Adenosine Kinase Inhibition Protects against Cranial Radiation-Induced Cognitive Dysfunction

Abstract

Clinical radiation therapy for the treatment of CNS cancers leads to unintended and debilitating impairments in cognition. Radiation-induced cognitive dysfunction is long lasting; however, the underlying molecular and cellular mechanisms are still not well established. Since ionizing radiation causes microglial and astroglial activation, we hypothesized that maladaptive changes in astrocyte function might be implicated in radiation-induced cognitive dysfunction. Among other gliotransmitters, astrocytes control the availability of adenosine, an endogenous neuroprotectant and modulator of cognition, via metabolic clearance through adenosine kinase (ADK). Adult rats exposed to cranial irradiation (10 Gy) showed significant declines in performance of hippocampal-dependent cognitive function tasks [novel place recognition, novel object recognition (NOR), and contextual fear conditioning (FC)] 1 month after exposure to ionizing radiation using a clinically relevant regimen. Irradiated rats spent less time exploring a novel place or object. Cranial irradiation also led to reduction in freezing behavior compared to controls in the FC task. Importantly, immunohistochemical analyses of irradiated brains showed significant elevation of ADK immunoreactivity in the hippocampus that was related to astrogliosis and increased expression of glial fibrillary acidic protein (GFAP). Conversely, rats treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 3.1 mg/kg, i.p., for 6 days) prior to cranial irradiation showed significantly improved behavioral performance in all cognitive tasks 1 month post exposure. Treatment with 5-ITU attenuated radiation-induced astrogliosis and elevated ADK immunoreactivity in the hippocampus. These results confirm an astrocyte-mediated mechanism where preservation of extracellular adenosine can exert neuroprotection against radiation-induced pathology. These innovative findings link radiation-induced changes in cognition and CNS functionality to altered purine metabolism and astrogliosis, thereby linking the importance of adenosine homeostasis in the brain to radiation injury.

Keywords: adenosine; adenosine kinase; astrogliosis; cancer therapy; cognition; neuroprotection; radiation.

FIGURE 1

Adenosine kinase (ADK) inhibition by systemic 5-iodotubercidin (5-ITU) treatment protects against radiation-induced cognitive dysfunction. Adult rats received 5-ITU (3.1 mg/kg, i.p., daily for 6 days) and were irradiated (0 or 10 Gy, head only) 1 h after the last injection. Animals were divided into four experimental groups: 0 or 10 Gy whole brain irradiated receiving either vehicle or 5-ITU (Con, Con + 5-ITU, IRR, IRR+5-ITU). (A,B) 1 month post-irradiation, animals were tested on spatial and episodic memory retention using the NPR and NOR tasks followed by fear conditioning (FC). The tendency to explore a novel place (NPR) or object (NOR) was derived from the Discrimination Index (DI). (A,B) Whole brain irradiation (IRR) shows significant behavioral deficits on NPR and NOR tasks compared to controls (Con and Con + 5-ITU) as indicated by impaired preference to a novel place or object. Irradiated animals treated with 5-ITU (IRR + 5-ITU) show significant preference for the novelty when compared with irradiated (IRR) animals receiving vehicle. (C) 5-ITU treatment also improves behavior on the hippocampal-dependent contextual FC task. The baseline freezing levels were comparable across groups, and all groups showed elevated freezing behavior following a series of 5 tone-shock pairings. The context test was administered 24 h later, and IRR animals showed significantly decreased freezing compared to controls (Con and Con + 5-ITU). Irradiated animals receiving 5-ITU showed a significant elevation in freezing behavior that was indistinguishable from the Con group. Data are presented as mean ± SEM. (N = 8–10 animals/group). P-values are derived from ANOVA and Bonferroni’s multiple comparisons test. ^∗∗∗P < 0.001; ^∗∗P < 0.01; ^∗P < 0.05 compared with the IRR group.

FIGURE 2

Cranial irradiation elevates adenosine kinase (ADK) immunoreactivity and astrogliosis. Immunofluorescence analysis demonstrates that at 1 month post-treatment, compared to controls (Con and Con + 5-ITU), exposure to cranial irradiation (10 Gy) leads to elevated ADK immunoreactivity (A,B; IRR group; ADK, green; DAPI nuclear counterstain, blue) that is reduced to control levels in irradiated animals treated with 5-ITU (IRR + 5-ITU). Representative confocal micrographs show the presence reactive astrocytic cell bodies (A,C; glial fibrillary acidic protein; GFAP, red) in the hippocampal dentate hilus (DH), sub-granular zone and granule cell layer (GCL) indicating astrogliosis. IRR + 5-ITU animals showed reduced ADK and GFAP immunoreactivity compared to IRR animals. Scale bar: 30 μm.

FIGURE 3

Work flow of quantitative immunofluorescence. Blinded deconvoluted volume of z stacks (step 1) were uploaded into Imaris (v8.1.2) for quantification of GFAP and ADK immunoreactivity (step 2). 3D algorithm-based Surface rendering of individual channels (pseudo-colored, steps 3–4) provide quantitative analyses of fluorescence intensity (GFAP, ADK) and Spot tool and Co-localization modules provide quantification of the number of astrocytes (steps 4–5). Data bin (step 6) provide quantitative immunofluorescence data for the individual channels. Scale bars: 5 μm.

FIGURE 4

Treatment with 5-iodotubercidin (5-ITU) attenuated radiation-induced elevation in adenosine kinase (ADK) and astrogliosis. 3D algorithm-based deconvolution and quantification (Imaris, Bitplane, Inc.) of ADK and glial fibrillary acidic protein (GFAP) immunoreactivity show that compared to controls (Con and Con + 5-ITU), irradiation significantly increased the ADK (A) and astrogliotic cell bodies (B) in the hippocampal dentate hilus, granule cell layer, sub-granular zone and CA3/CA1 subfields. Compared with the irradiated cohort (IRR), animals receiving 5-ITU (IRR + 5-ITU) had significantly lower ADK and GFAP immunoreactivity in all hippocampal subfields. The reduced ADK and GFAP immunofluorescence was comparable to controls (Con). The number of astrocytes per hippocampal section did not change after irradiation or 5-ITU treatment (C). All data are presented as mean ± SEM. (N = 4 animals per group). ^∗P < 0.02; ^∗∗P < 0.01; ^∗∗∗P < 0.001 compared with the IRR group (ANOVA and Bonferroni multiple comparisons test).