P5-TAT:a Novel Medicine Targeting CDK5 Over-activation For Radiation-induced Brain Injury

ObjectiveRadiation-induced brain injury is a common complication associated with cranial radiation therapy characterized by cognitive and emotional dysregulation.Hippocampal dysfunction,especially inhibition of hippocampal neurogenesis and proliferation,plays a critical role in radiation-induced brain injury.Radiation-induced hippocampal apoptosis may be significantly involved in this complication.Previous studies have demonstrated that hyperactivity of Cdk5 has a close relationship with hippocampal apoptosis.P5 is a 24-residue mimetic peptide of p35 and a specific inhibitor of p25/Cdk5.In our present study,we explored whether p5-TAT which can cross the blood-brain barrier would reduce hippocampal dysfunction and improved behavioral performance.Materials and MethodsIn the first part of the experiment,A total of 180 Sprague–Dawley male rats(21-day old)weighing ? 50-60 g were purchased from the Experimental Animal Center of Soochow University(Suzhou,China).Experimental animals were raised in temperatureand humidity-controlled animal with a 12h/12 h light/dark cycle(lights on at 7 AM)at 22 ± 2 C and free access to food and water ad libitum prior to the experiment.All the rules and regulations involving the care and protection of animals comply with Soochow University Animal Care and Ethics Guidelines,which are in agreement with national laboratory animal care.The animals were divided into four groups: the sham group(Sham),the sham with p5-TAT peptide injection(Sham +p5-TAT peptide),the irradiation group(IR),the irradiation and p5-TAT peptide injection group(IR +p5-TAT peptide).The rats were anesthetized with 3.6% chloral hydrate(360mg/kg)via intraperitoneal injection.The whole brain were received whole-brain irradiation(WBI)treatments with a single dose of 0(control)or 20 Gy by using 4 MeV electron beam,by a linear accelerator(Philips SL-18,UK).The dosage of the p5-TAT peptide used for each rat was 400ug/kg.Both of the second group and fourth group were injected with p5-TAT peptide for three days before radiation.Then the first part of the animals were sacrificed and the hippocampus tissues were collected for western blot detection at different time points(1h,3h,6h,12 h,24h,3d,7d)post-radiation.The second part of the animals continued to be treated with p5-TAT peptide for 3days post-radiation and then sacrificed for Immunofluorescence staining of hippocampus proliferating cells.The third part of the animals continued to be treated with p5-TAT peptide for 3days post-radiation and sacrificed for Immunofluorescence staining of hippocampal neurogenesis at 8weeks as well as hippocampus mature neurons at 4weeks after irradiation(n=3 per group for per time point).Immunofluorescence staining was used to detect hippocampal neurogenesis and proliferation.The behavior tests used here included open field,Morris water maze,novel object recognition.All experiments were performed during 08:00–16:00,and the experimenter was blinded to the treatment of the animals.Dates were expressed with mean ± SD and analyzed with two tiered Student's t test or one-oway ANOVA by GraphPad Prism(version 5.0;Graph Pad,La Jolla,USA).A value of P < 0.05 was considered to be statistically significant.Results1.Body weight and brain weight measurement:The average body weight of the irradiation group declined significantly at 1 month and at 2 months post-radiation versus the sham group(p <0.05)and the brain weight remained the same(p <0.05).Furthermore,p5-TAT peptide had no protective effect on either variable(p >0.05).2.WBI activated Cdk5 signals and induced apoptosis: We detected the expressions of Cdk5 activators,namely,p39,p35,andp29,p25,at different time points(1 h,3 h,6 h,12 h,24 h,3d,7d)post-radiation.Our data indicated that p25 increased at 1 h and peaked at 6 h post-radiation versus the sham group(P <0.05).The p35 level decreased noticeably at 1 h and reached its minimum level at 6 h post-radiation versus the sham group(P <0.05).Compared to p35/p25,p39/p29 demonstrated a similar variation trend.(P <0.05).We also examined the expression of Cdk5 at different time points(1 h,3 h,6 h,12 h,24 h,3d,7d)post-radiation.Our data indicated that Cdk5 expression did not noticeably change after radiation(P >0.05).Therefore,Cdk5 activity was further determined with the examination of its downstream substrates,phosphorylated Tau and GR,two markers of Cdk5 activity.Because of the remarkable level of p25 at 6 h post-radiation,we also analyzed the levels of p-GR and p-Tau,which significantly increased at 6 h post-radiation.(P <0.05).Previous studies indicated that over-activated Cdk5 induces cellular apoptosis.Therefore,we also detected cleaved caspase-3,a marker of apoptosis,at different time points(1 h,3 h,6 h,12 h,24 h,3d,7d)by Western blot.WBI induced a robust increase of cleaved caspase-3 at 6 h post-radiation(P <0.05).3.P5-TAT prevented radiation-induced apoptosis by inhibition of Cdk5 hyperactivity: Previous studies has proven that p5-TAT injection(50 ?g/kg)reduced Cdk5 activity without influencing the levels of Cdk5 activators including p35,p25,and p39,p29.Here,we further explored if p5-TAT injection(400 ?g/kg)had effect on the expression of these activators in radiation-induced brain injury.P5-TAT treatment did not affect the levels of p35 and p25(P >0.05).Since p5-TAT had no impact on the levels of Cdk5 activators p35,p25 and p39,p29,we alternatively explored the inhibition effect of p5-TAT on Cdk5 hyperactivity at three various doses(100 ?g/kg,200 ?g/kg,400 ?g/kg).Interestingly,we found that a dose of 400 ?g/kg seemed to be the most effective,so this dose was selected for the rest study.P5-TAT treatment significantly suppressed Cdk5 over-activation at 6 h post-radiation as demonstrated by the decreased levels of p-tau and p-GR(P <0.05).All of the results revealed the excellent inhibitory effect of p5-TAT on Cdk5 hyperactivity.Targeting the inhibition of Cdk5 signaling may be a novel potential intervention for radiation-induced apoptosis.Therefore,the next step in the study was the examination of neuronal apoptosis after p5-TAT treatment.We observed a remarkable reduction of cleaved caspase-3,a marker of apoptosis,at 6 h post-radiation by Western blot assay.(P <0.05),suggesting the excellent anti-apoptotic property of p5-TAT.4.P5-TAT peptide impeded radiation-induced injuries to hippocampal proliferation and neurogenesis as well as neuronal survival: The protection effect of p5-TAT peptide on radiation-induced brain injury was assessed by analysis of hippocampal neurogenesis and proliferation as well as neuronal survival.Our data showed that p5-TAT peptide increased the number of BrdU+ cells in the DG at 3 days post-radiation versus the irradiated group(P <0.05),suggesting protection of hippocampal proliferation provided by p5-TAT.Similarly,we observed an elevation of BrdU+/ NeuN+ cells in the DG at 2 month post-radiation versus the irradiated group due to protection of hippocampal neurogenesis(P <0.05).In addition,We found that the total number of mature neurons labelled with NeuN were preserved in the IR+p5-TAT peptide group in contrast to the IR group at 1 months post-radiation(P <0.05).5.P5-TAT peptide prevented radiation-induced cognitive deficits.In our study,p5-TAT demonstrated significant beneficial effects on radiation-induced cognitive impairment in behavioral tests,including the open field test,novel object recognition and the morris water maze test.Performance on learning and memory tests is greatly associated with different levels of anxiety.Therefore,we initially assessed anxiety with the open field test.There was an obvious difference in the total number of grids traveled and the time spent in the central region between the sham group and the irradiation group.However,p5-TAT treatment improved both performance variables of the open field test versus the irradiation group,revealing excellent remission of anxiety.(P < 0.05).Second,all rats demonstrated a preference for the novel object in the novel object recognition test.WBI clearly increased the total time of investigation of the familiar object in the irradiation group in contrast to the sham group.Our data indicated that the irradiation group failed to discriminate the novel object from the familiar object versus the other groups prior to p5-TAT treatment.(P <0.05).Third,in the training trials,WBI significantly increased escape latency compared with the sham group at 4 days and 5 days post-radiation.Furthermore,in the spatial probe test,the irradiation group exhibited poorer performance in crossing platform times and a lower percentage of time in the target quadrant versus the sham group.Our results sufficiently demonstrated that impaired learning and memory was improved with p5-TAT treatment.(P <0.05).Conclusions1.WBI activated the p25/Cdk5/caspase-3 pathway and subsequent hippocampal cell apoptosis.P5-TAT rescued radiation-induced apoptosis by inhibition of Cdk5 hyperactivity.2.WBI significantly induced dysfunction of hippocampal neurogenesis and proliferation as well as neuronal survival.p5-TAT protected subsequently hippocampal neurogenesis and proliferation as well as neuronal survival by the inhibition of Cdk5 hyperactivity.3.WBI significantly induced behavioral cognitive dysfunction.p5-TAT demonstrated excellent therapeutic effects on behavioral cognitive dysfunction by the inhibition of Cdk5 hyperactivity and protection of hippocampal neurogenesis and proliferation as well as neuronal survival.