Abstract
Our understanding of the molecular processes underlying Alzheimer’s disease (AD) is still limited, hindering the development of effective treatments and highlighting the need for human-specific models. The potential for human induced pluripotent stem cells (iPSCs) to provide a model for AD is an exciting advancement, and has great potential for investigating the mechanisms involved in neurodegeneration. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating β-amyloid (Aβ) toxicity. Mutations in CLU and APOE, major genetic AD risk factors, are known to have important roles in Aβ processing. This thesis aims to characterise the molecular cascade initiated by Aβ previously identified in rodent neurons. Aβ-mediated toxicity will be studied in cortical neurons differentiated from wild type iPSCs and a clusterin-knockout cell line generated by precise genome editing. This thesis also presents the process of developing an APOE mutant iPSC line.iPSCs from a healthy male with an APOE ε3/ε3 genotype were differentiated into cortical neurons and treated with Aβ25-35 peptides and Aβ1-42 oligomers. The downstream effects of Aβ exposure were measured using an automated imaging assay, optimised to identify changes in neuronal morphology. Western blotting and qPCR were used to assess changes in protein and gene expression. CRISPR/Cas9-mediated gene editing generated a CLU-knockout iPSC line. The morphological and transcriptomic effects of the CLU deletion were then investigated in differentiated neurons.
In wildtype cells imaging indicated that neuronal processes degenerate following Aβ treatment, in a dose dependent manner, and that intracellular levels of clusterin are increased following Aβ treatment, as occurs in rodent neurons. However, in CLU knockout neurons these morphological effects of Aβ neurotoxicity were absent, suggesting that clusterin is an important component of the amyloid cascade. Transcriptomic data from RNAseq were analysed to elucidate the pathways responsible for the altered response to Aβ in CLU knockout neurons.
An iPSC based isogenic model of AD has been established with different alleles of known risk genes. Determining the role of specific risk alleles on established measures of Aβ-neurotoxicity using a high throughput assay will be of great value to obtaining a better understanding of the underlying disease mechanisms and for the evaluation of compounds able to modulate these pathways.
| Date of Award | 1 Jun 2017 |
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| Original language | English |
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| Supervisor | Jack Price (Supervisor) |