Icariin ameliorates the cuprizone-induced demyelination associated with antioxidation and anti-inflammation.

The treatment of immunomodulation in multiple sclerosis (MS) can alleviate the severity and relapses. However, it cannot improve the neurological disability of patients due to a lack of myelin protection and regeneration. Therefore, remyelinating therapies may be one of the feasible strategies that can prevent axonal degeneration and restore neurological disability. Natural product icariin (ICA) is a flavonol compound extracted from epimedium flavonoids, which has neuroprotective effects in several models of neurological diseases. Here, we attempt to explore whether ICA has the potential to treat demyelination and its possible mechanisms of action using lipopolysaccharide-treated BV2 microglia, primary microglia, bone marrow-derived macrophages, and cuprizone-induced demyelination model. The indicators of oxidative stress and inflammatory response were evaluated using commercial kits. The results showed that ICA significantly reduced the levels of oxidative intermediates nitric oxide, hydrogen peroxide, malondialdehyde, and inflammatory cytokines TNF-α, IL-1ß, and increased the levels of antioxidants superoxide dismutase, catalase, glutathione peroxidase, and anti-inflammatory cytokines IL-10 and TGF-ß in vitro cell experiments. In vivo demyelination model, ICA significantly alleviated the behavioral abnormalities and enhanced the integrated optical density/mm2 of Black Gold II and myelin basic protein myelin staining, accompanied by the inhibition of oxidative stress/inflammatory response. Immunohistochemical staining showed that ICA significantly induced the expression of nuclear factor erythroid derived 2/heme oxygenase-1 (Nrf2/HO-1) and inhibited the expression of toll-like receptor 4/ nuclear factor kappa B (TLR4/NF-κB), which are two key signaling pathways in antioxidant and anti-inflammatory processes. Our results strongly suggest that ICA may be used as a potential agent to treat demyelination via regulating Nrf2/HO-1-mediated antioxidative stress and TLR4/NF-κB-mediated inflammatory responses.

Expression of Bruton´s tyrosine kinase in different type of brain lesions of multiple sclerosis patients and during experimental demyelination.

Background: Inhibition of Bruton's tyrosine kinase (BTK) is an emerging multiple sclerosis (MS) therapy. BTK inhibitors (BTKi) cross the blood-brain barrier and modulate B cells and microglia, major cellular players in active and chronic active lesions. Objective: To assess potential lesional and cellular targets of BTKi, we examined BTK expression in different type of MS white matter (WM) lesions, in unmanipulated CNS resident cells, and in a degenerative MS model associated with microglia activation in vivo. Methods: We examined BTK expression by next-generation RNA-sequencing in postmortem 25 control WM, 19 NAWM, 6 remyelinating, 18 active, 13 inactive and 17 chronic active lesions. Presence of B cells and microglia were examined by immunohistochemistry. CNS resident cells were isolated from the mouse brain by magnetic sorting. BTK expression was examined by quantitative PCR in isolated cells and dissected corpus callosum from mice treated with cuprizone (CPZ). Results: BTK expression was significantly increased in active and chronic active lesions with upregulated complement receptors and Fcγ receptors. Active lesions contained high number of perivascular B cells, microglia, and macrophages. Chronic active lesions were characterized by microglia/macrophages in the rim. Microglia expressed BTK at high level (120-fold) in contrast to other CNS cell types (2-4-fold). BTK expression was increasing during CPZ treatment reaching significance after stopping CPZ. Conclusion: Considering BTK expression in MS lesions and resident cells, BTKi may exert effect on B cells, microglia/macrophages in active lesions, and limit microglia activation in chronic active lesions, where tissue damage propagates.

Immunoglobulin directly enhances differentiation of oligodendrocyte-precursor cells and remyelination.

Multiple sclerosis (MS) is an inflammatory demyelinating disease characterized by multiple lesions in the central nervous system. Although the role of B cells in MS pathogenesis has attracted much attention, but the detailed mechanisms remain unclear. To investigate the effects of B cells on demyelination, we analyzed a cuprizone-induced demyelination model, and found that demyelination was significantly exacerbated in B cell-deficient mice. We next investigated whether immunoglobulin affected the myelin formation process using organotypic brain slice cultures and revealed that remyelination was improved in immunoglobulin-treated groups compared with the control group. Analysis of oligodendrocyte-precursor cell (OPC) monocultures showed that immunoglobulins directly affected on OPCs and promoted their differentiation and myelination. Furthermore, OPCs expressed FcγRI and FcγRIII, two receptors that were revealed to mediate the effects of IgG. To the best of our knowledge, this is the first study to demonstrate that B cells act in an inhibitory manner against cuprizone-induced demyelination, while immunoglobulins enhance remyelination following demyelination. Analysis of the culture system revealed that immunoglobulins directly act on OPCs to promote their differentiation and myelination. Future studies to elucidate the effects of immunoglobulins on OPCs in vivo and the detailed mechanisms of these effects may lead to new treatments for demyelinating diseases.

Pharmacological treatment promoting remyelination enhances motor function after internal capsule demyelination in mice.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system characterized by remyelination failure, axonal degeneration, and progressive worsening of motor functions. Animal models of demyelination are frequently used to develop and evaluate therapies for MS. We recently reported that focal internal capsule (IC) demyelination in mice with lysophosphatidylcholine injection induced acute motor deficits followed by recovery through remyelination. However, it remains unknown whether the IC demyelination mouse model can be used to evaluate changes in motor functions caused by pharmacological treatments that promote remyelination using behavioral testing and histological analysis. In this study, we examined the effect of clemastine, an anti-muscarinic drug that promotes remyelination, in the mouse IC demyelination model. Clemastine administration improved motor function and changed forepaw preference in the IC demyelinated mice. Moreover, clemastine-treated mice showed increased mature oligodendrocyte density, reduced axonal injury, an increased number of myelinated axons and thicker myelin in the IC lesions compared with control (PBS-treated) mice. These results suggest that the lysophosphatidylcholine-induced IC demyelination model is useful for evaluating changes in motor functions following pharmacological treatments that promote remyelination.

Sulforaphane, an Nrf-2 Agonist, Modulates Oxidative Stress and Inflammation in a Rat Model of Cuprizone-Induced Cardiotoxicity and Hepatotoxicity.

Cuprizone (CPZ) is a neurotoxic agent that is used to induce demyelination and neurotoxicity in rats. This study aimed to investigate the protective potential of sulforaphane (SF), nuclear factor E2 related factor (Nrf-2) activator, against CPZ-induced cardiotoxicity and hepatotoxicity. Male adult Wistar rats (n = 18) were fed with a regular diet or a CPZ-contained diet (0.2%) for four weeks. The rats were divided into three groups (n = 6): negative control rats, CPZ-exposed rats, and CPZ + SF treated rats. SF was intraperitoneally administrated (2 mg/kg/day) for two weeks. The anti-inflammatory and anti-oxidative functions of SF were investigated biochemically, histologically, and immunohistochemically. CPZ increased serum levels of cardiac troponin 1 (CTn1), aspartate amino transaminase (AST), alanine amino transaminase (ALT), and alkaline phosphatase (ALP). In addition, serum levels of inflammatory interferon-gamma (IFN-γ), and pro-inflammatory interleukin 1ß (IL-1ß) were significantly elevated. Moreover, CPZ administration provoked oxidative stress as manifested by declined serum levels of total antioxidant capacity (TAC), as well as, stimulated lipid peroxidation and decreased catalase activities in both cardiac and hepatic tissues. SF treatment reversed all these biochemical alterations through exerting anti-oxidative and anti-inflammatory activities, and this was supported by histopathological investigations in both cardiac and hepatic tissues. This SF-triggered modulation of oxidative stress and inflammation is strongly associated with Nrf-2 activation, as evidenced by activated immunoexpression in both cardiac and hepatic tissues. This highlights the cardioprotective and hepatoprotective activities of SF via Nrf-2 activation and enhancing catalase function.

Myelin repair is fostered by the corticosteroid medrysone specifically acting on astroglial subpopulations.

BACKGROUND: Multiple sclerosis is characterised by inflammation, oligodendrocyte loss and axonal demyelination and shows an additional impact on astrocytes, and their polarization. Although a certain degree of spontaneous myelin repair can be observed, disease progression, and aging impair regeneration efforts highlighting the need to better understand glial cell dynamics to establish specific regenerative treatments. METHODS: Applying a chronic demyelination model, we here analysed demyelination and remyelination related effects on astrocytes and stem cell niches and studied the consequences of medrysone application on myelin repair, and astrocyte polarization. FINDINGS: Medrysone induced recovery of mature oligodendrocytes, myelin expression and node formation. In addition, C3d/S100a10 co-expression in astrocytes was enhanced. Moreover, Timp1 expression in C3d positive astrocytes revealed another astrocytic phenotype with a myelination promoting character. INTERPRETATION: Based on these findings, specific astrocyte subpopulations are suggested to act in a myelin regenerative way and manner the regulation of which can be positively modulated by this corticosteroid. FUNDING: This work was supported by the Jürgen Manchot Stiftung, the Research Commission of the medical faculty of the Heinrich-Heine-University of Düsseldorf, the Christiane and Claudia Hempel Foundation for clinical stem cell research and the James and Elisabeth Cloppenburg, Peek and Cloppenburg Düsseldorf Stiftung.

Ginsenoside Rg1 promotes remyelination and functional recovery in demyelinating disease by enhancing oligodendrocyte precursor cells-mediated myelin repair.

BACKGROUND: Inefficient differentiation of oligodendrocyte precursor cells (OPCs) is one of the significant pathological obstacles of myelin repair and provides an essential therapeutic target against behavioral dysfunction in various neurodegenerative diseases, especially in secondary progressive multiple sclerosis (SPMS). Ginsenoside Rg1 (Rg1) has traditionally been recognized as a protector of neuronal damages, preventing its degeneration. PURPOSE: We investigated the effects of Rg1 on myelin regeneration-mediated by OPCs and its therapeutic significance in SPMS. METHODS: A cuprizone (CPZ) model was established and then administered with Rg1 specific for evaluations of functional recovery and remyelination. In vitro, the primary mouse OPCs were isolated and cultured for examining their ability of myelin repair. Furthermore, a chronic experimental autoimmune encephalomyelitis (EAE) model was utilized to assess the therapeutic value on SPMS. RESULTS: We found that Rg1 promoted functional recovery of the demyelinated mice, including spatial memory, motor function, and anxiety-like behavior. Histologically, Rg1 enhanced myelin-genesis as proven by myelin staining and microstructures of myelin observed by transmission electron microscope. Furthermore, Rg1 significantly increased Olig2+ oligodendrocyte lineage cells in callosum, implying that the pro-remyelination effect of Rg1 was closely correlated to the enhanced differentiation of OPCs. We further demonstrated that Rg1 increased the survival and proliferation of OPCs as well as induced maturation in oligodendrocytes (OLs). Molecular analysis showed that Rg1 transduced the pro-differentiation signaling programmed by the GSK3ß/ß-Catenin pathway. Notably, relying on its pro-remyelination effects, Rg1 ameliorated severity and histopathology of EAE disease. CONCLUSION: By paving the way for OPCs differentiation, Rg1 could maintain the integrity of myelin and is a promising candidate for functional recovery in demyelinating diseases.

Neuroprotective effect of liraglutide in an experimental mouse model of multiple sclerosis: role of AMPK/SIRT1 signaling and NLRP3 inflammasome.

The heterogeneous nature of multiple sclerosis (MS) and the unavailability of treatments addressing its intricate network and reversing the disease state is yet an area that needs to be elucidated. Liraglutide, a glucagon-like peptide-1 analogue, recently exhibited intriguing potential neuroprotective effects. The currents study investigated its potential effect against mouse model of MS and the possible underlying mechanisms. Demyelination was induced in C57Bl/6 mice by cuprizone (400 mg/kg/day p.o.) for 5 weeks. Animals received either liraglutide (25 nmol/kg/day i.p.) or dorsomorphin, an AMPK inhibitor, (2.5 mg/Kg i.p.) 30 min before the liraglutide dose, for 4 weeks (starting from the second week). Liraglutide improved the behavioral profile in cuprizone-treated mice. Furthermore, it induced the re-myelination process through stimulating oligodendrocyte progenitor cells differentiation via Olig2 transcription activation, reflected by increased myelin basic protein and myelinated nerve fiber percentage. Liraglutide elevated the protein content of p-AMPK and SIRT1, in addition to the autophagy proteins Beclin-1 and LC3B. Liraglutide halted cellular damage as manifested by reduced HMGB1 protein and consequently TLR-4 downregulation, coupled with a decrease in NF-κB. Liraglutide also suppressed NLRP3 transcription. Dorsomorphin pre-administration indicated a possible interplay between AMPK/SIRT1 and NLRP3 inflammasome activation as it partially reversed liraglutide's effects. Immunohistochemical examination of Iba+ microglia emphasized these findings. In conclusion, liraglutide exerts neuroprotection against cuprizone-induced demyelination via anti-inflammatory, autophagic flux activation, NLRP3 inflammasome suppression, and anti-apoptotic mechanisms, possibly mediated, at least in part, via AMPK/SIRT1, autophagy, TLR-4/ NF-κB/NLRP3 signaling. The potential mechanistic insight of Lira in alleviating Cup-induced neurotoxicity via: (1) AMPK/SIRT1 pathways activation resulting in the stimulation of brain autophagy flux (confirmed by lowering Beclin-1 and LC3-B protein expression). (2) Inhibition of NLRP3 inflammasome activation, as evidenced by reduced HMGB1, TLR-4, NF-κB and NLRP3 protein expression, alongside diminishing the activation of its downstream cascade as reflected by reduced levels of caspase-1 and IL-1ß protein expression. (3) A possible modulating interplay between the previously mentioned two pathways.

Increased Remyelination and Proregenerative Microglia Under Siponimod Therapy in Mechanistic Models.

BACKGROUND AND OBJECTIVES: Siponimod is an oral, selective sphingosine-1-phosphate receptor-1/5 modulator approved for treatment of multiple sclerosis. METHODS: Mouse MRI was used to investigate remyelination in the cuprizone model. We then used a conditional demyelination Xenopus laevis model to assess the dose-response of siponimod on remyelination. In experimental autoimmune encephalomyelitis-optic neuritis (EAEON) in C57Bl/6J mice, we monitored the retinal thickness and the visual acuity using optical coherence tomography and optomotor response. Optic nerve inflammatory infiltrates, demyelination, and microglial and oligodendroglial differentiation were assessed by immunohistochemistry, quantitative real-time PCR, and bulk RNA sequencing. RESULTS: An increased remyelination was observed in the cuprizone model. Siponimod treatment of demyelinated tadpoles improved remyelination in comparison to control in a bell-shaped dose-response curve. Siponimod in the EAEON model attenuated the clinical score, reduced the retinal degeneration, and improved the visual function after prophylactic and therapeutic treatment, also in a bell-shaped manner. Inflammatory infiltrates and demyelination of the optic nerve were reduced, the latter even after therapeutic treatment, which also shifted microglial differentiation to a promyelinating phenotype. DISCUSSION: These results confirm the immunomodulatory effects of siponimod and suggest additional regenerative and promyelinating effects, which follow the dynamics of a bell-shaped curve with high being less efficient than low concentrations.

Cuprizone-induced dopaminergic hyperactivity and locomotor deficit in zebrafish larvae.

Cuprizone (CPZ) is a copper-chelator and toxic to mitochondria. Recent studies have shown oligodendrocyte (OL) loss and demyelination along with dopamine (DA) increase and behavioral abnormalities in CPZ-exposed mice, demonstrating its application in schizophrenia research. This study examined effects of CPZ exposure on autonomous behavior and dopaminergic neurotransmission in larval zebra fish. CPZ exposure was found to reduce the swimming velocity of zebra fish thus decreased swimming distance during day and night time. Moreover, the treatment induced a movement response of zebra fish larvae reacting to light-on/off switch featured by swimming velocity increase and decrease during the first and second half of the light-on/off phase, respectively. But, it abolished responses of zebra fish to sound-on/off seen in Control group. HPLC analysis showed elevated DA levels in the zebra fish, no change in NE and 5-HT levels. Transcriptome analysis reported changes in gene expression related to dopaminergic synapse and oxidative phosphorylation in CPZ-exposed larvae relative to Control group. Of the gene expression changes, up-regulation of drd2a, drd2b, drd4a and drd4rs was confirmed by RT-PCR, although no difference existed between Control and CPZ groups in dopaminergic neuron numbers. These results demonstrated dopaminergic hyperactivity and locomotor deficit in CPZ-exposed zebra fish larvae, encouraging further application of this model in exploring neurotoxic effects of CPZ on mitochondria and dopaminergic neurotransmission in zebra fish.

Abnormal oxidative metabolism in the cuprizone mouse model of demyelination: An in vivo NIRS-MRI study.

Disruptions in oxidative metabolism may occur in multiple sclerosis and other demyelinating neurological diseases. The impact of demyelination on metabolic rate is also not understood. It is possible that mitochondrial damage may be associated with many such neurological disorders. To study oxidative metabolism with one model of demyelination, we implemented a novel multimodal imaging technique combining Near-Infrared Spectroscopy (NIRS) and MRI to cuprizone mouse model. The cuprizone model is used to study demyelination and may be associated with inhibition of mitochondrial function. Cuprizone mice showed reduced oxygen extraction fraction (-39.1%, p ≤ 0.001), increased tissue oxygenation (6.4%, p ≤ 0.001), and reduced cerebral metabolic rate of oxygen in cortical gray matter (-62.1%, p ≤ 0.001). These changes resolved after the cessation of cuprizone exposure and partial remyelination. A decrease in hemoglobin concentration (-34.4%, p ≤ 0.001), but no change in cerebral blood flow were also observed during demyelination. The oxidized state of the mitochondrial enzyme, Cytochrome C Oxidase (CCO) increased (46.3%, p ≤ 0.001) while the reduced state decreased (-34.4%, p ≤ 0.05) significantly in cuprizone mice. The total amount of CCO did not change significantly during cuprizone exposure. Total CCO did decline after recovery both in control (-23.1%, p ≤ 0.01) and cuprizone (-28.8%, p ≤ 0.001) groups which may relate to age. A reduction in the magnetization transfer ratio, indicating demyelination, was found in the cuprizone group in the cerebral cortex (-3.2%, p ≤ 0.01) and corpus callosum (-5.5%, p ≤ 0.001). In summary, we were able to detect evidence of altered CCO metabolism during cuprizone exposure, consistent with a mitochondrial defect. We observed increased oxygenation and reduced metabolic rate associated with reduced myelination in the gray and white matter. The novel multimodal imaging technique applied here shows promise for noninvasively assessing parameters associated with oxidative metabolism in both mouse models of neurological disease and for translation to study oxidative metabolism in the human brain.

Encapsulation of bryostatin-1 by targeted exosomes enhances remyelination and neuroprotection effects in the cuprizone-induced demyelinating animal model of multiple sclerosis.

Demyelination is a critical neurological disease, and there is still a lack of effective treatment methods. In the past two decades, stem cells have emerged as a novel therapeutic effector for neural regeneration. However, owing to the existence of the blood-brain barrier (BBB) and the complex microenvironment, targeted therapy still faces multiple challenges. Targeted exosome carriers for drug delivery may be considered a promising therapeutic method. Exosomes were isolated from mice neural stem cells. To develop targeting exosomes, we generated a lentivirus armed PDGFRα ligand that could anchor the membrane. Exosome targeting tests were carried out in vitro and in vivo. The modified exosomes showed an apparent ability to target OPCs in the lesion area. Next, the exosomes were loaded with Bryostatin-1 (Bryo), and the cuprizone-fed mice were administered with the targeting exosomes. The data show that Bryo exhibits a powerful therapeutic effect compared with Bryo alone after exosome encapsulation. Specifically, this novel exosome-based targeting delivery of Bryo significantly improves the protection ability of the myelin sheath and promotes remyelination. Moreover, it blocks astrogliosis and axon damage, and also has an inhibitory effect on pro-inflammatory microglia. The results of this investigation provide a straightforward strategy to produce targeting exosomes and indicate a potential therapeutic approach for demyelinating disease.

A novel probiotic strain exerts therapeutic effects on mouse model of multiple sclerosis by altering the expression of inflammasome and IDO genes and modulation of T helper cytokine profile.

Multiple sclerosis is an inflammatory demyelinating disease that commences to neuronal cell destruction. Recently, a promising evidence of synergic effects of combined supplementation with vitamin D and probiotics in modulating the gut microbiota and metabolome is emerging. Bacillus Coagulans IBRC-M10791 as a novel strain was chosen, prevention and treatment impacts of regular administered were studied in Cuprizone-induced C57bl/6 mouse of demyelination. The mice were divided into six groups and received a daily dose of cuprizone or probiotics. To investigate the effect of probiotic, the IDO-1, CYP27B1, NLRP1, NLRP3, and AIM2 expression were estimated by Real-Time PCR, and IL-4, IL-17, IFN-gamma, and TGF-beta cytokines were measured by ELISA. The results showed that there was significant decrease in IL-17 and IFN-γ and modulatory effects on IL-4 and TGF-ß. On the other hand, we demonstrated that there are significant decrease for expression of IDO-1, CYP27b1, NLRP1, NLRP3 and AIM2 genes in prevention and treatment groups compared to cuprizone group. Also, a significant enhancement in rate of remyelination and alternations proved by LFB staining and Y-Maze test. In conclusion, our study provides insight into how the therapeutic effect of the chosen strain of probiotic was correlated with the modulation of the level of inflammatory and anti-inflammatory cytokines. Further, we demonstrated that the expression of genes related to Tryptophan, Vitamin D and Inflammasome pathways could be affected by B.coagulans. Our study could be beneficial to provide a novel Co-therapeutic strategy for Multiple sclerosis.

Rg1 exerts protective effect in CPZ-induced demyelination mouse model via inhibiting CXCL10-mediated glial response.

Myelin damage and abnormal remyelination processes lead to central nervous system dysfunction. Glial activation-induced microenvironment changes are characteristic features of the diseases with myelin abnormalities. We previously showed that ginsenoside Rg1, a main component of ginseng, ameliorated MPTP-mediated myelin damage in mice, but the underlying mechanisms are unclear. In this study we investigated the effects of Rg1 and mechanisms in cuprizone (CPZ)-induced demyelination mouse model. Mice were treated with CPZ solution (300 mg· kg-1· d-1, ig) for 5 weeks; from week 2, the mice received Rg1 (5, 10, and 20 mg· kg-1· d-1, ig) for 4 weeks. We showed that Rg1 administration dose-dependently alleviated bradykinesia and improved CPZ-disrupted motor coordination ability in CPZ-treated mice. Furthermore, Rg1 administration significantly decreased demyelination and axonal injury in pathological assays. We further revealed that the neuroprotective effects of Rg1 were associated with inhibiting CXCL10-mediated modulation of glial response, which was mediated by NF-κB nuclear translocation and CXCL10 promoter activation. In microglial cell line BV-2, we demonstrated that the effects of Rg1 on pro-inflammatory and migratory phenotypes of microglia were related to CXCL10, while Rg1-induced phagocytosis of microglia was not directly related to CXCL10. In CPZ-induced demyelination mouse model, injection of AAV-CXCL10 shRNA into mouse lateral ventricles 3 weeks prior CPZ treatment occluded the beneficial effects of Rg1 administration in behavioral and pathological assays. In conclusion, CXCL10 mediates the protective role of Rg1 in CPZ-induced demyelination mouse model. This study provides new insight into potential disease-modifying therapies for myelin abnormalities.

Cuprizone feed formulation influences the extent of demyelinating disease pathology.

Cuprizone is a copper-chelating agent that induces pathology similar to that within some multiple sclerosis (MS) lesions. The reliability and reproducibility of cuprizone for inducing demyelinating disease pathology depends on the animals ingesting consistent doses of cuprizone. Cuprizone-containing pelleted feed is a convenient way of delivering cuprizone, but the efficacy of these pellets at inducing demyelination has been questioned. This study compared the degree of demyelinating disease pathology between mice fed cuprizone delivered in pellets to mice fed a powdered cuprizone formulation at an early 3 week demyelinating timepoint. Within rostral corpus callosum, cuprizone pellets were more effective than cuprizone powder at increasing astrogliosis, microglial activation, DNA damage, and decreasing the density of mature oligodendrocytes. However, cuprizone powder demonstrated greater protein nitration relative to controls. Furthermore, mice fed control powder had significantly fewer mature oligodendrocytes than those fed control pellets. In caudal corpus callosum, cuprizone pellets performed better than cuprizone powder relative to controls at increasing astrogliosis, microglial activation, protein nitration, DNA damage, tissue swelling, and reducing the density of mature oligodendrocytes. Importantly, only cuprizone pellets induced detectable demyelination compared to controls. The two feeds had similar effects on oligodendrocyte precursor cell (OPC) dynamics. Taken together, these data suggest that demyelinating disease pathology is modelled more effectively with cuprizone pellets than powder at 3 weeks. Combined with the added convenience, cuprizone pellets are a suitable choice for inducing early demyelinating disease pathology.

PARP1-mediated PARylation activity is essential for oligodendroglial differentiation and CNS myelination.

The function of poly(ADP-ribosyl) polymerase 1 (PARP1) in myelination and remyelination of the central nervous system (CNS) remains enigmatic. Here, we report that PARP1 is an intrinsic driver for oligodendroglial development and myelination. Genetic PARP1 depletion impairs the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes and impedes CNS myelination. Mechanistically, PARP1-mediated PARylation activity is not only necessary but also sufficient for OPC differentiation. At the molecular level, we identify the RNA-binding protein Myef2 as a PARylated target, which controls OPC differentiation through the PARylation-modulated derepression of myelin protein expression. Furthermore, PARP1's enzymatic activity is necessary for oligodendrocyte and myelin regeneration after demyelination. Together, our findings suggest that PARP1-mediated PARylation activity may be a potential therapeutic target for promoting OPC differentiation and remyelination in neurological disorders characterized by arrested OPC differentiation and remyelination failure such as multiple sclerosis.

Temporal and spatial evolution of various functional neurons during demyelination induced by cuprizone.

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS). Here we report the temporal and spatial evolution of various functional neurons during demyelination in a cuprizone (CPZ)-induced mouse model. CPZ did not significantly induce the damage of axons and neurons after 2 wk of feeding. However, after 4-6 wk of CPZ feeding, axons and neurons were markedly reduced in the cortex, posterior thalamic nuclear group, and hippocampus. Simultaneously, the expression of TPH+ tryptophan neurons and VGLUT1+ glutamate neurons was obviously decreased, and the expression of TH+ dopaminergic neurons was slightly decreased in the tail part of the substantia nigra striatum, whereas the number of ChAT+ cholinergic neurons was not significantly different in the brain. In the second week of feeding, CPZ caused a higher level of glutamate secretion and upregulated the expression of EAAT2 on astrocytes, which should contribute to rapid and sufficient glutamate uptake and removal. This finding reveals that astrocyte-driven glutamate reuptake protected the CNS from excitotoxicity by rapid reuptake of glutamate in 4-6 wk of CPZ feeding. At this stage, although NG2+ oligodendroglia progenitor cells (OPCs) were enhanced in the demyelination foci, the myelin sheath was still absent. In conclusion, we comprehensively observed the temporal and spatial evolution of various functional neurons. Our results will assist with understanding how demyelination affects neurons during CPZ-induced demyelination and provide novel information for neuroprotection in myelin regeneration and demyelinating diseases.NEW & NOTEWORTHY Our results further indicate temporal and spatial evolution of various functional neurons during the demyelination in a cuprizone (CPZ)-induced mouse model, which mainly occur 4-6 wk after CPZ feeding. At the same time, the axonal compartment is damaged and, consequently, neuronal death occurs, while glutamate neurons are lost obviously. The astrocyte-mediated glutamate reuptake could protect the neurons from the excitatory effects of glutamate.

Metformin Therapy Attenuates Pro-inflammatory Microglia by Inhibiting NF-κB in Cuprizone Demyelinating Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic disorder characterized by reactive gliosis, inflammation, and demyelination. Microglia plays a crucial role in the pathogenesis of MS and has the dynamic plasticity to polarize between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Metformin, a glucose-lowering drug, attenuates inflammatory responses by activating adenosine monophosphate protein kinase (AMPK) which suppresses nuclear factor kappa B (NF-κB). In this study, we indirectly investigated whether metformin therapy would regulate microglia activity in the cuprizone (CPZ)-induced demyelination mouse model of MS via measuring the markers associated with pro- and anti-inflammatory microglia. Evaluation of myelin by luxol fast blue staining revealed that metformin treatment (CPZ + Met) diminished demyelination, in comparison to CPZ mice. In addition, metformin therapy significantly alleviated reactive microgliosis and astrogliosis in the corpus callosum, as measured by Iba-1 and GFAP staining. Moreover, metformin treatment significantly downregulated the expression of pro-inflammatory associated genes (iNOS, H2-Aa, and TNF-α) in the corpus callosum, whereas expression of anti-inflammatory markers (Arg1, Mrc1, and IL10) was not promoted, compared to CPZ mice. Furthermore, protein levels of iNOS (pro-inflammatory marker) were significantly decreased in the metformin group, while those of Trem2 (anti-inflammatory marker) were increased. In addition, metformin significantly increased AMPK activation in CPZ mice. Finally, metformin administration significantly reduced the activation level of NF-κB in CPZ mice. In summary, our data revealed that metformin attenuated pro-inflammatory microglia markers through suppressing NF-κB activity. The positive effects of metformin on microglia and remyelination suggest that it could be used as a promising candidate to lessen the incidence of inflammatory neurodegenerative diseases such as MS.

A higher proportion of ermin-immunopositive oligodendrocytes in areas of remyelination.

Incomplete remyelination is frequent in multiple sclerosis (MS)-lesions, but there is no established marker for recent remyelination. We investigated the role of the oligodendrocyte/myelin protein ermin in de- and remyelination in the cuprizone (CPZ) mouse model, and in MS. The density of ermin+ oligodendrocytes in the brain was significantly decreased after one week of CPZ exposure (p < 0.02). The relative proportion of ermin+ cells compared to cells positive for the late-stage oligodendrocyte marker Nogo-A increased at the onset of remyelination in the corpus callosum (p < 0.02). The density of ermin-positive cells increased in the corpus callosum during the CPZ-phase of extensive remyelination (p < 0.0001). In MS, the density of ermin+ cells was higher in remyelinated lesion areas compared to non-remyelinated areas both in white- (p < 0.0001) and grey matter (p < 0.0001) and compared to normal-appearing white matter (p < 0.001). Ermin immunopositive cells in MS-lesions were not immunopositive for the early-stage oligodendrocyte markers O4 and O1, but a subpopulation was immunopositive for Nogo-A. The data suggest a relatively higher proportion of ermin immunopositivity in oligodendrocytes compared to Nogo-A indicates recent or ongoing remyelination.

Neuroprotective effect of newly synthesized 4-aminopyridine derivatives on cuprizone-induced demyelination in mice-a behavioral and immunohistochemical study.

The aim of this study was to assess the effect of newly synthesized derivatives of 4-aminopyridine (4-AP) on cuprizone-induced model of brain demyelination in mice. 4-AP is already approved for the treatment of walking difficulties in patients with multiple sclerosis. The model of demyelination was carried out by the administration of cuprizone to the drinking water of the experimental mice. Besides cuprizone, 4-AP derivatives and 4-AP were administered to the groups in order to assess their protective effect on the demyelination. We used immunohistochemistry for visualization of changes in corpus callosum. Memory storage processes were also assessed with the passive avoidance test on the last two days of the experiment. The experimental mice treated with compounds 4b and 4c increased significantly their latency time on the second day in comparison to the control group which indicated an improved memory process. The number of mature oligodendrocytes in the groups treated with compounds 4b, 4c and 4-AP is closer to those in the control group. The results of our studies showed that the newly synthesized compounds 4b and 4c reverse the effect of cuprizone. These groups also showed increased latency time in the passive avoidance test in comparison to the control group.