Research Paper Volume 10, Issue 11 pp 3397—3420

Cell cycle-dependent and -independent telomere shortening accompanies murine brain aging

Figure 6. Age-associated changes in TERT protein content, telomerase activity and relative Rela and c-Rel expression levels in the murine cortex. (A) Telomerase activity assessed from cortical isolates as a function of age. Telomerase activity in the mature neocortex remained unchanged with aging, displaying activity levels approximately 20 times lower than in E13 brain. Bars of probe samples represent means ± SEM (n = 5). HI E13, heat inactivated E13 protein extract. (B-D) Relative TERT protein levels in different subcellular compartments of murine cortical tissue at young (Y) and aged (A) states. (B) Left blot: Conventional Western blot. Purity of the cellular subfractions is illustrated by the enriched levels of the nuclear and cytoplasmic marker proteins Histone H3 and GAPDH in the chromatin-bound nuclear (cNE) and cytoplasmic (CP) fractions, respectively. Right blot: TERT product at the appropriate molecular weight of ~ 125 kDa in whole cell (WC) lysates from ovary (Ov) and cortex of young (Y) and aged (A) mice. (C) Simple Western™ technique. Little or no TERT protein was expressed in the soluble nuclear (sNE) and chromatin-bound nuclear (cNE) fraction, respectively, at both age categories, whereas TERT protein was found at significantly higher levels in the cytoplasmic (CP) and membrane (ME) fractions at 27 months of age. Specificity of the TERT antibody was proven by the detection of different TERT protein levels in whole cell (WC) lysates prepared from small intestine (In), ovary (Ov), muscle (Mu) and cortex of young mice (Y). All cortical samples were assayed in the same run, as were all the control samples. (D) Quantification of the compartment-specific TERT protein levels for young and old cortices as illustrated in (C). Bars represent means ± SEM (n = 3), and were reproduced in two independent runs. (E) Age-associated changes in relative mRNA levels of the subunits Rela and c-Rel of canonical NF-κB.Rela transcription levels increased significantly at advanced ages, whereas levels of c-Rel remained unaltered. Ratios represent geometric means of n = 5 animals ± SEM. P-values were calculated using Student’s t-test (A) and Two-way ANOVA (D and E).