Analysis of the subcellular localization of proteins implicated in Alzheimer’s disease

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder involving neuropathological changes including the presence of amyloid plaques in the brain. Amyloid plaques are comprised mainly of the Aβ peptide, formed by the cleavage of the AMYLOID BETA A4 PRECURSOR PROTEIN (APP) by the enzyme complex γ-secretase. The catalytic component of the γ-secretase complex is PRESENILIN (PSEN1 or PSEN2). Mutations in the human PSEN1 gene have been identified in AD. Some mutations have been found to cause frame shifts leading to premature stop codons that produce transcripts encoding truncated PSEN1 protein. The subcellular location of the PSENs has been controversial, with studies detecting these proteins in multiple areas in the cell. However recent research has shown that the PSENs are enriched in the mitochondria associated membranes (MAM). The zebrafish is a small freshwater fish that is a useful animal model for the study of human diseases. Zebrafish have genes that are orthologous to human genes that play a role in AD, including those that encode the components of the γ-secretase complex. However, the zebrafish orthologue of the NICASTRIN gene has not been studied in detail. In Chapter II, we characterize in zebrafish the orthologue of the human NICASTRIN (NCSTN) gene that encodes a protein component of the γ-secretase. We demonstrate the spatial and temporal expression level of zebrafish ncstn in embryos and various adult tissues. This work provides the basic knowledge required to further the understanding of the role of Ncstn in the γ-secretase complex using the zebrafish animal model. Several truncations of the human PSEN1 and zebrafish Psen1 proteins appear to have differential effects on Notch signalling and Appa cleavage. The basis for these differences is still unclear. Chapter III examines the subcellular localization of fusions of truncations of zebrafish Psen1 protein with green fluorescent protein (GFP) using a simple protocol to obtain single cells from zebrafish embryos for microscopy analysis. We show that the different truncations analysed appear to have similar distributions, suggesting that the differential effects on γ-secretase substrates are likely not due to different subcellular localisations of these truncated forms of Psen1. This chapter contributes a new method of viewing a single layer of zebrafish cells with confocal microscopy. The mitochondria-associated membranes (MAM) are a subcompartment of the endoplasmic reticulum (ER) which interacts with mitochondria. The MAM is highly enriched in PSEN1 and PSEN2. Mitochondrion-ER appositions are increased in Psen1⁻ʹ⁻/Psen2⁻ʹ⁻ mouse embryonic fibroblasts (MEFs) and also in familial AD (FAD) and sporadic AD (SAD) patient cells. These results, which were performed using mammalian cells, have not been studied in the zebrafish animal model. Therefore, Chapter IV of this thesis examines the effect of inhibiting zebrafish psen1 and psen2 activity on mitochondrion-ER appositions in 24 hpf embryos. The findings in this study differ from those done in mammalian cells. In Chapter V we use Western immunoblot analysis to determine if the MAM contains several proteins of interest. The results of this study show that SORL1 protein, which has been linked to SAD, is present in the MAM. The work in Chapters IV and V will help to give deeper insight into the role MAM plays in both SAD and FAD. Autophagy is a regulated catabolic process in which the cell digests cytoplasmic constituents by lysosomal degradation. The role of autophagy in AD continues to be investigated, as dysfunction in any of the stages of autophagosome formation could lead to neurodegeneration. In Chapter VI we examine the ability of the antihistamine drug Latrepirdine to induce autophagy in zebrafish larvae. The findings of this chapter indicate Latrepirdine is capable of inducing autophagy in 72 hpf larvae, making it a suitable model to study the mechanisms of this drug.