Non-Enzymatic Fragmentation Of The Amino Terminus Of Notch3 In Inherited Cerebral Small Vessel Disease

Introduction: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common genetic cause of cSVD, stroke, and vascular dementia. CADASIL is characterized by NOTCH3 mutations, NOTCH3 protein accumulation in arteries, and granular osmiophilic material. The mechanisms underlying CADASIL protein accumulation are currently unknown. We test the hypothesis that post-translational modification of NOTCH3 populations occurs within CADASIL cerebral vasculature. Methods: We generated monoclonal antibodies against the N-terminal region of NOTCH3. Antibodies were used for immunohistochemistry on 19 CADASIL and 6 age-matched control brains. Epitope mapping was performed on defined NOTCH3 fragments. Proteins treated with reducing agents were scored for antibody reactivity by western blotting. NOTCH3 complex formation was tested by co-immunoprecipitation. Results: NOTCH3 ectodomain undergoes cleavage into an N-terminal fragment (NTF) in diseased human cerebral vessels. The site of fragmentation was mapped to the peptide bond following D80. Mutagenesis of P81 abolished fragmentation. Both acidic and reducing conditions promote NOTCH3 cleavage in vitro . Finally, NTF is capable of forming dimers and detergent-resistant complexes with full length NOTCH3. Conclusion: Our studies demonstrate non-enzymatic fragmentation of NOTCH3 in CADASIL that could drive protein aggregation events that hallmark CADASIL. As in Alzheimer’s disease, protein fragmentation may contribute to neuropathology. Future studies will include testing the pathogenicity of fragmented NOTCH3, a potential novel target for cSVD therapy.