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  • Anne Joutel - CADASIL, Mechanisms of Disease

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    Fondation Singer-Polignac
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    Among the important advances in the field of small vessel disease (SVD) of the brain has been the identification of genes involved in mendelian forms of SVD. CADASIL, with an autosomal dominant mode of transmission, is an archetypal SVD that emerges as the most common heritable cause of stroke and vascular dementia worldwide. The disease is caused by highly stereotyped missense mutations that alter the number of cysteine residues in the extracellular domain of Notch3, a transmembrane receptor that undergoes a series of proteolytic cleavages upon ligand binding that release the extracellular domain (Notch3ECD) and ultimately enable the intracellular domain to translocate to the nucleus, where it acts as a transcriptional activator. Notch3 is predominantly expressed in smooth muscle cells and is critical for the maturation and function of small vessels of the brain. CADASIL mutations differentially affect Notch3 transcriptional activity, however, it is still highly debated whether this may contribute to the disease process. On the other hand, all CADASIL-associated Notch3 mutations are associated with early accumulation of Notch3ECD at the plasma membrane of smooth muscle cells, in extracellular deposits called Granular Osmiophilic Material. Importantly, recent studies lend support to a Notch3ECD cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3ECD is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Finally, a unique collection of mouse models, including one (TgPAC-Notch3R169C) that recapitulates the preclinical stage of the disease, has been generated. Analysis of these mice provides compelling evidence for cerebrovascular dysfunction and microcirculatory failure as the earliest consequences of pathogenic mutant Notch3, culminating in progressive hypoperfusion and white matter damages.

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    Among the important advances in the field of small vessel disease (SVD) of the brain has been the identification of genes involved in mendelian forms of SVD. CADASIL, with an autosomal dominant mode of transmission, is an archetypal SVD that emerges as the most common heritable cause of stroke and vascular dementia worldwide. The disease is caused by highly stereotyped missense mutations that alter the number of cysteine residues in the extracellular domain of Notch3, a transmembrane receptor that undergoes a series of proteolytic cleavages upon ligand binding that release the extracellular domain (Notch3ECD) and ultimately enable the intracellular domain to translocate to the nucleus, where it acts as a transcriptional activator. Notch3 is predominantly expressed in smooth muscle cells and is critical for the maturation and function of small vessels of the brain. CADASIL mutations differentially affect Notch3 transcriptional activity, however, it is still highly debated whether this may contribute to the disease process. On the other hand, all CADASIL-associated Notch3 mutations are associated with early accumulation of Notch3ECD at the plasma membrane of smooth muscle cells, in extracellular deposits called Granular Osmiophilic Material. Importantly, recent studies lend support to a Notch3ECD cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3ECD is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Finally, a unique collection of mouse models, including one (TgPAC-Notch3R169C) that recapitulates the preclinical stage of the disease, has been generated. Analysis of these mice provides compelling evidence for cerebrovascular dysfunction and microcirculatory failure as the earliest consequences of pathogenic mutant Notch3, culminating in progressive hypoperfusion and white matter damages.

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    Attribution Non-Commercial (BY-NC)
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    2K vizualizări35 pagini

    Anne Joutel - CADASIL, Mechanisms of Disease

    Încărcat de

    Fondation Singer-Polignac
    Among the important advances in the field of small vessel disease (SVD) of the brain has been the identification of genes involved in mendelian forms of SVD. CADASIL, with an autosomal dominant mode of transmission, is an archetypal SVD that emerges as the most common heritable cause of stroke and vascular dementia worldwide. The disease is caused by highly stereotyped missense mutations that alter the number of cysteine residues in the extracellular domain of Notch3, a transmembrane receptor that undergoes a series of proteolytic cleavages upon ligand binding that release the extracellular domain (Notch3ECD) and ultimately enable the intracellular domain to translocate to the nucleus, where it acts as a transcriptional activator. Notch3 is predominantly expressed in smooth muscle cells and is critical for the maturation and function of small vessels of the brain. CADASIL mutations differentially affect Notch3 transcriptional activity, however, it is still highly debated whether this may contribute to the disease process. On the other hand, all CADASIL-associated Notch3 mutations are associated with early accumulation of Notch3ECD at the plasma membrane of smooth muscle cells, in extracellular deposits called Granular Osmiophilic Material. Importantly, recent studies lend support to a Notch3ECD cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3ECD is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Finally, a unique collection of mouse models, including one (TgPAC-Notch3R169C) that recapitulates the preclinical stage of the disease, has been generated. Analysis of these mice provides compelling evidence for cerebrovascular dysfunction and microcirculatory failure as the earliest consequences of pathogenic mutant Notch3, culminating in progressive hypoperfusion and white matter damages.

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    din 35

    CADASIL

    Mechanisms of disease
    Anne JOUTEL

    Mechanisms of CADASIL: key issues

    ?
    SMC/pericytes

    a-Notch3ECD

    1. The earliest cerebrovascular changes?


    2. Notch3 receptor malfunction? 3. Mechanisms of Notch3ECD/GOM deposits formation? 4. Novel function gained by mutant Notch3?

    Modeling CADASIL in the mouse to define the earliest cerebrovascular changes


    Background Strain(s) Mouse (K-in) R142C Mouse (K-in) R170C TgSM22ahumN3R90C TgSM22ahumN3C428S humN3C455RStop/ lox; TgSM22aCre humN3R1031CStop /lox; TgSM22aCre TgPACratN3R169C
    ? 129/swiss C57BL/6

    Notch3 expression level


    endogenous endogenous Overexpression SMC promoter (~1 fold) SMC promoter Overexpression (~1.5 fold) SMC promoter Overexpression ? SMC promoter overexpression ? Notch3 promoter overexpression (~4 fold)

    Notch3ECD / GOM
    +/+ +/+

    Migraine/ CSD
    nd nd +

    White matter lesions


    -

    Lacunar infarcts
    (?) 7% -

    Motor / cognitive deficits


    ? 12% -/nd

    C57BL/6

    +/+

    nd

    -/nd

    129/BL6

    nd/+

    nd

    -/nd

    129/BL6

    nd/+

    nd

    -/nd

    FVB

    +/+

    nd

    -/nd

    Joutel; bioessays, 2011

    TgPAC-Notch3R169C mice recapitulate the presymptomatic stage of the disease


    White matter changes GOM deposits Notch3ECD deposits 0 1 6 12 20 months

    a-N3ECD

    a-MBP Joutel et al, J. Clin Invest. 2010 Cognat et al, in preparation

    Ultrastructural correlate of initial white matter lesions: intramyelinic oedema


    E

    F
    Myelin- associated vacuoles (%)

    Cognat et al, in preparation

    Vacuoles (%)

    Secondary axonal degeneration

    a-MBP a-NF

    Cognat et al, in preparation

    Preserved integrity of Blood-Brain-Barrier

    Joutel et al, J. Clin Invest. 2010

    No overt arterial stenosis

    Joutel et al, J. Clin Invest. 2010

    Impaired adaptative responses of CBF prior to the occurrence of brain lesions

    A- CBF autoregulation

    B- Functional hyperemia

    Joutel et al, J. Clin Invest. 2010

    Arterial & arteriolar myogenic responses are attenuated


    A B- pial arteries

    C- intraparenchymal arterioles

    Joutel et al, J. Clin Invest. 2010 Dabertrand, Joutel & Nelson, in preparation

    The earliest cerebrovascular changes in TgPAC-Notch3R169C mice

    Myelin vacuolation > axonal degeneration GOM deposits

    Notch3ECD deposits

    12 Cerebral hypoperfusion

    20 months

    Capillary regression Cerebrovascular dysfunction

    Key issues

    ?
    SMC/pericytes

    a-Notch3ECD

    The earliest cerebrovascular changes?


    Notch3 receptor malfunction? Mechanisms of Notch3ECD/GOM deposits formation? Novel function gained by mutant Notch3?

    Notch3 plays a key role in the structure and function of small arteries

    Ligands (Jagged1)

    Arterial morphogenesis (brain)


    Notch3 S1 S2 S3 NICD

    Arterial SMC differentiation and maturation Arterial tone Transcriptional activator: Notch3, Grip2, Nrip2, HeyL, Kv1.5...

    RBPJ GTGGGAA

    Domenga et al, Genes & Dev 2004 Prowler et al, PNAS 2007 Belin de Chantemle et al, ATVB 2008 Fouillade et al, Cardiovasc Res. 2012 Fouillade et al, ATVB 2013

    A few CADASIL mutations in the ligand binding domain abolish Notch3 activity
    15

    % of Mutations

    10

    Ligand binding

    34 EGFR

    RBPJ-LacZ

    Monet et al, HMG 2007 Monet et al, Brain 2009

    Knock-in mice with the archetypal Arg170/169Cys mutation retain normal Notch3 signaling in the brain arteries A

    Cognat et al, submitted

    Elimination of wildtype Notch3 in TgN3R169C mice does not exacerbate white matter lesions

    > Reduced Notch3 activity is unlikely the driving force in CADASIL


    Cognat et al, submitted

    Key issues

    ?
    SMC/pericytes

    a-Notch3ECD

    The earliest cerebrovascular changes?


    Notch3 receptor malfunction? Mechanisms of Notch3ECD/GOM deposits formation? Novel function gained by mutant Notch3?

    Notch3ECD deposits occur independently of ligand activation of the receptor

    Ligands (Jagged1)

    Notch3C428S
    S1

    TghN3WT,N3+/+
    S2

    TghN3C428S,N3+/+

    TghN3C428S,N3-/-

    S3

    RBPJ

    Notch3ECD is a component of GOM deposits


    a-Notch3ECD

    SMC

    Joutel et al, JCI 2000 Ishiko et al, Acta Neuropathol 2006 Monet-Leprtre et al, Brain 2013

    Mutant Notch3ECD accumulates in disulfide cross-linked, detergent-insoluble aggregates


    A B

    Brain arteries RIPA 25,000g S1 P1 SDS2% 25,000g S2 P2 Laemmli+me 25,000g S3 P3

    Notch3ECD

    RIPA

    SDS 2%

    laemmli+ me

    S1

    S2

    S3

    Nano LC MS/MS
    Monet-Leprtre et al, Brain 2013

    TIMP3 accumulates in the brain vessels of mouse models and patients with CADASIL

    nonTg

    TgN3R169C

    control

    patient

    a-Notch3ecd

    a-TIMP3 a-SMA

    TIMP3 deposits with Notch3ECD in GOM

    A a-TIMP3

    control

    patient

    patient

    patient

    patient

    a-TIMP3

    a-N3ecd

    merge

    a-TIMP3

    Vitronectin abnormally deposits in Notch3EC aggregates in CADASIL brain vessels


    TgNotch3WT

    a-Vtn

    a-N3ecd

    merge

    TgNotch3R169C a-Vtn control a-Vtn a-N3ecd patient

    merge patient

    Monet-Leprtre et al, Brain 2013

    Excess Notch3ECD promotes TIMP3-Notch3ECD interaction and TIMP3 accumulation


    A B C

    Monet-Leprtre et al, Brain 2013

    TIMP-3 interacts with Vitronectin and promotes Notch3-Vitronectin complex formation


    A B

    Monet-Leprtre et al, Brain 2013

    Proposed model for Notch3ECD/GOM deposits formation

    (1)

    (2)

    (3)

    (4)

    WT

    Mutant Notch3

    TIMP3

    Vitronectin

    Others

    Key issues

    ?
    SMC/pericytes

    a-Notch3ECD

    The early (potentially causative) cerebrovascular changes? Notch3 receptor malfunction? Mechanisms of Notch3ECD/GOM deposits formation? Novel function gained by mutant Notch3?

    General properties of the four Tissue inhibitor metalloproteinases (TIMPs)


    Location TIMP1 TIMP2 Secreted in the ECM Secreted in the ECM Bound to the ECM MMPs inhibited All MMPs ADAM10 All MMPs Other functions Strong inhibitor of MMP9 Activates MMP2 Genetic disorder

    TIMP3

    All MMPs ADAM10 ADAM17 (TACE) All

    - Apoptosis - Inhibits angiogenesis (VEGFR2) Inhibits angiogenesis

    Sorbsby fundus dystrophy

    TIMP4

    Secreted in the ECM

    From Rosenberg GA, Lancet Neurol 2009

    Increasing TIMP3 expression impairs cerebrovascular responses


    A
    Hum BAC 419C14 (193kB) TIMP3

    B
    a-TIMP3

    30-kDa 20-kDa

    a-SMA

    40-kDa

    TIMP3 protein level (fold change)

    non-Tg TgBAC-TIMP3(2)

    8 7 6 5 4 3 2 1 0 non-Tg TgBAC-TIMP3

    C. Capone & A. Joutel

    Reducing TIMP3 rescues cerebrovascular dysfunction in TgNotch3R169C mice


    A

    B
    Myogenic responses (% passive diameter)

    pressure

    C. Capone, C. Baron & A. Joutel

    Superfusion of TIMP3, but neither of TIMP1 nor of TIMP2, impairs neurovascular function
    A B Notch3R169C TIMP3

    ?
    MMPs C
    40

    TIMPs

    rTIMP2 topical (1mg/mL)

    D
    40

    rTIMP1 topical (1mg/mL)

    CBF (% increase)

    30 20 10 0

    CBF (% increase)
    Whisker Ringer
    n=5

    30 20 10 0

    ACh

    Adenosine Timp2(1mg/mL)

    Whisker Ringer
    n=5

    ACh

    Adenosine Timp1(1mg/mL)

    C. Capone & A. Joutel

    ADAM-17 (TACE) superfusion ameliorates neurovascular dysfunction in TgTIMP3 and TgNotch3R169C mice
    A Notch3R169C TIMP3 B

    ?
    ADAM17 (TACE) C D

    C. Capone & A. Joutel

    The Notch3ECD cascade hypothesis


    Cysteine mutation

    Notch3ECD deposits

    Matrisome of the neurovascular unit (TIMP3..)

    Cerebrovascular dysfunction

    Reduction Capillary bed

    Structural alterations Brain arteries

    Energetics/ionic homeostasis failure

    CADASIL team- 2013

    ALUMNI B. Bardot (post-doc), S. Cleophax (tech Staff), C. Dussaule (MSc student), P. Fardoux (PhD student), C. Fouillade (PhD student), P. Lacombe (Scientist), B. Lemaire (tech Staff), M. Monet-Leprtre (PhD student, post-doc), M. Fouillot-Pancha (tech Staff), M. Riani (MSc student)

    Collaborators
    I Haddad & J. Vinh (ESPCI, Paris, France) M. Cohen-Tannoudji & C. Babinet+ (Pasteur Institute, Paris, France) L Mesnard & E. Rondeau (Inserm, Paris, France) D. Henrion (Inserm, Angers, France) M Dewerchin (VIB, Leuven, Belgium) C. Gosele & N. Hbner (MDC Berlin, Germany) C. Haffner & M. Dichgans (Ludwig-Maximilians-University, Munich, Germany) H. Stoehr & B. Weber (Regensburg University, Germany) S. Christensen, J. Stavenhagen & J. Pedersen (Lundbeck, Valbi, Denmark) T. Gridley (Jackson Lab, Bar Harbor, USA) K. Takamiya & R. Huganir (The Johns Hopkins University School of Medicine, Baltimore, USA)F. Cenk Ayata & M. Moskowitz (Harvard Medical School, Charlestown, USA) Dabertrand & MT Nelson (University of Vermont, Burlington, USA) Brain banks : S. Turbant (GIE-NeuroCeb, Paris), JJ. Hauw (CHU Pitie-Salpetrire, Paris), F. Chapon (CHU Caen), C. Godfraind (Clinique St Luc, Bruxelles, Belgium), H. Kalimo (University of Turku, Finland), T. Arzberger (Brain Net, Munich, Germany) Facilities: C. Martin (Transgenic Mouse facility, TAAM, Villejuif), C. Thibault (MicroArray facility, IGBMC, Illkirch), J.P. Rio (Electron microscopy facility, IFM, Paris) Funding : National Institute Health (NINDS), European Community (Marie Curie ITN), Agence Nationale de la Recherche, Fondation Leducq, H Lundbeck A/S, Inserm, Universit Paris 7, Gis Maladies Rares, Fondation pour la Recherche Mdicale, Fdration pour la Recherche pour le Cerveau

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