Nerve growth factor-induced glutamate release is via p75 receptor,ceramide, and Ca(2+) from ryanodine receptor in developing cerebellar neurons

Very little is known about the contribution of a low affinity neurotrophin receptor, p75, to neurotransmitter release. Here we show that nerve growth factor (NGF) induced a rapid release of glutamate and an increase of Ca2+ in cerebellar neurons through a p75-dependent pathway. The NGF-induced release occurred even in the presence of the Trk inhibitor K252a. The release caused by NGF but not brain-derived neurotrophic factor was enhanced in neurons overexpressing p75. Further, after transfection of p75-small interfering RNA, which down-regulated the endogenous p75 expression, the NGF-induced release was inhibited, suggesting that the NGF-induced glutamate release was through p75. We found that the NGF-increased Ca2+ was derived from the ryanodine-sensitive Ca2+ receptor and that the NGF-increased Ca2+ was essential for the NGF-induced glutamate release. Furthermore, scyphostatin, a sphingomyelinase inhibitor, blocked the NGF-dependent Ca2+ increase and glutamate release, suggesting that a ceramide produced by sphingomyelinase was required for the NGF-stimulated Ca2+ increase and glutamate release. This action of NGF only occurred in developing neurons whereas the brain-derived neurotrophic factor-mediated Ca2+ increase and glutamate release was observed at the mature neuronal stage. Thus, we demonstrate that NGF-mediated neurotransmitter release via the p75-dependent pathway has an important role in developing neurons.     

Ceramide inhibits axonal growth and nerve growth factor uptake without compromising the viability of sympathetic neurons.

Ceramide inhibits axonal growth of cultured rat sympathetic neurons when the ceramide content of distal axons, but not cell bodies, is increased (Posse de Chaves, E. I., Bussiere, M. Vance, D. E., Campenot, R. B., and Vance, J.E. (1997) J. Biol. Chem. 272, 3028-3035). We now report that inhibition of growth does not result from cell death since although ceramide is a known apoptotic agent, C(6)-ceramide given to the neurons for 24 h did not cause cell death but instead protected the neurons from death induced by deprivation of nerve growth factor (NGF). We also find that a pool of ceramide generated from sphingomyelin in distal axons, but not cell bodies, inhibits axonal growth. Analysis of endogenous sphingomyelinase activities demonstrated that distal axons are rich in neutral sphingomyelinase activity but contain almost no acidic sphingomyelinase, which is concentrated in cell bodies/proximal axons. Together, these observations are consistent with the idea that generation of ceramide from sphingomyelin by a neutral sphingomyelinase in axons inhibits axonal growth. Furthermore, we demonstrate that treatment of distal axons with ceramide inhibits the uptake of NGF and low density lipoproteins by distal axons by approximately 70 and 40%, respectively, suggesting that the inhibition of axonal growth by ceramide might be due, at least in part, to impaired endocytosis of NGF. However, inhibition of endocytosis of NGF by ceramide could not be ascribed to decreased phosphorylation of TrkA.     

Nogo-A interacts with TrkA to alter nerve growth factor signaling in Nogo-A-overexpressing PC12 cells

The Nogo-A protein, originally discovered as a potent myelin-associated inhibitor of neurite outgrowth, is also expressed by certain neurons, especially during development and after injury, but its role in neuronal function is not completely known. In this report, we overexpressed Nogo-A in PC12 cells to use as a model to identify potential neuronal signaling pathways affected by endogenously expressed Nogo-A. Unexpectedly, our results show that viability of Nogo-A-overexpressing cells was reduced progressively due to apoptotic cell death following NGF treatment, but only after 24 h. Inhibitors of neutral sphingomyelinase prevented this loss of viability, suggesting that NGF induced the activation of a ceramide-dependent cell death pathway. Nogo-A over-expression also changed NGF-induced phosphorylation of TrkA at tyrosines 490 and 674/675 from sustained to transient, and prevented the regulated intramembrane proteolysis of p75^NTR, indicating that Nogo-A was altering the function of the two neurotrophin receptors. Co-immunoprecipitation studies revealed that there was a physical association between TrkA and Nogo-A which appeared to be dependent on interactions in the Nogo-A-specific region of the protein. Taken together, our results indicate that Nogo-A influences NGF-mediated mechanisms involving the activation of TrkA and its interaction with p75^NTR.     

Biological evaluation of sphingomyelin analogues as inhibitors of sphingomyelinase

Seeking neutral sphingomyelinase inhibitors, we designed and synthesized hydrolytically stable analogues of sphingomyelin. These novel analogues replace the phosphodiester moiety of sphingomyelin with carbamate and urea moiety, resulting in inhibition of neutral sphingomyelinase. Compound 1 prevented ceramide generation and apoptotic neuronal cell death in a model of ischemia based on organotypic hippocampal slice cultures.     

p75 neurotrophin receptor is required for constitutive and NGF-induced survival signalling in PC12 cells and rat hippocampal neurones

We have previously shown that nerve growth factor (NGF)-induced activation of nuclear factor-kappaB increased neuronal expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. In the present study we determined the role of the p75 neurotrophin receptor in constitutive and NGF-induced survival signalling. Treatment of rat pheochromocytoma (PC12) cells with a blocking anti-rat p75 antibody or inhibition of p75 expression by antisense oligonucleotides reduced constitutive and NGF-induced bcl-xL expression. Treatment with the blocking anti-p75 antibody also inhibited NGF-induced activation of the survival kinase Akt. Inhibition of phosphatidylinositol-3-kinase (PI3 kinase) activity or overexpression of a dominant-negative mutant of Akt kinase inhibited NGF-induced nuclear factor-kappaB activation. Activation of Akt kinase by NGF was also observed in PC12nnr5 cells and cultured rat hippocampal neurones which both lack significant TrkA expression. Treatment of hippocampal neurones with the blocking anti-p75 antibody inhibited constitutive and NGF-induced Bcl-xL expression, activation of Akt, and blocked the protective effect of NGF against excitotoxic and apoptotic injury. Our data suggest that the p75 neurotrophin receptor mediates constitutive and NGF-induced survival signalling in PC12 cells and hippocampal neurones, and that these effects are mediated via the PI3-kinase pathway.     

Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway

C(2)-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C(2)-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C(2)-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C(2)-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk, was without effect. Immunoblot analysis of protein extracts from C(2)-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C(2)-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover, ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells, while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C(2)-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis.     

Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.     

Fibrillar amyloid-beta peptides kill human primary neurons via NADPH oxidase-mediated activation of neutral sphingomyelinase. Implications for Alzheimer's disease

Alzheimer's disease is a major illness of dementia characterized by the presence of amyloid plaques, neurofibrillary tangles, and extensive neuronal apoptosis. However, the mechanism behind neuronal apoptosis in the Alzheimer's-diseased brain is poorly understood. This study underlines the importance of neutral sphingomyelinase in fibrillar Abeta peptide-induced apoptosis and cell death in human primary neurons. Abeta1-42 peptides induced the activation of sphingomyelinases and the production of ceramide in neurons. Interestingly, neutral (N-SMase), but not acidic (A-SMase), sphingomyelinase was involved in Abeta1-42-mediated neuronal apoptosis and cell death. Abeta1-42-induced production of ceramide was redox-sensitive, as reactive oxygen species were involved in the activation of N-SMase but not A-SMase. Abeta1-42 peptides induced the NADPH oxidase-mediated production of superoxide radicals in neurons that was involved in the activation of N-SMase, but not A-SMase, via hydrogen peroxide. Consistently, superoxide radicals generated by hypoxanthine and xanthine oxidase also induced the activation of N-SMase, but not A-SMase, through a catalase-sensitive pathway. Furthermore, antisense knockdown of p22phox, a subunit of NADPH oxidase, inhibited Abeta1-42-induced neuronal apoptosis and cell death. These studies suggest that fibrillar Abeta1-42 peptides induce neuronal apoptosis through the NADPH oxidase-superoxide-hydrogen peroxide-NS-Mase-ceramide pathway.     

Ceramide Prevents Neuronal Programmed Cell Death Induced by Nerve Growth Factor Deprivation

Abstract: We examined the ability of ceramide and sphingomyelinase (SMase) to prevent neuronal programmed cell death (PCD). We found that a cell-permeable ceramide analogue prevented neuronal PCD when applied to established sympathetic neuron primary cultures at the time of nerve growth factor (NGF) deprivation. Other amphiphilic lipids such as oleic acid failed to prevent cell death. Exogenous SMase also showed the same effect, probably by raising the intracellular ceramide level by sphingomyelin (SM) breakdown. Phosphocholine, another hydrolytic product of SM by SMase, did not prevent cell death. Other phospholipases, such as phospholipase C and phospholipase A₂, could not prevent cell death. Given the recent findings that the SM cycle is activated to increase the intracellular ceramide level on NGF binding to the low-affinity NGF receptor (LNGFR) and that NGF binding to LNGFR suppresses apoptosis in neural cell lines, our results suggest the possibility of the SM cycle as a signaling mechanism transducing the PCD-preventing activity of NGF.     

Programmed cell death in the developing somites is promoted by nerve growth factor via its p75(NTR) receptor

Neurotrophins control neuron number during development by promoting the generation and survival of neurons and by regulating programmed neuronal death. In the latter case, the cell death induced by nerve growth factor (NGF) in the developing chick retina is mediated by p75(NTR), the common neurotrophin receptor (J. M. Frade, A. Rodriguez-Tebar, and Y.-A. Barde, 1996, Nature 383, 166-168). Here we show that NGF also induces the programmed death of paraxial mesoderm cells in the developing somites. Both NGF and p75(NTR) are expressed in the somites of chick embryos at the time and the place of programmed cell death. Moreover, neutralizing the activity of endogenous NGF with a specific blocking antibody, or antagonizing NGF binding to p75(NTR) by the application of human NT-4/5, reduces the levels of apoptotic cell death in both the sclerotome and the dermamyotome by about 50 and 70%, respectively. Previous data have shown that Sonic hedgehog is necessary for the survival of differentiated somite cells. Consistent with this, Sonic hedgehog induces a decrease of NGF mRNA in somite explant cultures, thus showing the antagonistic effect of NGF and Sonic hedgehog with respect to somite cell survival. The regulation of programmed cell death by NGF/p75(NTR) in a mesoderm-derived tissue demonstrates the capacity of neurotrophins and their receptors to influence critical developmental processes both within and outside of the nervous system.     

Small molecule, non-peptide p75 ligands inhibit Abeta-induced neurodegeneration and synaptic impairment

The p75 neurotrophin receptor (p75(NTR)) is expressed by neurons particularly vulnerable in Alzheimer's disease (AD). We tested the hypothesis that non-peptide, small molecule p75(NTR) ligands found to promote survival signaling might prevent Abeta-induced degeneration and synaptic dysfunction. These ligands inhibited Abeta-induced neuritic dystrophy, death of cultured neurons and Abeta-induced death of pyramidal neurons in hippocampal slice cultures. Moreover, ligands inhibited Abeta-induced activation of molecules involved in AD pathology including calpain/cdk5, GSK3beta and c-Jun, and tau phosphorylation, and prevented Abeta-induced inactivation of AKT and CREB. Finally, a p75(NTR) ligand blocked Abeta-induced hippocampal LTP impairment. These studies support an extensive intersection between p75(NTR) signaling and Abeta pathogenic mechanisms, and introduce a class of specific small molecule ligands with the unique ability to block multiple fundamental AD-related signaling pathways, reverse synaptic impairment and inhibit Abeta-induced neuronal dystrophy and death.     

The p75 neurotrophin receptor: effects on neuron survival in vitro and interaction with death domain-containing adaptor proteins

The p75 neurotrophin receptor (p75NTR) is a death domain (DD) containing receptor of the TNF/FAS(APO-1) family. p75NTR has recently been shown to mediate apoptosis in certain types of neurons as well as in oligodendrocytes. The molecular mechanisms by which p75NTR stimulates apoptosis are still unknown. Here, we have tested whether overexpression of p75NTR could modulate survival of sympathetic neurons cultured in the presence or absence of NGF. Moreover, using the yeast two-hybrid system, we tested whether p75NTR intracellular domain was able to dimerize or interact with known DD-containing proteins including FADD, RIP, RAIDD and TRADD. We found that over-expression of p75NTR had no effect on the survival of sympathetic neurons cultured in the presence of NGF but instead delayed neuronal death following NGF deprivation. These results strongly support the finding that p75NTR is not involved in the apoptosis process induced by NGF deprivation in sympathetic neurons. We also foun d that the intracellular domain of p75NTR failed to associate either with itself or with other known DD-containing proteins. This suggests that the mechanisms by which p75NTR triggers apoptosis in certain cell types are different from those used by other receptors of the TNF/FAS family.     

Activation of neutral-sphingomyelinase,MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester–induced apoptosis in C6 glioma cells

Background

Caffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells.

Results

C6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869.

Conclusions

Taken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma.     

Nerve growth factor, sphingomyelins, and sensitization in sensory neurons

Because nerve growth factor(NGF)is elevated during inflammation,plays a causal role in the initiation of hyperalgesia     

Nerve Growth Factor-Induced Differentiation in Neuroblastoma Cells Expressing TrkA but Lacking p75NGFR

Abstract: Nerve growth factor (NGF) binds to two distinct cell surface receptors, TrkA, which is a receptor tyrosine kinase, and p75^NGFR, whose role in NGF-induced signal transduction remains unclear. We have found that human neuroblastoma IMR-32 cells express TrkA, but p75^NGFR expression was not detectable in these cells by northern blot analysis, immunoblotting, or chemical crosslinking experiments. Despite the lack of p75^NGFR expression, subnanomolar concentrations of recombinant human NGF induced neurite outgrowth, tyrosine phosphorylation, and immediate early gene expression in these cells. These results strongly suggest that NGF-induced neuronal differentiation in IMR-32 cells is initiated through TrkA in the absence of p75^NGFR. Thus, IMR-32 cells may provide a model for studying neurotrophic effects of NGF on adult striatal cholinergic neurons, which also lack p75^NGFR expression.     

Mechanisms of p75-mediated death of hippocampal neurons. Role of caspases

Neurotrophins support neuronal survival and differentiation via Trk receptors, yet can also induce cell death via the p75 receptor. In these studies, we investigated signaling mechanisms governing p75-mediated death of hippocampal neurons, specifically the role of caspases. Although p75 is structurally a member of the Fas/TNFR1 receptor family, caspase-8 was not required for p75-mediated death, unlike other members of this receptor family. In contrast, p75-mediated neuronal death was associated with mitochondrial loss of cytochrome c and required Apaf-1 and caspase-9, -6, and -3. In particular, caspase-6 plays a central role in mediating neurotrophin-induced death, illuminating a novel role for this caspase. Inhibition of DIABLO/Smac, which blocks inhibitor of apoptosis proteins, protected cells from death, whereas simultaneous inhibition of both DIABLO/Smac and MIAP3 allowed trophin-induced death to proceed. In vivo, pilocarpine-induced seizures, previously shown to up-regulate p75 expression and increase neurotrophin production, caused activation of caspase-6 and -3 and cleavage of poly(ADP-ribose) polymerase in p75-expressing hippocampal neurons. In p75(-/-) mice, no activated caspase-3 was detected, and there was a marked reduction in the number of dying neurons after pilocarpine treatment compared with wild type mice. Neurotrophin-induced p75-mediated death is likely to play an important role in mediating neuronal loss consequent to brain injury.     

P53 Is Essential for Developmental Neuron Death as Regulated by the TrkA and p75 Neurotrophin Receptors

Naturally occurring sympathetic neuron death is the result of two apoptotic signaling events: one normally suppressed by NGF/TrkA survival signals, and a second activated by the p75 neurotrophin receptor. Here we demonstrate that the p53 tumor suppressor protein, likely as induced by the MEKK-JNK pathway, is an essential component of both of these apoptotic signaling cascades. In cultured neonatal sympathetic neurons, p53 protein levels are elevated in response to both NGF withdrawal and p75NTR activation. NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax. Functional ablation of p53 using the adenovirus E1B55K protein inhibits neuronal apoptosis as induced by either NGF withdrawal or p75 activation. Direct stimulation of the MEKK-JNK pathway using activated MEKK1 has similar effects; p53 and Bax are increased and the subsequent neuronal apoptosis can be rescued by E1B55K. Expression of p53 in sympathetic neurons indicates that p53 functions downstream of JNK and upstream of Bax. Finally, when p53 levels are reduced or absent in p53+/− or p53−/− mice, naturally occurring sympathetic neuron death is inhibited. Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.     

Neutral sphingomyelinase: Localization in rat liver nuclei and involvement in regeneration/proliferation

We have studied the localization of neutral sphingomyelinase (N-SMase) in rat liver nuclei. The levels of neutral sphingomyelinase in regenerating liver nuclei were also assessed.We found that rat liver nuclei contain a sphingomyelinase having a pH optima of 7.2 and a kDa of 92. In intact nuclei, neutral sphingomyelinase was associated predominantly with the nuclear envelope. In regenerating/proliferating rat liver (during DNA synthesis), neutral sphingomyelinase was translocated from the nuclear envelope to the nuclear matrix. The levels of sphingomyelin in whole nuclei decreased in reverse proportion to an increase in the levels of neutral sphingomyelinase. By contrast, there was a corresponding increase in the levels of ceramide and sphingosine during cell regeneration/proliferation. Thus, endogenous nuclear neutral sphingomyelinase may play a role in the regulation of sphingomyelin levels and in relevant signal transduction reactions involving cell regeneration/proliferation. The potential significance of ceramide generation may be aimed at programmed cell death to allow the regeneration of liver mediated via target proteins such as, ceramide activated protein kinases/phospholipases or other unknown mechanisms.Abbreviations N-SMase neutral sphingomyelinase - A-SMase acid sphingomyelinase     

APP Regulates NGF Receptor Trafficking and NGF-Mediated Neuronal Differentiation and Survival

β-amyloid precursor protein (APP) is a key factor in Alzheimer''s disease (AD) but its physiological function is largely undetermined. APP has been found to regulate retrograde transport of nerve growth factor (NGF), which plays a crucial role in mediating neuronal survival and differentiation. Herein, we reveal the mechanism underlying APP-mediated NGF trafficking, by demonstrating a direct interaction between APP and the two NGF receptors, TrkA and p75NTR. Downregulation of APP leads to reduced cell surface levels of TrkA/p75NTR and increased endocytosis of TrkA/p75NTR and NGF. In addition, APP-deficient cells manifest defects in neurite outgrowth and are more susceptible to Aβ-induced neuronal death at physiological levels of NGF. However, APP-deficient cells show better responses to NGF-stimulated differentiation and survival than control cells. This may be attributed to increased receptor endocytosis and enhanced activation of Akt and MAPK upon NGF stimulation in APP-deficient cells. Together, our results suggest that APP mediates endocytosis of NGF receptors through direct interaction, thereby regulating endocytosis of NGF and NGF-induced downstream signaling pathways for neuronal survival and differentiation.