resulted in inhibition of ligand-stimulated HES-luciferase activa- tion. The transfection of either R90C or C49Y mutant NOTCH3 more potently inhibited Delta1-dependent NOTCH activation compared to wildtype NOTCH3. NOTCH3 overexpression failed to suppress the activity of constitutively active NOTCH3 lacking ectodomain sequences (NICD; Figure S2). In A7R5 cells (Figure 7B), ligand-stimulated HES-luciferase activity was inhibited by wildtype and mutant full-length NOTCH3. There was a trend towards stronger suppression of Notch responses with expression of mutant NOTCH3 (compared to WT), but this did not reach statistical significance. Overall, both wildtype and mutant NOTCH3 repressed canonical Notch transcriptional function in a cell and ligand specific manner. The inhibition of the reporter construct was also seen after overexpression of NOTCH3 ectodomain (Figure S3). Since levels of critical smooth muscle mRNAs were repressed by full-length NOTCH3 overexpression (Figure 6), we tested whether transfected NOTCH3 inhibits smooth muscle promoters normally targeted by Notch. In coculture assays, overexpression of all forms of full-length NOTCH3 resulted in inhibition of smooth muscle actin, smooth muscle myosin heavy chain, and SM22-luciferase constructs in A7R5 cells (Figures 7C–E). Although mutant NOTCH3 more potently inhibited the activity of the smooth muscle actin promoter (compared to wildtype NOTCH3; Figure 7D), there was no significant difference between the inhibitory properties of the wildtype and mutant NOTCH3 acting on the SM-MHC and SM22 promoters. All ligand-dependent promoter function was blocked by co-transfection with a DN- MAML peptide that has been shown to inhibit Notch signaling (not shown). Moreover, mutagenesis of the Notch-responsive element from the SM22 promoter construct fully blocked the inhibitory function of NOTCH3 overexpression (Figure 7E). Co-transfection of NOTCH3 cDNA potentially causes aggre- gation of proteins in the intracellular compartment. To test whether secreted NOTCH3 ectodomain is sufficient to block Notch signaling, we prepared recombinant NOTCH3 ectodomain proteins purified from stable cell lines to determine their effects in Notch signaling. Both wild type and mutant NOTCH3 proteins added at nanomolar concentrations to the culture media were capable of potently inhibiting Notch signaling in A7R5 cells (Figure 8). Discussion We report four novel findings regarding NOTCH3:1) both wildtype and mutant NOTCH3 ectodomains form complexes with NOTCH1, 3, and 4 ectodomains via EGF-repeats; 2) CADASIL mutations decrease NOTCH3 detergent solubility; 3) CADASIL mutant protein interferes with cellular clearance of NOTCH3; 4) accumulated NOTCH3 represses Notch-stimulated expression of smooth muscle genes by interfering with promoter activation. NOTCH3 Ectodomain Protein Complexes The appearance of GOM and granular NOTCH3 accumula- tion in blood vessels strongly suggests that vascular protein aggregation is a major pathological feature of CADASIL. Other studies [36,38] have examined potential protein interactions between a number of other CADASIL mutants of NOTCH3 (R133C, C185R, C183R, and C455R). Here, we show novel, unequivocal evidence that both wildtype and two additional mutant NOTCH ectodomains (R90C and C49Y) form stable complexes within transfected cells and that the EGF-repeats are sufficient for protein complex formation. Importantly, we demon- Cellular Effect of CADASIL Mutants of NOTCH3 strate for the first time that the NOTCH3 ectodomain is able to interact with ectodomains of NOTCH1 and NOTCH4, which are also expressed in the vascular wall. Our data demonstrate that NOTCH3 homomultimerization requires expression in the same cell, since lysate mixing did not result in multimer formation. Previous experiments have also suggested that NOTCH3 multi- mers form when expressed in cells [36,38]. The significance of these protein interactions in CADASIL is two-fold. First, our interaction studies suggest that accumulated NOTCH3 could interfere not only with NOTCH3, but also with other NOTCH proteins in vessels. Second, the ectodomains of new binding partners NOTCH1 and NOTCH4 consist of primarily EGF-repeats; notably, all of the other known NOTCH3 partners (Jagged, Delta, the thrombospondins [42] and LRP1 [49]) contain EGF-repeats as well. Thus, it is probable that other EGF repeat containing proteins form complexes with NOTCH3. This possibility provides a framework to identify additional proteins that could interact with NOTCH3 in the pathogenesis of CADASIL. Solubility of NOTCH3 Mutants We show new data that full-length mutant proteins are notably less soluble than wildtype proteins. A likely explanation for this is that mutant protein complexes form due to new protein-protein interactions, which has been suggested in an independent study of other CADASIL mutants [36]. Our experiments show that detergent resistance of the mutant protein was associated with the presence of intracytoplasmic perinuclear aggregates. In total, the results are consistent with the long held hypothesis that binding of NOTCH3 to other proteins form aggregates that could lead to disruptions on normal cell signaling required for smooth muscle homeostasis [36,60,61]. The relevance of perinuclear aggregates of NOTCH3 to CADASIL is unclear but similar staining has been observed by Karlstrom et al. [57]. Joutel et al. have found very similar intense aggregates of Notch3 in pericytes of the brain in juvenile transgenic mouse models of CADASIL [62], but not in earlier transgenic mice that failed to show white matter changes. The authors discussed the possibility that these aggregates were a consequence of overexpression of Notch3 in the vasculature, but, interestingly, rather than increasing over time, these aggregates disappear later in life despite continued expression of the protein in adult mice. The striking changes in distribution of these aggregates suggests that the localization of mutant Notch3 evolves over time, and raises the possibility that mutant Notch3 mutations may have different accumulation patterns and functional conse- quences over time. Overall, all studies which have expressed NOTCH3 in vitro, including the current one, have demonstrated significant intracellular expression. Despite significant efforts, we have been unable to express NOTCH3 at the cell surface without intracellular accumulation. Therefore, an important caveat to our study, and others, is that the effects of NOTCH3 overexpression on signaling may be a result of either effects inside the cell, or, potentially at the cell surface. In experiments reported in this study, we were able to inhibit global Notch signaling with purified, recombinant NOTCH3 ectodomain applied at nanomolar con- centrations. Thus, NOTCH3 protein expressed outside the cell is sufficient to interfere with cell signaling. Since we are able to control the solubility and quantity of Notch3 aggregates by altering cell culture density, this culture model of NOTCH3 expression may be useful in defining the regulation and significance of perinuclear Notch3 accumulation. The finding that low density cell plating results in increased aggregation and decreased solubility of NOTCH3 is consistent with prior findings that in CADASIL, protein accumulation PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e44964