Rachael Hanson

Introduction. Alzheimer’s disease is a progressive neurodegenerative disease that affects nearly 50 million people worldwide and is the leading cause of dementia among elderly individuals.¹ FAD makes up approximately 5% of AD cases, and is usually caused by a mutation in the APP gene.² An A673T mutation in the APP gene was shown to decrease β-secretase cleavage of APP by 40% in vitro.³ The A673T mutation was five times more common in dementia-free elderly individuals than in people with AD, and was found to protect against age-associated cognitive decline.³ The goal was to investigate the effects of the A673T mutation on Aβ production, to potentially target and prevent development of FAD through the use of gene therapy. Methods. The APP gene was inserted into a pcDNA6/V5-His plasmid and mutated, then repeated for 29 different forms of FAD and mutated with the A673T variant. Concentrations of Aβ40 and Aβ42 peptides were measured in the neuroblastoma culture medium and compared between plasmids containing solely an FAD mutation and those containing both FAD and A673T mutations.³ The size of oligomers made from synthetic human WT and A673T mutant Aβ proteins was determined via Western blot, and binding to neurons measured using immunofluorescence.⁴ Cas9 nickase was used to induce the cytosine to thymine mutation and the levels of Aβ40 and Aβ42 peptides were measured against APP WT and APP V717I cells in the culture media to assess the efficacy of the Cas9n system.⁵ Results. The insertion of the A673T mutation decreased the concentration of Aβ40/Aβ42 peptides produced by the WT APP and 7 out of 29 FAD mutation plasmids.³ A673T mutant Aβ forms approximately 50% fewer high molecular weight oligomers compared to WT Aβ. Assembly of A673T mutant Aβ oligomers plateau faster than WT, and exhibit a decreased propensity to aggregate. A673T oligomers bound to fewer neurons and with less intensity than the WT oligomers, even at saturating levels.⁴ APP WT and APP V717I cell lines treated with the Cas9n system had statistically significant decreases in Aβ peptide concentrations.⁵ Conclusions. The decrease in Aβ peptides in plasmids containing A673T and FAD mutations as well as the WT APP plasmid present a potential target for gene therapy. By learning the mechanism by which the A673T mutation decreases Aβ production, more therapeutic targets can be discovered that would mimic the effects of an A673T mutation.

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