Luca Scorrano, Convegno Mitocon 2015

Luca Scorrano

Keeping mitochondria in shape: a matter of life or death

Scorrano et al Dev. Cell 2002

Palade, 1953

The current model of mitochondrial ultrastructure

1. Any proposed mechanism for cytochrome c release should account

for the very complex ultrastructure of the organelle and for the

submitochondrial compartimentalization of cytochrome c

2. Mitochondria are not “isolated” organelles. The fact that they can

take up Ca2+ released by the neighboring ER highlights the intimate

connection between these two organelles, raising the question of

whether their cross-talk can control cell physiology and pathology

3. Ultimately, what is the role of mitochondria-shaping proteins in cell

physiology? In other words, what is the functional outcome of

mitochondrial shape regulation in health and disease, with a

particular outlook on autophagy?

Outstanding questions in my lab

• OPA1 is a dynamin-related protein located in the IMM, facing the IMS

• OPA1 not only promotes fusion, but it has an additional functions in the control of apoptosis, keeping in check the cristae remodelling pathway and in mitocondrial metabolism by favoring the assembly and stability of supercomplexes

HSHS GTPaseGTPase MiddleMiddle GEDGEDMTSMTS

Cipolat et al., Cell 2006; Frezza et al., Cell 2006

Mitochondria-shaping proteins have genetically distinguishable functions in morphology, apoptosis, organelle tethering and mitochondrial physiology

Cipolat et al., Cell 2006; Frezza et al., Cell 2006

Mitochondria-shaping proteins have genetically distinguishable functions in morphology, apoptosis, organelle tethering and mitochondrial physiology

Respiratory chain complexes

OPA1Respiratory ChainSupercomplexes

Cogliati et al, Cell 2013

MitCare-2:Treat cristae to

treat mitochondrial

diseases

Scorranomanipulate

cristae

Bernardi/Rizzuto

PT, Ca2+ and cristae

SalviatiCoQ and cristae

CarelliLHON, cristae

and ROS

MitCare-2: a Telethon funded research project to tackle mitochondrial diseases

Primary OXPHOS

deficiency

Cristae alteration

Ca2+ overload PTP opening

CoQ deficienc

y

ROS production

Mitochondrial disease

MitCare-2: a Telethon funded research project to tackle mitochondrial diseases

Generation of an Opa1 overexpressing mouse

HPRT :human hypoxanthine phosphoribosyltransferase; mouse PRT locus lies on Xq26.1

4 CCmts TMOPA1-isoform 1

h b-actin promoter OPA1 pA H P R

Targeting vector

P R T ES cell

Selection and microinjection of ES clones

OPA1 overexpressing mouse

h b-actin promoter OPA1 H P R TpA

OPA1 is mildly overexpressed in various tissues from Opa1tg animals

OPA1

GRP75

HeartLiver MuscleW

t

Opa

1tg

Wt

Opa

1tg

Wt

Opa

1tg

Brain

Wt

Opa

1tg

Wt

Opa

1tg

Cerebellum

Opa1tg mice are viable and grow normally Males Females

10 20 30 40 50 60 70

4

6

8

10

12

14

16

18

20

22

24 W

eigh

t (gr

)

Time (d)

10 20 30 40 50 60 70

4

6

8

10

12

14

16

18

20

22

Wei

ght (

gr)

Time (d)

Wt

Opa1tg

5

10

15

20

25

30

35

*

**

Wt Opa1tg

Bod

y w

eigh

t (g

)*

5 months 9 months

Wt

Cardiac fibrosis and mitochondrial ultrastructural changes are absent in 18-months old Opa1tg mice

Opa1tgOpa1tgWt

Denervation-induced atrophy

5

40

60

80

100

120

Sham 10 d. Den.

Cro

ss-s

ecti

on

al a

rea(

%)

WtOpa1tg

n=5 n=5

*

mean ±SEM (*p˂ 0.001 ANOVA)

Opa1tg gastrocnemius is protected from denervation-induced atrophy

Wt tg

Den

.C

ontr

o l Opa1tg Wt

Fox

O1

con

Fox

O1

den

Fox

O3

con

Fox

O3

den

Fox

O4

con

Fox

O4

den

Act

RIIB

con

Act

RIIB

den

AIk

3 co

nA

Ik3

den

Myo

stat

in c

onM

yost

atin

den

0

1

2

3

4

5

6 Wt

Opa1tg

Atrop

hy rel

ated

gen

es (fo

ld in

duct

ion)

*

LC3

con

LC3

den

p62

con

p62

den

Bni

p3 c

on

Bni

p3 d

en

Mul

1 co

n

Mul

1 de

n

Cat

hL c

on

Cat

hL d

en

0

1

2

3

4

5

6

7

8

*

Wt

Opa1tg

*

Aut

opha

gic

gene

s (f

old

indu

ctio

n)

Atrophy and autophagy genes are normally induced in denervated Opa1tg gastrocnemius

Denervated Opa1tg gastrocnemius is protected from mitochondrial dysfunction

0 10 20 30 40 50 60 70

0.2

0.4

0.6

0.8

1.0

1.2

TM

RM

fluo

resc

ence

(no

rmal

ized

to in

itial

val

ue)

Time (min)

wt wt denervated

Opa1tg

Opa1tg denervated

oligomycin FCCP

GAPDH

long

short

OPA1

Wt Opa1 tg

Den

erv a

t ed

c ont

r ol

Den

erv a

t ed

c ont

r ol

• Ablation of Opa1 in the adult mouse is sufficient to cause mitochondrial degradation, UPR induction, FGF21 production and release

• This ER sensor of mitochondrial dysmorphology is essential to mount a whole body response mediate by the starvation FGF21 cytokine

• Is protection granted by Opa1 expression confined to skeletal muscular atrophy?

• Can we exploit the Opa1tg model to test the role of Opa1 in other tissues and pathological conditions?

Conclusions 2

Outstanding Questions

Ischemia 40’ Reperfusion 15’

95% O2

5% CO2

LDHCoronary effluent

Male Female

20

25

30

35

40

45

*

rele

ased

LD

H (

% o

f tot

al)

WT

Opa1tg*

mean+SEM(*p<0,05)

Male Female

20

25

30

35

40

45

rele

ased

LD

H (

% o

f tot

al)

mean+SEM(*p<0,05)

*

*

WT

Opa1tg

C57Bl/6 Sv/129

n=10n=8 n=8 n=10

Opa1tg hearts are protected from ischemia-reperfusion

Wt

Opa

1

tg

Wt

Opa

1tg

Wt

Opa

1tg

Ischemia/Reperfusion

Actin

short

longOPA1

Opa1tg brains are protected from ischemia induced by MCAo

Wt Opa1tg

Wt

Opa1tg

6

8

10

12

14

mean+SEM(*p<0,05)

Infa

rct V

olu

me (

mm

3)

*

Wt

Opa1tg

Opa1tg brains are protected from ischemia induced by MCAo

Wt Opa1tg

Opa1tg livers are protected from Fas induced apoptosis


Wt Opa1tg


Wt Opa1tg

1

2

3

4

5

6

% o

f TU

NE

L po

sitiv

e he

pato

cyte

s

Wt

Opa1tg

*

Wt Opa1tg

Opa1tg hepatocytes are protected from Fas-induced cytochrome c release

Wt

***

Opa1tg

Opa1tg hepatocytes are protected from Fas-induced cytochrome c release

0 h 24 h

500

1000

1500

2000

2500

AS

T (

U/L

)

Wt

Opa1tg

*

0 h 24 h

500

1000

1500

2000

2500

3000

3500

*

ALT

(U

/L)

Wt

Opa1tg

Opa1tg hepatocytes are protected from Fas-induced damage

Mitochondria are slightly more elongated in primary Opa1tg myoblasts

Opa1tgWt

Complex I dependent respiration is more efficient in Opa1tg mitochondria

wt Opa1tg

0.4

2

3

4

5

6

7

8

Glutamate/Malate Rotenone/Succinate

Res

pira

tory

Con

trol

ratio

R

CR

Cogliati et al, Cell 2013

Glucose Galactose0.8

0.9

1.0

1.1

1.2

1.3

Mito

SO

X F

luor

esce

nce

(A.U

.)

Wt

Opa1tg

-- +AA1.0

1.1

1.2

1.3

mito

-roG

FP

1 (4

20/4

92 n

m)

Wt

Opa1tg

Opa1tg mitochondria accumulate less ROS

• Mild Opa1 overexpression is compatible with life• The Opa1-dependent cristae remodeling pathway is

crucial for response to atrophic, apoptotic and ischemic damage in vivo

• Can we exploit Opa1 overexpression as a therapeutic tool to ameliorate mitochondrial dysfunction, for example in mouse models of mitochondrial disease?

• If we have time, why don’t we have constitutively elevated Opa1 levels?

Conclusions 1

Outstanding Questions

Opa1 stabilizes RCS, rescues life span and motor performance of a Complex IV defect mouse model

Opa1 stabilizes RCS, rescues life span and motor performance of a Complex IV defect mouse model

wt Cox15sm/sm

IV

RCS

Opa1tg

+ +

WTOpa1

tg

Cox15sm

/sm

Cox15sm

/sm Opa1tg

1

2

3

4

5

RC

R

Cox15sm/sm Cox15sm/sm::Opa1tg

• Mild Opa1 overexpression is compatible with life• The Opa1-dependent cristae remodeling pathway is

crucial for response to atrophic, apoptotic and ischemic damage in vivo

• We exploited it to successfully correct mouse models of mitochondrial diseases

• Why don’t we have constitutively high levels of Opa1?

Conclusions 1

Outstanding Question

OPA1 is overexpressed (>2 folds) in human cancers

The trade off of Opa1 overexpression is increased prevalence of cancer and reduced lifespan

• The Opa1-dependent cristae remodeling pathway is crucial in vivo

• Induced Opa1 expression represent a viable strategy to counteract heart dysfunction and to treat mitochondrial diseases

• Opa1 overexpression however in the long run leads to cancer

Take home messages

Elisa Barbieri Maya Chergova Mauro Corrado Marta GiacomelloGianmaria GuidoChristina GlytsouFrancesca GrespiStephanie HerkenneSowmya LakshminaranayanDomenico MiglioriniDeborah NaonAlice NardinLena PernasAkiko OmoriRuben Quintana Charlotte QuirinDijana SamardzicEmilie SchrepferMartina SemenzatoAnnalisa SerafiniNorihito ShintaniFabrizio SoffiatoTatiana VaranitaMarta Zaninello

Stephan Frank, University of BaselGerald Dorn, Washington UniversityTonio Enriquez, CNIC MadridAntonio Zorzano IRB BarcelonaTania Zaglia and Marco Mongillo, U. Padova Marco Sandri, VIMM, PadovaGabriele Civiletto, Carlo Viscomi, Massimo

Zeviani, MBU MRC, CambridgeElena Ziviani, University of PadovaMaria Eugenia Soriano, University of Padova

9 months5 months

Cardiomegaly in 9 months old Opa1tg mice

Wt Opa1tg

WtOpa1tg

Cardiomyocyte cross-sectional area is increased in Opa1tg heart

2040

775

800

825

850

875

900

Cro

ss s

ectio

nal a

rea

(m

)

Wt

Opa1tg

RV IVS LV

Opa1tg

Opa1tg cardiac hypertrophy is not pathological

LVRV IVSLVRV IVS

Collagen I

Wt Opa1tg

Lack of myocardial interstitial fibrosis in Opa1tg mice

Wt Opa1tg

Cardiac function is preserved in Opa1tg mice

FE FS

10

20

30

40

50

60

70 Wt

Opa1tg

%

WT

Opa1

tg

Mitochondrial ultrastructure in Opa1tg hearts

Mitochondrial Bcl-2 dependent apoptosis regulates melanocyte survival

TGFb

MITF

MSCs

c-KitSMAD2-P

PAX3

Melanocytes

MITFTRP2TYR

S100Bcl-2

Apoptosis

MBs

bcl2-/-

Korsmeyer Lab. (1993)

MITF V/V

Lerner (1986)

tgfbRII -/-

Nishimura (2010)

8.23 kB

2Intron 1UTR 1 3

FRT PGK-Neo FRT

4432 5 Intron 5

Intron 1UTR 1 3 44 5 Intron 5

5.2 kB

2Intron 1UTR 1 4432 5 Intron 5FRT PGK-Neo FRT

Selection and microinjection of ES clones

Opa1flx/flx mouse

Generation of a conditional Opa1 knockout

WT Opa1Dm/Dm

Opa1Dm/Dm display a pigmentation defect

Matrix melanin is reduced in Opa1Dm/Dm adult mice

WTOpa1Dm/Dm

10.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

WT Opa1Dm/DmAlbino

Matrix melanin is reduced in Opa1Dm/Dm adult mice

2 Weeks 1 Month 2 Months 4 Months0.00

4.00

8.00

WTOpa1Dm/DmRe

lativ

e am

ount

of M

elan

in

Melanocyte markers are lost in Opa1Dm/Dm mice

WT

Hoechest Trp2 S100

Opa1Dm/DmWT

Hoechestc-Kit

Opa1Dm/Dm

Ectopic expression of NRasQ61K in melanocytes leads to hyperpigmentation and melanoma

WT

Tyr-NrasQ61K



Opa1 ablation reverts the effect of ectopic melanocyte NRasQ61K expression

WT

Tyr-NrasQ61K

Opa1Dm/Dm Tyr-NrasQ61K

Tyr-NrasQ61K

The cristae remodeling pathway of apoptosis


The remodeled cristae


Apoptotic stimulus

Cytochrome c release

Apoptosis

Cristae remodelling

Mitochondria undergo structural changes during reversible and irreversible cell damage

fission

• Mild Opa1 overexpression is compatible with life• Increased Opa1 levels protect from age-associated

heart fibrosis and from denervation-induced muscular atrophy

• Is Opa1 ablation in the adult skeletal muscle sufficient to trigger atrophy?

•

Conclusions 1

Outstanding Question

2Intron 1UTR 1 4432 5 Intron 5FRTPGK-NeoFRT

Opa1DSM/DSM mouse

CRE ER-T2

TAM CRE

Generation of a conditional inducible skeletal muscle Opa1 knockout

Human skeletal actin (HSA)

Inducible Opa1 skeletal muscle ablation causes muscle atrophy and massive weight loss

Wei

ght (

%)

Days

WTOpa1DSM/DSM

Opa1DSM/DSMWT

Induced Opa1 ablation causes mitochondrial depolarization and degradation

WT KO

TMRM

(4X)

TMRM

(20X

)To

m20

(63X

)

WT KO

**

GAPDH

Tom20

WT KO

UPS and autophagy genes are induced upon Opa1 ablation

Atrogin1 MuRF1

mR

NA

leve

ls

mR

NA

leve

ls

LC3P62

BNIP3

Cathep

sinL

Gabar

apL

WT

KO

Opa1 ablation causes UPR activation

p eIF2 alpha

eIF2 alpha

GAPDH

**

**

Opa1DSM/DSMWT

The «mitochondrial damage» cytokine FGF21 is upon Opa1 ablation

0 5 10 15 20 25

50

60

70

80

90

100

Time(days)

Clim

bing

act

ivity

(%

of t

otal

flie

s)

w1118

mXBP1s

MarfRNAi

mXBP1s;MarfRNAi

0 5 10 15 20 25

20

40

60

80

100

Clim

bin

g a

ctiv

ity (%

of to

tal fl

ies)

Time (days)

W1118

W1118+TUDCA

MarfRNAi

MarfRNAi+TUDCA

Compensation of ER stress restores locomotor function of Marf (Mfn) deficient flies

genetic pharmacological

Debattisti et al, J Cell Biol 2014

The chemical ER chaperone TUDCA blunts FGF21 induction and reduces weight loss upon Opa1

ablation

Time (w)

Wei

ght c

hang

es(%

of i

nitia

l val

ue)

0 5 10 15 20 25 30 35

10

20

30

40

50

60

70

80

Wt

Opa1tg

rel

ease

d C

ytoc

hrom

e c,

% o

f tot

al

time,min

0’ 5’ 15’ 30’ 0’ 5’ 15’ 30’cBIDWt Opa1tg

BMH

21-30-

46-66- *

BAK

Cytochrome c release by cBID is reduced in Opa1tg mitochondria

C-II

OPA1

EDC EDC

Wt Opa1tg

460-

268-

171-117- 71-

70-

cBID 0’ 0’15’30’0’ 0’15’30’

*

Mobilization of cytochrome c from cristae is reduced in Opa1tg liver mitochondria

Mobilization of cytochrome c from cristae is reduced in Opa1tg liver mitochondria

- - cBID

0.02

0.03

0.04

0.05

0.06

0.07

0.08 Wt

Opa1tg

asc

orba

te/ T

MP

D-d

riven

resp

iratio

n

*

RCS assembly are assembled faster and are more stable in Opa1tg mitochondria

chase (h) 0 480 24 48

wt Opa1tg

IV

VIII

I

24

RCS 1236

720

242

1048

Health & Medicine

Luca Scorrano, Convegno Mitocon 2015