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Genetics, Vol. 171, 1239-1246, November 2005, Copyright © 2005
doi:10.1534/genetics.105.044487
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* Departments of Orthopedic Surgery-Campbell Clinic and Pathology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, Department of Biology, University of Memphis, Memphis, Tennessee 38163, Departments of Neurology and Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, The Jackson Laboratory, Bar Harbor, Maine 04609, ** Functional Genomics Facility, University of Chicago, Chicago, Illinois 60637 and Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019
1 Corresponding author: University of Tennessee Health Science Center, A331 Coleman Bldg., 956 Court Ave., Memphis, TN 38163.
E-mail: wgu{at}utmem.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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-subunit of voltage-gated sodium channel) in MedJ mice (BURGESS et al. 1995), Grid2 (delta2 glutamate receptor) in lurcher mice (ZUO et al. 1997), Kcnj6 (potassium inwardly rectifying channel subfamily J member 6) in weaver mice (PATIL et al. 1995), Agtpbp1 (ATP/GTP-binding protein 1) in Purkinje cell degeneration mice (FERNANDEZ-GONZALEZ et al. 2002), and Cacna1a (P/Q type calcium channel -1A subunit) in tottering mice (FLETCHER et al. 1996). Although these models have shed considerable light on signaling and second messenger pathways, the relationship between cellular dysfunction due to these mutations and motor control at the systems level remains unclear. This disconnect is largely due to the ubiquitous expression of the encoded proteins within neural tissues. Furthermore, many mutant rodents with movement disorders exhibit overt structural and histological abnormalities of the central nervous system, extraneural disease, or early death. Thus, in many cases, the mutant gene may have deleterious effects on several local-area neural networks in addition to systemic effects that severely compromise attempts to correlate genotype with particular motor phenotypes (e.g., ataxia, Parkinsonism, dystonia, spasticity, or myoclonus). Identification of additional mutant genes with restricted expression in neural tissue and causally associated with distinct motor syndromes may help to bridge the gaps between the molecular and systems neurobiology of movement disorders. As part of this effort, we chose to study waddles (wdl) mice. The wdl mutation was discovered at The Jackson Laboratory (TJL) in 1995 in C57BL/KS mice (http://www.jax.org/mmr/waddler.html). The wdl phenotype is very similar to that of another model, waddler (wd), which was discovered in 1959 and is now thought to be extinct (YOON 1959). The gait of wdl mice is characterized by wobbly side-to-side ataxic movements that are readily seen when mice reach 2 weeks of age. The gait disorder of wdl mice persists throughout their life span.
In addition to ataxia, wdl mice exhibit frequent tail elevation and intermittent Straub tail. During ambulation, the trunks of wdl mice are abnormally elevated, particularly their caudal portions. Resting forelimb and hindlimb tone is normal. However, action dystonia with apparent cocontraction of knee and elbow flexors and extensors is exacerbated by ambulation (JINNAH and HESS 2004). This appendicular dystonia produces nearly straight limbs with minimal flexion at the knee and elbow joints, elevation of the pelvis, and a "bouncy" or "waddling" motion during ambulation, particularly at higher velocities. Occasionally, wdl mice fall to their sides.
Pathological examination of wdl mice at TJL was unremarkable except for one isolated case of hydrocephalus. In addition, vision and hearing were normal in the mutants. An early genetic study indicated that wdl mice were autosomal recessive mutants. Linkage mapping at TJL placed the wdl mutation in close approximation to the wd locus on mouse chromosome 4, although tests for allelism were not conducted since wd is extinct.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Motor function examination:
Adult (2–4 months) wdl/wdl mice and wild-type (+/+) littermates were used for quantitative analyses of motor function. The open field behavior of additional wdl mice along with heterozygote (+/wdl) and +/+ littermates was observed in both home cages and bedless arenas. Digital videos were analyzed for the presence of distinguishing behaviors. Analysis of variance was conducted for the significance between the homozygous wdl (wdl/wdl) and their normal littermates (+/wdl or +/+) whenever necessary.
Rotarod:
Mice were acclimated to a rotarod (San Diego Instruments) rotating at 5 rpm for 5 min prior to data acquisition. Three 2-min trials were performed at each target speed (5, 10, 20, 30, 40, and 50 rpm) with an intertrial interval of 5 min. The rotarod was accelerated to target speeds >1 min. Median values were used for statistical comparisons.
Footprint analysis:
Mouse forepaws and hindpaws were dipped in nontoxic water-based paints. Mice were then allowed to run down a runway lined with white paper. Three trials were performed with intertrial intervals of at least 5 min. Two to four steps from the middle portion of each run were measured for (1) stride length, (2) hind-base width (the distance between the right and left hindlimb strides), (3) front-base width (the distance between the right and left forelimb strides), and (4) overlap between forepaw and hindpaw placement. At least seven steps were measured for each mouse. Mean values were used for statistical analyses.
Tail suspension:
This test involved the response of each mouse to 1 min of suspension from the tail. Some mice with neurological dysfunction exhibited hindlimb and/or forelimb clasping during this maneuver.
Righting reflex:
To obtain righting reflex times, mice were placed in the supine position and then released. The time required for all four limbs to contact the tabletop was measured for three trials. Median values were used for statistical comparisons.
Vertical rope climbing:
Mice were acclimated to a vertical 40-cm-long, 10-mm-thick rope prior to testing. The bottom of the rope was suspended 15 cm above a padded base and the top entered an escape box. Three trials with a 5-min intertrial interval were completed for each mouse and median times were used for statistical analyses.
Raised-beam task:
Mice were acclimated to an 80-cm-long, 26-mm-wide beam elevated 50 cm above a padded foundation. A 60-W lamp at the start served as an aversive stimulus whereas the opposite end of the beam entered an escape box. Slips were counted as mice traversed the beam. Falls were counted as five slips.
Genome information:
Information on microsatellite markers and their locations were obtained from the Mouse Genome Database search forms (http://www.informatics.jax.org/searches/markerform.shtml). The locations of microsatellite markers on genome sequences and physical chromosomal distances were obtained by searching the Ensembl mouse genome database (http://www.ensembl.org/Mus_musculus); data used in this article are from its updated information as of March 20, 2005.
High-throughput screening of the wdl locus:
Liver genomic DNA (gDNA) from +/+, +/wdl, and homozygous (wdl/wdl) mice was extracted for temperature-gradient capillary electrophoresis (TGCE) and sequence analysis. On the basis of the Ensembl and National Center for Biotechnology Information (NCBI) databases, primer pairs flanking the exons of known and predicted genes (including ESTs) within the wdl locus were designed with Primer3 software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Primers were located 
100 bp 5' or 3' to each exon and, in general, produced 300- to 400-bp DNA fragments. Several primer pairs were required for complete coverage of some exons. PCR amplification of gDNA was performed in a 96-well plate format and consisted of 30–35 cycles at three temperatures: strand denaturation at 96° for 30 sec, primer annealing at 54°–60° for 60 sec, and primer extension at 72° for 120 sec. TGCE (SpectruMedix, State College, PA) was used to analyze amplicons from +/+, +/wdl, and wdl/wdl mice mixtures. The SpectruMedix system includes a high-throughput capillary electrophoresis instrument capable of analyzing 96 samples every 140 min. Heteroduplex analysis was subsequently performed offline using SpectruMedix software. Amplicons from wdl mice were sequenced if they differed from normal.
RT-PCR:
Total RNA was extracted from cerebellum and liver with Trizol reagent (Invitrogen, San Diego). Total RNA integrity was confirmed with the Agilent Bioanalyzer 2100. Reverse transcription and PCR were conducted using a one-step RT-PCR kit from Invitrogen. Reactions were performed in a total volume of 50 µl with 8 ng/µl of total RNA and 0.2 µM forward (CCAAAACAATTCCATGCTTTAAT) and reverse (GTATGAATTCCAGAAGCTGTGGT) primers used to amplify exons 6–9 of Car8. First, cDNA synthesis and predenaturation were performed in single cycles at 50° for 40 min and 94° for 2 min. Next, PCR amplification was performed for 35 cycles: 94° for 30 sec, 54°–58° for 36 sec, and 72° for 2 min.
DNA sequencing:
DNA sequencing was conducted to verify the deletion in the gDNA and cDNA of Car8. PCR products from both genomic and cDNA were purified using an AMPure PCR Purification Kit (Agencourt Beverly, MA) and the purified products were sequenced using a BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems, Foster City, CA). A total of 5-µl sequencing reactions, including 2-µl of Big Dye (plus Half-BD), 10–23 ng of purified DNA template, and 1–3 pmol of either forward or reverse universal sequencing primers, were incubated for 37 cycles at 96° for 180 sec, 50° for 30 sec, and 60° for 180 sec. Unreacted primers were removed by ethanol-acetate precipitation (3.75% 3 M NaOAc, 87.5% nondenatured 100% EtOH, and 8.75% dH2O, pH 4.6). The labeled products were dissolved in 0.02 mM EDTA in HiDi formamide prior to electrophoretically loading onto the SpectruMedix 96 capillary sequencing system. The same primers in the amplification of DNA fragments from either genomic DNA or mRNA were also used in the sequencing. Sequencing was conducted two times to verify the result for either gDNA or cDNA.
Expression of recombinant CAR8:
Normal (accession no. NM_007592) and mutant Car8 cDNA was amplified (forward primer, CACCATGGCTGACCTGAGCTTCATTG; reverse primer, CTGAAAGGCCGCTCGGATGACTCTAT) and cloned into Invitrogen's pET102/D-TOPO vector in-frame for transcription. A single colony of Escherichia coli BL21 (DE3) transformed with the Car8 expression vector was inoculated into 10 ml of Luria-Bertani medium containing 100 µg/ml ampicillin; this was incubated at 37° for 10 hr. Then 0.5 ml of the culture was inoculated into 50 ml of Luria-Bertani medium containing 100 µg/ml ampicillin. This culture was incubated at 37° until it reached an OD600 of 0.7. At that point, isopropyl-D-thiogalactoside was added to a final concentration of 0.5 mM and incubation was continued at 37° for 3 hr; cells were harvested by centrifugation. Protein products were analyzed on 8% SDS-PAGE gels. Calculated molecular weights for normal and mutant CAR8 fusion proteins are 46.09 and 45.49 kD, respectively.
Northern blot hybridization:
After isolation of total RNA from mouse cerebella, mRNA (from 20 mice: +/+, 7; wdl/wdl, 7; +/wdl, 6) was extracted and purified with the MicroPoly(A)Purist kit from Ambion (Austin, TX). The mRNA was electrophoretically resolved on denaturing gels and transferred to positively charged nylon membranes. Radiolabeled ([32P]UTP) complementary RNA (cRNA) probes were generated by in vitro transcription using T7 RNA polymerase. The location of Car8 probe in the cDNA of Car 8 gene is from 205 to 650 bp. After ultraviolet crosslinking, blots were prehybridized and then hybridized overnight with both Car8 and ß-actin cRNA probes. After washing, blots were exposed to Kodak Biomax MR radiographic film prior to development.
Antibody production:
A rabbit polyclonal antibody to CAR8 was generated by immunizing rabbits with a peptide sequence unique to CAR8 (DANGEYQSPINLNSREC) and encoded by nucleotides 27–71 from the second exon of Car8 (AnaSpec, San Jose, CA). This peptide shows no sequence similarity with other carbonic anhydrases. Serum was immunoaffinity purified.
Western blot analysis:
Cerebellar cortex was harvested from mice, rinsed in PBS, and homogenized in chilled NP-40 lysis buffer containing a protease inhibitor cocktail (Sigma, St. Louis). Lysates were clarified by centrifugation. Protein concentrations were determined with the Bio-Rad (Hercules, CA) DC protein assay kit using BSA standards. Equal amounts of proteins were electrophoretically resolved on 4–20% Tris-HCl Criterion precast gels (Bio-Rad) and then transferred onto Immunoblot PVDF membranes (Bio-Rad). Membranes were washed in Tris-buffered saline with 0.1% Tween, blocked in 5% nonfat dry milk, and then incubated in affinity-purified rabbit anti-CAR8 (1:2000) or rabbit anti-inositol 1,4,5-triphosphate receptor (IP3R1) (1:2000, A. G. Scientific, San Diego) antibodies. After washing, blots were incubated in horseradish-peroxidase-conjugated secondary antibodies (1:5000, Amersham, Buckinghamshire, UK). Targeted proteins were visualized with the ECL Plus chemiluminescent kit from Amersham. For loading controls, membranes were stripped and reprobed with a mouse anti-ß-tubulin antibody (1:5000; Chemicon, Temecula, CA).
Immunocytochemistry:
Mice and rhesus monkeys were perfusion fixed with saline/4% paraformaldehyde. Brains were postfixed and then cryoprotected in 30% sucrose/phosphate buffer. Mouse brains and monkey cerebella were sectioned parasagitally on a cryostat and collected onto Superfrost-Plus glass slides (Fisher). Mouse spinal cords were sectioned horizontally. Brain sections were processed for immunocytochemical detection of CAR8 (affinity-purified rabbit antibody, 1:20,000), calbindin (rabbit polyclonal antibody, 1:5000; Chemicon), and IP3R1 (rabbit polyclonal antibody, 1:2000). For light microscopic visualization, tissues were sequentially exposed to a biotinylated goat anti-rabbit polyclonal antibody (1:500; Vector, Burlingame, CA), streptavidin, and diaminobenzidine. Cy2- and rhodamine red-X-conjugated (Jackson ImmunoResearch, West Grove, PA) secondary antibodies were applied for confocal microscopy.
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RESULTS |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Our molecular analysis of the wdl mouse model has shown that CAR8 is essential for motor control. Moreover, the relative preponderance of CAR8 in cerebellar Purkinje cells suggests that the wdl mouse can be used as a tool to precisely investigate the effects of Purkinje cell dysfunction on local and wide-area motor networks. In contrast to several other mouse mutants with ataxia, there is no loss or overt morphological abnormalities of Purkinje cells in wdl mice. Although CAR8 is a member of the carbonic anhydrase family of zinc metalloenzymes that catalyze the reversible hydration of CO2, CAR8 lacks catalytic activity by virtue of missing critical amino acid residues required for zinc binding (SJOBLOM et al. 1996; TANIUCHI et al. 2002). Recombinant CAR8 generated by introducing R117H and E115Q mutations into the wild-type protein is able to bind zinc and catalyze the hydration of CO2. CAR8 may contribute to the pathophysiology of ataxia in other mouse models. For instance, KELLY et al. (1994) reported the absence of Car8 transcript in the cerebella of lurching mice years before the actual causal mutation was identified.
The reduced Car8 transcript and barely detectable CAR8 protein in wdl mice is indicative of nonsense-mediated decay (NMD). NMD, first documented by LOSSON and LACROUTE (1979) >20 years ago, is a proofreading mechanism that enables eukaryotic cells to detect and degrade mRNAs that contain premature termination codons (PTCs). About one-third of inherited genetic disorders and many forms of cancer are caused by frameshift or nonsense mutations, which result in the generation of PTCs (FRISCHMEYER and DIETZ 1999; HOLBROOK et al. 2004). Although mRNA containing a PTC may initially be translated into a truncated protein, cells can initiate the NMD mechanism to recognize and degrade the mutant transcripts if the truncated protein is deleterious (WAGNER and LYKKE-ANDERSEN 2002).
With respect to the cellular effects of CAR8 deficiency, it has been shown that CAR8 binds to the modulatory domain of IP3R1, which is an intracellular IP3-gated Ca2+ channel (HIROTA et al. 2003). CAR8 inhibits IP3 binding to IP3R1 by reducing the affinity of the receptor for IP3 (HIROTA et al. 2003). IP3 is an intracellular second messenger for calcium release. Increased cytosolic-free calcium concentration is a stimulatory signal for diverse calcium-dependent mechanisms such as secretion, contraction, or alterations in membrane excitability. Therefore, CAR8 deficiency may cause ataxia by altering calcium homeostasis and disturbing the normal physiology of cerebellar Purkinje cells.
Our study also demonstrates a useful strategy for simplifying positional cloning. Most procedures employed in traditional positional cloning have been labor intensive and time consuming. As described in MATERIALS AND METHODS, our strategy includes identifying a target genomic region on the basis of linkage mapping, identifying every gene and biologically functional element within the candidate region, TGCE heteroduplex analysis (CHOU et al. 2005; GIRALD-ROSA et al. 2005), confirmation of suspected mutations by cDNA sequencing, and, finally, investigatation of the encoded protein. Using a similar approach, we recently identified the causal mutation in a mouse disease model of spontaneous fractures (JIAO et al. 2005). In that study, mapping of the mutation locus was followed by positional cloning. In this study, mapping information on the TJL webpage allowed us to define a target genomic region. The mutant gene was discovered in less than half a year from the start of the project, thereby offering the possibility of rapid identification of mutations with only crude mapping data. We believe that our strategy will be particularly useful for familial human diseases with small kindreds. Furthermore, in the setting of rodent models with spontaneous mutations, mutations can be rapidly identified without the excessive cost and time associated with the extensive breeding programs required for high-resolution mapping.
In summary, we have identified a 19-bp deletion in exon 8 of Car8 in wdl mice using a positional candidate cloning approach. This loss-of-function mutation of Car8 may underlie the ataxic phenotype of wdl mice. Since we did not find an obvious change in the morphology of Purkinje cells or the distribution of the CAR8-binding target, IP3R1, the cellular pathways by which CAR8 deficiency causes ataxia and dystonia remain uncertain. Thus, the role of CAR8 cerebellar neurophysiology warrants additional study.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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