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Latanoprost, a synthetic derivative of the natural prostaglandin F(2a) (PGF(2a)), is a powerful antiglaucoma agent with ocular hypotensive and neuroprotective effects. However, the neuroregenerative effect and signaling pathway of latanoprost in retinal ganglion cells (RGCs) are still unknown. The purpose of this study is to investigate the regenerative effect of latanoprost in differentiated RGC-5 cells and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay and neurite length was examined by ArrayScan HCS Reader and Neurite outgrowth BioApplication. Expressions of Akt phosphorylation (p-Akt) and mammalian target of rapamycin phosphorylation (p-mTOR) were investigated by Western blot analysis. The results indicated that 0.1 μM latanoprost (at a clinically therapeutic concentration) significantly increased cell viability as compared with control. Meanwhile, 0.1 μM latanoprost resulted in the obvious promotion of neurite outgrowth similar to ciliary neurotrophic factor (CNTF) and simultaneously increased the levels of p-Akt and p-mTOR expression. The effects of latanoprost were blocked by the Prostaglandin F receptor (FP receptor) inhibitor AL8810, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 and the mTOR inhibitor rapamycin. This study presents novel in vitro evidence that latanoprost could promote neurite outgrowth through an FP receptor-mediated modulation of the PI3K-Akt-mTOR signaling pathway. This finding may provide insight into a better understanding of a new mechanism of latanoprost for glaucoma therapy and into the physiological-modulating activities of prostaglandins.

Neuroprotection offers potential as an alternative therapy for glaucoma. Pilocarpine, as a typical muscarinic receptor agonist, remains among the major intraocular pressure lowering drugs for the conventional treatment of glaucoma. However, whether pilocarpine also possesses neuroprotection against glutamate cytotoxicity in retinal neurons is still unknown. In rat primary retinal cultures, identification of neuron, cell viability, apoptosis, intracellular Ca(2+) concentration, mitochondrial membrane potential, gene expression were studied by immunofluorescence, MTT, High Content Scanning, confocal microscopy, reverse-transcription PCR, and western blot analysis, respectively. Pretreatment of pilocarpine could prevent glutamate-induced neuron death, which was blocked by the non-selective antagonist atropine and the M1-selective muscarinic receptor antagonist pirenzepine. The antiapoptotic effect of pilocarpine was associated with maintaining calcium homeostasis, recovering mitochondrial membrane potential, and regulating the expression of Bcl-2 and Caspase-3. These studies demonstrated that pilocarpine had effective protection against glutamate-induced neuronal apoptosis through M1 muscarinic receptor. The results may provide an insight into the new mechanism of glaucoma therapy that pilocarpine may potentially act as a retinal neuroprotectant.

Hypoxia-induced retinal ganglion cell (RGC) death has been proposed to be the critical event in the pathophysiology of glaucoma. Therefore, delaying or halting RGC degeneration, known as neuroprotection, is a novel and promising approach with potential clinical applications for treating glaucoma. In this study, we investigate hypoxia-induced cell death of RGCs and the underlying mechanisms of N-acetylcysteine (NAC) as a neuroprotectant. To establish a model for chemical hypoxia-induced cell death, RGC-5 cells were treated with the hypoxia mimetic cobalt chloride (CoCl2). Following CoCl2 exposure, significant levels of apoptotic and autophagic cell death were observed in RGC-5 cells, evidenced by lysosome dysfunction and autophagosome formation. Pretreating RGC-5 cells with NAC significantly counteracted the autophagic cell death. NAC-mediated neuroprotection was attributed to the direct scavenging of reactive oxygen species and was mediated by targeting the hypoxia-inducible factor-1α pathway via the BNIP3 and PI3K/Akt/mTOR pathways. These results provide insights into the degeneration of RGCs and present a potential clinical application for NAC as a neuroprotectant.

The retina is the most metabolically active tissue in the human body and hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. The aim of this study is to determine whether muscarinic receptor agonist pilocarpine, a classic antiglaucoma drug, possesses neuroprotection against cobalt chloride (CoCl(2))-mimetic hypoxia-induced apoptosis of rat retinal ganglion cells (RGC-5 cells) and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 assay and apoptosis was examined by annexin V and mitochondrial membrane potential (MMP) assays. Expressions of hypoxia-induced factor-1 alpha (HIF-1 alpha), p53, and BNIP3 were investigated by quantitative real-time PCR and western blot analysis. After treatment of 200 microM CoCl(2) for 24 h, RGC-5 cells showed a marked decrease of cell viability by approximately 30%, increased apoptosis rate and obvious decline in MMP, which could largely be reversed by the pretreatment of 1 microM pilocarpine mainly via the activation of muscarinic receptors. Meanwhile, pretreatment of 1 microM pilocarpine could significantly prevent CoCl(2)-induced HIF-1 alpha translocation from cytoplasm to nucleus and down-regulate the expression of HIF-1 alpha, p53, and BNIP3. These studies demonstrated that pilocarpine had effective protection against hypoxia-induced apoptosis in RGCs via muscarinic receptors and HIF-1 alpha pathway. The findings suggest that HIF-1 alpha pathway as a "master switch" may be used as a therapeutic target in the cholinergic treatment of glaucoma.

Amyloid-beta (Abeta) plays a central role in the neuroinflammation and cholinergic neuronal apoptosis in Alzheimer's disease, and thus has been considered as a main determinant of this disease. In the previous study, we reported that PMS777, a novel bis-interacting ligand for acetylcholinesterase (AChE) inhibition and platelet-activating factor (PAF) receptor antagonism, could significantly attenuate PAF-induced neurotoxicity. Continuing our efforts, we further investigated the protective effect of PMS777 on Abeta-induced neuronal apoptosis in vitro and neuroinflammation in vivo. PMS777 (1-100 microM) was found to inhibit Abeta-induced human neuroblastoma SH-SY5Y cell apoptosis in a concentration-dependent manner. Concurrently, PMS777 increased ratio of bcl-2 to bax mRNA, and inhibited both mRNA expression and activity of caspase-3 in SH-SY5Y cells after the exposure with Abeta. In vivo experimental study demonstrated that PMS777 could attenuate Abeta-induced microglial and astrocytic activation in the rat hippocampus after systemic administration. These results suggest that PMS777 potently protects against Abeta-induced neuronal apoptosis and neuroinflammation, and warrants further investigations in connection with its potential value in the treatment of Alzheimer's disease.

(1) HIV-1 and viral proteins-evoked chronic brain inflammation, which is characterized by microglial activation, is the pivotal neuropathogenesis of HIV-1-associated dementia (HAD). Platelet-activating factor (PAF), mainly released from activated microglia and acts as a high potent inflammatory mediator and a neurotoxin, is indicated to be a principle initiator of neuroinflammation, neuronal dysfunction, and apoptosis related to HAD. Thus, bis-interacting ligands of acetylcholinesterase (AChE) inhibition and PAF receptor antagonism would be of great interest in the therapeutic potential of HAD not only for improvement of cognitive performance, but also for disease-modifying. (2). We have previously reported that a novel tetrahydrofuran-derived bis-interacting ligand PMS777 had satisfying potencies for PAF receptor blockade and AChE inhibition, and markedly improved cholinergic dysfunction-induced cognitive impairment in mice. Continuing with our research, we further investigated the neuroprotective activities of PMS777 on PAF-triggered neuronal injury in human neuroblastoma SH-SY5Y cells. (3) The bis-interacting ligand PMS777 (10 muM) obviously alleviated PAF-induced cell apoptosis in SH-SY5Y cells. Pretreatment with PMS777 also markedly inhibited intracellular Ca(2+) overload, down-regulation of anti-apoptotic bcl-2 mRNA, stimulation of pro-apoptotic bax mRNA expression and activation of caspase-3 pathway. Also, PMS777 could fine-tune pro-inflammatory cyclooxygenase-2 (cox-2) mRNA expression in PAF-treated cells. (4) These results suggest that PMS777 possesses a neuroprotective profile via anti-apoptotic/inflammatory signaling and warrant further investigations in connection with the potential value of this compound in HAD treatment.