Fig 2: Pharmacological inhibition of sortilin prevents mechanical allodynia.(A) PWT to tactile stimuli of WT and Sort1-/- mice before and after SNI and intrathecal AF38469 (active) or AF38540 (inactive) injection. ****P < 0.0001 versus the ipsilateral side of WT + AF38540; n = 13 to 17, two-way RM ANOVA with post hoc Dunnett’s test [F(5,66) = 17.75, P < 0.0001], means ± SEM. (B) PWT to tactile stimuli of WT mice after intrathecal AF38469 (active) or AF38540 (inactive) injection 2 days after SNI. (C) Percentage of anti-allodynia by AF38469 1, 2, and 3 hours versus pre-injection baseline (day 2 of the same mouse). ***P < 0.0007; n = 9, one-way RM ANOVA with post hoc Dunnett’s test [F(3,24) = 8.597, P = 0.0005], means ± SEM. (D) Percentage of anti-allodynia by AF38540 1, 2, and 3 hours versus pre-injection baseline (day 2 of the same mouse). n = 6, one-way RM ANOVA with post hoc Dunnett’s test, means ± SEM. (E) PWT to tactile stimuli of WT mice before and after intrathecal BDNF ± AF38469 (active) injection. *P < 0.03, ***P < 0.0004, and ****P < 0.0001; n = 6 to 7, two-way RM ANOVA with post hoc Sidak’s test [F(1,11) = 19, P = 0.0011], means ± SEM. (F) PWT to tactile stimuli of hum-sortilin-KI mice after intrathecal AF38469 (active) or AF38540 (inactive) injection 2 days after SNI. ****P < 0.0001 versus ipsilateral side with AF38540; n = 8, two-way RM ANOVA with post hoc Dunnett’s test [F(3,28) = 45.67, P < 0.0001, means ± SEM.

Fig 3: Schwann cells of the sciatic nerve express sortilin. (A) Double-labeled immunofluorescence microscopy of sortilin (green) and s100ß (red) in longitudinal sections of wild-type (WT) naïve sciatic nerve. Nuclei are labeled with Hoechst (blue). White arrows demonstrate Schwann cells expressing sortilin. Scale bar 50 µm. (B) Double-label immunofluorescence microscopy of sortilin (green) and s100ß (red; Schwann cell marker) in longitudinal sections of naïve sciatic nerves and 5 days after crush injury in WT and Sort1–/– distal sciatic nerves. Nuclei are labeled with Hoechst (blue). The white arrow points to a sortilin-positive (green) s100ß-positive (red) WT Schwann cell. Scale bar 100 µm upper images and 50 µm lower images.

Fig 4: KCC2 down-regulation is prevented in sortilin-deficient mice.(A) Paw withdrawal threshold (PWT) to tactile stimuli of ipsilateral versus contralateral sides of WT and Sort1-/- mice before and after SNI (day 0). *P < 0.02, **P < 0.009, and ****P < 0.0001; n.s., not significant; n = 7 to 8, two-way repeated measures (RM) analysis of variance (ANOVA) with post hoc Tukey’s test [F(3,26) = 31.52, P < 0.0001], means ± SEM. (B) Representative Western blot of KCC2 in L3-L5 SDH 6 days after SNI. (C) KCC2 levels in L3-L5 SDH quantified by Western blot and normalized to WT contralateral 6 days after SNI. n = 6, one-way RM ANOVA with post hoc Tukey’s test [F(1.997,9.985) = 15.06, P = 0.001], means ± SEM. (D and E) IHC analysis showing IB4, NeuN, and KCC2 expression in the ipsilateral and contralateral SDH of WT and Sort1-/- mice. Scale bar, 100 µm. (F and G) Comparisons of average pixel intensity are shown across SNI animals of WT versus Sort1-/- mice in the region of interest (ROI). Nerve injury resulted in decreased IB4 intensity in the ROI in WT mice (contralateral versus ipsilateral: paired t test, t = 3.749; df = 18, P = 0.0015; n = 19) as in Sort1-/- mice (contralateral versus ipsilateral: paired t test, t = 4; df = 8, P = 0.004; n = 9). Nerve injury caused the down-regulation of KCC2 expression in the dorsal horn of WT mice but not in Sort1-/- mice [contralateral versus ipsilateral: (WT mice) paired t test, t = 6.24; df = 18, P < 0.0001; n = 19; and (Sort1-/- mice) t = 0.2093; df = 8, P = 0.839; n = 9]. No loss of neurons, measured as the difference in the average NeuN immunostaining intensities, was observed between ipsilateral and contralateral sides in both WT and Sort1-/- mice [contralateral versus ipsilateral: (WT mice) paired t test, t = 1.206; df = 18, P = 0.2436; n = 19; and (Sort1-/- mice) t = 0.3838; df = 8, P = 0.7111; n = 9]. **P < 0.01 and ***P < 0.0001; intensity units (i.u.) are shown as means ± SEM. (H) BDNF levels 6 days after SNI in L3-L5 SDH relative to WT contralateral [n = 3, pooled samples from eight mice for each run, paired t test within genotype (WT: t = 13.42, df = 2; Sort1-/-: t = 4.62, df = 2) and unpaired t test between genotypes (means ± SEM)].

Fig 5: Sortilin-KO mice have accelerated prion disease with earlier accumulation of PrPSc in their brains.(A) Kaplan-Meier survival curves for Sort1-/- and Sort1+/+ female mice inoculated with RML prions. Data was statistically analyzed using the log-lank test (Sort-/-, n = 24; Sort+/+, n = 19). *** p < 0.001. (B) Western blotting for PrPSc in the brains of Sort1-/- and Sort1+/+ female mice 45, 60, 90 dpi and at terminal stage. Sortilin was also detected by Western blotting. (C) Quantification of PrPSc in (B) after normalization against ß-actin. Signal intensity in Sort1-/- mice was evaluated against that in Sort1+/+ mice. Data are means ± SD of 4–5 independent brains. n.s, not significant; * p < 0.05, ** p < 0.01. (D) Immunohistochemical staining of PrPSc in the hippocampus of Sort1-/- and Sort1+/+ female mice at 60 and 90 dpi and at terminal stage. PBS-inoculated mice brain hippocampus was also stained as a control. The brains removed from PBS-inoculated Sort1-/- mice aged 16, 20, and 30 weeks were used as controls for 60 and 90 dpi and terminal stage, respectively. Bar, 300 µm.