Figure 2.

Antisense or chemical knockdown of N-SMase, but not A-SMase, protects neurons from Aβ-activated primary human astrocytes in astrocyte–neuron transwell cultures. A, B, Primary human neurons were incubated with ASOs and ScOs against N-SMase (A) and A-SMase (B). After 48 h, neurons were analyzed for protein expression of N-SMase and A-SMase. C, Primary human astrocytes seeded in inserts were stimulated by the combination of Aβ and IL-1β as described above. After 24 h, media were removed, and inserts were washed and placed on neurons that were already pretreated for 40 h with 1 μm ASO or ScO against N-SMase and A-SMase. After 6 h of treatment with activated astrocytes, apoptotic events in neurons were detected by TUNEL. D, TUNEL-positive cells were counted manually in four different images of each of three coverslips by three individuals blinded to the experiment. E, After 18 h of stimulation, cell viability was examined by the metabolism of MTT and the release of LDH. Activated astrocytes were placed on neurons that were pretreated with different concentrations of GW4869 (GW) and imipramine. F, G, Cell viability was checked by MTT (F) and LDH (G). Values obtained from the control group served as 100%, and data obtained in other groups were calculated as a percentage of control accordingly. Results are the mean ± SD of three different experiments. ^ap < 0.001 versus control; ^bp < 0.001 versus FAA. FAA, Fibrillar Aβ+IL-1β-activated astrocytes.