α-Synuclein aggregation intermediates form fibril polymorphs with distinct prion-like properties

Abstract

α-Synuclein (α-Syn) amyloid fibrils in synucleinopathies (such as Parkinson's disease (PD), multiple system atrophy (MSA)) are structurally and functionally different, reminiscent of prion-like strains. However, how a single protein can form different fibril polymorphs in various synucleinopathies is not known. Here, we demonstrate the structure-function relationship of two distinct α-Syn fibril polymorphs, the pre-matured fibrils (PMF) and helixmatured fibrils (HMF) based on α-Syn aggregation intermediates. These polymorphs not only display the structural differences, including their fibril core structure as demonstrated by solidstate nuclear magnetic resonance (NMR) spectroscopy and H/D-exchange coupled with mass spectrometry but also possess different cellular activities such as seeding, cellular internalization, and cell-to-cell transmission. The HMF with a compact core structure exhibits low seeding potency in cells but readily internalizes and transmits from one cell to another.Whereas the less structured PMF lacks the cell-to-cell transmission ability but induces abundant α-Syn pathology and triggers the formation of aggresomes in cells. Overall, the study highlights how the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior in PD. IntroductionSynucleinopathies are the group of neurological disorders, which are characterized by the presence of intracellular inclusion bodies composed of α-synuclein (α-Syn) amyloid fibrils 1 . Although α-Syn amyloids in neuronal inclusions termed as Lewy bodies (LBs) and Lewy neurites (LNs) are the characteristic feature of Parkinson's disease (PD), the α-Syn inclusions in oligodendrocytes (glial cell inclusions; GCIs) are predominant in multiple system atrophy (MSA). Previous studies have suggested that amyloid fibrils associated with various neurodegenerative disorders are infectious and exhibit 'prion-like' behavior 2,3 . 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 4,5 . Moreover, the LB/LN-like inclusions are also observed in animals upon receiving intracerebral injections of synthetic α-Syn fibrils or brain homogenates from old transgenic (Tg) mice with α-Syn pathology 6,7 . This suggests prion-like cellular transmission and propagation of α-Syn amyloids in PD and related disorders 8 . In this context, it has been hypothesized that α-Syn assembles into polymorphs, which may account for distinct disease phenotypes observed amongst the synucleinopathies. α-Syn fibrils from GCIs in oligodendrocytes and LBs in neurons of diseased brain samples differ significantly in their structure and exhibit distinct seeding propensity 9 .Although α-Syn fibril polymorphs mostly differ in their fibril diameter, presence of twists, and the number of protofilaments 10,11 , they share a common cross-β-sheet structure with different packing and inter-protofilament interface 12,13 as shown by cryo-...

Structural and Functional Insights into α-Synuclein Fibril Polymorphism

“…These contain highly exposed hydrophobic surfaces and are potentially more toxic than less ordered PMFs. These polymorphs display not only structural differences but also exhibit different biological activities [252]. A similar study involving the identification of the aggregation intermediates in the presence of phospholipid membrane revealed that prefibrillar species contain two loop regions with residues 57 to 61 and 71 to 80 [305].…”

“…Moreover, the structured β-sheet from the residues 74-79 is absent from its NAC domain (residues [65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80], suggesting PMFs to be less ordered fibril types. On the contrary, morphologically and structurally distinct HMFs are more compact and well-ordered with a stable fibril core [252]. These contain highly exposed hydrophobic surfaces and are potentially more toxic than less ordered PMFs.…”

“…In cases where the criteria to be called a 'strain', i.e., should be a structural variant of the protein aggregates, exhibit the ability to self-propagate and serially transmit the disease over the next generations and cause clinical and phenotypical disease variations, are not completely fulfilled, it would be more appropriate to call the fibrils as 'polymorphs.' These polymorphs may show striking differences in their morphology and structure [28,56,251], nucleation rates [252], seeding and membrane binding ability in cells [36], etc. However, as a functional consequence of these structural variations in polymorphs, they may or may not result in distinct clinical subtypes of diseases.…”

“…The mapping of the conformational space of α-Syn monomer reveals a structural subpopulation of α-Syn monomer that promotes its binding with membranes and induces the formation of various oligomers and fibrils [170]. Our lab recently demonstrated that the heterogeneous nucleation during the aggregation pathway forms the basis of the origin of polymorphism [252]. We generated two different polymorphs, HMFs (Helix matured fibrils) and PMFs (Pre-matured fibrils), from the aggregation intermediates formed under the same assembly conditions.…”

Cross-seeding Controls Aβ Fibril Populations and Resulting Function

“…13 Because fibrils are polymorphic, the number of conformations available to a single peptide sequence can be extremely large. 13 As recent studies have shown that structural variants can display differing phenotypes and disease subtypes, [23][24][25][26][27] the large number of possible fibril conformations complicates the development of effective therapeutics and hinders our understanding of the role of fibrils in disease progression.…”