Weiming Fu

Activation of NF-κB protects hippocampal neurons against oxidative stress-induced apoptosis: Evidence for induction of manganese superoxide dismutase and suppression of peroxynitrite production and protein tyrosine nitration

The transcription factor NF‐κB is expressed in neurons wherein it is activated in response to a variety of stress‐ and injury‐related stimuli including exposure to cytokines such as tumor necrosis factor‐α (TNFα), and excitotoxic and oxidative insults. NF‐κB may play a role in the anti‐death actions of TNFα in cultured hippocampal neurons exposed to metabolic and oxidative insults. We now report that pretreatment of hippocampal cell cultures with agents that activate NF‐κB (TNFα and C2‐ceramide) confers resistance of neurons to apoptosis induced by the oxidative insults FeSO4 and amyloid β‐peptide (Aβ25–35). The neuroprotective actions of TNFα and ceramide were abolished in cultures cotreated with κB decoy DNA demonstrating a requirement for NF‐κB activation for prevention of cell death. Levels of manganese superoxide dismutase (Mn‐SOD) in neurons were increased following exposure of cultures to TNFα and ceramide in control cultures, but not in cultures cotreated with κB decoy DNA. FeSO4and Aβ25–35 induced accumulation of mitochondrial peroxynitrite, and membrane lipid peroxidation, in neurons. Peroxynitrite accumulation and lipid peroxidation were largely prevented in neurons pretreated with TNFα and ceramide prior to exposure to FeSO4 and Aβ25–35, an effect blocked by κB decoy DNA. Immunoreactivity of neurons with an anti‐nitrotyrosine antibody was increased following exposure to FeSO4 and Aβ25–35; TNFα and C2‐ceramide suppressed protein tyrosine nitration, and κB decoy DNA blocked the effects of TNFα and C2‐ceramide. Finally, the peroxynitrite scavenger uric acid protected neurons against apoptosis induced by FeSO4 and Aβ, and suppressed peroxynitrite accumulation. We conclude that, by inducing production of Mn‐SOD and suppressing peroxynitrite formation and membrane lipid peroxidation, NF‐κB plays an anti‐apoptotic role in neurodegenerative conditions that involve oxidative stress. The data further suggest important roles for peroxynitrite and NF‐κB in the pathogenesis of neuronal degeneration in Alzheimers disease. J. Neurosci. Res. 49:681–697, 1997.

Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock-in mice.

Excitotoxicity, a form of neuronal injury in which excessive activation of glutamate receptors results in cellular calcium overload, has been implicated in the pathogenesis of Alzheimer disease (AD), although direct evidence is lacking. Mutations in the presenilin-1 (PS1) gene on chromosome 14 are causally linked to many cases of early-onset inherited AD (refs. 5,6). We generated PS1 mutant mice (PS1M146VKI) that express the PS1 M146V targeted allele at normal physiological levels. Although PS1M146VKI mice have no overt mutant phenotype, they are hypersensitive to seizure-induced synaptic degeneration and necrotic neuronal death in the hippocampus. Cultured hippocampal neurons from PS1M146VKI mice have increased vulnerability to death induced by glutamate, which is correlated with perturbed calcium homeostasis, increased oxidative stress and mitochondrial dysfunction. Agents that suppress calcium influx or release and antioxidants protect neurons against the excitotoxic action of the PS1 mutation. These findings establish a direct link between a genetic defect that causes AD and excitotoxic neuronal degeneration, and indicate new avenues for therapeutic intervention in AD patients.

The endoplasmic reticulum stress-responsive protein GRP78 protects neurons against excitotoxicity and apoptosis: Suppression of oxidative stress and stabilization of calcium homeostasis

The 78-kDa glucose-regulated protein (GRP78) is localized in the endoplasmic reticulum (ER), and its expression is increased by environmental stressors in many types of nonneuronal cells. We report that levels of GRP78 are increased in cultured rat hippocampal neurons exposed to glutamate and oxidative insults (Fe2+ and amyloid beta-peptide) and that treatment of cultures with a GRP78 antisense oligodeoxynucleotide increases neuronal death following exposure to each insult. GRP78 antisense treatment enhanced apoptosis of differentiated PC12 cells following NGF withdrawal or exposure to staurosporine. Pretreatment of hippocampal cells with 2-deoxy-d-glucose, a potent inducer of GRP78 expression, protected neurons against excitotoxic and oxidative injury. GRP78 expression may function to suppress oxidative stress and stabilize calcium homeostasis because treatment with GRP78 antisense resulted in increased levels of reactive oxygen species and intracellular calcium following exposure to glutamate and oxidative insults in hippocampal neurons. Dantrolene (a blocker of ER calcium release), uric acid (an antioxidant), and zVAD-fmk (a caspase inhibitor) each protected neurons against the death-enhancing action of GRP78 antisense. The data suggest that ER stress plays a role in neuronal cell death induced by an array of insults and that GRP78 serves a neuroprotective function.

Anti-apoptotic Role of Telomerase in Pheochromocytoma Cells

Telomerase is a protein-RNA enzyme complex that adds a six-base DNA sequence (TTAGGG) to the ends of chromosomes and thereby prevents their shortening. Reduced telomerase activity is associated with cell differentiation and accelerated cellular senescence, whereas increased telomerase activity is associated with cell transformation and immortalization. Because many types of cancer have been associated with reduced apoptosis, whereas cell differentiation and senescence have been associated with increased apoptosis, we tested the hypothesis that telomerase activity is mechanistically involved in the regulation of apoptosis. Levels of telomerase activity in cultured pheochromocytoma cells decreased prior to cell death in cells undergoing apoptosis. Treatment of cells with the oligodeoxynucleotide TTAGGG or with 3,3′-diethyloxadicarbocyanine, agents that inhibit telomerase activity in a concentration-dependent manner, significantly enhanced mitochondrial dysfunction and apoptosis induced by staurosporine, Fe2+ (an oxidative insult), and amyloid β-peptide (a cytotoxic peptide linked to neuronal apoptosis in Alzheimer’s disease). Overexpression of Bcl-2 and the caspase inhibitor zVAD-fmk protected cells against apoptosis in the presence of telomerase inhibitors, suggesting a site of action of telomerase prior to caspase activation and mitochondrial dysfunction. Telomerase activity decreased in cells during the process of nerve growth factor-induced differentiation, and such differentiated cells exhibited increased sensitivity to apoptosis. Our data establish a role for telomerase in suppressing apoptotic signaling cascades and suggest a mechanism whereby telomerase may suppress cellular senescence and promote tumor formation.

Bcl-2 Protects Isolated Plasma and Mitochondrial Membranes Against Lipid Peroxidation Induced by Hydrogen Peroxide and Amyloid β-Peptide

Abstract: The bcl‐2 protooncogene product possesses antiapoptotic properties in neuronal and nonneuronal cells. Recent data suggest that Bcl‐2s potency as a survival factor hinges on its ability to suppress oxidative stress, but neither the subcellular site(s) nor the mechanism of its action is known. In this report electron paramagnetic resonance (EPR) spectroscopy analyses were used to investigate the local effects of Bcl‐2 on membrane lipid peroxidation. Using hydrogen peroxide (H2O2) and amyloid β‐peptide (Aβ) as lipoperoxidation initiators, we determined the loss of EPR‐detectable paramagnetism of nitroxyl stearate (NS) spin labels 5‐NS and 12‐NS. In intact cell preparations and postnuclear membrane fractions, Aβ and H2O2 induced significant loss of 5‐NS and 12‐NS signal amplitude in control PC12 cells, but not PC12 cells expressing Bcl‐2. Cells were subjected to differential subcellular fractionation, yielding preparations of plasma membrane and mitochondria. In preparations derived from Bcl‐2‐expressing cells, both fractions contained Bcl‐2 protein. 5‐NS and 12‐NS signals were significantly decreased following Aβ and H2O2 exposure in control PC12 mitochondrial membranes, and Bcl‐2 largely prevented these effects. Plasma membrane preparations containing Bcl‐2 were also resistant to radical‐induced loss of spin label. Collectively, our data suggest that Bcl‐2 is localized to mitochondrial and plasma membranes where it can act locally to suppress oxidative damage induced by Aβ and H2O2, further highlighting the important role of lipid peroxidation in apoptosis.

Catecholamines Potentiate Amyloid β-Peptide Neurotoxicity: Involvement of Oxidative Stress, Mitochondrial Dysfunction, and Perturbed Calcium Homeostasis ☆

Oxidative stress and mitochondrial dysfunction are implicated in the neuronal cell death that occurs in physiological settings and in neurodegenerative disorders. In Alzheimers disease (AD) degenerating neurons are associated with deposits of amyloid beta-peptide (A beta), and there is evidence for increased membrane lipid peroxidation and protein oxidation in the degenerating neurons. Cell culture studies have shown that A beta can disrupt calcium homeostasis and induce apoptosis in neurons by a mechanism involving oxidative stress. We now report that catecholamines (norepinephrine, epinephrine, and dopamine) increase the vulnerability of cultured hippocampal neurons to A beta toxicity. The catecholamines were effective in potentiating A beta toxicity at concentrations of 10-200 microM, with the higher concentrations (100-200 microM) themselves inducing cell death. Serotonin and acetylcholine were not neurotoxic and did not modify A beta toxicity. Levels of membrane lipid peroxidation, and cytoplasmic and mitochondrial reactive oxygen species, were increased following exposure to neurons to A beta, and catecholamines exacerbated the oxidative stress. Subtoxic concentrations of catecholamines exacerbated decreases in mitochondrial energy charge and transmembrane potential caused by A beta, and higher concentrations of catecholamines alone induced mitochondrial dysfunction. Antioxidants (vitamin E, glutathione, and propyl gallate) protected neurons against the damaging effects of A beta and catecholamines, whereas the beta-adrenergic receptor antagonist propanolol and the dopamine (D1) receptor antagonist SCH23390 were ineffective. Measurements of intracellular free Ca2+ ([Ca2+]i) showed that A beta induced a slow elevation of [Ca2+]i which was greatly enhanced in cultures cotreated with catecholamines. Collectively, these data indicate a role for catecholamines in exacerbating A beta-mediated neuronal degeneration in AD and, when taken together with previous findings, suggest roles for oxidative stress induced by catecholamines in several different neurodegenerative conditions.

4-Hydroxynonenal, a product of lipid peroxidation, inhibits dephosphorylation of the microtubule-associated protein tau.

IN Alzheimers disease (AD) the microtubule-associated protein tau is excessively phosphorylated in degenerating neurons, but the mechanisms underlying the increased phosphorylation are unknown. Recent findings suggest that oxidative stress, and membrane lipid peroxidation in particular, contributes to the neurodegenerative process in AD. We now report that following exposure of cultured rat hippocampal neurons to 4-hydroxy-nonenal (HNE), an aldehydic product of membrane lipid peroxidation, tau is resistant to dephoshorylation. Immunocytochemical and Western blot analyses using phosphorylation-sensitive tau antibodies showed that HNE treatment causes a moderate increase in basal levels of tau phosphorylation, and prevents tau dephosphorylation by alkaline phosphatase in neurons pretreated with the phosphatase inhibitor okadaic acid. Studies with anti-HNE antibodies showed that HNE binds directly to tau, and that HNE immunoreactivity localizes to cell bodies and axons, cell compartments that contain tau. These data suggest a role for HNE in altered tau phosphorylation and neurofibrillary degeneration in AD.

The catalytic subunit of telomerase is expressed in developing brain neurons and serves a cell survival-promoting function

Telomerase, a specialized reverse transcriptase (RT) linked to cell immortalization and cancer, has been thought not to be expressed in postmitotic cells. We now report that telomerase activity and its essential catalytic subunit, telomerase reverse transcriptase (TERT), are expressed in neurons in the brains of rodents during embryonic and early postnatal development, and are subsequently downregulted. Suppression of TERT expression in cultured embryonic hippocampal neurons in creases their vulnerability to apoptosis and excitotoxicity. Overexpression of TERT in PC12 cells suppresses apoptosis induced by trophic factor withdrawal. TERT exerts its anti-apoptotic action at an early stage of the cell death process prior to mitochondrial dysfunction and caspase activation. TERT may serve a neuron survival-promoting function in the developing brain, and downregulation of TERT in the adult brain may contribute to increased neuronal vulnerability in various age-related neurodegenerative disorders.

Herp Stabilizes Neuronal Ca2+ Homeostasis and Mitochondrial Function during Endoplasmic Reticulum Stress

In response to endoplasmic reticulum (ER) stress, cells launch homeostatic and protective responses, but can also activate cell death cascades. A 54 kDa integral ER membrane protein called Herp was identified as a stress-responsive protein in non-neuronal cells. We report that Herp is present in neurons in the developing and adult brain, and that it is regulated in neurons by ER stress; sublethal levels of ER stress increase Herp levels, whereas higher doses decrease Herp levels and induce apoptosis. The decrease in Herp protein levels following a lethal ER stress occurs prior to mitochondrial dysfunction and cell death, and is mediated by caspases which generate a 30-kDa proteolytic Herp fragment. Mutagenesis of the caspase cleavage site in Herp enhances its neuroprotective function during ER stress. While suppression of Herp induction by RNA interference sensitizes neural cells to apoptosis induced by ER stress, overexpression of Herp promotes survival by a mechanism involving stabilization of ER Ca2+ levels, preservation of mitochondrial function and suppression of caspase 3 activation. ER stress-induced activation of JNK/c-Jun and caspase 12 are reduced by Herp, whereas induction of major ER chaperones is unaffected. Herp prevents ER Ca2+ overload under conditions of ER stress and agonist-induced ER Ca2+ release is attenuated by Herp suggesting a role for Herp in regulating neuronal Ca2+ signaling. By stabilizing ER Ca2+ homeostasis and mitochondrial functions, Herp serves a neuroprotective function under conditions of ER stress.

Autoantibodies to amyloid β-peptide (Aβ) are increased in Alzheimer’s disease patients and Aβ antibodies can enhance Aβ neurotoxicity

Studies of amyloid precursor protein transgenic mice suggest that immune responses to amyloid β peptide (Aβ) may be instrumental in the removal of plaques from the brain, but the initial clinical trial of an Aβ vaccine in patients with Alzheimer’s disease (AD) was halted as the result of serious neurological complications in some patients. We now provide evidence that AD patients exhibit an enhanced immune response to Aβ and that, contrary to expectations, Aβ antibodies enhance the neurotoxic activity of the peptide. Serum titers to Aβ were significantly elevated in AD patients and Aβ antibodies were found in association with amyloid plaques in their brains, but there was no evidence of cell-mediated immune responses to Aβ in the patients. Aβ antibodies were detected in the serum of old APP mutant transgenic mice with plaque-like Aβ deposits, but not in the serum of younger transgenic or nontransgenic mice. Serum from APP mutant mice potentiated the neurotoxicity of Aβ. Our data suggest that a humoral immune response to Aβ in AD patients may promote neuronal degeneration, a process with important implications for the future of vaccine-based therapies for AD.