The amyloid hypothesis as the cause for Alzheimer's Disease (AD) has recently come under fire due to the failure of so many clinical trials for amyloid peptide (A)-targeting therapies. However, it may be that amyloid precursor protein (APP) itself, or the C-terminal fragment- (CTF) produced by -secretase (BACE1) cleavage, is the actual culprit in AD, having a more direct role in AD than previously thought. In the current proposal, we will study a novel approach to reducing early-stage AD by targeting the protein-protein interaction of a complex (km23-1?Rab6) that we propose controls the trafficking of APP and the rate-limiting enzyme (BACE1) in APP processing. More specifically, we will test the novel hypothesis that APP and BACE1 are transported in km23-1?Rab6 vesicles to the Golgi, where CTF is produced to play a role in neurite outgrowth and dendritic branching. We hypothesize, further, that APP or CTF accumulation here in AD causes exuberant and aberrant hippocampal axodentritic sprouting, eventually leading to diminish dendritic arbor complexity and breakage of neuronal branches. We will examine the km23-1?Rab6-mediated transport of APP and BACE1 to the Golgi, as well as the effects of the resulting APP fragments on dendritic branching. The results will provide a stronger basis for targeting the physical association of APP and BACE1 at Golgi sites, to reduce BACE1 activity and the subsequent production of APP intermediates. ? greater understanding of the mechanisms underlying km23-1?Rab6 regulation of APP and BACE1 interactions at the Golgi to control dendritic arborization should facilitate targeting this key trafficking event to reduce early AD-associated causal events. Our structural modeling of the km23-1?Rab6 complex revealed ?hot spots? for the precise sites of protein- protein interaction.
In Aim 1, we will examine the effects of site-specific mutants of the proteins on the spatiotemporal regulation of km23-1?Rab6 complexes, as well as on APP/BACE1 association and compartment location. The focus will be on APP and BACE1 association at soma Golgi, as well as at Golgi outposts (GOs) in developing dendrites, in order to better understand the mechanisms underlying APP trafficking, processing, and signaling at these specific regions.
In Aims 2 and 3, we will us in silico modeling and apply a novel Protein Painting technology to reveal the unique interface by which km23-1 interacts with Rab6 in regulating APP trafficking and processing. The precise interaction region will be used to design corresponding peptide inhibitors to be tested for inhibitory effects on km23-1?Rab6 complex formation, APP processing, and AD-associated pathologies. While the majority of previous studies have used rodent familial AD (FAD) models, here we will apply human models that recapitulate sporadic AD (sAD) to test our novel km23-1?Rab6 inhibitors. The use of the hidden contact regions between these critical interacting proteins as drug targets will lead to paradigm-shifting therapies, overcoming the limitations with past therapeutic strategies. The novel therapeutic agents for AD developed as a result of the proposed studies will be among critical members of the next generation of AD-targeted therapeutics.
The accumulation of sticky amyloid peptides (A) in the brain of Alzheimer's Disease (AD) patients is a major component of the early AD process. It is thought that the breakdown of a protein called amyloid precursor protein (APP) results in A accumulation. Among other evidence, this hypothesis is based on the identification of rare genetic mutations in enzymes involved in APP processing in patients, on studies in mice engineered to carry these familial AD (FAD) alterations, and on the increased copy number for APP in Down syndrome patients who often develop A plaques by age 40. However, the location and regulation of APP processing may differ in sporadic AD (sAD), which accounts for more than 90% of all cases. Thus, it is critical to investigate the precise control of key steps in APP processing in models that recapitulate sAD. We will focus on the rate-limiting step in APP processing to better understand where in the cell and precisely how APP itself, or its products, may contribute to the growth of nerve branches too rapidly, which may ultimately cause the nerve cells to die. Targeting the proteins that link APP to the transport vesicles within cells should lead to novel ways too prevent APP from getting to the site of processing, thereby reducing aberrant signaling events that contribute to AD phenotypes. In our studies, we will also use ?protein paints? to coat the exposed surface of our critical protein complex involved in the control of these outgrowths. The paints can be utilized to predict a new peptide therapeutic agent based on the hidden region where the proteins interact. The results of our studies will lead to novel therapies and prevention strategies for patients with early stage Alzheimer's Disease.
