Mesencephalic locomotor region stimulation-cuneiform or pedunculopontine?

Roussel et al.1 provide new insight into mecencephalic locomotor region (MLR) stimulation to treat spinal cord injury in mice. Previously, it was unclear which part of the MLR to target. Now, evidence converges on cuneiform nucleus activation.

Chronic muscle recordings reveal recovery of forelimb function in spinal injured female rats after cortical epidural stimulation combined with rehabilitation and chondroitinase ABC.

Cervical level spinal cord injury (SCI) can severely impact upper limb muscle function, which is typically assessed in the clinic using electromyography (EMG). Here, we established novel preclinical methodology for EMG assessments of muscle function after SCI in awake freely moving animals. Adult female rats were implanted with EMG recording electrodes in bicep muscles and received bilateral cervical (C7) contusion injuries. Forelimb muscle activity was assessed by recording maximum voluntary contractions during a grip strength task and cortical motor evoked potentials in the biceps. We demonstrate that longitudinal recordings of muscle activity in the same animal are feasible over a chronic post-injury time course and provide a sensitive method for revealing post-injury changes in muscle activity. This methodology was utilized to investigate recovery of muscle function after a novel combination therapy. Cervical contused animals received intraspinal injections of a neuroplasticity-promoting agent (lentiviral-chondroitinase ABC) plus 11 weeks of cortical epidural electrical stimulation (3 h daily, 5 days/week) and behavioral rehabilitation (15 min daily, 5 days/week). Longitudinal monitoring of voluntary and evoked muscle activity revealed significantly increased muscle activity and upper limb dexterity with the combination treatment, compared to a single treatment or no treatment. Retrograde mapping of motor neurons innervating the biceps showed a predominant distribution across spinal segments C5-C8, indicating that treatment effects were likely due to neuroplastic changes in a mixture of intact and injured motor neurons. Thus, longitudinal assessments of muscle function after SCI correlate with skilled reach and grasp performance and reveal functional benefits of a novel combination therapy.

Chondroitin sulfate proteoglycans prevent immune cell phenotypic conversion and inflammation resolution via TLR4 in rodent models of spinal cord injury.

Chondroitin sulfate proteoglycans (CSPGs) act as potent inhibitors of axonal growth and neuroplasticity after spinal cord injury (SCI). Here we reveal that CSPGs also play a critical role in preventing inflammation resolution by blocking the conversion of pro-inflammatory immune cells to a pro-repair phenotype in rodent models of SCI. We demonstrate that enzymatic digestion of CSPG glycosaminoglycans enhances immune cell clearance and reduces pro-inflammatory protein and gene expression profiles at key resolution time points. Analysis of phenotypically distinct immune cell clusters revealed CSPG-mediated modulation of macrophage and microglial subtypes which, together with T lymphocyte infiltration and composition changes, suggests a role for CSPGs in modulating both innate and adaptive immune responses after SCI. Mechanistically, CSPG activation of a pro-inflammatory phenotype in pro-repair immune cells was found to be TLR4-dependent, identifying TLR4 signalling as a key driver of CSPG-mediated immune modulation. These findings establish CSPGs as critical mediators of inflammation resolution failure after SCI in rodents, which leads to prolonged inflammatory pathology and irreversible tissue destruction.

An active vesicle priming machinery suppresses axon regeneration upon adult CNS injury.

Axons in the adult mammalian central nervous system fail to regenerate after spinal cord injury. Neurons lose their capacity to regenerate during development, but the intracellular processes underlying this loss are unclear. We found that critical components of the presynaptic active zone prevent axon regeneration in adult mice. Transcriptomic analysis combined with live-cell imaging revealed that adult primary sensory neurons downregulate molecular constituents of the synapse as they acquire the ability to rapidly grow their axons. Pharmacogenetic reduction of neuronal excitability stimulated axon regeneration after adult spinal cord injury. Genetic gain- and loss-of-function experiments uncovered that essential synaptic vesicle priming proteins of the presynaptic active zone, but not clostridial-toxin-sensitive VAMP-family SNARE proteins, inhibit axon regeneration. Systemic administration of Baclofen reduced voltage-dependent Ca2+ influx in primary sensory neurons and promoted their regeneration after spinal cord injury. These findings indicate that functional presynaptic active zones constitute a major barrier to axon regeneration.

RhoA drives actin compaction to restrict axon regeneration and astrocyte reactivity after CNS injury.

An inhibitory extracellular milieu and neuron-intrinsic processes prevent axons from regenerating in the adult central nervous system (CNS). Here we show how the two aspects are interwoven. Genetic loss-of-function experiments determine that the small GTPase RhoA relays extracellular inhibitory signals to the cytoskeleton by adapting mechanisms set in place during neuronal polarization. In response to extracellular inhibitors, neuronal RhoA restricts axon regeneration by activating myosin II to compact actin and, thereby, restrain microtubule protrusion. However, astrocytic RhoA restricts injury-induced astrogliosis through myosin II independent of microtubules by activating Yes-activated protein (YAP) signaling. Cell-type-specific deletion in spinal-cord-injured mice shows that neuronal RhoA activation prevents axon regeneration, whereas astrocytic RhoA is beneficial for regenerating axons. These data demonstrate how extracellular inhibitors regulate axon regeneration, shed light on the capacity of reactive astrocytes to be growth inhibitory after CNS injury, and reveal cell-specific RhoA targeting as a promising therapeutic avenue.

Combined treatment with enteric neural stem cells and chondroitinase ABC reduces spinal cord lesion pathology.

BACKGROUND: Spinal cord injury (SCI) presents a significant challenge for the field of neurotherapeutics. Stem cells have shown promise in replenishing the cells lost to the injury process, but the release of axon growth-inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) by activated cells within the injury site hinders the integration of transplanted cells. We hypothesised that simultaneous application of enteric neural stem cells (ENSCs) isolated from the gastrointestinal tract, with a lentivirus (LV) containing the enzyme chondroitinase ABC (ChABC), would enhance the regenerative potential of ENSCs after transplantation into the injured spinal cord. METHODS: ENSCs were harvested from the GI tract of p7 rats, expanded in vitro and characterised. Adult rats bearing a contusion injury were randomly assigned to one of four groups: no treatment, LV-ChABC injection only, ENSC transplantation only or ENSC transplantation+LV-ChABC injection. After 16 weeks, rats were sacrificed and the harvested spinal cords examined for evidence of repair. RESULTS: ENSC cultures contained a variety of neuronal subtypes suitable for replenishing cells lost through SCI. Following injury, transplanted ENSC-derived cells survived and ChABC successfully degraded CSPGs. We observed significant reductions in the injured tissue and cavity area, with the greatest improvements seen in the combined treatment group. ENSC-derived cells extended projections across the injury site into both the rostral and caudal host spinal cord, and ENSC transplantation significantly increased the number of cells extending axons across the injury site. Furthermore, the combined treatment resulted in a modest, but significant functional improvement by week 16, and we found no evidence of the spread of transplanted cells to ectopic locations or formation of tumours. CONCLUSIONS: Regenerative effects of a combined treatment with ENSCs and ChABC surpassed either treatment alone, highlighting the importance of further research into combinatorial therapies for SCI. Our work provides evidence that stem cells taken from the adult gastrointestinal tract, an easily accessible source for autologous transplantation, could be strongly considered for the repair of central nervous system disorders.

Moving beyond the glial scar for spinal cord repair.

Traumatic spinal cord injury results in severe and irreversible loss of function. The injury triggers a complex cascade of inflammatory and pathological processes, culminating in formation of a scar. While traditionally referred to as a glial scar, the spinal injury scar in fact comprises multiple cellular and extracellular components. This multidimensional nature should be considered when aiming to understand the role of scarring in limiting tissue repair and recovery. In this Review we discuss recent advances in understanding the composition and phenotypic characteristics of the spinal injury scar, the oversimplification of defining the scar in binary terms as good or bad, and the development of therapeutic approaches to target scar components to enable improved functional outcome after spinal cord injury.

ErbB receptor signaling directly controls oligodendrocyte progenitor cell transformation and spontaneous remyelination after spinal cord injury.

We recently discovered a novel role for neuregulin-1 (Nrg1) signaling in mediating spontaneous regenerative processes and functional repair after spinal cord injury (SCI). We revealed that Nrg1 is the molecular signal responsible for spontaneous functional remyelination of dorsal column axons by peripheral nervous system (PNS)-like Schwann cells after SCI. Here, we investigate whether Nrg1/ErbB signaling controls the unusual transformation of centrally derived progenitor cells into these functional myelinating Schwann cells after SCI using a fate-mapping/lineage tracing approach. Specific ablation of Nrg1-ErbB receptors in central platelet-derived growth factor receptor alpha (PDGFRα)-derived lineage cells (using PDGFRαCreERT2/Tomato-red reporter mice crossed with ErbB3fl/fl/ErbB4fl/fl mice) led to a dramatic reduction in P0-positive remyelination in the dorsal columns following spinal contusion injury. Central myelination, assessed by Olig2 and proteolipid protein expression, was unchanged. Loss of ErbB signaling in PDGFRα lineage cells also significantly impacted the degree of spontaneous locomotor recovery after SCI, particularly in tests dependent on proprioception. These data have important implications, namely (a) cells from the PDGFRα-expressing progenitor lineage (which are presumably oligodendrocyte progenitor cells, OPCs) can differentiate into remyelinating PNS-like Schwann cells after traumatic SCI, (b) this process is controlled by ErbB tyrosine kinase signaling, and (c) this endogenous repair mechanism has significant consequences for functional recovery after SCI. Thus, ErbB tyrosine kinase receptor signaling directly controls the transformation of OPCs from the PDGFRα-expressing lineage into PNS-like functional remyelinating Schwann cells after SCI.

Immune-evasive gene switch enables regulated delivery of chondroitinase after spinal cord injury.

Chondroitinase ABC is a promising preclinical therapy that promotes functional neuroplasticity after CNS injury by degrading extracellular matrix inhibitors. Efficient delivery of chondroitinase ABC to the injured mammalian spinal cord can be achieved by viral vector transgene delivery. This approach dramatically modulates injury pathology and restores sensorimotor functions. However, clinical development of this therapy is limited by a lack of ability to exert control over chondroitinase gene expression. Prior experimental gene regulation platforms are likely to be incompatible with the non-resolving adaptive immune response known to occur following spinal cord injury. Therefore, here we apply a novel immune-evasive dual vector system, in which the chondroitinase gene is under a doxycycline inducible regulatory switch, utilizing a chimeric transactivator designed to evade T cell recognition. Using this novel vector system, we demonstrate tight temporal control of chondroitinase ABC gene expression, effectively removing treatment upon removal of doxycycline. This enables a comparison of short and long-term gene therapy paradigms in the treatment of clinically-relevant cervical level contusion injuries in adult rats. We reveal that transient treatment (2.5 weeks) is sufficient to promote improvement in sensory axon conduction and ladder walking performance. However, in tasks requiring skilled reaching and grasping, only long term treatment (8 weeks) leads to significantly improved function, with rats able to accurately grasp and retrieve sugar pellets. The late emergence of skilled hand function indicates enhanced neuroplasticity and connectivity and correlates with increased density of vGlut1+ innervation in spinal cord grey matter, particularly in lamina III-IV above and below the injury. Thus, our novel gene therapy system provides an experimental tool to study temporal effects of extracellular matrix digestion as well as an encouraging step towards generating a safer chondroitinase gene therapy strategy, longer term administration of which increases neuroplasticity and recovery of descending motor control. This preclinical study could have a significant impact for tetraplegic individuals, for whom recovery of hand function is an important determinant of independence, and supports the ongoing development of chondroitinase gene therapy towards clinical application for the treatment of spinal cord injury.

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

BACKGROUND: There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. METHODOLOGY/PRINCIPAL FINDINGS: To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of ß-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. CONCLUSION/SIGNIFICANCE: Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.