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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.

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.

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 (H 2 O 2 ). 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.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Piano, Corsi, Polini and Gargini.)

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.

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.

Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsin gene that can be activated by brief exposure to very intense light. Here, we test the possibility of an anatomical, metabolic, and functional recovery by delivering to degenerating Tvrm4 animals, Myriocin, an inhibitor of ceramide de novo synthesis previously shown to effectively slow down retinal degeneration in rd10 mutants (Strettoi et al., 2010; Piano et al., 2013). Different routes and durations of Myriocin administration were attempted by using either single intravitreal (i.v.) or long-term, repeated intraperitoneal (i.p.) injections. The retinal function of treated and control animals was tested by ERG recordings. Retinas from ERG-recorded animals were studied histologically to reveal the extent of photoreceptor death. A correlation was observed between Myriocin administration, lowering of retinal ceramides, and preservation of ERG responses in i.v. injected cases. Noticeably, the i.p. treatment with Myriocin decreased the extension of the retinal-degenerating area, preserved the ERG response, and correlated with decreased levels of biochemical indicators of retinal oxidative damage. The results obtained in this study confirm the efficacy of Myriocin in slowing down retinal degeneration in genetic models of RP independently of the underlying mutation responsible for the disease, likely targeting ceramide-dependent, downstream pathways. Alleviation of retinal oxidative stress upon Myriocin treatment suggests that this molecule, or yet unidentified metabolites, act on cellular detoxification systems supporting cell survival. Altogether, the pharmacological approach chosen here meets the necessary pre-requisites for translation into human therapy to slow down RP., (Copyright © 2020 Piano, D’Antongiovanni, Novelli, Biagioni, Dei Cas, Paroni, Ghidoni, Strettoi and Gargini.)

Sirtuin 1 (SIRT1) enzyme plays a pivotal role in the regulation of many physiological functions. In particular, it is implicated in ageing-related diseases, such as cardiac hypertrophy, myocardial infarct, and endothelial dysfunction; moreover, its expression decreases with age. Therefore, an effective strategy to extend the lifespan and improve cardiovascular function is the enhancement of the expression/activity of SIRT1 with exogenous agents. The Citrus flavonoid naringenin (NAR) presents structural similarity with the natural SIRT1 activator resveratrol. In this study, we demonstrate through in vitro assays that NAR significantly activates SIRT1 enzyme and shows antisenescence effects. The binding mode of NAR into SIRT1 was detailed investigated through in silico studies. Moreover, chronic administration (for six months) of NAR (100 mg/kg/day) to 6-month-old mice leads to an enhancement of SIRT1 expression and a marked reduction of reactive oxygen species production in myocardial tissue. Furthermore, at the end of the treatment, the plasma levels of two well-known markers of cardiovascular inflammation, TNF- α and IL6, are significantly reduced in 12-month-old mice treated with NAR, as well as the cardiovascular risk (total cholesterol/HDL ratio) compared to control mice. Finally, the age-associated fibrotic remodeling, which is well detected through a Mallory trichrome staining in the vehicle-treated 12-month-old mice, is significantly reduced by the chronic treatment with NAR. Moreover, an improvement of myocardium functionality is highlighted by the enhancement of citrate synthase activity and stabilization of the mitochondrial membrane potential after NAR treatment. Taken together, these results suggest that a nutraceutical approach with NAR may have positive impacts on many critical hallmarks of myocardial senescence, contributing to improve the cardiac performance in aged subjects., Competing Interests: The authors declare no conflict of interests., (Copyright © 2020 Lara Testai et al.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Retinal degeneration 9 (rd9) mice carry a mutation in the retina specific "Retinitis Pigmentosa GTPase Regulator (RPGR)" Open Reading Frame (ORF) 15 gene, located on the X chromosome and represent a rare model of X-linked Retinitis Pigmentosa (XLRP), a common and severe form of retinal degeneration (Wright et al., 2010; Tsang and Sharma, 2018). The rd9 RPGR-ORF15 mutation in mice causes lack of the protein in photoreceptors and a slow degeneration of these cells with consequent decrease in Outer Nuclear Layer (ONL) thickness and amplitude of ERG responses, as previously described (Thompson et al., 2012). However, relative rates of rod and cone photoreceptor loss, as well as secondary alterations occurring in neuronal and non-neuronal retinal cell types of rd9 mutants remain to be assessed. Aim of this study is to extend phenotype analysis of the rd9 mouse retina focusing on changes occurring in cells directly interacting with photoreceptors. To this purpose, first we estimated rod and cone survival and its degree of intraretinal variation over time; then, we studied the morphology of horizontal and bipolar cells and of the retinal pigment epithelium (RPE), extending our observations to glial cell reactivity. We found that in rd9 retinas rod (but not cone) death is the main cause of decrease in ONL thickness and that degeneration shows a high degree of intraretinal variation. Rod loss drives remodeling in the outer retina, with sprouting of second-order neurons of the rod-pathway and relative sparing of cone pathway elements. Remarkably, despite cone survival, functional defects can be clearly detected in ERG recordings in both scotopic and photopic conditions. Moderate levels of Muller cells and microglial reactivity are sided by striking attenuation of staining for RPE tight junctions, suggesting altered integrity of the outer Blood Retina Barrier (BRB). Because of many features resembling slowly progressing photoreceptor degeneration paradigms or early stages of more aggressive forms of RP, the rd9 mouse model can be considered a rare and useful tool to investigate retinal changes associated to a process of photoreceptor death sustained throughout life and to reveal disease biomarkers (e.g., BRB alterations) of human XLRP., (Copyright © 2019 Falasconi, Biagioni, Novelli, Piano, Gargini and Strettoi.)

Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by progressive degeneration of the visual cells and abnormalities in retinal pigment epithelium, the vision is lost slowly, and the final outcome is total blindness. RP primarily affects rods, but cones can also be affected as a secondary effect. Photoreceptor cell death is usually triggered by apoptosis, however the molecular mechanisms linking the rod degeneration to the secondary cone death are poorly understood. Possible causes of the secondary cone death are oxidative stress and/ or the release of toxic factors from dying rods. The aim of this study is to analyze the effect of nutraceutical molecules with antioxidant properties, on the progression of the disease in an established animal model of RP, and rd10 mice. We show that chronic treatment per os with a flavanone (naringenin) or a flavonol (quercetin) present in citrus fruits, grapes and apples, preserves retinal morphology, and ameliorates functionality. These actions are associated with a significant reduction of stress-oxidative markers, such as the detoxifying enzymes Sod1 and Sod2. In addition, naringenin and quercetin treatment reduces the levels of acrolein staining associated with a reduction of ROS in the cellular environment. The study demonstrates the beneficial effects of naringenin and quercetin, two molecules that possess antioxidant properties, limiting neurodegeneration, and thus preventing cone damage.

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., Competing Interests: The authors declare no conflict of interest.

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 (MT 1 and MT 2 ) in 661W cells, then whether MEL can prevent H 2 O 2 -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 H 2 O 2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H 2 O 2 -mediated cell death, a Fas/FasL antagonist was used before the exposure to H 2 O 2 . Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H 2 O 2 and/or MEL. Cell viability was analyzed by using Trypan Blue., Results: Both MEL receptors (MT 1 and MT 2 ) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H 2 O 2 -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 H 2 O 2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H 2 O 2 , and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H 2 O 2 ., Conclusions: The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H 2 O 2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.

Hallmarks of Retinitis Pigmentosa (RP), a family of genetic diseases, are a typical rod-cone-degeneration with initial night blindness and loss of peripheral vision, followed by decreased daylight sight and progressive visual acuity loss up to legal blindness. Great heterogeneity in nature and function of mutated genes, variety of mutations for each of them, variability in phenotypic appearance and transmission modality contribute to make RP a still incurable disease. Translational research relies on appropriate animal models mimicking the genetic and phenotypic diversity of the human pathology. Here, we provide a systematic, morphological and functional analysis of Rho Tvrm4 /Rho + rhodopsin mutant mice, originally described in 2010 and portraying several features of common forms of autosomal dominant RP caused by gain-of-function mutations. These mice undergo photoreceptor degeneration only when exposed briefly to strong, white light and allow controlled timing of induction of rod and cone death, which therefore can be elicited in adult animals, as observed in human RP. The option to control severity and retinal extent of the phenotype by regulating intensity and duration of the inducing light opens possibilities to exploit this model for multiple experimental purposes. Altogether, the unique features of this mutant make it an excellent resource for retinal degeneration research.

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., Competing Interests: The authors declare no conflict of interest.

Background and Purpose: Incidence of cardiovascular disorders increases with age, because of a dramatic fall of endogenous self-defense mechanisms and increased vulnerability of myocardium. Conversely, the effectiveness of many cardioprotective drugs is blunted in hearts of 1 year old rat. The Citrus flavanone naringenin (NAR) was reported to promote cardioprotective effects against ischemia/reperfusion (I/R) injury, through the activation of mitochondrial large conductance calcium-activated potassium channel (mitoBK). These effects were observed in young adult rats, but no data are available about the possible cardioprotective effects of NAR in aged animals. Experimental Approach: This study aimed at evaluating the potential cardioprotective effects of NAR against I/R damage in 1 year old rats, and the possible involvement of mitoBK. Key Results: Naringenin protected the hearts of 1 year old rats in both ex vivo and in vivo I/R protocols. Noteworthy, these effects were antagonized by paxilline, a selective BK-blocker. The cardioprotective effects of NAR were also observed in senescent H9c2 cardiomyoblasts. In isolated mitochondria from hearts of 1 year old, NAR exhibited the typical profile of a mitoBK opener. Finally, Western Blot analysis confirmed a significant (albeit reduced) presence of BK-forming alpha and beta subunits, both in cardiac tissue of 1 year old rats and in senescent H9c2 cells. Conclusion and Implications: This is the first work reporting cardioprotective effects of NAR in 1 year old rats. Although further studies are needed to better understand the whole pathway involved in the NAR-mediated cardioprotection, these preliminary data represent a promising perspective for a rational nutraceutical use of NAR in aging.

Retinitis pigmentosa (RP) comprises a group of inherited pathologies characterized by progressive photoreceptor degeneration. In rodent models of RP, expression of defective genes and retinal degeneration usually manifest during the first weeks of postnatal life, making it difficult to distinguish consequences of primary genetic defects from abnormalities in retinal development. Moreover, mouse eyes are small and not always adequate to test pharmacological and surgical treatments. An inducible paradigm of retinal degeneration potentially extensible to large animals is therefore desirable. Starting from the serendipitous observation that intraocular injections of a Rho GTPase activator, the bacterial toxin Cytotoxic Necrotizing Factor 1 (CNF1), lead to retinal degeneration, we implemented an inducible model recapitulating most of the key features of Retinitis Pigmentosa. The model also unmasks an intrinsic vulnerability of photoreceptors to the mechanism of CNF1 action, indicating still unexplored molecular pathways potentially leading to the death of these cells in inherited forms of retinal degeneration.

The notion that diabetic retinopathy (DR) is essentially a micro-vascular disease has been recently challenged by studies reporting that vascular changes are preceded by signs of damage and loss of retinal neurons. As to the mode by which neuronal death occurs, the evidence that apoptosis is the main cause of neuronal loss is far from compelling. The objective of this study was to investigate these controversies in a mouse model of streptozotocin (STZ) induced diabetes. Starting from 8 weeks after diabetes induction there was loss of rod but not of cone photoreceptors, together with reduced thickness of the outer and inner synaptic layers. Correspondingly, rhodopsin expression was downregulated and the scotopic electroretinogram (ERG) is suppressed. In contrast, cone opsin expression and photopic ERG response were not affected. Suppression of the scotopic ERG preceded morphological changes as well as any detectable sign of vascular alteration. Only sparse apoptotic figures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and glia was not activated. The physiological autophagy flow was altered instead, as seen by increased LC3 immunostaining at the level of outer plexiform layer (OPL) and upregulation of the autophagic proteins Beclin-1 and Atg5. Collectively, our results show that the streptozotocin induced DR in mouse initiates with a functional loss of the rod visual pathway. The pathogenic pathways leading to cell death develop with the initial dysregulation of autophagy well before the appearance of signs of vascular damage and without strong involvement of apoptosis.

Photoreceptors rely upon highly specialized synapses to efficiently transmit signals to multiple postsynaptic targets. Calcium influx in the presynaptic terminal is mediated by voltage-gated calcium channels (VGCC). This event triggers neurotransmitter release, but also gates calcium-activated chloride channels (TMEM), which in turn regulate VGCC activity. In order to investigate the relationship between VGCC and TMEM channels, we analyzed the retina of wild type (WT) and Cacna2d4 mutant mice, in which the VGCC auxiliary α2δ4 subunit carries a nonsense mutation, disrupting the normal channel function. Synaptic terminals of mutant photoreceptors are disarranged and synaptic proteins as well as TMEM16A channels lose their characteristic localization. In parallel, calcium-activated chloride currents are impaired in rods, despite unaltered TMEM16A protein levels. Co-immunoprecipitation revealed the interaction between VGCC and TMEM16A channels in the retina. Heterologous expression of these channels in tsA-201 cells showed that TMEM16A associates with the CaV1.4 subunit, and the association persists upon expression of the mutant α2δ4 subunit. Collectively, our experiments show association between TMEM16A and the α1 subunit of VGCC. Close proximity of these channels allows optimal function of the photoreceptor synaptic terminal under physiological conditions, but also makes TMEM16A channels susceptible to changes occurring to calcium channels.

Protease activated receptors (PARs) are G-protein coupled receptors that are activated by an unique proteolytic mechanism. These receptors play crucial roles in hemostasis and thrombosis but also in inflammation and vascular development. PARs have also been implicated in tumor progression, invasion and metastasis. In this study, we investigated expression and signaling of PAR1 in nonmalignant pleural mesothelial (Met-5A) and malignant pleural mesothelioma (NCI-H28) cells. We found that the expression level of PAR1 was markedly higher in NCI-H28 cells compared to Met-5A and human primary mesothelial cells. Other three malignant pleural mesothelioma cell lines, i.e. REN, Ist-Mes2, and Mero-14, did not show any significant PAR1 over-expression compared to Met-5A cell line. Thrombin and PAR1 activating peptides enhanced Met-5A and NCI-H28 cell proliferation but in NCI-H28 cells higher thrombin concentrations were required to obtain the same proliferation increase. Similarly, thrombin caused extracellular signal-regulated kinase 1/2 activation in both cell lines but NCI-H28 cells responded at higher agonist concentrations. We also determined that PAR1 signaling through Gq and G12/13 proteins is severely altered in NCI-H28 cells compared to Met-5A cells. On the contrary, PAR1 signaling through Gi proteins was persistently maintained in NCI-H28 cells. Furthermore, we demonstrated a reduction of cell surface PAR1 expression in NCI-H28 and malignant pleural mesothelioma REN cells. Thus, our results provide evidences for dysfunctional PAR1 signaling in NCI-H28 cells together with reduced plasma membrane localization. The role of PAR1 in mesothelioma progression is just emerging and our observations can promote further investigations focused on this G-protein coupled receptor.

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., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Rod and cone photoreceptors are coupled by gap junctions (GJs), relatively large channels able to mediate both electrical and molecular communication. Despite their critical location in our visual system and evidence that they are dynamically gated for dark/light adaptation, the full impact that rod-cone GJs can have on cone function is not known. We recorded the photovoltage of mouse cones and found that the initial level of rod input increased spontaneously after obtaining intracellular access. This process allowed us to explore the underlying coupling capacity to rods, revealing that fully coupled cones acquire a striking rod-like phenotype. Calcium, a candidate mediator of the coupling process, does not appear to be involved on the cone side of the junctional channels. Our findings show that the anatomical substrate is adequate for rod-cone coupling to play an important role in vision and, possibly, in biochemical signaling among photoreceptors. DOI: http://dx.doi.org/10.7554/eLife.01386.001.

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 ∼3%. This allowed us to obtain direct functional evidence suggestive of rod–cone 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 rod–rod 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.

Slow, progressive rod degeneration followed by cone death leading to blindness is the pathological signature of all forms of human retinitis pigmentosa (RP). Therapeutic schemes based on intraocular delivery of neuroprotective agents prolong the lifetime of photoreceptors and have reached the stage of clinical trial. The success of these approaches depends upon optimization of chronic supply and appropriate combination of factors. Environmental enrichment (EE), a novel neuroprotective strategy based on enhanced motor, sensory and social stimulation, has already been shown to exert beneficial effects in animal models of various disorders of the CNS, including Alzheimer and Huntington disease. Here we report the results of prolonged exposure of rd10 mice, a mutant strain undergoing progressive photoreceptor degeneration mimicking human RP, to such an enriched environment from birth. By means of microscopy of retinal tissue, electrophysiological recordings, visual behaviour assessment and molecular analysis, we show that EE considerably preserves retinal morphology and physiology as well as visual perception over time in rd10 mutant mice. We find that protective effects of EE are accompanied by increased expression of retinal mRNAs for CNTF and mTOR, both factors known as instrumental to photoreceptor survival. Compared to other rescue approaches used in similar animal models, EE is highly effective, minimally invasive and results into a long-lasting retinal protection. These results open novel perspectives of research pointing to environmental strategies as useful tools to extend photoreceptor survival.

This study investigates the role of two different HCN channel isoforms in the light response of the outer retina. Taking advantage of HCN-deficient mice models and of in vitro (patch-clamp) and in vivo (ERG) recordings of retinal activity we show that HCN1 and HCN2 channels are expressed at distinct retinal sites and serve different functions. Specifically, HCN1 operate mainly at the level of the photoreceptor inner segment from where, together with other voltage sensitive channels, they control the time course of the response to bright light. Conversely, HCN2 channels are mainly expressed on the dendrites of bipolar cells and affect the response to dim lights. Single cell recordings in HCN1⁻/⁻ mice or during a pharmacological blockade of I(h) show that, contrary to previous reports, I(kx) alone is able to generate the fast initial transient in the rod bright flash response. Here we demonstrate that the relative contribution of I(h) and I(kx) to the rods' temporal tuning depends on the membrane potential. This is the first instance in which the light response of normal and HCN1- or HCN2-deficient mice is analyzed in single cells in retinal slice preparations and in integrated full field ERG responses from intact animals. This comparison reveals a high degree of correlation between single cell current clamp data and ERG measurements. A novel picture emerges showing that the temporal profile of the visual response to dim and bright luminance changes is separately determined by the coordinated gating of distinct voltage dependent conductances in photoreceptors and bipolar cells.

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.

Purpose: To elucidate short- and long-term effects of ivabradine, an inhibitor of the hyperpolarization-activated current (I(f)) recently approved for treatment of stable angina, on retinal function and integrity. As careful ivabradine administration is recommended for patients with retinitis pigmentosa, an additional objective was to test the consequences of repeated ivabradine delivery on retinal integrity in the rd10 mouse, an animal model of the human degenerative disease., Methods: The electroretinogram (ERG) was recorded in intact anesthetized animals in response to flashes or time-varied sinusoidal light stimuli of different frequency. Retinal integrity and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel distribution were assessed by immunocytochemistry, confocal microscopy, and Western blot analysis., Results: Neither a- nor b-waves of the flash-ERG were significantly affected by ivabradine administration. Conversely, reversible changes in the response to sinusoidal stimuli were observed during both acute and continued treatment. HCN inhibition enhanced the gain of frequency-response curves (FRCs) at the lowest stimulus frequencies and reduced it in the 1- to 7-Hz range. These effects were dose dependent and reverted to normal 1 week after discontinuation of ivabradine. Retinal morphology and distribution of HCN were preserved and no signs of retinal damage were observed in healthy animals. HCN inhibition in dystrophic mice had no effect on either extent or progression of retinal degeneration., Conclusions: The results are consistent with the hypothesis that the visual symptoms reported by patients during prolonged treatment with ivabradine are due only to a reversible pharmacologic effect.

Light adaptation in vertebrate photoreceptors is mediated by multiple mechanisms, one of which could involve nuclear feedback and changes in gene expression. Therefore, we have investigated light adaptation-associated changes in gene expression using microarrays and real-time PCR in isolated photoreceptors, in cultured isolated retinas and in acutely isolated retinas. In all three preparations after 2 h of an exposure to a bright light, we observed an up-regulation of almost 100% of three genes, Sag, Guca1a and Guca1b, coding for proteins known to play a major role in phototransduction: arrestin, GCAP1 and GCAP2. No detectable up-regulation occurred for light exposures of less than 1 h. Functional in vivo electroretinographic tests show that a partial recovery of the dark current occurred 1-2 h after prolonged illumination with a steady light that initially caused a substantial suppression of the photoresponse. These observations demonstrate that prolonged illumination results in the up-regulation of genes coding for proteins involved in the phototransduction signalling cascade, possibly underlying a novel component of light adaptation occurring 1-2 h after the onset of a steady bright light.

Purpose: To evaluate in mammalian rod photoreceptors the selectivity for hyperpolarization-activated cyclic nucleotide-gated (Hcn1, coded by Hcn1) over potassium-selective (Kir 2.4, coded by Kcnj14) channels of ivabradine, a selective inhibitor of the cardiac "funny" current (I(f))., Methods: Rods were isolated from the mouse retina and voltage clamped by the perforated-patch technique. The hyperpolarization-activated current (I(h)) was blocked by ivabradine during repetitive stimulation with activating/deactivating voltage steps from -80 to -30 mV, from a holding of -35 mV., Results: Full inhibition was observed at a high concentration of ivabradine (30 microM), with intermediate effects at 3 and 0.3 microM. Steady state activation and activation kinetics of the ivabradine- and CsCl-blocked currents were similar, consistent with the block by ivabradine of ion permeation through Hcn1 channels. Hcn1 blockade was also consistent with the lack of current reactivation during long steps at -110 mV. At doses that fully block I(h), ivabradine does not affect the inward rectifier current through potassium-selective Kir 2.4 channels or the outward currents evoked by stepping up from -80 to 50 mV., Conclusions: In mammalian rods, ivabradine is a selective inhibitor of Hcn1 channels. Phosphenes perception in response to abrupt changes in luminance, which has been transiently reported in a dose-dependent way by few patients treated with ivabradine, was consistent with Hcn1 inhibition in rods.

Hyperpolarization-activated cyclic nucleotide-sensitive (HCN) channels mediate the I(f) current in heart and I(h) throughout the nervous system. In spiking neurons I(h) participates primarily in different forms of rhythmic activity. Little is known, however, about its role in neurons operating with graded potentials as in the retina, where all four channel isoforms are expressed. Intriguing evidence for an involvement of I(h) in early visual processing are the side effects reported, in dim light or darkness, by cardiac patients treated with HCN inhibitors. Moreover, electroretinographic recordings indicate that these drugs affect temporal processing in the outer retina. Here we analyzed the functional role of HCN channels in rod bipolar cells (RBCs) of the mouse. Perforated-patch recordings in the dark-adapted slice found that RBCs exhibit I(h), and that this is sensitive to the specific blocker ZD7288. RBC input impedance, explored by sinusoidal frequency-modulated current stimuli (0.1-30 Hz), displays band-pass behavior in the range of I(h) activation. Theoretical modeling and pharmacological blockade demonstrate that high-pass filtering of input signals by I(h), in combination with low-pass filtering by passive properties, fully accounts for this frequency-tuning. Correcting for the depolarization introduced by shunting through the pipette-membrane seal, leads to predict that in darkness I(h) is tonically active in RBCs and quickens their responses to dim light stimuli. Immunohistochemistry targeting candidate subunit isoforms HCN1-2, in combination with markers of RBCs (PKC) and rod-RBC synaptic contacts (bassoon, mGluR6, Kv1.3), suggests that RBCs express HCN2 on the tip of their dendrites. The functional properties conferred by I(h) onto RBCs may contribute to shape the retina's light response and explain the visual side effects of HCN inhibitors.

Purpose: To assess neurotrophic factor upregulation in the retina after damage to the optic nerve and relate that regulation to changes in photoreceptor stability and function., Methods: Retinas of adult pigmented (Long-Evans) rats were examined at successive times (1-60 days) after unilateral optic nerve section. The distribution and expression of ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (FGF-2) and their receptor elements FGFR1 and CNTFRalpha were studied with immunohistochemistry and Western blot analysis. FGF-2 and CNTF mRNA levels were also assessed, with semiquantitative reverse transcription-PCR. Levels and localization of the intracellular signaling molecule ERK and its activated, phosphorylated form pERK, were examined by immunohistochemistry. To assess the correlation between neurotrophic factor levels and their protective effect against light damage, albino (Sprague-Dawley) rats were exposed to bright continuous light (1000 lux) for 24 or 48 hours at successive times after nerve section. The TUNEL technique was used to visualize neuronal cell death in the retina., Results: CNTF upregulation was detected 1 week after optic nerve section, peaked at 2 weeks, and fell to control levels at 4 weeks. CNTF appeared first in the inner retina in the ganglion cells, then in the Muller cells in which it became prominent at the outer limiting membrane (OLM) and in the outer segment (OS) region of photoreceptors. FGF-2 upregulation became prominent, particularly in photoreceptors, 21 to 28 days after surgery, continued to 2 months, and slowly declined thereafter. Double labeling with antibodies to ligand and the receptor showed colocalization of CNTF to its receptor at the OS region, whereas FGF-2-to-FGFR1 binding was found in the outer nuclear (ONL) and outer plexiform (OPL) layers. Optic nerve section provided a significant protective effect against light-induced damage in the first 2 weeks. There was no protection when animals were exposed to damaging light 1 month after nerve section., Conclusions: The upregulation of CNTF 7 to 14 days after nerve section correlates with a reduction in the a-wave described previously. Colocalization of CNTF and CNTFRalpha on the outer segments suggests that CNTF acts at the photoreceptor membrane. The slower upregulation of FGF-2 correlates with a reduction of the b-wave. FGF-2/FGFR1 colocalization in the OPL suggests that this factor acts at the synaptic terminals of photoreceptors, modulating the release of neurotransmitters. The time course of pERK upregulation suggests that the successive upregulation of CNTF and FGF-2 activates the ERK pathway. Based on the time course of protection against bright continuous light, it seems that CNTF plays a major role in this effect, and FGF-2 has a less important role in the protection against light-induced damage.

The temporal properties of the electroretinogram (ERG) recorded from cat eyes were analyzed in the presence of either Cs+ or zatebradine which are known to inhibit the hyperpolarization activated current (Ih) in retinal rods. Both Cs+ and zatebradine reduce the ERG response to high-frequency sinusoidal stimuli of high mean luminance and contrast. Conversely, blockade of Ih has no effect on the frequency response characteristics of the isolated receptor component (PIII). These observations support the idea that Ih plays an important role in the transfer of signals from photoreceptors to second order neurons by suppressing the slow components originated in the phototransductive cascade. The result of this operation is an enhancement of the light response in a range of temporal frequencies relevant to vision.

An harmonic analysis was applied to the electroretinogram (ERG) measured in intact cat eyes in control conditions and after pharmacological isolation of the components attributed to photoreceptors (PIII) and bipolar neurons (PII). The frequency response curves obtained in various conditions showed that the bandwidth of the PII component extends over a range of stimulus frequencies higher than the bandwidth of PIII. The enhancement of the PII response to stimuli of high temporal frequency suggests the presence of a frequency dependent gain control located either pre- and/or post-synaptically in the transmission line between the phototransductive cascade and bipolar neurons. A possible role of these processes is to enhance relevant visual information whilst selectively attenuating low frequency signals originating in the transductive cascade.

The dendrites of ganglion cells initially ramify throughout the inner plexiform layer of the developing retina before becoming stratified into ON or OFF sublaminae. This ontogenetic event is thought to depend on glutamate-mediated afferent activity, because treating the developing retina with the glutamate analog 2-amino-4-phosphonobutyrate (APB), which hyperpolarizes ON cone bipolar cells and rod bipolar cells, thereby preventing their release of glutamate, effectively arrests the dendritic stratification process. To assess the functional consequences of this manipulation, extracellular recordings were made from single cells in the A laminae of the dorsal lateral geniculate nucleus and from the optic tract in mature cats that had received intraocular injections of APB during the first postnatal month. Such recordings revealed that stimulation of the APB-treated eye evoked both ON as well as OFF discharges in 37% of the cells tested. (As expected, when the normal eye was activated, virtually all cells yielded only ON or OFF responses.) The proportion of ON-OFF cells found here corresponds closely to the incidence of multistratified dendrites observed previously in anatomical studies of APB-treated cat retinas. This suggests that the ganglion cells with multistratified dendrites receive functional inputs from ON as well as OFF cone bipolar cells. This interpretation is further supported by the finding that the proportion of ON-OFF cells was very similar in the geniculate layer innervated by the treated eye and in the optic tract. The cells activated by the APB-treated eye were also found not to show response suppression when flashing stimuli of increasing size were used. This suggests that exposing the developing retina to APB perturbs the neural circuitry mediating the antagonistic center-surround organization found in normal receptive fields. The functional changes evident after treating the developing retina with APB suggest that it should now be feasible to assess how the segregation of ON and OFF retinal pathways relates to organizational features at higher levels of the visual system, such as orientation selectivity in cortical cells.