X-ray irradiation promotes apoptosis of hippocampal neurons through up-regulation of Cdk5 and p25

Abstract

Background: Cranial radiation therapy has been used for the treatment of primary and metastatic brain tumors. A prominent feature of brain injury induced by the radiation therapy is hippocampal dysfunction, characterized by a decline in memory. Cdk5 plays an important role in memory formation. Abnormal Cdk5 activity is associated with neuronal apoptosis induced by neurotoxic stimuli. However, the roles of Cdk5 in hippocampal apoptosis in response to X-ray irradiation have not been explored.

Methods: The expression of Cdk5 activators, p35 and p25, in hippocampal neurons was tested in both in vivo animal and in vitro couture after X-ray irradiation.

Results: After X-ray irradiation at 20 Gy and 30 Gy in rats, the number of hippocampal neuronal pyknosis was increased, but the number of hippocampal neuron was decreased, in the hippocampal CA1 region of rats. In these animals undergone with X-ray irradiation, the expression of p35 was significantly down-regulated, but it was up-regulated in p25. These opposite expressions were also shown in the primary cultured hippocampal neurons with 30 Gy irradiation. The apoptosis induced by X-ray irradiation were significantly prevented by the pretreatment of Cdk5 inhibitor, roscovitine, in both in vivo and in vitro settings.

Conclusions: X-ray irradiation resulted in a hippocampal neuronal apoptosis through up-regulation of p25, the Cdk5 activator. Hyperactivity of Cdk5 was involved in the pathogenesis of X-ray irradiation-induced hippocampal neuronal apoptosis. Blockade of Cdk5 signal pathway effectively protected neurons from the irradiation-induced brain injury.

Figure 1

A-F Hippocampal neuronal apoptosis in CA1 region in rats after irradiation. DAPI fluorescent images showing hippocampal neuronal apoptosis in rats with sham-irradiated control (A), irradiation of 10 Gy (B), 20 Gy (C), 30 Gy (D), and 30 Gy+R (E). The cells were counted in a 1 mm length of the middle portion of hipocampal CA1 region under bright-filed microscopy. n=6 for each group. *p<0.05 in 20 Gy and 30 Gy vs. control, *p<0.05 in 30 Gy+R vs. 30 Gy+S. R=Roscovitine, S=Saline.

Figure 2

A-C Western blot analysis of p35 and p25 expression in rats after irradiation. Representative Western blot showing the expression of p35 and p25 in rat hippocampi. Lane 1, sham-irradiation; lane 2, 10 Gy irradiation; lane 3, 20 Gy irradiation; lane 4, 30 Gy irradiation (A). Quantitative Western blot analysis of p35 (B) and p25 (C) expression in rats with sham-irradiation. β-actin was a loading control. n=3. *p<0.05 vs. control.

Figure 3

A-C Western blot analysis of p35 and p25 expression in cultured hippocampal neurons at different time after irradiation. Representative Western blot showing the expression of p35 and p25 in cultured hippocampal neurons exposed to a single dose of 30 Gy irradiation, and tested at 3.5 h, 4 h, 5 h, and 6 h after irradiation (A). Expression of p35 and p25 was tested at3.5 h after sham-irradiation as control. Quantitative Western blot analysis of p35 (B) and p25 (C) expression in cultured hippocampal neurons with sham-irradiation, and with 30 Gy irradiation at 3.5, 4 h, 5 h, and 6 h after irradiation. β-actin was a loading control. n=4. *p<0.05 vs. control.

Figure 4

A-E Hippocampal neuronal apoptosis in both in vitro and in vivo hippocampal neurons after irradiation. DAPI fluorescent images showing pyknosis of in vitro hippocampal neurons exposed to sham-irradiation (A), 30 Gy (B), 30 Gy with roscovitine (in vitro) (C), and 30 Gy with roscovitine (in vivo) (D). Percentage of in vitro and in vivo pyknosis with and without administration of roscovitine (E). *p<0.05 vs. control, #p<0.05 vs. 30 Gy, **p<0.05 vs. 30 Gy+S. S=Saline, R=Roscovitine.