Muiño Elena, Maisterra Olga, Jiménez-Balado Joan, Cullell Natalia, Carrera Caty, Torres-Aguila Nuria P, Cárcel-Márquez Jara, Gallego-Fabrega Cristina, Lledós Miquel, González-Sánchez Jonathan, Olmos-Alpiste Ferran, Espejo Eva, March Álvaro, Pujol Ramón, Rodríguez-Campello Ana, Romeral Gemma, Krupinski Jurek, Martí-Fàbregas Joan, Montaner Joan, Roquer Jaume, Fernández-Cadenas Israel
Stroke Pharmacogenomics and Genetics Group, Institut de Recerca de l`Hospital de la Santa Creu i Sant Pau, C/Sant Antoni María Claret 167, Barcelona, Spain.
Neurovascular Research Laboratory, Vall d'Hebron Institute of Research, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain.
Sci Rep. 2021 Mar 25;11(1):6846. doi: 10.1038/s41598-021-86349-1.
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 and PDCD6IP, p-value = 8.36 × 10) were selected for the validation study by qRT-PCR. Additionally, we selected two more mRNAs (CAMK2G, p-value = 4.52 × 10 and E2F4, p-value = 4.77 × 10) 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), and it was upregulated in CADASIL cases. Furthermore, higher E2F4 expression was associated with worse executive function (p = 2.04 × 10) and attention and information processing speed (IPS) (p = 8.73 × 10). 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.
伴有皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病（CADASIL）是一种小血管疾病，由NOTCH3基因突变引起，该突变导致表皮生长因子（EGF）样重复结构域中的半胱氨酸数量为奇数，从而引起蛋白质错误折叠和聚集。主要症状为偏头痛、精神障碍、复发性中风和痴呆，其中执行功能通常受损。这种受体聚集所改变的分子途径需要进一步研究。除了用于验证目的的定量逆转录聚合酶链反应（qRT-PCR）（10例新病例和8例新对照）外，还进行了一项全基因组转录组研究（4例病例与3名健康同胞配对）。为了按细胞类型研究所发现的显著信使核糖核酸（mRNA）的表达谱，我们进行了原位杂交（ISH）（9例病例和8例对照）以及在单核脑RNA测序表达浏览器（SNBREB）中的研究。使用基因本体论和Reactome进行通路分析富集。对qRT-PCR检测的5例病例进行了神经心理学测试。选择两个最显著差异表达的mRNA（BANP，p值 = 7.23×10；PDCD6IP，p值 = 8.36×10）用于通过qRT-PCR进行验证研究。此外，由于它们与缺血性神经元死亡相关，我们又选择了另外两个mRNA（CAMK2G，p值 = 4.52×10；E2F4，p值 = 4.77×10）。E2F4在全基因组转录组研究和qRT-PCR中均显示出差异表达（p = 1.23×10），并且在CADASIL病例中上调。此外，E2F4表达升高与更差的执行功能（p = 2.04×10）以及注意力和信息处理速度（IPS）（p = 8.73×10）相关。原位杂交显示E2F4在内皮细胞和血管平滑肌细胞中表达。计算机模拟研究表明E2F4在脑内皮细胞中也有表达。在所分析的最显著通路中，血管发育、细胞黏附、囊泡机制术语和自噬过程出现富集。与对照组相比，E2F4在CADASIL患者的皮肤活检中表达更高，并且其表达存在于内皮细胞和平滑肌细胞中。需要进一步研究以了解E2F4是否可用作生物标志物、监测疾病或用作治疗靶点。
