Les mécanismes moléculaires impliqués dans la dystrophie musculaire oculopharyngée et traitements pharmacologiques sur le modèle drosophile.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant genetic disease characterized by a progressive muscle degeneration leading to ptosis (eyelid drooping), dysphagia (swallowing difficulty) and proximal limb weakness. OPMD is due to (GCN) repeat expansion in the gene encoding the poly(A) binding protein nuclear 1 (PABPN1) protein. These mutations result in expansion of an alanine stretch (11 to 18 alanines) at the N-terminus of PABPN1, leading to misfolding and aggregation of mutant PABPN1 in muscle nuclei. PABPN1 has several functions in RNAs biogenesis, including key roles in mRNA polyadenylation and in the turn-over of non-coding RNAs.Several molecular mechanisms involved in OPMD have been discovered such as mitochondrial dysfunction and oxidative stress, as well as increased proteasome activity leading to the degradation of myofibrillar proteins.The aim of my thesis was to identify other molecular pathways deregulated in OPMD. We use Drosophila models of OPMD which express the alanine-expanded PABPN1 (PABPN1-17ala) specifically in Drosophila muscles. These models recapitulate OPMD features such as progressive muscle degeneration and PABPN1 nuclear aggregation.We discovered that the Unfolded Protein Response (UPR) is activated during OPMD pathogenesis. UPR is activated by the endoplasmic reticulum (ER) stress when misfolded proteins accumulate in the ER. Analysis of molecular markers of the UPR shows that this pathway is activated in muscles expressing PABPN1-17ala. In addition, the IFB-088 molecule (developed by Inflectis BioScience), that targets the UPR to maintain its activation and translation inhibition, is beneficial in the Drosophila OPMD model. The genetic approach allowed us to demonstrate the functional role of the UPR in OPMD and the effect of IFB-088 through targeting its known target in the UPR.We also found that ribosomal RNAs (rRNAs) have an increased number of polyadenylation sites and longer poly(A)-tails, indicating a defect in rRNA processing and turn-over. Moreover, small RNA fragments accumulate in muscles of the OPMD Drosophila model, likely resulting from defective rRNA processing and degradation. Our hypothesis is that these rRNA fragments could impede the RNA interference pathway through their loading into Argonaute proteins.Another objective of my thesis was to test new compounds and identify active molecules in Drosophila OPMD models. Positive results were obtained with molecules showing antiaggregation properties and with antioxidant molecules. Further analysis of these molecules could lead to identifying potential candidates for future pharmacological treatments of OPMD.Molecular defects involved in OPMD are also often observed in other proteinopathies. Understanding the mechanisms involved in OPMD allows us to identify new active molecules in OPMD that might also prove to be efficient in other diseases.La dystrophie musculaire oculopharyngée (DMOP) est une maladie génétique autosomale dominante. Elle est caractérisée par une dégénérescence progressive de certains muscles squelettiques conduisant à une ptose (chute des paupières), une dysphagie (problème de déglutition) et une faiblesse des muscles proximaux. Une extension de triplet (GCN) au niveau du gène codant pour la poly(A) binding protein nuclear 1 (PABPN1) est responsable de la maladie. Cette mutation se traduit par une extension de 11 à 18 alanines au niveau de la partie N-terminale de la protéine. PABPN1 mutante adopte une conformation anormale qui conduit à son agrégation dans les noyaux des cellules musculaires. PABPN1 a plusieurs fonctions nucléaires dans la régulation de la biogénèse des ARNs, principalement dans la réaction de polyadénylation des ARNm, et dans le turn-over d’ARNs non-codants.Plusieurs mécanismes moléculaires responsables de la DMOP ont été découverts, en particulier un dysfonctionnement mitochondrial et le stress oxydatif, ainsi qu’une dérégulation de l’activité du protéasome conduisant à la dégradation des protéines myofibrillaires.L’objectif de ma thèse a été d’identifier d’autres voies moléculaires dérégulées et responsables de la DMOP. Pour cela, nous utilisons des modèles drosophiles de la DMOP qui expriment la protéine mutante PABPN1 avec une extension de 17 alanines (PABPN1-17ala), spécifiquement dans les muscles de la drosophile.Nous avons ainsi découvert l’activation de la voie « Unfolded Protein Response » (UPR) dans la DMOP. L’UPR est activée par le stress du réticulum endoplasmique (RE) lorsque des protéines malformées s’accumulent dans le RE. L’analyse de marqueurs moléculaires montre que la voie UPR est activée dans les muscles exprimant PABPN1-17ala. De plus, la molécule IFB-088 (développée par Inflectis BioScience), qui agit au niveau de la voie UPR afin de maintenir son activation et l’inhibition de la traduction, est bénéfique dans le modèle drosophile de la DMOP. L’approche génétique a permis de montrer le rôle fonctionnel de l’UPR dans la DMOP et l’effet de la molécule IFB-088 via le ciblage de sa cible connue dans la voie UPR.D’autre part, nous avons observé une augmentation d’utilisation de sites de polyadénylation le long de l’ARN ribosomique (ARNr) ainsi qu’une augmentation de la taille des queues poly(A) des ARNr dans le modèle drosophile de la DMOP. De plus, des petits fragments d’ARNs s’accumulent dans les muscles de drosophiles DMOP, provenant potentiellement de défauts de maturation et turn-over des ARNr. Notre hypothèse est que ces fragments d’ARNr pourraient perturber la voie de l’ARN interférence en se chargeant dans les protéines Argonautes.Le deuxième objectif de ma thèse a été d’identifier de nouvelles molécules actives dans les modèles drosophiles de la DMOP, dont l’analyse pourrait être poursuivie pour identifier des candidats potentiels pour de futurs traitements pharmacologiques de la DMOP. Des résultats positifs ont été obtenus avec des molécules possédant des propriétés anti-agrégation, et avec des molécules antioxydantes.Les défauts responsables de la DMOP sont souvent observés dans d’autres protéinopathies. La compréhension des mécanismes mis en jeu dans la DMOP nous permet d’identifier des molécules actives pour la DMOP et qui pourraient être aussi efficace pour d’autres maladies

The small compound Icerguastat reduces muscle defects in oculopharyngeal muscular dystrophy through the PERK pathway of the unfolded protein response

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. Here, using Drosophila OPMD models, we show that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. We show that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals a major contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD

Supplementary figures from The small compound Icerguastat reduces muscle defects in oculopharyngeal muscular dystrophy through the PERK pathway of the unfolded protein response

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. Here, using Drosophila OPMD models, we show that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. We show that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals the key contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD

Activation of the ubiquitin-proteasome system contributes to oculopharyngeal muscular dystrophy through muscle atrophy

International audienceOculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD

The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm

International audiencePiwi-interacting RNAs (piRNAs) and PIWI proteins are essential in germ cells to repress transposons and regulate mRNAs. In Drosophila, piRNAs bound to the PIWI protein Aubergine (Aub) are transferred maternally to the embryo and regulate maternal mRNA stability through two opposite roles. They target mRNAs by incomplete base pairing, leading to their destabilization in the soma and stabilization in the germ plasm. Here, we report a function of Aub in translation. Aub is required for translational activation of nanos mRNA, a key determinant of the germ plasm. Aub physically interacts with the poly(A)-binding protein (PABP) and the translation initiation factor eIF3. Polysome gradient profiling reveals the role of Aub at the initiation step of translation. In the germ plasm, PABP and eIF3d assemble in foci that surround Aub-containing germ granules, and Aub acts with eIF3d to promote nanos translation. These results identify translational activation as a new mode of mRNA regulation by Aub, highlighting the versatility of PIWI proteins in mRNA regulation

Transcriptional differences between the two host strains of Spodoptera frugiperda (Lepidoptera: Noctuidae)

International audienceSpodoptera frugiperda, the fall armyworm (FAW), is an important agricultural pest in the Americas and an emerging pest in sub-Saharan Africa, India, East-Asia and Australia, causing damage to major crops such as corn, sorghum and soybean. While FAW larvae are considered polyphagous, differences in diet preference have been described between two genetic variants: the corn strain (sf-C) and the rice strain (sf-R). These two strains are sometimes considered as distinct species, raising the hypothesis that host plant specialization might have driven their divergence. To test this hypothesis, we first performed controlled reciprocal transplant (RT) experiments to address the impact of plant diet on several traits linked to the fitness of the sf-C and sf-R strains. The phenotypical data suggest that sf-C is specialized to corn. We then used RNA-Se to identify constitutive transcriptional differences between strains, regardless of diet, in laboratory as well as in natural populations. We found that variations in mitochondrial transcription levels are among the most substantial and consistent differences between the two strains. Since mitochondrial genotypes also vary between the strains, we believe the mitochondria may have a significant role in driving strain divergence

Anti-prion Drugs Targeting the Protein Folding Activity of the Ribosome Reduce PABPN1 Aggregation

International audiencePrion diseases are caused by the propagation of PrP Sc , the pathological conformation of the PrP C prion protein. The molecular mechanisms underlying PrP Sc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrP Sc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide, metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrP Sc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies