Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson's disease

Lewy bodies (LBs) are intraneuronal inclusions consisting primarily of fibrillized human α-synuclein (hα-Syn) protein, which represent the major pathological hallmark of Parkinson's disease (PD). Although doubling hα-Syn expression provokes LB pathology in humans, hα-Syn overexpression does not trigger the formation of fibrillar LB-like inclusions in mice. We hypothesized that interactions between exogenous hα-Syn and endogenous mouse synuclein homologs could be attenuating hα-Syn fibrillization in mice, and therefore, we systematically assessed hα-Syn aggregation propensity in neurons derived from α-Syn-KO, β-Syn-KO, γ-Syn-KO, and triple-KO mice lacking expression of all three synuclein homologs. Herein, we show that hα-Syn forms hyperphosphorylated (at S129) and ubiquitin-positive LB-like inclusions in primary neurons of α-Syn-KO, β-Syn-KO, and triple-KO mice, as well as in transgenic α-Syn-KO mouse brains in vivo. Importantly, correlative light and electron microscopy, immunogold labeling, and thioflavin-S binding established their fibrillar ultrastructure, and fluorescence recovery after photobleaching/photoconversion experiments showed that these inclusions grow in size and incorporate soluble proteins. We further investigated whether the presence of homologous α-Syn species would interfere with the seeding and spreading of α-Syn pathology. Our results are in line with increasing evidence demonstrating that the spreading of α-Syn pathology is most prominent when the injected preformed fibrils and host-expressed α-Syn monomers are from the same species. These findings provide insights that will help advance the development of neuronal and in vivo models for understanding mechanisms underlying hα-Syn intraneuronal fibrillization and its contribution to PD pathogenesis, and for screening pharmacologic and genetic modulators of α-Syn fibrillization in neurons.

Recapitulating De Novo Alpha-Synuclein Fibrillization in a Novel Neuronal Model of Parkinson's Disease

Mohamed Bilal Fares

Athough it has been established for over 100 years that Lewy bodies (LBs) represent the major pathological hallmark of Parkinson's disease (PD), we still do not know why these fibrillar intra-neuronal inclusions of the α-Synuclein (α-Syn) protein form, and how they contribute to disease progression. One of the major causes underlying this gap in knowledge is the unavailability of rodent animal models that truly reproduce the de novo formation of LBs. Although doubling α-Syn expression is sufficient to cause PD in humans, hα-Syn over-expression in rodents does not lead to the formation of authentic LB-like structures. This caveat has significantly hindered efforts towards understanding the mechanism underlying LB formation, their link to neurology, and the screening for drugs and pathways that block their formation. In the first chapter of this thesis, we show that the lack of hα-Syn fibrillization in rodent models can be attributed to interactions between hα-Syn and its endogenously expressed mouse α-Syn homologue. As such, hα-Syn over-expression on mouse α-Syn KO backgrounds provokes the formation of inclusions that reproduce several histological and biochemical features of LBs in cultured primary neurons, as well as in mouse brains in vivo. To unequivocally determine the authenticity of the formed inclusions, we performed correlative light/electron microscopy which established the fibrillar ultrastructure of the inclusions, and real-time FRAP/photo-conversion experiments which clearly revealed the incorporation of soluble protein into inclusions within living neurons. Moreover, we showed that the deletion of another endogenous α-Syn homologue (Î²-Syn) also provokes hα-Syn fibrillization, thereby proposing a role for these two homologous proteins as natural inhibitors of abnormal aggregation. These findings provide novel well-characterized primary neuronal and in vivo models which recapitulate some of the main molecular features of PD, including de novo α-Syn fibrillization. We believe that these models pave the way towards a systematic understanding the mechanisms underlying hα-Syn intra-neuronal fibrillization, as well as the contribution of this process to the pathogenesis of PD. These models also present powerful tools for screening pharmacologic and genetic modulators of fibrillization, monomer incorporation and clearance, all of which could help in generating novel therapeutic strategies for PD. In the second chapter of this thesis, we investigate how α-Syn PD-linked mutations cause pathology, independent from affecting α-Syn aggregation potential per se. Specifically, we explored how a novel α-Syn mutation (G51D) that has been recently identified in patients showing features of PD and multiple system atrophy, affects the biophysical and cellular properties of α-Syn. Indeed, our results suggest that unlike all other PD-linked mutations which have been shown to enhance α-Syn oligomerization and/or fibril formation, the G51D mutant aggregates significantly slower than WT α-Syn. In addition, the G51D mutant exhibits impaired membrane binding in vitro and in yeast, is secreted more rapidly by mammalian cells and is enriched in the nuclear compartment; an effect that is concomitant with enhanced S129 phosphorylation and exacerbated mitochondrial fragmentation. These results intriguingly suggest that PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be actually independent of α-Syn aggregation

EPFL2015

Elucidating the role of post-translational modifications of alpha-synuclein using semisynthesis

Mirva Hejjaoui

Alpha-synuclein (α-syn) is a natively unfolded protein that is closely linked to Parkinson’s disease (PD) by genetic, neuropathologic and biochemical evidence. Aggregated and fibrillar forms of α-syn are the main components of intracellular protein inclusions found in PD patients’ brains, termed Lewy Bodies (LB). Both in animal models and in vitro, α-syn forms fibrillar aggregates that resemble those observed in PD brain tissues. Although disease-associated mutations have been shown to promote the fibrillization of α-syn, the exact mechanisms responsible for triggering α-syn aggregation and toxicity in sporadic PD remain unknown. Addressing this gap of knowledge is crucial for understanding the molecular basis of the disease and developing effective therapies for the treatment of PD and other synucleinopathies. This project was initiated on the basis of the working hypothesis that post-translational modifications (PTM) may play important roles in modulating α-syn function and/or regulating its aggregation and toxicity. More specifically, α-syn is ubiquitously N-terminally acetylated, and phosphorylated (serines 87 and S129), ubiquitinated (lysines 12, 21 and 23) and truncated forms of α-syn have been observed in association with wild-type α-syn in LB and in brain tissues from PD patients and transgenic animals. Other modifications, such as phosphorylation at tyrosine 125 (Y125), were significantly reduced in diseased brains. Despite the discovery of candidate enzymes that mediate α-syn phosphorylation, ubiquitination and truncation, little is known about how each of these modifications alters α-syn structure, function, aggregation and toxicity in vivo. This is primarily due to the lack of tools that allow site-specific introduction of these modifications and the lack of natural mutations that can mimic the effect of these modifications, including phosphorylation. The primary focus of this thesis was to develop strategies to overcome these limitations so as to elucidate the effect of phosphorylation at Y125 and monoubiquitination at lysine 6 (K6) on α-syn structure, fibril formation, membrane binding and subcellular localization. Towards this goal, we developed two semisynthetic strategies that allow site-specific introduction of PTM in α-syn based on the ligation of synthetic peptides containing the desired modified amino acid with recombinantly expressed proteins using Expressed Protein Ligation. This approach enables the introduction of single or multiple PTM in the N- or C-terminal regions of α-syn, and the preparation of modified α-syn in milligram quantities. Using these approaches, we were able to show for the first time that ubiquitination stabilizes the monomeric form of the protein and inhibits, rather than promotes, α-syn aggregation, while phosphorylation at Y125 does not significantly change the structure and aggregation propensity of α-syn. With the semisynthetic pY125 α-syn in our hands, we were also able to investigate for the first time the sub-cellular localization of pY125 α-syn through its microinjection into primary neurons. This was not previously possible due to technical limitations related to the absence of appropriate antibodies against pY125 α-syn for immunocytochemical studies and difficulties in generating sitespecifically modified α-syn. Furthermore, we were able to investigate the effect of α-syn phosphorylation on its interactions with other proteins, by probing the effect of pS129 and pY125 on the binding to a nanobody that was specifically selected by phage-display to tightly bind to the C-terminal domain of α-syn. Accordingly, we demonstrated that phosphorylation at a single residue is capable of disrupting the binding of full-length α-syn to another protein. These results have wide-ranging implications for the potential role of phosphorylation and other PTM in regulating α-syn’s function(s). We also exploited our ability to combine semisynthetic and enzymatic approaches to investigate potential cross-talk between different PTM, namely phosphorylation at Y125 and S129 and monoubiquitination at K6. These advances would eventually allow investigating the effect of cross-talk between other N and C-terminal modifications on α-syn’s properties and to investigate PTM-dependent protein-protein and protein-ligand interactions in vitro and in cellular models of synucleinopathies. Together, our semisynthetic approaches provide novel means for the introduction of site-specific modifications in the N- and C-termini of α-syn. Current efforts in our laboratory are focused on extending these approaches to investigate the dynamics of PTM through the use of photocaged modified amino acids and to prepare novel fluorescently labeled α-syn variants to investigate its folding at the single-molecule level in living cells. The results presented in this thesis and preliminary studies from our group demonstrate that semisynthetic α-syn can provide unique opportunities to investigate structure-function of α-syn and the role of PTM in the biology of α-syn in health and disease.

EPFL2012

Elucidating the role of post-translational modifications of alpha-synuclein using semisynthesis

Mirva Hejjaoui

EPFL2012

Recapitulating De Novo Alpha-Synuclein Fibrillization in a Novel Neuronal Model of Parkinson's Disease

Mohamed Bilal Fares

EPFL2015