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SignificanceStudies in multiple experimental systems have demonstrated that an increase in proteolytic capacity of post-mitotic cells improves cellular resistance to a variety of stressors, delays cellular aging and senescence. Therefore, approaches to increase the ability of cells to degrade misfolded proteins could potentially be applied to the treatment of a broad spectrum of human disorders. An example would be retinal degenerations, which cause irreversible loss of vision and are linked to impaired protein degradation. This study suggests that chronic activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway in degenerating photoreceptor neurons could stimulate the degradation of ubiquitinated proteins and enhance proteasomal activity through phosphorylation.
Assuntos
Complexo de Endopeptidases do Proteassoma , Proteólise , Células Fotorreceptoras Retinianas Bastonetes , Retinose Pigmentar , Ubiquitina , Animais , Modelos Animais de Doenças , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismoRESUMO
Recombinant adeno-associated virus (rAAV) vectors are currently the only proven vehicles for treating ophthalmological diseases through gene therapy. A wide range of gene therapy programs that target ocular diseases are currently being pursued. Nearly 20 years of research have gone into enhancing the efficacy of targeting retinal tissues and improving transgene delivery to specific cell types. The engineered AAV capsid, AAV2.7m8 is currently among the best capsids for transducing the retina following intravitreal (IVT) injection. However, adverse effects, including intraocular inflammation, have been reported following retinal administration of AAV2.7m8 vectors in clinical trials. Furthermore, we have consistently observed that AAV2.7m8 exhibits low packaging titers irrespective of the vector construct design. In this report, we found that AAV2.7m8 packages vector genomes with a higher degree of heterogeneity than AAV2. We also found that genome-loaded AAV2.7m8 stimulated the infiltration of microglia in mouse retinas following IVT administration, while the response to genome-loaded AAV2 and empty AAV2.7m8 capsids produced much milder responses. This finding suggests that IVT administration of AAV2.7m8 vectors may stimulate retinal immune responses in part because of its penchant to package and deliver non-unit length genomes.
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Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. While the histopathology of the different disease stages is well characterized, the cause underlying the progression, from the early drusen stage to the advanced macular degeneration stage that leads to blindness, remains unknown. Here, we show that photoreceptors (PRs) of diseased individuals display increased expression of two key glycolytic genes, suggestive of a glucose shortage during disease. Mimicking aspects of this metabolic profile in PRs of wild-type mice by activation of the mammalian target of rapamycin complex 1 (mTORC1) caused early drusen-like pathologies, as well as advanced AMD-like pathologies. Mice with activated mTORC1 in PRs also displayed other early disease features, such as a delay in photoreceptor outer segment (POS) clearance and accumulation of lipofuscin in the retinal-pigmented epithelium (RPE) and of lipoproteins at the Bruch's membrane (BrM), as well as changes in complement accumulation. Interestingly, formation of drusen-like deposits was dependent on activation of mTORC1 in cones. Both major types of advanced AMD pathologies, including geographic atrophy (GA) and neovascular pathologies, were also seen. Finally, activated mTORC1 in PRs resulted in a threefold reduction in di-docosahexaenoic acid (DHA)-containing phospholipid species. Feeding mice a DHA-enriched diet alleviated most pathologies. The data recapitulate many aspects of the human disease, suggesting that metabolic adaptations in photoreceptors could contribute to disease progression in AMD. Identifying the changes downstream of mTORC1 that lead to advanced pathologies in mouse might present new opportunities to study the role of PRs in AMD pathogenesis.
Assuntos
Envelhecimento/patologia , Macula Lutea/patologia , Degeneração Macular/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Lâmina Basilar da Corioide/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Lipofuscina/metabolismo , Lipoproteínas/metabolismo , Macula Lutea/citologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Células Fotorreceptoras Retinianas Cones/metabolismo , Epitélio Pigmentado da Retina/metabolismoRESUMO
Purpose: The apoptotic mechanisms responsible for secondary cone death in retinitis pigmentosa (RP) remain largely unknown. The cone-enriched apoptotic protease caspase-7 (Casp7) is thought to be triggered by endoplasmic reticulum (ER) stress and plays a pivotal role in mice deficient in the cone cyclic nucleotide-gated channels, a deficiency that causes achromatopsia in humans and in mice with autosomal dominant rhodopsin mutations, in particular the T17M mutation. Thus, we tested in two mouse models of RP whether the cone-enriched Casp7 plays a role during secondary cone death. Methods: Casp7 knockout mice were crossed to two different RP mouse models with significantly different rod and cone death kinetics: the rd1 mouse model, which carries a mutation in the Pde6b gene, and the rhodopsin knockout mouse model (Rho-KO or Rho-/- ). In both models, cone survival was assessed on retinal flat mounts by quantifying the percentage of cone arrestin staining over the retinal surface area. The analyses were performed at two different time points for each model. Results: Loss of Casp7 did not alter cone survival in either of the two mouse models tested regardless of the time point analyzed. Rod survival was also not affected in either model nor did loss of Casp7 affect rod or cone function in a wild-type background as assessed with electroretinogram analyses. Conclusions: Secondary cone death in retinitis pigmentosa is unlikely to be triggered by ER stress and is likely independent of Casp7 activity.
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Apoptose/fisiologia , Caspase 7/fisiologia , Modelos Animais de Doenças , Células Fotorreceptoras Retinianas Cones/enzimologia , Células Fotorreceptoras Retinianas Cones/patologia , Retinose Pigmentar/patologia , Animais , Arrestinas/metabolismo , Western Blotting , Sobrevivência Celular , Eletrorretinografia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retina/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/metabolismoRESUMO
Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, ß-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy.
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Proteínas do Capsídeo/genética , Sistema Nervoso Central/metabolismo , Dependovirus/fisiologia , Vetores Genéticos/genética , Músculos/metabolismo , Transdução Genética , Tropismo Viral , Animais , Proteínas do Capsídeo/química , Dependovirus/classificação , Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter , Terapia Genética , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , TransgenesRESUMO
Retinitis pigmentosa (RP) is a family of inherited retinal degenerative diseases that leads to blindness. In many cases the disease-causing allele encodes for a gene exclusively expressed in the night active rod photoreceptors. However, because rod death always leads to cone death affected individuals eventually lose their sight. Many theories have been proposed to explain the secondary loss of cones in RP; however, most fail to fully explain the different pathological transition stages seen in humans. Incorporating experimental data of rod and cone death kinetics from two mouse models of RP, we use a mathematical model to investigate the interplay and role of energy consumption and uptake of the photoreceptors as well as nutrient availability supplied through the retinal pigment epithelium (RPE) throughout the progression of RP. Our data driven mathematical model predicts that the system requires a total reduction of approximately 27-31% in nutrients available to result in the complete demise of all cones. Simulations utilizing retinal degeneration 1 (rd1) mouse cell count data in which cone death was delayed by altering cell metabolism in cones show that preventing a 1-2% decrease in nutrients available can permanently halt cone death even when 90% have already died. Our results also indicate that the ratio of energy consumption to uptake of cones, Dc, is mainly disrupted during the death wave of the rods with negligible changes thereafter and that the subsequent nutrient decrease is mainly responsible for the demise of the cones. The change in this ratio Dc highlights the compensation that the cones must undergo during rod death to meet the high metabolic demands of the entire photoreceptor population. Global sensitivity analysis confirms the results and suggests areas of focus for halting RP, even at later stages of the disease, through feasible therapeutic interventions.
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Modelos Teóricos , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/patologia , Animais , Morte Celular , Modelos Animais de Doenças , Progressão da Doença , Metabolismo Energético , Alimentos , Humanos , Cinética , Camundongos , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismoRESUMO
Cones are the primary photoreceptor (PR) cells responsible for vision in humans. They are metabolically highly active requiring phosphoinositide 3-kinase (PI3K) activity for long-term survival. One of the downstream targets of PI3K is the kinase mammalian target of rapamycin (mTOR), which is a key regulator of cell metabolism and growth, integrating nutrient availability and growth factor signals. Both PI3K and mTOR are part of the insulin/mTOR signaling pathway, however if mTOR is required for long-term PR survival remains unknown. This is of particular interest since deregulation of this pathway in diabetes results in reduced PR function before the onset of any clinical signs of diabetic retinopathy. mTOR is found in two distinct complexes (mTORC1 & mTORC2) that are characterized by their unique accessory proteins RAPTOR and RICTOR respectively. mTORC1 regulates mainly cell metabolism in response to nutrient availability and growth factor signals, while mTORC2 regulates pro-survival mechanisms in response to growth factors. Here we analyze the effect on cones of loss of mTORC1, mTORC2 and simultaneous loss of mTORC1 & mTORC2. Interestingly, neither loss of mTORC1 nor mTORC2 affects cone function or survival at one year of age. However, outer and inner segment morphology is affected upon loss of either complex. In contrast, concurrent loss of mTORC1 and mTORC2 leads to a reduction in cone function without affecting cone viability. The data indicates that PI3K mediated pro-survival signals diverge upstream of both mTOR complexes in cones, suggesting that they are independent of mTOR activity. Furthermore, the data may help explain why PR function is reduced in diabetes, which can lead to deregulation of both mTOR complexes simultaneously. Finally, although mTOR is a key regulator of cell metabolism, and PRs are metabolically highly active, the data suggests that the role of mTOR in regulating the metabolic transcriptome in healthy cones is minimal.
Assuntos
Complexos Multiproteicos/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Sobrevivência Celular , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/fisiopatologia , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho/metabolismo , Imunossupressores/uso terapêutico , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/fisiologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Sirolimo/uso terapêuticoRESUMO
Previous lineage analyses have shown that retinal progenitor cells (RPCs) are multipotent throughout development, and expression-profiling studies have shown a great deal of molecular heterogeneity among RPCs. To determine if the molecular heterogeneity predicts that an RPC will produce particular types of progeny, clonal lineage analysis was used to investigate the progeny of a subset of RPCs, those that express the basic helix-loop-helix transcription factor, Olig2. The embryonic Olig2(+) RPCs underwent terminal divisions, producing small clones with primarily two of the five cell types being made by the pool of RPCs at that time. The later, postnatal Olig2(+) RPCs also made terminal divisions, which were biased toward production of rod photoreceptors and amacrine cell interneurons. These data indicate that the multipotent progenitor pool is made up of distinctive types of RPCs, which have biases toward producing subsets of retinal neurons in a terminal division, with the types of neurons produced varying over time. This strategy is similar to that of the developing Drosophila melanogaster ventral nerve cord, with the Olig2(+) cells behaving as ganglion mother cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Retina/citologia , Células-Tronco/metabolismo , Animais , Eletroporação , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Fator de Transcrição 2 de OligodendrócitosRESUMO
Inherited retinal dystrophies caused by dominant mutations in photoreceptor (PR) cell expressed genes are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets PR cell expressed genes. Here, we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best PR accumulation. A single intravitreal administration induces multimonths efficacy in rodent and porcine retinas while demonstrating a good safety profile. The data suggest that this configuration can treat retinal diseases caused by PR cell expressed genes with 1-2 intravitreal injections per year.
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Retinitis pigmentosa is characterized by loss of night vision, followed by complete blindness. Over 40 genetic loci for retinitis pigmentosa have been identified in humans, primarily affecting photoreceptor structure and function. The availability of excellent animal models allows for a mechanistic characterization of the disease. Metabolic dysregulation and oxidative stress have been found to correlate with the loss of vision, particularly in cones, the type of photoreceptors that mediate daylight and color vision. The evidence that these problems actually cause loss of vision and potential therapeutic approaches targeting them are discussed.
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Cegueira/metabolismo , Visão de Cores , Estresse Oxidativo , Retinose Pigmentar/metabolismo , Animais , Cegueira/genética , Cegueira/patologia , Cegueira/terapia , Loci Gênicos , Humanos , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Retinose Pigmentar/terapiaRESUMO
Purpose: To select individuals and families with a low genetic burden for age-related macular degeneration (AMD), to inform the clinical diagnosis of macular disorders, and to find novel genetic variants associated with maculopathies. Design: Genetic association study based on targeted and whole-exome sequencing. Participants: A total of 758 subjects (481 individuals with maculopathy and 277 controls), including 316 individuals in 72 families. Methods: We focused on 150 genes involved in the complement, coagulation, and inflammatory pathways. Single-variant tests were performed on 7755 variants shared among ≥ 5 subjects using logistic regression. Gene-based tests were used to evaluate aggregate effects from rare and low-frequency variants (at minor allele frequency [MAF] ≤ 5% or ≤ 1%) in a gene using burden tests. For families whose affected members had a low burden of genetic risk based on known common and rare variants related to AMD, we searched for rare variants (MAF < 0.001) whose risk alleles occurred in ≥ 80% of affected individuals but not in controls. Immunohistochemistry was performed to determine the protein expression of a novel gene (coagulation factor II thrombin receptor-like 2 [F2RL2]) in retinal tissues. Main Outcome Measures: Genotypes and phenotypes of macular degeneration. Results: We confirmed the association of a synonymous variant in complement factor H (Ala473, rs2274700, proxy to intronic rs1410996, r 2 = 1) with maculopathy (odds ratio, 0.64; P = 4.5 × 10-4). Higher AMD polygenic risk scores (PRSs) were associated with intermediate and advanced AMD. Among families with low PRSs and no known rare variants for maculopathy, we identified 2 novel, highly penetrant missense rare variants in ADAM15, A disintegrin and metalloprotease, metallopeptidase domain 15 (p.Arg288Cys) and F2RL2 (p.Leu289Arg). Immunohistochemistry analyses revealed F2RL2 protein expression in cone photoreceptor outer segments and Müller glia cells of human and pig retinas. Coagulation factor II thrombin receptor-like 2 expression appeared increased in fibrotic areas in advanced AMD samples with neovascularization, suggesting that F2RL2 may play a role in the progression to advanced macular disease. Conclusions: New missense rare variants in the genes ADAM15 and F2RL2 were associated with maculopathies. Results suggest that novel genes related to the coagulation and immune pathways may be involved in the pathogenesis of macular diseases.
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Familial dysautonomia (FD) is a rare neurodevelopmental and neurodegenerative disease caused by a splicing mutation in the Elongator Acetyltransferase Complex Subunit 1 (ELP1) gene. The reduction in ELP1 mRNA and protein leads to the death of retinal ganglion cells (RGCs) and visual impairment in all FD patients. Currently patient symptoms are managed, but there is no treatment for the disease. We sought to test the hypothesis that restoring levels of Elp1 would thwart the death of RGCs in FD. To this end, we tested the effectiveness of two therapeutic strategies for rescuing RGCs. Here we provide proof-of-concept data that gene replacement therapy and small molecule splicing modifiers effectively reduce the death of RGCs in mouse models for FD and provide pre-clinical foundational data for translation to FD patients.
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Disautonomia Familiar , Doenças Neurodegenerativas , Camundongos , Animais , Humanos , Células Ganglionares da Retina/metabolismo , Disautonomia Familiar/genética , Disautonomia Familiar/terapia , Disautonomia Familiar/metabolismo , Doenças Neurodegenerativas/metabolismo , Splicing de RNA , Terapia Genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Inherited retinal dystrophies caused by dominant mutations in photoreceptor-expressed genes, are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets photoreceptor-expressed genes. Here we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best photoreceptor accumulation. A single intravitreal administration induces multi-months efficacy in rodent and porcine retinas while showing a good safety profile. The data suggest that this configuration can treat retinal diseases caused by photoreceptor-expressed genes with 1-2 intravitreal injections per year.
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Familial dysautonomia (FD) is a rare neurodevelopmental and neurodegenerative disease caused by a splicing mutation in the Elongator Acetyltransferase Complex Subunit 1 ( ELP1 ) gene. The reduction in ELP1 mRNA and protein leads to the death of retinal ganglion cells (RGCs) and visual impairment in all FD patients. Currently, patient symptoms are managed, but there is no treatment for the disease. We sought to test the hypothesis that restoring levels of Elp1 would thwart the death of RGCs in FD. To this end, we tested the effectiveness of two therapeutic strategies for rescuing RGCs. Here we provide proof-of-concept data that gene replacement therapy and small molecule splicing modifiers effectively reduce the death of RGCs in mouse models for FD and provide pre-clinical data foundation for translation to FD patients.
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Introduction: Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. One-third of the genetic contribution to this disease remains unexplained. Methods: We analyzed targeted sequencing data from two independent cohorts (4,245 cases, 1,668 controls) which included genomic regions of known AMD loci in 49 genes. Results: At a false discovery rate of <0.01, we identified 11 low-frequency AMD variants (minor allele frequency <0.05). Two of those variants were present in the complement C4A gene, including the replacement of the residues that contribute to the Rodgers-1/Chido-1 blood group antigens: [VDLL1207-1210ADLR (V1207A)] with discovery odds ratio (OR) = 1.7 (p = 3.2 × 10-5) which was replicated in the UK Biobank dataset (3,294 cases, 200,086 controls, OR = 1.52, p = 0.037). A novel variant associated with reduced risk for AMD in our discovery cohort was P1120T, one of the four C4A-isotypic residues. Gene-based tests yielded aggregate effects of nonsynonymous variants in 10 genes including C4A, which were associated with increased risk of AMD. In human eye tissues, immunostaining demonstrated C4A protein accumulation in and around endothelial cells of retinal and choroidal vasculature, and total C4 in soft drusen. Conclusion: Our results indicate that C4A protein in the complement activation pathways may play a role in the pathogenesis of AMD.
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Exposure to daylight closes cyclic nucleotide-gated (CNG) and voltage-operated Ca(2+) -permeable channels in mammalian rods. The consequent lowering of the cytosolic calcium concentration ([Ca(2+)](i)), if protracted, can contribute to light-induced damage and apoptosis in these cells. We here report that mouse rods are protected against prolonged lowering of [Ca(2+)](i) by store-operated Ca(2+) entry (SOCE). Ca(2+) stores were depleted in Ca(2+)-free saline supplemented with the endoplasmic reticulum (ER) sequestration blocker cyclopiazonic acid. Store depletion elicited [Ca(2+)](i) signals that exceeded baseline [Ca(2+)](i) by 5.9 ± 0.7-fold and were antagonized by an inhibitory cocktail containing 2-APB, SKF 96365 and Gd(3+). Cation influx through SOCE channels was sufficient to elicit a secondary activation of L-type voltage-operated Ca2+ entry. We also found that TRPC1, the type 1 canonical mammalian homologue of the Drosophila photoreceptor TRP channel, is predominantly expressed within the outer nuclear layer of the retina. Rod loss in Pde6b(rdl) (rd1), Chx10/Kip1(-/-rdl) and Elovl4(TG2) dystrophic models was associated with â¼70% reduction in Trpc1 mRNA content whereas Trpc1 mRNA levels in rodless cone-full Nrl(-/-) retinas were decreased by â¼50%. Genetic ablation of TRPC1 channels, however, had no effect on SOCE, the sensitivity of the rod phototransduction cascade or synaptic transmission at rod and cone synapses. Thus, we localized two new mechanisms, SOCE and TRPC1, to mammalian rods and characterized the contribution of SOCE to Ca(2+) homeostasis. By preventing the cytosolic [Ca(2+)](i) from dropping too low under sustained saturating light conditions, these signalling pathways may protect Ca(2+)-dependent mechanisms within the ER and the cytosol without affecting normal rod function.
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Canais de Cálcio/fisiologia , Cálcio/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Canais de Cátion TRPC/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas do Olho/genética , Homeostase , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Células Fotorreceptoras Retinianas Cones/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
In 2001, the first large animal was successfully treated with a gene therapy that restored its vision. Lancelot, the Briard dog that was treated, suffered from a human childhood blindness called Leber's congenital amaurosis type 2. Sixteen years later, the gene therapy was approved by the U.S. Food and Drug Administration. The success of this gene therapy in dogs led to a fast expansion of the ocular gene therapy field. By now every class of inherited retinal dystrophy has been treated in at least one animal model and many clinical trials have been initiated in humans. In this study, we review the status of viral gene therapies for the retina, with a focus on ongoing human clinical trials. It is likely that in the next decade we will see several new viral gene therapies approved.
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Amaurose Congênita de Leber , Doenças Retinianas , Distrofias Retinianas , Animais , Cegueira/genética , Cegueira/terapia , Criança , Cães , Genes Virais , Terapia Genética , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/terapia , Retina , Doenças Retinianas/genética , Doenças Retinianas/terapia , Distrofias Retinianas/terapiaRESUMO
The DBA/2J (D2) and C57BL6 (B6) mouse strains are widely used in research as models for anxiety, addiction and chronic glaucoma. D2, but not B6, animals develop elevated intraocular pressure (IOP) that leads to progressive degeneration of retinal ganglion cell (RGC) axons and perikarya. Here we compare the expression and localization of intracellular ryanodine receptor (RyR) Ca(2+) store mechanisms in retinas from D2 and B6 animals. A subset of experiments included retinas from D2-Gpnmb(+) mice as strain-specific controls for D2s. RT-PCR analysis showed 6-8 -fold upregulation RyR1, but not RyR2 or RyR3 transcripts, in D2 retinas. The upregulation was more pronounced in D2 retinas categorized as exhibiting moderate or severe glaucoma eyes compared to eyes with no/little glaucoma. In B6 retinas, RyR1 was expressed in neuronal perikarya/processes across all three retinal layers whereas little labeling was observed in astrocyte, microglial or Müller cell processes. In contrast, RyR1 antibodies strongly labeled radial processes of in D2 Müller glia, in which the staining colocalized with the activated glial stress marker GFAP. RyR1 staining in 1 month-old D2-Gpnmb(+) strain resembled expression in B6 retinas whereas moderate RyR1, but not GFAP, localization to Müller glia was observed in 10-12 months - old D2-Gpnmb(+) eyes. Both RyR1-ir and GFAP-ir were augmented in the microbead injection model of acute experimental glaucoma. We conclude that RyR1 exhibits differential expression and localization in two ubiquitously used mouse lines. While RyR1 signals can be regulated in a strain-specific manner, our data also suggest that RyR1 transcription is induced by early glial activation and/or elevation in intraocular pressure.
Assuntos
Regulação da Expressão Gênica/fisiologia , Retina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Linhagem Celular , Técnica Indireta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos Específicos , Tonometria Ocular , Regulação para CimaRESUMO
Homeotic Hox selector genes encode highly conserved transcriptional regulators involved in the differentiation of multicellular organisms. Ectopic expression of the Antennapedia (ANTP) homeodomain protein in Drosophila imaginal discs induces distinct phenotypes, including an antenna-to-leg transformation and eye reduction. We have proposed that the eye loss phenotype is a consequence of a negative posttranslational control mechanism because of direct protein-protein interactions between ANTP and Eyeless (EY). In the present work, we analyzed the effect of various ANTP homeodomain mutations for their interaction with EY and for head development. Contrasting with the eye loss phenotype, we provide evidence that the antenna-to-leg transformation involves ANTP DNA-binding activity. In a complementary genetic screen performed in yeast, we isolated mutations located in the N terminus of the ANTP homeodomain that inhibit direct interactions with EY without abolishing DNA binding in vitro and in vivo. In a bimolecular fluorescence complementation assay, we detected the ANTP-EY interaction in vivo, these interactions occurring through the paired domain and/or the homeodomain of EY. These results demonstrate that the homeodomain supports multiple molecular regulatory functions in addition to protein-DNA and protein-RNA interactions; it is also involved in protein-protein interactions.