Structural studies of the in vitro assembly of tau and α-synuclein amyloids
Neurodegenerative diseases are characterised by the accumulation of filamentous protein aggregates, which are composed of amyloids. Cryo-EM studies of amyloid filaments isolated from human brains have revealed that specific conformers of tau and α-synuclein are associated with different diseases. These findings suggest that specific molecular mechanisms underlie the formation of filaments in the different diseases. However, studying the molecular mechanisms of amyloid assembly in *post mortem* brains is difficult to study. In my PhD research, I studied the molecular mechanisms of amyloid formation for tau and α-synuclein by developing in vitro amyloid assembly reactions that replicate the same structures as observed in diseased brains. My results are divided into three parts. First, I describe the seeded assembly of recombinant α-synuclein filaments with seeds from brains with Multiple System Atrophy (MSA), and show that seeded assembly does not necessarily replicate the structures of the seeds. The results in this section have important implications when interpreting seeded assembly assays. In the future, it will be important to identify the factors that determine which structures are formed in seeded aggregation experiments. Second, I focused on the *in vitro* assembly of tau. I identified truncated tau constructs, lacking the N- and C-termini, and the *in vitro* assembly conditions, which can accurately replicate disease-relevant folds observed in Alzheimer’s Disease (AD) and Chronic Traumatic Encephalopathy (CTE). These findings are the first to describe the formation of disease specific structures of any amyloid, using recombinant protein *in vitro*. The conditions identified in this study can be used for the development of high affinity binders which are specific to the AD and CTE folds. Finally, I studied the time-resolved filament formation of tau into AD and CTE filament folds. I show that tau filament formation is a step-wise and dynamic process, characterised by the formation of initial intermediate filaments, which I call First Intermediate Amyloids (FIAs), that subsequently mature into AD and CTE folds through a variety of later intermediate amyloid structures. This study is the first to provide a tangible structural characterisation of intermediates of amyloid filament formation.
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