This Program Project seeks to test the hypothese that non-fibrillar aggregated assemblies of amyloid beta (AB), which we have term Abeta-derived active ligands (ADALs), are responsible for triggering processes leading to the glial activation, neuronal plasticity malfunction, synaptic degeneration and cell death in Alzheimer's disease. Extending this hypothesis, it is proposed that particular glial proteins modulate competing Abeta assembly process-to enhance ADAL formation. In this program project we will prepare and characterize ADALs, definign conditions that promote their formation, and we will identify the specific responses activated by ADALs in neurons, glia, mixed cultures and organotypic brain slices. These studies will be coordinated with histochemical studies to establish the relevance of ADALs to AD pathology. The focus of project 1 is the preparation and characterization of ADALs and investigation of conditions reponsible for their formation. We will define optimal in vitro conditions for reproducible formation of ADALs,preparing sufficient quantities for structural, biochemical and biophysical characterization studies, and for investigations of their biological responses in collaboration with Projects 2 and 3. The mechanisms governing ADAL formation will be investigated, with an emphasis on the role played by selected glial co-factor proteins associated wit Abeta in AD. We will extend our studies involving atomic force microscopy (AFM) to provide more detailed analysis of ADAL morphology and distribution, and we will use techniques such as field flow fractionation and native gel electrophoresis for ADAL purification. We will employ laser light scattering, analytical ultracentrifugation and fluroescence polarization and energy transfer techniques to investigate ADAL assembly mechanisms. The major questions to be addressed in this project are: What experimental conditions favor the formation of ADALs and what techniques enable the isolation of homogeneous preparation of ADAL structures? What factors govern the formation of different ADAL structures, and inactive fibrillar or soluble Abeta assemblies? What are the biochemical and biophysical characteristics of ADALs? What specific features of the Abeta 1-42 primary structure are important for assembly into different ADAL and fibrillar structures? What amino acid residues are important for the biological activity of ADALs in neurons? in glia? The integration of these studies with the extensive biological capabilities within this Program Project will define the molecular and cellular processes that lead to the inflammatory pathology, synaptic degeneration and neuronal cell death that occurs in Alzheimer's disease.
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