Investigated By Sulforaphane In Photoreceptor Cell Survival/Protection In Tubby Mouse

PurposePhotoreceptor cell death is an irreversible event. With the inherent vulnerability of photoreceptor cells, it is not surprising that many different mutations and acquired insults lead to photoreceptor cell death . In post - mitotic photoreceptor cells, there is a significant increase in cell death when metabolic activity in the retina increases. This, together with the high focal concentrations of mitochondria in photoreceptors and the very high blood supply to the choriocapil-laris, suggests that there may be very little reserve in the energy supply to photoreceptor cells. The complex inter - relationships between photoreceptors, M Her cells and retinal pigment epithelium (RPE) add to the vulnerability in that failure of other cell types readily leads to secondary photoreceptor cell loss. It seems possible that relatively mild insults can significantly increase the probability of cell death. On the other hand, a relatively modest benefit to the cell may reduce the chances of cell death sufficiently to have an important clinical impact.The homozygous tubby mutant mouse has an early progressive hearing loss and progressive photoreceptor degeneration, the two phenotypic hallmarks seen in human sensorineural deafness/retinal dystrophic syndrome. The disease course in the homozygous tubby mice is similar to that in Usher type I patients. However, despite the similar phenotype, the tub/tub mouse is not currently being used as a model for Ushers Syndrome.Thioredoxin (Trx) is a small (13 kDa) ubiquitous protein with two redox - active cystine residues, - Cys - Gly - Pro - Cys - , in its active center. Hu-man Trx was originally cloned as a soluble factor released from human T cell leukemia virus type -1 transformed T cells. Trx has various biologic activities such as elimination of reactive oxygen species, activation of transcription factors, and regulation of the intracellular apoptotic pathway and is up - regulated in response to a wide variety of oxidative stresses, including viral infections and ultraviolet and X - ray irradiation. Thioredoxin reductase (TrxR) , a 55 kDa selenoprotein containing a penultimate C - terminal selenocysteine, catalyzes NADPH - dependent reduction of Trx, thus Trx sys tem is composed of Trx, TrxR, and NADPH. Current information suggests that imbalances in the tissue or cellular redox state are associated with various types of diseases, and normalization of the cellular redox state via intensification of endogenous and exogenous Trx expression prevents retinal photoreceptor cell damage. Thus, Trx system plays a crucial role in reduction/oxidation (redox) regulation of signal transduction and in cell defense against oxidative stresses.Sulforaphane (SF) is a naturally occurring isothiocyanate found as a precursor of glucosinolate in cruciferous vegetables like broccoli. SF inhibits phase I enzymes such as cytochrome P450, and induces phase II detoxification enzymes such as NADPH quinone oxidoreductase, glutathione S - transferase, UDP - glucuronosyl transferases, and Trx reductase. Transcription of phase II genes depends on activation of upstream regulation of antioxidant responsive elements ( ARE). Heterodimeric combination of the NF - E2 - related factor - 2 ( Nr￡2) with members of the small Maf family activates the NQO and glutathione S - transferase genes in mice. Hemin - induced expression of Trx in K562 eryth-roleukemia cells is mediated by binding of Nrf - 2/small Maf heterodimers to the ARE, and this ARE - mediated gene expression is controlled by Trx - dependent redox regulation. Intraperitoneal and oral administration of SF up - regulates Trx in retinal tissue and mediates cytoprotection against light - induced photoreceptor and retinal pigment epithelial (RPE) cell damage in mice. In cultured RPE cells, SF up -;regulates the Ti-x gene yia ARE,that, was regulated by Nr￡2, small Maf, and c - Jun proteins. Thus the Nr￡2 has been implicated as the central protein that interacts with the / RE to activate gene transcription by SF or in response to an oxidative stress signal.In this study, the neuroprotective effect of systemic administration of SF and the involvement of Trx redox system in photoreceptor survival/protection in tubby mouse were investigated. We have made novel observations showing that the retinal Trx system is impaired in tubby mice, and SF could restore the impaired Trx system and delay inherited photoreceptor degeneration via extracellular signal - regulated kinase (ERK) /Nr￡2 signal cascade.Methods1. Animal care and GenotypedThe homozygous tubby mouse (tub/tub) were purchased from Jackson Laboratory ( Bar Harbor, ME) , and bred with wild type C57BL/6J ( +/ + ). Obtained heterozygous tubby mice (tub/ + ) were used as breeding parents. All animals were born and raised in a 12 - hours - on and 12 - hours - off cyclic light environment at average illumination of 800 lux. All animals were genotyped with the Jackson Laboratory protocol .2. SF treatmentThe mice at postnatal days P10 were intraperitoneally injected with 0. 2 mg, 0. 35 mg, or 0. 5 mg of SF (S8046, LKT Laboratories Inc. , St. Paul, MN) once a day for up to P14;or the mice at postnatal days 14 ( P14) were intraperitoneally injected with 0. 2 mg of SF, once a day for up to P34;or 50 1 phosphate buffered saline (PBS) once a day for up to P34. In some animals, 0. 2 mg ERKs inhibitor PD98059 (sc - 13032, Santa Cruz Biotechnology, Inc, Santa Cruz, CA). All injections were performed at 10 AM.3. Semi quantitative RT - PCRAfter euthanasia by CO2 inhalation, the eyes were immediately enucleated, and the neural retinas were separated from the eyecups under a microscope. Total RNA was extracted using TRIzol reagent (Invitrogen Corp. , Carlsbad, CA) according to the manufacturer's protocol. Concentration of RNA was quantified by reading of 260nm on spectrophotometer. cDNA was synthesized using First -Strand Synthesis System for RT - PCR kit (Invitrogen) according to the manu-facturerS protocol, using 4 (xg of total RNA. PCR products were visualized by e-lectrophoresison 1.2% agarose gel (Invitrogen) , stained with 0.5 g/mlethidi-um bromide, and photographed under UV light. As an internal control, RT -PCR for -actinwas perfonned simultaneously.4. Quantitative real -time reverse transcription (RT) -PCRTotal RNA isolation and cDNA synthesis were described as above. All PCR reactions were at a final volume of 30 1 comprised of SYBR Green PCR mix (Invitrogen) , 600 M forward and reverse primers, and Ing of synthesized cDNA. Real - time PCR was performed in 96 - well plates using a Bio - Rad iCycler (Bio - Rad, Hercules, CA). We used the following PCR cycle parameters: polymerase activation for 15 min at 951!, 40 cycles of 95X1 for 15 sec, 60X1 for 30 sec, and 72^for 60 sec.5. Western blot analysisAfter euthanasia by CO2 inhalation, the eyes were immediately enucleated, and the neural retinas were separated from the eyecups under a microscope. Retinas were lysed in ice - cold tissue lysis buffer jxgeno Technology, Inc, St, Louis , MO) for 20 min. The preparation of retinal nuclear extracts and determination of the DNA binding activity were performed using a nuclear and cytoplasmic reagent kit (NE -PER;Pierce, Rockford, IL) according to the manufacturers protocols. The lysates were centrifuged at 12,000 g for 15 min and the superna-tants were collected as protein samples. Equal amounts of protein samples (10 -20 |xg) were loaded on to 12% SDS - poly aery lamide gel, electrophoresed at 100 V, and transferred onto a nitrocellulose membrane ( Bio - Rad). The membrane was blocked in 5% non - fat dry milk solution for 1 hour at room temperature , incubated with the primary antibody at4°C for overnight, and then incubated with horseradish peroxidase - conjugated secondary antibody for 1 hour at room temperature. Protein bands were detected with Enhanced Chemilumines-cence (ECL) Western blotting reagents (Amersham Biosciences).6. Morphological Analysis by Quantitative HistologyAfter euthanasia by CO2 inhalation, eyes were enucleated at the indicated time - points. Enucleated eyes were immersed in 4 % paraformaldehyde containing 20 % isopropanol, 2 % trichloroacetic acid, and 2 % zinc chloride, for 24 h, and then in 70 % ethanol for 24 ~ 60 h. After alcohol dehydration, the eyeswere embedded in paraffin and 5 m thick sagittal sections containing the whole retina including the optic disc were cut. The retinal sections were stained with hematoxylin - eosin (H&E). In each of the superior and inferior hemispheres, outer nuclear layer (ONL) thickness was measured at 9 defined points. Each point was centered on adjacent 220 m lengths of retina.7. Immunofluorescent StainingParaffin - embedded retinal sections were deparaffinized and rehydrated, subsequently antigen retrieval was performed by boiling the sections in 10 mM citrate buffer (pH 6. 0) for 20 min, washing twice with water and twice with PBS. Sections were blocked with normal horse serum for 20 minutes and then were incubated with the specific antibodies at 4t for overnight. After rinsing twice with PBS for 5 minutes, for analysis of Trx, the sections were incubated with biotinylated goat anti - rabbit immunoglobulin ( Biomeda Corp. , Foster Cit-y, CA) at room temperature for 1 hour, followed by incubation with fluorescein - labeled streptavidin ( Biomeda) (for Trx) , or the sections were incubated with fluorescein - labeled anti - rabbit IgG ( 1: 1000, Vector Laboratories, Inc. , Burlingame, CA) at room temperature for 1 h in a humidity chamber. The sections were finally rinsed twice for 5 minutes in PBS and mounted.8. Functional Evaluation by Electretinogram (ERG)The procedures for ERG testing were performed as described with modification. In this study, ERGs were recorded by using Espion ERG recording system ( Diagnosys, Littleton, MA) to test the retinal function. Animals were dark - a-dapted for overnight before being tested. After anesthesia was induced by intramuscular injection of a mixture of Ketamine and xylazine, the pupils were dilated with 1% atropine and 2.5% phenylephrine HCL eye drops. Gold electrodes were placed on the both eyes. An identical reference electrode was placed in the mouth and a ground electrode was placed on the footpad. The duration of light stimulation was 10 milliseconds. The band pass was set at 0.15 - 300 Hz. The intensity of stimuli used in this study was 400 cd s/m2. The b - wave amplitudes obtained from right and left eyes were averaged in each mouse.Results1. Morphological Evaluation of Photoreceptor Degeneration by Quantitative Histology and Functional Evaluation by Electroretinography (ERG) in tubby miceThe extent of photoreceptor degeneration in tubby mice at selected postnataldays (PIO......180) was evaluated histologically by measuring the thickness ofouter nuclear layers ( ONL). Similar time points from heterozygous littermates (tub/ + ) mice, which are morphologically and functionally normal, were also selected for morphological evaluation. No morphological difference in the ONL thickness between tubby mice and tub/.+ mice was seen before P12 (data not shown). Significant loss of photoreceptor cells was observed in tubby mice starting at P14 as indicated by reduction of ONL thickness. At P28, Approximate 50% loss of photoreceptor cells was seen.The ERG was performed at postnatal day 34 to evaluate and compare the retinal function among tub/tub, tub/ + , and +/ + mice. The data shows dramatically reduced a - and b - wave amplitudes in tubby mice.2. Retinal expressions of Trx and TrxR in tubby mouseWe first tested the gene and protein levels of Trx and TrxR in the retinas of tubby and tub/ + and wild type mice. By the semi - quantitative RT - PCR, gene expression levels for Trx and TrxR are clearly lower in retinas from tub/tub compared to +/ + or tub/ + at PIO and P14. Similarly, our quantitative real -time RT - PCR data showed that the gene expression levels of Trx and TrxR genes were also significantly lower in retinas from tub/tub compared to + / + (p<0.05 and p<0.05, respectively) or tub/+ (p<0.05 and p<0.05, respectively) at P14. Using the Western blot and subsequent densitometric analyses , the protein expression levels of Trx and TrxR were significantly lower in the retinas from tub/tub compared to +/ + (p <0.01 and p <0.01, respectively) or tub/ + (p <0.01 and p <0.01, respectively) at P14. These results clearly indicate the impaired expressions of Trx and TrxR genes and proteins in retinas of tubby mice during early postnatal periods before significant photoreceptor losscould be observed.3. Effect of SF on retinal levels of Trx and TrxR in tubby mouseIn set one of experiments, SF at different dose (0 mg, 0.2 mg, 0.35 mg, and 0.5 mg SF/mouse) or PBS was IP injected daily respectively from P10 to P14, the retinal levels of Trx and TrxR proteins at P14 were analyzed by Western blotting. Protein levels of Trx in retina of tub/tub were increased (p < 0. 05) , and those of TrxR were increased (p < 0.05) (compared to PBS - treated tub/ + at P14). The set two of experiments showed after a single injection of 0. 35 mg SF/mouse at age of P14, protein levels of Trx in retina of tub/tub were increased, and those of TrxR were increased. Significant differences were observed at 6 h (p <0.01 for Trx and p <0.01 for TrxR) and 12 h (p <0.01 for Trx and p <0. 05 for TrxR) compared to 0 h.Localizations of Trx and TrxR expressions in the retina were examined by immunofluorescent staining. In tub/ + , Trx staining was observed in inner segment (IS) , outer plexiform layer ( OPL) , inner plexiform layer (IPL) , and ganglion cell layer ( GCL) , and labeling of TrxR was observed in IS, outer nuclear layer (ONL) , OPL, inner nuclear layer (INL) , and GCL. Compared to tub/ + , intensities of Trx and TrxR labeling were remarkably lower on the retinal sections from PBS - treated tub/tub. Systemic administration of SF 0. 2 mg/ day for two weeks (from P10 - P28) clearly restored the intensities of Trx and TrxR labeling in almost equal to or higher than that observed in tub/ + mice.4. Effects of SF on retinal degeneration in tubby mouseThe daily injection of SF (0.2 mg/ day during P10 P28) has any protective effect on photoreceptor degeneration in tubby mouse. Remarkable loss of photo-receptor cell nuclei in ONL at P28 and P34 was observed in PBS - treated tub/ tub compared to tub/ + , whereas the loss of cell nuclei was less remarkable in SF - treated tub/tub compared to PBS - treated tub/tub. By the quantification of ONL thickness, ONL thickness was significantly higher in SF - treated tub/tub compared to PBS - treated tub/tub at P28 (p < 0.001) and P34 (p < 0.001). No statistically difference was noted between PBS - and SF - treated tub/ + indicating no toxic effect of SF on photoreceptor cells. By the ERG recordings, B - wave amplitude was significantly higher in SF - treated tub/tub compared toPBS - treated tub/tub at P34 (p <0.01) , whereas the amplitude was almost i-dentical between PBS - and SF - treated tub/ + .5. Effects of SF on retinal level of Nr￡2The gene expressions of Nrf2 exon 1-3 and Nr￡2 exon 5 tested by RT -PCR were significantly lower in retinas from PBS - treated tub/tub (p <0.001, and p < 0. 05, respectively) compared to PBS - treated tub/ + . Systemic administration of SF (0.2mg/day, from P10 to P14) clearly restored Nrf2 exon 1 - 3 and Nr￡2 exon 5 expression levels in the retinas of tub/tub. Similarly, Nr￡2 protein level was significantly lower in retinas of PBS - treated tub/tub (p <0. 01) compared to PBS - treated tub/ + .6. Roles of MAPK signal pathway in SF - mediated up - regulations of Nr￡2, Trx, and TrxRPhospholypated ERKs were significantly lower in the retinas of tub/tub without PD and SF treatment (p <0.05) compared to that of tub/ + . With the SF treatment, phospholylated ERKs were significantly increased in the retinas of tub/tub ( p < 0. 0001 ) compared to tub/ + , whereas this SF - mediated increase of phospholylated ERKs bands was not observed when the ERKs inhibitor PD98059 (0.2mg/day, from P10 to P14) was injected intraperitoneally simultaneously with SF in retinas from tub/tub [ PD ( + ) and SF ( + ) ].The role of ERKs phospholylation in SF - mediated up - regulations of Nr￡2, Trx, and TrxR in tubby mice retina was further examined. Retinal levels of Nrf2, Trx and TrxR proteins were significantly increased in the SF - treated tub/tub (p <0.001, p <0. 001, and p <0.01, respectively) compared to the un - treated tub/tub, whereas these up - regulations of proteins in the retinas of tub/tub by SF treatment were diminished when the PD98059 was injected simultaneously with SF [PD( +) and SF (+)].Retinal nuclear extracts levels of Nrf2 protein were significantly lower in the retinas of tub/tub (p <0.01) compared to tub/ +. With the SF treatment, Nr￡2 levels in the nuclear fraction of tub/tub retinas were significantly increased [ PD ( - ) and SF ( + ) ] compared to tub/ + , whereas this SF - mediated increase of Nrf2 protein in retinas nuclear extracts from tub/tub was blocked when the ERKs inhibitor PD98059 was injected simultaneously with SF [PD( + ) and SF( + ) ] (p < 0.05) compared to tub/ +. Furthermore, Nr￡2 protein level in retinas nuclear extracts from tub/tub was down - regulations when the PD98059 was injected but without SF [PD( + ) and SF ( - ) ] (p <0.01) compared to tub/ +.Conclusions1. At P10 and P14, retinal expressions of Trx and TrxR genes and proteins were clearly down - regulated in the retinas from tub/tub compared to tub/ + or+ / + tested. In tub/tub, significant loss of photoreceptor cells stared at P14.2. Down - regulations of Trx and TrxR observed during early postnatal period precede significant photoreceptor cell loss in tubby mouse, suggesting the underlying relations between attenuations of Trx and TrxR expressions and loss of photoreceptor cells in tubby mouse.3. We observed the significant increases of Trx and TrxR protein levels by SF treatment in retinas from tub/tub mice. Indicating clearly that SF has effect for delaying hereditary retinal degeneration in tubby mouse morphologically and functionally.4. We have demonstrated that levels of Nr￡2 in whole retinal lysate and nuclear fraction were significantly down - regulated in retinas from tub/tub compared to tub/ + , indicating that the expression and the activation of Nr￡2 are impaired in tubby retina. SF is able to up - regulate Nr￡2 levels in tubby retina.5. ERKs were down -regulated in retinas from tub/tub compared to tub/ + , and were significantly activated by SF treatment in tub/tub, whereas this SF- mediated activations of ERKs were inhibited by ERKs inhibitor.6. ERKs and Nr￡2 could be involved in the mechanism of SF - mediated up- regulation of Trx system in this model. Our data clearly demonstrated that ERKs signal transduction pathways is involved in SF - mediated up - regulations of Trx/TrxR/Nr￡2 expression in vivo.