Figure 5.

The oligomeric Aβ induced the TNF-α expression in glial primary cultures. A, Dose–response stimulation of the TNF-α expression by oligomeric and monomeric Aβ42 assessed in vitro in astromicroglial cultures. PBS and LPS (1 μg/ml) were used as negative and positive controls, respectively. Data are mean ± SD from three independent cultures and Aβ42 preparations. B, Stimulation of the glial cultures using the S1 soluble fractions. Increasing protein amounts (from 5 to 100 μg) of the different S1 fractions (6 and 18 months of age; WT and PS1xAPP; n = 3 per age and group) was added to the cultures. In parallel, PBS, monomeric (20 μm), oligomeric Aβ42 (20 μm) and LPS (1 μg/ml) were included as negative and positive controls, respectively. For each experiment, duplicate culture wells were used. This experiment was repeated twice, using independent cultures. Only the soluble extract from 18-month-old PS1xAPP produced the stimulation of the TNF-α expression in these experiments (asterisk; p < 0.05, Tukey's test). C, Immunodepletion experiments. In these experiments, the Aβ content from the S1 fractions (10 μg of protein) of three different 18-month-old PS1xAPP mice was immunodepleted by three sequential immunoprecipitations using either 6E10-Protein G-Sepharose or A11-Protein A-Sepharose complexes. After immunodepletion, the S1 fractions were used in stimulation experiments. In parallel, the different S1 fractions were treated with Protein G-Sepharose or Protein A-Sepharose. We observed no differences in the TNF-α stimulation between these control S1 fractions and the results were pooled. The immunodepletion of Aβ, by any of the antibodies used, precluded the stimulatory effect of the S1 fractions. PBS, oligomeric Aβ42 (20 μm) and LPS (1 μg/ml) were included as negative and positive controls, respectively.