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- Creators: Newbern, Jason
Description
Immediate early genes (IEGs) are rapidly activated in response to an environmental stimulus, and most code for transcription factors that mediate processes of synaptic plasticity, learning, and memory. EGR3, an immediate early gene transcription factor, is a mediator of biological processes that are disrupted in patients with schizophrenia (SCZ). A microarray experiment conducted by our lab revealed that Egr3 also regulates genes involved in DNA damage response. A recent study revealed that physiological neuronal activity results in the formation of DNA double-stranded breaks (DSBs) in the promoters of IEGs. Additionally, they showed that these DSBs are essential for inducing the expression of IEGs, and failure to repair these DSBs results in the persistent expression of IEGs. We hypothesize that Egr3 plays a role in repairing activity- induced DNA DSBs, and mice lacking Egr3 should have an abnormal accumulation of these DSBs. Before proceeding with that experiment, we conducted a preliminary investigation to determine if electroconvulsive stimulation (ECS) is a reliable method of inducing activity- dependent DNA damage, and to measure this DNA damage in three subregions of the hippocampus: CA1, CA3, and dentate gyrus (DG). We asked the question, are levels of DNA DSBs different between these hippocampal subregions in animals at baseline and following electroconvulsive stimulation (ECS)? To answer this question, we quantified γ-H2AX, a biomarker of DNA DSBs, in the hippocampal subregions of wildtype mice. Due to technical errors and small sample size, we were unable to substantiate our preliminary findings. Despite these shortcomings, our experimental design can be modified in future studies that investigate the role of Egr3 in activity-induced DNA damage repair.
ContributorsKhoshaba, Rami Samuel (Author) / Newbern, Jason (Thesis director) / Gallitano, Amelia (Committee member) / Marballi, Ketan (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Damage to DNA can affect the genes it encodes; if this damage is not repaired, abnormal proteins may be produced and cellular functions may be disturbed. DNA damage has been implicated in the initiation and progression of a variety of diseases. Conversely, DNA damage has also been discovered to contribute to beneficial biological processes. Madabhushi and colleagues (2015) determined that activity-dependent DNA double strand breaks (DSBs) in the promoter region of immediate early genes (IEGs) induced their expression. EGR3 is an IEG transcription factor which regulates the expression of growth factors and synaptic plasticity-associated genes. In a previously conducted microarray experiment, it was revealed that EGR3 regulates the expression of genes associated with DNA repair such as Cenpa and Nr4a2. These findings inspired us to investigate if EGR3 affects DNA repair in vivo. Before conducting this experiment, we sought to standardize and optimize a method of inducing DNA damage in the hippocampus. Electroconvulsive stimulation (ECS) is utilized to induce neuronal activity. Since neuronal activity leads to the formation of DNA DSBs, we theorized that ECS could be used to induce DNA DSBs in the hippocampus. We predicted that mice that receive ECS would have more DNA DSBs than those that receive the sham treatment. Gamma H2AX, a biomarker for DNA damage, was utilized to quantify DNA DSBs. Gamma H2AX expression in the dentate gyrus, CA1 and CA3 regions of the hippocampus was compared between mice that received the sham treatment and mice that received ECS. Mice that received ECS were sacrificed either 1 or 2 hours post-administration, constituting treatment conditions of 1 hr post-ECS and 2 hrs post-ECS. Our results suggest that ECS has a statistically significant effect exclusively in the CA1 region of the hippocampus. However, our analyses may have been limited due to sample size. A power analysis was conducted, and the results suggest that a sample size of n=4 mice will be sufficient to detect significant differences across treatments in all three regions of the hippocampus. Ultimately, future studies with an increased sample size will need to be conducted to conclusively assess the use of ECS to induce DNA damage within the hippocampus.
ContributorsAden, Aisha Abubakar (Author) / Newbern, Jason (Thesis director) / Gallitano, Amelia (Thesis director) / Marballi, Ketan (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05