G-quadruplexes Stabilization Upregulates CCN1 and Accelerates Aging in Cultured Cerebral Endothelial Cells

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.

Keywords: CCN1; G-quadruplex; aging; endothelial cells; senescence.

FIGURE 1

Cultured primary CEC from aged mice recapitulated senescence-associated phenotypes observed in vivo. CEC were isolated from young (3–4 m/o) and aged (18–20 m/o) female mice and cultured. Young and aged cultured CEC were stained for SA-β-galactosidase (A), with antibodies against the double-strand DNA damage marker γH2AX (C), or with antibodies against the tumor suppressor p16^INK4A (E). The nuclear Hoechst dye was used to stain nuclei and imaged with the DAPI channel (C,E). The percentage of SA-β-galactosidase-positive cells (B), the fluorescence intensity of γH2AX (D) and the fluorescence intensity of p16^INK4A (F) were quantified. Scale bar (A), 100 μm; scale bar (C), 25 μm; scale bar (E), 25 μm. t-test, ***p-value < 0.0001. Data were pooled from three independent experiments, 20 microscopic fields (A,B), or 50 cells (C–F) per each experiment and condition.

FIGURE 2

Cultured CEC from aged mice showed higher levels of RNA-G4 than cultured young CEC. (A) CEC were isolated from young (4-m/o) and aged (20-m/o) female mice and cultured. Cells were fixed and stained with antibodies against G4 (BG4) anti against actin, and with the nuclear Hoechst dye. (B) Cultured CEC from young and aged female mice were fixed and incubated with DNaseI to eliminate DNA G4, and then stained with antibodies against G4 (BG4) and against actin, and with the nuclear Hoechst dye. (C,D) BG4 fluorescence intensities were measured. (C) Quantification from (A). Scale bar (Yg), 20 μm; Scale bar (Ag), 40 μm. t-test, *p-value = 0.0116. (D) Quantification from (B). t-test, *p-value = 0.0435. Data were pooled from three independent experiments analyzing 50 cells per condition and experiment.

FIGURE 3

A G4-stabilizing compound exacerbated senescence-associated phenotypes in cultured CEC. (A) CEC isolated from aged (18–20 m/o) female mice and cultured were treated with pyridostatin (0.5 µM), or a vehicle (water), for 4 days. CEC were fixed and stained with antibodies against γH2AX and the nuclear Hoechst dye (DAPI channel). Scale bar, 50 µm (B) Quantification of the fluorescence intensity of γH2AX from (A). t-test, ***p-value = 0.0003. Data were pooled from three independent experiments analyzing 50 cells per condition and experiment. (C) Quantification of the nuclei size. t-test, ***p-value = 0.0001. Data were pooled from three independent experiments analyzing 50 cells per condition and experiment. (D) CEC isolated from aged (18–20 m/o) female mice and cultured were treated with pyridostatin (0.5 µM), or a vehicle (water), for 4 days. CEC were fixed and stained with the SA-β-galactosidase staining kit. Scale bar, 50 µm. (E) Quantification of the percentage of SA-β-galactosidase-positive cells. t-test, **p-value = 0.0015. Data were pooled from three independent experiments analyzing 50 cells (A–C) or 20 microscopic fields (D,E) per condition and experiment.

FIGURE 4

CCN1 was upregulated in the microvessels isolated from the brains of aged mice and in cultured CEC isolated from aged mice compared with young mice. (A) Representative image of microvessels isolated from the brains of young (3–4 m/o) and aged (18–20 m/o) female mice, and stained with antibodies against the endothelial marker CD31, with antibodies against CCN1, and with the nuclear Hoechst dye (DAPI channel). Scale bar, 25 µm. (B) CEC were isolated from young (3-4- m/o) and aged (18–20 m/o) female mice, cultured and maintained until 100% confluence. Cells were collected and lysed and processed for gel electrophoresis and analyzed for Western blotting. (C) Band intensities of CCN1 relative to tubulin. t-test, ** p-value < 0.0019. Data were obtained from four mice per age.

FIGURE 5

CCN1 induced senescence-associated phenotypes in cultured CEC. CEC were isolated from aged (18–20 m/o) female mice and cultured. Cultured CEC were stained with SA-β-galactosidase staining kit (A), or with antibodies against the double-strand DNA damage marker γH2AX (C), or with antibodies against CCN1 (E), and with the nuclear Hoechst dye (C,E). The percentage of SA-β-galactosidase-positive cells (B), the fluorescence intensity of γH2AX (D), and the fluorescence intensity of CCN1 (F) were quantified. Scale bar (A), 100 μm; scale bar (C), 50 μm; scale bar (E), 50 μm. t-test, ***p-value (B) = 0.0001, n = 4; *p-value (D) = 0.0164, ***p-value (F) = 0.0001. Data were pooled from three independent experiments analyzing 20 microscopic fields (A,B,E,F) or 50 cells (C,D) per condition and experiment.

FIGURE 6

The 3′-UTR Ccn1 mRNA folded into G4s. (A) Scheme of the mRNA Ccn1 where the 5′-UTR (green) does not contain any potential G4 motif, and the 3′-UTR (red) contains three potential G4 motifs. In the representation of a RNA G4 structure, blue dots indicate Gs and orange rectangles indicate planar rearrangements between Gs. (B) The RNA sequence containing the potential G4 in the 3′-UTR Ccn1 near the stop codon (3′-UTR) or the RNA sequence containing the 3′-UTR Ccn1 with a single mutation (mutant), as a negative control, were incubated at 90°C for 2 m, and cooled down at room temperature for 2 h. Then, the annealed RNA 3′-UTR and mutant sequences were mixed with a Thioflavin T (ThT) solution, and the fluorescence emission was measured. As controls we used the buffer alone, a ThT solution alone, and the 3′-UTR sequence alone. One-way ANOVA, ***p-value < 0.0001. Data were collected from four independent experiments. (C) CEC isolated from aged (18–20 m/o) female mice and cultured were treated with pyridostatin (0.5 µM), or a vehicle (water), for 4 days. CEC were fixed and stained with antibodies against CCN1 and with the nuclear Hoechst dye (DAPI channel). Scale bar, 50 µm. (D) Quantification of the fluorescence intensity of CCN1 from (C). t-test, *p-value = 0.0103. Data were pooled from three independent experiments analyzing 20 microscopic fields per condition and experiment. (E) CEC isolated from aged (18–20 m/o) female mice and cultured were treated with pyridostatin (0.5 µM), or a vehicle, for 4 days. RNA was isolated and qPCR was performed for the relative expression of Ccn1. We used Gapdh as a housekeeping gene. t-test, n.s., non-significant, p-value = 0.2424. Data were pooled from four independent experiments.