MitochondrialMitochondrial stressstress signalingsignaling-- reactivereactive oxygenoxygen speciesspecies as as signalingsignaling moleculesmolecules

2007–2013 m. Žmogiškųjų išteklių plėtros veiksmų programos 3 prioriteto „Tyrėjų gebėjimų stiprinimas“ VP1-3.1-ŠMM-05-K priemonės „MTTP tematinių tinklų, asociacijų veiklos stiprinimas“projektas „Lietuvos Biochemikų draugijos potencialo kurti žinių visuomenę didinimas“

(NR. VP1-3.1-ŠMM-05-K-01-022)

Laboratory of Bioenergetics and Biomembranes,Nencki Institute of Experimental Biology,

Polish Academy of Sciences

Dominika Malińska

Role of mitochondria in the cell

O2.-

H2O2

[Ca2+]cADP / ATP

∆∆∆∆ΨΨΨΨH+

Ca2+

ATP

Krebs cycle

Mitochondria in regulation of cytosolic calcium fluxes

- ATP supply for Ca2+ pumps in PM and ER

- special contact sites between mitochondrial membranes and PM or ER

Role of mitochondria in the cell

O2.-

H2O2

[Ca2+]cADP / ATP

∆∆∆∆ΨΨΨΨ

Ca2+-dependent signaling

redox signaling

oxidative stress

Ca2+

ATP

Krebs cycle

cell death

Regulation of mitochondrial function

ADP / ATP

∆∆∆∆ΨΨΨΨ

- ATP demand of the cell, intensity of ATP-consuming processes

- substrate supply

ATP

Krebs cycle

[Ca2+]cADP / ATP

∆∆∆∆ΨΨΨΨ

Influence of mitochondrial calcium uptake on respiratory chain functioning

Regulation of mitochondrial function

Ca2+

ATP

Krebs cycle

O2.-

H2O2

Mitochondrial retrograde signaling - feedback information to nucleus about functional state of mitochondria

Cellular response to mitochondrial stress

Regulation of mitochondrial function

Ca2+

O2

ATP

Krebs cycle

[Ca2+]cADP / ATP

∆∆∆∆ΨΨΨΨ

O2.-

Mitochondrial retrograde signalingcell nucleus

calcineurin A,CaMKAMPK

PGC1αNRF2NRF1

HIF1α

SIRT1

succ

inat

e,

oxo

-glu

tara

te

H2O2

↑ expression of:- respiratory chain

Ca2+

O2

ATP

Krebs cycle

[Ca2+]c

succ

inat

e,

2-o

xo

ADP / ATP↑ glycolysis,

↓OxPhos

- respiratory chainproteins- enzymes related to fatty acid oxidation- mitochondrialantioxidant defenceproteins (SOD2, mGPx)

∆∆∆∆ΨΨΨΨ

Cellular response to mitochondrial dysfunction

Osteosarcoma cells

- wild type

- cybrid cells with 98% NARP mutation (ATP synthase, subunit A6)

- rho0 cells (mitoDNA depletion)

http://www.columbiamitodiagnostics.org

Cellular response to mitochondrial dysfunction

Osteosarcoma cells

- wild type

- cybrid cells with 98% NARP mutation (ATP synthase, subunit A6)

- rho0 cells (mitoDNA depletion)

WT NARP Rho0

NARP Rho0

∆ψ

[Ca2+]c

[O2.-]m

↓

Cellular response to mitochondrial dysfunction

- mitochondrial membrane potential

Flu

ores

cenc

e [%

]

[O2.-]c

Szczepanowska et al. (2004) FEBS Lett. 578: 316-322

NARP Rho0

∆ψ

[Ca2+]c

[O2.-]m

↑

↓

↑↑

Cellular response to mitochondrial dysfunction

WT NARP Rho0

+thapsigargin

(sel

ectiv

e in

hibi

tor

of C

a2+

AT

Pas

e in

the

ER

mem

bran

e)

- cytosolic calcium levels

[O2.-]c

Flu

ores

cenc

e [%

]

+thapsigargin

(sel

ectiv

e in

hibi

tor

of C

aA

TP

ase

in th

e E

R m

embr

ane)

Szczepanowska et al. [unpublished observations]

NARP Rho0

∆ψ

[Ca2+]c

[O2.-]m

↑

↑

↓

↑↑

↓

Cellular response to mitochondrial dysfunction

- intracellular superoxide concentration

[O2.-]c ↑

Wojewoda et al. (2010) Biochim Biophys Acta 1797: 890-896

Cellular response to mitochondrial dysfunction

O2.-

ADP / ATP

mitochondrial dysfunction → changes in intracellular levels of

ATP, ROS and Ca2+ → compensatory response of the cell

cellular dysfunctionprevention of cell injury

Ca2+

O2

ATP

Krebs cycle

[Ca2+]cADP / ATP

∆∆∆∆ΨΨΨΨ

ROS synthesis in mitochondria

oxidative stress

cell death

redox signaling

proliferation

differentiation

cytoprotection

ROS

ROS synthesis in mitochondria

EXPERIMENTAL METHODS:

skel

etal

mu

scle

m

ito

cho

nd

ria

ROS synthesis in mitochondria

- comparison of different tissues

bra

in

mit

och

on

dri

ask

elet

al m

usc

le

mit

och

on

dri

a

Malinska et al. (2009) Meth Enz 456: 419-437

proliferating C2C12 myoblasts

mouse skeletal muscle homogenates

ROS synthesis in mitochondria

- changes during cellular differentiation process

C2C12 cells differentiated to myotubes

brain mitochondria redox titration of CoQ

- mouse and rat brain SMP

ROS synthesis in mitochondria

III

III IVQ

succ anti Amalon

Malinska et al. (2010) Biochim Biophys Acta 1797: 1163-1170

Influence of Ca2+ on mitochondrial ROS synthesis

brain mitochondria

+

-CaU

Ca2+

III

III IVQ

succ anti A

Ru 360

Malinska et al. (2010) Biochim Biophys Acta 1797: 1163-1170

brain mitochondria

ROS generation membrane potential

Influence of Ca2+ on mitochondrial ROS synthesis

+

-CaU

Ca2+

III

III IVQ

succ anti A

Ru 360

brain mitochondria

ROS generation membrane potential

Influence of Ca2+ on mitochondrial ROS synthesis

- Ru 360 + Ru 360

succ (RET to complex I)

succ + anti A(complex III)

∆Ψ∆Ψ∆Ψ∆Ψ

TTFB

brain mitochondria

Influence of Ca2+ on mitochondrial ROS synthesis

- Ru 360 + Ru 360

Ca2+

succ (RET to complex I)

succ + anti A(complex III)

∆Ψ∆Ψ∆Ψ∆Ψ

Influence of Ca2+ on mitochondrial ROS synthesis

complex I- and complex III- dependent superoxide synthesis differ in:

- sensitivity to changes in ∆pH and ∆ψ

- intracellular compartment to which they release O2.-

- dependence on respiratory chain redox state

Summary

∆ΨΨΨΨATP

synthesisrespiratory

chain

mitochondrialCa2+ uptake

mitochondrialROS synthesis

Krebs cycle

cytosolicCa2+ levels

intracellularATP/ ADP

intracellularROS

cell functioning retrograde signaling

Acknowledgements

Nencki Institute of Experimental Biology:

Laboratory of Bioenergetics and Biomembranes - prof. Jerzy Duszyński

Laboratory of Intracellular Ion Channels - prof. Adam Szewczyk

Bonn University

Department of Epileptology – prof. Wolfram S. Kunz

Laboratory of Bioenergetics and Biomembranes

Jerzy Duszyński, Lech Wojtczak,Joanna Szczepanowska,Marta Wojewoda,Mariusz Więckowski,Magdalena Lebiedzińska,Jan Suski,Dominika MalińskaMałgorzata Bejtka,Violetta Biernat

research techniques:- isolation of mitochondria and mitochondria-associated membranes- analysis of respiratory chain activity in isolated organelles and in whole cells (colorimetric activity determination after Blue Native gel electrophoresis; respirometric analysis)- visualisation of intracellular structures with laser scanning cytometry and confocal microscopy- fluorescent measurements of intracellular ROS levels, Ca2+ fluxes, Ca2+ levels in different organelles, mitochondrial membrane potential