Abstract
Selective neuronal loss is a hallmark of neurodegenerative diseases, which, counterintuitively, are often caused by mutations in widely expressed genes1. Charcot–Marie–Tooth (CMT) diseases are the most common hereditary peripheral neuropathies, for which there are no effective therapies2,3. A subtype of these diseases—CMT type 2D (CMT2D)—is caused by dominant mutations in GARS, encoding the ubiquitously expressed enzyme glycyl-transfer RNA (tRNA) synthetase (GlyRS). Despite the broad requirement of GlyRS for protein biosynthesis in all cells, mutations in this gene cause a selective degeneration of peripheral axons, leading to deficits in distal motor function4. How mutations in GlyRS (GlyRSCMT2D) are linked to motor neuron vulnerability has remained elusive. Here we report that GlyRSCMT2D acquires a neomorphic binding activity that directly antagonizes an essential signalling pathway for motor neuron survival. We find that CMT2D mutations alter the conformation of GlyRS, enabling GlyRSCMT2D to bind the neuropilin 1 (Nrp1) receptor. This aberrant interaction competitively interferes with the binding of the cognate ligand vascular endothelial growth factor (VEGF) to Nrp1. Genetic reduction of Nrp1 in mice worsens CMT2D symptoms, whereas enhanced expression of VEGF improves motor function. These findings link the selective pathology of CMT2D to the neomorphic binding activity of GlyRSCMT2D that antagonizes the VEGF–Nrp1 interaction, and indicate that the VEGF–Nrp1 signalling axis is an actionable target for treating CMT2D.
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Change history
20 January 2016
Nature 526, 710–714 (2015); doi:10.1038/nature15510. The Acknowledgements of this Letter should have included the following sentence: “This work was also supported in part by the University of Antwerp (TOP BOF 29069 to A.J.); the Fund for Scientific Research-Flanders (grant number G078414N to A.J.);the Association Belge contre les Maladies Neuromusculaire (to A.
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Acknowledgements
We thank P. Schimmel, J. Dolkas, C. Farrokhi, L. Lisowski, C. Ly, S. Chalasani, T. Liu, Q. Hu, P. Li, N. Sheng and members of The Scripps Laboratories of tRNA Synthetase Research and the Pfaff laboratory for advice and discussions on the experiments and manuscript. We are grateful to S. Ackerman, K.-F. Lee, D. O’leary and D. Ginty for providing mouse lines. W.H., H.Z. and N.W. were supported by fellowships from the National Foundation for Cancer Research. G.B. was supported by the Pioneer fund and Howard Hughes Medical Institute. S.L.P. is a Howard Hughes Medical Institute investigator and a Benjamin H. Lewis chair in Neuroscience. This research is supported by grants from the US National Institutes of Health (R01GM088278, R21NS084254 and R01NS054154), The Marshall Heritage Foundation and the Sol Goldman Trust, and by aTyr Pharma through an agreement with The Scripps Research Institute.
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Contributions
W.H., G.B., S.L.P. and X.-L.Y. designed the study, analysed the data, and prepared the manuscript. W.H. carried out molecular cloning, binding analyses, secretion studies, and other biochemical experiments. G.B. performed the mouse genetics, viral injections, behaviour testing, and histology experiments. H.Z. carried out Nrp1 domain mapping, GlyRS–VEGF competition, and additional pull-down assays, and contributed to study design and figure preparation. N.W. performed co-immunoprecipitation to detect aberrant GlyRS–Nrp1 interaction using CMT2D patient samples. A.J. and V.G. provided transformed lymphocytes samples from CMT2D patients. J.L. and P.R.G. performed the hydrogen–deuterium exchange analysis. N.M.W. and K.L. assisted with mouse studies. Y.S. and C.D.D. contributed to biochemical experiments. H.L. and V.S. contributed to histology experiments. R.W.B. provided mice, technical support, and scientific advice.
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X.-L.Y. is a scientific co-founder and consultant of aTyr Pharma.
Extended data figures and tables
Extended Data Figure 1 Hydrogen–deuterium exchange analysis to compare GlyRSCMT2D(P234KY) and GlyRSWT in solution.
A global increase (15%) in deuterium incorporation for the mutant GlyRS was observed, indicating overall structural opening. The regions having significant changes (>10%) in deuterium incorporation are highlighted under the human cytosolic GlyRS sequence with different colour codes (see box).
Extended Data Figure 2 Characterization of the binding activity of GlyRSCMT2D.
a, In vitro pull-down of GlyRSCMT2D(P234KY) proteins with the ectodomains of Nrp1, TrkB, Dcc, Robo1 and Unc5C proteins. Note the much stronger binding of GlyRSCMT2D with Nrp1 compared with other receptors. GlyRS was detected by immunoblot with anti-GlyRS antibody; similar amounts of input receptors were visualized by Coomassie blue staining. b, In vitro pull-down of GlyRSCMT2D proteins with the ectodomain of Nrp1. In addition to L129P and P234KY, direct binding to Nrp1 was detected for E71G and G240R GlyRSCMT2D. c, GST pull-down to confirm that b1 domain of Nrp1 is the main binding site of GlyRSCMT2D. The amount of GST and GST fusion proteins used for GlyRSCMT2D binding was visualized by Ponceau staining. d, e, In vitro pull-down assay showing the mutual competition between GlyRSCMT2D(L129P) and VEGF-A165 for Nrp1 binding.
Extended Data Figure 3 Detection of GlyRS proteins in the cell medium.
a, c, e, Western blot analysis of the GlyRS protein levels in NSC-34 motor neurons (a), C2C12 cell-differentiated myotubes (c) and undifferentiated C2C12 myoblasts (e). The observation that differentiated myotubes also secrete GlyRS raises the possibility that muscles, which are directly innervated by the peripheral motor neurons, might contribute to the disease pathology. The level of GlyRS proteins in cell medium is diminished by application of the exosome-pathway inhibitor GW4869, but not by brefeldin A (BFA), an inhibitor of the classical endoplasmic reticulum (ER)-to-Golgi secretory pathway. GAPDH (cytoplasmic protein), vWF (secretory protein through ER–Golgi pathway) and TSG101 (exosomal protein) are used as controls. b, d, Quantification of GlyRS protein level indicated in a, c. Data are presented as the mean ± s.e.m. of three independent experiments (*P < 0.05, t-test). f, Western blot analysis of the GlyRS protein level in NSC-34 motor neurons. The level of GlyRS proteins in the cell medium is increased by the treatment of monensin (MON), an activator for microvesicle release by regulating the intracellular calcium level40,41. Vehicle-treated cells were used as control (Ctrl). g, Western blot analysis of the GlyRS protein level in Cos7 cells transfected with plasmids encoding GlyRSWT and GlyRSCMT2D(P234KY). The expression of GlyRS proteins was detected by immunoblot with antibody to V5 epitope tag. GAPDH was used as control. Note the similar level of GlyRSCMT2D and GlyRSWT in the media of transfected Cos7 cells.
Extended Data Figure 4 Detection of GlyRS proteins in exosome-enriched fractions.
a, Diagram showing the procedure of exosome separation from the cell medium of NSC-34 cells by differential centrifugation. See Methods for details. b, Western blot analysis of proteins associated with various fractions. GlyRS proteins were detected in the exosome-enriched fractions but not in supernatant fractions. The quality of the exosome preparation was controlled by detection of TSG101 (exosomal protein), Bip (ER-associated protein), GAPDH (cytoplasmic protein), and vWF (secretory protein through ER–Golgi pathway).
Extended Data Figure 5 CMT2D mutant embryos have overall normal morphology but exhibit facial motor neuron migration defects.
a, Lateral view of wild-type and CMT2D mutant embryos at E12.5. Motor neurons are specifically labelled by a transgenic fluorescence reporter, Hb9:GFP (green). Note overall normal morphology of CMT2D mutant embryos (CMT) compared with their littermate controls (wild type). b, Western bolt analysis of protein expression in E12.5 mouse neural tissues. The expression levels of various neuronal proteins appear normal in CMT2D mutants compared with their littermate controls. c, Considering CMT2D mutants show varying degrees of morphological change of facial motor nucleus, the facial motor neuron migration phenotype is quantified by measuring the relative distance of the facial motor nucleus between wild-type and CMT littermate embryos (each dot represents one facial motor nucleus, n = 6 embryos for wild type; n = 8 embryos for CMT2D). We find that the migration of facial motor neurons is significantly disrupted in CMT embryos. Data are presented as the mean ± s.e.m. **P < 0.01 (t-test).
Extended Data Figure 6 Genetic interaction between Gars and Nrp1 in the early stage of CMT2D.
a, b, Hindlimb extension test of wild-type and mutant animals at 2 weeks. Note that two out of nine GarsCMT2D;Nrp1+/− (CMT;Nrp1+/−) mutants exhibit hindlimb weakness with significantly lower scores compared with GarsCMT2D (CMT), Nrp1+/− and wild-type littermate controls. c, Comparison of stride lengths in different CMT2D mutant mice at 4 weeks old: GarsCMT2D (CMT), GarsCMT2D;TrkB+/− (CMT;TrkB+/−), GarsCMT2D;Dcc+/− (CMT;Dcc+/−), GarsCMT2D;Robo1+/− (CMT;Robo1+/−), and GarsCMT2D;Unc5C+/− (CMT;Unc5C+/−). No significant differences were observed between compound heterozygotes and their littermate controls (CMT).
Extended Data Figure 7 Axonal dystrophy in CMT2D mice.
a–d, Histograms showing the axonal diameter frequencies in the sciatic nerves of 4-week-old wild-type (a), Nrp1+/− (b), GarsCMT2D (CMT; c), and CMT;Nrp1+/− (d) mutant mice. n = 3 mice per group. Note the decreased numbers of larger-diameter axons in CMT;Nrp1+/− mutants compared with CMT, Nrp1 heterozygous, and wild-type controls.
Extended Data Figure 8 Expression level of VEGF in mouse muscles.
The expression level of VEGF proteins in muscle fibres of mice injected with lentivirus expressing LV-VEGF165-IRES-GFP versus LV-GFP was determined by immunostaining with anti-VEGF antibodies. Note that the expression level of VEGF in LV-VEGF-infected muscles is significantly higher than in LV-GFP-infected control groups.
Extended Data Figure 9 VEGF treatment retains limb strength in CMT2D mice.
a, Diagram showing that lentiviral vectors encoding GFP (LV-GFP) or VEGF-A165 (LV-VEGF165) are injected unilaterally into each hindlimb of the same GlyRSCMT2D mutant mouse at P5. c, e, At 5 weeks, LV-GFP-injected legs (L, left) of CMT2D animals have largely lost their ability to extend, while LV-VEGF165-treated legs (R, right) retained more limb strength with significantly higher scores in the hindlimb extension test (three out of seven animals). P < 0.05 (permutation test). b, d, No significant difference was observed between both injected legs of wild-type animals in the hindlimb extension test. f, g, GDNF and VEGF-A121 treatments fail to improve stride length in CMT2D mice. Walking strides of 2-month-old CMT2D mice bilaterally injected with lentiviral vectors (LV) encoding GFP, GDNF or VEGF-A121. No significant difference of hindlimb stride length was observed between animals treated with LV-GDNF, LV-VEGF-A121, and LV-GFP controls.
Extended Data Figure 10 A simplified model for the neomorphic binding activity of GlyRSCMT2D.
Left, GlyRSWT is a multifunctional protein with both intracellular and extracellular distributions. VEGF–Nrp1 signalling is an essential pathway for survival and function of motor neurons. (Note that VEGF may also act synergistically with other trophic factors, and/or maintain motor function indirectly by acting on Nrp1 receptors on non-motor neurons.) Right, CMT2D mutations alter the conformation of GlyRS, enabling GlyRSCMT2D to bind Nrp1. This aberrant interaction antagonizes the binding of VEGF to Nrp1, contributing to motor defects in CMT2D. Our results do not exclude the possibility that GlyRSCMT2D may also interact with other extracellular and/or intracellular targets, related to CMT2D pathology.
Supplementary information
4-week-old Nrp1 heterozygous mouse
This video shows a 4-week-old control Nrp1+/- mouse. The mouse displays normal gait. (MOV 1243 kb)
4-week-old CMT2D mutant mouse
This video shows a 4-week-old GarsCMT2D mouse (CMT). The mouse displays mildly ataxic gait. (MOV 998 kb)
4-week-old CMT2D/Nrp1+/- compound mutant mouse
This video shows a 4-week-old GarsCMT2D;Nrp1+/- mouse (CMT;Nrp1+/-). The mouse displays severely impaired motor function and limb paralysis. (MOV 841 kb)
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He, W., Bai, G., Zhou, H. et al. CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase. Nature 526, 710–714 (2015). https://doi.org/10.1038/nature15510
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DOI: https://doi.org/10.1038/nature15510
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