Pin1-Catalyzed Protein Conformational Regulation in Alzheimer's Disease

描述（由申请人提供）：阿尔茨海默病（AD）的神经病理学特征是由过度磷酸化的tau （p- tau）组成的缠结和由β沉积组成的斑块。Tau蛋白的过度磷酸化，尤其是在丝氨酸或苏氨酸残基上，先于缠结形成（pSer/Thr-Pro）。我们最近发现某些pSer/Thr-Pro基序以两种不同的顺式和反式构象存在，并鉴定出一种独特的酶Pin1在体外特异性催化它们的异构化。重要的是，Pin1控制着一组关键的调节因子，以帮助协调它们的功能，并且它的放松可能导致某些疾病，特别是阿尔茨海默病。Pin1作用于p-tau蛋白中的pThr231-Pro基序以抑制tau蛋白相关病理（tauopathy），并作用于APP中的pThr668-Pro基序以减少A？——病理。Pin1敲除小鼠产生年龄依赖性tau-和A？而Pin1过表达抑制小鼠的牛头病。这些结果与人类AD相关，因为Pin1在MCI和AD神经元中通过多种机制受到抑制，但阻止Pin1抑制与AD的延迟发病有关。人类Pin1基因位于19p13.2，这是一个与晚发性AD相关的新位点。因此，我们提出Pin1可能通过调节蛋白质构象来帮助预防阿尔茨海默病的神经退行性变。然而，一个主要的挑战是缺乏任何可用的工具来区分天然蛋白中的顺式构象和trans - ser /Thr-Pro构象。因此，没有直接证据表明这两种p- tau构象的存在及其构象特异性功能或在tau病中的调节。此外，Pin1和p-tau构象是否在与AD相关的认知功能中发挥任何作用也尚不清楚。为了解决这些关键问题，我们开发了Pin1条件敲除小鼠和一种新技术，以产生能够特异性检测p-tau中顺式或反式pThr231-Pro基序的第一对抗体。我们的初步结果表明，在体外和小鼠中，Pin1增加了pThr231-tau的顺式到反式异构化，并且pThr231-tau在MCI大脑中显着升高，而不是反式，并在AD中进一步积累。本研究旨在验证我们的假设，即病理p-tau具有构象特异性，并且通过Pin1和其他构象调节这种构象会影响与AD相关的tau病和认知功能。目的1是使用AD小鼠模型和人类AD样本确定在tau病变期间哪种p-tau构象在病理学上更相关。目的2是确定通过Pin1等方法调节p-tau构象对小鼠tau病变的影响，以及人类AD中Pin1与tau构象的关系，以及Pin1在体外和细胞中作用的机制。目的3是利用神经元特异性Pin1敲除或过表达或其他方法来确定Pin1和p-tau构象在AD相关认知功能中的作用。这些实验将进一步确定Pin1及其催化的构象变化在AD中的作用，并可能导致治疗牛头病的新策略。这些研究将提供第一个在体内研究磷酸化后构象调节的方法。

DESCRIPTION (provided by applicant): The neuropathological hallmarks of Alzheimer's disease (AD) are tangles made of hyperphosphorylated tau (p- tau) and plaques consisted of Abeta deposits. Tau hyperphosphorylation especially on Ser or Thr residues before Pro (pSer/Thr-Pro) precedes tangle formation. We have recently found that certain pSer/Thr-Pro motifs exist in two distinct cis and trans conformations and identified a unique enzyme, Pin1 that specifically catalyzes their isomerization in vitro. Importantly, Pin1 controls a subset of key regulators to help coordinate their functions and its deregulation can contribute to certain diseases, notably AD. Pin1 acts on the pThr231-Pro motif in p-tau to inhibit tau-related pathology (tauopathy) and on the pThr668-Pro motif in APP to reduce A?- pathology. Pin1 knockout mice develop age-dependent tau- and A?-pathologies, whereas Pin1 overexpression inhibits tauopathy in mice. These results are relevant to human AD because Pin1 is inhibited in MCI and AD neurons by various mechanisms, but preventing Pin1 inhibition is associated with delayed onset of AD. Human Pin1 gene is located to 19p13.2, a new locus associated with late-onset AD. We thus proposed that Pin1 might help protect against neurodegeneration in AD by regulating protein conformations. However, a major challenge is the lack of any tool available that is able to distinguish cis from trans pSer/Thr-Pro conformation in a native protein. Thus there is no direct evidence for the presence of these two p- tau conformations and their conformation-specific functions or regulation in tauopathy. Moreover, whether Pin1 and p-tau conformations play any role in cognitive function relevant to AD is also not known. To address these critical issues, we developed Pin1 conditional knockout mice and a novel technology to generate a first pair of antibodies able to detect specifically cis or trans pThr231-Pro motif in p-tau. Our preliminary results suggest that Pin1 increased cis to trans isomerization of pThr231-tau in vitro and in mice, and that cis, but not trans, pThr231-tau was significantly elevated in MCI brains, and further accumulated in AD. This proposal is designed to test our hypotheses that the pathological p-tau is conformation-specific and that modulating such conformation by Pin1 and others would affect tauopathy and cognitive function relevant to AD. Aim 1 is to determine which p-tau conformations are pathologicaly more relevant during tauopathy using AD mouse models and human AD samples. Aim 2 is to determine the impact of modulating p-tau conformations by Pin1 and other methods on tauopathy in mice, and the relationship between Pin1 and tau conformations in human AD, and the mechanisms underlying the Pin1 action in vitro and in cells. Aim 3 is to use neuron-specific Pin1 knockout or overexpression or other methods to determine the role of Pin1 and p-tau conformations in cognitive function relevant to AD. These experiments would further define the role of Pin1 and its catalyzed conformational changes in AD, and could lead to new strategies for treating tauopathy. These studies would offer the first in vivo approach to study post-phosphorylation conformational regulation.