Tauopathies 635 central nervous system. Clusters (“tangles”) of paired-helical filaments containing phosphorylated tau are one of the characteristic features of certain myopathies such as the inclusion body myositis (Askanas and Engel, 1998) and myotonic dystrophy (Tolnay and Probst, 1999). Some adults with myotonic dystrophy type 1 (DM1) have been seen to develop dementia with aging, agreeing with recent studies doc- umenting an abnormal tau-protein expression in the brain tissues of patients with DM1 (Modoni et al., 2004). A possible distinct subclass of peripheral tauopathy has been postulated based on immunoblot studies (Maurage et al., 2004). This chapter, however, focuses on the vast majority of tauopathies that are well recognized. In most of them it so happens that cognitive and or motor impairments are the core clinical manifestation. 2 Biochemistry and Molecular Biology of Tau Microtubules (MT) are a major component of neuronal cell processes involved in maintaining the cell shape and axonal transport (Buee et al., 2000). It is probable that microtubule-associated proteins play a major role in this function (Wang and Liu, 2008). 2.1 Tau Gene Tau proteins are translated from a single gene located over 100 kilobases on the long arm of chromosome 17 (17q21.1) and consisting of 16 exons (Fig. 1a)(Kosik,1993; Neve et al., 1986; Andreadis, 2006). Expression of human tau complex regulation and regarding its control of gene expression, the presence of specific transcription factors (such as AP2/SP1) are involved, even though RNA-based control has been recently proposed that awaits more evidence (D’Souza and Schellenberg, 2006). The tau gene is transcribed into nuclear RNA, which by alternative splicing yields different RNA species (Fig. 1b) (Goedert et al., 1989b). Tau’s interactions with microtubules are mediated by the tubulin-binding domains/repeat at the C-terminal region (Fig. 1c) as detailed below. In vitro and transgenic animal models have demonstrated that different muta- tions impair protein function, promote tau fibrilization, or perturb tau gene splicing, leading to aberrant and distinct tau aggregates (Cairns et al., 2004). The mutations in the autosomal dominant tauopathies are of two types: intronic mutations that disrupt the splicing of tau and missense mutations that alter the function of tau. The splicing of tau is tightly regulated so as to maintain the relative proportion of the 3R-tau and 4R-tau isoforms. The function of tau is normally tightly regulated through phosphorylation. It is likely that loss of this normal regulation somehow results in tau aggregation, although it should be noted that, in vitro, the mutations also increase t au aggregation itself. Transgenic mice carrying tau mutations have been shown to exhibit behavioural and neuropathological correlates of the disease process. This indicates that tau aggregates are a sign of primary pathology. Tau aggregation without amyloid pathology is sufficient to cause dementia in mice and in 636 Mathew et al. Fig. 1 Schematic representation of the human tau gene (a), RNA (b), and brain tau isoforms (c). Alternatively spliced exons (2, 3, and 10) are shown in orange, cream, and green, respectively. The tandem repeats (3 or 4) are shown in white bars. The number of aminoacids is indicated on the right-hand side (modified from Spillantini and Goedert, 2000) humans and hence is likely to be a pathogenic protein (Lovestone and McLoughlin, 2002). 2.2 Structure, Cellular Localization, and Putative Functions of Tau Protein Tau protein was first discovered as an acid- and heat-stable protein essential for microtubule assembly. It was identified as a factor that lowered the concentration at which tubulin polymerizes into microtubules in the brain. Tau is one such neu- ronal MAP, which localizes primarily in the axon with a molecular tau weight of Tauopathies 637 approximately 50,000–64,000 Da. When purified from the brain it has very little secondary structure (Kosik, 1993). Because of its enormous molecular weights and meager tendency to form highly ordered 3D crystal lattices, tau had long evaded high-resolution structure determination (Margittai and Langen, 2004). Nevertheless, it is now known that several specific proteins serve to stabilize microtubules and tau is one such MAP. Tau organizes MTs into evenly spaced parallel assemblies known as MT-bundles. It is also well recognized that tau plays an important role in the assembly of tubulin monomers into MT to constitute the neuronal microtubule network, maintains the MT structure,(Alonso et al., 2001), and establishes links between microtubules and other cytoskeletal elements and proteins (Buee et al., 2000). It has been proposed that, in vivo, tau induces the bundling and stabilization of cellular microtubules, promotes neurite outgrowth, and establishes and maintains neuronal cell polarity. Tau fulfills several functions critical for neuronal formation and health. It dis- charges its functions by producing multiple isoforms via intricately regulated alternative splicing. These isoforms modulate tau function in the normal brain by altering the domains of the protein, thereby influencing its conformation and post- translational modifications and hence its affinity for microtubules and other ligands. Disturbances in tau expression result in disruption of the neuronal cytoskeleton and formation of pathological tau structures (e.g., neurofibrillary tangles in brains of patients with Alzheimer’s disease; Andreadis, 2006). It is not clear, however, how tau’s ability to decrease the dynamic instability of microtubules directly relates to these changes in microtubule organization and cell morphology (Leger et al., 1994). They are expressed predominantly in axons of central nervous system (CNS) neu- rons, and also are found in axons of peripheral nervous system (PNS) neurons, but are barely detectable in CNS astrocytes and oligodendrocytes (Trojanowski and Lee, 2002). It is one of the major and most studied MAPs in the central nervous system. Tau has been shown to be a highly asymmetric protein, compatible with the long rod structure, when observed by electron microscopy (Hirokawa et al., 1996). Earlier studies had suggested that it is a hydrophilic protein having a random coil structure. Attempts to crystallize tau alone or tau associated with microtubules have been unsuccessful and under normal conditions, tau demonstrates the properties of a highly soluble natively unfolded molecule, essentially devoid of secondary or tertiary structural elements (Mandelkow et al., 1996; Friedhoff et al., 2000). The above findings are in agreement with the detailed information of the structure of tau protein in its soluble state obtained through conventional methods such as electron microscopy, spectroscopy, and X-ray diffraction (Crowther et al., 1989, 1992, 1994; Schweers et al., 1994; Wille et al., 1992). However, tau molecule adopts specific secondary and tertiary structures that interact in an orderly fashion to produce the highly regular filaments in Alzheimer’s disease and many other neurodegenerative disorders (Gamblin et al., 2000). It is unlikely that random aggregation of tau in the disease state could lead to creation of these highly ordered structures. It can be understood that tau is partially folded when interacting with microtubules by a com- bined cryoelectron microscopy and tomographic 3-D analysis with freeze-drying and high-resolution unidirectional surface shadowing. 638 Mathew et al. In the adult human brain, six brain-specific isoforms are generated by alterna- tive mRNA splicing of 11 exons (Fig. 1b; Buee et al., 2000). Alternative splicing of exons 2 (E2), 3 (E3), and 10 (E10) give rise to six tau isoforms that range from 352 to 441 amino acids (Fig. 1c; Goedert et al., 1989a). The isoforms differ in whether they contain three (tau-3L, tau-3S, or tau-3: collectively 3R) or four (tau- 4L, tau-4S, or tau-4: collectively 4R) tubulin-binding domains/repeats (R) of 31 or 32 amino acids each at the C-terminal (i.e., the presence or absence of a fourth 31-amino acid repeat, coded by exon 10). They also differ on whether they have two (tau-3L, tau-4L), one (tau-3S, tau-4S), or no (tau-3, tau-4) repeats of 29 amino acids each in the N-terminal portion of the molecule (Fig. 1c; Trojanowski and Lee, 2002). Thus, exons 2 and 3 are alternatively spliced cassettes; exon 2 exists alone, but exon 3 never appears independent of exon 2 (Andreadis, 2006). As seen above, the isoforms are designated according to the number of MBDs they possess at the C-terminal. Each of these repeats can be divided into two parts, one composed of an 18-residue sequence that contains the minimal region with tubulin-binding capac- ity and the less conserved domain called the interrepeat. The proportion of these tau isoforms, as well as their phosphorylation status, changes during development (Kosik, 1990a, b; Kosik et al., 1986; Goedert et al., 1989a, b; Buee et al., 2000). In fact, in the adult human brain, the proportion of 3R-tau to 4R-tau isoforms is about 50% each, but that of tau-3L (or 4L), tau-3S (or 4S), and tau-3 (or 4) is about 54, 37, and 9%, respectively. Tau 4R binds microtubules with a greater affinity and can displace the previously bound tau 3R from microtubules that may produce physio- logical consequences in cells. As tau is developmentally regulated, only the shortest tau isoform (tau-3) is expressed in fetal brain, but all six isoforms are seen in the adult human brain (Trojanowski and Lee, 2002). Moreover, different neurons seem to have different tau isoforms. In the peripheral nervous system too, there is a high molecular weight tau isoform expressing the exon 4A, whose product forms a pro- tein known as big tau with an approximate size of 100 kDa (Couchie et al., 1990; Goedert et al., 1992). In summary, brain tau isoforms have been divided into two large domains such as projection domains (containing the amino terminal two-thirds of the molecule) and the microtubule binding domain (containing the carboxy terminal one-third of the molecule) (Avila et al., 2004). The projection domain has been further divided into two regions: the amino terminal region with a high proportion of acidic residues and the proline-rich region. The microtubule binding domain has also been subdivided into the basic, true tubulin-binding region and the acidic carboxy terminal region. Several distinct roles have been proposed for the projection domain including that of determining the spacing between axonal microtubules, interactions with other cytoskeletal proteins, or cation binding due to the presence of the acidic residues. The proline-rich domain plays an important role in interaction with proteins with SH3 domains, facilitating the binding of tau to the plasma membrane proteins (Brandt and Lee, 1993, 1994). A structure function relationship study by (Gamblin et al., 2000) points out that tau contains very few predicted structural elements, but these small structural units, whether predicted or measured through biochemical/biophysical methods, are likely Tauopathies 639 Table 1 Predictable Secondary Structure Forming Motifs in Tau Protein Sequence Possible Secondary Structure and Possible Implications 1 7 EFEVME 12 α-helix; tau aggregation is accompanied by a dramatic conformational change that brings the amino-terminus in close proximity to the microtubule-binding repeats. 2 31 MH 32 β-strand; imparts structural rigidity? 3 117 EAAGHVTQ 124 α-helix; region is adjacent to the second most hydrophobic region of the molecule; helical wheel analysis shows amphipathic making it a candidate to interact with the microtubule-binding repeats or the carboxy-terminus of tau. 4 226 VAVVR 230 β-strand; direct interaction of this region with the microtubule-binding repeats to strengthen the interaction of tau with microtubules. 5 275 VQII 278 and 306 VQIVY 310 β-strands; core structural elements for filament elongation. 6 315 LSKVTSKCGSL 325 α-helix; amphipathic in nature structural element that participates in tau–tau interactions in the aggregated state. 7 338 EVK 340 and 361 TH 362 β-strands; positioning in MTBR4 suggests that they could contribute to microtubule binding. 8 426 ATLADEVSASLA 437 α-helix; structural element can directly bind to some other element of tau and prevent the aggregation of the molecules. responsible for the normal and abnormal functions of tau by providing sites for specific interactions either with microtubules or other tau molecules. The study identifies certain structural elements that have a potential for adopting secondary structures which have a role in normal and pathological conditions (Table 1). The microtubule-associated tau protein participates in the organization and integrity of the neuronal cytoskeleton. Even though tau protein is mainly a neu- ronal MAP, localizing primarily in the axon, it has been demonstrated that tau is present within the somatodentritic compartment of neurons (Migheli et al., 1988). Tau that is present in the somatodendritic compartment is phosphorylated mainly in its proline-rich region, whereas when this region becomes dephosphorylated, it can be found principally in the distal region of the axon (Mandell and Banker, 1996). Additionally, presence of nuclear tau isoforms has been identified in human neurob- lastoma cells. Nuclear tau was found to be associated with both the fibrillar regions of interphase nucleoli and the nucleolar organizer regions of mitotic chromosomes; recent studies have also shown that nuclear tau is mainly present at the internal periphery of nucleoli, partially colocalizing with the nucleolar protein nucleolin and human AT-richα-satellite DNA sequences organized as constitutive heterochromatin (Sjoberg et al., 2006). The import of tau into the nucleus is possibly either by inter- acting with other nuclear proteins containing a nuclear import sequence or catalyzed by the basic 3/4 repeat MBDs. Because nuclear tau has also been found in neurons from patients with AD, aberrant nuclear tau could affect the nucleolar organization during the course of AD. Recent studies suggest that binding of tau to DNA was 640 Mathew et al. in an aggregation-dependent, and a phosphorylation-independent, manner. Tau has also been seen to localize on ribosomes. One possible function for this nonmicro- tubule, ribosome-associated tau is to target ribosomes to microtubules for transport into the somatodendritic compartment of neurons to facilitate local protein synthe- sis. The localization and the function of the different tau isoforms are regulated by its posttranslational modifications. 2.3 Posttranslational Modifications of Tau Heterogeneity of tau is due to several posttranslational modifications including phosphorylation, glycosylation, ubiquitinylation, oxidation, nitration, cross-linking, deamidation, glycation, truncation by protein cleavage (Avila et al., 2004), pro- lyl isomerization, association with heparan sulfate proteoglycan, and modification by advanced glycation end-products (Chen et al., 2004). Phosphorylation is of functional and clinical importance and therefore has been the most studied of all posttranslational modifications. 2.3.1 Phosphorylation Tau is a phosphoprotein and its biological activity is regulated by phosphorylation (Feijoo et al., 2005; Ihara et al., 1986; Grundke-Iqbal et al., 1986). Tau phosphory- lation is developmentally regulated and fetal tau is more highly phosphorylated in the embryonic compared to the adult CNS. The degree of phosphorylation of the six adult tau isoforms decreases with age. The tau phosphorylation sites are clustered in regions flanking the microtubule binding repeats. Phosphorylation at these sites has been reported in normal tau, however, the phosphorylation negatively regulates microtubule binding. Although the relative importance of individual sites for regu- lating the binding of tau to microtubules is unclear, phosphorylation of some sites such as Serine-262 and 396 has been reported to play a dominant role in reducing the binding of tau to microtubules. Both sites are phosphorylated in fetal tau and they are hyperphosphorylated in all six adult human brain tau isoforms that form paired helical filaments (PHFs) in Alzheimer’s disease. Other potentially important phos- phate acceptor sites also have been described and it is possible that phosphorylation at multiple phosphate acceptor sites regulates the binding of tau to microtubules (Trojanowski and Lee, 2002). Hyperphosporylation dislodges tau from the microtubule surface, resulting in compromised axonal integrity and accumulation of toxic tau peptides (Drewes, 2004). The definite role of tau in the neurodegenerative process is still not very clear. Recent studies suggest that, before forming fibrils but after becom- ing hyperphosphorylated, tau actively contributes to neurodgeneration (Takashima, 2008). There are 85 putative serine or threonine phosphorylation sites on the longest CNS tau isoform. Phosphorylation sites were characterized by phospho-dependent tau antibodies, phopho-peptide mapping, mass spectrometry, and NMR. Most of the phosphorylation sites surround the microtubule-binding domains in the proline-rich Tauopathies 641 region of the C terminal region of tau. A large number of serine/threonine pro- tein kinases have been suggested to play a role in regulating tau functions in vivo, however, this aspect of tau biology remains controversial. The major candi- date tau kinases include mitogen-activated protein kinase, glycogen synthase kinase 3β, cyclin-dependent kinase 2 (cdk2), cyclin-dependent kinase 5, cAMP-dependent protein kinase, Ca 2+ /calmodulin-dependent protein kinase II, microtubule-affinity regulating kinase, and stress-activated protein kinases (Trojanowski and Lee, 2002). The available evidence points to glycogen synthase kinase-3 being the predominant tau kinase in the brain, although other kinases also phosphorylate tau (Lovestone and McLoughlin, 2002). Protein phosphatases counterbalance the effects of tau kinases, although their role i n vivo is unclear. In vitro experiments showed that inhibition of protein phosphatases by okadaic acid in cultured human neurons was followed by increased tau phosphorylation, decreased tau binding to microtubules, selective destruction of stable microtubules and rapid axonal degeneration (Trojanowski and Lee, 2002). Today we know more than 20 protein kinases that can phosphorylate the tau protein and they are grouped into several different types as follows. 1. The proline-directed protein kinases (PDPK), which phosphorylate tau on serine or threonine residues that are followed by a proline residue. This group includes tau protein kinase II (cdk5), cdk2, MAP kinase (p38), JNK, and other SAPKs (Baumann et al., 1993; Holzer et al., 1994; Goedert et al., 1997). 2. The nonproline-directed protein kinases (NPDPKs) such as tau-tubulin kinase 1 and 2, protein kinase A (PKA), protein kinase C (PKC), PKB/AKT, calmodulin (CaM) kinase II, MARK kinases, or CK I and II that modify residues close to acidic residues mainly in exons 2 and 3. NPDPKs modify Ser or Thr residues that are not followed by prolines (Kitano-Takahashi et al., 2007; Sergeant et al., 2005). 3. The protein kinases that phosphorylates tau on serine or threonine residues, sometimes but not always, followed by a proline residue which includes tau protein kinase I (glycogen synthase kinase 3, GSK3; Hanger et al., 1992). 4. The tyrosine protein kinases such as Src kinases and c-abl. Along with tau kinases, several phosphatases, such as protein phosphatase [PP1, PP2A, PP2B (calcineurin), and PP2C] regulate the extent of tau phosphorylation (Goedert et al., 1992; Yamamoto et al., 1995) However, only PP1, PP2, and PP2B have been shown to dephosphorylate abnormally hyperphosphorylated tau (Gong et al., 1994a,b,c). PP2A binds to tau through its tubulin binding region (Sontag et al., 1999). Mutations in this region could decrease the capacity of PP2A to bind to tau and, as a consequence, produce an increase in tau phosphorylation, a feature that has been observed in some FTDP-17 patients bearing such mutations. The Physiological Role of Tau Phosphorylation The phosphorylation of tau at specific sites is the predominant mechanism by which tau function is r egulated. Dynamic, site-specific phosphorylation of tau is essential 642 Mathew et al. for its proper functioning. Interestingly, phosphorylation at different sites could take place in different tau isoforms. This could be due to the different cellular localiza- tion or subcellular compartmentalization of the different tau isoforms, or the fact that different kinases or phosphatases can modulate tau phosphorylation in many different ways (Avila et al., 2004) as described below. A tentative figure explaining the role of tau phosphorylation is depicted in Fig. 2. Microtubule Binding The ability to bind and stabilize microtubules is a hallmark of tau, and it is becoming obvious that phosphorylation of a few specific sites plays a significant role in reg- ulating tau–microtubule interactions. Phosphorylation of the KXGS motifs within the microtubule-binding repeats of tau strongly reduces the binding of tau to micro- tubules in vitro and probably in vivo (Biernat and Mandelkow, 1999; Drewes et al., 1995). The NPDPK phosphorylation mainly occurs at the tubulin-binding region of the tau molecule. Therefore, it has been suggested that this type of modification could result in a decrease in the binding of tau to microtubules whereas modification of tau by PDPK mainly affects tau self-aggregation. In vitro studies have shown that phosphorylation of Ser262 alone is sufficient to attenuate significantly the ability of tau to bind microtubules in vitro. GSK3 plays an important role in regulating tau phosphorylation under normal and pathologi- cal conditions. Two types of GSK3 phosphorylation have been proposed: primed (following prior phosphorylation of the substrate by another kinase) or unprimed phosphorylation. Primed phosphorylation appears to occur at Thr231 and affects microtubule binding (which means that Ser235 must be phosphorylated first to get efficient phosphorylation of Thr231), whereas unprimed phosphorylation can take place at serine-396 or -404 and does not appear to affect microtubule binding (Goedert et al., 1994). Although the relative importance of individual sites for reg- ulating the binding of tau to microtubules is unclear, phosphorylation of some sites such as Serine-262 and 396 has been reported to play a dominant role in reducing the binding of tau to microtubules. In addition to serine/threonine modifications, phosphorylation at tau tyrosines has been also reported. It is known that fetal tau is more extensively phosphorylated than adult tau and promotes microtubule assembly less efficiently than the latter. Altered Intracellular Trafficking/Polarity This occurs by two major mechanisms explained below. (a) Neurite outgrowth: Neuritic extension is essential for maintaining synaptic plas- ticity and in CNS repair. Evidence regarding the role of tau was obtained from earlier studies on cultured cerebellar neurons using antisense oligonucleotides (Kosik, 1990a,b). Primary cultures of hippocampal neurons lacking tau exhibit decreased rates of neurite extension and inhibited neuronal polarization (i.e., the development of axons and dendrites; Dawson et al., 2007), during axono- genesis, tau function appears to be locally regulated by phosphorylation. Tau mRNA may also play a role in the determination of polarity, inasmuch as it is Tauopathies 643 Fig. 2 Schematic representation of the physiological and pathological functions of tau phospho- rylation. When the phosphorylation state of tau is appropriately coordinated, it plays a role in regulating neurite outgrowth, axonal transport, and microtubule stability and dynamics. However, in pathological conditions in which there is an imbalance in the phosphorylation/dephosphorylation of tau, aberrant tau phosphorylation can cause tau/actin filament formation, disrupt microtubule- based processes owing to decreased microtubule binding, and perhaps even increase cell death (modified from Johnson et al., 2004 (129)) 644 Mathew et al. localized on microtubules in the proximal section of the axon. This implies that local translation of tau at a site determined by microtubular organization sites can lead to locally high levels of tau, which can theoretically cause bundling and forward movement of neurites (Billingsley and Kincaid, 1997). However, contrary to expectation, it was surprising to find that targeted deletions of tau protein led to only minor changes in the axonal calibre of small-fibre axons in restricted brain regions (Harada et al., 1994). (b) Axonal transport: Tau also regulates axonal transport. In mouse models in which tau are overexpressed in the central nervous system, there is almost always axonopathy, predominantly in spinal cord neurons. Tau can inhibit kinesin-dependent fast axonal transport in cell culture models (Ebneth et al., 1998), and this is probably the case in vivo when tau is overexpressed. The primary mechanism by which tau inhibits kinesin-dependent transport is by reducing the attachment frequency of the motors. Tatebayashi et al. (2004) recently demonstrated that, in cell culture models, GSK3β-mediated tau phos- phorylation is associated with proper anterograde organelle transport providing further evidence that the control of axonal transport by tau is regulated by GSK3β-mediated phosphorylation. Altered Proteolysis One key structural change that has been linked with the regulated phosphorylation of tau is altered turnover and proteolysis. The best characterized effect has been the reduction in tau cleavage by the calcium-activated protease calpain following pro- tein kinase A (PKA)-induced phosphorylation. Alterations in lysosomal trafficking of tau and/or loss of lysosomal function are thought to set off aberrant processing of tau. There is increasing evidence that inappropriate phosphorylation of tau, which leads to tau dysfunction, results in decreased cell viability. Indeed, in all neurode- generative diseases in which tau pathology has been observed, the tau is abnormally phosphorylated. 2.3.2 Other Modifications of Tau Proteins Glycosylation The presence of N-linked and (mucin-type) O-linked oligosaccharides on PHF- tau has been reported with N-glycosylation occurring in hyperphosphorylated tau (Wang et al., 1996a,b) whereas unmodified tau can be O-glycosylated (Arnold et al., 1996). O-glycosylation of cytosolic proteins is a dynamic and abundant posttranslational modification that is characterized by the addition of an O-linked N- acetylglucosamine (O-GlcNAc) in the serine or threonine in the vicinity of proline residues by an O-GlcNAc transferase. This relationship between phosphorylation and OGlcNAc glycosylation of tau proteins may play a role in transcriptional regulation, cell cycle regulation, protein degradation, cell activation, and the cor- rect assembly of multimeric protein complexes and in the nuclear localization of tau. . been divided into two large domains such as projection domains (containing the amino terminal two-thirds of the molecule) and the microtubule binding domain (containing the carboxy terminal one-third. subdivided into the basic, true tubulin-binding region and the acidic carboxy terminal region. Several distinct roles have been proposed for the projection domain including that of determining the spacing. microtubule-binding domains in the proline-rich Tauopathies 641 region of the C terminal region of tau. A large number of serine/threonine pro- tein kinases have been suggested to play a role in regulating