Unraveling the Impact of DNA Glycosylase NTHL1 on Mitochondrial Function and Dynamics

Every cell experiences a diverse array of dangerous DNA lesions daily. To counteract the resultant damage to vital genetic information, our cells deploy multiple DNA repair pathways, among which Base Excision Repair (BER) plays a pivotal role in eliminating oxidative lesions in the nucleus and in the mitochondria. Within BER, the initial step involves a DNA Glycosylase, and one such glycosylase, Nth-Like DNA Glycosylase 1 (NTHL1), takes center stage in this thesis, specifically focusing on its role in the mitochondria. The significance of BER and NTHL1 stems from emerging evidence suggesting that this theoretically beneficial DNA repair pathway may, paradoxically, contribute to harm during aging. This appears to be attributed to an imbalance in the processing speed of different pathway stages. While the initial BER steps efficiently excise damaged bases and generate DNA nicks—actions that occur at a consistent or accelerated rate with age—the subsequent steps, particularly end processing, slow down during aging. Consequently, the accumulation of single-strand breaks (ssBreaks) becomes inevitable, posing numerous challenges for cellular integrity (SenGupta et al., 2021). In this thesis we investigated the impact of NTHL1 knockout (NTHL1-/-) on mitochondrial DNA stability and mitochondrial dynamics in human HEK293 cells. Utilizing CRISPR-Cas9 technology, we established and validated NTHL1-/- cell lines and conducted a series of assays to assess mitochondrial function and DNA damage. Our investigations revealed that, in comparison to WT cells, NTHL1-/- cell lines are characterized by increased mitochondrial DNA (mtDNA) copy number and increased mtDNA lesions. Intriguingly, heightened levels of oxidative phosphorylation (OXPHOS) proteins suggested enhanced mitochondrial activity in the absence of NTHL1. These mitochondrial protein changes were accompanied by a substantial rise in both basal and maximum oxygen consumption rates (OCRs). Inspired by neuroprotection observed in Caenorhabditis elegans (C. elegans) following NTH-1 knockdown, a secondary objective was to establish a Parkinson's Disease (PD) model to investigate the potential effect of NTHL1 on the aggregation of alpha-synuclein (⍺-SYN), a protein associated with Parkinson’s Disease (PD) and expressed by the SNCA gene. Using the PiggyBac system we introduced GFP-tagged ⍺-SYN genes (GFP:SNCA) in the HEK293 genome. After selecting single clones highly expressing GFP:SNCA, we generated multiple GFP:SNCA NTHL1-/- cell lines via our established CRISPR Cas9 system. This model offers a platform to scrutinize the impact of NTHL1 on alpha-synuclein accumulation in future studies. These findings propose a broader role for NTHL1, extending beyond its conventional role in DNA repair. Further investigation is warranted to elucidate the precise mechanisms by which NTHL1 influences mitochondrial dynamics.