Fig 1: Effect of RAP1GDS1 transgene and knockdown on mouse brain aging.a Immunostaining by VDAC1 to stain mitochondria in the outer molecular layer of prefrontal region. Control (8 month-old) and RAP1GDS1 overexpression (8 month-old) is shown(left). Scale bar, 5 μm. The average mitochondrial size of control is set as 1, and the ratio of RAP1GDS1 overexpression is shown (right). Each mouse quantified 20 neurons. N = 5. Unpaired t-test. **P = 0.008. b The water Morris maze assay. Representative traces of mice with different age and RAP1GDS1 overexpression (8 month-old) of Morris water maze. The number of crosses of the platform in each mouse. N = 6, 6, 7, 7, and 8 (from left to right). One-way ANOVA with Tukey’s post hoc test. ****P < 0.0001, ****P < 0.0001. c Representative micrograph of Golgi staining to display the dendritic spines intensity in neurons in the outer molecular layer of prefrontal region. For wild type mice at different ages, N = 5. For RAP1GDS1 overexpression mice at 8 month-old, N = 6. One-way ANOVA with Tukey’s post hoc test. ****P < 0.0001, *P = 0.0383, ****P < 0.0001. d The survival curve of wild type and RAP1GDS1 overexpression mice. N = 5. long-rank(mantel-Cox) test. P = 0.0027. e Schematic time course of this experiment. 3 month-old heterozygous knockdown of RAP1GDS1 (MAP2-Cre-ERT2+/−; RAP1GDS1+/−) was injection D-galactose (100 mg/kg i.v.) from day 1 to day 70. At day 20, RAP1GDS1 knockdown was induced by tamoxifen (75 mg/kg i.p.) for 7 days. Behavioral assays were performed at day 90. These animals were sacrificed at day 120. f Representative traces of the open filed assay. Quantification of mouse stayed in the center zone, N = 6. Unpaired t-test. **P = 0.0022. g Representative micrograph of Golgi staining to display the dendritic spines intensity in neurons in the outer molecular layer of prefrontal region. N = 6. Error bars are mean ± SE. One-way ANOVA with Tukey’s post hoc test. ***P = 0.0004, **P = 0.0081. h Survival curve. Wild type mice treated with D-galactose, N = 9. Heterozygous RAP1GDS1 knockdown mice treated with D-galactose, N = 8. long-rank (mantel-Cox) test. P = 0.0285.
Fig 2: RAPGDS1 function on mitochondria in cultured cells.The U87-MG cells were transfected with a Pljm1-RAP1GDS1-eGFP vector which expresses RAP1GDS1 and eGFP independently. a Representative live image of mitochondrial morphology by Mito Tracker red. The green channel is RAP1GDS1; the red channel is mitochondria. Scale bar, 5 μm. The bar graph is the mitochondrial length quantified by ImageJ. Cell number N = 26 (control, without RAP1GDS1 expression) and 28 (test, with RAP1GDS1 expression). Unpaired t-test. ****P < 0.0001. b Representative live image of mitochondrial membrane potential stained by TMRM. Scale bar, 10 μm. GFP positive cells are RAP1GDS1 overexpressed. GFP negative is the control cells. The red intensity of TMRM was quantified by ImageJ. N = 5 (Control), 6 (RAP1GDS1 overexpression). Unpaired t-test. ****P < 0.0001. c Representative live image of ROS level stained by CellROX. Scale bar, 20 μm. The red intensity of cells was quantified by ImageJ. N = 15. Unpaired t-test. ****P < 0.0001. d Representative live image of mitochondrial calcium level stained by Rhod2-AM. Scale bar, 20 μm. In the same field, the GFP positive cells represent RAP1GDS1 overexpression; GFP negative cells are controls. Intensity of the red signal was quantified by ImageJ. N = 17 (control), 14 (RAP1GDS1 overexpression). Unpaired t-test. ****P < 0.0001. e Representative live image of mitochondrial morphology stained by Mito tracker red. The green channel is RAP1GDS1; the red channel is mitochondria. Scale bar, 5μm. Mitochondrial length was quantified by ImageJ. N = 11. Error bars are mean ± SE. Unpaired t-test. ****P < 0.0001. f The intensity of Rhod2-AM change of U87-MG cells treated with ionomycin. Each trace represents mitochondrial calcium level changes after ionomycin treatment. For control and RAP1GDS1 siRNA cells, N = 7 (control), N = 8 (RAP1GDS1 siRNA). Unpaired t-test. P < 0.0001.
Fig 3: RAP1GDS1/Miro1 and mitochondrial functional change mouse brain aging.a Western blot analysis of RAP1GDS1 (antibody from Abcam #ab188020) and Miro1 change during aging in the brain tissue of C57 mice. β-actin is the protein loading control. Uncropped blots are shown in Supplementary Fig. 8. With the 2 month-old sample set as 1. N = 6. One-way ANOVA with Tukey’s post hoc test. 2vs21, *P = 0.023; 2vs27, ****P < 0.0001; 16vs27, **P = 0.0035. b Co-Immunoprecipitation of RAP1GDS1 and Miro1 during aging. IP with RAP1GDS1 antibody (Santa #sc39003) to pull down Miro1. Total protein input is shown as the protein loading control. Uncropped blots are shown in Supplementary Fig. 8. The ratio of Miro1/RAP1GDS1 from the 2 month-old sample is set as 1, and the relative level of other samples are shown. N = 3. One-way ANOVA with Tukey’s post hoc test. **P = 0.0056. c Representative mitochondrial morphology stained by VDAC1 in neurons of prefrontal cortex. Scale bar, 5 μm. Mitochondrial size changes in brain neurons during aging. 2 month-old sample is set as 1. N = 10. One-way ANOVA with Tukey’s post hoc test. ****P < 0.0001. d Brain mitochondrial calcium concentration ([Ca2+]mito.) change during aging. The intensity of Rhod2-AM indicates [Ca2+]mito level. Each mouse brain quantified 20 neurons, the Rhod-2AM intensity from 2 month-old sample was set as 1, and the ratios of others are shown. N = 4. One-way ANOVA with Tukey’s post hoc test. ****P < 0.0001, ****P < 0.0001. e Cytosolic calcium ([Ca2+]cyto) change stained by BAPTA-1 AM in young (2 month-old) and senescent (27 month-old) mice. The BAPTA-1 AM intensity of 2 month-old is set as 1. N = 6. Error bars are mean ± SE. Unpaired t-test. ****P < 0.0001.
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