Hypoxia-inducible Factor 1α (HIF-1α)-mediated Hypoxia Increases BACE1 Expression and β-Amyloid Generation

Xian Zhang,Kun Zhou,Ruishan Wang,Jiankun Cui,Stuart A Lipton,Francesca-Fang Liao,Huaxi Xu,Yun-Wu Zhang

doi:10.1074/jbc.m608856200

Abstract

The incidence of Alzheimer disease (AD) and vascular dementia is greatly increased following cerebral ischemia and stroke in which hypoxic conditions occur in affected brain areas. beta-Amyloid peptide (Abeta), which is derived from the beta-amyloid precursor protein (APP) by sequential proteolytic cleavages from beta-secretase (BACE1) and presenilin-1 (PS1)/gamma-secretase, is widely believed to trigger a cascade of pathological events culminating in AD and vascular dementia. However, a direct molecular link between hypoxic insults and APP processing has yet to be established. Here, we demonstrate that acute hypoxia increases the expression and the enzymatic activity of BACE1 by up-regulating the level of BACE1 mRNA, resulting in increases in the APP C-terminal fragment-beta (betaCTF) and Abeta. Hypoxia has no effect on the level of PS1, APP, and tumor necrosis factor-alpha-converting enzyme (TACE, an enzyme known to cleave APP at the alpha-secretase cleavage site). Sequence analysis, mutagenesis, and gel shift studies revealed binding of HIF-1 to the BACE1 promoter. Overexpression of HIF-1alpha increases BACE1 mRNA and protein level, whereas down-regulation of HIF-1alpha reduced the level of BACE1. Hypoxic treatment fails to further potentiate the stimulatory effect of HIF-1alpha overexpression on BACE1 expression, suggesting that hypoxic induction of BACE1 expression is primarily mediated by HIF-1alpha. Finally, we observed significant reduction in BACE1 protein levels in the hippocampus and the cortex of HIF-1alpha conditional knock-out mice. Our results demonstrate an important role for hypoxia/HIF-1alpha in modulating the amyloidogenic processing of APP and provide a molecular mechanism for increased incidence of AD following cerebral ischemic and stroke injuries.

Highlights

An important pathologic feature of Alzheimer disease (AD)4 is formation of extracellular senile plaques in the brain, whose major components are small peptides called ␤-amyloid (A␤) derived from ␤-amyloid precursor protein (APP)
Several studies indicate that protein levels and BACE1 activity are elevated in brain regions affected by AD, suggesting that abnormal BACE1 activity contributes significantly to AD pathogenesis [19, 20]
Other studies report a drastic decrease in ADAM10 and TACE protein levels with unchanged BACE1 levels in human neuroblastoma SH-SY5Y cells subjected to chronic hypoxic treatments [42,43,44]

Summary

EXPERIMENTAL PROCEDURES

B, lysates of hypoxia-treated and untreated N2a-APP cells were assayed for ␤-secretase activity using. Pulse-Chase Experiments—To assay the kinetics of BACE1 metabolism, 4-h hypoxia-treated and untreated N2a-APP cells were labeled with [35S]methionine (500 ␮Ci/ml) for 5 or 10 min at 37 °C and collected for analysis. Equal amounts of protein in cell lysates were immunoprecipitated with an anti-BACE1 antibody, followed by SDS-PAGE analysis and autoradiography. B, luciferase reporter plasmids carrying the wild type or mutant mouse BACE1 promoter regions were co-transfected with the Renilla luciferase reporter plasmid into N2a-APP cells; and the cells were treated with or without hypoxia for 4 h. Quantitative real-time PCR analysis showed 1.5–2-fold increases in BACE1 mRNA following hypoxic treatment, confirming the effect of hypoxia on BACE1 transcription (Fig. 2B). Samples were either the BACE1 Promoter—Given that HIF-1 is a critical tranlysed in RIPA for immunoblotting or used for RNA extrac- scription factor activated in response to hypoxic stresses, we tion and quantitative RT-PCR

RESULTS

Findings

DISCUSSION