Chiarugi mitocon 2011

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Chiarugi mitocon 2011

  1. 1. Modulazione farmacologica dellomeostasi del NAD mitocondriale: implicazioni terapeutiche Strategie teraputiche Alberto Chiarugi I° Convegno Nazionale sulle Malattie Mitocondriali Roma, 21-22 Maggio 2011
  2. 2. 4 Nobel nella “storia del NAD”
  3. 3. Destino metabolico del NAD
  4. 4. Reazioni metaboliche che distruggono e riformano il NAD Tryptophan Kynurenine De novo pathway pathway NAADP NADPcADPR NADases NADK MARTs NAD PARPs PAR PARG ADPR ADPRPP NMNAT Sirtuins Nam ATP AADPR Rescue NPRT pathway ATP NMN
  5. 5. Alcuni ruoli metabolici aggiuntivi del NAD mitocondrialeOrganizzazione dei complessi Fe/SRigenerazione del glutatione ridottoAttività della piruvato deidrogenasi
  6. 6. Origine del NAD mitocondriale: un enigma ancora irrisolto NAD NAD
  7. 7. ….nascono quindi 2 nuovi settori della farmacologia direct NAD support therapy indirect NAD depletion therapy Occorre però porre attenzione a: Equilibri omeostatici Farmacocinetica
  8. 8. Effetti della deplezione di NAD cellulare sulla funzione mitocondriale
  9. 9. Effetti del blocco della resintesi del NAD sulla concentrazioni di NAD ATP Nam FK866 ARTSIRT1-7 Nampt CD38PARPs HeLa 110NAD NMN C6 100 MT2 (% of control) 90 Fibroblasts NMNAT NAD ATP 80 Glia 70 Neurons 60 C 1 10 100 µM, FK 866 Pittelli M. et al, JBC 2010
  10. 10. Ma la deplezione di NAD non avviene nei mitocondri 150 Cytosol Mitocondri Cell autofluorescence (NADH) 120 C FK100µM/1h FK100nM/24h% of control 90 ** NAD 60 30 0 C FK C FK 400 FK866 (arbitrary units) *** 300 *** ATP 200 * * 100 0 C PYR PYR C PYR PYR ADP ADP
  11. 11. Nampt is localized in citosol and not in mitochondria of HeLa cells TMRE Merge cytGFP Nampt TMRENamptGFP MitGFP TMRE MitGFP Pittelli M. et al, JBC 2010
  12. 12. Effetti del NAD esogeno sulla funzione mitocondriale
  13. 13. Effetti del NAD esogeno sulla funzione mitocondriale 400 10 mitochondria 350 300 8 **% of intracellular NAD nmol NAD/ mg prot 1µM 250 10µM 6 200 100µM 150 1mM 4 nuclei 100 1mM 4°C 2 * 50 0 0 0 1 2 6 12 C NAD C NAD hrs 250 Mit-Lucif CRL NAD 1mM (6h) ** 200 Cyt-Lucif Photon emission % of control 150 * 100 50 ** 0 ** C 0 5 C 0 5 h NAD 1mM NAD 1mM
  14. 14. Effetti del NAD esogeno sul consumo di ossigeno A B 235.0 4 230.0 ** Oxygen consumption ratenmol oxygen/ml 225.0 3 (nmol/ml/min) 220.0 2 215.0 210.0 Control 1 205.0 NAD 1mM 0 0 2 4 6 8 10 CTRL NAD min
  15. 15. Effetti del NAD esogeno sulla sopravvivenza cellulare CTRL STP STP + NAD 1 mM
  16. 16. Ruolo dell’enzima mitocondriale NMNAT3 NMN + ATP NAD + PPiTotal NAD+ cellular content in HEK293 shNMNAT3 stable cell line Total ATP cellular content in HEK 293 shNMNAT3 stable cell line (3 exp in duplicate) (2 exp in triplicate) 200 200 NAD+ (ABS/mg prot.) 150 ** 150 * ATP/mg prot. 100 100 50 50 0 0 D T3 LE D T3 A LE N B A M M N B N A M M sh R N A SC sh R SC sh sh NAD tot NMNAT3-flag (var.2) overexpression 48 h Total ATP cellular content overexpression NMNAT3-flag 48h HEK293 (1 exp. in duplicate) HEK 293 (1 exp in triplicate) 3 4.0×10 7 NAD+ (ABS/mg prot.) 3.0×10 7 ATP/mg prot. 2 ** 2.0×10 7 1 1.0×10 7 0 0 ol l g + tro tr a T3 -fl on on T3 A C N C A M N N M N
  17. 17. Strategie teraputiche
  18. 18. Potenziali precursori del NADAcido NicotinicoNicotinamideNicotinamide ribosideNicotinamide adenin mononucleotideIntermedi della via delle chinurenine
  19. 19. Potenziale terapeutico dei precursori del NAD
  20. 20. Conclusioni
  21. 21. Ringraziamenti Fondazione Giuseppe Tomasello Roberta FeliciLaura Formentini Elisa LanducciFrancesco Blasi Tania ScartabelliMirko Muzzi Elisabetta GeraceGiuseppe Faraco Alessio MasiAndrea Lapucci Maria SiliDaniela Buonvicino Andrea CozziLeonardo Cavone Vincenzo Carlà
  22. 22. PARP-1 and necrosis (the suicide hypothesis) DNA damage glucose NMN ADP ATP NMNAT PRT GAP ATP NADH NAD NA +PRPP 1,3-DPG NAD ATP pyruvate PARP1↓∆Ψ NADH I H+ II III IV NAD ADP H+ ATP CELL NECROSIS
  23. 23. NAD e NAMPT e degenerazione assonale
  24. 24. NAD e mitocondri concetto di saturazioneCRL NAD 1mM (6h) NADH (autofluorescenza cellulare)
  25. 25. A key role of PARP-1 in epigenetic chromatin remodeling
  26. 26. Poly(ADP-ribose) polymerase-1 (PARP-1) in nucleus-mitochondrial cross-talk under homeostatic or pathogenetic conditions Homeostatic role I III IV 3 mPARP1 BER I II 4 ROS Ca2+ III ANT IV IP3 Ca2+ I 1 II III IV ↓ ATP PLC Ca2+ 2 ↓ NAD GPR nPARP1 5 nPARP1 6 Detrimental role1 - nPARP-1 regulates nuclear respiratory chain gene expression2 - nPARP-1 regulates mitochondrial DNA repair gene expression3 - mPARP-1 regulates mitochondrial respiratory chain gene expression4 - mPARP-1 assists mitochondrial genome repair5 - mitochondrial ROS hyperactivate nuclear PARP-1 and prompt energy failure6 – mitochondrial ROS hyperactivate nuclear PARP-1 and alter epigenetic regulation
  27. 27. The DNA alkylating agent Methyl-Nitro-Nitrosoguanidine (MNNG) induces PARP-1 hyperactivation and a long-lasting depletion of NAD and ATP in HeLa cells O Working model O NH NH 2 MNNG Wash Benzamide (BZD) Phenanthridinone (PHE) IC50 = 30µM IC50 = 3µM 0 1h 100 PHE 100 PHE BZD BZD MNNG 100µM % of inhibition NAD depletion % of inhibition ATP depletion 110 75 75 100 50 50 90 NAD 80 % of control 25 25 70 ATP 0 0 60 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 50 40 30 20 10 0 0 15 30 45 60 2 4 6 8 min hrs C 5’ 10’ 15’
  28. 28. Mitochondrial membrane potential (∆Ψ) in HeLa cells undergoing PARP-1 hyperactivation Control MNNG MNNG 1h 160 * 140 MNNG 2hT M R E flu o r e s c e n c e 120 ( % o f c o n tr o l) 100 80 ** 60 ** ** Cell counts 40 MNNG 3h 20 0 0 1 2 3 4 5 6 MNNG hrs MNNG 4h TMRE fluorescence intensity
  29. 29. Poly(ADP-ribose) metabolism and catabolism 5-P ribose Phosphodiesterases ? AMP 5-P ribose ADP-ribose pyrophosphatases (NUDIX hydrolases) AMP 5-P ribose ADP-ribose PAR PARG ADPR pyrophosphorylaseNAD PARP ATP NA PPi PRT NMN NMNAT NAD ATP ATP NAD rescue pathway ATP ATP glycolysis mitochondria
  30. 30. Summary of the human Nudix genes and hydrolases From McLennan A.G., Cell. Mol. Life Sci. 63 (2006) 123–143
  31. 31. Inverted ADP/AMP ratio underlies PARP-1-dependent energy failure Control PARP-1 hyperactivation AMP ADPADP AMP ADP ADP = 6.3±0.9 = 0.35±0.05 AMP AMP
  32. 32. AMP reduces ADP uptake and ATP output from isolated mitochondria The Adenine Nucleotide Translocator ATP ATP MATRIX ADP ADP INTERMEMBRANE SPACE
  33. 33. Amino acid residues involved in the orientation and molecular constraint of ADP during its journey down to the ANT cavity - Non fix anti conformation Transported - NH in C6 Nucleotides - No sobstistution in C2 - 2 or 3 PO4 groups 125Ser227 Ser227 Arg235 Arg235 100 ADP Lys32 AMP Lys32 ( % o f c o n t r o l) 75 C PM A 50Gly224 Gly224 Arg137 Arg137 25 Glu29 Glu29 0 CRL ATR 0 1 10 100 1000 Arg279 Lys22 Arg279 Lys22 [AMP] µM
  34. 34. Conformational Energy Evaluation of ADP or AMP bound to ANT ADP AMPBioactive Conformation (ΔE) = +7.60 kcal/mole Bioactive Conformation (ΔE) = +3.25 kcal/mole
  35. 35. Conclusion: The Nudix Hypothesis AMP PARP NAD PAR ADP AK ATP NAD ANT rescue AK AK AK ADP AK s m oli e tab m ATP glycolysis EVOLUTIONARY IMPLICATIONSPARP-1-dependent energy failure helps disclosing some of the ancestral strategies adopted by eukaryotic cells to preserve bioenergetic exchanges with the protomitochondrion

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