An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot-Marie-Tooth type 2D peripheral neuropathy

Abstract

Mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system in humans, described clinically as Charcot-Marie-Tooth type 2D or distal spinal muscular atrophy type V. Here, we characterise a new mouse mutant, Gars(C201R), with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The Gars(C201R) mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons.

Fig. 1.

Motor deficits in Gars^C201R/+ mice. Four-paw grip strength of Gars^C201R/+ male mice compared with sex-matched wild-type littermates. (A) Mice with a C3H genetic background from 1 to 15 month of age. The numbers of mice sampled varied with the time point: Gars^C201R/+, n=5–11; wild-type littermates, n=4–10. (B) Mice with a C57BL/6 background at 3 months of age: Gars^C201R/+, n=23; wild-type littermates, n=14. (C) Mean grip strength values (units) for Gars^C201R/+ and wild-type littermates at 3 months of age on the C3H and the C57BL/6 genetic backgrounds. The average value of the heterozygote grip strength is also shown as a percentage of its wild-type littermate control cohort. (D–F) Reaching and grasping deficits in Gars^C201R/+ male mice (n=9) compared with sex-matched wild-type littermates (n=10), as detected by the MoRaG test. (D) The latency to the first reach (LFR) is the response time for reaching within the first session; the percentages of errors during reaching (E) and grasping (F) are shown for Gars^C201R/+ and wild-type controls. Levels of statistical significance are: *P<0.05 and **P<0.01.

Fig. 2.

The GARS protein. (A) The mutated residue in the Gars^C201R mouse is highly conserved from mammals through to Arabidopsis. (B) Known mutations in humans (in black) and mice (in red) are scattered throughout the GARS protein; the mouse mutation marked as P278K/Y is the Gars^Nmf249 mutation reported by Seburn and colleagues (Seburn et al., 2006). Note that there are discrepancies in the numbering of mutations in humans and mice –the protein shown here is the full-length mouse protein (Ensembl peptide ID ENSMUSP00000003572), which is the mitochondrial GARS; the cytosolic protein is shorter because it starts with an internal ATG. The mouse mutations are numbered according to the full-length form of the protein, whereas the human mutations are numbered according to the cytosolic form (Seburn et al., 2006). The Gars^C201R mutation lies within the catalytic domain, as described by Schimmel and colleagues (Xie et al., 2007).

Fig. 3.

GARS anomalies in the Gars^C201R mouse brain. Protein levels and enzyme activities of GARS in dissected brain homogenates from wild-type, Gars^C201R/+ and Gars^C201R/C201R mice at 15 days of age (n=3 for each cohort) and from wild-type and Gars^C201R/+ mice at 3 months of age (n=3 for each cohort); all mice were on the C3H background. (A) The dissected region, within the dotted lines, of 15-day- and 3-month-old Gars^C201R/+ and wild-type littermate brains, which includes the motor and sensory cortex (mouse atlas, www.mbl.org/ ). (B) Western blot showing levels of GARS protein in wild-type, heterozygous and homozygous littermates at 15 days of age. (C) Graph of quantified data from the western blot, normalised to the β-actin control. (D,E) Acylation activity of GARS in brain homogenates. (D) GARS activity in homogenates of the dissected motor and sensory cortex of wild-type, heterozygous and homozygous littermates at 15 days of age, and (E) wild-type and heterozygotes at 3 months of age.

Fig. 4.

Maximum TA muscle force and histopathological analysis of muscle fibres in 4-month-old wild-type and Gars^C201R/+ mice. The maximum twitch (A) and tetanic (B) force (grams) generated by TA muscles from wild-type (n=10) and Gars ^C201R/+ (n=10) littermates (***P<0.001). (C) The weight of TA muscles from wild-type (n=10) and Gars ^C201R/+ (n=10) littermates (***P<0.001). (D) Examples of TA muscle sections, stained for succinate dehydrogenase, an indicator of oxidative capacity, from (left) wild-type and (right) Gars^C201R/+ mice. Bar, 70 μm. Histograms representing the mean size of cross-sectional areas of TA muscle fibres (E) (***P<0.001) and the mean number of TA muscle fibres (F) from wild-type (n=3) and Gars^C201R/+ (n=3) littermates. Errors bars represent the standard error of the mean (s.e.m.). The data collected on muscle fibre sizes were sorted into frequency distribution histograms (G).

Fig. 5.

NMJ morphology and nerve occupancy. (A,B) NMJs in the EDL and TA of wild-type mice show typical morphologies. In the Gars^C201R/+ mice, NMJs in the EDL have variable geometries with some being relatively normal (C), whereas others were very small and much less elaborate (D). (E) In the TA, regions of partial innervation were observed (a subtle example is indicated by the blue arrow) and portions of the terminal arbor appear atrophied. Examples of clear denervation were also observed (red arrow). Bar, 7 μm.

Fig. 6.

Light microscopy of Toluidine Blue-stained, semi-thin resin section of the saphenous nerve from 3-month-old wild-type littermate control (A) and Gars^C201R/+ mutant mice (B) on the C57BL/6 background. There is no axonal loss, axon degeneration or demyelination, but quantification of axon diameters (C) reveals a shift towards a smaller diameter in the Gars^C201R/+ nerve. (D,E) Confocal fluorescent images of the subepidermal (yellow arrows) and epidermal (red arrows) axons innervating the glabrous skin of wild-type littermate control (D) and Gars^C201R/+ mutant (E) mice. Bars, 70 μm (A,B); 25 μm (D,E).

Fig. 7.

Testing conduction velocity and amplitude. (A) Schematic representation of the setup for nerve excitability testing of the saphenous nerve. (B) Stimulus paradigm for recording the recovery cycle. A single supramaximal conditioning stimulus or a train of seven conditioning stimuli is followed by the test stimulus at a variable latency of 0.7 to 200 milliseconds. (C) In animals carrying the Gars^C201R mutation, the magnitude of the superexcitability is lower and later in the recovery cycle, and the relative refractory period is longer compared with in wild-type controls. Using a single conditioning stimulus, superexcitability is defined as the decrease of the threshold below the line of neutrality (0 on the ordinate). The magnitude of the maximal threshold reduction is marked for wild-type (red arrow) and Gars^C201R/+ (blue arrow) by vertical arrows. The relative refractory period is defined as the time between zero and the point at which the excitability threshold crosses the line of neutrality. The horizontal arrows show this time period: wild-type (red arrow) and Gars^C201R/+ (blue arrow). (D) The magnitude (vertical arrows) and peak time (horizontal arrows) of the late subexcitability is not significantly different.