Mutations in the Cacnl1a4 calcium channel gene are associated with seizures, cerebellar degeneration, and ataxia in tottering and leaner mutant mice

Tottering and leaner, two mutations of the mouse tottering locus, have been studied extensively as models for human epilepsy. Here we describe the isolation, mapping, and expression analysis of Cacnl1a4, a gene encoding the alpha subunit of a proposed P-type calcium channel, and also report the physical mapping and expression patterns of the orthologous human gene. DNA sequencing and gene expression data demonstrate that Cacnl1a4 mutations are the primary cause of seizures and ataxia in tottering and leaner mutant mice, and suggest that tottering locus mutations and human diseases, episodic ataxia 2 and familial hemiplegic migraine, represent mutations in mouse and human versions of the same channel-encoding gene. Received: 1 November 1996 / Accepted: 20 November 1996     

Mapping of jog locus to the region between D6Mit104 and D6Mit336 on mouse chromosome 6.

The joggle mouse is a recessive ataxic mutant carrying an unknown mutation in a C3H/He (C3H)-derived chromosomal segment. Taking advantage of the mouse genome database, we selected 127 DNA microsatellite markers showing heterozygosity between C3H and C57BL/6J (B6) and a first round of screening for the joggle mutation was performed on B6-jog/+ partial congenic mice (N4). We identified 4 chromosomal regions in which 13 microsatellite markers show heterozygosity between C3H and B6. Then, we analyzed the genotype of these 4 chromosomal regions in mice that showed the joggle phenotype and mapped the jog locus between markers D6Mit104 (111.4 Mb) and D6Mit336 (125.1 Mb) (an interval of 13.7 Mb) on chromosome 6. By using a partial congenic strain together with the mouse genome database, we successfully mapped the chromosomal localization of the jog locus much more efficiently than by conventional linkage analysis.     

Frataxin knockin mouse

Friedreich ataxia is the consequence of frataxin deficiency, most often caused by a GAA repeat expansion in intron 1 of the corresponding gene. Frataxin is a mitochondrial protein involved in iron homeostasis. As an attempt to generate a mouse model of the disease, we introduced a (GAA)(230) repeat within the mouse frataxin gene by homologous recombination. GAA repeat knockin mice were crossed with frataxin knockout mice to obtain double heterozygous mice expressing 25-36% of wild-type frataxin levels. These mice were viable and did not develop anomalies of motor coordination, iron metabolism or response to iron loading. Repeats were meiotically and mitotically stable.     

Silencing Mutant Ataxin-3 Rescues Motor Deficits and Neuropathology in Machado-Joseph Disease Transgenic Mice

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein – ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.     

Expression of the zinc finger gene Gli3 is affected in the morphogenetic mouse mutant extra-toes (Xt).

Genetic analysis and homology between the phenotypic alterations of the human Greig Cephalopolysyndactyly Syndrome (GCPS) and the mouse mutant extra-toes (Xt) have suggested a dominant mutation in the same gene of both species. Recently, the GLI3 gene, a member of the Krüppel-related zinc finger genes, has been proposed as a candidate gene for GCPS. We examined the expression of the mouse Gli3 gene in both Xt mutant animals and during normal mouse development. Northern and RNAase protection analysis of embryos revealed that Gli3 expression was reduced about 50% in heterozygous Xt/+ mice and completely absent in homozygous Xt/Xt mice. In addition, in situ analysis of wild-type mice documented Gli3 expression in the developing limb and brain, structures affected in Xt mutant mice. This pattern suggests an important function of the Gli3 gene during morphogenesis.     

Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols

Endonuclease III, encoded by nth in Escherichia coli, removes thymine glycols (Tg), a toxic oxidative DNA lesion. To determine the biological significance of this repair in mammals, we established a mouse model with mutated mNth1, a homolog of nth, by gene targeting. The homozygous mNth1 mutant mice showed no detectable phenotypical abnormality. Embryonic cells with or without wild-type mNth1 showed no difference in sensitivity to menadione or hydrogen peroxide. Tg produced in the mutant mouse liver DNA by X-ray irradiation disappeared with time, though more slowly than in the wild-type mouse. In extracts from mutant mouse liver, we found, instead of mNTH1 activity, at least two novel DNA glycosylase activities against Tg. One activity is significantly higher in the mutant than in wild-type mouse in mitochondria, while the other is another nuclear glycosylase for Tg. These results underscore the importance of base excision repair of Tg both in the nuclei and mitochondria in mammals.     

Deletion of N-terminal residues 23-88 from prion protein (PrP) abrogates the potential to rescue PrP-deficient mice from PrP-like protein/doppel-induced Neurodegeneration

Accumulating evidence has suggested that prion protein (PrP) is neuroprotective and that a PrP-like protein/Doppel (PrPLP/Dpl) is neurotoxic. A line of PrP-deficient mice, Ngsk Prnp0/0, ectopically expressing PrPLP/Dpl in neurons, exhibits late-onset ataxia because of Purkinje cell death that is prevented by a transgene encoding wild-type mouse PrP. To elucidate the mechanisms of neurodegeneration in these mice, we introduced five types of PrP transgene, namely one heterologous hamster, two mouse/hamster chimeric genes, and two mutants, each of which encoded PrP lacking residues 23-88 (MHM2.del23-88) or with E199K substitution (Mo.E199K), into Ngsk Prnp0/0 mice. Only MHM2.del23-88 failed to rescue the mice from the Purkinje cell death. The transgenic mice, MHM2.del23-88/Ngsk Prnp0/0, expressed several times more PrP than did wild-type (Prnp+/+) mice and PrPLP/Dpl at an equivalent level to Ngsk Prnp0/0 mice. Little difference was observed in the pathology and onset of ataxia between Ngsk Prnp0/0 and MHM2.del23-88/Ngsk Prnp0/0. No detergent-insoluble PrPLP/Dpl was detectable in the central nervous system of Ngsk Prnp0/0 mice even after the onset of ataxia. Our findings provide evidence that the N-terminal residues 23-88 of PrP containing the unique octapeptide-repeat region is crucial for preventing Purkinje cell death in Prnp0/0 mice expressing PrPLP/Dpl in the neuron.     

"Skittish" Abca2 knockout mice display tremor, hyperactivity, and abnormal myelin ultrastructure in the central nervous system

The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein most highly expressed in the central and peripheral nervous system tissues and macrophages. Previous studies indicated its role in cholesterol/steroid (estramustine, estradiol, and progesterone) trafficking/sequestration, oxidative stress response, and Alzheimer's disease. Developmental studies have shown its expression during macrophage and oligodendrocyte differentiation, processes requiring membrane growth. To determine the in vivo function(s) of this transporter, we generated a knockout mouse from a gene-targeted disruption of the murine ABCA2 gene. Knockout males and females are viable and fertile. However, a non-Mendelian inheritance pattern was shown among male progeny of heterozygous crosses. Compared to wild-type and heterozygous littermates, knockout mice displayed a tremor without ataxia, hyperactivity, and reduced body weight; the latter two phenotypes were more marked in females than in males. This sexual disparity suggests a role for ABCA2 in hormone-dependent neurological and/or developmental pathways. Myelin sheath thickness in the spinal cords of knockout mice was greatly increased compared to that in wild-type mice, while a significant reduction in myelin membrane periodicity (compaction) was observed in both spinal cords and cerebra of knockout mice. Loss of ABCA2 function in vivo resulted in abnormal myelin compaction in spinal cord and cerebrum, an ultrastructural defect that we propose to be the cause of the phenotypic tremor.     

Association of a regulatory gene, slyA with a mouse virulence of Salmonella serovar Choleraesuis

The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated by using an slyA-defective mutant. The defective mutant was constructed by the insertion of a kanamycin-resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 10(5) higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild-type strain of S. serovar Choleraesuis was 40-fold higher than that of S. serovar Typhimurium. The time course of infection observed in the mice organs also proved the clear difference of the virulence between the parent and the mutant strains. These results suggested that the slyA gene product functions as a virulence-associated regulator also in S. serovar Choleraesuis.     

The cerebellar deficient folia (cdf) gene acts intrinsically in Purkinje cell migrations

Cerebellar deficient folia (cdf) is a recently identified mouse mutation causing ataxia and cerebellar abnormalities including lobulation defects and abnormal placement of a specific subset of Purkinje cells. To understand the etiology of the cerebellar defects in cdf mutant mice, we examined postnatal development of the cdf/cdf cerebellum. Our results demonstrate that Purkinje cell ectopia and foliation defects are apparent at birth, suggesting the cdf mutation disrupts the positioning of many, but not all, Purkinje cells during development. In addition to cerebellar abnormalities, we observed lamination defects in the hippocampus of cdf mutant mice, although neocortical defects were not seen. Furthermore, ectopic Purkinje cells in cdf/cdf mice express an increased level of Dab1 protein, as previously observed in mice with mutations in genes in the reelin signaling pathway. Lastly, analysis of cdf <-->ROSA26 chimeric mice demonstrated that the cdf mutation is intrinsic to Purkinje cells. We suggest that the cdf gene product is required in a subset of Purkinje cells, possibly to respond to Reelin signals.     

Analysis of the cerebellar proteome in a transgenic mouse model of inherited prion disease reveals preclinical alteration of calcineurin activity

Inherited prion diseases are linked to insertional and point mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. Transgenic (Tg) (PG14) mice express a mouse PrP homolog of a nine-octapeptide insertion associated with an inherited prion disorder. These mice develop a progressive neurological syndrome characterized by ataxia and cerebellar atrophy due to synaptic degeneration in the molecular layer and massive apoptosis of granule neurons. To investigate the molecular events that may contribute to neurological dysfunction, we carried out a differential proteomic analysis of cerebella from Tg(PG14) mice at the preclinical, onset, and symptomatic phases of their neurological illness. 2-D maps of cerebellar proteins from Tg(PG14) mice were compared to those obtained from age-matched Tg(WT) mice that express wild-type PrP and remain healthy. Proteins whose levels were significantly modified in at least one stage of the Tg(PG14) disease were identified by PMF. Analysis detected a preclinical decrease of the calcium/calmodulin-dependent phosphatase calcineurin (CaN) in granule neurons, suggesting that dysregulation of CaN activity induced by mutant PrP may be responsible for the cerebellar dysfunction in Tg(PG14) mice.     

Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development

Mammalian cells harbor three highly homologous and widely expressed members of the ras family (H-ras, N-ras, and K-ras), but it remains unclear whether they play specific or overlapping cellular roles. To gain insight into such functional roles, here we generated and analyzed H-ras null mutant mice, which were then also bred with N-ras knockout animals to ascertain the viability and properties of potential double null mutations in both loci. Mating among heterozygous H-ras(+/-) mice produced H-ras(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of H-ras did not interfere with embryonic and fetal viability in the uterus. Homozygous mutant H-ras(-/-) mice reached sexual maturity at the same age as their littermates, and both males and females were fertile. Characterization of lymphocyte subsets in the spleen and thymus showed no significant differences between wild-type and H-ras(-/-) mice. Analysis of neuronal markers in the brains of knockout and wild-type H-ras mice showed that disruption of this locus did not impair or alter neuronal development. Breeding between our H-ras mutant animals and previously available N-ras null mutants gave rise to viable double knockout (H-ras(-/-)/N-ras(-/-)) offspring expressing only K-ras genes which grew normally, were fertile, and did not show any obvious phenotype. Interestingly, however, lower-than-expected numbers of adult, double knockout animals were consistently obtained in Mendelian crosses between heterozygous N-ras/H-ras mice. Our results indicate that, as for N-ras, H-ras gene function is dispensable for normal mouse development, growth, fertility, and neuronal development. Additionally, of the three ras genes, K-ras appears to be not only essential but also sufficient for normal mouse development.     

C/EBPbeta, when expressed from the C/ebpalpha gene locus, can functionally replace C/EBPalpha in liver but not in adipose tissue

Knockout of C/EBPalpha causes a severe loss of liver function and, subsequently, neonatal lethality in mice. By using a gene replacement approach, we generated a new C/EBPalpha-null mouse strain in which C/EBPbeta, in addition to its own expression, substituted for C/EBPalpha expression in tissues. The homozygous mutant mice C/ebpalpha(beta/beta) are viable and fertile and show none of the overt liver abnormalities found in the previous C/EBPalpha-null mouse line. Levels of hepatic PEPCK mRNA are not different between C/ebpalpha(beta/beta) and wild-type mice. However, despite their normal growth rate, C/ebpalpha(beta/beta) mice have markedly reduced fat storage in their white adipose tissue (WAT). Expression of two adipocyte-specific factors, adipsin and leptin, is significantly reduced in the WAT of C/ebpalpha(beta/beta) mice. In addition, expression of the non-adipocyte-specific genes for transferrin and cysteine dioxygenase is reduced in WAT but not in liver. Our study demonstrates that when expressed from the C/ebpalpha gene locus, C/EBPbeta can act for C/EBPalpha to maintain liver functions during development. Moreover, our studies with the C/ebpalpha(beta/beta) mice provide new insights into the nonredundant functions of C/EBPalpha and C/EBPbeta on gene regulation in WAT.     

ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase

KK/San is a mutant mouse strain established in our laboratory from KK obese mice. KK/San mice show low plasma lipid levels compared with wild-type KK mice despite showing signs of hyperglycemia and hyperinsulinemia. Recently, we identified a mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/San mice, and injection of adenoviruses encoding Angptl3 or recombinant ANGPTL3 protein to mutant KK/San mice raised plasma lipid levels. To elucidate the regulatory mechanism of ANGPTL3 on lipid metabolism, we focused on the metabolic pathways of triglyceride in the present study. Overexpression of Angptl3 in KK/San mice resulted in a marked increase of triglyceride-enriched very low density lipoprotein (VLDL). In vivo studies using Triton WR1339 revealed that there is no significant difference between mutant and wild-type KK mice in the hepatic VLDL triglyceride secretion rate. However, turnover studies using radiolabeled VLDL revealed that the clearance of (3)H-triglyceride-labeled VLDL was significantly enhanced in KK/San mice, whereas the clearance of (125)I-labeled VLDL was only slightly enhanced. In vitro analysis of recombinant protein revealed that ANGPTL3 directly inhibits LPL activity. These data strongly support the hypothesis that ANGPTL3 is a new class of lipid metabolism modulator, which regulates VLDL triglyceride levels through the inhibition of LPL activity.     

Immunocytochemical analyses and targeted gene disruption of GTPBP1

We previously identified a gene encoding a putative GTPase, GTPBP1, which is structurally related to elongation factor 1alpha, a key component of protein biosynthesis machinery. The primary structure of GTPBP1 is highly conserved between human and mouse (97% identical at the amino acid level). Expression of this gene is enhanced by gamma interferon in a monocytic cell line, THP-1. Although counterparts of this molecule in Caenorhabditis elegans and Ascaris suum have also been identified, the function of this molecule remains to be clarified. In the present study, our immunohistochemical analyses on mouse tissues revealed that GTPBP1 is expressed in some neurons and smooth muscle cells of various organs as well as macrophages. Immunofluorescence analyses revealed that GTPBP1 is localized exclusively in cytoplasm and shows a diffuse granular network forming a gradient from the nucleus to the periphery of the cells in smooth muscle cell lines and macrophages. To investigate the physiological role of GTPBP1, we used targeted gene disruption in embryonic stem cells to generate GTPBP1-deficient mice. The mutant mice were born at the expected Mendelian frequency, developed normally, and were fertile. No manifest anatomical or behavioral abnormality was observed in the mutant mice. Functions of macrophages, including chemotaxis, phagocytosis, and nitric oxide production, in mutant mice were equivalent to those seen in wild-type mice. No significant difference was observed in the immune response to protein antigen between mutant mice and wild-type mice, suggesting normal function of antigen-presenting cells of the mutant mice. The absence of an eminent phenotype in GTPBP1-deficient mice may be due to functional compensation by GTPBP2, a molecule we recently identified which is similar to GTPBP1 in structure and tissue distribution.     

Characteristics of Gait Ataxia in δ2 Glutamate Receptor Mutant Mice,ho15J

The cerebellum plays a fundamental, but as yet poorly understood, role in the control of locomotion. Recently, mice with gene mutations or knockouts have been used to investigate various aspects of cerebellar function with regard to locomotion. Although many of the mutant mice exhibit severe gait ataxia, kinematic analyses of limb movements have been performed in only a few cases. Here, we investigated locomotion in ho15J mice that have a mutation of the δ2 glutamate receptor. The cerebellum of ho15J mice shows a severe reduction in the number of parallel fiber-Purkinje synapses compared with wild-type mice. Analysis of hindlimb kinematics during treadmill locomotion showed abnormal hindlimb movements characterized by excessive toe elevation during the swing phase, and by severe hyperflexion of the ankles in ho15J mice. The great trochanter heights in ho15J mice were lower than in wild-type mice throughout the step cycle. However, there were no significant differences in various temporal parameters between ho15J and wild-type mice. We suggest that dysfunction of the cerebellar neuronal circuits underlies the observed characteristic kinematic abnormality of hindlimb movements during locomotion of ho15J mice.     

The Spinner-IBMM mouse is a new spontaneous mutant in the Tmie gene

A recessively inherited, spontaneous mutation named Spinner-IBMM (SI) was identified in a transgenic mouse colony in our institute. SI mutant mice displayed hyperactivity, including a severe circling behavior, ataxia and inability to swim. Gene mapping revealed that the causative gene was located on a 35 Mb DNA fragment on chromosome 9. Candidate genes sequencing in this DNA fragment identified a new mutant allele in the Tmie gene. The identified mutant is characterized by a nucleotide deletion in exon 5, leading to a frameshift and a premature STOP codon. It has been reported that inactivating mutations in the mouse Tmie gene result in an identical phenotype, probably resulting from defects in the inner ear. However, the exact function of the Tmie protein in the ear and other organs is still unknown. The analysis of this new mouse mutant could contribute to a better understanding of Tmie functions in vivo in the ear and other organs.