Sobetirome rescues α‐synuclein‐mediated demyelination in an in vitro model of multiple system atrophy

Multiple system atrophy (MSA) is a rare and rapidly progressive atypical parkinsonian disorder characterized by oligodendroglial cytoplasmic inclusions containing α‐synuclein (α‐syn), demyelination, inflammation and neuronal loss. To date, no disease‐modifying therapy is available. Targeting α‐syn‐driven oligodendroglial dysfunction and demyelination presents a potential therapeutic approach for restricting axonal dysfunction, neuronal loss and disease progression. The present study investigated the promyelinogenic potential of sobetirome, a blood–brain barrier permeable and central nervous system selective thyromimetic in the context of an in vitro MSA model. Oligodendrocyte precursor cells (OPCs) were obtained from transgenic mice overexpressing human α‐syn specifically in oligodendrocytes (MBP29 mouse line), a well‐described MSA model, and non‐transgenic littermates. mRNA and protein expression analyses revealed a substantial rescue effect of sobetirome on myelin‐specific proteins in control and α‐syn overexpressing oligodendrocytes. Furthermore, myelination analysis using nanofibres confirmed that sobetirome increases both the length and number of myelinated segments per oligodendrocyte in primary murine α‐syn overexpressing oligodendrocytes and their respective control. These results suggest that sobetirome may be a promising thyromimetic compound targeting an important neuropathological hallmark of MSA.


| INTRODUCTION
Although oligodendroglial dysfunction and demyelination are early pathogenic events in multiple system atrophy (MSA), pharmacological approaches to induce remyelination appear to be a less studied target for modifying the disease course (Mész aros et al., 2020).In the central nervous system (CNS), oligodendrocytes produce myelin sheaths by concentrically wrapping their plasma membrane around axons with a diameter ranging from 0.4 to 1.2 μm (Nave & Werner, 2014).Thereby, oligodendrocytes begin to ensheath axons by lateral and radial expansion of their membranes (Figure 1).Interestingly, myelin basic protein (MBP) and proteolipid protein (PLP) are expressed in the final phase to commence myelin compaction by displacing the cytoplasm from the outer to the inner layers (Nave & Werner, 2014;Snaidero et al., 2014).
Remyelination is a continuous physiological process in adulthood, decreases during ageing and represents a repair mechanism restricted to certain regions, for example, inflammatory lesion borders (Mei et al., 2014).In the last years, many studies were conducted in models of inflammatory demyelinating autoimmune diseases such as multiple sclerosis (MS), by repurposing small compounds to test their potential for remyelination.A well-F I G U R E 1 Formation of the myelin sheath and experimental paradigm.(a) During maturation, oligodendrocytes ensheath nanofibres with their plasma membrane to form myelin sheaths; inspired by the review of Nave and Werner (2014).(b) Primary oligodendrocyte precursor cells (OPCs) were separated by shaking, seeded onto pre-coated nanofibres or cell culture plates and treated with proliferation medium (d À1 ).Starting on d 0 , OPCs were treated with sobetirome (SOB), triiodothyronine (T3) as positive control or dimethyl sulfoxide (DMSO) as vehicle control every other day.After 8 days of differentiation, oligodendrocytes were harvested for RNA or protein analysis or fixed with paraformaldehyde for immunocytochemistry (ICC) or with ITO buffer for transmission or scanning electron microscopy (TEM, SEM).
known inducer of myelination is the thyroid hormone triiodothyronine (T3) acting via the nuclear thyroid hormone receptor (TR) isoforms α1 and β1.Thus, it is frequently used as promyelinogenic agent for oligodendrocyte differentiation in cell culture (Barres et al., 1994;Baxi et al., 2014;Billon et al., 2001;Deshmukh et al., 2013;Lariosa-Willingham et al., 2016, 2022;Mei et al., 2014;Tokumoto et al., 2001).However, T3 is not suitable as a remyelinating approach in the clinic due to adverse systemic effects on heart, bone and skeletal muscle caused by its effects on TRα1 (Scanlan, 2010).Accordingly, scientists have searched for a TRβ1-selective thyroid hormone agonist devoid of aforementioned systemic side effects.In this way, sobetirome was found which was moreover blood-brain barrier permeable indicated by sufficiently high levels of sobetirome and upregulated thyroid hormone responsive genes in the brain of systemically treated rodents (Hartley et al., 2019;Saponaro et al., 2020;Takahashi et al., 2014;Trost et al., 2000).Sobetirome increased overall oligodendrocyte survival and remyelination in several in vivo models of MS, including the lysolecithin, cuprizone and experimental autoimmune encephalomyelitis (EAE) models (Chaudhary et al., 2021;Hartley et al., 2019).
Based on these promising in vitro and in vivo studies and considering the urgent need for the development of disease-modifying therapies in MSA, this study aimed at investigating the capacity of sobetirome to overcome the α-syn-induced oligodendroglial dysfunction.We therefore utilized primary α-syn overexpressing oligodendrocytes derived from the well-established and in depth characterized transgenic MBP29 mouse model presenting a severe oligodendroglial dysfunction and myelin deficit (Ettle et al., 2016;Hoffmann et al., 2019;Lambrecht et al., 2020;May et al., 2014).Besides analysing the expression level of late myelin markers MBP and PLP both on mRNA and protein level, we established an in vitro culture setup utilizing nanofibres mimicking an axon-like structure in combination with primary α-syn overexpressing oligodendrocytes in order to investigate the myelination ability upon exposure to sobetirome.

| Quantitative real-time PCR
The GoScript™ Reverse Transcription System (Promega) was used for reverse transcription of extracted RNA to cDNA.qPCRs were conducted using the Light Cycler 480 system (Roche).mRNA expression levels of genes of interest were calculated relative to the mean of housekeeping genes β-actin and 18S rRNA as previously described (Mész aros et al., 2021).

| Western blot analysis
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer.The protein concentration was measured using Pierce™ BCA Protein Assay Kit (Thermo Scientific) and CLARIOstar microplate reader (BMG LAB-TECH).Samples were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membrane (PVDF-FL, Merck).After blocking, membranes were incubated with primary and fluorescent secondary antibodies as previously described (Mész aros et al., 2021).Protein bands were detected and analysed using the FusionFx7 system (PeqLab) and Bio-1D software (Vilber Lourmat), respectively.

| Immunocytochemistry
After differentiation of OPCs on nanofibres, cells were fixed in 4% paraformaldehyde.Following blocking, primary and fluorescent secondary antibodies were incubated consecutively.Cells on nanofibres were visualized as z stack images using the Axio Observer fluorescence microscope using ZEN blue software (Carl Zeiss).The ImageJ software (National Institute of Health) was applied to measure the number and length of myelinated segments per oligodendrocyte using maximum intensity projections.

| Electron microscopy
After cultivation on nanofibres, cells were fixed with ITO buffer and dehydrated in ascending alcohol series for scanning and transmission electron microscopy (SEM, TEM).For SEM, samples were processed using the Q150T Turbo-pumped Sputter Coater (Quorum Technologies Inc) and visualized using the AURIGA CrossBeam scanning electron microscope (Carl Zeiss).For TEM, samples were embedded in epon 812, sectioned with Ultracut R (Eichert-Jung), contrasted with uranyl acetate and imaged with a JEM 1400 Plus transmission electron microscope (JEOL Germany).

| Statistical analysis
Data were analysed using GraphPad Prism software and visualized by CorelDRAW X6 software.The Shapiro-Wilk test was used in order to determine normal distribution.Accordingly, the Mann-Whitney test or unpaired t-test was used.p values < 0.05 were considered statistically relevant (*p < 0.05, **p < 0.01, ***p < 0.001).

| Sobetirome increases myelinspecific markers on mRNA and protein level
To investigate the efficacy of sobetirome, MBP29 and non-transgenic control mouse-derived OPCs were cultured and treated for 8 days prior to RNA analysis.As expected, T3 treatment increased mRNA expression levels of late myelin markers MBP and PLP in control mouse-derived oligodendrocytes by 9.4-and 9.2-fold, respectively (Figure 2b,c).The T3-mediated elevation of MBP and PLP levels was only marginally lower in α-syn overexpressing oligodendrocytes (7.9-and 8.1-fold, respectively).Interestingly, sobetirome-treated α-syn overexpressing oligodendrocytes showed increased MBP and PLP mRNA expression levels to a similar level as in control oligodendrocytes (MBP by 6.0-in MBP29 and 5.7-fold in non-transgenic mouse-derived oligodendrocytes, PLP by 6.2-and 6.8-fold in MBP29 mouse-derived oligodendrocytes, respectively).The observed promyelinogenic effect was furthermore confirmed by western blotting (Figure 2d).T3 administration increased both MBP and PLP protein levels (Figure 2e, MBP by 5.9-in MBP29 and 3.2-fold in control mouse-derived oligodendrocytes; Figure 2f, PLP by 3.4-and 2.0-fold in MBP29 and non-transgenic mouse-derived oligodendrocytes relative to DMSO-treated non-transgenic mouse-derived oligodendrocytes, respectively).Similar to mRNA expression analyses, treatment with sobetirome increased the protein levels of MBP and PLP in α-syn overexpressing oligodendrocytes to a similar level as in non-transgenic control oligodendrocytes (Figure 2e, MBP by 5.0-in MBP29 and 2.9-fold in control mouse-derived oligodendrocytes; Figure 2f, PLP by 3.2-and 2.5-fold in MBP29 mouse-derived oligodendrocytes relative to DMSOtreated non-transgenic mouse-derived oligodendrocytes, respectively).

| Sobetirome increases the length and number of myelinated segments per oligodendrocyte
The maturation of oligodendrocytes culminates in a complex process of myelination by concentrically wrapping axons with multilamellar sheaths.To investigate whether sobetirome not only drives maturation of OPCs to premyelinating oligodendrocytes but also induces myelination, an in vitro culture setup was established using nanofibres with 700 nm in diameter.Scanning electron microscopical analysis of primary OPCs differentiated for 8 days using T3 confirmed that oligodendrocytes were able to wrap nanofibres with their plasma membrane (Figure 3a-c).Moreover, TEM revealed intact and compact myelin sheaths circling nanofibres.Next, MBP29 and non-transgenic control mouse-derived OPCs were cultured on nanofibres and differentiated with sobetirome, T3 or DMSO.T3 promoted both the mean length and the number of myelinated segments per oligodendrocyte in MBP29 mouse-derived oligodendrocytes relative to DMSO-treated control mouse-derived oligodendrocytes by 1.7-and 2.0-fold, respectively (Figure 3g-i).Interestingly, comparable effects were achieved upon treatment with sobetirome, as the mean length and number of myelinated segments per oligodendrocyte enhanced by 1.5and 1.8-fold in MBP29 mice-derived oligodendrocytes in comparison with DMSO-treated control mouse-derived oligodendrocytes.

| DISCUSSION
Severe and widespread myelin deficits in MSA patients as well as MBP29 mice are probably linked to toxic α-syn aggregates, oligodendroglial dysfunction, lipid dysbalance and inflammatory response.Thus, promoting myelinogenesis represents a promising target to interfere with the vicious pathogenic circle in MSA (Ettle et al., 2016;Hoffmann et al., 2019;Mész aros et al., 2021).Known for its strong promyelinogenic effect, the thyroid hormone T3 has previously been established as a robust positive reference for several high-throughput screens in search for potential remyelinating small molecules in the context of MS (Barres et al., 1994;Deshmukh et al., 2013;Lariosa-Willingham et al., 2022, 2016;Mei et al., 2014).While the application of T3 is hampered in patients by severe systemic adverse effects akin to hyperthyroidism including tachycardia, arrhythmia, decreased bone density and muscular atrophy, the blood-brain barrier permeable and TRβ1-selective thyromimetic sobetirome does not induce peripheral thyrotoxic effects.Thus, sobetirome might be a promising therapeutic agent to overcome MSA-associated demyelination (Baxi et al., 2014;Scanlan, 2010).Here, we demonstrate that sobetirome is able to ameliorate α-syn-mediated myelin deficit indicated by substantially increased MBP and PLP mRNA and protein expression levels, comparable to T3 in MBP29 mouse-derived oligodendrocytes.In order to study the (re)myelination potential sobetirome, we established a nanofibre cell culture setup.Ultrastructural imaging clearly showed multilamellar myelin wrapping of nanofibres by primary oligodendrocytes.Subsequent immunofluorescence analyses enabled us to quantify the myelination potential of sobetirome in α-syn overexpressing oligodendrocytes cultured on nanofibres.Indeed, sobetirome not only accelerated oligodendroglial differentiation but moreover ameliorated myelination indicated by increased length and numbers of myelinated segments per oligodendrocyte.In respect to MS, sobetirome already showed promising remyelination effects in gliotoxin-induced demyelination models utilizing lysolecithin and cuprizone.Similar effects were seen in EAE mice, reflected by less degenerated axons, and an increase of intact myelin sheaths surrounding axons.It is noteworthy that Sob-AM2, the CNS-targeted prodrug of sobetirome, furthermore reduced the numbers of inflammatory CD4+ T cells in EAE mice compared to sobetirome and thus may be additionally considered for its anti-inflammatory potential in MSA as well (Chaudhary et al., 2021;Hartley et al., 2019;Hoffmann et al., 2019).
An important limitation of the model used in this study is that the effects of sobetirome on α-syn cannot be examined as the MBP29 mouse model expresses α-syn under the MBP promotera downstream target of sobetirome.Therefore, other models will be needed to study myelin-independent effects of sobetirome, for example, on α-syn pathology.
Overall, this study implies that thyromimetics as sobetirome might be promising disease-modifying candidates to overcome the α-syn-driven myelin deficit and thus set the basis for future studies in in vivo MSA models.Mész aros.All authors commented and approved the manuscript.

F
I G U R E 2 Promyelinogenic potential of sobetirome and triiodothyronine in α-syn overexpressing oligodendrocytes.(a) Structural formula of sobetirome (SOB) and triiodothyronine (T3).(b, c) Quantification of MBP (b) and PLP (c) mRNA levels relative to the housekeepers β-actin and 18S rRNA expressed as fold change of SOB-and T3-treated transgenic MBP29 (tg) or non-transgenic control (ctrl) mouse-derived OPCs over the mean level of dimethyl sulfoxide (DMSO)-treated control oligodendrocytes (n = 5).(d) Western blot analysis for myelin basic protein (MBP) and proteolipid protein (PLP) of control and MBP29 mouse-derived OPCs treated with SOB, T3 or DMSO as control for 8 days.(e, f) Quantification of MBP (e) and PLP levels (f) relative to the housekeeper β-actin (β-act) and expressed as fold change relative to DMSO-treated control-mouse derived oligodendrocytes (n = 5).Note that DMSO-treated control mouse-derived oligodendrocytes are presented as black dashed line, while DMSO-treated transgenic mouse-derived oligodendrocytes are presented as blue dashed line.Statistical analyses were performed using Mann-Whitney test; *p < 0.05, **p < 0.01.

F
I G U R E 3 Promyelinogenic compounds rescue myelination deficit in α-syn overexpressing oligodendrocytes.(a) SEM of polycaprolactone nanofibres with a diameter of 700 nm.(b, c) cultivation of primary oligodendrocytes on nanofibres after 8 days of differentiation shows that oligodendrocytes are able to wrap nanofibres with their plasma membrane.(d-f) Transmission electron microscopic images of a cross-sectioned myelinated nanofibre.Higher resolution of the myelin sheath (f) reveals several layers of myelin.Structure and density are comparable to myelin sheaths formed around axons.(g) Immunofluorescence staining of non-transgenic control mice-derived (ctrl) and transgenic MBP29 mice-derived oligodendrocytes (tg) which were cultured on nanofibres and treated for 8 days with sobetirome (SOB), triiodothyronine (T3) or dimethyl sulfoxide (DMSO) as control.Maximum intensity projection images are showing OLIG2 (blue) as oligodendrocyte-specific marker and MBP (red) as marker for myelin.(h) Quantification of the mean length of myelinated segments on nanofibres upon SOB or T3 treatment (n = 4).(i) Quantification of the number of myelinated segments on nanofibres per oligodendrocyte (n = 4).Note that DMSO-treated control mouse-derived oligodendrocytes are presented as black dashed line, while DMSOtreated transgenic mouse-derived oligodendrocytes are presented as blue dashed line.Statistical analyses were performed using unpaired ttest; *p < 0.05, **p < 0.01, ***p < 0.001.Scale bars: (a) 2 μm, (b) 1 μm, (c-e) 0.5 μm, (f) 0.2 μm, and (g) 20 μm.