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The role of Bax in glutamate-induced nerve cell death: Bax and nerve cell death
Journal of Neurochemistry
Abstract
The role of the Bax gene product was examined in three forms of cortical nerve cell death in primary cultures. These include spontaneous cell death, oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake resulting in toxic oxidative stress, and ionotropic glutamate receptor-mediated excitotoxicity following a brief exposure to 10 microM glutamate. Primary cortical and hippocampal neuron cultures were established from embryos of Bax -/+ x Bax -/+ matings and the embryos genotyped and assayed for cell death in the three experimental paradigms. Cell death induced by oxidative glutamate toxicity and glutamate-mediated excitotoxicity was not altered in the Bax -/- homozygous knockout animals. In contrast, there was an approximately 50% inhibition of spontaneous cell death. These results suggest that a classical Bax-dependent apoptotic pathway contributes to the spontaneous cell death that takes place when nerve cells are initially exposed to cell culture conditions. A Bax-dependent programmed cell death pathway is not, however, utilized in oxidative glutamate toxicity and NMDA receptor-mediated excitotoxicity following a brief exposure to low concentrations of glutamate.
... This suggests that, in addition to inducing apoptosis, AIF may also protect against several types of neuronal injury. To resolve the role of AIF in different mechanisms of neuronal injury, we asked whether AIF reduction in Hq mice could protect neurons against (1) BAX-mediated, DNA damage-induced apoptosis and (2) BAX-independent, NMDA receptor-mediated excitotoxicity (Miller et al., 1997; Dargusch et al., 2001). Using Hq/Apaf1 / mice, we show that reduced AIF confers sustained protection in DNA damage-induced neuronal cell death when caspase activity was inhibited. ...
... These results demonstrate that, by blocking both mitochondrial pathways, the caspase-dependent pathway (by BAF treatment or Apaf1 / ) and the AIF pathway (by Hq mutation), neurons can be significantly protected against DNA damage-induced cell death.Arundine and Tymianski, 2003; Yuan et al., 2003 ). Excitotoxicity can occur independent of BAX and caspase activation (Miller et al., 1997; Dargusch et al., 2001). To confirm that BAX is not involved in excitotoxic cell death, Bax / and wild-type neurons were exposed to 100 M glutamate or 200 M NMDA. ...
... During stroke or trauma, excitotoxic neuronal damage results from an excessive release and impaired reuptake of the neurotransmitter glutamate from synapses. There are several receptors for glutamate, but the key mediator in terms of excitotoxic cell death is the NMDA receptor (Arundine and Tymianski, 2003 ), which, in this system, functions in a BAX-independent (Miller et al., 1997; Dargusch et al., 2001) and caspase-independent (Fig. 5) manner. Once activated by excessive levels of glutamate, the NMDA receptors allow excessive calcium ion influx and elevated cytoplasmic calcium ion levels. ...
Mitochondria release proteins that propagate both caspase-dependent and caspase-independent cell death pathways. AIF (apoptosis-inducing factor) is an important caspase-independent death regulator in multiple neuronal injury pathways. Presently, there is considerable controversy as to whether AIF is neuroprotective or proapoptotic in neuronal injury, such as oxidative stress or excitotoxicity. To evaluate the role of AIF in BAX-dependent (DNA damage induced) and BAX-independent (excitotoxic) neuronal death, we used Harlequin (Hq) mice, which are hypomorphic for AIF. Neurons carrying double mutations for Hq/Apaf1-/- (apoptosis proteases-activating factor) are impaired in both caspase-dependent and AIF-mediated mitochondrial cell death pathways. These mutant cells exhibit extended neuroprotection against DNA damage, as well as glutamate-induced excitotoxicity. Specifically, AIF is involved in NMDA- and kainic acid- but not AMPA-induced excitotoxicity. In vivo excitotoxic studies using kainic acid-induced seizure showed that Hq mice had significantly less hippocampal damage than wild-type littermates. Our results demonstrate an important role for AIF in both BAX-dependent and BAX-independent mechanisms of neuronal injury.
... These observations are in line with the observation that caspase inhibitors that block apoptosis do not inhibit oxytosis (Tan et al., 1998b). In addition, the B-cell lymphoma protein 2 (Bcl-2) family member BAX has been excluded as part of the signaling cascade in oxytosis (Dargusch et al., 2001). BAX is transcriptionally upregulated by the tumor suppressor p53 (Selvakumaran et al., 1994) and upon death stimuli it dimerizes and inserts into the mitochondrial membrane to induce the mitochondrial pathway of apoptosis by mitochondrial outer membrane permeabilization (Cosentino and Garcia-Saez, 2017). ...
... BAX is transcriptionally upregulated by the tumor suppressor p53 (Selvakumaran et al., 1994) and upon death stimuli it dimerizes and inserts into the mitochondrial membrane to induce the mitochondrial pathway of apoptosis by mitochondrial outer membrane permeabilization (Cosentino and Garcia-Saez, 2017). Although spontaneous apoptosis was prominently decreased in primary neuronal cultures from BAX knock-out (BAX −/− ) cells, no changes in the susceptibility to oxytosis were observed in immature BAX −/− neurons compared to wild-type neurons (Dargusch et al., 2001). However, deficiency of another Bcl-2 family member, the antiapoptotic Bcl-x L, is associated with increased susceptibility to oxytosis although these cells show a compensatory increase in cellular GSH, most probably via an activated pentose phosphate pathway (Pfeiffer et al., 2017). ...
Although nerve cell death is the hallmark of many neurological diseases, the processes underlying this death are still poorly defined. However, there is a general consensus that neuronal cell death predominantly proceeds by regulated processes. Almost 30 years ago, a cell death pathway eventually named oxytosis was described in neuronal cells that involved glutathione depletion, reactive oxygen species production, lipoxygenase activation, and calcium influx. More recently, a cell death pathway that involved many of the same steps was described in tumor cells and termed ferroptosis due to a dependence on iron. Since then there has been a great deal of discussion in the literature about whether these are two distinct pathways or cell type- and insult-dependent variations on the same pathway. In this review, we compare and contrast in detail the commonalities and distinctions between the two pathways concluding that the molecular pathways involved in the regulation of ferroptosis and oxytosis are highly similar if not identical. Thus, we suggest that oxytosis and ferroptosis should be regarded as two names for the same cell death pathway. In addition, we describe the potential physiological relevance of oxytosis/ferroptosis in multiple neurological diseases.
... 28 Bax as a pro-apoptotic protein can be activated by Ca 2+ , leading to mitochondrial membrane permeability changes, release of cytochrome c and caspase activation. [29][30][31] The results of molecular docking suggested that polyphyllin VI and pennogenin 3-O-β-chacotrioside showed good docking abilities with these proteins, even better than their inhibitors. Combined with the above cell experiment results of TTM1, it can be concluded that polyphyllin VI and pennogenin 3-O-β-chacotrioside may be one of the material bases of T tschonoskii on anti-apoptosis. ...
Context
Among the Tujia people, the root or rhizome of Trillium tschonoskii Maxim.in Bull.Acad (TTM) is considered a miraculous herb for headaches. Previous studies have shown ethyl acetate extract (TTM1) can protect SH-SY5Y cells against glutamate injury.
Objective
This study clarified TTM1’s mechanism against glutamate-induced cell damage, focusing on the regulation of apoptosis. The compounds were separated, identified, and performed molecular docking with pro-apoptotic proteins.
Materials and Methods
SH-SY5Y cells were treated with glutamate (2 mM) for 12 hour, and the effect of TTM1 (2.5, 5, 10, and 20 μg/mL) was evaluated with MTT and LDH release assays, taking EGb761(40 μg/mL) as a control. Cell apoptosis was detected with Hoechst 33258 and Annexin V-FITC and measurements of intracellular calcium and caspase-3. The major components were separated and identified by LCMS-IT-TOF and NMR, then the proapoptotic activity of TTM1 was confirmed by molecular docking method.
Results
TTM1 protected SH-SY5Y cells by resisting apoptosis, TTM1 (10 and 20 μg/mL) decreased apoptotic bodies and nuclear fragments, increased the proportion of normal cells to 68.38 ± 5.63% and 92.80 ± .88%, decreased VA cells to 4.30 ± .76% and 3.58 ± .45% and caspase-3 to .365 ± .034 and .344 ± .047 ng/mL.TTM1 (10 μg/mL) decreased intracellular free calcium to 2.77 ± .40. Polyphyllin VI and pennogenin 3-O-β-chacotrioside were identified in TTM1 at 15.04% and 2.84%, and had potential anti-apoptosis activities.
Discussion and Conclusions
Folk records of TTM for headache may be related to its anti-apoptosis of nerve cells. Identification and content determination of index components based on effective extract provides research paradigms for rare and endangered ethnic plants.
... Cysteine proteases such as calpains and caspases, are activated by Ca 2+ that degrade various substrates, including cytoskeleton proteins, metabolic enzymes and membrane receptors. It triggers apoptosis, through activation of pro-apoptotic proteins such as Bax, Par-4 and p53 [154]. Mitochondria are responsible for the "fine tuning" of Ca 2+ . ...
Essential metals including iron (Fe) and manganese (Mn) with known physiological functions in human body play an important role in cell homeostasis. Excessive exposure to these essential as well as non-essential metals including mercury (Hg) and Aluminum (Al) may contribute to pathological conditions, including PD. Each metal could be toxic through specific pathways. Epidemiological evidences from occupational and ecological studies besides various in vivo and in vitro studies have revealed the possible pathogenic role and neurotoxicity of different metals. Pesticides are substances that aim to mitigate the harm done by pests to plants and crops, and are extensively used to boost agricultural production. This review provides an outline of our current knowledge on the possible association between metals and PD. We have discussed the potential association between these two, furthermore the chemical properties, biological and toxicological aspects as well as possible mechanisms of Fe, Mn, Cu, Zn, Al, Ca, Pb, Hg and Zn in PD pathogenesis. In addition, we review recent evidence on deregulated microRNAs upon pesticide exposure and possible role of deregulated miRNA and pesticides to PD pathogenesis.
... It might quench ROS through a GSHindependent way. It had been previously claimed the Bax expression is not required for oxidative stress-induced HT22 cell death and Bcl-2 can protect cells from oxidative stress as an anti-oxidant protein, so the ratio of the Bax/ Bcl-2 determines the cells' resistance to apoptosis triggered by different stimulus [48,49]. Our findings showed that gartanin could elevated the level of Bcl-2 in time-dependent manner and effectively preserving the Bcl-2 protein level down-regulated by glutamate, whereas it did not affect the expression of Bax, the Bcl-2/Bax protein ratio increased (Fig. 2b, c). ...
Oxidative stress mediates the pathogenesis of neurodegenerative disorders. Gartanin, a natural xanthone of mangosteen, possesses multipharmacological activities. Herein, the neuroprotection capacity of gartanin against glutamate-induced damage in HT22 cells and its possible mechanism(s) were investigated for the first time. Glutamate resulted in cell death in a dose-dependent manner and supplementation of 1–10 µM gartanin prevented the detrimental effects of glutamate on cell survival. Additional investigations on the underlying mechanisms suggested that gartanin could effectively reduce glutamate-induced intracellular ROS generation and mitochondrial depolarization. We further found that gartanin induced HO-1 expression independent of nuclear factor erythroid-derived 2-like 2 (Nrf2). Subsequent studies revealed that the inhibitory effects of gartanin on glutamate-induced apoptosis were partially blocked by small interfering RNA-mediated knockdown of HO-1. Finally, the protein expression of phosphorylation of AMP-activated protein kinase (AMPK) and its downstream signal molecules, Sirtuin activator (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), increased after gartanin treatment. Taken together, these findings suggest gartanin is a potential neuroprotective agent against glutamate-induced oxidative injury partially through increasing Nrf-2-independed HO-1 and AMPK/SIRT1/PGC-1α signaling pathways.
... However, it was revealed that a mechanism by which Kv prevented neuronal cell death in this study is through inhibition of Bax. It has been discussed that in neurons a wide variety of death stimuli like oxidative stress are associated with Bax translocation in mitochondria followed by caspase activation (Dargusch et al., 2001). The antiapoptotic potentials of Kv shown in this study may be explained by the downregulation of Bax, as neurons lacking Bax are protected against apoptosis induced by growth factor deprivation and axotomy (Culmsee and Mattson, 2005). ...
Identification of therapeutic targets following neurodegeneration is of major biomedical importance. Kolaviron (Kv) is a biflavonoid complex isolated from seeds of Garcina kola - a common oral masticatory agent in Nigeria known to hold medicinal value. Therefore this study evaluated the therapeutic potential of Kv on cells of the dorsolateral prefrontal cortex (DLPFC), before or after sodium azide (NaN3)-induced neurodegeneration. Rats were randomly assigned into 5 groups (6 each) and treated daily (orally) as follows: 1 ml of corn-oil (vehicle of Kv, 21 days); Kv only (200 mg/kg) for 21 days; NaN3 only (20 mg/kg for 5 days); NaN3 (20 mg/kg for 5 days) followed by Kv (200 mg/kg for 21 days); Kv (200 mg/kg for 21 days) followed by NaN3 (20 mg/kg for 5 days). After treatments, rats were sacrificed and perfused transcardially (with 4% PFA) with brains fixed in accordance with the technique to be used. The DLPFC was examined using histology (H&E), immunoperoxidase (GFAP), immunofluorescence (iNOS & nNOS) and Western blotting (MAPT, MAP2, Bax, BCL-2 and CAD). Quantitative analysis was done using ImageJ software and statistical analysis with Graphpad prism (ANOVA) at p<0.05. NaN3 treatment induced neuronal damage, characterized by reduced relative brain weight, pyknosis, karyorrhesis, astrogliosis, axonal/dendritic damage and cytoskeletal dysregualtion that subsequently resulted in increased expressions of apoptotic regulatory proteins. These degenerative changes were relatable to the observed iNOS and nNOS upregulations. However, Kv administration attenuated the NaN3- initiated destructive molecular cascades in the DLPFC of rats through mechanisms that involved inhibition of stressor molecules and toxic proteins, prevention of stress related biochemical redox, preservation of neuronal integrity, cytoskeletal framework and subsequently, reduced the level of apoptotic regulatory proteins. We conclude that Kv conferred therapeutic benefits on NaN3- induced neurodegeneration, particularly when administered before more than after the insult.
... Opened PTP are permeable to AIF and Cytochrome-C. An active involvement of Bax into Glu induced neuronal death has been demonstrated in experiments with Bax-knockout cortical neurons cultures [Dargusch et al., 2001]. Antibodies for Bax are now widely used in investigations of neurodegeneration [Zhang, Bhavnani, 2005;Raghupathi et al., 2003]. ...
Excitotoxicity is a term that describes the neuronal death caused by neurotoxic effects of glutamate, which is the most abundant excitatory neurotransmitter in the vertebrate central nervous system. Glutamate is well known to be involved in cognitive functions like learning and memory, but its excessive accumulation in extracellular space can lead to neuronal damages and eventual cell death via necrosis and apoptosis. As a result excitotoxicity contributes to pathogenesis of numerous neurodegenerative diseases. Both normal function and pathological action imply an activation of the same glutamate receptors particularly of NMDA- (N-methyl-D-aspartate), AMPA- (a-anino-3-hydroxyl-5-methyl-4-isoxazole-propionate) and KA- (kainate) subtypes. Many achievements in the mechanisms of neurodegeneration were obtained using different experimental approaches on primary neuronal cultures. Double successive acridine orange and ethidium bromide staining combined with confocal microscopy offers fast, easy, sensitive and reproducible method by which necrosis and apoptosis can be recognized and quantified in a population of living neurons. Together with immunostaining they provide many research advantages and allow analysis of protein expression patterns. The growing quantity of evidence reveals the diversity of apoptosis cascades. Whereas our data show the same profiles of excitotoxicity for NMDA and KA, we found receptor subtype specific differences in neuronal death mechanisms. For example, apoptosis caused by prolonged NMDA receptors activation develops through the caspase-independent cascades via release of apoptosis inducing factor (AIF) from mitochondria and its direct action on nuclear chromatin. In contrast AMPA and KA receptors mediated apoptosis includes caspase-dependent pathway. On the basis of our data and literature the chapter will review the contemporary state of research concerning the aspects of excitotoxicity mechanisms discussed above.
... Second, Ca 2+ induces oxidative stress through different mechanisms (Mattson and Sherman, 2003). Third, Ca 2+ triggers apoptosis by induction/activation of various pro-apoptotic proteins (Ankarcrona et al., 1995;Dargusch et al., 2001;Culmsee and Mattson, 2005). Furthermore, excessive Ca 2+ influx relates to a decreased ATP level and activated caspases and calpains, which contribute to impairment and inactivation of both PMCA and NCXs, thus exacerbating intracellular Ca 2+ overload (Schwab et al., 2002;Bano et al., 2005;Pottorf et al., 2006;Bruce, 2010). ...
Traumatic brain injury (TBI) is a major health and socioeconomic problem throughout the world. It is a complicated pathological process that consists of primary insults and a secondary insult characterized by a set of biochemical cascades. The imbalance between a higher energy demand for repair of cell damage and decreased energy production led by mitochondrial dysfunction aggravates cell damage. At the cellular level, the main cause of the secondary deleterious cascades is cell damage that is centred in the mitochondria. Excitotoxicity, Ca2+ overload, reactive oxygen species (ROS), Bcl‐2 family, caspases and apoptosis inducing factor (AIF) are the main participants in mitochondria‐centred cell damage following TBI. Some preclinical and clinical results of mitochondria‐targeted therapy show promise. Mitochondria‐ targeted multipotential therapeutic strategies offer new hope for the successful treatment of TBI and other acute brain injuries.
... ROS generated in response to Ca 2+ influx induced by glutamate includes superoxide, hydrogen peroxide, hydroxyl radical and peroxynitrite [148]. Finally, Ca 2+ induces apoptosis through activation of pro-apoptotic proteins such Bax, Par-4 and p53, enhancing mitochondrial membrane permeability and release of cytochrome c [149,150]. Oxidative stress plays a key role in the development of many neurodegenerative disorders such as Alzheimer's and Parkinson's disease. ...
... Among the multi-BH domain proteins, Bax has been shown to accumulate at mitochondria after seizures (Henshall et al., 2002) but we await in vivo functional studies. Some in vitro data support a role for Bax in glutamate-induced neuronal death (Xiang et al., 1998), but other studies have reported bax-deficient neurons are equally vulnerable to excitotoxicity (Dargusch et al., 2001; Cheung et al., 2005). Bak does not appear to promote neuronal death in adult brain (Fannjiang et al., 2003) and there are no in vivo data on the third multi-BH domain member, Bok. ...
Epilepsy is a complex disease, characterized by the repeated occurrence of bursts of electrical activity (seizures) in specific brain areas. The behavioral outcome of seizure events strongly depends on the brain regions that are affected by overactivity. Here we review the intracellular signaling pathways involved in the generation of seizures in epileptogenic areas. Pathways activated by modulatory neurotransmitters (dopamine, norepinephrine, and serotonin), involving the activation of extracellular-regulated kinases and the induction of immediate early genes (IEGs) will be first discussed in relation to the occurrence of acute seizure events. Activation of IEGs has been proposed to lead to long-term molecular and behavioral responses induced by acute seizures. We also review deleterious consequences of seizure activity, focusing on the contribution of apoptosis-associated signaling pathways to the progression of the disease. A deep understanding of signaling pathways involved in both acute- and long-term responses to seizures continues to be crucial to unravel the origins of epileptic behaviors and ultimately identify novel therapeutic targets for the cure of epilepsy.
... Multiple lines of evidence have demonstrated that Bax is a major transcriptional target of p53 in neuronal cell apoptosis induced by p53 overexpression in vitro or after traumatic or ischemic cell apoptosis in vivo (Xiang et al., 1998;Love, 2003;Raghupathi et al., 2004). Furthermore, activation of p53 in neurons is induced by a wide variety of death stimuli (for example, glutamate and oxidative stress) and activation is associated with Bax translocation to mitochondria, leading to disruption of mitochondrial membrane potential, opening of mitochondrial permeability transition pores (MPT), cytochrome c release, and caspase activation (Paradis et al., 1996;Dargusch et al., 2001;Polster and Fiskum, 2004). Inhibition of p53 can prevent Bax upregulation, mitochondrial damage and the downstream activation of caspases; conversely, Bax-deficient neurons are protected from cell apoptosis induced by adenovirus-mediated p53 overexpression (Xiang et al., 1996;Xiang et al., 1998;Cregan et al., 1999). ...
Aphanizomenon flos-aquae (A. flos-aquae), a cyanobacterium frequently encountered in water blooms worldwide, is source of neurotoxins known as PSPs or aphantoxins that present a major threat to the environment and to human health. Although the molecular mechanism of PSP action is well known, many unresolved questions remain concerning its mechanisms of toxicity. Aphantoxins purified from a natural isolate of A. flos-aquae DC-1 were analyzed by high-performance liquid chromatography (HPLC), the major component toxins were the gonyautoxins1 and 5 (GTX1 and GTX5, 34.04% and 21.28%, respectively) and the neosaxitoxin (neoSTX, 12.77%). The LD(50) of the aphantoxin preparation was determined to be 11.33 μg/kg (7.75 μg saxitoxin equivalents (STXeq) per kg) following intraperitoneal injection of zebrafish (Danio rerio). To address the neurotoxicology of the aphantoxin preparation, zebrafish were injected with low and high sublethal doses of A. flos-aquae DC-1 toxins 7.73 and 9.28 μg /kg (5.3 and 6.4 μg STXeq/kg, respectively) and brain tissues were analyzed by electron microscopy and RT-PCR at different timepoints postinjection. Low-dose aphantoxin exposure was associated with chromatin condensation, cell-membrane blebbing, and the appearance of apoptotic bodies. High-dose exposure was associated with cytoplasmic vacuolization, mitochondrial swelling, and expansion of the endoplasmic reticulum. At early timepoints (3 h) many cells exhibited characteristic features of both apoptosis and necrosis. At later timepoints apoptosis appeared to predominate in the low-dose group, whereas necrosis predominated in the high-dose group. RT-PCR revealed that mRNA levels of the apoptosis-related genes encoding p53, Bax, caspase-3, and c-Jun were upregulated after aphantoxin exposure, but there was no evidence of DNA laddering; apoptosis could take place by pathways independent of DNA fragmentation. These results demonstrate that aphantoxin exposure can cause cell death in zebrafish brain tissue, with low doses inducing apoptosis and higher doses inducing necrosis. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2011.
... There are several overlapping pathological Ca 2+ -mediated cascades, including activation of oxygenases (as in the arachidonic metabolic cascade) and cysteine proteases, i.e., calpains (which cleave cytoskeletal proteins, membrane receptors, and metabolic enzymes), matrix metalloproteases (which degrade the extracellular matrix), and caspases (which initiate apoptotic cascades) [49]. Impairments to calcium and energy metabolism in mitochondria induce the mechanisms of oxidative stress (formation of free radicals, i.e., superoxide anions and hydroxyl and peroxynitrite radicals, lipid peroxidation) [71,127] and the induction of apoptosis [11] (activation of proapoptotic proteins Bax, Par-4, and p53, as well as increases in mitochondrial membrane permeability and release of cytochrome c) [32]. ...
The current state of questions of neurodegeneration in demyelinating autoimmune diseases is reviewed. Experimental and clinical evidence for heterogeneity in the mechanisms of nervous tissue destruction is presented. Interactions between neuroinflammatory processes, excitotoxicity, and oxidative stress in producing damage to oligodendrocytes (myelin) and neurons (axons) are addressed.
... In primary cultures of mouse cortical cells, the non-NMDA glutamate receptor agonist kainic acid (KA) induces increased Bax protein, and bax gene ablation significantly protects cells against KA receptor toxicity [202]. However, NMDA receptor toxicity in mouse cerebellar granule cells [203] and mouse cortical cells [204] was not Bax-dependent. These results suggest that non-NMDA glutamate receptor excitotoxicity is more likely than NMDA receptor-mediated excitotoxicity to induce apoptosis or apoptosis-necrosis hybrid cell death [17,28,29]; however, species-specific responses might be operative. ...
Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) are the most common human adult-onset neurodegenerative diseases. They are characterized by prominent age-related neurodegeneration in selectively vulnerable neural systems. Some forms of AD, PD, and ALS are inherited, and genes causing these diseases have been identified. Nevertheless, the mechanisms of the neuronal cell death are unresolved. Morphological, biochemical, genetic, as well as cell and animal model studies reveal that mitochondria could have roles in this neurodegeneration. The functions and properties of mitochondria might render subsets of selectively vulnerable neurons intrinsically susceptible to cellular aging and stress and overlying genetic variations, triggering neurodegeneration according to a cell death matrix theory. In AD, alterations in enzymes involved in oxidative phosphorylation, oxidative damage, and mitochondrial binding of Aβ and amyloid precursor protein have been reported. In PD, mutations in putative mitochondrial proteins have been identified and mitochondrial DNA mutations have been found in neurons in the substantia nigra. In ALS, changes occur in mitochondrial respiratory chain enzymes and mitochondrial cell death proteins. Transgenic mouse models of human neurodegenerative disease are beginning to reveal possible principles governing the biology of selective neuronal vulnerability that implicate mitochondria and the mitochondrial permeability transition pore. This review summarizes how mitochondrial pathobiology might contribute to neuronal death in AD, PD, and ALS and could serve as a target for drug therapy.
... ROS generated in response to Ca 2+ influx induced by glutamate includes superoxide, hydrogen peroxide, hydroxyl radical and peroxynitrite [148]. Finally, Ca 2+ induces apoptosis through activation of pro-apoptotic proteins such Bax, Par-4 and p53, enhancing mitochondrial membrane permeability and release of cytochrome c [149,150]. Oxidative stress plays a key role in the development of many neurodegenerative disorders such as Alzheimer's and Parkinson's disease. ...
A number of disorders, such as Alzheimer disease and diabetes mellitus, have in common the alteration of the redox balance, resulting in an increase in reactive oxygen species (ROS) generation that might lead to the development of apoptosis and cell death. It has long been known that ROS can significantly alter Ca²+ mobilization, an intracellular signal that is involved in the regulation of a wide variety of cellular functions. Cells have a limited capability to counteract the effects of oxidative stress, but evidence has been provided supporting the beneficial effects of exogenous ROS scavengers. Here, we review the effects of oxidative stress on intracellular Ca²+ homeostasis and the role of antioxidants in the prevention and treatment of disorders associated to abnormal Ca²+ mobilization induced by ROS.
... The study of the role of different proteins in neuronal physiology or pathology requires an approach that should include the selective knockdown of such proteins to study a lack-of-function effect. One traditional approach is to generate knock-out mice for the selected protein (27), but this is a time-consuming method and, sometimes, the function of the lacking protein can be replaced by another protein during b development resulting in no change in phenotype. In addition, the alternate phenotype can be lethal very early in embryonic development preventing further studies as is the case for HIF-1α (12). ...
To study the effect of a non-viral vector (carbosilane dendrimer) to efficiently deliver small interfering RNA to postmitotic neurons to study the function of hypoxia-inducible factor-1alpha (HIF1-alpha) during chemical hypoxia-mediated neurotoxicity.
Chemical hypoxia was induced in primary rat cortical neurons by exposure to CoCl(2). HIF1-alpha levels were determined by Western Blot and toxicity was evaluated by both MTT and LDH assays. Neurons were incubated with dendriplexes containing anti-HIF1-alpha siRNA and both uptake and HIF1-alpha knockdown efficiency were evaluated.
We report that a non-viral vector (carbosilane dendrimer) can deliver specific siRNA to neurons and selectively block HIF1-alpha synthesis with similar efficiency to that achieved by viral vectors. Using this method, we have found that this transcription factor plays a neuroprotective role during the early phase of chemical hypoxia-mediated neurotoxicity.
This work represents a proof-of-concept for the use of carbosilane dendrimers to deliver specific siRNA to postmitotic neurons to block selected protein synthesis. This indicates that this type of vector is a good alternative to viral vectors to achieve very high transfection levels in neurons. This also suggests that carbosilane dendrimers might be very useful for gene therapy.
... To answer this question we killed HT22 hippocampal nerve cells with several neurotoxins and conditions which induce oxidative stress, and asked if these cell death pathways are blocked by reagents which inhibit a form of programmed cell death called oxytosis (Tan et al. 2002). Oxytosis is quite distinct from apoptosis in that the Bcl-2/Bax system is not involved (Dargusch et al. 2000), there is no DNA laddering and there are a number of morphological differences from apoptosis (Tan et al. 1998). A group of reagents have been identified which inhibit this pathway. ...
Two clonal nerve-like cell lines derived from HT22 and PC12 have been selected for resistance to glutamate toxicity and amyloid toxicity, respectively. In the following experiments it was asked if these cell lines show cross-resistance toward amyloid beta peptide (Abeta) and glutamate as well as toward a variety of additional neurotoxins. Conversely, it was determined if inhibitors of oxytosis, a well-defined oxidative stress pathway, also protect cells from the neurotoxins. It is shown that both glutamate and amyloid resistant cells are cross resistant to most of the other toxins or toxic conditions, while inhibitors of oxytosis protect from glutathione and cystine depletion and H2O2 toxicity, but not from the toxic effects of nitric oxide, rotenone, arsenite or cisplatin. It is concluded that while there is a great deal of cross-resistance to neurotoxins, the components of the cell death pathway which has been defined for oxytosis are not used by many of the neurotoxins.
... The PCR conditions were as follows: 30 s at 94°C, 30 s at 60°C, and 1.5 min at 72°C for 32 cycles (BT-IgSF) or 24 cycles (G3PDH). The neurons [10][11][12], glial cells [13][14][15], macrophages [9], or bone marrow fibroblastic stromal cells [16] were prepared, as described previously. ...
We have cloned and characterized a novel gene from both human and mouse that encodes a new member of the immunoglobulin superfamily. The gene is preferentially expressed in both brain and testis, and hence, termed BT-IgSF (brain- and testis-specific immunoglobulin superfamily). The predicted protein consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Human BT-IgSF protein (431 amino acids) is 88% identical to the mouse protein (428 amino acids) and both show significant homology to coxsackie and adenovirus receptor (CAR) and endothelial cell-selective adhesion molecule (ESAM). We examined the expression of BT-IgSF with various cultured cells and found that the gene was expressed in both neurons and glial cells in vitro. Furthermore, the expression was preferentially detected in pyramidal cell layers of the dentate gyrus and hippocampus and in commissure fibers of the corpus callosum, in brain tissue sections examined. These findings suggest that BT-IgSF plays a role in the development or function of the central nervous system.
... Although neither bax Ϫ/Ϫ nor bak Ϫ/Ϫ mice display any overt neurological phenotype, numerous studies have investigated cell death in the CNS of these mice. Variable effects on dependence of NGF, excitotoxins, staurosporine, chemotherapeutic agents, and ␥-irradiation have been reported previously (Deckwerth et al., 1996;Miller et al., 1997;Johnson et al., 1998;Xiang et al., 1998;Dargusch et al., 2001;Fannjiang et al., 2003). These data suggest a redundancy of bax and bak that was indeed discovered when mice deficient for both bax and bak were generated (Lindsten et al., 2000). ...
The proapoptotic Bcl-2 family members Bak and Bax play central and redundant roles in the regulation of apoptosis. In this study, we investigated the effect of loss of Bax and Bak in the CNS. The adult bax-/-bak-/- mice display masses of densely staining cells in the proliferative zones of the brain. These cells are shown to be a mix of neural progenitor cells and postmitotic cells at different stages of neural and glial differentiation. Both neural progenitor cells and mature neurons derived from bax-/-bak-/- mice were resistant to various apoptotic stimuli. Despite this resistance, postmitotic mature bax-/-bak-/- neurons remain as sensitive to excitoxic death as wild-type neurons. Thus, Bax and Bak play a critical role in regulating the number of neural progenitor cells in the adult brain but are not absolutely required for the initiation of neuronal cell death after neurotoxic injury.
... One possible explanation for cytochrome c release from mitochondria relates to the channel forming activity of the pro-apoptotic protein Bax. It has been demonstrated that the involvement of Bax on glutamate-induced apoptosis is dependent on the nature of the insult and the experimental conditions (Dargusch et al., 2001). Our results show that glutamate-induced apoptosis occurs independently of changes in Bax expression. ...
Neurotoxicity associated with increased glutamate release results in cell death through both necrotic and apoptotic processes. In addition, tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, is a strong modulator of apoptosis in several cell types. The aims of this study were to test the hypothesis that TUDCA reduces the apoptotic threshold induced by glutamate in rat cortical neurons and examine potential transduction pathways involved in both apoptotic signaling and neuroprotection by TUDCA. The results demonstrated that exposure of cortical neurons to glutamate induced cytochrome c release and caspase activation, as well as morphologic changes of apoptosis. These events were associated with down-regulation of antiapoptotic members of the Bcl-2 family, Bcl-2 and Bcl-x(L), and dephosphorylation of the serine/threonine protein kinase Akt. Pretreatment with TUDCA significantly reduced glutamate-induced apoptosis of rat cortical neurons. In addition, TUDCA induced marked phosphorylation and translocation of Bad from mitochondria to the cytosol. Moreover, inhibition of the phosphatidylinositol 3-kinase (PI3K) survival pathway abrogated the protective effects of TUDCA, including phosphorylation and translocation of Bad. In conclusion, TUDCA appears to modulate glutamate-induced neuronal apoptosis, in part, by activating a PI3K-dependent Bad signaling pathway. These data suggest that TUDCA may be beneficial in treating neurodegenerative disorders in which increased glutamate levels contribute to the pathogenesis of the disease.
... Multiple lines of evidence identified Bax as a major transcriptional target of p53 in neuronal cell death induced by DNA damage or p53 overexpression in vitro, and after traumatic or ischemic cell death in vivo [30,127,128]. In addition, in neurons a wide variety of death stimuli (for example, glutamate, Ab, and oxidative stress) are associated with Bax translocation to mitochondria followed by disruption of mitochondrial membrane potential, release of cytochrome c, and caspase activation [15,129,130]. Inhibition of p53 prevented Bax upregulation and mitochondrial damage as well as the downstream activation of caspases, and, in turn, Bax-deficient neurons were protected from cell death induced by adenovirusmediated p53 overexpression [29,30,32]. The activation of Bax is also essential for apoptosis in response to both ionizing radiation and camptothecin. ...
The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and activation of p53 can trigger apoptosis in many cell types including neurons. Apoptosis is a form of programmed cell death that occurs in neurons during development of the nervous system and may also be responsible for neuronal deaths that occur in neurological disorders such as stroke, and Alzheimer's and Parkinson's diseases. p53 production is rapidly increased in neurons in response to a range of insults including DNA damage, oxidative stress, metabolic compromise, and cellular calcium overload. Target genes induced by p53 in neurons include those encoding the pro-apoptotic proteins Bax and the BH3-only proteins PUMA and Noxa. In addition to such transcriptional control of the cell death machinery, p53 may more directly trigger apoptosis by acting at the level of mitochondria, a process that can occur in synapses (synaptic apoptosis). Preclinical data suggest that agents that inhibit p53 may be effective therapeutics for several neurodegenerative conditions.
... While excitotoxic cell death also converges on mitochondria, the upstream signalling events are quite distinct. We and others have shown that this mode of cell death occurs independent of Bax/Bak because deletion of these proteins does not affect the rate of cell death [8][9][10]. During acute brain injury such as stroke, oxygen levels are reduced, which leads to an abrupt decrease in ATP production and rapid depolarization of the plasma membrane which occurs early after the ischemic insult in stroke. ...
Mitochondria undergo continuous fission and fusion events in physiological situations. Fragmentation of mitochondria during cell death has been shown to play a key role in cell death progression, including release of the mitochondrial apoptotic proteins. Ultrastructural changes in mitochondria, such as cristae remodeling, is also involved in cell death initiation. Here, we emphasize the important role of mitochondrial fission/fusion machinery in neuronal cell death. Unlike many other cell types such as immortalized cell lines, neurons are distinct morphologically and functionally. We will discuss how this uniqueness presents special challenges in the cellular response to neurotoxic stresses, and how this affects the mitochondrial dynamics in the regulation of cell death in neurons.
All neurons in the central nervous system of mammals express receptors for the excitatory amino acid glutamate. Although glutamatergic neurotransmission is therefore essential for the functioning of neuronal circuits in the brain and spinal cord, under certain conditions activation of glutamate receptors can trigger the death of neurons. Such excitotoxicity most often occurs when cells are coincidentally subjected to reduced levels of oxygen or glucose, increased levels of oxidative stress, trauma, or exposure to toxins or other pathogenic agents. Excitotoxicity is mediated by excessive calcium influx and release from internal organelles, oxyradical production and the activation of a form of programmed cell death called apoptosis. Proteins such as p53, Bax and Par-4 induce mitochondrial membrane permeability changes resulting in the release of cytochrome c and the activation of proteases such as caspase-3. Essentially all subcellular compartments, including the endoplasmic reticulum, mitochondria and nucleus are involved in the excitotoxic process. Excitotoxic cascades are initiated in postsynaptic dendrites where glutamate receptors are most highly concentrated, and may either cause local degeneration or plasticity of those synapses or may propagate the signals to the cell body resulting in cell death. The nervous system protects itself against excitotoxicity by deploying multiple antiexcitotoxic signaling pathways including neurotrophic signaling pathways, intrinsic stress-response pathways, and survival proteins such as protein chaperones, calcium-binding proteins and inhibitor of apoptosis proteins. A rapid accumulation of information on the molecular underpinnings of the excitotoxic process is leading to the development of novel therapeutic approaches for neurodegenerative disorders, as well as unexpected insight into mechanisms of synaptic plasticity.
Introduction Perinatal hypoxia–ischemia (HI) and asphyxia due to umbilical cord prolapse, delivery complications, airway obstruction, asthma, drowning, and cardiac arrest are significant causes of brain damage, mortality, and morbidity in infants and young children. The incidence of HI encephalopathy (HIE), for example, is ∼ 2 to 4/1000 live term births. Term infants that experience episodes of asphyxia can have damage in the brainstem and forebrain, with the basal ganglia, particularly the striatum, and somatosensory systems showing selective vulnerability. Infants surviving with HIE can have long-term neurological disability, including disorders in movement, visual deficits, learning and cognition impairments, and epilepsy. Many of these neurological disabilities are contributors to the complex clinical syndrome of cerebral palsy. Neuroimaging studies of full-term neonates and experimental studies on animal models suggest that this pattern of selective vulnerability is related to local metabolism and brain regional interconnections that instigate and propagate the damage within specific neural systems. This idea has been called the “metabolism-connectivity concept”. The neurodegeneration is partly triggered by excitotoxic mechanisms resulting from excessive activation of excitatory glutamate receptors and oxidative stress. The ion channel N-methyl-d-aspartate (NMDA) receptor and intracellular signaling networks involving calcium, nitric oxide synthase (NOS), mitochondria, and reactive oxygen species (ROS), such as superoxide, nitric oxide (NO), peroxynitrite, and hydrogen peroxide (H2O2), appear to have instrumental roles in the neuronal cell death leading to perinatal HIE.
Introduction: Cell death is important in the normal histogenesis of organs, steady state kinetics of healthy adult tissues, pathogenesis of tissue damage and disease, and disease therapy. Pathologists conceived the concept of cell death as a mechanism for disease to aid in diagnosis and therapy (Virchow, 1858). Developmental biologists then realized the essential role of cell death in tissue and organ development (Glücksmann, 1951; Lockshin and Williams, 1964; Saunders, 1966). This idea was at first received with skepticism, but now it is dogma that cell number in tissues is controlled precisely in developing and adult tissues. The absence of this precise control of cell number in tissues causes cancer (impaired apoptosis is a central step toward neoplasia). Pathologic stimuli can be extrinsic or intrinsic and can inactivate normal cell death networks or can cause abrupt or delayed cell death. Cell demise can occur as multiple types of death (Schweichel and Merker, 1973; Lockshin and Zakeri, 2002). It is compelling that a goal of human disease therapy is, on the one hand, to prevent cell death in neurologic disease and, on the other hand, to stimulate cell death in malignancy. Thus, the study of cell death is fundamental to human pathobiology and disease treatment. In this chapter, recent critical views on the contributions of the different forms of cell death to human neurodegenerative diseases and their animal and cell models will be presented.
Historically, cell death has been divided into two generic categories: apoptosis, which requires energy and in which the cell
plays an active role, and necrosis, which occurs accidentally, does not require energy consumption and is considered as a
passive, uncontrolled cell death program. Among the conceptually opposite cell death forms, apoptosis is the best understood.
This death program has been defined as developmentally programmed and ordered cellular response. Apoptosis is initiated by
cell rounding and subsequent detachment from the surrounding cells. Chromatin condenses into “crescent-like” forms abutting
the inner nuclear membrane. Plasma membrane convolutes and gives rise to characteristic vesicles containing cellular organelles
and cytoplasm, known as the “apoptotic bodies.” Apoptosis is generally not accompanied by inflammation since macrophages or
neighbouring cells engulf the formed apoptotic bodies before the loss of plasma membrane integrity (Kerr et al. 1972). In
contrast to apoptosis, necrosis is characterized by disruption of the plasma membrane with a subsequent water influx and leakage
of cell content to the surroundings. Cell death by necrosis can elicit an inflammatory response (Edinger and Thompson 2004).
1. Excitotoxicity, a major cause of neuronal death in acute and chronic neurodegenerative diseases and conditions such as stroke and Parkinson’s disease, is initiated by overstimulation of glutamate receptors, leading to calcium overload in affected neurons. The sustained high concentration of intracellular calcium constitutively activates a host of enzymes, notably the calcium‐activated proteases calpains, neuronal nitric oxide synthase (nNOS) and NADPH oxidase (NOX), to antagonise the cell survival signalling pathways and induce cell death.
2. Upon overactivation by calcium, calpains catalyse limited proteolysis of specific cellular proteins to modulate their functions; nNOS produces excessive amounts of nitric oxide (NO), which, in turn, covalently modifies specific enzymes by S‐nitrosylation; and NOX produces excessive amounts of reactive oxygen species (ROS) to inflict damage to key metabolic enzymes. Presumably, key regulatory enzymes governing cell survival and cell death are aberrantly modified and regulated by calpains, NO and ROS in affected neurons; these aberrantly modified enzymes then cooperate to induce the death of affected neurons.
3. c‐Src, an Src family kinase (SFK) member, is one of the aberrantly regulated enzymes involved in excitotoxic neuronal death. Herein we review how SFKs are functionally linked to the glutamate receptors and the biochemical and structural basis of the aberrant regulation of SFKs.
4. Results in the literature suggest that SFKs are aberrantly activated by calpain‐mediated truncation and S‐nitrosylation. Thus, the aberrantly activated SFKs are targets for therapeutic intervention to reduce the extent of brain damage caused by stroke.
Pyrroloquinoline quinone (PQQ), a cofactor in several enzyme-catalyzed redox reactions, possesses a potential capability of scavenging reactive oxygen species (ROS) and inhibiting cell apoptosis. In this study, we investigated the effects of PQQ on glutamate-induced cell death in primary cultured hippocampal neurons and the possible underlying mechanisms. We found that glutamate-induced apoptosis in cultured hippocampal neurons was significantly attenuated by the ensuing PQQ treatment, which also inhibited the glutamate-induced increase in Ca²+ influx, caspase-3 activity, and ROS production, and reversed the glutamate-induced decrease in Bcl-2/Bax ratio. The examination of signaling pathways revealed that PQQ treatment activated the phosphorylation of Akt and suppressed the glutamate-induced phosphorylation of c-Jun N-terminal protein kinase (JNK). And inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt cascade by LY294002 and wortmannin significantly blocked the protective effects of PQQ, and alleviated the increase in Bcl-2/Bax ratio. Taken together, our results indicated that PQQ could protect primary cultured hippocampal neurons against glutamate-induced cell damage by scavenging ROS, reducing Ca²+ influx, and caspase-3 activity, and suggested that PQQ-activated PI3K/Akt signaling might be responsible for its neuroprotective action through modulation of glutamate-induced imbalance between Bcl-2 and Bax.
The non-Aβ component of Alzheimer's disease (AD) amyloid (NAC) is produced from the precursor protein NACP/α-synuclein (ASN) by till now unknown mechanism. Previous study showed that like ASN, NAC peptide induced oxidative/nitrosative stress and apoptosis. Our present study focused on the mechanisms of PC12 cells death evoked by NAC peptide, with particular consideration on the role of p53 protein. On the basis of molecular and transmission electron microscopic (TEM) analysis it was found that exogenous NAC peptide (10 μM) caused mitochondria dysfunction, enhanced free radical generation, and induced both apoptotic and autophagic cell death. Morphological and immunocytochemical evidence from TEM showed marked changes in expression and in translocation of proapoptotic protein Bax. We also observed time-dependent enhancement of Tp53 gene expression after NAC treatment. Free radicals scavenger N-tert-butyl-alpha-phenylnitrone (PBN, 1 mM) and p53 inhibitor (α-Pifithrin, 20 μM) significantly protected PC12 cells against NAC peptide-evoked cell death. In addition, exposure to NAC peptide resulted in higher expression of cyclin-dependent kinase 5 (Cdk5), one of the enzymes responsible for p53 phosphorylation and activation. Concomitantly, we observed the increase of expression of Cdk5r1 and Cdk5r2 genes, coding p35 and p39 peptides that are essential regulators of Cdk5 activity. Moreover, the specific Cdk5 inhibitor (BML-259, 10 μM) protected large population of cells against NAC-evoked cell death. Our findings indicate that NAC peptide exerts its toxic effect by activation of p53/Cdk5 and Bax-dependent apoptotic signaling pathway.
Mitochondrial dynamics have been extensively studied in the context of classical cell death models involving Bax-mediated cytochrome c release. Excitotoxic neuronal loss is a non-classical death signaling pathway that occurs following overactivation of glutamate receptors independent of Bax activation. Presently, the role of mitochondrial dynamics in the regulation of excitotoxicity remains largely unknown. Here, we report that NMDA-induced excitotoxicity results in defects in mitochondrial morphology as evident by the presence of excessive fragmented mitochondria, cessation of mitochondrial fusion, and cristae dilation. Up-regulation of the mitochondrial inner membrane GTPase, Opa1, is able to restore mitochondrial morphology and protect neurons against excitotoxic injury. Opa1 functions downstream of the calcium-dependent protease, calpain. Inhibition of calpain activity by calpastatin, an endogenous calpain inhibitor, significantly rescued mitochondrial defects and maintained neuronal survival. Opa1 was required for calpastatin-mediated neuroprotection because the enhanced survival found following NMDA-induced toxicity was significantly reduced upon loss of Opa1. Our results define a mechanism whereby breakdown of the mitochondrial network mediated through loss of Opa1 function contributes to neuronal death following excitotoxic neuronal injury. These studies suggest Opa1 as a potential therapeutic target to promote neuronal survival following acute brain damage and neurodegenerative diseases.
Increasing evidence demonstrates that stress or depression can lead to atrophy and cell loss in the hippocampus. In contrast, antidepressant treatment significantly reduces apoptosis in the dentate granule cell layer and subgranular zone in animal models of depression. In the present study, we investigated the neuroprotective action of SCLM, the total saponins extracted from Chaihu-jia-longgu-muli-tang, a traditional Chinese medicinal formula which was prescribed 1000 years ago, in the reduction of apoptosis in hippocampal neurons using an experimental chronic mild stress (CMS) model. Mice were subjected to the CMS procedure for a period of 21 consecutive days. SCLM (100 mg/kg, p.o.) or fluoxetine (20 mg/ kg, p.o.) was administered during the stress periods. CMS mice showed a decreased sucrose intake over 21 days, and an increase in the number of TUNEL-positive neurons as well as up-regulation of the apoptotic-related factors, such as Bax and caspase-3 in the hippocampus, compared with control mice. On the other hand, the administration of SCLM (100 mg/kg) and fluoxetine (20 mg/kg) reversed these effects induced by CMS, showing a significant increase of sucrose intake and a dramatic reduction of TUNEL-positive neurons and decreased expression of Bax and caspase-3 proteins. The present results suggest that SCLM possesses a significant antidepressant-like property, and this effect may be through protection against stress-induced neuronal apoptosis by affecting the expression of Bax and caspase-3 proteins in the hippocampus. These findings provide important information that the anti-apoptotic effect of herbal medicine therapy may be beneficial for the treatment of depression.
Achyranthes bidentata polypeptides (ABPP), the important constituents separated from the aqueous extract of Achyranthes bidentata, have been shown to attenuate N-methyl-D-aspartate (NMDA)-induced cell apoptosis in cultured hippocampal neurons through differential modulation of NR2A- and NR2B-containing NMDA receptors. The present study sought to investigate the possible mechanism underlying the neuroprotective effect of ABPP on NMDA-induced cell death. Western blot analysis and colorimetric enzymatic assay demonstrated that ABPP pretreatment inhibited NMDA-induced increase of Bax protein expression or caspase-3 activity in cultured hippocampal neurons. Fluorescence measurements after staining with 2,7-dichlorofluorescin diacetate and rhodamine 123 showed that ABPP treatment also reversed NMDA-induced intracellular radical oxygen species (ROS) elevation and mitochondrial membrane potential depression in cultured hippocampal neurons. Furthermore, the in vivo effects of ABPP on cerebral neuronal damage during focal ischemia-reperfusion were also investigated. In rat middle cerebral artery occlusion (MCAO) model, ABPP attenuated the increase in the neurological deficit and cerebral infarction induced by focal ischemia-reperfusion, showing in vivo neuroprotective effects. The results collectively suggest that ABPP might exert neuroprotective actions through inhibiting Bax protein expression, caspase-3 activity, ROS production, and mitochondrial dysfunction that are all caused by overstimulation of NMDA receptors.
Glutamate is an endogenous excitatory neurotransmitter. At high concentrations, it is neurotoxic and contributes to the development of certain neurodegenerative diseases. There is considerable controversy in the literature with regard to whether glutamate-induced cell death in cultured HT22 cells (an immortalized mouse hippocampal cell line) is apoptosis, necrosis, or a new form of cell death. The present study focused on investigating the mechanism of glutamate-induced cell death. We found that glutamate induced, in a time-dependent manner, both necrosis and apoptosis in HT22 cells. At relatively early time points (8-12 h), glutamate induced mostly necrosis, whereas at late time points (16-24 h), it induced mainly apoptosis. Glutamate-induced mitochondrial oxidative stress and dysfunction were crucial early events required for the induction of apoptosis through the release of the mitochondrial apoptosis-inducing factor (AIF), which catalyzed DNA fragmentation (an ATP-independent process). Glutamate-induced cell death proceeded independently of the Bcl-2 family proteins and caspase activation. The lack of caspase activation likely resulted from the lack of intracellular ATP when the mitochondrial functions were rapidly disrupted by the mitochondrial oxidative stress. In addition, it was observed that activation of JNK, p38, and ERK signaling molecules was also involved in the induction of apoptosis by glutamate. In conclusion, glutamate-induced apoptosis is AIF-dependent but caspase-independent, and is accompanied by DNA ladder formation but not chromatin condensation.
Glutamate-induced excitotoxicity has been implicated in the pathogenesis of various neurological damages and disorders. In the brain damage of immature animals such as neonatal hypoxic-ischemic brain injury, the excitotoxicity appears to be more intimately involved through apoptosis. Bax, a member of the Bcl-2 family proteins, plays a key role in the promotion of apoptosis by translocation from the cytosol to the mitochondria and the release of apoptogenic factors such as cytochrome c. Recently, Bax-inhibiting peptide (BIP), a novel membrane-permeable peptide which can bind Bax in the cytosol and inhibit its translocation to the mitochondria, was developed. To investigate the possibility of a new neuroprotection strategy targeting Bax translocation in glutamate-induced neuronal cell death, cerebellar granule neurons (CGNs) were exposed to glutamate with or without BIP. Pretreatment of CGNs with BIP elicited a dose-dependent reduction of glutamate-induced neuronal cell death as measured by MTT assay. BIP significantly suppressed both the number of TUNEL-positive cells and the increase in caspases 3 and 9 activities induced by glutamate. In addition, immunoblotting after subcellular fractionation revealed that BIP prevented the glutamate-induced Bax translocation to the mitochondria and the release of cytochrome c from the mitochondria. These results suggest that agents capable of inhibiting Bax activity such as BIP might lead to new drugs for glutamate-related diseases in the future.
Opiate addiction is a chronic medical disorder characterized by drug tolerance and dependence, behavioral sensitization, vulnerability to compulsive relapse, and high mortality. In laboratory animals, the potential effect of opiate drugs to induce cell death by apoptosis is a controversial topic. This postmortem human brain study examined the status of the extrinsic and intrinsic apoptotic pathways in the prefrontal cortex of a large group of well-characterized heroin or methadone abusers. In these subjects (n=36), the immunocontent of apoptosis-1 protein (Fas) death receptor did not differ from that in age-, gender-, and postmortem delay-matched controls. In contrast, Fas-associated protein with death domain (FADD), the mediator of the death signal, was significantly decreased in the same brain samples (all addicts: 30%, n=36; short-term abuse (ST): 31%, n=15; long-term abuse (LT): 29%, n=21). The initiator caspase-8 was not altered, but FLIP(L) (Fas-associated protein with death domain-like interleukin-1beta-converting enzyme-inhibitory protein), a dominant inhibitor of caspase-8, was increased in LT addicts (19%). In the intrinsic pathway, the pro-apoptotic mitochondrial proteins Bax (Bcl-2-associated X protein) and AIF (apoptosis-inducing factor) remained unchanged, but cytochrome c was decreased (all addicts: 25%; ST: 31%; LT: 20%) and anti-apoptotic B-cell leukemia 2 (Bcl-2) increased in LT addicts (24%). The content of executioner caspase-3 and the pattern of cleavage of the nuclear enzyme poly-(ADP-ribose)-polymerase-1 (PARP-1) were similar in opiate addicts and control subjects. Taken together, the data revealed that the extrinsic and intrinsic canonical apoptotic pathways are not abnormally activated in the prefrontal cortex of opiate abusers. Instead, the chronic modulation of some of their components (downregulation of FADD and cytochrome c; upregulation of FLIP(L) and Bcl-2) suggests the induction of non-apoptotic actions by opiate drugs related to phenomena of synaptic plasticity in the brain. These neurochemical adaptations could play a major role in the development of opiate tolerance, sensitization and relapse in human addicts.
Activation of the p53-stress response pathway has been implicated in excitotoxic neuronal cell death. Recent studies have demonstrated an age-dependent induction of both p53 mRNA and protein in the rat brain following lithium-pilocarpine-mediated status epilepticus (LPSE). We investigated whether other proteins that have been shown to participate in the p53 cascade are induced by LPSE. We used immunohistochemistry to examine the expression of Mdm2, Bax, CD95/Fas/APO-1, ATM, Ref-1 and ubiquitin. A significant increase in nuclear Mdm2 immunoreactivity, which colocalized with p53, was observed in cells within hippocampal pyramidal cell layers, dentate gyrus, piriform cortex, amygdala and thalamus. Dual immunofluorescence microscopy revealed a reduction in free ubiquitin expression in cells with p53 and Mdm2 accumulation. Increased immunoreactivity for CD95/Fas/APO-1 and Bax was also detected in the same p53-positive cells. Moreover, expression of Ref-1 and ATM, which are involved in the response to oxidative stress-induced DNA damage and regulation of p53 function, were increased. Colocalization of Ref-1 and p53 suggests that Ref-1 might activate p53 function in LPSE-induced neurodegeneration. In contrast, ATM immunoreactivity was predominantly cytoplasmic suggesting that ATM may not directly modulate p53 activity in injured neurons. These results extend our previous observations with regard to activation and stabilization of p53 in injured central nervous system neurons. The data indicate that p53 induction following LPSE may activate downstream pro-apoptotic genes leading to neurodegeneration.
A growing body of evidence suggests that impaired mitochondrial energy production and increased oxidative radical damage to
the mitochondria could be causally involved in motor neuron death in amyotrophic lateral sclerosis (ALS) and in familial ALS
associated with mutations of Cu,Zn superoxide dismutase (SOD1). For example, morphologically abnormal mitochondria and impaired
mitochondrial histoenzymatic respiratory chain activities have been described in motor neurons of patients with sporadic ALS.
To investigate further the role of mitochondrial alterations in the pathogenesis of ALS, we studied mitochondria from transgenic
mice expressing wild type and G93A mutated hSOD1. We found that a significant proportion of enzymatically active SOD1 was
localized in the intermembrane space of mitochondria. Mitochondrial respiration, electron transfer chain, and ATP synthesis
were severely defective in G93A mice at the time of onset of the disease. We also found evidence of oxidative damage to mitochondrial
proteins and lipids. On the other hand, presymptomatic G93A transgenic mice and mice expressing the wild type form of hSOD1
did not show significant mitochondrial abnormalities. Our findings suggest that G93A-mutated hSOD1 in mitochondria may cause
mitochondrial defects, which contribute to precipitating the neurodegenerative process in motor neurons.
Neuronal apoptosis is induced prominently in the newborn rodent brain by glutamate receptor excitotoxicity and related insults, including trauma and hypoxia-ischemia. However, the molecular mechanisms of this neurodegeneration are unclear. We tested the hypothesis that changes in the subcellular distribution of the proapoptotic protein Bax precede the activation of downstream apoptosis-effector mechanisms such as caspase-3 cleavage and endonuclease activation during the progression of excitotoxic neuronal apoptosis in the striatum of newborn rat. Kainic acid (4 nmol) was injected into striatum of anesthetized 7-day-old rats, and the animals were killed at 2, 6, 12, and 24 h postinsult. Controls were age-matched, vehicle-injected, or naive rats. Counts of ultrastructurally confirmed striatal neuron apoptosis in brain sections were highest at 24 h. Striatal tissue was microdissected and fractionated into cytosolic, mitochondrial-, and nuclear-enriched compartments. Immunoblots showed that Bax translocates from the cytosol fraction to the mitochondrial fraction, with maximal translocation by 2 h in the absence of changes in mitochondrial accumulation. Cleaved caspase-3 levels increase progressively in both cytosolic and mitochondrial fractions between 6 and 24 h. Cleaved caspase-3 accumulates in apoptotic striatal neurons as shown by immunolocalization. Internucleosomal fragmentation of DNA coincides with caspase-3 cleavage. We conclude that rapid translocation of Bax to mitochondria precedes caspase-3 and endonuclease activation during excitotoxic neuronal apoptosis in newborn rat brain and that initiation of this death cascade occurs within 2 h after glutamate receptor activation.
Activation of glutamate receptors can trigger the death of neurons and some types of glial cells, particularly when the cells are coincidentally subjected to adverse conditions such as reduced levels of oxygen or glucose, increased levels of oxidative stress, exposure to toxins or other pathogenic agents, or a disease-causing genetic mutation. Such excitotoxic cell death involves excessive calcium influx and release from internal organelles, oxyradical production, and engagement of programmed cell death (apoptosis) cascades. Apoptotic proteins such as p53, Bax, and Par-4 induce mitochondrial membrane permeability changes resulting in the release of cytochrome c and the activation of proteases, such as caspase-3. Events occurring at several subcellular sites, including the plasma membrane, endoplasmic reticulum, mitochondria and nucleus play important roles in excitotoxicity. Excitotoxic cascades are initiated in postsynaptic dendrites and may either cause local degeneration or plasticity of those synapses, or may propagate the signals to the cell body resulting in cell death. Cells possess an array of antiexcitotoxic mechanisms including neurotrophic signaling pathways, intrinsic stress-response pathways, and survival proteins such as protein chaperones, calcium-binding proteins, and inhibitor of apoptosis proteins. Considerable evidence supports roles for excitotoxicity in acute disorders such as epileptic seizures, stroke and traumatic brain and spinal cord injury, as well as in chronic age-related disorders such as Alzheimer's, Parkinson's, and Huntington's disease and amyotrophic lateral sclerosis. A better understanding of the excitotoxic process is not only leading to the development of novel therapeutic approaches for neurodegenerative disorders, but also to unexpected insight into mechanisms of synaptic plasticity.
Caspases are cysteine proteases that mediate apoptotic death in a variety of cellular systems, including neurons. Caspases are activated through extrinsic or intrinsic pathways. The latter is used by most neurons in most situations. In this pathway, release of mitochondrial cytochrome c into the cytoplasm induces formation of the apoptosome, which leads to the activation of caspase 9 and subsequently other caspases. Recent data demonstrate that when caspase activation is inhibited at or downstream of the apoptosome, neurons undergo a delayed, caspase-independent death. Furthermore, there are instances, most notably following excitotoxic injury and calcium overload, in which the direct cell death pathway elicited differs from classical apoptosis. The molecular and biochemical features of such caspase-independent, nonapoptotic forms of neuronal death are just beginning to be elucidated, but alterations at the level of the mitochondria and noncaspase proteases play significant roles. Mitochondrial alterations in caspase-independent death may include energy depletion, generation of free radicals, opening of the permeability transition pore, and release of cytotoxic proteins, such as apoptosis-inducing factor. The particular mechanisms employed can be context dependent. In disease states, in which a combination of apoptotic and nonapoptotic death occurs, therapeutic strategies need to take into account both caspase-dependent and -independent pathways.
The Bcl-2 family of apoptotic-regulating proteins plays important roles during both neural development and maintenance of tissue homeostasis. The major antiapoptotic family members, Bcl-x(L) and Bcl-2, and the major proapoptotic proteins, Bax and Bak, show distinct temporal and spatial patterns of expression in the developing brain. Targeted deletions of Bcl-x(L) and Bcl-2 as well as Bax and Bak have proven to be important tools in delineating the process of cell death in the nervous system. These genetic models show that Bcl-x(L) and Bax play crucial roles in regulating the survival of differentiating neurons. In contrast, Bax and Bak play redundant roles in regulating the size of the neural progenitor cell population in postnatal mice and in the normal development of the retinal layers of the eye. Bax, Bcl-x(L), and Bcl-2 regulate the apoptotic response to neurotrophic factor deprivation. In contrast, excitotoxic cell death is not dependent on either Bax or Bak. In fact, the absence of proapoptotic Bcl-2 proteins can enhance the toxicity of neuroexcitatory molecules. Together, these data establish the intrinsic apoptotic pathway regulated by Bcl-2 proteins as a critical but not exclusive regulator of neural cell survival.
During development, the survival of spinal motoneurons depends on the integrity of the connection to their peripheral targets. Peripheral nerve axotomy induces apoptosis in neonatal neurons supplying axons to the nerve. Bax is known to promote apoptosis among developing neurons. To examine the effect of axotomy on spinal motoneurons in Bax-deficient (Bax-/-) and wild-type neonatal mice (Bax+/+), the sciatic nerve was axotomized on postnatal day (P) 0, and motoneurons in the fourth lumbar (L4) segment were visualized at P7 by acetylcholinesterase (AChE) histochemical staining. Presumably due to the reduction in naturally occurring cell death resulting from the deficiency of Bax, there were about 50% more AChE-positive cells in Bax-/- than in Bax+/+. Motoneurons in the dorsolateral motor pool of L4 project through the sciatic nerve. In Bax+/+, axotomy of the sciatic nerve induced significant cell loss in the pool. Most motoneurons survived such axotomy in Bax-/-, although they appeared atrophic and their AChE expression was decreased. Motoneurons may receive vital support retrogradely from their targets, and loss of such support may lead to hypofunction of spinal motoneurons, as indicated by the reduced production of AChE by axotomized motoneurons and their small size in Bax-/-.
Naturally occurring cell death is a universal feature of developing nervous systems that plays an essential role in determining adult brain function. Yet little is known about the decisions that select a subset of CNS neurons for survival and cause others to die. We report that postnatal day 0 NMDA receptor subunit 1 (NMDAR1) knockout mice display an ≈2-fold increase in cell death in the brainstem trigeminal complex (BSTC), including all four nuclei that receive somatosensory inputs from the face (principalis, oralis, interpolaris, and caudalis). Treatment with the NMDA receptor antagonist dizocilpine maleate (MK-801) for 24 h before birth also caused an increase in cell death that reached statistical significance in two of the four nuclei (oralis and interpolaris). The neonatal sensitivity to NMDA receptor hypofunction in the BSTC, and in its main thalamic target, the ventrobasal nucleus (VB), coincides with the peak of naturally occurring cell death and trigeminothalamic synaptogenesis. At embryonic day 17.5, before the onset of these events, NMDAR1 knockout does not affect cell survival in either the BSTC or the VB. Immunostaining for active caspase-3 and the neuronal marker Hu specifically confirms the presence of dying neurons in the BSTC and the VB of NMDAR1 knockout neonates. Finally, genetic deletion of Bax rescues these structures from the requirement for NMDA receptors to limit naturally occurring cell death. Taken together, the results indicate that NMDA receptors play a survival role for somatosensory relay neurons during synaptogenesis by inhibiting Bax-dependent developmental cell death.
• brainstem
• neuroprotection
• sensory systems
• trophic
• ventrobasal
When properly controlled, Ca2+ fluxes across the plasma membrane and between intracellular compartments play critical roles in fundamental functions of neurons, including the regulation of neurite outgrowth and synaptogenesis, synaptic transmission and plasticity, and cell survival. During aging, and particularly in neurodegenerative disorders, cellular Ca2+-regulating systems are compromised resulting in synaptic dysfunction, impaired plasticity and neuronal degeneration. Oxidative stress, perturbed energy metabolism and aggregation of disease-related proteins (amyloid beta-peptide, alpha-synuclein, huntingtin, etc.) adversely affect Ca2+ homeostasis by mechanisms that have been elucidated recently. Alterations of Ca2+-regulating proteins in the plasma membrane (ligand- and voltage-gated Ca2+ channels, ion-motive ATPases, and glucose and glutamate transporters), endoplasmic reticulum (presenilin-1, Herp, and ryanodine and inositol triphosphate receptors), and mitochondria (electron transport chain proteins, Bcl-2 family members, and uncoupling proteins) are implicated in age-related neuronal dysfunction and disease. The adverse effects of aging on neuronal Ca2+ regulation are subject to modification by genetic (mutations in presenilins, alpha-synuclein, huntingtin, or Cu/Zn-superoxide dismutase; apolipoprotein E isotype, etc.) and environmental (dietary energy intake, exercise, exposure to toxins, etc.) factors that may cause or affect the risk of neurodegenerative disease. A better understanding of the cellular and molecular mechanisms that promote or prevent disturbances in cellular Ca2+ homeostasis during aging may lead to novel approaches for therapeutic intervention in neurological disorders such as Alzheimer's and Parkinson's diseases and stroke.
The aim of this study was to investigate the effects of breviscapine on cultured rat hippocampal neuronal toxicity induced by glutamate. Primary hippocampal neurons were prepared from 2 day-old SD rats. After 8 days cultured in vitro, the cultures subjected to 30 min treatment of 0.1, 0.5 and 1.0 mmol x L(-1) L-glutamate, separately. Breviscapine (10, 20 and 40 micromol x L(-1)) was added into the cultures during 30 min treatment of L-glutamate and for the following 24 h respectively. After 24 h of L-glutamate treatment, flow cytometric analysis of Annexin V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that L-glutamate dose-dependently induced hippocampal neuronal apoptosis and necrosis. In agreement with these results, RT-PCR experiments indicated a biphasic regulation of X-chromosome-linked inhibitor of apoptosis protein (XIAP) mRNA after L-glutamate treatment, i. e up-regulation by 0.1 mmol x L(-1) L-glutamate and down-regulation by 0.5 and 1.0 mmol x L(-1) L-glutamate. However, breviscapine markedly reduced apoptosis and necrosis due to toxicity of 0.5 mmol L(-1) L-glutamate. Compared with the vehicle-treated L-glutamate group, the apoptosis was reduced by 30.4% and 40.1%, and necrosis was reduced by 32.5% and 38.8%, after treatment by breviscapine of 20 and 40 micromol x L(-1). Meanwhile, breviscapine obviously reversed the down-regulation of XIAP expression induced by L-glutamate (up-regulation by 45.1% and 54.9% when compared with that of the vehicle-treated glutamate group). The results from the detection of confocal laser scanning microscopy with Fluo-3, a Ca2+ probe showed an obvious increase in intracellular Ca2+ during L-glutamate treatment; and breviscapine of 20 or 40 micromol x L(-1) significantly slowed down glutamate-induced Ca2+ influx and lowered the intracellular Ca2+ peak in hippocampal neurons (P < 0.01). These results suggest that neuroprotective effect of breviscapine against glutamate excitotoxicity was associated with inhibition of the accumulation of intracellular Ca2+ and up-regulation of XIAP expression in hippocampal neurons.
Excitotoxic neuronal death, associated with neurodegenerative disorders and hypoxic insults, results from excessive exposure to excitatory neurotransmitters. Glutamate neurotoxicity is triggered primarily by massive Ca ²⁺ influx arising from overstimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondria are primary targets in excitotoxicity by confocal imaging of intracellular Ca ²⁺ ([Ca ²⁺ ] i ) and mitochondrial membrane potential (ΔΨ) on cultured rat hippocampal neurons. Sustained activation of NMDA receptors (20 min) elicits reversible elevation of [Ca ²⁺ ] i . Longer activation (50 min) renders elevation of [Ca ²⁺ ] i irreversible (Ca ²⁺ overload). Susceptibility to NMDA-induced Ca ²⁺ overload is increased when the 20 min stimuli are applied to neurons pretreated with electron transport chain inhibitors, thereby implicating mitochondria in [Ca ²⁺ ] i homeostasis during excitotoxic challenges. Remarkably, ΔΨ exhibits prominent and persistent depolarization in response to NMDA, which closely parallels the incidence of neuronal death. Blockade of the mitochondrial permeability transition pore by cyclosporin A allows complete recovery of ΔΨ and prevents cell death. These results suggest that early mitochondrial damage plays a key role in induction of glutamate neurotoxicity.
Increasing evidence suggests that glutamate neurotoxicity is partly mediated by reactive oxygen species, formed as a consequence of several processes, including arachidonic acid metabolism and nitric oxide production. Here we used an oxidation-sensitive indicator, dihydrorhodamine 123, in combination with confocal microscopy, to examine the hypothesis that electron transport by neuronal mitochondria may be an important source of glutamate-induced reactive oxygen species (ROS). Exposure to NMDA, but not kainate, ionomycin, or elevated potassium stimulated oxygen radical production in cultured murine cortical neurons, demonstrated by oxidation of nonfluorescent dihydrorhodamine 123 to fluorescent rhodamine 123. Electron paramagnetic resonance spectroscopy studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a radical-trapping agent, also showed production of ROS by cortical neurons after NMDA but not kainate exposure. NMDA-induced ROS production depended on extracellular Ca2+, and was not affected by inhibitors of nitric oxide synthase or arachidonic acid metabolism. The increased production of ROS was blocked by inhibitors of mitochondrial electron transport, rotenone or antimycin, and mimicked by the electron transport uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These data support the possibility that NMDA receptor-mediated, Ca(2+)-dependent uncoupling of neuronal mitochondrial electron transport may contribute to the oxidative stress initiated by glutamate exposure.
The cellular mechanisms by which excess exposure to the excitatory neurotransmitter glutamate can produce neuronal injury are unknown. More than a decade ago it was hypothesized that glutamate neurotoxicity (GNT) is a direct consequence of excessive neuronal excitation (“excitotoxicity” hypothesis); more recently, it has been hypothesized that a Ca influx triggered by glutamate exposure might mediate GNT (Ca hypothesis). A basic test to discriminate between these hypotheses would be to determine the dependence of GNT on the extracellular ionic environment. The excitotoxicity hypothesis predicts that GNT should depend critically on the presence of extracellular Na, since that ion appears to mediate glutamate neuroexcitation in the CNS; the Ca hypothesis predicts that GNT should depend critically on the presence of extracellular Ca. The focus of the present experiments was to determine the effects of several alterations in the extracellular ionic environment upon the serial morphologic changes that occur after mouse neocortical neurons in cell culture receive toxic exposure to glutamate. The results suggest that GNT in cortical neurons can be separated into 2 components distinguishable on the basis of differences in time course and ionic dependence. The first component, marked by neuronal swelling, occurs early, is dependent on extracellular Na and Cl, can be mimicked by high K, and is thus possibly “excitotoxic.” The second component, marked by gradual neuronal disintegration, occurs late, is dependent on extracellular Ca, can be mimicked by A23187, and is thus possibly mediated by a transmembrane influx of Ca. While either component alone is ultimately capable of producing irreversible neuronal injury, the Ca-dependent mechanism predominates at lower exposures to glutamate. Glutamate exposure likely leads to a Ca influx both through glutamate-activated cation channels and through voltage- dependent Ca channels activated by membrane depolarization. Addition of 20 mM Mg, however, did not substantially block GNT; this finding, together with the observation that GNT is largely preserved in sodium- free solution, supports the notion that the activation of voltage- dependent Ca channels may not be required for lethal Ca entry. The possibility that N-methyl-D-aspartate receptors may play a dominant role in mediating glutamate-induced lethal Ca influx is discussed.
Stimulation of the Fas (APO-1, CD95) receptor, which is present on a variety of cells, usually triggers a process of programmed cell death. Systemic injection of anti-Fas antibody into mice leads to fulminant liver destruction resulting from massive hepatocyte apoptosis, and to rapid death. Hepatocytes bear Fas but do not express Bcl-2, a protein that plays, in a number of conditions , a protective role against apoptosis. We have generated mice whose liver expresses Bcl-2 as the result of a bcl-2 transgene placed under the control of the hepatocyte-specific otl-anti-trypsin gene promoter, but is otherwise not distinguishable from that of normal mice. These mice display a marked to almost total resistance to liver damage induced by anti-Fas antibody injection. This protective effect of Bcl-2 occurs in the absence of significant variations, in the stimulated livers, in the level of expression of other proteins also involved in resistance or sensitivity to apoptosis, namely Bcl-x, Bax, Bad, Bak, and p53. Mice with protected livers, however , die almost as rapidly as normal mice, which indicates that acute lethality results from stimulation of Fas receptors present on other target organs or cells.
The amyloid beta protein is deposited in the brains of patients with Alzheimer's disease but its pathogenic role is unknown. In culture, the amyloid beta protein was neurotrophic to undifferentiated hippocampal neurons at low concentrations and neurotoxic to mature neurons at higher concentrations. In differentiated neurons, amyloid beta protein caused dendritic and axonal retraction followed by neuronal death. A portion of the amyloid beta protein (amino acids 25 to 35) mediated both the trophic and toxic effects and was homologous to the tachykinin neuropeptide family. The effects of the amyloid beta protein were mimicked by tachykinin antagonists and completely reversed by specific tachykinin agonists. Thus, the amyloid beta protein could function as a neurotrophic factor for differentiating neurons, but at high concentrations in mature neurons, as in Alzheimer's disease, could cause neuronal degeneration.
The cellular mechanisms by which excess exposure to the excitatory neurotransmitter glutamate can produce neuronal injury are unknown. More than a decade ago it was hypothesized that glutamate neurotoxicity (GNT) is a direct consequence of excessive neuronal excitation ("excitotoxicity" hypothesis); more recently, it has been hypothesized that a Ca influx triggered by glutamate exposure might mediate GNT (Ca hypothesis). A basic test to discriminate between these hypotheses would be to determine the dependence of GNT on the extracellular ionic environment. The excitotoxicity hypothesis predicts that GNT should depend critically on the presence of extracellular Na, since that ion appears to mediate glutamate neuroexcitation in the CNS; the Ca hypothesis predicts that GNT should depend critically on the presence of extracellular Ca. The focus of the present experiments was to determine the effects of several alterations in the extracellular ionic environment upon the serial morphologic changes that occur after mouse neocortical neurons in cell culture receive toxic exposure to glutamate. The results suggest that GNT in cortical neurons can be separated into 2 components distinguishable on the basis of differences in time course and ionic dependence. The first component, marked by neuronal swelling, occurs early, is dependent on extracellular Na and Cl, can be mimicked by high K, and is thus possibly "excitotoxic." The second component, marked by gradual neuronal disintegration, occurs late, is dependent on extracellular Ca, can be mimicked by A23187, and is thus possibly mediated by a transmembrane influx of Ca. While either component alone is ultimately capable of producing irreversible neuronal injury, the Ca-dependent mechanism predominates at lower exposures to glutamate. Glutamate exposure likely leads to a Ca influx both through glutamate-activated cation channels and through voltage-dependent Ca channels activated by membrane depolarization. Addition of 20 mM Mg, however, did not substantially block GNT; this finding, together with the observation that GNT is largely preserved in sodium-free solution, supports the notion that the activation of voltage-dependent Ca channels may not be required for lethal Ca entry. The possibility that N-methyl-D-aspartate receptors may play a dominant role in mediating glutamate-induced lethal Ca influx is discussed.
Digital imaging of the Ca indicator fura-2 has been used to study the responses of developing granule cells in culture to depolarization and transmitter action. Unstimulated cells bathed in Krebs saline exhibited cytoplasmic Ca ion concentrations, [Ca2+], that were generally in the 30-60 nM range. Exposure of cells to high-potassium (25 mM) saline depolarized the membrane potential and produced an immediate rise in [Ca2+] that recovered within 2-3 min in normal saline. The response grew progressively larger over the first 20 d in culture. Transient increases in [Ca2+] to levels greater than 1 microM were observed after 12-14 d in vitro, at which time the cells displayed intense electrical activity when exposed to high K. At this stage, the increases were attenuated by blocking action potential activity with TTX. In TTX-treated or immature cells, in which the transient phase of the Ca change was relatively small, a second exposure to high K typically produced a much larger Ca response that the initial exposure. The duration of this facilitation of the response persisted for periods longer than 5 min. Application of the neurotransmitter GABA induced a transient increase in membrane conductance, with a reversal potential near resting potential (approx. -60 mV), and caused an intracellular Ca2+ increase that outlasted the exposure to GABA by several minutes. Glutamate, or kainate, induced an increase in membrane conductance but with a reversal potential more positive than spike threshold. These agents also elevated intracellular Ca2+, but unlike the case with GABA, this Ca response reversed rapidly upon removal of the transmitter. The facilitatory effect of repeated exposures to high-K saline, as well as the persistent Ca elevation following a brief GABA application, suggests that granule cells possess the capability of displaying activity-dependent changes in Ca levels in culture.
In the 15 years since the neurotoxic properties of glutamate and related amino acids were first described, there has been no thoroughly convincing explanation of the pathophysiology of excitatory amino acid-induced neuronal death. These substances depolarize central neurons, increase the frequency of neuronal discharge, and augment synaptic activity, leading to the suggestion that one or more of these properties may in some way be responsible for toxicity. More recently, an excessive calcium influx triggered by amino acids has been implicated in this process. As isolation of the different factors potentially involved in amino acid neurotoxicity is virtually impossible in vivo, dispersed hippocampal cultures were used to define the pathophysiology of this process in vitro. The toxicity of glutamate, N-methyl-D-aspartate, and kainate was unaffected when calcium was deleted and tetrodotoxin added to the balanced salt solution bathing the cultures. In parallel experiments, the calcium ionophore A23187 was not toxic in the presence of calcium. These experiments failed to confirm a role for neuronal activity or calcium influx in this process. However, when depolarization was blocked by deleting sodium from the control salt solution, neither glutamate, N-methyl-D-aspartate, nor kainate produced obvious changes. Alternately, when passive chloride influx was prevented by largely deleting chloride from the bath, the cells were also unchanged by the amino acids. Further experiments showed that depolarization produced by high external potassium concentrations or veratridine was also toxic, but only in the presence of external chloride. These experiments suggest that the pathophysiology of amino acid neurotoxicity may be rather straightforward.(ABSTRACT TRUNCATED AT 250 WORDS)
Increasing evidence suggests that glutamate neurotoxicity is partly mediated by reactive oxygen species, formed as a consequence of several processes, including arachidonic acid metabolism and nitric oxide production. Here we used an oxidation-sensitive indicator, dihydrorhodamine 123, in combination with confocal microscopy, to examine the hypothesis that electron transport by neuronal mitochondria may be an important source of glutamate-induced reactive oxygen species (ROS). Exposure to NMDA, but not kainate, ionomycin, or elevated potassium stimulated oxygen radical production in cultured murine cortical neurons, demonstrated by oxidation of nonfluorescent dihydrorhodamine 123 to fluorescent rhodamine 123. Electron paramagnetic resonance spectroscopy studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a radical-trapping agent, also showed production of ROS by cortical neurons after NMDA but not kainate exposure. NMDA-induced ROS production depended on extracellular Ca2+, and was not affected by inhibitors of nitric oxide synthase or arachidonic acid metabolism. The increased production of ROS was blocked by inhibitors of mitochondrial electron transport, rotenone or antimycin, and mimicked by the electron transport uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These data support the possibility that NMDA receptor-mediated, Ca(2+)-dependent uncoupling of neuronal mitochondrial electron transport may contribute to the oxidative stress initiated by glutamate exposure.
We have developed "pure" neuronal cultures (< 1% astrocytes) from mouse neocortex to study the effect of glial cells on the response of neurons to injury. Cortical neurons were found to require glial-conditioned medium to survive. Immature neurons, 2-4 d in vitro, deprived of glial-conditioned medium, underwent apoptosis over 48 hr, as suggested by condensed nuclear morphology, DNA fragmentation, and protection by inhibition of macromolecular synthesis. Apoptosis induced by trophic factor deprivation has been described for other neuronal populations, such as superior cervical ganglion and dorsal root ganglion cells. Cortical neurons in pure culture provide another neuronal population for the study of apoptosis induced by trophic factor deprivation. We then studied the interaction of neurons and glia under excitotoxic conditions. Experiments on mature cultures showed that pure neuronal cultures were at least 10-fold more sensitive to acute glutamate exposure than were neuronal-glial ("mixed") cocultures. The difference in sensitivity between pure neurons and mixed cultures was reduced when mixed cultures were treated with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). In 24 hr exposure to N-methyl-D-aspartate (NMDA), or oxygen, glucose deprivation, pure neurons were more sensitive than mixed cultures; trans-PDC again increased the sensitivity of mixed cultures to nearly that of pure neuronal cultures. In contrast, mixed and pure neuronal cultures exposed to NMDA for 10 min, or to kainate for 24 hr, had similar injury dose-response curves, suggesting that glial glutamate uptake is a less important protective mechanism in these excitotoxic injuries. Surprisingly, pure neurons were less sensitive than mixed cultures to (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) toxicity at concentrations up to 100 microM. This does not reflect astrocyte toxicity, as AMPA at concentrations to 1 mM did not injure astrocyte cultures. Glial cultures showed increased levels of glutamate in the extracellular medium in response to exposure to AMPA, but not NMDA or kainate. However, pure neuronal and mixed cultures exposed to the same concentration of AMPA did not have elevated levels of glutamate in the media. We found that glia were generally neuroprotective under excitotoxic conditions, likely through their ability to clear extracellular glutamate. However, the presence of glia exacerbated AMPA neurotoxicity.
During ischemic brain injury, glutamate accumulation leads to overstimulation of postsynaptic glutamate receptors with intracellular Ca2+ overload and neuronal cell death. Here we show that glutamate can induce either early necrosis or delayed apoptosis in cultures of cerebellar granule cells. During and shortly after exposure to glutamate, a subpopulation of neurons died by necrosis. In these cells, mitochondrial membrane potential collapsed, nuclei swelled, and intracellular debris were scattered in the incubation medium. Neurons surviving the early necrotic phase recovered mitochondrial potential and energy levels. Later, they underwent apoptosis, as shown by the formation of apoptotic nuclei and by chromatin degradation into high and low molecular weight fragments. These results suggest that mitochondrial function is a critical factor that determines the mode of neuronal death in excitotoxicity.
The bcl-2 protooncogene, which protects various cell types from apoptotic cell death, is expressed in the developing and adult nervous system. To explore its role in regulation of neuronal cell death, we generated transgenic mice expressing Bcl-2 under the control of the neuron-specific enolase promoter, which forced expression uniquely in neurons. Sensory neurons isolated from dorsal root ganglia of newborn mice normally require nerve growth factor for their survival in culture, but those from the bcl-2 transgenic mice showed enhanced survival in its absence. Furthermore, apoptotic death of motor neurons after axotomy of the sciatic nerve was inhibited in these mice. The number of neurons in two neuronal populations from the central and peripheral nervous system was increased by 30%, indicating that Bcl-2 expression can protect neurons from cell death during development. The generation of these transgenic mice suggests that Bcl-2 may play an important role in survival of neurons both during development and throughout adult life.
The mechanism by which the bcl-2 oncogene exerts its anti-apoptotic and antioxidant action is unknown. We found that expression of bcl-2 in superoxide dismutase-deficient (SOD-) Escherichia coli resulted in increased transcription of the KatG catalase-peroxidase, a 13-fold increase in KatG activity and a 100-fold increase in resistance to hydrogen peroxide. In addition, mutation rate was increased 3-fold, and katG and oxyR, a transcriptional regulator of katG induction, were required for aerobic survival. These data indicate that Bcl-2 acts as a pro-oxidant in E. coli, i.e. Bcl-2 generates reactive oxygen intermediates. In support of a pro-oxidant mechanism in eukaryotic cells, we found a 73% increase in superoxide dismutase activity in a murine B-cell line overexpressing Bcl-2. Increases in reduced glutathione and in oxyradical damage to DNA, previously observed in other overexpressing cell lines, are additional evidence for a pro-oxidant mechanism. Thus, Bcl-2 does not appear to be an antioxidant. Instead, Bcl-2 appears to influence levels of reactive oxygen intermediates that induce endogenous cellular antioxidants. This activity of Bcl-2 may control entry into apoptosis.
Stimulation of the Fas (APO-1, CD95) receptor, which is present on a variety of cells, usually triggers a process of programmed cell death. Systemic injection of anti-Fas antibody into mice leads to fulminant liver destruction resulting from massive hepatocyte apoptosis, and to rapid death. Hepatocytes bear Fas but do not express Bcl-2, a protein that plays, in a number of conditions, a protective role against apoptosis. We have generated mice whose liver expresses Bcl-2 as the result of bcl-2 transgene placed under the control of the hepatocyte-specific alpha1-anti-trypsin gene promoter, but is otherwise not distinguishable from that of normal mice. These mice display a marked to almost total resistance to liver damage induced by anti-Fas antibody injection. This protective effect of Bcl-2 occurs in the absence of significant variations, in the stimulated livers, in the level of expression of other proteins also involved in resistance or sensitivity to apoptosis, namely Bcl-x, Bax, Bad, Bak, and p53. Mice with protected livers, however, die almost as rapidly as normal mice, which indicates that acute lethality results from stimulation of Fas receptors present on other target organs or cells.
Excitotoxic neuronal death, associated with neurodegenerative disorders and hypoxic insults, results from excessive exposure to excitatory neurotransmitters. Glutamate neurotoxicity is triggered primarily by massive Ca2+ influx arising from overstimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondria are primary targets in excitotoxicity by confocal imaging of intracellular Ca2+ ([Ca2+]i) and mitochondrial membrane potential (delta psi) on cultured rat hippocampal neurons. Sustained activation of NMDA receptors (20 min) elicits reversible elevation of [Ca2+]i. Longer activation (50 min) renders elevation of [Ca2+]i irreversible (Ca2+ overload). Susceptibility to NMDA-induced Ca2+ overload is increased when the 20 min stimuli are applied to neurons pretreated with electron transport chain inhibitors, thereby implicating mitochondria in [Ca2+]i homeostasis during excitotoxic challenges. Remarkably, delta psi exhibits prominent and persistent depolarization in response to NMDA, which closely parallels the incidence of neuronal death. Blockade of the mitochondrial permeability transition pore by cyclosporin A allows complete recovery of delta psi and prevents cell death. These results suggest that early mitochondrial damage plays a key role in induction of glutamate neurotoxicity.
Cytosolic calcium ([Ca2+]i) is an important mediator of neuronal signal transduction, participating in diverse biochemical reactions that elicit changes in synaptic efficacy, metabolic rate, and gene transcription. Excessive [Ca2+]i also has been implicated as a cause of acute neuronal injury, although measurement of [Ca2+]i in living neurons by fluorescent calcium indicators has not consistently demonstrated a correlation between [Ca2+]i and the likelihood of neuronal death after a variety of potentially lethal insults. Using fluorescence videomicroscopy and microinjected calcium indicators, we measured [Ca2+]i in cultured cortical neurons during intense activation with either NMDA (300 microM) or AMPA (450 microM). At these concentrations NMDA killed >80% of the cultured neurons by the next day, whereas neuronal death from AMPA was <20%. Using the conventional calcium indicator, fura-2/AM, we estimated [Ca2+]i elevations to be approximately 300-400 nM during exposure to either glutamate agonist. In contrast, indicators with lower affinity for calcium, benzothiazole coumarin (BTC), and fura-2/dextran reported [Ca2+]i levels >5 microM during lethal NMDA exposure, but [Ca2+]i levels were <1.5 microM during nonlethal activation of AMPA receptors or voltage-gated calcium channels. Fura-2 reported [Ca2+]i responses during brief exposure to glutamate, NMDA, AMPA, kainate, and elevated extracellular K+ between 0.5 and 1 microM. With the use of BTC, only NMDA and glutamate exposures resulted in micromolar [Ca2+]i levels. Neurotoxic glutamate receptor activation is associated with sustained, micromolar [Ca2+]i elevation. The widely used calcium indicator fura-2 selectively underestimates [Ca2+]i, depending on the route of entry, even at levels that appear to be within its range of detection.
Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax -/- neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.
The tumor suppressor gene p53 has been implicated in the loss of neuronal viability, but the signaling events associated with p53-mediated cell death in cortical and hippocampal neurons are not understood. Previous work has shown that adenovirus-mediated delivery of the p53 gene causes cortical and hippocampal neuronal cell death with some features typical of apoptosis. In the present study we determined whether p53-initiated changes in neuronal viability were dependent on members of the Bcl-2 family of cell death regulators. Primary cultures of cortical neurons were derived from animals containing Bax (+/+ and +/-) or those deficient in Bax (-/-). Cell damage was assessed by direct cell counting and by measurements of MTT activity. Neurons containing at least one copy of the Bax gene were damaged severely by exposure to excitotoxins or by the induction of DNA damage. In contrast, Bax-deficient neurons (-/-) exhibited significant protection from both types of injury. Bax protein expression was elevated significantly by glutamate exposure, but not by camptothecin-induced DNA damage in wild-type neurons. The glutamate-induced increase in Bax protein was dependent on the presence of the p53 gene. However, increased p53 expression, using adenovirus-mediated transduction, was not sufficient by itself to elevate Bax protein levels. These results demonstrate that Bax is required for neuronal cell death in response to some forms of cytotoxic injury and further support the key role for p53 activation in response to excitotoxic and genotoxic injury.
Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5-10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200-400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5-10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1beta-converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.
In this study, we utilized potent antisense oligonucleotides to examine the role of two Bcl-2 family members found in human umbilical vein endothelial cells (HUVEC). The first, A1, is thought to be a TNF-α-inducible cytoprotective gene, and the second, Bcl-XL, is constitutively expressed.
Inhibition of the constitutive levels of Bcl-XL caused 10–25% of the cell population to undergo apoptosis and increased the susceptibility of cells to treatment with low concentrations of staurosporin or ceramide. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2 prevented DNA fragmentation and ΔYm loss caused by Bcl-XL inhibition or Bcl-XL inhibition combined with staurosporin. However, disruption of ΔYm caused by Bcl-XL inhibition combined with ceramide treatment was not inhibited by benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2, although DNA fragmentation was completely prevented. Taken together, these results demonstrate a direct protective role for Bcl-XL under normal resting conditions and under low level apoptotic challenges to HUVEC. Furthermore, Bcl-XL protects cells from caspase-dependent and -independent mechanisms of ΔYm disruption.
In contrast to Bcl-XL, A1 inhibition did not show a marked effect on the susceptibility of HUVEC to undergo apoptosis in response to TNF-α, ceramide, or staurosporin. These results demonstrate that although A1 may be a cytoprotective gene induced by TNF-α, it is not primarily responsible for HUVEC resistance to this cytokine.
Transport system xc- found in plasma membrane of cultured mammalian cells is an exchange agency for anionic amino acids with high specificity for anionic form of cystine and glutamate. We have isolated cDNA encoding the transporter for system xc- from mouse activated macrophages by expression in Xenopus oocytes. The expression of system xc- activity in oocytes required two cDNA transcripts, and the sequence analysis revealed that one is identical with the heavy chain of 4F2 cell surface antigen (4F2hc) and the other is a novel protein of 502 amino acids with 12 putative transmembrane domains. The latter protein, named xCT, showed a significant homology with those recently reported to mediate cationic or zwitterionic amino acid transport when co-expressed with 4F2hc. Thus xCT is a new member of a family of amino acid transporters that form heteromultimeric complex with 4F2hc, with a striking difference in substrate specificity. The expression of system xc- was highly regulated, and Northern blot analysis demonstrated that the expression of both 4F2hc and xCT was enhanced in macrophages stimulated by lipopolysaccharide or an electrophilic agent. However, the expression of xCT was more directly correlated with the system xc- activity.
p53 is a pivotal molecule regulating the death of neurons both after acute injury and during development. The molecular mechanisms by which p53 induces apoptosis in neuronal cells, however, are not well understood. We have shown previously that adenovirus-mediated p53 gene delivery to neurons was sufficient to induce apoptosis. In the present study we have examined the molecular mechanism by which p53 evokes neuronal cell death. Adenovirus-mediated delivery of p53 to cerebellar granule neurons resulted in caspase-3 (CPP32) activation followed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining and loss of viability as determined by an MTT survival assay. To determine whether Bax is essential for caspase-3 activation, p53 was expressed in Bax-deficient cells. Bax null neurons did not exhibit caspase-3 activation in response to p53 and were protected from apoptosis. To determine whether Bax-dependent caspase-3 activation was required in p53-mediated neuronal cell death, caspase-3-deficient neurons were examined. Our results indicate that caspase-3-deficient neurons exhibit a remarkable delay in apoptosis and a dramatic decrease in TUNEL-positive cells. These studies demonstrate that p53-induced cell death in postmitotic neurons involves a Bax-dependent caspase-3 activation, suggesting that these molecules are important determinants in neuronal cell death after injury.
Ataxia-telangiectasia is a hereditary multisystemic disease resulting from mutations of ataxia telangiectasia, mutated (ATM) and is characterized by neurodegeneration, cancer, immune defects, and hypersensitivity to ionizing radiation. The molecular
details of ATM function in the nervous system are unclear, although the neurological lesion in ataxia-telangiectasia becomes
apparent early in life, suggesting a developmental origin. The central nervous system (CNS) of Atm-null mice shows a pronounced
defect in apoptosis induced by genotoxic stress, suggesting ATM functions to eliminate neurons with excessive genomic damage.
Here, we report that the death effector Bax is required for a large proportion of Atm-dependent apoptosis in the developing
CNS after ionizing radiation (IR). Although many of the same regions of the CNS in both Bax−/− and Atm−/− mice were radioresistant, mice nullizygous for both Bax and Atm showed additional reduction in IR-induced apoptosis in the CNS. Therefore, although the major IR-induced apoptotic pathway
in the CNS requires Atm and Bax, a p53-dependent collateral pathway exists that has both Atm- and Bax-independent branches.
Further, Atm- and Bax-dependent apoptosis in the CNS also required caspase-3 activation. These data implicate Bax and caspase-3
as death effectors in neurodegenerative pathways.
There is an increasing amount of experimental evidence that oxidative stress is a causal, or at least an ancillary, factor in the neuropathology of several adult neurodegenerative disorders, as well as in stroke, trauma, and seizures. At the same time, excessive or persistent activation of glutamate-gated ion channels may cause neuronal degeneration in these same conditions. Glutamate and related acidic amino acids are thought to be the major excitatory neurotransmitters in brain and may be utilized by 40 percent of the synapses. Thus, two broad mechanisms--oxidative stress and excessive activation of glutamate receptors--are converging and represent sequential as well as interacting processes that provide a final common pathway for cell vulnerability in the brain. The broad distribution in brain of the processes regulating oxidative stress and mediating glutamatergic neurotransmission may explain the wide range of disorders in which both have been implicated. Yet differential expression of components of the processes in particular neuronal systems may account for selective neurodegeneration in certain disorders.
The BCL-2 gene was identified at the chromosomal breakpoint of t(14; 18)-bearing human follicular B cell lymphomas. BCL-2 proved to block programmed cell death rather than promote proliferation. Transgenic mice that overexpress Bcl-2 in the B cell lineage demonstrate extended cell survival and progress to high-grade lymphomas. Thus, BCL-2 initiated a new category of oncogenes, regulators of cell death. Bcl-2-deficient mice demonstrate fulminant apoptosis of lymphocytes, profound renal cell death and loss of melanocytes. BCL-2 protein duels with its counteracting twin, a partner known as BAX. When BAX is in excess, cells execute a death command; but, when BCL-2 dominates, the program is inhibited and cells survive. Bax-deficient mice display cellular hyperplasia, confirming its role as a proapoptotic molecule. An expanded family of BCL-2-related proteins shares homology clustered within four conserved regions termed BCL-2 homology 1 through 4 (BH1-4). These novel domains control the ability of these proteins to dimerize and function. An amphipathic alpha helix, BH3, is of particular importance for the proapoptotic family members. BID and BAD represent an evolving set of proapoptotic molecules, which bear sequence homology only at BH3. They appear to reside more proximal in the pathway serving as death ligands. BAD connects upstream signal transduction paths with the BCL-2 family, modulating this checkpoint for apoptosis. In the presence of survival factor interleukin-3, cells phosphorylate BAD on two serine residues. This inactivated BAD is held by the 14-3-3 protein, freeing BCL-XL and BCL-2 to promote survival. Activation of BAX results in the initiation of apoptosis. Downstream events in this program include mitochondrial dysfunction, as well as Caspase activation. The pro- and antiapoptotic BCL-2 family members represent central regulators in an evolutionarily conserved pathway of cell death. Aberrations in the BCL-2 family result in disordered homeostasis, a pathogenic event in diseases, including cancer.
Excitotoxicity refers to the ability of glutamate or related excitatory amino acids to mediate the death of central neurons under certain conditions, for example, after intense exposure. Such excitotoxic neuronal death may contribute to the pathogenesis of brain or spinal cord injury associated with several human disease states. Excitotoxicity has substantial cellular specificity and, in most cases, is mediated by glutamate receptors. On average, NMDA receptors activation may be able to trigger lethal injury more rapidly than AMPA or kainate receptor activation, perhaps reflecting a greater ability to induce calcium influx and subsequent cellular calcium overload. It is possible that excitotoxic death may share some mechanisms with other forms of neuronal death. © 1992 John Wiley & Sons, Inc.
Glutamate binds to both excitatory neurotransmitter binding sites and a Cl−-dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (N18-RE105) are susceptible to glutamate-induced cytotoxicity. The Cl−-dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Lowering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and Ca2+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like N18-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.
Brief exposure to glutamate produced widespread neuronal death in mature, but not young, cortical cell cultures. Extracellular sodium replacement or addition of tetrodotoxin produced only minor reduction in this toxic neuronal loss. However, removal of extracellular calcium markedly reduced neuronal loss, and elevation of extracellular calcium accentuated neuronal loss. These observations suggest that the toxicity of glutamate on cortical neurons may depend primarily on the presence of extracellular Ca, probably through a mechanism which is distinct from simple 'excitotoxicity'.
Information obtained over the past 25 years indicates that the amino acid glutamate functions as a fast excitatory transmitter in the mammalian brain. Studies completed during the last 15 years have also demonstrated that glutamate is a powerful neurotoxin, capable of killing neurons in the central nervous system when its extracellular concentration is sufficiently high. Recent experiments in a variety of preparations have shown that either blockade of synaptic transmission or the specific antagonism of postsynaptic glutamate receptors greatly diminishes the sensitivity of central neurons to hypoxia and ischemia. These experiments suggest that glutamate plays a key role in ischemic brain damage, and that drugs which decrease the accumulation of glutamate or block its postsynaptic effects may be a rational therapy for stroke.
Glutamate binds to both excitatory neurotransmitter binding sites and a Cl(-)-dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (N18-RE-105) are susceptible to glutamate-induced cytotoxicity. The Cl(-)-dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Lowering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and Ca2+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like N18-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.
In slices of developing rat cerebellum, a 30-min application of the excitatory amino acid receptor agonist, N-methyl-D-aspartate (NMDA), led to the necrosis of differentiating granule cells and deep nuclear neurones. The corresponding effect of another agonist, kainate, was the death of Golgi cells. The toxic effects of both agonists were prevented if the concentration of calcium in the exposing solution was reduced to 0.3 mM from the control level of 2.5 mM. A lesser reduction (to 1 mM) was enough to prevent 90% of the NMDA-induced necrosis of granule cells. The results indicate that an important component of the acute neurotoxic effects of excitatory amino acids is calcium-dependent and suggest reasons why this may not have been revealed in some previous studies.
Here I have reviewed evidence from electron microscopic studies showing that each of several sustained limbic seizure syndromes is associated with a type of acute brain damage which is ultrastructurally indistinguishable from the brain damage induced by Glu and other excitotoxins. In addition, I have presented evidence that persistent stimulation of specific axonal tracts that use Glu as transmitter results in Glu-like excitotoxic degeneration of postsynaptic neurons innervated by such tracts. Phencyclidine and ketamine, which powerfully block the neurotoxicity of the Glu analog NMA, protect against seizure-related brain damage. This may be explained by either an anticonvulsant or antiexcitotoxic mechanism, or both. Recent evidence suggests that an excitotoxic mechanism (excessive activation of Glu/Asp receptors) may underlie both seizure-mediated and anoxic brain damage. The acute fulminating type of neuronal degeneration induced by Glu is a Na+ and Cl- but not Ca2+ dependent phenomenon. According to a recent study, however, Glu may induce neuronal necrosis not only by an acute Ca2+ independent process but by a more slowly evolving Ca2+ dependent process. If, as these data suggest, an excitotoxic mechanism underlies brain damage associated with anoxia and epilepsy, a better understanding of excitotoxic mechanisms may lead eventually to prophylactic approaches for preventing such forms of brain damage.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Excerpt
The genetic control of tumorigenesis has been greatly advanced by the discovery of oncogenes. A central dogma holds that oncogenes induce an overt proliferation providing the driving force for oncogenesis. Cell death has long been recognized as a physiological event during embryonic development. Moreover, a distinct morphological form of cell destruction, apoptosis, had also been described even within cancer tissue (Kerr et al. 1972). In many respects, cancer can be viewed as a violation of normal tissue homeostasis. The maintenance of a relatively constant number of cells in normal tissues reflects a balanced equation between cell proliferation and cell death (Fig. 1). Aberrations of homeostasis that manifest as tumorigenesis would include events that promote proliferation or repress cell death. The history of cancer genetics is replete with examples of oncogenes that promote proliferation. However, bcl-2 provided the first certain example of an oncogene that regulated cell demise. The overexpression of...
There is an increasing amount of experimental evidence that oxidative stress is a causal, or at least an ancillary, factor
in the neuropathology of several adult neurodegenerative disorders, as well as in stroke, trauma, and seizures. At the same
time, excessive or persistent activation of glutamate-gated ion channels may cause neuronal degeneration in these same conditions.
Glutamate and related acidic amino acids are thought to be the major excitatory neurotransmitters in brain and may be utilized
by 40 percent of the synapses. Thus, two broad mechanisms--oxidative stress and excessive activation of glutamate receptors--are
converging and represent sequential as well as interacting processes that provide a final common pathway for cell vulnerability
in the brain. The broad distribution in brain of the processes regulating oxidative stress and mediating glutamatergic neurotransmission
may explain the wide range of disorders in which both have been implicated. Yet differential expression of components of the
processes in particular neuronal systems may account for selective neurodegeneration in certain disorders.
The 26-kDa protein encoded by the bcl-2 gene is a regulator of cell survival and blocks cell death induced by numerous stimuli. Amyloid beta protein (ABP) and glutamate are believed to play important roles in the neuronal cell death that occurs in Alzheimer's disease and stroke, respectively. Glutamate induces apoptosis in some neuronal cell systems, but it remains controversial whether ABP-mediated cell death occurs through apoptosis or necrosis. To further explore the pathways for cell death that are activated by these neurotoxins, we examined the effects of elevated levels of the p26-Bcl-2 protein on the susceptibility of neuronal cell lines to killing by glutamate and ABP. Gene transfer methods were used to elevate p26-Bcl-2 protein levels in the rat nerve lines PC-12 and B50 and the human neuroblastoma IMR-5. Bcl-2 protected all 3 cell lines from glutamate induced cell death but had no effect on killing mediated by ABP.
A neuronal cell line, HT-22, is sensitive to glutamate cytotoxicity via a non-receptor mediated oxidative pathway. 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, blocks this glutamate-induced cell death. Down-regulation of protein kinase C eliminates the protection against glutamate cytotoxicity afforded by TPA. The data suggest that protein kinase C activation blocks an early step in the cytotoxic pathway.
We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman, Brain Res 494: 65-74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement, B27, produced neuron survival above 60%, independent of plating density above 160 plated cells/mm2. For isolated cells (< 100 cells/mm2), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones.
We tested the pathogenic role of O2-) radicals in excitotoxic injury. Inactivation of the TCA cycle enzyme, aconitase, was used as a marker of intracellular O2- levels, and a porphyrin SOD mimetic was used to scavenge O2-. The selective, reversible, and SOD-sensitive inactivation of aconitase by known O2- generators was used to validate aconitase activity as a marker of O2- generation. Treatment of rat cortical cultures with NMDA, KA, or the intracellular O2- generator PQ2+ produced a selective and reversible inactivation of aconitase, which closely correlated with subsequent cell death produced by these agents. The SOD mimetic, but not its less active congener, attenuated both aconitase inactivation and cell death produced by NMDA, KA, and PQ2+. These results provide direct evidence implicating O2(-) generation in the pathway to excitotoxic injury.
Members of the BCL2-related family of proteins either promote or repress programmed cell death. BAX, a death-promoting member, heterodimerizes with multiple death-repressing molecules, suggesting that it could prove critical to cell death. We tested whether Bax is required for neuronal death by trophic factor deprivation and during development. Neonatal sympathetic neurons and facial motor neurons from Bax-deficient mice survived nerve growth factor deprivation and disconnection from their targets by axotomy, respectively. These salvaged neurons displayed remarkable soma atrophy and reduced elaboration of neurities; yet they responded to readdition of trophic factor with soma hypertrophy and enhanced neurite outgrowth. Bax-deficient superior cervical ganglia and facial nuclei possessed increased numbers of neurons. Our observations demonstrate that trophic factor deprivation-induced death of sympathetic and motor neurons depends on Bax.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is one of the most frequently used methods for measuring cell proliferation and neural cytotoxicity. It is widely assumed that MTT is reduced by active mitochondria in living cells. By using isolated mitochondria from rat brain and B12 cells, we indeed found that malate, glutamate, and succinate support MTT reduction by isolated mitochondria. However, the data presented in this study do not support the exclusive role of mitochondria in MTT reduction by intact cells. Using a variety of approaches, we found that MTT reduction by B12 cells is confined to intracellular vesicles that later give rise to the needle-like MTT formazan at the cell surface. Some of these vesicles were identified as endosomes or lysosomes. In addition, MTT was found to be membrane impermeable. These and other results suggest that MTT is taken up by cells through endocytosis and that reduced MTT formazan accumulates in the endosomal/lysosomal compartment and is then transported to the cell surface through exocytosis.
The BCL-2 family has various pairs of antagonist and agonist proteins that regulate apoptosis. Whether their function is interdependent is uncertain. Using a genetic approach to address this question, we utilized gain- and loss-of-function models of Bcl-2 and Bax and found that apoptosis and thymic hypoplasia characteristic of Bcl-2-deficient mice are largely absent in mice also deficient in Bax. A single copy of Bax promoted apoptosis in the absence of Bcl-2. In contrast, overexpression of Bcl-2 still repressed apoptosis in the absence of Bax. While an in vivo competition exists between Bax and Bcl-2, each is able to regulate apoptosis independently.
Bax, a family member of the survival protein Bcl-2, is expressed in the nervous system during development and throughout adulthood. Bax deficiency has been demonstrated to prevent developmental and trophic factor deprivation-induced neuronal death. To further clarify the role of Bax in naturally occurring neuronal death and in neuronal death following apoptotic stimuli, we generated several lines of transgenic mice expressing the human Bax protein specifically in neurons, under the control of the neuron-specific enolase promoter. Transgene expression was first detected around E10.5 and E12.5, depending on the transgenic line. The total number of ganglion cells in the retina and of pyramidal cells in the hippocampus, both expressing the transgene, was similar in control and transgenic mice. In addition, in our model system, Bax overexpression did not appear to influence the in vitro survival of sensory neurons isolated from dorsal root ganglia after nerve grwoth factor (NFG) deprivation or the apoptotic death of motor neurons following axotomy.
An expanding family of BCL-2 related proteins share homology, clustered within four conserved regions, namely BCL-2 homology (BH1-4) domains, which control the ability of these proteins to dimerize and function as regulators of apoptosis. Moreover, BCL-XL, BCL-2, and BAX can form ion-conductive pores in artificial membranes. The BCL-2 family, comprised of both pro-apoptotic and anti-apoptotic members, acts as a checkpoint upstream of CASPASES and mitochondrial dysfunction. BID and BAD possess the minimal death domain BH3, and the phosphorylation of BAD connects proximal survival signals to the BCL-2 family. BCL-2 and BCL-XL display a reciprocal pattern of expression during lymphocyte development. Gain- and loss-of-function models revealed stage-specific roles for BCL-2 and BCL-XL. BCL-2 can rescue maturation at several points of lymphocyte development. The BCL-2 family also reveals evidence for a cell-autonomous coordination between the opposing pathways of proliferation and cell death.
Oxidative stress is implicated in a number of neurological disorders including stroke, Parkinson's disease, and Alzheimer's disease. To study the effects of oxidative stress on neuronal cells, we have used an immortalized mouse hippocampal cell line (HT-22) that is particularly sensitive to glutamate. In these cells, glutamate competes for cystine uptake, leading to a reduction in glutathione and, ultimately, cell death. As it has been reported that protein kinase C activation inhibits glutamate toxicity in these cells and is also associated with the inhibition of apoptosis in other cell types, we asked if glutamate toxicity was via apoptosis. Morphologically, glutamate-treated cells underwent plasma membrane blebbing and cell shrinkage, but no DNA fragmentation was observed. At the ultrastructural level, there was damage to mitochondria and other organelles although the nuclei remained intact. Protein and RNA synthesis inhibitors as well as certain protease inhibitors protected the cells from glutamate toxicity. Both the macromolecular synthesis inhibitors and the protease inhibitors had to be added relatively soon after the addition of glutamate, suggesting that protein synthesis and protease activation are early and distinct steps in the cell death pathway. Thus, the oxidative stress brought about by treatment with glutamate initiates a series of events that lead to a form of cell death distinct from either necrosis or apoptosis.
The BCL2 family member BAX is required for the induction of apoptosis in neonatal sympathetic neurons after NGF withdrawal. Bax-deficient sympathetic neurons are NGF-independent for survival. To characterize the physiological state of neurons protected by BAX deficiency and to place BAX within the death pathway, we determine which of the molecular changes induced by NGF deprivation depend on BAX and compare the results with those for neurons protected by caspase inhibition. We find that neurons deficient in both Bax and Bcl2 resist NGF-deprivation similar to Bax-deficient neurons discounting a role for BCL2 in the mechanism by which Bax deficiency causes trophic factor independence. We identify two new molecular changes, phosphorylation of c-Jun on Ser63 and alpha-spectrin proteolysis, which precede and accompany apoptosis, respectively. Early reversible changes induced by NGF withdrawal, such as decreased protein synthesis and glucose uptake, increased c-Jun phosphorylation, increased steady state c-jun mRNA levels, and cellular atrophy, occur both in wild type and Bax-deficient neurons and thus are BAX-independent. In contrast to neurons protected by caspase inhibition, no c-fos induction occurs in Bax-deficient neurons. Terminal irreversible events of apoptosis such as caspase-mediated alpha-spectrin proteolysis are prevented by both Bax-deficiency and caspase inhibition. This places BAX downstream or in a different pathway of the early changes and upstream of the terminal events such as those leading to c-fos induction and caspase activation. This order indicates that the physiological state of NGF-deprived neurons protected by Bax deficiency may be less perturbed than that of caspase inhibitor-saved neurons.