Marion R. 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.

In vitro studies of phospholipid biosynthesis in Saccharomyces cerevisiae.

1. 1. Evidence is given for reactions leading to the formation of all the major glycerol containing phospholipids of Saccharomyces cerevisiae. A cell free particulate fraction prepared from S. cerevisiae is capable of incorporating sn-[14C]glycero-3-phosphoric acid into phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerophosphate, phosphatidylglycerol, cardiolipin, CDP-diglyceride and diglyceride. These products were separated by a two-dimensional chromatography system. 2. 2. The incorporation of sn-[14C]glycero-3-phosphoric acid into phosphatidic acid requires CoA and ATP and is greatly stimulated by the addition of divalent cations. The incorporation of this label into CDP-diglyceride, phosphatidylserine, phosphatidylinositol, phosphatidylglycerophosphate, phosphatidylglycerol and cardiolipin has an additional requirement for CTP. Inositol and serine enhance the incorporation of sn-[14C]glycero-3-phosphoric acid into phosphatidylinositol and phosphatidylserine, respectively. Evidence is given for two pathways for phosphatidylethanolamine synthesis, from the reaction of CDP-ethanolamine and diglyceride and by the decarboxylation of phosphatidylserine. There are also two pathways for the synthesis of phosphatidylcholine, the methylation of phosphatidylethanolamine and the reaction of CDP-choline and diglyceride. CDP-diglyceride may therefore be a precursor, directly or indirectly, of all the phospholipids under study. The direct conversion of labeled CDP-diglyceride to some of the phospholipids is demonstrated. 3. 3. Formation of CDP-diglyceride from endogenous precursors appears to be a major reaction.

Poly(ethylene glycol) (PEG) conjugated arginine deiminase: effects of PEG formulations on its pharmacological properties.

Some tumors, such as melanomas and hepatocellular carcinomas, have a unique nutritional requirement for arginine. Thus, enzymatic degradation of extracellular arginine is one possible means for inhibiting these tumors. Arginine deiminase is an arginine degrading enzyme (ADI) that has been studied as an anti-cancer enzyme. However, ADI has a short serum half-life and, as a microbial enzyme, is highly immunogenic. Formulation of other therapeutic proteins with poly(ethylene glycol) (PEG) has overcome these problems. Here, ADI-PEGs were synthesized using PEGs of varying size, structure (linear or branched chain) and linker chemistries. All ADI-PEGs retained approximately 50% of enzyme activity when PEG was covalently attached to approximately 40% of the primary amines irrespective of the PEG molecular weight or attachment chemistry used. However, it was observed that, as the PEG size increases to 20 kDa, there was a corresponding increase in the pharmacokinetic (pK) and pharmacodynamic (pD) properties of the formulation. Variation in PEG linker or structure, or the use of PEGs >20,000 mw, did not affect the pK or pD. As has been shown with other therapeutic proteins, repeated injection of ADI-PEG into experimental animals resulted in significantly lower titers of antibodies against this protein than unmodified ADI. These data suggest that formulation of ADI with PEG of 20,000 mw results is the optimal method for formulating this promising therapeutic agent.

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.

Histone deacetylase inhibitors induce reactivation of herpes simplex virus type 1 in a latency-associated transcript-independent manner in neuronal cells.

Histone acetylation is implicated in the regulation of herpes simplex virus type 1 (HSV-1) latency. However, the role of histone acetylation in HSV-1 reactivation is less clear. In this study, the well-established model system, quiescently infected, neuronally differentiated PC12 (QIF-PC12) cells, was used to address the participation of histone acetylation in HSV-1 reactivation. In this model, sodium butyrate and trichostatin A (TSA), two histone deacetylase inhibitors, stimulated production of infectious HSV-1 progeny from a quiescent state. To identify viral genes responsive to TSA, the authors analyzed representative α, β, and γ viral genes using quantitative real-time polymerase chain reaction. Only the latency-associated transcript (LAT) accumulated in response to TSA treatment, under culture conditions that restricted virus replication and spread. This led the authors to evaluate the importance of LAT expression on TSA-induced reactivation. In QIF-PC12 cells, the LAT deletion mutant virus dLAT2903 reactivated equivalently with its wild-type parental strain (McKrae) after TSA treatment, as well as forskolin and heat stress treatment. Both viruses also reactivated equivalently from latently infected trigeminal ganglia explants from rabbits. In contrast, there was a marked reduction in the recovery of dLAT2903, as compared to wild-type virus, from the eyes of latently infected rabbits following epinephrine iontophoresis. These combined in vitro, ex vivo, and in vivo data suggest that LAT is not required for reactivation from latently infected neuronal cells per se, but may enhance processes that allow for the arrival of virus at, or close to, the site of original inoculation (i.e., recrudescence).

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.

Responses of purified phospholipases A2 to phospholipase A2 activating protein (PLAP) and melittin

The role of the phospholipase A2 (PLA2) stimulating protein PLAP in the regulation of PLA2 activity was assessed by determination of the effects of PLAP on two purified PLA2s. An approx. 14 kDa enzyme was purified from mouse thymoma cells, EL-4 cells, by cation ion exchange HPLC and immunoaffinity HPLC (with antiserum to the N-terminal sequence of an inflammatory exudate PLA2). An approx. 110 kDa enzyme was purified from mouse mammary carcinoma derived cells by sequential hydrophobic, anion exchange, hydroxyapatite and gel filtration HPLC. Neither PLAP nor melittin, an immunologically related PLA2 stimulating peptide from bee venom, increased the activity of the high molecular weight enzyme. In contrast, there was more than a 20-fold stimulation of the low molecular weight PLA2 by PLAP and an approx. 5-fold stimulation by melittin. The stimulation of enzyme activity by PLAP was observed at a protein to phospholipid ratio of 1:10(6) while the ratio of melittin to phospholipid was 1:3. Thus, PLAP mediated stimulation of PLA2 activity may include an interaction between PLAP and the enzyme, in contrast to melittin stimulation, which involves interactions between melittin and phospholipid.