After 96 h of stimulation, cell lysates and CMs were harvested for APP and BACE1 immunoblot and Ab40 ELISA analyses, respectively. JAK I reduced the TNF a IFN g stimulated increase in astrocytic APP level in a dose dependent manner, but it did not block the elevations in astrocytic BACE1 or secreted Ab40. Unexpectedly, JAK I treatment Gefitinib buy with 1 uM and 5 uM appeared to elevate secreted Ab40 and BACE1 levels above 0 uM JAK I, respectively, but these increases were not significant. Although it is unclear why JAK I elevated astrocytic Ab40 and BACE1 at certain concentrations but not others, it is important to emphasize that JAK inhibition did not prevent the TNF a IFN g stimulated increase in BACE1 level, suggesting that JAK signaling may play a synergistic but not essential role in the TNF a IFN g stimulated BACE1 elevation.

Given that JAK I reduced the TNF a IFN g stimulated increase in astrocytic APP, it is not completely clear why secreted Ab40 levels were also not reduced by JAK inhibition. Secreted Ab40 levels appeared slow to change in response to TNF a IFN g stimulation, so we speculate that secreted Ab40 could have become significantly Inhibitors,Modulators,Libraries reduced with JAK I treatment times longer than 96 h. This Inhibitors,Modulators,Libraries is sup ported by an observed downward trend in secreted Ab40 with higher JAK I concentrations. Regardless, our JAK I results overall indicate that JAK signaling, at least in part, may play a role in elevating astrocytic APP levels and this might contribute to secreted Ab, although JAK signaling does not appear to contribute to an essential degree to BACE1 levels in astrocytes.

We also investigated signaling through iNOS, an inflammatory mediator induced by cytokine stimula tion, to explore its potential involvement in amyloido genic APP processing in astrocytes. Cell lysates from stimulated Inhibitors,Modulators,Libraries astrocytes were analyzed by immunoblot to determine iNOS levels. Paralleling the previously observed increases in endogenous APP, BACE1, and Ab40 levels, iNOS levels were dramatically induced by pro inflammatory Inhibitors,Modulators,Libraries agent combinations at all time points in stimulated astrocytes. With the exception of the bacterial endotoxin LPS, no single agent treatment induced appreciable iNOS expression in these cells. These results demonstrated that the eleva tions of endogenous APP, BACE1, and Ab40 correlated well with the induction of iNOS in cytokine stimulated astrocytes.

To determine whether iNOS played a role in the ele vation of astrocytic APP, BACE1, and Ab40 levels, we pre treated primary astrocytes cultures with the iNOS inhibitor 1400 W for Inhibitors,Modulators,Libraries 30 min followed by KPT-330 CRM1 stimulation with TNF a IFN g for 96 h. As expected, 1400 W pre treatment strongly inhibited iNOS activity as demonstrated by dose dependent suppression of astrocytic nitrite production without affect ing iNOS protein levels.