The Molecular Mechanism Of Hereditary Cerebellar Ataxia In Waddles Mouse

Objective: To discuss the molecular mechanism of hereditary cerebellar ataxia in waddles mouse.Methods: Heterozygous (+/Wdl) and homozygous (wd1/wd1) Waddles mice were purchased from Mouse Mutant Stock Resource colonies at JAX and a breeding colony was established at the UTHSC. Experimental animal procedures and mouse husbandry were performed in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals and approved by the UTHSC Institutional Animal Care and Use Committee. F2 generation are nomal (+/+) Heterozygous (+/Wdl) and homozygous Waddles (wdl/wdl) mice. 30 Adult (2-4 months) Wdl mice and 30 wide-type littermates were used for quantitative analyses of motor function, We detected the wdl locus on mouse Chr 4 by means of high-throughput temperation gradient capillary electrophoresis heteroduplex examination of PCR and RT-PCR and confirm the deletion mutation in Car8 exon 7 by sequencing and RT-PCR. We use Northern blotting and Western blotting examination to investigate whether the mutated Car8 gene affects the expression at transcriptional, translational, or both levels. Immunohistochemical approaches were then used to investigate the impact of Car8 deficiency on Wdl mice. To detect the molecules in the regulation of Car8 expression and its target genes, we conducted microarray analysis.Results:1. Behavioral analysis of Wdl mice and wild-type littermates. As quantified with footprint analysis, Wdl mice exhibit a wide-based, ataxic gait. Relative to wild-type littermates, both hind-base and front-base widths are increased in Wdl mice. However, there are no differences in either stride length or the distance of paw overlap between Wdl mice and wild-type mice. In comparison with wild-type littermates, Wdl mice perform poorly on rotarod testing. At each speed, mean latency is greater in wild-type than in Wdl mice.2. We found that the Car8 exon 7 amplicon was smaller in Wdl mice than in wild-type littermates. After verification of this difference by varying PCR conditions and examination of Car8 cDNA from normal and Wdl mice, we concluded the deletion mutation in Car8 exon 7. After sequencing Car8 cDNA from normal and Wdl mice, We definded a 19 nucleotide deletion within exon 7 of Car8.3. We use mRNA from wild-type and Wdl mice to do Northern blotting hybridization, The Wdl mice exhibit a much lower level of Car8 transcription and a slightly lower level of IP3R1 transcription. The amount of mRNA was intermediate in heterozygote animals. The Car8 protein from Wdl mice was not detectable in a western blot analysis using total protein extract from cerebellum, while the level of protein from homozygous normal and heterozygous mice is similar. Effect of mutation in Car8 on the translation level of IP3R1 appeared at a minimum level as there seemed no difference in the protein level between normal, heterozygous and Wdl mice in western blot analysis.4. To detect the molecules in the regulation of Car8 expression and its target genes, we conducted microarray analysis. Among genes showing altered expression were those involved in calcium homeostasis.Conclusion:1. The waddles mouse is a unique animal model that exhibits ataxia andappendicular dystonia without pathological abnormalities of the central nervous system. They may provide considerable insight into the fundamental pathophysiological mechanisms of these disorders.2. The deletion of 19 nucleotides within exon 7 alters the final 21 amino acids of the mutant protein. Furthermore, the mutant protein is shortened by 17 amino acids. First, the 6 amino acids coded by the deleted nucleotides eliminated from the protein. Next, the 19 nucleotides, one more nucleotide than the three coding group, cause a frame shift mutation. As the result, the translated amino acids after the deleted region are altered, instead of 38 amino adds, the Wdl mice produce a Car8 protein that is 17 amino acids shorter than wild-type.3. The mutated Car8 gene in Wdl mice reduced the expression at transcriptional and translational levels. Because CAR8 is a novel binding partner of IP3R1, CAR8 deficiency must alter the metabotropic glutamate receptor-IP3 -calcium signaling cascade. Waddles and other animal models indicate that this cascade is central to the pathophysiology of both ataxia and dystonia. Our results indicate that CAR8 plays a specific role in motor control.4. To detect the molecules in the regulation of Car8 expression and its target genes, we concluded that among genes showing altered expression were those involved in calcium homeostasis. In this regard, it should be emphasized that calcium dynamics are critical to Purkinje cell physiology and the pathophysiology of ataxia and other movement disorders.