Cellular Effect of CADASIL Mutants of NOTCH3 (Jagged1 or Delta1) stimulated activity was significantly suppressed by WT and mutant NOTCH3 transfection for every promoter tested (relative to control; p,0.05; the differences are not marked to simplify the presentation of data). Consistent and significant differences in Notch signaling were observed after coexpression of wildtype or mutant NOTCH3 in for H460 cells transfected with HES-luciferase (A) and for A7R5 cells transfected with all luciferase constructs except the mutant SM22 promoter. There were significant differences between the potency of inhibition of the mutant NOTCH3 proteins in selected groups. Each experiment was conducted three or more times; in each experiment, triplicate wells were analyzed. * indicates differences between NOTCH3 transfected cells versus control cells and # denotes differences between mutant NOTCH3 and wildtype NOTCH3 transfectants (p,0.05). Error bars represent standard deviations. doi:10.1371/journal.pone.0044964.g007 accompanies dissociation between smooth muscle cells. While a causal connection remains speculative, further experiments using this model system to understand the molecular basis of this finding may shed light on the pathways towards NOTCH3 accumulation in CADASIL. Mutations in NOTCH3 Affect Clearance of Protein Using a quantitative assay of protein clearance, we show that mutations in NOTCH3 significantly inhibit NOTCH3 ectodo- main protein clearance. Takahashi and colleagues [38] have also shown that NOTCH3 mutations markedly slow the rate of clearance of two independent mutants on NOTCH3; therefore, the impairment of clearance of mutated NOTCH3 is likely to be a general property of CADASIL proteins. Since WT and mutant proteins were both labeled with luciferase, these experiments do not definitively identify the protein species whose clearance is altered. Nevertheless, we can conclude that the presence of mutant protein sequences can dominantly slow NOTCH3 turnover, which supports the hypothesis of Joutel et al. [58] that the accumulation of NOTCH3 in CADASIL is the result of impairment of protein clearance and not increased production. We further submit, based on biochemical studies above, that the inhibited clearance may be caused by changes in solubility resulting from both homotypic and heterotypic complexes involving NOTCH3-binding proteins such as NOTCH1 and NOTCH4. We investigated whether mutants could dominantly affect other NOTCH3 ectodomain molecules by cotransfecting wildtype and mutant NOTCH3-luciferase fusions. Our new results indicated that the C49Y mutant did indeed dominantly affect clearance of other forms of NOTCH3, but that the R90C mutant did not. It remains to be tested whether dominant inhibition of clearance could be a more widespread property of CADASIL mutants and whether patients with mutations that dominantly affect NOTCH3 clearance have more severe disease and NOTCH3 accumulation. Excessive Full-length NOTCH3 Represses NOTCH Signaling Full-length NOTCH3 overexpression could be expected to increase potency of Notch signaling. However, our studies soundly demonstrate that NOTCH3 overexpression dominantly suppress- es Notch signaling in cells, including an arterial smooth muscle line. Both wild type and mutant NOTCH3 failed to activate smooth muscle promoters; but, instead, NOTCH3 overexpression suppressed Notch signaling; there was increased inhibition with mutant NOTCH3 in several assays, but this was not found with all mutants or promoters. Mutant NOTCH3 was able to inhibit signaling more strongly than wildtype NOTCH3, but this was not a consistent finding. Based on the increased ability to inhibit clearance and induce protein aggregation, mutants could in fact be more potent in inhibiting NOTCH signaling, but due to a ‘‘floor effect’’ in our Figure 8. Inhibition of Notch signaling by extracellular NOTCH3 ectodomain. As in Figure 7, A7R5 cells (n = 3 replicate wells per group) transfected with HES-luciferase reporter (but not NOTCH3 cDNA) were cocultured with ligand producing or control cells. Experiments were performed in the presence of 1 ug/ml (3 nM) recombinant NOTCH3-Fc protein (wildtype or R90C mutant ectodomains fused to Fc). Control studies were done with no added protein and with purified human IgG to control for the presence of immunoglobulin in the NOTCH3-Fc preparation. * indicates differences between NOTCH3-Fc treated cells versus control cells (p,0.05). Experiments were done three times with similar results. doi:10.1371/journal.pone.0044964.g008 PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e44964