MARKing tau for tangles and toxicity

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Vol.29 No.10 October 2004

MARKing tau for tangles and toxicity Gerard Drewes Cellzome AG, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

In healthy neurons, tau proteins regulate microtubule function in the axon. In the brains of individuals with Alzheimer’s disease, tau is hyperphosphorylated and aggregated into intraneuronal deposits called neurofibrillary tangles (NFTs). Hyperphosporylation dislodges tau from the microtubule surface, potentially resulting in compromised axonal integrity and the accumulation of toxic tau peptides. Recent biochemical and animal model studies have re-evaluated tau phosphorylation and other aspects of neurofibrillar pathology. The results indicate that phosphorylation of tau’s microtubule-binding domain by the protein kinase MARK primes tau for hyperphosphorylation by the kinases GSK-3 and Cdk5, which in turn triggers the aggregation of tau into filaments and tangles. Toxic consequences for the neuron might be exacerbated by tangle formation but are already evident during the early steps of the process. The loss of neurons is a hallmark of common neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease or ischaemic stroke, but the molecular events leading to cell death are not well understood. In Alzheimer’s disease, the amyloid cascade hypothesis, which is firmly built on genetic evidence, proposes that the overproduction of some secreted AMYLOID-b (Ab) PEPTIDES (see Glossary), derived from the proteolytic processing of b-amyloid precursor protein (b-APP), can initiate a cascade of events leading to neuronal death and dementia. This cascade links, by as yet unknown mechanisms, events such as disrupted Ca2C homeostasis, hyperphosphorylation and aggregation of TAU PROTEIN, neuronal dysfunction and death, and inflammatory responses [1,2]. The characteristic neuropathology of Alzheimer’s disease is defined by the presence of two types of lesion in the brain, which are usually diagnosed by post mortem microscopic examination: extracellular plaques containing aggregated Ab peptides, and intracellular NEUROFIBRILLARY TANGLES (Nfts) formed by twisted filaments mainly consisting of hyperphosphorylated tau protein. The presence of both plaques and NFTs is required for the unequivocal diagnosis of Alzheimer’s disease, and the amounts of each correlate with the degree of dementia [2]. This review focuses on recent findings that several distinct phosphorylation events contribute to the development of neurofibrillary pathology, discusses the evidence that implicates MARKs as a key factors early in the process Corresponding author: Gerard Drewes ([email protected]).

leading to neurotoxicity, and briefly reviews the biology of MARK kinases. The tau–tangle–toxicity connection A significant number of amyloid plaques is often diagnosed in healthy octagenarians and nonagenarians, suggesting that amyloid alone is not sufficient to cause dementia. Conversely, even in the absence of plaque formation the presence of NFTs is linked to neurodegeneration, as is observed in a few rare forms of tangle-only dementia that are now referred to as ‘tauopathies’, some of which are autosomal dominant syndromes caused by mutations in the gene encoding tau [3]. Moreover, the tangle load in the brains of individuals with Alzheimer’s disease shows a better correlation with dementia than does the amyloid plaque load [2,3]. Thus, as the integral component of NFTs, tau protein emerges as a primary suspect in neuronal death, although it is not clear whether it is the tau filaments themselves or other tau forms such as soluble oligomers or hyperphosphorylated species that are the main culprit. Notably, a similar debate has persisted for a long time in the amyloid field, where small oligomers rather than fibrils are now emerging as the prime mediators of neurotoxicity [1]. Obviously, this issue has important implications in terms of which molecular step to target in the development of disease-modifying therapeutic agents.

Glossary Amyloid-b (Ab) peptides: Chief components of the senile plaques found in the brains of individuals with Alzheimer’s disease. Ab peptides are generated from the transmembrane domain of b-amyloid precursor protein (b-APP) by two successive proteolytic steps. Cyclin-dependent kinase 5 (Cdk5): A predominantly neuronal member of the family of cyclin-dependent protein serine/threonine kinases. Glycogen synthase kinase 3 (GSK-3): A family of two serine/threonine kinases with pleiotropic functions in cellular growth, motility and apoptosis. Microtubules: Cytoskeletal polymers of tubulin that are crucial for maintaining cellular structure, transport processes and motility. Microtubule-associated proteins (MAPs): Proteins that regulate microtubule function, such as stability or organelle transport, by binding to microtubules. MAP/microtubule affinity regulating kinase (MARK): A family of four serine/ threonine kinases in mammals that are orthologues of yeast KIN1 and C. elegans and D. melanogaster PAR-1. Neurofibrillary tangles (NFTs): Bundles of aggregated paired helical or straight filaments found in the brains of individuals with Alzheimer’s disease and some other forms of dementia. Tangles consist mainly of hyperphosphorylated, partially degraded tau but might contain other proteins as well. Partitioning-defective (PAR) kinase family: Six structurally unrelated proteins discovered in the nematode C. elegans that are important for early asymmetric divisions of the zygote. PAR-1 and PAR-4 are protein serine/threonine kinases that are orthologous to mammalian MARK and LKB1. Tau protein: A neuron-specific microtubule-associated protein present in six isoforms localized in the axons of neurons, where it maintains, together with other MAPs, the integrity of the axon.

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The brains of transgenic mice overexpressing mutant b-APP contain amyloid plaques but no concomitant NFTs nor any evident synaptic dysfunction or neurotoxicity, indicating that NFTs – or hyperphosphorylated tau species as an early step in tangle formation – are prerequisite for neurotoxic effects. Recent mouse models overexpressing mutant forms of b-APP, presenilin and tau do, however, show plaque and tangle formation, as well as synaptic defects, suggesting that Ab peptides can trigger or accelerate a retrograde signalling cascade that leads to hyperphosphorylation of tau, with the consequences of tau aggregation and neurotoxicity [2,4]. Researchers have been intrigued by tau hyperphosphorylation for more than a decade, prompted by the availability of antibodies raised against tangle preparations that recognize defined serine or threonine phosphoepitopes on tau and can thereby implicate the causative protein kinases in the pathology. Furthermore, hyperphosphorylated tau purified from brain tissue from individuals with Alzheimer’s disease has lost its ability to bind to MICROTUBULES, a property that can be restored by dephosphorylation of tau [5,6]. Tau kinases: many suspects, few clues The list of around 20 kinases that have been reported to phosphorylate tau in biochemical studies has created a certain amount of confusion in the hunt for the actual culprit [3]. Because the mapping of most phosphoepitopes identified serine-proline or threonine-proline motifs, the proline-directed kinases of the mitogen-activated protein kinase (MAPK) families [7] – CYCLIN-DEPENDENT KINASE 5 (CDK5) [8,9] and the b-isoform of GLYCOGEN SYNTHASE KINASE 3 (GSK-3) [10,11] – feature prominently among the usual suspects, and compelling evidence indicating that Cdk5 and GSK-3b represent principal tau kinases in vivo has been provided [12]. To understand why hyperphosphorylation of tau might be bad for cells, it is necessary to contemplate the physiological consequences of tau phosphorylation. Tau proteins, which constitute a family of MICROTUBULE-ASSOCIATED PROTEINS (Maps) encoded by a single gene giving rise to six neuron-specific splice forms, strictly colocalize with microtubules mainly in the axon and are thought to be involved in maintaining axonal integrity and in regulating axonal transport [6,13]. Phosphorylation of tau controls tau binding to microtubules and, because bound tau stabilizes the microtubule polymer, this phosphorylation potentially provides a mechanism for regulating the stability of microtubules in a spatial and temporal fashion [13,14]. Mutational analysis of the distinct phosphorylation sites on tau has shown that the effect of the abovementioned serine-proline and threonine-proline phosphoepitopes on the affinity of tau for microtubules is less pronounced than is the effect of a different type of phosphoepitope located in the tubulin-binding domain of tau. This second type of phosphoepitope does not contain proline residues, but is characterized by the sequence Lys-(Ile/Cys)-Gly-Ser (KXGS). A single KXGS motif is located in each of the three or four (depending on the isoform) repeated sequences that constitute the www.sciencedirect.com

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tau microtubule-binding domain [13,15]. In addition to phosphorylation at the serine-proline and threonine-proline sites, phosphorylation of tau at these KXGS motifs has been found to be increased in brain tissue taken from individuals with Alzheimer’s disease. It is not surprising that the KXGS-type phosphorylation exerts a stronger effect on microtubule binding, because the serine-proline and threonine-proline sites are not part of microtubule-binding domain of tau, but cluster in adjacent sequence regions. Several kinases have been shown to phosphorylate the KXGS motifs in vitro, albeit mostly with low efficiency [16]. A major ‘KXGS kinase’ activity was purified from brain and was indeed shown to detach tau from microtubules and to destabilize microtubules in vitro and in cultured cells; thus, this kinase was called MAP/MICROTUBULE AFFINITY REGULATING KINASE (MARK) [15]. Among the four MARKs present in humans (Table 1), MARK4 represents the most intriguing candidate for a role in the pathological events leading to NFT formation, because its expression is relatively pronounced in brain tissue and is specifically localized along microtubules, and because it accumulates at the tips of neuritic processes, where the regulation of microtubule dynamics is crucial for process outgrowth [17]. It is thus tempting to speculate that in Alzheimer’s disease an increase in MARK activity might occur in response to some insult, resulting in the loss of axonal–dendritic polarity and eventually in cell death [18]. It has been recently shown that MARK4 is rapidly upregulated in neurons after induced focal ischaemia in mouse – an accepted model of stroke that has been shown to involve programmed cell death dependent on changes in gene expression [19]. Notably, another protein kinase, LKB1, that can also activate MARK4 (see below), was among the 4 protein kinase genes found to be upregulated out of a total of 56 known genes examined in this model [19]. New insight from mice and flies Important evidence further dissecting role of MARKs and the proline-directed kinases GSK-3 and Cdk5 in taurelated neurodegeneration has been recently presented from elegant models of tauopathies in the fruitfly Drosophila melanogaster [10,20–22]. In these models, overexpression of PAR-1, a member of the PARTITIONDEFECTIVE (PAR) KINASE FAMILY and the fly orthologue of mammalian MARKs, causes mild neuronal degeneration, which is manifest as a loss of photoreceptor cells. Bingwei Lu and colleagues [21] recently showed that this phenotype is dependent on the presence of endogenous fly tau, because overexpression of PAR-1 in heterozygous tau deletion mutants showed less pronounced degeneration. PAR-1 mutants lacking kinase activity did not induce neurotoxic effects, indicating that toxicity is due to a specific phosphorylation event rather than to an unspecific effect of overexpression. Lu and colleagues went on to overexpress PAR-1 in flies expressing human tau and observed that the moderate eye degeneration induced by exogenous expression of wild-type human tau, or a tau mutant associated with frontotemporal dementia [22], was exacerbated by overexpression of PAR-1.

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Table 1. Mammalian MARK kinases and their substratesa Isoform GenBank ID and human gene locus MARK1 AB040910 1q42.11 MARK2 X97630 11q12-q13

MARK3 U64205 14q32.3

Synonyms

Expression (by northern blotting) and representation in EST databases

Substrates

Functional consequence of phosphorylation

Refs

EMK3; hPAR-1c

High in brain, spleen, skeletal. muscle, pancreas, kidney and heart; low in lung and liver; 89 ESTs available Similar to MARK1: high in brain, spleen, skeletal muscle, pancreas, placenta, kidney and heart; low in lung and liver; 187 ESTs available

Tau, MAP2, MAP4

Loss of microtubule binding and thinning of microtubule array

[15]

Tau, MAP2, MAP4

Loss of microtubule binding and thinning of microtubule array

[15,23]

Dcx

Loss of Dcx-microtubule binding and increase microtubule dynamics in growth cone 14–3-3 binding , localization

[54]

EMK1; hPAR-1b

EMK2; hPAR-1a; KP78; C-TAK1

Highest in brain and pancreas; 417 ESTs available

PTPH1

Cdc25C KSR1 Plakophilin 2 Dishevelled (?) MARK4 AY057448 19q13.3

MARKL1; hPAR-1d

High in brain, glioma, testis; low in most other tissues; 162 ESTs available

tau, MAP2, MAP4

14–3-3 binding, localization 14–3-3 binding, localization 14–3-3 binding, localization Increased b-catenin-dependent signalling Microtubule bundling and thinning of microtubule array

[60]

[58] [59] [57] [56] [17,19,69]

a Abbreviations: Cdc25C, cell division cycle 25C; C-TAK1, Cdc25C-associated kinase 1; Dcx, Doublecortin; EMK, ELKL motif kinase; EST, expressed sequence tag; KSR1, kinase suppressor of Ras; MAP, microtubule-associated protein; MARK, MAP/microtubule affinity regulating kinase; PAR, partitioning-defective; PTPH1, protein tyrosine phosphatase H1.

Flies possess only a single PAR-1 kinase gene, which enabled Lu and colleagues [21] to strengthen further the physiological relevance of the interaction between PAR-1 and tau by a loss-of-function approach. Such an approach might not be feasible in mice, which, like humans, express four MARK/PAR-1 genes that probably have partially redundant functions. Indeed, mice null for MARK2 [23,24] or MARK3 (M. Darmon, pers. commun.) are viable, whereas flies lacking PAR-1 are not. Lu and co-workers [21] circumvented the embryonic lethality of PAR-1 deletion by inducing tissue clones of homozygous mutant cells in the brain of heterozygous flies that also expressed human mutant tau, and they observed that removing PAR-1 resulted in a threefold drop in neuronal death as compared with par-1C cells. A similar attenuation of neurotoxic effects was achieved by overexpressing tau carrying point mutations at two of the serines in the microtubule-binding domain that are targeted by PAR-1 [21]. Interestingly, the introduction of these mutations abolished not only phosphorylation by PAR-1, as expected, but also phosphorylation at the epitopes mediated by proline-directed kinases such as GSK-3 and Cdk5, implying that PAR-1 might trigger an ordered multistep phosphorylation cascade. Notably, this interdependency of the different phosphorylation events was not observed in previous in vitro studies, where tau was used as a free substrate rather than in its microtubule-bound form [14]. Because it is known that mammalian MARK/PAR-1 can dislodge tau from microtubules [25], it is likely that the regions of tau containing the sites for proline-directed kinases become accessible only after dissociation of tau from the microtubule. Alternatively, these sites might be generally inaccessible www.sciencedirect.com

in conformations of tau containing a dephosphorylated microtubule-binding domain, or MARK phosphorylation might create docking sites for other kinases on tau [21,26]. Importantly, Lu and colleagues [21] did not observe the formation of NFTs in their models, supporting the notion that NFTs, much like amyloid plaques, represent ‘signsignposts’ of damage that has been already done. Together with older biochemical studies on tau phosphorylation and function [14] and with reports describing the formation of NFTs caused by overexpression of the GSK-3 orthologue Shaggy in flies [10] and Cdk5 in mice [8], the PAR-1 fly model data are consistent with a cascade of events, which starts with the phosphorylation of microtubule-bound tau on its microtubule-binding domain by MARK/PAR-1 [Figure 1(i)]. Phosphorylation by MARK/PAR-1 triggers dissociation of tau from the microtubule surface, which, potentially, causes destabilization of the stable axonal microtubule system or changes the properties of the microtubule surface for cellular transport processes [27]. In addition, the phosphorylation by MARK/PAR-1and primes tau for further hyperphosphorylation, first by Cdk5 and then by GSK-3 [9,27] [Figure 1(ii)]; up to this stage, the process is probably reversible via the action of phosphatases. As a result of its dissociation from the microtubules, the hyperphosphorylated tau becomes delocalized to the somatodendritic compartment and, possibly catalysed through the formation of smaller, more aggregation-prone tau fragments through proteolytic cleavage [28] [Figure 1(iii)], begins to form aggregates. These aggregates then grow into filaments by further sequestration of soluble full-length tau [29] [Figure 1(iv)] and ‘mature’ by other covalent modifications, slowly rendering them insoluble [30]. It is unclear whether a proteolytic

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Cell body

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Insult: Aβ? Ca2+? 1 2 3 4

tau

1 2 3 4

Axon 1 2 3 4

GSK-3

1 2 3 4

1 2 3 4

Cdk5 1P 2 3 4P

(i)

P P 1P

(ii) P 1

2 3 P4

1 2 3 4

MARK

1 2 3 4

P

2 3 4P

P

P1

P

P

2 3 P4 P P

1 2 3 4

(iii) Microtubule Synapse

(vi)

Tangles

P 1 4 1 3 P2 1 34 2 2 3 31 4 1 42 4 3 3 24 P P4 1 3 23 2 4 1 P 2 P 1 13 2 42 2 3 1 4 3 31 4 24 P 1 P

Filaments

P

(v)

P 1 4 21

P

(iv)

P

Protease

1P 2 3 4P

2 23 3 3 4 P4 1 P

Oligomers

P

1P 2 3 4P Ti BS

Figure 1. Proposed cascade of events leading to neurofibrillary pathology. Axonal microtubules (red) function as tracks for synaptic vesicles. Microtubule function is regulated by bound tau protein (blue), which binds to the microtubules via repeats 1–4 in its microtubule-binding domain. (i) Activation of microtubule-associated protein (MAP)/microtubule affinity regulating kinase (MARK), possibly by an external insult to the cell such the disruption of intracellular Ca2C homeostasis by amyloid peptides, triggers phosphorylation of tau on repeats 1 and 4 of its microtubule-binding domain. (ii) Phosphorylation triggers the dissociation of tau from the microtubule surface, potentially, destabilizing the microtubule or changing the properties of the microtubule surface for transport processes. The phosphorylation by MARK primes tau for further hyperphosphorylation, first by cyclin-dependent kinase 5 (Cdk5) and then by glycogen synthase kinase-3 (GSK-3). Up to this stage, the process is probably reversible by the action of phosphatases. (iii) Unbound hyperphosphorylated tau delocalizes to the somatodendritic compartment and can be subject to proteolytic cleavage. (IV–VI) The resulting smaller, more aggregation-prone fragments of tau begin to aggregate into oligomers (iv), which might further sequester soluble full-length tau and form filaments (v), which are finally deposited as neurofibriallry tangles (vi). Abbreviation: Ab, amyloid-b peptide.

event is absolutely required for filament formation because filaments do contain full-length tau [3]. It is possible that other, as yet unidentified, components are necessary to trigger filament formation or even to coassemble with tau [31]. In contrast to what one might expect, purified bacterially expressed human tau has been shown to have a lower tendency to form filaments in vitro when phosphorylated at KXGS motifs [32]. Possibly, a distinct unknown combination of phosphorylation and other modifications, or a combination of phosphorylated and non-phosphorylated tau, is essential for initiating aggregation or, alternatively, the conditions in the aging neuron differ from those reconstructed in vitro. The fly and transgenic mouse models indicate that significant neurotoxic signals have been already transmitted before the stage of actual NFT formation, but it is not clear what the toxic species is at this stage, or to what degree and by what mechanism NFT formation might aggravate the damage [12]. Resolving this issue will be important for determining which molecular event to target for therapeutic intervention. If hyperphosphorylated tau itself, or the sarkosylinsoluble tau aggregates that precede actual tangles, www.sciencedirect.com

were to exert toxic effects by itself, for example, then a strategy aimed at dissolving NFTs would risk the release of significant amounts of toxic species. MARKs are evolutionarily conserved regulators of the cytoskeleton Important clues to the physiological roles of the MARK/PAR-1 kinases can be drawn from studies of orthologous genes from yeast to nematodes and flies [33–37]. The asymmetrically localized PAR proteins and protein complexes were first identified in Caenorhabditis elegans, and their concerted activities are important for cell polarity with roles in establishing the embryonic body axis and in cell fate decisions, as well as in maintaining functional differentiated cells. The emerging view is that PAR proteins cooperate to convert transient polarity cues into a stable polarized cellular axis by influencing diverse processes such as cytoskeletal dynamics or protein degradation [38,39]. Microtubules are important determinants of cell polarity in many cell types and, indeed, data from model organisms, and recently also from studies on polarized mammalian cell types, suggest that at least some of the effects of MARK/PAR-1 on cell polarity are mediated via

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microtubules [40–44]. For example, MARK2 is important for maintaining polarity in neuronal and epithelial cells and has been recently shown to promote process outgrowth in neuronal cells and reorganization of the microtubule network in developmental branching decisions of epithelial cells [45,46]. Two of the six nematode PAR proteins are protein kinases. In addition to PAR-1, the orthologue of the mammalian MARKs (Figure 2), PAR-4 is the orthologue of the mammalian serine/threonine kinase LKB1. In humans, loss-of-function mutations in LKB1 cause an autosomal dominant disorder characterized by gastrointestinal polyps and neoplasms, probably because the failure to establish cell polarity causes an improper (a)

(b)

AMPK subfamily hMARK1 hMARK3 hMARK2 hMARK4 dPAR1 CePAR1 SpKIN1 ScKIN1 ScKIN2

(c)

LKB1 PKC-λ

?

TAO-1 P

P

P

LDTFCGSPP

Catalytic

RSTFH

UBA 311 336 369

1 60

Spacer

KA1 685

793

14–3-3 binding Ti BS

Figure 2. Structure and regulation of microtubule-associate protein (MAP)/microtubule affinity regulating kinase (MARK) and partition-defective 1 (PAR-1) family kinases. (a) MARK/PAR-1 kinases form a subfamily of the AMP-dependent protein kinase (AMPK) family. In humans, four genes and 28 pseudogenes encode MARKs. The phylogenetic tree shows the relationship between the four human MARK gene products (GenBank accession numbers: MARK1, AB040910; MARK2, X97630; MARK3, U64205; MARK4, AY057448) and their orthologs from Drosophila melanogaster (PAR-1, AF258462), Caenorhabditis elegans (PAR-1, U22183), Schizosaccharomyces pombe (KIN1, M64999) and Saccharomyces cerevisiae (KIN1, M69017; KIN2, M69018). (b) Conserved domain structure of the MARK/PAR-1 kinases. A short diverse amino-terminal sequence is followed by the catalytic domain and a ubiquitin-associated (UBA) domain, which might be involved in interactions with other proteins in a ubiquitin-dependent fashion [50]. The carboxyterminal kinase-associated (KA1) domain contains two amphipathic helices (according to the NMR structure, PDB code: 1ul7) and is unique for this kinase family; its presence in some MARKs is dependant on alternative splicing [69]. The function of KA1 is unknown. All four human MARKs are activated by phosphorylation of a conserved threonine residue by PAR-4 (also known as LKB1) [49,50], and MARK2 can be also activated by the thousand and one amino acids kinase (TOA-1) [52]. The nearby serine-proline motif has been found to be phosphorylated by a unknown kinase in MARKs purified from brain [15], and this phosphorylation seems to confer inhibition [52]. The serine in the spacer domain is phosphorylated by protein kinase C-l (PKC-l) and regulates membrane localization, which can subsequently lead to inhibition [53]. In flies and in human cells, MARK/PAR-1 is in a complex with 14–3-3 family proteins, which bind to the catalytic domain with a region on 14–3-3 located outside the known phospholigand-binding pocket [50,62]. (c) Homology model of the catalytic domain of MARK1 created using Swissmodel [70], showing the activation loop (pink) and the position of the Thr215 and Ser219 side chains that regulate activation of the kinase. www.sciencedirect.com

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overgrowth of differentiated cells [47,48]. Intriguingly, it has been recently shown, in both biochemical studies and human cells, that LKB1 can interact with and activate all four MARKs by phosphorylating their catalytic domains directly on a conserved threonine residue in their regulatory loop [49,50] (Figure 2). LKB1 forms complexes with the pseudokinases STRAD or PAPK, and coexpression of LKB1 and either STRAD or PAPK redistributes the complex to areas of cell–cell contact that are known to contain PAR-1 family kinases and the polarity complex comprising PAR-3, PAR-6 and protein kinase C-l (PKC-l) [50,51]. These events constitute an emerging kinase cascade that could induce phosphorylation of tau but is likely to have other downstream effects. At least one other kinase, the thousand and one amino acids kinase TAO-1, has the ability to activate MARK2 and possibly MARK3, but its effect seems to be less pronounced, and in cultured mouse embryonic fibroblasts it does not seem to affect MARK1 or MARK4 [49,52]. Phosphorylation of the MARK regulatory loop by the LKB1 complex or by TAO-1 is not the only regulatory mechanism of MARK activity, because it has been recently shown that MARK4 can form a complex with PKC-l [50]. As mentioned above, PKC-l is also involved in cell polarity as a member of a polarity complex with PAR-3 and PAR-6, and it has been shown to phosphorylate a serine residue in the carboxy-terminal ‘spacer’ domain of MARK2 and MARK3, which, at least for MARK1 and MARK2, negatively regulates the peripheral membrane localization of the active kinases and, apparently as a consequence, induces their inactivation [53]. Whereas large parts of the ‘spacer’ domain show relatively little sequence conservation, the serine phosphorylated by PKC-l is present in all mammalian MARKs, and in the fly and nematode but not the yeast orthologues. Since the discovery of the mammalian MARKs as tau and MAP (microtubule-associated protein) kinases [15], several more putative substrates have been identified (Table 1). In an analogous way to tau and its paralogues MAP2 and MAP4, MARK phosphorylates and regulates the microtubule-binding activity of Doublecortin, which is involved in neuronal growth cone motility through its phosphorylation at a single serine residue. This residue is mutated in individuals affected with X-linked lissencephaly, a neuronal migration disorder [54]. In flies, PAR-1 can associate with and phosphorylate Dishevelled, a canonical component of the Wingless/Wnt signalling pathway. The phosphorylation of Dishevelled by PAR-1 increases activation of b-catenin and blocks relay of the signal to the Jun amino-terminal kinase (JNK) pathway [55]. A similar mechanism also seems to operate in mammalian cells, because the activation of a b-catenindependent transcriptional reporter by MARK3 has been recently shown in HeLa cells. The effect is suppressed by LKB1, indicating that the LKB1–MARK pathway is distinct from, and possibly competes with, the role of MARK/PAR-1 in Wnt signalling [56]. Intriguingly, the Wnt signalling pathway has been proposed to have a role in Alzheimer’s disease because GSK-3b and b-catenin bind to components of the b-APP processing machinery,

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although it is unclear which pathological molecular processes are actually affected by this binding [2,10]. MARKs regulate several 14–3-3 protein complexes A very interesting string of observations has come from Piwnica-Worms and colleagues [57–60], who have shown that MARK3 (also known as CTAK-1 or hPar-1b) phosphorylates 14–3-3-binding motifs on at least four different substrates: the tyrosine phosphatase PTPH1; the cellcycle phosphatase CDC25; the suppressor of Ras/MAPK signalling KSR; and the desmosomal protein plakophilin-2. In each case, phosphorylation of the substrate by MARK3 induces the formation of a complex between the substrate protein and 14–3-3, which changes the cellular localization of the substrate. 14–3-3 proteins are phosphoserine adaptor proteins with pleiotropic cellular functions in, for example, protein translocation and vesicle trafficking [61]. The functional link between phosphorylation by MARKs and the generation of 14–3-3-containing protein complexes becomes even more remarkable in light of the findings that one of the C. elegans PAR proteins, PAR-5, is a 14–3-3 protein, and that 14–3-3 proteins interact directly with MARK/PAR-1 in cultured human cells and in flies [50,62]. 14–3-3 proteins form dimers with a groove containing one phosphoserine-binding pocket per monomer to accommodate the interacting proteins [61]. Interestingly, binding of MARK/PAR-1 to 14–3-3 does not involve the phosphoserine-binding pocket; instead, the kinase binds via its catalytic domain to 14–3-3 outside the groove [62] and remains associated with its substrates (probably via 14–3-3) after the target site on the substrate is phosphorylated and 14–3-3 is bound. Thus, MARK/PAR-1 can target substrates to 14–3-3 and might be able to rephosphorylate sites quickly after their dephosphorylation and the disruption of 14–3-3 binding [57]. In line with these observations, MARK immunoreactivity has been found to colocalize with tau bearing phosphorylated KXGS epitopes in NFTs in brain tissue from individuals with Alzheimer’s disease [63]. It remains to be seen, however, whether the MARK-mediated phosphorylation of tau, after dislodging tau from microtubules, also induces the association of tau with 14–3-3 proteins. Intriguingly, it has been shown that tau itself is a 14–3-3-binding protein and that 14–3-3 stimulates the aggregation of tau into fibrils [64,65]. Moreover, a-synuclein, which is the main component of the Lewy-body-type deposits formed in Parkinson’s disease and has been identified as a component of NFTs [3], is a distant member of the 14–3-3 protein family. Nevertheless, we must await further in vivo data to clarify whether 14–3-3 proteins have a role in NFT formation. Inhibition of MARKs: a potential therapy for Alzheimer’s disease? Current medications for the treatment of Alzheimer’s disease are completely symptomatic: they do not treat the underlying disease pathology, they do not halt the progression of the disease, and they do not prolong life [1]. The main disease-modifying therapeutics in development are directed against targets involved in amyloid production, such as the b- or g-secretase inhibitors. So far, www.sciencedirect.com

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tau-targeted drug discovery projects have not advanced beyond preclinical development and are typically aimed at the inhibition of GSK-3 or Cdk5 [66]. For a GSK-3 inhibitor at least, there is a high mechanistic risk because the resulting activation of the oncogenic Wnt signalling pathway might cause severe side-effects. Although the balance between protein kinases and phosphatases is ultimately crucial for regulating the phosphorylation state of tau in the neuron [67], the activation of specific phosphatases could be potentially exploited for therapeutic benefit [5]. Small-molecule phosphatase agonists are, however, currently not available. What are our prospects for developing a MARK inhibitor for treating Alzheimer’s disease? The development of specific inhibitory compounds for different MARK paralogues – for example, for MARK4, which shows specific cytoskeletal localization and higher brain expression – might be considered to be advantageous [17]. Given the sequence conservation around the ATPbinding pockets in the catalytic domains of the human MARKs, however, it might be difficult to generate such isoform-specific compounds, at least as far as ATP competitive inhibitors (which comprise the vast majority of kinase inhibitors) are concerned. It is difficult to predict the risks of a chronically administered inhibitor of all four MARK kinases that could penetrate the central nervous system, despite the fact that mice lacking MARK2 or MARK3 are viable [23]. On a positive note, proof of concept studies to test the efficacy of specific inhibitors of GSK-3, Cdk5 and MARKs should be facilitated by recently developed mouse models, which for the first time recapitulate salient features of the pathology of Alzheimer’s disease [4]. Such programs not only would lead to the discovery of interesting drug candidates, but would also contribute to validating the role of tau phosphorylation in the development of Alzheimer’s disease. The fact that the first protein kinase inhibitors have been recently approved for clinical use [68] sparks some optimism that what lies ahead on this exciting therapeutic avenue might be revealed in the not too distant future. Concluding remarks The field of tau-dependent neurodegeneration has recently undergone significant progress, from studies based on in vitro and ex vivo systems, to the current animal models of tauopathies in transgenic fruit flies and in mice. The models predict the crucial involvement of the protein kinases MARK, cdk5 and GSK3 in tau-dependent neurotoxicity in human disease. However, if we are to base therapeutic approaches on a solid mechanistic understanding of the neurodegenerative process, open questions remain. Future research will be essential to determine the actual toxic species because it is not clear whether the modification of tau protein in the disease represents a toxic loss of function – related to its microtubule-regulating role in the axon – or, rather, a gain-of-function by an unknown mechanism leading to neuronal death. Pharmacological inhibition of tau phosphorylation and, if possible, tau-aggregation processes in the animal models should provide the necessary clues.

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