Furthermore, previous data have shown that 1 uM C16 markedly reduces levels of PT451 PKR and caspase 3 activity in Ab42 treated SH SY5Y cells. The T451 phosphorylated site in the PKR acti vation loop is required in vitro and in vivo for high level kinase activity. We first evaluated selleck products toxicity of compound C16 at 210 nM and 1 uM compared to its DMSO vehicle. By using scanning electron microscopy, Inhibitors,Modulators,Libraries we showed that the majority of cultured cells were neurons and astrocytic glial cells. Amongst these were some round cellular elements ranging from 10 to 15 um in diameter which were identified as microglia cells. In experimental conditions with DMSO or 210 nM C16, microglia looked like spherical smooth cells in contact with neurons and the astroglial layer. No reactive micro glia were observed in these control conditions.

However, Inhibitors,Modulators,Libraries 1 uM C16 greatly affected the integrity of Inhibitors,Modulators,Libraries cells in co cultures, with neuronal death, disruption Inhibitors,Modulators,Libraries of axonal net work and activated astrocytes. The microglia looked like macrophages. Based on these observations, further experiments were performed with the effective concentration 210 nM, corresponding to IC50 of com pound C16. Prevention of Ab induced PKR activation and NF B I B signaling pathway by compound C16 At its IC50, C16 significantly reduced by 33% the pro minent activation of PKR induced by 20 uM Ab42 over 72 h in the co cultures as shown by immunoblotting from nuclear extracts. Confocal staining of PT451 PKR confirmed the activation of PKR under Ab42 exposure compared to DMSO treated cells.

More over, co staining with the neuronal marker MAP2 indi cated that PT451 PKR was present in neurons, with intense perinuclear, nuclear and axonal staining, com pared to DMSO treated cells. Treatment with C16 decreased perinuclear and nuclear staining induced by Ab42, but some axons remained Inhibitors,Modulators,Libraries stained. The co cultures incu bated with compound C16 alone resembled those incu bated with DMSO alone. In astrocytes labeled by antibodies against GFAP, a diffuse cytoplasmic staining of PT451 PKR and a robust staining in spine like structures of astrocytic processes with Ab42 were observed and were well prevented by C16 treatment. Microglia stained with anti CD68 anti bodies displayed a high level of activated PKR after 72 h of Ab42 exposure compared to DMSO treated cells.

There was also a change in cellular morphology, microglia were activated with appearance of thick processes and irregular shape with Ab42 treatment. C16 partially rescued this activation of PKR in microglia. Furthermore, we found only microglia with no thick processes around cell bodies as with C16 alone. The same selleck chemicals Enzastaurin experimental conditions were followed to study activation of the NF B I B signaling pathway. Results obtained by immunoblotting from cell lysates are presented as the ratio of phospho protein total pro tein in order to evaluate the activation of both proteins.