Abstract
SLC7A10 (Asc-1) is a sodium-independent amino acid transporter known to facilitate transport of a number of amino acids including glycine, L-serine, L-alanine, and L-cysteine, as well as their D-enantiomers. It has been described as a neuronal transporter with a primary role related to modulation of excitatory glutamatergic neurotransmission. We find that SLC7A10 is substantially enriched in a subset of astrocytes of the caudal brain and spinal cord in a distribution corresponding with high densities of glycinergic inhibitory synapses. Accordingly, we find that spinal cord glycine levels are significantly reduced in Slc7a10-null mice and spontaneous glycinergic postsynaptic currents in motor neurons show substantially diminished amplitudes, demonstrating an essential role for SLC7A10 in glycinergic inhibitory function in the central nervous system. These observations establish the etiology of sustained myoclonus (sudden involuntary muscle movements) and early postnatal lethality characteristic of Slc7a10-null mice, and implicate SLC7A10 as a candidate gene and auto-antibody target in human hyperekplexia and stiff person syndrome, respectively.
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Introduction
Motor neuron activity is tonically regulated by inhibitory currents, mediated through spontaneous and evoked synaptic release of glycine and GABA. Binding of glycine to postsynaptic glycine receptors results in an influx of chloride anions, hyperpolarizing the target cell and increasing the threshold for action potential generation.
Synaptic concentrations of glycine are modulated by two sodium-dependent transporters, SLC6A9 (GLYT1) and SLC6A5 (GLYT2). GLYT1 is primarily expressed by astrocytes but is also found within pre- and post-synaptic terminals of glutamatergic synapses throughout the central nervous system (CNS), where it likely plays a role in modulating both excitatory and inhibitory synaptic activity. GLYT2 is solely expressed in presynaptic terminals of glycinergic inhibitory neurons. Genetic mouse models for both of these transporters have been valuable for understanding their synaptic functions. Complete GLYT1 deficiency is associated with hypotonia and respiratory arrest due to altered pattern generation in brainstem respiratory centers due to excessive, tonic inhibition; thus the presumptive normal function of GLYT1 is clearance of glycine from inhibitory synapses and termination of glycinergic signaling1,2. GLYT2 deficiency, in contrast, is associated with hypertonia, ataxia, exaggerated startle responses called hyperekplexia, and early postnatal lethality; this is thought to reflect impaired reuptake of vesicular glycine into glycinergic neurons following glycine release (presynaptic recycling), leading to insufficient inhibitory activity3,4.
SLC7A10 (Asc-1) is a sodium-independent amino acid transporter known as the primary mediator of D-serine transport within the central nervous system5. Its primary function has been thought to be related to regulation of NMDA receptor activity at glutamatergic synapses via D-serine clearance46. Male and female mice were used equally, and where possible all studies were conducted on matched littermates. Mice were genotyped by quantitative PCR through Transnetyx (Cordova, TN). Experiments were performed in accordance with protocols approved by the Animal Care and Use Committee at Johns Hopkins University.
Immunofluorescence
P2, P16-P21, or 8–10 week-old mice were deeply anesthetized with 0.4 mg/g 2,2,2-tribromoethanol (Avertin), then perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB), post-fixed overnight at 4 °C, and cryoprotected in 30% glycerol in 0.1 M PB overnight. 12–40 μm tissue sections were cut on a freezing microtome and processed free-floating. Primary antibodies were diluted in PBS containing 5% goat serum and 0.3% triton-X-100, and incubated with tissue sections for 18 hours at 4 °C in a humidified chamber. Primary antibodies included: chicken-anti-GFP (1:500, Aves or Abcam), mouse anti-NeuN (1:200, Millipore), mouse anti-GFAP (1:500, NeuroMab, clone N206A/8 or Millipore, clone GA5), rabbit anti-SLC7A10, N-term (1:250–1:500, Acris, lot #FGI263), rabbit anti-beta-galactosidase (1:500, MP/Cappel), mouse anti-GLYT2 (1:500, Millipore), mouse anti-OLIG2 (1:500, Millipore), mouse anti-PSD95 (1:2000, NeuroMab), and mouse anti-GPHN (1:200, Synaptic Systems). Secondary detection was conducted at room temperature for 2 hours, using the following antibodies: goat anti-rabbit Alexa Fluor 488, 568, or 647, goat anti-mouse Alexa Fluor 488 or 594, goat anti-chicken Alexa Fluor 488, goat anti-guinea pig Alexa Fluor 594 (1:500, all from Invitrogen). Images were acquired using Zeiss Meta 510, Zeiss Axiovis, or Zeiss 800 and 880 Airyscan microscopes.
Immunoblotting
Spinal cord homogenates were prepared in T-PER tissue protein extraction reagent (ThermoFisher Scientific) containing protease inhibitors (Roche). Protein concentration was measured by BCA assay (ThermoFisher Scientific). Protein equivalents from each sample were boiled in LDS (ThermoFisher Scientific) containing 2.5% β-mercaptoethanol and electrophoresed on 4–12% Bis-Tris acetate gels (BioRad). Protein was transferred to nitrocellulose (BioRad) in transfer buffer containing 25 mM Tris, pH 8.3, 192 mM glycine, 0.1% (w/v) SDS, and 20% methanol. Membranes were blocked for 1 h in 20 mM Tris, 500 mM NaCl, pH 7.5 (TBS) containing 5% milk, then incubated overnight at 4 °C with primary antibodies diluted in TBS-0.1% tween-20 (TBST) containing 5% milk. After washing in TBST, membranes were incubated with HRP-conjugated goat-anti-rabbit or goat-anti-mouse antibodies (GE Life Sciences) and detected using ECL reagents (GE Life Sciences). Primary antibodies included rabbit anti-beta-galactosidase (1:500, MP/Cappel), mouse anti-beta-tubulin (1:1000, Sigma), rabbit anti-SLC7A10, N-term (1:1000, Acris, lot #FGI263), mouse anti-GLYR (1:1000, Synaptic Systems), rabbit anti-VIAAT (1:1000, Aviva), rabbit anti-GLYT1 (1:1000, Aviva), and mouse anti-GLYT2 (1:1000, Millipore). Densitometry was performed using ImageJ.
Amino acid quantification
Amino acid profiling was conducted at the West Coast Metabolomics Center at the University of California, Davis, using ALEX-CIS GCTOF (Automated Liner Exchange-Cold Injection System Gas Chromatography Time-of-Flight) mass spectrometry. Relative normalization was achieved as follows: for analyte i of sample j, analyteij, normalized = [(peak height of analyteij, raw) / ∑ (peak heights of all annotated analytesj)] · [average peak height of all annotated analytes for all samples].
Acute slice preparation
P10-P13 mice were deeply anesthetized with isofluorane. A ventral laminectomy was performed in ice-cold ACSF containing (in mM) NaCl 120, KCl 2.5, CaCl2 2, MgCl2 2, NaHCO3 26, NaH2PO4 1, glucose 11. Thoracolumbar segments were rested in an agar block fixed vertically on a Leica vibratome; 400 μm transverse (axial) slices were cut and then incubated in ACSF for 1 h at 32 °C before transferring to the recording chamber. ACSF used for both dissection and recording was saturated with 95% O2 and 5% CO2.
Whole-cell patch-clamp recording
Whole-cell recordings were performed using an Axon 200B amplifier. To record and isolate mIPSCs, electrodes with a tip resistance of 3–5 MΩ were filled with a high-chloride internal solution containing (in mM) CsCl 147, Na2-phosphocreatine 5, HEPES 10, EGTA 1, MgATP 2, Na2GTP 0.3. Motor neurons were visualized by DIC optics using a 60x water-immersion objective. Cells were clamped at −70 mV and perfused with 1 μM TTX, 20 μM NBQX, and 50 μM AP5. To further isolate GABA-receptor-mediated or glycine receptor-mediated synaptic currents, 5 μM strychnine or 100 μM picrotoxin was perfused, respectively. To record and isolate AMPA/NMDA receptor-mediated excitatory synaptic currents (mEPSCs), electrodes were filled with an internal solution containing (in mM) Cs-methanesulfonate 115, CsCl 20, Na2-phosphocreatine 10, HEPES 10, EGTA 0.6, MgCl2 2.5, MgATP 2, and Na2GTP 0.3; neurons were clamped at −70 mV and perfused with 1 μM TTX, 5 μM strychnine, and 100 μM picrotoxin. Electrophysiological studies were conducted by an investigator (Y.W.) masked to genotype with recordings from at least 10 motor neurons (n ≥ 3 biological replicates for each genotype).
Statistical analysis
Quantitative experiments were conducted with n ≥ 3 age-matched biological replicates for each genotype, and littermates where possible. Cell counts were conducted using Imaris (Bitplane). Statistical analyses (one-way ANOVA with Bonferroni or Benjamini-Hochberg correction and Welch’s t-tests as appropriate) were conducted using Stata, version 12.1 (StataCorp, College Station, Texas) or R, version 3.1.3. mIPSCs were analyzed by Mini Analysis Program, version 6.0.3 (Synaptosoft, Georgia). α < 0.05 was considered statistically significant.
Additional Information
How to cite this article: Ehmsen, J. T. et al. The astrocytic transporter SLC7A10 (Asc-1) mediates glycinergic inhibition of spinal cord motor neurons. Sci. Rep. 6, 35592; doi: 10.1038/srep35592 (2016).
References
Gomeza, J. et al. Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition. Neuron 40, 785–96 (2003).
Alfadhel, M. et al. Mutation in SLC6A9 encoding a glycine transporter causes a novel form of non-ketotic hyperglycinemia in humans. Hum. Genet., doi: 10.1007/s00439-016-1719-x (2016).
Gomeza, J. et al. Deletion of the mouse glycine transporter 2 results in a hyperekplexia phenotype and postnatal lethality. Neuron 40, 797–806 (2003).
Gomeza, J., Ohno, K. & Betz, H. Glycine transporter isoforms in the mammalian central nervous system: structures, functions and therapeutic promises. Curr. Opin. Drug Discov. Devel. 6, 675–82 (2003).
Rutter, A. R. et al. Evidence from gene knockout studies implicates Asc-1 as the primary transporter mediating D-serine reuptake in the mouse CNS. Eur. J. Neurosci. 25, 1757–66 (2007).
**e, X. et al. Lack of the alanine-serine-cysteine transporter 1 causes tremors, seizures, and early postnatal death in mice. Brain Res. 1052, 212–21 (2005).
Rosenberg, D. et al. Neuronal D-serine and glycine release via the Asc-1 transporter regulates NMDA receptor-dependent synaptic activity. J. Neurosci. 33, 3533–44 (2013).
Fukasawa, Y. et al. Identification and characterization of a Na+-independent neutral amino acid transporter that associates with the 4F2 heavy chain and exhibits substrate selectivity for small neutral D- and L-amino acids. J. Biol. Chem. 275, 9690–8 (2000).
Helboe, L., Egebjerg, J., Moller, M. & Thomsen, C. Distribution and pharmacology of alanine-serine-cysteine transporter 1 (asc-1) in rodent brain. Eur. J. Neurosci. 18, 2227–38 (2003).
Matsuo, H. et al. High affinity D- and L-serine transporter Asc-1: cloning and dendritic localization in the rat cerebral and cerebellar cortices. Neurosci. Lett. 358, 123–6 (2004).
Shao, Z., Kamboj, A. & Anderson, C. M. Functional and immunocytochemical characterization of D-serine transporters in cortical neuron and astrocyte cultures. J. Neurosci. Res. 87, 2520–30 (2009).
McIntire, S. L., Reimer, R. J., Schuske, K., Edwards, R. H. & Jorgensen, E. M. Identification and characterization of the vesicular GABA transporter. Nature 389, 870–6 (1997).
Rousseau, F., Aubrey, K. R. & Supplisson, S. The glycine transporter GlyT2 controls the dynamics of synaptic vesicle refilling in inhibitory spinal cord neurons. J. Neurosci. 28, 9755–68 (2008).
Safory, H. et al. The alanine-serine-cysteine-1 (Asc-1) transporter controls glycine levels in the brain and is required for glycinergic inhibitory transmission. EMBO Rep. 16, 590–8 (2015).
Rees, M. I. et al. Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease. Nat. Genet. 38, 801–6 (2006).
Lovatt, D. et al. The transcriptome and metabolic gene signature of protoplasmic astrocytes in the adult murine cortex. J. Neurosci. 27, 12255–66 (2007).
Cahoy, J. D. et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–78 (2008).
Doyle, J. P. et al. Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell 135, 749–62 (2008).
Lovatt, D. & Nedergaard, M. The astrocyte transcriptome in Neuroglia (eds Kettenmann, H. & Ransom, B. R. ) (Oxford University Press, 2013).
Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–47 (2014).
Ottersen, O. P., Storm-Mathisen, J. & Laake, J. H. Cellular and subcellular localization of glycine studied by quantitative electron microscopic immunocytochemistry in Glycine Neurotransmission (eds Ottersen, O. P. & Storm-Mathisen, J. ) (Wiley, 1990).
Tibbetts, A. S. & Appling, D. R. Compartmentalization of mammalian folate-mediated one-carbon metabolism. Annu. Rev. Nutr. 30, 57–81 (2010).
Sato, K., Yoshida, S., Fujiwara, K., Tada, K. & Tohyama, M. Glycine cleavage system in astrocytes. Brain Res. 567, 64–70 (1991).
Ichinohe, A. et al. Glycine cleavage system in neurogenic regions. Eur. J. Neurosci. 19, 2365–70 (2004).
Lamers, Y. et al. Production of 1-carbon units from glycine is extensive in healthy men and women. J. Nutr. 139, 666–71 (2009).
Yang, J. H. et al. Brain-specific Phgdh deletion reveals a pivotal role for L-serine biosynthesis in controlling the level of D-serine, an N-methyl-D-aspartate receptor co-agonist, in adult brain. J. Biol. Chem. 285, 41380–90 (2010).
Bogdanik, L. P., Chapman, H. D., Miers, K. E., Serreze, D. V. & Burgess, R. W. A MusD retrotransposon insertion in the mouse Slc6a5 gene causes alterations in neuromuscular junction maturation and behavioral phenotypes. PLoS One 7, e30217 (2012).
Mohn, A. R., Gainetdinov, R. R., Caron, M. G. & Koller, B. H. Mice with reduced NMDA receptor expression display behaviors related to schizophrenia. Cell 98, 427–36 (1999).
Harvey, R. J. & Yee, B. K. Glycine transporters as novel therapeutic targets in schizophrenia, alcohol dependence and pain. Nat. Rev. Drug Discov. 12, 866–85 (2013).
Chen, L., Muhlhauser, M. & Yang, C. R. Glycine transporter-1 blockade potentiates NMDA-mediated responses in rat prefrontal cortical neurons in vitro and in vivo. J. Neurophysiol. 89, 691–703 (2003).
Kinney, G. G. et al. The glycine transporter type 1 inhibitor N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine potentiates NMDA receptor-mediated responses in vivo and produces an antipsychotic profile in rodent behavior. J. Neurosci. 23, 7586–91 (2003).
Gabernet, L. et al. Enhancement of the NMDA receptor function by reduction of glycine transporter-1 expression. Neurosci. Lett. 373, 79–84 (2005).
Brown, J. M. et al. In vitro characterization of a small molecule inhibitor of the alanine serine cysteine transporter-1 (SLC7A10). J. Neurochem. 129, 275–83 (2014).
Zeilhofer, H. U., Wildner, H. & Yevenes, G. E. Fast synaptic inhibition in spinal sensory processing and pain control. Physiol. Rev. 92, 193–235 (2012).
Harvey, R. J., Topf, M., Harvey, K. & Rees, M. I. The genetics of hyperekplexia: more than startle! Trends Genet. 24, 439–47 (2008).
Alexopoulos, H., Akrivou, S. & Dalakas, M. C. Glycine receptor antibodies in stiff-person syndrome and other GAD-positive CNS disorders. Neurology 81, 1962–4 (2013).
Shiang, R. et al. Mutations in the α1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia. Nat. Genet. 5, 351–8 (1993).
Rees, M. I., Andrew, M., Jawad, S. & Owen, M. J. Evidence for recessive as well as dominant forms of startle disease (hyperekplexia) caused by mutations in the alpha 1 subunit of the inhibitory glycine receptor. Hum. Mol. Genet. 3, 2175–9 (1994).
Rees, M. I. et al. Hyperekplexia associated with compound heterozygote mutations in the beta-subunit of the human inhibitory glycine receptor (GLRB). Hum. Mol. Genet. 11, 853–60 (2002).
Harvey, K. et al. The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering. J. Neurosci. 24, 5816–26 (2004).
Rees, M. I. et al. Isoform heterogeneity of the human gephyrin gene (GPHN), binding domains to the glycine receptor, and mutation analysis in hyperekplexia. J. Biol. Chem. 278, 24688–96 (2003).
Fritschy, J. M., Harvey, R. J. & Schwarz, G. Gephyrin: where do we stand, where do we go? Trends Neurosci. 31, 257–64 (2008).
Ohno, K., Koroll, M., El Far, O., Scholze, P., Gomeza, J. & Betz, H. The neuronal glycine transporter 2 interacts with the PDZ domain protein syntenin-1. Mol. Cell Neurosci. 26, 518–29 (2004).
Davies, J. S. et al. The glycinergic system in human startle disease: a genetic screening approach. Front. Mol. Neurosci. 3, doi: 10.3389/fnmol.2010.00008 (2010).
Zeilhofer, H. U. et al. Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J. Comp. Neurol. 482, 123–41 (2005).
Regan, M. R. et al. Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the develo** and mature CNS. J. Neurosci. 27, 6607–19 (2007).
Acknowledgements
This work was supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the Michael S. and Karen G. Ansari ALS Center for Cell Therapy and Regeneration Research, the National Institute on Aging Intramural Research Program, NIH grant #NS050275 to the MPI Microscopy Core Facility, and by the Medical Scientist Training Program, NIH grant #GM007309. We thank Dr. Solomon Snyder for providing Slc7a10 heterozygous mice; all members of the Höke lab and Neuromuscular Division for discussions and sharing of reagents; Dr. Xufeng Wu (NHLBI/NIH) for assistance and use of the Zeiss 880 Airyscan; Lisa Rein for statistical consultation; and Norman Barker for assistance with preliminary figure assembly.
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J.T.E., Y.L., Y.W., M.P.M. and A.H. designed experiments; J.T.E., Y.L., Y.W., N.P. and A.J. performed experiments; J.D.R. and S.d.L. contributed reagents; J.T.E. initiated the project and wrote the paper with contributions from all authors.
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Ehmsen, J., Liu, Y., Wang, Y. et al. The astrocytic transporter SLC7A10 (Asc-1) mediates glycinergic inhibition of spinal cord motor neurons. Sci Rep 6, 35592 (2016). https://doi.org/10.1038/srep35592
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DOI: https://doi.org/10.1038/srep35592
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