Sustained axon regeneration induced by co-deletion of PTEN and SOCS3

Abstract

A formidable challenge in neural repair in the adult central nervous system (CNS) is the long distances that regenerating axons often need to travel in order to reconnect with their targets. Thus, a sustained capacity for axon regeneration is critical for achieving functional restoration. Although deletion of either phosphatase and tensin homologue (PTEN), a negative regulator of mammalian target of rapamycin (mTOR), or suppressor of cytokine signalling 3 (SOCS3), a negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, in adult retinal ganglion cells (RGCs) individually promoted significant optic nerve regeneration, such regrowth tapered off around 2 weeks after the crush injury. Here we show that, remarkably, simultaneous deletion of both PTEN and SOCS3 enables robust and sustained axon regeneration. We further show that PTEN and SOCS3 regulate two independent pathways that act synergistically to promote enhanced axon regeneration. Gene expression analyses suggest that double deletion not only results in the induction of many growth-related genes, but also allows RGCs to maintain the expression of a repertoire of genes at the physiological level after injury. Our results reveal concurrent activation of mTOR and STAT3 pathways as key for sustaining long-distance axon regeneration in adult CNS, a crucial step towards functional recovery.

Figure 1. Synergistic effects of double deletion of PTEN and SOCS3 on axon regeneration observed at 4 weeks after injury

(a) Images of the optic nerve sections showing CTB-labeled axons in AAV-Cre-injected SOCS3^f/f with CNTF (SOCS3^−/−), PTEN^f/f (PTEN^−/−), or PTEN^f/f/SOCS3^f/f with CNTF (PTEN^−/−/SOCS3^−/−) mice. Asterisks: lesion sites. (b) High-magnification images of the boxed area in (a), which is about 1.5–2.0 mm from the lesion sites. (c) Regenerating axons at the optic chiasm. (d) Estimated numbers of regenerating axons. There was a significant difference between PTEN^−/−/SOCS3^−/− group and others. *: p<0.001, ANOVA, Bonferroni’s post hoc test. (e) Percentages of TUJ1-positive RGCs in each group compared to that in the intact retinas. *: p<0.001, ANOVA, Tukey’s post hoc test. N=7–8 per group. Error bars, s.d. Scale bars: 200 μm.

Figure 2. Synergistic effects of double deletion of PTEN and SOCS3 on optic nerve regeneration in a delayed treatment paradigm

(a) Scheme of the experiment. (b) Images of the optic nerve sections showing CTB-labeled axons in AAV-Cre-injected SOCS3^f/f with CNTF (SOCS3^−/−), PTEN^f/f (PTEN^−/−), or PTEN^f/f/SOCS3^f/f with CNTF (PTEN^−/−/SOCS3^−/−) mice. Asterisks: lesion sites. (c) Extensive axon regeneration is only evident in double mutants. Top panel shows the entire optic nerve up to the chiasm. Bottom panels show high-magnified areas (a and b) as indicated in the top panel. OX: optic chiasm. (d) Estimated numbers of regenerating axons. At all distances quantified, there was a significant difference between the PTEN^−/−/SOCS3^−/− group and the remaining groups. *: p<0.001, ANOVA, Bonferroni’s post hoc test. N=5–6 per group. Error bars, s.d. Scale bars: 100 μm.

Figure 3. STAT3 in axon regeneration induced by SOCS3 deletion

(a) Images showing signals detected with TUJ1 or anti-p-STAT3 antibodies in the retinal sections from intact or 1-day-post-injury mice. (b) Percentage of RGCs with nuclear phospho-STAT3 signals. N=3–4 per group. *: p<0.001, ANOVA, Dunnett’s post hoc test. (c, d) Images (c) and quantification (d) of optic nerve sections showing regenerating axons in each group at 14 days post-injury. By Bonferroni’s post hoc test, regenerating axons in SOCS3^−/−/STAT3^−/− double mutants were significantly less than those in the SOCS3^−/− mutants at 0.2–2.0 mm from lesion site (p<0.01; N=5 per group). Error bars, s.d. Scale bars: 50 μm in (a), 100 μm in (c).

Figure 4. Independence of PTEN- and SOCS3- regulated pathways

(a, b) Images of optic nerve sections from PTEN^−/− and various PTEN^−/− combined groups (a) or SOCS3^−/− mutants with or without rapamycin treatment (b) at 14 days post-injury. (c, d) Quantification of regenerating axons shown in (a) and (b) respectively. (c) Axon regeneration in either PTEN^−/−/gp130^−/− or PTEN^−/−/STAT3^−/− group was comparable to that in PTEN^−/−, but was significantly reduced in the PTEN^−/− group with rapamycin (p<0.01, ANOVA, Bonferroni’s post hoc test; N=5 per group). (d) Rapamycin treatment did not significantly reduce the number of regenerating axons in SOCS3^−/− mice (N=6 per group). Error bars, s.d. Scale bars: 100 μm.