Investigating β- and γ-secretase function and processing of APP with genetic and pharmacological tools

The sequential cleavage of Amyloid Precursor Protein (APP) by BACE1 and the gamma secretase complex (γS) generates amyloid-beta (Aβ), a small peptide that is highly implicated in the pathogenesis of Alzheimer’s Disease. Despite decades of research, there remains a lack of understanding of the endogenous functions of APP, BACE1, and γS in the brain, their subcellular distribution, and why the physiological production of Aβ becomes pathogenic, underscoring a critical need for further research into the basic molecular mechanisms that govern APP processing. Here, I demonstrate the endogenous tagging of Nicastrin, an integral subunit of γS, with the fluorescent protein mEmerald in human cells using CRISPR-Cas9, allowing for subcellular visualization of γS distribution in real time. I also test the ability of an optogenetic nanobody to perturb tagged γS function by light-induced clustering, and outline the creation of homology directed repair templates for endogenous tagging of the catalytic subunit of γS (PSEN1), as well as BACE1, with a genetic construct allowing for fluorescent visualization and optogenetic manipulation. I then create and test several genetic constructs that can potentially allow for manipulation of enzymatic function in a subcellular compartment-specific manner. Using confocal microscopy and quantitative image analysis, I demonstrate that the neurotrophin BDNF, previously shown to reduce BACE1 activity, alters the subcellular distribution of BACE1 in SH-SY5Y cells, increasing plasma membrane localization and reducing endosomal localization, and that this is coincident with reduced amyloidogenic processing of APP. Further, I show that this effect appears to be independent of clathrin-mediated endocytosis. I conclude that BDNF may reduce production of Aβ by altering BACE1 distribution, decreasing upstream β-cleavage. Lastly, BACE1 and γS play important roles in neural differentiation through regulation of Notch signalling. Here, I characterize the ability of several compounds with previously reported neurogenic effects, many of which are directly or indirectly linked to APP processing, to increase neuronal differentiation in two commonly used human neural cell lines by assessing the expression of neuron- and glia-specific markers. I also show that Neurogenin-2, a protein indirectly regulated by the same enzymes that process APP, can be used to improve the differentiation capacity of both cell types.

Name thesis:degree_name

Ph.D. Biological Sciences

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Faculty of Mathematics and Science

Department dc:contributor.department

Department of Biological Sciences

Grantor dc:publisher

Brock University

Year dc:date.issued

2025

Author dc:creator

• Hallam, Ryan

Advisor dc:contributor.advisor

• Necakov, Aleksandar

• NATURAL SCIENCES::Biology::Cell and molecular biology::Molecular biology
• NATURAL SCIENCES::Biology::Cell and molecular biology::Cell biology
• NATURAL SCIENCES::Biology::Cell and molecular biology::Neurobiology

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/10464/19656

OAI identifier oai:identifier

oai:brocku.scholaris.ca:10464/19656