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Functional Implications of Glycogen Synthase Kinase-3-Mediated Tau Phosphorylation

Functional Implications of Glycogen Synthase Kinase-3-Mediated Tau Phosphorylation SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2011, Article ID 352805, 11 pages doi:10.4061/2011/352805 Review Article Functional Implications of Glycogen Synthase Kinase-3-Mediated Tau Phosphorylation Diane P. Hanger and Wendy Noble Department of Neuroscience (P037), MRC Centre for Neurodegeneration Research, King’s College London, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK Correspondence should be addressed to Diane P. Hanger, diane.hanger@kcl.ac.uk Received 15 April 2011; Accepted 6 May 2011 Academic Editor: Adam Cole Copyright © 2011 D. P. Hanger and W. Noble. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tau is primarily a neuronal microtubule-associated protein that has functions related to the stabilisation of microtubules. Phosphorylation of tau is an important dynamic and regulatory element involved in the binding of tau to tubulin. Thus, highly phosphorylated tau is more likely to be present in the cytosolic compartment of neurons, whereas reduced phosphate burden allows tau to bind to and stabilise the microtubule cytoskeleton. Highly phosphorylated forms of tau are deposited in the brain in a range of neurodegenerative disorders including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal lobar degeneration associated with Pick bodies. A key candidate kinase for both physiological and pathological tau phosphorylation is glycogen synthase kinase-3 (GSK-3). Multiple phosphorylation sites have been identified on tau exposed to GSK-3 in vitro and in cells. In this review, we highlight recent data suggesting a role for GSK-3 activity on physiological tau function and on tau dysfunction in neurodegenerative disease. 1. Introduction predominantly of tau with reduced solubility and increased reactivity to phospho-specific tau antibodies. Determining The microtubule-associated protein tau is a normally soluble the key kinases that may be involved in the development and phosphoprotein found predominantly in neurons [1]. The progression of disease pathology is an important research structure of tau comprises three broadly defined regions, an goal. In this review, we highlight the links between glycogen N-terminal projection domain, that is thought to be respon- synthase kinase-3 (GSK-3) activity and tau function in sible for its interaction with membranes and other proteins, a normal and diseased brain. central proline-rich domain, and a C-terminal microtubule- binding repeat region (Figure 1). Phosphorylation of tau 2. The Microtubule-Associated Protein Tau is usually a very rapid and reversible process, which is mediated by the opposing actions of several protein kinases 2.1. Tau Function and Localisation. Tau is present in the and phosphatases [2]. Tau phosphorylation is increased adult human central nervous system (CNS) as six isoforms during embryonic development, and in neurodegenerative that are generated from alternative splicing of a single gene. conditions, in which tau deposition is a characteristic These isoforms differ from each other by the presence feature [3, 4]. Such disorders include Alzheimer’s disease, of none, one, or two inserts of 29 amino acids in the progressive supranuclear palsy (PSP), and frontotemporal N-terminus of the molecule, and the inclusion of either lobar degeneration associated with Pick bodies, amongst three (3R) or four (4R) repeated stretches of approximately others, collectively termed the “tauopathies”. A common 30 amino acids that comprise the microtubule-binding factor to all of these diseases is the presence of aggregated region of the molecule (Figure 1). Tau in embryonic brain and highly phosphorylated tau in the brain. These aggregates comprises primarily the smallest (0N3R) tau isoform, and characteristically form intracellular inclusions comprised this single isoform is gradually replaced with the six adult 2 International Journal of Alzheimer’s Disease 0N3R Proline enriched R1 R3 R4 352 1N Proline enriched R1 R3 R4 1N3R 381 Proline enriched 2N R1 R3 R4 2N3R 1N 0N4R Proline enriched 383 R1 R2 R3 R4 1N4R Proline enriched R1 R2 R3 R4 1N 2N Proline enriched 2N4R 1N R1 R2 R3 R4 441 T231 S46 T149 S195 S210 S258 S285 S352 T373 S404 T50 T153 S198 T212 S235 S262 S289 S356 S396 S409 S69 S199 S214 S237 S324 S400 S413 T175 S241 T71 T181 S202 T217 T205 T220 T245 S184 Figure 1: Tau isoforms in the human CNS and identified GSK-3 phosphorylation sites. The diagram illustrates the six isoforms of tau present in the human CNS. The longest tau isoform includes alternatively spliced exons 2, 3, and 10. Exons 2 and 3 encode two short amino acid inserts near the N-terminus of the molecule (1N and 2N, respectively). Exclusion of exons 2 and 3 gives rise to 0N tau isoforms, inclusion of exon 2 produces 1N tau isoforms, and inclusion of exons 2 and 3 results in the expression of 2N tau isoforms. Exon 10 encodes an additional microtubule-binding repeat domain (R2) that is present in 4R, but absent from 3R, tau isoforms. The number of amino acids in each tau isoform is indicated on the right. The centre of the molecule comprises a proline-enriched region that harbours the majority of the identified GSK-3 phosphorylation sites in 2N4R tau. Serine (S) and threonine (T) residues that have been identified as being phosphorylated by GSK-3 in vitro are indicated below. isoforms during development [5, 6]. In normal adult human [12], and some phosphorylated epitopes on tau (e.g., PHF- brain, the ratio of the tau isoforms harbouring three or 1, corresponding to phosphorylated Ser396/Ser404 in tau) four microtubule-binding repeats is approximately equal. have been associated with early stages of axon formation However, this ratio is altered in favour of expression of the 4R [13], indicating a role for tau in the development of neuronal tau isoforms in several neurodegenerative tauopathies [7], polarity. In disease, highly phosphorylated forms of tau although increased relative expression of 3R tau isoforms bind less well to microtubules, resulting in a loss of the has also been observed in frontotemporal lobar degeneration microtubule-stabilising properties of tau and ultimately the associated with Pick bodies [8]. Thus, abnormal tau splicing collapse of the neuronal cytoskeleton. This has the effect of and phosphorylation are both events that are closely associ- disrupting axonal transport and negatively impacting on the ated with neurodegeneration. delivery of organelles, neurotransmitters, and other proteins In neurons, the primary location of tau is in axons, to and from the cell body, with a consequent detrimental where it is presumed to act as a stabilising protein for the effect at synaptic termini [14–16]. microtubule cytoskeleton. Tau has an important function in Tau phosphorylation also influences the positioning of maintaining microtubule dynamic instability, through dual tau in dendrites, and the association of tau with plasma processes that result in the lengthening and shortening of membranes and nuclei. Elevated phosphorylation results in microtubules in response to external signals [9]. Increased the relocalisation of tau from axons into the somatodendritic tau phosphorylation leads to its detachment from tubulin, region of neurons. Interactions between tau and the non- thereby, enhancing microtubule disassembly and reducing receptor tyrosine kinase, Fyn, result in increased Fyn local- microtubule stability [10, 11]. In contrast, dephosphoryla- isation in dendrites [17]. In models of Alzheimer’s disease, tion of tau leads to an increase in its binding to tubulin, accel- increased Fyn activity in response to neurotoxic stimuli such erated microtubule growth, and stabilisation of the micro- as β-amyloid (Aβ), enables Fyn to phosphorylate subunit tubule cytoskeleton. Altered microtubule stability is partic- 2B (NR2B) of the N-methyl-D-aspartate (NMDA) receptor, ularly important during neurodevelopment when increased increasing the stability of this complex with the postsynaptic phosphorylation of tau reduces its binding to microtubules, density (PSD) protein, PSD-95, and ultimately enhancing allowing the rapid extension and retraction of exploratory neurotoxicity [17]. In contrast, dephosphorylation of tau neurites. Spatial and temporal changes in tau phosphory- increases its association with plasma membranes [18, 19], lation have been reported during neuronal differentiation which may also influence neurodegenerative processes by International Journal of Alzheimer’s Disease 3 Table 1: Comparison of phosphorylation sites in human Alzheimer Table 1: Continued. and control brain with recombinant tau phosphorylated by GSK-3 Tau residue number Alzheimer tau Control brain tau GSK-3 in vitro. Ser409 •• Tau residue number Alzheimer tau Control brain tau GSK-3 Ser412 •∗∗ Tyr18 A Ser413 •∗∗ • Ser46 A •• Thr414 ∗∗∗ Thr50 • Ser416 A • Ser68 • Ser422 • Thr69 •• Thr427 • Thr71 • Ser433 • Ser113 • Ser435 • Thr123 A Numbering of residues refers to 2N4R human tau, the largest isoform present in the human CNS. Ser131 • indicates tau phosphorylation sites identified by direct means. Thr149 • A indicates tau phosphorylation sites identified using phospho-specific tau Thr153 A • antibodies. ∗ indicates that one of the two closely spaced phosphorylation sites Thr175 •• indicated (Thr414, Ser416) is phosphorylated in tau from AD brain. Thr181 •• • ∗∗ indicates that two of the three closely spaced phosphorylation sites indicated (Ser412, Ser413, Thr414) are phosphorylated in tau from control Ser184 •• human brain. Ser185 • Ser191 • Ser195 • mediating the interactions of tau with membrane-associated Tyr197 • cell-signalling molecules such as Src-family kinases and Ser198 •• • phospholipase C-γ [20–22]. A pool of tau has also been identified in the nucleus Ser199 •• • of cultured cells and in brain tissue [23–28]. In human Ser202 •• • neuroblastoma cells, tau has been identified in the nucleolar Thr205 A •• organiser region [23], and, thus, tau-DNA binding may be Ser208 • involved in nucleolar organisation [27]. It has been reported Ser210 •• that the phosphorylation of tau, particularly in response to Thr212 •• • heat stress, increases the amount of tau found in neuronal Ser214 •• nuclei [29], although this finding is somewhat controversial Thr217 •• • [30]. The microtubule-binding domain of tau associates with RNA [31]and DNA[32], and the association of nuclear tau Thr220 • with DNA in response to stress is believed to play a role Thr231 •• • in protecting DNA from damage [29]. Tau has been found Ser235 •• • to be associated with the, predominantly nuclear protein, Ser237 •• mammalian solute transport protein-2 (SUT2). SUT2 is Ser238 • localised to SC35-positive speckles in the nucleus, and it Ser241 • may have a role in mRNA processing [33]. Expression of Thr245 • this protein in C. elegans potentiates tau neurotoxicity [34, 35]. In addition, mammalian SUT2 is reduced in disease- Ser258 •• affected regions of Alzheimer brain, under conditions where Ser262 •• tau phosphorylation is increased, and SUT2 expression is Ser285 • increased when tau is overexpressed in mammalian cells in Ser289 •• culture [34]. RNAi knockdown of SUT2 in cultured human Ser305 • cells overexpressing tau results in a marked decrease in tau Ser324 • aggregation, with no apparent effect on soluble tau species, Ser352 • suggesting a possible role for SUT2 in the pathogenesis of Ser356 •• tauopathies [34]. Thus, tau may mediate neurodegeneration by changes in phosphorylation that result in both losses and Thr373 • gains of tau function. Tyr394 • Ser396 •• • 2.2. Tau Phosphorylation. Tau possesses 80 phosphorylatable Ser400 •• • serine and threonine residues, approximately 20% of which Thr403 • have been identified as being phosphorylated in normal Ser404 •• • human brain (Table 1). A further five tyrosine residues in 4 International Journal of Alzheimer’s Disease tau are also amenable to phosphorylation by tyrosine kinases, 3.3. GSK-3 Regulation of Tau Splicing. In addition to phos- including Fyn, Syk, c-Abl, and Lck [36, 37]. A large number phorylating tau, GSK-3 can influence tau splicing. GSK-3 of serine/threonine kinases have been reported to phospho- has been shown to phosphorylate nuclear SC35, an enhancer rylate tau in vitro, but the identity of the protein kinase(s) of splicing elements that regulate exon 10 splicing in tau responsible for regulating physiological tau phosphorylation [51, 52]. Aβ application to cultured cells activates GSK-3 remains unknown [7]. Candidate serine/threonine-directed [53], leading to the phosphorylation of SC35 in parallel with tau kinases include GSK-3, casein kinase 1 (CK1), and cyclin- enhanced splicing of tau exon 10 and decreased expression dependent kinase-5 (cdk5), amongst others [7]. It seems of 4R tau [54]. These events can be suppressed by the likely that more than one kinase may be involved in the inhibition of GSK-3 activity with lithium or following siRNA phosphorylation of tau. However, substantial evidence exists knockdown of GSK-3 [54]. Despite these findings, most to support a major role for GSK-3 in both physiological and research investigating GSK-3 regulation of tau function has pathological tau phosphorylation. concentrated on GSK-3-mediated phosphorylation of tau. 3.4. GSK-3 Regulation of Tau Phosphorylation. Tau is a 3. Glycogen Synthase Kinase-3 good substrate for GSK-3 in vitro [39, 55], in cultured 3.1. GSK-3 Isoforms. GSK-3 exists as two isoforms, α and nonneuronal cells [56], and in transgenic mice overex- β, which share 85% sequence identity and are encoded by pressing GSK-3 [57]. In brain, tau exists in a complex distinct genes located on chromosomes 19 and 3, respectively with GSK-3 and the scaffolding protein 14-3-3 [58]. 14- 3-3 recognises GSK-3 phosphorylated at Ser9, and indeed [38]. GSK-3α and GSK-3β both phosphorylate tau in vitro GSK-3 in this complex is phosphorylated at Ser9 in brain and appear as granules with slightly differing morphologies [58, 59]. The association of tau with this complex is believed and densities in pyramidal cells of hippocampal neurons to regulate its phosphorylation by GSK-3, since in human [39]. There are two variants of GSK-3β,withGSK-3β2 embryonic kidney cells, tau phosphorylation by GSK-3 is differing from GSK-3β1 by the presence of an additional suppressed in the absence of 14-3-3, but GSK-3 is active and insertion of 13 amino acids. GSK-3β2 is enriched in neurons, phosphorylates tau if 14-3-3 is present [59]. where it is present in cell bodies, neuritis, and growth cones [40]. GSK-3α and GSK-3β share many substrates and appear 4. In vitro Phosphorylation of Tau by GSK-3 to be able to compensate partially for each other, although they also appear to have distinct functions [41]. A recent Tau is phosphorylated by GSK-3 on approximately 40 report has shown that, whereas GSK-3β is present in birds, different serine and threonine residues, at least in vitro GSK-3α is absent, indicating that the GSK-3α isoform is [60, 61]. The importance of this finding should not be not required either for viability or for normal physiological underestimated since few kinases have been shown to target function in birds [42]. Furthermore, knockout of GSK-3α in this number of sites in tau. Indeed, CK1 is the only other mice results in increased insulin sensitivity [43], suggesting kinase that has been reported to phosphorylate a similar arolefor GSK-3α in glucose metabolism that cannot be number of tau residues. Treatment of primary neuronal replaced by GSK-3β. In contrast, complete knockout of GSK- cortical cultures with specific inhibitors of either GSK-3 3β in mice is embryonic lethal [44]. or CK1 reduces tau phosphorylation, suggesting that these kinases could have functionally important roles in neurons 3.2. Regulation of GSK-3 Activity. The kinase activity of [7, 14, 60]. The phosphorylation of tau by GSK-3 or CK1 also GSK-3 is regulated by its phosphorylation at serine and reduces the ability of tau to promote microtubule assembly tyrosine residues. Phosphorylation of GSK-3α/β at Tyr216 in vitro and in cells [62, 63]. These results rank GSK-3 and and Tyr279 is believed to maintain the constitutive activity CK1 as targeting the greatest number of phosphorylatable of GSK-3 in neurons (reviewed in [45]), while the phospho- residues on tau and implicate these two protein kinases in rylation of Ser21 on GSK-3α or Ser9 on GSK-3β negatively physiological tau phosphorylation in neurons. It remains regulates GSK-3 activity [45], and phosphorylation at these to be seen whether the activities of GSK-3 and/or CK1 are residues is believed to the predominant mediator of GSK-3 modified in diseases in which increased phosphorylation of activity in vivo [46]. Both protein phosphatase 1, (PP1) and tau is a characteristic feature. PP2A are known to target Ser21/9 and thus regulate GSK-3 activity [47]. Indeed, PP activation with okadaic acid leads 4.1. Potential Priming of Tau for GSK-3 Phosphorylation. to increased phosphorylation of S9 and, hence, inhibition It is well established that GSK-3 preferentially phos- of GSK-3β, which results in reduced tau phosphorylation phorylates many of its substrates after they have been [48]. Other phosphatases could also be involved in GSK-3 prephosphorylated by other kinases, and this seems also to regulation since calcineurin (PP2B) also dephosphorylates be true for tau phosphorylation by GSK-3. Other examples and hence activates GSK-3 [49]. In addition, one of the many of GSK-3 substrates that require prephosphorylation by substrates of GSK-3 is the inhibitory subunit (inhibitor-2) another kinase before recognition by GSK-3 include glycogen of PP1. The phosphorylation of this subunit leads to its synthase, inhibitor-2 of PP1, the regulatory subunit of cyclic activation which results in the inhibition of PP1 [50], and AMP-dependent protein kinase (PKA), cAMP response decreased GSK-3 activity, demonstrating one mechanism element-binding (CREB), β-catenin, and kinesin light chain through which GSK-3 activity may be regulated. [46, 64]. Most priming phosphorylations for GSK-3 occur at International Journal of Alzheimer’s Disease 5 an amino acid located four residues C-terminal to the target Prephosphorylation of tau by PKA for subsequent GSK- residue [65]. However, there are some exceptions to this rule, 3 phosphorylation in rat brain appears to particularly and priming events have been reported that occur five or six enhance the overall amount of GSK-3 phosphorylation [73]. residues from the GSK-3 target site, for example, in collapsin Furthermore, the initial phosphorylation of tau by PKA response mediator protein-2 and also in tau [66, 67]. The results in a different spectrum of phosphorylation sites gen- GSK-3 priming phosphorylation is frequently provided by erated by the action of GSK-3 compared to those produced the activity of PKA, CK1, or CK2 on unphosphorylated when tau is exposed to GSK-3 alone, demonstrating that substrates, although other kinases, such as members of the prior phosphorylation by PKA alters the recognition of mitogen-activated protein kinase family, or cdk5, can also tau by GSK-3 [73]. Moreover, initial phosphorylation of initiate priming on some GSK-3 substrates [68, 69]. As has tau by PKA, which occurs primarily on Ser214, results in previously been shown, CK1 primes β-catenin for subse- the subsequent phosphorylation by GSK-3 of four closely quent phosphorylation by GSK-3 (reviewed in [69]), and spaced residues, namely Ser210, Thr205, Ser199, and Ser195, this might also occur on tau because the rate of GSK-3 phos- each separated by 4–6 amino acids [67]. Interestingly, this phorylation of tau is increased when it is first phosphorylated study also revealed an interaction between the low-density by CK1 [70]. Substrate priming, therefore, may represent an lipoprotein receptor-binding domain of apolipoprotein E important regulatory element of GSK-3 signalling since the (ApoE), a major genetic risk factor for Alzheimer’s disease, activity of GSK-3 has been reported to differ for its primed and GSK-3-phosphorylated tau [67]. Such an interaction and nonprimed substrates [68]. In the case of tau, this is was not observed with either nonphosphorylated tau or supported by the observation that targeting GSK-3 phos- following extensive phosphorylation of tau by PKA. These phorylation of tau to either unprimed or primed sites has results suggest that the pattern of phosphorylation sites adifferential impact on the binding of tau to microtubules generated by the action of GSK-3 on tau may be critical for [71, 72]. its interaction with ApoE. There are 24 short sequences of amino acids in tau that conform to the strict consensus sequence Ser/Thr-XXX- 4.2. GSK-3α and GSK-3β1/2 Isoforms Differentially Phospho- Ser/Thr (indicating pairs of serine or threonine residues rylate Tau. Suppressing the expression of individual GSK- separated by any three amino acids), that could be implicated 3α and GSK-3β isoforms results in differing tau phospho- in priming for GSK-3. Fourteen of these serine/threonine rylation patterns [74]. The induction of a phosphorylation- pairs have been found to be phosphorylated in vitro by induced shift in electrophoretic mobility of tau, follow- GSK-3 (Table 1), and six pairs contain a proline residue ing incubation with GSK-3, also appears to be favoured immediately C-terminal to the target sequence. Five of the preferentially by GSK-3β, rather than GSK-3α,asistau 10 remaining paired amino acids are phosphorylated by phosphorylation at the antibody epitopes recognised by tau- GSK-3 on only one of the two serine or threonine residues 1 (Ser199–Ser202) and 8D8 (Ser396) [39]. Together, these and five pairs have not been shown to be phosphorylated results support the view that GSK-3α and GSK-3β are likely by GSK-3. Relaxation of the consensus sequence to include to have differing preferences for tau that may be related to a separation of five or six amino acids between potential the distinct, but overlapping, intracellular locations of these priming sites would allow the inclusion of further sets kinases in neurons. of amino acids, including Ser214, Ser210, Thr205, and There is also a difference in the kinetics and sites of Ser199 (see below). Overall, therefore, tau appears to fulfil tau phosphorylation induced by the two GSK-3β isoforms, several of the requirements for GSK-3 substrates, including with GSK-3β2 appearing to phosphorylate tau more slowly multiple Ser/Thr-Pro sequences with nearby N-terminal than GSK-3β1 and on different, with some overlapping, phosphorylatable amino acids closely opposed, which could tau residues, even under conditions in which other GSK- allow for kinase priming, either by GSK-3 itself or by other 3β substrates, such as amyloid precursor protein, are phos- tau kinases. phorylated equally [75, 76]. For example, Ser396 in tau is Priming phosphorylation on tau residues Ser235 and phosphorylated by both splice variants, whereas Ser199 is a Ser404 by other kinases, including cdk5, has been shown to significant target of GSK-3β1, but not of GSK-3β2activity. It promote phosphorylation by GSK-3 at Thr231 and Ser400, has recently been suggested that the interaction of GSK-3β2 respectively [71, 72]. Interestingly, the stretch of amino acids with tau is weaker than that of GSK-3β1[76]. Taken together, in tau that includes the phosphorylatable residues, Ser396, these results suggest that, not only are there differential Ser400, and Ser404, can be directly phosphorylated by GSK- activities of GSK-3α and GSK-3β towards tau, there are 3 without the prior activity of other kinases [67]. However, also partially overlapping, but distinct, tau phosphorylation phosphorylation at Ser404 is critical to this process and sites recognised by each of the two isoforms of GSK-3β substitution of this residue by alanine ablates phosphoryla- [74–77]. tion of both Ser396 and Ser400. It appears, therefore, that the primary phosphorylation of Ser404 by GSK-3 can itself 5. Tau Phosphorylation in Human Brain serve as a primed residue for the subsequent sequential phosphorylation of tau at Ser400 and Ser396 by GSK-3. In the tauopathies, tau is present in both normally phospho- However, the functional significance of many of the other rylated and highly phosphorylated forms, the latter of which potential GSK-3 priming sites in tau has not been widely may be potentially pathological since it is most commonly investigated. observed as intraneuronal aggregates or glial inclusions. 6 International Journal of Alzheimer’s Disease Tau is normally a highly soluble protein under nonde- 6. Cellular Models of Tau naturing conditions, but the aggregated tau present in Phosphorylation by GSK-3 diseased brain displays a significantly reduced solubility and an increased reactivity to phospho-specific tau antibodies Further information regarding the role of GSK-3 in tau [78]. These characteristic features have facilitated purifica- phosphorylation and function has been gained from cell tion strategies for tau from diseased brain exhibiting tau models. In cells, the involvement of GSK-3 was first revealed pathology which yield relatively enriched preparations of when GSK-3 over-expression was shown to disrupt tau highly phosphorylated tau. Such preparations have resulted binding to microtubules, resulting in a diffuse cytoplasmic in the identification of phosphorylation sites on insoluble localisation of tau [56]. This staining pattern contrasts with tau isolated from human tauopathy brain, thereby providing the strongly fibrillar pattern displayed when tau is expressed clues to the identities of protein kinases that are potentially alone. Cellular phosphorylation of tau by GSK-3β over- involved in the pathogenesis or development of these expressed in mammalian cells decreases tau binding to neurodegenerative diseases [60, 79–81]. microtubules and influences microtubule organisation [87, In particular, these approaches have been used to identify 88]. This process is inhibited by mouse dishevelled-1, thereby phosphorylation sites on purified tau from Alzheimer’s potentially implicating the wingless signalling pathway in tau disease and PSP brain material, as well as soluble tau from phosphorylation mediated by GSK-3 [89]. control adult and foetal brain [60, 82, 83]. These studies, Investigations in human neuronal NT2N cells have combined with results obtained from immuno-labelling shown that tau phosphorylation at multiple sites recognised with phospho-specific tau antibodies, have led to the by phospho-specific tau antibodies could be prevented identification of approximately 45 phosphorylation sites on by lithium treatment, which inhibits GSK-3 activity [90]. tau from Alzheimer brain (Table 1). In comparison, only 15 Lithium also promotes tau binding to microtubules and sub- phosphorylation sites have been detected in insoluble tau sequent microtubule polymerisation, suggesting a role for from PSP brain and 16 sites on control brain tau, not all of GSK-3 in this normal physiological process [90]. It is impor- which overlap, and for some of which no kinase has yet been tant to note that lithium is not a specific inhibitor of GSK- identified [7]. 3 as it also effectively inhibits inositol monophosphatase, as A comparison of tau extracted from human brain with well as a family of structurally related phosphomonoesterases tau phosphorylated using protein kinases in vitro shows that (reviewed in [91]). However, the reported effects of lithium GSK-3 remains a principal candidate kinase for both phys- on tau phosphorylation in cells have subsequently been iological and pathological tau phosphorylation (Table 1). validated in studies using more selective GSK-3 inhibitors Although GSK-3 has been reported to be associated with [74, 92, 93]. The examination of endogenous tau phospho- neurofibrillary pathology in Alzheimer’s disease and related rylation by GSK-3 in primary rat neurons has revealed a neurodegenerative disorders [84, 85], otherstudieshavenot number of lithium-sensitive sites, suggesting that these might identified such colocalisation [39, 86], and it remains to represent the physiological targets of GSK-3 on tau [14, 94]. be seen if GSK-3 remains associated with tau pathology in Interestingly, in the latter study, the inhibition of GSK-3, diseased brain. In the case of tau extracted from neurolog- either by lithium or by the more specific GSK-3 inhibitor, ically normal human brain, all but two residues have been SB-415286, resulted not only in reduced tau phosphorylation shown to be phosphorylated by GSK-3. These two sites, but also in reduced axonal transport of tau [14]. This led to Ser412 and Thr414, are located within a group of three the idea that tau phosphorylation mediated by GSK-3 could closely spaced residues, including Ser413, of which only two influence tau axonal transport, at least in primary neuronal residues are phosphorylated in human brain. Since GSK- cultures [14]. In an ex vivo study, perfusion of preformed 3 phosphorylates Ser413, it is possible that only a single filaments of tau into isolated squid axoplasm resulted in site, either Ser412 or Thr414, remains unphosphorylated the inhibition of anterograde transport of membrane-bound by GSK-3, and, of these, Ser412 is phosphorylated by both organelles [95]. Interestingly, inhibiting GSK-3 activity, or CK1 and PKA, and potentially also by CK2. This suggests perfusion of inhibitor-2 to inhibit PP1 activity, overcame the that, under physiological conditions, tau in human brain detrimental effect of tau filaments on axonal transport in may be phosphorylated primarily by GSK-3, with potential this model [95]. These results, thus, support the notion that contributions from CK1, CK2, or PKA. Under pathological GSK-3 has an important role in relation to tau function in conditions such as Alzheimer’s disease, in which tau becomes neurons. deposited in the brain, GSK-3 accounts for some 27 of Inhibiting GSK-3 with SB-415286 also protects cultured the approximately 45 tau phosphorylation sites identified neurons from cell death induced by reduced phosphatidyli- in insoluble tau. The remaining phosphorylation sites are nositol 3-kinase activity, and this protection is paralleled by accounted for the activities of a combination of other kinases, decreased tau phosphorylation [92]. These results suggest including the tyrosine kinases, c-Abl, and Fyn, although there remain a few sites for which a kinase has not yet been that the state of tau phosphorylation by GSK-3 in cells is important for the maintenance of healthy functional identified. Thus GSK-3 plays a prominent role in both the physiological and pathological phosphorylation of tau in neurons, and changes in tau phosphorylation are likely be human brain. indicative of reduced neuronal viability. International Journal of Alzheimer’s Disease 7 7. Animal Models of Tau spatial learning deficits, and neuronal death [106, 107]. These features can be reversed by suppressing GSK-3β Phosphorylation by GSK-3 expression [108], indicating that tau is a prime substrate 7.1. Drosophila. Over-expression of tau together with GSK- for GSK-3 in vivo. Indeed, the axonopathy and motor 3 (the human orthologue of shaggy/Zw3 in Drosophila) impairment resulting from over-expression of human 2N4R has been demonstrated to exacerbate the neurodegenera- tau in mice is alleviated when GSK-3β is coexpressed [109], tion resulting from expression of tau alone in Drosophila with this beneficial effect believed to occur as a result of GSK- melanogaster [96], suggesting that the phosphorylation 3 phosphorylation of tau removing excess tau from micro- of tau by GSK-3 is involved in the neurodegenerative tubules. Further support for a potential pathological role of process. The presence of partition-defective 1 (PAR-1), the GSK-3-phosphorylated tau has come from studies showing Drosophila orthologue of mammalian microtubule affinity that GSK-3 inhibition in transgenic mice expressing disease- regulating kinase (MARK), has been shown to be required associated mutant human tau reduces tau phosphorylation to initiate GSK-3 and cdk5 phosphorylation of tau in and aggregation [110–114]aswellasaxonaldegeneration flies [97]. However, reports differ as to whether PAR- [111]. 1 activity enhances [97, 98] or suppresses [15, 99, 100] tau-induced toxicity, although PAR-1 over expression in 8. Conclusions Drosophila appears to increase neuronal loss [97]. MARK phosphorylates tau on Ser262 and Ser356 in vitro,bothof Changes in the phosphorylation state of tau have a major which are located within the microtubule-binding domain impact on its subcellular localisation and function in of tau, and this phosphorylation results in the disruption neurons. Major changes in the phosphorylation state of of microtubules [101]. MARK phosphorylation, therefore, tau are evident under two apparently unrelated conditions, results in the detachment of tau from microtubules, and that is, during early neuronal development and during this may be related to its involvement in microtubule-based neurodegenerative processes that lead to the deposition of axonal transport in neurons [102]. Interestingly, MARK itself highly phosphorylated pathological tau in the brain, such as is phosphorylated by GSK-3, resulting in MARK inactivation in Alzheimer’s disease. Therefore, determining the nature of [103], suggesting the possible involvement of multiple tau the kinases involved in this process in vivo is an important kinases in this model, however the relevance GSK-3 and research goal that will improve our understanding of these MARK interaction to tau phosphorylation has not yet been processes. determined. GSK-3 is a key candidate kinase for tau phosphorylation, More recent studies have found that tau phosphorylation under both physiological and pathological conditions. Multi- in Drosophila is not dependent on GSK-3 activity. A mutant ple serine and threonine residues on tau are targeted by GSK- form of tau, described as GSK-3 resistant, retained toxicity 3 in vitro, with many of these sites being coincident with in flies, and, under these conditions, mutant tau exhibited an those phosphorylated in vivo. Together with the, possibly increased affinity for binding to microtubules [98]. Notably, coordinated, activity of other kinases, including CK1, cdk5, while GSK-3 phosphorylation of tau was impaired in these PKA and/or MARK, GSK-3 is well positioned to act on tau flies, toxicity was unaffected, suggesting that the ability of tau in such a way that will result in significant and rapid tau to bind to microtubules, or possibly the propensity of GSK-3 phosphorylation and, hence, modulation of its function in to alter the oligomerisation state of tau, may be critical for neurons. Understanding the regulation of GSK-3 activity neurodegeneration. in neurons, including the possible differential effects of the Drosophila express a form of tau with limited homology related GSK-3α,GSK-3β1, and GSK-3β2 isoforms, should to human tau, especially in the microtubule-binding region lead to further elucidation of the mechanisms underlying tau [104]. There is less than 50% amino acid identity between phosphorylation and may ultimately lead to new therapeutic the presumed microtubule-binding domain of Drosophila targets for neurodegenerative disease. and mammalian tau. Coupled with the presence of an additional microtubule-binding repeat in flies, these consid- Abbreviations erable differences between invertebrate and mammalian tau suggest that the results obtained by overexpressing GSK-3 Aβ: β-amyloid in flies may differ from those obtained in the presence of ApoE: Apolipoprotein E human tau. The relationship, therefore, between tau toxicity cdk5: Cyclin-dependent kinase-5 and tau phosphorylation in Drosophila warrants further CK: Casein kinase investigation if it is to be applied to study the physiological CNS: Central nervous system relevance of GSK-3 phosphorylation of tau. CREB: Cyclic AMP response element binding GSK-3: Glycogen synthase kinase-3 7.2. Mice. 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Functional Implications of Glycogen Synthase Kinase-3-Mediated Tau Phosphorylation

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Copyright © 2011 Diane P. Hanger and Wendy Noble. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2011, Article ID 352805, 11 pages doi:10.4061/2011/352805 Review Article Functional Implications of Glycogen Synthase Kinase-3-Mediated Tau Phosphorylation Diane P. Hanger and Wendy Noble Department of Neuroscience (P037), MRC Centre for Neurodegeneration Research, King’s College London, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK Correspondence should be addressed to Diane P. Hanger, diane.hanger@kcl.ac.uk Received 15 April 2011; Accepted 6 May 2011 Academic Editor: Adam Cole Copyright © 2011 D. P. Hanger and W. Noble. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tau is primarily a neuronal microtubule-associated protein that has functions related to the stabilisation of microtubules. Phosphorylation of tau is an important dynamic and regulatory element involved in the binding of tau to tubulin. Thus, highly phosphorylated tau is more likely to be present in the cytosolic compartment of neurons, whereas reduced phosphate burden allows tau to bind to and stabilise the microtubule cytoskeleton. Highly phosphorylated forms of tau are deposited in the brain in a range of neurodegenerative disorders including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal lobar degeneration associated with Pick bodies. A key candidate kinase for both physiological and pathological tau phosphorylation is glycogen synthase kinase-3 (GSK-3). Multiple phosphorylation sites have been identified on tau exposed to GSK-3 in vitro and in cells. In this review, we highlight recent data suggesting a role for GSK-3 activity on physiological tau function and on tau dysfunction in neurodegenerative disease. 1. Introduction predominantly of tau with reduced solubility and increased reactivity to phospho-specific tau antibodies. Determining The microtubule-associated protein tau is a normally soluble the key kinases that may be involved in the development and phosphoprotein found predominantly in neurons [1]. The progression of disease pathology is an important research structure of tau comprises three broadly defined regions, an goal. In this review, we highlight the links between glycogen N-terminal projection domain, that is thought to be respon- synthase kinase-3 (GSK-3) activity and tau function in sible for its interaction with membranes and other proteins, a normal and diseased brain. central proline-rich domain, and a C-terminal microtubule- binding repeat region (Figure 1). Phosphorylation of tau 2. The Microtubule-Associated Protein Tau is usually a very rapid and reversible process, which is mediated by the opposing actions of several protein kinases 2.1. Tau Function and Localisation. Tau is present in the and phosphatases [2]. Tau phosphorylation is increased adult human central nervous system (CNS) as six isoforms during embryonic development, and in neurodegenerative that are generated from alternative splicing of a single gene. conditions, in which tau deposition is a characteristic These isoforms differ from each other by the presence feature [3, 4]. Such disorders include Alzheimer’s disease, of none, one, or two inserts of 29 amino acids in the progressive supranuclear palsy (PSP), and frontotemporal N-terminus of the molecule, and the inclusion of either lobar degeneration associated with Pick bodies, amongst three (3R) or four (4R) repeated stretches of approximately others, collectively termed the “tauopathies”. A common 30 amino acids that comprise the microtubule-binding factor to all of these diseases is the presence of aggregated region of the molecule (Figure 1). Tau in embryonic brain and highly phosphorylated tau in the brain. These aggregates comprises primarily the smallest (0N3R) tau isoform, and characteristically form intracellular inclusions comprised this single isoform is gradually replaced with the six adult 2 International Journal of Alzheimer’s Disease 0N3R Proline enriched R1 R3 R4 352 1N Proline enriched R1 R3 R4 1N3R 381 Proline enriched 2N R1 R3 R4 2N3R 1N 0N4R Proline enriched 383 R1 R2 R3 R4 1N4R Proline enriched R1 R2 R3 R4 1N 2N Proline enriched 2N4R 1N R1 R2 R3 R4 441 T231 S46 T149 S195 S210 S258 S285 S352 T373 S404 T50 T153 S198 T212 S235 S262 S289 S356 S396 S409 S69 S199 S214 S237 S324 S400 S413 T175 S241 T71 T181 S202 T217 T205 T220 T245 S184 Figure 1: Tau isoforms in the human CNS and identified GSK-3 phosphorylation sites. The diagram illustrates the six isoforms of tau present in the human CNS. The longest tau isoform includes alternatively spliced exons 2, 3, and 10. Exons 2 and 3 encode two short amino acid inserts near the N-terminus of the molecule (1N and 2N, respectively). Exclusion of exons 2 and 3 gives rise to 0N tau isoforms, inclusion of exon 2 produces 1N tau isoforms, and inclusion of exons 2 and 3 results in the expression of 2N tau isoforms. Exon 10 encodes an additional microtubule-binding repeat domain (R2) that is present in 4R, but absent from 3R, tau isoforms. The number of amino acids in each tau isoform is indicated on the right. The centre of the molecule comprises a proline-enriched region that harbours the majority of the identified GSK-3 phosphorylation sites in 2N4R tau. Serine (S) and threonine (T) residues that have been identified as being phosphorylated by GSK-3 in vitro are indicated below. isoforms during development [5, 6]. In normal adult human [12], and some phosphorylated epitopes on tau (e.g., PHF- brain, the ratio of the tau isoforms harbouring three or 1, corresponding to phosphorylated Ser396/Ser404 in tau) four microtubule-binding repeats is approximately equal. have been associated with early stages of axon formation However, this ratio is altered in favour of expression of the 4R [13], indicating a role for tau in the development of neuronal tau isoforms in several neurodegenerative tauopathies [7], polarity. In disease, highly phosphorylated forms of tau although increased relative expression of 3R tau isoforms bind less well to microtubules, resulting in a loss of the has also been observed in frontotemporal lobar degeneration microtubule-stabilising properties of tau and ultimately the associated with Pick bodies [8]. Thus, abnormal tau splicing collapse of the neuronal cytoskeleton. This has the effect of and phosphorylation are both events that are closely associ- disrupting axonal transport and negatively impacting on the ated with neurodegeneration. delivery of organelles, neurotransmitters, and other proteins In neurons, the primary location of tau is in axons, to and from the cell body, with a consequent detrimental where it is presumed to act as a stabilising protein for the effect at synaptic termini [14–16]. microtubule cytoskeleton. Tau has an important function in Tau phosphorylation also influences the positioning of maintaining microtubule dynamic instability, through dual tau in dendrites, and the association of tau with plasma processes that result in the lengthening and shortening of membranes and nuclei. Elevated phosphorylation results in microtubules in response to external signals [9]. Increased the relocalisation of tau from axons into the somatodendritic tau phosphorylation leads to its detachment from tubulin, region of neurons. Interactions between tau and the non- thereby, enhancing microtubule disassembly and reducing receptor tyrosine kinase, Fyn, result in increased Fyn local- microtubule stability [10, 11]. In contrast, dephosphoryla- isation in dendrites [17]. In models of Alzheimer’s disease, tion of tau leads to an increase in its binding to tubulin, accel- increased Fyn activity in response to neurotoxic stimuli such erated microtubule growth, and stabilisation of the micro- as β-amyloid (Aβ), enables Fyn to phosphorylate subunit tubule cytoskeleton. Altered microtubule stability is partic- 2B (NR2B) of the N-methyl-D-aspartate (NMDA) receptor, ularly important during neurodevelopment when increased increasing the stability of this complex with the postsynaptic phosphorylation of tau reduces its binding to microtubules, density (PSD) protein, PSD-95, and ultimately enhancing allowing the rapid extension and retraction of exploratory neurotoxicity [17]. In contrast, dephosphorylation of tau neurites. Spatial and temporal changes in tau phosphory- increases its association with plasma membranes [18, 19], lation have been reported during neuronal differentiation which may also influence neurodegenerative processes by International Journal of Alzheimer’s Disease 3 Table 1: Comparison of phosphorylation sites in human Alzheimer Table 1: Continued. and control brain with recombinant tau phosphorylated by GSK-3 Tau residue number Alzheimer tau Control brain tau GSK-3 in vitro. Ser409 •• Tau residue number Alzheimer tau Control brain tau GSK-3 Ser412 •∗∗ Tyr18 A Ser413 •∗∗ • Ser46 A •• Thr414 ∗∗∗ Thr50 • Ser416 A • Ser68 • Ser422 • Thr69 •• Thr427 • Thr71 • Ser433 • Ser113 • Ser435 • Thr123 A Numbering of residues refers to 2N4R human tau, the largest isoform present in the human CNS. Ser131 • indicates tau phosphorylation sites identified by direct means. Thr149 • A indicates tau phosphorylation sites identified using phospho-specific tau Thr153 A • antibodies. ∗ indicates that one of the two closely spaced phosphorylation sites Thr175 •• indicated (Thr414, Ser416) is phosphorylated in tau from AD brain. Thr181 •• • ∗∗ indicates that two of the three closely spaced phosphorylation sites indicated (Ser412, Ser413, Thr414) are phosphorylated in tau from control Ser184 •• human brain. Ser185 • Ser191 • Ser195 • mediating the interactions of tau with membrane-associated Tyr197 • cell-signalling molecules such as Src-family kinases and Ser198 •• • phospholipase C-γ [20–22]. A pool of tau has also been identified in the nucleus Ser199 •• • of cultured cells and in brain tissue [23–28]. In human Ser202 •• • neuroblastoma cells, tau has been identified in the nucleolar Thr205 A •• organiser region [23], and, thus, tau-DNA binding may be Ser208 • involved in nucleolar organisation [27]. It has been reported Ser210 •• that the phosphorylation of tau, particularly in response to Thr212 •• • heat stress, increases the amount of tau found in neuronal Ser214 •• nuclei [29], although this finding is somewhat controversial Thr217 •• • [30]. The microtubule-binding domain of tau associates with RNA [31]and DNA[32], and the association of nuclear tau Thr220 • with DNA in response to stress is believed to play a role Thr231 •• • in protecting DNA from damage [29]. Tau has been found Ser235 •• • to be associated with the, predominantly nuclear protein, Ser237 •• mammalian solute transport protein-2 (SUT2). SUT2 is Ser238 • localised to SC35-positive speckles in the nucleus, and it Ser241 • may have a role in mRNA processing [33]. Expression of Thr245 • this protein in C. elegans potentiates tau neurotoxicity [34, 35]. In addition, mammalian SUT2 is reduced in disease- Ser258 •• affected regions of Alzheimer brain, under conditions where Ser262 •• tau phosphorylation is increased, and SUT2 expression is Ser285 • increased when tau is overexpressed in mammalian cells in Ser289 •• culture [34]. RNAi knockdown of SUT2 in cultured human Ser305 • cells overexpressing tau results in a marked decrease in tau Ser324 • aggregation, with no apparent effect on soluble tau species, Ser352 • suggesting a possible role for SUT2 in the pathogenesis of Ser356 •• tauopathies [34]. Thus, tau may mediate neurodegeneration by changes in phosphorylation that result in both losses and Thr373 • gains of tau function. Tyr394 • Ser396 •• • 2.2. Tau Phosphorylation. Tau possesses 80 phosphorylatable Ser400 •• • serine and threonine residues, approximately 20% of which Thr403 • have been identified as being phosphorylated in normal Ser404 •• • human brain (Table 1). A further five tyrosine residues in 4 International Journal of Alzheimer’s Disease tau are also amenable to phosphorylation by tyrosine kinases, 3.3. GSK-3 Regulation of Tau Splicing. In addition to phos- including Fyn, Syk, c-Abl, and Lck [36, 37]. A large number phorylating tau, GSK-3 can influence tau splicing. GSK-3 of serine/threonine kinases have been reported to phospho- has been shown to phosphorylate nuclear SC35, an enhancer rylate tau in vitro, but the identity of the protein kinase(s) of splicing elements that regulate exon 10 splicing in tau responsible for regulating physiological tau phosphorylation [51, 52]. Aβ application to cultured cells activates GSK-3 remains unknown [7]. Candidate serine/threonine-directed [53], leading to the phosphorylation of SC35 in parallel with tau kinases include GSK-3, casein kinase 1 (CK1), and cyclin- enhanced splicing of tau exon 10 and decreased expression dependent kinase-5 (cdk5), amongst others [7]. It seems of 4R tau [54]. These events can be suppressed by the likely that more than one kinase may be involved in the inhibition of GSK-3 activity with lithium or following siRNA phosphorylation of tau. However, substantial evidence exists knockdown of GSK-3 [54]. Despite these findings, most to support a major role for GSK-3 in both physiological and research investigating GSK-3 regulation of tau function has pathological tau phosphorylation. concentrated on GSK-3-mediated phosphorylation of tau. 3.4. GSK-3 Regulation of Tau Phosphorylation. Tau is a 3. Glycogen Synthase Kinase-3 good substrate for GSK-3 in vitro [39, 55], in cultured 3.1. GSK-3 Isoforms. GSK-3 exists as two isoforms, α and nonneuronal cells [56], and in transgenic mice overex- β, which share 85% sequence identity and are encoded by pressing GSK-3 [57]. In brain, tau exists in a complex distinct genes located on chromosomes 19 and 3, respectively with GSK-3 and the scaffolding protein 14-3-3 [58]. 14- 3-3 recognises GSK-3 phosphorylated at Ser9, and indeed [38]. GSK-3α and GSK-3β both phosphorylate tau in vitro GSK-3 in this complex is phosphorylated at Ser9 in brain and appear as granules with slightly differing morphologies [58, 59]. The association of tau with this complex is believed and densities in pyramidal cells of hippocampal neurons to regulate its phosphorylation by GSK-3, since in human [39]. There are two variants of GSK-3β,withGSK-3β2 embryonic kidney cells, tau phosphorylation by GSK-3 is differing from GSK-3β1 by the presence of an additional suppressed in the absence of 14-3-3, but GSK-3 is active and insertion of 13 amino acids. GSK-3β2 is enriched in neurons, phosphorylates tau if 14-3-3 is present [59]. where it is present in cell bodies, neuritis, and growth cones [40]. GSK-3α and GSK-3β share many substrates and appear 4. In vitro Phosphorylation of Tau by GSK-3 to be able to compensate partially for each other, although they also appear to have distinct functions [41]. A recent Tau is phosphorylated by GSK-3 on approximately 40 report has shown that, whereas GSK-3β is present in birds, different serine and threonine residues, at least in vitro GSK-3α is absent, indicating that the GSK-3α isoform is [60, 61]. The importance of this finding should not be not required either for viability or for normal physiological underestimated since few kinases have been shown to target function in birds [42]. Furthermore, knockout of GSK-3α in this number of sites in tau. Indeed, CK1 is the only other mice results in increased insulin sensitivity [43], suggesting kinase that has been reported to phosphorylate a similar arolefor GSK-3α in glucose metabolism that cannot be number of tau residues. Treatment of primary neuronal replaced by GSK-3β. In contrast, complete knockout of GSK- cortical cultures with specific inhibitors of either GSK-3 3β in mice is embryonic lethal [44]. or CK1 reduces tau phosphorylation, suggesting that these kinases could have functionally important roles in neurons 3.2. Regulation of GSK-3 Activity. The kinase activity of [7, 14, 60]. The phosphorylation of tau by GSK-3 or CK1 also GSK-3 is regulated by its phosphorylation at serine and reduces the ability of tau to promote microtubule assembly tyrosine residues. Phosphorylation of GSK-3α/β at Tyr216 in vitro and in cells [62, 63]. These results rank GSK-3 and and Tyr279 is believed to maintain the constitutive activity CK1 as targeting the greatest number of phosphorylatable of GSK-3 in neurons (reviewed in [45]), while the phospho- residues on tau and implicate these two protein kinases in rylation of Ser21 on GSK-3α or Ser9 on GSK-3β negatively physiological tau phosphorylation in neurons. It remains regulates GSK-3 activity [45], and phosphorylation at these to be seen whether the activities of GSK-3 and/or CK1 are residues is believed to the predominant mediator of GSK-3 modified in diseases in which increased phosphorylation of activity in vivo [46]. Both protein phosphatase 1, (PP1) and tau is a characteristic feature. PP2A are known to target Ser21/9 and thus regulate GSK-3 activity [47]. Indeed, PP activation with okadaic acid leads 4.1. Potential Priming of Tau for GSK-3 Phosphorylation. to increased phosphorylation of S9 and, hence, inhibition It is well established that GSK-3 preferentially phos- of GSK-3β, which results in reduced tau phosphorylation phorylates many of its substrates after they have been [48]. Other phosphatases could also be involved in GSK-3 prephosphorylated by other kinases, and this seems also to regulation since calcineurin (PP2B) also dephosphorylates be true for tau phosphorylation by GSK-3. Other examples and hence activates GSK-3 [49]. In addition, one of the many of GSK-3 substrates that require prephosphorylation by substrates of GSK-3 is the inhibitory subunit (inhibitor-2) another kinase before recognition by GSK-3 include glycogen of PP1. The phosphorylation of this subunit leads to its synthase, inhibitor-2 of PP1, the regulatory subunit of cyclic activation which results in the inhibition of PP1 [50], and AMP-dependent protein kinase (PKA), cAMP response decreased GSK-3 activity, demonstrating one mechanism element-binding (CREB), β-catenin, and kinesin light chain through which GSK-3 activity may be regulated. [46, 64]. Most priming phosphorylations for GSK-3 occur at International Journal of Alzheimer’s Disease 5 an amino acid located four residues C-terminal to the target Prephosphorylation of tau by PKA for subsequent GSK- residue [65]. However, there are some exceptions to this rule, 3 phosphorylation in rat brain appears to particularly and priming events have been reported that occur five or six enhance the overall amount of GSK-3 phosphorylation [73]. residues from the GSK-3 target site, for example, in collapsin Furthermore, the initial phosphorylation of tau by PKA response mediator protein-2 and also in tau [66, 67]. The results in a different spectrum of phosphorylation sites gen- GSK-3 priming phosphorylation is frequently provided by erated by the action of GSK-3 compared to those produced the activity of PKA, CK1, or CK2 on unphosphorylated when tau is exposed to GSK-3 alone, demonstrating that substrates, although other kinases, such as members of the prior phosphorylation by PKA alters the recognition of mitogen-activated protein kinase family, or cdk5, can also tau by GSK-3 [73]. Moreover, initial phosphorylation of initiate priming on some GSK-3 substrates [68, 69]. As has tau by PKA, which occurs primarily on Ser214, results in previously been shown, CK1 primes β-catenin for subse- the subsequent phosphorylation by GSK-3 of four closely quent phosphorylation by GSK-3 (reviewed in [69]), and spaced residues, namely Ser210, Thr205, Ser199, and Ser195, this might also occur on tau because the rate of GSK-3 phos- each separated by 4–6 amino acids [67]. Interestingly, this phorylation of tau is increased when it is first phosphorylated study also revealed an interaction between the low-density by CK1 [70]. Substrate priming, therefore, may represent an lipoprotein receptor-binding domain of apolipoprotein E important regulatory element of GSK-3 signalling since the (ApoE), a major genetic risk factor for Alzheimer’s disease, activity of GSK-3 has been reported to differ for its primed and GSK-3-phosphorylated tau [67]. Such an interaction and nonprimed substrates [68]. In the case of tau, this is was not observed with either nonphosphorylated tau or supported by the observation that targeting GSK-3 phos- following extensive phosphorylation of tau by PKA. These phorylation of tau to either unprimed or primed sites has results suggest that the pattern of phosphorylation sites adifferential impact on the binding of tau to microtubules generated by the action of GSK-3 on tau may be critical for [71, 72]. its interaction with ApoE. There are 24 short sequences of amino acids in tau that conform to the strict consensus sequence Ser/Thr-XXX- 4.2. GSK-3α and GSK-3β1/2 Isoforms Differentially Phospho- Ser/Thr (indicating pairs of serine or threonine residues rylate Tau. Suppressing the expression of individual GSK- separated by any three amino acids), that could be implicated 3α and GSK-3β isoforms results in differing tau phospho- in priming for GSK-3. Fourteen of these serine/threonine rylation patterns [74]. The induction of a phosphorylation- pairs have been found to be phosphorylated in vitro by induced shift in electrophoretic mobility of tau, follow- GSK-3 (Table 1), and six pairs contain a proline residue ing incubation with GSK-3, also appears to be favoured immediately C-terminal to the target sequence. Five of the preferentially by GSK-3β, rather than GSK-3α,asistau 10 remaining paired amino acids are phosphorylated by phosphorylation at the antibody epitopes recognised by tau- GSK-3 on only one of the two serine or threonine residues 1 (Ser199–Ser202) and 8D8 (Ser396) [39]. Together, these and five pairs have not been shown to be phosphorylated results support the view that GSK-3α and GSK-3β are likely by GSK-3. Relaxation of the consensus sequence to include to have differing preferences for tau that may be related to a separation of five or six amino acids between potential the distinct, but overlapping, intracellular locations of these priming sites would allow the inclusion of further sets kinases in neurons. of amino acids, including Ser214, Ser210, Thr205, and There is also a difference in the kinetics and sites of Ser199 (see below). Overall, therefore, tau appears to fulfil tau phosphorylation induced by the two GSK-3β isoforms, several of the requirements for GSK-3 substrates, including with GSK-3β2 appearing to phosphorylate tau more slowly multiple Ser/Thr-Pro sequences with nearby N-terminal than GSK-3β1 and on different, with some overlapping, phosphorylatable amino acids closely opposed, which could tau residues, even under conditions in which other GSK- allow for kinase priming, either by GSK-3 itself or by other 3β substrates, such as amyloid precursor protein, are phos- tau kinases. phorylated equally [75, 76]. For example, Ser396 in tau is Priming phosphorylation on tau residues Ser235 and phosphorylated by both splice variants, whereas Ser199 is a Ser404 by other kinases, including cdk5, has been shown to significant target of GSK-3β1, but not of GSK-3β2activity. It promote phosphorylation by GSK-3 at Thr231 and Ser400, has recently been suggested that the interaction of GSK-3β2 respectively [71, 72]. Interestingly, the stretch of amino acids with tau is weaker than that of GSK-3β1[76]. Taken together, in tau that includes the phosphorylatable residues, Ser396, these results suggest that, not only are there differential Ser400, and Ser404, can be directly phosphorylated by GSK- activities of GSK-3α and GSK-3β towards tau, there are 3 without the prior activity of other kinases [67]. However, also partially overlapping, but distinct, tau phosphorylation phosphorylation at Ser404 is critical to this process and sites recognised by each of the two isoforms of GSK-3β substitution of this residue by alanine ablates phosphoryla- [74–77]. tion of both Ser396 and Ser400. It appears, therefore, that the primary phosphorylation of Ser404 by GSK-3 can itself 5. Tau Phosphorylation in Human Brain serve as a primed residue for the subsequent sequential phosphorylation of tau at Ser400 and Ser396 by GSK-3. In the tauopathies, tau is present in both normally phospho- However, the functional significance of many of the other rylated and highly phosphorylated forms, the latter of which potential GSK-3 priming sites in tau has not been widely may be potentially pathological since it is most commonly investigated. observed as intraneuronal aggregates or glial inclusions. 6 International Journal of Alzheimer’s Disease Tau is normally a highly soluble protein under nonde- 6. Cellular Models of Tau naturing conditions, but the aggregated tau present in Phosphorylation by GSK-3 diseased brain displays a significantly reduced solubility and an increased reactivity to phospho-specific tau antibodies Further information regarding the role of GSK-3 in tau [78]. These characteristic features have facilitated purifica- phosphorylation and function has been gained from cell tion strategies for tau from diseased brain exhibiting tau models. In cells, the involvement of GSK-3 was first revealed pathology which yield relatively enriched preparations of when GSK-3 over-expression was shown to disrupt tau highly phosphorylated tau. Such preparations have resulted binding to microtubules, resulting in a diffuse cytoplasmic in the identification of phosphorylation sites on insoluble localisation of tau [56]. This staining pattern contrasts with tau isolated from human tauopathy brain, thereby providing the strongly fibrillar pattern displayed when tau is expressed clues to the identities of protein kinases that are potentially alone. Cellular phosphorylation of tau by GSK-3β over- involved in the pathogenesis or development of these expressed in mammalian cells decreases tau binding to neurodegenerative diseases [60, 79–81]. microtubules and influences microtubule organisation [87, In particular, these approaches have been used to identify 88]. This process is inhibited by mouse dishevelled-1, thereby phosphorylation sites on purified tau from Alzheimer’s potentially implicating the wingless signalling pathway in tau disease and PSP brain material, as well as soluble tau from phosphorylation mediated by GSK-3 [89]. control adult and foetal brain [60, 82, 83]. These studies, Investigations in human neuronal NT2N cells have combined with results obtained from immuno-labelling shown that tau phosphorylation at multiple sites recognised with phospho-specific tau antibodies, have led to the by phospho-specific tau antibodies could be prevented identification of approximately 45 phosphorylation sites on by lithium treatment, which inhibits GSK-3 activity [90]. tau from Alzheimer brain (Table 1). In comparison, only 15 Lithium also promotes tau binding to microtubules and sub- phosphorylation sites have been detected in insoluble tau sequent microtubule polymerisation, suggesting a role for from PSP brain and 16 sites on control brain tau, not all of GSK-3 in this normal physiological process [90]. It is impor- which overlap, and for some of which no kinase has yet been tant to note that lithium is not a specific inhibitor of GSK- identified [7]. 3 as it also effectively inhibits inositol monophosphatase, as A comparison of tau extracted from human brain with well as a family of structurally related phosphomonoesterases tau phosphorylated using protein kinases in vitro shows that (reviewed in [91]). However, the reported effects of lithium GSK-3 remains a principal candidate kinase for both phys- on tau phosphorylation in cells have subsequently been iological and pathological tau phosphorylation (Table 1). validated in studies using more selective GSK-3 inhibitors Although GSK-3 has been reported to be associated with [74, 92, 93]. The examination of endogenous tau phospho- neurofibrillary pathology in Alzheimer’s disease and related rylation by GSK-3 in primary rat neurons has revealed a neurodegenerative disorders [84, 85], otherstudieshavenot number of lithium-sensitive sites, suggesting that these might identified such colocalisation [39, 86], and it remains to represent the physiological targets of GSK-3 on tau [14, 94]. be seen if GSK-3 remains associated with tau pathology in Interestingly, in the latter study, the inhibition of GSK-3, diseased brain. In the case of tau extracted from neurolog- either by lithium or by the more specific GSK-3 inhibitor, ically normal human brain, all but two residues have been SB-415286, resulted not only in reduced tau phosphorylation shown to be phosphorylated by GSK-3. These two sites, but also in reduced axonal transport of tau [14]. This led to Ser412 and Thr414, are located within a group of three the idea that tau phosphorylation mediated by GSK-3 could closely spaced residues, including Ser413, of which only two influence tau axonal transport, at least in primary neuronal residues are phosphorylated in human brain. Since GSK- cultures [14]. In an ex vivo study, perfusion of preformed 3 phosphorylates Ser413, it is possible that only a single filaments of tau into isolated squid axoplasm resulted in site, either Ser412 or Thr414, remains unphosphorylated the inhibition of anterograde transport of membrane-bound by GSK-3, and, of these, Ser412 is phosphorylated by both organelles [95]. Interestingly, inhibiting GSK-3 activity, or CK1 and PKA, and potentially also by CK2. This suggests perfusion of inhibitor-2 to inhibit PP1 activity, overcame the that, under physiological conditions, tau in human brain detrimental effect of tau filaments on axonal transport in may be phosphorylated primarily by GSK-3, with potential this model [95]. These results, thus, support the notion that contributions from CK1, CK2, or PKA. Under pathological GSK-3 has an important role in relation to tau function in conditions such as Alzheimer’s disease, in which tau becomes neurons. deposited in the brain, GSK-3 accounts for some 27 of Inhibiting GSK-3 with SB-415286 also protects cultured the approximately 45 tau phosphorylation sites identified neurons from cell death induced by reduced phosphatidyli- in insoluble tau. The remaining phosphorylation sites are nositol 3-kinase activity, and this protection is paralleled by accounted for the activities of a combination of other kinases, decreased tau phosphorylation [92]. These results suggest including the tyrosine kinases, c-Abl, and Fyn, although there remain a few sites for which a kinase has not yet been that the state of tau phosphorylation by GSK-3 in cells is important for the maintenance of healthy functional identified. Thus GSK-3 plays a prominent role in both the physiological and pathological phosphorylation of tau in neurons, and changes in tau phosphorylation are likely be human brain. indicative of reduced neuronal viability. International Journal of Alzheimer’s Disease 7 7. Animal Models of Tau spatial learning deficits, and neuronal death [106, 107]. These features can be reversed by suppressing GSK-3β Phosphorylation by GSK-3 expression [108], indicating that tau is a prime substrate 7.1. Drosophila. Over-expression of tau together with GSK- for GSK-3 in vivo. Indeed, the axonopathy and motor 3 (the human orthologue of shaggy/Zw3 in Drosophila) impairment resulting from over-expression of human 2N4R has been demonstrated to exacerbate the neurodegenera- tau in mice is alleviated when GSK-3β is coexpressed [109], tion resulting from expression of tau alone in Drosophila with this beneficial effect believed to occur as a result of GSK- melanogaster [96], suggesting that the phosphorylation 3 phosphorylation of tau removing excess tau from micro- of tau by GSK-3 is involved in the neurodegenerative tubules. Further support for a potential pathological role of process. The presence of partition-defective 1 (PAR-1), the GSK-3-phosphorylated tau has come from studies showing Drosophila orthologue of mammalian microtubule affinity that GSK-3 inhibition in transgenic mice expressing disease- regulating kinase (MARK), has been shown to be required associated mutant human tau reduces tau phosphorylation to initiate GSK-3 and cdk5 phosphorylation of tau in and aggregation [110–114]aswellasaxonaldegeneration flies [97]. However, reports differ as to whether PAR- [111]. 1 activity enhances [97, 98] or suppresses [15, 99, 100] tau-induced toxicity, although PAR-1 over expression in 8. Conclusions Drosophila appears to increase neuronal loss [97]. MARK phosphorylates tau on Ser262 and Ser356 in vitro,bothof Changes in the phosphorylation state of tau have a major which are located within the microtubule-binding domain impact on its subcellular localisation and function in of tau, and this phosphorylation results in the disruption neurons. Major changes in the phosphorylation state of of microtubules [101]. MARK phosphorylation, therefore, tau are evident under two apparently unrelated conditions, results in the detachment of tau from microtubules, and that is, during early neuronal development and during this may be related to its involvement in microtubule-based neurodegenerative processes that lead to the deposition of axonal transport in neurons [102]. Interestingly, MARK itself highly phosphorylated pathological tau in the brain, such as is phosphorylated by GSK-3, resulting in MARK inactivation in Alzheimer’s disease. Therefore, determining the nature of [103], suggesting the possible involvement of multiple tau the kinases involved in this process in vivo is an important kinases in this model, however the relevance GSK-3 and research goal that will improve our understanding of these MARK interaction to tau phosphorylation has not yet been processes. determined. GSK-3 is a key candidate kinase for tau phosphorylation, More recent studies have found that tau phosphorylation under both physiological and pathological conditions. Multi- in Drosophila is not dependent on GSK-3 activity. A mutant ple serine and threonine residues on tau are targeted by GSK- form of tau, described as GSK-3 resistant, retained toxicity 3 in vitro, with many of these sites being coincident with in flies, and, under these conditions, mutant tau exhibited an those phosphorylated in vivo. Together with the, possibly increased affinity for binding to microtubules [98]. Notably, coordinated, activity of other kinases, including CK1, cdk5, while GSK-3 phosphorylation of tau was impaired in these PKA and/or MARK, GSK-3 is well positioned to act on tau flies, toxicity was unaffected, suggesting that the ability of tau in such a way that will result in significant and rapid tau to bind to microtubules, or possibly the propensity of GSK-3 phosphorylation and, hence, modulation of its function in to alter the oligomerisation state of tau, may be critical for neurons. Understanding the regulation of GSK-3 activity neurodegeneration. in neurons, including the possible differential effects of the Drosophila express a form of tau with limited homology related GSK-3α,GSK-3β1, and GSK-3β2 isoforms, should to human tau, especially in the microtubule-binding region lead to further elucidation of the mechanisms underlying tau [104]. There is less than 50% amino acid identity between phosphorylation and may ultimately lead to new therapeutic the presumed microtubule-binding domain of Drosophila targets for neurodegenerative disease. and mammalian tau. Coupled with the presence of an additional microtubule-binding repeat in flies, these consid- Abbreviations erable differences between invertebrate and mammalian tau suggest that the results obtained by overexpressing GSK-3 Aβ: β-amyloid in flies may differ from those obtained in the presence of ApoE: Apolipoprotein E human tau. The relationship, therefore, between tau toxicity cdk5: Cyclin-dependent kinase-5 and tau phosphorylation in Drosophila warrants further CK: Casein kinase investigation if it is to be applied to study the physiological CNS: Central nervous system relevance of GSK-3 phosphorylation of tau. CREB: Cyclic AMP response element binding GSK-3: Glycogen synthase kinase-3 7.2. Mice. The influence of GSK-3-mediated tau phospho- MARK: Microtubule-affinity regulating kinase rylation in mammals has been studied in various mouse NMDAR: N-methyl-D-aspartate receptor models (reviewed in [105]). The postnatal over-expression NR2B: Subunit 2B of the NMDA receptor of GSK-3inmiceresults in GSK-3activationand the PAR-1: Partition-defective 1 development of several characteristics of human tauopathy PKA: Cyclic AMP-dependent protein kinase including elevated tau phosphorylation, reactive gliosis, PP: Protein phosphatase 8 International Journal of Alzheimer’s Disease [13] W. B. Pope, S. A. Enam, N. Bawa, B. E. Miller, H. A. Ghanbari, PP2B: Calcineurin and W. L. Klein, “Phosphorylated tau epitope of Alzheimer’s PSD: Postsynaptic density disease is coupled to axon development in the avian central PSP: Progressive supranuclear palsy nervous system,” Experimental Neurology, vol. 120, no. 1, pp. Ser: Serine 106–113, 1993. SUT2: Solute transport protein-2 [14] I. 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