Genetic defects in myelin formation and maintenance cause leukodystrophies, a group of white matter diseases whose mechanistic underpinnings are poorly understood1, 2. Hypomyelination and congenital cataract (HCC), one of these disorders, is caused by mutations in FAM126A, a gene of unknown function3. We show that FAM126A, also known as hyccin, regulates the synthesis of phosphatidylinositol 4-phosphate (PtdIns(4)P), a determinant of plasma membrane identity4, 5, 6. HCC patient fibroblasts exhibit reduced PtdIns(4)P levels. FAM126A is an intrinsic component of the plasma membrane phosphatidylinositol 4-kinase complex that comprises PI4KIIIα and its adaptors TTC7 and EFR3 (refs 5,7). A FAM126A–TTC7 co-crystal structure reveals an all-α-helical heterodimer with a large protein–protein interface and a conserved surface that may mediate binding to PI4KIIIα. Absence of FAM126A, the predominant FAM126 isoform in oligodendrocytes, destabilizes the PI4KIIIα complex in mouse brain and patient fibroblasts. We propose that HCC pathogenesis involves defects in PtdIns(4)P production in oligodendrocytes, whose specialized function requires massive plasma membrane expansion and thus generation of PtdIns(4)P and downstream phosphoinositides8, 9, 10, 11. Our results point to a role for FAM126A in supporting myelination, an important process in development and also following acute exacerbations in multiple sclerosis12, 13, 14.
At a glance
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- Supplementary Figure 1: FAM126A/B are identified as interaction partners of EFR3A/B, and FAM126A is expressed as two splice forms, with the 58 kD splice form the major form in mammalian brain. (240 KB)
(a) HeLa cells stably expressing EFR3A-GFP, EFR3B-GFP, or GFP only were subjected to GFP immunoprecipitation. The purified proteome was digested and analyzed by LC-MS/MS, and peptide and protein identities were determined using MaxQuant. Shown is the sequence coverage of FAM126A (left) and FAM126B (right) from two separate experiments: EFR3A-GFP vs. GFP IP (black bars) and EFR3B-GFP vs. GFP IP (white bars). (b) Top, Domain cartoon of the two splice forms of FAM126A (isoform 1, 58 kD corresponding to NCBI NP_115970.2; isoform 2, 47 kD, corresponding to NCBI XP_005249951.1), with regions common to both splice isoforms in cyan and regions unique to each splice isoform in yellow (isoform 1) and magenta (isoform 2). Bottom, Confirmation of the identity of the 47 kD splice form. Immunoprecipitations using an anti-FAM126A antibody raised against residues 2-308 of human FAM126A or a negative control (rabbit IgG) were performed from lysates of primary human skin fibroblasts and analyzed by SDS-PAGE followed by Coomassie staining. The band at ~47 kD in the anti-FAM126A sample was excised and analyzed by mass spectrometry. Shown is the list of identified peptides; those colored in magenta are unique to isoform 2 (XP_005249951.1). (c) Immunoblot analysis of FAM126A from lysates from lung of wild-type (WT) and FAM126A knockout (KO) mice. (d) Immunoblot analysis of FAM126A in lysates from gray matter (G) and adjacent white matter (W) samples from human or macaque brain (parietal cortex) and control (C) or patient-derived, FAM126A-deficient (P) primary human skin fibroblasts, demonstrating that isoform 1 is the major form in brain, and isoform 2 is the major form in skin fibroblasts. Arrowheads denote background bands at ~43 kD in mouse lung (c) and ~57 kD in fibroblasts (d). Shown are representative immunoblots from three independent experiments.
- Supplementary Figure 2: FAM126A is a soluble, cytosolic protein that is recruited to the plasma membrane by EFR3B and TTC7B. (420 KB)
COS-7 cells were transfected with the indicated combinations of the following plasmids: GFP-FAM126A-N, FAM126A-GFP, soluble mCherry (mCherry), a plasma membrane-targeted mCherry containing the first 11 residues of Lyn kinase (PM-mCherry) either alone (a) or in combination with EFR3B-HA and TTC7B-mTagBFP (b). The cells were imaged live by confocal microscopy; shown are single z-plane images. Scale bars, 20 μm.
- Supplementary Figure 3: Purification of PI4KIIIα complexes for in vitro kinase assay and representative electron density maps for the FAM126A-N/TTC7B crystal structure. (1,190 KB)
(a) 3xFLAG-tagged PI4KIIIα (wild-type or a kinase-dead point mutant) were expressed in Expi293 cells alone or co-expressed with TTC7B or both TTC7B and FAM126A-N. The kinases (or kinase complexes) were purified by anti-FLAG affinity chromatography and analyzed by SDS-PAGE, staining with Coomassie blue. Arrowhead indicates Hsp70, which partially co-purified with all samples (and whose identity was verified by mass spectrometry). (b) Top, Experimental electron density map, contoured at 1.0σ, into which the initial model was built. The map was calculated with phases from a SAD experiment after density modification and sharpened using B-factors (−30 Å2). The initial model before refinement is shown in green. Bottom, 2Fo-Fc map of the same region, contoured at 1.0σ, with B-factor sharpening (−30 Å2). The refined model is shown in green. (c) Point mutations in FAM126A that underlie HCC are indicated in the TTC7B/FAM126A-N structure. Most of the disease-causing mutations in FAM126A result in premature termination; two known disease-causing missense mutations (L53P and C57R), indicated here, likely cause FAM126A misfolding.
- Supplementary Figure 4: Biochemical evidence for defects in PI4KIIIα function in HCC fibroblasts and FAM126A KO mice. (214 KB)
(a) Quantification of changes in protein levels shown in Fig. 5. Shown are the ratios of protein levels between the two indicated samples from immunoblots in Fig. 5, quantified using densitometry. Top left, HCC patient and control fibroblasts (Fig. 5a). ∗, PI4KIIIα, p = 0.0049; FAM126A, p = 0.0026; FAM126B, p = 0.0008; TTC7A, p = 0.0003; TTC7B, p = 0.019; EFR3A, p = 0.0011; n = 3 independent experiments (with 3 total technical replicates for all, except 4 for PI4KIIIα). Top right, Primary cultured oligodendrocytes and cortical neurons (Fig. 5d). ∗, PI4KIIIα, p = 0.019; FAM126B, p = 0.043; TTC7A, p = 0.048; EFR3B, p = 0.027; n = 2 independent experiments (with 2 total technical replicates for all, except 5 for PI4KIIIα and 4 for FAM126A). Bottom left, FAM126A KO and WT mouse brain (Fig. 5e). ∗, FAM126A, p = 0.0020; TTC7A, p = 0.0003; EFR3A, p = 0.024; n = 3 independent experiments (with 3 total technical replicates for all, except 4 for PI4KIIIα and 5 for TTC7A). Bottom right, FAM126A KO and WT mouse optic nerve (Fig. 5f). ∗, FAM126A, p = 0.0008; TTC7A, p = 0.025; EFR3A, p = 0.0056; n = 2 independent experiments (with 2 total technical replicates for all, except 3 for EFR3A, EFR3B, and FAM126A). Significance was calculated using either an unpaired two-tailed Student’s t-test with equal variance (top left) or a two-tailed, paired ratio t-test (all others). Error bars represent standard deviation. (b) Rescue of PI4KIIIα complex levels in HCC patient fibroblasts by expression of FAM126A-GFP. Immunoblot (IB) analysis of PI4KIIIα complex components in lysates from control fibroblasts or HCC patient fibroblasts transduced with either a control lentivirus (−) or lentivirus containing a C-terminally tagged FAM126A construct corresponding to isoform 2, the major form in fibroblasts (see Supplementary Fig. 1d). Shown are representative immunoblots from three independent experiments. (c) FAM126A mRNA is more abundant than FAM126B mRNA in human fibroblasts. cDNA from human fibroblasts was analyzed by qRT-PCR using primers specific to FAM126A and FAM126B. Shown are relative amounts of FAM126A to FAM126B, with FAM126B value normalized to 1 (two-tailed, Student’s t-test, unequal variance, p = 0.0004; n = 3 independent experiments). Error bars represent standard deviation. (d) Plasma membrane PI4P levels are reduced in HCC fibroblasts. Immunofluorescence analysis of the plasma membrane pool of PI4P, using an anti-PI4P antibody, in control and HCC patient fibroblasts. Shown are representative average intensity projection images of a confocal z-stack. Quantification and statistical information is provided in Fig. 5c. (e) FAM126A KO results in a more severe impact on PI4KIIIα complex levels in oligodendrocytes compared to neuronal cells. Left, Immunoblot analysis of PI4KIIIα complex components in cells of the oligodendrocyte (Oligo) and neuronal (Neu) lineage that were immunoisolated from wild-type and FAM126A KO mice at postnatal day 8. Right, Quantification of these immunoblots, showing the relative amount of each PI4KIIIα complex component in the corresponding cell type in FAM126A KO compared to in wild-type. Black bars, oligodendrocytes; gray bars, neuronal cells. Two-tailed Student’s t-test, unequal variance, ∗, PI4KIIIα:p = 0.0068. FAM126B: p = 0.000050. TTC7A: p = 0.0457. TTC7B: p = 0.0411. EFR3A: p = 0.0162. EFR3B: p = 0.0611; n = 3 biological replicates (3 total technical replicates for all, except 4 for PI4KIIIα). Error bars represent standard deviation.
- Supplementary Figure 5: Morphological analysis of myelination in FAM126A KO and control mice. (2,167 KB)
Light and transmission electron micrographs of myelin in transverse sections of spinal cord (ventral funiculus cervical region), optic nerve, and sagittal sections of two regions of the central area of corpus callosum from WT and FAM126A KO male mice at age P15. No substantial differences in extent of myelination between the two genotypes can be observed. (a) Representative light microscopy images of semithin (1 μm) sections, stained with toluidine blue. (b) Representative transmission electron microscopy images of ultrathin sections (60 nm) contrasted with osmium tetroxide and uranyl acetate. Shown are representative images (n = 3 KO mice; n = 2 WT littermate control mice). Scale bars: 20 μm (a); 2 μm (b).
- Supplementary Information (1,400 KB)