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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 28, 2001 as doi:10.1096/fj.01-0691fje. |
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Department of Gastroenterology, Hepatology and Infectiology, and
* Department of Anatomy, Heinrich Heine University Düsseldorf, Düsseldorf, Germany; and
Retinal Electrodiagnostics Research Group, Department of Ophthalmology, University of Tübingen, Tübingen, Germany
3Correspondence: Department of Gastroenterology, Hepatology and Infectiology, Heinrich Heine University, Moorenstrasse 5, D-40225 Düsseldorf, Germany. E-mail: haeussin{at}uni-duesseldorf.de
SPECIFIC AIMS
In view of the immunomodulatory and cytoprotective effects of taurine, a mouse model with a disrupted gene coding for the Na+-dependent taurine transporter (taut-/- mice) was generated: besides reduced fertility and markedly decreased taurine levels in a variety of tissues, these mice show a loss of vision due to severe retinal degeneration resembling human retinitis pigmentosa. The study identifies an important role of the taurine transporter (TAUT) for the development and maintenance of retinal function and morphology.
PRINCIPAL FINDINGS
1) TAUT knockout mice (taut-/- mice) exhibit reduced fertility
An 18 kb genomic clone was isolated from a 
phage library containing mouse strain 129SvJ genomic DNA by screening with a cDNA taut probe. To disrupt the taut locus, an isogenic targeting vector was designed to delete exon 1 of taut. Embryonic stem (ES) cells were electroporated with the linearized vector and two ES clones containing the targeting event were injected into blastocysts of C57BL/6 mice to produce chimeras. Only one ES clone transmitted the taut mutation through the germ line. Heterozygous (taut+/-) mice with a C57BL/6 x 129/SvJ background were intercrossed to produce wild-type (taut+/+), heterozygous (taut+/-), and homozygous (taut-/-) animals in the F2 progeny. Offspring of matings between (taut+/-) females and (taut+/-) males were observed in 14 of 16 matings. The offspring showed a normal Mendelian ratio, suggesting no specific embryonic lethality in the mutants. Six of 13 (taut+/-) females became pregnant with (taut-/-) males, but none of 5 (taut-/-) females became pregnant by (taut+/-) males. Only 4 of 21 different breeding pairs of (taut-/-) mice produced offspring, but all 4 matings between (taut+/+) females and (taut-/-) males did.
2) Taurine uptake and taurine tissue levels are strongly decreased in (taut-/-) mice
Deletion of exon 1 of the taut gene led to a truncated protein of 450 amino acids (wild-type: 621 aa). Transmembrane domains 1 to 3 and part of the large extracellular region of the protein were no longer present. As shown by Northern blot analysis, no expression of the taut exon 1 was found in either kidney or liver of (taut-/-) animals, whereas wild-type mice showed exon 1 expression in both organs.
Loss of TAUT function in the null mutation was verified by measuring taurine uptake by fibroblasts in vitro. When primary fibroblasts were exposed to medium supplemented with taurine (200 µM), taurine uptake into fibroblasts isolated from (taut-/-) mice was 98% lower than normal.
(taut-/-) mice exhibited a lower body mass than (taut+/-) and wild-type mice. Further, (taut-/-) mice showed remarkably low taurine tissue levels when compared with wild-type mice. A decrease of taurine concentration by 
74% was observed in plasma, kidney, liver, and eye (Table 1
). In skeletal and heart muscle, taurine levels were decreased > 95%.
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3) Retinal degeneration in (taut-/-) mice
The most prominent morphological feature of (taut-/-) mice was severe and progressive retinal degeneration (Fig. 1
). In contrast to retinas of wild-type mice (Fig. 1a
, 1b
), no photoreceptor segments were discernible light microscopically in age-matched (taut-/-) mice beyond an age of 4 wk (Fig. 1d-h
). In 2-wk-old (taut-/-) mice, the outer nuclear layer (ONL) was largely preserved (Fig. 1c
), but the outer and inner photoreceptor segments were much smaller than those of 2-wk-old wild-type animals (Fig. 1b
). At 1–2 months of age, only one to three layers of nuclei were seen in the ONL (Fig. 1d, e
) and this layer disappeared completely in older animals (Fig. 1f-h
). Electron microscopic examination of retinas from (taut-/-) mice at 1–2 months revealed advanced stages of photoreceptor cell degeneration: many of the remaining nuclei were pyknotic, but ribbon synapses and remnants of outer segment disks could occasionally be seen.
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The effect of taut activity loss on retinal function was assessed by electroretinography (ERG). Beyond an age of 6 wk, ERG signals were completely absent in (taut -/-) mice. However, at ages between 2 and 4 wk, some residual electrical activity at both low and high stimulus intensities was detectable. Compared with controls, the b-wave amplitude of the ERG increased only insignificantly from postnatal wk 2 to 3, and decreased rapidly thereafter. Nevertheless, the double-peaked amplitude vs. intensity curves suggest that both the rod and the cone systems were somewhat functional.
The retinas of 2- and 6-wk-old (taut-/-) mice were analyzed for apoptosis using the TUNEL assay. TUNEL-positive nuclei were consistently present in the (taut-/-) animals (Fig. 2
). (taut -/-) mice display deterioration of inner and outer photoreceptor segments and a progressive reduction in ONL thickness from postnatal day 14 through 6 wk, where a single row of condensed photoreceptor nuclei was observed. No such nuclei were seen in our retinal sections from age-matched wild-type mice.
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CONCLUSIONS
As shown in this study, homozygous disruption of exon 1 of the taut gene results in plasma hypotaurinemia, a 98% decrease in taurine uptake by primary fibroblasts, and low levels of taurine in all tissues examined. These findings suggest that defective taurine uptake due to disruption of taut cannot be compensated for by taurine uptake via other transport systems such as the amino acid transport system A.
(taut-/-) mice exhibited a marked impairment of reproduction. Although taurine is not used for protein synthesis, it is normally present in high concentrations in mouse eggs and oviductal and uterine fluid, acts as an osmolyte in mouse oocytes and embryos, and promotes embryo development in a variety of species including mice and humans. Taurine is also present in human semen, maintains sperm motility, and is necessary for optimal fertilization. These roles of taurine at various levels of reproduction may explain the reduced fertility of (taut-/-) mice. However, TAUT is apparently much more crucial for fertility in females than in males, because (taut-/-) females exhibited extremely reduced fertility regardless of the taut status of the male partner.
Another prominent feature of (taut-/-) mice is the severe and progressive retinal degeneration. At 2 wk postnatally, the usual age of eye opening, there was still an approximately equal number of rows of photoreceptor nuclei in mutants and wild-type mice. Electrical retinal activity was detectable in the mutants at that stage, but the amplitudes were reduced to about one-third of that of age-matched controls. One week later, ERG amplitudes had dropped to 10–15% of normal. At 1 month of age, photoreceptor segments were largely absent. The ERG signals, even in response to bright stimuli, were close to noise level, and the further morphological deterioration was paralleled by a complete loss of retinal function. These findings indicate that photoreceptor cells initially develop but then undergo degeneration.
Apoptosis is apparently involved in retinal degeneration in (taut-/-) mice, since no macrophages were found in the subretinal space and the photoreceptor cell nuclei were TUNEL positive. In all mammalian species examined so far, high taurine concentrations are found in the retina, with about two-thirds of retinal taurine being localized in the photoreceptor layer. The absence of taurine as a radical scavenger may accelerate the programmed cell death.
The lack of light-induced retinal function and the marked morphological changes in (taut-/-) mice underscore the importance of an intact taurine transport system for the visual system. Studies of cats (for which taurine is an essential amino acid) have shown that a dietary deficiency in taurine leads to progressive retinal degeneration of the cone-rod type.
The importance of taurine for retinal function is underlined by the fact that ERGs are abnormal in cases of human taurine deficiency due to long-term parenteral nutrition. Cats are dietary dependents with regard to taurine. ERGs were also abnormal in cats fed taurine-deficient diets, and outer segment structure was severely disturbed. Mice are not dietary dependents, but their photoreceptor cells are evidently dependent on an intact taurine transport system for maintaining structurally and functionally sufficient levels of intracellular taurine. It remains to be determined whether retinal dystrophy in (taut-/-) mice is directly caused by a lack of taurine or whether taurine deficiency leads to disturbances in the osmotic balance specific to photoreceptor cells.
The pattern of retinal degeneration found in (taut-/-) mice resembles that seen in some forms of human retinitis pigmentosa (RP), a genetically heterogeneous group of hereditary retinal dystrophies. No mutated genes affecting taurine transport or metabolism have yet been identified in RP. Nonetheless, there is a longstanding and ongoing debate as to the role of taurine in RP. Although taurine supplementation in RP patients did not lead to any significant clinical improvement, there are reports of reduced taurine uptake into platelets and lymphoblastoid cells; in some RP patients, severely decreased plasma taurine levels were described. Thus, the present study suggests that taut might be another candidate gene whose mutation may underlie RP in a subgroup of patients.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0691fje; to cite this article, use FASEB J. (December 28, 2001) 10.1096/fj.01-0691fje 
2 These authors contributed equally to this work.
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