CR8, a selective and potent CDK inhibitor, provides neuroprotection in experimental traumatic brain injury

Abstract

Traumatic brain injury (TBI) induces secondary injury mechanisms, including cell cycle activation (CCA), that leads to neuronal death and neurological dysfunction. We recently reported that delayed administration of roscovitine, a relatively selective cyclin-dependent kinase (CDK) inhibitor, inhibits CCA and attenuates neurodegeneration and functional deficits following controlled cortical impact (CCI) injury in mice. Here we evaluated the neuroprotective potential of CR8, a more potent second-generation roscovitine analog, using the mouse CCI model. Key CCA markers (cyclin A and B1) were significantly up-regulated in the injured cortex following TBI, and phosphorylation of CDK substrates was increased. Central administration of CR8 after TBI, at a dose 20 times less than previously required for roscovitine, attenuated CCA pathways and reduced post-traumatic apoptotic cell death at 24 h post-TBI. Central administration of CR8, at 3 h after TBI, significantly attenuated sensorimotor and cognitive deficits, decreased lesion volume, and improved neuronal survival in the cortex and dentate gyrus. Moreover, unlike roscovitine treatment in the same model, CR8 also attenuated post-traumatic neurodegeneration in the CA3 region of the hippocampus and thalamus at 21 days. Furthermore, delayed systemic administration of CR8, at a dose 10 times less than previously required for roscovitine, significantly improved cognitive performance after CCI. These findings further demonstrate the neuroprotective potential of cell cycle inhibitors following experimental TBI. Given the increased potency and efficacy of CR8 as compared to earlier purine analog types of CDK inhibitors, this drug should be considered as a candidate for future clinical trials of TBI.

Fig.1

Traumatic brain injury (TBI) induces up-regulation of cyclins A and B1 and cyclin-dependent kinase (CDK) activation. (a-c) The expression of the 2 key cyclins (A and B1) was evaluated in cortical tissue following controlled cortical impact (CCI) by Western blot analysis. There was a significant up-regulation of cyclin A (a, b) (^**p < 0.01 vs sham) at 6 h, followed by a reduction at 24 h post-injury (^{^}p < 0.05 vs 6-h injured samples). The expression of cyclin B1 (a, c) (^*p < 0.05 vs sham) was significantly increased at 6 h after TBI. (d, e) To determine TBI-induced changes in CDK activity, levels of phospho-(Ser)-CDK substrates were evaluated. We observed a significant increase in phospho-(Ser)-CDK substrate levels (^*p < 0.05 vs sham; ^{^}p < 0.05 vs 6-h injured samples) at 24 h after TBI. Representative Western blots are shown. Analysis by one-way analysis of variance, followed by post-hoc adjustments using the Student-Newman-Keuls test. Mean ± standard error of the mean (n = 3-5/group)

Fig. 2

Central administration of CR8 inhibits cell cycle activation and apoptosis in cortical tissue after traumatic brain injury (TBI). (a-g) The effect of CR8 treatment on post-traumatic cell cycle activation (CCA) was evaluated by Western blot analysis of injured cortical tissue at 6 h after TBI. A significant increase resulted in cyclin A expression from TBI (a, b) (^***p < 0.001 vs sham), whereas CR8 treatment significantly attenuated the TBI-induced increase (⁺⁺p < 0.01 vs vehicle). TBI significantly increased (^***p < 0.001 vs sham) cyclin B1 expression (a, c), and the levels of the CDK1 substrate phospho-n-myc (a, d); CR8 treatment significantly attenuated levels of both CCA markers (cyclin B1: ⁺p < 0.05 vs vehicle; phospho-n-myc: ⁺⁺p < 0.01 vs vehicle). To be able to assess the effect of CR8 on apoptosis, the presence of 145/150 KDa cleaved fragment of fodrin was determined. TBI increased levels of the 145/150 kDa fodrin fragment (a, e) (^***p < 0.001 vs sham); CR8 treatment significantly the TBI-induced increase (p < 0.01 vs vehicle). Representative Western blots are shown. Analysis by one-way analysis of variance, followed by post-hoc adjustments using the Student-Newman-Keuls test. Mean ± standard error of the mean (n = 3-5/group)

Fig. 3

Central administration of CR8 improves sensorimotor function following traumatic brain injury (TBI). Fine motor coordination deficits were quantified using the beam walk test. Hind-limb foot placement was recorded and the number of mistakes (foot faults) was recorded from 50 steps. TBI induced significant impairment in motor outcomes at all time points (^**p < 0.01 vs sham). There was a statistically significant “post-injury day X groups” interaction (F(8,110) = 2.833) (p = 0.007). Central administration of CR8 at 3 h post-injury significantly improved fine motor coordination at 7 days (⁺⁺p = 0.002 vs vehicle), 14 days (⁺p = 0.014 vs vehicle), and 21 days (⁺⁺p = 0.002 vs vehicle) post-TBI. Analysis by repeated measures one-way analysis of variance, followed by post-hoc adjustments using the Student-Newman-Keuls test. Mean ± standard error of the mean (n = 10/group)

Fig. 4

Central administration of CR8 improves cognitive performance in the Morris water maze test following traumatic brain injury (TBI). (a) Spatial learning and memory was assessed using the Morris water maze (MWM) test. The factors of “post-injury days” (F(3,100) = 3.6) (p = 0.016) and “groups” (F(2,100) = 21.75) (p < 0.001) were statistically significant. TBI induced significant cognitive impairments at post-injury days 15, 16, and 17 (^**p < 0.01 vs sham). CR8-treated TBI mice had reduced latency to locate the submerged platform at day 15 (⁺p = 0.017 vs vehicle) and day 17 post-TBI (⁺⁺p = 0.0015 vs vehicle) when compared to vehicle-treated TBI mice. Analysis by repeated measures one-way analysis of variance, followed by post-hoc adjustments using the Student-Newman-Keuls test. Mean ± standard error of the mean (n = 10/group. (b, c) Reference memory was assessed using the probe trial of MWM test. TBI caused significant cognitive impairments in this test (^**p < 0.05 vs sham). CR8-treated TBI mice exhibited significant cognitive improvements in terms of number of entries (b) (⁺p < 0.05 vs vehicle) and latency to the first entry into the target quadrant (c) (⁺⁺⁺p < 0.001 vs vehicle) when compared to vehicle-treated TBI mice. Analysis by one-way analysis of variance, followed by post-hoc adjustments using the Student-Newman-Keuls test. (d) Cognitive performance was further evaluated by swim strategy analysis. Vehicle-treated TBI mice exhibited higher reliance on looping strategies rather than spatial and systematic patterns (p < 0.001 vs sham; χ² = 37.64). CR8-treated TBI mice showed significantly reduced usage of looping strategy and increased reliance on spatial and systematic swim strategies (p < 0.001 vs vehicle; χ² = 37.64). (e) The visual acuity of mice was evaluated by performing a visual cue test on post-finjury day 18. All mice performed well and there were no statistically significant differences between the groups. (f) The swim speeds (mm/s) of all mice was recorded all throughout the test and there were no statistically significant differences between the groups on all days

Fig. 5

Central administration of CR8 improves cognitive performance in the novel object recognition task following traumatic brain injury (TBI). (a, b) Retention or intact memory was assessed using the novel object recognition test. Vehicle-treated TBI mice showed significant cognitive impairments in this test (^**p < 0.01 vs sham). CR8-treated TBI mice had significant improvements in cognitive performance in terms of novel object exploration time (f) (⁺p < 0.05 vs vehicle) and discrimination index (g) (⁺p < 0.05 vs vehicle) when compared to vehicle-treated TBI mice. Analysis by one-way analysis of variance followed by post-hoc adjustments using the Student-Newman-Keuls test. Mean ± standard error of the mean (n = 10/group)

Fig. 6

Central administration of CR8 reduces lesion size following traumatic brain injury (TBI). (a) Unbiased stereological assessment of lesion volume at 21 days post-TBI was performed on cresyl violet stained brain sections. (b) Lesion quantification. CR8 treatment significantly reduced the lesion size at 21 days post-TBI (⁺⁺⁺p < 0.001 vs vehicle). Analysis by one-tailed paired Student’s t test versus sham and vehicle-treated groups. Mean ± standard error of the mean (n = 10/group). (c) Linear regression analysis comparing TBI-induced lesion volume with foot faults in the beam walk test at 21 days post-TBI. Linear regression model (n = 5/group) followed by determination of statistical significance and coefficient of correlation (p < 0.0001; r² = 0.96)

Fig. 7

Central administration of CR8 improves neuronal survival in the CA3 and DG after traumatic brain injury (TBI). (a, b) Unbiased stereological quantification of neuronal cell loss in the CA3 and dentate gyrus (DG) subregions of the hippocampus at 21 days post-TBI. Significant neuronal cell loss in the CA3 and DG caused by TBI (^**p < 0.01 vs sham). CR8 treatment significantly improved survival of neurons in the CA3 (a) and DG (b) regions (⁺⁺p < 0.01 vs vehicle). Analysis by one-way analysis of variance, followed by post-hoc adjustments using the Student-Newman-Keuls test. Mean ± standard error of the mean (n = 5/group). (c) Representative images of cresyl violet-stained CA3 hippocampal subregion from brain sections of vehicle- and CR8-treated TBI groups illustrate improved neuronal survival by CR8 treatment. (d) Linear regression analysis comparing TBI-induced neuronal loss in the CA3 with latency to reach the submerged platform on post-injury day 17 of the Morris water maze test followed by determination of statistical significance and coefficient of correlation (p < 0.0001; r² = 0.94). (e) Linear regression analysis comparing TBI-induced neuronal loss in the CA3 with “discrimination index” (%) in NOR test followed by determination of statistical significance and coefficient of correlation (p < 0.0001; r² = 0.96). (f) Linear regression analysis comparing TBI-induced neuronal loss in the DG with latency to reach the submerged platform on post-injury day 17 of the MWM followed by determination of statistical significance and coefficient of correlation (p < 0.0001; r² =0.95). (g) Linear regression analysis comparing TBI-induced neuronal loss in the DG with DI (%) in the novel object recognition test followed by determination of statistical significance and coefficient of correlation (p < 0.0001; r² = 0.96)

Fig. 8

Central administration of CR8 attenuates neuronal degeneration and neuronal cell loss in the cortex and thalamus after traumatic brain injury (TBI). (a, b) Qualitative assessment of neuronal degeneration at 24 h post-TBI using Fluoro-Jade B staining. Representative confocal images from vehicle-treated mice (a) demonstrate degenerating neurons (Fluoro-Jade B positive) around the lesion injury site (2), and in subcortical regions (1), whereas CR8-treated mice (b) had fewer Fluoro-Jade B positive neurons at the injury site (4), and in subcortical areas (3), indicating attenuated neuronal degeneration. Higher magnification images from the indicated regions are shown (n = 3/group). (c, d) Representative images of cresyl violet-stained brain sections illustrate morphological features of neurons, and improved neuronal survival in the cortex (c) and thalamus (d) by CR8 treatment at 21 days after injury. (e) Unbiased stereological quantification of neuronal cell loss in the cortex at 21 days post-TBI. CR8 treatment attenuated TBI-induced (^**p < 0.01 vs sham) neuronal loss in the cortex (⁺p < 0.05 vs vehicle) when compared to the vehicle-treated TBI group. (f) Unbiased stereological quantification of neuronal cell loss in the thalamus at 21 days post-TBI. CR8-treated TBI mice exhibited significantly improved neuronal survival in the thalamus (⁺⁺⁺p < 0.001 vs vehicle) when compared with vehicle-treated TBI mice (^**p < 0.01 vs sham). Analysis by one-way analysis of variance followed by post-hoc adjustments using the Student-Newman-Keuls test. Mean ± standard error of the mean (n = 5/group)

Fig. 9

Systemic administration of CR8 improves cognitive function after traumatic brain injury (TBI). (a) Fine motor coordination deficits were quantified using the beam walk test. TBI induced significant cognitive impairments on postinjury day 17 (^***p < 0.001 vs sham). CR8 treatment failed to cause significant improvement in motor performance. (b) Spatial learning and memory was assessed using the Morris water maze (MWM) test. The factors of “postinjury days” (F(3,84) = 10.125) (p < 0.001) and “groups” (F(2,84) = 7.931) (p < 0.001) were statistically significant. TBI induced significant cognitive impairments on postinjury day 17 (^**p < 0.01 vs sham). CR8-treated TBI mice had reduced latency to locate the submerged platform at day 17 post-TBI (⁺p = 0.022 vs vehicle) when compared to vehicle-treated TBI mice. Analysis by repeated measures one-way analysis of variance followed by post-hoc adjustments using the Student-Newman-Keuls test. (c) Reference memory was assessed using the probe trial of the MWM test. TBI caused significant cognitive impairments in this test (^**p < 0.01 and ^*p < 0.05 vs sham). CR8-treated TBI mice exhibited significant cognitive improvements in terms of number of entries (⁺⁺p < 0.01 vs vehicle). Analysis by one-way analysis of variance followed by post-hoc adjustments using the Student-Newman-Keuls test. (d) Cognitive performance was further evaluated by swim strategy analysis. Vehicle-treated TBI mice exhibited higher reliance on looping strategies rather than spatial and systematic patterns (p < 0.001 vs sham; χ² = 23.34). CR8-treated TBI mice showed significantly reduced usage of looping strategy and increased reliance on spatial and systematic swim strategies (p < 0.001 vs vehicle; χ² = 23.34). (e) Retention or intact memory was assessed using the novel object recognition test. Vehicle-treated TBI mice showed significant cognitive impairments in this test (^**p < 0.01 vs sham). CR8-treated TBI mice had significant improvements in cognitive performance in terms of discrimination index (⁺⁺p < 0.01 vs vehicle) when compared to vehicle-treated TBI mice. Analysis by one-way analysis of variance followed by post-hoc adjustments using the Student-Newman-Keuls test. Mean ± standard error of the mean (n = 7-8/group)