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Comparative Study
. 2005 Mar 2;25(9):2396-404.
doi: 10.1523/JNEUROSCI.4866-04.2005.

Cytoprotective effects of geranylgeranylacetone against retinal photooxidative damage

Masaki Tanito  1 , Yong-Won Kwon, Norihiko Kondo, Jie Bai, Hiroshi Masutani, Hajime Nakamura, Junichi Fujii, Akihiro Ohira, Junji Yodoi
Affiliations
Comparative Study

Cytoprotective effects of geranylgeranylacetone against retinal photooxidative damage

Masaki Tanito et al. J Neurosci. .

Abstract

Exposure to excessive light induces retinal photoreceptor cell damage, leading to development and progression of various retinal diseases. We tested the effect of geranylgeranylacetone (GGA), an acyclic polyisoprenoid, on light-induced retinal damage in mice. Oral treatment with GGA (1.0 mg/d) for 5 d induced thioredoxin (Trx) and heat shock protein 72 (Hsp72) predominantly in the retinal pigment epithelium (RPE). After white light exposure (8000 lux for 2 h), the percentage of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive photoreceptor cells decreased significantly at 24 and 96 h, and the number of photoreceptor cell nuclei at 96 h and the electroretinographic amplitudes of the a- and b-waves at 4 and 10 d increased significantly in GGA-pretreated mice compared with saline-pretreated mice. Light-induced upregulations of 8-hydroxy-2-deoxyguanosine and 4-hydroxy-2-nonenal-modified protein, markers of oxidative stress, were inhibited by GGA pretreatment. To elucidate the cytoprotective mechanism of GGA and Trx, we used human K-1034 RPE cells and mouse photoreceptor-derived 661W cells. In K-1034 cells, GGA (10 microM) induced intracellular Trx, Hsp72, and extracellular Trx but not extracellular Hsp72. Extracellular Trx (0.75 nM) attenuated H2O2 (200 microM)-induced cell damage in 661W cells. Pretreatment with GGA and overexpression of Trx in K-1034 cells counteracted H2O2 (50 microM)-induced attenuation of cellular latex bead incorporation. Protection of phagocytotic activity through induction of Trx and possibly Hsp72 in RPE cells and elimination of oxidative stress in the photoreceptor layer through release of Trx from RPE cells may be mechanisms of GGA-mediated cytoprotection. Therefore, Trx is a neurotrophic factor released from RPE cells and plays a crucial role in maintaining photoreceptor cell integrity.

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Figures

Figure 1.
Figure 1.
A-D, Representative immunohistochemistry for Trx (A, B) and Hsp72 (C, D) in murine retina. A, B, Expression of Trx in the RPE layer of saline-treated mice (A; arrowhead) and GGA-treated mice (B; arrow). C, D, Expression of Hsp72 in the RPE layer of saline-treated mice (C; white arrowhead) and GGA-treated mice (D; arrow). The results are representative of three independent experiments. INL, Inner nuclear layer; OPL, outer plexiform layer; RIS, rod inner segments; ROS, rod outer segments.
Figure 2.
Figure 2.
Western blotting for Trx and Hsp72 in murine retinal samples. A, Expression of Trx and Hsp72 in GGA-untreated [GGA(-)] and GGA-treated [GGA(+)] mice in RPE fraction. B, Expression of Trx and Hsp72 in GGA-untreated [GGA(-)] and GGA-treated [GGA(+)] mice in the neural retina. Samples from two independent mice in each group were analyzed by Western blotting in each experiment. The experiments were repeated three times, and the band intensities of GGA+ samples relative to GGA-samples are shown in the bar graph (mean ± SD; n = 6). Sample loading was monitored by staining with Coomassie Brilliant Blue R-250 (CBB).
Figure 3.
Figure 3.
Numbers of photoreceptor cell nuclei and percentages of TUNEL-positive cells in the ONL 24 and 96 h after light exposure. A, B, Hematoxylin-eosin (HE) staining (A) and TUNEL staining (B) of retinal specimens from mice not exposed to light (first and second panels) and from mice 24 h (panels 3 and 4) and 96 h (panels 5 and 6) after light exposure. Mice were pretreated with GGA (panels 2, 4, and 6) or saline (panels 1, 3, and 5). TUNEL-positive cells are observed in the ONL after light exposure (arrowheads). C, D, Quantification of the number of photoreceptor cell nuclei (C) and percentage of TUNEL-positive cells (D). *p < 0.05 and **p < 0.001 by unpaired t test comparison of the GGA-untreated and GGA-treated mice. Each bars are expressed as mean ± SD.
Figure 4.
Figure 4.
ERGs 4 and 10 d after light exposure. A, Representative ERG recordings before (top) and 10 d after (bottom) light exposure from GGA-untreated (left) and GGA-treated (right) mice. B, The a-wave (left) and b-wave (right) amplitudes are significantly higher in GGA-treated mice compared with GGA-untreated mice 4 and 10 d after light exposure (*p < 0.01 and **p < 0.001 by unpaired t test). Each bar is expressed as mean ± SD.
Figure 5.
Figure 5.
Representative immunohistochemistry for 8OHdG (A) and 4HNE-modified protein (B) in murine retina. A, In specimens from mice not exposed to light (top), no labeling or slight labeling of 8OHdG is observed. Twenty-four hours after light exposure, labeling intensity increases in most nuclei of the ONL, and some cells show strong labeling (arrowheads) in specimens from mice not treated with GGA (GGA-) (bottom left), but fewer cells are seen in specimens from GGA-treated mice (bottom right). B, In specimens from mice not exposed to light (top), no labeling or slight labeling of 4HNE-modified protein is observed. Twenty-four hours after light exposure, labeling intensity increases throughout the retina, and some cells show strong perinuclear or nuclear labeling in the inner nuclear layer (INL) (arrowheads), ONL (arrows), and RPE (white arrow) in specimens from mice not treated with GGA (GGA-) (bottom left), but fewer cells are seen in specimens from GGA-treated mice (bottom right).
Figure 6.
Figure 6.
Detection of intracellular and extracellular Trx and Hsp72 in cultured RPE cells, effect of extracellular Trx against H2O2-induced photoreceptor cell damage, and Prx expression in photoreceptor cells. A, Western blotting for Trx and Hsp72 in total cell lysates of GGA-treated human K-1034 RPE cells. The sample loading was monitored by staining with Coomassie Brilliant Blue R-250 (CBB). B, Western blotting for Trx and Hsp72 in concentrated culture medium of GGA-treated human K-1034 RPE cells. C, Sandwich ELISA for human Trx in nonconcentrated culture medium of GGA-treated human K-1034 RPE cells (**p < 0.001 by unpaired t test). Each bar is expressed as mean ± SD (n = 6 in each group). D, LDH-releasing assay for H2O2-treated mouse 661W photoreceptor-derived cells. Cell damage is significantly inhibited by the addition of rhTrx protein to the culture medium (*p < 0.01 and **p < 0.001, compared with 0 μm GGA by unpaired t test). Each bar is expressed as mean ± SD (n = 6 in each group). E, Western blotting for Prx-I, -IV, and -VI in total cell lysate of 661W cells.
Figure 7.
Figure 7.
Immunohistochemistry of Prx-I, -IV, and -VI in mouseretina. Clear expression of Prx-I (top left), Prx-IV (top right), and Prx-VI (bottom left) are observed in the GCL, IPL, outer plexiform layer (OPL), rod outer segment (ROS), and RPE. Nuclear staining of the Prx-I is observed in the inner nuclear layer (INL; arrows) and OPL (arrow heads). 1st ab,(-), Omitted the first antibodies as a negative control.
Figure 8.
Figure 8.
Effect of GGA on phagocytotic activity of cultured RPE cells. A, Representative phase-contrast microscopy (top row) and fluorescence microscopy (bottom row) of human K-1034 RPE cells treated with latex beads (red dots). The control cells (left), H2O2-treated and GGA-untreated cells (center), and H2O2-treated and GGA-treated cells (right) are shown. B, Quantification of latex bead incorporation into the cells by flow cytometry. In this measurement, free latex beads are excluded from the gate (panel 1), and almost all cells (>99%) not containing latex beads are gated into the lower left region (panel 2). H2O2-untreated and saline-pretreated (panel 3) or GGA-pretreated (panel 4) cells and H2O2-treated saline (panel 5) or GGA-pretreated (panel 6) cells are shown. x-axis, FL-1H; y-axis, FL-2H.
Figure 9.
Figure 9.
Effect of Trx overexpression on phagocytotic activity of RPE cells. A, Fluorescence microscopy in H2O2-pretreated K-1034 cells overexpressing Trx. Expressed GFP-Trx fusion protein and phagocytosed latex beads are seen as green and red, respectively. B, Pooled GFP-Trx transfected cells are subdivided into two populations (low- and high-Trx-expressing populations) referring to the upper limit of FL-1H intensity in parental nontransfected K-1034 cells. C, Quantification of latex bead incorporation into the cells by flow cytometry. H2O2-untreated (left) and H2O2-treated (right) cells are shown. x-axis, FL-1H; y-axis, FL-2H.
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