sirolimus and Retinal-Degeneration

Both photodamage and aberrant visual cycle contribute to disease progress of many retinal degenerative disorders, whereas the signaling pathways causing photoreceptor death remain unclear. Here we investigated the effects of intense photo-stress on (1) necrosome activation in wild-type and RPE65-null mice, (2) interaction of p62/Sequestosome-1 with the necrosome proteins, and (3) the effects of rapamycin on photodamage-induced necrosome activation and retinal degeneration in wild-type mice.. Dark-adapted rd12 mice and 129S2/Sv mice with or without rapamycin treatment were exposed to 15,000 lux light for different times. Expression levels and subcellular localization of proteins were determined through immunoblot and immunohistochemical analyses. Cone sheaths were stained with peanut agglutinin. Correlation between photoreceptor degeneration and receptor-interacting protein kinase-1 (RIPK1) expression was assessed with Spearman's correlation analysis. Protein-protein interaction was analyzed by immunoprecipitation.. Intense light caused rod and cone degeneration accompanied by a significant increase in RIPK1-RIPK3 expressions, mixed lineage kinase domain-like protein phosphorylation, damage-associated molecular patterns protein release, and inflammatory responses in wild-type mouse retina. The same intense light did not induce the necrosome activation in rd12 retina, but it did in rd12 mice that received 9-cis-retinal supply. RIPK1 expression levels are positively correlated with the degrees of rod and cone degeneration. Photodamage upregulated expression and interaction of the p62 autophagosome cargo protein with the necrosome proteins, whereas rapamycin treatment attenuated the light-induced necrosome activation and photoreceptor degeneration.. Necrosome activation contributed to photodamage-induced rod and cone degeneration. The visual cycle and autophagy are the important therapeutic targets to alleviate light-induced retinal necroptosis.

Topics: Animals; cis-trans-Isomerases; Eye Proteins; Mice; Mice, Knockout; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Sirolimus

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease that begins with defective rod photoreceptor function, followed by impaired cone function, and complete blindness in its late stage. To date, however, there is no effective treatment for RP. By carrying a nonsense mutation in the. 视网膜色素变性（RP）是一种遗传性视网膜变性疾病。RP最初影响视杆细胞的功能，随着病情的发展，视锥细胞也会受损，最终导致完全失明。该病目前没有有效的治疗方法。

Topics: Animals; Mice; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Mutations in the genes necessary for the structure and function of vertebrate photoreceptor cells are associated with multiple forms of inherited retinal degeneration. Mutations in the gene encoding RHO (rhodopsin) are a common cause of autosomal dominant retinitis pigmentosa (adRP), with the Pro23His variant of RHO resulting in a misfolded protein that activates endoplasmic reticulum stress and the unfolded protein response. Stimulating macroautophagy/autophagy has been proposed as a strategy for clearing misfolded RHO and reducing photoreceptor death. We found that retinas from mice heterozygous for the gene encoding the RHO

Topics: Animals; Autophagy; Autophagy-Related Protein 5; Beclin-1; Hydroxychloroquine; Mice, Inbred C57BL; Mutation; Photoreceptor Cells, Vertebrate; Proteasome Endopeptidase Complex; Protein Folding; Proteolysis; Retinal Degeneration; Rhodopsin; Sirolimus

Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.

Topics: Animals; Autophagy; Cell Death; Cell Dedifferentiation; Cell Movement; Cell Survival; Female; Glycolysis; Hepatocyte Growth Factor; Hypertrophy; Male; Mice; Mice, Transgenic; Oxidative Phosphorylation; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Retinal Degeneration; Retinal Pigment Epithelium; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Autophagy is a lysosomal machinery-dependent process that catabolizes cellular components/organelles and proteins in an autophagic vacuole (AV)-dependent and -independent manner, respectively. Short-term exposure of the retina to bright light results in shortening of the outer segments, concomitant with AV formation. Autophagy is also induced by continuous long-term light damage, leading to photoreceptor cell death. Here the authors examined two questions: is autophagy induced during light damage proapoptotic or antiapoptotic, and are rods and cones affected differently? To this end, Balb/c mice exposed to light damage were treated with rapamycin to increase autophagy.. Balb/c and GFP-LC3 mice were treated with rapamycin/vehicle. Photoreceptor degeneration was induced by 10-day light damage. Autophagy was documented by histologic, biochemical, and molecular tools; rod and cone survival was assessed by histology and electroretinography.. Light damage resulted in rod, but not cone, cell loss. Autophagy and AV formation was elicited in response to light damage, which was amplified by rapamycin. Rapamycin treatment significantly improved rod survival and function, reduced apoptosis, and normalized cytokine production that was increased in light damage. However, AV formation in GFP-LC3 mice revealed that light damage or rapamycin treatment induced AVs in cones, concomitant with reduced cone-mediated electroretinograms.. Systemic rapamycin treatment provided rod protection; however, AV formation was induced only in cones. Thus, rapamycin may act differentially in stressed photoreceptors; rapamycin might protect rods by normalizing cytokine production, removing damaged proteins by AV-independent autophagy, or both, whereas cones might be protected by AV-dependent autophagy, possibly involving reduced photon capture.

Topics: Animals; Apoptosis; Autophagy; Blotting, Western; Caspase 3; Cell Survival; Electroretinography; Female; Immunosuppressive Agents; Injections, Intraperitoneal; Light; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Radiation Injuries, Experimental; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus

To reevaluate the longevity and intraocular safety of recombinant adenovirus (rAd)-mediated gene delivery after subretinal injection, and to prolong transgene expression through the combination of 2 synergistic immunosuppressants.. An rAd vector carrying green fluorescent protein (GFP) gene was delivered subretinally in the rat eye. The GFP expression was monitored in real time by fundus fluorescent photography. Intraocular safety was examined by observation of changes of retinal pigmentation, cell infiltration in virus-contacted area, immunophenotyping for CD4(+) and CD8(+) cytotoxic T lymphocytes, and CD68(+) macrophages, histologic findings, and dark-adapted electroretinography. Two synergistic immunosuppressants, cyclosporine and sirolimus, were used alone or in combination to prolong transgene expression by temporary immunosuppression.. The GFP expression peaked on day 4, dramatically decreased on day 10, and was not detectable on day 14. The decreased GFP expression was coincident with cell infiltration in virus-contacted area. Immunostaining showed that the infiltrating cells were CD4(+) and CD8(+) cytotoxic T lymphocytes and CD68(+) macrophages. Clumped retinal pigmentation and decreased b wave of dark-adapted electroretinogram were observed at 3 to 4 weeks after injection. Histologic examination confirmed rAd-induced retinal degeneration. Transient immunosuppression by cyclosporine and sirolimus, either alone or in combination, improved transgene expression, with the combination being the most efficient. The combined immunosuppression attenuated but did not retard the rAd-induced retinal damage.. Transgene expression mediated by rAd after subretinal delivery is short-term and toxic to the retina. Combination of cyclosporine and sirolimus may act as an immunosuppressive adjunct to prolong rAd-mediated gene transfer.. The intraocular safety of rAd should be carefully considered before clinical trials are performed.

Topics: Adenoviridae; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cyclosporine; Drug Combinations; Electroretinography; Fluorescein Angiography; Fundus Oculi; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Green Fluorescent Proteins; Immunophenotyping; Immunosuppressive Agents; Luminescent Proteins; Macrophages; Rats; Rats, Mutant Strains; Retina; Retinal Degeneration; Sirolimus; T-Lymphocytes, Cytotoxic; Transgenes