Fig 1: Fabry Schwann cells release the protein p11 (S100A10), which alters isolated DRG neuron function and induces pain-like behaviors in rats.(A) NanoLC-MS/MS analysis was performed on Fabry- and WT-SCM. Fabry-SCM contained significantly more p11 (S100-A10) compared with WT samples; refer to Supplemental Table 4 for a list of the other significantly altered proteins. (B) Representative immunofluorescence images of DRG neuron soma incubated with 100 ng/mL of His-tagged recombinant p11 or without p11 (CTRL) (scale bar = 20 μm). (C) Representative calcium imaging traces of 0 ng/mL and 100 ng/mL p11 incubated neurons exposed to 20 mM KCl for 10 seconds, 50 mM KCl used as a positive control. Traces are representative from 3 animals. (D) Significantly more neurons incubated with soluble p11 at various concentrations (0.5–1,000 ng/mL) responded to mild depolarization with 20 mM KCl as assessed using calcium imaging. (E) Neurons incubated with soluble p11 exhibited increased 20 mM KCl–induced calcium influx. (F) Intraplantar injection of p11 into naive Sprague-Dawley rats decreased hind paw withdrawal thresholds to von Frey stimulation. (A) n = SCM samples from 5 animals per genotype, plotted per animal; (B) representative images, 4 independent DRG cultures; (C–E) n = 140–160 neurons per treatment from 3 animals; (F) n = 8 animals per dose. (A, E, and F) reported as mean ± SEM, (D) reported as mean. (A) Benjamini-Hochberg–corrected 2-tailed Student’s t test, (C) χ2 with corrected Fisher’s exact post hoc comparison, (E) 1-way ANOVA with Bonferroni post hoc comparison, (F) 2-way repeated measures ANOVA with Bonferroni post hoc comparison. * P < 0.05, ** P < 0.01, *** P < 0.001. BL, preinjection baseline behavioral measurements; SCM, Schwann cell–conditioned media.
Fig 2: p11 contributes to the resting membrane depolarization of naive DRG neurons treated with Fabry-SCM.(A) Naive neurons exposed to 100 ng/mL p11 exhibited significantly depolarized RMPs. (B) Incubation of p11 reduced rheobase in naive neurons. (C) Current protocol and representative traces of neurons undergoing current stimulation of 200 pA above rheobase for 500 ms. (D) Neurons exposed to p11 exhibit increased firing frequency to suprathreshold current stimulation compared with CTRL. (E and F) Incubation with 100 ng/mL p11 enhanced peak sodium current densities. (G) The maximum inward current density was higher in neurons exposed to p11, reported as absolute values. Mean capacitance values (pF ± SEM) for neurons tested were 25.5 ± 2.0 and 23.5 ± 1.9 for CTRL and p11-incubated neurons, respectively. (H) p11 immunodepletion protocol from Fabry-SCM media for electrophysiology studies in naive DRG neurons. (I) Immunodepletion reduced p11 concentration of Fabry-SCM by 86%, with WT-SCM exhibiting concentrations below the limit of detection as measured by ELISA. (J) Naive neurons exposed to Fabry-SCM-ID exhibited a hyperpolarized RMP compared with neurons treated with Fabry-SCM. Values reported as mean ± SEM. (A–D) n = 28 neurons per treatment from 7 animals; (E–G) n = 26 neurons per treatment from 8 animals; (I) n = 5 WT and Fabry Schwann cell cultures from individual animals; (J) n = 31 neurons per treatment from 8 animals. (A, B, G, and J) Unpaired Student’s t test, (D and F) 2-way repeated measures ANOVA with main effect of treatment, (I) 2-way ANOVA, Bonferroni post hoc comparison. * P < 0.05, ** P < 0.01. SCM, Schwann cell–conditioned media; SCM-ID, Schwann cell–conditioned media with immunodepleted p11; AP, action potential.
Supplier Page from Aviva Systems Biology for S100A10 Recombinant Protein (OPCD06771)