Restoration of skilled locomotion by sprouting corticospinal axons induced by co-deletion of PTEN and SOCS3 - PubMed (original) (raw)

Restoration of skilled locomotion by sprouting corticospinal axons induced by co-deletion of PTEN and SOCS3

Duo Jin et al. Nat Commun. 2015.

Abstract

The limited rewiring of the corticospinal tract (CST) only partially compensates the lost functions after stroke, brain trauma and spinal cord injury. Therefore it is important to develop new therapies to enhance the compensatory circuitry mediated by spared CST axons. Here by using a unilateral pyramidotomy model, we find that deletion of cortical suppressor of cytokine signaling 3 (SOCS3), a negative regulator of cytokine-activated pathway, promotes sprouting of uninjured CST axons to the denervated spinal cord. A likely trigger of such sprouting is ciliary neurotrophic factor (CNTF) expressed in local spinal neurons. Such sprouting can be further enhanced by deletion of phosphatase and tensin homolog (PTEN), a mechanistic target of rapamycin (mTOR) negative regulator, resulting in significant recovery of skilled locomotion. Ablation of the corticospinal neurons with sprouting axons abolishes the improved behavioural performance. Furthermore, by optogenetics-based specific CST stimulation, we show a direct limb motor control by sprouting CST axons, providing direct evidence for the reformation of a functional circuit.

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Figures

Figure 1

Figure 1. SOCS3 deletion in neonatal cortical neurons increases CST sprouting after unilateral pyramidotomy.

(a) Scheme of the experiments. AAVs were injected into the sensorimotor cortex of P1–2 SOCS3f/f mice, which then received a unilateral pyramidotomy (Py) or sham lesion at 8 weeks. BDA was injected into the contralateral sensorimotor cortex at 4 weeks post injury and the mice were terminated 2 weeks later. (b–d) Representative images (_n_=3) of cervical 7 (C7) spinal cord transverse sections from SOCS3f/f mice with cortical AAV-Cre injection and a sham injury (b) or with cortical AAV-GFP injection and a unilateral pyramidotomy (c) or cortical AAV-Cre injection and a unilateral lesion (d). (e) Quantification of sprouting axon density index (contralateral/ipsilateral). *P<0.01, ANOVA followed by Bonferroni's post hoc test. (f) Scheme of quantifying crossing axons at different regions of the spinal cord (Mid: midline, Z1 or Z2: different lateral positions). (g) Quantification of crossing axons counted in different regions of spinal cord normalized against the numbers of labelled CST axons. *P<0.01, ANOVA followed by Bonferroni's post hoc test. For e,g five mice used in each group. Three sections at the C7 level were quantified per mouse. Scale bar, 500 μm.

Figure 2

Figure 2. CNTF upregulated expression in denervated spinal cord neurons.

(a) Representative images (_n_=3) of spinal cord sections stained with anti-CNTF antibodies showing the spinal cord from the mice at 3 days post injury (lower panel), but not intact controls (upper panel) with higher immunoreactivity. Scale bar, 500 μm. (b) Cntf mRNA measurement by quantitative PCR with reverse transcription of the spinal cord at 1 and 3 days post injury, respectively. Cntf mRNA expression levels were normalized to Gapdh mRNA levels. **Student's t test, P<0.01 samples were from five animals, three replicates. (c) Representative images (_n_=3) from the spinal cord of intact (top) or 3 days post injury (the lower three panels) showing the co-staining of anti-CNTF with anti-NeuN (the second panel), but not with anti-CD68 (the third panel) or anti-GFAP (the fourth panel). Scale bars, 20 μm.

Figure 3

Figure 3. CNTF induces CST sprouting in intact spinal cord.

(a–c) Representative images (_n_=3) from the SOCS3f/f mice with neonatal cortical injection of AAV1-CreERT2 and tamoxifen (a,c) or oil (b) i.p. injection at the age of 6 weeks, intraspinal injection of AAV2-PLAP (a) or AAV2-CNTF (b,c) in the adult (∼20 weeks), and unilateral BDA injection at the somatosensory cortex 4 weeks post intraspinal injection. Scale bar, 500 μm. (d) Quantification of sprouting axon density index (contralateral/ipsilateral) in different groups. **P<0.01, ANOVA followed by Bonferroni's post hoc test. (e) Quantification of crossing axons counted in different regions of spinal cord normalized against the numbers of labelled CST axons. **P<0.01, *P<0.05 (_P_=0.038), ANOVA followed by Bonferroni's post hoc test. For d,e three mice used in each group. Three sections at the C6–7 levels were quantified per mouse.

Figure 4

Figure 4. CST sprouting induced by SOCS3 deletion is dependent on gp130.

(a–c) Representative images (_n_=3) from the SOCS3f/f (a), gp130f/f (b) or SOCS3f/f/gp130f/f (c) mice with neonatal cortical injection of AAV1-CreERT2 and AAV-ChR-YFP (as a tracer), tamoxifen induction at the age of 6 weeks, and unilateral pyramidotomy at the age of 8 weeks. The mice were sacrificed 12 weeks later. Scale bar, 500 μm. (d) Quantification of sprouting axon density index (contralateral/ipsilateral). *P<0.01, ANOVA followed by Bonferroni's post hoc test. (e) Quantification of crossing axons counted in different regions of spinal cord normalized against the numbers of labelled CST axons. *P<0.01, ANOVA followed by Bonferroni's post hoc test. For d,e five mice used in each group. Three sections at the C7 level were quantified per mouse.

Figure 5

Figure 5. Significant CST sprouting in SOCS3 and PTEN co-deleted mice after unilateral pyramidotomy.

(a–c) Representative images (_n_=3) of cervical 7 (C7) spinal cord transverse sections from SOCS3f/f/PTENf/f mice with cortical AAV-Cre injection and a sham injury (a) or with cortical AAV-GFP injection and a unilateral pyramidotomy (Py) (b) or cortical AAV-Cre injection and a Py (c). Scale bar, 500 μm. (d) Quantification of sprouting axon density index (contralateral/ipsilateral). *P<0.01, ANOVA followed by Bonferroni's post hoc correction. (e) Quantifications of crossing axons counted in different regions of spinal cord normalized against the numbers of labelled CST axons. *P<0.01, ANOVA followed by Bonferroni's post hoc correction. For d,e five mice were used in each group. Three sections at the C7 level were quantified per mouse.

Figure 6

Figure 6. Significant recovery of skilled locomotion in mice with neonatal SOCS3 and PTEN co-deletion after unilateral pyramidotomy.

(a) Experimental paradigm: mice were injected with AAV-GFP or AAV-Cre at neonatal stage and subjected to unilateral pyramidotomy in adults. (b–e) Performance on irregularly horizontal ladder of ipsilateral (b,d) and contralateral (c,e) forelimbs (b,c) and hindlimbs (d,e) to the lesion, respectively. P values: repeated-measures ANOVA, *P<0.05 (_P_=0.039, 0.019, 0.005, 0.015 for 8, 12, 16 and 20 week respectively), Bonferroni's post hoc correction (_n_=11 and 12 for AAV-GFP and AAV-Cre injected group, respectively). (f) BMS scores of intact mice and the PTENf/f/SOCS3f/f mice with AAV-GFP or AAV-Cre injection at 20 weeks after Py. _P_=0.25, one-way ANOVA (_n_=12, 11 and 12 for intact, PTENf/f/SOCS3f/f mice with AAV-GFP or AAV-Cre injection, respectively).

Figure 7

Figure 7. CST-specific optogenetic stimulation induces limb movement.

(a) Experimental paradigm of optogenetic stimulation. PTENf/f/SOCS3f/f mice received cortical injection of AAV1-ChR-YFP and AAV-CreERT2 at P1 (1), tamoxifen at 6 weeks (2), unilateral pyramidotomy at 8 weeks (3), and subjected to optogenetic stimulation at C5–C7 levels ∼12 weeks after injury (4). (b) Illustration of the optogenetic stimulation apparatus. White arrow indicates blue light stimuli. Red dots with connected lines delineate specific joints for trajectory analysis. (c) Representative trajectories (_n_=8 for control group, _n_=13 for experimental group) of paw movements induced by photostimulation in control and PTEN/SOCS3-deleted mice. In contrast to the control group (five out of eight animals showed only ipsilateral forelimb movement), the mice with PTEN/SOCS3 co-deletion showed different paw movement patterns, on CST specific, optogenetic stimulation. These include bilateral forelimb movement (3/13), ipsilateral forelimb and hindlimb movement (2/13), bilateral forelimb movement and ispilateral hindlimb movement (2/13) and bilateral forelimb and hindlimb movement (2/13). Representative movies are shown in Supplementary Material. (d–g) Quantification of paw movement patterns (d,f), paw placement (e,g), and latencies of the movement onset (h) for specific forepaws and hindpaws on photostimulation in two groups of mice. *P<0.01, Student's t test, NS, not significant, one-way ANOVA. Eight and thirteen animals were used for quantification in animals without or with tamoxifen induction, respectively. Three trajectories were quantified per mouse.

Figure 8

Figure 8. Ablation of cortical neurons with sprouting axons abolishes recovered skilled locomotion performance.

(a) Experimental paradigm: mice received cortical injection of AAV-CreERT2 at neonatal stage, tamoxifen at the age of 6 weeks, and unilateral pyramidotomy at 8–10 weeks, the same as Fig. 6a. (b,c) Performance on irregularly horizontal ladder of ipsilateral (b) and contralateral (c) forelimbs to the lesion, respectively. P values: repeated-measures ANOVA, *P<0.05 (_P_=0.03), Bonferroni's post hoc correction (_n_=9 and 10 for without and with tamoxifen injected group, respectively). (d) Experimental paradigm: the same group of animals used in a–c were further analysed here. At 20 weeks after pyramidotomy, tamoxifen was administrated again and lentivirus (HiRet-FLEX-DTR) was intraspinally injected into the denervated side of the cervical spinal cord (C5–C7). After 2 weeks, diphtheria toxin was administrated (i.p.). (e,f) Performance on irregularly horizontal ladder of ipsilateral (e) and contralateral (f) forelimbs to the lesion, respectively. **P<0.01, NS, not significant, Student's t test (_n_=6 and 4 for without and with tamoxifen injected group, respectively).

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