DNA breaks regulate neuronal activity: NMDA-receptor andionizing radiation-mediated DNA double-strand breaks governactivity regulating early-response gene expression

Abstract

DNA (Deoxyribonucleic acid) double-strand breaks (DSBs) are the hazardous form of DNA damage endured on multiple occasions by both proliferating and post-mitotic cells. Unlike replicating cells, which can employ sister chromatids as a repair template, post-mitotic cells such as adult neurons could suffer lethal effects from error-prone repair of non-homologous end-joining (NHEJ) machinery. Hence, until recently, DSBs were considered only the results of pathological activity in post-mitotic cells. However, recent shreds of evidence also show the presence of neuronal activity-induced and type 2 topoisomerase beta (Top2β) generated transient physiological DSBs, which regulate the expression of multi-functional early response or immediate early genes (IEGs). Till yet, these DSBs driven gene regulations have only been studied in cortical cultures. Among many, one of the functions of effectors IEGs is the regulation of neuronal activity by activation of secondary response genes. Therefore, hypothesizing that neuronal activity induced DSB promotes certain IEGs expression and further these genes could regulate the activity, we aimed to, i) first validate the correlation of neuronal activity generated DSBs with IEGs expression in neuronal model systems other than cortical cultures ii) investigate the effects of altering DNA DSB's status on neuronal network activity. As a model system for our analysis, we employed 14 days-in-vitro (DIV) mice hippocampal cultures (HCs), where the expression of IEGs has been reported before. As only specific neuronal activity could lead to induction of physiological DSBs, we primarily focused on the DNA breaks generated by N-Methyl-D-aspartate receptors (NMDARs) dependent cAMP (Cyclic adenosine monophosphate) response element-binding protein (CREB) activity. For our investigation, we employed a combination of Microelectrode arrays recordings (MEA) for neuronal network activity measurements, Gamma-H2AX staining to monitor DSBs' presence and messenger Ribonucleic acid (mRNA) and protein analysis for IEGs expression. Following NMDAR activation, we detected an increase in DSBs status, which correlated with the expression of IEGs- c-Fos, Egr1 and c-Jun and upregulation in neuronal activity. Additionally, the DSBs status and IEGs expression were also analyzed following the inhibition of essential synaptic plasticity-associated components, CREB and the GluN2B subunit of the NMDAR. Our results showed that the deficiency in any of these factors leads to a decrease in certain IEGs expression, likely by causing changes in DSBs status. Further, to investigate if DSBs status alteration can affect neuronal firing activity by perturbating IEGs expression, we incubated HCs with either etoposide, a chemotherapeutic agent that generates DSBs on the site of Top2β activity, Top2β (small interfering RNA) siRNA and NHEJ inhibitor (NU7441). Our data discovered that a perturbation in either the induction and repair of the DSBs following NMDAR activation contributes to both altered IEGs expression and neuronal activity. Furthermore, the IEGs expression was also monitored in the Severe combined immunodeficiency (SCID) mice model. Our novel results revealed that these mice models have a low level of IEGs expression, most likely due to the inherent deficiency in the components of NHEJ. This could lead to severe learning and memory problems that these mice possess. Thus, our study provides experimental evidence on the role of synaptic NMDARs mediated DSBs in mediating IEGs expression and further how a perturbation in the DSBs induction and repair could contribute to an altered pattern of neuronal activity in HCs. Furthermore, by assessing these connective links of receptor activity with gene expression and neuronal recordings, our study validates the multi-purpose use of such an in-vitro model to elucidate changes in cellular and electrophysiological levels. In the second part of this work, we investigated the effect of low-dose radiation exposure on IEGs expression. Ionizing radiation (IR) is an effective treatment for various tumors infested in the central nervous system (CNS). Proceeding the clinical trials, multiple literature pieces have shown how even low dose exposure could alter synaptic plasticity, a basis for cognitive functioning. However, it becomes complicated to assess the connective link between irradiation and synaptic plasticity-associated components due to IR's multi targets. Therefore, to understand how irradiation can have a lethal effect on cognitive domains, we focussed on the expression of the genes that are easily activated by various stimuli and play a role in learning and memory acquisition. For this, we studied the effect of low to moderate IR doses on the expression of IEGs in 14 DIV HCs. Results of immunostaining against Gamma-H2AX following radiation exposure revealed that a single 2 Gy dose generates many DSBs in the post-mitotic neuron post-60-minute (min) exposure. However, the expression of IEGs such as c-Fos and Egr1 as assessed by mRNA and protein analysis following the same dose radiation exposure is downregulated. As both of these genes are the regulator of neuronal activity, we next analyzed the neuronal network activity following irradiation. Our data revealed that a single dose of 2 Gy is sufficient to cause a decrease in neuronal network activity after 60 min of exposure. NMDAR plays a vital role in IEGs expression; therefore, we investigated if NMDA incubation can counteract the radiation alleviated IEGs expression level. Consequently, we further investigated the simultaneous impact of NMDAR activation and irradiation on the control of IEGs expression by DSB's and mRNA analysis. Our results revealed that 60 min of continuous NMDA treatment counteracts radiation-induced alleviation of IEGs expression. These findings are the first to reveal changes in IEGs expression and neuronal activity in HCs following 1-hour post- single-irradiation events. Further, it hints towards the interaction radiation has with the NMDAR pathway for the gene expression changes.