Il ruolo del PTP in apoptosi/necrosi

Some characteristics common to several diseases:

Oxidative damageChanges in Calcium homeostasisLoss of energy production Cell death

Tissue and organ dysfunction

Disease

The Mitochondrial connection.

Mitochondria are key integrators of cellular signals and stress: make life and death decisions

Apoptosis and necrosis are facilitated at the level of the mitochondria by the opening of large pores (excluding extrinsic apoptotic pathway)

c c

cc

c c

mPTPBax/Bcl2 Swelling, rupture

Release of proapoptotic factors

IMM

OMM

• Mitochondrial Permeability transition (mPT) characterized by:

• Loss of inner membrane potential;

• Release of Cytochrome C;

• Cell death by apoptosis or necrosis;

• Cessation of mitochondrial respiration;

• Release of mitochondrial Ca 2+;

• Mitochondrial swelling (rupture)

• The mPT can be caused by several agents/mechanisms (e.g. ROS, Ca2+ overload);

• The mPT, and as a consequence, mitochondrial dysfunction, is associated with the onset and progression of several diseases.

ANT: Adenine nucleotide transporter, VDAC: voltage dependent anion channel, HK: Hexokinase; CyD: Cyclophilin D, CK: Creatine kinase, PBR: Periferal benzodiazepin receptor

The Mitochondrial Permeability Transition.

Recenti sviluppi nella identificazione delle componenti del PTP

La ATP sintetasi, maggiore produttore cellulare di ATP nella cellula, è stata recentemente proposta come l’unico componente molecolare necessario per la formazione del PTP: questo dato è ancora da confermare in maniera definitiva, ma enfatizza la funzione duale dei componenti della catena respiratoria nel controllo di apoptosi e necrosi (che ritroveremo con il citocromo C)

Consequences of mPTP opening

mitochondria swell and outer membrane rupture

Ca++ andprotein release

(eg. Cytochrome c)

mPTP openingsolutes

across mPTPenter mitochondria

mitochondria swell and outer membrane

ruptures

stimulus

loss of membranepotential/energy

production

Cell death

• Opening of mPTP has been implicated in the aetiology and progression of several diseases including :

• Neurodegenerative diseases (Parkinsons, Alzheimers, MS, Huntingtons, ALS)

• Ischemia / reperfusion injury (AMI, Stroke, organ transplantation)

• Dystrophies (Bethlem, Ullrich, Duchene)

• Diabetic Complications (retinopathy, nephropathy)

• Oxidative damage, irregular calcium signalling and mitochondrial dysfunction is common to all of these diseases

The mPTP and Disease.

Take a closer look at Ischemia / reperfusion injury

(from a Mitochondrial standpoint only)

• Heart ischemia leads to infarct size of > 70%.

• Reperfusion of tissue can reduce infarct size.

• Paradoxically reperfusion also leads to tissue death and contributes to infarct size (LRI).

• Methods to reduce lethal reperfusion injury should have clinical benefit

Lethal reperfusion Injury.

PTP in Ischemia / Reperfusion Injury

What’s happening in the the cell and mitochondria:

Ca2+

Calcium enters mitochondria via the Calcium Uniporter (not saturated at High [Ca2+])

Ca2+ Uniporter

Ca2+

Normal conditions

Calcium is pumped out of the mitochondria via the Sodium / Calcium Antiporter (saturated at High [Ca2+])

Na/Ca2+ Antiporter

Mitochondria Cell

Under conditions of high cytoplasmic [Ca2+] the mitochondria can overload with calcium and this can cause the mPT.

PTP in Ischemia / Reperfusion Injury

Ischemic conditions

Mitochondria Cell

Loss of ATP production via respiration; increase in lactic acid and a drop in cellular pH.

pH [H+]

H+

H+

H+

H+

H+

H+

Mitochondria Cell

To counter the high [H+] the cell uses the Na+/H+ Antiporter. Due to lack of ATP the Na+ can not be pumped out and the cell loads with Sodium.

Na+/H+ Antiporter

Na+

Na+

Na+Na+

Na+Na+

Na+

Na+

Na+

H+

Ischemic conditions

Mitochondria Cell

High intracellular [Na+] causes reversal of the Na+/Ca2+ Antiporter and the cell loads with Ca2+.

Na+

Na+/Ca2+ Antiporter

Ischemic conditions

Ca2+

Ca2+Ca2+

Ca2+Ca2+

Ca2+

Ca2+Ca2+

Ca2+Ca2+

Ca2+

Cell

Restart to respiration. Large burst of ROS production. Mitochondria load with calcium via uniporter and saturate the Na+/Ca2+ antiporter.

Reoxygenation conditions

Ca2+

Ca2+

Ca2+ Uniporter

Na/Ca2+ Antiporter

Ca2+

Ca2+

Ca2+Ca2+ Ca2+

Mitochondria Mitochondria Cell

Calcium overload and oxidative damage from ROS production induce opening of the mPTP and mitochondrial dysfunction.Cell death via necrosis/apoptosis depending on damage.

Reoxygenation conditions

mPTP

Ca2+

Ca2+

Ca2+

CytC

CytC

CytC

Cell DeathNecrosisApoptosis

Mitochondria

Analisi di topi KO per la ciclofilina D

I topi KO hanno un difetto nell’apertura del PTP, ma sono normali

Apertura del PTP in mitocondri isolati

Cellule PTP -/- sono resistenti alla morte cellulare indotta da ROS (H2O2)

I topi KO sono resistenti al danno cardiaco indotto da ischemia-riperfusione

New cardioprotective strategies.

From Yellon and Hausenloy (2007 NEJM)

mPTP inhibitors could be a promising strategy for lethal reperfusion injury.

Inibitori del PTP (CsA) sono anche efficaci nella riduzione del danno post-ischemico dell’Uomo

Marker sierico Area infarto (NMR)

The mPTP is involved in the pathogenesis of several aging-associated diseases

• Studies using CsA and Ppif-/- mice (cyD null) have highlighted a potential role for the mPTP in the progression of several diseases.

Ppif -/- mice and CsA

Stroke

Korde et al, 2007

Coronary Artery Bypass GraftHeart Disease

Transplantation and Surgery

Reperfusion Injury

Alzheimer’s Disease ALS

Neurodegenerative Disease

Duchenne MD

Millay et al, 2008

Dystrophies

Diabetic Retinopathy HyperglycemiaDoxorubicin toxicity Platelet activationTraumatic brain injuryNon-alcoholic fatty liver disease

Other

Myocardial infarction

Piot et al 2008

Ullrich / Belthem

Du et al, 2009 Keep et al, 2001

Palma et al, 2009

L’identificazione di nuovi farmaci inibitori dell’apertura del PTP

A large network of industry/academia interactions

Padua:P BernardiF Di Lisa

Milan:CONGENIAPG PelicciR LatiniNikemNMS

HamburgOxford:EVOTECLyon:

M Ovize

Ca2+ 150 μM

1 mM CsA

ControlAbs

orba

nce

(A 5

40 n

m)

Time (mins)0 5 10

Innovative HTS

• A high pulse of Ca2+ (ca. 150 mM) given to purified mitochondria will cause mPTP opening and induce mitochondrial swelling (DA540nm).

• A Ca2+-induced swelling assay was developed suitable for HTS• Fresh Rat liver mitochondria were prepared daily and used in this assay

to screen >300,000 compounds • Counterscreens were run to elimate compounds that interfere with mitochondrial respiration.• Several chemical classes were identified

•Organelle based•Requiring the use of animals->liver mitochondria

unprecedented at this scale

Adaptation of mitochondrial assays to 1536 well format

• Parameters to optimize:• density / fitness of mitochondria

• concentration of stimulus (Ca++)

• assay sensitivity (CysA)

• reagents volumes / order of addition

• reagents incubation times

• reagents dispensing devices

• readout kinetics / stability of assay signal

• DMSO-sensitivity

• reader settings (i.e. OD filter sets)

Results of time-course experiment in 1536 well plates:

4 Fitzones Z‘: 0,74 / 0,76 / 0,70 / 0,61

Mitochondria: 1 mg/ml

Calcium: 300 µM

Incubation: 10 min

Cyclosporin A: 1 µM

HTS-Hardware in Operations

2 EVOscreen® Mark II: biochemical assays at 1 µL level 1 EVOscreen® Mark III: biochemical & cellular assays at 1-10 µL level

Class 6 compounds inhibit the mPT induced by:- Calcium overload- Oxidative damage- Protein crosslinkers- Respiration uncouplers

300μM Diamide,

40μM Ca2+, pH 7.2,

100μM Menadione

40μM Ca2+, pH 7.4,

50nM FCCP

40μM Ca2+, pH 7.4,

A 5

40 n

m

Time (mins)

Treated

GNX-A

Treated

GNX-A

Assay performed on prepared mouse hepatic mitochondria

Ab

sorb

ance

(A

540

nm

)

Time (mins)

Ca2+ 150μM, pH 7.4

Treated

GNX-A

Untreated

A 5

40 n

m

Time (mins)

Treated

GNX-A

A 5

40 n

m

Time (mins)

UntreatedUntreated Untreated

PTPi block mPTP openingby several stimuli

State 3 inhibition (with ADP)

[GNX-C] (mM)

% m

ax in

hib

ition

-30-20-10

0102030405060708090

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

State 4 inhibition

[GNX-C] (mM)

% m

ax in

hib

ition

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21-20-10

0102030405060708090

100

0 1 2

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

0 5 10 15 20 25

-10.0

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

State 3 inhibition (with ADP)

[GNX-B] (mM)

% m

ax in

hib

ition

State 4 inhibition

[GNX-B] (mM)

% m

ax in

hib

ition

PTPi do not have significant effects on mitochondrial respiration

The Calcium Retention Capacity (CRC) of purified mouse liver mitochondria is determined by measuring the point at which pulse-loaded calcium is released from the mitochondria. After the addition of a pulse of calcium (10 mM) the extramitochondrial fluorescence increases. As the mitochondria take-up the calcium the fluorescence signal decreases. Calcium is continually loaded into the mitochondria until there is a sudden, large, increase in calcium fluorescence which indicates complete release of the stored calcium due to opening of the mPTP. Inhibition of the mPTP, with Cyclosporin A or our proprietary inhibitors can increase the capacity of mitochondria to retain calcium and thus protect the mitochondria from calcium overload in stress situations. Extramitochondrial calcium is measured by the fluorescence of calcium green. Note, increasing the concentration of CsA does not increase the CRC due to saturation of its target (Cyclophilin D) and is one of its limitations.

Control CsA (1μM)GNX-B (0.1mM)

Ca2+ Pulses (10 mM)

Ext

ra-m

itoc

hond

rial

Cal

cium

flu

ores

cenc

e

Ca2+ Release mPTP open

GNX-B (1.0mM) GNX-B (5.0mM)

40 mM 120 mM 250 mM180 mM 380 mM

Time (min)

CR

C (m

M C

a2+)

GNX C

0.1mM

0.5mM

1.0mM

5.0mM

0.1mM

0.5mM

1.0mM

5.0mM

0

50

100

150

200

250

300

350

CsA

Control

The identified PTP inhibitors are >> than CsA

Class 6 compounds are able to increase the CRC (by mPTP inhibition) several fold over that of unprotected mitochondria.

Class 6 compounds increase the CRC significantly over that maximally attainable with CsA.

PTP inhibitors for Acute Myocardial Infarction

The highway to proof-of-concept:•unmet medical need•strong evidence for involvment of the mPTP•linear path for the design of a clinical study

Area at Risk (g)

Arae

Nec

rotic

(g)

vehicle A-B

GNX -B

CsA

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.2 0.4 0.6 0.8 1.0 1.2 1.4

No protection

Protection

.

. ..

. ..

. .

..

.

.. .

. ..

. ..

PTP inhibitors are cardioprotectivein animal models

New Zealand White Rabbits were subjected to Left Anterior Descending (LAD) coronary artery occlusion for 30 mins followed by 4 hrs of reperfusion. Area at Risk and infarcted area (area necrotic) was determined by Evans blue and TTC staining. GNX-B (15 mg /kg in 3 mls 40% PEG 400; 20% DMSO) and CsA (10 mg/kg in sandimune) were administered by i.v. bolus 5 mins prior to reperfusion. Note, as with CsA in the clinical proof of concept trial, there is greater protection when the area at risk is larger. (In collaboration with Prof Ovize, Ospice di Lyon)

Un ruolo inaspettato per p53 nell’induzione di necrosi

Dopo stress, p53 si trova localizzata insieme con la ciclofilina-D ai mitocondri

P53 è richiesta per l’apertura del PTP durante la necrosi da stress ossidativo

Viene misurata l’apertura del PTP

Lo stress ossidativo induce necrosi e non apoptosi che dipende da p53

Considerazioni

• P53 è un regolatore fondamentale dell’apoptosi, ma sembra giocare un ruolo altrettanto rilevante nella morte cellulare da necrosi dopo stress ossidativo– P53 è “good” e “bad”: in questo caso potrebbe essere

coinvolto nella patogenesi di molte malattie• Il coinvolgimento di p53 è un’altra dimostrazione

che anche la necrosi è sottoposta (almeno in alcuni casi) a un livello di regolazione impensabile fino a poco tempo fa