**2. Amyloid β**

A growing body of evidence suggests that altered processing of amyloid precursor protein (APP) is one of the early events in the pathogenesis of AD. APP is a transmembrane glycoprotein of type 1 from 120 to 200 kD wich are ubiquitously expressed, but are most abundant in the brain (Selkoe, 1994). The APP gene is located on the long arm of chromosome 21 in humans (Kang 1987) and contains 18 exons (figure 1A) through alternative splicing of exons 7, 8 and 15 are generated 8 isoforms that have pattern cell specific expression and are designated by the number of amino acids they contain. The size of the isoforms can vary from 563 to 770 amino acids (Coulson et al., 2000). In central nervous system express only 4 isoforms: PPA695, PPA714, PPA751 and PPA770, PPA695 of which is the most abundant isoform in neurons, while that PPA751 and PPA770 isoforms are expressed mainly in glial cells (Yoshikai et al., 1990; Zheng &Koo, 2006). Only isoforms PPA695, PPA751 and PPA770 contains the sequence encoding the amyloid-β (AB) peptide (Golde et al., 1990; Kang et al., 1987). The full-length APP (APP 770) consists of 18 exons, where exon 17 resembles the membrane spanning domain. APP 695 lacking both, exon 7 and exon 8 is primarily expressed by neurons and is the most abundant APP transcript in the brain (Neve 1988). APP 751 (lacking exon 8), APP 714 (lacking exon 7) and APP 770 were first identified in peripheral organs but are also expressed in brain glial cells. Other alternatively generated splice variants of APP involving exon 15 were recently discovered in peripheral leucocytes and in microglial cells and therefore denoted as leukocyte- derived APP (L-APP) (Konig et al., 1992). The AB fragment is part of exon 16 and 17 (figure 1B) and begins 99 amino acid residues from the carboxy terminus and extends 12-15 residues into the hydrophobic membrane domain (Estus et al., 1992). The APP protein is composed of three regions: a large extracellular domain in the N-terminal region that forms a globular structure, a transmembrane region, which contains part of the AB sequence, and a small

(Braak &Braak, 1991). About these lesions, Braak and Braak (1991) established a relationship between the appearance of NP and NFTs with cognitive decline in post-mortem studies

At the moment, AD can be diagnosed conclusively only post-mortem. However, advances in neuroimaging by magnetic resonance imaging (MRI) and by positron electron tomography (PET) allow researchers to see accumulation of amyloid plaques and NFTs in the living brain. In such a way, the course of the disease at various time points can be followed and an early diagnosis obtained. One, in principle, could even monitor the development/progression of the disease (Zerovnik, 2010). Several evidences demonstrate that under the formation of these lesions, are implicated in an important manner, conformational changes for Tau and amyloid β-peptide. Understanding the process at the molecular level is important for toxic aggregates could be stopped from forming or, alternatively, their removal could be accelerated. Antibodies directed against a common structural epitope shared by the prefibrillar oligomers could serve such a role. Common structural characteristics of amyloid fibrils are: predominantly β-sheet secondary structure, detected by specific dyes, and binding and a characteristic pattern seen by X-ray diffraction. By electron microscopy amyloigenic fibrils also are visualized allowing morphological studies (Zerovnik, 2010). In this chapter we focus on the importance of changes in

conformation of both proteins during abnormal aggregation associated to AD.

A growing body of evidence suggests that altered processing of amyloid precursor protein (APP) is one of the early events in the pathogenesis of AD. APP is a transmembrane glycoprotein of type 1 from 120 to 200 kD wich are ubiquitously expressed, but are most abundant in the brain (Selkoe, 1994). The APP gene is located on the long arm of chromosome 21 in humans (Kang 1987) and contains 18 exons (figure 1A) through alternative splicing of exons 7, 8 and 15 are generated 8 isoforms that have pattern cell specific expression and are designated by the number of amino acids they contain. The size of the isoforms can vary from 563 to 770 amino acids (Coulson et al., 2000). In central nervous system express only 4 isoforms: PPA695, PPA714, PPA751 and PPA770, PPA695 of which is the most abundant isoform in neurons, while that PPA751 and PPA770 isoforms are expressed mainly in glial cells (Yoshikai et al., 1990; Zheng &Koo, 2006). Only isoforms PPA695, PPA751 and PPA770 contains the sequence encoding the amyloid-β (AB) peptide (Golde et al., 1990; Kang et al., 1987). The full-length APP (APP 770) consists of 18 exons, where exon 17 resembles the membrane spanning domain. APP 695 lacking both, exon 7 and exon 8 is primarily expressed by neurons and is the most abundant APP transcript in the brain (Neve 1988). APP 751 (lacking exon 8), APP 714 (lacking exon 7) and APP 770 were first identified in peripheral organs but are also expressed in brain glial cells. Other alternatively generated splice variants of APP involving exon 15 were recently discovered in peripheral leucocytes and in microglial cells and therefore denoted as leukocyte- derived APP (L-APP) (Konig et al., 1992). The AB fragment is part of exon 16 and 17 (figure 1B) and begins 99 amino acid residues from the carboxy terminus and extends 12-15 residues into the hydrophobic membrane domain (Estus et al., 1992). The APP protein is composed of three regions: a large extracellular domain in the N-terminal region that forms a globular structure, a transmembrane region, which contains part of the AB sequence, and a small

(Braak &Braak, 1991).

**2. Amyloid β** 

cytoplasmic domain laregion C-terminal (figure 1B). The overall structure includes the position of a heparin binding domain, metal binding domains, sites of phosphorylation and glycosylation. Two of the isoforms (APP 751 and APP 770) have a domain Kunitz-type protease inhibior (KPI) and a signal peptide (De Strooper, 2010).

Fig. 1. Characteristics of APP and processing (A) Exon structure of human APP gene 23. Exons are indicated by rectangles. Alternatively spliced exons are 7, 8 and 15. Functional protein domains are indicated by different colors. Distances between exons are not representative. (B) Schematic representation of human APP protein including the relative position of the α-, β- and γ-secretase cleavage sites. KPI: Kunitz-type protease inhibitor domain, AICD: APP intracellular domain, YENPTY: motif that binds he phosphotyrosine binding (PTB) domain of X11. (C) Schematic diagram of APP processing pathways. APP proteolytic catabolism includes two different pathways: an amyloidogenic pathway and a non-amyloidogenic pathway (constitutive secretary pathway). The different APP fragments are generated after secretase cleavage.

In brain tissue, APP can be processed in two ways: non-amyloidogenic and amyloidogenic pathway. In the non-amyloidogenic pathway, APP is first cleaved by α-secretase within the AB sequence, which releases the sAPPα ectodomain. Further processing of the resulting carboxyl terminal by γ-secretase results in the release of the p3 fragment and AICD (figure 1C) (Greenwald &Riek, 2010). The most prevalent area of research in AD studies the proteolytic generation of AB from APP. The β-secretase and γ-secretase cleave APP in the so-called amyloidogenic pathway β-secretase release the ectodomain sAPPβ, and the

Tau and Amyloid-β Conformational Change to β-Sheet

residues were observed in crystal structures (Nerelius et al., 2010).

Also it has been suggested that the amphiphilicity of the peptide plays a significant role in determining whether peptide strands within the fibril are parallel or anti-parallel. Parallel β-

Structures as Effectors in the Development of Alzheimer's Disease 367

aggregation and eventually produce protofibrils fibrils, they acquire a conformation which β-pleated sheet. The AB peptide has a spontaneous tendency to oligomerization, forming toxic species which can even add intracellularly and therefore believed to play a role this fundamental neurotoxicity in AD (Klein et al., 2001; Murray et al., 2009). Recent experiments have established that the main structure adopted by these peptides depends on the environment. The monomers contain a amphipathic sequence that favors the α-helix structure in solution but preferred aqueous β-pleated structure (Greenwald &Riek, 2010). The structures of β-sheets are parallel or anti-parallel alignments of two or more β-stranded peptides that are held together by inter-strand hydrogen bonds. The β-sheet is not an unusual structure with more than 30% of the secondary structure of all proteins. The structure consisting of strands aligned in parallel feature twelve-membered hydrogen bonded rings, while those with strands in anti-parallel arrays are characterized by alternating 10- and 14-membered H-bonded rings. Dihedral angles commonly found in parallel (φ = –119°, ψ = 113°) and anti-parallel (φ = –139°, ψ = 135°) β-sheets are closer to those of the fully extended single-strand conformation (φ = ψ = ±180°) than β-turns or αhelices. The β-sheet usually acts as a scaffold to stabilize protein architecture, but it is also an important recognition motif in some protein–protein and protein–DNA interactions that mediate biological processes and some notable diseases (Harrison et al., 2007; Jenkins &Pickersgill, 2001). The peptide self-assembly to AB fibrils is a widespread phenomenon in nature; these structures are associated with protein aggregation pathologies such as AD. Therefore the structural analysis of AB fibrils is one of the most promising ways of revealing the mechanism involved in this event. Investigations showed a conserved evolutionarily protein motifs. The description of a common cross-β structure consists of β-strand peptides aligned in either parallel or antiparallel orientations to provide β-sheet structures that ultimately laminate to form fibrillar architectures. This process is mediated by noncovalent forces such as hydrophobic and hydrogen bonding interactions. However, the contribution of these forces is poorly understood (Bemporad et al., 2006; Tycko, 2011). Other critical interactions in cross-β peptide self-assembly are aromatic π-π, frequent in aromatic amino acids in the core of amyloid sequences, because mutations o deletions cause the loss of the structure (Bowerman et al., 2011; Marshall et al., 2011). Fibrils of AB42 and AB10-35 show a strong dependence of pH on morphology observed; at pH 7.4 protofilaments of AB42 exist singly and in pairs, but protofilaments of AB10-35 exist in pairs only. Studies on NMR indicate a parallel β-sheet organization on AB42 fibrils, consistent with supramolecular structures of other fibrils of AB10-35 and AB40. Although, there is a disagreement in the results obtained for fibrils formed by AB10-35 and AB40 with parallel organization, while the observations in AB42 fibril showed an antiparallel β-sheet structure. The explanation is that amyloid fibrils do not have a universal supramolecular organization. Also the results obtained of electronic microscopy and diffraction support a tubular structure for AB fibrils (Antzutkin et al., 2002). Analysis of the amyloidogenic regions in fibril-forming proteins showed a high occurrence of the aromatic residues phenylalanine and tyrosine, which have a high propensity to stack the delocalized π-electron rings in a parallel manner. Studies with short peptides have confirmed that even two consecutive phenylalanine residues are sufficient to facilitate assembly into nanotube-like structures, and stacks of aromatic

remaining APP carboxy-terminal fragment (C99) is subsequently cleaved by the γ-secretase liberating the secreted AB peptide(s) and the APP intracellular domain (AICD) (figure 1C). The biological functions of sAPPβ, AB, and the AICD remain rather elusive, although AB release is associated with synaptic activity, depression excitatory synaptic transmission onto neurons. The production of AB occurs naturally inside the human brain, these fragment possesses and amphiphilic structure with hydrophilic N- and hydrophobic C- terminus, however the C-terminal end is variable ranging at least from 37 to 42 residues; the most studied forms are AB 1-40 and AB 1-42 that consist of 40 and 42 residues respectively (Fandrich et al., 2011). Under physiological conditions, the ratio of AB42 to AB40 is about 1:10. AB42 plays a critical role in the pathogenesis of AD since its aggregative ability and neurotoxicity are much greater than those of AB40 (De Strooper, 2010).

About APP function, the analysis sequence and structure indicate that the protein is organized into different domains, because it is believed that APP is important for functions as neuronal survival, synaptogenesis, cell adhesion, inhibition of clotting factors, inhibiting platelet activation and in the modulation of copper homeostasis (Chow et al., 2010; De Strooper, 2010). The precise function of various APP isoforms in the brain is still unknown. It has been suggested that APP plays a role in establishing or maintaining cell-cell contacts which is also consistent with preferential localization of APP at nerve terminals, because have been shown their interaction with extracellular matrix proteins and heparin sulfate proteoglycans through E1 and E2 regions of APP (Schubert et al., 1995; Small et al., 1999). The function of APP as a cell surface receptor has been proposed for the evidence of AB could bind to APP and also the similarities of their secondary structure with Notch receptor (Zheng &Koo, 2011). About the neuronal survival and synaptogenesis functions of APP the reports showed that APP expression is upregulated during neuronal maturation and differentiation, also induced after traumatic brain injury, these results are reinforced because the reduction of APP is associated with neuronal impairment (Hung et al., 1992; Leyssen et al., 2005). The APP can be phosphorylated at multiple sites resulted in several consequence such as localization to the growth cones and neuritis (Muresan &Muresan, 2005). Other contrasting activity of soluble APP is the cytotoxic properties of C99 fragment as result of βsecretase cleaveage (Neve et al., 1996). Meanwhile, the functional properties of AB peptides have not been fully clarified to date, although numerous studies suggest that peptides have a number of neurotrophic and neurotoxic properties (Tycko, 2011). It is suggested that AB soluble plays an important role in neuronal growth, survival, and synaptic modulation, while the oligomers and fibrils have toxic properties (Kamenetz et al., 2003; Plant et al., 2003; Puzzo et al., 2008). Studies have shown that AB oligomers are able to induce increased cell death and apoptosis soluble or fibrillar forms, suggesting that the structural conformation peptide is important in determining its physiological action (Small et al., 2001).

#### **2.1 Amyloid β structure and aggregation**

Recent biophysical investigations using electron microscopy, Fourier Transform Infrared Resonance (FTIR) studies and Circular Dicroism (CD) spectroscopy showed that AB fibrils adopt a β-sheet structure (Serpell et al., 2000). However, a high-resolution structural analysis of AB fibrils has yet to be conducted since single crystal X-ray crystallography and solution nuclear magnetic resonance (NMR) cannot be applied to insoluble AB fibrils (Greenwald &Riek, 2010). AB peptides generated can exist as monomers, dimers and oligomers with

remaining APP carboxy-terminal fragment (C99) is subsequently cleaved by the γ-secretase liberating the secreted AB peptide(s) and the APP intracellular domain (AICD) (figure 1C). The biological functions of sAPPβ, AB, and the AICD remain rather elusive, although AB release is associated with synaptic activity, depression excitatory synaptic transmission onto neurons. The production of AB occurs naturally inside the human brain, these fragment possesses and amphiphilic structure with hydrophilic N- and hydrophobic C- terminus, however the C-terminal end is variable ranging at least from 37 to 42 residues; the most studied forms are AB 1-40 and AB 1-42 that consist of 40 and 42 residues respectively (Fandrich et al., 2011). Under physiological conditions, the ratio of AB42 to AB40 is about 1:10. AB42 plays a critical role in the pathogenesis of AD since its aggregative ability and

About APP function, the analysis sequence and structure indicate that the protein is organized into different domains, because it is believed that APP is important for functions as neuronal survival, synaptogenesis, cell adhesion, inhibition of clotting factors, inhibiting platelet activation and in the modulation of copper homeostasis (Chow et al., 2010; De Strooper, 2010). The precise function of various APP isoforms in the brain is still unknown. It has been suggested that APP plays a role in establishing or maintaining cell-cell contacts which is also consistent with preferential localization of APP at nerve terminals, because have been shown their interaction with extracellular matrix proteins and heparin sulfate proteoglycans through E1 and E2 regions of APP (Schubert et al., 1995; Small et al., 1999). The function of APP as a cell surface receptor has been proposed for the evidence of AB could bind to APP and also the similarities of their secondary structure with Notch receptor (Zheng &Koo, 2011). About the neuronal survival and synaptogenesis functions of APP the reports showed that APP expression is upregulated during neuronal maturation and differentiation, also induced after traumatic brain injury, these results are reinforced because the reduction of APP is associated with neuronal impairment (Hung et al., 1992; Leyssen et al., 2005). The APP can be phosphorylated at multiple sites resulted in several consequence such as localization to the growth cones and neuritis (Muresan &Muresan, 2005). Other contrasting activity of soluble APP is the cytotoxic properties of C99 fragment as result of βsecretase cleaveage (Neve et al., 1996). Meanwhile, the functional properties of AB peptides have not been fully clarified to date, although numerous studies suggest that peptides have a number of neurotrophic and neurotoxic properties (Tycko, 2011). It is suggested that AB soluble plays an important role in neuronal growth, survival, and synaptic modulation, while the oligomers and fibrils have toxic properties (Kamenetz et al., 2003; Plant et al., 2003; Puzzo et al., 2008). Studies have shown that AB oligomers are able to induce increased cell death and apoptosis soluble or fibrillar forms, suggesting that the structural conformation

neurotoxicity are much greater than those of AB40 (De Strooper, 2010).

peptide is important in determining its physiological action (Small et al., 2001).

Recent biophysical investigations using electron microscopy, Fourier Transform Infrared Resonance (FTIR) studies and Circular Dicroism (CD) spectroscopy showed that AB fibrils adopt a β-sheet structure (Serpell et al., 2000). However, a high-resolution structural analysis of AB fibrils has yet to be conducted since single crystal X-ray crystallography and solution nuclear magnetic resonance (NMR) cannot be applied to insoluble AB fibrils (Greenwald &Riek, 2010). AB peptides generated can exist as monomers, dimers and oligomers with

**2.1 Amyloid β structure and aggregation** 

aggregation and eventually produce protofibrils fibrils, they acquire a conformation which β-pleated sheet. The AB peptide has a spontaneous tendency to oligomerization, forming toxic species which can even add intracellularly and therefore believed to play a role this fundamental neurotoxicity in AD (Klein et al., 2001; Murray et al., 2009). Recent experiments have established that the main structure adopted by these peptides depends on the environment. The monomers contain a amphipathic sequence that favors the α-helix structure in solution but preferred aqueous β-pleated structure (Greenwald &Riek, 2010).

The structures of β-sheets are parallel or anti-parallel alignments of two or more β-stranded peptides that are held together by inter-strand hydrogen bonds. The β-sheet is not an unusual structure with more than 30% of the secondary structure of all proteins. The structure consisting of strands aligned in parallel feature twelve-membered hydrogen bonded rings, while those with strands in anti-parallel arrays are characterized by alternating 10- and 14-membered H-bonded rings. Dihedral angles commonly found in parallel (φ = –119°, ψ = 113°) and anti-parallel (φ = –139°, ψ = 135°) β-sheets are closer to those of the fully extended single-strand conformation (φ = ψ = ±180°) than β-turns or αhelices. The β-sheet usually acts as a scaffold to stabilize protein architecture, but it is also an important recognition motif in some protein–protein and protein–DNA interactions that mediate biological processes and some notable diseases (Harrison et al., 2007; Jenkins &Pickersgill, 2001). The peptide self-assembly to AB fibrils is a widespread phenomenon in nature; these structures are associated with protein aggregation pathologies such as AD. Therefore the structural analysis of AB fibrils is one of the most promising ways of revealing the mechanism involved in this event. Investigations showed a conserved evolutionarily protein motifs. The description of a common cross-β structure consists of β-strand peptides aligned in either parallel or antiparallel orientations to provide β-sheet structures that ultimately laminate to form fibrillar architectures. This process is mediated by noncovalent forces such as hydrophobic and hydrogen bonding interactions. However, the contribution of these forces is poorly understood (Bemporad et al., 2006; Tycko, 2011). Other critical interactions in cross-β peptide self-assembly are aromatic π-π, frequent in aromatic amino acids in the core of amyloid sequences, because mutations o deletions cause the loss of the structure (Bowerman et al., 2011; Marshall et al., 2011). Fibrils of AB42 and AB10-35 show a strong dependence of pH on morphology observed; at pH 7.4 protofilaments of AB42 exist singly and in pairs, but protofilaments of AB10-35 exist in pairs only. Studies on NMR indicate a parallel β-sheet organization on AB42 fibrils, consistent with supramolecular structures of other fibrils of AB10-35 and AB40. Although, there is a disagreement in the results obtained for fibrils formed by AB10-35 and AB40 with parallel organization, while the observations in AB42 fibril showed an antiparallel β-sheet structure. The explanation is that amyloid fibrils do not have a universal supramolecular organization. Also the results obtained of electronic microscopy and diffraction support a tubular structure for AB fibrils (Antzutkin et al., 2002). Analysis of the amyloidogenic regions in fibril-forming proteins showed a high occurrence of the aromatic residues phenylalanine and tyrosine, which have a high propensity to stack the delocalized π-electron rings in a parallel manner. Studies with short peptides have confirmed that even two consecutive phenylalanine residues are sufficient to facilitate assembly into nanotube-like structures, and stacks of aromatic residues were observed in crystal structures (Nerelius et al., 2010).

Also it has been suggested that the amphiphilicity of the peptide plays a significant role in determining whether peptide strands within the fibril are parallel or anti-parallel. Parallel β-

Tau and Amyloid-β Conformational Change to β-Sheet

Structures as Effectors in the Development of Alzheimer's Disease 369

width of 5–15 nm; the periodic twist often observed; and the conclusion that many amyloid fibers are made up of the bundling of thinner protofibrils. To better understand the higherorder assembly of amyloid fibers, scanning transmission electron microscopy (STEM) and tilted-beam transmission electron microscopy, both forms of electron microscopy, have provided data that describe the amount of mass-per-unit length (MPL) of amyloids. These data are particularly valuable in determining how many monomers make up a single layer of the fiber structure. AFM has also been used to determine the relative fiber rigidity by monitoring its propensity to bend. Electron microscopy and AFM both have the advantage of being single-fiber approaches, which are useful in visualizing structural heterogeneity

within a single-fiber preparation (Chen et al., 2009; Shirahama &Cohen, 1965).

Fig. 2. Characteristics of Amyloid β. (A) Structure diagram and amino acid sequence of AB42. (B) Amino acid distribution histogram of AB42. (C) Proposed AB42 structure based

Fiber X-ray diffraction was one of the first structural techniques that provided a substantial clue to the overall fold of amyloid fibers. In this method, fibrous samples are bombarded with X-ray radiation, and diffraction patterns result from interference patterns from any regularly spaced structural features present in the fibers. As alluded to above, this technique established the cross-β diffraction pattern interpreted as β-sheets parallel to the fiber axis, with β-strands perpendicular. It has also aided in testing the validity of particular model structures. Here, a theoretical Xray fiber diffraction pattern is calculated on the basis of a

on predicted β-strand propensities (Displayed in Jmol). (D) Electron Microscopy

photograph of *in vitro* AB42 aggregates.

sheets in proteins usually adjoin α-helices, suggesting that they are intrinsically less stable on their own than are anti-parallel β-sheets. The AB40 and AB10–35 peptides both have hydrophobic C-termini, and their reported assembly into parallel β-sheets allows the juxtaposition of the hydrophobic portions of the peptides, shielding them from aqueous solution. An anti-parallel disposition would be costly energetically because of the forced association of hydrophobic and hydrophilic regions. AB16–22 and AB34–42 both have a centrally located hydrophobic segment and thus no advantage of sequestration of hydrophobic regions provided by either a parallel or antiparallel arrangement. The antiparallel β-sheet arrangements found for these two peptides may then be the result of favorable charge interactions between side chains or termini, or improved H-bonding (Harrison et al., 2007). A model proposed for this structure is a parallel β-helix consisting of an extended polypeptide chain wrapping around a cylindrical template, where adjacent strands of the helix are connected through H-bonds and is based on two key features of the polyglutamine diffraction data; the absence of a 10-Å reflection and the presence of a weak, low-angle reflection of 31 Å (Perutz et al., 2002).

The crucial region for the formation of crossed-β fibrils is found in 15-23 residues (QKLVFFAED) (figure 2A), substitution of Thr for Phe19 can abolish plaque-forming competence of the mutant peptide and that the mutant peptide was significantly less folded in aqueous buffer than the wild-type peptide. Further, the Phe19 in the AB antiparallel βsheet is key in determining the properties of the fibril, because the observation that the Thr for Phe19 substitution lacks fibril-forming competence. Also the Lys16 in AB is positioned for electrostatic interaction with the charged sulfate groups of inductors of aggregation such as Congo Red (Carter &Chou, 1998). The KLVFFAED motif, a known amyloidogenic sequence of the AB, which contains positively and negatively charged residues (figure 2B) and has a high β-strand propensity (figure 2C). As mentioned above, minor alterations in sequence, such as replacement of Val or Phe with Leu or Ala, can abolish fibril formation and that the amyloidogenic properties of short peptides can be abolished by introduction of adjacent sequence motifs such as β-turns. These data indicate that both the amino acid sequence as such and its structural context affect the ability to form amyloid fibrils (Nerelius et al., 2010). In macromolecular level, aggregates are typically fibrillar in electron microscopy images generally with linear (figure 2D), unbranched fibrils of variable length. Each fibril is thought to consist of several protofilaments, the number being specific to the particular amyloid protein. Improved imaging of protofilaments has revealed that certain fibrils are clearly helical, with the protofilaments slowly twisting around each other. For Aβ34–42 fibrils, the twisted fibril "unwinds" under denaturing conditions and for Aβ40 at high pH, suggesting that these protofilaments are associated through both electrostatic and hydrophobic interactions (Fraser et al., 1992; Halverson et al., 1990). Amyloids are typically large (Megadalton) elongated structures with varying lengths and often varying ultrastructural appearances (Toyama &Weissman, 2011). Here we describe some of the most commonly used techniques employed in amyloid structure characterization. Electron microscopy and atomic force microscopy (AFM) are the two most widely used microscopy techniques employed in the study of amyloids. Both provide a nanometer-resolution perspective of the ultrastructural characteristics of amyloids. This includes amyloid fiber length and width, morphology such as curvature and persistence length, surface characteristics such as periodic twists, and higher-order assembly. Electron microscopy and AFM helped establish many of the common ultrastructural characteristics of amyloid fibers, such as the long, relatively straight and unbranched nature of the fibers; the typical fiber

sheets in proteins usually adjoin α-helices, suggesting that they are intrinsically less stable on their own than are anti-parallel β-sheets. The AB40 and AB10–35 peptides both have hydrophobic C-termini, and their reported assembly into parallel β-sheets allows the juxtaposition of the hydrophobic portions of the peptides, shielding them from aqueous solution. An anti-parallel disposition would be costly energetically because of the forced association of hydrophobic and hydrophilic regions. AB16–22 and AB34–42 both have a centrally located hydrophobic segment and thus no advantage of sequestration of hydrophobic regions provided by either a parallel or antiparallel arrangement. The antiparallel β-sheet arrangements found for these two peptides may then be the result of favorable charge interactions between side chains or termini, or improved H-bonding (Harrison et al., 2007). A model proposed for this structure is a parallel β-helix consisting of an extended polypeptide chain wrapping around a cylindrical template, where adjacent strands of the helix are connected through H-bonds and is based on two key features of the polyglutamine diffraction data; the absence of a 10-Å reflection and the presence of a weak,

The crucial region for the formation of crossed-β fibrils is found in 15-23 residues (QKLVFFAED) (figure 2A), substitution of Thr for Phe19 can abolish plaque-forming competence of the mutant peptide and that the mutant peptide was significantly less folded in aqueous buffer than the wild-type peptide. Further, the Phe19 in the AB antiparallel βsheet is key in determining the properties of the fibril, because the observation that the Thr for Phe19 substitution lacks fibril-forming competence. Also the Lys16 in AB is positioned for electrostatic interaction with the charged sulfate groups of inductors of aggregation such as Congo Red (Carter &Chou, 1998). The KLVFFAED motif, a known amyloidogenic sequence of the AB, which contains positively and negatively charged residues (figure 2B) and has a high β-strand propensity (figure 2C). As mentioned above, minor alterations in sequence, such as replacement of Val or Phe with Leu or Ala, can abolish fibril formation and that the amyloidogenic properties of short peptides can be abolished by introduction of adjacent sequence motifs such as β-turns. These data indicate that both the amino acid sequence as such and its structural context affect the ability to form amyloid fibrils (Nerelius et al., 2010). In macromolecular level, aggregates are typically fibrillar in electron microscopy images generally with linear (figure 2D), unbranched fibrils of variable length. Each fibril is thought to consist of several protofilaments, the number being specific to the particular amyloid protein. Improved imaging of protofilaments has revealed that certain fibrils are clearly helical, with the protofilaments slowly twisting around each other. For Aβ34–42 fibrils, the twisted fibril "unwinds" under denaturing conditions and for Aβ40 at high pH, suggesting that these protofilaments are associated through both electrostatic and hydrophobic interactions (Fraser et al., 1992; Halverson et al., 1990). Amyloids are typically large (Megadalton) elongated structures with varying lengths and often varying ultrastructural appearances (Toyama &Weissman, 2011). Here we describe some of the most commonly used techniques employed in amyloid structure characterization. Electron microscopy and atomic force microscopy (AFM) are the two most widely used microscopy techniques employed in the study of amyloids. Both provide a nanometer-resolution perspective of the ultrastructural characteristics of amyloids. This includes amyloid fiber length and width, morphology such as curvature and persistence length, surface characteristics such as periodic twists, and higher-order assembly. Electron microscopy and AFM helped establish many of the common ultrastructural characteristics of amyloid fibers, such as the long, relatively straight and unbranched nature of the fibers; the typical fiber

low-angle reflection of 31 Å (Perutz et al., 2002).

width of 5–15 nm; the periodic twist often observed; and the conclusion that many amyloid fibers are made up of the bundling of thinner protofibrils. To better understand the higherorder assembly of amyloid fibers, scanning transmission electron microscopy (STEM) and tilted-beam transmission electron microscopy, both forms of electron microscopy, have provided data that describe the amount of mass-per-unit length (MPL) of amyloids. These data are particularly valuable in determining how many monomers make up a single layer of the fiber structure. AFM has also been used to determine the relative fiber rigidity by monitoring its propensity to bend. Electron microscopy and AFM both have the advantage of being single-fiber approaches, which are useful in visualizing structural heterogeneity within a single-fiber preparation (Chen et al., 2009; Shirahama &Cohen, 1965).

Fig. 2. Characteristics of Amyloid β. (A) Structure diagram and amino acid sequence of AB42. (B) Amino acid distribution histogram of AB42. (C) Proposed AB42 structure based on predicted β-strand propensities (Displayed in Jmol). (D) Electron Microscopy photograph of *in vitro* AB42 aggregates.

Fiber X-ray diffraction was one of the first structural techniques that provided a substantial clue to the overall fold of amyloid fibers. In this method, fibrous samples are bombarded with X-ray radiation, and diffraction patterns result from interference patterns from any regularly spaced structural features present in the fibers. As alluded to above, this technique established the cross-β diffraction pattern interpreted as β-sheets parallel to the fiber axis, with β-strands perpendicular. It has also aided in testing the validity of particular model structures. Here, a theoretical Xray fiber diffraction pattern is calculated on the basis of a

Tau and Amyloid-β Conformational Change to β-Sheet

**3. Tau protein** 

Structures as Effectors in the Development of Alzheimer's Disease 371

mechanism. In cell culture models the exogenously applied AB in the form of oligomers can be trafficked on the neuronal membrane and accumulate in lipid rafts. The oligomers induced dynamic alterations in lipid raft protein composition were found to facilitate this movement. There is a clear association between AB accumulation and redistribution on the neuronal membrane and alterations in the protein composition of lipid rafts. Also the reports showed that fyn is a key protein on AB redistribution and accumulation in lipid rafts as and mediating the cell death induced by the AB oligomers and defines a mechanism by

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

which oligomers of AB and Tau accumulate in lipid rafts (Williamson et al., 2008).

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

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

mechanism. In cell culture models the exogenously applied AB in the form of oligomers can be trafficked on the neuronal membrane and accumulate in lipid rafts. The oligomers induced dynamic alterations in lipid raft protein composition were found to facilitate this movement. There is a clear association between AB accumulation and redistribution on the neuronal membrane and alterations in the protein composition of lipid rafts. Also the reports showed that fyn is a key protein on AB redistribution and accumulation in lipid rafts as and mediating the cell death induced by the AB oligomers and defines a mechanism by which oligomers of AB and Tau accumulate in lipid rafts (Williamson et al., 2008).
