Fig 2: Axonal FTO regulates local translation of GAP-43 in axons. (A and B) DRG neurons were cultured in vitro and bath application of rhein led to a decrease of GAP-43 IF signals in axons (Vehicle, n = 15 axons; Rhein, n = 14 axons). (C and D) Axon-specific treatment of rhein in severed axons resulted in a significant decrease of GAP-43 IF signals in axons (Vehicle, n = 10 axons; Rhein, n = 12 axons). (E and F) Loss-of-function of FTO by rhein (E) or siFto knockdown (F) did not change GAP-43 mRNA levels by qRT-PCR (n = 3). (G and H) Co-application of rhein with MG-132 in severed axons led to a significant decrease of GAP-43 IF signals in axons compared with MG-132 treatment alone (MG-132 + Vehicle, n = 11 axons; MG-132 + Rhein, n = 11 axons). Data are mean ± s.e.m. IF quantification data (B, D and H) are represented as box and whisker plots: 25th-75th percentiles (boxes), minimum and maximum (whiskers) and medians (horizontal lines). ***P = 2.76E-8 (B); ***P = 0.0002 (D); **P = 0.0026 (H); by unpaired t-test. qRT-PCR data (E and F) are represented as dot plots. n.s., not significant (unpaired t-test for E, one-way ANOVA followed by Tukey’s multiple comparison test for F). Each experiment was performed in triplicate (C, E, F and G) or quadruplicate (A). Scale bars, 10 µm (A, C and G).

Fig 3: Axonal FTO de-(m6A)methylates GAP-43 mRNA in axons. (A and B) Modification of GAP-43 mRNA by m6A. Total RNA from mouse DRG (A) or spinal cord (B) was immunoprecipitated with a specific m6A antibody or an IgG control. In either of these two tissues, GAP-43 mRNA was pulled down and detected by RT-PCR. (C) Mutation of m6A site eliminated m6A modification in GAP-43 mRNA. Wild-type (‘WT’) GAP-43 or a mutated form which has the predicted m6A site mutated (‘MTm6A’) were overexpressed in DRG neurons. RNA was purified after rhein treatment for 4 h. After anti-m6A IP, much less GAP-43 mRNA was detected from ‘MTm6A’ neurons, compared with ‘WT’ neurons. (D) Rhein treatment led to increase of m6A modification in GAP-43 mRNA. More GAP-43 mRNA was pulled down by anti-m6A IP in rhein-treated DRG neurons compared with vehicle control. (E and F) Mutation of m6A site in GAP-43 mRNA could rescue inhibition of GAP-43 local translation and axon elongation by rhein. ‘WT’ or ‘MTm6A’ GAP-43 was overexpressed in DRG neurons and rhein was specifically applied to axons in microfluidic chambers. Axon-specific treatment of rhein in ‘WT’ GAP-43-overexpressing DRG neurons led to significant decreases of GAP-43 protein levels (E) and axon growth rate (F) compared with vehicle control. These decreases were not observed with axonal rhein treatment of ‘MTm6A’ GAP-43-overexpressing DRG neurons (E and F). For E: WT, n = 15 axons; WT + Rhein, n = 10 axons; MTm6A + Rhein, n = 8 axons. For F: WT, n = 7 axons; WT + Rhein, n = 5 axons; MTm6A + Rhein, n = 6 axons. Data are mean ± s.e.m. Data are represented as box and whisker plots: 25th–75th percentiles (boxes), minimum and maximum (whiskers), and medians (horizontal lines). For E: WT versus WT + Rhein, *P = 0.01006; WT + Rhein versus MTm6A + Rhein, *P = 0.02977. For F: WT versus WT + Rhein, *P = 0.04229; WT + Rhein versus MTm6A + Rhein, *P = 0.01899; by one-way ANOVA followed by Tukey’s multiple comparison test. Each experiment was performed in triplicate.

Fig 4: Therapeutic potential of small-molecule FTO inhibitors.Equal number of CFPAC (A), AsPC-1 (B), and BxPC-3 (C) cells were subcutaneously transplanted into SCID-Beige mice. FB23-2 (10 mg/kg) or same volume of dimethyl sulfoxide was injected intraperitoneally into mice every other day. The mean tumor volume is plotted over time, and the tumor weight was measured at the end point. Photo credit, L. Dong. Error bars, means ± SEM; *P < 0.05, **P < 0.01, unpaired two tailed t test; n = 6.

Fig 5: Axon-specific loss-of-function of FTO increased m6A signals in axons and inhibited axon elongation. DRG explants were cultured in microfluidic chambers. After axons grew to the axonal compartment, different experiments were performed. (A and B) Axonal rhein treatment led to a significant increase of m6A signals in axons (vehicle, n = 12 axons; rhein, n = 10 axons). Before rhein treatment, axons were severed from explants to prevent any further transport of m6A modified RNA from soma. (C and D) Rhein treatment of intact axons resulted in a significant reduction of axon growth rate compared with vehicle treatment (vehicle, n = 16 axons; rhein, n = 17 axons). (E and F) Compartmentalized Fto knockdown in intact axons using two different siFto in axons led to significant increase of axonal m6A levels compared with siNC (siNC, n = 17 axons; siFto-1, n = 15 axons; siFto-2, n = 15 axons). (G) Compartmentalized Fto knockdown in intact axons significantly inhibited axon elongation (siNC, n = 8 axons; siFto-2, n = 10 axons). Data are mean ± s.e.m. All data are represented as box and whisker plots: 25th–75th percentiles (boxes), minimum and maximum (whiskers) and medians (horizontal lines). For B, D and G: *P = 0.0300 (B); ***P = 2.29E-6 (D); ***P = 1.089E-4 (G); by unpaired t-test. For F: *P = 0.038, ***P = 7.49E-4; by one-way ANOVA followed by Tukey’s multiple comparison test. Each experiment was performed in triplicate. Scale bars, 10 µm (A, C and E).