P2X7 receptor‐induced death of motor neurons by a peroxynitrite/FAS‐dependent pathway

The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase‐dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP. The P2X7 inhibitors BBG, oATP, and KN‐62 prevented 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP‐induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1‐100 μM). High concentrations of ATP (1 mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7‐induced motor neuron death was dependent on neuronal nitric oxide synthase‐mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well‐established peroxynitrite/FAS death pathway in motor neurons.     

ATP induces the death of developing avian retinal neurons in culture via activation of P2X7 and glutamate receptors

Previous data suggest that nucleotides are important mitogens in the developing retina. Here, the effect of ATP on the death of cultured chick embryo retina cells was investigated. In cultures obtained from retinas of 7-day-old chick embryos (E7) that were cultivated for 2 days (E7C2), both ATP and BzATP induced a ～30 % decrease in cell viability that was time- and dose-dependent and that could be blocked by 0.2 mM oxidized ATP or 0.3 μM KN-62. An increase in cleaved caspase-3 levels and in the number of TUNEL-positive cells was observed when cultures were incubated with 3 mM ATP and immunolabeling for cleaved-caspase 3 was observed over neurons but not over glial cells. ATP-dependent cell death was developmentally regulated, the maximal levels being detected by E7C2-3. Nucleotides were able to increase neuronal ethidium bromide and sulforhodamine B uptake in mixed and purified neuronal cultures, an effect that was blocked by the antagonists Brilliant Blue G and oxidized ATP. In contrast, nucleotide-induced cell death was observed only in mixed cultures, but not in purified cultures of neurons or glia. ATP-induced neuronal death was blocked by the glutamatergic antagonists MK801 and DNQX and activation of P2X7 receptors by ATP decreased the uptake of [³H]-d-aspartate by cultured glial cells with a concomitant accumulation of it in the extracellular medium. These results suggest that ATP induces apoptosis of chick embryo retinal neurons in culture through activation of P2X7 and glutamate ionotropic receptors. Involvement of a P2X7 receptor-mediated inhibition of the glial uptake of glutamate is suggested.     

Effects of Extracellular ATP on Fe2+-Induced Cytotoxicity in PC-12 Cells

Abstract: Extracellular ATP is known to cause a variety of changes, including the alteration of ion fluxes, cell growth, and other physiological activities. Recently, it has been suggested that ATP acts as an excitatory synaptic transmitter, which may produce a Ca²⁺ influx via the activation of a P_2y purinoceptor. Rat pheochromocytoma (PC-12) cells are known to resemble rat sensory neurons and to possess a P_2y purinoceptor. In this study, we demonstrated that extracellular ATP dose-dependently increased PC-12 cell death in the presence of ferrous ions. Voltage-sensitive calcium channel blockers and calpain and xanthine oxidase inhibitors were found to be effective at protecting PC-12 cells from Fe²⁺/ATP-induced lipid peroxidation and cell death. These results suggest that xanthine oxidase activation induced by calpains and subsequent free radical formation may be responsible for Fe²⁺/ATP-induced neuronal cell death.     

Role for apoptosis-inducing factor in the physiological death of cerebellar neurons

Apoptosis-inducing factor (AIF) is implicated in caspase-independent apoptotic-like death. AIF released from mitochondria translocates to the nucleus, where it mediates some apoptotic events such as chromatin condensation and DNA degradation. Here, the role of AIF in the neuronal death was studied under physiological conditions. When we analyzed the cellular localization of AIF during cerebellar development, we found a significant increase in the number of neurons with nuclear AIF localization in an age-dependent manner. On the other hand, cerebellar granule neurons (CGN) chronically cultured in low concentration of potassium (5 mM; K5) die with apoptotic-like characteristics after five days. In the present study we found that K5 induces a caspase-dependent apoptotic-like death of CGN as well as a late nuclear translocation of AIF. When CGN death induced by K5 was carried out in the presence of a general inhibitor of caspases, there was a slight decrement of cell death, but neurons eventually died by showing apoptotic-like features such as phosphatidylserine translocation and nuclear condensation. Besides, there was a significant increment of nuclear AIF translocation. These findings support the idea that AIF could be involved in apoptotic-like death of CGN and that it could be an alternative mechanism of neuronal death during cerebellar development.     

Mature neurons: equipped for survival

Neurons completely transform how they regulate cell death over the course of their lifetimes. Developing neurons freely activate cell death pathways to fine-tune the number of neurons that are needed during the precise formation of neural networks. However, the regulatory balance between life and death shifts as neurons mature beyond early development. Mature neurons promote survival at all costs by employing multiple, often redundant, strategies to prevent cell death by apoptosis. This dramatic shift from permitting cell death to ensuring cellular survival is critical, as these post-mitotic cells must provide neuronal circuitry for an organism''s entire lifetime. Importantly, as many neurodegenerative diseases afflict adult neuronal populations, the survival mechanisms in mature neurons are likely to be either reversed or circumvented during neurodegeneration. Examining the adaptations for inhibiting apoptosis during neuronal maturation is key to comprehending not just how neurons survive long term, but may also provide insight for understanding how neuronal toxicity in various neurodegenerative diseases may ultimately lead to cell death.     

Extracellular ATP and nerve growth factor intensify hypoglycemia-induced cell death in primary neurons: role of P2 and NGFRp75 receptors

In this study, we monitored the direct expression of P2 receptors for extracellular ATP in cerebellar granule neurons undergoing metabolism impairment. Glucose deprivation for 30–60 min inhibited P2Y₁ receptor protein, only weakly modulated P2X₁, P2X₂ and P2X₃, and up‐regulated by about two‐fold P2X₄, P2X₇ and P2Y₄. The P2X/Y antagonist basilen blue, protecting cerebellar neurons from hypoglycemic cell death, maintained within basal levels only the expression of P2X₇ and P2Y₄ proteins, but not P2X₄ or P2Y₁. Glucose starvation transiently increased (up to three‐fold) the expression of NGFRp75 receptor protein and strongly stimulated the extracellular release of nerve growth factor (NGF; about 10‐fold). Exogenously added NGF then augmented hypoglycemic neuronal death by about 60%, increasing the percentage of Höechst‐positive nuclei (from approximately 62 to 95%), reducing lactate dehydrogenase (LDH) release (from about 50 to 14%) and significantly overstimulating the hypoglycemia‐induced expression of P2X₇ and P2Y₄. Conversely, extracellular ATP augmented hypoglycemic neuronal death by about 80%, reducing the number of Höechst‐positive nuclei (from approximately 62% to 14%), augmenting LDH outflow (by about 30%) and further increasing the hypoglycemia‐induced expression of NGFRp75. Our results indicate that P2 and NGFRp75 receptors are modulated during glucose starvation and that extracellular ATP and NGF drive features of, respectively, necrotic and apoptotic hypoglycemic cell death, aggravating the consequences of metabolism impairment in cerebellar primary neurons.     

Lifeguard/neuronal membrane protein 35 regulates Fas ligand-mediated apoptosis in neurons via microdomain recruitment

Fas ligand (FasL)-receptor system plays an essential role in regulating cell death in the developing nervous system, and it has been implicated in neurodegenerative and inflammatory responses in the CNS. Lifeguard (LFG) is a protein highly expressed in the hippocampus and the cerebellum, and it shows a particularly interesting regulation by being up-regulated during postnatal development and in the adult. We show that over-expression of LFG protected cortical neurons from FasL-induced apoptosis and decreased caspase-activation. Reduction of endogenous LFG expression by small interfering RNA sensitized cerebellar granular neurons to FasL-induced cell death and caspase-8 activation, and also increased sensitivity of cortical neurons. In differentiated cerebellar granular neurons, protection from FasL-induced cell death could be attributed exclusively to LFG and appears to be independent of FLICE inhibitor protein. Thus, LFG is an endogenous inhibitor of FasL-mediated neuronal death and it mediates the FasL resistance of CNS differentiated neurons. Finally, we also demonstrate that LFG is detected in lipid rafts microdomains, where it may interact with Fas receptor and regulate FasL-activated signaling pathways.     

Endogenously released DOPA is a causal factor for glutamate release and resultant delayed neuronal cell death by transient ischemia in rat striata

Glutamate is implicated in neuronal cell death. Exogenously applied DOPA by itself releases neuronal glutamate and causes neuronal cell death in in vitro striatal systems. Herein, we attempt to clarify whether endogenous DOPA is released by 10 min transient ischemia due to four-vessel occlusion during rat striatal microdialysis and, further, whether DOPA, when released, functions to cause glutamate release and resultant delayed neuronal cell death. Ischemia increased extracellular DOPA, dopamine, and glutamate, and elicited neuronal cell death 96 h after ischemic insult. Inhibition of striatal L-aromatic amino acid decarboxylase 10 min before ischemia increased markedly basal DOPA, tripled glutamate release with a tendency of decrease in dopamine release by ischemia, and exaggerated neuronal cell death. Intrastriatal perfusion of 10-30 nM DOPA cyclohexyl ester, a competitive DOPA antagonist, 10 min before ischemia, concentration-dependently decreased glutamate release without modification of dopamine release by ischemia. At 100 nM, the antagonist elicited a slight ceiling effect on decreases in glutamate release by ischemia and protected neurons from cell death. Glutamate was released concentration-dependently by intrastriatal perfusion of 0.3-1 mM DOPA and stereoselectively by 0.6 mM DOPA. The antagonist elicited no hypothermia during and after ischemia. Endogenously released DOPA is an upstream causal factor for glutamate release and resultant delayed neuronal cell death by brain ischemia in rat striata. DOPA antagonist has a neuroprotective action.     

Glia maintain follower neuron survival during Drosophila CNS development

While survival of CNS neurons appears to depend on multiple neuronal and non-neuronal factors, it remains largely unknown how neuronal survival is controlled during development. Here we show that glia regulate neuronal survival during formation of the Drosophila embryonic CNS. When glial function is impaired either by mutation of the glial cells missing gene, which transforms glia toward a neuronal fate, or by targeted genetic glial ablation, neuronal death is induced non-autonomously. Pioneer neurons, which establish the first longitudinal axon fascicles, are insensitive to glial depletion whereas the later extending follower neurons die. This differential requirement of neurons for glia is instructive in patterning and links control of cell number with axon guidance during CNS development.     

Chemotherapy induces ATP release from tumor cells

Chemotherapy can induce anticancer immune responses. In contrast to a widely extended prejudice, apoptotic cell death is often more efficient in eliciting a protective anticancer immune response than necrotic cell death. Recently, we have found that purinergic receptors of the P2X7 type are required for the anticancer immune response induced by chemotherapy. ATP is the endogenous ligand that has the highest affinity for P2X7. Therefore, we investigated the capacity of a panel of chemotherapeutic agents to induce ATP release from cancer cells. Here, we describe that multiple distinct anticancer drugs reduce the intracellular concentration of ATP before and during the manifestation of apoptotic characteristics such as the dissipation of the mitochondrial transmembrane potential and the exposure of phosphatidylserine residues on the plasma membrane. Indeed, as apoptosis progresses, intracellular ATP concentrations decrease, although even advanced-stage apoptotic cells still contain sizeable ATP levels. Only when cells enter secondary necrosis, the ATP concentration falls to undetectable levels. Concomitantly, a wide range of chemotherapeutic agents causes the release of ATP into the extracellular space as they induce tumor cell death. Hence, ATP release is a general correlate of apoptotic cell death induced by conventional anticancer therapies.     

Maturation and maintenance of cholinergic medial septum neurons require glucocorticoid receptor signaling

Glucocorticoids have been shown to influence trophic processes in the nervous system. In particular, they seem to be important for the development of cholinergic neurons in various brain regions. Here, we applied a genetic approach to investigate the role of the glucocorticoid receptor (GR) on the maturation and maintenance of cholinergic medial septal neurons between P15 and one year of age by using a mouse model carrying a CNS-specific conditional inactivation of the GR gene (GRNesCre). The number of choline acetyltransferase and p75NTR immuno-positive neurons in the medial septum (MS) was analyzed by stereology in controls versus mutants. In addition, cholinergic fiber density, acetylcholine release and cholinergic key enzyme activity of these neurons were determined in the hippocampus. We found that in GRNesCre animals the number of medial septal cholinergic neurons was significantly reduced during development. In addition, cholinergic cell number further decreased with aging in these mutants. The functional GR gene is therefore required for the proper maturation and maintenance of medial septal cholinergic neurons. However, the loss of cholinergic neurons in the medial septum is not accompanied by a loss of functional cholinergic parameters of these neurons in their target region, the hippocampus. This pinpoints to plasticity of the septo-hippocampal system, that seems to compensate for the septal cell loss by sprouting of the remaining neurons.     

Role of DAPK in neuronal cell death

Neuronal cell death happens as a result of the normal physiological process that occurs during development, or as part of the pathological process that occurs during disease. Death-associated protein kinase (DAPK) is an intracellular protein that mediates cell death by its serine/threonine kinase activity, and transmits apoptotic cell death signals in various cells, including neurons. DAPK is elevated in injured neurons in acute models of injury such as ischemia and seizure. The absence of DAPK has been shown to protect neurons from a wide variety of acute toxic insults. Moreover, DAPK also regulates neuronal cell death during central nervous system development. Neurons are initially overproduced in the developing nervous system, following which approximately one-half of the original cell population dies. This “naturally-occurring” or “programmed” cell death is essential for the construction of the developing nervous system. In this review, we focus on the role of DAPK in neuronal cell death after neuronal injury. The participation of DAPK in developmental neuronal death is also explained.     

Characterization of ATP‐induced cell death in the GL261 mouse glioma

Gliomas have one of the worst prognosis among cancers. Their resistance to cell death induced by endogenous neurotoxic agents, such as extracellular ATP, seems to play an important role in their pathobiology since alterations in the degradation rate of extracellular ATP drastically affects glioma growth in rats. In the present work we characterized the mechanisms of cell death induced by extracellular ATP in a murine glioma cell line, GL261. ATP and BzATP, a P2X7 agonist, induced cell death at concentrations that are described to activate the P2X7 receptor in mouse. oATP, an antagonist of P2X7, blocked the ATP‐induced cell death. Agonists of purinergic receptors expressed in GL261 such as adenosine, ADP, UTP did not cause any cell death, even at mM concentrations. A sub‐population of cells more sensitive to ATP expressed more P2X7 when compared to a less sensitive subpopulation. Accordingly, RNA interference of the P2X7 receptor drastically reduced ATP‐induced cell death, suggesting that this receptor is necessary for this effect. The mechanism of ATP‐induced cell death is predominantly necrotic, since cells presented shrinkage accompanied by membrane permeabilization, but not apoptotic, since no phosphatidylserine externalization or caspase activity was observed. These data show the importance of P2X7 in ATP‐induced cell death and shed light on the importance of ATP‐induced cell death in glioma development. J. Cell. Biochem. 109: 983–991, 2010. © 2010 Wiley‐Liss, Inc.     

Prolonged expression of puma in cholinergic amacrine cells during the development of rat retina

During development of the nervous system, large numbers of neurons are overproduced and then eliminated by programmed cell death. Puma is a BH3-only protein that is reported to be involved in the initiation of developmental programmed cell death in rodent retinal neurons. The expression and cellular localization of Puma in retinal tissues during development are not, however, well known. Here the authors report the expression pattern of Puma during retinal development in the rat. During the period of programmed cell death in the retina, Puma was expressed in some members of each retinal neuron, including retinal ganglion cells, amacrine cells, bipolar cells, horizontal cells, and photoreceptor cells. Although the developmental programmed cell death of cholinergic amacrine cells is known to be independent of Puma, this protein was expressed in almost all their dendrites and somata of cholinergic amacrine cells at postnatal age 2 to 3 weeks, and it continued to be detected in cholinergic dendrites in the inner plexiform layer for up to 8 weeks after birth. These results suggest that Puma has some significant roles in retinal neurons after eye opening, especially that of cholinergic amacrine cells, in addition to programmed cell death of retinal neurons before eye opening.     

Cyclin D1 is an essential mediator of apoptotic neuronal cell death.

Many neurons in the developing nervous system undergo programmed cell death, or apoptosis. However, the molecular mechanism underlying this phenomenon is largely unknown. In the present report, we present evidence that the cell cycle regulator cyclin D1 is involved in the regulation of neuronal cell death. During neuronal apoptosis, cyclin D1-dependent kinase activity is stimulated, due to an increase in cyclin D1 levels. Moreover, artificial elevation of cyclin D1 levels is sufficient to induce apoptosis, even in non-neural cell types. Cyclin D1-induced apoptosis, like neuronal apoptosis, can be inhibited by 21 kDa E1B, Bcl2 and pRb, but not by 55 kDa E1B. Most importantly, however, overexpression of the cyclin D-dependent kinase inhibitor p16INK4 protects neurons from apoptotic cell death, demonstrating that activation of endogenous cyclin D1-dependent kinases is essential during neuronal apoptosis. These data support a model in which neuronal apoptosis results from an aborted attempt to activate the cell cycle in terminally differentiated neurons.     

Expression of CD47/integrin-associated protein induces death of cultured cerebral cortical neurons

The death and survival of neuronal cells are regulated by various signaling pathways during development of the brain and in neuronal diseases. Previously, we demonstrated that the neuronal adhesion molecule brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1 (BIT/SHPS-1) is involved in brain-derived neurotrophic factor (BDNF)-promoted neuronal cell survival. Here, we report the apoptosis-inducing effect of CD47/integrin-associated protein (IAP), the heterophilic binding partner of BIT/SHPS-1, on neuronal cells. We generated a recombinant adenovirus vector expressing a neuronal form of CD47/IAP, and found that the expression of CD47/IAP by infection with CD47/IAP adenovirus induced the death of cultured cerebral cortical neurons. The numbers of TdT-mediated biotin-dUTP nick-end labelling (TUNEL)-positive neurons and of cells displaying apoptotic nuclei increased by expression of CD47/IAP. Neuronal cell death was prevented by the addition of the broad-spectrum caspase inhibitor Z-VAD-fmk. Furthermore, we observed that co-expression of CD47/IAP with BIT/SHPS-1 enhanced neuronal cell death, and that BDNF prevented it. These results suggest that CD47/IAP is involved in a novel pathway which regulates caspase-dependent apoptosis of cultured cerebral cortical neurons. CD47/IAP-induced death of cultured cortical neurons may be regulated by the interaction of CD47/IAP with BIT/SHPS-1 and by BDNF.     

Rotenone Induces Cell Death of Cholinergic Neurons in an Organotypic Co-Culture Brain Slice Model

In Alzheimer and Parkinson’s disease cell death of cholinergic and dopaminergic neurons are characteristic hallmarks, respectively. It is well established that rotenone, an inhibitor of complex I of the electron transport chain, induces cell death of dopaminergic neurons, however, not much is known on the effects of rotenone on cholinergic neurons. The aim of the present study was to evaluate the effects of rotenone on cholinergic neurons in an organotypic in vitro brain co-slice model. When co-cultures were treated with 10 μM rotenone for 24 h a significantly decreased number of cholinergic neurons was found in the basal nucleus of Meynert but not in the dorsal striatum. This cell death exhibited apoptotic DAPI-positive malformed nuclei and enhanced TUNEL-positive cells. In summary, inhibition of complex I of the electron transport chain may play a role in neurodegeneration of cholinergic neurons.     

Clusterin levels increase during neuronal development

The expression of clusterin has been shown to be elevated in several models of experimentally induced programmed cell death and in association with a number of neurodegenerative conditions. In order to test whether this protein is expressed in neurons during development, the expression of clusterin was examined in the developing nervous system, using immunohistochemistry and mRNA analysis. Clusterin expression was observed in the earliest neurons of the cortical plate on embryonic day (E) 12. Thereafter, the intensity of clusterin staining continued to increase in an age-dependent manner, with the greatest intensity of staining being found in the postnatal mature brain. Virtually all neurons were clusterin-positive and there was no evidence for the appearance of clusterin-positive cells specifically during epochs of programmed neuronal death in the embryo. This study suggests that clusterin has a role in neuronal maturation and it is unlikely to be associated exclusively with neuronal cell death. © 1993 John Wiley & Sons, Inc.