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Neuropathology and treatment of Alzheimer disease: did we lose the forest for the trees? Rudy J Castellani, Xiongwei Zhu, Hyoung-gon Lee, Paula I Moreira, George Perry and Mark A Smith†

CONTENTS The problem with non-neoplastic conditions The light microscope as the window into pathogenesis Autopsy-proven AD Amyloid-β

Although amyloid-β-containing senile plaques and phospho-tau containing neurofibrillary tangles are hallmark lesions of Alzheimer disease (AD), neither is specific for AD, nor even a marker of AD. Rather, they are empirical lesions that require close correlation with age and clinical signs for optimal interpretation. In essence, these lesions represent the effect rather than the cause of disease. In this review, we discuss diagnostic criteria for AD, the relationship between pathology, pathogenesis and multiple treatment approaches that have so far been disappointing, including those that presume to address pathological lesions. An acceptance that lesion-based therapies do not address etiology or rate-limiting pathogenic factors is probably necessary for the best chance of significant advances that have thus far been elusive.

Oxidative stress Lipid metabolism Inflammation Hormonal changes with age Phosphorylated tau Excitotoxicity Cholinergic deficits Expert commentary & five-year view Key issues References Affiliations

†

Author for correspondence Case Western Reserve University, Department of Pathology, 2103 Cornell Road, Cleveland, OH 44106 USA Tel.: +1 216 368 3670 Fax: +1 216 368 8964 [email protected] KEYWORDS: Alzheimer disease, amyloid, cholinergic, oxidative stress, phosphorylation, tau, therapeutic

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Expert Rev. Neurotherapeutics 7(5), 473–485 (2007)

Alzheimer disease (AD) is the most prevalent neurodegenerative disease worldwide and affects nearly 25 million patients worldwide, with over 4 million in the USA alone. As the average life expectancy increases in the USA and throughout the world, so does the number of elderly men and women who become increasingly susceptible to AD with age. In fact, it is estimated that, by 2050, 14 million Americans will have AD if preventative treatments do not become available [1]. The cost of treatment is considerable: national direct and indirect costs of caring for AD patients have been estimated to be minimally upwards of US$100 billion per year, a figure that will rise as the prevalence increases [2]. In spite of these daunting statistics and frenzied activity in translational neurobiology, progress in terms of effective therapy for AD has been effectively stagnant, raising the possibility that the current obsession with a reductionist approach to translational research, coupled with the haste with which it is undertaken, has led to a superficial understanding of basic disease processes. Whether this is the case with ongoing treatment trials for AD remains to be seen, although results to 10.1586/14737175.7.5.473

date have not been promising [3]. The only reasonably effective therapy, which provides symptomatic relief only (without slowing disease progression), involves replacement of a cholinergic deficit in AD, while approaches based on ameliorating pathological lesions have failed miserably and require constant revision [3–5]. We suspect this is the case because, at present, the basic pathology of AD, on which such treatment strategies are based, is fundamentally misunderstood [6]. The problem with non-neoplastic conditions

The pathology of non-neoplastic diseases in general often represents expression of disease rather than cause and, importantly, our experience with neurodegenerative disease pathology is dominated by that expression of disease at the end point. To look at it another way, the bulk of pathology practice nowadays is dedicated to the diagnosis and the classification of tumors, in which case the etiology is always apparent. The pathologist is looking under the microscope at, in essence, the progeny of the offending agent, or thousands of clones of the original transformed cell. The same certainly cannot be said of non-neoplastic diseases,

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including neurodegenerative diseases. Indeed, the thoughtful pathologist who has had the occasion to interpret non-neoplastic conditions has some understanding of the limitations of light microscopy, particularly with respect to assigning etiology. In many instances, the interpretation is simply a description of the pattern of changes that ultimately require correlation with clinical or other data for the most complete explanation. Interstitial lung diseases, medical liver and kidney diseases, neuromuscular diseases, and toxic and inflammatory CNS diseases all come to mind. Neurodegenerative diseases are no exception; interpretation requires identification of a pattern of changes, and in many cases structural inclusions with different protein constituents, that ultimately require correlation with clinical data [7]. In AD, the interpretation can be even more challenging, since there is an overlay of the aging process that is distinguished only with difficulty from changes associated with disease [8]. The light microscope as the window into pathogenesis

Since the time of Alzheimer, the eyepiece of the light microscope has been regarded as a window into the pathogenesis of AD [9]. Almost without exception, all hypotheses related to AD pathogenesis, from the earliest concepts about senile plaque and neurofibrillary tangle morphology and disease kinetics, to present hypotheses encompassing molecular genetics, were pursued for no other reason than because two lesions associated with dementia were detectable by light microscopy. The existence of amyloid-β (Aβ) protein, for example, was determined by direct biochemical analysis of senile plaques [10,11], while localization of phosphorylated tau to neurofibrillary tangles was accomplished by direct examination of neurofibrillary tangles [12,13]. In the absence of these lesions, such concepts as Aβ oligomers, presenilins (PSENs), γ-secretase complex, and tau kinases and phosphatases, would likely not have been pursued or even suggested. Simply stated, the foundation for leading hypotheses regarding pathogenesis of AD is the microscopic pathology – the senile plaque and the neurofibrillary tangle. It is reasonable to conclude, therefore, that the validity of those hypotheses depends upon the validity of the construct that the microscopic lesions contain the cause of AD or, at minimum, some feature without which AD would not occur; that the pathology is not only a manifestation of disease, but also speaks directly to etiology and pathogenesis. If, on the other hand, senile plaques and neurofibrillary tangles are a priori epiphenomena, more extensive investigation would merely add more details to what is in essence a distraction, or worse provide the illusion of progress where, for practical purposes (i.e., therapeutic intervention based on those hypotheses), there is none [6]. Autopsy-proven AD

An important concept that is often overlooked, and that speaks to the relationship between etiology and pathology, is that ‘autopsy-proven AD’ is actually better described as ‘empirical

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microscopic changes associated with dementia.’ Diagnosis requires not only a clinical correlate, but also quantitation, which together indicate that an individual lesion is not only diagnostically meaningless (a senile plaque or a neurofibrillary tangle cannot predict the presence of dementia), but a marker of advanced age as much as disease [14,15]. In this context, it remains an open question whether a given lesion, or some constituent of that lesion, is driving the process [6]. It is true that AD patients tend to have more of such lesions, so accuracy of diagnosis is improved by correlating the quantity of senile plaques and neurofibrillary tangles with the presence or absence of dementia. Accuracy is not perfect, however, and standard approaches to diagnosis highlight the weakness in pathological assessment by using such terms as ‘probable’ and ‘intermediate likelihood’ [14,16]. It is also interesting that the most commonly used approaches [14,17] rely on different lesions, and thus accumulations of fundamentally different proteins, in effect testifying to our poor grasp of the pathology–pathogenesis connection. The National Institute on Aging-Reagan criteria [16], which is a composite of a senile plaque-dependent Consortium to Establish a Registry for Alzheimer Disease (CERAD) criteria and a neurofibrillary tangle-dependent Braak criteria, avoids offending the sensibilities of one camp or another [18], but is a further admission of the nebulous relationship between lesion and etiology. A closer examination of CERAD criteria for AD makes the point all the more clear. In this approach, diagnosis of AD at autopsy consists of semiquantitation of senile plaques, which is plotted into a scale of probability of AD as a function of age. According to CERAD, the older the patient, the more plaques are forgiven, and the more plaques are therefore required to diagnose AD. The precursor Khatchaturian criteria employed a similar approach [15]. It is thus a mathematical fact that instances exist in which the number of senile plaques that qualify for definite AD in one patient does not qualify for definite AD in another (older) patient, such as the autopsy-proven pathology in one patient may be identical to that in another patient, yet one would be diagnosed with AD and the other would not. A logical conclusion that one could therefore draw from CERAD, if CERAD indeed speaks to the issue of pathogenesis, is also a conclusion that few could, would or should utter with a straight face – that older patients tolerate senile plaques better than younger patients, or that some number of senile plaques are allowed as a sort of decoration in the brains of elderly where they are not allowed in middle age. In the Braak criteria for AD, more emphasis is placed on regional distribution of pathology, in particular neurofibrillary pathology, with a correlation established between pathology and stage of disease rather than likelihood of disease. This is a more sensitive approach since presumed early changes are designated AD; specificity suffers, however, as it is well known that early, intermediate, and occasionally even latestage AD changes by Braak criteria occur in the cognitively intact elderly [19]. Moreover, subjects with intermediate-stage disease may have profound dementia [20]. The shortcomings

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Alzheimer disease, neuropathology and treatment

of pathological assessment by Braak are thus similar to those of CERAD: pathological assessments may be the same for two given patients, yet one may have been demented during life, and the other may have been clinically normal. In any case, application of accepted pathological criteria for AD is a statistical exercise, based on comparisons to brain changes in populations of demented patients. They are useful as a standardizing tool for clinicopathological and applied studies. However, even a casual assessment of AD diagnosis at autopsy suggests that disease effect is much more relevant to diagnostic lesions than is cause. Treatment aimed at ameliorating these diagnostic lesions, therefore, should confront this simple fact. With this in mind, the following is an overview of treatment strategies for AD based on the targeted metabolic abnormality. Amyloid-β

While the amyloid cascade hypothesis (ACH) [21,22] appears to be based upon a fundamental misconception of the relationship between pathology and disease [23], those who favor this hypothesis often point to the existence of ‘familial AD’, in which germ line mutations in specific proteins lead to autosomal dominant, early-onset AD [21]. The specific proteins include Aβ protein precursor (AβPP), presenilin (PSEN)1, and PSEN2. All three mutations affect processing of the Aβ peptides, and all are associated with prodigious accumulations of Aβ as well as neurofibrillary tangles with disease. Down’s syndrome subjects, who have an extra copy of chromosome 21 and therefore constitutively increased synthesis of AβPP, are predisposed to AD changes at a relatively young age; the case of Down’s syndrome, therefore, represents another critical component of the ACH. At a glance, the ACH makes some mechanistic sense with a lesioned protein, in some rare cases a genetic mutation, and a resulting phenotype, and has been the dominant hypothesis for the last 20 years. The genetics of AD is often taken as prima facia evidence that Aβ is important in the disease. Indeed, it is known that all of the known autosomal dominant mutations that lead to AD involve genes related to Aβ (either directly as in the case of AβPP or, indirectly through AβPP-cleaving enzymes in the case of the PSENs). At a reductionist level, mutation affects amyloid that leads to disease. More complex interactions, though possible, are seemingly ignored to sway the argument that amyloid is a fundamental initiator of AD. Counter to this, however, recent findings show that mutations in AβPP and/or PSENs actually result in less, not more, Aβ [24,25]. Nevertheless, a large body of knowledge regarding Aβ processing has accumulated. The Aβ peptide is a proteolytic cleavage product of AβPP, a type-1 transmembrane protein of unknown function. The two aspartyl proteases responsible for cleavage and conversion of AβPP to Aβ are referred to as βand γ-secretases [26]. However, most AβPP molecules undergo cleavage by α-secretase that cleaves AβPP near the middle of the Aβ domain [27], resulting in a cleavage product with a large soluble ectodomain (AβPPs-α) that is released into the


extracellular space. The remaining C-terminal fragment can then be cleaved by γ-secretase to create a smaller fragment known as the p3 fragment. The function of such proteolytic processing in normal neurons has yet to be defined, although research has indicated that cleavage of AβPP by γ-secretase allows the release of the AβPP intracellular domain to the nucleus, where it is thought to participate in transcriptional signaling [26]. Cleavage of AβPP by β-secretase leaves a longer C-terminal fragment that is retained in the cellular membrane and is subjected to further cleavage by γ-secretase, which finally results in the Aβ peptide cleavage product. β-secretase has become an attractive therapeutic target as it initiates and is the rate-limiting step in the formation of Aβ (it is interesting to note that Aβ is constitutively secreted by mammalian cells and normally occurs in plasma and cerebrospinal fluid [CSF]) [28,29]. Interestingly, inhibitors of β-secretase have similar chemical structures to AβPP. Sinha and colleagues developed StatV, a transition state analogue that spans the cleavage site of Swedish mutant AβPP for use as a β-secretase inhibitor [30]. OM99-2, a similar AβPP analogue, was subsequently developed and is an even more potent β-secretase inhibitor. To date, however, available inhibitors are too large, and lack sufficient specificity to be suitable for therapeutic use [31]. Since the discovery of PSEN-linked familial AD, the relationship between mutation and mechanism of AβPP processing has been the study of intensive investigation [32]. Early studies suggested that PSEN is a cofactor for γ-secretase [33] or that PSEN is itself a γ-secretase [34]. Later interpretations of experimental data indicated that PSEN is an unusual and unprecedented intramembrane-cleaving aspartyl protease [34]. Most recently, investigations utilizing genetic analysis of Notch signaling in Caenorhabditis elegans have revealed that γ-secretase is a multiprotein complex that has multiple regulatory subunits, and that PSEN, nicastrin, aph-1, and pen-2 proteins are all required for γ-secretase activity [35,36]. Nevertheless, regulation of PSEN and γ-secretase are still under investigation, and whether our extensive knowledge of the γ-secretase complex and its relationship with AβPP will translate into productive therapy remains to be determined. As discussed, Aβ can normally be found in the CSF as a self-antigen and cell-mediated immune responses would not be traditionally predicted to this peptide. Self-reactive T cells mediate a number of autoimmune diseases; however, no evidence exists showing deleterious effects of T-cell reactivity against Aβ. It has been established that activated T cells penetrate the CNS [37] and that healthy elderly individuals and patients with AD have higher Aβ-reactive T-cell responses than middle-aged adults [38]. This suggests that the normal immune response includes an active response to remove excess Aβ from the brain. In light of this phenomenon, immunological approaches to clearing Aβ from the brain have been developed in recent years. Active immunization and passive immunization strategies have been studied although only active immunization has reached clinical testing. Briefly, findings regarding active immunization with

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synthetic intact Aβ1-42 were excellent in transgenic mouse models producing human Aβ. A significant reduction of Aβ plaques were reported in mice immunized before and after plaque development [39]. In addition, immunization allowed these animals to regain functional effects [40,41]. The underlying mechanism behind these beneficial reports has not been reported; however, one possibility is that reduction in Aβ deposition is due to Fc receptor-mediated imbroglio phagocytosis [39]. Passive immunization strategies with antibodies raised against Aβ have suggested that imbroglio are responsible for clearing Aβ plaques through Fc receptor-mediated phagocytosis and subsequent degradation [42]. Clinical trials were initiated to test tolerance to treatment in humans shortly after animal trials were completed. Synthetic peptide Aβ1-42 was combined with an adjuvant (QS-21) to create the immunogenic AN-1792 (Elan Pharmaceuticals/Wyeth). AN-1792 reached Phase IIa clinical trials in the USA and Europe; however, the program was suspended in January 2002 when approximately 5% of the patients in the active treatment group developed meningoencephalitis [43]. While the Aβ peptide continues to be a popular target for new therapies, it is relevant that Aβ plaques have not proved to correlate directly with dementia and that senile plaque-producing transgenic models are not directly associated with neurological deficits. These inconsistencies have lead to a modification of the ACH that seeks to establish the pathogenecity of Aβ [21]. Current research indicates that Aβ assembly of soluble oligomers provides the pathologenic molecules that previously were unidentified. Soluble oligomers have been implicated in the physical degeneration of synapses [44] and in vivo experiments have established that oligomers cause a failure in long-term potentiation (LTP) of neurons in the hippocampus. Exciting data have also come from hAPP transgenic mice and the ability of antibodies directed against pathogenic oligomers to reverse memory failure; this reversal of memory failure occurred without a reduction in senile plaques [45]. This phenomenon supported the concept that plaques are not responsible for neurological deficits, but rather oligomers are responsible for synaptic failure [46]. Where this then leaves plaque-focused strategies, such as AN-1792, is beyond the scope of this review [3,47]. The Aβ oligomers, shown to have the properties of disrupting LTP and synaptic degeneration, are often referred to as ‘oligomeric ligands’ or ‘amyloid-derived diffusible ligands’ (ADDLs). Given the nature of ADDLs, they appear to be an excellent therapeutic target for the prevention and treatment of AD. These ADDLs uniquely influence short-term synaptic plasticity and long-term neuronal survival. They are also considered excellent therapeutic targets due to the regional specificity that ADDLs possess. Parallel to the pathology seen in AD, ADDLs target hippocampal, but not cerebellar, neurons [48]. Characterization of oligomers has occurred through atomic force microscopy and immunoblot analyses [49]. Research on ADDLs has also shed light on the number of peptides involved in the globular structure and the length of

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peptide required. While Aβ1-40 can form oligomers [50], these products are unstable and require high peptide concentrations; ADDLs require Aβ1-42, the pathogenic Aβ species [51]. In summary, Aβ remains, despite its limitations as a putative pathogenic species highlighted earlier, the primary target for the developers of treatments for AD. The success of this approach will clearly depend on the validity of the ACH or, minimally, on the fact that Aβ plays an active role in disease progression [52,53]. If, on the other hand, Aβ is a marker of disease [6] or a protective consequence of disease [23], targeting Aβ will likely have little/no effect and could even exacerbate disease [4,5,54]. Oxidative stress

Multiple lines of evidence link oxidative stress and AD [55]. Reduced glucose metabolism and mitochondrial abnormalities are associated with AD as mitochondria are considered to be the source of oxidative stress [56,57]. Oxidative phosphorylation produces superoxide radicals as a by-product associated with electron transport. While most of these free radicals are sequestered in the mitochondria, oxidative insult is exacerbated by age, metabolic demand and disease [58]. Heightened superoxide radical formation in AD also correlates with heightened superoxide dismutase levels that may allow the release of H2O2 from the mitochondria to the cytoplasm [59,60]. Mitochondrial abnormalities have also been associated with deficiencies in enzyme activities, specifically α-ketoglutarate dehydrogenase complex, pyruvate dehydrogenase complex, and cytochrome oxidase in AD neurons [61–63]. Increased H2O2 in the cytoplasm may cause a localized increase in the concentration of reactive oxygen species (ROS), especially in the presence of redox-active metals [64]. It has been shown that metals are dysregulated in AD and that redox-active transition metals are aberrantly accumulated in the cytoplasm of AD-susceptible neurons [65–67]. A potential role for heavy metals in AD pathogenesis has been suggested in a number of studies; indeed, we have found a close association between redox-active iron and pathological lesions in AD [65]. It has also been shown that Aβ and AβPP exhibit physicochemical interactions with micromolar and submicromolar concentrations of metal ions [68–71]. The pharmacological basis of chelation therapy and concomitant side effects, however, is complex and multifaceted; to date, reproducible therapeutic efficacy has yet to be shown [72,73]. Also interesting with respect to metal ions is that the overall concentration of zinc is decreased in AD brains [74]; this metal has antioxidant activity, and has been evaluated as a therapeutic agent to prevent production of ROS. It is also interesting to note that the Aβ peptide is a strong redox-active agent capable of reducing transition metals in the cytoplasm and enabling the conversion of molecular oxygen to H2O2 [66,75,76]. Antioxidant projection via vitamin supplementation is another avenue for long-term and safe treatments of AD. The most notable antioxidant treatment that is currently available is taking vitamin E supplements. Chemically R,R,R-α-tocopherol



is the naturally occurring stereoisomer of vitamin E and is secreted by the liver due to specific binding by liver α-tocopherol transfer protein (α-TTP). Vitamin E has been demonstrated to be a lipid-soluble, chain-breaking antioxidant [77]. Vitamin regiments that include vitamin E and ascorbic acid (vitamin C) have indicated that consuming organic antioxidants may be beneficial. Some studies have found that antioxidants blunt the cognitive decline in AD or slow disease progression [78,79]. A large epidemiological study demonstrated that use of combination vitamin E and C was associated with reduced AD prevalence and incidence [80]. Lipid metabolism

Recent studies suggest that statins may afford protection against AD [81]. The mechanisms involved, however, are unclear, although the possibility that it may be related to apolipoprotein (Apo)E is intriguing. Unlike molecular gene mutations, Apo ε4 genotypes are not implicated in the generation of Aβ peptide; however, the ε4 allele is associated with lowering the age of AD onset. Mid-life high blood pressure and high cholesterol levels are associated with increased risk of developing AD later in life. While the data and mechanisms implicating cholesterol and lipid transport with development of AD are not well established, a number of studies indicate that the use of statins significantly reduces the risk of developing AD [81,82]. Experimentally, primary neurons treated with statins halt production of Aβ [83] and reduce the level of Aβ1-42, while other studies [84] indicate no relationship between the beneficial effects of statins and amyloid. As such, the mechanisms underlying the action of statins remain unclear. Statins also act to decrease cellular cholesterol by inhibition of methylglutaryl coenzyme A. Given these simultaneous actions, it cannot be strictly stated whether statins or the reduction in membrane cholesterol are responsible.

innate immune response may delay the onset and slow the progress of AD [92]. It appears that Aβ is responsible for activation of microglia and astrocytes since toxic forms of Aβ that are not completely removed induce chronic immune responses. NSAIDs have been used extensively in medical practice and are currently prescribed for a number of inflammation-related ailments. NSAIDs downregulate the proinflammatory signals sent out by glia and astrocytes. In addition to anti-inflammatory action, NSAIDs have been shown to alter the production of Aβ142. It has been shown that some NSAIDs may reduce the risk of AD by lowering Aβ1-42. Production of Aβ1-42 is mediated through a pathway that involves a small G protein called Rho. NSAIDs have been shown to inhibit the activity of Rho and decrease Aβ1-42 production in transgenic mice. It has also been shown that NSAIDs that do not block Rho activity also do not lower Aβ1-42 levels. The downstream effector proteins of Rho, Rock 1 and 2, are also pharmacological targets that could prevent Aβ1-42 production. Epidemiologically, a number of case–control and population-based cross-sectional studies have been conducted to evaluate the relationship between use of NSAIDs and risk of AD. Data from a co-twin control study found an inverse relationship between anti-inflammatory treatments and AD [93]. The follow-up was conducted with siblings with a high level of familial AD and found that NSAIDs have protective effects [94]. Population-based studies have also suggested that antiinflammatory drugs are protective against AD [95–97]. Whether these findings will translate into a change in the indications for NSAID therapy remains to be seen. Moreover, a thorough analysis of these data was undertaken by Launer, who concluded that no recommendation can be made concerning when or how a person can take NSAIDs to reduce the risk of developing AD [98]. Hormonal changes with age

Inflammation

Exciting findings regarding the use of non-steroidal antiinflammatory drugs (NSAIDs) were originally reported in epidemiological data showing that classical NSAIDs could prevent or retard AD [85,86]. Data based on positron emission tomography (PET) has shown that inflammation and activation of microglia are early pathogenic events that precede cerebral atrophy [87]. Additional evidence that inflammation mediates development of AD comes from studies demonstrating that inflammation from head trauma plays a role in initiating AD [88]. It is hypothesized that Aβ peptide is responsible for the innate immune response that occurs in AD patients. Additional findings suggest that Aβ deposition and senile plaque formation are associated with an innate immune response that includes activation of complement [89], secretion of proinflammatory cytokines, expression of chemokines and excretion of nitric oxide, which mediates apoptosis [90,91]. Experiments with transgenic mice show that the activation of the complement cascade may be beneficial for the brain [91]. However, a number of studies have shown that attenuating this inflammatory


Some epidemiological studies concerning the prevalence and incidence of AD have indicated that women have an almost twofold higher risk of developing AD [99]. Postmenopausal estrogen deficiency is hypothesized as a basis for the effect. This in turn has prompted the idea of hormone-replacement therapy (HRT) as possible treatment. Early studies concerning the use of HRT as a treatment for AD came to divergent conclusions concerning HRT-based therapies. These findings were reported for therapy that included estrogen in addition to progestin regardless of timecourse regiments. Evidence supporting protective effects of HRT in postmenopausal women were reported for a large epidemiological study [100], and further confirmed by studies indicating that women with high levels of endogenous estrogen were less prone to develop AD [101]. However, HRT was found to be ineffective in preventing AD when administered in postmenopausal women aged over 65 years [102]. The mechanistic basis for HRT in AD remains to be resolved, although progress is being made. Multiple studies have shown that terminally differentiated neurons display

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activation of cell cycle-related proteins in degenerating neurons [103,104]. Investigators have hypothesized that an unknown trigger pushes neurons back into the cell cycle but that death occurs before completion [105]. The concept that menopause- and andropause-related increase in gonadotropin hormones signals the activation of the cell cycle has thus been proposed as a pathogenic factor in AD [106]. Likewise, a novel therapy that uses leuprolide acetate, a gonadotropin-releasing hormone (GnRH) agonist, that suppresses production of luteinizing hormone by downregulating pituitary GnRH receptors, is currently being studied [107]. Phosphorylated tau

Abnormally phosphorylated tau is the major protein component of the neurofibrillary tangles, the first lesion to be characterized and associated with AD. Hyperphosphorylation causes disengagement of tau from microtubules and aggregation of the filamentous protein [12]. Importantly, hyperphosphorylated tau (phospho-tau) that accumulates in neurofibrillary tangles is not toxic to the neuron and does not induce the apoptotic cascade, and as much as 40% of phospho-tau is not aggregated into neurofibrillary tangles [108]. Pathologically active phospho-tau does not bind tubulin but instead sequesters normal tau in addition to other microtubule-associated proteins (MAP)s which interrupts assembly and disassembly of normal microtubules [109,110]. Tau is the substrate for a number of kinases, such as glycogen synthase kinase 3, cyclin-dependent protein kinase 5 and protein kinase A [111]. Dephosphorylation of phospho-tau protein by alkaline phosphatase, protein phosphatase (PP)-2A, PP-2B and PP-1 has been shown in vivo, all of which convert it into a normal state lacking toxic properties [112,113]. PP-2A and PP-1 are responsible for 90% of the serine/threonine protein phosphatase activity in mammalian cells [114]. Activities of PP2A and PP-1 have been shown to be compromised in AD [112,115], indicating that insufficient dephosphorylation could be responsible for the appearance of phospho-tau. Given these mechanisms for aberrant accumulation of phospho-tau, a number of therapeutic targets are presented. However, therapies that can address these targets are in the initial stages of design and testing. Either inhibiting the activity of PP-2A inhibitors, such that PP-2A activity is restored, or inhibiting the activity of the kinases that phosphorylate tau will prevent abnormal phosphorylation from occurring. A second strategy that emerges is preventing the ability of phospho-tau to sequester normal tau, which allows for normal function of other MAPs and preservation of cellular integrity. Drugs that are capable of achieving these goals are still in the early stages of development. High-throughput assays that are capable of measuring anti-sequestering effects are being addressed by researchers in the field [116]. Much like strategies focused on Aβ, the ultimate success of strategies against tau phosphorylation is dependent on the presumed importance of the pathological lesions [117], in this case, the neurofibrillary tangle. Should neurofibrillary tangles

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turn out to simply be markers of disease [6] or a protective consequence of disease [54,118,119], targeting tau will likely have little or no effect and could even exacerbate disease. Excitotoxicity

Almost all neurons of the CNS carry the N-methyl-D-aspartate (NMDA) subtype of ionotrophic L-glutamate receptors. These receptors mediate post-synaptic influx of Ca2+ after neurotransmission. It is theorized that excitotoxicity resulting from excessive activation of these receptor, increases the vulnerability of CNS neurons, leading to neuronal degeneration characteristic of the development of AD. In one school of thought, clinical manifestations early in disease are the result of synaptic failure [46], while disease severity is the result of synaptic loss [120]. Mementine, the most recently approved drug for AD treatment, prevents pathological excitotoxicity at the synapse, and is believed to preserve physiological activation [121–123]. Early pathological changes in AD apparently occur in the medial temporal lobe, including the transentorhinal allocortex [124] and hippocampal formation. Neocortical glutamatergic neurons in layers III and IV are also vulnerable [125]. Excitatory amino acids (EAAs), such as L-gluamate (L-Glu), are among the neurotransmitters used by these cells, although L-Glu distribution within the CNS is extensive. At basal conditions, the concentration of L-Glu in the synaptic cleft is around 0.6 µM [126], while synaptic transmission allows for a localized rise to 10 µM within the synaptic cleft [127]. Termination of excitation occurs by high affinity reuptake on both pre- and post-synaptic neuronal cell membranes and the membranes of adjacent glial cells [128]. When uptake mechanisms are impaired, EAAs cause selective neuronal toxicity, through NMDA-mediated Ca2+ influx [129]. A potentially important role of the astrocytes is to convert glutamate to glutamine by glutamine synthetase. Glutamine is then released by astrocytes and taken up by neurons where it is converted to glutamate in the mitochondria by the mitochondrial glutaminase. Recent studies have shown that glutamine synthetase is oxidized in the brains of individuals with AD [130]. This may explain the reported decrease in the activity of glutamine synthetase in AD patients [131]. While antioxidants may prove to be effective at preventing pathological oxidation, one therapy thought to block excitotoxicity through interaction with membrane receptors was also developed. Memantine is a blocker of glutamate-gated NMDA channels and has become a popular therapy in recent years [132]. Memantine is thought to prevent excitotoxicity mediated by glutamate-gated NMDA receptors by blocking pathological activation. This property of memantine has been shown to occur due to the rapid, voltage-dependent interaction between memantine and the NMDA receptor channel [133]. Memantine blocks the receptor channel under normal physiological conditions and leaves the receptor channel upon activation. In addition, memantine remains in the channel during



pathological activation and thus prevents excitotoxicity, but allows normal activation and memory formation. In animal models, memantine has been shown to prevent excitotoxic activation of glutamate receptors and preserve their physiological activation [121,122,134]. Clinical safety has been thoroughly studied in subjects diagnosed with mild-to-moderate dementia [135,136]. These studies have shown significant improvements in psychopathological, performance and behavior level. Additional studies have addressed the efficacy and effects of memantine use in a severely demented population [137]. Researchers found that memantine treatment significantly improved the functional capabilities of severely demented patients. Cholinergic deficits

AD can be characterized as a deficit in cholinergic neurotransmission due to loss of disease involving cholinergic neurons of the basal nucleus of Meynert [138]. Evidence has demonstrated that the enzymes involved in synthesis cholineacetyltransferase and degradation (acetylcholinesterase [AChE]) have reduced activity and may be responsible for this deficit [139,140]. In light of this neurotransmitter deficiency, strategies to boost the level of acetylcholine have been developed for the treatment of AD. This has lead to the development of AChE inhibitors (AChEIs) that prevent the breakdown of the neurotransmitter inside the synaptic cleft. The first drug approved for the treatment of AD in 1993 was 1,2,3,4-tetrahydro-9-aminoacridine (Tacrine) [141]. Currently, three other AChEIs are available on the market from different manufacturers. The second generation of drugs has proved to be more clinically effective and produce less severe side effects than the first generation. These drugs are currently prescribed for patients with mild-to-moderate progression of AD under the following names: Donepzil (Eisai Company and Pfizer Inc.), ENA-713 (Novartis Pharmaceuticals), and Galantamine (Hoechst Marion Roussel Inc., Shire Pharmaceutical Group and Janssen Pharmaceutical). Cholinergic therapy is considered a short-term intervention for the symptomatic treatment of AD. The effect of these inhibitors is to cognitively stabilize 50% of patients in treatment for 1 year. Clinical studies have indicated that beneficial effects can be maintained up to 36 months [142–144]. The stabilization of cognitive decline has led researchers to investigate whether AChEIs can affect the amyloid cascade or the neurotoxicity associated with Aβ [145]. Cholinesterase inhibitors are currently being investigated to find additional benefits that the drugs may yield in delay of onset and time of efficacy in AD. To test these criteria, research has moved from AD patients to patients with mild cognitive impairment (MCI) [146]. MCI patients are identified due to their high risk of developing dementia. Two major studies are underway in the USA and Europe to address cholinesterase inhibitor use in populations of MCI patients [147]. Long-term stabilization, which can occur when AD patients are treated with AChEI therapy, has been associated with the ability of AChEI to affect AβPP metabolism [142]. Evidence has


been published showing that AChEIs increase secretion of AβPP, thus slowing down the production of Aβ peptide and amyloidogenic compounds [148,149]. This suggests that AChEI could be administered at much earlier preclinical stages in at-risk individuals, and that combinatorial therapies represent future alternatives to AChEI monotreatment [142]. Neurotropins are recently being investigated as treatments that can supplement other therapeutic targets that have already been addressed. Neurotropins are of particular interest due to their ability to upregulate functions of cholinergic neurons [150]. Evidence demonstrates that neurotropins share the same signaling pathways as Aβ pathways mediating toxicity in neurons [151–153]. Aβ has been found to bind to the p75 neurotropin receptor (p75NTR) that causes p75NTR-mediated cell death [154]. A precursor of neuronal growth factor, proNGF, has been associated with signaling thorough p75NTR [155]. Research concerning proNGF in AD patients has shown the levels are increased in the brain [156]. Neurotropins are difficult to deliver effectively due to the short half-lives (in the order of minutes) thereby significantly hampering the ability of these compounds to reach the CNS [157]. In addition, neurotropins affect a broad range of biological signals, including sprouting of sympathetic nerves and upregulation of pain mechanisms. The development of neurotropin mimetics, which alter only a subset of signaling cascades, is a partial answer to these side effects. Investigations of NGF interaction with p75NTR have shown that regulation of different intracellular effector molecules occurs upon binding [152,158]. Studies concerning peptidomimetics corresponding to specific regions of NGF have shown that their effects can occur via p75NTR dependent mechanisms to prevent neuronal death. These studies have included NGF peptidomimetics corresponding to loops 1 and 4 of NGF globular protein, both preventing neuronal death mechanisms via p75NTR receptor [159–161]. Recent findings have shown that a number of adaptor proteins are involved in survival and death signaling mediated via p75NTR [162–164]. An interesting finding concerning these adaptors is that the selection of an adaptor is regulated by the nature of the ligand, which suggests that peptidomimetics may be able to regulate adaptor proteins [165]. These findings provide further support that peptidomimetics could possibly trigger p75NTR signaling which could to prevent cell death in AD patients. Expert commentary & five-year view

Sporadic AD is a complex process that develops over decades and involves biochemical dysfunction in multiple arenas. While Aβ and tau are attractive targets for therapy, both are not necessarily initiators of the disease process, particularly in sporadic disease. The lack of specificity of senile plaques and neurofibrillary tangles for AD highlight the need to look beyond Aβ and tau for early pathogenic factors and treatment. Oxidative stress, for example, is known to predate clinical symptoms and pathological accumulations of Aβ and tau [118]. More recently, based on evidence of inappropriate cell cycle

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activity in neurons in AD [166–168], we suggested this as an attractive therapeutic target [169,170]. Moreover, the strides that have been made in lipid metabolism with statins, and in neuroprotection from anti-inflammatory treatment, as well as antiexcitotoxic and cholinergic treatment, emphasizes the point that AD is complex and that any one strategy or construct may not be sufficient [171]. As knowledge expands, the list of avenues for treatment will only expand, and an open-minded approach to treatment will be all the more necessary. Much like the branches of a tree, consequences of disease are not never ending, but unlikely to have any real individual consequence. Real effect therapy can only come from attacking the tree trunk that connects all the branches. Current therapeutic

efforts are focused on pruning not cutting. Nonetheless, these authors are optimistic that we will eventually see the forest from the trees. Acknowledgements

Rudy J Castellani and Mark A Smith are both available for correspondence regarding this article, for contact details see ‘Affiliations’. Work in the authors’ laboratories is supported by the NIH, the Alzheimer’s Association, Philip Morris USA Inc. and Philip Morris International. Mark Smith and George Perry are, or were, compensated consultants to Voyager Pharmaceutical Corp. and own equity. Mark Smith is a compensated consultant and owns equity in NeuroPharm Ltd.

Key issues • Are amyloid-β or tau (senile plaques or neurofibrillary tangles) initiators, propagators or terminators in the disease process of Alzheimer disease (AD)? Knowing this is key to targeting them for therapeutic purposes. • Is treating AD as a unimodal disorder a good strategy? The possibility of multiple etiologies leading to a common phenotype might be dangerous. • Polypharmacy might be better than magic bullets. • Prevention is clearly better than the cure. • Preventative agents do not necessarily make good therapeutics.

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Xiongwei Zhu Case Western Reserve University, Department of Pathology, Cleveland, OH USA Tel.: +1 216 368 5903 Fax: +1 216 368 8964 [email protected]

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Hyoung-gon Lee Case Western Reserve University, Department of Pathology, Cleveland, OH USA Tel.: +1 216 368 6708 Fax: +1 216 368 8964 [email protected]

Affiliations •

Rudy J Castellani, MD University of Maryland, Department of Pathology, 22 South Greene Street, Baltimore, MD, 21201, USA Tel.: +1 410 328 5555 Fax: +1 410 328 5508 [email protected]


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Paula I Moreira University of Coimbra, Center for Neuroscience and Cell Biology of Coimbra, Coimbra, Portugal Tel.: +35 1 239 834729 Fax: +35 1 239 835812 [email protected]

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George Perry Case Western Reserve University, Department of Pathology, Cleveland, Ohio USA; College of Sciences, University of Texas, San Antonio, San Antonia, TX, USA Tel.: +1 210 458 4450 Fax: +1 210 458 4445 [email protected]

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Mark A Smith, PhD Case Western Reserve University, Department of Pathology, 2103 Cornell Road, Cleveland, OH 44106 USA Tel.: +1 216 368 3670 Fax: +1 216 368 8964 [email protected]

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