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Mol Cell 6:873–883 Oxidative Stress and Alzheimer Disease: Mechanisms and Therapeutic Opportunities Siddhartha Mondragón-Rodríguez, Francisco García-Sierra, Gemma Casadesus, Hyoung-gon Lee, Robert B. Petersen, George Perry, Xiongwei Zhu, and Mark A. Smith Abstract Oxidative stress is an early event in the development of Alzheimer dis- ease (AD), preceding classic fibril formation which eventually deposits as amyloid-β senile plaques and neurofibrillary tangles composed of tau protein. Mitochondrial and metallic abnormalities are likely precursors of oxidative stress during the early stages of AD and, under degenerative conditions, the capacity of neurons to main- tain redox balance decreases and results in mitochondrial dysfunction, a critical organelle involved in AD progression. Fibril formation, including amyloid-β pro- duction and tau phosphorylation, can be explained as a compensatory mechanism that may eventually enhance oxidative stress by increasing reactive oxygen species levels among many other free radicals. In this scenario, deposition of Aβ in the extracellular environment and tau protein in the intracellular environment can be explained as a redox imbalance with tragic consequences. If this hypothesis is cor- rect, pharmacological treatments directed against amyloid-β or tau may not provide a benefit. In contrast, antioxidant strategies may be helpful in treating AD symp- toms, although significant extended benefits have not been realized to date. In sum, the damage observed in the brain tissue of AD patients may be minimized with a healthy daily diet, exercise, and intellectual activities, factors that all reduce oxidative stress. Keywords Alzheimer disease · Amyloid-beta · Antioxidants · Fibrils · Mitochondria · Oxidative stress Contents 1 Introduction 608 2 Oxidative Stress, Genetics, and Alzheimer Disease Pathology 608 2.1 Fibrillary Aggregates and Oxidative Stress 610 2.2 Amyloid-β Peptide 610 M.A. Smith (B) Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA e-mail: mark.smith@case.edu 607 J.P. Blass (ed.), Neurochemical Mechanisms in Disease, Advances in Neurobiology 1, DOI 10.1007/978-1-4419-7104-3_18, C Springer Science+Business Media, LLC 2011 608 S. Mondragón-Rodríguez et al. 2.3 Tau Protein 612 3 Oxidative Stress and Metabolism 613 3.1 Energy Utilization 614 3.2 Mitochondria 614 3.3 Metals 615 4 Current and Future Pharmacological Treatments for Alzheimer Disease 616 5 Conclusion 619 References 619 1 Introduction Alzheimer disease (AD) is defined by insoluble filamentous aggregates known as senile plaques and neurofibrillary tangles (NFT), of which the major components are amyloid-β (Aβ) and tau protein, respectively (Wood et al., 1986; Arriagada et al., 1992; Goedert et al., 1998). These lesions accumulate in regions that are responsible for cognitive functions (Ball et al., 1988) and contribute towards t he declining activities of daily living as well as the neuropsychiatric symptoms and behavioral changes seen in patients with AD. Definitive risk factors for AD include genetic predisposition (i.e., apolipoprotein E (ApoE), amyloid-β protein precursor (AβPP), presenilins (PS)), medical conditions, environment, and lifestyle (Smith, 1998; Wang et al., 2004a,b; Williamson and LaRusse, 2004; Lemos et al., 2009; Zawia et al., 2009). Oxidative stress has also been strongly associated with the development of AD (Smith et al., 1997a, 1998, 1999; Paola et al., 2000; Smith et al., 2000a,b; Nunomura et al., 2001; Zhu et al., 2007). Therefore, it is not surprising that oxidative stress is related to the neurodegenerative process, inasmuch as it is known to affect, and result in, metabolic dysfunction, mitochondrial dysfunction, dysregulation of metal homeostasis, and alterations in the cell cycle, all of which contribute to the classi- cal fibril aggregations, Aβ plaques and NFT. These structures, counter-intuitively, may be compensatory responses mounted to combat such oxidative stress (Lee et al., 2005; Nunomura et al., 2006; Zhu et al., 2006; Nunomura et al., 2007a;Lee et al., 2009a; Lu et al., 2009). If this scenario is correct, pharmacological treat- ments reducing fibril aggregation may be detrimental (Perry et al., 2000; Smith et al., 2002a). The aim of this chapter is to evaluate the relationship between AD and oxidative stress and to consider how t his knowledge may dictate treatment options for the clinical symptoms of the disease. 2 Oxidative Stress, Genetics, and Alzheimer Disease Pathology Behavioral and cognitive decline in AD is accompanied by pathological accumu- lations of Aβ-containing senile plaques and tau-containing NFTs (Van Hoesen and Oxidative Stress and AD: Mechanisms and Therapeutics 609 Hyman, 1990; Van Hoesen et al., 1991). AβPP and PS1 mutations result in hetero- geneity in the clinical expression of neurological features during disease progression compared to sporadic AD, suggesting a genetic influence (Zekanowski et al., 2006; Larner and Doran, 2009). Although an accurate cascade that charts the effect of mutations through to the progression of dementia at the end-stage is an area of extensive study, one common feature in all AD cases is Aβ deposition resulting from the cleavage of AβPP (Younkin, 1994). Mutations are not just limited to AβPP and the PS genes. Recently, mutations in the tau gene, found in familial cases of frontotemporal dementia, which are characterized by an intracellular accumulation of polymerized tau as the primary cause of neurodegeneration, also exhibit increased Aβ 40 and Aβ 42 deposition. These data bring new support for a relationship between tau gene mutations and Aβ deposition (Vitali et al., 2004). Despite all of the evidence that links AD to a genetic component, a detailed mechanism leading from one event to the other remains elusive. In this regard, some authors have suggested that early life exposure to the xenobiotic metal lead (Pb) enhanced the expression of genes associated with AD, repressed the expression of others, and result in an increased burden of oxidative DNA damage in the aged brain; the mechanism acts through either hypomethylation or hypermethylation of DNA (Zawia et al., 2009). The association between genetics and neurodegeneration is also supported by predisposing risk factors such as the ε allele of the ApoE gene. In particular, the ε4 allele has been strongly correlated with increased risk of AD, whereas the ε3 allele is not (Basurto-Islas et al., 2008). In addition, the ε4 allele of the ApoE gene has been associated with increased vascular Aβ deposition, whereas, in contrast, the ε2 allele is associated with cerebral amyloid angiopathy (CAA) related to intracerebral hemorrhage (Hamaguchi and Yamada, 2008). However, the genetic association is not just the result of coding sequence changes; different levels of gene expression may also be involved, that is, the hypo- or hypermethylation of DNA, as mentioned earlier, that also contribute to the disease (de Carvalho et al., 2000; Speranca et al., 2008). Interestingly, deficient or altered energy metabolism that could change the overall oxidative microenvironment in neurons during the pathogenesis and progression of AD, leading to alterations in mitochondrial enzymes and in glucose metabolism in AD brain tissue, has been found in AD patients that also carried the ApoE ε4 allele (Mosconi et al., 2008). On the other hand, oxidative stress has also been found to induce PS1 transcrip- tion, thereby promoting production of pathological levels of Aβ in AD (Tamagno et al., 2008). Indeed, it has been proposed that the pathophysiology of oxidative stress is reflected in damage to tissue biomolecules, including lipids, proteins, and DNA by free radicals (Migliore and Coppede, 2009). Clearly, although not fully understood, the genetic component and oxidative stress may act synergistically or cooperatively, creating a pathological condition that contributes to the protein deposition seen in AD, although the precise mechanisms involved are unknown. 610 S. Mondragón-Rodríguez et al. 2.1 Fibrillary Aggregates and Oxidative Stress Senile plaques and NFTs are also present in a considerable percentage of elderly non-AD brains. Although these markers constitute the criteria for the diagnosis of AD, they do not always correlate with cognitive decline (Davis et al., 1999;Lee et al., 2007; Castellani et al., 2008). In fact, studies have appropriately raised the question whether senile plaque deposition has any relationship to the cognitive decline observed in AD (Dickson et al., 1992) inasmuch as Aβ deposition shows no correlation with neuronal loss (Gomez-Isla et al., 1997). In contrast, cognitive decline correlates well with NFT density (Garcia-Sierra et al., 2001), although the degree of neuronal loss greatly exceeds the amount of NFTs (Gomez-Isla et al., 1997). Nonetheless, neuronal loss has been halted and memory defects reversed in transgenic models of tau mutations by turning off mutant tau expression (Santacruz et al., 2005). Despite the debate over the relationship of fibril deposition and clinical symp- toms, their role in neurodegeneration remains as strong as ever. Regarding such a relationship, it has been found that Aβ-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neu- roblastoma cells, s uggesting an oxidative stress pathway as the molecular mediator (Guix et al., 2009). Indeed, it has been reported that behavioral stress aggravates AD pathology via generation of metabolic oxidative stress and MMP-2 downregulation in AD mouse models (Lee et al., 2009b). Clearly, oxidative stress plays a crucial role in neurodegeneration (Nunomura et al., 2007b; Sajad et al., 2009), however, the mechanism by which amyloid depo- sition causes oxidative stress is the subject of extensive study. In vitro studies have shown that monomeric Aβ1-40 and Aβ1-42 exhibit antioxidant activity in cultured neurons (Zou et al., 2002). Furthermore, Aβ was found to be one of the most impor- tant antioxidants in cerebrospinal fluid (CSF). Indeed, recent reports suggest that the fibrillary forms typically observed in senile plaques and NFTs may actually be neuroprotective, because Aβ seems to inhibit oxidation by chelating metal ions, a function that may also equally apply to tau protein (Kontush et al., 1996; Kontush, 2001; Smith et al., 2002b; Caughey and Lansbury, 2003; Walsh and Selkoe, 2004). Nevertheless, this fibrillar formation seems to be accompanied by further compen- satory changes that ultimately result in additional oxidative insult during the disease (Lee et al., 2004, 2005). Based on these observations, it is clear that a close and highly complicated relationship exists between fibril deposition and oxidative stress during neurode- generation. 2.2 Amyloid-β Peptide The Aβ plaque was one of the first identified hallmarks of AD. It has been proposed that these structures are the main cause of AD inasmuch as they appear in the limbic area, which is affected in AD (Giannakopoulos et al., 2003), and it is thought that Oxidative Stress and AD: Mechanisms and Therapeutics 611 Aβ binds to neurons activating apoptotic pathways that eventually contribute to neu- rodegeneration (Gamblin et al., 2003). Furthermore, Aβ1-42 has been determined to be the most toxic form to cultured neurons, because the Aβ1-42 oligomer was able to activate the apoptotic pathway leading to caspase activation (Yankner et al., 1990; Dahlgren et al., 2002; Gamblin et al., 2003; Yao et al., 2005). However, numerous studies support the idea that an oxidative event is critical for Aβ toxicity (Pratico et al., 2002): (1) Aβ staging does not distinguish between cognitive changes and dementia; and (2) Aβ shows overlap among the various clinical dementias (Gold et al., 2001). Along these lines, plaques have been further classified into subtypes such as senile, diffuse, and neuritic, and, in this regard, it is generally accepted that diffuse plaques (Aβ deposits without cores or a neuritic component) are merely dec- orative in nature, having little impact, if any, on cognitive function, whereas neuritic plaques are more pathogenic. It is thought that diffuse plaques appear during the early preclinical stages of the disease and eventually mature into a defined structure, the neuritic plaque. However, this hypothesis and the role of plaque maturation dur- ing AD pathogenesis remain controversial. Here again, no clinical correlation has been found between plaques and the degree of cognitive decline (Arriagada et al., 1992). In addition to being a pathological hallmark of AD, Aβ plays an important role in normal cell development and maintenance (Atwood et al., 2003). Some propose that diffuse amyloid plaques may be a compensatory response aimed at reducing oxidative stress, because there is a negative correlation between Aβ deposition and oxidative damage in Down syndrome patients, as well as AD patients (Nunomura et al., 2000; Smith et al., 2000b; Nunomura et al., 2001). Aβ is also highly involved in the neurodegenerative process, although its contribution remains debatable. Some authors have suggested that Aβ leads to depletion of cellular choline stores and con- sequently contributes to the selective vulnerability of cholinergic neurons in AD (Allen et al., 1997). Indeed, a pathological role has been attributed to Aβ accumula- tion in the brain of AD patients (Wang et al., 2007b). In point of fact, the majority of efforts for developing AD therapeutics have been directed towards eliminating the fibrillar form of aggregated Aβ (Asuni et al., 2006; Matsuda et al., 2009), although there is debate about the potential efficacy of this approach (Lee et al., 2006; Shah et al., 2008). Why should we doubt the effectiveness? The answer is far from sim- ple, however, growing evidence supports a nonpathological role for the Aβ peptide (Lee et al., 2007). In fact, it has been proposed that Aβ deposition may be a primary antioxidant defense indicating that A β expression is an adaptive response rather than a cause of AD (Castellani et al., 2006; Lee et al., 2007; Castellani et al., 2008). Chelation of metals by Aβ may play a role in oxidative stress (Dong et al., 2003; House et al., 2004). Specifically, the methionine at residue 35 of the Aβ sequence can scavenge free radicals and also reduce metals to their high-activity, low-valency form, showing both pro- and antioxidant properties (Cuajungco et al., 2005). On the other hand, Aβ is able to initiate oxidation of different biomolecules; for example, Aβ induces the peroxidation of membrane lipids and lipoproteins, which generates H 2 O 2 and hydroxynonenal in neurons, and damages DNA (Huang et al., 1999a;Xu et al., 2001; Butterfield et al., 2002). 612 S. Mondragón-Rodríguez et al. Despite the controversy over the toxicity of AβPP and Aβ, it can be inferred from the current data that Aβ may be playing, as proposed, a compensatory role that eventually becomes pathological by activating oxidative stress pathways, although more data are needed to support this hypothesis. 2.3 Tau Protein The main function of the tau protein is to stabilize microtubules; moreover, tau may also be involved in signal transduction, organelle transport, and cell growth, as well as anchoring of enzymes (Johnson and Hartigan, 1999; Johnson and Jenkins, 1999; Sontag et al., 1999). Furthermore, tau has a role in modulating axonal morphol- ogy and polarity (Buee et al., 2000). Therefore, it is not surprising that microtubule abnormalities and tau phosphorylation are also associated with AD because cell cycle re-entry, an early feature in AD (Webber et al., 2005; Evans et al., 2007;Lee et al., 2009a), and candidate tau kinases that have been implicated in cell cycle control such as Cdk2, Cdk5, Cdc2, and MAPK are all increased in AD in a topo- graphical manner that overlaps with hyperphosphorylated tau (Vincent et al., 1997; Swatton et al., 2004; Wang et al., 2004c, 2007a), which has also been proposed as an early event in tau-mediated pathology (Mondragon-Rodriguez et al., 2008a,b). A tau transgenic mouse line, THY-Tau22, expressing a mutated human tau pro- tein that has been linked to frontotemporal dementia with Parkinsonism linked to chromosome-17 displays increased neurogenesis associated with tau hyperphospho- rylation. Later, cell cycle events, abnormal tau phosphorylation, and tau aggregation occur preceding neuronal death and neurodegeneration (Schindowski et al., 2008). Tau is highly phosphorylated; at least 30 phosphorylation sites have been described, the majority are Ser-Pro and Thr-Pro motifs. Some of these motifs seem to be crucial to the development of AD. For example, phosphorylation at Ser262 mediated by P70S6 kinase dramatically reduces the affinity of tau for microtubules in vivo (Hamdane et al., 2003; Zhou et al., 2008b). Furthermore, phosphorylation at Ser202 appears to enhance tau polymerization; and phosphorylation at t wo sites (Ser202-Thr205) makes filament formation more sensitive to small changes in tau concentration (Rankin et al., 2005). Thus, phosphorylation outside the microtubule- binding domains, such as Ser202 and Thr205, may strongly influence tubulin assembly by modifying the affinity between microtubules and tau as well as tau itself (Alonso et al., 1996, 2001). Moreover, phosphorylation has been found to reg- ulate axonal transport by controlling tau binding to kinesin (Cuchillo-Ibanez et al., 2008). Regarding phosphorylation, members of the stress-activated protein kinase (SAPK) family have been shown to phosphorylate tau in vitro. SAPKIγ (or Jun N-Terminal kinase, JNK1), SAPK2a (p38), SAPK2β (p38 β), SAPK3 (p38γ), and SAPK4 can phosphorylate tau, although SAPK3 and SAPK4 are the most efficient in vitro (Goedert et al., 1997; Reynolds et al., 1997). In AD, hyperphosphorylated tau accumulates in neurons, and being a constitutive element of NFTs, eventually leads to degeneration (Alonso et al., 2001; Garcia and Cleveland, 2001). Oxidative Stress and AD: Mechanisms and Therapeutics 613 The abnormal phosphorylation of tau associated with AD may be related to either an increase in kinase activity (glycogen synthase kinase 3β, cyclin-dependent kinase-5, p42/44 MAP kinase, p38 MAPK, stress-activated protein kinases, mitotic protein kinases) or a decrease in phosphatase activity (protein phosphatases 1, 2a, 2b), suggesting soluble tau as a cause of neuronal degeneration (Buee et al., 2000; Tian and Wang, 2002; Chen et al., 2008; Liu et al., 2008; Yang et al., 2008; Zhou et al., 2008a). Hyperphosphorylation of tau has also been proposed to be protective (Lee et al., 2005); phosphorylation may prevent advanced tau processing, that is, cleavage of tau at site Asp421, an event that enhances fibril formation (Guillozet-Bongaarts et al., 2006). Phosphorylation plays a pivotal role in redox balance, so it is perhaps not surprising that oxidative stress, through activation of MAP kinase pathways, leads to phosphorylation of tau (Zhu et al., 2000, 2001a,b). MAP kinase activation and heme oxygenase (HO-1) induction may be but a few of the many responses that result from lipid peroxidation. Consequently, oxidative damage can no longer be considered an end-stage event, but rather a signal of an underlying change of state that is related to the phosphorylation of tau. 3 Oxidative Stress and Metabolism Oxidative damage has been found in several entities that are critical for neuronal structure and functional integrity. It is possible that under degenerative conditions the capacity of cells to maintain redox balance decreases resulting in mitochondrial dysfunction, a critical organelle involved in AD progression (Cash et al., 2002;Zhu et al., 2006). A significant body of evidence supports the hypothesis that mitochon- drial and metallic abnormalities are direct precursors of oxidative stress during the early stages of AD (Halliwell, 1999; Nunomura et al., 2000, 2001; Atamna, 2004; Zhu et al., 2004b, 2007). Also, increased intracellular iron may promote oxida- tive stress/free radical damage in vulnerable neurons (Casadesus et al., 2004;Zhu et al., 2004a, 2007; Dwyer et al., 2009). Interestingly, the loss of iron homeostasis directly affects mitochondrial function (Lu et al., 2009) and the proximal causes of mitochondrial abnormalities likely involve re-entry into the cell cycle (Cash et al., 2002; Zhu et al., 2006). Recent studies have also shown that reactive oxygen species generated by mitochondria regulate p53 activity, which in turn regulates cell-cycle progression and DNA repair and, in cases of irreparable DNA damage, executes programmed cell death (Holley and St Clair, 2009). Oxidative stress increases during aging, in parallel with the increased susceptibil- ity to several neurodegenerative diseases including AD. In AD, NFT accumulation within the neuronal cytoplasm is associated with impaired axonal transport of mitochondria between the cell nucleus and synapse, which leads to severe energy dysfunction and an imbalance in the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Smith et al., 1997b; Rapoport, 2003). DNA and RNA oxidation are marked by increased levels of 8-hydroxy-2-deoxyguanosine (8OHdG) and 8-hydroxyguanosine (8OHG), r espectively (Nunomura et al., 1999, 614 S. Mondragón-Rodríguez et al. 2000, 2001, 2004, 2007b). Meanwhile protein oxidation is marked by elevated levels of protein carbonyls and nitration of t yrosine residues (Smith et al., 1995). Modification of s ugars by glycation and glycoxidation is another component of the disease, although the levels of these modifications decrease as the disease progresses to advanced AD (Smith et al., 1994; Castellani et al., 2001; Perry et al., 2002). These data support the hypothesis that increased oxidative damage is an early event in the progression AD. 3.1 Energy Utilization The role of mitochondrial and redox metal ions as potential neuronal compen- satory responses against oxidative stress remains unclear, nevertheless, both are important elements of the energy metabolism deficiency in AD (Blass and Gibson, 1999; Blass et al., 2000). Glucose and oxygen are the primary sources of energy in neurons (Erecinska and Silver, 1989) and there i s reduced glucose metabolism in the tempoparietal and posterior cingulate cortex in AD (Drzezga et al., 2003). Furthermore, reduced glucose metabolism in limbic and associative areas of the brain have been reported in AD cases with ApoE ε4, reflecting its genetic influence (Kamino et al., 2000; Mosconi et al., 2004). Other features of AD are increased oxy- gen consumption (Hoyer, 1998), atrophy in the vasculature (Praprotnik et al., 1996; Perry et al., 1998, 2003), and reduced cerebral glucose transport activity (Kalaria et al., 1988; Perry et al., 2003). The reduction of ATP production from glucose by approximately 50% at the onset of sporadic AD is further evidence of the glucose metabolism imbalance (Hoyer, 1992). All these data support the involvement of altered glucose metabolism in the early pathophysiology of AD. Furthermore, the activity of many enzymes involved in metabolism is decreased in AD, such as glu- tamine synthetase, creatine kinase, and pyruvate dehydrogenase (Sorbi et al., 1983; Gibson et al., 2000). On the other hand, the activities of succinate dehydrogenase (complex II) and malate dehydrogenase have been reported to increase, suggest- ing a coordinated alteration of metabolic activity in the mitochondria (Bubber et al., 2005). 3.2 Mitochondria Due to the high oxygen consumption rate and relative paucity of antioxidant enzymes compared with other organs, the brain is especially vulnerable to free rad- ical damage (Floyd and Hensley, 2002; Mattson et al., 2002). The major source of free radicals [hydrogen peroxide (H 2 O 2 ), hydroxyl ( · OH) and superoxide (O 2 –· )] is oxidative phosphorylation (Wallace, 1999). The reactive oxygen species generated by mitochondria have many targets such as lipids, proteins, RNA, DNA, and mito- chondrial DNA (mtDNA), which due to the lack of histones, becomes a vulnerable target of oxidative stress. Indeed, nucleic acid oxidation is also deemed a hallmark of AD (Moreira et al., 2008). . controlling tau binding to kinesin (Cuchillo-Ibanez et al., 2008). Regarding phosphorylation, members of the stress-activated protein kinase (SAPK) family have been shown to phosphorylate tau in vitro cyclin-dependent kinase-5, p42/44 MAP kinase, p38 MAPK, stress-activated protein kinases, mitotic protein kinases) or a decrease in phosphatase activity (protein phosphatases 1, 2a, 2b), suggesting soluble. changes in tau concentration (Rankin et al., 2005). Thus, phosphorylation outside the microtubule- binding domains, such as Ser202 and Thr205, may strongly in uence tubulin assembly by modifying