The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients (original) (raw)
Notch3 expression is restricted to vascular smooth muscle cells in human adult tissues. We investigated the Notch3 expression pattern in a broad panel of human adult tissues from control individuals (n = 13), using both in situ hybridization (n = 7) and immunohistochemistry (n = 10). To avoid cross-hybridization with Notch1, Notch2, and Notch4 transcripts, we designed 2 sets of both sense and anti-sense riboprobes from regions of the Notch3 gene showing the lowest homology with these transcripts. Examination of sections of various parenchyma (Figure 1), including heart (a–c), kidney (d–f), brain (g–i), large artery (j–l), skeletal muscle, lung, urinary bladder, and large intestine (data not shown) from at least 2 distinct individuals per tissue indicated that Notch3 expression is restricted to the vasculature. The 2 antisense Notch3 riboprobes showed the same hybridization pattern (data not shown).
Notch3 expression is restricted to vascular smooth muscle cells in human adult tissues. Notch3 expression was examined on paraffin sections from control individuals by in situ hybridization (a–l) and immunohistochemistry (m–r). Sections of myocardium (a–c), kidney (d–f), brain white matter (g–i), and renal artery (j–l) were hybridized with Notch3 antisense riboprobe (b, e, h, and k) and Notch3 sense riboprobe (c, f, i, and l). Kidney sections were hybridized with the HN3X riboprobes; all other sections with the SHB1 riboprobes. Bright fields (a, d, g, and j) and corresponding dark-field micrographs are shown. Notch3 is detected exclusively in the vessels (arrows) within the various parenchyma (a–i), and primarily in the smooth muscle cell layer of the media or in vessels of the adventitia within the renal artery (j–l). Small arrows in k indicate the internal elastica lamina, the thick arrow shows the external elastica lamina, and the star marks the luminal side. The bar in l represents 125 μm for a–c, 100 μm for d–f, 50 μm for g–i, and 200 μm for j–l. (m–r) Adjacent paraffin sections of brain frontal lobe were immunostained with primary antibodies recognizing the smooth muscle myosin heavy chain (mhc) (n), Notch3 extracellular epitopes 1E4 (o and q) and 2E11 (p), and the vascular endothelial cell marker CD34 (r). In m, primary antibody was omitted. Anti-Notch3 antibodies exclusively immunostained the smooth muscle cells (o–q) but not the endothelial cells (arrowheads). Bar, 100 μm for m–p and 20 μm for q and r (higher magnification of the boxed area of the vessel wall in o). Counterstaining was with toluidine blue (a–l) and with hematoxylin (m–r).
Restriction of expression to the vasculature was confirmed by immunohistochemical analysis on paraffin sections with 2 anti-Notch3 antibodies that we raised against the extracellular domain, 1E4 and 2E11. Notch3 expression was further restricted to the smooth muscle cell layer within the vessels (Figure 1, o–q). Specificity of Notch3 immunoreactivity was confirmed by the control step of omitting each primary antibody (Figure 1m) and replacing it with an isotype-matched antibody (Figure 1r).
Anti-Notch3 antibodies raised against extracellular epitopes display an intense and granular immunoreactivity in CADASIL brains. Using the 1E4 anti-Notch3 monoclonal antibody raised against an extracellular epitope, we found a prominent increase and a granular appearance of Notch3 immunoreactivity in the vascular smooth muscle cells in CADASIL brains (n = 8) (Figure 2, b, d, f, and h) that contrasted with the weak and homogeneous staining in control brains (n = 5) (Figure 2, a, c, e, and g). Notch3 staining was specific based on 2 things: the absence of staining of vascular smooth muscle cells in the absence of primary antibody (Figure 2i) or in the presence of an isotype-matched antibody (Figure 2j); and the similarity of results obtained with 2E11, another anti-Notch3 antibody raised against an extracellular epitope (Figure 2l). This intense Notch3 immunoreactivity was observed in all vessels, including arteries, veins, and capillaries throughout CADASIL brain parenchyma, and was detected both in vascular smooth muscle cells (Figure 2, b, d, and f) and pericytes (Figure 2h), leading to a stippled decoration of the brain parenchyma. These data strongly suggested an abnormal accumulation of the Notch3 receptor.
Intense and granular appearance of Notch3 immunoreactivity in CADASIL brains. (a–h) Paraffin sections from control (C1–C3; a, c, e, and g) and CADASIL brains (cad1–cad4; b, d, f, and h) including sections from white matter (a and b), cortex (c, d, g, and h), and brainstem (e and f), were probed with the 1E4 anti-Notch3 antibody. The examples shown are from 3 control individuals and 4 CADASIL brains matched for formalin fixation conditions and duration: 48 hours for control 1 (a and g) and CADASIL patient 1 (b); 0.5 months and 3 months for control 2 (c) and CADASIL patient 2 (d), respectively; 8 years for control 3 (e) and CADASIL patient 3 (f), and 1 year for CADASIL patient 4 (h). Notch3 immunoreactivity is much more intense within smooth muscle cells of vessels from CADASIL brains than in control brains (a–h, right column compared with left column) for all durations of fixation. Granular and intense Notch3 immunostaining is also obvious within pericytes of capillaries (arrows, nuclei of pericytes) (h). Identical observations were obtained in brains from other donors (4 CADASIL patients, 2 controls). Bar, 50 μm for a–f and 20 μm for g and h. (i–l) Adjacent paraffin sections from CADASIL brain (patient 1) were probed with the 2E11 anti-Notch3 antibody (l), the anti-CD34 antibody (which is of the same isotype as the 1E4 and 2E11 antibodies) to identify the endothelial cells (j), the anti–smooth muscle α-actin antibody (to identify smooth muscle cells) (k), or without primary antibody (i). Bar, 50 μm. Counterstaining was with hematoxylin.
In contrast, using the 6A10 anti-Notch3 monoclonal antibody raised against an intracellular epitope, we found only weak immunoreactivity in the vascular smooth muscle cells in CADASIL brains, equivalent to that observed in control brains (data not shown).
Wild-type Notch3 is cleaved, producing 210-kDa and 97-kDa fragments. Discrepancy between immunostaining patterns obtained with the anti-intracellular and anti-extracellular Notch3 antibodies, and the possibility that Notch3 undergoes a proteolytic cleavage similar to that experienced by the other Notch receptors raised the question of the nature of the Notch3 immunoreactive material that accumulates in CADASIL brains. As a first step, we investigated the processing of the wild-type Notch3 receptor.
293T cells transfected with the full-length wild-type Notch3 cDNA expressed the full-length Notch3 precursor (∼280 kDa) detected both by 5G7 (anti-intracellular) and 5E1 (anti-extracellular) anti-Notch3 antibodies, as well as proteins of approximately 97 kDa and 210 kDa detected by 5G7 and 5E1 antibodies, respectively (Figure 3, a and b). Coimmunoprecipitation experiments provided evidence that the 97-kDa and the 210-kDa cleavage products were associated (Figure 3c).
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.
In control tissues, Notch3 was detected in extracts from arteries, including leptomeningeal, large cerebral, and extracerebral arteries, but not from brain parenchyma (Figure 3b and data not shown). The latter finding is probably explained by the weak expression level of this receptor and by the fact that smooth muscle cells and pericytes represent a minor cellular population within the brain parenchyma. Arteries expressed the 210-kDa and the 97-kDa fragments, but not the 280-kDa full-length protein (Figure 3b). These data strongly suggested that Notch3 undergoes a proteolytic cleavage identical to that of the Notch1 and Notch2 receptors.
Dramatic and selective accumulation of the 210-kDa Notch3 cleavage product in CADASIL patients. To further confirm Notch3 accumulation in CADASIL brains, and to investigate the nature of the Notch3 immunoreactive material, we performed Western blot analysis on lysates from CADASIL tissues. In CADASIL brains (n = 3), the 5E1 and 11A1 anti-extracellular antibodies detected a prominent 210-kDa protein; under the same experimental conditions using control brains (n = 2), Notch3 protein was not detected (Figure 4, a and b). The size of the Notch3 immunoreactive material that accumulated in CADASIL brains was identical to the 210-kDa Notch3 cleavage product, including the extracellular domain detected in Notch3-transfected cells and in control arterial tissue (Figure 4, a–c).
Selective accumulation of the 210-kDa cleavage product in CADASIL patients. (a and b) Western blot analysis of brain extracts from CADASIL and control individuals with antibodies raised against Notch3 extracellular domain. (a) Brain fragments were homogenized and centrifuged at 16,000 g; the resulting pellet and supernatant were adjusted to 1× SDS-Laemmli buffer. Approximately 100 μg of extracts were loaded per lane on a 6% SDS-PAGE gel, and were immunoblotted with the 5E1 antibody. Blots were stained with Ponceau S to confirm that equal amounts of proteins were loaded. Left panel, brain extracts (supernatant and pellet) from CADASIL patient 1 and control individuals 2 and 5. Middle panel, brain extract (pellet) from CADASIL patient 1 and extract from 293T cells transfected with Notch3 cDNA (N3). Right panel, brain extracts (pellet) from CADASIL patients 1, 8, and 9. Position of the 210-kDa Notch3 protein (open arrowhead) detected in CADASIL brain extracts is indicated. The asterisk marks a band of unknown significance (cross-reacting material or, most likely, post-lysis degradation product). (b) Brain extracts from CADASIL patient 1 (pellet) and control individual 2 (pellet), and extracts from 293T cells transfected with Notch3 cDNA (N3) were run on a 6% SDS-PAGE gel and immunoblotted with the 11A1 antibody (left panel) or without primary antibody (–) (right panel). (c) The 97-kDa Notch3 protein is not detected in either CADASIL or control brains. Whole lysates were prepared from brains of CADASIL patient 1 and control individual 5, and from a renal artery of a control subject. Approximately 100 μg of each extract was loaded on a 6% SDS-PAGE gel and immunoblotted with the 5G7 antibody (bottom) and the 5E1 antibody (top). Positions of the 210-kDa and the 97-kDa processing products are indicated (open arrowhead and small arrow, respectively). (d) Western blot analysis of extracts from CADASIL and control arterial tissues. Whole lysates were prepared from mesenteric arteries of CADASIL patient 1 and a renal artery of a control subject. Extracts were resolved on a 6% SDS-PAGE gel and immunoblotted with the 5G7 antibody (right) and the 5E1 antibody (left). The asterisk marks a band of unknown significance (cross-reacting material or, most likely, a post-lysis degradation product). Whole lysates were used in experiments shown in c and d to avoid missing any soluble Notch3 intracellular fragment. (e) Western blot analysis of extracts from CADASIL and control brains and from control arterial tissue under reducing and nonreducing conditions. Extracts from CADASIL and control brains and from a control artery were solubilized in SDS-Laemmli buffer with or without βME and were resolved on a 6% SDS-PAGE gel. Both stacking (bracket) and separating gels were immunoblotted with the 5E1 antibody. Under reducing conditions (βME+), the 210-kDa Notch3 protein is detected in both control artery and CADASIL brain (open arrowhead). Under nonreducing conditions (βME–), no band is detected in the CADASIL brain; a protein of approximately 280 kDa (large open arrow) is detected in the control artery. This protein probably corresponds to the associated extracellular and intracellular domains. Cross-reacting material is caught at the transition between the stacking and separating gels.
By contrast, both the 5G7 monoclonal antibody and the BC4 polyclonal anti-intracellular antibody failed to detect any signal in CADASIL brains, even though the 97-kDa fragment was detected successfully on the same Western blot in a control artery (Figure 4c and not shown). When using arterial tissues (considerably enriched in vascular smooth muscle cells) from either a control individual or a CADASIL patient, both 210-kDa and 97-kDa Notch3 cleavage products were detected. Comparative analysis of the relative amounts of the 210-kDa and 97-kDa proteins provided strong evidence of the accumulation of the 210-kDa fragment in the CADASIL patient. By contrast, an almost equivalent amount of the 97-kDa fragment was observed in both the control subject and a CADASIL patient. This 97-kDa fragment was of similar size in control and CADASIL arteries (Figure 4d).
The mutant Notch3 ectodomain probably undergoes an improper oligomerization. To investigate whether the odd number of cysteines in mutant Notch3 might favor its improper oligomerization and contribute to its accumulation, we performed Western blot analysis on lysates from CADASIL brains under nonreducing conditions with the 5E1 antibody. No band was detected in the CADASIL brain (Figure 4e, βME–) under these conditions, even though a prominent quantity of Notch3 protein was detected under reducing conditions (Figure 4e, βME+). These results strongly suggested that in the absence of reducing agents, it is likely that mutant Notch3 consists of large complexes that were unable to enter the SDS-PAGE gel. Under nonreducing conditions, the artery from a control individual, used as a positive control, expressed a band of approximately 280 kDa that may correspond to the tightly associated extracellular and intracellular cleavage products (Figure 4e, βME+), as reported previously for Notch1 (17).
Equivalent proteolytic processing and subcellular distribution of wild-type and mutated Notch3 proteins in 293T cells transfected with the wild-type and mutant Notch3 cDNAs. To further explore the mechanisms by which the Notch3 ectodomain abnormally accumulated, we examined the subcellular distribution of wild -type and mutant Notch3 (R90C and C212S) in 293T cells transfected with the wild-type or the mutant Notch3 cDNAs. Wild-type Notch3 protein was detected within the endoplasmic reticulum, the Golgi apparatus, and at the cell surface by immunofluorescence analysis (not shown). The 97-kDa and the 210-kDa cleavage products of the wild-type Notch3 were the major species detected at the cell surface using biotin labeling of surface proteins (Figure 5 and not shown). We found that proteolytic processing and subcellular distribution of mutant Notch3 were both equivalent to those of the wild-type Notch3 (Figure 5 and not shown). Furthermore, we did not obtain evidence of an abnormal accumulation of the 210-kDa cleavage product in 293T cells transfected with the mutant Notch3 cDNAs.
Wild-type and mutant Notch3 proteins expressed at the cell surface of transfected 293T cells are cleaved. 293T cells transfected with wild-type Notch3 (WT), mutant Notch3 (R90C and C212S), and vector alone (pSG5) were incubated with sulfo-NHS-biotin (+) or were mock treated. Cells were lysed in RIPA buffer, and extracts were loaded on a 6% SDS-PAGE gel either directly (T fraction, 15% of the extract) or after incubation on streptavidin-agarose beads (B fraction, 85% of the extract). Extracts were then immunoblotted with the 5E1 antibody. Positions of the 280-kDa full-length Notch3 extracellular domain (black arrow) and the 210-kDa Notch3 extracellular domain (arrowhead) are indicated.
Notch3 accumulates at the cytoplasmic membrane of vascular smooth muscle cells in close vicinity to the granular osmiophilic deposits. To search further for the subcellular compartment where Notch3 accumulated and to investigate whether Notch3 accumulation might account for the granular osmiophilic deposits, we performed immunoelectron microscopy analysis on sections from a CADASIL brain. Notch3 immunolabeling was detected at the cytoplasmic membrane of vascular smooth muscle cells (Figure 6). It was highly restricted to small areas located close to the granular osmiophilic deposits, which themselves were not labeled by the anti-Notch3 antibody .
Notch3 immunogold labeling is highly clustered at the cell surface of smooth muscle cells. (a) Vessel within the frontal lobe of CADASIL patient 1, processed for electron microscopy using standard procedure. Outline of a smooth muscle cell (m) is indicated by open small arrows. Granular osmiophilic deposits (large arrows) are observed within the basal lamina (square), in close contact with the cytoplasmic membrane of this smooth muscle cell. ×13,000. (b) Vessel within the occipital lobe from CADASIL patient 1, processed for immunoelectron microscopy with the 1E4 antibody. Numerous granular osmiophilic deposits (large arrows) are observed within the basal lamina (square). These are nested within invaginations of the smooth muscle cells (m), or are spread far away within the vessel wall (stars). Notch3 immunolabeling, which appears as black dots due to silver enhancement, is highly clustered at the cell membrane of smooth muscle cells (open small arrows) closely arranged around the deposits, which are not labeled. ×20,000. Ultrathin sections stained with uranyl acetate and lead citrate. ec, endothelial cell; Lu, lumen.