Lysophosphatidic acid induction of neuronal apoptosis and necrosis.

Lysophosphatidic acid (LPA) elicits a unique response in primary hippocampal neurons and sympathetic neuron-like cells, PC12 cells differentiated with nerve growth factor; LPA is cytotoxic. Treatment of rat hippocampal neurons with 50 microM LPA resulted in necrosis, as determined morphologically and by release of lactate dehydrogenase. Lower concentrations of LPA, 0.1, and 1 microM, induced neuronal apoptosis, as assessed by chromatin condensation, annexin V binding, TUNEL staining, and the caspase sensitivity of these events. In addition, 10 and 25 microM LPA induced apoptosis of PC12 cells. In order to define intracellular events associated with this neuronal apoptosis, protective agents were identified. Neurons and PC12 cells were protected against LPA-induced apoptosis by pretreatment with the antioxidant, propyl gallate, or with nitric oxide synthase inhibitors. PC12 cells were protected by insulin and insulin-like growth-factor-1 treatment. There is also evidence for mitochondrial participation in LPA-mediated apoptosis, including cyclosporin A-mediated protection. Thus, LPA-induced neuronal apoptosis is associated with mitochondrial alterations, the generation of reactive oxygen species and nitric oxide, and protection by pretreatment with a serum constituent, insulin-like growth factor 1.

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.

Superoxide mediates the cell-death-enhancing action of presenilin-1 mutations.

The mechanism whereby mutations in the presenilin-1 (PS-1) gene on chromosome 14 cause early-onset inherited Alzheimer's disease are unknown. We report that PC6 neural cells (a subclone of PC12 cells) expressing PS-1 mutations (M146V and L286V) exhibit increased superoxide production, nitrotyrosine accumulation, and membrane lipid peroxidation following exposure to amyloid beta-peptide 1-42 (Abeta). Mitochondrial calcium accumulation and membrane depolarization following exposure to Abeta were enhanced in cells expressing mutant PS-1. Overexpression of mitochondrial Mn-SOD greatly reduced superoxide production, nitrotyrosine formation, membrane lipid peroxidation, intramitochondrial calcium accumulation, and membrane depolarization following exposure to Abeta and conferred resistance to the apoptosis-enhancing action of the PS-1 mutations. Nitric oxide synthase inhibitors and the peroxynitrite scavenger uric acid blocked the apoptosis-enhancing action of PS-1 mutations. The data suggest pivotal roles for superoxide production and resulting peroxynitrite formation in the pathogenic mechanism of PS-1 mutations.

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.

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.

Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction.

Oxidative stress is implicated in neuronal apoptosis that occurs in physiological settings and in neurodegenerative disorders. Superoxide anion radical, produced during mitochondrial respiration, is involved in the generation of several potentially damaging reactive oxygen species including peroxynitrite. To examine directly the role of superoxide and peroxynitrite in neuronal apoptosis, we generated neural cell lines and transgenic mice that overexpress human mitochondrial manganese superoxide dismutase (MnSOD). In cultured pheochromocytoma PC6 cells, overexpression of mitochondria-localized MnSOD prevented apoptosis induced by Fe2+, amyloid beta-peptide (Abeta), and nitric oxide-generating agents. Accumulations of peroxynitrite, nitrated proteins, and the membrane lipid peroxidation product 4-hydroxynonenal (HNE) after exposure to the apoptotic insults were markedly attenuated in cells expressing MnSOD. Glutathione peroxidase activity levels were increased in cells overexpressing MnSOD, suggesting a compensatory response to increased H2O2 levels. The peroxynitrite scavenger uric acid and the antioxidants propyl gallate and glutathione prevented apoptosis induced by each apoptotic insult, suggesting central roles for peroxynitrite and membrane lipid peroxidation in oxidative stress-induced apoptosis. Apoptotic insults decreased mitochondrial transmembrane potential and energy charge in control cells but not in cells overexpressing MnSOD, and cyclosporin A and caspase inhibitors protected cells against apoptosis, demonstrating roles for mitochondrial alterations and caspase activation in the apoptotic process. Membrane lipid peroxidation, protein nitration, and neuronal death after focal cerebral ischemia were significantly reduced in transgenic mice overexpressing human MnSOD. The data suggest that mitochondrial superoxide accumulation and consequent peroxynitrite production and mitochondrial dysfunction play pivotal roles in neuronal apoptosis induced by diverse insults in cell culture and in vivo.

Lysophosphatidic acid induces necrosis and apoptosis in hippocampal neurons.

A diverse body of evidence indicates a role for the lipid biomediator lysophosphatidic acid (LPA) in the CNS. This study identifies and characterizes the induction of neuronal death by LPA. Treatment of cultured hippocampal neurons from embryonic rat brains with 50 microM LPA resulted in neuronal necrosis, as determined morphologically and by the release of lactate dehydrogenase. A concentration of LPA as low as 10 microM led to the release of lactate dehydrogenase. In contrast, treatment of neurons with 0.1 or 1.0 microM LPA resulted in apoptosis, as determined by chromatin condensation. In addition, neuronal death induced by 1 microM LPA was characterized as apoptotic on the basis of terminal dUTP nick end-labeling (TUNEL) staining, externalization of phosphatidylserine, and protection against chromatin condensation, TUNEL staining, and phosphatidylserine externalization by treatment with N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum inhibitor of caspases, i.e., members of the interleukin-1beta converting enzyme family. Studies with antagonists of ionotropic glutamate receptors did not indicate a significant role for these receptors in apoptosis induced by 1 microM LPA. LPA (1 microM) also induced a decrease in mitochondrial membrane potential. Moreover, pretreatment of neurons with cyclosporin A protected against the LPA-induced decrease in mitochondrial membrane potential and neuronal apoptosis. Thus, LPA, at pathophysiological levels, can induce neuronal apoptosis and could thereby participate in neurodegenerative disorders.

Lysophosphatidic acid-induced proliferation-related signals in astrocytes.

Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA-induced events in astrocytes were defined. As little as 1 nM LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca2+]i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca2+]i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca2+]i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca2+]i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA-induced increase in [Ca2+]i was inhibited by a protein kinase C inhibitor, Ro 31-8220, and a calcium-dependent protein kinase C inhibitor, Gö 6976. The increase in [Ca2+]i was important for the LPA-induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA-stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA-induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin-sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca2+]i, lipid peroxidation, and DNA synthesis.

Lysophosphatidic acid induces a sustained elevation of neuronal intracellular calcium.

Lysophosphatidic acid (LPA) is a lipid biomediator enriched in the brain. A novel LPA-induced response in rat hippocampal neurons is described herein, namely, a rapid and sustained elevation in the concentration of free intracellular calcium ([Ca2+]i). This increase is specific, in that the related lipids phosphatidic acid and lysophosphatidylcholine did not induce an alteration in [Ca2+]i. Moreover, consistent with a receptor-mediated process, there was no further increase in [Ca2+]i after a second addition of LPA. The LPA-induced increase in [Ca2+]i required extracellular calcium. However, studies with Cd2+, Ni2+, and nifedipine and nystatin-perforated patch clamp analyses did not indicate involvement of voltage-gated calcium channels in the LPA-induced response. In contrast, glutamate appears to have a significant role in the LPA-induced increase in [Ca2+]i, because this increase was inhibited by NMDA receptor antagonists and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonists. Thus, LPA treatment may result in an increased extracellular glutamate concentration that could stimulate AMPA/kainate receptors and thereby alleviate the Mg2+ block of the NMDA receptors and lead to glutamate stimulation of an influx of calcium via NMDA receptors.

Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors.

Brain injury, as occurs in stroke or head trauma, induces a dramatic increase in levels of tumor necrosis factor-alpha (TNF), but its role in brain injury response is unknown. We generated mice genetically deficient in TNF receptors (TNFR-KO) to determine the role of TNF in brain cell injury responses. Damage to neurons caused by focal cerebral ischemia and epileptic seizures was exacerbated in TNFR-KO mice, indicating that TNF serves a neuroprotective function. Oxidative stress was increased and levels of an antioxidant enzyme reduced in brain cells of TNFR-KO mice, indicating that TNF protects neurons by stimulating antioxidant pathways. Injury-induced microglial activation was suppressed in TNFR-KO mice, demonstrating a key role for TNF in injury-induced immune response. Drugs that target TNF signaling pathways may prove beneficial in treating stroke and traumatic brain injury.

Presence in human eosinophils of a lysophospholipase similar to that found in the pancreas.

The supernatant fraction from lysed human eosinophils, when separated by gel-filtration chromatography, contains a protein with lysophospholipase activity of approximate molecular mass 74 kDa. This mass differs substantially from the 17 kDa of a previously cloned eosinophil lysophospholipase (Charcot-Leyden crystal protein), but is similar to that reported for a pancreatic enzyme. We have therefore further characterized this pancreatic-like lysophospholipase in human eosinophils. A rabbit polyclonal antibody was produced against a synthetic peptide consisting of amino acids 325-349 from the 74 kDa rat pancreatic lysophospholipase. Western-blot analysis of eosinophil extracts indicate that this antibody recognizes a single 74 kDa band in these preparations. Incubation of the supernatant fraction from sonified eosinophils with this antibody, followed by precipitation of antibody-antigen complexes with Protein A, removes the majority of the lysophospholipase activity. Indirect immunofluorescence examination with this antibody indicates this protein to be localized to granules of eosinophils and not in other leucocytes. Moreover, reverse transcriptase PCR of polyadenylated RNA from eosinophils and from rat pancreatic tissue with primers to rat pancreatic lysophospholipase resulted in readily detectable 1 kb DNA products in both samples. Sequencing revealed this DNA fragment to be identical with the human pancreatic lysophospholipase cDNA sequence. Taken together, these data indicate that eosinophils contain a lysophospholipase that is similar to the human pancreatic enzyme.

Comparison of six mammalian lysophospholipases.

Lysophospholipases participate in the regulation of the levels of lysophospholipid, compounds with pleiotropic biological effects. Lysophospholipases were purified from a macrophage cell line (WEHI 265.1), a myelocytic leukemia cell line (HL-60) and peripheral blood eosinophils. WEHI 265.1 cells contain three lysophospholipases 28, 27 and 110 kDa as determined by polyacrylamide gel electrophoresis. The 110 kDa lysophospholipase also exhibits phospholipase A2 activity and appears to be identical to a previously described 110 kDa phospholipase A2. Similarly, the HL-60 cells have three lysophospholipases, the largest again a 110 kDa enzyme with phospholipase A2 activity and the smaller are 20 and 21 kDa. The low molecular mass lysophospholipases have distinctive chromatographic properties and amino acid compositions. However, the two low molecular mass enzymes from a given cell type are not radically different, e.g., 15 of the 20 amino acids of the C-terminal sequences of the HL-60 enzymes are identical. A single lysophospholipase, approx. 15 kDa, is a major eosinophil protein. This enzyme is different from those described above.

Butyric acid-induced differentiation of HL-60 cells increases the expression of a single lysophospholipase.

Treatment of HL-60 cells with 0.5 mM-butyric acid resulted in morphological changes, including the formation of cytoplasmic granules, nuclear condensation and segmentation. These differentiated cells had an elevated phospholipase A2 activity and an increased capacity to synthesize a variety of eicosanoids, including both lipoxygenase and cyclooxygenase products. Phospholipase A2-mediated release of arachidonic acid is accompanied by an equimolar production of potentially cytotoxic lysophospholipid. In association with the differentiation process, there was a 2-3-fold increase in lysophospholipase activity. Subsequent studies were undertaken to identify and characterize the lysophospholipases in this cell system, with 1-[1-14C]palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine as substrate. Hydrophobic chromatography of both undifferentiated and differentiated cell extracts revealed three peaks of enzyme activity. Extracts of differentiated cells contained a dramatic increase in activity contained in peak 2. The increase in enzymic activity of peak 2 appeared to account for the increase in total lysophospholipase activity found in the differentiated cell homogenates. The lysophospholipases contained in peaks 2 and 3 were purified to homogeneity and were 20 and 22 kDa respectively, as determined by denaturing polyacrylamide-gel electrophoresis. Peaks 2 and 3 were similar on the basis of amino acid composition, but had distinctive C-terminal peptide amino acid sequences. Enzymic characterization of these proteins demonstrated that there was no detectable level of non-specific esterase, acyltransferase or transacylase activity associated with these proteins. We concluded that peak 2 lysophospholipase is regulated by differentiation in HL-60 cells and may play an important role in protecting these cells from the cytolytic effects of the lysophospholipids produced by the activation of phospholipase A2.