Oligodendrocyte-like Cells Research Articles

Modification of the brain-derived neurotrophic factor gene: a portal to transform mesenchymal stem cells into advantageous engineering cells for neuroregeneration and neuroprotection

Multipotential mesenchymal stem cells (MSCs) are ideal seed cells for recruiting the loss of neural cells due to their strong proliferative capacity, easy acquisition, and considerable tolerance of genetic modifications. After transduction of brain-derived neurotrophic factor (BDNF) gene via recombinant retroviral vectors into the human MSCs, nearly 100% of cells expressed BDNF (which were therefore transformed into BNDF–MSCs) as detected by immunocytochemistry, and the quantity of BDNF in the culture medium was increased by approximately 20,000-fold. In spite of the genomic integration of an exogenous gene, BDNF–MSCs did not present any structural aberration in the chromosomes. All-trans-retinoic acid (RA) induction caused the BDNF–MSCs to differentiate into neural cells with significantly increased expressions of such neural-specific proteins as nestin, NeuN, O4, and glial fibrillary acidic protein (GFAP). The voltage-dependent K+/Ca2+ currents were recorded from the induced BDNF–MSCs using patch-clamp technique. Compared with the MSCs induced by both RA and BDNF, BDNF–MSCs survived in significantly greater number in the induction medium, and also more cells were induced into neuron-like cells (NeuN, P < 0.01) and oligodendrocyte-like cells (O4, P < 0.05). We suppose that, once engrafted into human central nervous system, the BDNF–MSCs would not only recruit the neuronal losses, but also provide, by way of paracrine, large quantities of BDNF that effectively perform the functions of neuroprotection and neuroregeneration, promoting the activation of endogenous neural stem/progenitor cells and their chemotactic migration. On the other hand, the BDNF–MSCs that can survive in the host environment and differentiate subsequently into functional mature cells may also serve as specifically targeting vectors for ex vivo gene therapy.

Dividing Olig2-expressing progenitor cells derived from ES cells.

G-Olig2 is a knock-in ES cell line with GFP inserted into the Olig2 gene so that ES cell-derived neural cells that express Olig2 also express GFP. This tool allows visualization of the subset of cells that differentiate along the Olig2-expressing pathway. By manipulating culture conditions, it is possible to induce Olig2 expression in rapidly dividing cells. These cells have many of the features of glial progenitor cells but, unlike other glial progenitors, are able to divide rapidly for at least 1 month while still expressing Olig2. Even after 1-month expansion, the cells differentiate readily into astrocyte-like and oligodendrocyte-like cells when switched to serum-containing medium. Cellular memory is the property whereby cells remain specified to a particular lineage or pathway while undergoing division. ES cell-derived neural cells show cellular memory for a glial progenitor phenotype and thus provide a new and tractable model for this basic feature of neural development.

Continued in situ DNA fragmentation of microglia/macrophages in white matter weeks and months after traumatic brain injury.

Paraffin-embedded material from the pons of head-injured patients whose disability could be attributed to diffuse traumatic axonal injury, and controls, was identified from the department's archive. The cases were divided into three groups based on survival, viz Group 1 (n = 5) who survived for between 4 and 8 weeks, Group 2 (n = 5) for between 3 and 9 months, and Group 3 (n = 5) who survived for more that 12 months. Sections were stained by the TUNEL (TdT-mediated UTP nick end labelling) technique, and by H&E, LFB/CV and immunohistochemically for astrocytes (GFAP) and microglia/macrophages (CD68). Microscopic abnormalities were mapped onto line diagrams of two levels of the pons and quantitation of the response determined by an eye-piece graticule placed over the medial lemmisci, cortico-spinal and transverse fiber tracts. Data were pooled by region of interest. In the H&E and LFB/CV stained sections, there was variable pallor of staining in ascending and descending fiber tracts due to loss of myelin: within these same tracts there was an astrocytosis and increased numbers of microglia/macrophages compared with controls. In the white matter tracts of the controls, there was on average 1-2 TUNEL+ cells per unit area. In contrast, there were on average 2-16 TUNEL+ cells in the cortico-spinal tracts and in the medial lemnisci of all groups of head-injured patients. CD68+ cells co-located with the TUNEL+, and their number mirrored the TUNEL + staining with on average 16-30 cells per unit area in Group 1, 14-27 cells per unit area in Group 2, and 12-14 cells per unit area in Group 3. There was a statistical association between the TUNEL+ and CD68+ cells. Few changes were seen in the transverse fiber tracts of the pons. These findings indicate that most of the in situ DNA fragmentation occurred in microglia/macrophages in ascending and descending fiber tracts of the brain stem in which by conventional light microscopy there is Wallerian degeneration. However, in addition, a few TUNEL+ oligodendrocyte-like cells were also seen.

Ethanol specifically decreases peroxisome proliferator activated receptor beta in B12 oligodendrocyte-like cells.

Peroxisome proliferator activated receptors (PPARs) are nuclear receptors that control important genes involved in lipid metabolism. Their role in nerve cells is uncertain, although anomalous myelination of the corpus callosum has been described in the PPARbeta-null mouse, and abnormalities of this tissue have been documented in fetal alcohol syndrome in humans. We report here that ethanol treatment of B12 oligodendrocyte-like cells induces a concentration- and time-dependent decrease in the mRNA and protein levels of PPARbeta, with no effect on PPARalpha or PPARgamma. The effect on PPARbeta is seen as an increase in mRNA degradation, as assessed by run-off assays, due to a significant decrease in PPARbeta mRNA half-life, with no observed changes in intracellular localization. Our results suggest a possible link between PPARbeta function and ethanol-induced abnormal myelination in oligodendrocytes.

Type-2 astrocyte-like cells are more resistant than oligodendrocyte-like cells against non-N-methyl-D-aspartate glutamate receptor-mediated excitotoxicity.

Glutamate causes excitotoxicity via non-N-methyl-D-aspartate (NMDA) glutamate receptors (GluR) in oligodendrocytes. Because both oligodendrocytes and type 2 astrocytes are differentiated from oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells, we investigated whether astrocytes are also vulnerable to non-NMDA GluR-mediated excitotoxicity. For these studies, oligodendrocyte-like cells (OLC) and type 2 astrocyte-like cells (2ALC) were derived from CG-4 cells, an immortalized rat O-2A progenitor cell line. About 50% of 2ALC were positive for glial fibrillary acidic protein and 90% were positive for A2B5, verifying that these cells have an type 2 astrocytic phenotype. A 24-hr exposure of OLC to 2 mM kainate, an activator of non-NMDA GluR, caused cell damage as shown by the release of lactate dehydrogenase. The extent of kainate-induced OLC damage was increased by cyclothiazide. In contrast, exposure of 2ALC to 2 mM kainate alone did not induce injury, though mild 2ALC injury was elicited by exposure to 2 mM kainate plus 100 microM cyclothiazide. Furthermore, we found that the kainate induced Ca(2+) uptake by 2ALC was 27.5% of that induced by kainate in OLC. Finally, both OLC and 2ALC expressed non-NMDA GluR subunit mRNAs, including GluR2, GluR3, GluR4, GluR6, GluR7, KA1, and KA2, but quantitative Western blot analysis revealed higher immunodetectable GluR2 and lower immunodetectable GluR3 and GluR4 in 2ALC than in OLC. Together, these results suggest that astrocytes are relatively resistant to non-NMDA GluR-mediated excitotoxicity because they have a higher expression of GluR2 and lower expression of GluR3 and GluR4.

A rosette-forming glioneuronal tumor of the fourth ventricle: infratentorial form of dysembryoplastic neuroepithelial tumor?

Eleven cases of a distinctive tumor of the posterior fossa are described. The patients (age range 12-59 years) presented with headache and/or ataxia. Neuroimaging revealed a relatively discrete, focally enhancing mass(es) primarily involving the aqueduct, fourth ventricle, and cerebellar vermis. Hydrocephalus was present in seven cases, and two lesions were multicentric. In two cases a significant increase in tumor size was documented. Gross total or subtotal resections were achieved in 10 cases. One patient underwent biopsy alone and another received postoperative irradiation. Histologically, two components were identified in all cases. One consisted of neurocytes forming neurocytic and/or perivascular pseudorosettes in a fibrillary, partly microcystic matrix. The second, astrocytic component resembled pilocytic astrocytoma in 10 cases and consisted of fibrillated spindle cells with oval nuclei associated with occasional Rosenthal fibers, granular bodies, glomeruloid capillaries, and microcalcifications. Regionally, this component was more diffuse and patternless, consisting of sheets of round to oval, oligodendrocyte-like cells. Rare ganglion cells were seen in four cases. The rosettes were consistently synaptophysin and MAP-2 immunoreactive, whereas the spindle cells were positive for S-100 protein and glial fibrillary acidic protein. Overall, atypia was minimal; no mitoses were found, and Ki67 labeling indices were low. Ultrastructurally, the neurocytic cells featured processes containing microtubules and occasional dense core granules. Mature synapses were found in one of the four cases studied. Although the histologic features of this unique tumor superficially resemble those of dysembryoplastic neuroepithelial tumor, rosette formation by neuronal cells, the frequent presence of a pilocytic astrocytoma component, and the growing nature of the lesion argue against that diagnosis, as does occasional multifocality.

Isolation and growth of a cytopathic agent from multiple sclerosis brain tissue.

Although many studies support a role for viruses in multiple sclerosis (MS) etiopathology, no specific agent has been consistently associated with significant numbers of MS patients without concomitant detection in non-MS controls. Previous studies have shown the presence of viral-like structures in MS plaques, although the specificity of these structures for MS has been questioned. The present study describes the use of polyclonal antisera against feline and human brain-derived cytopathic agents and immunoaffinity chromatography to purify and partially characterize possible virus-like structures from MS brain tissue. Chromatography eluates from 4 MS brains contained pleomorphic particles up to 350 nm in diameter and tubular/filamentous-like structures approximately 10-18 nm in thickness. Inoculation of primary rat glial cell cultures with chromatography eluates from MS brain tissue resulted in a reproducible pattern of cytopathic effects in the form of multinucleation in cells identified immunocytochemically as oligodendrocytes. Antisera raised against the feline and MS-derived cytopathic agents were used to successfully immunolabel infected oligodendrocyte-like cells and syncytia and to detect a 66,000 M(r) protein on Western blots of inoculated cultures or concentrated MS brain eluates. Similar structures, cytopathic effects (CPE) and protein expression were not observed in eluates from 5 control brains or in cultures inoculated with control brain eluates. These studies demonstrate that cytopathic, virus-like structures can be isolated from MS brain tissue using antisera raised against a cytopathic agent rescued from demyelinating brain lesions in cats. The identity of this agent and its possible role in MS aetio-pathology remains unknown.

Differentiation of bipolar CG-4 line oligodendrocytes is associated with regulation of CREB, MAP kinase and PKC signalling pathways.

Undifferentiated bipolar CG-4 cell line oligodendrocytes provide a model system for the O-2A progenitor cell from which oligodendrocytes are derived both in vivo and in vitro. The exchange of neuroblastoma conditioned basal media for basal media causes differentiation of undifferentiated bipolar CG-4 cells into multipolar oligodendrocyte-like cells whilst replacement with basal media containing 20% foetal bovine serum favours the formation of type-2 astrocyte-like cells. Here, we demonstrate that activation of these differentiation pathways correlates with distinct changes both in cell metabolism and in signal transduction. Exchange of neuroblastoma conditioned media for basal media correlates with stimulation of basal metabolic activity, reduced phosphorylation of p44/42 MAP kinase and reduced phosphorylation of the transcription factor CREB. In contrast, differentiation with basal medium containing 20% foetal bovine serum (FBS), into type 2 astrocyte-like cells, correlates with reduction in basal metabolic activity, increased phosphorylation of p44/42 MAP kinase and increased phosphorylation of the transcription factor CREB. Inhibition of protein kinase C blocked both the metabolic and morphological changes associated with differentiation towards mature multipolar oligodendrocyte-like cells. Inhibition of PKA and MEK did not effect metabolic activity. The rapid return of neuroblastoma conditioned basal media to cells treated with basal media, increased phosphorylation of CREB and MAP kinase. These results demonstrate that protein kinase C and p44/42 MAP kinase signalling pathways are modulated during bipolar CG-4 cell differentiation and demonstrate that the transcription factor CREB may play a pivotal role in differentiation along oligodendrocyte-or astrocyte-lineages.

Dysembryoplastic Neuroepithelial Tumor-Like Neoplasm of the Septum Pellucidum: A Lesion Often Misdiagnosed as Glioma

The authors report a series of 10 low-grade neoplasms arising in the midline anteriorly in the region of the septum pellucidum with many of the histologic features of dysembryoplastic neuroepithelial tumor (DNT). The patients (five female, five male) ranged in age from 6 to 35 years (mean age, 21.5 years). The most common presenting symptoms were headache, nausea and vomiting, and visual disturbances. Radiographically, the tumors extended into the lateral ventricles from the septal region and obstructed the foramen of Monro. Varying degrees of hydrocephalus were present. The lesions were lobular, well-delineated, hypointense to brain on T1-weighted magnetic resonance imaging, and hyperintense on T2-weighted images. They were uniformly nonenhancing or showed only minimal peripheral enhancement. The tumors, in aggregate, had the histologic features of DNT. These included a mucin-rich background, oligodendrocyte-like cells, "floating neurons," and a "specific glioneuronal element." Seven patients underwent gross total resection and two underwent subtotal resection. No patients received adjuvant chemotherapy or radiotherapy. On follow-up (n = 6; median, 14 months), all tumors had either not recurred or were radiologically stable. On the basis of both neuroimaging and histopathology, DNT-like lesions should be considered in the differential diagnosis of midline intraventricular tumors in children and young adults. Distinction from more aggressive neoplasms is essential because these tumors appear to behave in a benign fashion.

White matter damage following systemic injection of the mitochondrial inhibitor 3-nitropropionic acid in rat

Oxidative stress has been implicated as a pathogenic mediator of neuronal perikarya cell death. Axons and oligodendrocytes, components of white matter, could also be vulnerable to oxidative damage. An experimental model of oxidative stress was induced by systemic injection of 3-nitropropionic acid (3-NPA). Animals received an i.p. injection of 10, 15, 20 or 30 mg/kg 3-NPA or vehicle and were killed 24 h later. 3-NPA produced a concentration-dependent increase in axonal pathology within the striatum reflected by the amount of β-APP and SNAP-25 accumulation. Axonal damage was anatomically coincident with the neuronal lesion. There was no neuronal or axonal damage in the subcortical white matter or cerebral cortex in any of the animals treated with 3-NPA. Manganese superoxide dismutase (Mn-SOD) immunoreactivity was present in the vehicle and all 3-NPA treated groups. The amount of Mn-SOD cellular staining was concentration-dependently increased within the striatum supporting a role for oxidative stress in the mechanism of 3-NPA neurotoxicity. Oligodendrocyte-like cells within the subcortical white matter were immunopositive for calpain-mediated spectrin breakdown products and increased in a concentration-dependent manner. Therefore in this experimental model, mitochondrial inhibition may lead to the initiation of oxidative stress and calpain activation, which could mediate cytoskeletal breakdown in axons and oligodendrocytes suggesting an interaction between at least two pathogenic mechanisms.

Diminished calcium homeostasis and increased susceptibility to excitotoxicity of JS 3/16 progenitor cells after differentiation to oligodendroglia

JS 3/16, derived from passaged oligodendroglial cultures prepared from rat cerebral white matter, differentiate from progenitors (OP) into complex process-bearing, galactocerebroside-positive but myelin basic protein-negative immature oligodendrocyte-like cells (ImO) after withdrawal of trophic factors. We found that JS 3/16 ImO are markedly more susceptible than OP to cell death after sustained α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor (AMPA-GluR) activation. This excitotoxicity is preceded by loss of intracellular Ca2+ homeostasis, which is more marked in ImO than OP. We identified three factors likely to contribute to the diminished Ca2+ homeostatic capacity of ImO. First, signal intensities of immunoreactive GluR2, GluR3, and GluR4 AMPA-GluR subunits are increased 1.3- to 2.2-fold in ImO over OP without comparable changes in RNA editing and alternative splicing. Second, transcriptional levels of genes encoding Na+-Ca2+ exchanger proteins and a plasma membrane ATPase (PMCA1), which are necessary for Ca2+ extrusion across the plasma membrane, are lower in ImO than in OP. Third, ImO have more depolarized basal mitochondrial membrane potential (ΔΨ) than OP, and ΔΨ collapses within 15 min after onset of AMPA-GluR activation in almost all ImO, but not in the majority of OP. This ΔΨ collapse limits the capacity of ImO mitochondria to buffer the rise in intracellular Ca2+ caused by AMPA-GluR activation. The JS 3/16 line provides a valuable system for analysis of intracellular Ca2+ homeostasis and AMPA-GluR-mediated excitotoxicity in the oligodendroglial lineage. GLIA 31:165–180, 2000. © 2000 Wiley-Liss, Inc.

Diminished calcium homeostasis and increased susceptibility to excitotoxicity of JS 3/16 progenitor cells after differentiation to oligodendroglia

JS 3/16, derived from passaged oligodendroglial cultures prepared from rat cerebral white matter, differentiate from progenitors (OP) into complex process-bearing, galactocerebroside-positive but myelin basic protein-negative immature oligodendrocyte-like cells (ImO) after withdrawal of trophic factors. We found that JS 3/16 ImO are markedly more susceptible than OP to cell death after sustained alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor (AMPA-GluR) activation. This excitotoxicity is preceded by loss of intracellular Ca(2+) homeostasis, which is more marked in ImO than OP. We identified three factors likely to contribute to the diminished Ca(2+) homeostatic capacity of ImO. First, signal intensities of immunoreactive GluR2, GluR3, and GluR4 AMPA-GluR subunits are increased 1.3- to 2.2-fold in ImO over OP without comparable changes in RNA editing and alternative splicing. Second, transcriptional levels of genes encoding Na(+)-Ca(2+) exchanger proteins and a plasma membrane ATPase (PMCA1), which are necessary for Ca(2+) extrusion across the plasma membrane, are lower in ImO than in OP. Third, ImO have more depolarized basal mitochondrial membrane potential (Delta Psi) than OP, and Delta Psi collapses within 15 min after onset of AMPA-GluR activation in almost all ImO, but not in the majority of OP. This Delta Psi collapse limits the capacity of ImO mitochondria to buffer the rise in intracellular Ca(2+) caused by AMPA-GluR activation. The JS 3/16 line provides a valuable system for analysis of intracellular Ca(2+) homeostasis and AMPA-GluR-mediated excitotoxicity in the oligodendroglial lineage.

N-Cadherin Influences Migration of Oligodendrocytes on Astrocyte Monolayers

Oligodendrocyte cell migration is required for the development of the nervous system and the repopulation of demyelinated lesions in the adult central nervous system. We have investigated the role of the calcium-dependent adhesion molecules, the cadherins, in oligodendrocyte-astrocyte interaction and oligodendrocyte progenitor migration. Immunostaining demonstrated the expression of N-cadherin on the surfaces of both oligodendrocytes and astrocytes, and oligodendrocyte-like cells adhered to and spread on N-cadherin substrates. The blocking of cadherin function by antisera or specific peptides reduced adhesion of oligodendroglia to astrocyte monolayers, diminished contact time between oligodendrocyte processes and individual astrocytes, and significantly increased the migration of oligodendrocyte-like cells on astrocyte monolayers. Furthermore, a soluble cadherin molecule without adhesive properties increased oligodendroglial proliferation on various extracellular matrix substrates. These data suggest that cadherins are at least partially responsible for the poor migration-promoting properties of astrocytes and that decreasing cell–cell adhesion might effect repopulation of demyelinated multiple sclerosis lesions by oligodendrocyte progenitors.

Acrania: an autopsy case and review of the literature

ABSTRACT We report an autopsy case of acrania with a review of the literature. A 31-year-old woman was admitted in poor genaral condition and diagnosed as pregnant at the 26th week of gestation. The fetus was suspected to be anencephalic or hydrocephalic by ultrasonography. She delivered a stillbirth male baby at the 28th week of gestation. Autopsy showed the cranium lacked the cranial vault The scalp directly covered the hydrocephalic brain without calvaria. No cerebellum was found macroscopically. On histological examination, the thinned cerebral layer was composed of white matter-like brain tissue with glial and ependymal cells. In the cerebral layer near the base of the skull, a few NSE-positive neurons among many GFAP-positive glial cells and oligodendrocyte-like cells were observed. The choroid plexuses were observed inside the ependymal layers. The brain stem and the spinal cord were normally preserved with clusters of NSE-positive neurons. Agnethia and horseshoe kidney were combined as malformations other than the brain. We reviewed 42 cases of acrania from the literature.

Cyclic GMP/cyclic GMP-dependent protein kinase system prevents excitotoxicity in an immortalized oligodendroglial cell line.

Previously, we have demonstrated that excitotoxicity of oligodendrocyte-like cells (OLC), differentiated from immortalized rat O-2A progenitor cells (CG-4 cells), is prevented by cyclic AMP-elevating agents. We now report that some agents that elevate cyclic GMP prevent OLC excitotoxicity. Kainate-induced injury was prevented by cyclic GMP analogues (8-bromo-cyclic GMP and dibutyryl cyclic GMP), a guanylate cyclase activator [atrial natriuretic peptide (ANP)], and phosphodiesterase inhibitors [3-isobutyl-1-methylxanthine (IBMX), ibudilast, propentofylline, and rolipram]. When both forskolin and 8-bromo-cyclic GMP were added, kainate-induced injury was additively prevented. There was a strong positive correlation between suppression of kainate-induced Ca2+ influx and prevention of injury by these chemicals. The measurement of intracellular cyclic AMP and cyclic GMP by radioimmunoassay demonstrated the following: an increase of cyclic GMP with treatment with 8-bromo-cyclic GMP, dibutyryl cyclic GMP, and ANP; an increase of cyclic AMP with treatment with ibudilast and rolipram; and an increase of both cyclic AMP and cyclic GMP with treatment with IBMX and propentofylline. Kainate-induced Ca2+ influx was decreased by 8-(4-chlorophenylthiol)-guanosine-3',5'-monophosphate, an activator of cyclic GMP-dependent protein kinase (PKG), or okadaic acid, an inhibitor of protein phosphatases 1 and 2A. RT-PCR and westem blotting of OLC demonstrated transcription of PKG II gene and translation of PKG Ibeta mRNA, but no translation of PKG Ialpha mRNA. Therefore, we concluded that the cyclic GMP/PKG system prevents OLC excitotoxicity.

Bcl-2 immunoreactive cells with immature neuronal phenotype exist in the nonepileptic adult human brain.

Bcl-2, a cell death suppressor protein, is expressed during brain development but is largely down-regulated in the adult central nervous system. We previously reported strong expression of bcl-2 in small, "oligodendrocyte-like" cells (OLC) found in glioneuronal hamartias. These hamartias are microscopic cell rests found in temporal lobe resections from patients with intractable epilepsy and are considered a form of cerebral microdysgenesis. However, a causative relationship between these rests and seizures is not clear. We now report the identification, lineage characterization, and postnatal ontogeny of hamartia-like cell rests in temporal lobes of nonepileptic humans. Postmortem temporal lobes from 28 patients without history of neurologic disease (mean age = 53 years; range = 20 to 83 years) were studied. Microscopic cellular aggregates containing immature-appearing, bcl-2-immunoreactive cells (BIC) (identical to OLC) were observed in 23 of 28 (82%) temporal lobes from nonepileptic individuals. BIC were strongly immunoreactive for neuronal-specific class III beta tubulin, neuronal nuclear antigen, and MAP-2, but were consistently negative for neurofilament proteins and Ki67. Such cells were localized to subventricular regions of the caudal amygdala and often extended into the adjacent subcortical white matter and periamygdaloid cortex. BIC became less abundant with advancing age. These findings suggest that hamartia-like rests containing immature postmitotic neurons are normally present in the human brain and that glioneuronal hamartias may not always represent a maldevelopmental lesion associated with epilepsy.

Non- N-methyl- d-aspartate glutamate receptors mediate oxygen–glucose deprivation-induced oligodendroglial injury

Cells of oligodendroglial lineage are susceptible to oxygen and glucose deprivation. When oligodendrocyte-like cells differentiated from CG-4-immortalized rat O-2A progenitor cells were exposed to hypoxia alone or glucose deprivation alone for 48 h, release of lactate dehydrogenase (LDH) into the culture medium did not increase. However, when cells were deprived of both oxygen and glucose for 6 or 12 h preceding reoxygenation for 2 h, LDH release increased. Adding glucose to the medium protected against cell death and increased lactate production in a concentration-dependent manner. Cell damage induced by deprivation of oxygen and glucose was prevented by calcium-free medium or by non- N-methyl- d-aspartate glutamate receptor (GluR) antagonists, such as 6-cyano-7-nitroquinoxaline-2,3-dione or LY293558, but not by the voltage-dependent calcium channel blocker, nimodipine, or by the N-methyl- d-aspartate GluR antagonist, MK-801. The glutamate concentration in the medium from cells exposed to oxygen–glucose deprivation for 12 h was 49.70±3.04 μM/l, which is sufficient to activate GluRs during deprivation of oxygen and glucose. Apoptotic cells detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) or Hoechst 33258 staining did not increase in cells exposed to oxygen–glucose deprivation for 12 h and subsequent reoxygenation for 2 h. No DNA laddering was detected by agarose gel electrophoresis from cells exposed to deprivation of oxygen and glucose. Neither acetyl-YVAD-CHO, an inhibitor of caspase-1-like proteases, nor acetyl-DEVD-CHO, an inhibitor of caspase-3-like proteases, prevented oxygen–glucose deprivation-induced injury. Thus, oxygen and glucose deprivation causes calcium-influx-induced necrotic cell damage in cells of oligodendroglial lineage via non- N-methyl- d-aspartate GluR channels.

Upregulated expression of Fas and Fas ligand in brain through the spectrum of HIV-1 infection

Apoptosis of neurons and glial cells has been shown to occur in the brain of patients with the acquired immune deficiency syndrome (AIDS) and was postulated as contributing to brain atrophy and white matter damage in these patients. Since apoptotic events may be induced by the Fas-Fas ligand (FasL) system, we analyzed the relevance of these molecules to cell depletion in eight brains from HIV-1-infected patients and nine HIV-1-negative controls all of whom were analyzed histopathologically. The presence of Fas and FasL in brain tissue was analyzed by PCR amplification using Fas- and FasL-specific oligonucleotide primers and immunohistochemistry. The visualization of DNA fragmentation was used to evaluate apoptosis. Fas transcripts were detected in brains from each of four AIDS patients, each of three asymptomatic HIV-1 carriers and each of two HIV-1-negative controls. In the brains from AIDS patients the level of Fas expression was higher than in asymptomatic carriers and uninfected controls. FasL transcripts were seen in three of seven HIV-1-infected brains, two AIDS cases and one asymptomatic HIV-1 carrier. The predominant Fas-expressing cells were reactive astrocytes seen in each of two AIDS patients and one pre-AIDS case, but not in HIV-1-negative controls. Occasional Fas-positive oligodendrocyte-like cells were also seen in AIDS and pre-AIDS cases. No significant expression of Fas and FasL was seen in neurons. Fas-positive reactive astrocytes were more frequent in foci of HIV-1 encephalitis (HIVE). In the same area reactive apoptotic astrocytes were seen in close vicinity to FasL-expressing CD3 T lymphocytes, suggesting that apoptosis of astrocytes is mediated by Fas-FasL. The Fas expression on glial cells in asymptomatic HIV-1 infection may indicate apoptosis already in the asymptomatic stage of HIV-1 disease. In AIDS brains expression of Fas and FasL may contribute to the loss of glial cells and indirectly to the loss of neurons.

Expression of ferritin protein and subunit mRNAs in normal and iron deficient rat brain

In non-neuronal tissue, ferritin subunit mRNAs are regulated by post-transcriptional mechanisms leading to decreased ferritin protein synthesis during iron deficiency. Biochemical studies have demonstrated that the cerebral ferritin concentration declines during iron deficiency, suggesting that expression of ferritin subunit mRNAs in the brain may be regulated by mechanisms similar to those of non-neuronal tissue. However, as ferritin expression has been only vaguely studied in brain, this hypothesis remains to be tested. We investigated the influence of dietary iron deficiency on the cellular distribution of ferritin protein using immunohistochemistry and H- and L-ferritin subunit mRNAs by non-radioactive in situ hybridization. Pregnant rats were subjected to an iron depleted diet (6.4 mg/kg) from the day of conception. Litters were kept on the same diet until euthanized at the postnatal age of 10 weeks. This treatment reduced brain iron levels from approximately 57 to 26 μg/g. Reducing the iron stores reduced histochemical detectable iron and the expression of ferritin immunoreactivity in neurons, oligodendrocyte-like and microglia-like cells. In normal rats, H- and L-ferritin subunit mRNAs were expressed in virtually all neurons and non-neuronal cells. The cerebral expression of the ferritin subunit mRNAs was not affected by iron deficiency. The levels of ferritin subunit mRNAs in the brain were also unaltered from iron deficiency when examined by Northern blotting. In conclusion, brain levels of iron and ferritin protein are highly susceptible to dietary iron deficiency, whereas the cerebral expression of H- and L-ferritin subunit mRNAs remains unchanged.

Cyclic AMP-elevating agents prevent oligodendroglial excitotoxicity.

Previously, we have demonstrated that cells of the oligodendroglial lineage express non-NMDA glutamate receptor genes and are damaged by kainate-induced Ca2+ influx via non-NMDA glutamate receptor channels, representing oligodendroglial excitotoxicity. We find in the present study that agents that elevate intracellular cyclic AMP prevent oligodendroglial excitotoxicity. After oligodendrocyte-like cells, differentiated from the CG-4 cell line established from rat oligodendrocyte type-2 astrocyte progenitor cells, were exposed to 2 mM kainate for 24 h, cell death was evaluated by measuring activity of lactate dehydrogenase released into the culture medium. Released lactate dehydrogenase increased about threefold when exposed to 2 mM kainate. Kainate-induced cell death was prevented by one of the following agents: adenylate cyclase activator (forskolin), cyclic AMP analogues (dibutyryl cyclic AMP and 8-bromo-cyclic AMP), and cyclic AMP phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine, pentoxifylline, propentofylline, and ibudilast). Simultaneous addition of both forskolin and phosphodiesterase inhibitors prevented the kainate-induced cell death in an additive manner. A remarkable increase in Ca2+ influx (approximately 5.5-fold) also was induced by kainate. The cyclic AMP-elevating agents caused a partial suppression of the kainate-induced increase in Ca2+ influx, leading to a less prominent response of intracellular Ca2+ concentration to kainate. The suppressing effect of forskolin on the kainate-induced Ca2+ influx was partially reversed by H-89, an inhibitor of cyclic AMP-dependent protein kinase. In contrast to this, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, brought about a decrease in the kainate-induced Ca2+ influx. We therefore concluded that cyclic AMP-elevating agents prevented oligodendroglial excitotoxicity by cyclic AMP-dependent protein kinase-dependent protein phosphorylation, resulting in decreased kainate-induced Ca2+ influx.