FIGURE 3:

Neuron-bound AβOs are released into the media due to the action of astrocyte-derived insulin and IGF1. (A) Primary hippocampal neurons were grown on coverslips and exposed to AβOs. Unbound AβOs were quickly removed by submerging coverslips in excess MEM. Coverslips were transferred to new dishes containing MEM supplemented with growth factors. (B) Dot blot analysis of the media showed ACM contained AβOs released from neurons (red bar). This effect was also observed when MEM was supplemented with 300 nM insulin (blue bar) or IGF1 (green bar) or 10 μM demethylasterriquinone B1 (DB1). Significant release was not observed after supplementation with 300 nM EGF, NGF, or BDNF. Treatment with AG 1024, an antagonist of insulin and IGF1 receptors, prevented ACM from stimulating AβO release. (C) IDE treatment of ACM reduced its ability to stimulate AβO liberation by 56 ± 5%. (D) Insulin and IGF1 mRNAs were detectable in cultured astrocytes by RT-PCR at 1:100 and 1:1000 dilutions. RT-PCRs without cDNA did not yield a detectable product. (E, F) The detection of AβOs in the media was accompanied by a reduction in AβO immunofluorescence (NU1, green) along neurites (TuJ, red). ACM and insulin reduced neuritic AβO burden by 40 ± 15% and 37 ± 10%, respectively. For clarity, the AβO signal is shown in black and white next to each condition. (G) Half-maximal AβO liberation occurred at 1.6 ± 0.7 min and 3.4 ± 2.2 min for insulin and ACM treatments, respectively. (H) Insulin stimulated the removal of neuron-bound AβOs with an EC50 of 270 nM. *, p < 0.05, Mann-Whitney; **, p < 0.01, Mann-Whitney; ***, p < 0.0005, Mann-Whitney.