Limits...
HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix

View Article: PubMed Central - PubMed

ABSTRACT

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.

No MeSH data available.


Related in: MedlinePlus

Lack of toxicity, time course of disappearance and distribution of injected HB-GAM in adult mouse neocortex.(a) Cortical sections from adult C57Bl mice were processed for TUNEL staining (green) to mark apoptotic cells (note – not specific for neurons) at day 3 post-injection or 3 days after the prick-injury followed by microinjection of IgG or HB-GAM. Brain sections were treated with DNase I for positive control. DAPI (blue) was used to label cell nuclei. Scale bar, 30 μm. (b) The fraction of TUNEL+ cells was calculated (mean ± SEM) to quantify apoptosis following the microinjection of different HB-GAM concentrations (0.1, 1.0 and 10.0 mg/ml). There was no significant difference in the numbers of TUNEL+ cells between animals injected with PBS, IgG or with different concentrations of HB-GAM. In addition, quantification of TUNEL staining did not show a significant difference in HB-GAM-injected animals after prick-injury compared to IgG-injected animals (1.5 μl of 1.0 mg/ml either HB-GAM or IgG). (c) Western blotting showing HB-GAM protein levels in somatosensory cortex lysates from the injury site following IgG or HB-GAM microinjection. GAPDH was used for normalization. Error bars represent SEM. (d) Coronal sections from Thy1-YFP mouse brains (neurons - green) 3 hours, 3 and 20 days after the injury following IgG treatment (1.5 μl at 1 mg/ml). Microphotography of GFAP (magenta) and HB-GAM (red) immunoreactivity shows that astrocytes are activated in a substantial volume of the brain cortex 3 days after the injury. Astrocytic scarring is seen in the injury site on post-injury day 20, but immunohistochemistry did not detect injury-induced HB-GAM expression. (e) Coronal sections from Thy1-YFP mouse brains 20 days after the injury following HB-GAM injection (1.5 μl at 1 mg/ml). As in control animals in (d), GFAP immunoreactivity shows astrocyte activation and astrocytic scarring. Notice the HB-GAM distribution in the injury site on post-injury day 20. The scale bar in (a,d,e) is 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037378&req=5

f5: Lack of toxicity, time course of disappearance and distribution of injected HB-GAM in adult mouse neocortex.(a) Cortical sections from adult C57Bl mice were processed for TUNEL staining (green) to mark apoptotic cells (note – not specific for neurons) at day 3 post-injection or 3 days after the prick-injury followed by microinjection of IgG or HB-GAM. Brain sections were treated with DNase I for positive control. DAPI (blue) was used to label cell nuclei. Scale bar, 30 μm. (b) The fraction of TUNEL+ cells was calculated (mean ± SEM) to quantify apoptosis following the microinjection of different HB-GAM concentrations (0.1, 1.0 and 10.0 mg/ml). There was no significant difference in the numbers of TUNEL+ cells between animals injected with PBS, IgG or with different concentrations of HB-GAM. In addition, quantification of TUNEL staining did not show a significant difference in HB-GAM-injected animals after prick-injury compared to IgG-injected animals (1.5 μl of 1.0 mg/ml either HB-GAM or IgG). (c) Western blotting showing HB-GAM protein levels in somatosensory cortex lysates from the injury site following IgG or HB-GAM microinjection. GAPDH was used for normalization. Error bars represent SEM. (d) Coronal sections from Thy1-YFP mouse brains (neurons - green) 3 hours, 3 and 20 days after the injury following IgG treatment (1.5 μl at 1 mg/ml). Microphotography of GFAP (magenta) and HB-GAM (red) immunoreactivity shows that astrocytes are activated in a substantial volume of the brain cortex 3 days after the injury. Astrocytic scarring is seen in the injury site on post-injury day 20, but immunohistochemistry did not detect injury-induced HB-GAM expression. (e) Coronal sections from Thy1-YFP mouse brains 20 days after the injury following HB-GAM injection (1.5 μl at 1 mg/ml). As in control animals in (d), GFAP immunoreactivity shows astrocyte activation and astrocytic scarring. Notice the HB-GAM distribution in the injury site on post-injury day 20. The scale bar in (a,d,e) is 100 μm.

Mentions: Different concentrations (0.1, 1.0 and 10 mg/ml) of baculovirus-derived recombinant HB-GAM24 were microinjected (1.5 μl) in normal and injured cortex. We did not observe any effects on cell survival resulting from the treatment either in the non-injured or in the prick-injured cortex when assessed by TUNEL staining (Fig. 5a,b).


HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix
Lack of toxicity, time course of disappearance and distribution of injected HB-GAM in adult mouse neocortex.(a) Cortical sections from adult C57Bl mice were processed for TUNEL staining (green) to mark apoptotic cells (note – not specific for neurons) at day 3 post-injection or 3 days after the prick-injury followed by microinjection of IgG or HB-GAM. Brain sections were treated with DNase I for positive control. DAPI (blue) was used to label cell nuclei. Scale bar, 30 μm. (b) The fraction of TUNEL+ cells was calculated (mean ± SEM) to quantify apoptosis following the microinjection of different HB-GAM concentrations (0.1, 1.0 and 10.0 mg/ml). There was no significant difference in the numbers of TUNEL+ cells between animals injected with PBS, IgG or with different concentrations of HB-GAM. In addition, quantification of TUNEL staining did not show a significant difference in HB-GAM-injected animals after prick-injury compared to IgG-injected animals (1.5 μl of 1.0 mg/ml either HB-GAM or IgG). (c) Western blotting showing HB-GAM protein levels in somatosensory cortex lysates from the injury site following IgG or HB-GAM microinjection. GAPDH was used for normalization. Error bars represent SEM. (d) Coronal sections from Thy1-YFP mouse brains (neurons - green) 3 hours, 3 and 20 days after the injury following IgG treatment (1.5 μl at 1 mg/ml). Microphotography of GFAP (magenta) and HB-GAM (red) immunoreactivity shows that astrocytes are activated in a substantial volume of the brain cortex 3 days after the injury. Astrocytic scarring is seen in the injury site on post-injury day 20, but immunohistochemistry did not detect injury-induced HB-GAM expression. (e) Coronal sections from Thy1-YFP mouse brains 20 days after the injury following HB-GAM injection (1.5 μl at 1 mg/ml). As in control animals in (d), GFAP immunoreactivity shows astrocyte activation and astrocytic scarring. Notice the HB-GAM distribution in the injury site on post-injury day 20. The scale bar in (a,d,e) is 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037378&req=5

f5: Lack of toxicity, time course of disappearance and distribution of injected HB-GAM in adult mouse neocortex.(a) Cortical sections from adult C57Bl mice were processed for TUNEL staining (green) to mark apoptotic cells (note – not specific for neurons) at day 3 post-injection or 3 days after the prick-injury followed by microinjection of IgG or HB-GAM. Brain sections were treated with DNase I for positive control. DAPI (blue) was used to label cell nuclei. Scale bar, 30 μm. (b) The fraction of TUNEL+ cells was calculated (mean ± SEM) to quantify apoptosis following the microinjection of different HB-GAM concentrations (0.1, 1.0 and 10.0 mg/ml). There was no significant difference in the numbers of TUNEL+ cells between animals injected with PBS, IgG or with different concentrations of HB-GAM. In addition, quantification of TUNEL staining did not show a significant difference in HB-GAM-injected animals after prick-injury compared to IgG-injected animals (1.5 μl of 1.0 mg/ml either HB-GAM or IgG). (c) Western blotting showing HB-GAM protein levels in somatosensory cortex lysates from the injury site following IgG or HB-GAM microinjection. GAPDH was used for normalization. Error bars represent SEM. (d) Coronal sections from Thy1-YFP mouse brains (neurons - green) 3 hours, 3 and 20 days after the injury following IgG treatment (1.5 μl at 1 mg/ml). Microphotography of GFAP (magenta) and HB-GAM (red) immunoreactivity shows that astrocytes are activated in a substantial volume of the brain cortex 3 days after the injury. Astrocytic scarring is seen in the injury site on post-injury day 20, but immunohistochemistry did not detect injury-induced HB-GAM expression. (e) Coronal sections from Thy1-YFP mouse brains 20 days after the injury following HB-GAM injection (1.5 μl at 1 mg/ml). As in control animals in (d), GFAP immunoreactivity shows astrocyte activation and astrocytic scarring. Notice the HB-GAM distribution in the injury site on post-injury day 20. The scale bar in (a,d,e) is 100 μm.
Mentions: Different concentrations (0.1, 1.0 and 10 mg/ml) of baculovirus-derived recombinant HB-GAM24 were microinjected (1.5 μl) in normal and injured cortex. We did not observe any effects on cell survival resulting from the treatment either in the non-injured or in the prick-injured cortex when assessed by TUNEL staining (Fig. 5a,b).

View Article: PubMed Central - PubMed

ABSTRACT

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.

No MeSH data available.


Related in: MedlinePlus