Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease
2021; Springer Nature; Volume: 13; Issue: 11 Linguagem: Inglês
10.15252/emmm.202114434
ISSN1757-4684
AutoresAntonietta Tarallo, Carla Damiano, Sandra Strollo, Nadia Minopoli, Alessia Indrieri, Elena Polishchuk, Francesca Zappa, Edoardo Nusco, Simona Fecarotta, Caterina Porto, Marcella Coletta, Roberta Iacono, Marco Moracci, Roman Polishchuk, Diego L. Medina, Paola Imbimbo, Daria Maria Monti, Maria Antonietta De Matteis, Giancarlo Parenti,
Tópico(s)Biochemical and Molecular Research
ResumoArticle4 October 2021Open Access Source DataTransparent process Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease Antonietta Tarallo Antonietta Tarallo Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Federico II University, Naples, Italy These authors contributed equally to this work Search for more papers by this author Carla Damiano Carla Damiano Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Federico II University, Naples, Italy These authors contributed equally to this work Search for more papers by this author Sandra Strollo Sandra Strollo orcid.org/0000-0001-5397-2928 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Nadia Minopoli Nadia Minopoli orcid.org/0000-0003-1675-0538 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Alessia Indrieri Alessia Indrieri orcid.org/0000-0002-2325-0913 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Institute for Genetic and Biomedical Research (IRGB), National Research Council (CNR), Milan, Italy Search for more papers by this author Elena Polishchuk Elena Polishchuk Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Francesca Zappa Francesca Zappa Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Edoardo Nusco Edoardo Nusco Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Simona Fecarotta Simona Fecarotta Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Caterina Porto Caterina Porto Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Marcella Coletta Marcella Coletta Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Roberta Iacono Roberta Iacono orcid.org/0000-0002-3586-4322 Department of Biology, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, Naples, Italy Institute of Biosciences and BioResources - National Research Council of Italy, Naples, Italy Search for more papers by this author Marco Moracci Marco Moracci Department of Biology, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, Naples, Italy Institute of Biosciences and BioResources - National Research Council of Italy, Naples, Italy Search for more papers by this author Roman Polishchuk Roman Polishchuk orcid.org/0000-0002-7698-1955 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Diego Luis Medina Diego Luis Medina orcid.org/0000-0002-7347-2645 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Paola Imbimbo Paola Imbimbo Department of Chemical Sciences, Federico II University, Naples, Italy Search for more papers by this author Daria Maria Monti Daria Maria Monti Department of Chemical Sciences, Federico II University, Naples, Italy Search for more papers by this author Maria Antonietta De Matteis Maria Antonietta De Matteis orcid.org/0000-0003-0053-3061 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy Search for more papers by this author Giancarlo Parenti Corresponding Author Giancarlo Parenti [email protected] [email protected] orcid.org/0000-0002-6287-5748 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Antonietta Tarallo Antonietta Tarallo Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Federico II University, Naples, Italy These authors contributed equally to this work Search for more papers by this author Carla Damiano Carla Damiano Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Federico II University, Naples, Italy These authors contributed equally to this work Search for more papers by this author Sandra Strollo Sandra Strollo orcid.org/0000-0001-5397-2928 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Nadia Minopoli Nadia Minopoli orcid.org/0000-0003-1675-0538 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Alessia Indrieri Alessia Indrieri orcid.org/0000-0002-2325-0913 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Institute for Genetic and Biomedical Research (IRGB), National Research Council (CNR), Milan, Italy Search for more papers by this author Elena Polishchuk Elena Polishchuk Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Francesca Zappa Francesca Zappa Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Edoardo Nusco Edoardo Nusco Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Simona Fecarotta Simona Fecarotta Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Caterina Porto Caterina Porto Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Marcella Coletta Marcella Coletta Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Roberta Iacono Roberta Iacono orcid.org/0000-0002-3586-4322 Department of Biology, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, Naples, Italy Institute of Biosciences and BioResources - National Research Council of Italy, Naples, Italy Search for more papers by this author Marco Moracci Marco Moracci Department of Biology, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, Naples, Italy Institute of Biosciences and BioResources - National Research Council of Italy, Naples, Italy Search for more papers by this author Roman Polishchuk Roman Polishchuk orcid.org/0000-0002-7698-1955 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Diego Luis Medina Diego Luis Medina orcid.org/0000-0002-7347-2645 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Paola Imbimbo Paola Imbimbo Department of Chemical Sciences, Federico II University, Naples, Italy Search for more papers by this author Daria Maria Monti Daria Maria Monti Department of Chemical Sciences, Federico II University, Naples, Italy Search for more papers by this author Maria Antonietta De Matteis Maria Antonietta De Matteis orcid.org/0000-0003-0053-3061 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy Search for more papers by this author Giancarlo Parenti Corresponding Author Giancarlo Parenti [email protected] [email protected] orcid.org/0000-0002-6287-5748 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Federico II University, Naples, Italy Search for more papers by this author Author Information Antonietta Tarallo1,2, Carla Damiano1,2, Sandra Strollo1, Nadia Minopoli1,2, Alessia Indrieri1,3, Elena Polishchuk1, Francesca Zappa1,†, Edoardo Nusco1, Simona Fecarotta2, Caterina Porto2, Marcella Coletta2,†, Roberta Iacono4,5, Marco Moracci4,5, Roman Polishchuk1, Diego Luis Medina1,2, Paola Imbimbo6, Daria Maria Monti6, Maria Antonietta De Matteis1,7 and Giancarlo Parenti *,*,1,2 1Telethon Institute of Genetics and Medicine, Pozzuoli, Italy 2Department of Translational Medical Sciences, Federico II University, Naples, Italy 3Institute for Genetic and Biomedical Research (IRGB), National Research Council (CNR), Milan, Italy 4Department of Biology, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, Naples, Italy 5Institute of Biosciences and BioResources - National Research Council of Italy, Naples, Italy 6Department of Chemical Sciences, Federico II University, Naples, Italy 7Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy †Present address: Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA †Present address: IInd Division of Neurology, Multiple Sclerosis Center, University of Campania "Luigi Vanvitelli", Naples, Italy *Corresponding author. Tel: + 81 7463390 and +81 19230627; E-mails: [email protected]; [email protected] EMBO Mol Med (2021)13:e14434https://doi.org/10.15252/emmm.202114434 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pompe disease is a metabolic myopathy due to acid alpha-glucosidase deficiency. In addition to glycogen storage, secondary dysregulation of cellular functions, such as autophagy and oxidative stress, contributes to the disease pathophysiology. We have tested whether oxidative stress impacts on enzyme replacement therapy with recombinant human alpha-glucosidase (rhGAA), currently the standard of care for Pompe disease patients, and whether correction of oxidative stress may be beneficial for rhGAA therapy. We found elevated oxidative stress levels in tissues from the Pompe disease murine model and in patients’ cells. In cells, stress levels inversely correlated with the ability of rhGAA to correct the enzymatic deficiency. Antioxidants (N-acetylcysteine, idebenone, resveratrol, edaravone) improved alpha-glucosidase activity in rhGAA-treated cells, enhanced enzyme processing, and improved mannose-6-phosphate receptor localization. When co-administered with rhGAA, antioxidants improved alpha-glucosidase activity in tissues from the Pompe disease mouse model. These results indicate that oxidative stress impacts on the efficacy of enzyme replacement therapy in Pompe disease and that manipulation of secondary abnormalities may represent a strategy to improve the efficacy of therapies for this disorder. SYNOPSIS Enzyme replacement therapy (ERT) with recombinant human alpha-glucosidase (rhGAA) is currently the standard of care for the treatment of Pompe disease. However, this approach shows important limitations. We have tested whether modulation of oxidative stress may improve the efficacy of ERT. The impairment of the autophagic-lysosomal pathway in Pompe disease results into accumulation of dysfunctional mitochondria and defective clearance of reactive oxygen species (ROS), thus leading to increased oxidative stress. Oxidative stress is deleterious for the trafficking of the recombinant enzyme used for enzyme replacement therapy through the endocytic pathway. Antioxidants improve the amounts of the mannose-6-phosphate receptor available at the plasma membrane and improve recombinant enzyme trafficking to lysosomes. The paper explained Problem Pompe disease is a debilitating and progressive metabolic myopathy due to mutations of the GAA gene and functional deficiency of acid alpha-glucosidase, an enzyme involved in the lysosomal breakdown of glycogen. Enzyme replacement therapy with recombinant human alpha-glucosidase (rhGAA), currently the standard of care for Pompe disease patients, shows limitations. Despite treatment, some patients experience secondary decline after a few years, and some tissues, particularly skeletal muscles, appear to be relatively refractory to treatment. In addition to glycogen storage, secondary dysregulation of critical cellular pathways and functions contributes to the pathogenetic cascade of Pompe disease. We have tested whether these secondary abnormalities may represent novel therapeutic targets and whether modulation of these pathways is a potential adjunctive strategy to improve the efficacy of treatments for Pompe disease. Results We found elevated oxidative stress levels in tissues from the Pompe disease murine model and in patients’ cells. In cells, stress levels inversely correlated with the ability of rhGAA to correct the enzymatic deficiency. Correction of oxidative stress with antioxidants improved GAA activity in rhGAA-treated cells with a synergistic effect. Similar results were observed in the Pompe disease mouse model treated with rhGAA and antioxidants. Impact The results of our studies point to secondary cellular abnormalities as key therapeutic targets in Pompe disease. We showed that factors related to recipient tissues, in addition to the intrinsic properties of the recombinant enzyme, influence the response to enzyme replacement therapy. Thus, manipulation of secondary abnormalities may represent a strategy to improve the efficacy of therapies for this disorder. Introduction The pathology and the clinical manifestations of lysosomal storage diseases have been traditionally viewed as direct consequences of lysosomal engorgement with inert undegraded substrates. This concept has been challenged by the most recent vision of lysosomal functions. Together with the recognition of a central role of lysosomes in cellular homeostasis and metabolism (Ballabio & Bonifacino, 2020), multiple and diverse secondary events are now emerging as important players in the pathogenesis of these disorders. Among them, some have received particular attention and include autophagy impairment, jamming of intracellular vesicle trafficking, mitochondrial dysfunction, oxidative stress, abnormalities of calcium homeostasis, and dysregulation of signaling pathways (Platt et al, 2012; Parenti et al, 2021). Pompe disease (PD) pathophysiology is a paradigm of the role of secondary events in lysosomal storage diseases. PD is due to mutations of the GAA gene and functional deficiency of acid alpha-glucosidase, an enzyme involved in the lysosomal breakdown of glycogen (van der Ploeg & Reuser, 2008). PD phenotype is broad but is almost invariably highly debilitating and associated with premature death. Infantile-onset patients, presenting within the first months of life, are affected by severe hypertrophic cardiomyopathy, skeletal myopathy, recurrent respiratory infections, and, if untreated, die within the first year of life. Late-onset patients are affected by a progressive myopathy resulting in motor impairment, while heart involvement is usually asymptomatic (van der Ploeg & Laforet, 2016). Recent reports have further expanded PD phenotype with the demonstration of central nervous system involvement, particularly in severely affected patients (Korlimarla et al, 2019; Musumeci et al, 2019). A typical pathological hallmark of PD is generalized intra-lysosomal glycogen storage due to GAA deficiency. Several reports, however, have suggested that, in addition to glycogen storage, secondary dysregulation of critical cellular pathways and functions contributes to the pathogenetic cascade of the disease (Meena et al, 2020). Abnormalities of autophagy have been shown to play a primary role in the disease pathophysiology (Fukuda et al, 2006; Nascimbeni et al, 2012; Raben et al, 2012; Meena & Raben, 2020). Massive accumulation of autophagic material, due to upregulation of autophagy and/or impaired autophagic flux, has been mostly observed in skeletal muscles. A role of dysfunctional autophagy in PD pathophysiology has been further supported by studies based on modulation of autophagy by overexpression of either transcription factor EB (TFEB) or transcription factor binding to IGHM enhancer 3 (TFE3), two members of the MiT-TFE family of transcription factors that control lysosomal biogenesis and activation of the autophagosomal–lysosomal pathway. In these studies, overexpression of either TFEB or TFE3 resulted in improved cellular clearance of glycogen in vitro (Spampanato et al, 2013; Martina et al, 2014) and in some amelioration of physical performance in the murine model of PD (Gatto et al, 2017). Other studies have also shown that additional factors, such as altered calcium signaling, mitochondrial dysfunction and oxidative stress also take part in the pathophysiology of PD (Lim et al, 2015). The characterization of these secondary events has therapeutic implications. Currently, the only approved treatment for PD is enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) (Van den Hout et al, 2000; Van den Hout et al, 2004). Albeit effective on many aspects of PD, ERT has some limitations (Case et al, 2012; Kuperus et al, 2017; van der Meijden et al, 2018). A residual phenotype has been observed in infantile-onset patients (Prater et al, 2012), while many late-onset patients experience some secondary decline after a few years of therapy (Wyatt et al, 2012; Harlaar et al, 2019). There are several factors that affect ERT efficacy, such as the cross-reactive immunological material status of patients (Kishnani et al, 2010; van Gelder et al, 2015), age at start of treatment (Chien et al, 2013), and advancement of disease progression (van der Meijden et al, 2015). It is plausible that ERT limitations are in part due to the secondary cellular abnormalities triggered by storage. For example, in in vitro cellular systems and in the mouse model of PD rhGAA appears to be mistrafficked into areas of accumulation of autophagic material as a consequence of autophagy impairment and possibly of altered vesicle and membrane trafficking (Fukuda et al, 2006; Cardone et al, 2008; Spampanato et al, 2013). We speculated that secondary increase of oxidative stress is another factor limiting correction of GAA activity by ERT. Increased stress is expected to be one of the consequences of autophagy impairment and has been in fact observed in PD (Lim et al, 2015; Sato et al, 2017). The existence of a crosstalk between the autophagic pathway and oxidative stress has been clearly documented. On one hand, oxidative stress activates autophagy through reactive oxygen species (ROS) and reactive nitrogen species (RNS) that act as intracellular “alarm molecules” of cellular stress and of the availability of nutrients. This effect is mediated by mucolipin 1 (TRPML1) (Zhang et al, 2016) and mTOR complex 1 (mTORC1)-independent activation of TFEB and TFE3 transcription (Martina & Puertollano, 2018). On the other hand, autophagy is required for removing the conditions that cause oxidative stress (such as starvation), for clearing ROS and RNS from cells, and ultimately to allow cells to cope with stress (Filomeni et al, 2015). It has been shown that induction of oxidative stress in wild-type astrocytes results into defective clathrin-mediated endocytosis of transferrin (Volpert et al, 2017) and that in rat brain oxidative stress causes loss of soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins (Kaneai et al, 2013) that are crucial for vesicle docking and fusion (Lang et al, 2001). Thus, we have focused our attention on oxidative stress as a potential therapeutic target in PD. We have tested whether the ability of rhGAA in correcting GAA levels is affected by oxidative stress, and whether stress mitigation is a potential strategy to improve the lysosomal trafficking of the recombinant enzyme. Results Oxidative stress is increased in the Gaa KO mouse and in cells from PD patients We evaluated the levels of oxidative stress in PD using standard biochemical tests that measure ROS levels (2′,7′-dichloro-dihydrofluorescein, DCFDA), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), and intracellular glutathione (GSH) levels (5,5′-dithiobis-2-nitrobenzoic acid, DTNB). We first looked at tissues from a Gaa KO mouse, a murine model of PD generated by disruption of the Gaa gene exon 6 (Raben et al, 1998). We analyzed tissues that are most relevant for PD phenotype, specifically skeletal muscles (gastrocnemius, quadriceps, diaphragm), heart, and liver (Fig 1A). The results were compared to those obtained in the same tissues from wild-type animals that were arbitrarily taken as equal to 100. In muscles and heart from the Gaa KO mouse, we found significantly increased ROS levels (P ranging between 0.0001 and 0.0451 in the different tissues) and lipid peroxidation (P ranging between 0.0012 and 0.0092), while in liver only lipid peroxidation was significantly increased (P = 0.0202). Western blot analysis of the stress marker p-ERK (Fig 1B) in gastrocnemius also supported the presence of increased oxidative stress. Figure 1. Increased oxidative stress in PD A, B. Oxidative stress biochemical markers (lipid peroxidation, ROS production) (A) and p-ERK expression (B) in tissues from 3-month-old PD mice (KO) (n = 3) and from wild-type (WT) mice. C, D. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (C) and p-ERK expression (D) in PD and control (CNTR) fibroblasts (n = 6). E, F. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (E) and p-ERK expression (F) in PD and control (CNTR) myoblasts (n = 4). Data information: In all instances, indicators of stress were increased in PD, compared to the respective control samples. Data information: In each experiment, at least biological triplicates were analyzed for each cell line or tissue sample; each assay was performed at least in duplicate. Data are presented as mean ± SD. Student’s t-test was applied. Statistically significant comparison P-values are indicated. Fig 1B: brightness +20%, contrast +41%. Source data are available online for this figure. Source Data for Figure 1 [emmm202114434-sup-0003-SDataFig1.zip] Download figure Download PowerPoint We performed the same biochemical tests in PD fibroblasts from patients with different phenotypes (classic infantile-onset, intermediate, late-onset) and in myoblasts from late-onset patients (Table 1). The results were compared to those obtained in the respective control cell lines. Also in PD cells we found increased ROS levels (fibroblasts P = 0.0033; myoblasts P = 0.0044) and lipid peroxidation (fibroblasts P = 0.0014; myoblasts P = 0.0027), with reduced GSH levels (fibroblasts P = 0.0026; myoblasts P = 0.0373) and increased p-ERK (Fig 1C–F). ROS levels and lipid peroxidation were lower in fibroblasts from late-onset phenotypes (patients PD5, PD6) (Appendix Fig S1). The results obtained in cells were reflective of the in vivo findings, thus indicating that cultured cells, such as fibroblasts, are a reliable tool for further studies and for in vitro pharmacological manipulation of the oxidative stress pathway. Table 1. Cell lines used in the study, patient genotypes, and phenotypes. Pt ID Genotype (DNA) Genotype (protein) Phenotype PD1 fibroblasts c.1124G>T / c.2261dupC p.R375L / p.V755Sfs*41 Classic infantile PD2 fibroblasts c.1655T>C / c.1655T>C p.L552P / p.L5552P Intermediate PD3 fibroblasts c.1655T>C / c.236_246del p.L552P / p.P79Rfs*12 Classic infantile PD4 fibroblasts c.1101G>A / c.1927G>A p.W367X / p.G643R Classic infantile PD5 fibroblasts c.1655T>C / c.-35C>A p.L552P / abnormal splicing Intermediate / late onset PD6 fibroblasts c.-32-13T>G / unknown abnormal splicing / unknown Late onset PD7 myoblasts c.-32-13T>G / c.1551+1G>C abnormal splicing / abnormal splicing Late onset PD8 myoblasts c.-32-13T>G / c.525delT abnormal splicing / p.T175fsX46 Late onset PD9 myoblasts c.-32-13T>G / c.989G>A abnormal splicing / p.W330* Late onset PD10 myoblasts c.-32-13T>G /c.1927G>A abnormal splicing / p.G643R Late onset PD11 myoblasts c.-32-13T>G / c.2237G>A abnormal splicing / p.W746X Late onset In several disease conditions (Pieczenik & Neustadt, 2007; Stepien et al, 2017), mitochondrial dysfunction contributes to the increased oxidative stress. Indeed, an impairment of mitochondrial function and calcium signaling has also been extensively documented in PD (Lim et al, 2015; Sato et al, 2017; Meena & Raben, 2020). In line with these studies, we found abnormal mitochondrial morphology in the Gaa KO mouse. In gastrocnemii, in addition to the typical PD hallmarks (intra-lysosomal glycogen storage, expansion of the autophagic compartment), ultrastructural analysis showed abnormal mitochondria with altered cristae and electron-lucent matrix (Fig EV1A). A quantitative analysis of the number and morphology of mitochondria in 15 low-magnification (16,500×) electron microscopy fields showed that the number of abnormal mitochondria was significantly higher (P = 0.0038) in Gaa KO mice than in wild-type animals (Fig EV1B and C). In homogenates from Gaa KO gastrocnemii, we also performed a Western blot analysis of components of the 5 oxidative phosphorylation (OXPHOS) complexes (Uqcrc2, ATP5A, mtCOI, Sdhb, and Ndufb8) and of the mitochondrial outer membrane (Mfn2) (Fig EV1D). In the Gaa KO gastrocnemii, these markers were increased, as compared to control tissues, indicating an accumulation of mitochondrial proteins. Click here to expand this figure. Figure EV1. Characterization of mitochondria in gastrocnemii from the PD mouse model and in cultured PD patient cells A. Ultrastructural analysis of gastrocnemii from the Gaa KO mouse showed intra-lysosomal glycogen storage (white arrow), active mitophagy (arrowhead), abnormal mitochondria (black arrow). B, C. Quantitative analysis of the number of mitochondria (B) and of morphologically abnormal mitochondria (C) in 15 low-magnification (16×) electron microscopy fields showing significantly increased number of abnormal mitochondria (P = 0.0038) in Gaa KO compared to wild-type animals. Data presented as mean ± SD of at least 12 fields for each mouse muscle. Student’s t-test was applied. D. Western blot analysis of the levels of OXPHOS complexes in mitochondrial preparations from the Gaa KO gastrocnemii, showing increased levels of the markers tested. E, F. Number of mitochondria (E) and mitochondrial length (F) in PD fibroblasts and myoblasts compared to, respectively, control cells. Data presented as mean ± SD of at least 15 fields for each cell line. A Student’s t-test was applied. G, H. Co-staining of COX1 with LC3 (G) and quantitative analysis (H) showing significantly increased colocalization of these markers in PD cells compared to control cells under standard culture conditions. Data presented as mean ± SD of five images for each cell line. A Student’s t-test was applied. Confocal 63× images; scale bar 50 µm; contrast +15%; brightness +25%. Data information: In B, E, F, boxes include values between upper and lower quartiles, and central band corresponds to median, whiskers, and lower extremes to higher and lower values. Outlier values are indicated as dots. Source data are available online for this figure. Download figure Download PowerPoint In PD fibroblasts and myoblasts, ultrastructural abnormalities of mitochondria were also present, although less pronounced (Fig EV1E and F). In PD fibroblasts, co-staining of cytochrome c oxidase1 (COX1) with the autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) showed significantly increased colocalization of these markers in PD cells compared to control cells under standard culture conditions (Fig EV1G and H). These data (in combination with signs of activated autophagy) (Fig EV2A and B) are compatible with active but less efficient mitophagy, with reduced progression of aged or damaged mitochondria to the terminal part of autophagosomal–lysosomal pathway. Click here to expand this figure. Figure EV2. Autophagy markers Western blot analysis of p62 and LC3 in control (CNTR) (n = 2) and Pompe disease (PD) (n = 3) fibroblasts. Immunofluorescence analysis of LC3 in cultured CNTR and PD fibroblasts. Confocal 63× images; scale bar 50 µm; brightness +25%; contrast +20%. Western blot and quantitative analyses of autophagy marker LC3 in CNTR and in a PD cell line. The analysis was performed in untreated cells and after different treatments to modulate autophagy (starvation, STAR; rapamycin, RAPA; MK6-83; bafilomycin, BAFI). Western blot and quantitative analyses of autophagy marker LC3 in a PD cell line. The analysis was performed in untreated cells and after different treatments to induce stress (sodium arsenite, ARS; tert-butyl-peroxide, TBP). Western blot and quantitative analyses of autophagy marker LC3 in a PD cell line. The analysis was performed in untreated cells and after antioxidant treatments. Source data are available online for this figure. Download figure Download PowerPoint Modulation of autophagy impacts on stress in fibroblasts The increase of oxidative stress is expected to be a consequence of the impairment of autophagy typically observed in PD and of mitochondrial dysfunction. To support this hypothe
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