Kostja Renko

Margarethe Werner

Ingrid Renner-Müller

Trevor G Cooper

Ching Hei Yeung

Birgit Hollenbach

Marcus Scharpf

Josef Köhrle

Lutz Schomburg

Dr Ulrich Schweizer
email: ulrich.schweizer@charite.de

Hepatic selenoprotein P (Sepp) expression restores selenium transport and prevents infertility and motor-incoordination in Sepp-knockout mice

Keywords: trace element, brain, glutathione peroxidase, thioredoxin reductase, nutrition, fertility GPx, glutathione peroxidase, Se, selenium, Sec, selenocysteine, SECIS, selenocysteine insertion sequence, SePP, selenoprotein P, Sepp, mouse SePP gene, SEPP1, human SePP gene, Trsp, Secspecific tRNA

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction:

Fifty years ago, selenium (Se) was discovered as an essential trace element [START_REF] Schwarz | Selenium as an integral part of factor 3 against dietary necrotic liver degeneration[END_REF]. Low plasma Se levels have been associated with several disorders, including cancer, autoimmune disease, and others [START_REF] Rayman | The importance of selenium to human health[END_REF]. It is widely assumed that Se exerts its biological functions in the form of selenocysteine (Sec), the 21 st proteinogenic amino acid. This rare amino acid contains a selenol instead of a thiol in the side chain. Sec had initially been overlooked when the genetic code was elucidated, since it is encoded by the "STOP" codon UGA and is present in only a small number of proteins [START_REF] Hatfield | How selenium has altered our understanding of the genetic code[END_REF]. Selenoproteins are essential for mammals. Targeted inactivation of Trsp, the gene encoding the Sec-specific tRNA [Ser]Sec , leads to early embryonic lethality [START_REF] Bosl | Early embryonic lethality caused by targeted disruption of the mouse selenocysteine tRNA gene (Trsp)[END_REF][START_REF] Kumaraswamy | Selective removal of the selenocysteine tRNA [Ser]Sec gene (Trsp) in mouse mammary epithelium[END_REF]. The human and mouse genomes contain 25 and 24 genes encoding selenoproteins, respectively [6]. Gene targeting experiments in mice have shown that at least three of these, thioredoxin reductase 1 and 2 (Txnrd1, Txnrd2) and glutathione peroxidase 4 (GPx4), are essential [START_REF] Jakupoglu | Cytoplasmic thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development[END_REF][START_REF] Conrad | Essential role for mitochondrial thioredoxin reductase in hematopoiesis, heart development, and heart function[END_REF][START_REF] Yant | The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults[END_REF]. Selenoprotein P (SePP) is a plasma selenoprotein containing the unusual number of 8-17 Sec residues, depending on species [START_REF] Burk | Selenoprotein P: an extracellular protein with unique physical characteristics and a role in selenium homeostasis[END_REF]. The majority of plasma Se is bound to SePP [START_REF] Read | Selenium and amino acid composition of selenoprotein P, the major selenoprotein in rat serum[END_REF] and it was suggested very early that it could represent a Se transport protein [START_REF] Motsenbocker | A selenocysteine-containing selenium-transport protein in rat plasma[END_REF]. Targeted inactivation of the Sepp gene in the mouse has demonstrated that SePP is central to Se transport and storage [START_REF] Hill | Deletion of selenoprotein P alters distribution of selenium in the mouse[END_REF][START_REF] Schomburg | Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues[END_REF]. Expression of selenoenzymes such as glutathione peroxidase 1 (GPx1) or Txnrd1 is accordingly reduced in Seppdeficient tissues. Male Sepp -/-mice are infertile [START_REF] Olson | Selenoprotein P is required for mouse sperm development[END_REF] and, depending on dietary Se supply, Sepp -/-mice suffer from premature death before weaning, seizures, and ataxia [START_REF] Schweizer | Efficient selenium transfer from mother to offspring in selenoprotein-P-deficient mice enables dose-dependent rescue of phenotypes associated with selenium deficiency[END_REF][START_REF] Hill | Neurological dysfunction occurs in mice with targeted deletion of the selenoprotein p gene[END_REF]. Recently, the lipoprotein receptor ApoER2 has been characterized as a SePP-receptor in testis [START_REF] Olson | Apolipoprotein E receptor-2 (ApoER2) mediates selenium uptake from selenoprotein P by the mouse testis[END_REF]. However, Sepp mRNA is expressed in most tissues [START_REF] Dreher | Expression of selenoproteins in various rat and human tissues and cell lines[END_REF] including brain, where SePP immunoreactivity has been found on neurons and oligodendroglial cells [START_REF] Scharpf | Neuronal and ependymal expression of selenoprotein P in the human brain[END_REF]. Thus, the relative roles of locally expressed SePP versus circulating SePP remained unclear. When we made use of a recently developed mouse model [START_REF] Kumaraswamy | Selective removal of the selenocysteine tRNA [Ser]Sec gene (Trsp) in mouse mammary epithelium[END_REF][START_REF] Carlson | Specific excision of the selenocysteine tRNA[Ser]Sec (Trsp) gene in mouse liver demonstrates an essential role of selenoproteins in liver function[END_REF] in which biosynthesis of all hepatocytic selenoproteins was specifically abrogated by targeted deletion of the gene encoding tRNA [Ser]Sec , we realized that Se levels in serum and kidneys were reduced to the same levels as those in Sepp -/-mice. Nevertheless, neurological dysfunction was not observed [START_REF] Schweizer | Hepatically derived selenoprotein P is a key factor for kidney but not for brain selenium supply[END_REF].

In a further attempt to define the role of liver-derived plasma SePP, in contrast to locally expressed SePP, we have now generated Sepp -/-mice with liver-specific expression of human SePP (SEPP1). Here we show that transgenic expression of SEPP1 cDNA under control of a mouse transthyretin promoter construct resulted in SEPP1 expression in the liver only. Although serum Se levels increased only slightly in SEPP1-transgenic Sepp -/-mice (Sepp -/-; SEPP1 ), Se levels and selenoenzyme activities in the kidney, testis and brain were normalized, indicating improved Se traffic from liver to target tissues via hepatocytederived human SePP in plasma. In addition, Sepp -/-; SEPP1 mice did not suffer from neurological defects and the males were fertile and survived on Se-sufficient diet. These results suggest that plasma SePP derived from liver is the main transport form for Se supporting vitally important functions of Se in kidney, testis, and brain.

Materials and methods:

Animals: Mice were maintained according to local regulations as described for the Sepp-deficient mice generated in our laboratory [START_REF] Schomburg | Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues[END_REF]. All animal experiments were approved by the local authorities in Berlin and Munich. The SEPP1 minigene was cloned as an XhoI fragment into exon 2 of TTR1 exV3, an expression vector containing a 4 kb fragment of the mouse transthyretin gene including the promoter region, exon 1, intron 1, and exon 2. A cryptic ATG was mutated in exon 1 and a StuI site in exon 2 was used for cDNA insertion [START_REF] Yan | Distinct positive and negative elements control the limited hepatocyte and choroid plexus expression of transthyretin in transgenic mice[END_REF][START_REF] Wu | Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration[END_REF]. The minigene was liberated from the vector by HindIII-digestion, gelpurified, and microinjected into the male pronucleus of C57Bl/6 zygotes. Two oligonucleotides spanning the first SECIS element of human SEPP1, e5-3fwd (TGAATTGGCTTCCAGATTT) and e5-3rev (AGAAGAGTATGATACAACATTAG) produced a 491 bp PCR fragment specific for SEPP1 that was used for PCR-genotyping and as a human-specific hybridization probe. Of the 15 potential founder mice obtained, 4 expressed significant human SePP in serum biopsies (#5, #8, #9 and #12). Founder #8 died before reproduction and offspring from founder #12 did not express human SePP in serum, although the strain remained PCR-positive for SEPP1. Thus, two SEPP1-transgenic lines were established from founder mice #5 and #9, and were designated Tg(SEPP1)1 and Tg(SEPP1)3. All experiments reported here were performed with animals from Tg(SEPP1)1, unless otherwise indicated. Animals received a torula yeast-based diet with 0.16-0.2 mg Se/kg in the form of sodium selenite (Sniff, Hannover,

Germany).

RNA extraction: Tissue was snap-frozen in liquid nitrogen and stored at -80°C until use. For all subsequent sample preparations the tissue was powdered and homogenized under liquid nitrogen.

Total RNA extraction was performed using peqGOLD TriFast (PEQLAB, Erlangen, Germany) according to the instructions of the manufacturer. Isolated total RNA integrity was electrophoretically verified by ethidium bromide staining as described [START_REF] Schomburg | Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues[END_REF].

Semiquantitative RT-PCR: 1 µg of total RNA was reverse transcribed (iScript first strand cDNA synthesis kit, BioRad) and the resulting cDNA was diluted 1:10. 5µl of cDNA-dilution were taken for every single RT-PCR reaction. Absolute QPCR SYBR Green Fluoresceine Mix (Abgene, Epsom, UK) and the iCycler-System (BioRad) were used for transcript quantification.

All reactions were performed with an annealing temperature of 58°C and under standard conditions. Specificity of used primers was verified by melting curve analysis. Each sample was measured in duplicates. Quantitative data were calculated using the Relative Expression Software Tool-Multiple Condition Solver (REST-MCS, version 2, as previously described [START_REF] Pfaffl | Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR[END_REF]). Transgene-specific, intronspanning primers used were TTR-fwd1 (AGATCCACAAGCTCCTGACAGGATC) and SEPP1-rev2 Northern Blot: cDNA fragments encompassing the complete reading frame of murine SePP, and a fragment of the human SEPP1 3'UTR were amplified by PCR, subcloned into pGEM-T vectors (Promega, Mannheim, Germany) and verified by DNA-sequencing. The insert was prepared by restriction digest and preparative gel electrophoresis, radioactively labeled with 32P-dCTP (Hartmann Analytic, Braunschweig, Germany) using a random priming kit (High Prime, Roche Diagnostics GmbH, Mannheim, Germany). Hybridizations were conducted essentially as described [START_REF] Schomburg | Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues[END_REF] and specific signals were quantified employing a phosphoimager (Cyclone, Perkin Elmer, Köln, Germany).

Enzyme assays: GPx activity was determined in tissue homogenates as described [START_REF] Schomburg | Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues[END_REF]. In brief, NADPH consumption by glutathione reductase was monitored at 340 nm as a measure of the GSSG formation catalyzed by GPx. Enzymatic activity was recorded at 37°C in a buffer containing 0.02 M potassium phosphate, pH 7.0, 0.6 mM EDTA, 0.15 mM NADPH, 2 mM GSH and 4 units glutathione reductase (Calbiochem, Darmstadt, Germany). The reaction was started by addition of t-butylhydroperoxide (0.1 mM) as substrate. Background NADPH consumption was determined in the presence of 100 mM mercaptosuccinate and subtracted. Protein concentration was determined by the method of Bradford using IgG as a standard.

Selenium determination: Selenium measurements were performed by a fluorimetric assay as described [START_REF] Schweizer | Efficient selenium transfer from mother to offspring in selenoprotein-P-deficient mice enables dose-dependent rescue of phenotypes associated with selenium deficiency[END_REF]. A commercial Se standard (Aldrich #247928, Sigma, Munich, Germany) was used to construct a calibration curve and an independent seronorm standard (Sero AS, Billingstad, Norway) served as reference sample.

Rotarod: Rotarod analysis was done as described [START_REF] Schweizer | Efficient selenium transfer from mother to offspring in selenoprotein-P-deficient mice enables dose-dependent rescue of phenotypes associated with selenium deficiency[END_REF]. Animals were placed on the rotating drum at 4 rounds per min (rpm). After two min of accomodation, the rotation was linearly accelerated during 3 min to 40 rpm. The retention time spent on top of the rotating drum was recorded.

Western Blot: For GPx1 and GPx4 detection in brain, 100µg protein from cytosolic or membrane fraction, respectively, were separated in 12 % SDS-polyacrylamide gels. For GPx4 from testis, 50µg of membrane fractions were used. After electrotransfer, PVDF membranes were stained with Ponceau Red, photographed, and blocked with 5% BSA for 1 h at room temperature. Polyclonal antibodies against GPx1 and GPx4 (both from Abcam) were used at 1:1000 and 1:2000 dilution, respectively. Rabbit polyclonal anti--actin antiserum from Sigma was used at 1:1000 dilution. Antibodies against mouse SePP have been described [START_REF] Schweizer | Hepatically derived selenoprotein P is a key factor for kidney but not for brain selenium supply[END_REF]. Antibodies against human SePP were the same as used in the ELISA assay and described before [START_REF] Dumitrescu | Mutations in SECISBP2 result in abnormal thyroid hormone metabolism[END_REF].

S t a g e 2 ( a ) P O S T -P R I N T

Results

Generation of mice expressing human SePP specifically in liver

In order to complement Sepp -/-mice genetically, we wanted to express human SePP specifically in hepatocytes. The mouse transthyretin promoter has been successfully used to mediate liver-specific expression of cDNAs [START_REF] Wu | Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration[END_REF]. Thus, a human SEPP1 cDNA including the complete open reading frame and the two SECIS elements in the 3'-UTR were cloned into exon 2 of the vector TTR exV3 and the resulting minigene (Fig. 1A) was microinjected into mouse zygotes. Potential founder mice were genotyped via PCR for the presence of the human SEPP1 3'-UTR (Fig. 1B). In the next step, founders were screened for human SePP in blood samples using a recently developed human-specific SePP ELISA. Since our antisera are species-specific, we were able to test for human SePP in mice in the presence of the wild-type murine orthologue (Fig. 1C). Four founder mice (#5, #8, #9 and #12) turned out positive for human SePP in the murine serum samples. Next, we asked whether SEPP1 expression was confined to liver only. Therefore, we tested the species-specificity of our hybridization probes and verified transgene cDNA in offspring from founder mouse #5 (from now on designated Tg(SEPP1)1 and mouse #9 (now designated Tg(SEPP1)3 (Fig. 1D). Next, we used the human-specific probe to test SEPP1 expression in several tissues of the transgenic mice. We tested 16 tissues by Northern blotting in both transgenic lines (Fig. 1E).

In Tg(SEPP1)1, the liver was the only tissue expressing SEPP1. In Tg(SEPP1)3, low mRNA expression of SEPP1 was also observed in skeletal muscle. The transgenic SEPP1 mRNA from mouse liver migrated slightly slower than native human SEPP1 mRNA from the hepatoma cell line HepG2 providing an additional level of verification and specificity.

Selenium transport is restored by transgenic SEPP1 expression

Transgenic founders were crossed with Sepp +/-mice yielding Sepp +/-; SEPP1 offspring. These were crossed with Sepp +/-mice generating SEPP1-transgenic Sepp -/-mice (Sepp -/-; SEPP1 ). Serum samples from these mice were tested for transgene expression by Western blot. Using our human-specific antibodies, we detected full-length expression of human SePP in serum of transgenic mice, albeit at lower levels compared with human control serum (Fig. 2A). Although human SePP expression appeared lower in Tg(SEPP1)1 mice, this strain was selected for further experiments, since transgene expression was strictly limited to the liver (see Fig. 1E). In order to exclude gene-dose effects, we made sure that in subsequent matings only one parent was SEPP1-transgenic. In the next step we wanted to know whether expression of the SEPP1 transgene increased serum Se content in Sepp -/-mice. Therefore, we determined serum Se levels in all six genotypes of our mice. Interestingly, SEPP1 transgene expression doubled serum Se concentrations in Sepp -/-mice, but did not significantly affect Se levels in wild-type or heterozygous mice (Fig. 2B).

We have shown previously that renal Se content and selenoenzyme activities depend on SePP secreted from the liver [START_REF] Schweizer | Hepatically derived selenoprotein P is a key factor for kidney but not for brain selenium supply[END_REF]. We now wanted to know whether transgenic expression of human SePP exclusively in hepatocytes was able to increase Se content and selenoenzyme activities in SePP target tissues from Sepp -/- mice. As shown in figure 3, kidney Se content was significantly increased in SEPP1-transgenic compared with non-transgenic Sepp -/-mice (Fig. 3A). Similarly, renal GPx activity, a sensitive marker of tissue Se status, paralleled Se content and demonstrated partial restoration of selenoenzyme activity in Sepp -/-; SEPP1 mice (Fig. 3B). Moreover, cytosolic Txnrd activity was normalized in Sepp -/-kidneys by SEPP1 expression (not shown). Interestingly, activity of plasma GPx, an enzyme produced in the kidney and secreted into the blood was not restored in Sepp -/-; SEPP1 mice (Fig. 3C). This is in line with the wellestablished hierarchy of expression among selenoproteins under conditions of limited Se availability.

Restoration of male fertility in SEPP1 transgenic Sepp knockout mice

The testis is another SePP target tissue. Its Se content is reduced in Sepp -/-mice causing infertility [START_REF] Hill | Deletion of selenoprotein P alters distribution of selenium in the mouse[END_REF][START_REF] Schomburg | Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues[END_REF].

Recently it has been shown that the lipoprotein receptor ApoER2 functions as a SePP receptor in testis [START_REF] Olson | Apolipoprotein E receptor-2 (ApoER2) mediates selenium uptake from selenoprotein P by the mouse testis[END_REF]. However, since Sepp mRNA is also expressed in the testis [START_REF] Steinert | Analysis of the mouse selenoprotein P gene[END_REF], it is unclear whether ApoER2 can bind SePP from the circulation only or exclusively from within the testis.

Male infertility is associated with reduced testicular GPx4 expression during dietary Se-deficiency [START_REF] Ursini | Dual function of the selenoprotein PHGPx during sperm maturation[END_REF][START_REF] Olson | Sequential development of flagellar defects in spermatids and epididymal spermatozoa of selenium-deficient rats[END_REF], Sepp-deficiency [START_REF] Olson | Selenoprotein P is required for mouse sperm development[END_REF], or Apoer2-deficiency [START_REF] Andersen | Essential role of the apolipoprotein E receptor-2 in sperm development[END_REF][START_REF] Olson | Apolipoprotein E receptor-2 (ApoER2) mediates selenium uptake from selenoprotein P by the mouse testis[END_REF]. We have therefore determined GPx4 protein abundance in testis homogenates from our mice. As expected, GPx4 protein was lost from Sepp -/-testis and the SEPP1 transgene partially restored GPx4 expression (Fig. 4A). We determined GPx activity as a reliable surrogate marker of tissue Se content in the testis. The results were in line with our earlier findings that indicated reduced GPx activity in Sepp -/-testis [START_REF] Schomburg | Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues[END_REF]. Hepatic expression of SEPP1 fully restored Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2007 The Authors Journal compilation © 2007 Biochemical Society testicular GPx activity (Fig. 4B). Next, we performed qRT-PCR analysis with transgene-specific primers as indicated in figure 1A. No difference was observed in threshold cycle times whether the animals were SEPP1-transgenic or non-transgenic, ruling out ectopic expression of the SEPP1 transgene in the testis (Fig. 4C). In another attempt to test for ectopic testicular SEPP1-transgene expression, we stained tissue sections with an antibody specific for human SePP. Using this method, we were able to demonstrate human SePP in Leydig cells of normal human specimens, but not in SEPP1-transgenic murine testes (not shown).

Spermatozoa from the transgenic mice were almost as motile as those from the wild-type mice, swimming with the same vigour and attaining 90% the effectiveness of forward progression of the wild-type cells.

The Sepp -/-mice had much fewer motile sperm that showed markedly reduced swimming velocities and forward progression (Fig. 5A). Sperm motility was also increased by the SEPP1 transgene to levels indistinguishable from wild-type sperm. Morphological assessment of spermatozoa from the cauda epididymis showed that SEPP1 transgene expression prevented the formation of "hairpin-like" turns of the sperm tails (Fig. 5B). Sepp -/-mice maintained on a high Se diet (0.6 ppm) served as controls, since Sepp -/- mice maintained on Se adequate diet did not survive to reproductive age. For quantitative analysis, spermatozoa were fixed and processed with the Papanicolaou method (4 slides per animal, 200 spermatozoa were assessed). The fraction of spermatozoa with angulated flagella was 52.6±1.4% for Sepp - /-, 17.1±2.0% for Sepp +/+ , and 13.5±1.9% for Sepp -/-; SEPP1 (Sepp -/-was different from both other groups as assessed by ANOVA). Consistent with the biochemical, kinematic, and morphological rescue of their sperm phenotype, Sepp -/-; SEPP1 male mice were fertile as demonstrated by normal litter size when mated to Sepp +/-dams.

Rescue of neurological dysfunction in SEPP1-transgenic Sepp knockout mice

Gene targeting of Sepp using homologous recombination for the first time clearly revealed a role for Se in brain function [START_REF] Schweizer | Selenium and brain function: a poorly recognized liaison[END_REF]. Lack of Sepp leads to a reduction of brain cytosolic Gpx and Txnrd activities [13,14,].

Upon transgenic expression of SEPP1 in the liver, a significant increase in brain GPx activity in Sepp -/- mice is observed (Fig. 6A). In parallel, the increase of serum Se concentration in response to expression of SEPP1 is rather low (see Fig. 2B). These data support the idea that plasma SePP represents the preferred carrier of Se into the brain. Activity of Txnrd, an enzyme that occupies a high rank in the hierarchy among selenoproteins, was fully rescued in SEPP1 transgenic Sepp -/-brains (Fig. 6B). Similarly, expression of the strongly reduced GPx protein levels in Sepp -/-mice were partly (in the case of GPx1) or largely (GPx4)

restored by hepatic expression of SEPP1 (Fig. 6C). This differential rescue of GPx isoform expression is again well in agreement with the well-established hierarchical expression of different selenoproteins.

We have reported earlier that Sepp -/-mice suffer from movement incoordination that can be quantitatively assessed using the Rotarod apparatus [START_REF] Schweizer | Efficient selenium transfer from mother to offspring in selenoprotein-P-deficient mice enables dose-dependent rescue of phenotypes associated with selenium deficiency[END_REF]. When Sepp -/-mice were tested now along with SEPP1- expressing Sepp -/-littermates, Sepp -/-;SEPP1 mice behaved indistinguishable from wild-type mice (Fig. 7).

When Sepp -/-mice are challenged after weaning with a low Se diet, they rapidly deteriorated and died within 7-10 days on low Se diet [START_REF] Hill | Neurological dysfunction occurs in mice with targeted deletion of the selenoprotein p gene[END_REF]. Therefore, we challenged a small number of SEPP1-transgenic Sepp knockouts along with Sepp -/-littermates with a low Se diet (0.06 mg Se/kg). In contrast to their nontransgenic littermates, Sepp -/-;SEPP1 mice survived 4 weeks longer, but ultimately developed the usual Sepp - /-phenotype, including neurological dysfunction. We thus selected a more physiological setting and provided Se sufficient diet (0.16-0.2 mg Se/kg) after weaning. Again, we followed the development of the Sepp -/-phenotype. Under these conditions, Sepp -/-mice developed the neurodegenerative phenotype and had to be euthanized 2 weeks after diet change, while none of the Sepp -/-;SEPP1 mice showed any signs of neurological damage during the full observation period of 12 weeks after diet change (Fig. 8).

Discussion

Soon after the discovery of SePP, it was postulated that SePP might represent a Se transport protein in plasma [START_REF] Motsenbocker | A selenocysteine-containing selenium-transport protein in rat plasma[END_REF]. SePP is the only mammalian selenoprotein known to date that contains multiple Sec residues in the polypeptide chain [START_REF] Hill | The cDNA for rat selenoprotein P contains 10 TGA codons in the open reading frame[END_REF]. It is highly expressed in liver, and injection of radioactively labelled SePP into Se-deficient animals leads to accumulation of radioactivity in particular in the brain [START_REF] Read | Selenium and amino acid composition of selenoprotein P, the major selenoprotein in rat serum[END_REF]. However, expression of Sepp mRNA is widespread among tissues and sometimes specific for only one cell type within the organ, e.g. for Leydig cells in the testis [START_REF] Steinert | Analysis of the mouse selenoprotein P gene[END_REF]. Thus, the question remained, why so many tissues should express a plasma Se transport protein, if this was the only function of SePP. It has been proposed

that SePP detoxifies heavy metals [START_REF] Yoneda | Equimolar Hg-Se complex binds to selenoprotein P[END_REF] and protects proteins from oxidation [START_REF] Arteel | Protection by selenoprotein P in human plasma against peroxynitrite-mediated oxidation and nitration[END_REF], but both hypotheses remain to be supported in vivo. Gene targeting of Sepp in mice has revealed that SePP is required for Se distribution within the body [START_REF] Hill | Deletion of selenoprotein P alters distribution of selenium in the mouse[END_REF][START_REF] Schomburg | Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues[END_REF]. In addition, an essential role for SePP in privileged Se supply to the brain was established. Apparently, the top positions of brain and testes in the hierarchy of Se supply to organs [START_REF] Behne | Evidence for specific selenium target tissues and new biologically important selenoproteins[END_REF] were lost in Sepp -/-mice. Gene targeting of Sepp also demonstrated for the first time that Se was important for normal brain function [START_REF] Schweizer | Efficient selenium transfer from mother to offspring in selenoprotein-P-deficient mice enables dose-dependent rescue of phenotypes associated with selenium deficiency[END_REF][START_REF] Hill | Neurological dysfunction occurs in mice with targeted deletion of the selenoprotein p gene[END_REF]. Interestingly, reducing SePP expression in the liver through targeted deletion of tRNA [Ser]Sec , revealed that the brain is able to retain its Se provided local SePP expression is maintained [START_REF] Schweizer | Hepatically derived selenoprotein P is a key factor for kidney but not for brain selenium supply[END_REF]. Likewise, in dietary Se deficiency, liver SePP secretion is lost, while the brain maintains its Se content [START_REF] Behne | Evidence for specific selenium target tissues and new biologically important selenoproteins[END_REF]. Interestingly, SePP has been purified from serum as a neurotrophic factor for cortical neurons in culture [START_REF] Kaufman | Serum factor supporting long-term survival of rat central neurons in culture[END_REF][START_REF] Yan | Purification from bovine serum of a survival-promoting factor for cultured central neurons and its identification as selenoprotein-P[END_REF]. Therefore, it is possible that SePP expression not only provides a Se transport protein, but also generates a local (neuro)trophic molecule. We set out to test our hypothesis of a dual function of SePP [START_REF] Schomburg | Selenium and selenoproteins in mammals: extraordinary, essential, enigmatic[END_REF] by expressing SePP exclusively in liver of Sepp -/-mice. Here, we report that the selective transgenic expression of human SePP in hepatocytes only can already restore selenoprotein expression in known SePP target tissues and rescues male fertility, motor coordination, and survival of Sepp -/-mice. These findings lend further support to the hypothesis that SePP is an essential Se transport protein from liver to target tissues. However, upon severe dietary Se restriction, the neurological If SePP represented a transport protein, a receptor for SePP likely exists. This receptor should be preferentially expressed in SePP target tissues. In fact, ApoER2 has recently been biochemically purified as a SePP receptor from testis [START_REF] Olson | Apolipoprotein E receptor-2 (ApoER2) mediates selenium uptake from selenoprotein P by the mouse testis[END_REF]. In support of this, Apoer2-deficient mice are infertile [START_REF] Andersen | Essential role of the apolipoprotein E receptor-2 in sperm development[END_REF][START_REF] Olson | Selenoprotein P is required for mouse sperm development[END_REF] and their sperm phenotype resembles Sepp -/-spermatozoa with angulated flagella [START_REF] Andersen | Essential role of the apolipoprotein E receptor-2 in sperm development[END_REF]. Very recently, the group of Burk extended their findings and nicely demonstrated that Apoer2 -/-mice have reduced testis and brain Se content and display neurological defects similar to those in Sepp -/-mice [START_REF] Burk | Deletion of apolipoprotein E receptor-2 in mice lowers brain selenium and causes severe neurological dysfunction and death when a low-selenium diet is fed[END_REF]. Thus, biochemical and genetic evidence strongly suggest that ApoER2 is the postulated SePP receptor in the brain and testis.

Interestingly, GPx4 expression is more strongly reduced in testicular homogenates than GPx1 activity. It is known that GPx4 resides higher in the hierarchy among selenoproteins than GPx1. We thus speculate that the two proteins do not reside within the same cellular compartment with respect to Se supply, such that GPx4 expression is strictly Sepp/Apoer2-dependent, while GPx1 is expressed both in Apoer2dependent and -independent cells in testis. Detailed histological analyses of selenoprotein expression in testicular cell types may thus be required to explain this finding fully. Nevertheless, the restoration of sperm motility and normal sperm morphology in the transgenic mice along with rescued fertility argues for rescued GPx4 expression in spermatozoa, since spermatozoa with angulated flagella do not cross the uterotubal junction in mice [START_REF] Yeung | The cause of infertility of male c-ros tyrosine kinase receptor knockout mice[END_REF].

Expression of ApoER2 is not reported in the kidney, a clear SePP target tissue, and kidney Se is not reduced in Apoer2 -/-mice [START_REF] Burk | Deletion of apolipoprotein E receptor-2 in mice lowers brain selenium and causes severe neurological dysfunction and death when a low-selenium diet is fed[END_REF]. Thus, preferential Se supply to the testis and brain is reflected by ApoER2 expression in these organs, but this receptor may not represent the only SePP receptor.

In addition, we have shown here that liver-specific SEPP1 expression results in increased Se content in the brain and rescues the neurological deficits of Sepp -/-mice. Although at first glance this finding supports the transport hypothesis for SePP, new questions have been raised: how does Se contained in SePP cross the blood-brain-barrier (BBB)? Both brain endothelium and neurons are known to express ApoER2, but it is not clear whether SePP taken up at the BBB is transcytosed or whether Se derived from plasma SePP after intracellular degradation is used for de novo synthesis of SePP or another specific Se carrier protein behind the BBB. Since SePP-resynthesis at the BBB is unlikely in Sepp -/-;SEPP1 mice, our results favour transcytosis or synthesis of a second Se carrier as the transport mechanism of Se into the brain. Available data from Apoer2 -/-mice are compatible with both lack of SePP uptake at the BBB and lack of SePP uptake by neurons. It is now clear that dietary (inorganic) Se supplementation restores motor coordination in Sepp -/-mice [START_REF] Schweizer | Efficient selenium transfer from mother to offspring in selenoprotein-P-deficient mice enables dose-dependent rescue of phenotypes associated with selenium deficiency[END_REF], but not testicular Se content and normal sperm morphology. Thus, unlike the testis, the brain can make use of inorganic dietary Se sources to fulfill its essential functions. This notion is also substantiated by the finding that Apoer2 -/-mice do not show the neurological deficits of Sepp -/-mice, as The phenotypic similarities of Apoer2 -/-and Sepp -/-mice suggest that the main function of SePP in vivo is Se transport and storage -otherwise, phenotypic differences between mice lacking ligand and mice lacking receptor should be apparent. Since the phenotype of mice expressing a C-terminally truncated SePP fragment instead of full-length SePP is very similar to Sepp -/-mice [START_REF] Hill | The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium[END_REF], the current in vivo data do not favour a third prominent function of SePP.

What is the role of Se in the brain? It has been suggested that intractable seizures in children with low Se status depend on Se [START_REF] Weber | Glutathione peroxidase deficiency and childhood seizures[END_REF][START_REF] Ramaekers | Selenium deficiency triggering intractable seizures[END_REF]. Considering these data, it may be possible that children expressing low levels of SEPP1 of APOER2 in the brain may be particularly sensitive to low dietary Se intake. In fact, polymorphisms in the SEPP1 gene have recently been reported to impact on SePP biosynthesis and metabolism in humans [START_REF] Meplan | Genetic polymorphisms in the human selenoprotein P gene determine the response of selenoprotein markers to selenium supplementation in a gender-specific manner (the SELGEN study)[END_REF]. Thus it appears as if a peculiar system composed of SePP and ApoER2 is designed to channel Se selectively into brain and testis and thus promote survival of the individual and the species during times of Se shortage.

Acknowledgements:

The authors gratefully acknowledge the technical assistance of Antje Kretschmer, Vartitér Seher, Silke Kappler, and Tamara Hohly. We thank Dr. Terry VanDyke, Chapel Hill, for providing the TTR promoter construct. Funding for this study was provided by the Deutsche Forschungsgemeinschaft KO 922/8-3 SCHO 849/1-1, Zukunftsfonds EnForCé, and Deutsche Krebshilfe (10-1792 SchoI). (A) Kinematic data of motile spermatozoa isolated from the cauda epididymis. Liver-specific SEPP1 expression in Sepp -/-mice partially restores sperm swimming velocity and linearity of progression.

Motility in % (range) was 69 (66-72) for Sepp +/+ , 18 [START_REF] Schomburg | Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues[END_REF][START_REF] Olson | Selenoprotein P is required for mouse sperm development[END_REF][START_REF] Schweizer | Efficient selenium transfer from mother to offspring in selenoprotein-P-deficient mice enables dose-dependent rescue of phenotypes associated with selenium deficiency[END_REF][START_REF] Hill | Neurological dysfunction occurs in mice with targeted deletion of the selenoprotein p gene[END_REF][START_REF] Olson | Apolipoprotein E receptor-2 (ApoER2) mediates selenium uptake from selenoprotein P by the mouse testis[END_REF][START_REF] Dreher | Expression of selenoproteins in various rat and human tissues and cell lines[END_REF][START_REF] Scharpf | Neuronal and ependymal expression of selenoprotein P in the human brain[END_REF] for Sepp -/-, and 57 (41-72) for Sepp -/-;SEPP1 ,, respectively. * different from wild type; § different from wild type and transgenic mice as calculated by

One Way ANOVAR on Ranks followed by Dunn's test on all pair-wise comparisons. (B) Morphology of spermatozoa isolated from the cauda epididymis. Morphology was assessed by phase contrast microscopy in Sepp +/+ , Sepp -/-, and Sepp -/-;SEPP1 mice. Hepatic expression of SEPP1 rescues the structural defects of spermatozoa from Sepp -/-mice. The percentage of angulated flagella ("hairpin" spermatozoa; white arrows) in Sepp -/-;SEPP1 mice (13.5%) was similar to that in wild-type Sepp +/+ mice (17.1%), whereas the percentage of aberrant spermatozoa was 52.6% in non-transgenic Sepp -/-mice. Scale bars, 40µm (upper panels), 10µm (lower panels). Motor coordination was quantified with the Rotarod apparatus. When compared with Sepp +/+ mice (n=13), movement coordination is completely normal in Sepp -/-;SEPP1 mice (n=9), while Sepp -/-mice are not able to remain for more than one min on the rotating drum (n=14). Three Sepp -/-mice completely unable to remain on the rotating drum for one revolution were excluded from the analysis. Rotarod analysis was done on postnatal day 35.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 26 Oct 2007 as manuscript BJ20071172 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071172 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society (GGCTGGGGGTTGCTTACATAAG). 18S-rRNA primers from RTPrimerDB (http://medgen.ugent.be/rtprimerdb/) were used for normalization: Fw-m18S-TTGACGGAAGGGCACCACCAG and Rev-m18S-GCACCACCACCCAC GGAATCG.

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 26 Oct 2007 as manuscript BJ20071172 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071172 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 26 Oct 2007 as manuscript BJ20071172 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071172

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 26 Oct 2007 as manuscript BJ20071172 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071172 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 26 Oct 2007 as manuscript BJ20071172 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071172 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society phenotype of Sepp -/-mice develops even in SEPP1-transgenic mice, supporting in turn the postulated second role of SePP as a local Se storage protein in brain [39,40].

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 26 Oct 2007 as manuscript BJ20071172 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071172 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society long as they are not challenged by low Se diet [41,31]. It remains to be shown which chemical species and which mode of transport establishes this alternative route of Se entry into the brain.

Figure legends:Figure 1 :

 1 Figure legends: Figure 1: Generation of SEPP1 transgenic mice (A) Schematic representation of the transgene construct. A human SEPP1 cDNA including the 3'-UTR with both SECIS elements was inserted as an XhoI fragment into exon 2 of pTTR exV3 vector. The human-specific hybridization or PCR probe (491 bp) spanning the first SECIS element is indicated. The location of primers used for qRT-PCR is indicated as arrows. (B) Transgenic founder mice were screened by genomic PCR with primers spanning the first human SECIS element. (C) Expression of transgenic SePP in serum of founder animals was assessed by human-specific ELISA assay. Only 4 out of 15 mice expressed detectable amounts of SePP in serum. RLU, relative light units. (D) Specificity of hybridization probes. Northern hybridization of total liver RNA with probes directed against mouse Sepp (Sepp), human Sepp (SEPP1), and 18S rRNA. (E) Multiple tissue Northern blot analysis. 10-15 µg of total RNA from 16 tissues were prepared from F2 animals from lines Tg(SEPP1)1 and Tg(SEPP1)3. Transgenic mRNA was visualized with the SEPP1-specific probe. 18S rRNA served as loading control. Controls: human hepatoma HepG2 cells; Ht heart; Li liver; Ki kidney; Lu lung; Mu skeletal muscle; Ctx cerebral cortex; Crb cerebellum; Sp spleen; Th thymus; Co colon; Du duodenum; Cae caecum; St stomach; Ut uterus; Sa salivary gland; Pa pancreas.

Figure 2 :

 2 Figure 2: Human SePP is secreted into mouse serum. (A) Western blot on serum samples using antibodies specific for mouse and human SePP, respectively. Human serum (hum.) and serum from a non-transgenic Sepp +/+ mouse (+/+) served as controls. Human SePP from transgenic mouse serum co-migrates with SePP from regular human serum. Genotypes are indicated at the bottom. Tg1, Tg3, transgenic lines Tg(SEPP1)1 and Tg(SEPP1)3, respectively. The sizes of marker proteins are indicated at the right. One representative of three individual experiments is shown. (B) Serum Se content in transgenic mice. Expression of the SEPP1 transgene leads to significantly increased Se levels in Sepp -/-mice (n=5-17 for each genotype). Statistical significance versus wild-type was calculated with ANOVA followed by Bonferroni's post-hoc test. The horizontal bar indicates the comparison between Sepp -/-and Sepp -/-;SEPP1 . **p<0.01;***p<0.001.

Figure 3 :

 3 Figure 3: Se transport to kidney is partially restored in Sepp -/-by liver-specific expression of SEPP1. (A) Kidney Se content. Se content is significantly increased in Sepp -/-;SEPP1 kidneys as compared to Sepp -/- kidneys, n=5-16. (B) Cytosolic GPx activity. GPx activity, a sensitive marker of tissue Se status, is increased in Sepp -/-kidney by transgenic expression of SEPP1in liver, n=10-17. (C) Plasma GPx activity. GPx activity in serum is not increased in Sepp -/-;SEPP1 mice as compared to Sepp -/-, n=7-15. Error bars indicate SEM. Statistical significance of differences between Sepp -/-and Sepp -/-;SEPP1 was calculated with ANOVA followed by Dunnett's post-hoc test as indicated by the horizontal bars. The difference between S t a g e 2 ( a ) P O S T -P R I N T

Figure 4 :

 4 Figure 4: Hepatic SEPP1 expression restores fertility in Sepp -/-;SEPP1 mice. (A) GPx4 protein in testis. GPx4 expression in testis correlates with fertility of male mice. Western Blot demonstrates partial restoration of GPx4 expression in Sepp -/-; SEPP1 testis. -actin ( -Act.) served as loading control. Samples from three different animals were loaded per genotype and one representative of two experiments is shown. (B) Cytosolic GPx activity in testis homogenates. GPx activity, a sensitive probe of Se content, is restored in Sepp -/-;SEPP1 mice, n=3 per genotype. Statistical significance was calculated with ANOVA followed by Dunnett's post-hoc test as indicated by the horizontal bars. *p<0.05.(C) qRT-PCR with testis homogenates. The SEPP1 transgene is not expressed in testis from transgenic mice. Threshold cycle numbers (C T ) using testicular cDNAs are not different between SEPP1-transgenic and non-transgenic mice. cDNA from SEPP1-transgenic liver served as a standard in serial dilutions as indicated. 18S-rRNA served as control.

Figure 5 :

 5 Figure 5: Analysis of sperm phenotype.

Figure 6 :

 6 Figure 6: Brain selenoprotein expression is increased in SEPP1-transgenic Sepp -/-mice. (A) Cytosolic GPx activity. Hepatic expression of SEPP1 partially restores brain GPx activity in Sepp -/- ;SEPP1 mice as compared to Sepp -/-mice, n=10-15. (B) Cytosolic Txnrd activity. Brain Txnrd activity in Sepp -/-mice is less affected than brain GPx activity in Sepp -/-mice. Txnrd activity in brains of Sepp -/-mice is fully restored upon hepatic SEPP1 expression, n=9-13. Error bars indicate the SEM. Statistical significance was calculated with ANOVA followed by Dunnett's post-hoc test as indicated by the horizontal bars. *p<0.05; **p<0.01. (C) Western blot analysis of GPx1 and GPx4 expression. GPx1 S t a g e 2 ( a ) P O S T -P R I N T

Figure 7 :

 7 Figure 7: Motor impairment of Sepp -/-mice is prevented by hepatic SEPP1 expression.

Figure 8 .Figure 8 Sepp

 88 Figure 8. Rescue of neurological dysfunction of Sepp -/-by the SEPP1 transgene depends on dietary Se availability.Sepp -/-;SEPP1 mice survive without apparent phenotype for at least 12 weeks on Se-sufficient diet, but develop the neurological phenoytpe with atactic gait and occasional seizures after 4 weeks on Se-deficient diet. Mice were maintained either on Se-sufficient diet (RDA; 0.16-0.2 mg Se/kg) or on Se-deficient diet (low Se; 0.06 mg Se/kg) and a Kaplan-Meier plot was created demonstrating the time at which mice had to be euthanized, because of their severe neurological phenotype. Numbers of animals: low Se, Sepp -/- ;SEPP1 (n=5); Sepp -/-(n=9); Sepp +/+ (n=14). Adequate Se, Sepp -/-;SEPP1 (n=13); Sepp -/-(n=14); Sepp +/+ (n=32).S t a g e 2 ( a ) P O S T -P R I N T

  A human-specific SePP ELISA based on polyclonal sheep antisera was established and is described in detail elsewhere (B. Hollenbach, N.G. Morgenthaler, J. Struck, C. Alonso, A. Bergmann, J.

	Biochemical Journal Immediate Publication. Published on 26 Oct 2007 as manuscript BJ20071172
	SePP ELISA: Köhrle, and L. Schomburg, unpublished work).
	Sperm motility and morphology: Spermatozoa from the cauda epididymis were released into medium
	mimicking the osmolality of uterine fluid (330 mmol/kg) containing 12 mg/ml bovine serum albumin.
	After dispersion, motility was examined at 37°C in 40 µm deep siliconised slide chambers and video-
	recorded for analysis of kinematics using a computer system (IVOS version 10.8, Hamilton Thorne
	Research, Beverly, MA, USA). Motile spermatozoa were tracked for 1 s at 25 Hz and swimming
	velocities and forward progression efficiencies were measured [27]. Aliquots of this suspension were
	photographed in a Zeiss BX-40 microscope with phase contrast optics and assessed for the percentage of
	angulated flagella. Statistics: For all computations, Graph Pad Prism software was used with the tests indicated in the figure legends. Error bars represent the standard error of the mean, SEM.	THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071172
	Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.
	© 2007 The Authors Journal compilation © 2007 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2007 The Authors Journal compilation © 2007 Biochemical Society