Modulating autophagy affects neuroamyloidogenesis in an in vitro ischemic stroke model.
OBJECTIVE
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 6h oxygen-glucose deprivation/12h 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 12h. Cell viability and injury were investigated. The key proteins of nuclear factor kappa B (NF-κB) pathway and a key component of autophagy Beclin 1 were detected by Western blotting; immunofluorescence double-staining of amyloid-β (Aβ)1-42 with Beclin 1 was performed to investigate their cellular co-localization relationship; β-secretase and γ-secretase activity assay and Aβ1-42 enzyme-linked immunosorbent assay were performed to investigate the amyloidogenesis.


RESULTS
The results showed that, OGDR enhanced cell injury, autophagy activity, neuroinflammation and Aβ 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-κB pathway, suppressed β- and γ-secretase activities and Aβ generation; while up-regulating autophagy by rapamycin got the opposite results; immunofluorescence double-staining results showed elevated Aβ1-42(+) signal was co-localized with Beclin 1(+) signal.


CONCLUSIONS
Our data suggested that down-regulating autophagy may inhibit ischemia-induced neuroamyloidogenesis via suppressing the activation of NF-κB 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.
Department of Neurology, Shanghai Jiaotong University Affillilated Sixth People's Hospital, Shanghai 200233, China.
T
T
Zhang
Department of Neurology, Shanghai Jiaotong University Affillilated Sixth People's Hospital, Shanghai 200233, China.
H
H
Wang
Department of Neurology, Shanghai Jiaotong University Affillilated Nineth People's Hospital, Shanghai 200011, China.
Q
Q
Li
Department of Neurology, Shanghai Jiaotong University Affillilated Sixth People's Hospital, Shanghai 200233, China.
J
J
Huang
Department of Neurology, Shanghai Jiaotong University Affillilated Sixth People's Hospital, Shanghai 200233, China. Electronic address: sunxiaojiang155@sina.com.
X
X
Sun
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D001572:Benzofurans / Q000494:pharmacology; D002545:Brain Ischemia / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D051379:Mice; D009447:Neuroblastoma; D020123:Sirolimus / Q000494:pharmacology; D020521:Stroke / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D016564:Amyloid beta-Protein Precursor; D001572:Benzofurans; C025946:3-methyladenine; C027125:3-n-butylphthalide; D053829:Amyloid Precursor Protein Secretases; D000225:Adenine; D020123:Sirolimus
amyloid-β; autophagy; dl-3-n-butylphthalide; oxygen and glucose deprivation/reperfusion; post-stroke dementia