Cultured Rat Brain Oligodendrocytes Research Articles

HDAC6 inhibition results in tau acetylation and modulates tau phosphorylation and degradation in oligodendrocytes

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family. It is localized within the cytoplasm and has unique substrate specificities for nonhistone proteins, such as α-tubulin. Furthermore, it plays a major role in protein aggregate formation and recently was demonstrated to interact with the microtubule associated protein tau and tau was identified as a possible substrate for HDAC6 in neurons. This study was undertaken to investigate whether HDAC6 is present in oligodendrocytes and whether it is involved in tubulin and tau acetylation in these cells. We show for the first time that HDAC6 is expressed in cultured rat brain oligodendrocytes. Its inhibition by the specific HDAC6 inhibitor tubastatin A (TST) leads to morphological alterations, microtubule bundling, and tubulin acetylation, and changes in tau-isoform expression and phosphorylation. Furthermore, the microtubule binding activity of tau was reduced. Using the oligodendroglial cell lines OLN-t40 and OLN-t44, which were genetically engineered to express either the longest human tau isoform with four microtubule binding repeats (4R-tau), or the shortest tau isoform with three repeats (3R-tau), respectively, we demonstrate that tau is acetylated by HDAC6 within the 4R-binding domain. Tau acetylation reduced its turnover rate and acetylated tau was degraded slower in these cells. TST and shRNA-mediated knockdown of HDAC6 in oligodendroglia cells caused an increase in pathological hyperphosphorylated tau detectable with the 12E8 antibody. Hence HDAC6 and dysregulation of the deacetylation and acetylation process in oligodendrocytes may contribute to diseases with oligodendroglial pathology.

Involvement of Macroautophagy in Multiple System Atrophy and Protein Aggregate Formation in Oligodendrocytes

α-Synuclein-containing glial cytoplasmic inclusions (GCIs) originating in oligodendrocytes are the characteristic hallmark for neuropathological diagnosis of multiple system atrophy (MSA). α-Synuclein can be degraded either by the proteasomal machinery or by autophagy, a lysosomal pathway which involves the formation of autophagosomes. The autophagosome takes up polyubiquitinated proteins via the autophagosomal protein LC3 and the ubiquitin binding protein p62. In the present study, neuropathological examination of seven MSA cases revealed that LC3-immunoreactivity is found to be associated with α-synuclein-positive GCIs. These are also prominently stained by antibodies against p62 and ubiquitin, indicating that the autophagic pathway is upregulated during pathogenesis, which might be due to a persistent downregulation of proteasomal activity. To further address this question in a cellular context, we have investigated whether proteasomal inhibition in cultured rat brain oligodendrocytes promotes the recruitment of LC3 and p62 to protein aggregates. The data show that the autophagic marker LC3-II is upregulated and LC3 is recruited to the growing protein aggregates in cultured oligodendrocytes when the proteasome is impaired. However, aggregated proteins remain in the oligodendroglial cytoplasm and cannot be cleared efficiently. In conclusion, autophagy and the ubiquitin proteasome system are closely connected, and the presence of LC3-positive vesicles in GCIs indicates that macroautophagy participates in MSA pathogenesis.

The expression of tubulin polymerization promoting protein TPPP/p25α is developmentally regulated in cultured rat brain oligodendrocytes and affected by proteolytic stress

The tubulin polymerization-promoting protein (TPPP)/p25alpha was identified as a brain specific protein, is associated with microtubules (MTs) in vitro and can promote abnormal MT assembly. Furthermore it has aggregation promoting properties and is a constituent in pathological protein deposits of neurodegenerative diseases. In the brain, TPPP/p25alpha is present in myelinating oligodendrocytes. Here we show, using cultured rat brain oligodendrocytes, that TPPP/p25alpha expression is increasing during development in culture, and particularly in immature cells is associated with the centrosome. MT binding properties in oligodendrocytes are rather low, however, when MTs are disassembled by nocodazole, TPPP/p25alpha accumulates in the perinuclear region. Treatment of oligodendrocytes with the proteasomal inhibitor MG-132 (1 micaroM; 18 h) caused an increase in the amount of TPPP/p25alpha by about 40%, a decrease in its solubility, and led to the appearance of TPPP/p25alpha-positive cytoplasmic inclusions, which stained with thioflavin S and resembled inclusion bodies. Hence, it might be speculated that acute or chronic malfunction of the proteasomal degradation system, leading to the accumulation of aggregation prone proteins and the pro-aggregatory protein TPPP/p25alpha or to the aggregation of TPPP/p25alpha on its own, is causally related to the protein aggregation process in a variety of neurodegenerative diseases.

Transient overexpression of alpha-Synuclein in OLN-93 oligodendroglial cells is not cytotoxic and not affected by HSP70

Alpha-Synuclein (alpha-syn) accumulates in neurons and glia in a number of neurodegenerative disorders. The cytotoxicity and solubility of alpha-syn can be modified by the molecular chaperone HSP70. In multiple system atrophy (MSA), alpha-syn-positive glial cytoplasmic inclusions (GCIs) originating in oligodendrocytes are specifically prominent. Alpha-Synuclein is not detectable in mature oligodendrocytes in the normal brain, but we have shown previously that it is present in cultured rat brain oligodendrocytes [1] and down-regulated as the cultures mature. Hence, GCI formation might be causally related to a specific upregulation of the protein during pathological situations.

Proteasome inhibition by MG-132 induces apoptotic cell death and mitochondrial dysfunction in cultured rat brain oligodendrocytes but not in astrocytes

Proteasomal dysfunction has been implicated in neurodegenerative disorders and during aging processes. In frontotemporal dementias, corticobasal degeneration, and progressive supranuclear palsy, oligodendrocytes are specifically damaged. Application of proteasomal inhibitors to cultured oligodendrocytes is associated with apoptotic cell death. The present study was undertaken to investigate the death pathway activated in oligodendrocytes by proteasomal inhibition. Our data show that the proteasomal inhibitor MG-132 causes oxidative stress, as indicated by the upregulation of the small heat shock protein heme oxygenase-1 (HO-1) and the appearance of oxidized proteins. Activation of the mitochondrial pathway was involved in the apoptotic process. Mitochondrial membrane potential was disturbed, and cytochrome c was released from the mitochondria. Concomitantly, death-related caspases 3 and 9 were activated and poly(ADP-ribose)-polymerase cleavage occurred. MG-132-induced cell death, DNA-fragmentation, and caspase activation could be prevented by the broad caspase inhibitor zVAD-fmk. In contrast to oligodendrocytes, cultured astrocytes showed resistance to the treatment with proteasomal inhibitors and did not reveal cytotoxic responses. This was also observed in astrocytes differentiated in the presence of dibutyryl cyclic AMP. Hence, individual cells respond differently to proteasomal inhibition and the therapeutic use of proteasomal inhibitors, e.g. for the treatment of cancer or inflammatory diseases, needs to be carefully evaluated.

Proteolytic stress causes heat shock protein induction, tau ubiquitination, and the recruitment of ubiquitin to tau-positive aggregates in oligodendrocytes in culture.

Molecular chaperones and the ubiquitin-proteasome system are participants in the defense against unfolded proteins and provide an effective protein quality control system that is essential for cellular functions and survival. Ubiquitinated tau-positive inclusion bodies containing the small heat shock protein alphaB-crystallin in oligodendrocytes are consistent features of a variety of neurodegenerative diseases, and defects in the proteasome system might contribute to the aggregation process. Oligodendrocytes, the myelin-forming cells of the CNS, are specifically sensitive to stress situations. Here we can show that in cultured rat brain oligodendrocytes proteasomal inhibition by MG-132 or lactacystin caused apoptotic cell death and the induction of heat shock proteins in a time- and concentration-dependent manner. Specifically, alphaB-crystallin was upregulated, and ubiquitinated proteins accumulated. After incubation with MG-132 the tau was dephosphorylated, which enhanced its microtubule-binding capacity. Proteasomal inhibition led to ubiquitination of tau and its association with alphaB-crystallin and to the occurrence of thioflavine S-positive aggregates in the oligodendroglial cytoplasm. These aggregates were positive for tau and also contained ubiquitin and alphaB-crystallin; hence they resembled the glial cytoplasmic inclusions observed in white matter disease and frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17). In summary, the data underscore the specific sensitivity of oligodendrocytes to stress situations and point to a causal relationship of proteasomal impairment and inclusion body formation.

Mitochondrial pathway is involved in hydrogen-peroxide-induced apoptotic cell death of oligodendrocytes.

Oligodendrocytes, the myelin-forming cells of the CNS, are specifically sensitive to oxidative stress and respond by the onset of programmed cell death (PCD). To further unravel the molecular events underlying their enhanced susceptibility, we have investigated whether mitochondrial damage occurs during oxidative stress-induced PCD in cultured rat brain oligodendrocytes. Mitochondria are considered as a central control point of apoptosis, and mitochondrial dysfunction has been linked to neurodegenerative disease. Upon a number of stimuli through the release of cytochrome c, they coordinate caspase activation, causing morphological and biochemical changes associated with PCD. Oxidative stress was exerted by the application of hydrogen peroxide. The data show that hydrogen peroxide-induced apoptosis in oligodendrocytes involves mitochondrial damage and cytochrome c release and is accompanied by the activation of the death-related caspases 3 and 9. Concomitantly, the activation and nuclear translocation of extracellular signal regulated kinases ERK1,2 are observed, which have been implicated to participate in the regulation of cell death and survival. DNA fragmentation could not be attenuated by the ERK1,2 inhibitor PD 98059, indicating that the ERK1,2- pathway in oligodendrocytes may be involved in the initial survival response after exposure to stressful stimuli.

Activation of PP2A-like phosphatase and modulation of tau phosphorylation accompany stress-induced apoptosis in cultured oligodendrocytes.

In a number of neurodegenerative diseases, tau-positive glial cytoplasmic inclusions (GCIs), immunochemically labeled with antibodies to the small heat shock protein (HSP) alphaB-crystallin, occur in oligodendrocytes. The microtubule-associated protein tau is functionally modulated by phosphorylation. We have shown previously that oxidative stress (OS) and heat shock (HS) induce apoptotic cell death in oligodendrocytes. The present study was undertaken to test whether stress responses in oligodendrocytes cause abnormalities in the expression and posttranslational modification of tau proteins, and whether the dynamic phosphorylation and dephosphorylation of tau are involved in the pathogenesis of glial cells. Cultured rat brain oligodendrocytes were subjected to OS, exerted by hydrogen peroxide, or HS (44 degrees C, 30 min). Immunoblot analysis with a panel of phosphorylation-dependent antibodies shows that OS and HS caused the rapid dephosphorylation of tau proteins at multiple sites, before characteristic features of apoptosis were observed. Concomitantly, ERK1,2 (extracellular signal-regulated kinase) was activated. Tau phosphorylation and rephosphorylation after stress was mediated by glycogen synthase kinase 3beta (GSK-3beta), and not by ERK1,2 and could be suppressed by lithium chloride, a specific inhibitor of GSK-3beta. Stress-induced dephosphorylation could be mimicked by alkaline phosphatase and suppressed by the protein phosphatase inhibitor okadaic acid (OA), indicating that PP2A in oligodendrocytes is activated by stress. OA at low concentrations could prevent stress-induced DNA fragmentation, but eventually exerted cytotoxic effects. Hence, stress-induced activation of PP2A in oligodendrocytes and tau dephosphorylation constitute a major feature of the response to injury in these cells, which eventually undergo apoptotic cell death.

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 occurrence 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, alpha B-crystallin and HSP70 were up-regulated, while after oxidative stress the specific induction of HSP32 and alpha B-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.

Developmental changes of tau protein and mRNA in cultured rat brain oligodendrocytes.

Oligodendrocytes elaborate an extensive network of multibranched processes and flat membranous sheets. Microtubules (MT) participate in the elaboration and stabilization of myelin-forming processes and are essential for cellular sorting processes. Microtubule-associated proteins (MAPs) are involved in the regulation and stabilization of the dynamic MT network. It has been shown previously that oligodendrocytes express the MAP tau, a phosphoprotein most abundant in neurons of the CNS. In this article, we demonstrate for the first time that oligodendrocytes contain all six tau isoforms, and that tau mRNA and protein expression is developmentally regulated. Immunoblot analysis reveals that tau protein is more abundant, and mature isoforms are more prominent at later stages of development. During the first week of culture maturation, a marked decrease in phosphorylation is observable. Using an RT-PCR approach, we can show that oligodendrocytes express small amounts of exon 3 containing isoforms and that during culture maturation, tau mRNA splice products with 3 MT-binding domains (3R) decrease and mRNA with 4 MT-binding domains (4R) increase. In situ hybridization study demonstrates that tau mRNA is present in precursor cells and in mature oligodendrocytes. Tau mRNA is actively transported into the cellular processes, is specifically present in the primary and some of the secondary processes, enriched at the turning and branching points and the growing tips, and often appears as small patches. Hence, localized tau translation at specific sites in the cellular extensions might contribute to the regulation of MT stability during process formation, early axonal contact establishment, and 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. © 2000 Wiley-Liss, Inc.

Alpha-synuclein is developmentally expressed in cultured rat brain oligodendrocytes.

Although a neuronal protein, alpha-synuclein is a major component of glial cytoplasmic inclusions (GCIs) in oligodendrocytes of multiple system atrophy (MSA) brains. Because alpha-synuclein has not been identified in oligodendrocytes of normal brains, we examined cultured rat brain oligodendrocytes during in vitro development and showed that alpha-synuclein mRNA and protein are present in cultured oligodendrocytes. The expression of alpha-synuclein was developmentally regulated; it increased to peak levels at 2 or 3 days in culture but declined thereafter. Indirect immunofluorescence further shows that alpha-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 alpha-synuclein in MSA oligodendrocytes.

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

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.

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 (approximately 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.

The timely expression of myelin basic protein gene in cultured rat brain oligodendrocytes is independent of continuous neuronal influences

The developmental expression of the myelin basic protein (MBP) gene was studied in rat cultured oligodendrocytes using immunofluorescence and in situ hybridization. In newborn rat brain cultures, which contain only glial cells, large amounts of MBP-specific mRNA (as assayed by grain counts in autoradiograms) abruptly accumulated within immature oligodendrocytes 5 to 6 days postnatal. MBP always emerged 6 to 8 days after birth; thus, a week after, galactocerebroside (GC), an early oligodendrocyte marker, had appeared. The percentage of MBP mRNA and MBP-positive cells peaked at about 15 days postnatal and decreased thereafter. The time of emergence of MBP in these cultured oligodendrocytes appears to be determined at a very early stage in their development and independent of continuous neuronal influences. There is a striking correspondence between the times of appearance of MBP in cultured oligodendrocytes and those in the intact animal. Thus, primary cultures made from 5-day prenatal, newborn, and 2-day postnatal animals all express MBP at about the same developmental stage, namely, after 14, 8, and 6 days in culture, respectively. Furthermore, cultured oligodendrocytes obtained from the spinal cord express MBP before those obtained from midbrain or hemispheres, as they would in the intact animal. Thus, the developmental expression of the MBP gene occurs in a similar time frame in vitro and in vivo. In oligodendrocyte-enriched cultures, where 60% to 80% of the cells express MBP, in situ hybridization with the cDNA clone revealed MBP-specific mRNA in the cell body and sometimes in the processes of the differentiated oligodendrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)