Pamela Maher

A Role for 12-lipoxygenase in Nerve Cell Death Caused by Glutathione Depletion

An early and highly specific decrease in glutathione (GSH) in the substantia nigra is associated with Parkinsons disease, and low levels of GSH lead to the degeneration of cultured dopaminergic neurons. Using immature cortical neurons and a clonal nerve cell line, it is shown that a decrease in GSH triggers the activation of neuronal 12-lipoxygenase (12-LOX), which leads to the production of peroxides, the influx of Ca2+, and ultimately to cell death. The supporting evidence includes: 1) inhibitors of arachidonate metabolism and 12-LOX block cell death induced by GSH depletion; 2) there is an increase in 12-LOX activity and a membrane translocation in HT22 cells, and an induction of the enzyme in primary cortical neurons following the reduction of GSH; 3) 12-LOX is directly inhibited by GSH; and 4) exogenous arachidonic acid potentiates cell death. These data show that the LOX pathway is a critical intermediate in at least some forms of neuronal degeneration.

Oxidative Stress Induces a Form of Programmed Cell Death with Characteristics of Both Apoptosis and Necrosis in Neuronal Cells

Abstract: Oxidative stress is implicated in a number of neurological disorders including stroke, Parkinsons disease, and Alzheimers disease. To study the effects of oxidative stress on neuronal cells, we have used an immortalized mouse hippocampal cell line (HT‐22) that is particularly sensitive to glutamate. In these cells, glutamate competes for cystine uptake, leading to a reduction in glutathione and, ultimately, cell death. As it has been reported that protein kinase C activation inhibits glutamate toxicity in these cells and is also associated with the inhibition of apoptosis in other cell types, we asked if glutamate toxicity was via apoptosis. Morphologically, glutamate‐treated cells underwent plasma membrane blebbing and cell shrinkage, but no DNA fragmentation was observed. At the ultrastructural level, there was damage to mitochondria and other organelles although the nuclei remained intact. Protein and RNA synthesis inhibitors as well as certain protease inhibitors protected the cells from glutamate toxicity. Both the macromolecular synthesis inhibitors and the protease inhibitors had to be added relatively soon after the addition of glutamate, suggesting that protein synthesis and protease activation are early and distinct steps in the cell death pathway. Thus, the oxidative stress brought about by treatment with glutamate initiates a series of events that lead to a form of cell death distinct from either necrosis or apoptosis.

Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line

A neuronal cell line, HT-22, is sensitive to glutamate cytotoxicity via a non-receptor mediated oxidative pathway. 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, blocks this glutamate-induced cell death. Down-regulation of protein kinase C eliminates the protection against glutamate cytotoxicity afforded by TPA. The data suggest that protein kinase C activation blocks an early step in the cytotoxic pathway.

The effects of stress and aging on glutathione metabolism

Glutathione plays a critical role in many biological processes both directly as a co-factor in enzymatic reactions and indirectly as the major thiol-disulfide redox buffer in mammalian cells. Glutathione also provides a critical defense system for the protection of cells from many forms of stress. However, mild stress generally increases glutathione levels, often but not exclusively through effects on glutamate cysteine ligase, the rate-limiting enzyme for glutathione biosynthesis. This upregulation in glutathione provides protection from more severe stress and may be a critical feature of preconditioning and tolerance. In contrast, during aging, glutathione levels appear to decline in a number of tissues, thereby putting cells at increased risk of succumbing to stress. The evidence for such a decline is strongest in the brain where glutathione loss is implicated in both Parkinsons disease and in neuronal injury following stroke.

A comprehensive analysis of the distribution of FGF-2 and FGFR1 in the rat brain.

We have examined the cellular distribution of both FGF-2 and FGFR1 immunoreactivity and their mRNAs throughout the normal adult rat brain in order to reconcile numerous disparate findings in the published literature. The results confirm a widespread distribution of FGF-2 and FGFR1 in the rat brain, and different regions express distinct patterns of FGF-2 and FGFR1 mRNA and protein: neuronal and non-neuronal cells show different subcellular distributions that vary according to the area where they are located. The intensity of the staining and hybridization also varies according to the loci examined and the cell type involved. Astrocytes contain the highest levels of FGF-2 and FGFR1 mRNAs, and characteristically, possess high levels of immunoreactive FGF-2 within the nucleus. Amongst non-neuronal cells, oligodendrocytes do not synthesize or contain significant levels of FGF-2 immunoreactivity however, they do express FGFR1 mRNA. In these cells, immunoreactive FGFR1 is mainly associated with the myelin sheaths of neuronal fibers. In ventricular systems, ependymal cells synthesize and contain immunoreactive FGFR1. In contrast, only cells lining the lateral wall of the IIIrd ventricle express FGF-2 mRNA. Subependymal cells contain high levels of both FGF-2 and FGFR1 immunoreactivity. Neurons express low levels of FGF-2 mRNA and immunoreactive FGF-2 is localized predominantly to the perikaryon. However, selected populations of neurons, such as CA2 field of the hippocampus, show high levels of FGF-2 mRNA, in which the nucleus is strongly immunopositive. Similarly, high levels of FGFR1 mRNA are localized to select populations of neurons (e.g. amygdala). FGFR1 immunoreactivity is mainly associated with myelinated fiber tracts (e.g. striatum), and some neurons show immunoreactivity in the perikaryon (e.g. hippocampus), the nucleus (e.g. mesencephalic trigeminal nucleus), or in axonal projections (e.g. hypothalamus). Remarkably, in many of the areas studied, FGF-2 and FGFR1 mRNA and/or their translated protein do not co-localize in neurons (e.g. neo-cortices) or even in the same regions of the brain (e.g. substantia nigra). In other instances, mRNAs for both FGF-2 and FGFR1 colocalize (e.g. supraoptic nucleus). The brain, in contrast to peripheral tissues, contains high levels of FGF-2 and actively expresses its gene under normal physiological conditions. The highly specific anatomical distribution of immunoreactive FGF-2 in neuronal and non-neuronal brain cells, supports the notion that it plays a multifunctional role in the CNS under normal physiology. By correlating the localization and the synthesis of FGF-2 and one of its high affinity receptors, FGFR1, in the CNS, it should be possible to obtain a better understanding of the roles of FGF-2 in normal and pathological conditions.

Flavonoid fisetin promotes ERK-dependent long-term potentiation and enhances memory

Small molecules that activate signaling pathways used by neurotrophic factors could be useful for treating CNS disorders. Here we show that the flavonoid fisetin activates ERK and induces cAMP response element-binding protein (CREB) phosphorylation in rat hippocampal slices, facilitates long-term potentiation in rat hippocampal slices, and enhances object recognition in mice. Together, these data demonstrate that the natural product fisetin can facilitate long-term memory, and therefore it may be useful for treating patients with memory disorders.

The Cystine/Glutamate Antiporter System xc− in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities

The antiporter system x(c)(-) imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system x(c)(-) is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x(c)(-), including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x(c)(-). Moreover, the roles of system x(c)(-) in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system x(c)(-) inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System x(c)(-) is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x(c)(-) in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS.

The Regulation of Glucose Metabolism by HIF-1 Mediates a Neuroprotective Response to Amyloid Beta Peptide

It is frequently argued that both amyloid beta (Abeta) and oxidative stress are involved in the pathogenesis of Alzheimers disease (AD). We show here that clonal nerve cell lines and primary cortical neurons that are resistant to Abeta toxicity have an enhanced flux of glucose through both the glycolytic pathway and the hexose monophosphate shunt. AD brain also has increased enzymatic activities in both pathways relative to age-matched controls. The Abeta-induced changes in glucose metabolism are due to the activation of the transcription factor hypoxia inducible factor 1 (HIF-1). As a result of Abeta-induced changes in glucose metabolism, Abeta-resistant cells are more readily killed by glucose starvation and by classes of antipsychotic drugs that inhibit glucose uptake.

Cellular Mechanisms of Resistance to Chronic Oxidative Stress

Oxidative stress is implicated in several pathologies such as AIDS, Alzheimers disease, and Parkinsons disease, as well as in normal aging. As a model system to study the response of cells to oxidative insults, glutamate toxicity on a mouse nerve cell line, HT-22, was examined. Glutamate exposure kills HT-22 via a nonreceptor-mediated oxidative pathway by blocking cystine uptake and causing depletion of intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species and, ultimately, apoptotic cell death. Several HT-22 subclones that are 10-fold resistant to exogenous glutamate were isolated and the mechanisms involved in resistance characterized. The expression levels of neither heat shock proteins nor apoptosis-related proteins are changed in the resistant cells. In contrast, the antioxidant enzyme catalase, but not glutathione peroxidase nor superoxide dismutase, is more highly expressed in the resistant than in the parental cells. In addition, the resistant cells have enhanced rates of GSH regeneration due to higher activities of the GSH metabolic enzymes gamma-glutamylcysteine synthetase and GSH reductase, and GSH S-transferases activities are also elevated. As a consequence of these alterations, the glutamate resistant cells are also more resistant to organic hydroperoxides and anticancer drugs that affect these GSH enzymes. These results indicate that resistance to apoptotic oxidative stress may be acquired by coordinated changes in multiple antioxidant pathways.

p38 Mitogen-activated Protein Kinase Activation Is Required for Fibroblast Growth Factor-2-stimulated Cell Proliferation but Not Differentiation

Basic fibroblast growth factor (FGF-2) is a member of a family of polypeptides that have roles in a wide range of biological processes. To determine why different cell types show distinct responses to treatment with FGF-2, the array of FGF receptors present on the surface of a cell which differentiates in response to FGF-2 (PC12 cells) was compared with that present on the surface of a cell that proliferates in response to FGF-2 (Swiss 3T3 fibroblasts). Both cell types express exclusively FGFR1, suggesting that there are cell type-specific FGFR1 signaling pathways. Since mitogen-activated protein kinases function as mediators of cellular responses to a variety of stimuli, the roles of these proteins in FGF-mediated responses were examined. FGF-2 activates extracellular signal-regulated kinases with similar kinetics in both fibroblasts and PC12 cells, and a specific inhibitor of extracellular signal-regulated kinase activation blocks differentiation but has little effect on proliferation. In contrast, while p38 mitogen-activated protein kinase is activated weakly and transiently in PC12 cells treated with FGF-2, a much stronger and sustained activation of this kinase is seen in FGF-2-treated fibroblasts. Furthermore, specific inhibitors of this kinase block proliferation but have no effect on differentiation. This effect on proliferation is specific for FGF-2 since the same concentrations of inhibitors have little or no effect on proliferation induced by serum.