Role of the NF-κB pathway in the radiation response of primary murine astrocytes

Abstract

Exposure to ionizing radiation, as part of space radiation, is a major limiting factor for crewed space exploration. Astronauts will encounter different types of space radiation, which may cause cognitive damage causing detrimental effects on learning and attention, elevated anxiety and depression. Astrocytes, the most abundant glial cells of the CNS, have supportive function in the brain, for example in the immune response and sustaining the function of neurons. NF-κB is a transcription factor, regulating several different cellular and immunological processes, such as inflammation and apoptosis. NF-κB transcriptionally activates different genes that are involved in proliferation, angiogenesis, apoptosis, cell cycle, cytokine release, cell signalling and reactivity. Importantly, NF-κB is activated in response to ionizing radiation and but its function in the radiation response of astrocytes remains to be determined. In this study, primary murine astrocytes were irradiated with different doses of X-rays and 12C ions at two different heavy ion accelerator facilities, and various biological endpoints of the radiation response were studied. NF-κB pathway activation (p65), cell proliferation (Ki-67) and reactivity of astrocytes (GFAP) were analysed by immunofluorescence and fluorescence microscopy. Cell cycle progression was investigated on the basis of DNA content, using flow cytometry. Furthermore, the induction of gene expression changes, after exposure to ionizing radiation was investigated by RT-qPCR, for the genes of interest: CDKN1A, CDKN2A, GFAP, TNF, Il1β, Il6 and Tgfβ1. In addition, levels of the pro-inflammatory cytokine IL-6 was studied using ELISA. Our results show distinct responses of primary murine astrocytes to the two different radiation qualities, X-rays and carbon (12C) ions, tested. Analysis of NF-κB pathway activation did not reveal a clear dose-, time-, or LET-dependent activation. In contrast, a LET-dependent S phase delay in cell cycle was observed, as well as time-, but not dose-dependent increase of the cytokine IL-6 and GFAP protein expression. X-irradiation and 12C irradiation induced doseand time-dependent regulation of gene expression, while proliferation of astrocytes remained mostly unaffected. Astrocytes were shown to respond to ionizing radiation with regulation of gene expression and a S phase delay in cell cycle, while they were also observed to be relatively unresponsive to ionizing radiation with regards to NF-κB pathway activation, proliferation, production of cytokine Il-6 and reactivity.