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Title: The effects of Apolipoprotein E (APOE) polymorphism on in vitro human hippocampal neurogenesis
Author: Lee, Hyun Ah
ISNI:       0000 0004 9351 5774
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2020
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Hippocampal neurogenesis (HN) is a highly plastic mechanism of generating new neurons in the mammalian brain throughout adulthood and is also implicated in Alzheimer’s disease (AD). A growing body of evidence suggests that HN is altered during the earliest stages of AD even before the biological hallmarks of AD such as amyloid-β plaque or neurofibrillary tangle deposition begins, suggesting that HN can be a potential therapeutic target for early intervention of AD. Apolipoprotein E (APOE) ε4 genotype is a well-known genetic risk factor for late-onset sporadic AD which not only elevates the overall risk of developing AD but also significantly decreases the age of disease onset. Recent findings have demonstrated that APOE plays an important role in the regulation of HN and ε4 allele is associated with diminished HN. However, the impact of various APOE genotypes on HN in the human context has not been investigated to the full extent. In this study, the role of APOE polymorphism in HN was investigated using a newly established in vitro cellular model of HN that can generate dentate gyrus granule cell (DGC)-like neurons from pluripotent stem cells in total of 42 days. The effects of APOE genotype were studied by utilising a set of isogenic human induced pluripotent stem cell lines that harbour mutations only in the genomic regions responsible for giving rise to different APOE isoforms (rs429358 and rs7412). Time-course characterisation was performed on various neurogenic properties including the expression pattern of neural progenitor cell (NPC) markers known to play an important role in hippocampal cell fate determination, cell proliferation, apoptosis, and the capacity to differentiate into DGC-like neurons. In addition, the potential interaction between oxidative stress and APOE genotype on the course of HN was studied by treating the cells with tert-Butyl hydroperoxide (TBHP), an inducer of oxidative stress, at the NPC stage and examining changes in cell proliferation, apoptosis, and the capacity to differentiate into DGC-like neurons. Finally, RNA sequencing followed by differential gene expression analysis was performed to understand the effects of APOE genotype and TBHP on HN at the transcriptomic level. The results of time-course characterisation experiments show that the phenotype differences across the isogenic lines are subtle. The expression pattern of neural progenitor cell (NPC) markers known to play an important role in hippocampal cell fate determination was altered in ε4/ε4. However, the overall capacity to generate DGC-like neurons was comparable to ε3/ε3. Interestingly, a differential gene expression level for APOE was observed at the pluripotent stem cell stage prior to differentiation, but phenotype divergence across APOE genotypes became more clear at the neuronal stage. The results of APOE x TBHP experiments show that TBHP affects ε3/ε3 and ε4/ε4 cells at different stages of differentiation. However, ε4/ε4 did not display exacerbated phenotypes of HN compared to ε3/ε3. RNA sequencing and differential gene expression analysis on transcriptomic profiles of ε3/ε3 and ε4/ε4 cells revealed that the highest number of differentially expressed genes is found when NPCs differentiate into neurons, confirming the findings from time-course characterisation experiments. Furthermore, CHCHD2 gene was always found to be down-regulated in ε4/ε4 regardless of differentiation stage and TBHP treatment, suggesting that ε4 genotype is associated with mitochondrial dysfunction. Finally, control ε4/ε4 neurons shared many genes that were differentially expressed in TBHP-treated ε3/ε3 neurons such as IL33, a well-known pro-inflammatory gene. In conclusion, the effects of APOE genotype on HN might be subtle, but ε4 genotype seems to be associated with vulnerabilities that could potentially affect the function and health of newborn neurons in the DG if met with harsh environmental conditions. Future investigations on identifying the factors that can affect such vulnerabilities would facilitate the development of tailored therapeutic strategies for ε4 carriers and help the prevention of AD in this population.
Supervisor: Thuret, Sandrine ; Srivastava, Deepak Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available