A COBRA family protein, PtrCOB3, contributes to gelatinous layer formation of tension wood fibers in poplar.

Angiosperm trees usually develop tension wood (TW) in response to gravitational stimulation. TW comprises abundant gelatinous (G-) fibers with thick G-layers primarily composed of crystalline cellulose. Understanding of the pivotal factors governing G-layer formation in TW fiber remains elusive. This study elucidates the role of a Populus trichocarpa COBRA family protein, PtrCOB3, in the G-layer formation of TW fibers. PtrCOB3 expression was upregulated, and its promoter activity was enhanced during TW formation. Comparative analysis with wild-type trees revealed that ptrcob3 mutants, mediated by Cas9/gRNA gene editing, were incapable of producing G-layers within TW fibers and showed severely impaired stem lift. Fluorescence immunolabelling data revealed a dearth of crystalline cellulose in the tertiary cell wall (TCW) of ptrcob3 TW fibers. The role of PtrCOB3 in G-layer formation is contingent upon its native promoter, as evidenced by the comparative phenotypic assessments of pCOB11::PtrCOB3, pCOB3::PtrCOB3, and pCOB3::PtrCOB11 transgenic lines in the ptrcob3 background. Overexpression of PtrCOB3 under the control of its native promoter expedited G-layer formation within TW fibers. We further identified three transcription factors that bind to the PtrCOB3 promoter and positively regulate its transcriptional levels. Alongside the primary TCW synthesis genes, these findings enable the construction of a two-layer transcriptional regulatory network for the G-layer formation of TW fibers. Overall, this study uncovers mechanistic insight into TW formation, whereby a specific COB protein executes the deposition of cellulose, and consequently, G-layer formation within TW fibers.

Direct SUMOylation of M1 muscarinic acetylcholine receptor increases its ligand-binding affinity and signal transduction.

SUMOylation is a significant post-translational modification (PTM) by the small ubiquitin-related modifier (SUMO). Increasing evidence shows SUMOylation regulates GPCR signaling; however, very few GPCRs have been shown to be SUMOylation targets to date. In this study, we identified M1 muscarinic acetylcholine receptor (M1 mAChR), a member of the GPCRs, as a new SUMO substrate. When the mAChR was activated by the agonist carbachol, the colocalization of the M1 mAChR and SUMO-1 protein markedly decreased in immunoprecipitation and immunofluorescence assays. SUMOylation of the M1 mAChR played an important role in increasing the ligand-binding affinity to M1 mAChR, signaling efficiencies, and receptor endocytosis. Through the site-directed mutagenesis approach, K327 was identified as the SUMOylation site of the M1 mAChR. Mutation of the consensus SUMOylation site of the M1 mAChR reduces not only the colocalization of SUMO-1, but also the ligand-binding affinity and signal transduction. The function of M1 mAChR was regulated by SUMOylation through the stabilization of active-state conformation revealed by molecular dynamics simulations. Our results provide evidence that M1 SUMOylation is an important PTM involved in regulation of the affinity for agonists and for activation of signaling pathways.-Xu, J., Tan, P., Li, H., Cui, Y., Qiu, Y., Wang, H., Zhang, X., Li, J., Zhu, L., Zhou, W., Chen, H. Direct SUMOylation of M1 muscarinic acetylcholine receptor increases its ligand-binding affinity and signal transduction.

Involvement of HDAC6 in ischaemia and reperfusion-induced rat retinal injury.

BACKGROUND: The role of histone deacetylases 6 (HDAC6) has been elucidated in various neurodegenerative diseases. However, the effect of HDAC6 on retinal degenerative processes remains unknown. The aim of this study was to elucidate the potential role of HDAC6 in the retinal ischaemia and reperfusion (I/R) injury model. METHODS: The retinal pathological lesion was evaluated by haematoxylin and eosin (H&E) staining. HDAC expression or activity was detected by immunohistochemistry, Western blotting assays or colorimetric assays. The expression of apoptotic- and autophagic- related proteins were quantified by Western blotting and RT-PCR. The expression of peroxiredoxin 2 (Prx2) was determined by RT-PCR and ELISA. The levels of acetylated α-tubulin and acetylated histone 3 in the retina were assayed by Western blotting. RESULTS: We found that I/R-induced reduction of the retinal thickness was ameliorated, and the survival of RGCs was increased by the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) as well as by tubacin (an HDAC6 selective inhibitor). The decreased expression of THY (thymus cell antigen) in the I/R-induced retinas was also reversed by TSA and tubacin. Elevated HDAC6 expression and activity in the retina from I/R injury were significantly inhibited by tubacin, which also attenuated I/R-mediated apoptosis by decreasing TUNEL-positive RGCs and Bax expression and increasing Bcl-2 expression. Additionally, tubacin increased the expression of autophagy-related gene Beclin 1 and microtubule-associated protein 1 light chain 3B (LC3B) and the levels of Prx2. Furthermore, the protective effect of tubacin was associated with acetylated α-tubulin and was independent of acetylated histone 3. CONCLUSIONS: Our findings suggest that tubacin exhibits neuroprotective effects after I/R retinal injury, and HDAC6 may be a potential therapeutic target for the retinal neurodegenerative disease of glaucoma.

The activation of M3 mAChR in airway epithelial cells promotes IL-8 and TGF-ß1 secretion and airway smooth muscle cell migration.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) have been identified in airway epithelium, and epithelium-derived chemokines can initiate the migration of airway smooth muscle (ASM) cells. However, the mAChRs that are expressed in airway epithelium and the mechanism underlying the regulation of ASM cell migration are not clear. The aim of this study was to test whether the effects of the epithelium-derived chemokines on ASM cell migration could be modulated by mAChRs. METHOD: Human epithelial cells (A549 cells) were stimulated with cigarette smoke extract (CSE) or the mAChRs agonist carbachol. IL-8 and TGF-ß1 production were measured by ELISA, and human ASM cell migration was measured using the transwell migration assay and scratch assay. The mRNA levels of the mAChRs subtypes and the acetylcholine concentrations were measured using RT-PCR and LC-MS/MS, respectively. RESULTS: ASM cell migration toward CSE-stimulated A549 cells was markedly reduced by Ac-RRWWCR-NH2 (IL-8 inhibitor) and SB431542 (TGF-ß1 inhibitor). CSE-induced ASM cell migration was also suppressed by the mAChRs antagonist tiotropium. Interestingly, carbachol-stimulated A549 cells also induced ASM cell migration; this migration event was suppressed by tiotropium, Ac-RRWWCR-NH2 and SB431542. In addition, the effects of CSE on ASM cell migration were significantly and cooperatively enhanced by carbachol compared to CSE alone. Carbachol-induced ASM cell migration was reduced by selective inhibitors of PI3K/Akt (LY294002) and p38 (SB203580), suggesting that it occurred through p38 and Akt phosphorylation, which was inhibited by the M3 mAChR antagonist 4-DAMP. CONCLUSIONS: These findings indicate that M3 mAChR may be important therapeutic target for obstructive airway diseases, as it regulates the effects of the epithelial-derived chemokines on ASM cell migration, which results in lung remodeling.

A mucoactive drug carbocisteine ameliorates steroid resistance in rat COPD model.

Steroid insensitivity has been commonly found in chronic obstructive pulmonary disease (COPD) patients, which is mediated by the reduction of histone deacetylase (HDAC) 2. Here we aimed to establish a steroid resistant model on experimental COPD rats and evaluate the effect of carbocisteine (S-CMC), a mucoactive drug. Exposure to cigarette smoke (CS) caused marked pathological features of COPD which are insensitive to DEX associated with the down-regulation of HDAC2 expression/activity. The DEX insensitivity observed in COPD featured rats was improved by S-CMC in the aspects of inhibiting chronic lung inflammation (total and differential inflammatory cell counts, inflammatory cytokines release and inflammatory cells infiltration); ameliorating airway remodeling (thickness of airway epithelium and smooth muscle, airway fibrosis, and the level of α-SMA and TGF-ß1); improving emphysema (emphysema index D2, level of MMP-9 in BALF and the expression of alpha-1 antitrypsin) and preventing impairments of lung function (PEF, IP and IP-slope). Simultaneously, down-regulation of HDAC2 expression/activity was ameliorated by S-CMC treatment. These results indicate that the rat COPD model with steroid resistance was established by active smoking in a short time frame and demonstrate that the failure of steroid therapy can be restored by S-CMC accompanied by increasing HDAC2 expression/activity, providing additional evidence that S-CMC might be used for GC resistance in COPD.

Cu(II) enhances the effect of Alzheimer's amyloid-ß peptide on microglial activation.

BACKGROUND: Aggregated forms of amyloid-ß (Aß) peptides are important triggers for microglial activation, which is an important pathological component in the brains of Alzheimer's patients. Cu(II) ions are reported to be coordinated to monomeric Aß, drive Aß aggregation, and potentiate Aß neurotoxicity. Here we investigated whether Cu(II) binding modulates the effect of Aß on microglial activation and the subsequent neurotoxicity. METHODS: Aß peptides were incubated with Cu(II) at an equimolar ratio to obtain the Cu(II)-Aß complex. Primary and BV-2 microglial cells were treated with Cu(II)-Aß, Aß, or Cu(II). The tumor necrosis factor-α (TNF-α) and nitric oxide levels in the media were determined. Extracellular hydrogen peroxide was quantified by a fluorometric assay with Amplex Red. Mitochondrial superoxide was detected by MitoSOX oxidation. RESULTS: Incubation of Cu(II) with Aß confers different chemical properties on the resulting complex. At the subneurotoxic concentrations, Cu(II)-Aß (but not Aß or Cu(II) alone) treatment induced an activating morphological phenotype of microglia and induced the microglial release of TNF-α and nitric oxide as well as microglia-mediated neuronal damage. Cu(II)-Aß-triggered microglial activation was blocked by nuclear factor (NF)-κB inhibitors and was accompanied with NF-κB activation. Moreover, Cu(II)-Aß induced hydrogen peroxide release, which was not affected by NADPH oxidase inhibitors. Mitochondrial superoxide production was increased after Cu(II)-Aß stimulation. N-acetyl-cysteine, a scavenger of reactive oxygen species (ROS), inhibited Cu(II)-Aß-elicited microglial release of TNF-α and nitric oxide as well as the microglia-mediated neurotoxic effect. CONCLUSION: Our observations suggest that Cu(II) enhances the effect of Aß on microglial activation and the subsequent neurotoxicity. The Cu(II)-Aß-triggered microglial activation involves NF-κB activation and mitochondrial ROS production.

Carbocysteine restores steroid sensitivity by targeting histone deacetylase 2 in a thiol/GSH-dependent manner.

Steroid insensitivity is commonly observed in patients with chronic obstructive pulmonary disease. Here, we report the effects and mechanisms of carbocysteine (S-CMC), a mucolytic agent, in cellular and animal models of oxidative stress-mediated steroid insensitivity. The following results were obtained: oxidative stress induced higher levels of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α), which are insensitive to dexamethasone (DEX). The failure of DEX was improved by the addition of S-CMC by increasing histone deacetylase 2 (HDAC2) expression/activity. S-CMC also counteracted the oxidative stress-induced increase in reactive oxygen species (ROS) levels and decreases in glutathione (GSH) levels and superoxide dismutase (SOD) activity. Moreover, oxidative stress-induced events were decreased by the thiol-reducing agent dithiothreitol (DTT), enhanced by the thiol-oxidizing agent diamide, and the ability of DEX was strengthened by DTT. In addition, the oxidative stress-induced decrease in HDAC2 activity was counteracted by S-CMC by increasing thiol/GSH levels, which exhibited a direct interaction with HDAC2. S-CMC treatment increased HDAC2 recruitment and suppressed H4 acetylation of the IL-8 promoter, and this effect was further ablated by addition of buthionine sulfoximine, a specific inhibitor of GSH synthesis. Our results indicate that S-CMC restored steroid sensitivity by increasing HDAC2 expression/activity in a thiol/GSH-dependent manner and suggest that S-CMC may be useful in a combination therapy with glucocorticoids for treatment of steroid-insensitive pulmonary diseases.

Subneurotoxic copper(II)-induced NF-κB-dependent microglial activation is associated with mitochondrial ROS.

Microglia-mediated neuroinflammation and the associated neuronal damage play critical roles in the pathogenesis of neurodegenerative disorders. Evidence shows an elevated concentration of extracellular copper(II) in the brains of these disorders, which may contribute to neuronal death through direct neurotoxicity. Here we explored whether extracellular copper(II) triggers microglial activation. Primary rat microglia and murine microglial cell line BV-2 cells were cultured and treated with copper(II). The content of tumor necrosis factor-α (TNF-α) and nitric oxide in the medium was determined. Extracellular hydrogen peroxide was quantified by a fluorometric assay with Amplex Red. Mitochondrial superoxide was measured by MitoSOX oxidation. At subneurotoxic concentrations, copper(II) treatment induced a dose- and time-dependent release of TNF-α and nitric oxide from microglial cells, and caused an indirect, microglia-mediated neurotoxicity that was blocked by inhibition of TNF-α and nitric oxide production. Copper(II)-initiated microglial activation was accompanied with reduced IкB-α expression as well as phosphorylation and translocation of nuclear factor-κB (NF-κB) p65 and was blocked by NF-κB inhibitors (BAY11-7082 and SC-514). Moreover, copper(II) treatment evoked a rapid release of hydrogen peroxide from microglial cells, an effect that was not affected by NADPH oxidase inhibitors. N-acetyl-cysteine, a scavenger of reactive oxygen species (ROS), abrogated copper(II)-elicited microglial release of TNF-α and nitric oxide and subsequent neurotoxicity. Importantly, mitochondrial production of superoxide, paralleled to extracellular release of hydrogen peroxide, was induced after copper(II) stimulation. Our findings suggest that extracellular copper(II) at subneurotoxic concentrations could trigger NF-κB-dependent microglial activation and subsequent neurotoxicity. NADPH oxidase-independent, mitochondria-derived ROS may be involved in this activation.

M3 mAChR-mediated IL-8 expression through PKC/NF-κB signaling pathways.

OBJECTIVE: M3 muscarinic acetylcholine receptor (mAChR) plays an important role in the regulation of cytokine production in inflammatory diseases. In this study, we explored the precise role of M3 mAChR under stimulation with agonist in IL-8 expression and of the signaling pathway involved in this process. MATERIALS AND METHODS: Recombinant U2OS cells stably expressing M3 mAChR as a model system were stimulated by carbachol to evaluate the role of M3 mAChR in the expression of IL-8. RESULTS: Activation of M3 mAChR with carbachol increased both IL-8 mRNA and protein expression in a concentration-dependent manner. Elevated IL-8 expression was completely antagonized by atropine, 4-DAMP and tiotropium. M3 mAChR-mediated IL-8 expression was almost completely inhibited by the NF-κB inhibitor BAY11-7082 and, to a lesser extent, by U0126, SB203580, and SP600125, which are inhibitors for ERK1/2, p38, and JNK, respectively. Furthermore, M3 mAChR-mediated NF-κB activation and IL-8 expression were simultaneously attenuated by the PKC inhibitor calphostin C, whereas PMA, a PKC activator, mimicked the effects of carbachol, inducing IL-8 expression. CONCLUSIONS: Our findings offer insights into the specific and critical role of M3 mAChR in regulating inflammatory response and indicate M3 mAChR/PKC/NF-κB signaling axis driven by endogenous acetylcholine as a potential therapeutic targets for inflammatory diseases.

mAChRs activation induces epithelial-mesenchymal transition on lung epithelial cells.

BACKGROUND: Epithelial-mesenchymal transition (EMT) has been proposed as a mechanism in the progression of airway diseases and cancer. Here, we explored the role of acetylcholine (ACh) and the pathway involved in the process of EMT, as well as the effects of mAChRs antagonist. METHODS: Human lung epithelial cells were stimulated with carbachol, an analogue of ACh, and epithelial and mesenchymal marker proteins were evaluated using western blot and immunofluorescence analyses. RESULTS: Decreased E-cadherin expression and increased vimentin and α-SMA expression induced by TGF-ß1 in alveolar epithelial cell (A549) were significantly abrogated by the non-selective mAChR antagonist atropine and enhanced by the acetylcholinesterase inhibitor physostigmine. An EMT event also occurred in response to physostigmine alone. Furthermore, ChAT express and ACh release by A549 cells were enhanced by TGF-ß1. Interestingly, ACh analogue carbachol also induced EMT in A549 cells as well as in bronchial epithelial cells (16HBE) in a time- and concentration-dependent manner, the induction of carbachol was abrogated by selective antagonist of M1 (pirenzepine) and M3 (4-DAMP) mAChRs, but not by M2 (methoctramine) antagonist. Moreover, carbachol induced TGF-ß1 production from A549 cells concomitantly with the EMT process. Carbachol-induced EMT occurred through phosphorylation of Smad2/3 and ERK, which was inhibited by pirenzepine and 4-DAMP. CONCLUSIONS: Our findings for the first time indicated that mAChR activation, perhaps via M1 and M3 mAChR, induced lung epithelial cells to undergo EMT and provided insights into novel therapeutic strategies for airway diseases in which lung remodeling occurs.

Targeted inhibition of KCa3.1 channel attenuates airway inflammation and remodeling in allergic asthma.

KCa3.1 has been suggested to be involved in regulating cell activation, proliferation, and migration in multiple cell types, including airway inflammatory and structural cells. However, the contributions of KCa3.1 to airway inflammation and remodeling and subsequent airway hyperresponsiveness (AHR) in allergic asthma remain to be explored. The main purpose of this study was to elucidate the roles of KCa3.1 and the potential therapeutic value of KCa3.1 blockers in chronic allergic asthma. Using real-time PCR, Western blotting, or immunohistochemical analyses, we explored the precise role of KCa3.1 in the bronchi of allergic mice and asthmatic human bronchial smooth muscle cells (BSMCs). We found that KCa3.1 mRNA and protein expression were elevated in the bronchi of allergic mice, and double labeling revealed that up-regulation occurred primarily in airway smooth muscle cells. Triarylmethane (TRAM)-34, a KCa3.1 blocker, dose-dependently inhibited the generation and maintenance of the ovalbumin-induced airway inflammation associated with increased Th2-type cytokines and decreased Th1-type cytokine, as well as subepithelial extracellular matrix deposition, goblet-cell hyperplasia, and AHR in a murine model of asthma. Moreover, the pharmacological blockade and gene silencing of KCa3.1, which was evidently elevated after mitogen stimulation, suppressed asthmatic human BSMC proliferation and migration, and arrested the cell cycle at the G0/G1 phase. In addition, the KCa3.1 activator 1-ethylbenzimidazolinone-induced membrane hyperpolarization and intracellular calcium increase in asthmatic human BSMCs were attenuated by TRAM-34. We demonstrate for the first time an important role for KCa3.1 in the pathogenesis of airway inflammation and remodeling in allergic asthma, and we suggest that KCa3.1 blockers may represent a promising therapeutic strategy for asthma.

The effect of absolute configuration on activity, subtype selectivity (M3/M2) of 3α-acyloxy-6ß-acetoxyltropane derivatives as muscarinic M3 receptor antagonists.

Both enantiomers of 3α-acyloxy-6ß-acetoxyltropane derivatives 1-4 were prepared respectively and underwent functional studies and radioreceptor binding assays. 6S Enantiomers showed obvious muscarinic M3, M2 antagonistic activity, while the 6R ones elicited little muscarinic activity by functional studies. Besides, the affinity of 6S enantiomers to muscarinic M3 receptors of rat submandibulary gland, M2 receptors of rat left atria was much larger than that of corresponding 6R enantiomers. All these pharmalogical results indicated 6S configuration was favorable for 3α-acyloxy-6ß-acetoxyltropane derivatives to bind with muscarinic M3 or M2 receptors and elicited antagonistic activity. Furthermore, the muscarinic M3 activity and subtype selectivity (M3/M2) of 6S enantiomers could be improved by increasing the electron density of carbonyl oxygen or introducing methylene group between the carbonyl and phenyl ring in C-3α position. Understanding the effect of absolute configuration on activity, subtype selectivity (M3/M2) of 3α-acyloxy-6ß-acetoxyltropane derivatives will provide the clues for designing muscarinic M3 antagonists with high activity and low side effects or toxicity.

Protective effects of anisodamine on cigarette smoke extract-induced airway smooth muscle cell proliferation and tracheal contractility.

Anisodamine, an antagonist of muscarinic acetylcholine receptors (mAChRs), has been used therapeutically to improve smooth muscle function, including microvascular, intestinal and airway spasms. Our previous studies have revealed that airway hyper-reactivity could be prevented by anisodamine. However, whether anisodamine prevents smoking-induced airway smooth muscle (ASM) cell proliferation remained unclear. In this study, a primary culture of rat ASM cells was used to evaluate an ASM phenotype through the ability of the cells to proliferate and express contractile proteins in response to cigarette smoke extract (CSE) and intervention of anisodamine. Our results showed that CSE resulted in an increase in cyclin D1 expression concomitant with the G0/G1-to-S phase transition, and high expression of M2 and M3. Functional studies showed that tracheal hyper-contractility accompanied contractile marker α-SMA high-expression. These changes, which occur only after CSE stimulation, were prevented and reversed by anisodamine, and CSE-induced cyclin D1 expression was significantly inhibited by anisodamine and the specific inhibitor U0126, BAY11-7082 and LY294002. Thus, we concluded that the protective and reversal effects and mechanism of anisodamine on CSE-induced events might involve, at least partially, the ERK, Akt and NF-κB signaling pathways associated with cyclin D1 via mAChRs. Our study validated that anisodamine intervention on ASM cells may contribute to anti-remodeling properties other than bronchodilation.

Novel bis-(-)-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property.

The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may represent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(-)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal chelating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC(50) values of 9.63µM (for ZLA) and 8.64µM (for ZLB), and prevent AChE-induced amyloid-ß (Aß) aggregation with IC(50) values of 49.1µM (for ZLA) and 55.3µM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Aß aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treatment of AD.

N-acetylcysteine protects against hypoxia mimetic-induced autophagy by targeting the HIF-1α pathway in retinal ganglion cells.

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.

Latanoprost promotes neurite outgrowth in differentiated RGC-5 cells via the PI3K-Akt-mTOR signaling pathway.

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.

Huperzine A lowers intraocular pressure via the M3 mAChR and provides retinal neuroprotection via the M1 mAChR: a promising agent for the treatment of glaucoma.

BACKGROUND: Glaucoma is a neurodegenerative disease that shares similar pathological mechanisms with Alzheimer's disease (AD). Drug treatments for glaucoma increasingly rely upon both lowering of intraocular pressure (IOP) and optic nerve protection, as lowering of IOP alone has been unsatisfactory. Huperzine A (HupA) is an acetylcholinesterase inhibitor (AChEI) used for AD. This study investigated the potential of HupA as a treatment for glaucoma. METHODS: The ability of HupA to lower IOP via causing pupil constriction was assessed using New Zealand rabbits. The retinal neuroprotective effects of HupA were assessed in vivo using rat retinas subjected to ischemia-reperfusion (I/R) and in vitro using primary retinal neurons (PRNs) suffering from oxygen-glucose deprivation (OGD). RESULTS: HupA caused pupil constriction in a dose-time dependent manner which was reversed by the nonselective muscarinic acetylcholine receptor (mAChR) antagonist atropine and the selective M3 mAChR antagonist 4-DAMP. However, HupA had no effect on isolated iris muscle tension and calcium flow indicating an indirect M3 mAChR mediated effect. HupA exerted a neuroprotective effect against I/R and OGD to attenuate the retinal pathological lesion, improve retinal neuronal cell viability, reverse oxidative stress injury by increasing GSH levels and SOD activity, and decreasing MDA content and reduce the retinal neuronal apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 expression with no effect on the calcium flow tests. The effects were abolished by atropine and the selective M1 mAChR antagonist pirenzepine in OGD-induced PRNs suggesting an indirect M1 mAChR-mediated effect via inhibiting AChE activity to increase endogenous ACh level. Furthermore, HupA increased phosphorylated AKT level and decreased the levels of phosphorylated JNK, P38 MAPK and ERK via M1 mAChR antagonists indicating an involvement of activating the M1 mAChR and the downstream AKT/MAPK signaling pathway in the protective effects of HupA. CONCLUSIONS: HupA could significantly decrease IOP via activating M3 mAChR indirectly and produce retinal neuroprotective effect through M1 mAChR/AKT/MAPK by increasing endogenous ACh level. These investigations demonstrated that HupA was an effective drug in glaucoma treatment and the clinical application of HupA and other AChEIs for glaucoma patients should be further investigated.

Pilocarpine protects cobalt chloride-induced apoptosis of RGC-5 cells: involvement of muscarinic receptors and HIF-1 alpha pathway.

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.

Muscarinic activation attenuates abnormal processing of beta-amyloid precursor protein induced by cobalt chloride-mimetic hypoxia in retinal ganglion cells.

beta-Amyloid peptide (Abeta), the major pathological factor in Alzheimer's disease, has recently been reported to be implicated in the development of glaucoma. In this study, we explored the effect of muscarinic activation on abnormal processing of beta-amyloid precursor protein (APP) induced by a risk factor hypoxia in retinal ganglion cells. Hypoxia mimetic compound cobalt chloride could increase the generation of Abeta via up-regulating the expression of APP as well as the expression of beta-secretase and gamma-secretase, whereas muscarinic receptor agonist pilocarpine could significantly attenuate this abnormal pathway, thereby resulting in a decreased amyloidogenic cleavage of APP. This finding may provide an insight into better understanding of pathophysiology for the retinal neurodegenerative disease and searching for its new modifying approach.

Novel piperazine derivative PMS1339 exhibits tri-functional properties and cognitive improvement in mice.

Amyloid-beta-induced neuroinflammation plays a central role in the extensive loss of cholinergic neurons and cognitive decline in Alzheimer's disease. The acetylcholinesterase (AChE) inhibitors are the first class of drugs used to enhance surviving cholinergic activities. However, their limited effectiveness following long-term treatment raises a need for new multi-target therapies. We report herein a novel piperazine derivative compound PMS1339 possesses multifunctional properties including anti-platelet-activating factor, AChE inhibition, Abeta aggregation inhibition and cognitive improvement. PMS1339 could significantly inhibit both mice brain AChE (IC50=4.41+/-0.63 microM) and sera butyrylcholinesterase (BuChE, IC50=1.09+/-0.20 microM). PMS1339 was also found to inhibit neuronal AChE secreted by SH-SY5Y cell line (IC50=17.95+/-2.31 microM). Enzyme kinetics experiments performed on electric eel AChE indicated that PMS1339 acts as a mixed type competitive AChE inhibitor. Molecular docking studies using the X-ray crystal structure of AChE from Torpedo californica elucidated the interactions between PMS1339 and AChE: PMS1339 is well buried inside the active-site gorge of AChE interacting with Trp84 at the bottom, Tyr121 halfway down and Trp279 at the peripheral anionic site (PAS). Thioflavin T-based fluorimetric assay revealed the ability of PMS1339 to inhibit AChE-induced Abeta aggregation. In-vivo study indicated PMS1339 (1 mg/kg i.p.) reversed scopolamine-induced memory impairment in mice. Overall, these findings indicated that PMS1339 exhibits tri-functional properties in vitro and cognitive improvement in vivo, and revealed the emergence of a multi-target-directed ligand to tackle the determinants of Alzheimer's disease.