Loss of Thyroid Hormone Transporters MCT8 and OATP1C1 in Mouse Oligodendroglia Cells Results in a Delayed Oligodendrocyte Maturation and Myelination

Abstract

Background:

Due to an impaired thyroid hormone (TH) transport across brain barriers, inactivation of the murine TH transporters Mct8/Oatp1c1 causes a profound TH deficiency of the CNS that greatly impacts neural development and function. Since oligodendrocyte maturation and myelination are dependent on local TH signaling, Mct8/Oatp1c1 double knockout (DKO) mice exhibit a persistent state of hypomyelination. Yet, to which extent Mct8/Oatp1c1 inactivation also affects TH transport into oligodendroglia cells has not been addressed. Here, we studied oligodendrocyte maturation and myelination in mice lacking Mct8/Oatp1c1 specifically in oligodendroglia lineage (OL) cells and compared their phenotype with that of control and DKO animals.

Method:

Conditional Mct8/Oatp1c1 mutants were crossed with mice expressing constitutively Cre-recombinase under the control of the Olig2 promoter to inactivate both transporters in OL cells (so-called OL CKO mice). Neural maturation and myelination were assessed by immunofluorescence (IF) and fluorescence in situ hybridization (FISH) studies at different postnatal time points. Oligodendrocyte precursor cells (OPCs), premyelinating, and myelinating oligodendrocytes were visualized by coimmunolabeling.

Results:

OL CKO mice exhibited normal serum TH concentrations and hypothalamic Trh transcript levels. Quantification of neuronal TH-target gene transcript levels (Rc3; Klf9; Pde10a) revealed no alterations. Abundance of myelin sheaths-related proteins Mbp and Cnp was significantly reduced in OL CKO mice at postnatal day P12 but reached normal levels at P21 and P120. Quantification of OPC, premyelinating, and myelinating oligodendrocytes disclosed a strongly reduced number of mature OL at P6 and P12, while cell numbers normalized in adult OL CKO mice.

Conclusions:

Inactivation of murine TH transporters Mct8/Oatp1c1 in OL cells causes a delayed oligodendroglia maturation and myelination. These findings highlight a physiologically relevant function of Mct8/Oatp1c1 in developmental oligodendrogenesis and myelin formation. In contrast to the persistent myelination defect seen in central hypothyroid DKO mice, OL CKO mice exhibit only a transient oligodendrocyte differentiation impairment and transient hypomyelination. These observations indicate the presence of additional, yet unknown, TH transporters that ultimately enable cellular TH entry into oligodendroglia cells even in the absence of Mct8/Oatp1c1.

Keywords

SLC16A2 SLCO1C1 myelination Allan–Herndon–Dudley syndrome thyroid hormone transport

Introduction

Monocarboxylate transporter 8 (MCT8) represents a highly specific and widely expressed thyroid hormone (TH) transporter that mediates transmembrane passage of both thyroxine (T4) and triiodothyronine (T3) in humans and mice.^1,2 Loss-of-function mutations in the MCT8-encoding SLC16A2 gene lead to Allan–Herndon–Dudley syndrome (AHDS), a rare X-linked disorder characterized by profound neurocognitive deficits and severe motor abnormalities.^3–5 Affected individuals exhibit a markedly altered TH profile with highly elevated serum T3 along with clinical signs of thyrotoxicity, such as hypermetabolism, low body weight, and muscle wasting.⁶ In contrast, hypomyelination and impeded neural differentiation are indicative of a significant TH-deficient state in the patients’ brain.^7,8 Based on these observations, impaired TH transport across blood–brain barrier (BBB) cells has been suggested as a major pathogenic mechanism underlying the neurological symptoms of AHDS. However, whether MCT8 is also required for TH transport into other neural cell types is still a topic of intense research.

A strongly diminished TH delivery to the CNS is also a key feature of Mct8 knockout mice that additionally lack the T4-selective organic anion transporting polypeptide 1c1 (Oatp1c1).⁹ Of note, Oatp1c1 is coexpressed with Mct8 in rodent but not in primate BBB cells and thus can functionally compensate for a loss of Mct8 only in mice.^10,11 Accordingly, only Mct8/Oatp1c1 double knockout (DKO) mice exhibit a substantial TH deprivation of the CNS and a persistent hypomyelination as evidenced by a reduced expression of myelin-related proteins, decreased number of myelinated axons, as well as altered morphology of myelinating oligodendrocytes.^9,12,13 We recently studied oligodendroglia maturation in DKO mice in detail and observed an elevated number of oligodendrocyte precursor cells (OPCs) during postnatal development and a persistently reduced density of mature oligodendrocytes, whereas oligodendrogenesis was not disturbed in either Mct8 ko or Oatp1c1 ko mice.¹³ To which extent the oligodendroglia differentiation blockage only observed in DKO mice can be fully attributed to the profound TH-deficient state of the CNS in these animals, however, remained unclear. Of note, both Mct8 and Oatp1c1 expression has been detected in rodent OPCs and (pre)myelinating oligodendrocytes, suggesting that both transporters may also exert important cell-autonomous functions in controlling oligodendroglia cell maturation.^11,14,15 Hence, proper myelination in the CNS may not only depend on Mct8/Oatp1c1-mediated TH passage across brain barriers but also on Mct8/Oatp1c1-facilitated TH transport into oligodendroglia (precursor) cells.

To test this hypothesis, we generated mice that lack both Mct8/Oatp1c1 within the oligodendroglia cell lineage while keeping Mct8/Oatp1c1-facilitated TH transfer across brain barriers and into neurons intact. These so-called oligodendroglia lineage (OL) CKO mice exhibit a transient hypomyelination and a delayed oligodendrocyte maturation, thereby highlighting a relevant cell-autonomous function of Mct8/Oatp1c1 in myelination during development.

Material and Methods

Ethical approval

Mouse studies followed the European Union directive 2010/63/EU and were conducted in compliance with the Animal Welfare Committee of the Landesamt für Verbraucherschutz und Ernährung Nordrhein-Westfalen (Recklinghausen, Germany; approval codes AZ81-02.04.2021.A059 and AZ81-02.04.2022.A156).

Generation of OL CKO mice

Generation and genotyping of Mct8/Oatp1c1 DKO as well as of Mct8/Oatp1c1 double floxed mice have been described elsewhere.^16–18 Mct8/Oatp1c1 double floxed mice were crossed with animals expressing a constitutively active Cre-recombinase under the direction of the endogenous oligodendrocyte transcription factor Olig2 promoter (Olig2 tm1.1(cre)Wdr; JAX stock #025567)¹⁹ shown to be active in all cells of the oligodendrocyte lineage as well as in subsets of spinal cord motor neurons.^20,21 These so-called OL CKO mice were further interbred with a Cre reporter mouse (Gt(ROSA)26Sortm3(CAG-EYFP)Hze)²² harboring a loxP-flanked STOP cassette that prevents transcription of a CAG promoter-driven EYFP construct and that is inserted into the Rosa26 locus.

Animal studies

All experimental animals were kept on a C57BL/6 background and closely monitored for any undesirable germ-line deletion of the floxed alleles by PCR as described.¹⁷ Mct8/Oatp1c1 double floxed littermates without an Olig2-Cre knock-in construct were used as controls. For direct comparison, age-matched Mct8/Oatp1c1 DKO mice were included as well. At postnatal day P6, P12, and P21, mice of both sexes were analyzed, whereas at P120, only male mice were examined.

Tissue processing

For immunofluorescence (IF) studies, mice at P12, P21, and P120 were subjected to intracardial perfusion-fixation with 4% paraformaldehyde (4% PFA) in PBS under deep ketamine/xylazine-induced anesthesia, while mice at P6 were killed by decapitation and their brains were incubated in 4% PFA for at least 14 hours. After postfixation in 4% PFA, brains were cryoprotected in 30% sucrose, frozen in isopentane cooled on dry ice and then cut into 16-µm-thick coronal brain sections with a cryostat. Alternatively, perfusion-fixed brains were cut into 50-µm-thick coronal sections using a vibratome. For FISH studies, P21 old mice were killed by cervical dislocation and their brains were fresh-frozen in isopentane cooled on dry ice. Blood samples were collected by heart puncture, and total serum T4, T3, and rT3 concentrations were quantified by LC-MS/MS as described previously.^23,24

IF studies

Coronal forebrain cryosections between Bregma 1.045 and 0.38 were first incubated in blocking buffer (10% goat or donkey serum; 0.2% Triton X-100 in PBS) for 1 hour at RT, followed by incubation with primary antibodies listed in Supplementary Data at 4°C overnight. After washing, sections were treated for 1 hour at RT with the respective Alexa Fluor 488-, 555-, and 647-coupled secondary antibodies (1:500; Invitrogen) in blocking buffer containing Hoechst 33258 (1:10,000, Invitrogen). Imaging was conducted with a Leica SP8 confocal microscope. Z-stacks with 5 z-planes and a total depth of approximately 3.5 µm were used to visualize oligodendroglia cells in the corpus callosum between the midline and cingulum bundle. Free-floating vibratome sections were subjected to immunostaining to visualize neuronal and myelin marker proteins as described above.

Fluorescence in situ hybridization studies

Fluorescence in situ hybridization (FISH) experiments were carried out by applying the hybridization chain reaction method and protocols described elsewhere²⁵ and further outlined in Supplementary Data.

Image analysis

All images were processed and analyzed in a blinded manner using ImageJ version 1.54 (NIH). Oligodendroglia cells were quantified in the corpus callosum between midline and cingulum bundle, and cell numbers were normalized to the respective quantified area. Parv+ cells were enumerated in the somatosensory corteex and their cell numbers normalized to the analyzed area. Gad67 immunoreactivity was quantified by determining the mean gray values in the the area of the somatosensory cortex and subtracting background values measured in the corpus callosum. Mean gray values reflecting Mbp and Cnp immunoreactivity were quantified in the cerebral cortex and normalized to the respective area. For FISH experiments, first the background was subtracted using the ‘‘Subtract background’’ function in ImageJ (method “sliding paraboloid”) and then mean gray values were measured. Trh-specific FISH signals were quantified in the paraventricular hypothalamic nucleus (PVN), Klf9 in the somatosensory cortex, and Rc3, as well as Pde10a, in the striatum. At least 4 mice per genotype and time point were analyzed as biological replicates, and 4 images per animal were used for quantification in IF and FISH experiments. Control values were set as 1.0.

Statistics

Statistical analysis was conducted using R 4.2.2 integrated in BioRender Graph. Statistical significance between controls, OL CKO and DKO mice was calculated using one-way ANOVA followed by Tukey’s multiple comparison test. Results are presented as mean ± standard deviation. Differences were considered significant when α < 0.05. p-Values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Results

To explore a cell-autonomous function of Mct8/Oatp1c1 within the murine oligodendroglia cell lineage, we generated conditional Mct8/Oatp1c1 double floxed (dfl) mice that additionally express a constitutively active Cre-recombinase under the control of the oligodendrocyte transcription factor Olig2. The phenotype of these so-called OL CKO was compared with their cre-negative Mct8/Oatp1c1 double floxed littermates that served as controls (Fig. 1A). For direct comparison, we also included age-matched Mct8/Oatp1c1 DKO mice in our study.

OL CKO mice were born with the expected frequency and developed indistinguishably from their control littermates (Supplementary Fig. S1A,B). To monitor Cre-recombinase activity, we took advantage of a Stop-IRES-EYFP reporter construct and monitored EYFP protein expression in brain sections of OL CKO mice. Coimmunostainings revealed the presence of EYFP in more than 85% of Olig2-positive cells in white matter regions (Fig. 1B), confirming the presence of Cre-recombinase activity in the vast majority of oligodendroglia cells. To corroborate our hypothesis that cortical neuronal differentiation remains unaffected in OL CKO mice, Parvalbumin (Parv) immunopositive cells as well as Glutamate Decarboxylase (Gad67) immunoreactivity were quantified in the somatosensory cortex at postnatal day P21. As anticipated, these measurements did not reveal any changes in OL CKO mice, whereas DKO animals exhibited the expected significant reduction of both neuronal markers (Fig. 1C; Supplementary Fig. S1C,D). Next, FISH studies were conducted to assess transcript levels of well-established TH-regulated target genes as a proxy to evaluate intracellular neuronal TH status at P21. As demonstrated in Figure 1D, FISH signal intensities for Klf9, Rc3, and Pde10a were found to be strongly downregulated in the cortex and striatum of DKO mice, whereas no differences could be detected between control and OL CKO mice (Fig. 1D). We also quantified Trh mRNA expression in the paraventricular hypothalamic nucleus (PVN) at P21 and found normal levels in OL CKO mice in contrast to a strong increase in DKO animals (Fig. 2A). Likewise, serum T3, T4 and rT3 concentrations were similar in OL CKO and control mice, while DKO mice exhibited high T3, low T4, and low rT3 concentrations as expected. Altogether, these findings point to an undisturbed hypothalamus–pituitary–thyroid axis and an intact TH transfer into the CNS as well as into neurons of OL CKO mice.

To monitor the myelin status of OL CKO mice, perfusion-fixed brain vibratome sections were immunostained with antibodies against myelin basic protein (Mbp) present in myelinating oligodendrocytes and cyclic nucleotide phosphodiesterase (Cnp) as a marker for pre- and myelinating oligodendrodrocytes (Supplementary Fig. S2A) at different postnatal time points. At P12, OL CKO mice displayed a significantly decreased cortical Cnp protein expression and a profound reduction in cortical Mbp expression (Fig. 3A). However, at P21 (Fig. 3B) and P120 (Fig. 3C), both Mbp and Cnp IF signal intensities in OL CKO mice were similar as in control animals, indicating that, in contrast to DKO mice, myelin formation in OL CKO animals is only delayed but not persistently compromised.

Neural stem cells give rise to OPC which in turn differentiate into premyelinating oligodendrocytes and then mature into myelinating oligodendrocytes.²⁶ As this developmental program requires fine-tuned intracellular TH signaling, we studied oligodendroglia cell lineage progression in OL CKO mice by marker analysis (Supplementary Fig. S2A). In particular, OPCs were visualized by coimmunostaining for Olig2 and platelet-derived growth factor receptor alpha (Pdgfra) protein. Premyelinating oligodendrocytes were identified by their combined expression of Olig2 and breast carcinoma-amplified sequence 1 (Bcas1) protein. Myelinating oligodendrocytes were marked by coimmunostaining for Olig2 and Quaking 7, which is recognized by the monoclonal antibody CC1. We particularly focused our analysis on the corpus callosum (Supplementary Fig. S2B) as a prominent white matter tract and compared oligodendroglia cell composition in control, OL CKO, and DKO mice at postnatal P6, P12, P21, and P120 (Fig. 4; Supplementary Fig. S2C).

For OPC, the greatest difference in cell densities was observed at P6 when both OL CKO and DKO mice exhibited a 1.7-fold increase in Olig2/Pdgfra-positive cells compared with control mice. However, at later time points, only slight alterations in OPC densities between the different genotypes were noted (Fig. 4A). In comparison, differences in premyelinating Olig2/Bcas1-positive oligodendrocyte densities were most overt at P12 when both OL CKO and DKO mice exhibit an approximately 50% reduction in cell numbers compared with age-matched controls (Fig. 4B). Most importantly, numbers of myelinating OL were already significantly decreased in OL CKO and DKO mice at P6 and P12 (Fig. 4C). At later time points and in line with previous observations,¹³ Olig2/CC1 cell numbers remained low in DKO mice. In OL CKO mice, however, Olig2/CC1 cell numbers were already at P21 significantly higher compared with DKO animals and even reached control levels at P120 (Fig. 4C). In sum, these data indicate that OL CKO mice indeed exhibit a delayed postnatal oligodendrogenesis and a retarded myelin formation, but in contrast to DKO mice, myelination is fully recovered in adult OL CKO animals.

Discussion

Myelination represents a crucial process in brain development that is tightly controlled by TH on several levels. Upon binding to its nuclear TH receptors, T3 is sought to promote cell cycle arrest of proliferating OPCs and to drive their terminal differentiation by controlling the expression of oligodendroglia maturation factors.^27–30 In addition, T3 regulates the expression of prominent myelin-associated proteins, demonstrating that a precise local TH signaling is also required in mature oligodendrocytes to ensure proper myelin formation.³¹ Given the well-established role of TH in regulating myelinogenesis, MCT8 deficiency is frequently accompanied with a reduced white matter content particularly in the first years.³² Although longitudinal brain magnetic resonance imaging of MCT8-deficient patients often suggests myelination improvements with age, histomorphological studies of a deceased MCT8 patient disclosed a profound reduction in myelin content and a significantly lower number of large-caliber myelinated axons pointing to a persistent state of hypomyelination.^7,8

Hypomyelination represents also a characteristic feature of AHDS mouse models that exhibit a reduced number of myelinated axons, aberrant oligodendrocyte morphology, and a reduced expression of myelin-associated proteins not only during development but also in adulthood.^13,18 Yet, whether these myelin impairments in patients and mouse models can be solely explained by the profound TH deficiency of the CNS is still a matter of debate.

Studies using human embryonic stem cell-derived OPCs indeed revealed reduced maturation and increased apoptosis upon pharmacological and genetic blockage of MCT8 supporting an important cell-autonomous role of MCT8 in this in vitro system.³³ In contrast, Vatine et al. reported a normal differentiation pattern of human-induced (hi) OPC into mature oligodendrocytes in the presence or absence of MCT8 in vitro.³⁴ Moreover, upon transplantation into the corpus callosum of neonatal DKO mice, both MCT8-deficient and control hiOPC failed to mature into myelinating oligodendrocytes, highlighting the dominant impact of a TH-deficient CNS environment on myelination. Yet, transplantation of MCT8-deficient hiOPC into the euthyroid corpus callosum of an Mbp-depleted mouse model, impacted their myelination capacity, as well as suggesting that MCT8 indeed exerts cell-autonomous functions.³⁰

In light of these intriguing results, we aimed to clarify the impact of an oligodendroglia lineage (OL)-restricted TH transporter deficiency in mice. We anticipated that such information would not only help to define key components in TH-regulated myelin formation but may also be relevant for gene therapy studies intending to restore functional MCT8 in critical CNS cell types.^35,36 To this end, we generated mouse mutants that lack Mct8 and Oatp1c1 in oligodendroglia cells and compared their phenotype with that of control littermates as well as global DKO animals. As expected, OL CKO mice exhibited regular circulating TH parameters as well as an undisturbed neuronal development and normal mRNA expression of TH-regulated genes, suggesting that TH passage into the brain in OL CKO mice is unimpeded. Thus, we can assume that OL CKO mice also exhibit normal TH concentrations inside the brain parenchyma and, hence, an overall euthyroid CNS environment, which represents a major prerequisite for proper myelination.

However, despite their apparent normal appearance, OL CKO mice exhibited white matter abnormalities during early postnatal development. Quantification of OPC, as well as premyelinating and myelinating oligodendrocytes in the corpus callosum at P6, revealed similarly increased OPC numbers and similarly decreased numbers of myelinating oligodendrocyte densities in OL CKO and in DKO mice, indicating an intracellular TH deficit in oligodendroglia cells in both mouse models. Most likely, during the first postnatal week, TH cannot enter Mct8/Oatp1c1-deficient OPC to trigger cell-cycle arrest and to stimulate their differentiation. This early postnatal maturation impairment results in a visibly decreased myelination as seen by highly reduced Mbp and Cnp protein levels in OL CKO mice at P12, thus during the time when the murine brain is known to be most sensitive to changes in TH signaling.³⁷ Remarkably, myelination in OL CKO mice appears to be only transiently compromised as already at P21; thus upon weaning, numbers of mature oligodendrocytes gradually increased and finally reached normal levels in adulthood. Hence, in contrast to the permanent oligodendrocyte maturation blockage in DKO mice, oligodendrogenesis obviously can catch up in juvenile OL CKO mice, thereby preventing a persistent myelination defect.

Why can OL CKO mice overcome the transient state of hypomyelination? As the most straightforward explanation, one may envision the developmental upregulation of other TH transporters that facilitate TH access to oligodendroglia cells and thus compensate for a loss of Mct8/Oatp1c1. In fact, several studies have highlighted distinct changes in spatiotemporal TH transporter expression patterns both in murine and human CNS.^38,39 One intriguing candidate is Mct10 (encoded by Slc16a10 gene) that, apart from aromatic amino acids, also accepts TH as substrates.⁴⁰ Notably, Mct10-specific transcript levels were found to be elevated in murine white matter structures from P21 onward,³⁸ thus prior to P30 when the postnatal peak of myelination in mice is finished.⁴¹ Further, studies of Mct8 ko mice lacking additionally Mct10 disclosed a concerted action of both transporters in TH transmembrane passage in peripheral tissues.¹⁸ Whether Mct10 also exerts a compensatory TH transporting function in Mct8/Oatp1c1-deficient oligodendrocytes still needs to be tested in future studies, for example, by monitoring oligodendroglia maturation and myelination in Mct10-deficient OL CKO mice.

In conclusion, our studies clearly confirm a cell-autonomous function of Mct8/Oatp1c1 in murine OL progression. However, myelination impairment in OL CKO mice is only transient and thus less severe than in the TH-deficient CNS of DKO mice. In terms of treatment strategies for AHDS, our findings underscore the clinical relevance of restoring either TH access to the CNS^35,36 or applying CNS-entering thyromimetic substances,^33,42–44 ideally as early as possible, as such intervention might be sufficient to ensure regular myelination even if MCT8 is missing.⁴⁵

Data Availability Statement

Data are available from the corresponding author upon request.

Authors’ Contributions

J.-J.R.: Writing—original draft (supporting), methodology (equal), formal analysis (lead), writing, and review and editing (equal). B.M.: Writing—original draft (supporting), methodology (equal), formal analysis (supporting), and review and editing (equal). B.A.: Formal analysis (supporting) and review and editing (equal). A.B.: Formal analysis (supporting), writing—original draft (supporting), and review and editing (equal). D.F.: Writing—original draft (supporting) and review and editing (equal). S.M.: Conceptualization (equal), writing—original draft (supporting), formal analysis (supporting), methodology (equal), and review and editing (equal). H.H.: Conceptualization (equal), writing—original draft (lead), and review and editing (equal).

Supplemental Material

sj-docx-1-thy-10.1177_10507256261469832 — Supplemental material for Loss of Thyroid Hormone Transporters MCT8 and OATP1C1 in Mouse Oligodendroglia Cells Results in a Delayed Oligodendrocyte Maturation and Myelination

Supplemental material, sj-docx-1-thy-10.1177_10507256261469832 for Loss of Thyroid Hormone Transporters MCT8 and OATP1C1 in Mouse Oligodendroglia Cells Results in a Delayed Oligodendrocyte Maturation and Myelination by Joshua-Joel Richter, Boyka Markova, Beyza Atalay, Anita Boelen, Dagmar Führer, Steffen Mayerl, and Heike Heuer

Supplemental Material

sj-docx-2-thy-10.1177_10507256261469832 — Supplemental material for Loss of Thyroid Hormone Transporters MCT8 and OATP1C1 in Mouse Oligodendroglia Cells Results in a Delayed Oligodendrocyte Maturation and Myelination

Supplemental material, sj-docx-2-thy-10.1177_10507256261469832 for Loss of Thyroid Hormone Transporters MCT8 and OATP1C1 in Mouse Oligodendroglia Cells Results in a Delayed Oligodendrocyte Maturation and Myelination by Joshua-Joel Richter, Boyka Markova, Beyza Atalay, Anita Boelen, Dagmar Führer, Steffen Mayerl, and Heike Heuer

Supplemental Material

sj-docx-3-thy-10.1177_10507256261469832 — Supplemental material for Loss of Thyroid Hormone Transporters MCT8 and OATP1C1 in Mouse Oligodendroglia Cells Results in a Delayed Oligodendrocyte Maturation and Myelination

Supplemental material, sj-docx-3-thy-10.1177_10507256261469832 for Loss of Thyroid Hormone Transporters MCT8 and OATP1C1 in Mouse Oligodendroglia Cells Results in a Delayed Oligodendrocyte Maturation and Myelination by Joshua-Joel Richter, Boyka Markova, Beyza Atalay, Anita Boelen, Dagmar Führer, Steffen Mayerl, and Heike Heuer

Footnotes

Acknowledgments

The authors thank Markus Korkowski for excellent technical support. They also thank the Imaging Center Essen (IMCES) at the Faculty of Medicine of the University of Duisburg-Essen, Germany, for providing access to the Leica SP8 microscope (funded by DFG 234323630, INST 58219/26-1 FUGG) as well as Alexandra Brenzel, Anthony Squire, and Jan-Hagen Krohn for general support in its operation. All graphs and schemes were created in BioRender (Heuer, H. (2026) ).

Author Disclosure Statement

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding Information

J.-J.R., B.M., B.A., A.B.: no funding; D.F.: received funding by DFG (CRC/TR296; 424957847; Project P10); S.M.: received funding by DFG (CRC/TR296; 424957847; Project P19); and H.H.: received funding by DFG (CRC/TR296; 424957847; Project P09).

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