Lysosomes, weapons or shield in gentamicin-induced apoptosis.

Transcription

1 Lysosomes, weapons or shield in gentamicin-induced apoptosis. Hélène Servais Unité de Pharmacologie cellulaire et moléculaire Promoteur: Prof. M-P Mingeot-Leclercq Co-Promoteur: Prof. P.M. Tulkens

2 1. INTRODUCTION : Aminoglycosides (AG) history. Dr Waksman Kanamycin Amikacin Isepamicin «New aminoglycosides» Streptomycin Dibekacin Neomycin Paromomycin Tobramycin Netilmicin Gentamicin isolated from Micromonospora purpurea Based on the identification of the exact interactions between AG with the bacterial ribosome

3 1. INTRODUCTION : Aminoglycosides structure Mingeot-Leclercq and Tulkens 1999 Antimicrob Agents Chemother 43:

4 1. INTRODUCTION: Aminoglycosides mechanism of action «New aminoglycosides» Inducing miscoding during protein synthesis Davies et al., 1965, 1968 Binding to the A-site of the ribosome Moazed et al., 1987 Interaction of amine functions of with phosphate backbone of the RNA François et al., Interactions AG-A-Site similar changes induced that those observed in with corresponding trna-mrna 2000, 2002 Ogle et al., Gentamicin isolated from Micromonospora purpurea Interactions AG-A-Site 30S particle revealed by Crystal structure Moazed et al., 1987 Interactions AG-A- Site oligonucleotide Yoshizawa et al., 1998 Fourmy et al., 1998

5 1. INTRODUCTION: Aminoglycosides insertion inside the 16S rrna A-site Vicens and Westhof 2003 biopolymers 70: 42-57

6 1. INTRODUCTION: Aminoglycosides disturb protein synthesis Wrong AA added His Arg fmet 50S 50S 50S GUG GCG UAC A UG 30S CGC AG mrna Misreading codon for Arg A-site Wimberly et al., 2002 Nature 407: , Ogle et al., 2001 Science 292: , Ogle et al., 2002 Cell 111: Vicens and Westhof., 2003 Biopolymers 70:42-57

7 1. INTRODUCTION: Clinical indications of aminoglycosides Serious, life-threatening gram-negative infection Complicated skin, bone or soft tissue infection Complicated urinary tract infection Septicemia Peritonitis and other severe intra-abdominal infections Severe pelvic inflammatory disease Endocarditis Mycobacterium infection Neonatal sepsis

8 1. INTRODUCTION: Aminoglycosides are interesting drugs but Bactericidal Post-antibiotic effect Synergism with cell wall active antibacterials (penicillin, cephalosporin, monobactam, carbapenem and glycopeptide) But toxicity limits their clinical use - Choclear and vestibular toxicity - Nephrotoxicity: 5-25% (0-50%) * Risk factors nephrotoxicity: * Clinical features: Nonoliguric renal failure Slow rise in creatinine

9 1. INTRODUCTION: Aminoglycosides nephrotoxicity Evidence of and AG nephrotoxicity Falco et al., 1969 Inhibition of phosphatidyl inositol phospholipase C Lipsky et al., 1982 traffics Golgi-endoplasmic reticulum Sandoval et al., 2000 Kidney Lysosomes primary target of AG houghton et al., 1976 Inhibition of PI-PLC houghton et al., Gentamicin isolated from Micromonospora purpurea Lysosomal alterations accumulation phospholipids Kozek et al., 1974 confined proximal tubules mainly in lysosomes Silverblatt et al., 1979 Lysosomal phospholipidosis Aubert-Tulkens et al., 1979 Renal basolateral membrane binding Walker et al., 1987 AG-induced apoptosis In vivo and in vitro with clinicalrelevant low doses El Mouedden et al., 2000a,b Increase cellular calcium content Holohan et al., 1988 Inhibition of lysosomal sphingomyelinase and phospholipase A1 Laurent et al., 1982

10 1. INTRODUCTION: Gentamicin accumulation in renal cortex Accumulated in the renal cortex in proximal tubular epithelial cells 3 H-gentamicin collection of tissue 24hour after injection 40 µm 40 µm 20 µm 20 µm Schmitz et al., 2002 J Biol Chem 277:

11 . INTRODUCTION: Gentamicin uptake by kidney cells accumulation in lysosomes Gentamicin = 95% excreted glomerumar filtration Apical membrane Θ Θ Θ Θ Lumen- urine Endocytic Route G E N Proximal tubular epithelial cell Mitochondria Lysosomes Endoplasmic reticulum Golgi Nucleus 0.5 µm 0.5 µm Basolateral membrane Blood Schmitz et al., 2002 J Biol Chem 277:

12 1. INTRODUCTION: Lysosomal perturbations induced by gentamicin Proximal tubular cell - rat treated with 10 mg/kg.day gentamicin for 7 days Kosek et al 1974 Lab. Invest 30: Proximal tubular cell - rat treated with 4 mg/kg of gentamicin for 4 days Pictures from P.M. Tulkens & M.B. Carlier, adaptated from Tulkens 1986 Am J Med 80 Suppl 6B: Magnifications: X 75,000 (& 200,000, insert)

13 1. INTRODUCTION: Inhibition of lysosomal phospholipase and sphingomyelinase Cortex of rat treated with gentamicin 4 or 10 mg/kg.day Day of treatment Carlier et al 1982 Arch Toxicol Suppl. 5:

14 1. INTRODUCTION: Inhibition of lysosomal phospholipase and sphingomyelinase Adaptated from Mingeot-leclercd et al 1991 Biochem (Life Sci Adv). 10:

15 1. INTRODUCTION: Cellular alterations induced by Lipsky et al., 1982 Cronin et al., 1982 Schwertz et al., 1984 Levi et al., 1990 Apical membrane Lumen- urine Kozek et al., 1974 Houghton et al., 1976 Watanabe et al., 1978 Silverblatt et al., 1979 Hostetler et al., 1982 Endocytic Route Laurent et al., 1982 Viotte et al., 1982 Fillastre et al., 1983 Golgi Lysosomes Powell et al., 1983 Williams et al., 1985 Chatterjee et al., 1987 Mingeot-Leclercq et al., 1988 Vera-Roman et al., 1975 Mingeot-Leclercq et al., 1990a,b Bennett et al., 1988 Okuda et al., 1992 Sandoval et al., 1998 Endoplasmic reticulum Mingeot-Leclercq et al.,1991 Sundin et al., 2001 Mitochondria Vera-Roman et al., 1975 Walker et al., 1987 Rustenbeck et al., 1998 Nucleus Basolateral membrane Blood

16 1. INTRODUCTION: Gentamicin induces apoptosis in vivo 20 µm 20 µm Rat kidney cortical specimens of controls (A) and animal treated for 10 days with 10mg/kg of gentamicin. In vivo El Mouedden et al., 2000 Antimicrobial Agents and Chemother 44: Rat treated for 10 days with saline (control), 10mg/kg of gentamicin and netilmicin; and 40mg/kg isepamicin and amikacin. Apoptosis Nephrotoxicity

17 1. INTRODUCTION: Gentamicin induces apoptosis in vitro 4 days Closed symbols: 2 mm Open symbols: Control LLC-PK1 treated 4 days with 2 mm of In vitro El Moudden et al., 2000 Toxicological Sciences 56:

18 1. INTRODUCTION: Apoptotis or programmed cell death A. Apoptosis: Apoptosis: Programmed cell death (PCD) Role: Important in *Tissue Homeostasis *embryogenesis Diseases Toxicological involvement Neurodegenerative disorders Cancer Autoimmune disorders AIDS Excess of apoptotic process Liver CNS Kidney Decrease of apoptotic process

19 1. INTRODUCTION: Apoptotis and necrosis Cell death induced by a toxic Apoptosis Necrosis Cell suicide Cell murder Degree of insult by a toxic

20 1. INTRODUCTION: Morphological appearance of apoptotic cell Morphological changes in apoptotic cells - Cytoplasm shrink - Nuclear condensation margination fragmentation - Formation of apoptotic bodies - Results from the activation of special enzyme: CASPASE

21 1. INTRODUCTION: Apoptotic Pathways Fas Extrinsic pathway TNF-α caspase 8 Intrinsic pathway Mitochondria Lysosome Proteasome Golgi Endoplasmic Reticulum caspase 3 (6-7) Bax caspase 9 Apoptosome Cytochrome c D nucleus

22 2. AIM of the study: HOW INDUCES APOPTOSIS? CH3 Extrinsic pathway Fas TNF-α Intrinsic pathway Mitochondria caspase 8 Lysosome O H 2N R 1 O HO OH R O O 2 H 2N NH 2 Proteasome caspase 3 (6-7) OH H 3C HN Bax caspase 9 Apoptosome Cytochrome c Golgi Endoplasmic Reticulum D nucleus

23 3. RESULTS CH3 Part I: Are lysosomes involved in -induced apoptosis? Lysosome OH H 3C HN O H 2N R 1 O HO OH R 2 O O H 2N NH 2 Apoptosis

24 PI.1. Destabilization of lysosomal membrane by gentamicin: LLC-PK1cells: renal proximal tubular cells from pig Gm 1 mm Gm 3 mm Gm 2 mm MSDH 25 µm AO (530 nm/620 nm) % of control Time (hours) Lysosomal destabilization 2h 0h 6h 12h 18h 24h

25 Apoptotic signalling: CH3 Extrinsic pathway Fas TNF-α Intrinsic pathway Mitochondria caspase 8 Lysosome O H 2N R 1 O HO OH R O O 2 H 2N NH 2 Proteasome caspase 3 (6-7) OH H 3C HN Bax caspase 9 Apoptosome Cytochrome c Golgi Endoplasmic Reticulum D nucleus

26 PI.2. Disruption of mitochondrial membrane by Gm 2mM Gm 3mM Gm 1mM mcccp 20 µm JC-1 610/530 nm intensity ratio (% of control) Lysosomal destabilization Time (hours) Disruption Mitochondrial Ψ 24 0h 2h 10 h 6h 12h 18h 24h

27 PI.3. Release of cytochrome c : 2 mm 12h Ψ m PTP Cytochrome c Lysosomal destabilization Disruption mitochond rial Ψ Release of cyt.c 12 h 0h 2h 10 h 6h 12h 18h 24h

28 Apoptotic signalling: CH3 Extrinsic pathway Fas TNF-α Intrinsic pathway Mitochondria caspase 8 Lysosome O H 2N R 1 O HO OH R O O 2 H 2N NH 2 Proteasome caspase 3 (6-7) OH H 3C HN Bax caspase 9 Apoptosome Cytochrome c Golgi Endoplasmic Reticulum D nucleus

29 PI.4. Time-sequence of the development of apoptosis and caspase-3 activity: 1 mm 2 mm control 2 mm 3 mm 1 mm 3 mm Caspase-3 activity (% of control) Lysosomal destabilization * * * * * Time (hours) Disruption Mitochondrial Ψ Release of cyt.c 12 h % of apoptotic cells (DAPI) Caspase-3 activation 24h * * * Time (hours) Nuclear fragmentation 0h 2h 10 h 6h 12h 18h 24h

30 3. RESULTS : PART II Is able to destabilize membrane? Mather and Rottenberg 2001 Biochem Biophys Acta 1503: Van Bambeke et al., 1993 Eur J Pharmacol 247:

31 PII. Mimicking lysosomes and mitochondria : CHO PC SM PI CL SUV: Small Unilamellar vesicles Diameter: nm LYSO 5.4 Mimic the lysosomal membrane OUTER MITO 7.4 Mimic the outer mitochondrial membrane INNER MITO 7.4 Mimic the inner mitochondrial membrane 33.3 % 24.2 % 24.2 % 18.3 % % 24.2 % 24.2 % 18.3 % % 24.2 % 24.2 % % CHO: Cholesterol; PC: Phosphatidylcholine; SM: Sphingomyelin; PI: Phosphatidylinositol; CL: Cardiolipin

32 PII. Ability of gentamicin to destabilize liposomes : Lysosome Outer Mitoch Inner Mitoch % of calcein released mM PI (Lysosomes) ph 5.4 > PI (outer mitochondria) ph 7.4 > Cardiolipin (inner mitochondria) ph Time (hours)

33 3. RESULTS : PART III Is able to initiate apoptotic signalling if localized in the cytoplasm? Lysosome Apoptosis

34 Delivery of inside cellular cytosol Definition of Electroporation (EP): designates the use of SHORT HIGH VOLTAGE PULSE to OVERCOME the BARRIER of the CELL MEMBRANE This rupture is TRANSIENT and REVERSIBLE Lysosome?? Mitochondria Apoptotic cell death

35 PIII.1. EP cells showed apoptosis with lower concentration of Incubated cells Electroporated cells Incubation tme : 24 hours c 20 Apoptotic cells (%) b b f * * * f d a a d d a e e e e e e e mm mg/l gentamicin extracellular concentration

36 PIII.2. Apoptosis versus necrosis: a question of concentration in both cases (EP versus Inc) Electroporated cells Incubated cells Apoptotic cells Necrotic cells Apoptotic cells Necrotic cells 20 Incubation time : 24 hours Incubation time : 24 hours 80 Apoptotic cells (%) mm 1 mm LDH release (%) Apoptotic cells (%) mM 4mM LDH release (%) mm mm mg/l mg/l gentamicin extracellular concentration gentamicin extracellular concentration

37 How cytosolic induces apoptotic signalling? Electroporation of Low amount of cytosolic Intrinsic pathway about times than those needed in incubated cells (10-15 mg/l) Cytosolic apoptotic target? Bax Ψ m Cytochrome c Mitochondria Bax PTP Cytochrome c Apoptosome Nucleus D caspase 3 (6-7) caspase 9

38 PIII.3 Increase in Bax content after incubation or after electroporation of in LLC-PK1 cells Electroporated cells Incubated cells Bax 21kDa OD (% of control OD) mm mg/l Incubation time : 8 hours

39 Increase in Bax cellular content Bax Ubiquitinated Bax 3. Decrease degradation Increase transcription 1. Inhibitors proteasome Proteasomal degradation Increase traduction 2. Bax

40 PIII.4. Increase of ubiquitinated Bax content after incubation or after electroporation of in LLC-PK1 cells IP: Bax IB: Ubiquitinated-protein Electroporated cells Incubated cells Ubiquitinated Bax OD (% of control OD) OD (% of control OD) gentamicin extracellular concentration 0 2 mm mg/l Incubation tme : 8 hours

41 4.ERAL CONCLUSION AND PERSPECTIVES (1) Apical membrane Lumen- urine Endocytic Route Golgi 8h Bax Lysosomal destabilization Mitochondria 2h 10h Endoplasmic reticulum Cyt c 12h 24h Nuclear fragmentation Proteasome Nucleus Caspase-3 24h Basolateral membrane Blood

42 4. ERAL CONCLUSION AND PERSPECTIVES (2) Lipsky et al., 1982 Cronin et al., 1982 Schwertz et al., 1984 Levi et al., 1990 Apical membrane Lumen- urine Kozek et al., 1974 Houghton et al., 1976 Watanabe et al., 1978 Silverblatt et al., 1979 Hostetler et al., 1982 Endocytic Route Laurent et al., 1982 Viotte et al., 1982 Fillastre et al., 1983 Golgi Lysosomes Powell et al., 1983 Williams et al., 1985 Chatterjee et al., 1987 Mingeot-Leclercq et al., 1988 Vera-Roman et al., 1975 Mingeot-Leclercq et al., 1990a,b Bennett et al., 1988 Okuda et al., 1992 Sandoval et al., 1998 Endoplasmic reticulum Mingeot-Leclercq et al.,1991 Sundin et al., 2001 Vera-Roman et al., 1975 Mitochondria Walker et al., 1987 Rustenbeck et al., 1998 Nucleus Proteasome Basolateral membrane Blood

43 4.1 Short term perspectives Lysosomal destabilization Cytosolic Mitochondria permeabilization Ability of to destabilize directly mitochondria Mather and Rottenberg, 2001 Proteasome What about interaction with proteasomal precursor protein PSB-9 Horibe et al., 2004 Endoplasmic reticulum What about their ribosomal target? Ryu et al., 2001 Ryu and Rando, 2002 Apoptotis

44 4.2. Long term perspectives Streptomycin Kanamycin Amikacin isepamicin «New aminoglycosides» Netilmicin Neomycin Tobramycin Gentamicin isolated from Micromonospora purpurea Based on the identification of the exact interactions between AG with the bacterial ribosome New LESS TOXIC aminoglycosides

45 And finally Lysosomes. Shield? Weapons?.in gentamicin-induced apoptosis?

46 Acknowledgment - Pr M-P Mingeot-Leclercq, Pr P-M Tulkens and Dr F. Van Bambeke - Pr Devuyst, Pr Feron, Pr Hermans and Pr Renaud -PrOrtiz andprde Broe - Dr P. Van der Smissen and Pr Courtoy - Dr P. Jacquemin -Dr C. Dax - Dr Y. Jossin and Pr Goffinet - E. Delbecq, N. Duarte, G. Thirion, G Van der Essen. - N. Aguilera, M-C. Cambier, F. Renoird, M. Vergauwen - N. Mesaros and Dr N. Caceres - all the new and old «FACMists» -