UTILIZATION OF SELENIUM IN THE MOUSE BRAIN ......various brain diseases. Mouse models have been...
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UTILIZATION OF SELENIUM IN THE MOUSE BRAIN: IMPLICATIONS FOR NEUROLOGICAL DISEASE A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CELL AND MOLECULAR BIOLOGY (NEUROSCIENCES) AUGUST 2012 By Arjun Venkat Raman Dissertation Committee: Marla Berry, Chairperson Frederick Bellinger Scott Lozanoff Robert Nichols Bruce Shiramizu
Transcript of UTILIZATION OF SELENIUM IN THE MOUSE BRAIN ......various brain diseases. Mouse models have been...
UTILIZATION OF SELENIUM IN THE MOUSE BRAIN:
IMPLICATIONS FOR NEUROLOGICAL DISEASE
A DISSERTATION SUBMITTED TO
THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
CELL AND MOLECULAR BIOLOGY (NEUROSCIENCES)
AUGUST 2012
By
Arjun Venkat Raman
Dissertation Committee:
Marla Berry, Chairperson
Frederick Bellinger
Scott Lozanoff
Robert Nichols
Bruce Shiramizu
ii
ABSTRACT
Selenium is a chemical element that is an essential micronutrient associated with various
aspects of human health. The biochemical activity of selenium is mediated by proteins,
which incorporate it into the amino acid selenocysteine (Sec). There are 25 genes in
humans encoding Sec-containing selenoproteins. The functionally characterized
selenoproteins are oxidoreductase enzymes involved in cellular oxidation-reduction
reactions. Most selenoproteins are expressed in the mammalian brain, and dietary
selenium deficiency causes preferential retention in brain relative to other body organs.
Further, dietary selenium deficiency and specific selenoproteins are associated with
various brain diseases. Mouse models have been extensively used to study the function
and handling of selenium in mammals. Genetic deletion of a Sec-rich protein in mice
causes brain selenium deficiency, neurodegeneration and neurological impairment, and
disruption of a phospholipid hydroperoxidase selenoenzyme causes rapid
neurodegeneration. Therefore, selenoprotein expression and function promotes a healthy
nervous system. However, selenium metabolism and the function of several
selenoproteins in brain are not clearly defined.
The overall purpose of this work is to clarify the function and utilization of selenium in
the mammalian brain, to reveal implications for developmental and neurological diseases.
The goal of these studies is to investigate changes in brain function and selenoprotein
expression under conditions of altered selenium metabolism in mice. The research
presented in this dissertation covers three major topics that are separated into chapters. To
investigate selenium distribution in the brain, the neurological consequences of disrupting
selenium transport and recycling in mice are assessed and compared. Disruption of
selenium transport caused more profound neurological consequences than disruption of
selenium recycling. To investigate potential functions of selenium in the nervous system,
select selenoproteins were examined for cellular and subcellular expression in cells and
brain tissue from transgenic and control mice. Select selenoproteins and synthesis factors
were observed at synapses, suggesting localized expression and physiological relevance.
To investigate a potential interaction between selenium and methamphetamine,
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expression profiling of selenoproteins in mouse brain after exposure to methamphetamine
is described. Selenoprotein synthesis was adversely affected by methamphetamine
administration in mice. These results confirm the importance of selenium in the
mammalian brain.
iv
PAPERS ARISING FROM THIS DISSERTATION
Published, in press:
Raman, A. V., Pitts, M. W., Seyedali, A., Hashimoto, A. C., Seale, L. A., Bellinger, F. P.
and Berry, M. J. (2012), Absence of selenoprotein P but not selenocysteine lyase results
in severe neurological dysfunction. Genes, Brain and Behavior. doi: 10.1111/j.1601-
183X.2012.00794.x
In preparation for publication:
Raman, A. V., Pitts, M. W., Hashimoto, A. C., Nichols, R.A., Bellinger, F. P. and Berry,
M. J. (2012), Expression of selenoprotein W in neurons extends into processes and is
highly dependent on selenoprotein P. In preparation.
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DEDICATION
I would like to dedicate this dissertation directly to my parents, Neerja and Vasan Raman,
and indirectly to all of my ancestors, family, and friends. Needless to say, nothing about
me would have been possible without generations of love, dedication, and perseverance.
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TABLE OF CONTENTS
ABSTRACT........................................................................................................... ii
PAPERS ARISING FROM THIS DISSERTATION............................................ iv
DEDICATION....................................................................................................... v
LIST OF FIGURES................................................................................................ viii
CHAPTER 1: INTRODUCTION
1.1 Selenium in Chemistry and Biology.................................................... 1
1.2 Synthesis of Selenoproteins................................................................. 3
1.3 Selenoproteins as Oxidoreductase Enzymes........................................ 6
1.4 Selenoproteins and Metabolism........................................................... 9
1.5 The Selenoprotein Family.................................................................... 12
1.6 Selenoproteins in the Nervous System................................................. 18
1.7 References............................................................................................ 26
CHAPTER 2: ABSENCE OF SELENOPROTEIN P BUT NOT
SELENOCYSTEINE LYASE RESULTS IN SEVERE NEURLOGICAL
DYSFUNCTION
2.1 Abstract................................................................................................ 38
2.2 Introduction.......................................................................................... 39
2.3 Methods................................................................................................ 40
2.4 Results.................................................................................................. 46
2.5 Discussion............................................................................................ 51
2.6 References............................................................................................ 56
CHAPTER 3: EXPRESSION OF SELENOPROTEIN W IN NEURONS
EXTENDS INTO PROCESSES AND IS HIGHLY DEPENDENT ON
SELENOPROTEIN P
3.1 Abstract................................................................................................ 75
3.2 Introduction.......................................................................................... 76
3.3 Methods................................................................................................ 78
3.4 Results.................................................................................................. 82
3.5 Discussion............................................................................................ 85
3.6 References............................................................................................ 89
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CHAPTER 4: METHAMPHETAMINE-INDUCED ALTERATIONS IN
SELENOPROTEIN EXPRESSION IN MICE
4.1 Abstract................................................................................................ 101
4.2 Introduction.......................................................................................... 102
4.3 Methods................................................................................................ 104
4.4 Results.................................................................................................. 108
4.5 Discussion............................................................................................ 110
4.6 References............................................................................................ 112
CHAPTER 5: CONCLUSION
5.1 Summary and Discussion..................................................................... 123
5.2 Oxidative Stress and Selenoproteins.................................................... 128
5.3 Redox Systems and Selenoproteins in the Brain.................................. 130
5.4 References............................................................................................ 134
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LIST OF FIGURES
1.1 Assimilation of Se into selenoproteins 24
1.2 Selenoprotein localization to subcellular domains 25
2.1 Spontaneous activity and motor coordination is more reduced in male
than female Sepp1-/- mice compared to control mice fed a standard diet
65
2.2 High Se diet improves spontaneous activity and motor coordination
more in male than female Sepp1-/- mice compared to control mice
66
2.3 Generation of Scly-knockout mice 67
2.4 Spontaneous activity and motor coordination is normal in male and
female Scly-/- mice fed a low Se diet
68
2.5 Spatial learning and memory is disrupted in Sepp1-/- mice fed a
standard diet
69
2.6 Spatial learning and memory is not disrupted in Scly-/- mice fed a
standard diet
70
2.7 Spatial learning is mildly impaired in Scly-/- mice fed a low Se diet 71
2.8 Expression of selenoprotein transcripts is increased in Se-deficient
Scly-/- mice brains
72
2.9 Expression of selenoproteins is decreased in Se-deficient Scly-/- mice
brains
73
2.10 Glutathione peroxidase activity is decreased in Se-deficient Scly-/- mice
brains
74
3.1 Sepw1 is expressed in cell bodies and processes of cultured neurons 95
3.2 Sepw1 is expressed in cell bodies and processes of pyramidal neurons in
cortex and hippocampus
96
3.3 Regional expression of Sepw1 in neurons of mouse brain 97
3.4 Sepw1 is present in isolated nerve terminals 98
3.5 Sepw1 expression in isolated nerve terminals is greatly reduced in mice
lacking Sepp1
99
3.6 Several selenoprotein synthesis factors are present in isolated nerve
terminals
100
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4.1 Expression of selenoprotein mRNAs is altered by methamphetamine 116
4.2 Selenoprotein W and selenophosphate synthetase 2 mRNA are
upregulated in midbrain after two weeks of methamphetamine
administration
117
4.3 Selenoprotein expression following two weeks of methamphetamine
administration is not changed in mice brains
118
4.4 Long term methamphetamine administration dramatically reduces
selenoprotein mRNAs in striatum
119
4.5 Sepp1 impacts brain expression of selenoproteins more than
methamphetamine
120
4.6 Methamphetamine reduces tyrosine hydroxylase expression in
substantia nigra independently of Sepp1
121
4.7 Apoptotic cell death is not increased in methamphetamine-treated
selenoprotein P knockout mice
122
1
CHAPTER 1
INTRODUCTION
SELENIUM IN CHEMISTRY AND BIOLOGY
In 1817 the Swedish chemist Jöns Jacob Berzelius discovered the element selenium (Se)
after analyzing an impurity that was contaminating the sulfuric acid being produced at a
nearby factory. Since the factory workers were suffering an illness caused by Se, it was
initially considered to be a poisonous chemical. Nearly 140 years later, Se was identified
as an essential micronutrient in humans and livestock (Hatfield, 2006). Se is a trace
mineral present in the Earth’s crust and ocean water at wide ranging abundances,
averaging between 0.05 and 2.0 ppm. Perhaps due to low environmental quantities, Se
has a narrow concentration range from deficiency to toxicity in animals. The profound
biological effects of Se suggested that it was involved in enzymatic activity. This
speculation was confirmed when Se was discovered to be present as selenocysteine in the
active site of two unrelated enzymes (Behne et al., 1990, Berry et al., 1991b, Forstrom et
al., 1978, Rotruck et al., 1973b).
Like other group 16 elements oxygen and sulfur, Se has two unpaired electrons,
producing highly reactive atoms. Se is classified a nonmetal but has some metalloid
properties. For instance, it is a photoconductive semiconductor, meaning the electrical
conductivity is enhanced by light and somewhere in between that of a true conductor and
an insulator (Liao et al., 2010). This unique reactivity proved instrumental in production
of the first commercial photocopiers made by Xerox, and has been exploited for use in
solid-state electronics and photoelectric cells. Se and sulfur share some similar chemical
and physical properties, such as electronegativity and atomic radius, however selenium is
much heavier. Thus Se and sulfur compounds exist naturally as structurally analogous
molecules typically complexed with a metal. These inorganic and organic molecules exist
in a variety of oxidation states ranging from -2 to +6, and include selenides (Se2−
),
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selenites (SeO32−
), and selenates (SeO42–
), as well as the amino acids selenocysteine and
selenomethionine, and methylated forms (Johansson et al., 2005).
In biological systems, however, the substantial differences between Se and sulfur are
highlighted by their non-interchangeable nature. For example, sulfur represents about one
quarter of one percent of human body weight and possesses almost no toxicity risk even
at very high levels. In contrast, Se is required in nanomolar concentrations, and the
median lethal dose of in humans is 1.5 - 6.0 mg/kg body weight with inorganic salts
being more acutely toxic than organic forms (Koller & Exon, 1986). In vertebrates, Se is
obtained almost exclusively by ingestion of plants and animals. The amount of Se in
dietary sources depends on the concentration of environmental Se in the soil or water.
Currently, the recommended daily allowance of dietary Se for healthy adults is 55 µg/day
and the upper tolerable intake level is 400 µg/day (Monsen, 2000).
Most cellular biological functions of Se are attributed to selenoproteins, which
incorporate Se as the amino acid selenocysteine (Sec). Sec is structurally analogous to
cysteine with the exception of Se replacing sulfur in the side chain. However, the
similarity ends there. Se and sulfur have similar properties, but their differences generate
divergent character between Sec and cysteine. Having Sec in the active site of an enzyme
generally produces more efficient catalysis (Berry et al., 1992). The cause of the higher
catalytic capacity of Sec is still under investigation, and several ideas have been
proposed. For example, Sec is described as having stronger nucleophilic and electrophilic
character than cysteine, potentially aiding in electron and proton transfer during catalysis.
The acid dissociation constant for the selenol (pKa = 5.2) of Sec is lower than the thiol
(pKa = 8.3) of cysteine. This means that at physiological pH, Sec tends towards
ionization whereas cysteine tends towards protonation, and at low pH Sec retains much
higher reactivity than cysteine. The selenol is further thought to be a superior leaving
group due to higher acidity (Johansson et al., 2005).
The biological rationale for Sec utilization has been enigmatic. Due to the relative
stabilities of each oxidation state of Sec and cysteine near physiological pH, Sec has two
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main advantages over cysteine in terms of reaction with oxygen. Kinetic calculations
suggest that oxidized Sec, both selenenic and seleninic acid, will cycle more rapidly to
the reduced form than oxidized forms of cysteine, sulfenic and sulfinic acid. Second,
further oxidation to selenonic acid is extremely unfavorable, unlike irreversible oxidation
of cysteine to sulfonic acid. The actual reactivity of the residue will ultimately depend on
the context of the Sec within the local and global structure of the peptide and enzyme.
However, these unique reactive properties may potentially explain the biological pressure
to use Sec in proteins during evolution (Hondal & Ruggles, 2011).
SYNTHESIS OF SELENOPROTEINS
Beyond the chemistry, Sec is an extremely unusual amino acid, and can be considered an
anomaly in the genetic code. Roughly defined, the genetic code is the system in which
genetic information is first transcribed from deoxyribonucleic acid (DNA) to messenger
ribonucleic acid (mRNA), and subsequently interpreted by ribosomes and several transfer
RNAs (tRNAs) to produce polypeptides. Three-nucleotide sequences in mRNA termed
codons specify binding to various tRNAs, each carrying single amino acids to be
positioned during protein synthesis. This system of mRNA translation also uses initiation
and termination codons to determine the precise peptide sequence. Termination codons
typically recruit proteins called release factors rather than a tRNA, thus dissociating the
ribosomal subunits and releasing the peptide chain. In discordance with the standard rules
of the system, the opal termination codon specified by UGA can be recognized by a
specific tRNA (tRNASec) (Diamond et al., 1981). However, in order for tRNASec to
efficiently recognize the in-frame UGA codon, the mRNA must additionally contain a
Sec insertion sequence (SECIS). The SECIS element is a stem-loop structure downstream
of the UGA codon in selenoprotein mRNAs that recruits proteins, which facilitate
recoding of the UGA and incorporation of Sec (Fig. 1) (Berry et al., 1991a).
Although selenoproteins are present in eukarya, archaea, and bacteria, the mechanism of
Sec incorporation differs between domains in several respects. For example, the bacterial
SECIS element is located immediately following the Sec UGA codon, whereas the
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eukaryotic and archaeal SECIS elements are typically located much farther downstream
in the 3’ untranslated region (UTR) of the transcript. Additionally, the archaeal genus
Methanococcus has a 5’ UTR SECIS element, while eukaryotes have two distinct forms
of 3’ UTR SECIS elements (Kryukov et al., 2003, Kryukov & Gladyshev, 2004, Wilting
et al., 1997). An elongation factor specific to tRNASec has been identified as selB in
bacteria. In eukaryotes, the coordinated function of a SECIS binding protein (SBP2) and
a Sec-specific elongation factor (EFSec) allow the co-translational incorporation of Sec
instead of termination (Papp et al., 2007). Biosynthesis of Sec in eukarya and archaea is
accomplished by the sequential actions of O-phosphoseryl-tRNA kinase (PSTK) and O-
phosphoserine-tRNA:Sec-tRNA synthase (SEPSECS), which convert seryl-tRNASec to
phosphoseryl-tRNASec and ultimately to selenocysteinyl-tRNASec. Thus Sec formation
in archaea and eukarya is a two-step process, whereas bacterial selenocysteine synthase
(selA) can synthesize Sec directly from seryl-tRNASec. The Se donor utilized by both
SEPSECS and selA is selenophosphate, which is generated from selenide (H2Se) by
selenophosphate synthetase (SPS) enzymes. Selenophosphate is first transferred to O-
phosphoseryl-tRNASec by SEPSECS while displacing the phosphoseryl moiety, and
subsequent hydrolysis of the phosphate group yields Sec charged to its cognate tRNA
(Palioura et al., 2009). Bacteria have one SPS enzyme termed selD, while two (SPS1,
SPS2) have been identified in eukarya. Selenophosphate-synthetase 2 (SPS2) is a
eukaryotic selenoprotein that is required for the synthesis of all selenoproteins including
itself, and may provide feedback for global selenoprotein synthesis. Selenophosphate is
generated by SPS2 in the presence of selenide and ATP (Guimaraes et al., 1996). A
related protein called SPS1 contains a cysteine residue in place of Sec, but its
involvement in Sec and selenoprotein biosynthesis is uncertain (Low et al., 1995).
Intriguingly SPS2, but not SPS1, is required for selenoprotein synthesis in NIH3T3
mouse fibroblasts (Xu et al., 2007). Studies in Drosophila melanogaster, indicate that the
function of SPS1 is primarily in metabolism of vitamin B6, but whether this holds true
for vertebrates is unknown. It has alternatively been suggested that SPS2 assimilates
selenite, whereas SPS1 recycles Sec in a Se-salvage pathway (Tamura et al., 2004).
However an in vivo requirement of SPS2 and specificity of SPS1 and SPS2 with different
Se substrates have not been reported. Selenocysteine lyase (SCLY) is a putative Se
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recycling enzyme, which is able to catalyze the hydrolysis of Sec into selenide and
alanine (Esaki et al., 1982). It facilitates Se incorporation into selenoproteins when Sec is
the Se source, and may deliver selenide to SPS enzymes for phosphorylation and
subsequent insertion into nascent selenoproteins (Kurokawa et al., 2011, Tobe et al.,
2009). Pyridoxal phosphate is a prosthetic group derived from vitamin B6 and is required
by enzymes involved in metabolism of amino acids, glucose, lipids and
neurotransmitters. The enzymatic activity of both SCLY and SEPSECS are pyridoxal
phosphate-dependent, implying that Sec metabolism shares regulatory elements with
standard amino acid metabolic pathways (Lacourciere et al., 2000, Mihara et al., 2000,
Palioura et al., 2009).
Several steps in selenoprotein synthesis are regulated by Se availability (dietary or
environmental depending on the organism), as well as by the local oxidation state. For
example in addition to being a limiting substrate for SPS2, Se levels affect different
SECIS elements differentially, potentially regulating selenoprotein synthesis efficiency at
the level of translation (Papp et al., 2007). Oxidation of the cysteine-rich, redox-sensitive
domain of SBP2 masks the nuclear-export signal (NES) causing importation into the
nucleus. Subsequently it is either sequestered there during high oxidative burden, or else
reduced by nuclear-specific isoforms of selenoproteins, unmasking the NES for binding
and exportation by the nuclear export receptor CRM-1 (Papp et al., 2006). Individual
selenoprotein expression also responds differentially to Se-availability and oxidative
stress, providing another level of regulatory control for selenoprotein synthesis. Thus
there is a complex, nonlinear interaction between Se-status and oxidative burden that
coordinates the synthesis of the numerous selenoproteins. Despite a low environmental
availability of Se and the various elaborate mechanisms for Sec incorporation,
selenoproteins are widespread in organisms, with certain plants and fungi being the only
major exceptions that lack selenoproteins and an essential biological function for Se
(Lobanov et al., 2009).
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SELENOPROTEINS AS OXIDOREDUCTASE ENZYMES
The human genome codes for 25 selenoproteins, most of which have been identified
recently by bioinformatics approaches looking for SECIS elements downstream of in-
frame UGA codons (Kryukov et al., 2003). The various members display wide
subcellular and tissue distribution, and several are known to have multiple transcript
variants and protein isoforms. Functionally, selenoproteins are closely linked with the
cellular thiol-disulfide couples, particularly the glutathione (GSH) and thioredoxin (TXN)
couples. GSH is a tripeptide made of glycine, cysteine, and glutamate and is the most
abundant thiol in cells, present at millimolar concentrations. Oxidation of the cysteine
thiol links two molecules of GSH to form glutathione disulfide (GSSG). This reaction can
be spontaneous in the presence of electrophiles or alternatively can be catalyzed by a
number of enzymes that utilize GSH as an electron donor including glutathione
peroxidase (GPX), glutaredoxin, and glutathione S-transferase enzymes. Reduction of
GSSG, producing two molecules of GSH, is performed by the homodimeric flavoenzyme
glutathione reductase.
All of the characterized selenoproteins that function as enzymes are oxidoreductases that
catalyze thiol-disulfide oxidation-reduction (redox) reactions and contain Sec in the
active site. The first Se-dependent enzyme discovered was glutathione peroxidase 1
(GPX1), which catalyzes the reduction of hydrogen peroxide (H2O2) by oxidation of two
molecules of GSH to GSSG (Rotruck et al., 1973a). Cytosolic and mitochondrial forms
of the GPX1 enzyme are transcribed from the same gene containing one Sec-encoding
TGA. The enzyme functions as a homotetramer utilizing four Se atoms per active enzyme
(Esworthy et al., 1997). GPX1 is the most abundant glutathione peroxidase and the most
abundant selenoprotein in rats, representing a significant fraction of the total circulating
Se pool (Hawkes et al., 1985).
Four homologous GPX selenoproteins have subsequently been identified in humans.
GPX2, also known as gastrointestinal GPX, is a cytosolic enzyme that is specific to
epithelial cells and is abundant in the gut (Chu et al., 1997). The extracellular GPX3 has
7
broad substrate specificity and is found in most extracellular compartments but is
abundant in kidney and blood plasma (Olson et al., 2009). GPX6 is closely related to
GPX3 but is only expressed in the olfactory system, and exists as a cysteine homolog in
rodents (Kryukov et al., 2003). GPX4 is structurally and functionally different from the
other GPX enzymes because it is active as a monomer rather than a tetramer, and can
directly reduce membrane lipid hydroperoxides and free fatty acid hydroperoxides.
Alternative splicing and transcription initiation generates three distinct isoforms of GPX4
that localize to the cytosol, mitochondria, and nucleus (Maiorino et al., 2003).
Additionally, GPX4 translation is regulated by the mRNA binding proteins guanine-rich
sequence-binding factor 1 (Ufer et al., 2008) and PARK7/DJ-1 (Blackinton et al., 2009,
Van Der Brug et al., 2008). Genetic deletion of GPX4 in mice causes embryonic lethality
and knockdown of GPX4 in cells leads to rapid lipoxygenase-mediated lipid peroxidation
and subsequent apoptosis, suggesting that removal of lipid hydroperoxides by GPX4 is
essential for cell viability (Seiler et al., 2008, Yant et al., 2003). There are three
additional GPX enzymes (GPX5, GPX7, and GPX8) that are not selenoproteins in
humans.
TXN is a small protein of ~12 kDa and is present at concentrations several orders of
magnitude below GSH. It contains an active site dithiol that is highly conserved in
evolution and widely distributed among the TXN superfamily of proteins, which includes
protein disulfide isomerase enzymes. Through oxidation of the dithiol to a disulfide, TXN
can directly reduce cysteine sulfenic acids and control the state of dithiol-disulfide motifs
in target proteins, and can also serve as an electron donating cofactor for enzymes such as
ribonucleotide reductase, peroxiredoxins and methionine sulfoxide reductases (Arner &
Holmgren, 2000). In turn, reducing oxidized TXN is mediated exclusively by the
thioredoxin reductase (TXNRD) family of selenoproteins (Zhong et al., 2000). At least
four selenoproteins (GPX3, GPX4, SEPP1, SEPX1) can utilize TXN as a cofactor for
enzymatic reduction, and it is possible that others do as well (Bjornstedt et al., 1994,
Takebe et al., 2002). There are numerous thioredoxin-like proteins that may depend on
TXN or act in parallel to provide additional substrate specificity beyond that provided by
TXN. Several selenoproteins contain a TXN-like fold, which is a well described
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secondary/tertiary structure pattern with a conserved Cys-X-X-Cys or Cys-X-X-Ser/Thr
active-site motif characteristic of oxidoreductases (Dikiy et al., 2007), where X is any
amino acid. It is tempting to speculate that these selenoproteins operate similarly to TXN,
namely by controlling the redox state of cysteine residues and dithiol motifs, but there is
little evidence to support or deny this notion at present.
Three mammalian thioredoxin reductases are selenoenzymes encoded by individual
genes. TXNRD1, TXNRD2, and TXNRD3 encode homodimeric flavoproteins that
localize to the cytosol, mitochondria, and testes respectively. They are members of the
pyridine nucleotide-disulfide oxidoreductase family and contain two redox-sensitive sites
in the N- and C-terminus that interact in the head to tail dimer conformation of the active
enzyme. These enzymes are capable of reducing a number of substrates, but depend on
NADPH for donating electrons, which are first transferred to the FAD group, then passed
to the N-terminal dithiol of one subunit and subsequently to the C-terminal selenenyl-
sulfide of the other subunit. The highly conserved Sec-containing C-terminal motif is
absolutely critical for catalytic function of TXNRD enzymes (Arner & Holmgren, 2000,
Hatfield, 2006). The main substrate for TXNRDs is the small redox-sensitive protein
TXN, which is integral to physiological processes such as cell communication,
metabolism, proliferation, and apoptosis. In general, the reactive dithiol of TXN will
become oxidized to a disulfide during reduction of an oxidized target protein.
Regeneration of reduced TXN proteins requires TXNRD, and thus the TXN/TXNRD
system is completely dependent on Se in mammals. The importance of this system is
highlighted by the fact that knockout of either TXNRD1 or TXNRD2 is embryonic lethal
in mice (Conrad et al., 2004, Jakupoglu et al., 2005). It is worth noting that TXNRDs
from mammals differ from the Se-independent enzymes of archaea, bacteria, yeast, and
plants.
Reactions between target proteins and TXN can be spontaneous, but several enzymes can
catalyze the reduction of target proteins using TXN as an electron donating cofactor. The
human genome codes for four Methionine Sulfoxide Reductase (MSR) enzymes that
reduce oxidized methionine residues in proteins utilizing TXN as a cofactor. There is
9
now considerable evidence that like cysteine, reversible methionine oxidation can
regulate protein function (Levine et al., 2000). For example, calcium/calmodulin-
dependent protein kinase II and the phosphatase calcineurin, among many other proteins,
are regulated by methionine redox status (Agbas & Moskovitz, 2009, Erickson et al.,
2008). A single MSRA and three MSRB enzymes stereo-specifically reduce S- and R-
sulfoxidated methionines respectively. MSRB1 is a selenoprotein also known as
Selenoprotein R and SEPX1, while MSRA as well as MSRB2 and MSRB3 are Se-
independent enzymes. SEPX1 is a zinc-containing protein present in the cytosol and
nucleus and exhibits the highest methionine-R-sulfoxide reductase activity because of the
presence of Sec in its active site (Kim & Gladyshev, 2004). Interestingly, redox status of
the cysteine-rich metallothionein/thionein couple dictates zinc loading in that reduced
thionein binds zinc and oxidation of metallothionein releases it. Moreover, reduction of
non-selenoprotein MSRB3 by TXN, TXNRD, and NADPH is more efficient in the
presence of thionein (Sagher et al., 2006). Therefore regulation of specific kinases,
phosphatases, and other proteins by methionine-R-sulfoxide reduction is mediated by two
selenoproteins (MSRB1, TXNRD1) and NADPH.
SELENOPROTEINS AND METABOLISM
Specific selenoproteins function at the intersection of cellular and organism metabolism
by modulating insulin and thyroid hormone signaling. The iodothyronine deiodinases
(DIO) function in activation and deactivation of thyroid hormone and were the second
family of enzymes determined to be Sec-containing selenoenzymes (Berry et al., 1991b).
Thyroid hormone metabolism both at the level of production in the thyroid and local
hormone activity in the periphery is reliant on the DIO family of selenoenzymes. Most
vertebrates have three DIO enzymes that can deiodinate thyroid hormones to control local
availability. These integral membrane protein enzymes are thiol-requiring
oxidoreductases that remove iodine atoms from the aromatic rings of thyroxine (T4),
triiodothyronine (T3), and reverse triiodothyronine (rT3) (Bianco & Kim, 2006). DIO1 is
a plasma membrane protein found mainly in cells of the liver and kidney, is capable of
deiodinating both the inner and outer rings, and produces most of the circulating T3.
10
DIO2 is found in the endoplasmic reticulum of cells in several tissues including the
thyroid, heart, skeletal muscle, fat, and the central nervous system and selectively
removes the outer ring iodine, making it the primary tissue activator of thyroid hormone
by converting T4 to T3. DIO3 is also a plasma membrane protein, however it is mainly
found in fetal tissue and the placenta, selectively removes the inner ring iodine, and thus
contributes to thyroid hormone inactivation.
A specific role for a selenoprotein in redox regulation of insulin signaling was established
when it was found that overexpression of GPX1 causes hyperinsulinemia and insulin
resistance in mice (Mcclung et al., 2004). Moreover genetic deletion of GPX1 promotes
glucose tolerance and insulin sensitivity in mice on a high-fat diet by enhancement of
insulin-induced PI3K/Akt signaling (Loh et al., 2009). Dietary studies in humans have
further suggested that supranutritional levels of Se are associated with type II diabetes,
while animal studies confirm that both excessive dietary Se and GPX1 overexpression
lead to hyperinsulinemia and insulin resistance (Labunskyy et al., 2011, Lippman et al.,
2009, Stranges et al., 2007, Wang et al., 2008). Peroxide-induced oxidation of PTEN and
S-glutathionylation of protein tyrosine phosphatase 1B are affected by GPX1 activity,
which thereby modulates insulin receptor activation and insulin resistance (Mueller et al.,
2009).
Selenoprotein P (SEPP1) is a unique selenoprotein that contains multiple Sec residues
and is also implicated in insulin resistance. Diabetic patients display an increase in
hepatic SEPP1 mRNA and serum SEPP1 protein, and purified SEPP1 administered to
mice is able to induce insulin resistance and glucose intolerance. Furthermore,
knockdown or knockout of SEPP1 in mice improves glucose tolerance and insulin
sensitivity, and SEPP1 knockout mice are protected against glucose intolerance and
insulin resistance even when on an obesity-inducing diet (Misu et al., 2010). Since
SEPP1 expression can dictate expression of other selenoproteins including GPX1, its
effect on insulin resistance may be direct or indirect. Primate and rodent SEPP1 contains
up to ten Sec residues while as many as 17 Sec residues are present in zebrafish. SEPP1
is the most abundant selenoprotein in blood and accounts for as much as 65% of plasma
11
Se in rats (Burk & Hill, 1994). The abundant Sec residues of SEPP1 are divided into two
regions with the bulk being located in the C-terminal domain that is required for the Se
transport function. SEPP1 also contains an N-terminus Cys-X-X-Sec motif and catalyzes
the reduction of lipid hydroperoxides in vitro utilizing TXN as a cofactor (Saito et al.,
1999, Takebe et al., 2002). Aside from the high Se content in the form of Sec residues,
SEPP1 is distinct in that it is one of two extracellular selenoproteins (the other being
GPX3), and the carboxy-terminal Se transport domain appears to be a metazoan
adaptation (Lobanov et al., 2008). SEPP1 is abundantly produced by the liver and
secreted into blood, however local production and secretion in nearly all tissue systems
has been described (Burk & Hill, 2005). Bodily transport of Se to extrahepatic tissues,
particularly the brain, testes and kidneys, is facilitated by receptor-mediated uptake of
SEPP1 by the low-density lipoprotein receptor family members ApoER2 (LRP8) and
Megalin (LRP2) (Burk & Hill, 2009). In addition to a Se transport function and
peroxidase activity, SEPP1 exhibits pH-dependent heparin binding and heavy metal
binding that likely also function in redox-dependent processes.
In our lab, selenocysteine lyase (SCLY) has also shown to be involved in metabolic
syndrome in an in vivo mouse model. Although SCLY is not itself a selenoprotein, it is an
enzyme with high specific activity for Sec, and liberates selenide (Mihara et al., 2000).
Since selenoproteins are a reservoir of Sec and SEPP1 supplies Se in the form of Sec,
SCLY is a potentially relevant factor in the global distribution of Se and the expression of
all selenoproteins. Transgenic mice lacking SCLY were characterized and found to have
altered energy metabolism in the liver. When given adequate Se, the animals exhibit
hyperinsulinemia and mild hepatic steatosis, along with an increase in blood SEPP1.
Upon dietary Se restriction, the animals develop obesity, fatty liver, hypercholesteremia,
and insulin resistance (Seale, et al. 2012, unpublished). The results of studies on mice
with disrupted GPX1, SEPP1, and SCLY do not reconcile with a simple explanation.
Instead, they suggest a nonlinear, dynamic explanation. Regardless, these data
demonstrate the importance of proper Se handling in vertebrate carbohydrate and lipid
metabolism. Se utilization is tightly integrated with insulin and thyroid hormone
signaling, thus disruption of selenoproteins and Sec-related proteins alters vertebrate
12
metabolic systems. A connection between Se and metabolism in multicellular organisms
suggests that cell-autonomous regulation of redox systems and signaling may similarly
depend on one or more selenoproteins.
THE SELENOPROTEIN FAMILY
Several selenoproteins with uncertain functions could have a role in regulating target
protein oxidation state. The regulatory control of cellular redox signaling by
Selenoprotein W (SEPW1) will be discussed next as an example, bearing in mind that
selenoproteins with unknown roles can impact the cellular response to environmental
changes, particularly in relation to growth and stress. Following the section on SEPW1, a
brief summary of what is known of the remaining selenoproteins is presented, with the
biological functions described possibly owing to the activity of the selenoproteins in
undefined redox circuits.
SEPW1 was purified in the early 1990s but putatively identified much earlier due to its
absence in Se-deficient lambs suffering a myopathy called White Muscle Disease
(Vendeland et al., 1993). Mammalian SEPW1 is a highly conserved cytosolic protein of
just 87 amino acids, and SEPW1 orthologs are among the most widely distributed
selenoproteins in all species including prokaryotes (Kryukov & Gladyshev, 2004, Zhang
et al., 2005). The expression level of SEPW1 in vertebrates is very sensitive to dietary Se
intake as well as the expression level of SEPP1 (Hoffmann et al., 2007, Vendeland et al.,
1995, Yeh et al., 1995). Abundant SEPW1 expression is observed in muscle, and SEPW1
transcription during myocyte differentiation is maintained by binding of the myogenic
transcription factor MyoD to the SEPW1 promoter (Noh et al., 2010). A putative metal-
response element in the promoter of the SEPW1 gene was probed in vitro using a
luciferase reporter fusion construct, and luciferase specific activity was found to be
stimulated by copper and zinc, but not cadmium (Amantana et al., 2002). Although a
bona fide enzymatic activity has not been attributed to SEPW1, the presence of a Cys-X-
X-Sec motif in a thioredoxin-like fold may indicate thioredoxin-like redox activity (Dikiy
et al., 2007).
13
Recently, SEPW1 was shown to pull-down and co-immunoprecipitate with the beta and
gamma isoforms of 14-3-3 protein. This interaction was further confirmed by NMR
spectroscopy, and extended to identify three loops of SEPW1 that interact with 14-3-3
proteins (Aachmann et al., 2007). 14-3-3 beta and gamma proteins are scaffolding
proteins derived from the YWHAB and YWHAG genes respectively, and bind a diverse
assortment of proteins including kinases, phosphatases, and receptors. In this way 14-3-3
proteins coordinate molecular interactions and participate in cell cycle regulation,
metabolism, apoptosis, protein trafficking and gene transcription (Fu et al., 2000). A
computational study of SEPW1/14-3-3 interaction suggests that a conserved cysteine of
14-3-3 beta and gamma (Cys191 and Cys195 respectively) can be reversibly oxidized,
with SEPW1 acting as a reducing agent (Musiani et al., 2010). The oxidized cysteine
sulfenic acid of 14-3-3 can putatively react with Sec of SEPW1, producing a mixed
complex. Subsequently the formation of an intramolecular selenenyl-sulfide within
SEPW1 would result in 14-3-3 being fully reduced. Oxidized SEPW1 can then migrate
away and likely be reduced to its parent state by GSH. This speculated reduction is
supported by evidence that a SEPW1 cysteine residue conserved from rodents to primates
can be S-glutathionylated (Beilstein et al., 1996, Gu et al., 1999).
Redox regulation of 14-3-3 proteins by SEPW1 could serve several cellular functions, but
an intriguing possibility is presented by the in vitro finding that SEPW1 expression is
regulated by the cell-cycle, and knockdown of SEPW1 induces p53-dependent cell-cycle
arrest (Hawkes et al., 2009). Recently, it has been demonstrated that growth factor-
induced receptor tyrosine kinase phosphorylation, and downstream JNK and p38 MAPK
signaling, leading to cell proliferation requires SEPW1 (Hawkes, WC; personal
communication). Therefore SEPW1, through redox regulation of 14-3-3 proteins, may
coordinate Se availability and oxidative burden with cellular proliferation, differentiation
and death via the p53 pathway. Deficiency of SEPW1-mediated redox functionality may
serve as the basis for the myopathies in livestock (Whanger, 2009). Interestingly SEPW1
is also associated with multiple myeloma in humans, where SEPW1 overactivity may
promote malignancy and the overangiogenic phenotype of endothelial cells in active
14
disease (Ria et al., 2009). These studies further suggest a pivotal role for SEPW1 and Se
in regulatory control of the cell cycle.
A subset of selenoproteins is observed in mitochondrion to combat against electron leak
during oxidative respiration and phosphorylation. Mitochondria-specific isoforms of
GPX1, GPX4, and TXNRD2 regulate peroxide metabolism and oxidative tone within this
organelle. The selenoproteins GPX4, TXNRD1, MSRB1, and SELH have been shown to
exhibit varying degrees of nuclear localization (Fig. 2). Apart from SELH, the other three
selenoproteins are presumably involved in reduction of lipid peroxides, oxidized TXN,
and sulfoxidized methionine residues within the nuclear envelope.
SELH is the only DNA-binding selenoprotein described and has a role in regulation of
gene expression. Similar to SEPW1, Selenoprotein H (SELH) is a small selenoprotein
that is highly expressed during development and is sensitive to dietary Se intake (Kipp et
al., 2009, Novoselov et al., 2007). Like several selenoproteins it contains a Cys-X-X-Sec
sequence within a thioredoxin-like fold, but unlike any other selenoprotein described to
date, it is a DNA-binding protein of the AT-hook family. SELH is primarily located in
the nucleus and is implicated in redox-sensitive transcription of genes whose products are
involved in de novo glutathione synthesis and phase II detoxification (Panee et al., 2007).
Multiple metal-response elements are present in the SELH gene (Stoytcheva et al., 2010),
and one group has confirmed in vivo that SELH mRNA and protein are upregulated under
conditions of elevated copper in mouse liver (Burkhead et al., 2010). Although it is a
nuclear protein, mitochondrial biogenesis and function are also linked with SELH.
Overexpression of SELH in a transformed neuronal cell line attenuates the UVB-induced
increase of p53 protein and caspase-mediated apoptosis (Mendelev et al., 2009).
Additionally SELH overexpression increases mitochondrial size, cytochrome c content,
and expression of mitochondrial biogenesis proteins while boosting respiration
(Mendelev et al., 2011). Collectively these findings suggest that SELH is a Se- and
metal-regulated selenoprotein that is able to transduce oxidant signals by modulating
gene expression in conjunction with other redox-sensitive transcription factors. Further
15
investigation is warranted to determine if SELH modifies cysteine S-glutathionylation or
disulfide formation in target proteins such as p53 to regulate gene expression.
The endoplasmic reticulum (ER) regulates the synthesis, folding, and transport of
proteins, and additionally constitutes the main intracellular store for calcium ions, which
are integral in cell signaling. Seven selenoproteins are enriched in the ER (Fig. 2) and
some are postulated to have a role in protein folding and ER calcium handling, since
oxidative mechanisms within the ER are known to regulate these processes (Shchedrina
et al., 2010). The ER is a relatively oxidizing environment compared to other intracellular
organelles and contains oxidase enzymes to facilitate the formation of disulfide bonds in
proteins destined for export. Simultaneously GSH and TXN system components are
transported into the ER, providing both oxidation and reduction mechanisms for dynamic
redox regulation associated with protein processing and secretion. Redox state affects
calcium homeostasis by modulating ER calcium channels and chaperones, and oxidative
stress and ER-stress are intimately related in signaling for apoptosis (Gorlach et al.,
2006). Similar to the ER, the secretory and endosomal/lysosomal pathways are also more
oxidized than other subcellular compartments (Austin et al., 2005, Hwang et al., 1992).
Ligand binding to various receptors stimulates endocytosis of redox-active endosomes
whose luminal redox activity directs spatiotemporally-regulated signaling and prevents
nonspecific redox reactions (Li et al., 2006, Oakley et al., 2009). Therefore redox-
mediated processes are vital for secretory and endocytic function, and the ability of
selenoproteins to transmit oxidative signals from reactive intermediates to disulfide bonds
or exposed thiols of target proteins may help to explain the enrichment of selenoproteins
in the ER.
Selenoprotein T (SELT) is an ER- and Golgi-localized selenoprotein that is ubiquitously
expressed from development through adulthood, and shares some sequence similarity
with SEPW1 and SELH including the thioredoxin-like fold containing a Cys-X-X-Sec
motif (Dikiy et al., 2007). Deficiency of SELT in murine fibroblasts causes an
upregulation of SEPW1, in addition to altering cell adhesion and redox regulation
(Sengupta et al., 2009). A biological role for SELT in neuroendocrine secretion and
16
calcium mobilization in vitro has also been presented. SELT was identified as a target
gene of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP),
and manipulation of SELT expression altered PACAP-induced intracellular Ca2+
changes
and growth hormone secretion (Grumolato et al., 2008). Furthermore, PACAP regulation
of SELT is associated with ontogenesis, tissue maturation and regeneration of nervous,
endocrine, and metabolic tissues (Tanguy et al., 2011).
Two of the more abundant and ubiquitous ER selenoproteins, Selenoprotein M (SELM)
and 15-kDa selenoprotein (SEP15), are both 15-kDa proteins that share 31% sequence
homology and have Cys-X-X-Sec and Cys-X-Sec redox motifs respectively. SELM and
SEP15 have thioredoxin-like tertiary structure and homology to protein disulfide
isomerases that suggest oxidoreductase activity, but direct evidence in support of this
notion is lacking (Ferguson et al., 2006). SEP15 associates with UDP-
glucose:glycoprotein glucosyltransferase (UGTR), a protein involved in protein
conformation quality control, and has been suggested to facilitate proper protein folding
(Labunskyy et al., 2005). SEP15 knockout mice have been generated, and the only major
phenotype found was a predispostion to cataract formation that was suspected to be due
to improper folding of lens proteins. A role for SELM in protein folding has been
proposed, and recent evidence suggests that SELM may also be involved in regulating the
flux of calcium ions. Overexpression of SELM in a neuronal cell line in vitro reduced
peroxide-induced calcium influx, whereas knockdown of SELM increased the baseline
intracellular calcium concentration (Reeves et al., 2010).
Selenoprotein N (SEPN1) is a large (70 kDa) single-spanning transmembrane protein
localized to the ER membrane with two known isoforms generated by alternative splicing
of exon 3 (Moghadaszadeh et al., 2001). Several congenital muscular dystrophy
syndromes such as multiminicore disease, rigid spine muscular dystrophy, and desmin-
related myopathy with Mallory body-like inclusions are directly associated with
mutations in the SEPN1 gene and have been classified as SEPN1-related myopathies
(Ferreiro et al., 2004). Interestingly, mutations in SEPN1 leading to congenital fiber-type
disproportion are associated with insulin resistance (Clarke et al., 2006). To date, SEPN1
17
is the only selenoprotein gene in which mutations are directly and causatively linked to
human disease. An in vivo study using zebrafish determined that SEPN1 associates with
ER/SR ryanodine receptors, and that this interaction is necessary for the calcium-induced
release of calcium from intracellular stores (Jurynec et al., 2008). Ryanodine receptor
channels are homotetramers with several redox-regulated cysteine residues and SEPN1
contains a Cys-Sec-Gly-Ser motif, suggesting that SEPN1 regulates ryanodine receptor-
mediated calcium flux in muscle by redox-dependent signaling. Finally, SEPN1 is
integral to the generation and/or maintenance of skeletal muscle satellite cells, which are
an adult stem cell population involved in muscle growth and regeneration (Castets et al.,
2011).
Selenoprotein K (SELK) and Selenoprotein S (SELS) are also predominantly ER-
localized single-spanning transmembrane proteins, however they are much smaller than
SEPN1, and also show some localization to the plasma membrane (Chen et al., 2006,
Kryukov et al., 2003, Ye et al., 2004). Both are widely expressed in a variety of tissues
and have been implicated in the cellular response to ER-stress. Specifically, ER-stress
agents regulated the expression of SELK in HepG2 hepatoma cells, and knockdown of
SELK exacerbated cell death when challenged with ER-stress (Du et al., 2010). Genetic
deletion of SELK in mice decreases receptor-mediated calcium flux in immune cells,
impairs calcium-dependent immune cell function, and increases West Nile virus-induced
lethality (Verma et al., 2011). An interesting link between metabolism and inflammation
is presented in the case of SELS, which was originally identified as a glucose-regulated
protein in a rodent model of diabetes (Walder et al., 2002). The relationship between
SELS and type II diabetes was confirmed in humans, and there is evidence that SELS can
be secreted from liver and identified in blood sera where it associates with LDL (Gao et
al., 2007, Karlsson et al., 2004). SELS is now known to also be regulated by
inflammatory cytokines (Gao et al., 2006) and reciprocally, reduced expression of SELS,
due to polymorphisms in the gene promoter, influences the levels of IL-1, TNFα, and IL-
6 (Curran et al., 2005). SELS participates in removal of misfolded proteins from the ER
lumen (Ye et al., 2005, Ye et al., 2004) and was demonstrated to prevent ER-stress and
have anti-apoptotic function in macrophages and astrocytes (Fradejas et al., 2008, Kim et
18
al., 2007). Further work in mice indicates that in brain, SELS is mainly expressed
neuronally under basal conditions, but is intensely upregulated in reactive astrocytes
following brain injury (Fradejas et al., 2011).
Three selenoproteins remain largely unexplored with very little published data currently
available. The sequences of selenoprotein I (SELI), selenoprotein O (SELO), and
selenoprotein V (SELV) were identified in the human genome several years ago,
however almost no information is available on the cellular localizations or physiological
functions of these selenoproteins. SELI mRNA is known to be expressed in several
tissues, and is a putative transmembrane protein hypothesized to function in phospholipid
biosynthesis based on the presence of a CDP-alcohol phosphatidyltransferase motif that
is conserved in phospholipid synthases (Horibata & Hirabayashi, 2007, Kryukov et al.,
2003). SELO is predicted to be a 669 amino-acid selenoprotein containing a Cys-X-X-
Sec motif, but experimental data demonstrating a redox function is unavailable (Kryukov
et al., 2003). SELV appears to be a testes-restricted protein with a predicted thioredoxin-
like fold housing a Cys-X-X-Sec motif, and also has some sequence homology with
SEPW1, SELH, and SELT (Kryukov et al., 2003).
SELENOPROTEINS IN THE NERVOUS SYSTEM
The nervous system has a unique relationship with Se. This is most clearly demonstrated
by the fact that dietary Se deprivation causes a much greater drop in peripheral tissue Se
concentration than in brain Se concentration. In other words, the brain preferentially
retains Se compared to other organs during times of deficiency, suggesting that it has
some essential function in brain (Chen & Berry, 2003). Additionally, nearly all
selenoproteins are expressed in the brain, and neurons appear to be the major functional
sites of selenoprotein expression (Zhang et al., 2008).
This organ-specific prioritization of Se to the brain is at least partially explained by
receptor-mediated uptake of SEPP1 in a tissue specific manner. This unique
selenoprotein is only found in metazoans, and has high Se content with as many as 28
19
Sec residues in the sea urchin Strongylocentrotus purpuratus (Lobanov et al., 2008). The
C-terminal region of SEPP1, containing nine of the ten Sec residues in primates and
rodents, is involved in maintaining stable brain Se concentration during dietary variation.
SEPP1 is thought to be primarily produced in the liver and secreted to blood to transport
Se, however hepatic SEPP1 deficiency does not alter brain Se levels (Schweizer et al.,
2005). Rather, Se levels in brain, particularly hippocampus, are lowered by genetic
deletion of full-length SEPP1 or the C-terminus (Hill et al., 2007, Nakayama et al.,
2007). These mutations cause brain Se concentration to drop by a greater extent than can
be achieved by dietary Se deficiency, providing further evidence that SEPP1 facilitates a
steady supply of Se to the brain.
Mice lacking SEPP1 develop sensory, motor, and cognitive neurological impairment
including spasticity, hyperreflexia, gait disruption, and a spatial learning deficit. These
animals also display widespread neurodegeneration which, along with the behavioral
phenotype, is modulated by dietary Se status (Burk & Hill, 2009). However, Se-
supplemented SEPP1-deficient mice still exhibit a profound deficit in synaptic long term
potentiation (Peters et al., 2006). This suggests that beyond Se delivery, SEPP1 may
function in cell signaling, which is established for the SEPP1 receptor ApoER2.
ApoER2-deficient mice develop a phenotype similar to but less severe than SEPP1
knockout mice in terms of brain Se concentration and neurological impairment (Burk et
al., 2007). Alternatively, SEPP1 could be a preferred Se source within brain that is not
readily compensated for by dietary sources. Indeed Se supply to cultured Jurkat cells,
assessed by stimulation of GPX activity, is 5-100 times more efficient with SEPP1 than
other Se-containing proteins and compounds (Saito & Takahashi, 2002).
ApoER2 is a member of the low-density lipoprotein receptor-related protein family that is
highly expressed in brain. It exists in multiple isoforms, and has been observed in all
brain cell types (Fan et al., 2001, Korschineck et al., 2001). Expression at some
excitatory synapses, where it is associated with the postsynaptic densities, can cause
formation of a functional complex with N-methyl-D-aspartate (NMDA) receptors
(Beffert et al., 2005). This association allows ApoER2 to modulate NMDA receptor
20
activity, synaptic neurotransmission, and memory in mice. Although these effects have
been demonstrated using a different ApoER2 ligand, Reelin, alterations in synaptic
plasticity and memory in SEPP1-deficient mice suggest that both ligands can actively
modulate synaptic function.
The central nervous system is protected from drugs, peptides, and other substances in the
peripheral circulation by the blood-brain-barrier (BBB). It is chiefly composed of tight
junctions between endothelial cells of the cerebral vasculature. Additionally, the vast
majority of cerebral vessels are lined by astrocytic processes termed end-feet. This
anatomical microarchitecture provides a high trans-endothelial electrical resistance, and
prohibits passage of the vast majority of circulating blood components, except for small,
lipophilic molecules. Since blood constituents, chiefly oxygen and glucose, are absolutely
essential for nervous system function, the BBB contains an array of transmembrane
proteins that transport required substrates (Zlokovic, 2008).
Before the knowledge of particular receptors for SEPP1, endothelial cells of the cerebral
vasculature were observed to bind high quantities of this protein (Burk et al., 1997).
Recently, ApoER2 was identified as a specific component of the BBB based on its highly
enriched mRNA expression in mouse brain microvascular endothelial cells compared to
liver and lung endothelial cells (Daneman et al., 2010). However at the BBB, ApoER2 is
apparently present on the abluminal or basolateral side of endothelial cells, in between
the capillary lumen and the astrocytic endfeet (Elali & Hermann, 2010). In other words
ApoER2 is on the brain side of the cerebral microvascular endothelium. Thus, uptake of
SEPP1 within brain by endothelial ApoER2 may retain Se in brain. However, transfer of
SEPP1 across the BBB may involve additional receptors, such as Megalin. Megalin is the
primary SEPP1 receptor in kidney and is responsible for reabsorption of SEPP1 from the
glomerular filtrate by proximal convoluted tubule epithelial cells (Olson et al., 2008).
Interestingly, Megalin facilitates transcytosis of apolipoprotein J from blood to brain at
the cerebral vascular endothelium and the choroid plexus epithelium (Zlokovic et al.,
1996). Furthermore apolipoprotein J, a.k.a. Clusterin, associates with SEPP1 in high
molecular weight complexes in plasma, and Megalin-mutant mice have reduced Se in
21
brain and the periphery (Cheung et al., 2010, Chiu-Ugalde et al., 2010). Therefore, the
two receptors may work in tandem to take up blood SEPP1 and/or retain central SEPP1
depending on local demand, physiological activity, and developmental stage. Since
transgenic mice lacking SEPP1 or its receptors have some Se in brain, complementary
mechanisms affecting brain and whole body Se distribution are additionally inferred to
exist.
The choroid plexus is a heavily vascularized cuboidal epithelium protruding from the
ependymal walls of the cerebral ventricles. This structure produces cerebrospinal fluid
(CSF), the extracellular fluid bathing the nervous system, essentially by filtering blood.
SEPP1 is found in CSF, and choroid plexus highly expresses the mRNA, implying that it
is secreted (Scharpf et al., 2007). The ventricular regions are known to have high Se by
autoradiography, positioning the choroid plexus and CSF as primary sites of brain Se
homeostasis by virtue of SEPP1 expression and secretion (Kuhbacher et al., 2009).
Choroid plexus also expresses abundant mRNA for nearly all other selenoproteins, the
synthesis factors, the SEPP1 receptors ApoER2 and Megalin, and SCLY, further
suggesting a role in transfer of Se from peripheral blood to the central nervous system
(Lein et al., 2007).
The role of SCLY in the brain has received little attention. Although SEPP1 is
established in promoting brain Se concentration and selenoprotein expression, a complete
mechanism is lacking. This is because SEPP1 supplies Se in the form of Sec residues,
while selenoprotein synthesis is thought to require selenide to generate selenophosphate
and Sec co-translationally. The enzymatic activity of SCLY generates selenide from Sec,
and the protein has been suggested to associate with SPS enzymes to recycle Se from Sec
in support of selenoprotein synthesis (Tobe et al., 2009). SCLY protein and activity is
found in mouse brain, albeit at much lower levels than in kidney and liver (Mihara et al.,
2000). Curiously, the testes, which also has a blood-tissue barrier and relies on SEPP1 for
Se, show a pattern whereby cells expressing ApoER2 do not express SCLY and vice
versa (Kurokawa et al., 2011). Specifically, cells exposed to circulating SEPP1 express
ApoER2, whereas germ cells expressing other selenoproteins express SCLY. This
22
suggests that uptake of SEPP1 and utilization of the Sec residues may depend on multiple
interacting cell types, rather than cell autonomous utilization. In brain, where neurons,
astrocytes, oligodendrocytes, microglia and endothelial cells interact in a complex
system, Se distribution between cells and extracellular compartments may similarly occur
by cell-type and -context dependent expression of SEPP1, ApoER2, and SCLY. However
the exact nature of this system, both in body and brain, has not been fully elucidated.
23
FIGURE LEGENDS
Figure 1: Assimilation of Se into selenoproteins. Dietary forms of Se in vertebrates
include selenate, selenite, selenocysteine, and selenomethionine and are highlighted in
green. Dietary forms are converted to the intermediate metabolite selenide for
selenoprotein synthesis or Se excretion. Several factors are required to synthesize Sec on
its tRNA, and position it at UGA codons during translation.
Figure 2: Selenoprotein localization to subcellular domains. A partial list of mammalian
selenoproteins is depicted schematically in a prototypical cell. GPX isoforms are
distributed in the cytosolic, mitochondrial, nuclear, and extracellular compartments.
Seven selenoproteins display localization to the endoplasmic reticulum, and other
selenoproteins are also shown. Several steps involved in cellular signal transduction are
known to be redox-sensitive, and may be functionally affected by selenoproteins.
24
FIGURE 1
25
FIGURE 2
26
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37
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38
CHAPTER 2
ABSENCE OF SELENOPROTEIN P BUT NOT SELENOCYSTEINE LYASE
RESULTS IN SEVERE NEUROLOGICAL DYSFUNCTION
ABSTRACT
Dietary selenium restriction in mammals causes bodily selenium to be preferentially
retained in the brain relative to other organs. Almost all of the known selenoproteins are
found in brain, where expression is facilitated by selenocysteine-laden selenoprotein P.
The brain also expresses selenocysteine lyase, an enzyme that putatively salvages
selenocysteine and recycles the selenium for selenoprotein translation. We compared
mice with a genetic deletion of selenocysteine lyase to selenoprotein P knockout mice for
similarity of neurological impairments, and whether dietary selenium modulates these
parameters. We report that selenocysteine lyase knockout mice do not display
neurological dysfunction comparable to selenoprotein P knockout mice. Feeding a low-
selenium diet to selenocysteine lyase knockout mice revealed a mild spatial learning
deficit without disrupting motor coordination. Additionally, we report that the
neurological phenotype caused by the absence of selenoprotein P is exacerbated in male
versus female mice. These findings indicate that selenocysteine recycling via
selenocysteine lyase becomes limiting under selenium deficiency, and suggest the
presence of a complementary mechanism for processing selenocysteine. Our studies
illuminate the interaction between selenoprotein P and selenocysteine lyase 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.
39
INTRODUCTION
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 Sec synthetase
(SepSecS) in the presence of selenophosphate [reviewed in (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, 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 to 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., 2008,
Valentine et al., 2005).
If endocytosis of Sepp1 delivers Se to cells, the Sec residues from Sepp1 must be
processed for incorporation into selenoproteins. Sec lyase (Scly) catalyzes the
decomposition of Sec into alanine and hydrogen selenide (Esaki et al., 1982), and
40
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.
MATERIALS AND METHODS
Animals: Genetically modified male and female mice on a C57BL/6 background lacking
Sepp1 or Scly were bred on commercially available diets containing adequate Se (~0.25
ppm). Animals were given food and water ad libitum on a 12-hour light-cycle and group
housed until behavioral experimentation. All behavioral experiments were conducted on
single-housed adult mice aged 4-6 months during the light cycle. Male and female mice
of all genotypes were used in approximately equal numbers to examine sex differences
present in the animals. All animal procedures and experimental protocols were approved
by the University of Hawaii Institutional Animal Care and Use Committee.
Generation of Sepp1-/- and Scly-/- mice: Sepp1-/- mice were generated by
electroporating a construct into 129S9/SvEvH-derived embryonic stem (ES) cells that
were subsequently injected into C57BL/6 blastocysts. The resulting chimeric males were
bred with C57BL/6J females (Hill et al., 2003). Mutant mice were backcrossed to
C57BL/6J for at least 10 generations before arriving in our lab, and were bred with our
41
C57BL/6J colony to ensure congenic strains (Hoffmann et al., 2007). Sepp1+/- mice
were bred to generate littermate pups of Sepp1-/- knockout and Sepp1+/+ control mice.
Genotyping of the mice was carried out using methods previously described (Hoffmann
et al., 2007).
A targeting vector was generated by the NCRR-NIH supported KnockOut Mouse Project
(KOMP) Repository, and included an ~11-kb region of the wild type Scly locus
subcloned from a positively identified C57BL/6 BAC clone. The vector was designed
with one homology arm extending 5.5 kb 5’ to exon 4, and the other 5.5 kb homology
arm terminating 3’ to exon 7. A promoterless trapping cassette (L1L2_gt0) with flanking
Flp-recombinase target (FRT) sites was inserted in an intron 5’ of exon 4. Efficient
splicing to the reporter cassette results in truncation of the endogenous transcript, causing
a constitutive null mutation in the Scly gene. Cre-recombinase target loxP sites were
inserted 5’ and 3’ of critical coding exon 4. The total size of the targeting construct,
including vector backbone (L3L4_pZero_DTA_kan) and Neo cassette, was 21.642 kb.
The targeting vector was transfected into C57BL/6 embryonic stem cells by
electroporation. After selection with antibiotic, surviving clones were expanded and
analyzed by PCR to identify recombinant ES cell clones. ES cell clones were
microinjected into C57BL/6 blastocysts to produce chimeras with one wild-type and one
mutant Scly allele, which were then mated to generate Scly-/- mice on a pure C57BL/6
background. Mutant mice were backcrossed to C57BL/6J to ensure genetic comparability
with wild-type control C57BL/6J mice. The latter were maintained not more than five
generations after arrival from The Jackson Laboratory. Deletion of Scly was confirmed in
all offspring using PCR that amplified a 1.2-kb product in the targeted region present in
the wild-type allele (forward, 5’-CAC AGG TGC GGC CAT GAG GG-3’; reverse, 5’-
CTG GCT GTC CCT GAA CTA GCT TCA TA-3’) and a 233-bp product in the mutant
allele (forward, 5’-GAG ATG GCG CAA CGA AAT TAA T-3’; reverse, 5’- CTG GCT
GTC CCT GAA CTA GCT TCA TA -3’).
Diets: For dietary experiments, animals were switched from standard laboratory diets
containing ~0.25 mg/kg Se to defined diets at the time of weaning (3–4 wk of age). Mice
were fed Open Source Diets purchased from Research Diets containing either 0.08 mg/kg
42
(cat.#D19101) or 1.0 mg/kg (cat.#D05050403) Se. The diets were formulated with
purified ingredients and contained 20.3% protein, 66% carbohydrate, and 5% fat. The
protein source was casein, which was also the source of Se in the low Se diet. For high Se
diet, sodium selenite was added to achieve final Se levels. Multiple lots were
independently tested to confirm the Se concentration by inductively coupled plasma-MS
(Bodycote) with lot-to-lot variation at or below the detection limit of inductively coupled
plasma-MS testing (0.02 mg/kg). Mice were fed the defined diets from weaning until the
time of sacrifice. Se-adequate standard lab diets containing ~0.25 mg/kg Se are not
completely defined, and may be considered slightly supplemented compared to the rodent
RDA of 0.15 mg/kg Se. The low-Se diet containing 0.08 mg/kg Se is marginally
deficient, and causes moderate selenium deficiency in mice (Hoffmann et al., 2010). The
high-Se diet containing 1.0 mg/kg Se has been shown to prevent many of the
neurological symptoms in Sepp1-/- mice (Hill et al., 2003). This range of dietary Se
concentration reflects the global spectrum of human Se intake, from deficiency to
therapeutic supplementation.
Animal Behavior:
Spontaneous activity. Animals were placed in a transparent cylinder (20 cm diameter, 20
cm height) and activity was videotaped for three minutes. The cylinder was situated on
clear plexiglass with a mirror placed at an angle underneath for clear view of movement
along the ground as well as along the walls of the cylinder. The number of rears, forelimb
and hindlimb steps, and time spent grooming were measured. Videotapes were scored in
slow motion by an experimenter blind to the mouse genotype. A rear was counted when
an animal made a vertical movement with both forelimbs removed from the ground.
Forelimb and hindlimb steps were counted when an animal moved both forelimbs or both
hindlimbs across the floor of the cylinder. Number of steps, rears, and time spent
grooming were compared for wild-type and knockout mice (Fleming et al., 2004).
Open field. Animals were placed in a square box (50 cm sides, 40 cm walls) and
monitored by overhead camera linked to computer-assisted tracking software. During the
test, the mice were allowed to move freely around the open field and to explore the
environment for five minutes. The path of each mouse was automatically recorded, and
43
recordings were then analyzed. Total distance traveled, number of rears, time spent
grooming, and center time was compared between groups.
Vertical pole. The vertical pole descent test has been used to assess coordination and
basal ganglia related movement disorders in transgenic mice (Fleming et al., 2004).
Animals were placed head-up on top of a vertical wooden pole 50 cm long (1.2 cm in
diameter). The base of the pole was fixed in plexiglass and put in the home cage. When
placed on the pole, animals orient themselves downward and descend the length of the
pole back into their home cage. Knockout and wild-type mice received two days of
training consisting of five trials per day. On the third day, animals received five trials,
and time to orient downward (turn) and total time to descend (total) were measured with
a stopwatch. The best performance over the five trials was used for both wild-type and
knockout mice.
Inverted grid. The ability to hang upside down is a test of neuromuscular strength
(Crawley, 1999). Each mouse was placed on a wire grid (mesh, 12 cm2 with 0.5 cm
2
squares) 20 cm above a table top for 120 sec and videotaped. The lid was gently turned
upside down, 60 cm above a soft surface to avoid injuries. The latency to fall was timed.
Each mouse was given up to two attempts to hold on to the inverted grid for a maximum
of 120 seconds and the longest period was recorded.
Morris water maze. A circular pool (2 m in diameter, 1 m deep) surrounded by constant
external cues was located in an observation room and filled with 24°C water. White non-
toxic paint was added to make the water translucent. Tests were performed under dim
light conditions. Lights below the height of the tank, necessary for video capture, also
provided light for the swimming mouse. A small circular escape platform (7 cm diameter,
located just below the water surface, or protruding just above the water) was placed in a
constant location in the center of quadrant 1. Four equally spaced points around the wall
of the pool were used as starting points. The mice were given one block of four trials
each day with an inter-trial interval of 5 to 10 min. Each trial started from one of four
different points, in a semi-random order. The mouse was allowed to swim around until it
located the platform or 60 sec, at which point the mouse was placed on the platform by
the experimenter and allowed to stay on the platform for 15 sec. The time to locate the
platform was recorded as escape latency during training days. After sufficient training a
44
one-minute probe trial was performed, in which the platform was removed and the path
of each mouse on each trial was automatically recorded and then analyzed. Time
investigating the target, opposite, and adjacent quadrants, platform latency and platform
crossings were measured. Average swim speed was calculated from total distance
traveled per 60 sec trial. Animals that spent significant time floating, which occurred
sporadically in very few animals independent of genotype, were excluded from this
analysis.
Quantitative RT-PCR: Animals were sacrificed by CO2 asphyxiation, or deeply
anesthetized with tribromoethanol and decapitated, and the brains rapidly excised,
washed in PBS and snap-frozen in liquid nitrogen. Tissue was ground into powder, using
a mortar and pestle on dry ice, and collected into pre-chilled tubes. Total RNA from
tissue was prepared by Trizol extraction (Invitrogen, Carlsbad, CA, USA) followed by
purification using the RNeasy kit (QIAgen, Valencia, CA, USA). Concentration and
purity of extracted RNA and synthesized cDNA was determined using A260/A280 ratio
measured on an ND1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE,
USA). Synthesis of cDNA was carried out using High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Foster City, CA, USA), with 1 g RNA per 20 l
reaction. For real-time PCR, 100 ng of the cDNA was used in 5 l reactions with
Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Reactions were carried out in
triplicate or quadruplicate in a LightCycler 480 II thermal cycler (Roche, Indianapolis,
IN, USA). Cycling conditions followed the manufacturers suggestions in the SYBR
Green kit instructions. All qPCR results were normalized to 18S rRNA expression as a
housekeeping gene and analyzed using Absolute Quantification Software (Roche).
SDS-PAGE and Western blot: Total protein was extracted from powdered mouse tissues
by light sonication in CelLytic MT buffer (Sigma, St. Louis, MO, USA), followed by
centrifugation according to the manufacturers’ protocol. Protein was added to reduced
Laemmli buffer, boiled for 10 minutes, and loaded into 4-20% gradient polyacrylamide
gels (Bio-Rad, Hercules, CA, USA). Following electrophoresis, gel contents were
transferred to PVDF membranes, which were blocked with undiluted Odyssey Blocking
45
Buffer (Li-Cor Biosciences, Lincoln, NE, USA) for one hour. Membranes were then
probed for 90 minutes with the following primary antibodies: Goat-anti-GPX1 (R&D
Systems, Minneapolis, MN, USA), Rabbit-anti-GPX4 (AbFrontier, Seoul, Korea),
Rabbit-anti-SEPW1 (Rockland, Gilbertsville, PA, USA), and Mouse-anti-alpha Tubulin
(Novus, Littleton, CO, USA). After washing with PBS containing 0.05% Tween-20
(PBST), membranes were incubated in the dark in secondary antibodies labeled with
infrared fluorophores (Li-Cor Biosciences). After further washes in PBST, blots were
imaged and quantified with the Odyssey infrared imaging system (Li-Cor Biosciences).
Glutathione peroxidase activity assay: Total glutathione peroxidase activity was
measured using the Bioxytech GPx-340 Assay kit (Oxis International, Foster City, CA,
USA). Mouse tissues for the assay were collected in the same manner as above.
Powdered tissue was homogenized in lysis buffer by sonication and centrifuged at 15,000
x g. The resulting supernatant was serially diluted to determine the linear range of the
assay. 5 l of diluted sample (1:10 for brain, 1:200 for liver) was added to 25 l of assay
buffer and 25 l of NADPH reagent in a 96-well plate. 25 l of tert-butyl hydroperoxide
was added to initiate the reaction, which was monitored for 10 minutes at room
temperature by measuring kinetic absorbance at 340 nm on a SpectraMax M3 microplate
reader (Molecular Devices, Sunnyvale, CA, USA).
Statistical analysis: Data were analyzed using Microsoft Excel (Redmond, WA, USA),
and plotted using GraphPad Prism software (San Diego, CA, USA). Two-way ANOVA
was used to determine an interaction between genotype and sex for all experiments. If no
interaction effect was observed, male and female groups were sometimes combined.
Repeated measures ANOVA was used to assess training in the water maze, and genotype
and quadrant were the two factors for analyzing quadrant investigation on the probe trial.
Post hoc test using Bonferroni correction for multiple comparisons was used to determine
significance between individual groups. When male and female groups were combined,
unpaired t-tests comparing genotypes were performed for end-point measures in the
water maze and for biochemical experiments. The significance criteria were set at p <
0.05 for all statistical measures.
46
RESULTS
Neurological motor phenotype in Sepp1-/- mice is more pronounced in males
Genetic deletion of Sepp1 in mice results in neuromotor impairments (Hill et al., 2004,
Schomburg et al., 2003). We characterized the phenotype of Sepp1-deficient mice raised
on a standard lab diet using a battery of behavioral tests to assess motor function in male
and female mice. We found that motor coordination was worse in male knockout animals
compared to females. Spontaneous locomotor activity, as assessed using the cylindrical
chamber, was greatly decreased in the male Sepp1-deficient mice while being slightly
decreased in the female knockout animals, when compared to wild-type mice [Fig. 1A].
Two-way ANOVA revealed an effect of genotype on spontaneous rearing
(F(1,32)=13.06, p=0.0010) [Fig. 1A, left] and grooming (F(1,32)=16.93, p=0.0003) [Fig.
1A, center]. Post hoc analysis indicated a statistically significant decrease in rearing
(t(16)=3.464, p<0.01) and grooming (t(16)=2.532, p<0.05) in male Sepp1-/- mice,
whereas only grooming (t(16)=3.287, p<0.01) was decreased in female Sepp1-/- mice,
when compared to wild-type. Despite these differences, we did not observe any genotype
or sex interaction effects on total distance traveled in the open field [Fig. 1A, right]. To
assess motor coordination and strength, animals were subjected to the pole descent and
inverted hang tests. Two-way ANOVA revealed an interaction between genotype and sex
on time to turn (F(1,42)=16.57, p=0.0002) [Fig. 1B, left] and descend (F(1,42)=5.222,
p=0.0274) [Fig. 1B, center] a vertically oriented pole. Genotype strongly affected ability
to hang upside down for 120 seconds (F(1,32)=16.10, p=0.0003), however only male
Sepp1-/- mice were significantly different from wild-type control mice (t(16)=4.045,
p<0.001) [Fig. 1B, right].
Dietary Se supplementation alleviates motor deficits in Sepp1-/- mice
Previous studies have shown that Se supplementation can attenuate neuromotor
impairments in Sepp1-/- mice (Schweizer et al., 2004). We determined if gender could
influence this attenuation. Animals were supplemented with 1 mg/kg Se in the diet
starting at weaning, and assayed in the same manner as those fed a standard diet. We
found that Se-supplemented Sepp1-/- mice do not differ from wild-type mice in
47
spontaneous rearing [Fig. 2A, left], grooming [Fig. 2A, center] and distance traveled [Fig.
2A, right]. Performance on the pole descent test also improved in Se-supplemented mice,
however a main effect of genotype remained for the time to invert (F(1,33)=12.68,
p=0.0011) [Fig. 2B, left] and descend (F(1,33)=4.989, p=0.0324) [Fig. 2B, center] the
vertical pole, and post hoc test indicated male Sepp1-/- mice were slower than wild-type
littermates in turn time only (t(19)=4.004, p<0.001). Performance on the inverted hang
test also improved in the Se-supplemented Sepp1-/- mice, and an effect of genotype did
not reach statistical significance (F(1,31)=3.257, p=0.081) [Fig. 2B, right]. In all motor
tests, Se-supplemented Sepp1-/- mice showed subtle qualitative behavioral deficits in
both sexes, but the male bias was largely eliminated.
Generation and characterization of Scly-/- animals
Scly knockout mice were generated by a conditional knockout approach in the event that
deletion of Scly caused embryonic lethality. Mice were generated with a trapping cassette
inserted upstream of exon 4 of the Scly gene, resulting in a constitutive null mutation in
the whole animal [Fig 3A]. Mutation of the targeted region of the Scly gene was
confirmed by PCR of mouse tail DNA [Fig. 3B], and Scly mRNA was undetectable by
qPCR in all tissues examined from Scly-/- mice, including brain [Fig. 3C]. Scly-/- mice
were born at the expected Mendelian ratio, appeared generally healthy, and developed to
adulthood. Unlike Sepp1-/- mice, Scly-/- mice were fertile, producing viable offspring
that did not differ in size at birth compared to wild-type control mice.
Neurological motor phenotype is largely absent in Scly-/- mice
Scly is a relatively uncharacterized enzyme thought to be involved in recycling Se from
Sec in support of selenoprotein synthesis. Sepp1 contains up to 10 Sec residues and is
proposed to be a source of Se for the brain. We hypothesized that mice lacking Scly
would be unable to efficiently catalyze Sec degradation, and therefore would manifest a
phenotype similar to the Sepp1-/- mice. Contrary to our prediction, we found that Scly-/-
animals raised on a Se-adequate diet displayed no neurological phenotype. Spontaneous
rearing and movement were unaffected by Scly genotype, and performance on the
vertical pole descent was similar between Scly+/+ and Scly-/- mice [data not shown].
48
As no apparent behavioral phenotype presented in the Scly-/- mice raised on a standard
lab diet, we subjected the animals to a marginally low Se diet, containing 0.08 mg/kg Se,
to assess whether the animals display enhanced sensitivity to Se deficiency as measured
by locomotor behavior. Total distance traveled in the open field was unaffected by Scly
genotype under low-Se conditions, however two-way ANOVA revealed an interaction
between Scly genotype and sex on rearing activity (F(1,22)=4.937, p=0.0369) [Fig. 4A].
Motor coordination assayed by turn and total time in the vertical pole descent test
indicated no difference due to genotype in Se-deficient Scly-/- animals [Fig. 4B].
Spatial learning is sensitive to disruptions in Se availability
The Morris water maze is a paradigm for assessing spatial learning and memory in
rodents (Morris, 1984). Sepp1 knockout mice raised on a Se-supplemented diet have mild
impairments in learning measured with this paradigm, despite having a large deficit in
long-term potentiation, a cellular model for learning and memory (Peters et al., 2006). As
Se supplementation attenuates neurological impairments in Sepp1 knockout mice, we
questioned if learning deficits would be greater for Sepp1-/- mice raised on a normal Se
diet. Mice were initially trained over several days to locate a hidden platform in a large
tank of water. Subsequently the platform was removed for one final trial, in which the
amount of time the animal investigated the area where the platform used to be was
measured. We compared male and female Sepp1-/- and wild-type animals raised on
standard laboratory chow with adequate Se for learning deficits.
In contrast to our findings of gender differences in neuromotor function, we did not
observe significant learning differences between Sepp1-/- male and female mice.
Therefore male and female groups were combined after eliminating sex as an interacting
variable. We found that learning was impaired in Sepp1-/- mice fed a standard diet [Fig.
5A]. Two-way ANOVA revealed an interaction between genotype and training
(F(7,112)=2.361, p=0.0275). Escape latency over time was not substantially reduced in
Sepp1-/- as compared to control mice, suggesting the mice did not learn the spatial
location of the platform. The probe trial results are ambiguous since the training was
ineffective in Sepp1-/- mice; however a genotype x quadrant interaction effect was
49
observed (F(3,64)=3.277, p=0.0266) during quadrant investigation [Fig. 5B]. Sepp1-/-
mice spent significantly more time than controls investigating the opposite quadrant,
which was not due to uncoordinated swimming, and likely indicates failure to learn the
platform location. There was a strong trend towards reduced number of platform
crossings (t(16)=2.064, p=0.0557) [Fig. 5C], but swim speed (t(14)=0.7262, p=0.48)
[Fig. 5D] was not significantly different between genotypes by t-test. Similar to published
work on Se-supplemented Sepp1-/- mice in the water maze, we found a genotype
difference during training, but not on the probe trial in Sepp1-/- mice fed a high Se diet
[data not shown].
Although we found little difference between Scly-/- and control mice in locomotor
activity, we assayed the Scly-/- animals using the Morris water maze for comparison with
Sepp1-/- mice. Scly-/- mice fed standard chow performed like wild-type mice during
training [Fig. 6A], as the only effect observed by two-way ANOVA was for training day
(F(7,154)=24.07, p<0.0001). Similarly, in the probe trial we found an effect of quadrant
(F(3,88)=5.78, p=0.0012), but no interaction with genotype [Fig. 6B]. Platform crossings
(t(22)=0.5415, p=0.59) [Fig. 6C], latency to platform location [data not shown], and
swim speed (t(22)=0.4955, p=0.63) [Fig. 6D] were not significantly different between
genotypes when assessed by t-test. Scly-/- mice showed a trend towards delayed learning
on days three and four. The lack of spatial learning impairment in Se-adequate Scly-/-
mice starkly contrasted with Sepp1-/- mice fed the same diet.
The trend toward mild learning deficits in Scly-/- mice led us to question if a restricted Se
diet would result in greater learning impairments in these animals. When Scly-/- animals
fed a Se-deficient diet were assessed, two-way ANOVA revealed an interaction between
genotype and training (F(5,120)=2.496, p=0.0345), while post hoc tests indicated longer
latency times on days 2 (t(24)=2.942, p<0.05), 3 (t(24)=3.467, p<0.01), 5 (t(24)=2.895,
p<0.05), and 6 (t(24)=3.037, p<0.05) in the knockout mice [Fig. 7A], which suggests
reduced learning. However the mice learned the platform location and the deficit was not
as severe as in non-supplemented Sepp1-/- animals. Scly-/- animals performed as well as
wild-type animals in the probe trial. We found a main effect on quadrant investigation
50
(F(3,96)=11.19, p<0.0001) but no Scly genotype interaction [Fig. 7B]. We found no
difference in platform crossings (t(24)=1.089, p=0.29) [Fig. 7C], latency [data not
shown], or average swim speed (t(22)=1.60, p=0.124) [Fig. 7D] in Scly-/- mice on a low
Se diet compared to control mice.
Selenoprotein expression and Glutathione Peroxidase activity
When dietary Se is restricted in mammals, Sec-enriched Sepp1 helps maintain
selenoprotein expression in the brain (Hill et al., 2007). We therefore investigated the
expression of selenoproteins in brains of Scly-/- animals by quantitative RT-PCR,
western blot, and glutathione peroxidase (GPX) activity. We did not find a significant
change in the mRNA for Sepp1 (t(9)=1.536, p=0.1589, n=5-6), GPX1 (t(10)=1.056,
p=0.3157, n=6), GPX4 (t(10)=0.6899, p=0.5060, n=6), or selenoprotein W (Sepw1)
(t(8)=2.149, p=0.0639, n=5) in brains of Scly-/- mice fed normal chow [data not shown].
Thus Scly-/- mice on a Se-adequate diet show neither a behavioral phenotype, nor any
major changes in selenoprotein mRNA expression in brain. Therefore western blotting
and GPX activity assays to assess the severity of Se-deficiency were performed in mice
fed a low-Se diet only.
Scly-/- animals on a low-Se diet displayed significantly elevated Sepp1 mRNA
expression in brain compared to control mice (t(14)=2.686, p=0.0177) [Fig. 8A]. GPX1
(t(12)=3.40, p=0.0053) [Fig. 8B] and GPX4 (t(14)=3.093, p=0.0079) [Fig. 8C] mRNA
were also significantly increased in brain, while Sepw1 was not changed (t(14)=0.1380,
p=0.89) [Fig. 8D]. In the same mice, we did not detect an increase in mRNA expression
of Nfs1 [(t(14)=0.7154, p=0.4861, not pictured], a cysteine desulfurase enzyme with Sec
lyase activity.
In contrast to the selenoprotein mRNA expression data, the corresponding protein levels
assessed by western blot are consistently decreased in the brains of Se-deficient Scly-/-
mice. Both GPX1 (t(14)=12.39, p<0.0001) [Fig. 9A] and Sepw1 (t(14)=8.294,
p<0.0001)) [Fig. 9C] are expressed at ~37% of the wild-type level, while GPX4
expression (t(14)=5.365, p<0.0001) [Fig. 9B] is reduced to ~60% compared to control
51
brains. These results confirm that Scly contributes to selenoprotein expression in brain
during Se-deficiency.
GPX activity in brain was reduced to 43% of the wild-type control level (t(14)=4.495,
p=0.0005) [Fig. 10A], and was reduced to 54% in liver (t(14)=9.9297, p<0.0001) [Fig.
10B] of Scly-/- mice fed a low-Se diet. However, GPX activity in serum was not
significantly reduced [Fig. 10C]. These data indicate that Scly helps maintain
selenoprotein expression and activity when dietary Se availability is limiting. They
further suggest that Se status in tissues may be more affected than plasma in the absence
of Scly. The contrast of increased selenoprotein mRNA expression in the Scly-/- mouse
brain despite reduced selenoproteins and enzyme activity suggests that Scly is a
significant contributor to the Se pool for selenoprotein translation.
DISCUSSION
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 Se concentration 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
52
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, SBP2) and
nucleic acids (tRNA(Sec)
, SECIS-containing mRNA), in addition to ATP, 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 to 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.
ApoER2 is found at synaptic sites (Beffert et al., 2005), in cultured astrocytes,
oligodendrocytes, and microglia (Fan et al., 2001), and in the brain vasculature
(Korschineck et al., 2001), suggesting that all brain cells can take up Sepp1. In adult
brain, mRNA for Sepp1 is apparent in glia (Lein et al., 2007) but protein expression is
dominant in neurons (Bellinger et al., 2008, Scharpf et al., 2007). Sepp1 mRNA and
53
protein are abundant in choroid plexus epithelium (Bellinger et al., 2008, Steinert et al.,
1998, Zhang et al., 2008). Scly mRNA appears enriched in grey matter and neurons of
mouse brain (Lein et al., 2007). Further, a mouse proteomics study identified Scly in
synaptoneurosomes (Filiou et al., 2010).
We found that mRNA for Scly was not significantly changed in brain of Sepp1-/- mice
fed a standard diet. Since Sepp1-/- mice have depressed Se in brain, this finding suggests
that Scly is not Se-regulated in brain, 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 to 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 demonstrated 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
54
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 to 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,
55
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 that 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 impacts spatial learning.
56
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FIGURE LEGENDS
Figure 1: Spontaneous activity and motor coordination is more reduced in male than
female Sepp1-/- mice compared to control mice fed a standard diet. (A) Rearing and
grooming were measured in the cylinder, and total distance traveled in the open field.
Sepp1-/- mice were less active as measured by rearing (left, genotype **p<0.01) and
grooming (center, genotype ***p<0.001), but did not show decreased exploration of the
open field (right). (B) Motor coordination was measured using the pole test and inverted
hang test. Sepp1-/- mice took longer to turn (left, genotype x sex ***p<0.001) and
descend the pole (center, genotype x sex *p<0.05), and had reduced ability to suspend
themselves for two minutes (right, genotype ***p<0.001). Values are expressed as means
± SEM. n=8-12 per group, *p<0.05, **p<0.01, ***p<0.001, compared with control mice.
Figure 2: High Se diet improves spontaneous activity and motor coordination more in
male than female Sepp1-/- mice compared to control mice. (A) Rearing and grooming
were measured in the cylinder, and total distance traveled in the open field. Se-
supplemented Sepp1-/- mice were as active as controls when measured by rearing (left),
grooming (center), and exploration of the open field (right), and no sex differences were
observed. (B) Motor coordination was measured using the pole test and inverted hang
test. Se-supplemented Sepp1-/- mice took longer to turn (left, genotype **p<0.01) and to
descend the pole (center, genotype *p<0.05) when compared with control mice, but were
capable of suspending themselves for two minutes (right). Male and female Sepp1-/-
mice performed similarly except for turn time, and an interaction effect between
genotype and sex was not statistically evident in any test. Values are expressed as means
± SEM. n=8-10 per group, ***p<0.001, compared with control mice.
Figure 3: Generation of Scly-knockout mice. (A) Image from KOMP. A promoterless
trapping cassette was inserted upstream of exon 4 of the mouse Scly locus on
chromosome 1, causing splicing at the cassette and truncation of the endogenous
transcript. The cassette was flanked by FRT sites for conditional excision of the cassette
by breeding with FLP-recombinase transgenic mice in case of embryonic lethality.
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Presence of the loxP sites flanking exon 4 allowed excision of a coding exon critical for
enzymatic function by breeding with Cre-recombinase transgenic mice. (B) PCR
genotyping of mice tails was performed to detect presence of wild-type allele (1.2 kb) or
knockout allele (233 bp) using primers described in Materials and Methods. (C)
Quantitative RT-PCR analysis of brains from Scly+/+ and Scly-/- mice indicated no
detectable Scly mRNA in homozygous knockout mice.
Figure 4: Spontaneous activity and motor coordination is normal in male and female
Scly-/- mice fed a low Se diet. (A) Rearing and total distance traveled was measured in
the open field. Scly-/- mice were as active as controls when measured by exploration of
the open field (right), however an interaction between genotype and sex affected rearing
activity (left, genotype x sex *p<0.05). (B) Motor coordination was measured using the
pole test. Scly-/- mice and control mice performed similarly to turn (left) and descend the
pole (right). Male and female mice performed similarly, and no genotype effects were
present. Values are expressed as means ± SEM. n=6-7 per group.
Figure 5: Spatial learning and memory is disrupted in Sepp1-/- mice fed a standard diet.
(A) Average escape latency per training day over time was measured in the Morris Water
Maze. An interaction between Sepp1 genotype and training was present (*p<0.05) and
Sepp1-/- mice had significantly longer latency on days 7 and 8 (*p<0.05). (B) In the
probe trial, Sepp1-/- spent more time exploring the opposite (OP) quadrant (*p<0.05) and
we found a significant interaction effect (genotype x quadrant *p<0.05). They also had
non-significant trends toward fewer platform crossings (p=0.0557) (C) and a reduced
swim speed (p>0.05) (D). Values are expressed as means ± SEM. n=8-10 per group.
Figure 6: Spatial learning and memory is not disrupted in Scly-/- mice fed a standard diet.
(A) Average escape latency per training day over time was measured in the Morris Water
Maze. Scly-/- and control mice showed strongly reduced latency over time, and no
interaction between genotype and training was apparent. (B) In the probe trial at the end
of training, Scly-/- and control mice spent equal time exploring the target (TQ), left (LA)
and right adjacent (RA), and the opposite (OP) quadrants (p>0.05). Both genotypes had
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similar platform crossings (C) and swim speed (D) (p>0.05). Values are expressed as
means ± SEM. n=11-13 per group.
Figure 7: Spatial learning is mildly impaired in Scly-/- mice fed a low Se diet. (A)
Average escape latency per training day over time was measured in the Morris Water
Maze. An interaction between Scly genotype and training was present (*p<0.05), and
Scly-/- mice had significantly longer latency on days 2, 3, 5 and 6 (*p<0.05). (B) In the
probe trial at the end of training, Scly-/- and control mice spent equal time exploring the
target (TQ), adjacent (LA, RA) and opposite (OP) quadrants (p>0.05). Both genotypes
had similar platform crossings (C) and swim speed (D) (p>0.05) in the probe trial. Values
are expressed as means ± SEM. n=13 per group.
Figure 8: Expression of selenoprotein transcripts is increased in Se-deficient Scly-/- mice
brains. (A) Sepp1 mRNA level was measured in brains of Scly-/- mice (n=8). We
additionally measured GPX1 (n=7) (B), GPX4 (n=8) (C), and Sepw1 (n=8) (D) mRNA
expression in brain of Scly-/- mice fed a low Se diet. Sepp1, GPX1, and GPX4 were
increased (*p<0.05, **p<0.01), while Sepw1 was unchanged. Samples were assayed in
triplicate or quadruplicate and values were normalized to 18s rRNA and expressed as
means ± SEM.
Figure 9: Expression of selenoproteins is decreased in Se-deficient Scly-/- mice brains.
(A) GPX1, (B) GPX4, and (C) Sepw1 protein was measured by western blot and
quantified by integrated intensity (n=8). Below each graph is a representative sample of
the blot including loading equivalence determined by tubulin. GPX1, GPX4, and Sepw1
were drastically reduced in Scly-/- mice (***p<0.0001). All selenoprotein values are
normalized to relative amounts of tubulin, and expressed as means ± SEM.
Figure 10: Glutathione peroxidase activity is decreased in Se-deficient Scly-/- mice
brains. Total GPX activity in brain (n=8) (A), liver (n=8) (B), and serum (n=12-14) (C)
was assayed with a coupled reaction measuring NADPH oxidation. Mice were fed the
low Se diet from weaning until time of sacrifice, when tissues were harvested for
64
analysis. Although both brain and liver GPX activity were reduced by approximately half
(***p<0.001), serum GPX activity was not significantly reduced. Values are standardized
to total protein concentration (brain, liver) or volume of serum and expressed as means ±
SEM.
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FIGURE 1
Normal diet
66
FIGURE 2
High Se diet
67
FIGURE 3
68
FIGURE 4
Low Se diet
69
FIGURE 5
70
FIGURE 6
71
FIGURE 7
72
FIGURE 8
73
FIGURE 9
74
FIGURE 10
75
CHAPTER 3
EXPRESSION OF SELENOPROTEIN W IN NEURONS EXTENDS INTO
PROCESSES AND IS HIGHLY DEPENDENT ON SELENOPROTEIN P
ABSTRACT
During dietary selenium deprivation, bodily selenium is prioritized to the brain to
maintain selenoprotein expression by a process that depends on selenoprotein P.
Selenoprotein W is a small thioredoxin-like protein that is abundant in brain and muscle
tissues. Although peripheral expression of selenoprotein W is reduced by dietary
selenium deficiency, brain expression is maintained, suggesting it has an important
function in nervous tissue. We assessed the regional, cellular, and subcellular expression
of selenoprotein W in brains of wild-type mice and mice lacking selenoprotein P. We
found that selenoprotein W is widespread in neurons, processes, and neuropil of mouse
brain. Pyramidal neurons of somatosensory, motor, piriform, and cingulate cortex, and
CA1 and CA3 of hippocampus express high levels of selenoprotein W. Purkinje neurons
and their heavily branched dendritic arbors in cerebellum also express abundant
selenoprotein W. Analysis of synaptosome fractions indicated that selenoprotein W is
present at synapses, and expression is dramatically reduced in mice lacking selenoprotein
P. Several components of the selenoprotein synthesis machinery were also found in
isolated nerve terminals. These results indicate that widespread neuronal expression of
selenoprotein W relies on selenoprotein P for selenium. They further suggest that
selenoprotein W synthesis may occur in distal comparments of neurons, far removed
from the nucleus.
76
INTRODUCTION
Selenium (Se) is a trace micronutrient that is incorporated into antioxidant enzymes. Se is
unique among trace elements because it is covalently incorporated into proteins as the
amino acid selenocysteine (Sec). Biosynthesis of Sec and insertion of the residues into
polypeptides requires a Sec Insertion Sequence (SECIS) and several specific proteins to
reinterpret in-frame UGA codons in selenoprotein mRNAs as Sec incorporation sites
[reviewed in (Bellinger et al., 2009)]. Of the 25 primate (24 rodent) Sec-containing
selenoproteins that have been identified using bioinformatics, the glutathione peroxidase,
thioredoxin reductase, and iodothyronine deiodinase enzyme families are functionally
characterized.
Selenoprotein W (Sepw1) is the smallest mammalian selenoprotein and is one of the most
widely distributed selenoproteins across species in all domains of life (Lobanov et al.,
2009, Zhang & Gladyshev, 2008). It was putatively identified in the early 1970s as being
absent in muscle of myopathic lambs suffering from White Muscle disease, but was not
purified, cloned, and named until some 20 years later (Vendeland et al., 1993, Vendeland
et al., 1995, Whanger, 2000). White muscle disease is a Se-responsive muscular
dystrophy syndrome in ruminants, and was named because of the appearance of pale and
dry muscle, usually with longitudinal striations or chalky whiteness caused by abnormal
calcium deposition. Leg muscles are typically disrupted first, but all muscles, including
cardiac, can be affected.
Like most of the selenoproteins, Sepw1 is expected to be involved in oxidation-reduction
(redox) reactions. Indeed, it has been shown to act as a glutathione-dependent antioxidant
that protects cells from peroxide-mediated damage (Jeong et al., 2002). However, the
specific antioxidant function of Sepw1 has been disputed (Xiao-Long et al., 2010), and a
prominent role in cell signaling has also been proposed (Hawkes & Alkan, 2010). Sepw1
directly interacts with 14-3-3 proteins (Aachmann et al., 2007, Dikiy et al., 2007), and
siRNA knockdown of Sepw1 expression inhibits cell proliferation in a p53- and
p21-dependent mechanism (Hawkes et al., 2012).
77
In addition to expression in muscle and proliferating myoblasts, the Sepw1 gene is also
highly expressed in developing and adult mouse brain (Gu et al., 2000, Loflin et al.,
2006). However, unlike muscle, dietary Se depletion does not cause a reduction in Sepw1
levels in sheep or rat brain, despite reducing brain Se concentration and GPX activity
(Sun et al., 2001, Whanger, 2001). Selenoprotein P (Sepp1) supplies Se to the brain, and
mice deficient in Sepp1 have greatly reduced levels of Sepw1 mRNA and protein in brain
(Hoffmann et al., 2007). These data suggest that preferential retention of Sepw1 in brain
during dietary Se-deficiency is maintained by Sepp1. Regional analysis of Sepw1 mRNA
expression in mice brains suggests presence in neurons, with high expression in >90% of
brain regions (Zhang et al., 2008). Intriguingly, Sepw1 mRNA has also been identified in
processes of cultured central and peripheral neurons [(Willis et al., 2005, Willis et al.,
2007), see supplemental tables]. However, the protein expression and function of Sepw1
in brain remains largely unexplored.
In this report, we analyzed expression of Sepw1 protein in mouse brain and mouse
brain-derived primary cells. We report that Sepw1 protein expression is observed in
neurons of several brain regions including cortex, hippocampus, and cerebellum. Sepw1
immunoreactivity extends into the processes of these cells, and isolation of nerve
terminals by synaptosome preparations revealed the presence of Sepw1. We have also
identified several components of the selenoprotein synthesis machinery in isolated nerve
terminals. Additionally, expression of Sepw1 in synaptic and non-synaptic fractions was
reduced in Sepp1-deficient mice, despite no change in selenoprotein synthesis machinery.
Taken together these data suggest that Sepw1 is highly expressed in neurons and may be
locally synthesized in distal processes far removed from the nucleus, including synaptic
sites. The widespread neuronal expression of Sepw1 is dependent on Sepp1, although the
cells producing Sepp1 for Sepw1 expression are undetermined. The enzymatic role of
Sepw1 is unclear, but the combined data argue for a role in oxidant-mediated cell
signaling rather than detoxification of oxidizing agents.
78
MATERIALS AND METHODS
Primary cell culture: Glass bottom tissue culture plates (World Precision Instruments,
Sarasota, FL) were coated with 0.1 mg/ml laminin in 0.1 mg/ml poly-L-lysine solution
for 1 h, and then rinsed with PBS. Primary cells from cortex, hippocampus, and
cerebellum were harvested from postnatal day one C57BL/6 mice, gently dispersed by
trituration, and plated on coated dishes. Cultures were maintained at 5.0% CO2 and 5.0%
relative humidity in Neurobasal-A medium (Invitrogen) with 5% fetal bovine serum (FBS)
with the addition of 100 μM glutamate (Invitrogen) to reduce growth of glial cells and
enrich growth of neurons. B27 supplement (Invitrogen) was added to replace FBS after
24 h, and glutamate omitted from the media after 3 days. FBS lots were tested for Se
content (Bodycote, Santa Fe Springs, CA), and the selenium concentration of media
containing 10% FBS was 105 nM as determined using inductively coupled plasma-mass
spectrometry. B27 was tested for Se content (Bodycote) and the selenium concentration
of media containing 2% B27 was 93.8 nM by the same method.
Animals: C57BL/6 mice and genetically modified male mice on a C57BL/6 background
lacking Sepp1 were bred on commercially available diets containing adequate Se (~0.25
ppm). Animals were given food and water ad libitum on a 12-hour light-cycle and group
housed until experimentation. All experiments were conducted on adult mice aged 3-4
months during the light cycle. All animal procedures and experimental protocols were
approved by the University of Hawaii Institutional Animal Care and Use Committee.
Tissue Preparation: Mice were anesthetized with ketamine-xylazine, and sacrificed by
transcardial perfusion. Mice were initially perfused with phosphate-buffered saline (PBS)
to flush out blood, followed by perfusion with 4% paraformaldehyde (PFA) to fix the
tissue. The mice heads were cut off and the brains dissected out. Brains were post-fixed
in 4% PFA overnight, followed by cryoprotecting the tissue in 10% and 30% sucrose for
at least 4 hours each. The brains were then embedded in optimal cutting temperature
(OCT) compound and frozen until time of sectioning. 40 μm sections were cut on a Leica
CM1900 cryostat and saved in cryoprotectant solution, containing 0.1 M phosphate buffer,
79
30% sucrose (w/v), and 30% ethylene glycol (v/v), until time of further experimentation.
Immunohistochemistry: Primary cortical, hippocampal, and cerebellar cultures maintained
for three weeks in vitro were used for immunolabeling. Brain sections stored in
cryoprotectant in the freezer were warmed to room temperature, and sections containing
cortex and hippocampus were selected for analysis. After thorough washing, sections
were blocked in 5% normal goat serum with 0.3% Triton X-100 in PBS. After blocking,
the sections were incubated in diluted primary antibody solution overnight at 4C. The
following antibodies were used: Rabbit-anti-Sepw1 (Rockland) and Mouse-anti-Tuj1
(Covance). A control section where primary antibody was omitted was also included in
the procedure. After washing out primary antibody, sections were incubated in
species-matched secondary antibody. The secondary antibody was directly conjugated to
fluorophores (Alexa Fluor dyes, Invitrogen) for fluorescence imaging. Additonally, some
sections were dual-labeled with a fluorescent Nissl stain to label neurons (Neurotrace,
Invitrogen). Sections were then mounted onto slides, and coverslipped in VectaShield
containing DAPI for fluorescent labeling of nuclei. Additional sections were
colorimetrically developed using 3,3’-diaminobenzidene (DAB), after signal
amplification using the avidin-biotin complex method (Vector), and coverslipped using
Permount.
Synaptosome Preparation: Synaptosomes were prepared by the method of Dunkley
(Dunkley et al., 1986). Mice were anesthetized with Tribromoethanol and sacrificed by
decapitation. The brain was rapidly excised, rinsed in ice-cold 0.32 M sucrose, and
immersed in ice-cold 0.32 M sucrose with 1 mM EDTA. Brain tissue was homogenized
in 5 ml of ice-cold sucrose/EDTA by 10 strokes at 900 rpm using a pre-chilled
Teflon/glass homogenizer. The homogenate was centrifuged at 3,600 rpm for 10 minutes
at 4C in polycarbonate tubes. The resulting supernatant was collected and diluted with
sucrose/EDTA to a total volume of 9 ml, and the pellet was resuspended in sucrose/EDTA
and saved for whole cell lysis. Approximately 3 ml of diluted supernatant was loaded on
the top of a discontinuous three layer Percoll gradient. Three gradients per brain were
made by adding 2 ml of 23% Percoll to each polycarbonate tube, and slowly layering 2
80
ml each of 10% and 3% Percoll sequentially using a peristaltic pump. The gradients with
sample were centrifuged at 20,000 rpm for 5 minutes at 4C to isolate synaptosomes.
Isolated synaptosomes were collected from the interface band between the 23% and 10%
Percoll layers in each gradient, and transferred and pooled directly to a large
polycarbonate centrifuge tube. To wash synaptosomes, 25 ml of HEPES-buffered saline
(HBS) was added to the tube, and was centrifuged at 15,000 rpm for 10 minutes at 4C.
The pellet was resuspended in HBS, and centrifuged at 7,000 rpm for 7 minutes at 4C.
The final pellet was resuspended in HBS for analysis by SDS-PAGE followed by western
blotting for select selenoproteins and related factors.
SDS-PAGE and Western blot: Total protein was extracted from whole-cell lysates by light
sonication in CelLytic MT buffer (Sigma, St. Louis, MO, USA) containing DTT, EDTA,
and protease inhibitors, followed by centrifugation according to the manufacturers’
protocol. Synaptosomes were resuspeneded in CelLytic MT buffer without sonication or
centrifugation. Protein was added to reduced Laemmli buffer, boiled for 10 minutes, and
loaded into 4-20% gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA).
Following electrophoresis, gel contents were transferred to PVDF membranes, which
were blocked with undiluted Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE,
USA) for one hour. Membranes were then probed for 90 minutes with one of the
following primary antibodies: Rabbit-anti-GPX4 (AbFrontier, Seoul, Korea),
Rabbit-anti-SEPW1 (Rockland, Gilbertsville, PA, USA), Rabbit-anti-SEPHS2 (Rockland),
Rabbit-anti-SEPHS1 (Rockland), Rabbit-anti-SecP43 (Santa Cruz Biotech, Santa Cruz,
CA, USA), Goat-anti-SBP2 (Everest Biotech, Oxfordshire, UK), Rabbit-anti-EFSec
(AbCam, Cambridge, MA, USA), Mouse-anti-Syntaxin1 (Santa Cruz Biotechnology),
Mouse-anti-PSD95 (Thermo Scientific), Mouse-anti-TBP (AbCam), Mouse-anti-beta
actin (Sigma, St. Louis, MO, USA) and Mouse-anti-alpha Tubulin (Novus, Littleton, CO,
USA). After washing with PBS containing 0.05% tween-20 (PBST), membranes were
incubated in the dark in secondary antibodies labeled with infrared fluorophores (Li-Cor
Biosciences). After further washes in PBST, blots were imaged and quantified with the
Odyssey infrared imaging system (Li-Cor Biosciences).
81
Imaging: Fluorescence imaging of primary cultures and stained sections were performed
on a Zeiss LSM 5 Pascal laser confocal inverted microscope equipped Ar and HeNe
lasers. AlexaFluor-488, -546, and -633 secondary antibodies with directly conjugated
fluorophores were used to detect primary antibody signals. Images were acquired using
the included LSM software, and were analyzed using ImageJ. Bright-field imaging was
performed using an upright Zeiss AxioScope 2 Plus microscope equipped with an ASI
motorized stage and Zeiss Axiocam MRc camera.
Statistical analysis: Data were analyzed using Microsoft Excel (Redmond, WA, USA),
and plotted using GraphPad Prism software (San Diego, CA, USA). Unpaired t-tests
comparing genotypes were performed for protein expression in synaptosome experiments.
The significance criteria were set at p < 0.05 for statistical measures.
82
RESULTS
We sought to determine whether selenoprotein W (Sepw1) is expressed in neurons in
mice. To address this question, we first cultured primary cells harvested from neonatal
mouse brain, and assessed expression of Sepw1 along with a neuron-specific marker,
class III beta-tubulin (Tuj1). As seen in figure 1, primary cultures consisted primarily of
neurons, and Tuj1 immunoreactivity (magenta, middle) showed some overlap with
Sepw1 expression (green, left) in neurites. Primary neuronal cultures derived from
neonatal cortex (Fig. 1A), hippocampus (Fig. 1B) and cerebellum (Fig. 1C) all contained
robust Sepw1 expression, and some colocalization with Tuj1, as indicated by white color
in the merged panels (Fig. 1, right). Sepw1 immunoreactivity was also observed in some
Tuj1-negative cells, possibly indicating expression in glia.
To confirm that Sepw1 was expressed in adult mouse brain, we performed dual label
fluorescent imaging of fixed mouse brain sections using antibodies directed against
Sepw1 and Tuj1 or a fluorescent Nissl stain. In somatosensory cortex we observed robust
Sepw1 expression in large pyramidal neurons and extensive colocalization with Tuj1 (Fig.
2A). In CA1 of hippocampus (Fig. 2B), the pyramidal layer showed similar
immunolabeling of Sepw1. Intriguingly, Sepw1 expression extended into the apical and
basolateral dendrites of most pyramidal neurons, and was apparent in some axonal
compartments as well (Fig. 2, right). To further analyze regional expression of neuronal
Sepw1, we performed immunohistochemistry with DAB development. Extending the
previous results in somatosensory cortex and CA1, Sepw1 expression was observed in
somas and dendrites of motor cortex (Fig. 3A, left) and CA3 of hippocampus (Fig. 3A,
right). Additionally, piriform cortex (Fig. 3B) and cingulate cortex (Fig. 3C) displayed
high Sepw1 immunoreactivity in pyramidal neurons. Purkinje neurons of cerebellum (Fig.
3D), and their highly branched dendritic arbors, also showed abundant expression of
Sepw1. In fact, most neurons appeared to express Sepw1 to some degree and neuropil
generally appeared immunopositive for Sepw1. Conspicuously, large neurons showed
immunoreactivity in neuritic processes. The widespread expression in neurons of adult
mouse brain, along with the punctate staining in varicose segments of cultured neurons,
83
led us to question if Sepw1 is expressed in the synaptic compartment.
To assess if Sepw1 is expressed in synapses, we prepared synaptosomes from adult mice
and performed western blotting of the purified samples. After confirming the absence of
contaminating nuclear proteins by blotting for TATA-binding protein (TBP) (Fig. 4A), we
blotted for Sepw1 and found it to be present in synaptosomes (Fig. 4B). Sepw1 mRNA is
known to be reduced in the brain of Sepp1-/- mice (Hoffman, 2007). To determine if
synaptically expressed Sepw1 is reduced in the absence of Sepp1, we next prepared
synaptosomes from littermate Sepp1-/- and wild-type mice. We observed a dramatic
decrease in Sepw1 expression in synaptosomes isolated from Sepp1-/- mice compared to
control mice (Fig. 5A). Additionally, western blot analysis of Gpx4 showed presence in
wild-type synaptosomes, and slightly reduced expression in Sepp1-/- synaptosomes (Fig.
5B). We used beta actin to control for loading across samples (Fig. 5C). After
normalizing to Actin, quantification of selenoprotein expression revealed that Sepw1 was
significantly reduced to <25% of wild-type levels, while GPX4 was not significantly
reduced in synaptosomes (Fig. 5D-E).We did not observe a qualitative enrichment of
Sepw1 or Gpx4 in synaptosomes versus whole-cell lysate samples, nor did we observe
selective depletion of Sepw1 in either fraction in Sepp1 knockout mice. These findings
indicate that Sepw1 is roughly homogenously distributed throughout neurons, and all
compartments similarly depend on Sepp1 to maintain expression.
In addition to expression in the neuronal somata, Sepw1 mRNA has been detected in
axons, dendrites and neuropil, suggesting that it may be locally translated in neuronal
processes (Cajigas et al., 2012, Willis et al., 2007). However, selenoprotein synthesis is
unique, and requires several additional protein factors beyond the standard translation
machinery. We sought to assess if translation of selenoproteins can occur in distal
processes of neurons. Therefore, we did western blotting of synaptosomes for several
proteins involved in selenoprotein translation after confirming absence of nuclear
contamination by analyzing TBP (Fig. 6A). Both the Sec-specific elongation factor
(EFSec) (Fig. 6B) and the SECIS-binding protein 2 (Sbp2) (Fig. 6D) are absolutely
required for selenoprotein translation, and appeared to be present in synaptosomes in
84
addition to whole cell lysate samples. Selenophosphate, produced by the selenoenzyme
selenophosphate synthetase 2 (SPS2), acts as the Se-donor during selenoprotein
translation. We were able to detect the presence of SPS2 in synaptosomes (Fig. 6C).
Interestingly, EFSec and SPS2 appeared to be enriched in synaptosome fractions
compared to whole cell lysate samples, further suggesting the existence of selenoprotein
translation in this compartment. The Sec-tRNA associated-protein, SecP43, and
selenocysteine lyase (Scly) have been implicated in selenoprotein translation efficiency,
but are not absolutely required (Kurokawa et al., 2011, Squires & Berry, 2008). We were
unable to detect SecP43 in synaptosomes, despite robust expression in whole-cell lysates
(Fig. 6E). Conversely, Scly did appear to be present in synaptosomes in similar
abundance as whole cell lysates (Fig. 6F). Unlike Sepw1, and Gpx4 to a lesser degree,
none of the proteins involved in selenoprotein synthesis were altered in Sepp1-/- mice,
compared to wild-type controls. This is especially curious for SPS2, which is itself a
selenoprotein. The combination of presented data argues that translation of selenoproteins
Sepw1 and Gpx4 in synapses may be possible.
85
DISCUSSION
The results reported herein describe the first characterization of regional selenoprotein W
(Sepw1) localization in mouse brain, as well as selenoprotein and synthesis factor
expression in isolated nerve terminals. Sepw1 is abundantly expressed in neuronal somata
and neuropil, and is expressed along with several selenoprotein synthesis proteins in
synaptosome fractions. In all regions of brain in mice lacking selenoprotein P (Sepp1),
Sepw1 expression is drastically reduced without effect on synthesis factors, indicating
that Sepp1 facilitates Sepw1 synthesis.
Selenium (Se) is a trace micronutrient that is incorporated into the unique amino acid,
selenocysteine (Sec). Sec-containing selenoproteins are typically oxidoreductase enzymes
that play crucial roles in reducing reactive oxygen species and oxidized macromolecules.
A selenoprotein that is widely distributed across all domains of life, Sepw1, is particularly
abundant in brain and muscle of mammals (Gu et al., 2000). Sepw1 mRNA expression is
observed in cephalic neural folds and somites in developing rodents, with continued high
expression as they become the adult brain and skeletal muscles (Loflin et al., 2006).
Sepw1 was initially identified due to its absence in muscle of myopathic Se-deficient
lambs, however brain expression of Sepw1, unlike in muscle, is not depleted by dietary
Se deficiency (Whanger, 2001). However, Sepp1-deficient mice show reduced Sepw1
mRNA and protein in brain (Hoffmann et al., 2007).
Sepw1 is the smallest described mammalian selenoprotein at ~10 kDa and contains an
N-terminal thioredoxin-like Cys-X-X-Sec redox motif, where X is any amino acid
(Lobanov et al., 2009). As with all selenoproteins, the Sec residue is encoded by a UGA
codon in the mRNA. A Sec Insertion Sequence (SECIS) in the 3'UTR of the mRNA, the
SECIS binding protein SBP2, and the Sec-specific elongation factor EFSec help to
bypass translation termination and incorporate Sec during translation [reviewed in
(Squires & Berry, 2008)]. Sepw1 also has another conserved Cys residue in the
N-terminal region that is known to bind glutathione (GSH) (Beilstein et al., 1996, Gu et
al., 1999). Antioxidant function attributed to Sepw1 is GSH dependent. In vitro
86
experimental studies, which increased or decreased Sepw1 expression, have demonstrated
elevated and reduced resistance to oxidizing agents but only in the presence of reduced
GSH (Jeong et al., 2002). However, other studies demonstrate that siRNA knockdown of
Sepw1 causes increased enzyme activities of glutathione peroxidase, superoxide
dismutase, and catalase and total antioxidative capability and glutathione level in cultured
muscle cells, which prevents oxidant-induced apoptosis (Xiao-Long et al., 2010). These
authors suggested a role for Sepw1 in the antioxidative system that is not direct peroxide
detoxification. Therefore, the in situ enzymatic role of Sepw1 has remained elusive.
Sepw1 mRNA rapidly declines in response to peroxide, suggesting that it has a role in
oxidative metabolism. Similar to the metabolic enzyme glyceraldehyde phosphate
dehydrogenase (GAPDH), oxidative inactivation of Sepw1 may be involved in rerouting
carbohydrate flux from glycolysis to the pentose phosphate pathway, stimulating NADPH
generation and reducing the intracellular pool of GSH (Loflin et al., 2006).
A pull down experiment indicated that Sepw1 interacts with the cytoskeletal microtubule
protein tubulin (Dikiy et al., 2007). Our data show robust colocalization of Sepw1 with
the neuron-specific beta tubulin, Tuj1. Sepw1 was additionally shown to
immunoprecipitate specifically with the beta and gamma isoforms of the 14-3-3 family of
scaffolding proteins (Aachmann et al., 2007). A computational study explored a putative
reaction mechanism, whereby Sepw1 regulates the oxidation state of a conserved and
solvent exposed Cys residue of 14-3-3 beta and gamma. Sepw1 is suggested to reduce
oxidized Cys-Sulfenic acid of 14-3-3 back to its parental thiol using the Cys-X-X-Sec
motif in combination with the bound GSH moiety (Musiani et al., 2010). 14-3-3 proteins
are multifunctional proteins that coordinate the interaction of kinases and phosphatases
with other regulatory proteins, thereby affecting phosphorylation-dependent cellular
processes (Fu et al., 2000). Like SEPW1 gene expression, the YWHAG gene for 14-3-3
gamma, is highly expressed in brain, skeletal muscle, and heart in humans (Horie et al.,
1999).
Sepw1 has also been implicated in regulating growth factor-stimulated control of cell
cycle-entry in epithelial cells. Knockdown of Sepw1 by siRNA in breast and prostate
87
epithelial cells inhibits EGF-stimulated G1/S transition via nuclear accumulation of p53,
leading to induction of p21 and G1 arrest (Hawkes & Alkan, 2011, Hawkes et al., 2012).
Intriguingly, the stability of the EGFR as well as tyrosine phosphorylation was decreased
by knockdown of Sepw1 (Hawkes, personal communication). EGFR tyrosine
phosphorylation and reduction of the EGFR turnover rate is a process that depends on
EGF-induced hydrogen peroxide production, which may oxidatively inactivate protein
tyrosine phosphatases (Deyulia & Carcamo, 2005, Deyulia et al., 2005). Since 14-3-3
proteins are established in regulating phosphorylation-mediated cell signaling, Sepw1
may function in oxidative signal transduction reactions from receptors to target proteins
via reactive oxygen intermediates.
High muscle expression of Sepw1 mRNA is associated with myoblasts, and expression is
decreased in differentiated myotubes (Loflin et al., 2006). Thus, the abundance of Sepw1
mRNA and protein in post-mitotic neurons is mysterious. Sepw1 mRNA and protein are
widely expressed in neurons, including apparent expression in axonal and dendritic
compartments (Cajigas et al., 2012, Willis et al., 2005, Willis et al., 2007). Whether
translation of Sepw1 occurs in these distal cellular compartments is uncertain.
Selenoprotein translation in mammals specifically requires the proteins SBP2 and EFSec,
in addition to the standard translation machinery. Both of these proteins were identified in
synaptosomes, along with SPS2 and SCLY which are important in Sec metabolism. Thus,
the only major protein involved in selenoprotein translation that was not investigated in
this study is the Sec-synthetase enzyme, SepSecS. SepSecS is also known as soluble liver
antigen/liver pancreas antigen (SLA/LP) and is required to generate the Sec-loaded
tRNASec (Palioura et al., 2010). We were unable to test for the presence of SepSecS in
synaptosomes.
Selenoprotein mRNAs are thought to be packaged into mRNP complexes, which aid in
preventing nonsense codon-mediated decay (NMD) of transcripts with a Sec-specifying
UGA that could be interpreted as a premature termination codon. Two mRNA binding
proteins important for nervous system function, DJ-1/Park7 and Staufen 2, have been
experimentally demonstrated to bind Sepw1 mRNA (Blackinton et al., 2009, Furic et al.,
88
2008, Maher-Laporte & Desgroseillers, 2010, Van Der Brug et al., 2008). DJ-1 is a
multifunctional redox-sensitive protein that is associated with Parkinson’s disease, other
neurodegenerative disorders, and cancer (Kahle et al., 2009). Two isoforms of staufen,
Stau1 and Stau2, are known to direct subcellular localization of mRNAs, and have
recently been implicated in synaptic plasticity (Lebeau et al., 2011a, Lebeau et al., 2011b).
Both DJ-1 and Stau2 proteins have shown varying degrees of localization to synapses,
axons and dendrites, further suggesting the local regulation of Sepw1 expression in distal
compartments of neurons (Jeong et al., 2007, Olzmann et al., 2007, Price et al., 2006,
Usami et al., 2011). Lastly, Stau2 is reported to interact with the key NMD protein Upf1,
which is implicated in selenoprotein translation because their mRNAs contain in-frame
termination codons. Intriguingly, Stau2 may upregulate protein expression of target
mRNAs in a Upf1-dependent mechanism, while upregulation of the target mRNAs is
Upf1-independent (Miki et al., 2011). These mechanisms may ultimately provide high
specificity and sensitivity for the local expression of Sepw1 in neurons.
In sum, we have shown that Sepw1 expression in mouse is abundant in neurons of several
brain regions, including cingulate and piriform cortex, hippocampus, and Purkinje cells
of cerebellum. We also showed Sepw1 expression in neuronal processes and some
colocalization with tubulin. Analysis of isolated nerve terminals further revealed the
presence of Sepw1 and much of the selenoprotein synthesis machinery in synaptic
compartments. Sepw1 expression in synaptosomes and whole-cell lysates of brain was
drastically reduced in Sepp1 knockout mice. Combined with previous reports
documenting Sepw1 mRNA expression in neuronal processes and association of Sepw1
transcripts with mRNA-binding proteins, regulation of Sepw1 expression in pre- and/or
post-synaptic compartments is suggested and warrants further investigation.
89
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FIGURE LEGENDS
Figure 1: Sepw1 is expressed in cell bodies and processes of cultured neurons. Primary
cultures derived from neonatal mouse cortex (A), hippocampus (B), and cerebellum (C)
were grown on coverslips for 3 weeks and subsequently double immunolabeled for the
presence of Sepw1 (in green, left) and a neuronal marker, Tuj1 (in magenta, center).
Merged images show sporadic colocalization between Sepw1 and Tuj1 (in white, right) in
neuronal somata and neurites in cultures from all three braion regions.
Figure 2: Sepw1 is expressed in cell bodies and processes of pyramidal neurons in cortex
and hippocampus. Fixed, frozen mouse brain sections were immunolabeled for Sepw1 in
somatosensory cortex (A) and CA1 region of hippocampus (B). Cortex sections were
additionally dual immunolabled with Tuj1, while hippocampus sections were labeled with
a fluorescent Nissl stain. Large pyramidal neurons in both regions strongly displayed
Sepw1 immunoreactivity, which prominently extended into the apical and basolateral
dendrites. A few proximal axonal segments also showed Sepw1 immunoreactivity.
Figure 3: Regional expression of Sepw1 in neurons of mouse brain. (A) Motor cortex
(left) and CA3 region of hippocampus (right) displayed prominent Sepw1 staining in cell
bodies and processes. (B-D) Low magnification (left) and high magnification (right)
photomicrographs of piriform cortex (B), cingulate cortex (C), and cerebellum (D) show
Sepw1 immunoreactivity in neurons. Large pyramidal (B-C) and Purkinje (D) neurons
show high expression in dendritic arbors.
Figure 4: Sepw1 is present in isolated nerve terminals. Synaptosomes were prepared and
analyzed by SDS-PAGE followed by Western blot. Lanes 1,2 and 3,4 represent two
independent preparations from wild-type C57 mice run in duplicate. (A) Analysis of
TATA-binding protein (TBP) in synaptosome fractions indicated that the preparations
were free of nuclear contamination. (B) Western blot showed the presence of Sepw1 in
synaptosome fractions.
94
Figure 5: Sepw1 expression in isolated nerve terminals is greatly reduced in mice lacking
Sepp1. Additional synaptosome fractions were prepared from Sepp1-/- mice (KO) and
wild-type littermate conrols (WT). These fractions were analyzed in comparison to whole
cell lysate (WCL) and mitochondrial (Mito) fractions. Western blotting for Sepw1 (A)
and Gpx4 (B) revealed the presence of both selenoproteins, with Actin (C) used as a
loading-control. Quantitation of synaptosomal expression of Sepw1 (D) and Gpx4 (E)
revealed that Sepw1 was significantly decreased (p<0.01), while Gpx4 was not.
Figure 6: Several selenoprotein synthesis factors are present in isolated nerve terminals.
(A) TBP was analyzed to confirm that nuclear proteins, where selenoprotein synthesis
factors are known to be present, are not contaminating the synaptosome fractions. (B)
EFSec was found to be present in synaptosomes and qualitatively enriched compared to
whole cell lysates, and absent from mitochondria. (C) SPS2, the enzyme that generates
selenophosphate, showed a similar expression pattern, being apparently increased in
isolated nerve terminals, and absent from mitochondria. (D) SBP2, which is required for
selenoprotein translation, was found in similarly low abundance in synaptosomes and
whole cell lysates, but not mitochondria. (E) The tRNASec-associated protein SecP43
was found only in whole cell lysates, where as Sec lyase (Scly) (F) was found in both
synaptosome and whole cell lysate fracions.
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FIGURE 1
96
FIGURE 2
97
FIGURE 3
98
FIGURE 4
99
FIGURE 5
100
FIGURE 6
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CHAPTER 4
METHAMPHETAMINE-INDUCED ALTERATIONS IN SELENOPROTEIN
EXPRESSION IN MICE
ABSTRACT
Selenium is an essential micronutrient in mammals. It is primarily incorporated into the
amino acid selenocysteine, in a family of proteins termed selenoproteins. Some
selenoproteins are known antioxidant enzymes, and many of them are expressed in the
brain. Methamphetamine is a highly addictive drug with potentially neurotoxic side
effects. Previous studies have demonstrated modulatory effects of dietary selenium levels
on methamphetamine-induced neurotoxicity. Specifically, dietary selenium deficiency
promotes excessive neurotoxicity after methamphetamine administration, while dietary
selenium supplementation prevents it. Although the activity of cellular glutathione
peroxidase, a well characterized selenoprotein, is largely attributed to the effects of
dietary selenium, other selenoproteins may also influence the extent of
methamphetamine-induced neurotoxicity. To determine what selenoproteins may be
adversely impacted by methamphetamine, we screened for changes in brain selenoprotein
transcripts after exposure to methamphetamine in mice. We found an upregulation of
select selenoprotein mRNAs without effect on protein levels, possibly indicating that
methamphetamine causes increased transcription of selenoprotein genes, but an inability
to upregulate selenoprotein translation. We also looked at methamphetamine-induced
toxicity in mice lacking selenoprotein P, a selenium transport protein important for brain
selenium supply. Selenoprotein P knockout mice did not display exacerbated toxicity
after methamphetamine administration, compared to wild-type mice. These results may
indicate that methamphetamine alters the expression profile of selenoproteins in brain,
but less than selenoprotein P.
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INTRODUCTION
Methamphetamine (MA) is a psychostimulant drug with toxic side effects. Long term use
of MA leads to striatal depletion of dopamine (DA), metabolites, and proteins involved in
DA synthesis and transport (Cadet & Krasnova, 2009). It has also been associated with
persistent neurotoxicity towards dopaminergic terminals and striatal neurons (Tulloch et
al., 2011). Although the exact cellular and molecular mechanisms of toxicity are not fully
elucidated, several reports indicate a prominent role for oxidative signaling via reactive
oxygen species (ROS) including hydrogen peroxide and peroxynitrite (Cadet &
Brannock, 1998).
Selenium (Se) is a micronutrient that is incorporated into oxidoreductase enzymes with
potent antioxidant activity and prominent roles in cellular oxidation-reduction (redox)
balance (Kryukov et al., 2003). These enzymes use Se in the form of selenocysteine
(Sec). Sec is a unique amino acid because it is encoded by the UGA codon in mRNAs,
which typically signals for termination of protein synthesis. To recode the UGA for Sec
incorporation, the 3' untranslated regions of selenoprotein mRNAs are endowed with a
Sec Insertion Sequence (SECIS). The SECIS element is a stem-loop structure that binds
to specific proteins, SECIS binding protein 2 and the Sec-specific elongation factor,
which help bypass termination and promote insertion of Sec [reviewed in (Bellinger et
al., 2009)].
Sec is a highly reactive residue, thereby rendering enzymatic selenoproteins with high
catalytic activity. Cellular glutathione peroxidase 1 was the first discovered
selenoenzyme, and rapidly reacts with hydrogen peroxide and reduced glutathione
(GSH), releasing water and glutathione disulfide (GSSG) (Rotruck et al., 1973).
Subsequently, four other GPX isozymes were determined to be selenoproteins in humans.
GPX4 has high affinity for lipid and organic hydroperoxides, is essential for neuronal
survival, and is required for embryonic development (Seiler et al., 2008, Yoo et al.,
2012). GPX4 is associated with neuromelanin in dopaminergic neurons of human brain,
and in dystrophic axons of Parkinson's disease patients (Bellinger et al., 2011).
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Previous studies have shown that dietary Se status can profoundly alter the metabolism of
dopamine in brain and MA-induced toxicity to dopaminergic neurons. Specifically, a Se
deficient diet increases DA turnover in the substantia nigra, hippocampus and prefrontal
cortex (Castano et al., 1995, Castano et al., 1997, Castano et al., 1993). Further, Se
supplementation can prevent the MA-induced depletion of TH, DA and DA metabolites
in the nigrostriatal pathway (Imam & Ali, 2000, Imam et al., 1999, Kim et al., 1999).
GPX was suggested to be specifically inactivated by MA-induced production of
peroxynitrite, and was generally attributed as the cause for protective effects of Se
supplementation. However, individual selenoproteins were not examined. In this
research, we investigated the regulation of selenoproteins in response to MA by mRNA
and protein expression to assess the contribution of select selenoproteins.
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MATERIALS AND METHODS
Animals: Male and female C57BL/6 mice, and genetically modified mice on the same
background lacking Sepp1 were bred on commercially available diets containing
adequate Se (~0.25 ppm). Animals were given food and water ad libitum on a 12-hour
light-cycle and group housed until experimentation. All experiments were conducted on
adult mice aged 3-4 months during the light cycle. Male and female mice of both
genotypes were used in approximately equal numbers to examine sex differences present
in the animals. All animal procedures and experimental protocols were approved by the
University of Hawaii Institutional Animal Care and Use Committee.
Animal Procedures: Mice were administered methamphetamine by intraperitoneal (i.p.)
injection with various doses depending on the experiment. In initial studies,
methamphetamine was chronically administered to C57/Bl6 mice 5 days per week for 2
or 8 weeks, with the dosage escalating from 2mg/kg to 20 mg /kg and then 3 mg/kg to 30
mg/kg. In two subsequent studies using Sepp1-/- mice and wild-type littermate controls,
two different paradigms were used. The first study used two i.p. injections of 10 mg/kg
administered approximately 4 hours apart, while the second used a single bolus of 40
mg/kg. For qPCR and Western blot analysis, animals were sacrificed 24 hours after the
final injection. For histological analysis, animals were sacrificed 72 hours after the final
injection.
Tissue Preparation: For biochemistry, mice were sacrificed by CO2 asphyxiation, and the
brains rapidly excised, washed in PBS and snap-frozen in liquid nitrogen. The striatum
and ventral mesencephalon were dissected from frozen tissue in a cold chamber
maintained at -20C. Tissue was then cut into small pieces, using a razor blade, and
collected into pre-chilled tubes. Tissue was homogenized by sonication in the appropriate
buffer for the ensuing experiment. For histology, mice were anesthetized with ketamine-
xylazine, and sacrificed by transcardial perfusion. Mice were initially perfused with
phosphate-buffered saline (PBS) to flush out blood, followed by perfusion with 4%
paraformaldehyde (PFA) to fix the tissue. The mice heads were cut off and the brains
dissected out. Brains were post-fixed in 4% PFA overnight, followed by cryoprotecting
105
the tissue in 10% and 30% sucrose for at least 4 hours each. The brains were then
embedded in optimal cutting temperature (OCT) compound and frozen until time of
sectioning. 40 μm sections were cut on a Leica CM1900 cryostat and saved in
cryoprotectant solution, containing 0.1 M phosphate buffer, 30% sucrose (w/v), and 30%
ethylene glycol (v/v), until time of further experimentation.
Quantitative RT-PCR: Total RNA from tissue was prepared by Trizol extraction
(Invitrogen, Carlsbad, CA, USA) followed by purification using the RNeasy kit (QIAgen,
Valencia, CA, USA). Concentration and purity of extracted RNA and synthesized cDNA
was determined using A260/A280 ratio measured on an ND1000 Spectrophotometer
(NanoDrop Technologies, Wilmington, DE, USA). Synthesis of cDNA was carried out
using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City,
CA, USA), with 1 g RNA per 20 l reaction. For real-time PCR, 100 ng of the cDNA
was used in 5 l reactions with Platinum SYBR Green qPCR SuperMix-UDG
(Invitrogen). Reactions were carried out in triplicate or quadruplicate in a LightCycler
480 II thermal cycler (Roche, Indianapolis, IN, USA). Cycling conditions followed the
manufacturers suggestions in the SYBR Green kit instructions. All qPCR results were
normalized to 18S rRNA expression as a housekeeping gene and analyzed using Absolute
Quantification Software (Roche).
SDS-PAGE and Western blot: Total protein was extracted from powdered mouse tissues
by light sonication in CelLytic MT buffer (Sigma, St. Louis, MO, USA), followed by
centrifugation according to the manufacturers’ protocol. Protein was added to reduced
Laemmli buffer, boiled for 10 minutes, and loaded into 4-20% gradient polyacrylamide
gels (Bio-Rad, Hercules, CA, USA). Following electrophoresis, gel contents were
transferred to PVDF membranes, which were blocked with 5% milk in PBS containing
0.05% tween-20 (PBST) for one hour. Membranes were then probed for 90 minutes at
room temperature or overnight at +4C with the following primary antibodies: Rabbit-
anti-Tyrosine Hydroxylase (TH) (Pel-Freez, Rogers, AR, USA), Rabbit-anti-GPX4
(AbNova, Taipei, Taiwan), Rabbit-anti-SEPW1 (Rockland, Gilbertsville, PA, USA),
Rabbit-anti-SEPHS2 (Rockland) and Mouse-anti-beta actin (Sigma, St. Louis, MO,
USA). After washing with PBST, membranes were incubated in secondary antibodies
106
conjugated to horseradish peroxidase. After further washes in PBST, blots were
visualized by enhanced chemiluminesence reaction followed by exposure to film.
Immunohistochemistry: Brain sections stored in cryoprotectant in the freezer were
warmed to room temperature, and sections containing striatum and substantia nigra were
selected for analysis. After thorough washing, sections were blocked in 5% normal goat
serum with 0.3% Triton X-100 in PBS. After blocking, the sections were incubated in
diluted primary antibody solution overnight at +4C. The following antibodies were used:
Rabbit-anti-TH (Pel-Freez), Rabbit-anti-GPX4 (AbNova), and Rabbit-anti-SEPW1
(Rockland). A control section where primary antibody was omitted was also included in
the procedure. After washing out primary antibody, sections were incubated in species-
matched secondary antibody. The secondary antibody was either directly conjugated to
fluorophores (Alexa Fluor dyes, Invitrogen) for fluorescence imaging, or was biotinylated
for colorimetric visualization. After incubation in biotinylated secondary antibody, the
sections were then incubated in Avidin-Biotin Complex solution, followed by
visualization with 3,3-Diaminobenzidine (DAB) (Vector Labs, Burlingame, CA, USA).
Additionally, some sections were dual-labeled with a fluorescent Nissl stain to label
neurons (Neurotrace, Invitrogen), or phalloidin to label filamentous actin (Invitrogen).
Sections were then mounted onto slides, and either coverslipped in VectaShield
containing DAPI for fluorescence, or air-dried overnight and coverslipped using
Permount after ethanol dehydration and Xylene clearing.
TUNEL Histology: Apoptotic cell death in sections was assessed using an In Situ Cell
Death Detection Kit (Roche), using the Terminal deoxynucletidyl transferase (TdT)
dUTP Nick End Labeling (TUNEL) method. Sections were thoroughly washed, and then
blocked with 3% hydrogen peroxide in methanol for 10 minutes at room temperature.
Sections were then permeabilized by incubation in 0.5% Triton X-100 at +65C for 30
minutes. After washing in PBS, TUNEL labeling was performed for 60 minutes at +37C
in the dark. A negative control section lacking TdT, and a positive control section that
was incubated in DNAse I was also included. The fluorescent label was then converted
by incubating in POD conversion solution included in the kit for 30 minutes at +37C, and
was visualized by DAB reaction. The sections were mounted and air-dried overnight. The
107
sections were then counterstained with 0.5% Cresyl Violet after ethanol dehydration,
cleared in Xylene, and coverslipped using Permount.
Imaging: Both fluorescence and bright-field imaging of stained sections were acquired on
an upright Zeiss Axioscope 2 Plus microscope equipped with an ASI motorized stage and
Zeiss Axiocam MRc camera. Fluorescence imaging was done with an ultraviolet lamp
and appropriate filter sets for the fluorophores used, while bright-field imaging used
standard white-light illumination. Images were acquired using the included AxioVision
software, and were analyzed using ImageJ.
Statistical analysis: Data were analyzed using Microsoft Excel (Redmond, WA, USA),
and plotted using GraphPad Prism software (San Diego, CA, USA). Two-way ANOVA
was used to determine an interaction between MA administration and selenoprotein
expression. Post hoc test using Bonferroni correction for multiple comparisons was used
to determine significance between individual groups. Unpaired t-tests comparing control
and experimental groups were performed for some experiments. The significance criteria
were set at p < 0.05 for all statistical measures.
108
RESULTS
We sought to determine whether methamphetamine (MA) causes an alteration in
selenoprotein transcript expression in the substantia nigra and striatum of mice brains. To
achieve this, we administered MA to C57BL/6 mice for a period of 2 weeks, and
harvested their brains for analysis by quantitative real-time polymerase chain reaction
(qPCR). The majority of selenoprotein mRNAs were analyzed, and two-way ANOVA
revealed that MA had an effect on selenoprotein transcript expression in midbrain
(F(1,92)=6.329, p=0.0136) and striatum (F(1,92)=6.854, p=0.0103). Qualitatively,
selenoprotein expression appeared to increase in response to MA administration in both
brain regions (Fig. 1). Individual selenoprotein transcripts of interest were further
analyzed by t-test to compare groups. We found that selenoprotein W (Sepw1)
(t(4)=3.083, p=0.0368) and selenophosphate synthetase 2 (Sps2) (t(4)=2.809, p=0.0484)
mRNAs were significantly increased in midbrain, but not in striatum of MA-treated mice.
However, we did not find a significant increase in mRNA expression of glutathione
peroxidase 4 (Gpx4) or selenoprotein P (Sepp1) in either brain region (Fig. 2). These
results led us to question if selenoprotein expression was also increased in the mice
brains. In contrast to the mRNA data, western blot analysis of Sepw1, Sps2, and Gpx4
did not reveal an increase in selenoprotein expression in response to MA in either brain
region (Fig. 3).
We next sought to determine if prolonged, higher-dose MA exposure has a more dramatic
effect on selenoprotein expression. When mice were exposed to elevated doses of MA for
8 weeks, we found that selenoprotein mRNAs in midbrain were not increased, unlike
with short term MA administration. However, Sepw1 and Sps2 were decreased in
midbrain, contrasting their previously seen upregulation (Fig. 4, top). This may indicate
that expression of Sepw1 and Sps2 transcripts are regulated in midbrain by MA. In
striatum, qPCR showed that selenoprotein mRNA levels were dramatically reduced (Fig.
4, bottom). High-dose exposure to MA is known to cause degeneration of dopaminergic
terminals and medium spiny neurons in striatum (Zhu et al., 2006), which may explain
the reduction of selenoprotein transcripts in this region.
109
To determine if selenoprotein P augments selenoprotein expression in the context of MA-
induced changes, we next administered MA to Sepp1-/- and wild-type littermate control
mice. When administering two injections of 10 mg/kg MA and sacrificing 24 hours after
the second injection, we did not detect any major changes in selenoprotein expression due
to MA. However, the Sepp1-/- animals had reduced expression of several selenoprotein
transcripts, most notably with Sepw1 (data not shown).
As we observed little change in selenoprotein mRNA expression with 2 injections of 10
mg/kg MA, we next administered high-dose MA at 40 mg/kg. We investigated protein
expression by western blot and immunohistochemistry, and also performed TUNEL
histology to investigate cell death. Western blot analysis of striatum revealed that Sepp1-
/- mice have reduced levels of Sepw1 and Gpx4, but not Sephs2, and that MA did not
significantly influence the expression of selenoproteins in wild-type or knockout mice
(Fig. 5). Similar results were obtained in midbrain (data not shown). Tyrosine
hydroxylase (TH) expression was investigated by western blotting and by histology. MA
caused a slight upregulation of TH in midbrain lysates when measured by Western blot
(data not shown). However, an apparent decrease in TH staining in substantia nigra was
observed in brain sections (Fig. 6). TH expression did not show much change in striatum
in response to MA either by western blot or immunohistochemistry (data not shown).
Sepp1-/- mice did not respond differently than wild-type mice in terms of TH expression
in midbrain or striatum. In congruence with these findings, TUNEL histology revealed an
absence of apoptotic cell death in Sepp1-/- and wild-type mice exposed to MA (Fig. 7).
110
DISCUSSION
The results presented here show that MA affects both the short- and long-term mRNA
expression of selenoproteins in mammalian brain. These data are in agreement with
reports indicating that Se deficiency potentiates the neurotoxic profile of MA exposure,
while Se repletion attenuates it (Imam et al., 1999, Kim et al., 1999). Neurons appear to
be the key site of Se utilization, although these cells possess fairly low GPX activity
(Zhang et al., 2008). This suggests that selenoproteins in addition to GPX1 are involved
in protection from MA-induced neurotoxicity.
GPX4 is a phospholipid hydroperoxidase that is particularly important for neuronal
development and survival (Seiler et al., 2008). Conditional deletion of GPX4 in neurons
of adult mice causes rapid lipoxygenase-mediated apoptosis (Yoo et al., 2012). GPX4 is
found in dopaminergic neurons of human brain, where it has a conspicuous association
with neuromelanin. It has also been found in dystrophic neurites of individuals with
Parkinson’s disease (Bellinger et al., 2011). These findings strongly implicate GPX4 as a
necessary component of dopamine neurons.
Selenoprotein P (Sepp1) is an extracellular selenoprotein that functions in Se distribution,
and has roles in preventing neurodegeneration and deficits in synaptic plasticity (Caito et
al., 2011, Peters et al., 2006, Valentine et al., 2008, Valentine et al., 2005). The finding
that Sepp1-deficient mice did not display enhanced sensitivity to neurotoxicity in
dopaminergic brain regions has not been reported, but is probably due to the severe
pathology associated with deletion of Sepp1 alone. Although TUNEL labeling of
apoptotic cells revealed no increase in cell death in Sepp1 knockout mice, the mice are
unquestionably under more cellular stress than wild-type MA-treated control
counterparts.
Selenoprotein W (Sepw1) is a small thioredoxin-like protein that has a role in mediating
cell signaling and is particularly abundant in muscle and brain of mammals (Gu et al.,
2000, Whanger, 2009). Interestingly, dietary Se deficiency causes depletion of Sepw1 in
muscle, but not brain (Whanger, 2001). Maintenance of Sepw1 expression during dietary
Se deprivation is likely due to the Se-transport function of Sepp1, since mice deficient in
111
Sepp1 have drastically reduced expression of Sepw1 mRNA and protein in brain
(Hoffmann et al., 2007). Sepw1 mRNA expression was altered by both short and long
term MA administration, albeit in different directions, perhaps making it a specific target
in MA-induced neurotoxicity.
Lastly, selenophosphate synthetase 2 (Sps2) is a selenoprotein that generates
selenophosphate for selenocysteine biosynthesis (Xu et al., 2007). Although we initially
found that Sephs2 mRNA was acutely upregulated by MA, the effect on protein was
minimal. However, the mRNA was downregulated upon long-term MA treatment, similar
to Sepw1. Transcriptional regulation of Sepw1 and Sps2 suggests these two
selenoproteins may be specifically targeted in midbrain during MA exposure. Further, the
activity of the Sps2 enzyme was not assessed, and selenophosphate production may
indeed be compromised by MA treatment.
Collectively these data suggest that selenoproteins are neuroprotective in the context of
MA exposure. The reduction of selenoprotein mRNA expression in striatum after chronic
MA exposure indicates that Se availability may be limiting under MA-induced stress, and
therapeutic supplementation with dietary Se may be helpful. We did not detect dramatic
changes in selenoprotein expression by western blot, but it remains possible that the Sec
residue is oxidatively modified and that enzymatic catalysis is inactivated or reduced by
high levels of MA-induced ROS production.
112
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FIGURE LEGENDS
Figure 1: Expression of selenoprotein mRNAs is altered by methamphetamine. Following
two weeks of treatment with methamphetamine or vehicle, mice brains were analyzed for
expression of selenoprotein transcripts by qPCR. The regions principally affected by
methamphetamine were dissected from frozen brains. Methamphetamine administration
caused significant variance in expression of selenoprotein mRNAs both midbrain (top)
and striatum (bottom). Data are presented as fold-change relative to PBS treated-mice for
the sake of clarity, but raw data was analyzed by two-way ANOVA.
Figure 2: Selenoprotein W and selenophosphate synthetase 2 mRNA are upregulated in
midbrain after two weeks of methamphetamine administration. Select selenoprotein
mRNAs of interest were analyzed by t-test to determine significance between treatment
groups. Sepw1 and Sps2 had significantly elevated expression in midbrain (n=3, p<0.05),
but not in striatum of methamphetamine-treated mice. Trends towards increased
expression of Sepp1 and Gpx4 did not reach statistical significance in either brain region.
Figure 3: Selenoprotein expression following two weeks of methamphetamine
administration is not changed in mice brains. SDS-PAGE and Western blotting for
Sepw1 and Gpx4 was performed on the same samples as assessed by qPCR (n=3).
Qualitatively, expression of Sepw1 and Gpx4 appeared to be reduced, however
considerable variation and low sample size resulted in no statistically significant
alteration.
Figure 4: Long term methamphetamine administration dramatically reduces selenoprotein
mRNAs in striatum. Following eight weeks of treatment with methamphetamine or
vehicle, mice brains were analyzed by qPCR for expression of selenoprotein transcripts
(n=3-4). Sepw1 and Sps2 showed reduced expression in midbrain of methamphetamine
115
treated mice, but none of the other selenoproteins differed in midbrain, compared to
vehicle treated animals. In contrast, reduced expression of nearly all selenoprotein
mRNAs was observed in striatum.
Figure 5: Sepp1 impacts brain expression of selenoproteins more than methamphetamine.
Sepp1-deficient mice were administered a high-dose bolus of methamphetamine, and
expression of selenoproteins in striatum was investigated by Western blot (n=3-4).
Expression of Sepw1 and Gpx4 was reduced in brains of Sepp1 knockout mice, and did
not differ between methamphetamine and vehicle treatment groups. Expression of Sps2
was stable across all experimental groups.
Figure 6: Methamphetamine reduces tyrosine hydroxylase expression in substantia nigra
independently of Sepp1. Sepp1 knockout (KO) and wild-type (WT) control mice were
administered a bolus of MA or PBS as a vehicle control, and the brains were
subsequently analyzed by immunohistochemistry for the presence of tyrosine
hydroxylase (TH). An apparent decrease in TH-immunoreactivity in neurons of the
substantia nigra was observed in MA-treated animals. Sepp1 KO mice had similar
expression of TH as WT control mice, under both MA and PBS treatment conditions.
Figure 7: Apoptotic cell death is not increased in methamphetamine-treated selenoprotein
P knockout mice. Following a high-dose bolus administration of MA or vehicle control,
TUNEL labeling was performed on brain sections from Sepp1 KO and WT littermate
mice to investigate apoptosis. When compared to the negative (C) and positive (D)
control sections, both WT (A) and KO (B) methamphetamine-treated mice showed
virtually no TUNEL positive cells, indicating an absence of apoptotic cell death.
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FIGURE 1
117
FIGURE 2
118
FIGURE 3
119
FIGURE 4
120
FIGURE 5
121
FIGURE 6
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FIGURE 7
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CHAPTER 5
CONCLUSION
SUMMARY AND DISCUSSION
The overall goal of the research presented in this dissertation was to gain a deeper
understanding of selenoprotein expression and function in the mammalian brain. The first
section investigates the neurological consequences of genetic disruption of selenocysteine
lyase (SCLY), particularly in comparison to the behavioral phenotype of selenoprotein P
(SEPP1) knockout mice. The expression and activity of selenoproteins in SCLY
knockout mice was also analyzed. The second section describes the cellular and
subcellular expression of a relatively uncharacterized selenoprotein that is abundant in
brain, selenoprotein W (SEPW1). SEPW1 expression in brain was also examined in
SEPP1 knockout mice. The final section assesses and describes selenoprotein expression
in mouse brain after exposure to the drug methamphetamine. Methamphetamine-induced
pathology and alterations in proteins relevant to dopamine metabolism were also
investigated in the brains of mice lacking SEPP1.
The local and global distribution of bodily Se in mammals is a complex phenomenon that
is still mechanistically unclear. SEPP1 is certainly involved in the hierarchy of Se
distribution that prioritizes Se to the brain when dietary availability is limiting, however
the kinetics of Se utilization in the brain are unknown (Burk & Hill, 2009). The form of
Se in SEPP1 is Sec, but the presumed substrate for selenoprotein synthesis is selenide.
Therefore a complementary system to break down Sec is necessary to complete the Se
cycle and utilize Sec from SEPP1. To this end, SCLY has been proposed to close the loop
and recycle Sec residues within organisms as a source of Se (Mihara et al., 2000). To
investigate if SCLY is necessary to recycle Sec residues, we determined if genetic
deletion of SEPP1 and SCLY in mice produces similar phenotypes.
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On the contrary, we found that SCLY knockout mice display a minimal neurological
phenotype when contrasted to SEPP1 knockout mice. When given a Se-adequate diet, no
behavioral impairment was observed. Even when challenged with a Se-poor diet, the
SCLY knockout animals have only a slight learning impairment, and no motor
phenotype. This is highly discordant with the severe motor phenotype observed in SEPP1
knockout mice. This finding suggests that mice have one or more additional enzymes that
can break down Sec and provide usable Se, preventing the SEPP1 knockout phenotype.
The cysteine desulfurase enzymes NFS1 is known to have selenocysteine lyase acitivity,
and may be functionally compensating for the lack of SCLY (Lacourciere et al., 2000).
Despite a very mild behavioral phenotype in SCLY knockout mice fed a low Se diet, the
animals had drastically reduced expression of selenoproteins in brain, and GPX activity
in brain and liver was similarly reduced. This finding confirms that SCLY contributes to
the Se pool for selenoprotein translation, at least when dietary Se is limiting. Thus, both
SCLY and complementary enzymes such as NFS1 are likely to be involved in utilizing
Sec for synthesis of selenoproteins.
It is clear that Se is prioritized to the brain during dietary deficiency, indicating that
selenoproteins are important for nervous system function [reviewed in (Chen & Berry,
2003)]. Most of the discovered selenoproteins are found in the brain, but their cellular
functions and enzymatic activity remain elusive. SEPW1 is a small selenoprotein that is
highly expressed in muscle and nervous tissues, and is peculiarly absent in muscle of Se-
deficient lambs suffering a myopathy called White muscle disease. Unlike in muscle and
most tissues, SEPW1 expression is not diminished in brains of Se-deficient mammals
(Whanger, 2001). SEPW1 contains an N-terminal C-X-X-U motif in a thioredoxin-like
fold, where C is cysteine, U is Sec, and X is any amino acid. This structure is highly
conserved in thioredoxin superfamily proteins and usually is indicative of a redox
function (Dikiy et al., 2007). Biochemical and computational experiments indicate that
SEPW1 interacts with 14-3-3 beta and gamma proteins, possibly regulating the oxidation
state of a conserved cysteine residue (Aachmann et al., 2007, Musiani et al., 2010). 14-3-
3 proteins are multifunctional scaffolding proteins that coordinate the interaction of
receptors, kinases, and phosphatases, thereby modulating cell signaling. Using siRNA to
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reduce SEPW1, it has been demonstrated that cell cycle progression and proliferation
involves SEPW1. Knockdown of SEPW1 in prostate and breast epithelial cells results in
G1 arrest, and this effect is dependent on the tumor suppressor protein p53 (Hawkes &
Alkan, 2011, Hawkes et al., 2012). These results suggest that SEPW1 has an important
role in cell signaling related to cellular proliferation, differentiation, and death. However
the functional role of SEPW1 in the brain is unknown.
To investigate the expression of SEPW1 in the brain, we analyzed the localization of
SEPW1 in cultured cells, in mice brain sections, and in isolated nerve terminals from
mice brains. Intriguingly, SEPW1 is highly expressed in neurons in culture and adult
mouse brain, and expression is apparent in the processes of large neurons in multiple
brain regions. Analysis of isolated nerve terminals, or synaptoneurosomes, revealed the
presence of SEPW1. Previous studies from other labs have indicated that SEPW1 mRNA
is found in processes of neurons as well. This suggested that SEPW1 may be locally
translated in these subcellular neuronal compartments distal to the nucleus. However
selenoprotein synthesis requires specialized factors, some of which exhibit shuttling
between the nucleus and cytoplasm. Synaptoneurosomes contained several of these
factors, importantly including EFSec and SBP2, which are both required for
selenoprotein synthesis. Additionally, we found that SEPW1 expression is drastically
reduced in the brains of SEPP1 knockout mice, unlike in animals with dietary Se
deprivation. Similar results were obtained for other selenoproteins, but not for synthesis
factors. In sum, these results indicate that SEPW1 expression is widespread in neurons, it
may be synthesized locally, and its expression is dependent on SEPP1. The Sec residues
of SEPP1 are likely to supply Se for synthesis of SEPW1 and other selenoproteins, but a
signaling function of SEPP1 that stimulates transcription and translation of SEPW1
specifically cannot be ruled out.
The dopaminergic neurotransmitter system consists of neurons emanating from midbrain,
which synthesize dopamine and release it in several regions throughout the brain. The
two primary populations of dopaminergic neurons have cell somas in the substantia nigra
pars compacta and ventral tegmental area, located in the ventral mesencephalon. Neurons
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in the substantia nigra project mainly to the caudate nucleus and putamen, collectively
referred to as the striatum. The nigrostriatal pathway facilitates the initiation and smooth
control of movement. Extensive degeneration of nigrostriatal neurons is a canonical
feature of Parkinson's disease, and is responsible for motor symptoms including rigidity
and bradykinesia (Cookson, 2005). Parkinson's disease is a complex disorder with genetic
and environmental causes, and positive effects of Se-supplementation are reported in
animal models of the disease (Khan, 2010, Zafar et al., 2003). Research in our lab has
shown an association of GPX4 with neuromelanin in dopaminergic neurons of the
substantia nigra pars compacta (Bellinger et al., 2011). Lewy bodies, a pathological
hallmark of Parkinson's disease, are associated with SEPP1 in post-mortem tissue from
human patients (Bellinger, et al., unpublished).
The population of dopaminergic neurons in the ventral tegmental area mainly project to
the nucleus accumbens via the amygdala and hippocampus, in addition to frontal cortex.
These two pathways are referred to as the mesolimbic and mesocortical tracts
respectively, and are involved in diverse cognitive functions including pleasure and
reward. Both positive and negative deregulation of these fibers is observed in
schizophrenia and mood disorders, which are associated with Se-binding proteins and Se-
status (Benton, 2002, Glatt et al., 2005). Although these three primary dopaminergic
pathways are approximately separated anatomically, the distinction at the cellular level is
less discrete and considerable overlap between projection targets is observed.
Methamphetamine is a highly addictive psychostimulant drug of abuse that stimulates
dopamine release and reversal of the plasma and vesicular membrane dopamine
transporters, causing high levels of synaptic, cytosolic and extracellular dopamine (Cadet
& Krasnova, 2009). This is accompanied by regional changes in brain glucose
metabolism, the blood-brain barrier, and myelination (Bowyer et al., 2008, Volkow et al.,
2001a). Chronic abuse of methamphetamine is neurotoxic in humans, leading to loss of
dopaminergic terminals in the striatum and cortex (Sekine et al., 2003, Volkow et al.,
2001b). The exact cause of neurotoxicity is debated, but oxidative processes are strongly
implicated. In rodent models, Se supplementation can alleviate or prevent
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methamphetamine-induced neurotoxicity, but the contributions of specific selenoproteins
are uncertain. Dietary Se deficiency elevates and prolongs high potassium-induced
dopamine release in the striatum, and increases the turnover rate of dopamine in the
substantia nigra, prefrontal cortex and hippocampus (Castano et al., 1995, Castano et al.,
1997, Castano et al., 1993, Watanabe et al., 1997). Additionally, Se deficiency causes
induction of tyrosine hydroxylase and dopamine transporter mRNA in nigrostriatal
neurons, with concomitant increases in dopamine synthesis and uptake (Romero-Ramos
et al., 2000). Conversely, dietary Se supplementation reduces the activity of the
dopamine catabolic enzyme, monoamine oxidase (MAO) (Tang et al., 2008). Because
dopamine, and related precursors and breakdown products, can spontaneously oxidize to
electron-deficient quinones, oxidative processes partially determine survival of
dopaminergic neurons (Chen et al., 2008). The antioxidant activity of selenoproteins such
as GPX4 can inhibit oxidant-induced apoptosis (Seiler et al., 2008). Additionally,
dopamine deamination by MAO generates hydrogen peroxide, and MAO-catalyzed
peroxide generation is coupled to the enzymatic activity of the selenoprotein GPX1
(Maker et al., 1981). These studies suggest that the function and survival of dopamine
neurons rely on brain Se and one or more selenoproteins. Therefore toxicity caused by
methamphetamine may be partially due to deleterious effects on selenoproteins, and Se
supplementation may be of therapeutic benefit.
To determine if methamphetamine causes an alteration in selenoprotein expression, an
investigation of selenoprotein transcripts and proteins in striatum and midbrain after
methamphetamine administration in mice was performed. We found that selenoprotein
transcripts generally increased after acute methamphetamine exposure. Based on altered
SEPW1 and SPS2 mRNA levels in both MA-treatment paradigms, selenoprotein
expression may be altered in midbrain by MA. It seems probable that the oxidative
burden caused by methamphetamine is the primary cause of alterations in SEPW1 and
SPS2 transcripts. However in the same samples, SEPW1 protein was unchanged. This
may indicate that methamphetamine causes oxidation and degradation of the proteins, or
that the mRNA translation is inefficient, and the transcriptional increase is compensatory
to normalize protein expression.
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OXIDATIVE STRESS AND SELENOPROTEINS
An overarching theme surrounding the functions of selenium and selenoproteins in cell
biology is the relationship with oxidative stress and redox control systems. Oxidative
stress is a broadly used term, but generally signifies an imbalance in prooxidants and
antioxidants in cells and their environment, causing disruption of redox control and
macromolecular damage. Free radicals and nonradical oxidants operate differently as
prooxidants, because free radicals are promiscuously reactive while nonradical oxidants
typically react with specific targets. Additionally, macromolecular damage is a divergent
outcome from disrupted redox control and signaling. Macromolecular damage to DNA,
proteins, and lipids is caused by redox reactions with free radicals, which contain an
unpaired electron and are scarce in biological systems due to enzymes and chemicals that
rapidly scavenge them and typically generate nonradical oxidants (Jones, 2006). For
example the superoxide radical (O2-) is rapidly converted by superoxide dismutase to the
nonradical oxidant hydrogen peroxide (H2O2). Superoxide can also react with nitric oxide
(NO), another free radical, to produce the nonradical oxidant peroxynitrite (ONO2-). Free
radical-mediated macromolecular damage is a common end-point observed in aging and
disease. However, mechanistically it is likely that selenoproteins function in redox
systems control and signaling.
Due to rapid conversion in organisms, free radicals are quantitatively miniscule compared
to nonradical two-electron oxidants such as hydrogen peroxide, fatty acid
hydroperoxides, disulfides, peroxynitrite, aldehydes, and quinones. In prototypical redox
control systems, these nonradical oxidants reversibly react with protein thiols to elicit a
redox signal that functions in physiological regulation. The mechanisms of reversible
thiol oxidation as a biological control system generally include reaction with an active
site cysteine, modification of a distal allosteric cysteine, and alteration of macromolecular
interactions (Jones, 2008). If the relevant cysteine is in an active site such modifications
may serve as a simple binary on-off “switch” to activate or inactivate the protein.
Another scenario in which the cysteine residue is not in the active site allows for
129
allosteric regulation, which can throttle protein function along a continuous gradient, like
a “dimmer”. Additionally, oxidation of cysteine thiols is known to control intra- and
inter-molecular protein interactions by formation of disulfides. Among other things,
disulfide crosslinking is well established in maintaining proper protein structure,
regulating the viscosity of mucus, and connecting actin filaments and tethering proteins
to the cytoskeleton (Farah & Amberg, 2007, Moriarty-Craige & Jones, 2004).
The GSH and TXN redox couples are central to a global system of connected elements
involving reversible oxidation of proteins containing cysteine, methionine, and Sec
residues. GPX and TXNRD selenoproteins, being critical effectors of peroxide and
thioredoxin reduction, have a direct role in the redox systems biochemistry of organisms.
Oxidative stress in the form of disrupted redox control arises when cysteine (or Sec or
methionine) residues become abnormally oxidized or irreversibly modified, stripping the
reversible redox reactivity that is required for physiological processes. Several covalent
and noncovalent modifications to distal or active site cysteine residues modify protein
structure and function. A redox-active cysteine residue can become reversibly oxidized to
sulfenic acid, which can be reduced step-wise by two molecules of reduced GSH.
Additionally, cysteinyl-S-glutathione conjugates are semi-stable in certain proteins, and
S-glutathionylation can lead to altered protein function as observed with glyceraldehyde
3-phosphate dehydrogenase (GAPDH) and caspase 3 (Mohr et al., 1999, Sykes et al.,
2007). If two neighboring reduced cysteine residues (dithiol) are reactive, the pair can be
reversibly oxidized to a form a cysteine disulfide bridge, as with TXN. The gaseous
molecules, NO and hydrogen sulfide (H2S), also react with cysteine residues to produce
modified derivatives that are functionally relevant. For example, S-nitrosylation of
Cys118 in Ras activates the protein by stimulating guanine nucleotide exchange.
Similarly sulfhydration, the addition of a sulfhydryl group from hydrogen sulfide gas to a
cysteine thiol, has been demonstrated to augment the activity of GAPDH (Lander et al.,
1996, Mustafa et al., 2009). Proteins often contain multiple cysteine residues that can be
reversibly modified (e.g. dithiol-disulfide, S-glutathionylation, S-nitrosylation, S-
sulfhydration, etc.) to affect protein function and/or interactions. Therefore
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selenoproteins, with Sec, cysteine and methionine residues, are integral in redox control
systems and may be the most redox-active proteins in cells and organisms.
REDOX SYSTEMS AND SELENOPROTEINS IN THE BRAIN
The brain is an extremely oxygen-hungry organ. Therefore perhaps unsurprisingly,
oxidative stress is consistently implicated in brain dysfunction and disease. Despite being
roughly 2% of total body weight, it consumes approximately 20% of total body oxygen.
In other words, the rate of oxygen consumption by the brain is about ten times higher
than the rest of the body per gram of tissue. This staggering demand is highlighted by the
fact that oxygen deprivation to the brain causes loss of consciousness, brain damage, and
death within minutes (Lutz et al., 2003). The chemical reactivity between selenium,
oxygen, and sulfur produces a unique relationship between these chalcogen elements and
the mammalian nervous system.
Due to the high oxygen consumption of nervous tissue, reactive byproducts of oxygen
metabolism are continuously generated. Surprisingly however, antioxidant systems are
relatively sparse compared to other tissues. In particular, the brain has less catalase
activity and is more reliant on GPX1 for reduction of hydrogen peroxide (Dringen, 2000,
Dringen et al., 2000). However GPX activity is also low in brain compared to other
organs. The abundance of peroxide despite a relative lack of reducing capacity suggests
that peroxide may serve as an important regulatory molecule in the brain, while also
being a potentially toxic molecule when unregulated. Peroxide-mediated signaling via
effects on protein kinases, phosphatases, and transcription factors in neurons, glia, and
vascular cells is accepted, if not well understood (Bao et al., 2009). Nitric oxide is
another reactive product of oxygen metabolism with potent signaling properties. Three
isoforms of nitric oxide synthase, endothelial, inducible and neuronal, generate nitric
oxide in the presence of oxygen and arginine. The apposition of these isoforms in cell
types on both sides of the blood-brain barrier suggest that bidirectional regulation of
oxygen metabolism across the cerebral vasculature to the brain parenchyma is at least
partially mediated by nitric oxide (Zonta et al., 2003). Intriguingly SEPP1 is present on
131
both sides of the blood-brain barrier and scavenges nitric oxide and the reaction product
with superoxide, peroxynitrite (Arteel et al., 1998). Furthermore, SEPP1 knockout mice
exhibit degeneration of neurons in areas of the brain with high rates of blood perfusion
and glucose oxidation, such as the inferior colliculus (Valentine et al., 2008).
The nervous system also contains large quantities of oxidizable lipids and metals,
rendering it vulnerable to disruption of redox signaling and oxidant-mediated damage.
Genetic deletion of GPX4 in neurons causes rapid neurodegeneration, suggesting that
reduction of lipid hydroperoxides is essential for neuronal survival (Seiler et al., 2008,
Yoo et al., 2012). SEPP1 facilitates expression of GPX4 and other selenoproteins in
brain, and the neurodegeneration seen in SEPP1 knockout mice may be due to
insufficient GPX4 production. Additionally the metal-chelating properties of SEPP1 may
limit the availability of oxidizable metals, such as iron and copper, to catalyze the
nonspecific production of radical hydroxyl ions, which are highly reactive and
indiscriminately target nearby molecules (Sidenius et al., 1999). Collectively, these data
imply that SEPP1 promotes stable oxygen metabolism directly and indirectly to support a
healthy nervous system. While it directly reduces extracellular oxidants relevant to
neurovascular physiology, it indirectly stimulates the expression of other selenoproteins
involved in oxidative regulation of neuronal survival.
From a systems biology perspective, cellular redox status is understood in terms of redox
potential. The redox potential (Eh), or electron motive force, for an oxidation/reduction
couple depends on the inherent tendency of the molecule to accept/donate electrons
relative to the standard hydrogen electrode, and the concentrations of the acceptor and
donor (reduced and oxidized species of the couple). In biological systems the redox
potential of the major thiol/disulfide couples (GSH and TXN) is maintained at stable
nonequilibrium conditions, or in other words the mean Eh of the GSH and TXN couples
in subcellular compartments is nonzero. These redox couples, along with the Cys/CySS
(cysteine/cystine) couple and protein Cys residues involved in redox signaling, are
neither in equilibrium with each other nor the NADPH/NADP+ couple. In addition to
nonequilibrated redox potentials between couples within a subcellular compartment,
132
redox potential is also not equilibrated across subcellular and extracellular compartments.
The mitochondria and nucleus tend to be highly reduced (i.e. redox potential farther from
zero), while the extracellular compartment is relatively oxidized (i.e. redox potential
closer to zero), and the cytoplasm, endoplasmic reticulum, and lysosomes display
intermediate values. The steady-state Eh for thiol-disulfide couples range from -400 mV
for NADPH/NADP+ to -60 mV for plasma Cys/CySS (Kemp et al., 2008).
With an intracellular reduced GSH concentration of 1 mM, oxidation of just 18 µM of
GSH to GSSG (e.g. by GPX) will lower the GSH/GSSG redox potential (ΔEh) by ~60
mV. This relatively shallow redox potential gradient is sufficient to distinguish between
proliferating and apoptotic cells, and can theoretically drive a 100-fold change in the
dithiol:disulfide ratio in proteins with a reactive dithiol motif (Schafer & Buettner, 2001).
Therefore the current through a redox circuit need only be a fraction of the total electron
transfer in cells, provided that spatial or catalytic mechanisms are able to control reaction
rates. Under aerobic conditions oxygen and peroxide are always present in cells, therefore
coupling of electron transfer to oxidase or peroxidase (e.g. GPX) catalyzed reactions can
provide an additional energetic driving force to maintain function of low-current redox
control circuits. Kinetic and spatial insulation of reactive thiol and selenol couples allows
biological systems to be highly responsive and dynamic. Because noncatalyzed
oxidation-reduction and thiol-disulfide exchange reactions are relatively slow in the
reducing cellular environment, this enzymatic insulation concurrently provides specificity
and directionality to electron transfer reactions (Kemp et al., 2008).
S-glutathionylated Sec and diselenide bridges have not been described in mammals in
vivo, but transient and stable selenenyl-sulfide linkages are observed in selenoproteins as
either active intermediates or structural features, respectively. For example the reaction
mechanisms for GPX and TXNRD enzymes involve a transient mixed selenenyl-sulfide
bond, while rat selenoprotein P (SEPP1) has two such linkages that appear to be a
structural feature (Hatfield, 2006, Ma et al., 2005). Transient nitrosylation and
sulfhydration of Sec is thermodynamically feasible, but whether it occurs in cells and
organisms with physiological relevance is unknown. Sec is a low concentration amino
133
acid and free Sec is virtually absent from cells, implying that Sec/selenocystine and
Sec/cysteine redox couples are unlikely to be of functional importance. However
selenoproteins are present and may be maintained in a nonequilibrium steady-state
favoring the reduced form. Kinetic (i.e. reaction rate and substrate specificity) and spatial
(i.e. subcellular and tissue distribution) insulation of the various selenoproteins has been
described. Whether selenoproteins represent kinetically limiting sites of redox control
remains an important unanswered question. The standard reduction potential (E0) of
selenoproteins is unexplored to date. Determining whether the mean E0 of selenoproteins,
particularly SEPW1 and the other TXN-like selenoproteins, is maintained in a
nonequilibrium steady-state will help determine if they are central redox control points.
The Sec amino acid has known kinetic advantages compared to cysteine in catalysis, and
GPX and TXNRD selenoproteins occupy pivotal positions in respect to redox circuitry.
Therefore it is probable that more selenoproteins will be identified as kinetically limiting
redox control points that are rapidly disrupted during oxidative metabolism in the nervous
system. Several selenoproteins have unknown function but, given the reactivity of Sec
with oxygen and cysteine, they are aptly structured to transduce oxidant signals by
forming cysteine sulfenic acids and disulfide bonds in downstream target proteins, even
in the predominantly reducing cellular environment. Clarifying the role of selenoproteins
in oxygen metabolism will promote better therapies for developmental and neurological
diseases. Timely and localized administration of selenium in one form or another may in
fact be a cheap and effective treatment for a broad range of diseases.
134
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