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

Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1+ and Rab7+ vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1+perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis. DOI: http://dx.doi.org/10.7554/eLife.13023.001

FIG4 deficiency is particularly harmful for neural cells with elaborate morphologies, including projection neurons and myelinating glia. Mutations of human FIG4 result in neurological disorders including Charcot-Marie-Tooth type 4J, a severe form of peripheral neuropathy Nicholson et al., 2011), polymicrogyria with epilepsy (Baulac et al., 2014), and Yunis-Varon syndrome (Campeau et al., 2013). Mice null for Fig4 exhibit severe tremor, brain region-specific spongiform degeneration, hypomyelination, and juvenile lethality Ferguson et al., 2009;Winters et al., 2011). We previously demonstrated that a Fig4 transgene driven by the neuron-specific enolase (NSE) promoter rescued juvenile lethality and neurodegeneration in global Fig4 null mice, and that these phenotypes were not rescued by an astrocyte-specific Fig4 transgene eLife digest Neurons communicate with each other through long cable-like extensions called axons. An insulating sheath called myelin (or white matter) surrounds each axon, and allows electrical impulses to travel more quickly. Cells in the brain called oligodendrocytes produce myelin. If the myelin sheath is not properly formed during development, or is damaged by injury or disease, the consequences can include paralysis, impaired thought, and loss of vision.
Oligodendrocytes have complex shapes, and each can generate myelin for as many as 50 axons. Oligodendrocytes produce the building blocks of myelin inside their cell bodies, by following instructions encoded by genes within the nucleus. However, the signals that regulate the trafficking of these components to the myelin sheath are poorly understood.
Mironova et al. set out to determine whether signaling molecules called phosphoinositides help oligodendrocytes to mature and move myelin building blocks from the cell bodies to remote contact points with axons. Genetic techniques were used to manipulate an enzyme complex in mice that controls the production and turnover of a phosphoinositide called PI(3,5)P 2 . Mironova et al. found that reducing the levels of PI(3,5)P 2 in oligodendrocytes caused the trafficking of certain myelin building blocks to stall. Key myelin components instead accumulated inside bubble-like structures near the oligodendrocyte's cell body. This showed that PI(3,5)P 2 in oligodendrocytes is essential for generating myelin. Further experiments then revealed that reducing PI(3,5)P 2 in the neurons themselves indirectly prevented the oligodendrocytes from maturing. This suggests that PI(3,5)P 2 also takes part in communication between axons and oligodendrocytes during development of the myelin sheath.
A key next step will be to identify the regulatory mechanisms that control the production of PI (3,5)P 2 in oligodendrocytes and neurons. Future studies could also explore what PI(3,5)P 2 acts upon inside the axons, and which signaling molecules support the maturation of oligodendrocytes. Finally, it remains unclear whether PI(3,5)P 2 signaling is also required for stabilizing mature myelin, and for repairing myelin after injury in the adult brain. Further work could therefore address these questions as well. (Ferguson et al., 2012). The neuron-specific transgene also rescued conduction in peripheral nerves (Ferguson et al., 2012) and structural defects in CNS myelination (Winters et al., 2011). Conversely, inactivation of Fig4 specifically in neurons resulted in region-specific neurodegeneration (Ferguson et al., 2012).
The cellular and molecular mechanisms relating loss of Fig4 to hypomyelination are poorly understood. To further characterize the requirement of PI(3,5)P 2 for CNS myelination, we manipulated individual components of the PI(3,5)P 2 biosynthetic complex. Pikfyve and Vac14 global null mice die prematurely, before the onset of CNS myelination Ikonomov et al., 2011). To circumvent this limitation, we employed a combination of conditional null alleles and hypomorphic alleles in the mouse. Our study shows that multiple strategies to perturb the FIG4/PIKFYVE/VAC14 enzyme complex, and by extension the lipid product PI(3,5)P 2 , result in the common endpoints of arrested OL differentiation, impaired myelin protein trafficking through the LE/Lys compartment, and severe CNS hypomyelination. We demonstrate that these defects in myelin biogenesis are functionally relevant and result in faulty conduction of electrical impulses.

Conditional ablation of Fig4 in neurons or the OL lineage results in CNS hypomyelination
In the early postnatal brain, Fig4 is broadly expressed and enriched in oligodendrocyte progenitor cells (OPCs) and newly formed OLs (NFOs) . Mice in which exon 4 of the Fig4 gene is flanked by loxP sites (Ferguson et al., 2012) were used to generate . For a quantitative comparison of the myelination defects, whole brain membranes were prepared from P21 pups and analyzed by immunoblotting with antibodies specific for the myelin markers myelin-associated glycoprotein (MAG), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), proteolipid protein (PLP), and myelin basic protein (MBP) ( Figure 1E). Compared to Fig4 +/+ membranes, a significant reduction in myelin proteins was evident in Fig4 -/mice, Fig4 -/flox ,SynCre mice and Fig4 -/flox ,Olig2Cre mice ( Figure 1F -I). The finding that the neuronal marker classIII b-tubulin is not significantly decreased in any of these mice indicates that the decrease in CNS myelin is not secondary to neuronal loss. While the Olig2 promoter is highly active in the OL lineage, activity has also been reported in astrocytes and a subset of neurons (Dessaud et al., 2007;Zhang et al., 2014). To independently assess the role of Fig4 in the OL lineage, we generated Fig4 -/flox ,PdgfraCreER mice that permit tamoxifen inducible gene ablation. At postnatal-days (P)5 and 6, before the onset of CNS myelination, Fig4 -/flox ,PdgfraC-reER pups were injected with 4-hydroxytamoxifen and brains were analyzed at P20-P21. Inducible ablation of  (Vaccari et al., 2015). Analysis of Fig4 -/flox ,Hb9Cre spinal cord identified enlarged vacuoles within motoneuron axons, greatly extending their diameter and pushing the axoplasm into a thin peripheral Quantification of myelin protein signals is normalized to bIII Tub. Relative protein intensities compared to WT brain are shown as mean value ± SEM. For each of the four genotypes, three independent membrane preparations were carried out. One-way ANOVA with multiple comparisons, Dunnett posthoc test; **p<0.01, ***p<0.001 and ****p<0.0001. An independent strategy for OL-specific Fig4 deletion results in a similar phenotype as shown in To assess myelin health, we determined the g-ratio (the ratio of the inner axonal diameter to the total fiber diameter) of myelinated axons in the optic nerve of Fig4 control and conditional mutants. Compared to control mice, a small but significant increase in g-ratio was observed in Fig4 -/flox ,SynCre and Fig4 -/flox ,Olig2Cre mice, an indication of myelin thinning ( Figure 2E). To determine whether the optic nerve hypomyelination at P21 reflects a transient delay in myelin development, rather than a lasting defect, we repeated the analysis with adult mice. Similar to P21 optic nerves, ultrastructural analysis of both types of adult optic nerves revealed profound hypomyelination ( Figure

Conditional ablation of Fig4 in neurons or the OL lineage impairs nerve conduction
To determine whether the morphological defects in CNS myelin of Fig4 conditional mutants result in functional deficits, we performed electrophysiological recordings. We measured the conduction velocity and amplitude of compound action potentials (CAPs) in optic nerves acutely isolated from P21 mice. Global deletion of Fig4 (Fig4 -/-) results in a dramatic reduction in a population of fast conducting fibers and a corresponding increase in the proportion of slowly conducting fibers ( Figure 3A,B,E) (Winters et al., 2011). The average velocity of the largest peak in Fig4 control nerves carrying at least one intact allele of Fig4 is 1.9 ± 0.1 m/s but in Fig4 -/nerves this is reduced to 0.7 ± 0.2 m/s. A similar CAP redistribution was observed in optic nerves prepared from Fig4 -/flox , SynCre mice (0.7 ± 0.1 m/s) and Fig4 -/flox ,Olig2Cre mice (0.6 ± 0.03 m/s) ( Figure 3C,D,E). Thus, consistent with biochemical and morphological analyses (Figures 1 and 2), loss of Fig4 in neurons or in the OL-lineage results in slowed nerve conduction.  To assess the cellular basis of the CNS hypomyelination phenotype, we stained optic nerve cross sections from Fig4 conditional mutants for markers in the OL lineage. Compared to Fig4 control optic nerves, the diameter of nerves from P21 Fig4 -/flox ,SynCre and Fig4 -/flox ,Olig2Cre mice were each reduced by 20%. The density of NG2 + progenitor cells in optic nerve tissue sections is comparable among the three genotypes ( Figure 4A-A'' and D). The density of Olig2 + cells, a marker that labels immature and mature OLs, is reduced, as is labeling of Plp1, a mature OL marker ( Figure 4B-B'',C-C'',E and F). These studies indicate that OPCs are present at normal density and tissue distribution in the Fig4 conditional null optic nerves, but they fail to generate the normal population of mature myelin-forming OLs.

Loss of Fig4 attenuates OL differentiation in vitro
For a more detailed analysis of the OL lineage, we isolated primary OPCs from P6-P14 Fig4 pups by anti-PDGFRa immunopanning (Emery and Dugas, 2013). Yields of OPCs per brain did not differ between control and Fig4-deficient mice (data not shown). OPCs were cultured for two days in vitro (DIV2) under proliferating conditions, fixed and analyzed by double-immunofluorescence staining of Ki67 and PDGFRa. The density of Ki67 + /PDGFRa + cells in  Independent perturbation of three components of the PI(3,5)P 2 biosynthetic complex all result in severe CNS hypomyelination Together with the kinase PIKFYVE and the scaffolding protein VAC14, FIG4 forms a biosynthetic complex necessary for acute interconversion of PI(3) and PI(3,5)P 2 . The complex is located on the cytosolic surface of vesicles trafficking through the LE/Lys compartment (McCartney et al., 2014).
As an independent test of the effect of perturbation of the FIG4/PIKFYVE/VAC14 enzyme complex on CNS myelination, we generated Pikfyve flox/flox ,Olig2cre mice predicted to be more severely deficient in PI(3,5)P 2 than the FIG4 and VAC14 mutants. Consistent with this expectation, the phenotype of the Pikfyve mutant mice is much more severe, with a significant tremor (Videos 3 and 4) and death at 2 weeks of age (n = 16 pups). FluoroMyelin Green staining of P13 brain tissue revealed profound hypomyelination of the corpus callosum, internal capsule and cerebellar white matter of Pikfyve flox/flox ,Olig2cre pups ( Figure 6A Figure 6E-E' and G) and results in a 95 ± 1% reduction in cells that progress to the MBP + stage, compared with wildtype cells ( Figure 6F-F' and H). In addition to Fig4 and Pikfyve mutants, we also examined myelinogenesis in the well-characterized recessive Vac14 mouse mutant L156R (Vac14 L156R ) (Jin et al., 2008). The L156R missense mutation impairs the interaction of VAC14 with  PIKFYVE, but not with FIG4 ( Figure 7A). Similar to Fig4 -/mice, Vac14 L156R/L156R mice exhibit~50% reduction in PI(3,5)P 2 . Immunoblots of brain membranes prepared from Vac14 L156R/L156R mice showed significantly reduced levels of the myelin markers MAG, CNPase, and MBP ( Figure 7B-E). The electrical properties of optic nerve from Vac14 L156R homozygous mice were also impaired, with a significant increase in the population of slowly conducting fibers ( Figure 7F-H). Consistent with this observation, toluidine blue staining of optic nerve sections of adult wild-type mice revealed many myelinated fibers but optic nerves of adult Vac14 L156R/L156R mice showed few myelinated fibers ( Myelin proteins are present within enlarged LAMP1 + perinuclear vacuoles in primary OLs from Fig4 -/mice The FIG4/PIKFYVE/VAC14 biosynthetic complex regulates intracellular PI(3,5)P 2 and thereby influences membrane trafficking through the endo-lysosomal system. DIV2 primary OPC cultures  (3)P and PI(3,5)P 2 . The red asterisk in VAC14 indicates the L156R point mutation that perturbs the interaction with PIKfyve, but not with Fig4. (B) Western blot analysis of brain membranes prepared from adult (P90-120) WT and VAC14 L156R /VAC14 L156R littermate mice revealed a reduction in the myelin markers MAG, CNPase, and MBP. Anticlass III b-tubulin (bIII-Tub), a neuronal marker, is shown as a loading control. (C-E) Quantification of protein bands detected by Western blotting, shows a significant decrease in MAG, CNPase, and MBP in VAC14 mutant brain tissue (n = 3 independent blots per genotype). Unpaired Student's t-test; mean value ± SEM. ***p<0.001, **p=0.0015 and *p=0.0238. (F and G) Representative CAP traces recorded from acutely isolated optic nerves of WT and VAC14 L156T homozygous mice. (H) Quantification of average conduction velocity (CV) of largest amplitude peaks identified in F and G. Results are shown as mean value ± SEM, unpaired Student's t-test, **p=0.0063. WT   Cell surface derived MAG is trapped in large vacuoles in the LE/Lys compartment in Fig4 -/-OLs In developing OLs, myelin proteins such as MAG and PLP transiently accumulate on the plasma membrane (PM) at the cell soma, prior to undergoing endocytosis and LE/Lys dependent transport to the myelin sheet (Winterstein et al., 2008). To monitor trafficking of MAG, we used antibody tagging in live OL cultures. In wildtype OLs, anti-MAG-Alexa488 binds to MAG on the PM surface, undergoes endocytosis and is targeted to LAMP1 + vesicles in the LE/Lys compartment (Figure 8figure supplement 2B -B"). In these wildtype cultures, anti-MAG + vesicles are small, with a median volume of 0.3 ± 0.06 mm 3 , and partially overlap with LysoTracker + vesicles ( Figure 8A-A"). In contrast, in Fig4 -/-OLs, anti-MAG-Alexa488 is endocytosed and accumulates in LAMP1 + perinuclear vacuoles with greatly enlarged size (5 mm 3 , mean volume 94 ± 41 mm 3 ) and also in smaller MAG + / LAMP1 + vesicles with a median volume of 0.7 ± 0.25 mm 3 . The average size of all vesicles in Fig4 -/-OLs is 1.65 ± 0.32 mm 3 ( Figure 8B-B" and C, Figure 8-figure supplement 2C-C"). This suggests that independent of Fig4 genotype, MAG is transported to the PM and is rapidly endocytosed. In Fig4 -/-OLs, large MAG + /LAMP1 + vesicles rarely overlap with LysoTracker staining ( Figure 8B-8B"), suggesting that large vesicles may exhibit reduced acidification. As an independent approach to assess whether perturbation of PI(3,5)P 2 synthesis causes accumulation of MAG in large perinuclear vacuoles, wildtype OL cultures were treated with 1 mM apilimod, a potent inhibitor of PIKfyve (Cai et al., 2013). Treatment with apilimod for 90-120 min leads to the formation of large perinuclear vacuoles laden with MAG ( Figure 8D  OLs. Collectively, these studies indicate that PI(3,5)P 2 is critical for myelin protein trafficking through the LE/Lys compartment in developing OLs.

PI(3,5)P 2 is important for myelin membrane trafficking in live brain slices
Inter-cellular communication is critical for proper development of the axo-glial unit. To extend the studies of myelin protein trafficking to a system that contains intact axo-glial units, we prepared acute forebrain slices from P10-P14 mice and kept them in oxygenated artificial cerebrospinal fluid. Trafficking of MAG was monitored by bath application of mouse anti-MAG-Alexa555 for 2 hr at 32˚C. To distinguish between endocytosed MAG and PM localized MAG, brain slices were fixed and incubated with a secondary anti-mouse-Alexa488 conjugated antibody under non-permeabilizing conditions. Endocytosed MAG containing vesicles were prominently found in OL perinuclear regions and along cellular processes that form the myelin internode ( Figure 10A-A"). Only a small fraction of MAG is labeled with both antibodies, and thus localized to the PM on the cell surface ( Figure 10A-A"). To visualize cells in the OL lineage, we repeated MAG trafficking studies with brain slices from the ROSA-LacZ/EGFP,Olig2Cre reporter mouse. Vesicular MAG labeling was abundant in EGFP + cells, indicating that endocytosis of PM localized MAG does occur in cells of the OL-lineage and vesicular labeling is not the result of nonspecific antibody uptake by microglia or other cell types ( Figure 10-figure supplement 1A-C). To control for antibody specificity, brain slices from Mag -/mice were processed in parallel and revealed no significant labeling (Figure 10-figure supplement 1D-F). Thus, acute brain slices provide an opportunity to study myelin protein trafficking in live tissue. To assess whether PI(3,5)P 2 is required for endocytosis and trafficking of PM derived MAG in live brain tissue, the experiment was repeated with forebrain slices prepared from Pikfyve flox/flox , Olig2Cre pups. Strikingly, in the absence of PI(3,5)P 2 , MAG + labeling was restricted to abnormal perinuclear accumulations, and trafficking to cell processes was virtually absent ( Figure 10B'-B"). Figure 10. Impaired trafficking of MAG in Pikfyve flox/flox ,Olig2Cre brain slices. Confocal images of acute brain slices in oxygenated ACSF treated with bath-applied anti-MAG-Alexa555 antibody, fixed and stained with antimouse-Alexa488 secondary antibody to distinguish between endocytosed MAG (red) and PM localized MAG (green). (A) OLs in the striatum of Pikfyve control mice (P13) show punctate MAG labeling in the cell soma (arrows) and along processes that form internodes. Only few MAG + structures are also stained with anti-mouse-Alexa488, and thus, localized on the PM. (B-B") Limited perinuclear MAG labeling is observed in the Pikfyve flox/flox ,Olig2Cre striatum. Many MAG + structures are labeled red and green, and thus localized to the PM, however intracellular MAG is observed in some cells. Scale bar = 20 mm. Small inset shows a 3D view of the two cells labeled with arrows (B-B"). MAG + vesicles (red) only partially overlap with PM localized MAG (green). Alexa488 + isosurface transparency is adjusted to 50% to demonstrate intracellular Alexa555 + (red) and LiveNuc 647 + (blue) structures. Scale bar = 10 mm. To directly demonstrate that anti-MAG antibody labeled cells are OLs, parallel experiments were carried out with brain slices of LacZ/EGFP,Olig2Cre reporter mice. Anti-MAG antibody specificity is demonstrated with Mag -/slices in Figure 10 The data demonstrate that in brain slices, as well as cultured cells, PI(3,5)P 2 is required for proper membrane trafficking from the PM through the LE/Lys compartment.

Discussion
Multiple independent means of perturbing the Fig4/Vac14/PIKfyve enzyme complex all lead to profound CNS hypomyelination. Remarkably, conditional ablation of Fig4 either in neurons or OLs is sufficient to disrupt normal CNS myelination, indicating that both OL-intrinsic and OL-extrinsic mechanisms of OL maturation function in a Fig4-dependent manner. The hypomyelination phenotype in Fig4 conditional mutants and VAC14 L156R mice is physiologically relevant since it is associated with substantially reduced amplitude and conduction velocity of compound action potentials. Primary OPCs deficient in Fig4 progress normally to the stage of NFOs but their differentiation into mature OLs is impaired. In Fig4 -/-OLs, MAG is trafficked to the PM, undergoes endocytosis and is localized to enlarged LAMP1 + perinuclear vacuoles. The reduced motility of the enlarged MAG/ LAMP1 + vacuoles and their perinuclear position suggests that myelin building blocks are trapped in the LE/Lys compartment and cannot be delivered to the developing myelin sheath. Conditional deletion of Pikfyve in the OL lineage leads to more pronounced defects characterized by impaired OL differentiation, greatly reduced myelin membrane trafficking and profound CNS dysmyelination. Together, these studies firmly establish a critical role for the FIG4/PIKFYVE/VAC14 enzyme complex, and by extension its lipid product PI(3,5)P 2 , in myelin protein trafficking through the LE/Lys system in developing OLs and proper assembly of the axo-glial unit.

Impaired PI(3,5)P 2 metabolism attenuates OL differentiation
Immunohistological studies of Fig4 -/flox ,Olig2Cre optic nerves and experiments with Fig4 -/primary OLs did not detect a significant change in OPC density or reduction in viability. OPCs deficient for Fig4 progress and differentiate normally to the stage of newly formed OLs (NFOs), a postmitotic cell type characterized as PDGFRa -, GalC + , MOG - . However, differentiation of NFOs into mature OLs is PI(3,5)P 2 -dosage dependent. The arrest of OL differentiation becomes more severe as PI(3,5)P 2 levels are reduced to~50% of wildtype levels, in Fig4 and VAC14 mice, or completely depleted in Pikfyve mutant mice. OL maturation is highly regulated, and can be attenuated or blocked by perturbation of numerous signaling pathways and transcriptional programs (Emery et al., 2009;Bercury and Macklin, 2015;Marinelli et al., 2016). The fate of immature OLs that fail to progress to the mature stage remains unclear. However, these cells are likely to be shortlived and destined to die. The number of activated caspase-3 + cells in the OL lineage of Fig4 -/mice is not significantly increased (Winters et al., 2011), suggesting that immature OLs either do not die in large numbers or die in a caspase-independent manner. Additional studies are needed to determine exactly at which stage of OL lineage progression PI(3,5)P 2 deficiency impairs differentiation and how PI(3,5)P 2 regulates progression to a mature myelin producing cell.

Fig4-dependent trafficking of myelin building blocks through the LE/ Lys
Like epithelial cells, OLs are polarized, with the myelin sheath resembling the apical membrane domain and the membrane near the OL cell body the basolateral membrane domain (Salzer, 2003;Maier et al., 2008;Masaki, 2012). Myelin-producing OLs synthesize and transport large quantities of myelin building blocks (lipids and proteins) in order to segmentally ensheath multiple axons. Myelinogenesis also requires membrane sorting and trafficking to specific subdomains of the nascent myelin membrane sheath. Indeed, the final destination of myelin proteins may vary between compact myelin (e.g. PLP), peri-axonal loops (MAG) or abaxonal loops (MOG) of non-compact myelin (Arroyo andScherer, 2000, Salzer, 2003;White and Kramer-Albers, 2014). As in other polarized cells, OL proteins may be targeted through direct transport pathways from the Golgi to their final destination (Salzer, 2003). Alternative strategies are also employed to target key myelin constituents to their final destination. The mRNA for MBP, encoding a protein important for axon wrapping and myelin compaction, is packaged into RNA-granules and transported to distal sites within OL processes for regulated translation (Müller et al., 2013). MAG, PLP, and MOG are synthesized in the endoplasmatic reticulum and transported through the Golgi network to the PM near the OL cell body (analogous to the basolateral domain) as an intermediate target. From there MOG is targeted to the recycling endosome (RE) while MAG and PLP are targeted to the LE/Lys for delivery to the myelin sheath (analogous to the apical membrane domain) Maier et al., 2008;Winterstein et al., 2008).

LAMP1 is a marker for LE/Lys and we show that MAG is targeted to LAMP1 + vesicles in both Fig4 +/ + and Fig4 -/-OLs. A key feature of the MAG/LAMP1 double-labeled vesicles in Fig4 -/mutant OLs is
their greatly enlarged size and perinuclear position. The average velocity of these vesicles is significantly reduced, suggesting impaired membrane trafficking through the LE/Lys compartment. Trafficking defects in Fig4 -/-OLs are confined to the LE/Lys compartment as trafficking of MOG through RE occurs apparently normal, independent of Fig4 genotype. The severe CNS hypomyelination phenotype in Fig4 -/flox ,Olig2Cre mice is likely not the result of impaired MAG trafficking alone, but rather the result of mistrafficking of numerous myelin building blocks normally migrating through the LE/Lys compartment. For example, cholesterol (in part bound to PLP) and glycosphingolipids are endocytosed from the PM and stored in LE/Lys vesicles , Winterstein et al., 2008. During OL maturation, neuronal signals trigger a profound redistribution of PLP-containing membrane domains; endocytosis is reduced and PLP together with cholesterol and glycosphingolipids is moved from the LE/Lys to the PM . In humans, impaired trafficking of PLP due to mutation or altered dosage of the Plp1 gene, causes Pelizaeus-Merzbacher disease (PMD) and Spastic Paraplegia Type 2 (SPG2), developmental disorders with severe neurological impairment (Inoue, 2005). Overexpression of PLP in mice leads to accumulation of the protein in autophagic vesicles and LE/Lys, leading to reduction of other myelin proteins such MBP, MAG, and MOG (Karim et al., 2007). As in Fig4 -/mice, PMD results in reduced number of OLs and CNS dysmyelination. Failure of lysosomal trafficking or function is thus a common underlying mechanism for a growing number of hereditary disorders that cause CNS dysmyelination, including PMD, Niemann-Pick type C disease, and several lysosomal storage diseases (Folkerth, 1999;Yaghootfam et al., 2005;Prolo et al., 2009;Schweitzer et al., 2009;Faust et al., 2010;Grishchuk et al., 2014).

PI(3,5)P 2 -dependent trafficking of myelin membrane components in developing OLs
Different phosphoinositides exhibit unique distribution to intracellular membrane compartments and have been implicated as key regulators of membrane sorting and targeted vesicular trafficking (Mayinger, 2012). PI(3,5)P 2 , for example, decorates vesicles in the LE/Lys compartment and serves as a docking site for cytosolic proteins (Mayinger, 2012). PIP binding proteins frequently interact with small GTPases belonging to the Rab or Arf families, establishing a combinatorial code that defines membrane identity (Behnia and Munro, 2005;Stenmark, 2009;Jean and Kiger, 2012;Mayinger, 2012;Egami et al., 2014). The phosphorylation status of PIPs and the activation state of small GTPases can be rapidly modified, providing an identification code that is both unique and dynamic, two prerequisites for targeted membrane transport. In HeLa cells, for example, the lysosomal membrane is characterized by the presence of PI(3,5)P 2 and the small GTPases Rab7 and Arflike (Bucci et al., 2000;Hofmann and Munro, 2006). In fibroblasts cultured from Fig4 -/or VAC14 L156R/L156R mice, PI(3,5)P 2 levels are reduced by~50% leading to formation of greatly enlarged LAMP1 + vacuoles Jin et al., 2008;Zou et al., 2015). In Fig4 -/-OLs, Rab7-YFP localizes to large perinuclear vacuoles (Figure 8-figure supplement 2). In HeLa cells, overexpression of constitutively active Rab7 leads to formation of large LAMP1 + and LAMP2 + vacuoles (Bucci et al., 2000). A direct interaction of VAC14 with the Rab7 GTPase activating protein (GAP) TBC1D15 has recently been described in HeLa cells (Schulze et al., 2014). This suggests the existence of a large protein complex that controls the interconversion of PI(3)P and PI(3,5)P 2 and the activity of select Rab GTPases, an emerging theme for directed membrane trafficking (Jean et al., 2015). Rab GTPases constitute a large protein family whose members are localized to distinct intracellular membrane microdomains to coordinate vesicle trafficking (Stenmark, 2009;Hutagalung and Novick, 2011). The GTPase Rab3A is expressed in OLs and has been shown to participate in membrane trafficking and myelination (Schardt et al., 2009). As discussed above, transport of myelin membrane components, including PLP, cholesterol and MAG, involves membrane sorting and trafficking through the LE/Lys compartment prior to insertion into the nascent myelin sheath (White and Kramer-Albers, 2014). Thus, interference with PI(3,5)P 2 synthesis, turnover, or binding partners that define LE/Lys membrane identity results in impaired cargo delivery of key myelin membrane components required for membrane expansion and sheath formation.

Neuronal Fig4 participates in CNS myelination
The severe hypomyelination phenotype in Fig4 -/flox , SynCre mice suggests that Fig4-dependent neuronal signals are necessary for proper CNS myelination. When coupled with our previous finding that transgenic Fig4 directed by the NSE promoter on a Fig4 -/background (Fig4 -/-,NSE-Fig4) rescues the myelination defect (Winters et al., 2011;Ferguson et al., 2012), this suggests that normal levels of Fig4 in neurons is necessary for CNS myelination and that neuronal overexpression of recombinant Fig4 on a global Fig4 -/background is sufficient to drive CNS myelination. Multiple lines of evidence have demonstrated that neuron-derived signals regulate OL maturation and axon myelination (Coman et al., 2005;Trajkovic et al., 2006;Ohno et al., 2009;Winters et al., 2011;Yu and Lieberman, 2013;Yao et al., 2014). We speculate that neuronal Fig4 regulates LE/Lys-dependent transport and axonal presentation of a 'pro-myelination' signal(s) necessary for OL differentiation and CNS axon myelination and that transgenic overexpression of Fig4 in neurons (NSE-Fig4) leads to an elevated production of 'pro-myelination' signals(s) sufficient to rescue the deficiency of Fig4 in the OL lineage of the Fig4 -/-,NSE-Fig4 transgenic mice. Alternatively, neuronal Fig4 may accelerate the loss of 'anti-myelination' signal(s) on the axonal surface, e.g., through endocytosis. Inter-cellular communication may occur through paracrine action of secreted molecules or shedding vesicles. Exosomes are extracellular vesicles produced by many cells that facilitate transport and exchange of proteins, mRNAs and regulatory RNAs with important functions in cellular processes including myelination (Frühbeis et al., 2012;Pusic and Kraig, 2014). Because Fig4 plays an important role in membrane trafficking through the LE/Lys system, it is possible that protein secretion or the content and abundance of exosomes may be altered in the mutant mice. Two independent approaches to delete Fig4 in the OL lineage (Olig2Cre and PdgfraCreER) revealed that Fig4 is required in the OL lineage for proper CNS myelination. These data were corroborated by in vitro studies with primary OLs. Taken together, our observations suggest that endogenous levels of Fig4 gene expression in both neurons and OLs are necessary for normal CNS myelination.
Technical limitations in the specificity of transgene promoters may affect the interpretation of these experiments. For neuron-specific loss-of-function we employed female SynapsinCre/+ mice driven by a synapsin-1 gene (SYN1) promoter fragment (Rempe et al., 2006), and for neuron-specific gain-of-function studies we used a 4.6 kb NSE promoter fragment (Winters et al., 2011;Ferguson et al., 2012). While these are commonly used strategies, it is recognized that in the developing mouse the NSE (ENO2) and SYN1 promoters may have some leakiness that results in transient expression in non-neuronal cells including glia. A low level of expression of the endogenous SYN1 and ENO2 genes in OPCs/OLs has been reported , but it is not clear whether this expression is retained by the promoter fragments that were used to drive transgene expression. Independent of these technical limitations, we provide multiple lines of evidence that genetic manipulations that compromise PI(3,5)P 2 synthesis profoundly impact OL differentiation and CNS myelination.
Novel assay to monitor myelin protein trafficking in brain tissue Acutely prepared brain slices are viable for several hours when maintained in oxygenated ACSF, a method commonly used for electrophysiological recordings (Lee et al., 2008). Studies with primary OLs suggest that newly synthesized myelin proteins are initially transported to the PM near the cell soma where they interact with lipids and other myelin proteins (Winterstein et al., 2008). These myelin-like structures are then thought to be endocytosed and trafficked to specific subdomains of the nascent myelin membrane sheath. Using acute brain slices combined with genetic labeling of cell in the OL lineage and confocal microscopy, we show that antibody-labeled MAG on the PM becomes rapidly endocytosed and is found in small vesicles in the OL cell soma and long processes that form internodes. Since sorting and trafficking of myelin building blocks are key components of myelinogenesis, future studies using acute brain slices may be productively combined with pharmacological and genetic manipulations to obtain detailed understanding of membrane trafficking in developing OLs.

Transgenic mice
All mice were housed and cared for in accordance with NIH guidelines, and all research conducted was done with the approval of the University of Michigan Committee on Use and Care of Animals. The spontaneous Fig4 -/null mutation plt  is maintained as two congenic lines, C57BL/6J.plt/+ and C3HeB/FeJ.plt/+. F1 plt/plt homozygotes obtained from crosses between these lines survive to 30-45 days, permitting analysis of myelination, and these were used for most experiments. A subset of in vitro experiments was carried out on cells from the C3HeB/FeJ.plt congenic mice. The conditional Fig4 flox allele was described elsewhere (Ferguson et al., 2012) and is maintained on strain C3HeB/FeJ from which the retinal degeneration locus rd was removed by repeated backcrossing and selection. Neuron-specific conditional knockout mice (Fig4 -/flox ,SynCre) were generated and maintained as previously described (Ferguson et al., 2012). The Olig2Cre/+ line (Schüller et al., 2008) and the PdgfraCre-ER/+ (Kang et al., 2010) (Jackson Laboratory stock # 018280) were used to delete Fig4 in the OL lineage. For inducible gene ablation in Fig4 -/flox , PdfraCreER mice, 4-hydroxytamoxifen (4OH-tamoxifen) (Sigma-Aldrich, MO) was injected directly into the stomach of P5 pups, which is easily identified by its milky-white color. 4OH-tamoxifen was dissolved in 100% ethanol at 10 mg/ml and 5 ml/day were administered for 2 days. Fig4 -/flox ,Hb9Cre (Fig4 -/flox ,Mnx1Cre) mice have been described previously (Vaccari et al., 2015). The spontaneous point mutant VAC14 L156R is deficient in PIKfyve binding (Jin et al., 2008) and was maintained on a C3HeB/FeJ strain background from which the retinal degeneration locus rd was removed by repeated backcrossing. Pikfyve flox/flox mice were generated on the C57BL/6J strain background (Min et al., 2014) and were crossed with Olig2Cre/+ mice. Mag -/mice on a C57BL/6J background have been described elsewhere (Pan et al., 2005). LacZ/ EGFP reporter mice (Jackson laboratory stock #003920) were crossed with Olig2Cre/+ mice.

Transmission electron microscopy (TEM)
Postnatal day (P)21 and P60-P75 mice were deeply anesthetized with ketamine (200 mg/kg)/xylazine (20 mg/kg body weight) and perfused transcardially with ice-cold phosphate buffer saline (PBS) for 2 min, followed by 4% paraformaldehyde (PFA) and 2.5% glutaraldehyde in Sorensen's buffer and embedded in epoxy resin as described (Winters et al., 2011). Semi-thin sections were stained with toluidine blue for light microscopy. TEM micrographs were taken at 10,500-13,500x magnification with a Philips CM-100 or a JEOL 100CX microscope and analyzed using FIJI software.

Immunohistochemistry
Mice between P10 and adulthood were perfused transcardially with ice-cold 4% PFA in PBS. Brains were post-fixed in perfusion solution for 2 hr at 4˚C for in situ hybridization. For immunofluorescence labeling, brains were postfixed overnight and cryoprotected in 30% sucrose in PBS. For FluoroMyelin staining, brains were cryosectioned at 25-40 mm. Free-floating sections were rinsed 3x 5 min in PBS and then stained with FluoroMyelin Green (Millipore, MA,1:200) in PBS for 20 min. Sections were washed with PBS, mounted onto microscope slides, coverslipped with Prolong Gold antifade supplemented with DAPI (Life Technologies, CA) and imaged with an Olympus IX71 microscope attached to a DP72 camera. For immunofluorescence labeling of optic nerves, nerves were rapidly dissected, kept in perfusion solution for 30 min and cryoprotected in 30% sucrose in PBS. Cross sections (12-20 mm) were mounted onto microscope slides, rinsed 3x for 5 min in PBS and incubated for 1 hr in blocking solution: 1% horse serum and 0.1% Triton-X100 in PBS (anti-Olig2) or 4% normal goat serum and 0.3% Triton-X100 in PBS (anti-NG2). Primary antibody incubation was done overnight at 4˚C in blocking solution with rabbit anti-Olig2 (1:1000 Millipore) or rabbit anti-NG2 (1:800, Abcam, UK). The next day, sections were rinsed 3x 5 min with PBS, incubated with appropriate secondary antibodies for 1 hr at room temperature (1:1000, Alexa-conjugated, Life technologies), rinsed in PBS and mounted in Prolong Gold supplemented with DAPI.

RNA in situ hybridization
cDNA fragments of Mbp and Plp1 (Ye et al., 2009) were used to produce digoxigenin-labeled cRNA probe by run-off in vitro transcription. Brains were cryosectioned at 25 mm and mounted directly onto Superfrost + microscope slides (Fisher Scientific, MA). Optic nerve sections were prepared as described above and postfixed in 4% PFA/PBS overnight at 4˚C. The following day, sections were rinsed with 1x PBS and dehydrated with series of ethanol dilutions (50%, 70%, 95%, and 100%). Sections were then treated with 50mg/ml proteinase K in PBS/5mM EDTA for 15 min (optic nerves) and 30 min for brain sections. All subsequent steps were performed as described previously (Winters et al., 2011). Isolation of brain membranes P21 mouse brains were homogenized in a Wheaton Dounce tissue homogenizer cooled on ice. Brain membranes were isolated by centrifugation in a discontinuous sucrose gradient as described previously (Winters et al., 2011). Isolation of brain tissue P21 control littermate and Fig4 -/flox ,PdfraCreER brains were extracted and rapidly dissected on ice. Tissue was separated into two groups: 1) cerebellum + brainstem and 2) neocortex + hippocampus + thalamus ('forebrain'). Tissue was lysed in a radio-immunoprecipitation assay buffer (RIPA) using a tissue homogenizer and triturated with a 16G needle. Lysates were spun at 14,000 rpm for 15 min at 4˚C and supernatants were analyzed by Western blotting as described below.

Western blot analysis
Equal amounts of protein (7.5-15 mg) from brain membranes were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore). Membranes were blocked in 3% dry milk powder dissolved in Tris-HCl pH 7.4 buffered saline containing 0.3% Triton X-100 for at least 1 hr and incubated with primary antibody overnight at 4˚C. Primary antibodies included mouse anti-bIII tubulin (

Electrophysiology
Recordings were carried out as described elsewhere (Carbajal et al., 2015). Briefly, juvenile (P21-P23) and adult (3-4 months) mice were sacrificed by CO 2 inhalation. Optic nerves were rapidly dissected, incubated at room temperature in oxygenated artificial cerebrospinal fluid (ACSF) for 45 min and then transferred to a temperature-controlled recording chamber (held at 37 ± 0.4˚C) with oxygenated ACSF. Each end of the nerve was drawn into the tip of a suction pipette electrode. The stimulating electrode was connected to a constant-current stimulus isolation unit (WPI, FL) driven by Axon pClamp 10.3 software and a 50 ms pulse was applied to the retinal end of the nerve. The recording electrode was applied to the chiasmatic end of the nerve and connected to the input of a differential AC amplifier (custom-made). A second pipette, placed near the recording pipette but not in contact with the nerve, served to subtract most of the stimulus artifact from the recordings. Signals were digitized at 100 kHz through a data acquisition system (Axon Digidata 1440A, Axon pClamp 10.3, Molecular Devices, CA).
For live cell imaging, OPCs were switched to T3 supplemented differentiation medium and kept at 37˚C in a 5% CO 2 incubator equipped with an IncuCyte Zoom imaging system (Essen Bioscience, MI). Images were taken with a 20x objective every 2 hr for 3 days. Data were analyzed using the IncuCyte Zoom software and Fiji.

Live cell imaging
O4 + primary OLs were isolated by immunopanning as described above and cultured in 35 mm glass bottom dishes (Mattek, MA). After 2-3 days under differentiation conditions, anti-MAG-Alexa488 conjugated antibody (1:500, Millipore, MAB1567A4) was added to the culture medium for 12-14 hr. The following day, LysoTracker Deep Red (1:2000, Life Technologies) was added to the culture medium for 30-45 min. Fifteen minutes before imaging, the culture medium was replaced by 1x HBSS (Life Technologies) containing Prolong Live Antifade reagent (Life Technologies, 1:100) and Hoechst dye 33,342 (1:50,000) or NucRed Live 647 (Life Technologies). Cells were imaged at 37˚C and ambient CO 2 for 15-20 min/dish using a Leica SP5 confocal microscope. Confocal Z-stacks, xyt, and xyzt videos were acquired. As a specificity control for the anti-MAG-Alexa488 antibody, OLs were prepared from Mag -/and age-matched Mag +/+ pups and imaged under identical conditions. Mouse monoclonal anti-MOG antibody (Millipore) was conjugated with Alex555 using the Antibody Labeling Kit (Life Technologies). Some OL cultures were incubated with anti-MOG-Alexa555 (1:250) and anti-MAG-Alexa488 as described above. To some cultures 1 mM apilimod (Axon 1369; Axon Medchem BV) in DMSO was added 90-120 min prior to imaging. Images and videos were processed using Leica AS LF and Fiji. Tracking and movement analysis of anti-MAG-Alexa488 + particles in live cells was performed using Imaris (Bitplane, UK).

Ex vivo MAG labeling
To monitor MAG trafficking in acute brain tissue, sagittal slices were prepared from P13-P14 pups with the following genotypes, (i) Pikfyve control mice, (ii) littermates Pikfyve flox/flox ,Olig2Cre mice, (iii) Mag -/-, mice and (iv) P18 LacZ/EGFP, Olig2Cre reporter mice (Toth et al., 2013). Briefly, mice were decapitated, brains rapidly dissected and submerged in ice-cold ACSF (Toth et al., 2013). From forebrain tissue, hippocampi were removed and discarded. Cortex and striatum were sectioned at 300 mm using a tissue slicer (WPI, FL). Brain slices were kept in oxygenated (95% O 2, 5% CO 2 ) ACSF at RT for 40-60 min prior to incubation with anti-MAG-Alexa-555 (1:500) in oxygenated ACSF at 32˚C for 2 hr. Brain slices were then fixed in 4% PFA for 25 min, rinsed 3 times for 10 min each in PBS and incubated overnight with a goat anti-mouse Alexa-488 secondary antibody (1:1000) in 3% BSA at 4˚C. The following day, slices were rinsed 3 times for 10 min each in PBS, incubated with LiveRed 647 for 25 min at RT, rinsed 3 times for 10 min each in PBS, and mounted in Prolong antifade with DAPI. Individual MAG + cells in deep cortical layers and striatum were imaged using a Leica SP5 confocal microscope.

Primary OL transfection
For transfection of primary OPC/OLs, Lipofectamine2000 (Life Technologies) was used, following a protocol previously established for transfection of primary neurons (Duan et al., 2014). Briefly, 250 ng of LAMP1-mCherry or Rab7-YFP plasmid DNA were combined with 1 ml of Lipofectamine2000 (Invitrogen, CA) in optiMEM and mixed thoroughly. Transfection solution was added to OL culture medium and cells were incubated for 2.5 hr. Afterwards, the medium was completely replaced with fresh T3 supplemented medium. To visualize MAG trafficking, anti-MAG-Alexa-488 antibody was added to the culture medium as described above. The following day, live imaging of LAMP1-mCherry + /anti-MAG-Alexa-488 + OLs was carried out as described above.

Western blot analysis of OPC cultures
OPCs were allowed to expand in PDGF supplemented culture medium for 7-8 days, passaged and plated in 6-well culture dishes at a density of 200,000-300,000 cells/well and kept for 3 days in T3 supplemented medium. Cells were then processed for Western blotting as previously described (Raiker et al., 2010). Capillary immunoassays were performed using the automated Wes system (ProteinSimple, San Jose CA). All procedures were performed according to manufacturer's protocol. In brief, 0.8 mg of lysate (4 ml) were mixed with 2 ml of 5x fluorescent master mix and boiled for 5 min. These samples were dispensed into microplates along with blocking solution, primary and secondary antibodies and chemiluminescent substrate. After centrifugation, microplate was loaded into the Wes instrument for subsequent protein separation on capillaries and immuno-detection using the standard electrophores, immunolabeling, detection scheme of Wes. Data were analyzed by using Compass software (ProteinSimple) and peak areas were used for quantification. Erk1 peak area was used for normalization between samples. Three independent preparations were processed.

Statistical analysis
To assess myelination in the optic nerve, ten non-overlapping TEM images were randomly selected and the fraction of myelinated axons quantified as described (Winters et al., 2011). At least 600 axons were quantified per nerve. G-ratio analysis was performed as described previously (Winters et al., 2011). At least 100 axons per optic nerve were analyzed. For Western blot analysis, Western band intensity was measured using LI-COR Studio Image Software. All band intensities were normalized either to bIII-tubulin (brain lysates and membranes) or actin (OPC cultures). Normalized Western blot band intensity for control samples was set as 1 for each experiment. For optic nerve electrophysiology, data analysis was performed offline using Clampfit software. In order to analyze individual peaks, each trace was fitted as a sum of three or four Gaussians using Origin Pro software (Chen et al., 2004). A peak with the largest amplitude in each trace was used for conduction velocity analysis.
For quantification of Plp1, Olig2, and NG2 labeled cells, the number of respective positive cells was quantified per optic nerve cross section and normalized to the section area (arbitrary units in FIJI). At least four sections per nerve were analyzed.
For quantification of OL markers in vitro, ten non-overlapping images were taken at random positions for each coverslip/well and cells positive for a marker of interest counted and normalized to the number of Hoechst 33,342 dye positive cells in the same image. A minimum of 900 cells was quantified for each individual experiment with Fig4 cultures and a minimum of 120 cells was quantified for each individual experiment with Pikfyve cultures. GFAP + astrocytes were excluded from quantification. The analysis of actin/MBP postmitotic OL morphology was performed as characterized previously (Zuchero et al., 2015).
For cell viability experiments, the Live/Dead kit was used the number of live (green) and dead (red) cells was quantified and the live/total cell ratio was calculated. For all experiments, Hoechst 33,342 normalized cell density in control groups was set as 1. At least three independent experiments with duplicate coverslips were used for the analysis. For live imaging of MAG + vesicles in primary OLs, Imaris software (Bitplane) was used to calculate individual particle speed and size. Four independent experiments were analyzed for Fig4 +/+ and Fig4 -/cultures. MAG + particles of at least 0.01 mm 3 in volume were included in data analysis.
One-way ANOVA followed by Tukey posthoc was used for TEM optic nerve analysis. One-way ANOVA followed by Dunnett's posthoc was used for Western blot analysis and electrophysiology with more than two groups. The unpaired Student t-test was used for analysis in all experiments with two groups.

Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to protocols approved by the University committee on use and care for animals (UCUCA protocols: #00005863 and #00005902) of the University of Michigan.