NOVEL ALS MODELS · Developing novel models of Amyotrophic Lateral Sclerosis using motor neuron cultures and zebrafish

Developing novel models of Amyotrophic Lateral Sclerosis using motor neuron cultures and zebrafish

SCIENTIFIC REPORT This report acknowledges the results derived from the project «Novel ALS Models» funded by the Career Integration Grant 293848 from Marie Curie Actions. In this research project, we proposed five major objectives to develop novel in vivo and in vitro models to study pathophysiological mechanisms leading to motor neuron degeneration, the hallmark of Amyotrophic Lateral Sclerosis (ALS). Also, a major effort was to ascertain that these novel models of ALS would be capable to carry out translational studies, with knowledge derived from these results to provide therapeutic strategies for the patients’ diagnosed with ALS or related neurodegenerative diseases. Following identification of TDP-43 in ALS patients (Kabashi et al. Nat Genet 2008), we developedthe first zebrafish models for these mutations and described for the first time that mutant TDP-43 causes motor neuron degeneration through gain and loss of function mechanisms (Kabashi et al. HMG 2010). Similar pathogenic mechanisms were described for FUS mutations (Kabashi et al. 2011) identified in ALS patients (Kwiatowski et al. Science 2009). Recently, our team has established a C9orf72 knockdown zebrafish model that mirrors the reduction of C9orf72 at the transcript levels in ALS patients carrying the C9orf72 hexanucleotide repeats (Ciura et al. Ann Neurol 2013). Overall, the genetic models develop in zebrafish show major deficits of axonal projections from spinal motor neurons and major reduction of locomotion parameters at the level of evoked and spontaneous swimming. The team has also identified the prevalence of genetic markers in French ALS patients, including PFN1 and ATX2 (Lattante et al. Neurology 2014; Neurobio Aging 2013). Also, zebrafish models have been established for genes that harbor mutations that lead to motor neuron degeneration, such as VAPB and SQSTM1 (Kabashi et al. HMG 2013; Lattante et al. HMG 2015). Although we use cellular lines and primary cultures to study ALS mechanisms as described in Aim 1 of this research project, we have focused and optimized the usage of zebrafish as a vertebrate model to study motor neuron disease, with our particular focus the development of ALS genetic models in this model organism. Therefore, we have developed zebrafish models to study C9orf72 repeat expansion, and mutations in genes that encode TDP-43, FUS and SOD1 (see below). We have described and characterized functional interaction of TDP-43 and FUS described in Aim 2 and have identified common molecular partners for these mutants, such as intermediate repeat expansions of ATX2 (Lattante et al. Neurology 2014). Currently, we are determining whether two genes known to be involved in ALS, C9orf72 and SQSTM1 genetically interact with TDP-43 and FUS as described in Aim 3 similarly to functional interactions established between FUS and TDP-43 (Kabashi et al. PLoS Genet 2011). Currently, we are also establishing stable transgenic lines for the ALS genes using advanced genomic techniques. In Aim 4 of this project, we proposed to develop deletion mutants through zinc finger nucleases and TILLING. Deletion mutants for TDP-43 using this technology were recently published by our colleagues (Schmid et al. PNAS 2013). Since this proposal, more efficient genomic engineering tools to develop transgenic lines have been described, including TALE nucleases and the CRISPR/Cas9 system. We have designed appropriate tools to target these genes and are developing specific mutant transgenics for major ALS genes as described in Aim 4. Development and study using these models has significantly impacted and advanced the field. Indeed, the team has also participated in the identification of therapeutic strategies contributing with two major advances as described in Aim 5. Treatment with methylene blue, a compound known to reduce oxidative damage, leads to rescue of deficits in axonal projections linked to neurodegeneration in mutant TDP-43 and FUS zebrafish models (Vaccaro et al. PLoS One). Similarly, reduction of ER stress specifically by salubrinal and guanabenz reduces motor neuron degeneration in mutant TDP-43 and FUS transgenic zebrafish models. The subsequent objective of the Kabashi team will be to identify molecular cascades that can be modulated pharmacologically and to test these in murine models and eventually in ALS patients. For more details about this project, please contact: Scientific representative: Edor Kabashi Chef d’équipe / Group Leader Institut du Cerveau et de la Moelle Épinière (ICM) 47, bd de l'hôpital - 75013 Paris Phone: +33-1-5727-4311 Email: edor.kabashi@icm-institute.org Website : https://sites.google.com/site/kabashilab/

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