Insulin-like growth factor-1 inhibits mature oligodendrocyte apoptosis during primary demyelination.

Metabolic insult results in apoptosis and depletion of mature oligodendrocytes during demyelination. To examine the role of insulin-like growth factor-1 (IGF-1) during acute demyelination and remyelination in the adult CNS, we exposed transgenic mice that continuously express IGF-1 (IGF-1 tg) to cuprizone intoxication. Demyelination was observed within the corpus callosum in both wild-type and IGF-1 tg mice 3 weeks after exposure to cuprizone. Wild-type mice showed significant apoptotic mature oligodendrocytes and a dramatic loss of these cells within the lesion that resulted in near complete depletion and demyelination by week 5. In contrast, the demyelinated corpus callosum of the IGF-1 tg mice was near full recovery by week 5. This rapid recovery was apparently caused by survival of the mature oligodendrocyte population because apoptosis was negligible, and by week 4, the mature oligodendrocyte population was completely restored. Furthermore, despite demyelination in both wild-type and IGF-1 tg mice, oligodendrocyte progenitors accumulated only in the absence of mature oligodendrocytes and failed to accumulate if the mature oligodendrocytes remained as demonstrated in the IGF-1 tg mice. These results suggest that IGF-1 may be important in preventing the depletion of mature oligodendrocytes in vivo and thus facilitates an early recovery from demyelination.     

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.     

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.     

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.     

Thyroid hormones promote differentiation of oligodendrocyte progenitor cells and improve remyelination after cuprizone-induced demyelination

In the present work we analyzed the capacity of thyroid hormones (THs) to improve remyelination using a rat model of cuprizone-induced demyelination previously described in our laboratories. Twenty one days old Wistar rats were fed a diet containing 0.6% cuprizone for two weeks to induce demyelination. After cuprizone withdrawal, rats were injected with triiodothyronine (T3). Histological studies carried out in these animals revealed that remyelination in the corpus callosum (CC) of T3-treated rats improved markedly when compared to saline treated animals. The cellular events occurring in the CC and in the subventricular zone (SVZ) during the first week of remyelination were analyzed using specific oligodendroglial cell (OLGc) markers. In the CC of saline treated demyelinated animals, mature OLGcs decreased and oligodendroglial precursor cells (OPCs) increased after one week of spontaneous remyelination. Furthermore, the SVZ of these animals showed an increase in early progenitor cell numbers, dispersion of OPCs and inhibition of Olig and Shh expression compared to non-demyelinated animals. The changes triggered by demyelination were reverted after T3 administration, suggesting that THs could be regulating the emergence of remyelinating oligodendrocytes from the pool of proliferating cells residing in the SVZ. Our results also suggest that THs receptor β mediates T3 effects on remyelination. These results support a potential role for THs in the remyelination process that could be used to develop new therapeutic approaches for demyelinating diseases.     

Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination

We have documented changes in the oligodendrocyte population during demyelinating insult to the adult CNS. Feeding of cuprizone to adult mice led to apoptotic death of mature oligodendrocytes followed by profound demyelination of the corpus callosum. A regenerative response was initiated even during active demyelination. Oligodendrocyte progenitors have begun to proliferate and then accumulate within the lesion. Many of these cells may have migrated from the sub-ventricular zone and fornix before their accumulation in the demyelinating corpus callosum. The accumulation of differentiating oligodendrocyte progenitors was followed closely by the reappearance of mature oligodendrocytes and remyelination. Interestingly, an increase in IGF-1 mRNA was detected at Week 3 through Week 7, suggesting potential involvement in remyelination. Other factors, however, such as PDGF, NT3, FGF, jagged, and notch remained unchanged. These results suggest that the mature oligodendroglial population depleted by apoptosis is replaced by a newly formed oligodendroglial population derived from progenitors; these accumulate and seem to differentiate during remyelination.     

Long‐term impact of neonatal inflammation on demyelination and remyelination in the central nervous system

Perinatal inflammation causes immediate changes of the blood‐brain barrier (BBB) and thus may have different consequences in adult life including an impact on neurological diseases such as demyelinating disorders. In order to determine if such a perinatal insult affects the course of demyelination in adulthood as “second hit,” we simulated perinatal bacterial inflammation by systemic administration of lipopolysaccharide (LPS) to either pregnant mice or newborn animals. Demyelination was later induced in adult animals by cuprizone [bis(cyclohexylidenehydrazide)], which causes oligodendrocyte death with subsequent demyelination accompanied by strong microgliosis and astrogliosis. A single LPS injection at embryonic day 13.5 did not have an impact on demyelination in adulthood. In contrast, serial postnatal LPS injections (P0‐P8) caused an early delay of myelin removal in the corpus callosum, which was paralleled by reduced numbers of activated microglia. During remyelination, postnatal LPS treatment enhanced early remyelination with a concomitant increase of mature oligodendrocytes. Furthermore, the postnatal LPS challenge impacts the phenotype of microglia since an elevated mRNA expression of microglia related genes such as TREM 2, CD11b, TNF‐α, TGF‐β1, HGF, FGF‐2, and IGF‐1 was found in these preconditioned mice during early demyelination. These data demonstrate that postnatal inflammation has long‐lasting effects on microglia functions and modifies the course of demyelination and remyelination in adulthood. GLIA 2014;62:1659–1670     

Effects of acute and repeated exposure to lipopolysaccharide on cytokine and corticosterone production during remyelination

Chronic exposure to the copper-chelating agent, cuprizone (CPZ), is an increasingly popular model for producing demyelination. More importantly, cessation of cuprizone exposure allows for full remyelination, which represents a window of opportunity for determining the influence of environmental factors on regenerative processes. In the present study, CPZ-treated animals were assessed for functional status of systemic and central cytokine responsiveness to LPS, as well as assessment for signs of body weight changes. Exposure of male C57BL/6J mice to 5 weeks of 0.2% CPZ in the diet was optimal in producing demyelination and microglial activation, as measured by myelin basic protein, CD11b, and CD45 immunohistochemistry. Acute challenge with LPS at the end of 5 weeks CPZ treatment did not alter IL-1beta, IL-6, nor TNFalpha responses in the spleen and corpus callosum. Similarly, repeated exposure to LPS during the remyelination phase (CPZ removal) did not influence these measures to LPS. Plasma corticosterone was unaffected following acute challenge of CPZ-pretreated animals, but after repeated LPS treatment, there was a significant augmentation of the corticosterone response in CPZ-pretreated mice. Interestingly, the basal concentration of IL-1beta in the corpus callosum of CPZ treated animals was significantly increased, which was in keeping with the increase in activated microglial cells. In conclusion, the cuprizone model of demyelination and remyelination does not appear to influence the systemic nor central IL-1, IL-6, and TNF responses to acute nor repeated LPS. This opens up the possibility for studying the contribution of systemic inflammatory processes on remyelination after cessation of CPZ treatment.     

Clemastine rescues behavioral changes and enhances remyelination in the cuprizone mouse model of demyelination

Increasing evidence suggests that white matter disorders based on myelin sheath impairment may underlie the neuropathological changes in schizophrenia.But it is unknown whether enhancing remyelination is a beneficial approach to schizophrenia.To investigate this hypothesis,we used clemastine,an FDA-approved drug with high potency in promoting oligodendroglial differentiation and myelination,on a cuprizone-induced mouse model of demyelination.The mice exposed to cuprizone(0.2%in chow) for 6 weeks displayed schizophrenia-like behavioral changes,including decreased exploration of the center in the open field test and increased entries into the arms of the Y-maze,as well as evident demyelination in the cortex and corpus callosum.Clemastine treatment was initiated upon cuprizone withdrawal at 10 mg/kg per day for3 weeks.As expected,myelin repair was greatly enhanced in the demyelinated regions with increased mature oligodendrocytes(APC-positive) and myelin basic protein.More importantly,the clemastine treatment rescued the schizophrenia-like behavioral changes in the open field test and the Y-maze compared to vehicle,suggesting a beneficial effect via promoting myelin repair.Our findings indicate that enhancing remyelination may be a potential therapy for schizophrenia.     

Differential local tissue permissiveness influences the final fate of GPR17‐expressing oligodendrocyte precursors in two distinct models of demyelination

Promoting remyelination is recognized as a novel strategy to foster repair in neurodegenerative demyelinating diseases, such as multiple sclerosis. In this respect, the receptor GPR17, recently emerged as a new target for remyelination, is expressed by early oligodendrocyte precursors (OPCs) and after a certain differentiation stage it has to be downregulated to allow progression to mature myelinating oligodendrocytes. Here, we took advantage of the first inducible GPR17 reporter mouse line (GPR17‐iCreER^T2xCAG‐eGFP mice) allowing to follow the final fate of GPR17⁺ cells by tamoxifen‐induced GFP‐labeling to unveil the destiny of these cells in two demyelination models: experimental autoimmune encephalomyelitis (EAE), characterized by marked immune cell activation and inflammation, and cuprizone induced demyelination, where myelin dysfunction is achieved by a toxic insult. In both models, demyelination induced a strong increase of fluorescent GFP⁺ cells at damaged areas. However, only in the cuprizone model reacting GFP⁺ cells terminally differentiated to mature oligodendrocytes, thus contributing to remyelination. In EAE, GFP⁺ cells were blocked at immature stages and never became myelinating oligodendrocytes. We suggest these strikingly distinct fates be due to different permissiveness of the local CNS environment. Based on previously reported GPR17 activation by emergency signals (e.g., Stromal Derived Factor‐1), we propose that a marked inflammatory milieu, such as that reproduced in EAE, induces GPR17 overactivation resulting in impaired downregulation, untimely and prolonged permanence in OPCs, leading, in turn, to differentiation blockade. Combined treatments with remyelinating agents and anti‐inflammatory drugs may represent new potential adequate strategies to halt neurodegeneration and foster recovery.     

Investigation of Cuprizone Inactivation by Temperature

Animal models, such as cuprizone (bis-cyclohexanone oxaldihydrazone) feeding, are helpful to study experimental demyelination and remyelination in the context of diseases like multiple sclerosis. Cuprizone is a copper chelator, which when supplemented to the normal food of C57BL/6J mice in a concentration of 0.2% leads to oligodendroglial loss, subsequent microglia and astrocyte activation, resulting in demyelination. Termination of the cuprizone diet results in remyelination, promoted by newly formed mature oligodendrocytes. The exact mode of cuprizone’s action is not well understood, and information about its inactivation and cleavage are still not available. The knowledge of these processes could lead to a better understanding of cuprizone’s mode of action, as well as a safer handling of this toxin. We therefore performed experiments with the aim to inactivate cuprizone by thermal heating, since it was suggested in the past that cuprizone is heat sensitive. C57BL/6J mice were fed for 4 weeks with 0.2% cuprizone, either thermally pretreated (60, 80, 105, 121 °C) or not heated. In addition, primary rat oligodendrocytes, as a known selective toxic target of cuprizone, were incubated with 350 μM cuprizone solutions, which were either thermally pretreated or not. Our results demonstrate that none of the tested thermal pretreatment conditions could abrogate or restrict the toxic and demyelinating effects of cuprizone, neither in vitro nor in vivo. In conclusion, the current study rebuts the hypothesis of cuprizone as a heat-sensitive compound, as well as the assumption that heat exposure is a reason for an insufficient demyelination of cuprizone-containing pellets.     

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.     

Loss of Gas6 and Axl signaling results in extensive axonal damage,motor deficits,prolonged neuroinflammation,and less remyelination following cuprizone exposure

The TAM (Tyro3, Axl, and MerTK) family of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and ProS1, are important for innate immune responses and central nervous system (CNS) homeostasis. While only Gas6 directly activates Axl, ProS1 activation of Tyro3/MerTK can indirectly activate Axl through receptor heterodimerization. Therefore, we generated Gas6^–/–Axl^–/– double knockout (DKO) mice to specifically examine the contribution of this signaling axis while retaining ProS1 signaling through Tyro3 and MerTK. We found that naïve young adult DKO and WT mice have comparable myelination and equal numbers of axons and oligodendrocytes in the corpus callosum. Using the cuprizone model of demyelination/remyelination, transmission electron microscopy revealed extensive axonal swellings containing autophagolysosomes and multivesicular bodies, and fewer myelinated axons in brains of DKO mice at 3‐weeks recovery from a 6‐week cuprizone diet. Analysis of immunofluorescent staining demonstrated more SMI32+ and APP+ axons and less myelin in the DKO mice. There were no significant differences in the number of GFAP+ astrocytes or Iba1+ microglia/macrophages between the groups of mice. However, at 6‐weeks cuprizone and recovery, DKO mice had increased proinflammatory cytokine and altered suppressor of cytokine signaling (SOCS) mRNA expression supporting a role for Gas6‐Axl signaling in proinflammatory cytokine suppression. Significant motor deficits in DKO mice relative to WT mice on cuprizone were also observed. These data suggest that Gas6‐Axl signaling plays an important role in maintaining axonal integrity and regulating and reducing CNS inflammation that cannot be compensated for by ProS1/Tyro3/MerTK signaling.     

Demyelination of the superior cerebellar peduncle in the mouse induced by cuprizone

The superior cerebellar peduncles of mice fed a diet containing 0.5% cuprizone since weaning were examined. It was found that this tract was demyelinating after 4 weeks on the diet, a process which was almost complete after 5 weeks. Demyelination followed degeneration of oligodendrocytes. Extensive remyelination did not occur if the animals were kept on the diet. The reproducibility of demyelination was such that quantitative studies on remyelination can be undertaken. The genotype of the mice used in the present experiments was considered to be of importance in the aetiology of the lesion.     

Fibroblast growth factor 1 (FGFR1) modulation regulates repair capacity of oligodendrocyte progenitor cells following chronic demyelination

The adult mammalian brain contains multiple populations of endogenous progenitor cell types. However, following CNS trauma or disease, the regenerative capacity of progenitor populations is typically insufficient and may actually be limited by non-permissive or inhibitory signals in the damaged parenchyma. Remyelination is the most effective and simplest regenerative process in the adult CNS yet is still insufficient following repeated or chronic demyelination. Our previous in vitro studies demonstrated that fibroblast growth factor receptor 1 (FGFR1) signaling inhibited oligodendrocyte progenitor (OP) differentiation into mature oligodendrocytes. Therefore, we questioned whether FGFR1 signaling may inhibit the capacity of OP cells to generate oligodendrocytes in a demyelinating disease model and whether genetically reducing FGFR1 signaling in oligodendrocyte lineage cells could enhance the capacity for remyelination. FGFR1 was found to be upregulated in the corpus callosum during cuprizone mediated demyelination and expressed on OP cells just prior to remyelination. Plp/CreER^T:Fgfr1^fl/fl mice were administered tamoxifen to induce conditional Fgfr1 deletion in oligodendrocyte lineage cells. Tamoxifen administration during chronic demyelination resulted in reduced FGFR1 expression in OP cells. OP proliferation and population size were not altered one week after tamoxifen treatment. Tamoxifen was then administered during chronic demyelination and mice were given a six week recovery period without cuprizone in the chow. After the recovery period, OP numbers were reduced and the number of mature oligodendrocytes was increased, indicating an effect of FGFR1 reduction on OP differentiation. Importantly, tamoxifen administration in Plp/CreER^T:Fgfr1^fl/fl mice significantly promoted remyelination and axon integrity. These results demonstrate a direct effect of FGFR1 signaling in oligodendrocyte lineage cells as inhibiting the repair capacity of OP cells following chronic demyelination in the adult CNS.     

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.     

Proteomic Analysis of Demyelinated and Remyelinating Brain Tissue following Dietary Cuprizone Administration

Cuprizone intoxication is a commonly used model of demyelination that allows the temporal separation of demyelination and remyelination. The underlying biochemical alterations leading to demyelination, using this model, remain unclear and may be multifold. Analysis of proteomic changes within the brains of cuprizone-exposed animals may help elucidate key cellular processes. In the current study, we report the results of the liquid chromatography tandem mass spectrometry analysis of total protein from the brain hemispheres of control and toxin-exposed mice at 6 weeks of exposure and after 3 and 6 weeks of recovery to identify protein changes during the remyelination phase. We found that at 6 weeks of cuprizone exposure, myelin proteins were reduced compared to controls and increased throughout the course of recovery, as expected. In contrast, other protein groups, such as proteins related to mitochondrial function, were increased at 6 weeks of treatment compared to untreated controls and returned toward control levels following withdrawal of toxin. These results suggest that a global proteomic analysis of the brain tissue of cuprizone-treated mice can identify changes related to the demyelination/remyelination process.     

Golli myelin basic proteins stimulate oligodendrocyte progenitor cell proliferation and differentiation in remyelinating adult mouse brain

Golli myelin basic proteins are necessary for normal myelination, acting via voltage and store-dependent Ca(2+) entry at multiple steps during oligodendrocyte progenitor cell (OPC) development. To date nothing is known regarding the role of golli proteins in demyelination or remyelination events. Here the effects of golli ablation and overexpression in myelin loss and recovery were examined using the cuprizone (CPZ) model of demyelination/remyelination. We found severe demyelination in the corpus callosum (CC) of golli-overexpressing mice (JOE) during the CPZ treatment, which was accompanied by an increased number of reactive astrocytes and activation of microglia/macrophages. During demyelination of JOE brains, a significant increase in the number of proliferating OPCs was found in the CC as well as in the subventricular zone, and our data indicate that these progenitors matured and fully remyelinated the CC of JOE animals after CPZ withdrawal. In contrast, in the absence of golli (golli-KO mice) delayed myelin loss associated with a smaller immune response, and a lower number of OPCs was found in these mice during the CPZ treatment. Furthermore, incomplete remyelination was observed after CPZ removal in large areas of the CC of golli-KO mice, reflecting irregular recovery of the oligodendrocyte population and subsequent myelin sheath formation. Our findings demonstrate that golli proteins sensitize mature oligodendrocytes to CPZ-induced demyelination, while at the same time stimulate the proliferation/recruitment of OPCs during demyelination, resulting in accelerated remyelination.