J Biol Chem. 292:9760-9773 (2017) 

Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice

Somshuvra Mukhopadhyay


摘要:

Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging. To understand the consequences of loss of SLC30A10 function at the organism level, we generated Slc30a10 knock-out mice. During early development, knock-outs were indistinguishable from controls. Surprisingly, however, after weaning and compared with controls, knock-out mice failed to gain weight, were smaller, and died prematurely (by ∼6–8 weeks of age). At 6 weeks, manganese levels in the brain, blood, and liver of the knock-outs were ∼20–60-fold higher than controls. Unexpectedly, histological analyses revealed that the brain and liver of the knock-outs were largely unaffected, but their thyroid exhibited extensive alterations. Because hypothyroidism leads to growth defects and premature death in mice, we assayed for changes in thyroid and pituitary hormones. At 6 weeks and compared with controls, the knock-outs had markedly reduced thyroxine levels (∼50–80%) and profoundly increased thyroid-stimulating hormone levels (∼800–1000-fold), indicating that Slc30a10 knock-out mice develop hypothyroidism. Importantly, a low-manganese diet produced lower tissue manganese levels in the knock-outs and rescued the phenotype, suggesting that manganese toxicity was the underlying cause. Our unanticipated discovery highlights the importance of determining the role of thyroid dysfunction in the onset and progression of manganese-induced disease and identifies Slc30a10 knock-out mice as a new model for studying thyroid biology.The mouse Slc30a10 gene has four exons and encodes a protein of 470 amino acids. Exon 1 encodes the first 205 amino acids, which encompasses a significant portion of the predicted transmembrane domain (amino acid 1–299). Because the transmembrane domain is required for metal transport (22), large deletions in this domain should inactivate the protein. In contrast, a partially active protein could be produced if downstream exons were deleted. Therefore, deletion of exon 1 provided the best approach to secure complete loss of function of Slc30a10; hence, we targeted exon 1.We produced animals in which exon 1 of Slc30a10 was flanked by loxP sites, as described under “Experimental procedures.” Heterozygous and homozygous floxed mice appeared phenotypically normal. That is, unlike Slc30a10 knock-out mice, the body size and manganese levels of homozygous and heterozygous floxed animals were comparable with those of mice that were wild-type for the Slc30a10 gene (see Figs. 1 and 2 for phenotype of Slc30a10 knock-outs). Therefore, for this study, we considered homozygous and heterozygous floxed animals to be comparable with wild-types and designated these three genotypes as Slc30a10+/+.To delete exon 1, we bred floxed animals with transgenic Sox2Cre mice (see “Experimental procedures” for details about the Sox2Cre strain) and obtained animals in which exon 1 of one copy of Slc30a10 was deleted. These mice were designated as Slc30a10+/− and were heterozygous for the Slc30a10 knock-out. After another round of breeding, we obtained mice in which exon 1 of Slc30a10 was deleted in both chromosomes; these mice were the knock-out strain and designated Slc30a10−/−. Note that some Slc30a10+/− and Slc30a10−/− mice retained the Cre transgene; however, there was no effect of Cre expression on body size or manganese levels. Therefore, for these genotypes, we combined animals with or without Cre expression into one group.To routinely genotype animals, we performed PCR from genomic DNA. We ran two separate reactions designed to either amplify a product from the wild-type/floxed allele or from the knock-out allele (Fig. 1A). In Slc30a10+/+ animals, a positive product was detected for the wild-type/floxed allele but not the knock-out allele; in Slc30a10+/−, positive products were detected for both reactions; and in Slc30a10−/−, a positive PCR product was detected for the knock-out, but not the wild-type/floxed allele (Fig. 1A). For selected animals, we verified the PCR results by performing reverse transcription-PCR assays, which confirmed that Cre-mediated recombination led to a loss of Slc30a10 mRNA in Slc30a10−/− mice (Fig. 1B). Despite extensive efforts, we were unable to validate loss of Slc30a10 gene product at the protein level in knock-outs using immunoblots because we could not identify any commercial antibody that specifically detected SLC30A10 in rodent tissue. To get around this hurdle, we generated an antibody against the C-terminal domain of human SLC30A10. Unfortunately, we discovered that although this antibody specifically detected SLC30A10 in human cell lines, it lost specificity when mouse tissue samples were used. Despite the lack of immunoblot data, the results obtained from the genomic DNA PCR were unequivocal and confirmed by reverse transcription-PCR. Therefore, we used PCR as an accurate means to genotype animals for further studies.All experiments with mice were approved by the Institutional Animal Care and Use Committee of the University of Texas at Austin. The conditional gene targeting vector with loxP sites flanking exon 1 of Slc30a10 was constructed by Cyagen Biosciences (Santa Clara, CA). We electroporated the targeting vector into V6.5 embryonic stem cells (C57BL/6 × 129/Sv) (51). Electroporation, PCR screening, Southern analysis of embryonic stem cell clones, and blastocyst injection experiments were performed by the University of Texas at Austin Mouse Genetic Engineering Facility following standard methods (52). Germ-line chimeric males were bred with wild-type C57BL/6J mice, obtained from The Jackson Laboratory (Bar Harbor, ME), to produce mice heterozygous for the floxed allele. These heterozygous floxed mice were intercrossed to produce homozygous floxed animals. Because there was no detectable phenotype in heterozygous and homozygous floxed mice, we considered homozygous and heterozygous floxed animals to be comparable with wild-types and designated these three genotypes as Slc30a10+/+.To delete exon 1 of Slc30a10, we bred floxed animals with Sox2Cre transgenic mice. In this transgenic strain, Cre gene product induces recombination in all cells that arise from the epiblast by embryonic day 6.5 (53). Because of recombination at this early stage of life, the mutation is introduced into male and female gametes and is transmitted to future generations without further need for Cre expression. Through this breeding strategy, we obtained animals in which exon 1 of Slc30a10 was deleted in one chromosome. These animals were heterozygous for the Slc30a10 knock-out (designated Slc30a10+/−). A further round of breeding produced animals in which exon 1 was deleted in both chromosomes; these mice constituted the knock-out strain (designated Slc30a10−/−). Some Slc30a10+/− and Slc30a10−/− mice retained the Cre transgene. However, because there was no effect of Cre expression on body size or manganese levels, for these genotypes, we combined animals with or without Cre expression into one group. The Sox2Cre strain was obtained from The Jackson Laboratory.The mice were housed in the specific pathogen-free facility of the University of Texas at Austin in a room maintained at ∼21 °C with a 12-h light-dark cycle (lights on between 7 p.m. and 7 a.m.). After weaning, 3–4 littermates of the same sex were kept per cage. Animals had free access to food and water. The regular diet was PicoLab Rodent Diet 20, which contains ∼84 μg manganese/g chow. The low-manganese diet was AIN-93G, which contains ∼11 μg manganese/g chow.We thank Dr. Steven Vokes (University of Texas at Austin) for advice about knock-out animal generation; Dr. Anthony Hollenberg and Dr. Kristen Vella (Harvard Medical School) for technical assistance with thyroxine measurement assays; and Dr. Edward Mills (University of Texas at Austin) and Dr. Adam Linstedt (Carnegie Mellon University) for critical comments on the manuscript.
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