The Monocarboxylate Transporter 8 Linked to Human Psychomotor Retardation Is Highly Expressed in Thyroid Hormone-Sensitive Neuron Population
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内分泌学杂志 2005年第4期
Max Planck Institute for Experimental Endocrinology (H.H., M.K.M., S.I., J.M., K.B.), D-30625 Hannover, Germany; Institute of Molecular Biotechnology (H.H.), D-07745 Jena, Germany; and Department of Internal Medicine, Erasmus Medical Center (E.C.H.F., T.J.V.), 3015 GE Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Dr. Heike Heuer, Institute of Molecular Biotechnology, Beutenbergstrasse 11, D-07745 Jena, Germany. E-mail: hheuer@imb-jena.de.
Abstract
Recent genetic analysis in several patients presenting a severe form of X-linked psychomotor retardation combined with abnormal thyroid hormone (TH) levels have revealed mutations or deletions in the gene of the monocarboxylate transporter 8 (MCT8). Because in vitro MCT8 functions as a TH transporter, the complex clinical picture of these patients indicated an important role for MCT8 in TH-dependent processes of brain development. To provide a clue to the cellular function of MCT8 in brain, we studied the expression of MCT8 mRNA in the murine central nervous system by in situ hybridization histochemistry. In addition to the choroid plexus structures, the highest transcript levels were found in neo- and allocortical regions (e.g. olfactory bulb, cerebral cortex, hippocampus, and amygdala), moderate signal intensities in striatum and cerebellum, and low levels in a few neuroendocrine nuclei. Colocalization studies revealed that MCT8 is predominantly expressed in neurons. Together with the spatiotemporal expression pattern of MCT8 during the perinatal period, these results strongly indicate that MCT8 plays an important role for proper central nervous system development by transporting TH into neurons as its main target cells.
Introduction
THYROID HORMONE (TH) is essential for the metabolic homeostasis of almost all organs and tissues. Even more importantly, it is critically involved in the development and function of the central nervous system (CNS). In adults, individual TH deficiency may lead to a vast array of clinical manifestations, including neurological and psychiatric symptoms, which are usually reversible after adequate treatment. In contrast, TH deficiency during narrow developmental windows encompassing pre- and neonatal periods results in irreversible damage, notably in the development of the CNS (1, 2, 3). If TH replacement therapy is not instituted immediately after birth, severe forms of congenital hypothyroidism lead to the syndrome of cretinism, a disorder characterized by severe mental retardation, neurological deficits (e.g. movement disorders, spasticity, and speech problems), and hearing impairment. These deficiencies reflect the extensive structural alterations observed in brain development under hypothyroid conditions such as abnormalities in neuronal migration and differentiation, axonal and dendritic outgrowth, synaptogenesis, myelination, etc. (4, 5, 6). Unfortunately, these complex phenotypic changes are not well understood at the molecular level.
It is well established that the actions of TH are predominantly mediated by the binding of T3 to nuclear hormone receptors, thereby regulating gene expression in target tissues. T3, the principle bioactive form of TH, is produced by outer ring deiodination of T4, the main product of the thyroid gland. In the CNS, the conversion of T4 to T3 is carried out by the enzyme iodothyronine deiodinase type 2 (D2) whereas the inactivation of T4 and T3 by inner ring deiodination to rT3 and T2, respectively, is catalyzed by the iodothyronine deiodinase type 3 (D3) (7). Because both enzymes are located intracellularly, cellular entry is required for conversion of TH and for binding to the nuclear receptors.
Several classes of membrane iodothyronine transporters with different kinetics and substrate preferences have been identified recently (8, 9), questioning the hypothesis of passive TH diffusion into target cells. As a biochemically more extensively analyzed transport system, the organic anion-transporting polypeptide Oatp14 (Slc21a14) has been suggested to be important for the passage of T4 from the circulating blood system to the brain because it has been reported to be highly expressed in capillary endothelial cells (10). The monocarboxylate transporter 8 (MCT8; Slc16a2) was also identified as a very active and specific TH transporter highly expressed in brain and other TH-responsive tissues (11). Very recently, the physiological function of MCT8 as a TH transporter could be firmly established by the genetic analysis of children who carry mutations or deletions in the MCT8 gene and exhibit abnormal circulating TH concentrations in addition to severe neurological abnormalities, as observed under conditions of extreme iodine deficiency (12, 13).
To provide an anatomical basis for additional studies, we analyzed the mRNA expression pattern of MCT8 in the brains of wild-type and athyroid Pax8–/– mice in comparison with the expression pattern of D2 and Oatp14.
Materials and Methods
Experimental animals
Animals were maintained according to the regulations of the animal welfare committee of the Medizinische Hochschule Hannover. Standard laboratory chow and water were provided ad libitum. A temperature of 20 C and alternating 12-h light, 12-h dark cycles were controlled automatically. Pax8+/– male and female mice were used for mating, and the offspring were genotyped by Southern blot analysis as described recently (14). Wild-type and Pax8–/– littermates were decapitated between 1500 and 1700 h on selected postnatal days, and brains were removed and immediately frozen in isopentane cooled to –40 C on dry ice. Alternatively, animals were perfused transcardially with 4% paraformaldehyde in 0.1 M PBS solution (PBS), and their brains were then placed in 30% sucrose for dehydration and subsequently frozen on dry ice. Coronal 20-μm-thick sections were cut on a cryostat (Leica, Deerfield, IL), thaw-mounted on silane-treated slides, and then stored at –80 C until additional processing.
Probe synthesis
A cDNA fragment corresponding to nucleotides (nt) 1251–1876 of mouse MCT8 cDNA (GenBank accession code AF045692) and a fragment corresponding to nt 698-1165 of mouse Oatp14 cDNA (GenBank accession code NM_021471) were generated by PCR and subcloned into the pGEM-T easy vector (Promega Corp., Madison, WI). To detect D2 transcripts, a PCR fragment corresponding to nt 131-1045 of mouse D2 cDNA (GenBank accession code AF096875) was used as template. 35S-Labeled riboprobes were produced by in vitro transcription as described previously (15). Calculated specific activities were 5 x 105 Ci/mmol for mMCT8, 2.4 x 105 Ci/mmol for mOatp14, and 3.1 x 105 Ci/mmol for mD2. After synthesis, the probes were subjected to mild alkaline hydrolysis for calculated time periods, as described by Sch?fer and Day (16), to generate fragments of about 250 nt.
In situ hybridization (ISH)
ISH was carried out as previously described (15). Briefly, frozen sections were fixed with phosphate-buffered 4% paraformaldehyde solution (pH 7.4), then treated with 0.4% Triton in PBS, followed by an acetylation step. After dehydration, sections were covered with hybridization mix containing radioactive cRNA probes diluted in hybridization buffer [50% formamide, 10% dextran sulfate, 0.6 M NaCl, 10 mM Tris/HCl (pH 7.4), 1x Denhardt’s solution, 100 μg/ml sonicated salmon sperm DNA, 1 mM EDTA, and 10 mM dithiothreitol] to a final concentration of 2.5 x 104 cpm/μl. After incubation at 58 C for 16 h, coverslips were removed in 2x standard saline citrate (0.3 M NaCl and 0.03 M sodium citrate, pH 7.0). The sections were subsequently treated with ribonuclease A/T1 at 37 C for 30 min. Final washes were carried out in 0.2x standard saline citrate at 65 C for 1 h. After dehydration, the sections were exposed to x-ray film (BioMax MR, Eastman Kodak Co., Rochester, NY) for 18 h. For microscopic analysis, sections were dipped in Kodak NTB2 nuclear emulsion and stored for 8 d at 4 C. Autoradiograms were developed and analyzed under dark-field illumination using an Olympus AX microscope (New Hyde Park, NY). For bright-field illuminations, some sections were counterstained with cresyl violet.
Combined immunohistochemistry and ISH
For colocalization experiments, perfusion-fixed brain sections were stained with a monoclonal antibody against NeuN (1:100; Chemicon International, Temecula, CA) or with a monoclonal antibody against glial fibrillary acidic protein (GFAP; 1:500; Sigma-Aldrich Corp., St. Louis, MO) using the M.O.M.-Vectastain Kit-Peroxidase (Vector Laboratories, Inc., Burlingame, CA) according to the instructions of the manufacturer. To prevent RNA degradation, all incubation steps were carried out in the presence of 10 U/ml Superasein (Ambion, Inc., Austin, TX). After the peroxidase staining reaction with 3,3'-diaminobenzidine/H2O2 as substrates, MCT8 transcripts were localized by ISH as described above. For quantitative analysis, we determined the number of cells positive for MCT8 mRNA and NeuN or GFAP, respectively, in three different areas (retrosplenial cortex, stratum radiatum of the hippocampal formation, and amygdalo-hippocampal area) on three consecutive sections.
Results and Discussion
For a detailed analysis of the MCT8 expression pattern in mouse brain, we employed a highly sensitive ISH technique using 35S-labeled riboprobes. As a first approach, we determined the MCT8 mRNA distribution pattern in coronal sections through the CNS of 3-wk-old mice. Selected dark-field autoradiographs in rostral to caudal orientation are illustrated in Fig. 1. By microscopic analysis of photographic emulsion-coated slides, region-specific transcript levels were evaluated by three experienced investigators; the results are summarized in Table 1. The lack of any hybridization signal using the corresponding sense probes underlined the specificity of the hybridization experiments (Fig. 2D).
FIG. 1. Region-specific expression of MCT8 mRNA in the mouse brain on P21. A series of dark-field photomicrographs arranged from rostral (A) to caudal (H) illustrates the localization of MCT8 transcripts after hybridization with an antisense riboprobe. Acb, Nucleus accumbens; Arc, arcuate nucleus; AP, area postrema; Aq, aqueduct; AO, anterior olfactory nucleus; BM, basomedial amygdaloid nucleus; BL, basolateral amygdaloid nucleus; CA1, field CA1 (Ammon’s horn); CA3, field CA3 (Ammon’s horn); ChP, choroid plexus; Cl, claustrum; CPu, caudate putamen; DEn, dorsal endopiriform nucleus; DM, dorsomedial hypothalamic nucleus; DG, dentate gyrus; GrO, granular layer of the olfactory bulb; ICj, islands of Calleja; LV, lateral ventricle; ME, median eminence; Me, medial amygdaloid nucleus; MPA, medial preoptic area; Pa, paraventricular hypothalamic nucleus; PCL, Purkinje cell layer; Pe, periventricular hypothalamic nucleus; Pir, piriform cortex; PMCo, posteromedial cortical amygdaloid nucleus; S, subiculum; SFO, subfornical organ; Tu, olfactory tubercle; VMH, ventromedial hypothalamic nucleus. Scale bar, 2 mm.
TABLE 1. Subjective evaluation of regional MCT8 mRNA levels
FIG. 2. Illustration of MCT8 expression in the hypothalamus. Moderate MCT8 expression levels were only observed in the para- and periventricular hypothalamic nucleus. A, In contrast, scattered cells positive for Oatp14 mRNA were found throughout the hypothalamus, with an increased signal density in the PVN (B). Prominent expression of MCT8 was detected in the median eminence and in tanycytes lining the 3V (C). Hybridization experiments carried out with a sense probe for MCT8 did not reveal any specific labeling (D). AH, Anterior hypothalamic nucleus; ME, median eminence; Pe, periventricular nucleus; PVN, paraventricular hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus; 3V, third ventricle. Scale bar, 400 μm.
As illustrated in Figs. 1 and 3E, the highest transcript levels for MCT8 were detected in the choroid plexus of the lateral, third, and fourth ventricles. Moderate to low transcript levels for MCT8 were also found in circumventricular organs, namely, subfornical organ, median eminence, and area postrema. In addition, MCT8 mRNA expression was observed occasionally in larger blood vessels. The functional relevance of MCT8 expression in these structures remains elusive as yet, because previous studies in rats have strongly indicated that only a fraction of T4 in the brain passes through the choroid plexus-cerebrospinal fluid barrier (17).
FIG. 3. Cellular localization of MCT8 transcripts in various brain regions at higher magnification. A and B, Illustration of layer-specific MCT8 expression in the primary somatosensory cerebral cortex by bright-field (A) and dark-field (B) illumination. Strong signal intensities were observed over layers II/III and layer V. C and D, At the cellular level, clusters of silver grains (arrowhead) representing MCT8 mRNA could be detected over cell bodies that are immunoreactive for the neuronal marker NeuN (arrows), as shown exemplarily for the retrosplenial cortex (C). In contrast, MCT8 hybridization signals did not overlap with anti-GFAP-labeled cell bodies, implying that MCT8 is not expressed in astrocytes (D). Of all brain regions analyzed, the highest MCT8 transcript levels were detected in the choroid plexus, as shown in the bright-field micrograph of the dorsal part of the third ventricle (E). CC, Corpus callosum; ChP, choroid plexus; MHb, medial habenular nucleus; RSG, retrosplenial granular cortex; wm, white matter. Scale bar: A, B, and E, 140 μm; C and D, 14 μm.
According to these studies, the main entry of TH into the brain occurs via the blood-brain barrier. With the very limited expression of MCT8 found in blood vessels, MCT8, therefore, does not appear to be important for the transport of TH across the blood-brain barrier. Other proteins capable of transporting TH, notably Oatp14, may serve this function. In agreement with the detailed immunohistochemical and biochemical analysis by Sugyiama et al. (10) demonstrating the luminal localization of Oatp14 in rat capillary endothelial cells, we also observed that Oatp14 mRNA is selectively expressed in brain capillaries beside choroid plexus structures (Fig. 4, E and F). As a consequence, it is unlikely that impaired transport of TH via the choroid plexus-cerebrospinal fluid barrier could be responsible for the severe neurological deficits observed in patients with nonfunctional MCT8.
FIG. 4. MCT8 expression is not altered in athyroid Pax8–/– mice. ISH analysis in 15-d-old Pax8–/– mice (B, D, and F) and their wild-type littermates (A, C, and E) did not reveal significant changes in transcript levels for MCT8 (A and B) or Oatp14 (E and F), whereas in Pax8–/– mice, D2 signal intensities were significantly increased and were especially pronounced throughout cortical areas (C and D). Scale bar, 500 μm.
These deficits are more likely explained by the expression of MCT8 mRNA we observed in neuronal populations well acknowledged as being exquisitely dependent on proper TH supply. In the cerebral cortex, moderate to high signal intensities were observed throughout layers 2–5, whereas only scattered cells were labeled in layer 6 (Fig. 3, A and B). High transcript levels for MCT8 were also detected throughout the amygdala, in the pyramidal cell layer of the hippocampal formation, as well as in the granule cell layer of the dentate gyrus (Figs. 1 and 5). In support of our hypothesis, this distribution perfectly supports the findings that in brains of animals rendered hypothyroid, neocortical and hippocampal pyramidal cells display reduced dendritic aborization as well as abnormal spine disposition (18, 19). Furthermore, changes in the distribution of retrogradely labeled callosal neurons in both visual and auditory cortices have been described in rats made hypothyroid during embryonic stages (20, 21). Moreover, deficiencies in neuronal migration leading to a less defined cortical layering and a decreased number of granule cells in the hippocampus were also observed in the brains of hypothyroid animals (22). Finally, like patients with the syndrome of resistance to TH, who suffer from learning disorders and mental retardation (23), genetically engineered mice in which a T3 binding mutation was introduced into the TH receptor ? locus displayed learning deficits together with reduced BDNF (brain-derived neurotrophic factor) immunoreactivity in the hippocampus and a reduced number of granule cells and fibers in this brain area, especially in the CA3 region (24). These abnormalities fit well with the clinical picture of patients with mutated MCT8 who suffer from severe deficits in higher cognitive function and exhibit severe symptoms of mental retardation, with rudimentary communication skills and lack of speech development (12, 13).
FIG. 5. Spatiotemporal expression pattern of MCT8 in the CNS. High transcript levels of MCT8 were found in the cerebral cortex and in the hippocampal formation by embryonic d 18 (A). On P3 and P6, reduced transcript levels were observed in the parietal cortex, and increased intensities were found in neighboring cortical areas (B and C). These heterogeneities disappeared by P9 (D). In the pyramidal cell layer and granule cell layer of the hippocampal formation, the signals were diffuse during early periods of development. On P15, MCT8 expression reached adult levels (E and F). Scale bar, 500 μm.
MCT8 expression was also observed in brain areas closely associated with motor control. Hybridization signals were found throughout striatal areas, with high transcript levels in the islands of Calleja and olfactory tubercle and less prominent levels in scattered cells throughout the caudate-putamen and nucleus accumbens (Fig. 1, B and C). The interpretation that the striatum is dependent on proper TH supply, at least during development, is supported by the clinical picture of patients suffering from neurological cretinism. This syndrome is mostly caused by extreme iodine deficiency that affects both maternal and fetal TH production, with the result that TH is insufficiently supplied during the early phases of development. Consequently, brain development is more severely affected than under conditions of congenital hypothyroidism (3). It is therefore not surprising that neurological or endemic cretinism not only results in mental retardation, as observed in congenital hypothyroidism, but also leads to a striatopallidal syndrome with poor motor coordination and spastic diplegia (25), exactly the symptoms found in MCT8-affected patients (12, 13). Some of the children even suffer from spastic quadriplegia, which results from central hypotonia, followed by increased peripheral hypertonia.
According to the present concept of TH metabolism in brain, TH, preferentially T4, is taken up at the blood-brain barrier by transporters such as Oatp14, which indeed exhibits considerably higher transport capacity for T4 than for T3 (10). After release from endothelial cells, probably also mediated by Oatp14 (10), T4 has to be translocated (presumably mediated by another transporter, not yet identified) into astrocytes and subsequently converted to the biologically active T3 by the enzyme D2, which is known to be localized intracellularly in these cells (26). Consistent with this scheme (Fig. 6), our ISH analysis revealed an astrocytic distribution pattern of D2 hybridization signals (Fig. 4, C and D). After release from astrocytes, T3 has to enter neurons, the important target sites of T3 action, a process most likely mediated by MCT8.
FIG. 6. A schematic model outlining routes of TH transport in the brain. Oatp14 expressed by endothelial cells transports T4 from blood into extracellular fluid in the CNS. T4 then enters astrocytes by an as yet unknown mechanism to be converted to the active compound T3 by the intracellularly localized D2. After release, T3 is taken up by neurons, where it regulates gene expression and is finally inactivated by D3. This neuronal import is mediated by the transporter MCT8. BBB, Blood-brain barrier.
To prove this hypothesis, we stained perfusion-fixed brain sections first with an antibody against NeuN, a transcription factor expressed in a subset of neurons, or with an antibody against GFAP to label astrocytes. Thereafter, MCT8 mRNA was detected by ISH. Although the perfusion-fixation procedure necessary for the antibody staining results in an overall reduction of in situ hybridization signal intensity, cells positive for NeuN and MCT8 could be clearly observed in various cortical areas, as shown exemplarily for the retrosplenial cortex in Fig. 3C. Quantification of double-labeled cells in selected areas with high transcript levels of MCT8, such as the retrosplenial cortex and the amygdalo-hippocampal area, revealed that 62 ± 4% of all NeuN-positive cells were also covered with clusters of silver grains specific for MCT8 mRNA, whereas an overlapping expression pattern could not be found between GFAP-labeled cells and MCT8-positive cell bodies in the same areas. Furthermore, scattered cells labeled for MCT8 in the stratum radiatum of the hippocampal formation, a region enriched for astrocytes, were GFAP negative (Fig 3D), but positive for NeuN (89 ± 3%). Although the overall expression analysis indicated a predominant neuronal localization of MCT8, one exception was noted in hypothalamic areas: tanycytes, specialized glial cells lining the third ventricle that have been shown to express D2 (26), were labeled for MCT8 (Fig. 2C). Because these cells are mainly involved in the transport of hormones or other substances into the hypothalamus, MCT8 might offer a route for TH to enter hypothalamic regions.
In general, the MCT8 expression pattern provides strong evidence that MCT8 plays a decisive role in the transport of T3 into neurons. This idea is also supported by the finding that neurons are not only targets of T3 action, but also the site of TH metabolism, because they contain intracellularly the enzyme D3 that catalyzes the inactivation of T3 (27). Thus, a nonfunctioning neuronal TH transporter would also prevent the degradation of T3. In support of a neuronal localization of MCT8, it should be noted that all patients lacking a functional MCT8 transporter exhibit markedly elevated T3 levels (12, 13).
As evidenced by the deficits observed in conditions of congenital hypothyroidism, adequate concentrations of TH during perinatal periods are essential for proper brain development. By analyzing MCT8 mRNA expression during these time periods, we found that MCT8 mRNA is already highly expressed on embryonic d 18 in neo- and allocortical regions (Fig. 5A). On postnatal d 3 (P3) and P6, an intriguing expression pattern was found in the cerebral cortex, with a conspicuous absence of MCT8-specific labeling in the parietal cortex whereas neighboring cortical areas appeared to contain denser signal intensities (Fig. 5, B and C). In the pyramidal cell layer of the hippocampus, very broad and intense labeling was seen at P3. With the maturation of the hippocampal formation, labeling condensed between P3 and P9, reaching adult intensities around P15 (Fig. 5E). This developmental expression pattern fits well with the finding that TH deficiency during the first postnatal weeks affects pyramidal and granule cell dendritogenesis as well as granule cell migration (22, 28, 29). It is therefore tempting to speculate that the temporal regulation of MCT8 expression may reflect the time period during which T3 is especially required.
To address the question of whether MCT8 expression (like many processes regulating TH synthesis, metabolism, and function) is influenced by thyroidal status, we analyzed the MCT8 mRNA expression patterns in the CNS of wild-type animals compared with those of Pax8–/– mice. These mutant animals represent an ideal animal model of congenital hypothyroidism because they are born without a functional thyroid gland (30). As reported previously (7) and as shown in Fig. 4, C and D, for control and Pax8–/– mice, D2 mRNA expression in the brain is clearly up-regulated under hypothyroid conditions, which is especially obvious throughout the cerebral cortex. This increase in D2 expression has been discussed as a compensatory mechanism for preserving T3 concentrations in brain (31, 32). In contrast to D2, significant differences in the MCT8 mRNA expression pattern were not found in the CNS of wild-type and Pax8–/– mice (Fig. 4, A and B), indicating that under hypothyroid conditions the uptake of T3 into neurons is not compensated by changes in the transcript levels of MCT8. Our ISH analysis furthermore revealed that, like MCT8, the expression levels of Oatp14 in the brain are not significantly influenced by thyroidal status, because in athyroid Pax8–/– mice, Oatp14 signal intensities are comparable to those of wild-type animals (Fig. 4, E and F).
A preferential function of MCT8 in brain tissue has been suggested as the most likely explanation for the clinical picture of patients carrying mutations in the MCT8 gene. This idea is substantiated by the ISH analysis presented here. The neuronal localization of MCT8 in brain regions critically involved in motor control and mental development fits very well with the severe neurological phenotype of these patients. Although impaired TH transport into neuronal target cells seems to be the primary course for the patient’s phenotype, at present we cannot exclude the possibility that MCT8 might also be involved in the transport of other, not yet identified compounds that could be important for neuronal development and/or maintenance. Therefore, it will be important to generate MCT8-deficient mice to elucidate the role of MCT8 in TH metabolism during CNS development.
Acknowledgments
We thank M. Kraus and I. Bartholomaeus for excellent technical assistance, M. Piechotta for animal care, V. Ashe for linguistic help, and M. Sch?fer and G. Raivich for helpful discussion.
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Address all correspondence and requests for reprints to: Dr. Heike Heuer, Institute of Molecular Biotechnology, Beutenbergstrasse 11, D-07745 Jena, Germany. E-mail: hheuer@imb-jena.de.
Abstract
Recent genetic analysis in several patients presenting a severe form of X-linked psychomotor retardation combined with abnormal thyroid hormone (TH) levels have revealed mutations or deletions in the gene of the monocarboxylate transporter 8 (MCT8). Because in vitro MCT8 functions as a TH transporter, the complex clinical picture of these patients indicated an important role for MCT8 in TH-dependent processes of brain development. To provide a clue to the cellular function of MCT8 in brain, we studied the expression of MCT8 mRNA in the murine central nervous system by in situ hybridization histochemistry. In addition to the choroid plexus structures, the highest transcript levels were found in neo- and allocortical regions (e.g. olfactory bulb, cerebral cortex, hippocampus, and amygdala), moderate signal intensities in striatum and cerebellum, and low levels in a few neuroendocrine nuclei. Colocalization studies revealed that MCT8 is predominantly expressed in neurons. Together with the spatiotemporal expression pattern of MCT8 during the perinatal period, these results strongly indicate that MCT8 plays an important role for proper central nervous system development by transporting TH into neurons as its main target cells.
Introduction
THYROID HORMONE (TH) is essential for the metabolic homeostasis of almost all organs and tissues. Even more importantly, it is critically involved in the development and function of the central nervous system (CNS). In adults, individual TH deficiency may lead to a vast array of clinical manifestations, including neurological and psychiatric symptoms, which are usually reversible after adequate treatment. In contrast, TH deficiency during narrow developmental windows encompassing pre- and neonatal periods results in irreversible damage, notably in the development of the CNS (1, 2, 3). If TH replacement therapy is not instituted immediately after birth, severe forms of congenital hypothyroidism lead to the syndrome of cretinism, a disorder characterized by severe mental retardation, neurological deficits (e.g. movement disorders, spasticity, and speech problems), and hearing impairment. These deficiencies reflect the extensive structural alterations observed in brain development under hypothyroid conditions such as abnormalities in neuronal migration and differentiation, axonal and dendritic outgrowth, synaptogenesis, myelination, etc. (4, 5, 6). Unfortunately, these complex phenotypic changes are not well understood at the molecular level.
It is well established that the actions of TH are predominantly mediated by the binding of T3 to nuclear hormone receptors, thereby regulating gene expression in target tissues. T3, the principle bioactive form of TH, is produced by outer ring deiodination of T4, the main product of the thyroid gland. In the CNS, the conversion of T4 to T3 is carried out by the enzyme iodothyronine deiodinase type 2 (D2) whereas the inactivation of T4 and T3 by inner ring deiodination to rT3 and T2, respectively, is catalyzed by the iodothyronine deiodinase type 3 (D3) (7). Because both enzymes are located intracellularly, cellular entry is required for conversion of TH and for binding to the nuclear receptors.
Several classes of membrane iodothyronine transporters with different kinetics and substrate preferences have been identified recently (8, 9), questioning the hypothesis of passive TH diffusion into target cells. As a biochemically more extensively analyzed transport system, the organic anion-transporting polypeptide Oatp14 (Slc21a14) has been suggested to be important for the passage of T4 from the circulating blood system to the brain because it has been reported to be highly expressed in capillary endothelial cells (10). The monocarboxylate transporter 8 (MCT8; Slc16a2) was also identified as a very active and specific TH transporter highly expressed in brain and other TH-responsive tissues (11). Very recently, the physiological function of MCT8 as a TH transporter could be firmly established by the genetic analysis of children who carry mutations or deletions in the MCT8 gene and exhibit abnormal circulating TH concentrations in addition to severe neurological abnormalities, as observed under conditions of extreme iodine deficiency (12, 13).
To provide an anatomical basis for additional studies, we analyzed the mRNA expression pattern of MCT8 in the brains of wild-type and athyroid Pax8–/– mice in comparison with the expression pattern of D2 and Oatp14.
Materials and Methods
Experimental animals
Animals were maintained according to the regulations of the animal welfare committee of the Medizinische Hochschule Hannover. Standard laboratory chow and water were provided ad libitum. A temperature of 20 C and alternating 12-h light, 12-h dark cycles were controlled automatically. Pax8+/– male and female mice were used for mating, and the offspring were genotyped by Southern blot analysis as described recently (14). Wild-type and Pax8–/– littermates were decapitated between 1500 and 1700 h on selected postnatal days, and brains were removed and immediately frozen in isopentane cooled to –40 C on dry ice. Alternatively, animals were perfused transcardially with 4% paraformaldehyde in 0.1 M PBS solution (PBS), and their brains were then placed in 30% sucrose for dehydration and subsequently frozen on dry ice. Coronal 20-μm-thick sections were cut on a cryostat (Leica, Deerfield, IL), thaw-mounted on silane-treated slides, and then stored at –80 C until additional processing.
Probe synthesis
A cDNA fragment corresponding to nucleotides (nt) 1251–1876 of mouse MCT8 cDNA (GenBank accession code AF045692) and a fragment corresponding to nt 698-1165 of mouse Oatp14 cDNA (GenBank accession code NM_021471) were generated by PCR and subcloned into the pGEM-T easy vector (Promega Corp., Madison, WI). To detect D2 transcripts, a PCR fragment corresponding to nt 131-1045 of mouse D2 cDNA (GenBank accession code AF096875) was used as template. 35S-Labeled riboprobes were produced by in vitro transcription as described previously (15). Calculated specific activities were 5 x 105 Ci/mmol for mMCT8, 2.4 x 105 Ci/mmol for mOatp14, and 3.1 x 105 Ci/mmol for mD2. After synthesis, the probes were subjected to mild alkaline hydrolysis for calculated time periods, as described by Sch?fer and Day (16), to generate fragments of about 250 nt.
In situ hybridization (ISH)
ISH was carried out as previously described (15). Briefly, frozen sections were fixed with phosphate-buffered 4% paraformaldehyde solution (pH 7.4), then treated with 0.4% Triton in PBS, followed by an acetylation step. After dehydration, sections were covered with hybridization mix containing radioactive cRNA probes diluted in hybridization buffer [50% formamide, 10% dextran sulfate, 0.6 M NaCl, 10 mM Tris/HCl (pH 7.4), 1x Denhardt’s solution, 100 μg/ml sonicated salmon sperm DNA, 1 mM EDTA, and 10 mM dithiothreitol] to a final concentration of 2.5 x 104 cpm/μl. After incubation at 58 C for 16 h, coverslips were removed in 2x standard saline citrate (0.3 M NaCl and 0.03 M sodium citrate, pH 7.0). The sections were subsequently treated with ribonuclease A/T1 at 37 C for 30 min. Final washes were carried out in 0.2x standard saline citrate at 65 C for 1 h. After dehydration, the sections were exposed to x-ray film (BioMax MR, Eastman Kodak Co., Rochester, NY) for 18 h. For microscopic analysis, sections were dipped in Kodak NTB2 nuclear emulsion and stored for 8 d at 4 C. Autoradiograms were developed and analyzed under dark-field illumination using an Olympus AX microscope (New Hyde Park, NY). For bright-field illuminations, some sections were counterstained with cresyl violet.
Combined immunohistochemistry and ISH
For colocalization experiments, perfusion-fixed brain sections were stained with a monoclonal antibody against NeuN (1:100; Chemicon International, Temecula, CA) or with a monoclonal antibody against glial fibrillary acidic protein (GFAP; 1:500; Sigma-Aldrich Corp., St. Louis, MO) using the M.O.M.-Vectastain Kit-Peroxidase (Vector Laboratories, Inc., Burlingame, CA) according to the instructions of the manufacturer. To prevent RNA degradation, all incubation steps were carried out in the presence of 10 U/ml Superasein (Ambion, Inc., Austin, TX). After the peroxidase staining reaction with 3,3'-diaminobenzidine/H2O2 as substrates, MCT8 transcripts were localized by ISH as described above. For quantitative analysis, we determined the number of cells positive for MCT8 mRNA and NeuN or GFAP, respectively, in three different areas (retrosplenial cortex, stratum radiatum of the hippocampal formation, and amygdalo-hippocampal area) on three consecutive sections.
Results and Discussion
For a detailed analysis of the MCT8 expression pattern in mouse brain, we employed a highly sensitive ISH technique using 35S-labeled riboprobes. As a first approach, we determined the MCT8 mRNA distribution pattern in coronal sections through the CNS of 3-wk-old mice. Selected dark-field autoradiographs in rostral to caudal orientation are illustrated in Fig. 1. By microscopic analysis of photographic emulsion-coated slides, region-specific transcript levels were evaluated by three experienced investigators; the results are summarized in Table 1. The lack of any hybridization signal using the corresponding sense probes underlined the specificity of the hybridization experiments (Fig. 2D).
FIG. 1. Region-specific expression of MCT8 mRNA in the mouse brain on P21. A series of dark-field photomicrographs arranged from rostral (A) to caudal (H) illustrates the localization of MCT8 transcripts after hybridization with an antisense riboprobe. Acb, Nucleus accumbens; Arc, arcuate nucleus; AP, area postrema; Aq, aqueduct; AO, anterior olfactory nucleus; BM, basomedial amygdaloid nucleus; BL, basolateral amygdaloid nucleus; CA1, field CA1 (Ammon’s horn); CA3, field CA3 (Ammon’s horn); ChP, choroid plexus; Cl, claustrum; CPu, caudate putamen; DEn, dorsal endopiriform nucleus; DM, dorsomedial hypothalamic nucleus; DG, dentate gyrus; GrO, granular layer of the olfactory bulb; ICj, islands of Calleja; LV, lateral ventricle; ME, median eminence; Me, medial amygdaloid nucleus; MPA, medial preoptic area; Pa, paraventricular hypothalamic nucleus; PCL, Purkinje cell layer; Pe, periventricular hypothalamic nucleus; Pir, piriform cortex; PMCo, posteromedial cortical amygdaloid nucleus; S, subiculum; SFO, subfornical organ; Tu, olfactory tubercle; VMH, ventromedial hypothalamic nucleus. Scale bar, 2 mm.
TABLE 1. Subjective evaluation of regional MCT8 mRNA levels
FIG. 2. Illustration of MCT8 expression in the hypothalamus. Moderate MCT8 expression levels were only observed in the para- and periventricular hypothalamic nucleus. A, In contrast, scattered cells positive for Oatp14 mRNA were found throughout the hypothalamus, with an increased signal density in the PVN (B). Prominent expression of MCT8 was detected in the median eminence and in tanycytes lining the 3V (C). Hybridization experiments carried out with a sense probe for MCT8 did not reveal any specific labeling (D). AH, Anterior hypothalamic nucleus; ME, median eminence; Pe, periventricular nucleus; PVN, paraventricular hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus; 3V, third ventricle. Scale bar, 400 μm.
As illustrated in Figs. 1 and 3E, the highest transcript levels for MCT8 were detected in the choroid plexus of the lateral, third, and fourth ventricles. Moderate to low transcript levels for MCT8 were also found in circumventricular organs, namely, subfornical organ, median eminence, and area postrema. In addition, MCT8 mRNA expression was observed occasionally in larger blood vessels. The functional relevance of MCT8 expression in these structures remains elusive as yet, because previous studies in rats have strongly indicated that only a fraction of T4 in the brain passes through the choroid plexus-cerebrospinal fluid barrier (17).
FIG. 3. Cellular localization of MCT8 transcripts in various brain regions at higher magnification. A and B, Illustration of layer-specific MCT8 expression in the primary somatosensory cerebral cortex by bright-field (A) and dark-field (B) illumination. Strong signal intensities were observed over layers II/III and layer V. C and D, At the cellular level, clusters of silver grains (arrowhead) representing MCT8 mRNA could be detected over cell bodies that are immunoreactive for the neuronal marker NeuN (arrows), as shown exemplarily for the retrosplenial cortex (C). In contrast, MCT8 hybridization signals did not overlap with anti-GFAP-labeled cell bodies, implying that MCT8 is not expressed in astrocytes (D). Of all brain regions analyzed, the highest MCT8 transcript levels were detected in the choroid plexus, as shown in the bright-field micrograph of the dorsal part of the third ventricle (E). CC, Corpus callosum; ChP, choroid plexus; MHb, medial habenular nucleus; RSG, retrosplenial granular cortex; wm, white matter. Scale bar: A, B, and E, 140 μm; C and D, 14 μm.
According to these studies, the main entry of TH into the brain occurs via the blood-brain barrier. With the very limited expression of MCT8 found in blood vessels, MCT8, therefore, does not appear to be important for the transport of TH across the blood-brain barrier. Other proteins capable of transporting TH, notably Oatp14, may serve this function. In agreement with the detailed immunohistochemical and biochemical analysis by Sugyiama et al. (10) demonstrating the luminal localization of Oatp14 in rat capillary endothelial cells, we also observed that Oatp14 mRNA is selectively expressed in brain capillaries beside choroid plexus structures (Fig. 4, E and F). As a consequence, it is unlikely that impaired transport of TH via the choroid plexus-cerebrospinal fluid barrier could be responsible for the severe neurological deficits observed in patients with nonfunctional MCT8.
FIG. 4. MCT8 expression is not altered in athyroid Pax8–/– mice. ISH analysis in 15-d-old Pax8–/– mice (B, D, and F) and their wild-type littermates (A, C, and E) did not reveal significant changes in transcript levels for MCT8 (A and B) or Oatp14 (E and F), whereas in Pax8–/– mice, D2 signal intensities were significantly increased and were especially pronounced throughout cortical areas (C and D). Scale bar, 500 μm.
These deficits are more likely explained by the expression of MCT8 mRNA we observed in neuronal populations well acknowledged as being exquisitely dependent on proper TH supply. In the cerebral cortex, moderate to high signal intensities were observed throughout layers 2–5, whereas only scattered cells were labeled in layer 6 (Fig. 3, A and B). High transcript levels for MCT8 were also detected throughout the amygdala, in the pyramidal cell layer of the hippocampal formation, as well as in the granule cell layer of the dentate gyrus (Figs. 1 and 5). In support of our hypothesis, this distribution perfectly supports the findings that in brains of animals rendered hypothyroid, neocortical and hippocampal pyramidal cells display reduced dendritic aborization as well as abnormal spine disposition (18, 19). Furthermore, changes in the distribution of retrogradely labeled callosal neurons in both visual and auditory cortices have been described in rats made hypothyroid during embryonic stages (20, 21). Moreover, deficiencies in neuronal migration leading to a less defined cortical layering and a decreased number of granule cells in the hippocampus were also observed in the brains of hypothyroid animals (22). Finally, like patients with the syndrome of resistance to TH, who suffer from learning disorders and mental retardation (23), genetically engineered mice in which a T3 binding mutation was introduced into the TH receptor ? locus displayed learning deficits together with reduced BDNF (brain-derived neurotrophic factor) immunoreactivity in the hippocampus and a reduced number of granule cells and fibers in this brain area, especially in the CA3 region (24). These abnormalities fit well with the clinical picture of patients with mutated MCT8 who suffer from severe deficits in higher cognitive function and exhibit severe symptoms of mental retardation, with rudimentary communication skills and lack of speech development (12, 13).
FIG. 5. Spatiotemporal expression pattern of MCT8 in the CNS. High transcript levels of MCT8 were found in the cerebral cortex and in the hippocampal formation by embryonic d 18 (A). On P3 and P6, reduced transcript levels were observed in the parietal cortex, and increased intensities were found in neighboring cortical areas (B and C). These heterogeneities disappeared by P9 (D). In the pyramidal cell layer and granule cell layer of the hippocampal formation, the signals were diffuse during early periods of development. On P15, MCT8 expression reached adult levels (E and F). Scale bar, 500 μm.
MCT8 expression was also observed in brain areas closely associated with motor control. Hybridization signals were found throughout striatal areas, with high transcript levels in the islands of Calleja and olfactory tubercle and less prominent levels in scattered cells throughout the caudate-putamen and nucleus accumbens (Fig. 1, B and C). The interpretation that the striatum is dependent on proper TH supply, at least during development, is supported by the clinical picture of patients suffering from neurological cretinism. This syndrome is mostly caused by extreme iodine deficiency that affects both maternal and fetal TH production, with the result that TH is insufficiently supplied during the early phases of development. Consequently, brain development is more severely affected than under conditions of congenital hypothyroidism (3). It is therefore not surprising that neurological or endemic cretinism not only results in mental retardation, as observed in congenital hypothyroidism, but also leads to a striatopallidal syndrome with poor motor coordination and spastic diplegia (25), exactly the symptoms found in MCT8-affected patients (12, 13). Some of the children even suffer from spastic quadriplegia, which results from central hypotonia, followed by increased peripheral hypertonia.
According to the present concept of TH metabolism in brain, TH, preferentially T4, is taken up at the blood-brain barrier by transporters such as Oatp14, which indeed exhibits considerably higher transport capacity for T4 than for T3 (10). After release from endothelial cells, probably also mediated by Oatp14 (10), T4 has to be translocated (presumably mediated by another transporter, not yet identified) into astrocytes and subsequently converted to the biologically active T3 by the enzyme D2, which is known to be localized intracellularly in these cells (26). Consistent with this scheme (Fig. 6), our ISH analysis revealed an astrocytic distribution pattern of D2 hybridization signals (Fig. 4, C and D). After release from astrocytes, T3 has to enter neurons, the important target sites of T3 action, a process most likely mediated by MCT8.
FIG. 6. A schematic model outlining routes of TH transport in the brain. Oatp14 expressed by endothelial cells transports T4 from blood into extracellular fluid in the CNS. T4 then enters astrocytes by an as yet unknown mechanism to be converted to the active compound T3 by the intracellularly localized D2. After release, T3 is taken up by neurons, where it regulates gene expression and is finally inactivated by D3. This neuronal import is mediated by the transporter MCT8. BBB, Blood-brain barrier.
To prove this hypothesis, we stained perfusion-fixed brain sections first with an antibody against NeuN, a transcription factor expressed in a subset of neurons, or with an antibody against GFAP to label astrocytes. Thereafter, MCT8 mRNA was detected by ISH. Although the perfusion-fixation procedure necessary for the antibody staining results in an overall reduction of in situ hybridization signal intensity, cells positive for NeuN and MCT8 could be clearly observed in various cortical areas, as shown exemplarily for the retrosplenial cortex in Fig. 3C. Quantification of double-labeled cells in selected areas with high transcript levels of MCT8, such as the retrosplenial cortex and the amygdalo-hippocampal area, revealed that 62 ± 4% of all NeuN-positive cells were also covered with clusters of silver grains specific for MCT8 mRNA, whereas an overlapping expression pattern could not be found between GFAP-labeled cells and MCT8-positive cell bodies in the same areas. Furthermore, scattered cells labeled for MCT8 in the stratum radiatum of the hippocampal formation, a region enriched for astrocytes, were GFAP negative (Fig 3D), but positive for NeuN (89 ± 3%). Although the overall expression analysis indicated a predominant neuronal localization of MCT8, one exception was noted in hypothalamic areas: tanycytes, specialized glial cells lining the third ventricle that have been shown to express D2 (26), were labeled for MCT8 (Fig. 2C). Because these cells are mainly involved in the transport of hormones or other substances into the hypothalamus, MCT8 might offer a route for TH to enter hypothalamic regions.
In general, the MCT8 expression pattern provides strong evidence that MCT8 plays a decisive role in the transport of T3 into neurons. This idea is also supported by the finding that neurons are not only targets of T3 action, but also the site of TH metabolism, because they contain intracellularly the enzyme D3 that catalyzes the inactivation of T3 (27). Thus, a nonfunctioning neuronal TH transporter would also prevent the degradation of T3. In support of a neuronal localization of MCT8, it should be noted that all patients lacking a functional MCT8 transporter exhibit markedly elevated T3 levels (12, 13).
As evidenced by the deficits observed in conditions of congenital hypothyroidism, adequate concentrations of TH during perinatal periods are essential for proper brain development. By analyzing MCT8 mRNA expression during these time periods, we found that MCT8 mRNA is already highly expressed on embryonic d 18 in neo- and allocortical regions (Fig. 5A). On postnatal d 3 (P3) and P6, an intriguing expression pattern was found in the cerebral cortex, with a conspicuous absence of MCT8-specific labeling in the parietal cortex whereas neighboring cortical areas appeared to contain denser signal intensities (Fig. 5, B and C). In the pyramidal cell layer of the hippocampus, very broad and intense labeling was seen at P3. With the maturation of the hippocampal formation, labeling condensed between P3 and P9, reaching adult intensities around P15 (Fig. 5E). This developmental expression pattern fits well with the finding that TH deficiency during the first postnatal weeks affects pyramidal and granule cell dendritogenesis as well as granule cell migration (22, 28, 29). It is therefore tempting to speculate that the temporal regulation of MCT8 expression may reflect the time period during which T3 is especially required.
To address the question of whether MCT8 expression (like many processes regulating TH synthesis, metabolism, and function) is influenced by thyroidal status, we analyzed the MCT8 mRNA expression patterns in the CNS of wild-type animals compared with those of Pax8–/– mice. These mutant animals represent an ideal animal model of congenital hypothyroidism because they are born without a functional thyroid gland (30). As reported previously (7) and as shown in Fig. 4, C and D, for control and Pax8–/– mice, D2 mRNA expression in the brain is clearly up-regulated under hypothyroid conditions, which is especially obvious throughout the cerebral cortex. This increase in D2 expression has been discussed as a compensatory mechanism for preserving T3 concentrations in brain (31, 32). In contrast to D2, significant differences in the MCT8 mRNA expression pattern were not found in the CNS of wild-type and Pax8–/– mice (Fig. 4, A and B), indicating that under hypothyroid conditions the uptake of T3 into neurons is not compensated by changes in the transcript levels of MCT8. Our ISH analysis furthermore revealed that, like MCT8, the expression levels of Oatp14 in the brain are not significantly influenced by thyroidal status, because in athyroid Pax8–/– mice, Oatp14 signal intensities are comparable to those of wild-type animals (Fig. 4, E and F).
A preferential function of MCT8 in brain tissue has been suggested as the most likely explanation for the clinical picture of patients carrying mutations in the MCT8 gene. This idea is substantiated by the ISH analysis presented here. The neuronal localization of MCT8 in brain regions critically involved in motor control and mental development fits very well with the severe neurological phenotype of these patients. Although impaired TH transport into neuronal target cells seems to be the primary course for the patient’s phenotype, at present we cannot exclude the possibility that MCT8 might also be involved in the transport of other, not yet identified compounds that could be important for neuronal development and/or maintenance. Therefore, it will be important to generate MCT8-deficient mice to elucidate the role of MCT8 in TH metabolism during CNS development.
Acknowledgments
We thank M. Kraus and I. Bartholomaeus for excellent technical assistance, M. Piechotta for animal care, V. Ashe for linguistic help, and M. Sch?fer and G. Raivich for helpful discussion.
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