Israel Fernandez-Cadenas, Miquel Lledós, Ramon M. Pujol, Natalia Cullell, Jara Cárcel-Márquez, Olga Maisterra, Elena Muiño, Joan Jiménez-Balado, Cristina Gallego-Fabrega, Joan Montaner, Gemma Romeral, Jurek Krupinski, Ferran Olmos-Alpiste, Ana Rodríguez-Campello, Álvaro March, Eva Espejo, Jonathan González-Sánchez, Jaume Roquer, Caty Carrera, Nuria P. Torres-Aguila, Joan Martí-Fàbregas, University of St Andrews. School of Biology, [Muiño,E, Cullell,N, Carrera,C, Torres-Aguila,NP, Cárcel-Márquez,J, Gallego-Fabrega,C, Lledós,M, Fernández-Cadenas,I] Stroke Pharmacogenomics and Genetics Group, Institut de Recerca de l`Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. [Maisterra,O, Jiménez-Balado,J] Neurovascular Research Laboratory, Vall d’Hebron Institute of Research, Hospital Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain. [Gallego-Fabrega,C, González-Sánchez,J] Stroke Pharmacogenomics and Genetics, Fundació MútuaTerrassa per la Docència i la Recerca, Terrassa, Spain. [González-Sánchez,J] The Manchester Metropolitan University of All Saints, Manchester, UK. [Olmos-Alpiste,F, Espejo,E, March,A, Pujol,R] Dermatology Department, Hospital del Mar-Parc de Salut Mar, Barcelona, Spain. [Rodríguez-Campello,A, Romeral,G, Roquer,J] Neurology Department, IMIM-Hospital del Mar, Barcelona, Spain. [Krupinski,J] Neurology Department, Hospital Mútua Terrassa, Terrassa, Spain. [Martí-Fàbregas,J] Neurology Department, Hospital de la Santa Creu i Sant Pau, Biomedical Research Institute Sant Pau (IIB-Sant Pau), Barcelona, Spain. [Montaner,J] Biomedicine Institute of Seville, IBiS/Hospital Universitario Virgen del Rocío/CSIC, University of Seville, Seville, Spain. [Montaner,J] Department of Neurology, Hospital Universitario Virgen Macarena, Seville, Spain, This work was supported by a grant from the Carlos III Health Institute (PI 11/0176), Generación Project, Maestro Project, INVICTUS + network, Epigenesis Project (Marató de TV3), FEDER funds and economic donations from 'Asociación CADASIL España'. E. Muiño is supported by a Río Hortega Contract (CM18/00198) from the Carlos III Health Institute. J. Cárcel-Márquez is supported by an AGAUR Contract (agència de gestió d'ajuts universitaris i de recerca, FI_DGR 2019, grant number 2019_FI_B 00853) co-financed with Fons Social Europeu (FSE). M. Lledós is supported by a PFIS Contract (Contratos Predoctorales de Formación en Investigación en Salud) from the Carlos III Health Institute., Instituto de Salud Carlos III, Fundació La Marató de TV3, European Commission, Cadasil, Agència de Gestió d'Ajuts Universitaris i de Recerca, Institut Català de la Salut, [Muiño E, Carrera C, Torres-Aguila NP] Stroke Pharmacogenomics and Genetics Group, Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. [Maisterra O, Jiménez-Balado J] Laboratori de Recerca Neurovascular, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Vall d’Hebron Hospital Universitari, Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. [Cullell N] Stroke Pharmacogenomics and Genetics Group, Institut de Recerca de l`Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. Stroke Pharmacogenomics and Genetics, Fundació MútuaTerrassa per la Docència i la Recerca, Terrassa, Spain, and Vall d'Hebron Barcelona Hospital Campus
CADASIL is a small vessel disease caused by mutations in NOTCH3 that lead to an odd number of cysteines in the EGF-like repeat domain, causing protein misfolding and aggregation. The main symptoms are migraine, psychiatric disturbances, recurrent strokes and dementia, being executive function characteristically impaired. The molecular pathways altered by this receptor aggregation need to be studied further. A genome-wide transcriptome study (four cases paired with three healthy siblings) was carried out, in addition to a qRT-PCR for validation purposes (ten new cases and eight new controls). To study the expression profile by cell type of the significant mRNAs found, we performed an in situ hybridization (ISH) (nine cases and eight controls) and a research in the Single-nuclei Brain RNA-seq expression browser (SNBREB). Pathway analysis enrichment was carried out with Gene Ontology and Reactome. Neuropsychological tests were performed in five of the qRT-PCR cases. The two most significant differentially expressed mRNAs (BANP, p-value = 7.23 × 10–4 and PDCD6IP, p-value = 8.36 × 10–4) were selected for the validation study by qRT-PCR. Additionally, we selected two more mRNAs (CAMK2G, p-value = 4.52 × 10–3 and E2F4, p-value = 4.77 × 10–3) due to their association with ischemic neuronal death. E2F4 showed differential expression in the genome-wide transcriptome study and in the qRT-PCR (p = 1.23 × 10–3), and it was upregulated in CADASIL cases. Furthermore, higher E2F4 expression was associated with worse executive function (p = 2.04 × 10–2) and attention and information processing speed (IPS) (p = 8.73 × 10–2). In situ hibridization showed E2F4 expression in endothelial and vascular smooth vessel cells. In silico studies indicated that E2F4 is also expressed in brain endothelial cells. Among the most significant pathways analyzed, there was an enrichment of vascular development, cell adhesion and vesicular machinery terms and autophagy process. E2F4 is more highly expressed in the skin biopsy of CADASIL patients compared to controls, and its expression is present in endothelial cells and VSMCs. Further studies are needed to understand whether E2F4 could be useful as a biomarker, to monitor the disease or be used as a therapeutic target., This work was supported by a grant from the Carlos III Health Institute (PI 11/0176), Generación Project, Maestro Project, INVICTUS + network, Epigenesis Project (Marató de TV3), FEDER funds and economic donations from “Asociación CADASIL España”. E. Muiño is supported by a Río Hortega Contract (CM18/00198) from the Carlos III Health Institute. J. Cárcel-Márquez is supported by an AGAUR Contract (agència de gestió d'ajuts universitaris i de recerca; FI_DGR 2019, grant number 2019_FI_B 00853) co-financed with Fons Social Europeu (FSE). M. Lledós is supported by a PFIS Contract (Contratos Predoctorales de Formación en Investigación en Salud) from the Carlos III Health Institute.