The Beta Adrenergic Receptor Blocker Propranolol Counteracts Retinal Dysfunction in a Mouse Model of Oxygen Induced Retinopathy: Restoring the Balance between Apoptosis and Autophagy

Abstract

In a mouse model of oxygen induced retinopathy (OIR), beta adrenergic receptor (BAR) blockade has been shown to recover hypoxia-associated retinal damages. Although the adrenergic signaling is an important regulator of apoptotic and autophagic processes, the role of BARs in retinal cell death remains to be elucidated. The present study was aimed at investigating whether ameliorative effects of BAR blockers may occur through their coordinated action on apoptosis and autophagy. To this aim, retinas from control and OIR mice untreated or treated with propranolol, a non-selective BAR1/2 blocker, were characterized in terms of expression and localization of apoptosis and autophagy markers. The effects of propranolol on autophagy signaling were also evaluated and specific autophagy modulators were used to get functional information on the autophagic effects of BAR antagonism. Finally, propranolol effects on neurodegenerative processes were associated to an electrophysiological investigation of retinal function by recording electroretinogram (ERG). We found that retinas of OIR mice are characterized by increased apoptosis and decreased autophagy, while propranolol reduces apoptosis and stimulates autophagy. In particular, propranolol triggers autophagosome formation in bipolar, amacrine and ganglion cells that are committed to die by apoptosis in response to hypoxia. Also our data argue that propranolol, through the inhibition of the Akt-mammalian target of rapamycin pathway, activates autophagy which decreases retinal cell death. At the functional level, propranolol recovers dysfunctional ERG by recovering the amplitude of a- and b-waves, and oscillatory potentials, thus indicating an efficient restoring of retinal transduction. Overall, our results demonstrate that BAR1/2 are key regulators of retinal apoptosis/autophagy, and that BAR1/2 blockade leads to autophagy-mediated neuroprotection. Reinstating the balance between apoptotic and autophagic machines may therefore be viewed as a future goal in the treatment of retinopathies.

Keywords: apoptosis; autophagy; beta adrenergic receptors; electroretinogram; proliferative retinopathies; propranolol; retinal neurons.

FIGURE 1

Expression of apoptotic and autophagic markers in the retina. Representative blots for cytochrome c, cleaved Caspase 3, p62 and LC3 as evaluated by western blot analysis using β-actin as the loading control, were shown. Mice were sacrificed every day between PD13 and PD17.

FIGURE 2

Effects of propranolol on apoptotic and autophagic markers in the retina. Protein levels evaluated by the densitometric analysis of the blots depicted in Figure 1. Expression of cytochrome c (A) and cleaved Caspase 3 (B). In controls (white), their levels significantly decreased from PD13 to PD15 to then remain constant. In OIR mice (black), apoptotic markers were consistently higher than in controls. Propranolol (red) reduced the levels of apoptotic markers toward recovering their normoxic values. ^∗P < 0.05, ^∗∗P < 0.001 vs. the respective control values; ^†P < 0.05, ^††P < 0.001 vs. control values measured at PD14 or PD13, respectively (two-way ANOVA followed by Bonferroni’s multiple comparison post-test). Expression of p62 (C), LC3-I (D), and LC3-II (E). In controls, the levels of p62 remained low from PD13 to PD15 to then increase at PD16, whereas the levels of LC3-II progressively decreased from PD13 to PD15 to become almost undetectable at PD16. In OIR mice, levels of p62 were higher, while levels of LC3-II were lower, than in controls at PD13, PD14 and PD15. Propranolol reduced p62 while increased LC3-II at PD13, PD14, and PD15 toward recovering their control values. The levels of LC3-I remained constant over time in any experimental condition. ^∗P < 0.001 vs. the respective control values; ^†P < 0.001 vs. control values measured on the previous day (two-way ANOVA followed by Bonferroni’s multiple comparison post-test). Data are presented as scatter plots with mean ± SD.

FIGURE 3

Propranolol effects on the expression pattern of apoptosis and autophagy markers in the retina. Representative confocal images showing the pattern of cleaved Caspase 3 (A) and LC3 (B) immunofluorescence in retinal sections at PD13, both untreated OIR and propranolol-treated mice. Retinal layers are visualized with DAPI counterstain. Scale bar, 20 μm.

FIGURE 4

Propranolol effects on the expression pattern of apoptosis and autophagy markers in the retina. (A) Double-label immunofluorescence using antibodies directed to LC3 and cleaved Caspase 3. (B) Immunostaining showing the pattern of p62. Retinal layers are visualized with DAPI counterstain. The representative confocal images are collected in retinal sections at PD13, both untreated OIR and propranolol-treated mice. Scale bar, 20 μm.

FIGURE 5

Localization of LC3 staining in the retina. Double-label immunofluorescence using antibodies directed to LC3 and MAb115A10 (A) or LC3 and PKC (B). The representative confocal images are collected in retinal sections at PD13, both untreated OIR and propranolol-treated mice. Scale bar, 20 μm.

FIGURE 6

Localization of LC3 staining in the retina. Double-label immunofluorescence using antibodies directed to LC3 and GAT-1 (A) or LC3 and Dab1 (B). The representative confocal images are collected in retinal sections at PD13, both untreated OIR and propranolol-treated mice. Scale bar, 20 μm.

FIGURE 7

Localization of LC3 staining in the retina. Double-label immunofluorescence using antibodies directed to LC3 and β-tubulin III. The representative confocal images are collected in retinal sections at PD13, both untreated OIR and propranolol-treated mice. Scale bar, 20 μm.

FIGURE 8

Propranolol effects on autophagy signaling. Activation levels of anti- and pro-autophagic protein kinases were evaluated in the retina by western blot experiments (A,B) and their respective densitometric analysis (C). Propranolol reduced the levels of pAkt, pS6, p4EBP1, and pUlk1 at Ser757 while did not affect the levels of pAMPK and pUlk1 at Ser555. The ratio between the expression of phosphorylated protein and the respective total protein is presented as scatter plots with mean ± SD. ^∗P < 0.01 vs. OIR mice values (one-way ANOVA followed by Newman–Keuls’ multiple comparison post-test). β-actin was also used as the loading control.

FIGURE 9

LC3 and active Caspase 3 retinal staining in the presence of autophagy modulators. Immunofluorescence using antibodies directed to cleaved Caspase 3 and LC3 in retinas of OIR mice both in the absence and in the presence of the autophagy stimulator rapamycin or the autophagy inhibitor wortmannin. Propranolol was administered in untreated and wortmannin-treated OIR mice. The representative confocal images are collected in retinal sections at PD13. Scale bar, 20 μm.

FIGURE 10

Propranolol effects on retinal functions. (A) Representative ERG waveforms in controls, untreated OIR and propranolol-treated mice recorded at light intensities of –1, 0, and 1 log cd-s/m², at PD17. (B–D) a-wave, b-wave and SOP amplitudes in controls (white), untreated (black), propranolol-treated from PD12 to PD16 (red), and propranolol-treated at PD16 (gray) OIR mice at increasing light intensities. Repeated propranolol administration, from PD12 to PD16, restored a-, b-wave amplitudes and SOPs to levels that were not significantly different from those in controls. ^∗P < 0.01, ^∗∗P < 0.001 vs. controls (two-way ANOVA followed by Bonferroni’s multiple comparison post-test).

FIGURE 11

A schematic diagram representing the proposed mechanisms by which propranolol reduces apoptosis/autophagy neuronal ratio in the retina therefore recovering ERG dysfunction. Propranolol, by blocking BARs, causes a reduced phosphorylation of anti-autophagic molecules, including Akt, S6, 4EBP1, and Ulk1 at Ser 757 site that would result in reduced levels of p62 and increased levels/clustering of LC3-II both indicative of an increased autophagosome formation. BAR blockade would also cause a reduction in the levels of the apoptotic molecules cytochrome c and cleaved (active) Caspase 3 thus counteracting OIR-associated apoptotic processes. We hypothesize that the stimulated autophagy triggers anti-apoptotic events leading to ameliorative effects of propranolol on the damaged retina. In this scenario, the coordinated increase in autophagy and decrease in apoptosis may play a key role to reduce retinal cell death and ameliorate visual performance.