MAGDINIER Frédérique

Principal investigators: Patrice Roll, Elise Kaspi, Diane Frankel

In Human, the protein-coding sequences represent only ∼2% of the genome, which is very low regarding the complexity of the organism. Besides mRNAs, many transcripts called non-coding RNAs (ncRNAs) do not encode proteins but are functional. Among ncRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have become in recent years a new source of interest for the scientific community, allowing a better understanding of their mechanistic insights and biological functions. By regulating the expression of many target genes, miRNAs (small ncRNAs ∼20 nucleotides long) are considered as key actors in the pathophysiology of many human diseases and had become new therapeutic targets by mimic or antimiR approaches. lncRNAs (ncRNAs longer than 200 nucleotides) regulate gene expression at multiple levels: by interacting with DNA, RNA and proteins, they can modulate chromatin structure or affect RNA splicing, stability and translation. Some lncRNAs bearing miRNA-complementary sites can regulate gene expression as competitive endogenous RNAs or ‘sponges’ of miRNAs. Among the multiple molecular mechanisms which may contribute to aging modulation, miRNAs and lncRNAs are raising enormous interest due to their ability to influence several hallmarks of aging at the epigenetic level. In premature aging syndromes, few studies have focused on the role of ncRNAs, exclusively centered on miRNAs including as a therapeutic strategy to alleviate premature senescence and to increase longevity.

Over the last few years, our team has developed a new research axis supported by the AFM-Telethon, to evaluate the role of ncRNAs in HGPS and related progeroid syndromes. We have shown that the overexpression of miR-376a-3p and miR-376b-3p due to chromatin remodeling of the 14q32 locus, contributes to progerin accumulation by inhibiting autophagy level and leading to premature cellular senescence. Recently, we identified several other ncRNA candidates based on studies of patients’ fibroblasts and vascular smooth muscle cells (VSMCs) from LmnaG609G/G609G KI mice. In particular, we focused our attention on two miRNAs and several lncRNAs because of potential link between any of their targets and clinical disorders observed in these syndromes. We use the benefit of several different in vitro (human primary fibroblasts and hiPSC derived cells) and in vivo (LmnaG609G/G609G KI mice) models, as unique and complementary tools to study these new candidates.

Deciphering and elucidate the role of non-coding RNAs in HGPS and other related progeroid syndromes remain a challenge that will help to better understand the pathophysiology of these dramatic diseases, paving the way for new therapeutic proofs of concept.

Principal investigators: Annachiara De Sandre Giovannoli, Ali Badache

Hutchinson-Gilford Progeria (HGPS) is a rare, to date incurable, genetic disorder characterized by early and accelerated aging leading to the death of patients around the age of 13. We showed en 2014 that HGPS is due to an autosomal dominant de novo mutation in the LMNA gene resulting in the activation of a cryptic splicing site, which generates the synthesis of an abnormal and toxic nuclear protein, called progerin. This protein accumulates in vivo during patient aging and generates dose-dependent nuclear anomalies, both morphological and functional, such as DNA repair anomalies, deregulation of gene expression, associated with oxidative stress, all of which contribute to the induction of cellular premature senescence.

Mandibuloacral dysplasia (MAD) syndromes are rare, mostly autosomal recessive, progeroid disorders with clinical and genetic heterogeneity, characterized by growth

retardation, craniofacial dysmorphic features due to distal bone resorption, musculoskeletal and skin abnormalities associated with lipodystrophy, caused by mutations of the Lamin A/C gene or other genes contributing to Lamin A/C processing and to structure and function of the nuclear lamina. All Lamin-related MAD syndromes, just like HGPS, are characterized by nuclear morphological and functional abnormalities. We recently identified five homozygous null mutations in MTX2, encoding Metaxin-2 (MTX2), an outer mitochondrial membrane protein, in patients presenting with a severe laminopathy-like mandibuloacral dysplasia that we called MADaM, for Mandibuloacral dysplasia associated to MTX2 (MDPS; OMIM # 619127), characterized by growth retardation, bone resorption, arterial calcification, renal glomerulosclerosis and severe hypertension. At the cellular level, loss of MTX2 in patient’s primary fibroblasts leads to loss of its membrane partner Metaxin-1 (MTX1) and mitochondrial dysfunction, including network fragmentation and oxidative phosphorylation impairment. Importantly, patients’ fibroblasts are resistant to induced apoptosis, correlating with increased cell senescence and reduced cell proliferation. Interestingly, secondary nuclear morphological defects are observed in both MTX2-mutant fibroblasts and mtx-2-depleted C. elegans revealing an unsuspected pathophysiological link between mitochondrial composition and function and nuclear morphology.

We wish to deepen our understanding of the pathophysiological basis of MADaM Syndrome and explore its possible links with HGPS and Lamin-related MAD. For that purpose, we have assembled a collection of HGPS and MADaM Syndrome patient-derived fibroblasts, and generated patient-derived induced pluripotent stem cells (iPSCs), that could be differentiated into cell types of relevance for the disease. These, in conjunction with gene-edited iPSCs, constitute unique and precious tools to further explore, through multipronged cellular and molecular approaches, including genomic and epigenomic analyses, the mechanism whereby defects of the mitochondrial proteins MTX2 and MTX1 lead to “HGPS-like” progeroid symptoms. In turn, novel molecular insights should help identify potential therapeutic strategies for MADaM patients.

Principal investigator: Frédérique Magdinier

Facio Scapulo Humeral Dystrophy (FSHD) is a peculiar autosomal dominant form of muscular dystrophy with involvement of specific muscles of the face, shoulder and pelvic girdles and peroneal groups, with variable weakness. FSHD is considered as genetically heterogeneous but clinically homogenous with however incomplete penetrance. Two types of FSHD have been described. The first type, FSHD1 is the most frequent and associated with shortening of an array of D4Z4 macrosatellite repeats in the distal 4q35 locus. The second type, FSHD2 (5% of patients) is linked in 80% of cases to mutation in the SMCHD1 gene encoding a chromatin-associated factor which function is poorly understood. This gene is also mutated in an unrelated developmental disorder dubbed Bosma Arhinia and Microphtalmia Syndrome (BAMS). The current and most prevalent molecular model proposed as causative of FSHD associates reduction in the number of D4Z4 repeats (FSHD1) or loss of SMCHD1 function (FSHD2) to hypomethylation of D4Z4, chromatin relaxation and transcription of the most distal copy of the DUX4 gene. However, the molecular mechanisms resulting in the muscle weakness and involved in the muscle specificity are not at all understood.

For many years, we have been interested in understanding the regulation of the D4Z4 array and the contribution of epigenetics in FSHD, together with the role of SMCHD1 in this process. Thanks to our unique and close collaboration with the main center for the molecular diagnosis of FSHD in France and the Center of Reference for NMDs, we have access to hundreds of patients’ samples and medical record for clinical research. Over the years, we have developed a large number of tools for in vitro explorations in parallel to in-depth clinical investigations of FSHD patients, including through the development of induced pluripotent stem cells models.

Ongoing projects aim at:

o Deciphering the complexity of the 4q35 and homologous 10q26 loci in FSHD patients with atypical genomic features using a combination of genomic approaches including long read sequencing.

o Develop integrative tools for genotype-phenotype correlations in FSHD (collaboration with the systems biomedicine team headed by A. Baudot).

o Defining the contribution of non-muscular cells in the pathogenesis of FSHD. This part of the project includes cutting edge technologies of tissue bioengineering (microfluidics, production of organoids, tissue bioprinting, optogenetics).

o Investigates the dynamics of DNA methylation and the role of SMCHD1 in shaping the epigenome.

Principal investigator: Leslie Caron

Leslie Caron’s goal is to better understand the genetic and cellular mechanisms contributing to debilitating skeletal muscle diseases. Her current research is based on modeling neuromuscular disorders using patient’s induced pluripotent stem cells (hiPSC) to contribute to the identification of novel disease-causing genes patients in diagnosis deadlock. To this aim, our team continuously collaborate with hospital services and by combining state-of-the-art molecular and functional technologies (transcriptomics, multi-omics, NGS, calcium imaging and MEA), we are able to identify the genes, biological pathways and functional properties affected in these various neuromuscular diseases. Overall, through stem cells modeling, this project will offer new avenues for understanding muscle pathologies and will facilitate the development of innovative therapeutic approaches.

One of Leslie’s specific focus is also deciphering the pathomechanisms of Laminin a2 deficiency related muscular dystrophy (LAMA2-RD), an ECM-related neuromuscular disorder and one of the most common congenital muscular dystrophies for which there exists no cure.

Principal investigator: Stefano Testa

Nowadays there is growing interest in developing alternatives to animal testing for disease modeling. Tissue engineering, thanks to the combination of skills and notions from different disciplines, represents one of the most promising research fields in replicating the morphological and biological complexity typical of human tissues and organs.

In this project, human primary1 and iPS2 cells will be used together with the latest 3D bioprinting technologies3,4 to replicate the biological niche and architecture of skeletal muscle. The goal is to develop mature and functional human skeletal muscles to be used as a reliable platform for studying NMDs.

Starting from cells isolated from healthy donors and patients, pathologies such as FSHD will be modeled and subsequently studied directly on human muscles produced in vitro, offering on the one hand a solid alternative to animal models and on the other a powerful tool for personalized medicine.

(1. Testa S. et al., 2020. doi: 10.3389/fphys.2020.553198; 2. Caron L. et al., 2023. doi: 10.3233/JND-230076; 3. Costantini M. et al., 2021. doi: 10.15252/emmm.202012778; 4. Fornetti E. et al., 2023. doi: 10.1088/1758-5090/acb573.)