Molecular mechanisms of enhanced susceptibility to apoptosis in differentiating oligodendrocytes.

Several studies have shown that the progression of oligodendrocyte progenitors along the lineage correlates with increased susceptibility to death stimuli. The molecular basis of this phenomenon remains unclear. This study demonstrates that the protein levels of several proapoptotic molecules, including Bax, Bad (nonphosphorylated form), and certain caspase proforms, increase during oligodendrocyte development. In contrast, the steady-state levels of antiapoptotic molecules, such as Bcl2 and Bcl(XL), remain constant. This altered equilibrium between proapoptotic and antiapoptotic molecules correlates with increased cytochrome C in the cytosol. We conclude that, as oligodendrocytes mature, their susceptibility to apoptosis increases because of a change in the balance between protective mechanisms and proapoptotic pathways. This suggests the possible existence of a death susceptibility program, which is intrinsic to differentiating oligodendrocyte progenitors.

p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal.

Differentiation of most cell types requires both establishment of G1 arrest and the induction of a program related to achieving quiescence. We have chosen to study the differentiation of oligodendrocyte cells to determine the role of p27 and p21 in this process. Here we report that both p27 and p21 are required for the appropriate differentiation of these cells. p27 is required for proper withdrawal from the cell cycle, p21 is not. Instead, p21 is required for the establishment of the differentiation program following growth arrest. Similar observations were made in vivo. We show that p21-/- cells withdraw from the cell cycle similar to wild-type cells; however, early in animal life, the brain is hypomyelinated, inferring that the loss of p21 delayed myelination in the cerebellum. We found that we could complement or bypass the differentiation failure in p21-/- cells with either PD98059, an inhibitor of Mek1, or by transducing them with a tat-p16ink4a protein. We concluded that the two cdk inhibitors serve non-redundant roles in this program of differentiation, with p27 being responsible for arrest and p21 having a function in differentiation independent of its ability to control exit from the cell cycle.

Long-term maintenance of mature hippocampal slices in vitro.

Cultures of primary neurons or thin brain slices are typically prepared from immature animals. We introduce a method to prepare hippocampal slice cultures from mature rats aged 20-30 days. Mature slice cultures retain hippocampal cytoarchitecture and synaptic connections up to 3 months in vitro. Spontaneous epileptiform activity is rarely observed suggesting long-term retention of normal neuronal excitability and of excitatory and inhibitory synaptic networks. Picrotoxin, a GABAergic Cl(-) channel antagonist, induced characteristic interictal-like bursts that originated in the CA3 region, but not in the CA1 region. These data suggest that mature slice cultures displayed long-term retention of GABAergic inhibitory synapses that effectively suppressed synchronized burst activity via recurrent excitatory synapses of CA3 pyramidal cells. Mature slice cultures lack the reactive synaptogenesis, spontaneous epileptiform activity, and short life span that limit the use of slice cultures isolated from immature rats. Mature slice cultures are anticipated to be a useful addition for the in vitro study of normal and pathological hippocampal function.

Nerve growth factor activation of nuclear factor kappaB through its p75 receptor is an anti-apoptotic signal in RN22 schwannoma cells.

Recent evidence indicates that nerve growth factor (NGF) produces its effects through signaling contributions from both TrkA and the p75 receptor. In contrast to its trophic actions through TrkA, NGF binding to p75 has been shown to activate programmed cell death through a mechanism involving the stress kinase JNK. However, this receptor also activates nuclear factor kappaB (NF-kappaB), the role of which has yet to be determined. We investigated the function of p75-mediated NF-kappaB stimulation in regulating cell survival in the rat schwannoma cell line RN22, which expresses p75, but not TrkA. Gel shift assays demonstrated activation of NF-kappaB in response to NGF within 30 min and lasting at least 4 h. NGF also stimulated JNK in the cells (detected by in vitro kinase assays) with a similar time course. Preventing activation of NF-kappaB with the specific inhibitor SN50 resulted in NGF-induced cell loss. Similarly, transfection of the cells with a mutant form of the endogenous NF-kappaB inhibitor (IkappaBalphaDeltaN), which cannot be degraded and therefore remains bound to NF-kappaB, preventing its activation, resulted in a significant increase in the number of apoptotic cells following NGF treatment. These results suggest that NGF activation of NF-kappaB through the p75 receptor promotes survival, counterbalancing the pro-apoptotic signal.

Cell death in the oligodendrocyte lineage: a molecular perspective of life/death decisions in development and disease.

Cell death in the oligodendrocyte lineage occurs during development and in pathological conditions as the result of a balance between opposing molecular signals. This review focuses on the molecular mechanisms of activation of signal transduction pathways affecting life/death decisions in progenitor cells and in mature oligodendrocytes. Loss of trophic support, cytokine receptor activation, and oxidative stress may differentially contribute to the induction of cell death at specific stages of development and to the pathogenesis of demyelinating disorders. The execution of the death program leading to the morphological changes of apoptosis and/or necrosis is then determined by the generation of reactive oxygen species and the level of impairment of mitochondrial function. The final decision of a cell to die or survive is determined by a competition between survival and death signals. Depending on ligand availability, type, and levels of receptor expression and downstream cross-talks between distinct signaling pathways, the cell may activate a death execution program that will be affected by its stage of differentiation and its energetic metabolism.

Up-regulated p75NTR neurotrophin receptor on glial cells in MS plaques.

OBJECTIVE: To investigate the expression of the neurotrophin receptor p75NTR on glial cells within MS plaques. BACKGROUND: In recent studies on the pathogenesis of MS white matter plaques, we found large populations of inflammatory and resident glial cells, including oligodendrocytes undergoing cell death, and identified increased expression of Fas receptor and ligand death pathway signaling molecules on the same glial cell types. In another study, the p75NTR was shown to induce apoptotic death of maturing oligodendrocytes when exposed to NGF in vitro. METHODS: We used immunohistochemistry and in situ reverse-transcription PCR to detect p75NTR expression on inflammatory and resident glial cells in MS plaques and used TUNEL staining for fragmented DNA to detect cell death. RESULTS: Up-regulated p75NTR messenger RNA and protein were demonstrated in both oligodendrocytes and microglia/macrophages in MS plaques but not in control white matter. However, only a fraction of p75NTR expressing oligodendrocytes was also stained by TUNEL. CONCLUSIONS: Glial cell expression of p75NTR receptor is up-regulated during MS plaque formation. The exact role of this receptor in glial cell death and/or survival in MS remains to be elucidated.

Loss of p27Kip1 function results in increased proliferative capacity of oligodendrocyte progenitors but unaltered timing of differentiation.

In many tissues, progenitor cells permanently withdraw from the cell cycle prior to commitment towards a differentiated phenotype. In the oligodendrocyte lineage a counting mechanism has been proposed, linking the number of cell divisions to growth arrest and differentiation. A direct prediction of this model is that an increase in the number of cell divisions would result in a delayed onset of differentiation. Since the cell cycle inhibitor p27Kip1 is an essential component of the machinery leading to oligodendrocyte progenitor growth arrest, we examined the temporal relationship between cell cycle withdrawal and expression of late differentiation markers in vivo, in mice carrying a targeted deletion in the p27Kip1 gene. Using bromodeoxyuridine to label proliferating cells, quaking (QKI) to identify embryonic glial progenitors, NG2 to identify neonatal oligodendrocyte progenitors, and myelin basic protein to label differentiated oligodendrocytes, we found an increased number of proliferating QKI- and NG2-positive cells in germinal zones of p27Kip1(-/-) mice at the peak of gliogenesis. However, no delay was observed in these mice in the appearance of the late differentiation marker myelin basic protein in the developing corpus callosum and cerebellum. Significantly, a decrease in cyclin E levels was observed in the brain of p27Kip1 null mice coincident with oligodendrocyte growth arrest. We conclude that two distinct modalities of growth arrest occur in the oligodendrocyte lineage: a p27Kip1-dependent mechanism of growth arrest affecting proliferation in early phases of gliogenesis, and a p27Kip1-independent event leading to withdrawal from the cell cycle and differentiation.

p75 neurotrophin receptor as a modulator of survival and death decisions.

The p75 receptor is the founding member of the TNF receptor superfamily. Members in this receptor family share a common cysteine motif repeated two to six times that serves as the ligand binding domain. In addition, several members contain a cytoplasmic region designated the death domain. The neurotrophins NGF, BDNF, NT-3, and NT-4 each bind to the p75 receptor and also more selectively to members of the Trk family of receptor tyrosine kinases. Although the biological functions of p75 have been elusive, recent experimental evidence supports an involvement of this receptor in apoptosis. This presents a counter-intuitive function for neurotrophins, which are normally required for the survival of neurons during development. The life-and-death decisions by neurotrophins appear to be governed by the level of expression and signaling activities of the p75 and Trk tyrosine kinase receptors and their downstream effector molecules. The generation of the correct number of cells in the nervous system is a highly controlled and coordinated process that is the consequence of cell proliferation and cell death decisions. The appropriate number of neuronal and glial cells formed during development guarantees the establishment of proper innervation and functional synaptic connections. One common mechanism to account for the number of viable cells is the ability to form ligand-receptor complexes that promote cell survival under conditions of limiting concentrations of trophic factors. Another diametrically opposed mechanism is to produce ligand-receptor interactions that can activate programmed cell death directly.

Oligodendrocyte apoptosis mediated by caspase activation.

Treatment with NGF causes long-term cultures of oligodendrocytes to die via a yet undefined mechanism mediated by the p75 neurotrophin receptor. The p75 receptor belongs to the TNF receptor superfamily of molecules, which includes Fas and p55 TNF receptors. The Fas and TNF receptors use adaptor molecules to recruit and activate caspase-8 to the receptor. Using a combination of immunohistochemical and Western blotting assays, we have examined caspase activity during NGF-induced apoptosis. Interestingly, although caspase-1 [interleukin-1beta-converting enzyme (ICE)], caspase-2, caspase-3, and caspase-8 were expressed in oligodendrocytes, only caspase-1, -2, and -3 were activated after NGF treatment, whereas caspase-8 was not. These data suggest that the mechanism of apoptosis by NGF through the p75 receptor is different from TNF and Fas-mediated killing. gamma Radiation of oligodendrocytes also activated a similar subset of caspases as NGF, indicating that NGF-induced oligodendrocyte apoptosis uses a similar cell death execution mechanism as injury models. This consolidates a potential role of the p75 neurotrophin receptor during stress and inflammatory conditions.

Neurotrophins in cell survival/death decisions.

Neurotrophins are target-derived soluble factors required for neuronal survival. Nerve growth factor (NGF) the founding member of the neurotrophin family, binds to two types of receptors: Trk tyrosine kinase and the p75 neurotrophin receptor, which belongs to the Fas-tumor necrosis factor (TNF) receptor superfamily. Binding of neurotrophins to Trk receptor tyrosine kinases initiate signaling cascades that promote cell survival sand differentiation. In contrast, p75 NGFR has been shown to modulate the susceptibility to death of selective cellular populations--including differentiated rat oligodendrocytes--in specific conditions. Notably, NGF effect on viability was only observed in fully differentiated oligodendrocytes and not in oligodendrocyte progenitor cells. The effect of p75 activation on oligodendrocyte survival correlates with increased activity of the stress related kinase JNK-1 and cleavage of specific caspases. Indeed, activation of additional stress pathways or impairment of survival signals may be required for p75 mediated activation of cell death execution programs. Interestingly, co-expression of the TrkA receptor in the same cell type abolishes the JNK-1 mediated death signal and induces MAP kinase activity, resulting in cell survival. This suggests that glial cell survival results from a balance between positive and negative regulators modulated by selective signalling pathways by tyrosine kinases and cytokine receptors.

Two neuronal cell lines expressing the myelin basic protein gene display differences in their in vitro survival and in their response to glia.

We have generated two conditionally immortalized neuronal cell lines from primary cultures of embryonic day 13 (E13) and postmitotic (postnatal day 0; P0) cortical neurons transformed with the temperature-sensitive SV-40 large-T antigen. Two clonal cell lines (CN1.4 from E13 cultures and SJ3.6 from P0 cultures) were isolated and stable maintained in vitro. Both cell lines expressed a number of neuronal markers such as the neurofilaments, glutamic acid decarboxylase 67, neuron-specific enolase, and the BG21 isoform of the myelin basic protein gene. At 34 degrees C, the CN1.4 cell line had elaborated short processes, whereas the SJ3.6 cell line produced long processes that formed a delicate network. When these cell lines were cultured at 39 degrees C, some of the cellular processes grew longer, adopting a more mature neuronal morphology. Interestingly, at 39 degrees C, the in vitro survival of these cell lines differed significantly. Whereas the survival of CN1.4 cell line was greatly unaffected, SJ3.6 cells died soon after they were cultured at 39 degrees C. The cell death of SJ3.6 cells was accompanied by fragmentation and condensation of DNA in their nuclei, indicative of an apoptotic event. Under these conditions, SJ3.6 showed an upregulation of the p75 receptor. When this cell line was cocultured with oligodendrocytes, astrocytes, or glial conditioned media (GCM), there was a marked increase in survival. In contrast, little effect of glial cells or GCM was observed on the CN1.4 cell line. These lines appear to be useful models to study neuronal-glial interactions in addition to neuronal cell death and the effects of glial factors that promote the survival of neurons.

Ectopic expression of p27Kip1 in oligodendrocyte progenitor cells results in cell-cycle growth arrest.

Oligodendrocyte differentiation is a complex process believed to be controlled by an intrinsic mechanism associated with cell-cycle arrest. Recently, the cell-cycle inhibitor protein p27 Kip1 has been proposed as a key element in causing growth arrest of oligodendrocyte precursor cells. To investigate the effects of p27 upon oligodendrocyte cell development, we have introduced the p27 cDNA in oligodendrocyte progenitor cells using an adenovirus vector. Progenitor cells normally express low levels of p27. After adenoviral infection and p27 overexpression, progenitor cells were able to undergo cell-cycle arrest, even in the presence of strong mitogens. The effects of p27 were shown to be directly upon cyclin-dependent kinase-2 (CDK2), the protein kinase complex responsible for G1/S transition, as immunodepletion of oligodendrocyte extracts of p27 protein resulted in the activation of CDK2 activity. However, cells that became growth arrested owing to infection with p27 adenovirus did not display conventional oligodendrocyte differentiation markers, such as O4 or O1. Taken together, these data provide mechanistic evidence indicating that p27 is primarily involved in oligodendroglial progenitor proliferation by inhibiting CDK2 activity and inducing oligodendrocyte cell-cycle arrest.

Neurotrophin receptors: mediators of life and death.

The mechanism of action of NGF has continued to provide a challenging and formidable problem in signal transduction. NGF can bind independently to two different receptors, the trkA tyrosine kinase receptor and the p75 neurotrophin receptor, which are involved in many different signaling events. In addition to promoting cell differentiation survival, NGF can paradoxically be an inducer of cell death. Several receptor mediated mechanisms are proposed to explain how NGF might act as a trophic factor and as a cell killer. The survival and cell death properties of the receptors are dependent upon the relative ratio of receptors and the persistent nature of the signaling events.

Competitive signaling between TrkA and p75 nerve growth factor receptors determines cell survival.

In addition to its role as a survival factor, nerve growth factor (NGF) has been implicated in initiating apoptosis in restricted cell types both during development and after terminal cell differentiation. NGF binds to the TrkA tyrosine kinase and the p75 neurotrophin receptor, a member of the tumor necrosis factor cytokine family. To understand the mechanisms underlying survival versus death decisions, the TrkA receptor was introduced into oligodendrocyte cell cultures that undergo apoptosis in a p75-dependent manner. Here we report that activation of the TrkA NGF receptor in oligodendrocytes negates cell death by the p75 receptor. TrkA-mediated rescue from apoptosis correlated with mitogen-activated protein kinase activation. Concurrently, activation of TrkA in oligodendrocytes resulted in suppression of c-jun kinase activity initiated by p75, whereas induction of NFkappaB activity by p75 was unaffected. These results indicate that TrkA-mediated rescue involves not only activation of survival signals but also simultaneous suppression of a death signal by p75. The selective interplay between tyrosine kinase and cytokine receptors provides a novel mechanism that achieves alternative cellular responses by merging signals from different ligand-receptor systems.

Neurotrophins: the biological paradox of survival factors eliciting apoptosis.

Neurotrophins are target-derived soluble polypeptides required for neuronal survival. Binding of neurotrophins to Trk receptor tyrosine kinases initiate signaling cascades that promote cell survival and differentiation. All family members bind to another receptor (p75NTR), which belongs to the tumor necrosis factor superfamily. Hence, nerve growth factor (NGF) and related trophic factors are unique in that two separate receptor types are utilized. Although the biological function of p75NTR has been elusive, it has been suggested to mediate apoptosis of developing neurons in the absence of Trk receptors. This presents a tantalizing paradigm, in which life-death decisions of cells are dependent upon the expression and action of two different receptors with distinctive signaling mechanisms. In the presence of TrkA receptors, p75 can participate in the formation of high affinity binding sites and enhanced NGF responsiveness leading to a survival signal. In the absence of TrkA receptors, p75 can generate, in only specific cell populations, a death signal. Here we discuss the unique features and implications of this unusual signal transduction system.

Oligodendrocyte precursor differentiation is perturbed in the absence of the cyclin-dependent kinase inhibitor p27Kip1.

During development of the central nervous system, oligodendrocyte progenitor cells (O-2A) undergo an orderly pattern of cell proliferation and differentiation, culminating in the ability of oligodendrocytes to myelinate axons. Here we report that p27(Kip1), a cyclin-dependent kinase inhibitor, is an important component of the decision of O-2A cells to withdraw from the cell cycle. In vitro, accumulation of p27 correlates with differentiation of oligodendrocytes. Furthermore, only a fraction of O-2A cells derived from p27-knockout mice differentiate successfully compared to controls. Inability to differentiate correlates with continued proliferation, suggesting that p27 is an important component of the machinery required for the G1/G0 transition in O-2A cells. In vivo, expansion of O-2A precursors before withdrawal, in part, leads to a greater number of oligodendrocytes. Together these data indicate a role for p27 during the decision to withdraw from the cell cycle in the oligodendrocyte lineage.

Changes in cyclin-dependent kinase 2 and p27kip1 accompany glial cell differentiation of central glia-4 cells.

The generation of different glial cell types in the central nervous system depends upon a wide variety of proliferative and differentiative signals. Here we report that changes in the levels of cyclin-dependent kinase 2 (CDK2) and the cell cycle inhibitor p27kip1 accompany the differentiation of central glia-4 (CG-4) progenitor cells to an astrocytic cell phenotype in the presence of fetal calf serum. Although a decrease in CDK2 levels was observed in both oligodendrocyte and astrocyte cells derived from CG-4 cells, a striking increase in the levels of p27 was observed during the differentiation of astrocyte cells. In astrocyte cell extracts, inhibition of CDK2 activity could be overcome with exogenously added cyclin E. Furthermore, depletion of p27 from astrocyte extracts lowered the amount of cyclin E required for CDK2 activation. Taken together, these results suggest that the inhibitory action of p27 upon cyclin E-CDK2 may prevent entry of cells into the S phase and regulate the progression of CG-4 cells toward an astrocytic lineage.

Death of oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75.

Members of the nerve growth factor (NGF) family promote the survival of neurons during development. NGF specifically activates the receptor trkA, initiating a signal transduction cascade which ultimately blocks cell death. Here we show that NGF can have the opposite effect, inducing the death of mature oligodendrocytes cultured from postnatal rat cerebral cortex. This effect was highly specific, because NGF had no effect on oligodendrocyte precursors and astrocytes. Other neurotrophins such as brain-derived neurotrophin factor (BDNF) and neurotrophin-3 (NT-3) did not induce cell death. NGF binding to mature oligodendrocytes expressing the p75 neurotrophin receptor, but not trkA, resulted in a sustained increase of intracellular ceramide and c-Jun amino-terminal kinase (JNK) activity, which are thought to participate in a signal transduction pathway leading to cell death. Taken together, these results indicate that NGF has the ability to promote cell death in specific cell types through a ligand-dependent signalling mechanism involving the p75 neurotrophin receptor.

Central glial and neuronal populations display differential sensitivity to ceramide-dependent cell death.

Ceramide is a lipid second messenger implicated in the mechanism of apoptotic cell death. The effect of the cell-permeable ceramide analogue C2 has been tested on primary cortical cultures of neurons, astrocytes, and oligodendrocytes as well as on the bipotential glial precursor cell line CG-4. After 24 hr of treatment, C2 ceramide induced a dose-dependent cell death in primary oligodendrocytes and precursor cells, with a maximum effect at 10 microM. Commitment of oligodendrocytes to cell death occurred within the first 6 hr of treatment. Ultramicroscopic analysis of primary oligodendrocytes exposed to C2 ceramide for 3.5 hr revealed extensive membrane blebbing in the absence of nuclear condensation. In contrast, similar treatment of primary neuronal or astrocytic cortical cultures had no effect on cell survival. Neurons and astrocytes were resistant to 10 microM C2 ceramide. Furthermore, bipotential progenitors that were differentiated toward astrocytes also became resistant to ceramide treatment as they acquired a mature astrocytic phenotype. These experiments suggest that cell type specific factors are required for ceramide-mediated cell death in the nervous system.