SOCS3 deletion promotes optic nerve regeneration in vivo

Abstract

Axon regeneration failure accounts for permanent functional deficits following CNS injury in adult mammals. However, the underlying mechanisms remain elusive. In analyzing axon regeneration in different mutant mouse lines, we discovered that deletion of suppressor of cytokine signaling 3 (SOCS3) in adult retinal ganglion cells (RGCs) promotes robust regeneration of injured optic nerve axons. This regeneration-promoting effect is efficiently blocked in SOCS3-gp130 double-knockout mice, suggesting that SOCS3 deletion promotes axon regeneration via a gp130-dependent pathway. Consistently, a transient upregulation of ciliary neurotrophic factor (CNTF) was observed within the retina following optic nerve injury. Intravitreal application of CNTF further enhances axon regeneration from SOCS3-deleted RGCs. Together, our results suggest that compromised responsiveness to injury-induced growth factors in mature neurons contributes significantly to regeneration failure. Thus, developing strategies to modulate negative signaling regulators may be an efficient strategy of promoting axon regeneration after CNS injury.

Figure 1. SOCS3 deletion promotes RGC axon regeneration

(A–E) Confocal images of optic nerves showing CTB-labeled axons around the lesion sites at 14 days (A and B), 1 day (C), 3 day (D), 7 days (E) post crush injury (dpc) from SOCS3^f/f mice injected with AAV-GFP (A) or AAV-Cre (B–E). *: crush site. Scale bar: 100 µm. (F) Quantification of regenerating axons at different distances distal to the lesion sites at 14 days after crush injury. At least 5 different sections (every 4th section) from each animal were quantified. At 14 dpc, there were significant differences between control and SOCS3-deleted mice groups (ANOVA with Bonferroni’s post-test, p < 0.05 for each distance, 8 animals in each group). (G) Fluorescent photomicrographs of retinal whole-mounts showing surviving TUJ1⁺ RGCs at 14 days after injury. Scale bar: 50 µm. (H) Quantification of RGC survival at 14 dpc, expressed as a percentage of the total number of TUJ1⁺ RGCs in the contralateral (intact) retina. (n = 8 for each group). For each retina, 15–20 fields were chosen from different parts of the retina. The total viable RGC number was obtained by multiplying the average number per field of TUJ1⁺ cells in the ganglion cell layer by the retinal area. *: p < 0.01, Dunnett’s test.

Figure 2. Phospho-S6 levels in RGCs of SOCS3^f/f mice with AAV-GFP or AAV-Cre after optic nerve injury

(A, B) Immunofluorescence analysis with anti-p-S6 or TUJ-1 antibodies of the retinal sections from SOCS3^f/f mice injected with AAV-GFP (A) or AAV-Cre (B) at different time points post-crush. Scale bar, 50 µm. (C) Quantification of p-S6⁺ RGCs. Data is presented as mean percentages of p-S6⁺and TUJ1⁺ cells among total TUJ1⁺ cells in the ganglion cell layer of each retina. Cell counts were performed on at least 4 non-consecutive sections for each animal, from four or five mice per group. *: p<0.01 by Dunnett’s test.

Figure 3. gp130 co-deletion abolishes axon regeneration effects of SOCS3 knockout

(A–C) Confocal images of optic nerves showing CTB-labeled axons around the lesion sites at 14 days post crush injury (dpc) from SOCS3^f/f mice (A), gp130f/f mice (B), or SOCS3^f/f and gp130^f/f mice (C) injected with AAV-Cre. *: crush site. Scale bar: 100 µm. (D) Quantification of regenerating axons at different distances distal to the lesion sites at 14 days after crush injury. At least 5 different sections (every 4th section) from each animal were quantified. There were significant differences between the SOCS3^f/f group and the other two groups (ANOVA with Bonferroni’s post-tes,t p < 0.05 for each distance, 8 animals used in each groups). (E–G) Fluorescent photomicrographs of retinal whole-mounts showing surviving TUJ1⁺ RGCs at 14 days after injury in AAV-Cre-injected SOCS3^f/f (E), gp130^f/f (F) or SOCS3^f/f and gp130^f/f (G) mice. Scale bar: 50 µm. (H) Quantification of RGC survival at 14 dpc expressed as a percentage of the total number of TUJ1⁺ RGCs in the contralateral (intact) retina (n = 8 for each group). For each retina, 15–20 fields were chosen from different parts of the retina. The total viable RGC number was obtained by multiplying the average number per field of TUJ1⁺ cells in the ganglion cell layer by the retinal area. There is a significant difference between the SOCS3^f/f group and gp130^f/f group, but not between SOCS3^f/f group and SOCS3^f/f/gp130^f/f group (p < 0.05, ANOVA with post-hoc Tukey’s test).

Figure 4. Enhancement of axon regeneration in SOCS3-deleted mice by CNTF

(A–D) Confocal images of optic nerves showing CTB-labeled axons around the lesion ites at 14 days post crush injury (dpc) from SOCS3^f/f mice with AAV-GFP (A, B) or AV-Cre (C, D) and subsequent injection of PBS (A, C) or CNTF (B, D). *: crush site. Scale bar: 100 µm. (E, F) Quantification of regenerating axons at different distances (250–1000 µm in E, and 1500–2500 µm in F) distal to the lesion sites at 14 days after crush injury. At least 5 different sections (every 4th section) from each animal were quantified. There were significant differences between the SOCS3^f/f with CNTF group and the other three groups (ANOVA with Bonferroni’s post-test, p < 0.05 for each distance, 3 animals in each group).