Oligodendroglial fatty acid metabolism as a central nervous system energy reserve.

Brain function requires a constant supply of glucose. However, the brain has no known energy stores, except for glycogen granules in astrocytes. In the present study, we report that continuous oligodendroglial lipid metabolism provides an energy reserve in white matter tracts. In the isolated optic nerve from young adult mice of both sexes, oligodendrocytes survive glucose deprivation better than astrocytes. Under low glucose, both axonal ATP levels and action potentials become dependent on fatty acid ß-oxidation. Importantly, ongoing oligodendroglial lipid degradation feeds rapidly into white matter energy metabolism. Although not supporting high-frequency spiking, fatty acid ß-oxidation in mitochondria and oligodendroglial peroxisomes protects axons from conduction blocks when glucose is limiting. Disruption of the glucose transporter GLUT1 expression in oligodendrocytes of adult mice perturbs myelin homeostasis in vivo and causes gradual demyelination without behavioral signs. This further suggests that the imbalance of myelin synthesis and degradation can underlie myelin thinning in aging and disease.

CCR2+ monocytes promote white matter injury and cognitive dysfunction after myocardial infarction.

Survivors of myocardial infarction are at increased risk for vascular dementia. Neuroinflammation has been implicated in the pathogenesis of vascular dementia, yet little is known about the cellular and molecular mediators of neuroinflammation after myocardial infarction. Using a mouse model of myocardial infarction coupled with flow cytometric analyses and immunohistochemistry, we discovered increased monocyte abundance in the brain after myocardial infarction, which was associated with increases in brain-resident perivascular macrophages and microglia. Myeloid cell recruitment and activation was also observed in post-mortem brains of humans that died after myocardial infarction. Spatial and single cell transcriptomic profiling of brain-resident myeloid cells after experimental myocardial infarction revealed increased expression of monocyte chemoattractant proteins. In parallel, myocardial infarction increased crosstalk between brain-resident myeloid cells and oligodendrocytes, leading to neuroinflammation, white matter injury, and cognitive dysfunction. Inhibition of monocyte recruitment preserved white matter integrity and cognitive function, linking monocytes to neurodegeneration after myocardial infarction. Together, these preclinical and clinical results demonstrate that monocyte infiltration into the brain after myocardial infarction initiate neuropathological events that lead to vascular dementia.

Interferon-γ controls aquaporin 4-specific Th17 and B cells in neuromyelitis optica spectrum disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is a CNS autoimmune inflammatory disease mediated by T helper 17 (Th17) and antibody responses to the water channel protein, aquaporin 4 (AQP4), and associated with astrocytopathy, demyelination and axonal loss. Knowledge about disease pathogenesis is limited and the search for new therapies impeded by the absence of a reliable animal model. In our work, we determined that NMOSD is characterized by decreased IFN-γ receptor signalling and that IFN-γ depletion in AQP4201-220-immunized C57BL/6 mice results in severe clinical disease resembling human NMOSD. Pathologically, the disease causes autoimmune astrocytic and CNS injury secondary to cellular and humoral inflammation. Immunologically, the absence of IFN-γ allows for increased expression of IL-6 in B cells and activation of Th17 cells, and generation of a robust autoimmune inflammatory response. Consistent with NMOSD, the experimental disease is exacerbated by administration of IFN-ß, whereas repletion of IFN-γ, as well as therapeutic targeting of IL-17A, IL-6R and B cells, ameliorates it. We also demonstrate that immune tolerization with AQP4201-220-coupled poly(lactic-co-glycolic acid) nanoparticles could both prevent and effectively treat the disease. Our findings enhance the understanding of NMOSD pathogenesis and provide a platform for the development of immune tolerance-based therapies, avoiding the limitations of the current immunosuppressive therapies.

The bone transcription factor Osterix controls extracellular matrix- and node of Ranvier-related gene expression in oligodendrocytes.

Oligodendrocytes are the primary producers of many extracellular matrix (ECM)-related proteins found in the CNS. Therefore, oligodendrocytes play a critical role in the determination of brain stiffness, node of Ranvier formation, perinodal ECM deposition, and perineuronal net formation, all of which depend on the ECM. Nevertheless, the transcription factors that control ECM-related gene expression in oligodendrocytes remain unknown. Here, we found that the transcription factor Osterix (also known as Sp7) binds in proximity to genes important for CNS ECM and node of Ranvier formation and mediates their expression. Oligodendrocyte-specific ablation of Sp7 changes ECM composition and brain stiffness and results in aberrant node of Ranvier formation. Sp7 is known to control osteoblast maturation and bone formation. Our comparative analyses suggest that Sp7 plays a conserved biological role in oligodendrocytes and in bone-forming cells, where it mediates brain and bone tissue stiffness by controlling expression of ECM components.

Insights into the mechanism of oligodendrocyte protection and remyelination enhancement by the integrated stress response.

central nervous system (CNS) inflammation triggers activation of the integrated stress response (ISR). We previously reported that prolonging the ISR protects remyelinating oligodendrocytes and promotes remyelination in the presence of inflammation. However, the exact mechanisms through which this occurs remain unknown. Here, we investigated whether the ISR modulator Sephin1 in combination with the oligodendrocyte differentiation enhancing reagent bazedoxifene (BZA) is able to accelerate remyelination under inflammation, and the underlying mechanisms mediating this pathway. We find that the combined treatment of Sephin1 and BZA is sufficient to accelerate early-stage remyelination in mice with ectopic IFN-γ expression in the CNS. IFN-γ, which is a critical inflammatory cytokine in multiple sclerosis (MS), inhibits oligodendrocyte precursor cell (OPC) differentiation in culture and triggers a mild ISR. Mechanistically, we further show that BZA promotes OPC differentiation in the presence of IFN-γ, while Sephin1 enhances the IFN-γ-induced ISR by reducing protein synthesis and increasing RNA stress granule formation in differentiating oligodendrocytes. Finally, pharmacological suppression of the ISR blocks stress granule formation in vitro and partially lessens the beneficial effect of Sephin1 on disease progression in a mouse model of MS, experimental autoimmune encephalitis (EAE). Overall, our findings uncover distinct mechanisms of action of BZA and Sephin1 on oligodendrocyte lineage cells under inflammatory stress, suggesting that a combination therapy may effectively promote restoring neuronal function in MS patients.

Insights into the mechanism of oligodendrocyte protection and remyelination enhancement by the integrated stress response.

CNS inflammation triggers activation of the integrated stress response (ISR). We previously reported that prolonging the ISR protects remyelinating oligodendrocytes and promotes remyelination in the presence of inflammation (Chen et al., eLife , 2021). However, the exact mechanisms through which this occurs remain unknown. Here, we investigated whether the ISR modulator Sephin1 in combination with the oligodendrocyte differentiation enhancing reagent bazedoxifene (BZA) is able to accelerate remyelination under inflammation, and the underlying mechanisms mediating this pathway. We find that the combined treatment of Sephin1 and BZA is sufficient to accelerate early-stage remyelination in mice with ectopic IFN-γ expression in the CNS. IFN-γ, which is a critical inflammatory cytokine in multiple sclerosis (MS), inhibits oligodendrocyte precursor cell (OPC) differentiation in culture and triggers a mild ISR. Mechanistically, we further show that BZA promotes OPC differentiation in the presence of IFN-γ, while Sephin1 enhances the IFN-γ-induced ISR by reducing protein synthesis and increasing RNA stress granule formation in differentiating oligodendrocytes. Finally, the ISR suppressor 2BAct is able to partially lessen the beneficial effect of Sephin1 on disease progression, in an MS mouse model of experimental autoimmune encephalitis (EAE). Overall, our findings uncover distinct mechanisms of action of BZA and Sephin1 on oligodendrocyte lineage cells under inflammatory stress, suggesting that a combination therapy may effectively promote restoring neuronal function in MS patients.

Repurposing the cardiac glycoside digoxin to stimulate myelin regeneration in chemically-induced and immune-mediated mouse models of multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by inflammation, demyelination, and neurodegeneration. The ideal MS therapy would both specifically inhibit the underlying autoimmune response and promote repair/regeneration of myelin as well as maintenance of axonal integrity. Currently approved MS therapies consist of non-specific immunosuppressive molecules/antibodies which block activation or CNS homing of autoreactive T cells, but there are no approved therapies for stimulation of remyelination nor maintenance of axonal integrity. In an effort to repurpose an FDA-approved medication for myelin repair, we chose to examine the effectiveness of digoxin, a cardiac glycoside (Na+ /K+ ATPase inhibitor), originally identified as pro-myelinating in an in vitro screen. We found that digoxin regulated multiple genes in oligodendrocyte progenitor cells (OPCs) essential for oligodendrocyte (OL) differentiation in vitro, promoted OL differentiation both in vitro and in vivo in female naïve C57BL/6J (B6) mice, and stimulated recovery of myelinated axons in B6 mice following demyelination in the corpus callosum induced by cuprizone and spinal cord demyelination induced by lysophosphatidylcholine (LPC), respectively. More relevant to treatment of MS, we show that digoxin treatment of mice with established MOG35-55 -induced Th1/Th17-mediated chronic EAE combined with tolerance induced by the i.v. infusion of biodegradable poly(lactide-co-glycolide) nanoparticles coupled with MOG35-55 (PLG-MOG35-55 ) completely ameliorated clinical disease symptoms and stimulated recovery of OL lineage cell numbers. These findings provide critical pre-clinical evidence supporting future clinical trials of myelin-specific tolerance with myelin repair/regeneration drugs, such as digoxin, in MS patients.

Sensory neurons display cell-type-specific vulnerability to loss of neuron-glia interactions.

Peripheral nervous system (PNS) injuries initiate transcriptional changes in glial cells and sensory neurons that promote axonal regeneration. While the factors that initiate the transcriptional changes in glial cells are well characterized, the full range of stimuli that initiate the response of sensory neurons remain elusive. Here, using a genetic model of glial cell ablation, we find that glial cell loss results in transient PNS demyelination without overt axonal loss. By profiling sensory ganglia at single-cell resolution, we show that glial cell loss induces a transcriptional injury response preferentially in proprioceptive and Aß RA-LTMR neurons. The transcriptional response of sensory neurons to mechanical injury has been assumed to be a cell-autonomous response. By identifying a similar response in non-injured, demyelinated neurons, our study suggests that this represents a non-cell-autonomous transcriptional response of sensory neurons to glial cell loss and demyelination.

Resolving inflammatory links between myocardial infarction and vascular dementia.

Myocardial infarction is associated with increased risk for vascular dementia. In both myocardial infarction and vascular dementia, there is evidence that elevated inflammatory biomarkers are associated with worsened clinical outcomes. Myocardial infarction leads to a systemic inflammatory response, which may contribute to recruitment or activation of myeloid cells, including monocytes, microglia, and perivascular macrophages, within the central nervous system. However, our understanding of the causative roles for these cells linking cardiac injury to the development and progression of dementia is incomplete. Herein, we provide an overview of inflammatory cellular and molecular links between myocardial infarction and vascular dementia and discuss strategies to resolve inflammation after myocardial infarction to limit neurovascular injury.

Prolonging the integrated stress response enhances CNS remyelination in an inflammatory environment.

The inflammatory environment of demyelinated lesions in multiple sclerosis (MS) patients contributes to remyelination failure. Inflammation activates a cytoprotective pathway, the integrated stress response (ISR), but it remains unclear whether enhancing the ISR can improve remyelination in an inflammatory environment. To examine this possibility, the remyelination stage of experimental autoimmune encephalomyelitis (EAE), as well as a mouse model that incorporates cuprizone-induced demyelination along with CNS delivery of the proinflammatory cytokine IFN-γ were used here. We demonstrate that either genetic or pharmacological ISR enhancement significantly increased the number of remyelinating oligodendrocytes and remyelinated axons in the inflammatory lesions. Moreover, the combined treatment of the ISR modulator Sephin1 with the oligodendrocyte differentiation enhancing reagent bazedoxifene increased myelin thickness of remyelinated axons to pre-lesion levels. Taken together, our findings indicate that prolonging the ISR protects remyelinating oligodendrocytes and promotes remyelination in the presence of inflammation, suggesting that ISR enhancement may provide reparative benefit to MS patients.

CNS fibroblasts form a fibrotic scar in response to immune cell infiltration.

Fibrosis is a common pathological response to inflammation in many peripheral tissues and can prevent tissue regeneration and repair. Here, we identified persistent fibrotic scarring in the CNS following immune cell infiltration in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Using lineage tracing and single-cell sequencing in EAE, we determined that the majority of the fibrotic scar is derived from proliferative CNS fibroblasts, not pericytes or infiltrating bone marrow-derived cells. Ablating proliferating fibrotic cells using cell-specific expression of herpes thymidine kinase led to an increase in oligodendrocyte lineage cells within the inflammatory lesions and a reduction in motor disability. We further identified that interferon-gamma pathway genes are enriched in CNS fibrotic cells, and the fibrotic cell-specific deletion of Ifngr1 resulted in reduced fibrotic scarring in EAE. These data delineate a framework for understanding the CNS fibrotic response.

Evaluation of the potassium channel tracer [18F]3F4AP in rhesus macaques.

Demyelination causes slowed or failed neuronal conduction and is a driver of disability in multiple sclerosis and other neurological diseases. Currently, the gold standard for imaging demyelination is MRI, but despite its high spatial resolution and sensitivity to demyelinated lesions, it remains challenging to obtain specific and quantitative measures of molecular changes involved in demyelination. To understand the contribution of demyelination in different diseases and to assess the efficacy of myelin-repair therapies, it is critical to develop new in vivo imaging tools sensitive to changes induced by demyelination. Upon demyelination, axonal K+ channels, normally located underneath the myelin sheath, become exposed and increase in expression, causing impaired conduction. Here, we investigate the properties of the K+ channel PET tracer [18F]3F4AP in primates and its sensitivity to a focal brain injury that occurred three years prior to imaging. [18F]3F4AP exhibited favorable properties for brain imaging including high brain penetration, high metabolic stability, high plasma availability, high reproducibility, high specificity, and fast kinetics. [18F]3F4AP showed preferential binding in areas of low myelin content as well as in the previously injured area. Sensitivity of [18F]3F4AP for the focal brain injury was higher than [18F]FDG, [11C]PiB, and [11C]PBR28, and compared favorably to currently used MRI methods.

Pre-clinical and Clinical Implications of "Inside-Out" vs. "Outside-In" Paradigms in Multiple Sclerosis Etiopathogenesis.

Multiple Sclerosis (MS) is an immune-mediated neurological disorder, characterized by central nervous system (CNS) inflammation, oligodendrocyte loss, demyelination, and axonal degeneration. Although autoimmunity, inflammatory demyelination and neurodegeneration underlie MS, the initiating event has yet to be clarified. Effective disease modifying therapies need to both regulate the immune system and promote restoration of neuronal function, including remyelination. The challenge in developing an effective long-lived therapy for MS requires that three disease-associated targets be addressed: (1) self-tolerance must be re-established to specifically inhibit the underlying myelin-directed autoimmune pathogenic mechanisms; (2) neurons must be protected from inflammatory injury and degeneration; (3) myelin repair must be engendered by stimulating oligodendrocyte progenitors to remyelinate CNS neuronal axons. The combined use of chronic and relapsing remitting experimental autoimmune encephalomyelitis (C-EAE, R-EAE) ("outside-in") as well as progressive diphtheria toxin A chain (DTA) and cuprizone autoimmune encephalitis (CAE) ("inside-out") mouse models allow for the investigation and specific targeting of all three of these MS-associated disease parameters. The "outside-in" EAE models initiated by myelin-specific autoreactive CD4+ T cells allow for the evaluation of both myelin-specific tolerance in the absence or presence of neuroprotective and/or remyelinating agents. The "inside-out" mouse models of secondary inflammatory demyelination are triggered by toxin-induced oligodendrocyte loss or subtle myelin damage, which allows evaluation of novel therapeutics that could promote remyelination and neuroprotection in the CNS. Overall, utilizing these complementary pre-clinical MS models will open new avenues for developing therapeutic interventions, tackling MS from the "outside-in" and/or "inside-out".

m6A mRNA Methylation Is Essential for Oligodendrocyte Maturation and CNS Myelination.

The molecular mechanisms that govern the maturation of oligodendrocyte lineage cells remain unclear. Emerging studies have shown that N6-methyladenosine (m6A), the most common internal RNA modification of mammalian mRNA, plays a critical role in various developmental processes. Here, we demonstrate that oligodendrocyte lineage progression is accompanied by dynamic changes in m6A modification on numerous transcripts. In vivo conditional inactivation of an essential m6A writer component, METTL14, results in decreased oligodendrocyte numbers and CNS hypomyelination, although oligodendrocyte precursor cell (OPC) numbers are normal. In vitro Mettl14 ablation disrupts postmitotic oligodendrocyte maturation and has distinct effects on OPC and oligodendrocyte transcriptomes. Moreover, the loss of Mettl14 in oligodendrocyte lineage cells causes aberrant splicing of myriad RNA transcripts, including those that encode the essential paranodal component neurofascin 155 (NF155). Together, our findings indicate that dynamic RNA methylation plays an important regulatory role in oligodendrocyte development and CNS myelination.

Targeted Complement Inhibition at Synapses Prevents Microglial Synaptic Engulfment and Synapse Loss in Demyelinating Disease.

Multiple sclerosis (MS) is a demyelinating, autoimmune disease of the central nervous system. While work has focused on myelin and axon loss in MS, less is known about mechanisms underlying synaptic changes. Using postmortem human MS tissue, a preclinical nonhuman primate model of MS, and two rodent models of demyelinating disease, we investigated synapse changes in the visual system. Similar to other neurodegenerative diseases, microglial synaptic engulfment and profound synapse loss were observed. In mice, synapse loss occurred independently of local demyelination and neuronal degeneration but coincided with gliosis and increased complement component C3, but not C1q, at synapses. Viral overexpression of the complement inhibitor Crry at C3-bound synapses decreased microglial engulfment of synapses and protected visual function. These results indicate that microglia eliminate synapses through the alternative complement cascade in demyelinating disease and identify a strategy to prevent synapse loss that may be broadly applicable to other neurodegenerative diseases. VIDEO ABSTRACT.

The UPR-PERK pathway is not a promising therapeutic target for mutant SOD1-induced ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by motor neuron death in the brain and spinal cord. Mutations in the Cu/Zn superoxide dismutase (SOD1) gene account for ~20% of all familial ALS forms, corresponding to 1%-2% of all ALS cases. One of the suggested mechanisms by which mutant SOD1 (mtSOD1) exerts its toxic effects involves intracellular accumulation of abnormal mtSOD1 aggregates, which trigger endoplasmic reticulum (ER) stress and activate its adaptive signal transduction pathways, including the unfolded protein response (UPR). PERK, an eIF2α kinase, is central to the UPR and is the most rapidly activated pathway in response to ER stress. Previous reports using mtSOD1 transgenic mice indicated that genetic or pharmacological enhancement of the UPR-PERK pathway may be effective in treating ALS. We investigated the response to PERK haploinsufficiency, and the response to deficiency of its downstream effectors GADD34 and CHOP, in five distinct lines of mtSOD1 mice. We demonstrate that, in contrast to a previously published study, PERK haploinsufficiency has no effect on disease in all mtSOD1 lines examined. We also show that deficiency of GADD34, which enhances the UPR by prolonging the phosphorylation of eIF2α, does not ameliorate disease in these mtSOD1 mouse lines. Finally, we demonstrate that genetic ablation of CHOP transcription factor, which is known to be pro-apoptotic, does not ameliorate disease in mtSOD1 mice. Cumulatively, our studies reveal that neither genetic inhibition of the UPR via ablation of PERK, nor genetic UPR enhancement via ablation of GADD34, is beneficial for mtSOD1-induced motor neuron disease. Therefore, the PERK pathway is not a likely target for therapeutic intervention in mtSOD1-induced ALS.

Molecular Control of Oligodendrocyte Development.

Myelin is a multilayer lipid membrane structure that wraps and insulates axons, allowing for the efficient propagation of action potentials. During developmental myelination of the central nervous system (CNS), oligodendrocyte progenitor cells (OPCs) proliferate and migrate to their final destination, where they terminally differentiate into mature oligodendrocytes and myelinate axons. Lineage progression and terminal differentiation of oligodendrocyte lineage cells are under tight transcriptional and post-transcriptional control. The characterization of several recently identified regulatory factors that govern these processes, which are the focus of this review, has greatly increased our understanding of oligodendrocyte development and function. These insights are critical to facilitate efforts to enhance OPC differentiation in neurological disorders that disrupt CNS myelin.

Sephin1, which prolongs the integrated stress response, is a promising therapeutic for multiple sclerosis.

Multiple sclerosis is a chronic autoimmune demyelinating disorder of the CNS. Immune-mediated oligodendrocyte cell loss contributes to multiple sclerosis pathogenesis, such that oligodendrocyte-protective strategies represent a promising therapeutic approach. The integrated stress response, which is an innate cellular protective signalling pathway, reduces the cytotoxic impact of inflammation on oligodendrocytes. This response is initiated by phosphorylation of eIF2α to diminish global protein translation and selectively allow for the synthesis of protective proteins. The integrated stress response is terminated by dephosphorylation of eIF2α. The small molecule Sephin1 inhibits eIF2α dephosphorylation, thereby prolonging the protective response. Herein, we tested the effectiveness of Sephin1 in shielding oligodendrocytes against inflammatory stress. We confirmed that Sephin1 prolonged eIF2α phosphorylation in stressed primary oligodendrocyte cultures. Moreover, by using a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis, we demonstrated that Sephin1 delayed the onset of clinical symptoms, which correlated with a prolonged integrated stress response, reduced oligodendrocyte and axon loss, as well as diminished T cell presence in the CNS. Sephin1 is reportedly a selective inhibitor of GADD34 (PPP1R15A), which is a stress-induced regulatory subunit of protein phosphatase 1 complex that dephosphorylates eIF2α. Consistent with this possibility, GADD34 mutant mice presented with a similar ameliorated experimental autoimmune encephalomyelitis phenotype as Sephin1-treated mice, and Sephin1 did not provide additional therapeutic benefit to the GADD34 mutant animals. Results presented from the adoptive transfer of encephalitogenic T cells between wild-type and GADD34 mutant mice further indicate that the beneficial effects of Sephin1 are mediated through a direct protective effect on the CNS. Of particular therapeutic relevance, Sephin1 provided additive therapeutic benefit when combined with the first line multiple sclerosis drug, interferon ß. Together, our results suggest that a neuroprotective treatment based on the enhancement of the integrated stress response would likely have significant therapeutic value for multiple sclerosis patients.