Elucidating the biophysics of pre-fibrillar, toxic tau oligomers: from amino acid motifs to neuronal dysfunction

Abstract One of the most pressing questions in the study of Alzheimer’s disease (AD) and related dementias (ADRD) is how alterations in the amino-acid sequence of tau, along with post-translational modifications (PTMs) such as phosphorylation and cleavage, lead the protein to misfold and disrupt normal neuronal function. While much has been learned over decades of rigorous and focused research, there are currently no disease modifying therapies to treat AD or related tauopathies. Recently, the field has begun a complicated but promising shift from targeting large tau fibrils (e.g. PHFs and NFTs) to disrupting smaller, non-fibrillar tau oligomers. While late-stage tau fibrils have been studied extensively—including a flurry of recent high-resolution cryo- EM structures—there are few tools to study early-stage oligomers, especially in cells. As a result, almost nothing is known about 1) early misfolding events that produce toxic, non-fibrillar tau oligomers; nor 2) how these oligomers co-opt protein machinery to cause cellular distress. To begin to fill this void, our 2019 Alzheimer’s & Dementia paper established a set of high-resolution, lifetime-FRET based biosensors that monitor full-length tau oligomers in cells. Here, we present compelling preliminary data showing that these biosensors can delineate which folding motifs in the fibril structures, as well as PTMs, most affect early-stage tau oligomers. These biosensors have also enabled us to study two distinct pathological tau interactions in cells. First, co- Investigators Karen Ashe and Kathryn Nelson’s 2016 Nature Medicine paper showed that cleavage of tau by caspase-2 (Casp2) causes tau to mislocalize to dendritic spines, shut down AMPA receptors and promote cognitive defects in mice. We show intriguing evidence to suggest a complex feedback loop between cleavage, oligomerization and toxicity. Second, tau and alpha-Synuclein (aSyn) have well-known co-morbidity in multiple Alzheimer’s Disease related dementias, but the biophysics of their interaction in early-stage misfolding is poorly understood. We provide preliminary evidence of a preferred binding orientation between tau and aSyn, suggesting a stable and hence targetable binding motif. The two major goals of this proposal are to: 1) determine which structural motifs revealed in the available tau fibril structures, and which PTMs, contribute most to early-stage oligomerization in cells, and to pathology; and 2) to characterize and inhibit two pathogenic tau interactions: tau/Casp2 and tau/aSyn. In Aim 1, we analyze the recently available fibril structures and ask: how can these structures be used to unravel otherwise elusive structural details of non-fibrillar tau oligomers? Additionally, to deepen the impact of our investigations, and with the help of co-Investigator Shauna Yuan, we will develop new lines of iPSC-derived human cortical dopaminergic neurons expressing our biosensors. Then, in Aims 2 and 3, we study the biophysical interplay between tau oligomerization and toxicity of tau/Casp2 and tau/aSyn respectively. In each case, we will also perform high- throughput small-molecule screens to identify potent inhibitors of these two pathological, oligomeric assemblies.