Abstract
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 CCN1 mRNA has the ability to fold into G4s in vitro. 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 Ccn1 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.
Highlights
The blood-brain barrier (BBB) is the physical barrier between the brain parenchyma and the periphery (Wang et al, 2018)
We compared the transcriptional signature of our cerebral endothelial cells (CEC) culture obtained from RNA sequencing analysis with that from the microvasculature of mice (Song et al, 2020), and found that 41% of the 1,000 highest upregulated genes found in cultured CEC from young males corresponded with the most upregulated genes found in the microvessels of 6–7 weeks old male mice (Supplementary Table S1)
Aged CEC exhibited higher levels of both the DNA damage marker γH2AX (Figures 1C,D) and the tumor suppressor p16INK4a (Figures 1E,F), compared with young cells. These findings indicate that the age-dependent senescent phenotype is recapitulated in the primary CEC, and that this experimental approach can be useful in the study of cerebral endothelial aging
Summary
The blood-brain barrier (BBB) is the physical barrier between the brain parenchyma and the periphery (Wang et al, 2018) It is formed by cerebral endothelial cells (CEC), pericytes, astrocytes, and smooth muscle cells. CEC enter into a non-proliferative state named senescence This state is characterized by enhanced DNA damage (Lahteenvuo and Rosenzweig 2012) and secretion of proinflammatory cytokines that induce senescence in neighboring cells (Tchkonia et al, 2013). This “senescent-associated secretory phenotype (SASP)” in CEC contributes to cerebral endothelium dysfunction and dementia (Wang et al, 2018; Graves and Baker 2020). The agents that induce CEC senescence are poorly defined
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