Evaluating the effects of pro-myelinating drugs on motor function and myelination in a zebrafish model of genetic leukoencephalopathy.

Evaluating the Effects of Pro-Myelinating Drugs on Motor Function and Myelination in a Zebrafish Model of Genetic Leukoencephalopathy - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Neurosci Lett . Author manuscript; available in PMC: 2026 May 31. Published in final edited form as: Neurosci Lett. 2025 May 31;862:138280. doi: 10.1016/j.neulet.2025.138280 Evaluating the Effects of Pro-Myelinating Drugs on Motor Function and Myelination in a Zebrafish Model of Genetic Leukoencephalopathy Shreya Banerjee 1 , Yasmeen Berry 1 , Emily Fisher 1 , Ryan Thummel 1 Author information Article notes Copyright and License information 1 Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine, Detroit, MI 48201 Present/permanent address: 540 Canfield St., Detroit, MI 48201 Author Contributions: SB, YB, and EF performed all of the experiments, collected and analyzed the data, and performed the statistical analysis. SB and YB wrote the first draft of the manuscript. RT was involved in experimental design, provided reagents, and edited the manuscript. ✉ Corresponding Author: Ryan Thummel, Ph.D. Issue date 2025 Jul 27. PMC Copyright notice PMCID: PMC12289411  NIHMSID: NIHMS2088291  PMID: 40456507 The publisher's version of this article is available at Neurosci Lett Abstract Genetic leukoencephalopathy (gLE) is a white matter disorder affecting the central nervous system, causing hypomyelination, developmental delays, motor deterioration, and cognitive, visual, and hearing impairments. Its clinical variability makes diagnosis challenging. A novel homozygous missense mutation, p.Cys846Gly in vacuolar protein sorting 11 ( vps11 ), has been linked to infantile-onset gLE in humans. A zebrafish vps11 mutant model was developed to replicate gLE-like hypomyelination and sensorimotor deficits. This study investigates the effects of Clemastine, a pro-myelinating drug, on motor function and myelination in zebrafish larvae with mutations in the vps11 gene. We exposed zebrafish larvae to this drug during a critical period of early nervous system development, from 2 to 4 days post-fertilization (dpf), and assessed visuomotor responses at 7 dpf. Although Clemastine significantly increased the number of oligodendrocytes, it failed to improve visuomotor function in vps11 mutants. These findings imply that increasing oligodendrocyte numbers does not necessarily result in improved behavioral responses in vps11 mutants. Keywords: Vacuolar Protein Sorting 11 (Vps11), zebrafish, genetic leukoencephalopathy (gLE), neurodevelopment, visuomotor behavior, myelin, HOPS, CORVET INTRODUCTION Genetic leukoencephalopathy (gLE) refers to a diverse group of heritable central nervous system disorders marked by multifocal white matter abnormalities, often presenting in early childhood with symptoms like motor decline, cognitive impairment, and sensory neuropathy [ 1 ]. Diagnosing these heterogeneous conditions is challenging due to their varying clinical presentations and frequently unknown genetic causes [ 2 ]. Advances in MRI and next-generation sequencing, including whole exome sequencing, have improved diagnostic accuracy and the identification of new genetic variants [ 1 , 3 ]. A newly identified homozygous missense mutation, p.Cys846Gly in the Vacuolar Protein Sorting 11 ( VPS11 ) gene, has been associated with infantile-onset genetic leukoencephalopathy (gLE) in individuals of Ashkenazi Jewish (AJ) descent [ 4 , 5 ]. Affected patients display hallmark gLE symptoms, including hypomyelination, developmental delays, visuomotor deficits, hypotonia, dystonia, seizures, cortical blindness, optic atrophy, and cognitive impairment [ 5 , 6 ]. Moreover, the identical C846G mutation, with a carrier rate of 1:250 in the AJ population, suggests that it may be a founder mutation [ 5 ]. The discovery of a link between VPS11 dysfunction and myelin abnormalities highlights the importance of further research on the role of VPS11 in myelination. The Vps11 gene is highly conserved across species [ 7 ]. In vertebrates, the first mutant model was reported in zebrafish, known as the vps11 platinum (plt) line [ 5 , 8 ]. This model exhibits some gLE phenotypes, including progressive hypomyelination, visuomotor impairment, and loss of vision [ 5 ]. However, non-gLE systemic abnormalities are also observed, including a significantly shorter lifespan, pericardial edema, hepatomegaly, and a hypopigmentation phenotype [ 5 , 8 ]. These latter phenotypes likely result from differences in fish vs human embryonic development. Still, as a vertebrate model of loss of Vps11 function, the vps11(plt) model provides a unique opportunity for testing if pro-myelinating compounds can rescue the hypomyelination defect and help in functional recovery. Deficiencies in myelin and the cells in the CNS that produce it, oligodendrocytes, are key contributors to various neurodegenerative conditions, including leukoencephalopathies, multiple sclerosis, multiple system atrophy, Alzheimer’s disease, and Parkinson’s disease [ 9 , 10 ]. Given the complexity of myelin development and maintenance, different neurological disorders exhibit diverse disruptions in myelin function [ 9 ]. Studies investigating new drugs or repurposing existing ones have highlighted several potential treatments to improve myelination [ 11 – 14 ]. Clemastine, an FDA-approved antihistamine known for its muscarinic receptor antagonist properties, has been shown in previous studies to promote oligodendrocyte differentiation and myelin repair [ 15 ]. Specifically, Clemastine enhances functional recovery in demyelinated mouse models of multiple sclerosis, schizophrenia, and spinal cord injury [ 16 ]. Additionally, Clemastine has been effective in conditions like hypoxic brain injuries and demyelination from optic neuropathy [ 17 – 19 ]. Clemastine have been shown to partially alleviate hypomyelination in a zebrafish larval model but subsequent functional recovery has not been tested in progressive hypomyelination models in any species [ 20 ]. Herein, we tested this compound for its ability to rescue hypomyelination and associated sensorimotor defects caused by loss of Vps11 function in zebrafish. MATERIALS AND METHODS Animal models Zebrafish lines and maintenance: The following zebrafish lines were used for this study: vps11(plt) wsu1 (referred to as vps11(plt)) [ 8 ] and Tg(olig2:egfp);vps11(plt) mutant lines (referred to as olig2:egfp/plt )), their associated heterozygous and wild-type ( AB strain) siblings. Fish were fed a combination of brine shrimp and dried flake food three times daily and maintained at 28.5 °C on a 14 hr light (250 lux): 10 hr dark cycle [ 21 ]. All animal care and experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee at Wayne State University School of Medicine. Wholemount imaging Fish were bred from heterozygous parents and larvae were raised under standard conditions. 7 dpf larvae were anesthetized using 1:1000 2-Phenoxyethanol (2-PE) (Cat. no. 77699-250ML, Sigma-Aldrich) and mounted on 25 mm glass cover slips in 2% low melting point agarose (Cat. no. V2111, Promega Corporation, WI) dissolved in 1:1000 2-PE. Cover slips containing embedded larvae were immersed in 1:1000 2-PE, and images were collected using a Zeiss LSM 780 microscope. Clemastine treatment and Behavioral Analysis A 500 μM stock solution of Clemastine Fumerate (CLM; cat. no. S1847, Selleck Chemical, Houston, TX) in 0.1% DMSO was diluted in embryo medium to 500 nM Clemastine in 0.1% DMSO based on a previous zebrafish study [ 20 ]. Controls included untreated (i.e. embryo medium only) and 0.1% DMSO in embryo medium. 2 dpf larvae were segregated based on hypopigmentation phenotype under stereo microscope (Leica, M165 FC, Bannockburn, IL) and manually dechorionated. Equal number of mutants and siblings were incubated with the Clemastine and two controls in a 6-well plate for 2 days, with daily solution changes as previously described [ 20 ]. Larvae were then washed 3X with system water, maintained in system water until 6 dpf, and then transferred individually in 24-well plates for 24 hr acclimation prior to behavior analysis. Behavior analysis was performed at 7 dpf as previously described [ 8 , 21 ]. Briefly, fish were transferred in the 24-well plate into the DanioVision Observation Chamber, subjected to a 12 min acclimation period in the dark, followed by four alternating periods of 3 min light (10,500 lux) and 3 min dark. Response to stimulation was recorded as distance moved (cm) during each 3 min stimulus period using the EthoVision XT13 software. RNA isolation and quantitative Real-Time PCR RNA was isolated from 7 dpf mutant or control larvae (n = 10 per/sample) using the Direct-zol RNA MicroPrep kit (Zymo Research, Irvine CA), according to the manufacturer protocol. cDNA (SuperScript IV VILO Master Mix [Cat. no. 11756050, Invitrogen]) was diluted to a final concentration of 250 ng/μL in nuclease free water and 4 μL was used in the quantitative Real-Time PCR (qPCR) reaction. qPCR was carried out in technical and biological triplicate in a 20 μL reaction using SYBR green reagent (Cat. no. 4309155, Applied Biosystems, CA) on a MicroAmp Fast Optical 96-well reaction plate (Cat. no. 4346907, Applied Biosystems, CA), run on an Applied Biosystems 7500 Real-Time PCR system. qPCR primers used were as follows: mbpb Forward: 5’-GCGCTACCTAAAAGCAGGAA-3’, Reverse: 5’-AGATTTTGGCTGCTCGCCT-3’; gpia Forward: 5’-TCCAAGGAAACAAGCCAAGC-3’, Reverse: 5’-TTCCACATCACACCCTGCAC-3’. Statistical Analysis The number of EGFP+ cells in olig2:egfp/plt 7 dpf larvae were quantified manually; treatment groups were blinded to the viewer. Sample sizes included embryo medium (n = 7), 0.1% DMSO (n = 9), and 500 nM CLM in 0.1% DMSO (n = 9). Statistical differences between the groups were determined by a one-way ANOVA with a post-hoc Tukey test, using p<0.05 as a significant cut-off. The ΔΔCT method [ 22 ] was used to calculate mbp expression, with normalization to gpia as the housekeeping gene. Statistical differences between the groups were determined by a one-way ANOVA with a post-hoc Tukey test, using p<0.05 as a significant cut-off. For behavioral analysis, statistical differences between the mutants, siblings, and treated groups were analyzed using data from the first round of light and dark conditions. Statistical differences in Figure 1G were calculated using a Student’s t-test with a p<0.05 as a cutoff for significance. Statistical differences in Figure 1H – I were calculated using a one-way ANOVA in each light/dark condition followed by a post-hoc Tukey test, using p<0.05 as a cutoff for significance. Figure 1: Clemastine (CLM) effects on oligodendrocyte number, mbp expression, and visuomotor function in vsp11(plt) mutants. Open in a new tab (A) Schematic depiction of methods, created using BioRender. (B) Representative image of olig2:egfp/plt 7 dpf sibling larvae treated with embryo medium showing EGFP+ oligodendrocytes along the spinal column. (C) Representative image of olig2:egfp/plt 7 dpf sibling larvae treated with 0.1% DMSO showing EGFP+ oligodendrocytes along the spinal column. (D) Representative image of olig2:egfp/plt 7 dpf sibling larvae treated with 500 nM CLM in 0.1% DMSO showing EGFP+ oligodendrocytes along the spinal column. (E) Graph representing average number of EGFP+ cells in olig2:egfp/plt 7 dpf sibling larvae in each treatment group. Error bars indicate SEM. (F) Graph representing average fold-change of mbp expression in vps11(plt) larvae at 7 dpf post treatment with 500 nM CLM in 0.1% DMSO. Error bars indicate SEM. (G) Graph representing average distance travelled by vps11(plt) larvae (n=31) and siblings (n=32) at 7 dpf in 4 alternating light/dark cycles of 3 min each post treatment with embryo medium. (H) Graph representing average distance travelled by vps11(plt) larvae at 7 dpf in 4 alternating light/dark cycles of 3 min each post treatment with 500 nM Clemastine (CLM) in 0.1% DMSO. (I) Graph representing average distance travelled by siblings and vps11(plt) in the first light and dark period (DMSO siblings, n=31; DMSO mutants, n=32; CLM siblings, n=32; CLM mutants, n=22. RESULTS Clemastine treatment increased oligodendrocyte number, but did not increase mbp expression in vps11(plt) 7 dpf larvae Zebrafish larvae were incubated in 500 nM Clemastine for 2 days. The following treatment groups were used: olig2:egfp/plt mutant larvae incubated in embryo medium ( Fig. 1B ), 0.1% DMSO ( Fig. 1C ), or 500 nM Clemastine in 0.1% DMSO ( Fig. 1D ). Consistent with a previous report [ 20 ], we observed that 7 dpf larvae treated with Clemastine contained a significantly higher number of oligodendrocytes in the body flank when compared with the control treatment groups ( Fig. 1E , p=0.0025). However, mbp transcript levels between the treatment groups were not significantly different ( Fig. 1F , p=0.43307) . These data demonstrate that Clemastine treatment induced an increase in number of oligodendrocytes, but not an increase in mbp gene expression. Clemastine treatment did not rescue visuomotor responses in vps11(plt) 7 dpf larvae To test the effect of Clemastine on behavioral outcomes, 7 dpf vps11(plt) larvae and control animals were subjected to the light/dark visuomotor stimulation test. First, as we previously reported [ 21 ], vps11(plt) larvae raised in embryo media showed significantly reduced distance travelled in dark conditions compared with siblings raised in embryo media ( Fig. 1G , p<0.0001) . However, when we compared mutants and siblings treated with Clemastine or DMSO, we found that Clemastine treatment failed to increase the distance travelled in the dark in either the vps11(plt) mutants or the sibling controls ( Fig. 1H – I , p=0.5744 and p=0.9656, respectively) . These data suggest that the increase in oligodendrocyte number observed with Clemastine treatment did not translate into increased visuomotor activity. DISCUSSION Multiple previous studies have taken advantage of the zebrafish model, including rapid embryonic development and the ability to use pharmacological compounds in small volumes, to test pro-myelinated drugs [ 11 , 13 , 14 ]. Outcome measurements typically include oligodendrocyte number and or Mbp expression using qPCR or immunolocalization [ 11 ]. In using these approaches to screen over one thousand compounds, including Clemastine, it has been identified that some aspects of myelination increase in zebrafish [ 11 ]. However, very few of these studies performed functional behavioral assays. Using the transgenic line olig2:egfp/plt , we found that Clemastine induced a significant increase in EGFP-positive oligodendrocytes in mutant animals ( Fig. 1E ). This is consistent with previous findings in zebrafish [ 11 ] and strongly suggests that Clemastine was successfully delivered to the larvae. Despite increasing oligodendrocyte numbers, we found no increase in mbp expression following Clemastine treatment ( Fig. 1F ). This is consistent with multiple recent findings in mammalian models that point to a highly complex coordination of oligodendrocyte-mediated myelination of CNS axons [ 20 ]. In fact, Almeida and colleagues recently showed that excess oligodendrocytes and myelin in both zebrafish and mice resulted in myelin mistargeting [ 23 ], suggesting potential limitations for therapeutic approaches that rely solely on promoting oligodendrogenesis. Consistent with this, we observed that Clemastine treatment did not improve sensorimotor responses in vps11(plt) mutants or their siblings. Based on these findings, we suggest two considerations for future analysis. First, previous studies indicated that Clemastine was effective in improving myelination in a model of damage/repair [ 16 ]. It is possible that expanded treatment windows or doses could result in improvement in our model. It is also possible that there are fundamental differences between how oligodendrogenesis promotes improved outcomes in damage models vs models of hypomyelination. Second, these findings point to the critical need for behavioral testing following any pro-myelinating treatment. Ultimately, the minimum standard for effective compounds is improved behavioral outcomes, not simply increasing oligodendrocyte number or mbp expression. CONCLUSION While Clemastine shows promise in increasing oligodendrocyte number, further investigation is needed to understand its limited impact on myelination and sensorimotor function in vps11 -related hypomyelination. Understanding how vps11 dysfunction contributes to these defects will aid the development of more targeted therapies. Significance statement: While Clemastine shows promise in increasing oligodendrocyte number, it did not improve sensorimotor function in a model of hypomyelination. Acknowledgments: The authors would like to thank Xixia Luo for assistance in zebrafish husbandry. Funding Sources: Support for this project was provided by R01 EY026551 (RT), P30 EY004068 (PI: Linda Hazlett), and a Rumble Graduate Fellowship (YB). Footnotes Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. REFERENCES [1]. Vanderver A, et al. , Whole exome sequencing in patients with white matter abnormalities. Ann Neurol, 2016. 79(6): p. 1031–1037. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] [2]. Vanderver A, et al. , Case definition and classification of leukodystrophies and leukoencephalopathies. Mol Genet Metab, 2015. 114(4): p. 494–500. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] [3]. 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