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Gene Expression Profiling of Light-Induced Retinal Degeneration in Phototransduction Gene Knockout Mice

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Gene Expression Profiling of Light-Induced Retinal Degeneration in Phototransduction Gene Knockout Mice EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 40, No. 5, 495-504, October 2008 Gene expression profiling of light-induced retinal degeneration in phototransduction gene knockout mice Jayalakshmi Krishnan1*, Jiayan Chen2*, cades of gene components were induced or inhibited Kum-Joo Shin3*, Jong-Ik Hwang4, Sang-Uk Han1,5, as a result of corresponding gene knockout under spe- Gwang Lee1,6,8 and Sangdun Choi1,7,8 cific light conditions. Transducin deletion blocked the apoptotic signaling induced by exposure to low light 1Department of Molecular Science and Technology conditions, and it did not require c-Fos/AP-1. Deletion Ajou University of c-Fos blocked the apoptotic signaling induced by ex- Suwon 443-749, Korea posure to high intensity light. In the present study, we 2Zilkha Neurogenetic Institute identified many gene transcripts that are essential for University of Southern California the initiation of light-induced rod degeneration and Los Angeles, CA 90033, USA proposed several important networks that are involved 3Division of Biology in pro- and anti-apoptotic signaling. We also demon- California Institute of Technology strated the different cascades of gene components Pasadena, CA 91125, USA that participate in apoptotic signaling under specific 4Graduate School of Medicine light conditions. Korea University Seoul 136-705, Korea Keywords: arrestin; gene expression profiling; oli- 5Department of Surgery gonucleotide array sequence analysis; proto-onco- 6Brain Disease Research Center gene proteins c-fos; retinal degeneration; transducin Ajou University School of Medicine 7Department of Biological Sciences College of Natural Science, Ajou University Introduction Suwon 443-749, Korea 8Corresponding authors: Tel, 82-31-219-2600; Fax, 82-31-219-1615; Apoptosis is the final common pathway in many E-mail, [email protected] (S. Choi) cases of retinal degeneration or retinal disorders, Tel, 82-31-219-4554; Fax, 82-31-216-6381; such as retinitis pigmentosa (RP), and these E-mail, [email protected] (G. Lee) disorders might be ameliorated by interfering with *These authors contributed equally to this work. this process (Chang et al., 1993; Portera-Cailliau et DOI 10.3858/emm.2008.40.5.495 al., 1994). Retinal degeneration and its associated signaling networks have attracted much research interest for many years. Several reports have shown Accepted 15 May 2008 that light induces apoptosis in the retina and that Abbreviations: GPCR, G protein coupled receptor; Rhok, Rho- components of AP-1 are involved in this process dopsin kinase; Sag, S antigen (Arrestin) (Hafezi et al., 1997; Hao et al., 2002). However, the signaling networks that initiate the retinal degeneration cascade are not fully understood. Abstract Transducin is a heterotrimeric G protein expressed in the rods and cones of the vertebrate retina, and -/- Exposure to light can induce photoreceptor cell death knockout studies of transducin (Gnat1 ) showed no and exacerbate retinal degeneration. In this study, alteration in retinal morphology (Makino et al., 2003). However, there was an abrogation of mice with genetic knockout of several genes, including -/- -/- signaling from light-activated rhodopsin (Calvert et rhodopsin kinase (Rhok ), arrestin (Sag ), transducin al., 2000). Light activates rhodopsin, which is (Gnat1-/-), c-Fos (c-Fos-/-) and arrestin/transducin -/- -/- phosphorylated by rhodopsin kinase and the signal (Sag /Gnat1 ), were examined. We measured the ex- is terminated by arrestin, which binds specifically pression levels of thousands of genes in order to inves- with phosphorylated rhodopsin (Dolph, 2002; Miller tigate their roles in phototransduction signaling in and Lefkowitz, 2001). light-induced retinal degeneration using DNA micro- Rhodopsin signaling is prolonged in transgenic array technology and then further explored the gene mice with null mutations in the genes encoding network using pathway analysis tools. Several cas- rhodopsin kinase (Rhok-/-) (Chen et al., 1999a), 496 Exp. Mol. Med. Vol. 40(5), 495-504, 2008 arrestin (Sag-/-) (Chen et al., 1999b), or rhodopsin 5% Phenylephrine, Ciba Vision) was generated by kinase/arrestin (Rhok-/-/Sag-/-) (Choi et al., 2001). In diffused cool white fluorescent lamps and applied addition, c-Fos is known to be a mediator of for various time periods (24 h for 2,000 lux or 80 apoptosis, but its precise role in light-induced min for 6,000 lux). The temperature was kept at apoptosis is unclear. Therefore, we also used 25oC during irradiation. After light exposure, the c-Fos-/- mice to identify the genes affected. We mice were either analyzed immediately or after a screened the gene expression pattern in the retina given period in darkness. Retinas were removed of mice with knockout of key genes involved in rapidly through a slit in the cornea and frozen in phototransduction. Knockout strategies would liquid nitrogen until total RNA was extracted using provide a great deal of information regarding the the Trizol method (Invitrogen Life Technologies). functions of genes in mammals. Understanding the Retinas from three to four mice were pooled to mechanism of signaling networks in visual systems make the corresponding sample. will be greatly facilitated by the characterization of transcriptional regulation in genetic knockouts. There are two types of light-induced apoptotic Analysis of retinal morphology pathways: one is transducin-dependent and the Eyes obtained from anesthetized mice were other is transducin-independent (Hao et al., 2002). enucleated and dissected in 1/2 Karnovsky buffer The types of gene regulatory networks that are (2.5% glutaraldehyde, 2.0% paraformaldehyde in involved or predominant in these two types of 0.1 M cacodylate buffer, pH 7.2). Eyecups were apoptosis have not been clearly established. There- fixed overnight in 1/2 Karnovsky buffer and further fore, we aimed to delineate the signaling network fixed for 2 h in 1% osmium tetroxide. Eyecups in order to determine which network specifically were embedded in epoxy resin (EPON), and 1-μm participates in light-induced apoptosis. thick sections were cut along the vertical meridian at the optic nerve as described previously (Chen et al., 2006). Photomicrographs were acquired at Materials and Methods 40× magnification using AxioVision LE Rel. 4.1. software on a Zeiss Axioskop-2 microscope (Zeiss, Animals Göettingen, Germany). All procedures involving animals were performed in accordance with the Association for Research in Quantification of apoptosis in the retina Vision and Ophthalmology (ARVO) Statement on the use of animals in ophthalmic and vision Light-induced apoptosis in the retina was quantified research. Rhodopsin kinase (Rhok-/-), arrestin (Sag-/-), using a cell death detection ELISA assay kit transducin (Gna1-/-), and c-Fos (c-Fos-/-) knockout (Roche Molecular Biochemicals) that quantifies the mice were generated as described elsewhere soluble mono- and oligo-nucleosomes released in (Chen et al., 1999a, b; Hafezi et al., 1997; Calvert the cell lysate as a function of apoptosis (Leist et et al., 2000). Sag-/- and Gna1-/- mice were crossed al., 1994; Harada et al., 2000). One retina was to each other in order to obtain arrestin/ transducin homogenized with a 26-½ gauge needle in 200 μl double-deficient mice (Sag-/-/ Gna1-/-). All of the of PBS with 1 mM PMSF. The lysate was mice, including the wild type (WT) mice, were centrifuged at 15,000 × g for 10 min at room tem- reared in darkness until the given experiments perature. A total of 100 μl of supernatant was were performed. Wild type mice were derived from diluted 10 times with lysis buffer, and 20 μl of an initial cross of 129Sv and C57BL/6. The mice used diluted supernatant was used in the ELISA measu- in this study ranged from 6 to 8 weeks of age. rement according to the manufacturer's instructions. Light illumination Microarray analysis The mice reared in the dark were placed in First strand cDNA was synthesized using T7-oligo aluminum foil-wrapped polycarbonate cages that dT primer and SuperScript II (Invitrogen Life were covered with stainless steel wire tops in order Technologies) with 3 μg of total RNA from retinas. to protect them from uncontrolled light exposure. Second strand cDNA was synthesized with second Fluorescent lamps provided light from an opening strand buffer (Invitrogen Life Technologies), DNA at the top of the cage. The mice were supplied with polymerase I (New England Biolabs, Inc.), DNA food and water through the bottom of the cage. ligase (NEB) and RNase H (Invitrogen Life Techno- Constant illumination of 2,000 lux without dilation logies). cDNA was extracted using phenol:chloro- or 6,000 lux on dilated pupils (1% Cyclogyl, Alcon; form:isoamyl alcohol, precipitated with ethanol, Profiling of genes involved in retinal degeneration 497 washed with 80% and 100% cold ethanol, and air Results dried. The dried pellet was then dissolved in 22 μl of nuclease-free water and stored at -20oC. In vitro Morphological analysis transcription was performed using the RNA Trans- Figure 1 shows retinal sections from mice after cript Labeling Kit (Enzo Diagnostics) to produce exposure to light for various time periods. No hybridizable biotin-labeled RNA targets. The cDNA damage was detected in the wild type mice under was used as a template in the presence of a every illumination condition tested: 6,000 lux on mixture of unlabeled NTPs and biotinylated CTP dilated pupils for 80 min (data not shown), 2,000 and UTP. After in vitro transcription, cRNA was lux without dilation for 24
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