Cerebral cortex hyperthyroidism of newborn mct8-deficient mice transiently suppressed by lat2 inactivation

Abstract

Thyroid hormone entry into cells is facilitated by transmembrane transporters. Mutations of the specific thyroid hormone transporter, MCT8 (Monocarboxylate Transporter 8, SLC16A2) cause an X-linked syndrome of profound neurological impairment and altered thyroid function known as the Allan-Herndon-Dudley syndrome. MCT8 deficiency presumably results in failure of thyroid hormone to reach the neural target cells in adequate amounts to sustain normal brain development. However during the perinatal period the absence of Mct8 in mice induces a state of cerebral cortex hyperthyroidism, indicating increased brain access and/or retention of thyroid hormone. The contribution of other transporters to thyroid hormone metabolism and action, especially in the context of MCT8 deficiency is not clear. We have analyzed the role of the heterodimeric aminoacid transporter Lat2 (Slc7a8), in the presence or absence of Mct8, on thyroid hormone concentrations and on expression of thyroid hormone-dependent cerebral cortex genes. To this end we generated Lat2-/-, and Mct8-/yLat2-/- mice, to compare with wild type and Mct8-/y mice during postnatal development. As described previously the single Mct8 KO neonates had a transient increase of 3,5,3'-triiodothyronine concentration and expression of thyroid hormone target genes in the cerebral cortex. Strikingly the absence of Lat2 in the double Mct8Lat2 KO prevented the effect of Mct8 inactivation in newborns. The Lat2 effect was not observed from postnatal day 5 onwards. On postnatal day 21 the Mct8 KO displayed the typical pattern of thyroid hormone concentrations in plasma, decreased cortex 3,5,3'-triiodothyronine concentration and Hr expression, and concomitant Lat2 inactivation produced little to no modifications. As Lat2 is expressed in neurons and in the choroid plexus, the results support a role for Lat2 in the supply of thyroid hormone to the cerebral cortex during early postnatal development.

Figure 1. Lat2 expression.

A–D: P0: A and B are Nissl staining and C and D in situ hybridization radioautographs. E and F: P21 in situ hybridization radioautographs. Abbreviations: chp, choroid plexus; CTX, cerebral neocortex; CA, cornus Ammonis; CPu, caudate-putamen; DG, dentate gyrus; Hy, hypothalamus, Hip, hippocampus; Th, thalamus; PVT, thalamic paraventricular nucleus; LA, lateral amygdala. G: in situ hybridization (P21) with ³⁵S-Lat2 probe combined with immunohistochemistry for NeuN to reveal neurons. Hippocampal CA1 field. H: Similar field as G, but at lower magnification, with cells stained for glial fibrillary acidic protein (GFAP). The majority of the silver grains can be seen on the neuronal pyramidal layer (asterisk) with background signal on the astrocytes. Scale bars were 1 mm in C–D, E and F, and 50 µm in G and H.

Figure 2. Plasma T4 and T3 and cortex T3 concentrations in mice of different genotypes and different ages, as indicated.

Wt  =  wild type mice; M8 =  Mct8KO. L2 =  Lat2KO; M8L2 =  Mct8Lat2KO. Significance of differences was calculated by one way ANOVA, and the Tukey posthoc test. Only relevant significant comparisons are indicated. * P<0.05. ** P<0.01. *** P<0.001. P0, P5, and P21: postnatal days 0, 5, and 21, respectively.

Figure 3. Gene expression (mean ± SE) in the cerebral cortex and liver of mice of different genotypes and ages as indicated.

Wt =  wild type mice; M8  =  Mct8KO; L2 =  Lat2KO; M8L2 =  Mct8Lat2KO. Measurements were by qPCR, and the data expressed relative to 18S RNA. Significance of differences was calculated by one way ANOVA and the Tukey posthoc test. Only relevant significant comparisons are indicated. * P<0.05. ** P<0.01. *** P<0.001. Hr: Hairless mRNA. Sema7a: Semaphorin 7a mRNA. Klf9: Kruppel factor 9, or BTEB mRNA. P0, P5, P15, P21: postnatal days 0, 5, 15, or 21.

Figure 4. Type 1 deiodinase (Dio1) expression (mean ± SE) in the liver.

Wt =  wild type mice; M8 =  Mct8KO; L2 =  Lat2KO; M8L2 =  Mct8Lat2KO. Measurements were by qPCR, and the data expressed relative to 18S RNA. Note the different scale for the P0 data with respect to P5 and P21. Significance of differences was calculated by one way ANOVA and the Tukey posthoc test. Only relevant significant comparisons are indicated. ** P<0.01. *** P<0.001. P5, P21: postnatal days 5 or 21.