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Background The dietary consumption of cuprizone – a copper chelator – has long been known to induce demyelination of specific brain structures and is widely used as model of multiple sclerosis. Despite the extensive use of cuprizone, the mechanism by which it induces demyelination are still unknown. With this review we provide an updated understanding of this model, by showcasing two distinct yet overlapping modes of action for cuprizone-induced demyelination; 1) damage originating from within the oligodendrocyte, caused by mitochondrial dysfunction or reduced myelin protein synthesis. We term this mode of action ‘intrinsic cell damage’. And 2) damage to the oligodendrocyte exerted by inflammatory molecules, brain resident cells, such as oligodendrocytes, astrocytes, and microglia or peripheral immune cells – neutrophils or T-cells. We term this mode of action ‘extrinsic cellular damage’. Lastly, we summarize recent developments in research on different forms of cell death induced by cuprizone, which could add valuable insights into the mechanisms of cuprizone toxicity. With this review we hope to provide a modern understanding of cuprizone-induced demyelination to understand the causes behind the demyelination in MS.

Background Microglia regulate the response to injury and disease in the brain and spinal cord. In white matter diseases microglia may cause demyelination. However, how microglia respond and regulate demyelination is not fully understood. Methods To understand how microglia respond during demyelination, we fed mice cuprizone—a potent demyelinating agent—and assessed the dynamics of genetically fate-mapped microglia. We then used single-cell RNA sequencing to identify and track the microglial subpopulations that arise during demyelination. To understand how microglia contribute to the clearance of dead oligodendrocytes, we ablated microglia starting at the peak of cuprizone-induced cell death and used the viability dye acridine orange to monitor apoptotic and lytic cell morphologies after microglial ablation. Lastly, we treated serum-free primary microglial cultures to model distinct aspects of cuprizone-induced demyelination and assessed the response. Results The cuprizone diet generated a robust microglial response by week 4 of the diet. Single-cell RNA sequencing at this time point revealed the presence of several cuprizone-associated microglia (CAM) clusters. These clusters expressed a transcriptomic signature indicative of cytokine regulation and reactive oxygen species production with altered lysosomal and metabolic changes consistent with ongoing phagocytosis. Using acridine orange to monitor apoptotic and lytic cell death after microglial ablation, we found that microglia preferentially phagocytose lytic carcasses. In culture, microglia exposed to lytic carcasses partially recapitulated the CAM state, suggesting that phagocytosis contributes to this distinct microglial state during cuprizone demyelination. Conclusions Microglia serve multiple roles during demyelination, yet their transcriptomic state resembles other neurodegenerative conditions. The phagocytosis of cellular debris is likely a universal cause for a common neurodegenerative microglial state.

Heterozygous mutations in the granulin ( GRN ) gene, resulting in the haploinsufficiency of the progranulin (PGRN) protein, is a leading cause of frontotemporal lobar degeneration (FTLD). Complete loss of the PGRN protein causes neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder. Polymorphisms in the GRN gene have also been associated with several other neurodegenerative diseases, including Alzheimer’s disease (AD), and Parkinson’s disease (PD). PGRN deficiency has been shown to cause myelination defects previously, but how PGRN regulates myelination is unknown. Here, we report that PGRN deficiency leads to a sex-dependent myelination defect with male mice showing more severe demyelination in response to cuprizone treatment. This is accompanied by exacerbated microglial proliferation and activation in the male PGRN-deficient mice. Interestingly, both male and female PGRN-deficient mice show sustained microglial activation after cuprizone removal and a defect in remyelination. Specific ablation of PGRN in microglia results in similar sex-dependent phenotypes, confirming a microglial function of PGRN. Lipid droplets accumulate in microglia specifically in male PGRN-deficient mice. RNA-seq analysis and mitochondrial function assays reveal key differences in oxidative phosphorylation in male versus female microglia under PGRN deficiency. A significant decrease in myelination and accumulation of myelin debris and lipid droplets in microglia were found in the corpus callosum regions of FTLD patients with GRN mutations. Taken together, our data support that PGRN deficiency leads to sex-dependent alterations in microglia with subsequent myelination defects.

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 1β (IL-1β) 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.

Oligodendrocyte loss and myelin sheet destruction are crucial characteristics of demyelinating diseases. Phenytoin promotes the proliferation of endogenous neural precursor cells in the ventricular–subventricular zone in the postnatal brain that help restore the oligodendroglial population. This study aimed to evaluate whether phenytoin promotes myelin recovery of the corpus callosum of demyelinated adult mice. CD1 male mice were exposed to a demyelinating agent (0.2% cuprizone) for 8 weeks. We assembled two groups: the phenytoin-treated group and the control-vehicle group. The treated group received oral phenytoin (10 mg/kg) for 4 weeks. We quantified the number of Olig2 + and NG2 + oligodendrocyte precursor cells (OPCs), Rip + oligodendrocytes, the expression level of myelin basic protein (MBP), and the muscle strength and motor coordination. The oligodendroglial lineage (Olig2 + cells, NG2 + cells, and RIP + cells) significantly increases by the phenytoin administration when compared to the control-vehicle group. The phenytoin-treated group also showed an increased expression of MBP in the corpus callosum and better functional scores in the horizontal bar test. These findings suggest that phenytoin stimulates the proliferation of OPCs, re-establishes the oligodendroglial population, promotes myelin recovery in the corpus callosum, and improves motor coordination and muscle strength.

The TREM2 R47H variant is one of the strongest genetic risk factors for late-onset Alzheimer's Disease (AD). Unfortunately, many current Trem2 mouse models are associated with cryptic mRNA splicing of the mutant allele that produces a confounding reduction in protein product. To overcome this issue, we developed the Trem2 (Normal Splice Site) mouse model in which the Trem2 allele is expressed at a similar level to the wild-type Trem2 allele without evidence of cryptic splicing products. Trem2 mice were treated with the demyelinating agent cuprizone, or crossed with the 5xFAD mouse model of amyloidosis, to explore the impact of the TREM2 R47H variant on inflammatory responses to demyelination, plaque development, and the brain's response to plaques. Trem2 mice display an appropriate inflammatory response to cuprizone challenge, and do not recapitulate the null allele in terms of impeded inflammatory responses to demyelination. Utilizing the 5xFAD mouse model, we report age- and disease-dependent changes in Trem2 mice in response to development of AD-like pathology. At an early (4-month-old) disease stage, hemizygous 5xFAD/homozygous Trem2 (5xFAD/Trem2 ) mice have reduced size and number of microglia that display impaired interaction with plaques compared to microglia in age-matched 5xFAD hemizygous controls. This is associated with a suppressed inflammatory response but increased dystrophic neurites and axonal damage as measured by plasma neurofilament light chain (NfL) level. Homozygosity for Trem2 suppressed LTP deficits and loss of presynaptic puncta caused by the 5xFAD transgene array in 4-month-old mice. At a more advanced (12-month-old) disease stage 5xFAD/Trem2 mice no longer display impaired plaque-microglia interaction or suppressed inflammatory gene expression, although NfL levels remain elevated, and a unique interferon-related gene expression signature is seen. Twelve-month old Trem2 mice also display LTP deficits and postsynaptic loss. The Trem2 mouse is a valuable model that can be used to investigate age-dependent effects of the AD-risk R47H mutation on TREM2 and microglial function including its effects on plaque development, microglial-plaque interaction, production of a unique interferon signature and associated tissue damage.

Microglia are phagocytic cells that survey the brain and perform neuroprotective functions in response to tissue damage, but their activating receptors are largely unknown. Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial immunoreceptor whose loss-of-function mutations in humans cause presenile dementia, while genetic variants are associated with increased risk of neurodegenerative diseases. In myeloid cells, TREM2 has been involved in the regulation of phagocytosis, cell proliferation and inflammatory responses in vitro. However, it is unknown how TREM2 contributes to microglia function in vivo. Here, we identify a critical role for TREM2 in the activation and function of microglia during cuprizone (CPZ)-induced demyelination. TREM2-deficient (TREM2 −/− ) mice had defective clearance of myelin debris and more axonal pathology, resulting in impaired clinical performances compared to wild-type (WT) mice. TREM2 −/− microglia proliferated less in areas of demyelination and were less activated, displaying a more resting morphology and decreased expression of the activation markers MHC II and inducible nitric oxide synthase as compared to WT. Mechanistically, gene expression and ultrastructural analysis of microglia suggested a defect in myelin degradation and phagosome processing during CPZ intoxication in TREM2 −/− microglia. These findings place TREM2 as a key regulator of microglia activation in vivo in response to tissue damage.

•Oxidative metabolism is investigated in the cuprizone mouse model of demyelination.•Demyelination in gray matter is associated with reduced OEF and metabolic rate.•Cuprizone mitochondria show abnormal redox state and ATP-linked respiration.•Evidence for metabolic hypoxia is found during demyelination in gray matter.•Metabolic rate and mitochondrial status recover post cuprizone during remyelination. 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.

•IhMT is a sensitive biomarker of de-/remyelination in the acute cuprizone mouse model.•IhMT targeted toward long T1D components correlates with myelin histology.•IhMT targeted toward short T1D components correlates less with myelin histology.•Spatial-temporal extent of cuprizone-induced myelin alterations is best described by ihMT T1D-filters.•MPF correlates with myelin histology, but is less sensitive than ihMT to characterize the spatial extent of demyelination in the acute cuprizone model. To investigate the association of ihMT (inhom signals with the demyelination and remyelination phases of the acute cuprizone mouse model in comparison with histology, and to assess the extent of tissue damage and repair from MRI data. Acute demyelination by feeding 0.2% cuprizone for five weeks, followed by a four-week remyelination period was applied on genetically modified plp-GFP mice. Animals were scanned at different time points of the demyelination and remyelination phases of the cuprizone model using a multimodal MRI protocol, including ihMT T1D-filters, MPF (Macromolecular Proton Fraction) and R1 (longitudinal relaxation rate). For histology, plp-GFP (proteolipid protein – Green Fluorescent Protein) microscopy and LFB (Luxol Fast Blue) staining were employed as references for the myelin content. Comparison of MRI with histology was performed in the medial corpus callosum (mCC) and cerebral cortex (CTX) at two brain levels whereas ROI-wise and voxel-based analyses of the MRI metrics allowed investigating in vivo the spatial extent of myelin alterations. IhMT high-pass T1D-filters, targeted toward long T1D components, showed significant temporal variations in the mCC consistent with the effects induced by the cuprizone toxin. In addition, the corresponding signals correlated strongly and significantly with the myelin content assessed by GFP fluorescence and LFB staining over the demyelination and the remyelination phases. The signal of the band-pass T1D-filter, which isolates short T1D components, showed changes over time that were poorly correlated with histology, hence suggesting a sensitivity to pathological processes possibly not related to myelin. Although MPF was also highly correlated to histology, ihMT high-pass T1D-filters showed better capability to characterize the spatial-temporal patterns during the demyelination and remyelination phases of the acute cuprizone model (e.g., rostro-caudal gradient of demyelination in the mCC previously described in the literature). IhMT sequences selective for long T1D components are specific and sensitive in vivo markers of demyelination and remyelination and have successfully captured the spatially heterogeneous pattern of the demyelination and remyelination mechanisms in the cuprizone model. Interestingly, differences in signal variations between the ihMT high-pass and band-pass T1D-filter, suggest a sensitivity of the ihMT sequences targeted to short T1Ds to alterations other than those of myelin. Future studies will need to further address these differences by examining more closely the origin of the short T1D components and the variation of each T1D component in pathology.

Progressive phases of multiple sclerosis are associated with inhibited differentiation of the progenitor cell population that generates the mature oligodendrocytes required for remyelination and disease remission. To identify selective inducers of oligodendrocyte differentiation, we performed an image-based screen for myelin basic protein (MBP) expression using primary rat optic-nerve-derived progenitor cells. Here we show that among the most effective compounds identifed was benztropine, which significantly decreases clinical severity in the experimental autoimmune encephalomyelitis (EAE) model of relapsing-remitting multiple sclerosis when administered alone or in combination with approved immunosuppressive treatments for multiple sclerosis. Evidence from a cuprizone-induced model of demyelination, in vitro and in vivo T-cell assays and EAE adoptive transfer experiments indicated that the observed efficacy of this drug results directly from an enhancement of remyelination rather than immune suppression. Pharmacological studies indicate that benztropine functions by a mechanism that involves direct antagonism of M1 and/or M3 muscarinic receptors. These studies should facilitate the development of effective new therapies for the treatment of multiple sclerosis that complement established immunosuppressive approaches. Multiple sclerosis is associated with a deficiency in generation of mature oligodendroctyes; an image-based screen for oligodendrocyte differentiation inducers identified the compound benztropine, which enhances remyelination acting through muscarinic receptors and decreases clinical severity in a multiple sclerosis model system. Regenerative therapy for multiple sclerosis Peter Schultz and colleagues have performed a high-throughput, image-based screen with rat oligodendrocyte precursor cells (OPCs) of small molecules that enhance OPC in vitro differentiation. Using this approach they have identified and characterized small molecule drugs able to foster oligodendrocyte-based remyelination in disorders such as multiple sclerosis. The principal compound identified is benztropine, a drug licensed for the treatment of Parkinson's disease. Benztropine has efficacy in vivo in two models of demyelination disorders, with no measurable effects on the immune system.