Competing modes of assembly restrict amyloid formation by a neurodegenerative protein, TDP43

Abstract

The accumulation of aberrant deposits of specific protein(s) in the brain is a hallmark of many neurodegenerative diseases including amyotrophic lateral sclerosis (TDP43) and frontotemporal lobar degeneration (FUS and/or TDP43). These proteins exhibit “prion‐like” low complexity domains (LCDs), with the defining property of templating their own assembly into highly ordered aggregates known as amyloid. Structural biology investigations from other labs have revealed a multitude of TDP‐43 self‐assembly modes in vitro, ranging from cooperatively folding alpha‐helices to amyloid‐like beta‐sheets of varying stability. However, the extent to which these assembly modes occur in the intracellular environment, and their full relationship to physiological and/or pathological self‐assembly, have not yet been feasible to study.To interrogate mechanistic features of amyloid nucleation by the TDP43 LCD in the cellular milieu, I employed a recently developed FRET‐based flow cytometry approach along with rational mutagenesis informed by existing structural models. I observed that the LCD transitions from monomers to small condensates with a prominent phase boundary. I further found that these condensates were qualitatively distinct from amyloid, and that amyloid occurred probabilistically and only in cells harboring pre‐existing amyloids of other pathogenic proteins.Mutations designed to enhance or disrupt alpha‐helicity exhibited corresponding effects on non‐amyloid condensation, confirming the role of this assembly mode in non‐pathogenic aggregation. Remarkably, amyloid formation exhibited the opposite dependence, with strong alpha‐helix‐promoting mutations eliminating amyloid formation altogether. Mutations designed to probe the role of cooperative beta‐sheet formation revealed that amyloid‐like interactions either promote or inhibit amyloid formation depending on their stability. Importantly, I verified that the full‐length protein follows the same aggregation pathway, but does so at lower concentrations due to additional homotypic interactions mediated by non‐LCD domains. In summary, my data suggested the tendency of TDP43 toward amyloid is balanced by competing alternative modes of self‐assembly.Support or Funding InformationNIH Director’s Early Independence Award DP5‐OD009152 (to RH), the University of Kansas Alzheimer’s Disease CenterStowers Institute for Medical Research