The temporal profile and cell specificity of E2F1 and CDK1 expression after SCI.

<p><b>A–B</b>. Coronal section in intact spinal cord (A) showed that E2F1 is relatively weak and detected mainly in neurons of the gray matter. At 24 h after SCI, E2F1 immunoreactivity was upregulated not only in gray matter but also in lesion area (B). C. E2F1⁺ cells were also co-labelled with NeuN in the dorsal horn of the gray matter at 1 day after SCI. D–E. Only a small subset of E2F1⁺ cells in the lesion area were positive for OX42 at 24 h (D) and 7 d (E) after SCI. F–G. In the intact spinal cord (F), CDK1 immunoreactivity is relatively weak and detected mainly in motor neurons in the ventral horn and CC1⁺ oligodendrocytes. At 24 h after SCI (G), CDK1 immunoreactivity was upregulated not only in the ventral horn but also in the spared white matter, colocalized with CC1⁺ oligodendrocytes. CDK1⁺ cells also appeared in the lesion area. <b>H–I</b>. CDK1 was expressed by CC1⁺ oligodendrocytes in the white matter in the intact spinal cord (H) and at 1 day after SCI. <b>J</b>. Only a small subset of CDK1⁺ cells in the lesion area were positive for OX42 at 24 h after SCI. <b>K</b>. Coronal section in intact spinal cord (a) shows that E2F1 was expressed in the motor neurons in the ventral horn. Immunoreactivity of E2F1 (b–d) was increased at 5 h, and 1–3 days post injury, and highly expressed by motor neurons. L. In intact spinal cord (a), CDK1/NeuN was detected in the motor neurons in the ventral horn. At 5 h after injury, immunoreactivity of CDK1 (b) was increased and sustained until 3 days post injury (c–d), and highly expressed by motor neurons. All images were taken at 2 mm rostral to epicenter. Scale bar = 500 µm for A–B, F–G. Scale bar = 100 µm for C–E, H–J, K–L.</p

E2F1/CDK1 expression is necessary for trophic deprivation-induced neuronal apoptosis.

<p>Rat cortical neurons were co-transfected with expression plasmids for ß-galactosidase with, either empty vector or vector expressing E2F1 or CDK1 (A and B). Similarly, ß-galactosidase plasmid was co-transfected along with scrambled, E2F1 or CDK1 shRNAs (C, D and E) and the extent of apoptosis was examined 48 h after transfection, or after an additional 24 h of trophic deprivation (TD) induction post 48 h transfection. <b>A</b>. Neurons transfected with E2F1 vector (0.6 µg DNA/0.5×10⁶ neurons) increased basal apoptosis as compared to empty vector. <b>B</b>. Neurons transfected with CDK1 (0.8 µg DNA/0.5×10⁶ neurons) vector enhanced TD-induced apoptosis as compared to empty vector transfected cells. <b>C–D</b>. A quantitative assessment of the percentage of nuclei featuring chromatin condensation demonstrates a significant attenuation of TD-induced apoptosis in neurons transfected with two different shRNAs targeting either E2F1 or CDK1. <b>E</b>. Attenuation of TD-induced chromatin condensation in neurons transfected by CDK1 shRNA1 is shown. Representative photomicrographs of the shRNA transfected neurons, which also co-express GFP protein from the separate promoter are shown. In all experiments ß-galactosidase was co-transfected at 0.01 µg DNA/0.5×10⁶ neurons to visualize transfected neurons at later apoptotic stage. Neurons with either normal diffuse chromatin morphology or apoptotic condensation of nuclei (after Hoechst 33258 chromatin staining) are indicated by arrowheads or arrows, respectively. Statistical analysis was performed by Kruskal-Wallis one-way ANOVA on ranks, followed by post hoc adjustments using Dunnett's test. For A ^*p<0.001, vs. vector; For C ^*p<0.01, vs. TD vector; For D ^*p<0.05, vs. TD vector; Error bars are ± S.E.M. from three independent experiments.</p

CR8 administration reduces SCI-induced activation of the E2F1/CDK1 signaling pathway.

<p>Samples were obtained from rats exposed to spinal cord injury and CR8 treatment (1 mg/kg intraperitoneal administration) and analyzed by western blotting. Equal protein loading is demonstrated by consistent GAPDH levels. <b>A</b>. CR8 attenuated SCI mediated increase in E2F1 and its target cyclin A expression. <b>B–C</b>. Quantification of respective western blots in panel A. <b>D</b>. CR8 reduced SCI induced increase in phospho-(Ser54)-n-myc, phosphorylated CDK substrates and expression of cyclin B1. <b>E–G</b>. Quantification of respective western blots in panel E. <b>H–J</b>. Administration of CR8 significantly reduced Bim and c-Myb expression at 24 h after SCI. H shows representative Western blots for Bim, c-Myb, and the loading control, GAPDH. I and J show quantitative analysis of Bim and c-Myb expression. N = 4. ^*p<0.05 vs. vehicle group.</p

CR8 administration reduces SCI-induced neuronal death.

<p><b>A</b>. CR8 attenuated caspase 3 and fodrin cleavage, the latter as indicated by reduction of the 145/150 kDa fragment immunoreactivity. <b>B–C</b>. Signal quantification using densitometry and normalization relative to GAPDH levels demonstrated that the observed changes are significant. The figures reflect representative western blots. N = 4. ^*p<0.05 vs. sham. <b>D–E</b>. TUNEL analysis was performed on 20 µm sections from rats at 24 hours post-SCI using an ApopTag® Fluorescein/Red detection kit. TUNEL/NeuN staining revealed that CR8 effectively reduced neuronal death in the epicenter of the injury (p<0.05), as well as in rostral and caudal sections located in two consecutive 2 mm distance regions from the epicenter. Representative images in panel D were shown at 2 mm caudal to epicenter. Scale bar = 100 µm. <b>F</b>. Six to eight representative animals - were selected from each treatment group for quantification of neurons (NeuN⁺ cells). Unbiased stereology by optical fractionators method using StereoInvestigator Software (MBF Biosciences) indicates that CR8 administration significantly increased number of surviving neurons within 10 mm zone surrounding the injury epicenter (p<0.05, vs vehicle).</p

Pharmacological inhibition of CDK1 blocks neuronal apoptosis.

<p>Cortical neurons were pre-treated with Roscovitine or CR8 (CDK1 inhibitors) or vehicle and then exposed to TD-or campthotecin induced apoptosis. <b>A</b>. Representative photomicrographs of control and trophic deprived neurons treated with the indicated concentrations Roscovitine and CR8 are shown. Upper row presents phase contrast images (Healthy neurons are indicated by larger cell bodies and abundant processes; Apoptotic neurons display shrunken cell bodies and sparse or lost processes). Lower row shows chromatin staining with Hoechst 33258. Arrows and arrowheads indicate surviving and apoptotic neurons, respectively suggesting an attenuation of TD-induced neuronal death in neurons pre-treated with Roscovinine or CR8. <b>B</b>. A quantitative assessment of the percentage of nuclei featuring chromatin condensation demonstrates a significant attenuation of TD-induced apoptosis in neurons pre-treated with Roscovitine (10 µM; ^*p<0.05, vs. TD vehicle) whereas CR8 at concentrations as low as 1 µM (^***p<0.001, vs. TD vehicle) almost completely blocked development of apoptotic features in neuronal nuclei. <b>C</b>. Significant attenuation of campthotecin-induced apoptosis in neurons pre-treated with Roscovitine (50 µM; ^*p<0.001, vs. vehicle) and CR8 at concentrations as low as 1 µM (^***p<0.001, vs. vehicle).</p

Colocalization of E2F1/CDK1 upregulation with neuronal apoptosis in injured spinal cord.

<p><b>A–C</b>. Western blot analysis shows <b>a significant increase</b> in biochemical markers of apoptosis, active caspase-3 signal, as well as 145/150 kDa cleavage product of α-fodrin after SCI. N = 4 rats/time points. *<i>p</i><0.05 vs sham group. <b>D</b>. E2F1⁺ cells were co-label with cleaved caspase 3 (yellow, arrow heads) in the gray matter at 2 mm rostral to the epicenter at 1 day after SCI. Scale bar = 100 µm. <b>E</b>. Coronal section in intact spinal cord (top panel) shows that CDK1 was expressed in the motor neurons in the ventral horn (VH). At 1 day after injury, immunoreactivity of CDK1 (middle panel, green) was increased, and highly expressed by apoptotic motor neurons (red), as shown at 2 mm rostral to epicenter. CDK1 was rarely expressed by inter-neurons in the dorsal horn (DH) after SCI (bottom panel). Scale bar = 100 µm for D(a–h) and 500 µm for D(i–l).</p

SCI-induced immunoreactivity of E2F1 and CDK1 was attenuated by CR8 treatment.

<p><b>A</b>. Coronal section in intact spinal cord (a–c) shows that E2F1 was expressed in the motor neurons in the ventral horn. At 1 day after injury, immunoreactivity of E2F1 (d–f) was increased, and highly expressed by motor neurons. The upregulation of E2F1 was clearly attenuated by CR8 treatment (g–i). <b>B</b>. In intact spinal cord (a–c), CDK1/NeuN was detected in the motor neurons in the ventral horn. At 1 day after injury, immunoreactivity of CDK1 (d–f) was increased, and highly expressed by motor neurons. CDK1 upregulation was attenuated by CR8 treatment (g–i). All images were taken at 2 mm rostral to epicenter. Scale bar = 100 µm for C–F.</p

Spinal cord injury induces upregulation of expression of E2F1 and its downstream targets.

<p>Analysis of expression of E2F1 and its transcriptional target genes in intact and injured spinal cord were performed by western blotting. <b>A</b>. 5-mm-long segment centered at the injury epicenter was dissected and homogenized in RIPA buffer. Equal amounts of protein were electrophoretically separated on NuPAGE Novex Bis-Tris gradient gels, transferred to nitrocellulose membranes, and blotted with antibodies to E2F1 and CDK1. GAPDH signal served as a loading control. <b>B–C</b>. The signal quantifications for E2F1 (B) and CDK1 (C) using Gel-Pro Analyzer software are displayed. E2F1 and CDK1 expression level was upregulated as early as 15 min and sustained until 3 days after injury. <b>D–E</b>. Cyclin A expression was increased at all time points. <b>F</b>. Bim and c-Myb, downstream targets of E2F1 were upregulated as early as 5 h post-injury and sustained until day 3 after SCI. <b>G–H</b>. Quantification of respective western blots in panel D. N = 4 rats/time point. *P<0.05 vs sham group.</p

CDK1 activity is increased after spinal cord injury.

<p>Western blotting analysis of common CDK substrates, CDK1 co-activator cyclin B1 and phosphorylations of specific CDK1 substrate (Ser54)-n-myc was performed in homogenates obtained from intact and injured spinal cord. <b>A</b>. Cyclin B1 expression was upregulated at all time points tested. Phosphorylation (Ser54) of n-myc and phospho-CDK substrate motif signal levels were increased from 5 h to day 7. <b>B–D</b>. Quantification of respective western blots in panel A. n = 4 rats/time point. *P<0.05 vs sham group.</p

E2F1 gene silencing down-regulates endogenous CDK1 expression <i>in vitro</i>.

<p><b>A</b>. 27 mer siRNA duplexes for human E2F1 or trilencer-27 universal scrambled negative control siRNA duplex was transfected in the human neuroblastoma SH-SY5Y cells. Two days after transfection, the cells were harvested and subjected to western blotting using mouse monoclonal antibodies to E2F1 and CDK1. Transfection with shRNA against E2F1 resulted in reduction of E2F1 expression (58% to 66% of control), accompanied by 50% of reduction of CDK1 expression. <b>B</b>. Primary rat cerebral cortical neurons were transfected with shRNA against rat E2F1. E2F1 protein expression was robust reduced to 47% or 59% for shRNAs 1 and 2 respectively, and E2F1 knockdown resulted in reduction of CDK1 expression from 47% to 64% for shRNAs 1 and 2 respectively. N = 4 dishes from 3 independent culture. *P<0.05 vs sham group.</p