Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (36)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sprunger, L. K.
Right arrow Articles by Meisler, M. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sprunger, L. K.
Right arrow Articles by Meisler, M. H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics Pages 471-479  

Dystonia associated with mutation of the neuronal sodium channel Scn8a and identification of the modifier locus Scnm1 on mouse chromosome 3
Introduction
Results
   Dystonia in homozygous medJ mice
   Brain and muscle histopathology of dystonic medJ mice
   True null mutants of Scn8a exhibit only the lethal paralytic phenotype
   A low level of correctly spliced Scn8a transcript is present in medJ mice
   Splicing efficiency does not differ between paralyzed and dystonic mice
   Segregation of a modifier locus in crosses between strain C57BL/6J and C3H
   Localization of the modifier locus Scnm1 on mouse chromosome 3
   Mode of inheritance of Scnm1
   Other strains of mice carry the dominant allele of Scnm1
Discussion
Materials And Methods
   Animals
   Brain and spinal cord histology
   Skeletal muscle morphology
   Transcript analysis
   Microsatellite genotypes
Acknowledgements
References

Dystonia associated with mutation of the neuronal sodium channel Scn8a and identification of the modifier locus Scnm1 on mouse chromosome 3

Dystonia associated with mutation of the neuronal sodium channel Scn8a and identification of the modifier locus Scnm1 on mouse chromosome 3

Leslie K. Sprunger1, Andrew Escayg1, Sara Tallaksen-Greene2, Roger L. Albin2,3 and Miriam H. Meisler1,*

1Department of Human Genetics and 2Department of Neurology, University of Michigan and 3Geriatrics Research, Education and Clinical Center, VA Medical Center, Ann Arbor, MI 48109-0618, USA

Received October 13, 1998; Revised and Accepted December 21, 1998

The mouse mutant medJ contains a splice site mutation in the neuronal sodium channel Scn8a that results in a very low level of expression. On a C57BL/6J genetic background, medJ homozygotes exhibit progressive paralysis and juvenile lethality. The C3H genetic background has an ameliorating effect, producing viable adults with a novel dystonic phenotype. The dystonic mice exhibit movement-induced, sustained abnormal postures of the trunk and limbs. A dominant modifier locus responsible for the difference between strains was mapped to a 4.5 ± 1.3 cM interval on mouse chromosome 3. Our findings establish a role for ion channels in dystonia and demonstrate the impact of genetic background on its severity and progression. This new model suggests that SCN8A on chromosome 12q13 and SCNM1 on chromosome 1p21-1q21 may contribute to human inherited dystonia.

INTRODUCTION

Dystonia is a heterogeneous neurological disorder characterized by involuntary muscle contraction that produces twisting movements and sustained abnormal postures. The most common dystonias seen in clinical practice are idiopathic dystonia, with an estimated prevalence of 3/100 000, and focal dystonia, with an estimated prevalence of 30/100 000 (1). Many cases of dystonia are thought to be inherited. Mutations have been identified in two enzymes in the dopamine synthesis pathway (2,3) and in the torsin A gene at the DYT1 locus on chromosome 9 (4,5), and five additional dystonia loci have been mapped. The penetrance of torsin A mutations is only 30-40%, suggesting that genetic background, environmental influences and other factors may be important in the etiology of the human disorder. No morphological abnormalities have been observed in brains of patients with idiopathic dystonia. The currently available treatments for this disabling condition are palliative and development of effective therapy has been hindered by the absence of animal models that resemble the human disease (6).

Ion channel genes have been associated with inherited ataxia and epilepsy, but a role in dystonia has not previously been demonstrated. The voltage-gated sodium channels are responsible for the rapid depolarization phase of the action potential (7-9). The sodium channel Scn8a is expressed in neurons throughout the brain and spinal cord but not in muscle (10-12). Mutations in Scn8a are associated with neurological disease in the mouse (13). Two null mutations, med and medtg, result in progressive paralysis and juvenile lethality due to a failure of evoked transmitter release at the neuromuscular junction (10,14,15). One amino acid substitution in Scn8a causes ataxia (16). The fourth mutant allele, medJ, is a splice site mutation with a premature stop codon (17). On a C57BL/6J background, the phenotype of medJ homozygotes is nearly identical to the null mutants, except for a 1 week increase in survival time (17,18). Because of the phenotype and the premature stop codon, medJ was previously classified as a null allele (17).

It is not unusual for the severity of mouse mutant phenotypes to vary on different inbred backgrounds. This strain variation has been used to map modifier genes affecting spina bifida and neural tube defects (19,20), cystic fibrosis (21) and embryonic lethality of TGF[beta]1 deficiency (22). Enhancer/suppressor screens have been widely used in non-mammalian genetics to identify loci affecting a functional pathway (23,24). Anecdotal evidence that the survival of medJ mice is increased by several months in offspring of crosses with strain C3H (18,25) provided an opportunity to investigate a potential modifier of sodium channel function. We have demonstrated that strain C57BL/6J carries a recessive susceptibility allele at a major modifier locus. We describe a novel phenotype of the medJ mutation that has implications for the role of SCN8A in human dystonia and we provide evidence for the feasibility of cloning the modifier gene.

RESULTS

Dystonia in homozygous medJ mice

Sixty percent of the homozygous medJ F2 progeny of an intercross between strains C57BL/6J-medJ and C3H survive to adulthood and display a novel dystonic phenotype. Movement in these animals is accompanied by tremor and by severe twisting of the trunk (Fig. 1A). Abnormal postures of the trunk and limbs are sustained for periods of 2-10 s in younger animals and up to 1 min in older animals (Fig. 1B). The degree of flexion or extension at a given joint is variable and is often extreme. Dystonic posturing occurs frequently and is usually evident within a single traverse of the home cage. The dysfunction is persistent and coordinated gait is never observed. No abnormal movements are observed when the animals are at rest.


Figure 1. Dystonia in medJ mice. Homozygous medJ dystonic mice from the (C57BL/6J × C3H) F2 cross at 8 months of age. (A) Axial torsion; (B) dystonic postures of limbs; (C) muscle weakness.

In addition to this generalized kinesiogenic dystonia, affected animals exhibit muscle weakness and have difficulty supporting their head and body (Fig. 1C). The righting reflex is normal, but falling often induces axial twisting.

Dystonic mice are docile; they attempt to escape when handled but do not bite. Fighting has not been observed even among unrelated males housed together. Some females have reproduced, but homozygous males do not breed. More than 80% of the dystonic mice survive beyond 1 year of age.

This phenotype is quite different from the progressive paralysis and juvenile lethality previously described for medJ homozygotes on the C57BL/6J background (17). In these mice, tremor and ataxia are first observed at 10-12 days of age and muscle atrophy is severe by 3 weeks. Among 130 C57BL/6J-medJ/medJ animals examined, 90% died between 3 and 4 weeks of age and none survived >6 weeks.

Brain and muscle histopathology of dystonic medJ mice

No striking changes in morphology or neuronal cell number were observed in brain or spinal cord of the dystonic mice (Fig. 2). No signs of abnormal gliosis were detected in dystonic brain by immunohistochemistry with an antiserum to glial fibrillary acidic protein and no evidence of neurodegeneration was revealed by silver staining (data not shown).


Figure 2. Histology of brain and spinal cord from dystonic medJ mice. (A) Horizontal brain section (cresyl violet stain); (B) cortex; (C) striatum (scale bar 200 µm); (D) globus pallidus (scale bar 200 µm); (E) substantia nigra (scale bar 120 µm); (F) entopeduncular nucleus (scale bar 125 µm); (G) subthalamic nucleus (scale bar 100 µm); (H) spinal cord (scale bar 1 mm). Arrowheads in (G) define the dorsal border of the subthalamic nucleus. cp, cerebral peduncle; pc, substantia nigra pars compacta; pr, substantia nigra pars reticulata.

Although dystonic medJ homozygotes do not exhibit extensive muscle wasting, histological examination of biceps brachii and gastrocnemius muscles revealed a greater variation in fiber diameter than in wild-type muscle (Fig. 3A and C). Mutant muscle also contains many fibers with central nuclei (Fig. 3A and B, arrows). These observations are consistent with partial functional denervation of the mutant muscle leading to secondary muscle fiber regeneration. Succinate dehydrogenase and myofibrillar ATPase staining did not demonstrate a preferential effect on type I or type II fibers (data not shown). The histological changes in skeletal muscle are consistent with the weakness that is apparent in Figure 1C.


Figure 3. Skeletal muscle morphology from dystonic medJ mice. (A) Biceps brachii; (B) gastrocnemius. Muscle from five dystonic mutant mice and four controls was examined. Wide variations in fiber diameter, central nuclei (arrowheads) and numerous atrophied fibers typical of partially denervated muscle are evident. (C) A section from an age-matched C57BL/6J mouse gastrocnemius. The increased separation between muscle fascicles seen in (A) and (B) is a sample artifact, as it was not observed in frozen sections. Scale bars 20 µm.


True null mutants of Scn8a exhibit only the lethal paralytic phenotype

We examined the ability of the C3H genetic background to modify the lethal phenotype of two null mutants of Scn8a. The transgene-induced null allele Scn8amed-tg arose and is maintained in strain C57BL/6J (26). We examined 27 transgenic homozygotes from the F2 generation of the cross (C57BL/6J- medtg/+ × C3HeB/FeJ). All of the homozygotes were affected with the paralytic phenotype and died within 4 weeks after birth. We also analyzed the original med allele, a null mutation caused by insertion of a L1 element (17). This allele is routinely maintained in a C3H background. Among >100 affected med homozygotes examined, none survived beyond 1 month of age and none exhibited the dystonic phenotype. These observations reveal a fundamental difference between the true null mutations and medJ.

A low level of correctly spliced Scn8a transcript is present in medJ mice

Since the medJ mutation alters a splice donor site without changing the coding sequence of Scn8a, we tested the possibility that inefficient splicing of the mutant site generates a low level of full-length transcripts that protect medJ homozygotes from the inevitable lethality associated with the true null mutations. medJ homozygotes with both the paralytic and the dystonic phenotypes were examined. Due to the combined effect of the 4 bp deletion in the splice donor site of exon 3 and the non-consensus AT-AC splice sites of intron 2, the major Scn8a transcript in medJ brain is spliced from exon 1 to exon 4 and contains an in-frame stop codon within exon 4 (17; Fig. 4A and B). When RT-PCR is carried out with a forward primer from exon 1 and a reverse primer from exon 6, a 771 bp product is amplified from wild-type brain RNA and a 562 bp product lacking exons 2 and 3 is amplified from medJ brain (Fig. 4C). None of the correctly spliced 771 bp product could be detected in either dystonic or paralyzed mutants by ethidium bromide staining. This result is in agreement with previous experiments on paralyzed mutants (17).


Figure 4. Scn8a transcripts in medJ mice. (A) The 4 bp splice site deletion in the medJ allele of Scn8a (17). (B) PCR primers and lengths of predicted products and transcript-specific oligonucleotide probes A and B spanning exon junctions. (C) The major transcript of the medJ allele lacks exons 2 and 3. Total brain RNA from individual mice was amplified by RT-PCR using primers from exons 1 and 6. With ethidium bromide staining, only the mutant transcript is detected in RNA from two F2 medJ homozygotes with the paralytic phenotype and two with the dystonic phenotype. Age-matched C57BL/6J controls contain only the full-length transcript. (D) Detection of full-length transcript in medJ RNA. RT-PCR was carried out with primers in exons 2 and 4. The correctly spliced 243 bp product is amplified from homozygous paralytic and dystonic medJ mice and age-matched C57BL/6J controls. (E) Proportions of full-length and incorrectly spliced transcript in medJ homozyotes with the paralytic and dystonic phenotypes. RT-PCR was carried out using primers from exons 1 and 4. Products were transferred to a nylon filter and hybridized with oligo A and X-ray film was exposed for 48 h. The filter was washed and rehybridized with oligo B and film was exposed for 40 min. Two paralyzed F2 homozygotes and two dystonic F2 homozygotes were analyzed. Aliquots of diluted wild-type PCR product containing 1 or 2% of the total were included for comparison.

In order to specifically amplify any full-length transcript that might be present in the mutant, we carried out RT-PCR with primers from exons 2 and 4 (Fig. 4B). The predicted 243 bp product was obtained from both normal and mutant RNA (Fig. 4C), indicating that some correctly spliced transcripts are present in medJ homozygotes. Sequencing of gel purified fragments confirmed that the medJ product was correctly spliced and included exons 2 and 3. In this assay, the lack of competition from the abundant mutant transcript, which does not hybridize with the exon 2 primer, permits amplification of the full-length transcript in spite of its low abundance in medJ RNA.

Splicing efficiency does not differ between paralyzed and dystonic mice

To determine whether the proportion of correctly spliced transcript is different in mice with the paralyzed and dystonic phenotypes, we amplified both transcripts from each RNA sample using primers from exons 1 and 4. These primers amplify a 357 bp fragment from the full-length transcript and a 148 bp fragment from the incorrectly spliced transcript (Fig. 4B). By ethidium bromide staining only the 148 bp product was visible in the homozygous medJ samples (data not shown), as above with primers in exons 1 and 6. A more sensitive assay for the correctly spliced transcript was obtained by blotting the RT-PCR products onto nylon filters and hybridizing with a radiolabeled oligonucleotide that spans the exon 3-exon 4 junction and is specific for the full-length transcript (Fig. 4B, oligo A). A low level of the full-length 357 bp product hybridizing with oligo A could be detected in the medJ samples after a 48 h exposure (Fig. 4E). The blot was stripped and rehybridized with oligo B specific for the exon 1-exon 4 junction in the mutant transcript (Fig. 4B). The abundant, incorrectly spliced 148 bp product was detected after a 40 min exposure (Fig. 4E). The ratio of the full-length 357 bp product to the incorrectly spliced 148 bp product did not appear to differ in samples from two paralyzed and two dystonic medJ mice (Fig. 4E) and this was confirmed by densitometry (data not shown). Comparison with diluted aliquots of RT-PCR products amplified from +/+ RNA indicated that the abundance of correctly spliced transcripts in medJ homozygotes is ~2% of the wild-type level (Fig. 4E).

Segregation of a modifier locus in crosses between strain C57BL/6J and C3H

Among 854 F2 animals from an intercross between strains C57BL/6J-medJ/+ and C3H, there were 212 animals with visible neurological disease (24.8%), consistent with the expectation of 25% homozygosity for the recessive mutation. Among the homozygotes, two clearly distinct phenotypes were apparent. Sixty percent of the affected animals (n = 127) exhibited the dystonic phenotype and survived to adulthood (Fig. 5A, solid bars). The remaining F2 medJ homozygotes exhibit the paralyzed phenotype with juvenile lethality and died before 6 weeks of age (Fig. 5A, open bar).


Figure 5. Survival and neurological phenotype of homozygous medJ F2 mice exhibit linkage to a marker on mouse chromosome 3. (A) Length of survival of 206 F2 mice. All of these mice were clearly affected with neurological dysfunction at 21 days of age. Among the 108 mice that survived >4.5 months, 80% lived >12 months. (B) Genome scan. Genomic DNA from 19 paralyzed animals (from the group represented by the open bar) and 20 dystonic animals that survived for >6 months were combined into two pools and analyzed for 62 microsatellite markers on 20 chromosomes. A deficiency of C57BL/6J alleles in the dystonic pool was observed for marker D3Mit6.

The genetic basis for the segregation of two different phenotypes was examined by analysis of genomic DNA from a subset of 19 paralyzed mice (pool 1) and 20 dystonic mice (pool 2). The two pools were analyzed for 62 microsatellite markers from 20 chromosomes. We observed a deficiency of the C57BL/6J allele of the marker D3Mit6 in the dystonic pool and deficiency of the C3H allele in the paralyzed pool (Fig. 5B). This was confirmed by analysis of the individual samples. None of the other markers showed reproducible skewing. These data suggested that a modifier gene responsible for the phenotypic difference is present on chromosome 3.

Localization of the modifier locus Scnm1 on mouse chromosome 3

Genomic DNA samples from 111 mice with dystonia that had survived beyond 4 months of age were typed for microsatellite markers from the central region of chromosome 3 (Fig. 6A). All of the dystonic mice were either homozygous or heterozygous for the C3H allele at marker D3Mit141. The data are consistent with segregation of a dominant modifier locus, designated sodium channel modifier 1 (Scnm1). The modifer locus was mapped to the 4.5 cM interval between D3Mit40 and D3Mit101 (Fig. 6B).


Figure 6. Genetic mapping of the modifier locus Scnm1. (A) Genotypes of dystonic F2 mice from the cross between strains C57BL/6J-medJ/+ and C3HeB/FeJ. The number of animals with each genotype is indicated below the column. Scnm1 is non-recombinant with the microsatellite marker D3Mit141. The number of BB homozygotes at D3Mit141 (0) is significantly different from the Mendelian prediction of 25% BB (P < 10-13). Solid symbol, C57BL/6J homozygote; striped symbol, heterozygote; open symbol, C3H homozygote. (B) Localization of Scnm1 in the central region of mouse chromosome 3.


Mode of inheritance of Scnm1

The penetrance of the dominant dystonic phenotype in Scnm1H homozygotes and heterozygotes was estimated from the genotypes of the F2 animals. Among 55 F2 animals with juvenile lethality, 32 were BB, 16 were BH and 7 were HH at D6Mit141. Among 111 F2 mice with dystonia, 48 were HH and 63 were BH (Fig. 6A). We extrapolated these genotype frequencies to the total F2 population of 85 juvenile lethal and 127 adult dystonic mice. The penetrance of the dystonic phenotype was calculated by dividing the number of dystonic mice by the total number of medJ homozygotes with each Scnm1 genotype. The calculated penetrance for survival was 83% for HH homozygotes, 74% for BH heterozygotes and 0% for BB homozygotes. The genotype data are thus consistent with segregation of a single modifier locus with the C3H allele dominant to the C57BL/6J allele and with penetrance of ~80% for the dominant allele. The similar penetrance of the HH and BH genotypes suggests that gene dosage effects for the modifier locus are minimal. Since all dystonic mice carry at least one Scnm1H allele, this allele is necessary to prevent juvenile lethality. However, the early death of 20% of the mice carrying the Scnm1H allele demonstrates that this allele is not always sufficient for survival, due to the incomplete penetrance.

Other strains of mice carry the dominant allele of Scnm1

To determine whether the susceptibility phenotype of C57BL/6J mice was unique to that strain, we carried out two additional intercrosses of C57BL/6J-medJ/+ mice. In crosses with strains DBA/2J and A/J, both paralyzed and dystonic mice were observed in the F2 generation. The distribution of the two phenotypes was very similar to that in the C3H cross (Fig. 7A and B). To determine whether the phenotypic difference segregating in these crosses was linked to Scnm1, dystonic F2 mice were genotyped for markers on chromosome 3. The data demonstrated linkage of the dystonic phenotype to Scnm1 in both crosses (Fig. 7C and D). The results indicate that strains DBA/2J and A/J also carry the dominant, resistant allele of Scnm1.


Figure 7. Linkage of Scnm1 to neurological phenotype in crosses with strains DBA/2J and A/J. (A) Distribution of dystonic and paralyzed phenotypes in F2 animals from the cross between C57BL/6J- medJ/+ and DBA/2J. Among 193 F2 offspring in this cross there were 48 affected medJ homozygotes (25%). (B) Distribution of dystonic and paralyzed phenotypes in F2 animals from the cross between C57BL/6J-medJ/+ and A/J. Among 265 F2 offspring in this cross there were 58 affected medJ homozygotes (22%). (C) Genotypes of dystonic F2 animals from the cross with strain DBA/2J. (D) Genotypes of dystonic F2 mice from the cross with strain A/J.


DISCUSSION

We have described a novel dystonic phenotype associated with profound deficiency of the voltage-gated sodium channel Scn8a. This is the first demonstration of dystonia resulting from mutation of an ion channel. The medJ mouse differs from other animal models of dystonia in that the condition is persistent to adulthood and is not associated with neurodegeneration (27,28).

The detection of a low level of full-length Scn8a transcripts in medJ homozygotes demonstrates that the mutant splice donor site for exon 3, GTAACA, can be spliced at low efficiency. The medJ mutation should therefore be classified as a hypomorphic allele. medJ homozygotes carrying one or two copies of the modifier allele Scnm1H survive to adulthood and are not paralyzed. Homozygotes for true null alleles of Scn8a cannot survive even in the presence of two copies of the Scnm1H allele. Thus, prevention of paralysis and survival to adulthood requires both a low level of wild-type transcript and at least one copy of the dominant allele of Scnm1. C57BL/6J carries a recessive allele of Scnm1 that, in combination with a hypomorphic level of Scn8a, results in paralysis and lethality.

The lethal paralysis of medJ mice is caused by failure of evoked neurotransmitter release at the neuromuscular junction (29,30). To rescue the lethal paralysis, it is likely that Scnm1 functions within the motor neuron or in muscle to increase sensitivity in response to reduced neuronal activity. Scn8a is known to be the major carrier of sodium current in spinal motor neurons during the neonatal period (31). The severe muscle weakness in the dystonic mice is not surprising in view of the low level of correctly spliced Scn8a transcripts. The histological appearance of skeletal muscle suggests that some motor units fail while others remain functional in the dystonic animals.

Several molecular mechanisms of action for the modifier locus Scnm1 may be considered. It is unlikely that Scnm1 encodes a splicing factor that increases the efficiency of splicing of the mutant transcript, because the correctly spliced transcript accounted for only 2% of total Scn8a transcript in both paralyzed and dystonic mice. The modifier gene product could interact directly with Scn8a channels or could indirectly compensate for the low level of Scn8a by changing the excitability of neurons or skeletal muscle. If Scnm1 affects neuronal adaptation to low levels of Scn8a, it might also have a profound influence on the expression of other neuromuscular disorders. Having established the chromosomal location of Scnm1, it should be feasible to isolate this interesting gene by transgene complementation of the recessive lethal phenotype (32,33).

The dystonic phenotype of the medJ homozygotes can be considered a direct consequence of the low level of Scn8a expression and suggests an important role of this channel in neurons involved in motor control. Two brain regions that have been implicated in motor control and human dystonia are the basal ganglia and cerebellum (34-38). Cerebellar Purkinje cells isolated from Scn8a mutants are known to have reduced persistent sodium current and lack resurgent sodium current (39). The ability of Purkinje cells from mutant mice to fire repetitive action potentials of the type produced by climbing fiber stimulation in vivo is severely compromised and may contribute directly to the dystonia. The electrophysiological characteristics of neurons in the basal ganglia of Scn8a mutant mice have not yet been investigated. The dystonic phenotype of medJ mice suggests that the recently characterized human SCN8A gene (40) should be evaluated as a candidate gene for human inherited dystonias that are not linked to currently known loci.

Several examples of epistatic interaction between mammalian genes have recently been identified, including the digenic inheritance of retinitis pigmentosa due to mutations in peripherin/RDS and ROM1 (41), interaction between Pax2 and Pax5 during brain development (42) and the effect of secretory phospholipase (Mom) on tumor incidence in mice with ApcMin mutations (43). The dramatic effect of Scnm1 on the survival of animals with the hypomorphic Scn8a allele demonstrates the power of the enhancer/suppressor screen approach for identifying loci affecting specific functional pathways in the mouse. The effect of strain background on the phenotype of medJ mice also underscores the importance of using congenic lines for functional studies of new mutants (44). We are developing congenic lines that carry the medJ mutation on strain C57BL/6J (paralytic phenotype) and strain C3H (dystonic phenotype). Further analysis of the neurophysiology of dystonic medJ mice will expand our understanding of the etiology of movement disorders.

MATERIALS AND METHODS

Animals

The medJ allele of Scn8a arose at the Jackson Laboratory (Bar Harbor, ME) on a marker stock that carried a mutation at the Caracul locus (18) and was maintained on a (C57BL/6J × C3HeB/FeJ) F1 hybrid background. Heterozygous medJ/+ mice can be identified by the wavy hair produced by the dominant Caracul mutation that is located <0.3 cM from Scn8a on distal chromosome 15. B6C3Fe-a/a, Ca medJ/+ mice (N32) were obtained from the frozen embryo bank of the Jackson Laboratory in 1994 and have been maintained in our laboratory by crossing to strain C57BL/6J for eight generations. The inbred subline C3HeB/FeJ (C3H) was derived at the Jackson Laboratory in 1948 from strain C3H/HeJ by embryo transfer to strain C57BL/6J, which eliminated the mammary tumor virus carried by strain C3H/HeJ. The substrain C3HeB/FeJ-a/a, with black coat color, was generated at the Jackson Laboratory by crossing C3HeB/FeJ to C57BL/6J and backcrossing to C3HeB/FeJ.

The medtg mutation arose in 1991 as a result of non-targeted transgene insertion in a (C57BL/6J × C3H/HeJ) F2 mouse (26) and has been maintained in our colony by backcrossing to C57BL/6J for eight generations. Scn8amed arose on a mixed background that included strains C3H and 101 and has been maintained in our laboratory by backcrossing to C3H for three generations (17). C57BL/6J mice were obtained from the Jackson Laboratory.

Brain and spinal cord histology

Dystonic mice between 5 and 9 months of age were examined (n = 7). Control sections from strains C57BL/6J (3 months old, n = 2) and C3H (9 months old, n = 2) were processed in parallel. Tissues were processed for cresyl violet, cytochrome oxidase or immunohistochemical staining as described (45). The silver impregnation procedure to identify degenerating neurons was performed essentially as described (46).

Skeletal muscle morphology

Gastrocnemius muscle and biceps brachii muscle from dystonic (n = 5) and C57BL/6J (n = 2) mice were dissected, fixed for 1-3 days in 10% formalin in phosphate-buffered saline and processed for routine hematoxylin/eosin staining. For histochemical stains, the same muscles were dissected and immediately frozen in isopentane cooled with dry ice. Cryostat sections (14 µm) were stained for succinate dehydrogenase or myofibrillar ATPase(pH 3.8, 4.3, 4.5, 9.4 and 10.4) as described (47).

Transcript analysis

Total RNA was prepared from brain using the Trizol protocol (Life Technologies, Grand Island, NY). First strand cDNA was synthesized from 10 µg RNA with a random hexamer primer using the Superscript II RT Preamplification System (Life Technologies) in a 20 µl reaction according to the manufacturer’s instructions. First strand cDNA (1 µl) was used as template in 25 µl PCR reactions. PCR was carried out for 20-25 cycles using the following primers as previously described (17): for exon 1, nested primers a and b; for exon 2, primer d; for exon 3, primer i; for exon 4, primer k; for exon 6, nested primers m and n.

For oligonucleotide hybridization experiments, PCR products were separated on 1.5% agarose and transferred by Southern blotting to nylon membranes (Zetaprobe; Bio-Rad, Hercules, CA). Membranes were baked for 30 min at 80°C under vacuum, prehybridized for 30-60 min, hybridized for 3 h at 45°C, washed twice for 20 min at 45°C in 2× SSC, 0.1% SDS and autoradiographed. Oligonucleotide probes (5 pmol) were end-labeled by T4 polynucleotide kinase (New England Biolabs, Beverly, MA) in the presence of 30 µCi of [[alpha]-32P]ATP (6000 Ci/mmol; Amersham, Arlington Heights, IL). Oligo A (GTGGAGTACACATTCT) spans the junction between exons 3 and 4 in the wild-type transcript (Fig. 4A). Oligo B (CGCAGAAAGTACACAT) spans the junction between exons 1 and 4 in the mutant transcript. The specificity of hybridization was confirmed by testing products of known sequence. The weak cross-hybridization of oligo A with the incorrectly spliced transcript was prevented by including a 5-fold excess of unlabeled oligo B in the hybridization solution. MultiAnalyst software (Bio-Rad) was used for densitometric analysis of autoradiogram images.

Microsatellite genotypes

Genomic DNA was prepared from tail biopsies by proteinase K digestion, phenol-chloroform extraction and ethanol precipitation. Primers for microsatellite loci were obtained from Research Genetics (Huntsville, AL). PCR products were detected by incorporation of [[alpha]-32P]dCTP, electrophoresis on 6% polyacrylamide gels containing urea and autoradiography. (C57BL/6J × C3H) F1 parents of the F2 generation were confirmed to be heterozygous for markers on chromosome 3.

ACKNOWLEDGEMENTS

Muscle histochemistry was generously provided by Dr John Faulkner and Cheryl Hassett of the Department of Physiology and by the Reproductive Sciences Morphology Core Facility, University of Michigan. We are grateful to Sally A. Camper and Thomas Glaser for critical review of the manuscript. We thank Julie M. Jones and Daniel L. Burgess for many helpful discussions. This work was supported by NIH grants K08 HL02972 to L.K.S., NS34509 and GM24872 to M.H.M., AG08617 and a VA Merit Review to R.L.A., and a Basic Science Partnership Award from the University of Michigan School of Medicine.

REFERENCES

1. Nutt, J.G., Muenter, M.D., Aronson, A., Kurland, L.T. and Melton, L.J. (1988) Epidemiology of focal and generalized dystonia in Rochester, Minnesota. Mov. Disord., 3, 188-194. MEDLINE Abstract

2. Ichinose, H., Ohye, T., Takahashi, E., Seki, N., Hori, T., Segawa, M., Nomura, Y., Endo, K., Tanaka, H., Tsuij, S., Fujita, K. and Nagatsu, T. (1994) Hereditary progressive dystonia with marked diurnal fluctuation caused by mutations in the GTP cyclohydrolase I gene. Nature Genet., 8, 236-242. MEDLINE Abstract

3. Ludecke, B., Dworniczak, B. and Bartholomé, K. (1995) A point mutation in the tyrosine hydroxylase gene associated with Segawa's syndrome. Hum. Genet., 95, 123-125. MEDLINE Abstract

4. Ozelius, L.J., Hewett, J.W., Page, C.E., Bressman, S.B., Kramer, P.L., Shalish, C., de Leon, D., Brin, M., Raymond, D., Corey, D.P., Fahn, S., Risch, N.J., Buckler, A.J., Gusella, J.F. and Breakefield, X.O. (1997) The early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein. Nature Genet., 17, 40-48. MEDLINE Abstract

5. Klein, C., Brin, M.F., de Leon, D., Limborska, S.A., Ivanova-Smolenskaya, I.A., Bressman, S.B., Friedman, A., Markova, E.D., Risch, N.J., Breakefield, X.O. and Ozelius, L.J. (1998) De novo mutations (GAG deletion) in the DYT1 gene in two non-Jewish patients with early-onset dystonia. Hum. Mol. Genet., 7, 1133-1136. MEDLINE Abstract

6. Jankovic, J. (1997) In Watts, R.L. and Koller, W.C. (eds), Movement Disorders: Neurologic Principles and Practice. McGraw-Hill, New York, NY, pp. 443-454.

7. Hille, B. (1992) Ionic Channels of Excitable Membranes. Sinauer Associates, Sunderland, MA.

8. Goldin, A.L. (1995) In North, R.A. (ed.), Handbook of Receptors and Channels: Ligand- and Voltage-Gated Channels. CRC Press, Boca Raton, FL, pp. 73-113.

9. Plummer, N.W. and Meisler, M.H. (1999) Evolution and diversity of mammalian sodium channels. Genomics, 56, in press.

10. Burgess, D.L., Kohrman, D.C., Galt, J., Plummer, N.W., Jones, J.M., Spear, B. and Meisler, M.H. (1995) Mutation of a new sodium channel gene, Scn8a, in the mouse mutant `motor endplate disease'. Nature Genet., 10, 461-465. MEDLINE Abstract

11. Schaller, K.L., Krzemien, D.M., Yarowsky, P.J., Krueger, B.K. and Caldwell, J.H. (1995) A novel, abundant sodium channel expressed in neurons and glia. J. Neurosci., 15, 3231-3242. MEDLINE Abstract

12. Felts, P.A., Yokoyama, S., Dib-Hajj, S., Black, J.A. and Waxman, S.G. (1997) Sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1): different expression patterns in developing rat nervous system. Mol. Brain Res., 45, 71-82.

13. Meisler, M.H., Sprunger, L.K., Plummer, N.W., Escayg, A. and Jones, J.M. (1997) Ion channel mutations in mouse models of inherited neurological disease. Ann. Med., 29, 569-574. MEDLINE Abstract

14. Searle, A.G. (1962) New mutants report. Mouse Newslett., 27, 34-35.

15. Duchen, L.W., Searle, A.G. and Strich, S.J. (1967) An hereditary motor endplate disease in the mouse. J. Physiol. (Lond.), 189, 4P-6P.

16. Kohrman, D.C., Smith, M.R., Goldin, A.L., Harris, J. and Meisler, M.H. (1996) A missense mutation in the sodium channel Scn8a is responsible for cerebellar ataxia in the mouse mutant jolting. J. Neurosci., 16, 5993-5999. MEDLINE Abstract

17. Kohrman, D.C., Harris, J.B. and Meisler, M.H. (1996) Mutation detection in the med and medJ alleles of the sodium channel Scn8a-unusual splicing due to a minor class AT-AC intron. J. Biol. Chem., 271, 17576-17581. MEDLINE Abstract

18. Sidman, R.L., Cowen, J.S. and Eicher, E.M. (1979) Inherited muscle and nerve diseases in mice: a tabulation with commentary. Ann. NY Acad. Sci., 317, 497-505. MEDLINE Abstract

19. Helwig, U., Imai, K., Schmahl, W., Thomas, B.E., Varnum, D.S., Nadeau, J. and Balling, R. (1995) Interaction between undulated and Patch leads to an extreme form of spina bifida in double-mutant mice. Nature Genet., 11, 60-63. MEDLINE Abstract

20. Neumann, P.E., Frankel, W.N., Letts, V.A., Coffin, J.M., Copp, A.J. and Bernfield, M. (1994) Multifactorial inheritance of neural tube defects: localization of the major gene and recognition of modifiers in ct mutant mice. Nature Genet., 6, 357-362. MEDLINE Abstract

21. Rozmahel, R., Wilschanski, M., Matin, A., Plyte, S., Oliver, M., Auerbach, W., Moore, A., Forstner, J., Durie, P., Nadeau, J., Bear, C. and Tsui, L.-C. (1996) Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor. Nature Genet., 12, 280-287. MEDLINE Abstract

22. Bonyadi, M., Rusholme, S.A.B., Cousins, F.M., Su, H.C., Biron, C.A., Farrall, M. and Akhurst, R.J. (1997) Mapping of a major genetic modifier of embryonic lethality in TGF[beta]1 knockout mice. Nature Genet., 15, 207-211. MEDLINE Abstract

23. Simon, M.A., Bowtell, D.D.L., Dodson, G.S., Laverty, T.R. and Rubin, G.M. (1991) Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell, 67, 701-716. MEDLINE Abstract

24. Guarente, L. (1993) Synthetic enhancement in gene interaction: a genetic tool come of age. Trends Genet., 9, 362-366. MEDLINE Abstract

25. Ezine, S., Papiernik, M., Rieger, F. and Pinçon-Raymond, M. (1983) Modification of helper and suppressor/cytotoxic lymphocyte subsets in mice with motor end-plate disease. Clin. Exp. Immunol., 51, 475-478. MEDLINE Abstract

26. Kohrman, D.C., Plummer, N.W., Schuster, T., Jones, J.M., Jang, W., Burgess, D.L., Galt, J., Spear, B.T. and Meisler, M.H. (1995) Insertional mutation of the motor endplate disease (med) locus on mouse chromosome 15. Genomics, 26, 171-177. MEDLINE Abstract

27. Lorden, J.F. (1995) In Tsui, J.K.C. and Calne, D.B. (eds), Handbook of Dystonia. Marcel Dekker, New York, NY, Vol. 39, pp. 5-42.

28. Richter, A. and Löscher, W. (1998) Pathology of idiopathic dystonia: findings from genetic animal models. Prog. Neurobiol., 54, 633-677. MEDLINE Abstract

29. Duchen, L.W. and Stefani, E. (1971) Electrophysiological studies of neuromuscular transmission in hereditary `motor end-plate disease' of the mouse. J. Physiol., 212, 535-548. MEDLINE Abstract

30. Harris, J.B. and Pollard, S.L. (1986) Neuromuscular transmission in the murine mutants `motor endplate disease' and `jolting'. J. Neurosci., 76, 239-253.

31. Garcia, K.D., Sprunger, L.K., Meisler, M.H. and Beam, K.G. (1998) The sodium channel Scn8a is the major contributor to the postnatal developmental increase of sodium current density in spinal motoneurons. J. Neurosci., 18, 5234-5239. MEDLINE Abstract

32. Hamilton, B.A., Smith, D.J., Mueller, K.L., Kerrebrock, A.W., Bronson, R.T., van Berkel, V., Daly, M.J., Kruglyak, L., Reeve, M.P., Nemhauser, J.L., Hawkins, T.L., Rubin, E.M. and Lander, E.S. (1997) The vibrator mutation causes neurodegeneration via reduced expression of PITP alpha: positional complementation cloning and extragenic suppression. Neuron, 18, 711-722. MEDLINE Abstract

33. Probst, F.J., Fridell, R.A., Raphael, Y., Saunders, T.L., Wang, A., Liang, Y., Morell, R.J., Touchman, J.W., Lyons, R.H., Noben-Trauth, K., Friedman, T.B. and Camper, S.A. (1998) Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene. Science, 280, 1444-1447. MEDLINE Abstract

34. Bressman, S.B. and Fahn, S. (1997) In Watts, R.L. and Koller, W.C. (eds), Movement Disorders: Neurologic Principles and Practice. McGraw-Hill, New York, NY, pp. 419-428.

35. Tolosa, E.S. and Marti, M.J. (1997) In Watts, R.L. and Koller, W.C. (eds), Movement Disorders: Neurologic Principles and Practice. McGraw-Hill, New York, NY, pp. 429-441.

36. Lees, A. (1990) Dystonia and cerebellar ataxia. Mov. Disord., 5, 178.

37. Fletcher, N.A., Stell, R., Harding, A.E. and Marsden, C.D. (1988) Degenerative cerebellar ataxia and focal dystonia. Mov. Disord., 3, 336-342. MEDLINE Abstract

38. Berardelli, A., Rothwell, J.C., Hallett, M., Thompson, P.D., Manfredi, M. and Marsden, C.D. (1998) The pathophysiology of primary dystonia. Brain, 121, 1195-1212. MEDLINE Abstract

39. Raman, I.M., Sprunger, L.K., Meisler, M.H. and Bean, B.P. (1997) Altered subthreshold sodium currents and disrupted firing patterns in Purkinje neurons of Scn8a mutant mice. Neuron, 19, 881-891. MEDLINE Abstract

40. Plummer, N.W., Galt, J., Jones, J.M., Burgess, D.L., Sprunger, L.K., Kohrman, D.C. and Meisler, M.H. (1998) Exon organization, coding sequence, physical mapping and polymorphic intragenic markers for the human neuronal sodium channel gene SCN8A. Genomics, 54, 287-296. MEDLINE Abstract

41. Dryja, T.P., Hahn, L.B., Kajiwara, K. and Berson, E.L. (1997) Dominant and digenic mutations in the peripherin/RDS and ROM1 genes in retinitis pigmentosa. Invest. Ophthalmol. Visual Sci., 38, 1972-1982.

42. Urbanek, P., Fetka, I., Meisler, M.H. and Busslinger, M. (1997) Cooperation of Pax2 and Pax5 in midbrain and cerebellum development. Proc. Natl Acad. Sci. USA, 95, 5703-5708.

43. Cormier, R.T., Hong, K.H., Halberg, R.B., Hawkins, T.L., Richardson, P., Mulherkar, R., Dove, W.F. and Lander, E.S. (1997) Secretory phospholipase Pla2g2a confers resistance to intestinal tumorigenesis. Nature Genet., 17, 88-91. MEDLINE Abstract

44. Banbury Conference on Genetic Background in Mice (1997) Mutant mice and neuroscience: recommendations concerning genetic background. Neuron, 19, 755-759. MEDLINE Abstract

45. Ordway, J.M., Tallaksen-Greene, S., Gutekunst, C.A., Bernstein, E.M., Cearley, J.A., Wiener, H.W., Dure, L.S., Lindsey, R., Hersch, S.M., Jope, R.S., Albin, R.L. and Detloff, P.J. (1997) Ectopically expressed CAG repeats cause intranuclear inclusions and a progressive late onset neurological phenotype in the mouse. Cell, 91, 753-763. MEDLINE Abstract

46. Oo, T.F., Blazeski, R., Harrison, S.M.W., Henchcliffe, C., Mason, C.A., Roffler-Tarlov, S.K. and Burke, R.E. (1996) Neuron death in the substantia nigra of weaver mouse occurs late in development and is not apoptotic. J. Neurosci., 16, 6134-6145. MEDLINE Abstract

47. Larkin, L.M., Faulkner, J.A., Hinkle, R.T., Hassett, C.A., Supiano, M.A. and Halter, J.B. (1997) Functional deficits in medial gastrocnemius grafts in rats: relation to muscle metabolism and beta-AR regulation. J. Appl. Physiol., 83, 67-73. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +1 734 763 5546; Fax: +1 734 763 9691; Email: meislerm@umich.edu

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 6 Feb 1999
Copyright©Oxford University Press, 1999.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
M. S. Martin, B. Tang, L. A. Papale, F. H. Yu, W. A. Catterall, and A. Escayg
The voltage-gated sodium channel Scn8a is a genetic modifier of severe myoclonic epilepsy of infancy
Hum. Mol. Genet., December 1, 2007; 16(23): 2892 - 2899.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
R. Donato, K. M. Page, D. Koch, M. Nieto-Rostro, I. Foucault, A. Davies, T. Wilkinson, M. Rees, F. A. Edwards, and A. C. Dolphin
The ducky2J Mutation in Cacna2d2 Results in Reduced Spontaneous Purkinje Cell Activity and Altered Gene Expression
J. Neurosci., November 29, 2006; 26(48): 12576 - 12586.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
S. I. Levin, Z. M. Khaliq, T. K. Aman, T. M. Grieco, J. A. Kearney, I. M. Raman, and M. H. Meisler
Impaired Motor Function in Mice With Cell-Specific Knockout of Sodium Channel Scn8a (NaV1.6) in Cerebellar Purkinje Neurons and Granule Cells
J Neurophysiol, August 1, 2006; 96(2): 785 - 793.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
S. Saleh, S. Y. M Yeung, S. Prestwich, V. Pucovsky, and I. Greenwood
Electrophysiological and molecular identification of voltage-gated sodium channels in murine vascular myocytes
J. Physiol., October 1, 2005; 568(1): 155 - 169.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
L.S. Meadows and L.L. Isom
Sodium channels as macromolecular complexes: Implications for inherited arrhythmia syndromes
Cardiovasc Res, August 15, 2005; 67(3): 448 - 458.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
A. M. Swensen and B. P. Bean
Robustness of Burst Firing in Dissociated Purkinje Neurons with Acute or Long-Term Reductions in Sodium Conductance
J. Neurosci., April 6, 2005; 25(14): 3509 - 3520.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
M. T. H. Do and B. P. Bean
Sodium Currents in Subthalamic Nucleus Neurons From Nav1.6-Null Mice
J Neurophysiol, August 1, 2004; 92(2): 726 - 733.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
K Fukuchi, T Katsuya, K Sugimoto, M Kuremura, H D Kim, L Li, and T Ogihara
LMNA mutation in a 45 year old Japanese subject with Hutchinson-Gilford progeria syndrome
J. Med. Genet., May 1, 2004; 41(5): e67 - e67.
[Full Text] [PDF]

Home page
 ScienceHome page
D. A. Buchner, M. Trudeau, and M. H. Meisler
SCNM1, a Putative RNA Splicing Factor That Modifies Disease Severity in Mice
Science, August 15, 2003; 301(5635): 967 - 969.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
J. A. Kearney, D. A. Buchner, G. de Haan, M. Adamska, S. I. Levin, A. R. Furay, R. L. Albin, J. M. Jones, M. Montal, M. J. Stevens, et al.
Molecular and pathological effects of a modifier gene on deficiency of the sodium channel Scn8a (Nav1.6)
Hum. Mol. Genet., October 15, 2002; 11(22): 2765 - 2775.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
C. E. Pizoli, H. A. Jinnah, M. L. Billingsley, and E. J. Hess
Abnormal Cerebellar Signaling Induces Dystonia in Mice
J. Neurosci., September 1, 2002; 22(17): 7825 - 7833.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J. Barclay, N. Balaguero, M. Mione, S. L. Ackerman, V. A. Letts, J. Brodbeck, C. Canti, A. Meir, K. M. Page, K. Kusumi, et al.
Ducky Mouse Phenotype of Epilepsy and Ataxia Is Associated with Mutations in the Cacna2d2 Gene and Decreased Calcium Channel Current in Cerebellar Purkinje Cells
J. Neurosci., August 15, 2001; 21(16): 6095 - 6104.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
Y. De Repentigny, P. D. Cote, M. Pool, G. Bernier, S. Girard, S. M. Vidal, and R. Kothary
Pathological and genetic analysis of the degenerating muscle (dmu) mouse: a new allele of Scn8a
Hum. Mol. Genet., August 1, 2001; 10(17): 1819 - 1827.
[Abstract] [Full Text] [PDF]

Home page
 NeuroscientistHome page
M. H. Meisler, J. Kearney, A. Escayg, B. T. Macdonald, and L. K. Sprunger
Sodium Channels and Neurological Disease: Insights from Scn8a Mutations in the Mouse
Neuroscientist, April 1, 2001; 7(2): 136 - 145.
[Abstract] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. H. Caldwell, K. L. Schaller, R. S. Lasher, E. Peles, and S. R. Levinson
Sodium channel Nav1.6 is localized at nodes of Ranvier, dendrites, and synapses
PNAS, May 9, 2000; 97(10): 5616 - 5620.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (36)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sprunger, L. K.
Right arrow Articles by Meisler, M. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sprunger, L. K.
Right arrow Articles by Meisler, M. H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?