We observed that cytokines, especially combi nations containing TNF a IFN g, raised the levels of endogenous BACE1 and APP in C57BL 6J astrocytes and promoted the secretion of astrocytic Ab40. Inhibitor treatments suggested that iNOS signaling was not involved in cytokine stimulated selleck products astrocytic BACE1, APP, and Ab40 elevations, although JAK signaling appeared to have a role in the endogenous astrocytic APP increase. Similar to the effects of cytokine stimulation, Ab42 oligomers and fibrils elevated levels of endogenous BACE1 and APP in C57BL 6J astrocytes, and increased b secretase cleavage of APPsw in Tg2576 astrocytes. The astrocytic APP and BACE1 elevations for cytokine Inhibitors,Modulators,Libraries or Ab42 stimulations appeared in some cases to involve combined transcriptional and post transcriptional mechanisms, depending on the stimulation.
Overall, our results support the hypothesis that cytokine and Ab42 stimulated astrocytes could contribute significantly to the total burden of cerebral Ab in AD, potentially through Inhibitors,Modulators,Libraries elevated astrocytic b secretase processing of APP under neuroinflammatory conditions. Moreover, the similar effects of cytokine or Ab42 stimulation on astrocytic b secretase processing suggest a feed forward mechanism that might promote Ab generation in astrocytes. Methods Materials and reagents The bacterial endotoxin LPS purchased from Sigma Aldrich was from Salmonella typhimur ium. Stock solutions were prepared with sterile Dulbec cos phosphate buffered saline at a concentration of 1 mg ml. The recombinant murine cytokines TNF a, IL 1b, and IFN g were purchased from R D Systems and reconstituted in sterile 0.
1% bovine serum albumin in D PBS at a concentration of 10, 5, 50 ug ml, respectively. iNOS inhibitor was procured from Alexis Inhibitors,Modulators,Libraries Bio chemicals, JAK inhibitor was obtained from EMD Calbiochem. Inhibitors,Modulators,Libraries Ab42 peptide was purchased from Ameri can Peptide. Antibodies used for immu noblotting and fluorescence immunocytochemistry are listed in Table 1. The RNeasy Mini Kit from Qiagen was applied for astrocyte RNA isolation and real time PCR experiments. Primary astrocyte culture The wild type C57BL 6J and Tg2576 transgenic mice used in this study were purchased from Taconic and colonies of these mice were kept in the Northwestern University Center for Comparative Inhibitors,Modulators,Libraries Medicine animal facilities.
All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Northwestern University Animal Care and Use Committee. Mouse primary astrocyte cultures were established from cerebral selleck Sorafenib cortices of newborn mouse pups as pre viously described with some modifications. In brief, postnatal day 1 3 wild type C57BL 6J mouse brain cortices were harvested in ice cold D PBS, and meninges and blood vessels were removed. Tissues were digested in 0. 25% Trypsin containing 0.