Yeast-based chemobiological approaches of

mitochondrial diseases

- 2005: ANR «Maladies Rares» JP di Rago (yeast mitochondria)/M Blondel (yeast chemobiology)

- 2008: LM Steinmetz (yeast chemogenomics)

- 2011: FRM «Régulations Métaboliques»

A Roetig/JP di Rago/M Blondel/A Delahodde/G Dujardin

- 2014: AFM «Projet Stratégique»

A Roetig/JP di Rago/M Blondel/A Delahodde/G Dujardin/V Procaccio

LM Steinmetz/ML Jung

Yeast-based chemobiological approaches of

mitochondrial diseases

Marc Blondel (marc.blondel@univ-brest.fr)

Inserm UMR1078, Brest, France

Chemobiology at happy hour: yeast models for

human diseases

Yeast as a model for human diseases

Marc Blondel (marc.blondel@univ-brest.fr)

Inserm UMR1078, Brest, France

Marc Blondel,

Cécile Voisset, CR1, Inserm

Gaëlle Friocourt, CR1, Inserm

Déborah Tribouillard-Tanvier, CR2, Inserm

Olivier Billant, PhD, MRES

Maria-José Lista, PhD, La Ligue/Région

Marie-Astrid Contesse, IE, CDD UBO

Justine Evrard, IE, CDD Inserm

Nadège Loaëc, IE, CDD Inserm

Hélène Simon, AJT, UBO

Alice Léon, stage M2, UBO

modelling human diseases at happy hour…

yeast is an eukaryote as easy to handle as a prokaryote

cellular mechanisms & key players involved in most human diseases are conserved from yeast to humans therefore it is possible to create yeast models for human diseases… STARTING POINT: creating yeast models exhibiting a phenotype relevant for the pathology of interest

searching for modifiers + or - by screening:

• small MW compounds (identification of drug candidates) • genes/cDNA (identification of mechanisms)

validation of these modifiers (drugs, genes…): human cells, animal models… drugs are used for reverse screening: search for intracellular targets of drugs (identification of mechanisms)

drugs can be used as tools for basic research (chemical genetics)

yeast: a robust and versatile system to model human diseases

3 possible situations…

proofs of principle…

Yeast models for prion-based diseases (Bach et al. Nature Biotechnology 2003)

ex. of yeast-based drug screening

Yeast models for Huntington disease (Giorgini et al. Nature Genetics 2005)

ex. of yeast-based genetic screening

an example of a drug screening based on a yeast model for prion diseases:

active in vivo against

yeast prions

active ex vivo against

mammalian prion PrPSc

6-aminophenanthridine (6AP) & guanabenz (GA) are active against yeast &

mammalian prions both ex vivo & in vivo

active in vivo in a murine model for

prion-based diseases (collaboration

V. Beringue & H. Laude, INRA Jouy)

prion-controlling mechanisms are conserved from yeast to mammals

we identified the conserved target of 6AP & GA:

the protein folding activity of the ribosome (PFAR)

an example of a genetic screening based on a yeast

model for Huntington disease:

Brancusi

modelling human mitochondrial diseases in budding yeast:

from deciphering of physiopathological mechanisms

to isolation of candidate drugs:

a yeast-based model for the NARP syndrome

Marc Blondel

Inserm UMR1078

Faculté de Médecine et

des Sciences de la Santé

BREST

Jean-Paul di Rago

Institut de Biochimie et

Génétique Cellulaires

CNRS/Université Bordeaux2

UMR5095

BORDEAUX

a partnership between:

funded by ANR «Maladies Rares» 2006-8, AFM 2008-13 & FRM 2011-12

& in collab. with Lars Steinmetz,

EMBL Heidelberg, Germany

& Stanford University, USA

- Kucharczyk et al, 2009

- Kucharczyk et al, 2010

- Couplan et al, 2011

- Aiyar et al, 2014

NARP syndrome: a devastating mitochondrial disease

Neuropathy Ataxia Retinitis Pigmentosum

a maternally inherited mitochondrial disease (mutation within the mt DNA)

due to mutations in the mitochondrial ATP6 gene encoding a subunit of ATP

synthase, which is essential for the production of ATP by oxydative phosphorylation

(OXPHOS)

ADP ATP

F0

F1

H+ H+

O2

ATP synthase

respiratory

chain

Sous-unité

c

H+

H+

subunit a

(stator)

subunit c

(rotor) H2O

mitochondria =

cell powerhouse

(prod. ATP by OXPHOS)

reduction of O2 in H2O:

creation of a H+ gradient

used by ATP synthase

to produce ATP from ADP

ATP synthase =

nanomolecular engine,

fuel = H+ gradient

ATP synthase: a nano-molecular engine

NARP mutations:

lie in the stator, at the

interface with the rotor

Neuropathy Ataxia Retinitis Pigmentosum

a genetic disease: maternally inherited mitochondrial disease

due to mutations in the mitochondrial ATP6 gene encoding a subunit of the

stator of ATP synthase, these mutations block the rotor rotation

( like « a fly in the ointment »)

ADP ATP

F0

F1

H+ H+

O2

ATP synthase

respiratory

chain

Sous-unité

c

H+

H+

subunit a

(stator)

subunit c

(rotor)

deficiency in ATP production NARP syndrome

NARP syndrome: a devastating mitochondrial disease

like all eukaryotic cells, yeast cells do contain mitochondria (that have their own

mitochondrial genome) which are very similar to their human counterpart

why yeast

for modelling human mitochondrial diseases (myopathies, NARP, etc.)?

Cytosol

OM

IMS

IM

Matrix

14 subunits

Atp6p, Atp8p

ASSEMBLY

Nucleus

14 subunits

TOM

TIM

4

6 8

h

a

g

d e

d

9 9 9 9 9

oscp

i,f

b b

a

Human

mtDNA

ATP6 ATP8

Cytosol

OM

IMS

IM

Matrix

18 subunits

Atp6p, Atp8p, Atp9p

Nucleus

18 subunits

TOM

TIM

F1 : Atp11p,

Atp12p, Fmc1p

Fo : Atp23p,

Atp10p,Atp25

Expression of ATP6,

ATP8, ATP9

Nca1, Nca2, Nca3, Aep1,

Aep2, Aep3, Atp22, Atp25

4

6 8

h

a

g

d e

d

9 9 9 9 9

oscp

i,f

b b

a

ATP6

ATP8

ATP9

S.cerevisiae

mtDNA

ASSEMBLY

mitochondrial functions are well conserved

from yeast to human, in particular ATP synthase

like all eukaryotic cells, yeast cells do contain mitochondria (that have their own

mitochondrial genome) which are very similar to their human counterpart

budding yeast can survive w/o respiration (using fermentation to produce ATP)

why yeast

for modelling human mitochondrial diseases (myopathies, NARP etc.)?

GLUCOSE CO2 + H20

ADP+Pi ATP

Oxidative

Phosphorylation

Fermentation

ADP+Pi ATP GLUCOSE CO2 + EtOH

WT

yeast

respiratory

mutant

fermentable medium

(glucose)

respiratory medium

(glycerol)

budding yeast can survive w/o respiration (using fermentation to produce ATP): fermentation > very rapid but low yield / respiration > very slow but high yield

hence budding yeast can be used to perform pharmacological screening on NARP

yeast is one of the very few eucaryotes for which site-directed mutagenesis of the mitoch.

genome is possible (using a biolistic technique): introd. mut. synonymous to NARP mut.

like all eukaryotic cells, yeast cells do contain mitochondria (that have their own

mitochondrial genome) which are very similar to their human counterpart

budding yeast can survive w/o respiration (using fermentation to produce ATP)

why yeast

for modelling human mitochondrial diseases (myopathies, NARP etc.)?

ATP6

ATP8

ATP9

biolistic

site-directed

mutagenesis

JP di Rago

T8993G

T8993C

T9176G

T9176C

T8551C

NARP/MILS

S.cerevisiae

mtDNA

respiratory growth defect of yeast NARP mutants at 37°C

normal growth on glucose

ATP ↔ Fermentation

glucose

drops of serial dilutions of yeast cell liquid cultures

mutat. within

the yeast ATP6

gene

synonymous

to mutations

found in

NARP patients

T8993G

T8993C

T9176G

T9176C

T8851C

Wild type

fmc1

glycerol

growth defect on glycerol

ATP ↔ Respiration

ATP synthesis

% of WT

100

74

<5

6

8

7

50

perfect correlation with

severity of the mutations

in NARP patients!

perfect correlation > 1st validation of the yeast model for NARP…

genetic screening (JP di Rago & M Rigoulet)

ODC1 was isolated as a multicopy suppressor of NARP mutations in yeast (JP di Rago & M Rigoulet)

Odc1p

Porin

western-blot Glucose Glycerol

atp synthase

mutant

atp synthase

mutant + oe ODC1

ODC1 encodes oxodicarboxylate carrier

NARP/fmc1Δ

strain

in glucose

filters one drug

per filter

incubation

5-7 days at 35°C

active compound

negative control (DMSO)

NARP/fmc1Δ

strain spread

on respiratory

solid medium

(glycerol)

drug screening (E Couplan, JP di Rago & M Blondel)

compounds from chemical libraries are tested for their ability

to suppress the growth defect of yeast NARP mutants

pos. control = Di-Hydro Lipoïc Acid (DHLA) currently in clinic to treat

mitochondrial encephalopathies: 2nd validation of the yeast assay!

>12,000 molecules screened (including the Prestwick C.L.): ~20 active compounds

positive

control (DHLA)

HS

SH

OH

O

currently tested

in clinic to treat

mitochondrial

encephalopathies

example of an active compound: chlorhexidine (CH),

a compound able to suppress the respiratory phenotype

of NARP and ATP synthase mutants in yeast

T89

93

G

T91

76

G

Wil

d t

yp

e

T88

51

C

fmc1

NARP mut.

DMSO

CH 2 µM

…CH suppresses respiratory growth

defect when added directly in the medium…

& its effect is allele specific

CH suppresses respiratory

growth defect on solid medium…

3rd validation of the yeast assay: activity of DHLA & CH on NARP cybrids

NARPJCP239 cybrids mtDNA T8993G

cybrids = fusion between:

- rho0 osteosarcome (thus a cell line w/o mtDNA)

- platelets from NARP patients

(w/o nucleus & containing mtDNA T8993G )

hence resulting cybrids are cell lines containing NARP mitochondria

growth of NARPJCP239 cybrids in glucose similar to WT cybrids

because it relies on glycolysis (like most of tumoral cell lines)

w/o glucose: cybrids are forced to rely on oxidative phosphorylation

for their growth: pronounced growth defect of NARPJCP239 cybrids

compared to WT control cybrids (similar to the yeast situation)

drugs can be tested on primary cultures of fibroblasts from patients:

difficult to handle & no clear phenotype… thus we used another system:

effect of CH on the growth of NARP cybrids in a medium deprived of glucose

CH DHLA 200 µM

CH & DHLA also active on NARP cybrids: validation of the yeast assay & of CH

[CH] nM

Perc

enta

ge o

f gr

ow

th

0

100

200

DHLA 0 12.5 25 50 80

200 µM

mode of action of CH: acts on mitochondria

validation of the yeast-based assay & of CH

CH acts on mitochondria in yeast V

ox (

nA

tO2.m

in-1

.mg

-1)

0

100

200

300

400

500

600

EtOH TET CCCP

fmc1 + DMSO fmc1 + CH

FMC1+

CH increases respiration

rate of mutant cells…

fmc1

+

DM

SO

0.5 µm

V

N

0.2 µm0.2 µm

0.2 µm0.2 µm

m

m m

m

ATP synthase mutants:

accumulation of inclusion bodies

within mitochondria

& disapearance of cristae

fmc1

+

CH

0.5 µm

V

N

0.2 µm0.2 µm

0.2 µm0.2 µm

m

ATP synthase mutants + CH:

disapearance of inclusion bodies

within mitochondria

& reappearance of cristae

Cyt b

Atp9

Atp1

Cox2

Porin

Actin F

MC

1+

-

fmc1

+ - CH

ATP Synthase

subunits

(Complexe V)

Complex IV

subunit

Complex III

subunit

outer membr.

protein

CH restores levels

of several proteins of the

mitochondrial OXPHOS

pathway

determination of all genes whose expression is significantly affected

in ATP synthase mutant / WT strains & of CH effect on these genes…

global transcriptomic analysis by tilling arrays (R. Aiyar & L. Steinmetz, EMBL,

Heidelberg, Germany & Stanford University, USA)

most of the nuclear genes affected (down or upregulated)

in ATP synthase mutant / WT

are nuclear genes encoding mitochondrial proteins (~50)…

global transcriptomic analysis by tilling arrays (R. Aiyar & L. Steinmetz, EMBL,

Heidelberg, Germany & Stanford University, USA)

CH partially or totally restores expression of most of

these genes

QCR9 gene is the only one to be down-regulated in

ATP synthase mutants & very rapidly (after a few min.)

overexpressed when CH is added… thus QCR9 could

be a critical target for CH activity…

& indeed QCR9 overexpression partially suppresses the

respiratory growth defect of ATP synthase mutants…

& QCR9

conserved in

humans…

Sodium pyrithione (NaPT): another active compound

DMSO

CH

NaPT

DHLA

fmc1Δ @ 36°C

NaPT suppresses the respiratory growth defect of

an ATPsynthase mutant at 36°C

in a dose-dependent manner

quantity

0

20

40

60

80

100

120

140

160

180

200

DMSO 100 µM DHLA

20 nM C7

40 nM C7

60 nM C7

80 nM C7

100 nM C7

25 nM C1

Cell viability after a 6 days treatmentJC239 (NARP)

JC213 (WT)

NaPT is active on NARP cybrids

& has no effect on WT cybrids

CH

sodium pyrithione (NaPT)

NaPT mode of action? determination of its cellular target(s)

using chemogenomics (collab. L. Steinmetz, JP di Rago & M. van der Laan)

chemical genomics in yeast: finding drug targets via gene dosage G

row

th

Normal

Haploinsufficiency

Deletion

sensitivity

Inhibited

by drug

drug

method: take drug of interest at a concentration that partially inhibits cell growth

* when one copy of its target is deleted (haploinsufficiency), growth should be hypersensitive to the drug = deletion

sensitivity

* when its target is overexpressed from a plasmid, growth defect should be suppressed = multicopy suppression

Multicopy suppression

Overexpression

B. StOnge, Stanford

Haploinsufficiency (X axis) for every single yeast gene is

plotted as a function of Multicopy suppression (Y axis):

the most interesting genes are in the top right

Tim17, a component of the mitochondrial TIM

complex was identified as a putative target of NaPT

chemical genomics in yeast : identification of Tim17 as a target of NaPT collab. Bob StOnge (Stanford) & Lars Steinmetz (Heidelberg)

B. StOnge, Stanford

haploinsufficiency

overexpression

TIM

17 D

ele

tion

Se

nsitiv

ity

1000+ compounds (by date screened)

NaPT

this level of Tim17 sensitivity is by far the highest

observed among >1,000 compounds screened at Stanford.

TOM and TIM allow import within the mitochondria of

mitochondrial proteins synthesized in the cytoplasm

Tim17p is part of TIM23 complex, a mitochondrial

presequence translocase

NaPT affects import

through this TIM23

supercomplex

by favouring the

lateral sorting

what does Tim17 do?

- Tim17 is an essential component of the TIM23 supercomplex, which mediates mitochondrial import of all

presequence-carrying proteins, targeting them either to the matrix or inner membrane (lateral sorting) depending

on the substrate’s destination.

- Therefore the TIM23 complex exists in two forms: one that allows matrix import and the other (which contains

Tim21) responsible for the lateral sorting (that allows insertion in the inner membrane of proteins of the various

complexes of the respiratory chain).

TIML23 complex =

Tim23, Tim17,

Tim50 and Tim21

overexpression of

Tim21p also favours

the lateral sorting

overexpression of TIM21 which favours lateral sorting mimics the effect of C7

in yeast…

WT fmc1Δ R. Aiyar, Heidelberg

Re

sp

ira

tory

overexpression of TIM21 which favours lateral sorting mimics the effect of C7

in yeast… and also in NARP cybrids…

ODC1 was isolated as a multicopy suppressor of NARP mutations in yeast (JP di Rago & M Rigoulet)

Odc1p

Porin

western-blot Glucose Glycerol

atp synthase

mutant

atp synthase

mutant + oe ODC1

ODC1 gene and oleate (OA) effect on yeast and NARP cybrids

ODC1 gene and oleate (OA) effects on yeast and NARP cybrids

ODC1 was isolated as a multicopy suppressor gene of NARP mutations in yeast,

oleate (OA) is known to lead to overexpression of ODC1 gene in yeast,

therefore we tested the activity of OA in the yeast model for NARP: OA is active,

ODC1 gene is conserved in humans, thus we tested OA in NARP cybrids:

oleate (OA) is active in cybrids

Oleate

OH

O

conclusions:

- 4 drugs (CH, DHLA, NaPT & OA) active in both yeast & human models for NARP

(& DHLA tested in clinic on patients):

validation of the yeast model for NARP & of CH, NaPT & OA

- CH mode of action: increases respiration an restores mitochondrial morphology,

its effect may be mediated by QCR9 gene (for which CH induces an overexpres.)

- this yeast-based model for NARP = proof of principle

this type of method could be used for many other mitochondrial diseases

(myopathies, etc): within the Theramito consortium we are currently developing

yeast-based models for five other human mitochondrial diseases… both in

S. cerevisiae and in S. pombe…

… and many others can be envisioned: creation of a « target library »

(>100 ≠ mitoch. disorders, individually rare but altogether not neglectable)

- NaPT mode of action: potentially by favouring lateral sorting at TIM level,

highlighting the mitochondrial import as a potential relevant therapeutic target

the Theramito consortium: 8 teams developing an integrated (yeasts, nematode, cells from

patients, cybrids) approach for mitochondrial diseases (6 dif. diseases)

- 3 yeast labs:

- JP di Rago (IBGC, Bordeaux)

- G. Dujardin (CGM, Gif/Yvette)

- M. Blondel (Inserm UMR1078, Brest)

- 1 nematode lab:

- A. Delahodde (IGM, Orsay)

- 2 labs having access to patients and patients’ cells:

- A. Roetig (Hôp. Necker, Paris)

- V. Procaccio (CHU, Angers)

- 1 SME specialized in medicinal chemistry:

- Prestwick Chemical (Ilkirch)

- 1 lab of chemogenomics:

- L. M. Steinmetz (EMBL, Heidelberg)

dir.: Agnès Roetig

many thanks to ANR « Maladies Rares » , AFM & FRM

& thanks for

your attention…