Lack of the p50 subunit of nuclear factor-kappaB increases the vulnerability of hippocampal neurons to excitotoxic injury.

Nuclear factor-kappaB (NF-kappaB) is activated in brain cells after various insults, including cerebral ischemia and epileptic seizures. Although cell culture studies have suggested that the activation of NF-kappaB can prevent neuronal apoptosis, the role of this transcription factor in neuronal injury in vivo is unclear, and the specific kappaB subunits involved are unknown. We now report that mice lacking the p50 subunit of NF-kappaB exhibit increased damage to hippocampal pyramidal neurons after administration of the excitotoxin kainate. Gel-shift analyses showed that p50 is required for the majority of kappaB DNA-binding activity in hippocampus. Intraventricular administration of kappaB decoy DNA before kainate administration in wild-type mice resulted in an enhancement of damage to hippocampal pyramidal neurons, indicating that reduced NF-kappaB activity was sufficient to account for the enhanced excitotoxic neuronal injury in p50(-/-) mice. Cultured hippocampal neurons from p50(-/-) mice exhibited enhanced elevations of intracellular calcium levels and increased levels of oxidative stress after exposure to glutamate and were more vulnerable to excitotoxicity than were neurons from p50(+/+) and p50(+/-) mice. Collectively, our data demonstrate an important role for the p50 subunit of NF-kappaB in protecting neurons against excitotoxic cell death.

Molecular targets of opiate drug abuse in neuroAIDS.

Opiate drug abuse, through selective actions at mu-opioid receptors (MOR), exacerbates the pathogenesis of human immunodeficiency virus-1 (HIV-1) in the CNS by disrupting glial homeostasis, increasing inflammation, and decreasing the threshold for pro-apoptotic events in neurons. Neurons are affected directly and indirectly by opiate-HIV interactions. Although most opiates drugs have some affinity for kappa (KOR) and/or delta (DOR) opioid receptors, their neurotoxic effects are largely mediated through MOR. Besides direct actions on the neurons themselves, opiates directly affect MOR-expressing astrocytes and microglia. Because of their broad-reaching actions in glia, opiate abuse causes widespread metabolic derangement, inflammation, and the disruption of neuron-glial relationships, which likely contribute to neuronal dysfunction, death, and HIV encephalitis. In addition to direct actions on neural cells, opioids modulate inflammation and disrupt normal intercellular interactions among immunocytes (macrophages and lymphocytes), which on balance further promote neuronal dysfunction and death. The neural pathways involved in opiate enhancement of HIV-induced inflammation and cell death, appear to involve MOR activation with downstream effects through PI3-kinase/Akt and/or MAPK signaling, which suggests possible targets for therapeutic intervention in neuroAIDS.

4-Hydroxynonenal, a product of lipid peroxidation, damages cholinergic neurons and impairs visuospatial memory in rats.

The mechanisms that underlie cholinergic neuronal degeneration in Alzheimer disease (AD) are unclear, but recent data suggest that oxidative stress plays a role. We report that 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, damages and kills basal forebrain cholinergic neurons when administered intraparenchymally. Examination of Nissl-stained brain sections following unilateral HNE infusion revealed widespread neuronal loss in basal forebrain ipsilateral to the injection, but not on the contralateral side. Levels of choline acetyltransferase activity and immunoreactivity in the ipsilateral basal forebrain and hippocampus were significantly reduced by 60-80% seven days following HNE administration. Performance in Morris water maze tasks of visuospatial memory was severely impaired in a dose-dependent manner seven days following bilateral administration of HNE. Bilateral infusion of FeCl2 (an inducer of membrane lipid peroxidation) into the basal forebrain caused neuron loss and decreased choline acetyltransferease immunoreactivity and deficits in visuospatial memory. Additionally, FeCl2 infusion increased HNE immunoreactivity, implicating HNE in iron-induced oxidative damage. Because recent studies have demonstrated HNE adducts in degenerating neurons in AD brain, the present findings suggest a role for HNE in damage to cholinergic neurons in AD.

Antiinflammatory effects of estrogen on microglial activation.

In the present study the effects of 17beta-estradiol on microglial activation are described. Estrogen replacement therapy has been associated with decreased severity of age-related neurodegenerative diseases such as Alzheimer's disease, and estrogens have potent immunosuppressive properties outside of the brain. To determine the role that microglial cells might play in estrogen-mediated neuroprotection, primary rat microglia and N9 microglial cell lines were treated with increasing doses of 17beta-estradiol before or during immunostimulation by lipopolysaccharide, phorbol ester, or interferon-gamma. Pretreatment with 17beta-estradiol, but not 17alpha-estradiol or progesterone, dose dependently attenuated microglial superoxide release and phagocytic activity. Additionally, 17beta-estradiol attenuated increases in inducible nitric oxide synthase protein expression, but did not alter nuclear factor-KB activation. The antiinflammatory effects of 17beta-estradiol were blocked by the antiestrogen ICI 182,780. Additionally, 17beta-estradiol induced rapid phosphorylation of the p42/p44 mitogen-activated protein kinase (MAP kinase), and the MAP kinase inhibitor PD 98059 blocked the antiinflammatory effects of 17beta-estradiol. Overall, these results suggest that estrogen receptor-dependent activation of MAP kinase is involved in estrogen-mediated antiinflammatory pathways in microglial cells. These results describe a novel mechanism by which estrogen may attenuate the progression of neurodegenerative disease and suggest new pathways for therapeutic intervention in clinical settings.

Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor.

Ischemic and excitotoxic insults to the brain induce rapid production of tumor necrosis factor-alpha (TNF), but the role of TNF in neuronal responses to brain injury are unclear. Two different TNF receptors (p55 and p75) are expressed in neurons and glia. To understand the role of TNF in brain injury, we generated mice that lack p55, p75, or both receptors. We report that neuronal damage after focal cerebral ischemia-reperfusion is significantly increased in mice lacking p55 receptors (85+/-7 mm3 infarct volume; mean +/- SD) compared with wild-type mice (70+/-8 mm3) and mice lacking p75 receptors (72+/-6 mm3). Moreover, mice lacking p55 receptors exhibited increased degeneration of CA3 hippocampal neurons after administration of the excitotoxin kainic acid compared with wild-type mice and mice lacking p75 receptors. When taken together with recent data showing that TNF can prevent apoptosis of cultured neurons exposed to oxidative and metabolic insults, our findings suggest that TNF plays a neuroprotective role after acute brain insults.

Anti-death properties of TNF against metabolic poisoning: mitochondrial stabilization by MnSOD.

The cytokine tumor necrosis factor (TNF) is toxic to some mitotic cells, but protects cultured neurons from a variety of insults by mechanisms that are unclear. Pretreatment of neurons or astrocytes with TNF caused significant increases in MnSOD activity, and also significantly attenuated 3-nitropropionic acid (3-NP) induced superoxide accumulation and loss of mitochondrial transmembrane potential. In oligodendrocytes, however, MnSOD activity was not increased, and 3-NP toxicity was unaffected by TNF. Genetically engineered PC6 cells that overexpress MnSOD also were resistant to 3-NP-induced damage. TNF pretreatment and MnSOD overexpression prevented 3-NP induced apoptosis, and shifted the mode of death from necrosis to apoptosis in response to high levels of 3-NP. Mitochondria isolated from either MnSOD overexpressing PC6 cells or TNF-treated neurons maintained resistance to 3-NP-induced loss of transmembrane potential and calcium homeostasis, and showed attenuated release of caspase activators. Overall, these results indicate that MnSOD activity directly stabilizes mitochondrial transmembrane potential and calcium buffering ability, thereby increasing the threshold for lethal injury. Additional studies showed that levels of oxidative stress and striatal lesion size following 3-NP administration in vivo are increased in mice lacking TNF receptors.

Neuronal injury in hippocampus with human immunodeficiency virus transactivating protein, Tat.

Patients with human immunodeficiency virus infection may develop a dementing illness. Using both in vitro and in vivo models, we investigated the susceptibility of the hippocampal formation to the Tat protein of human immunodeficiency virus. We also determined the pattern of hippocampal injury in patients with human immunodeficiency virus encephalitis. Following exposure of hippocampal slices to Tat, marked susceptibility of CA3 region with relative insensitivity of the CA1/2 region was observed. Injection of Tat into different regions of the rat hippocampus produced similar neuronal loss in both CA3 region and the dentate gyrus. In animals administered Tat, lesions were dose-dependent and immunohistochemical staining showed marked gliosis and loss of microtubule associated protein-2 in the affected areas at 3 days post-injection. Interestingly, synaptophysin staining was relatively preserved. In hippocampal tissue from patients with human immunodeficiency virus encephalitis, loss of microtubule-associated protein-2 staining was reduced in the molecular layer of the dentate gyrus. The results of our experiments demonstrate a unique pattern of hippocampal injury in organotypic culture and rats exposed to Tat. Our observations that patients with human immunodeficiency virus reveal a similar pattern of damage suggests that Tat protein may be pathophysiological relevant in human immunodeficiency virus encephalitis.

Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury.

Glutamate-induced excitotoxicity involving the formation of reactive oxygen species (ROS) has been implicated in neuronal dysfunction and cell loss following ischemic and traumatic injury to the central nervous system (CNS). ROS are formed in mitochondria when energy metabolism is compromised, and are inactivated by the ROS scavengers superoxide dismutase (SOD), catalase, and glutathione (GSH). ROS can impair the function of several cellular components including proteins, nucleic acids, and lipids. In the present study, we measured indicators of mitochondrial metabolic activity, ROS formation, lipid peroxidation, and antioxidant enzyme activities in synaptosomes obtained from rat spinal cord at early times following traumatic injury. Mitochondrial metabolic activity was found to significantly decrease as early as 1 h following injury, and continued to be compromised over the remaining postinjury time points. ROS formation was found to be significantly increased at 4 and 24 h following injury, while lipid peroxidation levels were found to be significantly increased in the injured spinal cord at 1 and 24 h, but not 4 h following injury. SOD enzyme activity was unchanged at all postinjury time points, while catalase activity and GSH levels were significantly increased at 24 h following injury. These findings indicate that impaired mitochondrial function, ROS, and lipid peroxidation occur soon after traumatic spinal cord injury, while the compensatory activation of molecules important for neutralizing ROS occurs at later time points. Therapeutic strategies aimed at facilitating the actions of antioxidant enzymes or inhibiting ROS formation and lipid peroxidation in the CNS may prove beneficial in treating traumatic spinal cord injury, provided such treatments are initiated at early stages following injury.

Microglial-neuronal interactions in synaptic damage and recovery.

An understanding of the role of microglial cells in synaptic signaling is still elusive, but the neuron-microglia relationship may have important ramifications for brain plasticity and injury. This review summarizes current knowledge and theories concerning microglial-neuronal signaling, both in terms of neuron-to-microglia signals that cause activation and microglia-to-neuron signals that affect neuronal response to injury. Microglial activation in the brain involves a stereotypical pattern of changes including proliferation and migration to sites of neuronal activity or injury, increased or de novo expression of immunomodulators including cytokines and growth factors, and the full transformation into brain-resident phagocytes capable of clearing damaged cells and debris. The factors released from neurons that elicit such phenotypical and functional alterations are not well known but may include cytokines, oxidized lipids, and/or neurotransmitters. Once activated, microglia can promote neuronal injury through the release of low-molecular-weight neurotoxins and support neuronal recovery through the release of growth factors and the isolation/removal of damaged neurons and myelin debris. Because microglia respond quickly to neuronal damage and have robust effects on neurons, astrocytes, and oligodendrocytes, microglial cells could play potentially key roles in orchestrating the multicell cascade that follows synaptic plasticity and damage.

Compartmentalization of signaling in neurons: evolution and deployment.

The localization of signal transduction machinery at synapses is a fundamental organizational feature of the nervous system that allows for highly complex integration of information coding processes. Synaptic communication evolved as multicellular organisms became more complex, and as selection pressures were placed on such organisms such that those capable of responding rapidly and specifically to environmental demands survived. Two obvious advantages of synaptic transmission (as opposed to endocrine or paracrine signaling) are that it provides for rapid intercellular communication over great distances and that it provides a high level of spatial specificity. There are several structural and functional aspects of synapses that set them apart from other cellular compartments, with many of the specializations subserving roles in synaptic signal transduction (e.g., neurotransmitter release from the presynaptic terminal and postsynaptic receptor activation and second messenger production). However, studies of developing nervous systems have shown that many synaptic signaling mechanisms are operative prior to synaptogenesis and play important roles in regulating growth cone behaviors, synaptogenesis, and even programmed cell death. Indeed, the concept that "ontogeny recapitulates phylogeny" can be effectively applied to the evolution of the synapse. As the embryo rapidly grows, neurons must elaborate axons and dendrites, establish functional synaptic connections, and maintain and adjust those connections as the organism matures. The purpose of this introductory article is to set the stage for the following articles by briefly reviewing fundamental aspects of the molecular and cellular biology of synapses in an evolutionary context.

Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis.

Oxidative stress is believed to play important roles in neuronal cell death associated with many different neurodegenerative conditions (e.g., Alzheimer's disease, Parkinson's disease, and cerebral ischemia), and it is believed also that apoptosis is an important mode of cell death in these disorders. Membrane lipid peroxidation has been documented in the brain regions affected in these disorders as well as in cell culture and in vivo models. We now provide evidence that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, is a key mediator of neuronal apoptosis induced by oxidative stress. HNE induced apoptosis in PC12 cells and primary rat hippocampal neurons. Oxidative insults (FeSO4 and amyloid beta-peptide) induced lipid peroxidation, cellular accumulation of HNE, and apoptosis. Bcl-2 prevented apoptosis of PC12 cells induced by oxidative stress and HNE. Antioxidants that suppress lipid peroxidation protected against apoptosis induced by oxidative insults, but not that induced by HNE. Glutathione, which binds HNE, protected neurons against apoptosis induced by oxidative stress and HNE. PC12 cells expressing Bcl-2 exhibited higher levels of glutathione and lower levels of HNE after oxidative stress. Collectively, the data identify that HNE is a novel nonprotein mediator of oxidative stress-induced neuronal apoptosis and suggest that the antiapoptotic action of glutathione may involve detoxification of HNE.

Uric acid protects neurons against excitotoxic and metabolic insults in cell culture, and against focal ischemic brain injury in vivo.

Uric acid is a well-known natural antioxidant present in fluids and tissues throughout the body. Oxyradical production and cellular calcium overload are believed to contribute to the damage and death of neurons that occurs following cerebral ischemia in victims of stroke. We now report that uric acid protects cultured rat hippocampal neurons against cell death induced by insults relevant to the pathogenesis of cerebral ischemia, including exposure to the excitatory amino acid glutamate and the metabolic poison cyanide. Confocal laser scanning microscope analyses showed that uric acid suppresses the accumulation of reactive oxygen species (hydrogen peroxide and peroxynitrite), and lipid peroxidation, associated with each insult. Mitochondrial function was compromised by the excitotoxic and metabolic insults, and was preserved in neurons treated with uric acid. Delayed elevations of intracellular free calcium levels induced by glutamate and cyanide were significantly attenuated in neurons treated with uric acid. These data demonstrate a neuroprotective action of uric acid that involves suppression of oxyradical accumulation, stabilization of calcium homeostasis, and preservation of mitochondrial function. Administration of uric acid to adult rats either 24 hr prior to middle cerebral artery occlusion (62.5 mg uric acid/kg, intraperitoneally) or 1 hr following reperfusion (16 mg uric acid/kg, intravenously) resulted in a highly significant reduction in ischemic damage to cerebral cortex and striatum, and improved behavioral outcome. These findings support a central role for oxyradicals in excitotoxic and ischemic neuronal injury, and suggest a potential therapeutic use for uric acid in ischemic stroke and related neurodegenerative conditions.

Astrocytic gap junctional communication decreases neuronal vulnerability to oxidative stress-induced disruption of Ca2+ homeostasis and cell death.

We investigated the effect of uncoupling astrocytic gap junctions on neuronal vulnerability to oxidative injury in embryonic rat hippocampal cell cultures. Mixed cultures (neurons growing on an astrocyte monolayer) treated with 18-alpha-glycyrrhetinic acid (GA), an uncoupler of gap junctions, showed markedly enhanced generation of intracellular peroxides (2,7-dichlorofluorescein fluorescence), impairment of mitochondrial function [(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and cell death (lactate dehydrogenase release) following exposure to oxidative insults (FeSO4 and 4-hydroxynonenal). GA alone had little or no effect on basal levels of peroxides, mitochondrial function, or neuronal survival. Intercellular dye transfer analyses revealed extensive astrocyte-astrocyte coupling but no astrocyte-neuron or neuron-neuron coupling in the mixed cultures. Studies of pure astrocyte cultures and microscope analyses of neurons in mixed cultures showed that the increased oxidative stress and cell death in GA-treated cultures occurred only in neurons and not in astrocytes. Antioxidants (propyl gallate and glutathione) blocked the death of neurons exposed to FeSO4/GA. Elevations of neuronal intracellular calcium levels ([Ca2+]i) induced by FeSO4 were enhanced in neurons in mixed cultures exposed to GA. Removal of extracellular Ca2+ and the L-type Ca2+ channel blocker nimodipine prevented impairment of mitochondrial function and cell death induced by FeSO4 and GA, whereas glutamate receptor antagonists were ineffective. Finally, GA exacerbated kainate- and FeSO4-induced injury to pyramidal neurons in organotypic hippocampal slice cultures. The data suggest that interastrocytic gap junctional communication decreases neuronal vulnerability to oxidative injury by a mechanism involving stabilization of cellular calcium homeostasis and dissipation of oxidative stress.

2-Deoxy-D-glucose protects hippocampal neurons against excitotoxic and oxidative injury: evidence for the involvement of stress proteins.

Food restriction can extend life span in rodents and was recently reported to increase the resistance of neurons in the brain to excitotoxic and metabolic insults. In principle, administration to ad libitum fed rodents of an agent that reduces glucose availability to cells should mimick certain aspects of food restriction. We now report that administration of 2-deoxy-D-glucose (2DG), a non-metabolizable analog of glucose, to adult rats results in a highly significant reduction in seizure-induced spatial memory deficits and hippocampal neuron loss. Pretreatment of rat hippocampal cell cultures with 2DG decreases the vulnerability of neurons to excitotoxic (glutamate) and oxidative (Fe2+) insults. The protective action of 2DG is associated with decreased levels of cellular oxidative stress and enhanced calcium homeostasis. 2DG treatment increased levels of the stress-responsive proteins GRP78 and HSP70 in hippocampal neurons, without affecting levels of Bcl-2 or GRP75, suggesting that mild reductions in glucose availability can increase neuronal resistance to oxidative and metabolic insults by a mechanism involving induction of stress proteins. Our findings establish cell culture and in vivo models of "chemical food restriction" which may prove useful in elucidating mechanisms of neuroprotection and in developing preventive approaches for neurodegenerative disorders that involve oxidative stress and excitotoxicity.

Food restriction reduces brain damage and improves behavioral outcome following excitotoxic and metabolic insults.

Food restriction (FR) in rodents is known to extend life span, reduce the incidence of age-related tumors, and suppress oxidative damage to proteins, lipids, and DNA in several organ systems. Excitotoxicity and mitochondrial impairment are believed to play major roles in the neuronal degeneration and death that occurs in the brains of patients suffering from both acute brain insults such as stroke and seizures, and chronic neurodegenerative conditions such as Alzheimer's, Parkinson's, and Huntington's diseases. We now report that FR (alternate-day feeding regimen for 2-4 months) in adult rats results in resistance of hippocampal neurons to excitotoxin-induced degeneration, and of striatal neurons to degeneration induced by the mitochondrial toxins 3-nitropropionic acid and malonate. FR greatly increased the resistance of rats to kainate-induced deficits in performance in water-maze learning and memory tasks, and to 3-nitropropionic acid-induced impairment of motor function. These findings suggest that FR not only extends life span, but increases resistance of the brain to insults that involve metabolic compromise and excitotoxicity.

Pro-inflammatory and pro-oxidant properties of the HIV protein Tat in a microglial cell line: attenuation by 17 beta-estradiol.

Microglia are activated in humans following infection with human immunodeficiency virus (HIV), and brain inflammation is thought to be involved in neuronal injury and dysfunction during HIV infection. Numerous studies indicate a role for the HIV regulatory protein Tat in HIV-related inflammatory and neurodegenerative processes, although the specific effects of Tat on microglial activation, and the signal transduction mechanisms thereof, have not been elucidated. In the present study, we document the effects of Tat on microglial activation and characterize the signal transduction pathways responsible for Tat's pro-inflammatory effects. Application of Tat to N9 microglial cells increased multiple parameters of microglial activation, including superoxide production, phagocytosis, nitric oxide release and TNF alpha release. Tat also caused activation of both p42/p44 mitogen activated protein kinase (MAPK) and NF kappa B pathways. Inhibitor studies revealed that Tat-induced NF kappa B activation was responsible for increased nitrite release, while MAPK activation mediated superoxide release, TNF alpha release, and phagocytosis. Lastly, pre-treatment of microglial cells with physiological concentrations of 17 beta-estradiol suppressed Tat-mediated microglial activation by interfering with Tat-induced MAPK activation. Together, these data elucidate specific components of the microglial response to Tat and suggest that Tat could contribute to the neuropathology associated with HIV infection through microglial promulgation of oxidative stress.

Increased sensitivity to mitochondrial toxin-induced apoptosis in neural cells expressing mutant presenilin-1 is linked to perturbed calcium homeostasis and enhanced oxyradical production.

Many cases of autosomal dominant early onset Alzheimer's disease (AD) result from mutations in the gene encoding presenilin-1 (PS-1). PS-1 is an integral membrane protein expressed ubiquitously in neurons throughout the brain in which it is located primarily in endoplasmic reticulum (ER). Although the pathogenic mechanism of PS-1 mutations is unknown, recent findings suggest that PS mutations render neurons vulnerable to apoptosis. Because increasing evidence indicates that mitochondrial alterations contribute to neuronal death in AD, we tested the hypothesis that PS-1 mutations sensitize neurons to mitochondrial failure. PC12 cell lines expressing a PS-1 mutation (L286V) exhibited increased sensitivity to apoptosis induced by 3-nitropropionic acid (3-NP) and malonate, inhibitors of succinate dehydrogenase, compared with control cell lines and lines overexpressing wild-type PS-1. The apoptosis-enhancing action of mutant PS-1 was prevented by antioxidants (propyl gallate and glutathione), zVAD-fmk, and cyclosporin A, indicating requirements of reactive oxygen species (ROS), caspases, and mitochondrial permeability transition in the cell death process. 3-NP induced a rapid elevation of [Ca2+]i, which was followed by caspase activation, accumulation of ROS, and decreases in mitochondrial reducing potential and transmembrane potential in cells expressing mutant PS-1. The calcium chelator BAPTA AM and agents that block calcium release from ER and influx through voltage-dependent channels prevented mitochondrial ROS accumulation and membrane depolarization and apoptosis. Our data suggest that by perturbing subcellular calcium homeostasis presenilin mutations sensitize neurons to mitochondria-based forms of apoptosis that involve oxidative stress.

Lysophosphatidic acid and apoptosis of nerve growth factor-differentiated PC12 cells.

The lipid biomediator lysophosphatidic acid (LPA) elicits a unique response in hippocampal neurons, LPA induces neuronal apoptosis. This study explores the effects of LPA on cells with neuronal properties, nerve growth factor-differentiated PC6 cells, a clone of PC12 cells. LPA induced apoptosis in these cells as assessed by chromatin condensation, terminal dUTP nick end-labeling of DNA, protection against these nuclear alterations by a general caspase inhibitor and the lack of release of lactic dehydrogenase. LPA caused oxidative stress, namely a decreased reduction of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. This oxidative stress appears to be of functional significance, since cells were protected by pretreatment with the antioxidant propyl gallate and by stable transfection with cDNA encoding the antioxidant enzyme, manganese superoxide dismutase. Mitochondrial and nitric oxide participation in LPA-induced apoptosis are suggested by the protection afforded by pretreatment with either cyclosporin A, an inhibitor of mitochondrial permeability transition, or nitric oxide synthase inhibitors. The nitric oxide synthase inhibitor findings are novel, since to our knowledge, LPA has not heretofore been associated with an increase in nitric oxide. In addition, as observed for many neurotoxic agents, insulin-like growth factor I protected against LPA-induced apoptosis of PC6 cells.

Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury.

Tumor necrosis factor alpha (TNFalpha) is widely expressed in both neurons and glia and has been shown to be upregulated after traumatic brain injury (TBI). TNFalpha receptor activation results in activation of the transcription factor nuclear factor kappaB (NF-kappaB), which may serve an antiapoptotic role via the induction of target genes manganese superoxide dismutase (MnSOD) and/or calbindin. In the present study, we used a controlled cortical impact model of TBI with pertinent lines of transgenic mice to combine both morphological characterization and molecular analysis to elucidate the role of TNFalpha after TBI. Measurements of both the lesion volume and the blood-brain barrier breach indicated exacerbations in mice rendered genetically deficient in both the p55 and p75 TNFalpha receptors (TNFR-KO) compared with wild-type animals. Additionally, animals genetically altered to overexpress MnSOD showed a significant decrease in lesion volume compared with that of control littermates, whereas no alterations were observed in mice lacking the calcium-binding protein calbindin D28k. Analysis of NF-kappaB activation and relative levels of MnSOD revealed delayed responses in the injured cortex of TNFR-KO animals compared with wild-type animals, implying that endogenous TNFalpha may be neuroprotective after TBI.

Bcl-2 protects isolated plasma and mitochondrial membranes against lipid peroxidation induced by hydrogen peroxide and amyloid beta-peptide.

The bcl-2 protooncogene product possesses antiapoptotic properties in neuronal and nonneuronal cells. Recent data suggest that Bcl-2's potency as a survival factor hinges on its ability to suppress oxidative stress, but neither the subcellular site(s) nor the mechanism of its action is known. In this report electron paramagnetic resonance (EPR) spectroscopy analyses were used to investigate the local effects of Bcl-2 on membrane lipid peroxidation. Using hydrogen peroxide (H2O2) and amyloid beta-peptide (A beta) as lipoperoxidation initiators, we determined the loss of EPR-detectable paramagnetism of nitroxyl stearate (NS) spin labels 5-NS and 12-NS. In intact cell preparations and postnuclear membrane fractions, A beta and H2O2 induced significant loss of 5-NS and 12-NS signal amplitude in control PC12 cells, but not PC12 cells expressing Bcl-2. Cells were subjected to differential subcellular fractionation, yielding preparations of plasma membrane and mitochondria. In preparations derived from Bcl-2-expressing cells, both fractions contained Bcl-2 protein. 5-NS and 12-NS signals were significantly decreased following A beta and H2O2 exposure in control PC12 mitochondrial membranes, and Bcl-2 largely prevented these effects. Plasma membrane preparations containing Bcl-2 were also resistant to radical-induced loss of spin label. Collectively, our data suggest that Bcl-2 is localized to mitochondrial and plasma membranes where it can act locally to suppress oxidative damage induced by A beta and H2O2, further highlighting the important role of lipid peroxidation in apoptosis.