Live imaging of RNA dynamics for genetic forms of amyotrophic lateral sclerosis (ALS) in zebrafish

author: Raphael Munoz-Ruiz, Institut du Cerveau et de la Moelle épinière - ICM
published: July 21, 2017,   recorded: May 2017,   views: 4
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The recent identification of mutations in TDP-43, FUS, two RNA-binding proteins, altogether with C9orf72 and SOD1 representing more than half of genetic cases, and also SQSTM1 and VCP, seems to unveil a global mechanism of alteration of both protein and RNA homeostasis leading to toxicity and cell death in ALS. The lab has specialized in studying and developping ALS models in zebrafish. The revelance of this model in the field of motor neuron diseases relies on being a practical, convenient and large progeny generating vertebrate model. Furthermore, this model shows a great conservation of genetic mechanisms with mammals and humans. Moreover, the zebrafish allows the use of a wide range of genetic tools convenient to develop transient and stable transgenic lines. Also, this model shows transparency at the embryonic stage, making it particularly relevant to study, especially through imagery, pathogenic mechanisms in vivo. This project aims to study key ALS transcripts such as Sqstm1, VCP or C9orf72 in order to potentially comprehend their implication in the pathology through the use of zebrafish disease models already established in the lab. Two in vivo techniques will be developped: -the MS2 system which relies on the interaction between bacteriophage’s MCP protein (MS2 Coat Protein) and a specific “hairpin” RNA sequence called MBS (MS2-Binding site). The addition of several MBS sequences to an RNA sequence combined with the presence of the MCP protein fused to a fluorescent protein allows you to follow this particular exogenous RNA sequence at the cellular level (Buxbaum et al, 2015). -a modified Crispr system using an inactive Cas9 protein, fused with a fluorescent protein (“dead Cas9-eGFP”). Through modifications, the Cas9 is now adapted to target an endogenous mRNA thus making its visualization possible without modifying it (Nelles et al, 2016). The localization of transcripts through live-imaging will allow us the caracterization of key ALS RNA species metabolism and their defects in pathological mechanisms. All together with complementary experiments such as in situ colocalization with proteins, quantification of RNA levels, pull-down of interacting partners, novel aspects of disease pathogenesis leading to neuronal death would be unveiled. We believe a detailed study of RNA metabolism, especially through in vivo experiments, has the potential to durably impact our understanding of ALS and to a certain extent, other pathologies.

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