Fig. 5: pNF-associated NF1-mutant PNS neurons exhibit increased activity and COL1A2-dependent preneoplastic NF1^−/− Schwann cell growth.

A, B Nf1^+/neo, but not Nf1^+/1809, DRG neuron AP firing rates are elevated relative to WT DRG neurons, as measured by (A) multi-electrode array (WT, n = 24, Nf1^+/neo, n = 10; P = 0.0005, Nf1^+/1809 n = 10, ns), or (B) calcium imaging recordings (WT n = 8, Nf1^+/neo n = 5, P < 0.0001, Nf1^+/1809 n = 14, ns). C, D TTX (1 µM) and lamotrigine (LTR; 200 µM) reduce Nf1^+/neo DRG neuron AP firing rate as measured by multi-electrode array (vehicle n = 4, TTX n = 7, P < 0.0001; LTR n = 6, P < 0.0001) and calcium imaging (vehicle n = 23, TTX n = 9, P < 0.0001, LTR n = 14, P < 0.0001). The right panels show representative (A, C) spike plots of entire multi-electrode array well recordings over 30 s, and (B, D) traces of neuronal activity over 3 min. E Schematic illustrating treatment of human shNF1 Schwann cells with hiPSC-sensory neuron conditioned media (CM). NF1-deficient Schwann cell proliferation is increased after treatment with NF1^C383X, NF1^R681X, and NF1^E2207X mutant neuron CM (P < 0.0001), but not NF1^R1809C neuron CM relative to controls (CTL). n = 6 for all groups. F Analytical comparison of 2D gel electrophoresis (top-to-bottom: decreasing molecular weight; left-to-right: decreasing acidity) of NF1^R681X (left) and NF1^R1809C (right) CM relative to CTL hiPSC-sensory neuron CM. Red dots indicate proteins with increased expression, green dots indicate proteins with decreased expression, and yellow dots indicate unaltered proteins in NF1-mutant sensory neuron CM relative to CTL neuron CM. The six proteins uniquely increased more than 1.5-fold in NF1^R681X hiPSC-sensory neuron CM relative to CTL, but not in NF1^R1809C CM, relative to CTL are circled in blue and are listed in the lower panel. Representative CM from CTL, NF1^R1809C, and NF1^R681X sensory neurons was analyzed by 2D gel electrophoresis (n = 1). G, H COL1A2 levels are increased in (G) NF1^C383X, NF1^R681X, and NF1^E2207X mutant neuron CM (P < 0.0001), but not in NF1^R1809C neuron CM (n = 4 for all groups), as well as in (H) Nf1^+/neo mouse DRG neuron CM (P < 0.0001), but not in Nf1^+/1809 mouse DRG neuron CM (n = 6 for all groups). I Nf1-deficient DRG-NSC proliferation is increased after treatment with Nf1^+/neo DRG neuron CM (P < 0.0001), but not Nf1^+/1809 DRG neuron CM, relative to WT controls. n = 6 for all groups. Data are presented as the mean ± SEM. A–E, G–I One-way ANOVA with (A–D, G–I) Dunnett’s, or (E) Tukey’s multiple comparisons test. P values are indicated within each panel. ns, not significant. Source data are provided as a Source Data file.