Plexins receptors make guidance decisions for cell migration in cardiovascular and neuronal development, disease, and regeneration. Plexins are also associated with higher brain functions, memory and learning. A protein that has been associated with Alzheimer's, Parkinson's and other neuronal diseases and injuries is the Collapsin Response Mediator Protein (CRMP-2) which interacts with several kinases and becomes hyper- phosphorylated alongside their increased activation. The hyper-phosphorylated CRMP-2 then disrupts the formation of actin and microtubule cytoskeletal structures and it thought to impede A? and tau clearance. The intracellular region of plexin is known to interact directly with CRMP and the kinase, Fyn. CRMP can form a bigger complex with Cdk5 and GSK3? also involved in Alzheimer's. Our working hypothesis is that the Fyn- Plexin-CRMP interactions form a scaffold for the association and hyper-activation of several other kinases and that the formation of this complex could be used an early biomarker for the development of neuronal diseases. The proposal has three subaims.
Sub aim 1) seeks to establish the phosphorylation patters of various kinases on the intracellular domains of plexin-A1,-A2 and ?A4 and on CRMP2 (and various complexes) in vitro. 2) The effect that the corresponding phosphomimetic mutations have on the level of activity of the plexins and of the kinases will be studied in vitro. 3) The structure and dynamics of the reconstituted plexin-CRMP-kinase complexes will be examined by a number of biophysical techniques, ranging from 19F NMR, using ?CF3 labeled proteins, pulsed EPR, using spin-labeled proteins, to HD-MS (amide hydrogen exchange-mass spectrometry) as well as computational modeling and extensive all-atom/coarse grained molecular dynamics simulations. Finally, 4) the prominent linear phosphorylation motifs that are detected, will be the subject to a bioinformatics search for similar motifs and will be used as an antigen to generate monoclonal antibodies. The knowledge obtained with the plexin-CRMP-kinase complexes will likely provide a new perspective on AD and other neurodegenerative diseases. An antibody (and eventually complex-disrupting-peptides) will likely inform the future development of regeneration/AD-targeted diagnostics and/or therapeutics. As noted in the description of the R21 mechanism, we seek to establish a proof of concept and provide some of the knowledge, if not early leads towards a biomarker. A plexin project has been established in the Buck laboratory for more than a decade (currently in its 3rd R01 grant cycle), but work on CRMP and AD-associated kinases is an entirely new avenue of research in the applicant's laboratory.
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. A protein that is known to be involved in Alzheimer's, Parkinson's as well as other neuronal diseases is CRMP2 (Collapsin Response Mediator Protein-2). CRMP2 also interacts with several kinases and becomes hyper- phosphorylated alongside the increased activation of the kinases in diseased cellular states. However, how this is accomplished is not understood at the molecular level. Such a complex has been shown to disrupt actin base cytoskeletal and microtubule based structure formation, which in turn leads to a disruption of A? and tau clearance. The goal is to understand the effect of phosphorylation and of the plexin-CRMP2-kinase complex formation on the structural and functional behavior of the component proteins. The ultimate aim is to generate a diagnostic antibody (and/or eventually a peptide-based/derived agent) that detects what are thought to be early processes in the development of neuronal disease.
