Fig. 2.
Inhibition of PIKfyve but not of PI3K (Vps34) ameliorates myelin outfoldings in Mtmr2 KO Schwann cell/DRG neuron coculture explants. (A) Representative fluorescence microscopy images of Mtmr2 KO Schwann cell/DRG neuron coculture explants treated every other day for 14 d using 300 nM apilimod, which is the highest dose that does not interfere with myelination, as assessed by measuring the number of Mbp-positive segments and Schwann cell nuclei and quantified in A′ and A′′, respectively. Mbp-positive segments: n = 6 DRG/coverslips DMSO-treated and n = 6 DRG/coverslips apilimod-treated, two-tailed Mann–Whitney U test, P = 0.937. Schwann cell nuclei, n = 3 DRG/coverslips DMSO-treated and n = 3 apilimod-treated, two-tailed Mann–Whitney U test, P = 0.700. On the Left, representative images of the costaining of Lamp1 and Phalloidin (F-actin) after 3 d of ascorbic acid treatment in differentiating conditions and following 90 min of apilimod treatment qualitatively demonstrate efficacy of PIKfyve inhibition. Enlargement of the Lamp1-positive late endosome/lysosomal compartment was observed, which is a well-established readout of PIKfyve inhibition and of decreased PtdIns(3,5)P2 levels (20). (B) Representative confocal microscopy images of Mtmr2 KO Schwann cell/DRG neuron coculture explants with myelin outfoldings treated every other day using 300 nM apilimod for 14 d in differentiating conditions. Apilimod reduces the percentage of Mbp-positive fibers carrying myelin outfoldings on the total number of Mbp-positive fibers, as quantified in B′, n = 9 DRG/coverslips DMSO-treated, 325 fibers analyzed and n = 8 DRG/coverslips apilimod-treated, 296 fibers analyzed, two-tailed Mann–Whitney U test, ***P = 0.0003. Representative results from three independent experiments. (C) Representative bright-field images of cocultures treated using compound 19 (PI3K-VPS34 inhibitor 1) at 10 µM for 3 h qualitatively show efficacy of PI3K-Vps34 inhibition. Vacuolization-enlargement of endosomal compartments in a fibroblast as well as in Schwann cells can be observed, which is a well-established readout of PI3K inhibition. (D) Compound 19 treatment of Mtmr2 KO Schwann cell/DRG neuron coculture explants every other day for 14 d in differentiating conditions does not interfere with myelination as indicated by similar numbers of Mbp-positive fibers and Schwann cell nuclei between DMSO- and compound 19-treated cultures, and quantified in D′ and D′′, respectively. Mbp-positive segments: n = 4 DRG/coverslips DMSO-treated and n = 4 DRG/coverslips compound 19-treated, two-tailed Mann–Whitney U test, P > 0.9999. Schwann cell nuclei, n = 3 DRG/coverslips DMSO-treated and n = 3 compound19-treated, two-tailed Mann–Whitney U test, P = 0.700. (E) The percentage of Mbp-positive fibers carrying myelin outfoldings on the total number of Mbp-positive fibers in Mtmr2 KO Schwann cell/DRG neuron coculture explants were not reduced by compound 19 treatment, as quantified in E′ and displayed in confocal microscope representative images, n = 4 DRG/coverslips DMSO-treated, 207 fibers analyzed, and n = 4 DRG/coverslips compound 19-treated, 203 fibers analyzed, two-tailed Mann–Whitney U test, P = 0.1143. Results are mean ± SEM. Mbp, myelin basic protein, which stains myelinated segments; Nf, neurofilament, which stains axons and neurites. DAPI-positive nuclei are from Schwann cells, as DRG neurons are not dissociated and the cell bodies remain at the center of the organotypic explants, where myelination is not quantified. (Scale bar in A, 26 µm for LAMP1 staining and 116 µm for Mbp and DAPI staining; in B, 46 µm; in C, 64 µm; in D, 112 µm, and in E, 39 µm.)