Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

cells. Statistical analysis was performed on the samples with Student’s t -tests and one-way ANOVA followed by Bonferroni post test. All the groups were compared to each other, and statistical significance was set at P <0.05.

2.4Preparation of Membrane Fraction from 661W Cells and Assay of RDH Activity

Cells were resuspended in sucrose buffer (0.25 M sucrose, 10 mM Tris-Cl, pH 7.2, 1 mM EDTA), and a protease inhibitor mixture (2.0 μg/ml aprotinin, 5 μg/ml pepstatin A, 10 μg/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride) and disrupted using a Polytron. Cell homogenates were centrifuged at 800 X g for 15 min at 4◦C. The resulting supernatants were then centrifuged at 100,000 X g for 30 min at 4◦C, and membrane pellets were resuspended in storage buffer containing 50 mM Tris-Cl, pH 7.2, 1 mM EDTA, 20% glycerol and the protease inhibitor mixture described above. Membrane fractions were stored at –80◦C after snap freezing in liquid nitrogen. Reactions with all-trans-retinal were carried out in 1 ml reaction buffer (100 mM Tris-HCl at pH 7.2, 200 mM NaCl, 1 mM dithiothreitol, 2 mg/ml bovine serum albumin, 1% glycerol, and 150 μM of NADPH) containing 0 or 10 μg of proteins, and 0, 1.25, 2.5, 5, 10 and 20 μM of substrate. After 4 hours incubation at room temperature, reactions were terminated by the addition of 1 volume of cold methanol. Retinoids were then extracted with 2 volumes of hexane and analyzed by HPLC. Samples were dissolved in HPLC mobile phase (11.2% ethyl acetate/2.0% dioxane/1.4% octanol in hexane) and retinoids were separated by using a Lichrosphere Sil 60A (Phenomenex) 5-μm column. The retinal peaks were identified and quantified by comparison with pure retinoid isomeric standards. All procedures were performed under dim red light. Calculation of Km and Vmax were done by using the direct linear plot method.

3 Results

3.1Chromophore can Enhance Light Induced Apoptosis in 661W Cells

In the presence of 9-cis retinal and 30,000 lux of light, 661W cells undergo cell death. There is no change in viability at 1 and 2 hours after light exposure. At 3 hours after light stress however, 76% of the cells are viable, and at 4 and 5 hours after light stress, 50% and 27% of cells are viable, respectively, as shown in Fig. 1 Cells kept in the dark in the presence of 9-cis retinal for 5 hours were all viable. In the absence of chromophore, all the cells are viable at 5 hours after light stress and 68% of cells are viable after 6 hours of light stress (Kanan et al., 2007). Therefore, addition of 9-cis retinal, accelerates light induced damage to the cells.

3.3Expression of RDHs in 661W Cells Provides Protection Against Light Stress

661W cells express low levels of endogenous dehydrogenases (unpublished data). We transfected these cells with RDH8, RDH11 and RDH12 and isolated pure clones of stable transfectants. We investigated the susceptibility of these clones to light stress in comparison to their untransfected counterparts. We found that only 21% of untransfected 661W cells are viable after 5 hours of light stress while 69% of cells are viable after transfection with RDH8, 34% of cells are viable after transfection with RDH11, and 42% of cells are viable after transfection with RDH12. Therefore, all three RDHs offer protection to 661W cells with RDH8 appearing to offer the highest protection as shown in Fig. 3

It is possible that RDH8 has a higher catalytic activity (Vmax) and/or a higher affinity (Km) towards all-trans retinal compared to the RDH11 and RDH12. In Table 1 we compare the apparent Vmax and Km of these clones, using the membrane

Fig. 3 Viability of 661W cells after 5 hours of light induced apoptosis in the presence of 9-cis retinal. 661W cells transfected with RDH8, RDH11 and RDH12 were compared to non transfected 661W cells to assess protection. Statistical analysis using the Bonferroni multiple comparison was performed and significant probabilities with P ≤ 0.05, are denoted with asterisks

Table 1 Membrane fractions were prepared from the 4 clones used in Fig. 3. The total RDH activity was determined for each of them using 0 or 10 μg of protein in each reaction, and increasing concentrations of all-trans retinal. The reduction to all-trans retinol was quantified for each reaction, the protein-dependant production of all-trans retinol was calculated, and for each clone a saturation curve was plotted as well as the corresponding direct linear plot. Each clone was assayed in 3 independent experiments and the results shown are derived from one representative experiment

Susceptibility for light damage was not tested in the RDH8 and RDH11 knockout mice. We will study these mice to assess protection offered by these RDHs under light stress condition.

Acknowledgments This publication was made possible by NIH Grant Number P20 RR 017703 from the COBRE Program of the National Center for Research Resources.

References

Al-Ubaidi, M.R., Font, R.L., Quiambao, A.B., Keener, M.J., Liou, G.I., Overbeek, P.A., and Baehr,W., 1992, Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter. J. Cell Biol. 119:1681.

Cao, W., Tombran-Tink, J., Elias, R; Sezate, S., Mrazek, D., and McGinnis, J.F., 2001, In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor. Invest Ophthalmol. Vis. Sci. 42:1646.

Imai, D., Yoneya, S., Gehlbach, P.L., Wei, L.L., and Mori, K., 2005, Intraocular gene transfer of pigment epithelium-derived factor rescues photoreceptors from light-induced cell death. J. Cell Physiol. 202:570.

Janecke, A.R., Thompson, D.A., Utermann, G., Becker, C., Hubner, C.A., Schmid, E., McHenry, C.L., Nair, A.R., Ruschendorf, F., Heckenlively, J., Wissinger, B., Nurnberg, P., and Gal, A., 2004, Mutations in RDH12 encoding a photoreceptor cell retinol dehydrogenase cause childhood-onset severe retinal dystrophy. Nat. Genet. 36:850.

Kanan, K., Moiseyev, G., Agarwal, N., Ma., J.X., and Al-Ubaidi, M.R., 2007, Light induces programmed cell death by activating multiple independent proteases in a cone photoreceptor cell line. Invest. Ophthalmol. Vis. Sci 48:40.

Maeda, A., Maeda, T., Imanishi, Y., Sun, W., Jastrzebska, B., Hatala, D.A., Winkens, H.J., Hofmann, K.P., Janssen,J.J., Baehr, W., Driessen, C.A., and Palczewski, K., 2006, Retinol dehydrogenase (RDH12) protects photoreceptors from light-induced degeneration in mice.

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Role of BCL-XL in Photoreceptor Survival

Yun-Zheng Le, Lixin Zheng, Yuwei Le, Edmund B. Rucker III, and Robert E. Anderson

1 Introduction

Photoreceptors are post-mitotic neurons and understanding their survival mechanisms holds a key to the treatment of retinal degeneration. Many studies have demonstrated that phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol-3,4,5-trisphosphate (PIP3) are involved in the survival of neurons through growth factor receptor-mediated activation of the serine-threonine kinase, AKT (Barber et al., 2000; D’Mello et al., 1997; Yao and Cooper, 1995). Activation of AKT further activates a number of down-stream targets, including the anti-apoptotic protein BCL-XL, which can serve as a survival factor in a number of alternative pathways(Bui et al., 2001; Kim et al., 2005; Zha et al., 1996). For the past few years, we have investigated the roles of PI3K, insulin receptor, AKT, and BCL-XL in photoreceptor survival. BCL-XLwas postulated as a survival factor in photoreceptors, as it was up-regulated in the bright light-stressed retina (Zheng et al., 2006). To determine the significance of BCL-XLup-regulation in the bright light damage model, the Bcl-x gene was disrupted specifically in murine rod or cone photoreceptors using the Cre/lox-based conditional gene knockout strategy (Le and Sauer, 2000). Effects of Bcl-x disruption on photoreceptor function and morphology were characterized. This report reviews the results from rod-specific Bcl-x knockout mice and summarizes new information on cone-specific Bcl-x knockout mice.

2 Materials and Methods

2.1 Generation of Rodor Cone-specific Bcl-x Knockout Mice

Albino rodor cone-specific Bcl-x knockout mice were generated separately by mating floxed Bcl-x mice with Short Mouse Opsin Promoter-controlled cre (SMOPC1)

Y.-Z. Le

Departments of Medicine; Cell Biology; Dean A. McGee Eye Institute, Oklahoma City, OK, USA, Tel: 405-271-1087, Fax: 405-271-8128

e-mail: Yun-Le@ouhsc.edu

mice or Human Red Green Pigment Promoter-controlled cre (HRGPPC) mice, respectively,(Le et al., 2004; Le et al., 2006a; Rucker et al., 2000). The genotypes of these conditional Bcl-x knockout mice were verified according to the described methods (Zheng et al., 2006).

2.2 Bright Light Stress

All mice were maintained in 100-lux cyclic light after birth. Eight-week-old rodspecific Bcl-x knockout mice or cone-specific Bcl-x knockout mice were exposed to white light at an illumination of 7,000 lux for 48 h or up to 96 h, respectively. The mice were then maintained in 100-lux cyclic light. Gene expression, apoptosis, and DNA fragmentation assays were carried out 24 h after the light exposure. Morphological and functional analyses were carried out 7 days after the bright light exposure.

2.3 Analysis of the Conditional Bcl-x Knockout Mice

Immunostaining and immunoblotting for BCL-XL, TUNEL assay, DNA fragmentation assay, retinal morphological analysis, and electroretinography (ERG) were performed as described previously (Le et al., 2006a; Zheng et al., 2006). Cone density was determined with a lectin-staining assay according to the methods described previously (Le et al., 2004; Le et al., 2006b).

3 Results

3.1 Characterization of Rod-specific Bcl-x Knockout Mice

In the rod-specific Bcl-x knockout mice, the loss of BCL-XL expression was confirmed by Western blot analysis of retinal homogenates and immunohistochemistry (IHC) of retinal sections (Zheng et al., 2006). Examination of four-month-old rodspecific Bcl-x knockout mice demonstrated that the loss of BCL-XL did not cause any detectable changes in retinal apoptosis, morphology, or function under normal conditions (data not shown), suggesting that BCL-XL is not required for maintenance of the morphological and the functional integrity of the rod photoreceptors under normal conditions. Since BCL-XL was up-regulated under the bright light stress (7,000 lux for 48 h) in the retina of the wild-type mice, we then placed the 2-month-old rod-specific Bcl-x knockout mice and their littermate controls under bright light stress. Apoptosis was analyzed with TUNEL, nuclear staining, and DNA fragmentation assays. Our results suggest that there were significantly more apoptotic rod photoreceptors in the bright light-stressed, rod-specific Bcl-x knockout mice (Zheng et al., 2006). Examination of retinal morphology and function 7 days

after the bright light stress demonstrated that there was significantly greater loss of the photoreceptor outer nuclear layer (ONL) thickness and scotopic ERG amplitude in the rod-specific Bcl-x knockout mice (Zheng et al., 2006). In short, our results clearly demonstrated that the loss of BCL-XL caused increased susceptibility to the bright light stress in mouse rod photoreceptors.

3.2 Characterization of Cone-specific Bcl-x Knockout Mice

To address whether BCL-XL plays a protective role in the cone photoreceptors under bright light stress, we examined the effects of a genetic disruption of Bcl-x in mouse cone photoreceptors. Since mice have rod-dominant retinas and, since the methods to analyze both rod and cone photoreceptor-expressed genes in mouse cone photoreceptors have not been well established, it is difficult to analyze BCL- XL expression and apoptosis in the cone photoreceptors without interference from rod-expressed BCL-XL. To overcome this problem, we took advantage of our conespecific Cre mice that are capable of efficient Cre-mediated recombination in almost all S-cone and M-cone photoreceptors (Le et al., 2004). In a functional assay using double transgenic mice carrying cre and Cre-activatable lacZ reporter gene (Soriano, 1999), β-galactosidase was shown to be exclusively and efficiently expressed in the cone photoreceptors (Fig. 1). In addition, successful Cre-mediated recombination in many cell-type-specific Bcl-x knockout studies suggests that the chromosomal locus behaves normally and does not prevent Cre-mediated Bcl-x disruption in cone photoreceptors. Therefore, Bcl-x was disrupted genetically in cone photoreceptors in the cone-specific Bcl-x knockout mice.

Examination of four-month-old, albino cone-specific Bcl-x knockout mice demonstrated that the disruption of Bcl-x did not cause detectable changes in cone function or density under normal conditions (data not shown), suggesting that the BCL-XL, was not required for maintenance of the structural and the functional integrity of the cone photoreceptors under normal conditions. To determine if the BCL-XL had a protective effect under light stress in cone photoreceptors, the conespecific Bcl-x knockout mice were then subjected to bright light. Although bright

RPE

ONL

INL

GC

Fig. 1 Cre-mediated DNA recombination using F1 mice derived from cone-specific Cre mice and Cre-activatable lacZ reporter (R26R) mice. Arrows indicate a dark layer of β-galactosidase staining in a retinal section from a double transgenic mouse. ONL: outer nuclear layer; INL: inner nuclear layer; GC: ganglion cell. The scale bar represents 50 μm. Cre was efficiently expressed in the cone photoreceptors

is still not well understood, it is likely to serve as a cell death/survival checkpoint regulator of mitochondrial dysfunction. Recent evidences demonstrated that over-expression of BCL-XL protected photoreceptors from lead-induced apoptosis in mice through a blockage of the function of BAX, a pro-apoptotic protein (He et al., 2003). This is consistent with our result that loss of BCL-XL caused an increased susceptibility to bright light stress in rod photoreceptors, which is supported by the finding that loss of the pro-apoptotic proteins BAX and BAK protected photoreceptors from light damage in mice (Hahn et al., 2004). Interestingly, over-expression of BCL-XL did not appear to rescue either the inherited retinal degeneration caused by a Rd/Rd mutation or a dominant rhodopsin gene mutation (Joseph and Li, 1996). Therefore, the protective effect of BCL-XL is not universal and may be related to a specific stress condition. This may explain why we did not observe an increased susceptibility to bright light stress after disruption of Bcl-x in cone photoreceptors. In short, disruption of Bcl-x had no apparent effect on cone photoreceptor survival and thus BCL-XL may not play a protective role in cone photoreceptors under bright light stress.

Previous evidence demonstrates the presence of at least two independent apoptotic pathways for light-induced photoreceptor degeneration: a low light intensity caused phototransduction-related apoptosis and a high light intensity induced apoptosis via the transcription factor AP-1 (Hao et al., 2002). At present, the molecular mechanism governing bright light-induced photoreceptor protection caused by BCL-XLis not completely determined; however, BCL-XLis a down-stream target of the PI3K-AKT pathway. Genetic disruption of Akt also caused increased photoreceptor susceptibility to light damage (Li et al., 2007), suggesting that PI3K, AKT, and BCL-XL may act on the same pathway. BCL-XL is considered as an up-stream regulator of the caspase-dependent apoptotic pathways. Although the role of the caspase-dependent apoptotic pathways in light-induced photoreceptor degeneration is still controversial, our results suggest that BCL-XL, a widely regarded up-stream regulator of the caspase-dependent apoptotic pathway, is involved in the protection of rod photoreceptors from bright light-induced stress.

Acknowledgments The authors thank W. Zheng and M. Zhu for technical assistance; Dr. Lothar Hennighausen for providing the floxed Bcl-x mice. This study was supported by OCAST contracts HR01-083 and HR05-133; NIH grants EY00871, EY12190, EY04149, and RR17703; ADA Grant 1-06-RA-76; the Foundation Fighting Blindness, Inc; and Unrestricted Research Awards from Hope for Vision and Research to Prevent Blindness, Inc. Y.Z. Le is supported by a Young Investigator Travel Award to attend the XIIth International Symposium on Retinal Degenerations.

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