Animal models in mitochondrial disorders ... Massimo Zeviani, MD, PhD Dublin April 2013 Animal...

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Transcript of Animal models in mitochondrial disorders ... Massimo Zeviani, MD, PhD Dublin April 2013 Animal...

  • Massimo Zeviani, MD, PhD

    Dublin April 2013

    Animal models in mitochondrial disorders

    der bestirnte Himmel über mir, und das moralische Gesetz in mir

  • Q QH2

    DHODH

    uridine

    Q

    QH2

    cred

    QH2

    Ca2+

    H+

    Pyruvate

    Lactate

    NAD+

    NADH

    Alanine

    Glucose

    AIF CAD

    Apoptosis

    ADP ATP

    O2

    H2O

    NAD+

    NADH

    H+

    H+ Ac-CoA

    TCA cycle

    b-oxidn

    H+

    fumarate

    succinate

    NADH NAD+

    CO2

    Apaf-1

    inactive

    Apaf-1

    active

    Activated

    caspases

    H+

    Ca2+

    e -

    O2 -

    O2

    GPT

    H2O2

    Fe2+

    Fe3+

    OH -

    H2O

    GPx

    GSH

    GS-SG

    H+

    PDH

    LDH

    IV

    IV

    II

    III I

    I

    II

    III

    V

    III

    ADP

    ATP

    SOD2

    cox

    cred

    cred

  • 61%

    21% 18% mtDNA mutation

    nDNA mutation

    unknown

    Paediatric Patients with Respiratory Chain Deficiency

    0%

    20%

    40%

    60%

    80%

    100%

    C o m

    p le

    x I

    C o m

    p le

    x I I

    C o m

    p le

    x I II

    C o m

    p le

    x I V

    C o m

    p le

    x V

    M u lt ip

    le

    T o ta

    l

    D e p le

    ti o

    n

  • LINKAGE/HOMOZYGOSITY

    Linkage analysis or homozygosity mapping

    Candidate genes

    Families with (autosomal recessive) mitochondrial disorders

    Mutational screening

    Small families and single individuals

    Filtering strategies

    Candidate genes

    Exome sequencing Coding variants

    NGS

    Discovering new disease genes

  • ? Phenotype Function

    OXPHOS D2S2373

    D2S2259

    Locus

    I II III IV V

    G A A T G A G C A

    Gene

    Protein TTC19

    C #1-1 #1-2 i.v.

  • In silico

    Filtering genes Integrated genomics

    Phylogenetic analysis

    Systems analysis

    Omic- analysis

    Structure

  • Cell models

    patients

    MEFs

    yeast

    HeLa/cybrids

    iPS

    recombinant E. coli

  • In vivo

  • In vivo pathogenesis

    Experimental therapy

    Protein factory

    Clinical phenotype

    Organ pathophysiology

    Cell biology

    Systems biology and omics

    Gene replacement

    Cell replacement

    Drugs Organ replacement

    Other bioreactors

    Mutant proteins

    Tagged proteins

    Complexes

    Gene and protein interactions

  • Disease models

    SURF1 constitutive KO

    MPV17 constitutive KO

    ETHE1 constitutive and conditional KO

    COX15 constitutive KO (embryonic lethal) and conditionals (ACTA-COX15; GFAP-COX15)

    SCO2constitutive KOKI

    NDUFS1 constitutive KO

    NDUFS4 constitutive KO

    FASTKD2 constitutive KO

    TTC19 constitutive and conditional KO

    PARP1 constitutive KO

    FASTKD2 constitutive and conditional KO

    Transgenic mice

    PGC1

    TG CRE: general deleter

    ALB CRE: liver specific

    MYOD CRE: (muscle specific, early expressed)

    ACTA CRE: (skeletal muscle specific)

    TG FLPe (general deleter)

    GFAP-CRE: (brain specific)

    AOX (knockin in locus ROSA26)

    NDI1 (knockin in locus ROSA26)

    Mouse models

  • CNS •early-onset hypotonia. developmental delay •later spasticity, then global failure

    Vascular system •acrocyanosis •petechiae, microematuria, internal bleedings

    Gastrointestinal system •chronic diarrhoea

    Biochemistry •COX deficiency •lactic acidosis •ethylmalonic aciduria

    0

    1

    2

    3

    4

    5

    6

    7

    8

    9

    A b

    u n

    d an

    ce

    urea

    ethylmalonate

    I.S.

    2-ketoglutarate

    I.S.

    isobutyrylglycine

    isovalerylglycine

    Time

    Ethylmalonate Butyryl-CoA CO2 -> CH3-CH2-CH2-CO-SCoA + CH3-CH2-CH-COOH

    COOH

    CoASH

    Ethylmalonic Encephalopathy (EE)

  • 440del11

    3G>T

    113A>G 131delAG

    222insA

    406A>G

    488G>A

    487C>T

    604insG

    559insC 375+5G>A

    505+1G>A

    505+1G>T

    554T>G

    EX1-7del

    EX4del

    1 2 3 4 5 6 7

    1 Kb

    ETHE1 is the EE disease gene

    HeLa

    ETHE1 Mitotracker merge

    ETHE1p

    hom. sup. mitos matrix memb

    Works as a homodimer

    Binds 1 atom of iron/dimer

    Fe++

  • genotypes

    Lifespan

    days

    Behavior

    Creating an ETHE1-/- mouse E4

    NEO E1 E2 E3 E5 E6 E7

    LOXp LOXp frt frt

    0

    50

    100

    150

    200

    250

    300

    350

     g

    /m g

    c re

    a ti

    n in

    e

    CTR KO

    mouse CTR EE

    human

    Ethylmalonic acid

    (urine)

  • Respiratory chain complex activities

    n.s.

    ** **

    **

    **p

  • Phylogenetic search

    Bradyrhizobium japonicum 27376118 9

    Legionella pneumophila 54293763 9

    Legionella pneumophila 52841017 9 Danio rerio 47086181 9

    Caenorhabditis elegans 17538952 9 Apis mellifera 48140497 9

    Drosophila melanogaster 24652740 9

    Drosophila melanogaster 27819799 9 Anopheles gambiae 31210533 9 Anopheles gambiae 55240363 9

    Oryza sativa 56202167 9 Oryza sativa 34910930 9

    Arabidopsis thaliana 12324040 9 Arabidopsis thaliana 1644427 9 Arabidopsis thaliana 22655048 9

    Xenopus laevis 27371293 9

    Pan troglodytes 55649315 9 Homo sapiens ETHE1

    Mus musculus 53236965 9 Rattus norvegicus 27676450 9

    Burkholderia cepacia 46322841 9 F

    Burkholderia cepacia 46317848 9 F Ralstonia eutropha 45515657 9

    Ralstonia metallidurans 48771425 9 Nostoc punctiforme 23128919 9 F

    Synechococcus elongatus 56750614 9

    Nostoc sp. 17231396 9 Anabaena variabilis 45507470 9

    Synechocystis sp. 16330445 9

    Thermosynechococcus elongatus

    Polaromonas sp. 54032671 9 F

    ??

    Fusion events Operon

    ?? soxY soxA ?? soxB soxZ soxX

    ??

    ??

    ??

    Ethe1-related domain

    Rhodanese-related sulfur-transferase domain

    Domain of unknown function

    Predicted permease Glutathione S-transferase

    Sulfur oxidase gene cluster

    ETHE1-like proteins are present in operons containing Rhodanese-like proteins or

    as ETHE1/Rhodanese chimaeric proteins

    The Sörbo assay

  • Sulfur metabolism

    Szabo, Nat Rev Drug Discov 2007

    Thiosulfate in urines

    EE-Patients

    n m

    o l/ m

    g c

    re a ti n

    in e

    0

    500

    1000

    1500

    2000

    2500

    CTR 0

    2000

    4000

    6000

    8000

    10000

    12000

    14000

    16000

    18000

    20000

    n m

    o l/ m

    g c

    re a ti n

    in e

    CTR KO mice

    0

    40

    80

    120

    160

    200

    +/+ +/+ +/+ +/+ +/+ -/- -/- -/- -/- -/-

     M

    and tissues

  • LIVER

    0,0

    500,0

    1000,0

    1500,0

    2000,0

    2500,0

    3000,0

    3500,0

    MUSCLE

    0,0

    500,0

    1000,0

    1500,0

    2000,0

    2500,0

    CTR KO CTR KO

    p

  • H2S is a metabolically active compound

    Szabo, Nat Rev Drug Discov 2007

  • H2S inhibits COX and SCAD activities

    0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0

    0

    2 0

    4 0

    6 0

    8 0

    1 0 0

    1 2 0

    N a S H ( µ M )

    re si

    d u

    al a

    ct iv

    it y SCAD in liver COX/CS

    NaSH (µM)

    re s

    id u

    a l a

    c ti

    v it

    y

    120

    0 1 2 3 4 5 6 7 8 9 10 11 12 13

    0

    20

    40

    60

    80

    100

    NaCN

    MUSCLE

    BRAIN

    LIVER

    0 2 4 6 8 10 12

    0

    20

    40

    60

    80

    100

    120

    NaSH (µM)

    re s id

    u a

    l a c ti

    v it

    y

    CI and CS in muscle

    CI/CS

    CS

    Tiranti et al, Nat. Med. 2009

  • A mitochondrial pathway for H2S detoxification

    Jorns MS et al, 2012

  • 0

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