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- Materials and methods
Dietary selenium restriction in mammals causes bodily selenium to be preferentially retained in the brain relative to other organs. Almost all the known selenoproteins are found in brain, where expression is facilitated by selenocysteine (Sec)-laden selenoprotein P. The brain also expresses selenocysteine lyase (Scly), an enzyme that putatively salvages Sec and recycles the selenium for selenoprotein translation. We compared mice with a genetic deletion of Scly to selenoprotein P (Sepp1) knockout mice for similarity of neurological impairments and whether dietary selenium modulates these parameters. We report that Scly knockout mice do not display neurological dysfunction comparable to Sepp1 knockout mice. Feeding a low-selenium diet to Scly knockout mice revealed a mild spatial learning deficit without disrupting motor coordination. Additionally, we report that the neurological phenotype caused by the absence of Sepp1 is exacerbated in male vs. female mice. These findings indicate that Sec recycling via Scly becomes limiting under selenium deficiency and suggest the presence of a complementary mechanism for processing Sec. Our studies illuminate the interaction between Sepp1 and Scly in the distribution and turnover of body and brain selenium and emphasize the consideration of sex differences when studying selenium and selenoproteins in vertebrate biology.
Selenium (Se) is an essential dietary micronutrient with antioxidant properties, and the human health consequences of Se deficiency are under extensive study (Rayman 2000). Sex-specific differences are observed in Se status and metabolism, which has complicated this research (Combs et al. 2011; Galan et al. 2005). The functions of Se in biochemical reactions and cellular processes of organisms are principally mediated by selenoproteins that incorporate Se into the amino acid selenocysteine (Sec). Biosynthesis of Sec occurs on its UGA-recognizing tRNA and is catalyzed by selenocysteine synthetase (SepSecS) in the presence of selenophosphate (reviewed by Bellinger et al. 2009). Recoding the UGA stop codon for Sec incorporation requires a Sec-specific elongation factor (EFSec), an mRNA stem-loop termed a Sec insertion sequence (SECIS) and a SECIS-binding protein (SBP2). Among 25 human selenoproteins, the glutathione peroxidase (GPX), thioredoxin reductase and iodothyronine deiodinase families of enzymatic selenoproteins are relatively well characterized and crucial for the health of mammals.
Selenoprotein P (Sepp1) uniquely contains up to 10 Sec residues in primates and rodents. During Se deficiency, brain Se is maintained compared with other organs by tissue-specific uptake of Sepp1 by ApoER2 and Megalin (Burk et al. 2007; Chiu-Ugalde et al. 2010). Mutant mice lacking full-length Sepp1 or the Sec-rich C-terminus show a greater depletion of brain Se than can be achieved through dietary Se deprivation (Hill et al. 2007). Sepp1 gene disruption in mice additionally causes cognitive, motor and sensory symptoms that can be exacerbated by dietary Se restriction and diminished by Se supplementation. These mice present spatial learning deficits, spasticity and hyperreflexia that coincide with deficient synaptic plasticity and widespread neurodegeneration (Caito et al. 2011; Peters et al. 2006; Valentine et al. 2005, 2008).
If endocytosis of Sepp1 delivers Se to cells, then the Sec residues from Sepp1 must be processed for incorporation into selenoproteins. Selenocysteine lyase (Scly) catalyzes the decomposition of Sec into alanine and hydrogen selenide (Esaki et al. 1982) and promotes the production of selenophosphate in the presence of Sec and selenophosphate synthetase (SPS; Tobe et al. 2009). Scly mRNA and protein are expressed in mouse brain (Mihara et al. 2000), where it is posited to recycle Sec from Sepp1 for selenoprotein synthesis (Schweizer et al. 2005).
We hypothesized that Scly liberates Se from Sepp1 in brain and that deletion of Scly in mice would cause similar neurological deficits as observed in Sepp1−/− mice. To test this hypothesis, we assessed whether a novel transgenic mouse strain lacking functional Scly develops a phenotype similar to Sepp1-deficient mice. Here we report that, in contrast to Sepp1−/− mice, Scly−/− mice display few neurological abnormalities. However, spatial learning and selenoprotein expression are sensitive to Scly disruption when the mice are challenged with a low-Se diet. In addition, we extensively characterized sex differences in the behavioral phenotype of Sepp1−/− mice and report that male mice are more dependent on Sepp1 and Se than females for normal brain function.
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- Materials and methods
The results reported herein describe the first characterization of a novel mouse strain deficient in Scly. These mice exhibit minimal neurological deficits, an unexpected finding given the phenotypic effects of Sepp1 knockout and the proposed role of Scly in recycling the essential trace element Se from Sec. Unlike Sepp1−/− animals, deletion of Scly does not result in neuromotor impairments or spatial learning deficits, except under low-Se conditions. We also report sex differences in the motor phenotype of mice with genetic deletion of Sepp1, highlighting the importance of considering gender on studies addressing the biological functions of Se or selenoproteins in mammals.
Sepp1 is a plasma protein that is considered to be the physiological transporter of Se from liver to brain. Sepp1 is additionally found in grey and white matter and cerebrospinal fluid and may store Se within brain (Scharpf et al. 2007). Sepp1 and ApoER2 maintain a high concentration of Se in the testes and prioritize Se to brain albeit at a lower concentration (Burk & Hill 2009). Most tissues produce Sepp1 and Scly, and the whole body turnover rate of Sepp1 is high, fluxing a significant proportion of bodily Se even when dietary availability is limiting (Burk & Hill 2005). Brain Se level is not dependent on hepatic Sepp1 in Se-adequate adult animals (Schweizer et al. 2005). However, Se deficiency directs Se from liver-derived Sepp1 to the brain (Nakayama et al. 2007; Renko et al. 2008). The turnover rate of Sepp1 within the nervous system and the interaction with the circulation are uncertain.
Unlike most trace element transporters, Sepp1 cannot rapidly load and unload cargo because Se is covalently incorporated as the amino acid Sec, which must be degraded to supply Se. Biosynthesis and incorporation of Sec is a protracted, energy intensive process that requires organized interaction of specific proteins (SPS, SepSecS, EFSec and SBP2) and nucleic acids (tRNA(Sec), SECIS-containing mRNA), in addition to adenosine triphosphate, pyridoxal phosphate and the translation machinery. Receptor-mediated uptake by ApoER2 facilitates entry of Sepp1 into cells, but recycling of the Sec residues would depend on the lysosome or proteasome to hydrolyze peptide bonds followed by liberation of Se from Sec, presumably by Scly.
To test the hypothesis that Scly recycles Sec from Sepp1 in the brain, we investigated whether Scly−/− mice manifest a phenotype similar to Sepp1−/− mice. Surprisingly, very little neurological dysfunction was present in the Scly−/− mice, even when fed a diet low in Se. The lack of behavioral changes in Scly−/− mice, compared with Sepp1−/− mice, could be due to Nfs1 catalyzing Sec to selenide conversion for selenoprotein synthesis (Lacourciere et al. 2000). Although we did not detect increased Nfs1 mRNA in Scly−/− mice brains, the normal activity of the enzyme might be compensating for the absence of Scly. Alternatively, Sepp1 may have an acute function in brain not strictly related to Se delivery. Disrupted synaptic plasticity in Se-supplemented Sepp1−/− mice (Peters et al. 2006) supports the notion that Sepp1 has a role in cell signaling via its receptor, ApoER2.
We found that mRNA for Scly was not significantly changed in brain of Sepp1−/− mice fed a standard diet. As Sepp1−/− mice have depressed Se in brain, this finding suggests that Scly is not Se regulated, and the enzyme is minimally affected by dietary Se or tissue Se levels (Deagen et al. 1987). The mRNA expression of Sepp1 trended up in Scly−/− mice fed normal chow and was significantly increased in brain of Scly−/− mice fed low-Se chow. Se-deficient Scly−/− mice also had increased GPX1 and GPX4 mRNA, while that of Sepw1 was unchanged. However, GPX protein and activity in brain of the low-Se Scly−/− mice were dramatically reduced compared with wild-type animals. Liver GPX activity was similarly reduced, while serum activity was less affected. Therefore, Scly supports selenoprotein expression and function under conditions of dietary Se deficiency. Increased GPX mRNA despite reduced protein and activity in the Se-deficient Scly−/− brain could be a compensatory mechanism to boost inefficient selenoprotein translation. These findings extend a recent study, which showed reduced GPX1 expression and reduced incorporation of Se derived from radiolabeled Sepp1, in cells with Scly knocked down by siRNA (Kurokawa et al. 2011). In addition, we observe that tissues are more reliant on Scly than blood.
Our finding that Se-deficient Scly−/− mice manifest a subtle spatial learning deficit in the water maze corresponds with results on Se-supplemented Sepp1−/− mice (Peters et al. 2006). Spatial learning requires the hippocampus, which is more dependent on Sepp1 for optimal Se concentration than other brain regions (Nakayama et al. 2007). Therefore, Scly, Sepp1 and probably other selenoproteins in the hippocampus support spatial learning. It is likely that the kinetics of selenoprotein degradation and synthesis are even more disrupted in Scly−/− mice than the steady-state mRNA and protein expression levels. Brain regions with high metabolism or cellular turnover could be more dependent on a putative Sepp1–Scly recycling mechanism, while other cell populations might efficiently utilize an alternate Se source.
These results are the most extensive characterization of behavioral sex differences in Sepp1−/− mice to date. It has been suggested that the phenotype of Sepp1−/− mice is sex-dependent (Riese et al. 2006); however, studies on Sepp1−/− mice have focused on males and data regarding behavioral sex differences are limited. Despite variations in behavioral testing paradigms, our results showing impaired motor performance in male Sepp1−/− mice are in agreement with previous reports (Hill et al. 2004; Renko et al. 2008; Schweizer et al. 2004). We additionally assessed motor impairment in female Sepp1−/− mice and found it to be minimal compared with males. We also report that the spatial learning deficit in Sepp1−/− mice on a Se-adequate diet is worse than in Se-supplemented mice, building on a previous study that used only male mice on a high-Se diet (Peters et al. 2006).
Selenoprotein expression is modulated by sex in mammals (Meplan et al. 2007; Riese et al. 2006; Stoedter et al. 2010), and Sepp1 is an androgen responsive gene (Takahashi et al. 2006). Additionally, testosterone secretion declines during Se deficiency in male rats (Behne et al. 1996). Male mice displayed increased sensitivity to Sepp1 deletion, and male Sepp1−/− mice greatly improved when given supranutritional dietary Se, indicating that males have a higher demand than females for Sepp1 and Se in the nervous system. However, the phenotype is not exclusive to males, suggesting that some aspect of metabolism or development that is more prominent in males is dependent on Se.
Male gender is a risk factor for poor neurodevelopmental outcome after premature birth. Cerebral palsy and related developmental disorders are more common in males than females (Johnston & Hagberg 2007). The phenotype of Sepp1−/− mice resembles cerebral palsy in that the developmental onset of spasticity and ataxia often presents with intellectual impairment and seizures. Moreover, this phenotype is not rapidly progressive and remains stable in adulthood when given adequate Se. Perinatal infection and hypoxia-ischemia are synergistic risk factors for cerebral palsy (Johnston & Hagberg 2007; Mayoral et al. 2009), while Sepp1 is known to modulate immunity (Bosschaerts et al. 2008) and metabolism (Misu et al. 2010). Metabolically demanding brain regions and cells, with a presumably higher rate of selenoprotein synthesis, are susceptible to neurodegeneration in Sepp1−/− mice (Valentine et al. 2008). Moreover, an autosomal-recessive human disease termed progressive cerebellocerebral atrophy has been linked to mutations in SepSecS, which globally disrupt selenoprotein synthesis (Agamy et al. 2010). The sequelae of these patients, including mental retardation, spasticity and seizures, are similar to those found in Sepp1−/− mice and emphasize the importance of selenoproteins in the function and health of the nervous system.
In conclusion, these results indicate that a novel mouse strain lacking Scly does not develop a neurological phenotype similar to Sepp1−/− mice. A subtle learning deficit is observed when Scly−/− animals are fed a low-Se diet, and these animals also have reduced expression of selenoproteins in brain. We further report a male bias in the neurological motor phenotype of Sepp1−/− mice. The disparity of neurological problems in Scly−/− and Sepp1−/− mice suggests that Sepp1 is more critical than Scly for maintenance of brain Se but that recycling Se from Sec via Scly is physiologically important during dietary Se deficiency. Altogether these findings highlight that Se is critically important for the nervous system and that Se metabolism through Sepp1 and Scly affects spatial learning.