Student Theses and Dissertations

Date of Award

Spring 5-18-2024

Document Type

Thesis

Degree Name

B.A. with honors

Honors Designation

yes

Program of Study

Biology

Language

English

First Advisor

Pablo Peixoto

Second Advisor

Rebecca Spokony

Third Advisor

Krista C. Dobi

Abstract

Aerobic metabolism is known to generate damaging ROS, particularly hydrogen peroxide. Reactive oxygen species (ROS) are highly reactive molecules containing oxygen that have the potential to cause damage to cells and tissues in the body. ROS are highly reactive atoms or molecules that rapidly interact with other molecules within a cell. Intracellular accumulation can result in oxidative damage, dysfunction, and cell death. Due to the limitations of H2O2 (hydrogen peroxide) detectors, other impacts of ROS exposure may have been missed. HyPer7, a genetically encoded sensor, measures hydrogen peroxide emissions precisely and sensitively, even at sublethal levels, during regular neural activation. In this study, we used HyPer7 to measure sublethal hydrogen peroxide levels in mitochondria during brain activation. Mouse-derived neuroblastoma cell lines (NSC-34) were differentiated into a more advanced neuron-like phenotype and then transfected with HyPer7. The cells were then imaged using confocal fluorescence in the presence of high potassium (K+) medium. Neuronal excitation is initiated by a sudden rise in extracellular potassium (K+) concentrations, which is subsequently accompanied by an excessive depolarization. Depolarization initiates the activation of voltage-dependent calcium channels, leading to the influx of calcium ions into the cell. Ca2+ ions are transported from the cytoplasm into the mitochondria through voltage dependent anion channels (VDAC) or calcium uniporter. This process is commonly known as Ca2+ uptake. This mechanism occurs during ATP synthesis and leads to the creation of hydrogen peroxide. This preliminary work shows that mitochondrial hydrogen peroxide levels can be measured and detected during presynaptic excitation in cultured mammalian motor neurons.

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