NOTCH3 Variant Position Affects the Phenotype at the Pluripotent Stem Cell Level in CADASIL

Abstract

Disease modeling; Proteomic analysis; Stem cells

Modelització de malalties; Anàlisi proteòmica; Cèl·lules mare

Modelado de enfermedades; Análisis proteómico; Células madre

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common genetic form of stroke. It is caused by a cysteine-altering variant in one of the 34 epidermal growth factor-like repeat (EGFr) domains of Notch3. NOTCH3 pathogenic variants in EGFr 1–6 are associated with high disease severity, whereas those in EGFr 7–34 are associated with late stroke onset and increased survival. However, whether and how the position of the NOTCH3 variant directly affects the disease severity remains unclear. In this study, we aimed to generate human-induced pluripotent stem cells (hiPSCs) from patients with CADASIL with EGFr 1–6 and 7–34 pathogenic variants to evaluate whether the NOTCH3 position affects the cell phenotype and protein profile of the generated hiPSCs lines. Six hiPSCs lines were generated: two from patients with CADASIL with EGFr 1–6 pathogenic variants, two from patients with EGFr 7–34 variants, and two from controls. Notch3 aggregation and protein profiles were tested in the established six hiPSCs lines. Cell analysis revealed that the NOTCH3 variants did not limit the cell reprogramming efficiency. However, EGFr 1–6 variant position was associated with increased accumulation of Notch3 protein in pluripotent stem cells and proteomic changes related with cytoplasmic reorganization mechanisms. In conclusion, our analysis of hiPSCs derived from patients with CADASIL support the clinical association between the NOTCH3 variant position and severity of CADASIL.

This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project PI17/00540, PI20/01014, PI23/000890, RICORS-ICITUS RD21/0006/0003, RD21/0006/0004, RD24/0009/0022, RD24/0009/0017 and AC23-2/00029. AC23-2/00029 (named as CADANHIS) project has been supported by the EJP RD—European Joint Programme on Rare Diseases—Joint Transnational Call 2023 for Rare Diseases Research Project (JTC 2023). The EJP RD initiative has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N°825575. Finally, this work was supported by grants from the Instituto de Salud Carlos III, PReDICT Project (PMPER24/00021) together with Next-Generation EU funds that finance the actions of the Recovery and Resilience Mechanism.