**3. Role of T668 phosphorylation and APP conformation in AD: Pin1 as a molecular switch to regulate APP function**

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 hyperphosphorylated tau in AD is quite clear.

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 mutant mice in this study.

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], 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 phosphorylation.

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.
