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Endocrinology, doi:10.1210/en.2008-0378
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Endocrinology Vol. 149, No. 12 6251-6261
Copyright © 2008 by The Endocrine Society

Expression of the Thyroid Hormone Transporters Monocarboxylate Transporter-8 (SLC16A2) and Organic Ion Transporter-14 (SLCO1C1) at the Blood-Brain Barrier

Lori M. Roberts, Kathleen Woodford, Mei Zhou, Deborah S. Black, Jill E. Haggerty, Emily H. Tate, Kent K. Grindstaff, Wondwessen Mengesha, Chandrasekaran Raman and Noa Zerangue

Discovery Biology, XenoPort, Inc., Santa Clara, California 95051

Address all correspondence and requests for reprints to: Dr. Lori Roberts, Discovery Biology, XenoPort, Inc., 3410 Central Expressway, Santa Clara, California 95051. E-mail: Lori.Roberts{at}XenoPort.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormones require transport across cell membranes to carry out their biological functions. The importance of transport for thyroid hormone signaling was highlighted by the discovery that inactivating mutations in the human monocarboxylate transporter-8 (MCT8) (SLC16A2) cause severe psychomotor retardation due to thyroid hormone deficiency in the central nervous system. It has been reported that Mct8 expression in the mouse brain is restricted to neurons, leading to the model that organic ion transporter polypeptide-14 (OATP14, also known as OATP1C1/SLCO1C1) is the primary thyroid hormone transporter at the blood-brain barrier, whereas MCT8 mediates thyroid hormone uptake into neurons. In contrast to these reports, we report here that in addition to neuronal expression, MCT8 mRNA and protein are expressed in cerebral microvessels in human, mouse, and rat. In addition, OATP14 mRNA and protein are strongly enriched in mouse and rat cerebral microvessels but not in human microvessels. In rat, Mct8 and Oatp14 proteins localize to both the luminal and abluminal microvessel membranes. In human and rodent choroid plexus epithelial cells, MCT8 is concentrated on the epithelial cell apical surface and OATP14 localizes primarily to the basal-lateral surface. Mct8 and Oatp14 expression was also observed in mouse and rat tanycytes, which are thought to form a barrier between hypothalamic blood vessels and brain. These results raise the possibility that reduced thyroid hormone transport across the blood-brain barrier contributes to the neurological deficits observed in affected patients with MCT8 mutations. The high microvessel expression of OATP14 in rodent compared with human brain may contribute to the relatively mild phenotype observed in Mct8-null mice, in contrast to humans lacking functional MCT8.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE IS essential for cell growth and metabolism in all tissues. The developing central nervous system is particularly sensitive to thyroid hormone deficiency, and thyroid hormone insufficiency during brain development results in neurological deficits and cognitive impairment. Based on studies in rat, it is thought that the majority of active thyroid hormone in the brain is generated locally by the action of outer ring iodothyronine deiodinases, which convert the prohormone T4 to active thyroid hormone, T3 (1). T3 then binds to intracellular nuclear receptors to exert its biological effects (2). Inner ring iodothyronine deiodinases inactivate T4 and T3 by conversion to rT3 and 3,3'-diiodothyronine (T2), respectively (1). Although it was initially assumed that thyroid hormones could traverse cellular membranes by passive diffusion due to their lipophilicity, it has since been discovered that active transport is required for cellular uptake of both T3 and T4 (2). Functional studies of cloned transporters expressed in Xenopus laevis oocytes or mammalian cell lines have identified the monocarboxylate transporter (MCT) family member MCT8 (SLC16A2) as a transporter of T4 and T3 and their inactive metabolites rT3 and T2 (3), whereas the organic anion transporter polypeptide OATP14 (OATP1C1, encoded by the gene SLCO1C1) has been shown to transport T4, rT3, and to a much lesser extent T3 (4, 5, 6). In addition, MCT10 (SLC16A10) (7), the amino acid transporter LAT1 (SLC7A5) (8), several members of the SLCO organic anion transporter polypeptide (OATP) family (9), and fatty acid translocase (FAT/CD36) (10) are reported to be capable of specific uptake of T3 and T4.

The importance of thyroid hormone transport was revealed by the discovery that mutation of MCT8 causes Allan-Herndon-Dudley syndrome (AHDS), an X-linked developmental disorder characterized by hypotonia, spasticity, muscle weakness, neurological disorders, and cognitive impairment (11, 12, 13). Affected patients show elevated serum T3, normal to low serum T4 levels, and symptoms of hyperthyroidism in peripheral tissues combined with symptoms of hypothyroidism in the central nervous system. Similar to AHDS patients, Mct8-null mice exhibit elevated serum T3 and low serum T4 (14, 15). In addition, brain uptake of exogenous T3 is markedly reduced in mice lacking functional Mct8, whereas T4 uptake is reduced by 50% compared with wild type (14, 15). The diminished brain uptake of T3 and T4 in Mct8-deficient mice suggests that Mct8 transports thyroid hormones across the blood-brain barrier (BBB). Previous studies in mice have shown Mct8 mRNA is expressed primarily in neurons and choroid plexus (15, 16). Endothelial expression of Mct8 in mouse brain was reported to be limited to a subset of large capillaries, and thus it was concluded that Mct8 is not important for thyroid hormone transport across the BBB (16). Instead, it has been proposed that OATP14 is primarily responsible for T4 uptake from the blood into the brain, where it is locally converted to the active thyroid hormone T3, which is in turn transported into neurons by MCT8 (17).

In this study, we examined the BBB expression of the thyroid hormone transporters MCT8 and OATP14 in human, mouse, and rat by quantitative PCR (qPCR) analysis and immunofluorescence staining. We also examined the subcellular localization of MCT8 and OATP14 proteins in mouse and human choroid plexus epithelium and in isolated rat cerebral microvessels by immunofluorescence staining and confocal microscopy. In contrast to previous studies, we show that MCT8 mRNA and protein are expressed in microvessels of the BBB in human, mouse, and rat brain. In addition, we show that OATP14 mRNA and protein are highly expressed and enriched in brain microvessels in mouse and rat but to a much lesser extent in human brain microvessels. We discuss how these results relate to current models of thyroid hormone transport in the brain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
All protocols involving animals were approved by the XenoPort Animal Use Committee in accordance with the recommendations outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague Dawley rats and CD-1 mice were obtained from Charles River Laboratories (Hollister, CA).

Isolation of brain microvessel endothelial cells (BMEC)
Cerebral cortices from 10 adult rat or mouse brains were dounce-homogenized in isolation medium (DMEM containing 25 mM HEPES, 3% fetal bovine serum, and 10 U/ml heparin) by five strokes of a type A pestle. The homogenized tissue was pelleted by centrifugation for 5 min at 1000 x g, resuspended in 100 ml warm isolation medium containing 1 mg/ml collagenase/dispase (Roche Applied Science, Indianapolis, IN), and transferred to a 125-ml spinner flask. Tissue was incubated at 37 C, 5% CO2 with stirring at 50 RPM for 1 h. Digested tissue was divided between two 50-ml conical tubes, pelleted by centrifugation at 1000 x g, resuspended in 40 ml per tube of centrifugation medium (either 25% BSA or 12.5% dextran), and centrifuged for 20 min at 2500 x g. The resulting floating white matter and centrifugation medium were transferred to fresh tubes, and the microvessel pellets were resuspended in isolation medium. The remaining white matter and centrifugation medium were mixed again, the centrifugation step was repeated three times, and the microvessels from each centrifugation were pooled. Microvessels were pelleted and resuspended in isolation medium, and the BMEC were filtered through 100- and 40-µm nylon mesh and then either captured on 20-µm nylon mesh or centrifuged through a discontinuous 20/50% Percoll gradient at 1000 x g for 15 min to remove erythrocytes.

For immunofluorescence staining, microvessels were isolated using a nonenzymatic protocol to preserve vessel morphology. Rat brains were minced and dounce-homogenized as described above and then centrifuged through 15% dextran as described above for four spins total. The resulting microvessel pellets were pooled and filtered through 100- and 40-µm nylon mesh strainers. Microvessels were adhered to glass coverslips placed in six-well plates for 5 min at room temperature and then fixed and processed for immunofluorescence staining as described below.

Human tissue specimens
Specimens of human brain tissue and BMEC (isolated by collagenase digestion as described above) from epilepsy surgical patients were obtained from Dr. Thomas J. Feuerstein (Department of Neurosurgery, University Hospital Freiberg, Freiberg, Germany). Brain specimens used for qPCR analysis were obtained from two patients aged 5 and 29 yr. BMEC specimens used for qPCR were obtained from three patients aged 50, 41, and 32 yr. OCT frozen blocks containing cerebral cortex autopsy specimens from four male and five female donors ranging in age from 57–76 yr were purchased from Analytical Biological Services (Wilmington, DE). Frozen sections of fetal brain (region unspecified, female, 20 wk), fetal parietal lobe (female, 37 wk), hippocampus (female, 82 yr), and choroid plexus (female, 70 yr) were purchased from BioChain Institute (Hayward, CA).

qPCR
RNeasy RNA Isolation Kit and QIAshredder homogenizer columns (QIAGEN, Valencia, CA) were used to isolate total RNA from tissue samples. RNA integrity was verified on an agarose gel. Thermoscript reverse transcriptase (RT) kit (Invitrogen, Carlsbad, CA) was used to synthesize single-stranded cDNA from total RNA.

Gene-specific PCR primers were designed using GeneTools Software (Doubletwist, Oakland, CA). At least two primer sets against different DNA sequences were used for MCT8 and OATP14 qPCR from human, mouse, and rat samples with similar results, and data generated with the two primer sets were averaged. Five independent primer sets were used for human OATP14 qPCR. A single primer set was used for other genes. Primer sequences are listed in supplemental Table 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

PCR included 10 µl 2x SYBR Green Master Mix (Applied Biosystems, Foster City, CA), 0.5 µl single-stranded cDNA, and 10 pmol of each primer. Reactions were carried out in a 96-well format using an Opticon Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). Samples were denatured for 8 min at 95 C, followed by 35 amplification cycles: 30 sec denaturation at 95 C; 30 sec annealing at 53 C, 57 C, or 62 C, depending on the melting temperature of the specific primers used; and 1 min extension at 72 C. Fluorescent readings were taken after each extension period. Melting-curve analysis was performed upon completion of the 35 cycles to verify amplification of a single PCR product. In addition, PCR products were analyzed by agarose gel electrophoresis to confirm the correct size of the amplified targets. The cycle threshold value was designated for each reaction to be the cycle at which fluorescent signal exceeded 20 SD above the average signal between cycles 3–10. qPCR results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cycle threshold values.

Antibodies
Covance Research Products (Princeton, NJ) generated rabbit polyclonal antisera against the C-terminal 40 amino acids of human or rat MCT8 (100% conserved in mouse) and the N-terminal 39 amino acids of rat Oatp14 (95% conserved in mouse) fused to glutathione-S-transferase (GST). Cocalico Biologicals (Reamstown, PA) generated rabbit polyclonal antisera against the C-terminal 34 amino acids of human OATP14 or rat Oatp14 (94% conserved in mouse) fused to GST. BLAST searches of the epitope sequences against rat, mouse, or human translated nucleotide databases showed that they are specific to the proteins of interest (data not shown). Antisera against rMct8, hOATP14, rOatp14 N terminus, and rOatp14 C terminus were subtracted on immobilized GST to remove anti-GST antibodies and affinity purified on GST-Sepharose cross-linked to their respective GST-fusion protein epitopes using standard protocols. Whole anti-hMCT8 antiserum was used, because the affinity purification protocol abolished antibody reactivity. Rat anti-Mrp4 (clone M41-10; Axxora, San Diego, CA) was used at a 1:20 dilution. Mouse-anti-Pgp (clone C219; EMD Chemicals, San Diego, CA) was used at a 1:20 dilution. Mouse anti-aquaporin-4 (clone 4/18; Santa Cruz Biotechnology, Santa Cruz, CA) was used at a 1:50 dilution.

Transfected cells
TREx-HEK-293 cells (Invitrogen) stably express the tetracycline repressor, allowing for tetracycline-inducible gene expression when transfected with pTREx-hOATP14 or pTREx-rOatp14 vectors. TREx-HEK-293 cells were also used for transfection with non-tetracycline-regulated pMO-hMCT8 or pMO-rMct8 vectors. Cells were seeded at 104 cells per chamber on collagen-coated Lab-Tek II eight-chambered coverglasses (Nalge Nunc International, Rochester, NY) or at 106 cells per well in six-well tissue culture dishes in DMEM with 4.5 g/liter glucose, 10% FBS, 6 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Cells were transfected at the time of plating with 0.5–1 µg expression vector per 106 cells. For pTREx vectors, 1 µg/ml doxycycline was added at the time of transfection to induce transgene expression. Three days after plating, cells were rinsed three times with chilled Hanks’ balanced salt solution containing calcium and magnesium and fixed for immunofluorescence staining or lysed for Western blotting as described below.

Immunofluorescence staining and confocal microscopy
Tissues were frozen in OCT compound on dry ice, cryosectioned at 10 µm thickness, adhered to slides, air dried, and stored at –80 C. Before staining, slides were warmed to room temperature and air dried. Tissue sections and transfected cells were fixed in 4% paraformaldehyde in Hanks’ balanced salt solution for 15 min at 4 C. OATP14 staining was not detectable in paraformaldehyde-fixed tissue and required fixation with chilled methanol for 5 min at –20 C. For P-glycoprotein (PGP) staining, paraformaldehyde-fixed tissue sections were postfixed in 1:2 acetic acid ethanol at –20 C for 10 min (18); postfixation was not required when methanol fixation was used. Specimens were blocked and permeabilized in PBS containing 5% normal goat serum and 0.1% Triton X-100 for 30 min at room temperature. Primary antibodies were diluted in blocking/permeabilization buffer and added to specimens for 2 h at room temperature or overnight at 4 C. For preabsorption experiments, primary antibodies at final working concentration were preincubated with 20 µg/ml purified GST protein (Pierce Biotechnology, Rockford, IL) or the appropriate GST-fusion antigen used for immunization in blocking buffer for 30 min before staining. Specimens were washed three times for 10 min each in PBS and incubated with fluorescein isothiocyanate- or Cy3-conjugated secondary antibodies (final concentration 3.75 µg/ml) diluted in blocking/permeabilization buffer for 30 min to 2 h at room temperature. Specimens were washed three times for 20 min each in PBS. For some experiments, tissues were counterstained with the far-red fluorescent nucleic acid dye TOTO-3 iodide (Invitrogen) at 0.5 µg/ml in PBS for 5 min and rinsed several times in PBS. Human adult brain sections were stained for 10 min with 0.3% Sudan Black B in 70% ethanol to quench lipofuscin autofluorescence (19) and then rinsed several times with PBS. Specimens were mounted using SlowFade Gold mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen). Imaging was performed using a Zeiss Pascal 5 confocal imaging system or Zeiss Axiocam II color camera mounted on a Zeiss Axiovert 200 fluorescence microscope (Carl Zeiss, Thornwood, NY).

Western blotting
Transfected cells or microvessels were homogenized using 23- and 27-gauge needles in PBS containing 2% sodium dodecyl sulfate (SDS) and 1–2x protease inhibitor cocktail III (Calbiochem, San Diego, CA). Brain tissue was dounce-homogenized in PBS using a loose-fitting (type A) pestle and then lysed as described above. Samples were heated to 70 C for 10 min and then centrifuged at 16,000 x g in a tabletop microcentrifuge for 10 min at room temperature to pellet-insoluble material. Total protein concentration was determined by BCA assay (Pierce).

SDS-PAGE was performed using NuPage SDS-PAGE gels, reagents, and buffers according to manufacturer’s instructions (Invitrogen). Equal microgram amounts of total protein were separated 4–12% Bis-Tris polyacrylamide mini-gels using MES running buffer and transferred to nitrocellulose membranes. Membranes were blocked in TBS-Tween [20 mM Tris (pH 7.4), 500 mM NaCl, 0.1% Tween 20] containing 5% nonfat dry milk for 30 min at room temperature with rocking. Primary antibodies were diluted in blocking buffer and incubated with membranes overnight at 4 C or for 2 h at room temperature with rocking. For preabsorption experiments, primary antibodies at final working concentration were preincubated with 20 µg/ml purified GST protein (Pierce) or the appropriate GST-fusion antigen used for immunization in blocking buffer for 30 min before staining. Membranes were washed six times for 10 min each in TBS-Tween with rocking at room temperature and then incubated with horseradish peroxidase-conjugated secondary antibodies (Pierce) diluted in blocking buffer (8 ng/ml final concentration) for 30 min to 2 h at room temperature. Membranes were washed nine times for 10 min each in TBS-Tween at room temperature with rocking and then developed using SuperSignal Femto HRP chemiluminescent substrate (Pierce) and exposed to Biomax Light chemiluminescence film (Kodak, Rochester, NY).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
qPCR profiling of thyroid hormone transporters at the BBB
qPCR analysis of endothelial, neuronal, and glial marker expression in BMEC and brain tissue from human, mouse, and rat revealed that transcripts for the well-characterized BMEC marker GLUT1 (SLC2A1) (20) was enriched 27- to 59-fold in BMEC compared with whole brain (Fig. 1AGo). In addition, the endothelial cell marker PECAM-1/CD36 (21) was enriched 75-fold on average in BMEC isolates compared with whole brain (data not shown), whereas transcripts for the glial marker GFAP (22) and the neuronal marker Syntaxin 1a (SYN1a) (23) were reduced 80–90% on average in BMEC compared with brain (data not shown), indicating that BMEC isolates were strongly enriched in endothelial cells and depleted of other brain cell types.


Figure 1
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  		FIG. 1. qPCR analysis of MCT8 and OATP14 expression at the BBB. A, GLUT1. Transcripts for the well-characterized BMEC marker GLUT1 are abundant and highly enriched in BMEC (dark gray bars) from human, mouse, and rat compared with whole brain (light gray bars). BMEC:Brain represents the ratio of transcripts in BMEC compared with whole brain. Error bars represent the SEM of at least three independent qPCR and at least two independent tissue isolations in A–C. B, MCT8. MCT8 transcript levels are 5–10% the levels of the highly abundant BBB transporter GLUT1 in BMEC (% GLUT1). MCT8 shows strong enrichment in BMEC compared with whole brain (BMEC:Brain) in mouse and rat similar to the BMEC marker GLUT1, whereas more modest enrichment is seen in human BMEC compared with whole brain. C, OATP14. Oat14 transcripts are highly abundant in mouse and rat BMEC, near the levels of GAPDH expression, about 60% of GLUT1 levels. Oatp14 is strongly enriched in BMEC compared with whole brain in mouse and rat. OATP14 transcript levels are much lower in human BMEC compared with mouse and rat, and no enrichment is seen in human BMEC compared with whole brain.

 
As measured by qPCR, MCT8 was expressed in BMEC in all species examined, ranging in expression from 5–11% of GLUT1 levels (Fig. 1BGo). In addition, MCT8 was enriched in BMEC compared with whole-brain tissue in all species examined (Fig. 1BGo). In mouse and rat, Mct8 transcripts were enriched in BMEC compared with whole brain by 21-fold and 44-fold, respectively, comparable to the level of BMEC enrichment measured for Glut1 (Fig. 1AGo). Modest enrichment of 2.5-fold was observed for MCT8 in human BMEC compared with whole brain (Fig. 1AGo). In mouse and rat BMEC, Oatp14 transcripts were strikingly abundant; Oatp14 transcripts were detected at approximately the same level as GAPDH, around 60% of GLUT1 levels and enriched 38- to 46-fold in BMEC compared with whole brain (Fig. 1CGo). In contrast, OATP14 expression was much lower in human BMEC at less than 1% of GLUT1 expression, and OATP14 transcripts were not enriched in human BMEC compared with whole brain (Fig. 1CGo).

Antibody validation
To measure the expression of MCT8 and OATP14 proteins, we generated polyclonal antibodies against human and rat/mouse MCT8 as well as human and rat OATP14 (94% conserved in mouse). The specificity of these antibodies for Western blotting and immunofluorescence staining was confirmed using cells transfected with human or rat MCT8 or OATP14 clones (Fig. 2Go). Anti-MCT8 antibodies showed specific recognition of rat and human and rat MCT8 in transfected, but not untransfected, cells by immunofluorescence staining (Fig. 2Go, A–D). Preabsorption of anti-MCT8 with the appropriate GST-fusion protein used for immunization abolished immunofluorescence staining (Fig. 2Go, E and F).


Figure 2
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  		FIG. 2. Validation of anti-MCT8 and anti-OATP14 antibodies in transfected TREx-HEK cells. A–F, MCT8 immunofluorescence. No staining with anti-MCT8 antibodies is detected in untransfected cells (A and B, No tfx). Cells were identified by nuclear DAPI staining (not shown). Specific staining is observed in cells transfected (Tfx) with rat (C) or human (D) MCT8. Preabsorption of MCT8 antibodies with the GST-fusion proteins used for immunization abolishes staining of cells transfected with MCT8 (E and F, Preabs). Scale bars for A–F and K–S, 50 µm. G–J, MCT8 Western blotting. By Western blotting, MCT8 antibodies detect bands near 60 kDa, the predicted molecular mass of MCT8 (arrows) in transfected cells (G and H, + lanes). No bands are detected by antirat Mct8 in untransfected cells (G, – lane); minor bands are detected in untransfected cells (H, – lane) by antihuman MCT8. Higher molecular mass bands are also specifically detected in transfected cells (arrowheads), possibly due to aggregation or posttranslational modification of overexpressed MCT8. Preabsorption of antibodies with immunogen abolishes Western blot detection of MCT8 in transfected cells (I and J, Preabs). For G–J and T–Y, GAPDH loading controls are shown below each blot. The positions of molecular mass standards are marked on the left side of each blot. K–S, OATP14 immunofluorescence. No staining is detected in cells transfected with tetracycline-inducible rat or human OATP14 expression vectors in the absence of tetracycline (K–M, No tet). Cells were identified by nuclear DAPI staining (not shown). Specific staining of OATP14 is observed when expression of rat (N and O) or human (P) OATP14 is induced by addition of tetracycline (N–P, Plus tet). Preabsorption of antibodies with immunogen abolishes staining of cells induced by tetracycline to express OATP14 (Q–S, Preabs). T–Y, OATP14 Western blotting. By Western blotting, OATP14 antibodies detected a doublet of bands (arrows) that appear smaller than 79 kDa, the predicted molecular mass of OATP14 in cells induced to express OATP14 (T–V, + lanes) but not in uninduced cells (– lanes), which may represent proteolyzed OATP14. Preabsorption of antibodies with immunogen abolished Western blot detection of OATP14 (W–Y, Preabs). A minor nonspecific band is detected by antihuman OATP14 that is not blocked by preabsorption (arrowheads, V and Y).

 
By Western blotting, anti-MCT8 antibodies detected a band near the predicted molecular mass of 59 kDa in extracts of cells transfected with either rat or human MCT8, but not in untransfected extracts (arrows, Fig. 2Go, G and H). Anti-MCT8 antibodies also detected several higher molecular mass bands specifically in transfected cells (arrowheads, Fig. 2Go, G and H). These bands probably represented aggregates or posttranslational modifications of MCT8 that arise due to overexpression. Preabsorption of anti-MCT8 antibodies with the GST-fusion proteins used for immunization abolished Western blot signals (Fig. 2Go, I and J). Preabsorption of antibodies with purified GST not fused to immunogen peptide did not affect immunofluorescence or Western blot signals (data not shown).

Cells transfected with tetracycline-inducible OATP14 expression vectors exhibited staining with antibodies against rat and human OATP14 only when expression was induced by the addition of tetracycline (Fig. 2Go, K–P). Preabsorption of anti-OATP14 antibodies with the GST-fusion proteins used for immunization abolished immunofluorescence signals (Fig. 2Go, Q–S). Preabsorption of antibodies with purified GST not fused to immunogen peptide did not affect immunofluorescence signals (data not shown).

By Western blotting, antirat and antihuman OATP14 antibodies detected doublets of bands in cells induced to express OATP14, but not in uninduced cells (arrows, Fig. 2Go, T–V), indicating that the bands represent OATP14. The doublets appeared to be smaller than the predicted molecular mass of 79 kDa, suggesting that OATP14 may be susceptible to proteolysis. Because plasma membrane staining was observed with anti-OATP14 antibodies in transfected HEK cells (Fig. 2Go, N–P), it is likely that both antibodies detected intact Oatp14 in whole cells and that degradation of Oatp14 occurred during cell lysis. Preabsorption of anti-OATP14 antibodies with the GST-fusion proteins used for immunization abolished Western blot signals (Fig. 2Go, W–Y). A minor nonspecific band was detected by antihuman OATP14 antibody in both uninduced and induced samples and was not blocked by preabsorption (arrowheads, Fig 2Go, V and Y). Preabsorption of antibodies with purified GST not fused to immunogen peptide did not affect Western blot signals (data not shown).

Expression of MCT8 and OATP14 proteins in BMEC and brain
Western blotting of brain and BMEC.
Mct8 antibodies recognized a single band in mouse and rat brain and BMEC protein extracts near 59 kDa, the predicted molecular mass of Mct8 (arrows, Fig. 3Go, A and B). Consistent with qPCR analysis of Mct8 transcripts (Fig. 1BGo), Western blotting revealed enrichment of Mct8 protein in mouse and rat BMEC extracts compared with whole brain (Fig. 3Go, A and B). Preabsorption of anti-Mct8 with the GST-fusion protein used for immunization abolished Western blot detection of Mct8 in mouse and rat brain and BMEC (Fig. 3Go, C and D). Preabsorption of anti-Mct8 with purified GST (nonfusion protein) did not affect Western blot results (data not shown). Because of the limited availability of human brain surgery specimens, we were unable to perform conclusive Western blot analysis of MCT8 in isolated human BMEC.


Figure 3
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  		FIG. 3. Western blotting of Mct8 and Oatp14 in rat brain and BMEC. A and B, Anti-Mct8 antibodies recognize a single band (arrows) near 59 kDa, the predicted molecular mass for MCT8, in total protein lysates of mouse (A) and rat (B) brain (lanes marked B) and BMEC (lanes marked EC). Mct8 protein is enriched in BMEC compared with whole brain. Gapdh is shown as a control for equal protein loading between lanes, and molecular mass standards are marked on the left side of each set of panels for A–H. B, Brain; EC, brain microvessel endothelial cells. C and D, Preabsorbed anti-Mct8. Preabsorption (Preabs) of anti-Mct8 antibody with its GST-fusion protein immunogen abolishes Western blot detection of mouse (C) and rat (D) Mct8. E and F, Oatp14. In mouse (E) and rat (F) brain and BMEC, anti-Oatp14 (C terminus) antibodies recognize a band near the predicted molecular mass of 78 kDa (arrows) as well as bands at higher (arrowheads) and lower (open arrowheads) molecular weights, which may represent posttranslational modifications of Oatp14 such as glycosylation or proteolysis. The bands recognized by anti-Oatp14 (C terminus) are strongly enriched in BMEC compared with whole brain. G and H, Preabsorbed anti-Oatp14. Preabsorption (Preabs) of anti-Oatp14 (C terminus) with its GST-fusion protein immunogen abolishes Western blot detection of Oatp14 in mouse (G) and rat (H) brain and BMEC.

 
Anti-Oatp14 (C terminus) detected two or more indistinct bands near the predicted molecular mass of 79 kDa in mouse and rat brain and BMEC extracts (arrow, Fig. 3Go, E and F) as well as higher and lower molecular mass bands (arrowheads, Fig. 3Go, E and F) that may represent posttranslational modifications such as glycosylation or degradation of Oatp14. The detection of multiple bands by Western blotting of mouse brain and BMEC with an independently developed anti-Oatp14 (C terminus) antibody has been reported previously (6). Consistent with qPCR analysis of Oatp14 expression (Fig. 1CGo), the bands recognized by anti-Oatp14 (C terminus) were strongly enriched in BMEC compared with whole brain. Preabsorption of anti-Oatp14 (C terminus) with the GST-fusion protein used for immunization abolished Western blot detection of Oatp14 in mouse and rat brain and BMEC (Fig. 3Go, G and H). Preabsorption of anti-Oatp14 antibodies with purified GST (nonfusion protein) did not affect Western blot results (data not shown). Anti-Oatp14 (N terminus) antibodies detected only lower than expected molecular mass bands (data not shown), suggesting that the N terminus of Oatp14 may be susceptible to degradation. Because plasma membrane staining was observed with anti-OATP14 antibodies in transfected HEK cells (Fig. 2Go, N–P) as well as in choroid plexus (see Fig. 7Go) and isolated microvessels (see Fig. 8Go), it is likely that both antibodies detected intact Oatp14 in whole cells and that degradation of Oatp14 occurred during cell lysis. Because of the limited availability of human brain surgery specimens, we were unable to perform conclusive Western blot analysis of OATP14 in isolated human BMEC.


Figure 7
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  		FIG. 7. Immunofluorescence staining for MCT8 and OATP14 in choroid plexus. A and B, Mouse choroid plexus. Mct8 staining (red, A) is observed primarily on the apical surface of mouse choroid plexus, costained with Mrp4 (green). Crosses mark the cerebral spinal fluid space; Mrp4 staining (green, A and B) localizes primarily to the basal-lateral (blood-facing) epithelial surface (marked by asterisks). Oatp14 (C terminus) staining (red, B) appears on both apical and basal-lateral choroid plexus epithelial surfaces, with a bias toward basal-lateral localization. Extensive colocalization of Oatp14 and Mrp4 (green, B) is apparent on the basal-lateral surface (marked by asterisks). Areas of colocalization appear yellow (B). Scale bars for A–D, 20 µm. C and D, Human choroid plexus. MCT8 staining (red, C) is visible on the apical and basal surfaces of human choroid plexus. OATP14 staining (red, D) appears on both apical and basal-lateral choroid plexus epithelial surfaces. Nuclei are stained with TOTO-3 iodide, pseudocolored green. Asterisks mark basal surfaces; crosses mark cerebral-spinal fluid spaces.

 

Figure 8
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  		FIG. 8. Immunofluorescence localization of Mct8 and Oatp14 in isolated rat cerebral microvessels. A–C, Mct8 and Pgp. MCT8 staining in an isolated microvessel (red, A) overlaps strongly with staining for Pgp (green, B), a marker of the cerebral capillary lumen. Areas of overlap appear yellow in the merged image (C). Abluminal Mct8 staining is apparent adjacent to the endothelial cell nucleus (arrowhead, A) that appears distinctly red in the merged image (arrowhead, C). Scale bars for A–L, 20 µm. D–F, Mct8 and Aq-4. Mct8 staining (red, D) appears largely distinct from staining for Aq-4 (green, E), a marker of the glial endfeet that ensheath microvessels. Distinct red (arrow) and green (arrowhead) signals can be seen in the merged image (F). G–I, Oatp14 and Pgp. Oatp14 (N terminus) staining (red, G) partially overlaps with Pgp staining (green, H) in the microvessel lumen, indicated by areas of yellow indicated by arrow in the merged image (arrow, I). Distinct areas of abluminal staining appear red in the merged image (I, arrowhead). J–L, Oatp14 and Aq-4. Oatp14 staining (red, J) partially overlaps with Aq-4 staining (green, K) in glial endfeet, with areas of overlap appearing yellow in the merged image (L, arrow). Distinct areas of endothelial staining appear red in the merged image (L, arrowhead).

 
Brain localization.
Immunofluorescence staining of frozen sections of human, mouse, and rat brain revealed striking expression of MCT8 in cerebral microvessels (Fig. 4Go), identified by costaining with the microvessel markers multidrug resistance protein-4 (Mrp4; ABCC4) (24) in mouse (Fig. 4Go, A–C) and Pgp (ABCB1) (25) in rat (Fig. 4Go, D–F), and human adult (Fig. 4Go, G–I) and fetal (Fig. 4Go, J–L) brain. In mouse and rat striatum, Mct8 staining appeared to be strongly enriched in microvessels (arrows, Fig. 4Go, C and F), with little staining of surrounding brain tissue. In human cerebral cortex and human fetal brain, staining for MCT8 appeared to be strongly enriched in microvessels (arrows, Fig. 4Go, I and L). Similar results were observed in all samples of human cortex examined (supplemental Fig. 1) and in human hippocampus and fetal parietal lobe (data not shown). Preabsorption of MCT8 antibodies with the GST-fusion proteins used for immunization abolished staining with MCT8 antibodies (insets, Fig. 4Go, A, D, G, and J) but did not affect staining for Mrp4 or Pgp (data not shown) in brain sections. Minor staining remained in human cortex after preabsorption with immunogen (Fig. 4GGo, inset), but this staining did not localize to microvessels (data not shown). Preabsorption with GST (nonfusion protein) did not affect MCT8 staining (data not shown).


Figure 4
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  		FIG. 4. Immunofluorescence staining of MCT8 in brain. A–C, Mouse. Staining for mouse Mct8 (A, red) overlaps with staining for Mrp4 (B, green) in cerebral microvessels (arrows) in mouse striatum, with areas of overlap appearing yellow in the merged image (C). Preabsorption of Mct8 antibody with antigen abolishes staining (A, inset, Preabs). Scale bars for A–L, 20 µm (main panels) and 50 µm (insets). D–F, Rat. Staining for rat Mct8 (D, red) overlaps with staining for the BBB marker Pgp (E, green) in cerebral microvessels (arrows) in rat striatum, with areas of overlap appearing yellow in the merged image (F). Preabsorption of Mct8 antibody with antigen abolishes staining (D, inset). G–I, Adult human. Staining for human MCT8 (G, red) is apparent in microvessels (arrows), identified by staining for the microvessel marker PGP (H, green), with areas of overlap appearing yellow in the merged image (I, arrows). Minor staining that does not localize to microvessels remains after preabsorption of MCT8 antibody with immunogen (G, inset). The brain specimen was from a 57-yr-old male donor whose cause of death was renal failure; the specimen was obtained 5 h postmortem. J–L, Fetal human. MCT8 staining (J, red) is apparent in cerebral microvessels (arrows) stained for PGP (K, green) as well as in surrounding brain tissue. The merged image is shown in L. Preabsorption of MCT8 antibody with antigen abolishes staining (J, inset). The brain specimen (region unspecified) was obtained from a 20-wk-old female fetus whose cause of death was not reported.

 
As previously reported (5), strong staining for Oatp14 was observed in mouse and rat cerebral microvessels (arrows, Fig. 5Go, C and F). Results using the anti-Oatp14 (N terminus) are shown; similar results were obtained using anti-Oatp14 (C terminus) (data not shown). Diffuse staining for OATP14 was observed in human adult (Fig. 5Go, G–I) and fetal (Fig. 5Go, J–L) brain tissue including microvessels (arrows, Fig. 5Go, I and L). Consistent with qPCR profiling, low-level OATP14 staining that was not enriched in microvessels was observed in about half of human cortex specimens examined (supplemental Fig. 1, A–E) and also in adult hippocampus and fetal parietal lobe (data not shown), in contrast to MCT8 staining, which was strongly expressed and enriched in microvessels in all human brain specimens examined (Fig. 4Go, G–L, and supplemental Fig. 1). Low-level OATP14 staining appeared to be slightly enriched in microvessels in three specimens of human cortex (Fig. 5Go, G–I, and supplemental Fig. 1, F and G) and in fetal brain (Fig. 5Go, J–L). Preabsorption of OATP14 antibodies with the GST-fusion proteins used for immunization abolished staining with OATP14 antibodies (insets, Fig. 5Go, A, D, G, and J) but did not affect staining for Mrp4 or Pgp (data not shown) in brain sections. Preabsorption with GST (nonfusion protein) did not affect OATP14 staining (data not shown).


Figure 5
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  		FIG. 5. Immunofluorescence staining of OATP14 in brain. A–C, Mouse. Oatp14 staining (A, red) overlaps with staining for Mrp4 (B, green) in cerebral microvessels (arrows) in mouse striatum, with areas of overlap appearing yellow in the merged image (C). Preabsorption of Oatp14 antibody with antigen abolishes staining (A, inset, Preabs). Scale bars for A–L, 20 µm (main panels) and50 µm (insets). D–F, Rat. Oatp14 (N terminus) staining (D, red) overlaps with staining for the BBB marker Pgp (E, green) in cerebral microvessels in rat striatum as well as in surrounding brain tissue, with areas of overlap between OATP14 and PGP appearing yellow in the merged image (F). The microvessels (arrows) in these panels appear largely in cross-section. Preabsorption of Oatp14 antibody with antigen abolishes staining (D, inset). G–I, Adult human. Staining for OATP14 (G, red) appears weak in human cerebral cortex, and does not appear strongly enriched in microvessels (arrows), identified by staining for PGP (H, green). Preabsorption of OATP14 antibody with antigen abolishes staining (G, inset). The brain specimen was obtained from a 66-yr-old male donor whose cause of death was chronic obstructive pulmonary disease; the specimen was obtained 6.5 h postmortem. J–L, Fetal human. Staining for OATP14 (J, red) is observed throughout fetal brain, including microvessels (arrows) identified by PGP staining (K, green). Preabsorption of Oatp14 antibody with antigen abolishes staining (J, inset). The brain specimen (region unspecified) was obtained from a 20-wk-old female fetus whose cause of death was not reported.

 
Mct8 mRNA is reportedly expressed in neuronal populations throughout mouse brain, including cerebral cortex and hippocampus, as well as in choroid plexus epithelial cells (15, 16). We examined the expression of Mct8 and Oatp14 protein in different regions of adult (8-wk-old) mouse brain by immunofluorescence staining. In mouse striatum, Mct8 staining was concentrated in microvessels (arrows, Fig. 6AGo) with little staining of surrounding brain tissue (Fig. 6AGo; see also Fig. 4Go). Consistent with previous reports of Mct8 mRNA expression, we observed Mct8 staining throughout adult mouse cerebral cortex and hippocampus (Fig. 6Go, B and C). Mct8 staining appeared to localize to neuronal processes in hippocampus, with little staining in cell bodies (cell bodies are marked by asterisks in Fig. 6CGo). In all brain regions examined, Mct8 expression was detected in microvessels (arrows, Fig. 6Go, A–C). As previously reported (16, 26), Mct8 expression was also observed in tanycytes lining the third ventricle (arrows, Fig. 6DGo), which form a barrier between the brain and the portal capillaries of the hypothalamus and which are implicated in hypothalamic thyroid hormone homeostasis (26, 27). Oatp14 staining was enriched in microvessels throughout the brain (arrows, Fig. 6Go, E–G) and in tanycytes of the third ventricle (arrows, Fig. 6HGo). Microvessel staining was confirmed by colocalization with Mrp4 staining (data not shown). We observed similar results in 21-d-old mouse brain as well as in rat brain at 5 d, 21 d, and 8 wk of age (data not shown). Results using the anti-Oatp14 N terminus are shown; similar results were obtained using anti-Oatp14 C terminus (data not shown).


Figure 6
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  		FIG. 6. Immunofluorescence staining for Mct8 and Oatp14 in different regions of mouse brain. A–D, Mct8. Mct8 staining is largely restricted to microvessels in mouse striatum (A). Mct8 staining is apparent in microvessels (arrows) and surrounding neural tissue in cortex (B) and hippocampus (C). Asterisks mark the cell bodies of hippocampal neurons (C). Mct8 staining is observed in tanycytes lining the third ventricle (arrows, D). III marks the ventricular space. Scale bars for A–H, 20 µm. E–H, Oatp14. Staining with anti-Oatp14 (N terminus) is apparent in microvessels (arrows) and in surrounding tissue in mouse striatum (E), cortex (F), and hippocampus (G). Oatp14 staining is observed in tanycytes lining the third ventricle (arrows, H). III marks the ventricular space.

 
Choroid plexus localization.
In frozen sections of mouse choroid plexus epithelium, Mct8 staining localized to the apical [cerebrospinal fluid (CSF)-facing] cell surface (red in Fig. 7AGo; CSF spaces marked by crosses), in contrast to Mrp4 staining (green in Fig. 7AGo), which labeled the basal surface (marked by asterisks) as previously reported (24). Staining with anti-Oatp14 (C terminus) was observed on both basal and apical cell surfaces (red staining in Fig. 7BGo; crosses mark CSF; asterisks mark basal surface), with strong overlap between Oatp14 and Mrp4 (green in Fig. 7BGo) on the basal cell surface (areas of overlap appear yellow in Fig. 7BGo). Similar results were observed in rat choroid plexus (data not shown). Similar results were obtained using anti-Oatp14 (N terminus) (data not shown). In human choroid plexus, MCT8 staining (red, Fig. 7CGo) was observed primarily on the epithelial apical surface, with staining also apparent on the basal surface (marked by asterisks). Human OATP14 staining (red, Fig. 7DGo) was observed on both apical and basal epithelial surfaces.

Subcellular localization in isolated microvessels.
We examined the subcellular localization of rat Mct8 and Oatp14 (N terminus) in isolated rat cerebral microvessels. Mct8 staining (red, Fig. 8AGo) overlapped strongly with staining for the well-characterized luminal marker Pgp (25) (Fig. 8BGo, green; overlap appears yellow in Fig. 8CGo). Distinctly abluminal Mct8 staining was also observed (arrowheads, Fig. 8Go, A and C). Mct8 staining (Fig. 8Go, D and F) appeared distinct from staining for aquaporin-4 (Aq-4; Fig. 8Go, E and F), a marker for the glial endfeet that ensheath microvessels (28), indicating that Mct8 staining was localized in endothelial cells. Mct8 expression was not observed in vascular pericytes (data not shown). In contrast to the primarily luminal localization of Mct8, Oatp14 staining (red, Fig. 8GGo) appeared predominantly abluminal, although some overlap with Pgp staining (green, Fig. 8HGo) in the microvessel lumen was also observed (arrow, Fig. 8IGo). Oatp14 staining (Fig. 8Go, J and L) also partially overlapped with Aq-4 staining (Fig. 8Go, K and L), providing evidence of Oatp14 expression in glial endfeet and possibly the adjacent vascular pericytes in addition to endothelial cells. Similar results were observed in isolated mouse microvessels (data not shown), and with anti-Oatp14 (C terminus) (data not shown). Due to the limited availability of human brain surgery specimens, we were unable to perform immunofluorescence staining for MCT8 and OATP14 in isolated human microvessels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we show that MCT8 mRNA and protein are highly expressed in cerebral microvessels in human, mouse, and rat in addition to its previously reported expression in neurons and choroid plexus. In contradiction to previous models, strong expression of MCT8 in brain endothelial cells suggests a contribution of MCT8 to thyroid hormone transport across the BBB in addition to its role in thyroid hormone uptake by neurons. Previous in situ hybridization studies failed to detect Mct8 mRNA in cerebral microvessels in mouse brain (15, 16), possibly due to the difficulty of discerning cerebral microvessels in the low-magnification images of brain sections shown in these studies. Alternatively, strain differences in Mct8 expression between the CD-1 mice examined in this study and different mouse strains used for previous in situ hybridization studies may account for this discrepancy, as has been reported for expression of the ABC transporter Mrp2 (ABCC2) in mouse brain vasculature (29).

Generally, we found strong correlation between transporter expression levels measured by qPCR, Western blotting, and immunofluorescence staining. However, immunofluorescence staining of MCT8 revealed apparently stronger enrichment in human microvessels than did qPCR analysis. The discrepancy in the degree of MCT8 enrichment in human microvessels may be due to the fact that by necessity we analyzed small specimens of human brain that were collected from different regions of the brain, which may express variable levels of MCT8 in neurons compared with endothelial cells. Also, human brain and BMEC specimens used for qPCR were obtained from patients undergoing surgery for epilepsy, whereas human brain tissue used for immunofluorescence staining was obtained at autopsy from nonepileptic individuals, which could explain the difference in microvessel enrichment of MCT8. Analysis of additional human specimens representing multiple brain regions will be required to confirm the results presented here and fully characterize MCT8 expression in human brain. Despite these issues, qPCR and immunofluorescence analysis demonstrate expression of MCT8 in several regions of human adult and fetal brain and therefore is likely to play a role in thyroid hormone transport across the BBB. Similar to the thyroid hormone levels measured in the brains of Mct8-deficient mice, low T4 and normal T3 levels were detected in the CSF of a patient with AHDS (30), indicating that in addition to mediating neuronal T3 uptake, MCT8 may play a role in T4 transport across the BBB.

The MCT8 localization reported here is consistent with functional studies of thyroid hormone transport across the BBB. For example, Mct8 knockout mice show dramatically reduced uptake of peripherally dosed T3 into the brain (14, 15). Studies of thyroid hormone transport suggest that brain endothelium, not choroid plexus, is the major site of brain uptake of circulating thyroid hormones (31). Taken together, these data implicate Mct8 in thyroid hormone transport across brain microvessels in mice. Mct8-mediated T3 transport across the BBB is also consistent with the finding that brain development and function is normal in D2 deiodinase-deficient mice (32), which indicates that direct brain uptake of T3 from the circulation can compensate for the lack of local brain generation of T3 by deiodination of T4. However, the mechanism by which Mct8-deficient neurons take up T3 remains to be elucidated; species differences in the neuronal expression of other transporters capable of T3 transport may be responsible for the lack of motor deficits in Mct8-deficient mice.

We detected Mct8 and Oatp14 on both the luminal and abluminal membrane surfaces of rat microvessels, as previously reported for rat Oatp14 (5). Mct8 localized more strongly to the luminal (blood-facing) surface, and Oatp14 was more concentrated on the abluminal (brain-facing) surface. Based on these localization patterns, these transporters may function as a complementary pair to transport thyroid hormone into the brain. If Mct8 were involved primarily in transport from the blood into endothelial cells, and Oatp14 were responsible for efflux of thyroid hormones from endothelial cells into brain, then T4 would be predicted to be the primary form of thyroid hormone transported across the BBB, given the preferential transport of T4 by Oatp14 compared with T3 (4, 5, 6). This is consistent with data showing that up to 80% of T3 in rat cerebral cortex is generated locally (33) by D2 deiodinase in glial cells (1). The potential expression of Oatp14 that we observe in glial endfeet suggests that transport of T4 across the BBB and into glial cells may be mediated by Oatp14 expressed on both the basal surface of microvessels and the glial endfeet that intimately ensheath them.

In human, mouse and rat choroid plexus, MCT8 appears to be concentrated on the apical, CSF-facing, epithelial membrane, whereas OATP14 is detectable on both the apical and basal surfaces, with a potential bias toward basal localization, consistent with previous reports of Oatp14 localization in rat choroid plexus (6). Interestingly, the polarity of Mct8 and Oatp14 localization is reversed in choroid plexus compared with isolated cerebral microvessels. This pattern of localization suggests that in addition to transporting thyroid hormones across the BBB into the brain, Mct8 and Oatp14 may function as a pair to transport T4 from the blood into the CSF, to efflux thyroid hormones or their inactive metabolites rT3 and T2 from the CSF into the blood.

Interestingly, we found OATP14 to be highly expressed and enriched in cerebral microvessels in rodent, whereas OATP14 expression and localization was variable in human brain, with generally weak enrichment in microvessels. The physiological significance of the species differences in OATP14 expression is not clear. The CSF levels of free T4 in an AHDS patient were found to be about half of normal control levels, whereas free T3 levels were within the normal range (30), similar to the relative brain levels of these hormones in Mct8-null mice compared with wild type (14, 15). It is possible that CSF levels of thyroid hormones do not reflect the hormone levels in the brain parenchyma and that high expression of Oatp14 in rodent microvessels may permit higher levels of T4 to cross the BBB in the absence of functional MCT8 in mice compared with humans, thus contributing to the mild phenotype observed in Mct8-null mice compared with AHDS patients. Generating mice lacking both Mct8 and Oatp14 could test this hypothesis. Additional human brain specimens must be examined to confirm a species difference in OATP14 expression, particularly in developing brain where thyroid hormone may play a more essential role compared with adult brain.

Several AHDS patients have been treated with levothyroxine (L-T4) replacement therapy (50 µg/d or less) without significant improvement of psychomotor symptoms (11, 34). The failure of conventional T4 therapy has been attributed to poor hormone uptake in brain due to MCT8 deficiency (35), and our data suggest that OATP14 expression may be too low in human brain microvessels to compensate for the absence of MCT8-mediated T4 transport across the BBB. Slight improvement in motor control has been reported after treatment of a single AHDS patient with very high levels of L-T4 (up to 400 µg/d) beginning at age 3 (36); earlier intervention with supraphysiological doses of T4 has yet to be fully evaluated. Thyroid hormone analogs that bypass the requirement for MCT8 transport have been proposed as AHDS therapeutics (35); however, an additional challenge for AHDS treatment will be to restore thyroid hormone action in the brain without exacerbating symptoms such as muscle wasting that are thought to stem from abnormally high T3 in the periphery (13). Improvement of thyrotoxic symptoms has been reported in a 16-yr-old AHDS patient treated with a novel therapy of propylthiouracil to suppress T3 production in combination with high dose L-T4 (100 µg/d) (37). Direct intrathecal or intraventricular administration of T3 or thyroid hormone analogs that bypass the requirement for transport by MCT8 might improve the central nervous system defects in AHDS patients without exacerbating peripheral hyperthyroid symptoms. Understanding mechanisms of thyroid hormone transport and differences between human and rodent transport pathways will be critical to the design and testing of potential AHDS therapies.


   Acknowledgments
 
We thank Steven Cwirla for assistance with GST-fusion protein production.


   Footnotes
 
Disclosure Statement: The authors are current or former employees of, and may hold equity interests in, XenoPort Inc.

First Published Online August 7, 2008

Abbreviations: AHDS, Allan-Herndon-Dudley syndrome; Aq-4, aquaporin-4; BBB, blood-brain barrier; BMEC, brain microvessel endothelial cells; CSF, cerebrospinal fluid; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT1, glucose transporter 1; GST, glutathione-S-transferase; MCT, monocarboxylate transporter; Mrp4, multidrug resistance protein-4; OATP, organic anion transporter polypeptide; PGP, P-glycoprotein; qPCR, quantitative PCR; T2, 3,3'-diiodothyronine.

Received March 19, 2008.

Accepted for publication July 25, 2008.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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L. Mebis, D. Paletta, Y. Debaveye, B. Ellger, L. Langouche, A. D'Hoore, V. M Darras, T. J Visser, and G. Van den Berghe
Expression of thyroid hormone transporters during critical illness
Eur. J. Endocrinol., August 1, 2009; 161(2): 243 - 250.
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E. K. Wirth, S. Roth, C. Blechschmidt, S. M. Holter, L. Becker, I. Racz, A. Zimmer, T. Klopstock, V. Gailus-Durner, H. Fuchs, et al.
Neuronal 3',3,5-Triiodothyronine (T3) Uptake and Behavioral Phenotype of Mice Deficient in Mct8, the Neuronal T3 Transporter Mutated in Allan-Herndon-Dudley Syndrome
J. Neurosci., July 29, 2009; 29(30): 9439 - 9449.
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 EndocrinologyHome page
A. Ceballos, M. M. Belinchon, E. Sanchez-Mendoza, C. Grijota-Martinez, A. M. Dumitrescu, S. Refetoff, B. Morte, and J. Bernal
Importance of Monocarboxylate Transporter 8 for the Blood-Brain Barrier-Dependent Availability of 3,5,3'-Triiodo-L-Thyronine
Endocrinology, May 1, 2009; 150(5): 2491 - 2496.
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 EndocrinologyHome page
H. Heuer and T. J. Visser
Pathophysiological Importance of Thyroid Hormone Transporters
Endocrinology, March 1, 2009; 150(3): 1078 - 1083.
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