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. Author manuscript; available in PMC: 2022 Sep 3.
Published in final edited form as: Science. 2021 Sep 1;373(6559):1161–1166. doi: 10.1126/science.abb3356

tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase

Amila Zuko 1,†, Moushami Mallik 1,2,†, Robin Thompson 3, Emily L Spaulding 4,5,‡, Anne R Wienand 1, Marije Been 1, Abigail LD Tadenev 4, Nick van Bakel 1, Céline Sijlmans 1, Leonardo A Santos 3, Julia Bussmann 2, Marica Catinozzi 1,2, Sarada Das 3, Divita Kulshrestha 1,2, Robert W Burgess 4,5, Zoya Ignatova 3, Erik Storkebaum 1,2,*
PMCID: PMC8856733  NIHMSID: NIHMS1778363  PMID: 34516840

Abstract

Heterozygous mutations in six tRNA synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT-mutant tRNA synthetases inhibit protein synthesis by an unknown mechanism. Here, we found that CMT-mutant glycyl-tRNA synthetases (GlyRS) bound tRNAGly, but failed to release it, resulting in tRNAGly sequestration. This sequestration potentially depleted the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome. Accordingly, we found ribosome stalling at glycine codons and activation of the integrated stress response (ISR) in affected motor neurons. Moreover, transgenic overexpression of tRNAGly rescued protein synthesis, peripheral neuropathy, and ISR activation in Drosophila and mouse CMT2D models. Conversely, inactivation of the ribosome rescue factor GTPBP2 exacerbated peripheral neuropathy. Our findings suggest a molecular mechanism for CMT2D, and elevating tRNAGly levels may thus have therapeutic potential.

One Sentence Summary:

tRNAGly sequestration by mutant glycyl-tRNA synthetase triggers Charcot-Marie-Tooth peripheral neuropathy.

Heterozygous mutations in six genes encoding cytoplasmic aminoacyl-tRNA-synthetases (aaRSs) cause axonal and intermediate forms of CMT (1–3). aaRSs are ubiquitously expressed enzymes which covalently attach amino acids to their cognate tRNAs (tRNA aminoacylation) (4, 5). Aminoacylated tRNAs are used by the ribosome for mRNA translation (6). Interestingly, some CMT-aaRS mutations do not affect aminoacylation activity (7–11), indicating that loss of aminoacylation activity is not a prerequisite for disease-causality. Rather, a gain-of-toxic-function mechanism may underlie CMT associated with GlyRS mutations (CMT2D) (9, 12). In vivo cell-type-specific visualization of newly synthesized proteins in Drosophila (13) by fluorescent non-canonical amino acid tagging (FUNCAT) (14) revealed that each of six GlyRS or tyrosyl-tRNA synthetase (TyrRS) mutants substantially inhibited global protein synthesis in motor or sensory neurons (9), implicating impaired mRNA translation in CMT-aaRS. Here, we investigated the molecular mechanism by which CMT-mutant GlyRS variants inhibit translation. Manipulation of upstream regulatory pathways or translation initiation did not rescue inhibition of translation (Fig. S1), suggesting that CMT-mutant GlyRS may interfere with translation elongation. We thus evaluated the effect of tRNAGly overexpression, by generating Drosophila carrying a BAC transgene containing five tRNAGly genes with GCC anticodon (tRNAGly-GCC) (Fig. 1A). Flies with 10 or 20 additional tRNAGly-GCC gene copies displayed ~13% and ~25% increased tRNAGly-GCC levels, respectively (Fig. S2A,B). The 10xtRNAGly-GCC transgene partially rescued the translation defect (Fig. 1B, Fig. S3A) and peripheral neuropathy-like phenotypes induced by three CMT-mutant GlyRS proteins (E71G, G240R, G526R) (9) (Table S1), including larval muscle denervation (Fig. 1C, Fig. S3F), developmental lethality (Fig. S3B–E), adult motor deficits (Fig. 1D), sensory neuron morphology defects (Fig. S3G,H), and reduced life span (Fig. S3I). In general, phenotypic rescue was more pronounced for G240R and G526R than for E71G. tRNAGly-GCC overexpression did not alter GlyRS protein levels (Fig. S4), and did not rescue peripheral neuropathy phenotypes induced by CMT-mutant TyrRS (Fig. S5), indicating that only the cognate tRNA can rescue. Transgenic lines containing 10 different tRNAGly-GCC genes (tRNAGly-GCC ‘scramble’, Fig. 1A) induced a dosage-dependent increase in tRNAGly-GCC level, more pronounced than the BAC transgene (~30% for 10xtRNAGly-GCC, Fig. S2C,D), and a more substantial rescue of muscle denervation and motor performance (Fig. 1E,F). Thus, the degree of rescue correlated with tRNAGly-GCC overexpression level.

Fig. 1. tRNAGly overexpression rescues inhibition of protein synthesis and peripheral neuropathy phenotypes in Drosophila CMT2D models.

Fig. 1.

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(A) Schematic of the transgenes used for tRNAGly-GCC or tRNAGly-UCC overexpression. (B) Relative translation rate as determined by FUNCAT in motor neurons (OK371-GAL4) of larvae expressing E71G, G240R, or G526R GlyRS (2x: two transgene copies), in the presence or absence of the tRNAGly-GCC BAC transgene (10xtRNAGly-GCC). n=10–34 animals per genotype; ***p<0.001 by Kruskal-Wallis test. (C,E,G) Percentage of larvae with innervated muscle 24. GlyRS transgenes were expressed in motor neurons (OK371-GAL4), in the presence or absence of 10xtRNAGly-GCC BAC (C), 10xtRNAGly-GCC scramble (E), or 12xtRNAGly-UCC (G). n=19–26 (C), 8–22 (E), 12–27 (G) animals per genotype; *p<0.05; ***p<0.005 by Fisher’s exact test with Bonferroni correction. (D,F,H,I) Negative geotaxis climbing speed of 7-day-old female flies expressing GlyRS transgenes in motor neurons (OK371-GAL4), in the presence or absence of 10xtRNAGly-GCC BAC (D), 10xtRNAGly-GCC scramble (F), or 12xtRNAGly-UCC (H,I). n=11–13 (D), 8–52 (F), 7–19 (H,I) groups of 10 flies per genotype; **p<0.01; ***p<0.005 by two-way ANOVA (D) or Brown-Forsythe and Welch ANOVA (F,H,I). Controls in (B-I) are driver-only. Graphs represent mean ± SEM.

We next generated transgenic lines overexpressing the other tRNAGly isoacceptor, tRNAGly-UCC (Fig. 1A). 12xtRNAGly-UCC flies displayed ~75% increased tRNAGly-UCC levels (Fig. S6A,B). For E71G and G240R, tRNAGly-UCC overexpression partially rescued developmental lethality (Fig. S6C–F), muscle denervation (Fig. 1G), motor deficits (Fig. 1H,I), and life span (Fig. S6I). For G526R, tRNAGly-UCC overexpression partially rescued motor performance (Fig. 1I), but aggravated sensory neuron morphology defects (Fig. S6G,H) and further reduced life span (Fig. S6I). Thus, for E71G and G240R, both tRNAGly-GCC and tRNAGly-UCC partially rescued peripheral neuropathy phenotypes, while for G526R, the rescue was isoacceptor-specific.

To strengthen the potential relevance for human CMT2D, we evaluated the effect of tRNAGly-GCC overexpression in CMT2D mouse models. We generated transgenic mice with ~27 (tRNAGly-high) or two (tRNAGly-low) copies of a genomic transgene containing two tRNAGly-GCC genes (Fig. 2A, Fig. S7A). In spinal cord (SC), tibialis anterior muscle and sciatic nerve of tRNAGly-high mice, tRNAGly-GCC levels were increased by ~90 to 150% (Fig. S7B–G). Targeted locus amplification (TLA) revealed integration of all transgene copies in Stk38 on Chr17, with a ~7kb deletion at the integration site, deleting exons 8–12 of Stk38 (Fig. S8). In both male and female GarsC201R/+ mice (15) of 3 to 6 weeks of age, tRNAGly-GCC overexpression fully rescued the reduced body weight (Fig. S9A,B) and motor deficits (Fig. S9C–F). Reduced nerve conduction velocity (NCV) and compound muscle action potential (CMAP) amplitude in GarsC201R/+ mice was also fully rescued (Fig. S9G–J). Thus, increasing tRNAGly-GCC levels completely prevented peripheral neuropathy in GarsC201R/+ mice, without affecting Gars mRNA and GlyRS protein levels (Fig. S10).

Fig. 2. tRNAGly-GCC overexpression rescues peripheral neuropathy in CMT2D mouse models.

Fig. 2.

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(A) Schematic of the genomic fragment used for generation of tRNAGly-GCC transgenic mice. (B,M) Hanging time in the inverted grid test of male GarsC201R/+ x tRNAGly-high (B) or GarsΔETAQ/+ x tRNAGly-high (M) mice. n=8–9 (B), 11–13 (M) mice per genotype; ***p<0.0001 by one-sample t-test and two-tailed unpaired t-test with Bonferroni correction per time point. (C,I,N) 4-paw grip strength as measured by dynamometer. n=8–9 (C), 10–11 (I), 11–13 (N) mice per genotype; ***p<0.001 by two-way ANOVA with Tukey’s multiple comparisons test per time point (C,I) or Brown-Forsythe and Welch ANOVA (N). (D,E,J,K,O,P) Electromyography (EMG) at 12 (D,E,O,P) or 52 (J,K) weeks of age. (D,J,O) Latency time between sciatic nerve stimulation at sciatic notch level and detection of a compound muscle action potential (CMAP) in the gastrocnemius muscle. n=8–9 (D), 10–11 (J), 11–13 (O) mice per genotype; ***p<0.0001 by two-way ANOVA with Tukey’s multiple comparisons test (D) or Brown-Forsythe and Welch ANOVA (J,O). (E,K,P) CMAP amplitude in the gastrocnemius muscle. n=8–9 (E), 10–11 (K), 11–13 (P) mice per genotype; ***p<0.0005 by Brown-Forsythe and Welch ANOVA (E,P) or two-way ANOVA with Tukey’s multiple comparisons test (K). (F,L,Q) Ratio of muscle weight to body weight (MW:BW, shown as % of WT) of the gastrocnemius at 12 (F,Q) or 52 (L) weeks of age. n=8–9 (F), 10–11 (L), 11–13 (Q) mice per genotype; ***p<0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. (G,H,R) Representative images (G) and quantification (H,R) of NMJ innervation status in plantaris muscle. In (G), neurofilament (NF) and SV2 label presynaptic nerve endings, while TRITC-conjugated bungarotoxin (BTX) labels postsynaptic acetylcholine receptors. n=5 mice per genotype; ***p<0.005 by Fisher’s Exact test with Bonferroni correction. Scale bar: 25μm. Graphs represent mean ± SEM.

Follow-up of an independent cohort of GarsC201R/+ x tRNAGly-high mice from 4 to 12 weeks confirmed full rescue of motor performance (Fig. 2B,C) and neuromuscular transmission (Fig. 2D,E). At 12 weeks of age, tRNAGly-GCC overexpression fully rescued the reduced gastrocnemius muscle weight (Fig. 2F), and substantially mitigated muscle denervation (Fig. 2G,H). The rescuing effect persisted until 1 year of age in another cohort of GarsC201R/+ x tRNAGly-high mice. Body weight and motor performance were fully rescued from 4 to 52 weeks (Fig. 2I; Fig. S9K,L), as well as NCV, CMAP amplitude and gastrocnemius muscle weight (Fig. 2J–L). Thus, tRNAGly-GCC overexpression completely prevents peripheral neuropathy in GarsC201R/+ mice.

Finally, we crossed tRNAGly-high mice to another CMT2D mouse model carrying a patient mutation (245-248_delETAQ) in the mouse Gars gene (16). At 4, 8 and 12 weeks, tRNAGly-GCC overexpression fully rescued motor deficits, reduced NCV and CMAP amplitude, reduced gastrocnemius weight and muscle denervation (Fig. 2M–R). In tRNAGly-low mice, tRNAGly-GCC level was not altered (Fig. S11). GarsC201R/+; tRNAGly-low mice were indistinguishable from GarsC201R/+ mice for all parameters evaluated (Fig. S12), showing that tRNAGly-GCC overexpression and not the mere presence of the transgene is responsible for phenotypic rescue.

We next explored the molecular mechanism underlying the rescue of CMT2D phenotypes by tRNAGly overexpression. We hypothesized that CMT-mutant GlyRSs may exhibit altered kinetics of tRNAGly binding and release. Size-exclusion chromatography of various purified human GlyRS variants revealed that WT and E71G migrated predominantly as dimers, whereas L129P, C157R (≅mouse C201R), G240R, E279D and G526R partitioned between the monomer and dimer forms (Fig. 3A). All CMT-mutant GlyRS dimers bound tRNAGly-GCC (Kon) with a 2- to 10-fold lower affinity than WT dimers (Fig. 3B). L129P, C157R, G240R, E279D and G526R dimers displayed markedly slower tRNAGly-GCC release (Koff), with >80% of traces showing no tRNAGly-GCC release (Fig. 3B). In contrast, E71G dimers displayed tRNAGly-GCC release kinetics comparable to WT. L129P, C157R, G240R, E279D and G526R monomers bound tRNAGly-GCC with very low affinity, but once bound, the tRNAGly-GCC release was markedly inhibited (Fig. 3B). The tRNAGly-UCC isoacceptor displayed similar binding and release kinetics to GlyRS dimers and monomers (Table S2). The slow tRNAGly release by CMT-mutant GlyRS dimers and monomers suggests that mutant GlyRSs sequester a large fraction of cellular tRNAGly and thus deplete it for translation. To provide in vivo evidence for tRNAGly sequestration, we immunoprecipitated GlyRS from brains of GarsC201R/+ and WT littermate mice and quantified the amount of tRNAGly bound to GlyRS. The tRNAGly amount was ~65% higher in GarsC201R/+ versus WT (Fig. 3C, Fig. S13A), indicating stronger tRNAGly association with GlyRS-C201R. Because tRNAGly sequestration may lead to ribosome stalling at Gly codons, we performed ribosome profiling on SC extracts of GarsC201R/+ and WT littermate mice, revealing that Gly codons are more frequently found in the ribosomal A site in GarsC201R/+ SC relative to WT (cumulative increase of 79%, Fig. S13B).

Fig. 3. tRNAGly sequestration by CMT-mutant GlyRS induces ribosome stalling.

Fig. 3.

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(A) Size-exclusion chromatography of purified recombinant human GlyRS proteins. D:M= dimer:monomer ratio. (B) Kon and Koff values of tRNAGly-GCC binding and release to dimer and monomer forms of the indicated GlyRS variants. The (percentage) denotes the frequency of a measured value. (C) Quantification of tRNAGly bound to GlyRS in tRNAGly:GlyRS complexes immunoprecipitated from whole brains of GarsC201R/+ and WT littermate control mice. tRNAGly/GlyRS ratio of WT is set as 100%; n=5 independent experiments; *p<0.05 by one-sample t-test. (D) Hanging time in the inverted grid test of male Gtpbp2+/? or −/−; Gars+/+ (control), Gtpbp2+/?; GarsC201R/+, and Gtpbp2−/−; GarsC201R/+ littermate mice at 4, 5, 6, 7 and 8 weeks of age. n=15–28 mice per genotype group; ***p<0.0005 by one-sample t-test and two-tailed unpaired t-test with Bonferroni correction per time point. (E) Nerve conduction velocity of the sciatic nerve at 8 weeks of age. n=13–20 mice per genotype group; ***p<0.0001 by Brown-Forsythe and Welch ANOVA. (F) Axon number in the motor branch of the femoral nerve at 8 weeks of age. n=8–13 per genotype group; ***p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. Graphs represent mean ± SEM.

Prolonged ribosome dwelling at codons is resolved by ‘ribosome rescue’ pathways (17–19), and because Gly codons are frequent, ribosome stalling in CMT2D may deplete ribosome rescue factors and inactivation of a rescue factor may aggravate the phenotype of CMT2D mice. Indeed, inactivation of Gtpbp2, encoding the ribosome rescue factor GTPBP2, does not induce peripheral neuropathy by itself (20), but substantially enhanced peripheral neuropathy in GarsC201R/+ mice (Fig. 3D–F, Fig. S14A,B). Thus, ribosome stalling causally contributes to CMT2D pathogenesis. Because stalled ribosomes may activate the integrated stress response (ISR) through GCN2 (21–23) and ISR activation was implicated in CMT2D (24), we evaluated ISR induction in CMT2D mice intercrossed with tRNAGly-high mice. tRNAGly-GCC overexpression fully rescued increased phosphorylated eIF2α immunostaining intensity (~75%) in spinal motor neurons of GarsΔETAQ/+ mice (Fig. 4A,B), as well as the strong induction of ATF4 target genes Gdf15, Adm2, B4galnt2 and Fgf21 in motor neurons of GarsC201R/+ mice (Fig. 4C–I). Thus, tRNAGly-GCC overexpression abrogates ISR activation in CMT2D mice, indicating that depletion of the cellular tRNAGly pool and consequent ribosome stalling is upstream of ISR activation. When Gtpbp2 is inactivated in GarsC201R/+ mice, the percentage of motor neurons showing ISR activation did not change, nor did additional cell types show ISR activation, despite widespread Gtpbp2 expression in SC (Fig. S15). This suggests that tRNAGly levels are only below a critical threshold in affected motor and sensory neurons, leading to ribosome stalling selectively in these cell types. This may explain the relatively modest increase in ribosome dwelling at Gly codons in GarsC201R/+ SC (Fig. S13B).

Fig. 4. tRNAGly-GCC overexpression prevents ISR activation in CMT2D mouse models.

Fig. 4.

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(A,B) Representative images (A) and quantification (B) of immunostaining intensity of phosphorylated eIF2α in motor neuron cell bodies in the spinal cord ventral horn of GarsΔETAQ/+ x tRNAGly-high mice. Scale bar: 100μm. n=4–5 mice per genotype; **p<0.01, ***p<0.0005 by two-way ANOVA with Tukey’s multiple comparisons test. (C-F) Representative images (C) and quantification of fluorescent in situ hybridization (FISH) for ATF4 target genes Gdf15 (D), Adm2 (E), and B4galnt2 (F). Scale bar: 50μm. n=5–6 mice per genotype; *p<0.05 by two-tailed Welch’s t-test with Bonferroni correction. (G-I) mRNA levels of ATF4 target genes Gdf15 (G), Fgf21 (H) and B4galnt2 (I) in spinal cord of GarsC201R/+ x tRNAGly-high mice. n=3 per genotype; *p<0.05, **p<0.01 by Brown-Forsythe and Welch ANOVA. Graphs represent mean ± SEM.

In all, our data propose a detailed molecular mechanism underlying CMT2D (Fig. S16). Beyond the seven CMT2D mutations studied here, this mechanism may apply to additional CMT-mutant GlyRS proteins, because 14 out of 25 reported CMT2D mutations result in net addition of positive charge (Table S3), which could alter binding and release kinetics of the negatively charged tRNAGly. Similarly, the majority of CMT-causing mutations in TyrRS and AlaRS also result in net addition of positive charge (Table S4). Finally, our data indicate that increasing tRNAGly level may constitute a therapeutic approach for CMT2D.

Supplementary Material

1

Acknowledgments:

We thank Xiang-Lei Yang for GlyRS expression plasmids and the General Instruments Facility (Faculty of Science, Radboud University) for advice on image acquisition and analysis.

Funding:

This work was supported by the Max Planck Society, the Donders Center for Neuroscience, the Muscular Dystrophy Association (MDA479773), the EU Joint Programme – Neurodegenerative Disease Research (JPND; ZonMW 733051075 (TransNeuro) and ZonMW 733051073 (LocalNMD)), the Radala Foundation, an ERC consolidator grant (ERC-2017-COG 770244), Deutsche Forschungsgemeinschaft (DFG, IG73/14-2) to Z.I., and NIH grants to R.W.B. (R01 NS054154, U54 OD020351).

Footnotes

Competing interests: Patent application 2024840 with E.S. as inventor was submitted to the Netherlands Patent Agency. E.L.S. and R.W.B. have a pending patent application ‘GCN2 inhibitors for treating peripheral neuropathy’. R.W.B. is a member of the Scientific Advisory Board of the Charcot-Marie-Tooth Association and the Hereditary Neuropathy Foundation.

Supplementary Materials:

Materials and Methods

Figures S1–S16

Tables S1–S8

References (25–92)

Data and materials availability:

The ribosome profiling data are in GEO (accession number GSE160584). tRNAGly transgenic mice are available under a material transfer agreement.

References and Notes:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1778363-supplement-1.pdf (37MB, pdf)

Data Availability Statement

The ribosome profiling data are in GEO (accession number GSE160584). tRNAGly transgenic mice are available under a material transfer agreement.

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