The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients
Anne Joutel, Fréderic Andreux, Swann Gaulis, Valérie Domenga, Michaelle Cecillon, Nicole Battail, Nadia Piga, Françoise Chapon, Catherine Godfrain, Elisabeth Tournier-Lasserve
Anne Joutel, Fréderic Andreux, Swann Gaulis, Valérie Domenga, Michaelle Cecillon, Nicole Battail, Nadia Piga, Françoise Chapon, Catherine Godfrain, Elisabeth Tournier-Lasserve
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The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients

Abstract

Mutations in Notch3 cause CADASIL (cerebral autosomal dominant adult onset arteriopathy), which leads to stroke and dementia in humans. CADASIL arteriopathy is characterized by major alterations of vascular smooth muscle cells and the presence of specific granular osmiophilic deposits. Patients carry highly stereotyped mutations that lead to an odd number of cysteine residues within EGF-like repeats of the Notch3 receptor extracellular domain. Such mutations may alter the processing or the trafficking of this receptor, or may favor its oligomerization. In this study, we examined the Notch3 expression pattern in normal tissues and investigated the consequences of mutations on Notch3 expression in transfected cells and CADASIL brains. In normal tissues, Notch3 expression is restricted to vascular smooth muscle cells. Notch3 undergoes a proteolytic cleavage leading to a 210-kDa extracellular fragment and a 97-kDa intracellular fragment. In CADASIL brains, we found evidence of a dramatic and selective accumulation of the 210-kDa Notch3 cleavage product. Notch3 accumulates at the cytoplasmic membrane of vascular smooth muscle cells, in close vicinity to but not within the granular osmiophilic material. These results strongly suggest that CADASIL mutations specifically impair the clearance of the Notch3 ectodomain, but not the cytosolic domain, from the cell surface.

Authors

Anne Joutel, Fréderic Andreux, Swann Gaulis, Valérie Domenga, Michaelle Cecillon, Nicole Battail, Nadia Piga, Françoise Chapon, Catherine Godfrain, Elisabeth Tournier-Lasserve

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Proteolytic processing of wild-type Notch3. (a) Diagram of the Notch3 pr...
Proteolytic processing of wild-type Notch3. (a) Diagram of the Notch3 protein. Bars below the diagram indicate the various regions used as antigens to generate Notch3 antibodies (monoclonal antibodies 5E1, 11A1, and 5G7; polyclonal antibodies BC2 and BC4). TM, transmembrane domain. (b) Western blot analysis of transfected 293T cells and human control arterial tissue. Extracts were prepared from 293T cells transfected with human Notch1 (N1), Notch2 (N2), Notch3 (N3), or pSG5 vector (V). Fragment of a renal artery from a control individual was homogenized and then centrifuged at 16,000 g. The resulting pellet and supernatant were adjusted to 1× SDS-Laemmli buffer. Thirteen micrograms of transfected 293T cells and approximately 100 μg of pellet and supernatant (Sup) from the control artery were run on a 6% SDS-PAGE gel and incubated after transfer with 5E1 (left) and 5G7 (right). Positions of the 280-kDa full-length Notch3 (large black arrow) and the 210-kDa and 97-kDa processing products are indicated (open arrowhead and small arrow, respectively). Notice the weak expression level of Notch3 in arterial tissue, which required more than 1 hour of exposure as opposed to a few seconds for transfected cells. (c) Notch3 210-kDa and 97-kDa processing products are associated. Extracts from 293T cells transfected with Notch3 cDNA (N3) or vector alone (V) were immunoprecipitated with BC2 and BC4 polyclonal antibodies and with preimmune serum (PPI). Precipitated proteins were resolved by SDS-PAGE and immunoblotted with 5E1 and 5G7 antibodies. IP, immunoprecipitation. Migration of molecular-weight markers is shown to the left of each panel.