MODULATING AUTOPHAGY AFFECTS NEUROAMYLOIDOGENESIS IN AN IN VITRO ISCHEMIC STROKE MODEL

To explore the effects of modulating autophagy on neuroamyloidogenesis in an ischemic stroke model of cultured neuroblastoma 2a (N2a)/Amyloid precursor protein (APP)695 cells. Methods: The ischemic stroke model of N2a/APP695 cells was made by 6 h oxygen-glucose deprivation/12 h reperfusion (OGDR). Drug administration of 3-methyladenine (3-MA), rapamycin or DL-3-n-butylphthalide (NBP) was started at the beginning of the OGDR and lasted until the end of reperfusion, in order to explore their effects on N2a/APP695 cells under OGDR conditions. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 12 h. Cell viability and injury were investigated. The key proteins of nuclear factor kappa B (NF-kappa B) pathway and a key component of autophagy Beclin 1 were detected by Western blotting; immunofluorescence double-staining of amyloid-beta (A beta)(1-42) with Beclin 1 was performed to investigate their cellular co-localization relationship; beta-secretase and gamma-secretase activity assay and A beta(1-42) enzyme-linked immunosorbent assay were performed to investigate the amyloidogenesis. Results: The results showed that, OGDR enhanced cell injury, autophagy activity, neuroinflammation and A beta generation in N2a/APP695 cells; down-regulating autophagy by 3-MA and NBP increased cell viability, decreased lactate dehydrogenase (LDH) production, inhibited the activation of NF-kappa B pathway, suppressed beta- and gamma-secretase activities and A beta generation; while up-regulating autophagy by rapamycin got the opposite results; immunofluorescence double-staining results showed elevated A beta(+)(1-42) signal was co-localized with Beclin 1(+) signal. Conclusion: Our data suggested that down-regulating autophagy may inhibit ischemia-induced neuroamyloidogenesis via suppressing the activation of NF-kappa B pathway. This study might help us to find a new therapeutic strategy to prevent brain ischemic damage and depress the risk of post-stroke dementia. (C) 2014 Published by Elsevier Ltd. on behalf of IBRO.