Cuprizone induces similar demyelination in male and female C57BL/6 mice and results in disruption of the estrous cycle

Multiple sclerosis is a demyelinating neurological disease that is influenced by gender, primarily reflected in greater susceptibility to disease development in women than in men. Cuprizone intoxication, an animal model that is used to study demyelination and remyelination, has been extensively characterized in male C57BL/6 mice. Here, we have undertaken a comprehensive characterization of the morphological and cellular processes that occur in female C57BL/6J mice during cuprizone‐induced demyelination and subsequent remyelination and compared them with age‐matched male mice. We find that the pattern of demyelination and remyelination is similar between genders and that there is little or no difference in the loss or repopulation of mature oligodendrocytes or accumulation of reactive glia. Furthermore, examination of αERKO and βERKO mice suggests that estrogen receptors do not affect the outcome for demyelination or remyelination. Interestingly, we found that cuprizone treatment disrupts estrous cyclicity in female mice, possibly interfering with potential hormone influences on demyelination and remyelination. Therefore, cuprizone‐induced demyelination in C57BL/6J mice may have limitations as a model for the study of sex differences. © 2009 Wiley‐Liss, Inc.     

Lack of interferon-beta leads to accelerated remyelination in a toxic model of central nervous system demyelination

Interferon-beta (IFN-β) is a pleiotropic cytokine that is known to modulate the immune response in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). Spontaneous remyelination and repair mechanisms in MS are mostly insufficient and contribute to clinical disability. Here, we investigated whether IFN-β has a potential in modifying the extent of de- and remyelination in a toxic model of CNS demyelination induced by the copper chelator cuprizone. IFN-β deficient (k/o) mice showed an accelerated spontaneous remyelination. However, the amount of remyelination after 6 weeks did not differ between the two groups. Demyelination in IFN-β k/o mice was paralleled by a diminished astrocytic and microglia response as compared with wildtype controls, whereas the accelerated remyelination was paralleled by an increased number of oligodendrocyte precursor cells (OPC) within the demyelinated lesion at the beginning of the remyelination phase. We hypothesize that the absence of IFN-β leads to more efficient recruitment and proliferation of OPC already during demyelination, thus allowing early remyelination. These results demonstrate that IFN-β is able to alter remyelination in the absence of an immune-mediated demyelination. The first two authors contributed equally to this work.     

Leukemia inhibitory factor signaling modulates both central nervous system demyelination and myelin repair

Leukemia inhibitory factor (LIF) receptor signaling limits the severity of inflammatory demyelination in experimental autoimmune encephalomyelitis, a T-cell dependent animal model of multiple sclerosis (MS) [Butzkueven et al. (2002) Nat Med 8:613-619]. To identify whether LIF exerts direct effects within the central nervous system to limit demyelination, we have studied the influence of LIF upon the phenotype of mice challenged with cuprizone, a copper chelator, which produces a toxic oligodendrocytopathy. We find that exogenously administered LIF limits cuprizone-induced demyelination. Knockout mice deficient in LIF exhibit both potentiated demyelination and oligodendrocyte loss after cuprizone challenge, an effect that is ameliorated by exogenous LIF, arguing for a direct beneficial effect of endogenous LIF receptor signaling. Numbers of oligodendrocyte progenitor cells in cuprizone-challenged mice are not influenced by either exogenous LIF or LIF deficiency, arguing for effects directed to the differentiated oligodendrocyte. Studies on the influence of LIF upon remyelination after cuprizone challenge fail to reveal any significant effect of exogenous LIF. The LIF-knockout mice do, however, display impaired remyelination, although oligodendrocyte replenishment, previously identified to occur from the progenitor pool, is not significantly compromised. Thus endogenous LIF receptor signaling is not only protective of oligodendrocytes but can also enhance remyelination, and exogenous LIF has therapeutic potential in limiting the consequences of oligodendrocyte damage.     

Upregulation of the stress-associated gene p8 in mouse models of demyelination and in multiple sclerosis tissues

Cuprizone-induced demyelination is a mouse model of multiple sclerosis (MS) as cuprizone-fed mice exhibit neuroinflammation and demyelination in the brain. Upon removal of cuprizone from the diet, inflammation is resolved and reparative remyelination occurs. In an Affymetrix GeneChip analysis, the stress-associated gene p8 was strongly upregulated (>10x) during cuprizone-induced demyelination but not remyelination. We verified this upregulation (>15x) of p8 in the CNS during demyelination by real-time polymerase chain reaction (PCR). This upregulation is brain-specific, as p8 is not elevated in the liver, lung, kidney, spleen, and heart of cuprizone-treated mice. We also localized the cellular source of p8 during cuprizone treatment, and further found elevated expression during embryogenesis but not in normal adult brain. Compared with wild-type controls, the death of oligodendrocytes in p8-/- mice is delayed, as is microglial recruitment to areas of demyelination. The corpus callosum of p8-/- mice demyelinates at a slower rate than wild-type mice, suggesting that p8 exacerbates CNS inflammation and demyelination. Enhanced expression of p8 is also observed in the spinal cords of mice with acute experimental autoimmune encephalomyelitis (EAE) induced by PLP139-151 peptide (10x). Increased expression is detected during disease onset and expression wanes during the remission phase. Finally, p8 is found upregulated (8x) in post-mortem tissue from MS patients and is higher in the plaque tissue compared with adjacent normal-appearing white and gray matter. Thus, p8 is an excellent candidate as a novel biomarker of demyelination.     

Cuprizone-Induced Demyelination as a Tool to Study Remyelination and Axonal Protection

In the brain of multiple sclerosis (MS) patients, the conduction block of axons due to demyelination and inflammation underlies early neurological symptoms, whereas axonal transection accounts for permanent deficits occurring during later disease stages. The beneficial function of myelin for the protection of the axonal compartment and network stability between neurons has been shown in numerous studies. Thus, rapid and adequate remyelination is an important factor for axonal patronage during neuroinflammatory conditions. In this review article, we discuss frequently used experimental in vivo and in vitro animal models to examine remyelination and repair in MS. The focus of the discussion is the relevance of the toxin model ‘cuprizone’ to study the pathology of demyelination and the physiology of remyelination. This also includes recent findings in this animal model which implicate that axonal damage is an ongoing process independent of the initiation of endogenous remyelination.     

The chemokine receptor CXCR2 is differentially regulated on glial cells in vivo but is not required for successful remyelination after cuprizone-induced demyelination

Unravelling the factors that can positively influence remyelination is one of the major challenges in multiple sclerosis research. Expression of the chemokine receptor CXCR2 on oligodendrocytes both in vitro and in MS lesions has suggested a possible role for CXCR2 in the recruitment of oligodendrocyte precursor cells (OPC). To investigate the function of CXCR2 during remyelination in vivo, we studied this receptor in cuprizone-induced demyelination and subsequent remyelination. We found that CXCR2 is constitutively expressed on OPC, whereas on macrophages/microglia CXCR2 is upregulated upon activation during demyelination. Hence, the expression of CXCR2 is differentially regulated in oligodendrocytes and macrophages/microglia. Furthermore, we subjected CXCR2-/- mice to the cuprizone model demonstrating that remyelination was not altered in comparison to wildtype controls. In addition, the number of OPC and the amount of microglial accumulation were similar in both CXCR2-/- and wildtype animals during the whole demyelination and remyelination process. These results suggest that despite expression on OPC and microglia CXCR2 plays only a minor role during remyelination.     

Astrocyte‐targeted production of interleukin‐6 reduces astroglial and microglial activation in the cuprizone demyelination model: Implications for myelin clearance and oligodendrocyte maturation

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system. Interleukin (IL)−6 is a pleiotropic cytokine with a potential role in MS. Here we used transgenic mice with astrocyte‐targeted production of IL‐6 (GFAP‐IL6Tg) to study the effect of IL‐6 in the cuprizone‐induced demyelination paradigm, which is an experimental model of de‐ and re‐myelination, both hallmarks of MS. Our results demonstrated that cuprizone‐treated GFAP‐IL6Tg mice showed a significant reduction in astroglial and especially microglial activation/accumulation in the corpus callosum in comparison with the corresponding cuprizone‐treated wild type (WT). Production of a key microglial attracting chemokine CXCL10, as well as CXCL1 and CCL4 was lower in cuprizone‐treated GFAP‐IL6Tg mice compared with cuprizone‐treated WT. Reduced microglial cell accumulation was associated with inefficient removal of degraded myelin and axonal protection in cuprizone‐treated GFAP‐IL6Tg mice, compared with WT mice at the peak of demyelination. In addition, transgenic production of IL‐6 did not alter initial oligodendrocyte (OL) apoptosis and oligodendrocyte precursor cell recruitment to the lesion site, but it impaired early OL differentiation, possibly due to impaired removal of degraded myelin. Indeed, a microglial receptor involved in myelin phagocytosis, TREM2, as well as the phagolysosomal protein CD68 were lower in cuprizone‐treated GFAP‐IL6Tg compared with WT mice. Our results show for the first time that astrocyte‐targeted production of IL‐6 may play a role in modulating experimental demyelination induced by cuprizone. Further understanding of the IL‐6‐mediated molecular mechanisms involved in the regulation of demyelination is needed, and may have implications for the development of future therapeutic strategies for the treatment of MS. GLIA 2016;64:2104–2119     

Mesenchymal stem cells do not exert direct beneficial effects on CNS remyelination in the absence of the peripheral immune system

Remyelination is the natural repair mechanism in demyelinating disorders such as multiple sclerosis (MS) and it was proposed that it might protect from axonal loss. For unknown reasons, remyelination is often incomplete or fails in MS lesions and therapeutic treatments to enhance remyelination are not available. Recently, the transplantation of exogenous mesenchymal stem cells (MSC) has emerged as a promising tool to enhance repair processes. This included the animal model experimental autoimmune encephalomyelitis (EAE), a commonly used model for the autoimmune mechanisms of MS. However, in EAE it is not clear if the beneficial effect of MSC derives from a direct influence on brain resident cells or if this is an indirect phenomenon via modulation of the peripheral immune system. The aim of this study was to determine potential regenerative functions of MSC in the toxic cuprizone model of demyelination that allows studying direct effects on de- and remyelination without the influence of the peripheral immune system. MSC from three different species (human, murine, canine) were transplanted either intraventricularly into the cerebrospinal fluid or directly into the lesion of the corpus callosum at two time points: at the onset of oligodendrocyte progenitor cell (OPC) proliferation or the peak of OPC proliferation during cuprizone induced demyelination. Our results show that MSC did not exert any regenerative effects after cuprizone induced demyelination and oligodendrocyte loss. During remyelination, MSC did not influence the dynamics of OPC proliferation and myelin formation. In conclusion, MSC did not exert direct regenerative functions in a mouse model where peripheral immune cells and especially T lymphocytes do not play a role. We thus suggest that the peripheral immune system is required for MSC to exert their effects and this is independent from a direct influence of the central nervous system.     

Demyelination in the juvenile period, but not in adulthood, leads to long-lasting cognitive impairment and deficient social interaction in mice

Background

Dysmyelination is hypothesized to be one of the causes of schizophrenic symptoms. Supporting this hypothesis, demyelination induced by cuprizone was recently shown to cause schizophrenia-like symptoms in adult rodents [Xiao L, Xu H, Zhang Y, Wei Z, He J, Jiang W, et al. Quetiapine facilitates oligodendrocyte development and prevents mice from myelin breakdown and behavioral changes. Mol Psychiatry 2008;13:697–708]. The present study asked if the timing of demyelination (i.e., juvenile period or adulthood) influenced abnormal behavior.

Methods

B57BL/6 mice were fed with 0.2% cuprizone either from postnatal day 29 (P29) to P56 (early demyelination group) or from P57 to P84 (late demyelination group), and then returned to normal mouse chow until P126, when the behavioral analysis was initiated.

Results

In both groups, the intake of cuprizone for 28 days produced massive demyelination in the corpus callosum by the end of the treatment period, and subsequent normal feeding restored myelination by P126. In a Y-maze test, the spatial working memory was impaired in both groups right after the cuprizone feeding ceased, consistent with previous studies, whereas only the early demyelination group exhibited impaired working memory after remyelination took place. In an open field test, social interactions were decreased in the early demyelination group, but not in the late group. Novel cognition and anxiety-related behaviors were comparable between the two groups.

Conclusions

Our findings suggest that the timing of demyelination has substantial impacts on behaviors of adult mice.     

Astrocyte-specific expression of a soluble form of the murine complement control protein Crry confers demyelination protection in the cuprizone model

Complement has been implicated as a potential effector mechanism in neurodegeneration; yet the precise role of complement in this process remains elusive. In this report, we have utilized the cuprizone model of demyelination-remyelination to examine the contribution of complement to disease. C1q deposition was observed in the corpus callosum of C57BL/6 mice during demyelination, suggesting complement activation by apoptotic oligodendrocyte debris. Simultaneously, these mice lost expression of the rodent complement regulatory protein, Crry. A soluble CNS-specific form of the Crry protein (sCrry) expressed in a transgenic mouse under the control of an astrocyte-specific promoter was induced in the corpus callosum during cuprizone treatment. Expression of this protein completely protected the mice from demyelination. Interestingly, sCrry mice had low levels of demyelination at later times when control mice were remyelinating. Although the sCrry transgenic mice had lower levels of demyelination, there was no decrease in overall cellularity, however there were decreased numbers of microglia in the sCrry mice relative to controls. Strikingly, sCrry mice had early recovery of mature oligodendrocytes, although they later disappeared. TUNEL staining suggested that production of the sCrry protein in the transgenic mice protected from a late apoptosis event at 3 weeks of cuprizone treatment. Our data suggest complement provides some protection of mature oligodendrocytes during cuprizone treatment but may be critical for subsequent remyelination events. These data suggest that temporal restriction of complement inhibition may be required in some disease settings.     

mTOR Signaling Regulates Metabolic Function in Oligodendrocyte Precursor Cells and Promotes Efficient Brain Remyelination in the Cuprizone Model

In demyelinating diseases, such as multiple sclerosis, primary loss of myelin and subsequent neuronal degeneration throughout the CNS impair patient functionality. While the importance of mechanistic target of rapamycin (mTOR) signaling during developmental myelination is known, no studies have yet directly examined the function of mTOR signaling specifically in the oligodendrocyte (OL) lineage during remyelination. Here, we conditionally deleted Mtor from adult oligodendrocyte precursor cells (OPCs) using Ng2-Cre^ERT in male adult mice to test its function in new OLs responsible for remyelination. During early remyelination after cuprizone-induced demyelination, mice lacking mTOR in adult OPCs had unchanged OL numbers but thinner myelin. Myelin thickness recovered by late-stage repair, suggesting a delay in myelin production when Mtor is deleted from adult OPCs. Surprisingly, loss of mTOR in OPCs had no effect on efficiency of remyelination after lysophosphatidylcholine lesions in either the spinal cord or corpus callosum, suggesting that mTOR signaling functions specifically in a pathway dysregulated by cuprizone to promote remyelination efficiency. We further determined that cuprizone and inhibition of mTOR cooperatively compromise metabolic function in primary rat OLs undergoing differentiation. Together, our results support the conclusion that mTOR signaling in OPCs is required to overcome the metabolic dysfunction in the cuprizone-demyelinated adult brain.SIGNIFICANCE STATEMENT Impaired remyelination by oligodendrocytes contributes to the progressive pathology in multiple sclerosis, so it is critical to identify mechanisms of improving remyelination. The goal of this study was to examine mechanistic target of rapamycin (mTOR) signaling in remyelination. Here, we provide evidence that mTOR signaling promotes efficient remyelination of the brain after cuprizone-mediated demyelination but has no effect on remyelination after lysophosphatidylcholine demyelination in the spinal cord or brain. We also present novel data revealing that mTOR inhibition and cuprizone treatment additively affect the metabolic profile of differentiating oligodendrocytes, supporting a mechanism for the observed remyelination delay. These data suggest that altered metabolic function may underlie failure of remyelination in multiple sclerosis lesions and that mTOR signaling may be of therapeutic potential for promoting remyelination.     

Reduced axonopathy and enhanced remyelination after chronic demyelination in fibroblast growth factor 2 (Fgf2)-null mice: differential detection with diffusion tensor imaging

Chronic central nervous system demyelinating diseases result in long-term disability because of limited remyelination capacity and cumulative damage to axons. Corpus callosum demyelination in mice fed cuprizone provides a reproducible model of chronic demyelination in which the demyelinating agent can be removed to test modifications that promote recovery and to develop noninvasive neuroimaging techniques for monitoring changes in myelin and axons. We used the cuprizone model in mice with genetic deletion of fibroblast growth factor 2 (Fgf2) to determine the impact of FGF2 on axon pathology and remyelination after chronic demyelination. We also evaluated the ability of quantitative magnetic resonance diffusion tensor imaging (DTI) to distinguish the corresponding pathological changes in axons and myelin during the progression of demyelination and remyelination. During the recovery period after chronic demyelination, Fgf2-null mice exhibited enhanced remyelination that was detected using DTI measures of radial diffusivity and confirmed by electron microscopic analysis of the proportion of remyelinated axons. Ultrastructural analysis also demonstrated reduced axonal atrophy in chronically demyelinated Fgf2-null versus wild-type mice. This difference in axon atrophy was further demonstrated as reduced immunohistochemical detection of neurofilament dephosphorylation in Fgf2-null mice. Diffusion tensor imaging axial and radial diffusivity measures did not differentiate Fgf2-null mice from wild-type mice to correlate with changes in axonal atrophy during chronic demyelination. Overall, these findings demonstrate that attenuation of FGF2 signaling promotes neuroprotection of axons and remyelination, suggesting that FGF2 is an important negative regulator of recovery after chronic demyelination.     

Endogenous cell repair of chronic demyelination

In multiple sclerosis lesions, remyelination typically fails with repeated or chronic demyelinating episodes and results in neurologic disability. Acute demyelination models in rodents typically exhibit robust spontaneous remyelination that prevents appropriate evaluation of strategies for improving conditions of insufficient remyelination. In the current study, we used a mouse model of chronic demyelination induced by continuous ingestion of 0.2% cuprizone for 12 weeks. This chronic process depleted the oligodendrocyte progenitor population and impaired oligodendrocyte regeneration. Remyelination remained limited after removal of cuprizone from the diet. Fibroblast growth factor 2 (FGF2) expression was persistently increased in the corpus callosum of chronically demyelinated mice as compared with nonlesioned mice. We used FGF2 mice to determine whether removal of endogenous FGF2 promoted remyelination of chronically demyelinated areas. Wild-type and FGF2 mice exhibited similar demyelination during chronic cuprizone treatment. Importantly, in contrast to wild-type mice, the FGF2 mice spontaneously remyelinated completely during the recovery period after chronic demyelination. Increased remyelination in FGF2 mice correlated with enhanced oligodendroglial regeneration. FGF2 genotype did not alter the density of oligodendrocyte progenitor cells or proliferating cells after chronic demyelination. These findings indicate that attenuating FGF2 created a sufficiently permissive lesion environment for endogenous cells to effectively remyelinate viable axons even after chronic demyelination.     

Platelet-derived growth factor promotes repair of chronically demyelinated white matter

In multiple sclerosis, remyelination becomes limited after repeated or prolonged episodes of demyelination. To test the effect of platelet-derived growth factor-A (PDGF-A) in recovery from chronic demyelination we induced corpus callosum demyelination using cuprizone treatment in hPDGF-A transgenic (tg) mice with the human PDGF-A gene under control of an astrocyte-specific promoter. After chronic demyelination and removal of cuprizone from the diet, remyelination and oligodendrocyte density improved significantly in hPDGF-A tg mice compared with wild-type mice. In hPDGF-A tg mice, oligodendrocyte progenitor density and proliferation values were increased in the corpus callosum during acute demyelination but not during chronic demyelination or the subsequent recovery period, compared with hPDGF-A tg mice without cuprizone or to treatment-matched wild-type mice. Proliferation within the subventricular zone and subcallosal zone was elevated throughout cuprizone treatment but was not different between hPDGF-A tg and wild-type mice. Importantly, hPDGF-A tg mice had reduced apoptosis in the corpus callosum during the recovery period after chronic demyelination. Therefore, PDGF-A may support oligodendrocyte generation and survival to promote remyelination of chronic lesions. Furthermore, preventing oligodendrocyte apoptosis may be important not only during active demyelination but also for supporting the generation of new oligodendrocytes to remyelinate chronic lesions.     

Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre

Exposure of young adult C57BL/6 mice to cuprizone in the diet initiated profound and synchronous demyelination of the corpus callosum, which was virtually complete by 4 weeks of exposure. Interestingly, even in the face of a continued exposure to cuprizone, there was spontaneous remyelination 2 weeks later. This remyelination preferentially involved smaller calibre axons. There was a suggestion of yet another cycle of demyelination (at 10 weeks) and remyelination (at 12 weeks), but by 16 weeks of exposure, the regenerative capacity was exhausted and the animals were near death. The relapsing-remitting pattern suggests this may be a useful model for certain human demyelinating disorders. In contrast to the above chronic model, the corpus callosum from mice exposed to cuprizone for only 6 weeks continued to remyelinate, with 67% of the axons being myelinated or remyelinated at 10 weeks. Interestingly, a significant reduction in the mean value for axonal diameter was observed during acute demyelination. Upon remyelination, however, the axonal calibre distribution returned to near-normal. In contrast, when mice were maintained on a cuprizone diet for 16 weeks, the mean value for axonal diameter was reduced to 60% of normal. These results provide further evidence that the interactions between oligodendrocytes and axons alter axonal calibre.     

Cuprizone [Bis(Cyclohexylidenehydrazide)] is Selectively Toxic for Mature Oligodendrocytes

Cuprizone [bis(cyclohexylidenehydrazide)]-induced toxic demyelination is an experimental animal model commonly used to study de- and remyelination in the central nervous system. In this model, mice are fed with the copper chelator cuprizone which leads to oligodendrocyte death with subsequent demyelination. The underlying mechanisms of cuprizone-induced oligodendrocyte death are still unknown, and appropriate in vitro investigations to study these mechanisms are not available. Thus, we studied cuprizone effects on rat primary glial cell cultures and on the neuroblastoma cell line SH-SY5Y. Treatment of cells with different concentrations of cuprizone failed to show effects on the proliferation and survival of SH-SY5Y cells, microglia, astrocytes, and oligodendrocyte precursor cells (OPC). In contrast, differentiated mature oligodendrocytes (OL) were found to be significantly affected by cuprizone treatment. This was accompanied by a reduced mitochondrial potential in cuprizone-treated OL. These results demonstrate that the main toxic target for cuprizone is mature OL, whilst other glial cells including OPC are not or only marginally affected. This explains the selective demyelination induced by cuprizone in vivo.     

Abbreviated exposure to cuprizone is sufficient to induce demyelination and oligodendrocyte loss

Cuprizone intoxication is one of several animal models used to study demyelination and remyelination. Early treatment protocols exposed mice to cuprizone for 6 weeks to induce demyelination; however, more recent reports have varied exposure times from 4 to 5 weeks. The goal of this study was to determine the minimal exposure of cuprizone in C57BL/6 mice that would induce a pathology of robust demyelination and gliosis similar to that described for a 5‐ or 6‐week treatment. We found that an abbreviated insult of only 2 weeks of exposure to cuprizone induced significant demyelination 3 weeks later (5‐week time point) but was somewhat variable. Three weeks of exposure to cuprizone produced extensive demyelination by week 5, equivalent to that observed with 5 weeks of exposure. The depletion of mature oligodendrocytes, as well as microglia and astrocyte accumulation, showed trends similar to those with 5‐week exposure to cuprizone. Once mature oligodendrocytes are perturbed after a 3‐week treatment, the progression to demyelination occurs without requiring further exposure. Furthermore, the early removal of cuprizone did not accelerate remyelination, suggesting that other sequences of events must follow before repair can occur. Thus, a short, “hit and run” CNS insult triggers a cascade of events leading to demyelination 2–3 weeks later. © 2012 Wiley Periodicals, Inc.     

Effects of commissural de- and remyelination on motor skill behaviour in the cuprizone mouse model of multiple sclerosis

Feeding of copper chelator cuprizone induces reversible demyelination, predominantly of the corpus callosum in C57/Bl6 mice. With the availability of knockout and transgenic mice, this animal model of multiple sclerosis has increasingly attracted scientists to study the roles of various factors involved in de- and remyelination. However, central motor deficits have not been reported in this model so far. In the present study, we introduce a novel murine motor test, the motor skill sequence (MOSS). This test is designed to detect latent deficits in motor performance. In a first step, we habituated mice to training wheels composed of regularly spaced crossbars till maximal wheel-running performance was achieved. Subsequently, the animals were exposed to wheels with irregularly spaced crossbars demanding high-level motor coordination. This two-step approach minimized a contribution of cardiopulmonary and musculoskeletal training to any improvement of motor performance on the complex wheels. We applied the MOSS test under acute cuprizone-induced demyelination as well as in remyelinated mice after cuprizone withdrawal. Demyelinated animals on a cuprizone diet already showed reduced running performance on the training wheels as compared to control animals. This was even more pronounced when these mice were subsequently exposed to the complex wheels. In contrast, remyelinated animals after cuprizone withdrawal did not exhibit any functional impairment on the training wheels. Latent motor skill deficits were however revealed on the complex wheels, although clearly ameliorated as compared to acutely demyelinated mice. Our results show that latent motor deficits of cuprizone-induced demyelination and after remyelination can be quantified by MOSS. This motor test thus expands the usability of the cuprizone model to a functional level and might also be applicable to other animal models of human CNS diseases associated with subtle motor deficits of central origin.