Puma,but not noxa is essential for oligodendroglial cell death

The mechanisms involved in oligodendroglial cell death in human demyelinating diseases are only partly understood. Here, we demonstrate that the BH3 only protein Puma, but not Noxa, is essential for oligodendroglial cell death in toxic demyelination induced by the copper chelator cuprizone. Primary oligodendrocytes derived from Noxa‐ or Puma‐deficient mice showed comparable differentiation to wild‐type cells, but Puma‐deficient oligodendrocytes were less susceptible to spontaneous, staurosporine, or nitric oxide‐induced cell death. Furthermore, Puma was expressed in oligodendrocytes in multiple sclerosis (MS) lesions and Puma mRNA levels were upregulated in primary human oligodendrocytes upon cell death induction by staurosporine. Our data demonstrate that Puma is pivotal for oligodendroglial cell death induced by different cell death stimuli and might play a role in oligodendroglial cell death in MS. GLIA 2013;61:1712–1723     

Methamphetamine induces oligodendroglial cell death in vitro

We investigated whether the psychostimulant methamphetamine (METH) has a cytotoxic effect on oligodendrocytes and which cell-death pathways are involved in the cytotoxic process. METH caused concentration- and time-dependent cytotoxicity in rat oligodendrocyte cultures. METH induced apoptotic cell death and mRNA expression of pro-apoptotic proteins (bax and DP5), but not anti-apoptotic proteins (bcl-2 and bcl-XL). These results suggest that METH induces cytotoxicity in rat oligodendrocytes via the differential regulation of the expression of genes involved in the apoptotic process.     

XIAP protects oligodendrocytes against cell death in vitro but has no functional role in toxic demyelination

Oligodendroglial damage and loss are typical characteristics of demyelinating diseases such as multiple sclerosis (MS) and the leukodystrophies. Axonal loss is the underlying cause of permanent neurological deficits in MS and it is thought to arise from a combination of immune-mediated axonal damage and the loss of trophic support to axons from myelin sheaths after demyelination. Prevention of oligodendroglial damage or death and demyelination are therefore attractive neuroprotective treatment strategies. However, a better understanding of mechanisms leading to oligodendroglial damage and demyelination is a prerequisite for the development of such treatment options. Here, we demonstrate that X-linked inhibitor of apoptosis (XIAP), the most potent member of the inhibitor of apoptosis proteins (IAP) family is expressed in oligodendrocytes in vivo and in vitro. Increased expression of XIAP is associated with protection against selected cell death pathways, whereas decreased expression increases oligodendroglial cell death in vitro. However, lack of XIAP does not modulate oligodendroglial cell death in toxic demyelination in vivo.     

Amyloid-beta induced cell death is independent of free radicals

Acutely dissociated rat cerebellar granule cell neurons were incubated with amyloid-beta (1-42) and studied by flow cytometry. Amyloid-beta caused a dose-dependent loss of viability, as determined by intracellular accumulation of propidium iodide (PI),and that was not accompanied by significant elevation of intracellular calcium, measured by Fluo-3 or reactive oxygen species (ROS), measured by 2',7'-dihydro-dichlorfluorescein diacetate (DCF). Carnosine, a ROS scavenger and an inhibitor of non-enzymatic glycosylation, partially reduced cell death caused by amyloid-beta. We conclude that amyloid-beta causes a relatively acute loss of cell viability in cerebellar granule cell neurons, which does not result from either elevation of intracellular calcium concentration or generation of ROS.     

TGF-beta induces cell death in the oligodendroglial cell line OLI-neu

We have shown that TGF-beta plays an important role during the period of developmental cell death in the nervous system. Immunoneutralization of TGF-beta prevents ontogenetic neuron death in vivo. Like neurons, oligodendrocytes are generated in excess and eliminated by apoptosis. It has been shown that oligodendrocyte progenitors and newly formed oligodendrocytes are especially susceptible to apoptosis. We choose the oligodendrocyte precursor cell line OLI-neu to address the question if TGF-beta could play a role for the control of oligodendrocyte proliferation and cell death. Flow cytometric analysis revealed that OLI-neu cells arrested in the G1 phase of the cell cycle underwent apoptosis in response to TGF-beta. TUNEL assays, apoptosis ELISA, and caspase assays substantiated the finding that OLI-neu cells died after TGF-beta treatment. Cell death could be inhibited by application of pan-caspase or caspase 8 and 9 inhibitors, whereas the inhibition of calpain was unaffected. Furthermore, we found a reduction of bcl-X(L) at the protein as well as at the mRNA level, while p27 was upregulated. The Smad cascade was activated while TGF-beta reduced the activity of the p42/p44 MAP kinase pathway. Together, these data show that TGF-beta induced apoptotic cell death in cells of oligodendroglial origin, whereby the signaling cascade involved the downregulation of antiapoptotic signaling such as bcl-X(L) leading to the activation of caspases.     

Stem cell protein BMI-1 is an independent marker for poor prognosis in oligodendroglial tumours

Aims: The polycomb factor BMI‐1 has recently been implicated in tumorigenesis of the central nervous system in several experimental animal models. However, the significance of BMI‐1 in human glioma has not been investigated. Here we describe expression of the polycomb protein BMI‐1 and its downstream targets p16^Ink4a and MDM2 in both high‐ and low‐grade human glioma. Methods: Tumour samples were collected from 305 adult patients treated for primary grades 2–4 gliomas between 1980 and 2006 in Finland and Germany. BMI‐1, p16 and MDM2 expression was evaluated using immunohistochemistry in representative paraffin‐embedded tumour tissue. The significance of observed immunoreactivity, age at onset, gender, histopathological findings and proliferative index was analysed in univariate and multivariate survival models. Results: BMI‐1 was expressed in all histologic types of diffuse gliomas. We found a significant correlation (P = 0.007) between the frequency of BMI‐1 immunoreactive tumour cells and poor survival in World Health Organization grades II–III oligodendrogliomas and oligoastrocytomas (n = 62). The median survival of patients grouped by low, intermediate or high frequency of BMI‐1 immunoreactive tumour cells was 191 months, 151 months and 68 months, respectively. This association was also significant in the Cox multivariate regression model. Nuclear p16 immunopositivity predicted better survival in astrocytomas and an inverse correlation between p16 expression and the Ki‐67 mitotic index was also observed. Conclusions: BMI‐1 is found in all histological types of gliomas and the relative protein expression of BMI‐1 is a novel independent prognostic marker in oligodendroglial tumours.     

Apoptosis occurs in the oligodendroglial lineage,and is prevented by basic fibroblast growth factor

During the perinatal period, oligodendroglial precursor cells proliferate rapidly, then cease dividing and differentiate into oligodendroglia. Many of these newly formed oligodendroglia are destined to die. We now demonstrate that oligodendroglia generated in passaged cultures of rat forebrain oligodendroglial precursor cells after removal of basic fibroblast growth factor (basic FGF) from the medium often undergo internucleosomal DNA nicking and nuclear fragmentation, features characteristic of apoptosis. These alterations are rare in cultures maintained continuously in basic FGF. As in many other cellular lineages susceptible to apoptosis, these degenerative changes can be prevented by treatment with the endonuclease antagonist, aurintricarboxylic acid, or by inhibiting de novo RNA or protein synthesis. Supplementation of the basic FGF-free medium with insulin, insulin-like growth factor-1, platelet-derived growth factor, or ciliary neuronotrophic growth factor also diminishes DNA nicking. Both oligodendroglial differentiation and DNA nicking are induced in basic FGF-treated cultures by inhibiting DNA synthesis with aphidicholin or excess thymidine, thus suggesting a close linkage between the anti-apoptotic, anti-differentiation, and mitogenic effects of basic FGF on the oligodendroglial lineage. © 1995 Wiley-Liss, Inc.     

Serum-free induced neuronal apoptosis-like cell death is independent of caspase activity

Cultured cortical neurons survived in a density-dependent manner under serum-free conditions. Low-density cultured cells died in an aurintricarboxylic acid (ATA)-sensitive manner, which was accompanied with marked chromatin condensation and nuclear fragmentation. These features, characteristic for apoptosis, were not attenuated by DEVD-CHO, a caspase-3-specific inhibitor, or zVAD-FMK, a broad range caspase inhibitor, while zVAD-FMK showed a marked inhibition of camptothecin-induced cell death. Therefore, cortical neurons died in an apoptosis-like and a caspase-independent manner under serum-free conditions.     

Manganese-induced rat pheochromocytoma (PC12) cell death is independent of caspase activation

Manganese (Mn) is an essential mineral that at high concentrations can produce an irreversible syndrome resembling Parkinson's disease. To examine the mechanism by which Mn elicits its toxic response, we have selected the rat pheochromocytoma cells (PC12) as our model system because it possesses much of the biochemical machinery associated with dopaminergic neurons. Mn-induced PC12 cell death is both time and concentration dependent with approximately 50% cell survival at 48 hr in the presence of 0.3 mM Mn. To determine whether oxidative stress contributed to cytotoxicity induced by Mn, lipid peroxidation was assessed in Mn-treated in PC12 cells. The highly sensitive HPLC assay that measures the lipid peroxide product, 9-HODE, was used and results of these experiments demonstrate there was no increase in the lipid peroxidation in cells exposed to 0.3 mM Mn for 24 hr. Mn was found to stimulate the activation of the apoptotic marker proteins, p38 and caspase-3 within the first 24 hr of treatment. The selective inhibitor of caspase-3, DEVD-CHO, and the nonselective caspase inhibitor, Z-VAD-FMK, however, fail to prevent Mn-induced PC12 cell death. Studies were performed to determine the role of mitochondria in initiating or supporting Mn cytotoxicity, because Mn has been reported to cause changes in membrane permeability. Mn caused a decrease in ATP levels in PC12 cells in both a time and concentration dependent manner. We hypothesize that both apoptosis and necrosis contribute to PC12 cell death although the necrotic events prevail even when the apoptotic signaling is inhibited.     

p75NTR independent oligodendrocyte death in cuprizone-induced demyelination in C57BL/6 mice

Feeding C57Bl/6 J mice the copper chelator cuprizone leads to selective apoptosis of mature oligodendrocytes and concomitant demyelination predominantly in the corpus callosum. The process of oligodendrocyte apoptosis in this animal model for multiple sclerosis (MS) involves early microglial activation, but no infiltration of T-lymphocytes. Therefore, this model could mimic early stages of oligodendrocyte degeneration Affected oligodendrocytes express the common neurotrophin receptor, p75(NTR), a 'stress-receptor' which under certain circumstances can induce apoptosis. Only affected oligodendrocytes in MS lesions and MS animal models express this receptor. In order to study the significance of p75(NTR) in the fate of oligodendrocytes, we have exposed wild-type as well as p75(NTR)-knockout mice to a 0.2% (w/w) cuprizone diet and performed a comparative immunohistochemical analysis of the corpus callosum at various time points. Surprisingly, our results show that the absence of p75(NTR) did not alter cuprizone-induced oligodendrocyte death (and subsequent de- or remyelination). Apparently, intracellular apoptosis pathways in adult oligodendrocytes do not require p75(NTR) activated signal transduction in the absence of T-lymphocytes and T-lymphocyte derived cytokines.     

The link between excitotoxic oligodendroglial death and demyelinating diseases

Oligodendrocytes, the myelinating cells of CNS axons, are highly vulnerable to excitotoxic signals mediated by glutamate receptors of the AMPA and kainate classes. Receptors in these cells are commonly activated by glutamate that is released from axons and glial cells. In addition, oligodendrocytes contribute to the control of extracellular glutamate levels by means of their own transporters. However, acute and chronic alterations in glutamate homeostasis can result in overactivation of AMPA and kainate receptors and subsequent excitotoxic oligodendroglial death. Furthermore, demyelinating lesions caused by excitotoxins can be similar to those observed in multiple sclerosis. This, together with the effect of AMPA and kainate receptor antagonists in ameliorating the neurological score of animals with experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis), indicates that oligodendrocyte excitotoxicity could be involved in the pathogenesis of demyelinating disorders.     

Cytokine-induced cell death in human oligodendroglial cell lines: I. Synergistic effects of IFN-gamma and TNF-alpha on apoptosis

Multiple sclerosis is a chronic inflammatory disease of the central nervous system. Myelin and oligodendrocytes are considered the major targets of injury caused by a cell-mediated immune response. There is circumstantial evidence that proinflammatory cytokines like tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) could have disease-promoting roles in multiple sclerosis (MS). In the present study, the cytotoxic effects of IFN-gamma and TNF-alpha on the human oligodendroglial cell lines human oligodendroglioma (HOG) and MO3.13 were analyzed. When the oligodendroglial cell lines were cultured in the presence of IFN-gamma or TNF-alpha, apoptotic cell death was observed in both cell lines after >24 hr incubation. Apoptosis was evidenced by a decrease in cell viability, apoptotic changes in cell and nucleus morphology, and disruption of the membrane asymmetry. Our data show that TNF-alpha and IFN-gamma induce apoptosis in a dose-dependent fashion in both oligodendroglial cell lines and that their synergistic effect results in enhanced cell death. Understanding the regulation of cell death pathways in oligodendrocytes is critical for protecting myelin-producing cells and their associated axons during injury in patients with MS.     

Synthetic neuromelanin is toxic to dopaminergic cell cultures

Summary. In the present study, primary cultures of mesencephalic dopaminergic cells were exposed to synthetic dopamine neuromelanin (NM) for 48 hrs at concentrations of 0, 1, 10, 20, 50 and 100 μg NM/ml medium. Differently prepared synthetic NM with or without incorporated iron and NM oxidatively damaged by hydrogen peroxide were used. All NMs affected cellular structures e.g. as swelling of neural processes, rounding of cells, and occasional inclusion of neuromelanin particles. Cell numbers were uniformly and dose dependently reduced. Exposure to MPP⁺ and ferric iron led to cytotoxic changes which could be further aggravated by oxidatively damaged NM, suggesting cytotoxicity of soluble compounds of NM in predamaged neurons. Received February 4, 2002; accepted February 27, 2002     

Cyclic AMP differentiation of the oligodendroglial cell line N20.1 switches staurosporine-induced cell death from necrosis to apoptosis.

Understanding the regulation of cell death pathways is critical for protecting myelin-producing cells and their associated axons during injury resulting from multiple sclerosis and other degenerative diseases. The immortalized N20.1 oligodendroglial cell line provides a useful model for identifying mechanisms that can be exploited to attenuate cell death in myelin-producing cells and their precursors. In our hands, the N20.1 cell line exhibits different characteristics and morphology depending on temperature (permissive or non-permissive) and the presence of cAMP-elevating agents (Studzinski et al. [1998] Neurochem. Res. 23:435-441; Boullerne et al. [1999] J. Neurochem. 72:1050-1060; Studzinski et al. [1999] J. Neurosci. Res. 57:633-642). Our laboratory previously observed that NO donors cause primarily necrotic death in N20.1 cells grown at permissive temperature, but the NO donor SNP switched a portion of cell death to the apoptic pathway. We have continued our study of apoptotic death in these cells by comparing the effects of staurosporine, a known apoptotic agent, on cells grown at the permissive temperature ("undifferentiated") vs. the non-permissive temperature in the presence of forskolin ("differentiated"). Undifferentiated N20.1 cells exhibit maximal cell death after 24 hr of exposure to 50 nM staurosporine, whereas differentiated cells show delayed cell death, with maximal death seen after 48 hr. Pyknotic nuclei were observed in both growth conditions; however, differentiated cells were protected by caspase inhibitors, whereas undifferentiated cells were not. Increased ssDNA staining and DNA laddering were found following 24-hr staurosporine treatment in the differentiated cells only. These results support the conclusion that N20.1 cells can switch from necrotic to apoptotic cell death when cell division is slowed and cyclic AMP is elevated.     

Inflammatory demyelination and neurodegeneration in early multiple sclerosis

A number of recent magnetic resonance imaging studies have challenged the classical view of multiple sclerosis (MS) as a "two-stage" disease where an early inflammatory demyelinating phase with focal macroscopic lesions formed in the white matter (WM) of the central nervous system is followed by a late neurodegenerative phase, which is believed to be a mere consequence of repeated inflammatory insults and irreversible demyelination. These studies have consistently shown the presence of diffuse normal-appearing WM damage, marked gray matter involvement and significant cortical functional reorganization, as well as the occurrence of the neurodegenerative component of MS from the earliest clinical stages of the disease with only a partial relation to MRI markers of inflammatory demyelination. The present review argues that MS can no longer be viewed as a "two-stage" disease, which suggests that the two pathological components are dissociated in time, but rather as a "simultaneous two-component" disease, where the relative contributions of the various pathological processes of the disease to the development of "fixed" disability, their relationship and their evolution over time need to be clarified. This new view of MS should inform the development of future research protocols to define its actual physiopathology and prompt the institution of early treatment which should ideally target not only inflammatory demyelination, but also the neurodegenerative aspects of the disease, as well as promote neuroprotection and enhance reparative mechanisms and adaptive functional reorganization of the cortex.     

Cytokine-induced cell death in human oligodendroglial cell lines. II: Alterations in gene expression induced by interferon-gamma and tumor necrosis factor-alpha

Cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), can initiate dual effects resulting in either cell growth or cell death. In this study, the human oligodendroglial cell lines HOG and MO3.13 were used as a model to study the molecular mechanisms of cytokine-induced cell death in human oligodendrocytes. We have previously shown that TNF-alpha and IFN-gamma induce apoptosis in both oligodendroglial cell lines within 72 hr. In the present study, the cell death pathways operating within these cells were further investigated at the gene expression level. Both cell lines express a broad repertoire of caspases and apoptosis-related genes. Some of these genes are specifically up-regulated by cytokine treatment; e.g., caspase-1 is up-regulated by IFN-gamma. In addition to direct cytotoxic effects, IFN-gamma and TNF-alpha also enhance the expression of Fas, TNFR1, and MHC class I molecules in both cell lines. This suggests that cytokines can make oligodendrocytes more vulnerable to different cell death pathways in an inflammatory environment. cDNA microarray analysis of the HOG cell line revealed that TNF-alpha induces genes that regulate apoptosis, survival, inflammation, cell metabolism, and cell signaling. The data suggest that oligodendroglial cells activate both death and survival pathways upon cytokine challenges. However, the survival pathways seem to be unable to compete with the death signal after more than 24 hr of cytokine treatment. These results may contribute to the development of therapeutic strategies aimed at interfering with cytokine-induced cell death of oligodendrocytes in patients with multiple sclerosis.     

Astrogliopathy and oligodendrogliopathy are early events in CNS demyelination

We examined the phenotypic composition of cells and the underlying mechanisms of demyelination following injection of lipopolysaccharide (LPS) into the corpus callosum of rats. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed fragmented DNA, which co‐localized with oligodendrocytes in areas of demyelination following intracerebral injection with LPS. Immunostaining showed the presence of caspase 3 in cells which expressed the oligodendrocyte markers, suggesting activation of the apoptotic pathway. Commensurate reduction in glial fibrillary acid protein (GFAP)+/ gap junction protein connexin43+ (Cx43) cells, was also seen in the corpus callosum prior to histochemical evidence of demyelination. Expression of mRNA for proinflammatory cytokines was maximal 3 day postinjection, at a time when the numbers of TUNEL positive cells in the corpus callosum were declining and the total number of CD68+ cells peaked at day 14 postinjection. Our studies suggest that death of oligodendrocytes is an early event in LPS model of demyelination. We believe that the innate immune model of oligodendrocyte death will be useful in the development of neuroprotective agents capable of rescuing oligodendrocytes from apoptosis. GLIA 2013;61:1261–1273