The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells.

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.

S100B is expressed in, and released from, OLN-93 oligodendrocytes: Influence of serum and glucose deprivation.

S100B (member of a family of proteins that are 100% soluble in ammonium sulfate at neutral pH) has been widely used as astrocyte marker in animal models and in human brain diseases. Recent studies revealed S100B-immunopositivity in oligodendrocytes and O2A oligodendroglial progenitor cells. It is unknown, however, if oligodendrocytes produce S100B themselves, or if the S100B-immunolabeling is caused by binding or absorption of the protein. To address this question, S100B expression and protein release were analyzed in a highly pure oligodendrocytic OLN-93 cell line (from rat), in the astrocytic C6 cell line (from rat) and primary astrocytes. S100B was gene expressed in all cultures, as revealed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. OLN-93 cells and glial fibrillary acidic protein (GFAP)-negative astrocytes expressed the multiligand receptor for advanced glycation end products (RAGE). S100B protein levels were determined in supernatants and cell homogenates by immunoluminometry under normal conditions and after serum and glucose deprivation (SGD). SGD led to a several-fold increased release of S100B (after 6 and 24 h), which was particularly pronounced in primary astrocytes. Increased S100B in cell homogenates was most notable in OLN-93 cells under SGD, indicating activated S100B synthesis. These cells also showed the highest percentage of dead cells, as determined by propidium iodide-positivity, after SGD. Incubation with 0.5, 2 and 5 microg/l exogenous S100B was not toxic to OLN-93 cells. In conclusion, OLN-93 cells produce more S100B under SGD than astrocytes and are more susceptible to cell death upon SGD, which provokes leakage of S100B. Our data indicate active S100B secretion from astrocytes under SGD since highly elevated levels of S100B were detected in the supernatant despite a low percentage of dead cells. The experimental results provide further evidence for a production/release of S100B in/from oligodendrocytes, e.g. in metabolic stress conditions like cerebral ischemia. Studies on S100B in bodily fluids should be carefully interpreted in order to avoid misleading hypotheses concerning the specific involvement of astrocytes, due to the various cellular sources of S100B.

Distinct FTDP-17 missense mutations in tau produce tau aggregates and other pathological phenotypes in transfected CHO cells.

Multiple tau gene mutations are pathogenic for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), with filamentous tau aggregates as the major lesions in the CNS of these patients. Recent studies have shown that bacterially expressed recombinant tau proteins with FTDP-17 missense mutations cause functional impairments, i.e., a reduced ability of mutant tau to bind to or promote the assembly of microtubules. To investigate the biological consequences of FTDP-17 tau mutants and assess their ability to form filamentous aggregates, we engineered Chinese hamster ovary cell lines to stably express tau harboring one or several different FTDP-17 mutations and showed that different tau mutants produced distinct pathological phenotypes. For example, delta K, but not several other single tau mutants (e.g., V337 M, P301L, R406W), developed insoluble amorphous and fibrillar aggregates, whereas a triple tau mutant (VPR) containing V337M, P301L, and R406W substitutions also formed similar aggregates. Furthermore, the aggregates increased in size over time in culture. Significantly, the formation of aggregated delta K and VPR tau protein correlated with reduced affinity of these mutants to bind microtubules. Reduced phosphorylation and altered proteolysis was also observed in R406W and delta K tau mutants. Thus, distinct pathological phenotypes, including the formation of insoluble filamentous tau aggregates, result from the expression of different FTDP-17 tau mutants in transfected Chinese hamster ovary cells and implies that these missense mutations cause diverse neurodegenerative FTDP-17 syndromes by multiple mechanisms.

Glial cells as targets and producers of neurotrophins.

Glial cells fulfill important tasks within the neural network of the central and peripheral nervous systems. The synthesis and secretion of various polypeptidic factors (cytokines) and a number of receptors, with which glial cells are equipped, allow them to communicate with their environment. Evidence has accumulated during recent years that neurotrophins play an important role not only for neurons but also for glial cells. This brief update of some morphological, immunocytochemical, and biochemical characteristics of glial cell lineages conveys our present knowledge about glial cells as targets and producers of neurotrophins under normal and pathological conditions. The chapter discusses the presence of neurotrophin receptors on glial cells, glial cells as producers of neurotrophins, signaling pathways downstream Trk and p75NTR, and the significance of neurotrophins and their receptors for glial cells during development, in cell death and survival, and in neurological disorders.

Differential effects of two structurally related N6-substituted cAMP analogues on C6 glioma cells.

Membrane permeable derivatives of cAMP are widely used to investigate the role of cAMP in the regulation of cell growth and differentiation. To further investigate the molecular mechanisms, underlying the effects of cAMP analogues on growth control and differentiation, the concentration-dependent action of four structurally related cAMP analogues with substitutions at the N6-position in the adenine moiety, namely N6-benzyl-cAMP (Bn-cAMP), N6-benzoyl-cAMP (Bz-cAMP), N6-butyryl-cAMP (Bt-cAMP) and N6, O2'-cAMP (Bt2-cAMP), on C6 rat glioma cell proliferation was determined. The four analogues tested showed different specificities, and the order of growth inhibitory potency was: Bn-cAMP >> Bt-cAMP = Bt2-cAMP >> Bz-cAMP. Thus, although both derivatives have been described to equally bind and activate cAMP-dependent protein kinase (cAK) isozymes, Bn-cAMP most effectively inhibited C6 glioma cell proliferation with an IC50 of 25 microM, while Bz-cAMP was almost ineffective in C6 cells (IC50 >> 1000 microM). In vivo and in vitro studies using HPLC analysis, revealed that Bn-cAMP was subject to enzymatic degradation and that the metabolite Bn-adenosine (Bn-Ado) exerted growth inhibitory effects at a concentration even below 10 microM. Additionally, C6 glioma cells morphologically differentiated in the presence of Bn-cAMP (100 microM) and of Bn-Ado (10 microM), by extending long cellular processes. The growth inhibitory activity of Bn-Ado was not influenced, when dipyridamole, an inhibitor of adenosine uptake, was added to the incubation medium, indicating that adenosine action was mediated through a receptor-mediated mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

D2-protein in PC12 pheochromocytoma cells after nerve growth factor stimulation.

The rat brain D2-protein has previously been shown to play a role in interneuronal adhesion Monospecific antisera against this protein reacted with adrenal medulla cells and with PC12 pheochromocytoma cells as demonstrated by indirect immunofluorescence. Upon stimulation by nerve growth factor the PC12 cells extend neurites. These neurites were shown to contain D2-protein both by immunofluorescence and by crossed immunoelectrophoresis. The findings substantiate the close relationship between neurons from the central nervous system and both the PC12 cells line, as well as adrenal medulla cells.

Expression of protease-activated receptors (PARs) in OLN-93 oligodendroglial cells and mechanism of PAR-1-induced calcium signaling.

Protease-activated receptors (PARs) are a group of four members of the superfamily of G protein-coupled receptors that transduce cell signaling by proteolytic activity of extracellular serine proteases, such as thrombin. Possible expression and functions of PARs in oligodendrocytes, the myelin forming cells of the CNS, are still unclear. Here, the oligodendrocyte cell line OLN-93 was used to investigate the signaling of PARs. By reverse transcription-polymerase chain reaction (RT-PCR), immunostaining and Ca(2+) imaging studies, we demonstrate that OLN-93 cells functionally express PAR-1. PAR-3 seems to be expressed without apparent activity, and PAR-2 and PAR-4 cannot be detected. Short-term stimulation of the OLN-93 cells with PAR-1 agonists, such as thrombin, trypsin and PAR-1 activating peptide, dose-dependently induced a transient rise of [Ca(2+)](i). Concentration-effect curves display a sigmoidal concentration dependence. Elevation of [Ca(2+)](i) induced by PAR-1 mainly resulted from Ca(2+) release from intracellular stores. Studies on the effects of pertussis toxin (PTX), phospholipase C antagonist and 2-APB, showed that in OLN-93 cells (i). the calcium signaling cascade from PAR-1 was mediated through PTX-insensitive G proteins, (ii). activation of phospholipase C and liberation of InsP(3) were events upstream of the Ca(2+) release from the stores. In addition, the present study analyzed PAR-1 desensitization caused by exposure to thrombin, trypsin, and PAR-1 activating peptide, elucidated the influence of the protease cathepsin G on PAR-1 activation, and also characterized PAR-1 desensitization. This is the first study, which shows that OLN-93 oligodendrocytes functionally express PAR-1, and identifies the receptor coupling to mobilization of intracellular calcium. Moreover, the expression of PAR-1 was demonstrated by RT-PCR in primary oligodendrocytes from rat brain.

Organization and functional roles of the cytoskeleton in oligodendrocytes.

Mature oligodendrocytes are characterized by their numerous cytoplasmic extensions and flat membranous sheets. These sheets contain an extensive cytoskeletal network of microtubules (MTs) that maintain the cellular morphology, are specifically important for cellular sorting, and provide the rails for organelle trafficking. Mitochondria are localized in the primary and secondary processes and follow the tracks of the MTs in the cytoplasmic extensions. Oligodendrocytes express microtubule associated proteins (MAPs), specifically MAP2 and tau, which might be involved in the regulation and stabilization of the dynamic MT network in the myelin-containing cellular processes. Tau and MAP2 heterogeneity increases during oligodendroglia maturation, and in mature oligodendrocytes tau mRNA with four MT binding domains are more prominent than in progenitor cells. Filamentous cell inclusions are a unifying mechanism underlying a variety of late-onset neurodegenerative disorders and have mainly been viewed as neuron-specific. Recent evidence indicated that glial changes occur in CNS degenerative diseases and seem to be a more common feature than previously thought. Glial fibrillary tangles (GFTs) in oligodendrocytes were observed in familial multiple system tauopathy, and glial cytoplasmic inclusions (GCIs) and oligodendroglia degeneration are the histological hallmark of multiple system atrophy (MSA). GCIs are associated with MTs and contain stress proteins and MAPs. Thus, neurons and glial cells share common cytoskeletal pathologies. During health and disease, MAPs might be important regulators of the structural stability and plasticity of the oligodendroglia cytoskeleton.

Identification of a B-50-like protein in frog brain synaptosomes.

Vestibular compensation in the frog following unilateral labyrinthectomy is accompanied by distinct changes in the endogenous phosphoprotein patterns in total frog brain homogenate and isolated synaptosomes. The purpose of this study was to characterize one of these proteins, an acidic 45-kDa synaptosomal protein, resembling in some of its features the growth-associated protein GAP-43/B-50. Our results demonstrate by comparative analysis with purified rat B-50/GAP-43 that the 45-kDa protein (IP 4.8) in synaptosomal membranes of frog brain is phosphorylated by added purified PKC, cross-reacts with affinity-purified rabbit antibodies to rat B-50 and exhibits a Staphylococcus aureus V8 protease peptide digestion pattern corresponding to rat B-50. Therefore, we conclude that the acidic 45-kDa synaptosomal protein is a growth-associated B-50-like protein, probably involved in processes responsible for compensatory reorganization of the vestibular structures after hemilabyrinthectomy in the frog.

Expression of eight metabotropic glutamate receptor subtypes during neuronal differentiation of P19 embryocarcinoma cells: a study by RT-PCR and in situ hybridization.

Metabotropic glutamate receptors modulate neuronal activity but expression and alternative splicing of their subtypes (mGluR1-mGluR8) during early neuronal differentiation are essentially unknown. In the mouse embryocarcinoma cell line P19, one of the best established systems to study neurogenesis in vitro, it was shown by RT-PCR and in situ hybridization that the neuronal differentiation process, induced by retinoic acid, is characterized by an early increase in the expression of mGluR3, mGluR7 and mGluR8 and a late rise in the mRNA levels of mGluR1 and mGluR5, whereas mGluR2 and mGluR4 seem to be constitutively expressed. In comparison, in primary embryonic neurons all mGluR subtypes were detected at day 3 after plating while primary astrocytes and oligodendrocytes have diverging mGluR pattern. In addition, the splicing pattern of mGluR1 and mGluR5 transcripts differ remarkably between neural cells in vitro and brain tissue. These data, although not comparable to the situation in vivo, might be a hint on so far unknown functions of metabotropic glutamate receptors during neuronal differentiation.

Expression and mRNA splicing of glycine receptor subunits and gephyrin during neuronal differentiation of P19 cells in vitro, studied by RT-PCR and immunocytochemistry.

The mouse EC cell line P19, differentiating in vitro into neural cell types under the influence of retinoic acid, represents a well established model system for neurogenesis. In this system the expression of the alpha (alpha 1-alpha 3) and beta subunits of the inhibitory glycine receptor (GlyR) and of gephyrin as well as their mRNA splice variants was analyzed by RT-PCR and by immunocytochemistry. In the course of neuronal differentiation of P19 cells mRNA of GlyR beta is constitutively expressed, GlyR alpha 1 and alpha 2 are induced and GlyR alpha 3 was not detected. From the three gephyrin transcripts known to be differently spliced in the C3/C4 cassette region, the C3 transcript was found at all stages while the C4 transcript was not detectable. The insert-free form was measurable in P19 cells only 3-4 days post induction by retinoic acid. In addition a GlyR beta splice variant and a fourth gephyrin transcript were detected. Primary glial cells do not contain significant amounts of GlyR alpha subunits while in primary neuronal cells transcripts of GlyR alpha 2 were found as well as the mRNA of the GlyR beta subunit and of gephyrin. PC12 cells do not express glycine receptor genes but do express gephyrin. Immunocytochemistry confirmed the constitutive expression of gephyrin at the protein level, whereas GlyR antigens could only be detected in islets of the 'P19 neurons'. In conclusion, P19 and primary neuronal cells but not PC12 cells express the transcripts of glycine receptor components, necessary to generate functional receptors.

Rat brain primary neurons immobilized in basement membrane gel threads: an improved method for on-line 13C NMR spectroscopy of live cells.

In vivo MRS studies on intact brain reflect the metabolism of all cells present, but do not distinguish between different cell types. NMR studies of immobilized cultured primary cells, such as neurons and astrocytes, are a useful model to monitor the specific differences in metabolism of the various cell types in the brain. The present study shows that primary rat neuronal cells can be cultured in basement membrane gel threads. After 4 days of incubation the threads are filled with viable cells, and represent a population of morphologically differentiated neuronal cells with less than 5% of non-neuronal cells, i.e., astrocytes. These threads were placed into a NMR tube and used for on-line monitoring of neuronal metabolism. Under these conditions cells remained viable and metabolically active for several days. The energy status was monitored by using 31P NMR spectroscopy. To study neuronal glucose metabolism [1-13C]glucose was added to the perfusion medium and 30 min later 13C-labeled metabolites were detectable by 13C NMR spectroscopy. Immobilized neurons synthesized glycolytic products such as [3-13C]lactate and [3-13C]alanine, as well as several tricarboxylic acid cycle products, i.e., [2-13C]glutamate, [3-13C]glutamate, [4-13C]glutamate, [2-13C]aspartate, and [3-13C]aspartate. In summary, 31P and 13C NMR spectra can be recorded from live neuronal cells for up to 24 h using the newly designed procedure described in the present communication.

Stress response of lysosomal cysteine proteinases in rat C6 glioma cells.

Acid proteinases of C6 rat glioma cells were analyzed by means of gelatine polyacrylamide electrophoresis with respect to their responses to stress (heat shock and butanol). Proteinase activities on gelatine gels were characterized by their molecular masses. pH-optima, isoelectric points and reactions to inhibitors. Four bands of 25, 35 and 65/85 kDa most probably represent active and proforms as well as precursor complexes of lysosomal cysteine proteinases with pH optima between 4.0 and 5.0. The 25-kDa band seems to contain cathepsin L and B, the 35-kDa band proforms of cathepsin L and B and the 65/85-kDa bands possibly precursor complexes of cathepsin L and B. After 30-min heat shocks of different temperatures (40-50 degrees C), the 35-kDa activity increased, whereas the 65/85-kDa activity decreased after exposure to 42 and 44 degrees C, which also caused a strong increase in the level of the inducible heat shock protein of 68 kDa (HSP 68). The alterations of the proteinase activities and the increases of the HSP 68 levels occur at heat shock treatments that cause cell death in about 25-40% of the population as determined by Trypan blue staining. HSP 68 induction and proteinase activity changes were also observed 12 hr after a 1-hr treatment with different butanol concentrations (0.14-0.16 M). Kinetics of the response to a 30-min heat shock (44 degrees C) revealed a maximal decrease of the 35-kDa and a maximal increase of the 65/85-kDa activities after 12 hr recovery. When cells were exposed to repeated heat shocks (44 degrees C) at 12-hr intervals, the HSP 68 level further increased, whereas the 35-kDa and 65/85-kDa proteinase activities did not change. This result indicates a role of HSP 68 (or other HSPs) in the processing or stability of the putative cathepsin precursors (65/85-kDa complexes).

Haloperidol and clozapine decrease S100B release from glial cells.

Recent meta-analyses showed consistently elevated levels of S100B in serum and cerebrospinal fluid of schizophrenic patients. This finding has been attributed to glial pathology because S100B is produced by astrocytes and oligodendrocytes. However, S100B may be likewise associated with schizophrenia-related disturbances in glial cell as well as adipocyte energy supply and glucose metabolism. The influence of antipsychotic drugs on S100B levels remains unclear, and some studies have suggested that treatment with these drugs may actually contribute to the elevated S100B levels observed in schizophrenic patients. In this study, we explored the effects of the typical antipsychotic haloperidol and the atypical prototype drug clozapine on the release of S100B by astrocytic C6 cells and oligodendrocytic OLN-93 cells. Because of the association between schizophrenia and disturbances in energy metabolism, we assessed the effects of these drugs under basal condition (BC) compared to serum and glucose deprivation (SGD). We found that treatment of C6 and OLN-93 cells with haloperidol and clozapine reduced the release of S100B from C6 and OLN-93 cells under BC and SGD in vitro at a tissue concentration corresponding to the assumed therapeutic dose range of these drugs. These data suggest that elevated levels of S100B in bodily fluids of schizophrenic patients are normalized rather than increased by the effects of antipsychotic drugs on glial cells.

Nerve growth factor-inducible, large external (NILE) glycoprotein in developing rat cerebral cells in culture.

Fetal rat cerebral cells underwent neuronal differentiation in culture. This process was accompanied by distinct changes in the cellular glycoprotein pattern. The incorporation of [3H]-fucose into two proteins of apparent molecular weights of 30,000 and 60,000 daltons was significantly decreased and specific developmental changes were observed in a group of glycoproteins with high molecular weights (150,000-250,000 dalton). By means of indirect immunoprecipitation one of them was identified as NILE gp (nerve growth factor-inducible large external) glycoprotein (200,000 dalton), a marker of central and peripheral neurons. Its developmental expression on neurons of dissociated rat cerebral cultures was studied using the indirect immunofluorescence technique and compared to the fluorescent-labeling pattern of other neuronal markers. Neurons expressing NILE gp were detected as early as after one day in culture. No preferential staining of neurites versus cell bodies was observed. Two classes of NILE gp-positive cell-type, whereas the other group was represented by larger, more spindle-shaped neurons with a limited number of neuritic processes. In most cases one of these neuritic processes was preferentially labeled. Astroglia cells, as identified by immunolabeling with antisera against the glial acidic fibrillary protein, were observed to develop and mature after the first week in culture. NILE-positive neurons were found to be positioned in close association with glial cell processes.

The oligodendroglia cytoskeleton in health and disease.

Oligodendrocytes have a high rate of synthetic activity and produce vast amounts of myelin. The membrane production requires specific sorting and transport processes and structural support. In culture, oligodendrocytes extend flat membranous sheets containing an extensive cytoskeletal network of microtubules (MTs) and microfilaments (MFs). The microtubules participate in the elaboration and stabilization of the myelin-containing cellular processes and have an impact not only on the complex oligodendroglia architecture but also influence their functions. They participate in intracellular sorting processes and the translocation of myelin basic protein (MBP) mRNAs to the forming myelin sheath. The two major groups of neuronal microtubule-associated proteins (MAPs), MAP2 and tau are expressed in oligodendrocytes and might be involved in the regulation of MT stability and organization. Myelin-specific proteins, such as MBP and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), interact with the cytoskeleton. Glial changes occur in a variety of neurodegenerative diseases, and glial fibrillary tangles and glial cytoplasmic inclusions (GCls), containing abnormal microtubular structures which stain positively for stress proteins and microtubule-associated proteins, are found in oligodendrocytes of the affected brains. The role of MTs and their associated proteins in oligodendrocytes during normal development and pathological situations is specifically emphasized in this review.

Mode of cell injury and death after hydrogen peroxide exposure in cultured oligodendroglia cells.

Oxidative stress has been implicated as a causal factor in a wide variety of neurodegenerative diseases. To investigate the direct consequences of oxidative damage on myelin-forming cells, we have exposed oligodendrocytes to hydrogen peroxide. Cytotoxicity was assessed in glial cultures by neutral red (NR) and MTT assay, and half-maximal cytotoxicity was reached after a 30-min application with 100-200 microM H2O2 during a 16-24-h recovery period. The cytotoxic effect could be partly abolished by the simultaneous incubation with N-acetyl-l-cysteine, an antioxidant and precursor of glutathione. In purified mature oligodendroglia cultures (7 div), metabolic activity as determined by the MTT assay, was impaired directly after the treatment with H2O2, and only slightly further enhanced during the 24-h recovery period. Morphological inspection revealed that oligodendrocytes in either the presence or the absence of astrocytes were specifically susceptible to free radical damage, the membranous sheets were disrupted, membranous blebs appeared, and fragmented nuclei were seen. Similar changes were induced by treatment with menadione or staurosporine. The data show that brief exposure to H2O2 induced cell death via apoptosis. This death occurred over a period of 24 h and was accompanied by the appearance of fragmented and condensed DAPI-stained nuclei and internucleosomal DNA cleavage. Concomitantly, as investigated by RT-PCR analysis, the transcriptional activity of c-fos and c-jun was stimulated, without altering mRNA expression of the myelin-specific genes MBP, MAG, and PLP. Thus, oxidative stress in oligodendrocytes leads to the onset of programmed cell death, involving the transcriptional activation of the immediate-early genes c-fos and c-jun.

Developmental regulation of alternatively spliced isoforms of mRNA encoding MAP2 and tau in rat brain oligodendrocytes during culture maturation.

Oligodendrocytes are responsible for the formation and maintenance of the myelin sheaths in the central nervous system (CNS), and microtubules essentially participate in the elaboration and stabilization of myelin-containing cellular processes. We have shown before that the two major groups of neuronal microtubule-associated proteins (MAPs), MAP2 and tau, are expressed in the myelin forming cells of the CNS (Mueller et al. [1997] Cell Tissue Res. 288:239-249). Here we demonstrate for the first time that during culture maturation, changes in mRNA splicing and a shift from immature to mature MAP2 and tau mRNAs occur in oligodendrocytes. Similarly to neurons, a developmental shift from MAP2 isoforms with 3 microtubule (MT)-binding domains (3R) to the isoforms with 4 MT-binding domains (4R) is observable. MAP2c constitutes the major MAP2 isoform in oligodendrocytes. They contain tau mRNA splice products with both 3 and 4 MT-binding repeats (3R, 4R) with no amino terminal insert or with exon 2, and do not express isoforms containing exon 3. The shortest form tau 1 (3R; no inserts) representing the immature tau isoform is most prominently expressed in early progenitor cells and gradually decreases during culture maturation, while tau 5 (4R; with exon 2) appears later during in vitro differentiation. The product corresponding to tau 2 (3R; with exon 2) and tau 4 (4R; no inserts) remains approximately at the same level. Hence, the occurrence of MAPs in oligodendrocytes is developmentally regulated. While in progenitor cells, 3R- and 4R-MAP2c are expressed at approximately the same level, in mature oligodendrocytes after 12 days in vitro, the ratio of 4R- to 3R-MAP2c is nearly 2. In contrast, the ratio of 4R- to 3R-tau in progenitor cells is 1:3 and shifts to 1:1 after 12 days in culture.

Effects of organotins on rat brain astrocytes in culture.

The interaction of triethyltin (TET) and trimethyltin (TMT) with rat brain astrocytes in vitro was investigated. Both compounds are highly neurotoxic after in vivo application, cause neurobehavioral changes, and elicit neuronal and glial responses in the CNS. In this study, 5-week-old cultures were exposed to TMT or TET (0.1-2.5 microM) for 24 h. A concentration-dependent cytotoxicity was observed for both agents by vital dye uptake assay using neutral red (NR). The order of potency for half-maximal cytotoxicity (NR-50) was TET (0.7 microM) > TMT (2.5 microM), in agreement with results found after in vivo administration. TET and TMT caused similar morphological changes: large holes extending through the plasma membrane appeared initially in the flattened cell bodies, cytoplasmic extensions were retracted, and long cellular processes formed. Later, the cell bodies rounded up and had only a few extremely long and thin processes. Indirect immunofluorescence staining using anti-vimentin and anti-glial fibrillary acidic protein (GFAP) antibodies revealed that the orderly array of the intermediate filament system was severely disturbed. At lower concentrations, an increased bundling was observed, and at higher concentrations the disassembly of the intracellular framework was seen, and cellular staining appeared rather diffuse. Western blot analysis of cellular extracts was carried out to determine the protein levels of GFAP and vimentin. In this culture system, TET and TMT caused an almost two-fold increase in the levels of GFAP at concentrations around and below NR-50, indicating that astrocytes react to organotins independently of neuronal signals.(ABSTRACT TRUNCATED AT 250 WORDS)