Activation of mitochondrial calpain and release of apoptosis-inducing factor from mitochondria in RCS rat retinal degeneration.

The present study was performed to investigate changes of cytosolic and mitochondrial calpain activities, and effects of intravitreously injected calpain inhibitor on photoreceptor apoptosis in Royal College of Surgeon's (RCS) rats. Time courses of activities for both cytosolic and mitochondrial calpains and amount of calpastatin in RCS rat retina were analyzed by subcellular fractionation, calpain assay and western blotting. Calpain assay was colorimetrically performed using Suc-LLVY-Glo as substrate. Effects of intravitreously injected calpain inhibitor (ALLN and PD150606) on RCS rat retinal degeneration were analyzed by TUNEL staining. Effects of mitochondrial calpain activity on activation and translocation of apoptosis-inducing factor (AIF) were analyzed by western blotting. Mitochondrial calpain started to be significantly activated at postnatal (p) 28 days in RCS rat retina, whereas cytosolic micro-calpain was activated at p 35 days, although specific activity of mitochondrial calpain was 13% compared to cytosolic micro-calpain. Intravitreously injected ALLN and PD150606 effectively inhibited photoreceptor apoptosis only when injected at p 25 days, but did not inhibit photoreceptor apoptosis when injected at p 32 days. Parts of AIF were truncated/activated by mitochondrial calpains and translocated to the nucleus. These results suggest that 1), calpain presents not only in the cytosolic fraction but also in the mitochondrial fraction in RCS rat retina; 2), mitochondrial calpain is activated earlier than cytosolic calpain during retinal degeneration in RCS rats; 3), photoreceptor apoptosis may be regulated by not only calpain systems but also other mechanisms; 4), mitochondrial calpain may activate AIF to induce apoptosis; and 5), calpain inhibitors may be partially effective to inhibit photoreceptor apoptosis in RCS rats. The present study provides new insights into the molecular basis for photoreceptor apoptosis in RCS rats and the future possibility of new pharmaceutical treatments for retinitis pigmentosa.

S-opsin protein is incompletely modified during N-glycan processing in Rpe65(-/-) mice.

Retinal pigment epithelium-specific protein 65 kDa (RPE65) is a key enzyme for the visual cycle in the eye. Rpe65(-/-) mice lack 11-cis-retinal, and show early cone degeneration and mislocalization of cone opsins. The present study investigated whether abnormal modification of cone opsins at the protein level is present in Rpe65(-/-) mice. Retina-RPE-choroids of Rpe65(-/-) mice at 3, 5 and 7 weeks old were used. Immunohistochemistry of opsins was performed using cryosections and retinal flatmounts. We evaluated levels of mRNA for cone and rod opsin genes by RT-PCR and levels of proteins by western blotting. To examine modification patterns of N-glycan in Rpe65(-/-) mice, cone opsins were digested with peptide-N-glycosidase (PNGase) F. S-opsin protein was detected at approximately 40-kDa as a major band in wild-type mice, whereas approximately 42-kDa S-opsin protein was detected in Rpe65(-/-) mice. After PNGase F treatment, mobility of S-opsin protein in wild-type and Rpe65(-/-) mice on SDS-PAGE was similar. In addition, approximately 25-kDa S-opsin polypeptide was notably detected in Rpe65(-/-) mice. Conversely, M-opsin proteins were not observed by immunohistochemistry or western blotting in Rpe65(-/-) mice, but expression of M-opsin mRNA in Rpe65(-/-) mice did not differ significantly from that in wild-type mice at 3 and 5 weeks. Mobility of M-opsin protein in Rpe65(-/-) mice was unchanged. Our data suggest that S-opsin protein is incompletely modified during N-glycan processing in Rpe65(-/-) mice, whereas M-opsin protein is severely reduced by posttranslational degradation in the absence of incomplete N-glycan processing in Rpe65(-/-) mice.

Crystal deposits on the lens capsules in Bietti crystalline corneoretinal dystrophy associated with a mutation in the CYP4V2 gene.

PURPOSE: We report a patient (Case 1) with Bietti crystalline corneoretinal dystrophy (BCD) associated with previously unknown findings of crystal-like deposits on the anterior and posterior lens capsules. This patient is one of four (Cases 1-4) in whom we have found BCD associated with the same mutation in the CYP4V2 gene. METHODS: We present a case report with molecular diagnosis. A 45-year-old man (Case 1) was referred to our clinic with complaints of gradual progression of visual disturbances and night blindness. His visual acuity was limited to hand movement bilaterally. Slit-lamp biomicroscopy disclosed glistening, crystal-like deposits on the anterior and posterior lens capsules, as well as on the corneal stroma near the corneoscleral limbus. No such deposit was found in the lens stroma. Fundus examination disclosed profound chorioretinal atrophy with scarce crystal deposits. Full-field electroretinography showed extinguished responses of isolated rods, isolated cones, and mixed rods and cones. RESULTS: Molecular genetic analysis revealed that the subject had a homozygous mutation in the CYP4V2 gene (IVS6-8delTCATACAGGTCATCGCG/insGC), which is most commonly found in Japanese patients with BCD. Three other cases (Cases 2-4) of BCD associated with the same mutation did not show such crystal-like deposits on the lens surface. CONCLUSIONS: Although their exact origin remains unknown, crystal-like deposits may appear on the lens capsule of patients with BCD associated with a mutation in the CYP4V2 gene.

Indocyanine green as useful guide for retinal vein cannulation and injection of tissue plasminogen activator in rabbits.

Central retinal vein occlusion (CRVO) is caused by a fibrin clot in central retinal vein and is one of the intractable diseases that deteriorate visual acuities. Tissue plasminogen activator (TPA) is a thrombolytic agent that has a high affinity for fibrin and that activates plasminogen into plasmin. Injection of TPA into the retinal vein was helpful to treat CRVO. But TPA injection into retinal vein was difficult for clinical use, because TPA solution was transparent and confirmation of its injection was not easy. Indocyanine green (ICG) has been used as an angiographic agent and a tissue-marking agent in ocular surgeries. We studied the effectiveness and safety of ICG assisted injection of TPA into the retinal vein in rabbits. The major retinal vein was punctured using a micropipette fabricated from a glass tube, then TPA/ICG was injected. Total of 12 eyes and 5 eyes were enucleated 7 days and 1 month after injection of TPA/ICG for histological observations including immunostaining of glial fibrillary acidic protein (GFAP) and TUNEL staining, respectively. GFAP is expressed in Müller cells under pathologic conditions and indicates retinal damages. TUNEL indicates apoptosis of sensory retina cells. Retinal vein cannulation was easily performed, as retinal vein became green following injection of TPA/ICG. Histologically, no retinal damages, due to the TPA/ICG solution, were observed. GFAP and TUNEL staining were negative. TPA/ICG causes no disturbance in retinal circulation when performed correctly. Because of its safety, ICG is a useful agent as a guide for retinal vein injection of TPA.

Systemic administration of nilvadipine delays photoreceptor degeneration of heterozygous retinal degeneration slow (rds) mouse.

To investigate the effect of nilvadipine, a calcium channel blocker, upon the retina of retinal degeneration slow (rds) mouse, nilvadipine was intraperitoneally injected into heterozygous rds mice for up to 200 days. The effect of nilvadipine was evaluated by electroretinography (ERG), light and electron microscopies, DNA microarray, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and western-blot analysis. After nilvadipine treatment, both a- and b-waves of ERG were significantly higher than in the control group (p<0.01). Although there was no difference in histological findings by light microscopy between the nilvadipine treated group and control group, apparent preservation of photoreceptor disc was demonstrated by electron microscopy in the treated group. Rhodopsin level was also increased in the treated group comparing to the control group. The DNA microarray analysis detected increased expression of genes encoding proteins which function in protein synthesis, growth factors and neurotrophic factor like ciliary neurotrophic factor (CNTF) and fibroblast growth factors (FGFs22 and 13). Decreased expression of genes coding for proteins related to proteolysis, apoptosis and growth factor (FGF18) was also demonstrated. Increased expression of CNTF, FGF22 and FGF13 and decreased expression of FGF18 were confirmed by both quantitative RT-PCR and western-blot analysis. In addition, FGF2 was constitutively expressed in both treated and control groups. Since CNTF has been known to retard retinal degeneration by rds mouse or other models of inherited retinal degeneration, it is possible that nilvadipine has a photoreceptor survival effect on rds retinal degeneration partly by enhancing expression of endogenous CNTF in the retina.