Characterization of a murine high-affinity thiamine transporter, Slc19a2

Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity thiamine transporter. We hypothesize that thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the transporter further, we cloned the murine Slc19a2 locus and characterized the resulting protein. Murine Slc19a2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans approximately 13kb with 6 exons, structurally identical to that of the human homolog. We localized the Slc19a2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of Slc19a2 in HEK293T cells resulted in specific uptake of [3H] thiamine, confirming a thiamine transporter function. Western blot analysis of mouse tissues reveals a wide distribution of Slc19a2 protein. Immunohistochemistry studies indicate that Slc19a2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.     

Targeted disruption of Slc19a2, the gene encoding the high-affinity thiamin transporter Thtr-1, causes diabetes mellitus,sensorineural deafness and megaloblastosis in mice

Thiamin-responsive megaloblastic anemia syndrome (TRMA) is characterized by diabetes mellitus, megaloblastic anemia and sensorineural deafness. Mutations in the thiamin transporter gene SLC19A2 cause TRMA. To generate a mouse model of TRMA, we developed an Slc19a2 targeting construct using transposon-mediated mutagenesis and disrupted the gene through homologous recombination in embryonic stem cells. Erythrocytes from Slc19a2(-/-) mice lacked the high-affinity component of thiamin transport. On a thiamin-free diet, Slc19a2(-/-) mice developed diabetes mellitus with reduced insulin secretion and an enhanced response to insulin. The diabetes mellitus resolved after 6 weeks of thiamin repletion. Auditory-evoked brainstem response thresholds were markedly elevated in Slc19a2(-/-) mice on a thiamin-free diet, but were normal in wild-type mice treated on that diet as well as thiamin-fed Slc19a2(-/-) mice. Bone marrows from thiamin-deficient Slc19a2(-/-) mice were abnormal, with a megaloblastosis affecting the erythroid, myeloid and megakaryocyte lines. Thus, Slc19a2(-/-) mice have provided new insights into the TRMA disease pathogenesis and will provide a tool for studying the role of thiamin homeostasis in diabetes mellitus more broadly.     

Altered neurotensin mrna expression in mice lacking the dopamine transporter

Psychostimulants and antipsychotic drugs increase mRNA expression of the neuropeptide neurotensin (NT) in the striatum and nucleus accumbens. In the present study, we used mice lacking the dopamine transporter (DAT) to investigate the consequences of a chronic hyperdopaminergic state on NT gene expression. NT mRNA expression was examined under basal conditions and after administration of haloperidol or amphetamine using in situ hybridization with a digoxigenin-labeled NT cRNA probe. DAT-/- mice exhibited a striking increase in the number of NT mRNA-expressing perikarya in the substantia nigra and ventral tegmental area, as well as a less pronounced increase in the lateral septum compared with wild-type littermates. No changes were detected in other regions expressing NT mRNA. Acute administration of haloperidol (1 mg/kg) induced a significant increase in the number of NT mRNA-expressing neurons in the dorsomedial and dorsolateral striatum of wild-type mice but failed to stimulate NT gene expression in DAT mutants. In contrast, a higher dose of haloperidol (5 mg/kg) stimulated striatal NT mRNA expression both in DAT+/+ and DAT-/- mice. Amphetamine (10 mg/kg) increased the number of hybridized neurons in the nucleus accumbens shell and fundus striati of wild-type and DAT-/- mice, indicating that the drug acted through a target other than DAT, such as the serotonin or the norepinephrine transporters. The up-regulation of NT mRNA observed in DAT-/- mice may represent an adaptive mechanism in response to constitutive hyperdopaminergia. These results illustrate the profound alterations in the NT system induced by chronic stimulation of DA receptors and underscore the potential clinical relevance of NT/DA interactions in schizophrenia and drug abuse.     

The 5q- chromosome in a case of erythroid hypoplasia

A case of erythroid hypoplasia with refractory anemia and an interstitial deletion of the long arm of one chromosome No. 5 is described. This 5q- anomaly has been previously described in cases of refractory anemia with erythroid hypoplasia or hyperplasia, and acute myelogenous leukemia. The relationship between the 5q- chromosome and associated clinical features is discussed together with evidence against a geographical localization.     

Loss of autoreceptor functions in mice lacking the dopamine transporter.

Autoreceptors provide an important inhibitory feedback mechanism for dopamine neurons by altering neuronal functions in response to changes in extracellular levels of dopamine. Elevated dopamine may be a component of several neuropsychiatric disorders. However, evidence concerning the state of autoreceptors in such conditions has remained elusive. The function of dopamine autoreceptors was assessed in mice lacking the dopamine transporter (DAT). Genetic deletion of the DAT gene in mice results in a persistent elevation in levels of extracellular dopamine. Direct assessment of impulse-, synthesis- and release-regulating autoreceptors in these mice reveals a nearly complete loss of function. These findings may provide insight into the neurochemical consequences of hyperdopaminergia.     

Osteopenia and male-specific sudden cardiac death in mice lacking a zinc transporter gene,Znt5

We isolated a mammalian gene whose expression transiently increased in response to intimal denudation of rabbit aorta. It was identical to a gene encoding a zinc transporter, ZNT5, reported very recently by others. Mice deficient for this gene showed poor growth and a decrease in bone density due to impairment of osteoblast maturation to osteocyte. More than 60% of male null mice died suddenly because of the bradyarrhythmias. Analysis of gene-expression profiles in murine hearts by means of an oligonucleotide microarray disclosed that a subset of genes encoding immediate-early response factors (IEGs) and heat shock proteins (HSPs) were down-regulated in Znt5-null mice. These results indicate that Znt5 protein plays an important role in maturation of osteoblasts and in maintenance of the cells involved in the cardiac conduction system, partly owing to dysregulated expression of IEGs and HSPs.     

Analysis of slc19a2, on 1q23.3 encoding a thiamine transporter as a candidate gene for type 2 diabetes mellitus in pima indians

Mutations in the SLC19A2 gene cause thiamine-responsive megaloblastic anemia (TRMA) frequently combined with diabetes mellitus and deafness. Type 2 diabetes mellitus is heritable and a region on 1q21-q23 encompassing SLC19A2 was linked with the disease in Pima Indians and Caucasians. We therefore investigated this candidate gene in selected diabetic and nondiabetic Pimas and found no variants. We conclude that mutations in SLC19A2 do not contribute to type 2 diabetes in this population.     

Slc19a2: cloning and characterization of the murine thiamin transporter cDNA and genomic sequence, the orthologue of the human TRMA gene

Recently, our group and others cloned the TRMA disease gene, SLC19A2, which encodes a thiamin transporter. Here, we report the cloning and characterization of the full-length cDNA and genomic sequences of mouse Slc19a2. The Slc19a2 cDNA contained a 1494-bp open-reading frame, and had 5'- and 3'-untranslated regions of 189 and 1857 bp, respectively. A putative GC-rich, TATA-less promoter was identified in genomic sequence directly upstream of the identified 5' end. The Slc19a2 gene spanned 16.3 kb and was organized into six exons, a gene structure conserved with the human orthologue. The predicted Slc19a2 protein, like SLC19A2, was predicted to have 12 transmembrane domains and shared a number of other conserved sequence motifs with the human orthologue, including one potential N-glycosylation site (N(63)) and several potential phosphorylation sites. Comparison of the Slc19a2 amino acid sequence with those of the other known SLC19A solute carriers highlighted interesting patterns of conservation and divergence in various domains, allowing insight into potential structure-function relationships. The identification of the mouse Slc19a2 cDNA and genomic sequences will facilitate the generation of an animal model of TRMA, permitting future studies of disease pathogenesis.     

Mechanisms of preleukemic hypoplasia of the bone marrow erythroid stem in AKR/JY mice

During the preleukemic period, the expression of erythroblast receptors on bone marrow macrophages, the number of neutral red-stained hemopoietic islets and their erythroid and erythrogranulocytic types, and the content of erythrokaryocytes decreased in AKR/JY mice compared with (CBA×AKR/JY) F₁ mice. The concentration of erythroid colony-forming units increased. The impairment of qualitative (receptor) and quantitative properties of bone marrow resident macrophages inhibits the differentiation of erythroid cells from committed to mature forms and is probably one of the mechanisms of hypoplasia of the erythroid stem in AKR/JY mice. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 6, pp. 633–635, June, 1999     

Anxiety-like behaviors in mice lacking GIT2

G protein-coupled receptor kinase-interactor 2 (GIT2) is a signaling scaffold protein that also functions as GTPase-activating protein (GAPs) for ADP-ribosylation factor (Arf) small GTP-binding proteins. GIT2 has been implicated in the regulation of G protein-coupled receptor trafficking and cell adhesion and migration. To evaluate possible neurobehavioral functions of GIT2 in vivo, we evaluated GIT2-knockout (KO) mice for abnormalities in emotionality and mood. Male and female GIT2-KO mice presented with anxiety-like behaviors in the zero-maze and light–dark emergence tests. Immobility times in tail suspension were reduced in GIT2-KO males, but were normal in GIT2-KO females. Hence, GIT2-KO mice display anxiety-like behavior in an absence of depressive-like responses.     

Impaired ability to increase water excretion in mice lacking the taurine transporter gene TAUT

Summary Cellular taurine uptake or release counteracts alterations of cell volume. Na⁺-coupled taurine transporter TAUT mediates concentrative cellular uptake of taurine. Inhibition of vasopressin secretion by hypotonicity may involve taurine release from glial cells of supraoptic nucleus. We compared renal function of mice lacking TAUT (taut^–/–) and wild-type littermates (taut^+/+). We observed renal taurine loss and subsequent hypotaurinemia in taut^–/– mice. With free access to water, plasma and urine osmolality, urinary flow rate as well as urinary excretion and plasma concentrations of Na⁺ and K⁺ were similar in taut^–/– and taut^+/+ mice, whereas plasma concentrations of urea were enhanced in taut^–/– mice. An oral water load (1 ml/16 g body weight) induced a similar diuresis in both genotypes. Repeating the oral water load immediately after normalization of urine flow rate, however, resulted in delayed diuresis and higher urinary vasopressin/creatinine ratios in taut^–/– mice. In comparison, the repeated diuretic response to vasopressin V₂ receptor blockade was not different between genotypes. Water deprivation for 36 h led to similar antidiuresis and increases of urinary osmolality in both genotypes. Upon free access to water after deprivation, taut^–/– mice continued to concentrate urine up to 6 days, while taut^+/+ mice rapidly returned to normal urinary osmolality. Urinary vasopressin/creatinine ratios and plasma aldosterone concentrations were not different under basal conditions but were significantly higher in taut^–/– mice than in taut^+/+ mice at 6 days after water deprivation. In conclusion, taut^–/– mice suffer from renal taurine loss and impaired ability to lower urine osmolality and to increase urinary water excretion. The latter defect could reside extrarenally and result from a role of taurine in the suppression of vasopressin release which may be attenuated in taut^–/– mice.     

Male infertility and DNA damage in Doppel knockout and prion protein/Doppel double-knockout mice

The prion protein (PrP) and Doppel (Dpl) have many structural and biochemical properties in common, leading to the suggestion that the lack of an obvious phenotype in PrP-deficient mice maybe because of compensation by Dpl. To test this hypothesis and also investigate the function of Dpl we have generated Prnd(-/-) and Prnp(-/-)/Prnd(-/-) mouse lines. Both develop normally and display an identical male sterility phenotype that differs from that reported for another Prnd(-/-) mouse line. Sperm from both our mutant lines were present at normal concentrations, had normal motility, and no morphological abnormalities. Despite only rarely fertilizing oocytes in vivo, because of an inability to perform the acrosome reaction, mutant sperm were capable of fertilization in vitro, albeit at reduced rates compared to wild type. Elevated levels of oxidative DNA damage were found in both types of mutant sperm and resulting embryos failed at an early stage. Therefore we found no evidence that Dpl compensates for the loss of PrP function in mutant mouse lines, but it does have an important anti-oxidant function necessary for sperm integrity and male fertility.     

Male infertility risk factors in a French military population

We investigated infertility risk factors by conducting a population-based case-control study in the military population of the French town of Brest. Sixty couples who had sought medical advice for infertility of more than 12 months duration (cases) were compared with 165 couples who had had a child (controls). All the men in these couples had been employed by the military. The infertility risk factors studied were male and female medical factors, occupational and environmental exposures. We obtained age-adjusted odds ratios of 7.4 [95% confidence interval (CI): 1.4--39.5] for testis surgery, and 13.0 for varicocele (95% CI: 1.4--120.3) in men. In logistic regression, the age-adjusted odds ratio for men who had worked in a nuclear submarine was found to be 2.0 (95% CI: 1.0--3.7), and that for heat exposure was 4.5 (95% CI: 1.9--10.6). One limitation of this study is the lack of exposure measurements, especially for potential exposure to nuclear radiation (type of reactor used in nuclear-powered submarines, inability to obtain personal dosimeters worn by military personnel working in nuclear submarines). In conclusion, this study suggests that in this military population, having worked as a submariner in a nuclear-powered submarine, and having worked in very hot conditions, should be considered as risk factors for infertility.     

Mutation of the Na-K-Cl co-transporter gene Slc12a2 results in deafness in mice.

Hearing impairment is a common human condition, but we know little about the molecular basis of cochlear function. Shaker-with-syndactylism (sy) is a classic deaf mouse mutant and we show here that a second allele, sy(ns), is associated with abnormal production of endolymph, the fluid bathing sensory hair cells. Using a positional candidate approach, we demonstrate that mutations in the gene encoding the basolateral Na-K-Cl co-transporter Slc12a2 (Nkcc1, mBSC2) cause the deafness observed in sy and sy(ns) mice. This finding provides the molecular basis of another link in the chain of K+recycling in the cochlea, a process essential for normal cochlear function.     

Impaired glucose homeostasis,neutrophil trafficking and function in mice lacking the glucose-6-phosphate transporter

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT). In addition to disrupted glucose homeostasis, GSD-Ib patients have unexplained and unexpected defects in neutrophil respiratory burst, chemotaxis and calcium flux, in response to the bacterial peptide f-Met-Leu-Phe, as well as intermittent neutropenia. We generated a G6PT knockout (G6PT-/-) mouse that mimics all known defects of the human disorder and used the model to further our understanding of the pathogenesis of GSD-Ib. We demonstrate that the neutropenia is caused directly by the loss of G6PT activity; that chemotaxis and calcium flux, induced by the chemokines KC and macrophage inflammatory protein-2, are defective in G6PT-/- neutrophils; and that local production of these chemokines and the resultant neutrophil trafficking in vivo are depressed in G6PT-/- ascites during an inflammatory response. The bone and spleen of G6PT-/- mice are developmentally delayed and accompanied by marked hypocellularity of the bone marrow, elevation of myeloid progenitor cell frequencies in both organs and a corresponding dramatic increase in granulocyte colony stimulating factor levels in both GSD-Ib mice and humans. So, in addition to transient neutropenia, a sustained defect in neutrophil trafficking due to both the resistance of neutrophils to chemotactic factors, and reduced local production of neutrophil-specific chemokines at sites of inflammation, may underlie the myeloid deficiency in GSD-Ib. These findings demonstrate that G6PT is not just a G6P transport protein but also an important immunomodulatory protein whose activities need to be addressed in treating the myeloid complications in GSD-Ib patients.     

Adaptive immune response to Shigella flexneri 2a cydC in immunocompetent mice and mice lacking immunoglobulin A

Shigella flexneri cydC, which is deficient in cytochrome bd, was rapidly cleared from the lungs of intranasally inoculated mice and was Sereny negative, yet it induced 93% protection against challenge with wild-type S. flexneri. Mice that lack immunoglobulin A (IgA) were fully protected, suggesting that IgA may not be required for adaptive immunity in this model system.     

Inactivation of ether lipid biosynthesis causes male infertility,defects in eye development and optic nerve hypoplasia in mice

Although known for almost 80 years, the physiological role of plasmalogens (PLs), the major mammalian ether lipids (ELs), is still enigmatic. Humans that lack ELs suffer from rhizomelic chondrodysplasia punctata (RCDP), a peroxisomal disorder usually resulting in death in early childhood. In order to learn more about the functions of ELs, we generated a mouse model for RCDP by a targeted disruption of the dihydroxyacetonephosphate acyltransferase gene. The mutant mice revealed multiple abnormalities, such as male infertility, defects in eye development, cataract and optic nerve hypoplasia, some of which were also observed in RCDP. Mass spectroscopic analysis demonstrated the presence of highly unsaturated fatty acids including docosahexaenoic acid (DHA) in brain PLs and the occurrence of PLs in lipid raft microdomains (LRMs) isolated from brain myelin. In mutants, PLs were completely absent and the concentration of brain DHA was reduced. The marker proteins flotillin-1 and F3/contactin were found in brain LRMs in reduced concentrations. In addition, the gap junctional protein connexin 43, known to be recruited to LRMs and essential for lens development and spermatogenesis, was down-regulated in embryonic fibroblasts of the EL-deficient mice. Free cholesterol, an important constituent of LRMs, was found in these fibroblasts to be accumulated in a perinuclear compartment. These data suggest that the EL-deficient mice allow the identification of new phenotypes not related so far to EL-deficiency (male sterility, defects in myelination and optic nerve hypoplasia) and indicate that PLs are required for the correct assembly and function of LRMs.