Oligodendrocyte Cell Bodies Research Articles

Diverse injury responses of human oligodendrocyte to mediators implicated in multiple sclerosis.

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.

Detection of MOG-IgG by cell-based assay: moving from discovery to clinical practice.

Myelin oligodendrocyte glycoprotein (MOG) is a unique CNS-specific mammalian protein that is expressed on the surface of compact myelin and oligodendrocyte cell bodies. MOG is an accessible target for autoantibodies, associated with immune-mediated demyelination in the central nervous system. The identification of MOG reactive immunoglobulin G antibodies (MOG-IgG) helps to distinguish a subgroup of patients from multiple sclerosis and other CNS disorders, reducing the risk of clinical misdiagnosis. The development of the cell-based assays (CBA) improved the detection of clinically meaningful MOG-IgG binding to conformational MOG expressed in the cell membrane surface. In this review, we describe factors that impact on the results of CBA, such as MOG conformation, protein glycosylation, addition of fluorescent tags, serum dilution, secondary antibodies, and data interpretation.

Microtubule organization and dynamics in oligodendrocytes, astrocytes, and microglia.

Though much is known about microtubule organization and microtubule-based transport in neurons, the development and function of microtubules in glia are more enigmatic. In this review, we provide an overview of the literature on microtubules in ramified brain cells, including oligodendrocytes, astrocytes, and microglia. We focus on normal cell biology-how structure relates to function in these cells. In oligodendrocytes, microtubules are important for extension of processes that contact axons and for elongating the myelin sheath. Recent studies demonstrate that new microtubules can form outside of the oligodendrocyte cell body off of Golgi outpost organelles. In astrocytes and microglia, changes in cell shape and ramification can be influenced by neighboring cells and the extracellular milieu. Finally, we highlight key papers implicating glial microtubule defects in neurological injury and disease and discuss how microtubules may contribute to invasiveness in gliomas. Thus, future research on the mechanisms underlying microtubule organization in normal glial cell function may yield valuable insights on neurological disease pathology.

Gallic and vanillic acid suppress inflammation and promote myelination in an in vitro mouse model of neurodegeneration.

Neuroinflammation affects millions of people around the world as a result of injury or stress. Neuroinflammation represents almost all types of neurological diseases such as multiple sclerosis and Alzheimer's disease. Neurodegenerative diseases comprise demyelination and synaptic loss. The inflammatory response is further propagated by the activation of glial cells and modulation of constitutively expressed extracellular matrix proteins. The aim of the present study was to identify the anti-inflammatory effects of purified compounds gallic acid (GA, 1.0µM) and vanillic acid (VA, 0.2µM) on the lysolecithin (LPC, 0.003%)-induced model of inflammation. Hippocampal neurons were co-cultured with glial cells, and LPC was added to induce inflammation. Neurite outgrowth was measured by morphometry software. The level of myelination and demyelination was identified by immunostaining and sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting techniques using different antibodies. Whole-cell patch clamp recordings were used to observe the sustained repetitive firing pattern. The data showed that GA and VA significantly increased the neurite outgrowth after 48h in culture. Both compounds significantly reduced the expression of cyclooxygenase-2, NFκB, tenascin-C, chondroitin sulfate proteoglycans and glial fibrillary acidic protein in astrocytes in the LPC-induced model of inflammation. The level of myelin protein in neurites and oligodendrocyte cell bodies was significantly upregulated by GA and VA treatment. The reduction in sustained repetitive firing in the LPC-induced model of inflammation was reversed by both GA and VA treatment. This study supports the hypothesis that VA and GA have anti-inflammatory activities and could be regarded as potential treatments for neurodegenerative disease.

Localization of the zinc binding tubulin polymerization promoting protein in the mice and human eye

Tubulin Polymerization Promoting Protein (TPPP/p25) modulates the dynamics and stability of the microtubule network by its bundling and acetylation enhancing activities that can be modulated by the binding of zinc to TPPP/p25. Its expression is essential for the differentiation of oligodendrocytes, the major constituents of the myelin sheath, and has been associated with neuronal inclusions. In this paper, evidence is provided for the expression and localization of TPPP/p25 in the zinc-rich retina and in the oligodendrocytes in the optic nerve. Localization of TPPP/p25 was established by confocal microscopy using calbindin and synaptophysin as markers of specific striations in the inner plexiform layer (IPL) and presynaptic terminals, respectively. Postsynaptic nerve terminals in striations S1, S3 and S5 in the IPL and a subset of amacrine cells show immunopositivity against TPPP/p25 both in mice and human eyes. The co-localization of TPPP/p25 with acetylated tubulin was detected in amacrine cells, oligodendrocyte cell bodies and in synapses in the IPL. Quantitative Western blot revealed that the TPPP/p25 level in the retina was 0.05–0.13 ng/μg protein, comparable to that in the brain. There was a central (from optic nerve head) to peripheral retinal gradient in TPPP/p25 protein levels. Our in vivo studies revealed that the oral zinc supplementation of mice significantly increased TPPP/p25 as well as acetylated tubulin levels in the IPL. These results suggest that TPPP/p25, a microtubule stabilizer can play a role in the organization and reorganization of synaptic connections and visual integration in the eye.

Myelinosome formation represents an early stage of oligodendrocyte damage in multiple sclerosis and its animal model.

Oligodendrocyte damage is a central event in the pathogenesis of the common neuroinflammatory condition, multiple sclerosis (MS). Where and how oligodendrocyte damage is initiated in MS is not completely understood. Here, we use a combination of light and electron microscopy techniques to provide a dynamic and highly resolved view of oligodendrocyte damage in neuroinflammatory lesions. We show that both in MS and in its animal model structural damage is initiated at the myelin sheaths and only later spreads to the oligodendrocyte cell body. Early myelin damage itself is characterized by the formation of local myelin out-foldings—‘myelinosomes'—, which are surrounded by phagocyte processes and promoted in their formation by anti-myelin antibodies and complement. The presence of myelinosomes in actively demyelinating MS lesions suggests that oligodendrocyte damage follows a similar pattern in the human disease, where targeting demyelination by therapeutic interventions remains a major open challenge.

The oligodendrocyte-specific antibody 'CC1' binds Quaking 7.

The mouse monoclonal antibody marketed as anti-adenomatous polyposis coli clone CC1, often referred to as CC1, is the antibody most commonly used to specifically label mature oligodendrocytes without labeling myelin. Previous studies have shown that despite being raised against adenomatous polyposis coli, this antibody binds another unknown antigen. We show that the CC1 antibody binds Quaking 7, an RNA-binding protein that is highly up-regulated in myelinating oligodendrocytes in the central nervous system. The monoclonal antibody anti-adenomatous polyposis coli (APC) clone CC1, is the antibody most commonly used to specifically label the cell bodies of mature oligodendrocytes. Despite being raised against APC, previous studies showed this antibody binds another unknown antigen. We show that the CC1 antibody binds Quaking (QKI) 7, an RNA-binding protein which is highly up-regulated in myelinating oligodendrocytes.

The VF rat with abnormal myelinogenesis has a mutation in Dopey1

The vacuole formation (VF) rat is an autosomal recessive myelin mutant characterized by generalized tremor, hypomyelination, and periaxonal vacuole formation of the central nervous system (CNS). Here, we report the most likely causative gene for neurological disease in the VF rat and pursue its roles in the development and maintenance of the CNS myelin. We identified a nonsense mutation in the dopey family member 1 (Dopey1) located on rat chromosome 8. Expression level of Dopey1 mRNA was decreased and DOPEY1 protein was undetectable both in the white and gray matter of the spinal cords in the VF rats. Double immunohistochemistry demonstrated that DOPEY1 was mainly expressed in neurons and oligodendrocytes in the wild-type rats, whereas no positive cells were detected in the VF rats. We also demonstrated a marked reduction in myelin components both at mRNA and protein levels during myelinogenesis in the VF rats. In addition, proteolipid protein and myelin-associated glycoprotein accumulated in oligodendrocyte cell body, suggesting that Dopey1 is likely to be involved in the traffic of myelin components. Our results highlighted the importance of Dopey1 for the development and maintenance of the CNS myelin.

Assessing White Matter Ischemic Damage in Dementia Patients by Measurement of Myelin Proteins

White matter ischemia is difficult to quantify histologically. Myelin-associated glycoprotein (MAG) is highly susceptible to ischemia, being expressed only adaxonally, far from the oligodendrocyte cell body. Myelin-basic protein (MBP) and proteolipid protein (PLP) are expressed throughout the myelin sheath. We compared MAG, MBP, and PLP levels in parietal white matter homogenates from 17 vascular dementia (VaD), 49 Alzheimer's disease (AD), and 33 control brains, after assessing the post-mortem stability of these proteins. Small vessel disease (SVD) and cerebral amyloid angiopathy (CAA) severity had been assessed in paraffin sections. The concentration of MAG remained stable post-mortem, declined with increasing SVD, and was significantly lower in VaD than controls. The concentration of MBP fell progressively post-mortem, limiting its diagnostic utility in this context. Proteolipid protein was stable post-mortem and increased significantly with SVD severity. The MAG/PLP ratio declined significantly with SVD and CAA severity. The MAG and PLP levels and MAG/PLP did not differ significantly between AD and control brains. We validated the utility of MAG and MAG/PLP measurements on analysis of 74 frontal white matter samples from an Oxford cohort in which SVD had previously been scored. MAG concentration and the MAG/PLP ratio are useful post-mortem measures of ante-mortem white matter ischemia.

FAS-Dependent Cell Death in α-Synuclein Transgenic Oligodendrocyte Models of Multiple System Atrophy

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention.

Targeted Ablation of Oligodendrocytes Induces Axonal Pathology Independent of Overt Demyelination

The critical role of oligodendrocytes in producing and maintaining myelin that supports rapid axonal conduction in CNS neurons is well established. More recently, additional roles for oligodendrocytes have been posited, including provision of trophic factors and metabolic support for neurons. To investigate the functional consequences of oligodendrocyte loss, we have generated a transgenic mouse model of conditional oligodendrocyte ablation. In this model, oligodendrocytes are rendered selectively sensitive to exogenously administered diphtheria toxin (DT) by targeted expression of the diphtheria toxin receptor in oligodendrocytes. Administration of DT resulted in severe clinical dysfunction with an ascending spastic paralysis ultimately resulting in fatal respiratory impairment within 22 d of DT challenge. Pathologically, at this time point, mice exhibited a loss of ∼26% of oligodendrocyte cell bodies throughout the CNS. Oligodendrocyte cell-body loss was associated with moderate microglial activation, but no widespread myelin degradation. These changes were accompanied with acute axonal injury as characterized by structural and biochemical alterations at nodes of Ranvier and reduced somatosensory-evoked potentials. In summary, we have shown that a death signal initiated within oligodendrocytes results in subcellular changes and loss of key symbiotic interactions between the oligodendrocyte and the axons it ensheaths. This produces profound functional consequences that occur before the removal of the myelin membrane, i.e., in the absence of demyelination. These findings have clear implications for the understanding of the pathogenesis of diseases of the CNS such as multiple sclerosis in which the oligodendrocyte is potentially targeted.

Claudin expression profile separates Alzheimer's disease cases from normal aging and from vascular dementia cases

We have reported earlier that tight-junction proteins are detectable by standard immunohistochemistry in the brain parenchyma, namely in the cell bodies of neurons, astrocytes, and oligodendrocytes. Here we show, by projection to latent structures — discriminate analysis (PLS-DA), that the immunohistochemical detection profile of tight junction proteins clearly distinguishes the AD cases from healthy aging controls and from the cases of dementia with a predominantly vascular pathology underlying the symptoms (vascular dementia, VaD; cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, CADASIL; and cerebral amyloid angiopathy, CAA). Our findings might be valuable in the perspective of developing biomarkers for AD.

Axon Myelination and Electrical Stimulation in a Microfluidic, Compartmentalized Cell Culture Platform

Axon demyelination contributes to the loss of sensory and motor function following injury or disease in the central nervous system. Numerous reports have demonstrated that myelination can be achieved in neuron/oligodendrocyte co-cultures. However, the ability to selectively treat neuron or oligodendrocyte (OL) cell bodies in co-cultures improves the value of these systems when designing mechanism-based therapeutics. We have developed a microfluidic-based compartmentalized culture system to achieve segregation of neuron and OL cell bodies while simultaneously allowing the formation of myelin sheaths. Our microfluidic platform allows for a high replicate number, minimal leakage, and high flexibility. Using a custom built lid, fit with platinum electrodes for electrical stimulation (10-Hz pulses at a constant 3 V with ~190 kΩ impedance), we employed the microfluidic platform to achieve activity-dependent myelin segment formation. Electrical stimulation of dorsal root ganglia resulted in a fivefold increase in the number of myelinated segments/mm² when compared to unstimulated controls (19.6 ± 3.0 vs. 3.6 ± 2.3 MBP+ segments/mm²). This work describes the modification of a microfluidic, multi-chamber system so that electrical stimulation can be used to achieve increased levels of myelination while maintaining control of the cell culture microenvironment.

Extensive aspartoacylase expression in the rat central nervous system.

Aspartoacylase (ASPA) catalyzes deacetylation of N-acetylaspartate (NAA) to generate acetate and aspartate. Mutations in the gene for ASPA lead to reduced acetate availability in the CNS during development resulting in the fatal leukodystrophy Canavan disease. Highly specific polyclonal antibodies to ASPA were used to examine CNS expression in adult rats. In white matter, ASPA expression was associated with oligodendrocyte cell bodies, nuclei, and some processes, but showed a dissimilar distribution pattern to myelin basic protein and oligodendrocyte specific protein. Microglia expressed ASPA in all CNS regions examined, as did epiplexus cells of the choroid plexus. Pial and ependymal cells and some endothelial cells were ASPA positive, as were unidentified cellular nuclei throughout the CNS. Astrocytes did not express ASPA in their cytoplasm. In some fiber pathways and nerves, particularly in the brainstem and spinal cord, the axoplasm of many neuronal fibers expressed ASPA, as did some neurons. Acetyl coenzyme A synthase immunoreactivity was also observed in the axoplasm of many of the same fiber pathways and nerves. All ASPA-immunoreactive elements were unstained in brain sections from tremor rats, an ASPA-null mutant. The strong expression of ASPA in oligodendrocyte cell bodies is consistent with a lipogenic role in myelination. Strong ASPA expression in cell nuclei is consistent with a role for NAA-derived acetate in nuclear acetylation reactions, including histone acetylation. Expression of ASPA in microglia may indicate a role in lipid synthesis in these cells, whereas expression in axons suggests that some neurons can both synthesize and catabolize NAA.

Robust In Vivo Transduction of Nervous System and Neural Stem Cells by Early Gestational Intra Amniotic Gene Transfer Using Lentiviral Vector

Presently, in vivo methods to efficiently and broadly transduce all major cell types throughout both the central (CNS) and peripheral adult nervous system (PNS) are lacking. In this study, we hypothesized that during early fetal development neural cell populations, including neural stem cells (NSCs), may be accessible for gene transfer via the open neural groove. To test this hypothesis, we injected lentiviral vectors encoding a green fluorescent protein (GFP) marker gene into the murine amniotic cavity at embryonic day 8. This method (i) efficiently and stably transduced the entire nervous system for at least 80% of the lifespan of the mice, (ii) transduced all major neural cell types, and (iii) transduced adult NSCs of the subventricular zone (SVZ) and subgranular zones (SGZs). This simple approach has broad applications for the study of gene function in nervous system development and adult NSCs and may have future clinical applications for treatment of genetic disorders of the nervous system.

Oligodendrocyte degeneration and recovery after focal cerebral ischemia

The vulnerability of oligodendrocytes to ischemic injury may contribute to functional loss in diseases of central white matter. Immunocytochemical methods to identify oligodendrocyte injury in experimental models rely on epitope availability, and fail to discriminate structural changes in oligodendrocyte morphology. We previously described the use of a lentiviral vector (LV) carrying enhanced green fluorescent protein (eGFP) under the myelin basic protein (MBP) promoter for selective visualization of oligodendrocyte cell bodies and processes. In this study, we used LV-MBP-eGFP to label oligodendrocytes in rat cerebral white matter prior to transient focal cerebral ischemia, and examined oligodendrocyte injury 24 h, 48 h and 1 week post-reperfusion by quantifying cell survival and assaying the integrity of myelin processes. There was progressive loss of GFP+ oligodendrocytes in ischemic white matter at 24 and 48 h. Surviving GFP+ cells had non-pyknotic nuclear morphology and were terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-negative, but there was marked fragmentation of myelin processes as early as 24 h after stroke. One week after stroke, we observed a restoration of GFP+ oligodendrocytes in ischemic white matter, reflected both by cell counts and by structural integrity of myelin processes. Proliferating cells were not the main source of GFP+ oligodendrocytes, as revealed by bromodeoxyuridine (BrdU) incorporation. These observations identify novel transient structural changes in oligodendrocyte cell bodies and myelinating processes, which may have consequences for white matter function after stroke.

Axon–Myelin Interactions during a Viral Infection of the Central Nervous System

Theiler's virus offers a remarkable example of a pathogen that navigates the various cells of the organism to evade immune responses and establish a persistent infection. Here, we discuss the transition from neuron to myelin and oligodendrocyte infection, a step that is crucial for the persistence of this virus in the central nervous system (CNS). CNS myelin is an extension of the cytoplasmic membrane of oligodendrocytes wrapped numerous times around axons. An oligodendrocyte sends many such extensions and can myelinate up to 50 different axons. Myelinated axon segments are separated by short unmyelinated regions called nodes of Ranvier. Cytoplasm is totally extruded from myelin except in areas where it forms channels that are in continuity with the oligodendrocyte cell body. These channels form the so-called ad-axonal inner loop and the paranodal loops at the level of the nodes of Ranvier. Inner and paranodal loops are in close contact with the axon membrane (Figure 1). (For a review of myelin and node organization, see [1].) Figure 1 Diagrammatic view of CNS myelin.

Kif1b is essential for mRNA localization in oligodendrocytes and development of myelinated axons

The kinesin motor protein Kif1b has previously been implicated in the axonal transport of mitochondria and synaptic vesicles1,2. More recently kif1b has been linked with susceptibility to Multiple Sclerosis (MS) 3. Here we show that Kif1b is required for the localization of myelin basic protein mRNA to processes of myelinating oligodendrocytes in zebrafish. We observe the ectopic appearance of myelin-like membrane in kif1b mutants, coincident with the ectopic localization of myelin proteins in kif1b mutant oligodendrocyte cell bodies. These observations suggest the hypothesis that oligodendrocytes localize certain mRNA molecules, namely those encoding small basic proteins such as mbp, to prevent aberrant effects of these proteins elsewhere in the cell. We also find that Kif1b is required for outgrowth of some of the longest axons in the peripheral and central nervous systems. Our data demonstrate new functions of kif1b in vivo and provide insights into its possible roles in Multiple Sclerosis.

Focal adhesion kinase (FAK): A regulator of CNS myelination

The formation of the myelin sheath is a crucial step during development because it enables fast and efficient propagation of signals within the limited space of the mammalian central nervous system (CNS). During the process of myelination, oligodendrocytes actively interact with the extracellular matrix (ECM). These interactions are considered crucial for proper and timely completion of the myelin sheath. However, the exact regulatory circuits involved in the signaling events that occur between the ECM and oligodendrocytes are currently not fully understood. Therefore, in the present study we investigated the role of a known integrator of cell-ECM signaling, namely, focal adhesion kinase (FAK), in CNS myelination via the use of conditional (oligodendrocyte-specific) and inducible FAK-knockout mice (Fak(flox/flox): PLP/CreER(T) mice). When inducing FAK knockout just prior to and during active myelination of the optic nerve, we observed a significant reduction in the number of myelinated fibers on postnatal day 14. In addition, our data revealed a decreased number of primary processes extending from oligodendrocyte cell bodies at this postnatal age and on induction of FAK knockout. In contrast, myelination appeared normal on postnatal day 28. Thus, our data suggest that FAK controls the efficiency and timing of CNS myelination during its initial stages, at least in part, by regulating oligodendrocyte process outgrowth and/or remodeling.

Disposition of axonal caspr with respect to glial cell membranes: Implications for the process of myelination

Neurofascin-155 (NF155) and caspr are transmembrane proteins found at discrete locations early during development of the nervous system. NF155 is present in the oligodendrocyte cell body and processes, whereas caspr is on the axonal surface. In mature nerves, these proteins are clustered at paranodes, flanking the node of Ranvier. To understand how NF155 and caspr become localized to the paranodal regions of myelinated nerves, we have studied their distribution over time in myelinating cultures. Our observations indicate that these two proteins are recruited to the cell surface at the contact zone between axons and oligodendrocytes, where they trans-interact. This association explains the early pattern of caspr distribution, a helical coil that winds around the axon, resembling the turns of the myelin sheath. Caspr, an axonal membrane protein, therefore seems to move in register with the overlying myelinating cell via its interactions with myelin proteins. We suggest that NF155 is the glial cell membrane protein responsible for caspr distribution. The pair act as interacting partners on either side of the axoglial contact area. Most likely, there are other proteins on the axonal surface whose distribution is equally influenced by interaction with the nascent myelin sheath. The fact that caspr follows the movement of the spiraling membrane has a direct affect on the interpretation of the way in which myelin is formed.