Exploiting PrP for the diagnosis and treatment of protein aggregation diseases

描述（由申请人提供）：神经退行性疾病是影响数千万美国人的主要公共卫生问题，令人信服的证据表明，特定蛋白质的寡聚化和纤维化是几乎所有此类疾病的共同方面。参与神经变性的两种最广泛研究的蛋白质是朊病毒蛋白（PrP）和淀粉样蛋白b-蛋白（Ab）。在争议中，挑衅性的数据已经出现，这表明PrP可能作为介导抗体毒性的受体。虽然Ab与PrP结合的病理学意义仍然存在争议，但完全一致的是，PrP以高亲和力结合Ab寡聚体，并且PrP是Ab的亚化学计量抑制剂 聚合来具有此类性质的试剂具有用于阿尔茨海默病的诊断和治疗的潜力，但迄今为止，靶向和检测疾病相关蛋白寡聚体的努力在很大程度上依赖于抗体的使用。基于我们证明PrP在亲和力、特异性和免疫原性方面优于先导构象特异性抗体的上级证据， 抗聚集活性，我们建议利用PrP产生高效的抗寡聚体试剂。然而，很少有人知道的基础PrP的能力，结合抗体寡聚体和防止抗体单体的聚集的分子基础。因此，我们建议确定参与识别抗体的PrP中的氨基酸，并修改这些以进一步增强PrP结合抗体寡聚体和抑制抗体聚集的能力。PrP内的两段序列涉及寡聚体结合，我们将集中精力研究这些。关键残基的鉴定和PrP与Ab寡聚体结合的进一步优化将通过在这两个位点引入“设计突变”的迭代过程来实现。在每个步骤中，我们将测试诱变的PrP是否可以与Ab寡聚体结合或防止Ab聚集。这种方法应该产生一种重组PrP衍生物-一种可用于定量和靶向毒性Ab寡聚体的“超级结合剂”。然而，PrP衍生物的潜在用途可能超越阿尔茨海默病。由于PrP是已知的结合寡聚体形式的某些设计的b-片层丰富的肽，我们假设，PrP将能够结合到其他蛋白质寡聚体。因此，我们将研究PrP是否可以结合寡聚体和/或防止与帕金森病相关的α-突触核蛋白和与额颞叶痴呆相关的tau蛋白的聚集。在这样做的时候，我们将确定PrP的活性是否对Ab具有特异性，或者是延伸到其他关键疾病相关蛋白质寡聚体的特性。我们还将两个PrP寡聚体结合位点的序列移植到IgG中，以产生独特类型的嵌合抗体，并测试该抗体是否可以识别和保护从人脑中分离的Ab寡聚体。最后，我们将产生含有我们在PrP诱变研究中优化的PrP序列的额外抗体，并针对α-突触核蛋白和tau寡聚体测试这些抗体。通过这种方式，我们将产生新的工具来检测和中和与人类神经变性有关的毒性寡聚体。

DESCRIPTION (provided by applicant): Neurodegenerative diseases are a major public health problem that affects tens of millions of Americans and compelling evidence indicates that oligomerization and fibrillization of specific proteins is a common facet of almost all such disorders. Two of the most widely studied proteins involved in neurodegeneration are the prion protein (PrP) and the amyloid b-protein (Ab). Amid controversy, provocative data have emerged which suggest that PrP may serve as a receptor that mediates Ab toxicity. While the pathological significance of Ab binding to PrP remains contentious, there is complete agreement that PrP binds Ab oligomers with high affinity and that PrP is a sub-stoichiometric inhibitor of Ab aggregation. Agents with such properties have potential for use in both the diagnosis and treatment of Alzheimer's disease, but until now efforts to target and detect disease- associated protein oligomers have largely depended on the use of antibodies. Based on our demonstration that PrP is superior to lead conformation-specific antibodies in terms of affinity, specificity and anti-aggregation activity, we propose to exploit PrP to generate highly potent anti-oligomer reagents. However, little is known about the molecular basis that underlies PrP's ability to bind Ab oligomers and prevent the aggregation of Ab monomer. Therefore, we propose to identify the amino acids in PrP that are involved in the recognition of Ab and to modify these to further enhance PrP's ability to bind Ab oligomers and inhibit Ab aggregation. Two stretches of sequence within PrP are implicated in oligomer binding and we will focus our efforts on these. Identification of the key residues and further optimization of PrP's binding to Ab oligomers will be achieved by an iterative process of introducing "design mutations" in these two sites. At each step we will test if the mutagenized PrP can bind to Ab oligomers or prevent Ab aggregation. This approach should produce a recombinant PrP derivative - a 'super-binder' that can be used for both the quantification and the targeting of toxic Ab oligomers. However, the potential uses of PrP derivatives may go beyond Alzheimer's disease. Since PrP is known to bind oligomeric forms of certain designed b-sheet-rich peptides, we hypothesize that PrP will be capable of binding to other protein oligomers. Consequently we will investigate if PrP can bind to oligomers and/or prevent the aggregation of a-synuclein which is associated with Parkinson's disease, and of tau which is associated with frontotemporal dementia. In so doing, we will determine whether PrP's activity is specific for Ab or is a property that extends to other key disease-implicated protein oligomers. We will also transplant sequences from the two PrP oligomer-binding sites into an IgG to produce a unique type of chimeric antibody and test if this antibody can recognize and protect against Ab oligomers isolated from human brain. Finally, we will generate additional antibodies containing PrP sequences we optimize in our PrP mutagenesis studies and test these against a-synuclein and tau oligomers. In this way we will generate new tools to detect and neutralize toxic oligomers centrally implicated in human neurodegeneration.