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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.


HB-GAM improves dendrite regeneration in injury site after prick-injury in vivo.(a) Experimental timeline. (b) The maximum projection of orthogonal reconstructions (side view) from a stack acquired by in vivo two-photon microscopy at 0 d after injury mark with dashed lines corresponding to the injury site (red), perilesional (green) and remote (white) areas. (c–f) The maximum projection (top view) of YFP-labelled cortical neurons dendritic tuft (yellow) 3 hours and 20 days after injury following IgG treatment in (c,d) or HB-GAM treatment in (e,f). Injury site (red dashed line) was defined from second harmonic signal in pia mater that indicates borders of injury. Notice the limited fraction of the dendritic tuft number regenerated in the injury site in response to IgG treatment in (d) compared to HB-GAM treatment in (f). (g–j) 3D reconstruction of YFP-labelled apical dendrites 3 hours and 20 days after injury following IgG treatment in (g,h) or HB-GAM treatment in (i,j). Apical dendrites in the injury core are color-coded in red and in the perilesion area in green. (k–n) Average density of dendritic tufts in (k) and average number of apical dendrites in (l) in injury site normalized to the time point of 3 h following control (blue column) and HB-GAM treatment (red column). Average ratio of the numbers of apical dendrites in the injury site in (m) and perilesional area in (n) compared to remote area over time in control experiments (blue line) and following HB-GAM treatment (red line) (p-value from Mann-Whitney-U test). Error bars, SEM (n = 6 control; n = 7 HB-GAM). (o) The maximum projection (top view) of dendritic tuft (yellow) in remote area 3 hours and (p) 20 days after injury.
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f6: HB-GAM improves dendrite regeneration in injury site after prick-injury in vivo.(a) Experimental timeline. (b) The maximum projection of orthogonal reconstructions (side view) from a stack acquired by in vivo two-photon microscopy at 0 d after injury mark with dashed lines corresponding to the injury site (red), perilesional (green) and remote (white) areas. (c–f) The maximum projection (top view) of YFP-labelled cortical neurons dendritic tuft (yellow) 3 hours and 20 days after injury following IgG treatment in (c,d) or HB-GAM treatment in (e,f). Injury site (red dashed line) was defined from second harmonic signal in pia mater that indicates borders of injury. Notice the limited fraction of the dendritic tuft number regenerated in the injury site in response to IgG treatment in (d) compared to HB-GAM treatment in (f). (g–j) 3D reconstruction of YFP-labelled apical dendrites 3 hours and 20 days after injury following IgG treatment in (g,h) or HB-GAM treatment in (i,j). Apical dendrites in the injury core are color-coded in red and in the perilesion area in green. (k–n) Average density of dendritic tufts in (k) and average number of apical dendrites in (l) in injury site normalized to the time point of 3 h following control (blue column) and HB-GAM treatment (red column). Average ratio of the numbers of apical dendrites in the injury site in (m) and perilesional area in (n) compared to remote area over time in control experiments (blue line) and following HB-GAM treatment (red line) (p-value from Mann-Whitney-U test). Error bars, SEM (n = 6 control; n = 7 HB-GAM). (o) The maximum projection (top view) of dendritic tuft (yellow) in remote area 3 hours and (p) 20 days after injury.

Mentions: Since the injected HB-GAM targets the astrocytic scar region, likely due to its avid binding to the dense deposition of the CS chains of CSPGs at the scar347, reversal of the CSPG inhibition might allow for dendritic regeneration in the injured cortex. In our prick-injury model we followed the HB-GAM effect on dendritic regeneration in the injury site, in the perilesional area, and in an area remote from the lesion site (experimental protocol summarized in Fig. 6a,b). In vivo two-photon microscopy revealed robust regeneration of the dendritic tuft and of apical dendrites compared to the start of the experiment (3 h from the acute injury) within 2–3 weeks in the core and perilesional area in HB-GAM-injected cortical injury sites compared to the controls (Fig. 6c–j; optical sections included in the merged image stacks shown in Video 1). Quantification of the experiment showed significant regeneration based on the density of the dendritic tuft (Fig. 6k) and on the number of apical dendrites normalized to the corresponding distant area of the cortex (Fig. 6l).


HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix
HB-GAM improves dendrite regeneration in injury site after prick-injury in vivo.(a) Experimental timeline. (b) The maximum projection of orthogonal reconstructions (side view) from a stack acquired by in vivo two-photon microscopy at 0 d after injury mark with dashed lines corresponding to the injury site (red), perilesional (green) and remote (white) areas. (c–f) The maximum projection (top view) of YFP-labelled cortical neurons dendritic tuft (yellow) 3 hours and 20 days after injury following IgG treatment in (c,d) or HB-GAM treatment in (e,f). Injury site (red dashed line) was defined from second harmonic signal in pia mater that indicates borders of injury. Notice the limited fraction of the dendritic tuft number regenerated in the injury site in response to IgG treatment in (d) compared to HB-GAM treatment in (f). (g–j) 3D reconstruction of YFP-labelled apical dendrites 3 hours and 20 days after injury following IgG treatment in (g,h) or HB-GAM treatment in (i,j). Apical dendrites in the injury core are color-coded in red and in the perilesion area in green. (k–n) Average density of dendritic tufts in (k) and average number of apical dendrites in (l) in injury site normalized to the time point of 3 h following control (blue column) and HB-GAM treatment (red column). Average ratio of the numbers of apical dendrites in the injury site in (m) and perilesional area in (n) compared to remote area over time in control experiments (blue line) and following HB-GAM treatment (red line) (p-value from Mann-Whitney-U test). Error bars, SEM (n = 6 control; n = 7 HB-GAM). (o) The maximum projection (top view) of dendritic tuft (yellow) in remote area 3 hours and (p) 20 days after injury.
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f6: HB-GAM improves dendrite regeneration in injury site after prick-injury in vivo.(a) Experimental timeline. (b) The maximum projection of orthogonal reconstructions (side view) from a stack acquired by in vivo two-photon microscopy at 0 d after injury mark with dashed lines corresponding to the injury site (red), perilesional (green) and remote (white) areas. (c–f) The maximum projection (top view) of YFP-labelled cortical neurons dendritic tuft (yellow) 3 hours and 20 days after injury following IgG treatment in (c,d) or HB-GAM treatment in (e,f). Injury site (red dashed line) was defined from second harmonic signal in pia mater that indicates borders of injury. Notice the limited fraction of the dendritic tuft number regenerated in the injury site in response to IgG treatment in (d) compared to HB-GAM treatment in (f). (g–j) 3D reconstruction of YFP-labelled apical dendrites 3 hours and 20 days after injury following IgG treatment in (g,h) or HB-GAM treatment in (i,j). Apical dendrites in the injury core are color-coded in red and in the perilesion area in green. (k–n) Average density of dendritic tufts in (k) and average number of apical dendrites in (l) in injury site normalized to the time point of 3 h following control (blue column) and HB-GAM treatment (red column). Average ratio of the numbers of apical dendrites in the injury site in (m) and perilesional area in (n) compared to remote area over time in control experiments (blue line) and following HB-GAM treatment (red line) (p-value from Mann-Whitney-U test). Error bars, SEM (n = 6 control; n = 7 HB-GAM). (o) The maximum projection (top view) of dendritic tuft (yellow) in remote area 3 hours and (p) 20 days after injury.
Mentions: Since the injected HB-GAM targets the astrocytic scar region, likely due to its avid binding to the dense deposition of the CS chains of CSPGs at the scar347, reversal of the CSPG inhibition might allow for dendritic regeneration in the injured cortex. In our prick-injury model we followed the HB-GAM effect on dendritic regeneration in the injury site, in the perilesional area, and in an area remote from the lesion site (experimental protocol summarized in Fig. 6a,b). In vivo two-photon microscopy revealed robust regeneration of the dendritic tuft and of apical dendrites compared to the start of the experiment (3 h from the acute injury) within 2–3 weeks in the core and perilesional area in HB-GAM-injected cortical injury sites compared to the controls (Fig. 6c–j; optical sections included in the merged image stacks shown in Video 1). Quantification of the experiment showed significant regeneration based on the density of the dendritic tuft (Fig. 6k) and on the number of apical dendrites normalized to the corresponding distant area of the cortex (Fig. 6l).

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.