Pin1-Catalyzed Protein Conformational Regulation in Alzheimer's Disease

描述(申请人提供)：阿尔茨海默病(AD)的神经病理特征是由过度磷酸化的tau(p-tau)组成的缠结和由Abeta沉积组成的斑块。在形成缠结之前，tau过度磷酸化，特别是在Pro(pSer/Thr-Pro)之前的丝氨酸或苏氨酸残基上。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，并在体外发现了一种独特的酶Pin1，它特异性地催化它们的异构化。重要的是，Pin1控制着一组关键调节因子的子集，以帮助协调它们的功能，而它的放松管制可能会导致某些疾病，特别是AD。Pin1作用于p-tau中的pThr231-Pro基序，以抑制tau相关的病理(tau病)，并作用于APP中的pThr668-Pro基序，以减少A？-病理。Pin1基因敲除小鼠出现与年龄相关的tau-和A？-病理，而Pin1的过度表达抑制了小鼠的tau病。这些结果与人类AD有关，因为Pin1在MCI和AD神经元中通过不同的机制被抑制，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相关的新基因座。因此，我们提出Pin1可能通过调节蛋白质构象来帮助预防AD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质中的顺式和反式pSer/Thr-Pro构象。因此，没有直接证据表明这两种p-tau构象的存在及其在肌病中的构象特异性功能或调节。此外，Pin1和p-tau构象是否在与AD相关的认知功能中发挥作用也是未知的。为了解决这些关键问题，我们开发了Pin1条件基因敲除小鼠和一种新的技术来生成第一对抗体，该抗体能够检测p-tau中特异性的顺式或反式pThr231-Pro基序。我们的初步结果表明，Pin1在体外和小鼠体内增加了pThr231-tau的顺式到反式异构化，并且顺式而不是反式的pThr231-tau在MCI脑中显著升高，并在AD中进一步积累。这一建议旨在检验我们的假设，即病理的p-tau是构象特异性的，并且Pin1和其他人调节这种构象会影响与AD相关的肌松和认知功能。目的1是利用AD小鼠模型和人类AD样本，确定哪些p-tau构象在肌萎缩侧索硬化症期间病理上更相关。目的2确定Pin1和其他方法调节p-tau构象对小鼠的影响，以及在人类AD中Pin1和tau构象的关系，以及Pin1在体外和细胞内作用的机制。目的3是利用神经元特异性的Pin1基因敲除或过度表达或其他方法来确定Pin1和p-tau构象在与AD相关的认知功能中的作用。这些实验将进一步确定Pin1在AD中的作用及其催化的构象变化，并可能导致治疗相互作用的新策略。这些研究将为研究磷酸化后构象调控提供第一种活体方法。

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.