CD95 maintains stem cell-like and non-classical EMT programs in primary human glioblastoma cells.

Glioblastoma (GBM) is one of the most aggressive types of cancer with limited therapeutic options and unfavorable prognosis. Stemness and non-classical epithelial-to-mesenchymal transition (ncEMT) features underlie the switch from normal to neoplastic states as well as resistance of tumor clones to current therapies. Therefore, identification of ligand/receptor systems maintaining this privileged state is needed to devise efficient cancer therapies. In this study, we show that the expression of CD95 associates with stemness and EMT features in GBM tumors and cells and serves as a prognostic biomarker. CD95 expression increases in tumors and with tumor relapse as compared with non-tumor tissue. Recruitment of the activating PI3K subunit, p85, to CD95 death domain is required for maintenance of EMT-related transcripts. A combination of the current GBM therapy, temozolomide, with a CD95 inhibitor dramatically abrogates tumor sphere formation. This study molecularly dissects the role of CD95 in GBM cells and contributes the rational for CD95 inhibition as a GBM therapy.

CD95 promotes metastatic spread via Sck in pancreatic ductal adenocarcinoma.

Cancer stem cells (CSCs) have been implicated in the initiation and maintenance of tumour growth as well as metastasis. Recent reports link stemness to epithelial-mesenchymal transition (EMT) in cancer. However, there is still little knowledge about the molecular markers of those events. In silico analysis of RNA profiles of 36 pancreatic ductal adenocarcinomas (PDAC) reveals an association of the expression of CD95 with EMT and stemness that was validated in CSCs isolated from PDAC surgical specimens. CD95 expression was also higher in metastatic pancreatic cells than in primary PDAC. Pharmacological inhibition of CD95 activity reduced PDAC growth and metastasis in CSC-derived xenografts and in a murine syngeneic model. On the mechanistic level, Sck was identified as a novel molecule indispensable for CD95's induction of cell cycle progression. This study uncovers CD95 as a marker of EMT and stemness in PDAC. It also addresses the molecular mechanism by which CD95 drives tumour growth and opens tantalizing therapeutic possibilities in PDAC.

Control of neuronal branching by the death receptor CD95 (Fas/Apo-1).

The CD95 (Apo-1/Fas)/CD95 ligand (CD95L) system is best characterized as a trigger of apoptosis. Nevertheless, despite broad expression of CD95L and CD95 in the developing brain, absence of functional CD95 (lpr mice) or CD95L (gld mice) does not alter neuronal numbers. Here, we report that in embryonic hippocampal and cortical neurons in vivo and in vitro CD95L does not induce apoptosis. Triggering of CD95 in cultured immature neurons substantially increases neurite branches by promoting their formation. The branching increase occurs in a caspase-independent and death domain-dependent manner and is paralleled by an increase in the nonphosphorylated form of Tau. Most importantly, lpr and gld mutants exhibit a reduced number of dendritic branches in vivo at the time when synapse formation takes place. These data reveal a novel function for the CD95 system and add to the picture of guidance molecules in the developing brain.

[Prehospital management of spinal cord injuries]. / Präklinisches Management von Rückenmarkverletzungen.

In both the United States and Europe about 10,000 patients suffer from spinal cord injury (SCI) each year and 20% die before being admitted to hospital. Prehospital management of SCI is very important since 25% of SCI damage may occur after the initial event. Emergency treatment includes examination of the patient, spinal immobilization, careful airway management, cardiovascular stabilization (maintenance of mean arterial blood pressure above 90 mmHg) and glucose levels within the normal range. From an evidence-based point of view, it is still not known whether additional specific therapy is useful and studies have not convincingly demonstrated that methylprednisolone (MPS) or other substances have clinically important benefits. Recently published statements from the US do not support the therapeutic use of MPS in patients suffering from SCI in the prehospital setting. Moreover, it is not known whether hypothermia or any other pharmacological interventions have beneficial effects. Networks for clinical studies in SCI patients should be established as a basic requirement for further improvement in outcome in these patients.

Therapeutic neutralization of CD95-ligand and TNF attenuates brain damage in stroke.

Stroke is the third most common cause of death in the Western world. The mechanisms of brain damage in the affected areas are largely unknown. Hence, rational treatment strategies are limited. Previous experimental evidence suggested that cerebral lesions were less prominent in CD95 (APO-1/Fas)-deficient (lpr) than in wild-type mice. Additional results strongly suggested that the CD95-ligand (CD95L) was a major cause of neuronal autocrine suicide in the penumbra. These data and the assumption that death-receptor systems might determine stroke-related damage in the brain prompted us to examine these systems in in vitro and in vivo models of ischemia. We showed that hybrids of TNF-deficient and gld mice were strongly resistant towards stroke-induced damage. To determine the mechanism of action of TNF and CD95L, we separately investigated their influence on primary ischemic death and secondary inflammatory injury. Inhibition of both TNF and CD95L in vitro prevented death of primary neurons induced by oxygen-glucose deprivation and reperfusion. The recruitment of inflammatory cells to the ischemic hemisphere was abrogated in the absence of both TNF and CD95L. Significantly, mice injected with a mixture of neutralizing anti-TNF and anti-CD95L antibodies 30 min after induction of stroke showed a marked decrease in both infarct volumes and mortality. Accordingly, the locomotor performance of these animals was not significantly impaired in comparison to sham-operated animals. These data reveal that inhibition of TNF and CD95L blocks stroke-related damage at two levels, the primary ischemic and the secondary inflammatory injury. These results offer new approaches in stroke treatment.

Involvement of CD95/Apo1/Fas in cell death after myocardial ischemia.

BACKGROUND: The death of cardiac cells during ischemia and reperfusion is partially mediated by apoptosis, as seen, eg, in autopsy material of patients after acute myocardial infarction. METHODS AND RESULTS: To study the role of CD95/Fas/Apo1 for induction of postischemic cell death, we used an ischemia/reperfusion model of isolated rat and mouse hearts in Langendorff perfusion. In this model, caspase-dependent apoptosis occurred during postischemic reperfusion. Moreover, soluble CD95 ligand/Fas ligand was released by the postischemic hearts early after the onset of reperfusion. In addition, this ligand was synthesized de novo under these circumstances. Similar findings were observed for other "death-inducing" ligands, such as tumor necrosis factor (TNF)-alpha and TNF-related apoptosis-inducing ligand. In primary adult rat myocyte culture, hypoxia and reoxygenation caused a marked increase in sensitivity to the apoptotic effects of CD95 ligand. Isolated hearts from mice lacking functional CD95 (lpr) display marked reduction in cell death after ischemia and reperfusion compared with wild-type controls. CONCLUSIONS: These data suggest that CD95/Apo1/Fas is directly involved in cell death after myocardial ischemia. The CD95 system might thus represent a novel target for therapeutic prevention of postischemic cell death in the heart.

FK506 prevents stroke-induced generation of ceramide and apoptosis signaling.

Ceramide is a key mediator of apoptosis during the cellular stress response which is also involved in stroke-induced death. Transient occlusion of the middle cerebral artery (MCA) in rats led to a strong generation of ceramide as measured in thalamus and entorhinal cortex of the ischemic brain tissue. Enhanced levels of ceramide may be involved in apoptosis signaling following stroke since exogenously added synthetic C2-ceramide increased expression of c-jun and the death-inducing ligands (DILs) CD95-L, TRAIL and TNF-alpha in neuroblastoma cells. DILs in turn mediated death via binding to their respective receptors as concluded from diminished apoptosis upon blocking of the common pathway by dominant negative FADD. C2-ceramide induced both necrosis and apoptosis in a concentration-dependent manner corresponding to the situation present in the ischemic brain. The immunosuppressant FK506 inhibited the release of ceramide, expression of CD95-L and apoptosis in an in vitro and in vivo model for ischemia/reperfusion. These data suggest that ceramide is a crucial initiator of death, e.g., by induction of DILs following stroke.

CD95 ligand (Fas-L/APO-1L) and tumor necrosis factor-related apoptosis-inducing ligand mediate ischemia-induced apoptosis in neurons.

Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined, in vivo and in vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cells in vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands both in vivo and in vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, in lpr mice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease.

NF-kappaB is activated and promotes cell death in focal cerebral ischemia.

The transcription factor NF-kappaB is a regulator of cell death or survival. To investigate the role of NF-kappaB in neuronal cell death, we studied its activation in a rodent model of stroke. In the ischemic hemisphere, NF-kappaB was activated, as determined by increased expression of an NF-kappaB-driven reporter transgene, nuclear translocation of NF-kappaB in neurons and enhanced DNA binding of NF-kappaB subunits RelA and p50. In p50 knockout mice, ischemic damage was significantly reduced. This indicates a cell death-promoting role of NF-kappaB in focal ischemia. NF-kappaB may provide a new pharmacological target in neurologic disease.

Rapid and long-lasting suppression of the ATF-2 transcription factor is a common response to neuronal injury.

The activating transcription factor 2 (ATF-2) protein, a neuronal constitutively expressed CRE-binding transcription factor, is essential for the intact development of the mammalian brain. ATF-2 is activated by c-Jun N-terminal kinases and modulates both the induction of the c-jun gene and the function of the c-Jun protein, a mediator of neuronal death and survival. Here we show by immunocytochemistry and Western blotting that ATF-2 is rapidly suppressed in neurons within 1-4 h following neuronal stress such as transient focal ischemia by occlusion of the medial cerebral artery, mechanical injury of the neuroparenchym, stimulation of adult dorsal root ganglion neurons in vitro by doxorubicin as well as within 24 h following nerve fiber transection. ATF-2 reappears and regains basal levels between 12 h and 72 h following ischemia, between 50 and 100 days following axotomy, but remains absent around the site of mechanical injury during the process of degeneration. Following ischemia and tissue injury, ATF-2-IR also disappeared in areas remote from the affected brain compartments indicating the regulation of its expression by diffusible molecules. These findings demonstrate that the rapid and persistent down-regulation of ATF-2 is a constituent of the long-term neuronal stress response and that the reappearance of ATF-2 after weeks is a marker for the normalization of neuronal gene transcription following brain injury.

Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury.

Transcription factor c-Jun is proposed to control neuronal cell death and survival, but its activation by N-terminal phosphorylation and the underlying activity of the c-Jun N-terminal kinases (JNKs) remain to be elucidated in the adult mammalian brain. We generated a polyclonal antiserum that specifically recognizes c-Jun phosphorylated at its serine 73 (S73) residue after UV irradiation of 3T3 cells. Disruption of the c-jun locus in 3T3 cells abolished this reaction, and retransfection of the human c-jun at the c-jun-/- background restored it. The phospho-c-Jun antiserum was used to visualize N-terminally phosphorylated c-Jun in the adult rat brain with cellular resolution. Prolonged c-Jun S73 phosphorylation was detected in affected neurons up to 5 d after transient occlusion of medial cerebral artery or up to 50 d after transection of central nerve fiber tracts. After cerebral ischemia-reperfusion, phosphorylation of c-Jun was linked with induced expression of Fas-ligand (APO-1, CD95-ligand), whose gene is a putative c-Jun/AP-1 target, and with terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reactivity, a marker for apoptosis. After nerve fiber transection, however, lasting c-Jun phosphorylation occurred in axotomized neurons negative for Fas-ligand or TUNEL and regardless of degeneration or survival. In contrast to these lasting phosphorylation patterns, transient seizure activity by pentylenetetrazole provoked only a brief c-Jun phosphorylation and JNK activation. In extracts from ischemic or axotomized brain compartments, c-Jun phosphorylation correlated with enhanced long-term JNK activity, and in-gel kinase assays visualized proteins with sizes corresponding to JNK isoforms as the only c-Jun N-terminally phosphorylating enzymes. These results demonstrate that lasting c-Jun S73 phosphorylation and JNK activity are part of neuronal stress response after neurodegenerative disorders in the adult mammalian brain with Fas-ligand as a putative apoptotic effector.

Expression of Jun, Fos, and ATF-2 proteins in axotomized explanted and cultured adult rat dorsal root ganglia.

The expression of c-Jun, JunB, JunD, c-Fos and ATF-2 transcription factors was studied in L4/L5 dorsal root ganglion neurons of adult rats, in order to determine the extent to know to which extend the expression of transcription factors in vitro parallels the pathophysiological expression in vivo. First, dorsal root ganglia were dissociated and cultured for up to 15 days in vitro (culture). Second, the dorsal root and the peripheral nerve fibres were transected close at the dorsal root ganglia, and the completely axotomized dorsal root ganglia were kept in artificial cerebrospinal fluid for up to 24 h. This procedure (explantation) preserves the intraganglionic morphology intact. Culture evoked a persistent expression of c-Jun and JunD in the majority of small neurons independent on neurite extension, In contrast, the number of large neurons with c-Jun decreased and with JunD increased with incubation time. JunB and c-Fos, which were also visible in the majority of neurons, strongly decreased with culture time in both small and large neurons. ATF-2 was visible in the vast majority of neurons and did not change during the observation period. Incubation with brain-derived neurotrophic factor for 15 days reduced JunB expression and raised c-Fos expression, but did not affect c-Jun or JunD labelings. Explantation of dorsal root ganglia evoked a dramatic and rapid induction of c-Jun in neurons located in the periphery of the ganglia, an area that showed prominent apoptosis as visualized by transferase dUTP nick end-labelling, followed by a delayed increase in neurons of the central parts of dorsal root ganglia. Expression of JunB showed a dramatic increase within 2 h in the whole ganglion, but disappeared within the following hours. JunD dropped from its basal levels within 4 h and was almost absent after 8 h. c-Fos did not appear until 6 h, when transferase dUTP nick end-labelling also became detectable, and remained visible in a rather small number of neurons. As with culture, incubation of explanted dorsal root ganglia with brain-derived neurotrophic factor prevented the initial rise in JunB, accelerated and enhanced c-Fos expression, but did not alter c-Jun and JunD expression. Immunoreactivity of ATF-2 declined or disappeared in those dorsal root ganglia compartments that showed a rise in c-Jun and transferase dUTP nick end-labelling. These findings demonstrate that inducible transcription factors such as Jun and Fos proteins are differentially expressed in adult neurons in vitro when compared to pathophysiological conditions in vivo such as nerve fibre transection (axotomy or rhizotomy). Moreover, the comparison between the explantation and culture experiments suggests that it is the complete axotomy of neurons that provokes those expression patterns found in neuronal cultures of adult neurons. The rapid and persisting expression of c-Jun during neurite extension and apoptosis points at the activation of a pivotal program that might be determined by the presence or absence of ATF-2 and that is involved in regeneration or degeneration.

Transection of rat fimbria-fornix induces lasting expression of c-Jun protein in axotomized septal neurons immunonegative for choline acetyltransferase and nitric oxide synthase.

The fimbria-fornix (FF) fiber tract was unilaterally transected in adult rats by a stereotaxic knife cut. In the axotomized neurons of the medial septal nucleus (MS) and ventral diagonal band of Broca (VDB), the expression of Jun, Fos, Krox, CREB transcription factors, choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) were studied by immunocytochemistry. In addition, NADPH-diaphorase (NDP) and acetylcholine esterase (AChE) were visualized by activity assays. For retrograde tracing of axotomized neurons, either HRP-coupled gold was injected in the entorhinal cortex prior to axotomy, or Fast Blue was injected into the transection site subsequently to FF transection. Following FF transection c-Jun and in a less extend JunD were expressed in axotomized MS and VDB neurons. Expression levels rose at 24 h, but not at 18 h, postaxotomy, reached their maximal levels between 5 and 7 days, and then gradually declined. Up to 100 days, c-Jun was still present in a substantial number of septal neurons. JunB, Krox-20, Krox-24, c-Fos, and pan-Fos immunoreactivities (IR) were not detectable in axotomized septal neurons and CREB-IR did not change compared to the intact contralateral side. ChAT-IR dramatically declined over 36 h, and furthermore AChE reactivity had substantially fallen after 5 days. The number and intensity of cytoplasmic neuronal NOS-IR and NDP which generated congruent temporospatial patterns gradually fell between 3 and 5 days postaxotomy. The surviving neurons labeled by NOS and NDP showed a high coexpression of c-Jun, whereas c-Jun was almost completely absent in neurons stained for ChAT and AChE. Finally, ChAT-IR and NDP reaction labeled different subpopulations. Our findings demonstrate a lasting expression of the c-Jun transcription factor in axotomized MS and VDB neurons that might indicate the regenerative propensity of damaged neurons. The decrease of NOS and NDP in MS and VDB neurons demonstrates that neuronal populations respond to axotomy with an individual regulation of NOS expression.