Two novel alleles of tottering with distinct Ca(v)2.1 calcium channel neuropathologies.

The calcium channel CACNA1A gene encodes the pore-forming, voltage-sensitive subunit of the voltage-dependent calcium Ca(v)2.1 type channel. Mutations in this gene have been linked to several human disorders, including familial hemiplegic migraine, episodic ataxia 2 and spinocerebellar ataxia type 6. The mouse homologue, Cacna1a, is associated with the tottering, Cacna1a(tg), mutant series. Here we describe two new missense mutant alleles, Cacna1a(tg-4J) and Cacna1a(Tg-5J). The Cacna1a(tg-4J) mutation is a valine to alanine mutation at amino acid 581, in segment S5 of domain II. The recessive Cacna1a(tg-4J) mutant exhibited the ataxia, paroxysmal dyskinesia and absence seizures reminiscent of the original tottering mouse. The Cacna1a(tg-4J) mutant also showed altered activation and inactivation kinetics of the Ca(v)2.1 channel, not previously reported for other tottering alleles. The semi-dominant Cacna1a(Tg-5J) mutation changed a conserved arginine residue to glutamine at amino acid 1252 within segment S4 of domain III. The heterozygous mouse was ataxic and homozygotes rarely survived. The Cacna1a(Tg-5J) mutation caused a shift in both voltage activation and inactivation to lower voltages, showing that this arginine residue is critical for sensing Ca(v)2.1 voltage changes. These two tottering mouse models illustrate how novel allelic variants can contribute to functional studies of the Ca(v)2.1 calcium channel.

Differential expression of cellular prion protein in mouse brain as detected with multiple anti-PrP monoclonal antibodies.

The normal cellular prion protein (PrP(C)) plays an essential role in the development of prion diseases. Indirect evidence has suggested that different PrP(C) glycoforms may be expressed in different brain regions and perform distinct functions. However, due to a lack of monoclonal antibodies (Mabs) that are specific for mouse PrP(C), the expression of PrP(C) in the mouse brain has not been studied in great detail. We used Mabs specific for either the N-terminus or the C-terminus of the mouse PrP(C) to study its expression in the mouse brain by immunoblotting and immunohistochemistry. Immunoblotting studies demonstrated that the expression of PrP(C) differed quantitatively as well as qualitatively in different regions of the brain. The anti-C-terminus Mabs reacted with all three molecular weight bands of PrP(C); the anti-N-terminus Mabs only reacted with the 39-42 kDa PrP(C). The results from immunohistochemical staining revealed the spatial distribution of PrP(C) in the mouse brain, which were consistent with that from immunoblotting. Although expression of PrP(C) has been reported to be required for long-term survival of Purkinje cells, we were unable to detect PrP(C) in the Purkinje cell layer in the cerebellum with multiple anti-PrP Mabs. Our findings suggest that PrP(C) variants, i.e. various glycoforms and truncated forms, might be specifically expressed in different regions of mouse brain and might have different functions.

Rocker is a new variant of the voltage-dependent calcium channel gene Cacna1a.

Rocker (gene symbol rkr), a new neurological mutant phenotype, was found in descendents of a chemically mutagenized male mouse. Mutant mice display an ataxic, unstable gait accompanied by an intention tremor, typical of cerebellar dysfunction. These mice are fertile and appear to have a normal life span. Segregation analysis reveals rocker to be an autosomal recessive trait. The overall cytoarchitecture of the young adult brain appears normal, including its gross cerebellar morphology. Golgi-Cox staining, however, reveals dendritic abnormalities in the mature cerebellar cortex characterized by a reduction of branching in the Purkinje cell dendritic arbor and a "weeping willow" appearance of the secondary branches. Using simple sequence length polymorphism markers, the rocker locus was mapped to mouse chromosome 8 within 2 centimorgans of the calcium channel alpha1a subunit (Cacna1a, formerly known as tottering) locus. Complementation tests with the leaner mutant allele (Cacna1a(la)) produced mutant animals, thus identifying rocker as a new allele of Cacna1a (Cacna1a(rkr)). Sequence analysis of the cDNA revealed rocker to be a point mutation resulting in an amino acid exchange: T1310K between transmembrane regions 5 and 6 in the third homologous domain. Important distinctions between rocker and the previously characterized alleles of this locus include the absence of aberrant tyrosine hydroxylase expression in Purkinje cells and the separation of the absence seizures (spike/wave type discharges) from the paroxysmal dyskinesia phenotype. Overall these findings point to an important dissociation between the seizure phenotypes and the abnormalities in catecholamine metabolism, and they emphasize the value of allelic series in the study of gene function.

ATM is a cytoplasmic protein in mouse brain required to prevent lysosomal accumulation.

We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm-/-) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm-/- mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm-/- mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm-/- mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers.

Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice.

mGluR1 mutant mice are viable but show characteristic cerebellar symptoms such as ataxic gait and intention tremor. The anatomy of the cerebellum is not overtly disturbed. Excitatory synaptic transmission from parallel fibers (PFs) to Purkinje cells and that from climbing fibers (CFs) to Purkinje cells appear to be functional, and voltage-gated Ca2+ channels of Purkinje cells are normal. Both PF and CF synapses display normal short-term synaptic plasticity to paired stimuli. By marked contrast, long-term depression (LTD) is clearly deficient and conditioned eyeblink response is impaired. We conclude that mGluR1 is required for the induction of LTD and that the ataxic behavior and impaired eyeblink conditioning of the mGluR1 mutant mice are primarily due to deficient LTD.

Transcription of circular and noncircular forms of Sry in mouse testes.

Although its expression in adult testis was immediately apparent, the role for Sry (sex determining region, Y) in testicular function remains elusive. We have performed transcriptional studies in an effort to elucidate potential roles of Sry by studying the time and location of its transcription in mouse testes. Northern analyses and more sensitive nuclease protection assays detected transcripts in 28-day-old testes and beyond. The highly sensitive technique of reverse transcription polymerase chain reaction (RTPCR) could not detect Sry expression in 14-day testes when primers for the most conserved portion of the gene, the high mobility group (HMG) box, were used, but primers for the circular form detected Sry transcription at all postnatal stages studied. The same HMG box primers were able to detect expression of Sry in XX, Sxra or Sxrb testes. This suggested that Sry is expressed in cells other than germ cells, which was confirmed with studies on fractionated cells--RTPCR detected transcription of Sry in the highly pure interstitial cell fraction. However, Leydig cells and a Leydig cell tumor were negative for Sry expression. We performed in situ studies in an attempt to localize the expression of Sry in the testes. Abundant expression of an Sry cross-hybridizing transcript was found in spermatogonia, in early spermatocytes, and in some interstitial cells with antisense probes to the HMG box or a more specific, 3' region, whereas the sense probe gave little or no hybridization. It is probable that the circular transcripts, which are seen in reverse transcriptase positive (RT+) and RT- reactions by PCR because of the RT activity of Taq polymerase, are responsible for the hybridization seen in spermatogonia and spermatocytes, whereas linear and circular forms are detected later. Thus Sry is expressed in pre- and postmeiotic germ cells and in somatic cells of the testes.

Zfy is transcribed in the normal mouse epididymis and in the XXSxr ("sex reversed") testis.

The presence of the mutation Sex reversed (Sxr), a copy of a Y-chromosomal segment that gets transferred to an X chromosome, causes the resulting XXSxr mice to develop as apparent males. However, several features of male sexual development are abnormal in these animals. The testes are small and aspermatogenic, and the epididymides lack the initial segment. Testes and epididymides show abnormalities of extracellular matrix. In this study we examined transcription of the conserved Y chromosomal gene Zfy, which has an X-chromosomal homologue (Zfx). Northern blotting showed Zfy to be expressed in the testes of XXSxr animals, except for those that carry the coat-marker gene Tabby (Ta), despite the lack of germ cells in XXSxr mice. Reverse transcription polymerase chain reaction (RT-PCR) studies detected Zfy in mRNA in testes even when Ta was present. RT-PCR also demonstrated Zfy transcription in epididymides of normal males, though not in XXSxr mice. Previous authors reported an absence of Zfy transcription in XXSxr testes; Zfy transcription in normal testes has been ascribed to germ cells. Our observation indicates that this idea requires re-evaluation. The occurrence of Zfy transcription in the normal epididymis is similarly a novel finding that may help explain those aspects of epididymal development that occur in the absence of androgen.

Non-disjunction in human sperm: results of fluorescence in situ hybridization studies using two and three probes.

Fluorescence in situ hybridization using two or three probes was utilized to estimate the incidence of diploidy, the incidence of disomy for the sex chromosomes and chromosomes 16 and 18, and the proportion of Y- and X-chromosome bearing sperm, in a series of normal males. Our results demonstrate the importance of using an approach capable of distinguishing disomy from diploidy, as most donors had levels of diploidy higher than the disomy levels of individual chromosomes. Our analyses suggest the existence of chromosome-specific mechanisms of paternal non-disjunction, as sex chromosome disomy was approximately 1.5 times as common as disomy 16, and over two times as common as disomy 18. In studies of gametic sex ratio, we found little evidence for marked deviation from an expected 1:1 ratio.

Developmentally regulated transcription of the four liver-specific genes for inter-alpha-inhibitor family in mouse.

The inter-alpha-inhibitor family is composed of the plasma-protease inhibitors inter-alpha-inhibitor, pre-alpha-inhibitor and bikunin. Inter-alpha-inhibitor and pre-alpha-inhibitor are distinct assemblies of bikunin with distinct sets from three heavy (H) chains designated H1, H2 and H3. These H chains are encoded by a set of three evolutionarily related H genes, and bikunin by an alpha-1-microglobulin/bikunin precursor gene (AMBP). This precursor is cleaved to yield bikunin, a member of the Kunitz-type protease-inhibitor superfamily, and alpha-1-microglobulin, which belongs to the lipocalin superfamily. Northern-blot experiments with RNAs obtained from various tissues in fetal and in adult mice indicated that the transcription of the four AMBP and H genes is liver-restricted, although there is expression of H3 in brain. An analysis of the H1, H2, H3 and AMBP transcripts, as well as of transcripts for other control genes, in liver during development showed a progressive increase in the amounts of the H1, H2, H3 and AMBP RNAs, which all peak transiently at day 5 after birth. This was shown by a nuclear run-on experiment to originate from a change in transcription rate. The transient and postnatal increase in transcription could be explained neither by the liver-restricted expression nor by a common origin of these four genes, nor by a perinatal requirement for many lipocalins or protease inhibitors. This suggests that all four genes are perinatally triggered at the level of similar elements in their transcriptional regulatory regions, a conclusion strengthened by the weak expression of the four genes that is seen in a mutant mouse strain (albino) that is deficient in some liver-specific transcription factors.

Gene expression, X-inactivation, and methylation during spermatogenesis: the case of Zfa, Zfx, and Zfy in mice.

While it has become clear that X-inactivation in the female soma is complete in mouse (in contrast to being "patchy" in man), the degree of X-inactivation in the testes has not been ascertained. We have compared autosomal and X-linked zinc finger homolog expression and X-linked and Y-linked zinc finger homolog methylation in an attempt to elucidate this question. Using RTPCR, we have extended earlier studies of Zfx and Zfa expression in developing testes and find that Zfa expression starts at the time of X-inactivation while Zfx expression is continuous. Cell separation studies did not preclude continued expression of Zfx in adult germ cells. The methylation status of four CCGG residues in the Zfx promoter was studied using PCR bridging this region before and after DNA digestion with the isoschizomers Msp I and Hpa II, the latter being methylation sensitive. Hpa II resistant Zfx promoter DNA was found in all female tissues, but not in male tissues, including the testes. Previous studies have shown that Zfy is expressed at meiosis (like Zfa and unlike Zfx). Despite its expression, the Zfy gene is adjacent to, or contains, highly methylated CCGG sites since hybridization after Msp I digestion detected multiple small fragments that were not released after DNA digestion with Hpa II. Thus, Zfx is not methylated in sperm, while Zfy is, in contrast to their apparent patterns of expression.

Transcription of the sex-determining region genes Sry and Zfy in the mouse preimplantation embryo.

We have confirmed the faster growth of male preimplantation mouse embryos. We have also studied the transcription of Y chromosomal genes postulated to have a role in sex determination, using the highly sensitive technique of reverse-transcription polymerase chain reaction at these early stages. We find that two sex-determining region genes, Sry and Zfy, are transcribed during mouse preimplantation development, while the Zfy homologs Zfx and Zfa and a sex-determining region gene originally called A1s9 (now called Ube1y-1) are not. We also show that the anti-Müllerian hormone gene, which contains a Sry consensus binding element in its 5' promoter region, is not transcribed at this time. Developmental curves show that Sry and Zfy are expressed commencing at the two-cell stage. These results suggest that mammalian sex determination starts prior to gonad differentiation.

End-stage renal disease and primary hypogonadism associated with a 46,XX karyotype.

OBJECTIVE: To determine the cause of absent sexual development in a 17-year-old girl with end-stage renal disease. DESIGN: Case study. PARTICIPANT: Seventeen-year-old girl with end-stage renal failure. INTERVENTIONS: None. MEASUREMENTS/MAIN RESULTS: The patient had phenotypically normal external female genitalia, müllerian duct hypoplasia, and no ovaries. Her serum gonadotropin levels were in the castrate range at baseline and after gonadotropin-releasing hormone stimulation. Her karyotype, in lymphocytes and cultured fibroblasts, was 46,XX. Analysis of genomic DNA, following polymerase chain reaction-amplication with oligonucleotide primers corresponding to the Y-encoded zinc finger protein ZFY and the testis-determining SRY gene, showed Y chromosome material in a male control but none in the patient. CONCLUSIONS: The results suggest a diagnosis of Frasier syndrome, a disorder characterized by true gonadal dysgenesis and end-stage renal disease occurring in normal phenotypic girls. Although previously reported only in individuals with a 46,XX karyotype, our studies indicate that Frasier syndrome may also occur in 46,XX girls. Delayed puberty is not uncommon in renal failure. This case illustrates the importance of measuring gonadotropin levels in teenage girls with delayed puberty and renal failure, particularly if the origin of the renal disease is obscure.