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Increasing evidence indicates heterogeneity in functional and molecular properties of oligodendrocyte lineage cells both during development and under pathologic conditions. In multiple sclerosis, remyelination of grey matter lesions exceeds that in white matter. Here we used cells derived from grey matter versus white matter regions of surgically resected human brain tissue samples, to compare the capacities of human A2B5-positive progenitor cells and mature oligodendrocytes to ensheath synthetic nanofibers, and relate differences to the molecular profiles of these cells. For both cell types, the percentage of ensheathing cells was greater for grey matter versus white matter cells. For both grey matter and white matter samples, the percentage of cells ensheathing nanofibers was greater for A2B5-positive cells versus mature oligodendrocytes. Grey matter A2B5-positive cells were more susceptible than white matter A2B5-positive cells to injury induced by metabolic insults. Bulk RNA sequencing indicated that separation by cell type (A2B5-positive vs mature oligodendrocytes) is more significant than by region but segregation for each cell type by region is apparent. Molecular features of grey matter versus white matter derived A2B5-positive and mature oligodendrocytes were lower expression of mature oligodendrocyte genes and increased expression of early oligodendrocyte lineage genes. Genes and pathways with increased expression in grey matter derived cells with relevance for myelination included those related to responses to external environment, cell-cell communication, cell migration, and cell adhesion. Immune and cell death related genes were up-regulated in grey matter derived cells. We observed a significant number of up-regulated genes shared between the stress/injury and myelination processes, providing a basis for these features. In contrast to oligodendrocyte lineage cells, no functional or molecular heterogeneity was detected in microglia maintained in vitro, likely reflecting the plasticity of these cells ex vivo. The combined functional and molecular data indicate that grey matter human oligodendrocytes have increased intrinsic capacity to myelinate but also increased injury susceptibility, in part reflecting their being at a stage earlier in the oligodendrocyte lineage., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Multiple lines of evidence suggest a role of inflammation in epilepsy. Seizure incidence in patients with multiple sclerosis (MS) is twofold to threefold higher than the age-matched general population., Objectives: To explore the association of MS disease-modifying therapies (DMTs) and FDA-approved Bruton tyrosine kinase inhibitors (for lymphocytic malignancies) with the occurrence of epilepsy using the US Food and Drug Administration Adverse Event Reporting System (FAERS) database., Design: Secondary analysis of the FAERS database., Methods: We conducted a disproportionality analysis of FAERS between 2003-Q4 and 2023-Q3. MS DMTs and the Bruton tyrosine kinase inhibitor, ibrutinib, were included in the analysis. An inverse association was defined by a 95% confidence interval (CI) upper limit of reporting odds ratio (ROR) <1., Results: We found an inverse association of ibrutinib, ocrelizumab, ofatumumab, rituximab, and teriflunomide with epilepsy. The strongest inverse association was seen with ibrutinib (ROR: 0.338; 95% CI: 0.218-0.524)., Conclusion: Our findings suggest the possibility of considering these medications for repurposing opportunities in epilepsy and support a potential pathogenic role of leukocyte subsets in seizure perpetuation., (© The Author(s), 2024.)

Mature myelinating oligodendrocytes, the cells that produce the myelin sheath that insulates axons in the central nervous system, have distinct energetic and metabolic requirements compared to neurons. Neurons require substantial energy to execute action potentials, while the energy needs of oligodendrocytes are directed toward building the lipid-rich components of myelin and supporting neuronal metabolism by transferring glycolytic products to axons as additional fuel. The utilization of energy metabolites in the brain parenchyma is tightly regulated to meet the needs of different cell types. Disruption of the supply of metabolites can lead to stress and oligodendrocyte injury, contributing to various neurological disorders, including some demyelinating diseases. Understanding the physiological properties, structures, and mechanisms involved in oligodendrocyte energy metabolism, as well as the relationship between oligodendrocytes and neighboring cells, is crucial to investigate the underlying pathophysiology caused by metabolic impairment in these disorders. In this review, we describe the particular physiological properties of oligodendrocyte energy metabolism and the response of oligodendrocytes to metabolic stress. We delineate the relationship between oligodendrocytes and other cells in the context of the neurovascular unit, and the regulation of metabolite supply according to energetic needs. We focus on the specific bioenergetic requirements of oligodendrocytes and address the disruption of metabolic energy in demyelinating diseases. We encourage further studies to increase understanding of the significance of metabolic stress on oligodendrocyte injury, to support the development of novel therapeutic approaches for the treatment of demyelinating diseases., (© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Oligodendrocytes (OLs) are key players in the central nervous system, critical for the formation and maintenance of the myelin sheaths insulating axons, ensuring efficient neuronal communication. In the last decade, the use of human induced pluripotent stem cells (iPSCs) has become essential for recapitulating and understanding the differentiation and role of OLs in vitro. Current methods include overexpression of transcription factors for rapid OL generation, neglecting the complexity of OL lineage development. Alternatively, growth factor-based protocols offer physiological relevance but struggle with efficiency and cell heterogeneity. To address these issues, we created a novel SOX10-P2A-mOrange iPSC reporter line to track and purify oligodendrocyte precursor cells. Using this reporter cell line, we analyzed an existing differentiation protocol and shed light on the origin of glial cell heterogeneity. Additionally, we have modified the differentiation protocol, toward enhancing reproducibility, efficiency, and terminal maturity. Our approach not only advances OL biology but also holds promise to accelerate research and translational work with iPSC-derived OLs., (© 2024 The Authors. GLIA published by Wiley Periodicals LLC.)

Background: Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R., Methods: Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL., Results: The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam 3 CSK 4 . Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1R WT/KO and CSF1R WT/E633K iMGL compared to their respective isogenic controls., Conclusions: We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities., (© 2024. The Author(s).)

Metformin restores the myelination potential of aged rat A2B5+ oligodendrocyte progenitor cells and may enhance recovery in children with post-radiation brain injury. Human late progenitor cells (O4+A2B5+) have a superior capacity to ensheath nanofibres compared to mature oligodendrocytes, with cells from paediatric sources exceeding adults. In this study, we assessed the effects of metformin on ensheathment capacity of human adult and paediatric progenitors and mature oligodendrocytes and related differences to transcriptional changes. A2B5+ progenitors and mature cells, derived from surgical tissues by immune-magnetic separation, were assessed for ensheathment capacity in nanofibre plates over 2 weeks. Metformin (10 µM every other day) was added to selected cultures. RNA was extracted from treated and control cultures after 2 days. For all ages, ensheathment by progenitors exceeded mature oligodendrocytes. Metformin enhanced ensheathment by adult donor cells but reduced ensheathment by paediatric cells. Metformin marginally increased cell death in paediatric progenitors. Metformin-induced changes in gene expression are distinct for each cell type. Adult progenitors showed up-regulation of pathways involved in the process of outgrowth and promoting lipid biosynthesis. Paediatric progenitors showed a relatively greater proportion of down- versus up-regulated pathways, these involved cell morphology, development and synaptic transmission. Metformin-induced AMP-activated protein kinase activation in all cell types; AMP-activated protein kinase inhibitor BML-275 reduced functional metformin effects only with adult cells. Our results indicate age and differentiation stage-related differences in human oligodendroglia lineage cells in response to metformin. Clinical trials for demyelinating conditions will indicate how these differences translate in vivo ., Competing Interests: There are no conflicts of interest to report., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis 1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY + p300 + memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY + p300 + astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Oligodendrocyte (OL) injury and subsequent loss is a pathologic hallmark of multiple sclerosis (MS). Stress granules (SGs) are membrane-less organelles containing mRNAs stalled in translation and considered as participants of the cellular response to stress. Here we show SGs in OLs in active and inactive areas of MS lesions as well as in normal-appearing white matter. In cultures of primary human adult brain derived OLs, metabolic stress conditions induce transient SG formation in these cells. Combining pro-inflammatory cytokines, which alone do not induce SG formation, with metabolic stress results in persistence of SGs. Unlike sodium arsenite, metabolic stress induced SG formation is not blocked by the integrated stress response inhibitor. Glycolytic inhibition also induces persistent SGs indicating the dependence of SG formation and disassembly on the energetic glycolytic properties of human OLs. We conclude that SG persistence in OLs in MS reflects their response to a combination of metabolic stress and pro-inflammatory conditions., (© 2024. The Author(s).)

Mer tyrosine kinase (MerTK) is a receptor tyrosine kinase that mediates non-inflammatory, homeostatic phagocytosis of diverse types of cellular debris. Highly expressed on the surface of microglial cells, MerTK is of importance in brain development, homeostasis, plasticity and disease. Yet, involvement of this receptor in the clearance of protein aggregates that accumulate with ageing and in neurodegenerative diseases has yet to be defined. The current study explored the function of MerTK in the microglial uptake of alpha-synuclein fibrils which play a causative role in the pathobiology of synucleinopathies. Using human primary and induced pluripotent stem cell-derived microglia, the MerTK-dependence of alpha-synuclein fibril internalization was investigated in vitro. Relevance of this pathway in synucleinopathies was assessed through burden analysis of MERTK variants and analysis of MerTK expression in patient-derived cells and tissues. Pharmacological inhibition of MerTK and siRNA-mediated MERTK knockdown both caused a decreased rate of alpha-synuclein fibril internalization by human microglia. Consistent with the non-inflammatory nature of MerTK-mediated phagocytosis, alpha-synuclein fibril internalization was not observed to induce secretion of pro-inflammatory cytokines such as IL-6 or TNF, and downmodulated IL-1β secretion from microglia. Burden analysis in two independent patient cohorts revealed a significant association between rare functionally deleterious MERTK variants and Parkinson's disease in one of the cohorts (P = 0.002). Despite a small upregulation in MERTK mRNA expression in nigral microglia from Parkinson's disease/Lewy body dementia patients compared to those from non-neurological control donors in a single-nuclei RNA-sequencing dataset (P = 5.08 × 10-21), no significant upregulation in MerTK protein expression was observed in human cortex and substantia nigra lysates from Lewy body dementia patients compared to controls. Taken together, our findings define a novel role for MerTK in mediating the uptake of alpha-synuclein fibrils by human microglia, with possible involvement in limiting alpha-synuclein spread in synucleinopathies such as Parkinson's disease. Upregulation of this pathway in synucleinopathies could have therapeutic values in enhancing alpha-synuclein fibril clearance in the brain., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Mature oligodendrocytes form myelin sheaths that are crucial for the insulation of axons and efficient signal transmission in the central nervous system. Recent evidence has challenged the classical view of the functionally static mature oligodendrocyte and revealed a gamut of dynamic functions such as the ability to modulate neuronal circuitry and provide metabolic support to axons. Despite the recognition of potential heterogeneity in mature oligodendrocyte function, a comprehensive summary of mature oligodendrocyte diversity is lacking. We delve into early 20 th -century studies by Robertson and Río-Hortega that laid the foundation for the modern identification of regional and morphological heterogeneity in mature oligodendrocytes. Indeed, recent morphologic and functional studies call into question the long-assumed homogeneity of mature oligodendrocyte function through the identification of distinct subtypes with varying myelination preferences. Furthermore, modern molecular investigations, employing techniques such as single cell/nucleus RNA sequencing, consistently unveil at least six mature oligodendrocyte subpopulations in the human central nervous system that are highly transcriptomically diverse and vary with central nervous system region. Age and disease related mature oligodendrocyte variation denotes the impact of pathological conditions such as multiple sclerosis, Alzheimer's disease, and psychiatric disorders. Nevertheless, caution is warranted when subclassifying mature oligodendrocytes because of the simplification needed to make conclusions about cell identity from temporally confined investigations. Future studies leveraging advanced techniques like spatial transcriptomics and single-cell proteomics promise a more nuanced understanding of mature oligodendrocyte heterogeneity. Such research avenues that precisely evaluate mature oligodendrocyte heterogeneity with care to understand the mitigating influence of species, sex, central nervous system region, age, and disease, hold promise for the development of therapeutic interventions targeting varied central nervous system pathology., (Copyright © 2025 Copyright: © 2025 Neural Regeneration Research.)

Background: B cells can be enriched within meningeal immune-cell aggregates of multiple sclerosis (MS) patients, adjacent to subpial cortical demyelinating lesions now recognized as important contributors to progressive disease. This subpial demyelination is notable for a 'surface-in' gradient of neuronal loss and microglial activation, potentially reflecting the effects of soluble factors secreted into the CSF. We previously demonstrated that MS B-cell secreted products are toxic to oligodendrocytes and neurons. The potential for B-cell-myeloid cell interactions to propagate progressive MS is of considerable interest., Methods: Secreted products of MS-implicated pro-inflammatory effector B cells or IL-10-expressing B cells with regulatory potential were applied to human brain-derived microglia or monocyte-derived macrophages, with subsequent assessment of myeloid phenotype and function through measurement of their expression of pro-inflammatory, anti-inflammatory and homeostatic/quiescent molecules, and phagocytosis (using flow cytometry, ELISA and fluorescently-labeled myelin). Effects of secreted products of differentially activated microglia on B-cell survival and activation were further studied., Findings: Secreted products of MS-implicated pro-inflammatory B cells (but not IL-10 expressing B cells) substantially induce pro-inflammatory cytokine (IL-12, IL-6, TNFα) expression by both human microglia and macrophage (in a GM-CSF dependent manner), while down-regulating their expression of IL-10 and of quiescence-associated molecules, and suppressing their myelin phagocytosis. In contrast, secreted products of IL-10 expressing B cells upregulate both human microglia and macrophage expression of quiescence-associated molecules and enhance their myelin phagocytosis. Secreted factors from pro-inflammatory microglia enhance B-cell activation., Interpretation: Potential cross-talk between disease-relevant human B-cell subsets and both resident CNS microglia and infiltrating macrophages may propagate CNS-compartmentalized inflammation and injury associated with MS disease progression. These interaction represents an attractive therapeutic target for agents such as Bruton's tyrosine kinase inhibitors (BTKi) that modulate responses of both B cells and myeloid cells., Funding: Stated in Acknowledgments section of manuscript., Competing Interests: Declaration of interests H.T. and L.Z. received consulting fees from EMD Serono outside of the submitted work. D.J. has received consulting fees from Biogen, Genentech, Novartis, EMD Serono, Banner Life Sciences, Bristol Meyers Squibb, Horizon, Cycle and Sanofi Genzyme outside of the submitted work. A.BO. has received fees for advisory board participation and/or consulting from Abata, Accure, Atara Biotherapeutics, Biogen, BMS/Celgene/Receptos, GlaxoSmithKline, Gossamer, Immunic, Janssen/Actelion, Medimmune, Merck/EMD Serono, Novartis, Roche/Genentech, Sangamo, Sanofi-Genzyme, Viracta; and has received grant support to the University of Pennsylvania from Biogen Idec, Roche/Genentech, Merck/EMD Serono and Novartis. L.P. has recived grant support from Alector on projects not related to this work., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Oligodendrocyte (OL) injury and loss are central features of evolving lesions in multiple sclerosis. Potential causative mechanisms of OL loss include metabolic stress within the lesion microenvironment. Here we use the injury response of primary human OLs (hOLs) to metabolic stress (reduced glucose/nutrients) in vitro to help define the basis for the in situ features of OLs in cases of MS. Under metabolic stress in vitro, we detected reduction in ATP levels per cell that precede changes in survival. Autophagy was initially activated, although ATP levels were not altered by inhibitors (chloroquine) or activators (Torin-1). Prolonged stress resulted in autophagy failure, documented by non-fusion of autophagosomes and lysosomes. Consistent with our in vitro results, we detected higher expression of LC3, a marker of autophagosomes in OLs, in MS lesions compared to controls. Both in vitro and in situ, we observe a reduction in nuclear size of remaining OLs. Prolonged stress resulted in increased ROS and cleavage of spectrin, a target of Ca 2+ -dependent proteases. Cell death was however not prevented by inhibitors of ferroptosis or MPT-driven necrosis, the regulated cell death (RCD) pathways most likely to be activated by metabolic stress. hOLs have decreased expression of VDAC1, VDAC2, and of genes regulating iron accumulation and cyclophilin. RNA sequencing analyses did not identify activation of these RCD pathways in vitro or in MS cases. We conclude that this distinct response of hOLs, including resistance to RCD, reflects the combined impact of autophagy failure, increased ROS, and calcium influx, resulting in metabolic collapse and degeneration of cellular structural integrity. Defining the basis of OL injury and death provides guidance for development of neuro-protective strategies., (© 2023. The Author(s).)

Efforts to understand microglia function in health and diseases have been hindered by the lack of culture models that recapitulate in situ cellular properties. In recent years, the use of serum-free media with brain-derived growth factors (colony stimulating factor 1 receptor [CSF1R] ligands and TGF-β1/2) have been favored for the maintenance of rodent microglia as they promote morphological features observed in situ. Here we study the functional and transcriptomic impacts of such media on human microglia (hMGL). Media formulation had little impact on microglia transcriptome assessed by RNA sequencing which was sufficient to significantly alter microglia capacity to phagocytose myelin debris and to elicit an inflammatory response to lipopolysaccharide. When compared to immediately ex vivo microglia from the same donors, the addition of fetal bovine serum to culture media, but not growth factors, was found to aid in the maintenance of key signature genes including those involved in phagocytic processes. A phenotypic shift characterized by CSF1R downregulation in culture correlated with a lack of reliance on CSF1R signaling for survival. Consequently, no improvement in cell survival was observed following culture supplementation with CSF1R ligands. Our study provides better understanding of hMGL in culture, with observations that diverge from those previously made in rodent microglia., (© 2023 The Authors. GLIA published by Wiley Periodicals LLC.)

Multiple sclerosis (MS) is an autoimmune disease of the CNS, featuring inflammation and demyelination with variable recovery. In this issue of the JCI, Kapell, Fazio, and authors address the potential for targeting neuron-oligodendrocyte potassium shuttling at the nodes of Ranvier as a neuroprotective strategy during inflammatory demyelination of the CNS in experimental MS. Their extensive and impressive study could serve as a template for defining the physiologic properties of a putative protective pathway. The authors examined MS features in existent disease models, investigated the impact of pharmacologic intervention, and evaluated its status in tissues from patients with MS. We await future studies that will tackle the challenge of translating these findings into a clinical therapy.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Early multiple sclerosis lesions feature relative preservation of oligodendrocyte cell bodies with dying back retraction of their myelinating processes. Cell loss occurs with disease progression. Putative injury mediators include metabolic stress (low glucose/nutrient), pro-inflammatory mediators (interferon γ and tumour necrosis factor α), and excitotoxins (glutamate). Our objective was to compare the impact of these disease relevant mediators on the injury responses of human mature oligodendrocytes. In the current study, we determined the effects of these mediators on process extension and survival of human brain derived mature oligodendrocytes in vitro and used bulk RNA sequencing to identify distinct effector mechanisms that underlie the responses. All mediators induced significant process retraction of the oligodendrocytes in dissociated cell culture. Only metabolic stress (low glucose/nutrient) conditions resulted in delayed (4-6 days) non-apoptotic cell death. Metabolic effects were associated with induction of the integrated stress response, which can be protective or contribute to cell injury dependent on its level and duration of activation. Addition of Sephin1, an agonist of the integrated stress response induced process retraction under control conditions and further enhanced retraction under metabolic stress conditions. The antagonist ISRIB restored process outgrowth under stress conditions, and if added to already stressed cells, reduced delayed cell death and prolonged the period in which recovery could occur. Inflammatory cytokine functional effects were associated with activation of multiple signalling pathways (including Jak/Stat-1) that regulate process outgrowth, without integrated stress response induction. Glutamate application produced limited transcriptional changes suggesting a contribution of effects directly on cell processes. Our comparative studies indicate the need to consider both the specific injury mediators and the distinct cellular mechanisms of responses to them by human oligodendrocytes to identify effective neuroprotective therapies for multiple sclerosis., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Myelin, the membrane surrounding neuronal axons, is critical for central nervous system (CNS) function. Injury to myelin-forming oligodendrocytes (OL) in chronic neurological diseases (e.g. multiple sclerosis) ranges from sublethal to lethal, leading to OL dysfunction and myelin pathology, and consequent deleterious impacts on axonal health that drive clinical impairments. This is regulated by intrinsic factors such as heterogeneity and age, and extrinsic cellular and molecular interactions. Here, we discuss the responses of OLs to injury, and perspectives for therapeutic targeting. We put forward that targeting mature OL health in neurological disease is a promising therapeutic strategy to support CNS function., (© 2022. The Author(s).)

Morphological and emerging molecular studies have provided evidence for heterogeneity within the oligodendrocyte population. To address the regional and age-related heterogeneity of human mature oligodendrocytes (MOLs) we applied single-cell RNA sequencing to cells isolated from cortical/subcortical, subventricular zone brain tissue samples, and thoracolumbar spinal cord samples. Unsupervised clustering of cells identified transcriptionally distinct MOL subpopulations across regions. Spinal cord MOLs, but not microglia, exhibited cell-type-specific upregulation of immune-related markers compared to the other adult regions. SVZ MOLs showed an upregulation of select number of development-linked transcription factors compared to other regions; however, pseudotime trajectory analyses did not identify a global developmental difference. Age-related analysis of cortical/subcortical samples indicated that pediatric MOLs, especially from under age 5, retain higher expression of genes linked to development and to immune activity with pseudotime analysis favoring a distinct developmental stage. Our regional and age-related studies indicate heterogeneity of MOL populations in the human CNS that may reflect developmental and environmental influences., (© 2022 Wiley Periodicals LLC.)

Objective: Myelin regeneration in the human central nervous system relies on progenitor cells within the tissue parenchyma, with possible contribution from previously myelinating oligodendrocytes (OLs). In multiple sclerosis, a demyelinating disorder, variables affecting remyelination efficiency include age, severity of initial injury, and progenitor cell properties. Our aim was to investigate the effects of age and differentiation on the myelination potential of human OL lineage cells., Methods: We derived viable primary OL lineage cells from surgical resections of pediatric and adult brain tissue. Ensheathment capacity using nanofiber assays and transcriptomic profiles from RNA sequencing were compared between A2B5+ antibody-selected progenitors and mature OLs (non-selected cells)., Results: We demonstrate that pediatric progenitor and mature cells ensheathed nanofibers more robustly than did adult progenitor and mature cells, respectively. Within both age groups, the percentage of fibers ensheathed and ensheathment length per fiber were greater for A2B5+ progenitors. Gene expression of OL progenitor markers PDGFRA and PTPRZ1 were higher in A2B5+ versus A2B5- cells and in pediatric A2B5+ versus adult A2B5+ cells. The p38 MAP kinases and actin cytoskeleton-associated pathways were upregulated in pediatric cells; both have been shown to regulate OL process outgrowth. Significant upregulation of "cell senescence" genes was detected in pediatric samples; this could reflect their role in development and the increased susceptibility of pediatric OLs to activating cell death responses to stress., Interpretation: Our findings identify specific biological pathways relevant to myelination that are differentially enriched in human pediatric and adult OL lineage cells and suggest potential targets for remyelination enhancing therapies. ANN NEUROL 2022;91:178-191., (© 2021 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

The migration of circulating leukocytes into the central nervous system (CNS) is a key driver of multiple sclerosis (MS) pathogenesis. The monoclonal antibody natalizumab proved that pharmaceutically obstructing this process is an effective therapeutic approach for treating relapsing-remitting MS (RRMS). Unfortunately, the clinical efficacy of natalizumab is somewhat offset by its incapacity to control the progressive forms of MS (PMS) and by life-threatening side effects in RRMS rising from the expression of its molecular target, very late antigen 4 (VLA4), on most immune cells and consequent impairment of CNS immunosurveillance. Here, we identified dual immunoglobulin domain containing cell adhesion molecule (DICAM) as a cell trafficking molecule preferentially expressed by T helper 17 (T H 17)–polarized CD4 + T lymphocytes. We found that DICAM expression on circulating CD4 + T cells was increased in patients with active RRMS and PMS disease courses, and expression of DICAM ligands was increased on the blood-brain barrier endothelium upon inflammation and in MS lesions. Last, we demonstrated that pharmaceutically neutralizing DICAM reduced murine and human T H 17 cell trafficking across the blood-brain barrier in vitro and in vivo, and alleviated disease symptoms in four distinct murine autoimmune encephalomyelitis models, including relapsing-remitting and progressive disease models. Collectively, our data highlight DICAM as a candidate therapeutic target to impede the migration of disease-inducing leukocytes into the CNS in both RRMS and PMS and suggest that blocking DICAM with a monoclonal antibody may be a promising therapeutic approach.

Neuroimmune interaction during development is strongly implicated in the pathogenesis of neurodevelopmental disorders, but the mechanisms that cause neuronal circuit dysregulation are not well understood. We performed in vivo imaging of the developing retinotectal system in the larval zebrafish to characterize the effects of immune system activation on refinement of an archetypal sensory processing circuit. Acute inflammatory insult induced hyperdynamic remodeling of developing retinal axons in larval fish and increased axon arbor elaboration over days. Using calcium imaging in GCaMP6s transgenic fish, we showed that these morphologic changes were accompanied by a shift toward decreased visual acuity in tectal cells. This finding was supported by poorer performance in a visually guided behavioral task. We further found that the pro-inflammatory cytokine, interleukin-1β (IL-1β), is upregulated by the inflammatory insult, and that downregulation of IL-1β abrogated the effects of inflammation on axonal dynamics and growth. Moreover, baseline branching of the retinal ganglion cell arbors in IL-1β morphant animals was significantly different from that in control larvae, and their performance in a predation assay was impaired, indicating a role for this cytokine in normal neuronal development. This work establishes a simple and powerful non-mammalian model of developmental immune activation and demonstrates a role for IL-1β in mediating the pathologic effects of inflammation on neuronal circuit development. SIGNIFICANCE STATEMENT Maternal immune activation can increase the risk of neurodevelopmental disorders in offspring; however, the mechanisms involved are not fully understood. Using a non-mammalian vertebrate model of developmental immune activation, we show that even brief activation of inflammatory pathways has immediate and long-term effects on the arborization of axons, and that these morphologic changes have functional and behavioral consequences. Finally, we show that the pro-inflammatory cytokine IL-1β plays an essential role in both the effects of inflammation on circuit formation and normal axonal development. Our data add to a growing body of evidence supporting epidemiological studies linking immune activation to neurodevelopmental disorders, and help shed light on the molecular and cellular processes that contribute to the etiology of these disorders., (Copyright © 2021 the authors.)

Introduction: The Coronavirus disease-19 (COVID-19) pandemic continues to expand across the world. This pandemic has had a significant impact on patients with chronic diseases. Among patients with demyelinating diseases of the central nervous system (CNS), such as Multiple Sclerosis (MS) or Neuromyelitis Optica Spectrum Disorder (NMOSD), concerns remain about the potential impact of COVID-19 on these patients given their treatment with immunosuppressive or immunomodulatory therapies. In this study, we review the existing literature investigating the impact of disease-modifying therapies(DMT) on COVID-19 risks in this group of patients., Method: For this systematic review, we searched PubMed from January 1, 2020, to December 3, 2020. The following keywords were used: "COVID-19" AND "Multiple Sclerosis" OR "Neuromyelitis Optica." Articles evaluating COVID-19 in patients with demyelinating diseases of CNS were included. This study evaluates the different aspects of the DMTs in these patients during the COVID-19 era., Results and Conclusion: A total of 262 articles were found. After eliminating duplicates and unrelated research papers, a total of 84 articles met the final inclusion criteria in our study. Overall, the experiences of 2493 MS patients and 37 NMOSD patients with COVID-19 were included in this review. Among them, 46(1.8%) MS patients died(the global death-to-case ratio of Covid-19 was reported about 2.1%). Among DMTs, Rituximab had the highest mortality rate (4%). Despite controversies, especially concerning anti-CD20 monoclonal antibody therapies, a relation between DMT-use and COVID-19 disease- course was not found in many studies. This observation reinforces the recommendation of not stopping current DMTs. Other variables such as age, higher expanded disability status scale (EDSS) scores, cardiac comorbidities, and obesity were independent risk factors for severe COVID-19. Despite the risks of infection, most patients were willing to continue their DMT during the pandemic because of more significant concern about the risk of relapse or worsening MS symptoms. After the infection, an immune response's attenuation was seen in the patients on Fingolimod and anti-CD20 monoclonal antibodies. This may be a critical finding in future vaccinations., (Copyright © 2021. Published by Elsevier B.V.)

Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: In multiple sclerosis loss of myelin and oligodendrocytes impairs saltatory signal transduction and leads to neuronal loss and functional deficits. Limited capacity of oligodendroglial precursor cells to differentiate into mature cells is the main reason for inefficient myelin repair in the central nervous system. Drug repurposing constitutes a powerful approach for identification of pharmacological compounds promoting this process., Methods: A phenotypic compound screening using the subcellular distribution of a potent inhibitor of oligodendroglial cell differentiation, namely p57kip2, as differentiation competence marker was conducted. Hit compounds were validated in terms of their impact on developmental cell differentiation and myelination using both rat and human primary cell cultures and organotypic cerebellar slice cultures, respectively. Their effect on spontaneous remyelination was then investigated following cuprizone-mediated demyelination of the corpus callosum., Findings: A number of novel small molecules able to promote oligodendroglial cell differentiation were identified and a subset was found to foster human oligodendrogenesis as well as myelination ex vivo. Among them the steroid danazol and the anthelminthic parbendazole were found to increase myelin repair., Interpretation: We provide evidence that early cellular processes involved in differentiation decisions are applicable for the identification of regeneration promoting drugs and we suggest danazol and parbendazole as potent therapeutic candidates for demyelinating diseases., Funding: This work was supported by the Jürgen Manchot Foundation, Düsseldorf; Research Commission of the Medical Faculty of Heinrich-Heine-University Düsseldorf; Christiane and Claudia Hempel Foundation; Stifterverband/Novartisstiftung; James and Elisabeth Cloppenburg, Peek and Cloppenburg Düsseldorf Stiftung and International Progressive MS Alliance (BRAVEinMS)., Competing Interests: Declaration of Competing Interest Dr Healy reports consultancy fees from Merck, and funding from Canadian Stem Cell Network, CDQM, Quantum Leap and Bouchard Foundation outside of the submitted work. The rest of the authors declare that there is no conflict of interests regarding the publication of this article., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Mitochondria are dynamic organelles that produce energy and molecular precursors that are essential for myelin synthesis. Unlike in neurons, mitochondria in oligodendrocytes increase intracellular movement in response to glutamatergic activation and are more susceptible to oxidative stress than in astrocytes or microglia. The signaling pathways that regulate these cell type-specific mitochondrial responses in oligodendrocytes are not understood. Here, we visualized mitochondria migrating through thin cytoplasmic channels crossing myelin basic protein-positive compacted membranes and localized within paranodal loop cytoplasm. We hypothesized that local extracellular enrichment of netrin-1 might regulate the recruitment and function of paranodal proteins and organelles, including mitochondria. We identified rapid recruitment of mitochondria and paranodal proteins, including neurofascin 155 (NF155) and the netrin receptor deleted in colorectal carcinoma (DCC), to sites of contact between oligodendrocytes and netrin-1-coated microbeads in vitro. We provide evidence that Src-family kinase activation and Rho-associated protein kinase (ROCK) inhibition downstream of netrin-1 induces mitochondrial elongation, hyperpolarization of the mitochondrial inner membrane, and increases glycolysis. Our findings identify a signaling mechanism in oligodendrocytes that is sufficient to locally recruit paranodal proteins and regulate the subcellular localization, morphology, and function of mitochondria., (© 2020 Wiley Periodicals LLC.)

Myelin destruction and oligodendrocyte (OL) death consequent to metabolic stress is a feature of CNS disorders across the age spectrum. Using cells derived from surgically resected tissue, we demonstrate that young (

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8 + T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8 + T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8 + T cells that recirculate via TDL., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Remyelination failure in multiple sclerosis (MS) is associated with a migration/differentiation block of oligodendroglia. The reason for this block is highly debated. It could result from disease-related extrinsic or intrinsic regulators in oligodendroglial biology. To avoid confounding immune-mediated extrinsic effect, we used an immune-deficient mouse model to compare induced pluripotent stem cell-derived oligodendroglia from MS and healthy donors following engraftment in the developing CNS. We show that the MS-progeny behaves and differentiates into oligodendrocytes to the same extent as controls. They generate equal amounts of myelin, with bona fide nodes of Ranvier, and promote equal restoration of their host slow conduction. MS-progeny expressed oligodendrocyte- and astrocyte-specific connexins and established functional connections with donor and host glia. Thus, MS oligodendroglia, regardless of major immune manipulators, are intrinsically capable of myelination and making functional axo-glia/glia-glia connections, reinforcing the view that the MS oligodendrocyte differentiation block is not from major intrinsic oligodendroglial deficits., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Introduction: Coronavirus disease-19 (COVID-19) pandemic continues to grow all over the world. Several studies have been performed, focusing on understanding the acute respiratory syndrome and treatment strategies. However, there is growing evidence indicating neurological manifestations occur in patients with COVID-19. Similarly, the other coronaviruses (CoV) epidemics; severe acute respiratory syndrome (SARS-CoV-1) and Middle East respiratory syndrome (MERS-CoV) have been associated with neurological complications., Methods: This systematic review serves to summarize available information regarding the potential effects of different types of CoV on the nervous system and describes the range of clinical neurological complications that have been reported thus far in COVID-19., Results: Two hundred and twenty-five studies on CoV infections associated neurological manifestations in human were reviewed. Of those, 208 articles were pertinent to COVID-19. The most common neurological complaints in COVID-19 were anosmia, ageusia, and headache, but more serious complications, such as stroke, impairment of consciousness, seizures, and encephalopathy, have also been reported., Conclusion: There are several similarities between neurological complications after SARS-CoV-1, MERS-CoV and COVID-19, however, the scope of the epidemics and number of patients are very different. Reports on the neurological complications after and during COVID-19 are growing on a daily basis. Accordingly, comprehensive knowledge of these complications will help health care providers to be attentive to these complications and diagnose and treat them timely., Competing Interests: Declaration of Competing Interest Authors have no relevant financial disclosure to report., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Characterizing the developmental trajectory of oligodendrocyte progenitor cells (OPC) is of great interest given the importance of these cells in the remyelination process. However, studies of human OPC development remain limited by the availability of whole cell samples and material that encompasses a wide age range, including time of peak myelination. In this study, we apply single cell RNA sequencing to viable whole cells across the age span and link transcriptomic signatures of oligodendrocyte-lineage cells with stage-specific functional properties. Cells were isolated from surgical tissue samples of second-trimester fetal, 2-year-old pediatric, 13-year-old adolescent, and adult donors by mechanical and enzymatic digestion, followed by percoll gradient centrifugation. Gene expression was analyzed using droplet-based RNA sequencing (10X Chromium). Louvain clustering analysis identified three distinct cellular subpopulations based on 5,613 genes, comprised of an early OPC (e-OPC) group, a late OPC group (l-OPC), and a mature OL (MOL) group. Gene ontology terms enriched for e-OPCs included cell cycle and development, for l-OPCs included extracellular matrix and cell adhesion, and for MOLs included myelination and cytoskeleton. The e-OPCs were mostly confined to the premyelinating fetal group, and the l-OPCs were most highly represented in the pediatric age group, corresponding to the peak age of myelination. Cells expressing a signature characteristic of l-OPCs were identified in the adult brain in situ using RNAScope. These findings highlight the transcriptomic variability in OL-lineage cells before, during, and after peak myelination and contribute to identifying novel pathways required to achieve remyelination., (© 2020 Wiley Periodicals, Inc.)

Cells of the adaptive and innate immune systems in the brain parenchyma and in the meningeal spaces contribute to physiologic functions and disease states in the central nervous system (CNS). Animal studies have demonstrated the involvement of immune constituents, along with major histocompatibility complex (MHC) molecules, in neural development and rare genetic disorders (e.g., colony stimulating factor 1 receptor [CSF1R] deficiency). Genome wide association studies suggest a comparable role of the immune system in humans. Although the CNS can be the target of primary autoimmune disorders, no current experimental model captures all of the features of the most common human disorder placed in this category, multiple sclerosis (MS). Such features include spontaneous onset, environmental contributions, and a recurrent/progressive disease course in a genetically predisposed host. Numerous therapeutic interventions related to antigen and cytokine specific therapies have demonstrated effectiveness in experimental autoimmune encephalomyelitis (EAE), the animal model used to define principles underlying immune-mediated mechanisms in MS. Despite the similarities in the two diseases, most treatments used to ameliorate EAE have failed to translate to the human disease. As directly demonstrated in animal models and implicated by correlative studies in humans, adaptive and innate immune constituents within the systemic compartment and resident in the CNS contribute to the disease course of neurodegenerative and neurobehavioral disorders. The expanding knowledge of the molecular properties of glial cells provides increasing insights into species related variables. These variables affect glial bidirectional interactions with the immune system as well as their own production of "immune molecules" that mediate tissue injury and repair., (© 2019 Wiley Periodicals, Inc.)

Objective: To determine whether there are nuclear depletion and cellular mislocalization of RNA-binding proteins (RBPs) transactivation response DNA-binding protein of 43 kDa (TDP-43), fused in sarcoma (FUS), and polypyrimidine tract-binding protein (PTB) in MS, as is the case in amyotrophic lateral sclerosis (ALS) and oligodendrocytes infected with Theiler murine encephalomyelitis virus (TMEV), we examined MS lesions and in vitro cultured primary human brain-derived oligodendrocytes., Methods: Nuclear depletion and mislocalization of TDP-43, FUS, and PTB are thought to contribute to the pathogenesis of ALS and TMEV demyelination. The latter findings prompted us to investigate these RBPs in the demyelinated lesions of MS and in in vitro cultured human brain-derived oligodendrocytes under metabolic stress conditions., Results: We found (1) mislocalized TDP-43 in oligodendrocytes in active lesions in some patients with MS; (2) decreased PTB1 expression in oligodendrocytes in mixed active/inactive demyelinating lesions; (3) decreased nuclear expression of PTB2 in neurons in cortical demyelinating lesions; and (4) nuclear depletion of TDP-43 in oligodendrocytes under metabolic stress induced by low glucose/low nutrient conditions compared with optimal culture conditions., Conclusion: TDP-43 has been found to have a key role in oligodendrocyte function and viability, whereas PTB is important in neuronal differentiation, suggesting that altered expression and mislocalization of these RBPs in MS lesions may contribute to the pathogenesis of demyelination and neurodegeneration. Our findings also identify nucleocytoplasmic transport as a target for treatment., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Multiple sclerosis is a chronic inflammatory disease of the CNS 1 . Astrocytes contribute to the pathogenesis of multiple sclerosis 2 , but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.

Metabolism has been shown to control peripheral immunity, but little is known about its role in central nervous system (CNS) inflammation. Through a combination of proteomic, metabolomic, transcriptomic, and perturbation studies, we found that sphingolipid metabolism in astrocytes triggers the interaction of the C2 domain in cytosolic phospholipase A2 (cPLA2) with the CARD domain in mitochondrial antiviral signaling protein (MAVS), boosting NF-κB-driven transcriptional programs that promote CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis. cPLA2 recruitment to MAVS also disrupts MAVS-hexokinase 2 (HK2) interactions, decreasing HK enzymatic activity and the production of lactate involved in the metabolic support of neurons. Miglustat, a drug used to treat Gaucher and Niemann-Pick disease, suppresses astrocyte pathogenic activities and ameliorates EAE. Collectively, these findings define a novel immunometabolic mechanism that drives pro-inflammatory astrocyte activities, outlines a new role for MAVS in CNS inflammation, and identifies candidate targets for therapeutic intervention., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Astrocytes are increasingly recognized as active contributors to the disease process in multiple sclerosis (MS), rather than being merely reactive. We investigated the expression of a selected microRNA (miRNA) panel that could contribute both to the injury and to the recovery phases of the disease. Individual astrocytes were laser microdissected from brain sections. We then compared the miRNAs' expressions in MS and control brain samples at different lesional stages in white versus grey matter regions. In active MS lesions, we found upregulation of ischemia-related miRNAs in white but not grey matter, often with reversion to the normal state in inactive lesions. In contrast to our previous findings on MS macrophages, expression of 2 classical inflammatory-related miRNAs, miRNA-155 and miRNA-146a, was reduced in astrocytes from active and chronic active MS lesions in white and grey matter, suggesting a lesser direct pathogenetic role for these miRNAs in astrocytes. miRNAs within the categories regulating aquaporin4 (-100, -145, -320) and glutamate transport/apoptosis/neuroprotection (-124a, -181a, and -29a) showed some contrasting responses. The regional and lesion-stage differences of expression of these miRNAs indicate the remarkable ability of astrocytes to show a wide range of selective responses in the face of differing insults and phases of resolution., (© 2019 American Association of Neuropathologists, Inc. All rights reserved.)