Investigating the Mechanisms and Impact of Amyloid-Beta Mediated Disruption of Synaptic Nanoachitecture

Project Summary The nanoscale organization of key synaptic proteins is now considered an important contributor to synaptic activity and plasticity. However, there is a lack of research on how such delicate nano-organization might be involved in brain diseases, which often directly impact synaptic function. Alzheimer’s Disease (AD) is associated with brain accumulation of the Amyloid-Beta (Aß) peptide. Furthermore, oligomeric assemblies of the 42 amino acid variant, Aß42, which directly target synapses, have been found especially pernicious toward synaptic function. In preliminary research, I have observed through multiple replicates that a brief, 15-minute application of 500 nM Aß is sufficient to disrupt existing synaptic nanostructure. This manifests as the addition of nanoscale objects of the PSD95 scaffolding protein, and of GluA1 subunit-containing AMPA-type glutamate receptors, a phenomenon we have titled, “fragmentation”. As this observation occurs along the same timescale as Aß- mediated NMDA-type glutamate receptor dysfunction and deficits to LTP (Long-Term Potentiation), I hypothesize that Aß-mediated fragmentation of synaptic nanostructure ultimately leads to deficits in synapse function and plasticity. In this proposal, I aim to thoroughly characterize both the kinetics, and dose-response of Aß-mediated fragmentation, as well as to what extent this fragmentation occurs (i.e., does fragmentation include select synaptic proteins, or completely restructure synaptic nanoarchitecture). I also plan to assess what signaling mechanisms underlie Aß-mediated fragmentation and whether these are similar, or distinct from already established mechanisms causing Aß-mediated synaptic dysfunction. Lastly, my preliminary data show that I can prevent the fragmentation of AMPAR nanostructure at the synapse by crosslinking receptors with a GluA1 antibody prior to Aß exposure. Using this method, as well as others our lab has developed, I will then test whether Aß-mediated synaptic nanostructure fragmentation is responsible for LTP deficits and synapse loss. Importantly, this fellowship proposal provides the opportunity for my extensive training in multiple experimental techniques including: super-resolution microscopy (multicolor and live-cell STED imaging, including preparation of STED-dye conjugated primary antibodies), writing computer based imaging-analyses, receptor signaling pharmacology, molecular cloning (CRISPR-based knockout / knockin strategies, optogenetic / chemogenetic construct design), electrophysiology and Ca2+ imaging, and 2-photon glutamate uncaging.