NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells (2016)
Figure 10

Figure 10. PTPσ is involved in the signaling cascade mediated by NB-3 trans-homophilic interactions.

A Co-immunostaining of PTPσ (blue), NB-3 (green), and BDA (red) at spinal lesion sites of NB-3+/+ (A1) and NB-3−/− (A6) mice. (A2–A5 and A7–A10) High-magnification images of boxed areas in (A1 and A6) showing PTPσ expression at the terminations of BDA-labeled corticospinal axons, respectively. Images in (A2 and A7) are presented as single-channel fluorescence images in (A3–A5 and A8–A10), respectively.

B Quantification of fluorescence intensity of PTPσ at BDA-labeled axonal terminations of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05; one-way ANOVA followed by Bonferroni post-test. n = 12 mice per group.

C Co-immunostaining of PTPσ and NB-3 in the FG-labeled corticospinal neurons (arrowheads) of NB-3+/+ and NB-3−/− mice 14 days post-injury (dpi). Insets, only the signals from PTPσ and FG in NB-3+/+ corticospinal neurons (red arrowheads) were visualized.

D Quantification of fluorescence intensity of PTPσ in (C). *p < 0.05; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.

E Co-immunoprecipitation of NB-3 and PTPσ from sensorimotor cortical lysates of NB-3+/+ mice after SCI.

F Quantification of immunoprecipitated PTPσ by NB-3 in (E). **p < 0.01; one sample t-test. Data were analyzed from 3 independent experiments each including 15 mice per group.

G PTPσ expression in NB-3+/+ and NB-3−/− pyramidal neurons when co-culturing with NB-3–expressing astrocytes (dashed lines and asterisks) or astrocytes that didn't express NB-3 (dashed lines). Upper inset, high-magnification view of the soma (white arrowhead). Lower inset, high-magnification view of the axon tip (green arrowhead).

H Quantification of fluorescence intensity of PTPσ in (G). n.s., not significant, and *p < 0.05; one-way ANOVA followed by Bonferroni post-test.

I Expression of PTPσ, p-Akt, p-mTOR, and p-S6 (S240/244) in pyramidal neurons that expressed PTPσ shRNA-GFP. A control plasmid expressing GFP was transfected as the negative control (nc).

J Quantification of fluorescence intensity of PTPσ, p-Akt, p-mTOR, and p-S6 (S240/244) in (I). *p < 0.05; one sample t-test. The intensities of more than 300 pyramidal neurons from 4 independent experiments in each group in (H) and (J) were quantified, respectively.

K–M Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (K) and LV-PTPσ shRNA-GFP (L) into the sensorimotor cortex. High-magnification image (M) of the area in (L) (asterisk). The arrows indicate the lesion sites; the asterisk indicates the lesion epicenter.

N Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (K and L). *p < 0.01; two-way ANOVA followed by Fisher’s LSD. n = 8 mice in (K), and n = 10 mice in (L).

O A model illustrating the signal transduction between scar-forming cells and post-traumatic axons mediated by trans-homophilic NB-3 interactions.

Data are represented as mean ± SEM (B, D, F, H, J, and N). Scale bars, 500 µm (A1 and A6), 40 µm (A2–A5 and A7–A10), 20 µm (C), 10 µm (G), 10 µm (I), 400 µm (K and L), and 40 µm (M).