HIV-gp120 affects the functional activity of oligodendrocytes and their susceptibility to complement

The aim of this study was to assess whether the HIV protein gp120 can induce direct or/and indirect damage to oligodendrocytes (OL). Using highly purified cultures of rat OL, we report that gp120 binds to OL and induces functional alterations in these cells. Indeed, the percentage of cells expressing myelin basic protein (MBP) and the levels of all four MBP isoforms were substantially reduced after a 3-day treatment with 10 nM gp120. As gp120 depressed the ability of OL to reduce the tetrazolium salt MTT (a sign of mitochondrial impairment), the alteration of MBP production may be a consequence of decreased metabolic activity. The above effects were accompanied by a small increase in the number of apoptotic nuclei (from 4.3% in controls to 17.6% in cells treated for 3 days with gp120). As complement can lyse OL and gp120 is known to activate complement, we also studied the interaction between these two factors using OL cultures. The viral protein potentiated (by about 25%) the lytic effect of complement, when administered to the cultures 5 hr after complement, and depressed it (by about 30–40%), when added 5 hr before complement. Heat denaturation and anti-gp120 antibodies prevented the direct effect of gp120 on OL, but did not influence the interactions between gp120 and complement. Some gp120 non glycosylated peptides (V3 loop, 254-274 and 415-435 peptides) mimicked the ability of gp120 to antagonize the lytic effect of complement, but not that of potentiating complement lytic activity. In conclusion, our study indicates that gp120 can alter OL functional activity directly and can interfere with OL susceptibility to complement mediated lysis. J. Neurosci. Res. 50:946–957, 1997. © 1997 Wiley-Liss, Inc.     

Differentiation of induced pluripotent stem cells into functional oligodendrocytes

The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells.     

Alpha‐synuclein transfers from neurons to oligodendrocytes

The origin of α‐synuclein (α‐syn)‐positive glial cytoplasmic inclusions found in oligodendrocytes in multiple system atrophy (MSA) is enigmatic, given the fact that oligodendrocytes do not express α‐syn mRNA. Recently, neuron‐to‐neuron transfer of α‐syn was suggested to contribute to the pathogenesis of Parkinson's disease. In this study, we explored whether a similar transfer of α‐syn might occur from neurons to oligodendrocytes, which conceivably could explain how glial cytoplasmic inclusions are formed. We studied oligodendrocytes in vitro and in vivo and examined their ability to take up different α‐syn assemblies. First, we treated oligodendrocytes with monomeric, oligomeric, and fibrillar forms of α‐syn proteins and investigated whether α‐syn uptake is dynamin‐dependent. Second, we injected the same α‐syn species into the mouse cortex to assess their uptake in vivo. Finally, we monitored the presence of human α‐syn within rat oligodendroglial cells grafted in the striatum of hosts displaying Adeno‐Associated Virus‐mediated overexpression of human α‐syn in the nigro‐striatal pathway. Here , we show that oligodendrocytes take up recombinant α‐syn monomers, oligomers and, to a lesser extent, fibrils in vitro in a concentration and time‐dependent manner, and that this process is inhibited by dynasore. Further, we demonstrate in our injection model that oligodendrocytes also internalize α‐syn in vivo. Finally, we provide the first direct evidence that α‐syn can transfer to grafted oligodendroglial cells from host rat brain neurons overexpressing human α‐syn. Our findings support the hypothesis of a neuron‐to‐oligodendrocyte transfer of α‐syn, a mechanism that may play a crucial role in the progression and pathogenesis of MSA. GLIA 2014;62:387–398     

Plasma membrane of cultured oligodendrocytes: II. Possible structural and functional domains

An oligodendrocyte plasma membrane-rich fraction, F2.2, was resolved by equilibrium density centrifugation on a linear sucrose gradient from 0.5 M to 1.3 M into three fractions, F2.2a, F.2.2b, F2.2c, and a pellet F2.2p. F2.2a and F.2.2b were enriched 1.5-fold relative to F2.2 in plasma membrane markers at the expense of F2.2c and F2.2p, which became correspondingly impoverished. This gave F2.2a and F2.2b a 42-fold and 37-fold enrichment, respectively, in plasma membrane markers relative to the initial cell homogenate. F2.2c had a sevenfold enrichment in a Golgi marker; together with F2.2p, they contained all the Golgi marker initially present in F2.2. Preliminary data indicated that the F2.2-subfractions differed from one another in their molar ratios of cholesterol to phospholipids and protein to lipids but had similar protein profiles when examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Their content of fucosylated glycoproteins appeared also to be different. Morphologically, F2.2a and F2.2b were very similar: they contained large membrane vesicles, membrane sheets, and vesicles entrapped within other vesicles. Membrane-membrane interaction was apparent in these fractions. F2.2c had many of the same elements, but most of the membrane structures contained amorphous material. F2.2p differed morphologically from the other fractions in that it had principally electron-dense structures. It is postulated that F2.2a, F2.2b, and perhaps F2.2c represent different domains of oligodendrocyte plasma membrane. Alternatively, these fractions might correspond to the plasma membrane of oligodendrocyte subtypes.     

Immune response to isolated oligodendrocytes

Oligodendrocytes were isolated from bovine white matter and were injected with complete Freund's adjuvant (CFA) into experimental animals. Indirect immunofluorescence studies using fluoresceinated goat anti-rabbit or anti-guinea pig immunoglobulin (GARIg; GAGPIg) showed that rabbit and guinea pig anti-oligodendrocyte (RAO, GPAO) sera reacted specifically with the surface of isolated oligodendrocytes in suspension, as well as with oligodendroglia in bovine and human brain sections, and in mouse cerebellum cultures. This activity of RAO was blocked by non-fluoresceinated GARIg and by GPAO, and absorbed by oligodendrocyte preparation (OP) or whole white matter, but not by purified myelin, neuroblastoma or non-brain tissue. Low levels of anti-basic protein antibodies were found in many RAO (but not GPAO) sera by radioimmunoassay, and a few showed significant anti-galactocerebroside antibody by agglutination and radioimmunoprecipation techniques. Guinea pigs sensitized with isolated oligodendrocytes in CFA showed cell-mediated immunity (CMI) to OP as manifested by delayed type skin test and induced in vitro lymphocyte transformation. CMI to purified myelin basic protein was not detected. The demonstration of humoral and CMI to the cell responsible for the production of CNS myelin may be related to some aspects of the immunopathogenesis of demyelinating disorders.     

Cellular iron status influences the functional relationship between microglia and oligodendrocytes

Previously, we have reported that there is a spatiotemporal relationship between iron accumulation in microglia and oligodendrocytes during normal development and in remyelination following injury. This in vivo observation has prompted us to develop a cell culture model to test the relationship between iron status of microglia and survival of oligodendrocytes. We found that conditioned media from iron-loaded microglia increases the survival of oligodendrocytes; but conditioned media from iron loaded activated microglia is toxic to oligodendrocytes. In the trophic condition, one of the proteins released by iron-loaded microglia is H-ferritin, and transfecting the microglia with siRNA for H-ferritin blocks the trophic response on oligodendrocytes. Lipopolysaccharide (LPS) activation decreases the amount of H-ferritin that is released from microglia and increases the release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1. LPS activation of iron-enriched microglia results in the activation of NF-kB and greater release of cytokines when compared with that of control microglia; whereas treating microglia with an iron chelator is associated with less NF-kB activation and less release of cytokines. These results indicate that microglia play an important role in iron homoeostasis and that their iron status can influence how microglia influence growth and survival of oligodendrocytes. The results further indicate that ferritin, released by microglia, is a significant source of iron for oligodendrocytes.     

CNTF protects oligodendrocytes from ammonia toxicity: intracellular signaling pathways involved

In pediatric patients, hyperammonemia can provoke irreversible damages to developing CNS like cortical atrophy, ventricular enlargement, demyelination or gray and white matter hypodensities which are concordant with alterations of neurons and oligodendrocytes. Cerebral injury triggers endogenous protective mechanisms that can prevent or limit brain damage. Understanding these mechanisms may lead to new therapeutic strategies. We investigated whether ciliary neurotrophic factor (CNTF), a cytokine-like protein expressed by astrocytes and described as an injury-associated survival factor, was up-regulated by ammonia in developing reaggregated 3D brain cell cultures. We showed that CNTF is up-regulated by ammonia exposure, through mediation of p38 MAPK activation in astrocytes. We also observed that SAPK/JNK and Erk1/2 activations in oligodendrocytes and neurons, respectively, also play indirect roles in CNTF synthesis by astrocytes. Co-treatment with exogenous CNTF demonstrated strong protective effects on oligodendrocytes, but not on neurons, against ammonia toxicity. These protective effects involved JAK/STAT, SAPK/JNK and c-jun proteins.     

Corticosteroids protect oligodendrocytes from cytokine-induced cell death

The present data show that the simultaneous exposure to tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) induces cell death with characteristics of apoptosis in cultured rat oligodendrocytes; TNFalpha alone was ineffective. We have also demonstrated that different corticosteroids (aldosterone, deoxycorticosterone, dexamethasone and corticosterone) protect rat oligodendrocytes in culture from apoptosis induced by TNFalpha plus IFNgamma. This effect seems to be exerted via the interaction with both type I and type II corticosteroid receptors since all steroids considered are effective. Since oligodendrocyte apoptosis represents an important event in multiple sclerosis and in several demyelinating diseases, the present observations might be considered an interesting background for further researches directed to the possibility of controlling in vivo the death of these cells.     

Glial conditioned medium enables jimpy oligodendrocytes to express properties of normal oligodendrocytes: production of myelin antigens and membranes

Cultures of cerebral cells from 2-day-old jimpy (jp) and normal animals were immunocytochemically stained with antibodies to galactocerebroside, 2', 3'-cyclic nucleotide 3'-phosphohydrolase and proteolipid protein (PLP). As the normal cultures mature, oligodendrocytes express these markers in cell bodies, in processes and in expansive membrane sheets produced along the lengths of cell processes. In contrast, oligodendrocytes from jp cultures do not produce large membrane sheets and their total numbers are reduced. Only rarely do jp oligodendrocytes exhibit immunostaining for PLP. Thus, jp oligodendrocytes grown in vitro mimic deficiencies of jp CNS in situ. In order to examine the effect of environment and cell-cell interactions on expression of the jp mutation we grew jp cerebral cells in the presence of medium conditioned for short periods by normal cerebral cells. Under these conditions jp oligodendroglia appeared nearly normal by immunostaining criteria. Their numbers were increased; they were able to produce and maintain membrane sheets; and some cells expressed PLP. These results show that jp oligodendrocytes have the capacity to express certain normal phenotypic parameters, and when given an appropriate environment they do so. The effects of normal conditioned medium may be due to a secreted factor, possibly produced by the astrocytes, which constitute the vast majority of cells in the conditioning cultures. The dramatic effect of normal glial conditioned medium on jp oligodendrocytes suggests that the steps leading to hypomyelination in jp are exceedingly complex and may involve glial-glial interactions.     

Stimulation of oligodendrocytes by extracts from astrocyte-enriched cultures

A soluble, heat-labile, trypsin-sensitive factor present in extracts of astrocyte-enriched cultures enhances the expression of myelination-associated events in rat brain cultures enriched in oligodendrocytes. Both the number of oligodendrocytes detected by immunofluorescence microscopy and the levels of biochemical activities associated with oligodendrocyte differentiation increased in the cultures in response to the extract in a time- and concentration-dependent fashion. The possible significance of these studies for normal oligodendrocytes function, and thus myelination, is discussed.     

Myelin and oligodendrocytes: recent data

Oligodendrocytes are the myelin forming cells of the central nervous system. They are derived from a precursor cell, named O-2A. This precursor cell is bipotential, and can differentiate in vitro into either an oligodendrocyte or a certain type of astrocyte, named type 2 astrocyte, depending on the presence or absence of extracellular factors. The chronology of glial differentiation is now better understood. Some factors influencing the choice of the differentiation pathway have been described. Most of these factors seem to be secreted in vitro by an other type of astrocyte, type 1 astrocyte. The succession of events leading to myelination can now be analyzed at molecular level. These very recent results have mostly been obtained in vitro. Their significance in vivo has now to be ascertained.     

Plasma membrane of cultured oligodendrocytes: III. Relatedness to myelin

We have compared highly purified fractions of oligodendrocyte plasma membrane to myelin by one- and two dimensional gel electrophoresis and found them to be distinct. The major myelin proteins--proteolipid protein (PLP), DM-20, and myelin basic protein (MBP), which dominate the sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern of myelin--were minor components of the plasmalemma. However, 2',3', cyclic nucleotide phosphodiesterase (CNPase) and myelin-associated glycoprotein (MAG) were represented equally in both membranes. Labeling the cells with various precursors followed by isolation of plasmalemma revealed that newly synthesized PLP, DM-20, CNPase, and MAG were incorporated into the plasma membrane of "floating" oligodendrocytes (i.e., nonattached to substratum). This was not so with MBP. Nevertheless, scattered patches of MBP were localized on the plasma membrane of intact cells using the immunogold method at the electron microscopic level. The data are consistent with the notion that MBP is not a constituent of the plasma membrane of mature oligodendrocytes (the MBP patches on intact cells are likely remnants from past association with myelin) but is rapidly associated with the plasmalemma of myelinating oligodendrocytes (i.e., attached cells). It is suggested that phosphorylation of MBP provides the triggering signal for plasma membrane association. In order to analyze the minor proteins in myelin and compare them to the plasma membrane by two-dimensional gel electrophoresis, myelin was extracted with chloroform:methanol to remove PLP, DM-20, and MBP. Even in the absence of PLP, DM-20, and MBP the pattern of extracted myelin still differed from that of plasmalemma indicating that their minor protein compositions were not the same. Myelin was characterized by a group of proteins that clustered at pI 5.5-6.5 and Mr 40,000-60,000 of which alpha-tubulins, beta-tubulins, and actin are part: the plasmalemma had tubulins and actin but in different proportions. Our findings indicate that in addition to PLP, DM-20, and MPB, myelin is also enriched relative to the plasmalemma in another group of proteins.     

Lymphocyte responsiveness to oligodendrocytes in multiple sclerosis

Lymphocyte responsiveness to oligodendrocytes has been examined with a modification of the antigen-reactive early T cell test in a total of 206 blood samples [104 multiple sclerosis (MS); 62 other neurological diseases (OND); 14 non-neurologic diseases; and 26 normals]. Oligodendrocyte reactivity was detectable more frequently in MS (49%) than in OND (23%) and normals (8%) but was absent from patients with non-neurologic diseases. Percentages of lymphocytes binding oligodendrocyte antigens were determined by direct and indirect immunofluorescence and immunocyto-adherence. Comparable results were obtained from all 3 systems. Percentages of oligodendrocyte antigen-binding lymphocytes were higher in MS than in OND and normals. With the present battery of techniques, lymphocyte responsiveness to oligodendrocytes has been shown to be more common in MS but since it is also found among OND, it is not MS-specific and most likely represents an epiphenomenon associated with white matter destruction.     

Can oligodendrocytes attached to myelin proliferate?

The uptake of thymidine by oligodendrocytes in the brains of adult mice was examined after the induction of cortical traumatic lesions in an attempt to determine whether mature oligodendrocytes actively attached to myelin sheaths were capable of proliferating. In view of the great difficulty in visualizing the connection between a given oligodendrocyte and a myelin sheath even in the normal adult animal, the neuropil was made edematous in the traumatized animals in order to separate out the components and to study the oligodendrocyte processes. Uptake of tritiated thymidine was found in oligodendrocytes, as well as in endothelial cells, astrocytes, and microglia. The percentage of labeled oligodendrocytes appeared low in relation to the total number of the oligodendrocytes. In addition, in a few labeled cells, cytoplasmic processes could be seen extending to and apparently forming the myelin sheath. The possibility that a differentiated cell still attached to myelin may at the same time be able to proliferate is of great significance in understanding the potential for remyelination and recovery, in the adult, because it would imply that any oligodendrocyte is a candidate for proliferation, rather than only uncommitted or immature glial cells, which may be limited in the mature brain.     

少突胶质细胞抗氧化损伤能力的成熟依赖性研究

目的 探讨凋亡相关蛋白Bcl-2/Bax、Caspase-3的表达和抗氧化能力与少突胶质细胞(OL)对氧化损伤的成熟依赖性关系.方法 用不同浓度过氧化氢(H2O2)刺激培养OL前体及成熟OL,应用MTT法检测细胞活力.应用Western blot分别检测两种细胞Bcl-2/Bax和Caspase-3的蛋白表达.并用比色法检测细胞总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(GSH-px)和过氧化氢酶(CAT)的活性以及丙二醛(MDA)的含量.结果 在不同H2O2浓度的刺激下,OL前体的细胞活力均明显低于成熟OL.OL前体的Bax和Caspase-3蛋白表达明显高于成熟OL,Bcl-2蛋白表达则明显低于成熟OL:其T-SOD、GSH-1ax和CAT的活性亦明显弱于成熟OL,但MDA含量明显升高,差异有统计学意义(P<0.05).结论 OL对氧化损伤的成熟依赖性与其Bcl-2/Bax、Caspase-3在不同阶段中的表达差异以及抗氧化能力不同有关.     

From neural stem cells to myelinating oligodendrocytes

The potential to generate oligodendrocytes progenitors (OP) from neural stem cells (NSCs) exists throughout the developing CNS. Yet, in the embryonic spinal cord, the oligodendrocyte phenotype is induced by sonic hedgehog in a restricted anterior region. In addition, neuregulins are emerging as potent regulators of early and late OP development. The ability to isolate and grow NSCs as well as glial-restricted progenitors has revealed that FGF2 and thyroid hormone favor an oligodendrocyte fate. Analysis of genetically modified mice showed that PDGF controls the migration and production of oligodendrocytes in vivo. Interplay between mitogens, thyroid hormone, and neurotransmitters may maintain the undifferentiated stage or result in OP growth arrest. Notch signaling by axons inhibits oligodendrocyte differentiation until neuronal signals--linked to electrical activity-trigger initiation of myelination. To repair myelin in adult CNS, multipotential neural precursors, rather than slowly cycling OP, appear the cells of choice to rapidly generate myelin-forming cells.     

Hypoxic injury to oligodendrocytes: reversible inhibition of ATP-dependent transport of ceramide from the endoplasmic reticulum to the Golgi.

Previous studies have shown that gradual progressive hypoxia specifically inhibits the synthesis of the major myelin lipid galactosylceramide (GalCer) in cultured neonatal rat oligodendrocytes (OLG) (Kendler and Dawson, J Biol Chem 265:12259-12266, 1990). The inhibition of de novo synthesized GalCer (measured by [3H]palmitate incorporation) was accompanied by an increase in the [3H]labeled pool of nonhydroxy fatty acid ceramide, the precursor of GalCer. The decreased galactosylation of NFACer was not due to an inhibition of UDP-Gal:ceramide:galactosyltransferase activity or to a depletion in available UDP-Gal. Analysis of subcellular fractionations of OLG membranes on Percoll gradients indicated that NFA ceramide was accumulating in the endoplasmic reticulum (ER) during hypoxia, suggesting that the transport of NFACer from its site of synthesis (ER) to its site of galactosylation, presumably the Golgi, was blocked by hypoxia. This accumulation of ceramide was replicated by lowering ATP levels to 80-90% of control by treating OLG with 12 nM oligomycin, and was reversed by reoxygenation of the cells. Conversion of [3H]palmitate-labeled NFACer to GalCer in semi-intact OLG required both exogenous UDP-Gal and ATP, further suggesting that the transport of NFACer from the ER to its site of synthesis (cis-Golgi) is an energy-dependent step that is highly susceptible to relatively minor ATP depletion associated with early hypoxic injury. Our results further suggest that ceramide appears to be a good marker for ER and GalCer is a good marker for the cis-Golgi.