The analysis of Presenilin processing and activity with a focus on its implications for Alzheimer's disease pathogenesis using Danio Rerio as a model organism.

Abstract

Aberrant proteolytic processing of AMYLOID BETA PRECURSOR PROTEIN(AβPP) may result in an imbalance between production and clearance of the amyloid-β (Aβ) peptide proteolytic product and promote neuronal dysfunction and death. β-site amyloid-β A4 precursor protein-cleaving enzyme 1 (BACE1) with γ-secretase are responsible for the cleavage of AβPP to produce Aβ peptide. Presenilin proteins form the catalytic core of γ-secretase complexes. PRESENILIN1 (PSEN1) is the major locus for mutations causing familial Alzheimer’s disease (FAD) and is also mutated in Pick disease of brain, familial acne inversa and dilated cardiomyopathy. It is a critical facilitator of Notch signalling. The zebrafish, Danio rerio, is a versatile vertebrate model for investigating the molecular bases of Alzheimer’s disease (AD) pathology. It possesses genes orthologous to human PSEN1 and PSEN2, and the genes appa and appb that are duplicates of an ancestral AβPP orthologue). This thesis primarily utilizes zebrafish as a system to investigate AD pathogenesis.. Chapter I describes an assay in which the level of a γ-secretase substrate (a modified form of Appa protein) is observed in zebrafish embryos by western immunoblotting relative to a co-expressed protein not subject to γ-secretase activity. Prior to the development of this assay there existed no in vivo assay appropriate for directly monitoring γ-secretase activity. The assay was subsequently used to analyse the effects on γ-secretase activity of blocking translation of zebrafish psen1 and/or psen2. Chapter II explores various truncations of human PSEN1 (or zebrafish Psen1) protein that have differential effects on Notch signalling and cleavage of zebrafish Appa (a paralogue of human AβPP). Different truncations can suppress or stimulate Notch signalling but not Appa cleavage and vice versa. The results show that the truncated protein potentially translated from these transcripts incorporates into stable Psen1-dependent higher molecular weight complexes and suppresses cleavage of Appa but not Notch signalling. In contrast, the truncated protein potentially produced by the P242LfsX11 acne inversa mutation has no effect on Appa cleavage but, unexpectedly, enhances Notch signalling. The results suggest novel hypotheses for the pathological mechanisms underlying AD. Chapter III investigates truncated isoforms of PRESENILIN known to form naturally. In particular a truncated PSEN2 isoform “PS2V” has been previously identified. PS2V is formed by exclusion of exon 5 from PSEN2 transcripts leading to a frameshift after exon 4 sequence and a premature stop codon. This truncates the ORF/protein after PSEN2’s first transmembrane domain. The K115Efx10 mutation in PSEN2 is the only completely truncating mutation of the PRESENILIN genes that is thought to cause AD. K115Efx10 is especially interesting since, if expressed, it would generate a truncated protein very similar to PS2V and would be expected to boost Aβ production. Zebrafish possess an isoform of Psen1 that has a similar role to PS2V and zebrafish Psen1 truncated after exon 4 sequence behaves in a similar manner to PS2V. We have modeled human and zebrafish PS2V and K115Efx10-like mutations in zebrafish to investigate their effect on gene expression profiles, γ-secretase activity and complex constitution.