A MusD Retrotransposon Insertion in the Mouse Slc6a5 Gene Causes Alterations in Neuromuscular Junction Maturation and Behavioral Phenotypes

Glycine is the major inhibitory neurotransmitter in the spinal cord and some brain regions. The presynaptic glycine transporter, GlyT2, is required for sustained glycinergic transmission through presynaptic reuptake and recycling of glycine. Mutations in SLC6A5, encoding GlyT2, cause hereditary hyperekplexia in humans, and similar phenotypes in knock-out mice, and variants are associated with schizophrenia. We identified a spontaneous mutation in mouse Slc6a5, caused by a MusD retrotransposon insertion. The GlyT2 protein is undetectable in homozygous mutants, indicating a null allele. Homozygous mutant mice are normal at birth, but develop handling-induced spasms at five days of age, and only survive for two weeks, but allow the study of early activity-regulated developmental processes. At the neuromuscular junction, synapse elimination and the switch from embryonic to adult acetylcholine receptor subunits are hastened, consistent with a presumed increase in motor neuron activity, and transcription of acetylcholine receptors is elevated. Heterozygous mice, which show no reduction in lifespan but nonetheless have reduced levels of GlyT2, have a normal thermal sensitivity with the hot-plate test, but differences in repetitive grooming and decreased sleep time with home-cage monitoring. Open-field and elevated plus-maze tests did not detect anxiety-like behaviors; however, the latter showed a hyperactivity phenotype. Importantly, grooming and hyperactivity are observed in mouse schizophrenia models. Thus, mutations in Slc6a5 show changes in neuromuscular junction development as homozygotes, and behavioral phenotypes as heterozygotes, indicating their usefulness for studies related to glycinergic dysfunction

Charcot-Marie-Tooth–Linked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels

Charcot-Marie-Tooth disease type 2D (CMT2D) is a dominantly inherited peripheral neuropathy caused by missense mutations in the glycyl-tRNA synthetase gene (GARS). In addition to GARS, mutations in three other tRNA synthetase genes cause similar neuropathies, although the underlying mechanisms are not fully understood. To address this, we generated transgenic mice that ubiquitously over-express wild-type GARS and crossed them to two dominant mouse models of CMT2D to distinguish loss-of-function and gain-of-function mechanisms. Over-expression of wild-type GARS does not improve the neuropathy phenotype in heterozygous Gars mutant mice, as determined by histological, functional, and behavioral tests. Transgenic GARS is able to rescue a pathological point mutation as a homozygote or in complementation tests with a Gars null allele, demonstrating the functionality of the transgene and revealing a recessive loss-of-function component of the point mutation. Missense mutations as transgene-rescued homozygotes or compound heterozygotes have a more severe neuropathy than heterozygotes, indicating that increased dosage of the disease-causing alleles results in a more severe neurological phenotype, even in the presence of a wild-type transgene. We conclude that, although missense mutations of Gars may cause some loss of function, the dominant neuropathy phenotype observed in mice is caused by a dose-dependent gain of function that is not mitigated by over-expression of functional wild-type protein

Neuromuscular junction phenotypes.

<p><b>A.</b> Representative pictures of P7 NMJs in WT (left) and mutant (right) triangularis sterni muscles. The axons are labeled by the Thy1-YFP16 transgene (green) and the acetylcholine receptor plaques by fluorescent alpha-bungarotoxin (red). NMJs innervated by more than one axon terminal (arrowheads) are more abundant in the WT than in the mutant. <b>B.</b> Time course of the synapse elimination<b>.</b> The curves represent the average percentage of polyinnervated NMJs in wild-type (gray) and mutant (black) triangularis sterni muscles at ages P3 to P12. a: Wilcoxon signed-rank paired test, p = 0.09. The bars represent the average percentage of polyinnervated NMJs in the P3-P5 and P6-P9 age groups. * : Mann-Whitney test, p<0.05. Error bars represent the SEM. <b>C.</b> Schematic representation of the molecular switch from gamma to epsilon AChR subunits in the muscle pentameric acetylcholine receptor, also indicating the constitutive subunits alpha, beta and delta. <b>D.</b> Quantification of the fold-change of AChR subunits alpha, gamma and epsilon, and myogenin, in P5 and two week old synaptic regions of the mutant diaphragm, expressed as a ratio to age-matched WT values ( = 1.0). Biological replicates: 5 for P5, 7 for two week. Error bars represent the confidence interval. * : Mann-Whitney test, p<0.05.</p

Nocturnal behavioral phenotypes in the home cage monitoring system.

<p><b>A.</b> Table of the major behaviors that did not show any change between genotypes, in % of the total nocturnal time (mean and SEM). <b>B.</b> Behaviors with a significant change. <i>a</i> : p = 0.08 for the amount of sleep and b : p = 0.06 for the amount of grooming in the 7 months old mutants, * : p<0.05, ** : p<0.01, Mann-Whitney test. See “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030217#s4" target="_blank">Methods</a>” section for more detailed description. 6 female mice per age group and genotype were observed.</p

Characterization of the new <i>Slc6a5</i> mutation as a null-allele.

<p><b>A.</b> Schematic of the <i>Slc6a5</i> gene organization, with boxes and lines representing exons and introns respectively, and boundaries of PCRs (1 to 4) run on reverse-transcribed cDNAs. <b>B.</b> Agarose gel of the PCR amplification products from cDNA. Note the extra band in PCR 2 on mutant brain cDNAs (arrow). <b>C.</b> Schematic representation of intron 5, and location of the insertion and splice donor and acceptor sites in the retrotransposon sequence. The 10 base pairs immediately 5′ of the insertion site, 1833 bp into intron 5, are shown, as well as the first base-pairs of the LTR element of the retrotransposon. The 183 bp sequence of the transposon that is spliced-in <i>Slc6a5</i> mRNA is indicated with its 10 first and last base-pairs and the inferred acceptor and donor splice sites. The primers used to localize the insertion (int5F and MusR), and to amplify the transposon from mutant genomic DNA (int5F and int5R) are indicated by arrows. <b>D.</b> Agarose gel of the products of the PCR on genomic DNA with primers flanking the insertion site (int5F/R of C.). A 3 kb product was amplified from wild-type gDNA, but only a 9 kb product was amplified from mutant gDNA. <b>E.</b> Anti-GLYT2 Western blot on spinal cord cell membrane preparations, with beta-dystroglycan used as a loading control. Genotypes are indicated above the blot. <b>F.</b> Anti-GLYT2 immunocytochemistry (DAB) on brain sagittal sections and spinal cord cross sections. Nuclei were counterstained with hematoxylin before mounting. The lateral brain stem is shown in the top panels with the ventral lobules of the cerebellum visible in the top right corners and the ventral horn of the spinal cord in shown in the bottom panels.</p

Overt phenotype and mapping of the new mutation.

<p><b>A.</b> P15 mutant (left, white) and unaffected littermate (right, brown) in the NOD x DBA/2 mapping cross. Picture was taken at the time of a generalized tremor of the mutant. <b>B.</b> Growth curve for wild-type, heterozygous and homozygous mutants, completed once the genotyping assay was established. <b>C.</b> Haplotype of affected (left) and unaffected (right) mice at three loci of chromosome 7 flanking the <i>Slc6a5 (Slc6a5)</i> gene. Simple sequence length polymorphism (<i>Mit</i> markers) different in NOD and DBA/2 and their positions in megabases (Mb) are indicated. The number of mice of each genotype is indicated below the diagram. <b>D.</b> Schematic representation of chromosome 7 with centromere at the top, location of MIT markers used and <i>Slc6a5</i>. Positions are given in megabases (Mb) on the left and centimorgans (cM) on the right.</p

Normal thermal nociception in heterozygotes.

<p>Results of the hot-plate assay. Reaction time before flickering/retraction of one paw after mice have been placed on a 50 deg hot plate. Individual values are shown by circles, mean +/- SEM are shown by the chart and error bars. The number of mice tested is indicated.</p

Precision mouse models of Yars/dominant intermediate Charcot-Marie-Tooth disease type C and Sptlc1/hereditary sensory and autonomic neuropathy type 1

Animal models of neurodegenerative diseases such as inherited peripheral neuropathies sometimes accurately recreate the pathophysiology of the human disease, and sometimes accurately recreate the genetic perturbations found in patients. Ideally, models achieve both, but this is not always possible; nonetheless, such models are informative. Here we describe two animal models of inherited peripheral neuropathy: mice with a mutation in tyrosyl tRNA-synthetase, YarsE196K, modeling dominant intermediate Charcot-Marie-Tooth disease type C (diCMTC), and mice with a mutation in serine palmitoyltransferase long chain 1, Sptlc1C133W, modeling hereditary sensory and autonomic neuropathy type 1 (HSAN1). YarsE196K mice develop disease-relevant phenotypes including reduced motor performance and reduced nerve conduction velocities by 4 months of age. Peripheral motor axons are reduced in size, but there is no reduction in axon number and plasma neurofilament light chain levels are not increased. Unlike the dominant human mutations, the YarsE196K mice only show these phenotypes as homozygotes, or as compound heterozygotes with a null allele, and no phenotype is observed in E196K or null heterozygotes. The Sptlc1C133W mice carry a knockin allele and show the anticipated increase in 1-deoxysphingolipids in circulation and in a variety of tissues. They also have mild behavioral defects consistent with HSAN1, but do not show neurophysiological defects or axon loss in peripheral nerves or in the epidermis of the hind paw or tail. Thus, despite the biochemical phenotype, the Sptlc1C133W mice do not show a strong neuropathy phenotype. Surprisingly, these mice were lethal as homozygotes, but the heterozygous genotype studied corresponds to the dominant genetics seen in humans. Thus, YarsE196K homozygous mice have a relevant phenotype, but imprecisely reproduce the human genetics, whereas the Sptlc1C133W mice precisely reproduce the human genetics, but do not recreate the disease phenotype. Despite these shortcomings, both models are informative and will be useful for future research

Metabolite profile of a mouse model of Charcot-Marie-Tooth type 2D neuropathy: implications for disease mechanisms and interventions.

Charcot-Marie-Tooth disease encompasses a genetically heterogeneous class of heritable polyneuropathies that result in axonal degeneration in the peripheral nervous system. Charcot-Marie-Tooth type 2D neuropathy (CMT2D) is caused by dominant mutations in glycyl tRNA synthetase (GARS). Mutations in the mouse Gars gene result in a genetically and phenotypically valid animal model of CMT2D. How mutations in GARS lead to peripheral neuropathy remains controversial. To identify putative disease mechanisms, we compared metabolites isolated from the spinal cord of Gars mutant mice and their littermate controls. A profile of altered metabolites that distinguish the affected and unaffected tissue was determined. Ascorbic acid was decreased fourfold in the spinal cord of CMT2D mice, but was not altered in serum. Carnitine and its derivatives were also significantly reduced in spinal cord tissue of mutant mice, whereas glycine was elevated. Dietary supplementation with acetyl-L-carnitine improved gross motor performance of CMT2D mice, but neither acetyl-L-carnitine nor glycine supplementation altered the parameters directly assessing neuropathy. Other metabolite changes suggestive of liver and kidney dysfunction in the CMT2D mice were validated using clinical blood chemistry. These effects were not secondary to the neuromuscular phenotype, as determined by comparison with another, genetically unrelated mouse strain with similar neuromuscular dysfunction. However, these changes do not seem to be causative or consistent metabolites of CMT2D, because they were not observed in a second mouse Gars allele or in serum samples from CMT2D patients. Therefore, the metabolite \u27fingerprint\u27 we have identified for CMT2D improves our understanding of cellular biochemical changes associated with GARS mutations, but identification of efficacious treatment strategies and elucidation of the disease mechanism will require additional studies. Biol Open 2016 Jun 10 [Epub ahead of print

Data from: Metabolite profile of a mouse model of Charcot-Marie-Tooth type 2D neuropathy: implications for disease mechanisms and interventions

Charcot-Marie-Tooth disease encompasses a genetically heterogeneous class of heritable polyneuropathies that result in axonal degeneration in the peripheral nervous system. Charcot-Marie-Tooth type 2D neuropathy (CMT2D) is caused by dominant mutations in glycyl tRNA synthetase (GARS). Mutations in the mouse Gars gene result in a genetically and phenotypically valid animal model of CMT2D. How mutations in GARS lead to peripheral neuropathy remains controversial. To identify putative disease mechanisms, we compared metabolites isolated from the spinal cord of Gars mutant mice and their littermate controls. A profile of altered metabolites that distinguish the affected and unaffected tissue was determined. Ascorbic acid was decreased fourfold in the spinal cord of CMT2D mice, but was not altered in serum. Carnitine and its derivatives were also significantly reduced in spinal cord tissue of mutant mice, whereas glycine was elevated. Dietary supplementation with acetyl-L-carnitine improved gross motor performance of CMT2D mice, but neither acetyl-L-carnitine nor glycine supplementation altered the parameters directly assessing neuropathy. Other metabolite changes suggestive of liver and kidney dysfunction in the CMT2D mice were validated using clinical blood chemistry. These effects were not secondary to the neuromuscular phenotype, as determined by comparison with another, genetically unrelated mouse strain with similar neuromuscular dysfunction. However, these changes do not seem to be causative or consistent metabolites of CMT2D, because they were not observed in a second mouse Gars allele or in serum samples from CMT2D patients. Therefore, the metabolite 'fingerprint' we have identified for CMT2D improves our understanding of cellular biochemical changes associated with GARS mutations, but identification of efficacious treatment strategies and elucidation of the disease mechanism will require additional studies