Inducing mitophagy in diabetic platelets protects against severe oxidative stress

• DOI: 10.15252/emmm.201506046
• Publication Date: 2016-05-25T00:45:39Z
• Authors: Seung Hee Lee; Jing Du; Jeremiah Stitham; Gourg Atteya; Suho Lee; Yaozu Xiang; Dandan Wang; Yu Jin; Kristen L Leslie; Geralyn Spollett; Anup Srivastava; Praveen Mannam; Allison Ostriker; Kathleen A Martin; Wai Ho Tang; John Hwa
• Keywords: Haven

Research Article24 May 2016Open Access Source DataTransparent process Inducing mitophagy in diabetic platelets protects against severe oxidative stress Seung Hee Lee Section of Cardiovascular Medicine, Department Internal Yale Center, University School New Haven, CT, USA Search for more papers by this author Jing Du Jeremiah Stitham Gourg Atteya Suho Departments Neurology and Neurobiology, Cellular Neuroscience, Neurodegeneration Repair Program, Yaozu Xiang Dandan Wang Yu Jin Kristen L Leslie Geralyn Spollett Endocrinology & Metabolism, Anup Srivastava Pulmonary, Critical Care Sleep Praveen Mannam Allison Ostriker Kathleen A Martin Wai Ho Tang Corresponding Author Guangzhou Institute Pediatrics, Women Children's Medical Centre, University, Guangzhou, China John Hwa orcid.org/0000-0001-7366-2628 Information Lee1, Du1, Stitham1, Atteya1, Lee2, Xiang1, Wang1, Jin1, Leslie1, Spollett3, Srivastava4, Mannam4, Ostriker1, Martin1, 1,5 1 1Section 2Departments 3Section 4Department 5Guangzhou *Corresponding author. Tel: [phone]; Fax: 6118; E-mail: [email protected] EMBO Mol Med (2016)8:779-795https://doi.org/10.15252/emmm.201506046 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures Info Abstract Diabetes mellitus (DM) is growing international concern. Considerable mortality morbidity associated with diabetes arise predominantly from thrombotic cardiovascular events. Oxidative stress-mediated mitochondrial damage contributes significantly enhanced thrombosis DM. basal autophagy has recently been described as playing an important role normal platelet activation. We now report substantial induction (above levels) platelets, suggesting alternative roles pathology. Using combination molecular, biochemical, imaging studies on human DM we that serves protective mechanism responds through JNK By removing damaged mitochondria (mitophagy), phosphorylated p53 reduced, preventing progression apoptosis, preserving function. The absence results failure protect stress, leading increased thrombosis. Surprisingly, removal does not require contributions transcription, lack nucleus. considerable energy resources expended "prepackaging" complex machinery short-lived support critical role, anticipation exposure stress. Synopsis Under conditions commonly patients, apoptosis preserves Autophagy are platelets. Mitophagy induced ROS/JNK-mediated pathway. stress-induced apoptosis. mellitus. Introduction Platelets (7–10 days) circulating anucleate cytoplasmic fragments (1.5–3 μm) containing factors required regulation thrombus formation, vascular homeostasis, immune response (Lindemann et al, 2001; Leytin 2007; Alexandru 2012; Leytin, 2012). capable many fundamental cellular functions despite being anucleate, de novo protein synthesis (Weyrich 1998; Pabla 1999; Lindemann 2001) programmed cell death (Vanags 1997; Mason 2007). only (Feng 2014; Cao 2015; Ouseph 2015). Macroautophagy (hereafter referred autophagy) ubiquitous, evolutionarily conserved, tightly regulated eukaryotic cells serving degrade components using lysosomal (Yu 2010; Choi 2013). plays essential growth, development, recycling (Choi targeting invading bacteria, aggregates, organelles such endoplasmic reticulum (ER) (Hanna Kubli Gustafsson, Recent investigations have identified organelle-specific selectivity recognition substrates turnover quality control (Kubli highly ordered, initial phagophore formation (nucleation) requiring assembly complex. Subsequent expansion membrane mediated ubiquitin-like conjugating systems, microtubule-associated light chain 3 (LC3), system (ATGs). expands, completely surrounding its target, followed fusion lysosome, content degradation enzymes (Shintani Klionsky, 2004; While importance nucleus regulating highlighted elegant review (Fullgrabe 2013), autophagy/mitophagy can also occur appears play activation progressive chronic metabolic disorder characterized hyperglycemia caused impaired insulin levels, sensitivity, and/or action. Currently, over 19.7 million adults diagnosed estimated 8.2 undiagnosed (Go 2013a,b). Sixty-five percent patients will die events heart attacks strokes (Ferreiro Angiolillo, 2011). key roles, occlusion major vessels tissue death. Of great concern 10–40% biochemically insensitive most used drug prevent treat (heart strokes), aspirin (Price Holman, 2009; Pignone 2010a,b; Ferreiro Compounding insensitivity, recognized be hyperactive 2011; 2011) arising (Kaneto 2010). therapies underlying mechanisms urgently warranted, particularly prevalence (38.2% US adult population prediabetes abnormal fasting glucose) In examining mellitus, discovered specifically were upregulated. disease never before reported. This could provide mechanistic insights into other potential health disease. set out discover how was why there intense upregulation. Clearly, must reason possess all undertake energy-requiring process. present first intrinsic Results phosphorylation, dysfunction, Hyperglycemia (associated mellitus—DM) lead phosphorylation p53, dysfunction damage, resulting (Polyak von Harsdorf Li 2014). Diabetic subject hyperglycemia, lipids, (e.g. inflammation) Indeed, ROS when compared healthy controls (Fig 1A). To demonstrate increases having effects assessed modifications directly 3-nitrotyrosine (3-NT), aldehyde adducts (4-HNE), carbonyl derivatives (dinitrophenyl (DNP)-derivatized carbonyl), polyubiquitination (Dhiman Silva Analysis HC (pooled, n = 4) vs. 8) demonstrated 5.3-fold 1.2-fold 1.6-fold (DNP derivatized 1.5-fold (polyubiquitination) (Appendix Fig S1). Severe leads proteins Western blot analysis vs 1B ). Confocal microscopy visualize (normally located nucleus) anuclear 1C). translocation indicator instigator (Tang 2014) organs (Tasdemir 2008; Hoshino Indicative (or loss mitochondria), substantially reduced MitoTracker fluorescence 1D) flow cytometry 1E—tetramethylrhodamine methyl ester, TMRM fluorescence). Associated increase annexin V 1E), marker shows hallmarks release cytochrome c active caspase-3 1F G). then visualized [disrupted internal outer membranes (Boland 2013) (Ding 2012)] randomly selected electron (EM) (HC DM). 1H—HC numbers per field 27% (average number HC, 3.2 ± 1.6 (100%) 33; DM, 1.7 0.7 (52.6%) 17), supporting significant 1I). constituted 75.8% 24.1% ultrastructure 85.0% 14% 1J). Taken together, these previous demonstrating markers next question address whether able launch itself damaging Figure 1. levels measured after staining specific detection dyes (CellROX green, Molecular Probes, USA) (n (*P 0.032 HC). downstream signaling molecules (#1–3) (#1–8) patient Quantification 5) 18) individuals (**P 0.0025 GAPDH served loading control. Fluorescent immunostaining (arrows). Graph indicates positive signal. y-axis percentage p53-positive total 0.0064 Representative figure 5. Mitochondrial (ΔΨm) (50 nM 15 min) signal intensity fold 1.1608E-05 Signal each group converted change values. 6) 4). ΔΨm analysis. detected TMRM, (PS externalization). 3. apoptosis-related pp53, c, Tubulin showing (Cyto c) cytosol EM examples typical mitochondrion (HC). Arrows indicate disrupted membranes. 33) 17) average single-platelet view 5.50811E-05 Data information: All data expressed mean SD. available online figure. [emmm201506046-sup-0002-SDataFig1.pdf] Download PowerPoint intrigued what appeared distinct Beclin1, ATG3, ATG7, ATG12-5, LC3II consistently above (HC) 2A B). Moreover, examination versus marked differences vacuolation S2). Detailed vacuoles confirmed ultrastructural characteristics autophagosomes 2C) early-stage phagophores 2C DM1), mid-stage DM2), late-stage autolysosomes DM3). further confirm vacuolated structures indeed autophagosomes, performed immunogold labeling (immuno-EM) autophagosome LC3 (Kabeya 2000). contrast (gold particles) which localized accumulated around 2D). 2. activated LC3I/II, mitophagy-related Parkin PINK1 (#1–8). (#1–5) (#1–18) (Beclin1, **P 0.0003; ATG12, *P 0.0388; 0.0213; P 0.4051; LC3, 0.0298; PINK, 0.0155; Parkin, 0.0103 three panels represent stages found Autophagosome containing/enclosing indicated black box. Shown panel enlargement (DM1 2) or autolysosome (DM3). Dashed outline immuno-EM immunogold-labeled clusters. No clusters areas gold (DM1–3) presented. insets enlargements adjacent mitochondria-like structures. Double (mitochondria potential) (autophagy) Boxed enlarged (F). high low representative LC3. autophagosome-positive 6 4 samples CoxIV (mitochondria) (autophagy marker). LC3-positive 0.006 2 [emmm201506046-sup-0003-SDataFig2.pdf] provides means visualizing individual steps process, allowing colocalization taking place Dissipation (reduced observed 2E low-powered field, 2F single 2G quantitation—reduced red green platelets), mitophagy. later autophagy, fuse lysosomes generate autolysosomes, autophagosomal components. High-resolution confocal immunofluorescence revealed LAMP1 (lysosomal autolysosomes) enriched relative S3A). As function severely 1), based upon our localization mitochondrial-like 2D), Parkin. recruited where it ubiquitinates responsible recruiting LC3-conjugated (Narendra Muller-Rischart Both translocated S3B) colocalized S3C) S3D). Appropriate secondary antibody showed no labeling. BNIP3L/NIX shown involved reticulocytes (Zhang Increases S4A DNA assess reduction (Suliman Dasgupta There decrease both mouse S5) consistent 1I. use multiple biochemical 2015), along direct visualization supports platelet. (biochemical detection, EM, immuno-EM) It intriguing cell. postulated serve pathway understand platelet, autophagy-regulating signals mTOR/AKT Kroemer 2010) (Wei Ravikumar Haberzettl Hill, Although AKT borderline significance, mTOR changes 3A). activation, pUlk (757), pp70S6K, pS6 known (Kim These S6A However, (Thr183/Tyr185) (approximately twofold) (P 0.0001) Increased (as under hyperglycemia) trigger Kaneto Thus, may possibly linking (pJNK, JNK, pAKT, AKT, pmTOR, mTOR) upstream (