Biochimie | EPFL Graph Search

vignette|Représentation tridimensionnelle de la neuraminidase. Les coordonnées des atomes ont été obtenues par cristallographie aux rayons X sur un échantillon de protéine cristallisée. vignette| Relations schématiques entre la biochimie, la génétique et la biologie moléculaire. La biochimie est l'étude des réactions chimiques qui se déroulent au sein des êtres vivants, et notamment dans les cellules. La complexité des processus chimiques biologiques est contrôlée à travers la signalisation cellulaire et les transferts d'énergie au cours du métabolisme. Depuis un demi-siècle, la biochimie est parvenue à rendre compte d'un nombre considérable de processus biologiques, au point que pratiquement tous les domaines de la biologie, depuis la botanique jusqu'à la médecine, sont aujourd'hui engagés dans la recherche biochimique, voire biotechnologique. L'objectif principal de la biochimie de nos jours est de comprendre, en intégrant les données obtenues au niveau moléculaire, comment les biom

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: What you see is not always what you get

Abdelrahman Mohammed Adel Elsaeed Mansour AlOkda, Bryan Frey, Hilal Lashuel, Nathan Alain Denis Riguet

Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (alpha-syn) oligomerization in intact cells. Since then, several alpha-syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased alpha-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that (1) validate the extent to which the BiFC fluorescent signal correlates with alpha-syn oligomerization at the biochemical level; (2) provide a structural characterization of the oligomers and aggregates formed by the BiFC. To address this knowledge gap, we first analysed the expression level and oligomerization properties of the individual constituents of alpha-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 & SH-SY5Y cells from three different laboratories using multiple biochemical approaches and techniques. Next, we investigated the biochemical and aggregation properties of alpha-syn upon co-expression of both BiFC fragments. Our results show that (1) the C-terminal-Venus fused to alpha-syn (alpha-syn-Vc) is present in much lower abundance than its counterpart with N-terminal-Venus fused to alpha-syn (Vn-alpha-syn); (2) Vn-alpha-syn exhibits a high propensity to form oligomers and higher-order aggregates; and (3) the expression of either or both fragments does not result in the formation of alpha-syn fibrils or cellular inclusions. Furthermore, our results suggest that only a small fraction of Vn-alpha-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of alpha-syn-Vc in Vn-alpha-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate alpha-syn oligomerization in cells or in vivo. Altogether, our results suggest that cellular and animal models of oligomerization, aggregation and cell-to-cell transmission based on the alpha-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.

2020

Capturing the heterogeneity of alpha-synuclein pathology in synucleinopathies

Melek Firat Altay

Synucleinopathies such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy (MSA) are detrimental neurodegenerative diseases. Alpha-synuclein (aSyn), the main aggregating protein found in the pathological inclusions of these ailments, exists in numerous modified forms and conformations, leading to neuropathological heterogeneity in synucleinopathies. The functional, pathological and clinical implications of this heterogeneity remain unclear. Responding this knowledge gap requires the development of novel toolsets capable of capturing the biochemical and structural diversity of aSyn pathology in the brain and peripheral tissues.To address this challenge, we describe in Chapter 2 the development and characterisation of antibodies that cover all three regions and post-translational modifications (PTMs) of aSyn. We assess these antibodies through a comprehensive validation pipeline, and select the best performing antibody subset to capture the neuropathological diversity of aSyn across Lewy body diseases (LBDs). We demonstrate that these antibodies could be used to achieve an in-depth characterization of the biochemical and morphological diversity of aSyn aggregates in the neuronal and in vivo models of aSyn seeding. Our results suggest that aSyn pathology is rich in Serine 129 phosphorylation (pS129) and in previously under-described PTMs, including nitration and phosphorylation at Tyrosine 39 (nY39, pY39) and C-terminal tyrosine phosphorylations (pY133, pY136) across neuronal and glial populations in LBDs. We also report that aSyn is hyperphosphorylated in the cellular and in vivo seeding models. In Chapter 3, we demonstrate the power of our antibodies to achieve an unprecedented analysis of astrocytic aSyn pathology in LBDs. We characterise the biochemical properties, post-translational modifications and the aggregation state of astrocytic aSyn accumulations in the limbic regions across sporadic and familial LBDs. The astrocytic aSyn is revealed distinctively by antibodies against the late amino-terminus (N-terminus) and non-amyloid-beta component (NAC) region of aSyn, but not by the extreme N-terminal or carboxy-terminal (C-terminal) antibodies, suggesting that these aSyn species are truncated at both ends. The astrocytic aSyn accumulations are negative for the key markers of Lewy bodies (aSyn pS129, Amytracker, p62- and ubiquitin-positivity and proteinase K resistance), and positive for aSyn nY39 and pY39. These findings suggest that the astrocytic accumulations may be composed of non-fibrillar, possibly oligomeric conformers of aSyn. Chapter 4 focuses on deciphering the biochemical and morphological signatures of aSyn pathology in MSA. The antibodies described in Chapter 2 are applied to high and low pathology regions of MSA brains by immunohistochemistry (IHC). We also biochemically profile the aSyn species, and report an abundance of aSyn pS129-positive glial and neuronal inclusions that correspond to insoluble high molecular weight species by Western blot. We detect an enrichment of insoluble and truncated aSyn in the MSA cerebellum. By IHC, there is an enhancement of aSyn nY39, pY136 and of other aSyn PTMs.Collectively, the new tools and findings described here may pave the way for future studies elucidating the role of these neuronal and glial aSyn proteoforms in disease pathogenesis and propagation in synucleinopathies.

EPFL2022