Targeting PTEN but not SOCS3 resists an age-dependent decline in promoting axon sprouting

Abstract

Axonal repair is critical for functional recovery after injury of the CNS. We previously reported that neuronal PTEN deletion exhibits an age-dependent decline in promoting axon regeneration from the corticospinal tract (CST). How sprouting of uninjured axons, a naturally occurring form of axonal repair, is impacted by age is unknown. We assessed CST sprouting after unilateral pyramidotomy in PTEN and/or SOCS3-deleted mice at different ages. While PTEN deletion enhances sprouting independently of age, SOCS3 deletion loses its sprouting-promoting effect with age. The synergistic effect of PTEN/SOCS3 co-deletion on CST sprouting is rapidly lost with increased age. Overall, promoting sprouting appears more robust across age than regeneration, yet distinct molecular pathways are differentially impacted by age. Importantly, six-week delayed PTEN deletion promotes CST sprouting across age groups, supporting a clinically relevant time frame for this neural repair strategy independently of age.

Keywords: Cellular neuroscience; Molecular neuroscience.

Graphical abstract

Figure 1

PTEN but not SOCS3 deletion enhances sprouting independently of age (A and F) Schematic representations of the experimental design; gene deletion was induced at P1 (A) or 12 months (F), 6 weeks before pyramidotomy, with AAV-Cre cortical injections. Sprouting was assessed 4 weeks after pyramidotomy. (B–D) Representative images of CST sprouting phenotype at C7 after postnatal day 1 (P1) single gene deletion 6 weeks before pyramidotomy, targeting PTEN (C-C′) or SOCS3 (D-D′); PTEN or SOCS3 deletion significantly enhanced sprouting compared to WT control (B-B′). (E) Sprouting axon number indices on the contralateral, denervated side of the spinal cord expressed as a function of the distance to midline after AAV-Cre injection at P1 and pyramidotomy at 6 weeks of age. There was a significant increase in sprouting in the PTEN KO group compared to WT from midline to 450 μm, and in the SOCS3 KO group compared to WT from 150 to 350 μm. Stats: two-way ANOVA: Tukey’s multiple comparisons test was used to determine the differences between the groups. Data presented as means ± SEM, N = 7 (WT P1), 8 (PTEN KO P1), 4 (SOCS3 KO P1). WT P1 vs. PTEN KO P1: ∗p = 0.01–0.05; ∗∗p = 0.001–0.01; WT P1 vs. SOCS3 KO P1: ^@p = 0.01–0.05. (G–I) Representative images of CST sprouting phenotype at C7 after middle-age (12-months-old) gene deletion 6 weeks before pyramidotomy, targeting PTEN (H-H′) or SOCS3 (I-I′); PTEN deletion but not SOCS3 deletion, significantly enhanced sprouting. (J) Sprouting axon number indices on the contralateral, denervated side of the spinal cord expressed as a function of the distance to midline after AAV-Cre injection at 12 months and pyramidotomy at 13.5 months of age. There was a significant increase in sprouting in the PTEN KO group compared to WT from 50 to 450 μm, and compared to SOCS3 KO at 350 and 450 μm. Stats: two-way ANOVA: Tukey’s multiple comparisons test was used to determine the differences between the groups. Data presented as means ± SEM, N = 3 (WT 12m), 4 (PTEN KO 12m), 4 (SOCS3 KO 12m). WT 12m vs. PTEN KO 12m: ∗p = 0.01–0.05; ∗∗p = 0.001–0.01; SOCS3 KO 12m vs. PTEN KO 12m: ^@p = 0.01–0.05; ^@@p = 0.001–0.01.See also Figure S1.

Figure 2

SOCS3 deletion rapidly loses effectiveness in synergizing with PTEN deletion to enhance CST sprouting with increased age (A) Schematic representation of the experimental design; gene deletion was induced with AAV-Cre cortical injections at postnatal day 1 (P1), 10 weeks or 12 months, 6 weeks before pyramidotomy (at 6 weeks, 16 weeks and 13.5 months of age respectively). Sprouting was assessed 4 weeks after pyramidotomy. (B–F) Representative images of CST sprouting phenotype at C7 after PTEN;SOCS3 co-deletion 6 weeks before pyramidotomy at postnatal day 1 (D-D′), 10 weeks (E-E′) or middle-age (12-months-old, F-F′); PTEN;SOCS3 co-deletion at P1, but not at 10 weeks or 12 months, synergized with PTEN deletion in enhancing CST sprouting. (G) Sprouting axon number indices on the contralateral, denervated side of the spinal cord, expressed as a function of the distance to midline. PTEN;SOCS3 co-deletion at postnatal day 1 (P1) significantly increased sprouting compared to WT mice from midline to 650 μm; PTEN;SOCS3 co-deletion significantly increased sprouting compared to PTEN;SOCS3 co-deletion at 10 weeks and 12 months (significance stated in table H). (H) Table of statistical significance from (G). Stats: two-way ANOVA: Tukey’s multiple comparisons test was used to determine the differences between the groups. Data presented as means ± SEM, N = 7 (WT P1), 3 (WT 12m), 11 (PTEN;SOCS3 KO P1), 7 (PTEN;SOCS3 KO 10w), 8 (PTEN;SOCS3 KO 12m). WT P1 vs. PTEN;SOCS3 KO P1:∗p = 0.01–0.05; ∗∗p = 0.001–0.01; ∗∗∗p = 0.001–0.01, ∗∗∗∗p < 0.0001; PTEN;SOCS3 P1 vs. PTEN;SOCS3 10w: ^@p = 0.01–0.05; ^@@@p = 0.001–0.01; ^@@@p = 0.001–0.01; PTEN;SOCS3 P1 vs. PTEN;SOCS3 12m: ^#p = 0.01–0.05; ^##p = 0.001–0.01; ^###p = 0.001–0.01. See also Figure S1.

Figure 3

Delayed PTEN deletion enhances CST sprouting independently of age (A) Schematic representation of the experimental design; gene deletion was induced at 3 or 8 months, 6 weeks after pyramidotomy in adult mice, with AAV-Cre cortical injections. Sprouting was assessed 7 weeks after cortical injections. (B-E) Representative images of CST sprouting phenotype at C7 after PTEN (C-C’), SOCS3 (E-E′) single deletion or PTEN;SOCS3 co-deletion (D-D′) in 3-months-old adult, 6 weeks after pyramidotomy; PTEN single deletion and PTEN;SOCS3 co-deletion enhanced CST sprouting to the same level, while SOCS3 deletion did not appear to have any overt effect compared to WT (B-B′). (F) Sprouting axon number indices on the contralateral, denervated side of the spinal cord expressed as a function of the distance to midline after pyramidotomy at 1.5 months and AAV-Cre injection at 3 months of age. PTEN single deletion and PTEN;SOCS3 co-deletion significantly increased sprouting compared to WT and SOCS3 deletion from midline to 450 μm. Stats: two-way ANOVA: Tukey’s multiple comparisons test was used to determine the differences between the groups. Data presented as means ± SEM, N (at 3months) = 14 (WT), 9 (PTEN KO), 6 (PTEN;SOCS3 KO), 4 (SOCS3 KO). WT and SOCS3 KO vs. PTEN KO: ∗p = 0.01–0.05; ∗∗p = 0.001–0.01; ∗∗∗p = 0.001–0.01; ∗∗∗∗p < 0.0001; WT and SOCS3 KO vs. PTEN;SOCS3 KO: ^@p = 0.01–0.05; ^@@p = 0.001–0.01; ^@@@p = 0.001–0.01; ^@@@@p < 0.0001. (G-I) Representative images of CST sprouting phenotype at C7 after PTEN (H-H′) or PTEN;SOCS3 co-deletion (I-I′) in 8-months-old mice, 6 weeks after pyramidotomy; PTEN single deletion and PTEN;SOCS3 co-deletion significantly enhanced CST sprouting compared to WT (G-G′), to similar levels. (J) Sprouting axon number indices on the contralateral, denervated side of the spinal cord expressed as a function of the distance to midline after pyramidotomy at 6.5 months and AAV-Cre injection at 8 months of age. PTEN single deletion and PTEN;SOCS3 co-deletion significantly increased sprouting compared to WT up to 450 μm. Data presented as means ± SEM, N (at 8 months) = 8 (WT), 11 (PTEN KO), 6 (PTEN;SOCS3 KO). WT and SOCS3 KO vs. PTEN KO: ∗p = 0.01–0.05; ∗∗p = 0.001–0.01; ∗∗∗p = 0.001–0.01; ∗∗∗∗p < 0.0001; WT and SOCS3 KO vs. PTEN;SOCS3 KO: ^@p = 0.01–0.05; ^@@p = 0.001–0.01; ^@@@p = 0.001–0.01; ^@@@@p < 0.0001. See also Figure S1.

Figure 4

PTEN;SOCS3 deletion increased STAT3 signaling independently of age (A–D) Representative images of pSTAT3 (at Tyr705) and td-Tomato (tdT) in layer V of the right sensorimotor cortex of 4-week-old (young), 3-month-old (full adult) and 12-month-old (middle-aged) mice (the age at which AAV-Cre was injected, with mice sacrificed 4 weeks later). The control mouse shown was from the 3-month-old group. Scale bars: 50 μm. (E–G) Quantification of the relative pSTAT3-Y705 immunoreactivity (IR) in mice of different genotypes and ages. N = 2 per condition (genotype/age combination). 150 cells were quantified per mouse. Stats: D’Agostino Normality Test; Student’s t test (for normally distributed data) or a Mann–Whitney U test (for non-normally distributed data). Data presented as means ± SEM Significance level: ∗p = 0.01–0.05; ∗∗p = 0.001–0.01; ∗∗∗p = 0.001–0.01, ∗∗∗∗p < 0.0001). Of note, ANOVA did not show significant differences between the three age groups.