Characterization of a caspase-cleavage resistant tau knock-in mouse

描述（由申请人提供）：阿尔茨海默病（AD）是世界上最常见的老年痴呆原因，与脑淀粉样斑块和神经原纤维缠结（nft）有关。淀粉样斑块主要由ß肽组成，而nft则由细胞骨架蛋白tau过度磷酸化形式的异常聚集体组成。β肽是由淀粉样蛋白前体蛋白（APP）的双重裂解产生的。BACE1裂解产生c端片段ß-CTF，然后被γ-分泌酶加工成几个ase1亚型。遗传数据表明APP加工的调控有助于AD的发生。目前阿尔茨海默病的药物发现方法主要集中在预防ß形成或去除现有的淀粉样蛋白沉积。针对淀粉样蛋白的化合物/生物制剂在临床试验中的反复失败，要么归因于纳入了疾病进展太迟而无法从治疗干预中获益的AD患者，要么归因于asas不是AD病理生理学的主要原因。AD最先进的替代假说是tau毒性假说。与这一假设相一致的是，减少内源性tau的表达可以防止具有家族性AD突变的表达人APP的转基因小鼠的行为缺陷，而不改变asβ水平。我们的初步数据表明，tau也介导家族性丹麦痴呆（FDDKI小鼠）的行为缺陷，这是一种ad样痴呆，与APP加工BRI2/ITM2B的调节因子突变相关，也以tau病为特征。一些证据表明，半胱天蛋白酶在阿尔茨海默病的早期被激活，并可能在神经元丢失和NFT病理中发挥作用。Tau蛋白在D421位点（ΔTau）被刽子手caspases切割。在caspase裂解后，ΔTau促进成核依赖的细丝形成，并采用MC1识别的构象变化，MC1是早期病理性tau标志物。ΔTau可以被磷酸化并被NFT抗体PHF-1识别。在AD大脑中，ΔTau与nft标志物相关，与认知能力下降相关。此外，ΔTau启动NFT的形成，并可以发挥毒性作用。这些发现导致了ΔTau是神经退行性变的关键毒性部分的假设。为了直接验证这一理论，我们产生了敲入小鼠，其中编码D421的12外显子内源性tau密码子GAC已经突变为AAC，现在编码天冬酰胺(N)。这些被称为TauD421N的敲入小鼠表达一种tau突变体，即不能被半胱天冬酶切割的tau突变体，因此不能产生ΔTau。TauD421N小鼠将用于确定ΔTau是否介导AD和FDD动物模型中的突触可塑性和记忆缺陷，这两种神经退行性疾病中tau起致病作用。我们的研究将讨论ΔTau形成的调节是否是治疗AD和其他由神经毒性tau形式介导的神经退行性疾病的潜在策略。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common cause of ageing-dependent dementia in the world and is associated with cerebral amyloid plaques and neurofibrillary tangles (NFTs). Amyloid plaques are mostly composed of Aß peptides while NFTs are composed of abnormal aggregates of hyperphosphorylated forms of the cytoskeletal protein tau. Aß peptides are produced by a double cleavage of the amyloid precursor protein (APP). BACE1 cleavage produces the C-terminal fragment, ß-CTF, which is then processed into several Aß isoforms by γ-secretase. Genetic data suggest that regulation of APP processing contributes to AD. Current drug discovery approaches in AD have focused on preventing Aß formation or removing existing amyloid deposits. The repeated failures of compounds/biologics targeting amyloid in clinical trials can either be attributed to the inclusion of AD patients who were too advanced in their disease progression to benefit from therapeutic intervention, or to the fact that Aß is not the main cause of AD patho-physiology. The most advanced alternative hypothesis of AD is that of tau toxicity. Consistent with this hypothesis, reducing endogenous tau expression prevents behavioral deficits in transgenic mice expressing human APP with familial AD mutations, without altering Aß levels. Our preliminary data indicate that tau also mediates behavioral deficits in Familial Danish Dementia (FDDKI mice), an AD-like dementia associated with mutations in the regulator of APP processing BRI2/ITM2B, also characterized by tauopathy. Some evidence suggests that caspases are activated early in the progression of AD and may play a role in neuronal loss and NFT pathology. Tau is cleaved at D421 (ΔTau) by executioner caspases. Following caspase-cleavage, ΔTau facilitates nucleation-dependent filament formation and adopts a conformational change recognized by MC1, an early pathological tau marker. ΔTau can be phosphorylated and recognized by the NFT antibody PHF-1. In AD brains, ΔTau associates with markers of NFTs and correlates with cognitive decline. Furthermore, ΔTau initiates NFT formation and can exert toxic effects. These findings have led to the hypothesis that ΔTau is a critical toxic moiety underlying neurodegeneration. To directly test this theory, we have generated knock-in mice in which the endogenous tau codon GAC in exon 12, encoding for D421, has been mutated into AAC, which now encodes for an asparagine (N). These knock-in mice, called TauD421N, express a tau mutant, taucas/res that cannot be cleaved by caspases and therefore cannot generate ΔTau. TauD421N mice will be used to determine whether ΔTau mediates synaptic plasticity and memory deficits in animal models of AD and FDD, two neurodegenerative disorders in which tau plays a pathogenic role. Our studies will speak to whether modulation of ΔTau formation is a potential strategy for the treatment of AD and other neurodegenerative disorders mediated by neuro-toxic tau forms.