Neuropilin 1 sequestration by neuropathogenic mutant glycyl-tRNA synthetase is permissive to vascular homeostasis

Figures

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  Figure 1. Mutant GlyRS aberrantly binds to the transmembrane receptor neuropilin 1. (A) Schematic of Nrp1 and one of its principal co-receptors, VEGFR2, which together bind to the secreted glycoprotein VEGF-A165 (on the left)11. VEGF-A functions in vasculogenesis, angiogenesis, and arteriogenesis, with the latter two processes occurring through Nrp1 and VEGFR2 signalling17, 18. VEGF-A is also critical for nervous system development and maintenance15. VEGF-A165 binding to Nrp1 is mainly dependent by the b1 domain12. CMT2D-associated mutations in GARS cause a conformational opening of GlyRS, allowing the aberrant binding of mutant GlyRS to the b1 domain of Nrp1 (on the right)10. This competitively antagonises Nrp1/VEGF-A signalling, which contributes to motor deficits observed in CMT2D mice10. Schematics are not drawn to scale and adapted from Plein et al.18 and He et al.10. (B) Co-immunoprecipitation of endogenous Nrp1 showing aberrant interaction with P234KY, L129P, and C157R GlyRS (ectopically expressed with a V5-tag) in NSC-34 cells. Wild-type GlyRS shows no significant binding to Nrp1. The aberrant interaction is weaker with C157R than with P234KY GlyRS, which correlates with the…

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  Figure 2. Nrp1 localises to skeletal muscle blood vessels but not motor neurons. Representative Nrp1 staining in one month old wild-type lumbrical muscles. (A) Nrp1 (white) localises to structures surrounding, but not perfectly overlapping, lower motor neurons (SV2/2H3, green), and in SV2/2H3 negative areas. α-bungarotoxin (α-BTX, magenta) identifies post-synaptic acetylcholine receptors at the neuromuscular junction. (B) Nrp1 staining is not wholly contiguous with the Schwann cell marker S100 (blue) either. This was confirmed with a second glial cell marker, myelin basic protein (Mbp) in Fig. S1C. (C) Nrp1 co-localises with IB4 (red), an endothelial cell marker, indicating that Nrp1 is found in muscle blood vessels. Arrows highlight Nrp1 staining associated with motor neurons/myelin, while arrowheads indicate Nrp1+ blood vessels. A similar staining pattern was observed at three months and in the TVA muscle at both time points (data not shown). A-B are collapsed Z-stack images and C is a single plane image. Scale bars = 20 μm. See also Fig. S1.

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  Figure 3. Capillaries appear unaffected in Gars C201R/+ skeletal muscles. (A,B) Representative single plane (A, top) and collapsed Z-stack (A, bottom and B) images of IB4 (green) staining in one month lumbrical (A) and TVA muscles (B). Scale bars = 1 mm (A, top) and 20 μm (A, bottom and B). (C,E,G) No defects in capillary diameter (C, age, P = 0.007; genotype, P = 0.090; interaction, P = 0.508), density (E, age, P < 0.001; genotype, P = 0.290; interaction, P = 0.625), or branching density (F, age, P < 0.001; genotype, P = 0.468; interaction, P = 0.721) were observed in Gars C201R/+ mouse lumbrical muscles at one or three months. (D,F,H) The TVA muscle also showed no difference in capillary diameter (D, age, P = 0.875; genotype, P = 0.264; interaction, P = 0.359), density (F, age, P < 0.001; genotype, P = 0.223; interaction, P = 0.889), or branching density (H, age, P = 0.020; genotype, P = 0.086; interaction, P = 0.340). All data sets were analysed with two-way ANOVAs. n = 5. See also Fig. S2.

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  Figure 4. Pathways integral to vascular homeostasis appear unperturbed in mutant Gars muscle. (A) Representative Nrp1 (white) and VEGFR2 (red) staining in one month old wholemount wild-type and Gars C201R/+ TVA muscles. Nrp1 and VEGFR2 colocalise with the endothelial marker IB4 (green) in both genotypes, and show no obvious differences. A similar pattern was seen at three months and in the lumbrical muscles at both time points (data not shown). Both panels are single plane images. Scale bars = 200 μm. (B,C) qPCR analysis of a series of genes integral to the maintenance of blood vessels indicates very little perturbation in gene expression in one month Gars C201R/+ tibialis anterior muscles. This was confirmed using either Gapdh (top graphs) or endothelium-specific Pecam1 (bottom graphs) as the reference gene. All individual gene data sets were separately analysed with unpaired, two-tailed t-tests. n = 6 wild-type and 4 Gars C201R/+.

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  Figure 5. The blood vessel network is unperturbed in Gars C201R/+ retinas. (A) Representative single plane (top) and collapsed Z-stack (bottom) images of IB4 (green) staining in one month retina. Scale bars = 1 mm (top) and 20 μm (bottom). (B–E) No defects in capillary diameter (B, age, P = 0.823; genotype, P = 0.112; interaction, P = 0.105), density (C, age, P = 0.878; genotype, P = 0.638; interaction, P = 0.504), branching density (D, age, P = 0.042; genotype, P = 0.548; interaction, P = 0.345), or major radial branch (arteries and veins, arrow in A) number (E, age, P = 0.541; genotype, P = 0.395; interaction, P = 0.188) were observed in Gars C201R/+ mouse retinas at one or three months. n = 5. (F) Representative single plane images of IB4 (i) and αSMA (red, ii) staining in P6-7 retina. Letters a and b (i) indicate how vascular extension was assessed (a /a + b) and the arrowhead (ii) highlights an αSMA+ artery. Scale bars = 1 mm. (G,H) There is no difference in IB4+ vascular extension (G, P = 0.818) or αSMA+ artery number (H, P = 0.967) between wild-type and Gars C201R/+ P6-7 retinas. n = 3 wild-type and 5 Gars C201R/+. Data sets were analysed with either a two-way ANOVA (B–E) or an…

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  Figure 6. Gars mutant embryonic hindbrains and sciatic nerves show no vascular defects. (A,B) Representative single plane (A, top and B) and collapsed Z-stack (A, bottom) images of IB4 and Pecam1 staining (green) in E13.5 hindbrain (A) and one month sciatic nerve (B), respectively. Scale bars = 1 mm (A, top) and 20 μm (A, bottom, and B). (C–E) Capillary diameter (C, P = 0.593), density (D, P = 0.456), and branching density (E, P = 0.278) are all unimpaired in Gars C201R/+ E13.5 hindbrains. n = 9 wild-type and 5 Gars C201R/+. (F) Mutant sciatic nerves show no difference in capillary density at one month (P = 0.111). n = 4 wild-type and 5 Gars C201R/+. All data were analysed with unpaired, two-tailed t-tests. See also Fig. S2.