Christiane Richter-Landsberg

OLN-93 : A NEW PERMANENT OLIGODENDROGLIA CELL LINE DERIVED FROM PRIMARY RAT BRAIN GLIAL CULTURES

We have established a new permanent cell line (OLN‐93), derived from spontaneously transformed cells in primary rat brain glial cultures. In growth medium supplemented with 10% fetal calf serum a doubling time of 16–18 hr was determined. OLN‐93 cells in their antigenic properties resemble primary oligodendrocytes in culture. As analyzed by indirect immunofluorescence, the A2B5 surface marker is absent, they express galactocerebroside and myelin‐specific proteins, such as myelin basic protein (MBP), myelin‐associated glycoprotein (MAG), proteolipidprotein (PLP), and Wolfgram protein (WP), but do not exhibit astrocytic properties, such as the expression of vimentin or the glial fibrillary acidic protein (GFAP). In their morphological features they resemble bipolar O‐2A‐progenitor cells and, when grown at low density or on poly‐L‐lysine‐coated culture dishes under low serum conditions, immature oligodendrocytes with a more arborized cell morphology. The cellular processes contain microfilaments, while N‐CAM/D2 immunoreactivity is localized on the cell surface of the somata and processes. Immunoblot analysis further confirmed the presence of MAG, WP and MBP immunoreactivity, and the absence of vimentin and GFAP. Only a single MBP isoform (∼14 kDa) was detectable in the cellular extracts. PLP mRNA expression was studied by RT‐PCR. The two proteolipid‐specific mRNAs, DM20 and PLP, were present in OLN‐93 cell extracts. Comparisons with embryonic rat cerebral cells in culture and primary oligodendrocytes suggest that OLN‐93 cells in their morphological features and their antigenic properties resemble 5‐ to 10‐day‐old (postnatal time) cultured rat brain oligodendrocytes. Thus, the new cell line described in this study should provide a useful model system to investigate the specific mechanisms regulating the proliferation and differentiation of oligodendrocytes in vitro, and the molecular interactions with other cells of the nervous system.

Activation of AP-1 and Nuclear Factor-κB Transcription Factors Is Involved in Hydrogen Peroxide-Induced Apoptotic Cell Death of Olligodendrocytes

Abstract : H2O2‐induced onset and execution of programmed cell death in mature rat brain oligodendrocytes in culture is accompanied by the induction and nuclear translocation of the transcription factors AP‐1 and nuclear factor‐κB (NF‐κB), both of which have been discussed as regulators of cell death and survival. Supershift analysis of nuclear extracts indicated that the AP‐1 complex consists of c‐Jun, c‐Fos, JunD, and possibly JunB proteins, and that the NF‐κB complex contains p50, p65, and c‐Rel proteins. The first signs of DNA fragmentation were seen already during the first hour after the treatment. DNA fragmentation could be prevented by the antioxidants pyrrolidine dithiocarbamate and vitamin E, by the nuclease inhibitor aurintricarboxylic acid, and by preincubation with the iron chelator deferoxamine (DFO). Additionally, DFO protected oligodendrocytes from H2O2‐induced cytotoxic effects as assessed by the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay, and suppressed the formation of free radicals. DFO alone led to a slight increase and in combination with H2O2 synergistically induced DNA‐binding activities of AP‐1 and NF‐κB in oligodendrocytes. Our data suggest that although low levels of H2O2 directly activate AP‐1 and NF‐κB and might contribute to signal transduction pathways promoting cell survival, the formation and action of hydroxyl radicals promote cell death mechanisms that can be attenuated by the iron chelator DFO.

Nitration of Tau Protein Is Linked to Neurodegeneration in Tauopathies

Oxidative and nitrative injury is implicated in the pathogenesis of Alzheimers disease (AD) and Down syndrome (DS), but no direct evidence links this type of injury to the formation of neurofibrillary tau lesions. To address this, we generated a monoclonal antibody (mAb), n847, which recognizes nitrated tau and alpha-synuclein. n847 detected nitrated tau in the insoluble fraction of AD, corticobasal degeneration (CBD), and Picks disease (PiD) brains by Western blots. Immunohistochemistry (IHC) showed that n847 labeled neurons in the hippocampus and neocortex of AD and DS brains. Double-label immunofluorescence with n847 and an anti-tau antibody revealed partial co-localization of tau and n847 positive tangles, while n847 immunofluorescence and Thioflavin-S double-staining showed that a subset of n847-labeled neurons were Thioflavin-S-positive. In addition, immuno-electron microscopy revealed that tau-positive filaments in tangle-bearing neurons were also labeled by n847 and IHC of other tauopathies showed that some of glial and neuronal tau pathologies in CBD, progressive supranuclear palsy, PiD, and frontotemporal dementia with parkinsonism linked to chromosome 17 also were n847-positive. Finally, nitrated and Thioflavin-S-positive tau aggregates were generated in a oligodendrocytic cell line after treatment with peroxynitrite. Taken together, these findings imply that nitrative injury is directly linked to the formation of filamentous tau inclusions.

Role of the oligodendroglial cytoskeleton in differentiation and myelination

Oligodendrocytes, the myelin‐forming cells of the central nervous system, are in culture characterized by an elaborate process network, terminating in flat membranous sheets that are rich in myelin‐specific proteins and lipids, and spirally wrap axons forming a compact insulating layer in vivo. By analogy with other cell types, maintenance and stability of these processes, as well as the formation of the myelin sheath, likely rely on a pronounced cytoskeleton consisting of microtubules and microfilaments. While the specialized process of wrapping and compaction forming the myelin sheath is not well understood, considerably more is known about how cytoskeletal organization is mediated by extracellular and intracellular signals and other interaction partners during oligodendrocyte differentiation and myelination. Here, we review the current state of knowledge on the role of the oligodendrocyte cytoskeleton in differentiation with an emphasis on signal transduction mechanisms and will attempt to draw out implications for its significance in myelination.

α-synuclein is developmentally expressed in cultured rat brain oligodendrocytes

Although a neuronal protein, α‐synuclein is a major component of glial cytoplasmic inclusions (GCIs) in oligodendrocytes of multiple system atrophy (MSA) brains. Because α‐synuclein has not been identified in oligodendrocytes of normal brains, we examined cultured rat brain oligodendrocytes during in vitro development and showed that α‐synuclein mRNA and protein are present in cultured oligodendrocytes. The expression of α‐synuclein was developmentally regulated; it increased to peak levels at 2 or 3 days in culture but declined thereafter. Indirect immunofluorescence further shows that α‐synuclein was localized predominantly in cell bodies and primary processes of oligodendroglia. Thus, GCIs may be a consequence of altered rather than de novo expression of α‐synuclein in MSA oligodendrocytes. J. Neurosci. Res. 62:9–14, 2000.

Stress proteins in oligodendrocytes: differential effects of heat shock and oxidative stress.

Heat shock proteins (HSP) or stress proteins serve as biomarkers to identify the contribution of stress situations underlying the pathogenesis of degenerative diseases of the CNS. We have analyzed by immunoblot technique the constitutive and inducible occurance of stress proteins in cultured rat brain oligodendrocytes subjected to heat shock or oxidative stress exerted by hydrogen peroxide, or a combination of both. The data demonstrate that oligodendrocytes constitutively express HSP32, HSP60 and the cognate form of the HSP70 family of proteins, HSC70. After heat shock, HSP25, αB‐crystallin and HSP70 were up‐regulated, while after oxidative stress the specific induction of HSP32 and αB‐crystallin was observed. HSP32 represents heme oxygenase 1 (HO‐1), a small stress protein with enzymatic activity involved in the oxidative degradation of heme which participates in iron metabolism. The presence of the iron chelators phenanthroline or deferoxamine (DFO), which previously has been shown to protect oligodendrocytes from oxidative stress‐induced onset of apoptosis, caused a marked stimulation of HSP32 without affecting HSP70. This indicates that DFO possibly exerts its protective role by directly influencing the antioxidant capacity of HO‐1. In summary, HSP in oligodendrocytes are differentially stimulated by heat stress and oxidative stress. Heme oxygenase‐1 has been linked to inflammatory processes and oxidative stress, its specific up‐regulation after oxidative stress in oligodendrocytes suggests that it is an ideal candidate to investigate the involvement of oxidative stress in demyelinating diseases.

Stress proteins in neural cells: functional roles in health and disease

Abstract. Heat shock proteins (HSPs) or stress proteins participate in protein synthesis, protein folding, transport and translocalization processes. Stress situations trigger a heat shock response leading to their induction. Similarly, they can be upregulated by impairment of the proteasomal degradation pathway. The upregulation of stress proteins is an important step in prevention of protein aggregation and misfolding after stress, and also is essential during development and differentiation. A number of HSPs are constitutively or inducibly expressed in the nervous system and connected to protection of nerve cells and glia. The cytoskeleton is affected by stress, and HSPs have been shown to interact with the cytoskeleton in normal cells and to assist proper assembly, spatial organization and cross-linking properties. The integrity of the cytoskeleton is disturbed in many neurodegenerative disorders, and filamentous cytoplasmic inclusion bodies, containing a variety of HSPs, are observed. This review summarizes the recent literature on the presence and induction of HSPs in neural cells, and their possible functional roles in health and disease are discussed.

13C isotopomer analysis of glucose and alanine metabolism reveals cytosolic pyruvate compartmentation as part of energy metabolism in astrocytes

After incubation of glial cells with both 13C‐labeled and unlabeled glucose and alanine, 13C isotopomer analysis indicates two cytosolic pyruvate compartments in astrocytes. One pyruvate pool is in an exchange equilibrium with exogenous alanine and preferentially synthesizes releasable lactate. The second pyruvate pool, which is of glycolytic origin, is more closely related to mitochondrial pyruvate, which is oxidized via tri carbonic acid (TCA) cycle activity. In order to provide 2‐oxoglutarate as a substrate for cytosolic alanine aminotransferase, glycolytic activity is increased in the presence of exogenous alanine. Furthermore, in the presence of alanine, glutamate is accumulated in astrocytes without subsequent glutamine synthesis. We suggest that the conversion of alanine to releasable lactate proceeds at the expense of flux of glycolytic pyruvate through lactate dehydrogenase, which is used for ammonia fixation by alanine synthesis in the cytosol and for mitochondrial TCA cycle activity. In addition, an intracellular trafficking occurs between cytosol and mitochondria, by which these two cytosolic pyruvate pools are partly connected. Thus, exogenous alanine modifies astrocytic glucose metabolism for the synthesis of releasable lactate disconnected from glycolysis. The data are discussed in terms of astrocytic energy metabolism and the metabolic trafficking via a putative alanine‐lactate shuttle between astrocytes and neurons. GLIA 34:200–212, 2001.

Expression of microtubule-associated proteins MAP2 and tau in cultured rat brain oligodendrocytes

Abstract.Oligodendrocytes in culture are characterized by large membranous sheets containing an elaborate network of microtubules. Microtubule-associated proteins (MAPs) participate in microtubule stability and the regulation of the cellular architecture. We have investigated the expression of two major groups of MAPs, MAP2 and tau, in cultured rat brain oligodendrocytes. Alternatively spliced isoforms of mRNAs encoding MAP2 and tau were assessed by means of reverse transcription and polymerase chain reaction using a newly designed set of MAP2- and tau-specific primers. The data were compared with data obtained with cultures of rat brain astrocytes and rat cerebral neurons, and adult rat brain. The results show that oligodendrocytes, similarly to neurons, express mainly MAP2c transcripts containing three microtubule-binding repeats. They also contain small amounts of MAP2b mRNA. Six low molecular weight tau isoforms, namely tau 1–6, have been described in the brain (Goedert et al. 1991). The major isoform of tau mRNA in oligodendrocytes was found to be tau 1, which represents a marker typical for immature neurons. Tau 2 and tau 4 isoforms were also detected, albeit at a very low level. Immunoblot analysis of oligodendroglia cell extracts confirmed the presence of tau protein. It migrates as a single polypeptide with an apparent molecular weight of approximately 55 kDa. In addition, oligodendrocytes express MAP2c protein, which migrates as a close double band with an apparent molecular weight around 70 kDa. Indirect immunofluorescence staining indicated that tau and MAP2 immunoreactivity was expressed in oligodendrocytes of immature and mature morphologies in the cell somata and cellular processes. Tau was particularly found in the end of the cellular extensions, and both proteins exhibited a distribution similar to myelin basic protein. Thus, oligodendroglia, like neuronal cells, contain microtubule-associated proteins, mainly MAP2c and the tau 1 isoform, although at a much lower level. The presence of these MAPs in myelin-forming cells further points to the functional significance of the cytoskeleton during oligodendrocyte differentiation, process outgrowth, and myelin formation.

Alpha-synuclein aggregation and Ser-129 phosphorylation-dependent cell death in oligodendroglial cells.

Multiple system atrophy is a neurodegenerative disorder characterized by accumulation of aggregated Ser-129-phosphorylated α-synuclein in oligodendrocytes. p25α is an oligodendroglial protein that potently stimulates α-synuclein aggregation in vitro. To model multiple system atrophy, we coexpressed human p25α and α-synuclein in the rat oligodendroglial cell line OLN-93 and observed a cellular response characterized by a fast retraction of microtubules from the cellular processes to the perinuclear region followed by a protracted development of apoptosis. This response was dependent on phosphorylation at Ser-129 in α-synuclein as demonstrated by site-directed mutagenesis. Treatment of the cells with the kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole that targets kinases like casein kinase 2, and polo-like kinases abrogated the toxicity. The polo-like kinase inhibitor BI 2536 caused apoptosis in the model. Ser-129 phosphorylation was linked to the formation of phosphorylated oligomers detectable by immunoblotting, and their formation was inhibited by 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole. The process of microtubule retraction was also dependent on aggregation as demonstrated by the protective effect of treating the cells with the specific peptide inhibitor of α-synuclein aggregation ASI1D and the non-selective inhibitors Congo Red and baicalein. The fast microtubule retraction was followed by the development of the apoptotic markers: activated caspase-3, phosphatidylserine externalization, nuclear condensation, and fragmentation. These markers could all be blocked by the inhibitors of phosphorylation, aggregation, and caspase-3. Hence, the model predicts that both Ser-129 phosphorylation and aggregation control the toxic α-syn pathway in oligodendroglial cells and may represent therapeutic intervention points in multiple system atrophy.