Dissertations, Theses, and Capstone Projects

Date of Degree

2-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Patricia Cortes

Advisor

Gonzalo Torres

Committee Members

Jun Yoshioka

Ashiwel Undieh

Aneel Aggarwal

Venetia Zachariou

Subject Categories

Biological Phenomena, Cell Phenomena, and Immunity | Biology | Cell Biology | Genetics | Laboratory and Basic Science Research | Macromolecular Substances | Molecular and Cellular Neuroscience | Other Cell and Developmental Biology | Other Chemicals and Drugs | Other Neuroscience and Neurobiology | Substance Abuse and Addiction

Keywords

methamphetamine, DNA damage, double stranded breaks, dopamine, substance use disorder, comet assay

Abstract

Methamphetamine (METH) abuse remains a global health concern, with emerging evidence highlighting its genotoxic potential. In the central nervous system METH enters dopaminergic cells primarily through the dopamine transporter (DAT), which controls the dynamics of dopamine (DA) neurotransmission by driving the reuptake of extracellular DA into the presynaptic neuronal cell. Additional effects of METH on the storage of DA in synaptic vesicles lead to the dysregulated cytosolic accumulation of DA. Previous studies have shown that after METH disrupts intracellular vesicular stores of DA, the excess DA in the cytosol is rapidly oxidized. This generates an abundance of reactive oxygen species (ROS) that promote oxidative stress leading to cellular damage, including DNA damage.

Our investigation begins by establishing a cellular system that overexpresses a functional DAT (GFP-DATC2) and elucidating the role of DAT and DA in METH-induced nuclear DNA damage, with a focus on nuclear DNA double-stranded breaks (DSBs). DNA DSBs, which represent the most deleterious form of DNA damage, were measured using two classical and well established approaches namely, detection of phosphorylated H2AX (gH2AX) and the neutral comet assay. Immunohistochemistry (IHC) experiments reveal an increase in

g-H2AX expression in METH-treated N2A GFP-DATC2 cells compared to control N2A GFP cells, highlighting the link between DAT expression and DNA DSB formation. IHC analyses also demonstrated that the DAT blocker GBR 12909 significantly reduces METH-induced DNA DSBs in GFP-DATC2 cells, providing further evidence of DAT's pivotal role in this genotoxic process. Interestingly, the DNA DSBs induced by exposing cells to low concentrations of METH were repaired after METH removal.

Investigation using the comet assay to functionally characterize nuclear DNA DSBs shows that co-treatment with METH and inhibitors targeting cytoplasmic ROS production, from DA, such as N-acetyl-l-cysteine (NAC), substantially decreases METH-induced DNA DSBs. Also in regard to DA regulation such as, pargyline and tetrabenazine, inhibitors of monoamine oxidase B (MAO-B) and vesicular monoamine transporter 2 (VMAT2) respectively showed a strong inhibition of METH-induced DNA DSBs. Intriguingly, inhibition of monoamine oxidase A (MAO-A) on the other hand fails to elicit a similar effect, suggesting the distinctive contributions of MAO-B in this context. Our findings also shed light on L-DOPA's dual role in DNA damage, which includes a protective effect in preventing METH-induced DNA DSBs at low concentrations.

In summary, our studies unravel the intricate mechanisms underpinning METH-induced DNA DSBs in N2A cells overexpressing DAT. Highlighting the central roles of DAT, DA, ROS, MAO-B, and VMAT2 in the genotoxic processes induced by METH. These insights into the molecular pathways of METH-induced DNA DSBs may guide future therapeutic strategies to mitigate the detrimental effects of METH abuse on genomic stability.

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