Alpha aynuclein conformers and their interaction with FcγRIIb: implications for Parkinson’s Disease.

Abstract

Evidence suggests that alpha-synuclein (αSN) aggregates in patients living with different synucleinopathies such as Parkinson’s Disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) have a unique structure. It is hypothesized that the structure of the aggregates may contribute to the clinical heterogeneity of these conditions. Fibrils, ribbons and pre-formed fibrils (PFFs) have been used to model these aggregates in experimental models. In addition, the mechanisms behind αSN cell-to-cell transmission are still elusive, but evidence suggests Fc gamma receptor IIb (FcγRIIb) might be involved.
This PhD project will investigate whether the structure of the aggregate would influence the biological properties of αSN. To assess this, fibrils, ribbons and PFFs were generated using human recombinant full-length wild-type αSN and characterized using biophysical experiments. To study αSN cell-to-cell transmission, aggregates were injected into the right striatum of adult wild-type mice. SS-NMR, UV CD and TEM data confirmed a unique structure and conformation for fibrils, ribbons and PFFs. In vitro work shows cell cytotoxicity for SH-SY5Y cells treated with fibrils, ribbons and PFFs, whereas ribbons show higher phosphorylated αSN fluorescence intensity compared to fibrils and PFFs. In vivo work indicates a significantly higher % of Lewy neurites at the right striatum for mice injected with ribbons compared to mice injected with fibrils. In addition, in vivo work shows a trend towards a higher % of Lewy neurites in brain regions distant from the right striatum for mice injected with ribbons and PFFs compared to mice injected with fibrils.
To investigate if the differences observed in the biological properties of αSN ribbons, fibrils and PFFs could be explained by their interaction with FcγRIIb, docking studies, a pull-down assay and SS-NMR were used. To investigate αSN cell-to-cell transmission, aggregates were injected into adult FcγRIIb-/- knock-out mice (mFcγRIIb-/-). Data from the pull-down assay show that PFFs, ribbons and fibrils bind to FcγRIIb. Docking studies suggest that ribbons and PFFs bind to both immunoglobulin-like (Ig) domains on FcγRIIb whereas fibrils bind only to the second Ig-like domain. Information on the binding affinity for each aggregate and FcγRIIb was inconclusive. In vivo data shows that fibrils, ribbons and PFFs are transmitted from cell-to-cell through FcγRIIb since knocking down the receptor expression results in lower % of Lewy neurites at the right striatum.
To conclude, the αSN aggregates have a unique conformation and structure which could potentially explain different biological properties in vitro and in vivo. Data in this thesis suggest that αSN conformation and/or their interaction with FcγRIIb may be used to explain differences in disease severity between individuals and synucleinopathies.

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Identifiers

ORCID for Christina Georgiadou: orcid.org/0000-0001-5331-1978

ORCID for Phil Williamson: orcid.org/0000-0002-0231-8640

ORCID for Jessica Teeling: orcid.org/0000-0003-4004-7391

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