**1. Introduction**

#### **1.1. Plaque and tangle pathology in AD**

Alzheimer's disease (AD] is the most common form of dementia, and it accounts for more than 60% of all cases of dementia. Although many factors may increase the risk for AD, the only cause so far known is aging [1]. Most of the cases are sporadic, as only less than 0.1% of the cases occur because of inherited mutations on genes directly involved in the disease (familial AD, FAD] [2].

AD is caused by progressive and irreversible neurodegeneration. At the moment, there is no cure for AD. Therapies available are only aimed at lessening the progression of the cognitive decline and neurodegeneration and do not target pathways directly causative of the disease [3]. These include the acetylcholinesterase inhibitors (Aricept] [4] or inhibitors of the gluta‐ matergic NMDA receptor (Namenda] [5] and were shown to be mostly effective when administered at early stages [6-8]. However, a proper diagnostic approach able to identify AD early in the development is still missing, and this reduces the efficacy of the treatments available. Therefore, there is the need to develop both diagnostic tools able to detect early stages of the disease, and to generate effective treatments targeting the early pathogenic events in AD. This is becoming increasingly important also considering that the population affected by AD will dramatically increase in the years to come. Numbers are in fact dramatic: 10 million baby boomers may develop AD within the next 10-20 years [9]. Currently, in the United States alone there are more than 5 million AD patients, and the costs to the US government exceeds the 200 billion/year.These numbers are expected to quadruple in the next 40 years, causing unsustainable costs for the care of these patients and their caregivers, who could not receive support and care and would then have to face undignified life conditions.

© 2013 Pastorino et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Studying the molecular mechanisms responsible for the neurodegeneration in AD can help identify new effective therapeutic targets. Two main pathways are identified in AD. They involve two proteins, the amyloid precursor protein APP and the microtubule-associated protein tau, as they are responsible for the formation of the two characteristic lesions, the extracellular plaques and the intracellular neurofibrillary tangles (NFTs], respectively [10, 11]. Both plaques and tangles are considered causative of the disease; they deposit following the progression of the disease, and they could contribute to alter neuronal morphology leading to neuronal death [12-16].

during the disease [23, 37-40]. Hence, the identification of molecular pathways that can control non physiologic phosphorylation of both tau and APP in the disease could help identify targets

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We found that the enzyme Pin1 protects from both tangle and Abeta pathology, since a genetically modified animal model lacking Pin1 (Pin1KO] developed age-dependent tauop‐ athy and was characterized by increased production of Abeta, deposited in form of intracel‐ lular aggregates [41, 42]. This seems to be due to the capability of Pin1 to regulate the conformation of cis and trans isomers of both phosphorylated tau and APP, as shown using

Pin1 (Protein interacting with NIMA (never in mitosis A]-1] is a prolyl isomerase, which regulates the function of phosphorylated protein substrates by regulating their cis/trans isomerization [43, 44]. Pin1 belongs to the family of PPIase (peptidyl prolyl cis trans isomerase], enzymes that are evolutionary conserved. Unlike other PPIases, Pin1 specifically regulates the conformation of substrates phosphorylated at specific serine or threonine residues preceding a proline (S/T-P motifs] [45-47]. The stereochemistry of Proline allows the protein to undergo two different conformations (cis and trans], which could be determined by the presence of a phospho group on the S or T residue [43, 48]. Since Proline-directed phosphorylation regulates key cellular mechanisms, by maintaining the equilibrium between the two conformations, Pin1

The structure of Pin1 consists of two domains, an N-terminal WW domain comprised of the first 40 aminoacids which is responsible for Pin1 binding to its substrates, and a larger PPIase domain that spans the remaining part of the protein and catalyzes the substrate's isomerization [49]. Of note, although mostly in the nucleus, Pin1 subcellular localization is driven by the presence of its substrates [50], to extranuclear compartments, with obvious expression in the plasma membrane, cytosol and cytosolic organelles involved in endocytosis [41, 51]. The ubiquitous expression of Pin1 allows the protein to control the isomerization of multiple substrates in different cellular compartments, including cytosolic proteins like NF-KappaB [52], p53 [53], beta-catenin [54], IRAK1 [55] and others [46], or protein that localize at different compartments like APP [41, 51] and tau [42, 56]. This determines a crucial role for Pin1 in controlling the physiological activity of proteins involved in diverse functions, such as protein transcription and stability, and protein interaction, by regulating the aforementioned sub‐

Notably, Pin1 function is highly regulated and its aberration affects Pin1's ability to isomerize its substrates with consequences on their function, contributing to an increasing number of pathological conditions, including Alzheimer's disease, cancer and immunologic disorders and aging. Lack of Pin1 function was found to impair immune responses in Pin1KO animal models [55], due to lack of activation of IRAK1, which is involved in the regulation of the TLR signaling [57]. In cancer, Pin1 levels are increased due to transcriptional activation and loss of inhibitory phosphorylation and other mechanisms [45, 58]. This leads to up-regulated isomerization of substrates involved in hyperproliferative processes, activating two dozens of oncogenes and inactivating a dozen of tumor suppressors [46, 59, 60]. On the contrary, in AD brain Pin1 activity is reduced due to decreased protein level and to oxidation [56, 61, 62]. Some

to tackle at the same time both tangle and plaque pathologies.

conformation specific antibodies for tau, and by means of NMR.

may dramatically contribute to the maintenance of vital cellular functions.

strates [43].

The origin and composition of plaques and tangles are quite different. Plaques are forms of aggregated, fibrillar material called amyloid, insoluble fibrous protein aggregations organized in β-sheet strands that deposits in the outer part of the brain [17-19]. Their core is mainly composed of Abeta (beta-amyloid], a peptide of small molecular weight deriving from APP, which tends to form small size aggregates called oligomers with known toxic properties [20]. Oligomers are found intracellularly, but can be secreted to the extracellular space, where they will aggregate into larger structures called fibrils, forming the core of the plaque [18, 21, 22].

Similarly, tangles are formed by insoluble structures organized into fibrils, the pair helical filaments (PHFs], which eventually organize and aggregate into larger structures, the tangles [19]. The main component is hyperphosphorylated protein tau, which in this form becomes insoluble and tends to form aggregates [13, 23].

The biological functions of APP and tau are very different [13, 24], but during the disease both the beta amyloid product and the hyperphosphorylated tau become toxic to the neuron, causing neurodegeneration. However, the mechanisms by which tau and Abeta may be toxic differ. In fact, as a microtubule stabilizing protein, tau can become toxic to the cytoskeleton when hyperphosphorylated, as in this form it would detach from the microtubules destabi‐ lizing them. Hyperphosphorylated tau would also tend to aggregate into NFTs, impairing cellular functions [23]. As to the plaques, their mechanism of toxicity is still under debate. Although they cause the formation of dystrophic neuritis [18], it is still unclear whether they are really toxic or rather protecting, by sequestering Abeta oligomers from the environment. In facts, Abeta is sequestered from the extracellular space to form the plaque [25]. Indeed, oligomers are considered toxic: they form early in the pathology [26], associate with impaired cognitive functions in mice [27] and in AD patients [28], and impair neurotransmission [29-33]. Therefore, identifying the pathways that lead to both increased Abeta production and/or tau hyperphosphorylation and also regulate their aggregation into organized insoluble structure may dramatically help find a cure to treat AD.

#### **1.2. Pin1–regulated protein isomerization as a mechanism to control tangle and plaque pathologies**

Protein phosphorylation seems to be a common feature of both plaque and tangle pathologies. In fact, changes in the levels of phosphorylated APP seems to influence APP function and toxicity in the pathology, as increased phosphorylation of APP at specific domains positively regulates Abeta production [34-36]. Of note, both APP and tau can be phosphorylated by the same kinases, such as cdc2, CDK5 and GSK3, and such kinases seem to be particularly active during the disease [23, 37-40]. Hence, the identification of molecular pathways that can control non physiologic phosphorylation of both tau and APP in the disease could help identify targets to tackle at the same time both tangle and plaque pathologies.

Studying the molecular mechanisms responsible for the neurodegeneration in AD can help identify new effective therapeutic targets. Two main pathways are identified in AD. They involve two proteins, the amyloid precursor protein APP and the microtubule-associated protein tau, as they are responsible for the formation of the two characteristic lesions, the extracellular plaques and the intracellular neurofibrillary tangles (NFTs], respectively [10, 11]. Both plaques and tangles are considered causative of the disease; they deposit following the progression of the disease, and they could contribute to alter neuronal morphology leading to

The origin and composition of plaques and tangles are quite different. Plaques are forms of aggregated, fibrillar material called amyloid, insoluble fibrous protein aggregations organized in β-sheet strands that deposits in the outer part of the brain [17-19]. Their core is mainly composed of Abeta (beta-amyloid], a peptide of small molecular weight deriving from APP, which tends to form small size aggregates called oligomers with known toxic properties [20]. Oligomers are found intracellularly, but can be secreted to the extracellular space, where they will aggregate into larger structures called fibrils, forming the core of the plaque [18, 21, 22]. Similarly, tangles are formed by insoluble structures organized into fibrils, the pair helical filaments (PHFs], which eventually organize and aggregate into larger structures, the tangles [19]. The main component is hyperphosphorylated protein tau, which in this form becomes

The biological functions of APP and tau are very different [13, 24], but during the disease both the beta amyloid product and the hyperphosphorylated tau become toxic to the neuron, causing neurodegeneration. However, the mechanisms by which tau and Abeta may be toxic differ. In fact, as a microtubule stabilizing protein, tau can become toxic to the cytoskeleton when hyperphosphorylated, as in this form it would detach from the microtubules destabi‐ lizing them. Hyperphosphorylated tau would also tend to aggregate into NFTs, impairing cellular functions [23]. As to the plaques, their mechanism of toxicity is still under debate. Although they cause the formation of dystrophic neuritis [18], it is still unclear whether they are really toxic or rather protecting, by sequestering Abeta oligomers from the environment. In facts, Abeta is sequestered from the extracellular space to form the plaque [25]. Indeed, oligomers are considered toxic: they form early in the pathology [26], associate with impaired cognitive functions in mice [27] and in AD patients [28], and impair neurotransmission [29-33]. Therefore, identifying the pathways that lead to both increased Abeta production and/or tau hyperphosphorylation and also regulate their aggregation into organized insoluble structure

**1.2. Pin1–regulated protein isomerization as a mechanism to control tangle and plaque**

Protein phosphorylation seems to be a common feature of both plaque and tangle pathologies. In fact, changes in the levels of phosphorylated APP seems to influence APP function and toxicity in the pathology, as increased phosphorylation of APP at specific domains positively regulates Abeta production [34-36]. Of note, both APP and tau can be phosphorylated by the same kinases, such as cdc2, CDK5 and GSK3, and such kinases seem to be particularly active

neuronal death [12-16].

110 Understanding Alzheimer's Disease

insoluble and tends to form aggregates [13, 23].

may dramatically help find a cure to treat AD.

**pathologies**

We found that the enzyme Pin1 protects from both tangle and Abeta pathology, since a genetically modified animal model lacking Pin1 (Pin1KO] developed age-dependent tauop‐ athy and was characterized by increased production of Abeta, deposited in form of intracel‐ lular aggregates [41, 42]. This seems to be due to the capability of Pin1 to regulate the conformation of cis and trans isomers of both phosphorylated tau and APP, as shown using conformation specific antibodies for tau, and by means of NMR.

Pin1 (Protein interacting with NIMA (never in mitosis A]-1] is a prolyl isomerase, which regulates the function of phosphorylated protein substrates by regulating their cis/trans isomerization [43, 44]. Pin1 belongs to the family of PPIase (peptidyl prolyl cis trans isomerase], enzymes that are evolutionary conserved. Unlike other PPIases, Pin1 specifically regulates the conformation of substrates phosphorylated at specific serine or threonine residues preceding a proline (S/T-P motifs] [45-47]. The stereochemistry of Proline allows the protein to undergo two different conformations (cis and trans], which could be determined by the presence of a phospho group on the S or T residue [43, 48]. Since Proline-directed phosphorylation regulates key cellular mechanisms, by maintaining the equilibrium between the two conformations, Pin1 may dramatically contribute to the maintenance of vital cellular functions.

The structure of Pin1 consists of two domains, an N-terminal WW domain comprised of the first 40 aminoacids which is responsible for Pin1 binding to its substrates, and a larger PPIase domain that spans the remaining part of the protein and catalyzes the substrate's isomerization [49]. Of note, although mostly in the nucleus, Pin1 subcellular localization is driven by the presence of its substrates [50], to extranuclear compartments, with obvious expression in the plasma membrane, cytosol and cytosolic organelles involved in endocytosis [41, 51]. The ubiquitous expression of Pin1 allows the protein to control the isomerization of multiple substrates in different cellular compartments, including cytosolic proteins like NF-KappaB [52], p53 [53], beta-catenin [54], IRAK1 [55] and others [46], or protein that localize at different compartments like APP [41, 51] and tau [42, 56]. This determines a crucial role for Pin1 in controlling the physiological activity of proteins involved in diverse functions, such as protein transcription and stability, and protein interaction, by regulating the aforementioned sub‐ strates [43].

Notably, Pin1 function is highly regulated and its aberration affects Pin1's ability to isomerize its substrates with consequences on their function, contributing to an increasing number of pathological conditions, including Alzheimer's disease, cancer and immunologic disorders and aging. Lack of Pin1 function was found to impair immune responses in Pin1KO animal models [55], due to lack of activation of IRAK1, which is involved in the regulation of the TLR signaling [57]. In cancer, Pin1 levels are increased due to transcriptional activation and loss of inhibitory phosphorylation and other mechanisms [45, 58]. This leads to up-regulated isomerization of substrates involved in hyperproliferative processes, activating two dozens of oncogenes and inactivating a dozen of tumor suppressors [46, 59, 60]. On the contrary, in AD brain Pin1 activity is reduced due to decreased protein level and to oxidation [56, 61, 62]. Some genetic polymorphisms on the Pin1 gene were found to associate with forms of late onset AD [63-65]. Interestingly, a polymorphism that associated with increased Pin1 levels by regulating AP-4 mediated transcription, was found to be protective as it correlates with delayed disease onset in a Chinese cohort [66]. In AD, the changes in Pin1 levels and activity prevent from an effective isomerization of the phosphorylated APP and tau [41, 56]. As a consequence, the equilibrium between the cis and trans conformation is not maintained and the proteins exist in the pathogenic cis conformation: APP will generate more Abeta and tau will lose normal microtubule function and become toxic, leading to both plaque and tangle pathologies.

portion of the trasmembrane domain [71]. Three isoforms of APP exists characterized by different molecular weight, the result of alternative RNA splicing, APP751, APP750 and APP695 [72]. Since the splicing occurs in the most N-terminal region of the protein, all the three isoforms express the domains for both Abeta and the intracellular domain [72]. APP isoforms may be differently expressed in the various organs. AP770 is for example mostly present in the heart and in peripheral cells, whereas APP695 is the only form expressed in the brain and therefore linked to Abeta generation in AD [72]. For this reason, the APP isoform considered in AD studies is APP695, and the numbering of the aminoacids follows this sequence.

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Within the cell, APP localization is not limited to a single part, as it undergoes trafficking through different compartments. Upon synthesis in the ER, APP travels through the Golgi compartment where it undergoes glycosylation, to finally reach the plasma membrane. It eventually will recycle to the Golgi, following internalization from the plasma membrane and trafficking through the endosomal pathway [70, 73, 74]. Of note, the significance of APP physiological function may depend on the compartment where APP localizes during the life of the cell. In fact, depending on whether APP is retained at the plasma membrane or it is internalized to the endosomes, it will generate different metabolites with diverse function, either neurotrophic and therefore protective from AD, or toxic. More in details, at the plasma membrane APP will undergo a processing pathway called non-amyloidogenic [75, 76], in which metalloproteases of the ADAMs family and others (ADAM10, ADAM17 and TACE [77-81]], called alpha secretase, will cleave APP in the middle of the sequence for Abeta, generating the extracellular stub alphaAPPs with known neurotrophic properties [82], and a C-terminal stub called C83. C83 will be further cleaved in the late endosomes by a complex of four proteins called gamma-secretase, to generate a small fragment called p3 with no amyloi‐ dogenic properties. This pathway is called non-amyloidogenic, as it prevents the formation of intact Abeta peptides. The amount of APP at the plasma membrane that does not undergo alpha-secretase cleavage will internalize in the cell through the endocytic pathway [70, 73, 74]. This occurs thanks to the binding of proteins such as Fe65 to the 682YNPTY687 motif at the intracellular, C-terminal domain of APP [83-85]. Once in the early endosomes, full length APP is cleaved by BACE or beta secretase [86, 87], an aspartyl protease that cuts APP at the beginning of the sequence for Abeta. Such cleavage generates a soluble stub called betaAPPs with known apoptotic properties in the neuron [88], and a C-terminal stub called C99 which still contains the intact sequence for Abeta. C99 will traffic to the late endosomes, where it will be cleaved by gamma-secretase to generate intact Abeta [17, 89]. This pathway is called

amyloidogenic as it produces Abeta peptides, and is increased in AD [90, 91].

**2.2. APP phosphorylation and conformation to regulate APP processing**

It is clear that the intracellular localization of APP will determine whether APP will be amyloidogenic or not. Therefore, any mechanism that may help APP stay retained at the plasma membrane will protect from Abeta production and AD, whereas those that help APP internalize to the endosomes will favor the amyloidogenic processing and Abeta formation.

One such mechanism is APP phosphorylation. In fact, it was shown that the Y682 residue can be phosphorylated by different kinases such as abl and TrkA [92, 93]. Phosphorylation at this

In this book chapter we will discuss findings from our and other labs that point to a crucial role of Pin1 in protecting against AD by regulating diverse cellular pathways using multiple mechanisms. We will specifically highlight how Pin1 regulates protein conformation of APP and tau to control APP trafficking, APP stability and Abeta production as well as tau phos‐ phorylation, microtubule function, stabilization and aggregation in vivo and in vitro. We will also emphasize the importance of Pin1-mediated regulation of APP and tau conformation as a modulator of pathogenic mechanisms that might occur early in the development of the disease. Finally, we will also discuss how Pin1 is emerging as a novel diagnostic and thera‐ peutic tool for early intervention to tackle both tau and Abeta pathologies in AD.
