luca scorrano, convegno mitocon 2015
TRANSCRIPT
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
Opa1tg livers are protected from Fas induced apoptosis
Wt Opa1tg
Opa1tg livers are protected from Fas induced apoptosis
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
Ectopic expression of NRasQ61K in melanocytes leads to hyperpigmentation and melanoma
Ectopic expression of NRasQ61K in melanocytes leads to hyperpigmentation and melanoma
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
Scorrano et al Dev. Cell 2002
The remodeled cristae
Scorrano et al Dev. Cell 2002
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