Delayed expression of cell cycle proteins contributes to astroglial scar formation and chronic inflammation after rat spinal cord contusion

Background Traumatic spinal cord injury (SCI) induces secondary tissue damage that is associated with astrogliosis and inflammation. We previously reported that acute upregulation of a cluster of cell-cycle-related genes contributes to post-mitotic cell death and secondary damage after SCI. However, it remains unclear whether cell cycle activation continues more chronically and contributes to more delayed glial change. Here we examined expression of cell cycle-related proteins up to 4 months following SCI, as well as the effects of the selective cyclin-dependent kinase (CDKs) inhibitor CR8, on astrogliosis and microglial activation in a rat SCI contusion model. Methods Adult male rats were subjected to moderate spinal cord contusion injury at T8 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 weeks or 4 months post-injury, and processed for protein expression and lesion volume. Functional recovery was assessed over the 4 months after injury. Results Immunoblot analysis demonstrated a marked continued upregulation of cell cycle-related proteins − including cyclin D1 and E, CDK4, E2F5 and PCNA − for 4 months post-injury that were highly expressed by GFAP+ astrocytes and microglia, and co-localized with inflammatory-related proteins. CR8 administrated systemically 3 h post-injury and continued for 7 days limited the sustained elevation of cell cycle proteins and immunoreactivity of GFAP, Iba-1 and p22PHOX − a key component of NADPH oxidase − up to 4 months after SCI. CR8 treatment significantly reduced lesion volume, which typically progressed in untreated animals between 1 and 4 months after trauma. Functional recovery was also significantly improved by CR8 treatment after SCI from week 2 through week 16. Conclusions These data demonstrate that cell cycle-related proteins are chronically upregulated after SCI and may contribute to astroglial scar formation, chronic inflammation and further tissue loss

Delayed expression of cell cycle proteins contributes to astroglial scar formation and chronic inflammation after rat spinal cord contusion

Abstract Background Traumatic spinal cord injury (SCI) induces secondary tissue damage that is associated with astrogliosis and inflammation. We previously reported that acute upregulation of a cluster of cell-cycle-related genes contributes to post-mitotic cell death and secondary damage after SCI. However, it remains unclear whether cell cycle activation continues more chronically and contributes to more delayed glial change. Here we examined expression of cell cycle-related proteins up to 4 months following SCI, as well as the effects of the selective cyclin-dependent kinase (CDKs) inhibitor CR8, on astrogliosis and microglial activation in a rat SCI contusion model. Methods Adult male rats were subjected to moderate spinal cord contusion injury at T8 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 weeks or 4 months post-injury, and processed for protein expression and lesion volume. Functional recovery was assessed over the 4 months after injury. Results Immunoblot analysis demonstrated a marked continued upregulation of cell cycle-related proteins − including cyclin D1 and E, CDK4, E2F5 and PCNA − for 4 months post-injury that were highly expressed by GFAP+ astrocytes and microglia, and co-localized with inflammatory-related proteins. CR8 administrated systemically 3 h post-injury and continued for 7 days limited the sustained elevation of cell cycle proteins and immunoreactivity of GFAP, Iba-1 and p22PHOX − a key component of NADPH oxidase − up to 4 months after SCI. CR8 treatment significantly reduced lesion volume, which typically progressed in untreated animals between 1 and 4 months after trauma. Functional recovery was also significantly improved by CR8 treatment after SCI from week 2 through week 16. Conclusions These data demonstrate that cell cycle-related proteins are chronically upregulated after SCI and may contribute to astroglial scar formation, chronic inflammation and further tissue loss.</p

Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

Apoptosis of post-mitotic neurons plays a significant role in secondary tissue damage following traumatic spinal cord injury (SCI). Activation of E2F1-dependent transcription promotes expression of pro-apoptotic factors, including CDK1; this signal transduction pathway is believed to represent an important mechanism for the physiological or pathological neuronal cell death. However, a specific role for this pathway in neuronal apoptosis induced by SCI has not yet been reported. Here we demonstrate up-regulation of the E2F1/CDK1 pathway that is associated with neuronal apoptosis following impact SCI in rats. Expression of E2F1 and CDK1 were robustly up-regulated as early as 15 min after injury and sustained until 3 days post-injury. CDK1 activity and E2F1 downstream targets bim and c-Myb were significantly increased after SCI. Activation of E2F1/CDK1 signaling also was associated with death of neurons in vitro; this was attenuated by shRNA knockdown or pharmacological inhibition of the E2F1/CDK1 pathway. CR8, a novel and potent CDK1 inhibitor, blocked apoptosis of primary cortical neurons at low-micromolar concentrations. Moreover, SCI-induced up-regulation of E2F1/CDK1 and associated neuronal apoptosis was significantly attenuated by systemic injection of CR8 (1 mg/kg, i.p.) at 5 min after injury. CR8 significantly decreased posttraumatic elevation of biochemical markers of apoptosis, such as products of caspase-3 and α-fodrin cleavage, as well as neuronal cell death, as indicated by TUNEL staining. Importantly, CR8 treatment also increased the number of surviving neurons at 5 weeks after injury. Together, these findings indicate that activation of the E2F1/CDK1 pathway contributes to the pathophysiology of SCI and that selective inhibition of this signaling cascade may represent an attractive therapeutic strategy

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

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 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

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

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

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