Cognitive impairments following cranial irradiation can be mitigated by treatment with a tropomyosin receptor kinase B agonist

Abstract

Brain radiotherapy is frequently used successfully to treat brain tumors. However, radiotherapy is often associated with declines in short-term and long-term memory, learning ability, and verbal fluency. We previously identified a downregulation of the brain-derived neurotrophic factor (BDNF) following cranial irradiation in experimental animals. In the present study, we investigated whether targeting the BDNF high affinity receptor, tropomysin receptor kinase B (TrkB), could mitigate radiation-induced cognitive deficits. After irradiation, chronic treatment with a small molecule TrkB agonist, 7,8-dihydroxyflavone (DHF) in mice led to enhanced activation of TrkB and its downstream targets ERK and AKT, both important factors in neuronal development. DHF treatment significantly restored spatial, contextual, and working memory, and the positive effects persisted for at least 3months after completion of the treatment. Consistent with preservation of cognitive functions, chronic DHF treatment mitigated radiation-induced suppression of hippocampal neurogenesis. Spine density and major components of the excitatory synapses, including glutamate receptors and postsynaptic density protein 95 (PSD-95), were also maintained at normal levels by DHF treatment after irradiation. Taken together, our results show that chronic treatment with DHF after irradiation significantly mitigates radiation-induced cognitive defects. This is achieved most likely by preservation of hippocampal neurogenesis and synaptic plasticity.

Keywords: Cognitive function; Hippocampus; Irradiation; Neurogenesis; Synaptic plasticity; TrkB.

Fig. 1

In vivo activation of TrkB and its downstream targets. Hippocampal formation collected from the four experimental cohorts after irradiation and DHF treatment were examined for the extent of TrkB activation. A, experimental timeline for irradiation, DHF treatment, and tissue collection. B, the ratio of phosphoTrkB (pTrkB) and total TrkB. C and D, the ratio of phosphoErk (pErk) and phosphoAkt (pAkt) to total Erk and Akt, respectively. Results from two-way ANOVA with Bonferroni’s post-hoc analysis are shown. *, p<0.05. N=5 each. Western blot lanes 1-4: Veh/Sham; Veh/Irr; DHF/Sham; DHF/Irr.

Fig. 2

Examination of cognitive functions following irradiation and DHF treatment. A, experimental timeline showing the chronological order of different treatments and behavioral tests. B, success rate of SAB in the Y maze. Mice were tested at 8 and 14 weeks after irradiation. C, Barnes maze test for spatial learning and memory approximately 16-19 weeks after irradiation. Escape latencies from the final trial were plotted. D, experimental design for contextual fear conditioning. E and F, cued (E) and contextual (F) freezing measured approximately 9 and 15 weeks after irradiation. Results from two-way ANOVA with Bonferroni’s post-hoc analysis are shown. *, p<0.05; **, p<0.01. N=8 per group for Barnes maze and 12 per group for all other tests.

Fig. 3

Hippocampal neurogenesis following irradiation and DHF treatment. A, experimental timeline. Long-term and short-term study indicates the long and short interval, respectively, between BrdU injection and tissue collection. The long interval allowed examination of the long-term survival of newborn cells and neurons in the SGZ, while the short interval allowed assessment of progenitor cell proliferation at the time of BrdU administration. B and C, total number of newborn cells (BrdU+, B) and newborn mature neurons (BrdU+/NeuN+, C) in the SGZ of hippocampal dentate gyrus from the long-term study. D and E, total number of newborn cells (BrdU+, D) and immature neurons (Dcx+, E) in the SGZ of hippocampal dentate gyrus. Results from two-way ANOVA with Bonferroni’s post-hoc analysis are shown. *, p<0.05; **, p<0.01; ***, p<0.001. N=6 each.

Fig. 4

Changes in neuronal activities and synaptic proteins following irradiation and DHF treatment. A, experimental timeline highlighting time points of irradiation, DHF treatment, and tissue collection. B, expression of the immediate early gene, c-Fos, upon exposure to a novel environment. The number of c-Fos positive cells in the dentate gyrus was normalized to the area examined. C-E, the levels of synaptophysin (C), PSD-95 (D), and glutamate receptor (GluN2B) (E) in the synaptosomes isolated from the hippocampal formation. Results from two-way ANOVA with Bonferroni’s post-hoc analysis are shown. *, p<0.05; **, p<0.01. N=6 each. Western blot lanes 1-4: Veh/Sham; Veh/Irr; DHF/Sham; DHF/Irr.