Thesis defense – Ludivine Sabatier

Venue: CARF

Thesis defended in french

IMN
Team : SynTeam

Thesis directed by Florent Laferrière

Title

Alpha-synuclein cerebral amyloids in synucleinopathies : quantification, bioactivity and strain properties

Abstract

The presence of proteinaceous inclusions primarily composed of alpha-synuclein (a-syn), a protein naturally present in the brain, is the hallmark of synucleinopathies, a group of neurodegenerative diseases. These include multiple system atrophy (MSA), Parkinson’s disease (PD), and dementia with Lewy bodies. Within inclusion bodies, a-syn is found in the form of amyloid fibrils. By analogy with prion diseases, it is now generally accepted that synucleinopathies represent distinct a-syn strains. The molecular basis of their divergent phenotypes lies in the structure of the protein’s amyloid assemblies, which differs across these pathologies. The bioactivity of an amyloid – its ability to recruit normal a-syn, induce its aggregation and propagate the pathology – constitutes, alongside structure, a strain property. However, the precise links between structure and bioactivity remain to be established.

Numerous studies have shown that fibrils derived from MSA samples are more bioactive than those from PD samples. Although these studies attempted to normalize or quantify amyloids, we identified several biases related to their identification and quantification, such that these bioactivity measurements may reflect differences in dose rather than in specific bioactivity – i.e. relative to the amount of amyloids. Furthermore, a structural heterogeneity of fibrils exists within MSA itself, but the functional consequences of this heterogeneity remain to be determined.

To this end, our study first focused on the identification of methodological biases, before developing procedures enabling unbiased measurement of the amount of amyloid a-syn in biological samples. We established native blotting methods (drop-blot and filter-blot), which allow evaluation of the relative amyloid load in samples through immunolabelling with specific antibody pairs that we identified. Moreover, as some commercial ELISAs do not allow accurate measurement of a-syn amyloids, due to the use of inappropriate antibodies, we developed two in-house ELISA assays: one detecting all a-syn species, and a second specifically and absolutely quantifying amyloid assemblies.

Thanks to the development of these tools, we were able to accurately quantify amyloid a-syn present in brain samples from synucleinopathy patients, in order to determine their specific bioactivity. We chose to use a HEK-hSyn-YFP reporter cell line as a model.

These in vitro studies showed that fibrils contained in MSA brains are more bioactive than PD fibrils, both when using crude brain homogenates or amyloid-enriched samples obtained by biochemical fractionation from the same brain tissues. Remarkably, by extending these measurements to different brain regions across different patients, we were able to demonstrate that the bioactivity of MSA fibrils was distinct depending on the patient from whom they were derived, while this property was conserved across brain regions within a given patient. These data document the existence of amyloid sub-strain populations within a single strain. This corroborates studies that revealed structural diversity among MSA filaments from different patients. Our work further indicates the absence of notable structural evolution during the course of cerebral disease progression.

Biochemical and functional characterization of the in vitro-generated aggregates will determine whether the strain properties of the brain-derived amyloids are conserved upon replication.

Key words

Alpha-synuclein, protein aggregates, amyloids, neurodegenerative diseases

Publications

Biases in α-synuclein immuno-quantitation: a core problem for basic and ancillary studies of Parkinson’s disease and multiple system atrophy.
Laferrière F, Sabatier L, Claverol S, De Giorgi F, Ichas F. Transl Neurodegener. 2024 Mar 25;13(1):15. doi: 10.1186/s40035-024-0040. PMID: 38528639

Jury

Dr. REZAEI Human, DR INRAE, Université Paris-Saclay Rapporteur
Dr. TORRENT Joan, CRHC INSERM, Université de Montpellier Rapporteur
Dr. LECOMTE Sophie, DR CNRS, Université de Bordeaux Examinatrice
Dr. EL MAMMERI Nadia, CR CNRS, Université de Bordeaux Examinatrice

• Place
  CARF
  
• Dates
  On 23 September 2026 at 14:00