Carmen Grijota-Martinez

Importance of Monocarboxylate Transporter 8 for the Blood-Brain Barrier-Dependent Availability of 3,5,3′-Triiodo-l-Thyronine

Mutations of the gene expressing plasma membrane transporter for thyroid hormones MCT8 (SLC16A2) in humans lead to altered thyroid hormone levels and a severe neurodevelopmental disorder. Genetically engineered defect of the Mct8 gene in mice leads to similar thyroid hormone abnormalities but no obvious impairment of brain development or function. In this work we studied the relative role of the blood-brain barrier and the neuronal plasma cell membrane in the restricted access of T(3) to the target neurons. To this end we compared the effects of low doses of T(4) and T(3) on cerebellar structure and gene expression in wild-type (Wt) and Mct8 null male mice [Mct8-/y, knockout (KO)] made hypothyroid during the neonatal period. We found that compared with Wt animals, T(4) was considerably more potent than T(3) in the Mct8KO mice, indicating a restricted access of T(3), but not T(4), to neurons after systemic administration in vivo. In contrast, T(3) action in cultured cerebellar neurons was similar in Wt cells as in Mct8KO cells. The results suggest that the main restriction for T(3) entry into the neural target cells of the mouse deficient in Mct8 is at the blood-brain barrier.

Thyroid Hormone-Regulated Mouse Cerebral Cortex Genes Are Differentially Dependent on the Source of the Hormone: A Study in Monocarboxylate Transporter-8- and Deiodinase-2-Deficient Mice

Thyroid hormones influence brain development through the control of gene expression. The concentration of the active hormone T(3) in the brain depends on T(3) transport through the blood-brain barrier, mediated in part by the monocarboxylate transporter 8 (Mct8/MCT8) and the activity of type 2 deiodinase (D2) generating T(3) from T(4). The relative roles of each of these pathways in the regulation of brain gene expression is not known. To shed light on this question, we analyzed thyroid hormone-dependent gene expression in the cerebral cortex of mice with inactivated Mct8 (Slc16a2) and Dio2 genes, alone or in combination. We used 34 target genes identified to be controlled by thyroid hormone in microarray comparisons of cerebral cortex from wild-type control and hypothyroid mice on postnatal d 21. Inactivation of the Mct8 gene (Mct8KO) was without effect on the expression of 31 of these genes. Normal gene expression in the absence of the transporter was mostly due to D2 activity because the combined disruption of Mct8 and Dio2 led to similar effects as hypothyroidism on the expression of 24 genes. Dio2 disruption alone did not affect the expression of positively regulated genes, but, as in hypothyroidism, it increased that of negatively regulated genes. We conclude that gene expression in the Mct8KO cerebral cortex is compensated in part by D2-dependent mechanisms. Intriguingly, positive or negative regulation of genes by thyroid hormone is sensitive to the source of T(3) because Dio2 inactivation selectively affects the expression of negatively regulated genes.

Thyroid Hormone Action in the Adult Brain: Gene Expression Profiling of the Effects of Single and Multiple Doses of Triiodo-L-Thyronine in the Rat Striatum

Thyroid hormones have profound effects on mood and behavior, but the molecular basis of thyroid hormone action in the adult brain is relatively unknown. In particular, few thyroid hormone-dependent genes have been identified in the adult brain despite extensive work carried out on the developing brain. In this work we performed global analysis of gene expression in the adult rat striatum in search for genomic changes taking place after administration of T(3) to hypothyroid rats. The hormone was administered in two different schedules: 1) a single, large dose of 25 microg per 100 g body weight (SD) or 2) 1.5 microg per 100 g body weight once daily for 5 d (RD). Twenty-four hours after the single or last of multiple doses, gene expression in the striatum was analyzed using Codelink microarrays. SD caused up-regulation of 149 genes and down-regulation of 88 genes. RD caused up-regulation of 18 genes and down-regulation of one gene. The results were confirmed by hybridization to Affymetrix microarrays and by TaqMan PCR. Among the genes identified are genes involved in circadian regulation and the regulation of signaling pathways in the striatum. These results suggest that thyroid hormone is involved in regulation of striatal physiology at multiple control points. In addition, they may explain the beneficial effects of large doses of thyroid hormone in bipolar disorders.

Lack of Action of Exogenously Administered T3 on the Fetal Rat Brain Despite Expression of the Monocarboxylate Transporter 8

Mutations of the monocarboxylate transporter 8 gene (MCT8, SLC16A2) cause the Allan-Herndon-Dudley syndrome, an X-linked syndrome of severe intellectual deficit and neurological impairment. Mct8 transports thyroid hormones (T4 and T3), and the Allan-Herndon-Dudley syndrome is likely caused by lack of T3 transport to neurons during critical periods of fetal brain development. To evaluate the role of Mct8 in thyroid hormone action in the fetal brain we administered T4 or T3 to thyroidectomized pregnant dams treated with methyl-mercapto-imidazol to produce maternal and fetal hypothyroidism. Gene expression was then measured in the fetal cerebral cortex. T4 increased Camk4, Sema3c, and Slc7a3 expression, but T3 was without effect. To investigate the cause for the lack of T3 action we analyzed the expression of organic anion transport polypeptide (Oatp14, Slco1c1), a T4 transporter, and Mct8 (Slc16a2), a T4 and T3 transporter, by confocal microscopy. Both proteins were present in the brain capillaries forming the blood-brain barrier and in the epithelial cells of the choroid plexus forming the blood-cerebrospinal fluid barrier. It is concluded that T4 from the maternal compartment influences gene expression in the fetal cerebral cortex, possibly after transport via organic anion transporter polypeptide and/or Mct8, and conversion to T3 in the astrocytes. On the other hand, T3 does not reach the target neurons despite the presence of Mct8. The data indicate that T4, through local deiodination, provides most T3 in the fetal rat brain. The role of Mct8 as a T3 transporter in the fetal rat brain is therefore uncertain.

Thyroid Hormone Regulation of Gene Expression in the Developing Rat Fetal Cerebral Cortex: Prominent Role of the Ca2+/Calmodulin-Dependent Protein Kinase IV Pathway

Thyroid hormones influence brain development through regulation of gene expression mediated by nuclear receptors. Nuclear receptor concentration increases rapidly in the human fetus during the second trimester, a period of high sensitivity of the brain to thyroid hormones. In the rat, the equivalent period is the last quarter of pregnancy. However, little is known about thyroid hormone action in the fetal brain, and in rodents, most thyroid hormone-regulated genes have been identified during the postnatal period. To identify potential targets of thyroid hormone in the fetal brain, we induced maternal and fetal hypothyroidism by maternal thyroidectomy followed by antithyroid drug (2-mercapto-1-methylimidazole) treatment. Microarray analysis identified differentially expressed genes in the cerebral cortex of hypothyroid fetuses on d 21 after conception. Gene function analysis revealed genes involved in the biogenesis of the cytoskeleton, neuronal migration and growth, and branching of neurites. Twenty percent of the differentially expressed genes were related to each other centered on the Ca(2+) and calmodulin-activated kinase (Camk4) pathway. Camk4 was regulated directly by T(3) in primary cultured neurons from fetal cortex, and the Camk4 protein was also induced by thyroid hormone. No differentially expressed genes were recovered when euthyroid fetuses from hypothyroid mothers were compared with fetuses from normal mothers. Although the results do not rule out a specific contribution from the mother, especially at earlier stages of pregnancy, they indicate that the main regulators of thyroid hormone-dependent, fetal brain gene expression near term are the fetal thyroid hormones.

Mutations of the Thyroid Hormone Transporter MCT8 Cause Prenatal Brain Damage and Persistent Hypomyelination

CONTEXT Mutations in the MCT8 (SLC16A2) gene, encoding a specific thyroid hormone transporter, cause an X-linked disease with profound psychomotor retardation, neurological impairment, and abnormal serum thyroid hormone levels. The nature of the central nervous system damage is unknown. OBJECTIVE The objective of the study was to define the neuropathology of the syndrome by analyzing brain tissue sections from MCT8-deficient subjects. DESIGN We analyzed brain sections from a 30th gestational week male fetus and an 11-year-old boy and as controls, brain tissue from a 30th and 28th gestational week male and female fetuses, respectively, and a 10-year-old girl and a 12-year-old boy. METHODS Staining with hematoxylin-eosin and immunostaining for myelin basic protein, 70-kDa neurofilament, parvalbumin, calbindin-D28k, and synaptophysin were performed. Thyroid hormone determinations and quantitative PCR for deiodinases were also performed. RESULTS The MCT8-deficient fetus showed a delay in cortical and cerebellar development and myelination, loss of parvalbumin expression, abnormal calbindin-D28k content, impaired axonal maturation, and diminished biochemical differentiation of Purkinje cells. The 11-year-old boy showed altered cerebellar structure, deficient myelination, deficient synaptophysin and parvalbumin expression, and abnormal calbindin-D28k expression. The MCT8-deficient fetal cerebral cortex showed 50% reduction of thyroid hormones and increased type 2 deiodinase and decreased type 3 deiodinase mRNAs. CONCLUSIONS The following conclusions were reached: 1) brain damage in MCT8 deficiency is diffuse, without evidence of focal lesions, and present from fetal stages despite apparent normality at birth; 2) deficient hypomyelination persists up to 11 years of age; and 3) the findings are compatible with the deficient action of thyroid hormones in the developing brain caused by impaired transport to the target neural cells.

In Vivo Activity of the Thyroid Hormone Receptor β- and α-Selective Agonists GC-24 and CO23 on Rat Liver, Heart, and Brain

Thyroid hormone analogs with selective actions through specific thyroid hormone receptor (TR) subtypes are of great interest. They might offer the possibility of mimicking physiological actions of thyroid hormone with receptor subtype or tissue specificity with therapeutic aims. They are also pharmacological tools to dissect biochemical pathways mediated by specific receptor subtypes, in a complementary way to mouse genetic modifications. In this work, we studied the in vivo activity in developing rats of two thyroid hormone agonists, the TRβ-selective GC-24 and the TRα-selective CO23. Our principal goal was to check whether these compounds were active in the rat brain. Analog activity was assessed by measuring the expression of thyroid hormone target genes in liver, heart, and brain, after administration to hypothyroid rats. GC-24 was very selective for TRβ and lacked activity on the brain. On the other hand, CO23 was active in liver, heart, and brain on genes regulated by either TRα or TRβ. This compound, previously shown to be TRα-selective in tadpoles, displayed no selectivity in the rat in vivo.

Increased Oxidative Metabolism and Neurotransmitter Cycling in the Brain of Mice Lacking the Thyroid Hormone Transporter Slc16a2 (Mct8)

Mutations of the monocarboxylate transporter 8 (MCT8) cause a severe X-linked intellectual deficit and neurological impairment. MCT8 is a specific thyroid hormone (T4 and T3) transporter and the patients also present unusual abnormalities in the serum profile of thyroid hormone concentrations due to altered secretion and metabolism of T4 and T3. Given the role of thyroid hormones in brain development, it is thought that the neurological impairment is due to restricted transport of thyroid hormones to the target neurons. In this work we have investigated cerebral metabolism in mice with Mct8 deficiency. Adult male mice were infused for 30 minutes with (1-13C) glucose and brain extracts prepared and analyzed by 13C nuclear magnetic resonance spectroscopy. Genetic inactivation of Mct8 resulted in increased oxidative metabolism as reflected by increased glutamate C4 enrichment, and of glutamatergic and GABAergic neurotransmissions as observed by the increases in glutamine C4 and GABA C2 enrichments, respectively. These changes were distinct to those produced by hypothyroidism or hyperthyroidism. Similar increments in glutamate C4 enrichment and GABAergic neurotransmission were observed in the combined inactivation of Mct8 and D2, indicating that the increased neurotransmission and metabolic activity were not due to increased production of cerebral T3 by the D2-encoded type 2 deiodinase. In conclusion, Mct8 deficiency has important metabolic consequences in the brain that could not be correlated with deficiency or excess of thyroid hormone supply to the brain during adulthood.

L1CAM Binds ErbB Receptors through Ig-Like Domains Coupling Cell Adhesion and Neuregulin Signalling

During nervous system development different cell-to-cell communication mechanisms operate in parallel guiding migrating neurons and growing axons to generate complex arrays of neural circuits. How such a system works in coordination is not well understood. Cross-regulatory interactions between different signalling pathways and redundancy between them can increase precision and fidelity of guidance systems. Immunoglobulin superfamily proteins of the NCAM and L1 families couple specific substrate recognition and cell adhesion with the activation of receptor tyrosine kinases. Thus it has been shown that L1CAM-mediated cell adhesion promotes the activation of the EGFR (erbB1) from Drosophila to humans. Here we explore the specificity of the molecular interaction between L1CAM and the erbB receptor family. We show that L1CAM binds physically erbB receptors in both heterologous systems and the mammalian developing brain. Different Ig-like domains located in the extracellular part of L1CAM can support this interaction. Interestingly, binding of L1CAM to erbB enhances its response to neuregulins. During development this may synergize with the activation of erbB receptors through L1CAM homophilic interactions, conferring diffusible neuregulins specificity for cells or axons that interact with the substrate through L1CAM.

Uridine 5′-Triphosphate Promotes In Vitro Schwannoma Cell Migration through Matrix Metalloproteinase-2 Activation

In response to peripheral nerve injury, Schwann cells adopt a migratory phenotype and modify the extracellular matrix to make it permissive for cell migration and axonal re-growth. Uridine 5′-triphosphate (UTP) and other nucleotides are released during nerve injury and activate purinergic receptors expressed on the Schwann cell surface, but little is known about the involvement of purine signalling in wound healing. We studied the effect of UTP on Schwannoma cell migration and wound closure and the intracellular signaling pathways involved. We found that UTP treatment induced Schwannoma cell migration through activation of P2Y2 receptors and through the increase of extracellular matrix metalloproteinase-2 (MMP-2) activation and expression. Knockdown P2Y2 receptor or MMP-2 expression greatly reduced wound closure and MMP-2 activation induced by UTP. MMP-2 activation evoked by injury or UTP was also mediated by phosphorylation of all 3 major mitogen-activated protein kinases (MAPKs): JNK, ERK1/2, and p38. Inhibition of these MAPK pathways decreased both MMP-2 activation and cell migration. Interestingly, MAPK phosphorylation evoked by UTP exhibited a biphasic pattern, with an early transient phosphorylation 5 min after treatment, and a late and sustained phosphorylation that appeared at 6 h and lasted up to 24 h. Inhibition of MMP-2 activity selectively blocked the late, but not the transient, phase of MAPK activation. These results suggest that MMP-2 activation and late MAPK phosphorylation are part of a positive feedback mechanism to maintain the migratory phenotype for wound healing. In conclusion, our findings show that treatment with UTP stimulates in vitro Schwannoma cell migration and wound repair through a MMP-2-dependent mechanism via P2Y2 receptors and MAPK pathway activation.