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In vivo analysis of neuromuscular junction stability in zebrafish models of amyotrophic lateral sclerosis

Periodic Reporting for period 1 - NMJALS (In vivo analysis of neuromuscular junction stability in zebrafish models of amyotrophic lateral sclerosis)

Reporting period: 2015-11-16 to 2017-11-15

Amyotrophic lateral sclerosis (ALS) is a late onset, lethal neurodegenerative disease of motor neurons that affects about 2 in 100,000 individuals per year. Different genetic mutations have been described in patients, but the genes involved seem unrelated and the causes of the disease appear complex and are still largely not understood. Of relevance, the destabilisation of neuromuscular junctions (NMJ) may be an early event, preceding neuronal death, making them an interesting therapeutic target to slow down disease progression. The goal of the study was to analyse the precise organisation of the NMJ, in particular of cell adhesion molecules and motor complexes linked to it, in order to better understand the mechanisms of maintenance of NMJ by analysing early defects in motor neuron degenerative contexts.
In this project, we combined genetic engineering, neuron biology and latest fluorescent imaging tools in zebrafish, an excellent vertebrate model for live imaging.

First, we highly improved CRISPR/Cas9 based genetic engineering in zebrafish to generate mutations in genes having a potential role in motor neuron organisation and synapse stabilisation and to fluorescently label endogenous proteins. The CRISPR/Cas9 technology is a recently developed tool that enables to precisely target a small region of the DNA and generate local breakage. These breaks can be used to generate early stop in a gene or to precisely modify the gene of interest by the insertion of a supplied small DNA molecule. We managed to increase the efficiency of targeted DNA breakage in vivo in zebrafish eggs to 90-100%. This enables us to create several mutations in genes coding for motor proteins implicated in motor neuron differentiation and synapse organisation.

Second, we analysed the localisation in the NMJ of adhesion molecules fluorescently labelled, in particular molecules of the cadherin family. We could then compare the localisation of this molecule in normal conditions and in zebrafish mutated for the motor neuron proteins of interest. This work is still ongoing in zebrafish SOD1 model of ALS to decipher whether some of these adhesion molecules are differently localised compared to wild type conditions.

Third, we have characterised motor neuron formation, arborisation and NMJ establishment and maintenance in a mutant of a subunit of a motor complex known to be implicated in some forms of ALS. This enabled to propose a new function of this protein in NMJ organisation and dynamics that suggests a new mechanism for the participation of this protein in ALS disease progression.

Overall, the project will result in at least 5 publications in international scientific journals, one being already published and 3 submitted.
1- The project has achieved a clear technological advance with the development and improvement of the revolutionary CRISPR/Cas9 technique for in vivo genetic engineering. This rapidly advancing technique is promising for many fields including medical and farming and opens great potential of improvement for the society, being health or food production.

2- For the biological and medical impacts, the project has made significant advance for the field of the neuro-degenerative disease ALS by proposing a new unexpected function for a protein whose expression is reduced or that is found mutated in some forms of this syndrome. Moreover, it will go further with the analysis of other models mutated in genes potentially involved in ALS. Finally, for a collaboration with a team working on a rare child genetic disease, the use of the advance made in the CRISPR/Cas9 technique has enabled to generate an animal model of the disease mimicking the genetic mutation of the patient. This new model has the expected phenotype and will highly advance our understanding of this rare disease.

3- Finally, the project has resulted and benefited from collaborations with French and European teams and is as such participating to the creation of a European excellence scientific network. It will in the coming month result in several publications in renowned international journals further contributing to European scientific excellence.