Abstract
Oxidative DNA damage induces changes in the neuronal cell cycle and activates a DNA damage response (DDR) to promote repair, but these processes may be altered under a chronic oxidative environment, leading to the accumulation of unrepaired DNA damage and continued activation of a DDR. Failure to repair DNA damage can lead to apoptosis or senescence, which is characterized by a permanent cell cycle arrest. Increased oxidative stress and accumulation of oxidative DNA damage are features of brain ageing and neurodegeneration, but the effects of persistent DNA damage in neurons are not well characterized. We developed a model of persistent oxidative DNA damage in immortalized post-mitotic neurons in vitro by exposing them to a sublethal concentration of hydrogen peroxide following a 'double stress' protocol and performed a detailed characterization of the neuronal transcriptome using microarray analysis. Persistent DNA damage significantly altered the expression of genes involved in cell cycle regulation, DDR and repair mechanisms, and mitochondrial function, suggesting an active DDR response to replication stress and alterations in mitochondrial electron transport chain. Quantitative polymerase chain reaction (qPCR) and functional validation experiments confirmed hyperactivation of mitochondrial Complex I in response to persistent DNA damage. These changes in response to persistent oxidative DNA damage may lead to further oxidative stress, contributing to neuronal dysfunction and ultimately neurodegeneration.
Highlights
Neurons are long-lived terminally differentiated cells that are vulnerable to oxidative stress due to their high metabolic rate and limited antioxidant mechanisms (Dringen et al, 1999, 2005)
Exposure to a single dose of a sublethal concentration of hydrogen peroxide (H2O2) promoted an oxidative environment and caused oxidative DNA damage in the form of double-strand breaks (DSBs) that were detected through γH2AX foci formation very early after treatment. γH2AX is widely used as a marker of DSBs, as the number of phosphorylated H2AX molecules correlates with the quantity of DNA damage (Paull et al, 2000; Rogakou et al, 1998)
The results of this study demonstrate that acute and persistent oxidative DNA damage have different effects on the transcriptomic profile of immortalized post-mitotic neurons
Summary
Neurons are long-lived terminally differentiated cells that are vulnerable to oxidative stress due to their high metabolic rate and limited antioxidant mechanisms (Dringen et al, 1999, 2005). If the DNA damage is not completely repaired, resulting in a persistent DDR, it could result in a senescent-like phenotype in neurons, a mechanism that was thought to only occur in mitotic cells (Jurk et al, 2012; Simpson et al, 2015). These data point to the involvement of persistent DNA damage and a DDR in the early stages of neurodegeneration, and a more detailed characterization is needed to identify their impact on neuronal function. To determine how persistent oxidative DNA damage affects neurons and could potentially lead to neuronal dysfunction, we developed an in vitro model of persistent oxidative stress in immortalized post-mitotic neurons and conducted transcriptomic profiling using microarray technology to determine alterations of cell pathways under this condition
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