Human Molecular Genetics, 2002, Vol. 11, No. 22 2765-2775
© 2002 Oxford University Press
Molecular and pathological effects of a modifier gene on deficiency of the sodium channel Scn8a (Nav1.6)
1Department of Human Genetics, 2Department of Neurology and 3Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA, 4Ann Arbor Veterans Affairs Medical Center Geriatric Research, Education, and Clinical Center, Ann Arbor, MI 48105, USA and 5Division of Biology, Section of Neurobiology, University of California at San Diego, La Jolla, CA 92093, USA
Received June 26, 2002; Accepted August 16, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Scn8a encodes an abundant, widely distributed voltage-gated sodium channel found throughout the central and peripheral nervous systems. Mice with different mutant alleles of Scn8a provide models of the movement disorders ataxia, dystonia, tremor and progressive paralysis. We previously reported that the phenotype of the hypomorphic allele of Scn8a, medJ, is dependent upon an unlinked modifier locus, Scnm1. Strain C57BL/6J carries a sensitive allele of the modifier locus that results in juvenile lethality. We now provide evidence that the modifier acts on the splicing efficiency of the mutant splice donor site. Mutant mice display either 90% or 95% reduction in the proportion of correctly spliced mRNA, depending on modifier genotype. The abundance of the channel protein, Nav1.6, is also reduced by an order of magnitude in medJ mice, resulting in delayed maturation of nodes of Ranvier, slowed nerve conduction velocity, reduced muscle mass and reduction of brain metabolic activity. medJ mice provide a model for the physiological effects of sodium channel deficiency and the molecular mechanism of bigenic disease.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Scn8a encodes a voltage-gated neuronal sodium channel, Nav1.6, that is widely distributed in neurons of the central and peripheral nervous systems (CNS and PNS) (1–4). Mutations of Scn8a in the mouse produce neurological phenotypes ranging in severity from mild tremor to lethality (1,5). The null alleles Scn8amed, Scn8amedTg and Scn8admu produce progressive paralysis and juvenile death (6–8). The muscle atrophy and paralysis is secondary to the absence of evoked neurotransmitter release at the neuromuscular junction (5). Scn8amedJ (abbreviated medJ) is a hypomorphic allele with a splice donor mutation in intron 3 (6). The overall abundance of mRNA is normal, but the majority of transcripts are incorrectly spliced as a result of exon skipping (6). The phenotype of medJ homozygotes depends upon the unlinked modifier gene Scnm1 (9). The common allele of the modifier is carried by strains C3HeB/FeJ, A/J, DBA/2J and 129S6. On these strains, medJ homozygotes survive to adulthood and display a movement disorder that resembles human kinesiogenic dystonia, with sustained abnormal postures, torsional movements of the trunk and limbs, and muscle weakness (9). A recessive allele of Scnm1 is carried by strain C57BL/6J. On this strain, homozygous medJ mice resemble null mice with paralysis and juvenile lethality. The mechanism of action of the modifier has not previously been determined.
Nav1.6 is localized at multiple subcellular sites, including dendrites, unmyelinated axons, presynaptic and postsynaptic membranes, and Purkinje cell axon initial segments (10–13). It is also the predominant channel at the nodes of Ranvier of myelinated axons (10). Abnormal sodium currents have been recorded in several classes of neurons from Nav1.6 mutant mice, including cerebellar Purkinje cells, cortical pyramidal cells and spinal motor neurons (14–16). Deficiency of Nav1.6 decreases repetitive firing of action potentials in cerebellar Purkinje cells and reduces the level of resurgent and persistent current in Purkinje cells and cortical pyramidal neurons (14,16).
Concentration of sodium channels at the nodes of Ranvier makes possible the rapid saltatory conduction of action potentials along myelinated axons. Early in postnatal development, the immature nodes of Ranvier contain the channel Nav1.2, which is replaced by Nav1.6 during nodal maturation (17). The transition from Nav1.2 to Nav1.6 requires formation of compact myelin, and does not occur in mice lacking myelin basic protein (17). The transition is likely to be mediated by protein–protein interactions, rather than differences in protein synthesis, since the abundance of Nav1.2 and Nav1.6 mRNAs increase during postnatal development. Potential protein binding partners for Nav1.6 in mature nodes include ankyrin G, Nr-CAM and neurofascin (13,18).
The role of modifier genes in modulating the severity of genetic disease is an important factor in understanding and treating human disease. In this report, we provide evidence that the Scnm1 modifier acts at the level of mRNA splicing. In order to investigate the pathophysiology of mice carrying the common allele of the modifier, we generated a new congenic line, C3HeB/FeJ-medJ. On the C3H background, medJ homozygotes can survive to adulthood. These mice were used to study the effect of the medJ mutation on peripheral nerve function.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Characterization of adult medJ mice
To examine the phenotype of medJ homozygotes surviving beyond 3 weeks of age, we crossed the two congenic lines, B6-medJ/+ and C3H-medJ/+. One quarter of the offspring are homozygous medJ/medJ, on a background that is heterozygous at all other loci including the modifier locus Scnm1. More than 90% of the F1 medJ homozygotes survive to adulthood, compared with 60% of the medJ homozygotes on the C3H congenic background. The genetic uniformity and better viability of the F1 mice make them ideal for physiological analysis of the medJ phenotype.
The body length of medJ homozygotes is normal, but body weight is significantly reduced. At 3 weeks of age, medJ homozygotes weighed 8.7±1.6 g (n=6), compared with 22.9±2.7 g (n=6) for +/+ littermates. At 3 months of age, medJ homozygotes weighed 18.6±4.0 g (n=10) compared with 39.5±4.8 g (n=7) for +/+ littermates. The low body weight persists and is accounted for by a severe reduction in muscle mass, as is evident from the appearance of the hind limb muscles (Fig. 1).
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Reduced muscle mass and poor coordination result in impaired performance of medJ/medJ mice on tests of motor function. Their gait is grossly abnormal, with hind limb dragging and twisting of the trunk and limbs, as demonstrated in the accompanying video (http://www.sitemaker.umich.edu/meislerm/medj_video). Dystonic postures increase in frequency and severity with age. In 6-month-old mice, these abnormal postures are often sustained for periods of several minutes. medJ mice are unable to walk on a 1/4 inch diameter beam, and they fall immediately when placed on a slowly rotating rotorod (4 r.p.m.). At 3 weeks of age, medJ mice are able to swim, but by 3 months of age, their uncoordinated thrashing prevents them from remaining upright in the water.
Since Scn8a is an abundant sodium channel, we evaluated its contribution to overall synaptic activity in the brain by measuring [14C]-2-deoxyglucose uptake (19). Homozygous medJ and control mice were injected intraperitoneally with 100 µCi/kg of [14C]-2-deoxyglucose. After 60 minutes, the glucose uptake in brain of homozygous mutants was 0.15±0.05 µCi/g (n=12) compared with 0.26±0.1 µCi/g (n=14) in wild-type controls (P<0.002). The data demonstrate that Scn8a is a major contributor to synaptic activity in the brain.
Low level of correctly spliced Scn8a mRNA and Nav1.6 protein in medJ mice
The total amount of Scn8a mRNA in medJ brain is normal (6), but as a result of a splice site mutation, the majority of Scn8a transcripts skip exons 2 and 3 (Fig. 2A and B). We developed a quantitative RT–PCR/primer extension assay to determine the proportion of correctly spliced transcripts in medJ mice carrying the common H allele of the modifier locus.
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The full-length and mutant transcripts are both amplified from brain RNA with primers a and b from exon 1 and exon 6 (Fig. 2B). The RT–PCR products are subjected to primer extension from primer c in the presence of [
-33P]ddGTP and unlabeled dATP, dTTP and dCTP (Fig. 2B). The wild-type transcript generates a 34 nt extension product terminating in exon 2, while the mutant transcript generates a 28 nt product terminating in exon 4. The radiolabeled 34 and 28 nt primer extension products are separated on 12% polyacrylamide gels and quantitated on a phosphoimager. As expected, the 28 nt product predominates in mutant brain (Fig. 2D). To test the linearity of the assay, standards containing known ratios of cloned full-length and mutant fragments were subjected to PCR and primer extension reactions (Fig. 2C). The percentage of radioactivity in the 34 and 28 nt products was directly proportional to the percentage of each clone in the mixture (Fig. 2E). Analysis of RNA from medJ homozygotes carrying the common H allele of the modifier locus indicated that the full-length transcript accounts for 12%±2% of the total Scn8a transcript in mutant brain (Fig. 2E). We were interested in determining whether the Nav1.6 protein is stabilized in mutant brain, as a compensatory mechanism for the low level of full-length mRNA. Nav1.6 protein was assayed by western blotting with an antibody specific for Nav1.6. The amount of Nav1.6 protein in adult brain is greatly reduced in medJ mice in comparison with wild-type littermates (Fig. 3A). Based on diluted samples of wild-type protein, we estimate that the amount of Nav1.6 protein in medJ brain is slightly greater than 10% (Fig. 3B). This is comparable to the amount of correctly spliced transcript. In younger mice, at 3 weeks of age, Nav1.6 was barely detectable in mutant extracts, although it was abundant in age-matched wild-type mice (data not shown).
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Normal levels of sodium channels Nav1.1 and Nav1.2 in medJ mice
Little is known about the dynamic regulation of sodium channel gene expression, or the ability of neurons to compensate for channel deficiencies. We therefore measured the levels of the other two major neuronal sodium channels, Nav1.1 and Nav1.2, in brain of medJ homozygotes deficient in Nav1.6. Northern blots revealed comparable levels of transcripts in RNA from mutant and wild-type brains (Fig. 4A). Western blot analysis demonstrated comparable levels of channel protein (Fig. 4B). These related sodium channels are not upregulated to compensate for the deficiency of Nav1.6.
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A single copy of the medJ allele is not sufficient for survival
To determine the effect of an additional 2-fold reduction of Nav1.6 expression below that in the medJ homozygotes described above, we crossed C57BL/6J-medJ/+ heterozygotes with heterozygotes carrying a null allele of Scn8a, C3H-med/+ (6). Offspring were genotyped at 3 weeks of age. Twenty-five percent of offspring (22/95) were compound heterozygotes, with one medJ allele and one null allele, consistent with predicted Mendelian ratios. The phenotype of the Scn8amedJ/- compound heterozygotes closely resembles that of homozygous null mice (6), with severe neurological impairment and juvenile lethality. The average survival of the compound heterozygotes was 30±2 days (n=22) (range 25–34 days). Since the compound heterozygotes express only one copy of the medJ gene, the lethal phenotype indicates that the predicted 6% of wild-type channel level is insufficient for survival.
The modifier locus, Scnm1, influences splicing of the Scn8a transcript
To determine whether genotype at the Scnm1 locus influences the proportion of correctly spliced transcript, brain RNA was prepared from affected (C57BL/6JxC3H) F2-medJ/medJ mice. On gels containing RT–PCR/primer extension products, the proportion of wild-type transcript was consistently lower in Scnm1B/B mice than in Scnm1H/H (Fig. 5). Quantitation indicated that Scnm1B/B mice contain 6.4%± 1.4% of correctly spliced transcript (Table 1), which is half of the level in the Scnm1H/H mice (Table 1 and Fig. 2E). The level of transcript in Scnm1B/H heterozygotes was identical to that in Scnm1H/H homozygotes (Table 1), indicating that the B allele is recessive at the cellular level (Table 1).
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The measured level of wild-type transcript in the Scn8amedJ/medJ, Scnm1B/B mice is the same as that predicted for the Scn8amedJ/- compound heterozygotes described above. Both genotypes exhibit the lethal, paralyzed phenotype, confirming that 6% of wild-type channel is insufficient for survival.
Delayed developmental switch of sodium channels at nodes of Ranvier in myelinated nerves of the PNS and CNS
The sodium channel Nav1.2 is localized at immature nodes of Ranvier, where it is normally replaced by Nav1.6 during the first weeks of postnatal life (17). To determine the effect of Nav1.6 deficiency on nodal maturation, we examined sciatic nerve from medJ mice between 3 weeks and 6 months of age. Teased nerves were doubly labeled with antibodies to Nav1.6 (red) and the paranodal protein Caspr (contactin-associated protein) (green). Nav1.6 was not detectable in mutant nodes at 3 weeks of age, although the nodes of wild-type mice were strongly positive (Fig. 6: upper panels). The medJ nodes were wider than controls, confirming their immaturity. By 6 weeks of age, Nav1.6 reached detectable levels in the majority of sciatic nerve nodes of the medJ mice (Table 2). However, the staining intensity of mutant nodes remained consistently lower than that of wild type (Fig. 6: middle and lower panels), reflecting the reduction in Nav1.6 protein observed on western blots.
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Nodal Nav1.2 was examined by double labeling with an antibody specific for Nav1.2 (green) and a ‘pan-
-subunit’ antibody that detects all nodal sodium channels (red). By three weeks of age, Nav1.2 was completely replaced in wild-type nodes (0/103), but 25% of mutant nodes were still strongly positive for Nav1.2 (28/106) (Fig. 7). By 6 weeks of age, Nav1.2 was no longer detected in mutant nodes (Fig. 7). Thus, there is a delay of several weeks in the replacement of nodal Nav1.2 by Nav1.6 in medJ sciatic nerve. The appearance of Nav1.6 in nodes of the optic nerve, containing myelinated neurons of the CNS, is also delayed (Fig. 8).
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Reduced motor nerve conduction velocity in medJ mice
In view of the evidence that the abundance at Scn8a at the nodes of Ranvier is greatly reduced in the mutant mice, we investigated the effect on motor neuron function. Conduction velocity was measured in sciatic nerve from adult animals. Motor nerve conduction velocity was reduced by 50% compared with unaffected littermate controls (Fig. 9). This reduction is likely to result in reduced signalling at the neuromuscular junction, contributing to the severe muscle weakness of the mutant mice.
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Effect of a null allele of Nav1.2 on the medJ phenotype
The presence of Nav1.2 in immature nodes of Ranvier suggested that deficiency of Nav1.2 might exacerbate the phenotype of medJ mice. To test this hypothesis, we crossed C3H-medJ/+ mice with Scn2a+/- mice carrying a targetted null allele of Scn2a (23). The double heterozygous offspring (Scn2a+/-, Scn8amedJ/+) did not exhibit any visible abnormalities. An F2 generation was produced by crossing the double heterozygotes. As expected, the Scn2a-/- offspring did not survive (20). Among the medJ homozygotes, the proportion of Scn2a+/+ and Scn2a+/- mice was in agreement with Mendelian expectation (Table 3). The severity of the dystonic phenotype was not affected by Scn2a genotype. There was thus no deleterious effect of combining heterozygosity for a null allele of Scn2a with homozygosity for the hypomorphic allele of Scn8a.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Analysis of the modifier locus, Scnm1
It is widely appreciated that variation in genetic modifiers can alter the severity of monogenic disorders. We previously mapped the sodium channel modifier locus Scnm1, and showed that inheritance of two copies of the B allele from strain C57BL/6J prevents survival of medJ mice (9). The four other inbred strains that were tested carry the dominant H allele. Since medJ is a splice site mutation, we hypothesized that the B allele might reduce the efficiency of splicing of the Scn8a transcript. In our previous study, we used a semiquantitative RT–PCR–filter hybridization assay that did not detect any difference between the strains. In order to test the splicing hypothesis more rigorously, we developed a quantitative RT–PCR/primer extension assay that made it possible to detect a 2-fold reduction in the proportion of correctly spliced transcript in medJ mice that are homozygous for the B allele of Scnm1, from 12% to 6% of total Scn8a transcript. A similar 2-fold reduction in level of correctly spliced transcripts in medJ/null compound heterozygotes results in a lethal phenotype, indicating that 6% is insufficient for survival (Table 4). It thus appears likely that a 50% reduction of correct splicing of the mutant splice site accounts for the lethality of the B/B genotype. Interestingly, the proportion of correctly spliced transcripts is identical in heterozygous B/H and homozygous H/H mice, consistent with the dominant effect of the H allele on survival (9).
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Regulation of pre-mRNA splicing has been proposed as the mechanism for genetic modification of phenotypic severity in several human disorders (reviewed in 21). In one well-studied example, the severity of cystic fibrosis is inversely correlated with the level of correctly spliced cystic fibrosis transmembrane receptor (CFTR) transcript (22–24). The ability of the Scnm1 locus to reduce the splicing efficiency of the Scn8amedJ transcript by 2-fold in medJ homozygotes demonstrates the direct effect of a modifier on a primary mutation affecting a splice site. Since all C57BL/6J mice carry the B allele of Scnm1, it is likely that splice site mutations in other genes will also produce a more severe phenotype on this strain. C57BL/6J may thus be considered a sensitized strain for certain splice site mutations. Identification of the modifier genes responsible for variation in splicing efficiency could provide novel therapeutic targets and contribute to understanding the mechanisms of polygenic disease. Because Scnm1 segregates in crosses between inbred strains of mice, positional cloning of this modifier should be feasible.
RT–PCR/primer extension assay for quantitation of alternatively spliced products
The combination of RT–PCR with single-nucleotide primer extension has been used previously to compare the expression of alleles differing by a single nucleotide (25). We developed the method to permit comparison of alternatively spliced transcripts, using primer extension plus chain termination by a single dideoxynucleotide. The quantitation of radiolabeled primer extension products permits accurate and reproducible measurement of the proportion of alternative splice products, provided that PCR conditions are adjusted to prevent preferential amplification of one product. Known mixtures of cloned fragments representing the alternative transcripts can be used to develop appropriate conditions and to validate the linearity of the assay. RT–PCR/primer extension was at least as reliable as real-time PCR, and more convenient for optimization of conditions in this experiment, which involved multiple primers, products of different lengths and a relatively small 2-fold difference between samples. With appropriate validation, RT–PCR/primer extension is an excellent method for quantitation of the relative proportions of differentially spliced transcripts in RNA samples.
Neurological function in mice with low levels of Scn8a
Reduction of the expression of the sodium channel Nav1.6 by an order of magnitude in medJ mice provides an opportunity to identify the in vivo functions of this widespread, abundant sodium channel. The visible abnormalities of motor function in these mice are likely to result from combined effects on peripheral nerve function and central motor control. Specific deficits that we have identified include reduced motor nerve conduction velocity, loss of muscle mass and reduced glucose uptake indicative of a widespread reduction in synaptic activity in the brain.
During the second week of postnatal development, the sodium channel Nav1.2 in immature nodes of Ranvier in the sciatic nerve is normally replaced by Nav1.6 (17). By postnatal day 8, Nav1.6 is the major carrier of sodium current in spinal motor neurons (15), and at 3 weeks, Nav1.6 is readily detected at sciatic nerve nodes in wild-type mice (10). In previous work on C57BL/6J-medJ homozyzgotes, nodal staining of Nav1.6 could not be detected at 3 weeks of age (10). In the new C3H-medJ strain, Nav1.6 protein was detectable in nodes of adult medJ mice, with greatly reduced staining intensity. We found no evidence for retention of Nav1.2 in mature nodes, or for upregulation of Nav1.2 protein, a possibility that had been suggested by others (26).
Motor nerve conduction velocity is reduced by 50% in adult medJ mice. Nerve conduction velocity is a function of the conduction capacity of the largest myelinated fibers, and is influenced by sodium channel density along the axon. Saltatory conduction from node to node is crucial for rapid propagation of action potentials along large myelinated fibers. The decreased abundance of Nav1.6 at the nodes of Ranvier is the likely cause of reduced motor nerve conduction velocity, which in turn reduces muscle stimulation and generates the morphological features of functional denervation, such as variable muscle fiber diameter and centrally located nuclei (9). Dystonic contractions in medJ mice may reflect central dysregulation exacerbated by slow and incomplete peripheral responses. The contributions of CNS and PNS to the medJ dystonia may be dissected in the future using genetic models with region-specific inactivation of Scn8a.
[14C]-2-deoxyglucose uptake in brain is driven largely by synaptic activity in regions dense in nerve terminals, where glucose is required to produce ATP for regeneration of resting ion gradients after depolarizations. Analysis of [14C]-2-deoxyglucose uptake in homozygous mutant mice revealed a 40% reduction in overall brain glucose utilization. This large effect indicates that Nav1.6 is a major contributor to synaptic activity throughout the brain. Diminished Nav1.6 expression in nerve terminals and dendrites would be expected to directly affect synaptic activity. Reduced expression in soma could constrain the generation of action potentials, as demonstrated by the loss of repetitive spiking in cerebellar Purkinje cells (14). Reduction of Nav1.6 in nodes of myelinated axons would also contribute to the overall depression of activity by impairment of action potential propagation.
Sodium channels and disease
We tested the possibility of genetic interaction between Nav1.2 and Nav1.6 by crossing medJ mice with Scn2a+/- mice. The resultant double heterozygotes, with 
50% of the normal levels of Nav1.2 and Nav1.6, did not exhibit neurological deficits. We also generated med J/med J, Scn2a+/- mice that contain 12% of normal Nav1.6 levels and 50% of normal Nav1.2 levels. These mice were not more severely affected than their medJ/medJ, Scn2a+/+ littermates. These experiments indicated that Scn2a is not haploinsufficient in the mouse, even when Scn8a expression is greatly reduced. In contrast, human patients heterozygous for null alleles of Scn1a exhibit severe myoclonic epilepsy infancy (SMEI) (27).
Small contributions from many loci contribute to the hybrid vigor observed in heterozygous F1 mice from crosses between inbred strains (28). Crosses between the two medJ congenic lines, on strains C3H and C57BL/6J, produces medJ homozygotes on an F1 background that exhibit reproducible phenotypes, genetic uniformity and improved viability. Genetically matched +/+ littermates are produced by the same cross. These advantages make medJ F1 homozygotes ideal for physiological studies of Nav1.6 deficiency.
By combining different alleles of Scn8a, we were able to titrate the effects of reduced expression levels (Table 4). Null heterozygotes with 50% of normal activity have no clinical impairment, but reduction to 12% results in serious neurological disease. Further reduction to 6% results in lethality. The threshold for survival is thus between 6% and 12% of wild-type Scn8a levels. The abnormalities in medJ mice with reduced Scn8a expression suggest that it would be worthwhile to screen patients with movement disorders such as ataxia and dystonia for quantitative reduction in Scn8a expression. The RT–PCR/primer extension reaction described here could be modified for this purpose to detect underexpressed alleles in RNA samples from heterozygotes for common single-nucleotide polymorphisms (SNPs). The spastic paraplegia locus SPG10 was recently mapped close to SCN8A on chromosome 12q13, but we have excluded Scn8a as a candidate gene for this disorder (29).
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MATERIALS AND METHODS |
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Animals
The med J mutation arose on a Caracul stock at the Jackson Laboratory 30 years ago (6). Since l995, we have maintained the mutant by backcrossing to strain C57BL/6J, producing the congenic line C57BL/6J-med J/+, now in generation N15 (6,9). Homozygous mutants from this line do not survive beyond postnatal day 30. In order to study the adult phenotype, we developed a second congenic line, C3HeB/FeJ-med J/+, now at generation N12, by backcrossing to strain C3HeB/FeJ. During development of this strain, med J/+ offspring were identified by inheritance of the closely linked visible marker Caracul, and heterozygosity was confirmed by intercrosing. Crosses between medJ heterozygotes from the two congenic strains produces (C3HeB/FeJxC57BL/6J)F1-medJ/medJ homozygous offspring that have a survival rate of >90%. These mice were used for most experiments. Rotorod and swim tests were carried out as previously described (30).
RT–PCR/primer extension assay for quantitation of alternatively spliced Scn8a transcripts
Total brain RNA was isolated from 1-month-old mice. First-strand cDNA was prepared by reverse transcription with Superscript II (Invitrogen) using random hexamer primers. PCR amplification was carried out with forward primer a from exon 1 (ACCCC CGAGT CGCTG GCAAA CATC) and reverse primer b from exon 6 (GGGCG AAGAC ACTCA GGCAG AACA), generating products that span exon 2 and exon 3. Exons 2 and 3 are skipped as a result of the splice donor site mutation in intron 3 of the medJ allele of Scn8a (6). After 30 cycles of amplification with a l.5 min extension time, the amplification of the 539 bp mutant fragment (M) and the 748 bp full-length fragment (WT) was comparable. Single-stranded DNA and dNTPs were removed from the PCR products by treatment with ExoSAP-IT (USB). Primer extension was carried out with Thermosequenase (USB) using PAGE-purified primer c (TTTGA CCCGT ACTAT TTGAC GCAGA) (Fig. 1) in the presence of [-33P]ddGTP and unlabeled dATP, dTTP and dCTP. After 30 cycles of the extension reaction, the radiolabeled products were separated on 12% denaturing acrylamide gels and visualized using the Bio-Rad Personal molecular imager FX system. The bands were quantitated with Quantity One software version 4.2 (Bio-Rad). To generate standards of known composition, the PCR fragments of 748 bp (WT) and 539 bp (M) were cloned into the vector pGEM T-easy (Promega). The clones were combined in known ratio, diluted to 1 pg/µl, and analyzed as described above by PCR and primer extension. The standard curve in Figure 2E was generated by the Excel program using regression analysis.
Antibodies
The antibody to Nav1.6 used for western blots (WB) and immunocytochemistry (ICC) was rabbit polyclonal anti-Scn8a lot AN-01 (Alomone Labs, 1 : 100 for ICC and 1 : 200 for WB) prepared against a 20-residue peptide from the intracellular loop between domains 2 and 3. (Lot AN-02 from the same supplier was found to react with an extraneous protein of 160 kb on western blots in addition to the Nav1.6 protein migrating at 250 kDa.) Rabbit polyclonal anti-SP19 (pan-) (1 : 100 for ICC) and anti-Scn1a (1 : 200 for WB) were from Alomone Labs, and rabbit polyclonal anti-Scn2a (1 : 100 for ICC and 1 : 200 for WB) was from Sigma. Mouse monoclonal anti-Scn2a and anti-Caspr for immunocytochemistry (1 µg/ml) were kindly provided by Dr James Trimmer.
Immunocytochemistry
Nerves were dissected, immersion-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 30 min at room temperature, and washed twice with 0.1 M PB for 30 min each. Optic nerves were cryoprotected with 20% sucrose for 24 h, frozen in OCT mounting medium, cut into 30 µm sections, and mounted on poly-L-lysine-coated coverglass. Sciatic nerves were teased into individual fibers on poly-L-lysine-coated coverglass. Tissues were permeabilized for 2 h in PB, pH 7.4, containing 10% normal goat serum and 0.3% Triton X-100 (PBTGS), and incubated with primary antibodies diluted in PBTGS overnight at room temperature. Between incubation steps, tissues were washed twice with PB, then once with PBTGS. Secondary antibodies were biotinylated goat anti-rabbit IgG (1 : 500; Jackson) and anti-mouse-Alexa-488 (1 : 300; Molecular Probes). Polyclonal antibody labeling was visualized with strepavidin–Alexa-568 (1 : 800; Molecular Probes). After the final incubation, tissues were washed three times with PB and mounted onto slides with gel mount. Images were captured with a Bio-Rad MRC 600 laser scanning confocal microscope in the University of Michigan Microscopy and Image Analysis Laboratory.
Western blots
Isolation of membrane proteins from brain homogenates and Western blotting were carried out as described previously (31). Membranes were incubated overnight with primary antibody and stained with peroxidase-conjugated mouse anti-rabbit (1 : 200 000; Jackson). Signal was visualized by chemiluminescent detection with SuperSignal West Femto (Pierce).
Northern blots
RNA was isolated from adult brain at 3 and 10 months of age. Poly(A)+ RNA was prepared and hybridziation was carried out as previously described (30). Probes were prepared by amplification of 0.5 kb fragments of the 3'-UTR of Scn1a (Nav1.1) and Scn2a (Nav1.2) from genomic DNA using the following primers: (Scn1a forward) GCCAA AGGGA AATGA ACAAA; (Scn1a reverse) ACACC GGGAA AAGAG TTCCT; (Scn2a forward) TGGAA CATTT ACAGG CACATT; (Scn2a reverse) GGAGA GCAAT GGCTA CTCAG A. Amplified fragments were labeled with [32P]dCTP using the Megaprime kit (Amersham).
Motor nerve conduction velocity
Sciatic nerve was stimulated proximally at the sciatic notch and distally at the ankle via bipolar electrodes with supramaximal stimuli. The latencies of the compound muscle action potentials were recorded via bipolar electrodes from the first interosseous muscle of the hindpaw and measured from stimulus artifact to the onset of the negative M-wave deflection. Motor nerve conduction velocity (MNCV) was calculated by subtracting the distal latency from the proximal latency and the result was divided into the distance between the stimulating and recording electrodes as described previously (32).
[14C]-2-deoxyglucose metabolic mapping
Homozygous medJ mice 4–6 months of age received an intraperitoneal injection of [14C]-2-deoxyglucose (l00 µCi/kg; ARC, St Louis, MO). After 1 h, mice were sacrificed by decapitation and brains were processed for autoradiography as described previously (19). Autoradiograms were analyzed by quantitative densitometry using an MCID image analysis system (Imaging Research, St Catherines, ONT). Film optical density was converted to bound radioactivity using a standard curve of optical densities from co-exposed standards. The values for medJ and control mice were compared with a t-test.
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ACKNOWLEDGEMENTS |
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We thank J. Malhotra, N. Fettman and L. Argetsinger for advice on western blots, J. Lehozsky for assistance with primer selection, J. Trimmer for the gift of antibodies, and L. Isom for review of the manuscript. This work was supported by NIH research grants NS34509 (M.M.) and NS38166 (R.L.A.) and a VA Merit Review Grant (R.L.A.). J.A.K. acknowledges NRSA fellowship NS10692. Support was provided by the following NIH training programs: Sensory Mechanisms and Disorders T32 DC00011 (G. deH.), Predoctoral Genetics T32 GM07544 (D.A.B.), Genomic Sciences T32 HG 00040 (D.A.B.) and Biomedical Research Training for Veterinary Scientists T32 RR07008 (S.I.L.)
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FOOTNOTES |
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* To whom correspondence should be addressed at: University of Michigan, 4808 Medical Science II, Ann Arbor, MI 48109-0618, USA. Tel: +1 7347635546; Fax: +1 7347639691; Email: meislerm{at}umich.edu
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REFERENCES |
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