Date of Award

Fall 11-16-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Science and Nanotechnology

First Advisor

Gergana Nestorova

Abstract

Extracellular vesicles (EVs) play a major role in cell-to-cell communication via the horizontal transfer of RNA, DNA, proteins, and lipids that affect the physiological response of the recipient cells. Astrocytes are a type of glial cell that exerts a protective effect on neurons and brain endothelial cells. The astrocytes and the endothelial cells form the blood-brain barrier. Due to their nano-size and non-complex structure, EVs can efficiently cross the blood-brain barrier. This study investigated and assessed the impact of EVs on reducing oxidative DNA damage in human brain endothelial cells (HBECs). The protective potential of astrocyte-derived EVs was determined by assessing the changes in nuclear 8-OHdG concentration and OGG1 mRNA levels in HBECs treated with sodium dichromate to induce oxidative DNA damage. Exposure to sodium dichromate for 5 and 16 hours induced oxidative DNA damage, as reflected in increased genomic 8-OHdG levels, while the EVs mitigated the accumulation of the adduct, reducing the levels to the normal physiology range. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to assess the gene expression levels of OGG1, an enzyme involved in the excision of the 8-OHdG adducts. A neurotoxic environment caused an upregulation of the DNA repair enzyme OGG1 in HBECs while the astrocyte-derived EVs reduced its expression. An MTT assay, conducted to evaluate cell proliferation and cytotoxicity, revealed that EVs were associated with increased cell proliferation and reduced cytotoxicity in sodium dichromate-treated cells. Mito Tracker Green fluorescent analyses were performed to evaluate the impact of EVs on mitochondrial mass and copy numbers in human brain endothelial cells exposed to oxidative stress. Previous studies indicate that oxidative stress leads to increased mitochondrial mass, decreased oxidative activity, and reduced mtDNA copy numbers in the cells.[1] Mitochondrial dysfunction, including reduced copy number and circular morphology induced by neurotoxic exposure, was reversed by astrocyte-derived EVs, leading to elongated and interconnected mitochondria. High-throughput RNA sequencing revealed that exposure to sodium dichromate without EVs suppressed immune response genes. The addition of astrocyte-derived EVs resulted in the dysregulation of long noncoding RNAs impacting genes associated with brain development and angiogenesis. These findings reveal the positive impact of astrocyte-derived EVs in mitigating neurotoxicity and as potential therapeutic avenues for neurodegenerative diseases.

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