RESUMO
Saffron treatment is a broad-spectrum therapy used for several retinal diseases, and its effectiveness depends on a particular molecular composition (REPRON® saffron). Its production requires specific crops and procedures that, together with low yields, make this spice expensive. To reduce costs, the use of hydroponic crops is gradually increasing. In this study, we tested the protective properties of a hydroponic saffron (sH) batch in models of retinal pigmented epithelium (RPE) degeneration. ARPE-19 cells were pretreated with 40 µg/mL saffron and exposed to different types of damage: excess light and retinol (LE + RET) or oxidative stress (H2O2). After analyzing the composition of all saffron types with spectroscopy, we performed cell viability and immunofluorescence analysis for both protocols. We compared the sH results with those of a validated batch of saffron REPRON® (sR) and those of a saffron non-REPRON® (sNR) batch. sH and sR, which we found had the same chemical composition, were more effective than sNR in increasing cell survival and attenuating the morphological changes related to apoptosis. In conclusion, hydroponic culturing is a suitable strategy to produce high-quality saffron to reduce costs and increase the accessibility of this promising treatment for retinal degeneration.
Assuntos
Crocus , Crocus/metabolismo , Peróxido de Hidrogênio/farmacologia , Epitélio Pigmentado da Retina/metabolismo , Linhagem Celular , Estresse Oxidativo , Corantes/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The mammalian retina contains an autonomous circadian clock system that controls many physiological functions within this tissue. Previous studies on young mice have reported that removal of the key circadian clock gene Bmal1 from the retina affects the circadian regulation of visual function, but does not affect photoreceptor viability. Because dysfunction in the circadian system is known to affect cell viability during aging in other systems, we compared the effect of Bmal1 removal from the retina on visual function, inner retinal structure, and photoreceptor viability in young (1 to 3 months) and aged (24 to 26 months) mice. We found that removal of Bmal1 from the retina significantly affects visual information processing in both rod and cone pathways, reduces the thickness of inner retinal nuclear and plexiform layers, accelerates the decline of visual functions during aging, and reduces the viability of cone photoreceptors. Our results thus suggest that circadian clock dysfunction, caused by genetic or other means, may contribute to the decline of visual function during development and aging.
Assuntos
Fatores de Transcrição ARNTL/fisiologia , Envelhecimento/patologia , Ritmo Circadiano , Retina/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Visão Ocular , Envelhecimento/metabolismo , Animais , Relógios Circadianos , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismoRESUMO
Aldose reductase is a key enzyme in the development of long term diabetic complications and its inhibition represents a viable therapeutic solution for people affected by these pathologies. Therefore, the search for effective aldose reductase inhibitors is a timely and pressing challenge. Herein we describe the access to a novel class of oxyimino derivatives, obtained by reaction of a 1,5-dicarbonyl substrate with O-(arylmethyl)hydroxylamines. The synthesised compounds proved to be active against the target enzyme. The best performing inhibitor, compound (Z)-8, proved also to reduce both cell death and the apoptotic process when tested in an in vitro model of diabetic retinopathy made of photoreceptor-like 661w cell line exposed to high-glucose medium, counteracting oxidative stress triggered by hyperglycaemic conditions.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Antioxidantes/farmacologia , Inibidores Enzimáticos/farmacologia , Iminas/química , Açúcares/química , Inibidores Enzimáticos/química , Estrutura MolecularRESUMO
Diabetes is a multi-factorial disorder that should be treated with multi-effective compounds. Here we describe the access to polyhydroxylated pyrrolidines, belonging to the d-gluco and d-galacto series, through aminocyclization reactions of two differentially protected d-xylo-hexos-4-ulose derivatives. The prepared compounds proved to inhibit both alpha-glucosidase, responsible for the emergence of hyperglycemic spikes, and aldose reductase, accountable for the development of abnormalities in diabetic tissues. Accordingly, they show the dual inhibitory profile deemed as ideal for diabetes treatment. Significantly, compound 17b reduced the process of cell death and restored the physiological levels of oxidative stress when tested in the photoreceptor-like 661w cell line, thus proving to be effective in an in vitro model of diabetic retinopathy.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pirrolidinas/farmacologia , alfa-Glucosidases/metabolismo , Aldeído Redutase/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Pirrolidinas/síntese química , Pirrolidinas/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
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
Rod-cone gap junctions mediate the so-called "secondary rod pathway", one of three routes that convey rod photoreceptor signals across the retina. Connexin 36 (Cx36) is expressed at these gap junctions, but an unidentified connexin protein also seems to be expressed. Cx36 knockout mice have been used extensively in the quest to dissect the roles in vision of all three pathways, with the assumption, never directly tested, that rod-cone electrical coupling is abolished by deletion of this connexin isoform. We previously showed that when wild type mouse cones couple to rods, their apparent dynamic range is extended toward lower light intensities, with the appearance of large responses to dim flashes (up to several mV) originating in rods. Here we recorded from the cones of Cx36del[LacZ]/del[LacZ] mice and found that dim flashes of the same intensity evoked at most small sub-millivolt responses. Moreover, these residual responses originated in the cones themselves, since: (i) their spectral preference matched that of the recorded cone and not of rods, (ii) their time-to-peak was shorter than in coupled wild type cones, (iii) a pharmacological block of gap junctions did not reduce their amplitude. Taken together, our data show that rod signals are indeed absent in the cones of Cx36 knockout mice. This study is the first direct demonstration that Cx36 is crucial for the assembly of functional rod-cone gap junctional channels, implying that its genetic deletion is a reliable experimental approach to eliminate rod-cone coupling.
Assuntos
Conexinas/metabolismo , Junções Comunicantes/fisiologia , Potenciais da Membrana/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Animais , Eletrofisiologia , Feminino , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estimulação Luminosa , Proteína delta-2 de Junções ComunicantesRESUMO
In this work, we reported the application and validation of an improved high-performance liquid chromatography method coupled with a fluorimetric detector (HPLC-FL) to screen the activity of two heterocyclic derivatives reported as serine palmitoyl transferase (SPT) inhibitors. The analytical conditions were optimized in terms of the derivatization procedure, chromatographic condition, extraction procedure, and method validation according to EMEA guidelines. Once fully optimized, the method was applied to assess the SPT-inhibitory activity of the above-mentioned derivatives and of the reference inhibitor myriocin. The obtained results, expressed as a percentage of residual SPT activity, were compared to those obtained with the reference radio immune assay (RIA). The good correlation between the two types of assay demonstrated that the improved HPLC-FL method is suitable for a preliminary and rapid screening of potential SPT-inhibitors.
Assuntos
Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fluorometria , Serina C-Palmitoiltransferase/antagonistas & inibidores , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Relação Dose-Resposta a Droga , Fluorometria/métodos , Fluorometria/normas , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Reprodutibilidade dos Testes , Serina C-Palmitoiltransferase/química , Especificidade por SubstratoRESUMO
The endogenous gasotransmitter hydrogen sulphide (H2S) is an important regulator of the cardiovascular system, particularly of myocardial function. Moreover, H2S exhibits cardioprotective activity against ischemia/reperfusion (I/R) or hypoxic injury, and is considered an important mediator of "ischemic preconditioning", through activation of mitochondrial potassium channels, reduction of oxidative stress, activation of the endogenous "anti-oxidant machinery" and limitation of inflammatory responses. Accordingly, H2S-donors, i.e. pro-drugs able to generate exogenous H2S, are viewed as promising therapeutic agents for a number of cardiovascular diseases. The novel H2S-donor 4-carboxy phenyl-isothiocyanate (4CPI), whose vasorelaxing effects were recently reported, was tested here in different experimental models of myocardial I/R. In Langendorff-perfused rat hearts subjected to I/R, 4CPI significantly improved the post-ischemic recovery of myocardial functional parameters and limited tissue injury. These effects were antagonized by 5-hydroxydecanoic acid (a blocker of mitoKATP channels). Moreover, 4CPI inhibited the formation of reactive oxygen species. We found the whole battery of H2S-producing enzymes to be present in myocardial tissue: cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST). Notably, 4CPI down-regulated the post-ischemic expression of CSE. In Langendorff-perfused mouse hearts, 4CPI reduced the post-ischemic release of norepinephrine and the incidence of ventricular arrhythmias. In both rat and mouse hearts, 4CPI did not affect the degranulation of resident mast cells. In isolated rat cardiac mitochondria, 4CPI partially depolarized the mitochondrial membrane potential; this effect was antagonized by ATP (i.e., the physiological inhibitor of KATP channels). Moreover, 4CPI abrogated calcium uptake in the mitochondrial matrix. Finally, in an in vivo model of acute myocardial infarction in rats, 4CPI significantly decreased I/R-induced tissue injury. In conclusion, H2S-donors, and in particular isothiocyanate-based H2S-releasing drugs like 4CPI, can actually be considered a suitable pharmacological option in anti-ischemic therapy.
Assuntos
Cardiotônicos/farmacologia , Sulfeto de Hidrogênio/metabolismo , Isotiocianatos/farmacologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Canais de Potássio/metabolismo , Animais , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Cistationina beta-Sintase/metabolismo , Cistationina beta-Sintase/farmacologia , Cistationina gama-Liase/metabolismo , Cisteína/análogos & derivados , Cisteína/farmacologia , Ácidos Decanoicos/farmacologia , Coração/efeitos dos fármacos , Hidroxiácidos/farmacologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
The parathyroid glands play an overall regulatory role in the systemic calcium (Ca(2+)) homeostasis. The purpose of the present study was to demonstrate the presence of the Ca(2+) channels transient receptor potential vanilloid (TRPV) 5 and TRPV6 in human parathyroid glands. Semi-quantitative and quantitative PCR was carried out to evaluate the presence of TRPV5 and TRPV6 mRNAs in sporadic parathyroid adenomas and normal parathyroid glands. Western blot and immunocytochemical assays were used to assess protein expression, cellular localization and time expression in primary cultures from human parathyroid adenoma. TRPV5 and TRPV6 transcripts were then identified both in normal and pathological tissues. Predominant immunoreactive bands were detected at 75-80 kD for both vanilloid channels. These channels co-localized with the calcium-sensing receptor (CASR) on the membrane surface, but immunoreactivity was also detected in the cytosol and around the nuclei. Our data showed that western blotting recorded an increase of protein expression of both channels in adenoma samples compared with normal glands suggesting a potential relation with the cell calcium signalling pathway and the pathological processes of these glands.
Assuntos
Adenoma/patologia , Canais de Cálcio/metabolismo , Transformação Celular Neoplásica/patologia , Hiperparatireoidismo Primário/patologia , Glândulas Paratireoides/metabolismo , Canais de Cátion TRPV/metabolismo , Adenoma/genética , Adenoma/metabolismo , Western Blotting , Cálcio/metabolismo , Canais de Cálcio/genética , Membrana Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Humanos , Hiperparatireoidismo Primário/genética , Hiperparatireoidismo Primário/metabolismo , Técnicas Imunoenzimáticas , Glândulas Paratireoides/citologia , Técnicas de Patch-Clamp , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV/genéticaRESUMO
Photoreceptor death causes blinding inheritable retinal diseases, such as retinitis pigmentosa (RP). As disease progression often outpaces therapeutic advances, finding effective treatments is urgent. This study focuses on developing a targeted approach by evaluating the efficacy of small peptides derived from pigment epithelium-derived factor (PEDF), known to restrict common cell death pathways associated with retinal diseases. Peptides with affinity for the PEDF receptor, PEDF-R, (17-mer and H105A) delivered via eye drops reached the retina, efficiently promoted photoreceptor survival, and improved retinal function in RP mouse models based on both the rd10 mutation and the rhodopsin P23H mutation. Additionally, intravitreal delivery of AAV-H105A vectors delayed photoreceptor degeneration in the latter RP mouse model. Furthermore, peptide H105A specifically prevented photoreceptor death induced by oxidative stress, a contributing factor to RP progression, in human retinal organoids. This promising approach for peptide eye drop delivery holds significant potential as a therapeutic for preventing photoreceptor death in retinal disorders, offering a high safety profile, low invasiveness and multiple delivery options.
RESUMO
Inherited retinal diseases, which include retinitis pigmentosa, are a family of genetic disorders characterized by gradual rod-cone degeneration and vision loss, without effective pharmacological treatments. Experimental approaches aim to delay disease progression, supporting cones' survival, crucial for human vision. Histone deacetylases (HDACs) mediate the activation of epigenetic and nonepigenetic pathways that modulate cone degeneration in RP mouse models. We developed new HDAC inhibitors (5a-p), typified by a tetrahydro-γ-carboline scaffold, characterized by high HDAC6 inhibition potency with balanced physicochemical properties for in vivo studies. Compound 5d (repistat, IC50 HDAC6 = 6.32 nM) increased the levels of acetylated α-tubulin compared to histone H3 in ARPE-19 and 661W cells. 5d promoted vision rescue in the atp6v0e1-/- zebrafish model of photoreceptor dysfunction. A single intravitreal injection of 5d in the rd10 mouse model of RP supported morphological and functional preservation of cone cells and maintenance of the retinal pigment epithelium array.
RESUMO
The prevention of cone loss during retinal degeneration is a major goal of most therapeutic strategies in retinal degenerative diseases. An intriguing issue in the current research in this field is to understand why a genetic mutation that affects rods eventually leads to cone death. The main objective of the present study was to investigate to what extent rescuing rods from degeneration affects the survival of cones and prevents functional impairment of the visual performance. To this purpose, we compared rod and cone viabilities by both ex vivo and in vivo determinations in the rd10 mutant mouse, a validated model of human retinitis pigmentosa. The ex vivo experiments included morphological and biochemical tests, whereas in vivo studies compared the rod-mediated scotopic with the cone-mediated photopic electroretinogram. We also determined the overall visual performance by behaviorally testing the visual acuity (VA). The electroretinogram measurements showed that the kinetics of the photopic response in rd10 mice was slowed down with respect to the age-paired wild-type at a very early stage of the disease, when rods were still present and responsive. We then tested cone viability and function under a pharmacological scheme previously shown to prolong rod survival. The treatment consisted of eye drop administration of myriocin, an inhibitor of the biosynthesis of ceramide, a powerful proapoptotic messenger. The results of biochemical, morphological and functional assays converged to show that, in treated rd10 mice cone photoreceptors, the inner retina and overall visual performance were preserved well after rod death.
Assuntos
Células Fotorreceptoras Retinianas Cones/patologia , Retinose Pigmentar/fisiopatologia , Acuidade Visual , Animais , Apoptose , Sobrevivência Celular , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Monoinsaturados/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/tratamento farmacológico , Retinose Pigmentar/patologiaRESUMO
Retinitis pigmentosa (RP) is a genetic disease causing progressive apoptotic death of photoreceptors and, ultimately, incurable blindness. Using the retinal degeneration 10 (rd10) mouse model of RP, we investigated the role of ceramide, a proapoptotic sphingolipid, in retinal degeneration. We also tested the possibility that photoreceptor loss can be slowed or blocked by interfering with the ceramide signaling pathway of apoptosis in vivo. Retinal ceramide levels increased in rd10 mice during the period of maximum photoreceptor death. Single intraocular injections of myriocin, a powerful inhibitor of serine palmitoyl-CoA transferase, the rate-limiting enzyme of ceramide biosynthesis, lowered retinal ceramide levels to normal values and rescued photoreceptors from apoptotic death. Noninvasive treatment was achieved using eye drops consisting of a suspension of solid lipid nanoparticles loaded with myriocin. Short-term noninvasive treatment lowered retinal ceramide in a manner similar to intraocular injections, indicating that nanoparticles functioned as a vector permitting transcorneal drug administration. Prolonged treatment (10-20 d) with solid lipid nanoparticles increased photoreceptor survival, preserved photoreceptor morphology, and extended the ability of the retina to respond to light as assessed by electroretinography. In conclusion, pharmacological targeting of ceramide biosynthesis slowed the progression of RP in a mouse model, and therefore may represent a therapeutic approach to treating this disease in humans. Transcorneal administration of drugs carried in solid lipid nanoparticles, as experimented in this study, may facilitate continuous, noninvasive treatment of patients with RP and other retinal pathologies.
Assuntos
Ceramidas/biossíntese , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/fisiologia , Retinose Pigmentar/tratamento farmacológico , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/administração & dosagem , Ácidos Graxos Monoinsaturados/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Células Fotorreceptoras de Vertebrados/patologia , Retinose Pigmentar/patologia , Retinose Pigmentar/fisiopatologia , Serina C-Palmitoiltransferase/antagonistas & inibidoresRESUMO
A mechanism shared by most neurodegenerative diseases, like Alzheimer's disease (AD) and Parkinson's disease (PD), is neuroinflammation. It has been shown to have a link between cognitive impairment and retinal function under neuroinflammatory conditions, confirming the essential role of the retina as a window to the brain. Here, we characterize a mouse model of LPS-induced neuroinflammation describing the parallel deterioration of both memory and visual function. Then, we demonstrate, using the Novel Object Recognition test (NOR) and electroretinogram (ERG) recordings, that preventive, chronic treatment with saffron Repron® is able to reduce the neuroinflammation process and prevent the impairment of both cognitive and visual function. The improvement in behavioral and visual function is confirmed by the pattern of expression of neuroinflammation-related genes and related proteins where pre-treatment with Repron® saffron presents a positive modulation compared with that obtained in animals treated with LPS alone. These results hold for retinal tissue and partially in the brain, where it appears that the onset of damage was delayed. This trend underlines the critical role of the retina as a most sensitive portion of the central nervous system to LPS-induced damage and could be used as a "sensor" for the early detection of neurodegenerative diseases such as Alzheimer's.
RESUMO
Research on photoreceptors has led to important insights into how light signals are detected and processed in the outer retina. Most information about photoreceptor function, however, comes from lower vertebrates. The large majority of mammalian studies are based on suction pipette recordings of outer segment currents, a technique that doesn't allow examination of phenomena occurring downstream of phototransduction. Only a small number of whole-cell recordings have been made, mainly in the macaque. Due to the growing importance of the mouse in vision research, we have optimized a retinal slice preparation that allows the reliable collection of perforated-patch recordings from light responding rods and cones. Unexpectedly, the frequency of cone recordings was much higher than their numeric proportion of â¼3%. This allowed us to obtain direct functional evidence suggestive of rodcone coupling in the mouse. Moreover, rods had considerably larger single photon responses than previously published for mammals (3.44 mV, SD 1.37, n = 19 at 24°C; 2.46 mV, SD 1.08, n = 10 at 36°C), and a relatively high signal/noise ratio (6.4, SD 1.8 at 24°C; 6.8, SD 2.8 at 36°C). Both findings imply a more favourable transmission at the rodrod bipolar cell synapse. Accordingly, relatively few photoisomerizations were sufficient to elicit a half-maximal response (6.7, SD 2.7, n = 5 at 24°C; 10.6, SD 1.7, n = 3 at 36°C), leading to a narrow linear response range. Our study demonstrates new features of mammalian photoreceptors and opens the way for further investigations into photoreceptor function using retinas from mutant mouse models.
Assuntos
Adaptação à Escuridão/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Potenciais de Ação/fisiologia , Animais , Luz , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The aim of this study was to investigate the distribution of serotonin (5-HT) receptors of type 6 (5-HT(6)) in postmortem human prefrontal cortex, striatum and hippocampus. The brain samples were obtained from 6 subjects who had died for causes not involving primarily or secondarily the CNS. The 5-HT(6) receptor distribution was explored by the [(125)I]SB-258585 binding to brain membranes followed by the pharmacological characterization, where possible, and by autoradiographic, immunohistochemical and immunofluorescence evaluations. A specific and saturable [(125)I]SB-258585 binding was detected in striatum only, with a pharmacological characterization consistent with that of a 5-HT(6) receptor. The autoradiography showed the presence of a specific [(125)I]SB-258585 binding distributed homogeneously in caudate, putamen and accumbens. The immunohistochemistry, carried out in the striatum only, coupled with the immunofluorescence with glial fibrillary acidic protein (GFAP) and parvalbumin (PV) showed the co-localization of 5-HT(6) receptor with PV, while indicating that this receptor subtype was expressed in neurons and not in astrocytes. Taken together, the present findings showed the presence of a higher density of 5-HT(6) receptors, as labeled by [(125)I]SB-258585, in striatum than in hippocampus and prefrontal cortex, and specifically within the neuronal body. In addition, they would suggest that striatum is one of the major potential CNS targets linked to 5-HT(6) receptor modulation.
Assuntos
Encéfalo/metabolismo , Receptores de Serotonina/metabolismo , Autorradiografia , Humanos , Imuno-Histoquímica , Radioisótopos do Iodo/metabolismo , Piperazinas/metabolismo , Mudanças Depois da Morte , Ensaio Radioligante , Sulfonamidas/metabolismoRESUMO
Rhodopsin (RHO) mutations are responsible for 25-40% of the dominant cases of retinitis pigmentosa (RP) with different severity and progression rates. The Tvrm4 mice, heterozygous for an I307N dominant mutation of RHO, display a normal retinal phenotype when raised in ambient light conditions, but undergo photoreceptor degeneration when briefly exposed to strong white light. Here, The Tvrm4 mice is pre-treated with naringenin 100 mg/kg/die, quercetin 100 mg/kg/die, naringenin 50 + quercercetin 100 mg/kg/die or vehicle dimethyl sulfoxide (DMSO 0.025%) in the drinking water for 35 days. On the 30th day, retinal degeneration was induced by exposure for 1 min to the white light of 12,000 lux intensity, and the treatment was repeated for another 5 days. At the end of the protocol retinal functionality was tested by recording an electroretinogram (ERG). The retinal tissue was collected and was used for further analyses, including immunohistochemically, biochemical, and molecular biology assays. The data obtained show that treatment with nutraceutical molecules is effective in counteracting retinal degeneration by preserving the functionality of photoreceptors and increasing the antioxidant and anti-apoptotic pathways of retinal cells. The present data confirm that nutraceutical molecules are effective in slowing photoreceptor degeneration in a mutation-independent way by modulating the antioxidant response of the retina at the gene expression level.
RESUMO
The 18 kDa translocator protein (TSPO) is predominantly located in the mitochondrial outer membrane, playing an important role in steroidogenesis, inflammation, survival, and cell proliferation. Its expression in the CNS, and mainly in glial cells, is upregulated in neuropathologies and brain injury. In this study, the potential of targeting TSPO for the therapeutic treatment of inflammatory-based retinal neurodegeneration was evaluated by means of an in vitro model of lipopolysaccharide (LPS)-induced degeneration in 661 W cells, a photoreceptor-like cell line. After the assessment of the expression of TSPO in 661W cells, which, to the best of our knowledge, was never investigated so far, the anti-inflammatory and cytoprotective effects of a number of known TSPO ligands, belonging to the class of N,N-dialkyl-2-arylindol-3-ylglyoxylamides (PIGAs), were evaluated, using the classic TSPO ligand PK11195 as the reference standard. All tested PIGAs showed the ability to modulate the inflammatory and apoptotic processes in 661 W photoreceptor-like cells and to reduce LPS-driven cellular cytotoxicity. The protective effect of PIGAs was, in all cases, reduced by cotreatment with the pregnenolone synthesis inhibitor SU-10603, suggesting the involvement of neurosteroids in the protective mechanism. As inflammatory processes play a crucial role in the retinal neurodegenerative disease progression toward photoreceptors' death and complete blindness, targeting TSPO might represent a successful strategy to slow down this degenerative process that may lead to the inexorable loss of vision.
Assuntos
Doenças Neurodegenerativas , Degeneração Retiniana , Humanos , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/patologia , Lipopolissacarídeos/farmacologia , Receptores de GABA/metabolismo , Inflamação/metabolismo , Apoptose , Proteínas de Transporte , LigantesRESUMO
Overproduction of reactive oxygen species (ROS) and alterations in metallostasis are common and related hallmarks in several neurodegenerative diseases (NDDs). Nature-based derivatives always represent an attractive tool in MTDL drug design, especially against ROS in NDDs. On this notion, we designed a new series of 8-quinoline-N-substituted derivatives with a natural antioxidant portion (i.e., lipoic, caffeic, and ferulic acids). These compounds were shown to chelate copper, a metal involved in ROS-induced degeneration, and scavenger oxygen radicals in DPPH assay. Then, selected compounds 4 and 5 were evaluated in an in vitro model of oxidative stress and shown to possess cytoprotective effects in 661W photoreceptor-like cells. The obtained results may represent a starting point for the application of the proposed class of compounds in retinal neurodegenerative diseases such as retinitis pigmentosa (RP), comprising a group of hereditary rod-cone dystrophies that represent a major cause of blindness in patients of working age, where the progression of the disease is a multifactorial event, with oxidative stress contributing predominantly.
RESUMO
Retinal diseases can be induced by a variety of factors, including gene mutations, environmental stresses and dysmetabolic processes. The result is a progressive deterioration of visual function, which sometimes leads to blindness. Many treatments are under investigation, though results are still mostly unsatisfactory and restricted to specific pathologies, particularly in the case of gene therapy. The majority of treatments have been tested in animal models, but very few have progressed to human clinical trials. A relevant approach is to study the relation between the type of treatments and the degenerative characteristics of the animal model to better understand the effectiveness of each therapy. Here we compare the results obtained from different animal models treated with natural compounds (saffron and naringenin) to anticipate the potentiality of a single treatment in different pathologies.