Macroautophagy is impaired in old murine brain tissue as well as in senescent human fibroblasts

Abstract

The overall decrease in proteolytic activity in aging can promote and accelerate protein accumulation and metabolic disturbances. To specifically analyze changes in macroautophagy (MA) we quantified different autophagy-related proteins (ATGs) in young, adult and old murine tissue as well as in young and senescent human fibroblasts. Thus, we revealed significantly reduced levels of ATG5-ATG12, LC3-II/LC3-I ratio, Beclin-1 and p62 in old brain tissue and senescent human fibroblasts. To investigate the role of mTOR, the protein itself and its target proteins p70S6 kinase and 4E-BP1 were quantified. Significant increased mTOR protein levels were determined in old tissue and cells. Determination of phosphorylated and basal amount of both proteins suggested higher mTOR activity in old murine tissue and senescent human fibroblasts. Besides the reduced levels of ATGs, mTOR can additionally reduce MA, promoting further acceleration of protein accumulation and metabolic disturbances during aging.

Keywords: ATGs; Aging; Autophagy-lysosome pathway; Fibroblasts; MTOR; Senescence.

Fig. 1

Examination of autophagy-associated proteins in young, adult and old C57BL/6J male mice. Panels depict immunoblot analyses of (A) p62, (B) ATG5-ATG12 and (C) Beclin-1 in young, adult and old brain tissue from C57BL/6J male mice. Four lanes represent four individual mice per age group. All data were analyzed in relation to GAPDH and the young control was set to 100%. Statistical significant differences between young, adult and old tissue were calculated, using one-way ANOVA, followed by Tukey's post hoc test and are shown by *p<0.05, **p<0.01, all compared to young control.

Fig. 2

Relative quantification of mTOR protein levels in brain tissue of young, adult and old C57BL/6J mice. To quantify mTOR protein levels samples were prepared as described above (see methods) and all data were analyzed in relation to GAPDH, setting the young control at 100%. Statistical significant differences between young, adult and old murine brain tissue samples were obtained, using one-way ANOVA, followed by Tukey's post hoc test and are shown by **p<0.01, all compared to young control.

Fig. 3

Determination of mTOR activity by p70S6K and 4E-BP1 in brain tissue of young, adult and old C57BL/6J mice. Panel (A) shows the amount of p-p70S6K, while panel (B) demonstrates the basal amount of the protein in young, adult and old murine brain tissues from C57BL/6J male mice. The immunoblots are shown for four individual mice per age group. In panel (C) the ratio of p-p70S6K to basal p70S6K is given. Panels (D-F) show the analysis of 4E-BP1. Panel (D) demonstrates the amount of the p-4E-BP1, followed by panel (E), indicating the amount of the basal 4E-BP1 protein. The ratio of the p-4E-BP1 to 4E-BP1 is given in panel (F). All data were analyzed in relation to GAPDH and data are shown as percentages in relation to the young control. Statistical significant differences between young, adult and old murine brain samples were calculated, using one-way ANOVA, followed by Tukey's post hoc test and are indicated by *p<0.05 and **p<0.01, all compared to young control..

Fig. 4

Steady state levels of ferritin H in brain tissue of young, adult and old C57BL/6J mice. In panel (A) representative immunohistochemistry of the distribution of ferritin H in young and old murine brain sections is shown. Panel (B) displays the protein expression of ferritin H in young, adult and old murine brain tissues from C57BL/6J male mice. The immunoblots are shown for four individual mice per age group. All data are protein levels in relation to GAPDH, setting the young control at 100%, statistical significant differences between young, adult and old murine brain tissue were calculated, using one-way ANOVA, followed by Tukey's post hoc test and are shown by **p<0.01, all compared to young control.

Fig. 5

Examination of autophagy-associated proteins in young and old cells. Relative quantification of ATGs were performed in young and old fibroblasts with and without 24 h incubation of Concanamycin A (24 h ConA). Analyses were performed by immunoblotting in cell lysates (see methods), always comparing untreated and 24 h ConA treated samples. Panels show: (A) quantification of LC3-II/LC3-I ratio, (B) the amount of p62, (C) protein levels of ATG5-ATG12, (D) the amount of Beclin-1. All data were analyzed in relation to GAPDH, setting the corresponding control young at 100%. Statistical significant differences between controls and treated cells is shown by #p<0.05, **p<0.01, compared to the corresponding control.

Fig. 6

Determination of mTOR amount and its activity by p70S6K and 4E-BP1 in young and old human fibroblasts. Panels show the levels of (A) phosphorylated p-p70S6K and (B) basal p70S6K in young and old human fibroblast. Panel (C) shows the calculated ratio of p-p70S6K to basal p70S6K. In panels (D-F), the analyses of the second target protein 4E-BP1 is shown, while panel (D) shows the amount of the p-4E-BP1, panel (E) demonstrates the amount of the basal 4E-BP1 protein. The calculated ratio of p-4E-BP1 to 4E-BP1 is given in panel (F). mTOR analysis is presented in panel G. All data were analyzed in relation to GAPDH and data are shown as percentages, setting the young cells at 100%. Statistical significant differences between control and treated cells were calculated, using Turkey's post hoc test, followed by Tukey's post hoc test and are shown by *** p<0.001, all compared to the young.