Amyloid formation plays an important role in a broad range of human diseases, including Alzheimer's disease (AD), Parkinson's disease, and type 2 diabetes (T2D). Islet amyloid polypeptide (known as amylin or IAPP) is an endocrine hormone responsible for pancreatic amyloid formation in T2D. The process of amyloid formation by human amylin (h-amylin) promotes b-cell death thereby contributing to the loss of 2-cell mass associated with advanced stages of T2D. There is increasing evidence that islet amyloid formation is an important complicating factor in islet cell transplantation, and we have recently shown that prevention of islet amyloid can contribute significantly to graft survival. My goals are to define the nature of the toxic species produced during amyloid formation by h-amylin and to identify the mechanism of islet amyloid induced toxicity in T2D. Biochemical and physiological studies have shown that b-sheet containing amyloid precursors can bind and activate RAGE (receptor for advanced glycation end-products). RAGE is a multi-ligand receptor involved in a diverse range of chronic human disorders including cancers, AD and diabetes. RAGE is a potential cell-surface acceptor site for the neurotoxic amyloid-b (Ab) peptides in the brain of individuals with AD, and plays an important role in the pathogenesis of neurological dysfunction and death. Ab amyloid accumulation in the brain increases with RAGE expression, and activation of RAGE is associated with sustained cellular oxidative stress which adversely affects cellular function and organic homeostasis. Given the similar polypeptide sequences and aggregation kinetics of h-amylin and Ab, I hypothesize that RAGE may also be a pathological receptor for h-amylin;and that activation of RAGE by h-amylin binding is a mechanism of islet amyloid toxicity in T2D. To test this hypothesis, I will apply a multidisciplinary approach which utilizes biological, biophysical and biochemical methods. We have recruited the assistance of a world leader in amylin biophysics to assist us and help guide my studies. My preliminary data show that h-amylin binds RAGE in vitro. I will follow up these results by determining which form of h-amylin (monomer, oligomer and/or amyloid) binds to RAGE and/or has the strongest interaction with RAGE. As part of this aim, I will also identify which extracellular domain(s) of RAGE interact(s) with h-amylin using recombinant soluble RAGE (sRAGE) mutants with one or more domain deletions. Independent of the first aim, I will determine whether h-amylin cytotoxicity is RAGE-mediated. To do this I will first identify the form of h-amylin (monomer, oligomer and/or amyloid) is responsible for cytotoxicity during amyloid formation by using thioflavin-T fluorescence assays, far-UV CD, light scattering and TEM in conjunction with time-dependent assays of cytotoxicity. I will determine the role of RAGE in h-amylin mediated cytotoxicity. These studies will make use of inhibitors of h-amylin toxicity and amyloid formation, as well as mutants of h-amylin which oligomerize but are not toxic. Molecules which trap specific toxic conformations populated by h-amylin during amyloid formation will also be used. The ability of these species to bind the soluble extracellular domain of recombinant human sRAGE will be tested and their effects on cultured RAGE +/+ and RAGE -/- primary cells will be analyzed. This work will provide a molecular level understanding of a receptor-mediated mechanism of islet amyloid toxicity in T2D. The studies will provide a nonbiased strategy for investigating amyloid-RAGE interactions that can be applied to other systems. The lessons learned will not only provide insight into potential strategies for the treatment and prevention of T2D, but may also be translated to better controlling pathological amyloidosis associated with other diseases. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page
The goal of this project is to determine whether RAGE plays a role in the pathological amyloid formation and toxicity of human amylin, which is associated with pancreatic islet amyloid and type 2 diabetes. The lessons learned from the proposed studies will provide a nonbiased strategy for investigating amyloid-RAGE interactions that can be applied to other systems. The outcomes of the proposed studies will provide insight into potential strategies for the treatment and prevention of T2D in particular, but may also be translated to better controlling pathological amyloidosis associated with other diseases. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page
