Combining expansion microscopy and CRISPR-tagging to understand amyloid-induced synaptic pathology.

Memory loss in Alzheimer’s disease (AD) is thought to be due synaptic dysfunction and synapse loss, pathogenesis initiated by oligomeric amyloid beta. However, there is currently a paucity in our understanding of what happens to not only the structure of synapses, but also to the proteins that localise to them. Moreover, while we know that synapses change shape and number in response to synaptic stimuli, how this is altered in AD is also unclear.
One of the major challenges of studying synapses is the ability to accurately resolve the morphology of synapses as well as the protein that reside within them. This is because the size of synapses falls below the resolution limits of traditional microscopy. A second challenge is the ability to label synaptic proteins in order to visualise the proteins and to subsequently assess their nanoscopic localisation.
Advances in optical microscopy techniques has led to the development of expansion microscopy (ExM) – an approach that allows nanoscale imaging of biological specimens through the physically magnification of biological specimen. Advances in the field of genome editing, and specifically the development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 approaches now allows for the tagging of endogenous proteins in living cells.
This PhD project aims to combine both ExM with CRISPR/Cas9-tagging of synaptic proteins to examine synaptic structures and proteins at the nanoscale. The first part of the project will focus on optimising ExM and CRISPR-Cas9-tagging approaches to visualise synaptic proteins in primary rodent neuronal cultures, ex vivo brain tissue as well as human iPSC-derived neurons. Subsequently, these approaches will be used in combination to examine the nanoscale consequence of amyloid-driven synaptotoxicity, and to subsequently examine how activity(synaptic)-dependent remodelling of synapses is altered in AD.