*2.1.1. Tau's interaction with microtubule*

The expression of tau protein varies in different tissues, but the brain has the most abundant level. In brains, tau is predominately expressed in neurons but can also be detected in glial cells, especially in oligodendrocytes [11]. In neurons, tau proteins are mainly localized in axons, but they are not excluded from dendrites [12]. Functional analyses in vitro demonstrated that tau plays critical roles in both microtubules assembly and maintaining the structural stabilization [7, 13]. But not until recently, researchers are starting to understand the interactions between tau and microtubule in real-time by adopting different newly developed techniques. By fusing a Halo-tag, a dehalogenase modified to bind certain fluorescent ligand; tau could be labeled and monitored in live imaging [14]. With tubulins being labeled with photoactivatable green fluorescent protein (PAGFP), the interactions between tau and tubulins are viewed under total internal reflection fluorescence microscope (TIRF). With such high resolution and relatively short time frame, the live imaging revealed that tau moves on microtubule quickly without direction. In authors' words, it could "hops on and off" to another microtubule in milliseconds and moves along a microtubule with little dwindle time [14, 15]. In another study, under transmission electron microscope (TEM), it was found that tau could promote the microtubule assemble by laterally crosslinking protofilaments [13]. Moreover, tau showed a preference to bind GDP-tubulin over GTP-tubulin, but the reason behind it is not understood [13].

#### *2.1.2. Tau structures and functions*

involvement of tau protein in a range of neurological disorders makes it one of the most studied proteins. However, despite it has been studied for approximately 30 years since tau is coined as the major component of fibrillary tangles, concrete evidence that detail tauopathy in molecular and cellular levels is still limited [3], and most of the pathological data are obtained from studies using postmortem brain. Therefore, how tau protein and its dynamic changes affect the pathogenesis of various neurodegenerative diseases is still a mystery. Many outstanding questions are being stressed, including which posttranslational modifications are critical for it to gain toxicity, does other neurodegenerative diseases involved proteins interact with tau protein, which brain regions or cell types are most susceptive to the toxicity of tau and how the aggregates cause cellular dysfunction, and which forms of tau during the tangle formation process are toxic. In this process, some widely accepted concepts are being challenged. For example, traditionally, it is believed that hyperphosphorylation of tau induces it to detach from microtubule and increases its toxicity, but recent findings suggest some phosphorylations are protective instead [4, 5]. These findings will be emphasized in the following sections. Apparently, more efforts are needed before we can reach a definitive answer for these questions. Some exciting technological advances promise further exploration of some exist

Tau pathology, namely fibrillary tangles, was observed way before the protein was identified. In fact, it was Alois Alzheimer who first described the heavy burdens of this never reported feature in his demented patient back to 1906 [6]. Seventy years later, tau protein was isolated as a factor that is critical for the re-polymerization of some depolymerized tubulins to form microtubules in vitro [7]. After another 10 years, a series studies confirmed that the tangles observed in AD brain are composed of tau [3, 8–10]. Since then, tau received significant attention in AD research. Nevertheless, as researchers soon realized tau pathology in a panel of neurological dysfunction, solving the underlying mechanism of tauopathy has been regarded

The expression of tau protein varies in different tissues, but the brain has the most abundant level. In brains, tau is predominately expressed in neurons but can also be detected in glial cells, especially in oligodendrocytes [11]. In neurons, tau proteins are mainly localized in axons, but they are not excluded from dendrites [12]. Functional analyses in vitro demonstrated that tau plays critical roles in both microtubules assembly and maintaining the structural stabilization [7, 13]. But not until recently, researchers are starting to understand the interactions between tau and microtubule in real-time by adopting different newly developed techniques. By fusing a Halo-tag, a dehalogenase modified to bind certain fluorescent ligand; tau could be labeled and monitored in live imaging [14]. With tubulins being labeled

questions and more unexplored fields involving tau protein.

**2. Tau biology and pathology**

as a unique field of neurobiology.

*2.1.1. Tau's interaction with microtubule*

**2.1. Tau biology**

34 Cognitive Disorders

Tau is encoded by the gene MAPT (microtubule-associated protein tau), which is located on chromosome 17q21. *MAPT* has 15 exons, and the alternative splicing of the mRNA resulted in six different isoforms. The longest one among these has 441 amino acids, which is often referred as full-length tau. In late 1980s, the basic protein structure of tau was defined [16, 17], and it was realized that the C-terminus of tau protein contains repeated domains responsible for the binding of microtubules [17]. The basic structure and functions of each functional region of it are summarized as follow:

On the N terminal side (1–150), it has two N-terminal domains. Each has 29 amino acids, one from 45 to 74 and another from 75 to 103. The physiological functions of N terminal domain are largely unknown, and speculations on that including it could play roles in signal transduction as tau could co-immunoprecipitated with phospholipase C gamma through the binding sites within N terminus [18].

The N terminal side is followed by two proline-rich domains, one from 151 to 198 followed by another from 199 to 243. Studies showed that this region interacts with src kinase family members, such as fyn serving for signal transductions [19, 20]. Furthermore, it was shown that tau could interact with beta and gamma actins, which are the subtypes of actins commonly seen in neurons [21]. A panel of different truncated and or mutated tau was generated to test the interactions of it with actins, and it was found that the proline-rich regions were responsible for this interaction [21].

The proline-rich region is followed by microtubule binding region (244–370), which is composed of four repeated domains and each one of them contains 18 highly conserved amino acids. The microtubule binding region directly binds to tubulin, which plays the most critical role in microtubule interaction [22]. Because the N terminal side does not bind to microtubule and was thought to interact with other proteins, the N terminal side plus the first proline-rich domain is often referred as projection domain of tau. The rest residues, including the second proline-rich domain and microtubule binding domains as well as the C terminal tail, are often referred as microtubule assembly domain [23].

Because the full-length tau has all these N terminal regions (2 N) and C terminal repeats (4R), it is also referred as "2N4R" tau. The rest of other tau isoforms found in brain are the combinations of either lacks one (the second N terminal domain; 1 N) or two N terminal domains (0 N) or does not contain the second microtubule-binding domain (3R). Therefore, isoforms "1N4R", "0N4R", "2N3R", "1N3R," and "0N3R" are simply denoting the major functional domains of tau [22, 23]. The dominate forms found in human brain are 2N4R and 2N3R. Under physiological conditions, the ratio of isoforms with 4R and 3R is around 1 [24]. While in pathological conditions, the expression of tau isoforms could favor one form, especially 2N4R for most tauopathies. Researchers have long noticed the ratio alterations in disease conditions, but the exact meaning and the reason behind this change are still unknown [25].

**Step 1.** Conformational change of tau: Certain sites phosphorylation and other posttranslational modifications or genetic mutation of MAPT rendering tau bind to tubulins with reduced affinity and detaches from microtubules no longer support the microtubule

Tauopathy

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http://dx.doi.org/10.5772/intechopen.73198

**Step 2.** Oligomer formation: The detached tau proteins form globular oligomers, which are composed of 40 monomers in vitro in the presence of heparin [37]. The mainstream opinion speculates that the detached/modified tau proteins, especially the hyperphosphorylated types, are prone to interact with each other. The phosphorylation on the residues of C-terminal and proline-rich domains neutralizes the charges of the region and reduces the net charge of the protein by which it may contribute to losing the natively unfolded property and prohibit the intramolecular interactions of tau protein [22, 38, 39]. On the other hand, it was reported that the two cysteine residues on tau, cysteine-291 and cysteine-322, play a pivotal role in the tau dimerization [40] because oxidation of the residues may lead to the disulfide bond formation between tau monomers, which potentially seeds for the oligomerization process. This theory is supported by the frequent observation of oxidative stress in tauopathies [41]. Moreover, a recent study showed a compound which could effectively inhibit heparin-induced tau oligomerization was through its interaction with the cysteine residues [37]. However, tau can also form cysteine-independent oligomers [40]. Another recent study adopted tau fragment (aa. 297–391), which is the core of PHF, to study the role of cysteine in the polymerization of tau in the absence of heparin [42]. The results showed that replacement of the cysteine residue or in the presence of reducing

agents, the polymerization process was accelerated rather than decelerated [42].

in tau oligomers and PHF could be detected with thioflavin T or S staining [28].

It is noteworthy that most of these studies used anionic agents like heparin to induce the oligomerization of different recombinant tau isoforms or fragments to test the intrinsic properties and the effects of the modifications in vitro. But to what extent could this artificially induced tau oligomerization reflects the real pathological process is questionable. A recent study showed that heparin-induced recombinant tau tangles have little seeding ability in the wild-type mouse. In contrast, tau tangles isolated from patients have a strong seeding ability, and the pathology could spread quickly to different brain regions, shedding lights on the difference between in vitro generated tau tangles and in situ harvested tau tangles [43]. **Step 3.** PHF formation: Tau oligomers may further develop into more complex structures like PHF or straight filaments (SF) [44]. It has been shown that in different tauopathies, the ratio of PHF/SF and their sizes may vary [45, 46]. For example, in Alzheimer's disease, PHF is more commonly observed than SF [45]. On the other hand, in progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and pick's disease (PiD), SF is predominately found [46]. In certain cell types beyond neurons, tau inclusions may exhibit different morphologies and have been given different names to describe their shapes [47]. With the help of Cryo-Electron Microscopy (Cryo-EM), we are now able to identify the structures with high resolutions [48]. It becomes clear that the cores of the filaments are composed of eight β-sheets which requires some hydrophobic interactions between individual peptides; the difference between PHF and SF is due to the lateral interactions between the sheets. However, disulfide bonds formed in the structures remain to be determined [48]. The β-sheets formed

assemble, increasing the free tau protein pool [22, 36].

Under physiological condition, tau exists in an unfolded state, and 80% of the proteins interact with microtubule in neurons [22, 26]. When tau is not interacting with other proteins, it may curl on its own, and this random curled state is believed important for preventing interactions with other tau proteins by masking the possible interacting sites [27, 28]. The protein itself is bipolar; the N-terminal side is highly negatively charged in normal physiology, while the proline-rich domain and C-terminal end are positively charged, allowing it to interact with the negatively charged C-terminal of tubulins [22, 29]. Various posttranslational modifications could alter its charge. Paired helical filaments (PHFs) are relatively acidic compared to normal full-length tau, which is believed due to the phosphorylation of the amino acid residues [30, 31]. Tau is also very hydrophilic, containing only a small portion of hydrophobic residues [27]. Both the net charge changes and a possible shift from being hydrophilic to hydrophobic are speculated of contributing to its aggregation behavior under pathological conditions [28, 31]. Also, normal tau proteins only exhibit transient secondary structures [27]. Phosphorylation of certain residues may prompt tau to form secondary structures, which is revealed by pseudophosphorylation of all the residues that could be recognized by phosphotau specific antibodies AT8, AT100, and PHF1 and are shown by the structural changes in nuclear magnetic resonance spectroscopy [32]. But how normal tau proteins are transformed to form aggregates remains a mystery.

Given that tau may exist in various forms and structures, one shall be mindful not to overstate the possible role of tau based on data derived from truncated/engineered tau, which may only have the N terminal side or the C terminal side [18, 33–35]. Nevertheless, we have gained more understanding of tau and its function from previous works, but better manipulations are needed before we can be comfortable about applying those bench-side results to tauopathies treatment.
