Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults, characterised by myotonia, muscle weakness and progressive muscle atrophy. DM1 is caused by the expression of mutated RNAs containing expansions of CUG repeats (CUGexp-RNA) that form nuclear aggregates and alter the activity of RNA-binding proteins and the metabolism of certain RNAs. Although a number of muscular symptoms such as myotonia have been associated with abnormalities in the regulation of alternative splicing of target transcripts, the mechanisms involved in the dystrophic process are still poorly understood and animal models of DM1 do not reproduce, or only partially reproduce, this most disabling phenotype of the disease. Using new mouse models enabling cell/tissue-specific expression of the DM1 mutation in adults, we will study muscle changes following specific expression of CUGexp-RNA in muscle fibres and/or in muscle stem cells in order to determine their respective contribution to the dystrophic process that progressively develops in DM1 patients. Physiological and histological analyses of the muscles will be supplemented by transcriptomic analyses using RNAseq and also at the level of single nuclei using snRNAseq in order to identify and characterize the molecular alterations induced by the expression of CUGexp-RNA that will affect their cellular fate. Spatial transcriptomic or RNAscope analyses will complement these analyses in order have a comprehension view of the cellular mechanisms involved in the degenerative process found in the skeletal muscle of DM1 patients.

Autosomal dominant Centronuclear myopathy (CNM) is a rare neuromuscular disorder due to mutations in the DNM2 gene coding for Dynamin 2 (DNM2). We have demonstrated that the ubiquitous clathrin heavy chain (CHC) and DNM2, well characterized for their role in endocytosis of clathrin-coated vesicles, regulate the formation of large clathrin-coated plaques. These structures are localized at plasma membrane (PM) adhesion sites known as costameres where they associate with branched actin filaments. Clathrin plaques are absolutely required for organizing the actin cytoskeleton at the costameres and for subsequent formation of the contractile apparatus. Both in vitro and in vivo depletion of clathrin disrupts actin scaffolding, costamere formation and the capacity of muscle tissue to generate mechanical force. This project aims at understanding the articulated mechanisms of clathrin-coated plaque regulation and to identify the signalling cascades initiated at these structures which contribute to transduce mecanical stimuli into biochemical responses. We hypothesize that CHC and DNM2 sense mechanical forces through interactions with actin binding proteins (Hip1, Hip1R, cortactin), and adjust actin scaffolding. We will use both in vitro and in vivo approaches coupled to high resolution electron microscopy, intravital imaging and biochemical analysis in order to decipher the contribution of CHC, DNM2, and several actin binding proteins to plaque assembly and costamere organization. We will test the possibility that CHC and DNM2 phosphorylation by the Src and FAK kinases induces assembly of clathrin-coated plaques at the plasma membrane and we will analyze the effect of stretching and contractions on clathrin-coated plaque abundance. In light of our identification of DNM2 gene mutations as the cause for autosomal dominant centronuclear myopathy (CNM), we will address the effect of CNM causing DNM2 mutants on clathrin-coated plaque assembly and costamere integrity. Experiments will be conducted on several experimental models including DNM2 knock-in/knock-out mice and myogenic cells from CNM patients. Our proposal will establish a novel unconventional role for endocytosis proteins in cellular architecture and mechanical force transduction. This project may also prove relevant to additional tissues and to the pathophysiology of diseases such as CNM, other neuromuscular disorders where costamere integrity is compromised and cancer where abnormal clathrin plaque assembly could perturb adhesion leading to aberrant cellular migration.

Amyotrophic Lateral Sclerosis (ALS) is an incurable disorder, characterized by degeneration of motor neurons (MN) leading to progressive paralysis and death usually within 3 to 5 years after diagnosis. ALS is a rare disorder, however, a recent epidemiological study predicts that, along with aging of the population, ALS cases could increase by 69% in the upcoming years. Over the past decade, a breakthrough for the treatment of neurological conditions was the demonstration that viral vectors, derived from Adeno-Associated Virus (AAV) can efficiently transduce cells in the central nervous system. Different applications for MN disorders followed this discovery and an AAV-mediated gene transfer approach has been tested in patients affected by Spinal Muscular Atrophy type 1, with encouraging results. ALS is epidemiologically classified into sporadic (90%) and familial forms (10%, fALS). We recently developed an AAV-mediated gene therapy for fALS, caused by mutations in the SOD1 gene (representing about 20% of fALS cases). Several pre-clinical tests in rodent models overexpressing mutant forms of human SOD1 (hSOD1) have proven the efficacy of knockdown strategies for the treatment of SOD1-ALS. Furthermore, the intrathecal administration of antisense (AS) oligonucleotides against hSOD1 is under clinical trial in SOD1-ALS patients and various AAV-mediated silencing approaches are currently under pre-clinical development. Taking advantage of AAV serotype 10 vectors and the U7 small nuclear RNA particle, we designed and tested AS sequences inducing hSOD1 silencing through exon skipping (AAV10-U7-hSOD1). The result we obtained in SOD1G93A mice (model of SOD1-ALS) was reported as the most favorable therapeutic effect to date and the preclinical work aimed at the clinical translation of this approach to humans is undergoing. The overall objective of this proposal is to advance the development of gene therapy strategies for fALS. To this aim, we intend to address the effects of the exon-skipping strategy on the silencing of the endogenous non-mutated hSOD1 gene (Objective 1). In addition, we plan to test a similar therapeutic approach for the most common genetic form of fALS and frontotemporal dementia (FTD), caused by hexanucleotide repeat expansion (HRE) in the C9 gene (Objective 2). Objective 1- For the advancement of SOD1-silencing strategies is important to consider that the long-term removal of the SOD1 enzyme could be detrimental to cell physiology due to its essential role in protecting cells from oxidative stress. Furthermore, data from literature revealed that functional loss of SOD1 worsens ALS disease severity in animal models. Therefore, we propose to test in patient-derived cells the nonspecific silencing strategy based on the AAV10-U7-hSOD1 vector and to compare its effects with a newly generated vector that allows for both hSOD1 silencing (Erase) and re-expression of a functional wild-type hSOD1 (Replace). In parallel, we plan to assess the potential therapeutic effect of the Erase/Replace vector in SOD1G93A mice. The results of these experiments will be pivotal to predict outcomes of undergoing pre-clinical and clinical trials. Objective 2- The HRE G4C2 in intron 1 of the C9 gene is responsible of ALS, FTD and ALS/FTD through three non-exclusive pathological mechanisms including loss of protein function, toxicity of nuclear HRE RNA and accumulation of dipeptide repeats. The potential therapeutic effect of AS oligonucleotides for the treatment of C9-ALS entered in a phase I/II clinical trial. The main goal here is to prove the therapeutic efficacy of an AAV-U7-mediated AS delivery for C9-ALS for durable and widespread transduction of affected tissues. We plan to test this approach in human cell models and in pre-clinical setting in vivo. The results of this study will be a prelude to the development of a pre-clinical therapeutic strategy for patients with ALS, FTD and ALS/FTD.

Fibrosis is defined as the excessive accumulation of extracellular matrix (ECM) components as a result of a failed tissue-repair process. Fibrosis can occur in any organ and is responsible for nearly 45% of all deaths in the industrialized world. In muscle, fibrosis is one of the main complications in many myopathies. In this project, we will use as paradigms COL6-related (COL6-RD), LAMA2-related (LAMA2-RD) and Duchenne (DMD) muscular dystrophies which, although clinically and genetically different, are all characterized by the presence of fibrosis that provokes the disruption of muscle architecture, and ultimately its functional failure. Fibro-adipogenic progenitors (FAPs), the main cell actors of fibrosis, are the major ECM collagen-producing cells within the stromal tissue environment. While they have been extensively described in mouse, very few studies have analysed their profile in human dystrophic/fibrotic conditions and a comprehensive study comparing different muscle pathologies is lacking. While there is an urgent need for strategies to ameliorate the efficacy of therapeutic options, in particular based on gene therapy, which have been shown to be less efficient in fibrotic tissue due to the increased extracellular microenvironment reducing the accessibility of viral vectors to their target, today there is no efficient treatment to cure muscle fibrosis. Data on human samples to define muscle fibrosis are therefore essential. The goal of the FibroDys project is to compare in parallel three different fibrotic muscle diseases using already available human samples to investigate which molecular pathways and cellular actors are unique and which ones are shared among these diseases, with the final goal to pave the way towards appropriate and effective therapeutic avenues. Three specific aims are therefore developed. A first aim based on high-dimensional omics analyses on human biopsies will characterise the heterogeneity of the resident cell populations and their interactions in the intricate ECM fibrotic muscle (snRNAseq, Imaging Mass Cytometry) and identify the ECM molecular components altered in each fibrotic muscle (proteomic approach). A second aim will study the secretome and proteome of FAPs and the cross-talk between FAPs and ECM/muscle cells, revealing the exact contribution of FAPs from each pathology to the secretome in muscle fibrosis. This aim will also dissect how ECM proteins influence FAPs behaviour and how ECM produced by FAPs impact muscle differentiation (co-culture experiments). A third aim will screen an anti-fibrotic drugs library (Medium-Throughput screening) to identify a list of compounds active on muscle FAPs secretion and proliferation, and a list of signalling pathways implicated in FAPs-mediated fibrosis. This list will be further refined and validated using a pseudo-3D in vitro system (scar-in-a-jar system) and in vivo models (xenotransplantation of human FAPs in immunodeficient mice) to identify potentially shared or disease-specific therapeutic candidates.

Myotonic dystrophy type 1 (DM1) is a RNA gain of function disorder due to the abnormal expansion of CTG repeats in the 3’UTR of the DMPK gene. Toxic mutated RNA aggregates in the nucleus and sequester MBNL RNA binding proteins leading to RNA metabolism abnormalities. The principal aim of this research project is to analyze the role of the motor unit and particularly the communication between motor neurons (MN) and the skeletal muscle via neuromuscular junctions in muscle dysfunction in DM1. Using mice and cellular models, associated with CLIP and high throughput RNA sequencing, we will identify molecular targets that are regulated by MBNL in MN and determine the consequences of MBNL functional loss in MN on motor unit development and function knowing that MN controls skeletal muscle activity. The proposal will a better understanding of MBNL loss of function in MN and its contribution to DM1 muscle pathophysiology.