Sheldon M. Steiner

Nordihydroguaiaretic acid protects hippocampal neurons against amyloid β-peptide toxicity, and attenuates free radical and calcium accumulation

Recent findings indicate that amyloid beta-peptide (A beta) can be neurotoxic by a mechanism involving an increase in the concentration of intracellular free Ca2+ ([Ca2+]i) and the generation of free radicals. In the present study, the lipoxygenase inhibitor/antioxidant nordihydroguaiaretic acid (NDGA) protected cultured rat hippocampal neurons against the toxicity of A beta in a concentration-dependent manner. Measurements of cellular oxidation (using the oxidation-sensitive dye 2,7-dichlorofluorescin) and intracellular free Ca2+ levels (using the Ca2+ indicator dye fura-2), showed that NDGA suppressed A beta-induced accumulation of reactive oxygen species (ROS) and Ca2+; Ca2+ responses to glutamate were also suppressed by NDGA. NDGA prevented neuronal injury and accumulation of ROS induced by iron, indicating a role for NDGA as an antioxidant in NDGA-mediated neuroprotection. Another lipoxygenase inhibitor (AA861) also protected against A beta and iron toxicity whereas the the 5-lipoxygenase-activating protein inhibitor L655,238 and the cyclooxygenase inhibitor indomethacin were ineffective. These findings suggest that NDGA can interupt a neurodegenerative pathway relevant to the pathophysiology of Alzheimers disease.

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.

Effects of 2-deoxy-d-glucose on herpes simplex virus replication☆

Abstract The replication of herpes simplex virus (HSV) was studied in BSC1 cells cultured in the presence of various concentrations of 2-deoxy- d -glucose. At all concentrations tested, the yield of infectious virus was inhibited to a greater degree than the yield of physical particles, while the percentage of the total particles which were enveloped remained constant. Analysis of HSV-induced glycoproteins on SDS-polyacrylamide gels (SDS-PAGE) indicated that as the concentration of 2-deoxy- d -glucose increased, there was a decrease in the apparent molecular weights of the viral glycoproteins. Analysis on SDS-PAGE of the viral-induced proteins indicated no significant alterations with the exception of an additional component detectable only in HSV-infected cultures containing the higher concentrations of 2-deoxy- d -glucose. Preliminary SDS-PAGE analysis indicated that 2-deoxy- d -glucose is incorporated into the HSV-induced glycoproteins.

Lysophosphatidic acid induces necrosis and apoptosis in hippocampal neurons

Abstract: 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 µM LPA resulted in neuronal necrosis, as determined morphologically and by the release of lactate dehydrogenase. A concentration of LPA as low as 10 µM led to the release of lactate dehydrogenase. In contrast, treatment of neurons with 0.1 or 1.0 µM LPA resulted in apoptosis, as determined by chromatin condensation. In addition, neuronal death induced by 1 µM 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‐1β converting enzyme family. Studies with antagonists of ionotropic glutamate receptors did not indicate a significant role for these receptors in apoptosis induced by 1 µM LPA. LPA (1 µM) 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.

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 Alzheimers 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 β‐peptide 1–42 (Aβ). Mitochondrial calcium accumulation and membrane depolarization following exposure to Aβ 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 Aβ 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. J. Neurosci. Res. 56:457–470, 1999. 

Lysophosphatidic Acid-Induced Proliferation-Related Signals in Astrocytes

Abstract: 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 decreases glutamate and glucose uptake by astrocytes

Abstract: The brain is a rich source of the lipid biomediator lysophosphatidic acid, and lysophosphatidic acid levels can significantly increase following brain trauma. Responses of primary rat brain astrocytes to this novel lipid are defined in the current study. Treatment of cells with lysophosphatidic acid resulted in a time‐ and dose‐dependent inhibition of glutamate uptake. Inhibition of glutamate uptake was specific because the related phospholipids, phosphatidic acid, lysophosphatidylcholine, and lysophosphatidylglycerol, did not inhibit this uptake under comparable conditions, i.e., treatment with 10 µM lipid for 30 min. Lysophosphatidic acid treatment of cells resulted in an increase in lipid peroxidation, as measured by the thiobarbituric acid assay. This increase in content of thiobarbituric acid‐reactive substances was largely inhibited by treatment with dithiothreitol or propyl gallate; however, such treatment did not affect the lysophosphatidic acid‐induced inhibition of glutamate uptake. Lysophosphatidic acid also inhibited glucose uptake with a dose‐response curve that paralleled the inhibition of glutamate uptake. By impairing uptake of glutamate by astrocytes, lysophosphatidic acid may exacerbate excitotoxic processes in various neurodegenerative conditions.

Multiple astrocyte responses to lysophosphatidic acids

Lysophosphatidic acid (LPA) and LPA receptors are enriched in the brain. Moreover, the levels of these receptors and ligand are modulated during brain development and injury, respectively, suggesting multiple roles for LPA in the brain. In cultured astrocytes and glioma-derived cells, LPA increases intracellular calcium concentrations and causes morphological changes. LPA also induces glioma cell migration. In normal astrocytes, LPA stimulates reactive oxygen species synthesis, activation of multiple protein kinases and expression of c-fos and c-jun. It is noteworthy that LPA-induced astrocyte responses vary as a function of the specific brain region of origin of the astrocytes. This may be one factor in the finding of LPA-stimulated proliferation in some, but not all, astrocyte studies. The species and/or developmental stage also differed in many of the astrocyte proliferation analyses. Micromolar LPA is required to elicit some astrocyte responses, including the stimulation of cytokine expression and inhibition of glutamate uptake. These events could significantly impact on survival of injured neurons and micromolar LPA concentrations are likely in diverse brain pathologies. There are important aspects of astrocyte LPA responses still to be fully evaluated, including functions in development and activation, synergy between LPA and other biomediators, and astrocyte interactions with other cells.

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. J. Neurosci. Res. 53:685–696, 1998.

Incorporation of 2-deoxy-d-glucose into glycolipids of normal and SV40-transformed hamster cells

Abstract 1. 1. Normal hamster embryo fibroblast cells and SV40-transformed hamster cells grown in the presence of radioactive 2-deoxyglucose, a glucose and mannose analogue, incorporate the 2-deoxyglucose into membrane glycolipids. Hydrolysis of the 2-deoxyglucose-labeled lipids reveals that 2-deoxyglucose is incorporated intact. 2. 2. Short term labeling experiments reveal that both 2-deoxyglucose and glucosamine are rapidly incorporated into glycolipid and in significant amounts. 3. 3. The above results demonstrate that 2-deoxyglucose can be metabolized beyond 2-deoxyglucose 6-phosphate and 2-deoxygluconic acid 6-phosphate in both normal and SV40-transformed hamster cells.