PI(3,5)P₂ biosynthesis regulates oligodendrocyte differentiation by intrinsic and extrinsic mechanisms

Essential revisions:

1) No g ratio analysis is shown to make it clear whether Fig4 null oligodendrocytes form normal myelin sheath.

We agree with the reviewers’ comment and have included g-ratio analysis for the Fig4 conditional knock-out (cKO) mice lacking Fig4 in neurons (SynCre) or in the OL lineage (Olig2Cre) mice. In both cKO mice, g ratios are increased, indicating that myelin sheath thickness is reduced. These data are now shown in Figure 2E.

2) The paper demonstrates that absence of other components of the Fig4 complex, PIKFYVE and VAC14 may also lead to hypomyelination, although the data here are in a way premature and should include convincing morphological analysis.

Morphological studies of epoxy resin embedded and toluidine blue stained optic nerve sections from P14 Pikfyve^flox/flox, Olig2Cre mice (Figure 6—figure supplement 1) and VAC14^L156R/L156R (Figure 7—figure supplement 1) are now included in the manuscript. Loss of Pikfyve in the OL lineage leads to a complete loss of myelin. Consistent with electrophysiological and biochemical studies, toluidine blue labeling of VAC14^L156R/L156R optic nerve sections revealed severe hypomyelination.

3) Altogether, the data supports a role for the PI(3,5)P2 biosynthesis complex in myelination by regulating membrane trafficking in oligodendrocytes, but additional experiments would make the story more convincing.

We agree with the reviewers’ comment. In addition to the membrane trafficking studies in primary OLs, shown in Figures 8 and 9 of the original submission, we now included OL membrane trafficking studies in acute forebrain tissue from P14 mice (Figure 10 and Figure 10—figure supplement 1). We show that in acute wildtype brain slices (kept in oxygenated artificial cerebrospinal fluid) plasma membrane derived MAG is rapidly endocytosed and localized to small vesicular structures that accumulate in the cell soma and OL processes that form internodes. To visualize MAG trafficking, brain slices were incubated with mouse anti-MAG-Alexa555 antibody for two hours. To distinguish between surface-localized MAG and endocytosed MAG, brain slices were fixed and incubated with an anti-mouse Alexa488 secondary antibody under non-permabilizing conditions. As shown in Figure 10A-A”, the majority of MAG in wildtype slices is endocytosed and only labeled by anti-MAG-Alexa555. To demonstrate antibody specificity, parallel studies with Mag^-/-brain slices were carried out (Figure 10—figure supplement 1D-E) and revealed no staining above background. To demonstrate anti-MAG-Alexa555 antibody is taken up by OLs, cells in the OL lineage were genetically labeled (LacZ/EGFP, Olig2Cre). Three-dimensional rendering of EGFP⁺ cells (OLs) revealed accumulation of MAG in small intracellular vesicles (see Insert in Figure 10—figure supplement 1C). Thus far these experiments show that we can monitor MAG endocytosis and vesicular localization in acute brain slices of wildtype mice. To demonstrate a role for the lipid PI(3,5)P₂ in this process, experiments were repeated with acute brain slices from Pikfyve^flox/flox, Olig2Cre mice. As shown in Figure 10B-B”, MAG trafficking is impaired. Endocytosis of MAG from the plasma membrane does occur in some cells (see insert in Figure 10B”), vesicle accumulation is perinuclear and no labeling of endocytosed MAG in cellular processes is observed. Together, these studies show that PI(3,5)P₂ plays a critical role in mobilization of plasma membrane derived MAG in brain slices.

4) Although the discovery of the Fig4/Vac14/PIKfyve enzyme complex for normal myelination is significant, these results still don't tell us anything about the nature of what is being trafficked in neurons or immature OLs that leads to the hypomyelination phenotypes. Of course, this is very hard to do and will no doubt be the focus of the next paper.

These are important questions and the focus of ongoing studies. In the revised manuscript we have added one additional piece of data. We now show that trafficking of MAG through LE/Lys is impaired, as plasma membrane derived MAG accumulates in large perinuclear vesicles in Fig4^-/-OLs. Trafficking of plasma membrane derived MOG occurs through recycling endosomes (RE) and is not affected by Fig4 deficiency. This shows that PI(3,5)P₂ is required for membrane trafficking through the LE/Lys compartment but not through RE.