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

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

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.,

Tau and Amyloid-β Conformational Change to β-Sheet

Peña et al., 2009; Wischik et al., 1996).

Structures as Effectors in the Development of Alzheimer's Disease 375

indicating that pre-phosphorylation at certain sites can alter the conformation such that other phosphorylation reactions are no longer possible (Friedhoff et al., 2000). Tau abnormal phosphorylation at specific sites are strongly associated to other post-translational modifications such endogenous proteolysis or truncation. This abnormal process is defined as a protein cutting that could also promote aberrant aggregation (Ávila et al., 2004) In the case of Tau molecule, the lost of any of its C- and N-extremes leads to a change of properties of the molecule. The lost of the N-terminal has been proposed as an early event in Tau aggregation process because of the capability of this extreme of inhibit Tau oligomerization *in vitro* (Ghoshal et al., 2002; Horowitz et al., 2006) On the other hand, C-terminal proteolysis is strongly correlated with neuropathological lesions and cognitive impairment (García-Sierra et al., 2001; Meraz-Ríos et al., 2010). Latest years many reports have demonstrated the relationship between site-specific phosphorylation at sites Ser199, Ser202, Thr205, Thr212, Ser214, located in proline-rich region, and Ser396 and Ser 404, located near C-termini. These impact in conformation of Tau, and perhaps expose sites for further specific proteolysis in Asp421 and Glu391 (Fischer et al., 2009; Jeganathan et al., 2008a; Luna-Muñoz et al., 2007; Mondragon-Rodriguez et al., 2008). Specific Tau-phosphorylation also alters microtubulebinding capacity and increase rate of filament nucleation (Chang et al., 2011; Fischer et al., 2009). With this issue, is proposed the sequential relationship between phosphorylation→ conformational change→ truncation, which can be repeated until Tau protein lost their Nand C-termini, exposing a minimal resistance protease region named PHF-core (figure 3) (Binder et al., 2005; García-Sierra et al., 2008; Wischik et al., 1985; Wischik et al., 1996). The dense core of PHFs is compound of the repeat region of the Tau protein, forming a fuzzy coat beside the filament with N- and C-termini of the protein(Skrabana et al., 2004). According to *in vitro* assays, this PHF-core is able to promote Tau aggregation (Campos-

Considering these data, is important to use diverse strategies for the study of this phenomena. For elucidated this issue is used conformational-specific antibodies. The Alz-50 and MC-1, two conformational sensitive antibodies, have been demonstrated to recognize a pathological conformation of Tau molecule in NFTs (Jicha et al., 1997; Lin et al., 2007). Either antibody recognizes the N-termini nearby C-termini, determinate by fluorescence lifetime imaging and other techniques (Hyman et al., 2005). Even these antibodies recognize a region in the N-termini (7-9) and C-termini (312-342), both conserved in all Tau human isoforms, in tissue they have a distinct pattern of signal (Jicha et al., 1997). In AD brain, MC1 stains "pretangle neurons", and its immunopositivity is indicative of very early neuronal pathological changes. Alz50 stains both pretangle neurons, as well as early-formed pairedhelical filaments (Nogalska et al., 2011). Moreover, a number of studies have proposed that the phosphorylation modification at S-P or T-P motifs in Tau might cause Tau conformational change and the *cis-trans* isomerisation, made by the prolyl-peptide isomerise Pin-1, might regulate Tau function. This association is dependent on a folding of the Tau molecule, this bend occurring locally in the vicinity of Thr231-P motif, and actually recognized by TG3 antibody (Friedhoff et al., 2000; Lin et al., 2007; Luna-Muñoz et al., 2005; Weaver et al., 2000). The antibody Tau-66 is another conformational-associated antibody, recognizing residues between 155-244 and 305-314. This antibody recognizes NFT, and also cytoplasmic points on some non-fibrillary neurons, mostly associated to truncated N- and Ctermini (García-Sierra et al., 2003). During this study, they suggest there is a progression from Alz-50 to Tau-66 conformation during NFTs evolution. Related to this, Goux based on PHFs extraction and CD assays, mention that Tau has a structure containing 31-37% helix,

2010). Tau protein is not the exception. Despite its random coil appearance in solution, Tau assembles into well-defined fibbers, the Paired Helical Filaments (PHFs) (von Bergen et al., 2000). The appearance of conformational changes in Tau as early alterations in AD neuropathology has been eloquently confirmed in several studies (Ávila et al., 2002). Even when is reported that α-helix structures are present in PHFs (Sadqi et al., 2002), the β-sheet structure seems to be the major structure involved in Tau aggregation. Two hexapeptides, 275VQJINK280 and 306VQJVYK311 which are respectively located at the beginning of the second and third MTBR are prominent in generating β-sheet structures during Tau aggregation process (Martin et al., 2011). These hexapeptides structure are well characterized by several methodologies, like FTIR, AFM and CD (Chaudhary et al., 2009). Another reports mention that proline is frequently involved in a β-turn by reason of its restricted ø angle and that the Pro-Gly motif is favored to make a Type II β-turn and the SPXX motif is known to stabilize β-turns, independent of X. Also is predicted that Tau contains 54 β-turns involving about 35% the protein sequence (Goux, 2002). For the conformational studies, Goux (2002) mentions that is important to consider factors as temperature or solvent-induced changes in Tau to the *cis-trans* proline distribution of isomers, resulting in a change in protein conformation, also mentioned by other authors for the two hexapeptides mentioned before (Chaudhary et al., 2009; Goux, 2002).

As a general view for aggregation studies, conformational changes have been studied by the use of aggregations inducers or punctual mutations in the MTBR. The other manner is by promoting post-translational modifications in Tau molecule, such as phosphorylations or truncations or by the analysis of PHFs. In the presence of pro-aggregatory factors as polyanions, stimuli the assembly of full lenght Tau, 3R or 4R, and when mutations forms associated to other Tauopathies seem to be a faster process of filament formation. In both are reported the presence of β-sheet enriched structures (Friedhoff et al., 2000). The β-strands usually run perpendicular to the helix axis forming a cross-β motif. By *in vitro* assays, is suggested that β-sheet structures might be stabilized by the presence of salt bridges or by hydrogen bonds, which promote forming a continuous β-sheet extending along the axis (Berriman et al., 2003; Jeganathan et al., 2008b). Such structures can be stained with certain dyes such as Congo red (CR), Thioflavine S (TS) or Thiazine Red (TR) which are thought to interact with the repeating β strands (Glenner et al., 1972; Luna-Muñoz et al., 2008; Zilka et al., 2006). Planar aromatic dyes as CR, TR, and TS also are capable of inducing Tau fibrillization *in vitro*. In our group we developed a cellular model to study the particularities of these dyes at *in vivo* Tau aggregation. The SH-SY5Y cells were incubated with each dye and our results showed that three dyes gave rise to aggregates of Tau with different morphological characteristics. This method can be used to test related drugs with inhibitory potential for Tau abnormal polymerization (Lira-De León et al, unpublished data).

Alternatively, Tau can indeed adopt distinct conformations has been observed in sequential phosphorylation reactions. This theory is supported by biophysical evidence showing that Tau paracrystals become longer and stiffer following phosphorylation, suggesting a conformational change of the protein upon phosphorylation; specifically NMR and CD evidence shows that a Tau peptide undergoes a conformational change following phosphorylation at Thr231 and Ser235 (Goux, 2002). Moreover, where the AD-specific phosphoepitope of antibody AT-100 in the proline-rich region can only be generated by a sequential phosphorylation of Tau first by GSK-3β (at Thr212) and then by PKA (at Ser214),

2010). Tau protein is not the exception. Despite its random coil appearance in solution, Tau assembles into well-defined fibbers, the Paired Helical Filaments (PHFs) (von Bergen et al., 2000). The appearance of conformational changes in Tau as early alterations in AD neuropathology has been eloquently confirmed in several studies (Ávila et al., 2002). Even when is reported that α-helix structures are present in PHFs (Sadqi et al., 2002), the β-sheet structure seems to be the major structure involved in Tau aggregation. Two hexapeptides, 275VQJINK280 and 306VQJVYK311 which are respectively located at the beginning of the second and third MTBR are prominent in generating β-sheet structures during Tau aggregation process (Martin et al., 2011). These hexapeptides structure are well characterized by several methodologies, like FTIR, AFM and CD (Chaudhary et al., 2009). Another reports mention that proline is frequently involved in a β-turn by reason of its restricted ø angle and that the Pro-Gly motif is favored to make a Type II β-turn and the SPXX motif is known to stabilize β-turns, independent of X. Also is predicted that Tau contains 54 β-turns involving about 35% the protein sequence (Goux, 2002). For the conformational studies, Goux (2002) mentions that is important to consider factors as temperature or solvent-induced changes in Tau to the *cis-trans* proline distribution of isomers, resulting in a change in protein conformation, also mentioned by other authors for

the two hexapeptides mentioned before (Chaudhary et al., 2009; Goux, 2002).

potential for Tau abnormal polymerization (Lira-De León et al, unpublished data).

Alternatively, Tau can indeed adopt distinct conformations has been observed in sequential phosphorylation reactions. This theory is supported by biophysical evidence showing that Tau paracrystals become longer and stiffer following phosphorylation, suggesting a conformational change of the protein upon phosphorylation; specifically NMR and CD evidence shows that a Tau peptide undergoes a conformational change following phosphorylation at Thr231 and Ser235 (Goux, 2002). Moreover, where the AD-specific phosphoepitope of antibody AT-100 in the proline-rich region can only be generated by a sequential phosphorylation of Tau first by GSK-3β (at Thr212) and then by PKA (at Ser214),

As a general view for aggregation studies, conformational changes have been studied by the use of aggregations inducers or punctual mutations in the MTBR. The other manner is by promoting post-translational modifications in Tau molecule, such as phosphorylations or truncations or by the analysis of PHFs. In the presence of pro-aggregatory factors as polyanions, stimuli the assembly of full lenght Tau, 3R or 4R, and when mutations forms associated to other Tauopathies seem to be a faster process of filament formation. In both are reported the presence of β-sheet enriched structures (Friedhoff et al., 2000). The β-strands usually run perpendicular to the helix axis forming a cross-β motif. By *in vitro* assays, is suggested that β-sheet structures might be stabilized by the presence of salt bridges or by hydrogen bonds, which promote forming a continuous β-sheet extending along the axis (Berriman et al., 2003; Jeganathan et al., 2008b). Such structures can be stained with certain dyes such as Congo red (CR), Thioflavine S (TS) or Thiazine Red (TR) which are thought to interact with the repeating β strands (Glenner et al., 1972; Luna-Muñoz et al., 2008; Zilka et al., 2006). Planar aromatic dyes as CR, TR, and TS also are capable of inducing Tau fibrillization *in vitro*. In our group we developed a cellular model to study the particularities of these dyes at *in vivo* Tau aggregation. The SH-SY5Y cells were incubated with each dye and our results showed that three dyes gave rise to aggregates of Tau with different morphological characteristics. This method can be used to test related drugs with inhibitory indicating that pre-phosphorylation at certain sites can alter the conformation such that other phosphorylation reactions are no longer possible (Friedhoff et al., 2000). Tau abnormal phosphorylation at specific sites are strongly associated to other post-translational modifications such endogenous proteolysis or truncation. This abnormal process is defined as a protein cutting that could also promote aberrant aggregation (Ávila et al., 2004) In the case of Tau molecule, the lost of any of its C- and N-extremes leads to a change of properties of the molecule. The lost of the N-terminal has been proposed as an early event in Tau aggregation process because of the capability of this extreme of inhibit Tau oligomerization *in vitro* (Ghoshal et al., 2002; Horowitz et al., 2006) On the other hand, C-terminal proteolysis is strongly correlated with neuropathological lesions and cognitive impairment (García-Sierra et al., 2001; Meraz-Ríos et al., 2010). Latest years many reports have demonstrated the relationship between site-specific phosphorylation at sites Ser199, Ser202, Thr205, Thr212, Ser214, located in proline-rich region, and Ser396 and Ser 404, located near C-termini. These impact in conformation of Tau, and perhaps expose sites for further specific proteolysis in Asp421 and Glu391 (Fischer et al., 2009; Jeganathan et al., 2008a; Luna-Muñoz et al., 2007; Mondragon-Rodriguez et al., 2008). Specific Tau-phosphorylation also alters microtubulebinding capacity and increase rate of filament nucleation (Chang et al., 2011; Fischer et al., 2009). With this issue, is proposed the sequential relationship between phosphorylation→ conformational change→ truncation, which can be repeated until Tau protein lost their Nand C-termini, exposing a minimal resistance protease region named PHF-core (figure 3) (Binder et al., 2005; García-Sierra et al., 2008; Wischik et al., 1985; Wischik et al., 1996). The dense core of PHFs is compound of the repeat region of the Tau protein, forming a fuzzy coat beside the filament with N- and C-termini of the protein(Skrabana et al., 2004). According to *in vitro* assays, this PHF-core is able to promote Tau aggregation (Campos-Peña et al., 2009; Wischik et al., 1996).

Considering these data, is important to use diverse strategies for the study of this phenomena. For elucidated this issue is used conformational-specific antibodies. The Alz-50 and MC-1, two conformational sensitive antibodies, have been demonstrated to recognize a pathological conformation of Tau molecule in NFTs (Jicha et al., 1997; Lin et al., 2007). Either antibody recognizes the N-termini nearby C-termini, determinate by fluorescence lifetime imaging and other techniques (Hyman et al., 2005). Even these antibodies recognize a region in the N-termini (7-9) and C-termini (312-342), both conserved in all Tau human isoforms, in tissue they have a distinct pattern of signal (Jicha et al., 1997). In AD brain, MC1 stains "pretangle neurons", and its immunopositivity is indicative of very early neuronal pathological changes. Alz50 stains both pretangle neurons, as well as early-formed pairedhelical filaments (Nogalska et al., 2011). Moreover, a number of studies have proposed that the phosphorylation modification at S-P or T-P motifs in Tau might cause Tau conformational change and the *cis-trans* isomerisation, made by the prolyl-peptide isomerise Pin-1, might regulate Tau function. This association is dependent on a folding of the Tau molecule, this bend occurring locally in the vicinity of Thr231-P motif, and actually recognized by TG3 antibody (Friedhoff et al., 2000; Lin et al., 2007; Luna-Muñoz et al., 2005; Weaver et al., 2000). The antibody Tau-66 is another conformational-associated antibody, recognizing residues between 155-244 and 305-314. This antibody recognizes NFT, and also cytoplasmic points on some non-fibrillary neurons, mostly associated to truncated N- and Ctermini (García-Sierra et al., 2003). During this study, they suggest there is a progression from Alz-50 to Tau-66 conformation during NFTs evolution. Related to this, Goux based on PHFs extraction and CD assays, mention that Tau has a structure containing 31-37% helix,

Tau and Amyloid-β Conformational Change to β-Sheet

.

Tau aggregates induced by the presence of the PHF core.

Structures as Effectors in the Development of Alzheimer's Disease 377

associated with enriched β-sheet content, can be driven by small diffusible ligands in a

Pathologic Tau fibrils show diverse morphologies in diseased brains, where transmission electron microscopy (TEM) images typically reveal PHFs in AD NFTs or neuropil threads, although straight filaments (SFs) and twisted ribbons (TRs) are also seen (Xu et al., 2010). Those protofibrils seem to be wound around one another exposing a crossover repeat of~80 nm, a maximal width of ~22 nm, and a narrow waist of ~12 nm. The twisted appearance is variable as follows: ~10% of Tau fibrils are straight filaments (Wegmann et al., 2010). Additional morphologic variants of Tau fibrils have been observed in other Tauopathies

Fig. 4. Characteristics of PHF-core. (A) Structure diagram and amino acid sequence of PHF core (Tau441). Although Tau presents a secondary random coil structure, the R2 and R3 of MTBD showed a motif with regional trend to β-sheet structure. (B) Amino acid distribution histogram of PHF core; they are partially hydrophobic; (C) Proposed core structure based on predicted β-strand propensities (Displayed in Jmol). (D) Electron Microscopy photograph of

such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease, and FTDP-17 syndromes. It seems that in these pathologies the crossover repeat is ~160 nm. Likewise, Tau fibrils reassembled *in vitro* can display a variable twist between filaments assembled from different variants of Tau proteins (e.g. different splicing variants) or even within a given filament (Wegmann et al., 2010). In the cellular environment protein expression, protein folding, and protein degradation are highly regulated processes. Protein

process that approximates homogeneous nucleation (Congdon et al., 2008).

15-20% β-sheet, 20-23% turn, and 26-29% unordered structure (Goux, 2002), perhaps influencing in this "specific-antibody" conformation. It was also mentioned by Barghorn and co-workers that for aggregation issues, Tau must adopt a specific conformation that allows a subsequent fibril formation (Barghorn &Mandelkow, 2002). Recent studies use mAb423 antibody, which recognize truncated Tau in Glu391, as a tool for structural studies directed to the PHF core (from 297-391 of 441 isoform), allowing to estimate the enrichment of β-sheet structures (Sevcik et al., 2007; Skrabana et al., 2010; Wischik et al., 1985). In this aim, antibodies have probe been an important tool for recognize conformational changes in Tau during aggregation events.

#### **3.2 Impact of Tau conformational change during aggregation process**

Partially folded or misfolded states often tend to aggregate because these forms typically expose hydrophobic amino acid residues and regions of unstructured polypeptide backbone, features that are largely buried in the native state. Like intramolecular folding, aggregation, i.e., the association of two or more non-native protein molecules, is driven by hydrophobic forces and predominantly results in the formation of amorphous structures that lack long range order. Although the toxic principle operating in the age-of-onset diseases is far from being understood, a consensus is emerging that oligomeric, soluble states of the respective disease protein are the primary cytotoxic species (Vabulas et al., 2010). Tau proteins fibrillize efficiently *in vitro* into β-pleated sheet structures with biochemical and biophysical properties of amyloid fibrils (figure 4A-B), but they do so only in the presence of negatively charged cofactors such as heparin, RNA, or DNA. The kinetic profile of Tau fibrillization *in vitro* generally resembles that of other amyloid proteins, when is important an increase of β-sheet structures (figure 4C), with a lag phase during which time nucleating units are believed to be formed followed by rapid fibril growth (Congdon et al., 2008; Matthes et al., 2011; von Bergen et al., 2000; Xu et al., 2010). Covalently linked dimers can be formed by disulfide cross-linking and by electronic microscopy it seems as rod-like particles (Friedhoff et al., 2000). The dimers of Tau molecules become subunits of filaments, forming protomers, adopting the parallel, in register cross-β-sheet structure typical of amyloid aggregates. On the basis of morphology and mass-per-unit length measurements, mature Tau filaments (PHFs) consist of two protofilaments wound around each other (Congdon et al., 2008). This has led to several proposed ''nucleation-elongation'' models of Tau fibril assembly (figure 5). In the classic equilibrium nucleation-elongation model elaborated for linear polymer formation, assembly-competent monomer is in rapid equilibrium with a thermodynamically unstable species termed the nucleus, in Tau case, probably the PHF-core (figure 4D). Once assembly-competent conformations are adopted (Weaver et al., 2000), the rate-limiting step in the reaction becomes dimerization, with subsequent aggregate growth occurring through monomer addition. When the critical nucleus cluster size is reached, subsequent additions to the nascent filament end are energetically favorable, and elongation proceeds efficiently (figure 5). This pathway leads to peaked distributions of filament lengths early in the reaction time series followed by slow relaxation toward exponential distributions at equilibrium. Finally, aggregation rates can be limited by secondary nucleation events, which occur on existing aggregates (Congdon et al., 2008). In addition, an alternative linear colloidal aggregation model has been proposed for Tau in which the protein forms colloidal spheres that serve as nucleation units that, through charge-dipole and dipole-dipole interactions, go on to form linear fibers (Xu et al., 2010). Either pathway seems that adoption of assembly competent conformations, perhaps

15-20% β-sheet, 20-23% turn, and 26-29% unordered structure (Goux, 2002), perhaps influencing in this "specific-antibody" conformation. It was also mentioned by Barghorn and co-workers that for aggregation issues, Tau must adopt a specific conformation that allows a subsequent fibril formation (Barghorn &Mandelkow, 2002). Recent studies use mAb423 antibody, which recognize truncated Tau in Glu391, as a tool for structural studies directed to the PHF core (from 297-391 of 441 isoform), allowing to estimate the enrichment of β-sheet structures (Sevcik et al., 2007; Skrabana et al., 2010; Wischik et al., 1985). In this aim, antibodies have probe been an important tool for recognize conformational changes in

Partially folded or misfolded states often tend to aggregate because these forms typically expose hydrophobic amino acid residues and regions of unstructured polypeptide backbone, features that are largely buried in the native state. Like intramolecular folding, aggregation, i.e., the association of two or more non-native protein molecules, is driven by hydrophobic forces and predominantly results in the formation of amorphous structures that lack long range order. Although the toxic principle operating in the age-of-onset diseases is far from being understood, a consensus is emerging that oligomeric, soluble states of the respective disease protein are the primary cytotoxic species (Vabulas et al., 2010). Tau proteins fibrillize efficiently *in vitro* into β-pleated sheet structures with biochemical and biophysical properties of amyloid fibrils (figure 4A-B), but they do so only in the presence of negatively charged cofactors such as heparin, RNA, or DNA. The kinetic profile of Tau fibrillization *in vitro* generally resembles that of other amyloid proteins, when is important an increase of β-sheet structures (figure 4C), with a lag phase during which time nucleating units are believed to be formed followed by rapid fibril growth (Congdon et al., 2008; Matthes et al., 2011; von Bergen et al., 2000; Xu et al., 2010). Covalently linked dimers can be formed by disulfide cross-linking and by electronic microscopy it seems as rod-like particles (Friedhoff et al., 2000). The dimers of Tau molecules become subunits of filaments, forming protomers, adopting the parallel, in register cross-β-sheet structure typical of amyloid aggregates. On the basis of morphology and mass-per-unit length measurements, mature Tau filaments (PHFs) consist of two protofilaments wound around each other (Congdon et al., 2008). This has led to several proposed ''nucleation-elongation'' models of Tau fibril assembly (figure 5). In the classic equilibrium nucleation-elongation model elaborated for linear polymer formation, assembly-competent monomer is in rapid equilibrium with a thermodynamically unstable species termed the nucleus, in Tau case, probably the PHF-core (figure 4D). Once assembly-competent conformations are adopted (Weaver et al., 2000), the rate-limiting step in the reaction becomes dimerization, with subsequent aggregate growth occurring through monomer addition. When the critical nucleus cluster size is reached, subsequent additions to the nascent filament end are energetically favorable, and elongation proceeds efficiently (figure 5). This pathway leads to peaked distributions of filament lengths early in the reaction time series followed by slow relaxation toward exponential distributions at equilibrium. Finally, aggregation rates can be limited by secondary nucleation events, which occur on existing aggregates (Congdon et al., 2008). In addition, an alternative linear colloidal aggregation model has been proposed for Tau in which the protein forms colloidal spheres that serve as nucleation units that, through charge-dipole and dipole-dipole interactions, go on to form linear fibers (Xu et al., 2010). Either pathway seems that adoption of assembly competent conformations, perhaps

**3.2 Impact of Tau conformational change during aggregation process** 

Tau during aggregation events.

associated with enriched β-sheet content, can be driven by small diffusible ligands in a process that approximates homogeneous nucleation (Congdon et al., 2008).

Pathologic Tau fibrils show diverse morphologies in diseased brains, where transmission electron microscopy (TEM) images typically reveal PHFs in AD NFTs or neuropil threads, although straight filaments (SFs) and twisted ribbons (TRs) are also seen (Xu et al., 2010). Those protofibrils seem to be wound around one another exposing a crossover repeat of~80 nm, a maximal width of ~22 nm, and a narrow waist of ~12 nm. The twisted appearance is variable as follows: ~10% of Tau fibrils are straight filaments (Wegmann et al., 2010). Additional morphologic variants of Tau fibrils have been observed in other Tauopathies

Fig. 4. Characteristics of PHF-core. (A) Structure diagram and amino acid sequence of PHF core (Tau441). Although Tau presents a secondary random coil structure, the R2 and R3 of MTBD showed a motif with regional trend to β-sheet structure. (B) Amino acid distribution histogram of PHF core; they are partially hydrophobic; (C) Proposed core structure based on predicted β-strand propensities (Displayed in Jmol). (D) Electron Microscopy photograph of Tau aggregates induced by the presence of the PHF core.

.

such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease, and FTDP-17 syndromes. It seems that in these pathologies the crossover repeat is ~160 nm. Likewise, Tau fibrils reassembled *in vitro* can display a variable twist between filaments assembled from different variants of Tau proteins (e.g. different splicing variants) or even within a given filament (Wegmann et al., 2010). In the cellular environment protein expression, protein folding, and protein degradation are highly regulated processes. Protein

Tau and Amyloid-β Conformational Change to β-Sheet

reveal novel insights into diseases pathogenesis.

finally ends in the formations of fibrills or pores (5).

CONACyT- México to MD-H and KL-D grants.

Biophys Acta 1739(2-3): 91-103.

**5. Acknowledgment** 

**6. References** 

Structures as Effectors in the Development of Alzheimer's Disease 379

Analysis of the amyloidogenic regions showed a high occurrence of the aromatic residues phenylalanine and tyrosine, which have a high propensity to stack the delocalized π-electron rings. Studies with short peptides have confirmed that even two consecutive phenylalanine residues are sufficient to facilitate assembly into nanotube-like structures. Protofibrils have been identified which appear to be fibrillar precursors to amyloidogenic fibril formation, and these are preceded by the appearance of small oligomers of Tau and AB. Whether these small intermediates interact directly to form protofibrils and then these elongate to form the fully formed fibril (figure 5). This process can be favored by interaction with membranes rich in negative charged surface, whereas the hydrophobic interior strengthens electrostatic secondary interactions and favors monomer recruitment with increase of local concentration, resulting in aggregate nucleation and membrane disassembly. Developing techniques for directly visualizing amyloidogenic fibrils at high resolution is promising solution to answer many key questions remain to be addressed. Elucidating these questions will undoubtedly

Fig. 5. Common fibrillization pathway of amyloid-β and Tau protein. Under physiological conditions, both proteins adopt a basal conformation (1), but when this is altered, they can adopt a pro-aggregatory conformation (enriched in β-sheet structures). This abnormal conformation (2) allows the formation of dimers and the beginning of a nucleation phase (3). Continuous recruitment of pro-aggregatory forms lead to an elongation phase (4); which

To Carmen Silva-Lucero, MSc. for the assistant of this writing. Financials support by

Andreadis, A. (2005). Tau gene alternative splicing: expression patterns, regulation and

modulation of function in normal brain and neurodegenerative diseases. Biochim

over-expression can lead to protein aggregation, which also is a regulated process. The molecular machines, from the microtubular transport system which assists protein folding and prevents aggregation, to the ubiquitin proteasome system and autophagic vacuoles, which degrade normal and aggregated proteins all work in concert. In a healthy cell they can maintain the equilibrium between synthesis, folding, function and degradation (Zerovnik, 2010). Recent evidence suggests that proteins that are able to constrain the structural freedom of Tau are essential for Tau processing and participate in its accumulation. Interactions of Tau with a specific family of proteins termed *cis-trans* peptidyl-prolyl isomerases (PPIases) has already revealed the importance of this protein family to regulate Tau phosphorylation cycles and its overall stability (Koren et al., 2011). Pin-1 is demostrated to interact with Tau through Thr231-P, promoting a conformational change that allows the action of phospatase PP2A over Tau (Bulbarelli et al., 2009). Recent findings suggest that FKBP51 has a similar activity to Pin1; however, unlike Pin1, FKBP51 coordinates with Hsp90 to isomerize Tau. It also cooperates with distinct protein phosphatases that may also be novel participants in Tau biology. Recently is also suggested the participation of another isomerases like cyclophilin family, but is under research (Koren et al., 2011). In this matter chaperones, specially Hsp90, also can be necessary to maintain Tau in a non-aggregated state, a consequence that may ultimately be deleterious for the brain under pathological conditions (Koren et al., 2009).

The phenomena are not stopped there. The accumulation and propagation of amyloidogenic proteins like Tau are believed to occur through nucleation-dependent polymerization and propagate in an extracellular manner. Tau may be released to the extracellular space upon neuron degeneration, where it could be toxic to other neurons (Ávila, 2010; Gómez-Ramos et al., 2006). This suggests that high molecular weight protein aggregates or amyloid seeds shed from one cell may easily be propagated to others (e.g. neurons or glial cells) under pathological conditions (e.g. alteration in membrane permeability due to aging or virus infection, impairment of membrane function as a result of physical interaction or abnormal membrane depolarization) that favor intracellular deposition of protein fibrils (Nonaka et al., 2010). Further investigation about this must be done. Until now, there is still an incomplete understanding of the sequence of events leading to Tau fibrillization, and it is unclear how the different structural variants of Tau fibrils arise or if these variants (PHFs, SFs, TRs) are interconvertible (Xu et al., 2010). And as a recent issue, the importance of extracellular misfolded Tau during aggregation issues and cellular components such as plasma membrane or mislocalization (Campos-Peña et al., 2009; Lira-De León, 2009), need more investigations. With this last purpose, in cell-models we noticed the enrichment of βsheet structures in cells expressing PHF-core with a signal promoting the localization of it into Plasma membrane associated to presence of 441-Tau (Campos-Peña et al., 2009). Further investigation in this aim needs complementary studies.

#### **4. Conclusion**

Structural studies of amyloidogenic fibrils are crucial. The insolubility and stability of fibrils makes difficult to work with by conventional methods. The research has concentrated on the use of peptides for structural studies, but the information obtained is complex for the different conditions used in the assembly of amyloidogenic fibrils in Tau or AB proteins. There is a minimum sequence with amino acids partially hidrofobic that trend to form a β-sheet structure, this region nucleating amyloidogenic fibril formation and is extremely insoluble.

over-expression can lead to protein aggregation, which also is a regulated process. The molecular machines, from the microtubular transport system which assists protein folding and prevents aggregation, to the ubiquitin proteasome system and autophagic vacuoles, which degrade normal and aggregated proteins all work in concert. In a healthy cell they can maintain the equilibrium between synthesis, folding, function and degradation (Zerovnik, 2010). Recent evidence suggests that proteins that are able to constrain the structural freedom of Tau are essential for Tau processing and participate in its accumulation. Interactions of Tau with a specific family of proteins termed *cis-trans* peptidyl-prolyl isomerases (PPIases) has already revealed the importance of this protein family to regulate Tau phosphorylation cycles and its overall stability (Koren et al., 2011). Pin-1 is demostrated to interact with Tau through Thr231-P, promoting a conformational change that allows the action of phospatase PP2A over Tau (Bulbarelli et al., 2009). Recent findings suggest that FKBP51 has a similar activity to Pin1; however, unlike Pin1, FKBP51 coordinates with Hsp90 to isomerize Tau. It also cooperates with distinct protein phosphatases that may also be novel participants in Tau biology. Recently is also suggested the participation of another isomerases like cyclophilin family, but is under research (Koren et al., 2011). In this matter chaperones, specially Hsp90, also can be necessary to maintain Tau in a non-aggregated state, a consequence that may ultimately be deleterious for the

The phenomena are not stopped there. The accumulation and propagation of amyloidogenic proteins like Tau are believed to occur through nucleation-dependent polymerization and propagate in an extracellular manner. Tau may be released to the extracellular space upon neuron degeneration, where it could be toxic to other neurons (Ávila, 2010; Gómez-Ramos et al., 2006). This suggests that high molecular weight protein aggregates or amyloid seeds shed from one cell may easily be propagated to others (e.g. neurons or glial cells) under pathological conditions (e.g. alteration in membrane permeability due to aging or virus infection, impairment of membrane function as a result of physical interaction or abnormal membrane depolarization) that favor intracellular deposition of protein fibrils (Nonaka et al., 2010). Further investigation about this must be done. Until now, there is still an incomplete understanding of the sequence of events leading to Tau fibrillization, and it is unclear how the different structural variants of Tau fibrils arise or if these variants (PHFs, SFs, TRs) are interconvertible (Xu et al., 2010). And as a recent issue, the importance of extracellular misfolded Tau during aggregation issues and cellular components such as plasma membrane or mislocalization (Campos-Peña et al., 2009; Lira-De León, 2009), need more investigations. With this last purpose, in cell-models we noticed the enrichment of βsheet structures in cells expressing PHF-core with a signal promoting the localization of it into Plasma membrane associated to presence of 441-Tau (Campos-Peña et al., 2009). Further

Structural studies of amyloidogenic fibrils are crucial. The insolubility and stability of fibrils makes difficult to work with by conventional methods. The research has concentrated on the use of peptides for structural studies, but the information obtained is complex for the different conditions used in the assembly of amyloidogenic fibrils in Tau or AB proteins. There is a minimum sequence with amino acids partially hidrofobic that trend to form a β-sheet structure, this region nucleating amyloidogenic fibril formation and is extremely insoluble.

brain under pathological conditions (Koren et al., 2009).

investigation in this aim needs complementary studies.

**4. Conclusion** 

Analysis of the amyloidogenic regions showed a high occurrence of the aromatic residues phenylalanine and tyrosine, which have a high propensity to stack the delocalized π-electron rings. Studies with short peptides have confirmed that even two consecutive phenylalanine residues are sufficient to facilitate assembly into nanotube-like structures. Protofibrils have been identified which appear to be fibrillar precursors to amyloidogenic fibril formation, and these are preceded by the appearance of small oligomers of Tau and AB. Whether these small intermediates interact directly to form protofibrils and then these elongate to form the fully formed fibril (figure 5). This process can be favored by interaction with membranes rich in negative charged surface, whereas the hydrophobic interior strengthens electrostatic secondary interactions and favors monomer recruitment with increase of local concentration, resulting in aggregate nucleation and membrane disassembly. Developing techniques for directly visualizing amyloidogenic fibrils at high resolution is promising solution to answer many key questions remain to be addressed. Elucidating these questions will undoubtedly reveal novel insights into diseases pathogenesis.

Fig. 5. Common fibrillization pathway of amyloid-β and Tau protein. Under physiological conditions, both proteins adopt a basal conformation (1), but when this is altered, they can adopt a pro-aggregatory conformation (enriched in β-sheet structures). This abnormal conformation (2) allows the formation of dimers and the beginning of a nucleation phase (3). Continuous recruitment of pro-aggregatory forms lead to an elongation phase (4); which finally ends in the formations of fibrills or pores (5).
