Figure 1.
8-OH DPAT reduces lipofuscin accumulation in cultured RPE cells.
A - The effect of different concentrations of 8-OH DPAT on lipofuscin accumulation from autophagy in human ARPE19 cells. Cells were maintained in basal medium and received 10 µM 8-OH DPAT every two days for four weeks. B - Autofluorescent intensity of ARPE19 cells treated with 10 µM 8-OH DPAT every two days for up to 4 weeks. Control cells received vehicle alone. C - Fluorescent micrographs of ARPE19 cells as described in B showing reduced lipofuscin granules in 8-OH DPAT treated cells compared to controls. D - Autofluorescent intensity of ARPE19 cells fed POS treated with 10 µM 8-OH DPAT every two days for up to 14 days. Control cells received POS and saline vehicle alone. E - Fluorescent micrographs of ARPE19 cells described in D showing reduced lipofuscin granule accumulation in 8-OH DPAT treated cells fed POS compared to untreated controls. Fluorescence intensity was determined by flow cytometric analysis. Data represent the mean of three experiments. Bar marker is 50 µM.
Figure 2.
The effect of 8-OH DPAT is sustained following discontinuation but 8-OH DPAT has only a minimal effect on pregenerated lipofuscin.
A, B - The effect of the selective 5-HT1A receptor antagonist, UH-301 (5 µM) on 8-OH DPAT (10 uM) reduction in autophagy- (A) and POS-derived (B) lipofuscin accumulation in cultured RPE. C, D - To determine if the effect of 8-OH DPAT was sustained following discontinuation of 8-OH DPAT treatment, 8OH DPAT treatment was discontinued after 28 days and the cells maintained in basal medium (C) or fed POS (D) for a further 28 days. E, F, G - To assess the ability of 8-OH DPAT (10 uM) to remove pregenerated lipofuscin, 8-OH DPAT was added every second day for 28 days after: E – 28 days accumulation of autophagy-derived lipofuscin; F - cultures were fed mature lipofuscin granules; G - 14 days accumulation of POS-derived lipofuscin. Data represent the mean of three experiments.
Figure 3.
8-OH DPAT reduces lipid peroxidation and peroxynitrite accumulation and protects against mitochondrial damage in cultured RPE cells exposed to H2O2.
Cells were exposed to H2O2 (200 µM) for 1 hour and either pre-or post treated with 8-OH DPAT (10 µM) for 24 hours. In the case of pretreatment all measurements were made 24 hr after H2O2 and for post treatment 8-OH DPAT was added immediately following H2O2 exposure. A - The level of lipid peroxides (4HNE/MDA) was determined using a commercially available kit from OxisResearch. B – Nitrotyrosine levels were assessed by Western blotting using a polyclonal antibody and band intensities were normalized against β-actin. C - Cytochome c oxidase levels were determined using a Complex IV ELISA assay. D - Mitochondrial DNA damage was analyzed as previously described using the long chain PCR reaction. Data represent the mean of three experiments.
Figure 4.
8-OH DPAT reduces superoxide anion generation and increases antioxidant capacity in cultured RPE cells.
Cells were exposed to H2O2 (200 µM) for 1 hour and either pre-or post treated with 8-OH DPAT (10 µM) for 24 hours. In the case of pretreatment all measurements were made 24 hr after H2O2 and for post treatment 8-OH DPAT was added immediately following H2O2 exposure. A - Superoxide generation was measured using FACS analysis following staining with MitoSOX and results are expressed as the mean fluorescence intensity. B - SOD2 levels were determined by Western blot analysis. C - The ratio of reduced glutathione (GSH) to the oxidized form of glutathione (GSSG) was measured by ELISA. Data represent the mean of three experiments.
Figure 5.
Extent of retinal transduction with AAV-VMD2-SOD2 Rz.
Digital fundus images were made 4 months following subretinal injection of the AAV-VMD2-SOD2 Rz, which also contained the mCherry gene as a marker of genetic transduction. As previously reported [15] we routinely observe 60–80% transduction of the RPE. This was revealed as red-fluorescence using a long wave length filter (A) and as thinning of the retina (hypopigmentation) as a consequence of knockdown of MnSOD (B).
Figure 6.
ERG a-wave and b-wave amplitudes and retinal thickness measured in AAV-ribozyme SOD2 knockdown eyes and control eyes treated with 8-OH DPAT.
Eyes received injection of AAV-SOD2 ribozyme or AAV-mCherry and animals received subcutaneous 8-OH DPAT or saline control for up to 4 months. ERGs were obtained at 1 and 4 months following virus injection: A & B - ERG wave-forms from mice treated with AAV-mCherry (A) or AAV-VMD2-SOD2 Rz (B). Simultaneous full-field ERG measurements were recorded in dark-adapted mice four months after injection with AAV. The scale units on the ordinate are 100 microvolts. The graphs show wave forms for mice treated with saline, blue lines, low dose (0.5 mg/kg) 8-HO-DPAT, red lines, or high dose (5 mg/kg) 8-HO-DPAT, green lines. C – a-wave; D – b-wave. E - ONL thickness measured by SD-OCT at 1 and 4 months. Untreated wild type animals acted as the baseline control. (10 animals per group, P≤0.01 for all doses and time points).
Figure 7.
8-OH DPAT decreases oxidative stress in the RPE of SOD2 knockdown eyes of mice.
Eyes received injection of AAV-SOD2 ribozyme or AAV-mCherry and animals received subcutaneous 8-OH DPAT or saline control for up to 4 months. Sections were stained for 8-hydroxydeoxyguanosine (8OHdG) (green). A – Representative sections of 8OHdG expression in the RPE of control and SOD2 eyes receiving different concentrations of 8-OH DPAT. B - Graph shows quantitation of 8OHdG fluorescence in the RPE layer.
Figure 8.
8-OH DPAT decreases RPE autofluorescence in SOD2 knockdown eyes of mice.
Eyes received injection of AAV-SOD2 ribozyme or AAV-mCherry and animals received subcutaneous 8-OH DPAT or saline control for up to 4 months. A - Confocal fluorescence microscopy of the RPE layers from AAV-mCherry and AAV-ribozyme treated eyes treated with saline, low dose or high dose of 8-OH DPAT. B - Treatment with 8-OH DPAT lowered autofluorescence in oxidatively stressed RPE in mice. Mice were injected with AAV-mCherry or AAV-RzSOD2-mCherry and analyzed after 4 months of daily injections of saline (no treatment) low dose or high dose of 8-OH DPAT. * P<0.05, **P<0.01 (compared to AAV-RzSOD2 control).