Are reactive oxygen species involved in Alzheimer's disease?

Neurobiology of Aging, Vol. 16, No. 4, pp. 661-674, 1995 Copyright © 1995 Elsevier Science Ltd. Printed in the USA. All rights reserved 0197-4580/95 $9.50 + .00

Pergamon 0197-4580(95)00066-6

O P E N PEER C O M M E N T A R Y

Are Reactive Oxygen Species Involved in Alzheimer's Disease? GIANNI

BENZI AND ANTONIO

MORETTI

Institute of Pharmacology, Faculty of Science, University of Pavia, Piazza Botta 11, 27100 Pavia, Italy R e c e i v e d 20 D e c e m b e r 1993; R e v i s e d 18 A u g u s t 1994; A c c e p t e d 22 F e b r u a r y 1995 BENZI, G. AND A. IVIORETTI. Are reactiveoxygenspeciesinvolvedin Alzheimer'sdisease?NEUROBIOL AGING 16(4) 661674, 1995.--Alzheimer's disease has a multifactorial pathogenesis. Among the various factors involved, this review examines, in particular, the possibility of oxidative stress, meaning an imbalance between the formation and spread of reactive oxygen species (ROS) and the antioxidant defenses. This theory is supported by the following observations: (a) the alteration of mitochondrial function, which is likely to lead to the electron leakage in the respiratory chain and the consequent formation of superoxide radicals; (b) the unbalanced high activity of superoxide dismutase and monoamine oxidase B which causes the production of more H202; (c) the. alteration of iron homeostasis which, in combination with the superoxide and H202, gives rise to the most deleterious hydroxyl radicals; (d) the increased lipid peroxidation and membrane alterations; (e) the pro-aggregating effect of ROS on ~/A4 protein and the C-terminal fragment of amyloid precursor (A4CT). Most of these changes are already present in the normal aging brain but are aggravated in AD presumably over a number of years. However, further investigations are needed to confirm these theories particularly regarding the alterations of another target of ROS, the proteins. Peroxidative stress is presumably present in the AD brain. This stress might not be a primary factor in the pathogenesis of AD, but a consequence of the tissue injury. In any case, it could contribute considerably to the pathology, in a vicious cycle of actions and reactions resulting in a critical mass of metabolic errors, responsible in the end for this disease. Alzheimer's disease

Reactive oxygen species

IT is a m o o t question whether the formation of free radicals and the resulting toxicity might be responsible for the brain degeneration in Alzheimef's disease (AD). This hypothesis is based: (a) on the observation that some of the processes which lead to free radical formation, e.g., brain trauma, aging, are risk factors for AD, and (b) on the similarity between A D and Down's syndrome where free radical formation is known to be increased (188). This review focusses on the role of some factors related to free radicals (hereafter: reactive oxygen species, ROS) in the pathogenesis of AD, as they result from the recent literature. The reader is referred to some recent reviews on ROS and their meaning for the CNS (15,26,89). We begin by examining various points related to brain ROS formation in aging and AD, particularly the roles o f the electron transfer chain, superoxide dismutase, the monoamine oxidases and iron. We then consider the evidence for oxidative stress (i.e., the imbalance between ROS generation and antioxidant

defenses) in the A D brain, focusing on lipid peroxidation, protein oxidation, and D N A damage. THE ELECTRON TRANSFER CHAIN AS A SITE OF SUPEROXIDE RADICAL FORMATION The mitochondrial transfer of electrons from the donors produced in the tricarboxylic acid cycle ( N A D H and succinate) to molecular oxygen causes the release o f considerable amounts o f energy for A T P synthesis, ion translocation, protein importation, and so on. Electron transfer arises with the vectorial translocation of protons, and a series o f molecular complexes (consisting of various equipotential subunits located in the mitochondrial inner membrane) ensure both the transduction of oxidative energy to proton-motive force and the use of proton energy in ATP synthesis (Scheme 1). The complexes that are functionally connected to mitochondrial energy transduction include: Complex I = N A D H :ubiq-

IRequests for reprints should be addressed to Gianmartino Benzi, M.D., Ph.D., Istituto di Farmacologia, Facolt~ di Scienze, Universit~ di Pavia, Piazza Botta 11, 27100 Pavia, Italy. 661

662

BENZI AND MORETTI SCHEME 1 MITOCHONDRIALENERGY TRANSDUCINGSYSTEM

The mitochondrial energy transduction system works in the presence of the following mobile or constituent molecules: Extra-respiratory chain mobile molecules: N A D H and succinate; Electron transfer chain or respiratory chain: Complexes I, II, III, and IV;

Intra-respiratory chain mobile molecules: ubiquinone population and cytochrome c;

Phosphorylating system: Complex V; Mobile molecule outside the chain correlated with the surroundings: oxygen.

uinone oxido-reductase; Complex II -- succinate:ubiquinone oxido-reductase; Complex I I I = ubiquinol:ferricytochrome c oxido-reductase; Complex IV = ferrocytochrome c:oxygen oxidoreductase; Complex V = ATP-synthase. These complexes are made up of more than sixty polypeptides, but the mitochondrial synthesis controlled by the mitochondrial DNA is only known for a few of them. In fact, only 13 out of 60 polypeptides are known to be encoded by the mammalian mitochondrial DNA and synthesized within the mitochondria. In the inner mitochondrial membrane, within Complexes I, III, and IV the energy transduced from electron transfer is conserved by coupled vectorial proton translocation which generates a membrane electrochemical potential of protons [A#H +] used in ATP synthesis. The whole electron transfer system is reversible and an electron flow can be generated against the current. However, the final stage of electron transfer (cytochrome aa 3 in Complex IV - , oxygen) is irreversible so the equilibrium in the system is shifted toward ATP synthesis. Cytochrome aa3 in Complex IV retains all the partially reduced oxygen intermediates bound to its active sites until the O2 itself is completely reduced to water (oxygen with four electrons). However, through auto-oxidation affecting their reduced forms, other elements in the mitochondrial electron transfer chain (ubiquinones and cytochrome b family) may transfer the electrons directly to oxygen, but do not retain the partially reduced oxygen intermediates in their active sites until the O2 is completely reduced to water. Because oxygen accepts only one electron at a time, the superoxide radical (oxygen with one electron) is released. In the cytochrome b family, it should be stressed that cytochrome b566 is closely involved with the processes of energy transduction in Complex III, wavering continually between a very low potential state ( ~ = - 3 0 mV) and a very high one ( ~ = 245 mV). Cytochrome b566's low potential may play a prime role in the formation of mitochondrial superoxide radicals because: (a) an increase in its redox potential inhibits the univalent transfer of electrons to oxygen and (b) the intervention of the molecular species with a potential of - 3 0 mV characterizes the release of superoxide radicals. Therefore, electron leakage from the cytochrome b566 would appear to be a real possibility, already accompanying the electron flow of perfectly coupled young mitochondria which can thus bring about a continuous release of oxygen free radicals. During aging, the increasing amounts of these radicals that manage to escape the local defense mechanisms (e.g., scavengers, electron-trapping agents, etc.) may lead to multiple changes in the chemical and physical state of the membranes. As a matter of fact, superoxide generation (161,172) is significantly greater in the brain mitochondria of aged rats rather than young rats.

This may be related to the fact that, with the exception of cytochrome aa3 in Complex IV, the content of the electron carriers undergoes no great change with aging. However, the decrease in either the content of cytochrome aa3 (cyt aa3) or its catalytic cytochrome oxidase activity (COX) (1,14,15,20,80) in synaptic mitochondria from some cerebral regions (frontal cortex, parieto-temporal cortex, hippocampus, cerebellum, etc.) (53,54) may account for the finding that stoichiometric calculations show aging is related to an increase in the percentage of ubiquinones and cytochrome b family. Although this increase is not dramatic in any absolute sense, in the relative sense, it does explain how electrons can "escape" the electron transfer sequence from electron donors to oxygen. The Km for cyt c is unchanged in old rats but the Vm~x decreases (53). Moreover, the COX activity: (a) drops significantly less in cortical synaptic mitochondria from old rats fed a hypocaloric diet (3), and (b) declines in the homogenate of old rat cerebral cortex (9) and in insect mitochondria (171). Electrons can leak from the energy-transducing sequences even in young animals, which indicates that the formation of superoxide radicals could be associated with the normal process of mitochondrial respiration. The production of these radicals causes cell damage because of the dismutase reaction in which hydrogen peroxide [H 202 -- oxygen with two electrons) is formed and which, through the intervention of low-molecular-weight iron and copper complexes, leads to the highly dangerous hydroxyl radical (oxygen with three electrons). The catalytic activity of Complex IV, namely COX activity, is low in three cortical areas but not in putamen and hippocampus of AD patients compared with age-matched controls (105, 106). There is no correlation between the changes in COX and those of other marker mitochondrial enzymes, i.e., glutamate dehydrogenase and citrate synthase, so possibly the decrease is not related to the loss of mitochondria (105). The decrease in COX activity in cortical areas and in the hippocampus from AD patients (155,166) suggests a primary defect of Complex IV, resulting in more O~- released. A specific decrease in COX in the platelets of AD patients has also been reported (144) but not confirmed (185). COX is heterogeneously distributed in the CNS (192). Expression of mRNA for COX in normal human and monkey brain is high in those regions which are most vulnerable to AD pathology and is particularly reduced in the same regions from AD patients (42,43). In the mid-temporal gyrus (but not in the primary motor cortex) of AD patients there is a 50%-65% specific decrease in the mRNA levels of the mitochondrial DNA (mtDNA)-encoded COX subunits I and III (41). However, the mitochondrial-encoded 12S ribosomal RNA (a mitochondrial transcript) does not change, suggesting that the observed reduction of COX I and III mRNA is not due to loss of mitochondria but to a specific alteration of the transcription regulation. A behavioral study in rats treated with the selective COX inhibitor sodium azide showed significant inhibition of a low-threshold form of hippocampal long-term potentiation and impaired spatial learning (13). This finding: (a) lends further, though indirect, support to the theory that Complex IV alteration is somehow involved in the pathogenesis of AD and (b) raises the possibility of developing an animal model reflecting this aspect of AD provided it is specific. Superoxide radicals are described as having considerable reactivity, short half-life and limited diffusion through membranes. However, the latter property has been questioned, as these radicals can appear in the brain extracellular space (109, 130). O~- have a dual effect: they help protect against infectious microorganisms, but they can also be harmful as they par-

OXYGEN RADICALS AND A L Z H E I M E R ' S DISEASE

663

ticipate in the formation of the most noxious hydroxyl radicals "OH (see below). Moreover, they can inactivate a number of useful enzymes: (a) antioxidant enzymes [catalase (108) and glutathione peroxidase (25)]; (b) enzymes involved in neurotransmission [glutamine synthetase (162)], in signal transduction [adenylate cyclase, 140)], and in energy transduction [creatine phosphokinase (121), NADFI dehydrogenase and ATPase (200)]. The GTP-binding proteins are more sensitive to superoxideinduced injury than the catalytic site of adenylate cyclase (140). Finally, superoxide radicals can react with nitric oxide (NO) giving peroxynitrite, eliciting vasoconstriction or inducing cytotoxic effects (discussed in 8:9). In AD brain and fibroblasts there is evidence of partial uncoupling of mitochondrial oxidation and phosphorylation (24,167). Apart from the neurodegeneration induced by the impairment of energy metabolism, these oxidative abnormalities could contribute to the accumulation of cytoskeletal material. Addition of an uncoupler to cultured fibroblasts from normal subjects causes the appearance of epitopes recognized by antibodies to paired helical filaments (PHF) and Alz-50 monoclonal antibodies (24),. thus reproducing a pattern characteristic of fibroblasts from AD patients (8). HYDROGEN PEROXIDE AND HYDROXYL RADICAL FORMATION AND REMOVAL

Role of Superoxide Dismutase and Metal Ions Oxygen accepts (or prefers to accept) one electron at a time. As previously discussed, w!hen molecular oxygen (O2) accepts an electron from a reducing agent, the primary product is the superoxide anion (O~-) that, in aqueous environments, is in equilibrium with its protonated form ('O2H). When the reduced form of molecular oxygen (O~-) and the protonated form of the superoxide anion approach equal molar concentrations, spontaneous dismutation occurs, and hydrogen peroxide (H202) plus O2 or singlet oxygen (102) are generated. 20~- + 2H + ~ H202 +

0 2

[or IO2]

[l]

In reaction [1], the superoxide radical can be converted into hydrogen peroxide by a dismutation of O~- catalyzed by the superoxide dismutase (SOD) that is present in varying concentrations in neural cells. Thus, the conversion removes O~- and prevents its direct toxic action as well as its interaction with metal ions to increase the production of hydroxyl radicals as indicated by reaction [9]. The rate constant for SOD-catalyzed dismutation [1] is approximately four magnitudes greater than for the spontaneous dismutation of O~- a! physiological pH. Obviously, for SOD protection to work properly, it is absolutely vital for other enzymes (e.g., catalase, glutathione peroxidase, etc.) to convert hydrogen peroxide immediately into water, thus preventing the intervention of metal-ion complexes transforming hydrogen peroxide into the highly toxic hydroxyl radical ('OH), by reaction [9]. In this last case, the intervention of SOD may paradoxically be dangerous for neural cells. There are two types of SOD: one is manganese-dependent (Mn SOD) and is located in the mitochondria, where it interreacts with the superoxide radicals derived from the electron transfer chain. The other is copper- and zinc-dependent (Cu-Zn SOD) and is located in the neural cytosol where it carries out a more generic catalytic function. Auto-oxidizable electron carriers on the internal mitochondrial membrane can generate O~- which is enzymatically dis-

mutated to H 2 0 2 . However, some reactions catalyzed by several enzymes (e.g., monoamine oxidase and L-aminoacid oxidase) can produce hydrogen peroxide directly. Thus, hydrogen peroxide may be generated either as a direct product or as a SODcatalyzed dismutation product from each of the various sources of O ) - : (a) by autooxidation of a variety of low-molecularweight molecules; (b) as by-products of various enzyme-substrate reactions such as between xanthine and xanthine oxidase; (c) by the mitochondrial electron-transfer system. The hydroxyl radical ('OH) is one of molecular oxygen's most potent reactive metabolites produced in brain systems. This radical, O2, and O H - are all products of reaction [2] when H 2 0 2 is directly reduced by O~-. O ~ - + H 2 0 2 ~, 0 2 [or I o 2 ] "~- " O H -3L O H -

[2]

H 2 0 2 c a n cross cell membranes directly whereas O~- crosses cell membranes through anion channels. Although hydrogen peroxide cannot be classified as a radical because it contains no unpaired electrons, it is still potentially dangerous on two accounts: (a) it easily permeates cell membranes and can thus migrate from where it is first formed to other organic compartments; (b) it can interact with the reduced forms of some metal ions (generally, bivalent iron, or monovalent copper) which decompose into the highly reactive hydroxyl radical ('OH) and the hydroxyl ion (OH-), according to the following reactions [3] and [4]: H 2 0 2 + Fe 2+ ~ Fe 3+ + "OH + O H -

[3]

H 2 0 2 + C u + ~ C u 2+ + ' O H + O H -

[4]

The formation of "OH thus requires reduced forms of metal ions, such as Fe E+ or Cu ÷. Now the superoxide radical O~- can give rise to Fe 2÷ or Cu + by reducing Fe 3+ or Cu E+ according to the following reactions [5] and [6]: O~- + Fe 3+ ~ 02 + Fe 2+

I51

O-~- -[- C u 2+ ~. 0 2 + C u +

[61

Reaction [2] is extremely slow at physiological pH and would thus require steady-state concentrations of the reaction partners far beyond those found in cerebral mitochondria to account for detectable amounts of the highly unstable radical. As previously shown in reactions [3], [4], [5], and [6], metal ions (generically labeled as M n+) accelerate reaction [2] by catalyzing two intermediate reactions [7] and [8]: O~- + M n+ ~ 0 2 + M (n-l)+

[7]

M (n-l)+ + H 2 0 2 ~ M n+ + " O H + O H -

[81

Mn+

O'~- + H202 '

' 0 2

[ or IO2] + "OH + O H -

[9]

In reaction [9], O~- reduces trace metals (Fe 3+, Cu2+), and generates oxygen or singlet oxygen. The reduced form of the metal then reacts with H202 to produce the initial oxidized form of the metal, the hydroxide ion, and the hydroxyl radical. In view of the evidence that in neural systems reaction [2] pro-

664 ceeds very slowly, the hydroxyl radicals are possibly produced through reaction [9]. An additional mechanism by which "OH may be generated is suggested by the observation that incubation of H202 with ferrous ion (Fe 2÷) and iodide ions ( I - ) gives rise to a potent oxidant molecule that is inhibited by scavengers of the hydroxyl radical (e.g., mannitol and ethanol). The Fe2÷-H202-I system iodinates unsaturated fatty acids (including arachidonic acid) at double bonds. Iodide radicals ( ' I ) and hypoiodous acid (HOI) are each capable of initiating iodination on target molecules. Singlet oxygen (102) is generated by the intervention of SOD in reaction [2] or by the intervention of metal ions in reaction [9] when one of the two unpaired electrons of molecular oxygen acquires sufficient energy to undergo spin inversion or both spin inversion and orbital transition. There are two distinct forms of singlet oxygen, namely A and ~, depending respectively on whether the excited electron forms an electron pair in the same orbital or remains unpaired in a different orbital. The A form of singlet oxygen is stabler than the ~; form. Singlet oxygen is highly electrophilic and reacts with electron-rich compounds such as tryptophan, methionine, and molecules containing unsaturated double bonds. The effect of physiological aging on brain SOD is controversial, though most reports describe some age-related decline, mainly of the Cu-Zn form (19,77,86,151,164,184), probably related to the decline of mRNA SOD. However, other studies report no change of this form and an increase in Mn-SOD (38,55). Besides being a physiological antioxidant, SOD, when injected IV as a pharmacological agent, exerts a protective effect on the CNS in some experimental pathologic conditions where ROS formation is enhanced, e.g., in postischemic reperfusion (reviewed in 180). Thus, it would appear that more SOD is beneficial and less SOD is detrimental. However, if the activity of SOD is increased without a concomitant enhancement of the activity of the enzymes which dispose of H202 (mainly glutathione peroxidase) and the concentration of reduced glutathione, then H202 accumulates and reacts with O~- and Fe E+ to form the very dangerous hydroxyl radical ('OH). Thus, the imbalance between "SOD-and-H202 converting enzymes" results in a toxic effect of SOD by "OH generation inducing DNA fragmentation, protein denaturation, activation of the autocatalytic process of lipid peroxidation, etc.. Down's syndrome (trisomy 21) has provided some provocative clues on the balance between SOD, ROS, and antioxidants. Human Cu-Zn SOD is encoded by a gene located on chromosome 21 and Down patients have a 50% increase in the activity of this enzyme secondary to gene dosage effect (168). The increase in SOD, not accompanied by a concomitant adaptative rise in glutathione peroxidase (30), might induce oxidative damage to the CNS, including lipid peroxidation and this might explain some of the neurobiological abnormalities found in Down's syndrome, such as accelerated aging and Alzheimer-type neuropathology. This is supported by the results in an animal model of gene dosage effect, i.e., in transgenic mice carrying the human CuZn SOD gene. The Cu-Zn SOD protein and mRNA are preferentially expressed in the large pyramidal neurons of Ammon's horn and granule cells of the dentate gyrus, which are particularly susceptible to degenerative processes in AD (40). Brain lipid peroxidation is also increased. In seeming contrast with this finding, increased Cu-Zn SOD in transgenic mice makes the hippocampus more resistant to the neurotoxicity induced by amyloid fl/A4 (74).

BENZI AND MORETTI The levels of Cu-Zn SOD protein and mRNA in the vulnerable hippocampal neurons of AD patients (39), their association with neurofibrillar degeneration (PHF) (56), and the observation that the cell distribution of Cu-Zn SOD mRNA in the human hippocampus is the same as amyloid mRNA (7) suggest that high levels of antioxidant enzymes are needed to remove excess superoxide radicals but could also indicate that ROS contribute to the degenerative processes leading to neuropathology in AD. Cu-Zn SOD activity is also higher in the temporal cortex and nucleus basalis Meynert (128,141) and in the fibroblasts of AD and Down patients (198). Cultured skin fibroblasts from both familial and sporadic AD patients are more susceptible to ROSinduced damage then from age-matched controls (179). Finally, high immunoreactivity for SOD and catalase in the AD brain is associated with some neurofibrillar tangles and senile plaques (142). This immunoreactivity is absent in tangle-free neurons of AD and all neurons of normal control brains.

Role of Catalase and Monoamine Oxidases Another interesting enzyme is catalase which decomposes hydrogen peroxide by the following reaction: 2H202 ~, 2H20 +

0 2

[10]

Catalase is particularly active in the liver, kidney, and erythrocytes but only slightly in the brain. The brain areas richest in catalase are the hypothalamus and the substantia nigra where the enzyme is located in small subcellular particles called peroxisomes. Brain peroxisomes are very much smaller than liver peroxisomes, so they are called "microperoxisomes." Catalase and its expression seem to decrease with age (86,151,164). Hydrogen peroxide is also formed during the oxidative deamination of monoamines (catecholamines, serotonin) catalyzed by MAO, an enzyme associated with the outer mitochondrial membrane: RCH2NH 2 +

0 2

"1- H20 -~ RCHO + NH 3 + H202

[11]

There are two MAO isoenzymes, A and B, with different substrate specificities, inhibitor sensitivities, and cellular localization. MAO B, but not MAO A, activity apparently increases with age in various regions of the human and rat brain (reviewed in ref. 31). This is due to increased enzyme protein rather than to changes of its catalytic properties. This age-related increase in the MAO B activity of various brain regions is more marked in AD (99,137,156,199). As MAO B is found mostly in glial cells (158), its increase could conceivably be associated with the proliferation, mainly of astrocytes, which accompanies neuronal loss in aging and particularly in AD. The activity of MAO B coexists with neuritic plaques (199) and is expressed in fibrillar astrocytes in or around these plaques (130). From the above findings, it can be inferred that an excess of H202 is formed in the AD brain.

Role of Enzymes Related to the Glutathione Cycle The hydrogen peroxide formed through the action of superoxide dismutase and MAO is removed by the important catalytic intervention of glutathione peroxidase in the presence of glutathione (glutamyl-cysteinyl-glycine), a tripeptide of nonprotein origin containing a glutamic acid residue linked by an unusual peptide bond, in which its -¢-carboxylic group participates.

OXYGEN RADICALS AND A L Z H E I M E R ' S DISEASE

665

Glutathione peroxidase :requires selenium to act. The result of its action is the conversion of reduced glutathione (GSH) to oxidized glutathione (GSSG) which, in its turn, must be reduced by the catalytic intervention of glutathione reductase. 2GSH + H202 ~-~2H20 + GSSG

[121

This reduction of GSSG to GSH requires N A D P H which is produced in the pentose phosphate pathway [glucose = ribose 5-phosphate] in two reactions catalysed by glucose-6-phosphate dehydrogenase:

(153). After GSH oxidation, mitochondria, unlike cytoplasm, are unable to export GSSG and this may be an important mechanism of neuronal derangement or death. There may be a compensatory increase in GSH in the AD brain (2) concomitant with an even larger increase in GSSG, particularly in the caudate nucleus and hippocampus. At least in these two brain regions, the glutathione redox index is lower in AD patients than in agedmatched healthy subjects. The brain concentrations of c~-tocopherol are unaffected (2,125) despite low plasma levels in AD patients (95,196). However, there is poor correspondence between the plasma levels of a-tocopherol (10-40 #M) and its concentrations in tissues.

glucose 6-phosphate + NADP + --* 6-phosphogluconolactone + N A D P H

[13]

and by 6-phosphogluconate dehydrogenase: 6-phosphogluconate + NADP + ribulose 5-phosphate + N A D P H

[141

Any event involving an increase in the oxidation of catecholamines by monoamine oxidase results in acceleration of the metabolic flow of the pentose phosphate pathway. This is probably due to the increased use of N A D P H in reactions catalysed by glutathione reductase [GSSG = 2GSH] and by aldehyde reductase [aldeyde = alcohol]. Apart from its antioxidant characteristics, the role of reduced glutathione is to keep the ionic cell balance constant. Changes in the concentration of GSH cause major changes in the distribution of ions in cells: GSH even functions as a modulator of the cell redox state. Therefore, considerable fluctuations in the intracellular concentration of GSH interfere both with the distribution of the electrical clharge of ceils, and with the ratio of reducing equivalents to oxidizing equivalents. Reduced glutathione increases in the mitotic phase and decreases in the cell redifferentiation phase. The marker enzyme catalysing the GSH oxidation, 3,-glutamyl-transpeptidase, decreases in developing systems, reaching its lowest levels in differentiated cells. The rate of fall of the concentration of reduced glutathione is inversely proportional to the rate of differentiation. The increased production of free radicals during differentiation can lead to the oxidation of glutathione and its extrusion from cell elements. Agents raising or lowering reduced glutathione cell concentrations cause a corresponding decrease or increase in the rate of cell differentiation. GSH decreases in the aging rat brain, glutathione peroxidase and its expression remain constant (19,77,151), whereas GSSG remains practically constant or drops only slightly thus lowering the glutathione redox index during aging ( 16,18,44,154). Peroxidative stress (e.g., induced by H202 or electrophilic agents) causes the glutathione redox index to drop more in aged rat brains than in young ones (17,19). The severe depletion of GSH reduces protein synthesis and alters DNA synthesis because of its pivotal role as reducing equivalent for the glutaredoxin system supplying electrons to ribonucleotide reductase. GSH depletion affects some GSH-depende,nt enzymes (glutathione synthetase, glutathione peroxidase, glutathione transferase, leukotriene C4 synthetase, glutaredoxin system, glyoxylase I and II), making cells more susceptible to arty further challenge. GSH depletion in the rat led to striking degeneration of brain mitochondria (96). Most GSH is localized in the cytoplasm, where it is the main reducing power, and only 10%-15 % is present in mitochondria

Specific Role of Iron As previously discussed, transition metals, especially iron, in their free form play an essential role in many processes related to ROS generation. Organisms take great care in handling iron, using transport proteins (e.g., transferrin) and storage proteins (e.g., ferritin) and minimizing the size of the intracellular iron pool (91). This iron sequestration can be regarded as a contribution to antioxidant defenses. However, oxidant stress can itself provide the iron necessary for the reaction [9], for example, by mobilizing iron from ferritin or by degrading heme proteins (e.g., hemoglobin) to release iron (91). In normal conditions, the availability of free iron to stimulate "OH generation in vivo is very limited (not more than 3 t~M in human samples) and the antioxidant defense systems can remove 02 and H202 before they give rise to "OH. However, tissue injury (typically brain ischemia and trauma) can greatly stimulate the reaction [9] by releasing substantial amounts of nonprotein-bound iron from the damaged cells into the surrounding environment. In AD brains, various mechanisms besides aging could conceivably activate oxidative stress. First, head trauma appears to be a risk factor for AD although its role has not been well defined (4,159,188). Trauma to the brain promotes lipid peroxidation in vivo (reviewed in ref. 28) and triggers the deposition of amyloid/3/A4 in the human cerebral cortex in an astonishingly short time (76,159) and of the amyloid precursor protein [APP] in the rodent brain (75). Second, variable degrees of hypoperfusion are likely at least in some areas of the AD brain on account of the altered microcirculation resulting from the deformation of brain capillaries (60), the amyloid angiopathy (187), and the marked proliferation of reactive astrocytes (73). Vascular amyloid fibrils are associated with and formed within the basement membrane of meningeal and cortical microvessels (145,193) and senile plaques grow in association with degenerated cerebral capillaries (126). This whole process appears to be self-destructive and progressive (60). A history of myocardial infarct might constitute a risk factor for senile dementia (6) and is related to abundant senile plaque deposition even in the brains of nondemented subjects (173). An experimental model of chronic cerebrovascular insufficiency in aged rats reproduces some of the biochemical, neuropathological, and behavioral alterations of AD at its onset (59). Focal cerebral ischemia in rats induces accumulation of A P P (the amyloid precursor), particularly in dystrophic axons and neuronal perikarya (100,177). Thus localized ischemic insults or chronic hypoperfusion may lead to increased expression of APP in the surviving brain cells and this could be related to the A P P mobilization in AD (100). A third important factor is the alteration of iron homeostasis in the aging brain (12) which is further exacerbated in AD, prob-

666 ably because of iron accumulation consistently with the reactive proliferation o f microglia associated with the neuritic plaques (48). Ferritin, a marker for microglia (101), is also greatly increased in these cells around neuritic (but not diffuse) plaques and blood vessels (48,85). This might be the request of increased reactive ferritin synthesis in glial cells in an attempt to detoxify the excess iron originating from blood or from cell degeneration (48). Alternatively, iron could be mobilized from ferritin after a reduction induced by superoxide anion, nitric oxide, or other reductants, a process accelerated by acidosis (27). In any case, the accumulation of iron and ferritin in microglia is important, as these cells appear to play an active role in forming amyloid fibers in neuritic plaques (191). Finally, in addition to more iron, the ferritin isolated from AD brains also contains more aluminum than age-matched controls (67). It is beyond the scope of this review to examine the role of aluminum in the pathogenesis of AD, a question that is still controversial. It might accumulate in the brain during the progression of AD because of alteration of the blood-brain barrier. Aluminum is present in the core of mature plaques of AD patients (22,36,197) though this is not always confirmed (see ref. I 10) and is co-localized with the neurofibrillar tangles (82,115) and iron (82). One of the possible mechanisms of Al-induced neurotoxicity (98) is based on its ability to stimulate ROS generation and lipid peroxidation (65,72) and to accelerate iron-induced lipid peroxidation (87,134,139). Interestingly, the Al-induced alteration of membranes and enhancement of iron's effect on lipid peroxidation are more pronounced at an acidic pH (87,139), such as in the AD brain (194). In an experiment with liposomes, the Al and Fe 2÷ effect on lipid peroxidation was much more evident at a higher phosphatidylserine/phosphatidylcholine ratio (139). The negatively charged phosphatidylserine is one of the few phospholipids elevated in the AD brain. Most of the other phospholipids, including phosphatidylcholine, are reduced (147). In vitro aluminum interferes with iron metabolism by affecting its storage and transport by ferritin (68), thus, conceivably making more free iron available for ROS production and ferritin synthesis. Aluminum also enhances the oxidation of NADH by a source of O~-, indicating the formation of an oxidizing complex between A1 and O~- which could contribute to the deleterious biological effects of A1 (107). The suggestion that aluminum and/or iron might somehow be involved in AD is corroborated by a clinical study which found that desferrioxamine, a chelator, slows the clinical progression of AD dementia (52). In any case, this result should be confirmed in other specific studies. Evidence for Oxidative Stress in the A D Brain Lipids, proteins, and DNA are the main targets of ROS, especially hydroxyl radicals. Because the brain membranes are particularly rich in polyunsaturated fatty acids, lipid peroxidation is an obvious consequence of ROS action. Lipid Peroxidation The effect of aging on lipid peroxidation (measured as TBAreactive substances, TBARS) in the rat brain is controversial. Some reports describe an increase either in the whole brain (58,151,161 ) or in various brain regions (77,86,127,133,165,186), but there are also reports of either no change (45,114) or even a decrease (61). The age-related increase in plasma membrane TBARS is preceded by a rise in superoxide radical levels and accompanied by a decrease in membrane fluidity (161). The agerelated increase in lipid peroxidation and lipofuscin concentra-

BENZI AND MORETTI tion in the rat hippocampus significantly correlate with the decline of neuronal electrical activity recorded in this area and the biochemical and electrophysiological alterations are corrected by long-term treatment with centrophenoxine (165). The proposal that aging is associated with altered membrane properties and receptor function in the brain has been confirmed (reviewed in refs. 5,183). Basal and iron-stimulated lipid peroxidation appears to rise in the cerebral cortex (e.g., the frontal, temporal, and parietal cortices) of AD patients (84,88,122,178). Peroxidation is not increased in the occipital cortex or in the cerebellum, two brain areas that are less affected by the disease (178). Iron-stimulated cortical peroxidation is inhibited by adding a 21-aminosteroid (an antioxidant, iron-chelator compound) to the incubation medium. The ICs0 is somewhat higher for AD (10/~M) than for control samples (2.5 ~M) (178). A logical consequence of increased lipid peroxidation would be membrane alteration. There is, in fact, substantial evidence of membrane derangement in the AD brain as shown by lipid composition and metabolism studies (66,102,116,132,148,169), membrane microviscosity (201), magnetic resonance spectroscopy (147), and differential scanning calorimetry (84). In AD patients, cell membranes from the mid-temporal cortex (but not from a less affected area such as cerebellum or from either area in normal elderly subjects) have an inherent tendency to destabilization (79). This change predictably causes the membrane lipids to shift from a unilamellar state to a multibilayer, a transformation which may induce structural defects with potential serious consequences for cellular function (79). Protein Oxidation The second class of substances oxidized in aging, albeit not confirmed in rats (4,5), are the proteins as supported by studies in gerbils (37,174). The cerebral cortex of aged gerbils has significantly higher levels of oxidized proteins (as carbonyl protein content) and lower glutamine synthetase (GS) and protease activities than the cortex of young animals. GS is a key enzyme in brain nitrogen metabolism as it synthesizes glutamine from glutamate, ammonia, and ATP. Thus, a low level could lead to increased glutamate and ammonia, two potentially neurotoxic compounds. The protease reduction might impair the removal of oxidized, inactivated proteins. Moreover, in behavioral experiments, old gerbils make more errors than young animals in a radial arm maze test for temporal and spatial memory. Finally, it should be stressed that these alterations in aged gerbils are corrected by treating them with the spin-trapping compound c~-phenyl-tert-butyl nitrone (PBN). Old gerbils given PBN for 14 days are biochemically and behaviorally indistinguishable from the young ones. The biochemical effects of PBN are reversible: when it is stopped, the oxidized proteins and the GS and protease activities gradually return to their original status. Similar findings of protein oxidation, GS inactivation and their correction with PBN are reported in gerbil brains after ischemia followed by reperfusion (69,136). Thus, an acute pathologic event causes the same oxidative damage as a chronic one and both are reversed by a spin-trapping compound. In the human brain, the content of oxidized proteins rises exponentially with aging especially in the frontal cortex, less in the occipital cortex. The activity of GS and creatine kinase are markedly reduced in both regions. The age-related enhancement of protein oxidation is not restricted to the brain, but it occurs also in cultured human dermal fibroblasts, where it is even more evident in premature aging, such as progeria and Werner disease (135), in human erythrocytes as a function of cell age (135), and

OXYGEN RADICALS AND ALZHEIMER'S DISEASE in the rat liver (175), where protein or caloric restriction both reduce the accumulation of oxidatively damaged proteins (195). In AD patients, protein oxidation in the frontal cortex is not further enhanced, hut GS activity is reduced compared with old non-AD subjects (170). The loss of GS in the brain of old humans and gerbils, and even more so in AD patients, may be interpreted in the light of this enzyme's vulnerability to ROS (162).

DNA Damage DNA damage is almost iinvariably observed in a wide range of mammalian cells exposed to ROS (90). The mitochondrial DNA is particularly susceptible to oxidative stress because: (a) mitochondria have high 02 consumption and are the source of a continuous flux of oxygen radicals; (b) mtDNA is not protected by histones and is close to the inner membrane where ROS are produced; (c) mitochondria may repair DNA damage less efficiently than the nucleus. Thus, oxidative damage to DNA may be responsible for the age-related decline in oxidative phosphorylation especially in postmitotic cells, namely the neurons which have high respiratory rates and slow mitochondrial turnover (113,181,190). Indeed, mtDNA deletions, assessed by the polymerase chain reaction, progressively increase with aging in various human brain regions (23,49,50). Although the ratio of deleted DNA is markedly higher at the age of 80, it is not known whether the absolute value, approximately 0.1 °70, is sufficient to cause deleterious physiological effects. An age-related increase in oxidative damage to mtDNA has also been found (124). A three-fold enhancement of oxidative damage to DNA has been recently reported in the AD brain (123). A specific mtDNA point mutation is described too (112) but not confirmed (146). Direct assay of nuclear DNA damage in the cortex of AD patients indicated it is double that in controls (129), a finding which could be attributed to the effect of ROS on DNA. REACTIVEOXYGENSPECIESAND fl/A4 PROTEIN The/3/A4 protein is crucial in the pathogenesis of AD. It is the major constituent of amyloid plaque cores and amyloid congophilic angiopathy. "Diffuse" plaques consisting of focal extracellular deposits of nonfibrillar fl/A4 protein not surrounded by degenerating neurites may be the early lesion in the disease (reviewed in ref. 97). However, the nonfibrillar nature of the preamyloid deposits and their presence in regions where the plaques are rare (e.g., the cerebellum) suggest they are not inevitably precursors of the plaques. Variable deposits of fl/A4 protein have been seen in nondemented centenarians who might, however, be in a preclinical stage of AD (57). The fl/A4 protein is believed to derive from the degradation of the amyloid precursor protein (APP) leading to amyloidogenic fragments and subsequent proteolysis. The protein is then released and aggregates in the brain parenchyma into amyloid fibrillar deposits which form the dense core of the compacted senile plaques surrounded by dystrophic neurite processes and glial cells (reviews in 70,97,143,163,191). Thus, the deposition of amyloid fibrils in classic senile plaques appears to depend not only on the rates of production and removal of the fl/A4 protein hut also on the rate of formation of insoluble from soluble protein. Aggregation of/3/A4 protein is essential for its in vitro and in vivo neurotoxicity (33,51,71,94,120,149,150,182,189). ROS could play a significant role in this connexion, as shown by the observation that in vitro iron-catalyzed oxidation systems transform the nonaggregated into aggregated/3/A4 protein, i.e., into 16 and 32 kDa forms (152). The characteristics of this aggre-

667 gation are very similar to those of synthetic fl/A4 protein in various experimental conditions (10,94) and also to the fl/A4 protein isolated from AD plaque cores (117). A similar proaggregating effect is elicited by ROS-inducing systems on the Cterminal fragment of the A P P containing 100 residues and beginning with the fl/A4 sequence at the N-terminus (A4CT). This fragment is a potential intermediate in amyloid formation from APP (64,81). Radical scavengers, such as ascorbic acid and a water-soluble a-tocopherol analogue, inhibit the pro-aggregating effect of the ROS on A4CT (62). The observation that these aggregates of the fl/A4 protein and A4CT are stable in the presence of 6M urea suggests they are formed by tightly bound molecules. Thus, ROS induces cross-linking of the fl/A4 and APP fragments that are then able to aggregate further. An important point is that the aggregation of synthetic fl/A4 protein is fostered by an acid pH (10,32), which can be achieved in the AD brain (194), possibly due to hypoxia and ischemia and to other disorders found in elderly patients. Moreover, in vitro ROS promote aggregation and neurotoxicity of fl/A4 protein (92) and aggregation of amyloid plaque components (47). A possible interaction between ROS and the amyloid is supported in vitro by the observation that /3/A4 protein potentiates H202 toxicity on neuronal cultures (160) and by the following findings obtained with PC12 cell lines and rat cortical primary cultures (11,35): (a) the fl/A4 protein and its biologically active fragment/325-35 cause increased production of intracellular peroxides (mostly H202) in a time- and concentration-dependent manner and this production is highly correlated with peptide toxicity; (b) cells selected for resistance to (3/A4 are also highly resistant to H202 toxicity; (c) catalase and various antioxidants and free radical scavengers inhibit peroxide accumulation and reduce the peptide toxicity; (d) the/3/A4 also enhances lipid peroxidation and antioxidants suppress this effect. Thus, both H202 and "OH can be implicated in fl/A4induced neurotoxicity. The in vitro protection by vitamin E against the neurotoxicity induced by/325-35 on hippocampal cultures has been confirmed (111). These observations are corroborated by the finding that the /3/A4 protein in aqueous solution fragments and generates free radical peptides in a reaction requiring O2 and possibly involving oxidation of the methionine residue 35 of fl/A4 to sulfoxide. These fragments, which are neurotoxic toward neuronal cultures, inactivate GS and CK (93). The same effects are also elicited by the active fragment (25-35) but much more promptly than by fl/A4 (min vs. h). The (25-35) fragment also produces lipoperoxidation (34). Great progress toward the interpretation of the relationship between ROS and amyioid toxicity comes from the following results obtained in rat hippocampai cell cultures: (a) fl/A4 potentiates the iron-induced oxidative injury to neurons; (b) fl/A4 induces an increase in intracellular Ca 2+ levels; (c) vitamin E protects neurons against both neurotoxicity and Ca 2+ elevation; (d) the secreted forms of APP (Apps695 and Apps751) attenuate the fl/A4-induced ROS formation, neuronal injury, and Ca 2+ elevation as well as the neuronal injury caused by iron (83,118). These findings, therefore, suggest a model for fl/A4 neurotoxicity in which initial perturbation of proteolytic or other pathways promotes cleavage of amyloid precursor protein and release of/3/A4 into the extraneuronal space. Such primary fl/A4 may then fragment to form smaller, toxic oligopeptide radicals. These radicals could attack cell membranes, initiating lipoperoxidation and damaging sensitive membrane proteins and altering the homeostasis of Ca 2+.

668

BENZI AND MORETTI

It is not known whether these two mechanisms of interaction between ROS and fl/A4 protein (the former in which ROS stimulate the aggregation and neurotoxicity of fl/A4 and the latter in which ROS production and neurotoxicity are elicited by j3/A4) coexist and potentiate each other. It must be underlined that all these results are obtained in vitro and should be confirmed in many other specific research. CONCLUSIONS

Any hypothesis on the pathogenesis of AD must take into consideration the fact that aging is the primary risk factor (103) and the manifestations of AD presumably exacerbate those induced by aging over a number of years (21,29). Histological manifestations of AD (e.g., neuritic plaques) may also be seen in the brain of normal aged persons, though to a lesser extent and with different localizations. Among the various factors involved in the pathogenesis of AD, there may be a metabolic derangement in the brain over an extended period of time. Oxidative stress, involved in the aging of the brain, can be considered at least a contributing factor. That this stress is somehow implicated in AD is indicated by the coalition of the various factors described in the previous sections and summarized in Table 1. The strongest evidence stems from the changes in the brain resulting in more ROS being produced than in healthy age-matched individuals (decrease in COX activity, unbalanced increases in SOD and MAO, alteration of iron homeostasis and probable association with Al deposition) and from reduced antioxidative defenses. Subtle, local ischemia and head traumas are other possible contributors. Thus, in the AD brain a number of biochemical conditions appear to favor mitochondrial electron leakage and oxygen freeradical production, i.e., a condition in which the pro-oxidant status has risen to such a level that it can no longer be counterbalanced by antioxidants. This condition is not necessarily severe and may even be mild. Nevertheless, if this subtle shift in the "oxidative index" is prolonged enough, cumulative alterations

TABLE 1 EVIDENCE IN FAVOR OF A ROLE OF OXIDATIVE STRESS IN T H E AD BRAIN

I. Deficiency of Complex IV favoring electron leakage with ROS release. 2. Higher unbalanced activity of SOD and MAO B, probably associated with some neuropathological hallmarks of AD. 3. Possible history of head trauma and variable focal hypoperfusion. 4. Alteration of iron homeostasis resulting in a potential excess of ROS generation. 5. Association of aluminum with neurofibrillar tangles and neuritic plaques; Al-induced ROS generation and lipid peroxidation (enhanced by an acidic pH). 6. Reduced antioxidative defenses. 7. Increase in lipid peroxidation and membrane alterations. 8. In vitro pro-aggregating effect of ROS on fl/A4 protein and A4CT, fostered by lowered pH. 9. Increased in vitro production of H 2 0 2 and lipid peroxidation induced by fl/A4 concomitantly with neurotoxicity. Protection by antioxidants. 10. Fragmentation and generation of free radical peptides from fl/A4 protein. Potentiation by fl/A4 of iron-induced neurotoxicity; alteration of Ca 2+ homeostasis; protection by vitamin E. 11. Possible effect of desferrioxamine in AD (?).

of molecular and cellular targets of ROS are likely. Although it is not easy to assess all the above observations together, various explanations can be put forward for the mechanism(s) by which oxidative stress contributes to the pathogenesis of AD and its site(s) of action. The possible AD-dependent increase in the formation of superoxide radical and its dismutase product (hydrogen peroxide) is based on the concept that the Complex IV deficiency modifies the electron flux in the mitochondrial respiratory chain, thus making it easier for electrons to escape the normal flow sequence. This condition, in turn, weakens the protective mechanism that works to prevent the autooxidation of electrontransferring ubiquinone and cytochrome b populations, allowing an increase in electron leakage outside the chain. The damage caused by peroxidation to the structure of the mitochondrial inner membrane affects the phospholipid layer, also increasing the hydrophilic properties of the mitochondrial inner membrane; the resulting endless loop creates the thermodynamic conditions for destabilization of ubisemiquinones by auto-oxidation that easily occurs in aqueous phases. Besides ubisemiquinones, cytochrome b566 too is very important because it is strongly auto-oxidizing and may be held responsible for the formation of mitochondrial ROS, thus confirming that superoxide radical formation in the mitochondria is correlated with the existence of reduced forms of cytochrome b566 rather than with a redox-cycle linked only to the ubiquinone family. Because membranes appear to be the main target of ROS and APP is located in the plasma membrane, it is tempting to speculate that the attack by ROS is involved in one or more steps of amyloidogenesis (63). Membrane damage could result in the production of more easily proteolyzable substrates, thus favoring proteolytic processing of APP. The acidosis-facilitated reduction by Al of the activity of protease inhibitors, such as a~antichymotrypsin (ACT), may help accelerate the proteolytic processing of APP (46). As shown in vitro, the possibility also exists that the attack by ROS is involved in the aggregation of ~/A4 protein, a condition that seems critical for its neurotoxicity. Alternatively (or possibly concomitantly) ROS can be generated by fl/A4 fragmentation. Destabilization of calcium homeostasis, perhaps secondary to a defect in membrane structure and function, is another possible mechanism for neurotoxicity in the AD brain (78,104). Among the possible causes, either fl/A4 protein (119) or ROS (138) or both could be important. A clue to understand these mechanisms is provided by the above-mentioned recent studies indicating that fl/A4 exerts its toxic action by increasing ROS production and [Ca2+]i. The resulting overload of Ca 2+, if intense and prolonged enough, will in turn activate various catabolic processes, leading eventually to cell death. The pro-oxidantinduced calcium release from mitochondria could in fact be related to apoptosis (152,157). Though tempting, this scenario is hypothetical. Arguing against it or calling for closer examination are the observations listed in Table 2, particularly the lack of any definite demonstration that a molecular target of ROS, i.e., the proteins, is damaged in the AD brain, although membrane, enzyme and oxidative phosphorylation function might be impaired enough in AD due to the damage accumulated over a lifetime. The correlation between the biochemical and neuropathological alterations in the various areas of the AD brain also need investigating further, employing more specific and extensive assays of oxygen radical activity. In apparent conflict with the above theory is also the finding that fl/A4 protein does not potentiate ischemia-inducedbrain

OXYGEN RADICALS AND ALZHEIMER'S DISEASE

669

AGING ~[ OXIDATIVEPHOSPHORYLATIONDYSFUNCTION T MAO B ANTIOXIDANTS free iron acidosis

ROS

~ MEMBRANE ~ ' DAMAGE

T Ca2.

1"Ca-HYDROLASES

T SOD ALUMINUM

[3/ A4 AGGREGATION AMYLOIDDEPOSITION

1 AMYLOIDANGIOPATHY FOCALISCHEMIA ,...-~- Fe, acidosis SPREADINGDAMAGE .91

ROS

NEUROTOXICITY ~

CELLDEATH RELEASEOF PROTEASES DIGESTION OF METALLOPROTEINS

FREE IRON

FIG. 1. Hypothetical mechanisms for the contribution of oxidative stress to brain damage in AD. t increase; ~ decrease; see text for abbreviations.

damage in rodents (176). This, however, needs to be substantiated on account of the known variability of in vivo fl/A4 neurotoxicity. A final, though minor reason for caution is that ct-tocopherol deficiency is riot known to be associated with AD nor does its level seem to be reduced in the brain of AD patients. In conclusion, evidence of a state of oxidative stress in the AD brain is now growing, though still incomplete. It may not be a primary pathogenic factor, as tissue injury itself might induce oxidative stress. However, regardless of the primary mechanism, oxidative stress could greatly contribute by participating in a vicious circle of actions and reactions which intensify each other and result in the accumulation of a critical mass of metabolic errors (46). A hypothetical schematic portrayal of these events, which takes a number of observations into account, is given in Fig. 1. This provocative presentation w i l l - it is hoped stimulate further endeavours to clarify the pathogenesis of the disease, hence, to envisage new strategies for its treatment.

"FABLE 2 EVIDENCE AGAINST OR LACK OF SUFFICIENT PROOF OF OXIDATIVE STRESS IN THE AD BRAIN 1. The apparent lack of increased protein oxidation. 2. The need for more extensive studies on lipid peroxidation, protein oxidation and DNA damage in the brain areas mainly affected in AD. 3. The need for a better understanding of the alteration of glutamine synthetase (discrepancy between the decrease in its activity and glial proliferation). 4. The apparent failure of the 3/A4 protein to potentiate ischemiainduced damage. 5. The normal level of a-tocnpherol. 6. The need for more clinical studies on antioxidants in AD.

ACKNOWLEDGEMENTS We thank Gianluigi Forloni for his useful suggestions and Judy Baggott for the English revision. The secretarial work of Gianfranca Corbellini is greatly appreciated.

Note added in proof. Since the preparation of this manuscript, a number of pertinent studies have been published. The depression of cytochrome oxidase activity (COX, Complex IV) in AD has been confirmed in cortical homogenates (Mutisya et al., J. Neurochem. 63:2179-2184, 1994; Chagnon et al., Neuroreport 6:711715, 1995) and brain mitochondria (Parker et al., Neurology 44:10901096, 1994). Interestingly, patients with a shorter delay between the onset of the disease and death had lower COX activity (Chagnon et al., see above reference). Another study, on only three patients, suggests that the AD COX is not lowered but rather kinetically abnormal due to structural alteration (Parker and Parks, Neurology 45:482-486, 1995). Furthermore, inhibition of energy metabolism markedly enhances the formation of potentially amyloidogenic COOH-terminal derivatives from APP (Gabudza et al., J. Biol. Chem. 269:13623-13628, 1994). The enhanced formation of H202 in the AD brain is further indicated by the increase in: (a) SOD activity (Balazs and Leon, Neurochem. Res. 19:1131-1137, 1994), (b) SOD/catalase ratio (Gsell et al., J. Neurochem. 64:1216-1223, 1995), and (c) MAO B activity, especially in patches corresponding to GFAP and senile plaques (Saura et al., Neuroscience 62:15-30, 1994). That the AD cortex has higher lipid peroxidation has been confirmed by Balazs and Leon (see above ref.) and Palmer and Burns (Brain Res. 645:338-342, 1994), although with some discrepancies regarding the different cortical areas. A marked increase in mtDNA deletion has been described by CorralDebrinski et al. (Genomics 23:471-476, 1994). Finally, further support to the generation of free radicals by 3/A4 has been provided by the findings that: (a) synthetic fl/A4(25-35) is able to cleave the C = N bond of the spin trap PBN in a radical additionfragmentation reaction involving the formation of a B/A4-peptidil peroxy radical species (Hensley et al., Neuroreport 6:489-492 and 493-496, 1995); (b) the ability of 3/A4 to generate PBN-radical adducts is correlated with the intensity of protein oxidation, enzyme inactivation, changes in intracellular Ca 2+ and neurotoxicity (Harris, Exp. Neurol. 13 l:193-202, 1995).

670

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