Targeting membrane fragility in LGMD R2 through pharmacological autophagy induction

Targeting membrane fragility in LGMD R2 through pharmacological autophagy induction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting membrane fragility in LGMD R2 through pharmacological autophagy induction Noella Grossi, Emilie Pellier, Quentin Miagoux, Manon Benabides, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6671973/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of genetic disorders characterized by progressive weakening of the limb-girdle muscles. Among LGMDs, Limb-girdle muscular dystrophy type R2 (LGMDR2) is a rare condition affecting fewer than 1 in 100,000 individuals caused by mutations in the gene encoding dysferlin. Recent in vitro studies have suggested that autophagy flux is impaired in LGMDR2. Based on this evidence, we hypothesized that enhancing autophagy could provide therapeutic benefits for this condition. Autophagy plays a critical role in maintaining muscle integrity by clearing damaged cellular components, thus improving the defective dysferlin-mediated membrane repair mechanism. In this study, we performed a multiparametric screening of seventeen autophagy inducers in immortalized myoblasts derived from LGMDR2 patients to identify novel pharmacological compounds capable of enhancing membrane repair. Among the drugs tested, six were found to effectively stimulate autophagy and improve membrane resistance. Our findings demonstrate that inhibition of the mTOR pathway improves the cellular phenotype and underscore the potential of autophagy activators as a promising therapeutic target for LGMDR2. Biological sciences/Cell biology Biological sciences/Drug discovery Biological sciences/Molecular biology Health sciences/Diseases limb-girdle muscular dystrophies dysferlinopathy drug screening autophagy membrane resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Autophagy is a highly conserved process that involves the sequestration of cytoplasmic components into autophagosomes, which subsequently fuse with lysosomes for degradation and recycling. This process is essential for cellular maintenance, particularly under conditions of stress 1 . Dysregulation of autophagy has been implicated in several diseases, including neurodegenerative disorders, cancer and muscle diseases 2 . In recent years, there has been an explosion in the use of autophagy inducers in clinical medicine 3 . Several studies have particularly highlighted the potential of autophagy activation as a therapeutic strategy in muscular dystrophies, suggesting that enhancing autophagic flux may alleviate muscle damage and improve function in diseased states 4 . Among the different drugs inducing autophagy, rapamycin is one of the most extensively studied. As previously reported in Duchenne muscular dystrophy (DMD), systemic administration of rapamycin improves muscle function and reduces fibrosis in the mdx mouse by inhibiting the mTOR pathway 5 . In the same way, other studies described that activating the AMPK pathway in mdx mice using AICAR or metformin treatments, respectively improves on one hand diaphragm histopathology and maximal force-generating capacity 6 and on the other hand muscle strength and fiber membrane integrity 7 . The benefit of autophagy induction has also been evaluated in multiple other muscular dystrophies as reported in COLVI bethlem muscular dystrophy 8 – 10 , EDMD 11 , 12 or centronuclear myopathies 13 – 15 , revealing a reduction in the dystrophic phenotype, restoration of muscle strength, improvement in mitochondrial function and survival in mouse models 16 . In the past decade, several hypotheses have emerged to explain the mechanisms by which autophagy delays progression and reduces the severity of muscular diseases. Although the exact processes remain unclear, one leading hypothesis involves detoxification, where autophagy clears damaged proteins and dysfunctional organelles, thereby reducing cellular stress and preventing further muscle cell degeneration 17 . Another hypothesis emphasizes autophagy's role in facilitating the recruitment of intracellular vesicles for the repair of damaged muscle fiber membranes. Muscle fibers, due to their continuous cycles of contraction and relaxation, are especially prone to membrane damage. Autophagy contributes to this repair process by directing autophagosomes or lysosomes to the site of injury via the ESCRT transport machinery, thereby preserving the membrane integrity and functionality of muscle cells 18 , 19 . LGMDR2 or dysferlinopathy presents a unique set of characteristics that make it an ideal model for investigating the effect of autophagy inducers in muscular dystrophies. This disease affects approximately 1.63 people per 100,000 and is caused by a mutation in the DYSF gene, which encodes the dysferlin protein. Dysferlin shares structural similarity with the SYT protein family, which includes vesicle-anchored proteins involved in calcium sensing, synaptic vesicle fusion, and lysosomal exocytosis 20 . Dysferlin plays a critical role in membrane repair, and its absence or dysfunction leads to the progressive degeneration of muscle fibers and subsequent loss of muscle function. While the mechanisms by which dysferlin regulates intracellular membrane dynamics are complex and multifaceted, numerous studies have highlighted its primary role in the docking and fusion of vesicles carrying membrane components to sites of injury 20 . This vesicle fusion creates a "membrane patch" that effectively seals the damaged plasma membrane, restoring its integrity 21 , 22 . Here we report a comprehensive multiparametric pharmacological screening of seventeen autophagy-activating molecules in immortalized myoblasts derived from LGMDR2 patients. This library was assessed for its ability to stimulate autophagy by measuring p62, a classical receptor of autophagy. We also monitored their capacity to improve membrane resistance following osmotic shock. By focusing on these three readouts, our study narrowed down the six most potent inducers of autophagy as promising therapeutic candidates for the treatment of dysferlinopathy. RESULTS To investigate the therapeutic potential of autophagy inducers for dysferlinopathy, we screened a library of seventeen pharmacological compounds previously reported to enhance autophagy 23 . To do so, we utilized dysferlin-deficient immortalized myoblasts (DYSF S1173X ), a well-established cellular model that recapitulates key hallmarks of the disease 24 – 26 . Drug efficacy was evaluated in DYSF S1173X cells following 24 hours of treatment. Parallel analyses were conducted to measure the autophagy marker SQSTM1/p62 protein (hereafter referred to as p62), lysosomal activity using the Lysosensor probe, and membrane resistance following hypo-osmotic shock. Bazedoxifene was used as positive control 27 , and the control condition treated with 0.1% DMSO was used as negative control. Secondary assays were performed to comprehensively characterize the most promising candidates identified during the primary screen. The workflow is shown in Fig. 1 . Pharmacological induction of autophagy in dysferlin-deficient immortalised myoblasts. To minimize bias related to drug dosage and toxicity, the concentrations of the tested drugs were determined based on literature and cytotoxicity assessments. Cell viability was evaluated by comparing treated cells to the control condition (0.1% DMSO) using Incucyte® Nuclight Rapid Red Dye for live-cell nuclear labelling (Figure S1 A and S1B). For certain drugs, toxicity was observed at the reported doses, leading to a reduction in their concentrations. The following experiments were conducted at the adjusted doses listed in Table S1 B. To assess the ability of these drugs at the selected doses to induce autophagy in LGMDR2 myoblasts, we used LysoSensor™ dyes, acidotropic probes that accumulate in acidic organelles such as lysosomes following protonation, along with p62 immunolabeling. Bazedoxifene 2µM was used as a positive control, as we have previously reported that it induces an increase in the LysoSensor signal 27 . This analysis showed that all tested molecules were able to enhance the quantity of acidic organelles in LGMDR2 cells after 24 hours of treatment, as demonstrated by the representative images (Figure S2A). Quantifications (Fig. 2 A and 2 B) revealed that the drugs that induced the greatest increase in the LysoSensor signal were tomatidine, bazedoxifene, MF-094 and ambroxol. Because this result could reflect a decrease in lysosomal pH or an increase in lysosome volume, we further assessed autophagy activity by measuring the abundance of the autophagic receptor p62 28 . Our results revealed that all molecules showed a varying degree of increase in p62 fluorescence intensity, as shown by the representative images (Figure S2B) and quantitative graphs (Fig. 2 C and 2 D). Spermidine, actinonin, trehalose, and tomatidine induced the greatest increase in p62 signal intensity after 24 hours of treatment. Collectively, these findings suggest that the tested molecules induce autophagy at different levels in our model. Interestingly, the molecules showing more intense LysoSensor labelling were not necessarily the same ones showing a strong increase in p62, indicating that these drugs act at different stages of the autophagy process. As a control, we then determined whether the effect of these drugs was specific to mutated cells by evaluating their impact on autophagy induction in healthy muscle cells. The results showed that the trends were similar between the healthy and mutated cell lines, except for a few molecules that showed no increase in LysoSensor signal in healthy cells, such as nicotinamide mononucleotide, myo-inositol, carbamazepine, and metformin (Figure S3 and S4). Measurement of membrane resistance following autophagy induction in LGMDR2 To evaluate the therapeutic potential of autophagy inducers in dysferlinopathy, we then tested their ability to enhance membrane resistance following a hypo-osmotic shock assay. Briefly, for the osmotic shock assay, myoblasts were treated with the drugs for 24h and incubated with the Incucyte® Nuclight Rapid Red Dye to assess the toxicity of the compounds relative to the DMSO negative control and to establish a baseline of 0% for osmotic shock-induced mortality. Then, a hypo-osmotic shock was induced with a solution of 25% PBS and 75% water, along with the incubation of the Incucyte® Caspase-3/7 Green Dye. As previously reported, under these conditions, we can indirectly assess membrane fragility and membrane resistance in dysferlin-deficient cells in real-time by comparing the ratio of green to red fluorescence relative to the negative control (0.1% DMSO) 27 . Membrane resistance to osmotic shock was tested following treatment with the same library and bazedoxifene 2µM as a positive control (Fig. 3 A). Among these molecules, six compounds showed a beneficial effect on cell membranes: rapamycin, torkinib, metformin, actinonin, resveratrol, and spermidine, with respective mortality rates of 3%, 8%, 9%, 13%, 17%, and 30% in DYSF S1173X myoblasts (Fig. 3 B, 3 C and 3 D). Unexpectedly, other molecules had the opposite effect, such as trehalose, tomatidine, USP30 inhibitor, sorafenib, and nicotinamide riboside, which increased the percentage of cell death, reflecting a worsening of the phenotype. These results suggest that while certain autophagy-inducing drugs can enhance membrane resistance in dysferlinopathy, others may exacerbate cell death, highlighting the need for careful selection of therapeutic candidates and further investigation into their distinct pathways. Assessment of autophagy induction by the six drugs that enhance membrane resistance in LGMDR2 muscle cells. We then investigated several parameters reflecting autophagy induction following treatment with the six drug candidates that improve membrane resistance. To do so, we first confirmed drugs’ effects using Lysosensor (Fig. 4 A and 4 B) and p62 immunostaining through confocal imaging (Fig. 4 C and 4 D). To further characterize the effect of these drugs on autophagy, we then used a tandem fluorescence RFP-GFP-LC3B reporter system. This tandem RFP-GFP sensor capitalizes on the pH difference between the acidic autolysosome and the neutral autophagosome to stain autophagosome in yellow and the autolysosome in red. Accordingly, DYSF S1173X myoblasts were transiently transduced with the RFP-GFP-LC3B tandem sensor and treated with each of the six drugs or DMSO 0.1% for 24h. Our results revealed that all these molecules increased the accumulation of both autophagosomes and the autolysosomes except torkinib (Fig. 5 A and 5 B). This result was then confirmed by measuring LC3 and p62 levels by Western blot (Fig. 5 C). The Western blot analysis allows us to distinguish the effects of the drugs on the two forms of LC3, LC3B-I and LC3B-II. Indeed, the conversion of LC3B from its cytosolic form (LC3B-I) to the membrane-bound form (LC3B-II) is a key marker of autophagy modulation and the formation of autophagosomes. Our results showed that the treatments with the six drugs were capable of increasing the proportion of LC3B-II and p62, reflecting an accumulation of autophagosomes in immortalized myoblasts. Finally, we extended our analysis by measuring CytoID autophagy activity via flow cytometry. This assay allows for the quantification of autophagic vacuoles in live cells. For this assay, chloroquine, a known autophagy inhibitor that exacerbates the accumulation of vesicles, was used as a positive control. The results of this analysis revealed that the autophagic signal, calculated as median fluorescence intensity (MFI), was increased with the different treatments, with torkinib showing the most pronounced effect (Fig. 5 D and 5 E). Altogether, our data reveal that a subset of autophagy inducers improves membrane resistance in LGMDR2 muscle cells. Based on their known targets and reported mechanisms of action, three hypotheses emerge from these results to explain these effects on muscle cells: 1) that the increased autophagic vesicles serve as a pool of membrane material recruited by the membranes repair machinery, restoring cellular integrity; 2) that the increased number of autolysosomes improves muscle regeneration and contraction by degrading and recycling cellular debris; and 3) that the removal of damaged organelles, like mitochondria, through mitophagy reduces cellular stress and supports mitochondrial remodelling necessary for muscle regeneration (Fig. 6 ). DISCUSSION The present study identifies, on the one hand, tomatidine and MF-094 as the most effective autophagy inducers in LGMDR2 muscle cells based on lysosome quantification, and on the other hand, actinonin and spermidine based on p62 quantification. Notably, our findings also demonstrate that autophagy activation can enhance membrane resistance, a key phenotype in dysferlinopathy. Among the tested compounds, torkinib, resveratrol, spermidine, actinonin, metformin, and rapamycin showed significant potential in improving this phenotype. These results provide a promising therapeutic perspective for the treatment of LGMDR2. From this screening, we identified six compounds (torkinib, resveratrol, spermidine, actinonin, metformin, and rapamycin) that effectively induce autophagy and enhance membrane resistance in muscle cells affected by dysferlinopathy. These drugs can be categorized into two groups based on whether they modulate autophagy via mTOR-dependent mechanisms or not. A common feature of rapamycin, torkinib, and metformin is their ability to directly or indirectly target the mTOR (mammalian target of rapamycin) signalling pathway. As previously described, rapamycin specifically inhibits mTORC1 by binding to the FKBP12 protein, preventing its activation 29 . Torkinib, a direct inhibitor of mTOR kinase activity, targets both mTORC1 and mTORC2 complexes. While metformin primarily activates AMPK (AMP-activated protein kinase), this drug can also indirectly inhibit the mTOR pathway. The mTOR pathway is a crucial regulator of autophagy, which is vital for maintaining cellular homeostasis and degrading damaged proteins and organelles. By inhibiting mTOR, these compounds promote autophagy, aiding in the removal of defective cellular components and potentially restoring muscle function. In contrast, resveratrol and spermidine offer promising alternatives to direct mTOR inhibition for inducing autophagy in muscle cells 30 , 31 . Both compounds demonstrated efficacy in improving the membrane repair phenotype. Resveratrol, a polyphenol with antioxidant properties, activates autophagy through modulation of the AMPK/SIRT1 signalling pathway 32 . Spermidine, an endogenous polyamine, activates autophagy through inhibition of acetyltransferase, in particular E1A-binding protein p300 (EP300), an endogenous repressor of autophagy 33 , 34 . Interestingly, both resveratrol and spermidine also offer additional benefits, such as anti-inflammatory and cytoprotective effects, making them attractive candidates for therapies to enhance muscle regeneration and function 35 – 38 without the risks associated with mTOR long-term treatment 39 – 41 . Lastly, actinonin activates mitophagy 42 , a selective form of autophagy responsible for the removal of damaged mitochondria, essential for maintaining cellular health. Regardless of the mechanisms underlying the effects of these drugs, our study demonstrates that several potent autophagy inducers enhance the lysosomal pool and promote the membrane repair process in LGMDR2 muscle cells. These findings align with multiple other studies showing that lysosomes are the primary vesicular population responsible for repairing the plasma membrane and highlight the critical role of dysferlin in mediating their exocytosis toward membrane lesions. Autophagy induction has been explored as a therapeutic avenue in a wide range of diseases, including cancers, infections, neurodegenerative disorders, metabolic diseases, inflammatory conditions, and muscular diseases. Over the past decades, several FDA-approved drugs have been shown to enhance autophagy and yield beneficial effects in various clinical contexts. For instance, mTOR inhibitors have demonstrated therapeutic potential in degenerative diseases such as Huntington’s disease 43 , while the tyrosine kinase inhibitor erlotinib has shown efficacy in diabetic nephropathy 44 , and nilotinib has been studied in Alzheimer's disease 45 . Other drugs, such as carbamazepine for alpha1-antitrypsin deficiency 46 , trifluoperazine for salmonella infection 47 , and statins for Mycobacterium tuberculosis infection 48 , also modulate autophagy and have demonstrated therapeutic benefits. However, it remains unclear whether the positive regulation of autophagy is the primary mechanism underlying the therapeutic effects of these agents, or if they exert their benefits through other pleiotropic actions. Notably, resveratrol has been extensively studied in clinical trials for its potential to prevent and manage a broad spectrum of diseases such as diabetes mellitus, obesity, colorectal cancer, breast cancer, multiple myeloma, metabolic syndrome, hypertension, Alzheimer's disease, stroke, cardiovascular disease, kidney disease, inflammatory disorders, and nasopharyngitis 49 . The beneficial effects of autophagy in muscle cells extend beyond enhancing membrane repair following muscle contraction; they also involve its potent detoxifying action. In the muscles, pathological conditions often lead to progressive mitochondrial dysfunction, which causes chronic bioenergetic inefficiency and the accumulation of cytotoxic aggregates or redox-active proteins (ROS). While ROS can exacerbate cell damage through oxidative stress, autophagy plays a protective role by eliminating damaged mitochondria and proteins, thereby reducing cellular injury and promoting cell survival 50 . Furthermore, autophagy counteracts cellular senescence and regulated cell death by facilitating the repair of damaged DNA 51 . Autophagic responses are crucial for maintaining intracellular homeostasis, particularly in stem cell compartments. Activated autophagy supports cellular energy requirements during the activation and proliferation of myogenic stem cells following muscle injury 52 . Additionally, autophagy declines with age, and the pharmacological compounds used in this study are known to extend lifespan and protect against degenerative diseases. In general, these compounds promote healthy aging by sustaining cellular function and mitigating age-related decline. In summary, this study advances our understanding of how autophagy activation can improve membrane repair process in dysferlinopathy and identifies pharmacological compounds with potential as therapeutic interventions. These compounds are not only well-characterized but also generally well-tolerated in humans, positioning them as strong candidates for therapies aimed at modulating autophagy in dysferlinopathies. Our results also underscore the need for in vivo studies to evaluate the long-term effects of these compounds and to further elucidate the molecular mechanisms underlying their autophagic activities and benefits. Materials and Methods Cell culture and treatments. In this study, we use immortalized myoblasts derived from either LGMDR2 patients or healthy controls. These myoblasts were isolated from human muscle biopsy and obtained from the human cell immortalization platform Myoline at the Institut de Myologie (Paris, France), with the consent of the subjects by signing an informed consent form and anonymization before immortalization, according to the EU GDPR regulation. The healthy line is AB1079, and the mutated line is AB320, which carries the heterozygous nonsense mutation DYSF c.342-1G > A/c.3516_3517delTT; p.Ser1173X (DYSF S1173X ). Myoblasts were cultured in growth medium consisting of 1 volume of 199 medium (Invitrogen, United States) for 4 volumes of Dulbecco’s modified Eagle’s medium (Invitrogen, United States), supplemented with 20% fetal bovine serum (Sigma-Aldrich, United States), 25 µg/ml fetuin (Life Technologies, United States), 5 ng/ml epidermal growth factor (Life Technologies, United States), 0.5 ng/ml basic fibroblast growth factor (Life Technologies, United States), 0.2 µg/ml dexamethasone (Sigma-Aldrich, United States) and 5 µg/ml insulin (Sigma-Aldrich, United States). Cells were seeded on plates coated with 0.1% of gelatin and maintained in a humidified atmosphere of 5% CO 2 at 37°C. Reagents and antibodies. The antibodies used in immunofluorescence were as follows: anti-p62/SQSTM1 (Abcam, Cambridge, UK, #ab56416) primary antibodies, Alexa Fluor 555-conjugated goat anti-mouse IgG (Invitrogen, Thermo Fisher Scientific, Carlsbad, USA, #A-31570) secondary antibody. Antibodies used in western blot: anti-LC3B (Novus Biological, USA, NB600-1384), anti-p62/SQSTM1 (Abcam, Cambridge, UK, #ab56416), anti-β-actin (LI-COR Biosciences, Lincoln, USA, #926-42210), IRDye 800CW donkey anti-rabbit (LI-COR Biosciences, Lincoln, USA, #926-32213), IRDye 800CW donkey anti-rabbit (LI-COR Biosciences, Lincoln, USA, #926-32213), IRDye 680RD donkey anti-mouse (LI-COR Biosciences, Lincoln, USA, #926-68072). The reagents used in the study were: Spermidine (MedChemExpress, Monmouth Junction, USA, #HY-B1776 ), Nicotinamide riboside (TargetMol, Massachusetts, USA, #T13795), Nicotinamide mononucleotide (TargetMol, Massachusetts, USA, #T4721), Actinonin (MedChemExpress, Monmouth Junction, USA, #HY-113952 ), Tomatidine (MedChemExpress, Monmouth Junction, USA, #HY-N2149), D-(+)-Trehalose (MedChemExpress, Monmouth Junction, USA, #HY-N1132), I-Inositol (MedChemExpress, Monmouth Junction, USA, #HY-B1411), Resveratrol (MedChemExpress, Monmouth Junction, USA, #HY-16561), Rapamycine (Selleck Chemicals LLC, Houston, USA, #S1039), Carbamazepine (Selleck Chemicals LLC, Houston, USA, #S1693), Metformin (MedChemExpress, Monmouth Junction, USA, #HY-B0627), Ambroxol (MedChemExpress, Monmouth Junction, USA, #HY-B1039), MF-094 (TargetMol, Massachusetts, USA, #282T12024), USP30inhibitor 18 (TargetMol, Massachusetts, USA, #282T36682), Sorafenib (MedChemExpress, Monmouth Junction, USA, #HY-10201), Torkinib (MedChemExpress, Monmouth Junction, USA, #HY-10474), Bazedoxifene HCL (Selleck Chemicals LLC, Houston, USA, #S2128). Osmotic shock assay. The phenotypic membrane resistance assay was used as previously described 27 (Bruge et al., 2025). Cells were seeded using a Bravo Automated Liquid Handling platform (Agilent, United States). DYSF S1173X myoblasts were seeded at 8300 cells/cm 2 in 38 µl of culture medium in black clear-bottom 384-well plates. After 72 hours, the cells were treated with 2 µl of 20x concentrated solutions of each drug at the concentration shown in Table S1 , along with a negative control (0.1% dimethyl sulfoxide, DMSO, VWR), or a positive control (2µM Bazedoxifene). Following 24 hours of treatment, cells were incubated for 3 hours with a red viability probe (1/3000, Incucyte® Nuclight Rapid Red Dye - Sartorius, Germany), which labels the nuclei of live cells. This step was performed to assess the toxicity of the compounds relative to the DMSO control. Then, a hypo-osmotic shock was induced by exposing the cells to a solution containing 25% PBS and 75% water, along with a green mortality probe (1/3000, Incucyte® Caspase-3/7 Green Dye - Sartorius, Germany). Green fluorescence was monitored every 4 hours using an Incucyte® S3 Live-Cell Analysis system (Sartorius, Germany). The resulting images were analysed with Incucyte software, to assess the cell resistance to osmotic shock by comparing the green-to-red fluorescence ratio relative to the negative control (DMSO). Immunostaining assay. Immunofluorescence staining was carried out to evaluate the ability of the molecules to induce autophagy in our cell models. After 24 hours of drug treatment, myoblasts were fixed in 4% paraformaldehyde for 10 minutes at room temperature. Permeabilization was done using 0.5% Triton X-100 (Thermo Scientific, United States) for 10 minutes at room temperature, followed by blocking with 1% bovine serum albumin (BSA; Sigma-Aldrich, United States) for 1 hour at room temperature. The cells were then incubated overnight at 4°C with primary antibody: mouse anti-P62 antibody (1:250 dilution), diluted in 1% BSA. After three washes in PBS, cells were incubated with secondary antibody: Alexa fluor 555 goat anti-mouse (1:1,000 dilution), with Hoechst solution (Invitrogen, United States) for nuclear staining, in the dark for 1 hour at room temperature. Following additional washing steps, cells were visualized under a LSM 800 confocal microscope (Zeiss, Germany) with a 40x oil immersion high-resolution objective and imaged using Zen software (Zeiss, Germany). P62 puncta quantification. Myoblasts were seeded in 96-well plates and treated with the different drugs or negative control DMSO for 24 hours. After fixation with 4% PFA, immunostaining was carried out, and image of p62 expression was captured using an LSM-800 confocal microscope. For quantification, p62 expression was analysed using a 20x high-resolution objective of the CellInsight CX7 HCS Platform (Cellomics Inc.). The p62 puncta area was quantified for each well (n = 3 fields) by applying thresholding. Total cell number was determined by counting the Hoechst-stained cells per well, allowing normalization of the total p62 puncta area to the number of nuclei. To confirm the results with confocal image replicates, a second quantification was carried out in Fig. 4 . The relative fluorescence expression area was analysed using the ImageJ2 software (Fuji). Lysosomal pH measurement. LysoSensor Green DND-189 dye (L7535, ThermoFisher) was employed to measure the lysosomal pH in cells. Myoblasts were grown in 96-well plates and treated with the different drugs or negative control DMSO for 24 hours. Preheated 1 µM LysoSensor Green DND-189 dye (37°C) was added in culture medium for 2 hours at 37°C. We fixed cells with 4% formaldehyde and counterstained with Hoechst 33342 solution (Invitrogen, United States), and images were acquired using a LSM-800 confocal microscope (Zeiss, Germany). For quantification, the plate was analysed by a 20x high-resolution objective of the ImageXpress Micro XL System imager (Molecular Devices). Lysosensor fluorescence area was quantified for each well (n = 4 fields) by applying thresholding. Total cell number was determined by counting Hoechst-stained cells per well, allowing normalization of the total lysosensor fluorescence to the number of nuclei. To confirm the results with confocal image replicates, a second quantification was carried out in Fig. 4 . The relative fluorescence expression area was analysed using the ImageJ2 software (Fuji). Western blot analysis. Immortalized myoblasts were collected after 24 hours of drug treatment. Cells were lysed in a solution containing NP40 lysis buffer (Thermo Scientific, United States), 1X Proteases and phosphatase Inhibitors (Complete PIC, Roche, Switzerland), to extract total protein. The lysate was centrifuged for 15 minutes at 4°C, and the supernatant was collected as total cellular protein. Their concentrations were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, United States). Samples were heated at 95°C for 5 min, and 20 µg of protein was loaded onto 4–15% Criterion™ XT tris-glycine protein gel (BioRad, United States) for electrophoresis and transferred to nitrocellulose membrane (BioRad, United States) using a Trans-Blot Turbo Transfert system (BioRad, United States). Blocked in Odyssey blocking buffer (Li-Cor, United States) for 1h at room temperature, the membrane was then incubated with primary antibodies in blocking buffer supplemented with 0.1% Tween 20 (VWR, United States) overnight at 4°C for the rabbit anti-LC3B 1:1,000 (NB600-1384, Novus), the mouse anti-p62/SQSTM1 1:1,000 (Abcam, ab56416), and the mouse anti—β-actin 1:1,000 (Li-Cor, 926-42210). After three washes for 10 min at room temperature with Tris-buffered saline with 0.1% Tween 20 (VWR, United States), the membrane was incubated with fluorescent secondary antibodies donkey anti-mouse antibody IRDye-680 1:10,000 (926-32222, Li-Cor) or a donkey anti-rabbit antibody IRDye-800 1:5,000 (926-32213, Li-Cor) for 1 hour at room temperature. The membrane was then washed again, and protein bands were visualized by fluorescence using the Odyssey CLx system (Li-Cor, United States). Protein expression levels were quantified by measuring the intensity of bands using Image Studio™ software. The fold change was determined after normalizing to the loading control. Flow cytometry measurement of autophagy flux. We use CYTO-ID® Autophagy Detection Kit (Enzo Life Sciences) to measure autophagy flux. This kit detects all autophagic vesicles in cells, including pro-autophagosomes, autophagosomes, and autophagic lysosomes. Immortalized myoblasts were cultured on 0.1% gelatin-coated 6-well plates until they reached 80% confluence and then treated with pharmacological compounds for 24 hours. After treatment, cells were collected by trypsin-EDTA (Gibco, United States) dissociation, followed by centrifugation at 1200 rpm for 5 minutes. Cells were then washed with 300 µl of 1X Assay Buffer, centrifuged, and the supernatant discarded by inverting the plate onto laboratory paper. A solution with CYTO-ID® Green Detection Reagent was added to the cells for 30 min at 37°C in the dark. After two successive washings, the cells were resuspended in 100 µl of 1X Assay Buffer and analysed using a MACSquant analyser (Miltenyi Biotec, Germany) with a 488 nm laser source and the green fluorescence channel. A minimum of 30,000 cells was measured per sample. Data analysis was performed using FlowJo Software (BD Biosciences, United States). Intact cells were gated based on FSC-A vs SSC-A, and the median fluorescence intensity (MFI) of eGFP for each sample was calculated. The baseline autophagic flux, represented by the eGFP-MFI of the DMSO control, was subtracted to determine the autophagic flux shift (eGFP-MFI shift). Quantification was performed with five measures of independent experiments. RFP-GFP-LC3B Tandem Sensor Assay. Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B kit (Thermo Scientific, United States) was used to monitor autophagic flux. Reagent was added to immortalized myoblasts at a concentration of 30 particles per cell according to the manufacturer’s instructions. After 24 hours, culture medium was replaced, and cells were treated with the different drugs or 0.1% DMSO for an additional 24 hours. The cells were then fixed with 4% formaldehyde and counterstained with Hoechst 33342 solution 1:1,000 (Invitrogen, United States) for 10 minutes at room temperature. Finally, coverslips were mounted for fluorescence microscopy. Images were acquired using an LSM-800 confocal microscope. Quantification was performed with twenty images of three independent experiments. The degree of fluorescence co-localization and relative fluorescence expression area were analysed using the ImageJ2 software (Fuji). Statistical analysis. Statistical analyses were performed using one-way analysis of variance (ANOVA) to perform the comparisons between multiple groups. Results from each experiment were expressed as the mean ± standard deviation. Statistical significance was defined as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. All graphs were plotted and analyzed using GraphPad Prism Software (v9.2.0). Declarations Ethical statement All methods were performed in accordance with the relevant guidelines and French regulations. The experimental protocols and ethical authorization were approved by the comité de protection des personnes under the reference: Mecamedirare 2019-A02599-48. Competing interests The author(s) declare no competing interests. Funding Istem/CECS is supported by the Association Française contre les Myopathies (AFM-Téléthon). This project was also supported by grants from Laboratoire d’Excellence Revive (Investissement d’Avenir; ANR-10-LABX-73), the IDEX Paris-Saclay (for Initiative D’EXcellence), the Region Ile-de-France via the doctoral school « Structure et dynamique des systèmes vivants » (ED 577) from Paris Saclay University. The stemCARE platform is supported by AFM-Téléthon, GIS IBISA, Région Ile de France, BPI, INSERM and UEVE for staff and equipments. The stemCARE platform is part of GENOPOLE and GENOTHER bioclusters. This study was part of the DREAMS project. Funded by the European Union under 101080229-2. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union (EU) or European Research Executive Agency (REA). Neither the EU nor REA can be held responsible for them. Author Contribution X.N. was responsible for the experimental design and project management. N.G. performed the cell culture experiments, drug treatments, carried out screening analysis and realized functional characterization of the drugs. C.B. and E.P. developed the osmotic shock method. C.L. and J.P. developed imaging analysis of p62 puncta and lysosensor puncta. Q.M. contributed to the statistical analyses and provided his expertise in autophagy measurement tools. M.B. provided technical assistance for flow cytometry experiments. E.P., M.B., C.B. provided technical assistance for cell culture and pharmacological studies. N.G. prepared the figures. N.G. and X.N. wrote the manuscript. All authors reviewed and edited the paper. Acknowledgement The authors thank Marc Peschanski (IStem), Isabelle Richard (Genethon) for helpful discussions and Marc Bartoli (MMG, Marseille) for his expertise in osmotic shock. We are grateful to the Platform for Immortalization of Human Cells “Myoline” from the Centre of Research in Myology (Institute of Myology, Paris) for providing immortalized myoblasts used in this study. Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files References Ryter, S. W., Bhatia, D. & Choi, M. E. Autophagy: A Lysosome-Dependent Process with Implications in Cellular Redox Homeostasis and Human Disease. Antioxid Redox Signal 30 , 138–159 (2019). Schneider, J. L. & Cuervo, A. M. Autophagy and human disease: emerging themes. Curr Opin Genet Dev 26 , 16–23 (2014). Levine, B., Packer, M. & Codogno, P. Development of autophagy inducers in clinical medicine. The Journal of Clinical Investigation 125 , 14 (2015). Li, H., Zhang, L., Zhang, L. & Han, R. Autophagy in striated muscle diseases. Front Cardiovasc Med 9 , 1000067 (2022). Eghtesad, S., Jhunjhunwala, S., Little, S. R. & Clemens, P. R. Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle. Mol Med 17 , 917–924 (2011). Pauly, M. et al. AMPK activation stimulates autophagy and ameliorates muscular dystrophy in the mdx mouse diaphragm. Am J Pathol 181 , 583–592 (2012). Dong, X. et al. Metformin Increases Sarcolemma Integrity and Ameliorates Neuromuscular Deficits in a Murine Model of Duchenne Muscular Dystrophy. Front Physiol 12 , 642908 (2021). Grumati, P. et al. Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration. Nat Med 16 , 1313–1320 (2010). Chrisam, M. et al. Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice. Autophagy 11 , 2142–2152 (2015). Castagnaro, S. et al. Autophagy activation in COL6 myopathic patients by a low-protein-diet pilot trial. Autophagy 12 , 2484–2495 (2016). Choi, J. C. & Worman, H. J. Reactivation of autophagy ameliorates LMNA cardiomyopathy. Autophagy 9 , 110–111 (2013). Ramos, F. J. et al. Rapamycin reverses elevated mTORC1 signaling in lamin A/C-deficient mice, rescues cardiac and skeletal muscle function, and extends survival. Sci Transl Med 4 , 144ra103 (2012). Tasfaout, H., Cowling, B. S. & Laporte, J. Centronuclear myopathies under attack: A plethora of therapeutic targets. J Neuromuscul Dis 5 , 387–406. Massana-Muñoz, X. et al. Inactivating the lipid kinase activity of PI3KC2β is sufficient to rescue myotubular myopathy in mice. JCI Insight 8 , e151933 (2023). Ji, J. et al. BIN1 gene replacement reverses BIN1-related centronuclear myopathy. Mol Ther S1525-0016(25)00315–6 (2025) doi:10.1016/j.ymthe.2025.04.036. Castets, P., Frank, S., Sinnreich, M. & Rüegg, M. A. ‘Get the Balance Right’: Pathological Significance of Autophagy Perturbation in Neuromuscular Disorders. J Neuromuscul Dis 3 , 127–155 (2016). Xia, Q. et al. The Role of Autophagy in Skeletal Muscle Diseases. Front Physiol 12 , 638983 (2021). Claude-Taupin, A. et al. ATG9A protects the plasma membrane from programmed and incidental permeabilization. Nat Cell Biol 23 , 846–858 (2021). Lu, T.-W., Frost, A. & Moss, F. R. Organelle homeostasis requires ESCRTs. Current Opinion in Cell Biology 93 , 102481 (2025). Glover, L. & Brown, R. H. Dysferlin in membrane trafficking and patch repair. Traffic 8 , 785–794 (2007). Han, R. & Campbell, K. P. Dysferlin and Muscle Membrane Repair. Curr Opin Cell Biol 19 , 409–416 (2007). Bansal, D. et al. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature 423 , 168–172 (2003). Aman, Y. et al. Autophagy in healthy aging and disease. Nat Aging 1 , 634–650 (2021). Cea, L. A. et al. The absence of dysferlin induces the expression of functional connexin-based hemichannels in human myotubes. BMC Cell Biol 17 Suppl 1 , 15 (2016). Báez-Matus, X. et al. Defects in G-Actin Incorporation into Filaments in Myoblasts Derived from Dysferlinopathy Patients Are Restored by Dysferlin C2 Domains. Int J Mol Sci 21 , 37 (2019). Mamchaoui, K. et al. Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders. Skelet Muscle 1 , 34 (2011). Bruge, C. et al. High-throughput screening identifies bazedoxifene as a potential therapeutic for dysferlin-deficient limb girdle muscular dystrophy. Br J Pharmacol (2025) doi:10.1111/bph.70017. Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8 , 445–544 (2012). Harris, T. E. & Lawrence, J. C. TOR signaling. Sci STKE 2003 , re15 (2003). Morselli, E. et al. Autophagy mediates pharmacological lifespan extension by spermidine and resveratrol. Aging (Albany NY) 1 , 961–970 (2009). Morselli, E. et al. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. J Cell Biol 192 , 615–629 (2011). Bastin, J. & Djouadi, F. Resveratrol and Myopathy. Nutrients 8 , 254 (2016). Galasso, L. et al. Polyamines and Physical Activity in Musculoskeletal Diseases: A Potential Therapeutic Challenge. Int J Mol Sci 24 , 9798 (2023). Pietrocola, F. et al. Spermidine induces autophagy by inhibiting the acetyltransferase EP300. Cell Death Differ 22 , 509–516 (2015). Gerner, E. W. & Meyskens, F. L. Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer 4 , 781–792 (2004). Deng, K. et al. Increased synthesis of spermidine as a result of upregulation of arginase I promotes axonal regeneration in culture and in vivo. J Neurosci 29 , 9545–9552 (2009). LaRocca, T. J., Gioscia-Ryan, R. A., Hearon, C. M. & Seals, D. R. The autophagy enhancer spermidine reverses arterial aging. Mech Ageing Dev 134 , 314–320 (2013). Eisenberg, T. et al. Cardioprotection and lifespan extension by the natural polyamine spermidine. Nat Med 22 , 1428–1438 (2016). Pallet, N. & and Legendre, C. Adverse events associated with mTOR inhibitors. Expert Opinion on Drug Safety 12 , 177–186 (2013). Stallone, G., Infante, B., Grandaliano, G. & Gesualdo, L. Management of side effects of sirolimus therapy. Transplantation 87 , S23-26 (2009). Gyurus, E., Kaposztas, Z. & Kahan, B. D. Sirolimus therapy predisposes to new-onset diabetes mellitus after renal transplantation: a long-term analysis of various treatment regimens. Transplant Proc 43 , 1583–1592 (2011). Kshirsagar, S., Sawant, N., Morton, H., Reddy, A. P. & Reddy, P. H. Mitophagy enhancers against phosphorylated Tau-induced mitochondrial and synaptic toxicities in Alzheimer disease. Pharmacol Res 174 , 105973 (2021). Sarkar, S. Chemical screening platforms for autophagy drug discovery to identify therapeutic candidates for Huntington’s disease and other neurodegenerative disorders. Drug Discovery Today: Technologies 10 , e137–e144 (2013). Zhang, M.-Z., Wang, Y., Paueksakon, P. & Harris, R. C. Epidermal growth factor receptor inhibition slows progression of diabetic nephropathy in association with a decrease in endoplasmic reticulum stress and an increase in autophagy. Diabetes 63 , 2063–2072 (2014). Lonskaya, I., Hebron, M. L., Desforges, N. M., Schachter, J. B. & Moussa, C. E.-H. Nilotinib-induced autophagic changes increase endogenous parkin level and ubiquitination, leading to amyloid clearance. J Mol Med (Berl) 92 , 373–386 (2014). Hidvegi, T. et al. An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis. Science 329 , 229–232 (2010). Conway, K. L. et al. Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection. Gastroenterology 145 , 1347–1357 (2013). Parihar, S. P. et al. Statin therapy reduces the mycobacterium tuberculosis burden in human macrophages and in mice by enhancing autophagy and phagosome maturation. J Infect Dis 209 , 754–763 (2014). Singh, A. P. et al. Health benefits of resveratrol: Evidence from clinical studies. Med Res Rev 39 , 1851–1891 (2019). Ureshino, R. P., Rocha, K. K., Lopes, G. S., Bincoletto, C. & Smaili, S. S. Calcium signaling alterations, oxidative stress, and autophagy in aging. Antioxid Redox Signal 21 , 123–137 (2014). Karantza-Wadsworth, V. et al. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev 21 , 1621–1635 (2007). Salemi, S., Yousefi, S., Constantinescu, M. A., Fey, M. F. & Simon, H.-U. Autophagy is required for self-renewal and differentiation of adult human stem cells. Cell Res 22 , 432–435 (2012). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6671973","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483186718,"identity":"05756eb8-0c16-4961-b20d-f5751a4554d4","order_by":0,"name":"Noella Grossi","email":"","orcid":"","institution":"Institut des Cellules Souches pour le Traitement et l'Étude des Maladies Monogéniques","correspondingAuthor":false,"prefix":"","firstName":"Noella","middleName":"","lastName":"Grossi","suffix":""},{"id":483186719,"identity":"5c6ff087-fe0b-4b1a-9da1-bb1a2e8b3f5b","order_by":1,"name":"Emilie Pellier","email":"","orcid":"","institution":"Institut des 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Biorender).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6671973/v1/2b3c0cb77909c1c9acbe00ca.png"},{"id":86667224,"identity":"0da9b471-113a-474f-aef0-a54e4f4d9c6a","added_by":"auto","created_at":"2025-07-14 11:14:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":245419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbility of the molecules tested to modulate autophagy on S1173X dysferlin\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emyoblasts. (A)\u003c/strong\u003e Primary screen cell-based assay for lysosome expression. Dot plot representation of the effects of the 17 compounds on lysosome expression after 24 hours of treatment and cell viability. \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of total lysosome puncta area following treatment relative to the control DMSO 0.1%, using MetaXpress software. Each bar represents the mean ± SD (n=3). \u003cstrong\u003e(C)\u003c/strong\u003e Secondary screen cell-based assay for p62 expression. Dot plot representation of the effects of the 17 compounds on p62 expression after 24 hours of treatment and cell viability. \u003cstrong\u003e(D)\u003c/strong\u003eQuantification of total p62 puncta area following treatment relative to the control DMSO 0.1%, using HCS Studio software. Each bar represents the mean ± SD (n=3). Abbreviations: NR, Nicotinamide riboside; NMN, Nicotinamide mononucleotide.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6671973/v1/01c84fa69f881ff23b835cfe.png"},{"id":86667226,"identity":"ac04b338-244e-46cd-9c76-aeb7eef23d5a","added_by":"auto","created_at":"2025-07-14 11:14:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":365734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMeasurement of membrane resistance following autophagy induction in LGMDR2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Cell-based screen for drug efficacy on membrane repair phenotype. Measurement of cell mortality after hypo-osmotic shock on \u003cem\u003eDYSF\u003c/em\u003e\u003csup\u003e\u003cem\u003eS1173X\u003c/em\u003e\u003c/sup\u003e immortalized myoblasts pretreated for 24 hours with the 17 compounds. Each point represents the mean ± SD (n=4) of a representative experiment from three independent experiments. \u003cstrong\u003e(B)\u003c/strong\u003e Dot plot of the effects of the 17 compounds on cell mortality after 24 hours of hypo-osmotic shock and cell viability. \u003cstrong\u003e(C)\u003c/strong\u003e Graphical representation of cell mortality after hypo-osmotic shock with the 6 most effective treatments, i.e. torkinib (10 µM, purple curve), resveratrol (300 µM, dark blue curve), spermidine (100 µM, light blue curve), actinonin (10 µM, yellow curve), metformin (200 µM, pink curve), rapamycin (20 µM, brown curve) compared with 0.1% DMSO (red curve). \u003cstrong\u003e(D)\u003c/strong\u003e Table summarizing cell viability after 24 hours of treatment and cell mortality after 24 hours of osmotic shock.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6671973/v1/ecd8ba73974300dfa6a1d26e.png"},{"id":86668706,"identity":"83a01a37-fb99-4e35-9d8c-e8a915b7c421","added_by":"auto","created_at":"2025-07-14 11:22:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":937643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of autophagic fluxes on the S1173X dysferlin\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emyoblasts treated with the 6 effective compounds.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative confocal images depicting lysosomes (green) puncta in \u003cem\u003eDYSF\u003c/em\u003e\u003csup\u003e\u003cem\u003eS1173X\u003c/em\u003e\u003c/sup\u003e immortalized myoblasts treated with 0.1% DMSO or each 6 effective compounds for 24 hours. Hoechst staining (blue) labels nuclei. Scale bar = 50 µm.\u003cstrong\u003e (B)\u003c/strong\u003e Quantification of total lysosome puncta area following treatment relative to the control DMSO 0.1%, using Fiji software. Each bar represents the mean ± SD of 15 images in 3 independent experiments (5 images per experiment). *\u003cem\u003ep\u003c/em\u003e≤0.05, **\u003cem\u003ep\u003c/em\u003e≤ 0.01, ***\u003cem\u003ep\u003c/em\u003e≤0.001 versus DMSO group (one-way ANOVA with Dunn's multiple comparisons test). \u003cstrong\u003e(C)\u003c/strong\u003e Representative confocal images depicting p62 (red) puncta in \u003cem\u003eDYSF\u003c/em\u003e\u003csup\u003e\u003cem\u003eS1173X\u003c/em\u003e\u003c/sup\u003e immortalized myoblasts treated with 0.1% DMSO or each 6 effective compounds for 24 hours. Hoechst staining (blue) labels nuclei. Scale bar = 50 µm. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of total p62 puncta area following treatment relative to the control DMSO 0.1%, using Fiji software. Each bar represents the mean ± SD of 15 images in 3 independent experiments (5 images per experiment). *\u003cem\u003ep\u003c/em\u003e≤0.05, **\u003cem\u003ep\u003c/em\u003e≤ 0.01, ***\u003cem\u003ep\u003c/em\u003e≤0.001 versus DMSO group (one-way ANOVA with Dunn's multiple comparisons test).\u0026nbsp;\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6671973/v1/fcea3ed7b6455ce256204974.png"},{"id":86668708,"identity":"1c3e4b44-95ad-4fab-b68b-fc6afdcfd901","added_by":"auto","created_at":"2025-07-14 11:22:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":677521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSecondary tests on autophagic fluxes with the 6 effective compounds.\u003c/strong\u003e \u0026nbsp;\u003cstrong\u003e(A)\u003c/strong\u003e Representative confocal images depicting GFP-LC3 (green) and RFP-LC3 (red) puncta colocalization in \u003cem\u003eDYSF\u003c/em\u003e\u003csup\u003eS1173X\u003c/sup\u003e myoblasts transiently expressing GFP-RFP-LC3B and treated with 0.1% DMSO or each 6 effective compounds for 24 hours. Nuclei are labelled by Hoechst staining (blue). Scale bar = 20 µm. \u003cstrong\u003e(B) \u003c/strong\u003eQuantification of autophagosomes and autolysosomes in myoblasts transiently expressing GFP-RFP-LC3B, following treatment relative to the control DMSO 0.1%, using Fiji software. Each bar represents the mean ± SD of 15 images in 3 independent experiments (5 images per experiment). *\u003cem\u003ep\u003c/em\u003e≤0.05, **\u003cem\u003ep\u003c/em\u003e≤ 0.01, ***\u003cem\u003ep\u003c/em\u003e≤0.001 versus DMSO group (one-way ANOVA with Dunn's multiple comparisons test). \u003cstrong\u003e\u0026nbsp;(C)\u003c/strong\u003e Representatives immunoblot analysis of LC3-I, LC3-II and p62 expression in DYSF\u003csup\u003eS1173X\u003c/sup\u003e myoblasts treated with DMSO (0.1%) or each 6 effective compounds for 24 hours. The values below the blot show relative levels of LC3-II/β-actin and p62/β-actin protein after normalization to DMSO-treated cells. Full-length gels are presented in the Supplementary File S5. \u003cstrong\u003e(D)\u003c/strong\u003e Representative FACS plots showing eGFP fluorescence detected in \u003cem\u003eDYSF\u003c/em\u003e\u003csup\u003eS1173X\u003c/sup\u003e myoblasts, treated with 0.1% DMSO or each 6 effective compounds for 24 hours, using a CYTO‑ID® Autophagy detection kit. \u003cstrong\u003e(E)\u003c/strong\u003e Quantification of autophagic vesicles with the CYTO‑ID® Autophagy detection kit. MFI from FACS experiments showing eGFP fluorescence, following treatment relative to the control DMSO 0.1%. Each bar represents the mean ± SD of 5 measures of independent analysis. *\u003cem\u003ep\u003c/em\u003e≤0.05, **\u003cem\u003ep\u003c/em\u003e≤ 0.01, ***\u003cem\u003ep\u003c/em\u003e≤0.001 versus DMSO group (one-way ANOVA with Dunn's multiple comparisons test).\u0026nbsp;\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6671973/v1/bca724e236240732e507caeb.png"},{"id":86667231,"identity":"5ef68eea-c10f-4d8e-941c-94e686f859f3","added_by":"auto","created_at":"2025-07-14 11:14:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":376752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchema of the known mechanism of action of the six effective compounds and the resulting hypotheses on the phenotype of dysferlinopathy\u003c/strong\u003e (created with Biorender).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6671973/v1/4ea9425fee1b5e29ed72a216.png"},{"id":89323944,"identity":"50d5b0f8-87d7-43e0-8e4a-0daeeb5b8992","added_by":"auto","created_at":"2025-08-18 19:46:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3588039,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6671973/v1/bd8e39e7-11ca-4d10-85b6-652f722d7e84.pdf"},{"id":86667222,"identity":"a2b0f993-6375-4026-8859-3ae4013601f5","added_by":"auto","created_at":"2025-07-14 11:14:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":823145,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6671973/v1/ba1eba8750b6d82229a31998.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting membrane fragility in LGMD R2 through pharmacological autophagy induction","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAutophagy is a highly conserved process that involves the sequestration of cytoplasmic components into autophagosomes, which subsequently fuse with lysosomes for degradation and recycling. This process is essential for cellular maintenance, particularly under conditions of stress\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Dysregulation of autophagy has been implicated in several diseases, including neurodegenerative disorders, cancer and muscle diseases\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In recent years, there has been an explosion in the use of autophagy inducers in clinical medicine\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Several studies have particularly highlighted the potential of autophagy activation as a therapeutic strategy in muscular dystrophies, suggesting that enhancing autophagic flux may alleviate muscle damage and improve function in diseased states\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Among the different drugs inducing autophagy, rapamycin is one of the most extensively studied. As previously reported in Duchenne muscular dystrophy (DMD), systemic administration of rapamycin improves muscle function and reduces fibrosis in the mdx mouse by inhibiting the mTOR pathway\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In the same way, other studies described that activating the AMPK pathway in mdx mice using AICAR or metformin treatments, respectively improves on one hand diaphragm histopathology and maximal force-generating capacity\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and on the other hand muscle strength and fiber membrane integrity\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The benefit of autophagy induction has also been evaluated in multiple other muscular dystrophies as reported in COLVI bethlem muscular dystrophy\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, EDMD\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e or centronuclear myopathies\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, revealing a reduction in the dystrophic phenotype, restoration of muscle strength, improvement in mitochondrial function and survival in mouse models\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn the past decade, several hypotheses have emerged to explain the mechanisms by which autophagy delays progression and reduces the severity of muscular diseases. Although the exact processes remain unclear, one leading hypothesis involves detoxification, where autophagy clears damaged proteins and dysfunctional organelles, thereby reducing cellular stress and preventing further muscle cell degeneration\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Another hypothesis emphasizes autophagy's role in facilitating the recruitment of intracellular vesicles for the repair of damaged muscle fiber membranes. Muscle fibers, due to their continuous cycles of contraction and relaxation, are especially prone to membrane damage. Autophagy contributes to this repair process by directing autophagosomes or lysosomes to the site of injury via the ESCRT transport machinery, thereby preserving the membrane integrity and functionality of muscle cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eLGMDR2 or dysferlinopathy presents a unique set of characteristics that make it an ideal model for investigating the effect of autophagy inducers in muscular dystrophies. This disease affects approximately 1.63 people per 100,000 and is caused by a mutation in the DYSF gene, which encodes the dysferlin protein. Dysferlin shares structural similarity with the SYT protein family, which includes vesicle-anchored proteins involved in calcium sensing, synaptic vesicle fusion, and lysosomal exocytosis\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Dysferlin plays a critical role in membrane repair, and its absence or dysfunction leads to the progressive degeneration of muscle fibers and subsequent loss of muscle function. While the mechanisms by which dysferlin regulates intracellular membrane dynamics are complex and multifaceted, numerous studies have highlighted its primary role in the docking and fusion of vesicles carrying membrane components to sites of injury\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This vesicle fusion creates a \"membrane patch\" that effectively seals the damaged plasma membrane, restoring its integrity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Here we report a comprehensive multiparametric pharmacological screening of seventeen autophagy-activating molecules in immortalized myoblasts derived from LGMDR2 patients. This library was assessed for its ability to stimulate autophagy by measuring p62, a classical receptor of autophagy. We also monitored their capacity to improve membrane resistance following osmotic shock. By focusing on these three readouts, our study narrowed down the six most potent inducers of autophagy as promising therapeutic candidates for the treatment of dysferlinopathy.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eTo investigate the therapeutic potential of autophagy inducers for dysferlinopathy, we screened a library of seventeen pharmacological compounds previously reported to enhance autophagy\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To do so, we utilized dysferlin-deficient immortalized myoblasts (DYSF\u003csup\u003eS1173X\u003c/sup\u003e), a well-established cellular model that recapitulates key hallmarks of the disease\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Drug efficacy was evaluated in DYSF\u003csup\u003eS1173X\u003c/sup\u003e cells following 24 hours of treatment. Parallel analyses were conducted to measure the autophagy marker SQSTM1/p62 protein (hereafter referred to as p62), lysosomal activity using the Lysosensor probe, and membrane resistance following hypo-osmotic shock. Bazedoxifene was used as positive control\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and the control condition treated with 0.1% DMSO was used as negative control. Secondary assays were performed to comprehensively characterize the most promising candidates identified during the primary screen. The workflow is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePharmacological induction of autophagy in dysferlin-deficient immortalised myoblasts.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo minimize bias related to drug dosage and toxicity, the concentrations of the tested drugs were determined based on literature and cytotoxicity assessments. Cell viability was evaluated by comparing treated cells to the control condition (0.1% DMSO) using Incucyte\u0026reg; Nuclight Rapid Red Dye for live-cell nuclear labelling (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B). For certain drugs, toxicity was observed at the reported doses, leading to a reduction in their concentrations. The following experiments were conducted at the adjusted doses listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB. To assess the ability of these drugs at the selected doses to induce autophagy in LGMDR2 myoblasts, we used LysoSensor\u0026trade; dyes, acidotropic probes that accumulate in acidic organelles such as lysosomes following protonation, along with p62 immunolabeling. Bazedoxifene 2\u0026micro;M was used as a positive control, as we have previously reported that it induces an increase in the LysoSensor signal\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This analysis showed that all tested molecules were able to enhance the quantity of acidic organelles in LGMDR2 cells after 24 hours of treatment, as demonstrated by the representative images (Figure S2A). Quantifications (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) revealed that the drugs that induced the greatest increase in the LysoSensor signal were tomatidine, bazedoxifene, MF-094 and ambroxol. Because this result could reflect a decrease in lysosomal pH or an increase in lysosome volume, we further assessed autophagy activity by measuring the abundance of the autophagic receptor p62 \u003csup\u003e28\u003c/sup\u003e. Our results revealed that all molecules showed a varying degree of increase in p62 fluorescence intensity, as shown by the representative images (Figure S2B) and quantitative graphs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Spermidine, actinonin, trehalose, and tomatidine induced the greatest increase in p62 signal intensity after 24 hours of treatment. Collectively, these findings suggest that the tested molecules induce autophagy at different levels in our model. Interestingly, the molecules showing more intense LysoSensor labelling were not necessarily the same ones showing a strong increase in p62, indicating that these drugs act at different stages of the autophagy process. As a control, we then determined whether the effect of these drugs was specific to mutated cells by evaluating their impact on autophagy induction in healthy muscle cells. The results showed that the trends were similar between the healthy and mutated cell lines, except for a few molecules that showed no increase in LysoSensor signal in healthy cells, such as nicotinamide mononucleotide, myo-inositol, carbamazepine, and metformin (Figure S3 and S4).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of membrane resistance following autophagy induction in LGMDR2\u003c/h2\u003e\u003cp\u003eTo evaluate the therapeutic potential of autophagy inducers in dysferlinopathy, we then tested their ability to enhance membrane resistance following a hypo-osmotic shock assay. Briefly, for the osmotic shock assay, myoblasts were treated with the drugs for 24h and incubated with the Incucyte\u0026reg; Nuclight Rapid Red Dye to assess the toxicity of the compounds relative to the DMSO negative control and to establish a baseline of 0% for osmotic shock-induced mortality. Then, a hypo-osmotic shock was induced with a solution of 25% PBS and 75% water, along with the incubation of the Incucyte\u0026reg; Caspase-3/7 Green Dye. As previously reported, under these conditions, we can indirectly assess membrane fragility and membrane resistance in dysferlin-deficient cells in real-time by comparing the ratio of green to red fluorescence relative to the negative control (0.1% DMSO)\u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMembrane resistance to osmotic shock was tested following treatment with the same library and bazedoxifene 2\u0026micro;M as a positive control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Among these molecules, six compounds showed a beneficial effect on cell membranes: rapamycin, torkinib, metformin, actinonin, resveratrol, and spermidine, with respective mortality rates of 3%, 8%, 9%, 13%, 17%, and 30% in DYSF\u003csup\u003eS1173X\u003c/sup\u003e myoblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Unexpectedly, other molecules had the opposite effect, such as trehalose, tomatidine, USP30 inhibitor, sorafenib, and nicotinamide riboside, which increased the percentage of cell death, reflecting a worsening of the phenotype. These results suggest that while certain autophagy-inducing drugs can enhance membrane resistance in dysferlinopathy, others may exacerbate cell death, highlighting the need for careful selection of therapeutic candidates and further investigation into their distinct pathways.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssessment of autophagy induction by the six drugs that enhance membrane resistance in LGMDR2 muscle cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe then investigated several parameters reflecting autophagy induction following treatment with the six drug candidates that improve membrane resistance. To do so, we first confirmed drugs\u0026rsquo; effects using Lysosensor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and p62 immunostaining through confocal imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). To further characterize the effect of these drugs on autophagy, we then used a tandem fluorescence RFP-GFP-LC3B reporter system. This tandem RFP-GFP sensor capitalizes on the pH difference between the acidic autolysosome and the neutral autophagosome to stain autophagosome in yellow and the autolysosome in red. Accordingly, DYSF\u003csup\u003eS1173X\u003c/sup\u003e myoblasts were transiently transduced with the RFP-GFP-LC3B tandem sensor and treated with each of the six drugs or DMSO 0.1% for 24h. Our results revealed that all these molecules increased the accumulation of both autophagosomes and the autolysosomes except torkinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This result was then confirmed by measuring LC3 and p62 levels by Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The Western blot analysis allows us to distinguish the effects of the drugs on the two forms of LC3, LC3B-I and LC3B-II. Indeed, the conversion of LC3B from its cytosolic form (LC3B-I) to the membrane-bound form (LC3B-II) is a key marker of autophagy modulation and the formation of autophagosomes. Our results showed that the treatments with the six drugs were capable of increasing the proportion of LC3B-II and p62, reflecting an accumulation of autophagosomes in immortalized myoblasts.\u003c/p\u003e\u003cp\u003eFinally, we extended our analysis by measuring CytoID autophagy activity via flow cytometry. This assay allows for the quantification of autophagic vacuoles in live cells. For this assay, chloroquine, a known autophagy inhibitor that exacerbates the accumulation of vesicles, was used as a positive control. The results of this analysis revealed that the autophagic signal, calculated as median fluorescence intensity (MFI), was increased with the different treatments, with torkinib showing the most pronounced effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Altogether, our data reveal that a subset of autophagy inducers improves membrane resistance in LGMDR2 muscle cells. Based on their known targets and reported mechanisms of action, three hypotheses emerge from these results to explain these effects on muscle cells: 1) that the increased autophagic vesicles serve as a pool of membrane material recruited by the membranes repair machinery, restoring cellular integrity; 2) that the increased number of autolysosomes improves muscle regeneration and contraction by degrading and recycling cellular debris; and 3) that the removal of damaged organelles, like mitochondria, through mitophagy reduces cellular stress and supports mitochondrial remodelling necessary for muscle regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study identifies, on the one hand, tomatidine and MF-094 as the most effective autophagy inducers in LGMDR2 muscle cells based on lysosome quantification, and on the other hand, actinonin and spermidine based on p62 quantification. Notably, our findings also demonstrate that autophagy activation can enhance membrane resistance, a key phenotype in dysferlinopathy. Among the tested compounds, torkinib, resveratrol, spermidine, actinonin, metformin, and rapamycin showed significant potential in improving this phenotype. These results provide a promising therapeutic perspective for the treatment of LGMDR2.\u003c/p\u003e\u003cp\u003eFrom this screening, we identified six compounds (torkinib, resveratrol, spermidine, actinonin, metformin, and rapamycin) that effectively induce autophagy and enhance membrane resistance in muscle cells affected by dysferlinopathy. These drugs can be categorized into two groups based on whether they modulate autophagy via mTOR-dependent mechanisms or not. A common feature of rapamycin, torkinib, and metformin is their ability to directly or indirectly target the mTOR (mammalian target of rapamycin) signalling pathway. As previously described, rapamycin specifically inhibits mTORC1 by binding to the FKBP12 protein, preventing its activation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Torkinib, a direct inhibitor of mTOR kinase activity, targets both mTORC1 and mTORC2 complexes. While metformin primarily activates AMPK (AMP-activated protein kinase), this drug can also indirectly inhibit the mTOR pathway. The mTOR pathway is a crucial regulator of autophagy, which is vital for maintaining cellular homeostasis and degrading damaged proteins and organelles. By inhibiting mTOR, these compounds promote autophagy, aiding in the removal of defective cellular components and potentially restoring muscle function. In contrast, resveratrol and spermidine offer promising alternatives to direct mTOR inhibition for inducing autophagy in muscle cells\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Both compounds demonstrated efficacy in improving the membrane repair phenotype. Resveratrol, a polyphenol with antioxidant properties, activates autophagy through modulation of the AMPK/SIRT1 signalling pathway\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Spermidine, an endogenous polyamine, activates autophagy through inhibition of acetyltransferase, in particular E1A-binding protein p300 (EP300), an endogenous repressor of autophagy\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Interestingly, both resveratrol and spermidine also offer additional benefits, such as anti-inflammatory and cytoprotective effects, making them attractive candidates for therapies to enhance muscle regeneration and function\u003csup\u003e\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e without the risks associated with mTOR long-term treatment\u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Lastly, actinonin activates mitophagy\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, a selective form of autophagy responsible for the removal of damaged mitochondria, essential for maintaining cellular health. Regardless of the mechanisms underlying the effects of these drugs, our study demonstrates that several potent autophagy inducers enhance the lysosomal pool and promote the membrane repair process in LGMDR2 muscle cells. These findings align with multiple other studies showing that lysosomes are the primary vesicular population responsible for repairing the plasma membrane and highlight the critical role of dysferlin in mediating their exocytosis toward membrane lesions.\u003c/p\u003e\u003cp\u003eAutophagy induction has been explored as a therapeutic avenue in a wide range of diseases, including cancers, infections, neurodegenerative disorders, metabolic diseases, inflammatory conditions, and muscular diseases. Over the past decades, several FDA-approved drugs have been shown to enhance autophagy and yield beneficial effects in various clinical contexts. For instance, mTOR inhibitors have demonstrated therapeutic potential in degenerative diseases such as Huntington\u0026rsquo;s disease\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, while the tyrosine kinase inhibitor erlotinib has shown efficacy in diabetic nephropathy\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and nilotinib has been studied in Alzheimer's disease\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Other drugs, such as carbamazepine for alpha1-antitrypsin deficiency\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, trifluoperazine for salmonella infection\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, and statins for Mycobacterium tuberculosis infection\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, also modulate autophagy and have demonstrated therapeutic benefits. However, it remains unclear whether the positive regulation of autophagy is the primary mechanism underlying the therapeutic effects of these agents, or if they exert their benefits through other pleiotropic actions. Notably, resveratrol has been extensively studied in clinical trials for its potential to prevent and manage a broad spectrum of diseases such as diabetes mellitus, obesity, colorectal cancer, breast cancer, multiple myeloma, metabolic syndrome, hypertension, Alzheimer's disease, stroke, cardiovascular disease, kidney disease, inflammatory disorders, and nasopharyngitis\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The beneficial effects of autophagy in muscle cells extend beyond enhancing membrane repair following muscle contraction; they also involve its potent detoxifying action. In the muscles, pathological conditions often lead to progressive mitochondrial dysfunction, which causes chronic bioenergetic inefficiency and the accumulation of cytotoxic aggregates or redox-active proteins (ROS). While ROS can exacerbate cell damage through oxidative stress, autophagy plays a protective role by eliminating damaged mitochondria and proteins, thereby reducing cellular injury and promoting cell survival\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Furthermore, autophagy counteracts cellular senescence and regulated cell death by facilitating the repair of damaged DNA\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Autophagic responses are crucial for maintaining intracellular homeostasis, particularly in stem cell compartments. Activated autophagy supports cellular energy requirements during the activation and proliferation of myogenic stem cells following muscle injury\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Additionally, autophagy declines with age, and the pharmacological compounds used in this study are known to extend lifespan and protect against degenerative diseases. In general, these compounds promote healthy aging by sustaining cellular function and mitigating age-related decline.\u003c/p\u003e\u003cp\u003eIn summary, this study advances our understanding of how autophagy activation can improve membrane repair process in dysferlinopathy and identifies pharmacological compounds with potential as therapeutic interventions. These compounds are not only well-characterized but also generally well-tolerated in humans, positioning them as strong candidates for therapies aimed at modulating autophagy in dysferlinopathies. Our results also underscore the need for in vivo studies to evaluate the long-term effects of these compounds and to further elucidate the molecular mechanisms underlying their autophagic activities and benefits.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eCell culture and treatments.\u003c/b\u003e In this study, we use immortalized myoblasts derived from either LGMDR2 patients or healthy controls. These myoblasts were isolated from human muscle biopsy and obtained from the human cell immortalization platform Myoline at the Institut de Myologie (Paris, France), with the consent of the subjects by signing an informed consent form and anonymization before immortalization, according to the EU GDPR regulation. The healthy line is AB1079, and the mutated line is AB320, which carries the heterozygous nonsense mutation \u003cem\u003eDYSF\u003c/em\u003e c.342-1G\u0026thinsp;\u0026gt;\u0026thinsp;A/c.3516_3517delTT; p.Ser1173X (DYSF\u003csup\u003eS1173X\u003c/sup\u003e). Myoblasts were cultured in growth medium consisting of 1 volume of 199 medium (Invitrogen, United States) for 4 volumes of Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (Invitrogen, United States), supplemented with 20% fetal bovine serum (Sigma-Aldrich, United States), 25 \u0026micro;g/ml fetuin (Life Technologies, United States), 5 ng/ml epidermal growth factor (Life Technologies, United States), 0.5 ng/ml basic fibroblast growth factor (Life Technologies, United States), 0.2 \u0026micro;g/ml dexamethasone (Sigma-Aldrich, United States) and 5 \u0026micro;g/ml insulin (Sigma-Aldrich, United States). Cells were seeded on plates coated with 0.1% of gelatin and maintained in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReagents and antibodies.\u003c/b\u003e The antibodies used in immunofluorescence were as follows: anti-p62/SQSTM1 (Abcam, Cambridge, UK, #ab56416) primary antibodies, Alexa Fluor 555-conjugated goat anti-mouse IgG (Invitrogen, Thermo Fisher Scientific, Carlsbad, USA, #A-31570) secondary antibody. Antibodies used in western blot: anti-LC3B (Novus Biological, USA, NB600-1384), anti-p62/SQSTM1 (Abcam, Cambridge, UK, #ab56416), anti-β-actin (LI-COR Biosciences, Lincoln, USA, #926-42210), IRDye 800CW donkey anti-rabbit (LI-COR Biosciences, Lincoln, USA, #926-32213), IRDye 800CW donkey anti-rabbit (LI-COR Biosciences, Lincoln, USA, #926-32213), IRDye 680RD donkey anti-mouse (LI-COR Biosciences, Lincoln, USA, #926-68072).\u003c/p\u003e\u003cp\u003eThe reagents used in the study were: Spermidine (MedChemExpress, Monmouth Junction, USA, #HY-B1776 ), Nicotinamide riboside (TargetMol, Massachusetts, USA, #T13795), Nicotinamide mononucleotide (TargetMol, Massachusetts, USA, #T4721), Actinonin (MedChemExpress, Monmouth Junction, USA, #HY-113952 ), Tomatidine (MedChemExpress, Monmouth Junction, USA, #HY-N2149), D-(+)-Trehalose (MedChemExpress, Monmouth Junction, USA, #HY-N1132), I-Inositol (MedChemExpress, Monmouth Junction, USA, #HY-B1411), Resveratrol (MedChemExpress, Monmouth Junction, USA, #HY-16561), Rapamycine (Selleck Chemicals LLC, Houston, USA, #S1039), Carbamazepine (Selleck Chemicals LLC, Houston, USA, #S1693), Metformin (MedChemExpress, Monmouth Junction, USA, #HY-B0627), Ambroxol (MedChemExpress, Monmouth Junction, USA, #HY-B1039), MF-094 (TargetMol, Massachusetts, USA, #282T12024), USP30inhibitor 18 (TargetMol, Massachusetts, USA, #282T36682), Sorafenib (MedChemExpress, Monmouth Junction, USA, #HY-10201), Torkinib (MedChemExpress, Monmouth Junction, USA, #HY-10474), Bazedoxifene HCL (Selleck Chemicals LLC, Houston, USA, #S2128).\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsmotic shock assay.\u003c/b\u003e The phenotypic membrane resistance assay was used as previously described\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e (Bruge et al., 2025). Cells were seeded using a Bravo Automated Liquid Handling platform (Agilent, United States). DYSF\u003csup\u003eS1173X\u003c/sup\u003e myoblasts were seeded at 8300 cells/cm\u003csup\u003e2\u003c/sup\u003e in 38 \u0026micro;l of culture medium in black clear-bottom 384-well plates. After 72 hours, the cells were treated with 2 \u0026micro;l of 20x concentrated solutions of each drug at the concentration shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e, along with a negative control (0.1% dimethyl sulfoxide, DMSO, VWR), or a positive control (2\u0026micro;M Bazedoxifene). Following 24 hours of treatment, cells were incubated for 3 hours with a red viability probe (1/3000, Incucyte\u0026reg; Nuclight Rapid Red Dye - Sartorius, Germany), which labels the nuclei of live cells. This step was performed to assess the toxicity of the compounds relative to the DMSO control. Then, a hypo-osmotic shock was induced by exposing the cells to a solution containing 25% PBS and 75% water, along with a green mortality probe (1/3000, Incucyte\u0026reg; Caspase-3/7 Green Dye - Sartorius, Germany). Green fluorescence was monitored every 4 hours using an Incucyte\u0026reg; S3 Live-Cell Analysis system (Sartorius, Germany). The resulting images were analysed with Incucyte software, to assess the cell resistance to osmotic shock by comparing the green-to-red fluorescence ratio relative to the negative control (DMSO).\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunostaining assay.\u003c/b\u003e Immunofluorescence staining was carried out to evaluate the ability of the molecules to induce autophagy in our cell models. After 24 hours of drug treatment, myoblasts were fixed in 4% paraformaldehyde for 10 minutes at room temperature. Permeabilization was done using 0.5% Triton X-100 (Thermo Scientific, United States) for 10 minutes at room temperature, followed by blocking with 1% bovine serum albumin (BSA; Sigma-Aldrich, United States) for 1 hour at room temperature. The cells were then incubated overnight at 4\u0026deg;C with primary antibody: mouse anti-P62 antibody (1:250 dilution), diluted in 1% BSA. After three washes in PBS, cells were incubated with secondary antibody: Alexa fluor 555 goat anti-mouse (1:1,000 dilution), with Hoechst solution (Invitrogen, United States) for nuclear staining, in the dark for 1 hour at room temperature. Following additional washing steps, cells were visualized under a LSM 800 confocal microscope (Zeiss, Germany) with a 40x oil immersion high-resolution objective and imaged using Zen software (Zeiss, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eP62 puncta quantification.\u003c/b\u003e Myoblasts were seeded in 96-well plates and treated with the different drugs or negative control DMSO for 24 hours. After fixation with 4% PFA, immunostaining was carried out, and image of p62 expression was captured using an LSM-800 confocal microscope. For quantification, p62 expression was analysed using a 20x high-resolution objective of the CellInsight CX7 HCS Platform (Cellomics Inc.). The p62 puncta area was quantified for each well (n\u0026thinsp;=\u0026thinsp;3 fields) by applying thresholding. Total cell number was determined by counting the Hoechst-stained cells per well, allowing normalization of the total p62 puncta area to the number of nuclei. To confirm the results with confocal image replicates, a second quantification was carried out in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The relative fluorescence expression area was analysed using the ImageJ2 software (Fuji).\u003c/p\u003e\u003cp\u003e\u003cb\u003eLysosomal pH measurement.\u003c/b\u003e LysoSensor Green DND-189 dye (L7535, ThermoFisher) was employed to measure the lysosomal pH in cells. Myoblasts were grown in 96-well plates and treated with the different drugs or negative control DMSO for 24 hours. Preheated 1 \u0026micro;M LysoSensor Green DND-189 dye (37\u0026deg;C) was added in culture medium for 2 hours at 37\u0026deg;C. We fixed cells with 4% formaldehyde and counterstained with Hoechst 33342 solution (Invitrogen, United States), and images were acquired using a LSM-800 confocal microscope (Zeiss, Germany). For quantification, the plate was analysed by a 20x high-resolution objective of the ImageXpress Micro XL System imager (Molecular Devices). Lysosensor fluorescence area was quantified for each well (n\u0026thinsp;=\u0026thinsp;4 fields) by applying thresholding. Total cell number was determined by counting Hoechst-stained cells per well, allowing normalization of the total lysosensor fluorescence to the number of nuclei. To confirm the results with confocal image replicates, a second quantification was carried out in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The relative fluorescence expression area was analysed using the ImageJ2 software (Fuji).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot analysis.\u003c/b\u003e Immortalized myoblasts were collected after 24 hours of drug treatment. Cells were lysed in a solution containing NP40 lysis buffer (Thermo Scientific, United States), 1X Proteases and phosphatase Inhibitors (Complete PIC, Roche, Switzerland), to extract total protein. The lysate was centrifuged for 15 minutes at 4\u0026deg;C, and the supernatant was collected as total cellular protein. Their concentrations were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, United States). Samples were heated at 95\u0026deg;C for 5 min, and 20 \u0026micro;g of protein was loaded onto 4\u0026ndash;15% Criterion\u0026trade; XT tris-glycine protein gel (BioRad, United States) for electrophoresis and transferred to nitrocellulose membrane (BioRad, United States) using a Trans-Blot Turbo Transfert system (BioRad, United States). Blocked in Odyssey blocking buffer (Li-Cor, United States) for 1h at room temperature, the membrane was then incubated with primary antibodies in blocking buffer supplemented with 0.1% Tween 20 (VWR, United States) overnight at 4\u0026deg;C for the rabbit anti-LC3B 1:1,000 (NB600-1384, Novus), the mouse anti-p62/SQSTM1 1:1,000 (Abcam, ab56416), and the mouse anti\u0026mdash;β-actin 1:1,000 (Li-Cor, 926-42210). After three washes for 10 min at room temperature with Tris-buffered saline with 0.1% Tween 20 (VWR, United States), the membrane was incubated with fluorescent secondary antibodies donkey anti-mouse antibody IRDye-680 1:10,000 (926-32222, Li-Cor) or a donkey anti-rabbit antibody IRDye-800 1:5,000 (926-32213, Li-Cor) for 1 hour at room temperature. The membrane was then washed again, and protein bands were visualized by fluorescence using the Odyssey CLx system (Li-Cor, United States). Protein expression levels were quantified by measuring the intensity of bands using Image Studio\u0026trade; software. The fold change was determined after normalizing to the loading control.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFlow cytometry measurement of autophagy flux.\u003c/b\u003e We use CYTO-ID\u0026reg; Autophagy Detection Kit (Enzo Life Sciences) to measure autophagy flux. This kit detects all autophagic vesicles in cells, including pro-autophagosomes, autophagosomes, and autophagic lysosomes. Immortalized myoblasts were cultured on 0.1% gelatin-coated 6-well plates until they reached 80% confluence and then treated with pharmacological compounds for 24 hours. After treatment, cells were collected by trypsin-EDTA (Gibco, United States) dissociation, followed by centrifugation at 1200 rpm for 5 minutes. Cells were then washed with 300 \u0026micro;l of 1X Assay Buffer, centrifuged, and the supernatant discarded by inverting the plate onto laboratory paper. A solution with CYTO-ID\u0026reg; Green Detection Reagent was added to the cells for 30 min at 37\u0026deg;C in the dark. After two successive washings, the cells were resuspended in 100 \u0026micro;l of 1X Assay Buffer and analysed using a MACSquant analyser (Miltenyi Biotec, Germany) with a 488 nm laser source and the green fluorescence channel. A minimum of 30,000 cells was measured per sample. Data analysis was performed using FlowJo Software (BD Biosciences, United States). Intact cells were gated based on FSC-A vs SSC-A, and the median fluorescence intensity (MFI) of eGFP for each sample was calculated. The baseline autophagic flux, represented by the eGFP-MFI of the DMSO control, was subtracted to determine the autophagic flux shift (eGFP-MFI shift). Quantification was performed with five measures of independent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRFP-GFP-LC3B Tandem Sensor Assay.\u003c/b\u003e Premo\u0026trade; Autophagy Tandem Sensor RFP-GFP-LC3B kit (Thermo Scientific, United States) was used to monitor autophagic flux. Reagent was added to immortalized myoblasts at a concentration of 30 particles per cell according to the manufacturer\u0026rsquo;s instructions. After 24 hours, culture medium was replaced, and cells were treated with the different drugs or 0.1% DMSO for an additional 24 hours. The cells were then fixed with 4% formaldehyde and counterstained with Hoechst 33342 solution 1:1,000 (Invitrogen, United States) for 10 minutes at room temperature. Finally, coverslips were mounted for fluorescence microscopy. Images were acquired using an LSM-800 confocal microscope. Quantification was performed with twenty images of three independent experiments. The degree of fluorescence co-localization and relative fluorescence expression area were analysed using the ImageJ2 software (Fuji).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis.\u003c/b\u003e Statistical analyses were performed using one-way analysis of variance (ANOVA) to perform the comparisons between multiple groups. Results from each experiment were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical significance was defined as *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001. All graphs were plotted and analyzed using GraphPad Prism Software (v9.2.0).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical statement\u003c/h2\u003e\u003cp\u003eAll methods were performed in accordance with the relevant guidelines and French regulations. The experimental protocols and ethical authorization were approved by the comit\u0026eacute; de protection des personnes under the reference: Mecamedirare 2019-A02599-48.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eIstem/CECS is supported by the Association Fran\u0026ccedil;aise contre les Myopathies (AFM-T\u0026eacute;l\u0026eacute;thon). This project was also supported by grants from Laboratoire d\u0026rsquo;Excellence Revive (Investissement d\u0026rsquo;Avenir; ANR-10-LABX-73), the IDEX Paris-Saclay (for Initiative D\u0026rsquo;EXcellence), the Region Ile-de-France via the doctoral school \u0026laquo; Structure et dynamique des syst\u0026egrave;mes vivants \u0026raquo; (ED 577) from Paris Saclay University. The stemCARE platform is supported by AFM-T\u0026eacute;l\u0026eacute;thon, GIS IBISA, R\u0026eacute;gion Ile de France, BPI, INSERM and UEVE for staff and equipments. The stemCARE platform is part of GENOPOLE and GENOTHER bioclusters. This study was part of the DREAMS project. Funded by the European Union under 101080229-2. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union (EU) or European Research Executive Agency (REA). Neither the EU nor REA can be held responsible for them.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.N. was responsible for the experimental design and project management. N.G. performed the cell culture experiments, drug treatments, carried out screening analysis and realized functional characterization of the drugs. C.B. and E.P. developed the osmotic shock method. C.L. and J.P. developed imaging analysis of p62 puncta and lysosensor puncta. Q.M. contributed to the statistical analyses and provided his expertise in autophagy measurement tools. M.B. provided technical assistance for flow cytometry experiments. E.P., M.B., C.B. provided technical assistance for cell culture and pharmacological studies. N.G. prepared the figures. N.G. and X.N. wrote the manuscript. All authors reviewed and edited the paper.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Marc Peschanski (IStem), Isabelle Richard (Genethon) for helpful discussions and Marc Bartoli (MMG, Marseille) for his expertise in osmotic shock. We are grateful to the Platform for Immortalization of Human Cells \u0026ldquo;Myoline\u0026rdquo; from the Centre of Research in Myology (Institute of Myology, Paris) for providing immortalized myoblasts used in this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRyter, S. W., Bhatia, D. \u0026amp; Choi, M. E. Autophagy: A Lysosome-Dependent Process with Implications in Cellular Redox Homeostasis and Human Disease. \u003cem\u003eAntioxid Redox Signal\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 138\u0026ndash;159 (2019).\u003c/li\u003e\n\u003cli\u003eSchneider, J. L. \u0026amp; Cuervo, A. M. Autophagy and human disease: emerging themes. \u003cem\u003eCurr Opin Genet Dev\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 16\u0026ndash;23 (2014).\u003c/li\u003e\n\u003cli\u003eLevine, B., Packer, M. \u0026amp; Codogno, P. Development of autophagy inducers in clinical medicine. \u003cem\u003eThe Journal of Clinical Investigation\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 14 (2015).\u003c/li\u003e\n\u003cli\u003eLi, H., Zhang, L., Zhang, L. \u0026amp; Han, R. Autophagy in striated muscle diseases. \u003cem\u003eFront Cardiovasc Med\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1000067 (2022).\u003c/li\u003e\n\u003cli\u003eEghtesad, S., Jhunjhunwala, S., Little, S. R. \u0026amp; Clemens, P. R. Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle. \u003cem\u003eMol Med\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 917\u0026ndash;924 (2011).\u003c/li\u003e\n\u003cli\u003ePauly, M. \u003cem\u003eet al.\u003c/em\u003e AMPK activation stimulates autophagy and ameliorates muscular dystrophy in the mdx mouse diaphragm. \u003cem\u003eAm J Pathol\u003c/em\u003e \u003cstrong\u003e181\u003c/strong\u003e, 583\u0026ndash;592 (2012).\u003c/li\u003e\n\u003cli\u003eDong, X. \u003cem\u003eet al.\u003c/em\u003e Metformin Increases Sarcolemma Integrity and Ameliorates Neuromuscular Deficits in a Murine Model of Duchenne Muscular Dystrophy. \u003cem\u003eFront Physiol\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 642908 (2021).\u003c/li\u003e\n\u003cli\u003eGrumati, P. \u003cem\u003eet al.\u003c/em\u003e Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1313\u0026ndash;1320 (2010).\u003c/li\u003e\n\u003cli\u003eChrisam, M. \u003cem\u003eet al.\u003c/em\u003e Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice. \u003cem\u003eAutophagy\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 2142\u0026ndash;2152 (2015).\u003c/li\u003e\n\u003cli\u003eCastagnaro, S. \u003cem\u003eet al.\u003c/em\u003e Autophagy activation in COL6 myopathic patients by a low-protein-diet pilot trial. \u003cem\u003eAutophagy\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 2484\u0026ndash;2495 (2016).\u003c/li\u003e\n\u003cli\u003eChoi, J. C. \u0026amp; Worman, H. J. Reactivation of autophagy ameliorates LMNA cardiomyopathy. \u003cem\u003eAutophagy\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 110\u0026ndash;111 (2013).\u003c/li\u003e\n\u003cli\u003eRamos, F. J. \u003cem\u003eet al.\u003c/em\u003e Rapamycin reverses elevated mTORC1 signaling in lamin A/C-deficient mice, rescues cardiac and skeletal muscle function, and extends survival. \u003cem\u003eSci Transl Med\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 144ra103 (2012).\u003c/li\u003e\n\u003cli\u003eTasfaout, H., Cowling, B. S. \u0026amp; Laporte, J. Centronuclear myopathies under attack: A plethora of therapeutic targets. \u003cem\u003eJ Neuromuscul Dis\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 387\u0026ndash;406.\u003c/li\u003e\n\u003cli\u003eMassana-Mu\u0026ntilde;oz, X. \u003cem\u003eet al.\u003c/em\u003e Inactivating the lipid kinase activity of PI3KC2\u0026beta; is sufficient to rescue myotubular myopathy in mice. \u003cem\u003eJCI Insight\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e151933 (2023).\u003c/li\u003e\n\u003cli\u003eJi, J. \u003cem\u003eet al.\u003c/em\u003e BIN1 gene replacement reverses BIN1-related centronuclear myopathy. \u003cem\u003eMol Ther\u003c/em\u003e S1525-0016(25)00315\u0026ndash;6 (2025) doi:10.1016/j.ymthe.2025.04.036.\u003c/li\u003e\n\u003cli\u003eCastets, P., Frank, S., Sinnreich, M. \u0026amp; R\u0026uuml;egg, M. A. \u0026lsquo;Get the Balance Right\u0026rsquo;: Pathological Significance of Autophagy Perturbation in Neuromuscular Disorders. \u003cem\u003eJ Neuromuscul Dis\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 127\u0026ndash;155 (2016).\u003c/li\u003e\n\u003cli\u003eXia, Q. \u003cem\u003eet al.\u003c/em\u003e The Role of Autophagy in Skeletal Muscle Diseases. \u003cem\u003eFront Physiol\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 638983 (2021).\u003c/li\u003e\n\u003cli\u003eClaude-Taupin, A. \u003cem\u003eet al.\u003c/em\u003e ATG9A protects the plasma membrane from programmed and incidental permeabilization. \u003cem\u003eNat Cell Biol\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 846\u0026ndash;858 (2021).\u003c/li\u003e\n\u003cli\u003eLu, T.-W., Frost, A. \u0026amp; Moss, F. R. Organelle homeostasis requires ESCRTs. \u003cem\u003eCurrent Opinion in Cell Biology\u003c/em\u003e \u003cstrong\u003e93\u003c/strong\u003e, 102481 (2025).\u003c/li\u003e\n\u003cli\u003eGlover, L. \u0026amp; Brown, R. H. Dysferlin in membrane trafficking and patch repair. \u003cem\u003eTraffic\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 785\u0026ndash;794 (2007).\u003c/li\u003e\n\u003cli\u003eHan, R. \u0026amp; Campbell, K. P. Dysferlin and Muscle Membrane Repair. \u003cem\u003eCurr Opin Cell Biol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 409\u0026ndash;416 (2007).\u003c/li\u003e\n\u003cli\u003eBansal, D. \u003cem\u003eet al.\u003c/em\u003e Defective membrane repair in dysferlin-deficient muscular dystrophy. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e423\u003c/strong\u003e, 168\u0026ndash;172 (2003).\u003c/li\u003e\n\u003cli\u003eAman, Y. \u003cem\u003eet al.\u003c/em\u003e Autophagy in healthy aging and disease. \u003cem\u003eNat Aging\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 634\u0026ndash;650 (2021).\u003c/li\u003e\n\u003cli\u003eCea, L. A. \u003cem\u003eet al.\u003c/em\u003e The absence of dysferlin induces the expression of functional connexin-based hemichannels in human myotubes. \u003cem\u003eBMC Cell Biol\u003c/em\u003e \u003cstrong\u003e17 Suppl 1\u003c/strong\u003e, 15 (2016).\u003c/li\u003e\n\u003cli\u003eB\u0026aacute;ez-Matus, X. \u003cem\u003eet al.\u003c/em\u003e Defects in G-Actin Incorporation into Filaments in Myoblasts Derived from Dysferlinopathy Patients Are Restored by Dysferlin C2 Domains. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 37 (2019).\u003c/li\u003e\n\u003cli\u003eMamchaoui, K. \u003cem\u003eet al.\u003c/em\u003e Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders. \u003cem\u003eSkelet Muscle\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 34 (2011).\u003c/li\u003e\n\u003cli\u003eBruge, C. \u003cem\u003eet al.\u003c/em\u003e High-throughput screening identifies bazedoxifene as a potential therapeutic for dysferlin-deficient limb girdle muscular dystrophy. \u003cem\u003eBr J Pharmacol\u003c/em\u003e (2025) doi:10.1111/bph.70017.\u003c/li\u003e\n\u003cli\u003eKlionsky, D. J. \u003cem\u003eet al.\u003c/em\u003e Guidelines for the use and interpretation of assays for monitoring autophagy. \u003cem\u003eAutophagy\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 445\u0026ndash;544 (2012).\u003c/li\u003e\n\u003cli\u003eHarris, T. E. \u0026amp; Lawrence, J. C. TOR signaling. \u003cem\u003eSci STKE\u003c/em\u003e \u003cstrong\u003e2003\u003c/strong\u003e, re15 (2003).\u003c/li\u003e\n\u003cli\u003eMorselli, E. \u003cem\u003eet al.\u003c/em\u003e Autophagy mediates pharmacological lifespan extension by spermidine and resveratrol. \u003cem\u003eAging (Albany NY)\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 961\u0026ndash;970 (2009).\u003c/li\u003e\n\u003cli\u003eMorselli, E. \u003cem\u003eet al.\u003c/em\u003e Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e192\u003c/strong\u003e, 615\u0026ndash;629 (2011).\u003c/li\u003e\n\u003cli\u003eBastin, J. \u0026amp; Djouadi, F. Resveratrol and Myopathy. \u003cem\u003eNutrients\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 254 (2016).\u003c/li\u003e\n\u003cli\u003eGalasso, L. \u003cem\u003eet al.\u003c/em\u003e Polyamines and Physical Activity in Musculoskeletal Diseases: A Potential Therapeutic Challenge. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 9798 (2023).\u003c/li\u003e\n\u003cli\u003ePietrocola, F. \u003cem\u003eet al.\u003c/em\u003e Spermidine induces autophagy by inhibiting the acetyltransferase EP300. \u003cem\u003eCell Death Differ\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 509\u0026ndash;516 (2015).\u003c/li\u003e\n\u003cli\u003eGerner, E. W. \u0026amp; Meyskens, F. L. Polyamines and cancer: old molecules, new understanding. \u003cem\u003eNat Rev Cancer\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 781\u0026ndash;792 (2004).\u003c/li\u003e\n\u003cli\u003eDeng, K. \u003cem\u003eet al.\u003c/em\u003e Increased synthesis of spermidine as a result of upregulation of arginase I promotes axonal regeneration in culture and in vivo. \u003cem\u003eJ Neurosci\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 9545\u0026ndash;9552 (2009).\u003c/li\u003e\n\u003cli\u003eLaRocca, T. J., Gioscia-Ryan, R. A., Hearon, C. M. \u0026amp; Seals, D. R. The autophagy enhancer spermidine reverses arterial aging. \u003cem\u003eMech Ageing Dev\u003c/em\u003e \u003cstrong\u003e134\u003c/strong\u003e, 314\u0026ndash;320 (2013).\u003c/li\u003e\n\u003cli\u003eEisenberg, T. \u003cem\u003eet al.\u003c/em\u003e Cardioprotection and lifespan extension by the natural polyamine spermidine. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 1428\u0026ndash;1438 (2016).\u003c/li\u003e\n\u003cli\u003ePallet, N. \u0026amp; and Legendre, C. Adverse events associated with mTOR inhibitors. \u003cem\u003eExpert Opinion on Drug Safety\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 177\u0026ndash;186 (2013).\u003c/li\u003e\n\u003cli\u003eStallone, G., Infante, B., Grandaliano, G. \u0026amp; Gesualdo, L. Management of side effects of sirolimus therapy. \u003cem\u003eTransplantation\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, S23-26 (2009).\u003c/li\u003e\n\u003cli\u003eGyurus, E., Kaposztas, Z. \u0026amp; Kahan, B. D. Sirolimus therapy predisposes to new-onset diabetes mellitus after renal transplantation: a long-term analysis of various treatment regimens. \u003cem\u003eTransplant Proc\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 1583\u0026ndash;1592 (2011).\u003c/li\u003e\n\u003cli\u003eKshirsagar, S., Sawant, N., Morton, H., Reddy, A. P. \u0026amp; Reddy, P. H. Mitophagy enhancers against phosphorylated Tau-induced mitochondrial and synaptic toxicities in Alzheimer disease. \u003cem\u003ePharmacol Res\u003c/em\u003e \u003cstrong\u003e174\u003c/strong\u003e, 105973 (2021).\u003c/li\u003e\n\u003cli\u003eSarkar, S. Chemical screening platforms for autophagy drug discovery to identify therapeutic candidates for Huntington\u0026rsquo;s disease and other neurodegenerative disorders. \u003cem\u003eDrug Discovery Today: Technologies\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e137\u0026ndash;e144 (2013).\u003c/li\u003e\n\u003cli\u003eZhang, M.-Z., Wang, Y., Paueksakon, P. \u0026amp; Harris, R. C. Epidermal growth factor receptor inhibition slows progression of diabetic nephropathy in association with a decrease in endoplasmic reticulum stress and an increase in autophagy. \u003cem\u003eDiabetes\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 2063\u0026ndash;2072 (2014).\u003c/li\u003e\n\u003cli\u003eLonskaya, I., Hebron, M. L., Desforges, N. M., Schachter, J. B. \u0026amp; Moussa, C. E.-H. Nilotinib-induced autophagic changes increase endogenous parkin level and ubiquitination, leading to amyloid clearance. \u003cem\u003eJ Mol Med (Berl)\u003c/em\u003e \u003cstrong\u003e92\u003c/strong\u003e, 373\u0026ndash;386 (2014).\u003c/li\u003e\n\u003cli\u003eHidvegi, T. \u003cem\u003eet al.\u003c/em\u003e An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e329\u003c/strong\u003e, 229\u0026ndash;232 (2010).\u003c/li\u003e\n\u003cli\u003eConway, K. L. \u003cem\u003eet al.\u003c/em\u003e Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection. \u003cem\u003eGastroenterology\u003c/em\u003e \u003cstrong\u003e145\u003c/strong\u003e, 1347\u0026ndash;1357 (2013).\u003c/li\u003e\n\u003cli\u003eParihar, S. P. \u003cem\u003eet al.\u003c/em\u003e Statin therapy reduces the mycobacterium tuberculosis burden in human macrophages and in mice by enhancing autophagy and phagosome maturation. \u003cem\u003eJ Infect Dis\u003c/em\u003e \u003cstrong\u003e209\u003c/strong\u003e, 754\u0026ndash;763 (2014).\u003c/li\u003e\n\u003cli\u003eSingh, A. P. \u003cem\u003eet al.\u003c/em\u003e Health benefits of resveratrol: Evidence from clinical studies. \u003cem\u003eMed Res Rev\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 1851\u0026ndash;1891 (2019).\u003c/li\u003e\n\u003cli\u003eUreshino, R. P., Rocha, K. K., Lopes, G. S., Bincoletto, C. \u0026amp; Smaili, S. S. Calcium signaling alterations, oxidative stress, and autophagy in aging. \u003cem\u003eAntioxid Redox Signal\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 123\u0026ndash;137 (2014).\u003c/li\u003e\n\u003cli\u003eKarantza-Wadsworth, V. \u003cem\u003eet al.\u003c/em\u003e Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. \u003cem\u003eGenes Dev\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 1621\u0026ndash;1635 (2007).\u003c/li\u003e\n\u003cli\u003eSalemi, S., Yousefi, S., Constantinescu, M. A., Fey, M. F. \u0026amp; Simon, H.-U. Autophagy is required for self-renewal and differentiation of adult human stem cells. \u003cem\u003eCell Res\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 432\u0026ndash;435 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"limb-girdle muscular dystrophies, dysferlinopathy, drug screening, autophagy, membrane resistance","lastPublishedDoi":"10.21203/rs.3.rs-6671973/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6671973/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLimb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of genetic disorders characterized by progressive weakening of the limb-girdle muscles. Among LGMDs, Limb-girdle muscular dystrophy type R2 (LGMDR2) is a rare condition affecting fewer than 1 in 100,000 individuals caused by mutations in the gene encoding dysferlin. Recent \u003cem\u003ein vitro\u003c/em\u003e studies have suggested that autophagy flux is impaired in LGMDR2. Based on this evidence, we hypothesized that enhancing autophagy could provide therapeutic benefits for this condition. Autophagy plays a critical role in maintaining muscle integrity by clearing damaged cellular components, thus improving the defective dysferlin-mediated membrane repair mechanism. In this study, we performed a multiparametric screening of seventeen autophagy inducers in immortalized myoblasts derived from LGMDR2 patients to identify novel pharmacological compounds capable of enhancing membrane repair. Among the drugs tested, six were found to effectively stimulate autophagy and improve membrane resistance. Our findings demonstrate that inhibition of the mTOR pathway improves the cellular phenotype and underscore the potential of autophagy activators as a promising therapeutic target for LGMDR2.\u003c/p\u003e","manuscriptTitle":"Targeting membrane fragility in LGMD R2 through pharmacological autophagy induction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 11:14:44","doi":"10.21203/rs.3.rs-6671973/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af6177fe-d70a-4ae4-85e5-4098bbccfbaf","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51304966,"name":"Biological sciences/Cell biology"},{"id":51304967,"name":"Biological sciences/Drug discovery"},{"id":51304968,"name":"Biological sciences/Molecular biology"},{"id":51304969,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2025-08-18T19:38:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 11:14:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6671973","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6671973","identity":"rs-6671973","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}