Mice

Global deletion of Mfsd7c has been described previously.2 To generate the postnatal deletion of Mfsd7c in the BBB, Mfsd7cf/f was crossed with Cdh5-CreERT2 mice40 to generate Mfsd7cf/fCdh5-CreERT2 mice (EcMfsd7c-KO). At about 2 months of age, mice were injected daily with 5 doses of 200 µg/g bodyweight tamoxifen prepared in corn oil via oral gavage.41 Male and female mice with age more than 8 weeks old were used for experiments. Mice were maintained at a constant temperature of 20 °C with 12-h light/12-h dark cycle on normal chow diets. All experimental protocols and procedures in protocol R19-0567 and R23-0973 were approved by IACUC committees under National University of Singapore.

Plasmids

All plasmids were constructed in pcDNA3.1 vector for overexpression unless stated otherwise. hChKA (D10704.1), ETNK1, CHAT (NM_020986.4) genes were synthesized from GenScript and inserted into pcDNA3.1, respectively. pcDNA3.1-hMfsd7c, pcDNA3.1-hMfsd7cT430M and S203Y were generated previously.2 The other missense mutations including hMfsd7cT430A, P276S, L483R, P340L, and T430R were generated by mutagenesis and confirmed by Sanger sequencing. Zebrafish (DaMfsd7c_a, XM_688497.7; DaMfsd7c_b: XM_003200755.5), medaka fish (MeMfsd7c, XM_004082328.4), and frog Mfsd7c (XeMfsd7c, NM_001016982.2) coding sequences were synthesized by GenScript and cloned into pcDNA3.1 plasmid for overexpression, respectively. The pESC-HIS-hMfsd7c plasmid (GenScript) was used for overexpression in S. cerevisiae.

Untargeted metabolite analysis

Untargeted metabolomics analysis was performed at the Genome Analysis Center, Research Unit Molecular Endocrinology and Metabolism, Helmholtz Center Munich. Frozen WT and Mfsd7c KO embryonic brain and liver samples at E14.5 were weighed and placed into pre-cooled (dry ice) 2 mL homogenization tubes containing ceramic beads with a diameter of 1.4 mm (Precellys® Keramik-Kit 1.4 mm). Pre-cooled water with a ratio of 5 µL/mg tissue was added into the tubes. The samples were then homogenized in Precellys 24 homogenizer (PEQLAB Biotechnology GmbH, Germany) equipped with an integrated cooling unit 3 times for 20 s at 5500  rpm each, with 30 s intervals (to ensure freezing temperatures in sample vials) between the homogenization steps. 100 µL of the brain and liver homogenates were loaded onto two separate 2 mL 96-deep well plates, one plate for the brain sample set and the other for the liver samples. Three types of quality control samples were analyzed in concert with the experimental samples in each sample set: samples generated from a pool of human plasma; samples generated from a small portion of each experimental sample served as technical replicates throughout the dataset; and extracted water samples served as process blanks. Experimental samples and controls were randomized across the metabolomics analysis.

The homogenates in each well of the 2 mL 96-deep well plate were extracted with 500 µL methanol, containing four recovery standards to monitor the extraction efficiency. After centrifugation, the supernatant was split into aliquots in two 96-well microplates. The first 2 aliquots of each sample set were used for ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis in positive electrospray ionization modes (i.e., early and late eluting compounds). 1 aliquot of each sample set was used for UPLC-MS/MS analysis in negative ionization mode, and the rest were kept as reserve for backup. The extract aliquots were dried on a TurboVap 96 (Zymark).

Prior to UPLC-MS/MS analysis, the dried extract samples were reconstituted in acidic or basic LC-compatible solvents, each of which contained eight or more standard compounds at fixed concentrations to ensure injection and chromatographic consistency. The UPLC-MS/MS platform utilized a Waters Acquity UPLC with Waters UPLC BEH C18-2.1 × 100 mm, 1.7 μm columns and Q-Exactive high resolution/accurate mass spectrometer (ThermoFisher Scientific) interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. Extracts reconstituted in acidic conditions were gradient eluted using water and methanol containing 0.1% formic acid, while the basic extracts, which also used water/methanol, contained 6.5 mM ammonium bicarbonate. The MS analysis alternated between MS and data-dependent MS2 scans using dynamic exclusion, and the scan range was from 80 to 1000 m/z.

Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra and curated by visual inspection for quality control using software developed at Metabolon. Chromatographic peaks were quantified using area-under-the-curve.

In vivo transport of radiolabelled choline

To study the uptake of choline into the brain, control (Mfsd7cf/f) and EcMfsd7c-KO mice aged 4–6 months were intravenously injected with 2 mM radiolabelled choline (stock: 333 mM choline with 0.05 µCi/µL). After 5 min of injection, 50 µL blood was collected and the radioactive levels were quantified to monitor the initial radiolabelled levels in the circulation. After 30 min of injection, mice were perfused with 20 mL of PBS. Then, brains, lungs, hearts, kidneys, and livers were collected for quantification of radioactive signals. A similar amount of tissues was homogenized in RIPA buffer and the radioactive signals were quantified by a liquid scintillation counter. The radioactive signals from tissue from each mouse were normalized to radioactive signals from blood collected at 5 min post-injection.

Choline-d9 and LPC-d49 experiments

For stable isotopic tracing with choline-d9 (containing 9 deuterium atoms) injection, pregnant Mfsd7c+/– mice at 12.5 days of gestation were injected daily by intravenous route with an amount of 2 mM choline-d9/day until gestation day 14.5 (total of 3 doses of choline-d9 from E12.5–14.5). The embryos were collected at E15.5 after the last dose at E14.5 and genotyped. The brains and livers of embryos were harvested for lipid extraction for lipidomic analysis. Briefly, an equal amount of brain and liver lysates from WT, heterozygous and KO embryos were extracted with 1-butanol:methanol (ratio 1:1) method for choline and lipid analyses by LC-MS/MS.

For stable isotopic LPC-d49 (870308P-5MG, Sigma-Aldrich, containing choline-d13, glycerol-d5, and palmitate-d31) injection, EcMfsd7c-KO and control mice aged 7–10 months were injected with a dose of 300 µM LPC-d49 in 12% BSA. After 2 h of injection, plasma was collected from the blood; then the mice were perfused with cold PBS to remove blood. After that the brains and livers were collected. Brains and livers were homogenized in PBS and the same amount of tissue lysates were used for lipid and choline extraction with chloroform:methanol (1:2 v/v) method.42 Organic phase was used for lipid analysis and the aqueous phase (upper phase) was used for choline analysis by LC-MS/MS.

Lipids and choline quantification by LC-MS/MS

Samples were randomized before extraction and analysis. Blank samples (10 µL MilliQ water), matrix blanks (samples extracted without spiking internal standards, see below), and pooled QC samples were used to assess method performance. In addition, diluted pooled QC samples were used to assess response linearity. Blanks, blank extracts, QC, and diluted QC were interspersed with study samples throughout the analytical run. Phospholipids and choline were separated using HILIC column (Kinetex 2.6 µm HILIC 100 Å, 150 × 2.1 mm, Phenomenex, Torrance, CA, USA) on Agilent 6495A and 6495C triple quadrupole mass spectrometers (Agilent Technologies). Mobile phases A (50% acetonitrile (LC-MS grade, ThermoFisher Scientific) and 50% 25 mM ammonium formate (Sigma-Aldrich), pH 3.5) and B (95% acetonitrile and 5% of 25 mM ammonium formate, pH 3.5) were mixed at the following gradient: 0–6 min, 99%–75% B; 6–7 min, 75%–10% B; 7–7.1 min, 10%–99% B; 7.1–10.1 min, 99% B. The flow rate was 0.6 mL/min, and the sample injection volume was 1 µL. MS parameters were as follows: electrospray ionization, gas temperature 200 °C, gas flow 12 L/min, sheath gas flow 12 L/min, and capillary voltage 3500 V. Phospholipids were quantified at the sum composition level using multiple reaction monitoring (MRM) using transition to phosphocholine headgroup (m/z 184 for endogenous lipids and adjusted to appropriate m/z for deuterated lipids). Choline was quantified using MRM using a transition from 104 to 60 (adjusted to appropriate m/z for deuterated choline). These internal standards (ISs) were used for lipidomics and choline analysis by LC-MS/MS. IS solutions were prepared in butanol/methanol (BuMe) (1:1, v/v) or chloroform/methanol (1:2) containing phospholipid IS SPLASH Mix (Avanti, 330707) (containing 5.3 µM 15:0–18:1(d7) PC, 0.19 µM 15:0–18:1(d7) PE, 0.13 µM 15:0–18:1(d7) PS (Na Salt), 0.93 µM 15:0–18:1(d7) PG (Na Salt), 0.27 µM 15:0–18:1(d7) PI (NH4 Salt), 0.27 µM 15:0–18:1(d7) PA (Na Salt), 1.20 µM 18:1(d7) Lyso PC, 0.27 µM 18:1(d7) Lyso PE, 13.32 µM 18:1(d7) d18:1–18:1(d9) SM). Choline-d9 was used as IS in choline analysis.

Transport assays in HEK293 cells

hMfsd7c or mouse mMfsd7c cDNA constructed in pcDNA3.1 was transfected to HEK293 cells using lipofectamine 2000 (ThermoFisher Scientific). Mock was treated with an empty plasmid. For experiments in which hChKA was used, 1–1.5 µg pcDNA3.1-hChKA plasmid was co-transfected with 2–2.5 µg plasmid of hMfsd7c or mutant plasmids. Cells transfected with 1–1.5 µg pcDNA3.1-hChKA plasmid were used as control. After 18–30 h post-transfection, cells were used for transport assays with 100 µM [3H] choline (ARC: ART 0197) in DMEM containing 10% FBS. The transport assays were stopped after 1 h incubation at 37 °C by washing once with cold plain DMEM medium. Cell pellets were lyzed in RIPA buffer and transferred to scintillation vials for quantification of the radioactive signal using Tricarb liquid scintillation counter. The transport activity of MFSD7c was expressed as DMP for [3H] isotopes and CPM [14C] isotopes. For dose-dependent transport activity, HEK293 cells were similarly transfected with hMfsd7c or hMfsd7cS203Y mutant plasmid alone or co-transfected with hChKA. Transport activity was conducted with 20, 45, 90, and 250 µM [3H] choline in DMEM containing 10% FBS for 1 h at 37 °C. For the time-dependent assay, hMfsd7c was transfected or co-transfected with hChKA. Transfected HEK293 cells were incubated with 100 µM [3H] choline in DMEM containing 10% FBS for 15, 30, 60, and 120 min at 37 °C. For transport assay with [14C] ethanolamine, [3H] L-carnitine, and [3H] acetyl-carnitine, [3H] palmitoyl-carnitine, [14C] octanoyl-carnitine, HEK293 cells were co-transfected with MFSD7c and hChKA as described above. Transport assays were performed with 100 µM [14C] ethanolamine, 1 mM [3H] L-carnitine, 1 mM [3H] acetyl-carnitine, 10 µM [3H] palmitoyl-carnitine, or 100 µM [14C] octanoyl-carnitine in DMEM with 10% FBS for 1 h. Cells were washed once with cold plain DMEM and lyzed with RIPA buffer for radioactive quantification. For import assays with the missense mutants, these plasmids were co-transfected with hChKA to HEK293 cells. Import assays were performed with 100 µM [3H] choline in DMEM containing 10% FBS for 60 min. Radioactive signals from cells were quantified and converted to a percentage of WT activity.

For export assays, HEK293 cells were transfected with empty, hMfsd7c, or mMfsd7c plasmids. After 24 h of transfection, cells were loaded with 500 µM [3H] choline for 1 h. The cells were then washed to remove the remaining [3H] choline in the medium and incubated with plain DMEM medium to stimulate the release. The cells were harvested at 0 (before release) and 1 h (after release) after incubation with DMEM for radioactive quantification as described above.

Sodium and pH-dependent transport assays

For sodium-dependent assays, sodium is replaced with lithium in transport buffer (NaCl buffer: 150 mM NaCl, 5 mM KCl, 10 mM HEPES-Na, pH 7.4; LiCl buffer: 150 mM LiCl, 5 mM KCl, 10 mM HEPES, pH 7.4 with HCl). For pH-dependent assays, NaCl buffer was adjusted to pH 6.5 and pH 8.5, respectively. For transport assay conditions, overnight hMfsd7c and hChKA co-transfected HEK293 cells were washed once with the same buffer before being incubated with 100 µM [3H] choline for 15 min at 37 °C. Cells were lyzed in RIPA buffer and mixed with scintillation fluid for radioactive quantification.

Electrophysiology

For single-cell recording of membrane potential alteration during transport of choline, HEK293FT cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin and streptomycin and maintained in 5% CO2 incubator at 37 °C. For transfection, cells were seeded on the Petri dishes and grown overnight. Subsequently, 2 µg of empty pIRES2-EGFP (empty plasmid), pIRES2-EGFP-hMfsd7c or mutant pIRES2-EGFP-S203Y plasmid was co-transfected with 2 µg of pIRES2-RFP-hChKA plasmid using lipofectamine 2000. The transfected cells were incubated for 24 h in 5% CO2 incubator at 37 °C. At 48 h post-transfection, the cells were split and seeded on poly-D-lysine-coated coverslips for one more day before recording.

Whole-cell patch clamp recordings and data analysis

To record the changes in resting membrane potential, the internal solution (pipette solution) contained 130 mM K-gluconate, 10 nM KCl, 5 mM EGTA, 10 mM HEPES, 1 mM MgCl2, 0.5 mM Na3GTP, 4 mM Mg-ATP, 10 mM Na-phoshocreatine, pH 7.4 (adjusted with KOH) was filled in the pipette tip. The external solution contained: 10 mM Glucose, 125 mM NaCl, 25 mM NaHCO3, 1.25 mM NaH2PO4·2H2O, 2.5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, pH 7.4 (300–310 mOsm). Single cells with both EGFP (empty, hMfsd7c or S203Y plasmid) and RFP (hChKA) fluorescence were visualized under a fluorescent microscope for patching. Resting membrane potential was recorded under the current clamp with an Axopatch200B or multiclamp 200B amplifier (Molecular Device) with 0 pA current injection. Subsequently, 200 µM choline was perfused to the cells after 2 min of stable baseline recording. After 5 min of recording, the ligand was washed away. Approximately, 10 individual cells were recorded for each condition. The data were analyzed using GraphPad Prism V software (San Diego, CA) and Microsoft (Seattle, WA) Excel. Data are expressed as mean ± SEM.

Expression of hMfsd7c in S. cerevisiae Hnm1 mutants

S. cerevisiae acquires choline via HNM1. The S. cerevisiae Hnm1 mutants were acquired from Dharmacons. WT and Hnm1 mutant yeasts were grown in Yeast extract-Peptone-Dextrose (YPD) medium at 30 °C overnight before transformation. The pESC-HIS-hMfsd7c plasmid was transformed into WT and Hnm1 mutant yeasts following the previously described protocol.43 After heat shock at 42 °C for 30 min and recovery in YPD for 2 h, the yeasts were spread on 2% agar plates prepared in yeast nitrogen base (YNB) medium (Sigma-Aldrich) supplemented with 2% glucose and yeast synthetic drop-out medium supplements without histidine (Sigma-Aldrich). Positive colonies were picked up randomly and further confirmed by transport assay and western blot.

Transport assay for yeast cells

WT, Hnm1 mutant and transformed yeast cells were grown in YNB medium with 2% glucose and drop-out medium supplements without histidine at 30 °C overnight followed by induction of hMfsd7c expression in YNB medium with 2% galactose for at least 5 h at 30 °C. An 800 µL of yeast cells at OD600 = 1 was centrifuged, washed twice with distilled water, then incubated in PBS containing 100 µM [3H] choline for 10 min. The transport assay was stopped by washing twice with PBS containing 100 µM choline. Yeast cells were lyzed with lysis buffer containing 2% Triton-X, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0) at 95 °C for 5 min for radioactive quantification by liquid scintillation counter.

Western blot analysis

For protein extraction, transfected HEK293 cells with hMfsd7c, mMfsd7c, hMfsd7c mutants were lysed with RIPA buffer, respectively. For validation of deletion of Mfsd7c in mice, micro-vessels from PBS-perfused brains of WT and EcMfsd7c-KO mice were isolated, homogenized and lyzed with RIPA buffer. For Western blot analysis of hMfsd7c expression in yeasts, 1 mL of yeast culture at OD600 = 1 from WT, Hnm1 mutant yeasts or transformed yeasts was used for total protein extraction. BCA assay was used for total protein quantification. These antibodies were used: VDAC (Cell Signaling, CS4866), CoxIV (Invitrogen, MA5-15686), OPA1 (BD biosciences, 612602), MRPS35 (Protein biotech, 16457), CHKA (Cell Signaling, CS13422S), HMOX1 (Proteintech, 10701-1-AP), NDUFS1 (Proteintech, 12444-1-AP). Western blot analysis was conducted as previously described.2

Fowler patient recruitment

A 5-month-old infant born at 39w1d with multifocal epilepsy, ventriculomegaly, developmental delay, and lissencephaly was identified via GeneMatcher collaboration.44 She was born to a 38-year-old mother. Pregnancy was complicated by gestational diabetes and maternal hypertension. There was concern for lissencephaly on prenatal ultrasound and MRI. Prenatal CMA and karyotype were normal. She was delivered vaginally complicated by shoulder dystocia. She required brief positive pressure ventilation and was admitted to the NICU on NIPPV. Apgars at 1 and 5 min were 4 and 9, respectively. Birth parameters include length of 49 cm (47th percentile WHO), weight of 2.86 kg (20th percentile WHO), and head circumference of 32 cm (6th percentile WHO). Her whole exome sequencing at birth showed two variants of uncertain significance in FLVCR2, c.826C>T (Maternal) and c.1448T>G (Paternal). The missense mutations were confirmed by Sanger sequencing. There was no family history of a similar presentation. This was the first pregnancy between this patient’s parents. Mother had a previous miscarriage and a healthy older daughter. Parents report no consanguinity. The IRB approval for this case study was exempted by the Human Research Protection Office (HRPO) of Washington University in St. Louis.

RNA sequencing and analysis

For bulk RNA sequencing of whole brains from E14.5 WT and KO embryos from normal chow diet, this dataset has been deposited in GEO database with the accession number: GSE148854. For RNA sequencing of isolated endothelial cells from heterozygous and KO embryos at E14.5, we re-analyzed the dataset GSE129838.7 For RNA sequencing of whole brains from E14.5 WT embryos from CDD, pregnant WT mice were fed with CDD at the mating day until embryo collection. The whole brain of E14.5 embryos was collected for bulk RNA sequencing. This dataset has been deposited in the GEO database with the following accession number: GSE239589.

Immunofluorescence staining for neocortical vessels

Mfsd7c+/– female mice were fed on CDD for 2 weeks and then time-mated with the heterozygous male. E14.5 embryos were dissected from the pregnant female for collection of brains for immunostaining of CNS vessel morphology. Brain section was permeabilized in 0.5% triton X-100 (PBST), blocked in 5% normal goat serum and then incubated with anti-mouse GLUT1 (Abcam, ab40084,1:200) at 4 °C overnight, followed by 3 washes in PBS at 10-min intervals. Then, the goat anti-mouse antibody (Alexa Fluor 488, ThermoFisher Scientific, A11034,1:500) was added to the slides for 1-h incubation at room temperature to visualize the GLUT1 signal. Images were taken by the confocal microscope Zeiss LSM710. Vessel phenotypes were analyzed using measurement tools in ImageJ.

Choline and acetylcholine assays

Choline and acetylcholine from plasma, liver, and brain samples were measured by fluorometric assay (Abcam: ab65345). Tissues were homogenized in choline buffer. Choline and acetylcholine were assayed according to the manufacturer’s instructions.

Transmission electron microscope (TEM)

WT (n = 3) and Mfsd7c–/– (n = 3) embryos at E14.5 were collected from the same pregnant mice. Samples were fixed with a solution containing 2.5% glutaraldehyde in 0.1 M PBS buffer (pH 7.3). Samples were then washed and post-fixed with 1% buffered osmium. The samples were dehydrated in increasing concentrations of ethanol and then infiltrated and embedded in Araldite medium. The samples were then polymerized in a 60 °C oven for approximately 2 days. Ultrathin sections were cut using a Leica Ultracut microtome and then stained with lead citrate. The stained samples were examined in a JEM 1400 transmission electron microscope (JEOL USA, Inc., Peabody, MA) using an accelerating voltage of 100 kV. Digital images were obtained using a CMOS Matataki Flash 2K CCD camera.

Statistical analysis

Data were analyzed using GraphPrism9 software. Statistical significance was calculated using parametric and non-parametric t-tests, one- or two-way ANOVA as indicated in the figure legends. P < 0.05 was considered as statistically significant.