Impaired glycine neurotransmission causes adolescent idiopathic scoliosis

Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity, affecting millions of adolescents worldwide, but it lacks a defined theory of etiopathogenesis. Because of this, treatment of AIS is limited to bracing and/or invasive surgery after onset. Preonset diagnosis or preventive treatment remains unavailable. Here, we performed a genetic analysis of a large multicenter AIS cohort and identified disease-causing and predisposing variants of SLC6A9 in multigeneration families, trios, and sporadic patients. Variants of SLC6A9, which encodes glycine transporter 1 (GLYT1), reduced glycine-uptake activity in cells, leading to increased extracellular glycine levels and aberrant glycinergic neurotransmission. Slc6a9 mutant zebrafish exhibited discoordination of spinal neural activities and pronounced lateral spinal curvature, a phenotype resembling human patients. The penetrance and severity of curvature were sensitive to the dosage of functional glyt1. Administration of a glycine receptor antagonist or a clinically used glycine neutralizer (sodium benzoate) partially rescued the phenotype. Our results indicate a neuropathic origin for “idiopathic” scoliosis, involving the dysfunction of synaptic neurotransmission and central pattern generators (CPGs), potentially a common cause of AIS. Our work further suggests avenues for early diagnosis and intervention of AIS in preadolescents.


Cell surface biotinylation assay
HEK293T cells were plated onto 6-well plates at 70%-80% confluence and transfected with indicated plasmids, as well as an unrelated membrane protein HA-tagged Vangl2 as an internal transfection control.48 hours after transfection, cells were rinsed with ice-cold PBS three times and incubated for 30 minutes at 4°C in 0.5 mL PBS containing the 1 mg/mL nonpermeable EZ-Link™ Sulfo-NHS-SS-Biotin reagent (ThermoFisher Scientific, #21331, U.S.).The cells were then incubated with 100 mM L-lysine (Sigma Aldrich, #L5501, U.S.) in PBS for 30 minutes at 4°C to quench the biotinylation reaction.After three additional washing with ice-cold PBS, the cells were lysed with 1 mL of modified RIPA lysis buffer (150 mM NaCl, 5 mM EDTA, 25mM Tris, 0.5% deoxycholate, 1%Triton X-100, 1% NP-40 and 0.1% SDS, pH 7.4) for 30 minutes on ice.Cell lysates were cleared by centrifugation at 14000 rpm at 4°C for 30 minutes, and 200 μL of cell lysates were collected and served as total cell lysate fraction.The biotinylated proteins were recovered by incubating the remaining cell lysates for 2 hours at room temperature with Pierce™ high-capacity streptavidin agarose resin (ThermoFisher Scientific, #20359, U.S.).After three washing of the agarose resin with 1 mL of lysis buffer, the biotinylated proteins were eluted with 2X Laemmli sample buffer (4% SDS, 20% Glycerol, 120 mM Tris, 0.02% bromophenol blue, 100mM dithiothreitol, pH 7.4).Total cell lysates and the biotinylated cytomembrane fraction were stored at -80°C until further use.

Western blotting
The harvested protein samples were loaded on SDS-PAGE gel (4% stacking and 8% or 10% separating gels).The samples were transferred to polyvinylidene difluoride membranes (PVDF, Merck, #IPVH00010, U.S.) by a wet western blot transfer apparatus and then blocked with 5% Bovine Serum Albumin (BSA, Sigma Aldrich, #A3912, U.S.) in PBS containing 0.1% Tween 20.The PVDF membrane was incubated with indicated primary antibodies overnight at 4°C and then secondary antibodies for 2 hours at room temperature.Super Signal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, #34580, U.S.) was used to detect immunoblot signals.

Immunofluorescence and confocal microscopy
MDCK cells were seeded onto slides in 24 well plates and grown to 50%-60% confluence.Cells were transfected with indicated plasmids and incubated for 48 hours before fixation.Cells were rinsed three times with cold PBS and then fixed with 4% paraformaldehyde (PFA, Sigma Aldrich, #158127, U.S.) in PBS at room temperature for 10 minutes, followed by permeabilization with 0.5% Triton X-100/PBS for 5 minutes.After washing with 0.1% Triton X-100/PBS twice, cells were blocked with 3% BSA in 0.1% Triton X-100/PBS for 1 hour at room temperature, and incubated with indicated primary antibodies overnight at 4°C.After rinsing 3 times for 5 minutes each with 1 X PBS with 0.1%

Zebrafish brain disassociation for extracellular glycine level measurement
At 7 dpf, zebrafish embryos were anesthetized in 0.01% MS222 (pH=7.0,Sigma Aldrich, #10521, U.S.) in 1X PBS.The fish's whole brain was dissected out under a stereo microscope by using pairs of ultra fine forceps.Ten brains were collected in one tube and PBS was removed by low-speed centrifugation (1500 rpm for 5 minutes at 4 °C).100 μL TrypLE™ Express Enzyme (1X, ThermoFisher Scientific, #AC 12604013, U.S.) was added to dissociate brains, followed by a 10-minute incubation in a 37 °C water bath.Gentle pipetting up and down was applied to facilitate dissociation, and the reaction was stopped by adding 100 μL of ddH2O.The sample was then centrifuged at 1500 rpm for 5 minutes at 4 °C, and the cell-free supernatant was collected as the extracellular fluid of the zebrafish.The glycine concentration was measured using the fluorometric glycine assay kit (Abcam, #ab211100, UK), according to the manufacturer's instructions.Fluorescence was measured at Ex/Em 535/587nm in endpoint mode using a microplate reader (Varioskan Flash, ThermoFisher Scientific, U.S.).

Antibody staining and imaging of zebrafish
The fertilized eggs were collected in the morning and cultured in E3 medium at 28.5 °C for 12 hours, and then 1-phenyl 2-thiourea (PTU, ThermoFisher Scientific, #AC207250050, U.S.) was added into E3 medium at a final concentration 0.003% to prevent pigmentation.At 24 hours post-fertilization (hpf), the unhatched embryos were manually dechorionated using ultra-fine-tip forceps under a stereomicroscope.The embryos were fixed with 4% PFA in PBS for 1-2 hours at room temperature, followed by washing three times in 1% Triton-X/PBS for 5 minutes each.The fixed embryos were blocked in 10% goat serum (Sigma Aldrich, #G9023, U.S.) in 1% Triton-X/PBS for 1-2 hours at room temperature and were then incubated with primary antibody diluted in blocking solution overnight at 4 °C with rotation.
Next day, the embryos were washed five times in 1% Triton-X/PBS for 10 minutes each at room temperature with rocking, and then incubated with secondary antibody diluted in blocking solution for 2 hours at room temperature (or overnight at 4 °C).After three times washing of 1% Triton-X/PBS, the embryos were embedded with 0.8-1% low-melting agarose gel (SeaPlaque™ Agarose, Lonza Bioscience, #50101, U.S.) and then transferred onto the 6 cm confocal dishes with desired direction, which were later subjected to Zeiss LSM800 confocal microscopy.Images were analyzed by Zeiss Zen software.
To quantify the total level of activated spinal cord neurons in wild-type and slc6a9 m/m mutant zebrafish, embryos with a Tg(elavl3-H2B-GCaMP6s) transgenic background were collected at 24 hpf and fixed in 4% PFA in 1X PBS for 1-2 hours at room temperature.After three washes with 1X PBS, the embryos were embedded in 0.8-1% low-melting agarose gel and transferred onto 6 cm confocal dishes with the desired orientation.Snap captures of the dorsal view were obtained for both wild-type and slc6a9 m/m mutant embryos using the same confocal settings (Laser wavelength: 488nm; 0.94%; Pinhole: 32µm; Detector Gain: 700V) in a Zeiss LSM800 confocal microscope.The images were analyzed using ImageJ software, with the spinal cord neuronal GCaMP6s signals used for quantification.

Live staining and imaging of zebrafish
Fertilized zebrafish eggs were collected in the morning and cultured in E3 medium until the desired developmental stage.Live staining was performed on zebrafish embryos using vital dyes to visualize cell internal membranes and acidic notochord vacuoles.Specifically, 48 hpf wild-type and mutant embryos were stained with 100 μM BODIPY TR Methyl Ester (MED, Invitrogen, # C34556, U.S.) for 1 hour, followed by three washes with E3 medium.At 6 dpf, LysoTracker GreenDND-26 (Invitrogen, # L7526, U.S.) was used to live-label acidic notochord vacuoles.Wild-type and mutant zebrafish were stained with 50 μM LysoTracker GreenDND-26 for 2 hours, followed by three washes with E3 medium.The stained embryos were anesthetized with MS222, mounted in 0.8-1% low-melting agarose gel, and transferred onto 6 cm confocal dishes with the desired orientation.Confocal imaging was performed using a Zeiss LSM800 confocal microscope, and images were analyzed with Zeiss Zen software.Zebrafish live skeletal staining was performed by incubating embryos in a 0.2% Calcein (pH 7.0, Sigma Aldrich, #C0875, U.S.) solution for 10 minutes, followed by three washes with E3 medium.Calcein-stained embryos were imaged using an Olympus IX71 microscope (Olympus, Japan).

Zebrafish mRNA microinjection
The zygotes from slc6a9 m/+ and slc6a9 m/+ mating pairs were used for mRNA microinjection.The SLC6A9 mRNA encoding human wild type, Y206F or R662W variant GLYT1 was synthesized using the mMESSAGE mMACHINE™ T3 Transcription Kit (ThermoFisher Scientific, #AM1348, U.S.).60, 120, 200, and 400 pg of wild type GLYT1 mRNA were respectively injected into the zygotes to test the optimal rescue dosage.As the injection of 200 pg of wild type GLYT1 mRNA significantly rescued the curvature phenotype of slc6a9 m/m fish, the same dosage of Y206F and R662W variant mRNA was used for rescue experiments.At 7 dpf, all fish were examined for axial phenotype and then lysed for genotyping.

Swimming behavior
At 7 dpf, zebrafish larvae were individually transferred into 48-well plate.The swimming distance and trajectory over 10 minutes was recorded under Leica MZ10F Fluorescence Stereomicroscope with a vision recording system.Analysis was performed by using idTracker in MATLAB (9).After recording, all the fish were sacrificed for genotyping.
Chen S, Zhou Y, Chen Y, and Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor.Bioinformatics.Supplemental analysis of AIS Family 1 and 2. (A) Workflow of genetic analysis.WGS data of Family 1 and 2 were analyzed independently.Linkage analysis identified a locus in chromosome 1 containing SLC6A9, in which two SLC6A9 variants, c.1984C>T, p.R662W and c.617A>T, p.Y206F are associated with Family 1 and 2, respectively.(B) Linkage analysis of Family 1 and 2. The X-axis represents the relative location on the chromosome.One single centimorgan (cM) is approximately equal to 1 million base pairs (bps).The Y-axis represents the exponential LOD (logarithm of the odds) scores from nonparametric linkage analyses.

Vendor Catalog. No. WB IF
Triton X-100, secondary antibodies coupled to Alexa Fluor 488 or 568 (1:1000, Life Technologies, U.S.) were applied at the room temperature for 1 hour.After rinsing 3 times with 1 X PBS with 0.1% Triton X-100, slides were mounted with ProLong Diamond Antifade Mountant with DAPI (ThermoFisher Scientific, #P36962, U.S.) and subjected to Zeiss LSM800 confocal microscopy.Images were analyzed by Zeiss Zen software.ImageJ software was used to quantify the cytomembrane and cytosolic signals of GLYT1.Signal intensities of the entire cell and intracellular fluorescence were respectively measured after calibration of background signals around the cell.The membrane signal intensities were calculated by subtracting the intracellular intensities from the total cell intensities.

. Spinal radiographs of familial and sporadic AIS patients.
Patients were subjected to full spine X-ray scanning in a relaxed and standing posture.The posteroanterior whole spine radiographs are shown.DKCH: Duchess of Kent Children's Hospital; PUMCH: Peking Union Medical College Hospital.

Figure 4 Supplemental Figure 4. Quantification of functional assays of GLYT1 variants
.(A) Statistical ratio of cytomembrane to total cell signal intensities of GLYT1 wild type and variants.The number of calculated cells of each group is indicated on the bottom of each box.Boxes show the median and IQRs with all individual data points superimposed.1-way ANOVA test, ****P < 0.0001; ns, no significance.(B) The normalized glycine uptake activities of GLYT1 variants.Glycine uptake activities of SLC6A9 variants were normalized to cytomembrane protein expression levels.Each data dot represents one independent experiment.Data are shown as means ±SEM.1way ANOVA test.ns, no significance.
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