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

#### **4.1. Prolyl isomerization of tau**

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

**3. Role of T668 phosphorylation and APP conformation in AD: Pin1 as a**

The question how increased APP phosphorylation at T668 is linked to AD has raised a real debate in the field. Many evidences point to a role in the disease. In fact, not only phospho-T668 levels were increased in AD brains [34], also many studies in vitro and in vivo point to a role of T668 phosphorylation by different kinases in altered protein transport in neurons [115, 116], also associated with increased amyloidogenic processing and Abeta production in cellular and animal models [34, 36, 39, 117]. In these studies, a non-phosphorylatable mutant T668A was used as an experimental paradigm to compare the effects of phosphorylated endogenous APP to the non-phosphorylated T668A form. Interestingly, a knock-in T668A animal model did not show any age-dependent alteration in APP processing [118], since in this study levels of Abeta, alphaAPPs and betaAPPs were comparable in both T668A and wild type mice during aging. Although at a first glance these data seem to challenge the hypothesis that regulation of T668 phosphorylation might be involved in AD, from these data it cannot be concluded that such phosphorylation is irrelevant to AD progression. It is not by abolishing APP phosphorylation at T668 by either knocking out APP or knocking in a non-phosphory‐ latable version of it that we can exclude a role for such pathway in the disease. APP KO mice are viable and their development and aging does not rely on alterations of APP processing [119, 120], and yet a role for APP in the development of AD is not disputed. Similarly, tau KO mice develop properly, reach adulthood and age normally [121-123], however a role for

It is possible that the controversy around the role of T668 phosphorylation in AD lies in the fact that physiologic phosphorylation at this domain is low, and it is only elevated in AD. This would explain why the T668A knock in mice did not show any difference from the wild type [118], because basal levels of phosphorylated T668 APP are very low, and phosphorylationmediated regulation of APP activity in wild type mice was therefore comparable to the T668A

Based on our data in vivo in Pin1KO mice and in vitro in Pin1KD cells [41, 51], we could hypothesize that the relevance of physiological T668 phosphorylation could be to maintain allow the equilibrium between cis and trans conformation. We could also assume that reduced Pin1 levels or increased T668 phosphorylation, both conditions associated with AD [34, 56],

possibility to consider Pin1 as a valid tool to target Abeta pathologies in AD.

**molecular switch to regulate APP function**

118 Understanding Alzheimer's Disease

hyperphosphorylated tau in AD is quite clear.

mutant mice in this study.

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 tangle formation. In particular, cis-trans isomerization around these prolines modulates protein phosphatase binding and activity at specific S/T sites. We discovered that Pin1 regulates tau phosphorylation in concert with protein phosphatase 2A (PP2A) especially at T231 [42, 56, 128-130]. Findings by Dickey's group suggest that FKBP51 has a similar activity to Pin1; but unlike Pin1, FKBP51 coordinates with Hsp90 to isomerize tau [131].

tau dephosphorylation because PP2A is trans-specific [42, 128], and (iii) to promote p-tau degradation [164] (Fig.2). Indeed, Pin1 overexpression promotes tau dephosphorylation selectively on pT231 in neurons and mouse brains [167, 168]. Pin1 has no effect on T231A mutant tau in vitro or vivo [56, 128, 139, 164]. Furthermore, Pin1 deficiency (Pin1-KO) mice display age-dependent tauopathy [42], whereas Pin1 overexpression in postnatal neurons effectively suppresses tauopathy induced by human wild-type (WT) tau in mice, albeit it

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pT231-tau protein exists in the two completely different cis and trans conformations, as depicted in cartoons of the primary backbone structures. Cis, but not trans, pT231-tau loses normal function and also gains toxic function. Pin1 inhibits the accumulation of the pathogenic cis pT231-tau conformation in AD by converting it to the nonpathogenic trans form. Conformation-specific antibody and/or vaccines against the pathogenic cis pT231-tau might be developed

**Figure 2.** Pin1 inhibits the accumulation of the pathogenic cis pT231-tau conformation in AD by converting it to the

These findings are highly relevant to human AD. We found that Pin1 localize to tangle and is depleted in AD brains [56], which has been confirmed and expanded to other tauopathies including FTD [61, 62, 169-172]. Furthermore, Pin1 is induced by neuron differentiation and highly expressed in most normal neurons, but inhibited by various mechanisms in AD neurons [44, 54, 56, 59, 62, 167, 169, 171-175]. For example, Pin1 expression is especially low in AD

enhances tauopathy induced by the P301L mutant tau [164].

for the diagnosis and treatment of AD, especially during its early stages.

nonpathogenic trans form

#### **4.2. Deletion of Pin1 causes tauopathy in mice**

Pin1 knockout (KO) mice [42] serve as a good model to investigate the effect of Pin1 on endogenous tau *in vivo*. Pin1-deficient mice showed progressive age-dependent motor and behavioral deficits including abnormal limb-clasping reflexes, hunched postures and reduced mobility [42] similar to tau transgenic mice [132, 133]. These phenotypes in Pin1 mutant mice are significant because the total level of NFTs correlates with the degree of cognitive impair‐ ment [134, 135]. Pioneering studies that used immunohistochemical techniques to determine the level of both NFTs and senile plaques in different brain regions of AD patients, as well as non-demented elderly individuals, demonstrated that the number of NFTs, but not the numbers of senile plaques, correlates with the degree of cognitive impairment [134, 135].

#### **4.3. Pin1 acts on the pT231–P motif in p–tau to protect against tauopathy in AD**

It is increasingly evident that tauopathy in AD may result from the combination of toxic gainsof function acquired by phosphorylated tau (p-tau) aggregates, and the malignant effects from loss of tau normal function, including the failure of p-tau to bind and promote microtubule (MT) assembly [15]. Interestingly, a common characteristic event that disrupts tau MT function and precedes tangle formation is increased phosphorylation of tau especially on S/T-P motifs [11, 126, 136-141]. Indeed, tau kinases, such as mitogen-activated protein (MAP) kinases, cyclin-dependent protein kinase 5 (CDK5) and glycogen synthase 3 (GSK3) [142-144] or phosphatases, such as protein phosphatase 2A (PP2A) [145, 146] are deregulated in AD and modulating these enzymes can affect tauopathy [23, 39, 147-155]. Moreover, recent GWAS studies identify several new AD genes that might modulate tau phosphorylation and/or cytoskeleton, including MARK4 (pro-directed tau kinase) and BIN1 [156], and CD2AP [157, 158]. Notably, T231 phosphorylation in tau is reported to be the first of a number of tau phosphoepitopes appearing sequentially during early stages in pretangle AD neurons: pT231 → TG3 → AT8 → AT100 → Alz50 →→ NFTs. However, it is still unclear how this phos‐ phorylation leads to tau misfolding, aggregation and tangle formation [130, 159-161]. The results from our group and others support a critical role for Pin1 in protecting against tauopathy in AD by acting on the phosphorylated T231-P (pT231-P)motif (Fig.2). We discov‐ ered that pT231-P motif in tau exists in distinct cis and trans conformations, and that the cis to trans conversion is accelerated by a unique isomerase, Pin1 [44, 56, 128, 162], thereby protecting against tangle formation in AD [42, 48, 56, 163, 164]. pT231 is the first of sequential tau phosphoepitopes appearing in pretangle neurons in prodromal AD [159, 160] and its cerebro‐ spinal fluid (CSF) level is an early biomarker that tracks MCI (mild cognitive impairment) conversion to AD, albeit with wide interindividual variations [165, 166]. Pin1 acts on the pT231- P motif in tau (i) to restore the ability of p-tau to promote MT assembly [56], (ii) to facilitate ptau dephosphorylation because PP2A is trans-specific [42, 128], and (iii) to promote p-tau degradation [164] (Fig.2). Indeed, Pin1 overexpression promotes tau dephosphorylation selectively on pT231 in neurons and mouse brains [167, 168]. Pin1 has no effect on T231A mutant tau in vitro or vivo [56, 128, 139, 164]. Furthermore, Pin1 deficiency (Pin1-KO) mice display age-dependent tauopathy [42], whereas Pin1 overexpression in postnatal neurons effectively suppresses tauopathy induced by human wild-type (WT) tau in mice, albeit it enhances tauopathy induced by the P301L mutant tau [164].

tangle formation. In particular, cis-trans isomerization around these prolines modulates protein phosphatase binding and activity at specific S/T sites. We discovered that Pin1 regulates tau phosphorylation in concert with protein phosphatase 2A (PP2A) especially at T231 [42, 56, 128-130]. Findings by Dickey's group suggest that FKBP51 has a similar activity

Pin1 knockout (KO) mice [42] serve as a good model to investigate the effect of Pin1 on endogenous tau *in vivo*. Pin1-deficient mice showed progressive age-dependent motor and behavioral deficits including abnormal limb-clasping reflexes, hunched postures and reduced mobility [42] similar to tau transgenic mice [132, 133]. These phenotypes in Pin1 mutant mice are significant because the total level of NFTs correlates with the degree of cognitive impair‐ ment [134, 135]. Pioneering studies that used immunohistochemical techniques to determine the level of both NFTs and senile plaques in different brain regions of AD patients, as well as non-demented elderly individuals, demonstrated that the number of NFTs, but not the numbers of senile plaques, correlates with the degree of cognitive impairment [134, 135].

to Pin1; but unlike Pin1, FKBP51 coordinates with Hsp90 to isomerize tau [131].

**4.3. Pin1 acts on the pT231–P motif in p–tau to protect against tauopathy in AD**

It is increasingly evident that tauopathy in AD may result from the combination of toxic gainsof function acquired by phosphorylated tau (p-tau) aggregates, and the malignant effects from loss of tau normal function, including the failure of p-tau to bind and promote microtubule (MT) assembly [15]. Interestingly, a common characteristic event that disrupts tau MT function and precedes tangle formation is increased phosphorylation of tau especially on S/T-P motifs [11, 126, 136-141]. Indeed, tau kinases, such as mitogen-activated protein (MAP) kinases, cyclin-dependent protein kinase 5 (CDK5) and glycogen synthase 3 (GSK3) [142-144] or phosphatases, such as protein phosphatase 2A (PP2A) [145, 146] are deregulated in AD and modulating these enzymes can affect tauopathy [23, 39, 147-155]. Moreover, recent GWAS studies identify several new AD genes that might modulate tau phosphorylation and/or cytoskeleton, including MARK4 (pro-directed tau kinase) and BIN1 [156], and CD2AP [157, 158]. Notably, T231 phosphorylation in tau is reported to be the first of a number of tau phosphoepitopes appearing sequentially during early stages in pretangle AD neurons: pT231 → TG3 → AT8 → AT100 → Alz50 →→ NFTs. However, it is still unclear how this phos‐ phorylation leads to tau misfolding, aggregation and tangle formation [130, 159-161]. The results from our group and others support a critical role for Pin1 in protecting against tauopathy in AD by acting on the phosphorylated T231-P (pT231-P)motif (Fig.2). We discov‐ ered that pT231-P motif in tau exists in distinct cis and trans conformations, and that the cis to trans conversion is accelerated by a unique isomerase, Pin1 [44, 56, 128, 162], thereby protecting against tangle formation in AD [42, 48, 56, 163, 164]. pT231 is the first of sequential tau phosphoepitopes appearing in pretangle neurons in prodromal AD [159, 160] and its cerebro‐ spinal fluid (CSF) level is an early biomarker that tracks MCI (mild cognitive impairment) conversion to AD, albeit with wide interindividual variations [165, 166]. Pin1 acts on the pT231- P motif in tau (i) to restore the ability of p-tau to promote MT assembly [56], (ii) to facilitate p-

**4.2. Deletion of Pin1 causes tauopathy in mice**

120 Understanding Alzheimer's Disease

pT231-tau protein exists in the two completely different cis and trans conformations, as depicted in cartoons of the primary backbone structures. Cis, but not trans, pT231-tau loses normal function and also gains toxic function. Pin1 inhibits the accumulation of the pathogenic cis pT231-tau conformation in AD by converting it to the nonpathogenic trans form. Conformation-specific antibody and/or vaccines against the pathogenic cis pT231-tau might be developed for the diagnosis and treatment of AD, especially during its early stages.

**Figure 2.** Pin1 inhibits the accumulation of the pathogenic cis pT231-tau conformation in AD by converting it to the nonpathogenic trans form

These findings are highly relevant to human AD. We found that Pin1 localize to tangle and is depleted in AD brains [56], which has been confirmed and expanded to other tauopathies including FTD [61, 62, 169-172]. Furthermore, Pin1 is induced by neuron differentiation and highly expressed in most normal neurons, but inhibited by various mechanisms in AD neurons [44, 54, 56, 59, 62, 167, 169, 171-175]. For example, Pin1 expression is especially low in AD vulnerable neurons or actual degenerated neurons in AD [42]. Pin1 can be inactivated by oxidation in mild cognitive impairment (MCI) and AD neurons [61, 169, 170]. Pin1 is seques‐ tered into tangles [56] or tangle or Ab-free granulovacuolar bodies with increasing tauopaty [176]. The human Pin1 gene is located at 19p13.2, a new late onset AD locus distinct from ApoE4 [173] and Pin1 promoter SNPs that reduce Pin1 expression [177] are associated with increased risk for AD in some cohorts [174], although not in others [65, 178]. A different Pin1 promoter SNP that prevents its suppression by AP4 is associated with delayed onset of AD [178]. Our findings of the opposite effects of Pin1 on cancer and AD [43, 45] have been supported by genetic association studies [174, 177, 179] and epidemiological studies [180-182]. Our analysis of the Framingham Study has further shown that cancer patients have decreased risk of AD, that AD patients have reduced cancer risk, and importantly, that this inverse relationship in not due to selective mortality or underdiagnosis [183]. Thus, we recently proposed that lack of sufficient Pin1 to catalyze cis to trans isomerization of pT231-tau might be a pathogenic mechanism leading to tauopathy in AD [48, 163].

ylation by PP2A or Pin1 [129]. Importantly, the ability of Pin1 to restore p-tau MT function is fully blocked by incubation of Pin1-treated p-tau with trans antibodies, but not cis antibodies. Furthermore, trans pT231-tau peptide is readily dephosphorylated by the tau phosphatase PP2A, which dephosphorylate on trans pS/T-P motifs [128]. Moreover, cis pT231-tau is much more stable than the trans both in cells and in Tau-transgenic (Tg) mouse brain slice cultures. Finally cis pT231-tau is much more prone to aggregation than the trans in Tau-Tg mouse brains and human MCI brains, as detected by sarcosyl fractionation experiments [42, 164, 187]. Thus, cis, but not trans, p-tau loses normal MT

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**5.3. Pin1 overexpression increases cis to trans pT231–P conversion in WT tau–Tg mice**

**5.4. Cis, but not trans, pT231–tau is significantly elevated and localized to dystrophic**

To examine p-tau conformational changes during the development of AD, we analyzed different Braak brain tissues with *cis* or *trans-*specific tau antibodies. There is little *cis* or *trans* pT231-tau in normal brains [129]. In AD cortex, *trans* pT231-tau is very low, but the *cis* is readily detected, even at Braak stages III and IV (MCI), and further accumulates as the Braak stage increases [129]. Notably, *cis*, but not *trans*, is localized to the dystrophic neurites [129], an early hallmark change highly correlating with synaptic and cognitive loss in AD [188-194]. Given Pin1 inhibition by oxidation in MCI brains [169, 170, 195], Pin1 might act at a very early step

**5.5. Cis pT231–tau fully overlaps with neurofibrillary degeneration and correlates with**

As shown [42, 134, 196, 197], Pin1 is highly expressed in the CA2 region of the hippocampus, but dramatically reduced in the CA1 region, whereas PHF-1, a solid neurofibrillary neurodegenera‐ tion marker [161], is prevalent in the CA1, but not in CA2 region [129]. Importantly, transpositiveneuronsaredominantintheCA2region.However,intheCAregion,cis-positiveneurons are greatly increased. All cis-positive neurons are also positive for PHF-1 in both CA2 and CA1 regions. However, 74% of trans-positive cells in the CA2 region are negative for PHF-1. Thus, cis,

but not trans, pT231-tau is fully overlapped with neurofibrillary degeneration.

can differentiate AD form frontotemporal dementia[165, 166].

**neurites in MCI and further accumulated in AD**

**reduced Pin1 levels in the AD hippocampus**

to inhibit tauopathy in AD.

To detect the ability of Pin1 to increase cis to trans isomerization, we showed that Pin1 reduces cis, but increases trans pT231-tau in vitro [129]. Moreover, Pin1 overexpression in Tau-Tg mice (Tau+Pin1) reduces the cis content, but increases the trans content, whereas Pin1 deficiency in Tau-Tg mice (Tau+Pin1-KO) has the opposite effects, as shown by immunoblots and immu‐ nostains [129]. In contrast to Wt tau-Tg mice, Pin1 overexpression increased cis, but decreased trans pT231-tau in P301L tau Tau-Tg mice because the P301L tau mutation greatly reduced cis, but not trans, pT231-tau, as we hypothesized [164]. These results explain why Pin1 has the opposite effects on WT tau and P301L tau [164] and are consistent with that CSF pT231-tau

functions, and gains toxic functions.
