To overcome this problem, we designed an experiment to directly m

To overcome this problem, we designed an experiment to directly measure BACE1 pro cessing of APP, which positively correlates with Ab pro duction in cells. In Paclitaxel this experiment, we investigated the effects of Ab42 oligomers and fibrils on primary astro cytes cultured from Tg2576 transgenic mice that overex press APPsw, which is a superior BACE1 substrate as compared to wild type APP. As a consequence, Tg2576 neurons and astrocytes Inhibitors,Modulators,Libraries exhibit rates of APPsw amyloidogenic processing and Ab production that are substantially higher than those of non transgenic cells. BACE1 cleavage of APPsw generates an N terminal ectodomain Inhibitors,Modulators,Libraries fragment of APPsw that is named APPsbsw. To measure levels of APPsbsw, we generated an anti body that specifically recognizes the cleaved C terminal neo epitope of APPsbsw following BACE1 processing.

We used this anti APPsbsw neo epitope antibody to Inhibitors,Modulators,Libraries perform immunoblots Inhibitors,Modulators,Libraries of cell lysates from Tg2576 primary astrocytes that were stimulated with Ab42 oli gomers or fibrils for 24, 48, or 72 h. Tg2576 astrocytes expressed several fold more APP than non transgenic astrocytes, demonstrating that the Tg2576 transgene promoter was active in astrocytes. Moreover, stimulation with Ab42 oligomers and fibrils caused levels of both transgenic and endogenous APP to signifi cantly increase in Tg2576 and non transgenic astrocytes, respectively, at 24 and 48 h time points, simi lar to results obtained with Ab42 treated C57BL 6J astrocytes. Most importantly, robust APPsbsw signals on immunoblots indicated that Ab42 stimulation of Tg2576 astrocytes caused dramatic increases in BACE1 cleavage of APPsw at all treatment time points.

Both oligomeric and fibrillar Ab42 stimulation elevated APPsbsw levels to similar extents at the earlier time points, Inhibitors,Modulators,Libraries although the potency of Ab42 oli gomers appeared to decrease somewhat relative to Ab42 fibrils by 72 h of treatment. APPsbsw signals were absent in immunoblot lanes of lysates from vehicle control treated Tg2576 astrocytes, indi cating that Ab42 may have induced non amyloidogenic astrocytes to initiate BACE1 cleavage of APP. Taken together, these results demonstrated that Ab42 oligo mers and fibrils are not only capable of elevating levels of astrocytic APP and BACE1, but they could also increase BACE1 cleavage of APP in astrocytes, a prere quisite of Ab synthesis. Discussion Are astrocytes a significant source of Ab in AD Is a feed forward vicious cycle involved in AD pathogen esis These are underappreciated yet critical questions that have important mechanistic and therapeutic impli cations for AD.

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