NEMF mutations that impair ribosome-associated quality control are associated with neuromuscular disease

Abstract

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.

Fig. 1. Mutations in mouse Nemf lead to deficits in body weight, motor function, and lifespan.

a, b A 18-day-old R86S homozygous mutant (R86S; a) and 55-week-old R487G homozygous mutant (R487G; b) mice are smaller compared to wild-type (WT) littermates. c Body weight curves of male and female wild-type (orange squares), R487G (purple circles), and R86S mice (green triangles). Mean ± SD are indicated for each timepoint. Source data are provided as a source data file. d Kaplan–Meier survival curves of males and females combined WT (orange line, n = 33), R86S (green line, n = 34; median survival: 20 days) and R487G (purple line, n = 10; median survival 601 days) mice. Statistical analysis was performed by Log-rank (Mantel–Cox) test with two-tailed p value reported R86S vs. WT: p ≤ 0.0001 and R487G vs. WT: p = 0.0074. e Motor function of 8-week-old WT (orange squares), R487G (purple circles), and R86S mice (green triangles) (n = 16, 10, and 3, respectively) was measured by wire hang assay. Individual data points and mean ± SD are indicated. Statistical analysis was performed by Kruskal–Wallis test followed by Dunn’s multiple comparisons, R487G vs. WT: p = 0.0712 (n.s.), R86S vs. WT: p = 0.0018(**), R86S vs. R487G: p = 0.1748. Source data are provided as a source data file. For growth curve (c) n per genotype per time-point: Male: WT (n = 5, 6, 7, 6, 5 for weeks 1–6, 7, 8, 20, 55), R86S (n = 5, 4, 3 for weeks 1–2, 3–6, 7–8), R487G (n = 4, 6, 5, 3, 4, 5, 4 for weeks 2, 3, 4–6, 7, 8, 20, 55). Female: WT (n = 4, 3, 5, 4 3,4, 5 for weeks 1, 2, 3, 4, 5, 6, 8–55), R86S (n = 3, 5, 1 for weeks 1, 2, 3–5), R487G (n = 2, 5, 3, 4, 5, 4 for weeks 2, 3, 4, 5, 6–20, 55).

Fig. 2. Nemf ENU mutations cause progressive neuromuscular degeneration.

a–e Representative images of neuromuscular junction (NMJ) staining of medial gastrocnemius (MG) muscle from mice of indicated genotypes and ages. Tissues were stained for presynaptic vesicles (anti-SV2, green), neurofilaments (anti-neurofilament 2H3, green), and acetylcholine receptor (α-bungarotoxin, red). Unoccupied (arrow) and partially occupied (arrowhead) junctions are highlighted. Scale bar, 60 µm. Inset scale bar, 30 µm. f Quantification of fully innervated, partially innervated or denervated NMJs based on presynaptic and postsynaptic staining overlap. Eight-week-old WT, R487G and R86S mice (n = 4, 5, and 3 respectively), and 55-week-old WT and R487G mice (n = 4 and 3, respectively) were analyzed. By 8 weeks R487G or R86S mice compared to WT (****p < 0.0001), but were not different from one another (p = 0.4391). Fifty-five weeks of age, R487G vs. WT (****p < 0.0001). Data are presented as mean percent occupancy value ± SD. Statistical analysis was performed by two-way ANOVA followed by Tukey’s multiple comparison, for % fully occupied. g–n Representative cross-sections of femoral nerve motor branches from mice of indicated genotypes and ages. Scale bar, 20 µm. o Total myelinated axon number in cross-section of WT and Nemf mutant mice at 8-week-old wild-type (WT), R487G and R86S mice (n = 9, 7, and 4, respectively), and 55-week-old WT and R487G mice (n = 6 and 4, respectively). Eight weeks (WT vs. R487G ****p < 0.0001, WT vs. R86S ****p < 0.0001 and R487G vs. R86S ***p = 0.0005). Fifty-five weeks (WT vs. R487G ****p < 0.0001. R487G 8 weeks vs. 55 weeks ^n.s.p = 0.9767). p Myelinated axon number in cross-sections of L4 ventral roots of WT and Nemf ENU mice. Analysis of 8-week-old WT, R487G and R86S mice (n = 5, 3, and 4, respectively), and 55-week-old WT and R487G mice (n = 4 each). At 8 weeks of age R487G vs. WT *p = 0.0136, R86S vs. WT ****p < 0.0001, R487G vs. R86S **p = 0.0063, by 55 weeks of age R487G vs. WT ****p < 0.0001, R487G 8 weeks vs. R487G 55 weeks **p = 0.0055. o, p Individual data points and mean ± SD are indicated. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison (wild-type, orange squares; R487G, purple circles; R86S, green triangles). Source data for f, o, p are provided as a source data file.

Fig. 3. Nemf D106* mice display perinatal neurodegeneration and lethality.

a Western blot analysis of brain lysates from 10-day-old wild-type (WT), and Nemf D106* hetero- and homozygous mice using anti-NEMF antibody, and anti-GAPDH antibody as loading control. b Eleven-day-old D106* homozygous mice are smaller than WT littermates. Mutant mice display wasting and are unable to upright themselves at this timepoint. c Body weight of WT (green circle, n = 8 day 1, n = 5 days 7 and 11) and D106* homozygous (purple circle, n = 4 days 1 and 7, n = 7 day 11) mice for WT vs. D106* at each 1, 7, and 11 days of age, respectively (n.s.p > 0.9999, ****p < 0.0001, ****p < 0.0001). Mean ± SD is indicated. Statistical analysis was performed by two-way ANOVA followed by Sidak’s multiple comparison. d D106* mice have shortened lifespan. Kaplan–Meier survival curves of D106* homozygous (purple line, n = 9; median survival: 11 days) and WT (green line, n = 9) mice p = 0.0022. Statistical analysis was performed by Log-rank (Mantel–Cox) test with two-tailed p value reported (e), myelinated axon numbers from whole cross-sections of femoral nerve motor branches in 9- to 11-day-old WT and D106* homozygous mice. For motor (WT: n = 3; Nemf D106*: n = 4, ****p < 0.0001) and sensory (WT: n = 3; Nemf D106*: n = 6, **p = 0.0031). Individual data points and mean ± SD are indicated. Statistical analysis was performed by a two-sided unpaired t test. Source data for c, e are provided as a source data file.

Fig. 4. Nemf ENU mutations selectively affect the CATylation function of Rqc2.

a Domain diagram and partial alignment of mouse NEMF and homologs—human NEMF, fly Caliban, and yeast Rqc2. Sequences flanking residues affected by the ENU mutations are shown. Domains and motifs depicted: NFACT-N (N), helix–hairpin–helix (H), coiled-coil (cc), middle domain (M), NFACT-R (R), and NFACT-C (C). NEMF Arg86 and Arg487 mutated in Nemf ENU mice (* and red rectangles), and yeast Rqc2 Asp98 and Arg99 residues previously implicated in CAT tail synthesis (green rectangle) are indicated. b WT, ltn1Δ, or ltn1Δ rqc2Δ cells were transformed with the GFP-R12-RFP (GRR) stalling reporter and plasmids encoding Rqc2-FLAG WT, CATylation-deficient D98Y mutant, or ENU-equivalent mutants (R88S and K534G). Immunoblots: anti-GFP and anti-FLAG to monitor reporter and Rqc2 levels, respectively, and anti-Pgk1 as loading control. c Strains transformed with Rqc2-FLAG and the GRR stalling reporter, as indicated. Immunoblot anti-GFP monitored reporter modification and aggregation. d Wild-type (WT) and RQC2-deleted (rqc2Δ) cells were transformed with the GRR stalling reporter, and indicated Rqc2-FLAG constructs and analyzed as in “b”. e Kless-K2, -K27, or -K36 reporters (see Supplementary Fig. 8) expressed in rqc2Δ cells together with Rqc2-FLAG WT, the CATylation-deficient Rqc2 aaa mutant (Asp9, Asp98, and Arg99 mutated to Ala), or Rqc2-equivalent ENU mutants as indicated. Anti-HA and anti-FLAG blots monitored reporter and Rqc2 expression, respectively. f As in panel “f”. Quantification of reporter levels relative to the Pgk1 internal control from three biological replicates, individual values with means ± SEM. For each blot b–e MW markers in kDa are indicated.

Fig. 5. NEMF variants are associated with neuromuscular disease in humans.

a Pedigrees of identified families having affected individuals and NEMF variants. “−” indicates wild-type allele, “+” indicates variant allele, arrowhead indicates proband. Different font colors are used to facilitate visualization of the distribution of wild-type (“−”) and unique mutant (“+”) alleles in the pedigrees. b–f Clinical photography of 7-year-old subject AUS1-II:3. Asymmetric pectus excavatum, scoliosis (b), and characteristic adducted thumb posture are evident (c). Right foot showing surgical scars with residual equinovarus foot deformity and toe clawing (d). Images showing pectus excavatum, kyphoscoliosis, hip, and knee flexor contractures with distal muscle wasting and residual equinovarus foot posturing despite previous orthopedic surgery (e, f).