Together with aspartate, glutamate is the major excitatory neurotransmitter in the brain. Glutama... more Together with aspartate, glutamate is the major excitatory neurotransmitter in the brain. Glutamate binds and activates both ligand-gated ion channels (ionotropic glutamate receptors) and a class of G-protein coupled receptors (metabotropic glutamate receptors). Although the intracellular glutamate concentration in the brain is in the millimolar range, the extracellular glutamate concentration is kept in the low micromolar range by the action of excitatory amino acid transporters that import glutamate and aspartate into astrocytes and neurons. Excess extracellular glutamate may lead to excitotoxicity in vitro and in vivo in acute insults like ischemic stroke via the overactivation of ionotropic glutamate receptors. In addition, chronic excitotoxicity has been hypothesized to play a role in numerous neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer's disease and Huntington's disease. Based on this hypothesis, a good deal of effort has been devoted to develop and test drugs that either inhibit glutamate receptors or decrease extracellular glutamate. In this review, we provide an overview of the different pathways that are thought to lead to an over-activation of the glutamatergic system and glutamate toxicity in neurodegeneration. In addition, we summarize the available experimental evidence for glutamate toxicity in animal models of neurodegenerative diseases.
Over the last few years, we have identified an orally active, novel neuroprotective and cognition... more Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglia and astrocytes and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their by-products. However, key questions about its targets and brain penetration remain. In this study, we used label-free two-photon microscopy of intrinsic fisetin fluorescence to examine the localization of fisetin in living nerve cells and the brains of living mice. In cells, fisetin but not structurally related flavonols with different numbers of hydroxyl groups, localized to the nucleoli suggesting that key targets of fisetin may reside in this organelle. In the mouse brain, following intraperitoneal injection and oral administration, fisetin rapidly distributed to the blood vessels of the brain followed by a slower dispersion into the brain parenchyma. Thus, these results provide further support for the effects of fisetin on brain function. In addition, they suggest that label-free two-photon microscopy may prove useful for studying the intracellular and tissue distribution of other intrinsically-fluorescent flavonoids.
It is becoming increasingly clear that neurological diseases are multi-factorial involving disrup... more It is becoming increasingly clear that neurological diseases are multi-factorial involving disruptions in multiple cellular systems. Thus, while each disease has its own initiating mechanisms and pathologies, certain common pathways appear to be involved in most, if not all, neurological diseases. Thus, it is unlikely that modulating only a single factor will be effective at either preventing disease development or slowing disease progression. A better approach is to identify small (< 900 daltons) molecules that have multiple biological activities relevant to the maintenance of brain function. We have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity and inhi...
There is growing evidence for alterations in iron homeostasis during aging that are exacerbated i... more There is growing evidence for alterations in iron homeostasis during aging that are exacerbated in neurodegenerative diseases such as Alzheimer's disease. However, since essentially all neurodegenerative diseases are multi-factorial in the sense that there are a large number of mechanisms that can be identified as contributing to nerve cell death, iron chelators that have additional activities might be the most useful for the treatment of age-related CNS diseases. We have described a series of cell culture-based assays that define molecular toxicity pathways relevant to neurodegenerative diseases and have used these assays to identify potential therapeutic compounds for the treatment of these diseases. Deferiprone is a blood brain barrier permeable, low molecular weight iron chelator that has been used for many years to treat systemic iron disease. In this study, we describe the use of our cell culture-based screening assays to identify deferiprone derivatives with the greatest ...
Neurotrophic factors promote the development, maintenance and regeneration of nerve cells. Classi... more Neurotrophic factors promote the development, maintenance and regeneration of nerve cells. Classical neurotrophic factors are proteins and thus not well-suited for therapeutic purposes. Recently, we showed that specific flavonoids such as fisetin (3, 7, 3&amp;amp;amp;amp;amp;amp;amp;amp;#39;, 4&amp;amp;amp;amp;amp;amp;amp;amp;#39; tetrahydroxyflavone) promote the differentiation of nerve cells in culture through the activation of extracellular signal-regulated kinase (ERK) suggesting that flavonoids could substitute for neurotrophic factors. It has also been shown that fisetin promotes nerve cell survival following exposure to toxic oxidative insults. To determine whether or not this is unique to fisetin, a series of related compounds were assayed for neurotrophic activities. Many of these related compounds also promote nerve cell differentiation and are neuroprotective against toxic oxidative insults. However, the mechanisms underlying these neurotrophic effects differ among the compounds.
The role of the proteasome in neurodegenerative diseases is controversial. On the one hand, there... more The role of the proteasome in neurodegenerative diseases is controversial. On the one hand, there is evidence that a dysfunction of proteasome activity can lead to neurodegeneration but there is also data showing that proteasome inhibition can protect nerve cells from a variety of insults. In an attempt to clarify this issue, we studied the effects of four different proteasome inhibitors in a well characterized model of oxidative stress-induced nerve cell death. Consistent with the hypothesis that proteasome inhibition can be neuroprotective, we found that low concentrations of proteasome inhibitors were able to protect nerve cells from oxidative stress-induced death. Surprisingly, the neuroprotective effects of the proteasome inhibitors appeared to be at least partially mediated by the induction of NF-kappaB since protection was significantly reduced in cells expressing a specific NF-kappaB repressor. The activation of NF-kB by proteasome inhibitors was mediated by IkappaB alpha and IKK and was blocked by antioxidants and inhibitors of mitochondrial reactive oxygen species production. These data suggest that low concentrations of proteasome inhibitors induce a moderate level of mitochondrial oxidative stress which results in the activation of neuroprotective pathways.
To explore the possibility that specific flavonoids can substitute for neurotrophic factors, we e... more To explore the possibility that specific flavonoids can substitute for neurotrophic factors, we examined the ability of the flavonol fisetin and several related flavonoids to support the survival of low density, serum-free cultures of rat cortical neurons. Normally these cells die within 24h in the absence of trophic factors but in the presence of fisetin and several related flavonoids the cells survive and produce long neurites. While the survival-promoting effect of several of the fisetin-related flavonoids was partially dependent on ERK activation, the effect of fisetin was not. Fisetin can enhance glutathione synthesis but the survival-promoting effect of fisetin was also not dependent on glutathione. However, proteasome inhibitors almost completely blocked the ability of fisetin to promote survival. Consistent with this observation, fisetin increased proteasome activity. Together these results demonstrate a new activity for fisetin and tie this activity to its neurotrophic effects.
Protein tyrosine phosphorylation may be an important indicator of both the proliferative status a... more Protein tyrosine phosphorylation may be an important indicator of both the proliferative status and differentiation status of cells during embryonic development. To determine how each of these factors contributes to the level of phosphotyrosine-containing proteins detectable in embryonic tissues we have used immunohistochemistry with anti-phosphotyrosine antibodies on sections of developing chicken embryos. In contrast to an earlier study (Takata and Singer, 1988) we found proteins phosphorylated on tyrosine residues to be present in many different cells of the developing chicken embryo. The successful detection of phosphotyrosine-containing proteins in many cell types required the presence of sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, during fixation. Despite the fact that the majority of tyrosine kinases identified to date are growth factor receptors, the highest levels of phosphotyrosine-containing proteins in many tissues were localized to populations of cells which were differentiating or migrating rather than dividing.
The ganglioside GM1 has neuroprotective effects but is not of therapeutic value because of its la... more The ganglioside GM1 has neuroprotective effects but is not of therapeutic value because of its lack of bioavailability. Thus, molecules that mimic GM1 represent a novel approach to neuroprotection. We have synthesized 19 small GM1-like analogues whose simplified structure includes a hydrophobic saturated or unsaturated moiety linked to a hydrophilic moiety. We report their neuroprotective effects in two distinct models of nerve cell death using hippocampus-derived HT22 cells. We found that several analogues protected the HT22 cells from death at concentrations ranging from 2 to 5 microM. Additional neuroprotective assays using cortical slices injured by glutamate confirmed these results. Since members of the MAP kinase family are known to be key players in nerve cell survival and death, we characterized the role of these kinases in the neuroprotective mechanisms of the GM1-like analogues. Interestingly, the results indicate that the compounds provide neuroprotection through distinct mechanisms of action.
In their interactions with membranes, amphipathic small molecules exhibit detergent-like properti... more In their interactions with membranes, amphipathic small molecules exhibit detergent-like properties. At sufficiently high concentrations (above their critical micelle concentrations, if they form micelles), they substantially dissolve membranes. At lower concentrations, between maximally antihemolytic and lytic, we show here that the amphipaths significantly perturb membrane structure. Each of six small-molecule amphipaths was shown by hygroscopic desorption filtration to induce the extraction of small but significant amounts of membrane components, partly in the form
Peroxynitrite-mediated damage has been linked to numerous neurological and neurodegenerative dise... more Peroxynitrite-mediated damage has been linked to numerous neurological and neurodegenerative diseases, including stroke, Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s and Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s Diseases, amyotrophic lateral sclerosis and multiple sclerosis. Studies on the toxic effects of peroxynitrite in neurons have focused primarily on adverse effects resulting from the nitration of cellular proteins as the principal mode of toxicity while the consequences of the modulation of kinase pathways by peroxynitrite have received relatively less attention. Our results show that treatment of primary rat neurons with the peroxynitrite donor, SIN-1, leads to decreases in glutathione (GSH) levels and cell viability via a novel extracellular-signal-related kinase (ERK)/c-Myc phosphorylation pathway and a reduction in the nuclear expression of NF-E2-related factor-2 (Nrf2) that down-regulate the expression of glutamate cysteine ligase, the rate limiting enzyme for GSH synthesis. The flavonoid fisetin protects against the SIN-1-mediated alterations in ERK/c-Myc phosphorylation, nuclear Nrf2 levels, glutamate cysteine ligase levels, GSH concentration and cell viability. We also show that inhibition of mitogen-activated protein kinase kinase or Raf kinase can increase GSH levels in unstressed primary rat neurons through the same ERK/c-Myc phosphorylation pathway. Together, these results demonstrate that distinct signaling pathways modulate GSH metabolism in unstressed and stressed cortical neurons.
The Journal of Neuroscience the Official Journal of the Society For Neuroscience, May 1, 2001
Oxidative stress is implicated in the nerve cell death that occurs in a variety of neurological d... more Oxidative stress is implicated in the nerve cell death that occurs in a variety of neurological disorders, and the loss of protein kinase C (PKC) activity has been coupled to the severity of the damage. The functional relationship between stress, PKC, and cell death is, however, unknown. Using an immortalized hippocampal cell line that is particularly sensitive to oxidative stress, I show that activation of PKC by the phorbol ester tetradecanoylphorbol acetate (TPA) inhibits cell death via the stimulation of a complex protein phosphorylation pathway. TPA treatment leads to the rapid activation of extracellular signalregulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), the inactivation of p38 mitogen-activated protein kinase (MAPK), and the downregulation of PKC␦. Inhibition of either ERK or JNK activation blocks TPA-mediated protection, whereas p38 MAPK and PKC␦ inhibitors block stress-induced nerve cell death. Both p38 MAPK inactivation and JNK activation appear to be downstream of ERK because an agent that blocks ERK activation also blocks the modulation of these other MAP kinase family members by TPA treatment. Thus, the protection from oxidative stress afforded nerve cells by PKC activity requires the combined modulation of multiple enzyme pathways and suggests why the loss of PKC activity contributes to nerve cell death.
Micromolar conccntrations of sodium orthovanadate 5timulated the proliferation of bovine capillar... more Micromolar conccntrations of sodium orthovanadate 5timulated the proliferation of bovine capillary endothelial cclls, but no1 bovine aortic endothelial cells. Vanadate was equally potent at inducing protein tyrosine phosphorylation and changes in morphology in both types of cells. However, vanadatc treatment lead to an inhibition of protein tyrosine kinase activity in the aortic endothelial cells, but not the capillary endothelial cells. In capillary endothelial cells, the effect of vanadatc was additive with basic FGF (bFGF) at low concentrations of bFGF. There was no interaction between bFGF and vanadate in aortic endothelial cells. TGF-p, which inhibits the induction of endothelial cell proliferation by bFGF, appeared to shift the dose response curve to vanadate in capillary endothelial cells, increasing the proliferative effect of vanadate at low vanadate concentrations, but decreasing the proliferative effect a1 higher vanadate concentrations.
A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101... more A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.
Herein we report the synthesis and neuroprotective effects of new N-alkyl-1,2,4-oxadiazolidine-3,... more Herein we report the synthesis and neuroprotective effects of new N-alkyl-1,2,4-oxadiazolidine-3,5-diones and their corresponding synthetic intermediates, N-alkylhydroxylamines and N-1-alkyl-3-carbonyl-1-hydroxyureas, in an in vitro model of ischemia. We found five analogues that protect HT22 cells from death in the concentration range of 1-5 muM. Because members of the MAP kinase family are known to be key players in nerve cell survival and death, we characterized the role of these kinases in the neuroprotective mechanisms of the newly synthesized analogues. The results indicate that these compounds provide neuroprotection through distinct mechanisms of action.
Together with aspartate, glutamate is the major excitatory neurotransmitter in the brain. Glutama... more Together with aspartate, glutamate is the major excitatory neurotransmitter in the brain. Glutamate binds and activates both ligand-gated ion channels (ionotropic glutamate receptors) and a class of G-protein coupled receptors (metabotropic glutamate receptors). Although the intracellular glutamate concentration in the brain is in the millimolar range, the extracellular glutamate concentration is kept in the low micromolar range by the action of excitatory amino acid transporters that import glutamate and aspartate into astrocytes and neurons. Excess extracellular glutamate may lead to excitotoxicity in vitro and in vivo in acute insults like ischemic stroke via the overactivation of ionotropic glutamate receptors. In addition, chronic excitotoxicity has been hypothesized to play a role in numerous neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer&#39;s disease and Huntington&#39;s disease. Based on this hypothesis, a good deal of effort has been devoted to develop and test drugs that either inhibit glutamate receptors or decrease extracellular glutamate. In this review, we provide an overview of the different pathways that are thought to lead to an over-activation of the glutamatergic system and glutamate toxicity in neurodegeneration. In addition, we summarize the available experimental evidence for glutamate toxicity in animal models of neurodegenerative diseases.
Over the last few years, we have identified an orally active, novel neuroprotective and cognition... more Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglia and astrocytes and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their by-products. However, key questions about its targets and brain penetration remain. In this study, we used label-free two-photon microscopy of intrinsic fisetin fluorescence to examine the localization of fisetin in living nerve cells and the brains of living mice. In cells, fisetin but not structurally related flavonols with different numbers of hydroxyl groups, localized to the nucleoli suggesting that key targets of fisetin may reside in this organelle. In the mouse brain, following intraperitoneal injection and oral administration, fisetin rapidly distributed to the blood vessels of the brain followed by a slower dispersion into the brain parenchyma. Thus, these results provide further support for the effects of fisetin on brain function. In addition, they suggest that label-free two-photon microscopy may prove useful for studying the intracellular and tissue distribution of other intrinsically-fluorescent flavonoids.
It is becoming increasingly clear that neurological diseases are multi-factorial involving disrup... more It is becoming increasingly clear that neurological diseases are multi-factorial involving disruptions in multiple cellular systems. Thus, while each disease has its own initiating mechanisms and pathologies, certain common pathways appear to be involved in most, if not all, neurological diseases. Thus, it is unlikely that modulating only a single factor will be effective at either preventing disease development or slowing disease progression. A better approach is to identify small (< 900 daltons) molecules that have multiple biological activities relevant to the maintenance of brain function. We have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity and inhi...
There is growing evidence for alterations in iron homeostasis during aging that are exacerbated i... more There is growing evidence for alterations in iron homeostasis during aging that are exacerbated in neurodegenerative diseases such as Alzheimer's disease. However, since essentially all neurodegenerative diseases are multi-factorial in the sense that there are a large number of mechanisms that can be identified as contributing to nerve cell death, iron chelators that have additional activities might be the most useful for the treatment of age-related CNS diseases. We have described a series of cell culture-based assays that define molecular toxicity pathways relevant to neurodegenerative diseases and have used these assays to identify potential therapeutic compounds for the treatment of these diseases. Deferiprone is a blood brain barrier permeable, low molecular weight iron chelator that has been used for many years to treat systemic iron disease. In this study, we describe the use of our cell culture-based screening assays to identify deferiprone derivatives with the greatest ...
Neurotrophic factors promote the development, maintenance and regeneration of nerve cells. Classi... more Neurotrophic factors promote the development, maintenance and regeneration of nerve cells. Classical neurotrophic factors are proteins and thus not well-suited for therapeutic purposes. Recently, we showed that specific flavonoids such as fisetin (3, 7, 3&amp;amp;amp;amp;amp;amp;amp;amp;#39;, 4&amp;amp;amp;amp;amp;amp;amp;amp;#39; tetrahydroxyflavone) promote the differentiation of nerve cells in culture through the activation of extracellular signal-regulated kinase (ERK) suggesting that flavonoids could substitute for neurotrophic factors. It has also been shown that fisetin promotes nerve cell survival following exposure to toxic oxidative insults. To determine whether or not this is unique to fisetin, a series of related compounds were assayed for neurotrophic activities. Many of these related compounds also promote nerve cell differentiation and are neuroprotective against toxic oxidative insults. However, the mechanisms underlying these neurotrophic effects differ among the compounds.
The role of the proteasome in neurodegenerative diseases is controversial. On the one hand, there... more The role of the proteasome in neurodegenerative diseases is controversial. On the one hand, there is evidence that a dysfunction of proteasome activity can lead to neurodegeneration but there is also data showing that proteasome inhibition can protect nerve cells from a variety of insults. In an attempt to clarify this issue, we studied the effects of four different proteasome inhibitors in a well characterized model of oxidative stress-induced nerve cell death. Consistent with the hypothesis that proteasome inhibition can be neuroprotective, we found that low concentrations of proteasome inhibitors were able to protect nerve cells from oxidative stress-induced death. Surprisingly, the neuroprotective effects of the proteasome inhibitors appeared to be at least partially mediated by the induction of NF-kappaB since protection was significantly reduced in cells expressing a specific NF-kappaB repressor. The activation of NF-kB by proteasome inhibitors was mediated by IkappaB alpha and IKK and was blocked by antioxidants and inhibitors of mitochondrial reactive oxygen species production. These data suggest that low concentrations of proteasome inhibitors induce a moderate level of mitochondrial oxidative stress which results in the activation of neuroprotective pathways.
To explore the possibility that specific flavonoids can substitute for neurotrophic factors, we e... more To explore the possibility that specific flavonoids can substitute for neurotrophic factors, we examined the ability of the flavonol fisetin and several related flavonoids to support the survival of low density, serum-free cultures of rat cortical neurons. Normally these cells die within 24h in the absence of trophic factors but in the presence of fisetin and several related flavonoids the cells survive and produce long neurites. While the survival-promoting effect of several of the fisetin-related flavonoids was partially dependent on ERK activation, the effect of fisetin was not. Fisetin can enhance glutathione synthesis but the survival-promoting effect of fisetin was also not dependent on glutathione. However, proteasome inhibitors almost completely blocked the ability of fisetin to promote survival. Consistent with this observation, fisetin increased proteasome activity. Together these results demonstrate a new activity for fisetin and tie this activity to its neurotrophic effects.
Protein tyrosine phosphorylation may be an important indicator of both the proliferative status a... more Protein tyrosine phosphorylation may be an important indicator of both the proliferative status and differentiation status of cells during embryonic development. To determine how each of these factors contributes to the level of phosphotyrosine-containing proteins detectable in embryonic tissues we have used immunohistochemistry with anti-phosphotyrosine antibodies on sections of developing chicken embryos. In contrast to an earlier study (Takata and Singer, 1988) we found proteins phosphorylated on tyrosine residues to be present in many different cells of the developing chicken embryo. The successful detection of phosphotyrosine-containing proteins in many cell types required the presence of sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, during fixation. Despite the fact that the majority of tyrosine kinases identified to date are growth factor receptors, the highest levels of phosphotyrosine-containing proteins in many tissues were localized to populations of cells which were differentiating or migrating rather than dividing.
The ganglioside GM1 has neuroprotective effects but is not of therapeutic value because of its la... more The ganglioside GM1 has neuroprotective effects but is not of therapeutic value because of its lack of bioavailability. Thus, molecules that mimic GM1 represent a novel approach to neuroprotection. We have synthesized 19 small GM1-like analogues whose simplified structure includes a hydrophobic saturated or unsaturated moiety linked to a hydrophilic moiety. We report their neuroprotective effects in two distinct models of nerve cell death using hippocampus-derived HT22 cells. We found that several analogues protected the HT22 cells from death at concentrations ranging from 2 to 5 microM. Additional neuroprotective assays using cortical slices injured by glutamate confirmed these results. Since members of the MAP kinase family are known to be key players in nerve cell survival and death, we characterized the role of these kinases in the neuroprotective mechanisms of the GM1-like analogues. Interestingly, the results indicate that the compounds provide neuroprotection through distinct mechanisms of action.
In their interactions with membranes, amphipathic small molecules exhibit detergent-like properti... more In their interactions with membranes, amphipathic small molecules exhibit detergent-like properties. At sufficiently high concentrations (above their critical micelle concentrations, if they form micelles), they substantially dissolve membranes. At lower concentrations, between maximally antihemolytic and lytic, we show here that the amphipaths significantly perturb membrane structure. Each of six small-molecule amphipaths was shown by hygroscopic desorption filtration to induce the extraction of small but significant amounts of membrane components, partly in the form
Peroxynitrite-mediated damage has been linked to numerous neurological and neurodegenerative dise... more Peroxynitrite-mediated damage has been linked to numerous neurological and neurodegenerative diseases, including stroke, Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s and Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s Diseases, amyotrophic lateral sclerosis and multiple sclerosis. Studies on the toxic effects of peroxynitrite in neurons have focused primarily on adverse effects resulting from the nitration of cellular proteins as the principal mode of toxicity while the consequences of the modulation of kinase pathways by peroxynitrite have received relatively less attention. Our results show that treatment of primary rat neurons with the peroxynitrite donor, SIN-1, leads to decreases in glutathione (GSH) levels and cell viability via a novel extracellular-signal-related kinase (ERK)/c-Myc phosphorylation pathway and a reduction in the nuclear expression of NF-E2-related factor-2 (Nrf2) that down-regulate the expression of glutamate cysteine ligase, the rate limiting enzyme for GSH synthesis. The flavonoid fisetin protects against the SIN-1-mediated alterations in ERK/c-Myc phosphorylation, nuclear Nrf2 levels, glutamate cysteine ligase levels, GSH concentration and cell viability. We also show that inhibition of mitogen-activated protein kinase kinase or Raf kinase can increase GSH levels in unstressed primary rat neurons through the same ERK/c-Myc phosphorylation pathway. Together, these results demonstrate that distinct signaling pathways modulate GSH metabolism in unstressed and stressed cortical neurons.
The Journal of Neuroscience the Official Journal of the Society For Neuroscience, May 1, 2001
Oxidative stress is implicated in the nerve cell death that occurs in a variety of neurological d... more Oxidative stress is implicated in the nerve cell death that occurs in a variety of neurological disorders, and the loss of protein kinase C (PKC) activity has been coupled to the severity of the damage. The functional relationship between stress, PKC, and cell death is, however, unknown. Using an immortalized hippocampal cell line that is particularly sensitive to oxidative stress, I show that activation of PKC by the phorbol ester tetradecanoylphorbol acetate (TPA) inhibits cell death via the stimulation of a complex protein phosphorylation pathway. TPA treatment leads to the rapid activation of extracellular signalregulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), the inactivation of p38 mitogen-activated protein kinase (MAPK), and the downregulation of PKC␦. Inhibition of either ERK or JNK activation blocks TPA-mediated protection, whereas p38 MAPK and PKC␦ inhibitors block stress-induced nerve cell death. Both p38 MAPK inactivation and JNK activation appear to be downstream of ERK because an agent that blocks ERK activation also blocks the modulation of these other MAP kinase family members by TPA treatment. Thus, the protection from oxidative stress afforded nerve cells by PKC activity requires the combined modulation of multiple enzyme pathways and suggests why the loss of PKC activity contributes to nerve cell death.
Micromolar conccntrations of sodium orthovanadate 5timulated the proliferation of bovine capillar... more Micromolar conccntrations of sodium orthovanadate 5timulated the proliferation of bovine capillary endothelial cclls, but no1 bovine aortic endothelial cells. Vanadate was equally potent at inducing protein tyrosine phosphorylation and changes in morphology in both types of cells. However, vanadatc treatment lead to an inhibition of protein tyrosine kinase activity in the aortic endothelial cells, but not the capillary endothelial cells. In capillary endothelial cells, the effect of vanadatc was additive with basic FGF (bFGF) at low concentrations of bFGF. There was no interaction between bFGF and vanadate in aortic endothelial cells. TGF-p, which inhibits the induction of endothelial cell proliferation by bFGF, appeared to shift the dose response curve to vanadate in capillary endothelial cells, increasing the proliferative effect of vanadate at low vanadate concentrations, but decreasing the proliferative effect a1 higher vanadate concentrations.
A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101... more A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.
Herein we report the synthesis and neuroprotective effects of new N-alkyl-1,2,4-oxadiazolidine-3,... more Herein we report the synthesis and neuroprotective effects of new N-alkyl-1,2,4-oxadiazolidine-3,5-diones and their corresponding synthetic intermediates, N-alkylhydroxylamines and N-1-alkyl-3-carbonyl-1-hydroxyureas, in an in vitro model of ischemia. We found five analogues that protect HT22 cells from death in the concentration range of 1-5 muM. Because members of the MAP kinase family are known to be key players in nerve cell survival and death, we characterized the role of these kinases in the neuroprotective mechanisms of the newly synthesized analogues. The results indicate that these compounds provide neuroprotection through distinct mechanisms of action.
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Papers by Pamela Maher