**2. Pin1 as a crucial regulator of APP trafficking and stabilization to protect from Abeta pathology in AD**

Although both tau and Abeta pathologies define AD, only Abeta is the characteristic feature that distinguishes AD from other forms of dementia. In fact, only the presence of plaques containing AbetapeptideallowsadefiniteADdiagnosis[10,67-69],whereasthepresenceofPHFalonecould be related also to other forms of tauopathies, like FTPD, Pick disease and others [13]. The specific‐ ityofAbetapathologytoADmakesofAbetaanditsprecursorAPPidealtherapeutictargets.Here we will review the role of APP in AD, the molecular mechanisms that regulate Abeta formation, focusing on the role of Pin1 as a post-phosphorylative event to regulate both APP intracellular localization and trafficking, and also turnover, preventing Abeta formation. These topics are of particular relevance for the understanding of the mechanisms underlying Abeta production in AD. In fact, the intracellular localization of APP will determine whether APP will be toxic influencing the production of beta-amyloid peptides. Moreover, impaired APP turnover will cause APP stabilization, which will lead to increased levels of both APP and beta-amyloid peptides. Thisphenomenon is particularly consistent with pathologies associated with higher levels of APP and development of AD, such as Down syndrome.

#### **2.1. APP trafficking and processing pathways**

APP is a type 1 transmembrane protein that is ubiquitously expressed. APP is characterized by a long extracellular domain, a short transmembrane domain and a small intracellular domain that regulates APP phosphorylation and trafficking [68, 70]. The domain that contains the sequence for Abeta spans a region of approximately 40 aminoacids across the N-terminal 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.

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.

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‐

**2. Pin1 as a crucial regulator of APP trafficking and stabilization to protect**

Although both tau and Abeta pathologies define AD, only Abeta is the characteristic feature that distinguishes AD from other forms of dementia. In fact, only the presence of plaques containing AbetapeptideallowsadefiniteADdiagnosis[10,67-69],whereasthepresenceofPHFalonecould be related also to other forms of tauopathies, like FTPD, Pick disease and others [13]. The specific‐ ityofAbetapathologytoADmakesofAbetaanditsprecursorAPPidealtherapeutictargets.Here we will review the role of APP in AD, the molecular mechanisms that regulate Abeta formation, focusing on the role of Pin1 as a post-phosphorylative event to regulate both APP intracellular localization and trafficking, and also turnover, preventing Abeta formation. These topics are of particular relevance for the understanding of the mechanisms underlying Abeta production in AD. In fact, the intracellular localization of APP will determine whether APP will be toxic influencing the production of beta-amyloid peptides. Moreover, impaired APP turnover will cause APP stabilization, which will lead to increased levels of both APP and beta-amyloid peptides. Thisphenomenon is particularly consistent with pathologies associated with higher

APP is a type 1 transmembrane protein that is ubiquitously expressed. APP is characterized by a long extracellular domain, a short transmembrane domain and a small intracellular domain that regulates APP phosphorylation and trafficking [68, 70]. The domain that contains the sequence for Abeta spans a region of approximately 40 aminoacids across the N-terminal

peutic tool for early intervention to tackle both tau and Abeta pathologies in AD.

levels of APP and development of AD, such as Down syndrome.

**2.1. APP trafficking and processing pathways**

**from Abeta pathology in AD**

112 Understanding Alzheimer's Disease

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].

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.

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

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 level can regulate the association of APP to binding partners such as Fe65, X11/MINTs and Shc [94-97], ultimately controlling APP trafficking, processing and function also associated with cell movement and axonal branching [98, 99], and with NGF activity [100]. Tyr phosphoryla‐ tion at Y682 motif has also been associated with increased Abeta production and amyloido‐ genic processing in vitro [101], in vivo [102] and in AD [103].

Pin1 were elevated beyond physiologic, APP amyloidogenic processing would be reduced, as Abeta levels were decreased in the media of the cultured cells. On the contrary, lack of Pin1 expression in cultured Pin1KO breast cancer cells resulted in decreased alphaAPPs secretion and increased Abeta production. Similarly, in the brain of Pin1KO mice we could observe agedependent increase of Abeta production, since levels of aggregated insoluble Abeta were

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We then crossed PinKO animals to APPtg2576 and studied the processing of APP. We observed an age-dependent shift in the processing of APP that would result in an increase of the amyloidogenic versus the non-amyloidogenic, paralleled by the accumulation of Abeta42 deposits in multivesicular bodies, a form of deposited Abeta associated with early stages of AD [32, 33]. This led us to hypothesize a model in which Pin1 would protect against neuro‐ degeneration possibly by maintaining the equilibrium between the cis and the trans confor‐ mation of APP. In particular, in physiological conditions, Pin1 would favor the trans conformation of APP, increasing the non-amylodogenic processing. Vice versa in the absence of Pin1, the cis form would accumulate as the isomerization between the two forms would be

Because Pin1 was found to localize with full length APP at the plasma membrane, we specu‐ lated that Pin1 may be involved in APP trafficking and internalization, regulating the amount of APP that undergoes amyloidogenic processing. We therefore tested the hypothesis whether Pin1 protects from Abeta formation by inhibiting APP internalization to amyloidogenic compartments [51]. For this purpose we used brain derived human H4 neuroglioma cells expressing APP either at endogenous level or stably overexpressing it, and Pin1 expression was knocked down by RNAi. We found that lower Pin1 levels associated with i) decreased levels of APP at the plasma membrane, ii) increased levels of betaAPPs and decreased alphaAPPs and iii) increased kinetic of internalization, as evidenced by means of immunocy‐ tochemistry in both fixed and living cells [51]. Levels of APP phosphoryated at T668 seemed to be elevated too. These data are in agreement with data from other groups that propose a toxic role of T688 phosphorylated APP [34], and may suggest that reduced Pin1 levels could be toxic in the same pathways. Interestingly, Ando and colleagues suggested that phosphor‐ ylation at T668 may affect APP conformation to ultimately alter the capability of APP to bind to partners such as Fe65 regulating APP trafficking, even if such interaction occurs at the 682YNPTY687 domain, further C-terminal than T668 [106]. This effect could be related to Pin1 mediated changes in APP conformation that could change the 682YNPTY687 stereochemistry. Of note, in Pin1 KD treated cells that were also overexpressing Fe65, we found that higher amounts of Fe65 associated with APP and that C99 accumulated, as compared to wild type cells. This was probably due to stabilization of Fe65 under these conditions, since Fe65 levels were elevated at the steady state in Pin1KD cells. Together with our immunocytochemistry data, under conditions that promote Fe65/APP interaction, these results suggest that reduced Pin1 expression may be linked to fastened internalization of APP to amyloidogenic compart‐

elevated in 18 months old mice when compared to 5 months old mice.

lost, ultimately favoring the amyloidogenic processing.

**2.4. Pin1 inhibits APP trafficking and internalization**

ments, where C99 is produced and accumulates (Fig. 1).

Interestingly, APP can be phosphorylated at a further N-terminal part of the intracellular domain, the 668Thr-669Pro residue [104], and phosphorylation at this domain has been associated with increased amyloidogenic processing of APP, both in vivo [39, 40] and in vitro [34]. The kinases involved in such phosphorylation are GSK3beta, CDK5, cdc2, known to be overactive in AD and responsible also for tau phosphorylation [23, 38, 39, 104, 105]. Of note, T668 phosphorylation was found to be elevated in AD brain [34], suggesting that it might induce toxic mechanisms linked to Abeta production. Such mechanisms seemed to relate to conformational changes affecting the 682YNPTY687 motif and therefore its ability to interact with binding partners [106, 107]. In support of this hypothesis, T668 has been linked to specific isomer formation. In fact, by means of NMR studies, it was observed that phosphorylation at the Thr668 residue causes an isomerization of APP from trans to cis. In fact, non-phosphory‐ lated APP retains 100% trans conformation, and upon phosphorylation at T668 approximately 10% of the population turns to cis [108, 109].

Altogether, these findings draw attention to the role of T668 phosphorylation as an initiator of molecular pathways that lead to Abeta production by regulating APP conformation, trafficking and processing. They also suggest that different cis and trans APP isomers may contribute to shift the processing of APP towards either the amyloidogenic or the nonamyloidogenic processing, and therefore T668 phosphorylation may emerge as a potential target to halt amyloidogenic pathways in AD.

#### **2.3. Pin1 to protect from Abeta pathology in animal models**

Based on these findings and on the capability of Pin1 to protect from tau pathology by regulating tau conformation [42, 56], we hypothesized that Pin1 might regulate also the conformation of APP protecting from Abeta pathology. We found that Pin1 can bind to phosphorylated APP at T668, maintains the equilibrium between cis and trans conformation, ultimately shifting the processing of APP from the toxic amyloidogenic to the protective nonamyloidogenic [41].

More in detail, by means of pull down experiments, we observed that Pin1 can bind to APP only if phosphorylated at T668 [41]. Such interaction regulates APP isomerization. In fact, using a pentapetide containing part of the C-terminal domain and the T668-P motif, we observed that Pin1 isomerizes the conformation of this peptide from cis to trans 1000 times faster than the reversed equilibrium, suggesting that shifting the isomerization towards the trans conformation may be crucial for APP function, and that Pin1 might be key to regulate APP physiologic activity. We then tested whether altering the equilibrium between cis and trans conformation might result in changes of APP functions. For this purpose, we manipulated Pin1 cellular levels either by knocking Pin1 out in genetically modified animals (Pin1KO], or by overexpressing Pin1 in cultured cells. Our in vitro experiments showed that when levels of Pin1 were elevated beyond physiologic, APP amyloidogenic processing would be reduced, as Abeta levels were decreased in the media of the cultured cells. On the contrary, lack of Pin1 expression in cultured Pin1KO breast cancer cells resulted in decreased alphaAPPs secretion and increased Abeta production. Similarly, in the brain of Pin1KO mice we could observe agedependent increase of Abeta production, since levels of aggregated insoluble Abeta were elevated in 18 months old mice when compared to 5 months old mice.

We then crossed PinKO animals to APPtg2576 and studied the processing of APP. We observed an age-dependent shift in the processing of APP that would result in an increase of the amyloidogenic versus the non-amyloidogenic, paralleled by the accumulation of Abeta42 deposits in multivesicular bodies, a form of deposited Abeta associated with early stages of AD [32, 33]. This led us to hypothesize a model in which Pin1 would protect against neuro‐ degeneration possibly by maintaining the equilibrium between the cis and the trans confor‐ mation of APP. In particular, in physiological conditions, Pin1 would favor the trans conformation of APP, increasing the non-amylodogenic processing. Vice versa in the absence of Pin1, the cis form would accumulate as the isomerization between the two forms would be lost, ultimately favoring the amyloidogenic processing.

#### **2.4. Pin1 inhibits APP trafficking and internalization**

level can regulate the association of APP to binding partners such as Fe65, X11/MINTs and Shc [94-97], ultimately controlling APP trafficking, processing and function also associated with cell movement and axonal branching [98, 99], and with NGF activity [100]. Tyr phosphoryla‐ tion at Y682 motif has also been associated with increased Abeta production and amyloido‐

Interestingly, APP can be phosphorylated at a further N-terminal part of the intracellular domain, the 668Thr-669Pro residue [104], and phosphorylation at this domain has been associated with increased amyloidogenic processing of APP, both in vivo [39, 40] and in vitro [34]. The kinases involved in such phosphorylation are GSK3beta, CDK5, cdc2, known to be overactive in AD and responsible also for tau phosphorylation [23, 38, 39, 104, 105]. Of note, T668 phosphorylation was found to be elevated in AD brain [34], suggesting that it might induce toxic mechanisms linked to Abeta production. Such mechanisms seemed to relate to conformational changes affecting the 682YNPTY687 motif and therefore its ability to interact with binding partners [106, 107]. In support of this hypothesis, T668 has been linked to specific isomer formation. In fact, by means of NMR studies, it was observed that phosphorylation at the Thr668 residue causes an isomerization of APP from trans to cis. In fact, non-phosphory‐ lated APP retains 100% trans conformation, and upon phosphorylation at T668 approximately

Altogether, these findings draw attention to the role of T668 phosphorylation as an initiator of molecular pathways that lead to Abeta production by regulating APP conformation, trafficking and processing. They also suggest that different cis and trans APP isomers may contribute to shift the processing of APP towards either the amyloidogenic or the nonamyloidogenic processing, and therefore T668 phosphorylation may emerge as a potential

Based on these findings and on the capability of Pin1 to protect from tau pathology by regulating tau conformation [42, 56], we hypothesized that Pin1 might regulate also the conformation of APP protecting from Abeta pathology. We found that Pin1 can bind to phosphorylated APP at T668, maintains the equilibrium between cis and trans conformation, ultimately shifting the processing of APP from the toxic amyloidogenic to the protective non-

More in detail, by means of pull down experiments, we observed that Pin1 can bind to APP only if phosphorylated at T668 [41]. Such interaction regulates APP isomerization. In fact, using a pentapetide containing part of the C-terminal domain and the T668-P motif, we observed that Pin1 isomerizes the conformation of this peptide from cis to trans 1000 times faster than the reversed equilibrium, suggesting that shifting the isomerization towards the trans conformation may be crucial for APP function, and that Pin1 might be key to regulate APP physiologic activity. We then tested whether altering the equilibrium between cis and trans conformation might result in changes of APP functions. For this purpose, we manipulated Pin1 cellular levels either by knocking Pin1 out in genetically modified animals (Pin1KO], or by overexpressing Pin1 in cultured cells. Our in vitro experiments showed that when levels of

genic processing in vitro [101], in vivo [102] and in AD [103].

10% of the population turns to cis [108, 109].

target to halt amyloidogenic pathways in AD.

amyloidogenic [41].

114 Understanding Alzheimer's Disease

**2.3. Pin1 to protect from Abeta pathology in animal models**

Because Pin1 was found to localize with full length APP at the plasma membrane, we specu‐ lated that Pin1 may be involved in APP trafficking and internalization, regulating the amount of APP that undergoes amyloidogenic processing. We therefore tested the hypothesis whether Pin1 protects from Abeta formation by inhibiting APP internalization to amyloidogenic compartments [51]. For this purpose we used brain derived human H4 neuroglioma cells expressing APP either at endogenous level or stably overexpressing it, and Pin1 expression was knocked down by RNAi. We found that lower Pin1 levels associated with i) decreased levels of APP at the plasma membrane, ii) increased levels of betaAPPs and decreased alphaAPPs and iii) increased kinetic of internalization, as evidenced by means of immunocy‐ tochemistry in both fixed and living cells [51]. Levels of APP phosphoryated at T668 seemed to be elevated too. These data are in agreement with data from other groups that propose a toxic role of T688 phosphorylated APP [34], and may suggest that reduced Pin1 levels could be toxic in the same pathways. Interestingly, Ando and colleagues suggested that phosphor‐ ylation at T668 may affect APP conformation to ultimately alter the capability of APP to bind to partners such as Fe65 regulating APP trafficking, even if such interaction occurs at the 682YNPTY687 domain, further C-terminal than T668 [106]. This effect could be related to Pin1 mediated changes in APP conformation that could change the 682YNPTY687 stereochemistry. Of note, in Pin1 KD treated cells that were also overexpressing Fe65, we found that higher amounts of Fe65 associated with APP and that C99 accumulated, as compared to wild type cells. This was probably due to stabilization of Fe65 under these conditions, since Fe65 levels were elevated at the steady state in Pin1KD cells. Together with our immunocytochemistry data, under conditions that promote Fe65/APP interaction, these results suggest that reduced Pin1 expression may be linked to fastened internalization of APP to amyloidogenic compart‐ ments, where C99 is produced and accumulates (Fig. 1).

as it is known that higher APP levels correlate with AD. In fact, genetic modifications causing either duplication of the APP gene [111] or increased expression [112] were found to cause familial early onset AD. In addition, in Down syndrome patients, the triplication of the APP gene associates with the development of AD after age 40 [113], with the exception of those individuals affected by partial trisomy excluding the APP region [114]. In our experimental paradigm, such APP stabilization is caused by the lack of GSK3beta inhibition under condi‐ tions of impaired Pin1 activity. This may suggest that lack of Pin1 in AD impacts Abeta pathology by targeting multiple pathways, from APP trafficking to APP stabilization via

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More in details, we found that GSK3beta inhibitory mechanism was decreased in Pin1KO mice [110], since phoshorylation at S9, a mechanism that inhibits the kinase's activity, was decreased in these mice. We speculated that GSK3beta, which contains several Ser-Pro motifs, might serve as a substrate of Pin1 and that, by regulating GSK3beta conforma‐ tion, Pin1 could control GSK3beta activity. This mechanism would contribute to the understanding of a link between loss of Pin1 activity and APP and tau pathologies in AD. GSK3beta in fact is responsible for the phosphorylation of both T668 in APP and T231 in tau, crucial in determining toxic conformations of both proteins in the disease. We found that Pin1 binds to GSK3beta at the T330 residue, and that lack of phosphorylation at this site using a T330A mutant would prevent such interaction. Of note, changes in Pin1 levels would affect GSK3beta activity, in vivo and in vitro. In fact, in crude brain lysates from Pin1Tg mice, GSK3beta activity was decreased, whereas it increased in Pin1KO mice. Similarly, overexpression of a wild type form of Pin1 reduced GSK3beta activity in H4 cells, whereas overexpression of mutants in regulatory regions of Pin1 such as the WW (W34A) or at the PPIase (K63A) domains, or at the Pin1 binding site (T330A) would not induce any change in the kinase activity, suggesting that by binding to T330, Pin1 is a

We then tested the hypothesis whether the Pin1-mediated control of GSK3beta activity could help prevent APP from entering the amyloidogenic processing. We found that lack of Pin1 mediated regulation of GSK3beta activity in T330A mutant expressing H4 cells reduced the levels of the non-amyloidogenic alphaAPPs, whereas it increased overall T668 APP phos‐ phorylation. Of note, under these conditions, levels of APP were elevated at the steady state in cells, as well as in mice Pin1 concentration regulated APP levels. In fact, APP was reduced in Pin1Tg mice, whereas it accumulated in the brain of Pin1KO mice, similarly to what we had previously observed [41]. We found that APP accumulation in Pin1KO mice or in Pin1KD cells is the result of a failed physiologic degradation of APP, which is stabilized under these conditions. In fact, by means of cycloheximide treatment, we tested APP turnover under conditions of either reduced Pin1 expression (Pin1KD cells) or in absence of Pin1-mediated regulation of GSK3beta activity, in cells overexpressing the T330A mutant. We found not only that APP was stabilized in Pin1 KD cells as compared to wild type cells, also such stabilization seemed to depend on Pin1-mediated regulation of GSK3beta activity, since APP was stabilized in cells overexpressing the GSK3beta mutant T330A as compared to cells GSK3beta wild type.

GSK3beta activation.

crucial negative regulator of GSK3beta activity.

Our hypothesis is that Pin1 binds to and isomerizes the phosphorylated T668-Pro motif in full length APP, resulting in protein conformational changes that ultimately affect APP intracellular trafficking. (A, B]. In the presence of proper Pin1 function, the equilibrium between the cis and the trans form of phosphorylated APP is maintained [41] (A], and this may help APP stay anchored at the plasma membrane where it will undergo the non-amyloidogenic processing (B]. On the contrary, when Pin1 function is reduced, as in AD, the equilibrium between the cis and the trans form of phosphorylated APP will be disrupted, as the cis form of phosphorylated APP would not be isomerized to trans in a timely manner (C], and the levels of Fe65 will be stabilized. Moreover, reduced Pin1 function will enhance GSK3beta activity, leading to overall increase of phosphorylated APP at T668 and inhibiting APP turnover. These effects may lead to overall increased levels of APP undergoing internalization, trafficking and amyloidogenic processing (D]. PM: Plas‐ ma membrane. EE: Early Endosomes. LE: Late Endosomes.

**Figure 1.** A model for the role of Pin1 in inhibiting APP accumulation and amyloidogenic processing.

We had previously observed that reduced Pin1 expression is linked to Abeta production [41], and we found that Pin1KD may increase gamma-secretase cleavage of APP to generate AICD [51]. Hence, we could assume that AD-associated reduced Pin1 expression is linked to increased amyloidogenic processing, promoting both internalization and gamma-secretase dependent cleavage of APP.

#### **2.5. Pin1 increases APP protein turnover**

Recent findings from our lab link Pin1 deficit and amyloidogenic processing in AD to increased APP stabilization [110]. This is particularly relevant to a role of increased APP in the disease, as it is known that higher APP levels correlate with AD. In fact, genetic modifications causing either duplication of the APP gene [111] or increased expression [112] were found to cause familial early onset AD. In addition, in Down syndrome patients, the triplication of the APP gene associates with the development of AD after age 40 [113], with the exception of those individuals affected by partial trisomy excluding the APP region [114]. In our experimental paradigm, such APP stabilization is caused by the lack of GSK3beta inhibition under condi‐ tions of impaired Pin1 activity. This may suggest that lack of Pin1 in AD impacts Abeta pathology by targeting multiple pathways, from APP trafficking to APP stabilization via GSK3beta activation.

**Presence of Pin1 function Absence of Pin1 function**

**Non-amyloidogenic processing Amyloidogenic processing**

<sup>N</sup> -Pin1 <sup>C</sup> <sup>N</sup> C

> **P Thr668 Thr668 Pin1 P**

**Figure 1.** A model for the role of Pin1 in inhibiting APP accumulation and amyloidogenic processing.

pThr668

**A C**

**B D**

**alphaAPPs**

**PM PM**

**EE**

**LE**

ma membrane. EE: Early Endosomes. LE: Late Endosomes.

**2.5. Pin1 increases APP protein turnover**

dependent cleavage of APP.

pThr668

**APP**

**Thr668**

Pro669

116 Understanding Alzheimer's Disease

Pro669 *trans cis*

+Pin1

N

pThr668

**APP**

**Thr668**

Our hypothesis is that Pin1 binds to and isomerizes the phosphorylated T668-Pro motif in full length APP, resulting in protein conformational changes that ultimately affect APP intracellular trafficking. (A, B]. In the presence of proper Pin1 function, the equilibrium between the cis and the trans form of phosphorylated APP is maintained [41] (A], and this may help APP stay anchored at the plasma membrane where it will undergo the non-amyloidogenic processing (B]. On the contrary, when Pin1 function is reduced, as in AD, the equilibrium between the cis and the trans form of phosphorylated APP will be disrupted, as the cis form of phosphorylated APP would not be isomerized to trans in a timely manner (C], and the levels of Fe65 will be stabilized. Moreover, reduced Pin1 function will enhance GSK3beta activity, leading to overall increase of phosphorylated APP at T668 and inhibiting APP turnover. These effects may lead to overall increased levels of APP undergoing internalization, trafficking and amyloidogenic processing (D]. PM: Plas‐

We had previously observed that reduced Pin1 expression is linked to Abeta production [41], and we found that Pin1KD may increase gamma-secretase cleavage of APP to generate AICD [51]. Hence, we could assume that AD-associated reduced Pin1 expression is linked to increased amyloidogenic processing, promoting both internalization and gamma-secretase

Recent findings from our lab link Pin1 deficit and amyloidogenic processing in AD to increased APP stabilization [110]. This is particularly relevant to a role of increased APP in the disease,

**APP**

Pro669

<sup>C</sup> <sup>N</sup>

**GSK3beta**

**P P**

Pro669 *trans cis*

pThr668

**APP**

**C99**

**Fe65**

**Abeta**

**EE**

**LE**

C

**betaAPPs**

More in details, we found that GSK3beta inhibitory mechanism was decreased in Pin1KO mice [110], since phoshorylation at S9, a mechanism that inhibits the kinase's activity, was decreased in these mice. We speculated that GSK3beta, which contains several Ser-Pro motifs, might serve as a substrate of Pin1 and that, by regulating GSK3beta conforma‐ tion, Pin1 could control GSK3beta activity. This mechanism would contribute to the understanding of a link between loss of Pin1 activity and APP and tau pathologies in AD. GSK3beta in fact is responsible for the phosphorylation of both T668 in APP and T231 in tau, crucial in determining toxic conformations of both proteins in the disease. We found that Pin1 binds to GSK3beta at the T330 residue, and that lack of phosphorylation at this site using a T330A mutant would prevent such interaction. Of note, changes in Pin1 levels would affect GSK3beta activity, in vivo and in vitro. In fact, in crude brain lysates from Pin1Tg mice, GSK3beta activity was decreased, whereas it increased in Pin1KO mice. Similarly, overexpression of a wild type form of Pin1 reduced GSK3beta activity in H4 cells, whereas overexpression of mutants in regulatory regions of Pin1 such as the WW (W34A) or at the PPIase (K63A) domains, or at the Pin1 binding site (T330A) would not induce any change in the kinase activity, suggesting that by binding to T330, Pin1 is a crucial negative regulator of GSK3beta activity.

We then tested the hypothesis whether the Pin1-mediated control of GSK3beta activity could help prevent APP from entering the amyloidogenic processing. We found that lack of Pin1 mediated regulation of GSK3beta activity in T330A mutant expressing H4 cells reduced the levels of the non-amyloidogenic alphaAPPs, whereas it increased overall T668 APP phos‐ phorylation. Of note, under these conditions, levels of APP were elevated at the steady state in cells, as well as in mice Pin1 concentration regulated APP levels. In fact, APP was reduced in Pin1Tg mice, whereas it accumulated in the brain of Pin1KO mice, similarly to what we had previously observed [41]. We found that APP accumulation in Pin1KO mice or in Pin1KD cells is the result of a failed physiologic degradation of APP, which is stabilized under these conditions. In fact, by means of cycloheximide treatment, we tested APP turnover under conditions of either reduced Pin1 expression (Pin1KD cells) or in absence of Pin1-mediated regulation of GSK3beta activity, in cells overexpressing the T330A mutant. We found not only that APP was stabilized in Pin1 KD cells as compared to wild type cells, also such stabilization seemed to depend on Pin1-mediated regulation of GSK3beta activity, since APP was stabilized in cells overexpressing the GSK3beta mutant T330A as compared to cells GSK3beta wild type. Altogether, these data suggest that Pin1 regulates APP turnover by inhibiting GSK3beta activation and therefore contributes to lower T668 phosphorylation, which is responsible for toxic conformations of APP, as previously discussed in this chapter. These evidences suggest that in the pathology, mechanisms that favor the accumulation of APP will be toxic by increasing the amount of APP that will undergo amyloidogenic processing, and lack of Pin1 function could be one of these (Fig.1). Therefore, Pin1-mediated GSK3beta activity is an additional mechanism that Pin1 uses to protect from Abeta pathology, and strengthen the possibility to consider Pin1 as a valid tool to target Abeta pathologies in AD.

may disturb such equilibrium leading to increased Abeta production. Of note, the overex‐ pression in Pin1KO breast cancer cells of a T668A APP mutant, which retains 100% trans conformation, rescued the amount of APP anchored at the plasma membrane, and also the levels of alphaAPPs [51]. These data may suggest that the protective non-amyloidogenic processing of APP is maintained only if APP is in the trans conformation, a conditions that associates with physiologic low levels of phosphorylated T668 and to physiologic levels of Pin1. This poses attention on protein isomerization and Pin1 as a fine post-phosphorylative tool to regulate a protein function, bypassing the regulation of the kinases. Targeting abnormal protein isomerization and Pin1 function may therefore offer a preferred approach in AD to halt the toxic effects of hyperphosphorylated proteins, such as phosphorylated T668 APP and T231 tau, instead of the pharmacological inhibition of the many kinases responsible for their

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Altogether, the data discussed here emphasize a role for Pin1-mediated isomerization of APP and GSK3beta as a mechanism to control APP physiologic function, shifting APP processing towardthenon-amyloidogenicpathway(Fig.1).Suchregulationprevents the formationoftoxic species produced downstream the amyloidogenic pathway, such as Abeta and betaAPPs, by regulating both APP trafficking and stabilization, and occurs as a post-phosphorylative event to maintain the equilibrium between cis and trans conformations. Therefore, Pin1 and regula‐ tion of APP conformation emerge as ideal candidates in the search of therapeutic targets for AD.

Tau-mediated neurodegeneration may result from the combination of toxic gains-of-function acquired by the aggregates or their precursors and the detrimental effects that arise from the loss of the normal function(s) of tau in the disease state [15]. The toxic gains-of-function includes sequestration of normal tau function by NFTs made of hyperphosphorylated tau. NFTs also become physical obstacles to the transport of vesicles and other cargos[15]. The loss of the normal function of tau includes detachment of tau from microtubules that causes loss of microtubule-stabilizing function [124]. Although dynamic tau phosphorylation occurs during embryonic development [125], aberrant tau phosphorylation in mature neurons is harmful to the neuron [126]. Tau hyperphosphorylation is a key regulatory mechanism that leads to both such toxic gains-of-function and the loss of the normal function(s) of tau [15].

A high proportion of prolines residues are common to intrinsically disordered proteins, and tau is no exception [127]. Nearly 10% of full-length tau is composed of proline residues and around 20% of the residues between I151 and Q244 are proline. Many functions of tau are mediated through microtubule (MT) binding domains distal to this proline-rich domain. Interestingly, many disease-associated phosphorylation events that seed tau tangle formation occur at proline-directed serine (S) and threonine (T) residues in this proline-rich region. This indicates that important structural changes in the proline-rich region of tau are regulating

**4. Pin1 and tau pathology in animal models and in AD**

phosphorylation.

**4.1. Prolyl isomerization of tau**
