ASsembly and phase Transitions of Ribonucleoprotein Aggregates in neurons: from physiology to pathology.

Project Information

ASTRA

Grant agreement ID: 855923

DOI

10.3030/855923

EC signature date 26 February 2020

Start date 1 March 2020

End date 31 August 2027

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 7 741 799,00

EU contribution

€ 7 741 799,00

Coordinated by

UNIVERSITA DEGLI STUDI DI ROMA LA SAPIENZA
Italy

Periodic Reporting for period 3 - ASTRA (ASsembly and phase Transitions of Ribonucleoprotein Aggregates in neurons: from physiology to pathology.)

Reporting period: 2023-03-01 to 2024-08-31

Summary of the context and overall objectives of the project

·What is the problem/issue being addressed?
The aim of the project stands on the evidence that altered RNA metabolism and aberrant ribonucleoprotein (RNP) assembly are causatively linked to several neurodegenerative diseases, such as Amyotrophic lateral sclerosis. The experimental plan is aimed at unraveling the composition and dynamics of RNP assemblies in normal and pathological conditions, to understand the role of non coding RNAs (ncRNAs) in the control of their structure and function and finally to reveal the molecular determinants of liquid phase separation and liquid-to-solid phase transition.
The knowledge about the composition of pathological aggregates in ALS should enable the setting up of methodologies and approaches that could be applied to other neurodegenerative diseases characterized by the pathological aggregates.
The objectives revolve around the main idea that once the determinants of aggregation are identified, intervention by using a molecular approach, such as RNA aptamers, can be exploited for therapeutic intervention.
·Why is it important for society?
Many neurodegenerative disorders are still lacking effective therapeutic treatments and drugs that can counteract the progression of the disease. Even if these diseases are characterized by different genetic alterations, they share the common feature of being causatively linked to the formation of pathological aggregates. These aggregates can exert their pathological role by sequestering factors important for the activity of neuronal cells and preventing their correct function. Therefore, the deep characterization of the RNA and protein aggregates in different neurodegenerative disorders has a strong impact on human health. Our effort is mainly aimed to study the formation of aggregates in ALS; the knowledge of how they are made and how they are regulated will offer a unique opportunity for the identification of reagents able to interfere with their formation or even to direct their disassembly. Beyond the scientific relevance of this problem, there is the important question we intend to answer of which disease-related genomic alterations predispose individuals to contract neurodegenerative diseases such as ALS.
-What are the overall objectives?
We plan to develop novel advanced microscopy methods to monitor formation of aberrant RNPs in vivo as a novel early diagnostic predictive tool; moreover, we will explore new molecules to impede pathological cascades driven by aberrant RNP assemblies. In conclusion, ASTRA will allow us to gain a comprehensive understanding of RNP function and dysfunction; we will use this knowledge to develop novel therapeutic strategies that will impact on several protein-misfolding neurodegenerative diseases.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The main results so far obtained can be summarized in:
● Identification of ncRNAs which are expressed in motor neurons (MN) and characterization of their functional role in physiological and pathological conditions. We are searching for species which control aggregate formation and phase transition in pathological conditions.
● In situ detection of ncRNAs and analysis of their altered localization in pathological conditions.
● Setting up of cellular systems expressing mutant FUS proteins, as models for the study of chemico-physical properties of FUS aggregates (Brillouin) and for their purification (STOMP, APEX and IP through protein tagging).
● Identification of a list of RNA candidates ready to be analyzed for protein-RNA networks to unravel links among molecules, with special focus on long non coding and circular RNAs.
● Design of small RNA molecules (aptamers) with high binding affinity for TDP-43 and test of their activities in cell lines.
● Development of a high-throughput computational protocol to assess interactions of ncRNAs with proteins.
● Brillouin microscopy improvement. All the hardware has been purchased, the Mach-Zehnder interferometer for filtering the elastic radiation has been set and tested. The heterodyne detection scheme based on an electro-optic modulator is under test. The RF cavity (as an alternative to the electro-optic modulator) has been constructed and will be tested soon.
● Brillouin microscopy. The present microscope is in use for testing the rigidity of stress granules (FUS) and aggregates (TDP43).
● SAI microscope. The present on-bench setup is being used to test its performance on post mortem pig’s eyes.
● Fluorophores for SAI microscope. The BT1 molecule (patent pending as TAU fibrils marker) has been designed and synthesized. Nowadays it is under test for safety in in-vivo cell culture.
● STOM/STONT. The hardware (specifically the fs pulsed laser soiurce) has been delivered only recently. The set-up is under construction.
● Bio-computation. The software (based on the Zernike polynomial expansion of the protein surface) has been developed and extensively tested. This software will be delivered open source in the next few months.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

These are the major progresses and expected results:
·The setting up of computational and experimental activities to unravel how nucleic acid mutations lead to the assembly of proteins in large macromolecular complexes characterized by different physical properties.
·The development of high-throughput strategies for predicting interactions of proteins with lncRNAs or circRNAs.
·The characterization of ncRNA species which control liquid phase separation and liquid-to-solid phase transition. The availability of these data will provide interesting novel candidate molecules that can be targeted in a putative novel ALS therapy aimed to prevent the formation of pathological inclusions.
·The setting up of imaging methodologies that enable ncRNA detection in cells and how their compartmentalization is affected in pathological conditions
·Identification of the composition of ribonucleoprotein aggregates. The hypothesis is that RNA and protein components of RNP granules change in ALS-related variants. This knowledge will allow to find relevant candidates, both proteins and RNAs, that can be targeted in order to control liquid phase separation and liquid-to-solid phase transition
·Development of RNA aptamers and demonstration that they can be used either to detect aggregates through a type of super resolution microscopy or to inhibit progressive protein accumulation in the test tube. There is a great potential for two major applications that will push the scientific boundaries: diagnostics and therapeutics. A patent is about to be filed to further exploit these results.
·Upgrading the Brillouin Microscope for the fast scan mode (heterodyne detection) for in vivo mechanical measurements of the rigidity and viscosity of the stress granules.
·Building the STOMP/STONT set-up for spatially selective pull-down (stress granules).
·Installing the SAI (Scattering Assisted Imaging) microscope on a commercial ophthalmoscope for in-vivo human retina detection of protein aggregates.
·Developing and testing the TAU-specific fluorescent ligand BT1
·Improving (introduction of the electrostatic terms) the protein-protein in silico interaction predictor based on the orthogonal polynomial expansion of the proteins surface’.

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