Ablation of the transcription factors E2F1-2 limits neuroinflammation and associated neurological deficits after contusive spinal cord injury

Abstract

Traumatic spinal cord injury (SCI) induces cell cycle activation (CCA) that contributes to secondary injury and related functional impairments such as motor deficits and hyperpathia. E2F1 and E2F2 are members of the activator sub-family of E2F transcription factors that play an important role in proliferating cells and in cell cycle-related neuronal death, but no comprehensive study have been performed in SCI to determine the relative importance of these factors. Here we examined the temporal distribution and cell-type specificity of E2F1 and E2F2 expression following mouse SCI, as well as the effects of genetic deletion of E2F1-2 on neuronal cell death, neuroinflammation and associated neurological dysfunction. SCI significantly increased E2F1 and E2F2 expression in active caspase-3(+) neurons/oligodendrocytes as well as in activated microglia/astrocytes. Injury-induced up-regulation of cell cycle-related genes and protein was significantly reduced by intrathecal injection of high specificity E2F decoy oligodeoxynucleotides against the E2F-binding site or in E2F1-2 null mice. Combined E2F1+2 siRNA treatment show greater neuroprotection in vivo than E2F1 or E2F2 single siRNA treatment. Knockout of both E2F1 and E2F2 genes (E2Fdko) significantly reduced neuronal death, neuroinflammation, and tissue damage, as well as limiting motor dysfunction and hyperpathia after SCI. Both CCA reduction and functional improvement in E2Fdko mice were greater than those in E2F2ko model. These studies demonstrate that SCI-induced activation of E2F1-2 mediates CCA, contributing to gliopathy and neuronal/tissue loss associated with motor impairments and post-traumatic hyperesthesia. Thus, E2F1-2 provide a therapeutic target for decreasing secondary tissue damage and promoting recovery of function after SCI.

Keywords: E2F1; E2F2; astrocytes; cell cycle pathways; contusive spinal cord injury; inflammation; motor function; neuropathic pain; neuroprotection.

Figure 1.

Spinal cord injury induces up-regulation of E2F1 and E2F2 transcription factors. (A-B) Expression of E2F1 and E2F2 mRNA in the mouse injured spinal cord was elevated at 6 and 24 hours after SCI. n=3-4 mice per time point. (C) Representative immunoblots for E2F1, E2F2, and the loading control (β-actin). (D-E) Expression levels of E2F1 and E2F2 protein were normalized by GAPDH, as estimated by optical density measurements, and expressed as a fold of sham spinal cord. Quantification of western blot showed significantly increased E2F1 and E2F2 expression at 24 hours post-injury. n=4-5 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham group.

Figure 2.

Upregulated E2F1 and E2F2 are associated with cleaved caspase-3⁺ neurons at 24 hours after SCI. (A-B) E2F1 and E2F2 are induced in neurons of the ventral horn (VH) gray matter at day 1 after SCI. (C) Most of E2F1⁺ cells (red) in the gray matter were also co-labeled with E2F2 (green), which had neuronal morphology. Arrows indicated E2F1⁺/E2F2⁺ cells with z-stacks (x-z axis, y-z axis). (D-E) Co-localization between cleaved caspase-3 (green) and E2F1 (red) or E2F2 (red) is apparent in the VH at day 1 after SCI. Arrows indicated caspase-3⁺/E2F1-2⁺; Arrowheads indicated caspase-3⁻/E2F1-2⁺. Scale bar is 100 μm.

Figure 3.

Increased E2F1 and E2F2 are associated with activated microglial and astrocytes at 7 days after SCI. (A-D) E2F1 (red in A) and E2F2 (red in C) are co-labeled with Iba-1⁺ microglia (green) in the ventral media whiter matter at 1 mm rostral to the epicenter. High magnifications in B,D (from insert in A,C) showed their highly co-localization. (E-H) A subtype of GFAP⁺ astrocytes (green) expressed E2F1 (red in E) and E2F2 (red in G) in the ventral lateral white matter at 1 mm rostral to the epicenter. F (from insert in E) and H (from insert in G) indicated their strong co-localization. (I) Almost all E2F1⁺/ED1⁺ microglia/macrophages also expressed E2F2. Arrows indicated E2F1⁺(red)/E2F2⁺(green)/ED1⁺(blue) cells with z-stacks (x-z axis, y-z axis). Scale bar is 100 μm.

Figure 4.

SCI-induced upregulation of cell cycle-relayed proteins at lesion site was significantly attenuated by intrathecal injection of E2Fs decoy oligodeoxynucleotides (ODNs) at day 1 post-injury. (A) Representative western blots of cell cycle-related proteins including E2F1, E2F2, CDK1, and PCNA. (B) Quantification of western blot from A. Veh: Vehicle; SC: scrambled ODN; ODNs: E2Fs ODNs. n=3-5 mice/group. *p <0.05, **p<0.01, ***p< 0.001 vs. sham group; ^#p<0.05, ^##p<0.01 versus SC group.

Figure 5.

Expression of cell cycle-related genes in the spinal cord from WT, E2F2ko, and E2Fdko mice at 24 hours after SCI. All these CCA genes were up-regulated at 24 h after SCI. Among them, cyclins A1 (A) and A2 (B) were robust down-regulated in injured mice with deletion of E2F2 gene or E2F1+2 genes. Notably, cyclins B2, D1, D2, D3, CDK1, and PCNA (C-H) were significantly reduced in E2F dko mice, but not in the E2F2 ko mice. E2Fdko = E2F1-2 double ko. n=5-8 mice/group. **p <0.01, ***p< 0.001 vs. Sham; ^#p<0.05, ^##p<0.01, ^###p<0.001 versus WT group.

Figure 6.

E2F1-2 siRNA reduces neuronal death at 24 h after SCI. E2F1, E2F2, E2F1+2, or scrambled siRNA were intrathecally administrated at 5 min post-injury and cleaved or uncleaved fragments of a-fodrin were assessed in the injured spinal cord at 24 h post-injury by Western blotting analysis. (A) Representative western blots for α-fodrin proteins and the loading control (β-actin). (B-D) Expression levels of α-fodrin (240, 150, 145 kDa) were normalized by β-actin, as estimated by optical density measurements and expressed as a fold of sham spinal cord. Quantitative analysis of western blots showed that 145 or 150 kDa cleavage fragment of the a-fodrin was increased 7- and 13- fold, respectively, whereas uncleaved fragment was decreased 60 %, as compared to sham-injury. These markers of apoptosis were significantly reversed by E2F1, E2F2, or E2F1+2 siRNA. Notably, E2F1+2 siRNA has greater effect than E2F1 or E2F2 single siRNA. n= 4-5 mice/group. ***p <0.001 vs. sham; ^##p<0.01, ^###p<0.001 versus SCI/Veh; ^$$$p<0.001 vs. E2F1+2 siRNAs.

Figure 7.

E2F1-2 ablation improves motor functional recovery and limits hyperesthesia after SCI. (A) Basso Mouse Scale (BMS) locomotor scores were significantly improved up to 6 weeks post-SCI in E2Fdko (E2F1-2 double ko) mice as compared to the wild-type (WT) controls. Deletion of E2F2 did not affect BMS scores. n=10 (WT), 8 (E2Fdko), 5 (E2F2ko) mice. *p < 0.05, **p < 0.01, versus WT group. (B) E2Fdko mice significantly decreased grid walk errors at 4 weeks after SCI. n=6 (WT), 6 (E2Fdko), 5 (E2F2ko) mice. *p < 0.05 vs. WT group. (C) E2Fdko mice increased stride length of the hindlimbs at weeks 4 post-injury. n=6 (WT), 6 (E2Fdko), 5 (E2F2ko) mice. *p < 0.05 versus WT mice. (D) Von Frey mechanical test. No significant difference in withdrawal threshold was found in sham groups between the genotypes. On day 28 after SCI, the E2Fdko mice had a higher mechanical threshold than the WT mice. n=5 mice/group. *p < 0.05 vs. sham/WT mice; ^#p < 0.05 versus SCI/WT mice. (E-F) Thermal test. SCI caused increased thermal sensitivity in WT mice, whereas E2Fdko mice had similar thermal withdrawal threshold compared to sham uninjured mice. n=5 mice/group. *p < 0.05 vs. sham/WT mice; ^#p < 0.05 versus SCI/WT mice.

Figure 8.

E2F1-2 ablation protects against tissue damage induced by SCI. (A-B) Representative sections show the lesion epicenter stained with eriochrome cyanine (ECRC) for residual white matter (WM) area. Bar graph shows that E2Fdko (E2F1-2 double ko) mice appeared increased spared tissue in whiter matter at epicenter. WM area is expressed as a percentage of ECRC-positive area in total area of each section. (C-D) Assessment of lesion volume at 6 weeks post-injury was performed on GFAP/DAB stained coronal sections. Representative images showed lesion cavity at the injury center. Quantification analysis showed significantly reduced lesion volume in E2Fdko mice. Lesion volume is expressed as a percentage of GFAP absent area in total area of each section. n=6 mice/group. *p < 0.05 vs. WT group.

Figure 9.

E2F1-2 ablation reduces cell death and increases neuronal survival at 24 hours after SCI. (A) Representative images of IHC staining for cleaved caspase-3 in lateral whiter matter and ventral horn of the gray matter from WT and E2Fdko (E2F1-2 double ko) mice. The images indicated 1 mm rostral to epicenter at 24 hours post-injury. Arrows show caspase-3⁺ cells (green) in nuclei (blue). (B) Quantification of cleaved caspase-3⁺ cells in both of gray matter (GM) and whiter matter (WM) at 1 mm rostral to epicenter. Deleting E2F1-2 genes in E2Fdko mice decreases numbers of caspase-3⁺ cells at day 1 after SCI compared with WT mice (arrows). n=5 mice/group. *p <0.05, **p < 0.01 versus WT group. (C) IHC showed that cleaved caspasae-3⁺ cells (green) presented in the WM are double immunostained for CC1⁺ oligodendrocytes (red, arrows). (D) In the GM, most of caspasae-3⁺ cells (green) are also NeuN⁺ (red, arrows). (E) Representative immunoblots for cleaved caspase-3 and the loading control (GAPDH). (F) Quantification of western blot showed that E2F1-2 ablation (dko mice) significantly reduced cleaved caspase-3 expression levels induced by SCI. n=3-4 mice/group. ***p < 0.001 vs. sham/WT mice; ^##p < 0.01 versus SCI/WT mice. (G) Quantification of NeuN⁺ cells showed that significantly more neurons survived in the ventral gray matter of the E2Fdko mice at epicenter as well as 1.2 mm rostral or caudal to it compared to their WT littermates; n=5 (WT), 6 (E2Fdko) mice. *p < 0.05, ***p < 0.001 vs. WT. Scale bar is 100 μm.

Figure 10.

E2F1-2dko mice have reduced microglial activation in the injured spinal cord. (A) Representative immunoblots for Iba-1 and the loading control (GAPDH) at day 1 after SCI. (B) Quantification of western blot from A showed that the E2Fdko mice had significantly less upregulation of Iba-1 protein expression compared with WT mice. (C) Iba-1⁺ cells in the white matter area were quantified at 1 mm rostral to the epicenter after 7 days post-injury. No significant difference was found between the genotypes in sham groups. The E2Fdko mice had significantly less number of Iba-1⁺ cells compared with WT mice. n=5 mice/group. ***p < 0.001 versus sham/WT, ^###p < 0.001 vs. SCI/WT. (D) Representative images with Iba-1 staining in WT and E2Fdko mice at 7 days post-injury. Scale bar is 100 μm.

Figure 11.

Genetic deletion of E2F1-2 reduces astrogliosis at 7 days after SCI. (A) Quantitative image analysis at 0.5mm rostral to epicenter showed a significant reduction of GFAP⁺ astrocytes in the white matter from the E2F1-2dko mice compared with WT mice. In contrast, no significant difference was found between the genotypes in sham groups. n=5 mice/group. ***p < 0.001 versus sham/WT, ^###p < 0.001 vs. SCI/WT. (B) Representative images with GFAP staining in WT and E2Fdko mice at 7 days post-injury. Scale bar is 100 μm.