Is synuclein aggregation a derived or ancestral trait? Ancestral sequence reconstruction uncovers stepwise evolution of synuclein aggregation

Abstract

Protein aggregation drives many neurodegenerative diseases, including Parkinsons disease, where misfolded -synuclein (Syn) forms fibrillar assemblies that accumulate as Lewy bodies. Although Syn aggregation has been extensively characterized, its evolutionary origins and sequence determinants remain unresolved. Here, we use ancestral sequence reconstruction (ASR) to trace the emergence of fibril-forming ability in the synuclein family. We inferred synuclein phylogeny and experimentally resurrected common ancestors, including ROOT synuclein, the last common ancestor of all synucleins, and key intermediates along the Syn lineage. Strikingly, ROOT synuclein is non-aggregating, demonstrating that fibril formation is an evolved, rather than ancestral property. Aggregation first emerges at the ancestral {beta} node, is retained in Syn, and suppressed in {beta}-synuclein. Biophysical analyses including mass spectrometry and NMR reveal that aggregation aligns with greater complexity and heterogeneity in the monomer conformational ensemble, suggesting that evolutionary sequence changes progressively remodel monomer landscapes to favor fibril formation. Complementing these insights, comparative sequence analysis reveals that the transition from ROOT to -WT is marked by the stepwise acquisition of residues critical for stabilizing the fibril core. Early mutations stabilized the {beta}-arch core, enabling the onset of fibril formation, followed by substitutions that reinforce protofilament-protofilament interactions. Together, ASR defines an evolutionary framework for synuclein aggregation linking progressive sequence evolution and conformational complexity to the molecular origins of Syn fibril formation.