Neuronal Cell Death Pathways Research Articles

Neuroprotective Role of a Proline-Rich Akt Substrate in Apoptotic Neuronal Cell Death after Stroke: Relationships with Nerve Growth Factor

The Akt signaling pathway contributes to regulation of apoptosis after a variety of cell death stimuli. A novel proline-rich Akt substrate (PRAS) was recently detected and found to be involved in apoptosis. In our study, Akt activation was modulated by growth factors, and treatment with nerve growth factor (NGF) reduced apoptotic cell death after ischemic injury. However, the role of the PRAS pathway in apoptotic neuronal cell death after ischemia remains unknown. Phosphorylated PRAS (pPRAS) and the binding of pPRAS/phosphorylated Akt (pPRAS/pAkt) to 14-3-3 (pPRAS/14-3-3) were detected, and their expression transiently decreased in mouse brains after transient focal cerebral ischemia (tFCI). Liposome-mediated pPRAS cDNA transfection induced overexpression of pPRAS, promoted pPRAS/14-3-3, and inhibited apoptotic neuronal cell death after tFCI. The expression of pPRAS, pPRAS/pAkt, and pPRAS/14-3-3 increased in NGF-treated mice but decreased with inhibition of phosphatidylinositol-3 kinase and the NGF receptor after tFCI. These results suggest that PRAS phosphorylation and its interaction with pAkt and 14-3-3 might play an important role in neuroprotection mediated by NGF in apoptotic neuronal cell death after tFCI.

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Gene expression in TUNEL-positive neurons in human immunodeficiency virus-infected brain

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) results in neuronal damage and apoptosis, and both in vitro models and pathological studies suggest that a variety of neurotoxins released by HIV-infected and -activated macrophages/microglia selectively damage susceptible subsets of neurons. Confirmation of in vitro findings of mechanisms of neurodegeneration and neuronal cell dysfunction in vivo has been approached through detailed pathological analysis of regional structural damage, immunohistochemical detection of selected antigens in damaged cells, and, more recently, analysis of gene expression in whole tissue blocks or pooled populations (hundreds/thousands) of microdissected cells. Recently developed techniques of gene expression analysis through antisense mRNA amplification (aRNA) at the single-cell level may offer the potential to study pathways of neuronal cell death and to determine patterns of coordinated gene expression that may more specifically identify susceptible neuronal subclasses in vivo. Utilizing this unique technique, the authors have demonstrated, for the first time, RNA amplification and gene expression profiling in individual deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-reactive neurons microdissected from fixed, archival human brain tissue. RNA amplification was successful in >80% of TUNEL-positive neurons, and quantitative aRNA/cDNA hybridization slot-blot analysis demonstrated similar levels of actin RNA but significant differences in caspase-2 RNA expression between TUNEL-reactive and -nonreactive neurons. Reliable quantitative comparisons were achieved with modest numbers of sampled neurons (approximately 10). These studies suggest that analysis of coordinated gene expression in individual damaged neurons in vivo can be reliably used to identify neuronal subclasses that express certain susceptibility- or survival-promoting genes that may be targeted for more specific neuroprotective strategies against HIV.

Methods for culturing primary sympathetic neurons and for determining neuronal viability.

Developing nerve growth factor (NGF)-dependent sympathetic neurons are one of the best-studied in vitro models of neuronal apoptosis and have been used to identify key components of the neuronal cell death pathway. This chapter describes how to prepare purified cultures of primary sympathetic neurons and how to induce apoptosis by NGF deprivation. In addition, a simple method for measuring neuronal viability based on the live/dead assay is also described. This can be used for assessing the effect of small molecule inhibitors of protein kinases, caspases and other enzymes, on NGF withdrawal-induced death.

Fundamental role of the Rip2/caspase-1 pathway in hypoxia and ischemia-induced neuronal cell death.

Caspase-1 plays a key role in inflammatory pathways by processing pro-IL-1beta into the active cytokine mature IL-1beta. Given its sequence similarity with the Caenorhabditis elegans cell death gene ced-3,it has long been speculated that caspase-1 may also play a role in cell death. However, an unequivocal role for caspase-1 in cell death has been questioned, and not definitively demonstrated. Furthermore, if caspase-1 does play a role in cell death, its position in the apoptotic hierarchy has not been clearly defined. Previous studies have shown that caspase-1 knockout (KO) mice and transgenic mice expressing a dominant-negative caspase-1 construct are resistant to ischemic brain injury. We provide direct evidence that caspase-1 plays a key role in neuronal cell death and that caspase-1 is an apical activator of the cell death pathway in the premitochondrial collapse stage. Furthermore, we demonstrate that Rip2/Cardiak/Rick is a stress-inducible upstream modulator of pro-caspase-1 apoptotic activation. We provide evidence that Bid cleavage appears to be an important downstream effector of caspase-1-mediated cell death. Our data demonstrate that caspase-1 is an apical mediator of neuronal cell death during in vitro hypoxia, and confirmed in vivo in ischemia, and provide insights into the sequence of events involved in this pathological cell death process.

Inhibition of CDK5 is protective in necrotic and apoptotic paradigms of neuronal cell death and prevents mitochondrial dysfunction

Previous studies suggested that pro-apoptotic stimuli may trigger a fatal reactivation of cell cycle elements in postmitotic neurons. Supporting this hypothesis, small molecule inhibitors of cyclin-dependent kinases (CDKs), which are known primarily as cell cycle regulators, are neuroprotective. However, available CDK inhibitors cannot discriminate between the different members of the CDK family and inhibit also CDK5, which is not involved in cell cycle control. Testing a new class of CDK inhibitors, we find that inhibitory activity against CDK5, but not cell cycle-relevant CDKs, confers neuroprotection. Moreover, we demonstrate that cleavage of the CDK5 activator protein p35 to p25 is associated with CDK5 overactivation after focal cerebral ischemia, but not in other models used in this study. We find that blocking CDK5 activity, but not caspase inhibition, protects mitochondrial integrity of lesioned neurons. Thus, in our models, CDK5, rather than cell cycle-relevant CDKs, activates neuronal cell death pathways upstream of mitochondrial dysfunction, and inhibition of CDK5 may promote functional long-term rescue of injured neurons. Moreover, we present the first CDK5-selective small molecule inhibitor, lacking unwanted cytostatic effects due to cross-inhibition of mitotic CDKs.

Cell cycle events distinguish sensory neuronal death from motoneuron death as a result of trophic factor deprivation

The clarification of mechanisms of developmental cell death may provide hints in the prevention of pathological neuronal death. The execution phase of cell death has been extensively characterized; however, events that occur prior to this phase are less well understood. Previous studies have suggested that terminally differentiated neurons induced to die in various experimental paradigms may be making an abortive attempt to reenter the cell cycle. We have examined this process in postmitotic motoneurons and dorsal root ganglia sensory neurons in the developing chick embryo in vitro and in vivo. An examination of the programmed cell death of postmitotic motoneurons does not implicate a role for cell cycle-related proteins. We did, however, observe a decrease in the amount of cell death in dorsal root ganglion cells of embryos treated with cell cycle inhibitors. These results indicate that upstream initiators of the neuronal cell death pathway vary between phenotypes.

Cell death, Bcl-2, Bax, and the cerebellum.

Bax and Bcl-2 are prototypical members of a large family of Bcl-2-like proteins that play a key role in regulating programmed cell death in many cell types. The purpose of this review is to summarize recent findings about the role of Bcl-2 and Bax in regulating programmed cell death in the cerebellum during normal development and in a mouse model of cell autonomous and target-related cell death, the Lurcher mutant. Both Bcl-2 and Bax are expressed in the developing cerebellum and recent studies of Bcl-2 transgenic mice and Bcl-2 or Bax knock-out mutants have shown that both proteins are likely to play a role in regulating cell death among Purkinje cells, granule cells and olivary neurons. However, the evidence suggests that there are diverse cell death pathways in cerebellar neurons that vary depending on the cell type and cell death stimulus. For example, the number of Purkinje cells is increased by over 30% in one line of Bcl-2 overexpressing transgenics and in Bax knock-out mutants, suggesting that both proteins may be involved in naturally occurring Purkinje cell death. However, overexpression of Bcl-2 or deletion of Bax expression in heterozygous Lurcher mutants delays but does not prevent the cell autonomous death of Lurcher Purkinje cells. The deletion of Bax expression from granule cells does not affect their number in adult Bax knock-out mutants, suggesting that Bax is not involved in naturally occurring granule cell death. However, Bax does appear to be involved in granule cell target-related cell death since substantially more granule cells survive in Bax -/-: Lurcher double mutants than in control Lurcher mutants. In contrast, deletion of Bax expression in Lurcher mutants does not prevent olivary neuron target-related cell death in the Lurcher mutant while overexpression of Bcl-2 in olivary neurons rescues them from both target-related and naturally occurring cell death.

Selective protection by phosphatidic acid against staurosporine-induced neuronal apoptosis.

Phosphatidic acid, the main product of lipid breakdown through phospholipase D activation, has been implicated in important signal transduction pathways able to influence cell fate in many ways. The purpose of this work was to determine possible effects of phosphatidic acid on neuronal cell death pathways. Here we used cerebellar granular cell cultures and cell death was triggered with either staurosporine or H(2)O(2). Cell viability was quantified by spectrophotometry, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. Staurosporine (1-3 microM) or H(2)O(2) (50-800 microM) induced cell death in a dose-dependent manner. Using fluorescent staining (propidium iodide or annexin V-Cy3/6-carboxyfluorescein) we showed that cell death was mostly apoptotic in staurosporine treated cells and mostly non-apoptotic (necrotic) in H(2)O(2) treated cells. Phosphatidic acid was able to increase cell viability in staurosporine-, but not in H(2)O(2) - treated cells. We therefore conclude that phosphatidic acid has neuroprotective potential in neurons exposed to stimuli that trigger apoptosis.

Peg3/Pw1 Is a Mediator between p53 and Bax in DNA Damage-induced Neuronal Death

Neuronal cell death after DNA damage requires p53 and Bax, but the mechanism by which p53 activation leads to Bax translocation and cell death in neurons is not known. We report here that Peg3/Pw1 is up-regulated after DNA damage in cortical neurons in a p53-dependent manner. Overexpression of Peg3/Pw1 leads to decreased neuronal viability. The deleterious effect of Peg3/Pw1 on neuronal survival is abrogated by deletion of either p53 or Bax, indicating an essential role for both in Peg3/Pw1-mediated neuronal death. Moreover, overexpression of a Peg3/Pw1 dominant negative protein inhibits Bax translocation and neuronal cell death after DNA damage. These findings implicate Peg3/Pw1 as a mediator between p53 and Bax in a neuronal cell death pathway activated by DNA damage.

Differential induction of p53 in immature and adult rat brain following lithium–pilocarpine status epilepticus

Activation of the tumor suppressor gene, p53, has been strongly implicated in selective neuronal cell death. This study investigated p53 expression in the immature and adult rat brain following status epilepticus induced by the administration of lithium–pilocarpine (LPSE). Both p53 mRNA and protein were examined in relation to neuronal degeneration using in situ hybridization and immunohistochemistry, respectively. Injured cells with eosinophilic cytoplasm with increased p53 mRNA were observed in hippocampal subfields, piriform cortex, amygdala and thalamus. p53 mRNA levels reached a peak by 8 h and returned to baseline by 24 h after the onset of LPSE. The magnitude of p53 mRNA induction was greatest in 21-day-old rats. In contrast to the cellular expression pattern of p53 mRNA, immunohistochemistry demonstrated that p53 protein was increased in all of the eosinophilic cells. Further, double-labeling studies revealed that p53 protein was elevated in neurons that were degenerating. This was supported by colocalization of activated caspase 3 in some cells with damaged DNA. These results provide additional evidence for a critical role for the p53 pathway in excitotoxic neuronal cell death due to status epilepticus.

Activation of divergent neuronal cell death pathways in different target cell populations during neuroadapted sindbis virus infection of mice.

Infection of adult mice with neuroadapted Sindbis virus (NSV) results in a severe encephalomyelitis accompanied by prominent hindlimb paralysis. We find that the onset of paralysis parallels morphologic changes in motor neuron cell bodies in the lumbar spinal cord and in motor neuron axons in ventral nerve roots, many of which are eventually lost over time. However, unlike NSV-induced neuronal cell death found in the brain of infected animals, the loss of motor neurons does not appear to be apoptotic, as judged by morphologic and biochemical criteria. This may be explained in part by the lack of detectable caspase-3 expression in these cells.

Activation of Divergent Neuronal Cell Death Pathways in Different Target Cell Populations during Neuroadapted Sindbis Virus Infection of Mice

Activation of Divergent Neuronal Cell Death Pathways in Different Target Cell Populations during Neuroadapted Sindbis Virus Infection of Mice

Vitamin E and other antioxidants in neuroprotection.

Several pathological conditions are believed to be causally related to the generation of reactive oxygen species and free radicals including various neurodegenerative disorders. In the histopathology of Alzheimer's disease (AD) many signs of oxidative reactions can be found building the basis of the oxidative stress hypothesis of AD. One major player in the generation of an overall oxidative microenvironment for the nerve cells is the amyloid beta protein (A beta) of the senile plaques in brain areas affected in AD. A beta can be neurotoxic and this toxicity is mediated by peroxides and by the peroxidation of membrane lipids leading to the lysis of the cell. Consequently, lipophilic free radical scavengers such as vitamin E and the recently discovered antioxidant activity of the female sex hormone estrogen protects neurons against the oxidative toxicity of A beta and other AD-related oxidative insults. In a first clinical trial using vitamin E in therapy, this antioxidant could slow down the course of the disease launching further clinical investigations. Although antioxidants act as non-specific protective chemical shields for neurons and do not target specific pathological events, they are highly effective and further investigations on their activity might lead to an even more effective application of antioxidants. Since the knowledge of the pathways of neuronal cell death that occur during oxidative challenges is increasing, it will be of central interest how antioxidants can interfere with signal transduction mechanisms and therefore also modify genetic programs. As long as specific interventions are not available the optimistic data concerning the neuroprotective activity of antioxidants in vitro and in vivo underline an important role for antioxidative acting compounds for the prevention and therapy of oxidative stress-related conditions including AD.