**5. Regulation of tau conformation as an early change in AD pathology: Implications for Pin1–catalyzed protein conformational regulation as a therapeutic and diagnostic tool**

#### **5.1. Cis pT231–tau is extremely early pathogenic conformation that is accumulated in MCI and AD**

To analysis Pin1-catalyzed cis/trans protein conformational regulation and conformationspecificfunctionorregulation,wehavedevelopedanoveltechnologytogeneratethefirstcisand trans-specific antibodies. We discovered that cis, but not trans, pT231-tau is extremely early pathogenic conformation in AD due to lack of Pin1 convert it to the physiological trans [129].

We found that replacing the five membered ring of Pro232 with the six membered ring homoproline (Pip) increases cis to 74%, since ~90% of pT231-P motif in a synthetic peptide in trans. Polyclonal antibodies raised against a pT231-Pip tau peptide recognize human and mouse p-tau. Resulting cis-specific antibody recognize regular pT231-Pro tau and cis pT231-5,5-demethylproline (Dmp) tau peptide, whereas trans-specific antibody recognizes regular pT231-Pro and trans pT231-Ala tau peptide. Neither antibody recognizes the nonphosphopeptide. Furthermore, both antibodies recognize tau, but not its T231A mutant expressed in SY5Y cells. Thus, cis- or trans- antibodies are highly specific [129]. Antibodies against cis pT231-tau might provide opportunity for efficient immunotherapy and diagnostic tools against early pathogenic tau conformation, raising the possibility of preventing tauop‐ athy in AD patients at early stages.

#### **5.2. Cis pT231–tau not only loses normal MT functions, but also gains toxic functions**

As we and other groups had been shown [56, 184-186], phosphorylation of tau on T231 by Ccd2 abolishes its ability to promote MT assembly, which can be restored after dephosphor‐ 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 functions, and gains toxic functions.

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

**5. Regulation of tau conformation as an early change in AD pathology: Implications for Pin1–catalyzed protein conformational regulation as a**

**5.1. Cis pT231–tau is extremely early pathogenic conformation that is accumulated in MCI**

To analysis Pin1-catalyzed cis/trans protein conformational regulation and conformationspecificfunctionorregulation,wehavedevelopedanoveltechnologytogeneratethefirstcisand trans-specific antibodies. We discovered that cis, but not trans, pT231-tau is extremely early pathogenic conformation in AD due to lack of Pin1 convert it to the physiological trans [129].

We found that replacing the five membered ring of Pro232 with the six membered ring homoproline (Pip) increases cis to 74%, since ~90% of pT231-P motif in a synthetic peptide in trans. Polyclonal antibodies raised against a pT231-Pip tau peptide recognize human and mouse p-tau. Resulting cis-specific antibody recognize regular pT231-Pro tau and cis pT231-5,5-demethylproline (Dmp) tau peptide, whereas trans-specific antibody recognizes regular pT231-Pro and trans pT231-Ala tau peptide. Neither antibody recognizes the nonphosphopeptide. Furthermore, both antibodies recognize tau, but not its T231A mutant expressed in SY5Y cells. Thus, cis- or trans- antibodies are highly specific [129]. Antibodies against cis pT231-tau might provide opportunity for efficient immunotherapy and diagnostic tools against early pathogenic tau conformation, raising the possibility of preventing tauop‐

**5.2. Cis pT231–tau not only loses normal MT functions, but also gains toxic functions**

As we and other groups had been shown [56, 184-186], phosphorylation of tau on T231 by Ccd2 abolishes its ability to promote MT assembly, which can be restored after dephosphor‐

be a pathogenic mechanism leading to tauopathy in AD [48, 163].

**therapeutic and diagnostic tool**

122 Understanding Alzheimer's Disease

athy in AD patients at early stages.

**and AD**

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

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

#### **5.4. Cis, but not trans, pT231–tau is significantly elevated and localized to dystrophic neurites in MCI and further accumulated in AD**

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 to inhibit tauopathy in AD.

#### **5.5. Cis pT231–tau fully overlaps with neurofibrillary degeneration and correlates with reduced Pin1 levels in the AD hippocampus**

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.

### **5.6. Potential novel cis and trans conformation–specific disease diagnoses and therapies**

however they may [216] or may not [219] undergo neurodegeneration, which is a feature of AD. Moreover, they may not be representative of the way the disease progresses in sporadic AD, which affects the vast majority of AD patients, as they may represent only those familial cases of AD caused by those same mutations. Furthermore, these models may not be all specific for AD, since tau hyperphosphorylation and tangle formation occur also in other neurodege‐ nerative diseases, and some of the tau mutations used to generate animal models for AD do not associate with AD, but with other neurodegenerative diseases, such as frontotemporal

Pin1 Protects Against Alzheimer's Disease: One Goal, Multiple Mechanisms

http://dx.doi.org/10.5772/55085

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Both APP and tau are phosphorylated by protein kinase (PKs) as part of their normal function. The trans-conformation of phosphorylated APP and tau may present the physiological conformation that promotes their normal function (green boxes). Pin1 expression is induced during neuron differentiation and necessary to maintain normal neuronal function by preventing the unscheduled activation of mitotic events and/or controlling the function of phosphopro‐ teins in the event that they become abnormally phosphorylated. For example, by catalyzing isomerization of the cis to trans conformation, Pin1 might promote non-amyloidogenic APP processing reducing Abeta production, as well as promote tau dephosphorylation and restore tau function. However in AD, a loss of Pin1 function, either through downregulation of Pin1 function, oxidative inactivation, phosphorylation or possible genetic alterations, can lead to build-up of cis-pS/T –P motifs. Cis-p-tau and cis-p-APP are proposed to represent pathological conformations (red ovals). Cis-p-APP is processed by the amyloidogenic pathway, which lead to a build-up of amyloid beta-42 (Abeta42), decreased levels of neurotropic alphaAPPs and the resultant formation of amyloid plaques. Cis-p-tau is resistant to protein phosphatases, which leads to a loss of MT binding, hyperphosphorylated tau an the formation of neurofibril‐ lary tangles. The formation of tangles and plaques might further reduce Pin1 function by sequestering Pin1 and induc‐ ing oxidative modifications, respectively, in a positive feedback loop. In addition, a lack of proper Pin1 function leads to activation of kinases such as GSK3beta, which further increases both phosphorylation of tau and APP and also in‐ hibits APP turnover, contributing to both tau and Abeta pathologies and causing neuronal death. Therefore, Pin1 de‐

dementia associated with parkinsonism FTDP [17, 220, 221].

regulation might act on multiple pathways to contribute to AD development.

**Figure 3.** The regulation of APP processing and tau function by Pin1 in healthy and Alzheimer's neuron.

Our exciting new insight into the role and regulation of p-tau conformations in AD might have important and novel therapeutic implications. For example, it has been shown that Thr231 phosphorylation is the earliest detectable tau phosphorylation event in human AD [130, 159, 160, 198] and its levels are elevated in cerebrospinal fluids and tracks AD progression, but with large individual variations [199, 200], making it difficult to become a standardized test. Our findings that the *cis* conformation appears earlier in MCI and is pathologically more relevant suggest that *cis* pThr231-tau and especially its ratio with *trans* might be a better and easier standardized diagnostic marker, especially for early diagnosis and patient comparison. Furthermore, the findingsthatPin1overexpressionconverts*cis*to*trans*,promotestaudegradationandinhibitstau pathology and neurodegeneration in AD mouse models [201] and that Pin1 SNPs preventing its inhibitionbybrain-specifictranscriptionfactorAP-4isassociatedwithdelayedonsetofAD[202] suggestthatoverexpressingPin1orpreventingPin1inhibitionmightbeanewapproachtoreduce the *cis* to *trans* pThr231-tau ratio to block tau pathology at early stages. Finally, active or passive immunizationagainstsomepSer/Thr-PromotifsintauincludingthepThr231-Promotifhasbeen shown to reduce tau aggregates and improve memory deficits in mouse models [203-209]. However, we have here shown that only ~10% of regular synthetic pThr231-tau peptides is in the pathologicallyrelevant*cis*conformationandtheremaining90%isin*trans*,whichcanstillpromote MT assembly and is not related to neurofibrillary degeneration. Therefore, immunotherapies either using conformation-specific vaccines or antibodies specifically against the pathological‐ ly relevant *cis* pT231-tau conformation might be more specific and effective and safer in treating AD. Given the critical role of Pin1 and other isomerases in controlling the function of many other key regulators in the pathogenesis of human disease, notable Alzheimer's disease, cancer, viral infection, inflammation and autoimmune disorders [210-213], it would be interesting to deter‐ mine whether prolyl isomerization regulates the cellular function of these proteins and whether these conformational switches might be explored for developing novel diagnoses and therapies.
