PhD title: Fibrillation of proteins involved in neurodegenerative diseases: influence of mRNA

We are looking for candidate who will represent the team and participate to the doctoral school contest (end of June) to obtain his PhD funding for 3 years (oct 2018 – Sept 2021). Candidate should be Biologist interested in Biophysical approaches. Skills in protein production and purification are welcome. Candidates are invited to apply (CV + motivation letter) until the end of April at this e-mail address: loic.hamon@nulluniv-evry.fr

Keywords:
RNA/protein complexes, fibrillation, neurodegenerative diseases, structure, prion like domain, mutation, phosphorylation

Localisation:
The PhD will be held in the Laboratoire Structure Activité des Biomolecules Normales Et Pathologiques which belong to the Paris-Saclay University. The Lab is localized in a recent building in Evry (25 kms in the south of Paris) and is under the scientific direction of Pr D. Pastré. Three main thematic are developed in the lab: i) mRNA translational control by mRNA binding proteins, ii) mRNA splicing, iii) mRNA binding protein aggregation in neurodegenerative diseases. Our research projects beneficiate from several facilities that are present in the lab including NMR spectroscopy, AFM microscopes, fluorescent microscopies, RT-PCR, cell culture and protein production and purification facilities. The team is large with 25 members and international with 12 different nationalities from all over the world.

State of art:
TDP-43 and FUS, two RNA-binding proteins (RBPs), have been recently the subject of increased attention due to their role in Amyotrophic Lateral Sclerosis (ALS) and FrontoTemporal Lobar Degeneration (FTLD). In non-pathological conditions, these proteins are mostly found in the nucleus where they participate to mRNA biogenesis and regulation of alternative splicing. However, TDP-43 and FUS are also present in the cytoplasm and they are able to shuttle from one compartment to another. TDP-43 and FUS notably participate in cytoplasmic functions such as mRNA transport and localized translation, which are of critical importance for neuron physiology. In neurons of ALS and FTLD patients, TDP-43 and FUS form insoluble cytoplasmic aggregates with fibrillar structure that can further spread the disease to other areas of the brain.

The prion-like behavior of TDP-43 and FUS has been attributed to their long self-attracting domains of low complexity (LCD) that enable weak and multivalent homotypic interactions mediating the formation of liquid droplets and fibril formation. In line with the role of LCD in ALS and FTLD, many mutations identified as genetic factors driving these diseases are found within this domain of TDP-43 and FUS. Consequently, preventing the fibrils formation of free TDP-43 and FUS has been considered as a putative therapeutic route for disease treatments but has been so far disappointing as many weak interactions in the LCD are involved. Exploring the role of mRNA in the formation of TDP-43 and FUS fibrillar inclusions may provide a new angle to tackle this issue. Indeed, mRNA is known to favor phase separation of RBPs with LCD, then, the toxicity of TDP-43 and FUS requires their intact mRNA binding capacity and last, but not least, stress granules (SGs) which are large mRNA-rich cytoplasmic granules formed during cellular stress  also sequester FUS and TDP-43. Thereby, stress granules are considered as putative crucibles for the genesis of TDP-43 and FUS fibrillar inclusions, although this hypothesis remains to be further supported by experimental evidences.

To better document the role of protein mutations and mRNA in TDP-43 and FUS fibrils formation, we propose a single molecule approach using Atomic Force Microscopy (AFM). Most in vitro investigations have been rather focused on phase separation observations by fluorescence microscopy. Although very useful, resolution limit precludes structural analysis of fibrillar structures that would precise the molecular mechanism underlying liquid phase formation and possibly the transition toward insoluble aggregates. Then, regarding classical biochemical methods such as high-speed centrifugation, chromatographies and gel-shift assays, these methods are not appropriate to explore dynamic structures such as mRNA granules which result from the permanent exchange of mRNA and proteins with the environment. The thermodynamic equilibrium is indeed disrupted whenever mRNA granules are separated from their environment by classical biochemical methods. In line with this, SG integrity is disrupted upon cell permeabilization with detergents, which renders their isolation a challenging issue. In addition, while centrifugation has been used successfully to identify proteins enriched in mRNA granules, the structural composition of the pellet could be heterogeneous as mRNA granules and protein aggregates can coexist. Finally, the exploration by our team, using high resolution AFM (Abrakhi et al. ACS Nano, 2017), of the interaction between various RBPs and TDP-43- and FUS-RNA complexes has enabled to distinguish the structural signatures of protein aggregates, granules containing mRNA (mRNA granules) and isolated RBP-RNA complexes without any separation step.

This last point clearly confirms that the characterization of fibrils structures in the TDP-43 and FUS aggregates and the detection of subtle changes in these structures due to RNA and/or protein mutation, truncation or phosphorylation could be obtain using AFM microscopy.

Research Program
Objective #1: Structural characterization of fibrillar aggregates of TDP-43 and FUS (Full length, truncated, mutated and phosphorylated). Numerous studies evidence that the cytoplasmic accumulation of TDP-43 and FUS proteins is related to their modification (mutation, deletion, phosphorylation) and the fibrils dynamics and structure could be modified according to the protein constructs. Indeed, it has been demonstrated that, for example, the C-ter domain of TDP-43 is enriched in cytoplasmic inclusion in ALS patient cells. Concerning FUS, mutation in the LCD induce further phase transition into poorly soluble fibrillar hydrogel. Last but not least, phosphorylation of both FUS and TDP-43 has been recently found to impede protein polymerization. At the end of this objective, we will have a clear overview of the different structures obtained by the aggregation of these proteins and especially, the impact of mutation, deletion… on the fibril structure and stability.

Task 1-1: production and purification of the different TDP-43 and FUS construction (full length, truncated, mutated and phosphorylated).

Task 1-2: biochemical characterization of the propensity of each protein construct toward aggregation.

Task 1-3: Structural analysis at nanometric scale of the different fibrils obtained depending on protein sequences.

Objective #2: To determine the influence of mRNA on both the kinetics of assembly of the fibrils and their structure. We will also explore the consequence of specific RNA sequence that may impact the affinity between RNA and proteins, on protein fibrillation. Finally, we will test the effect of drugs developed by our collaborators in Taïwan on fibril structure and stability in order to validate their efficiency and their universality.

Task 2-1: design and synthesis of different mRNAs

Task 2-2: effect of mRNA on fibrils structure and dynamics by AFM corroborated by biochemical approaches

Task 2-3: effect of drugs on fibril structure and dynamics in the presence or not of mRNA.

Originality, Feasibility and perspectives:
Originality: this project aims at deciphering the stability of key structures (fibrils) involved in many neurodegenerative diseases. The approach (AFM) will bring out information at nanometer scale that are out of reach from methods used generally in the literature.

Feasibility:
This approach has been validated very recently by a publication in a high impact journal [28] using the full length TDP-43 and FUS protein. Furthermore, these 2 proteins and some constructs of interest for this new project have already been produced in the SABNP. The PhD student will be in a very comfortable position to start his research program.

Perspectives:
ALS and FTLD are two common neurodegenerative diseases worldwide that affect people of all races and ethnic backgrounds with major social impact. The prevalence of ALS is about 4 cases per 100,000 persons and 10 per 100,000 persons for FTLD. In these two diseases, the presence of fibrillar aggregated of one of these proteins has been detected in the neurons for a vast majority of patients. The study of their functional properties represents a putative target for developing original therapeutic options that are still lacking. The collaboration between the SABNP lab and a private company of Taïwan that is developing drugs against TDP-43 fibrillation is a chance in this project.

Contact:
loic.hamon@nulluniv-evry.fr
alexandre.maucuer@nullinserm.fr 
UEVE
Batiment Maupertuis, rue du Père Jarlan
Evry, France