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

描述(申请人提供)：阿尔茨海默病(AD)表现为细胞外淀粉样沉积(斑块)和称为神经原纤维缠结(NFT)的细胞内聚集体的发展。斑块是由淀粉样蛋白前体蛋白(APP)蛋白分解释放的不溶性多肽淀粉样蛋白B(A2)积聚而成。NFTs源于细胞骨架蛋白tau的异常过度磷酸化，它组装成聚集的成对螺旋细丝(PHF)。越来越多的证据表明，抗体和过度磷酸化的tau之间的协同作用导致AD症状的充分发展。在过去的几年里，在细胞内蛋白质上添加O-连接的N-乙酰氨基葡萄糖单元(O-GlcNAc)可以调节其活性和稳定性。最近，一种调节tau磷酸化的机制被证明涉及O-GlcNAc单位的添加。阿尔茨海默病患者脑组织中tau O-GlcNAc水平明显低于正常脑组织，过度磷酸化的tau蛋白几乎不存在O-GlcNAc。这些结果和其他结果表明，tau O-GlcNAc水平和tau过度磷酸化以一种相互关联的方式联系在一起。在这两种修饰之间保持适当的平衡可能对于避免与AD疾病进展相关的致病tau物种的发展至关重要。这些观察表明，阻止O-GlcNAc从tau中移除将防止过度磷酸化，进而阻止tau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水平会影响APP的处理。因此，药理学上有可能提高tau的O-GlcNAc水平，在较小程度上，APP可以作为一种方法来防止tau过度磷酸化和斑块形成的病理过程。我们最近设计了几种有效的和选择性的O-GlcNAcase小分子抑制剂，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。我们的目标是建立一个原则证明，药物阻断O-GlcNAcase可以防止tau过度磷酸化，从而阻止AD样症状的发展。这项实验将使用阿尔茨海默病的动物模型在体内进行。转基因TAPP小鼠(包含突变形式的人tau和APP)将被口服抑制剂36周，治疗效果将通过蛋白质印迹分析、免疫细胞化学和组织学进行评估。从第12周开始，运动技能和认知能力将通过行为测试进行评估。小鼠将在14、24和36周时被处死，并测试tau磷酸化、NFT形成和抗体沉积的水平。剂量组的结果将与对照组的结果进行比较，仅接受赋形剂，以评估抑制剂对疾病进展的影响。与公共卫生相关：阿尔茨海默病的定义特征是形成致病形式的tau蛋白和淀粉样肽在大脑中沉积。这项拟议的工作将在转基因小鼠身上测试一种参与调节这些蛋白质的酶的抑制剂。转基因小鼠是指出现类似阿尔茨海默病症状的小鼠。如果接受抑制剂的小鼠表现出延缓疾病进展，这将验证这种酶作为药物靶点的有效性，并可能导致一种新的、更有效的治疗阿尔茨海默病的药物类别。

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.