RESUMO
Chronic inflammation is a constitutive component of many age-related diseases, including age-related macular degeneration (AMD). Here, we identified interleukin-1 receptor-associated kinase M (IRAK-M) as a key immunoregulator in retinal pigment epithelium (RPE) that declines during the aging process. Rare genetic variants of IRAK3, which encodes IRAK-M, were associated with an increased likelihood of developing AMD. In human samples and mouse models, IRAK-M abundance in the RPE declined with advancing age or exposure to oxidative stress and was further reduced in AMD. Irak3-knockout mice exhibited an increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M led to a disruption in RPE cell homeostasis, characterized by compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of adeno-associated virus (AAV)-expressing human IRAK3 rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in Irak3-knockout mice. Our data show that replenishment of IRAK-M in the RPE may redress dysregulated pro-inflammatory processes in AMD, suggesting a potential treatment for retinal degeneration.
Assuntos
Quinases Associadas a Receptores de Interleucina-1 , Camundongos Knockout , Estresse Oxidativo , Degeneração Retiniana , Epitélio Pigmentado da Retina , Animais , Humanos , Masculino , Camundongos , Senescência Celular , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/genética , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologiaAssuntos
Hemorragia Retiniana , Epitélio Pigmentado da Retina , Tomografia de Coerência Óptica , Acuidade Visual , Degeneração Macular Exsudativa , Humanos , Degeneração Macular Exsudativa/diagnóstico , Hemorragia Retiniana/diagnóstico , Hemorragia Retiniana/etiologia , Hemorragia Retiniana/cirurgia , Doença Aguda , Epitélio Pigmentado da Retina/transplante , Epitélio Pigmentado da Retina/patologia , Masculino , Seguimentos , Feminino , Angiofluoresceinografia/métodos , Idoso , Células-Tronco Embrionárias Humanas/transplante , Fatores de Tempo , Gravidade do Paciente , Transplante de Células-Tronco/métodosRESUMO
Unchecked, chronic inflammation is a constitutive component of age-related diseases, including age-related macular degeneration (AMD). Here we identified interleukin-1 receptor-associated kinase (IRAK)-M as a key immunoregulator in retinal pigment epithelium (RPE) that declines with age. Rare genetic variants of IRAK-M increased the likelihood of AMD. IRAK-M expression in RPE declined with age or oxidative stress and was further reduced in AMD. IRAK-M-deficient mice exhibited increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M disrupted RPE cell homeostasis, including compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of AAV-expressing IRAK-M rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in IRAK-M-deficient mice. Our data support that replenishment of IRAK-M expression may redress dysregulated pro-inflammatory processes in AMD, thereby treating degeneration.
RESUMO
War and conflict are global phenomena, identified as stress-inducing triggers for epigenetic modifications. In this state-of-the-science narrative review based on systematic principles, we summarise existing data to explore the outcomes of these exposures especially in veterans and show that they may result in an increased likelihood of developing gastrointestinal, auditory, metabolic and circadian issues, as well as post-traumatic stress disorder (PTSD). We also note that, despite a potential "healthy soldier effect", both veterans and civilians with PTSD exhibit the altered DNA methylation status in hypothalamic-pituitary-adrenal (HPA) axis regulatory genes such as NR3C1. Genes associated with sleep (PAX8; LHX1) are seen to be differentially methylated in veterans. A limited number of studies also revealed hereditary effects of war exposure across groups: decreased cortisol levels and a heightened (sex-linked) mortality risk in offspring. Future large-scale studies further identifying the heritable risks of war, as well as any potential differences between military and civilian populations, would be valuable to inform future healthcare directives.
RESUMO
Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKß/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKß/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKß is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKß signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKß is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKß signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK.
Assuntos
Actomiosina , Miotonina Proteína Quinase , Fagocitose , Actinas/metabolismo , Actomiosina/metabolismo , Miosina Tipo II/metabolismo , Miotonina Proteína Quinase/metabolismo , Fagocitose/fisiologia , Proteínas Tirosina Quinases , Receptores Fc , c-Mer Tirosina Quinase/metabolismoRESUMO
Leber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents.
Assuntos
Amaurose Congênita de Leber , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/genética , Criança , Códon sem Sentido , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Guanosina Monofosfato , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/terapia , Organoides/metabolismo , Oxidiazóis , Diester Fosfórico Hidrolases/genéticaRESUMO
Diabetic retinopathy is the most common microvascular complication of diabetes and is a major cause of blindness, but an understanding of the pathogenesis of the disease has been hampered by a lack of accurate animal models. Here, we explore the dynamics of retinal cellular changes in the Nile rat (Arvicanthis niloticus), a carbohydrate-sensitive model for type 2 diabetes. The early retinal changes in diabetic Nile rats included increased acellular capillaries and loss of pericytes that correlated linearly with the duration of diabetes. These vascular changes occurred in the presence of microglial infiltration but in the absence of retinal ganglion cell loss. After a prolonged duration of diabetes, the Nile rat also exhibits a spectrum of retinal lesions commonly seen in the human condition including vascular leakage, capillary non-perfusion, and neovascularization. Our longitudinal study documents a range and progression of retinal lesions in the diabetic Nile rat remarkably similar to those observed in human diabetic retinopathy, and suggests that this model will be valuable in identifying new therapeutic strategies.
Assuntos
Capilares/patologia , Retinopatia Diabética/patologia , Retina/patologia , Animais , Progressão da Doença , Edema/patologia , Estudos Longitudinais , MurinaeRESUMO
Choroideremia (CHM) is an x-linked recessive chorioretinal dystrophy, with 30% caused by nonsense mutations in the CHM gene resulting in an in-frame premature termination codon (PTC). Nonsense-mediated mRNA decay (NMD) is the cell's natural surveillance mechanism that detects and destroys PTC-containing transcripts, with UPF1 being the central NMD modulator. NMD efficiency can be variable amongst individuals with some transcripts escaping destruction, leading to the production of a truncated non-functional or partially functional protein. Nonsense suppression drugs, such as ataluren, target these transcripts and read-through the PTC, leading to the production of a full length functional protein. Patients with higher transcript levels are considered to respond better to these drugs, as more substrate is available for read-through. Using Quantitative reverse transcription PCR (RT-qPCR), we show that CHM mRNA expression in blood from nonsense mutation CHM patients is 2.8-fold lower than controls, and varies widely amongst patients, with 40% variation between those carrying the same UGA mutation [c.715 C>T; p.(R239*)]. These results indicate that although NMD machinery is at work, efficiency is highly variable and not wholly dependent on mutation position. No significant difference in CHM mRNA levels was seen between two patients' fibroblasts and their induced pluripotent stem cell-derived retinal pigment epithelium. There was no correlation between CHM mRNA expression and genotype, phenotype or UPF1 transcript levels. NMD inhibition with caffeine was shown to restore CHM mRNA transcripts to near wild-type levels. Baseline mRNA levels may provide a prognostic indicator for response to nonsense suppression therapy, and caffeine may be a useful adjunct to enhance treatment efficacy where indicated.
Assuntos
Coroideremia/tratamento farmacológico , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Helicases/genética , RNA Mensageiro/sangue , Transativadores/genética , Cafeína/administração & dosagem , Coroideremia/sangue , Coroideremia/genética , Coroideremia/fisiopatologia , Códon sem Sentido/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Degradação do RNAm Mediada por Códon sem Sentido/efeitos dos fármacos , Oxidiazóis/administração & dosagem , Fenótipo , Células-Tronco Pluripotentes/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismoRESUMO
Age-related macular degeneration (AMD) remains a major cause of blindness, with dysfunction and loss of retinal pigment epithelium (RPE) central to disease progression. We engineered an RPE patch comprising a fully differentiated, human embryonic stem cell (hESC)-derived RPE monolayer on a coated, synthetic basement membrane. We delivered the patch, using a purpose-designed microsurgical tool, into the subretinal space of one eye in each of two patients with severe exudative AMD. Primary endpoints were incidence and severity of adverse events and proportion of subjects with improved best-corrected visual acuity of 15 letters or more. We report successful delivery and survival of the RPE patch by biomicroscopy and optical coherence tomography, and a visual acuity gain of 29 and 21 letters in the two patients, respectively, over 12 months. Only local immunosuppression was used long-term. We also present the preclinical surgical, cell safety and tumorigenicity studies leading to trial approval. This work supports the feasibility and safety of hESC-RPE patch transplantation as a regenerative strategy for AMD.
Assuntos
Células-Tronco Embrionárias Humanas/transplante , Degeneração Macular/terapia , Epitélio Pigmentado da Retina/transplante , Acuidade Visual/fisiologia , Idoso , Animais , Membrana Basal/diagnóstico por imagem , Membrana Basal/crescimento & desenvolvimento , Diferenciação Celular/genética , Feminino , Humanos , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Epitélio Pigmentado da Retina/diagnóstico por imagem , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Transplante de Células-Tronco/efeitos adversos , Suínos , Tomografia de Coerência ÓpticaRESUMO
Ciliary trafficking defects are the underlying cause of many ciliopathies, including Retinitis Pigmentosa (RP). Anterograde intraflagellar transport (IFT) is mediated by kinesin motor proteins; however, the function of the homodimeric Kif17 motor in cilia is poorly understood, whereas Kif7 is known to play an important role in stabilizing cilia tips. Here we identified the ciliary tip kinesins Kif7 and Kif17 as novel interaction partners of the small GTPase Arl3 and its regulatory GTPase activating protein (GAP) Retinitis Pigmentosa 2 (RP2). We show that Arl3 and RP2 mediate the localization of GFP-Kif17 to the cilia tip and competitive binding of RP2 and Arl3 with Kif17 complexes. RP2 and Arl3 also interact with another ciliary tip kinesin, Kif7, which is a conserved regulator of Hedgehog (Hh) signaling. siRNA-mediated loss of RP2 or Arl3 reduced the level of Kif7 at the cilia tip. This was further validated by reduced levels of Kif7 at cilia tips detected in fibroblasts and induced pluripotent stem cell (iPSC) 3D optic cups derived from a patient carrying an RP2 nonsense mutation c.519C > T (p.R120X), which lack detectable RP2 protein. Translational read-through inducing drugs (TRIDs), such as PTC124, were able to restore Kif7 levels at the ciliary tip of RP2 null cells. Collectively, our findings suggest that RP2 and Arl3 regulate the trafficking of specific kinesins to cilia tips and provide additional evidence that TRIDs could be clinically beneficial for patients with this retinal degeneration.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas do Olho/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Ribosilação do ADP/genética , Cílios/metabolismo , Proteínas do Olho/genética , Proteínas de Ligação ao GTP , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesinas/genética , Cinesinas/metabolismo , Proteínas de Membrana/genética , Transporte Proteico , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismoRESUMO
Inherited retinal dystrophies are an important cause of blindness, for which currently there are no effective treatments. In order to study this heterogeneous group of diseases, adequate disease models are required in order to better understand pathology and to test potential therapies. Induced pluripotent stem cells offer a new way to recapitulate patient specific diseases in vitro, providing an almost limitless amount of material to study. We used fibroblast-derived induced pluripotent stem cells to generate retinal pigment epithelium (RPE) from an individual suffering from retinitis pigmentosa associated with biallelic variants in MERTK. MERTK has an essential role in phagocytosis, one of the major functions of the RPE. The MERTK deficiency in this individual results from a nonsense variant and so the MERTK-RPE cells were subsequently treated with two translational readthrough inducing drugs (G418 & PTC124) to investigate potential restoration of expression of the affected gene and production of a full-length protein. The data show that PTC124 was able to reinstate phagocytosis of labeled photoreceptor outer segments at a reduced, but significant level. These findings represent a confirmation of the usefulness of iPSC derived disease specific models in investigating the pathogenesis and screening potential treatments for these rare blinding disorders.
Assuntos
Gentamicinas/farmacologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Oxidiazóis/farmacologia , Fagocitose , Retinose Pigmentar/terapia , c-Mer Tirosina Quinase/metabolismo , Adulto , Humanos , Masculino , Elongação Traducional da Cadeia Peptídica , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , c-Mer Tirosina Quinase/genéticaRESUMO
The photoreceptor cells in the retina have a highly specialised sensory cilium, the outer segment (OS), which is important for detecting light. Mutations in cilia-related genes often result in retinal degeneration. The ability to reprogramme human cells into induced pluripotent stem cells and then differentiate them into a wide range of different cell types has revolutionised our ability to study human disease. To date, however, the challenge of producing fully differentiated photoreceptors in vitro has limited the application of this technology in studying retinal degeneration. In this review, we will discuss recent advances in stem cell technology and photoreceptor differentiation. In particular, the development of photoreceptors with rudimentary OS that can be used to understand disease mechanisms and as an important model to test potential new therapies for inherited retinal ciliopathies.
Assuntos
Ciliopatias/patologia , Ciliopatias/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Retina/patologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Cílios/metabolismo , Cílios/patologia , Ciliopatias/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Degeneração Retiniana/terapiaRESUMO
Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare, early-onset retinal dystrophy characterised by distinct bands of circumferential pigmentary degeneration in the peripheral retina and developmental eye defects. ADVIRC is caused by mutations in the Bestrophin1 (BEST1) gene, which encodes a transmembrane protein thought to function as an ion channel in the basolateral membrane of retinal pigment epithelial (RPE) cells. Previous studies suggest that the distinct ADVIRC phenotype results from alternative splicing of BEST1 pre-mRNA. Here, we have used induced pluripotent stem cell (iPSC) technology to investigate the effects of an ADVIRC associated BEST1 mutation (c.704T > C, p.V235A) in patient-derived iPSC-RPE. We found no evidence of alternate splicing of the BEST1 transcript in ADVIRC iPSC-RPE, however in patient-derived iPSC-RPE, BEST1 was expressed at the basolateral membrane and the apical membrane. During human eye development we show that BEST1 is expressed more abundantly in peripheral RPE compared to central RPE and is also expressed in cells of the developing retina. These results suggest that higher levels of mislocalised BEST1 expression in the periphery, from an early developmental stage, could provide a mechanism that leads to the distinct clinical phenotype observed in ADVIRC patients.
RESUMO
Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290, which causes missplicing and premature termination, but the basis of this sensitivity is unclear. Here, we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups, despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups, explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290, restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention.
Assuntos
Cegueira/patologia , Cegueira/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Padrões de Herança/genética , Disco Óptico/citologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Cílios/efeitos dos fármacos , Cílios/metabolismo , Proteínas do Citoesqueleto , Éxons/genética , Proteínas do Olho/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Amaurose Congênita de Leber/patologia , Masculino , Morfolinos/farmacologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Opsinas/metabolismo , Organogênese/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/ultraestrutura , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/ultraestrutura , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Retinal degeneration arises from the loss of photoreceptors or retinal pigment epithelium (RPE). It is one of the leading causes of irreversible blindness worldwide with limited effective treatment options. Generation of induced pluripotent stem cell (IPSC)-derived retinal cells and tissues from individuals with retinal degeneration is a rapidly evolving technology that holds a great potential for its use in disease modelling. IPSCs provide an ideal platform to investigate normal and pathological retinogenesis, but also deliver a valuable source of retinal cell types for drug screening and cell therapy. In this review, we will provide some examples of the ways in which IPSCs have been used to model diseases of the outer retina including retinitis pigmentosa (RP), Usher syndrome (USH), Leber congenital amaurosis (LCA), gyrate atrophy (GA), juvenile neuronal ceroid lipofuscinosis (NCL), Best vitelliform macular dystrophy (BVMD) and age related macular degeneration (AMD).
RESUMO
Affinity reagent pairs that recognize distinct epitopes on a target protein can greatly improve the sensitivity and specificity of molecular detection. Importantly, such pairs can be conjugated to generate reagents that achieve two-site "bidentate" target recognition, with affinities greatly exceeding either monovalent component. DNA aptamers are especially well-suited for such constructs, because they can be linked via standard synthesis techniques without requiring chemical conjugation. Unfortunately, aptamer pairs are difficult to generate, primarily because conventional selection methods preferentially yield aptamers that recognize a dominant "hot spot" epitope. Our array-based discovery platform for multivalent aptamers (AD-MAP) overcomes this problem to achieve efficient discovery of aptamer pairs. We use microfluidic selection and high-throughput sequencing to obtain an enriched pool of aptamer sequences. Next, we synthesize a custom array based on these sequences, and perform parallel affinity measurements to identify the highest-affinity aptamer for the target protein. We use this aptamer to form complexes that block the primary binding site on the target, and then screen the same array with these complexes to identify aptamers that bind secondary epitopes. We used AD-MAP to discover DNA aptamer pairs that bind distinct sites on human angiopoietin-2 with high affinities, even in undiluted serum. To the best of our knowledge, this is the first work to discover new aptamer pairs using arrays. We subsequently conjugated these aptamers with a flexible linker to construct ultra-high-affinity bidentate reagents, with equilibrium dissociation constants as low as 97 pM: >200-fold better than either component aptamer. Functional studies confirm that both aptamers critically contribute to this ultrahigh affinity, highlighting the promise of such reagents for research and clinical use.
Assuntos
Angiopoietina-2/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Microfluídica/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Técnica de Seleção de Aptâmeros/métodos , Angiopoietina-2/genética , Aptâmeros de Nucleotídeos/química , Sítios de Ligação , Fluorescência , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study, we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519C>T (p.R120X) into induced pluripotent stem cells (iPSC), and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells, suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization, Golgi cohesion and Gß1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren), we were able to restore up to 20% of endogenous, full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Células-Tronco Pluripotentes Induzidas/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Mutação , Biossíntese de Proteínas , Epitélio Pigmentado da Retina/citologia , Diferenciação Celular , Reprogramação Celular , Cílios/metabolismo , Cílios/patologia , Proteínas do Olho/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Oxidiazóis/farmacologia , Fenótipo , Biossíntese de Proteínas/efeitos dos fármacos , Transporte Proteico , Adulto JovemRESUMO
Human embryonic stem cells (hESCs) offer a potentially unlimited supply of cells for emerging cell-based therapies. Unfortunately, the process of deriving distinct cell types can be time consuming and expensive. In the developed world, age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with more than 7.2 million people afflicted in the U.S. alone. Both hESC-derived retinal pigmented epithelium (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) are being developed for AMD therapies by multiple groups, but their potential for expansion in culture is limited. To attempt to overcome this passage limitation, we examined the involvement of Rho-associated, coiled-coil protein kinase (ROCK) in hESC-RPE and iPSC-RPE culture. We report that inhibiting ROCK1/2 with Y-27632 allows extended passage of hESC-RPE and iPSC-RPE. Microarray analysis suggests that ROCK inhibition could be suppressing an epithelial-to-mesenchymal transition through various pathways. These include inhibition of key ligands of the transforming growth factor-ß pathway (TGFB1 and GDF6) and Wnt signaling. Two important processes are affected, allowing for an increase in hESC-RPE expansion. First, ROCK inhibition promotes proliferation by inducing multiple components that are involved in cell cycle progression. Second, ROCK inhibition affects many pathways that could be converging to suppress RPE-to-mesenchymal transition. This allows hESC-RPE to remain functional for an extended but finite period in culture.