Fig. 6: Elevated intracellular Ca2+ levels rescue the axonal and mitochondrial phenotypes in Cyfip1+/- neurons. | Nature Communications

Fig. 6: Elevated intracellular Ca2+ levels rescue the axonal and mitochondrial phenotypes in Cyfip1+/- neurons.

From: CYFIP1 governs the development of cortical axons by modulating calcium availability

Fig. 6

a Left, representative images of axons growing in the axonal compartment of microfluidic devices at day in vitro 3 (DIV 3) and DIV 4 for WT and Cyfip1+/- neurons treated with DMSO or Ionomycin (IONO, 1 µM). Scale bar 200 µm. Axonal growth rate was quantified (black line, DIV 3; red line, DIV 4). Right, average axonal growth rate in WT and Cyfip1+/- neurons treated with DMSO or Ionomycin, expressed as a percentage over WT-DMSO (WT n = 7 embryos, 149 DMSO-treated axons and 143 Ionomycin-treated axons; Cyfip1+/- n = 6 embryos, 121 DMSO-treated axons and 148 Ionomycin-treated axons; mean ± SEM; Two-Way ANOVA, F(1,22) = 4.326, p = 0.0494; genotype effect p < 0.0001, treatment effect p = 0.0420, interaction p = 0.0494; Sidak’s multiple comparisons test, WT-DMSO vs. Cyfip1+/--DMSO p = 0.0392, Cyfip1+/--DMSO vs. Cyfip1+/--IONO p < 0.0001, WT-DMSO vs. WT-IONO p = 0.0027). b Left, representative kymographs showing mitochondrial transport along axons of WT and Cyfip1+/- DIV 4 cortical neurons treated with DMSO or Ionomycin (1 µM). Scale bar 10 μm. Right, histogram shows percentage of motile mitochondria in WT and Cyfip1+/- axons treated with DMSO or Ionomycin (WT n = 3 embryos, 195 DMSO-treated mitochondria and 157 Ionomycin-treated mitochondria; Cyfip1+/- n = 7 embryos, 393 DMSO-treated mitochondria and 398 Ionomycin-treated mitochondria; mean ± SEM; Two-Way ANOVA, F(1;16) = 8.386, p = 0.0105; treatment effect p = 0.0134, genotype effect p = 0.0091, interaction p = 0.0105; WT-DMSO vs. Cyfip1+/--DMSO p = 0.0045, WT-IONO vs. Cyfip1+/--IONO p > 0.9999). c Left, representative images of TMRE intensity in axonal mitochondria of WT and Cyfip1+/- neurons treated with DMSO or Ionomycin (1 µM). Scale bar 2.5 μm. Right, average TMRE intensity expressed as a percentage over WT-DMSO (WT n = 3 embryos, Cyfip1+/- n = 5 embryos; mean ± SEM; Two-Way ANOVA, F(1,12) = 16.87, p = 0.0015; treatment effect p = 0.0866, genotype effect p = 0.0014, interaction p = 0.0015; WT-DMSO vs. Cyfip1+/--DMSO p = 0.0004, WT-IONO vs. Cyfip1+/--IONO p > 0.9999). d Left, representative images of axons at DIV 3 and DIV 4 for WT and Cyfip1+/- neurons treated with DMSO, (S)-(-)-Bay-K-8644 (50 µM) or Nefiracetam (10 µM). Scale bar 200 µm. Right, average axonal growth rate expressed as a percentage over WT-DMSO (WT n = 5 embryos, 142 DMSO-treated axons, 190 Bay-K-8644-treated axons and 189 Nefiracetam-treated axons; Cyfip1+/- n = 7 embryos, 150 DMSO-treated axons, 258 Bay-K-8644-treated axons and 207 Nefiracetam-treated axons; mean ± SEM; Two-Way ANOVA, F(2,30) = 5.8, p = 0.0070; genotype effect p = 0.0003, treatment effect p < 0.0001, interaction p = 0.0070; Sidak’s multiple comparisons test, WT-DMSO vs. Cyfip1+/--DMSO p = 0.0002, Cyfip1+/--DMSO vs. Cyfip1+/--Bay-K-8644 p < 0.0001, Cyfip1+/--DMSO vs. Cyfip1+/--Nefiracetam p < 0.0001, WT-Bay-K-8644 vs. Cyfip1+/--Bay-K-8644 p = 0.9824, WT- Nefiracetam vs. Cyfip1+/--Nefiracetam p = 0.8057). Source data are provided as a Source Data file.

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