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1.
J Neurosci ; 36(11): 3254-67, 2016 Mar 16.
Article in English | MEDLINE | ID: mdl-26985035

ABSTRACT

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.


Subject(s)
Charcot-Marie-Tooth Disease/pathology , Disease Models, Animal , Glycine-tRNA Ligase/genetics , Mutation/genetics , Neuromuscular Junction/pathology , Synaptic Transmission/genetics , Age Factors , Aminopyridines/pharmacology , Animals , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/physiopathology , Electric Stimulation , Imaging, Three-Dimensional , Mice , Mice, Transgenic , Motor Endplate/genetics , Motor Endplate/physiopathology , Muscle Strength/genetics , Muscle, Skeletal/pathology , Muscle, Skeletal/ultrastructure , Nerve Tissue Proteins/metabolism , Neuromuscular Junction/drug effects , Neuromuscular Junction/genetics , Neuromuscular Junction/metabolism , Patch-Clamp Techniques , Receptors, Cholinergic/metabolism , Synaptic Potentials/drug effects , Synaptic Potentials/genetics , Synaptic Vesicles/pathology , Synaptic Vesicles/ultrastructure
2.
PLoS Genet ; 8(8): e1002853, 2012.
Article in English | MEDLINE | ID: mdl-22912588

ABSTRACT

Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wld(s) and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.


Subject(s)
Adenosine Triphosphatases/genetics , Axons/enzymology , Neurodegenerative Diseases/genetics , Phospholipid Transfer Proteins/genetics , Wallerian Degeneration/genetics , Adenosine Triphosphatases/metabolism , Alleles , Animals , Axons/pathology , Base Sequence , Brain/enzymology , Brain/pathology , Genotype , Mice , Mice, Transgenic , Molecular Sequence Data , Mutation , Neurodegenerative Diseases/enzymology , Neurodegenerative Diseases/pathology , Phenotype , Phospholipid Transfer Proteins/metabolism , Retina/enzymology , Retina/pathology , Spinal Cord/enzymology , Spinal Cord/pathology , Wallerian Degeneration/enzymology , Wallerian Degeneration/pathology
3.
Hum Mol Genet ; 21(12): 2807-14, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22442204

ABSTRACT

Infantile neuroaxonal dystrophy (INAD; OMIM #no. 256600) is an inherited degenerative nervous system disorder characterized by nerve abnormalities in brain, spinal cord and peripheral nerves. About 85% of INAD patients carry mutations in the PLA2G6 gene that encodes for a Ca(2+)-independent phospholipase A(2) (VIA iPLA(2)), but how these mutations lead to disease is unknown. Besides regulating phospholipid homeostasis, VIA iPLA(2) is emerging with additional non-canonical functions, such as modulating store-regulated Ca(2+) entry into cells, and mitochondrial functions. In turn, defective Ca(2+) regulation could contribute to the development of INAD. Here, we studied possible changes in ATP-induced Ca(2+) signaling in astrocytes derived from two mutant strains of mice. The first strain carries a hypomorphic allele of the Pla2g6 that reduces transcript levels to 5-10% of that observed in wild-type mice. The second strain carries a point mutation in Pla2g6 that results in inactive VIA iPLA(2) protein with postulated gain in toxicity. Homozygous mice from both strains develop pathology analogous to that observed in INAD patients. The nucleotide ATP is the most important transmitter inducing Ca(2+) signals in astroglial networks. We demonstrate here a severe disturbance in Ca(2+) responses to ATP in astrocytes derived from both mutant mouse strains. The duration of the Ca(2+) responses in mutant astrocytes was significantly reduced when compared with values observed in control cells. We also show that the reduced Ca(2+) responses are probably due to a reduction in capacitative Ca(2+) entry (2.3-fold). Results suggest that altered Ca(2+) signaling could be a central mechanism in the development of INAD pathology.


Subject(s)
Astrocytes/metabolism , Calcium/metabolism , Group VI Phospholipases A2/genetics , Mutation , Neuroaxonal Dystrophies/genetics , Adenosine Triphosphate/pharmacology , Animals , Astrocytes/drug effects , Calcium Signaling/drug effects , Cells, Cultured , Disease Models, Animal , Gene Expression , Genotype , Group VI Phospholipases A2/metabolism , Humans , Mice , Mice, Inbred C3H , Mice, Knockout , Neuroaxonal Dystrophies/metabolism , Neuroaxonal Dystrophies/pathology , Reverse Transcriptase Polymerase Chain Reaction
4.
Hum Mol Genet ; 21(4): 730-50, 2012 Feb 15.
Article in English | MEDLINE | ID: mdl-22048958

ABSTRACT

We have identified a point mutation in Npc1 that creates a novel mouse model (Npc1(nmf164)) of Niemann-Pick type C1 (NPC) disease: a single nucleotide change (A to G at cDNA bp 3163) that results in an aspartate to glycine change at position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the NPC1 protein and is highly similar to commonly occurring human mutations. Genetic and molecular biological analyses, including sequencing the Npc1(spm) allele and identifying a truncating mutation, confirm that the mutation in Npc1(nmf164) mice is distinct from those in other existing mouse models of NPC disease (Npc1(nih), Npc1(spm)). Analyses of lifespan, body and spleen weight, gait and other motor activities, as well as acoustic startle responses all reveal a more slowly developing phenotype in Npc1(nmf164) mutant mice than in mice with the null mutations (Npc1(nih), Npc1(spm)). Although Npc1 mRNA levels appear relatively normal, Npc1(nmf164) brain and liver display dramatic reductions in Npc1 protein, as well as abnormal cholesterol metabolism and altered glycolipid expression. Furthermore, histological analyses of liver, spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol accumulation, glial activation and Purkinje cell loss at a slower rate than in the Npc1(nih) mouse model. Magnetic resonance imaging studies also reveal significantly less demyelination/dysmyelination than in the null alleles. Thus, although prior mouse models may correspond to the severe infantile onset forms of NPC disease, Npc1(nmf164) mice offer many advantages as a model for the late-onset, more slowly progressing forms of NPC disease that comprise the large majority of human cases.


Subject(s)
Carrier Proteins/genetics , Disease Models, Animal , Membrane Glycoproteins/genetics , Niemann-Pick Disease, Type C/genetics , Point Mutation/genetics , Age of Onset , Alleles , Animals , Astrocytes/pathology , Brain/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cholesterol/metabolism , DNA Mutational Analysis , Disease Progression , Endoplasmic Reticulum Stress , Gangliosides/metabolism , Homozygote , Humans , Intracellular Signaling Peptides and Proteins , Lipid Metabolism , Lung/cytology , Macrophages/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Mice , Microglia/pathology , Myelin Sheath , Niemann-Pick C1 Protein , Niemann-Pick Disease, Type C/metabolism , Niemann-Pick Disease, Type C/pathology , Niemann-Pick Disease, Type C/physiopathology , Phenotype , Proteostasis Deficiencies , Purkinje Cells/pathology , RNA, Messenger/analysis , RNA, Messenger/genetics , Reflex, Startle , Survival Rate
5.
PLoS Genet ; 7(12): e1002399, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22144914

ABSTRACT

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.


Subject(s)
Charcot-Marie-Tooth Disease/genetics , Glycine-tRNA Ligase/genetics , Peripheral Nervous System/metabolism , Animals , Axons/metabolism , Disease Models, Animal , Glycine-tRNA Ligase/metabolism , Heterozygote , Homozygote , Humans , Mice , Mice, Transgenic , Mutation, Missense/genetics , Neurons/metabolism , Neurons/pathology , Peripheral Nervous System/pathology , Schwann Cells/metabolism , Sciatic Nerve/metabolism
6.
Biology (Basel) ; 12(7)2023 Jul 03.
Article in English | MEDLINE | ID: mdl-37508383

ABSTRACT

Mitochondrial fission and fusion are required for maintaining functional mitochondria. The mitofusins (MFN1 and MFN2) are known for their roles in mediating mitochondrial fusion. Recently, MFN2 has been implicated in other important cellular functions, such as mitophagy, mitochondrial motility, and coordinating endoplasmic reticulum-mitochondria communication. In humans, over 100 MFN2 mutations are associated with a form of inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 2A (CMT2A). Here we describe an ENU-induced mutant mouse line with a recessive neuromuscular phenotype. Behavioral screening showed progressive weight loss and rapid deterioration of motor function beginning at 8 weeks. Mapping and sequencing revealed a missense mutation in exon 18 of Mfn2 (T1928C; Leu643Pro), within the transmembrane domain. Compared to wild-type and heterozygous littermates, Mfn2L643P/L643P mice exhibited diminished rotarod performance and decreases in activity in the open field test, muscular endurance, mean mitochondrial diameter, sensory tests, mitochondrial DNA content, and MFN2 protein levels. However, tests of peripheral nerve physiology and histology were largely normal. Mutant leg bones had reduced cortical bone thickness and bone area fraction. Together, our data indicate that Mfn2L643P causes a recessive motor phenotype with mild bone and mitochondrial defects in mice. Lack of apparent nerve pathology notwithstanding, this is the first reported mouse model with a mutation in the transmembrane domain of the protein, which may be valuable for researchers studying MFN2 biology.

7.
Genesis ; 50(12): 882-91, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22926980

ABSTRACT

The three adducin proteins (α, ß, and γ) share extensive sequence, structural, and functional homology. Heterodimers of α- and ß-adducin are vital components of the red cell membrane skeleton, which is required to maintain red cell elasticity and structural integrity. In addition to anemia, targeted deletion of the α-adducin gene (Add1) reveals unexpected, strain-dependent non-erythroid phenotypes. On an inbred 129 genetic background, Add1 null mice show abnormal inward curvature of the cervicothoracic spine with complete penetrance. More surprisingly, a subset of 129-Add1 null mice develop severe megaesophagus, while examination of peripheral nerves reveals a reduced number of axons in 129-Add1 null mice at four months of age. These unforeseen phenotypes, described here, reveal new functions for adducin and provide new models of mammalian disease.


Subject(s)
Calmodulin-Binding Proteins/genetics , Cytoskeletal Proteins/genetics , Esophageal Achalasia/genetics , Kyphosis/genetics , Animals , Cytoskeletal Proteins/metabolism , Esophageal Achalasia/pathology , Gene Deletion , Kyphosis/pathology , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Penetrance , Species Specificity
8.
Neurobiol Dis ; 48(1): 132-40, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22750521

ABSTRACT

This study examined whether activity is a contributing factor to motor terminal degeneration in mice that overexpress the G93A mutation of the SOD1 enzyme found in humans with inherited motor neuron disease. Previously, we showed that overload of muscles accomplished by synergist denervation accelerated motor terminal degeneration in dogs with hereditary canine spinal muscular atrophy (HCSMA). In the present study, we found that SOD1 plantaris muscles overloaded for 2months showed no differences of neuromuscular junction innervation status when compared with normally loaded, contralateral plantaris muscles. Complete elimination of motor terminal activity using blockade of sciatic nerve conduction with tetrodotoxin cuffs for 1month also produced no change of plantaris innervation status. To assess possible effects of activity on motor terminal function, we examined the synaptic properties of SOD1 soleus neuromuscular junctions at a time when significant denervation of close synergists had occurred as a result of natural disease progression. When examined in glucose media, SOD1 soleus synaptic properties were similar to wildtype. When glycolysis was inhibited and ATP production limited to mitochondria, however, blocking of evoked synaptic transmission occurred and a large increase in the frequency of spontaneous mEPCs was observed. Similar effects were observed at neuromuscular junctions in muscle from dogs with inherited motor neuron disease (HCSMA), although significant defects of synaptic transmission exist at these neuromuscular junctions when examined in glucose media, as reported previously. These results suggest that glycolysis compensates for mitochondrial dysfunction at motor terminals of SOD1 mice and HCSMA dogs. This compensatory mechanism may help to support resting and activity-related metabolism in the presence of dysfunctional mitochondria and prolong the survival of SOD1 motor terminals.


Subject(s)
Glycolysis/physiology , Motor Neuron Disease/physiopathology , Motor Neurons/pathology , Neuromuscular Junction/pathology , Superoxide Dismutase/genetics , Animals , Dogs , Mice , Mice, Transgenic , Motor Neuron Disease/genetics , Motor Neuron Disease/metabolism , Motor Neuron Disease/pathology , Motor Neurons/metabolism , Muscle, Skeletal/innervation , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiopathology , Neuromuscular Junction/metabolism , Superoxide Dismutase/metabolism , Superoxide Dismutase-1
9.
Mol Cell Neurosci ; 46(2): 432-43, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21115117

ABSTRACT

Mutations in glycyl-, tyrosyl-, and alanyl-tRNA synthetases (GARS, YARS and AARS respectively) cause autosomal dominant Charcot-Marie-Tooth disease, and mutations in Gars cause a similar peripheral neuropathy in mice. Aminoacyl-tRNA synthetases (ARSs) charge amino acids onto their cognate tRNAs during translation; however, the pathological mechanism(s) of ARS mutations remains unclear. To address this, we tested possible mechanisms using mouse models. First, amino acid mischarging was discounted by examining the recessive "sticky" mutation in alanyl-tRNA synthetase (Aars(sti)), which causes cerebellar neurodegeneration through a failure to efficiently correct mischarging of tRNA(Ala). Aars(sti/sti) mice do not have peripheral neuropathy, and they share no phenotypic features with the Gars mutant mice. Next, we determined that the Wallerian Degeneration Slow (Wlds) mutation did not alter the Gars phenotype. Therefore, no evidence for misfolding of GARS itself or other proteins was found. Similarly, there were no indications of general insufficiencies in protein synthesis caused by Gars mutations based on yeast complementation assays. Mutant GARS localized differently than wild type GARS in transfected cells, but a similar distribution was not observed in motor neurons derived from wild type mouse ES cells, and there was no evidence for abnormal GARS distribution in mouse tissue. Both GARS and YARS proteins were present in sciatic axons and Schwann cells from Gars mutant and control mice, consistent with a direct role for tRNA synthetases in peripheral nerves. Unless defects in translation are in some way restricted to peripheral axons, as suggested by the axonal localization of GARS and YARS, we conclude that mutations in tRNA synthetases are not causing peripheral neuropathy through amino acid mischarging or through a defect in their known function in translation.


Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Nerve Degeneration/genetics , Peripheral Nervous System Diseases/genetics , Animals , Axons/pathology , Charcot-Marie-Tooth Disease/enzymology , Charcot-Marie-Tooth Disease/genetics , Disease Models, Animal , Femoral Nerve/pathology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Microscopy, Confocal , Mutation , Nerve Degeneration/enzymology , Nerve Degeneration/pathology , Neuromuscular Junction/pathology , Peripheral Nervous System Diseases/enzymology , Peripheral Nervous System Diseases/pathology , Phenotype , Protein Biosynthesis , Purkinje Cells/pathology
10.
Genetics ; 218(1)2021 05 17.
Article in English | MEDLINE | ID: mdl-33734376

ABSTRACT

The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.


Subject(s)
Proline/biosynthesis , Pyrroline Carboxylate Reductases/genetics , Animals , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mutation , Phenotype , Proline/chemistry , Proline/genetics , Pyrroline Carboxylate Reductases/metabolism
11.
Neuron ; 51(6): 715-26, 2006 Sep 21.
Article in English | MEDLINE | ID: mdl-16982418

ABSTRACT

Of the many inherited Charcot-Marie-Tooth peripheral neuropathies, type 2D (CMT2D) is caused by dominant point mutations in the gene GARS, encoding glycyl tRNA synthetase (GlyRS). Here we report a dominant mutation in Gars that causes neuropathy in the mouse. Importantly, both sensory and motor axons are affected, and the dominant phenotype is not caused by a loss of the GlyRS aminoacylation function. Mutant mice have abnormal neuromuscular junction morphology and impaired transmission, reduced nerve conduction velocities, and a loss of large-diameter peripheral axons, without defects in myelination. The mutant GlyRS enzyme retains aminoacylation activity, and a loss-of-function allele, generated by a gene-trap insertion, shows no dominant phenotype in mice. These results indicate that the CMT2D phenotype is caused not by reduction of the canonical GlyRS activity and insufficiencies in protein synthesis, but instead by novel pathogenic roles for the mutant GlyRS that specifically affect peripheral neurons.


Subject(s)
Charcot-Marie-Tooth Disease/genetics , Disease Models, Animal , Glycine-tRNA Ligase/genetics , Mutation/genetics , Peripheral Nervous System Diseases/genetics , Amino Acid Sequence , Animals , Axons/metabolism , Axons/pathology , Axons/ultrastructure , Base Sequence , Charcot-Marie-Tooth Disease/classification , Charcot-Marie-Tooth Disease/enzymology , Chromosome Mapping , Female , Genes, Dominant/genetics , Glycine-tRNA Ligase/metabolism , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Molecular Sequence Data , Neuromuscular Junction/metabolism , Neuromuscular Junction/pathology , Neuromuscular Junction/physiopathology , Peripheral Nervous System Diseases/enzymology , Peripheral Nervous System Diseases/pathology , Sciatic Nerve/metabolism , Sciatic Nerve/pathology , Sciatic Nerve/ultrastructure , Sequence Homology, Amino Acid
12.
Physiol Behav ; 96(2): 350-61, 2009 Feb 16.
Article in English | MEDLINE | ID: mdl-19027767

ABSTRACT

The use of a treadmill to gather data for gait analysis in mice is a convenient, sensitive method to evaluate motor performance. However, evidence from several species, including mice, shows that treadmill locomotion is a novel task that is not equivalent to over ground locomotion and that may be particularly sensitive to the test environment and protocol. We investigated the effects of age, genetic background and repeated trials on treadmill walking in mice and show that these factors are important considerations in the interpretation of gait data. Specifically we report that as C57BL/6J (B6) mice age, the animals use progressively longer, less frequent strides to maintain the same walking speed. The increase is most rapid between 1 and 6 months of age and is explained, in part, by changes in size and weight. We also extended previous findings showing that repeat trials cause mice to modify their treadmill gait pattern. In a second trial B6 mice consistently walk with a shorter swing phase and greater duty factor. Also, with the shortest retest interval (3 min) mice use shorter more frequent steps but the response varies with the number and timing of trials. Finally, we compared the gait pattern of an additional seven inbred strains of mice and found significant variation in the length and frequency of strides used to maintain the same walking speed. The combined results offer the bases for further mechanistic studies and can be used to guide optimal experimental design.


Subject(s)
Aging/physiology , Exercise Test/methods , Learning/physiology , Locomotion/genetics , Age Factors , Animals , Female , Male , Mice , Mice, Inbred Strains , Posture/physiology , Species Specificity
13.
Cell Rep ; 18(13): 3178-3191, 2017 03 28.
Article in English | MEDLINE | ID: mdl-28355569

ABSTRACT

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of inherited polyneuropathies. Mutations in 80 genetic loci can cause forms of CMT, resulting in demyelination and axonal dysfunction. The clinical presentation, including sensory deficits, distal muscle weakness, and atrophy, can vary greatly in severity and progression. Here, we used mouse models of CMT to demonstrate genetic interactions that result in a more severe neuropathy phenotype. The cell adhesion molecule Nrcam and the Na+ channel Scn8a (NaV1.6) are important components of nodes. Homozygous Nrcam and heterozygous Scn8a mutations synergized with both an Sh3tc2 mutation, modeling recessive demyelinating Charcot-Marie-Tooth type 4C, and mutations in Gars, modeling dominant axonal Charcot-Marie-Tooth type 2D. We conclude that genetic variants perturbing the structure and function of nodes interact with mutations affecting the cable properties of axons by thinning myelin or reducing axon diameter. Therefore, genes integral to peripheral nodes are candidate modifiers of peripheral neuropathy.


Subject(s)
Charcot-Marie-Tooth Disease/genetics , Demyelinating Diseases/genetics , Peripheral Nerves/pathology , Animals , Axons/metabolism , Carrier Proteins/genetics , Cell Adhesion Molecules/genetics , Charcot-Marie-Tooth Disease/pathology , Demyelinating Diseases/pathology , Disease Models, Animal , Heterozygote , Intracellular Signaling Peptides and Proteins , Mice, Inbred C57BL , Mutation/genetics , NAV1.6 Voltage-Gated Sodium Channel/genetics , Neuromuscular Junction/metabolism
14.
Brain Res ; 1091(1): 16-26, 2006 May 26.
Article in English | MEDLINE | ID: mdl-16516865

ABSTRACT

The auditory brainstem response (ABR) is an evoked potential response of auditory activity in the auditory nerve and subsequent fiber tracts and nuclei within the auditory brainstem pathways. The threshold, amplitude, and latency analysis of the ABR provides information on the peripheral hearing status and the integrity of brainstem pathways. In this study, we compared the threshold, amplitude, and latency of ABRs recorded from 149 mice of 10 commonly used inbred strains (BALB/cJ, C3HeB/FeJ, C3H/HeJ, CAST/EiJ, CBA/CaJ, CBA/J, FVB/NJ, MRL/MpJ, NZB/BlNJ, and SJL/J) using clicks of different intensities. The ABR thresholds of these strains ranged from 32 to 43 dB SPL. The amplitude of both waves I and IV of ABRs, which increased monotonically with click intensity in most strains, differed significantly among different strains at each intensity tested. Moreover, the amplitude of both waves was inversely correlated with the body weight of each strain at most intensities tested. In general, the amplitude of wave IV was smaller than that of wave I resulting in the IV/I amplitude ratio of <1.0 in all strains. The peak latency of both waves I and IV decreased significantly with click intensity in each strain. However, this intensity-dependent decrease was greater for wave IV than for wave I such that the wave I-IV inter-peak latency also decreased significantly with increasing intensity. I-IV inter-peak latencies for MRL/MpJ, C3HeB/FeJ, NZB/BlNJ, and C3H/HeJ strains are longer than FVB/NJ, SJL/J, or CAST/EiJ. This work is the first step to study the genetic basis underlying strain-related differences in auditory pathway.


Subject(s)
Auditory Pathways/physiology , Brain Stem/physiology , Evoked Potentials, Auditory, Brain Stem/physiology , Acoustic Stimulation/methods , Analysis of Variance , Animals , Auditory Threshold/physiology , Cochlear Nerve/physiology , Dose-Response Relationship, Radiation , Mice , Mice, Inbred Strains , Reaction Time/physiology
15.
Exp Neurol ; 275 Pt 1: 172-81, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26416261

ABSTRACT

In mice that express SOD1 mutations found in human motor neuron disease, degeneration begins in the periphery for reasons that remain unknown. At the neuromuscular junction (NMJ), terminal Schwann cells (TSCs) have an intimate relationship with motor terminals and are believed to help maintain the integrity of the motor terminal. Recent evidence indicates that TSCs in some SOD1 mice exhibit abnormal functional properties, but other aspects of possible TSC involvement remain unknown. In this study, an analysis of TSC morphology and number was performed in relation to NMJ innervation status in mice which express the G93A SOD1 mutation. At P30, all NMJs of the fast medial gastrocnemius (MG) muscle were fully innervated by a single motor axon but 50% of NMJs lacked TSC cell bodies and were instead covered by the processes of Schwann cells with cell bodies located on the preterminal axons. NMJs in P30 slow soleus muscles were also fully innervated by single motor axons and only 5% of NMJs lacked a TSC cell body. At P60, about 25% of MG NMJs were denervated and lacked labeling for TSCs while about 60% of innervated NMJs lacked TSC cell bodies. In contrast, 96% of P60 soleus NMJs were innervated while 9% of innervated NMJs lacked TSC cell bodies. The pattern of TSC abnormalities found at P30 thus correlates with the pattern of denervation found at P60. Evidence from mice that express the G85R SOD1 mutation indicate that TSC abnormalities are not unique for mice that express G93A SOD1 mutations. These results add to an emerging understanding that TSCs may play a role in motor terminal degeneration and denervation in animal models of motor neuron disease.


Subject(s)
Motor Neuron Disease/pathology , Motor Neurons/pathology , Nerve Degeneration/pathology , Neuromuscular Junction/pathology , Schwann Cells/pathology , Superoxide Dismutase/metabolism , Animals , Cell Shape , Mice , Motor Neuron Disease/genetics , Motor Neuron Disease/metabolism , Motor Neurons/metabolism , Nerve Degeneration/genetics , Nerve Degeneration/metabolism , Neuromuscular Junction/genetics , Neuromuscular Junction/metabolism , Schwann Cells/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase-1
16.
Methods Mol Biol ; 1438: 349-94, 2016.
Article in English | MEDLINE | ID: mdl-27150099

ABSTRACT

Neuromuscular diseases can affect the survival of peripheral neurons, their axons extending to peripheral targets, their synaptic connections onto those targets, or the targets themselves. Examples include motor neuron diseases such as Amyotrophic Lateral Sclerosis, peripheral neuropathies such as Charcot-Marie-Tooth diseases, myasthenias, and muscular dystrophies. Characterizing these phenotypes in mouse models requires an integrated approach, examining both the nerve and muscle histologically, anatomically, and functionally by electrophysiology. Defects observed at these levels can be related back to onset, severity, and progression, as assessed by "Quality of life measures" including tests of gross motor performance such as gait or grip strength. This chapter describes methods for assessing neuromuscular disease models in mice, and how interpretation of these tests can be complicated by the inter-relatedness of the phenotypes.


Subject(s)
Motor Neurons/pathology , Neuromuscular Diseases/physiopathology , Animals , Disease Models, Animal , Humans , Mice , Neuromuscular Diseases/pathology , Phenotype , Quality of Life
17.
Exp Neurol ; 278: 116-26, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26853136

ABSTRACT

In several animal models of motor neuron disease, degeneration begins in the periphery. Clarifying the possible role of Schwann cells remains a priority. We recently showed that terminal Schwann cells (TSCs) exhibit abnormalities in postnatal mice that express mutations of the SOD1 enzyme found in inherited human motor neuron disease. TSC abnormalities appeared before disease-related denervation commenced and the extent of TSC abnormality at P30 correlated with the extent of subsequent denervation. Denervated neuromuscular junctions (NMJs) were also observed that lacked any labeling for TSCs. This suggested that SOD1 TSCs may respond differently than wildtype TSCs to denervation which remain at denervated NMJs for several months. In the present study, the response of SOD1 TSCs to experimental denervation was examined. At P30 and P60, SC-specific S100 labeling was quickly lost from SOD1 NMJs and from preterminal regions. Evidence indicates that this loss eventually becomes complete at most SOD1 NMJs before reinnervation occurs. The loss of labeling was not specific for S100 and did not depend on loss of activity. Although some post-denervation labeling loss occurred at wildtype NMJs, this loss was never complete. Soon after denervation, large cells appeared near SOD1 NMJ bands which colabeled for SC markers as well as for activated caspase-3 suggesting that distal SOD1 SCs may experience cell death following denervation. Denervated SOD1 NMJs viewed 7 days after denervation with the electron microscope confirmed the absence of TSCs overlying endplates. These observations demonstrate that SOD1 TSCs and distal SCs respond abnormally to denervation. This behavior can be expected to hinder reinnervation and raises further questions concerning the ability of SOD1 TSCs to support normal functioning of motor terminals.


Subject(s)
Disease Models, Animal , Gene Expression Regulation/physiology , Motor Neuron Disease/pathology , Schwann Cells/pathology , Age Factors , Animals , Antigens, Differentiation/metabolism , Gene Expression Regulation/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Neuron Disease/genetics , Muscle Denervation/methods , Mutation/genetics , Nerve Regeneration/physiology , Neuromuscular Junction/metabolism , Neuromuscular Junction/pathology , Neuromuscular Junction/ultrastructure , Receptor, Nerve Growth Factor/metabolism , Receptors, Cholinergic/metabolism , S100 Proteins/metabolism , Schwann Cells/metabolism , Sciatic Neuropathy/metabolism , Sciatic Neuropathy/pathology , Superoxide Dismutase/genetics
18.
J Neural Eng ; 13(4): 046012, 2016 08.
Article in English | MEDLINE | ID: mdl-27265358

ABSTRACT

OBJECTIVE: A significant challenge in rehabilitating upper-limb amputees with sophisticated, electric-powered prostheses is sourcing reliable and independent channels of motor control information sufficient to precisely direct multiple degrees of freedom simultaneously. APPROACH: In response to the expressed needs of clinicians, we have developed a miniature, batteryless recording device that utilizes emerging integrated circuit technology and optimal impedance matching for magnetic resonantly coupled (MRC) wireless power transfer to improve the performance and versatility of wireless electrode interfaces with muscle. MAIN RESULTS: In this work we describe the fabrication and performance of a fully wireless and batteryless EMG recording system and use of this system to direct virtual and electric-powered limbs in real-time. The advantage of using MRC to optimize power transfer to a network of wireless devices is exhibited by EMG collected from an array of eight devices placed circumferentially around a human subject's forearm. SIGNIFICANCE: This is a comprehensive, low-cost, and non-proprietary solution that provides unprecedented versatility of configuration to direct myoelectric prostheses without wired connections to the body. The amenability of MRC to varied coil geometries and arrangements has the potential to improve the efficiency and robustness of wireless power transfer links at all levels of upper-limb amputation. Additionally, the wireless recording device's programmable flash memory and selectable features will grant clinicians the unique ability to adapt and personalize the recording system's functional protocol for patient- or algorithm-specific needs.


Subject(s)
Electromyography/instrumentation , Prosthesis Design/methods , Animals , Artificial Limbs , Computer Systems , Electrodes, Implanted , Electronics , Humans , Magnetic Fields , Mice , Mice, Inbred C57BL , Microelectrodes , Reproducibility of Results , Telemetry , Wireless Technology
19.
Biol Open ; 5(7): 908-20, 2016 Jul 15.
Article in English | MEDLINE | ID: mdl-27288508

ABSTRACT

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.

20.
Genetics ; 168(2): 953-9, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15514066

ABSTRACT

Chemical mutagenesis of the mouse is ongoing in several centers around the world, with varying estimates of mutation rate and number of sites mutable to phenotype. To address these questions, we sequenced approximately 9.6 Mb of DNA from G1 progeny of ethylnitrosourea-treated mice in a large, broad-spectrum screen. We identified 10 mutations at eight unique sites, including six nonsynonymous coding substitutions. This calibrates the nucleotide mutation rate for two mutagenesis centers, implies significance criteria for positional cloning efforts, and provides working estimates of effective genetic target sizes for selected phenotypes.


Subject(s)
Alkylating Agents/pharmacology , Ethylnitrosourea/pharmacology , Genetic Testing , Mutation/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Female , Homozygote , Male , Mice , Molecular Sequence Data , Mutagenesis , Phenotype , Phylogeny , Predictive Value of Tests , Sequence Homology, Amino Acid
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