In vitro caloric restriction induces protective genes and functional rejuvenation in senescent SAMP8 astrocytes

Abstract

Astrocytes are key cells in brain aging, helping neurons to undertake healthy aging or otherwise letting them enter into a spiral of neurodegeneration. We aimed to characterize astrocytes cultured from senescence-accelerated prone 8 (SAMP8) mice, a mouse model of brain pathological aging, along with the effects of caloric restriction, the most effective rejuvenating treatment known so far. Analysis of the transcriptomic profiles of SAMP8 astrocytes cultured in control conditions and treated with caloric restriction serum was performed using mRNA microarrays. A decrease in mitochondrial and ribosome mRNA, which was restored by caloric restriction, confirmed the age-related profile of SAMP8 astrocytes and the benefits of caloric restriction. An amelioration of antioxidant and neurodegeneration-related pathways confirmed the brain benefits of caloric restriction. Studies of oxidative stress and mitochondrial function demonstrated a reduction of oxidative damage and partial improvement of mitochondria after caloric restriction. In summary, caloric restriction showed a significant tendency to normalize pathologically aged astrocytes through the activation of pathways that are protective against the age-related deterioration of brain physiology.

Keywords: RNA microarrays; SAMP8; astrocytes; caloric restriction; mitochondria; oxidative stress.

Fig 1

Gene pathways of biological processes were modulated differentially in SAMP8 vs. SAMR1 astrocytes, and these differences were attenuated by caloric restriction. (A) Hierarchical cluster of the 100 gene pathways of gene ontology (GO) gene sets for biological processes most differentially modulated between the experimental groups. (B) Venn diagrams showing the number of pathways upregulated (upward arrow, red number), downregulated (downward arrow, green number) or with opposed changes (black number). (C) Histograms indicating the GO terms for the top five upregulated and downregulated biological pathways between the experimental groups.

Fig 2

Senescence phenotype of SAMP8 astrocytes was ameliorated by caloric restriction. (A) No distinctive morphological changes were observed by phase contrast microscopy (PhC) in SAMP8 as compared to control strain SAMR1, but β-galactosidase (β-GAL) blue staining of the same microscopy fields indicated the presence of replicative senescence in SAMP8 astrocytes treated with ad libitum serum (SAMP8-AL), whereas staining was greatly reduced in those treated with caloric restriction serum (SAMP8-CR). Mitotracker green FM (MTG) stained a similar mitochondrial pattern in the different cultures (green fluorescence). Immunostaining of nitrotyrosinated proteins (NT) was only visible (green fluorescence spots) in SAMP8-AL astrocytes, whereas that of GFAP (red fluorescence) showed similar astrocyte morphology in the different cultures; nuclei were counterstained with TO-PRO-3 iodide (confocal images). Representative images, n = 4. (B) Gene expression of mitochondrial and antioxidant genes measured by real-time qPCR showed a moderate increase in SAMP8 astrocytes after CR treatment. Nudfa1, Uqcrh, Cox6b1, Apt5e, and Mrpl14 encode proteins of mitochondrial complexes I, III, IV, V, and mitochondrial ribosomes, respectively; Sod1, Gpx3, Gpx8, and Atox1 encode a variety of soluble and membrane antioxidants, and Nudt1 a selective nucleic acid antioxidant. See Table S7 (Supporting information) for full name of genes and oligomers used. Statistics: **P < 0.01 overall effect of CR treatment according to ANOVA, without post hoc Bonferroni's test significance.

Fig 3

Caloric restriction reduced the oxidative stress suffered by SAMP8 astrocytes. (A) SAMP8 astrocytes generated more reactive oxygen species (ROS) than SAMR1 astrocytes in control conditions as measured by increased dichlorofluorescein (DCF) fluorescence units (FU), whereas differences between the two cell types were minor in the presence of hydrogen peroxide. (B) Caloric restriction (CR) reduced the oxygen consumption of the astrocyte cells compared with control conditions (AL). (C) CR barely decreased the excess ROS generated by SAMP8 astrocytes, whereas the decrease it induced in the ROS generated by hydrogen peroxide was only significant in control SAMR1 astrocytes. (D) CR reduced the increased level of carbonylated proteins of SAMP8 astrocytes, as shown in representative oxyblots. Coomassie blue staining was used to normalize protein oxidation in an integrated area including the two main oxidation bands (arrows). Statistics: *P < 0.05 vs. SAMR1; #P < 0.05, and ##P < 0.01, vs. control ad libitum (AL) conditions by Bonferroni's test.

Fig 4

SAMP8 astrocytes showed mitochondrial impairment that was partially ameliorated by caloric restriction. (A) SAMP8 astrocyte mitochondria had reduced membrane potential (MMP) as measured by rhodamine 123 (Rh123). (B) Mitochondrial uncouplers and specific inhibitors showed the differential sensitivity of SAMP8 astrocytes against antimycin A, an inhibitor of complex III, compared with SAMR1 astrocytes. (C) Caloric restriction (CR) barely modified MMP in comparison with the control ad libitum (AL) treatment, measured according to the ratio of tetramethylrhodamine methyl ester to nonyl acridine orange (TMRM/NAO). (D) Mitochondrial complex proteins showed a general trend to decrease in SAMP8 in comparison with the SAMR1 levels. CR improved the level of the mitochondrial complex III protein marker in SAMP8, which was significantly decreased, as shown in representative immunoblots and densitometry analysis of proteins from the respiratory chain complexes. The immunoreactivity of the protein bands of adenosine triphosphate (ATP) synthase subunit α (ATP5A, complex V), ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2, complex III), cytochrome c oxidase I (COX1, complex IV), and NADH dehydrogenase [ubiquinone] 1 β subcomplex subunit 8 (NDUFB8, complex I) was normalized to that of succinate dehydrogenase [ubiquinone] iron-sulfur subunit (SDHB, 30 kDa subunit, complex II) protein. (E) CR barely modified the mitochondrial mass as measured by NAO. Statistics: *P < 0.05 vs. SAMR1 by Bonferroni's test.