The BACE1-PSEN-AβPP Regulatory Axis has an Ancient Role in Response to Low Oxygen/Oxidative Stress

Abstract

Oxygen homeostasis is essential for the development and normal physiology of an organism. Hypoxia causes the mitochondrial electron transport chain to generate higher levels of reactive oxygen species resulting in oxidative stress. Hypoxia can be a direct consequence of hypoperfusion, a common vascular component among Alzheimer's disease (AD) risk factors, and may play an important role in AD pathogenesis. Beta-site amyloid-β A4 precursor protein-cleaving enzyme 1 (BACE1) is responsible, with γ-secretase, for cleavage of the amyloid-β protein precursor (AβPP) to produce amyloid-β (Aβ) peptide. A recent study observed that oxidative stress increases BACE1 expression via a regulatory pathway dependent on γ-secretase cleavage of AβPP and this increases Aβ peptide production. Zebrafish embryos represent normal cells in which complex and subtle manipulations of gene activity can be performed to facilitate analysis of genes involved in human disease. Here we identify and describe the expression of bace1, the zebrafish ortholog of human BACE1. We observe that the zebrafish AD-related genes bace1, psen1, psen2, appa, and appb all show increased mRNA levels under hypoxia. A dominant negative form of psen1 putatively blocking γ-secretase activity blocks bace1 upregulation under hypoxia. Hypoxia increases catalase gene mRNA indicating increased oxidative stress but we did not observe increased levels of F2-isoprostanes that indicate peroxidation of arachidonic acid, possibly due to relatively low levels of arachidonic acid in zebrafish. Our results demonstrate that upregulation of PSEN1 & 2, AβPP and the γ-secretase-dependent upregulation of BACE1 is an ancient, conserved, and thus selectively advantageous response to hypoxia/oxidative stress.

Using the zebrafish model for Alzheimer’s disease research

“…Zebrafish embryos and adults can be exposed to real hypoxia by depleting their water environment of oxygen or to chemical mimicry of hypoxia through, e.g., sodium azide treatment (Moussavi Nik et al, 2011). Similarly to what is observed in humans (Lukiw et al, 2001; Pluta, 2005; De Gasperi et al, 2010; Zhang and Le, 2010), hypoxia upregulates psen1 , psen2 , appa , appb , and bace1 in zebrafish adult brain and larvae (Moussavi Nik et al, 2012). This suggests that Aβ is produced as a protective response to hypoxia in both human and zebrafish cells – a response conserved over 450 Mya of evolutionary time.…”

“…Zebrafish hold orthologous genes for the components of the gamma-secretase complex, PSENEN ( psenen ; Francis et al, 2002; Campbell et al, 2006), NCTN ( ncstn ; Strausberg et al, 2002) and APH1b ( aph1b ; Francis et al, 2002). While orthologs of β-secretase ( BACE1 and BACE2 ) have also recently been identified in zebrafish: bace1 (Moussavi Nik et al, 2012) and bace2 (van Bebber et al, 2013). The microtubule-associated protein tau ( MAPT ) gene in humans encodes the tau protein and our laboratory identified co-orthologs of this gene in zebrafish, mapta , and maptb (Chen et al, 2009).…”

Analysis of Oxidative Stress in Zebrafish Embryos

“…Recently reported experiments of gene knock-down and microinjection of capped mRNA in zebrafish embryos established different roles for the antioxidant Nrf2a and Nrf2b genes in protecting cells from oxidative stress during development. Approaches of gene expression modulation assessed an evolutionary conserved axis between mammalian and zebrafish to hypoxia response and oxidative stress in neuronal cells 38,48 . In addition, a great opportunity to study the oxidative stress is offered by several zebrafish mutant lines.…”

Identification and expression analysis of the zebrafish orthologues of the mammalian MAP1LC3 gene family

“…For PCR amplification, 5 μL of the cDNA was used with the primers: map1lc3a F (5′-CGAGTCGACCGACAATTTAGC-3′) and map1lc3a R (5′-TCCTTGCAACGATCAGCGAA-3′); map1lc3b F (5′-AATGTGACGATTGGACACGAGT -3′) and map1lc3b R (5′-AGTACAACAGCTCACGGTTATGC -3′). Control PCR primers for zebrafish β-actin were: β-actin F (5′-TTCTGGTCGGTACTGGTATTGTG -3′) and β-actin R (5′-ATCTTCATCAGGTAGTCTGTCAGGTT -3′) [21].…”

“…Gene specific primers were designed for amplification of map1lc3a (Qmap1lc3a.F: 5′-CGAGTCGACCGACAATTTAGC-3′ and Qmap1lc3aR: 5′-TCCTTGCAACGATCAGCGAA-3′), map1lc3b (Qmap1lc3bF:5'-AATGTGACGATTGGACACGAGT -3' and Qmap1lc3bR: 5' -AGTACAACAGCTCACGGTTATGC -3') and for the ubiquitously expressed control genes eef1a1a (Qeef1a1aF: 5′-CCAACTTCAACGCTCAGGTCA-3′ and Qeef1a1aR: 5′-CAAACTTGCAGGCGATGTGA-3′) and β-actin (Q β-actinF: 5′-TTCTGGTCGGTACTGGTATTGTG -3′and β-actinR : 5′-ATCTTCATCAGGTAGTCTGTCAGGTT -3′) [21]. cDNA was serially diluted (100 ng, 50 ng, 25 ng, and 12.5 ng per reaction) to generate the standard curve.…”