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

ARTICLE Received 28 Apr 2015 | Accepted 15 Jul 2015 | Published 24 Nov 2015 DOI: 10.1038/ncomms9074 OPEN Restoration of skilled locomotion by sprouting corticospinal axons induced by co-deletion of PTEN and SOCS3 Duo Jin1, Yuanyuan Liu1, Fang Sun1, Xuhua Wang1, Xuefeng Liu1 & Zhigang He1 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. 1 F.M. Kirby Neurobiology Center, Children’s Hospital and Department of Neurology, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. Correspondence and requests for materials should be addressed to D.J. (email: ) or to Z.H. (email: ). NATURE COMMUNICATIONS | 6:8074 | DOI: 10.1038/ncomms9074 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9074 S pontaneous sprouting of spared axons may innervate denervated targets and contribute to naturally occurring functional recovery after injury. However this appears to be very limited in the adult central nervous system (CNS)1–5. A number of studies have attempted to explore the underlying mechanisms and several manipulations have also been developed to enhance such collateral sprouting1–12. For example, both neutralizing extrinsic inhibitory activities and task-specific rehabilitation training are able to promote functional recovery by increasing axonal sprouting in different injury models5–10. However, how the sprouting response is initiated after injury remains poorly understood. On the other hand, observed motor function recovery after these treatments are still at most partial. Very often, only the trained motor tasks, but not other motor behaviours, are improved with such manipulations6,10,12,13. At some cases, training one task can even have detrimental effects on another task6,10,12. A possible explanation is that rehabilitation training might enhance the functional performance of task-specific circuits at the expense of reducing spared axons available for other behaviours. Therefore, available sprouting axons might be a likely limiting factor in mediating functional recovery. To develop new strategies of boosting robust sprouting of corticospinal tract (CST) axons, we here attempted to explore how spared (intact) axons sense injury and respond with extensive sprouting. In this study, we show that ciliary neurotrophic factor (CNTF) and perhaps other cytokines, are induced in denervated neurons, might serve as molecular triggers of CST sprouting. By manipulating CNTF and other pathways, we achieved robust CST sprouting, forming new circuits almost as strong as intact ones and significant behavioural functional recovery. Furthermore, by both optogenetic and behavioural approaches, we demonstrated that sprouting CST axons could relay the cortical signals to the spinal cord, controlling the limb movement. Results SOCS3 deletion in cortical neurons promotes CST sprouting. Our previous studies showed that deleting SOCS3 in retinal ganglion neurons promotes regeneration of injured optic nerve axons after injury14,15. We thus asked whether SOCS3 deletion could affect the sprouting of CST axons after unilateral pyramidotomy (Fig. 1a). In this injury paradigm, CST axons from a unilateral cortical hemisphere are transected at the medullary pyramid above the pyramidal decussation. To monitor collateral sprouting from uninjured CST axons, biotinylated dextran amines (BDAs) are injected into the intact side of sensorimotor cortex at indicated post-injury time points, and transverse sections from different levels of the spinal cord are analysed after an additional 2 weeks (Fig. 1a). In control mice, most of the labelled axons are on the contralateral side of the spinal cord, with minimal labelling on the ipsilateral side7,16–18. Thus, the number of labelled axons on the denervated side of the spinal cord originating from the intact CST in the contralateral side can be used to quantify the extent of CST compensatory sprouting. To delete SOCS3 in cortical neurons, we first injected Cre-expressing adeno-associated virus (AAV2-Cre) into the intact side of the sensorimotor cortex (contralateral to the injury) of homozygous conditional SOCS3 mutants (SOCS3f/f, ref. 19) on postnatal day 1 (P1). This approach has been previously shown to induce efficient Cre-dependent recombination in neurons throughout the sensorimotor cortex18. Deletion of SOCS3 at this stage did not appear to change the pattern of CST projections in the adult (Fig. 1b). A unilateral pyramidotomy was performed at 8 weeks of age, and sprouting responses were analysed 6 weeks 2 post injury. SOCS3 deletion significantly increased sprouting from the spared (intact, left) half of the CST into the denervated (right) side of the spinal cord (Fig. 1d,e), comparing to the controls (Fig. 1c,e). In the denervated (right) side of the spinal cord, the labelled axons could be seen in different regions of the grey matter, with the most abundant projections in the intermediate and dorsal spinal cord (Fig. 1d–g). The density of compensatory/collateral sprouting axons amounted to 25% of the uncrossing CST (Fig. 1e). These results suggest that the signalling pathway(s) regulated by SOCS3 regulate the capacity for compensatory sprouting of spared CST axons. Spinal CNTF upregulation after unilateral pyramidotomy. SOCS3 is a negative regulator of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, which is often activated by cytokines such as CNTF20. Enhanced CST sprouting from intact cortical neurons after SOCS3 deletion suggests that axonal sprouting responses might be regulated by access to extrinsic cytokines which activate the SOCS3-regulated JAK/STAT pathway. Because the pyramidotomy is performed on one side of the medullary pyramid, we examined the expression of CNTF in the cortex (where CST axons originate) and in the spinal (...truncated)