Preclinical testing of an O-GlcNAcase inhibitor to block neurodegeneration for AD

描述（由申请人提供）：阿尔茨海默病（AD）表现为细胞外淀粉样蛋白沉积（斑块）和称为神经原纤维缠结（nft）的细胞内聚集物的发展。斑块是由淀粉样蛋白b前体蛋白（APP）水解释放的不溶性肽淀粉样蛋白b （A2）的积累引起的。nft源于细胞骨架蛋白tau的异常过度磷酸化形式，其组装成聚集的成对螺旋细丝（phf）。越来越多的证据表明，Ab和过度磷酸化的tau之间的协同相互作用导致AD症状的全面发展。在过去的几年里，人们发现在细胞内蛋白上添加O-linked N-acetylglucosamine units （O-GlcNAc）可以调节它们的活性和稳定性。最近，一种调节tau磷酸化的机制已被证明与O-GlcNAc单位的添加有关。AD患者大脑中Tau O-GlcNAc水平明显低于正常大脑，过度磷酸化的Tau携带少量O-GlcNAc。这些结果和其他结果表明，tau O-GlcNAc水平和tau过度磷酸化以相互的方式联系在一起。维持这两种修饰之间的适当平衡可能对于避免与AD疾病进展相关的致病性tau物种的发展至关重要。这些观察结果表明，阻断从tau蛋白中去除O-GlcNAc将防止过度磷酸化，进而阻止tau蛋白聚集体的形成。APP也被证明是O-GlcNAc修饰的，初步报告表明O-GlcNAc水平影响APP的加工。因此，通过药物增强tau蛋白的O-GlcNAc水平具有明显的潜力，在较小程度上，APP可以作为防止tau蛋白过度磷酸化和斑块形成病理过程的一种方法。我们最近设计了几种有效的选择性小分子O-GlcNAcase抑制剂，O-GlcNAcase是负责从蛋白质中去除O-GlcNAc的酶。我们还证明，口服这些抑制剂中的几种可以显著降低大鼠的tau磷酸化水平。我们的目标是建立原理证明，药物阻断O-GlcNAcase可防止tau过度磷酸化，从而阻断ad样症状的发展。本实验将采用AD动物模型在体内进行。转基因TAPP小鼠（含有人类tau和APP的突变形式）将口服抑制剂36周，并使用Western blot分析、免疫细胞化学和组织学评估治疗效果。从第12周开始，将使用行为测试评估运动技能和认知表现。小鼠将在第14、24和36周处死，并检测tau磷酸化、NFT形成和Ab沉积的水平。给药组的结果将与对照组的结果进行比较，以评估抑制剂对疾病进展的影响。公共卫生相关性：阿尔茨海默病的决定性特征是致病性tau蛋白的形成和淀粉样肽在大脑中的沉积。拟议的工作将在转基因小鼠中测试参与这些蛋白质调节的酶的抑制剂；出现类似阿尔茨海默病症状的老鼠如果接受抑制剂的小鼠表现出疾病进展延迟，这将证实这种酶是一种药物靶点，并可能导致一种新的更有效的治疗阿尔茨海默病的药物。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) manifests both by the development of extracellular amyloid deposits (plaques) and intracellular aggregates known as neurofibrillary tangles (NFTs). The plaques result from accumulation of the insoluble peptide amyloid b (A2) that is proteolytically released from the amyloid b precursor protein (APP). The NFTs arise from an abnormally hyperphosphorylated form of the cytoskeletal protein tau that assembles into aggregated, paired helical filaments (PHFs). Accumulating evidence suggests that a synergistic interplay between Ab and hyperphosphorylated tau results in the full development of AD symptoms. It has emerged over the past number of years that the addition of O-linked N-acetylglucosamine units (O-GlcNAc) on intracellular proteins can regulate their activities and stabilities. Recently, one mechanism for regulating tau phosphorylation has been shown to involve addition of O-GlcNAc units. Tau O-GlcNAc levels in human AD brains are markedly lower than in normal brains and hyperphosphorylated tau bears little O- GlcNAc. These results, and others, demonstrate that tau O-GlcNAc levels and tau hyperphosphorylation are linked in a reciprocal manner. Maintenance of an appropriate balance between these two modifications may be critical to avoid the development of the pathogenic tau species that are associated with disease progression in AD. These observations suggest that blocking the removal of O-GlcNAc from tau will prevent hyperphosphorylation and, in turn, arrest the formation of tau aggregates. APP has also been shown to be O-GlcNAc modified, and preliminary reports indicate that O-GlcNAc levels affect APP processing. Accordingly, there is clear potential for pharmacological enhancement of O-GlcNAc levels of tau and, to a lesser extent, APP as a method to prevent the pathological processes of tau hyperphosphorylation and plaque formation. We have recently devised several potent and selective small-molecule inhibitors of O-GlcNAcase, the enzyme responsible for removal of O-GlcNAc from proteins. We have also demonstrated that oral dosing with several of these inhibitors dramatically lowers tau phosphorylation levels in rats. We aim to establish proof-of- principle that pharmacologic blockade of O-GlcNAcase prevents tau hyperphosphorylation and thereby blocks the development of AD-like symptoms. This experiment will be carried out in vivo using an animal model of AD. Transgenic TAPP mice (containing mutant forms of human tau and APP) will be dosed orally with inhibitor for 36 weeks and the effects of treatment will be assessed using Western blot analyses, immunocytochemistry, and histology. Motor skills and cognitive performance will be assessed using behavioral tests from week 12 onward. Mice will be sacrificed at weeks 14, 24, and 36, and tested for levels of tau phosphorylation, NFT formation, and Ab deposition. Results for the dosed groups will be compared to those for control groups, receiving vehicle alone, to assess the effects of inhibitor on disease progression. PUBLIC HEALTH RELEVANCE: The defining features of Alzheimer's disease are the formation of pathogenic forms of tau protein and deposition of amyloid peptide in the brain. The proposed work will test an inhibitor of an enzyme involved in the regulation of these proteins in transgenic mice; mice that develop symptoms similar to Alzheimer's disease. If the mice receiving inhibitor show delayed disease progression, this will validate this enzyme as a drug target and may lead to a new and more effective class of drugs for treatment of Alzheimer's disease.