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.

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.

Carbocisteine Improves Histone Deacetylase 2 Deacetylation Activity via Regulating Sumoylation of Histone Deacetylase 2 in Human Tracheobronchial Epithelial Cells.

Histone deacetylase (HDAC) 2 plays a vital role in modifying histones to mediate inflammatory responses, while HDAC2 itself is commonly regulated by post-translational modifications. Small ubiquitin-related modifier (SUMO), as an important PTM factor, is involved in the regulation of multiple protein functions. Our previous studies have shown that carbocisteine (S-CMC) reversed cigarette smoke extract (CSE)-induced down-regulation of HDAC2 expression/activity in a thiol/GSH-dependent manner and enhanced sensitivity of steroid therapy. However, the mechanism by which S-CMC regulates HDAC2 is worth further exploring. Our study aimed to investigate the relationships between HDAC2 sumoylation and its deacetylase activity under oxidative stress and the molecular mechanism of S-CMC to regulate HDAC2 activity that mediates inflammatory responses in human bronchial epithelial cells. We found that modification of HDAC2 by SUMO1 and SUMO2/3 occurred in 16HBE cells under physiological conditions, and CSE induced SUMO1 modification of HDAC2 in a dose and time-dependent manner. K462 and K51 of HDAC2 were the two major modification sites of SUMO1, and the K51 site mediated deacetylation activity and function of HDAC2 on histone H4 that regulates IL-8 secretion. S-CMC inhibited CSE-induced SUMO1 modification of HDAC2 in the presence of thiol/GSH, increased HDAC activity, and decreased IL-8 expression. Our study may provide novel mechanistic explanation of S-CMC to ameliorate steroid sensitivity treatment in chronic obstructive pulmonary disease.

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.

L-Satropane Prevents Retinal Neuron Damage by Attenuating Cell Apoptosis and Aß Production via Activation of M1 Muscarinic Acetylcholine Receptor.

Muscarinic acetylcholine receptor (mAChR) agonists have been used to treat glaucoma due to their intraocular pressure-lowering effects. Recently, it has been reported that retinal mAChRs activation can also stimulate neuroprotective pathways. PURPOSE: In our study, we evaluated the potential neuroprotective effect of L-satropane, a novel mAChR agonist, on retinal neuronal injury induced by cobalt chloride (CoCl2) and ischemia/reperfusion (I/R). METHODS: CoCl2-induced hypoxia injury in cultured cell models and I/R-induced retinal neuronal damage in rats in vivo were used to evaluate the abilities of L-satropane. In detail, we measured the occurrence of retinal pathological changes including molecular markers of neuronal apoptosis and Aß expression. RESULTS: Pretreatment with L-satropane protects against CoCl2-induced neurotoxicity in PC12 and primary retinal neuron (PRN) cells in a dose-dependent manner by increasing retinal neuron survival. CoCl2 or I/R-induced cell apoptosis by upregulating Bax expression and downregulating Bcl-2 expression, which resulted in an increased Bax/Bcl-2 ratio, and upregulating caspase-3 expression/activity was significantly reversed by L-satropane treatment. In addition, L-satropane significantly inhibited the upregulation of Aß production in both retinal neurons and tissue. We also found that I/R-induced histopathological retinal changes including cell loss in the retinal ganglion cell layer (GCL) and increased TUNEL positive retinal ganglion cells in GCL and thinning of the inner plexiform layer (IPL) and inner nuclear layer (INL) were markedly improved by L-satropane. The effects of L-satropane were largely abolished by the nonselective mAChRs antagonist atropine and M1-selective mAChR antagonist pirenzepine. CONCLUSION: These results demonstrated that L-satropane might be effective in preventing retinal neuron damage caused by CoCl2 or I/R. The neuroprotective effects of L-satropane may be attributed to decreasing cell apoptosis and Aß production through activation of M1 mAChR.

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.

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.

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.

Potentiated interaction between ineffective doses of budesonide and formoterol to control the inhaled cadmium-induced up-regulation of metalloproteinases and acute pulmonary inflammation in rats.

The anti-inflammatory properties of glucocorticoids are well known but their protective effects exerted with a low potency against heavy metals-induced pulmonary inflammation remain unclear. In this study, a model of acute pulmonary inflammation induced by a single inhalation of cadmium in male Sprague-Dawley rats was used to investigate whether formoterol can improve the anti-inflammatory effects of budesonide. The cadmium-related inflammatory responses, including matrix metalloproteinase-9 (MMP-9) activity, were evaluated. Compared to the values obtained in rats exposed to cadmium, pretreatment of inhaled budesonide (0.5 mg/15 ml) elicited a significant decrease in total cell and neutrophil counts in bronchoalveolar lavage fluid (BALF) associated with a significant reduction of MMP-9 activity which was highly correlated with the number of inflammatory cells in BALF. Additionally, cadmium-induced lung injuries characterized by inflammatory cell infiltration within alveoli and the interstitium were attenuated by the pre-treatment of budesonide. Though the low concentration of budesonide (0.25 mg/15 ml) exerted a very limited inhibitory effects in the present rat model, its combination with an inefficient concentration of formoterol (0.5 mg/30 ml) showed an enhanced inhibitory effect on neutrophil and total cell counts as well as on the histological lung injuries associated with a potentiation of inhibition on the MMP-9 activity. In conclusion, high concentration of budesonide alone could partially protect the lungs against cadmium exposure induced-acute neutrophilic pulmonary inflammation via the inhibition of MMP-9 activity. The combination with formoterol could enhance the protective effects of both drugs, suggesting a new therapeutic strategy for the treatment of heavy metals-induced lung 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.

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.

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.

Activation of muscarinic receptors protects against retinal neurons damage and optic nerve degeneration in vitro and in vivo models.

AIMS: Muscarinic acetylcholine receptor agonist pilocarpine reduces intraocular pressure (IOP) of glaucoma mainly by stimulating ciliary muscle contraction and then increasing aqueous outflow. It is of our great interest to know whether pilocarpine has the additional properties of retinal neuroprotection independent of IOP lowering in vitro and in vivo models. METHODS: In rat primary retinal cultures, cell viability was measured using an MTT assay and the trypan blue exclusion method, respectively. Retinal ganglion cells (RGCs) were identified by immunofluorescence and quantified by flow cytometry. For the in vivo study, the retinal damage after retinal ischemia/reperfusion injury in rats was evaluated by histopathological study using hematoxylin and eosin staining, transmission electron microscopy, and immunohistochemical study on cleaved caspase-3, caspase-3, and ChAT. RESULTS: Pretreatment of pilocarpine attenuated glutamate-induced neurotoxicity of primary retinal neurons in a dose-dependent manner. Protection of pilocarpine in both retinal neurons and RGCs was largely abolished by the nonselective muscarinic receptor antagonist atropine and the M1-selective muscarinic receptor antagonist pirenzepine. After ischemia/reperfusion injury in retina, the inner retinal degeneration occurred including ganglion cell layer thinning and neuron lost, and the optic nerve underwent vacuolar changes. These degenerative changes were significantly lessened by topical application of 2% pilocarpine. In addition, the protective effect of pilocarpine on the ischemic rat retina was favorably reflected by downregulating the expression of activated apoptosis marker cleaved caspase-3 and caspase-3 and upregulating the expression of cholinergic cell marker ChAT. CONCLUSIONS: Taken together, this highlights pilocarpine through the activation of muscarinic receptors appear to afford significant protection against retinal neurons damage and optic nerve degeneration at clinically relevant concentrations. These data also further support muscarinic receptors as potential therapeutic neuroprotective targets in glaucoma.

A new motif in the N-terminal of acetylcholinesterase triggers amyloid-ß aggregation and deposition.

BACKGROUND AND PURPOSE: As a molecular chaperone, acetylcholinesterase (AChE; EC 3.1.1.7) plays a critical role in the pathogenesis of Alzheimer's disease (AD). The peripheral anionic site (PAS) of AChE has been indicated as the amyloid-ß (Aß) binding domain. The goal of this study was to determine other motifs in AChE involved in Aß aggregation and deposition. METHODS AND RESULTS: The ß-hairpin in monomeric Aß is the key motif of nucleation-dependent Aß self-aggregation. As AChE could induce Aß aggregation and deposition, we searched AChE for ß-hairpin structures. In A11-specific dot blot assay, AChE was detected by an oligomer-specific antibody A11, implying the existence of ß-hairpin structures in AChE as ß-hairpin was the core motif of oligomers. A molecular superimposing approach further revealed that the N-terminal region, from Glu7 to Ile20, in AChE (AChE 7-20) was similar to the ß-hairpin domain in Aß. The results of further dot blot assays, thioflavin T fluorescence assays, and electron microscopy imaging experiments, indicated that the N-terminal synthetic peptide AChE7-20 had nearly the same ability as AChE with regard to triggering Aß aggregation and deposition. CONCLUSIONS: AChE 7-20, a ß-hairpin region in AChE, might be a new motif in AChE capable of triggering Aß aggregation and deposition. This finding will be helpful to design new and more effective Aß aggregation inhibitors for AD treatment.

Non-neuronal release of gamma-aminobutyric Acid by embryonic pluripotent stem cells.

γ-Aminobutyric acid (GABA), the principle inhibitory transmitter in the mature central nervous system, is also involved in activities outside the nervous system. Recent studies have shown that functional GABA receptors are expressed in embryonic stem (ES) cells and these receptors control ES cell proliferation. However, it is not clear whether ES cells have their own GABAergic transmission output machinery that can fulfill GABA release or whether the cells merely process the GABA receptors by receiving and responding to the diffused GABA released elsewhere. To get further insight into this unresolved problem, we detected the repertoire of components for GABA synthesis, storage, reaction, and termination in ES and embryonal carcinoma stem cells by biological assays, and then directly quantified released GABA in the intercellular milieu from these pluripotent stem (PS) cells by an analytical chemical assay based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). We found that embryonic PS cells processed a GABAergic circuit machinery and spontaneously released GABA, which suggests the potential that embryonic PS cells could autonomously establish a GABA niche via release of the transmitter.

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.

Bis(9)-(-)-nor-meptazinol as a novel dual-binding AChEI potently ameliorates scopolamine-induced cognitive deficits in mice.

Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder which is characterized by the progressive deterioration of cognition and the emergence of behavioral and psychological symptoms in aging patients. Given that the clinical effectiveness of acetylcholinesterase inhibitors (AChEIs) has still been questioned due to dubious disease-modifying effects, the multi-target directed ligand (MTDL) design has become an emerging strategy for developing new drugs for AD treatment. Bis(9)-(-)-nor-meptazinol (Bis-Mep) was firstly reported by us as a novel MTDL for both potent cholinesterase and amyloid-ß aggregation inhibition. In this study, we further explored its AChE inhibition kinetic features and cognitive amelioration. Bis-Mep was found to be a mixed-type inhibitor on electric eel AChE by enzyme kinetic study. Molecular docking revealed that two "water bridges" located at the two wings of Bis-Mep stabilized its interaction with both catalytic and peripheral anionic sites of AChE. Furthermore, subcutaneous administration of Bis-Mep (10, 100 or 1000 ng/kg) significantly reversed the scopolamine-induced memory deficits in a typical bell-shaped dose-response manner. The maximal cognitive amelioration of Bis-Mep was achieved at 100 ng/kg, comparable with the effect of a reference drug Huperzine A at 1 mg/kg and also the relevant AChE inhibition in brain. These findings suggested that Bis-Mep might be a promising dual-binding AChE inhibitor for potential AD therapeutics.