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.
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