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CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro.

Stepanek L, Sun QL, Wang J, Wang C, Bixby JL - J. Cell Biol. (2001)

Bottom Line: We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons.This chemorepulsive effect could be regulated by the level of cGMP in the growth cone.Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.

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Summary of growth cone turning in response to cPTPRO. (A) Superimposed traces of growth cone trajectories in response to gradients of either cPTPRO–Fc or mIgG-1. The origin represents the position of the growth cone at the gradient onset, and the vertical line represents the original direction of growth (before gradient onset). Large arrows represent the position of the pipette at ∼45° to the original direction of growth. Almost all growth cones tested turned away from the source of cPTPRO–Fc. In contrast, growth cones maintained their original heading in the presence of an IgG gradient. Addition of Sp-cAMPs had no effect on the repulsive effect of cPTPRO, but the addition of 8-Br-cGMP converted cPTPRO-mediated repulsion into attraction. (B) Quantification of the mean final turning angle (± SEM) of the growth cones after a 1-h gradient application. The repulsive effects of cPTPRO–Fc, and the attraction in the presence of 8-Br-cGMP, are clearly shown. *P < 0.05, significantly different from IgG control; **P < 0.01, significantly different from control. Numbers in parentheses indicate the number of growth cones examined.
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fig10: Summary of growth cone turning in response to cPTPRO. (A) Superimposed traces of growth cone trajectories in response to gradients of either cPTPRO–Fc or mIgG-1. The origin represents the position of the growth cone at the gradient onset, and the vertical line represents the original direction of growth (before gradient onset). Large arrows represent the position of the pipette at ∼45° to the original direction of growth. Almost all growth cones tested turned away from the source of cPTPRO–Fc. In contrast, growth cones maintained their original heading in the presence of an IgG gradient. Addition of Sp-cAMPs had no effect on the repulsive effect of cPTPRO, but the addition of 8-Br-cGMP converted cPTPRO-mediated repulsion into attraction. (B) Quantification of the mean final turning angle (± SEM) of the growth cones after a 1-h gradient application. The repulsive effects of cPTPRO–Fc, and the attraction in the presence of 8-Br-cGMP, are clearly shown. *P < 0.05, significantly different from IgG control; **P < 0.01, significantly different from control. Numbers in parentheses indicate the number of growth cones examined.

Mentions: The ability of the cPTPRO ECD to inhibit neurite growth when present on the substrate, combined with its growth cone collapsing activity, suggests the possibility that cPTPRO is a repulsive guidance cue for retinal neurons. To test this possibility directly, we used a growth cone steering assay designed to examine growth cone guidance in vitro. This assay has been used to demonstrate growth cone steering by a variety of guidance proteins, including one type II RPTP (Gundersen and Barrett, 1980; Zheng et al., 1994; Ming et al., 1997; Song et al., 1997; Sun et al., 2000b). Because both cPTPRO and its putative binding partner are expressed by RGCs, we wished to examine the influence of cPTPRO on the growth cones of RGC axons. Therefore, we used retinal explants rather than dissociated neurons for these experiments; the neurites leaving these explants are mainly the axons of RGCs (Barnstable and Drager, 1984; Akagawa and Barnstable, 1986; Sheppard et al., 1988; McLoon and Barnes, 1989). RGC growth cones were subjected to a stable gradient of soluble cPTPRO–Fc using the methods described previously for the type II RPTP, PTP-δ (Sun et al., 2000b). Within 15 min of the cPTPRO gradient being established, growth cones turned away from the source of the cPTPRO gradient (Fig. 9) . Measurement of individual growth cone trajectories revealed that this repulsive turning response was quite consistent (Fig. 10 A), and quantification demonstrated an average turning angle of ∼−20° (n = 22; Fig. 10 B), similar to earlier data using semaphorin IIIA (Song et al., 1998). Control experiments with gradients of mIgG showed no turning effect, demonstrating that the repulsive effect of cPTPRO–Fc was due to the cPTPRO ECD (Fig. 10, A and B).


CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro.

Stepanek L, Sun QL, Wang J, Wang C, Bixby JL - J. Cell Biol. (2001)

Summary of growth cone turning in response to cPTPRO. (A) Superimposed traces of growth cone trajectories in response to gradients of either cPTPRO–Fc or mIgG-1. The origin represents the position of the growth cone at the gradient onset, and the vertical line represents the original direction of growth (before gradient onset). Large arrows represent the position of the pipette at ∼45° to the original direction of growth. Almost all growth cones tested turned away from the source of cPTPRO–Fc. In contrast, growth cones maintained their original heading in the presence of an IgG gradient. Addition of Sp-cAMPs had no effect on the repulsive effect of cPTPRO, but the addition of 8-Br-cGMP converted cPTPRO-mediated repulsion into attraction. (B) Quantification of the mean final turning angle (± SEM) of the growth cones after a 1-h gradient application. The repulsive effects of cPTPRO–Fc, and the attraction in the presence of 8-Br-cGMP, are clearly shown. *P < 0.05, significantly different from IgG control; **P < 0.01, significantly different from control. Numbers in parentheses indicate the number of growth cones examined.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196468&req=5

fig10: Summary of growth cone turning in response to cPTPRO. (A) Superimposed traces of growth cone trajectories in response to gradients of either cPTPRO–Fc or mIgG-1. The origin represents the position of the growth cone at the gradient onset, and the vertical line represents the original direction of growth (before gradient onset). Large arrows represent the position of the pipette at ∼45° to the original direction of growth. Almost all growth cones tested turned away from the source of cPTPRO–Fc. In contrast, growth cones maintained their original heading in the presence of an IgG gradient. Addition of Sp-cAMPs had no effect on the repulsive effect of cPTPRO, but the addition of 8-Br-cGMP converted cPTPRO-mediated repulsion into attraction. (B) Quantification of the mean final turning angle (± SEM) of the growth cones after a 1-h gradient application. The repulsive effects of cPTPRO–Fc, and the attraction in the presence of 8-Br-cGMP, are clearly shown. *P < 0.05, significantly different from IgG control; **P < 0.01, significantly different from control. Numbers in parentheses indicate the number of growth cones examined.
Mentions: The ability of the cPTPRO ECD to inhibit neurite growth when present on the substrate, combined with its growth cone collapsing activity, suggests the possibility that cPTPRO is a repulsive guidance cue for retinal neurons. To test this possibility directly, we used a growth cone steering assay designed to examine growth cone guidance in vitro. This assay has been used to demonstrate growth cone steering by a variety of guidance proteins, including one type II RPTP (Gundersen and Barrett, 1980; Zheng et al., 1994; Ming et al., 1997; Song et al., 1997; Sun et al., 2000b). Because both cPTPRO and its putative binding partner are expressed by RGCs, we wished to examine the influence of cPTPRO on the growth cones of RGC axons. Therefore, we used retinal explants rather than dissociated neurons for these experiments; the neurites leaving these explants are mainly the axons of RGCs (Barnstable and Drager, 1984; Akagawa and Barnstable, 1986; Sheppard et al., 1988; McLoon and Barnes, 1989). RGC growth cones were subjected to a stable gradient of soluble cPTPRO–Fc using the methods described previously for the type II RPTP, PTP-δ (Sun et al., 2000b). Within 15 min of the cPTPRO gradient being established, growth cones turned away from the source of the cPTPRO gradient (Fig. 9) . Measurement of individual growth cone trajectories revealed that this repulsive turning response was quite consistent (Fig. 10 A), and quantification demonstrated an average turning angle of ∼−20° (n = 22; Fig. 10 B), similar to earlier data using semaphorin IIIA (Song et al., 1998). Control experiments with gradients of mIgG showed no turning effect, demonstrating that the repulsive effect of cPTPRO–Fc was due to the cPTPRO ECD (Fig. 10, A and B).

Bottom Line: We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons.This chemorepulsive effect could be regulated by the level of cGMP in the growth cone.Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.

Show MeSH
Related in: MedlinePlus