INHIBITING AGGREGATION WITH PHAGE DISPLAY ANTIBODIES

与年轻细胞相比，包括错误折叠和聚集在内的蛋白质加工改变在衰老神经元中经常发生。许多神经系统疾病如阿尔茨海默病（AD）和帕金森病（PD）与特定蛋白质或肽的错误折叠和聚集增加有关。β-淀粉样蛋白（Abeta）通过形成细胞外无定形斑块和神经毒性原纤维参与AD的进展，而α-突触核蛋白通过形成细胞内原纤维聚集体参与PD的进展。Abeta和α-突触核蛋白的不同变体和形态与神经毒性聚集体的形成增加相关。早期检测和随后抑制神经毒性聚集体的形成可以减缓或阻止这些疾病的进展。该项目的长期目标是开发可用于鉴定促进神经毒性聚集体形成的关键蛋白质形态的抗体片段;并设计这些抗体，使其能够在体内抑制这些聚集体的形成，作为潜在的治疗方法。噬菌体展示抗体文库将用于分离结合特定形态的Abeta和alpha突触核蛋白的单链抗体片段（scFv）的库。本提案的具体目的是利用噬菌体展示抗体库; 1）分离对各种长度、构象和形态的Abeta特异性的scFv抗体片段，2）分离对各种形态的野生型和突变体α-突触核蛋白特异性的scFv，3）鉴定这些scFv抗体中的哪一种可以在体外抑制Abeta或α-突触核蛋白聚集和原纤维形成，这些scFv抗体中的哪一种可以在体外抑制A β或α-突触核蛋白聚集和原纤维形成，和4）根据需要增加这些抗体的特异性，用于在体内条件下成像和抑制A β或α-突触核蛋白的聚集。通过对分离的亲本抗体进行靶向抗体盲区的随机诱变，可提高抗体特异性。从该第二代抗体库中，将分离具有增加的特异性的scFv片段。将测试这些高特异性抗体在体内条件下抑制神经毒性聚集体形成的能力。将使用不同的技术监测蛋白质聚集，包括硫磺素T染色、原子显微镜和电子显微镜。

Altered protein processing including misfolding and aggregation is a frequent occurrence in aging neurons compared to younger cells. A number of neurological diseases such as Alzheimer's Disease (AD) and Parkinson's Disease (PD) have been connected with increased misfolding and aggregation of specific proteins or peptides. Beta-amyloid (Abeta) is involved in the progression of AD through formation of extracellular amorphous plaques and neurotoxic fibrils, while alpha-synuclein is involved in the progression of PD through formation of intracellular fibrillar aggregates. Different variants and morphologies of Abeta and alpha-synuclein have been correlated with increased formation of the neurotoxic aggregates. Early detection and subsequent inhibition of neurotoxic aggregate formation can slow or stop the progression of such diseases. The long term goal of this project is to develop antibody fragments which can be used to identify critical protein morphologies which promote neurotoxic aggregate formation; and to engineer these antibodies so they can inhibit formation of these aggregates in vivo as a potential treatment. Phage display antibody libraries will be utilized to isolate pools of single chain antibody fragments (scFvs) which bind to particular morphologies of Abeta and alpha synuclein. The specific aims of this proposal are to use phage display antibody libraries to; 1) isolate scFv antibody fragments specific to various lengths, conformations and morphologies of Abeta, 2) isolate scFvs specific to various morphologies of wild-type and mutant alpha-synuclein proteins, 3) identify which of these scFv antibodies can inhibit either Abeta or alpha-synuclein aggregation and fibril formation in vitro, which of these scFv antibodies can inhibit either Abeta or alpha-synuclein aggregation and fibril formation in vitro, and 4) increase the specificity of these antibodies as needed for imaging and inhibiting aggregation of Abeta or alpha- synuclein under in vivo conditions. Antibody specificity will be increased by subjecting the isolated parent antibody to random mutagenesis of targeted antibody blinding regions. From this pool of second generation antibodies, scFv fragments with increased specificity will be isolated. These high-specificity antibodies will be tested for their ability to inhibit formation of neurotoxic aggregates under in vivo conditions. Protein aggregation will be monitored using different techniques including Thioflavin T staining, atomic microscopy, and electron microscopy.