Nerve Terminal Degeneration Is Independent of Muscle Fiber Genotype in SOD1G93A Mice

Background: Motor neuron degeneration in SOD1 G93A transgenic mice begins at the nerve terminal. Here we examine whether this degeneration depends on expression of mutant SOD1 in muscle fibers. Methodology/Principal Findings: Hindlimb muscles were transplanted between wild-type and SOD1 G93A transgenic mice and the innervation status of neuromuscular junctions (NMJs) was examined after 2 months. The results showed that muscles from SOD1 G93A mice did not induce motor terminal degeneration in wildtype mice and that muscles from wildtype mice did not prevent degeneration in SOD1 G93A transgenic mice. Control studies demonstrated that muscles transplanted from SOD1 G93A mice continued to express mutant SOD1 protein. Experiments on wildtype mice established that the host supplied terminal Schwann cells (TSCs) at the NMJs of transplanted muscles. Conclusions/Significance: These results indicate that expression of the mutant protein in muscle is not needed to cause motor terminal degeneration in SOD1 G93A transgenic mice and that a combination of motor terminals, motor axons and Schwann cells, all of which express mutant protein may be sufficient

Three ENU-induced neurological mutations in the pore loopof sodium channel Scn8a (Na v 1.6) and a genetically linkedretinal mutation, rd13

The goal of The Jackson Laboratory Neuroscience Mutagenesis Facility is to generate mouse models of human neurological disease. We describe three new models obtained from a three-generation screen for recessive mutations. Homozygous mutant mice from lines nmf2 and nmf5 exhibit hind limb paralysis and juvenile lethality. Homozygous nmf58 mice exhibit a less severe movement disorder that includes sustained dystonic postures. The mutations were mapped to the distal region of mouse Chromosome (Chr) 15. Failure to complement a mutant allele of a positional candidate gene, Scn8a , demonstrated that the mutations are new alleles of Scn8a . Missense mutations of evolutionarily conserved residues of the sodium channel were identified in the three lines, with the predicted amino acid substitutions N1370T, I1392F, and L1404H. These residues are located within the pore loop of domain 3 of sodium channel Na v 1.6. The lethal phenotypes suggest that the new alleles encode proteins with partial or complete loss of function. Several human disorders are caused by mutation in the pore loop of domain 3 of paralogous sodium channel genes. Line nmf5 contains a second, independent mutation in the rd13 locus that causes a reduction in cell number in the outer nuclear layer of the retina. rd13 was mapped to the distal 4 Mb of Chr 15. No coding or splice site mutations were detected in Pde1b , a candidate gene for rd13 . The generation of three independent Scn8a mutations among 1100 tested G3 families demonstrates that the Scn8a locus is highly susceptible to ENU mutagenesis. The new alleles of Scn8a will be valuable for analysis of sodium channel physiology and disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46985/1/335_2004_Article_2332.pd

A Novel ENU-Induced

The fission and fusion of mitochondria are important processes for maintaining mitochondrial health. One of the proteins responsible for mediating mitochondrial fusion, mitofusin 2 (MFN2), has over 100 known mutations that cause Charcot–Marie–Tooth disease type 2A (CMT2A). This disease causes the nerves that control your muscles to degenerate, leading to muscle atrophy and weakness, problems walking, and other related symptoms. In this paper, we describe a mouse line with a recessive mutation in the Mfn2 gene (Leu643Pro) that causes a similar set of symptoms, including abnormal gait, weight loss, and decreased muscular endurance. However, further analysis of these mice revealed signs of skeletal muscle dysfunction (including smaller mitochondria) and bone abnormalities, with little evidence of axon degeneration typical of CMT2A. While this makes these mice a poor model for CMT2A, they are the first reported mouse line with a mutation in the transmembrane domain, a region critical for MFN2′s role in mitochondrial fusion. For this reason, we believe these mice will be a valuable tool for scientists interested in studying the biological functions of MFN2

Synaptic Deficits at Neuromuscular Junctions in Two Mouse Models of Charcot-Marie-Tooth Type 2d

Patients with Charcot–Marie–Tooth Type 2D (CMT2D), caused by dominant mutations in Glycl tRNA synthetase (GARS), present with progressive weakness, consistently in the hands, but often in the feet also. Electromyography shows denervation, and patients often report that early symptoms include cramps brought on by cold or exertion. Based on reported clinical observations, and studies of mouse models of CMT2D, we sought to determine whether weakened synaptic transmission at the neuromuscular junction (NMJ) is an aspect of CMT2D. Quantal analysis of NMJs in two different mouse models of CMT2D (Gars(P278KY), Gars(C201R)), found synaptic deficits that correlated with disease severity and progressed with age. Results of voltage-clamp studies revealed presynaptic defects characterized by: (1) decreased frequency of spontaneous release without any change in quantal amplitude (miniature endplate current), (2) reduced amplitude of evoked release (endplate current) and quantal content, (3) age-dependent changes in the extent of depression in response to repetitive stimulation, and (4) release failures at some NMJs with high-frequency, long-duration stimulation. Drugs that modify synaptic efficacy were tested to see whether neuromuscular performance improved. The presynaptic action of 3,4 diaminopyridine was not beneficial, whereas postsynaptic-acting physostigmine did improve performance. Smaller mutant NMJs with correspondingly fewer vesicles and partial denervation that eliminates some release sites also contribute to the reduction of release at a proportion of mutant NMJs. Together, these voltage-clamp data suggest that a number of release processes, while essentially intact, likely operate suboptimally at most NMJs of CMT2D mice. SIGNIFICANCE STATEMENT We have uncovered a previously unrecognized aspect of axonal Charcot–Marie–Tooth disease in mouse models of CMT2D. Synaptic dysfunction contributes to impaired neuromuscular performance and disease progression. This suggests that drugs which improve synaptic efficacy at the NMJ could be considered in treating the pathophysiology of CMT2D patients

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

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

Alteration of the unfolded protein response modifies neurodegeneration in a mouse model of Marinesco–Sjögren syndrome

Endoplasmic reticulum (ER) stress has been linked to the onset and progression of many diseases. SIL1 is an adenine nucleotide exchange factor of the essential ER lumen chaperone HSPA5/BiP that senses ER stress and is involved in protein folding. Mutations in the Sil1 gene have been associated with Marinesco–Sjögren syndrome, hallmarks of which include ataxia and cerebellar atrophy. We have previously shown that loss of SIL1 function in mouse results in ER stress, ubiquitylated protein inclusions, and degeneration of specific Purkinje cells in the cerebellum. Here, we report that overexpression of HYOU1/ORP150, an exchange factor that works in parallel to SIL1, prevents ER stress and rescues neurodegeneration in Sil1−/− mice, whereas decreasing expression of HYOU1 exacerbates these phenotypes. In addition, loss of DNAJC3/p58IPK, a co-chaperone that promotes ATP hydrolysis by BiP, ameliorates ER stress and neurodegeneration in Sil1−/− mice. These findings suggest that alterations in the nucleotide exchange cycle of BiP cause ER stress and neurodegeneration in Sil1-deficient mice. Our results present the first evidence of important genetic modifiers of Marinesco–Sjögren syndrome, and provide additional pathways for therapeutic intervention for this, and other ER stress-induced, diseases

A Spontaneous Mutation in Contactin 1 in the Mouse

Mutations in the gene encoding the immunoglobulin-superfamily member cell adhesion molecule contactin1 (CNTN1) cause lethal congenital myopathy in human patients and neurodevelopmental phenotypes in knockout mice. Whether the mutant mice provide an accurate model of the human disease is unclear; resolving this will require additional functional tests of the neuromuscular system and examination of Cntn1 mutations on different genetic backgrounds that may influence the phenotype. Toward these ends, we have analyzed a new, spontaneous mutation in the mouse Cntn1 gene that arose in a BALB/c genetic background. The overt phenotype is very similar to the knockout of Cntn1, with affected animals having reduced body weight, a failure to thrive, locomotor abnormalities, and a lifespan of 2–3 weeks. Mice homozygous for the new allele have CNTN1 protein undetectable by western blotting, suggesting that it is a null or very severe hypomorph. In an analysis of neuromuscular function, neuromuscular junctions had normal morphology, consistent with previous studies in knockout mice, and the muscles were able to generate appropriate force when normalized for their reduced size in late stage animals. Therefore, the Cntn1 mutant mice do not show evidence for a myopathy, but instead the phenotype is likely to be caused by dysfunction in the nervous system. Given the similarity of CNTN1 to other Ig-superfamily proteins such as DSCAMs, we also characterized the expression and localization of Cntn1 in the retinas of mutant mice for developmental defects. Despite widespread expression, no anomalies in retinal anatomy were detected histologically or using a battery of cell-type specific antibodies. We therefore conclude that the phenotype of the Cntn1 mice arises from dysfunction in the brain, spinal cord or peripheral nervous system, and is similar in either a BALB/c or B6;129;Black Swiss background, raising a possible discordance between the mouse and human phenotypes resulting from Cntn1 mutations

The effect of the manipulation of blood lactate on the integrated EMG of the vastus lateralis muscle during incremental exercise

This study was designed to test the hypothesis that the electromyographic signal recorded from a working muscle reflects changes in blood lactate concentrations. A group of trained cyclists performed two incremental exercise tests on a cycle ergometer. The Control Trial was a incremental test with power increments of 23.5 watts per minute. Cadence was monitored and maintained at 90+/-1 revolutions per minute. The Experimental Trial consisted of a high intensity arm exercise protocol designed to elevate blood lactate above 8 mmol/1. The arm protocol was followed by five minutes of rest and the incremental exercise protocol used in the Control Trial. Expired gases were sampled every fifteen seconds and calculated values for oxygen uptake, ventilation, excess CO₂, and R.Q. were averaged to give a mean value for each minute in both trials. Heart rate was monitored and recorded every minute for both trials. Electromyographic data were sampled from the vastus lateralis of the right leg for the final eight seconds of each workload in both trials. The data were integrated for each pedal cycle and averaged to give a mean integrated value for each cycle (CIEMG) for each workload. During both trials blood samples were drawn from the cephalic vein of the left arm during the last ten seconds of each workload. The anaerobic threshold (Tlac) was determined using the log-log transformation as outlined by Beaver et al., (1985). Control Trial lactate concentration showed a marked inflection point after an initial slow increase. The mean maximal lactate concentration was 18.21 +/- 5.54 in the Control Trials. This inflection point occurred at a mean lactate concentration of 5.58 +/- 1.05 mmol/1. The mean oxygen uptake at the inflection point was 2.28 +/- 0.37 1/min which represented a mean of 72.6 +/- 7.20 % of maximum. Experimental Trial mean plasma lactate at the beginning of incremental exercise was 26.61 +/- 8.86 mmol/1. The plasma lactate concentration decreased steadily for the initial loads to a mean low concentration of 10.78 +/- 5.78 mmol/1 at Tlac and then increased to a mean of 19.08 +/- 6.66 mmol/1 at test completion. Plasma lactate concentration was greater in the Experimental Trial at all workloads though the values tended to converge once Tlac was surpassed. No visually identifiable inflection point in the plot of CIEMG vs Power could be determined in any of the plots. An analysis of the slope of the CIEMG vs. Power relationship was therefore performed. An analysis of variance demonstrated no significant difference in the slope of the relationship within or between trials in three different comparisons. The slope of the line was not statistically different when compared over: (a) the entire sample (b) pre Tlac and (c) post Tlac. Correlations performed between plasma lactate concentrations and CIEMG were significant in five of six subjects during the Control Trial (r = 0.57 to 0.97). During the Experimental Trial only three of the six subjects showed significant correlations and they were in the opposite direction (r = -0.62 to -0.96). Correlations between power output and CIEMG were for all subjects in both trials (r = 0.92 to 0.99 Control, r = 0.91 to 0.99 Experimental). The increase seen in CIEMG with increased power output reflects poorly the changes in blood lactate concentrations under the conditions of this investigation. Plasma lactate showed a dramatic increase in the Control Trial and a steady decrease from an initial high concentration followed by a marked increase in the final workloads of the Experimental Trial. In contrast the CIEMG increased in a near linear fashion for all subjects in both trials. The changes in CIEMG showed highly significant correlations with changes in VO₂ or power output in both trials for all subjects. These results indicate that changes in the surface electromyogram are highly related to changes in power output. However the surface electromyogram changes are not driven by changes in lactate concentration under the conditions of this investigation and may not be a sensitive enough indicator of these changes to be employed in the determination of Tlac.Education, Faculty ofCurriculum and Pedagogy (EDCP), Department ofGraduat

Low-Frequency Depression of the Monosynaptic Reflex is Not Altered By Tetrodotoxin-Induced Nerve Conduction Blockade

The present study is part of ongoing investigations into activity-related synaptic plasticity in the intact animal. In this investigation we sought to determine whether the previously reported increase in synaptic efficacy at the Ia-motoneuron connection following nerve conduction blockade could be attributed to changes in circuitry external to the monosynaptic pathway. Specifically, we used the phenomena of low-frequency depression of the extracellularly recorded group I monosynaptic reflex (MSR) as an indirect measure of presynaptic inhibition. Tibial nerve conduction blockade was achieved by superfusion of the sodium channel blocker tetrodotoxin (TTX). An osmotic pump delivered the TTX to the tibial branch of the sciatic nerve for a period of either 3 or 10 days. Control rats were either unoperated or received implants of pumps not containing TTX. Data collection consisted of tibial nerve stimulation (0.1-20 Hz) with bilateral recordings of the MSR from the L5 ventral roots. The extent of low-frequency depression was compared between treated and untreated sides of TTX-treated animals and between treated and untreated animals. Results showed that the extent of low-frequency depression was unchanged by either 3 or 10 days of complete blockade of tibial afferents. On the basis of this finding, it is concluded that the previously reported TTX-induced increase in Ia excitatory postsynaptic potential amplitude is unlikely to be due to changes in presynaptic inhibitory pathways