Synucleinopathies are a group of neurodegenerative disorders that are marked by the presence of intracellular inclusion bodies composed of α-synuclein (α-Syn) amyloid fibrils.1 In Parkinson's disease (PD), these inclusion bodies are deposited in the neurons of the patients and termed ‘Lewy bodies (LBs) and Lewy neurites (LNs),’ whereas, in multiple system atrophy (MSA), these are predominant in oligodendrocytes and termed ‘glial cytoplasmic inclusions (GCIs).’ Previous studies have suggested that amyloid fibrils associated with various neurodegenerative disorders are transmissible and exhibit prion-like behavior.2 For example, exogenously added α-Syn fibrils readily internalize in cells and induce the aggregation of endogenous soluble α-Syn into pathogenic insoluble LB-like inclusions.3, 4 Similar inclusions are also observed in animals upon receiving intracerebral injections of synthetic α-Syn fibrils or brain homogenates from transgenic (Tg) mice with α-Syn pathology.5, 6

Further, it has been postulated that α-Syn assembles into fibril polymorphs, which accounts for distinct disease phenotypes observed among synucleinopathies. Polymorphism is a key feature of amyloid fibril structures, but it is challenging to explain the origin of these variations. Polymorphism exists in recombinant fibrils,7, 8, 9, 10 and fibrils extracted from a diseased animal or patient's brain.11, 12, 13 For instance, α-Syn fibrils from GCIs in oligodendrocytes and LBs in neurons extracted from diseased brain samples differ significantly and exhibit distinct seeding propensity.11 α-Syn fibril polymorphs mainly show differences in their fibril diameter, the presence of twists, and the number of protofilaments.7, 14 However, in some cases, they might share a common cross-β-sheet structure with different packing and inter-protofilament interfaces,15, 16 as shown by cryo-EM studies.

Several experimental conditions have been employed to develop α-Syn fibril polymorphs in vitro,7, 8, 17, 18, 19 exhibiting distinct clinical and pathological phenotypes in the tested model system.9, 10 However, the origin of the fibril polymorphism and the structure-pathogenic relationship of these polymorphs are not well understood. The stochastic nature of the aggregation process and the existence of multiple inter-convertible forms of the same protein adds another layer of complexity to fibril polymorphism.20, 21 The formation of fibrils polymorphs is even more complicated in vivo due to complex interactions between the biomolecules and conditions defined by the cellular milieu.11, 19, 22, 23

Since α-Syn aggregation involves a heterogeneous population of intermediate species,21, 24, 25 we hypothesize that these intermediate species are capable of forming α-Syn fibril polymorphs even under identical assembly conditions. To test the same, we adopt a unique approach wherein we isolate the aggregation intermediates and generate two fibrillar polymorphs, named the pre-matured fibrils (PMFs) and helix-matured fibrils (HMFs). These polymorphs show differences in fibril packing, secondary structure, and prion-like properties. HMFs are more compact with a stable fibril core, readily internalize in different cells, and show an increased ability for intercellular transfer. In contrast, PMFs, without a robust protease-resistant fibril core, lack the intercellular transfer ability but show high seeding potency both in vitro and in cells compared to HMFs. The present data support that the relative population and/or type of intermediate species might dictate the property of fibrils polymorphs and their biological activities associated with pathological traits in synucleinopathies.