"The use of patient's fibroblasts in the evaluation of different therapeutic approaches

including enzyme replacement therapy “

Ann Saada (Reisch) PhD

Metabolic and Enzyme LaboratoryDepartment of Genetics and Metabolic Diseases

Hadassah Medical Center & Hebrew University Medical School

Jerusalem

1956 1966 1976 1986 1996 2006 20160

500

1000

1500

2000

2500

3000

3500

4000

4500

Year

num

ber o

f pub

licati

ons (

PubM

ed)

Human genome project WES(WGS)

SNP arrays/automated sequencing

mtDNA sequence

mtDNA Mutation

1988Luft’sDisease

1962

nDNA Mutation

1995

Respiratory chain biochemistry

Prevalence ~1:5000 ~1:8000 ~1:10000 ~1:4300

Mitochondrial disease are increasingly common inborn disorders

mtDNA encodedMRC subunits

mtDNA encodedt-RNA r-RNA

mtDNA

deletion/duplication

SECONDARY DEFECTScofactor bio synthesis/transport

lipid biosynthesismitochondrial fission/fusion

detoxificationapoptotic factors

agingneurodegenerative disease

environment

mtDNA-nDNA communicationreplication fork/nucleotide metabolism

nDNA encodedtranslation factors/ribosomal proteins/

t-RNA modifying enzymes

nDNA encoded

assembly factors

nDNA encoded

MRC subunits

mtDNA Depletion

mtDNA multiple deletions

SINGLE DEFECTCI,CII,CIII,CIV,CV

COMBINED DEFECTCI+CIII+CIV+CV

Mitochondrial diseases are caused by many factors

MGM2012

mtDNA encodedMRC subunits

mtDNA encodedt-RNA r-RNA

mtDNA

deletion/duplication

SECONDARY DEFECTScofactor bio synthesis/transport

lipid biosynthesismitochondrial fission/fusion

detoxificationapoptotic factors

agingneurodegenerative disease

environment

mtDNA-nDNA communicationreplication fork/nucleotide metabolism

nDNA encodedtranslation factors/ribosomal proteins/

t-RNA modifying enzymes

nDNA encoded

assembly factors

nDNA encoded

MRC subunits

mtDNA Depletion

mtDNA multiple deletions

SINGLE DEFECTCI,CII,CIII,CIV,CV

COMBINED DEFECTCI+CIII+CIV+CV

Treatment and Clinical trials are limited

Kanabus M, Heales SJ, Rahman S.Br J Pharmacol. 2014 171:1798-817

patient derived cells

microorganisms

animal models

MOUSE

C.elegans

D.melongaster

+ manipulation- phenotype

- maintenance

+mammal+ manipulation+/-phenotype- maintenance

- cost

+ manipulation+ eukaryote

- phenotype - maintenance

E.coli

S.cerevisiae

+ manipulation- prokaryote

- phenotype

MODEL systems For testing treatments

iPSCs

+ tissue specificity- generation/growth

- normal controls- ethics- cost

cybrids

+ mtDNA+/- tissue specificity- generation/growth - nuclear background

LYMHOCYTES/ LYMHOBLASTS

+ Accessible- phenotype

- manipulation

FIBROBLASTS(SKIN)

+ accessible

-limited passages (- )phenotype

+ growth, maintenance

MYOBLASTS

+ phenotype/muscle- sampling /timing

- growth- normal controls

STEMCELLS

+ tissue specificity-sampling

- generation/growth - -genetic instability

- normal controls- ethics- cost

IJCBC 2014

MITOCHONDRIAL DISEASE models

ROS? oxidative stress

ΔΨ(disruption of mitochondrial

membrane potential)

ATP depletionenergy deficit

Many parameters/consequences=what to measure?

Ca2+

(disrupted calcium

homeostasis)

Oxidative damage(lipids, proteins, nucleic acids)

DEFECTIVE OXPHOS

SECONDARY DEFECT

Vicious?

Circle

CELLULAR DYSFUNCTION

AUTOPHAGY/MITOPHAGYCELL DEATH/APOPTOSIS

mtDNA nDNA mutations

Patient’s FIBROBLASTS

Many “therapeutic approaches “

IMPROVED OXPHOS

IMPROVED CELL FUNCTION

Protein replacement therapy?? Mitochondrial therapy???Small molecules?

Patient’s FIBROBLASTS

Heteroplasmic shifting ?

Gene therapy ?Detoxification ?

PGC1α

INCREASE MITOCHONDRIAL

BIOGENESIS

Many “molecules and pathways“

ANTIOXIDANTS

COFACTORS, VITAMINS

IMPROVED OXPHOS

IMPROVED CELL FUNCTIONCa2+

Channelblockers

AUTOPHAGY/MITOPHAGYmodulators

APOPTOSIS inhibitors

PPARγactivators

AMPKactivators

Chemical chaperonesNucleotides etc.

Small molecules?

Patient’s FIBROBLASTS

PGC1α

INCREASE MITOCHONDRIAL

BIOGENESIS ANTIOXIDANTS

COFACTORS, VITAMINS

IMPROVED OXPHOS

IMPROVED CELL FUNCTIONCa2+

Channelblockers

AUTOPHAGY/MITOPHAGYmodulators

APOPTOSIS inhibitors

PPARγactivators

AMPKactivators

Chemical chaperonesNucleotides etc.

ROS? oxidative stress

ΔΨ(disruption of mitochondrial

membrane potential)

ATP depletionenergy deficit

Ca2+

(disrupted calcium

homeostasis)

Oxidative damage(lipids, proteins, nucleic acids) CELLULAR DYSFUNCTION

AUTOPHAGY/MITOPHAGYCELL DEATH/APOPTOSIS

DEFECTIVE OXPHOS

RiboflavinNiacin

ThiaminLipoate

AscorbateCoenzymeQ

Vitamin-EAICAR

bezafibrateResveratrol

genistein

Sodium phenylbutyrateUridine

OltiprazECGCNAC

Devorah SoifermanLiza DouievAnna Golubitzky

Small molecules?

Many “molecules and pathways“& Limited amount of cells/passages

PGC1α

INCREASE MITOCHONDRIAL

BIOGENESIS ANTIOXIDANTS

COFACTORS, VITAMINS

IMPROVED OXPHOS

IMPROVED CELL FUNCTIONCa2+

Channelblockers

AUTOPHAGY/MITOPHAGYmodulators

APOPTOSIS inhibitors

PPARγactivators

AMPKactivators

Chemical chaperonesNucleotides etc.

ROS? oxidative stress

ΔΨ(disruption of mitochondrial

membrane potential)

ATP depletionenergy deficit

Ca2+

(disrupted calcium

homeostasis)

Oxidative damage(lipids, proteins, nucleic acids) CELLULAR DYSFUNCTION

AUTOPHAGY/MITOPHAGYCELL DEATH/APOPTOSIS

DEFECTIVE OXPHOS

RiboflavinNiacin

ThiaminLipoate

AscorbateCoenzymeQ

Vitamin-EAICAR

bezafibrateResveratrol

genistein

Sodium phenylbutyrateUridine

OltiprazECGCNAC

Devorah SoifermanLiza DouievAnna Golubitzky

Small molecules?

Many “molecules and pathways“& Limited amount of cells/passages

x

0 20 40 60 80 100 120 1400.1

0.2

0.3

0.4

0.5

0.6

0.7

MB A620 Cont-GLU

Cont-GAL

NDUFS2-GLU

NDUFS2-GAL

time (hrs)

E

Methylene blue

5000 cells 72 hours

Patient’s fibroblasts that grow normally in high glucose medium show defective growth in glucose free medium (GAL)

Controls (n=5)

CI (NDUFS2)

CI (FOXRED1)

CI (LHON)

CIV (COX 6B1)

Trans. (EFTs)

Trans. (GFM1) Trans. (MRPS22)

Sec. (DNM1L)

0

0.1

0.2

0.3

0.4

0.5

0.6

GLUGAL

E

GROWTH

Most Patient’s fibroblasts show defective growth in GAL medium

nDNACI subunits

mtDNACI subunit

nDNACIV subunits

nDNAtranslation

nDNAsecondary

MB A620

E

Controls (n=5)

CI (NDUFS2)

CI (FOXRED1)

CI (LHON)

CIV (COX 6B1)

Trans. (EFTs)

Trans. (GFM1) Trans. (MRPS22)

Sec. (DNM1L)

0

500

1000

1500

2000

2500

3000

3500

GLUGAL

ROS

GROWTH

Controls (n=5)

CI (NDUFS2)

CI (FOXRED1)

CI (LHON)

CIV (COX 6B1)

Trans. (EFTs)

Trans. (GFM1) Trans. (MRPS22)

Sec. (DNM1L)

0

0.1

0.2

0.3

0.4

0.5

0.6

GLUGAL

MB A620

DCF RFU:MB A620

Most Patient’s fibroblasts show defective growth in GAL mediumMany show increased ROS and decreased ATP production

E

Controls (n=5)

CI (NDUFS2)

CI (FOXRED1)

CI (LHON)

CIV (COX 6B1)

Trans. (EFTs)

Trans. (GFM1) Trans. (MRPS22)

Sec. (DNM1L)

0

500

1000

1500

2000

2500

3000

3500

GLUGAL

ROS

GROWTH

Controls (

n=5)

CI

(NDUFS2

)

CI (FO

XRED1)

CI

(LH

ON)

CIV

(COX 6B1)

Trans.

(E

FTs)

Trans. (

GFM1)

Trans. (

MRPS22)

Sec.

(D

NM1L)0

40000

80000

120000 ATP-content (GAL)

Controls (n=5)

CI (NDUFS2)

CI (FOXRED1)

CI (LHON)

CIV (COX 6B1)

Trans. (EFTs)

Trans. (GFM1) Trans. (MRPS22)

Sec. (DNM1L)

0

0.1

0.2

0.3

0.4

0.5

0.6

GLUGAL

MB A620

DCF RFU:MB A620

ATP RLU:MB A620

Most Patient’s fibroblasts show defective growth in GAL mediumMany show increased ROS and decreased ATP production

E

Screening 10 compounds on 6 complex I deficient cellsAICAR has a positive effect on some cells

control NDUFS20

0.1

0.2

0.3

0.4

0.5

MB A620GLU GAL GAL+AICAR

control NDUFS20

500

1000

1500

2000

2500

3000

DCF RFU:MB A620

GLU GAL GAL+AICAR

control NDUFS20

100000

200000

300000

400000

500000

600000

ATP RLU:MB A620

GLU GAL GAL+AICAR

ROS

PLOSone2011

GROWTH

ATP-content (GAL)

Small moleculesNuclear encoded

CI subunits

E

AICAR enhances mito-biogenesis via AMPK

control NDUFS20

500

1000

1500

2000

2500

3000

DCF RFU:MB A620

GLU GAL GAL+AICAR

control NDUFS20

100000

200000

300000

400000

500000

600000

ATP RLU:MB A620

GLU GAL GAL+AICAR

ROS

ATP-content (GAL)

Small moleculesNuclear encoded

CI subunits

Mitochondrial contentNDUFS2 NDUFS2+AICAR

PLOSone2011

Phospho-AMPK

GROWTH

ATP-content (GAL)

Average Cell Growth

0.000.200.400.600.801.001.201.401.601.80

Ave

fold

GAL

no

addi

tive

Ave ROS Production/Cell

0.000.200.400.600.801.001.201.401.601.80

Ave

fold

GAL

no

addi

tive

Ave ATP Content/Cell

0.000.200.400.600.801.001.201.40

Ave

fold

GAL

no

addi

tive

EJHG 2016

MB A620

DCF RFU:MB A620

ATP RLU:MB A620

ROS

Small moleculesNuclear encoded

COX subunit COX6B1

Ascorbate, AICAR and resveratrol are beneficial to complex IV (COX6B1) deficient cells

ATP-content (GAL)

Ave ROS Production/Cell

0.000.200.400.600.801.001.201.401.601.80

Ave

fold

GAL

no

addi

tive

Ave ATP Content/Cell

0.000.200.400.600.801.001.201.40

Ave

fold

GAL

no

addi

tive

EJHG 2016

DCF RFU:MB A620

ATP RLU:MB A620

ROS

Small moleculesNuclear encoded

COX subunit COX6B1

Ascorbate, decrease ROS, increases ATP, mito-content and oxygen consumption

Ave Mitochondrial Content

0.000.200.400.600.801.001.201.401.60

GAL

AICAR 0.5mM

Ascorb

ate 10u

M

Bezafib

rate

0.1mM

NAC 2mM

Oltipraz

20uM

RSV 25uM

Ave

fold

GAL

no

addi

tive

Mitochondrial content(mitotracker green)

No a

dditi

ve

No a

dditi

ve

+ AI

CAR

0.5m

M

+ As

corb

ate

10uM

+ Re

sver

a-tr

ol

0.02

5mM

Control COX6B1

0.0000

0.0050

0.0100

0.0150

0.0200

0.0250 Maximal OCR

pMO

2/ce

ll/m

in

Oxygen consumption

ATP-content (GAL)

Ave ROS Production/Cell

0.000.200.400.600.801.001.201.401.601.80

Ave

fold

GAL

no

addi

tive

Ave ATP Content/Cell

0.000.200.400.600.801.001.201.40

Ave

fold

GAL

no

addi

tive

EJHG 2016

DCF RFU:MB A620

ATP RLU:MB A620

ROS

Small moleculesNuclear encoded

COX subunit COX6B1

BUT !! N-acetyl cysteine (NAC) reduces ROSNOT ONLYbut also ATP and mitochondrial content

Ave Mitochondrial Content

0.000.200.400.600.801.001.201.401.60

GAL

AICAR 0.5mM

Ascorb

ate 10u

M

Bezafib

rate

0.1mM

NAC 2mM

Oltipraz

20uM

RSV 25uM

Ave

fold

GAL

no

addi

tive

Mitochondrial content(mitotracker green)

Mito-translation disorders show individual results

Biochim2013

Small moleculesMitochondrial translation

Bezafibrate and idebenone are beneficial for Mitochondrial fission in DNM1L mutated cells

AJMG 2016 and in preparation

Small moleculesFission defect

DNM1L FOXRED1 C6ORF66 NDUFS4 NDUFS2 COX6B1 TRMU MRPS22 GFM1 EFTs

+/+/- ns + +/- +/+ +/-/+ ns +/+/ns +/ns +/ns BEZA

ns + +/+ - +/+/+ + +/ns +/ns +/ns ns AICAR

ns + ns +/+ ns +/ns ns ns ns OLTI

-/+/+ + + ns +/- +/-/- +/+ ns ns +/+ SBP

-/+ +/-/+ -/+ +/- +/+/- +/+ +/+ ns +/- +/+ RSV

-/-/+ +/- +/+ +/ns/- +/- +/- NAC

+/+/- +/+ +/ns +/ns ns +/+ Asc

+ -/+ +/+/- +/- GENI

+/- +/- GSE

The effect of ANTIOXIDANTS is variable Decreased ROS combined with decreased, unaffected, increased ATP

Small moleculesSummary

“beneficial” = green “mixed response”=gray“detrimental”= red ns=not significant

DNM1L FOXRED1 C6ORF66 NDUFS4 NDUFS2 COX6B1 TRMU MRPS22 GFM1 EFTs

+/+/ns ns + +/- +/+ +/-/+ ns +/+/ns +/ns +/ns BEZA

ns + +/+ - +/+/+ + +/ns +/ns +/ns ns AICAR

ns + ns +/+ ns +/ns ns ns ns OLTI

-/+/+ + + ns +/- +/-/- +/+ ns ns +/+ SBP

-/+ +/-/+ -/+ +/- +/+/- +/+ +/+ ns +/- +/+ RSV

-/-/+ +/+ +/+ +/ns/- +/- +/- NAC

+/+/- +/+ +/ns +/ns ns +/+ Asc

+ -/+ +/+/- +/- GENI

+/- +/- GSE

“beneficial” = green “mixed response”=gray“detrimental”= red ns=not significant

The effect of MITOBIOGENESIS inducers is variable Is increased biogenesis beneficial or does it “exacerbate stress” ?

Small moleculesSummary

DNM1L FOXRED1 C6ORF66 NDUFS4 NDUFS2 COX6B1 TRMU MRPS22 GFM1 EFTs

+/+/ns ns + +/- +/+ +/-/+ ns +/+/ns +/ns +/ns BEZA

ns + +/+ - +/+/+ +/+/+ +/ns +/ns +/ns ns AICAR

ns + ns +/+ ns +/ns ns ns ns OLTI

-/+/+ + + ns +/- +/+ ns ns +/+ SBP

-/+ +/-/+ -/+ +/- +/+/- +/+ +/+ ns +/- +/+ RSV

-/-/+ +/+ +/+ +/ns/- +/- +/- NAC

+/+/- +/+ +/ns +/ns ns +/+ Asc

+ -/+ +/+/- +/- GENI

+/- +/- GSE

“beneficial” = green “mixed response”=gray“detrimental”= red ns=not significant

The response is individual

Unpublished, EJHG 2015, Biochim2013, PLOSone2011

Small moleculesSummary

DNM1L FOXRED1 C6ORF66 NDUFS4 NDUFS2 COX6B1 TRMU MRPS22 GFM1 EFTs

+/+/ns ns + +/- +/+ +/-/+ ns +/+/ns +/ns +/ns BEZA

ns + +/+ - +/+/+ +/+/+ +/ns +/ns +/ns ns AICAR

ns + ns +/+ ns +/ns ns ns ns OLTI

-/+/+ + + ns +/- +/+ ns ns +/+ SBP

-/+ +/-/+ -/+ +/- +/+/- +/+ +/+ ns +/- +/+ RSV

-/-/+ +/+ +/+ +/ns/- +/- +/- NAC

+/+/- +/+ +/ns +/ns ns +/+ Asc

+ -/+ +/+/- +/- GENI

+/- +/- GSE

“beneficial” = green “mixed response”=gray“detrimental”= red ns=not significant

The response is individual Could results obtained in fibroblasts be relevant for personalized treatment?

Unpublished, EJHG 2015, Biochim2013, PLOSone2011

Small moleculesConclusion?

?

Some other “therapeutic approaches “

IMPROVED OXPHOS

IMPROVED CELL FUNCTION

Protein replacement therapy??

Patient’s FIBROBLASTS

Prof. Haya Lorberboum-GalskiMatan RapoportDana Marcus

Small molecules

Protein replacement therapies are currently used in some diseasesBut there are still obstacles

Protein replacement

COMMON OBSTACLES: delivery by IV infusion

mis-targeting of recombinant enzymeDifficulty reaching target tissue

Immune reactions

Enzyme replacement therapy (ERT)In lysosomal storage diseases (LSD’s):

Gaucher diseasePompe (GSDII) disease

Fabry diseaseMucopolysaccharidoses I,II, IV

Protein replacement

A recombinant mitochondrial protein should: Penetrate not only cellular membrane,

BUT also both the OUTER and INNER mitochondrial membranes AND integrate into a multi-subunit complex

COMMON OBSTACLES: delivery by IV infusion

mis-targeting of recombinant enzymeDifficulty reaching target tissue

Immune reactions

Protein replacement in mitochondrial diseasesis even more complex

Protein replacement

A recombinant mitochondrial protein should: Penetrate not only cellular membrane,

BUT also both the OUTER and INNER mitochondrial membranes AND integrate into a multi-subunit complex

Protein replacement in mitochondrial diseasesis even more complex

PROTEIN TRANSDUCTION DOMAINS PTD’s

Amino acid domains serving asDelivery vectors

Protein replacement

A recombinant mitochondrial protein should: Penetrate not only cellular membrane,

BUT also both the OUTER and INNER mitochondrial membranes AND integrate into a multi-subunit complex

Protein replacement in mitochondrial diseasesis even more complex

MITOCHONDRIAL protein+ mitochondrial target sequence

MTS

Trans Activator of Transcription TAT

11 amino acids from HIV-1(Arginine rich, positively charged)

TAT+MTS+MITOPROTEINFUSIONPROTEIN produced in vitro

Protein replacement

A recombinant mitochondrial protein should: Penetrate not only cellular membrane,

BUT also both the OUTER and INNER mitochondrial membranes AND integrate into a multi-subunit complex

Protein replacement in mitochondrial diseasesis even more complex

Trans Activator of Transcription TAT

11 amino acids from HIV-1(Arginine rich, positively charged)

MITOCHONDRIAL protein+ mitochondrial target sequence

MTS

TAT+MTS+MITOPROTEINFUSIONPROTEIN produced in vitro

Protein replacement-LAD

Lipoamide dehydrogenase (PHDc-E3 aKDH-E3 BCKA-E3)The pure TAT-MTS-LAD recombinant protein is active

TAT+MTS+LADFUSIONPROTEIN

Molec Ther 2008

PDHcE3

Protein replacement-LAD

TAT+MTS+LADFUSIONPROTEIN

(native protein)(control )

Molec Ther 2008

Lipoamide dehydrogenase (PHDc-E3 aKDH-E3 BCKA-E3)The pure TAT-MTS-LAD recombinant protein is active

The TAT-MTS-LAD recombinant protein enters mitochondria is processed and

improves activity in LAD deficient Patient’s fibroblasts

Patient (G229C/Y35X )Fibroblasts

TAT+MTS+LADFUSIONPROTEIN

-processed TAT-LAD and native mutant LAD-TAT-LAD

LAD activity

Protein replacement-LAD

Molec Ther 2008

The TAT-MTS-LAD recombinant protein enters mitochondria co-localized with PDHc

and improves PDHc activity in LAD deficient Patient’s fibroblasts

Molec Ther 2008

TAT+MTS+LADFUSIONPROTEIN

Patient (G229C/Y35X )Fibroblasts

anti-PDHc1α FITC- TAT-LAD Overlay

PDHc activity

Protein replacement-LAD

In theory, intermittent treatment couldSuffice in mild cases/during crisis

BBRC 2000

Muscle/LiverIntermittent“Normal life”

Liver dysfunctionInfantile, fatalLiver failureencephalopathy

ATP-production

Protein replacement-LAD

Protein replacement-CI

TAT+MTS+C6ORF66 (NDUFAF4)FUSIONPROTEIN

The TAT-MTS-NDUFAF4 recombinant protein enters mitochondria and improves CI activity

and mitochondrial functions in Patient’s fibroblasts

GROWTH

ATP-content (GAL)

ROS

FITC- TAT-NDUFAF4

1h 3h

Mol Med 2013

Enzymatic activity

TAT+MTS+LADFUSIONPROTEIN

Next step-proof of concept in animal models

Rapoport et al J Mol Med 2011,

Protein replacement-next step

Another “therapeutic approach “

IMPROVED OXPHOS

Mitochondrial therapy???

Patient’s FIBROBLASTS

Protein replacement-next step

Eyal Kesner

NATURE1982

Mitochondrial therapy?

IMPROVED OXPHOS

Mitochondrial therapy???

Patient’s FIBROBLASTS

Eyal Kesner

NATURE1982

Sci Rep 2016,

Isolated mitochondria from HeLa cells quicklyenter recipient cells

HeLa-dsRed2

Recipient cells

Sci Rep 2016,

Mitochondrial therapy???

NATURE1982

Isolated mitochondria from HeLa cells quicklyenter fibroblasts, maintaining identity & improves growth

GROWTH(GAL)

Enzymatic activity

Mitochondrial therapy???

Sci Rep 2016,

Isolated mitochondria from HeLa cells quicklyenter fibroblasts, maintaining identity & improves growth

Mechanism of mitochondrial import: Intact mitochondrial membrane

MacropinocytosisProteoglycan involvement

Mitochondrial therapy???

Sci Rep 2016,

Biochemical Diagnostics

Corinne BelaicheRosi Shwartz

Sarah weissmanPolina Ilin

Elena fatale

Molecular diagnostics Prof.Orly Elpeleg

Dr.Avraham ShaagProf.Vardiella Meiner

Collaborators

Prof. Chaya Lorberboum-Galski

Dr. Matan RapaportEyal Kesner

Dana Markus

Experimental Devorah Soiferman

Lisa DouievDr Chaya Miller

Anna Golubitzky (alumni)Dr Phyllis Dan (alumni)

Dr. Maskit Bar-Meir (alumni)

Clinical collaboratorsProf.Orly Elpeleg

Dr.Simon EdvardsonDr. Ruth Sheffer

Prof.Ronen SpiegelDr Itai Beger

Protein replacement therapy?? Mitochondrial therapy???Small molecules?

Thank you …………….

COFACTORS, VITAMINS

RiboflavinNiacin

ThiaminLipoate

COFACTORS, VITAMINS

One fibroblast one parameter-riboflavin beneficial

AscorbateCoenzymeQ

Vitamin-E

ANTIOXIDANTS

Bar-Meir J Ped 2001

Small molecules

Mitochondrion 2009

the response is individual depending on the defect

+gentamicin +gentamicin +gentamicin

nDNAtranslation Individual mito translation disorders are sensitive to

translation targeted antibiotics

AICARAminoImidazole Carboxamide Ribonucleotide

Adapted from: Scarpulla RC. 2011 BBA :1813

Resveratrol

GenisteinBezafibrate

Same pathway DIFFERENT EFFECTS Mitochondrial BIOGENESIS

Nicotinamide(Ribo nucleoside)