Jürgen Götz

Amadeus Gladbach

Luis Pennanen

Janet Van Eersel

Andreas Schild

Della David

Lars M Ittner

'Mouse Models of Human Neurological Disorders' (BBADIS-09-30) Animal models reveal role for tau phosphorylation in human disease

Keywords: Alzheimer's disease, amyloid, Drosophila, frontotemporal dementia, kinase, mice, nematode, phosphatase, phosphorylation, tau, transgenic

Many proteins that are implicated in human disease are posttranslationally modified. This includes the microtubule-associated protein tau that is deposited in a hyperphosphorylated form in brains of Alzheimer's disease patients. The focus of this review article is on the physiological and pathological phosphorylation of tau, the relevance of aberrant phosphorylation for disease, the role of kinases and phosphatases in this process, its modeling in transgenic mice, flies and worms, and implications of phosphorylation for therapeutic intervention.

Introduction

The Alzheimer's disease (AD) brain is characterized by massive neuronal and synaptic loss at specific predilection sites [START_REF] Selkoe | Alzheimer's disease is a synaptic failure[END_REF]. Together with tau-containing neurofibrillary tangles (NFTs) and β-amyloid (Aβ) plaques, they are the defining histopathological hallmarks of AD. Neurofibrillary lesions are found in cell bodies and apical dendrites as NFTs, in distal dendrites as neuropil threads, and in the abnormal neurites that are associated with some β-amyloid plaques (neuritic plaques) [START_REF] Goedert | Tau protein in Alzheimer's disease[END_REF]. NFTs are also abundant, in the absence of overt plaques, in certain subsets of frontotemporal dementia (FTD) and other tauopathies [START_REF] Lee | Neurodegenerative tauopathies[END_REF]. The neurofibrillary lesions contain aggregates of the microtubule-associated protein tau that in the course of disease is redistributed from the mainly axonal to the somatodendritic compartment of neurons [START_REF] Goedert | Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau[END_REF]. In tauopathies such as Progressive Supranuclear Palsy (PSP) or Corticobasal Degeneration (CBD), tau forms aggregates also in non-neuronal cells [START_REF] Gotz | Tau and transgenic animal models[END_REF].

Tau is a phosphoprotein owing to its unusually high serine and threonine content. Under pathological conditions, tau is hyperphosphorylated, meaning that it is phosphorylated to a higher degree at normal, physiological sites, and at additional "pathological" sites [START_REF] Chen | Posttranslational modifications of tau -Role in human tauopathies and modeling in transgenic animals[END_REF] (Fig. 1).

Phosphorylation of tau tends to dissociate it from microtubules. Tau also undergoes a conformational change which may assist in differential phosphorylation, or vice versa [START_REF] Jicha | A conformation-and phosphorylation-dependent antibody recognizing the paired helical filaments of Alzheimer's disease[END_REF].

The MAPT (microtubule-associated protein tau) gene that encodes tau is located on human chromosome 17q21; in the central nervous system it contains 15 exons, with the major tau protein isoform being encoded by 11 exons [START_REF] Andreadis | Structure and novel exons of the human tau gene[END_REF]. By alternative mRNA splicing of exons 2, 3, and 10, six major tau isoforms are produced in the adult human brain. They differ by the presence or absence of one or two short inserts in the amino-terminal half (0N, 1N, or 2N, respectively), and have either three or four microtubule-binding repeat motifs in the carboxyterminal half (3R or 4R) (Fig. 1). In contrast to humans, mice express only four-repeat tau isoforms (4R0N, 4R1N, or 4R2N) [START_REF] Goedert | Expression of separate isoforms of human tau protein: correlation with the tau pattern in brain and effects on tubulin polymerization[END_REF].

In AD, no mutations were found in the MAPT gene; these were identified in FTD with Parkinsonism linked to chromosome 17 (FTDP-17) [START_REF] Hutton | Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17[END_REF][START_REF] Poorkaj | Tau is a candidate gene for chromosome 17 frontotemporal dementia[END_REF][START_REF] Spillantini | Mutation in the tau gene in familial multiple system tauopathy with presenile dementia[END_REF] (Fig. 1). This established that dysfunction of tau in itself can cause neurodegeneration and lead to dementia. In a subgroup of FTD, termed FTLD-U (frontotemporal lobar degeneration

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with ubiquitin-immunoreactive lesions) or FTDU-17, tau-negative, ubiquitin-positive lesions are prominent. In these patients, mutations were found in the PGRN gene encoding progranulin, a growth factor involved in multiple physiological and pathological processes including tumorigenesis [START_REF] Baker | Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17[END_REF][START_REF] Cruts | Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21[END_REF]. It was found that the TAR DNA-binding protein of 43 kDa (TDP-43) is a primary constituent of the ubiquitinpositive inclusions in FTLD-U and amyotrophic lateral sclerosis [START_REF] Neumann | Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF]. Under pathological conditions, TDP-43 is, similar to tau, hyperphosphorylated, ubiquitinated, and carboxyterminally truncated [START_REF] Cook | TDP-43 in neurodegenerative disorders[END_REF].

Serine-and threonine-directed phosphorylation of tau

Of all post-translational modifications, protein phosphorylation in general receives particular attention as it is an important cellular regulatory mechanism [START_REF] Chen | Posttranslational modifications of tau -Role in human tauopathies and modeling in transgenic animals[END_REF]. It determines enzymatic activity, protein stability, folding properties including protein aggregation, binding of other bio-molecules and subcellular localization. Phosphorylation occurs mainly on serine, threonine and tyrosine residues, although proteins can be phosphorylated also on histidine, lysine and arginine residues. Phosphorylation depends on a balanced interplay between kinases and phosphatases that is disturbed under pathological conditions. Phosphorylation can also be affected by O-glycosylation since some serine and threonine residues either being Oglycosylated or phosphorylated [START_REF] Guevara | Altered glycosylation pattern of proteins in Alzheimer disease[END_REF][START_REF] Yao | Reduction of O-linked N-acetylglucosamine-modified assembly protein-3 in Alzheimer's disease[END_REF][START_REF] Robertson | The potential role of tau protein Oglycosylation in Alzheimer's disease[END_REF][START_REF] Liu | O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer's disease[END_REF].

Phosphorylation of normal, fetal and 'PHF'-tau.

Tau is a phosphoprotein with a remarkably high number of potential phosphorylation sites. The longest human tau isoform in brain, htau40, contains 45 serines and 35 threonines, of which 17 are followed by prolines (so-called SP/TP-sites). A KXGS motif is found in each microtubule binding domain making this site a substrate of MARK kinase [START_REF] Mandelkow | MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons[END_REF]. In addition, there are 4 tyrosine residues [START_REF] Chen | Posttranslational modifications of tau -Role in human tauopathies and modeling in transgenic animals[END_REF]. Overall, 20% of the protein is composed of amino acids that can be potentially phosphorylated. Therefore, it is no surprise that differential phosphorylation of tau is crucial for its physiological and pathological activities. It is then also logical that phosphorylation of tau within the microtubule-binding domain (e.g. at the KXGS motives recognized by an antibody such as

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12E8 specific for pSer262/pSer3561 , see below) has functional consequences that are different from those of phosphorylation of tau within the flanking sequences (Fig. 1).

Fetal brain contains only 3R tau that is phosphorylated at more sites than tau from adult brain (that contains an equal mix of 3R and 4R tau), implying selective dephosphorylation of the short isoforms during brain maturation [START_REF] Goedert | Molecular dissection of the paired helical filament[END_REF]. In the developing axon, tau forms a smooth proximo-distal phosphorylation gradient: Tau in the soma, immature dendrites and the proximal axon is phosphorylated up to 80% at the tau-1 antibody site (Ser198/Ser199/Ser202 based on htau40), compared to only 20% in the axonal growth cone [START_REF] Tanaka | Abnormally phosphorylated tau in SY5Y human neuroblastoma cells[END_REF][START_REF] Mandell | A spatial gradient of tau protein phosphorylation in nascent axons[END_REF].

In the AD brain, tau is hyperphosphorylated meaning that it is phosphorylated to a higher degree at physiological sites as well as, in addition, at pathological sites (Fig. 1). This tau associates to form paired helical filaments (PHFs) within NFTs. Thus pathologically phosphorylated tau is also called "PHF"-tau [START_REF] Grundke-Iqbal | Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology[END_REF][START_REF] Ihara | Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer's disease[END_REF]. Pathological sites include pSer422, pThr212/pSer214 (AT100), and pThr231/pSer235 (recognized by the conformation-and phosphorylation-dependent antibody PHF-27/TG3) [START_REF] Jicha | A conformation-and phosphorylation-dependent antibody recognizing the paired helical filaments of Alzheimer's disease[END_REF][START_REF] Hasegawa | Characterization of mAb AP422, a novel phosphorylation-dependent monoclonal antibody against tau protein[END_REF][START_REF] Hoffmann | Unique Alzheimer's disease paired helical filament specific epitopes involve double phosphorylation at specific sites[END_REF][START_REF] Bussiere | Phosphorylated serine422 on tau proteins is a pathological epitope found in several diseases with neurofibrillary degeneration[END_REF]. Luckily, the tau research community early on benefited from an excellent collection of epitope-specific antibodies although in recent years the costs for some commercial antibodies have become exorbitantly high (Fig. 1).

Compared to normal tau, PHF-tau displays a reduced electrophoretic mobility reflecting an increased phosphorylation state. As phosphorylation of 'PHF'-tau is very heterogeneous, it runs as a smear on SDS-PAGE gels [START_REF] Flament | Direct demonstration of abnormal phosphorylation of Tau microtubular proteins in Alzheimer's disease[END_REF][START_REF] Hanger | Tau in Alzheimer's disease and Down's syndrome is insoluble and abnormally phosphorylated[END_REF][START_REF] Goedert | Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms[END_REF][START_REF] Greenberg | Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau[END_REF][START_REF] Garver | Tau phosphorylation in human, primate, and rat brain: evidence that a pool of tau is highly phosphorylated in vivo and is rapidly dephosphorylated in vitro[END_REF]. Upon dephosphorylation, the smear can be resolved into discrete bands of a reduced mobility. PHF-tau also has a lower range of pI values, indicating that it is more negatively charged than normal tau [START_REF] Ksiezak-Reding | Alzheimer disease proteins (A68) share epitopes with tau but show distinct biochemical properties[END_REF]. It has a reduced capacity to bind tubulin and to promote the polymerization and stabilization of microtubules. Upon dephosphorylation, PHF-tau binds to tubulin in a manner that is identical to that of normal tau [START_REF] Drewes | Dephosphorylation of tau protein and Alzheimer paired helical filaments by calcineurin and phosphatase-2A[END_REF][START_REF] Iqbal | Alzheimer paired helical filaments. Restoration of the biological activity by dephosphorylation[END_REF]. Using mass spectrometry and phospho-specific antibodies, an increasing number of potential phosphorylation sites have been confirmed in the AD brain, suggesting that as many as 45 of the 84 possible sites of tau are phosphorylated in PHF-tau, compared to 15 in control and up to 20 in fetal brain [START_REF] Hasegawa | Protein sequence and mass spectrometric analyses of tau in the Alzheimer's disease brain[END_REF][START_REF] Morishima-Kawashima | Proline-directed and non-proline-directed phosphorylation of PHF-tau[END_REF][START_REF] Morishima-Kawashima | Hyperphosphorylation of tau in PHF[END_REF][START_REF] Hanger | New phosphorylation sites identified in hyperphosphorylated tau (paired helical filament-tau) from Alzheimer's disease brain using nanoelectrospray mass spectrometry[END_REF][START_REF] Friedhoff | Guidebook to the cytoskeletal and motor proteins[END_REF][START_REF] Hanger | Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis[END_REF]. Obviously, there may be sites that have been missed due to a lack of sensitivity [START_REF] Hanger | Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis[END_REF], however, the question is whether these additional phospho-sites are relevant for disease initiation and/or progression.

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Many phosphorylation sites have been confirmed with phosphorylation-dependent and epitope-specific anti-tau antibodies (Fig. 1). This assisted in correlating differential phosphorylation with disease progression. It was shown that in the pre-NFT state, when tau has not yet formed filaments, neurons were stained specifically with antibody TG3 (conformational and pThr231/pSer235-specific), and the phosphorylation-dependent antibodies pSer262, and pThr153. Intracellular NFTs were most prominently stained with antibodies against pSer46, pThr175/pThr181, pSer214, pSer262/pSer356 (12E8) and pSer422, whereas extracellular NFTs, the so-called ghost tangles, were most prominently stained with the AT8 (pSer202/pThr205), AT100 (pThr212/pSer214) and PHF-1/AD2 (pSer396/pSer404) antibodies, which also stain intraneuronal NFTs. The different states of tau phosphorylation associated with the different stages in the progression of the disease suggest a sequential phosphorylation process which ultimately leads to NFT formation [START_REF] Augustinack | Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer's disease[END_REF]. Sequential phosphorylation is also evident with regards to individual phospho-epitopes. E.g., the AT100 epitope is generated in vitro by sequential phosphorylation, first of Ser199, Ser202 and Thr205

(flanking the AT8 epitope), then of Thr212 (by GSK-3β)' and finally of Ser214 (by PKA) [START_REF] Zheng-Fischhofer | Sequential phosphorylation of Tau by glycogen synthase kinase-3beta and protein kinase A at Thr212 and Ser214 generates the Alzheimerspecific epitope of antibody AT100 and requires a paired-helical-filament-like conformation[END_REF].

Tyrosine-directed phosphorylation of tau

Tau in NFTs is also phosphorylated at tyrosine residues (Fig. 1). There are only four tyrosines in tau compared to 80 serines and threonines, which may reflect the relative importance of tyrosine phosphorylation in physiological and pathological functions, as well as the low number of studies. As for the Ser/Thr-epitopes, there are specific antibodies for the Tyr epitopes (reviewed in [START_REF] Chen | Posttranslational modifications of tau -Role in human tauopathies and modeling in transgenic animals[END_REF]). c-Abl has been suggested as a candidate kinase for Tyr394 phosphorylation, a site phosphorylated in both fetal and PHF-tau [START_REF] Derkinderen | Tyrosine 394 is phosphorylated in Alzheimer's paired helical filament tau and in fetal tau with c-Abl as the candidate tyrosine kinase[END_REF].

Tyrosine phosphorylation plays a pivotal role in the association of tau with the plasma membrane, which is mediated by the amino-terminal projection domain of tau [START_REF] Brandt | Interaction of tau with the neural plasma membrane mediated by tau's amino-terminal projection domain[END_REF].

Association of tau with the plasma membrane may play a crucial role in relaying extra-cellular signals. With its proline rich sequences in the amino-terminus, tau interacts with the SH3 domains of the tyrosine kinase Fyn that phosphorylates tau at Tyr18 [START_REF] Lee | Tau interacts with src-family non-receptor tyrosine kinases[END_REF][START_REF] Lee | Phosphorylation of tau by fyn: implications for Alzheimer's disease[END_REF]. In a study that goes far beyond the mapping of phosphorylation sites, Bhaskar and colleagues addressed the question why an altered ratio of 3R to 4R tau might cause disease in FTD [START_REF] Bhaskar | Disease-related modifications in tau affect the interaction between Fyn and Tau[END_REF]. They used the

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surface plasmon resonance biosensor technique on a panel of tau constructs and found that the interaction between the SH3 domain of Fyn and 3R-tau was 20-fold higher than that for 4Rtau. In addition, the affinity between 4R-tau and Fyn SH3 was increased 25-45-fold by phosphorylation-mimicking mutations or by FTDP-17 mutations, supporting a role for the Fyntau interaction in neurodegeneration [START_REF] Bhaskar | Disease-related modifications in tau affect the interaction between Fyn and Tau[END_REF].

Tau phosphorylation in tau transgenic mice

Classical transgenic approaches continue to be instrumental in dissecting pathogenic mechanisms and testing therapies for AD and FTD [START_REF] Ittner | Pronuclear injection for the generation of transgenic mice[END_REF][START_REF] Gotz | Animal models of Alzheimer's disease and frontotemporal dementia[END_REF]. There are reports however of viral delivery, such as with the Sindbis virus which was employed to express tau in selected hippocampal sites, inducing a pathological conformation of tau as shown by Alz50/MC1-reactivity, causing an accumulation of insoluble tau and inducing a regionspecific neurodegeneration [START_REF] Shahani | Tau aggregation and progressive neuronal degeneration in the absence of changes in spine density and morphology after targeted expression of Alzheimer's diseaserelevant tau constructs in organotypic hippocampal slices[END_REF].

The first published tau transgenic mouse model expressed the longest human fourrepeat (2N4R) tau isoform under control of the neuron-specific human Thy1 promoter [START_REF] Gotz | Somatodendritic localization and hyperphosphorylation of tau protein in transgenic mice expressing the longest human brain tau isoform[END_REF].

Reflecting the approximately tenfold lower levels of human relative to endogenous murine tau, the phosphorylation-specific anti-tau antibodies AT8 and PHF-1/AD2 revealed strongly labeled neurons in many brain areas, but their numbers were small. However, as in the AD brain, tau was re-localized to the somatodendritic domain, in addition to tau's physiological axonal localization. Gallyas silver impregnation failed to reveal tau filament and NFT formation, and tau staining appeared homogenous or at the most, granular [START_REF] Gotz | Somatodendritic localization and hyperphosphorylation of tau protein in transgenic mice expressing the longest human brain tau isoform[END_REF]. A comparable tau phenotype was achieved in mice which expressed the shortest human tau isoform, using the murine 3hydroxy-methyl-glutaryl CoA reductase promoter. Here, tau was phosphorylated at the AT180 (pThr231/pSer235), AT270 (pThr181), AD2 (pSer396/pSer404), 12E8 (pSer262/pSer356), but not the AT8 (pSer202/pThr205) epitopes [START_REF] Brion | Transgenic expression of the shortest human tau affects its compartmentalization and its phosphorylation as in the pretangle stage of Alzheimer's disease [see comments[END_REF] (Fig. 1).

Subsequently, a more advanced phenotype was achieved in three wild-type tau transgenic strains, by choosing better expression vectors [START_REF] Ishihara | Age-dependent emergence and progression of a tauopathy in transgenic mice overexpressing the shortest human tau isoform[END_REF][START_REF] Spittaels | Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein[END_REF][START_REF] Probst | Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein[END_REF]. Tau was found to be phosphorylated at several phospho-epitopes, but NFTs did not develop until the mice reached a very old age [START_REF] Ishihara | Age-Dependent Induction of Congophilic Neurofibrillary Tau Inclusions in Tau Transgenic Mice[END_REF]. In all three mouse strains, neurofilament-containing axonal spheroids formed in brain and spinal cord, representing focal axonal dilatations. Orthograde fast axonal transport

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was reduced as the mice developed a progressive motor phenotype [START_REF] Ishihara | Age-dependent emergence and progression of a tauopathy in transgenic mice overexpressing the shortest human tau isoform[END_REF]. Phosphorylation of tau was analyzed in more detail in one of the strains, ALZ17, where it turned out to be compartmentalized [START_REF] Gotz | Compartmentalized tau hyperphosphorylation and increased levels of kinases in transgenic mice[END_REF]. In the axons of CA1 pyramidal neurons, tau was specifically phosphorylated at the AD2 epitope, concomitant with increased levels of cyclin-dependent kinase-5 (cdk5). In contrast, the 12E8 (pSer262/pSer356) and AT180 (pThr231/pSer235) epitopes were specifically phosphorylated in dendrites, and co-localized with increased levels of GSK-3β [START_REF] Gotz | Compartmentalized tau hyperphosphorylation and increased levels of kinases in transgenic mice[END_REF]. Kinase activities have also been monitored in htau mice, which express genomic wild-type tau (the 8c line [START_REF] Duff | Characterization of pathology in transgenic mice over-expressing human genomic and cDNA tau transgenes[END_REF]), on a tau knockout background [START_REF] Tucker | Neurotrophins are required for nerve growth during development[END_REF]. The authors found a significant correlation between specific phosphorylation changes and the amount of aggregated tau. In the htau line, but not in non-NFT-forming control mice, there were increased levels in phosphorylated (i.e. activated) p38 and the neuronal cdk5 activators, p35 and p25, with aging, which in turn phosphorylate tau. Changes in tau kinases in the htau mice correlated with the amount of tau present in abnormal conformations and with insoluble tau [START_REF] Kelleher | Kinase activities increase during the development of tauopathy in htau mice[END_REF].

Together this shows that tau is subject to differential phosphorylation, due to differential activities and compartmentalization of kinases and phosphatases.

The tau field experienced a major advance with the identification of both exonic and intronic mutations in the MAPT gene encoding tau in FTDP-17, a familial dementia related to AD [START_REF] Hutton | Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17[END_REF][START_REF] Poorkaj | Tau is a candidate gene for chromosome 17 frontotemporal dementia[END_REF][START_REF] Spillantini | Mutation in the tau gene in familial multiple system tauopathy with presenile dementia[END_REF]. The mutations affect the ratio of 3R to 4R tau isoforms and the propensity of tau to form fibrils. By expressing FTDP-17 mutant tau in transgenic mice, many models were established that represented NFT formation in both neuronal and glial cells [START_REF] Gotz | Oligodendroglial tau filament formation in transgenic mice expressing G272V tau[END_REF][START_REF] Gotz | A Decade of Tau Transgenic Animal Models and Beyond[END_REF]. The transgenic models were examined using transcriptomic and proteomic technology, including the assessment of posttranslational modification [START_REF] Hoerndli | Reference genes identified in SH-SY5Y cells using custom-made gene arrays with validation by quantitative polymerase chain reaction[END_REF][START_REF] David | Functional Genomics meets neurodegenerative disorders Part I: Transcriptomic and proteomic technology[END_REF][START_REF] Hoerndli | Functional genomics meets neurodegenerative disorders. Part II: Application and data integration[END_REF][START_REF] David | ß-Amyloid treatment of two complementary P301L tau-expressing Alzheimer's disease models reveals similar deregulated cellular processes[END_REF][START_REF] Sowell | Proteomics in animal models of Alzheimer's and Parkinson's diseases[END_REF], and subjected to behavioral analyses [START_REF] Pennanen | Accelerated extinction of conditioned taste aversion in P301L tau transgenic mice[END_REF][START_REF] Pennanen | Impaired spatial reference memory and increased exploratory behavior in P301L tau transgenic mice[END_REF]. Phosphorylation patterns, however, are difficult to compare as each laboratory uses its own set of phospho-tau-specific antibodies [START_REF] Gotz | Tau and transgenic animal models[END_REF]. In P301S mutant tau transgenic mice, neuronal loss was more pronounced than in the P301L tau models, consistent with the early onset of FTD in patients carrying the P301S mutation [START_REF] Allen | Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein[END_REF]. In the P301S mice, the largest number of stained neurons was observed with AT8, followed by AP422 (pSer422), AT180

(pThr231/pSer235) and PG5 (pSer409) [START_REF] Allen | Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein[END_REF]. Fewer neurons were labeled by AT100 (pThr212/ pSer214) and CP3 (pSer214), with the smallest number of neurons being stained by 12E8 (pSer262/pSer356) and PHF1 (pSer396/pSer404). Western blotting of perchloric acid-soluble tau demonstrated that the human tau band was strongly immunoreactive with all tested

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antibodies, but AT100 (pThr212/pSer214) and CP3 (pSer214). In contrast, sarkosyl-insoluble human tau protein reacted with all phosphorylation-dependent anti-tau antibodies, including AT100 and CP3. This indicates that immunoreactivity for pSer214 (part of epitope AT100) closely mirrors the presence of filaments, suggesting that phosphorylation of this site occurs in the course of, or after, filament assembly. The first P301L transgenic strain generated, JNPL3, is characterized by a pronounced motor phenotype that is not seen in the related P301L transgenic pR5 mice [START_REF] Lewis | Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein[END_REF][START_REF] Gotz | Tau Filament Formation in Transgenic Mice Expressing P301L Tau[END_REF].

When we monitored NFT formation and tau phosphorylation in these pR5 mice for up to 24 months of age we found that NFTs first appeared in the amygdala at 6 months, while at 24 months the only other site with overt NFTs was the CA1 region of the hippocampus [START_REF] Deters | Divergent phosphorylation pattern of tau in P301L tau transgenic mice[END_REF]. A histological analysis revealed an increase in phosphorylation at the AT180 (pThr231/pSer235), AT270 (pThr181) and 12E8 (pSer262/pSer356) epitopes with ageing whilst the AT8 (pSer202/ pThr205) and pS422 epitopes behaved differently. Firstly, whereas AT8 reactivity was found in both the soma and dendritic branch of CA1 pyramidal neurons, pS422 staining was more confined to the soma. Secondly, numbers of AT8-positive neurons increased from 3 to 6 months of age, but at 20 months of age, the only neurons left with AT8-reactivity were those which had undergone NFT formation. In contrast, pS422-reactivity came up only late and concomitantly with NFT formation [START_REF] Deters | Divergent phosphorylation pattern of tau in P301L tau transgenic mice[END_REF]. As far as the disappearance in our pR5 mice of the AT8 epitope in all but NFT-bearing neurons is concerned, one explanation may be that with advanced pathology, neurons learn to cope with AT8 phosphorylation and the activities of the kinase and/or phosphatase regulating phosphorylation of this epitope become balanced resulting in a zero net phosphorylation at this epitope [START_REF] Chen | Posttranslational modifications of tau -Role in human tauopathies and modeling in transgenic animals[END_REF][START_REF] Iqbal | Mechanisms of neurofibrillary degeneration and the formation of neurofibrillary tangles[END_REF]. This balancing act seems to be specific for the AT8 epitope and the enzymes governing its phosphorylation, as other epitopes such as AT180 or AT270 continue to be phosphorylated. In NFT-bearing neurons, however, the AT8 phospho-epitope becomes stabilized suggesting that either a tau phosphatase may not be able to access the phosphorylation site and/or the kinase/phosphatase equilibrium in the dysfunctional neuron may be out of balance. Our AT8 findings are supported by data in P301S transgenic mice which, although analyzed only until 6 months of age, reveal an increased pS422, AT8 and AT180 phosphorylation in RIPA-extracted brains that peaks at 4-5 months and then declines significantly at 6 months. This decline was not seen with AT270 or phosphorylation-independent tau antibodies [START_REF] Delobel | Analysis of tau phosphorylation and truncation in a mouse model of human tauopathy[END_REF]. The authors state that the reduction at 5

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months was particularly marked for the AT8 epitope [START_REF] Delobel | Analysis of tau phosphorylation and truncation in a mouse model of human tauopathy[END_REF]. The importance of both the AT8 and pS422 epitope in tauopathies is also demonstrated by the fact that in both the JNPL3 and the rTg4510 strain discussed below, a 170 kDa band linked to clinical features was preferentially detected by AT8 and pS422, and this, possibly oligomeric, species negatively correlated with memory [START_REF] Berger | Accumulation of pathological tau species and memory loss in a conditional model of tauopathy[END_REF].

To determine whether NFTs are central to the neurotoxic cascade in AD or represent a protective neuronal response, P301L transgenic rTg4510 mice were generated [START_REF] Santacruz | Tau suppression in a neurodegenerative mouse model improves memory function[END_REF]. Here, transgene overexpression could be reduced by adding doxycycline to the drinking water [START_REF] Santacruz | Tau suppression in a neurodegenerative mouse model improves memory function[END_REF]. Despite the fact that even under suppressed conditions tau levels were higher than what is generally achieved by conventional transgenic approaches, memory function recovered and number of neurons stabilized, while NFTs continued to accumulate under these conditions. These data show that dysfunction of tau impairs memory, when mutant tau is massively overexpressed. They further imply that soluble tau rather than NFTs themselves are neurotoxic [START_REF] Santacruz | Tau suppression in a neurodegenerative mouse model improves memory function[END_REF]. Tau phosphorylation in the rTg4510 brain was addressed in an accompanying paper, monitoring the mice from 1.3 to 8.5 months of age [START_REF] Ramsden | Age-dependent neurofibrillary tangle formation, neuron loss, and memory impairment in a mouse model of human tauopathy (P301L)[END_REF]. Differences were found between the hippocampus and cortex as AT8 reactivity was preceded by TG3 in the hippocampal CA1 region in an order which is opposite to that found in the cortex. In the CA1 region the epitopes appeared in the following order: CP13 (pSer202), MC1 (conformational) and TG3 (conformational & pThr231/pSer235) at 1.3 months, followed by PG5 (pSer409), AT8 (pSer202/pThr205), PHF-1 (pSer396/pSer404), followed by Bielschowsky reactivity at 4-5.5 months. For the cortex, MC1, CP-13, AT8 and PHF-1 appeared first, followed by TG3, PG5 and Bielschowsky reactivity [START_REF] Ramsden | Age-dependent neurofibrillary tangle formation, neuron loss, and memory impairment in a mouse model of human tauopathy (P301L)[END_REF]. This appearance of phosphorylation sites raises the question as to whether for tau to aggregate there needs to be a specific order of phosphorylation events for the formation of tau aggregates, an issue discussed below in more detail.

An interesting model of the specific phosphorylation profile that characterizes

Pick's disease is the K3 strain that expresses human tau carrying the FTD mutation K369I [START_REF] Ittner | Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia[END_REF]. K3 mice develop a progressive histopathology that is reminiscent of human FTD with the K369I mutation [START_REF] Neumann | Pick's disease associated with the novel Tau gene mutation K369I[END_REF]. Specifically, as in human Pick's disease [START_REF] Van Eersel | Phosphorylation of soluble tau differs in Pick's disease and Alzheimer's disease brains[END_REF] and in the human K369I patient where the tau-containing Pick bodies are 12E8

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(pSer262/pSer356)-negative [START_REF] Neumann | Pick's disease associated with the novel Tau gene mutation K369I[END_REF], K3 mice also develop ovoid tau inclusions that are 12E8-negative, while additional phospho-epitopes of tau (as in humans) are strongly phosphorylated [START_REF] Ittner | Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia[END_REF]. K3 mice show an early-onset memory impairment and amyotrophy, in the absence of overt neurodegeneration. However, as the mice age, neurodegeneration becomes evident. Different from our previously generated tau transgenic strains, the K3 mice express the transgene in the substantia nigra (SN) and show an early-onset motor phenotype that reproduces Parkinsonism with tremor, bradykinesia, abnormal gait and postural instability. Interestingly, motor performance of young, but not old K3 mice improved upon L-dopa treatment, which bears similarities to Parkinsonism in FTD. The early-onset symptoms in the K3 mice are mechanistically related to selectively impaired anterograde axonal transport of distinct cargos, which precedes the loss of dopaminergic SN neurons that occurs in aged mice [START_REF] Ittner | Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia[END_REF]. The impaired axonal transport in SN neurons affects, among others, vesicles containing the dopamine-synthesizing enzyme tyrosine hydroxylase (TH) [START_REF] Ittner | Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia[END_REF]. We found that phosphorylated tau interacts pathologically with the kinesin-associated adapter protein JIP1 both, in the mice and in AD brain [START_REF] Ittner | Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia[END_REF][START_REF] Ittner | Phosphorylated Tau Interacts with c-Jun N-terminal Kinase-interacting Protein 1 (JIP1) in Alzheimer Disease[END_REF].

We proved that phosphorylation of tau is required for this pathogenic effect [START_REF] Ittner | Phosphorylated Tau Interacts with c-Jun N-terminal Kinase-interacting Protein 1 (JIP1) in Alzheimer Disease[END_REF] suggesting a pathological interaction of JIP1 and phosphorylated tau as a general pathomechanism in tauopathies including AD.

Using an elegant stereotaxic injection approach, tau toxicity was shown to bear resemblance to prions and to spread through the brain [START_REF] Clavaguera | Transmission and spreading of tauopathy in transgenic mouse brain[END_REF]. The study used two mouse strains: ALZ17 mice that express high levels of wild-type human tau but reveal only a modest pathology: amyotrophy in the absence of obvious neuronal cell loss and, despite a massive hyperphosphorylation of tau, no formation of NFTs [START_REF] Probst | Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein[END_REF][START_REF] Gotz | Compartmentalized tau hyperphosphorylation and increased levels of kinases in transgenic mice[END_REF]. In contrast, P301S

mice express, at levels comparable to the ALZ17 mice, a mutant form of tau found in familial cases of FTD; the mice present with a particularly robust phenotype, characterized by neurodegeneration in the spinal cord and an abundance of NFTs [START_REF] Allen | Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein[END_REF].

When diluted brain extracts from 6 month-old P301S mice were intracerebrally injected into 3 month-old ALZ17 mice and the injected mice analyzed up to 15 months postinjection, NFT formation was found as revealed by Gallyas silver impregnation, and reactivity with antibody AT100 (pThr212/pSer 214).

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Interestingly, Clavaguera and colleagues found that Gallyas reactivity (i.e. NFT formation) was not confined to the site of injection, but rather induced up to 2 mm distant of the injection site [START_REF] Clavaguera | Transmission and spreading of tauopathy in transgenic mouse brain[END_REF]. A time-course analysis suggests a stereotypical mode of spreading (a feature characteristic of AD). Another remarkable finding was that insoluble rather than soluble tau was responsible for the induction of a tau pathology. Also, induction of the tau pathology seemed to be mediated by oligodendroglia (a cell type not affected by tau pathology in the parental P301S mice).

Additional tau transgenic models have been discussed by us in detail elsewhere [START_REF] Gotz | Tau and transgenic animal models[END_REF][START_REF] Gotz | A Decade of Tau Transgenic Animal Models and Beyond[END_REF][START_REF] Gotz | Transgenic animal models of Alzheimer's disease and related disorders: Histopathology, behavior and therapy[END_REF]. Taken together, tau transgenic models have proven in vivo that the presence of familial FTD-associated tau mutations causes tau hyperphosphorylation, aggregation, nerve cell dysfunction as well as neuronal and glial cell loss. They have revealed that a glial tau pathology can affect neuronal functioning and that a tau pathology in general causes behavioural impairment [START_REF] Kurosinski | Glial cells under physiologic and pathological conditions[END_REF]. Finally, the models highlight distinct phosphorylation sites such as pS422, pThr212/pSer214 (AT100), pThr231/pSer235 (TG3) and pSer202/pThr205 (AT8) in disease initiation and progression and hence, provide a means to target distinct kinases for therapeutic intervention.

Tau phosphorylation in kinase transgenic mice

In principal, tau phosphorylation can be brought about by the upregulation of kinases, the downregulation of phosphatases, or both. A major question asked in the field is which kinase(s) and phosphatase(s) in brain bring about the pathological changes that characterize AD-tau and how these are related to neuronal demise. The human genome encodes a total of 516 protein kinases, with numbers comparable in mice. Of these kinases, several have been identified as potential tau kinases, mostly by in vitro assays incubating recombinant tau protein with the respective kinases. These include GSK-3β; Cdk5; the MAP kinases JNK, ERK and p38; MARK; CK2; DYRK1A; TTBK1 and P70S6 kinase [START_REF] Hanger | Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis[END_REF][START_REF] Hanger | Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase[END_REF][START_REF] Mandelkow | Glycogen synthase kinase-3 and the Alzheimerlike state of microtubule-associated protein tau[END_REF][START_REF] Baumann | Abnormal Alzheimer-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5[END_REF][START_REF] Hyman | Extracellular signal regulated kinases. Localization of protein and mRNA in the human hippocampal formation in Alzheimer's disease[END_REF][START_REF] Trinczek | Domains of tau protein, differential phosphorylation, and dynamic instability of microtubules[END_REF][START_REF] Reynolds | Stressactivated protein kinase/c-jun N-terminal kinase phosphorylates tau protein[END_REF][START_REF] Reynolds | Reactivating kinase/p38 phosphorylates tau protein in vitro[END_REF][START_REF] Guimera | Human minibrain homologue (MNBH/DYRK1): characterization, alternative splicing, differential tissue expression, and overexpression in Down syndrome[END_REF][START_REF] Ferrer | Current advances on different kinases involved in tau phosphorylation, and implications in Alzheimer's disease and tauopathies[END_REF][START_REF] Pei | P70 S6 kinase mediates tau phosphorylation and synthesis[END_REF][START_REF] Sato | Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation[END_REF]. These experiments assisted in determining which kinases phosphorylate which phospho-epitopes of tau and whether there is a sequential phosphorylation of sites, such as for phosphorylation of Thr231, Ser396 and Ser400 by GSK-3β that depends on a previous phosphorylation of Ser235, Ser400 and Ser404, respectively, to provide one example [START_REF] Li | Glycogen synthase kinase 3beta phosphorylates Alzheimer's disease-specific Ser396 of microtubule-associated protein tau by a sequential mechanism[END_REF][START_REF] Li | Cyclin-dependent protein kinase 5 primes microtubule-associated protein tau site-specifically for glycogen synthase kinase 3beta[END_REF]. A few of these candidate kinases

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have been expressed in transgenic mice and tau phosphorylation has been analyzed, either in single-transgenic mice or after crossbreeding with tau transgenic mice. The findings confirmed that kinases play an important role in NFT formation and neurodegeneration. The bulk of studies concentrated on two kinases, GSK-3β and Cdk5, as outlined below.

The kinase GSK-3β is inactivated by phosphorylation of residue Ser9. Mice with a constitutive active S9A form of GSK-3β showed increased activity, in the absence of neurofibrillary pathology. Interestingly, crossing of these mice with human wild-type tau transgenic mice markedly improved the axonopathy and motor deficits that characterize the latter strain [START_REF] Spittaels | Glycogen synthase kinase-3beta phosphorylates protein tau and rescues the axonopathy in the central nervous system of human four-repeat tau transgenic mice[END_REF]. These findings would therefore suggest that GSK-3β is protective. However, additional studies using different models reached the opposite conclusion. When mice with an inducible expression of GSK-3β were crossed with mice that express tau with three FTDP-17 mutations combined (VLW mice), the double-transgenic mice developed thioflavin S-positive tau aggregates and tau filaments. Moreover, the atrophy of the dentate gyrus of the hippocampus, which was present in the single-transgenic GSK-3β mice, was accelerated in the double-transgenic mice [START_REF] Engel | Cooexpression of FTDP-17 tau and GSK-3beta in transgenic mice induce tau polymerization and neurodegeneration[END_REF]. To explore whether the phenotype resulting from increased GSK-3β activity could be reverted following restoration of normal GSK-3β levels, transgene expression was shut down in symptomatic mice. This led to normal GSK-3β activity, normal phospho-tau levels, diminished neuronal cell death and suppression of the cognitive deficits [START_REF] Engel | Full reversal of Alzheimer's diseaselike phenotype in a mouse model with conditional overexpression of glycogen synthase kinase-3[END_REF]. When an inducible system was used to express a dominant negative mutant form of GSK-3β, this caused apoptosis and a reversible motor deficit [START_REF] Gomez-Sintes | Neuronal apoptosis and reversible motor deficit in dominant-negative GSK-3 conditional transgenic mice[END_REF]. In these mice, AT8 phosphorylation (pSer202/pThr205) was reduced by 72% and PHF-1 phosphorylation (pSer396/pSer404) by 46%. So in conclusion, these data identify GSK-3β as a tau kinase in vivo.

Another kinase that has been expressed in transgenic mice is Cdk5. To address its role in tau pathogenesis, P301L tau transgenic JNPL3 mice were crossed with mice transgenic for the Cdk5 activator p25, the latter being expressed under control of the NSE promoter. This caused a fivefold increase in NFT numbers in double-transgenic mice, along with hyperphosphorylation of tau at the putative cdk5 epitopes pThr181 (AT270), pSer202, pThr231, and pSer396/pSer404 (AD2/PHF1) [START_REF] Noble | Cdk5 is a key factor in tau aggregation and tangle formation in vivo[END_REF]. On the other hand, a p25-mediated overactivation of Cdk5 in a CaMKII promoter-driven p25 transgenic strain did not induce tau hyperphosphorylation at a young age per se, possibly because Cdk5 over-activation inhibited

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GSK-3β, by phosphorylating its inhibitory Ser9 site [START_REF] Plattner | The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation[END_REF]. However, as the mice became older, this inhibition was lost, resulting in increased GSK-3β activity that was associated with tau hyperphosphorylation at the AT8 (pSer202/pThr205) and PHF1 (pSer396/pSer404) sites.

Together with pharmacological and co-immunoprecipitation experiments this would suggest that GSK-3β is a key mediator of tau hyperphosphorylation, while Cdk5 acts as a modulator of tau hyperphosphorylation via the inhibitory regulation of GSK-3β [START_REF] Plattner | The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation[END_REF]. These findings were confirmed in a second p25 transgenic model [START_REF] Wen | Interplay between cyclin-dependent kinase 5 and glycogen synthase kinase 3 beta mediated by neuregulin signaling leads to differential effects on tau phosphorylation and amyloid precursor protein processing[END_REF]. It was found that phosphorylation of residue Ser9 of GSK-3β was mediated by an enhanced activity of the neuregulin receptor complex, ErbB, and by activation of the downstream phosphatidylinositol 3 kinase/Akt pathway. While young p25 mice had elevated Aβ levels, levels of phosphorylated tau were decreased. Thus, Cdk5 appears to play a dominant role in the regulation of amyloidogenic APP processing, whereas GSK-3β seems to play a dominant role in overall tau phosphorylation [START_REF] Wen | Interplay between cyclin-dependent kinase 5 and glycogen synthase kinase 3 beta mediated by neuregulin signaling leads to differential effects on tau phosphorylation and amyloid precursor protein processing[END_REF].

Inducible systems were employed that caused pronounced phenotypic changes.. For example, transgenic mice with an inducible p25 expression in the postnatal forebrain revealed a massive neuronal loss in the cortex and hippocampus, along with forebrain atrophy, astrogliosis, and caspase-3 activation [START_REF] Cruz | Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles[END_REF]. At only 5 weeks of age, endogenous tau was hyperphosphorylated at many epitopes, as shown for AT8 (pSer202/pThr205) and PHF1 (pSer396/pSer404). In one year-old mice, immuno-electron microscopy of sarkosyl-insoluble fractions revealed tau filaments that were phosphorylated at the AT8, PHF1, TG3 (pThr231/pSer235) and AT100 (pThr212/pSer214) epitopes. Also, Gallyas silver impregnation revealed NFT formation in these mice [START_REF] Cruz | Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles[END_REF]. In a second inducible mouse strain, neuronal p25 triggered a similar neurodegeneration and marked neuronal loss, causing brain atrophy with a 40% loss at 5 months of age resulting in an almost complete elimination of the hippocampus.

Interestingly, this type of neurodegeneration was not associated with hyperphosphorylation of tau or Aβ generation [START_REF] Muyllaert | Neurodegeneration and neuroinflammation in cdk5/p25-inducible mice: a model for hippocampal sclerosis and neocortical degeneration[END_REF]. One likely explanation is that p25 neuro-toxicity is related to substrates other than tau or APP.

The tau-tubulin kinase (TTBK) family consists of TTBK1 and TTBK2 that belong to the casein kinase 1 superfamily. Different from TTBK2, TTBK1 is specifically expressed in neurons. It can phosphorylate tau directly at multiple Ser/Thr residues that are found in PHFtau in AD brain. Furthermore, TTBK1 is expressed in NFT-bearing neurons in the AD cortex

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ACCEPTED MANUSCRIPT [START_REF] Sato | Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation[END_REF]. Transgenic mice expressing full-length human TTBK1 show an age-dependent memory impairment accompanied by increased phosphorylation of tau and neurofilaments, and increased levels of p25 and p35 [START_REF] Sato | Spatial learning impairment, enhanced CDK5/p35 activity, and downregulation of NMDA receptor expression in transgenic mice expressing tau-tubulin kinase 1[END_REF].

Finally, another interesting kinase is DYRK1A (dual specificity tyrosine-regulated kinase-1A) that has been implicated in Down's syndrome (DS), due to the location of the Dyrk1a gene in the critical region of human chromosome 21 [START_REF] Guimera | Human minibrain homologue (MNBH/DYRK1): characterization, alternative splicing, differential tissue expression, and overexpression in Down syndrome[END_REF]. DS is a genetic disorder in humans caused by partial or complete trisomy of chromosome 21. People with DS show a tendency towards premature aging and an increased risk for AD [START_REF] Zigman | Alzheimer's disease in Down syndrome: neurobiology and risk[END_REF]. DS patients show an early-onset tauopathy that resembles AD in many ways. In addition to mental problems, motor dysfunction is highly prevalent in DS. Dyrk1a transgenic mice express the transgene in several areas of the mid-and hindbrain [START_REF] Martinez De Lagran | Motor phenotypic alterations in TgDyrk1a transgenic mice implicate DYRK1A in Down syndrome motor dysfunction[END_REF]. When assessed in motor tests such as the treadmill, the mice showed impairment in some parameters; in particular, they required longer training periods [START_REF] Martinez De Lagran | Motor phenotypic alterations in TgDyrk1a transgenic mice implicate DYRK1A in Down syndrome motor dysfunction[END_REF]. A transcriptomic analysis revealed upregulation of the NMDA receptor subunit 2A which may explain the altered excitatory transmission reported in humans with DS and in DS mouse models [START_REF] Altafaj | Increased NR2A expression and prolonged decay of NMDA-induced calcium transient in cerebellum of TgDyrk1A mice, a mouse model of Down syndrome[END_REF]. Altered synaptic plasticity along with learning and memory deficits was reported for a second strain, that used a BAC clone of Dyrk1a [START_REF] Ahn | DYRK1A BAC transgenic mice show altered synaptic plasticity with learning and memory defects[END_REF]. A similar memory phenotype was found in Dyrk1a haplo-insufficient mice [START_REF] Arque | Impaired spatial learning strategies and novel object recognition in mice haploinsufficient for the dual specificity tyrosine-regulated kinase-1A (Dyrk1A)[END_REF]. In one of the mouse models, tau phosphorylation was addressed: tau in Dyrk1a transgenic mice was hyperphosphorylated at Thr212, Ser202 and Ser404; furthermore, phosphorylation by DYRK1A strongly inhibited the ability of tau to promote microtubule assembly [START_REF] Ryoo | DYRK1A-mediated hyperphosphorylation of Tau. A functional link between Down syndrome and Alzheimer disease[END_REF]. The finding that gene dosage of Dyrk1 affects tau splicing may explain the early onset tauopathy in individuals with DS [START_REF] Shi | Increased dosage of Dyrk1A alters alternative splicing factor (ASF)-regulated alternative splicing of tau in Down syndrome[END_REF] Whilst these studies always only test a subset of tau phosphorylation sites, they still highlight a role for cdk5 and its activator p25 in Aβ pathology, and for GSK-3β, DYRK1A and TTBK1 in tau aggregation. Whether targeting a specific kinase in the development of an AD therapy will be a fruitful approach is still a matter of debate. It may well be that in a human setting a general subtle down-regulation of kinase(s) in the brain may be more beneficial.

Tau phosphorylation in phosphatase transgenic mice

Of the serine/threonine-specific phosphatases that are abundant in the brain, protein phosphatase 2A (PP2A) is a major phosphatase implicated in tau dephosphorylation [START_REF] Goedert | Protein phosphatase 2A is the major enzyme in brain that dephosphorylates tau protein phosphorylated by proline-directed protein kinases or cyclic AMP-dependent protein kinase[END_REF],

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perhaps more so because PP2A can bind directly to tau [START_REF] Sontag | Regulation of the phosphorylation state and microtubule-binding activity of Tau by protein phosphatase 2A[END_REF][START_REF] Sontag | Molecular interactions among protein phosphatase 2A, tau, and microtubules. Implications for the regulation of tau phosphorylation and the development of tauopathies[END_REF][START_REF] Liu | Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation[END_REF]. PP2A is a trimeric holoezyme that consists of a catalytic subunit C and a scaffolding subunit A. The A/C core enzyme associates with variable regulatory subunits of the PR55(B), PR56/61(B'), PR59/72/130(B'') and PR93/110(B''') families to form heterotrimers [START_REF] Janssens | Protein phosphatase 2A: a highly regulated family of serine/ threonine phosphatases implicated in cell growth and signalling[END_REF]. Recruitment of regulatory B subunits into the holoenzyme is dependent upon the highly conserved DYFL motif in the carboxy-terminus of subunit C that undergoes methylation at Leu309, thereby affecting subunit B recruitment [START_REF] Xie | Protein phosphatase 2A is reversibly modified by methyl esterification at its C-terminal leucine residue in bovine brain[END_REF][START_REF] Bryant | Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Balpha subunit[END_REF]. The resolution of the structure of the B subunit provides significant insight into how PP2A dephosphorylates tau [START_REF] Xu | Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated Tau dephosphorylation[END_REF]. Interestingly, methylation of the C subunit does not seem to be required for in vitro assembly of the PP2A holoenzyme involving either B or B'. The authors argue that the regulatory subunits may be sequestered in a specific cellular compartment and that the methylated carboxy-terminus of the C subunit may allow subcellular targeting for holoenzyme assembly [START_REF] Xu | Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated Tau dephosphorylation[END_REF].

Dephosphorylation of tau can be blocked in cells by okadaic acid (OA), an inhibitor of the two phosphatases PP1 and PP2A [START_REF] Arendt | Paired helical filament-like phosphorylation of tau, deposition of beta/A4-amyloid and memory impairment in rat induced by chronic inhibition of phosphatase 1 and 2A[END_REF][START_REF] Planel | Inhibition of protein phosphatase 2A overrides tau protein kinase I/glycogen synthase kinase 3 beta and cyclindependent kinase 5 inhibition and results in tau hyperphosphorylation in the hippocampus of starved mouse[END_REF]. When rat brain slices were incubated with OA, tau became phosphorylated at multiple sites [START_REF] Pei | Okadaic-acid-induced inhibition of protein phosphatase 2A produces activation of mitogen-activated protein kinases ERK1/2, MEK1/2, and p70 S6, similar to that in Alzheimer's disease[END_REF]. A role for PP2A in AD is implicated by the finding that its activity is reduced in AD brain [START_REF] Gong | Phosphoprotein phosphatase activities in Alzheimer disease brain[END_REF]; furthermore, association analyses indicate that a CAG repeat polymorphism in one of the B subunits may confer susceptibility to AD [START_REF] Chen | PPP2R2B CAG repeat length in the Han Chinese in Taiwan: Association analyses in neurological and psychiatric disorders and potential functional implications[END_REF].

Transgenic strategies targeting PP2A include gene knockouts, overexpression of regulatory subunits and dominant negative mutant approaches [START_REF] Gotz | Transgenic and knockout models of PP2A[END_REF]. A knockout of the major catalytic subunit Cα caused delayed embryonic lethality [START_REF] Gotz | Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Calpha[END_REF], as the highly homologous Cβ subunit failed to complement the lack of Cα in mesoderm formation [START_REF] Gotz | The role of protein phosphatase 2A catalytic subunit Calpha in embryogenesis: evidence from sequence analysis and localization studies[END_REF], due to a different subcellular localization of the two catalytic subunits [START_REF] Gotz | Distinct role of protein phosphatase 2A subunit calpha in the regulation of E-cadherin and beta-catenin during development[END_REF]. Under physiological conditions, the A and C subunits are ubiquitously expressed, whereas the regulatory B subunits show a tissuespecific expression; they also reveal a differential expression pattern in brain and during neuronal differentiation [START_REF] Schmidt | Diversity, developmental regulation and distribution of murine PR55/B subunits of protein phosphatase 2A[END_REF][START_REF] Martens | Genomic organisation, chromosomal localisation, tissue distribution and developmental regulation of the PR61/B' regulatory subunits of protein phosphatase 2A in mice[END_REF][START_REF] Schild | Altered levels of PP2A regulatory B/PR55 isoforms indicate role in neuronal differentiation[END_REF].

To address the role of PP2A in tau phosphorylation in vivo, transgenic mice were generated that express a dominant negative mutant form of the catalytic subunit Cα of PP2A, L199P, in neurons. The transgenic mice have a reduced PP2A activity resulting in a pre-NFT phenotype, with phosphorylation of endogenous murine tau at the AT8 (pSer202/pThr205) and

Ser422 epitopes [START_REF] Kins | Reduced PP2A activity induces hyperphosphorylation and altered compartmentalization of tau in transgenic mice[END_REF]. This effect may be directly mediated by PP2A, but could also occur

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indirectly, via deregulated kinases such as ERK and JNK, that are themselves substrates of PP2A [START_REF] Kins | Activation of the ERK and JNK signaling pathways caused by neuron specific inhibition of PP2A in transgenic mice[END_REF]. The carboxy-terminal DYFL motif of the catalytic subunit has a role in the recruitment of B subunits into the PP2A complex, by methylating leucine 309 and phosphorylating tyrosine 307 [START_REF] Janssens | Protein phosphatase 2A: a highly regulated family of serine/ threonine phosphatases implicated in cell growth and signalling[END_REF]. To determine the role of the DYFL motif in PP2A activity in vivo, a second dominant negative mutant strain was established that expressed the L309A mutant form of Cα in neurons. This caused an altered subunit composition of the PP2A holoenzyme in vivo. In the brain, tau was hyperphosphorylated at Ser202/Thr205 (AT8/CP13); furthermore, there was an impaired dephosphorylation of the intermediate filament protein

vimentin [START_REF] Schild | Altered phosphorylation of cytoskeletal proteins in mutant protein phosphatase 2A transgenic mice[END_REF]. The additional expression of the L309A transgene expression in the Harderian (lacrimal) gland caused a delayed postnatal development and hypoplasia of this gland, causing enophthalmos [START_REF] Schild | Impaired development of the Harderian gland in mutant protein phosphatase 2A transgenic mice[END_REF]; .

When the PP2A L309A mutant mice were crossed with P301L tau mutant pR5 mice, this caused a seven-fold increased number of hippocampal neurons that specifically s phosphorylated the Ser422 epitope of tau. The double-transgenic mice showed eight-fold increased numbers of NFTs compared to pR5 mice, in agreement with the previous finding that NFT formation is correlated with and preceded by phosphorylation of tau at the Ser422 epitope [START_REF] Deters | Substrate-specific reduction of PP2A activity exaggerates tau pathology[END_REF]. The critical role of the DYFL motif in PP2A function, with particular regards to the cytoskeleton, is further demonstrated by cell culture experiments, which showed that expression of mutants such as L309Δ induces a loss of microtubules [START_REF] Nunbhakdi-Craig | Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules[END_REF]. As mentioned above, in addition to methylation at the carboxy-terminal Leu309, phosphorylation at Tyr307 also regulates PP2A function [START_REF] Janssens | Protein phosphatase 2A: a highly regulated family of serine/ threonine phosphatases implicated in cell growth and signalling[END_REF]. PP2A phosphorylated at Tyr307 associates with pretangles and NFTs in areas such as the entorhinal cortex and the hippocampus, brain areas where the neurofibrillary changes are initiated [START_REF] Liu | Phosphorylated PP2A (tyrosine 307) is associated with Alzheimer neurofibrillary pathology[END_REF]. Together, these studies demonstrate a crucial role for PP2A in tau phosphorylation. As many kinases are themselves substrates of PP2A, the control of PP2A goes beyond the reversible phosphorylation of its non-enzymatic substrates. It would not be surprising to find that targeting of PP2A, in particular of its regulatory subunits, represents a valid means of treating AD.

Tau phosphorylation in mice with an amyloid pathology

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Tau phosphorylation is part of the phenotypic characterization of APP mutant mouse models.

In the following, we selected a few examples to demonstrate how Aβ affects endogenous tau phosphorylation in vivo. For example, in one of the earliest reports of a strong Aβ-plaque forming mouse model, the APP23 mice, increased tau phosphorylation was found to parallel Aβ peptide deposition [START_REF] Sturchler-Pierrat | Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology[END_REF]. APP23 mice have been thoroughly investigated, recently by using 3D reconstruction techniques to determine how small Aβ aggregates associated with the microvasculature lead to morphological and architectural alterations of the vasculature, thus resulting in an altered local blood flow [START_REF] Meyer | Altered morphology and 3D architecture of brain vasculature in a mouse model for Alzheimer's disease[END_REF]. Even more studies have been conducted with the APP mutant mouse strain Tg2576 in which an anti-Aβ treatment provided evidence for a slow seeding mechanism preceding a rapid fibrillogenesis in determining the extent of Aβ deposition [START_REF] Karlnoski | Suppression of amyloid deposition leads to long-term reductions in Alzheimer's pathologies in Tg2576 mice[END_REF]. Formation of AT8 (pSer202/pThr205)-positive dystrophic neurites was found to occur simultaneously with Congo red-positive plaque development [START_REF] Noda-Saita | Exclusive association and simultaneous appearance of congophilic plaques and AT8-positive dystrophic neurites in Tg2576 mice suggest a mechanism of senile plaque formation and progression of neuritic dystrophy in Alzheimer's disease[END_REF]. Oxidative stress seems to play an important role in the Aβ pathology. Superoxide dismutase 2 (SOD2) is an enzyme which detoxifies reactive oxygen species that are produced predominantly by mitochondria.

Mice lacking SOD2 die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress [START_REF] Melov | Mitochondrial disease in superoxide dismutase 2 mutant mice[END_REF]. By crossing the Tg2576 mice onto an SOD2 heterozygous knockout background, it was found that this mitochondrial SOD2 deficiency both exacerbated Aβ plaque burden and tau phosphorylation at Ser396 [START_REF] Melov | Mitochondrial oxidative stress causes hyperphosphorylation of tau[END_REF].

Combinatorial approaches were particularly helpful in elucidating the role of Aβ in tau phosphorylation and aggregation. When P301L tau transgenic JNPL3 mice were crossed with Aβ plaque-forming Tg2576 mice, a sevenfold increase in NFT induction was seen [START_REF] Lewis | Enhanced neurofibrillary degeneration in transgenic mice expressing mutant Tau and APP[END_REF]. In a parallel study with P301L tau transgenic pR5 mice, we found that stereotaxic injections with Aβ 42 fibrils caused a fivefold increase in NFT formation compared to uninjected mice [START_REF] Gotz | Formation of neurofibrillary tangles in P301L tau transgenic mice induced by Abeta 42 fibrils[END_REF].

NFT formation was tightly correlated with phosphorylation of tau at Ser422 and AT100 (pThr212/pSer214), but not the AT8 (pSer202/pThr205) epitope. Mutagenesis of phospho-sites in a human SH-SY5Y tissue culture system revealed that the Ser422 epitope is required for the Aβ-mediated formation of tau filaments [START_REF] Ferrari | Beta-amyloid induces PHF-like tau filaments in tissue culture[END_REF][START_REF] Gotz | Amyloid-induced neurofibrillary tangle formation in Alzheimer's disease: insight from transgenic mouse and tissue-culture models[END_REF][START_REF] Pennanen | Different tau epitopes define Abeta(42)-mediated tau insolubility[END_REF]. Aβ further exacerbates a mitochondrial dysfunction that characterizes the pR5 mice [START_REF] David | Proteomic and functional analysis reveal a mitochondrial dysfunction in P301L tau transgenic mice[END_REF][START_REF] Eckert | Oligomeric and fibrillar species of βamyloid (Aβ42) both impair mitochondrial function in P301L tau transgenic mice[END_REF]. In a third study, Aβ plaque and NFT pathology was combined in a single animal by a triple transgenic approach [START_REF] Oddo | Triple-transgenic model of Alzheimer's disease with plaques and tangles. Intracellular abeta and synaptic dysfunction[END_REF]. In these 3xtg-AD mice (that express P301L mutant tau as well as mutant APP and PS1), synaptic

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dysfunction, including deficits in long-term potentiation, was found to precede Aβ plaque and NFT formation. The mice showed a remarkable phosphorylation pattern in that AT8 (pSer202/ pThr205) staining was only evident after 6 months and PHF1 (pSer396/pSer404) only at around 18 months of age, as shown for the hippocampus and cortex, despite a markedly high transgene expression and prominent HT7 (human tau) reactivity [START_REF] Oddo | Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer's disease[END_REF]. In a follow-up of the stereotaxic injection approach, Bolmont and colleagues diluted brain extracts from aged Aβ plaque-forming APP23 transgenic mice and intracerebrally infused these in young P301L tau transgenic mice. They found that six months after the infusion a tau pathology was induced in the injected hippocampus but also in brain regions well beyond the injection sites such as the entorhinal cortex and amygdala, areas with neuronal projection to the injection site [START_REF] Bolmont | Induction of tau pathology by intracerebral infusion of amyloid-beta -containing brain extract and by amyloid-beta deposition in APP x Tau transgenic mice[END_REF]. This is similar to findings in pR5 mice where Aβ while injected into the hippocampus and somatosensory cortex, induced NFT formation in the amygdala, aan area with neuronal projections to the injection site [START_REF] Gotz | Formation of neurofibrillary tangles in P301L tau transgenic mice induced by Abeta 42 fibrils[END_REF]. Together, this demonstrates that Aβ can induce tau hyperphosphorylation and augment a pre-existing tau pathology in mice.

Tau phosphorylation in flies and worms

More recently, invertebrate species have gained considerable attention. Despite their reduced complexity, they offer distinct advantages compared to mice. The life span of both the nematode Caenorhabditis (C.) elegans and the fruitfly Drosophila melanogaster is short. Costs for maintenance are low, handling is relatively easy, mass production is possible and screening for mutations simple. Also, working with these two species does not need approval from animal ethics committees.

Several of the genes that are implicated in AD pathogenesis have been expressed in flies and worms. For example, both wild-type and FTDP-17 mutant human tau has been expressed in the fly. These reproduced key features of the human disease, including adult onset, progressive neurodegeneration, enhanced toxicity of mutant tau, accumulation of abnormal tau and anatomical selectivity. Interestingly, immunoreactivity for phospho-epitopes such as 12E8 (pSer262/pSer356), AT100 (pThr212/pSer214), and AT180 (pThr231/pSer235) increased as the flies aged, whereas immunoreactivity using a phosphorylation-independent antibody was unaltered. Neurodegeneration, however, can occur without NFT formation [START_REF] Wittmann | Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles[END_REF],

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which is consistent with studies in mice [START_REF] Santacruz | Tau suppression in a neurodegenerative mouse model improves memory function[END_REF]. When wild-type human tau was expressed in combination with Shaggy, the Drosophila GSK-3β homolog and wnt signaling pathway component, this lead to a neurofibrillary pathology with tau filaments [START_REF] Jackson | Human wild-type tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila[END_REF].

When going through the vast literature on animal models of AD, it becomes obvious that most of the studies addressing the role of tau phosphorylation in tau aggregation and neurodegeneration are descriptive, although a few mutagenesis studies have been performed.

To address the role of distinct tau phosphorylation sites in controlling tau neurotoxicity, a mutagenesis study was performed in Drosophila which indicates that here, no single phosphorylation residue plays a dominant role in controlling tau toxicity, but rather that tau phosphorylation sites work in concert to promote neurotoxicity in vivo [START_REF] Steinhilb | Tau phosphorylation sites work in concert to promote neurotoxicity in vivo[END_REF].

The 12E8 epitope, located in the microtubule-binding domain of tau, is a substrate of MARK [START_REF] Augustinack | Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer's disease[END_REF][START_REF] Nishimura | PAR-1 kinase plays an initiator role in a temporally ordered phosphorylation process that confers tau toxicity in Drosophila[END_REF]. Work in Drosophila showed that the MARK homologue PAR-1 is a physiological tau kinase that plays a central role in regulating tau phosphorylation and toxicity, without promoting NFT formation [START_REF] Nishimura | PAR-1 kinase plays an initiator role in a temporally ordered phosphorylation process that confers tau toxicity in Drosophila[END_REF]. Mutating the PAR-1 phosphorylation site Ser262/Ser356 (12E8) by alanine substitution abolished tau toxicity. When human tau was expressed in the Drosophila eye along with either GSK-3β/Shaggy or Cdk5, this enhanced toxicity of tau [START_REF] Chau | Biochemical investigation of Tau protein phosphorylation status and its solubility properties in Drosophila[END_REF]. Lithium administration inhibited GSK-3β activity and thus reduced tau phosphorylation but did not ameliorate tau-induced toxicity, possibly reflecting high levels of tau expression [START_REF] Chau | Biochemical investigation of Tau protein phosphorylation status and its solubility properties in Drosophila[END_REF].

Compared to Drosophila, the nematode is even easier to work with [START_REF] Morcos | The Model Caenorhabditis elegans in Diabetes Mellitus and Alzheimer's Disease[END_REF]. What is unique to C. elegans is that wild-type adult worms contain a constant 959 somatic cells. Not only is the cell number constant but also the position of each cell. Moreover, the worm is transparent and hence, it is easy to track cells and follow cell lineages. The nervous system of an adult hermaphrodite C. elegans consists of only 302 neurons that form approximately 7000 synapses. Transgenic animals can be generated either using microinjection or ballistic approaches [START_REF] Green | Expression and imaging of fluorescent proteins in the C. elegans gonad and early embryo[END_REF]. Increasingly, C. elegans is employed for studies into diabetes and AD.

Regarding the latter, research currently progresses along two lines, firstly the characterization of C. elegans homologues of AD-related genes, and secondly the expression of human AD genes in C. elegans [START_REF] Morcos | The Model Caenorhabditis elegans in Diabetes Mellitus and Alzheimer's Disease[END_REF].

Expression of P301L and V337M mutant tau in C. elegans lead to behavioral, synaptic and pathological abnormalities, and caused an earlier and more severe

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phenotype than over-expression of wild-type human tau. Substantial neurodegeneration followed by loss of neurons occurred after insoluble tau began to accumulate. Tau was phosphorylated at the PHF1 (pSer396/pSer404), AT270 (pThr181), and 12E8 (pSer262/pSer356) epitopes in both the soluble and insoluble fraction, and at Ser422 and AT8 (pSer202/pThr205) only in the soluble fraction. However, tau phosphorylation did not appear to correlate with the severity of the phenotype [START_REF] Kraemer | From the Cover: Neurodegeneration and defective neurotransmission in a Caenorhabditis elegans model of tauopathy[END_REF]. The fact that neurological symptoms were apparent before insoluble tau accumulated, indicates that toxicity does not depend on the formation of large tau aggregates. To determine the role of tau phosphorylation in disease more directly, a short, fetal isoform of human tau was expressed, with a total of ten serines and threonines replaced by either glutamate (a socalled pseudophosphorylation or PHP construct) or alanine [START_REF] Brandt | A Caenorhabditis elegans model of tau hyperphosphorylation: induction of developmental defects by transgenic overexpression of Alzheimer's disease-like modified tau[END_REF]. Both, wild-type and PHP tau induced a progressive age-dependent phenotype of uncoordinated locomotion (unc) in the absence of neuronal degeneration. In comparison, the alanine mutant transgenic worms displayed a reduced survival and developed an earlier unc phenotype,

indicating that phosphorylation at these sites alone is not the cause of the observed defects [START_REF] Brandt | A Caenorhabditis elegans model of tau hyperphosphorylation: induction of developmental defects by transgenic overexpression of Alzheimer's disease-like modified tau[END_REF]. However, it is difficult to draw any conclusions with regards to the role of single phospho-epitopes in pathogenesis, as in the PHP construct, ten sites were phosphorylated simultaneously.

Implications of animal work for pathogenesis and therapy

Which phosphorylation sites are critical in triggering the cascade of neuronal dysfunction and eventually, neuronal loss, in tauopathies? What are the implications of data obtained in animal models for the development of therapies? Obviously, the promiscuity of kinases and phosphatases and the interdependence of phosphorylation sites, along with a profile that can differ from one neuron to the next, renders it difficult at present to pinpoint specific phosphorylation sites and their regulation [START_REF] Hernandez | Transgenic mouse models with tau pathology to test therapeutic agents for Alzheimer's disease[END_REF][START_REF] Li | Stabilization of the cyclin-dependent kinase 5 activator, p35, by paclitaxel decreases beta-amyloid toxicity in cortical neurons[END_REF][START_REF] Phiel | GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides[END_REF].

We have found in vivo, that NFT formation is associated with the phosphorylation of the pSer422 and pThr212/pSer214 (AT100) epitope [START_REF] Gotz | Formation of neurofibrillary tangles in P301L tau transgenic mice induced by Abeta 42 fibrils[END_REF] (Fig. 1). This and additional studies including site-directed mutagenesis of tau phosphorylation sites suggest a role for specific phospho-epitopes and hence kinases and phosphatases, in tau aggregation and

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neuronal dysfunction [START_REF] Kelleher | Kinase activities increase during the development of tauopathy in htau mice[END_REF][START_REF] Ferrari | Beta-amyloid induces PHF-like tau filaments in tissue culture[END_REF][START_REF] Bussiere | Phosphorylated serine422 on tau proteins is a pathological epitope found in several diseases with neurofibrillary degeneration[END_REF][START_REF] Schneider | Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments[END_REF]. Other studies support the notion that a generally increased phosphorylation rather than phosphorylation of specific sites is needed [START_REF] Pennanen | Different tau epitopes define Abeta(42)-mediated tau insolubility[END_REF][START_REF] Steinhilb | Tau phosphorylation sites work in concert to promote neurotoxicity in vivo[END_REF]. This concept is also supported by a recent analysis of tau aggregation and the role of phosphorylation in this process in an inducible N2a cell line that expresses ΔK280 full-length tau with and without truncated forms of tau [START_REF] Wang | Stepwise proteolysis liberates tau fragments that nucleate the Alzheimer-like aggregation of full-length tau in a neuronal cell model[END_REF]. While this study is restricted to 4R tau and needs to be validated in vivo, interestingly, aggregation of fulllength tau was found to be triggered by truncated fragments and was associated with the phosphorylation of most of the few sites analyzed. These were distributed between both soluble and aggregated tau, suggesting that none of the sites determines aggregation in an all-or-none manner [START_REF] Wang | Stepwise proteolysis liberates tau fragments that nucleate the Alzheimer-like aggregation of full-length tau in a neuronal cell model[END_REF]. Overall, whereas phosphorylation in the repeat domain tends to inhibit aggregation, other sites show a tendency to support aggregation. Together, this has obvious implications for treatment strategies as it would imply that phosphorylation in general, rather than specific tau kinases, needs to be blocked [START_REF] Gotz | Transgenic animal models of Alzheimer's disease and related disorders: Histopathology, behavior and therapy[END_REF].

Other take home lessons are that transgenic animal models prove that expression of FTDP-17 mutant tau accelerates tau pathology, causing NFT formation within the lifetime of mice. They underscore the role of serine/threonine-specific phosphorylation in tau aggregation. Strong evidence has been provided for a role of the tau phospho-epitopes AT100 (pThr212/pSer214) and S422 in tau aggregation as indicated above, although a map of "necessary" and "sufficient" epitopes of tau is still lacking. Based on the currently available data it appears that phosphorylation of tau is closely linked to tau aggregation and fibril formation.

A tau-directed treatment strategy may target any step involved in converting tau to a toxic species [START_REF] Ballatore | Tau-mediated neurodegeneration in Alzheimer's disease and related disorders[END_REF]. Alternatively, tau levels (and hence levels of phosphorylated tau) may be altogether reduced as it has been shown that Aβ toxicity is exerted via tau and that excitotoxicity can be rescued when tau levels are reduced [START_REF] Roberson | Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model[END_REF].

Several therapeutic strategies for treating AD on the basis of tau hyperphosphorylation are available as discussed recently [START_REF] Gong | Hyperphosphorylation of microtubule-associated protein tau: a promising therapeutic target for Alzheimer disease[END_REF]: these include (1) inhibition of GSK-3β, Cdk5 and other tau kinases; (2) restoration of PP2A activity; and (3) targeting O-glycosylation of tau [START_REF] Gong | Hyperphosphorylation of microtubule-associated protein tau: a promising therapeutic target for Alzheimer disease[END_REF].

A more recent study evaluated the concept of the promiscuity of kinases by using an orally bioavailable and blood-brain barrier (BBB)-penetrating analog of the relatively non-

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specific protein kinase inhibitor K252a [START_REF] Le Corre | An inhibitor of tau hyperphosphorylation prevents severe motor impairments in tau transgenic mice[END_REF]. This compound prevented motor deficits in the P301L tau transgenic mouse line JNPL3 and reduced levels of soluble aggregated hyperphosphorylated tau. Interestingly, NFT numbers were not reduced, suggesting that the main cytotoxic effects of tau are not exerted by NFTs, but by lower molecular mass aggregates.

This finding is in line with studies where a reduction in the expression of transgenic human P301L tau led to a recovery of memory function and stabilization of neuron numbers, despite the continued accumulation of NFTs [START_REF] Santacruz | Tau suppression in a neurodegenerative mouse model improves memory function[END_REF].

In many studies the GSK-3β inhibitor lithium has been evaluated. For example, in 3xTg-AD mice that develop both plaques and tangles, lithium chloride reduced tau phosphorylation but it did not significantly alter the Aβ load [START_REF] Caccamo | Lithium reduces tau phosphorylation but not A beta or working memory deficits in a transgenic model with both plaques and tangles[END_REF]. In a second mutant human tau transgenic mouse model, treatment with lithium chloride resulted in a significant inhibition of GSK-3 activity. After normalization to total levels of tau, lithium chloride treatment was found to result in a significantly decreased phosphorylation at putative GSK-3-directed sites, including pSer202 (CP13) and pSer396/pSer404 (PHF-1). Phosphorylation at sites not recognized by GSK-3 such as pSer422 or pSer262 appeared not to be affected. There were significantly reduced levels of aggregated, insoluble tau. Administration of another GSK-3 inhibitor also correlated with reduced insoluble tau levels, supporting the idea that lithium exerts its effect through GSK-3 inhibition. Levels of aggregated tau correlated strongly with the degree of axonal degeneration, and lithium-chloride-treated mice showed less degeneration when administration was started during early stages of NFT development [START_REF] Noble | Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo[END_REF]. Very recently, a zebrafish model has been established which reproduced tau hyperphosphorylation, NFT formation, neuronal and behavioral disturbances as well as cell death. Of the many inhibitors of GSK-3β tested in the fish model, a compound called AR-534 turned out to reduce tau hyperphosphorylation in vivo, without causing toxic side-effects [START_REF] Paquet | A zebrafish model of tauopathy allows in vivo imaging of neuronal cell death and drug evaluation[END_REF].

Furthermore, the protein kinase inhibitor rapamycin has been reported to reduce toxicity in Drosophila expressing either wild-type or mutant forms of tau, probably by reducing the amount of insoluble tau [START_REF] Berger | Rapamycin alleviates toxicity of different aggregate-prone proteins[END_REF]. Rapamycin induces autophagy through inhibition of the protein kinase mammalian target of rapamycin (mTOR) [START_REF] Noda | Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast[END_REF]. It remains to be seen whether the beneficial effects of rapamycin are related to TOR-dependent abnormal cell cycle activation that has been described in Drosophila tauopathy models.

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While these data encourage the use of kinase inhibitors, at the same time, microtubules, known to be stabilized by tau, have also emerged as a drug target, as they can be strengthened by drugs such as paclitaxel [START_REF] Zhang | Microtubule-binding drugs offset tau sequestration by stabilizing microtubules and reversing fast axonal transport deficits in a tauopathy model[END_REF]. Which of the approaches will be translated into human practice remains to be seen. As selective vulnerability characterizes the AD brain, different brain areas may be differently susceptible to any of these treatments [START_REF] Gotz | Alzheimer's Disease Selective Vulnerability and Modelling in Transgenic Mice[END_REF].

Figure 1

 1 Figure 1

For clarity, subsequently first the phosphorylated tau epitope (pS202 or S202) is listed followed by the phospho tau-specific antibody in brackets.

Acknowledgements

JG is a Medical Foundation Fellow. This work has been supported by the University of Sydney, the National Health & Medical Research Council (NHMRC), the Australian Research Council (ARC), the New South Wales Government through the Ministry for Science and Medical Research (BioFirst Program), the Nerve Research Foundation, the Medical Foundation (University of Sydney) and the Judith Jane Mason & Harold Stannett Williams Memorial Foundation. LI has been supported by the NHMRC and ARC.