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Structural characterization of alpha-synuclein aggregates seeded by patient material


Strohäker,  T.
Research Group of Protein Structure Determination using NMR, MPI for Biophysical Chemistry, Max Planck Society;

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Strohäker, T. (2018). Structural characterization of alpha-synuclein aggregates seeded by patient material. PhD Thesis, Georg-August-Universität, Göttingen. doi:10.53846/goediss-7768.

Cite as: https://hdl.handle.net/21.11116/0000-0005-5C44-8
Neurodegenerative diseases share a common underlying pathologic hallmark, the appearance of insoluble protein aggregates in diverse tissues of the nervous system. For many neurodegenerative diseases a common temporal and spatial spreading of the pathology is proposed in analogy to prion disease and discussed under the term “prion-like”. For many diseases the major component of the insoluble protein aggregates is known and aggregation into higher molecular weight amyloid fibrils with intermolecular beta-sheet rich cores can be studied in vitro. The aggregation process involves the templated misfolding and aggregation of native monomeric proteins, involving severe conformational changes. An important family of neurodegenerative diseases is caused by the misfolding and aggregation of the protein alpha-synuclein, the so-called synucleinopathies. Alpha-synuclein, which in vivo forms disease-specifically the main component of intracellular inclusions such as Lewy bodies in neurons and cytoplasmatic inclusions in glial cells, undergoes in vitro dramatic conformational changes from a monomeric intrinsically disordered state over transient oligomeric beta-sheet rich species into highly ordered alpha-synuclein fibrils. Alpha-synuclein pathology in patients is diverse and there are clinically distinct disease entities with defined pathologic phenotypes among those Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are the best characterized. Similar to prion diseases, key differences within the broad clinical representation of synucleinopathies are thought to be structurally encoded by distinct protein aggregate conformations, referred to as alpha-synuclein polymorphs. The aim of the study was to amplify alpha-synuclein aggregates from brain extracts of patients thoroughly diagnosed on the basis of the molecular pathology as well as the clinical symptoms as PD, DLB and MSA, using the established protocol of protein misfolding cyclic amplification (PMCA). A combination of hydrogen-deuterium (HD) exchange coupled to nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) and the specific binding of fluorescent probes to amyloid fibrils was chosen to obtain single-residue resolution of the conformational properties of brain-extract seeded alpha-synuclein fibrils. The same approach was also applied to two well-characterized in vitro alpha-synuclein polymorphs, their aggregation was performed in the absence of brain extract seeds following published aggregation procedures and they acted as internal references for benchmarking the methodological approach. On the other hand, the availability of a high-resolution cryo-electron microscopy model of the fibrillar core for one of the in vitro alpha-synuclein polymorphs obtained under high salt conditions, allowed direct correlation of the residue-specific conformational restraints to a structural model, both for in vitro polymorphs of alpha-synuclein as well as brain-extract amplified alpha-synuclein fibrils of PD, MSA and DLB. Distinct highly ordered conformational features of in vitro alpha-synuclein fibrils were successfully reproduced, detecting solvent-protected residues with high precision and in agreement with published data. In contrast, alpha-synuclein fibrils amplified from brain extracts were more flexible and differed structurally from in vitro fibrils. Hydrogen-deuterium exchange coupled to NMR spectroscopy identified a common solvent-protected core shared among all patient brain derived alpha-synuclein fibrils for the synucleinopathies PD, MSA and DLB. The solvent-protected fibrillar core was formed by the most hydrophobic residues of alpha-synuclein. Outside the common core structure, alpha-synuclein fibrils derived from brain extracts differed disease-specifically in the conformation. Residue-specific conformational differences in core-flanking residues of alpha-synuclein as well as in defined N-terminal regions were observed. This study establishes a strong correlation between alpha-synuclein aggregate structure and the disease phenotype for the synucleinopathies Parkinson’s disease, Dementia with Lewy bodies and multiple system atrophy and the data provide further insight in “prion-like” features of neurodegenerative diseases in general and synucleinopathies in particular. The work presented here is a step forward towards the understanding of alpha-synuclein pathology and hopefully contributes to improved disease diagnosis and treatment of synucleinopathies.