Defense Date

6-18-2024

Graduation Date

Summer 8-10-2024

Availability

One-year Embargo

Submission Type

thesis

Degree Name

MS

Department

Pharmaceutics

School

School of Pharmacy

Committee Chair

Devika Soundara Manickam

Committee Member

Wilson S. Meng

Committee Member

Lauren O’Donnell

Committee Member

James K. Drennen III

Keywords

extracellular vesicles uptake, brain endothelial cells, endocytosis, membrane fusion, amyotrophic lateral sclerosis, mitochondria, motor neurons

Abstract

Extracellular vehicles (EVs) are an emerging class of drug carriers and are primarily reported to be internalized into recipient cells via a combination of endocytic routes such as clathrin-mediated, caveolae-mediated and macropinocytosis pathways. In part one of this thesis, (1) we investigated potential effects of homotypic vs. heterotypic interactions by studying the cellular uptake of homologous EVs (EV donor cells and recipient cells of the same type) vs. heterologous EVs (EV donor cells and recipient cells of different types) and (2) determined the route of EV internalization into low pinocytic/hard-to-deliver cell models such as brain endothelial cells (BECs). We used BECs and macrophages as low-pinocytic and phagocytic cell models, respectively, to study the effect of homotypic vs. heterotypic interactions on EV uptake in the recipient cells. We studied both medium to large EVs (m/lEVs) as well as small EVs (sEVs). Homotypic interactions led to a greater extent of uptake into the recipient BECs compared to heterotypic interactions. However, we did not see a complete reduction in EV uptake into recipient BECs when endocytic pathways were blocked using pharmacological inhibitors. Our results suggest that EVs primarily use membrane fusion to enter low-pinocytic recipient BECs instead of relying on endocytosis. Lipophilic PKH67 dye-labeled EVs but not intravesicular esterase-activated calcein ester-labeled EVs severely reduced particle uptake into BECs while phagocytic macrophages internalized both types of EV-labeled particles to comparable extents. Our results also highlight the importance of carefully choosing labeling dye chemistry to study EV uptake, especially in the case of low pinocytic cells such as BECs. In part two of this work, we developed neuron-derived EVs as a potential treatment for amyotrophic lateral sclerosis (ALS). ALS is fatal condition characterized by progressive degeneration of motoneurons with a survival time of ~three years after diagnosis. With no curative therapies available, ALS leads to muscle weakness, progressive motor disability, and finally death by respiratory failure or an associated infection. Rilutek, Relyvrio and Radicava slow down progression of ALS, however, their efficacy is limited, they pose numerous side effects and fail to prolong the life expectancy of the patients. Pre-clinical and human patient data reveal that mitochondrial dysfunction is an early pathological event in ALS. Therefore, increasing mitochondrial function may potentially be therapeutic in ALS. EVs naturally contain cargoes such as nucleic acids, lipids and proteins and mitochondria and are capable of transferring these cargoes to the recipient cells. We have previously demonstrated that BEC-derived m/lEVs can increase the ATP levels and mitochondrial bioenergetics in the recipient BECs and shows potential for neuroprotection in a mouse model of stroke. Here, we investigated the potential of harnessing EV mitochondria to increase mitochondrial function in motor neurons under oxidative stress. Here, we developed and characterized neuron-derived EVs from NSC-34, a Neuroblastoma x spinal cord hybrid cell line (a mouse neural hybrid cell line developed by the fusion of neuroblastoma with motor neuron-enriched embryonic day 12-14 spinal cord cells). We demonstrated that large EVs but not small EVs transferred their innate mitochondrial content to the recipient motor neurons. Additionally, we observe increased cellular ATP levels in the motor neurons under oxidative stress when treated with m/lEVs. Our results suggest that m/lEV-mediated mitochondrial transfer resulted in a functional increase in the recipient motor neuron ATP levels. Additional work will be instrumental in developing m/lEVs as a therapeutic modality to treat damaged motor neurons in ALS.

Language

English

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