Triac Treatment Prevents Neurodevelopmental and Locomotor Impairments in Thyroid Hormone Transporter Mct8/Oatp1c1 Deficient Mice

Abstract

Patients with inactive thyroid hormone (TH) transporter MCT8 display intellectual disability due to compromised central TH transport and action. As a therapeutic strategy, application of thyromimetic, MCT8-independent compounds Triac (3,5,3'-triiodothyroacetic acid), and Ditpa (3,5-diiodo-thyropropionic acid) was proposed. Here, we directly compared their thyromimetic potential in Mct8/Oatp1c1 double knock-out mice (Dko) modeling human MCT8 deficiency. Dko mice received either Triac (50 ng/g or 400 ng/g) or Ditpa (400 ng/g or 4000 ng/g) daily during the first three postnatal weeks. Saline-injected Wt and Dko mice served as controls. A second cohort of Dko mice received Triac (400 ng/g) daily between postnatal weeks 3 and 6. Thyromimetic effects were assessed at different postnatal stages by immunofluorescence, ISH, qPCR, electrophysiological recordings, and behavior tests. Triac treatment (400 ng/g) induced normalized myelination, cortical GABAergic interneuron differentiation, electrophysiological parameters, and locomotor performance only when administered during the first three postnatal weeks. Ditpa (4000 ng/g) application to Dko mice during the first three postnatal weeks resulted in normal myelination and cerebellar development but only mildly improved neuronal parameters and locomotor function. Together, Triac is highly-effective and more efficient than Ditpa in promoting CNS maturation and function in Dko mice yet needs to be initiated directly after birth for the most beneficial effects.

Keywords: Allan-Herndon-Dudley syndrome; Ditpa; SLC16A2; SLCO1C1; Triac; thyroid hormone analog; thyroid hormone transport.

Figure 1

TH-analog treatment initiated after birth improves brain development. Newborn mice received a daily injection of saline (control) or different doses (in ng/g bw) of Triac or Ditpa between postnatal days 1 (P1) and P11, as indicated. (A) At P12, cerebellar Purkinje cell development was monitored by calbindin immunostaining and myelination in the cerebral cortex by MBP immunoreactivity, and PV+ interneurons were visualized in the somatosensory cortex (sc) and retrosplenial cortex (rc). (B) Dimension of the Purkinje cell dendritic tree (arrow in (A)) was measured highlighting a reduced thickness in Dko animals that was restored dose-dependently by Triac and Ditpa treatment. (C) MBP integrated signal density was determined in the inner layers of the cerebral cortex (box in (A)) showing reduced levels in Dko animals that normalized only upon high-dose Triac or Ditpa administration. PV+ interneurons were enumerated in the sc (D) and rc (E) (boxes in (A)). PV+ cells were almost absent in Dko mice and increased dose-dependently following Triac or high-dose Ditpa application. n = 3–5. Scale bars 50 µm (Calbindin), 500 µm (MBP), 250 µm (PV). **, p < 0.01; ***, p < 0.001.

Figure 2

Triac executes stronger thyromimetic actions on the HPT axis than Ditpa. Dko mice were injected daily with either a saline solution or different concentrations of Triac and Ditpa between P1 and P20. At P21, activity of the HPT axis was monitored. (A) Saline-treated Dko mice exhibited strongly increased Trh-specific ISH signals in PVN neurons, which were reduced in intensity following Triac application in a dose-dependent manner. Ditpa had only little effect on Trh expression. Bright-field images are depicted while dark-field illuminations were employed for quantification. (B) Dark-field autoradiograms illustrate alterations in Tshb transcripts in the pituitary with increased levels in Dko mice that were almost completely suppressed by Triac application, but only moderately so by Ditpa treatment. (C) Hr-specific hybridization signals were used to examine thyromimetic effects of TH analogs in the brain. Centrally severely hypothyroid Dko mice present with strongly decreased hairless expression that was restored by Ditpa and, even more efficiently, by Triac application. (D) Hepatic and renal Dio1 expression were studied by qPCR. Mice receiving the low dose of Triac showed a reduction in Dio1 expression in both organs while high-dose Triac maintained elevated Dio1 transcript levels as seen in saline-treated Dko mice. Ditpa decreased Dio1 expression dose-dependently. n = 4–10. Scale bars 200 µm (Trh), 500 µm (Tshb), 1 mm (Hairless). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 3

Transient early TH-analog treatment improves locomotion in adulthood. Dko mice received daily injections with saline, 400 ng/g bw Triac (Dko + T (400)) or 4000 ng/g bw Ditpa (Dko + D (4000)) between P1 and P20. Thereafter, treatment was ceased, and locomotor behavior was addressed at the age of 7–9 weeks (A) In contrast to Dko mice receiving saline and showing severe locomotor deficiencies, Ditpa application moderately and Triac treatment fully normalized performance on an accelerating rotarod. (B) Neuromuscular abnormalities were further examined by a hanging wire test. Dko mice clung to the wire for only a short time-period, which was significantly improved in TH analog-treated experimental groups. n = 10–14. *, p < 0.05; **, p < 0.01; ***, p < 0.001 significant difference to Wt and Dko + T (400) mice.

Figure 4

Long-term morphological alterations are induced by early postnatal TH-analog application. Following transient treatment with TH analogs Triac and Ditpa (in ng/g bw) between P1 and P20, brain parameters were examined at 10 weeks of age. (A) Myelination was evaluated in the corpus callosum (cc) by FluoroMyelin staining. TH-analog treatment could rescue the hypomyelination phenotype of Dko mice. (B) PV+ interneurons were visualized in the retrosplenial cortex (rc) and somatosensory cortex (sc). PV cell numbers were low in saline injected Dko animals, but fully restored in both areas upon Triac treatment. Ditpa only mildly improved PV+ numbers in the sc. (C) GAD67 immuno-reactivity analyzed in the same cortical regions demonstrated reduced integrated densities in saline injected Dko mice that was unaffected by Ditpa, but fully restored by Triac application. n = 3–4. Scale bars 500 µm (FluoroMyelin), 250 µm (PV, GAD67). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5

Deletion of Mct8/Oatp1c1 modulates GABAergic transmission in the cortex. Electrophysiological parameters were recorded on coronal forebrain sections using patch-clamp technique. (A) Representative traces of mIPSCs of Wt, Dko, and Dko + T (400) recorded from pyramidal neurons of the somatosensory cortex. (B) Cumulative plot and bar chart of mIPSC frequencies demonstrating a significant increase in Dko animals that can be rescued by application of Triac for 3 weeks. (C) Amplitude, rise time, half-width, time constant of decay, and transported electric charges are not modified in neurons from Dko animals. n = 17–18. *, p < 0.05; **, p < 0.01.

Figure 6

Delayed onset of Triac application compromises efficacy of the treatment. Dko mice were injected with saline or 400 ng/g bw Triac either between P1 and P20 or P22–P42 and analyzed at P43. (A) PV+ interneurons were visualized in the retrosplenial cortex (rc) or somatosensory cortex (sc) and myelination was assessed in the corpus callosum (cc) by FluoroMyelin staining. Pictures of animals treated with Triac between P22–P42 are shown. Low numbers of PV+ neurons (normalized to respective Wt numbers) in saline-injected Dko mice were fully restored following early onset Triac treatment while upon late onset only a partial recovery was observed in the rc (B) and sc (C,D) FluoroMyelin integrated density in the cc indicated rescue of Dko hypomyelination phenotype by Triac application between P1–P20 only. n = 3–4. Scale bars 250 µm (PV), 500 µm (FluoroMyelin). *, p < 0.05; **, p < 0.01; ***, p < 0.001.