Sciatic nerve analysis in thyroid hormone transporters Mct8 and Oatp1c1 knockout mice

in European Thyroid Journal
Authors:
Steffen Mayerl Department of Endocrinology, Diabetes & Metabolism, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Center for Translational Neuro- and Behavioral Sciences (C-TNBS), Medical Faculty, University of Duisburg-Essen, Essen, Germany

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Andrea Alcaide Martin Department of Endocrinology, Diabetes & Metabolism, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Center for Translational Neuro- and Behavioral Sciences (C-TNBS), Medical Faculty, University of Duisburg-Essen, Essen, Germany

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Reinhard Bauer Institute of Molecular Cell Biology, Jena University Hospital, Jena, Germany

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Heike Heuer Department of Endocrinology, Diabetes & Metabolism, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Center for Translational Neuro- and Behavioral Sciences (C-TNBS), Medical Faculty, University of Duisburg-Essen, Essen, Germany

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Correspondence should be addressed to S Mayerl: steffen.mayerl@uk-essen.de
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Objective

Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause Allan–Herndon–Dudley syndrome (AHDS), a severe form of psychomotor retardation with muscle hypoplasia and spastic paraplegia as key symptoms. These abnormalities have been attributed to impaired TH transport across brain barriers and into neural cells, thereby affecting brain development and function. Likewise, Mct8/Oatp1c1 (organic anion-transporting polypeptide 1c1) double knockout (M/Odko) mice, a well-established murine AHDS model, display a strongly reduced TH passage into the brain as well as locomotor abnormalities. To which extent the peripheral nervous system is affected by combined MCT8/OATP1C1 deficiency has not been addressed.

Methods

Using the sciatic nerve as a model, we studied the spatiotemporal expression of TH transporters as well as the sciatic thyroidal state, sciatic nerve myelination and function in M/Odko mice by immunofluorescence, qPCR, Western blotting and electrophysiology.

Results

We detected MCT8 protein expression in sciatic nerve axons, whereas OATP1C1 expression was observed in a subset of endothelial cells early in postnatal development. The absence of MCT8 and OATP1C1 did not alter the thyroidal state of isolated nerves at P12. Moreover, electrophysiological studies did not disclose any significant alteration in sciatic nerve signal propagation parameters in adult M/Odko mice. Although Schwann cell numbers were similar, Western blot analysis showed a mild form of hypermyelination in adult M/Odko mice.

Conclusions

Altogether, our data point to a largely unaffected sciatic nerve structure and function in the absence of MCT8 and OATP1C1.

Abstract

Objective

Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause Allan–Herndon–Dudley syndrome (AHDS), a severe form of psychomotor retardation with muscle hypoplasia and spastic paraplegia as key symptoms. These abnormalities have been attributed to impaired TH transport across brain barriers and into neural cells, thereby affecting brain development and function. Likewise, Mct8/Oatp1c1 (organic anion-transporting polypeptide 1c1) double knockout (M/Odko) mice, a well-established murine AHDS model, display a strongly reduced TH passage into the brain as well as locomotor abnormalities. To which extent the peripheral nervous system is affected by combined MCT8/OATP1C1 deficiency has not been addressed.

Methods

Using the sciatic nerve as a model, we studied the spatiotemporal expression of TH transporters as well as the sciatic thyroidal state, sciatic nerve myelination and function in M/Odko mice by immunofluorescence, qPCR, Western blotting and electrophysiology.

Results

We detected MCT8 protein expression in sciatic nerve axons, whereas OATP1C1 expression was observed in a subset of endothelial cells early in postnatal development. The absence of MCT8 and OATP1C1 did not alter the thyroidal state of isolated nerves at P12. Moreover, electrophysiological studies did not disclose any significant alteration in sciatic nerve signal propagation parameters in adult M/Odko mice. Although Schwann cell numbers were similar, Western blot analysis showed a mild form of hypermyelination in adult M/Odko mice.

Conclusions

Altogether, our data point to a largely unaffected sciatic nerve structure and function in the absence of MCT8 and OATP1C1.

Introduction

Thyroid hormone (TH) is a critical regulator of nervous system development (1). TH uptake into target cells is mediated by specialized transmembrane transporters belonging to different gene families, for example the monocarboxylate transporter (MCT) family, which include MCT8 and MCT10; L-type amino acid transporters (LATs), such as LAT1 and LAT2; or organic anion-transporting proteins (OATPs), such as OATP1C1 (2). To date, MCT8 encoded by the X-linked SLC16A2 gene has been considered as the most specific TH transporter exhibiting the highest substrate specificity toward the prohormone 3,3′,5,5′-tetraiodothyronine (thyroxine; T4) and the receptor-active form 3,3′,5-triiodothyronine (T3). Inactivating mutations in MCT8 cause Allan–Herndon–Dudley syndrome (AHDS) (3, 4, 5). AHDS is characterized by severe intellectual and motor disabilities that most likely result from impaired TH transport across brain barriers and thus from a profound TH deficiency in the developing central nervous system (CNS). In addition, patients exhibit a unique TH profile in the circulation with highly elevated T3 that in turn is considered as the major cause of signs of peripheral thyrotoxicosis, including low body weight, hypermetabolism and skeletal muscle atrophy. Moreover, key clinical symptoms are spastic tetraplegia and dystonic movements. Yet, to which extent central and/or peripheral alterations in TH homeostasis contribute to this prominent neuromuscular phenotype is still poorly understood.

To elucidate the pathogenic mechanisms driving AHDS, Mct8 knockout (ko) mice have been generated and studied (6, 7, 8). Although Mct8ko mice replicate the abnormal serum TH profile together with hallmarks of a peripheral hyperthyroid state, the CNS of these animals is only in a mild TH-deficient state. This phenotype can be explained by the presence of the T4-specific TH transporter OATP1C1 at murine brain barriers that can compensate for the absence of MCT8 in mice. Consequently, a concomitant ablation of Mct8 and Oatp1c1 in mice (M/Odko) results in a profound central TH deprivation that leads to compromised neuronal differentiation, hypomyelination and locomotor impairments (9).

For voluntary locomotion, central commands are relayed from upper motor neurons in the primary motor cortex through lower motor neurons in the brainstem and spinal cord to skeletal muscle fibers (10). Lower motor neurons utilize the neurotransmitter acetylcholine, and although their cell bodies are located in the spinal cord, their axons constitute a part of the peripheral nervous system (PNS). These axons are subject to myelination by Schwann cells, a specialized glia cell type that is necessary for both signal propagation and survival of axons (11). Motor neuron synapses on muscle fibers, neuromuscular junctions, play an extraordinary role for all voluntary movements (12).

Whether neuromuscular impairments in M/Odko mice involve local alterations in peripheral nerves has not been specifically studied before. In fact, very little information on the expression pattern of TH signaling regulators in peripheral nerves is currently available. Here, we investigated the sciatic nerve as a model system and established the expression profile of well-known TH transporter candidates therein at different developmental stages. We next assessed sciatic nerve structure and function in M/Odko mice by qPCR, immunohistochemistry and electrophysiology. Our findings point to a mild PNS hypermyelination in MCT8/OATP1C1 deficiency, whereas the overall sciatic nerve function was found to be unaffected.

Materials and methods

Animals

Wt, Mct8ko, Oatp1c1ko and M/Odko mice on a C57BL/6 background were bred and genotyped as described elsewhere (7, 9, 13). Animals were housed under a 12 h light:12 h darkness cycle at a constant temperature (22 °C) and were provided with standard laboratory chow and water ad libitum. All animal studies were executed in accordance with the European Union (EU) directive 2010/63/EU on the protection of animals used for scientific purposes and in compliance with local regulations by the Animal Welfare Committee of the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (Germany) as well as the Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz (TLLV; Germany; approval code 02-004/12). Efforts were made to reduce the number of animals and their suffering. For all tissue analyses, mice were sacrificed by dislocation of the neck. Animals of both sexes were used at P8 and P12, whereas only female mice were employed at 6 months (P180) and 1 year of age. Of note, adult female mice replicate the abnormal serum TH parameters and peripheral thyrotoxicosis as seen in male MCT8/OATP1C1dko mice and thus constitute a valid model for AHDS research (14).

Electrophysiological measurements

Electrophysiological measurements on mouse sciatic nerves and their analyses were performed as described elsewhere (15). In brief, female mice at 1 year of age were anesthetized by isoflurane/oxygen inhalation and kept under constant anesthesia. Using a heating plate with an integrated sensor and a rectal thermometer, body temperature was kept stable at 38 °C. Heart rate was monitored using three electrocardiogram recording electrodes placed under the skin of each forelimb as well as in the neck area. Fur was removed using an electric razor and hair removal cream. A sensing ring electrode was positioned at the point of thickest gastrocnemius muscle diameter, and a reference ring electrode was placed just beneath it. For measurements, stimulation by needle electrodes was performed either 4 mm away from the sensing electrode (distal) or 16 mm away (proximal). Nerves were stimulated using repetitive single square-wave pulses (repetition rate 200 ms and duration 0.1 ms). Stimulation signals and neuromuscular function response curves were recorded simultaneously, and a series of maximal responses was averaged. Three independent measurements were taken per site, position and animal. Anesthetized mice were killed by dislocation of the neck. For analysis of electrophysiological measurements, the AtisaPro software package (www.atisa.de) was utilized. Latency, defined as the time delay between stimulation and onset of compound motor action potential (CMAP), was determined. The difference between recorded latencies over the distance between the two stimulation sites was used to calculate nerve conduction velocity. Moreover, CMAP amplitudes as the maximum differences between positive and negative turnaround points were calculated.

Immunofluorescence studies

For TH transporter analysis, sciatic nerves were isolated, embedded in NEG-50 frozen section medium (Thermo Scientific, USA), and frozen on dry ice. Cross sections (20 μm) were produced using a cryostat (Leica, Germany) and thaw-mounted on superfrost slides (Thermo Scientific). In order to visualize MCT8 and OATP1C1, sciatic nerve sections were fixed in methanol for 10 min at −20 °C, air-dried, blocked in 1% milk powder in PBS containing 0.2% Triton X-100 and incubated with primary antibodies overnight at 4 °C. Following washing with PBS, sections were incubated with Alexa Fluor 488, 555 or 647-labeled secondary antibodies raised in goat (Invitrogen, USA; 1:1000) and with Hoechst33258 (1:10,000; Invitrogen) in 1% milk powder in PBS containing 0.2% Triton X-100. To assess the expression of LAT1 and MCT10, slides were post-fixed in 4% paraformaldehyde (PFA) for 10 min and permeabilized with 0.1% Triton X-100/0.1 M glycine. Sections were blocked with blocking buffer (PBS containing 10% goat serum and 0.2% Triton X-100) and subsequently incubated with primary antibodies in blocking buffer overnight at 4 °C. After washing with PBS, sections were incubated with Alexa Fluor 488, 555 or 647-labeled secondary antibodies raised in goat (Invitrogen; 1:1000) and with Hoechst33258 (1:10,000; Invitrogen) to label cell nuclei in blocking buffer. All pictures were obtained using a Leica SP8 confocal microscope as a z-stack of varying thickness (optical depth 0.333 μm per image). For presentation, five consecutive z-stack images were merged (max intensity).

For Schwann cell analysis, sciatic nerves were isolated, fixed in 4% PFA in PBS overnight, cryo-protected in 30% sucrose in PBS (w/v) and frozen on dry ice. Sections (16 μm) were produced, stained and imaged as for LAT1. Five z-stack images (optical thickness: 0.346 μm per z-stack image) were merged for analysis and display. SOX10 and YY1 positive cells were enumerated using ImageJ (NIH, USA; https://imagej.net/nih-image/), and densities per area were calculated. For YY1, elongated nuclei at the fascicle membrane were not included. Four pictures from different sciatic nerve sections per animal were used.

The following primary antibodies were utilized: rat anti-CD31 (1:100; BD Pharmingen, USA), rabbit anti-LAT1 (1:500; TransGenic Inc., Japan), rat anti-MBP (1:250; Millipore, USA), rabbit anti-MCT8 (1:500; Sigma-Aldrich, USA), rabbit anti-MCT10 (1:50; MyBioSource, USA), chicken anti-NFH (1:250; BioLegend, USA), rabbit-anti OATP1C1 (1:100; gift from Prof. Theo Visser, Rotterdam), rabbit anti-SOX10 (1:250; Abcam, UK) and rabbit anti-YY1 (1:1000; ProteinTech, USA).

Information on qPCR, reverse transcription (RT)-PCR and Western blotting can be found in the Supplementary information (see section on Supplementary materials given at the end of the article).

Statistical analysis

Using GraphPad Prism 7 (https://www.graphpad.com/), the normality of the data was confirmed (Shapiro–Wilk test), while no outliers were identified (ROUT method). All data are presented as mean ± SD. To compare Wt, Mct8ko, Oatp1c1ko and M/Odko mice, two-way ANOVA followed by Bonferroni post hoc testing was performed using GraphPad Prism 7. Differences were considered significant when P < 0.05 and are marked as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Results

TH signaling regulator expression in the sciatic nerve

Anatomically, the sciatic nerve comprises axons of spinal cord-residing motor neurons and sensory fibers originating from neurons in dorsal root ganglia organized in fascicles as well as myelinating and non-myelinating Schwann cells as major components (16). Although pro-myelinating Schwann cells are detectable already in the E18.5 murine sciatic nerve, the process of Schwann cell myelination starts around the first postnatal week (17).

Only sparse information is available regarding the cell type- and stage-specific expression of TH transporters, deiodinases and TH receptors in the sciatic nerve. To elucidate the spatiotemporal expression of well-known TH transporters, we conducted immunofluorescence studies on sciatic nerve sections of animals at P8 and P12 as well as adult female mice of 6 months of age (Fig. 1A). At P8, no specific MCT8 immunostaining could be detected, whereas at P12 and P180, MCT8 exhibited an axonal staining pattern as demonstrated by co-localization with an antibody against neurofilament heavy chain Nfh. Axonal MCT8 staining was strongly reduced in adult Mct8ko mice, although a background staining pattern remained noticeable (Suppl. Fig. 1). We then compared our results to a recent single-cell RNA sequencing atlas of the P1 and P60 murine sciatic nerve (18). Although axons are not represented in that dataset, prominent Mct8 transcript expression was found in epineurial and endoneurial cells (known as fibroblast-like cells) (Fig. 1B). We cannot fully exclude that our observed MCT8 protein staining at least partially reflects its presence in the endoneurial intrafascicular tissue.

Figure 1
Figure 1

MCT8 expression in the sciatic nerve. (A) Immunohistochemical analysis on Wt sciatic nerve cryo-sections obtained from P8, P12 and P180 mice following fixation in methanol. Axonal marker NFH is depicted in yellow, myelin marker MBP in blue, Hoechst33258 to label cell nuclei in cyan, and MCT8 in magenta. Single-channel grayscale pictures are shown for NFH and MCT8. n = 3. Scale bar: 10 μm. (B) Visualization of Mct8 (Slc16a2) transcript expression in single-cell RNA sequencing data of the P1 and P60 sciatic nerve. pmSC, pro-myelinating Schwann cells; mSC, myelinating Schwann cells; nm(R)SC, non-myelinating (Remak) Schwann cells; iSC, immature Schwann cells; prol. SC, proliferating Schwann cells; IC, immune cells; Prol. Fb, proliferating fibroblast-like cells; Fb.Rel*, fibroblast-related cluster (tentative cluster); EnC, endoneurial cells; PnC, perineurial cells; EpC, epineurial cells; EC, endothelial cells; Per, pericytes; and VSMC, vascular smooth muscle cells.

Citation: European Thyroid Journal 14, 1; 10.1530/ETJ-24-0248

OATP1C1 positive signals were clearly detected in the P8 sciatic nerve, but neither co-localized with NFH nor MBP (Fig. 2A). In the previously published scRNAseq dataset (18) OATP1C1 transcripts clustered in endothelial cells (Fig. 2B). To unravel whether the OATP1C1 immunopositive structures indeed constitute blood vessels, we performed a co-staining with the endothelial cell marker CD31 at P8 (Fig. 2C). Although the majority of CD31-positive cells were OATP1C1 negative, both staining overlapped in roughly 10% of all vessels. Vice versa, some OATP1C1-positive structures were devoid of CD31 signals and may represent perineurial cells according to scRNAseq data (18) (Fig. 2B).

Figure 2
Figure 2

OATP1C1 expression in a subset of sciatic nerve endothelial cells. (A) Wt sciatic nerve sections were fixed in methanol and co-stained for OATP1C1 (in magenta), MBP (in blue), NFH (in yellow) and Hoechst33258 (in cyan). Single-channel grayscale pictures are shown for NFH and OATP1C1. n = 3. Scale bar: 10 μm. (B) Visualization of Oatp1c1 (Slc01c1) transcript expression in single-cell RNA sequencing data of the P1 and P60 sciatic nerve. pmSC, pro-myelinating Schwann cells; mSC, myelinating Schwann cells; nm(R)SC, non-myelinating (Remak) Schwann cells; iSC, immature Schwann cells; prol. SC, proliferating Schwann cells; IC, immune cells; Prol. Fb, proliferating fibroblast-like cells; Fb.Rel*, fibroblast-related cluster (tentative cluster); EnC, endoneurial cells; PnC, perineurial cells; EpC, epineurial cells; EC, endothelial cells; Per, pericytes; and VSMC, vascular smooth muscle cells. (C) Sciatic nerve sections obtained from P8 Wt animals were stained for OATP1C1 (in green) and CD31 (in magenta). Representative pictures for double-positive blood vessels (left) and structures positive for only one of these proteins (right) are displayed. Hoechst33258 counter-stained cell nuclei appear blue. Single-channel grayscale pictures are presented for CD31 and OATP1C1. n = 3. Scale bar: 10 μm.

Citation: European Thyroid Journal 14, 1; 10.1530/ETJ-24-0248

We also addressed the expression pattern of other TH transporters in the sciatic nerve. Immunofluorescence studies were conducted for the aromatic amino acid transporter MCT10 (encoded by the Slc16a10 gene), which also accepts TH as substrates (2, 19). MCT10 protein was undetectable at P8 but localized distinctly around a few axons at P12 and P180 (Suppl. Fig. 2A). Results from single-cell RNA sequencing (18) point to immune cells as the main MCT10-expressing population (Suppl. Fig. 2B), in agreement with a recently identified role of MCT10 for TH supply to macrophages (20). We also conducted immunofluorescence staining for the TH-transporting L-type amino acid transporters LAT1 (SLC7A5) and LAT2 (SLC7A8) (2, 21). LAT1-positive signals appeared in elongated structures resembling blood vessels at every analyzed time point (Suppl. Fig. 3A), which was subsequently confirmed by co-staining with the blood vessel marker CD31 (Suppl. Fig. 3B) in accordance with scRNAseq data (18) (Suppl. Fig. 3C). For LAT2, we failed to detect a specific immunostaining (8, 22). However, scRNAseq data (18) suggests that LAT2 is expressed in immune cells in the sciatic nerve (Suppl. Fig. 4A).

To substantiate our findings, we further evaluated the presence of TH transporter transcripts by RT-PCR analysis in the sciatic nerve using the same time points as before (Suppl. Fig. 4B). Mct8, Mct10, Lat1 and Lat2 mRNA were detectable at all developmental and adult stages. Yet, Oatp1c1 transcript was absent from P180 samples, which aligns with our immunofluorescence assessments.

By taking advantage of a published scRNAseq atlas (18), we further searched for the presence of additional TH transporter candidates that are listed in Groeneweg et al. (2) and Chen et al. (23) (gene names: Slco1a2, Slco1b1, Slco1b3, Slco2b1, Slco3a1, Slco4a1, Slco4c1, Slc10a1, Slc17a4, Slc22a8, Slc22a27, Slc22a28, Slc22a29 and Slc22a30) (2, 23). Of all these candidates, only Oatp2b1 (Slco2b1) and Oatp3a1 (Slco3a1) transcripts could be found in the database. At P1, Oatp2b1 transcripts were exclusively detected in immune and endothelial cells, while at P60, Oatp2b1 mRNA was present in all cell populations with the exception of Schwann cells (Suppl. Fig. 5A). Oatp3a1 transcripts were found in all cell populations, including Schwann cells, to a varying degree (Suppl. Fig. 5B) at both time points.

We also explored the presence of deiodinase and TH receptor transcripts in the scRNAseq atlas (18). Low Dio2 and Dio3 transcript levels were found in epineurial cells (Suppl. Fig. 6A and B). Similarly, TH receptor beta (Thrb) transcript expression was sparse and primarily noticeable in epineurial cells (Suppl. Fig. 7B). In contrast, TH receptor alpha (Thra) expression was prominent and ubiquitously found in all sciatic nerve cell populations at both P1 and P60 (Suppl. Fig. 7A), suggesting that these Thra-expressing cells are indeed sensitive to TH.

Sciatic nerves compensate for the loss of MCT8 and OATP1C1

Global absence of MCT8 results in elevated T3 and reduced T4 concentrations in the circulation in both Mct8ko and M/Odko mice (6, 7, 9). Recently, we have demonstrated that these abnormal TH concentrations are present in the serum as early as P12 and thus during a critical phase of sciatic nerve development (24).

To assess whether these abnormal TH concentrations are sensed in the sciatic nerve in MCT8/OATP1C1 deficiency, we performed qPCR studies on sciatic nerve samples obtained from P12 animals (Fig. 3), thus matching our previous serum analysis (24). We first investigated mRNA expression of deiodinases and detected only a tendency toward elevated Dio2 transcript levels in Mct8ko and M/Odko mice, while Dio3 mRNA levels were not altered (Fig. 3A). Likewise, sciatic transcript levels of Thra and Thrb, as well as mRNA levels of well-known TH responsive genes that are either positively (Aldh1a1, Klf9 and Me1) or negatively TH-regulated (Crym) were unchanged in all ko mice at P12 (Fig. 3B). Together, these observations point to a rather euthyroid situation in the sciatic nerve in MCT8/OATP1C1 deficiency.

Figure 3
Figure 3

Sciatic nerve thyroidal state. Sciatic nerves from P12 mice were subjected to qPCR analysis, and the expression of TH signaling components (A) and TH-responsive marker genes (B) was evaluated. Expression levels were normalized to Gapdh, cyclophilin A and cyclophilin D expression as housekeeping genes. Wt values were set as 1.0. n = 4–5.

Citation: European Thyroid Journal 14, 1; 10.1530/ETJ-24-0248

Sciatic nerve structure and function in MCT8/OATP1C1 deficiency

The locomotor impairments observed in M/Odko mice prompted us to investigate whether signal propagation along peripheral nerves is affected. To this end, we determined in vivo sciatic nerve conduction properties in Wt, M/Odko, as well as in the single TH transporter-deficient animals (Fig. 4). CMAPs that result from a summation of many individual action potentials were recorded at different distances to the stimulation site as before (15) but did not reveal any significant differences between Wt, Mct8ko, Oatp1c1ko and M/Odko mice (Fig. 4A and B). Although CMAP values measured 4 mm away from the stimulus showed a tendency toward lower values in the latter two genotypes, no significant differences were found upon post hoc testing (Fig. 4B). Determination of motor nerve conduction velocity did not disclose any significant differences between the genotypes as well (Fig. 4C).

Figure 4
Figure 4

Electrophysiological properties of the sciatic nerve. Sciatic nerves of 1-year-old female mice were stimulated using repetitive single square-wave pulses. Signals were recorded at different distances to the stimulus, and maximum responses were averaged. CMAP amplitudes at the proximal (A) and distal (B) positions as well as maximum nerve conduction velocity (C) are displayed. n = 8 (Wt, Mct8ko); 5 (Oatp1c1ko); 7 (M/Odko).

Citation: European Thyroid Journal 14, 1; 10.1530/ETJ-24-0248

To further assess the impact of MCT8/OATP1C1 deficiency on peripheral nerve myelination, we enumerated SOX10-positive Schwann cells in sciatic nerve cross sections obtained from P12 animals (Fig. 5A and B). Although a tendency toward higher SOX10+ cell numbers was observed in M/Odko mice, post hoc testing did not reveal any statistical significance. Of note, SOX10 labels both myelinating and non-myelinating Schwann cells. To further test if specifically myelinating Schwann cell numbers are altered, we employed the marker Yy1 that labels this Schwann cell subpopulation (17). Again, no differences between the groups were detected (Fig. 5A and C).

Figure 5
Figure 5

Schwann cell analysis in developing animals. Cross sections of PFA-fixed sciatic nerves obtained from P12 animals were immunostained for the Schwann cell marker SOX10 and the myelinating Schwann cell marker YY1 (both in green) (A). Hoechst33258-labeled cell nuclei appear in blue. Numbers of SOX10+ cells (B) and YY1+ cells (C) were quantified and are shown as density per mm2. n = 3. Scale bar: 100 μm.

Citation: European Thyroid Journal 14, 1; 10.1530/ETJ-24-0248

We performed the same staining on sciatic nerve cross sections from P180 females (Fig. 6A, B and C). As before, neither SOX10-positive (Fig. 6A and B) nor Yy1-positive (Fig. 6A and C) cell populations were altered between the different genotypes. Finally, we aimed to evaluate the overall myelin content of sciatic nerve samples. We decided to investigate the 6-month time point when the sciatic nerve and thus sciatic myelin are fully mature. To this end, we performed Western blot analysis and analyzed levels of the mature myelin marker P0 (myelin protein zero) in sciatic nerve homogenates (Fig. 6D). P0 relative integrated densities were unaltered in single TH transporter ko mice, but exhibited higher levels in M/Odko mice (Fig. 6E). Collectively, our data suggest that, while Schwann cell numbers are unaltered, loss of MCT8/OATP1C1 results in mildly increased mature myelin levels.

Figure 6
Figure 6

Schwann cell and myelination analysis in adult animals. Cross sections of PFA-fixed sciatic nerves obtained from P180 female mice were immunostained for the Schwann cell marker SOX10 and for YY1 (both in green) (A). Hoechst33258-labeled cell nuclei are shown in blue. Cells positive for SOX10 (B) and YY1 (C) were enumerated and are presented as density per mm2. n = 5. Scale bar: 100 μm. (D) Sciatic nerves of 6-month-old females were homogenized and subjected to Western blot analysis. The mature myelin marker P0 was assessed. Protein levels were quantified by measuring the integrated density of the respective bands and normalizing it to Vinculin as a housekeeping protein. Wt values were set as 1.0. n = 3. *P < 0.05.

Citation: European Thyroid Journal 14, 1; 10.1530/ETJ-24-0248

Discussion

One of the most prominent hallmarks of AHDS is a profound neuromuscular phenotype that includes muscle weakness, dystonia and spastic tetraplegia (2, 3, 4, 5). The precise underlying cellular alterations have not been fully unraveled. Persistent hypomyelination in CNS white matter regions is a consistent observation and may contribute to the neurological deficits (25, 26). To which extent myelination and function of the PNS are affected in human MCT8 patients or murine AHDS mouse models has not been evaluated.

Here, we provide the first description of sciatic nerve structure and function in MCT8/OATP1C1-deficient mice, thus in an animal model that faithfully replicates the central TH deficiency and the abnormal serum TH profile seen in MCT8 patients. Moreover, M/Odko mice display persistent CNS hypomyelination due to impaired TH transport across brain barriers and into oligodendrocytes, where proper intracellular TH signaling is required for differentiation and myelination (9, 27). In our study, we focused primarily on the analysis of mice at P12 and P180, two time points well established in AHDS research (9, 24, 27, 28, 29). In addition, for our TH transporter expression analysis, we included mice at P8, when the majority of Schwann cells are still immature and proliferative and yet before Dio2 mRNA levels are known to rise in the rat sciatic nerve (30, 31). Notably, at adult stages, we employed female mice, which exhibit the same abnormal serum TH levels and peripheral thyrotoxic state as seen in males and are therefore suited for such baseline analyses (14).

Our immunofluorescence studies revealed MCT8 expression in NFH-positive axons in sciatic nerves of adult Wt mice, suggesting that MCT8 may contribute to the sensing of local TH levels by mature sensory and motor neurons. This finding aligns with previous observations in zebrafish carrying an mct8 reporter system (mct8:GAL4Xuas:SYP-EGFP) (32). In those fish, prominent reporter signals were found in descending motor neurons in the spinal cord that send axons into the periphery. Yet, we failed to detect MCT8 protein expression in the sciatic nerve at postnatal day P8. The presence of MCT8 in the P12 sciatic nerve, however, suggests an upregulation to ensure cellular TH entry precisely during the critical period of peripheral nerve myelination that peaks at around 2 weeks of age (33). Expression of OATP1C1, in contrast, was only observed at P8 in a subset of endothelial cells as well as in cells of still unknown identity. These observations point to a role of OATP1C1 in sciatic TH supply in Wt mice, even before the critical period at early postnatal stages. Along this line, we noticed prominent LAT1 expression in CD31+ endothelial cells, suggesting that TH may enter the sciatic nerve through LAT1. However, in contrast to a restricted permeability across the adult blood–nerve barrier, endoneurial vessels are rather leaky in newborn mouse sciatic nerves, which may further contribute to the TH supply during early stages (34). Subsequently, MCT8 may mediate the T3 uptake into axons and possibly even further into endosomal/non-degradative lysosomes, as recently demonstrated in brain-derived neuronal cultures (35). This would allow spinal cord-residing motor neurons to integrate information about both central and peripheral TH concentrations.

A cell type in the PNS that is thought to be sensitive to TH during a defined developmental time window are Schwann cells (16, 36). Schwann cells and satellite glia cells express TH receptors, which serves as a proxy for TH sensitivity, from late gestation till the end of the second postnatal week, a period characterized by intense Schwann cell proliferation and myelin sheath formation (16, 37). Although the abnormal serum TH profile in the absence of MCT8 is already fully established at P12 (24), we observed an euthyroid situation in the sciatic nerve of Mct8ko and M/Odko mice at P12. According to a recent scRNAseq atlas of the sciatic nerve (18), our selected TH marker genes cover the majority of sciatic cell types, as Klf9 mRNA is ubiquitously expressed, while Me1, Crym and Aldh1a1 are present in epineurial, endoneurial, perineurial and some Schwann cells. Noteworthy, these TH responsive genes were selected based on our experience in the liver and brain, yet the regulation may differ in the PNS. As one possible explanation for the apparent euthyroid state, the sciatic nerve may rather sense the drop in serum T4 in the absence of MCT8. We hypothesize that the trend to elevated Dio2 transcript levels in the absence of MCT8 causes an increase in sciatic DIO2 activity, which may then produce sufficient amounts of T3 locally. Such a scenario was seen before in the brown adipose tissue (38). Alternatively, an upregulation of other TH transporters may occur in the MCT8-deficient sciatic nerve, as we have detected in a recent brain proteomic analysis for LAT1 (39). In contrast, the adult sciatic nerve appears rather insensitive to TH, with a reported low to undetectable expression of deiodinases and TH receptors in rats (31, 40, 41).

Along these lines, our analysis of SOX10-positive total Schwann cell numbers and YY1-positive myelinating Schwann cell numbers revealed normal values at P12 and P180, arguing that at least in mice, the time window during which Schwann cell proliferation is driven by TH signaling might be earlier and/or shorter compared to rats. Given their described TH-sensitivity, the presence of other TH transporters in Schwann cells must be assumed. Investigating transcript expression of TH transporter candidates in a scRNAseq atlas, we only unraveled the presence of the T4-specific transporter Oatp3a1 in Schwann cells (2, 18, 23). It still remains enigmatic by which transporters Schwann cells are supplied with T3.

It also needs to be considered that peripheral nerves are not anatomically separate entities but intertwined with the spinal cord where motor neuron soma are located. Our findings indicate rather negligible alterations in the PNS, arguing that the locomotor impairments seen in M/Odko mice are of central origin in the brain and/or spinal cord. Like the brain, the spinal cord is protected by the semi-permeable blood–spinal cord barrier. We hypothesize that, similar to the brain, the spinal cord in M/Odko mice is in a much more TH-deficient state than in single ko mice. Altered axonal signals emanating from such a strongly TH-deficient spinal cord could further explain why the mild hypermyelination seen in our Western blot analysis was specific to M/Odko mice and not replicated in Mct8ko mice. However, like for the PNS, only sparse information is currently available about the expression of TH transporters and TH signaling components in the spinal cord and specifically the blood–spinal cord barrier. Similarly, motor neuron axons synapse on muscle fibers and muscles are well-known TH-sensitive tissues (42, 43). Whether and how modulation of TH levels on one site of the synapse, the neuromuscular junction, affects the other site remains to be seen and will be a target of future studies.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/ETJ-24-0248. Updated version attached

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work.

Funding

This work was supported by grants of the DFG to SM (CRC/TR296-P19) and HH (CRC/TR296-P01, P09). Funding was further provided by the BMBF (01GM1401) and Sherman Family to HH.

Author contribution statement

SM and HH devised the experiments and wrote the manuscript. SM and AAM conducted and analyzed the experiments in collaboration with RB (electrophysiological measurements).

Acknowledgments

We thank the University Hospital Essen imaging facility (IMCES; Alexandra Brenzel and Anthony Squire) for their support. We further thank Markus Korkowski and Natalie Sadowski for their excellent work.

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Supplementary Materials

 

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  • Figure 1

    MCT8 expression in the sciatic nerve. (A) Immunohistochemical analysis on Wt sciatic nerve cryo-sections obtained from P8, P12 and P180 mice following fixation in methanol. Axonal marker NFH is depicted in yellow, myelin marker MBP in blue, Hoechst33258 to label cell nuclei in cyan, and MCT8 in magenta. Single-channel grayscale pictures are shown for NFH and MCT8. n = 3. Scale bar: 10 μm. (B) Visualization of Mct8 (Slc16a2) transcript expression in single-cell RNA sequencing data of the P1 and P60 sciatic nerve. pmSC, pro-myelinating Schwann cells; mSC, myelinating Schwann cells; nm(R)SC, non-myelinating (Remak) Schwann cells; iSC, immature Schwann cells; prol. SC, proliferating Schwann cells; IC, immune cells; Prol. Fb, proliferating fibroblast-like cells; Fb.Rel*, fibroblast-related cluster (tentative cluster); EnC, endoneurial cells; PnC, perineurial cells; EpC, epineurial cells; EC, endothelial cells; Per, pericytes; and VSMC, vascular smooth muscle cells.

  • Figure 2

    OATP1C1 expression in a subset of sciatic nerve endothelial cells. (A) Wt sciatic nerve sections were fixed in methanol and co-stained for OATP1C1 (in magenta), MBP (in blue), NFH (in yellow) and Hoechst33258 (in cyan). Single-channel grayscale pictures are shown for NFH and OATP1C1. n = 3. Scale bar: 10 μm. (B) Visualization of Oatp1c1 (Slc01c1) transcript expression in single-cell RNA sequencing data of the P1 and P60 sciatic nerve. pmSC, pro-myelinating Schwann cells; mSC, myelinating Schwann cells; nm(R)SC, non-myelinating (Remak) Schwann cells; iSC, immature Schwann cells; prol. SC, proliferating Schwann cells; IC, immune cells; Prol. Fb, proliferating fibroblast-like cells; Fb.Rel*, fibroblast-related cluster (tentative cluster); EnC, endoneurial cells; PnC, perineurial cells; EpC, epineurial cells; EC, endothelial cells; Per, pericytes; and VSMC, vascular smooth muscle cells. (C) Sciatic nerve sections obtained from P8 Wt animals were stained for OATP1C1 (in green) and CD31 (in magenta). Representative pictures for double-positive blood vessels (left) and structures positive for only one of these proteins (right) are displayed. Hoechst33258 counter-stained cell nuclei appear blue. Single-channel grayscale pictures are presented for CD31 and OATP1C1. n = 3. Scale bar: 10 μm.

  • Figure 3

    Sciatic nerve thyroidal state. Sciatic nerves from P12 mice were subjected to qPCR analysis, and the expression of TH signaling components (A) and TH-responsive marker genes (B) was evaluated. Expression levels were normalized to Gapdh, cyclophilin A and cyclophilin D expression as housekeeping genes. Wt values were set as 1.0. n = 4–5.

  • Figure 4

    Electrophysiological properties of the sciatic nerve. Sciatic nerves of 1-year-old female mice were stimulated using repetitive single square-wave pulses. Signals were recorded at different distances to the stimulus, and maximum responses were averaged. CMAP amplitudes at the proximal (A) and distal (B) positions as well as maximum nerve conduction velocity (C) are displayed. n = 8 (Wt, Mct8ko); 5 (Oatp1c1ko); 7 (M/Odko).

  • Figure 5

    Schwann cell analysis in developing animals. Cross sections of PFA-fixed sciatic nerves obtained from P12 animals were immunostained for the Schwann cell marker SOX10 and the myelinating Schwann cell marker YY1 (both in green) (A). Hoechst33258-labeled cell nuclei appear in blue. Numbers of SOX10+ cells (B) and YY1+ cells (C) were quantified and are shown as density per mm2. n = 3. Scale bar: 100 μm.

  • Figure 6

    Schwann cell and myelination analysis in adult animals. Cross sections of PFA-fixed sciatic nerves obtained from P180 female mice were immunostained for the Schwann cell marker SOX10 and for YY1 (both in green) (A). Hoechst33258-labeled cell nuclei are shown in blue. Cells positive for SOX10 (B) and YY1 (C) were enumerated and are presented as density per mm2. n = 5. Scale bar: 100 μm. (D) Sciatic nerves of 6-month-old females were homogenized and subjected to Western blot analysis. The mature myelin marker P0 was assessed. Protein levels were quantified by measuring the integrated density of the respective bands and normalizing it to Vinculin as a housekeeping protein. Wt values were set as 1.0. n = 3. *P < 0.05.

  • 1

    Bernal J . Thyroid hormones and brain development. Vitam Horm 2005 71 95122. (https://doi.org/10.1016/s0083-6729(05)71004-9)

  • 2

    Groeneweg S , van Geest FS , Peeters RP , et al. Thyroid hormone transporters. Endocr Rev 2020 41 146201. (https://doi.org/10.1210/endrev/bnz008)

  • 3

    Friesema EC , Grueters A , Biebermann H , et al. Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation. Lancet 2004 364 14351437. (https://doi.org/10.1016/s0140-6736(04)17226-7)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4

    Dumitrescu AM , Liao XH , Best TB , et al. A novel syndrome combining thyroid and neurological abnormalities is associated with mutations in a monocarboxylate transporter gene. Am J Hum Genet 2004 74 168175. (https://doi.org/10.1086/380999)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5

    Schwartz CE , May MM , Carpenter NJ , et al. Allan-Herndon-Dudley syndrome and the monocarboxylate transporter 8 (MCT8) gene. Am J Hum Genet 2005 77 4153. (https://doi.org/10.1086/431313)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6

    Dumitrescu AM , Liao XH , Weiss RE , et al. Tissue-specific thyroid hormone deprivation and excess in monocarboxylate transporter (mct) 8-deficient mice. Endocrinology 2006 147 40364043. (https://doi.org/10.1210/en.2006-0390)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7

    Trajkovic M , Visser TJ , Mittag J , et al. Abnormal thyroid hormone metabolism in mice lacking the monocarboxylate transporter 8. J Clin Invest 2007 117 627635. (https://doi.org/10.1172/jci28253)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8

    Wirth EK , Roth S , Blechschmidt C , 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 2009 29 94399449. (https://doi.org/10.1523/jneurosci.6055-08.2009)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9

    Mayerl S , Muller J , Bauer R , et al. Transporters MCT8 and OATP1C1 maintain murine brain thyroid hormone homeostasis. J Clin Invest 2014 124 19871999. (https://doi.org/10.1172/jci70324)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10

    Javed K & Daly DT . Neuroanatomy, lower motor neuron lesion. In StatPearls. Treasure Island, FL: StatPearls Publishing, 2025. (https://www.ncbi.nlm.nih.gov/books/NBK539814/)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11

    Nave KA . Myelination and the trophic support of long axons. Nat Rev Neurosci 2010 11 275283. (https://doi.org/10.1038/nrn2797)

  • 12

    Rudolf R , Khan MM & Witzemann V . Motor endplate-anatomical, functional, and molecular concepts in the historical perspective. Cells 2019 8 387. (https://doi.org/10.3390/cells8050387)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    Mayerl S , Visser TJ , Darras VM , et al. Impact of Oatp1c1 deficiency on thyroid hormone metabolism and action in the mouse brain. Endocrinology 2012 153 15281537. (https://doi.org/10.1210/en.2011-1633)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    Mayerl S , Schmidt M , Doycheva D , et al. Thyroid hormone transporters MCT8 and OATP1C1 control skeletal muscle regeneration. Stem Cell Rep 2018 10 19591974. (https://doi.org/10.1016/j.stemcr.2018.03.021)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15

    Schulz A , Walther C , Morrison H , et al. In vivo electrophysiological measurements on mouse sciatic nerves. J Vis Exp 2014 86 51181. (https://doi.org/10.3791/51181)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    Barakat-Walter I & Kraftsik R . Stimulating effect of thyroid hormones in peripheral nerve regeneration: research history and future direction toward clinical therapy. Neural Regen Res 2018 13 599608. (https://doi.org/10.4103/1673-5374.230274)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

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