Emmanuelle M. Blanc

4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes

Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimers, Parkinsons and Huntingtons diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4-hydroxynonenal potentiates oxidative cascades. Collectively, these findings suggest that 4-hydroxynonenal plays important roles in oxidative impairment of synaptic functions that would be expected to promote excitotoxic cascades.

Astrocytic Gap Junctional Communication Decreases Neuronal Vulnerability to Oxidative Stress-Induced Disruption of Ca2+ Homeostasis and Cell Death

Abstract: We investigated the effect of uncoupling astrocytic gap junctions on neuronal vulnerability to oxidative injury in embryonic rat hippocampal cell cultures. Mixed cultures (neurons growing on an astrocyte monolayer) treated with 18‐α‐glycyrrhetinic acid (GA), an uncoupler of gap junctions, showed markedly enhanced generation of intracellular peroxides (2,7‐dichlorofluorescein fluorescence), impairment of mitochondrial function [(dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction], and cell death (lactate dehydrogenase release) following exposure to oxidative insults (FeSO4 and 4‐hydroxynonenal). GA alone had little or no effect on basal levels of peroxides, mitochondrial function, or neuronal survival. Intercellular dye transfer analyses revealed extensive astrocyte‐astrocyte coupling but no astrocyte‐neuron or neuron‐neuron coupling in the mixed cultures. Studies of pure astrocyte cultures and microscope analyses of neurons in mixed cultures showed that the increased oxidative stress and cell death in GA‐treated cultures occurred only in neurons and not in astrocytes. Antioxidants (propyl gallate and glutathione) blocked the death of neurons exposed to FeSO4/GA. Elevations of neuronal intracellular calcium levels ([Ca2+]i) induced by FeSO4 were enhanced in neurons in mixed cultures exposed to GA. Removal of extracellular Ca2+ and the L‐type Ca2+ channel blocker nimodipine prevented impairment of mitochondrial function and cell death induced by FeSO4 and GA, whereas glutamate receptor antagonists were ineffective. Finally, GA exacerbated kainate‐ and FeSO4‐induced injury to pyramidal neurons in organotypic hippocampal slice cultures. The data suggest that interastrocytic gap junctional communication decreases neuronal vulnerability to oxidative injury by a mechanism involving stabilization of cellular calcium homeostasis and dissipation of oxidative stress.

Amyloid β‐Peptide Induces Cell Monolayer Albumin Permeability, Impairs Glucose Transport, and Induces Apoptosis in Vascular Endothelial Cells

Abstract: Amyloid β‐peptide (Aβ) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimers disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood‐brain barrier occur in AD. Here we report that Aβ25‐35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to Aβ25‐35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by Aβ25‐35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of Aβ25‐35 were specific because Aβ1‐40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of Aβ1‐40 aggregates and because astrocytes did not undergo similar changes after exposure to Aβ25‐35. Damage and death of ECs induced by Aβ25‐35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that Aβ induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then Aβ and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.

4-hydroxynonenal, a lipid peroxidation product, impairs glutamate transport in cortical astrocytes.

Astrocytes possess plasma membrane glutamate transporters that rapidly remove glutamate from the extracellular milieu and thereby prevent excitotoxic injury to neurons. Cellular oxidative stress is increased in neural tissues in a variety of acute and chronic neurodegenerative conditions. Recent findings suggest that oxidative stress increases neuronal vulnerability to excitotoxicity and that membrane lipid peroxidation plays a key role in this process. We now report that 4‐hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, impairs glutamate transport in cultured cortical astrocytes. Impairment of glutamate transport occurred within 1–3 h of exposure to HNE; FeSO4, an inducer of membrane lipid peroxidation, also impaired glutamate transport. Vitamin E prevented impairment of glutamate transport induced by FeSO4, but not that induced by HNE, consistent with HNE acting as an effector of lipid peroxidation‐induced impairment of glutamate transport. Glutathione, which binds and thereby detoxifies HNE, prevented HNE from impairing glutamate transport. Western blot, immunoprecipitation, and immunocytochemical analyses using an antibody against HNE‐protein conjugates provided evidence that HNE covalently binds to many different astrocytic proteins including the glutamate transporter GLT‐1. Data further suggest that HNE promotes intermolecular cross‐linking of GLT‐1 monomers to form dimers. HNE also induced mitochondrial dysfunction and accumulation of peroxides in astrocytes. Impairment of glutamate transport and mitochondrial function occurred with sublethal concentrations of HNE, concentrations known to be generated in cells exposed to various oxidative insults. Collectively, our data suggest that HNE may be an important mediator of oxidative stress‐induced impairment of astrocytic glutamate transport and may thereby play a role in promoting neuronal excitotoxicity. GLIA 22:149–160, 1998.

4-Hydroxynonenal, an aldehydic product of lipid peroxidation, impairs signal transduction associated with muscarinic acetylcholine and metabotropic glutamate receptors : Possible action on Gαq/11

Abstract: Considerable data indicate that oxidative stress and membrane lipid peroxidation contribute to neuronal degeneration in an array of age‐related neurodegenerative disorders. In contrast, the impact of subtoxic levels of membrane lipid peroxidation on neuronal function is largely unknown. We now report that 4‐hydroxy‐nonenal (HNE), an aldehydic product of lipid peroxidation, disrupts coupling of muscarinic cholinergic receptors and metabotropic glutamate receptors to phospholipase C‐linked GTP‐binding proteins in cultured rat cerebrocortical neurons. At subtoxic concentrations, HNE markedly inhibited GTPase activity, inositol phosphate release, and elevation of intracellular calcium levels induced by carbachol (muscarinic agonist) and (RS)‐3,5‐dihydroxyphenyl glycine (metabotropic glutamate receptor agonist). Maximal impairment of agonist‐induced responses occurred within 30 min of exposure to HNE. Other aldehydes, including malondialdehyde, had little effect on agonist‐induced responses. Antioxidants that suppress lipid peroxidation did not prevent impairment of agonist‐induced responses by HNE, whereas glutathione, which is known to bind and detoxify HNE, did prevent impairment of agonist‐induced responses. HNE itself did not induce oxidative stress. Immunoprecipitation‐western blot analysis using an antibody to HNE‐protein conjugates showed that HNE can bind to Gαq/11. HNE also significantly suppressed inositol phosphate release induced by aluminum fluoride. Collectively, our data suggest that HNE plays a role in altering receptor‐G protein coupling in neurons under conditions of oxidative stress that may occur both normally, and before cell degeneration and death in pathological settings.

Amyloid β-peptide and oxidative cellular injury in Alzheimer's disease

Alzheimer’s disease is a progressive neurodegenerative disorder that affects primarily learning and memory functions. There is significant neuronal loss and impairment of metabolic functioning in the temporal lobe, an area believed to be crucial for learning and memory tasks. Aggregated deposits of amyloid β-peptide may have a causative role in the development and progression of AD. We review the cellular actions of Aβ and how they can contribute to the cytotoxicity observed in AD. Aβ causes plasma membrane lipid peroxidation, impairment of ionmotive ATPases, glutamate uptake, uncoupling of a G-protein linked receptor, and generation of reactive oxygen species. These effects contribute to the loss of intracellular calcium homeostasis reported in cultured neurons. Many cell types other than neurons show alterations in the Alzheimer’s brain. The effects of Aβ on these cell types is also reviewed.

Calcium Homeostasis and Free Radical Metabolism as Convergence Points in the Pathophysiology of Dementia

Realization that calcium and free radicals are key mediators of neuronal injury and death initially came from studies of acute neurodegenerative insults, such as ischemia or excitotoxic injury (see refs. 1 and 2 for review). At that time, many were skeptical (and some remain so) regarding the relevance of ischemic and excitotoxic injury to such disorders as Alzheimer’s disease (AD). Nevertheless, it is becoming increasingly appreciated that “final common pathways” of cell death are very similar in both acute and chronic neurodegenerative conditions. Central to such final common pathways are calcium and free radicals, which can be considered transducers of cell death in both acute and chronic neurodegenerative conditions. There also existed somewhat of a dichotomy among researchers focusing on mechanisms of “necrotic” and “apoptotic” cell death, wherein “apoptologists” believed that there existed fundamental mechanistic differences that distinguished apoptosis from necrosis. That is, apoptosis was considered a process of cellular suicide involving induction of the expression of “cell death genes,” whereas necrosis was a passive process resulting from an uncontrollable avalanche of ion influx and cell lysis (see ref. 3 for review). However, the more that mechanisms of cell death were studied, the more evident it became that calcium and free radicals are key mediators of both necrosis and apoptosis, and that the distinction between the two manifestations of cell death depended more on the quantity (severity and duration of the insult) than the quality of the insult. It is therefore critical that we understand the various genetic and environmental factors that influence neural calcium homeostasis and free radical metabolism. This translates into the following tacks of investigation: 1. Determining how mutations linked to specific neurodegenerative disorders impact on calcium regulation and free radical metabolism; 2. Identifying environmental factors that may compromise calcium homeostasis and promote free radical accumulation, and determining the specific molecular cascades involved; and 3. Elucidating the mechanisms whereby the brain normally resists neuronal degeneration (e.g., neurotrophic factor signal transduction pathways and acute response pathways).