Noh Brian, Blasco-Conesa Maria P, Lai Yun-Ju, Ganesh Bhanu Priya, Urayama Akihiko, Moreno-Gonzalez Ines, Marrelli Sean P, McCullough Louise D, Moruno-Manchon Jose Felix
Department of Neurology, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States.
Solomont School of Nursing, Zuckerberg College of Health Sciences, University of Massachusetts Lowell, Lowell, MA, United States.
Front Aging. 2022 Jan 12;2:797562. doi: 10.3389/fragi.2021.797562. eCollection 2021.
Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. However, whether CCN1 contributes to senescence in CEC and how this is regulated requires further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. Stabilization of the G4 structures regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and these mice exhibited enhanced cognitive deficits. A sequence in the 3'-UTR of the human mRNA has the ability to fold into G4s . We hypothesize that G4 stabilization regulates CCN1 in cultured primary CEC and induces endothelial senescence. We used cerebral microvessel fractions and cultured primary CEC from young (4-months old, m/o) and aged (18-m/o) mice to determine CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA-G4s for fluorescence spectra. We found that cultured CEC from aged mice exhibited enhanced levels of SA phenotypes, and higher levels of CCN1 and G4 stabilization. In cultured CEC, CCN1 induced SA phenotypes, such as SA β-galactosidase activity, and double-strand DNA damage. Furthermore, CCN1 levels were upregulated by a G4 ligand, and a G-rich motif in the 3'-UTR of the mRNA was folded into a G4. In conclusion, we demonstrate that CCN1 can induce senescence in cultured primary CEC, and we provide evidence that G4 stabilization is a novel mechanism regulating the SASP component CCN1.
脑内皮细胞衰老被认为是一种可导致脑血管功能障碍的机制，而脑血管功能障碍早于血管性痴呆。富含半胱氨酸的血管生成诱导因子61（Cyr61/CCN1）是脑内皮细胞（CEC）分泌的一种基质细胞蛋白。CCN1可诱导成纤维细胞衰老。然而，CCN1是否会导致CEC衰老以及其调控方式仍需进一步研究。衰老与DNA和RNA富含鸟嘌呤基序中四链体鸟嘌呤（G4s）的形成有关。G4结构的稳定通过上调或下调来调节转录和翻译，具体取决于基因靶点。此前，我们发现用G4稳定化合物处理的老年小鼠大脑中衰老相关（SA）表型增强，且这些小鼠表现出更严重的认知缺陷。人mRNA 3'-UTR中的一个序列能够折叠成G4s。我们推测G4稳定可调节原代培养CEC中的CCN1并诱导内皮细胞衰老。我们使用来自年轻（4个月大，m/o）和老年（18个月大）小鼠的脑微血管组分和原代培养CEC来测定CCN1水平。通过高分辨率荧光显微镜在原代培养CEC中测定SA表型，我们使用硫黄素T识别RNA-G4s以进行荧光光谱分析。我们发现老年小鼠原代培养的CEC表现出增强的SA表型水平，以及更高水平的CCN1和G4稳定。在原代培养CEC中，CCN1诱导SA表型，如SAβ-半乳糖苷酶活性和双链DNA损伤。此外，G4配体上调CCN1水平，且mRNA 3'-UTR中的富含G基序折叠成G4。总之，我们证明CCN1可诱导原代培养CEC衰老，并提供证据表明G4稳定是调节衰老相关分泌表型成分CCN1的新机制。
