Insights into the mode of action of flunarizine on microRNAs and/or targets n rare motor neurondiseases – MiRrOR

Amyotrophic lateral sclerosis (ALS) and Spinal muscular atrophy (SMA) are rare neurodegenerative diseases characterized by a progressive loss of motor neurons. A marked improvement of SMA patients is observed with recent innovative therapies, but the clinical outcomes are unpredictable and might show detrimental long-term effects. ALS patients have fewer therapeutic options with very limited efficacy, in part, because the disease mechanisms are poorly understood. Thus, great improvements are needed for therapies in both ALS and SMA. These diseases reveal altered RNA metabolism, but the detailed molecular commonalities remain elusive. However, a shared feature of ALS and SMA is a deficiency of SMN-positive nuclear bodies. Indeed, a severe SMN loss or reduction is associated respectively with SMA or ALS patients carrying either TDP43 or FUS mutations. TDP43 and FUS are ubiquitously expressed nucleic-acid-binding proteins recruited by SMN to nuclear bodies. SMN assembles diverse ribonucleoproteins (RNPs), including splicing snRNPs which final biogenesis steps take place in SMN nuclear bodies. Consistently, RNA expression and splicing patterns are altered in SMA and ALS. We identified by a chemical screen that flunarizine (Fz) increases SMN and TDP43 co-localization in nuclear bodies of SMA patient fibroblasts, even though the SMN protein levels remain low. Remarkably, another group independently reported that Fz modulates splicing, a key function regulated by SMN. In collaboration, we also showed that Fz enhances motor neuron survival, decreases muscle atrophy and prolongs life span in an SMA mouse model. Moreover, our preliminary results showed that Fz modulates the RNA levels of non-coding small RNAs, including microRNAs. We here propose to challenge the hypothesis that identifying the downstream effectors of Fz could provide key insights into the molecular mechanisms underlying motor neuron survival/degeneration along 3 main axes: 1) uncovering Fz-signaling pathway, 2) developing therapeutic tools based upon Fz-signaling and 3) providing preclinical evidence in zebrafish and mouse ALS and SMA models. First, we propose to develop a biochemical pull-down assay to isolate Fz-interacting protein(s) and to determine the impact of Fz on ALS and SMA transcriptome keeping it focused on interspecies conserved microRNA/mRNA pairs. This aim will highlight the molecular mechanism(s) modulated by the drug and will open on new therapeutic opportunities. In the second axe, we will chemically modify Fz and product AAV constructs for the microRNA manipulation of a selected target to improve and mimic the Fz activity. Finally, building on this knowledge we will evaluate in zebrafish and mouse ALS and SMA models the application of these new tools to provide the first evidence for translational potential from the lab to the clinic. “MIRROR” is a multidisciplinary project that brings together experts on the biology of ALS and SMA and chemistry for biomedical research in France. Our strategy is an integrated approach between 5 partners with complementary expertise and competence aiming at filling the gaps in the understanding of rare motor neuron diseases and hopefully, to propose new therapeutic options for the unmet medical needs in rare diseases.