Expression of Sirtuins in the Retinal Neurons of Mice, Rats, and Humans

Abstract

Sirtuins are a class of histone deacetylases (HDACs) that have been shown to regulate a range of pathophysiological processes such as cellular aging, inflammation, metabolism, and cell proliferation. There are seven mammalian Sirtuins (SIRT1-7) that play important roles in stress response, aging, and neurodegenerative diseases. However, the location and function of Sirtuins in neurons are not well defined. This study assessed the retinal expression of Sirtuins in mice, rats, and humans and measured the expression of Sirtuins in aged and injured retinas. Expression of all 7 Sirtuins was confirmed by Western blot and Real-Time PCR analysis in all three species. SIRT1 is highly expressed in mouse, rat, and human retinas, whereas SIRT2-7 expression was relatively lower in human retinas. Immunofluorescence was also used to examine the expression and localization of Sirtuins in rat retinal neurons. Importantly, we demonstrate a marked reduction of SIRT1 expression in aged retinal neurons as well as retinas injured by acute ischemia-reperfusion. On the other hand, none of the other Sirtuins exhibit any significant age-related changes in expression except for SIRT5, which was significantly higher in the retinas of adults compared to both young and aged rats. Our work presents the first composite analysis of Sirtuins in the retinal neurons of mice, rats, and humans, and suggests that increasing the expression and activity of SIRT1 may be beneficial for the treatment of glaucoma and other age-related eye dysfunction.

Keywords: Sirtuins; aging; animal; human; retinal neuron.

FIGURE 1

Expression of Sirtuin (SIRT1-7) genes in adult mouse, rat, and human retinal neurons. Histograms showing the SIRT1-7 mRNA levels in the retinas of mice (A), rats (B), and humans (C) as determined by qRT-PCR. The y-axis shows the Sirtuin expression levels relative to the SIRT1 gene. Data shown are based on the linear conversion of delta CT values for each sample (n = 5); error bars denote SEM. Statistical comparisons between the retinal expression levels of the different Sirtuins in each species are presented as a table (D).

FIGURE 2

Sirtuin (SIRT1-7) protein levels in mouse, rat, and human retinal neurons. Representative western blots showing the protein levels for SIRT1 (A), SIRT2 (B), SIRT3 (C), SIRT4 (D), SIRT5 (E), SIRT6 (F), and SIRT7 (G) in the retinas of each species. The histograms in each panel show the densitometric mean and SEM normalized to the corresponding level of the loading control protein β-Tubulin (n = 4 independent subjects). Statistical comparisons between the expression levels of the different Sirtuin proteins in the retinas of adult mouse, rat, and human are presented as a table (H).

FIGURE 3

Relative Sirtuin protein to mRNA ratios in mouse, rat, and human retinal neurons. The ratios of protein levels from western blot to mRNA levels from Real-Time PCR are denoted on the Y-axis for each Sirtuin family member (X-axis) in the retinas of mice (A), rats (B), and humans (C). Supplemental data about PCR and protein/mRNA are displayed (D).

FIGURE 4

Immunolocalization of Sirtuin proteins in retinal cross-sections of rat. Retinal cross sections prepared from rat eyes were immunostained with Sirtuin antibodies. The results are displayed as negative control (without primary antibody), SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7 in adult rat retinas. All photographs were taken at 40× magnification. Con, control; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

FIGURE 5

Protein levels of SIRT1 in aging and injured rat retinas. Representative Western blot showing SIRT1 protein levels in aged (old) retinas compared to young and adult retinas (A). SIRT1 protein levels in rat retinas following transient retinal ischemia-reperfusion injury (B). Densitometry analysis of SIRT1 in aged and injured retinas and normalized to β-tubulin is shown below the representative blots (^∗P < 0.05, n = 4).

FIGURE 6

SIRT2-7 protein levels in aging rat retinal neurons. Representative western blots showing the protein levels for SIRT2 (A), SIRT3 (B), SIRT4 (C), SIRT5 (D), SIRT6 (E) and SIRT7 (F) in the retinas of young, adult, and old rats. The histograms in each panel show the densitometric mean and SEM normalized to the corresponding level of the loading control protein β-Tubulin (^∗P < 0.05, n = 4 independent subjects).