Investigation of the Effects of a Novel NOX2 Inhibitor, GLX7013170, against Glutamate Excitotoxicity and Diabetes Insults in the Retina.

Glutamate excitotoxicity and oxidative stress represent two major pathological mechanisms implicated in retinal disorders. In Diabetic Retinopathy (DR), oxidative stress is correlated to NADPH oxidase (NOX), a major source of Reactive Oxygen Species (ROS), and glutamate metabolism impairments. This study investigated the role of NOX2 and the novel NOX2 inhibitor, GLX7013170, in two models of a) retinal AMPA excitotoxicity [AMPA+GLX7013170 (10-4 M, intravitreally)] and b) early-stage DR paradigm (ESDR), GLX7013170: 14-day therapeutic treatment (topically, 20 µL/eye, 10 mg/mL (300 × 10-4 M), once daily) post-streptozotocin (STZ)-induced DR. Immunohistochemical studies for neuronal markers, nitrotyrosine, micro/macroglia, and real-time PCR, Western blot, and glutamate colorimetric assays were conducted. Diabetes increased NOX2 expression in the retina. NOX2 inhibition limited the loss of NOS-positive amacrine cells and the overactivation of micro/macroglia in both models. In the diabetic retina, GLX7013170 had no effect on retinal ganglion cell axons, but reduced oxidative damage, increased Bcl-2, reduced glutamate levels, and partially restored excitatory amino acid transporter (EAAT1) expression. These results suggest that NOX2 in diabetes is part of the triad, oxidative stress, NOX, and glutamate excitotoxicity, key players in the induction of DR. GLX7013170 is efficacious as a neuroprotective/anti-inflammatory agent and a potential therapeutic in retinal diseases, including ESDR.

Investigating the Effects of Exogenous and Endogenous 2-Arachidonoylglycerol on Retinal CB1 Cannabinoid Receptors and Reactive Microglia in Naive and Diseased Retina.

The endocannabinoid system (ECS) is a new target for the development of retinal disease therapeutics, whose pathophysiology involves neurodegeneration and neuroinflammation. The endocannabinoid 2-arachidonoylglycerol (2-AG) affects neurons and microglia by activating CB1/CB2 cannabinoid receptors (Rs). The aim of this study was to investigate the effects of 2-AG on the CB1R expression/downregulation and retinal neurons/reactive microglia, when administered repeatedly (4 d), in three different paradigms. These involved the 2-AG exogenous administration (a) intraperitoneally (i.p.) and (b) topically and (c) by enhancing the 2-AG endogenous levels via the inhibition (AM11920, i.p.) of its metabolic enzymes (MAGL/ABHD6). Sprague Dawley rats were treated as mentioned above in the presence or absence of CB1/CB2R antagonists and the excitatory amino acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Immunohistochemistry, Western blot and a 2-AG level analyses were performed. The 2-AG repeated treatment (i.p.) induced the CB1R downregulation, abolishing its neuroprotective actions. However, 2-AG attenuated the AMPA-induced activation of microglia via the CB2R, as concurred by the AM630 antagonist effect. Topically administered 2-AG was efficacious as a neuroprotectant/antiapoptotic and anti-inflammatory agent. AM11920 increased the 2-AG levels providing neuroprotection against excitotoxicity and reduced microglial activation without affecting the CB1R expression. Our findings show that 2-AG, in the three paradigms studied, displays differential pharmacological profiles in terms of the downregulation of the CB1R and neuroprotection. All treatments, however, attenuated the activation of microglia via the CB2R activation, supporting the anti-inflammatory role of 2-AG in the retina.

Topically Administered NOX4 Inhibitor, GLX7013114, Is Efficacious in Treating the Early Pathological Events of Diabetic Retinopathy.

NADPH oxidases (NOXs) are major players in generating reactive oxygen species (ROS) and are implicated in various neurodegenerative ocular pathologies. The aim of this study was to investigate the role of a NOX4 inhibitor (GLX7013114) in two in vivo, experimental streptozotocin (STZ) paradigms depicting the early events of diabetic retinopathy (DR). Animals in the diabetic treated group received GLX7013114 topically (20 µL/eye, 10 mg/mL, once daily) for 14 days (paradigm A: preventive) and 7 days (paradigm B: treated) at 48 h and 4 weeks after STZ injection, respectively. Several methodologies were used (immunohistochemistry, Western blot, real-time PCR, ELISA, pattern electroretinography [PERG]) to assess the diabetes-induced early events of DR, namely oxidative stress, neurodegeneration, and neuroinflammation, and the effect of GLX7013114 on the diabetic insults. GLX7013114, administered as eye drops (paradigms A and B), was beneficial in treating the oxidative nitrative stress, activation of caspase-3 and micro- and macroglia, and attenuation of neuronal markers. It also attenuated the diabetes-induced increase in vascular endothelial growth factor, Evans blue dye leakage, and proinflammatory cytokine (TNF-α protein, IL-1ß/IL-6 mRNA) levels. PERG amplitude values suggested that GLX7013114 protected retinal ganglion cell function (paradigm B). This study provides new findings regarding the pharmacological profile of the novel NOX4 inhibitor GLX7013114 as a promising therapeutic candidate for the treatment of the early stage of DR. ARTICLE HIGHLIGHTS: NADPH oxidases (NOXs) are implicated in the early pathological events of diabetic retinopathy (DR). The NOX4 inhibitor GLX7013114, topically administered, reduced oxidative damage and apoptosis in the rat streptozotocin model of DR. GLX7013114 protected retinal neurons and retinal ganglion cell function and reduced the expression of pro-inflammatory cytokines in the diabetic retina. GLX7013114 diminished the diabetes-induced increase in vascular endothelial growth factor levels and Evans blue dye leakage in retinal tissue. GLX7013114 exhibits neuroprotective, anti-inflammatory, and vasculoprotective properties that suggest it may have a role as a putative therapeutic for the early events of DR.

Blockade of CB1 or Activation of CB2 Cannabinoid Receptors Is Differentially Efficacious in the Treatment of the Early Pathological Events in Streptozotocin-Induced Diabetic Rats.

Oxidative stress, neurodegeneration, neuroinflammation, and vascular leakage are believed to play a key role in the early stage of diabetic retinopathy (ESDR). The aim of this study was to investigate the blockade of cannabinoid receptor 1 (CB1R) and activation of cannabinoid receptor 2 (CB2R) as putative therapeutics for the treatment of the early toxic events in DR. Diabetic rats [streptozotocin (STZ)-induced] were treated topically (20 µL, 10 mg/mL), once daily for fourteen days (early stage DR model), with SR141716 (CB1R antagonist), AM1710 (CB2R agonist), and the dual treatment SR141716/AM1710. Immunohistochemical-histological, ELISA, and Evans-Blue analyses were performed to assess the neuroprotective and vasculoprotective properties of the pharmacological treatments on diabetes-induced retinal toxicity. Activation of CB2R or blockade of CB1R, as well as the dual treatment, attenuated the nitrative stress induced by diabetes. Both single treatments protected neural elements (e.g., RGC axons) and reduced vascular leakage. AM1710 alone reversed all toxic insults. These findings provide new knowledge regarding the differential efficacies of the cannabinoids, when administered topically, in the treatment of ESDR. Cannabinoid neuroprotection of the diabetic retina in ESDR may prove therapeutic in delaying the development of the advanced stage of the disease.

The endocannabinoid 2-arachidonoylglycerol and dual ABHD6/MAGL enzyme inhibitors display neuroprotective and anti-inflammatory actions in the in vivo retinal model of AMPA excitotoxicity.

The endocannabinoid system has been shown to be a putative therapeutic target for retinal disease. Here, we aimed to investigate the ability of the endocannabinoid 2-arachidonoylglycerol (2-AG) and novel inhibitors of its metabolic enzymes, α/ß-hydrolase domain-containing 6 (ABHD6) and monoacylglycerol lipase (MAGL), a) to protect the retina against excitotoxicity and b) the mechanisms involved in the neuroprotection. Sprague-Dawley rats, wild type and Akt2-/- C57BL/6 mice were intravitreally administered with phosphate-buffered saline or (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA). 2-AG was intravitreally co-administered with AMPA in the absence and presence of AM251 or AM630 (cannabinoid 1 and 2 receptor antagonists, respectively) or Wortmannin [Phosphoinositide 3-Kinase (PI3K)/Akt inhibitor]. Inhibitors of ABHD6 and dual ABHD6/MAGL (AM12100 and AM11920, respectively) were co-administered with AMPA intravitreally in rats. Immunohistochemistry was performed using antibodies raised against retinal neuronal markers (bNOS), microglia (Iba1) and macroglia (GFAP). TUNEL assay and real-time PCR were also employed. The CB2 receptor was expressed in rat retina (approx. 62% of CB1 expression). 2-AG attenuated the AMPA-induced increase in TUNEL+ cells. 2-AG activation of both CB1 and CB2 receptors and the PI3K/Akt downstream signaling pathway, as substantiated by the use of Akt2-/- mice, afforded neuroprotection against AMPA excitotoxicity. AM12100 and AM11920 attenuated the AMPA-induced glia activation and produced a dose-dependent partial neuroprotection, with the dual inhibitor AM11920 being more efficacious. These results show that 2-AG has the pharmacological profile of a putative therapeutic for retinal diseases characterized by neurodegeneration and neuroinflammation, when administered exogenously or by the inhibition of its metabolic enzymes.

Effect of acute and subchronic administration of (R)-WIN55,212-2 induced neuroprotection and anti inflammatory actions in rat retina: CB1 and CB2 receptor involvement.

Cannabinoids have been shown to protect the retina from ischemic/excitotoxic insults. The aim of the present study was to investigate the neuroprotective and anti-inflammatory properties of the synthetic cannabinoid (R)-WIN55,212-2 (CB1/CB2 receptor agonist) when administered acutely or subchronically in control and AMPA treated retinas. Sprague-Dawley rats were intravitreally administered (acutely) with vehicle or AMPA, in the absence or presence of (R)-WIN55,212-2 (10-7-10-4M) alone or in combination with AM251 [CB1 receptor antagonist/inverse agonist,10-4M] and AM630 (CB2 receptor antagonist,10-4M). In addition, AMPA was co-administered with the racemic (R,S)-WIN55,212 (10-4Μ). (R)-WIN55,212-2 was also administered subchronically (25,100 µg/kg,i.p.,4d) in control and AMPA treated rats. Immunohistochemical studies were performed using antibodies against the CB1R, and retinal markers for retinal neurons (brain nitric oxide synthetase, bNOS) and microglia (ionized calcium binding adaptor molecule 1, Iba1). ELISA assay was employed to assess TNFα levels in AMPA treated retinas. Intravitreal administration of (R)-WIN55,212-2 reversed the AMPA induced loss of bNOS expressing amacrine cells, an effect that was blocked by both AM251 and AM630. (R,S)WIN55,212 had no effect. (R)-WIN55,212-2 also reduced a) the AMPA induced activation of microglia, by activating CB2 receptors that were shown to be colocalized with Iba1+ reactive microglial cells, and b) TNFα levels in retina. (R)-WIN55,212-2 administered subchronically led to the downregulation of CB1 receptors at the high dose of 100 µg/kg(i.p.), and to the attenuation of the WIN55,212-2 induced neuroprotection of amacrine cells. At the same dose, (R)-WIN55,212-2 did not attenuate the AMPA induced increase in the number of reactive microglia cells, suggesting CB2 receptor downregulation under subchronic conditions. This study provides new findings regarding the role of CB1 and CB2 receptor activation by the synthetic cannabinoid (R)-WIN55,212-2, administered acutely or sub-chronically, on neuron viability and microglia activation in healthy and diseased retina.

Effect of NADPH oxidase inhibitors in an experimental retinal model of excitotoxicity.

NADPH oxidases (NOX) are activated in ischemic conditions leading to increases in reactive oxygen species (ROS) and neurotoxicity. The aim of the present study was to investigate the role of NOX in the development of retinal pathologies, associated with excitotoxicity and the evaluation of NOX inhibitors as putative therapeutic agents. Sprague-Dawley rats were used for the induction of the in vivo retinal model of (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA) excitotoxicity. Rats were intravitreally administered with PBS, AMPA (42 nmoles) or AMPA + NOX inhibitors, VAS2870 (pan-NOX inhibitor, 10-6-10-4 M), ML171 (NOX1 inhibitor, 10-5, 10-4 M), and GLX7013114 (NOX4 inhibitor, 10-4 M). Immunohistochemical studies were performed using antibodies raised against nitrotyrosine, a ROS/oxidative stress marker, bNOS, a neuronal marker for nitric oxide synthase and the macro and microglia markers, glial fibrillary acidic protein and ionized calcium-binding adaptor molecule-1, respectively. VAS2870 and ML171 showed neuroprotective and anti-inflammatory actions reversing the AMPA induced reduction of bNOS expressing amacrine cells and attenuating macro/microglial activation. GLX7013114 (10-4 M) did not protect bNOS expressing amacrine cells, but it did attenuate the AMPA induced increase in nitrotyrosine positive cells and activation of glial cells. These results suggest that NOX1, NOX4 and possibly NOX2 (due to the actions of VAS2870) play an important role in the pathophysiology of the retina and that NOX inhibitors are putative neuroprotective and anti-inflammatory agents against retinal abnormalities caused by excitotoxicity.

Differential effects of chronic voluntary wheel-running on morphine induced brain stimulation reward, motor activity and striatal dopaminergic activity.

Physical exercise could be a protective factor against the development of substance use disorders; however, a number of preclinical studies report reward-enhancing effects of exercise for various drugs of abuse. We examined the effects of chronic wheel-running on brain reward sensitivity, reaction to novelty, reward-facilitating and locomotor-stimulating effects of morphine, using the intracranial self-stimulation (ICSS) and the open field test (OFT). Male Sprague-Dawley rats were randomly assigned to a sedentary or exercised group. For the ICSS procedure, rats were implanted with electrodes and trained to respond for electrical stimulation. Several indices were recorded in the training phase to estimate brain reward sensitivity. Once responding was stable, the animals of both groups received systemic injections of morphine and their ICSS thresholds were measured with the curve-shift paradigm. Employing the OFT, basal and morphine-induced locomotor activity was measured. Finally, basal and morphine-evoked tissue levels of dopamine and its metabolites were determined in the striatum using gas chromatography/mass spectrometry. Chronic wheel-running decreased brain reward sensitivity and subsequently increased the reward-facilitating effect of morphine. Exercised animals demonstrated a decreased reaction to novelty and reduced morphine-induced locomotion. Lastly, dopaminergic activity was decreased in the striatum of exercised animals under basal conditions, whereas morphine administration led to an increase in dopamine turnover. These findings indicate that chronic voluntary exercise exerts divergent effects on reward function, psychomotor activity and the reward-facilitating and locomotor-activating effects of opioids during adulthood. Our results provide insights into the increased non-medical use of opioids among young athletes reported in the literature.

Effect of topical administration of the microneurotrophin BNN27 in the diabetic rat retina.

PURPOSE: Diabetic retinopathy (DR) is a complex eye disease associated with diabetes mellitus. It is characterized by three pathophysiological components, namely microangiopathy, neurodegeneration, and inflammation. We recently reported that intraperitoneal administration of BNN27, a novel neurosteroidal microneurotrophin, reversed the diabetes-induced neurodegeneration and inflammation in rats treated with streptozotocin (STZ), by activating the NGF TrkA and p75 receptors. The aim of the present study was to investigate the efficacy of BNN27 to protect retinal neurons when applied topically as eye drops in the same model. METHODS: The STZ rat model of DR was employed. BNN27 was administered as eye drops to diabetic Sprague-Dawley rats for 7 days, 4 weeks post-STZ (70 mg/kg) injection. Immunohistochemistry and western blot analyses were employed to examine the viability of retinal neurons in control, diabetic, and diabetic-treated animals and the involvement of the TrkA receptor and its downstream signaling ERK1/2 kinases, respectively. RESULTS: BNN27 reversed the STZ-induced attenuation of the immunoreactive brain nitric oxide synthetase (bNOS)- and tyrosine hydroxylase (TH)-expressing amacrine cells and neurofilament (NFL)-expressing ganglion cell axons in a dose-dependent manner. In addition, BNN27 activated/phosphorylated the TrkA receptor and its downstream prosurvival signaling pathway, ERK1/2 kinases. CONCLUSIONS: The results of this study provide solid evidence regarding the efficacy of BNN27 as a neuroprotectant to the diabetic retina when administered topically, and suggest that its pharmacodynamic and pharmacokinetic profiles render it a putative therapeutic for diabetic retinopathy.

Endogenous and synthetic cannabinoids induce the downregulation of cannabinoid CB1 receptor in retina.

Endogenous and synthetic cannabinoids have been shown to provide neuroprotection to retinal neurons in acute animal models of retinopathy. Chronic exposure to cannabinoid receptor (CB1R) agonists has been reported to induce downregulation of the CB1R in brain and behavioral tolerance. The aim of this study was to investigate the effect of subchronic/chronic cannabinoid administration on CB1R downregulation in normal rat retina, its downstream prosurvival signaling and subsequent effect on retinal neuroprotection against AMPA excitotoxicity. Sprague-Dawley rats were administered intraperitoneally with vehicle (Control), the endogenous N-arachidonoyl ethanolamine (AEA), and the synthetic cannabinoids R-(+)-Methanandamide (MethAEA) and HU-210 daily (25, 50, 100 µg/kg) for four or fourteen days (4d/14d, subchronic/chronic administration, respectively). HU-210 was also administered acutely as follows, vehicle injection for 13 days and a single dose of HU-210 on the 14th day. Immunohistochemistry studies and Western blot analysis were employed to assess CB1R expression in control and AMPA treated retinas and cannabinoid induced changes in Akt and ERK1/2 phosphorylation (ph). Real time PCR was employed to examine the effect of MethAEA (50 mg/kg,4d) on CB1R mRNA expression. AEA, MethAEA and HU-210 attenuated CB1R expression in a dose-dependent manner (25, 50, 100 µg/kg), after subchronic and chronic administration. No effect was observed at the lower dose of 25 µg/kg. MethAEA (50 mg/kg,4d) attenuated CB1R mRNA expression. AM251 (CB1 antagonist/inverse agonist, 0.5 mg/kg,4d), administered prior to HU-210 (50 µg/kg,4d) inhibited CB1R downregulation. Chronic/subchronic treatments (50 µg/kg) of HU-210 and MethAEA reduced levels of ph-Akt and ph-Akt/ph-ERK1/2, respectively. AEA had no effect on ph-Akt nor ph-ERK1/2. All three cannabinoids (50 µg/kg,4d) failed to protect brain nitric oxide synthetase (bNOS) expressing amacrine cells against AMPA excitotoxicity, in agreement with the downregulation of CB1 receptor. At the lower doses of 12.5 and 25 µg/kg, HU-210 protected bNOS-expressing amacrine cells. This study provides novel information regarding agonist-induced CB1R downregulation in rat retina after subchronic/chronic cannabinoid treatment, and its effect on downstream prosurvival signaling and neuroprotection.

The Synthetic Microneurotrophin BNN27 Affects Retinal Function in Rats With Streptozotocin-Induced Diabetes.

BNN27, a C17-spiroepoxy derivative of DHEA, was shown to have antiapoptotic properties via mechanisms involving the nerve growth factor receptors (tropomyosin-related kinase A [TrkA]/neurotrophin receptor p75 [p75NTR]). In this study, we examined the effects of BNN27 on neural/glial cell function, apoptosis, and inflammation in the experimental rat streptozotocin (STZ) model of diabetic retinopathy (DR). The ability of BNN27 to activate the TrkA receptor and regulate p75NTR expression was investigated. BNN27 (2,10, and 50 mg/kg i.p. for 7 days) administration 4 weeks post-STZ injection (paradigm A) reversed the diabetes-induced glial activation and loss of function of amacrine cells (brain nitric oxide synthetase/tyrosine hydroxylase expression) and ganglion cell axons via a TrkA receptor (TrkAR)-dependent mechanism. BNN27 activated/phosphorylated the TrkAY490 residue in the absence but not the presence of TrkAR inhibitor and abolished the diabetes-induced increase in p75NTR expression. However, it had no effect on retinal cell death (TUNEL+ cells). A similar result was observed when BNN27 (10 mg/kg i.p.) was administered at the onset of diabetes, every other day for 4 weeks (paradigm B). However, BNN27 decreased the activation of caspase-3 in both paradigms. Finally, BNN27 reduced the proinflammatory (TNFα and IL-1ß) and increased the anti-inflammatory (IL-10 and IL-4) cytokine levels. These findings suggest that BNN27 has the pharmacological profile of a therapeutic for DR, since it targets both the neurodegenerative and inflammatory components of the disease.

Endogenous and Synthetic Cannabinoids as Therapeutics in Retinal Disease.

The functional significance of cannabinoids in ocular physiology and disease has been reported some decades ago. In the early 1970s, subjects who smoked Cannabis sativa developed lower intraocular pressure (IOP). This led to the isolation of phytocannabinoids from this plant and the study of their therapeutic effects in glaucoma. The main treatment of this disease to date involves the administration of drugs mediating either the decrease of aqueous humour synthesis or the increase of its outflow and thus reduces IOP. However, the reduction of IOP is not sufficient to prevent visual field loss. Retinal diseases, such as glaucoma and diabetic retinopathy, have been defined as neurodegenerative diseases and characterized by ischemia-induced excitotoxicity and loss of retinal neurons. Therefore, new therapeutic strategies must be applied in order to target retinal cell death, reduction of visual acuity, and blindness. The aim of the present review is to address the neuroprotective and therapeutic potential of cannabinoids in retinal disease.

Dendrimers as tunable vectors of drug delivery systems and biomedical and ocular applications.

Dendrimers are large polymeric structures with nanosize dimensions (1-10 nm) and unique physicochemical properties. The major advantage of dendrimers compared with linear polymers is their spherical-shaped structure. During synthesis, the size and shape of the dendrimer can be customized and controlled, so the finished macromolecule will have a specific "architecture" and terminal groups. These characteristics will determine its suitability for drug delivery, diagnostic imaging, and as a genetic material carrier. This review will focus initially on the unique properties of dendrimers and their use in biomedical applications, as antibacterial, antitumor, and diagnostic agents. Subsequently, emphasis will be given to their use in drug delivery for ocular diseases.

Trans-Modulation of the Somatostatin Type 2A Receptor Trafficking by Insulin-Regulated Aminopeptidase Decreases Limbic Seizures.

Within the hippocampus, the major somatostatin (SRIF) receptor subtype, the sst2A receptor, is localized at postsynaptic sites of the principal neurons where it modulates neuronal activity. Following agonist exposure, this receptor rapidly internalizes and recycles slowly through the trans-Golgi network. In epilepsy, a high and chronic release of somatostatin occurs, which provokes, in both rat and human tissue, a decrease in the density of this inhibitory receptor at the cell surface. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. In addition, IRAP ligands display anticonvulsive properties. We therefore sought to assess by in vitro and in vivo experiments in hippocampal rat tissue whether IRAP ligands could regulate the trafficking of the sst2A receptor and, consequently, modulate limbic seizures. Using pharmacological and cell biological approaches, we demonstrate that IRAP ligands accelerate the recycling of the sst2A receptor that has internalized in neurons in vitro or in vivo. Most importantly, because IRAP ligands increase the density of this inhibitory receptor at the plasma membrane, they also potentiate the neuropeptide SRIF inhibitory effects on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures and possibly for other neurological conditions in which downregulation of G-protein-coupled receptors occurs. SIGNIFICANCE STATEMENT: The somatostatin type 2A receptor (sst2A) is localized on principal hippocampal neurons and displays anticonvulsant properties. Following agonist exposure, however, this receptor rapidly internalizes and recycles slowly. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. We therefore assessed by in vitro and in vivo experiments whether IRAP could regulate the trafficking of this receptor. We demonstrate that IRAP ligands accelerate sst2A recycling in hippocampal neurons. Because IRAP ligands increase the density of sst2A receptors at the plasma membrane, they also potentiate the effects of this inhibitory receptor on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures.

Synthetic and endogenous cannabinoids protect retinal neurons from AMPA excitotoxicity in vivo, via activation of CB1 receptors: Involvement of PI3K/Akt and MEK/ERK signaling pathways.

Cannabinoids have been suggested to protect retinal ganglion cells in different models of toxicity, but their effects on other retinal neurons are poorly known. We investigated the neuroprotective actions of the endocannabinoid N-arachidonoyl ethanolamine (Anandamide/AEA) and the synthetic cannabinoids R1-Methanandamide (MethAEA) and HU-210, in an in vivo retinal model of AMPA excitotoxicity, and the mechanisms involved in the neuroprotection. Sprague-Dawley rats were intravitreally injected with PBS or AMPA in the absence or presence of the cannabinoid agonists. Brain nitric oxide synthase (bNOS) and choline acetyltransferase (ChAT) immunoreactivity (IR), as well as TUNEL staining, assessed the AMPA-induced retinal amacrine cell loss and the dose-dependent neuroprotection afforded by cannabinoids. The CB1 receptor selective antagonist AM251 and the PI3K/Akt inhibitor wortmannin reversed the cannabinoid-induced neuroprotection, suggesting the involvement of CB1 receptors and the PI3K/Akt pathway in cannabinoids' actions. Experiments with the CB2 agonist JWH015 and [(3)H]CP55940 radioligand binding suggested that the CB2 receptor is not involved in the neuroprotection. AEA and HU-210 induced phosphorylation of Akt but only AEA induced phosphorylation of ERK1/2 kinases, as revealed by western blot analysis. To investigate the role of caspase-3 in the AMPA-induced cell death, the caspase-3 inhibitor Z-DEVD-FMK was co-injected with AMPA. Z-DEVD-FMK had no effect on AMPA excitotoxicity. Moreover, no difference was observed in the phosphorylation of SAPK/JNK kinases between PBS- and AMPA-treated retinas. These results suggest that endogenous and synthetic cannabinoids protect retinal amacrine neurons from AMPA excitotoxicity in vivo via a mechanism involving the CB1 receptors, and the PI3K/Akt and/or MEK/ERK1/2 signaling pathways.

Effects of octreotide and insulin on colon cancer cellular proliferation and correlation with hTERT activity.

Peptide hormone somatostatin and its receptors have a wide range of physiological functions and play a role in the treatment of numerous human diseases, including colorectal cancer. Octreotide, a synthetic somatostatin-analog peptide, inhibits growth of colonic cancer cells primarily by binding to G-protein coupled receptors and elicits cellular responses through second-messenger systems. Insulin also initiates mitogenic signals in certain cell types. The objective of the present study was to explore the effects of octreotide with or without insulin treatment, on Caco-2 and HT-29 human colon-cancer cell proliferation and to correlate their effects with the activation of telomerase reverse transcriptase (hTERT). The involvement of protein tyrosine phosphatases in the regulation of the anti-proliferative effect of octreotide was also evaluated. Sodium orthovanadate was used to reverse the anti- proliferative effect of octreotide. Telomerase activity was determined for each time point under octreotide and/or insulin treatment. Elevated expression of sst1, sst2 and sst5 was confirmed in both cell lines by RT-PCR. Immunocytochemistry detected sst1, sst2A, sst2B, sst3, sst4 and sst5 protein expression in the membranes of both cell lines. Octreotide inhibited the proliferation of Caco-2 and HT-29 cells in a time and dose-dependent manner. Insulin exerted proliferative effects in Caco-2 cells and octreotide reversed its effect in both cell lines. Sodium orthovanadate suppressed the anti-proliferative effect of octreotide both in Caco-2 and HT-29 cells. Telomerase activity was significantly reduced when Caco-2 cells were exposed to octreotide, under serum-free cultured medium. On the other hand, telomerase attenuation after octreotide treatment could not counteract the actions of insulin on both cells. Our data indicate that the use of octreotide could provide a possible therapeutic approach to the management of certain patients who suffer from colon cancer.

Neurosteroids and microneurotrophins signal through NGF receptors to induce prosurvival signaling in neuronal cells.

The neurosteroid dehydroepiandrosterone (DHEA) exerts a portion of its neuroprotective effects by directly interacting with the nerve growth factor (NGF) receptors TrkA and p75(NTR) to induce prosurvival signaling. DHEA is an intermediate in the biosynthesis of estrogens and androgens that affects the endocrine system and potentially increases the risk for developing estrogen- and androgen-dependent tumors. We have synthesized 17-spiro analogs of DHEA that lack estrogenic or androgenic properties and bind to and activate NGF receptors, thus exerting potent neuroprotective effects without the tumor risk. These synthetic DHEA derivatives may serve as lead molecules to develop small agonists of NGF receptors that can penetrate the blood-brain barrier (microneurotrophins) with potential applications in the treatment of neurodegenerative diseases. The neuroprotective properties of microneurotrophins are now being tested in various animal models of neurodegenerative diseases.

The neurosteroid dehydroepiandrosterone (DHEA) protects the retina from AMPA-induced excitotoxicity: NGF TrkA receptor involvement.

The aim of the present study was to investigate the neuroprotective properties of the endogenous neurosteroid dehydroepiandrosterone (DHEA) in an in vivo model of retinal excitotoxicity, and the involvement of Nerve Growth Factor (NGF) in its actions. Adult Sprague-Dawley rats (250-300 g) received intravitreally (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA; 42 nmol/eye) alone or in combination with DHEA (10(-8), 10(-7), 10(-6) M), or PBS (50 mM, control group). To examine the involvement of NGF and its TrkA receptor in the pharmacological effects of DHEA, animals received AMPA and NGF (60 pg/eye) in the absence or presence of a TrkA receptor inhibitor (Calbiochem 648450, 10(-6) M) or AMPA, DHEA (10(-6) M) and TrkA receptor inhibitor (10(-6), 10(-5) M). Immunohistochemistry studies [choline acetyltransferase (ChAT), brain nitric oxide synthetase (bNOS), calbindin, and TUNEL] and fluorescence-activated cell sorting (FACS) were used to examine retinal cell loss and protection. TrkA receptor immunoreactivity (-IR) and colocalization studies with relevant markers were also performed. AMPA (42 nmol) treatment resulted in a loss of bNOS, ChAT and calbindin immunoreactivities 24 h after its administration. DHEA, administered intravitreally, protected the retina from excitotoxicity in a dose-dependent manner. This effect was mimicked by NGF, and reversed by the NGF TrkA receptor inhibitor. The TrkA receptor is expressed in ganglion cells of rat retina. TUNEL staining and FACS analysis substantiated the neuroprotective actions of DHEA. These results demonstrate for the first time that the neurosteroid DHEA, administered intravitreally, protects the retina from AMPA excitotoxicity. An NGF TrkA receptor mechanism appears to be involved in this neuroprotection.

Somatostatin receptor activation (sst(1) -sst(5) ) differentially influences human retinal pigment epithelium cell viability.

PURPOSE: To investigate the differential effects of somatostatin and its receptors (sst(1-5) ) on the viability of cultured human retinal pigment epithelium (hRPE) cells. METHODS: MTT [3 (4, 5-dimethylthiazol-2yl)-2, 5 diphenyltetrazolium bromide], APO Percentage(TM) and trypan blue assays were performed to assess the mechanisms via which somatostatin (10(-10) -10(-4) m) and selective receptor (sst(1-5) ) ligands (10(-12) -10(-4) m) affect cell viability. The effect of orthovanadate (phosphatase inhibitor, 10(-7) -10(-5) m) on somatostatin's (10(-5) m) actions was examined, and western blot analysis was employed to determine the presence of ssts and phosphotyrosine phosphatase SHP-1 in human RPE cells. RESULTS: Somatostatin and selective ligands for the five somatostatin receptor subtypes (sst(1-5) ) decreased cell viability in a concentration-dependent manner. The observed decrease in cell number was partly because of apoptosis via the activation of sst(1) and sst(5) receptors. Activation of sst(2) , sst(3) and sst(4) receptors led to inhibition of cell growth that did not involve apoptosis, but rather antiproliferative actions. SHP-1 was found in the human RPE cells and sodium orthovanadate reversed somatostatin's actions. CONCLUSIONS: This study provides new information regarding the involvement of ssts in human RPE cell viability and suggests that a pathway involving the phosphotyrosine phosphatase may mediate somatostatin's actions.