Spironolactone Targets Retinoid X Receptor γ to Promote Myelin Sheath Regeneration

Abstract

Background and Objectives

Demyelinating diseases are neurologic disorders characterized by the loss of the myelin sheath and impaired regeneration. Retinoid X receptor γ (RXRγ) is a member of the nuclear receptor superfamily and plays a crucial role in oligodendrocyte biology and myelin formation. However, the clinical application of drugs targeting RXRγ for demyelinating diseases is limited. Selecting small-molecule drugs approved by the U.S. Food and Drug Administration (FDA) that have high binding activity to RXRγ may be an effective strategy for treating demyelinating disorders.

Methods

We used an online molecular docking tool to predict that spironolactone (SPIR), an FDA-approved drug, displays strong binding activity to RXRγ. Subsequently, we verified the impact of SPIR on oligodendrocyte precursor cell (OPC) differentiation and myelin sheath formation through in vitro OPC culture and pharmacologic experiments in mice. Furthermore, using genetic models with CRISPR-LSL-Cas9, we confirmed that the effect of SPIR on OPCs relies on RXRγ.

Results

In this study, we identified that SPIR, an FDA-approved drug, functions as an RXRγ agonist in OPCs. RXRγ was identified as a crucial factor of myelin production. Its activation promotes the differentiation of OPCs and enhances myelin generation. We confirmed the specificity of SPIR's target, demonstrating that SPIR facilitates OPC differentiation and myelin generation in a RXRγ-dependent manner. Our findings not only identify the RXRγ agonist to promote OPC differentiation but also provide new experimental evidence for expanding the clinical indications of SPIR.

Discussion

The promotion of OPC differentiation by SPIR in animal models suggests its potential for treating demyelinating diseases.

Introduction

Myelin, the multilamellar axonal sheath in the CNS and peripheral nervous system, enables saltatory conduction. Its destruction in demyelinating disorders such as multiple sclerosis (MS) causes debilitating neurologic deficits.¹ Remyelination, via recruitment, differentiation, and maturation of oligodendrocyte precursor cells (OPCs) into myelin-forming oligodendrocytes, provides endogenous repair.^2,3 However, this process fails in chronic disease, leaving axons demyelinated. Deciphering molecular checkpoints governing myelin regeneration is thus critical for remyelination therapeutics.

Retinoid X receptor γ (RXRγ), activated by retinoic acid (vitamin A–derived), drives oligodendrocyte development and myelination.⁴ Yet, retinoic acid's poor solubility, photosensitivity, and toxicity limit its therapeutic use,^5,6 highlighting the need for synthetic, oligodendrocyte-selective RXRγ agonists with improved safety profiles.

Drug repurposing accelerates therapy development for demyelinating diseases. The success story of clemastine exemplifies this approach and provides a valuable roadmap for investigating other potential remyelinating agents. Clemastine exemplifies the preclinical studies in experimental autoimmune encephalomyelitis (EAE). The lysolecithin (LPC)-induced and cuprizone (CPZ)-induced demyelination models^7,8 demonstrated enhanced remyelination, improved visual evoked potential latencies, and increased optic nerve remyelination.^9,10 Critically, its Phase II ReBUILD trial showed sustained visual acuity gains in patients with MS after treatment.^11,12 Clemastine also pioneered quantitative remyelination imaging (myelin water fraction) while bexarotene validated magnetization transfer ratio, establishing clemastine as the repurposing benchmark.^13,14

Building on this, spironolactone (SPIR), an FDA-approved diuretic and antiandrogen medication,¹⁵ emerges as a candidate remyelinating agent via its novel RXRγ agonism, governing oligodendrocyte differentiation.¹⁶ Previous research has shown that SPIR modulates NKG2D ligand and metastasis-suppressor genes via RXRγ in cancer cells,¹⁷ suggesting potential CNS promyelinating actions. Despite extensive clinical evaluation, no trials target demyelinating diseases,¹⁸ highlighting the need to investigate SPIR's RXRγ-mediated myelin repair.

Given the urgent need for remyelinating therapies, we explore SPIR's effects on OPC differentiation and myelination via RXRγ activation. Using CRISPR-Cas9 and in vitro pharmacology, we elucidate the molecular mechanisms underlying SPIR's remyelination potential.

Methods

Mice

The animal strains used in this study are all C57BL/6 mice, with adult mice being 6–8 weeks old, purchased from the Experimental Animal Center of Shaanxi Normal University. Neonatal mice experiments were conducted using mice within 4 days of birth, weighing 2–3 g, bred in the laboratory. LSL-Cas9 mice (stock no. 026175) were obtained from The Jackson Laboratory (Bar Harbor, ME). LSL-Cas9 mice injected with AAV-sgRxrg-P_Olig2-Cre virus were named RXRγ CKO mice (AAV-sgRxrg-P_Olig2-Cre-KO). LSL-Cas9 mice injected with control AAV-sgScramble-P_Olig2-Cre virus were named Ctrl mice (AAV-sgScramble-P_Olig2-Cre-KO). The mice were housed in a 12-hour light/dark cycle, at a temperature of 22–26°C, with regular diet and free access to water.

Ethics

All experimental procedures and protocols of mice were approved by the Committee on the Ethics of Animal Experiments of Shaanxi Normal University (No. SCXK-2022-043) and were performed in accordance with the approved institutional guidelines and regulations.

SPIR Treatment

SPIR (Sigma-Aldrich) was administered via drinking water, dissolved in 3% dimethyl sulfoxide +10% Kolliphor (Sigma-Aldrich) and then diluted with 5% dextrose to working concentration. Vehicle contained 3% dimethyl sulfoxide/10% Kolliphor/87% dextrose (w/v). Canrenone (Merck; 1 mM ddH₂O stock), and HX531 (TOCRIS; 2 mM ddH₂O stock) were diluted to 1 μM and 2 μM working concentrations, respectively.

LPC-Induced Demyelinating Injury

LPC-induced demyelination was performed in the corpus callosum (CC) of 6-week-old C57BL/6 mice as described.^19,20 Brain tissue carrying the lesions was harvested at 7 (prophylactic) and 14 (therapeutic) days postlesion (DPL).

CPZ-Induced Demyelinating Injury

Adult mice (6–8 weeks) received 0.2% CPZ chow for 6 weeks to induce acute demyelination.²¹ After 6 weeks, normal chow was provided for 2 weeks to initiate spontaneous remyelination.

Behavioral Assessments

Beam Walking

Mice walked along a 100-cm-long, 2-cm-diameter stainless-steel beam elevated 60 cm; latency to traverse (60 seconds max) was recorded and averaged across 3 trials.

Wire Hang Test

Mice were placed at the midpoint of a 2-mm cotton rope suspended 1 m between 2 platforms; latency to reach either platform (max 60 seconds) was recorded, and the mean of 3 trials was calculated.

Rotarod

Before formal testing on mice using the fatigue rod apparatus, a 3-day training period was implemented at a speed of 5 rpm. During the testing phase, mice were placed on an accelerating rotarod that increased by 1 rpm each second for 5 minutes; latency to fall was recorded, and the average of 3 trials was calculated.

Cell Culture

Primary OPCs were isolated and cultured as previously described.^20,22,23 Cells were maintained in a proliferation medium containing Dulbecco's Modified Eagle Medium/F12 (11320033, ThermoFisher Scientific) supplemented with 20 ng/mL of PDGF-AA, 2% B27, 20 ng/mL of bFGF, 2 mM GlutaMAX, and 1% N2. For differentiation, the proliferation medium was replaced with the differentiation medium consisting of Dulbecco's Modified Eagle Medium/F12 supplemented with 30 ng/mL of thyroid hormone (T3), 2% B27, 50 ng/mL of sonic hedgehog (Shh), 1% N2, and 50 ng/mL of Noggin. OPCs were treated with vehicle or SPIR for 72 hours prior to immunostaining analysis.

Histology and Immunofluorescent Analysis

Immunohistochemistry

Brain tissues were sectioned coronally (6–8 μm) using a freezing microtome (Leica). Sections were blocked for 60 minutes and then incubated overnight (4°C) with the following primary antibodies: rabbit anti-myelin basic protein (MBP) (PA1-10008, ThermoFisher), rabbit anti-PDGFRα (ab203491, Abcam), mouse anti-CNPase (ab6319, Abcam), rabbit anti-Olig2 (ab109186, Abcam), mouse anti-CC1 (ab16794, Abcam), guinea pig anti-Sox10 (OB-GP001-01, Oasis), mouse anti-glial fibrillary acidic protein (GFAP) (MAB360, Millipore), and rabbit anti-IBA1 (019–19741, Wako Pure Chemical Industries). After thorough washing, sections were incubated with appropriate Alexa Fluor 488, 594, or 405 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc). Myelin was visualized using the FluoroMyelin fluorescent myelin stain kit (F34651, Invitrogen). Demyelination was quantified on a scale of 0–3 as previously described.²⁴

Immunocytochemistry

OPCs on coverslips were fixed with 4% paraformaldehyde (30 minutes, room temperature), blocked in phosphate buffered saline containing 10% horse serum (60 minutes), and incubated overnight with primary antibodies (4°C). Secondary antibody incubation was performed the next day.

Imaging and Analysis

Fluorescent images were captured using a Nikon Eclipse Ci-s inverted fluorescence microscope. Quantitative image analysis was performed using Image-Pro or Fiji software.

Transmission Electron Microscopy

For transmission electron microscopy (TEM), brain tissue underwent primary fixation in 3% glutaraldehyde and secondary fixation in 1% osmium tetroxide. Subsequent dehydration was performed using an acetone gradient. Tissues were then infiltrated and embedded using a blend of dehydrating solvent and Epon812 epoxy resin. Sections of 60–90 nm thickness were generated with an ultramicrotome, mounted onto copper grids, and contrasted with uranyl acetate followed by lead citrate. Grids were imaged using a JEM-1400-FLASH transmission electron microscope.

Molecular Docking

The molecular docking was performed as previously reported.²⁵ The protein structure of mus RXRγ (Swiss Prot database ID: P626MJ7) and the compound structure of SPIR were used for molecular docking.

gRNA Design

Two guide RNAs (gRNAs) were designed as previously described.²⁶ The sequences were 5′-CAC​CGA​GAG​CTC​ATC​TAC​ACC​TGT-3′ and 5′-CAC​CGG​TAC​TGG​CAG​CGG​TTG​CGC-3′, which were cloned into a vector.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 9.0. Data are presented as mean ± SEM. Significance (p < 0.05) was determined by 2-tailed t tests or analysis of variance (ANOVA) with appropriate multiple comparisons.

Single-Cell Data Sets

Human single-cell/nucleus RNA sequencing data were obtained from published data sets with accession number GSE118257. The mouse single-cell/nucleus RNA sequencing data were obtained from Mouse Cell Atlas²⁷ and Single-Cell Portal.²⁸ Dot plot visualization was performed in R (v4.4.3).

Data Availability

All data needed to evaluate the conclusions in the article are presented here.

Results

Expression Mode of RXRγ in the Process of Myelin Sheath Development and Demyelinating Lesions

To clarify the expression profile of RXRγ in myelin development and demyelinating conditions, we reanalyzed the GSE118257 data sets.²⁹ The findings revealed upregulated RXRG in MS-derived oligodendrocyte lineage cells, particularly in normal appearing white matter and active stages (Figure 1A). Mouse studies^27,28 confirmed developmental downregulation of Rxrg (Figure 1B), but upregulation in Alzheimer OPCs (eFigure 1A), indicating regenerative potential. We previously identified RXRγ as a PPARγ co-activator in remyelination.²² CC analysis at postnatal day 7 (P7), P14, and P60 (Figure 1C) showed that RXRγ and PPARγ double-positive cells declined from 80% (P7) to 40% (adulthood) in the oligodendrocyte lineage (Figure 1D). In LPC demyelination (eFigure 1, B and C), RXRγ and PPARγ double-positive cells increased at 7 DPL vs control and remained elevated at 14 DPL (eFigure 1, D and E), indicating that RXRγ⁺ OLs critically regulate myelination and remyelination.

Figure 1. Expression Mode of RXRγ and PPARγ in the Process of Myelin Sheath Development and Demyelinating Lesions.

(A) A dot plot showing expressions of RXRG, PPARG, and PPARGC1A in the cerebrocortex of the human brain based on the snRNA-seq data extracted from the GSE118257. RXRG is indicated with asterisk (*). (B) Representative graph showing the Rxrg expression across mouse development stages. Data were extracted from the Mouse Cell Atlas.²⁷ (C and D) Representative image showing PPARγ (red) and RXRγ (blue) expression in oligodendrocyte lineage cells (Olig2, green) across young and adult mice. Scale bar, 20 μm. (N = 3 mice for each group). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. One representative experiment of 3 is shown. Statistical significance was determined using the unpaired Student t test. RXRγ = retinoid X receptor γ.

SPIR Acts as an RXRγ Agonist, Promoting the Generation of Myelin Sheaths

SPIR, an FDA-approved diuretic with established safety, was investigated as an RXRγ agonist for remyelination. Molecular docking revealed a strong SPIR-RXRγ binding affinity (−10.2 kcal/mol), forming hydrogen bonds with ARG394, alkyl interactions with LEU420/PRO424/LEU423, and pi-alkyl interactions with TYR5/PHE8 (eFigure 2A). OPC differentiation can be divided into 4 stages in the cultured model, including proliferation, migration, differentiation, and ability to myelinate, which can be distinguished by their morphologic features.³⁰ In our in vitro study, SPIR dose-dependently doubled the CNPase⁺/PDGFRα⁺ cell ratio (Figure 2, A and B) and increased MBP⁺ cells (eFigure 2, B and C), indicating enhanced OPC differentiation. SPIR reduced stage 1–2 cells but increased stage 3–4 cells, confirming maturation promotion.

Figure 2. Spironolactone Promoting the OPC Differentiation and Generation of Myelin Sheaths.

(A) Immunostaining image showing PDGFRα (green) and CNPase (red) in cultured oligodendrocyte lineage cells after treatment with SPIR for 3 days. Scale bar, 50 μm (N = 8 cultures for each group). (B) Quantitative data of (A) showing the percent of PDGFRα⁺ cells and CNPase⁺ cells in the cultured medium after SPIR treatment. (C) Representative images showing FluoroMyelin staining (green) in the spinal cord slice and brain slice on several dosages. Scale bar, 200 μm (overview) and 50 μm (enlarged) (N = 5 mice for each group). (D) Quantitation of FluoroMyelin staining in the spinal cord and brain slice in (C). Data are represented as mean ± SEM. One representative experiment of 3 is shown. Statistical significance was determined using the unpaired t test, *p < 0.05, **p < 0.01. OPC = oligodendrocyte precursor cell; SPIR = spironolactone.

Given SPIR's dose-dependent promotion of OPC differentiation in vitro, we assessed its effects on oligodendrocyte development in vivo. Guided by the established interspecies dose conversion between humans and mice,³¹ we evaluated 3 doses of SPIR, 6.25 mg/kg (low), 31.25 mg/kg (medium), and 62.5 mg/kg (high), for their effects on myelin sheath regeneration in the mouse experiments. FluoroMyelin staining revealed that SPIR significantly enhanced myelin generation in both the brain and spinal cord of P14 mice, in a clear dose-dependent manner (Figure 2, C and D). In addition, high-dose SPIR increased CC1⁺ mature oligodendrocytes without altering PDGFRα⁺ OPCs in the brain and spinal cord (eFigure 3, A–H), indicating enhanced OPC differentiation. Consequently, myelin matured and motor performance on wire hang, beam walking, and rotarod improved (eFigure 4, A–C).

SPIR Promotes Remyelination in Both LPC-Induced and CPZ-Induced Demyelination Models

To explore the therapeutic potential of SPIR in promoting remyelination, we used both LPC-induced and CPZ-induced demyelination models. In the LPC-induced mouse model, we implemented 2 distinct dosing regimens: prophylactic and therapeutic (Figure 3A). In the prophylactic group, SPIR pretreatment reduced MBP⁺ demyelinated areas (eFigure 5, A and B) and increased Olig2⁺ lineage cells/CC1⁺ mature oligodendrocytes in the CC (eFigure 5, C–E), indicating RXRγ-dependent protection of oligodendrocytes. In the therapeutic group, SPIR administration after demyelination significantly enhanced remyelination, as evidenced by increased FluoroMyelin staining intensity in lesion areas (Figure 3B) and the number of SOX10⁺CC1⁺ double-positive mature oligodendrocytes (Figure 3C). These findings collectively demonstrate that SPIR promotes both neuroprotection and active myelin repair in demyelinating conditions.

Figure 3. SPIR Promotes Remyelination in the LPC-Induced Demyelination Model.

(A) LPC-induced demyelination workflow. (B) FluoroMyelin staining showing remyelination in the lesion core at 14 days after injury, along with SPIR treatment 5 days after LPC lesion. Scale bar, 200 μm. Vehicle, N = 3 mice for each group; post-SPIR, N = 6 mice for each group. (C) Quantitation of FluoroMyelin intensity in the lesion core region in (B). (D) Representative image showing the SOX10 (green) and CC1 (red) expressing cells in the lesion core site. Scale, 20 μm. Vehicle, N = 3 mice for each group; post-SPIR, N = 6 mice for each group. Data are represented as mean ± SEM. One representative experiment of 3 is shown. Statistical significance was determined using the unpaired t test, *p < 0.05, **p < 0.01. LPC = lysolecithin; SPIR = spironolactone.

In the CPZ demyelination model, we concurrently compared myelin alterations during demyelination and remyelination stages (Figure 4A). FluoroMyelin staining at week 6 showed that SPIR co-treatment significantly reduced demyelination in CPZ-exposed mice while increasing CC1⁺ oligodendrocytes, indicating protection against CPZ-induced damage (Figure 4, B and C). During week 6 + 2 of SPIR treatment, demyelination scores based on FluoroMyelin staining were lower in the SPIR-treated group (eFigure 6A), with more CC⁺ cells (eFigure 6B), indicating better remyelination in the mice after SPIR treatment. Moreover, electron microscopy revealed better remyelination and higher myelin integrity in mice treated with SPIR (Figure 4D). Motor function tests also suggested improved coordination in mice treated with SPIR (eFigure 6, C–E). These results indicate that treatment with the RXRγ agonist SPIR during demyelination in mice promotes OPC differentiation and remyelination.

Figure 4. SPIR Promotes Remyelination in the CPZ-Induced Demyelination Model.

(A) The workflow of the CPZ-induced demyelination model. (B) FluoroMyelin staining showing myelin sheath in the CPZ-induced model after SPIR treatment; CC1 staining showing mature oligodendrocyte cell numbers in the corpus callosum. Scale bar, 200 μm of FluoroMyelin staining and 50 μm of CC1 staining (N = 5 mice per group). (C) Quantitation of the FluoroMyelin-positive area in (H). (D) Transmission electron microscope image showing the myelin. The red arrow indicates myelin sheath. (N = 5 mice per group). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01. One representative experiment of 3 is shown. Statistical significance was determined using the unpaired Student t test. CPZ = cuprizone; SPIR = spironolactone.

Microglia and astrocyte activation served as damage indicators. In the CPZ model, SPIR-treated mice showed significantly reduced Iba-1⁺ microglial and GFAP⁺ astrocytic activation in the CC during peak demyelination (Figure 5, A and B) and remyelination (Figure 5C) vs controls, suggesting that SPIR mitigates CPZ-induced glial reactivity and confers partial protection against myelin loss.

Figure 5. SPIR Reduced Astrocyte and Microglia Overactivation During Both Demyelination and Remyelination Stages.

(A) Representative image showing GFAP (red) and IBA1 (green) expressions at the demyelinating stage in the CPZ-induced model. Scale bar, 50 μm. N = 5 mice per group. (B) Quantitation of GFAP+ cell numbers in (A). (C) Quantitation of IBA1⁺ cell numbers in (A). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01. One representative experiment of 3 is shown. Statistical significance was determined using the one-way ANOVA. CPZ = cuprizone; SPIR = spironolactone.

SPIR Acts to Promote Myelin Sheath Formation in an RXRγ-Dependent Manner

To confirm RXRγ-dependent myelination by SPIR, we compared effects of the mineralocorticoid receptor (MR) antagonist canrenone and RXRγ antagonist HX531 on OPC differentiation in vitro (Figure 6A). SPIR enhanced OPC differentiation into CNPase⁺ oligodendrocytes, an effect that was inhibited by RXRγ antagonist HX531 but unaffected by MR antagonist canrenone, confirming RXRγ-dependent action (Figure 6B). To further investigate this, we generated an RXRγ conditional knockout (CKO) mouse model using AAV-sgRXRγ-P_Olig1-Cre in LSL-Cas9-GFP mice and induced demyelination with CPZ (eFigure 7A). Immunostaining for Sox10 showed that AAV-sgRXRγ-P_Olig1-Cre was effective in oligodendrocyte lineage cells, and RXRγ was knocked out in Sox10⁺ cells (eFigure 7, B and C). In addition, CC1 staining revealed that SPIR promoted OPCs to differentiate into mature oligodendrocytes, but this effect was abolished in the remyelination stage of the CPZ-induced model in the RXRγ CKO mice (eFigure 7, D and E).

Figure 6. SPIR Acts to Promote Myelin Sheath Formation in an RXRγ-Dependent Manner.

(A) Representative image showing the PDGFRα and CNPase expression and relative percentage in primary OPCs after challenged with SPIR, canrenone, and HX531. Scale bar, 50 μm. (N = 8 per group). (B) Quantitative data of (A) showing the percent of PDGFRα⁺ cells and CNPase⁺ cells in the cultured medium. (C) MBP staining showing myelin sheath in RXRγ CKO and SPIR-treated mice. Scale bar, 200 μm. (N = 5 mice per group). (D) Quantitation of MBP-positive area in (C). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One representative experiment of 3 is shown. Statistical significance was determined using the one-way ANOVA. OPC = oligodendrocyte precursor cell; RXRγ = retinoid X receptor γ; SPIR = spironolactone.

MBP staining at 8 weeks showed modest remyelination in scramble-virus controls, markedly enhanced by SPIR, whereas RXRγ CKO mice failed to remyelinate despite SPIR treatment (Figure 6, C and D). Concordantly, behavioral assays (wire hang, beam walking, rotarod) revealed SPIR-mediated motor recovery that was abolished in RXRγ CKO mice (eFigure 7, F and H), demonstrating that SPIR promotes OPC differentiation and remyelination in an RXRγ-dependent manner.

Discussion

MS is a chronic inflammatory demyelinating disorder in adults, and progressive forms remain inadequately treated.³² Demyelination also contributes to neurodegenerative diseases, including Alzheimer disease,³³ suggesting that OPC gene dysregulation is a shared oligodendrocyte lineage signature. Therapeutic strategies, therefore, encompass anti-inflammatory, neuroprotective, and remyelination-promoting approaches.³⁴ We demonstrate that the FDA-approved drug SPIR, via RXRγ activation in OPCs, directly drives their differentiation and myelin regeneration (Figure 7), offering a clinically translatable remyelination therapy.

Figure 7. Schematic of Spironolactone Target to RXRγ to Promote Myelin Sheath Regeneration.

During early development, RXRγ is highly expressed but declines as myelin matures. After demyelinating injury, RXRγ is upregulated, indicating its critical role in myelin formation and regeneration. The FDA-approved drug SPIR acts as an RXRγ agonist, promoting oligodendrocyte precursor cell differentiation and myelin regeneration in an RXRγ-dependent manner (by Figdraw). RXRγ = retinoid X receptor γ; SPIR = spironolactone.

Clinically approved SPIR, safe in conditions such as heart failure,³⁵ binds RXRγ with high affinity. It dose-dependently accelerates OPC differentiation and enhances myelin formation in developing and adult demyelinated mice, without affecting OPC numbers, yielding more mature oligodendrocytes and improved myelin completeness. The myelination-promoting dose in mice (31.25 mg/kg, ∼200 mg/d human equivalent) falls within the established clinical range (50–400 mg). Crucially, SPIR's prodifferentiation effect requires RXRγ (absent in RXRγ-deficient mice) and is independent of its known MR antagonism.¹⁵ In addition, SPIR reduces microglial and astrocyte activation/clustering, suggesting that it mitigates neuroinflammation and stabilizes CNS homeostasis, consistent with reports of alleviating Aβ-induced deficits and inhibiting microglial overactivation.^36,37 The optimal dose for patients with MS requires clinical confirmation.

Our findings were obtained in the LPC-induced and CPZ-induced demyelinating models, which recapitulate demyelination but lack the chronic inflammatory milieu characteristic of MS. Consequently, the next critical step is to validate the remyelinating efficacy and safety of SPIR in EAE mice. This evaluation should extend beyond standard locomotor assays to include visual electrophysiology, thereby providing a functional readout of myelin repair within inflammatory lesions. Parallel translational biomarkers are also warranted. Recent work has established neurofilament light chain (NfL) as a sensitive, blood-accessible indicator of neuroaxonal injury and demyelination severity; incorporating longitudinal plasma NfL measurements will strengthen the preclinical-clinical bridge.¹² Finally, mechanistic depth is essential. Elucidating the downstream effectors of RXRγ—specifically, the molecular intermediates through which SPIR/RXRγ drives OPC differentiation and subsequent remyelination—will refine our understanding and guide rational therapeutic optimization.

Glossary

CC

corpus callosum

CPZ

cuprizone

DPL

days postlesion

EAE

experimental autoimmune encephalomyelitis

gRNA

guide RNA

LPC

lysolecithin

MR

mineralocorticoid receptor

MS

multiple sclerosis

NfL

neurofilament light chain

OPC

oligodendrocyte precursor cell

RXRγ

retinoid X receptor γ

SPIR

spironolactone

TEM

transmission electron microscopy

Author Contributions

Q-Q. Sun: drafting/revision of the manuscript for content, including medical writing for content. R-S. Ai: major role in the acquisition of data. N-N. Chai: drafting/revision of the manuscript for content, including medical writing for content; analysis or interpretation of data. B. Han: analysis or interpretation of data. M-Y. Bao: analysis or interpretation of data. Y-B. Li: analysis or interpretation of data. G-X. Ma: analysis or interpretation of data. L-J. Wang: analysis or interpretation of data. Z. Qian: analysis or interpretation of data. X. Li: study concept or design. Y. Zhang: study concept or design.

Study Funding

This study was supported by the Chinese National Natural Science Foundation (Grant Nos. 92268118, 82271199, and 82471421), the Fundamental Research Funds for the Central Universities (Grant Nos. GK202501001 and GK202506007), Shaanxi Provincial Department of Education Basic Science Research Project (Grant No. 23HQ057), and Innovation Capability Support Program of Shaanxi (Program No. 2021PT-055).

Disclosure

The authors report no relevant disclosures. Go to Neurology.org/NN for full disclosures.