Fig 2: Effect of EA on GAP-43 in rats with cerebral ischemia injury. The protein expression and mRNA levels of GAP-43 in ischemic cerebral tissues on day 3, 7 and 14 were determined by western blot and reverse-transcription quantitative polymerase chain reaction analyses. Values are expressed as the mean ± standard deviation from 6 independent experiments. #P<0.05 vs. SC group; *P<0.05 vs. IC group. SC, sham-operated control; IC, ischemic control; EA, electroacupuncture; GAP, growth-associated protein.

Fig 3: Model depicting the proposed molecular mechanism underlying HuD regulation by FMRP and mutant FUS.The figure depicts a model of the competition between mutant FUS and FMRP for HuD 3'UTR binding. In FUSWT MNs, the FUS protein is predominantly localized in the nucleus. In the cytoplasm, FMRP binds HuD 3'UTR repressing its translation. NRN1 mRNAs are destabilized. In FUSP525L MNs, mutant FUS is partially delocalized to the cytoplasm and outcompetes FMRP binding on the HuD 3'UTR. As a consequence, increased HuD protein levels accumulate in FUS mutant MNs. HuD binding to NRN1 and GAP43 3'UTR leads to stabilization of these transcripts and higher protein levels. NRN1 increase underlies the aberrant axonal growth phenotypes.

Fig 4: GAP43 levels are increased in mutant FUS MNs.a GAP43 mRNA analysis by FISH (red) in FUSWT, FUSP525L, and FUSWT overexpressing HuD under the Syn1 promoter (FUSWT + HuD) hiPSC-derived spinal MNs. The graphs show the average count of HuD mRNA puncta per cell from three independent differentiation experiments, error bars indicate the standard error of the mean (Student’s t-test; unpaired; two tails). DAPI (blue) was used for nuclear staining. Scale bar: 10 µm. b GAP43 protein levels analysis by western blot in FUSWT and FUSP525L hiPSC-derived spinal MNs. The molecular weight (kDa) is indicated on the left. The graph shows the average from three independent differentiation experiments and error bars indicate the standard deviation (Student’s t-test; paired; two tails). Values in the y-axis represent GAP43/TUBB3 signal intensity. c Gap43 protein level analysis by western blot in mouse primary spinal MNs (P81). The molecular weight (kDa) is indicated on the left. The graphs show the average from three mice and error bars indicate the standard deviation. The differences are not significant for all pairs (ordinary one-way ANOVA; multiple comparisons). Values in the y-axis represent Gap43/Tubb3 signal intensity. d Immunostaining analysis in FUSWT and FUSP525L hiPSC-derived spinal MNs growth cones. GAP43 signal is magenta; PHALLOIDIN signal (marking growth cones) is green, TYR-TUBULIN (tyrosinated alpha-tubulin; marking axons) is white. Scale bar: 10 µm. The graph shows the GAP43 signal intensity from three differentiation experiments, error bars indicate the standard error of the mean (Student’s t-test; paired; two tails).