Overexpression of Sox11 promotes corticospinal tract regeneration after spinal injury while interfering with functional recovery

Abstract

Embryonic neurons, peripheral neurons, and CNS neurons in zebrafish respond to axon injury by initiating pro-regenerative transcriptional programs that enable axons to extend, locate appropriate targets, and ultimately contribute to behavioral recovery. In contrast, many long-distance projection neurons in the adult mammalian CNS, notably corticospinal tract (CST) neurons, display a much lower regenerative capacity. To promote CNS repair, a long-standing goal has been to activate pro-regenerative mechanisms that are normally missing from injured CNS neurons. Sox11 is a transcription factor whose expression is common to a many types of regenerating neurons, but it is unknown whether suboptimal Sox11 expression contributes to low regenerative capacity in the adult mammalian CNS. Here we show in adult mice that dorsal root ganglion neurons (DRGs) and CST neurons fail to upregulate Sox11 after spinal axon injury. Furthermore, forced viral expression of Sox11 reduces axonal dieback of DRG axons, and promotes CST sprouting and regenerative axon growth in both acute and chronic injury paradigms. In tests of forelimb dexterity, however, Sox11 overexpression in the cortex caused a modest but consistent behavioral impairment. These data identify Sox11 as a key transcription factor that can confer an elevated innate regenerative capacity to CNS neurons. The results also demonstrate an unexpected dissociation between axon growth and behavioral outcome, highlighting the need for additional strategies to optimize the functional output of stimulated neurons.

Keywords: Sox11; axon regeneration; gene therapy; spinal cord injury; transcription factor.

Figure 1.

Sox11 overexpression reduces net retraction in centrally injured DRG axons. A, B, Cervical spinal cords 8 weeks after dorsal injury (arrowheads) and 9 weeks after intrathecal delivery of AAV8-EBFP-2A-mCherry control (A) or AAV8-Sox11-2A-mCherry (B). Viral transduction is confirmed by mCherry expression in DRG cell bodies (red, insets), and Sox11 expression is confirmed by immunohistochemistry (blue, insets). Ascending DRG axons were labeled by dextran-Alexa Fluor 488 (green) to visualize the position of injured axons (arrows) relative to the lesion center (arrowhead), outlined by GFAP immunoreactivity (white). C, Quantification shows a significant reduction in the distance between the lesion center and the tip of the nearest injured axon at both 5 d and 8 weeks postinjury in Sox11-treated animals. Scale bars: A, B, 200 μm; insets, 50 μm. N = 8 animals per group, *p < 0.05, paired t test. Error bars show SEM.

Figure 2.

Sox11 is developmentally downregulated and is not re-expressed by spinally injured CST neurons. A, Sox11 (blue) is readily detected by immunohistochemistry in the embryonic cortex. B, C, Adult cortex 3 weeks after cortical injection of AAV8-EBFP (B) or AAV-Sox11 (C), and 2 weeks after cervical spinal injury and injection of retrograde CTB-647 (purple). Sox11 is not detected in injured cortex that received control virus (B) but is detected in the vicinity of the AAV8-Sox11 injection (C). Scale bars: A–C, 500 μm; insets, 20 μm.

Figure 3.

Viral overexpression of Sox11 promotes the growth of injured CST axons. A–C, Transverse sections of cervical spinal cord 8 weeks after left pyramidotomy and right unilateral cortical AAV injection. Sprouting across the midline (insets) is significantly elevated in Sox11-treated animals. D–F, Sagittal sections of cervical spinal cord 8 weeks postinjury and 9 weeks after cortical AAV injection. GFAP reactivity (blue) indicates the injury site (arrowheads) and transduced CST axons are green. The number of EGFP⁺ CST axons distal to the injury, normalized to the total number of transduced CST axons quantified in transverse sections of the medullary pyramid (insets), is significantly elevated in Sox11-treated animals (arrows, E, and quantified in F). G–I are similar to D and E except viral treatment was delayed until 8 weeks postinjury. N ≥ 8 animals per group. *p < 0.05, **p < 0.01, two-way repeated-measures ANOVA with Bonferroni's post hoc correction. Scale bars: A–I, 500 μm; insets, 50 μm. Error bars show SEM.

Figure 4.

Viral overexpression of Sox11 in the cortex interferes with forelimb function after injury without affecting cell survival. A, B, Adult mice were subjected to pyramidotomy or spinal injury and were cortically injected with AAV-EBFP control or AAV-Sox11, and then were tested weekly on a pellet retrieval task. Injury caused a persistent reduction in successful retrievals that was unaffected by viral treatment (p > 0.05, two-way repeated-measures ANOVA with Bonferroni's post hoc correction). C, D, Mice received spinal injury, and 8 weeks later received cortical injection of AAV-EBFP control of AAV-Sox11. Quantification of footfall errors by the right forelimb on a horizontal task revealed a significant reduction in accuracy in Sox11-treated animals that emerged 5 weeks after viral injection. *p < 0.05, two-way repeated-measures ANOVA with Bonferroni's post hoc correction. E, Animals were tested 8 weeks after viral treatment on a horizontal ladder with unevenly spaced rungs. Sox11-treated animals showed significantly reduced accuracy of forelimb placement in all three injury paradigms. N ≥ 8 animals per group. *p < 0.05, paired t test. F–K, Sox11-transduced cortical cells (F, H, red) display no TUNEL reactivity either 1 week (F, G) or 4 weeks (H, I) after viral injection and cervical injury (G, I, arrows). J, As a control, staurosporine toxin causes robust TUNEL signal (green). I, Quantification reveals no difference in TUNEL signal between EBFP- or Sox11-injected animals. N = 3 animals per group. *p < 0.05, ANOVA with Tukey's HSD test. Error bars show SEM.