Disruption of Brain Cell Ion Homeostasis in Alzheimer's Disease by

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Disruption of Brain Cell Ion Homeostasis in Alzheimer’s Disease by Oxy Radicals, and Signaling Pathways That Protect Therefrom Mark P. Mattson,* Robert J. Mark, Katsutoshi Furukawa, and Annadora J. Bruce Sanders-Brown Research Center on Aging and Department of Anatomy & Neurobiology, University of Kentucky, Lexington, Kentucky 40536-0230 Received July 30, 1996

Evolutionary Perspective: Oxy Radicals, Ion Homeostasis, and the Nervous System Since the time that cells first arose from the “primordial soup” they have existed in an oxidizing environment. It is therefore no wonder that cells have devised an array of molecular tools with which to prevent accumulation of free radicals and to limit damage to vital cellular constituents. When viewed this way, oxy radicals are considered a powerful force in the process of natural selection. The fact that enzymes such as catalase, glutathione peroxidase, and Cu/Zn-superoxide dismutase (Cu/Zn-SOD)1 are present in organisms from flies to man attests to their importance in cell survival (1). As multicellular organisms evolved they developed intercellular signaling mechanisms that allowed them to pass on “signals of impending doom” from cell to cell. As will be discussed below, when cells are injured they produce trophic factors that signal adjacent cells of imminent danger (2). The adjacent cells respond by increasing their expression of antioxidant enzymes and other cytoprotective proteins. Oxy radicals take on particularly important roles in the nervous system, from both physiological and pathophysiological perspectives. There is increasing evidence to support a role for oxy radicals as mediators of signaling in neuronal networks. For example, nitric oxide is believed to play an important role in the process of long-term potentiation, a cellular correlate of learning and memory (3). Evolutionary pressure also favored cells with mechanisms to maintain ion gradients across their cell membranes and to use those ion gradients to their advantage. Such ion-regulating systems are most intricate and refined in nerve cells, a cell type which has harnessed ion movements to initiate and propogate rapid information flow in neural circuits. Ironically, some of the ionregulating mechanisms that are unique to neurons can contribute to their demise in neurodegenerative disorders. For example, glutamate, the major excitatory neurotransmitter in the mammalian brain, may promote neuronal degeneration in a variety of disorders ranging from stroke to epilepsy to Alzheimer’s disease (AD) by inducing massive influx of Ca2+ (4, 5). Indeed, neurons that express glutamate receptors are among the first to * Address for correspondence: 211 Sanders-Brown Building, 800 S. Limestone, Lexington, KY 40536-0230. Phone: (606) 257-6040. Fax: (606) 323-2866. E-mail: [email protected]. 1Abbreviations: Aβ, amyloid β-peptide; AD, Alzheimer’s disease; βAPP, β-amyloid precursor protein; bFGF, basic fibroblast growth factor; BDNF, brain-derived neurotrophic factor; ER, endoplasmic reticulum; HNE, 4-hydroxy-2,3-nonenal; IGF, insulin-like growth factor; IP3, inositol triphosphate; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate; NS, nitroxyl stearate; sAPPR, secreted form of β-amyloid precursor protein; SOD, superoxide dismutase; TNF, tumor necrosis factor; VDCC, voltage-dependent calcium channel.

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degenerate in many different neurodegenerative conditions. Whereas many cell types in the body are mitotic and therefore capable of replacing lost neighbors, neurons are not capable of cell division and are therefore irreplaceable, and it is remarkable that neurons can exist and function normally for over 100 years. It is, conversely, no surprise that many neurons do not survive the adverse consequences of brain aging including increased oxidative stress. The present article will review our current level of understanding of why neurons die in AD and will focus on evidence linking oxidative stress and disruption of ion homeostasis to neuronal degeneration in AD. Any theory of AD has to account for the fact that some cases of AD involve mutations in specific genes. This article will therefore include pertinent information concerning the role of genetic mutations, particularly those in the β-amyloid precursor protein (βAPP), in the pathogenesis of AD. The genetic data will be placed in the context of age-related metabolic and oxidative alterations, with specific consideration of the free radical (6, 7) and calcium (4) hypotheses of AD.

Neuronal Oxy Radical Metabolism Mitochondria are a major intrinsic source of oxy radicals (6). Superoxide anion radical (O2•-) is constantly generated during the process of electron transport in mitochondria. Neurons contain two different superoxide dismutases (SOD) which convert O2•- to H2O2 (Figure 1). Cu/Zn-SOD is localized in the cytoplasm and within some organelles, while Mn-SOD is confined largely to mitochondria. H2O2 itself is not a free radical but is a major source for generation of hydroxyl radical (OH•) which is formed in a reaction (Fenton reaction) catalyzed by ionic (ferrous) iron and copper. Other pathways for oxy radical production in neurons include generation of nitric oxide (NO•) which results from calcium/calmodulin-stimulated activation of nitric oxide synthase (NO• further reacts with O2•- to form peroxynitrite); release of arachidonic acid from membrane phospholipids and subsequent metabolism by cyclooxygenases and lipoxygenases; and the xanthine-xanthine/oxidase and other oxidase pathways (e.g., monoamine oxidase). Regenerative cascades of oxy radical production can occur at several sites within cells, with the classic example being the process of lipid peroxidation in membranes initiated by OH• attack at double bonds of membrane fatty acids. Lipid peroxidation is likely a key event leading to disruption of ion homeostasis in neurons subjected to oxidative stress because the process leads to impairment of ion-motive ATPases and other ion-regulating membrane proteins (see below). © 1997 American Chemical Society

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Figure 1. Sources of reactive oxygen species in neurons and mechanisms for their removal. Superoxide anion radical (O2•-) is produced during the electron transport process in the mitochondria. Superoxide dismutase (SOD) converts O2•- to H2O2 which is converted to water by the enzymes catalase and glutathione peroxidase (GSHPx). Alternatively, highly reactive OH• can be produced via Fenton chemistry in the presence of iron (Fe). Elevation of [Ca2+]i induced by glutamate or amyloid β-peptide, for example, promotes production of several different reactive oxygen species. Ca2+ binds calmodulin and thereby activates nitric oxide synthase (NOS) resulting in the generation of nitric oxide (NO•); NO• interacts with O2•- resulting in formation of peroxynitrite which can damage proteins. Ca2+ also promotes phospholipid hydrolysis via activity of phospholipase A2 (PLA2). PLA2 induces release of arachidonic acid which is then attacked by lipoxygenases (LOX) and cyclooxygenases (COX) with resultant oxy radical formation. Elevated [Ca2+]i also reduces mitochondrial transmembrane potential which can lead to increased O2•- production. Other abbreviations: LTs, leukotrienes; PGs, prostaglandins; THRs, thromboxanes.

As indicated in the evolutionary perspective above, eukaryotes have evolved genes encoding proteins whose function is to prevent free radical formation or to destroy free radicals once they have formed (Figure 1). Particularly important are enzymes that convert H2O2 to H2O and thereby prevent OH• formation, namely, catalase and glutathione peroxidase. Glutathione reductase serves the important function of reducing oxidized glutathione so that it can be used as a substrate for glutathione peroxidase. Reduced glutathione is a very important antioxidant and is, in fact, quite effective in protecting neurons from being damaged and killed by a variety of insults including exposure to excitotoxins (8) and amyloid β-peptide (Aβ) (Figure 2). Additional antioxidant enzymes are Cu/Zn-SOD and Mn-SOD. Nonenzyme antioxidants include vitamin E (R-tocopherol), vitamin C, and ubiquinone. The importance of each of the antioxidant enzymes and small molecule antioxidants can be appreciated when one begins to study the effects of oxidative insults on neurons in experimental settings. For example, overexpression of Cu/Zn-SOD in transgenic mice confers resistance to ischemic brain injury (9), and overexpression of the same enzyme in Drosophila extends the lifespan of that organism greatly (10). Cultured brain nerve cells pretreated with antioxidants such as vitamin E, propyl gallate, nordihydroguaiaretic acid, and n-tertbutylphenylnitrone were protected against degeneration otherwise caused by excitotoxins, metabolic insults, or exposure to Aβ (11-13). Neuroblastoma cell lines resistant to Aβ toxicity were shown to express higher levels of antioxidant enzymes than cell lines vulnerable to Aβ toxicity (14).

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Figure 2. Glutathione protection of neurons against Aβ toxicity and HNE toxicity. Embryonic rat hippocampal cell cultures were pretreated for 2 h with vehicle or 1 mM cell permeant glutathione ethyl ester and then exposed to 0.1% ethanol (control), 50 µM Aβ, or 10 µM HNE, and neuronal survival was determined 20 h later. Values are the mean and SEM (n ) 3 cultures).

Pathological Disturbances in Oxy Radical Metabolism There is now compelling evidence that oxy radicals are generated whenever the brain is injured. This is very clear in ischemic brain injury (stroke) where massive increases of lipid peroxidation and protein oxidation occur within the injury region (15). Increased levels of lipid peroxidation have also been reported to occur in vulnerable regions of AD brain such as the hippocampus (16). Moreover, the identification of advanced glycation end products in both senile plaques and neurofibrillary tangles strongly implicates the presence of oxidative stress associated with those structures (7). Alterations in antioxidant enzyme levels occur in AD brain tissue and may reflect cellular responses to oxidative stress or could conceivably contribute to the neurodegenerative process (17). For example, catalase activity and protein levels are greatly reduced in AD brain tissues, while levels of Cu/Zn-SOD are increased (Figure 3). In addition, there is evidence for increased protein oxidation in regions of AD brain in which neurodegeneration occurs (18). It was recently shown that advanced glycation end products are associated with both amyloid plaques and neurofibrillary tangles in AD (19). Glycation is a process in which sugars conjugate to proteins; this process is promoted by, and propogates, free radical production. Within the context of this review article, there is not space to provide a detailed consideration of the ways in which oxidative stress is induced in various neurodegenerative conditions, and we will therefore only briefly consider some of the most common mechanisms before moving on to a more in-depth consideration of mechanisms believed operative in AD. An important instigator of free radical production is metabolic impairment induced by conditions such as ischemia or mitochondrial abnormalities (20). In neurons a particularly important mechanism leading to free radical formation is activation of glutamate receptors. Studies have shown that glutamate, acting mainly at the NMDA receptor, can induce accumulation of O2•- (21), H2O2 (22), and per-

Forum on Free Radical Mechanisms of Neurotoxicity

Figure 3. Catalase levels decreased and Mn-SOD levels increased in AD brain. Protein from hippocampus (Hippo) and cerebellum (Cblm) from Alzheimer’s disease patients (n ) 6) and age-matched neurologically normal controls (n ) 5) was separated by SDS-PAGE, transferred to nitrocellulose sheets, and immunoblotted with antibodies to catalase or manganeseSOD (Mn-SOD). Densitometry was used to quantify relative levels of catalase and Mn-SOD. Bars represent the mean and SEM. The decreases in catalase levels in hippocampus and cerebellum in AD brain were significant (p < 0.01), as was the increase in Mn-SOD level in hippocampus.

oxynitrite (23). As described above, activation of glutamate receptors appears to be a key factor in rendering neurons vulnerable in a variety of neurodegenerative disorders. Exposure of neurons to Fe2+ induces massive production of OH• and lipid peroxidation; increased levels of iron have been shown to occur in the neurofibrillary tangles of AD (24) and in neurons degenerating in Parkinson’s/ALS/dementia complex of Guam (25). It is believed that Aβ can induce generation of free radicals (11, 12, 26), perhaps by forming free radical moieties on the peptide itself (27). However, it should be noted that Aβ is only one of many possible sources of oxidative challenge in AD (see refs 6 and 7 for review).

Neuronal Ion Homeostasis Ion-motive ATPases (Na+/K+- and Ca2+-ATPases) expel Na+ and Ca2+ and keep levels of these two ions inside the cell low, typically within the range of 50-200 nM. Na+ and Ca2+ concentrations outside cells are typically 130-150 and 1-2 mM, respectively. These ion gradients have been adapted for use in responses of cells to a variety of environmental stimuli. In neurons, the cellular focus of this article, the ion gradients provide the driving force for fundamental intra- and intercellular communications in neural circuits. Thus, Na+ influx mediates propagation of action potentials, and Ca2+ influx mediates neurotransmitter release, as well as longlasting changes in synaptic plasticity (learning and memory) and gene expression (28). Voltage- and ligandactivated proteinaceous ion channels that span the plasma membrane phospholipid bilayer provide conduits for the rapid flux of Na+ and Ca2+ into cells. Repolarization of the membrane potential following (Na+-mediated) depolarization is rapidly restored by efflux of K+ (normally at high levels inside the cell) through one or more types of specific channels.

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Among cellular systems regulating ion homeostasis, those that control the level of intracellular free Ca2+ ([Ca2+]i) in neurons are particularly complex (Figure 4). Within the plasma membrane are a variety of voltageand ligand-activated Ca2+ channels (29). There are several types of voltage-dependent Ca2+ channels in the central nervous system that differ in their pharmacology, physiological functions, and distribution. In addition to flux through plasma membrane channels, Ca2+ can be released into the cytoplasm from intracellular stores. Ca2+ channels within the endoplasmic reticulum (ER) provide an important route for elevation of [Ca2+]i in response to ligands that activate cell surface receptors linked to certain GTP-binding proteins (30). For example, activation of muscarinic cholinergic receptors by acetylcholine leads to stimulation of a GTP-binding protein called Gq11 which, in turn, activates phospholipase C resulting in hydrolysis of phosphatidylinositol bisphosphate and release of inositol triphosphate (IP3). IP3 then diffuses to the ER where it activates Ca2+ channels resulting in elevation of [Ca2+]i. Once [Ca2+]i is elevated, cells remove that free Ca2+ by several mechanisms. The plasma membrane Ca2+-ATPase is the main mechanism for maintenance of rest [Ca2+]i (31), while the Na+/Ca2+ exchanger in the plasma membrane has a very high capacity for Ca2+ removal and is thought to play a major role in rapidly restoring the transmembrane calcium gradient following a stimulus. Ca2+ is also pumped into ER via a specific ER Ca2+-ATPase, and Ca2+ is also (more slowly) taken up by mitochondria, an organelle that typically contains millimolar levels of Ca2+. A final example of mechanisms for reducing [Ca2+]i in neurons are Ca2+-binding proteins such as calbindin and parvalbumin. These Ca2+-binding proteins contain typical EFhand motifs that serve as Ca2+-trapping pockets. Ca2+binding proteins may be present in neurons at concentrations exceeding 10 mM and are thought to act as Ca2+ “sponges”, although there is evidence that they may also function as effector proteins by analogy with calmodulin, the well-known mediator of Ca2+-responsive signaling pathways. The reason that there are so many control mechanisms for Ca2+ in neurons is almost surely that this ion plays vital roles in many different physiological processes; we will present just a few examples. One neuron in a circuit rapidly communicates information to the next neuron at a structural specialization called the chemical synapse. At the synapse, membrane depolarization leads to release of a neurotransmitter which then diffuses across the small extracellular synaptic cleft where it binds to specific receptors on the postsynaptic cell. Ca2+ influx is the trigger for neurotransmitter release and is therefore essential for synaptic transmission. Ca2+ also plays a major role in responses of the postsynaptic cell to neurotransmitters. For example, a process called longterm potentiation, in which synaptic transmission is “strengthened”, is mediated by Ca2+ influx into the postsynaptic cell (28). Another example of a fundamental process in neurons regulated by Ca2+ comes from studies of brain development. In order to form neural circuits, neurons must grow axons and dendrites and these neurites must “find” their synaptic partners and then form stable synapses. Ca2+ plays major roles in regulation of both neurite outgrowth and synaptogenesis (32).


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Figure 4. Neuronal signal transduction pathways involved in regulation of ion homeostasis and mechanisms that may disrupt ion homeostatic systems in Alzheimer’s disease. The extracellular calcium concentration is normally 10000-fold greater than [Ca2+]i. This gradient is maintained, and restored following excitatory stimuli, by the plasma membrane Ca2+-ATPase and Na+/Ca2+ exchanger. The Na+/K+-ATPase also plays an important role in Ca2+ homeostasis by maintaining and restoring membrane potential. Ca2+ is also removed from the cytoplasm by sequestration in endoplasmic reticulum (ER) and mitochondria and by binding to calciumbinding proteins. Elevation of [Ca2+]i is induced by excitatory transmitters such as glutamate, by reduced energy (glucose and oxygen) availability, and by amyloid β-peptide (Aβ). Glutamate induces Ca2+ influx by activating NMDA receptors, which themselves conduct calcium, and by activating AMPA and kainate receptors which depolarize the membrane resulting in opening of voltage-dependent calcium channels (VDCC). In Alzheimer’s disease reduced glucose availability may lead to ATP depletion which impairs function of ion-motive ATPases and may thereby increase vulnerability to excitotoxicity. Aβ arises from βAPP via enzymatic processing involving β-secretase (β). Aβ forms insoluble aggregates, and chemical reactions involving oxy radicals that are involved in the aggregation process also induce membrane oxidation resulting in lipid peroxidation and impairment of Na+/K+- and Ca2+-ATPase activities; this results in membrane depolarization, calcium influx, and increased sensitivity to excitotoxicity. Calcium influx induces several reactive oxygen species (ROS) including superoxide anion radical, hydrogen peroxide, and nitric oxide. Calcium and ROS both contribute to damage to proteins, lipids, and DNA and ultimately cell death. Subtoxic levels of membrane oxidation, as induced by Aβ, can impair coupling of receptors to GTP-binding proteins (g), an example being disruption of coupling of muscarinic acetylcholine receptors (Ach R) to Gq11. Neurotrophic factors (NTF) activate receptor tyrosine kinases (tk R) resulting in a cascade of phosphorylation reactions involving intermediate kinases (IKs) and mitogen-activated protein kinases (MAPK) and transcription factors. Tumor necrosis factor (TNF) binds to a receptor (R), the activation of which results in hydrolysis of sphingomyelin, release of ceramide, and activation of the transcription factor NFκB. The expression of genes involved in regulating [Ca2+]i and antioxidant enzymes can be induced by NTFs and TNF. Secreted forms of βAPP (sAPP) are released from βAPP as the result of neuronal activity and bind to a putative receptor linked to elevation of cyclic GMP levels. cGMP activates a cGMP-dependent protein kinase (PKG) which may then activate a protein phosphatase resulting in dephosphorylation and activation of K+ channels. In this way sAPPs hyperpolarize the membrane and counteract the depolarizing actions of glutamate, energy deprivation, and Aβ.

Pathological Disruptions of Ion Homeostasis [Ca2+]i

Whereas transient elevations of play vital roles in the normal development and function of the nervous system, sustained increases of [Ca2+]i can result in cell injury and death (28, 32). While neurodegenerative actions of Ca2+ can easily be shown in experimental settings (e.g., Ca2+ ionophores kill cells and removal of extracellular Ca2+ prevents their death), links between dysregulation of Ca2+ homeostasis and specific neurodegenerative disorders have only recently been demonstrated. Although the present article focuses mainly on AD, it is conceptually important to consider other neurodegenerative conditions in which Ca2+ may play pathological roles. A now classic example is stroke, a condition in which the blood supply to brain cells is disrupted. The resulting lack of glucose and oxygen results in ATP depletion, impairment of ion-motive ATPases, and elevation of [Ca2+]i. One of the most important contributors to [Ca2+]i in neurons exposed to ischemic conditions is activation of receptors for the excitatory neurotransmitter

glutamate (33). A particular subtype of glutamate receptor called the N-methyl-D-aspartate (NMDA) receptor fluxes massive amounts of Ca2+; this receptor is activated when the membrane depolarizes as occurs secondary to Na+/K+-ATPase failure. Drugs that block glutamate receptors or buffer Ca2+ can reduce neuronal damage in animal models of stroke. Another neurodegenerative condition in which Ca2+ almost surely plays a role is severe temporal lobe epilepsy. In this condition, neurons are excessively excited resulting in sustained Ca2+ influx which is evident from cytoskeletal alterations clearly linked to activation of calcium-dependent proteases (34). Again, drugs that reduce membrane excitability and Ca2+ influx can protect neurons against seizure-induced injury. Sustained elevation of [Ca2+]i promotes neuronal degeneration in several different ways. Ca2+-dependent proteases such as the calpains degrade cytoskeletal proteins and some regulatory enzymes and can thereby mediate structural and functional damage to cells. Calpains may play important roles in neuronal degenera-


tion in stroke and epilepsy, and calpain inhibitors have been reported to protect brain cells in animal models of these disorders (35). Interestingly, calpains are in the same class of cysteine proteases shown to effect apoptosis (programmed cell death) in certain neuronal cell systems (36). Another way in which Ca2+ promotes neuronal degeneration is by activating endonucleases which cleave DNA. Ca2+ is also known to promote activation of several enzymes linked to neurodegenerative processes including phospholipase A2 and protein kinase C (37). Mitochondrial function can be severely compromised when cytoplasmic [Ca2+]i is excessively elevated, and this action of Ca2+ leads to ATP depletion and the cascade of untoward events that result therefrom (38). The evidence for disruption of neuronal ion homeostasis as an important event in the neurodegenerative process is less clear, but fairly convincing. Two main histopathological lesions are observed upon inspection of brain tissue from an AD patient. One lesion is the neurofibrillary tangle which consists of abnormal accumulations of cytoskeletal proteins that form filaments, often arranged in a paired-helical conformation (39). The major component of the abnormal filaments is the microtubule-associated protein tau, and indeed, many antibodies that bind the filaments recognize epitopes in tau. Experimental studies in cell culture and in adult rodents have shown that conditions that induce oxidative stress and elevate [Ca2+]i can induce changes in tau and other cytoskeletal components similar to those seen in the neurofibrillary tangles of AD (40-42). The conditions include exposure to excess levels of glutamate, energy deprivation, and exposure to amyloid β-peptide (Aβ). In AD, only certain populations of neurons in particular brain regions degenerate. For example, pyramidal neurons in the hippocampus (a brain region that functions in learning and memory processes) degenerate, whereas neurons in the motor cortex and cerebellum do not. Studies of the distribution of Ca2+-regulating proteins in the different neuronal populations reveal interesting relationships between a cell’s repertoire of Ca2+-regualting mechanisms and its vulnerability to degeneration in AD. For example, within the population of hippocampal neurons in culture, those containing the Ca2+-binding protein calbindin are relatively resistant to excitotoxicity (43). In general, neurons that degenerate also express very high levels of glutamate receptors, particularly the NMDA subtype. A link between disruption of ion homeostasis and neuronal degeneration and AD comes from data showing that Aβ, believed to be a central instigator of neuronal injury and death in AD, disrupts neuronal ion homeostasis and increases neuronal vulnerability to excitotoxicity (4, 44, 45). The remainder of this article considers the hypothesis that disruption of ion homeostasis in AD is initiated and propogated by oxy radicals which attack key ion-regulating systems in neural cells.

Inter-Relationships of Oxy Radicals and Ion Homeostasis Oxidative stress can disrupt ion homeostasis, and conversely, increases in [Ca2+]i promote free radical production. As studies of the specific mechanisms of interactions of free radicals with ion-regulating systems and of Ca2+ with free radical-generating systems proceed, it is becoming clear that such interactions are very complex. Conditions that induce lipid peroxidation impair functions of several proteins that are vital for


Figure 5. Effects of Aβ on function of ion-motive ATPases and Na+/Ca2+ exchanger in human brain synaptosomes. Synaptosomes prepared from postmortem hippocampus of neurologically normal adult humans (n ) 4) were exposed for 1 h to vehicle (control), 50 µM Aβ, or 100 µM FeSO4. Activity levels of plasma membrane Na+/K+-ATPase, Ca2+-ATPase, and Na+/Ca2+ exchanger were then quantified. Aβ and FeSO4 significantly reduced Na+/K+- and Ca2+-ATPase activities without affecting Na+/Ca2+ exchange activity.

maintenance of neuronal ion homeostasis. For example, oxidative insults have been shown to impair Na+/K+- and Ca2+-ATPase activities in a variety of cell types including neurons (14, 46). The way in which oxy radicals impair the ion-motive ATPases is not fully established, but evidence supports several mechanisms including alteration of the lipid microenvironment of the proteins, direct oxidative damage to the proteins, and modification of the proteins by aldehydic products of lipid peroxidation such as 4-hydroxy-2-nonenal (HNE) (47). Impairment of ionmotive ATPases in neurons results in membrane depolarization and Ca2+ influx through NMDA receptors and VDCC. Other ion-regulating proteins appear less sensitive to damage by oxy radicals; the plasma membrane Na+/Ca2+ exchanger is relatively resistant to impairment by oxidative insults (Figure 5) (14). Oxy radicals can also damage mitochondria and disrupt the ability of that organelle to sequester Ca2+ (48). Interestingly, some effects of oxy radicals in neurons may protect against elevation of [Ca2+]i. For example, it has been shown that function of the NMDA receptor is sensitive to redox modulation such that oxidation inhibits channel opening (49). Elevation of [Ca2+]i promotes oxy radical production in many different ways (7, 22). For example, Ca2+-calmodulin activates nitric oxide synthase resulting in NO• production. NO• can then interact with O2•- to form peroxynitrite. Peroxynitrite induces irreversible nitration of proteins on tyrosine residues, an event that may impair the function of the many different proteins regulated by tyrosine phosphorylation. Ca2+ promotes activation of phospholipase A2 resulting in release of arachidonic acid; oxy radicals are generated by the activity of oxygenases that metabolize arachidonic acid. By disrupting mitochondrial transmembrane potential, Ca2+ promotes generation of O2•-.


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Figure 6. Proteolytic processing pathways of βAPP. Following synthesis in the rough endoplasmic reticulum (RER) and passage through the Golgi complex, βAPP is inserted into membranes of vesicles. βAPP may be proteolytically processed by R- and β-secretases in vesicles prior to their fusion with the plasma membrane or may be incorporated into the plasma membrane. Cleavage by R-secretase results in the release of sAPPR into the extracellular milieu (pathway 1); this pathway is activated by electrical activity (EA), excitatory neurotransmitters (ET), and protein kinase C (PKC). Because the R-secretase cleaves in the middle of Aβ, it precludes release of intact (potentially amyloidogenic) Aβ. Cleavage by β-secretase releases sAPPβ and leaves behind a C-terminal membrane-spanning fragment that contains intact Aβ (pathway 2). The C-terminal product of β-secretase cleavage may be endocytosed and further proteolyzed in a subcellular compartment(s) resulting in the release of intact Aβ from cells, which then has the potential to form amyloid fibrils (pathway 3).

“Amyloid Hypothesis” A histological hallmark of AD is the presence of amyloid deposits in many different brain regions (39). The amyloid deposits are particularly concentrated in vulnerable brain regions in which neurodegeneration occurs. The major protein component of the amyloid deposits is Aβ, a 40-42 amino acid peptide that arises from a much larger membrane-spanning β-amyloid precursor protein (βAPP) which consists of a large extracellular N-terminus, a single membrane-spanning domain, and a relatively short cytoplasmic C-terminus (Figure 6). The Aβ sequence is situated partially in the extracellular space and partially within the membrane bilayer. βAPP can be enzymatically processed in several different ways. A cleavage within the Aβ sequence by an enzyme called R-secretase (between amino acids 16 and 17) releases a large N-terminal portion of βAPP; this secreted form of βAPP (sAPPR) may play important intercellular signaling roles in the nervous system (50, 51). The R-secretase cleavage prevents release of intact, potentially amyloidogenic Aβ. Another enzyme (β-secretase) cleaves βAPP at the N-terminus of Aβ and thereby generates a C-terminal βAPP fragment containing intact Aβ; further processing of this C-terminal fragment by γ-secretase results in release of intact Aβ from cells. Both Aβ and sAPPR are produced, in varying amounts, during normal metabolism of neural cells (52). Aβ is normally soluble, but for reasons now being elucidated, it forms insoluble fibrillar aggregates in neural tissue and cerebral blood vessels in AD. In brain regions in which neurons degenerate and die in AD, such as those involved in learning and memory (e.g., hippocampus), deposits of

Aβ are often surrounded by degenerated neurons suggesting a causal role for Aβ in the neurodegenerative process. However, because studies of postmortem tissue cannot reveal the dynamics of the events occurring, it had been unclear until recently whether Aβ deposits were the byproduct of neuronal degeneration or preceded the degenerative process. The data described below link Aβ deposition with neuronal degeneration and suggest that oxy radicals play a key role in this degenerative process. It should be noted that the amyloid hypothesis is but one of several hypotheses that have been forwarded to explain the mechanism of neuron degeneration in AD. Other hypotheses include the tau hypothesis, the calcium hypothesis, the excitotoxicity hypothesis, the metabolic hypothesis, and the vascular hypothesis (see ref 53 for review). Most likely, these different hypotheses are not mutually exclusive, and in fact, there is considerable evidence that they are mechanistically inter-related (53).

Genetic Links AD can be considered a syndrome of diseases with autosomal dominant forms that harbor a variety of mutations including those in βAPP and late-onset familial and sporadic forms associated with apolipoprotein E genotype but not with βAPP mutations. Molecular genetic analyses have clearly shown that mutations in βAPP can cause AD. Several mutations have been identified that cause AD in the families which bear them. Interestingly, the mutations are located immediately adjacent to or within the Aβ sequence (Figure 6). Experimental data in which mutated forms of βAPP were expressed in cultured cells showed that at least some of



the mutations increase production of Aβ. For example, one mutation results in decreased R-secretase cleavage and increased β-secretase cleavage (54). In addition to promoting increased production of potentially toxic Aβ, βAPP mutations may reduce levels of sAPPR, a product which has been shown to promote neuronal survival and plasticity (4, 50, 51). Another genetic link between βAPP and AD can be appreciated when one considers the fact that essentially all persons with Down’s sydrome develop the histopathological features of AD including massive Aβ deposition and neurofibrillary tangles (55); the βAPP gene is located on chromosome 21, the one for which there is an extra copy in Down’s syndrome. Recently, it was reported that cortical neurons from Down’s syndrome fetuses undergo spontaneous apoptosis in culture, apparently the result of increased oxidative stress (56). Experimental genetic studies also support a causal role for Aβ in the pathogenesis of AD. Expression of a mutated human βAPP gene in transgenic mice resulted in Aβ deposition in some of the same brain regions vulnerable in AD and evidence for degeneration of neurons associated with the Aβ deposits (57).

Mechanisms of Amyloid Cytotoxicity With the cloning of the βAPP gene came the ability to synthesize large amounts of Aβ for use in experimental studies aimed at determining if and how Aβ damages and kills neurons. Exposure of cultured brain cells to Aβ at concentrations in the 1-50 µM range can induce neuronal degeneration (58, 59) and greatly increase neuronal vulnerability to excitotoxic (44, 45), metabolic (60), and oxidative (11) insults. Behl et al. (11) showed that Aβ can induce lipid peroxidation in cultured neurons, and Butterfield et al. (26) used EPR analyses with nitroxyl stearate (NS) spin labels to show that Aβ induces lipid peroxidation in synaptosomes. Exposure of cultured hippocampal neurons to Aβ induced malondialdehyde accumulation (61) and conjugation of HNE to cellular proteins (62) (Figure 7), indicating induction of lipid peroxidation by Aβ. Levels of cellular H2O2 were greatly increased in cultured neurons following exposure to Aβ (11). Oxy radical production appears to play a major role in the neurotoxicity of Aβ because pretreatment of cells with antioxidants such as vitamin E and propyl gallate protects them against Aβ toxicity (11, 13). Interestingly, glutathione appears to serve as a key “detoxifier” of HNE (63), and we have found that glutathione is very effective in protecting cultured hippocampal neurons against Aβ toxicity (Figure 2) (62). On the basis of the available data, we propose that the following sequence of events may constitute the mechanism of Aβ toxicity (Figure 8). Aβ induces membrane lipid peroxidation which results in release of HNE from unsaturated fatty acids. HNE conjugates to lysine, histidine, and cysteine residues of plasma membrane proteins including Na+/K+- and Ca2+-ATPases. Impairment of ion-motive ATPases results in membrane depolarization, elevation of [Ca2+]i, and increased vulnerability of neurons to excitotoxicity. A cascade is thereby propogated in which oxy radicals promote increased [Ca2+]i and increased [Ca2+]i promotes oxy radical production. Both the oxy radicals and the Ca2+ induce irreversible damage to proteins, nucleic acids, and membrane lipids. Whereas high levels of Aβ can kill neurons, lower levels may induce low levels of oxidation at the plasma membrane that impair the function of important

Figure 7. Aβ induction of HNE production and conjugation to proteins in neurons. (A) HPLC traces of derivatized extracts from a control hippocampal culture (upper chromatogram) and a culture that had been exposed for 2 h to 50 µM Aβ (lower chromatogram). The HNE and hexanal (Hex) peaks are labeled. Levels of HNE were increased in neurons exposed to Aβ. (B) Cultures were exposed for 3 h to vehicle (CON), 50 µM FeSO4 (FE), 10 µM HNE, or 50 µM Aβ. Total cell protein (50 µg) from each culture was separated by SDS-PAGE gel, transferred to a nitrocellulose sheet, and immunoreacted with an antibody that recognizes HNE-conjugated proteins. Note that FeSO4, HNE, and Aβ each caused a massive increase in protein-bound HNE in the neurons. Numbers at the left indicate molecular weight in kDa.

signal transduction pathways. For example, exposure of cultured rat neocortical neurons to subtoxic levels of Aβ impaired muscarinic receptor-G protein coupling (64). In the latter study, agonist-induced GTPase activity, inositol phosphate production, and Ca2+ release from ER were all impaired in cells pretreated with Aβ. Aβ did not affect ligand binding to muscarinic receptors indicating that the site of action of Aβ was at the level of receptor-G protein interaction. Antioxidants abrogated the disruptive effect of Aβ on muscarinic cholinergic signal transduction demonstrating that oxy radicals were mechanistically involved in this action of Aβ (64). Additional membrane systems disrupted by Aβ include glutamate transporters, which serve the important function of removing glutamate from the extracellular space, and glucose transporters, which are necessary for cellular uptake of glucose, the key substrate for ATP production (R. J. Mark, E. M. Blanc, and M. P. Mattson, manuscript in preparation).

Neuroprotective Signal Transduction The fact that brain cells must exist in an oxidizing environment demonstrates that mechanisms must exist


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Figure 8. Mechanism of Aβ toxicity. In the presence of an oxidizing environment, Aβ forms free radical peptides which promote cross-linking into amyloid fibrils. When this process occurs in proximity to neuronal cell membranes, oxidation of membrane polyunsaturated fatty acids (PUFAs) occurs resulting in further production of oxy radicals and release of aldehydes including 4-hydroxynonenal (HNE). HNE (and probably oxy radicals) damages ion-motive ATPases resulting in dysregulation of ion homeostasis, membrane depolarization, and Ca2+ influx through glutamate receptor channels and voltage-dependent Ca2+ channels. Ca2+ further potentiates oxy radical production, and both Ca2+ and oxy radicals contribute to cell injury and death.

to suppress oxy radical production and accumulation. Described above were some of the enzymatic and nonenzymatic antioxidant mechanisms. In the present section we consider novel, and highly dynamic, neuroprotective signaling pathways that are activated in response to oxidative stressors (and other stressors as well). These pathways include both intercellular signaling molecules, such as neurotrophic factors and cytokines, and intracellular signal transduction pathways involving kinases and transcription factors. In recent years it has been shown that a remarkably broad array of growth factors can protect neurons against oxidative (e.g., exposure to Fe2+, H2O2, or Aβ), metabolic (glucose deprivation or mitochondrial toxins), and excitotoxic insults (see refs 2 and 5 for review). The list of growth factors includes basic fibroblast growth factor (bFGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and insulin-like growth factor (IGF). Brain injuries (e.g., ischemia, epileptic seizures, and trauma) induce increased expression of each of these growth factors. The signal transduction pathways that mediate neuroprotective actions of growth factors are beginning to be understood (Figure 4). Plasma membrane receptors for these neurotrophic factors possess intrinsic tryosine kinase activity; ligand binding induces receptor dimerization and autophosphorylation. Specific cytoplasmic proteins then associate with the receptors, are phosphorylated, and then activate additional kinases which ultimately leads to activation of a transcription factor. Genes induced by neurotrophic factors which likely play roles in their neuroprotective actions include those encoding antioxidant enzymes (22) and calcium-binding proteins (65). Pretreatment of neuronal cultures with bFGF resulted in reduced [Ca2+]i responses to glutamate (66) and attenuated the elevation of [Ca2+]i otherwise induced by Aβ (45). Most of the growth factors mentioned above have also been shown to stabilize [Ca2+]i in neurons

exposed to glucose deprivation, glutamate, or mitochondrial toxins (38). In addition to inducing calcium-binding protein expression, neurotrophic factors may reduce expression of glutamate receptor proteins such as particular subunits of the NMDA receptor (66). Pretreatment of neuronal cultures with bFGF, NGF, and BDNF resulted in a significant attenuation of H2O2 accumulation following exposure to glutamate demonstrating an antioxidant action of these factors. Activity levels of Cu/ Zn-SOD and GSH reductase were increased in hippocampal cell cultures treated with bFGF, while levels of catalase were greatly increased in similar cultures exposed to NGF (22). Thus, two general ways in which growth factors protect neurons are by stabilizing ion homeostasis and suppressing accumulation of oxy radicals. Cytokines constitute another class of endogenous, injury-induced, neuroprotective factors. Exemplary is tumor necrosis factor-R (TNF). We found that the vulnerability of cultured hippocampal neurons to glutamate toxicity and glucose deprivation is reduced in cultures pretreated with TNF (64). TNF also protected hippocampal neurons against Aβ toxicity and other oxidative insults; increases of H2O2, lipid peroxidation products, and Ca2+ induced by Aβ were suppressed in neurons pretreated with TNF (67). The TNF receptor that may transduce the neuroprotective signal is linked to a signal transduction pathway in which membraneassociated sphingomyelin is hydrolyzed resulting in the release of ceramide, which then induces activation of the transcription factor NFκB (Figure 4). TNF induces κB DNA binding activity in cultured hippocampal neurons, and ceramide and other manipulations that activate NFκB mimic the neuroprotective actions of TNF (60, 67). TNFs and ceramide induced expression of calbindin and Mn-SOD in hippocampal neurons (64, 68). Collectively, these data suggest that TNF induces both calcium-


stabilizing and antioxidant responses in neurons via an NFκB-mediated mechanism. Another neuroprotective signal transduction pathway is activated by sAPPR (Figure 4). The putative pathway involves activation of a sAPPR receptor linked to elevation of cGMP levels (69). cGMP then activates cGMPdependent protein kinase which, in turn, activates a protein phosphatase. The protein phosphatase then causes dephosphorylation of high-conductance K+ channels resulting in membrane hyperpolarization (51). This pathway may account for the ability of sAPPR to protect neurons against metabolic and excitotoxic insults (4, 70). These kinds of data strengthen the case for altered enzymatic processing of APP being a key event in the pathogenesis of AD (see above).

Pharmacological Means of Suppressing Neurodegenerative Cascades Involving Oxy Radical and Perturbed Ion Homeostasis In this last section, we focus on the different links in the neurodegenerative cascade in AD from the perspective of interrupting the cascade with clinically-relevant agents. One class of such agents, currently in development, is compounds that affect βAPP processing so as to reduce β-secretase cleavage and increase R-secretase cleavage. Agents that activate protein kinase C can induce R-secretase cleavage and thereby reduce release of intact Aβ from cells (71, 72). Agents that prevent or reverse aggregation of Aβ are certainly of potential utility in treating AD. Examples include Congo red which prevents Aβ fibril formation and protects cultured neurons against Aβ toxicity (73) and small-molecule anionic sulfonates or sulfates (74). Because oxidative stress may contribute to Aβ aggregation in the aging brain and AD (75), and antioxidants have been shown to protect neurons against Aβ toxicity (11, 14), antioxidants could interrupt the amyloid cascade at the initial step of peptide aggregation. Agents that protect neurons against Aβ neurotoxicity are many and varied in action, although almost all act by either suppressing free radical accumulation or stabilizing ion homeostasis. Antioxidants effective in experimental paradigms of Aβ toxicity include vitamin E, the spin-trapping compound PBN, and nordihydroguaiaretic acid (11-13). Neurotrophic factors exhibit antioxidant properties indirectly by inducing expression of antioxidant enzymes in brain cells (see above). Interestingly, estrogens exhibit intrinsic antioxidant activity and can protect neurons against Aβ toxicity and other oxidative insults (76, 77). Clinical trials of several antioxidants in AD patients are in progress. Blockers of voltage-dependent sodium (78) and calcium (79) channels protected cultured neurons against Aβ toxicity. Potassium channel activators such as diazoxide, pinacidil, and levocromakalim were very effective in protecting cultured hippocampal neurons against Aβ toxicity (80). Additional calcium-stabilizing agents are being identified that have intriguing mechanisms of action. For example, actin-depolymerizing agents such as cytochalasins protected neurons against excitotoxicity and Aβ toxicity (81). The mechanism of action of cytochalasins involves depolymerization of actin and suppression of calcium influx through NMDA receptors and voltage-dependent calcium channels. The microtubulestabilizing agent taxol was also effective in protecting cultured hippocampal neurons against excitotoxicity (82).


Many laboratories are working to identify compounds that activate neuroprotective signal transduction pathways or increase neurotrophic factor production. Receptor tryosine kinase cascades and cytokine signaling pathways involving NFκB can activate a host of cellular defense mechanisms in neurons. Administration of neurotrophic factors themselves is one approach to activating these pathways in brain cells of AD patients, and indeed, clinical trials of neurotrophic factors are in progress (83). Another approach is to identify small lipophilic compounds that activate neuroprotective signaling pathways. Examples include bacterial alkaloids K252a and staurosporine which exhibit neuroprotective activities related to activation of receptor tyrosine kinase pathways (84). Another example is ceramide which activates NFκB and can protect cultured hippocampal neurons against excitotoxicity and oxidative insults including Aβ toxicity (60). Finally, several compounds that induce neurotrophic factor production in brain have been described (85). Collectively, the emerging data clearly indicate that multiple targets exist for preventing or slowing degeneration of neurons that results from oxidative disruption of ion homeostasis.

Acknowledgment. We thank S. W. Barger, B. Cheng, and V. L. Smith-Swintosky for contributions to original research from this laboratory. Collaborations with D. A. Butterfield, J. Carney, S. Christakos, R. E. Rydel, and R. M. Sapolsky are greatly appreciated. Research was supported by grants to M.P.M. from the NIH (NINDS and NIA), the Alzheimer’s Association, and the Metropolitan Life Foundation. R.J.M. was supported by a Neurobiology of Aging Training Grant from the NIA.

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