RESUMO
Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6ßrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.
Assuntos
Modelos Animais de Doenças , Reposicionamento de Medicamentos , Retinose Pigmentar , Animais , Camundongos , Cães , Retinose Pigmentar/tratamento farmacológico , Retinose Pigmentar/genética , Mutação , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Camundongos Knockout , Amaurose Congênita de Leber/tratamento farmacológico , Amaurose Congênita de Leber/genética , Bromocriptina/farmacologia , Bromocriptina/uso terapêutico , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo , Humanos , Quimioterapia Combinada , Camundongos Endogâmicos C57BL , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Feminino , AMP Cíclico/metabolismo , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/genética , Masculino , Cálcio/metabolismoRESUMO
Animal models of retinal degeneration are critical for understanding disease and testing potential therapies. Inducing degeneration commonly involves the administration of chemicals that kill photoreceptors by disrupting metabolic pathways, signaling pathways, or protein synthesis. While chemically induced degeneration has been demonstrated in a variety of animals (mice, rats, rabbits, felines, 13-lined ground squirrels (13-LGS), pigs, chicks), few studies have used noninvasive high-resolution retinal imaging to monitor the in vivo cellular effects. Here, we used longitudinal scanning light ophthalmoscopy (SLO), optical coherence tomography, and adaptive optics SLO imaging in the euthermic, cone-dominant 13-LGS (46 animals, 52 eyes) to examine retinal structure following intravitreal injections of chemicals, which were previously shown to induce photoreceptor degeneration, throughout the active season of 2019 and 2020. We found that iodoacetic acid induced severe pan-retinal damage in all but one eye, which received the lowest concentration. While sodium nitroprusside successfully induced degeneration of the outer retinal layers, the results were variable, and damage was also observed in 50% of contralateral control eyes. Adenosine triphosphate and tunicamycin induced outer retinal specific damage with varying results, while eyes injected with thapsigargin did not show signs of degeneration. Given the variability of damage we observed, follow-up studies examining the possible physiological origins of this variability are critical. These additional studies should further advance the utility of chemically induced photoreceptor degeneration models in the cone-dominant 13-LGS.
Assuntos
Células Fotorreceptoras Retinianas Cones , Degeneração Retiniana , Sciuridae , Tomografia de Coerência Óptica , Animais , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Modelos Animais de Doenças , Injeções Intravítreas , Oftalmoscopia , Nitroprussiato/farmacologia , Feminino , MasculinoRESUMO
Highly expressed in the retinal pigment epithelium (RPE), the RPE-specific 65-kDa (RPE65) enzyme is indispensable to generate 11-cis-retinal (11cRAL), a chromophore for rhodopsin and cone photopigments. RPE65 deficiency can lead to Leber congenital amaurosis type 2 (LCA2), in which the isomerization of photobleached all-trans-retinal into photosensitive 11cRAL is blocked, ultimately causing severe retinal dysfunction and degeneration. The related mouse models, which are constructed through gene knockout or caused by spontaneous mutations, morphologically present with early-onset and rapid retinal cone cells degeneration, including loss of short-wavelength-sensitive cone opsins (S-opsins) and mislocalization of medium-wavelength-sensitive cone opsins (M-opsins). Studies have shown that routine Rpe65 gene replacement therapy, mediated by an adeno-associated virus (AAV) vector, can restore RPE65 protein. However, AAV transfection and Rpe65 transgene expression require at least one to two weeks, and the treatment cannot fully block the early-onset cone degeneration. To determine the feasibility of delaying cone degeneration before gene therapy, we investigated the impact of 11cRAL treatment in an early-age LCA2 retinal degeneration 12 (rd12) mouse model. Similar to human patients, the mouse model carries a spontaneous mutation in the Rpe65 gene, which results in disrupted endogenous 11cRAL regeneration. We found that RPE65 deficiency did not notably affect rodent retinal vessels. Under red light illumination, the rd12 mice were intraperitoneally injected with exogenous 11cRAL from postnatal day (P) 14 to P21. Three days after the last injection, a notable recovery of retinal function was observed using scotopic and photopic electroretinograms. Using optical coherence tomography and histological analyses of the deficient retinas, we found changes in the thickness of the photoreceptor outer segment (OS); this change could be rescued by early 11cRAL treatment. In addition, the treatment notably preserved M- and S-opsins, both of which maintained appropriate localization inside cone cells, as shown by the wild-type mice. In contrast, the age-matched untreated rd12 mice were characterized by retinal S-opsin loss and M-opsin mislocalization from the photoreceptor OS to the inner segment, outer nuclear layer, or outer plexiform layer. Notably, 11cRAL treatment could not maintain retinal function for a long time. Ten days after the last injection, the rod and M-cone electroretinograms significantly decreased, and S-cone responses almost extinguished. Our findings suggest that early 11cRAL treatment is useful for restoring retinal function and rescuing morphology in the rd12 mouse model, and the early-onset and rapid cone degeneration can be delayed before gene therapy.
Assuntos
Amaurose Congênita de Leber/tratamento farmacológico , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/etiologia , Retinaldeído/administração & dosagem , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eletrorretinografia , Injeções Intraperitoneais , Amaurose Congênita de Leber/complicações , Amaurose Congênita de Leber/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/diagnóstico , Degeneração Retiniana/metabolismo , Tomografia de Coerência Óptica/métodosRESUMO
Inherited retinal dystrophies (IRDs) are a group of rare eye diseases caused by gene mutations that result in the degradation of cone and rod photoreceptors or the retinal pigment epithelium. Retinal degradation progress is often irreversible, with clinical manifestations including color or night blindness, peripheral visual defects and subsequent vision loss. Thus, gene therapies that restore functional retinal proteins by either replenishing unmutated genes or truncating mutated genes are needed. Coincidentally, the eye's accessibility and immune-privileged status along with major advances in gene identification and gene delivery systems heralded gene therapies for IRDs. Among these clinical trials, voretigene neparvovec-rzyl (Luxturna), an adeno-associated virus vector-based gene therapy drug, was approved by the FDA for treating patients with confirmed biallelic RPE65 mutation-associated Leber Congenital Amaurosis (LCA) in 2017. This review includes current IRD gene therapy clinical trials and further summarizes preclinical studies and therapeutic strategies for LCA, including adeno-associated virus-based gene augmentation therapy, 11-cis-retinal replacement, RNA-based antisense oligonucleotide therapy and CRISPR-Cas9 gene-editing therapy. Understanding the gene therapy development for LCA may accelerate and predict the potential hurdles of future therapeutics translation. It may also serve as the template for the research and development of treatment for other IRDs.
Assuntos
Amaurose Congênita de Leber/genética , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Dependovirus/genética , Proteínas do Olho/genética , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Humanos , Amaurose Congênita de Leber/terapia , Mutação , RNA , Retina/efeitos dos fármacos , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismoRESUMO
Rhegmatogenous retinal detachment (RD) is a threatening visual condition and a human disease model for retinal degenerations. Despite successful reattachment surgery, vision does not fully recover, due to subretinal fluid accumulation and subsequent photoreceptor cell death, through mechanisms that recapitulate those of retinal degenerative diseases. Hydrophilic bile acids are neuroprotective in animal models, but whether they can be used orally for retinal diseases is unknown. Ursodeoxycholic acid (UDCA) being approved for clinical use (e.g., in cholestasis), we have evaluated the ocular bioavailability of oral UDCA, administered to patients before RD surgery. The level of UDCA in ocular media correlated with the extent of blood retinal barrier disruption, evaluated by the extent of detachment and the albumin concentration in subretinal fluid. UDCA, at levels measured in ocular media, protected photoreceptors from apoptosis and necrosis in rat retinal explants, an ex vivo model of RD. The subretinal fluid from UDCA-treated patients, collected during surgery, significantly protected rat retinal explants from cell death, when compared to subretinal fluid from control patients. Pan-transcriptomic analysis of the retina showed that UDCA upregulated anti-apoptotic, anti-oxidant, and anti-inflammatory genes. Oral UDCA is a potential neuroprotective adjuvant therapy in RD and other retinal degenerative diseases and should be further evaluated in a clinical trial.
Assuntos
Apoptose/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Colagogos e Coleréticos/farmacologia , Retina/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Degeneração Retiniana/terapia , Descolamento Retiniano/terapia , Ácido Ursodesoxicólico/farmacologia , Administração Oral , Albuminas/metabolismo , Animais , Disponibilidade Biológica , Linhagem Celular , Colagogos e Coleréticos/metabolismo , Criocirurgia , Feminino , Humanos , Técnicas In Vitro , Terapia a Laser , Masculino , Pessoa de Meia-Idade , Necrose , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Ratos , Retina/patologia , Retina/cirurgia , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Descolamento Retiniano/metabolismo , Descolamento Retiniano/patologia , Líquido Sub-Retiniano/química , Ácido Ursodesoxicólico/metabolismo , VitrectomiaRESUMO
Endoplasmic reticulum (ER) stress is a common threat to photoreceptors during the pathogenesis of chronic retinopathies and often results in irreversible visual impairment. 2,3,5,6-Tetramethylpyrazine (TMP), which possesses many beneficial pharmacological activities, is a potential drug that could be used to protect photoreceptors. In the present study, we found that the cellular growth rate of 661 W cells cultured under low glucose conditions was lower than that of control cells, while the G2/M phase of the cell cycle was longer. We further found that the mitochondrial membrane potential (ΔΨm) was lower and that ER stress factor expression was increased in 661 W cells cultured under low glucose conditions. TMP reversed these trends. Visual function and cell counts in the outer nuclear layer (ONL) were low and the TUNEL-positive rate in the ONL was high in a C3H mouse model of spontaneous retinal degeneration. Similarly, visual function was decreased, and the TUNEL-positive rate in the ONL was increased in fasted C57/BL6j mice compared with control mice. On the other hand, ER stress factor expression was found to be increased in the retinas of both mouse models, as shown by reverse transcription real-time PCR (RT-qPCR) and western blotting. TMP reversed the physiological and molecular biological variations observed in both mouse models, and ATF4 expression was enhanced again. Further investigation by using western blotting illustrated that the proportion of insoluble prion protein (PRP) versus soluble PRP was reduced both in vitro and in vivo. Taken together, these results suggest that TMP increased the functions of photoreceptors by alleviating ER stress in vitro and in vivo, and the intrinsic mechanism was the ATF4-mediated inhibition of PRP aggregation. TMP may potentially be used clinically as a therapeutic agent to attenuate the functional loss of photoreceptors during the pathogenesis of chronic retinopathies. KEY MESSAGES: ⢠Already known: TMP is a beneficial drug mainly used in clinic to enhance organ functions, and the intrinsic mechanism is still worthy of exploring. ⢠New in the study: We discovered that TMP ameliorated retinal photoreceptors function via ER stress alleviation, which was promoted by ATF4-mediated inhibition of PRP aggregation. ⢠Application prospect: In prospective clinical practices, TMP may potentially be used in the clinic as a therapeutic agent to attenuate the photoreceptors functional reduction in chronic retinopathies.
Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas Priônicas/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/prevenção & controle , Pirazinas/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Degeneração Retiniana/prevenção & controle , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Eletrorretinografia , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Jejum , Feminino , Glucose/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas Priônicas/química , Agregação Patológica de Proteínas/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/fisiopatologia , Método Simples-Cego , Solubilidade , Organismos Livres de Patógenos Específicos , Transcrição Gênica/efeitos dos fármacosRESUMO
Here, we evaluated the effects of PEDF (pigment epithelium-derived factor) and PEDF peptides on cone-photoreceptor cell damage in a mouse model of focal LED-induced phototoxicity (LIP) in vivo. Swiss mice were dark-adapted overnight, anesthetized, and their left eyes were exposed to a blue LED placed over the cornea. Immediately after, intravitreal injection of PEDF, PEDF-peptide fragments 17-mer, 17-mer[H105A] or 17-mer[R99A] (all at 10 pmol) were administered into the left eye of each animal. BDNF (92 pmol) and bFGF (27 pmol) injections were positive controls, and vehicle negative control. After 7 days, LIP resulted in a consistent circular lesion located in the supratemporal quadrant and the number of S-cones were counted within an area centered on the lesion. Retinas treated with effectors had significantly greater S-cone numbers (PEDF (60%), 17-mer (56%), 17-mer [H105A] (57%), BDNF (64%) or bFGF (60%)) relative to their corresponding vehicle groups (≈42%). The 17-mer[R99A] with no PEDF receptor binding and no neurotrophic activity, PEDF combined with a molar excess of the PEDF receptor blocker P1 peptide, or with a PEDF-R enzymatic inhibitor had undetectable effects in S-cone survival. The findings demonstrated that the cone survival effects were mediated via interactions between the 17-mer region of the PEDF molecule and its PEDF-R receptor.
Assuntos
Proteínas do Olho/farmacologia , Fatores de Crescimento Neural/farmacologia , Peptídeos/farmacologia , Retina/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Serpinas/farmacologia , Animais , Córnea/efeitos dos fármacos , Córnea/crescimento & desenvolvimento , Córnea/metabolismo , Dermatite Fototóxica , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Humanos , Camundongos , Fatores de Crescimento Neural/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos/genética , Fotoperíodo , Receptores de Neuropeptídeos/genética , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Serpinas/metabolismoRESUMO
All neurodegenerative diseases involve a relatively long period of timeframe from the onset of the disease to complete loss of functions. Extending this timeframe, even at a reduced level of function, would improve the quality of life of patients with these devastating diseases. The retina, as the part of the central nervous system and a frequent site of many distressing neurodegenerative disease, provides an ideal model to investigate the feasibility of extending the functional timeframe through pharmacologic intervention. Retinitis Pigmentosa (RP) is a group of blinding diseases. Although the rate of progression and degree of visual loss varies, there is usually a prolonged time before patients totally lose their photoreceptors and vision. It is believed that inhibitory mechanisms are still intact and may become relatively strong after the gradual loss of photoreceptors in RP patients. Therefore, it is possible that light-evoked responses of retinal ganglion cells and visual information processes in retinal circuits could be "unmasked" by blocking these inhibitory mechanisms restoring some level of visual function. Our results indicate that if the inhibition in the inner retina was unmasked in the retina of the rd10 mouse (the well-characterized RP mimicking, clinically relevant mouse model), the light-evoked responses of many retinal ganglion cells can be induced and restore their normal light sensitivity. GABA A receptor plays a major role in this masking inhibition. ERG b-wave and behavioral tests of spatial vision partly recovered after the application of PTX. Hence, removing retinal inhibition unmasks signalling mediated by surviving cones, thereby restoring some degree of visual function. These results may offer a novel strategy to restore the visual function with the surviving cones in RP patients and other gradual and progressive neurodegenerative diseases.
Assuntos
Neurônios/fisiologia , Picrotoxina/farmacologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Degeneração Retiniana/tratamento farmacológico , Células Ganglionares da Retina/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Visão Ocular/efeitos dos fármacos , Animais , Comportamento Animal , Modelos Animais de Doenças , Luz , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacosRESUMO
Purpose: Retinitis pigmentosa (RP) refers to a group of inherited blinding retinal diseases, whereby the death of mutated rod photoreceptors is followed closely by the death of cone photoreceptors. Cone cell death can be hugely debilitating as color/daytime vision becomes impaired. Thus, treatments that are effective against cone cell death are urgently needed. Our research has been working toward development of a neuroprotective treatment for RP. We have previously demonstrated significant neuroprotective properties of norgestrel, a progesterone analogue, in the mouse retina. The current study further investigates the potential of norgestrel as a treatment for RP, with a focus on long-term preservation of cone photoreceptors. Methods: Using the well-established rd10 mouse model of RP, we administered a norgestrel-supplemented diet at postnatal day (P)30, following widespread loss of rod photoreceptors and at the outset of cone degeneration. We subsequently assessed cone cell morphology and retinal function at P50, P60, and P80, using immunohistochemistry, electroretinograph recordings, and optomotor testing. Results: While cone cell degeneration was widespread in the untreated rd10 retina, we observed profound preservation of cone photoreceptor morphology in the norgestrel-treated mice for at least 50 days, out to P80. This was demonstrated by up to 28-fold more cone arrestin-positive photoreceptors. This protection transpired to functional preservation at all ages. Conclusions: This work presents norgestrel as an incredibly promising long-term neuroprotective compound for the treatment of RP. Crucially, norgestrel could be used in the mid-late stages of the disease to protect remaining cone cells and help preserve color/daytime vision.
Assuntos
Neuroproteção/efeitos dos fármacos , Norgestrel/farmacologia , Progesterona/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Retinose Pigmentar/tratamento farmacológico , Animais , Modelos Animais de Doenças , Eletrorretinografia , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Progestinas/farmacologia , Células Fotorreceptoras Retinianas Cones/patologia , Retinose Pigmentar/patologia , Retinose Pigmentar/fisiopatologia , Transdução de SinaisRESUMO
BACKGROUND AND PURPOSE: Simvastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor with multiple targets and effects. It protects neurons in the brain, but its protective effects on photoreceptors are unclear. In this study, we evaluated the neuroprotective effect of simvastatin on photoreceptors exposed to stress induced by all-trans-retinal (atRAL). EXPERIMENTAL APPROACH: AlamarBlue and LDH assays were used to evaluate the viability and metabolic activity of Y79 cells (a retinoblastoma cell line) exposed to atRAL-induced stress with or without simvastatin pretreatment. Changes in cellular ROS were evaluated using flow cytometry and mitochondrial stress markers JC-1 and HSP60. Changes in levels of two photoreceptor-specific markers, cone-rod homeobox protein (CRX) and interphotoreceptor retinoid binding protein (IRBP), were evaluated with western blot. The results were validated in ex vivo human retinal explants and a mouse model of photoreceptor degeneration. KEY RESULTS: Simvastatin improved mitochondrial function, alleviated oxidative stress and up-regulated the photoreceptor-specific markers IRBP and its upstream regulator CRX in Y79 cells and ex vivo human retinal explants under atRAL-induced stress. Simvastatin attenuated photoreceptor degeneration in association with up-regulation of IRBP and CRX expression after knockdown of IRBP in a murine model. CONCLUSION AND IMPLICATIONS: Our findings suggest that simvastatin has a novel role in protecting photoreceptors from atRAL-induced stress. Simvastatin treatment resulted in up-regulation of IRBP and its upstream transcription factor CRX in Y79 cells, ex vivo human retinal explants, and murine retinas in vivo. Further studies of simvastatin to treat photoreceptor degeneration are warranted.
Assuntos
Substâncias Protetoras/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Proteínas de Ligação ao Retinol/metabolismo , Sinvastatina/farmacologia , Animais , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Injeções Intraperitoneais , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/administração & dosagem , Células Fotorreceptoras Retinianas Cones/metabolismo , Sinvastatina/administração & dosagem , Relação Estrutura-Atividade , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologia , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Retinal degeneration is a major cause of severe vision loss and irreversible blindness and is characterized by progressive damage to retinal photoreceptor cells. Resveratrol (RSV) serves as an activator of the histone deacetylase, Sirt1, and has been shown to exert anti-oxidative properties. In this study, we mimicked retinal degeneration by subjecting photoreceptors (661â¯W cells) to glucose deprivation (GD) or light exposure. Under these conditions, we investigated the mechanisms underlying GD- or light exposure-induced cell death and the protective effect of RSV. We found that GD and light exposure resulted in mitochondrial dysfunction, oxidative stress, and cell death. Treatment of injured cells with RSV decreased the production of reactive oxygen species (ROS), improved the ratio of reduced/oxidized glutathione (GSH/GSSG), mitochondrial membrane potential and morphology, and reduced apoptosis. We used the caspase inhibitor, z-VAD-fmk, and a lentiviral-mediated shRNA knockdown of PARP-1 to reveal that GD and light exposure-induced cell death have different underlying mechanisms; GD triggered a caspase-dependent cell death pathway, whereas light exposure triggered a PARP-dependent cell death pathway. The level of caspase-9 and caspase-3, upregulated following GD, were reduced by treatment with RSV. Similarly, the level of PARP-1 and AIF, upregulated following light exposure, were decreased by treatment with RSV. Additionally, treatment with RSV elevated the protein expression and enzymatic activity of Sirt1 and a Sirt1 inhibitor reduced the protective effect of RSV against insult-induced cellular injuries, indicating that RSV's protective effect may involve Sirt1 activation. Finally, we investigated the neuroprotection of RSV in vivo. Administration of RSV to mice under extreme light exposure led to a suppression of the light-induced thinning of the outer nuclear layer (ONL) detected by hematoxylin and eosin (H&E) staining and restored retinal function evaluated by electroretinography (ERG). Taken together, our findings provide evidence that treatment with RSV has neuroprotective effects on both GD and light exposure-induced cell death pathways in photoreceptor cells.
Assuntos
Antioxidantes/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Espécies Reativas de Oxigênio/antagonistas & inibidores , Resveratrol/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Inibidores de Caspase/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Glucose/deficiência , Glucose/farmacologia , Glutationa/metabolismo , Luz/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Transdução de Sinais/genética , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
Achromatopsia is an autosomal recessively inherited congenital defect characterized by a lack of cone photoreceptor function, leading to severely impaired vision. In this clinical study, achromatopsia patients were treated with a single subretinal injection of rAAV.hCNGA3 to restore cone function. The focus of this trial was on the safety of the treatment. After surgery, patients were monitored in eight extensive visits during the first year, followed by a 4-year follow-up period with annual visits. For essential complementation of the standard ophthalmological and systemic examinations, disease-specific methods were developed to assess the safety, efficacy, and patient-reported outcomes in this trial.
Assuntos
Defeitos da Visão Cromática/genética , Defeitos da Visão Cromática/terapia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Terapia Genética/efeitos adversos , Adulto , Idoso , Defeitos da Visão Cromática/patologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/administração & dosagem , Canais de Cátion Regulados por Nucleotídeos Cíclicos/efeitos adversos , Dependovirus/genética , Relação Dose-Resposta a Droga , Feminino , Vetores Genéticos/administração & dosagem , Humanos , Injeções , Masculino , Pessoa de Meia-Idade , Mutação , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/patologiaRESUMO
Retbindin (Rtbdn) is a novel protein of unknown function found exclusively in the retina. Recently, our group has suggested, from in silico analysis of the peptide sequence and in vitro binding data, that Rtbdn could function to bind riboflavin (RF) and its derivatives flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), collectively known as flavins. Here we confirm that Rtbdn is capable of flavin binding and that this characteristic can protect photoreceptors from flavin-sensitized light damage.
Assuntos
Proteínas do Olho/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Animais , Células COS , Morte Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Meios de Cultivo Condicionados , Humanos , Camundongos , Radiossensibilizantes/farmacologia , Proteínas Recombinantes/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Riboflavina/farmacologiaRESUMO
Studies form our laboratory and others show that the oncogenic tyrosine kinase and serine threonine kinase signaling pathways are essential for cone photoreceptor survival. These pathways are downregulated in mouse models of retinal degenerative diseases. In the present study, we found that activation mutants of mTOR delayed the death of cones in a mouse model of retinal degeneration. These studies suggest that oncogenic protein kinases may be useful as therapeutic agents to treat retinal degenerations that affect cones.
Assuntos
Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Retinose Pigmentar/terapia , Serina-Treonina Quinases TOR/uso terapêutico , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Sobrevivência Celular , Modelos Animais de Doenças , Proteínas do Olho , Genes Sintéticos , Injeções Intraoculares , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Mutantes , Regiões Promotoras Genéticas , Receptor de Insulina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Serina-Treonina Quinases TOR/genéticaRESUMO
Retinal degenerative diseases are a major cause of untreatable blindness due to a loss of photoreceptors. Recent advances in genetics and gene therapy for inherited retinal dystrophies (IRDs) showed that therapeutic gene transfer holds a great promise for vision restoration in people with currently incurable blinding diseases. Due to the huge genetic heterogeneity of IRDs that represents a major obstacle for gene therapy development, alternative therapeutic approaches are needed. This review focuses on the rescue of cone function as a therapeutic option for maintaining central vision in rod-cone dystrophies. It highlights recent developments in better understanding the mechanisms of action of the trophic factor RdCVF and its potential as a sight-saving therapeutic strategy.
Assuntos
Distrofias de Cones e Bastonetes/terapia , Terapia Genética , Vetores Genéticos/uso terapêutico , Células Fotorreceptoras Retinianas Cones/fisiologia , Tiorredoxinas/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Comunicação Celular , Dependovirus/genética , Proteínas do Olho/fisiologia , Heterogeneidade Genética , Glicólise , Humanos , Modelos Moleculares , Medicina de Precisão , Conformação Proteica , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/uso terapêutico , Pesquisa Translacional Biomédica , Resultado do TratamentoRESUMO
Usher syndrome type 1 (USH1) is a major cause of inherited deafness and blindness in humans. The eye disorder is often referred to as retinitis pigmentosa, which is characterized by a secondary cone degeneration following the rod loss. The development of treatments to prevent retinal degeneration has been hampered by the lack of clear evidence for retinal degeneration in mutant mice deficient for the Ush1 genes, which instead faithfully mimic the hearing deficit. We show that, under normal housing conditions, Ush1g-/- and Ush1c-/- albino mice have dysfunctional cone photoreceptors whereas pigmented knockout animals have normal photoreceptors. The key involvement of oxidative stress in photoreceptor apoptosis and the ensued retinal gliosis were further confirmed by their prevention when the mutant mice are reared under darkness and/or supplemented with antioxidants. The primary degeneration of cone photoreceptors contrasts with the typical forms of retinitis pigmentosa. Altogether, we propose that oxidative stress probably accounts for the high clinical heterogeneity among USH1 siblings, which also unveils potential targets for blindness prevention.
Assuntos
Antioxidantes/uso terapêutico , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/prevenção & controle , Animais , Antioxidantes/farmacologia , Apoptose , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto , Escuridão , Dieta , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Abrigo para Animais , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Opsinas/metabolismo , Fenótipo , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Degeneração Retiniana/patologia , Taurina/administração & dosagemRESUMO
Purpose: Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MT1 and MT2) in 661W cells, then whether MEL can prevent H2O2-induced cell death, and last, through which pathway MEL confers protection. Methods: The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with H2O2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H2O2-mediated cell death, a Fas/FasL antagonist was used before the exposure to H2O2. Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H2O2 and/or MEL. Cell viability was analyzed by using Trypan Blue. Results: Both MEL receptors (MT1 and MT2) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H2O2-mediated cell death (20-25%). This effect was replicated with IIK7 (a melatonin receptor agonist) when used at a concentration of 1 µM. Preincubation with luzindole (a melatonin receptor antagonist) blocked MEL protection. Kp7-6, an antagonist of Fas/FasL, blocked cell death caused by H2O2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H2O2, and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H2O2. Conclusions: The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H2O2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.
Assuntos
Antioxidantes/farmacologia , Caspase 3/metabolismo , Proteína Ligante Fas/antagonistas & inibidores , Peróxido de Hidrogênio/toxicidade , Melatonina/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Receptor fas/antagonistas & inibidores , Animais , Caspase 3/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Oxidantes/toxicidade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/genética , Receptor MT2 de Melatonina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Retinal diseases, such as hereditary retinitis pigmentosa and age-related macular degeneration, are characterized by the progressive loss of photoreceptors. Histone deacetylase 6 (HDAC6) is considered as a stress surveillance factor and a potential target for neuroprotection and regeneration. Overexpression of HDAC6 has been connected to neurodegenerative disorders, and its suppression may provide protection. Here we show that HDAC6 is constitutively present in the mouse retina, and in the cone-like mouse cell line 661W. In 661W cells HDAC6 inhibition by the specific inhibitor tubastatin A (TST) led to the acetylation of α-tubulin, which is a major substrate for HDAC6. After oxidative stress, exerted by hydrogen peroxide, TST promoted cell survival and the upregulation of heat-shock proteins HSP70 and HSP25 by activation of heat-shock transcription factor 1. Furthermore, in response to oxidative stress the redox regulatory protein peroxiredoxin 1 (Prx1) was modulated in 661W cells by HDAC6 inhibition. The peroxide reducing activity of Prx1 is dependent on its acetylation, which is mediated by HDAC6. Pre-incubation with TST prevented the inactivation of Prx1 and its preserved activity may exert protective effects in photoreceptor cells. To determine whether TST treatment has a therapeutic effect on visual function, the dyeucd6 zebrafish model of inherited sight loss was utilized. Zebrafish have developed as a suitable model system for pharmacological testing. In vivo application of TST caused the hyperacetylation of α-tubulin, indicating that HDAC6 is active in this model. Furthermore, TST was sufficient to rescue visual function and retinal morphology. Hence, HDAC6 inhibition and the regulation of peroxiredoxin activity may play a significant role in protecting retinal cells and in particular photoreceptors, which are exposed to high levels of reactive oxygen species derived from oxidative stress-induced injuries.
Assuntos
Desacetilase 6 de Histona/genética , Inibidores de Histona Desacetilases/farmacologia , Peróxido de Hidrogênio/antagonistas & inibidores , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Amaurose Congênita de Leber/tratamento farmacológico , Tubulina (Proteína)/genética , Acetilação , Animais , Linhagem Celular , Sobrevivência Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/patologia , Camundongos , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Transdução de Sinais , Tubulina (Proteína)/metabolismo , Peixe-ZebraRESUMO
Retinal degeneration and in particular retinitis pigmentosa (RP) is associated to ceramide (Cer) accumulation and cell death induction. Cer and sphingosine-1-phosphate (S1P) belong to the sphingolipids class and exert a pro-apoptotic and pro-survival activity, respectively. Our aim is to target sphingolipid metabolism by inhibiting S1P lyase that regulates one of the S1P degradation pathways, to reduce retinal photoreceptor damage. The murine 661W cone-like cell line was pretreated with THI, an inhibitor of S1P lyase and exposed to H2O2-induced oxidative stress. 661W cell viability and apoptosis were evaluated by Trypan Blue and TUNEL assay, respectively. Protein expression of mediators of the survival/death pathway (ERK1/2, Akt, Bcl-2, Bax) was analyzed by Western blotting. RT-PCR was performed to establish HO-1 transcript changes and LC-MS analysis to measure Cer intracellular content. THI rescues inhibitory H2O2-effect on 661W cell viability and impairs H2O2-induced apoptosis by increasing Bcl-2/Bax ratio. THI administration counteracts the oxidative stress effects of H2O2 on 661W cells by activating the Nrf2/HO-1 pathway, regulating ERK and Akt phosphorylation levels, and decreasing Cer intracellular content. We conclude that sphingolipid metabolism manipulation can be considered a therapeutic target to promote photoreceptor survival.
Assuntos
Aldeído Liases/antagonistas & inibidores , Imidazóis/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Heme Oxigenase-1/fisiologia , Peróxido de Hidrogênio/toxicidade , Proteínas de Membrana/fisiologia , Camundongos , Fator 2 Relacionado a NF-E2/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Células Fotorreceptoras Retinianas Cones/metabolismo , Esfingolipídeos/metabolismo , Proteína X Associada a bcl-2/análiseRESUMO
The endocannabinoid system is involved in some neurodegenerative diseases such as Alzheimer's disease. An endogenous constellation of proteins related to cannabinoid1 receptor signaling, including free fatty acids, diacylglycerol lipase, and N-acylethanolamine-hydrolyzing acid amidase, are localized in the murine retina. Moreover, the expression levels of endogenous agonists of cannabinoid receptors are changed in the vitreous fluid. However, the role of the endocannabinoid system in the retina, particularly in the light-induced photoreceptor degeneration, remains unknown. Therefore, we investigated involvement of the cannabinoid1 receptor in light-induced retinal degeneration using in vitro and in vivo models. To evaluate the effect of cannabinoid1 receptors in light irradiation-induced cell death, the mouse retinal cone-cell line (661W) was treated with a cannabinoid1 receptor antagonist, rimonabant. Time-dependent changes of expression and localization of retinal cannabinoid1 receptors were measured using Western blot and immunostaining. Retinal damage was induced in mice by exposure to light, followed by intravitreal injection of rimonabant. Electroretinograms and histologic analyses were performed. Rimonabant suppressed light-induced photoreceptor cell death. Cannabinoid1 receptor expression was upregulated by light exposure. Treatment with rimonabant improved both a- and b-wave amplitudes and the thickness of the outer nuclear layer. These results suggest that the cannabinoid1 receptor is involved in light-induced retinal degeneration and it may represent a therapeutic target in the light-induced photoreceptor degeneration related diseases.