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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 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In multiple sclerosis, cerebellar symptoms are associated with clinical impairment and an increased likelihood of progressive course. Cortical atrophy and synaptic dysfunction play a prominent role in cerebellar pathology and although the dentate nucleus is a predilection site for lesion development, structural synaptic changes in this region remain largely unexplored. Moreover, the mechanisms leading to synaptic dysfunction have not yet been investigated at an ultrastructural level in multiple sclerosis. Here, we report on synaptic changes of dentate nuclei in post-mortem cerebella of 16 multiple sclerosis patients and eight controls at the histological level as well as an electron microscopy evaluation of afferent synapses of the cerebellar dentate and pontine nuclei of one multiple sclerosis patient and one control. We found a significant reduction of afferent dentate synapses in multiple sclerosis, irrespective of the presence of demyelination, and a close relationship between glial processes and dentate synapses. Ultrastructurally, we show autophagosomes containing degradation products of synaptic vesicles within dendrites, residual bodies within intact-appearing axons and free postsynaptic densities opposed to astrocytic appendages. Our study demonstrates loss of dentate afferent synapses and provides, for the first time, ultrastructural evidence pointing towards neuron-autonomous and neuroglia-mediated mechanisms of synaptic degradation in chronic multiple sclerosis. [ABSTRACT FROM AUTHOR]

Objective: Degeneration of oligodendroglial distal processes has been identified as an early event in multiple sclerosis (MS) lesion development. Our objective was to further define the development of the "dying-back" oligodendrocyte lesion in situ and to model the development and potential reversibility of such responses using dissociated cultures of adult human brain-derived oligodendrocytes.Methods: In situ analyses were performed on glutaraldehyde-fixed thin sections of clinically acute and pathologically active cases of MS. In vitro studies were conducted using adult human brain-derived oligodendrocytes challenged by metabolic stress conditions (low nutrient/glucose).Results: In situ analyses indicated a spectrum of myelin changes in the presence of morphologically intact oligodendrocytes; these included degeneration of the inner cytoplasmic tongue with increasing sizes of intramyelinic bleb formation that could result in radial fractures of the myelin sheath. Macrophages with ingested myelin fragments were identified only once the fragmentation was established. In vitro studies indicated that oligodendrocyte process retraction, which was linked to reduced glycolytic respiratory activity, is reversible until a critical time point. Subsequent cell death was not linked to caspase-3-dependent programs. Gene expression studies conducted at the latest reversible time point revealed reduced expression of pathways associated with cell process outgrowth and myelination, as well as with metabolic activity.Interpretation: Our findings reveal the potential to protect and possibly restore myelin elaborated by existent oligodendrocytes in early and evolving MS lesions, and suggest the necessity of ongoing studies of the mechanisms underlying subsequent adult human oligodendrocyte cell death. Ann Neurol 2017;81:811-824. [ABSTRACT FROM AUTHOR]

Objective Dimethyl fumarate ( DMF), a therapy for relapsing-remitting multiple sclerosis ( RRMS), is implicated as acting on inflammatory and antioxidant responses within both systemic immune and/or central nervous system ( CNS) compartments. Orally administered DMF is rapidly metabolized to monomethyl fumarate ( MMF). Our aim was to analyze the impact of fumarates on antiinflammatory and antioxidant profiles of human myeloid cells found in the systemic compartment (monocytes) and in the inflamed CNS (blood-derived macrophages and brain-derived microglia). Methods We analyzed cytokine and antioxidant expression in monocytes from untreated or DMF-treated RRMS patients and controls, and in monocyte-derived macrophages (MDMs) and microglia isolated from adult and fetal human brain tissue. Results Monocytes from multiple sclerosis ( MS) patients receiving DMF had reduced expression of the proinflammatory micro- RNA miR-155 and of antioxidant genes HMOX1 and OSGIN1 compared to untreated MS patients; similar changes were observed in patients receiving FTY720 and/or natalizumab. In vitro addition of DMF but not MMF to MDMs and microglia inhibited lipopolysaccharide-induced production of inflammatory cytokines and increased expression of the antioxidant gene HMOX1 in the absence of significant cytotoxicity. Interpretation Our in vivo-based observations that effects of DMF therapy on systemic myeloid cell gene expression are also observed with FTY720 and natalizumab therapy suggests that the effect may be indirect, reflecting reduced overall disease activity. Our in vitro results demonstrate significant effects of DMF but not MMF on inflammation and antioxidant responses by MDMs and microglia, questioning the mechanisms whereby DMF therapy would modulate myeloid cell properties within the CNS. [ABSTRACT FROM AUTHOR]

Objective To define the functional significance of increased miR-155 expression in myeloid cells in multiple sclerosis (MS). Methods miR-155 expression levels were measured in CD14+ monocytes from untreated relapsing-remitting MS patients and compared to healthy controls. Similar microRNA (miRNA) analyses were performed in laser-captured CD68+ cells from perivascular (blood-derived macrophages) and parenchymal (microglia) brain regions in both active MS lesions and noninflammatory cases. Using human adult blood-derived macrophages and brain-derived microglia, in vitro experiments were performed to demonstrate how miR-155 influences the polarization state, phenotype, and functional properties of myeloid cells, in addition to their ability to subsequently impact adaptive T-cell responses. Results In MS, miR-155 expression was significantly increased in both peripheral circulating CD14+ monocytes and active lesions (CD68+ cells) compared to control donor monocytes and parenchymal microglia, respectively. In vitro, miR-155 was significantly increased in both M1-polarized primary human macrophages and microglia. Transfection of an miR-155 mimic increased proinflammatory cytokine secretion and costimulatory surface marker expression in both cell types; an miR-155 inhibitor decreased proinflammatory cytokine expression. Coculture experiments demonstrated that allogeneic T-cell responses were significantly enhanced in the presence of miR-155-transfected myeloid cells compared to controls. Interpretation Our results demonstrate that miR-155 regulates proinflammatory responses in both blood-derived and central nervous system (CNS)-resident myeloid cells, in addition to impacting subsequent adaptive immune responses. Differential miRNA expression may therefore provide insight into mechanisms responsible for distinct phenotypic and functional properties of myeloid cells, thus impacting their ability to influence CNS injury and repair. Ann Neurol 2013;74:709-720 [ABSTRACT FROM AUTHOR]

Neuroaxonal damage and loss are increasingly recognized as disability determining features in multiple sclerosis (MS) pathology. However, little is known about the long-term sequelae of inflammatory demyelination on neurons and axons. Spinal cord tissue of 31 MS patients was compared to three amyotrophic lateral sclerosis (ALS) and 10 control subjects. MS lesions were staged according to the density of KiM-1P positive macrophages and microglia and the presence of myelin basic protein (MBP) positive phagocytes. T cells were quantified in the parenchyma and meninges. Neuroaxonal changes were studied by immunoreactivity (IR) for amyloid precursor protein (APP) and variably phosphorylated neurofilaments (SMI312, SMI31, SMI32). Little T cell infiltration was still evident in chronic inactive lesions. The loss of SMI32 IR in ventral horn neurons correlated with MS lesion development and disease progression. Similarly, axonal loss in white matter (WM) lesions correlated with disease duration. A selective reduction of axonal phosphorylated neurofilaments (SMI31) was observed in WM lesions. In ALS, the loss of neuronal SMI32 IR was even more pronounced, whereas the relative axonal reduction resembled that found in MS. Progressive neuroaxonal neurofilament alterations in the context of chronic inflammatory demyelination may reflect changes in neuroaxonal metabolism and result in chronic neuroaxonal dysfunction as a putative substrate of clinical progression. [ABSTRACT FROM AUTHOR]

Fingolimod (FTY720) is a sphingosine 1-phosphate (S1P) receptor modulator that regulates lymphocyte trafficking and exerts pleiotropic actions on oligodendrocytes (OLGs) and other neural cells. The purpose of this study was to investigate the role of S1P receptors in a non-T-cell model of demyelination, the cuprizone (cupr) model in C57BL/6 mice. Treatment with FTY720 (1 mg/kg) led to attenuated injury to OLGs, myelin, and axons in the corpus callosum (percentage of myelinated fibers was 44.7% in cupr-water and 63% in cupr-FTY720). Reactive astrogliosis and microgliosis were ameliorated when FTY720 was given from d 1, but astrogliosis was augmented when FTY720 was given from wk 4-9. FTY720 did not promote remyelination in this model. The protective effect of FTY720 was associated with decreased interleukin-1β and CCL2 transcripts in the corpus callosum, as well as altered S1P1 expression. Targeted deletion of S1P1 in OLG lineage cells did not lead to obvious clinical phenotype, but resulted in subtle abnormalities in myelin and an increased susceptibility to cupr-induced demyelination. We conclude that S1P receptors expressed by neuroglia are involved in regulating the response to injury, and CNS effects of FTY720 could contribute to its favorable therapeutic response in multiple sclerosis. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 113, 1589–1597. The guidance cue netrin-1 and its receptor Deleted in Colorectal Cancer play distinct roles during different stages of oligodendrocyte development. A gradient of netrin-1 repels migrating oligodendrocyte precursor cells (OPCs) in the embryonic spinal cord by promoting process collapse, but later in development netrin-1 increases oligodendrocyte process extension and branching. Here we investigate the intracellular mechanism that governs this switch in response to netrin-1, and focus on the role of the GTPase RhoA and its effector Rho Kinase (ROCK) downstream of netrin-1 in OPCs and maturing oligodendrocytes. In OPCs, we show that netrin-1 induces a sustained increase in RhoA activity that requires Deleted in Colorectal Cancer function. Furthermore, we demonstrate that activation of RhoA and ROCK is required for the reduction in OPC process length triggered by netrin-1, and for the chemorepellent response made by OPCs to netrin-1. Unlike OPCs, application of netrin-1 to oligodendrocytes decreases RhoA activity. We demonstrate that inactivation of RhoA is essential for netrin-1 to increase oligodendrocyte process branching. We conclude that netrin-1 induces distinct morphological responses in OPCs and oligodendrocytes through differential regulation of RhoA activity. [ABSTRACT FROM AUTHOR]

Objective The serum of most neuromyelitis optica (NMO) patients contains autoantibodies (NMO-IgGs) directed against the aquaporin-4 (AQP4) water channel located on astrocyte foot processes in the perivessel and subpial areas of the brain. Our objectives were to determine the source of central nervous system (CNS) NMO-IgGs and their role in disease pathogenesis. Methods Fluorescence-activated cell sorting and single-cell reverse transcriptase polymerase chain reaction were used to identify overrepresented plasma cell immunoglobulin (Ig) sequences in the cerebrospinal fluid (CSF) of an NMO patient after a first clinical attack. Monoclonal recombinant antibodies (rAbs) were generated from the paired heavy and light chain sequences and tested for target specificity and Fc effector function. The effect of CSF rAbs on CNS immunopathology was investigated by delivering single rAbs to rats with experimental autoimmune encephalomyelitis (EAE). Results Repertoire analysis revealed a dynamic, clonally expanded plasma cell population with features of an antigen-targeted response. Using multiple independent assays, 6 of 11 rAbs generated from CSF plasma cell clones specifically bound to AQP4. AQP4-specific rAbs recognized conformational epitopes and mediated both AQP4-directed antibody-dependent cellular cytotoxicity and complement-mediated lysis. When administered to rats with EAE, an AQP4-specific NMO CSF rAb induced NMO immunopathology: perivascular astrocyte depletion, myelinolysis, and complement and Ig deposition. Interpretation Molecular characterization of the CSF plasma cell repertoire in an early NMO patient demonstrates that AQP4-specfic Ig is synthesized intrathecally at disease onset and directly contributes to CNS pathology. AQP4 is now the first confirmed antigenic target in human demyelinating disease. Ann Neurol 2009;66:617-629 [ABSTRACT FROM AUTHOR]

Research in multiple sclerosis (MS) has recently been focusing on the extent of neuroaxonal damage and its contribution to disease outcome. In the present study, we examined spinal cord tissue from 30 clinically well-characterized MS patients. MS, amyotrophic lateral sclerosis (ALS), and control spinal cord tissue were subjected to morphometric analysis and immunohistochemistry for markers of cell damage and regeneration. Data were related to disease duration and age at death. Here, we present evidence for substantial, nonprogressive neuronal loss on the cervical and lumbar levels early in the disease course of MS. Chromatolytic neurons and immunoreactivity for c-Jun and GAP43 were observed in the ventral gray matter in and adjacent to actively demyelinating lesions, pointing toward neuronal damage and regeneration as an early response to lesion formation. Ann Neurol 2009;66:698-704 [ABSTRACT FROM AUTHOR]

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro. In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo, but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution. [ABSTRACT FROM AUTHOR]

Objective FTY720, a sphingosine-1-phosphate (S1P) receptor agonist that crosses the blood-brain barrier, is a potential immuno-therapy for multiple sclerosis. Our objective was to assess the effect of FTY720 on process extension, differentiation, and survival of human oligodendrocyte progenitor cells (OPCs), and link the functional effects with S1P receptor expression and signaling. Methods Functional assays and receptor expression studies were conducted on A2B5+ OPCs derived from the human fetal central nervous system. Cells were treated with physiologically relevant concentrations of the active phosphorylated form of FTY720. S1P receptor/signaling modulators were used to elucidate the basis of the FTY720-induced functional responses. Results Short-term (1 day) FTY720 treatment caused initial process retraction that was reversed by uncoupling S1P3 and 5 from their G protein using suramin, and with a Rho-kinase inhibitor H1152. Retraction was associated with RhoA-mediated cytoskeletal signaling and with inhibition of OPC differentiation into more mature phenotypes. Continued FTY720 treatment (2 days) induced process extension and enhanced cell survival associated with increased extracellular signal-regulated kinases 1 and 2 phosphorylation, mimicked with the S1P1-specific agonist SEW2871, but not reversed with suramin. Quantitative real-time polymerase chain reaction showed that FTY720 induced reciprocal and cyclic modulation of S1P1 and S1P5 messenger RNA levels. The observed initial downregulation of S1P5 and subsequently of S1P1 messenger RNA supports functional responses being mediated sequentially by S1P5- and later S1P1-associated signaling. Interpretation FTY720 induces time-dependent modulation of S1P receptors on human OPCs with consequent functional responses that are directly relevant for the remyelination process. Ann Neurol 2007 [ABSTRACT FROM AUTHOR]

Abstract: Oligodendrocytes (OLs) and their myelin membranes are the primary targets in the autoimmune disease multiple sclerosis (MS). The inflammatory cytokine tumor necrosis factor-α (TNF-α) has been implicated as a mediator of OL cell injury. TNF-α is detectable within MS lesions and induces apoptosis of mature human OLs in vitro. One possible mechanism by which TNF-α mediates cell death is through the activation of c-jun N-terminal kinase (JNK). We have previously shown that treatment of human OLs with TNF-α leads to activation of JNK. Here we provide evidence that p53, a regulator of the cell cycle and apoptosis, is a mediator of TNF-α-induced apoptosis of OLs. Although p53 was undetectable by western blot analysis in adult human OLs, its levels increased within 24 h after TNF-α treatment (100 ng/ml). The induced p53 was immunolocalized to the nucleus prior to the appearance of significant numbers of apoptotic cells. Overexpression of p53 by adenovirus-mediated gene transfer into human OLs in vitro resulted in marked apoptosis as revealed by in situ cleavage of DNA (TUNEL positive), decreased mitochondrial function, and release of lactate dehydrogenase into the culture medium. These in vitro studies demonstrate that increased p53 levels are associated with apoptosis of human OLs. The findings further implicate p53 as a target for the JNK pathway activated during TNF-α-mediated cell death of human adult OLs. [ABSTRACT FROM AUTHOR]