The abnormal assembly of tau protein in neurons is the pathological hallmark of multiple neurodegenerative diseases, including Alzheimer’s disease (AD). In AD, tau assembly begins in the locus coeruleus and transentorhinal cortex, and progresses over years to connected regions in the limbic system and neocortex. Cellular and in vivo models of tau assembly suggest that prion-like propagation may underlie this progression, including the intracellular trafficking, intercellular transfer and seeded aggregation of assembled tau. However, the underlying molecular mechanisms are poorly understood. The goal of my thesis was to investigate tau propagation in situ. I focussed on characterising the ultrastructural context of assembled tau filaments at different stages of propagation, including their subcellular localisation and molecular interactions. To achieve this, I employed electron cryo-tomography (cryo-ET), a powerful technique that allows the visualization of biological samples at molecular resolution in near-native conditions. I wanted to study tau propagation in a disease relevant context. I first focused on human tissue and then developed a neuronal cell model of propagation to enable further investigation.

In the first chapter of my thesis, I investigated the association of assembled tau with brain extracellular vesicles (EVs). This association has been documented in AD and has been linked to the clearance and propagation of assembled tau. However, the molecular species of assembled tau and how they associate are not known. By performing cryo-ET on EVs isolated from AD patient brain, I discovered tau filaments enclosed within the lumen of large EVs. I also observed multiple novel molecular interactions of filaments, including molecules that tether filaments to the EV limiting membrane. These findings suggest that tau filaments are selectively packaged within EVs in AD. Using single particle electron cryo-microscopy (cryo-EM), I found that the filaments contained distinct anionic molecules compared to tau filaments within intracellular inclusions. These results reveal the molecular species and structures of assembled tau associated with brain EVs in AD, as well as how they associated with EVs. This will guide future investigations of the mechanisms of assembled tau secretion, and inform biomarker and therapeutic strategies targeting extracellular tau.

In the second chapter, I investigated tau assembly at the synapse in AD. Synaptic dysfunction is the earliest manifestation of neurodegeneration. Evidence suggests that this may be driven by tau assembly directly at the synapse. How assembled tau contributes to synaptic dysfunction is not known. In addition, synapses may mediate the intercellular transfer of assembled tau during propagation. I analysed synaptosomes isolated from AD patient brain. Using cryo-ET, I found tau filaments in the cytoplasm of pre-synapses. The filaments associated with one another, as well as with vesicles. The filaments were also tethered to the plasma membrane and were decorated with globular densities, similar to the interactions that I observed in brain EVs. These results provide insights into disease mechanisms of tau assembly and guide future studies to ultimately understand synapse dysfunction in AD.

In the third chapter of my thesis, I established a neuronal cell culture model of tau propagation compatible with in situ studies using cryo-ET. Observations made in AD patient brain tissue relate directly to disease. However, they only provide static snap shots of tau assembly and propagation, whereas these are dynamic processes. Therefore, a model system that recapitulates tau propagation is important to further investigate observations made in human brain. I induced tau propagation in primary mouse hippocampal neurons by incubation with purified tau filaments from AD patient brain. I introduced strategies for the fluorescent labelling of tau filaments and for neuronal culture on EM grids in order to make this model system compatible with fluorescent live cell imaging, correlative light and electron cryomicroscopy and cryo-ET. These results lay the foundation for future in situ studies of dynamic propagation events.

During the course of this thesis, I established cryo-ET of human brain tissue to investigate the subcellular localisation of assembled tau and its molecular interactions at different stages of propagation. This revealed tau filaments within EVs and pre-synapses, and identified their tethering to the limiting membranes of these compartments. I then developed a model system to enable in situ studies of dynamic tau propagation events. This work sets the foundations for future studies of the molecular mechanisms of tau assembly and will guide therapeutic strategies and biomarker development to target assembled tau in AD.