Unraveling the role of NOTCH3 dysfunction in disease and for therapy development : a focus on CADASIL

The Notch signaling pathway, including the Notch3 receptor, is involved in many pivotal contexts of cell signaling and cell fate determination processes during development and in adulthood. Different mutations in the NOTCH3 receptor, which is highly expressed in vascular smooth muscle cells (VSMC), are associated with various diseases, including developmental disorders, several forms of cancer, and vascular dementia. In some of these diseases, the underpinning disease-mechanism is relatively well-understood whereas in other cases the role of the NOTCH3 mutations in the disease pathogenesis remains more obscure. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of familial small vessel disease and is a monogenic disorder caused by mutations in the NOTCH3 gene. CADASIL-causing mutations lead to misfolding and aggregation of the NOTCH3 receptor extracellular domain (ECD) around VSMC, which degenerates and plays a pivotal role in disease pathogenesis. Currently, there are no therapies available that could prevent or ameliorate CADASIL. However, given that NOTCH3 aggregation and accumulation is a hallmark of the disease, therapeutic strategies that aim to inhibit NOTCH3 aggregation and/or to clear pre-formed NOTCH3 aggregates hold great promise as novel treatments for CADASIL.

In this thesis, I have worked on the development of novel therapies targeting NOTCH3 misfolding in CADASIL and in exploring the underpinning molecular mechanisms that link NOTCH3 mutations to diseases other than CADASIL. More specifically, I have used biochemical, cellular, and animal studies to develop two new therapeutic strategies aimed at preventing the formation of or promoting the clearance of CADASIL-associated misfolded NOTCH3 ECD. Furthermore, I have conducted clinical and preclinical research to identify and characterize a novel disease-associated NOTCH3 mutant, which is linked to a CADASIL-like small vessel disease, and to characterize the signaling of the NOTCH3 L1519P mutant which causes infantile myofibromatosis (IMF), but where the impact of the L1519P mutation on NOTCH3 signaling has remained poorly understood.

In paper I, we showed that the IMF causing NOTCH3 L1519P mutation results in a hyperactive ligand-independent NOTCH3 receptor and increased PDGFRB expression. Increased Notch3 signaling activity is consistent with an overproduction of NOTCH3 intercellular domain (ICD), increased reporter activity, and an increase in the downstream expression of Notch3 target genes. Our study establishes a connection between NOTCH3 and PDGFRB in IMF, indicating that Notch3 signaling may be epistatic to PDGFRB and that activating mutations in both NOTCH3 and PDGFRB could be disease-causative. In paper II, we identified a novel NOTCH3 cysteine-sparing mutation, NOTCH3 A1604T, in a family suffering from migraine and white matter lesions, which are also hallmarks of CADASIL. Additionally, in cell culture studies we showed that this mutation affects NOTCH3 receptor stability and processing and result in decreased NOTCH3 signaling. These data identify NOTCH3 A1604T as a novel dysfunctional NOTCH3 mutant associated with cerebral small vessel disease. In paper III, we developed an active immunization therapy targeting aggregated NOTCH3 ECD for the treatment of CADASIL. Four months treatment caused a reduction in NOTCH3 ECD deposits in both size and quantity around brain capillaries in a CADASIL mouse model. These data indicate that active immunization may be a viable disease modifying therapeutic strategy for CADASIL and support its further development towards clinical usage. In paper IV, we showed that the CADASIL-associated NOTCH3 multimerization/aggregation could be inhibited by the molecular chaperone Bri2 BRICHOS, which has been shown to inhibit multiple misfolding and aggregation reactions. We demonstrated that Bri2 BRICHOS stabilized the mutant NOTCH3 protein in a monomeric form and lowered the aggregation kinetics of the NOTCH3 mutant, suggesting that Bri2 BRICHOS could be used to as a novel therapeutic entity to prevent NOTCH3 ECD aggregation in CADASIL.

In conclusion, I believe the work of my thesis has increased our understanding of the role of NOTCH3 dysregulation in vascular disease and in the developmental disorder IMF as well as provided important preclinical proof of concept data for novel therapeutic strategies aimed to interfere with the NOTCH3 misfolding and aggregation in CADASIL.

List of scientific papers

I. Dan Wu, Sailan Wang, Daniel V. Oliveira, Francesca Del Gaudio, Michael Vanlandewijck, Thibaud Lebouvier, Christer Betsholtz, Jian Zhao, ShaoBo Jin, Urban Lendahl, Helena Karlström. The infantile myofibromatosis NOTCH3 L1519P mutation leads to hyperactivated ligand-independent Notch signaling and increased PDGFRB expression. Dis Model Mech. 2021 Jan 28;14(2):dmm046300.
https://doi.org/10.1242/dmm.046300

II. Snjolaug Arnardottir, Francesca Del Gaudio, Stefanos Klironomos, Eike-Benjamin Braune, Ariane A. Lombraña, Daniel V. Oliveira, ShaoBo Jin, Helena Karlström, Urban Lendahl, Christina Sjöstrand. Novel Cysteine-Sparing Hypomorphic NOTCH3 A1604T Mutation Observed in a Family With Migraine and White Matter Lesions. Neurol Genet. 2021 Apr 22;7(3):e584.
https://doi.org/10.1212/NXG.0000000000000584

III. Daniel V. Oliveira, Kirsten Coupland, ShaoBo Jin, Francesca Del Gaudio, Sailan Wang, Rhys Fox, Julie W. Rutten, Johan Lundkvist, Saskia A. J. Lesnik Oberstein, Urban Lendahl, and Helena Karlström. NOTCH3 active immunotherapy decreases NOTCH3 deposition in capillaries in a CADASIL mouse model. [Manuscript]

IV. Daniel V. Oliveira, Julia Svensson, Xueying Zhong, Henrik Biverstål, Gefei Chen and Helena Karlström. Molecular chaperone BRICHOS inhibits CADASIL-mutated NOTCH3 aggregation in vitro. [Manuscript]