Beta-amyloid toxicity and reversal in embryonic rat septal neurons.

Alzheimer's disease is characterized mainly by loss of neurons from the septal nucleus. In this study, neurons from the septal nucleus of the embryonic day 16 (E16) rat were grown in culture with a plane of astrocytes from the embryonic rat and in a defined medium in the absence of serum. Neurons were treated with beta-amyloid (Abeta: 0.1, 1 and 10 microM) on day in vitro (DIV) 1 and DIV 4 and fluorescent microscopy was used to measure survival and apoptosis following exposure of the treated cells on DIV 7. Reversal of neurotoxicity was studied using the potentially neuroprotective agents nerve growth factor (NGF, 100 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml), insulin-like growth factors (IGF1 and IGF2, 10 ng/ml) and estrogen (10 nM), administered on DIV 4 and DIV 5, that is, subsequent to the Abeta (10 microM)-induced neurotoxicity. Abeta caused a significant decrease in survival at 10 microM, and a significant increase in apoptosis at 0.1 and 10 microM. IGF1, IGF2 and bFGF all caused a reversal of the Abeta-induced neurotoxic effect on survival while NGF and estrogen did not under these experimental conditions.

Beta-amyloid toxicity in embryonic rat astrocytes.

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.

Reversal of ethanol toxicity in embryonic neurons with growth factors and estrogen.

Prenatal exposure to ethanol is the cause of fetal alcohol syndrome, which is characterized by brain abnormalities and decreased mental capacity. In the current study, cultured neurons from embryonic rat cortices were used to study the reversal of ethanol toxicity on neuronal survival and neurite outgrowth. Ethanol treatment followed by treatment with estrogen and certain growth factors were used to assess the potential of these growth factors and estrogen to reverse the effects of ethanol damage. Cortical neurons from embryonic day (E) 16 rats were grown in defined medium with a glial plane at a distance of 1mm from the neurons. Ethanol (45 mM) was administered on day in vitro 1 (DIV 1) and DIV 4. Insulin-like growth factor-I (IGF-I, 10 ng/ml), insulin-like growth factor-II (IGF-II, 10 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml), nerve growth factor (NGF, 100 ng/ml), and estrogen (Es, 10 ng/ml) were administered on DIV 4 and DIV 5. Cell viability was determined on DIV 6 using the intravital dyes fluorescein diacetate and propidium iodide. IGF-I and bFGF reduced ethanol's toxic effect on neuronal survival. Estrogen, bFGF, and NGF increased total neurite length after ethanol treatment. Although none of the treatments had a statistically significant effect on the mean number of primary neurites, all caused a statistically significant increase in the mean number of secondary neurites per cell (a measure of neuritic branching) relative to the ethanol treatment alone.

Effects of ethanol on cultured embryonic neurons from the cerebral cortex of the rat.

Fetal alcohol syndrome is a serious disorder that causes lifelong learning, memory, and behavioral problems. In the current study, we determined the ethanol concentrations that produced detrimental effects on the development of embryonic cortical neurons because mental capacity seems to be proportional to the level of dendritic arborization. Neurons from fetal rat cortices were grown in culture in close proximity to a glial plane. The cells were treated with concentrations of ethanol ranging from 450 nM to 45 mM, and neurite outgrowth was subsequently quantified. A significant decrease in dendritic branching was observed at ethanol concentrations as low as 45 microM after 6 days of ethanol exposure in vitro, whereas changes in primary neurite outgrowth were observed at an ethanol concentration of 4.5 microM. This finding is of particular interest as it seems to indicate that occasional ethanol exposure is detrimental to cortical development at very low concentrations of ethanol.

Tyrosine kinase activity of nerve growth factor and estrogen in embryonic septal neurons cultured from the rat.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia, senile plaques, fibrillary tangles, and a reduction of cholinergic neurons in the septal nucleus of the brain. Nerve growth factor (NGF) and estrogen were studied to observe effects on tyrosine kinase activity in septal neurons. The time course of tyrosine kinase activation and number of cells in which tyrosine kinase was activated were measured. Tissue from embryonic day 16 rats was microdissected and the septal neurons obtained were treated with estrogen (10 microM) or NGF (100 ng/mL) at intervals of 1, 2, 3, 4, 5, or 10 min. Immunostaining for phosphotyrosine revealed that cells treated with NGF showed an increase in phosphotyrosine activity within 2-4 min followed by a decline to control levels of enzyme activity. Treatment with estrogen led to an increase in phosphotyrosine immunostaining within 2-3 min followed by a decline to control levels. This time course suggests a mechanism for estrogen activity other than the traditional method involving binding to nuclear receptors followed by protein synthesis.