Molecular responses to low oxygen levels/oxidative stress in zebrafish.

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with pathologies such as neuron loss, glial cell proliferation, extracellular deposition of senile plaques from the accumulation of amyloid beta (Aβ) peptides and deposition of intracellular neurofibrillary tangles. Aβ is created from the cleavage of the Amyloid Precursor Protein (APP) by two different types of aspartyl proteases, β- and γ-secretase. The majority of AD cases are sporadic and have a late onset.Mutations in the genes encoding APP, PRESENILIN1 and 2 (PSEN1 and PSEN2) genes cause an autosomal dominant inherited form of the disease with an early onset known as familial AD. In some sporadic cases an aberrant splice variant of PSEN2named PS2V is formed that can be found in inclusion bodies in the brain. PS2V results from the binding of the High Mobility Group A1a (HMGA1a) protein close to the splice donor site of exon 5 of PSEN2. The High Mobility Group A1 protein,HMGA1, is widely expressed during embryo development but not in adults. Its expression can be induced in adult neurons by hypoxia/oxidative stress and it is commonly reactivated in many types of cancer. Hypoxia can be a direct consequence of hypoperfusion, a common vascular component among Alzheimer’s disease risk factors and may play an important role in AD pathogenesis. BETA-SITE AMYLOID BETA A4 PRECURSOR PROTEIN-CLEAVING ENZYME 1, BACE1 is responsible, with γ-secretase, for cleavage of AMYLOID PRECURSOR PROTEIN, APP to produce Aβ peptide. A recent study observed that oxidative stress upregulates BACE1 expression via a regulatory pathway that is dependent on γ-secretase cleavage of APP and that results in increased Aβ peptide production. In this thesis, we define strategies for exposure of zebrafish to hypoxia and “chemical hypoxia”. We identifiyendogenous zebrafish hmga1 in an attempt to investigate PS2V formation in fish. We also demonstrate that responses to low oxygen/oxidative stress by genes involved in Alzheimer’s disease are evolutionarily conserved in fish. Paper1 (thesis chapter in the form of a manuscript) describes the identification of the hmga1 gene in zebrafish which is an orthologue of human HMGA1. It also examines the regulation of this gene under hypoxia/oxidative stress conditions and demonstrates thathmga1 expression is induced under these conditions. However no PS2V-like splice variant of zebrafish psen2 is observed. Paper 2 (thesis chapter in the form of a manuscript) describes the identification of the zebrafish bace1 gene which is orthologous to human BACE1. It also examines the regulation of AD-related genes under hypoxia/oxidative stress. We show that the response of the BACE1-PSEN-APPregulatory axisto hypoxia/oxidative stress is evolutionarily conserved between fish and mammals. Therefore, we also demonstrate that zebrafish are a valid model system for analysis of the effects of hypoxia/oxidative stress on genes associated with Alzheimer’s disease.