Robert J. Mark

A Role for 4‐Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, in Disruption of Ion Homeostasis and Neuronal Death Induced by Amyloid β‐Peptide

Abstract: Peroxidation of membrane lipids results in release of the aldehyde 4‐hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimers disease (AD) and because amyloid β‐peptide (Aβ) can promote free radical production, we tested the hypothesis that HNE mediates Aβ25‐35‐induced disruption of neuronal ion homeostasis and cell death. Aβ induced large increases in levels of free and protein‐bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time‐ and concentration‐dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na+,K+‐ATPase activity and induced an increase of neuronal intracellular free Ca2+ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both Aβ and HNE. The antioxidant propyl gallate protected neurons against Aβ toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates Aβ‐induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.

CALCIUM, FREE RADICALS, AND EXCITOTOXIC NEURONAL DEATH IN PRIMARY CELL CULTURE

Publisher Summary Elucidation of cellular and molecular mechanisms of excitotoxic neuronal injury have been facilitated by the development of several cell biological technologies—namely, (1) neural cell culture, (2) quantitative assays of neuronal injury and death, (3) fluorescence imaging of intracellular free calcium levels ([Ca 2+ ] i ), (4) evaluation of mitochondrial function, (5) quantification of reactive oxygen species, (6) assessment of cytoskeletal alterations elicited by excitotoxic/metabolic insults, and (7) detection of “stress response” proteins. This chapter presents protocols for each of these technical approaches, as applied to embryonic rat and human brain neurons in dissociated cell culture. One strategy employed to study neuronal death is to elucidate the mechanisms that normally protect neurons from adverse environmental conditions. These studies have shown that many cellular signaling mechanisms exist that are designed to protect neurons against adverse environmental conditions such as metabolic and excitotoxic insults. Many of the neuroprotective strategies acquired during evolution involve systems that control calcium and free radical metabolism.

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.

Cellular signaling roles of TGFβ, TNFα and βAPP in brain injury responses and Alzheimer's disease

beta-Amyloid precursor protein (beta APP), transforming growth factor beta (TGF beta), and tumor necrosis factor-alpha (TNF alpha) are remarkably pleiotropic neural cytokines/neurotrophic factors that orchestrate intricate injury-related cellular and molecular interactions. The links between these three factors include: their responses to injury; their interactive effects on astrocytes, microglia and neurons; their ability to induce cytoprotective responses in neurons; and their association with cytopathological alterations in Alzheimers disease. Astrocytes and microglia each produce and respond to TGF beta and TNF alpha in characteristic ways when the brain is injured. TGF beta, TNF alpha and secreted forms of beta APP (sAPP) can protect neurons against excitotoxic, metabolic and oxidative insults and may thereby serve neuroprotective roles. On the other hand, under certain conditions TNF alpha and the fibrillogenic amyloid beta-peptide (A beta) derivative of beta APP can promote damage of neuronal and glial cells, and may play roles in neurodegenerative disorders. Studies of genetically manipulated mice in which TGF beta, TNF alpha or beta APP ligand or receptor levels are altered suggest important roles for each factor in cellular responses to brain injury and indicate that mediators of neural injury responses also have the potential to enhance amyloidogenesis and/or to interfere with neuroregeneration if expressed at abnormal levels or modified by strategic point mutations. Recent studies have elucidated signal transduction pathways of TGF beta (serine/threonine kinase cascades), TNF alpha (p55 receptor linked to a sphingomyelin-ceramide-NF kappa B pathway), and secreted forms of beta APP (sAPP; receptor guanylate cyclase-cGMP-cGMP-dependent kinase-K+ channel activation). Knowledge of these signaling pathways is revealing novel molecular targets on which to focus neuroprotective therapeutic strategies in disorders ranging from stroke to Alzheimers disease.

4‐Hydroxynonenal, a Lipid Peroxidation Product, Rapidly Accumulates Following Traumatic Spinal Cord Injury and Inhibits Glutamate Uptake

Abstract: Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult. One such event is the accumulation of free radicals that damage lipids, proteins, and nucleic acids. A major reactive product formed following lipid peroxidation is the aldehyde, 4‐hydroxynonenal (HNE), which cross‐links to side chain amino acids and inhibits the function of several key metabolic enzymes. In the present study, we used immunocytochemical and immunoblotting techniques to examine the accumulation of protein‐bound HNE, and synaptosomal preparations to study the effects of spinal cord injury and HNE formation on glutamate uptake. Protein‐bound HNE increased in content in the damaged spinal cord at early times following injury (1–24 h) and was found to accumulate in myelinated fibers distant to the site of injury. Immunoblots revealed that protein‐bound HNE levels increased dramatically over the same postinjury interval. Glutamate uptake in synaptosomal preparations from injured spinal cords was decreased by 65% at 24 h following injury. Treatment of control spinal cord synaptosomes with HNE was found to decrease significantly, in a dose‐dependent fashion, glutamate uptake, an effect that was mimicked by inducers of lipid peroxidation. Taken together, these findings demonstrate that the lipid peroxidation product HNE rapidly accumulates in the spinal cord following injury and that a major consequence of HNE accumulation is a decrease in glutamate uptake, which may potentiate neuronal cell dysfunction and death through excitotoxic mechanisms.

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.

Basic FGF attenuates amyloid β-peptide-induced oxidative stress, mitochondrial dysfunction, and impairment of Na+/K+-ATPase activity in hippocampal neurons

Basic fibroblast growth factor (bFGF) exhibits trophic activity for many populations of neurons in the brain, and can protect those neurons against excitotoxic, metabolic and oxidative insults. In Alzheimers disease (AD), amyloid beta-peptide (A beta) fibrils accumulate in plaques which are associated with degenerating neurons. A beta can be neurotoxic by a mechanism that appears to involve induction of oxidative stress and disruption of calcium homeostasis. Plaques in AD brain contain high levels of bFGF suggesting a possible modulatory role for bFGF in the neurodegenerative process. We now report that bFGF can protect cultured hippocampal neurons against A beta25-35 toxicity by a mechanism that involves suppression of reactive oxygen species (ROS) accumulation and maintenance of Na+/K+-ATPase activity. A beta25-35 induced lipid peroxidation, accumulation of H2O2, mitochondrial ROS accumulation, and a decrease in mitochondrial transmembrane potential; each of these effects of A beta25-35 was abrogated in cultures pre-treated with bFGF. Na+/K+-ATPase activity was significantly reduced following exposure to A beta25-35 in control cultures, but not in cultures pre-treated with bFGF. bFGF did not protect neurons from death induced by ouabain (a specific inhibitor of the Na+/K+-ATPase) or 4-hydroxynonenal (an aldehydic product of lipid peroxidation) consistent with a site of action of bFGF prior to induction of oxidative stress and impairment of ion-motive ATPases. By suppressing accumulation of oxyradicals, bFGF may slow A beta-induced neurodegenerative cascades.

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.

Aβ25-35 induces rapid lysis of red blood cells: Contrast with aβ1-42 and examination of underlying mechanisms

Abstract Amyloid β-peptide (Aβ) is produced by many different cell types and circulates in blood and cerebrospinal fluid in a soluble form. In Alzheimers disease (AD), Aβ forms insoluble fibrillar aggregates that accumulate in association with cells of the brain parenchyma and vasculature. Both full-length Aβ (Aβ1–40/42) and the Aβ25–35 fragment can damage and kill neurons by a mechanism that may involve oxidative stress and disruption of calcium homeostasis. Circulating blood cells are exposed to soluble Aβ1–40/42 and may also be exposed to Aβ aggregates associated with the luminal surfaces of cerebral microvessels. We therefore examined the effects of Aβ25–35 and Aβ1–42 on human red blood cells (RBCs) and report that Aβ25–35, in contrast to Aβ1–42, induces rapid (10–60 min) lysis of RBCs. The mechanism of RBC lysis by Aβ25–35 involved ion channel formation and calcium influx, but did not involve oxidative stress because antioxidants did not prevent cell lysis. In contrast, Aβ1–42 induced a delayed (4–24 h) damage to RBCs which was attenuated by antioxidants. The damaging effects of both Aβ25–35 and Aβ1–42 towards RBCs were completely prevented by Congo red indicating a requirement for peptide fibril formation. Aβ1–42 induced membrane lipid peroxidation in RBC, and basal levels of lipid peroxidation in RBCs from AD patients were significantly greater than in age-matched controls, suggesting a possible role for Aβ1–42 in previously reported alterations in RBCs from AD patients.