and oxidative stress (Abramov et al, 2003(Abramov et al, , 2004; the Ab was also reported to trigger apoptosis in cultured embryonic astrocytes (Assis-Nascimento et al, 2007) and in human AD brains (Kobayashi et al, 2004). Reactive astrocytes were postulated to accumulate large amounts of Ab (Nagele et al, 2003(Nagele et al, , 2004 forming astroglial Ab deposits, although this ability has not been conclusively confirmed.…”