In situ Structural Studies of α-Synuclein Fibrils

by Kevin Sicking
Doctoral thesis
Date of Examination:2025-10-27
Date of issue:2025-11-06
Advisor:Prof. Dr. Rubén Fernández-Busnadiego
Referee:Prof. Dr. Rubén Fernández-Busnadiego
Referee:Prof. Dr. Christian Griesinger
Persistent Address: http://dx.doi.org/10.53846/goediss-11598

 

 

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Abstract

English

Protein aggregation into amyloid fibrils is a hallmark of many human diseases, yet the structural principles that govern fibril organization and their physiological consequences remain incompletely understood. This thesis examined how amyloid fibrils fold, interact with their surroundings, and may be detected in material derived from patients. The first project investigated cardiac amyloid fibrils (AL59) from a patient with systemic light chain amyloidosis. Cryo-electron tomography showed that AL59 amyloid fibrils are consistently decorated by the extracellular matrix protein collagen VI. Interestingly, collagen appeared to helically wrap around the fibrils, revealing a case of chirality transfer from amyloids to matrix components. This finding provides one of the clearest structural examples of how amyloids can impose order on their environment, raising broader questions about how fibril–matrix interactions contribute to tissue pathology. The second project focused on the structural consequences of novel α-synuclein (aSyn) mutations, K58N and G14R, which were recently identified in patients with Parkinson’s disease (PD) and atypical parkinsonism. Single-particle cryo-electron microscopy (cryo-EM) showed that these variants alter the protofilament balance in distinct ways: while wild-type aSyn forms a mixture of single- and double-protofilament fibrils (1PF and 2PF, respectively), K58N stabilises a 2PF arrangement, whereas G14R exclusively forms 1PF fibrils. Notably, G14R fibrils closely resemble pathogenic folds observed in multiple system atrophy and juvenile-onset synucleinopathy, suggesting a structural link between sequence variation and disease phenotype. Together, these results illustrate how single amino acid changes can reweight the structural landscape of aSyn fibrils and bias them towards disease-associated folds. The third project explored whether fibrillar aSyn can be detected ex vivo from living patients by analysing patient-derived biological material using negative-stain transmission electron microscopy and cryo-EM. Despite their low abundance, fibrillar structures were detected that bore morphological resemblance to recombinant aSyn fibrils. This proof-of-concept demonstrates that such samples could provide a minimally invasive route to access native aSyn aggregates in patients. Together, these studies demonstrate the value of structural approaches for bridging the gap between recombinant fibrils, patient-derived material, and physiological context. They reveal how amyloids can shape their environment, how disease mutations bias structural ensembles, and how native fibrils might be sampled from living patients.
Keywords: Alpha-synuclein; Cryo-electron tomography
 
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