Drugs Aging 2010; 27 (5): 351-365 1170-229X/10/0005-0351/$49.95/0

LEADING ARTICLE

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Targeting Tau Protein in Alzheimer’s Disease Cheng-Xin Gong, Inge Grundke-Iqbal and Khalid Iqbal Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, USA

Abstract

Alzheimer’s disease (AD) is characterized histopathologically by numerous neurons with neurofibrillary tangles and neuritic (senile) amyloid-b (Ab) plaques, and clinically by progressive dementia. Although Ab is the primary trigger of AD according to the amyloid cascade hypothesis, neurofibrillary degeneration of abnormally hyperphosphorylated tau is apparently required for the clinical expression of this disease. Furthermore, while approximately 30% of normal aged individuals have as much compact plaque burden in the neocortex as is seen in typical cases of AD, in several tauopathies, such as cortical basal degeneration and Pick’s disease, neurofibrillary degeneration of abnormally hyperphosphorylated tau in the absence of Ab plaques is associated with dementia. To date, all AD clinical trials based on Ab as a therapeutic target have failed. In addition to the clinical pathological correlation of neurofibrillary degeneration with dementia in AD and related tauopathies, increasing evidence from in vitro and in vivo studies in experimental animal models provides a compelling case for this lesion as a promising therapeutic target. A number of rational approaches to inhibiting neurofibrillary degeneration include inhibition of one or more tau protein kinases, such as glycogen synthase kinase-3b and cyclin-dependent protein kinase 5, activation of the major tau phosphatase protein phosphatase-2A, elevation of b-N-acetylglucosamine modification of tau through inhibition of b-N-acetylglucosaminidase or increase in brain glucose uptake, and promotion of the clearance of the abnormally hyperphosphorylated tau by autophagy or the ubiquitin proteasome system.

Alzheimer’s disease (AD) is a common neurodegenerative disease that is characterized clinically by a progressive decline of cognitive function, leading to dementia. It eventually leads to the death of affected individuals on an average of 9 years after diagnosis.[1] AD is the single most common cause of dementia in adults, affecting approximately 27 million individuals worldwide[2] (see also http://en.wikipedia.org[3]). Because the disease mechanism is still unknown,

there is no cure for AD at the present time. If no new treatments become available, the number of affected individuals is predicted to reach over 100 million by 2050[2] (see also http://en.wikipedia. org[3]). In the last 3 decades, the standard treatment for AD has been acetylcholinesterase inhibitors to improve cognitive function, and other drugs to manage the mood disturbance, agitation and psychosis that often occur in the later stages of the disease. In more recent years, memantine,

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an NMDA receptor antagonist and a potentially neuroprotective agent, has been widely used. However, all of these treatments show only modest symptomatic effects that generally last around 6–12 months. Research on AD has progressed significantly in the last 3 decades. Recent studies on the possible disease mechanisms have led to a new era in developing therapeutic treatments for AD. As a result, numerous new therapeutic drugs for AD are at various stages of development. Several clinical trials putatively targeting the mechanisms fundamental to the disease process have been conducted. Most of these so-called diseasemodifying drugs are based on the amyloid cascade hypothesis of AD, according to which amyloid-b (Ab) toxicity is believed to play a primary role in the development of the disease.[4,5] However, despite enormous amounts of investment, none of these clinical trials has been successful so far. It has become very clear that in order to fight AD, possible disease mechanisms other than those indicated by the amyloid cascade hypothesis have to be considered. Increasing evidence supports a crucial role of tau protein abnormalities in AD neurodegeneration and suggests that tau could be a promising therapeutic target for developing disease-modifying drugs for AD. Indeed, a shift in AD research and drug development from Ab to tau has become apparent recently. In this article, we describe the rationale for targeting tau pathology for the development of therapeutic drugs for AD, followed by a discussion of the molecular mechanism of neurofibrillary degeneration in which tau abnormality plays a central role. We detail the possible therapeutic strategies that are based on reversal of tau pathology in AD. Finally, we conclude this article with a brief perspective on the development of AD therapy. 1. Rationale for Targeting Tau Protein for the Treatment of Alzheimer’s Disease Tau is the major neuronal microtubuleassociated protein (MAP)[6] and is coded by a single gene on chromosome 17.[7] In the adult ª 2010 Adis Data Information BV. All rights reserved.

human brain, six isoforms of tau are expressed as a result of alternative splicing of the tau gene.[8] Tau has little secondary structure; it is a mostly random coil with b-structure in the second and third microtubule-binding repeats. The major biological function of tau is to stimulate microtubule assembly and to stabilize microtubule structure. This biological activity of tau is regulated by its degree of phosphorylation. Normal brain tau contains 2–3 moles of phosphate per mole of the protein.[9] Hyperphosphorylation of tau depresses its microtubule assembly-promoting activity.[10,11] In the AD brain, tau is abnormally hyperphosphorylated and, in this form, it is the major protein subunit of paired helical filaments (PHFs) and straight filaments (SFs), which form neurofibrillary tangles (NFTs), neuropil threads and plaque dystrophic neurites in AD.[12-16] Besides abnormal hyperphosphorylation, tau also undergoes several other abnormal post-translational modifications.[17] The abnormal modifications of tau, especially hyperphosphorylation, appear to lead to neurofibrillary degeneration in AD. Studies of the correlation of the cognitive impairment to the histopathological changes have consistently demonstrated that the number of NFTs, and not the plaques, correlates best with the presence and/or the degree of dementia in AD.[18-20] Neurofibrillary degeneration appears to be required for the clinical expression of the disease, i.e. the dementia. b-amyloidosis alone in the absence of neurofibrillary degeneration does not produce the disease clinically. In fact, some normal elderly individuals have as much Ab plaque burden in the brain as is seen in typical cases of AD; however, in the former case, the plaques lack dystrophic neurites with neurofibrillary changes surrounding the Ab cores.[19-23] On the other hand, neurofibrillary degeneration of the AD type, in the absence of b-amyloidosis, is seen in the neocortex in several tauopathies such as Guam parkinsonism-dementia complex, dementia pugilistica, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration and Pick’s disease (table I). All of these neurodegenerative disorders Drugs Aging 2010; 27 (5)

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Table I. Diseases characterized by neurofibrillary degeneration of abnormally hyperphosphorylated tau but without amyloid-b plaques Diseases Guam parkinsonism-dementia complex Dementia pugilistica Pick’s disease Dementia with argyrophilic grains Frontotemporal dementia with tau mutations Corticobasal degeneration Pallido-ponto-nigral degeneration Progressive supranuclear palsy Gerstmann-Straussler-Scheinker disease with tangles

are clinically characterized by dementia. In progressive supranuclear palsy, the neurofibrillary degeneration is primarily in the brain stem and is associated with motor dysfunction. The discovery of certain missense mutations in the tau gene, which lead to FTDP-17, clearly indicates that abnormalities of tau itself can cause neurodegeneration and dementia.[24-26] The crucial role of tau in neurodegeneration is further supported by the results of recent in vivo studies. In a transgenic mouse model of amyloid pathology and cognitive deficits, the phenotype almost disappeared when tau was knocked out,[27] indicating the essential role of tau in mediating neurodegeneration in this model. In another study, reduction of soluble Ab and tau, but not Ab alone, ameliorated cognitive decline in a triple transgenic mouse model of AD that develops amyloid plaques and NFTs.[28] Taken together, tau pathology and the consequent neurofibrillary degeneration appear to be the central mechanism leading to dementia in AD and probably in other related tauopathies. It has become clear that targeting tau protein could be a promising approach for developing therapeutic treatments for AD and related tauopathies. 2. Mechanism of Neurofibrillary Degeneration The detailed mechanism of neurofibrillary degeneration has been reviewed recently[29] and, thus, is not discussed here in detail. Instead, we outline only the major stages of tau pathology (figure 1). ª 2010 Adis Data Information BV. All rights reserved.

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In sporadic AD, multiple aetiological factors cause abnormal hyperphosphorylation of tau via various pathways, including dysregulation of signal transduction leading to phosphorylation/ dephosphorylation imbalance and impaired brain glucose metabolism. The abnormally hyperphosphorylated tau not only loses its biological activity and disassociates from microtubules, but also promotes its self-polymerization. The cytosolic abnormally hyperphosphorylated tau/oligomeric tau, instead of stimulating microtubule assembly, sequesters normal tau and other MAPs and causes disruption of microtubules.[11,30-32] Probably as a defence mechanism of the neuron, the abnormal tau, which is resistant to proteolysis,[33] polymerizes into highly aggregated PHFs/NFTs. The NFTs are inert and have no effect on microtubule assembly,[34,35] but they may finally choke the affected neurons and facilitate cell death by acting as a space-occupying lesion. Besides abnormal hyperphosphorylation, other tau abnormalities also appear to contribute to tau pathology and neurofibrillary degeneration. Mutations of the tau gene, as seen in FTDP-17, cause neurofibrillary degeneration and dementia.[24-26] Truncation of tau has been demonstrated in the AD brain.[36-39] In both cases, the pathological tau is always hyperphosphorylated. Thus, the mutation and truncation of tau appear to result in neurofibrillary degeneration by promoting its abnormal hyperphosphorylation. Several FTDP17 mutations of tau have been demonstrated to promote its hyperphosphorylation.[40] Transgenic rats expressing human tau truncated both N- and C-terminally (tau151–391) show a marked neurofibrillary degeneration of abnormally hyperphosphorylated tau.[41] 3. Therapeutic Targets for Inhibition of Neurofibrillary Degeneration Since tau abnormalities are crucial to neurofibrillary degeneration, tau protein could be a promising target for developing disease-modifying therapy for AD. According to the molecular mechanism of tau-mediated neurofibrillary degeneration (figure 1), the therapeutic strategies may include the following: (i) inhibition of abnormal Drugs Aging 2010; 27 (5)

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Multiple aetiological factors Normal tau Inhibit tau hyperphosphorylation

Dysregulation of phosphorylation/dephosphorylation Tau O-GlcNAcylation ↓ ← Glucose uptake/metabolism ↓

Abnormally hyperphosphorylated tau Inhibit tau misfolding/aggregation

Misfolded/oligomeric tau

Detached from MTs

Other modifications such as ubiquitination, glycation, polyamination, nitration, truncation

Intraneuronal tau ↑

se

rea

Inc

ce

ran

lea

c tau

Neurotoxicity

PHFs/NFTs

Neurodegeneration

Dementia Fig. 1. Mechanism of tau-mediated neurofibrillary degeneration and tau-based therapeutic targets for Alzheimer’s disease (AD). Multiple aetiological factors cause abnormal hyperphosphorylation of tau via various pathways, including dysregulation of signal transduction leading to phosphorylation/dephosphorylation imbalance and impaired brain glucose metabolism. The abnormally hyperphosphorylated tau not only loses its biological activity and disassociates from microtubules (MTs), but also promotes its self-polymerization. This cytosolic abnormal tau sequesters normal MT-associated proteins, forming oligomers that disrupt MTs and lead to neuronal degeneration and dementia. Probably as a result of the defence mechanism of the neuron, abnormal tau further polymerizes into highly aggregated paired helical filaments/ neurofibrillary tangles (PHFs/NFTs) that are inert to MTs but finally choke the affected neurons to death, probably acting as a space-occupying lesion. Thus, tau protein is obviously a promising target for developing disease-modifying therapy for AD. The grey arrows indicate some of the therapeutic strategies targeting neurofibrillary degeneration. O-GlcNAcylation = b-N-acetylglucosamine modification.

tau hyperphosphorylation; (ii) inhibition of tau misfolding/aggregation; and (iii) increasing tau clearance. 3.1 Inhibition of Abnormal Tau Hyperphosphorylation

Dynamic regulation of tau kinases and tau phosphatases determines the level of tau phosphorylation, and abnormal hyperphosphorylation of tau could result from the dysregulation of this dynamic. Much effort was made in the last decade to identify the kinases and phosphatases that regulate tau phosphorylation in the brain. Accumulated studies have demonstrated that the major tau kinases include glycogen synthase kinase-3b (GSK-3b), cyclin-dependent protein kinase 5 (cdk5), cyclic adenosine monophosphate ª 2010 Adis Data Information BV. All rights reserved.

(cAMP)-dependent protein kinase (PKA), stressactivated protein kinases and MAP/microtubule affinity-regulating kinase 1 (MARK1).[42,43] Unlike protein kinases, protein phosphatases (PPs) usually have broad substrate specificities. The authors and others[44-47] have found that PP2A is by far the most important and major tau phosphatase in the brain. Therefore, targeting these enzymes is a logical approach to inhibiting and/ or reversing abnormal hyperphosphorylation of tau. 3.1.1 Glycogen Synthase Kinase-3b Inhibitors

The majority of AD drug developments targeting tau phosphorylation focus on kinase inhibitors rather than tau phosphatase activators. General pharmacological experience indicates that enzyme inhibition by small molecules is much Drugs Aging 2010; 27 (5)

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more easily realized than activation, especially if the target enzyme has a natural small-molecular binding site, as kinases do for adenosine triphosphate. Most of the tau kinase-targeting drugs considered to date have been GSK-3b inhibitors because this kinase appears to play a critical role in AD pathogenesis.[48,49] A recent study showed that GSK-3b activity may increase with aging,[50] which is consistent with the fact that aging is the most important risk factor for AD. We have found that GSK-3b is associated with all Braak stages of neurofibrillary pathology,[51] and its activity is increased in the AD brain compared with age-matched controls (unpublished observations). Both in vitro and in vivo studies have demonstrated that inhibition of GSK-3b, by either pharmacological or genetic means, can reverse hyperphosphorylation of tau and prevent behavioural impairments in mice.[52-56] These studies make GSK-3b inhibition very attractive as a therapeutic target for AD. Another important reason is that the human body tolerates GSK-3b inhibition well. Lithium, which inhibits GSK-3b both directly by competition with magnesium and indirectly via the phosphoinositide 3-kinase pathway,[57] has been used for the long-term treatment of bipolar disorders for decades. With the potentially pivotal role of GSK-3b in the pathogenesis of AD as well as in several other diseases such as type 2 diabetes mellitus and cancer, much effort directed at identifying selective GSK-3b inhibitors has been made and recently reviewed.[48,58-61] Several GSK-3b inhibitors, including lithium, aloisines, flavopiridol (alvocidib), hymenialdisine, paullones (e.g. NSC641166) and staurosporine, are under active investigation and development, and have been reviewed in detail.[43,62] Recent findings that GSK-3b inhibition may also inhibit Ab production and help cell survival[49,63] make the development of GSK-3b inhibitors for the treatment of AD even more attractive. However, critical questions regarding potential mechanism-based undesirable effects of the GSK-3b inhibitors linked to the regulation of glucose metabolism and tumourigenesis remain. A 10-week randomized, single-blind, placebo-controlled, multicen-

tre trial of lithium in patients with mild AD failed to show any significant alteration in cerebrospinal fluid (CSF) biomarkers, total tau, phosphotau and Ab1–42 as primary outcome measures or in global cognitive performance as measured by the Alzheimer’s Disease Assessment Scalecognitive subscale as a secondary outcome measure.[64] This negative outcome could have been the result of a relatively short duration of treatment, i.e. 10 weeks, and/or an inability of lithium to inhibit GSK-3b sufficiently to produce the therapeutic effect. GSK-3b and cdk5 phosphorylate tau largely at the same phosphorylation sites.[65] Thus, it is plausible that either persistent GSK-3b inhibition over a much longer period than 10 weeks or inhibition of both GSK-3b and cdk5 might be required to show significant therapeutic effect. Successful clinical development of a GSK-3b inhibitor for treating AD is both challenging and eagerly awaited.

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3.1.2 Cyclin-Dependent Protein Kinase 5 Inhibitors

Cdk5 was reported to be upregulated in the AD brain,[66] although this observation has been challenged by other investigators.[67-70] Nevertheless, cdk5 is another target kinase toward which pharmacological inhibitors are under development for the treatment of AD. Many inhibitors of cdk5 have been described across several structural classes, although their selectivity for cdk5 over other cdk kinases important for cell-cycle control is either poor or has not been reported.[71,72] One of these inhibitors, roscovitine, inhibits 50% of cdk5/p25 at a concentration of 0.16 mmol/L,[73,74] has useful selectivity for cdk5 over other kinases and can pass through the blood-brain barrier (BBB).[75] Administration of roscovitine resulted in reduced hyperphosphorylation of tau and other neuronal proteins in two different transgenic mouse models and in transient ischaemic rats.[76-78] Other cdk5 inhibitors include olomoucine, flavopiridol, aloisines and indirubins.[62] Many of these compounds are non-selective cdk inhibitors and have shown efficacy as anti-proliferative agents. Their efficacy in inhibiting tau hyperphosphorylation has not been well studied, and their utility may be compromised by their cell-cycle Drugs Aging 2010; 27 (5)

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effects. Further truncation of p25, a potent cdk5 activator, was found to yield a cdk5 inhibitory peptide that specifically inhibits cdk5/p25 activity without affecting cdk5/p35 or mitotic cdk5 activities.[79] This inhibitory peptide was able to reduce tau hyperphosphorylation and neuronal death induced by cdk5/p25 and, thus, might be used for developing a specific cdk5 inhibition strategy in the treatment of neurodegeneration. An in vivo study demonstrated that inhibition of cdk5 could cause activation of GSK-3b, which plays a more dominant role in overall tau phosphorylation than does cdk5.[50] Thus, cdk5 inhibitors might be unable to reverse abnormal hyperphosphorylation of tau and treat neurofibrillary degeneration. The same study also demonstrated that cdk5 inhibitors can reduce Ab level in the mouse brain. Therefore, cdk5 inhibitors deserve further investigation as potential therapeutic agents for AD. 3.1.3 Inhibition of Other Tau Kinases

Besides GSK-3b and cdk5, other protein kinases that can catalyze tau phosphorylation have also been considered as targets for inhibiting tau hyperphosphorylation. These kinases include mitogen-activated protein kinases, casein kinases, Ca2+/calmodulin-dependent protein kinase II, MARK1 and PKA, and the development of inhibitors of these kinases has been reviewed.[62] A common problem of the kinase inhibitors is their insufficient selectivity. However, a study using a less selective inhibitor, SRN-003-556, targeting GSK-3b, extracellular signal-regulated kinase 2/cell division control 2, PKA and protein kinase C, demonstrated efficacy in reducing soluble aggregated hyperphosphorylated tau and delaying the motor deficits in JNPL3 tau transgenic mice.[56] This study suggests that nonspecific kinase inhibitors might be considered as AD drugs, as more than one kinase is probably involved in the abnormal hyperphosphorylation of tau.[65] Because many protein kinases are dynamically regulated and are critical to many vital signalling pathways, any short- and long-term adverse effects of using kinase inhibitors will have to be investigated during development of these therapies. ª 2010 Adis Data Information BV. All rights reserved.

3.1.4 Restoration of Tau Phosphatase

It is most likely that abnormal hyperphosphorylation of tau in AD is caused by multiple factors and mechanisms. One of these mechanisms is downregulation of PP2A, the major tau phosphatase in the brain.[44-47,80] We first observed this downregulation in the early 1990s, and these findings were subsequently confirmed in various approaches by several laboratories.[47,81-85] Consistent with the relatively broad substrate specificity of PP2A, several other neuronal proteins in addition to tau, such as neurofilaments, MAP1B, b-tubulin and b-catenin, are also hyperphosphorylated in the AD brain.[86-89] Reversal of the downregulation of tau phosphatase is certainly another approach to inhibiting abnormal tau hyperphosphorylation. One important consideration of this approach is that PPs, including PP2A, have much broader substrate specificities than protein kinases. Thus, more undesirable effects might be expected than when using kinase inhibitors. Because PP2A is downregulated in the AD brain, these concerns may be eliminated if only the pathological PP2A downregulation is corrected. The activity, substrate specificity and subcellular localization of PP2A are regulated by various regulatory subunits. To date, the cellular and subcellular distribution of the PP2A downregulation in AD remains elusive. Activating the right pool of PP2A to the right extent is certainly a challenge for drug development. Two endogenous protein inhibitors, I1 PP2A and I2 PP2A, regulate PP2A activity.[90,91] These two inhibitors appear to be dysregulated in the AD brain,[92,93] contributing to PP2A inhibition. Targeting these inhibitors may serve as another approach to restoring PP2A activity in AD. Restoration of PP2A activity might be partially involved in the therapeutic efficacy of memantine, a low- to moderate-affinity antagonist of the NMDA receptor, which has been approved for treating AD and is clinically beneficial for moderate to severe AD. We found that memantine reverses okadaic acid-induced PP2A inhibition and prevents tau hyperphosphorylation in hippocampal slice cultures from adult rats.[94] As other NMDA receptor antagonists Drugs Aging 2010; 27 (5)

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failed to show similar effects to memantine, its efficacy for treating AD might be involved, at least partly, in PP2A restoration. Similarly, melatonin has also been shown to restore PP2A activity and reverse tau hyperphosphorylation, both in vitro and in animal studies.[95-98] Memantine apparently restores PP2A activity by inhibiting I2 PP2A.[99] However, the detailed mechanism by which memantine restores PP2A activity remains to be determined. The fact that clinical studies to date mainly support only a symptomatic action of memantine is probably because of the limited entry of this drug into neurons other than those in a state of excitotoxicity; in addition, on binding to the NMDA receptor, memantine blocks calcium channels and the entry of calcium into the neurons. It is probably for this reason that memantine has therapeutic benefit in moderate to severe AD where there is a more persistent excitotoxicity. A significant decrease in the CSF level of phosphorylated threonine 181 phosphotau has been found in patients with AD after 1 year of treatment with memantine.[100]

lished for decades, contributes to neurodegeneration.[107,108] In the AD brain, impaired glucose metabolism may cause decreased tau O-GlcNAcylation which, in turn, facilitates hyperphosphorylation of tau contributing to neurofibrillary degeneration. On the basis of these new observations, restoration of normal brain glucose metabolism could help reverse tau hyperphosphorylation in the AD brain. Rosiglitazone, a well known insulin sensitizer commonly used for treating type 2 diabetes, has been shown to reduce tau phosphorylation in cultured cells[109] and in obese rats,[110] and to attenuate learning and memory deficits in Tg2567 mice that overexpress mutated amyloid precursor protein.[111] It is possible that rosiglitazone achieves its efficacy by increasing brain insulin sensitivity, restoring brain glucose metabolism, increasing tau O-GlcNAcylation and, finally, inhibiting tau hyperphosphorylation. A phase II clinical trial showed therapeutic efficacy of rosiglitazone in patients with AD who did not carry the apolipoprotein E4 (apoE4) allele.[112] However, a follow-up 24-week, double-blind, placebo-controlled study of 553 patients with mild to moderate AD, genotyped and stratified into apoE4-positive and apoE4-negative groups, failed to show any significant effects of rosiglitazone on cognition or global function as outcome measures.[113] Unfortunately, at present it is not known whether 24 weeks of treatment with rosiglitazone produced a significant increase in brain glucose level and reduced the CSF level of phosphotau. Tau O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase and b-N-acetylglucosaminidase (O-GlcNAcase). This regulation offers the possibility of altering tau phosphorylation via tau O-GlcNAcylation by targeting these two enzymes. Small inhibitory compounds that selectively inhibit O-GlcNAcase, the enzyme that removes O-GlcNAc from proteins, have been developed recently.[114,115] A new O-GlcNAcase inhibitor, thiamet-G, has been shown to decrease tau phosphorylation at pathologically relevant sites both in phaeochromocytoma cell line PC12 cells and in rat brains.[116] These inhibitors and their future derivatives have the potential to be

3.1.5 Upregulation of Brain Glucose Metabolism and b-N-Acetylglucosamine Modification of Tau

In addition to tau kinases and phosphatases, alterations of tau itself, the substrate of these enzymes, also play a role in its hyperphosphorylation and aggregation. Besides phosphorylation, the serine/threonine residues of tau are also modified by a monosaccharide called b-N-acetylglucosamine (GlcNAc) via a glycosidic bond, and this modification is called O-GlcNAcylation.[101-103] Most interestingly, O-GlcNAcylation regulates phosphorylation of tau inversely both in vitro and in vivo.[102-106] Tau O-GlcNAcylation was found to be decreased in the AD brain, and this decrease correlates to tau hyperphosphorylation.[105] A similar phenomenon has also been seen for neurofilaments.[89] Because tau O-GlcNAcylation is directly regulated by glucose metabolism supplying uridine diphosphateGlcNAc, a donor for protein O-GlcNAcylation, the abovementioned observations led us to propose a novel hypothesis that explains the molecular mechanism by which impaired glucose uptake/metabolism in the AD brain, well estabª 2010 Adis Data Information BV. All rights reserved.

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used to inhibit tau hyperphosphorylation via an increase in tau O-GlcNAcylation. 3.2 Inhibition of Tau Misfolding/Aggregation

In addition to PHF-tau and normal tau, the AD brain also contains abnormally hyperphosphorylated tau as oligomers in the cytosol.[9,14,117-119] Tau pathology may consist of several stages, including abnormal hyperphosphorylation, misfolding/oligomerization and polymerization into PHFs/SFs.[120] Earlier studies showing a strong correlation between the numbers of NFTs in the brain and the severity of dementia[18-20] suggested that the aggregated NFTs might cause neurodegeneration. This view was challenged recently by studies demonstrating that the unpolymerized abnormal tau or its oligomers, rather than the highly polymerized PHFs, is toxic. In a conditional transgenic mouse model that expressed tau with FTDP-17 P301L mutation and showed NFT pathology together with neuronal loss and behavioural deficits, when the mutant tau expression was turned off at 4 months of age, the removal of mutant tau led to reversal of the behavioural deficits, whereas NFT pathology continued to progress.[121] In vitro, polymerization of hyperphosphorylated tau into PHFs also abolishes its toxic activity to sequester other MAPs.[35] Thus, in the same way that Ab plaques are viewed, NFTs could be regarded as markers of damage that has already occurred rather than as a primary cause of neurodegeneration. Indeed, polymerization of toxic abnormal tau into PHFs/NFTs could even be a defence mechanism by which neurons attempt to reduce the toxic activity of the abnormal cytosolic tau. The fact that the tangle-bearing neurons seem to survive for many years[122] and that the decrease in microtubule density in the AD brain is unrelated to PHF accumulation[123] is consistent with such a self-defence role for the formation of tangles. Andorfer et al.[124] showed that in human tau transgenic mice, while there was widespread neurodegeneration, the PHF-containing neurons appeared ‘healthy’ in terms of nuclear morphology, which also suggests that the polymerization of abnormal tau was probably neuroprotective. ª 2010 Adis Data Information BV. All rights reserved.

This phenomenon is apparently common in other diseases characterized by abnormal protein aggregates, such as Huntington’s disease and cardiomyopathy, in which the abnormal non-fibrillar protein oligomers, rather then the protein aggregates themselves, appear to be pathogenic.[125-127] Efforts have been made to find small-molecule compounds that can inhibit tau polymerization as another approach to treating AD. These compounds include methylene blue (methylthioninium chloride) [phenothiazines], N774, daunorubicin (anthracyclines), phenylthiazolyl-hydrazides, Nphenylamines, rhodanines, exifone (polyphenols), quinoxalines and aminothienopyridazines, and have recently been reviewed by Brunden et al.[128] The first compound that was reported to inhibit tau aggregation was the dye methylene blue, which was also shown to alter the structure of existing PHFs isolated from the AD brain.[129] A recent phase II clinical trial for AD indicated a positive therapeutic effect of this compound.[130] A larger phase III study is required to prove its efficacy. High-throughput screening of compound libraries has been used to search for inhibitors of tau aggregation. Using thioflavine S fluorescence to monitor fibrillization of a three-repeat tau fragment, the Mandelkow group has screened approximately 200 000 compounds.[131] This led to the identification of a number of anthraquinone inhibitors of tau fibril formation, including daunorubicin and adriamycin (doxorubicin). Another group has screened >290 000 compounds at six concentrations.[132] A total of 285 compounds showed complete dose-dependent inhibition of tau assembly, and a unique set of aminothienopyridazine inhibitors with drug-like physical and chemical attributes was identified. Targeting tau aggregation for the treatment of AD is intriguing but faces many challenges. First, as already discussed, recent studies have suggested that polymerization of tau into NFTs is probably a defence mechanism of the affected neurons. Inhibition of tau polymerization into NFTs and disassociation of NFTs might actually do more harm than benefit. However, if we can find some compounds that inhibit the initial oligomerization instead of the final polymerization Drugs Aging 2010; 27 (5)

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of tau, these inhibitors would hold some promise. Most investigations to date have not differentiated between inhibition of early oligomerization and inhibition of late polymerization. Second, most studies so far have used artificial tau or tau fragment polymerization assays in which the polymerization is induced by polyanions, such as heparin. Although these assays have facilitated screening, the co-assembly of tau with polyanions appears to involve a mechanism different from that seen in AD and in tauopathies. Polyanion-induced assembly of tau is very slow and does not result either in the lateral association of filaments into tangles or the formation of any protofilaments seen in AD PHFs. Furthermore, unlike AD and related tauopathies and transgenic animal models, in vitro polyanion-induced assembly of tau into filaments is inhibited and not promoted by phosphorylation.[133] Thus, the compounds that inhibit polyanion-induced assembly of tau might not inhibit tau aggregation in tauopathies, including AD. Third, many of the existing tau assembly inhibitors have chemical or biological properties that probably make them unsuitable for use in vivo, and at least some may act through the generation of reactive species or by covalent modifications that increase the potential for offtarget effects. 3.3 Increasing Tau Clearance

Degradation of tau, especially misfolded/ aggregated tau, may occur through two major pathways: (i) the ubiquitin-proteasome system (UPS); and (ii) macroautophagy. With the UPS, misfolded proteins are first tagged with polyubiquitins and subsequently degraded by the proteasome complex. Large multimeric tau aggregates are unlikely to be degraded by this mechanism because it requires the threading of the targeted protein into a narrow opening of the proteasome. These tau aggregates are thus degraded through macroautophagy, which requires encapsulation by an autophagosome and subsequent fusion with a hydrolase-containing lysosome.[134] Both the UPS and macroautophagy systems appear to be affected and to contribute to ª 2010 Adis Data Information BV. All rights reserved.

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the accumulation of aggregated tau in the AD brain.[134-138] Thus, promoting one or both of these catabolic systems could theoretically lead to a reduction in pathological tau in AD and other tauopathies. Inhibitors of heat shock protein 90 (HSP90) have been shown to promote tau clearance. In cells overexpressing mutated human tau, several HSP90 inhibitors reduce the levels of phosphorylated tau.[139] Treating transgenic mice that express human tau with BBB-permeable HSP90 inhibitors reduces the amount of hyperphosphorylated tau in the mouse brains.[140,141] This action of HSP90 inhibitors might be because HSP90, as a molecular chaperone, prevents protein degradation via the UPS. Although HSP90 inhibitors appear to hold promise for reducing phosphorylated and misfolded tau through the UPS, it is unlikely that this strategy would help clear larger tau oligomers and aggregates because of their multimeric size. Large protein aggregates, including those involved in neurodegenerative diseases, can be removed via the autophagic clearance system.[142,143] It has been shown that treatment of flies that express wild-type or mutated tau with rapamycin results in a reduction of insoluble tau and associated toxicity by inducing macroautophagy.[144] In contrast, the addition of the autophagy inhibitor 3-methyladenine led to enhanced tau accumulation and aggregation.[145] Thus, upregulation of the autophagy-lysosomal system with drugs such as rapamycin might be a potential strategy for the treatment of AD. However, rapamycin affects mammalian target of rapamycin (mTOR) signalling and, thus, has pleiotropic effects, including immunosuppression. In this regard, inhibition of an mTOR-independent target, such as inositol monophosphatase, with lithium has been shown to cause upregulation of autophagy and an increased clearance of asynuclein that forms intracellular inclusions in the Parkinson’s disease brain.[146] Tau vaccination is another approach that was investigated recently for the removal of abnormal tau in the diseased brain. The first tau vaccination study was carried out in transgenic mice overexpressing mutated tau P301L.[147] The investigators Drugs Aging 2010; 27 (5)

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used a peptide corresponding to tau379–408, with the phosphorylation of serine 396 (Ser396) and Ser404. Animals were immunized with the immunogen for 2–5 months while the controls received the adjuvant alone. These investigators found reduced tau pathology and improved behavioural performance as tested using the rotarod and traverse beam compared with controls treated with adjuvant alone. Another study in two-tangle mouse models indicated that immunization with a phosphotau derivative reduces aggregated tau in the brain and slows progression of the tangle-related behavioural phenotype.[148] Using antibodies against misfolded tau to clear the pathological tau has also been proposed.[149] However, at present it is not understood how tau vaccination might work, because, unlike amyloid, pathological tau deposits are intraneuronal, and it was assumed that antibodies directed at tau would not be internalized into neurons and would therefore fail to come into contact with their target. However, extracellular toxic tau has been described in the AD brain recently.[150,151] Since the tau immunotherapy approach is only in the early stages of investigation, it remains to be studied whether it works by removal of extracellular tau. 4. Perspective To date, most efforts at developing diseasemodifying drugs for AD have been directed at Ab-based targets. Overall, the results have been very disappointing. It is time to rethink and invest more on other hypotheses and pathways than the amyloid cascade hypothesis. The AD field has now started to recognize tau-mediated neurodegeneration as a promising target for investigation, and more studies are expected in this area in the near future. It is obvious that many challenges in targeting tau for the development of treatments for AD will need to be overcome on the road to success. For instance, as most kinases regulate several cellular processes, potential adverse effects of tau kinase inhibitors could be a serious challenge to overcome. Although kinase inhibitors have been used successfully in oncology, it remains to be determined whether these ª 2010 Adis Data Information BV. All rights reserved.

molecules can be safely administered on a longterm basis for the treatment of tauopathies. Sporadic AD is multifactorial and heterogeneous. Although it ends up with the same main pathologies and similar clinical symptoms, various sporadic AD cases could have different causes and develop the disease via different pathways. Based on CSF levels of proteins associated with plaques and tangles, five different subgroups of AD have been identified.[152] It is possible that one drug might be effective for only one or two, but not all, subgroups of AD.[153] Thus, stratifying the patient population into various AD subgroups could greatly facilitate the success of AD clinical trials. An example is a recent clinical trial with rosiglitazone that showed some efficacy only in a subset of patients who did not carry the apoE4 allele.[112] Because of the multifactorial nature of sporadic AD, it is possible that treating only a single target might not be sufficient to yield significant benefit. As multiple insults during aging reach the level of the threshold that collectively results in sporadic AD, simultaneous treatment with drugs for different targets might be needed for significant therapeutic efficacy. Acknowledgements Studies carried out in our laboratories were supported in part by the New York State Office of Mental Retardation and Developmental Disabilities and National Institutes of Health grants AG019158, AG028538, AG27429, AG031969 and TW008123. The authors have no conflicts of interest that are directly relevant to the content of this article. The authors are grateful to Janet Murphy for secretarial assistance.

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Correspondence: Dr Khalid Iqbal, Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA. E-mail: [email protected]

Drugs Aging 2010; 27 (5)