Date of Award
Doctor of Philosophy (PhD)
Molecular Science and Nanotechnology
Gergana G. Nestorova
The high lipid content of the brain, coupled with its heavy oxygen dependence and relatively weak antioxidant system, makes it highly susceptible to oxidative DNA damage that contributes to neurodegeneration. This study assesses and compares the neurotoxic effects of proton and photon radiation on mitochondrial function and DNA repair capabilities of human astrocytes. Human astrocytes received either proton (0.5 Gy and 3 Gy), photon (0.5 Gy and 3 Gy), or sham-radiation treatment. The mRNA expression level of the human base-excision repair protein, 8-deoxyguanosine DNA glycosylase 1 (hOGG1) was determined via RT-qPCR. Radiation-induced changes in mitochondrial mass and oxidative activity were assessed using fluorescent imaging with MitoTracker™ Green FM and MitoTracker™ Orange CM-H2TMRos dyes, respectively. A significant increase in mitochondrial mass and levels of reactive oxygen species was observed after radiation treatment. This was accompanied by a decreased OGG1 mRNA expression. These results are indicative of a radiation-induced dose-dependent decrease in mitochondrial function, an increase in senescence and astrogliosis, and impairment of the DNA repair capabilities in healthy glial cells. Photon irradiation was associated with a more significant disruption in mitochondrial function and base-excision repair mechanisms in vitro in comparison to the same dose of proton treatment. This study further identifies specific ROS-responsive miRNAs that modulate the expression and activity of the DNA repair proteins in human astrocytes, which could lead to the development of targeted therapeutic strategies for neurological diseases. Oxidative DNA damage was established after treatment of human astrocytes with 10 μM sodium dichromate for 16 hours. Comet assay analysis indicated a significant increase in oxidized guanine lesions. PCR analysis confirmed that sodium dichromate reduced the mRNA expression levels of hOGG1. Small RNAseq was performed on an Ion Torrent™ system and the differentially expressed miRNAs were identified using Partek Flow® software. The biologically significant miRNAs were selected using miRNet 2.0. Oxidative-stressinduced DNA damage was associated with a significant decrease in miRNA expression: 231 downregulated miRNAs and 2 upregulated miRNAs (p < 0.05; > 2-fold). In addition to identifying multiple miRNA-mRNA pairs involved in DNA repair processes, this study uncovered two novel miRNA-mRNA pairs interactions: miR-1248:OGG1 and miR-103a- OGG1. Inhibition of miR-1248 and miR-103a via the transfection of their inhibitors restored the increased expression levels of hOGG1. Therefore, targeting the identified microRNAs could ameliorate the nuclear DNA damage caused by exposure to mutagens. The miRNA candidates identified in this study could serve as potential biomarkers and therapeutics for oxidative stress in the brain to reduce the incidence and improve the treatment of cancer and neurodegenerative disorders.
In a parallel but closely related study, we report a direct, one-step exosome sampling technology, for selective capture of CD63+ exosome subpopulations using an immune-affinity protocol. The ExoPRIME microprobe provides a Precise Rapid Inexpensive Mild (non-invasive) and Efficient (i.e. PRIME) alternative to the conventional polymer precipitation-based methods by enriching a comparatively more homogenous exosome population. The tool consists of an inert Serin™ stainless steelz microneedle (300 μm in diameter × 30 mm in height), pre-coated with a thin-film polyelectrolyte layer that serves as a substrate for covalent bonding of biotin. An anti-CD63 steptavidin-conjugated antibody that selectively binds to the corresponding tetraspanin embedded in the lipid bilayer of exosomes was immobilized to the outer surface of the probe. The feasibility of the ExoPRIME technology was validated using two types of biological samples: conditioned astrocyte medium (CAM) and astrocyte-derived exosome suspension (EXO). The study investigated the impact of the temperature (4°C and 22°C) and incubation duration (2h and 16h) on the capture efficiency of the ExoPRIME tool. A fluorescence-based enzymatic assay for exosome quantification was used to assess the probe’s exosomes capture efficiency and the reproducibility of the technology. The low level of non-specific binding initially observed in non-functionalized microneedles was drastically minimized by blocking the ExoPRIME probe with 0.1% BSA. The ExoPRIME microprobe captured exponentially more exosomes than the non-functionalized microneedle that indicates enrichment of CD63-expressing exosomes.
A major advantage provided by the ExoPRIME technology over existing platforms is its applicability over a broad dynamic range of temperature and incubation parameters without compromising the purity and viability of exosomal cargoes. The loading capacity of the probe increased after incubation for 16 h at 40C in exosome suspension (24Å~106 exosomes per probe) while the efficiency decreased 10 folds after 2 h at 40C (24Å~105 exosomes per probe). The increase in temperature had an impact on the stability of the reagents that contributed to a 2-fold efficiency reduction after incubation in exosome suspension for 16 h at 220C (12Å~106 exosomes per probe). However, the 2-hour roomtemperature incubation (2 h at 220C) of the ExoPRIME probe yielded an increased capture efficiency (12Å~106 exosomes per probe) when compared to the 2 h at 4°C incubation (24Å~105 exosomes per probe). These results suggest that lower temperatures with extended incubation times constitute the most optimal parameters that ensure high probe loading capacity.
Another advantage of the ExoPRIME microprobe is that it captures antigen-specific subpopulation of exosomes directly from conditioned astrocyte medium (CAM), eliminating the requirements for additional filtration and pre-concentration, and thereby cutting down costs and handling time. Besides the relatively reduced number of enriched exosomes, the CAM results are consistent with the trend obtained for EXO incubations, a phenomenon that could be attributed to the presence of various extracellular proteins and cellular debris, which could mask antibodies and compete physically with exosomes for binding. The capabilities to integrate different incubation times, temperatures, and biofluid type thus present exosome researchers with the flexibility to choose the combined parameters that best suit their purpose, the desired factor in clinical and laboratory applications.
The developed tool requires very low amounts of antibody, permits the use and reuse of minimal sample volumes (≤ 200 μL), can be multiplexed in arrays to diagnostically profile multiple exosome classes and is amenable to integration into a lab-on-a-chip platform to achieve parallel, high-throughput isolation in a [semi]-automated workstation. Moreover, this platform could provide direct exosomal analysis of biological fluids since it can elegantly interface with existing picomolar-range nucleic acid assays to provide a clinical diagnostic tool at the point of care and facilitate fundamental studies in exosomes functions.
Nwokwu, Chukwumaobim Daniel Obiwulu, "" (2021). Dissertation. 911.