Date of Award
Doctor of Philosophy (PhD)
Alzheimer’s disease (AD) is the most common cause of dementia worldwide, and is now the 5th leading cause of death in the United States. The pathologic hallmarks of AD include the deposition of extracellular plaques of aggregated amyloid-β (Aβ) and intracellular neurofibrillary tangles of tau aggregates (NFTs). Autosomal dominant inheritance of AD has been attributed to genetic mutations in three key genes: amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN-2). Together, these pathologic findings and genetics provided the framework for the amyloid cascade hypothesis, which states that Aβ deposition is a necessary, early event that is upstream of the formation of NFTs, and is causative of AD. Despite this seminal work, the mechanisms underlying the clinical progression of AD is still poorly understood. This gap in our knowledge is due, in large part, to the lack of appropriate AD disease models. Specifically, rodent models of human neurodegenerative disease fail to completely recapitulate disease phenotypes. In the body of work that follows, we utilized recent advances in induced pluripotent stem cell (iPSC) and genome editing technologies to investigate two separate AD disease mechanisms: tau spreading and Aβ production in familial AD (FAD) PSEN1 mutants. Using TALENs, we generated a transgenic donor iPSC line that harbors a transgene for inducing the expression of fluorescently tagged tau protein and a recipient iPSC line that expresses membrane anchored YFP. These cell lines, when differentiated into human cortical neurons and cultured together, demonstrated that tau is transferred between neurons. However, similar protein spreading was observed for the control cell line expressing only mCherry, suggesting that tau did not transfer by a unique mechanism in this culture system. With the hope of revealing new insights into AD mechanisms, we next used CRISPR/Cas9 to produce a series of isogenic iPSC lines that harbor discrete FAD PSEN1 mutations. These mutations in PSEN1 alter the relative amount of Aβ peptides, specifically increasing the ratio of Aβ 42:40 and Aβ 42:38. Our results demonstrate that each mutation causes a reduction in the levels of both Aβ40 and Aβ38, as well as an increase in Aβ42. These results support the model that FAD PSEN1 mutations cause a loss of protein function, in that PSEN1 cannot properly process Aβ42 into smaller, less aggregation prone peptides. Consistent with this, we found that C-terminal fragments of APP (β-CTF) accumulate in neurons with homozygous FAD mutations in PSEN1. Additionally, we also observed defects in the processing of other g-secretase substrates such as N-Cadherin. Intriguingly, the Aβ 42:40 ratio and Aβ40 levels correlated with disease onset in heterozygous mutants, while Aβ42 levels correlated with disease onset in homozygous mutants, suggesting that these values could be predictive of disease progression in culture. These results are all consistent with a partial loss in PSEN1 function, extending our current understanding of how FAD PSEN1 mutations affect PSEN1 function, and, perhaps more importantly, identifying a mechanistic perturbation that is common to the tested FAD mutations. Taken together, PSEN1 dysfunction results in production of larger, aggregation prone Aβ peptides, as well as CTFs. These insights may be important for ultimately understanding how these mutations cause FAD, which will be critical for developing effective therapeutics that slow or prevent progression of this devastating disease.
Gregg, Andrew, "Modeling Alzheimer's Disease in Induced Pluripotent Stem Cells" (2018). Student Theses and Dissertations. 428.