**3. Tau protein**

370 Neuroscience – Dealing with Frontiers

particular model and then is compared to an actual diffraction pattern (Makin et al., 2005). FTIR and CD are absorptive spectroscopic techniques that measure nonsymmetrical or chiral molecular systems in bulk, FTIR spectroscopy measuring molecular bond vibrational frequencies and CD the differential absorption of left versus right circular polarized light. Both of these properties are highly sensitive to secondary structure, and therefore, deconvoluted FTIR and CD spectrum provide accurate estimations of the contribution of βsheets, α-helices, and loops to the overall structure (Berthomieu &Hienerwadel, 2009; Ranjbar &Gill, 2009). Cryoelectron Microscopy: is a technique that positions itself as a capable alternative to conventional X-ray crystallography and solution NMR in amyloid structure determination. Unlike X-ray crystallography, proteins of interest need not be in a crystalline form for cryoelectron microscopy; rather, structural data can be acquired on single particles. Furthermore, amyloid preparations do not need to be labeled with stable isotopes, is the case with NMR, nor do they even need to be highly pure preparations. This is particularly advantageous owing to the inherent heterogeneous nature of the amyloid conformation (Mizuno et al., 2011). X-Ray Crystallography: although the large and heterogeneous nature of amyloid fibers would seem to be incompatible with X-ray crystallography, Eisenberg and colleagues have been able to acquire adequate diffraction data from microcrystals of short peptides (6–7 residues) that formed amyloid-like structures. Based on X-ray crystallography showed that structures bore strong resemblance to many existing models of amyloid fibers, suggesting they may in fact represent the structure of the amyloid fold. Since this initial study, multiple amyloidogenic peptides have been successfully crystallized and their structures

AB early aggregates also can showed a globular appearance that further organize into beaded chains, globular annular "doughnut" shaped assemblies eventually giving mature protofilaments and fibrils. Pre-fibrilar aggregates may interact with reconstituted phospholipid membranes and with cell membranes where they form aspecific channels (pores) disrupting cellular homeostasis (figure 5). The latter possible mechanism of toxicity is similar to that displayed by antimicrobial peptides, pore-forming eukaryotic proteins and bacterial toxins and newly synthesised cyclic peptide antibiotics (Stefani &Dobson, 2003).

The cell membrane could be a nucleating center for amyloid aggregation. The evidence showed that AB species are tight binding to GM1 ganglioside (GM1), in the brain showing early pathological changes of AD. The ganglioside-bound AB (GAB) possessed unique characteristics, including its altered immunoreactivity, which suggests its distinct conformation from native AB, and its strong potency to accelerate Aβ assembly into fibrils. The hypothesis is that AB adopts an altered conformation following interaction with GM1, leading to the generation of GAB, and then GAβ acts as an endogenous seed for Alzheimer amyloid in the brain. GAB is favorably generated in the unique ganglioside-enriched (clustered), raft-like microdomains; moreover, amyloid fibrils formed in the presence of gangliosides are neurotoxic. Probably the ganglioside binding is the initial and common step in the development of a part of human misfolding-type amyloidoses, including AD (Matsuzaki et al., 2010). Recent reports supports that soluble oligomers of AB may be the key neurotoxic species associated with the progression of AD and that the process of AB aggregation may drive this event. Recent data obtained in our laboratory suggest that the presence of soluble oligomers in rat hippocampus promotes localized mechanism of inflammatory responses (unpublished data). Is alspo reported that soluble oligomers of AB and Tau accumulate in the lipid rafts of brains from AD patients through an as yet unknown

solved (Nelson et al., 2005; Wiltzius et al., 2009).

Tau is a microtubule-associated protein (MAP) that is believed to stabilise microtubules and to promote microtubule assembly. Of the neuronal MAPs, it is one of the most abundant (Goedert &Spillantini, 2011). Tau protein is found in many animal species such as *Caenorhabditis elegans*, *Drosophila*, goldfish, bullfrog, rodents, bovines, goat, monkeys and humans (Buee et al., 2000). In humans, Tau is encoded by a single-copy gene located on chromosome 17q21.1 in humans (figure 3A). It produces three transcripts of 2, 6 and 9 kb which are differentially expressed in the nervous system, depending upon stage of neuronal maturation and neuron type. The 2 and 6 kb Tau mRNAs arise from utilization of two alternative polyadenylation sites separated by ~4 Kbp. So far, one promoter has been mapped for the Tau gene in both human and rat, located directly upstream of Tau exon -1. However, the 6 kb Tau transcript is responsive to NGF, whereas the 9 kb one is not. Also, the 6 and 9 kb transcripts are restricted to neuronal tissues, whereas the 2 kb Tau transcript is ubiquitous (Andreadis, 2005; Andreadis et al., 1996). Tau primary transcript contains 16 exons, but two of them (exons 4A and 8) are not present in mRNA in human brain. Exon 4A is present in peripheral nervous system not only in humans, also in bovine and rodent peripherial tissues. On the other hand, exon 8 has not been described in humans. Exon -1 is part of the promoter, transcribed but not translated, just like exon 14 (Buee et al., 2000). Exon 3 is never found without exon 2, and as exon 10, are present in an adult-specific manner (figure 1B), but their ratios differ in various central nervous system compartments (Andreadis, 2011; Takuma et al., 2003). All six possible product combinations of the 2/3/10 splicing events have been observed (figure 3B), indicating that separate factors govern their splicing (Andreadis, 2011). Alternative splicing of hinge-region exon 6 gives rise to Tau variants that lack the domain responsible of microtubule binding, and is mentioned that alters Tau function (Andreadis, 2011; Luo et al., 2004). Saitohin (STH), an intronless gene encoding an open reading frame of 128 amino acids, is located in the intron between exons 9 and 10 of the human Tau gene (Wang et al., 2011). Recently has been associated the existence of a polymorphic form with AD, but is still under research. In the 6 principal human Tau protein isoforms are present two domains (Figure 2C): the projection domain located in the amino-terminal and is composed of an acidic region and a proline rich region (PRR). The other domain is named microtubule binding domain (MTBD), which contain the C-termini *per se*, and a microtubule binding region (MTBR), conformed indeed by the presence from three (3R) to four repeats (4R) (Buee et al., 2000). They differ from each other by the presence or absence of 29- or 58-amino acid inserts located in the amino-terminal half and an additional 31-amino acid repeat in the carboxy-terminal half (Goedert &Spillantini, 2011). Tau is enriched in axons of growing and mature neurons and is critical for neuronal

Tau and Amyloid-β Conformational Change to β-Sheet

this phenomena needs further research.

protein kinase 2 (Meraz-Ríos et al., 2010)

Structures as Effectors in the Development of Alzheimer's Disease 373

proposed that the motif KKXK is involved in heparing binding (Ávila et al., 2002) and actin interaction (Zmuda &Rivas, 2000). In this regions is present various PPXXP or PXXP motifs which allow Tau protein to interacts with SH3 domains of diverse proteins such as Src family members like Fyn, Lck, Src (Lee, 2005; Lee et al., 1998; Scales et al., 2011) and others like PCL-γ (Hwang et al., 1996; Jenkins &Johnson, 1998). Is also mentioned that STH interacts with Tau and Abl and influences in Abl phosphorylation (Wang et al., 2011), but

The efficacy of Tau to binding MTs is influenced by the degree of phosphorylation and the exclusion/inclusion of exon 10. The affinity of Tau against MTs is weaker in 3R isoforms than 4R isoforms, perhaps leading to differential regulation of microtubule behaviour (Eckermann et al., 2007; Goode et al., 2000; Lu &Kosik, 2001). Another important factor influences Tau-MTs interaction: site-specific phosphorylation. This is due to the high frequency of phosphorylatable residues (45 S, 35 T and 5 Y in the largest isoform), combined with the open structure of Tau which renders it accessible to many kinases (Mandelkow et al., 2007). The majority of the phosphorylation sites are Serine or Threonine residues followed by Prolines (SP/TP motifs) that lie in the domains flanking the repeats and can be phosphorylated by several Proline-directed kinases and by neuronal kinases MARK (affecting the KXGS motifs within Tau's repeat domain) (Eckermann et al., 2007; Trinczek et al., 1995). Among the kinases responsible for Tau phosphorylation are Glycogen Synthase Kinase 3β (Lin et al., 2007), cyclin-dependent kinase 5 (Cdk5), MT-affinity regulatory kinase, cAMP-dependent protein kinase (Johnson &Stoothoff, 2004), dual-specificity tyrosinephosphorylated and regulated kinase 1A, Tau–tubulin kinase 1, and calmodulin-dependent

**3.1 Tau conformational changes in physiological and pathological conditions** 

(Hirokawa et al., 1988; Mandelkow et al., 2007; Sillen et al., 2007).

Tau is a highly flexible and extended protein, natively unfolded and with hydrophilic and basic character, highly soluble shown by sequence analysis and CD experiments (Eckermann et al., 2007; Shkumatov et al., 2011). Firstly was suggested that Tau in solution may be as much as 77% random coil and behave like "Gaussian coils" or "worm likechains", in which the direction of the polypeptide backbone chain varies in a more-or-less random fashion (Goode et al., 2000). However, a potential β-pleated region has been proposed for the microtubule binding region (Ávila et al., 2002). When Tau is attached to MTs, is suggested to assume an "L" conformation, meaning that MTBR interacts with the MTs and the N-termini is projected to cytosol, where can interacts with other proteins

On the other hand, is suggested by fluorescence resonance energy transfer (FRET), electron paramagnetic resonance (EPR) and small-angle X-ray scattering that Tau can adopt a global hairpin (paperclip) structure in solution (when is not attached to MTs) (Jeganathan et al., 2008a; Mandelkow et al., 2007; Mylonas et al., 2008). From a structural perspective, the folding of the N- and C-domains over the repeat domain would be expected to protect against aggregation, and indeed Tau forms aggregates more readily when the non-repeat domains are cleaved off (Mylonas et al., 2008). The conversion of certain soluble peptides and proteins into insoluble filaments or misfolded amyloid proteins is believed to be the central event in the etiology of a majority of neurodegenerative diseases (Nonaka et al.,

function. Among its many roles, Tau promotes neurite outgrowth, organizes axonal microtubules (MTs) and is involved in kinesin-dependent axonal transport (Andreadis, 2011; Hirokawa et al., 1988; LaPointe et al., 2009). Additionally, the interaction of Tau with diverse structural and functional proteins suggests that Tau may play crucial roles not only in normal architecture but also in signal transduction of the neurons (Wang &Liu, 2008). The acidic sequences may be involved in cation binding and in iron binding site motif, also is

Fig. 3. Characteristics of human Tau protein. (A) Human Tau gene presents 14 exons and -1 is part of the Tau gen promotor: -1 and 14 can be transcribed but not translated; in human CNS are not normally expressed exons 4A, 6 and 8. In all isoforms are expressed exons 1, 4, 5, 7, 9, 11, 12 and 13. Exons 2, 3 and 10 can be excluded depending of Tau isoform. In exon 9 is codified the protein Saitohin. Exons are indicated by rectangles. Distances between exons are representative. (B) Tau isoforms expressed in human CNS, showing number of aminoacids and inserts that differ each of them. Functional protein domains are indicated by different colors (C) Domains contained in most of the isoforms of human Tau protein: the Nterminal is named projection domain and is subdivided in acidic domain and proline rich regions; also here is a PXXP motif for SH3 domain interactions. The C-terminal, also named microtubule binding domain (MTBD) contains the MTBR and the C-terminus *per se*; in 4R isoforms is present 2 sequences with β sheet structure tendency adjacent to the second and third repeats. P= phosphorylation sites; = truncations sites.

function. Among its many roles, Tau promotes neurite outgrowth, organizes axonal microtubules (MTs) and is involved in kinesin-dependent axonal transport (Andreadis, 2011; Hirokawa et al., 1988; LaPointe et al., 2009). Additionally, the interaction of Tau with diverse structural and functional proteins suggests that Tau may play crucial roles not only in normal architecture but also in signal transduction of the neurons (Wang &Liu, 2008). The acidic sequences may be involved in cation binding and in iron binding site motif, also is

Fig. 3. Characteristics of human Tau protein. (A) Human Tau gene presents 14 exons and -1 is part of the Tau gen promotor: -1 and 14 can be transcribed but not translated; in human CNS are not normally expressed exons 4A, 6 and 8. In all isoforms are expressed exons 1, 4, 5, 7, 9, 11, 12 and 13. Exons 2, 3 and 10 can be excluded depending of Tau isoform. In exon 9 is codified the protein Saitohin. Exons are indicated by rectangles. Distances between exons

aminoacids and inserts that differ each of them. Functional protein domains are indicated by different colors (C) Domains contained in most of the isoforms of human Tau protein: the Nterminal is named projection domain and is subdivided in acidic domain and proline rich regions; also here is a PXXP motif for SH3 domain interactions. The C-terminal, also named microtubule binding domain (MTBD) contains the MTBR and the C-terminus *per se*; in 4R isoforms is present 2 sequences with β sheet structure tendency adjacent to the second and

are representative. (B) Tau isoforms expressed in human CNS, showing number of

third repeats. P= phosphorylation sites; = truncations sites.

proposed that the motif KKXK is involved in heparing binding (Ávila et al., 2002) and actin interaction (Zmuda &Rivas, 2000). In this regions is present various PPXXP or PXXP motifs which allow Tau protein to interacts with SH3 domains of diverse proteins such as Src family members like Fyn, Lck, Src (Lee, 2005; Lee et al., 1998; Scales et al., 2011) and others like PCL-γ (Hwang et al., 1996; Jenkins &Johnson, 1998). Is also mentioned that STH interacts with Tau and Abl and influences in Abl phosphorylation (Wang et al., 2011), but this phenomena needs further research.

The efficacy of Tau to binding MTs is influenced by the degree of phosphorylation and the exclusion/inclusion of exon 10. The affinity of Tau against MTs is weaker in 3R isoforms than 4R isoforms, perhaps leading to differential regulation of microtubule behaviour (Eckermann et al., 2007; Goode et al., 2000; Lu &Kosik, 2001). Another important factor influences Tau-MTs interaction: site-specific phosphorylation. This is due to the high frequency of phosphorylatable residues (45 S, 35 T and 5 Y in the largest isoform), combined with the open structure of Tau which renders it accessible to many kinases (Mandelkow et al., 2007). The majority of the phosphorylation sites are Serine or Threonine residues followed by Prolines (SP/TP motifs) that lie in the domains flanking the repeats and can be phosphorylated by several Proline-directed kinases and by neuronal kinases MARK (affecting the KXGS motifs within Tau's repeat domain) (Eckermann et al., 2007; Trinczek et al., 1995). Among the kinases responsible for Tau phosphorylation are Glycogen Synthase Kinase 3β (Lin et al., 2007), cyclin-dependent kinase 5 (Cdk5), MT-affinity regulatory kinase, cAMP-dependent protein kinase (Johnson &Stoothoff, 2004), dual-specificity tyrosinephosphorylated and regulated kinase 1A, Tau–tubulin kinase 1, and calmodulin-dependent protein kinase 2 (Meraz-Ríos et al., 2010)
