Toll-like receptors 2 and 3 agonists differentially affect oligodendrocyte survival, differentiation, and myelin membrane formation

Toll-like receptors (TLRs) play a key role in controlling innate immune responses to a wide variety of pathogen-associated molecules as well as endogenous signals. In addition, TLR expression within nonimmune cells has been recognized as as modulator of cell behavior. In this study we have addressed the question of whether functional TLRs are expressed on oligodendrocytes, the myelinating cells of the central nervous system. Primary cultures of rat oligodendrocytes at different maturation stages were found to express TLR2 and, to lesser extent, TLR3. Immunocytochemical analysis revealed that both TLRs were localized at the cell body and primary processes and were excluded from myelin-like membranes. Interestingly, innate immune receptor ligands were able to modulate oligodendrocyte survival, differentiation, and myelin-like membrane formation, indicating that TLRs on oligodendrocytes are functional. In highly purified oligodendrocytes cultures, the TLR2 agonist zymosan promoted survival, differentiation, and myelin-like membrane formation, whereas poly-I:C, a TLR3 ligand, was a potent inducer of apoptosis. Together, these data indicate that, in addition to other neural cell types, also oligodendrocytes express functional TLRs, which play a role in regulating various aspects of oligodendrocyte behavior.     

Interaction of mTOR and Erk1/2 signaling to regulate oligodendrocyte differentiation

A multitude of factors regulate oligodendrocyte differentiation and remyelination, and to elucidate the mechanisms underlying this process, we analyzed the interactions of known signaling pathways involved in these processes. Previous work from our lab and others shows that Akt, mTOR, and Erk 1/2 are major signaling pathways regulating oligodendrocyte differentiation and myelination in vitro and in vivo. However, the relative contribution of the different pathways has been difficult to establish because the impact of inhibiting one pathway in in vitro cell culture models or in vivo may alter signaling through the other pathway. These studies were undertaken to clarify the interactions between these major pathways and understand more specifically the crosstalk between them. Oligodendrocyte differentiation in vitro required Akt, mTOR, and Erk 1/2 signaling, as inhibition of Akt, mTOR, or Erk 1/2 resulted in a significant decrease of myelin basic protein mRNA and protein expression. Interestingly, while inhibition of the Erk1/2 pathway had little impact on Akt/mTOR signaling, inhibition of the Akt/mTOR pathways significantly increased Erk1/2 signaling, although not enough to overcome the loss of Akt/mTOR signaling in the regulation of oligodendrocyte differentiation. Furthermore, such crosstalk was also noted in an in vivo context, after mTOR inhibition by rapamycin treatment of perinatal pups. GLIA 2014;62:2096–2109     

Maintenance of high proteolipid protein level in adult central nervous system myelin is required to preserve the integrity of myelin and axons

Proteolipid protein (PLP) is the most abundant integral membrane protein in central nervous system (CNS) myelin. Expression of the Plp-gene in oligodendrocytes is not essential for the biosynthesis of myelin membranes but required to prevent axonal pathology. This raises the question whether the exceptionally high level of PLP in myelin is required later in life, or whether high-level PLP expression becomes dispensable once myelin has been assembled. Both models require a better understanding of the turnover of PLP in myelin in vivo. Thus, we generated and characterized a novel line of tamoxifen-inducible Plp-mutant mice that allowed us to determine the rate of PLP turnover after developmental myelination has been completed, and to assess the possible impact of gradually decreasing amounts of PLP for myelin and axonal integrity. We found that 6 months after targeting the Plp-gene the abundance of PLP in CNS myelin was about halved, probably reflecting that myelin is slowly turned over in the adult brain. Importantly, this reduction by 50% was sufficient to cause the entire spectrum of neuropathological changes previously associated with the developmental lack of PLP, including myelin outfoldings, lamellae splittings, and axonal spheroids. In comparison to axonopathy and gliosis, the infiltration of cytotoxic T-cells was temporally delayed, suggesting a corresponding chronology also in the genetic disorders of PLP-deficiency. High-level abundance of PLP in myelin throughout adult life emerges as a requirement for the preservation of white matter integrity.     

Erk1/2 MAPK and mTOR signaling sequentially regulates progression through distinct stages of oligodendrocyte differentiation

Myelination is the culmination of a complex process in which oligodendrocyte (OL) progenitors transition through defined stages in a well-coordinated differentiation program. The signaling mechanisms that regulate this progression are poorly understood. Here we investigate the role of extracellular signal-regulated-kinase-1,-2 (Erk1/2) and the mammalian target of rapamycin (mTOR), downstream effectors of the Ras/Raf/Mek/Erk and PI3K/Akt/mTOR pathways, at specific stages of OL development in vitro. Using a panel of developmental stage-specific antigenic markers and pharmacological inhibitors, we provide evidence that Erk1/2 signaling regulates transition of early progenitors to the late progenitor stage and, as a consequence, to the immature OL stage, but not the transition of immature OL to the mature OL stage. In contrast, mTOR signaling is not required for early progenitor transition to late progenitor stage. Surprisingly, it is also not required for the transition of late progenitors to terminally differentiated immature OLs, as has been reported previously, but is required for the next sequential transition of immature OLs to the mature OL stage. Furthermore, mTOR signaling regulates OL cytoskeletal organization and major myelin protein expression. These in vitro findings correlate with our in vivo data showing that inhibition of mTOR by rapamycin injection attenuated the onset of myelination in the early postnatal brain. Thus, these studies demonstrate that Erk1/2 and mTOR signaling sequentially regulates distinct stages of OL progenitor differentiation and suggest that cells in the OL-lineage require distinct signaling mechanisms to transition through specific stages of their development.     

Ablation of neuronal ADAM17 impairs oligodendrocyte differentiation and myelination

Myelin, one of the most important adaptations of vertebrates, is essential to ensure efficient propagation of the electric impulse in the nervous system and to maintain neuronal integrity. In the central nervous system (CNS), the development of oligodendrocytes and the process of myelination are regulated by the coordinated action of several positive and negative cell-extrinsic factors. We and others previously showed that secretases regulate the activity of proteins essential for myelination. We now report that the neuronal α-secretase ADAM17 controls oligodendrocyte differentiation and myelin formation in the CNS. Ablation of Adam17 in neurons impairs in vivo and in vitro oligodendrocyte differentiation, delays myelin formation throughout development and results in hypomyelination. Furthermore, we show that this developmental defect is, in part, the result of altered Notch/Jagged 1 signaling. Surprisingly, in vivo conditional loss of Adam17 in immature oligodendrocytes has no effect on myelin formation. Collectively, our data indicate that the neuronal α-secretase ADAM17 is required for proper CNS myelination. Further, our studies confirm that secretases are important post-translational regulators of myelination although the mechanisms controlling CNS and peripheral nervous system (PNS) myelination are distinct.     

Identification of GTP-binding proteins in myelin and oligodendrocyte membranes

Myelin membranes purified from mouse and rat brain are associated with alpha subunits of four signal transducing guanosine triphosphate (GTP)-binding proteins: Go, Gi, Gs, and ras. Four low-molecular-weight (Mr) GTP-binding proteins are also present, as demonstrated by the binding of GTP to proteins immobilized in nitrocellulose. This latter group is more prominent at early stages of myelination and remains associated with isolated myelin membranes despite repetitive cycles of purification. At least one nonmyelin subcellular membrane fraction possesses the same proteins. The total membrane fraction of cultured oligodendrocytes is associated with both groups of GTP-binding proteins. None of the well-known myelin proteins bound GTP by the procedure described.     

Quantitation of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and central nervous system and its relationship to the clinicopathologic features of autoimmune encephalomyelitis

There is controversy whether the amount of autoantigens expressed in the thymus regulates negative selection of autoreactive T cells and determine susceptibility or resistance to experimental autoimmune encephalomyelitis (EAE). In the present study, we have addressed this issue by quantifying neuroantigens in the thymus of two EAE-susceptible (LEW and LEW.1AV1) and one EAE-resistant (BN) rat strains. We further examined whether amounts of neuroantigens in various parts of the central nervous system (CNS) affect the clinical course and lesion distribution of acute and chronic EAE. Real-time PCR and histologic analyses showed that there was no significant difference in the amount and distribution of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and CNS among the three strains and that both acute and chronic EAE lesions in the CNS were preferentially distributed in the area where neuroantigens were abundantly present. These findings suggest that susceptibility or resistance to EAE is not regulated by the amount of the neuroantigens expressed in the thymus. Furthermore, the lesion distribution, but not the clinical course, of EAE is related to the neuroantigen expression in the CNS.     

Transplantation of oligodendrocyte progenitor cells into the rat retina: Extensive myelination of retinal ganglion cell axons

In most mammals, retinal ganglion cell axons are unmyelinated in the retina. The same axons become myelinated in the optic nerve. Various studies suggest that retinal ganglion cell axons are also in principle, myelination competent intraretinally and that non-neuronal factors at the retinal end of the optic nerve prevent the migration of oligodendrocyte progenitor cells into the retina. To test this hypothesis directly, we injected oligodendrocyte progenitor cells into the retina of young postnatal rats. We observed massive myelination of ganglion cell axons in the retina 1 month after cell transplantation. Electron microscopic analysis revealed that intraretinal segments of ganglion cell axons were surrounded by central nervous system myelin sheaths with a normal morphology. Our results thus provide direct evidence for the myelination competence of the intraretinal part of rat retinal ganglion cell axons. © 1996 Wiley-Liss, Inc.     

Autophagy is essential for oligodendrocyte differentiation,survival, and proper myelination

Deficient myelination, the spiral wrapping of highly specialized membrane around axons, causes severe neurological disorders. Maturation of oligodendrocyte progenitor cells (OPC) to myelinating oligodendrocytes (OL), the sole providers of central nervous system (CNS) myelin, is tightly regulated and involves extensive morphological changes. Here, we present evidence that autophagy, the targeted isolation of cytoplasm and organelles by the double-membrane autophagosome for lysosomal degradation, is essential for OPC/OL differentiation, survival, and proper myelin development. A marked increase in autophagic activity coincides with OL differentiation, with OL processes having the greatest increase in autophagic flux. Multiple lines of evidence indicate that autophagosomes form in developing myelin sheathes before trafficking from myelin to the OL soma. Mice with conditional OPC/OL-specific deletion of the essential autophagy gene Atg5 beginning on postnatal Day 5 develop a rapid tremor and die around postnatal Day 12. Further analysis revealed apoptotic death of OPCs, reduced differentiation, and reduced myelination. Surviving Atg5^−/− OLs failed to produce proper myelin structure. In vitro, pharmacological inhibition of autophagy in OPC/dorsal root ganglion (DRG) co-cultures blocked myelination, producing OLs surrounded by many short processes. Conversely, autophagy stimulation enhanced myelination. These results implicate autophagy as a key regulator of OPC survival, maturation, and proper myelination. Autophagy may provide an attractive target to promote both OL survival and subsequent myelin repair after injury.     

Basic fibroblast growth factor down-regulates myelin basic protein gene expression and alters myelin compaction of mature oligodendrocytes in vitro

The effects of basic fibroblast growth factor (bFGF) on myelin basic protein (MBP) gene expression and myelin-like membrane formation were investigated in oligodendrocyte cultures containing mainly mature oligodendrocytes expressing MBP. These cultures were obtained by selective detachment of the cells of the oligodendrocyte lineage from 40-day-old mixed cultures derived from newborn rat brain. They were further purified by a 3-day pretreatment with cytosine arabinoside (ARA-C) in order to kill cycling cells. After withdrawal of ARA-C, daily treatment of the cells with bFGF for 3 days induced a drastic decrease in MBP mRNA level compared to control cultures treated only with ARA-C. Moreover, the percentage of oligodendrocytes labelled with anti-MBP antibodies decreased by 50%, as well as that of oligodendrocytes expressing myelin oligodendrocyte glycoprotein (MOG), whereas proteolipid protein (PLP) immunolabelled cells were less affected. At the ultrastructural level, myelin-like membranes were still abundant in the ARA-C-and bFGF-treated cultures, but they were conspicuously uncompacted compared to cultures only pretreated with ARA-C. These results bring the first evidence that bFGF is able to down-regulate myelin protein gene expression in mature oligodendrocytes and to alter myelin structure. They imply that if bFGF is secreted after a demyelinating lesion of the central nervous system (CNS), this plasticity of mature oligodendrocytes will allow final remyelination of axons to complete only after this factor has returned to low levels. © 1995 Wiley-Liss, Inc.     

The remyelinating potential and in vitro differentiation of MOG-expressing oligodendrocyte precursors isolated from the adult rat CNS

There is a long-standing controversy as to whether oligodendrocytes may be capable of cell division and thus contribute to remyelination. We recently published evidence that a subpopulation of myelin oligodendrocyte glycoprotein (MOG)-expressing cells in the adult rat spinal cord co-expressed molecules previously considered to be restricted to oligodendrocyte progenitors [G. Li et al. (2002) Brain Pathol., 12, 463-471]. To further investigate the properties of MOG-expressing cells, anti-MOG-immunosorted cells were grown in culture and transplanted into acute demyelinating lesions. The immunosorting protocol yielded a cell preparation in which over 98% of the viable cells showed anti-MOG- and O1-immunoreactivity; 12-15% of the anti-MOG-immunosorted cells co-expressed platelet-derived growth factor alpha receptor (PDGFRalpha) or the A2B5-epitope. When cultured in serum-free medium containing EGF and FGF-2, 15-18% of the anti-MOG-immunosorted cells lost anti-MOG- and O1-immunoreactivity and underwent cell division. On removal of these growth factors, cells differentiated into oligodendrocytes, or astrocytes and Schwann cells when the differentiation medium contained BMPs. Transplantation of anti-MOG-immunosorted cells into areas of acute demyelination immediately after isolation resulted in the generation of remyelinating oligodendrocytes and Schwann cells. Our studies indicate that the adult rat CNS contains a significant number of oligodendrocyte precursors that express MOG and galactocerebroside, molecules previously considered restricted to mature oligodendrocytes. This may explain why myelin-bearing oligodendrocytes were considered capable of generating remyelinating cells. Our study also provides evidence that the adult oligodendrocyte progenitor can be considered as a source of the Schwann cells that remyelinate demyelinated CNS axons following concurrent destruction of oligodendrocytes and astrocytes.     

IL‐17A activates ERK1/2 and enhances differentiation of oligodendrocyte progenitor cells

Inflammatory signals present in demyelinated multiple sclerosis lesions affect the reparative remyelination process conducted by oligodendrocyte progenitor cells (OPCs). Interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α), and interleukin (IL)?6 have differing effects on the viability and growth of OPCs, however the effects of IL‐17A are largely unknown. Primary murine OPCs were stimulated with IL‐17A and their viability, proliferation, and maturation were assessed in culture. IL‐17A‐stimulated OPCs exited the cell cycle and differentiated with no loss in viability. Expression of the myelin‐specific protein, proteolipid protein, increased in a cerebellar slice culture assay in the presence of IL‐17A. Downstream, IL‐17A activated ERK1/2 within 15 min and induced chemokine expression in 2 days. These results demonstrate that IL‐17A exposure stimulates OPCs to mature and participate in the inflammatory response. GLIA 2015;63:768–779     

Cdon,a cell surface protein,mediates oligodendrocyte differentiation and myelination

During central nervous system development, oligodendrocyte progenitors (OLPs) establish multiple branched processes and axonal contacts to initiate myelination. A complete understanding of the molecular signals implicated in cell surface interaction to initiate myelination/remyelination is currently lacking. The objective of our study was to assess whether Cdon, a cell surface protein that was shown to participate in muscle and neuron cell development, is involved in oligodendrocyte (OLG) differentiation and myelination. Here, we demonstrate that endogenous Cdon protein is expressed in OLPs, increasing in the early differentiation stages and decreasing in mature OLGs. Immunocytochemistry of endogenous Cdon showed localization on both OLG cell membranes and cellular processes exhibiting puncta‐ or varicosity‐like structures. Cdon knockdown with siRNA decreased protein levels by 62% as well as two myelin‐specific proteins, MBP and MAG. Conversely, overexpression of full‐length rat Cdon increased myelin proteins in OLGs. The complexity of OLGs branching and contact point numbers with axons were also increased in Cdon overexpressing cells growing alone or in coculture with dorsal root ganglion neurons (DRGNs). Furthermore, myelination of DRGNs was decreased when OLPs were transfected with Cdon siRNA. Altogether, our results suggest that Cdon participates in OLG differentiation and myelination, most likely in the initial stages of development. GLIA 2016;64:1021–1033     

Evaluation of diagnostic criteria and red flags of myelin oligodendrocyte glycoprotein encephalomyelitis in a clinical routine cohort

AimsMyelin oligodendrocyte glycoprotein antibodies (MOG‐IgG) have been proposed to define “MOG encephalomyelitis” (MOG‐EM), with published diagnostic and “red flag” criteria. We aimed to evaluate these criteria in a routine clinical setting.MethodsWe retrospectively analyzed patients with borderline/positive MOG‐IgG and applied the diagnostic and red flag criteria to determine likelihood of MOG‐EM diagnosis. Para‐/clinical parameters were described and analyzed with chi‐square test.ResultsIn total, 37 patients fulfilled MOG‐EM diagnostic criteria (female‐to‐male ratio: 1.6:1, median onset age: 28.0 years [IQR 18.5‐40.5], n = 8 with pediatric onset). In 24/37, red flags were present, predominantly MOG‐IgG at assay cutoff and/or MRI lesions suggestive of multiple sclerosis (MS). As proposed in the consensus criteria, these patients should rather be described as “possible” MOG‐EM. Of these, we classified 13 patients as “unlikely” MOG‐EM in the presence of the red flag “borderline MOG‐IgG” with negative MOG‐IgG retest or coincidence of ≥1 additional red flag. This group mainly consisted of patients diagnosed with MS (n = 11). Frequency of cerebrospinal fluid (CSF‐)—specific oligoclonal bands (OCB) is significantly lower in definite vs possible and unlikely MOG‐EM (P = .0005).ConclusionEvaluation of diagnostic and red flag criteria, MOG‐IgG retesting (incl. change of assay), and CSF‐specific OCB are relevant in clinical routine cohorts to differentiate MOG‐EM from MS.     

Platelet-derived growth factor delays oligodendrocyte differentiation and axonal myelination in vivo in the anterior medullary velum of the developing rat

The AA dimeric form of platelet-derived growth factor (PDGF-AA) is implicated in the differentiation of cells of the oligodendrocyte lineage, which express PDGF receptors of the alpha subunit type (PDGF-αR). In the present study, we show that a single injection of PDGF-AA into the cerebrospinal fluid of neonatal rats delays oligodendrocyte differentiation and interrupts the progress of myelination in the anterior medullary velum (AMV), a white matter tract roofing the IVth ventricle of the brain. PDGF-AA or saline was injected intrathecally in postnatal day (P) 7 rats, and the AMV was subsequently removed and immunolabelled with the oligodendrocyte-specific antibody Rip, at P9, P12, and P21, corresponding to postinjection days (PID) 2, 5, and 14. At P9 (PID2), myelination was retarded in PDGF-AA-treated rats as opposed to saline-treated controls but progressed rapidly after P12 (PID5). Quantification supported the qualitative observations that PDGF-AA mediated an acute decrease in the number of Rip+ oligodendrocytes at P9–12, which largely recovered by P21, suggesting that PDGF-AA may have delayed recruitment of myelinating oligodendrocytes. However, the definitive number of Rip+ oligodendrocytes in the AMV was not increased, suggesting that its action as a promoter of early oligodendrocyte survival may not ultimately affect the definitive number of myelinating oliogdendrocytes in vivo. We discuss the possibilities that excess PDGF-AA may have acted on early oligodendrocytes (precursors or preoligodendrocytes) to either (1) delay their differentiation by maintaining them in the cell cycle or (2) accelerate their differentiation, which may result in premature cell death in the absence of synchronised survival signals. This study supports a role for PDGF-AA in the timing of oligodendrocyte differentiation in vivo, as has been shown in vitro. J. Neurosci. Res. 48:588–596, 1997. © 1997 Wiley-Liss Inc.