Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Anatomy and Cell Biology

Supervisor

Whitehead, Shawn N.

2nd Supervisor

Walton, Paul A.

Joint Supervisor

Abstract

A core pathological feature of AD is the over-production of reactive oxygen species (ROS), which mediate oxidative stress in the brain. Peroxisomes play a crucial role in mitigating ROS accumulation due to the presence of catalase, an antioxidant enzyme found within the organelle. As the brain ages, progressive dysfunction of peroxisomes and decreased localization of catalase into the organelle contributes to elevated ROS. A suitable therapy to address the increased accumulation of ROS in AD has yet to be identified. Our team has engineered a recombinant derivative of the antioxidant enzyme catalase (CAT-SKL) that specifically targets peroxisomes, thereby providing powerful organelle-based antioxidant and anti-inflammatory effects. In the present study, we aimed to (i) establish the packaging CAT-SKL into macrophage-derived exosomes — endogenous membrane vesicles for the transport of proteins between cells—to effectively target our therapeutic to cells undergoing oxidative stress while minimizing degradation in the bloodstream, (ii) determine the safety and biodistribution in vivo, and (iii) assess the protective effects of prophylactic intranasal administration of exosomes loaded with CAT-SKL (exoCAT-SKL). CAT-SKL was packaged into macrophage-derived exosomes via sonication, and loaded exosomes were purified using size exclusion chromatography or ultracentrifugation. Male and female, control, and 3xTG AD transgenic mice were intranasally administered exoCAT-SKL, and tissues of interest were investigated. The diffuse labelling of CAT- SKL in these mice's brains supports the intranasal administration of exosomes as an effective delivery method to bypass issues surrounding bioavailability, antigenicity, and the blood-brain barrier. Histopathological assessments of the brain and off-target tissue demonstrated no toxicity. Further behavioural and immunohistochemical analysis is required to determine whether exoCAT- SKL can prevent and ameliorate pathological and behavioural outcomes in 3xTG AD mice.

Summary for Lay Audience

This project investigated the use of nanosized vesicles called exosomes to carry and deliver CAT-SKL, an antioxidant molecule, into a preclinical transgenic mouse model of Alzheimer’s disease (AD). Human patients with AD demonstrate progressive dysfunction of peroxisome- mediated lipid oxidation leading to excessive accumulation of reactive oxygen species (ROS). CAT-SKL specifically targets these peroxisomes and provides antioxidant, anti-inflammatory and anti-ageing effects. Intranasal administration of CAT-SKL loaded exosomes can penetrate the brain, potentially preventing and ameliorating pathological, biochemical, and behavioural outcomes. This project also provided further validation for the use of nanoparticles to deliver therapeutics to the brain.

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