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Background and Purpose In the spinal cord injury (SCI) axon regeneration is inhibited by the glial scar, which contains reactive astrocytes that secrete inhibitory chondroitin sulphate proteoglycan (CSPG). We previously reported that a novel compound, denosomin, promotes axonal growth under degenerative conditions in cultured cortical neurons. In this study, we investigated the effects of denosomin on functional recovery in SCI mice and elucidated the mechanism though which denosomin induces axonal growth in the injured spinal cord. Experimental Approach Denosomin was administered p.o. for 7 or 14 days to contusion mice. Behavioural evaluations and immunohistochemistry were done. Primary cultured cortical neurons and astrocytes were treated with denosomin to investigate the mechanism of axonal growth facilitation. Key Results Denosomin improved hind limb motor dysfunction and axonal growth, especially in the 5‐HT‐positive tracts across the scar and increased the density of astrocytes. Denosomin increased astrocyte proliferation, inhibited astrocytic death and increased the expression and secretion of vimentin in cultured astrocytes. Furthermore, vimentin increased axonal outgrowth in cultured neurons, even in the presence of inhibitory CSPG. Denosomin increased the number of vimentin‐expressing astrocytes inside glial scars of SCI mice, and 5‐HT‐positive axonal growth occurred in a vimentin‐associated manner. Conclusion and Implications Denosomin increased the ratio of astrocytes that secrete vimentin as an axonal growth facilitator, which, we propose enhances axonal growth beyond the glial scar and promotes functional recovery in SCI mice. This study is the first to demonstrate this novel role of vimentin in SCI and drug‐mediated modification of the inhibitory property of reactive astrocytes.

Vimentin, an intermediate filament protein, is generally recognised as an intracellular protein. Previously, we reported that vimentin was secreted from astrocytes and promoted axonal growth. The effect of extracellular vimentin in neurons was a new finding, but its signalling pathway was unknown. In this study, we aimed to determine the signalling mechanism of extracellular vimentin that facilitates axonal growth. We first identified insulin-like growth factor 1 receptor (IGF1R) as a receptor that is highly phosphorylated by vimentin stimulation. IGF1R blockades diminished vimentin- or IGF1-induced axonal growth in cultured cortical neurons. IGF1, IGF2 and insulin were not detected in the neuron culture medium after vimentin treatment. The combined drug affinity responsive target stability method and western blotting analysis showed that vimentin and IGF1 interacted with IGF1R directly. In addition, immunoprecipitation and western blotting analyses confirmed that recombinant IGF1R bound to vimentin. The results of a molecular dynamics simulation revealed that C-terminal residues (residue number 330-407) in vimentin are the most appropriate binding sites with IGF1R. Thus, extracellular vimentin may be a novel ligand of IGF1R that promotes axonal growth in a similar manner to IGF1. Our results provide novel findings regarding the role of extracellular vimentin and IGF1R in axonal growth.

Vimentin, an intermediate filament protein, is an intracellular protein that is involved in various cellular processes. Several groups have recently reported that vimentin also appears in the extracellular space and shows novel protein activity. We previously reported that denosomin improved motor dysfunction in mice with a contusive spinal cord injury (SCI). At the injured area, astrocytes expressing and secreting vimentin were specifically increased, and axonal growth occurred in a vimentin-dependent manner in denosomin-treated mice. However, the axonal growth that was induced by extracellular vimentin was only investigated in vitro in the previous study. Here, we sought to clarify whether increased extracellular vimentin can promote the axonal extension related to motor improvement after SCI in vivo. Extracellular vimentin treatment in SCI mice significantly ameliorated motor dysfunction. In vimentin-treated mice, 5-HT-positive axons increased significantly at the rostral and central areas of the lesion, and the total axonal densities increased in the central and caudal parts of the lesioned area. This finding suggests that increased axonal density may contribute to motor improvement in vimentin-treated mice. Thus, our in vivo data indicate that extracellular vimentin may be a novel neurotrophic factor that enhances axonal growth activity and motor function recovery after SCI.

BackgroundAmong the variety of methods used to evaluate locomotor function following a spinal cord injury (SCI), the Basso Mouse Scale score (BMS) has been widely used for mice. However, the BMS mainly focuses on hindlimb movement rather than on graded changes in body support ability. In addition, some of the scoring methods include double or triple criteria within a single score, which likely leads to an increase in the deviation within the data. Therefore we aimed to establish a new scoring method reliable and easy to perform in mice with SCI.FindingsOur Toyama Mouse Score (TMS) was established by rearranging and simplifying the BMS score and combining it with the Body Support Scale score (BSS). The TMS reflects changes in both body support ability and hindlimb movement. The definition of single score is made by combing multiple criteria in the BMS. The ambiguity was improved in the TMS. Using contusive SCI mice, hindlimb function was measured using the TMS, BMS and BSS systems. The TMS could distinguish changes in hindlimb movements that were evaluated as the same score by the BMS. An analysis of the coefficient of variation (CV) of score points recorded for 11 days revealed that the CV for the TMS was significantly lower than the CV obtained using the BMS. A variation in intra evaluators was lower in the TMS than in the BMS.ConclusionThese results suggest that the TMS may be useful as a new reliable method for scoring locomotor function for SCI models.