Case report: CADASIL

Background and aim: Notch signaling pathway is important for the regulation of cell fate decisions and cellular differentiation in many organs. NOTCH3 belongs to a family of single-pass transmembrane receptors. In response to interaction with ligands, it undergoes a set of proteolytic events releasing a signaling molecule to the nucleus where it interacts with the DNA-binding protein RBP-Jk. Point mutations in NOTCH3 lead to a disorder called CADASIL, resulting in adult onset of migraine, recurrent strokes and vascular dementia in humans. Our objective is to create transgenic cell lines that stably express CADASILassociated mutations to induce higher production of NOTCH3 proteins. Methods: CADASIL mutations, R142C and C456R, were introduced using site-directed mutagenesis into mouse NOTCH3 (mN3). Different mN3 constructs were transfected into C2C12 mouse myoblast cell line to induce high NOTCH3 expression. The entire gene was sequenced and immonublotting was carried out to verify expression of the protein. Real-time RT-PCR was conducted to determine level of expression before clones were selected. Results:We have established transgenic lines of C2C12 cells expressingmN3 with CADASILmutations, R142C andC456R.R142C is amutation locatedonexon 4 of mN3 gene corresponding to CADASIL mutation R141C in human NOTCH3 (hN3), whereas C456R is a mutation on exon 8 corresponding to CADASIL mutation C455R in hN3. We have chosen several lines that showed higher expression compared to the endogenous expression of mN3 in C2C12. Conclusion: Thus far, studies showing that NOTCH signaling pathway is impaired in cells carrying a typical CADASIL mutation have been inconclusive. Wepostulate thatCADASILmutationshave adirect consequenceon theNOTCH3 protein foldingmachinery as a result of the loss or gain of cysteine residues. Our present plan is to harvest NOTCH3 proteins from the transgenic cell lines for protein chemistry analyses and protein expression studies.