Hypoxia Affects Neprilysin Expression Through Caspase Activation and an APP Intracellular Domain-dependent Mechanism

Abstract

While gene mutations in the amyloid precursor protein (APP) and the presenilins lead to an accumulation of the amyloid β-peptide (Aβ) in the brain causing neurodegeneration and familial Alzheimer's disease (AD), over 95% of all AD cases are sporadic. Despite the pathologies being indistinguishable, relatively little is known about the mechanisms affecting generation of Aβ in the sporadic cases. Vascular disorders such as ischaemia and stroke are well established risk factors for the development of neurodegenerative diseases and systemic hypoxic episodes have been shown to increase Aβ production and accumulation. We have previously shown that hypoxia causes a significant decrease in the expression of the major Aβ-degrading enzyme neprilysin (NEP) which might deregulate Aβ clearance. Aβ itself is derived from the transmembrane APP along with several other biologically active metabolites including the C-terminal fragment (CTF) termed the APP intracellular domain (AICD), which regulates the expression of NEP and some other genes in neuronal cells. Here we show that in hypoxia there is a significantly increased expression of caspase-3, 8, and 9 in human neuroblastoma NB7 cells, which can degrade AICD. Using chromatin immunoprecipitation we have revealed that there was also a reduction of AICD bound to the NEP promoter region which underlies the decreased expression and activity of the enzyme under hypoxic conditions. Incubation of the cells with a caspase-3 inhibitor Z-DEVD-FMK could rescue the effect of hypoxia on NEP activity protecting the levels of AICD capable of binding the NEP promoter. These data suggest that activation of caspases might play an important role in regulation of NEP levels in the brain under pathological conditions such as hypoxia and ischaemia leading to a deficit of Aβ clearance and increasing the risk of development of AD.

Keywords: AICD; APP; caspases; hypoxia; neprilysin.

Figure 1

The effects of hypoxia on cell viability and GLUT1 protein levels in NB7 cells. (A) A cell viability assay carried out in NB7 cells subjected to 24 h normoxia or hypoxia (1% O₂). (B) Representative Western blot and calculation of GLUT1 levels in whole cell lysates of NB7 cells subjected to 24 h normoxic or hypoxic (1% O₂) conditions. Bars represent mean ± SEM, ^*p < 0.05 (n = 3).

Figure 2

The effects of hypoxia on NEP expression and activity. (A) NEP mRNA expression levels normalized to U6; (B) NEP protein levels showing a representative blot with CD10 antibody against human NEP and β-actin as loading control; (C) NEP enzyme activity (nmol/min/mg) in NB7 cells subjected to 24 h normoxia or 24 h hypoxia (1% O₂). Bars represent mean ± SEM, ^*p < 0.05 (n = 5).

Figure 3

The effect of hypoxia on caspase-1,-8, and -9 mRNA expression and caspase -3/-7 activity levels. Expression levels of (A) caspase-1, (B) caspase-8, (C) caspase-9 normalized to U6 in NB7 cells subjected to 24 h normoxia or 24 h hypoxia (1% O₂). (D) Caspase-3/-7 activity levels in NB7 cells subjected to 24 h normoxia or 24 h hypoxia (1% O₂). Bars represent mean ± SEM, ^*p < 0.05, ^**p < 0.01.

Figure 4

The effect of a caspase-3 inhibitor on NEP activity and AICD binding to the NEP promoter in hypoxia. (A) NEP enzyme activity (nmol/min/mg) and (B) enrichment of AICD on the NEP promoter analyzed by ChIP in NB7 cells subjected to 24 h normoxia or 24 h hypoxia (1% O₂) with or without the caspase-3 inhibitor, Z-DEVD-FMK (100 μM, 24 h). Bars represent n of 3 ± SEM, ^*p < 0.05 compared to control, ^#p < 0.05 compared to hypoxia.