Structure and function of α-synuclein fibril polymorphs in Lewy body dementia

Abstract

Accumulation of α-synuclein (α-syn) fibrils throughout the nervous system has been suggested to play a crucial role in the development and progression of synucleinopathies including Parkinson’s disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB). α-Syn fibrils have the propensity to propagate and form cytoplasmic deposits in a prion-like manner. Intracranial injections of synucleinopathy-affected brain extracts containing α-syn fibrils into the model mice initiate the formation of α-syn-enriched cytoplasmic inclusions followed by a neurological dysfunction in the brain. Recent reports discovered specific structural topologies of α-syn fibrils extracted from different synucleinopathies that may underlie distinct pathological patterns. A prevailing hypothesis suggests that the structural composition of α-syn fibrils encodes a crucial functional component that may contribute to disease progression and severity. To explore the structural frustrations that might lead to a gain of adventitious pathogenicity, we first selected two variants of recombinant α-syn fibrils with reported prominent functional differences. Recombinant fibrils generated with mouse α-syn are widely utilized in neuronal and animal models of α-syn pathology formation and spreading models, while human α-syn fibrils poorly template rodents α-syn. Dissection of structural features in a scope of pathogenic traits could illuminate the origins of the observed variations in propagation profiles between two recombinant fibril species. Although the high-resolution structures are available for recombinant human α-syn fibrils, molecular arrangement of mouse α-syn fibrils remains elusive despite their widespread use in mechanistic studies, drug discovery and validation efforts. Using cryo-electron (cryo-EM) microscopy, we procured atomical maps of mouse α-syn fibril structures together with human α-syn fibrils produced under identical buffer conditions. We found that structural arrangement of mouse fibrils closely aligns with MSA-associated fibrils. Mouse fibrils lack characteristic surface hydrophobicity, lack resistance to mild detergents, and are prone to fracturing into shorter fibrils. As opposed to human α-syn fibrils, mouse α-syn fibrils fail to evoke robust cytokine or lysosomal damage responses in myeloid cells in vitro. However, mouse fibrils robustly and more efficiently spread cross-seeded α-syn pathology in neurons expressing human α-syn than equivalent concentrations of human fibrils. Unilateral inoculation with mouse fibrils demonstrate elevated levels of α-syn pathology prorogation as compared to human variant indicating higher pathogenicity profiles. Resultant chimeric fibrils from mouse-to-human α-syn cross-seeding demonstrate amyloid dye and amplification profiles closely matching the original fibril template patterns despite the variations in primary sequence of monomeric protein. Overall, these results provide fundamental insights into pathophysiological properties associated with commonly utilized mouse α-syn fibrils that can be typified by weak β-sheets between fibril rungs, low surface hydrophobicity, weak immunogenicity, but strong potency in spreading templated pathology in neurons. These findings might offer an insight into α-syn fibril structure-to-function relationships driving the pathobiology and underscore the critical importance to develop preclinical models that reproduce the structural and biochemical properties of diseases-associated brain-derived fibrils as an essential step to developing more effective α-syn targeting therapies and diagnostics. The pathophysiological heterogeneity observed within the synucleinopathy subtype, along with the collected findings of variability in propagation profiles of isolated α-syn fibrils across patients, suggests the presence of multiple conformations in a scope of synucleinopathy subtype. Following the approach employed for recombinant model fibrils, we investigated the DLB-associated fibrils harbored from post-mortem cerebrospinal fluid (CSF). Using α-syn seed aggregation assay (SAA) capable of amplifying circulating α-syn fibrils present in DLB CSF, we increased a pool of aggregates for subsequent cryo-EM and functional analysis. We discovered more than 10 different structural conformers across and among the patient-derived fibrils and developed a classification based on interprotofilament interaction, β-sheet stacking, and the geometry of the hydrophobic cavity. To further investigate the functional capabilities of the discovered structures, we selected two homogeneous preparations each sharing a unique structural arrangement. Two conformers with closely matching interprotofilament and hydroponic pocket arrangement, exhibited a distinct β-sheet stacking architecture. These two α-syn conformers possessed antipodal profiles in amyloid dye binding and variable sensitivity to fragmentation by computed mechanistical stress, sonication, and denaturation indicating the significant conformational fluctuation driven by distinct β-sheet stacking. Moreover, we found an α-syn fibril conformer that was more prone to fracture, also responsible for elevated levels of propagation in cellular models and in fibril exposed rodents. Overall variety in structural arrangement of SAA-amplified DLB CSF α-syn fibrils between and within specimens might underlie the heterogeneity in pathophysiological features of DLB highly enriched in α-syn fibrils. Comparison of two conformers differing by exclusively β-sheet stacking facilitated understanding its impact to pathogenicity in propagation levels. In agreement with the previous findings, we confirmed that the β-sheet stacking might control the fibril core stability associated with propagation profiles. These findings highlight the crucial need to further explore the specific structural configurations required for adventitious spread of α-syn-associated pathology and underlying cytotoxicity. Herein, we found that α-syn fibrils of higher susceptibility to fragmentation are more adept at spreading the pathology, as demonstrated with both recombinant and patient-associated fibrils. Furthermore, the discovery of multiple structural variants across CSF samples from DLB specimens may indicate a high degree of conformational plasticity of disease-associated α-syn fibrils. Therefore a more nuanced understanding of the structural diversity of α-syn fibrils in disease context is warranted.