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Current Medicinal Chemistry, 2013, 20, 4648-4664

Oxidative Stress and Amyloid Beta Toxicity in Alzheimer’s Disease: Intervention in a Complex Relationship by Antioxidants S. Chakrabarti*, M. Sinha, I.G. Thakurta, P. Banerjee and M. Chattopadhyay Department of Biochemistry, Institute of Postgraduate Medical Education and Research, 244, Acharya J.C. Bose Road, Kolkata 700020, India Abstract: The elucidation of the intriguing relationship between oxidative stress and Alzheimer’s disease is crucial to understand the pathogenesis of the disease as also to design a suitable drug trial with antioxidants against this condition. We begin by reviewing the basic facts about Alzheimer’s disease and the chemistry and biology of oxygen free radicals with particular reference to the cellular adaptive response through redox-signalling pathways. The post-mortem evidence of oxidative damage in the brain of Alzheimer’s disease patients is overwhelming which is also supported by the similar changes in transgenic mice models of this disease. However, the causal relationship of oxidative stress with amyloid beta pathology or the genesis of Alzheimer’s disease is not clear. Considering the available evidence the review suggests that the oxidative stress could be an early event in the disease process and may trigger various adaptive responses such as the alterations of amyloid beta metabolism and the activation of stress responsive kinases which can subsequently lead to neuronal degeneration and AD pathology. Further, we have presented a large body of evidence from various studies to highlight the beneficial effects of antioxidants against amyloid beta toxicity or AD pathology in animal or cell based models of AD. The failure of clinical trials with antioxidants against AD has been mentioned and the possible causes of such failures have been analysed.

Keywords: Alzheimer’s disease, amyloid beta protein, oxidative stress, antioxidant, redox signalling, lipid peroxidation, protein oxidation, DNA damage. ALZHEIMER’S DISEASE: SOME FACTS AND FIGURES Alzheimer’s disease (AD) is the commonest form of dementia found in elderly people across the world accounting for nearly 70-75% of all dementia subjects above the age group of 65 years [1]. Other major types include vascular dementia, dementia with Lewy bodies and fronto temporal dementia. AD subjects present an insidious development of memory impairment with a steady decline of MMSE (Mini Mental State Examination) score, multiple cognitive deficits like aphasia, apraxia, agnosia and behavioral changes like agitation, depression, social withdrawal, mood swings [2, 3]. With the disease progression, characteristic brain cortical atrophy and shrinkage of the hippocampus can be observed by MRI. The diagnosis of the disease at present is based on extensive neuropsychiatric evaluations for which several diagnostic criteria are available and radio-imaging studies can further support the diagnosis. However, the confirmation of the diagnosis can be made only by examining the postmortem brain. The therapeutic management of AD is terribly inadequate, and the patient follows a progressively downhill course with devastating loss of memory and cognition and succumbs to the illness within 5 -9 years of diagnosis.

*Address correspondence to this author at the Department of Biochemistry, Institute of Postgraduate Medical Education and Research, 244, Acharya J.C. Bose Road, Kolkata 700020, India; Tel: +91-33-22234413 / +919874489805; E-mail: profschakrabarti95@gmail.com / [email protected] 1875-533X/13 $58.00+.00

With the progressive increase in the life span of the general population in developed countries as also in some developing countries, AD is becoming an enormous challenge to the healthcare system worldwide. In 2010, Alzheimer’s Disease International has estimated that there are 35.6 million people living with dementia worldwide out of which more than 26 million are estimated to be afflicted with AD [4]. The prevalence of AD has been estimated in many different studies, and in developed countries the prevalence estimates in 65 year olds are around 4.4%, but lower in Asian countries and sub-Saharan Africa [1, 4-6]. AD manifests in two major forms called the familial and the sporadic with the latter accounting for 90-95% of the AD patients. In familial AD, the age of onset of the disease is early (below 65 years), and 3 clear gene mutations have been identified: APP (amyloid precursor protein) gene in chromosome no 21, Presenilin 1(PS1) gene in chromosome no 14 and Presenilin 2 (PS2) gene in chromosome no 1 [1, 3]. The vast majority of AD patients, however, suffer from the sporadic variety of the disease (sporadic AD) which is multifactorial in origin with aging being the most prominent risk factor. Pathologically, familial AD and sporadic AD do not differ from each other although the exact pathways of brain damage are not clear in either case [3, 7]. The characteristic pathological features of AD are the extracellular deposition of amyloid plaques, the intraneuronal formation of neurofibrillary tangles and the diffuse loss of neurons with degeneration of axons and dendrites affecting predominantly the brain regions like entorhinal cortex, hippocampus, parahip© 2013 Bentham Science Publishers

Oxidative Stress and Amyloid Beta Toxicity in Alzheimer’s Disease

pocampal gyrus, amygdala, frontal, temporal, parietal and occipital association cortices [7]. The amyloid plaques contain depositions of amyloid  peptide in the form of insoluble fibrils, while the neurofibrillary tangles are formed by paired helical filaments composed of hyperphosphorylated tau protein [8]. The typical AD pathology is, however, often complicated by various other features like the presence of vascular pathology characteristic of vascular dementia. Neurofibrilliary tangles occupy much of the perinuclear cytoplasm of neurons and consist of pairs of 10 nm filaments wound into helices (paired helical filaments) as observed under the electron microscope [3, 7, 8]. Paired helical filaments are composed of aggregated hyperphosphorylated forms of tau which is a microtubule-associated protein having multiple functions [3, 7, 8]. Amyloid plaques contain extracellular deposits of amyloid  protein (A42) which is a peptide of 42 amino acids derived from a larger precursor protein APP (amyloid precursor protein) by sequential cleavage by ß-secretase and -secretase [3, 7, 9]. The chain length of amyloid beta peptide can vary from 39 to 42 residues with A40 and A42 being the predominant species. The amyloid peptide is deposited in the form of insoluble fibrils where the peptide has a crossed  strand conformation which leads to the binding of specific dyes like Thioflavin T, Thioflavin S, Congo Red etc. [3, 7, 9]. Neurochemical alterations are also quite characteristics of AD brain, and the cholinergic neurons undergo severe degeneration in this disease. However, post-mortem examination reveals the loss of many other neurotransmitter systems such as GABA (gamma-aminobutyric acid), somatostatin, excitatory amino acid neurotransmitters etc., and the reduction in acetylcholine, dopamine and serotonin receptor levels in many brain areas [10-12]. A decreased glucose metabolism in the brain is an early feature of AD which can be monitored by PET (Positron Emission Tomography) study in vivo using 2-18 F- fluorodeoxyglucose [13]. ETIOPATHOGENESIS OF AD Familial Alzheimer's disease (FAD) is inherited in an autosomal dominant manner. FAD is diagnosed in families with multiple affected individuals with a mean age of onset before 65 years, but in many cases the disease appears in the fourth or the fifth decade of life. The diagnosis of FAD is confirmed by the identification of a disease causing mutation. Various mutations including sequence variation, deletion, partial or complete gene duplication have been identified in APP, PS1 and PS2 [7, 14, 15]. The FAD mutations in general lead to an overproduction of amyloid beta 42 or alter the profile of amyloid beta peptide formation with changes in the ratio of A42 and A40 or lead to the mistrafficking and mislocalization of amyloid precursor protein [16-19]. In sporadic AD, interplay of extra-genetic and genetic mechanisms also leads to an increased accumulation of A42 and apart from aging several risk factors like hyperhomocysteinemia, type 2 diabetes, the presence of APOE4 isoform, Al toxicity etc. have been implicated in this disease condition [1, 20, 21]. Amyloid Cascade Hypothesis The dominant hypothesis of AD pathogenesis revolves around the over-production of A42 from the precursor pro-

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tein, the polymerization of amyloid beta peptide to form soluble oligomers, protofibrils and finally to insoluble fibrils. The wide-spread and inter-dependent toxic actions of soluble oligomers on neurons and glia include mitochondrial dysfunction, oxidative stress, Ca2+ dysregulation, inflammatory reactions etc. and these together constitute the so-called ‘ amyloid cascade mechanism’[22, 23]. The overwhelming importance given to ‘amyloid cascade mechanism’ in explaining AD pathogenesis has resulted in a detailed study of APP biology including APP synthesis, processing and intracellular trafficking. APP and A Human APP gene, a 240kb stretch of DNA located on chromosome 21 consists of 18 exons, and alternate splicing gives rise to several mRNA species which are translated to proteins of different chain lengths which further undergo post-translational modifications [24]. Three principal varieties of APP with amino acid residues of 695, 751, 770 contain A peptide with APP 695 predominantly found in neurons [24, 25]. APP, class I integral membrane protein belongs to a family whose members are distributed in diverse species from C. elegans to mammals [24]. In mammals, three members of APP family, e.g. APP, APP like protein 1 (APLP1) and APP like protein 2 (APLP2) exist [24, 26]. The N-terminal portion of APP is projected extracellularly when the peptide is present on the plasma membrane with a short intracellular segment representing the C terminal end [27]. APP is also localized in several intracellular compartments like endoplasmic reticulum (ER), trans-Golgi network (TGN), endosomes, lysosomal vesicles, autophagosomes and possibly mitochondria [23]. The intracellular trafficking of APP from ER to the plasma membrane is still not clearly known, but APP probably follows the normal active secretory pathway of proteins after post-translational N- and Oglycosylation in ER and TGN respectively to reach the plasma membrane from where it is endocytosed again [28, 29]. The processing of APP in amyloidogenic and nonamyloidogenic pathways is now well-established and has been detailed in many published articles. Suffice it to say for our present purpose that, in the amyloidogenic pathway, APP is first cleaved at a -secretase site by the enzyme BACE (site APP cleaving enzyme) releasing a soluble -cleaved APP fragment (sAPP) and leaving a membrane-bound 99 amino acid C-terminal fragment (C 99) which is subsequently cleaved by a -secretase/presenilin complex within its intramembrane region to release the A peptide [9, 24]. In the non-amyloidogenic pathway, APP is processed by secretase and  secretase without producing A42. Although several forms of amyloid  peptide like A40, A42 etc. are present in AD brains, A 42 is considered as the most toxic species, and this protein has a high propensity for oligomerization and aggregation [22, 30, 31]. Although A42 is produced and released extracellularly from the plasma membrane bound APP, there is considerable evidence that the peptide is also produced intracellularly in different compartments of the secretory pathway and specially in TGN and endosomal vesicles [28, 32] Both  and  secretases have been identified in TGN and endosomes which also provide acidic pH for optimal  secretases activity [28, 32]. Apart

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from BACE1, cathepsin B has also been shown to possess  secretases activity and to contribute to intracellular generation of amyloid beta peptide within secretory vesicles [33]. The extra-cellular A42 peptide also can re-enter the cell via a diverse group of receptors like cholinergic nicotinic, NMDA (N-Methyl-D-aspartic acid), LRP1 (LDL receptorrelated protein 1), APOE receptor, RAGE (receptor for advanced glycation end products) etc. to produce an intracellular pool of A42 which is also contributed by A42 derived intracellularly from APP present in different membranous compartments [27, 34]. It is presumable that the intracellular pool of A42 like its extracellular counterpart, gives rise to different oligomeric forms, and the peptide in the monomeric or the oligomeric form gets localized to different organelles. However, the intracellular trafficking of A42 and especially how and where the peptide undergoes oligomerization within the cell and gains entry to different organelles are far from understood. Likewise, the relationship of intracellular and extracellular pools of A42 vis-a-vis the neurotoxic effects of A42 is not firmly established. The dynamics of these two pools in relation to clearance of the peptide from the brain or the enzymatic degradation of the peptide is also not clearly elucidated. However, many details about amyloid beta degradation by neprilysin, insulin degrading enzyme (IDE), endothelin converting enzyme (ECE), plasmin etc. have been revealed [35]. The clearance of A42 alone or in complexes with APOE or  2 - macroglobulin from the brain and CSF to blood involving LRP1 and the uptake of A42 in the brain from blood across the blood-brain barrier involving RAGE are also being investigated intensively could be important therapeutic targets in the near future [36, 37]. In comparison to amyloid beta peptide, the accumulation of phosphorylated tau in AD brain as a primary mechanism of pathogenesis is much less popular, although some studies have shown that the former causes microtubular disassembly resulting in dysfunction of axoplasmic flow, axonal transport and alterations in cytoskeletal structure and stability which are linked to AD pathogenesis [38, 39]. Several kinases like GSK3 (glycogen synthase kinase 3), cdk5 (cyclin dependent kinase 5), PKA, p38 kinase, MARK2(microtubule affinity regulating kinase 2), calcium calmodulin kinase 2 and check point kinase 2 are known to cause phosphorylation of tau while protein phosphatise 2A is responsible for dephosphorylation of this protein, but how these enzymes are involved in AD is not yet understood [38-40]. On the other hand, the central importance of APP and amyloid beta in AD pathogenesis is recognized from multiple lines of evidence. Although the clinical severity of AD does not correlate with amyloid plaque burden of the brain, recent studies have revealed that the soluble A42 oligomers are primarily responsible for the disease pathogenesis [22, 41]. The central importance of APP and amyloid beta peptide in AD pathology is borne by the post-mortem findings of the accumulation of A in AD brain or the FAD mutations giving rise to excess amyloid beta production and deposition. Moreover, diverse experimental results in transgenic animals or cell based models of AD and findings from in vitro experiments of amyloid beta toxicity on brain subcellular fractions have supported this. It is also interesting in this context that subjects with Downs’s syndrome (trisomy 21) possess an extracopy of APP gene, and they invariably develop AD by the fourth decade of life [42]. It is not known with certainty

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whether the molecular pathogenic mechanisms operative in FAD and sporadic AD are identical, but post-mortem findings in the brain of sporadic AD subjects reveal many features that are also observed in transgenic AD animals carrying single or multiple FAD mutations [43]. This obviously implies that both FAD and sporadic AD have some common triggers and underlying pathways. Apart from the accumulation of amyloid beta peptides and hyperphosphorylated tau proteins, the AD pathology forms a spectrum of abnormalities in the brain like microvascular alterations, hypometabolism of glucose, oxidative damage, structural and functional alterations of mitochondria, inflammatory reactions, calcium dysregulation, ER stress, synaptic alterations, altered functions of MAM (mitochondria associated endoplasmic reticulum membrane) and changes in the activities of various kinases [38, 40, 44-47, 16, 48, 49]. It appears rather improbable that all these diverse changes arise from amyloid beta accumulation as suggested by the ‘ amyloid cascade hypothesis’. On the contrary, it is quite logical to believe that a network of reinforcing and interdependent damage pathways triggered by genetic mutations in FAD or multiple risk factors including aging and environmental toxins in sporadic AD lead to gross neuronal degeneration and death in AD brain with features of apoptosis, necrosis and autophagy [50]. The various early and late triggers of sporadic AD have been explored in different experimental models or hypothesized from epidemiological or experimental data. Several attractive paradigms such as vascular insufficiency with cerebral hypoperfusion, oxidative stress with metabolic derangement, impaired insulin and insulin like growth factor signalling, early life epigenetic changes predisposing to late life AD (LEARn or Latent Early Life Associated Regulation model) have been forwarded to explain the genesis of sporadic AD.[47, 48, 51-54]. The detailed discussion of these pathogenic mechanisms are not in the purview of this article, and we will restrict ourselves mostly to a systematic dissection of the complex relationship of oxidative stress and AD. OXIDATIVE STRESS AND AD The role of oxidative stress in the genesis and progression of AD is complex and not fully understood, but it is worthwhile to dissect out this multi-faceted relationship systematically to understand the disease process and the therapeutic potential of antioxidants in AD. There is ample evidence of an accumulation of oxidative damage markers in post-mortem AD brain and in the brains of AD transgenic mice with some studies showing also an improvement in amyloid pathology in the latter after anti-oxidant treatment [45, 55-58]. On the other hand, the clinical evidence of improvement in AD patients after anti-oxidant treatment is generally not convincing [59]. Thus, the issue of oxidative stress has remained contentious in the context of AD pathogenesis and the present review will summarise the available facts and the opposing views in this regard and thereafter discuss the prospects and the problems of anti-oxidant supplementation therapy in AD. Oxidative Stress Oxidative stress is often defined as an imbalance between reactive oxygen species (ROS) formation and its removal by

Oxidative Stress and Amyloid Beta Toxicity in Alzheimer’s Disease

the anti-oxidative systems existing in the cell leading to accumulation of toxic oxy-radicals with indiscriminate damage to various biomolecules and subcellular organelles, but by itself this definition is rather incomplete and not particularly revealing either. It may be more meaningful to think that a complex network of reactions exists within the cell as a part of normal metabolism under physiological conditions which comprises of both enzymatic and non-enzymatic free radical reactions and involves ROS, reactive nitrogen species (RNS), carbon-centred radicals, various lipid and protein derived radicals and transition metals [60, 61]. The ROS constitute the most prominent group of this system and comprises of both radical and non-radical members like O2.- (superoxide), H2O2, hydroxyl radical (.OH) and singlet oxygen. Moreover, many growth factors (EGF - Epidermal growth factor, PDGF - platelet-derived growth factor, VEGF - Vascular endothelial growth factor, etc.), cytokines (IL1, IL6, TNF- etc.) and hormones (insulin, angiotensin etc.) during physiological cell signalling give rise to ROS formation usually through NADPH oxidase reaction [60, 62]. The radicalgenerating reactions are counterbalanced by an array of antioxidants, both enzymic and non-enzymic, resulting in a low level of intracellular ROS and other free radicals which under physiological conditions take part in cell signalling pathways and eventually modulate the gene expression patterns through activation of various cell signalling stress kinases like ERK (Extracellular signal-regulated kinases), JNK (c-Jun N-terminal kinases), p38 etc. or Src (Protooncogene tyrosine-protein kinase) family of kinases or redox-sensitive transcription factors like NF- (Nuclear Factor-Kappa ), AP-1 (activator protein 1), Nrf 1 (nuclear factor (erythroid-derived 2)-like 1), Nrf 2 (nuclear factor (erythroid-derived 2)-like 2) etc. [60, 62, 63]. This results in the induction of various antioxidant and detoxification enzyme genes or cytoprotective genes or genes responsible for growth, differentiation, inflammation and apoptosis [62, 64]. Under pathological conditions, however, ROS (reactive oxygen species) and other reactive radicals may accumulate in the cells through overproduction as well as defective removal by the anti-oxidants causing not only the alterations in signalling pathways but also a widespread direct damage to subcellular components [61, 62, 65]. The varied pathways of such direct oxidative damage include membrane lipid peroxidation, protein and DNA oxidation and affect enzyme activities, functions of ion channels and receptors, permeability and fluidity of biomembranes as also genomic structure and stability leading to widespread derangement of cellular functions [61, 65, 66]. It is a distinct possibility that in the pathological scenario initially the oxidative stress through redox-signalling pathways induce stress-responsive and cytoprotective genes to produce the so-called ‘hormetic’ effect, but in the subsequent phase the latter effect is overshadowed by the direct radical damage to important biomolecules and subcellular components setting in cell death programmes like apoptosis and necrosis [61, 67]. There are other twists in this scheme of things; for example, the activation of NF- by ROS is generally thought to produce the beneficial ‘hormetic’ effect, but it also leads to induction of pro-inflammatory cytokine genes like IL-1, IL-6, TNF- etc. which can promote tissue damage [60, 62]. Likewise, a moderate degree of oxidative stress may lead to activation and nuclear translocation of NF-, Nrf2 or AP1, but a

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higher load of ROS may inactivate them by interfering with their DNA binding or dimerization or protein-protein interactions [60, 62, 67]. Many excellent reviews have described the intracellular origin of ROS and various processes related to oxidative stress in considerable details, which will not be repeated here, but a schematic summary is presented (Figs. 1, 2 & 3) to highlight the salient aspects before we proceed to discuss the role of ROS in brain especially in the context of AD pathogenesis. Oxidative Stress in the Brain and CNS Oxidative injury has been linked with various acute and chronic diseases of the brain and CNS. The vulnerability of the brain to oxidative stress stems from various reasons such as an increased oxygen consumption compared to other tissues, an abundance of oxidizable substrates like polyunsaturated fatty acids and catecholamines, an enrichment of several brain areas with transition metals and a deficient antioxidant defence system [61, 68]. The brain utilizes nearly 20% of total body oxygen composition because of its high demand for ATP to maintain intracellular ionic homeostasis, and a part of it roughly about 0.2 % goes to produce ROS at different respiratory chain complexes. The production of ROS in vitro from brain mitochondria in the presence of various respiratory substrates and inhibitors have been convincingly demonstrated and quantitated, although it is virtually impossible to estimate the in vivo rate of ROS production from mitochondria [69-71]. Although complex I and complex III of the respiratory chain are generally considered as the most important sites of ROS production, various other enzyme complexes like pyruvate dehydrogenase complex, ketoglutarate dehydrogenase, dihydroorotate dehydrogenase, monoamine oxidase, -glycerophosphate dehydrogenase, cytochrome b5 reductase etc. also contribute substantially to mitochondrial ROS production [72]. The CNS also has high NADPH oxidase activity which is another important source of intracellular ROS [73]. The multi-subunit NOX family of NADPH oxidase, originally associated with respiratory bursts of neutrophils, has since been identified in many tissues where it takes part in a myriad of physiological functions like host defence, immune response, cell proliferation, cell senescence, oxygen sensing etc. [73, 74]. In brain NADPH oxidase (NOX) is present in astrocytes, microglia and possibly in neurons predominantly as NOX 2, but smaller amounts of other isoforms like NOX4 and NOX5 also exist [73, 74]. There is possibly a regional variation in NOX2 distribution in the brain, which may also be species dependent, but this aspect needs more elaborate studies [73, 74]. NOX2 isoform remains in association with membranebound p22 phox and requires interactions with a number of cytosolic proteins like p40phox, p47phox and p67phox before becoming catalytically active and brain tissue is shown to be immunoreactive for all these proteins [73, 74]. In a variety of pathological conditions like ischemia, hypoglycaemia, inflammation, neurodegenerative diseases as also aging overproduction of ROS occurs in the brain which includes increased mitochondrial production of ROS, as well as an augmented activity of NADPH oxidase [70, 74-77]. The neuronal membrane is highly enriched in polyunsaturated fatty acids especially eicosapentaenoic and docosa hexaenoic acids which are highly vulnerable to free radical

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Fig. (1). Intracellular sources of ROS. NOS, nitric oxide synthase; NOX, NADPH oxidase, KDGH,-ketoglutarate dehydrogenase; PDH, pyruvate dehydrogenase; Asc, ascorbate; ETC, electron transport chain; MAO, monoamine oxidase; cyt b5 red; cytochrome b5 reductase; cyt P450; cytochrome P450.

Fig. (2). ROS mediated damage to lipid, protein and DNA.

Fig. (3). Redox-signalling pathways: a simplified scheme.

Oxidative Stress and Amyloid Beta Toxicity in Alzheimer’s Disease

attack by ROS [68]. The brain homogenate in vitro readily undergoes lipid peroxidation and protein oxidation especially in the presence of added transition metals, and the in vivo levels of lipid peroxidation end products like malondialdehyde (MDA) and 4-hydroxy nonenal (4-HNE) or the protein oxidation markers are elevated in the brain following ischemic injuries and in aging and neurodegenerative disorders [45, 77-80]. The transition metals like Fe2+, Cu+ etc. which are essential for normal metabolism and are important components of many enzymes and proteins, can, however, take part in oxidative damage reactions by catalyzing the formation of initiating free radicals like ROS, the breakdown of alkoxy and peroxy radicals, lipid hydroperoxides etc. during lipid peroxidation and the generation of protein derived free radicals (Figs. 1 & 2) [61, 78]. Several regions of the brain like the caudate nucleus, putamen, substantia nigra, globus pallidus are enriched in iron which remains as a ‘labile form’ (Fe2+) taking part in free radical reactions, and also in the redox-inactive (Fe3+) form in association with an iron storage protein ‘ferritin’ [61, 81]. However, iron can be released from the ferritin store in the redox active form by ascorbate, reduced glutathione, superoxide radical etc. to promote oxidative damage in the brain, and it is indeed interesting to note that iron enriched regions of the brain are most vulnerable to neurodegenerative diseases [61, 81, 82]. The brain content of ascorbate (2-3 mM) is much higher than that of plasma (0.05 mM), being taken up actively at the bloodbrain barrier across the epithelium of the choroid plexus to reach a concentration of 0.2 - 0.4 mM in rat cerebrospinal fluid or CSF (approximately 0.16 mM in human CSF) and brain interstitial fluid, and thereafter ascorbate is transported in brain cells against a stiff concentration gradient by active transport by means of sodium dependent vitamin C transporter type 2 [83]. Ascorbate is an important cofactor for several enzymes including dopamine  hydroxylase, which is required for the biosynthesis of epinephrine and norepinephrine, and it also exhibits strong anti-oxidant functions both in vitro and in vivo, but in the presence of transition metals ascorbate can act as a pro-oxidant by redox recycling of Fe3+ or Cu2+ [61, 78]. It is difficult to predict whether ascorbate in vivo acts primarily as a pro-oxidant or an anti-oxidant especially in pathological conditions, but its presence in the brain in high concentration is intriguing.The other reasons for increased vulnerability of the brain to oxidative stress include the availability of catecholamines with a propensity for autoxidation. These catecholamines are catabolized by MAO-A and MAO-B generating H2O2, and they can also undergo autoxidation to produce O2 .- radicals. Dopamine autoxidation in particular generates both superoxide radicals and toxic quinones which have been linked with the dopaminergic neuronal death in Parkinson’s disease [84, 85]. The excitatory neurotransmitter glutamate, which is used by nearly 40% of all CNS synapses, can lead to excitotoxic death of neurons and glia through activation of NMDA receptors primarily but also of AMPA (2-amino-3-(3-hydroxy5-methyl-isoxazol-4-yl)propanoic acid), and metabotropic glutamate receptors leading to Ca2+ influx from outside as well as mobilization of the ion from the intracellular stores [86]. The rise in intracellular Ca2+ leads to a prolonged increase in superoxide radicals and NO production apart from producing other deleterious effects [68, 86]. Excitotoxic damage is implicated in several acute conditions like an epi-

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leptic seizure, ischemic and traumatic brain injury as also in neurodegenerative disorders including AD and PD (Parkinson’s disease) [86, 87]. The role of glia and proinflammatory cytokines in eliciting oxidative stress in the brain has been studied intensively in the context of several pathological states. Activated microglia cells in the brain are one of the most robust source of free radicals such as superoxide, nitric oxide, hydrogen peroxide and peroxynitrite [88, 89]. It also should be noted that microglial have efficient antioxidative defense mechanisms in the form of high concentrations of glutathione and the various antioxidative enzymes like SOD, catalase, glutathione peroxidase and glutathione reductase, as well as NADPH-regenerating enzymes [90]. The production of ROS in phagocytes is derived from peroxidases inside the cell, NADPH oxidase on the membrane surface and the mitochondrial respiratory chain. NADPH oxidase activation and subsequent ROS dependent signaling involving various downstream molecules such as protein kinase C, MAPK (Mitogen-activated protein kinases) and NF- etc. form the basis of proinflammatory response by the microglia triggering changes in morphology, upregulation of genes for proinflammatory cytokines like IL-1, IL-6 and TNF-, activation of phagocytosis and overexpression of immune response related surface markers of microglia. Microglial activation has been implicated in neurotoxicity through extracellular release of ROS, NO (nitric oxide) and proinflammatory cytokines in various pathological conditions [91, 92]. In the context of vulnerability of the brain to oxidative stress, it is important to realize that neurons in several regions are more vulnerable than those in other regions, and specially neurons in CA 1 (cortical area 1) of hippocampus, cerebellar granule cells and dopaminergic neurons of the substantia nigra pars compacta are particularly susceptible to oxidative injury. The phenomenon has been ascribed to a combination of factors like high basal level of oxidative stress, low ATP production, mitochondrial dysfunction, low intracellular calcium buffering, deficient DNA repair etc., and interestingly these susceptible neurons also exhibit a high transcriptional activity of redox - responsive genes [93]. Evidence of Oxidative Damage in Brain of AD Many different studies have provided evidence implicating oxidative stress as a major pathogenic mechanism in AD [45, 58]. Oxidative damage involves all classes of organic molecules and excessive lipid peroxidation, protein oxidation, DNA and RNA oxidation and glycooxidation have all been documented in AD brains [94-96]. The evidence of increased lipid peroxidation is seen most prominently in the regions of AD brain with most severe degenerative changes [55, 97]. The peroxidation of polyunsaturated fatty acids produces several aldehydes including 4-hydroxynonenal (HNE) and acrolein which are reactive cytotoxic compounds and can cause death of cultured primary hippocampal neuron [98]. Acrolein and 4-hydroxynonenal levels are increased in CSF and in the post-mortem brain samples of AD patients and HNE - adducts are present in NFTs (neurofibrillary tangles) and senile plaques [99-101]. The F2-isoprostanes are prostaglandin - like compounds derived from free radicalinduced oxidation of arachidonic acid and elevated levels of F2 - isoprostanes are seen in postmortem ventricular CSF of AD subjects [102].

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The typical damage marker of protein oxidation such as protein carbonyls, nitrotyrosine, HNE - protein adducts etc. accumulate in AD brain [45, 103]. Immunohistochemistry, immunoblotting, 2D - gel electrophoresis, mass spectrometry have all been used very successfully to demonstrate protein oxidation in AD brain, and several enzymes like glutamine synthetase, creatine kinase, triose phosphate isomerase, enolase etc. and some other proteins like -actin, DRP 2 (dystrophin related protein 2) etc. are oxidatively modified in AD brain compared to that in age-matched control [45, 103, 104]. Pathological aggregation of proteins into insoluble fibrils is a characteristic of AD. Oxidative modifications can cause crosslinking of proteins by covalent bonds leading to aggregation, fibril formation and insolubility. Oxidatively modified A42 is found in senile plaques, while NFT contain nitrotyrosine, protein carbonyls and HNE-protein adducts [58, 105, 106]. The importance of oxidative changes in NFT formation gains support by the presence of active aldehydes like HNE, acrolein etc. as well as the advanced glycation end products (AGE), and hemeoxygenase-1 (an antioxidant enzyme) in these lesions [106, 107]. Oxidation of DNA can produce single or double-stranded breaks, sister chromatid exchange, DNA-protein crosslinking, and base modifications. Several studies demonstrate an increase in oxidative DNA damage in the brains of subjects with AD [108, 109]. The most common DNA damage marker described is 8-hydroxy deoxyguanosine (8-OHdG), which is increased in nuclear and more prominently in mitochondrial DNA [109]. Elevations of 5-hydroxyuracil, 8hydroxyadenine, 5-hydroxycytosine and other modified bases also have been observed in the brain in AD subjects [110]. Advanced glycation end products are post-translational modifications of proteins that are formed when the amino groups of lysine or arginine react nonenzymatically with monosaccharides, and these may play a role in AD that is linked to oxidative modifications of  amyloid peptides and tau [96, 111, 112]. Advanced glycation end products (AGE) are present in senile plaques and NFTs in subjects with AD, and AGE - modified  amyloid peptides tend to aggregate in protease-resistant deposits [96, 112, 113]. The receptor for advanced glycation end products (RAGE) has been demonstrated to play a central role in the pathogenic mechanisms of AD [114]. Increased RAGE expression is shown to be present on microglia and neurons of the hippocampus, entorhinal cortex, and superior frontal gyrus of AD brain [115]. RAGE, a multi-ligand receptor in the Ig superfamily functions as a cell surface binding site for A and mediates the toxicity of the latter [114]. The binding of A to RAGE generates ROS through activation of NADPH oxidase and also leads to activation of ERK and phospholipase A2 [116]. Source of Oxidative Stress in AD Brain Although the enhanced oxidative stress as a pathogenic mechanism is well recognized in AD, the underlying mechanisms creating a pro-oxidative milieu in the disease affected brain are less clear. Several possibilities, however, can be envisaged. First and foremost is the dysregulation of transition metal metabolism in AD brain. Secondly, the enhanced production of ROS in mitochondria or by NADPH oxidase

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in the aged brain may provide the pro-oxidative trigger for AD pathogenesis. Thirdly, the pro-oxidative action of amyloid beta peptide may be the predominant mechanism of oxidative stress in AD brain. Metal Dysregulation An excess accumulation of transition metals like Fe, Cu and Zn occurs in AD brain. The transition metals like Fe and Cu in the redox-active form can give rise to ROS like O2.- or OH. or else can take part in radical propagation reactions of lipid and protein oxidation as indicated earlier. Although the role of Cu+ in inducing oxidative stress in AD brain has been highlighted in some studies, iron appears to be the most important metal in this regard [81, 82, 117, 118]. An increased accumulation of iron occurs in and around senile plaques and neurofibrillary tangles in AD brain and senile plaques of Tg2576 transgenic AD mice [82, 118, 119]. The neuropil in AD brain is also enriched in iron which is particularly more in regions such as the hippocampus and cerebral cortex etc. A variety of biochemical, histochemical and spectroscopic methods has been used to demonstrate iron accumulation in post-mortem AD brain [82, 118]. MRI has been used to show the increased accrual of iron in the brain of AD transgenic mice in vivo [120]. The entry of Fe3+ in the brain is regulated by the BBB, (Blood Brain Barrier) and a major part of the iron uptake in the brain occurs at brain vascular endothelial cells (BVEC) through transferrin dependent receptor mediated as well as transferrin independent vesicular and non-vesicular pathways [121]. The latter pathways may involve other iron transport proteins like lactoferrin, melanotransferrin, DMLT 1 (Divalent Metal Transporter 1) etc. [121, 122]. The migration of iron through BVEC, its escape at the abluminal surface of the endothelial cells in to interstitial fluid of the brain, the uptake of the metal by neurons and glia, storage of iron within these cells, removal of excess iron into CSF and final clearance in to blood through CSF choroid plexus interphase require a variety of proteins other than transferrin and transferrin receptors [121, 122]. These proteins include several transport proteins like DMLT 1 and ferroportin, enzymes like ceruloplasmin (converts Fe2+ to Fe3), Steap 3 (converts Fe3+ to Fe2+) and Hephaestin (converts Fe2+ to Fe3+), and storage protein like ferritin [121123]. The levels of some of these proteins involved in iron metabolism are regulated by IRP (Iron Regulatory Protein) which by binding to IRE (Iron Responsive Element) present at 5’-UTR or 3’-UTR of mRNA causes a translational inhibition or increased mRNA stability respectively [122, 123]. It is not clear how in AD brain these proteins are affected to bring about iron de-homeostasis and consequent oxidative stress, but limited information so far available indicate that alterations in the levels and mislocalizations of several such proteins such as transferrin, transferrin receptor, ferritin, ceruloplasmin, lactoferrin, melanotransferrin etc. take place in the brain of AD subjects [82, 122-125]. An abnormal accumulation of IRP2 has also been shown in senile plaques and also an increased binding affinity of IRP to IRE, which may result in decreased ferritin and increased transferrin availability in the brain, have been implicated in AD [82, 122, 123]. Another link between AD and iron de-homeostasis is indicated by the presence of HFE (Human hemochromatosis protein) mutations, responsible for inherited disorder called

Oxidative Stress and Amyloid Beta Toxicity in Alzheimer’s Disease

hemochromatosis, in the former disease state [119]. Thus, one source of oxidative stress in AD brain could be the increased availability of labile Fe pool from an altered metabolism and transport of iron. ROS from the Aging Brain The aging of the brain is accompanied by the accumulation of oxidative damage markers of phospholipid, protein and DNA and oxidative stress is recognized as a major contributor to the biochemical and functional deficits of the aged brain in various species [61, 126]. It has been further shown that enhanced mitochondrial production of ROS occurs in the aged brain which also has a high level of NADPH oxidase activity [75]. Additionally, an increased accumulation of transition metal like iron occurs during brain aging in areas like putamen, substantia nigra, globus pallidus, caudate nucleus etc. [61, 119]. The activation of microglia which is a typical characteristic of the aging brain also leads to an increased release of ROS and pro-inflammatory cytokines from the former [88, 89, 127, 128]. The activation of microglia is associated with marked morphological changes, upregulation of surface complement receptors and MHC (major histocompatibility complex) molecules and the extracellular release of ROS from activation of NADPH oxidase. On the other hand, the activation of NADPH oxidase also leads to an increased intracellular level of hydroxyl radicals, H2O2 and lipid derived radicals within microglia which turn on the expression of pro-inflammatory cytokine genes [127]. The pro-inflammatory cytokines like IL1, IL6 and TNF- in turn can induce further production of ROS [60]. There are also variable reports of a diminished anti-oxidant defence in the aging brain through a decrease in antioxidant enzymes like SOD, glutathione peroxidase and catalase as also a lowering of reduced glutathione level [80, 129, 130]. Thus, the aged brain provides a pro-oxidative backdrop for the genesis of AD pathology. Pro-oxidative Property of A42 The pro-oxidative function of A42 has been reported in numerous studies, in animal and cell based models of AD, as well as in various in vitro experiments with brain subcellular fractions. Under such conditions, the amyloid beta peptide has been shown to increase lipid and protein oxidation, and some of the toxic actions of amyloid beta peptide are ascribed to this pro-oxidative property [131-134]. In brief, the amyloid beta peptide is thought to bind transition metals like Fe3+ or Cu2+ in a redox-active state. The detailed coordination chemistry of metal binding to A42 is still not completely established, but requires three histidine residues (His 6, His 13, His14), and another unidentified ligand [135137]. The peptide bound metal ion initiates redox-cycling reactions, which probably require the involvement of a methionine residue (Met 35) of the peptide or the presence of an exogenous reducing agent in the medium such as ascorbate or dopamine, generating ROS [136-139]. The reactive free radicals of oxygen in turn not only causes damage to various tissue components, but also oxidatively modifies the peptide leading to changes in its aggregation properties and toxicity [140, 141]. Other important mechanisms operative in the pro-oxidative functions of A42 are the microglial activa-

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tion, mitochondrial dysfunction or calcium dysregulation caused by the peptide in vivo [142-144]. THE CONTROVERSY: AN ALTERNATIVE HYPOTHESIS The description so far presented highlight two important aspects of AD pathology such as oxidative stress and amyloid beta peptide accumulation, but it fails to address an important question whether the oxidative stress is the trigger for or the consequence of the altered amyloid beta metabolism and accumulation in AD brain. This issue is not superfluous and indeed it could be a determining factor in searching for the therapeutic targets and understanding the disease progression. There is now clear evidence of a temporal separation between oxidative stress and A42 accumulation in the AD brain. The post-mortem evidence is indicative that the oxidative damage occurs more in the early phase of AD, but is diminished as the deposition of amyloid peptide builds up [145]. In different transgenic AD models, the evidence of lipid or protein oxidation appears in the brain much before any significant accumulation or deposition of amyloid beta occurs [146]. The subjects of Down’s syndrome show evidence of oxidative stress in the brain as an accumulation of 8-hydroxy guanosine (8-OHG), the oxidation product of RNA, in the early part of life well before the deposition of amyloid beta peptide which takes place much later after the age of 30 years [147]. Moreover, in such cases the depositions of A42 is associated with a diminished level of 8OHG [147]. Studies with patients of MCI (mild cognitive impairment) also indicate that the oxidative stress is an early event in AD pathogenesis [148]. Several cell based studies have demonstrated that the induction of oxidative stress leads to increased production of A42 through APP overexpression and increased activity of  secretase [149, 150]. A decreased level of neprilysin, an important A42 degrading peptidase, is also noticed in human neuroblastoma cell lines and primary culture of rat cortical neurons following an oxidative insult [151]. Thus, the possibility of oxidative stress acting as the initial trigger for altered amyloid metabolism and accumulation of A42 is quite compelling. Several studies in contrast to the existing view have shown that A42 behaves as an anti-oxidant in cell free as well as biological systems providing protection to cultured cells or incubated brain mitochondria or intact animals exposed to oxidative injury [152-157]. The antioxidative function of A42 is presumably related to its metal-chelating property and whatever has so far been stated about transition metals and oxidative stress in the context of AD pathogenesis, it is plausible, though not confirmed, that the increased synthesis and accumulation of A42 in AD brain is a cellular defence against persistent oxidative stress and metal de-homeostasis in this disease condition [157]. However, in the long run this cellular defence leads to an array of toxic events mediated by oligomers of accumulated A42 culminating in neuronal death and degeneration. Other kinds of adaptive changes such as the activation of stress-responsive kinases may also occur in the face of persisting oxidative stress taking place early in the disease process, and this may usher in AD pathology subsequently [107]. The use of antioxidants and metal-chelators in the treatment of AD would be discussed in the following section in the light of this model.

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THERAPEUTIC INTERVENTION IN AD The complexity of AD pathogenesis and the inadequate understanding of the molecular mechanisms have led to different types of drug treatment strategies which have been tested in animal and cell -based models and in clinical trials. However, the treatment of AD in general is terribly inadequate at present, and the disease progresses relentlessly with devastating failure of memory and cognition till the patient succumbs to the illness usually by 5- 9 years after the diagnosis. A major focus of the drug treatment for AD is to improve cognitive abilities, such as memory and thinking and slow the progression of these symptoms. Four drugs are currently approved by the U.S. Food and Drug Administration (FDA) for treating cognitive symptoms of AD. Three of them (Galantamine, Rivastigmine, Donepezil) act as anticholinesterase agents while Memantine acts by preventing excitatory neuronal damage [158]. In general Galantamine, Rivastigmine and Donepezil hydrochloride are most effective at early stages while memantine hydrochloride is effective for the later stages of the disease. Many other drugs have shown promising beneficial effects in AD models or experimental systems and are in the different phases of clinical trial, but quite a few of them are already rejected in the process [158-160]. In general several strategies have been evolved to halt the progression of the disease such as increasing the clearance of amyloid beta peptide from the brain by active or passive immunization against the peptide or promoting enzymatic degradation of the peptide, diminishing the synthesis of A42 by inhibition of  - secretases or  secretases, activation of non-amyloidogenic processing of APP through modulation of  - secretase action, preventing the aggregation and fibrilization of A42, inhibiting the phosphorylation of tau, preventing inflammation or oxidative stress or excitotoxicity and so on [159, 160]. Cathepsin B inhibitors could be potential therapeutic candidates for AD and have been shown to reduce amyloid beta load and to improve learning and memory impairments in transgenic AD mice [161]. Several compounds like melatonin, estrogens, statins, monoamine oxidase inhibitor etc. have exhibited potential beneficial effects in AD animal models and also in clinical trials with AD subjects [162-164]. The tryptophan derivative from the pineal gland melatonin which regulates sleep - wake rhythm of animals and human beings is grossly diminished in AD brain compared to age-matched controls and AD patients suffer from severe sleep deprivation with altered sleep - wake cycles [162, 165]. These findings have led to clinical trials with melatonin for amelioration of AD symptoms, and several reports have claimed improvements in sleep disturbance and cognitive decline in AD by melatonin treatment [165]. However, a recent paper has reviewed a large number of clinical studies using melatonin and concluded that the efficacy of melatonin as a potential drug against AD needs to be further evaluated in larger randomized studies [166]. Estrogens have antioxidative functions, but also numerous other actions on the brain and neural tissue including neurotrophic, neuroprotective, antiamyloidogenic and anti-apoptotic actions, and in population based studies hormone replacement therapy in women with estrogen reduces the risk of AD depending on the timing of initiation of the replacement therapy and its duration [163, 167]. Although estrogen replacement therapy is generally

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considered to reduce the risk of AD in women based on epidemiological data, several randomized control clinical trials have shown no beneficial effects of estrogen in AD [168170]. Since this review specifically deals with the relationship of AD and oxidative stress, we would restrict ourselves in analyzing the prospects and problems of antioxidant therapy for AD. In this context, it will be worthwhile to mention the protective action of antioxidant supplementation against biochemical and physiological alterations associated with normal brain aging, because of many commonalities between the latter and the AD pathogenesis [171, 172]. Numerous attempts have been made to halt the oxidative damage, mitochondrial dysfunction and proinflammatory state in the aged brain in normal animals as well as in senescence accelerated mice by antioxidant supplementation with varying degree of success [75, 172, 173]. Different treatment protocols and various antioxidants such as vitamin E or a combination of acetyl L- carnitine and -lipoic acid or N-acetyl cysteine with  - tocopherol plus -lipoic acid or plant derived products or even crude preparations from plants or fruits have been used [75, 172, 173]. In many such cases, the antioxidant supplementation has provided some significant protection against age-related cognitive decline [171-174]. ANTIOXIDANTS AND AD Many studies with experimental models of AD and small scale clinical trials have revealed that various antioxidants like lipoic acid, ubiquinones (coenzyme Q10), vitamins A, C, E, N-acetylcysteine, green tea polyphenols and Ginko biloba extract could be potentially beneficial against AD pathogenesis. Several animal models of AD such as APP transgenic mice, PS1 transgenic mice or tau transgenic mice or transgenic double mutants or pharmacological models show strong behavioral deficits when tested in Morris water maze, open field, radial arm maze, operant bar pressing, and visual object recognition tasks as compared to age-matched controls [175, 176]. These behavior alterations correlate with the development of amyloid plaques and with impaired longterm potentiation in both the CA 1 and dentate gyrus regions of the hippocampus and such models have been extensively used for testing the beneficial effects of antioxidants against AD pathogenesis. The other experimental models used are primary cultures of cortical or hippocampal neurons or cultured cell lines of neural origin exposed to toxic actions of amyloid beta peptide. The end points of testing in animal models have generally been the improvement of memory and learning tasks with or without a decrease in amyloid beta accumulation or tau phosphorylation in the brain, while in cell based models, a decrease in neurodegenerative parameters is considered as positive. In experimental studies vitamin E has been shown to attenuate protein oxidation, ROS formation and neurotoxicity mediated by amyloid beta peptide in primary culture of hippocampal neurons and PC12 cells [177, 178]. In experimental animals toxicity of injected amyloid beta to hippocampus has been shown to be prevented by vitaminE, while in tau transgenic mice, the latter also delays the development of neurodegenerative pathology and attenuates the motor weakness [179, 180]. Early supplementation with vitamin E

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shows a significant reduction in amyloid beta levels and amyloid deposition in Tg 2576 AD transgenic mice [181]. Further, vitamin E in combination with anti inflammatory agent indomethacin can suppress the inflammatory response and oxidative stress with a significant reduction of amyloid beta accumulation in the hippocampus and neocortex in transgenic AD mice [182]. In patients with moderately severe AD, the treatment with - tocopherol slows the progression of the disease and delays the functional disability [183]. Vitamin C is a water-soluble antioxidant which is also a powerful inhibitor of lipid peroxidation and acts as a major defense against free radicals in whole blood and plasma. Vitamin C reduces -tocopheroxyl radicals rapidly in membranes regenerating -tocopherol and inhibiting tocopheroxyl mediated propagation of radical reactions [61]. A population based study has shown that the use of vitamin E and vitamin C in combination reduces the prevalence and the incidence of AD [184]. Several studies have shown that lipoic acid or dihydrolipoic acid protects against amyloid beta or H2O2 mediated toxicity on primary cultures of cortical or hippocampal neurons [185, 186]. Lipoic acid and its reduced form are also known to inhibit oligomerization of amyloid beta peptide in vitro [187]. In transgenic AD mice, lipoic acid has been shown to prevent oxidative damage but without any decrease in  amyloid load or any improvement in Y maze performance [188]. On the other hand, another study has reported that chronic dietary supplementation of lipoic acid improves memory and learning tasks on Morris water maze of Tg2576 transgenic AD mice, but does not affect amyloid beta deposition or the accumulation of oxidative damage markers in the brain of the latter [189]. An initial small study with AD patients has shown that the administration of 600 mg of lipoic acid daily for 337 days leads to a stabilization of cognitive functions compared to patients without treatment or on anticholinesterase therapy [190]. Mitochondria targeted antioxidant like MitoQ prevents amyloid beta toxicity in mouse cortical neurons in cell culture and in a triple transgenic mouse model of AD, the drug prevents amyloid beta accumulation, astrogliosis, loss of synapses and oxidative stress in the brain and further ameliorates cognitive decline [191]. Another mitochondria-targeted plastoquinone antioxidant has been shown to prevent the A42 induced impairment of hippocampal long term potentiation in rats [192]. In mouse neuroblastoma cells, mitochondria-targeted antioxidants prevent amyloid beta induced mitochondrial structural and functional anomalies and also significantly improve neurite outgrowth [193]. Likewise, SS31, a mitochondria-targeted peptide antioxidant prevents many of the abnormalities of mitochondrial structure and dynamics seen in primary culture of neurons from transgenic AD mouse brain [194]. N-acetylcysteine, a potent antioxidant widely used in experimental studies prevents the learning and memory deficits of mice injected intracerebroventricularly with aggregated amyloid beta peptide and also reverses the increased malondialdehyde level and decreased levels of reduced glutathione, acetylcholine and choline acetyltransferase in the brain of these animals [195]. In APP / PS1 mice, N-acetyl cysteine (NAC) supplementation prevents the loss of glutathione peroxidase and glutathione reductase activitise and also reverses the increase in lipid peroxidation and protein oxidation in the brain [196]. Proteomic analysis reveals that,in APP/PS1 transgenic double mutant,

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the expressions of many proteins involved in energy-related pathways, excitotoxicity, cell cycle signalling, synaptic functions are altered as a result of A deposition and oxidative stress and these phenomena are prevented by long-term supplementation with NAC [197]. Senescence accelerated mice (SAMP8) overexpress APP resulting in elevated levels of A in the brain along with oxidative damage which may contribute to the memory and behavioral deficits, and the administration of NAC to such animals improves cognition in the T maze [173, 198]. Another group has demonstrated that a cocktail of antioxidants containing NAC, -lipoic acid, curcumin, epigallocatechin gallate, B vitamins, folate, ascorbic acid, piperine improves learning and memory in Tg2576 mice with significant decreases in soluble amyloid beta oligomer levels [199]. Melatonin inhibits A-induced microglial ROS production, and it improves learning and memory deficits and decreases oxidative damage and A burden in APP transgenic mice [200, 201]. Melatonin has potent antioxidant and radical-scavencing properties which are supplemented by its ability to prevent amyloid fibril formation and to inhibit apoptosis and in different AD models melatonin has exhibited neuroprotective functions [162]. Long-term supplementation with more potent antioxidants like tocopherol, -lipoic acid, vitamin C and acetyl L- carnitine along with behavioral enrichment has been shown to reduce amyloid plaque burden and the accumulation of A42 and other other peptides derived from APP in brain as also to improve behavioral and cognitive tests in aged canine [202]. Some herbal products such as the alkaloids from coptidis rhizoma may have potential anti-AD effects through multiple mechanisms including antioxidant properties [203]. Likewise, silibinin, a flavonoid derived from the herb milk thistle has been shown to have antioxidative properties and to prevent memory impairment and oxidative damage induced by A in mice [204]. Ginkgo biloba which is rich in flavonoid antioxidants shows cognitive improvement in Tg2576 mice but without any effect on brain A levels or senile plaque size or oxidative protein damage [205]. Caffeine, on the other hand, decreases A accumulation, phosphorylation of tau and oxidative stress in cholesterol - fed rabbit model of AD [206]. Long-term oral caffeine administration reduces soluble and insoluble amyloid beta burden in the brain transgenic AD mice, but it is unclear if it is related to its antioxidant property of caffeine [207]. Several compounds present in green tea, spices and other food additives like I- theanine, rosamarinic acid, nordihydroguiaretic acid have strong antioxidant properties [208-211]. These compounds prevent toxic effects of amyloid beta protein on cell lines or primary neuronal culture, and also in experimental animals the former attenuates neurodegeneration and oxidative protein or lipid damage in the brain after intracerebroventricular injection of A42 or other amyloid peptides [208-211]. The involvement of transition metals Fe2+ or Cu+ in oxidative damage in the brain has already been highlighted in this review, and especially it has been emphasized that metal dyshomeostasis with oxidative stress could be the early trigger for AD pathogenesis. Iron in particular can also affect the translational rate of APP mRNA by binding at the iron responsive elements at 5’ UTR and thus intracellular iron can directly regulate the level of APP much in the same fashion as it does with ferritin and transferrin receptor [82, 122, 123].

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The metal chelators, thus, have long been thought of as potential disease modifying agents for AD. Conventional metal chelators like desferrioxamine and EDTA have been used in AD clinical trials and met with some success [82, 212, 213]. Another metal chelator clioquinol has shown promise in delaying cognitive decline and preventing amyloid beta accumulation in the brain in AD transgenic mice, and it also has shown moderate beneficial effects in AD clinical trials [82, 212, 213]. Although a very significant body of evidence is available on the benefits of antioxidants in various AD models and the protection these agents provide against amyloid beta toxicity in different experimental systems, the clinical trials in AD with these compounds have proved to be less than satisfactory [214, 215]. Several antioxidants like -tocopherol, ascorbate, selegiline, acetyl-L-carnitine, coenzyme Q, lipoic acid etc. have often shown promising beneficial effects in AD in clinical trials, and several studies have also shown that the use of antioxidant supplementation reduces the risk of AD [216-220]. However, some placebo-controlled clinical trials have shown that antioxidants do not improve the cognitive decline in AD and may even worsen it in some patients [221, 222]. In fact, a recent randomized, placebo-controlled large clinical trial has reported a greater decline of cognitive functions in AD subjects in the antioxidant supplemented group with no improvement of CSF biomarker levels [59]. Other studies also tend to imply that the antioxidant supplementation can often produce deleterious effects on clinical outcome of some disease conditions [223]. Apart from the clinical trials, recent experimental evidence in Proteus anguinus, C. elegans and mice with single or multiple antioxidant enzyme genes knocked out has also cast some doubt on the prime importance of oxidative damage in the aging process [224-226]. Thus taking everything in to consideration, there is a legitimate suspicion that antioxidants will at all provide a therapeutic window for AD. On the other hand, the overwhelming body of robust evidence in the post-mortem brain of AD subjects or in the brain of AD transgenic animals linking oxidative stress with this disease pathology cannot be brushed aside. In fact, many factors including the nature and the bio-availability of the antioxidants are to be taken in to account to interpret the failure of clinical trials with antioxidants in AD [227, 228]. First and foremost is the fact that the sporadic AD which is multi-factorial in origin, is unlikely to be halted to a noticeable degree by any one group of drugs targeted to one underlying mechanism. Secondly, clinical trials with a single antioxidant instead of a cocktail of such drugs are likely to fail. Oxidative damage pathways form a network of radical reactions encompassing the different aqueous and hydrophobic compartments of the cells. A combination of antioxidants, both water-soluble and lipidsoluble varieties and acting by different mechanisms at different subcellular sites, could possibly bring about a significant inhibition of the oxidative injury and also ensure the optimum bio-availability at cellular and subcellular levels. Moreover, we have mentioned earlier that oxidative stress in the brain is probably an initial trigger for sporadic AD which leads to an accumulation of APP and amyloid beta or activation of stress kinases as cellular defence reactions which subsequently usher in AD pathology [107, 157] and as such the clinical trial should begin in the very early or prodromal

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stage of the disease process (e.g. MCI). Finally, the interference of exogenously administered antioxidants in redox signaling pathways and redox- responsive gene transcription may block the physiological adaptive response or the ‘hormetic’ effect of oxidative stress which may undermine the possible beneficial effects of antioxidants against AD. These considerations when taken in to the account will certainly help us to design better clinical trials with antioxidants against AD than those conducted so far. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS We thank the Department of Science and Technology, Govt. of India and the Indian Council of Medical Research, New Delhi for continued financial support for our research in brain aging and Alzheimer’s disease. The authors declare no conflict of interest. REFERENCES [1] [2]


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