Glial-neuron crosstalk in the regulation of neurofilament dynamics

Projektinformationen

GliaNFish

ID Finanzhilfevereinbarung: 101109363

DOI

10.3030/101109363

Projekt abgeschlossen

EK-Unterschriftsdatum 5 Mai 2023

Startdatum 1 Juni 2023

Enddatum 31 Mai 2025

Finanziert unter

• Marie Skłodowska-Curie Actions (MSCA)

Gesamtkosten

Keine Daten

EU-Beitrag

€ 195 914,88

Koordiniert durch

INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
France

Periodic Reporting for period 1 - GliaNFish (Glial-neuron crosstalk in the regulation of neurofilament dynamics)

Berichtszeitraum: 2023-06-01 bis 2025-05-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Every cell in the human body contains an internal scaffold known as the cytoskeleton, which plays a vital role in maintaining cellular structure, stability, and function. The cytoskeleton consists of three major components: actin filaments, microtubules, and intermediate filaments. In neurons, the intermediate filament network is mainly composed out of neurofilaments, which ensures the integrity and organisation of the neuronal cytoskeleton.
Increasing evidence suggests that neurodegenerative diseases are not only associated with abnormalities in the cytoskeletal network, but are also driven by them. Disorganisation and dysregulation of neurofilaments are common pathological features in a wide range of neurodegenerative conditions, even in cases where there are no mutations in the gene encoding neurofilaments itself. Furthermore, experimental studies have shown that targeted manipulation of neurofilaments can improve disease outcomes, highlighting it as a promising and underexplored therapeutic target. Despite this, the precise role of neurofilaments in healthy neuronal physiology and neurological disease remains poorly understood.
The goal of the GliaNFish project was to investigate the dynamics and organisation of neurofilaments in vivo, both during normal neuronal development and under pathological conditions. Using zebrafish as a model organism, the project aimed to visualise and characterise neurofilament transport and organization in real time, allowing to study its function in the context of a living, developing nervous system. To study the impact of disease, the project focussed on two disease-related genetic alterations: 1) mutations in frataxin, known to cause the autosomal recessive ataxia Friedreich’s ataxia and 2) a recently discovered gene duplication in a gene previously associated with inherited neuropathies. By creating and characterising zebrafish models that replicate these human disease mutations, the project aims to gain a better understanding of the disease pathology and explore how genetic disruptions influence neurofilament organization.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

The overarching goal of the project was to understand to the role of neurofilament organization and dynamics during normal nervous system physiology and disease. To address this, the project was structured in three main parts.
In the first part, we focused on understanding the dynamics of neurofilaments during the normal development of neurons in a living organism. We used a zebrafish model in which the expression of neurofilaments can be regulated by temperature. To track the protein’s movement, we tagged neurofilaments with a fluorescent marker that changes colour after exposure to blue light. This allowed us to alter the colour of neurofilaments in a specific region of the neuron and then monitor its transport using microscopy. By performing these experiments at different stages of zebrafish development, we observed that the transport of neurofilaments changes over time. Specifically, the proportion of neurofilament proteins that is actively being transported at a given time decreases as the nervous system matures, and the direction of transport also shifts. These findings reveal how neurofilament protein transport is regulated during development.
In the second part, we investigated the role of neurofilaments in the context of Friedreich’s ataxia, a disorder caused by mutations in the frataxin gene. Our aim was to create zebrafish models in which both frataxin mutations and neurofilament organization could be studied. We initiated a collaboration with a research group in the United States that had recently developed a zebrafish model of Friedreich’s. Our first major finding was that loss of frataxin results in significant changes in the shape and cellular composition of the cerebellum of zebrafish larvae. Our second achievement was the successful application of genome editing techniques to generate new zebrafish models with targeted mutations and/or labeling in the frataxin and neurofilament genes. These novel models will enable future research into the mechanisms by which neurofilaments contributes to disease pathology, offering potential insights into therapeutic strategies for Friedreich’s ataxia.
In the third part of the project, we examined the potential pathogenic role of a recently identified gene duplication involving a previously known neuropathy gene. To study this, we developed new experimental tools to model the gene duplication and began analysing its effects on the organisation and function of neurofilaments. This ongoing work aims to clarify whether and how the gene duplication alters neuronal function, potentially contributing to neurodegenerative disease mechanisms.
Overall, the project has advanced our understanding of neurofilament biology in normal physiology, established valuable experimental models, and laid the groundwork for further exploration of molecular mechanisms involved in neurological disorders.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

Using zebrafish models, we investigated the transport dynamics of neurofilaments in a living vertebrate organism. This represents an advancement in the field, as previous studies of neurofilament dynamics have primarily been conducted in cell cultures or ex vivo tissues. By enabling in vivo analysis, our approach offers new insights into the physiological regulation of neurofilament protein transport during the development of the nervous system.
In parallel, we successfully established and initiated the characterisation of a zebrafish model for Friedreich’s ataxia. In collaboration with an international research team, we demonstrated that loss of frataxin results in cell type-specific alterations in the cerebellum. Additionally, we applied knock-in gene editing techniques to generate a novel disease model that mimics a recently discovered pathogenic point-mutation mutation. After further development, these complementary models will provide a valuable platform to dissect how distinct domains or mutations of the frataxin protein contribute to disease pathology, and will thus advance mechanistic understanding of Friedreich’s ataxia. In addition, the newly developed tools for studying neurofilaments are not only relevant for research on Friedreich’s ataxia, but also offer a broader applicability for the neuroscience community as a whole.
Third, we developed a new experimental tool to investigate the pathogenicity of a recently identified gene duplication in a known neuropathy gene. This tool will allow us to explore how the duplication affects the organisation of neurofilaments, and whether it contributes to previously unrecognised disease mechanisms.
Although this research is still in its early stages, the zebrafish models and experimental tools developed through the GliaNFish project hold strong potential for future translational applications. These include the identification of novel disease mechanisms, preclinical testing of therapeutic compounds, and broader applicability to other neurodegenerative and neurodevelopmental disorders.