Alzheimer's disease (AD) and related dementias, such as Parkinson's, are devastating and ultimately fatal conditions for which currently there are no cures. Extensive research efforts have been focused on the study of high risk AD proteins, such as ApoE variants and on the proteins that form amyloid aggregates in the brain, specifically A?. The fundamental molecular mechanisms of AD are still debated. Not only is the characterization of the disease causing agents important, but also how amyloidogenic proteins such as A? interfere with the normal functions of neuronal cells. Since Plexin and Neuropilins (Nrps) are clearly involved in the development and maintenance of higher brain functions, a number of recent reports of their involvement in AD are highly significant. However, early co- immuno-precipitation experiments suggest, rather than prove, a direct interaction between PlexinA4/Nrp1 and A?. Thus, we believe a biophysical approach is needed ?using purified proteins- to directly reveal the interacting protein regions. The proposed side-project is a timely addition to the parent NEI R01 grant as this grant involves Plexin-Neuropilin complexes and their interactions with ligands (such as Semaphorin) and other receptors (specifically cMet and VEGFR2). The study of Plexin- Neuropilin complexes from an AD angle is a new opportunity in our laboratory and it is also the first time we will work on A? a. We posit that the interactions of A? with the Neuropilin co-receptor are likely part of the pathology of AD and the interaction may eventually serve as a biomarker for the early detection of the disease, thus falling within the scope of the Aging Institute's Alzheimer's initiative. The working hypothesis of this supplemental project is that the CUB Neuropilin extracellular domain interacts directly with A? and that this interaction inhibits the function of Plexin (and later VEGFR2). The former receptor is involved in the formation and maintenance of neuronal and vascular cell-cell contacts, while the latter plays a role in angiogenesis, including in pathogenic blood vessel formation in eye diseases such as age related macular degeneration (AMD) and retinal degeneration in diabetes. Indeed, the A? protein is elevated in the retina in both AD and AMD and likely contributes to AMD and other retinal pathologies itself. The synergistic subaims for this supplement request are a) the in vitro validation of A?? interactions between Neuropilin extracellular domains and alongside, b) an in silico discovery/modeling of potential interactions via molecular dynamics simulations. The modeling will inform on the design of mutations to test the interactions, also for c) experiments in model cells where we will examine how functions of the Plexin-Neuropilin system are affected by A? binding. Anticipated outcome and impact: We will understand which protein regions are involved in the interactions, either from experiments and/or from simulations and this information will eventually be useful for raising Nrp1- A? binding site specific antibodies. Mutations in these regions will then be used for further validation of the protein complexes and for functional studies. The newly characterized interaction will add to a growing list of proteins which directly interact with A? / A? precursor, further raising our understanding of the behavior of A? a in AD. As a longer term project (potential R01 proposal) we aim to use the preliminary data generated from this supplement is to solve the structure of Neuropilin domain A? complex by x-ray crystallography, and if higher order oligomers are obtained, possibly by cryo-EM.
Plexins are receptors that make cell guidance decisions in cardiovascular and neuronal development, disease, and regeneration. Plexins are also associated with higher brain functions, memory and learning. These normal functions appear to be compromised by the presence of amyloidogenic A? peptides which likely account for some of the pathology seen in Alzheimer's Disease (AD). Recent data suggest that PlexinA4 with its co- receptor Neuropilin-1 can form complexes with A?. The goal of this supplement proposal is to characterize the A? binding region on Neuropilin and Plexin at the molecular level and to study the effect of A? binding on Plexin-Neuropilin function in a simple model cells. The outcome of the work will likely strengthen Neuropilin as a marker for AD onset. Relevance to the Eye Institute's mission is further provided by the already published finding that A? is elevated in eye diseases such as age related macular degeneration (AMD). The proposed pilot project is within the scope of the parent grant on the molecular level characterization of Plexin-Neuropilin co-receptor interactions.
