HDAC-Mediated Glial Crosstalk Mediates Radiation Induced Memory Changes from Whole Brain Radiation in a Mouse Model

Abstract

Prior work demonstrated radiation sensitivity can be regulated via Class III HDAC-mediated intracellular DNA damage response coordinated with Wnt-beta catenin signaling. We hypothesized that radiation induced functional alterations in HDAC mediated glial crosstalk in the CNS produce intercellular signaling alterations leading to phenotypic changes in cognition following whole brain radiation exposure. Primary human astrocytes, human astrocytoma (U251), and immortalized human microglia (HMC3) were examined in vitro in response to treatment with conditioned media isolated from irradiated glial cells. Prior to treatment with either single-dose or fractionated radiation schedules, cells were transfected with siRNA expression vector for Sirt2, a class III HDAC, versus scrambled controls. Quantitative PCR and immunoblots for neurotransmitters, metabolism, and inflammatory markers were obtained. Cells exposed to conditioned media were examined by immunofluorescence for cytoskeletal alterations and changes in junctional proteins. The findings were correlated with in vivo qPCR and immunohistochemical changes in brain tissue and functional memory changes in Sirt2 knockout versus wild type mice after whole brain radiation using novel object recognition testing at 2 weeks and 6 month post radiation timepoints. Statistical analysis was performed using paired Students T test between treated and control groups. Cells treated with conditioned media produced from irradiated U251, astrocytes and microglia produced increased expression of inflammatory cytokines IL-6, IL-8, GM-CSF and VEGF. However, immunofluorescence of cells treated with conditioned media from irradiated Sirt2 knockdown microglia and primary astrocytes demonstrated qualitative disruptions in glutamate neurotransmitter metabolism and actin cytoskeletal alterations with changes in pseudopodia and lamellipodia after staining with phalloidin and neurofilament L, suggesting altered intercellular communication. Connexin 43 in gap junctions was increased >2-fold (p<0.05) after exposure to conditioned media, whereas E-cadherin in adherens junctions was not significantly affected. Novel object recognition testing of Sirt2 knockout mice demonstrated resistance to radiation induced memory decline from whole brain radiation at pre radiation day 5 versus post radiation days 5 and 185 compared to controls (p<0.05). Glial crosstalk can be mediated via elimination of a Class III HDAC and appears to be a key mediator of radiation induced disruptions of intercellular communication in the CNS, connecting radiation to structural changes on the cell surface to synaptic activity and neurotransmitter metabolism, leading to functional disputations in recognition memory similar to what is experienced by patients receiving brain radiotherapy. These data suggest glial cross talk as a new therapeutic avenue to combat radiation induced cognitive dysfunction.