| Summary: | 碩士 === 國立陽明大學 === 腦科學研究所 === 105 === Alzheimer’s disease (AD) is the most common type of dementia in the elderly. Accumulation of extracellular senile plaques mainly composed of amyloid beta-peptide (Aβ) is one of the major pathological hallmarks observed in the AD brains. Aβ-induced neurotoxicity is mediated by multiple mechanisms including enhanced production of hydrogen peroxide causing oxidative stress. Sestrin2 is a stress-responsive protein that has been suggested to play a role in both antioxidation and autophagy induction. Our laboratory has previously demonstrated that sestrin2 may be induced by Aβs to promote autophagy with neuroprotective effects (Chen et al., 2014). However, how Aβs may induce sestrin2 expression and whether antioxidation, in addition to autophagy, is also involved in sestrin2-dependent neuroprotection against Aβ toxicity remains unknown.
Primary fetal rat cortical neurons exposed to Aβ25-35 were used as the in vitro AD model. Pharmacological inhibitors and siRNAs were used to investigate the molecular mechanisms underlying sestrin2 induction by Aβs. To confirm the antioxidative effects of sestrin2, immunocytochemistry with the antibody against oxidative DNA damage marker 8-OH-dG, CellROX® reagents to measure reactive oxygen species (ROS), and glutathione colorimetric assay to measure the content of reduced form glutathione (GSH) were applied.
We found that Aβ enhanced nuclear translocation of the transcription factor nuclear factor-kappaB (NF-κB); more importantly, Aβ-induced sestrin2 expression was suppressed by SN50, the NF-κB inhibitor, and the siRNA targeting at the NF-κB subunit p65 at protein or mRNA level. Pharmacological inhibitors of nitric oxide synthase (NOS), cGMP-dependent protein kinase (PKG), phosphatidylinositide 3-kinases (PI3K) and protein kinase B (Akt) also suppressed Aβ-induced sestrin2 expression at protein level. Aβ enhanced oxidative stress based on the extents of ROS production and oxidative DNA damages. Knockdown of sestrin2 by its siRNA showed the tendency towards aggravating oxidative DNA damage, increasing ROS generation, and decreasing the GSH content caused by Aβ25-35; however, statistical significance is not achieved yet.
Aβ-induced expression of sestrin2 is mediated, at least in part, by NF-κB; in addition, NOS/PKG and PI3K/Akt pathways are also involved. Whether antioxidation contributes to sestrin2-dependent neuroprotection against Aβ requires further investigation.
|
|---|
