RESUMO
Retinitis pigmentosa (RP), an inherited retinal disease, affects 1,5 million people worldwide. The initial mutation-driven photoreceptor degeneration leads to chronic inflammation, characterized by Müller cell activation and upregulation of CD44. CD44 is a cell surface transmembrane glycoprotein and the primary receptor for hyaluronic acid. It is involved in many pathological processes, but little is known about CD44's retinal functions. CD44 expression is also increased in Müller cells from our Pde6bSTOP/STOP RP mouse model. To gain a more detailed understanding of CD44's role in healthy and diseased retinas, we analyzed Cd44-/- and Cd44-/-Pde6bSTOP/STOP mice, respectively. The loss of CD44 led to enhanced photoreceptor degeneration, reduced retinal function, and increased inflammatory response. To understand the underlying mechanism, we performed proteomic analysis on isolated Müller cells from Cd44-/- and Cd44-/-Pde6bSTOP/STOP retinas and identified a significant downregulation of glutamate transporter 1 (SLC1A2). This downregulation was accompanied by higher glutamate levels, suggesting impaired glutamate homeostasis. These novel findings indicate that CD44 stimulates glutamate uptake via SLC1A2 in Müller cells, which in turn, supports photoreceptor survival and function.
Assuntos
Células Ependimogliais , Receptores de Hialuronatos , Retinose Pigmentar , Transdução de Sinais , Animais , Receptores de Hialuronatos/metabolismo , Receptores de Hialuronatos/genética , Camundongos , Células Ependimogliais/metabolismo , Transdução de Sinais/fisiologia , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Retinose Pigmentar/genética , Camundongos Knockout , Camundongos Endogâmicos C57BL , Células Fotorreceptoras de Vertebrados/metabolismo , Sobrevivência Celular/fisiologia , Camundongos Transgênicos , Retina/metabolismo , Retina/patologiaRESUMO
Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the centre of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signalling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC- sequencing identified the GR as a master regulator of Müller cell response to diabetic conditions. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Animais , Camundongos , Diabetes Mellitus/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Células Ependimogliais/metabolismo , Neuroglia/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Retina/metabolismoRESUMO
Patients with progressive neurodegenerative disorder retinitis pigmentosa (RP) are diagnosed in the midst of ongoing retinal degeneration and remodeling. Here, we used a Pde6b-deficient RP gene therapy mouse model to test whether treatment at late disease stages can halt photoreceptor degeneration and degradative remodeling, while sustaining constructive remodeling and restoring function. We demonstrated that when fewer than 13% of rods remain, our genetic rescue halts photoreceptor degeneration, electroretinography (ERG) functional decline and inner retinal remodeling. In addition, in a water maze test, the performance of mice treated at 16 weeks of age or earlier was indistinguishable from wild type. In contrast, no efficacy was apparent in mice treated at 24 weeks of age, suggesting the photoreceptors had reached a point of no return. Further, remodeling in the retinal pigment epithelium (RPE) and retinal vasculature was not halted at 16 or 24 weeks of age, although there appeared to be some slowing of blood vessel degradation. These data suggest a novel working model in which restoration of clinically significant visual function requires only modest threshold numbers of resilient photoreceptors, halting of destructive remodeling and sustained constructive remodeling. These novel findings define the potential and limitations of RP treatment and suggest possible nonphotoreceptor targets for gene therapy optimization.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Terapia Genética/métodos , Doenças Neurodegenerativas/metabolismo , Mutação Puntual , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Modelos Animais de Doenças , Eletrorretinografia/métodos , Camundongos , Camundongos Transgênicos , Teste do Labirinto Aquático de Morris/efeitos dos fármacos , Doenças Neurodegenerativas/genética , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Vasos Retinianos/metabolismo , Retinose Pigmentar/metabolismo , Tamoxifeno/administração & dosagemRESUMO
Retinitis pigmentosa (RP) is an inherited neurodegenerative disease, in which the death of mutant rod photoreceptors leads secondarily to the non-cell autonomous death of cone photoreceptors. Gene therapy is a promising treatment strategy. Unfortunately, current methods of gene delivery treat only a fraction of diseased cells, yielding retinas that are a mosaic of treated and untreated rods, as well as cones. In this study, we created two RP mouse models to test whether dying, untreated rods negatively impact treated, rescued rods. In one model, treated and untreated rods were segregated. In the second model, treated and untreated rods were diffusely intermixed, and their ratio was controlled to achieve low-, medium-, or high-efficiency rescue. Analysis of these mosaic retinas demonstrated that rescued rods (and cones) survive, even when they are greatly outnumbered by dying photoreceptors. On the other hand, the rescued photoreceptors did exhibit long-term defects in their outer segments (OSs), which were less severe when more photoreceptors were treated. In summary, our study suggests that even low-efficiency gene therapy may achieve stable survival of rescued photoreceptors in RP patients, albeit with OS dysgenesis.
Assuntos
Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Animais , Morte Celular , Modelos Animais de Doenças , Terapia Genética/métodos , Camundongos , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Rodopsina/metabolismoRESUMO
Small molecule pharmacological inhibition of dominant human genetic disease is a feasible treatment that does not rely on the development of individual, patient-specific gene therapy vectors. However, the consequences of protein inhibition as a clinical therapeutic are not well-studied. In advance of human therapeutic trials for CAPN5 vitreoretinopathy, genetic inactivation can be used to infer the effect of protein inhibition in vivo. We created a photoreceptor-specific knockout (KO) mouse for Capn5 and compared the retinal phenotype to both wild-type and an existing Capn5 KO mouse model. In humans, CAPN5 loss-of-function (LOF) gene variants were ascertained in large exome databases from 60,706 unrelated subjects without severe disease phenotypes. Ocular examination of the retina of Capn5 KO mice by histology and electroretinography showed no significant abnormalities. In humans, there were 22 LOF CAPN5 variants located throughout the gene and in all major protein domains. Structural modeling of coding variants showed these LOF variants were nearby known disease-causing variants within the proteolytic core and in regions of high homology between human CAPN5 and 150 homologs, yet the LOF of CAPN5 was tolerated as opposed to gain-of-function disease-causing variants. These results indicate that localized inhibition of CAPN5 is a viable strategy for hyperactivating disease alleles.
Assuntos
Calpaína/genética , Doenças da Coroide/genética , Oftalmopatias Hereditárias/genética , Mutação , Degeneração Retiniana/genética , Tamoxifeno/farmacologia , Animais , Calpaína/química , Calpaína/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Inativação Gênica , Humanos , Masculino , Camundongos , Modelos Moleculares , Células Fotorreceptoras de Vertebrados/metabolismoRESUMO
Retinal gene therapy has yet to achieve sustained rescue after disease onset - perhaps because transduction efficiency is insufficient ("too little") and/or the disease is too advanced ("too late") in humans. To test the latter hypothesis, we used a mouse model for retinitis pigmentosa (RP) that allowed us to restore the mutant gene in all diseased rod photoreceptor cells, thereby generating optimally treated retinas. We then treated mice at an advanced disease stage and analyzed the rescue. We showed stable, sustained rescue of photoreceptor structure and function for at least 1 year, demonstrating gene therapy efficacy after onset of degeneration. The results suggest that RP patients are treatable, even when the therapy is administered at late disease stages.
Assuntos
Terapia Genética , Retinose Pigmentar/terapia , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/biossíntese , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Modelos Animais de Doenças , Eletrorretinografia , Indução Enzimática/efeitos dos fármacos , Integrases , Camundongos , Mutação Puntual , Proteínas Recombinantes/biossíntese , Células Fotorreceptoras Retinianas Cones/enzimologia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/enzimologia , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Organismos Livres de Patógenos Específicos , Tamoxifeno/farmacologia , Resultado do TratamentoRESUMO
OBJECTIVE: Retinitis pigmentosa (RP) is a hereditary retinal disease characterized by progressive photoreceptor degeneration, leading to vision loss. The best hope for a cure for RP lies in gene therapy. However, given that RP patients are most often diagnosed in the midst of ongoing photoreceptor degeneration, it is unknown how the retinal proteome changes as RP disease progresses, and which changes can be prevented, halted, or reversed by gene therapy. METHODS: Here, we used a Pde6b-deficient RP gene therapy mouse model and performed untargeted proteomic analysis to identify changes in protein expression during degeneration and after treatment. RESULTS: We demonstrated that Pde6b gene restoration led to a novel form of homeostatic plasticity in rod phototransduction which functionally compensates for the decreased number of rods. By profiling protein levels of metabolic genes and measuring metabolites, we observed an upregulation of proteins associated with oxidative phosphorylation in mutant and treated photoreceptors. CONCLUSION: In conclusion, the metabolic demands of the retina differ in our Pde6b-deficient RP mouse model and are not rescued by gene therapy treatment. These findings provide novel insights into features of both RP disease progression and long-term rescue with gene therapy.
RESUMO
In retinal degenerative diseases, such as retinitis pigmentosa (RP), the characteristic photoreceptor cell death is associated with changes of microglia and macroglia cells. Gene therapy, a promising treatment option for RP, is based on the premise that glial cell remodeling does not impact vision rescue. However, the dynamics of glial cells after treatment at late disease stages are not well understood. Here, we tested the reversibility of specific RP glia phenotypes in a Pde6b-deficient RP gene therapy mouse model. We demonstrated an increased number of activated microglia, retraction of microglial processes, reactive gliosis of Müller cells, astrocyte remodelling and an upregulation of glial fibrillary acidic protein (GFAP) in response to photoreceptor degeneration. Importantly, these changes returned to normal following rod rescue at late disease stages. These results suggest that therapeutic approaches restore the homeostasis between photoreceptors and glial cells.
Assuntos
Degeneração Retiniana , Retinose Pigmentar , Animais , Camundongos , Microglia/metabolismo , Retinose Pigmentar/terapia , Retina/metabolismo , Neuroglia/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Modelos Animais de DoençasRESUMO
Hereditary retinal degenerative diseases, such as retinitis pigmentosa (RP), are characterized by the progressive loss of rod photoreceptors followed by loss of cones. While retinal gene therapy clinical trials demonstrated temporary improvement in visual function, this approach has yet to achieve sustained functional and anatomical rescue after disease onset in patients. The lack of sustained benefit could be due to insufficient transduction efficiency of viral vectors ("too little") and/or because the disease is too advanced ("too late") at the time therapy is initiated. Here, we tested the latter hypothesis and developed a mouse RP model that permits restoration of the mutant gene in all diseased photoreceptor cells, thereby ensuring sufficient transduction efficiency. We then treated mice at early, mid, or late disease stages. At all 3 time points, degeneration was halted and function was rescued for at least 1 year. Not only do our results demonstrate that gene therapy effectively preserves function after the onset of degeneration, our study also demonstrates that there is a broad therapeutic time window. Moreover, these results suggest that RP patients are treatable, despite most being diagnosed after substantial photoreceptor loss, and that gene therapy research must focus on improving transduction efficiency to maximize clinical impact.