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Background: miR-210 is one of the most evolutionarily conserved microRNAs. It is known to be involved in several physiological and pathological processes, including response to hypoxia, angiogenesis, cardiovascular diseases and cancer. Recently, new roles of this microRNA are emerging in the context of eye and visual system homeostasis. Recent studies in Drosophila melanogaster unveiled that the absence of miR-210 leads to a progressive retinal degeneration characterized by the accumulation of lipid droplets and disruptions in lipid metabolism. However, the possible conservation of miR-210 knock-out effect in the mammalian retina has yet to be explored., Results: We further investigated lipid anabolism and catabolism in miR-210 knock-out (KO) flies, uncovering significant alterations in gene expression within these pathways. Additionally, we characterized the retinal morphology of flies overexpressing (OE) miR-210, which was not affected by the increased levels of the microRNA. For the first time, we also characterized the retinal morphology of miR-210 KO and OE mice. Similar to flies, miR-210 OE did not affect retinal homeostasis, whereas miR-210 KO mice exhibited photoreceptor degeneration. To explore other potential parallels between miR-210 KO models in flies and mice, we examined lipid metabolism, circadian behaviour, and retinal transcriptome in mice, but found no similarities. Specifically, RNA-seq confirmed the lack of involvement of lipid metabolism in the mice's pathological phenotype, revealing that the differentially expressed genes were predominantly associated with chloride channel activity and extracellular matrix homeostasis. Simultaneously, transcriptome analysis of miR-210 KO fly brains indicated that the observed alterations extend beyond the eye and may be linked to neuronal deficiencies in signal detection and transduction., Conclusions: We provide the first morphological characterization of the retina of miR-210 KO and OE mice, investigating the role of this microRNA in mammalian retinal physiology and exploring potential parallels with phenotypes observed in fly models. Although the lack of similarities in lipid metabolism, circadian behaviour, and retinal transcriptome in mice suggests divergent mechanisms of retinal degeneration between the two species, transcriptome analysis of miR-210 KO fly brains indicates the potential existence of a shared upstream mechanism contributing to retinal degeneration in both flies and mammals., (© 2024. The Author(s).)

Pituitary neuroendocrine tumors (PitNETs) are generally benign but comprise an aggressive, invasive, therapy-resistant, metastatic subset, underpinning a need for novel therapeutic targets. PitNETs exhibit low mutation rates but are associated with conditions linked to alternative splicing, an alternative oncogene pathway activation mechanism. PitNETs express the neurotrophin receptor TrkA, which exhibits oncogenic alternative TrkAIII splicing in other neuroendocrine tumors. We, therefore, assessed whether TrkAIII splicing represents a potential oncogenic participant in PitNETs. TrkAIII splicing was RT-PCR assessed in 53 PitNETs and TrkA isoform(s) expression and activation were assessed by confocal immunofluorescence. TrkAIII splicing was also compared to HIF1α, HIF2α, SF3B1, SRSF2, U2AF1, and JCPyV large T antigen mRNA expression, Xbp1 splicing, and SF3B1 mutation. TrkAIII splicing was detected in all invasive and most non-invasive PitNETs and was significantly elevated in invasive cases. In PitNET lineages, TrkAIII splicing was significantly elevated in invasive PIT1 PitNETs and high in invasive and non-invasive SF1 and TPIT lineages. Immunoreactivity consistent with TrkAIII activation characterized PitNET expressing TrkAIII mRNA, and invasive Pit1 PitNETs exhibited elevated HIF2α expression. TrkAIII splicing did not associate with SF3B1 mutations, altered SF3B1 , SRSF2 , and U2AF1 or JCPyV large T antigen expression, or Xbp1 splicing. Therefore, TrkAIII splicing is common in PitNETs, is elevated in invasive, especially PIT1 tumors, can result in intracellular TrkAIII activation, and may involve hypoxia. The data support a role for TrkAIII splicing in PitNET pathogenesis and progression and identify TrkAIII as a novel potential target in refractory PitNETs.

Age-related macular degeneration (AMD) is a complex, multifactorial disease leading to progressive and irreversible retinal degeneration, whose pathogenesis has not been fully elucidated yet. Due to the complexity and to the multiple features of the disease, many efforts have been made to develop animal models which faithfully reproduce the overall AMD hallmarks or that are able to mimic the different AMD stages. In this context, light damage (LD) rodent models of AMD represent a suitable and reliable approach to mimic the different AMD forms (dry, wet and geographic atrophy) while maintaining the time-dependent progression of the disease. In this review, we comprehensively reported how the LD paradigms reproduce the main features of human AMD. We discuss the capability of these models to broaden the knowledge in AMD research, with a focus on the mechanisms and the molecular hallmarks underlying the pathogenesis of the disease. We also critically revise the remaining challenges and future directions for the use of LD models., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Resolvin E1 (RvE1) is an eicosapentaenoic acid-derived lipid mediator involved in the resolution of inflammation. Here, we investigated whether RvE1 alterations may occur in an animal model of age-related macular degeneration (AMD). To this end, Sprague Dawley albino rats underwent light damage (LD), and retinas and serum were analyzed immediately or seven days after treatment. Western blot of retinas showed that the RvE1 receptor ChemR23 and the RvE1 metabolic enzymes 5-LOX and COX-2 were unchanged immediately after LD, but they were significantly up-regulated seven days later. Instead, the RvE1 receptor BLT1 was not modulated by LD, and neither was the RvE1 degradative enzyme 15-PGDH. Moreover, ChemR23, 5-LOX, COX-2 and BLT1 were found to be more expressed in the inner retina under all experimental conditions, as observed through ImageJ plot profile analysis. Of note, amacrine cells highly expressed BLT1, while ChemR23 was highly expressed in the activated microglia of the outer retina. ELISA assays also showed that LD rats displayed significantly higher circulating levels and reduced retinal levels of RvE1 compared to controls. Altogether, our data indicate that RvE1 metabolism and signaling are modulated in the LD model, suggesting a potentially relevant role of this pathway in AMD.

Post-therapeutic relapse, poor survival rates and increasing incidence justify the search for novel therapeutic targets and strategies in cutaneous malignant melanoma (CMM). Within this context, a potential oncogenic role for TrkA in CMM is suggested by reports of NTRK1 amplification, enhanced TrkA expression and intracellular TrkA activation associated with poor prognosis. TrkA, however, exhibits tumour-suppressing properties in melanoma cell lines and has recently been reported not to be associated with CMM progression. To better understand these contradictions, we present the first analysis of potential oncogenic alternative TrkA mRNA splicing, associated with TrkA immunoreactivity, in CMMs, and compare the behaviour of fully spliced TrkA and the alternative TrkAIII splice variant in BRAF(V600E) -mutated A375 melanoma cells. Alternative TrkA splicing in CMMs was associated with unfolded protein response (UPR) activation. Of the several alternative TrkA mRNA splice variants detected, TrkAIII was the only variant with an open reading frame and, therefore, oncogenic potential. TrkAIII expression was more frequent in metastatic CMMs, predominated over fully spliced TrkA mRNA expression in ≈50% and was invariably linked to intracellular phosphorylated TrkA immunoreactivity. Phosphorylated TrkA species resembling TrkAIII were also detected in metastatic CMM extracts. In A375 cells, reductive stress induced UPR activation and promoted TrkAIII expression and, in transient transfectants, promoted TrkAIII and Akt phosphorylation, enhancing resistance to reductive stress-induced death, which was prevented by lestaurtinib and entrectinib. In contrast, fully spliced TrkA was dysfunctional in A375 cells. The data identify fully spliced TrkA dysfunction as a novel mechanism for reducing melanoma suppression, support a causal relationship between reductive stress, UPR activation, alternative TrkAIII splicing and TrkAIII activation and characterise a targetable oncogenic pro-survival role for TrkAIII in CMM.

Opposing dose-dependent effects of curcumin (Cur) have been documented in Retinal Pigment Epithelium (RPE); therefore, to shed the light on the mechanisms of action is crucial for ophthalmic applications. On this basis we explored new insights about the dose-dependent mechanisms triggered by Cur in human retinal pigment epithelial cells (ARPE-19). Three concentrations (0.01 mM; 0.05 mM; 0.1 mM) of Cur were tested, followed by morphological, molecular, and functional analysis of the cells. Cur 0.01 mM promotes a significant increase in cell proliferation, not affecting cell cycle progression and apoptosis; by contrast, Cur 0.05 mM and 0.1 mM block cellular proliferation and trigger S-phase cell cycle arrest without inducing apoptosis. The observation of neuronal-like morphological changes in Cur 0.05 mM and 0.1 mM were not associated with neuronal differentiation, as observed by the quantification of Neurofilament-200 and by the analysis of voltage-dependent currents by patch clamp. Evaluation of autophagic markers LC3BII and p62 revealed significant modulations, suggesting an important activation of autophagy in ARPE-19 cells treated with Cur 0.05 mM and Cur 0.1 mM; conversely, Cur 0.01 mM did not affect autophagy. Altogether, our findings show new dose-dependent mechanisms of action of Cur that suggest a wide therapeutic application in ocular diseases with different pathogenesis (i.e., proliferative vitreoretinopathy or Age-Related Macular Degeneration).

The mammalian target of rapamycin (mTOR) signaling plays a critical role in cell homeostasis, growth and survival. Here, we investigated the localization of the main mTOR signaling proteins in the organ of Corti of normal-hearing and deafened guinea pigs, as well as their possible modulation by exogenously administered brain-derived neurotrophic factor (BDNF) in deafened guinea pigs. Animals were ototoxically deafened by systemic administration of kanamycin and furosemide, and one week later, the right cochleas were treated with gelatin sponge soaked in rhBDNF, while the left cochleas were used as negative controls. Twenty-four hours after treatment, animals were euthanized, and the cochleas were processed for subsequent analysis. Through immunofluorescence, we demonstrated the localization of AKT, pAKT, mTOR, pmTOR and PTEN proteins throughout the cochlea of guinea pigs for the first time, with a higher expression in supporting cells. Moreover, an increase in mTOR immunostaining was observed in BDNF-treated cochleas by means of fluorescence intensity compared to the other groups. Conversely, Western blot analysis showed no significant differences in the protein levels between groups, probably due to dilution of proteins in the neighboring tissues of the organ of Corti. Altogether, our data indicate that mTOR signaling proteins are expressed by the organ of Corti (with a major role for supporting cells) and that the modulation of mTOR may be a protective mechanism triggered by BDNF in the degenerating organ of Corti.

Retinal degeneration is the major and principal cause behind many incurable blindness diseases. Several studies indicated the neuroprotective effect of Curcuma longa in eye pathologies, specifically retinopathy. However, the molecular mechanism behind its effect has not been completely elucidated. Using an ex vivo model of retinal degeneration obtained from an ex vivo optic nerve cut (ONC), we demonstrated that Curcuma extract (Cur) exerted a neuroprotective effect. Importantly, Cur was able to modulate apoptosis and MAPK signaling pathway activation and prevent retinal ganglion cell (RGC) loss. Other well-known neuroprotective pharmacological tools, including memantine (Mem), citicoline (Cit), and ginkgolic acid (GA), were used to compare the potential mechanisms of Cur. The antioxidant activity of retinas treated with Cur following optic nerve cut was significantly higher than control, but Cur failed to change the retina glutamate content. Considering the antioxidant effect of Cur and taking advantage of our recent findings on the crosstalk between oxidative stress and post-translational protein modifiers, in particular, small ubiquitin-related modifier (SUMO), we were interested in exploring the effect of Cur on SUMOylation. We found that Cur significantly prevented the increase of protein SUMOylation, confirming our previous in vitro data indicating the cytoprotective effect of curcumin through modulating the oxidative stress and SUMO-JNK axis. Altogether, these results suggest that Curcuma protects the retina from degeneration via antioxidant activity and targets SUMOylation. Therefore, it might be considered for the combination therapy with other neuroprotective agents with different mechanisms in preclinical studies on retinal degeneration., Competing Interests: The authors declare that they have no conflicts of interest regarding the publication of this paper., (Copyright © 2022 Kambiz Hassanzadeh et al.)

In this study, we investigated whether cerium oxide nanoparticles (CeO 2 -NPs), a promising antioxidant nanomaterial, may contrast retinal vascular alterations induced by oxidative damage in vitro and in vivo. For the in vivo experiments, the light damage (LD) animal model of Age-Related Macular Degeneration (AMD) was used and the CeO 2 -NPs were intravitreally injected. CeO 2 -NPs significantly decreased vascular endothelial growth factor (VEGF) protein levels, reduced neovascularization in the deep retinal plexus, and inhibited choroidal sprouting into the photoreceptor layer. The in vitro experiments were performed on human retinal pigment epithelial (ARPE-19) cells challenged with H 2 O 2 ; we demonstrated that CeO 2 -NPs reverted H 2 O 2 -induced oxidative stress-dependent effects on this cell model. We further investigated the RPE-endothelial cells interaction under oxidative stress conditions in the presence or absence of CeO 2 -NPs through two experimental paradigms: (i) treatment of human umbilical vein endothelial cells (HUVECs) with conditioned media from ARPE-19 cells, and (ii) coculture of ARPE-19 and HUVECs. In both experimental conditions, CeO 2 -NPs were able to revert the detrimental effect of H 2 O 2 on angiogenesis in vitro by realigning the level of tubule formation to that of the control. Altogether, our results indicate, for the first time, that CeO 2 -NPs can counteract retinal neovascularization and may be a new therapeutic strategy for the treatment of wet AMD.

Purpose: Recent studies have shown the presence of SARS-CoV-2 entry factors on the ocular surface, identifying the eye as an additional entry route for the virus. Moreover, the coexpression of angiotensin-converting enzyme 2 (ACE2) with other SARS-CoV-2 entry factors [transmembrane protease serine 2 (TMPRSS2), transmembrane protease serine 4 (TMPRSS4), and dipeptidyl peptidase-4 (DPP4)] facilitates the virus infection. Methods: Here, we performed a study over 10 adult corneal and limbal tissues from human donors, both male and female between 58 and 85 years of age. Some of the main virus entry factors were analyzed and their expression was quantified and correlated with the age and sex of the donors through western blot. The receptors' localization was investigated through immunofluorescence. Results: Immunofluorescence confirmed the localization of ACE2 and TMPRSS2 on the ocular surface and showed, for the first time, the localization of TMPRSS4 and DPP4 in limbal and corneal epithelial superficial cells. The quantitative analysis showed that the expression of SARS-CoV-2 entry factors on corneal and limbal cells is likely to be modulated in an age-dependent manner, in agreement with the increased susceptibility to COVID-19 in the elderly. Moreover, we found a relationship between the expression of TMPRSS proteases with the activation state of limbal cells in 80-year-old donors. Conclusion: This study provides information on the expression of SARS-CoV-2 entry factors on the ocular surface of 10 adult human donors and is a first observation of a possible age-dependent modulation on corneal and limbal tissues. Our data pave the way to further investigate the susceptibility to the infection through the ocular surface in the elderly.

We investigated whether treatment with brain-derived neurotrophic factor (BDNF), which is known to protect spiral ganglion cells (SGCs), could also protect hair cells (HCs) and supporting cells (SCs) in the organ of Corti of a guinea pig model of sensorineural hearing loss. Hearing loss was induced by administration of kanamycin/furosemide and two BDNF treatments were performed: (1) by gelatin sponge (BDNF-GS) with acute cochlear implantation (CI), and (2) through a mini-osmotic pump (BDNF-OP) with chronic CI. Outer HCs (OHCs), inner HCs (IHCs), Border, Phalangeal, Pillar, Deiters', and Hensen's cells were counted. The BDNF-GS cochleas had significantly fewer OHCs compared to the untreated ones, while the IHC and SC numbers did not differ between treated and untreated cochleas. The BDNF-OP group showed similar cell numbers to the untreated group. SGC packing density was not correlated with the total number of SCs for either BDNF group. Our data suggest that: (1) BDNF does not prevent cell death in the organ of Corti, and that the protection of SGCs could result from a direct targeting by BDNF; (2) BDNF might induce a different function/activity of the remaining cells in the organ of Corti (independently from cell number).

Retinal ganglion cell (RGC) loss is a pathologic feature common to several retinopathies associated to optic nerve damage, leading to visual loss and blindness. Although several scientific efforts have been spent to understand the molecular and cellular changes occurring in retinal degeneration, an effective therapy to counteract the retinal damage is still not available. Here we show that eyeballs, enucleated with the concomitant optic nerve cut (ONC), when kept in PBS for 24 h showed retinal and optic nerve degeneration. Examining retinas and optic nerves at different time points in a temporal window of 24 h, we found a thinning of some retinal layers especially RGC's layer, observing a powerful RGC loss after 24 h correlated with an apoptotic, MAPKs and degradative pathways dysfunctions. Specifically, we detected a time-dependent increase of Caspase-3, -9 and pro-apoptotic marker levels, associated with a strong reduction of BRN3A and NeuN levels. Importantly, a powerful activation of JNK, c-Jun, and ERK signaling (MAPKs) were observed, correlated with a significant augmented SUMO-1 and UBC9 protein levels. The degradation signaling pathways was also altered, causing a significant decrease of ubiquitination level and an increased LC3B activation. Notably, it was also detected an augmented Tau protein level. Curcumin, a powerful antioxidant natural compound, prevented the alterations of apoptotic cascade, MAPKs, and SUMO-1 pathways and the degradation system, preserving the RGC survival and the retinal layer thickness. This ex vivo retinal degeneration model could be a useful method to study, in a short time window, the effect of neuroprotective tools like curcumin that could represent a potential treatment to contrast retinal cell death., (© 2021. The Author(s).)

Parkinson's disease (PD) and diabetes mellitus share similar pathophysiological characteristics, genetic and environmental factors. It has been reported that people with diabetes mellitus appear to have a remarkable higher incidence of PD than age matched non diabetic individuals. Evidences suggest that use of antidiabetic glitazone is associated with a diminished risk of PD incidence in patients with diabetes. This study examined the effect of lobeglitazone, a member of thiazolidinedione class, in rat model of Parkinson's disease with diabetes co-morbidity. Rats received either rotenone and/or a combination of streptozocin and a high calorie diet for disease induction and they were treated with different doses of lobeglitazone or its vehicle. Behavioral tests comprising rotarod, bar test and rearing test were conducted to evaluate the motor function. Changes in the level tyrosine hydroxylase, TNF-α and NF-κB were analyzed using ELISA. In the same brain regions the possible changes in PPAR-γ receptor level were evaluated. Findings showed that although lobeglitazone tends to reverse the effect of rotenone in animals with diabetes, it was just able to prevent partly the motor defect in rearing test. Furthermore, lobeglitazone (1 mg/kg) reversed, in substantia nigra and striatum, the changes in tyrosine hydroxylase, TNF-α, NF-κB and PPAR-γ receptor content induced by rotenone in rats with diabetic condition. Although other preclinical studies are needed, these findings suggest that lobeglitazone is a promising neuroprotective candidate for clinical trials for PD patients with diabetes co-morbidity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The blood retinal barrier (BRB) is a fundamental eye component, whose function is to select the flow of molecules from the blood to the retina and vice-versa, and its integrity allows the maintenance of a finely regulated microenvironment. The outer BRB, composed by the choriocapillaris, the Bruch's membrane, and the retinal pigment epithelium, undergoes structural and functional changes in age-related macular degeneration (AMD), the leading cause of blindness worldwide. BRB alterations lead to retinal dysfunction and neurodegeneration. Several risk factors have been associated with AMD onset in the past decades and oxidative stress is widely recognized as a key factor, even if the exact AMD pathophysiology has not been exactly elucidated yet. The present review describes the BRB physiology, the BRB changes occurring in AMD, the role of oxidative stress in AMD with a focus on the outer BRB structures. Moreover, we propose the use of cerium oxide nanoparticles as a new powerful anti-oxidant agent to combat AMD, based on the relevant existing data which demonstrated their beneficial effects in protecting the outer BRB in animal models of AMD.

Merkel cell carcinomas (MCCs) are rare, aggressive, cutaneous neuroendocrine tumours, approximately 80% of which are caused by the genomic integration of Merkel cell polyomavirus (MCPyV). MCPyV-positive MCCs carry poor prognosis in approximately 70% of cases, highlighting the need for greater understanding of the oncogenic mechanisms involved in pathogenesis, progression and post-therapeutic relapse, and translation into novel therapeutic strategies. In a previous pilot study, we reported a potential relationship between MCPyV gene expression and oncogenic alternative Δ exon 6-7 TrkAIII splicing in formalin-fixed paraffin-embedded (FFPE) MCC tissues from a 12-patient cohort of >90% MCPyV-positive MCCs, diagnosed at San Salvatore Hospital, L'Aquila, Italy, characterising a new MCC subgroup and unveiling a novel potential MCPyV oncogenic mechanism and therapeutic target. This, however, could not be fully verified due to poor RNA quality and difficulty in protein extraction from FFPE tissues. Here, therefore, we extend our previous observations to confirm the relationship between MCPyV and oncogenic alternative Δ exon 6-7 TrkAIII splicing in fresh, nonfixed, MCPyV-positive MCC metastasis by detecting sequence-verified RT-PCR products, including full-length Δ exon 6-7 TrkAIII, and by Western blot detection of a 100 kDa TrkA protein isoform of identical size to 100 kDa Δ exon 6-7 TrkAIII expressed by stable transfected SH-SY5Y cells. We also report that in three MCC patients submitted for multidisciplinary treatment, including locoregional chemotherapy, MCPyV large T-antigen mRNA expression, Δ exon 6-7 TrkAIII mRNA expression and intracellular indirect immunofluorescence (IF) TrkA and phosphorylation protein isoform(s) immunoreactivity in FFPE tissues were not reduced in postchemotherapeutic-relapsed MCCs compared to pretherapeutic MCCs, extending the possible roles of this novel potential MCPyV oncogenic mechanism from MCC pathogenesis to post-therapeutic relapse and progression. Detection of alternative Δ exon 6-7 TrkAIII splicing in MCC, therefore, not only characterises a new MCPyV-positive MCC subgroup and unveils a novel potential MCPyV oncogenic mechanism but also identifies patients who may benefit from inhibitors of MCPyV T-antigen and/or TrkAIII expression or clinically approved Trk kinase inhibitors such as larotrectinib or entrectinib, which are known to inhibit activated TrkA oncogenes and to elicit durable responses in TrkA-fusion oncogene-driven cancers, supporting the call for a large-scale multicentre clinical study.

Retinal pigment epithelium (RPE) dysfunction and degeneration underlie the development of age-related macular degeneration (AMD), which is the leading cause of blindness worldwide. In this study, we investigated whether cerium oxide nanoparticles (CeO 2 -NPs or nanoceria), which are anti-oxidant agents with auto-regenerative properties, are able to preserve the RPE. On ARPE-19 cells, we found that CeO 2 -NPs promoted cell viability against H 2 O 2 -induced cellular damage. For the in vivo studies, we used a rat model of acute light damage (LD), which mimics many features of AMD. CeO 2 -NPs intravitreally injected three days before LD prevented RPE cell death and degeneration and nanoceria labelled with fluorescein were found localized in the cytoplasm of RPE cells. CeO 2 -NPs inhibited epithelial-mesenchymal transition of RPE cells and modulated autophagy by the down-regulation of LC3B-II and p62. Moreover, the treatment inhibited nuclear localization of LC3B. Taken together, our study demonstrates that CeO 2 -NPs represent an eligible candidate to counteract RPE degeneration and, therefore, a powerful therapy for AMD.

Cerium oxide nanoparticles (CeO 2 -NPs; or nanoceria) have been largely studied for biomedical applications due to their peculiar auto-regenerative antioxidant activity. This review focuses on ophthalmic applications of nanoceria. Many in vivo data indicate that nanoceria protect the retina from neurodegeneration. In particular, they have been tested in animal models of age-related macular degeneration and retinitis pigmentosa and their neuroprotective properties have been shown to persist for a long time, without any collateral effects. In vitro cytotoxicity studies have shown that CeO 2 -NPs could be safe for lens cells and could represent a new therapy for cataract treatment, but further studies are needed. To date, different pharmaceutical formulations based on nanoceria have been created looking at future clinical ophthalmic applications, such as water-soluble nanoceria, glycol chitosan-coated ceria nanoparticles (GCCNPs), and alginate-gelatin hydrogel loaded GCCNPs. GCCNPs were also effective in preventing choroidal neovascularization in vivo . Based on the nanosize of nanoceria, corneal permeation could be achieved to allow topical treatment of nanoceria. PEGylation and encapsulation in liposomes represent the main strategies to support corneal permeation, without altering nanoceria chemical-physical properties. Based on their great antioxidant properties, safety, and nanosize, nanoceria represent a new potential therapeutic for the treatment of several eye disorders.

Background: Merkel cell carcinomas (MCCs) are rare, aggressive, therapeutically-challenging skin tumours that are increasing in incidence and have poor survival rates. The majority are caused by genomic Merkel cell polyomavirus (MCPyV) integration and MCPyV T-antigen expression. Recently, a potential oncogenic role for the tropomyosin-related tyrosine kinase A receptor (TrkA) has been proposed in MCC. Alternative TrkAIII splicing is a TrkA oncogenic activation mechanism that can be promoted by SV40 large T-antigen, an analogue of MCPyV large T-antigen. In this pilot study, therefore, we have evaluated TrkAIII splicing as a novel potential oncogenic mechanism and therapeutic target in MCPyV positive MCC., Methods: Formalin-fixed paraffin-embedded MCC tissues, consisting of 10 stage IV, 1 stage IIIB, 1 stage IIB, 4 stage IIA and 2 stage I tumours, from patients diagnosed and treated from September 2006 to March, 2019, at the University of L'Aquila, L'Aquila, Italy, were compared to 3 primary basal cell carcinomas (BCCs), 3 primary squamous cell carcinomas (SCCs) and 2 normal skin samples by RT-PCR for MCPyV large T-antigen, small T-antigen, VP-1 expression and alternative TrkAIII splicing and by indirect IF for evidence of intracellular TrkA isoform expression and activation., Results: 9 of 10 Recurrent stage IV MCCs were from patients (P.1-3) treated with surgery plus loco-regional Melphalan chemotherapy and remaining MMCs, including 1 stage IV tumour, were from patients treated with surgery alone (P. 4-11). All MCPyV positive MCCs exhibiting MCPyV large T-antigen expression (17 of 18MCCs, 90%) exhibited alternative TrkAIII mRNA splicing (100%), which was exclusive in a significant number and predominant (> 50%) in all stage IV MCCs and the majority of stage 1-III MCCs. MCCs with higher TrkAIII to 18S rRNA expression ratios also exhibited strong or intermediate immunoreactivity to anti-TrkA antibodies, consistent with cytoplasmic TrkAIII expression and activation. In contrast, the MCPyV negative MCC, BCCs, SCCs and normal skin tissues all exhibited exclusive fully-spliced TrkA mRNA expression, associated with variable immunoreactivity for non-phosphorylated but not phosphorylated TrkA., Conclusions: MCPyV positive MCCs but not MCPyV negative MCC, BCCs and SCCs exhibit predominant alternative TrkAIII splicing, with evidence of intracellular TrkAIII activation. This establishes a new potential MCC subset, unveils a novel potential MCPyV oncogenic mechanism and identifies TrkAIII as a novel potential therapeutic target in MCPyV positive MCC.

The degeneration of photoreceptors in the retina is one of the major causes of adult blindness in humans. Unfortunately, no effective clinical treatments exist for the majority of retinal degenerative disorders. Here we report on the fabrication and functional validation of a fully organic prosthesis for long-term in vivo subretinal implantation in the eye of Royal College of Surgeons rats, a widely recognized model of retinitis pigmentosa. Electrophysiological and behavioural analyses reveal a prosthesis-dependent recovery of light sensitivity and visual acuity that persists up to 6-10 months after surgery. The rescue of the visual function is accompanied by an increase in the basal metabolic activity of the primary visual cortex, as demonstrated by positron emission tomography imaging. Our results highlight the possibility of developing a new generation of fully organic, highly biocompatible and functionally autonomous photovoltaic prostheses for subretinal implants to treat degenerative blindness.

We investigated the effects of continuous artificial light exposure on the mouse substantia nigra (SN). A three month exposure of C57Bl/6J mice to white fluorescent light induced a 30% reduction in dopamine (DA) neurons in SN compared to controls, accompanied by a decrease of DA and its metabolites in the striatum. After six months of exposure, neurodegeneration progressed slightly, but the level of DA returned to the basal level, while the metabolites increased with respect to the control. Three month exposure to near infrared LED light (∼710nm) did not alter DA neurons in SN, nor did it decrease DA and its metabolites in the striatum. Furthermore mesencephalic cell viability, as tested by [ 3 H]DA uptake, did not change. Finally, we observed that 710nm LED light, locally conveyed in the rat SN, could modulate the firing activity of extracellular-recorded DA neurons. These data suggest that light can be detrimental or beneficial to DA neurons in SN, depending on the source and wavelength., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Experimental studies demonstrated that saffron (Crocus sativus) given as a dietary supplement counteracts the effects of bright continuous light (BCL) exposure in the albino rat retina, preserving both morphology and function and probably acting as a regulator of programmed cell death [1]. The purpose of this study was to ascertain whether the neuroprotective effect of saffron on rat retina exposed to BCL is associated with a modulation of the endocannabinoid system (ECS). To this aim, we used eight experimental groups of Sprague-Dawley rats, of which six were exposed to BCL for 24 hours. Following retinal function evaluation, retinas were quickly removed for biochemical and morphological analyses. Rats were either saffron-prefed or intravitreally injected with selective type-1 (CB1) or type-2 (CB2) cannabinoid receptor antagonists before BCL. Prefeeding and intravitreally injections were combined in two experimental groups before BCL. BCL exposure led to enhanced gene and protein expression of retinal CB1 and CB2 without affecting the other ECS elements. This effect of BCL on CB1 and CB2 was reversed by saffron treatment. Selective CB1 and CB2 antagonists reduced photoreceptor death, preserved morphology and visual function of retina, and mitigated the outer nuclear layer (ONL) damage due to BCL. Of interest, CB2-dependent neuroprotection was more pronounced than that conferred by CB1. These data suggest that BCL modulates only distinct ECS elements like CB1 and CB2, and that saffron and cannabinoid receptors could share the same mechanism in order to afford retinal protection., Competing Interests: Competing Interests: This investigation was partially supported by the commercial companies Essse Caffè S.p.A. and Hortus Novus s.r.l. We confirm that this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Replacement strategies arise as promising approaches in case of inherited retinal dystrophies leading to blindness. A fully organic retinal prosthesis made of conjugated polymers layered onto a silk fibroin substrate is engineered. First, the biophysical and surface properties are characterized; then, the long-term biocompatibility is assessed after implantation of the organic device in the subretinal space of 3-months-old rats for a period of five months. The results indicate a good stability of the subretinal implants over time, with preservation of the physical properties of the polymeric layer and a tight contact with the outer retina. Immunoinflammatory markers detect only a modest tissue reaction to the surgical insult and the foreign body that peaks shortly after surgery and progressively decreases with time to normal levels at five months after implantation. Importantly, the integrity of the polymeric layer in direct contact with the retinal tissue is preserved after five months of implantation. The recovery of the foreign-body tissue reaction is also associated with a normal b-wave in the electroretinographic response. The results demonstrate that the device implanted in nondystrophic eyes is well tolerated, highly biocompatible, and suitable as retinal prosthesis in case of photoreceptor degeneration., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In human SH-SY5Y neuroblastoma (NB) cells, nascent immature N-glycosylated 110kDa TrkA moves rapidly from the endoplasmic reticulum (ER) to the Golgi Network (GN), where it matures into the 140kDa receptor prior to being transported to the cell surface, creating GN and cell surface pools of inactive receptor maintained below the spontaneous activation threshold by a full compliment of inhibitory domains and endogenous PTPases. In contrast, the oncogenic alternative TrkAIII splice variant is not expressed at the cell surface but re-localises to intracellular membranes, within which it exhibits spontaneous ERGIC/COPI-associated activation and oncogenic Akt signalling. In this study, we characterise the mechanism responsible for TrkAIII re-localisation. Spontaneous TrkAIII activation, facilitated by D4 IG-like domain and N-glycosylation site omission, increases spontaneous activation potential by altering intracellular trafficking, inhibiting cell surface expression and eliminating an important inhibitory domain. TrkAIII, spontaneously activated within the permissive ERGIC/COPI compartment, rather than moving in an anterograde direction to the GN exhibits retrograde transport back to the ER, where it is inactivated. This sets-up self-perpetuating TrkAIII re-cycling between the ERGIC and ER, that ensures continual accumulation above the spontaneous activation threshold of the ERGIC/COPI compartment. This is reversed by TrkA tyrosine kinase inhibitors, which promote anterograde transport of inactivated TrkAIII to the GN, resulting in GN-associated TrkAIII maturation to a 120kDa species that is degraded at the proteasome.

The first target of any therapy for retinal neurodegeneration is to slow down the progression of the disease and to maintain visual function. Cerium oxide or ceria nanoparticles reduce oxidative stress, which is known to play a pivotal role in neurodegeneration. Our aim was to investigate whether cerium oxide nanoparticles were able to mitigate neurodegeneration including microglial activation and related inflammatory processes induced by exposure to high intensity light. Cerium oxide nanoparticles were injected intravitreally or intraveinously in albino Sprague-Dawley rats three weeks before exposing them to light damage of 1000 lux for 24 h. Electroretinographic recordings were performed a week after light damage. The progression of retinal degeneration was evaluated by measuring outer nuclear layer thickness and TUNEL staining to quantify photoreceptors death. Immunohistochemical analysis was used to evaluate retinal stress, neuroinflammatory cytokines and microglial activation. Only intravitreally injected ceria nanoparticles were detected at the level of photoreceptor outer segments 3 weeks after the light damage and electoretinographic recordings showed that ceria nanoparticles maintained visual response. Moreover, this treatment reduced neuronal death and "hot spot" extension preserving the outer nuclear layer morphology. It is noteworthy that in this work we demonstrated, for the first time, the ability of ceria nanoparticles to reduce microglial activation and their migration toward outer nuclear layer. All these evidences support ceria nanoparticles as a powerful therapeutic agent in retinal neurodegenerative processes.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

Age-related macular degeneration (AMD) is a retinal neurodegenerative disease whose development and progression are the results of a complex interaction between genetic and environmental risk factors. Both oxidative stress and chronic inflammation play a significant role in the pathogenesis of AMD. Experimental studies in rats with light-induced photoreceptors degeneration demonstrated that saffron may protect photoreceptor from retinal stress, preserving both morphology and function and probably acting as a regulator of programmed cell death, in addition to its antioxidant and anti-inflammatory properties. Recently, a randomized clinical trial showed that in patients with early AMD, dietary supplementation with saffron was able to improve significantly the retinal flicker sensitivity suggesting neuroprotective effect of the compound. Here, we examine the progress of saffron dietary supplementation both in animal model and AMD patients, and discuss the potential and safety for using dietary saffron to treat retinal degeneration.

Background: To determine whether the functional effects of oral supplementation with Saffron, a natural compound that proved to be neuroprotective in early age-related macular degeneration, are influenced by complement factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) risk genotypes., Methods: Thirty-three early AMD patients, screened for CFH (rs1061170) and ARMS2 (rs10490924) polymorphisms and receiving Saffron oral supplementation (20 mg/day) over an average period of treatment of 11 months (range, 6-12), were longitudinally evaluated by clinical examination and focal electroretinogram (fERG)-derived macular (18°) flicker sensitivity estimate. fERG amplitude and macular sensitivity, the reciprocal value of the estimated fERG amplitude threshold, were the main outcome measures., Results: After three months of supplementation, mean fERG amplitude and fERG sensitivity improved significantly when compared to baseline values (p < 0.01). These changes were stable throughout the follow-up period. No significant differences in clinical and fERG improvements were observed across different CFH or ARMS2 genotypes., Conclusions: The present results indicate that the functional effect of Saffron supplementation in individual AMD patients is not related to the major risk genotypes of disease.

The Ikaros gene encodes a Krüppel-like zinc-finger transcription factor involved in hematopoiesis regulation. Ikaros has been established as one of the most clinically relevant tumor suppressors in several hematological malignancies. In fact, expression of dominant negative Ikaros isoforms is associated with adult B-cell acute lymphoblastic leukemia, myelodysplastic syndrome, acute myeloid leukemia and adult and juvenile chronic myeloid leukemia. Here, we report the isolation of a novel, non-canonical Ikaros splice variant, called Ikaros 11 (Ik11). Ik11 is structurally related to known dominant negative Ikaros isoforms, due to the lack of a functional DNA-binding domain. Interestingly, Ik11 is the first Ikaros splice variant missing the transcriptional activation domain. Indeed, we demonstrated that Ik11 works as a dominant negative protein, being able to dimerize with Ikaros DNA-binding isoforms and inhibit their functions, at least in part by retaining them in the cytoplasm. Notably, we demonstrated that Ik11 is the first dominant negative Ikaros isoform to be aberrantly expressed in B-cell lymphoproliferative disorders, such as chronic lymphocytic leukemia. Aberrant expression of Ik11 interferes with both proliferation and apoptotic pathways, providing a mechanism for Ik11 involvement in tumor pathogenesis. Thus, Ik11 could represent a novel marker for B-cell lymphoproliferative disorders.

Interfacing organic electronics with biological substrates offers new possibilities for biotechnology due to the beneficial properties exhibited by organic conducting polymers. These polymers have been used for cellular interfaces in several fashions, including cellular scaffolds, neural probes, biosensors and actuators for drug release. Recently, an organic photovoltaic blend has been exploited for neuronal stimulation via a photo-excitation process. Here, we document the use of a single-component organic film of poly(3-hexylthiophene) (P3HT) to trigger neuronal firing upon illumination. Moreover, we demonstrate that this bio-organic interface restored light sensitivity in explants of rat retinas with light-induced photoreceptor degeneration. These findings suggest that all-organic devices may play an important future role in sub-retinal prosthetic implants.

This study explores the effect of continuous exposure to bright light on neuromelanin formation and dopamine neuron survival in the substantia nigra. Twenty-one days after birth, Sprague-Dawley albino rats were divided into groups and raised under different conditions of light exposure. At the end of the irradiation period, rats were sacrificed and assayed for neuromelanin formation and number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. The rats exposed to bright light for 20 days or 90 days showed a relatively greater number of neuromelanin-positive neurons. Surprisingly, TH-positive neurons decreased progressively in the substantia nigra reaching a significant 29% reduction after 90 days of continuous bright light exposure. This decrease was paralleled by a diminution of dopamine and its metabolite in the striatum. Remarkably, in preliminary analysis that accounted for population density, the age and race adjusted Parkinson's disease prevalence significantly correlated with average satellite-observed sky light pollution.

Purpose: To evaluate the functional effect of short-term supplementation of saffron, a spice containing the antioxidant carotenoids crocin and crocetin, in early age-related macular degeneration (AMD)., Methods: Twenty-five patients with AMD were randomly assigned to oral saffron 20 mg/d or placebo supplementation over a 3-month period and then reverted to placebo or saffron for a further 3 months. Focal electroretinograms (fERGs) and clinical findings were recorded at baseline and after 3 months of saffron or placebo supplementation. fERGs were recorded in response to a sinusoidally modulated (41 Hz), uniform field presented to the macular region (18°) at different modulations between 16.5% and 93.5%. Main outcome measures were fERG amplitude (in microvolts), phase (in degrees), and modulation thresholds., Results: After saffron, patients' fERGs were increased in amplitude, compared with either baseline or values found after placebo supplementation (mean change after saffron, 0.25 log μV; mean change after placebo, -0.003 log μV; P < 0.01). fERG thresholds were decreased after saffron supplementation but not placebo, compared with baseline (mean change after saffron, -0.26 log units; mean change after placebo, 0.0003 log units)., Conclusions: The results indicate that short-term saffron supplementation improves retinal flicker sensitivity in early AMD. Although the results must be further replicated and the clinical significance is yet to be evaluated, they provide important clues that nutritional carotenoids may affect AMD in novel and unexpected ways, possibly beyond their antioxidant properties. (ClinicalTrials.gov number, NCT00951288.).

Calmodulin-dependent phosphodiesterase (PDE1) is a key enzyme in cyclic nucleotides metabolism. We studied its gene expression and protein localization during retinal development in chick embryos. Western blot and densitometric analysis demonstrated that the expression of the three isoforms changed during development. PDE1A was highly expressed at the early stages and decreased as development proceeded. PDE1B expression remained relatively low and constant over time. PDE1C showed a prominent increase (13-fold) between embryonic day (E) 7 and E13, followed by a moderate increase between E13 and postnatal day (P) 1. The presence of the enzyme in the different retinal locations was strongly modulated by development. PDE1A immunostaining was first detected at the ganglion cell level (E7), then in the outer retina (E15-E21). At P5, the immunostaining was confined in the optic fiber layer. Isoform C immunolocalization followed the same inner-outer pattern as isoform A. At 5 days posthatching (P5), the immunoreactivity was restricted, as well as for the isoform A, in the optic fiber layer. The isoform B immunolabelling was low and evenly distributed across the retina at all stages. The different developmental profiles of PDE1A, PDE1B, and PDE1C induced a temporal modulation in cyclic nucleotides concentration, suggesting specific roles of this enzyme in the morphofunctional development of retinal circuitry.

Purpose: To test whether the saffron extract (Crocus sativus L.) given as a dietary supplement counteracts the effects of continuous light exposure in the albino rat retina., Methods: Three experimental groups of Sprague-Dawley rats were used. Experimental animals were prefed either saffron or beta-carotene (1 mg extract/kg/d) before they were exposed to bright continuous light (BCL) for 24 hours. Flash electroretinograms (fERGs) were recorded in control and treated rats the day before and 1 week after light exposure. At the end of the second recording session, the animals were killed and the retinas were quickly removed, fixed, cryosectioned, and labeled so that the thickness of the outer nuclear layer (ONL) could be analyzed. Changes in protein level and cellular localization of fibroblast growth factor (FGF)2 were determined by Western blot analysis and retinal immunohistochemistry, respectively. In a second series of experiments, rats were killed at the end of light exposure, and the amount of apoptotic figures in the ONL was assessed by terminal transferase-mediated deoxyuridine triphosphate (d-UTP)-biotin nick-end labeling (TUNEL). BCL induced DNA fragmentation, characteristic of dying cells, almost exclusively in the photoreceptor layer. The rate of photoreceptor death induced by BCL is expressed as the frequency of TUNEL-positive profiles per millimeter., Results: The photoreceptor layer was largely preserved in saffron-treated animals because it was the fERG response. In addition, the rate of photoreceptor death induced by BCL appeared drastically reduced in treated animals. In beta-carotene prefeeding experiments, morphologic analysis showed preservation of the ONL similar to that obtained with saffron prefeeding, whereas the fERG response was unrecordable. Western blot analysis showed that exposure to light induced a strong upregulation of FGF2 in control and beta-carotene-treated rats, but s no change was noted in saffron-treated rats., Conclusions: These results show that saffron may protect photoreceptors from retinal stress, maintaining both morphology and function and probably acting as a regulator of programmed cell death.

Recent data challenged the assumption that light has little effect on retina development. Here, we report evidence that dark rearing permanently changes the synaptic input to GCs. A reduced spontaneous postsynaptic currents (SPSCs) frequency was found in retinal GCs from rats born and raised in the dark for three months. Glutamate antagonists (CNQX and AP-5) reversibly reduced SPSCs frequency in control and dark-reared (DR) retinae. The GABA antagonist picrotoxin (PTX) reduced SPSCs frequency in control retinas, but increased SPSCs frequency in DR, mainly by presynaptic action on excitatory currents. In DR animals exposed to normal cyclic light for 3 months, SPSCs frequency remained lower then in control rats and increased following PTX, suggesting that long-term dark rearing induces permanent modifications of the retinal circuitry. Our results strongly support the idea that light stimulation plays a role in establishing normal synaptic input to GCs.

Calcium calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) was identified in crude extract and immunolabeled sections of rat retina. Both cAMP and cGMP PDE activities were stimulated by calcium-calmodulin (4.7-fold and 2.3-fold, respectively). To characterize PDE1 isoforms in retinal cells further, we used antibodies that specifically recognize PDE1 gene products. PDE1B antibody stained a band at molecular mass of 63 kDa whereas PDE1C antibody recognized two bands at 74- and 70-kDa molecular masses. Two PDE1A antibodies (against N-terminal and C-terminal peptides) detected a band at 79 kDa never described before. Immunohistochemical analysis showed a distribution of PDE1A in the outer retina with a bright fluorescence in the outer segments of photoreceptors. PDE1B is uniformly distributed across the retina. PDE1C is confined mainly to the inner retina, with a precise localization in the inner nuclear layer. Immunostaining with choline acetyltransferase antibody indicates localization in cholinergic amacrine cell. The present data provide evidence of expression of PDE1 isoforms in mammalian retina with a complementary distribution of PDE1A and PDE1C, suggesting different roles in retinal function., (Copyright 2006 Wiley-Liss, Inc.)

We investigated the rate of cell proliferation and death in the retina of the Monodelphis opossum during its postnatal development and the influence of early monocular enucleation on these processes. Our results show that in the opossum, as in other marsupials, the peak of the retinal cells divisions occurs postnatally and that generation of retinal cells continues till the time of eye opening (P34), except of the marginal rim, where it continued till P60. Ganglion and amacrine cells are generated between postnatal days (P) P4 and P9, while bipolar cells and photoreceptors are generated simultaneously between P14 and P25. The peak of ganglion cell death as detected by the TUNEL method occurs around P14-19 in the center of retina. The second peak of apoptosis appears in the inner nuclear layer (INL) at P19-25. Gliogenesis takes place between P25 and P34. We also found that monocular enucleation performed during the early period of retinal development (P0-P7) did not influence proliferation, developmental apoptosis or other developmental processes in the retina of the remaining eye.

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 gradual restriction of initially multistratified retinal ganglion cell (RGC) dendrites into ON and OFF sublaminae of the inner plexiform layer (IPL) can be effectively blocked by treating the developing retina with 2-amino-4-phosphonobutyrate (APB), the metabotropic glutamate agonist, or by light deprivation. Previous studies have focused on the short-term consequences of such manipulations, so the long-term effects of arresting dendritic stratification on the structural development of RGCs are as yet unknown. In the present study, we have addressed this issue by performing a morphological analysis of alpha RGCs labeled by retrograde transport of horseradish peroxidase injected into the dorsal lateral geniculate nucleus of adult cats that received monocular injections of APB from postnatal (P) day 2 until P30. A large proportion of the alpha cells in the APB-treated eye (44%) were found to have multistratified dendrites that terminated in both the ON and OFF sublaminae of the IPL. The dendritic arborization pattern in the sublaminae of the IPL of these cells was asymmetric, showing a variety of forms. Immunolabeling of retinal cross-sections showed that mGLUR6 receptors appeared normal in density and location, while qualitative observation suggested an increase in the axonal arborization of rod bipolar cells. These findings indicate that long-term treatment of the neonatal retina with APB induces a long- lasting structural reorganization in retinal circuitry that most likely accounts for some of the previously described changes in the functional properties of RGCs., (Copyright 2004 S. Karger AG, Basel.)

We analysed the specific effects of IL-1beta immunoneutralization on the expression of IL-6 in different pure cultures of neurones and glia after both experimental subliminal hypoxia and recovery. Whereas the IL-1beta-deprivation signal induced a decrease in IL-6 expression and release of normoxic neurones, it provoked an increase in IL-6 protein in hypoxic neurones. Moreover, the direct correlation between IL-1beta and IL-6, observed in normal and recovering neuronal cultures, was reversed in hypoxic conditions. These reversals were not observed in glial cells, in which IL-1beta immunosuppression led to a decrease in IL-6 under all conditions considered. In conclusion, the IL-1beta modulates IL-6 in different ways according to the ambient physiological or pathological conditions, and also acts via different mechanisms, depending on the cellular phenotype.