Conditional Sox9 ablation improves locomotor recovery after spinal cord injury by increasing reactive sprouting

doi:10.1016/j.expneurol.2016.05.028

Experimental Neurology

Volume 283, Part A, September 2016, Pages 1-15

https://doi.org/10.1016/j.expneurol.2016.05.028 Get rights and content

Highlights

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Cord-injured Sox9 knock outs recover more inputs caudal to the lesion than controls.
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The increased neural inputs are not due to increased sparing or regeneration.
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Sox9 knock out mice have more reactive sprouting than controls.
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Doxycycline stabilizes CSPG levels and diminishes locomotor recovery in these mice.

Abstract

The absence of axonal regeneration after spinal cord injury (SCI) has been attributed to the up-regulation of axon-repelling molecules, such as chondroitin sulfate proteoglycans (CSPGs) present in the glial scar that forms post-SCI. We previously identified the transcription factor SOX9 as a key up-regulator of CSPG production and also demonstrated that conditional Sox9 ablation leads to decreased CSPG levels and improved recovery of hind limb function after SCI. We herein demonstrate increased neural input onto spinal neurons caudal to the lesion in spinal cord injured Sox9 conditional knock out mice as indicated by increased levels of the presynaptic markers synaptophysin and vesicular glutamate transporter 1 (VGLUT1) compared to controls. Axonal sparing, long-range axonal regeneration and reactive sprouting were investigated as possible explanations for the increase in neural inputs caudal to the lesion and for the improved locomotor outcomes in spinal cord-injured Sox9 conditional knock out mice. Whereas retrograde tract-tracing studies failed to reveal any evidence for increased axonal sparing or for long-range regeneration in the Sox9 conditional knock out mice, anterograde tract-tracing experiments demonstrated increased reactive sprouting caudal to the lesion after SCI. Finally we demonstrate that application of a broad spectrum MMP inhibitor to reduce CSPG degradation in Sox9 conditional knock out mice prevents the improvements in locomotor recovery observed in untreated Sox9 conditional knock out mice. These results suggest that improved recovery of locomotor function in Sox9 conditional knock out mice after SCI is due to increased reactive sprouting secondary to reduced CSPG levels distal to the lesion.

Introduction

Spinal cord injury (SCI) is a catastrophic event that is a major health care issue, causing lifelong disability. The absence of axonal regeneration after SCI has been attributed to axon-repelling molecules in the damaged myelin and in the scar (Thuret et al., 2006). Amongst the most important of the nerve growth inhibiting molecules in the scar are chondroitin sulfate proteoglycans (CSPGs) produced by reactive astrocytes responding to the injury (Asher et al., 2001). CSPGs also play a major role in neurodevelopment as key components of perineuronal nets (PNNs). PNNs are a highly condensed matrix that surrounds the cell bodies and dendrites of some classes of neurons (Celio and Blumcke, 1994). CSPGs and other components of the PNNs are produced by both neurons and glia (Galtrey and Fawcett, 2007). One suggested function of the CSPGs in PNNs is to stabilize synapses during development by preventing axonal sprouting onto inappropriate targets after appropriate connections have been made (Galtrey and Fawcett, 2007). The role of PNN CSPGs in limiting synaptic plasticity is well illustrated in the development of ocular dominance columns where it has been shown that treating the visual cortex with chondroitinase reactivates ocular dominance plasticity in adolescent rats (Pizzorusso et al., 2002, Pizzorusso et al., 2006). Thus, reducing CSPG levels at the glial scar or at more distant sites may be expected to increase axonal growth and improve recovery from SCI. This prediction is supported by the observation that CSPG digestion by the enzyme chondroitinase (Barritt et al., 2006, Bradbury et al., 2002, Zuo et al., 1998b), or interference with CSPG synthesis (Grimpe and Silver, 2004, Takeuchi et al., 2013) or blocking the CSPG receptor PTPσ (Lang et al., 2014) improve recovery in rodent models of SCI.

We have previously identified SOX9 as a transcription factor that up-regulates the expression of CSPGs in astrocyte cultures (Gris et al., 2007). We subsequently demonstrated reduced expression of known SOX9 target gene mRNAs in the spinal cord-injured conditional Sox9 knock out mice including: XT-I (xylosyltransferase-I), GFAP and three CSPG core proteins (aggrecan, brevican and neurocan) (Gris et al., 2007, McKillop et al., 2013). The reduction in mRNA expression of these genes was accompanied by reductions in CSPG protein levels both in the glial scar and in PNNs distant to the injury (McKillop et al., 2013). In addition to reduced CSPG levels, the Sox9 conditional knock out mice also demonstrated improved recovery of hind limb function as assessed by frequency of plantar placement, the Basso Mouse Scale (BMS) for hind limb function and overall mobility as assessed by activity boxes (McKillop et al., 2013). Sox9 conditional knock out mice also demonstrated increased 5-HT immunoreactivity caudal to the lesion site suggesting that the improved locomotor recovery could be due to increased neuronal inputs below the level of the lesion. In the present study we investigate sparing, long-range regeneration and reactive sprouting of spared axons as possible explanations for the improved locomotor outcomes in Sox9 conditional knock out mice after SCI. We herein report that neither long-range regeneration nor sparing of axons contributes significantly to the improved recovery of spinal cord injured Sox9 conditional knock out mice. Rather, our studies demonstrate that the improved outcomes of Sox9 conditional knock out mice after SCI is due to increased reactive sprouting that can be directly attributed to reduced levels of CSPGs in these mice.

Section snippets

Sox9 conditional knock out mice

Mice homozygous for floxed Sox9 (exons 2 and 3 of Sox9 surrounded by loxP sites) alleles (Bi et al., 1999) and hemizygous for Cre recombinase fused to the mutated ligand binding domain of the human estrogen receptor (ER) under the control of a chimeric cytomegalovirus immediate-early enhancer/chicken β–actin promoter (Hayashi and McMahon, 2002) (Sox9^flox/flox;CAGGCre-ER referred to as Sox9^flox/flox;Cre) were used as Sox9 conditional knock out mice. The mutated ER ligand binding domain of the

Lumbar enlargement ventral horn PNN matrix post-SCI

Although we have previously demonstrated decreased CSPG levels 0.8–1.6 mm caudal to the lesion site in the Sox9 conditional knock out mice after a T9, 70 kdyne contusion SCI (McKillop et al., 2013), arguably the most critical level of the spinal cords to investigate for changes in CSPG levels and markers of synaptic plasticity is the lumbar enlargement where the spinal circuits and primary motor neurons innervating hind limb musculature are found. Thus, we investigated CSPG levels in the PNNs in

Discussion

Degradation of CSPG side chains by administration of the bacterial enzyme chondroitinase has been shown to enhance recovery following SCI in rodents (Barritt et al., 2006, Bradbury et al., 2002). We previously proposed that SOX9 inhibition could serve as an alternate strategy for reducing CSPG levels in the injured spinal cord by reducing the expression of CSPG biosynthetic enzymes and core proteins (Gris et al., 2007, McKillop et al., 2013). This proposition was supported by our demonstration

Conflict of interest

A.B. and T.H. are named as inventors on patents and patent applications related to SOX9 inhibition.

Acknowledgments

The Sox9^flox/flox mice were kindly provided by Dr. Andreas Schedl. This work was supported by grants from the Canadian Institutes of Health Research (CIHR, RFN# 133460) and the International Foundation of Research in Paraplegia (IFP P93). WMM was supported by a doctoral scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC). We thank Dr. Lynne C. Weaver for critical review of the manuscript.

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Increasing evidence indicates that innate immune receptors play important, yet controversial, roles in traumatic central nervous system (CNS) injury. Despite many advances, the contributions of toll-like receptors (TLRs) to spinal cord injury (SCI) remain inadequately defined. We previously reported that a toll-like receptor 9 (TLR9) antagonist, oligodeoxynucleotide 2088 (ODN 2088), administered intrathecally, improves the functional and histopathological outcomes of SCI. However, the molecular and cellular changes that occur at the injury epicenter following ODN 2088 treatment are not completely understood.
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The anterograde labeling studies also showed increased numbers of corticospinal fibers projecting into the contralesional, denervated cervical spinal cord. It should also be noted that while the relationships shown in this study between conditional Sox9 ablation, reductions in PNN matrix and increased reparative sprouting after MCAO are only correlative, we have previously shown in a model of spinal cord injury that improved recovery in Sox9 conditional knockout mice is dependent on the reduction in PNN matrix levels (McKillop et al., 2016). In addition to increasing neuroplasticity after MCAO, Sox9 ablation also decreased tissue loss.
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Research PaperConditional Sox9 ablation improves locomotor recovery after spinal cord injury by increasing reactive sprouting

Highlights

Abstract

Introduction

Section snippets

Sox9 conditional knock out mice

Lumbar enlargement ventral horn PNN matrix post-SCI

Discussion

Conflict of interest

Acknowledgments

Exp. Neurol.

Prog. Brain Res.

Cell

Exp. Neurol.

Pharmacol. Res.

Brain Res. Brain Res. Rev.

Trends Neurosci.

Neurosci. Lett.

J. Biol. Chem.

Brain Res. Rev.

Dev. Biol.

Trends Neurosci.

Exp. Neurol.

Mol. Cell. Neurosci.

Exp. Neurol.

Neurobiol. Dis.

Exp. Neurol.

Neurosci. Lett.

Neuron

Brain Res.

Neurotherapeutics

Exp. Neurol.

Tenascin-R restricts posttraumatic remodeling of motoneuron innervation and functional recovery after spinal cord injury in adult mice

J. Neurosci.

The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats

Nat. Neurosci.

Chondroitinase ABC promotes sprouting of intact and injured spinal systems after spinal cord injury

J. Neurosci.

A sensitive and reliable locomotor rating scale for open field testing in rats

J. Neurotrauma

Sox9 is required for cartilage formation

Nat. Genet.

Chondroitinase ABC promotes functional recovery after spinal cord injury

Nature

Research Paper
Conditional Sox9 ablation improves locomotor recovery after spinal cord injury by increasing reactive sprouting