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Negatively targeting the tumor suppressor and phosphoinositide phosphatase PTEN (phosphatase and tensin homologue) promotes axon regrowth after injury. How PTEN functions in axon guidance has remained unknown. Here we report the differential role of PTEN in chemotactic guidance of axonal growth cones. Down-regulating PTEN expression in Xenopus laevis spinal neurons selectively abolished growth cone chemorepulsion but permitted chemoattraction. These findings persisted during cAMP-dependent switching of turning behaviors. Live cell imaging using a GFP biosensor revealed rapid PTEN-dependent depression of phosphatidylinositol 3,4,5-trisphosphate levels in the growth cone induced by the repellent myelin-associated glycoprotein. Moreover, down-regulating PTEN expression blocked negative remodeling of β1-integrin adhesions triggered by myelin-associated glycoprotein, yet permitted integrin clustering by a positive chemotropic treatment. Thus, PTEN negatively regulates growth cone phosphatidylinositol 3,4,5-trisphosphate levels and mediates chemorepulsion, whereas chemoattraction is PTEN-independent. Regenerative therapies targeting PTEN may therefore suppress growth cone repulsion to soluble cues while permitting attractive guidance, an essential feature for re-forming functional neural circuits.
FIGURE 1. Down-regulating PTEN selectively blocks MAG-induced repulsion.
A, Xenopus growth cones immunostained for Akt substrate phosphorylation (pAkt sub) under control conditions, with PTEN-MO alone and PTEN-MO plus wild-type rescue (PTEN-MO + WT). Scale bar, 5 μm. B, quantification of relative Akt substrate phosphorylation immunofluorescence normalized to the control condition. C and D, example images and summary plots depict the final position of wild-type and PTEN-MO growth cones relative to the starting position (origin) after 30 min of exposure to a gradient (arrows) of MAG (C) or BDNF (D). Scale bar, 10 μm. Asterisks denote growth outside of the plot. E, cumulative distribution of growth cone turning angles in response to a gradient of medium (Control), MAG, or BDNF either alone or with PTEN-MO and PTEN-MO plus wild-type PTEN (MO + WT). F, mean turning angles and growth rates from all experiments. All data are expressed as mean ± S.E. (n = number associated with each bar; *, p < 0.05; NS, no significant difference, t test (B) and Mann-Whitney U test (F)).
FIGURE 2. Differential function of PTEN during cAMP-dependent conversion of turning responses.
A and B, summary plots depict the final position of wild-type and PTEN-MO growth cones relative to the starting position (origin) after 30 min of exposure to a gradient of MAG (A) and BDNF (B) in the presence of (Sp)-cAMPS and (Rp)-cAMPS, respectively. Scale bar, 10 μm. Asterisks denote growth outside of the plot. C, cumulative distribution of growth cone turning angles for all experiments as in A and B. D, mean turning angles and growth rates for all experiments. All data are mean ± S.E. (n = number associated with each bar; *, p < 0.05; NS, no significant difference, Mann-Whitney U test). The control group in C and D is the same as in Fig. 1, E and F.
FIGURE 3. MAG induces PTEN-dependent depression of PIP3 levels.
A–C, pseudocolor time-lapse confocal images of PHAkt-GFP standard-corrected for growth cone thickness during basal conditions (A, n = 6; see also supplemental Movie S1) and with MAG treatment (150 ng/ml; starting at time = 0) either alone (B, n = 6; see also supplemental Movies S2–S4) or after down-regulating PTEN expression (C, PTEN-MO, n = 7; see also supplemental Movie S5). Time is in minutes. White is highest fluorescence intensity. Scale bar, 5 μm. D, quantification of change in fluorescence intensity as compared with the mean intensity prior to treatment (F/F0). Treatment groups are significantly different (p = 0.0029, repeated measures two-way ANOVA).
FIGURE 4. MAG-induced β1-integrin internalization and inhibition of clustering is PTEN-dependent.
A, representative immunofluorescence images show the distribution of β1-integrin in the growth cone of wild-type and PTEN-MO neurons after experimental treatments: control (BSA), MAG (1 μg/ml; 5 min), or (Sp)-cAMPS (20 μm) + MAG. Arrowheads designate clustered β1-integrin. Scale bar, 5 μm. B and C, summary graphs show the quantification of β1-integrin surface levels (B) and β1-integrin clustering (C) for all experimental conditions. Data are the mean ± S.E. (n > 150, *, p < 0.05, one-way ANOVA with a Tukey's post hoc analysis).
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