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Crmp4 deletion promotes recovery from spinal cord injury by neuroprotection and limited scar formation.

Nagai J, Kitamura Y, Owada K, Yamashita N, Takei K, Goshima Y, Ohshima T - Sci Rep (2015)

Bottom Line: We found increases in the inhibitory and toxic forms of CRMP4 in injured spinal cord.Notably, CRMP4 expression was evident in inflammatory cells as well as in neurons after spinal cord transection.Crmp4-/- mice displayed neuroprotection against SCI and reductions in inflammatory response and scar formation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, TWIns, Waseda University, Tokyo, 162-8480 Japan [2] Research Fellow of Japan Society for the Promotion of Science.

ABSTRACT
Axonal outgrowth inhibitors and scar formation are two major obstacles to central nervous system (CNS) repair. No target molecule that regulates both axonal growth and scarring has been identified. Here we identified collapsin response mediator protein 4 (CRMP4), a common mediator of inhibitory signals after neural injury, as a crucial factor that contributes to both axonal growth inhibition and scarring after spinal cord injury (SCI). We found increases in the inhibitory and toxic forms of CRMP4 in injured spinal cord. Notably, CRMP4 expression was evident in inflammatory cells as well as in neurons after spinal cord transection. Crmp4-/- mice displayed neuroprotection against SCI and reductions in inflammatory response and scar formation. This permissive environment for axonal growth due to CRMP4 deletion restored locomotor activity at an unusually early phase of healing. These results suggest that deletion of CRMP4 is a unique therapeutic strategy that overcomes two obstacles to CNS repair after SCI.

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Expression changes of CRMP4 in spinal motoneuron and glial cells after SCI.Immunohistochemical analysis of the expression of CRMP4 of the intact and injured spinal cords at 1 week after transection (SCI 1 W). (a) Representative images from cross sections of spinal cord show double-immunofluorescent staining for Nissl (the marker for neurons; red) and CRMP4 (green). CRMP4 staining was apparent in Nissl-positive motoneuron in the ventral horn (solid arrowheads) both in intact and injured spinal cords from control Crmp4+/+ mice. This CRMP4 signal was undetectable in intact Crmp4−/− spinal cord. (b) Co-localization of CRMP4 (green) and MAP2 (red) immunopositive structures, which labeled neuronal cell bodies and their dendrites, in the ventral horn of intact and transected spinal cords. (c–d) Immunohistochemical analysis of the expression of CRMP4 in microglia/macrophage and astrocytes. (c) Images of sagittal sections show double immunofluorescent staining for CRMP4 (green) and red signals of OX-41, the marker for microglia/macrophage (c), or GFAP, the marker for normal and reactive astrocytes (d). In the intact spinal cord, red signals in the resting OX-41-positive microglia/macrophage and GFAP-positive astrocytes did not co-localized with green signals of CRMP4 (open arrowheads). However, at 1 week after SCI, CRMP4 signals are evident in activated these cells (arrowheads) adjacent lesion epicenter and astroglial scar. Nuclei were counterstained with DAPI (blue) in the same view in each section. Asterisks in d indicate lesion site. W, weeks. Scale bars: 100 μm.
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f2: Expression changes of CRMP4 in spinal motoneuron and glial cells after SCI.Immunohistochemical analysis of the expression of CRMP4 of the intact and injured spinal cords at 1 week after transection (SCI 1 W). (a) Representative images from cross sections of spinal cord show double-immunofluorescent staining for Nissl (the marker for neurons; red) and CRMP4 (green). CRMP4 staining was apparent in Nissl-positive motoneuron in the ventral horn (solid arrowheads) both in intact and injured spinal cords from control Crmp4+/+ mice. This CRMP4 signal was undetectable in intact Crmp4−/− spinal cord. (b) Co-localization of CRMP4 (green) and MAP2 (red) immunopositive structures, which labeled neuronal cell bodies and their dendrites, in the ventral horn of intact and transected spinal cords. (c–d) Immunohistochemical analysis of the expression of CRMP4 in microglia/macrophage and astrocytes. (c) Images of sagittal sections show double immunofluorescent staining for CRMP4 (green) and red signals of OX-41, the marker for microglia/macrophage (c), or GFAP, the marker for normal and reactive astrocytes (d). In the intact spinal cord, red signals in the resting OX-41-positive microglia/macrophage and GFAP-positive astrocytes did not co-localized with green signals of CRMP4 (open arrowheads). However, at 1 week after SCI, CRMP4 signals are evident in activated these cells (arrowheads) adjacent lesion epicenter and astroglial scar. Nuclei were counterstained with DAPI (blue) in the same view in each section. Asterisks in d indicate lesion site. W, weeks. Scale bars: 100 μm.

Mentions: Next, we examined which cell types expressed CRMP4 after SCI. A markedly increased CRMP4 expression level has been reported in spinal motoneurons in the mutant SOD1 mouse model31 and in adult sensory neurons after sciatic nerve injury32. We first conducted double immunostaining for neuronal marker and CRMP4 in cross sections of spinal cords. We detected CRMP4 expression and found that it was co-localized with Nissl-positive neuronal cell bodies and MAP2-positive dendrites and somata of motoneurons in the ventral horn of intact and injured spinal cords (Fig. 2a,b). The fraction of neurons expressing these levels of CRMP4 was significantly above background in Crmp4−/− spinal cords (Fig. 2a). To analyze CRMP4 protein expression in microglia/macrophages and reactive astrocytes before, during, and after inflammatory responses to SCI, we double immunostained parasagittal sections of spinal cords for CRMP4 and glial fibrillary acidic protein (GFAP), a marker for normal as well as reactive astrocytes, or OX-41, a marker for microglia/macrophages33. The CRMP4 staining signal was weak in both GFAP-positive and OX-41-positive cells in the gray matter of intact spinal cord (Fig. 2c,d; open arrowheads). However, CRMP4 immunoreactivity was remarkably enhanced in both types of glial cells adjacent to the lesion site and in the astroglial scar after SCI (Fig. 2c,d; solid arrowheads).


Crmp4 deletion promotes recovery from spinal cord injury by neuroprotection and limited scar formation.

Nagai J, Kitamura Y, Owada K, Yamashita N, Takei K, Goshima Y, Ohshima T - Sci Rep (2015)

Expression changes of CRMP4 in spinal motoneuron and glial cells after SCI.Immunohistochemical analysis of the expression of CRMP4 of the intact and injured spinal cords at 1 week after transection (SCI 1 W). (a) Representative images from cross sections of spinal cord show double-immunofluorescent staining for Nissl (the marker for neurons; red) and CRMP4 (green). CRMP4 staining was apparent in Nissl-positive motoneuron in the ventral horn (solid arrowheads) both in intact and injured spinal cords from control Crmp4+/+ mice. This CRMP4 signal was undetectable in intact Crmp4−/− spinal cord. (b) Co-localization of CRMP4 (green) and MAP2 (red) immunopositive structures, which labeled neuronal cell bodies and their dendrites, in the ventral horn of intact and transected spinal cords. (c–d) Immunohistochemical analysis of the expression of CRMP4 in microglia/macrophage and astrocytes. (c) Images of sagittal sections show double immunofluorescent staining for CRMP4 (green) and red signals of OX-41, the marker for microglia/macrophage (c), or GFAP, the marker for normal and reactive astrocytes (d). In the intact spinal cord, red signals in the resting OX-41-positive microglia/macrophage and GFAP-positive astrocytes did not co-localized with green signals of CRMP4 (open arrowheads). However, at 1 week after SCI, CRMP4 signals are evident in activated these cells (arrowheads) adjacent lesion epicenter and astroglial scar. Nuclei were counterstained with DAPI (blue) in the same view in each section. Asterisks in d indicate lesion site. W, weeks. Scale bars: 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f2: Expression changes of CRMP4 in spinal motoneuron and glial cells after SCI.Immunohistochemical analysis of the expression of CRMP4 of the intact and injured spinal cords at 1 week after transection (SCI 1 W). (a) Representative images from cross sections of spinal cord show double-immunofluorescent staining for Nissl (the marker for neurons; red) and CRMP4 (green). CRMP4 staining was apparent in Nissl-positive motoneuron in the ventral horn (solid arrowheads) both in intact and injured spinal cords from control Crmp4+/+ mice. This CRMP4 signal was undetectable in intact Crmp4−/− spinal cord. (b) Co-localization of CRMP4 (green) and MAP2 (red) immunopositive structures, which labeled neuronal cell bodies and their dendrites, in the ventral horn of intact and transected spinal cords. (c–d) Immunohistochemical analysis of the expression of CRMP4 in microglia/macrophage and astrocytes. (c) Images of sagittal sections show double immunofluorescent staining for CRMP4 (green) and red signals of OX-41, the marker for microglia/macrophage (c), or GFAP, the marker for normal and reactive astrocytes (d). In the intact spinal cord, red signals in the resting OX-41-positive microglia/macrophage and GFAP-positive astrocytes did not co-localized with green signals of CRMP4 (open arrowheads). However, at 1 week after SCI, CRMP4 signals are evident in activated these cells (arrowheads) adjacent lesion epicenter and astroglial scar. Nuclei were counterstained with DAPI (blue) in the same view in each section. Asterisks in d indicate lesion site. W, weeks. Scale bars: 100 μm.
Mentions: Next, we examined which cell types expressed CRMP4 after SCI. A markedly increased CRMP4 expression level has been reported in spinal motoneurons in the mutant SOD1 mouse model31 and in adult sensory neurons after sciatic nerve injury32. We first conducted double immunostaining for neuronal marker and CRMP4 in cross sections of spinal cords. We detected CRMP4 expression and found that it was co-localized with Nissl-positive neuronal cell bodies and MAP2-positive dendrites and somata of motoneurons in the ventral horn of intact and injured spinal cords (Fig. 2a,b). The fraction of neurons expressing these levels of CRMP4 was significantly above background in Crmp4−/− spinal cords (Fig. 2a). To analyze CRMP4 protein expression in microglia/macrophages and reactive astrocytes before, during, and after inflammatory responses to SCI, we double immunostained parasagittal sections of spinal cords for CRMP4 and glial fibrillary acidic protein (GFAP), a marker for normal as well as reactive astrocytes, or OX-41, a marker for microglia/macrophages33. The CRMP4 staining signal was weak in both GFAP-positive and OX-41-positive cells in the gray matter of intact spinal cord (Fig. 2c,d; open arrowheads). However, CRMP4 immunoreactivity was remarkably enhanced in both types of glial cells adjacent to the lesion site and in the astroglial scar after SCI (Fig. 2c,d; solid arrowheads).

Bottom Line: We found increases in the inhibitory and toxic forms of CRMP4 in injured spinal cord.Notably, CRMP4 expression was evident in inflammatory cells as well as in neurons after spinal cord transection.Crmp4-/- mice displayed neuroprotection against SCI and reductions in inflammatory response and scar formation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, TWIns, Waseda University, Tokyo, 162-8480 Japan [2] Research Fellow of Japan Society for the Promotion of Science.

ABSTRACT
Axonal outgrowth inhibitors and scar formation are two major obstacles to central nervous system (CNS) repair. No target molecule that regulates both axonal growth and scarring has been identified. Here we identified collapsin response mediator protein 4 (CRMP4), a common mediator of inhibitory signals after neural injury, as a crucial factor that contributes to both axonal growth inhibition and scarring after spinal cord injury (SCI). We found increases in the inhibitory and toxic forms of CRMP4 in injured spinal cord. Notably, CRMP4 expression was evident in inflammatory cells as well as in neurons after spinal cord transection. Crmp4-/- mice displayed neuroprotection against SCI and reductions in inflammatory response and scar formation. This permissive environment for axonal growth due to CRMP4 deletion restored locomotor activity at an unusually early phase of healing. These results suggest that deletion of CRMP4 is a unique therapeutic strategy that overcomes two obstacles to CNS repair after SCI.

Show MeSH
Related in: MedlinePlus