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BRAINA JOURNAL OF NEUROLOGY

Abnormal recruitment of extracellular matrixproteins by excess Notch3ECD: a newpathomechanism in CADASILMarie Monet-Lepretre,1,2 Iman Haddad,3 Celine Baron-Menguy,1,2 Maı Fouillot-Panchal,1,2

Meriem Riani,1,2 Valerie Domenga-Denier,1,2 Claire Dussaule,1,2 Emmanuel Cognat,1,2

Joelle Vinh3 and Anne Joutel1,2

1 INSERM, U740, Paris, F-75010, France

2 Univ Paris Diderot, Sorbonne Cite, UMR S740, Paris, F-75010, France

3 CNRS ESPCI ParisTech, USR3149, SMBP, Paris France

Correspondence to: Anne Joutel

Faculte de Medecine Paris Diderot,

site Villemin,

10 av de Verdun,

75010 Paris,

France

E-mail: [email protected]

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most

common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and

extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the

NOTCH3 receptor (Notch3ECD) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 muta-

tions differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3ECD-

containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel

pathology. Herein, we hypothesized that excess Notch3ECD could recruit and sequester functionally important proteins within

small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immuno-

histochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL

that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and

artery samples demonstrated that mutant Notch3ECD accumulates in disulphide cross-linked detergent-insoluble aggregates in

mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important

extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into

Notch3ECD-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the

formation of Notch3ECD–TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex

formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of

both insoluble cross-linked and soluble TIMP3 species. Moreover, reverse zymography assays show a significant elevation of

TIMP3 activity in the brain vessels from mice and patients with CADASIL. Collectively, our findings lend support to a Notch3ECD

cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3ECD in the brain

vessels is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that

may ultimately cause multifactorial toxicity. Specifically, our results suggest a dysregulation of TIMP3 activity, which could

contribute to mutant Notch3ECD toxicity by impairing extracellular matrix homeostasis in small vessels.

doi:10.1093/brain/awt092 Brain 2013: 136; 1830–1845 | 1830

Received October 3, 2012. Revised January 21, 2013. Accepted February 20, 2013. Advance Access publication May 6, 2013

� The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.

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Keywords: CADASIL; Notch3; protein aggregation; extracellular matrix proteins; cerebrovasculature

Abbreviations: CADASIL = cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy;Notch3ECD = NOTCH3 extracellular domain; RIPA = radio-immunoprecipitation assay

IntroductionSmall vessel disease of the brain is a major contributor to stroke

and a leading cause of vascular cognitive impairment in human

adults. Clinical manifestations result from the occurrence of

multiple subcortical lacunar infarctions and extensive white

matter injuries (Dichgans, 2007; Pantoni, 2010). CADASIL is the

most common causative diagnosis of hereditary small vessel dis-

ease of the brain caused by dominant mutations in the NOTCH3

receptor (Joutel et al., 1996; Chabriat et al., 2009). The under-

lying vasculopathy involves primarily the leptomeningeal and small

penetrating arteries and is characterized by progressive loss of

vascular smooth muscle cells, prominent thickening of the vessel

wall by various types of collagens and extracellular accumulation

of the so-called granular osmiophilic material (Ruchoux et al.,

1995; Tikka et al., 2009; Dong et al., 2012).

NOTCH3 receptor exists at the plasma membrane as a hetero-

dimer, which consists of a 210 kDa ectodomain (Notch3ECD) con-

taining 34 epidermal growth factor-like repeats, non-covalently

attached to a 97 kDa membrane tethered intracellular domain

(Notch3TMIC) (Joutel et al., 2000). In response to ligand binding,

NOTCH3, like the other Notch receptors, undergo sequential pro-

teolytic cleavages that release the Notch intracellular domain,

which translocates to the nucleus where it binds to the transcrip-

tion factor RBPJ and co-activators to activate the transcription of

genes (Kopan and Ilagan, 2009). Notch3 is predominantly

expressed in vascular smooth muscle cells of small arteries and

pericytes of brain capillaries. Studies in the mouse have demon-

strated a critical role for Notch3 in mural cell investment, arterial

differentiation and maturation of vascular smooth muscle cells of

small arteries (Domenga et al., 2004; Liu et al., 2010; Fouillade

et al., 2012).

Patients with CADASIL harbour highly stereotyped NOTCH3

mutations. The vast majority of mutations are missense mutations

and virtually all mutations hitherto reported result in an odd

number of cysteine residues within a given EGF-like repeat

(Joutel et al., 1997; Peters et al., 2005). Yet, the connection be-

tween NOTCH3 mutations and small vessel pathology is un-

known. On one hand, cell-based systems and in vivo studies

have shown that mutations differentially affect NOTCH3–RBPJ

activity, with a few mutations behaving as loss of function or

hypomorphic mutations and many others appearing not to

impair NOTCH3 receptor function and canonical signalling

(Joutel et al., 2004; Peters et al., 2004; Monet et al., 2007,

2009; Arboleda-Velasquez et al., 2011). Moreover, whether

impaired NOTCH3 activity might contribute to the disease pheno-

type is still a matter of debate because total loss of NOTCH3

in the mouse is not associated with a CADASIL phenotype

and genotype–phenotype correlation analyses suggest that

CADASIL-associated loss of function mutations are associated

with an attenuated clinical phenotype (Domenga et al., 2004;

Monet-Lepretre et al., 2009). On the other hand, the common

denominator to CADASIL-associated mutations is the presence of

non-fibrillar deposits of Notch3ECD and granular osmiophilic ma-

terial in the vessel wall. Notch3ECD accumulates as microscopic

extracellular aggregates around vascular smooth muscle cells and

brain pericytes and has been recently recognized as a component

of granular osmiophilic material (Joutel et al., 2000, 2001; Lesnik

Oberstein et al., 2003; Ishiko et al., 2006; Monet et al., 2007,

2009; Tikka et al., 2009; Joutel et al., 2010). The observation in

transgenic mouse models that Notch3ECD accumulation is one of

the earliest events in pathogenesis suggests that it might be the

proximate cause of cellular pathology (Monet et al., 2007, 2009;

Joutel et al., 2010). Herein we tested the hypothesis that excess

Notch3ECD can recruit and sequester functionally important pro-

teins. To achieve this, we used a combination of cerebral and

arterial tissue derived from patients with CADASIL and mouse

models of CADASIL to perform biochemical, nano-liquid chroma-

tography-tandem mass spectrometry (nanoLC-MS/MS) and

immunohistochemical analyses. Post-mortem CADASIL brain tis-

sues exhibit robust pathology, although in the end-stage of the

disease process, with prominent vascular smooth muscle cell

degeneration and vessel fibrosis, whereas material derived from

CADASIL mouse models shows fewer lesions, but, in the early

stage of the disease.

Materials and methodsAdditional information is provided in the Supplementary material.

Brain and artery samples

Human brain tissue

We used frozen or paraffin embedded samples (frontal, temporal or

occipital lobes) from seven deceased elderly patients with CADASIL

(mean age 62.8, range 49–70 years) with molecular genetically

confirmed NOTCH3 mutations (R153C, C1261R, R169C, R133C and

R110C) and nine deceased elderly control subjects (mean age 68,

range 48–86 years) with no known cerebrovascular disorders. All

human samples were stored and handled in accordance with the

French bioethics laws and the study was approved by the

Institutional Review Board of INSERM.

Human brain vessels

Microvessels were isolated from occipital or frontal lobes as described

previously (Yousif et al., 2007). Briefly, brain tissue was weighed

(1–2 g), minced with a scalpel and homogenized in cold PBS (20 ml)

with 20–40 up-and-down strokes in a glass homogenizer.

Homogenate was mixed with an equal volume of cold PBS containing

30% dextran. The suspension was then centrifuged at 6000g for

20 min at 4�C. The supernatant was discarded and the pellet was

resuspended in PBS containing 1% bovine serum albumin. The sus-

pension was then poured through a 40 mm nylon mesh, triturated and

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abundantly washed with PBS. Microvessels were collected in PBS by

inversion of the nylon mesh and pelleted by centrifugation. Purity of

microvessel preparations was monitored by phase-contrast microscopy.

Murine brain vessels

We used TghNotch3(WT), TghNotch3(R90C) and TghNotch3(C428S)

mice, which express a human NOTCH3 transgene, with the wild-type

or mutant sequence, in the C57BL/6J background (Monet et al., 2007,

2009). The transgene in the TghNotch3(WT) and TghNotch3(R90C)

mice is expressed at the homozygous state. TgNotch3WT and

TgNotch3R169C are transgenic mice overexpressing the wild-type or

mutant rat Notch3 locus, respectively, at the heterozygous state, in

the FVB/N background (Joutel et al., 2010). Cerebral arteries, includ-

ing arteries of the circle of Willis and the medium-sized branches, were

dissected under microscope, immediately snap frozen in liquid nitrogen

and stored at �80�C until use. The experimental procedures con-

formed to the national guidelines for the use of animals in research

and were approved by the Ethics committee on animal experiment

(Local committee of University Paris Diderot, Lariboisiere-Villemin).

Biochemical proceduresAll extraction buffers contained a mixture of protease inhibitors

(CompleteTM

protease inhibitor mixture; Roche). For experiments

using quantitative amounts of protein, total protein concentration

was determined with the BCA protein assay kit (Pierce) using bovine

serum albumin as a standard.

Total vessel lysate

Brain vessels were homogenized using a ground glass homogenizer in

SDS buffer (2% SDS, 50 mM Tris, pH 7.4) with �30 strokes and

rotated at room temperature for 1 h.

Sequential biochemical fractionation

Human brain tissue samples (1.5–2.0 g) were homogenized on ice in

10 ml/g of buffer A (2 M NaCl, 10 mM HEPES/NaOH pH 7.4, 1 mM

EDTA, 10 mM N-methylmaleimide) using a Potter-Elvehjem glass/glass

homogenizer (30 strokes) and sedimented by ultracentrifugation at

100 000g for 1 h. The supernatant was saved as the high-salt (S1)

fraction and the resulting pellet (P1) was then homogenized in

10 ml/g of buffer B (100 mM NaCl, 10 mM HEPES/NaOH pH 7.4,

1 mM EDTA, 10 mM N-methylmaleimide) containing 2% SDS and

rotated at room temperature for 1 h before sedimentation at

100 000g at 15�C for 1 h. The supernatant was saved as the SDS

(S2)-soluble fraction and the remaining pellet (P2) was re-extracted

in SDS-Laemmli buffer (0.44 ml/g) with 10% 2-mercaptoethanol for

at least 24 h before centrifugation at 25 000g for 30 min at 12�C and

saved as the ‘SDS + ß-mercaptoethanol’ fraction. In another set of

experiments, the pellet P1 was resuspended in buffer B containing

either 1% TritonTM X-100 or 4 M urea or 6 M guanidine hydrochloride

or a combination of 7 M urea, 2 M thiourea and 3% CHAPS or was

extracted in 99% formic acid. All resuspended P1 were rotated for 1 h

and sedimented at 100 000g for 1 h. The supernatants were saved as

the S2 fractions and a final extraction using SDS-Laemmli buffer with

10% 2-mercaptoethanol was conducted on the resulting pellets (P2).

Human or murine brain vessels were homogenized on ice using a

ground glass homogenizer in radio-immunoprecipitation assay (RIPA)

buffer (150 mM NaCl, 50 mM Tris–HCl, 1% NP-40, 0.1% SDS, 0.5%

sodium deoxycholate supplemented with 10 mM N-methylmaleimide)

and centrifuged at 25 000g for 30 min at 4�C. The supernatant was

retained (S1), and the resultant insoluble pellet (P1) was extracted for

several hours in SDS buffer (3% SDS, 125 mM Tris–HCl, 10 mM

N-methylmaleimide) and centrifuged at 25 000g for 30 min at 11�C.

The supernatant was saved as the S2 fraction and a final extraction

using SDS-Laemmli buffer with 10% 2-mercaptoethanol or 20 mM

dithiothreitol was conducted on the resulting pellet (P2) before centri-

fugation at 25 000g for 30 min at 11�C. S1 and S2 fractions were

concentrated by acetone precipitation.

Western blot analyses

Samples were mixed with SDS-Laemmli buffer (4% SDS, 125 mM Tris–

HCl, pH 6.8, 20% glycerol) containing 10% 2-mercaptoethanol or

20 mM DTT, passed through a 29 gauge needle 15–20 times, clarified

by centrifugation at 25 000g for 30 min at 11�C and heated for 5 min

at 95�C before SDS-PAGE. Protein extracts were electrophoresed on a

6% Tris–glycine SDS-PAGE or 4–12% Bis-tricine NUPAGE (Invitrogen)

and transferred to a nitrocellulose membrane. The following primary

antibodies were used: mouse monoclonal anti-Notch3ECD (clone 5E1,

dilution 1:500; clone 11A1, dilution 1:1000; Joutel et al., 2000),

mouse monoclonal anti-vitronectin (Clone BV2, Chemicon, dilution

1:200), rabbit monoclonal anti-TIMP3 (clone D74B10, Cell Signaling,

dilution 1:2500), mouse monoclonal anti-smooth muscle alpha actin

(clone 1A4, Dako, dilution 1:25 000) and mouse monoclonal anti-ß

actin (clone AC15, Sigma, dilution 1:10 000). The blots were incubated

with the primary antibody at 4�C overnight followed by secondary

polyclonal anti-mouse or anti-rabbit immunoglobulins conjugated

with horseradish peroxidase and enhanced chemoluminescence detec-

tion (Thermo Fisher Scientific). Densitometric quantification of band

intensity was performed using ImageJ (version 10.2, NIH).

Reverse zymography assay

Equal amounts of total brain vessel lysates (8 mg) were mixed with

non-reducing Laemmli sample buffer and loaded onto a 12% SDS-

PAGE gel containing 1 mg/ml gelatin and conditioned medium from

baby hamster kidney cells expressing gelatinase A (MMP2), which is

inhibited by all four TIMP proteins (a generous gift from Dylan

Edwards). The gel was washed, incubated for 24 h in rinse buffer

(50 mM Tris pH 7.5, 5 mM CaCl2, 2.5% TritonTM X-100) at room

temperature, then incubated for 18 h in regenerating buffer (50 mM

Tris pH 7.5, 5 mM CaCl2) at 37�C, washed in water and stained with

Coomassie blue. Lysates of cells transfected with human TIMP1,

TIMP2 or TIMP3 were used as controls. Under these conditions,

TIMPs inhibit gelatin digestion by activated gelatinase A, producing

dark blue bands against a lighter background. Identical samples were

run in parallel onto a 4–12% NUPAGE gel, transferred and immuno-

blotted with ß-actin antibody. Densitometric quantification of band

intensity was performed using ImageJ and normalized against ß-actin

signal.

Sample preparation and massspectrometry analysisHuman brain samples and murine brain artery samples were fractio-

nated using RIPA and then SDS-containing buffer. Each murine sample

was prepared with vessels pooled from four transgenic mice. The final

pellet was extracted in 40 ml of Laemmli buffer containing 20 mM

dithiothreitol between 20–48 h and clarified by centrifugation. Thirty

microlitres of protein extracts were denatured by incubation at 95�C

for 5 min, electrophoresed on a 4–12% NUPAGE Bis-tricine gel run for

12 min at 200 V. The gel was recovered, washed in water, stained with

Coomassie blue (Bio-Rad) and extensively washed in water for 2 to 3 h

at room temperature (three changes). Each lane was divided in three

(murine samples) or five (human samples) parts based on size, cut into

1832 | Brain 2013: 136; 1830–1845 M. Monet-Lepretre et al.


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1 mm cubes and put into 1% acetic acid. Hence, each sample further

comprised three or five subsamples.

In-gel tryptic digestion method was used on the purified samples as

described (Shevchenko, 2001). Briefly, after reduction–alkylation

(5 mM dithiothreitol in 50 mM NH4HCO3, 30 min at 56�C; 25 mM

iodoacetamide in 50 mM NH4HCO3, 20 min in dark at room tempera-

ture), gel pieces were digested by incubation with 12.5 ng/ml Trypsin

(modified sequencing grade, Roche) in sodium carbonate, overnight at

37�C with gentle shaking. The reaction was stopped with one volume

(50 ml) 5% formic acid. Subsamples were sonicated for 10 min in an

ultrasonic bath at room temperature and processed for nanoLC-MS/

MS analysis as described in the Supplementary material or stored at

�20�C until use.

Immunohistochemical analysesThe following antibodies (mouse monoclonal anti-vitronectin, Clone

BV2, Chemicon, dilution 1:200; mouse monoclonal anti-TIMP3,

Clone 13613H4, Chemicon, dilution 1:1000) were applied to paraffin

sections of human brain as described in the Supplementary material.

The following antibodies (rabbit polyclonal anti-vitronectin, Oxford

biomedical research, dilution 1:1000 and Genway, dilution 1:1000;

mouse monoclonal anti-TIMP3, Clone 13613H4, Chemicon, dilution

1:200; mouse monoclonal anti-Notch3ECD, clone 5E1, dilution 1:2;

polyclonal anti-Notch3ECD, BC2, dilution 1:1000) were used on

frozen brain sections as described in the Supplementary material.

Immuno-electron microscopy analysisFrozen brain sections were incubated with the mouse monoclonal anti-

TIMP3 (Clone 13613H4, dilution 1:50) and processed as described in

the Supplementary material.

In situ hybridizationA 951 bp human TIMP3 probe (nucleotides, 3964–4915 gi: 75905820)

was made by PCR amplification and cloned into pBluescript�. In situ

hybridization was performed on brain paraffin sections from patients

with CADASIL (n = 3) and control individuals (n = 3) as described in

the Supplementary material.

Expression plasmids, cell culture andco-immunoprecipitation analysis

Expression plasmids

Full-length wild-type NOTCH3, NOTCH3 deletion and point muta-

tions, full-length TIMP3 and deletion mutants, and vitronectin con-

structs (Supplementary Fig. 5) were generated as described in the

Supplementary material.

Cell culture and transfection

HEK 293T cells were grown in GlutamaxTM supplemented with 10%

foetal bovine serum and 1% penicillin–streptomycin. Human coronary

artery smooth muscle cells (Cascade Biologics) were grown in Medium

231 supplemented with Smooth Muscle Growth Supplement (Cascade

Biologics). Plasmids were transfected into subconfluent 293T cells by

using the calcium phosphate precipitation method as described previ-

ously (Joutel et al., 2004). For co-immmunoprecipitation, 293T cells

were grown in 50 cm2 plates and transfected with 20 ng of haem-

agglutinin-tagged TIMP3, 1 mg of haemagglutinin or V5-tagged

vitronectin and 1 mg of FLAG�-tagged NOTCH3 plasmids. For

co-expression experiments, 293T cells were transfected with 200 ng

haemagglutinin-tagged TIMP3 and 1 mg of FLAG�-tagged NOTCH3

plasmids. At 48 h after transfection, the cells were harvested and the

extracts prepared for downstream assays.

Co-immunoprecipitation, cellular fractions and immu-noblot analyses

293T cells were lysed in immunoprecipitation buffer (0.5% TritonTM

X-100, 10 mM TrisCl pH 8, 140 mM NaCl) supplemented with a cock-

tail of protease inhibitors. Immunoprecipitation was performed using

anti-FLAG M2, anti-haemagglutinin or anti-V5 affinity gel (Sigma-

Aldrich). Endogenous NOTCH3 and TIMP3 were immunoprecipitated

from human coronary artery smooth muscle cells with anti-Notch3ECD

rabbit polyclonal antibody (BC2) (Joutel et al., 2000), rabbit immuno-

globulins were used as a control. To prepare cellular fractions, 293T

cells were dislodged from the culture plate in Ca2 + , Mg2 + free PBS,

harvested and lysed in immunoprecipitation or RIPA buffer. The cul-

ture plate was then rinsed several times in PBS, incubated in PBS

containing 5 mM EDTA for 15 min, followed by a final rinse in PBS

and the extracellular matrix was scraped in a small volume of Laemmli

buffer.

Protein extracts were separated on 4–12% NUPAGE gels (Invitrogen)

and transferred onto nitrocellulose membranes. Epitope tags were de-

tected with rabbit polyclonal anti-FLAG� (Sigma-Aldrich dilution,

1:50,000), rabbit polyclonal anti-haemagglutinin (Sigma-Aldrich, dilu-

tion 1:5000) or rabbit polyclonal anti-c-myc, peroxydase conjugate

(Sigma-Aldrich, dilution 1:5,000), vitronectin with rabbit polyclonal

anti-vitronectin (GenWay, dilution 1:5000), followed by secondary poly-

clonal anti-rabbit or anti-mouse immunoglobulins conjugated with

horseradish peroxidase and enhanced chemoluminescence detection

(Thermo Fisher Scientific). Samples of extracellular matrix were run in

parallel on 4–12% NUPAGE gels stained with silver stain (Pierce Silver

stain, Thermo Fisher Scientific). Densitometric quantification of band in-

tensity was performed using ImageJ.

Statistical analysisData are expressed as mean � standard error of the mean (SEM).

Two-group comparisons were analysed by the two-tailed t-test for

independent samples. Multiple comparisons were evaluated by one-

factor ANOVA followed by post hoc test. Results with a P-value

50.05 were considered statistically significant.

Results

Mutant Notch3ECD accumulates indisulphide cross-linkeddetergent-insoluble aggregatesWe first analysed the biochemical properties of Notch3ECD in

human derived material. We performed serial extractions on

brain samples from patients with CADASIL and control subjects

using buffers of increasing protein extraction strength (Fig. 1A).

Consistent with our previous report (Joutel et al., 2000),

Notch3ECD was almost undetectable in the control whereas it ro-

bustly accumulated in the CADASIL sample. In the patient, little or

no Notch3ECD was detected in the high salt and SDS-soluble frac-

tions, wheras a substantial amount was present in the SDS plus



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Figure 1 Biochemical distribution of Notch3ECD in human brain samples and mouse brain vessels. (A) Frontal lobe from a human control

individual and a patient with CADASIL were homogenized and subjected to sequential biochemical extractions using high salt (HS) buffer,

2% SDS and Laemmli + ß-mercaptoethanol (bme) (left). The equivalent of 1.7 mg of brain tissue was loaded per lane for high salt (S1),

2% SDS (S2) and Laemmli + ß-mercaptoethanol (S3) fractions, subjected to 6% SDS-PAGE under reducing conditions and analysed by

immunoblotting with the 5E1 anti-Notch3ECD antibody (right). The blot shown is representative from experiments using post-mortem

brain tissue samples from five patients with CADASIL and three control subjects. (B) Frontal lobe from a patient with CADASIL was

subjected to sequential biochemical extractions using high salt buffer, various detergents or denaturants including 1% TritonTM X-100,

4 M urea, a combination of 7 M urea, 2 M thiourea, 3% CHAPS (UTC), 6 M guanidine (Gnd), or 99% formic acid (FA), followed by a final


(continued)


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reducing agent (Laemmli + ß-mercaptoethanol) fraction (Fig. 1A).

The data were confirmed using brain samples from two other

control subjects and four patients (Supplementary Fig. 1). We

then assessed buffers containing other detergents and denatur-

ants, including TritonTM X-100 (1%), formic acid, urea (4 M),

guanidine (6 M), or a mixture of urea (7 M), thiourea (2 M) and

CHAPS (3%), for their ability to extract Notch3ECD. Strikingly, we

found that none of these detergent/denaturants could solubilize

Notch3ECD whereas Notch3ECD was recovered from the resultant

pellet in SDS buffer containing a reducing agent (Fig. 1B).

We extended our analysis to isolated brain arteries of CADASIL

mouse models. TgNotch3WT and TgNotch3R169C mice overexpress

a rat Notch3 transgene �4-fold that of the endogenous Notch3

(Joutel et al., 2010). In TgNotch3WT mice, a low amount of

Notch3ECD was equally extractable in RIPA and SDS containing

ß-mercaptoethanol buffers. In contrast, in TgNotch3R169C mice,

there was a dramatic accumulation of Notch3ECD in the SDS con-

taining ß-mercaptoethanol fraction and to a lesser extent in the

SDS-soluble fraction whereas Notch3ECD was barely extractable in

RIPA buffer alone (Fig. 1C).

We also analysed biochemical properties of Notch3ECD in

TghNotch3(R90C) and TghNotch3(C428S) mutant mice and the

control TghNotch3(WT) mice that express physiological levels of a

human NOTCH3 transgene (�1.5-fold over the endogenous

mouse Notch3). R90C and C428S are active and inactive

NOTCH3–RPBJ mutants, respectively (Joutel et al., 2004; Monet

et al., 2007, 2009). Of interest, the transgene in these mice can

be specifically recognized using a monoclonal antibody specific to

human Notch3ECD (Ruchoux et al., 2003). We found that, in wild-

type arteries, Notch3ECD was predominantly recovered in RIPA

buffer and that the majority of both human and endogenous

Notch3ECD was detected in this fraction (Supplementary Fig. 2A

and B). By contrast, in both TghNotch3(R90C) and

TghNotch3(C428S) mice, mutant Notch3ECD was poorly extract-

able in RIPA buffer whereas it accumulated in the SDS + ß-mer-

captoethanol fraction (Supplementary Fig. 2B). Taken together,

the data suggest that mutant Notch3ECD is incorporated into

higher order multimers, cross-linked by disulphide bonds.

TIMP3 and vitronectin are enriched inthe Notch3ECD-enriched fraction ofCADASIL samplesNext, we determined the proteome of the Notch3ECD-enriched frac-

tion, hypothesizing that proteins sequestered by Notch3ECD should

be enriched in this fraction. To achieve this, we performed a sensitive

and semi-quantitative proteomic analysis using nanoLC-MS/MS and

the spectral counting approach (Liu et al., 2004). We first conducted

a pilot proteomic study using post-mortem brain tissue from one

patient with CADASIL and one control subject. Consistent with the

claim that we were surveying the Notch3ECD enriched fraction, the

CADASIL sample contained 18 distinct peptides of NOTCH3, exclu-

sively derived from the Notch3ECD sequence, whereas the control

sample contained none. We identified a total of 323 and 281 proteins

in the control and CADASIL samples, respectively, 104 of which were

enriched in the CADASIL sample. Among these, 72 proteins, includ-

ing predominantly extracellular matrix proteins, were present in the

CADASIL sample and absent in the control (Supplemental Table 1).

To identify proteins that were differentially expressed at the

early stage of the disease process, we repeated the proteomic ana-

lysis using brain arteries from 10–12 month old wild-type

TghNotch3(WT) and mutant transgenic TghNotch3(R90C) mice.

Proteomic experiments conducted on three pairs of wild-type and

mutant samples identified 39 and 48 proteins in wild-type and

mutant samples, respectively, proteins being identified by the con-

sensus of two or more unique peptides in at least two of three bio-

logical replicates (Supplementary Table 2). Again, we identified eight

distinct peptides of NOTCH3, exclusively derived from the

Notch3ECD sequence, in the mutant samples and none in the wild-

type samples. Importantly, two proteins, TIMP3 and VTN, identified

as strongly enriched in the human CADASIL sample, were present in

the mutant arteries and almost absent in the control arteries

(Supplementary Table 2 and Supplementary Fig. 3).

Immunoblot analysis confirmed that TIMP3 and VTN were en-

riched in the SDS plus reducing agent (Laemmli + ß-mercap-

toethanol) fraction of human CADASIL brains when compared

with analogous fraction of control brains (Supplementary Fig. 4A

and B). Likewise, Laemmli + ß-mercaptoethanol fractions prepared

from arterial samples derived from 10–12 month old mice showed

an accumulation of TIMP3 and VTN in the mutant mice compared

with control mice (Supplementary Fig. 4C and D).

TIMP3 and vitronectin are incorporatedinto Notch3ECD-containing depositsWe next evaluated the expression pattern of TIMP3 and VTN in

brain tissue from patients and control subjects and from mouse

models by immunohistochemistry. Examination of CADASIL brain

tissue revealed robust and granular immunoreactivity in the media

of pial and penetrating white matter arteries, with both anti-vitro-

nectin (Fig. 2B and C) and anti-TIMP3 (Fig. 3B and C) antibodies,

bearing a striking resemblance to that seen with anti-Notch3ECD

Figure 1 Continuedextraction with Laemmli + ß-mercaptoethanol. The detergent/denaturant (S2) and corresponding Laemmli + ß-mercaptoethanol (S3)

fractions were analysed by immunoblotting with the 5E1 antibody. The equivalent of 34 mg of brain tissue was loaded per lane for the

formic acid-soluble and insoluble fractions and equivalent of 1.7 mg of brain tissue for all other fractions. (C) Brain arteries from non-

transgenic (non-Tg), TgNotch3WT and TgNotch3R169C mice aged 6 months were subjected to sequential biochemical extractions using

RIPA, 2% SDS and Laemmli + ß-mercaptoethanol (left). Ten micrograms of proteins were loaded per lane for RIPA fractions (S1), 4 mg for

2% SDS fractions (S2) and the resultant 2% SDS-insoluble/Laemmli + ß-mercaptoethanol-extractable fractions (S3) were loaded directly,

and subjected to immunoblot with the 5E1 antibody (upper and middle panels) and smooth muscle actin antibody (SMA, lower panel). A

representative blot from at least two independent experiments is shown.



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antibody (Joutel et al., 2000). In contrast, vessels of control sub-

jects exhibited a weak and homogeneous staining pattern (Figs 2A

and 3A). Likewise, immunofluorescence analysis revealed the pres-

ence of VTN accumulation in the form of dot-like structures within

the brain vessels of TgNotch3R169C mice that was absent in control

TgNotch3WT mice (Fig. 2D–L). Granular VTN immunoreactivity

was detectable in the pial and intracerebral arteries as early as

12 months of age (data not shown), but was marked at 20

months of age (Fig. 2G), whereas it was already obvious in the

brain capillaries at 12 months of age (Fig. 2J). Of note, Notch3ECD

starts to form microscopic aggregates earlier in these mice,

between 1 and 6 months of age (Joutel et al., 2010).

Importantly, we found significant co-localization of VTN and

TIMP3 with Notch3ECD-positive aggregates in brain vessels of

TgNotch3R169C mice and patients with CADASIL, respectively

(Fig. 2I, L and 3F). Owing to the lack of suitable antibodies, we

Figure 2 VTN deposits colocalize with Notch3ECD aggregates. (A–C) Paraffin brain sections from control (A) and CADASIL subjects (B and

C) were stained with the monoclonal anti-VTN antibody (�-Vtn), which robustly detects VTN deposits into the media of CADASIL brain

vessels. The images shown are representative of at least two sections from an experiment using four patients with CADASIL and four

control subjects. (D–L) Frozen brain sections from TgNotch3WT (D–F) and TgNotch3R169C mice (G–L) were stained with anti-Vitronectin

and anti-Notch3ECD antibodies and analysed by immunofluorescence. Representative sections of a pial artery from a 20-month-old

TgNotch3WT mouse (D–F), of two adjacent pial arteries cut longitudinally from a 20-month-old TgNotch3R169C mouse (G–I) and from

capillaries of a 12-month-old TgNotch3R169C mouse (J–L) are shown. Panels are representative of at least six sections from two experi-

ments using three mutant and three control mice. Scale bars: 50 mm (A–C), 26 mM (D–I) and 15 mM (J–L).



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could not analyse TIMP3 distribution in murine tissues, nor

could we examine Notch3ECD and VTN co-localization in human

tissues.

To assess the relationship between Notch3ECD and TIMP3

deposits in more detail, brain sections were processed for

immuno-electron microscopy analysis. Fine localization of VTN

by this technique was hampered by a lack of suitable antibodies.

As shown in Fig. 3H, immunogold labelling of Notch3ECD was

visible at the plasma membrane of vascular smooth muscle cells

in the vicinity of granular osmiophilic material and also within the

granular osmiophilic material deposits, consistent with a recent

report suggesting that Notch3ECD can accumulate into the granu-

lar osmiophilic material (Ishiko et al., 2006). Notably, TIMP3

immunogold labelling was detected in the granular osmiophilic

material deposits, although the silver-enhanced gold particles

were seen mainly at the perimeters of granular osmiophilic mater-

ial and almost absent from the core area (Fig. 3G). Nevertheless,

we acknowledge that the pre-embedding method used for this

Figure 3 TIMP3 and Notch3ECD are incorporated into granular osmiophilic material deposits. (A–C) Paraffin brain sections from control

(A) and CADASIL subjects (B and C) were stained with the monoclonal anti-TIMP3 antibody, which reliably detects TIMP3 deposits in

CADASIL brain vessels. The images shown are representative of at least two sections from an experiment using four patients with

CADASIL and four control subjects. (D–F) Frozen brain sections from CADASIL subjects were stained with anti-TIMP3 and anti-Notch3ECD

antibodies and analysed by immunofluorescence. The images shown are representative of at least four sections from two experiments

using material from three patients with CADASIL. (G and H) Frozen brain sections from a CADASIL subject were processed for immuno-

electron microscopy with anti-TIMP3 (G) or anti-Notch3ECD (H) antibodies. Asterisks indicate granular osmiophilic material deposits.

Immunolabelling appears as black dots (arrows). Panels are representative of at least three tissue blocks from two experiments.

SMC = smooth muscle cell. Scale bars: 50 mm (A–C), 60 mM (D–F) and 1 mM (G and H).



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experiment may have restricted the accessibility of the antibody

and may have not provided with the optimal signal. These results,

taken together, indicate that VTN and TIMP3 are recruited into

CADASIL deposits in vivo.

Increased levels or aggregation ofNOTCH3 enhances NOTCH3–TIMP3complex formation and TIMP3 promotescomplex formation including NOTCH3and VTNNext, we investigated whether TIMP3 and VTN complex with

Notch3ECD, using co-immunoprecipitation assays. We found that a

robust amount of haemagglutinin-tagged TIMP3 (TIMP3-HA) was

pulled down by anti-FLAG antibody in the presence of FLAG-tagged

wild-type NOTCH3 (Flag-N3-WT) but not in the presence of the

NOTCH3 deletion mutant lacking all 34 EGF-like repeats (Flag-

N3�EGFR1-34) (Fig. 4A). Similar results were obtained in the reverse

co-immunoprecipitation experiments (Fig. 4D). Significantly, the

CADASIL-associated mutations in NOTCH3 (R90C, C212S and

C428S) complexes with TIMP3, in a manner similar to wild-type

NOTCH3 (Fig. 4B). In contrast, a very low amount of wild-type

and mutant NOTCH3 co-immunoprecipitated with V5-tagged vitro-

nectin (VTN-V5) using anti-V5 antibody (Fig. 4C). Moreover, no

Flag-N3-WT co-immunoprecipitated with haemagglutinin-tagged

vitronectin (VTN-HA) using the anti-haemagglutinin (HA) antibody

whereas a large amount of Flag-N3-WT co-immunoprecipitated

with TIMP3-HA, confirming that VTN had a much lower affinity

for Notch3ECD than TIMP3 in this assay (Fig. 4D).

We then tested the possibility that TIMP3 complexes with VTN.

We found that anti-V5 could pull down TIMP3-HA in the pres-

ence of VTN-V5 but not in its absence, (Fig.4E). Importantly, Flag-

N3FL-WT could pull down a larger amount of VTN-V5 in the

presence of exogenous TIMP3-HA (Fig. 4E).

It is worth considering that there is an inevitable accumulation of

intracellular Notch3 aggregates in cells transfected with wild-type or

mutant Notch3, even at low expression levels (Opherk et al., 2009).

To determine whether NOTCH3 and TIMP3 associate in the same

complex when expressed at physiological levels, we performed co-

immunoprecipitation of cultured human coronary smooth muscle

cells using anti-Notch3ECD antibody; we were unable to perform

the reverse co-immunoprecipitation assay owing to the lack of suit-

able TIMP3 antibodies. We found that Notch3ECD was able to co-

immunoprecipitate a tiny amount of TIMP3 (Fig. 4F). Collectively the

results suggest that increased levels or aggregation of Notch3ECD

enhances NOTCH3–TIMP3 complex formation and that TIMP3 pro-

motes complex formation including NOTCH3 and VTN. Hence,

TIMP3 was selected for further studies.

Increased levels or aggregation ofNotch3ECD promote TIMP3 recruitmentand accumulationWe next asked whether increased levels or aggregation of

Notch3ECD could influence the expression level and subcellular

localization of TIMP3 using HEK293T cells expressing TIMP3-HA

alone or in combination with FLAG-tagged NOTCH3. It is

important to consider that in this assay NOTCH3 is primarily over-

expressed intracellularly (data not shown). Upon overexpression of

full-length wild-type NOTCH3 we found that the steady-state

level of TIMP3 protein was increased in the cell lysate by 2.6-

fold relative to cells expressing TIMP3 alone (Fig. 5A). TIMP3 is

a secreted protein that distinguishes itself from TIMP1, TIMP2 and

TIMP4 by its ability to bind the extracellular matrix (Lee et al.,

2007). Quantification of TIMP3 bound to the extracellular matrix,

where Notch3ECD is not present (data not shown), revealed a 2.3-

fold decrease in cells overexpressing NOTCH3 versus cells express-

ing TIMP3 alone (Fig. 5B). Significantly, the familial linked

CADASIL R90C mutant modulated TIMP3 protein levels in a

manner similar to wild-type NOTCH3 (Fig. 5A and B).

Interestingly, NOTCH3 protein level was unchanged on overex-

pression of TIMP3 (Supplementary Fig. 6). Importantly, we

checked that transiently transfected cells expressed comparable

levels of TIMP3-HA messenger RNA in these assays (Fig. 5A

and data not shown). Thus, these data indicate that excess

NOTCH3 can promote the recruitment and accumulation of

TIMP3 protein, in the cellular compartment where it is

overexpressed.

We next examined whether NOTCH3-induced TIMP3 protein

level changes required NOTCH3–TIMP3 complex formation.

TIMP3 protein level was unaffected on overexpression of the

NOTCH3 deletion mutant, N3�EGFR1-34, which is unable to

complex with TIMP3 (Fig. 5C). On the other hand, a NOTCH3

construct containing the ectodomain only (Notch3ECD) strongly

upregulated the levels of TIMP3 (Supplementary Fig. 7).

Moreover, a TIMP3 mutant lacking the N-terminal half (TIMP-

3�NTD-HA), which is unable to complex with NOTCH3

(Supplementary Fig. 8), had unchanged expression on overexpres-

sion of NOTCH3 (Fig. 5D), whereas the steady-state level of a

TIMP3 mutant lacking the C-terminal half (TIMP-3�CTD-HA),

which retains the ability to form a complex with NOTCH3, was

increased on NOTCH3 overexpression (Fig. 5E and Supplementary

Fig. 8). Hence, these data suggest that NOTCH3-induced

TIMP3 accumulation requires NOTCH3–TIMP3 complex forma-

tion and that NOTCH3 signalling activity is dispensable for this

effect.

TIMP3 protein level and biologicalactivity in CADASIL brain vesselsWe assessed whether Notch3ECD accumulation/aggregation af-

fects TIMP3 expression and activity in vivo, in brain vessels.

First, microvessels were isolated from post-mortem brain samples

of patients with CADASIL and control subjects and analysed by

western blot. Quantification of total levels of TIMP3 in lysates

from brain vessels revealed a 14.4-fold increase in patients with

CADASIL compared with control subjects. Notably, there was a

dramatic accumulation of high molecular weight TIMP3 species

ranging from 30 kDa to the top of the gel, which likely correspond

to TIMP3 aggregates that were not detected in control subjects

(Fig. 6A). Sequential extractions in RIPA buffer, then SDS buffers



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Figure 4 TIMP3 forms complexes with Notch3ECD and Vitronectin. (A–D) FLAG-tagged wild-type NOTCH3 (Flag-N3-WT) and mutant

NOTCH3 (�EGF1-34, R90C, C212S, C428S, TMIC) were cotransfected with haemagglutinin-tagged TIMP3 (TIMP3-HA) (A and B) or V5-

tagged Vitronectin (Vtn-V5) (C) in 293T cells. Forty-eight hours after transfection cells were harvested and immunoprecipitated (IP) with

anti-FLAG antibody (A and B) or anti-V5 antibody (C) and subjected to immunoblot (IB) with the indicated antibodies. (D) FLAG-tagged

wild-type NOTCH3 (Flag-N3-WT) and FLAG-tagged NOTCH3 deletion mutant lacking the 34 EGF-like repeats (Flag-N3�EGF1-34) were

co-transfected with TIMP3-HA or haemagglutinin-tagged VTN (Vtn-HA). Immunoprecipitation was performed with anti-haemagglutinin

(HA) antibody. (E) Flag-N3-WT was co-transfected with TIMP3-HA and Vtn-V5 and IP was performed with anti-V5 antibody. This panel is

representative of three independent experiments. (F) Interaction between endogenous NOTCH3 and TIMP3 in coronary artery smooth

muscle cells. NOTCH3 immunoprecipitation was performed using anti-Notch3ECD rabbit polyclonal antibody (BC2), normal rabbit IgGs were

used as controls. Asterisk indicates non-specific labelling. A representative gel of at least two independent experiments is shown.



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showed, as expected, an accumulation of TIMP3 in the SDS-in-

soluble fraction from brain vessels of patients with CADASIL.

Strikingly, we also detected elevated levels of RIPA and

SDS-soluble forms of TIMP3 in the vessels of patients with

CADASIL (Fig. 6B). Of note, in these later vessels, Notch3ECD

accumulated exclusively in the SDS-insoluble fraction.

In situ hybridization on human brain sections from control indi-

viduals revealed that vascular cells are the predominant source of

TIMP3 messenger RNA in the brain, the highest expression being

detected in the small penetrating vessels. Importantly, we found

no overt difference in the level of TIMP3 messenger RNA in the

vessels from patients with CADASIL versus vessels from control

individuals indicating that the observed increase in TIMP3 protein

levels was unlikely due to a transcriptional effect but rather

resulted from increased translational efficiency or compromised

protein degradation (Fig. 6C).

Figure 5 NOTCH3 influences TIMP3 protein levels in a manner requiring NOTCH3–TIMP3 complex formation. (A and B)

Haemagglutinin-tagged TIMP3 (TIMP3-HA) was transfected alone or in combination with wild-type (Flag-N3-WT) or mutant R90C

FLAG-tagged NOTCH3 (Flag-N3-R90C). Forty-eight hours after transfection cells (A) and extracellular matrix (B) were harvested. Cell

lysates and extracellular matrix were subjected to immunoblot analysis with the indicated antibodies or to silver staining (top panels).

Relative TIMP3-HA messenger RNA and TIMP3-HA protein levels were determined (bottom panels). Shown are relative TIMP3-HA

messenger RNA levels normalized to G6PD (A, left), relative TIMP3-HA protein levels in the cell lysate normalized to ß-actin (A, middle) or

NOTCH3 (A, right) and relative TIMP3-HA protein levels in the extracellular matrix normalized to total protein content (B) (n = 3) (S,

molecular weight standard). (C) FLAG-tagged NOTCH3 deleted of the 34 EGF-like repeats (Flag-N3�EGF1-34) does not affect TIMP3-

HA steady-state levels in the cell lysate compared with cells transfected with TIMP3-HA alone and to cells co-expressing TIMP3-HA and

Flag-N3-WT. Bottom: Relative TIMP3-HA messenger RNA levels normalized to G6PD (C, left) and relative TIMP3-HA protein levels in the

cell lysate normalized to ßactin (C, right) (n = 3). (D and E) Flag-N3-WT does not affect TIMP3�NTD-HA steady-state levels (D) but

increases TIMP3�CTD-HA steady-state levels (E). Bottom panels, relative messenger RNA levels of TIMP3�NTD-HA (D, left) and of

TIMP3�CTD-HA (E, left) normalized to G6PD and relative intracellular protein levels of TIMP3�NTD-HA (D, right) and of TIMP3�CTD-

HA (E, right) normalized to ß-actin (n = 3). *P50.05, **P5 0.01 and ***P50.001, ANOVA with Tukey post hoc analysis (A and C) or

student’s test (D and E). ns = not significant.



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Figure 6 TIMP3 protein levels and biological activity in brain vessels from patients with CADASIL. (A) Total lysates of brain vessels

isolated from CADASIL and control subjects were subjected to immunoblot with the indicated antibodies (left panel). Bottom: Relative

TIMP3 protein levels were normalized to ß-actin levels (n = 4 patients with CADASIL and n = 4 control subjects). (B) Brain vessels from

control subjects and patients with CADASIL were subjected to sequential biochemical extractions using RIPA, 2% SDS and Laemmli + ß-

mercaptoethanol. Thirty micrograms of total proteins were loaded per lane for RIPA fractions, the resulting 2% SDS-soluble and 2% SDS-

insoluble but Laemmli + ß-mercaptoethanol-extractable fractions were loaded directly, and subjected to immunoblot with the indicated

antibodies. A representative blot from at least two independent experiments using brain vessels from two control and two patients with

CADASIL is shown. (C) Paraffin brain sections from control and patients with CADASIL were hybridized with TIMP3 complementary RNA

probe and examined with dark field (left) and bright field illumination (right). The images shown (white matter with penetrating vessels)


(continued)


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We further analysed the TIMP3 activity levels in human brain

vessels by reverse zymography, which measures the ability of

TIMP3 to inhibit matrix metalloproteinases (Stetler-Stevenson,

2008). Strikingly, in human brain vessels, TIMP3 accounts for

the majority of gelatinase inhibitory activity (Fig. 6D and data

not shown). Importantly, TIMP3 activity was 2.5-fold higher in

patients with CADASIL compared with controls (Fig. 6D).

Next, we repeated the same analyses using brain arteries

isolated from TgNotch3R169C and control TgNotch3WT mice.

Immunoblot analysis revealed a 1.9-fold increase in total levels

of TIMP3 in TgNotch3R169C mice compared with TgNotch3WT

control mice (Fig. 7A). Notably, TgNotch3WT and non-transgenic

mice express comparable protein levels of TIMP3 (Supplementary

Fig. 9). Sequential extractions in TritonTM X-100, RIPA then

SDS + ß-mercaptoethanol buffers revealed more TIMP3 in all

three fractions, including the TritonTM X-100 and RIPA-soluble

fractions, in the TgNotch3R169C mice (Fig. 7B). Reverse zymogra-

phy assay showed that TIMP3 is the predominant endogenous

matrix metalloproteinases inhibitor also in murine brain arteries.

Significantly, gelatinase inhibitory activity was 1.8-fold higher in

TgNotch3R169C mice compared with TgNotch3WT mice (Fig. 7C).

Collectively, these data demonstrate that brain vessels from pa-

tients and mice with CADASIL accumulate a range of insoluble

and soluble TIMP3 species and suggest that this is associated

with an abnormally elevated TIMP3 activity.

DiscussionHere we demonstrate that, in brain vessels from patients with

CADASIL and transgenic mice, Notch3ECD accumulates in aggre-

gates that contain functionally important matricellular proteins,

including TIMP3 and VTN. Importantly, TIMP3 and VTN have

not been previously implicated in CADASIL and were not known

as interaction partners of NOTCH3. By focusing our further

analysis on these two proteins, we could decipher some of the

mechanisms of CADASIL deposit formation and the possible con-

sequences of their abnormal recruitment.

Previously, we and others have reported that the addition of

both detergent and reducing agent were necessary to solubilize

Notch3ECD from CADASIL brains (Joutel et al., 2000; Duering

et al., 2011). Moreover, in vitro studies using purified recombin-

ant NOTCH3 fragments containing the first five EGF-like repeats

showed that fragments bearing a cysteine mutation spontaneously

form high order multimers, cross-linked by disulphide bonds

(Duering et al., 2011). Here, our biochemical analyses using

brain and artery samples from patients with CADASIL and

mouse models extend these findings by showing that mutant

Notch3ECD is incorporated into denaturant and detergent-insoluble

aggregates, from which it can be recovered by the addition of a

reducing agent. Notably, NOTCH3 molecules bearing either RBPJ-

active or -inactive mutations behave similarly. Thus, these data

strongly argue that cysteine modifications are critical determinants

of the altered conformation of mutant Notch3ECD molecules,

which ultimately results in the formation of microscopically dis-

cernible Notch3ECD aggregates. Yet, the exact pairing and contri-

bution of individual cysteine residues in this process remains to be

determined. The possibility that Notch3ECD aggregates occur first

in vivo receives some support from the observations in transgenic

mutant mice that Notch3ECD aggregates can be detected as early

as 1 month of age and that aggregation of TIMP3 and VTN occurs

afterward. Next, our experimental results showed that increased

levels or aggregation of Notch3ECD, rather than the unpaired cyst-

eine residue within mutant Notch3ECD, strongly enhanced TIMP3–

Notch3ECD complex formation, resulting in the recruitment and

accumulation of TIMP3, which in turns promotes complex forma-

tion including NOTCH3 and VTN. This suggests that aggregated

or excess levels of Notch3ECD may form a platform or act as a

seed that influences interactions with extracellular matrix proteins

like TIMP3, that in turn can bind and recruit more and more

proteins, like VTN, generating new interaction surfaces and mag-

nifying the toxic potential of aggregates in a snowball effect. This

is highly reminiscent of what occurs in many neurodegenerative

diseases with inappropriate deposition of protein aggregates

(Wolfe and Cyr, 2011).

So far, only two other proteins, clusterin, an extracellular chap-

erone, and endostatin, a naturally-occurring proteolytic fragment

derived from the collagen alpha-1(XVIII) chain, have been identi-

fied as granular osmiophilic material components using laser cap-

ture microdissection on post-mortem brain tissue from patients

with CADASIL (Arboleda-Velasquez et al., 2011). Interestingly,

our proteomic analyses revealed additional proteins, including

mostly extracellular matrix proteins, which were enriched in the

CADASIL samples that warrant further investigation. It is import-

ant to note that the human-derived CADASIL sample was strongly

enriched with clusterin and endostatin, supporting the validity and

value of our approach. Nonetheless, abundance of these two pro-

teins seemed comparable in wild-type and mutant brain arteries of

transgenic mice, suggesting that clusterin and endostatin may ac-

cumulate at an advanced stage of the disease. This may apply to

many other proteins identified through our proteomic analyses, a

finding that would be consistent with the snowball model pro-

posed above. Thus, comparative proteomic analysis of the

Notch3ECD enriched fraction prepared from mutant and wild-

type samples, at various stages of the disease process, may rep-

resent an interesting approach to identifying proteins that co-ag-

gregate with Notch3ECD during the pathogenesis.

Figure 6 Continuedare representative of at least four sections from an experiment using three patients with CADASIL and three control subjects. (D) Lysates

of brain vessels isolated from four patients with CADASIL and four control subjects were subjected in parallel to reverse zymography and

to immunoblot with ß-actin. Lysates of cells transfected with TIMP3 or empty plasmid served as internal controls for reverse zymography.

Shown is a representative reverse zymogram from two independent experiments. Right: Relative TIMP3 activities, normalized to ß-actin

levels, were determined from n = 4 patients with CADASIL and n = 4 control subjects. SMA = smooth muscle actin.



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Recruitment and further sequestration of proteins into

aggregates usually result in the collapse of their biological function

(Olzscha et al., 2011). Nevertheless, our reverse zymography

assays raised the intriguing possibility that TIMP3 activity is abnor-

mally elevated in CADASIL brain vessels. This is supported by the

observation that brain vessels from patients with CADASIL and

mice exhibit, in addition to elevated levels of cross-linked deter-

gent-insoluble TIMP3 species, an increase in the levels of soluble

TIMP3 intermediates. However, it is worthy to note that one limi-

tation of the reverse zymography assay is the use of detergents

that may solubilize TIMP3 species that would be otherwise insol-

uble or inactive in situ. Therefore, additional experiments are

needed to assess the functional role of increased TIMP3 protein

levels in CADASIL brain vessels.

TIMP3 has multiple biological activities that are directly relevant to

CADASIL pathogenesis. In the brain, vascular cells of small penetrat-

ing vessels are the primary source of TIMP3. TIMP3 dysregulation

may contribute to small vessel pathology through its well-known

matrix metalloproteinase inhibitory activity. Homeostasis of the

extracellular matrix is maintained by a balance in the functions of

matrix metalloproteinases that degrade extracellular matrix compo-

nents and TIMP proteins, their endogenous inhibitors. Notably, our

results indicate that, in the brain vessels, TIMP3 is the major regulator

of metalloproteinase activities. As such, an abnormally elevated

Figure 7 TIMP3 protein levels and biological activity in brain vessels from CADASIL mouse model. (A) Total lysates of brain vessels

isolated from TgNotch3WT and TgNotch3R169C mice aged 20 months were subjected to immunoblot with the indicated antibodies. Shown

is a representative blot from two brain artery samples from TgNotch3WT and TgNotch3R169C mice. Right: Relative TIMP3 protein levels,

normalized to smooth muscle actin levels, were determined from n = 4 wild-type and n = 4 mutant samples prepared from eight

TgNotch3WT mice and eight TgNotch3R169C mice, respectively. (B) Brain vessels from TgNotch3WT and TgNotch3R169C were subjected to

sequential biochemical extractions using TritonTM X-100, RIPA and Laemmli + ß-mercaptoethanol (ßme). Twenty-five micrograms of total

proteins were loaded per lane for Triton-soluble fractions, the resulting RIPA-soluble and RIPA-insoluble but Laemmli + ß-mercap-

toethanol-extractable fractions were loaded directly, and subjected to immunoblot with the indicated antibodies. A representative blot

from at least two independent experiments using brain vessels from control and mutant mice is shown. (C) Total lysates of brain vessels

isolated from TgNotch3WT and TgNotch3R169C mice aged 20 month-old were subjected to reverse zymography. Lysates of cells trans-

fected with TIMP1, TIMP2 and TIMP3 or empty plasmid served as internal controls. Shown is a representative reverse zymography from

two independent experiments. Right panel, relative TIMP3 activities, normalized to smooth muscle actin levels, were determined from two

independent experiments using n = 4 TgNotch3WT samples and n = 4 TgNotch3R169C samples prepared from eight TgNotch3WT mice and

eight TgNotch3R169C mice, respectively.



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TIMP3 activity is anticipated to result in vessel fibrosis. Fibrotic

thickening of the arteriolar walls has been amply demonstrated in

CADASIL and recently, an increase in types I, III, IV and VI collagens

has been documented in all calibres of brain vessels (Dong et al.,

2012). Noteworthy, a number of non-extracellular matrix molecules

are also potential substrates of matrix metalloproteinases, whereby

elevated TIMP3 activity could exert a deleterious effect. TIMP3 dys-

regulation could also contribute to pathology through its anti-angio-

genic activity (Qi et al., 2003; Ebrahem et al., 2011). Particularly,

TIMP3 can suppress vascular endothelial growth factor-mediated

angiogenesis independently of its matrix metalloproteinases inhibi-

tory activity. Previously, we have documented a substantial reduc-

tion of capillary density in the TgNotch3R169C CADASIL mouse

model. Also, TIMP3 is the only TIMP that can inhibit ADAM17/

TNF converting enzyme, which mediates ectodomain shedding of

transmembrane receptors and ligands including TNF and ligands of

the epidermal growth factor receptor (Nagase et al., 2006). Of inter-

est, recent work suggests a role for heparin-binding EGF-like growth

factor in the pericyte recruitment process that leads to vessel matur-

ation and stabilization (Stratman et al., 2010). Finally, mutations in

TIMP3 cause Sorsby fundus dystrophy, an adult-onset hereditary

macular degenerative disease characterized by abnormal deposition

of TIMP3, macular atrophy and choroidal neovascularization (Weber

et al., 1994; Fariss et al., 1998; Qi et al., 2003). Disturbed homeo-

stasis in extracellular matrix remodelling is likely involved in Sorsby

fundus dystrophy pathology, although the mechanisms remain un-

clear. On the other hand, it’s conceivable that the diverse biological

activities of TIMP3 are differentially affected by TIMP3 accumula-

tion. Further studies are required to test these hypotheses.

Likewise, abnormal recruitment and sequestration of VTN

may impair its functions. While VTN is known as an abundant

circulating plasma protein, we found that, in the brain vessels,

VTN is abundantly transcribed (unpublished) and is a normal com-

ponent of the extracellular matrix. By its ability to bind to integrin-

type cell adhesion receptors, urokinase receptor and extracellular

matrix proteins, VTN has the ability to regulate cell adhesion, sig-

nalling and cytoskeletal reorganization. Moreover, by its ability to

bind and stabilize plasminogen activation inhibitor type 1, VTN can

modulate the balance of the fibrinolytic system, and consequently,

the extracellular matrix homeostasis (Preissner and Reuning, 2011).

Further studies are required to examine whether and how focal

VTN deposits can alter these biological activities.

In summary our results lend support to a Notch3ECD cascade hy-

pothesis, which posits that the aggregation of Notch3ECD in the brain

vessels is a central event in CADASIL disease pathology, promoting

the abnormal recruitment and potential dysregulation of functionally

important proteins of the extracellular matrix that may ultimately

cause multifactorial toxicity. Identifying the CADASIL–Notch3ECD

interactome may represent an interesting approach to further delin-

eate the molecular mechanisms of this toxicity.

AcknowledgementsWe are grateful to the laboratory of Dr. Dylan Edwards for the

TIMP activity assay, to Drs. Hannu Kalimo, Francoise Chapon,

Jean-Jacques Haw, Catherine Godfraind and the GIE-NeuroCeb

brain bank (Paris, France) for human brain samples, and to

Barbara Lemaire-Carrette for technical assistance. We thank

Electron microscopy Core at Institut du Fer-a-Moulin and

TAAM-Orleans (Karine Jambou) for animal housing.

FundingThis work was supported by grants from the French National

Research Agency (grant number ANR Genopath 2009-

RAE09011HSA), National Institutes of Health (grant number R01

NS054122) and the Fondation Leducq (Transatlantic Network of

Excellence on the Pathogenesis of Small Vessel Disease of the

Brain) to A.J. M.R. is a recipient of a fellowship from the

European community (Marie Curie Initial Training Network). FT

ICR MS acquisition was supported by investment grant from the

contrat de plan Etat-Region (CPER) Fonds Recherche et

Technologie (2002-06). Financial support from the TGE FT-ICR

for conducting the research is gratefully acknowledged.

Supplementary materialSupplementary material is available at Brain online.

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Vanderburg CR, et al. Hypomorphic Notch 3 alleles link Notch signal-

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