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

University Of Colorado Denver, Aurora CO

Investigators

Abstract

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.