Sprague-Dawley embryonic cultured neurons from the hippocampi were used for experiments. This cell population exhibited large pyramidal neurons that are the target in AD pathogenesis.
Phosphorylation of protein kinase A (PKA) was assayed by harvesting cultured neuronal cells after treatment with amyloid beta protein for 3,6,12 and 24 hrs. Cells were harvested in RIPA buffer and centrifuged. The supernatant was assayed for basal PKA activity with kemptide (Sigma) as substrate. Protein extracts were incubated with kemptide, gamma p32ATP and ATP with or without PKA inhibitor (PKI, Sigma).
Immunoblotting was performed on supernates from RIPA treated neurons. The antibody (Santa Cruz) recognizes an epitope at the C terminus of isoform IIa of PKA regulatory subunit.
Measurements of phosphorylated CREB were obtained after exposing the cultured neurons to glutamate. These neurons had been preincubated with either amyloid-beta alone or with amyloid-beta and rolipram (Sigma). The cells were harvested in RIPA and immunoblotted using antibody for CREB phosphorylated at Ser-133, 1:1000 dilution (Upstate Biotechnology).
Long-term potentiation (LTP) was measured on hippocampal slices perfused with saline solution and 95% oxygen/5% carbon dioxide. Electrodes, stimulation and recording, were used to record the postsynaptic potential. Either amyloid-beta, forskolin, rolipram, H89 were added to the perfused slices and measurements taken for one hour after treatment. Data analyzed with ANOVA.