Autophagy protects neuron from Aβ-induced cytotoxicity

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimer's disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ_25-35, Aβ_1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ_1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.     

Nicotine promotes atherosclerosis development in apolipoprotein E-deficient mice through α1-nAChR

α1 Nicotinic acetylcholine receptor (α1nAChR) is an important nicotine receptor that is widely distributed in vascular smooth muscle cells, macrophages, and endothelial cells. However, the role of α1nAChR in nicotine-mediated atherosclerosis remains unclear. The administration of nicotine for 12 weeks increased the area of the atherosclerotic lesion, the number of macrophages infiltrating the plaques, and the circulating levels of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α, in apolipoprotein E-deficient (ApoE^−/−) mice fed a high-fat diet. Nicotine also increased α1nAChR, calpain-1, matrix metalloproteinase-2 (MMP-2), and MMP-9 expression in the aortic tissue. Silencing of α1nAChR with an adenoassociated virus decreased the atherosclerotic size, lesion macrophage content, and circulating levels of inflammatory cytokines and suppressed α1nAChR, calpain-1, MMP-2, and MMP-9 expression in the nicotine group. In vitro, nicotine-induced α1nAChR, calpain-1, MMP-2, and MMP-9 expression in mouse vascular smooth muscle cells (MOVAS) and macrophages (RAW264.7), and enhanced the migration and proliferation of these cells. The silencing of α1nAChR inhibited these effects of nicotine MOVAS and RAW264.7 cells. Thus, we concluded that nicotine promoted the development of atherosclerosis partially by inducing the migration and proliferation of vascular smooth muscle cells and macrophages and inducing an inflammatory reaction. The effect of nicotine on atherogenesis may be mediated by α1nAChR-induced activation of the calpain-1/MMP-2/MMP-9 signaling pathway.     

Differential involvement of α4β2, α7 and α9α10 nicotinic acetylcholine receptors in B lymphocyte activation in vitro

Mouse B lymphocytes express several nicotinic acetylcholine receptor (nAChR) subtypes, their exact functions being not clearly understood. Here we show that α7 nAChR was present in about 60%, while α4β2 and α9(α10) nAChRs in about 10% and 20% of mouse spleen B lymphocytes, respectively; Balb/c and C57Bl/6 mice possessed different relative amounts of these nAChR subtypes. α4β2 and α7, but not α9(α10) nAChRs, were up-regulated upon B lymphocyte activation in vitro. Flow cytometry and sandwich ELISA studies demonstrated that α7 and α9(α10) nAChRs are coupled to CD40, whereas α4β2 nAChR is coupled to IgM. B lymphocytes of both α7(-/-) and β2(-/-) mice responded to anti-CD40 stronger than those of the wild-type mice, whereas the cells of β2(-/-) mice responded to anti-IgM worse than those of the wild-type or α7(-/-) mice. Inhibition of α7 and α9(α10) nAChRs with methyllicaconitine resulted in considerable augmentation of CD40-mediated B lymphocyte proliferation in cells of all genotypes; stimulation of α4β2 nAChRs with epibatidine increased the IgM-mediated proliferation of the wild-type and α7(-/-), but not β2(-/-) cells. Inhibition of α9(α10) nAChRs with α-conotoxin PeAI exerted weak stimulating effect on CD40-mediated proliferation. This nAChR subtype was up-regulated in α7(-/-) B-cells. α7 nAChRs were found recruited to immune synapses between human T and B lymphocytes, both of which produced acetylcholine. It is concluded that α7 nAChR fulfills inhibitory CD40-related mitogenic function, α4β2 nAChR produces a stimulatory IgM-related effect, while α9α10 nAChR is a "reserve" receptor, which partly compensates the absence of α7 nAChR in α7(-/-) cells. Acetylcholine is an additional mediator to modulate activation of interacting T and B lymphocytes.     

Nicotine Elicits Prolonged Calcium Signaling along Ventral Hippocampal Axons

Presynaptic nicotinic acetylcholine receptors (nAChRs) have long been implicated in the modulation of CNS circuits. We previously reported that brief exposure to low concentrations of nicotine induced sustained potentiation of glutamatergic transmission at ventral hippocampal (vHipp)-striatal synapses. Here, we exploited nAChR subtype-selective antagonists and agonists and α7*nAChR knockout mutant mice (α7-/-) to elucidate the signaling mechanisms underlying nAChR-mediated modulation of synaptic transmission. Using a combination of micro-slices culture from WT and α7-/-mice, calcium imaging, and immuno-histochemical techniques, we found that nicotine elicits localized and oscillatory increases in intracellular Ca²⁺ along vHipp axons that persists for up to 30 minutes. The sustained phase of the nicotine-induced Ca²⁺ response was blocked by α-BgTx but not by DHβE and was mimicked by α7*nAChR agonists but not by non-α7*nAChR agonists. In vHipp slices from α7-/- mice, nicotine elicited only transient increases of axonal Ca²⁺ signals and did not activate CaMKII. The sustained phase of the nicotine-induced Ca²⁺ response required localized activation of CaMKII, phospholipase C, and IP₃ receptor mediated Ca²⁺-induced Ca²⁺ release (CICR). In conclusion, activation of presynaptic nAChRs by nicotine elicits Ca²⁺ influx into the presynaptic axons, the sustained phase of the nicotine-induced Ca²⁺ response requires that axonal α7*nAChR activate a downstream signaling network in the vHipp axons.     

Repositioning of Memantine as a Potential Novel Therapeutic Agent against Meningitic E. coli–Induced Pathogenicities through Disease-Associated Alpha7 Cholinergic Pathway and RNA Sequencing-Based Transcriptome Analysis of Host Inflammatory Responses

Neonatal sepsis and meningitis (NSM) remains a leading cause worldwide of mortality and morbidity in newborn infants despite the availability of antibiotics over the last several decades. E. coli is the most common gram-negative pathogen causing NSM. Our previous studies show that α7 nicotinic receptor (α7 nAChR), an essential regulator of inflammation, plays a detrimental role in the host defense against NSM. Despite notable successes, there still exists an unmet need for new effective therapeutic approaches to treat this disease. Using the in vitro/in vivo models of the blood-brain barrier (BBB) and RNA-seq, we undertook a drug repositioning study to identify unknown antimicrobial activities for known drugs. We have demonstrated for the first time that memantine (MEM), a FDA-approved drug for treatment of Alzheimer’s disease, could very efficiently block E. coli-caused bacteremia and meningitis in a mouse model of NSM in a manner dependent on α7 nAChR. MEM was able to synergistically enhance the antibacterial activity of ampicillin in HBMEC infected with E. coli K1 (E44) and in neonatal mice with E44-caused bacteremia and meningitis. Differential gene expression analysis of RNA-Seq data from mouse BMEC infected with E. coli K1 showed that several E44-increased inflammatory factors, including IL33, IL18rap, MMP10 and Irs1, were significantly reduced by MEM compared to the infected cells without drug treatment. MEM could also significantly up-regulate anti-inflammatory factors, including Tnfaip3, CISH, Ptgds and Zfp36. Most interestingly, these factors may positively and negatively contribute to regulation of NF-κB, which is a hallmark feature of bacterial meningitis. Furthermore, we have demonstrated that circulating BMEC (cBMEC) are the potential novel biomarkers for NSM. MEM could significantly reduce E44-increased blood level of cBMEC in mice. Taken together, our data suggest that memantine can efficiently block host inflammatory responses to bacterial infection through modulation of both inflammatory and anti-inflammatory pathways.     

Calcium activity in response to nAChR ligands in murine bone marrow granulocytes with different Gr-1 expression

Polymorphonuclear neutrophilic granulocytes (PMNs) are the largest proportion of leukocytes in adult human blood that perform numerous functions, including phagocytosis, degranulation, generation of reactive oxygen species, and NETosis. Excessive neutrophil activity associates with hyperinflammation and tissue damage during pathologies such as inflammatory bowel disease, diabetes mellitus, tuberculosis, and coronavirus disease 2019. Nicotinic acetylcholine receptors (nAChRs) can modulate immune cells, including neutrophils, functions, therefore, nAChR ligands are considered as the potent agents for therapy of inflammation. Earlier it was shown, that about 30% of PMNs from the acute inflammatory site responded to nicotine by calcium spikes. In this study, we studied the generation of calcium spikes in murine granulocytes with different maturity level (evaluated by Gr-1 expression) isolated from bone marrow in response to ligands of nAChRs in control and under chronic nicotine consumption. It was found that nearly 20%–25% cells in the granulocyte population responded to nicotine or selective antagonists of different type of nAChRs (α-cobratoxin, GIC, and Vc1.1). We demonstrated that in the control group Ca²⁺-mobilizing activity was regulated through α7 and α9α10 nAChRs in immature granulocytes (Gr-1^int), whereas in mature granulocytes (Gr-1^hi) it was regulated through α7, α3β2, and α9-contained nAChRs. Sensitivity of PMNs to nicotine depended on their maturity level after chronic nicotine consumption. Gr-1^int cells responded to nicotine through α7 and α9-contained nAChRs, while Gr-1^hi did not respond to nicotine. Thus, calcium response to nAChR ligands in bone marrow PMNs depends on their maturity level.     

Nicotinic receptor agonists and antagonists increase sAPPα secretion and decrease Aβ levels in vitro

We have earlier reported that Aβ were significantly reduced in brains of smoking Alzheimer patients and control subjects compared with non-smokers, as well as in nicotine treated APPsw transgenic mice. To examine the mechanisms by which nicotine modulates APP processing we here measured levels of secreted amyloid precursor protein (sAPPα), total sAPP, Aβ40 and Aβ42 in different cell lines expressing different nicotinic receptor (nAChR) subtypes or no nAChRs. Treatment with nicotine increased release of sAPPα and at the same time lowered Aβ levels in both SH-SY5Y and SH-SY5Y/APPsw cells expressing α3 and α7 nAChR subtypes. These effects could also be evoked by co-treatment with the competitive α7 nAChR antagonists α-bungarotoxin and methyllycaconitine (MLA), and by these antagonists alone, suggesting that binding to the agonist binding site, rather than activation of the receptor, may be sufficient to trigger changes in APP processing. The nicotine-induced increase in sAPPα could only be blocked by co-treatment with the open channel blocker mecamylamine. In addition to nicotine, the agonists epibatidine and cytisine both significantly increased the release of sAPP in M10 cells expressing the α4/β2 nAChR subtype, and this effect was blocked by co-treatment with mecamylamine but not by the α4/β2 competitive antagonist dihydro-β-erythroidine. The lack of effect of nicotine on sAPPα and Aβ levels in HEK 293/APPsw cells, which do not express any nAChRs, demonstrates that the nicotine-induced attenuation of β-amyloidosis is mediated by nAChRs and not by a direct effect of nicotine. Our data show that nicotinic compounds stimulate the non-amyloidogenic pathway and that α4 and α7 nAChRs play a major role in modulating this process. Nicotinic drugs directed towards specific nAChR subtypes might therefore be beneficial for the treatment of AD not only by lowering Aβ production but also by enhance release of neuroprotective sAPPα.     

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

Background and aimsCigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine – which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH).MethodshHSC were isolated from healthy human livers and nAChR expression analyzed – RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-α2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-β1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH).ResultshHSCs express muscle type (α1, β1, delta and epsilon) and neuronal type (α3, α6, α7, β2 and β4) nAChR subunits at the mRNA level. Among these subunits, α3, α7, β1 and ε were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-α2 and TGF-β1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. α1 and α3-nAChR mRNA expression was significantly upregulated in NASH fibrosis compared to normal livers.ConclusionNicotine at levels in smokers’ blood is pro-fibrogenic, through actions on hHSCs expressed nAChRs. Therefore, CS, via its nicotine content, may worsen liver fibrosis. Moreover, nicotinic receptor antagonists may have utility as novel anti-fibrotic agents.     

α5‐nAChR contributes to epithelial‐mesenchymal transition and metastasis by regulating Jab1/Csn5 signalling in lung cancer

Recent studies have showed that α5 nicotinic acetylcholine receptor (α5‐nAChR) is closely associated with nicotine‐related lung cancer. Our previous studies also demonstrated that α5‐nAChR mediates nicotine‐induced lung carcinogenesis. However, the mechanism by which α5‐nAChR functions in lung carcinogenesis remains to be elucidated. Jab1/Csn5 is a key regulatory factor in smoking‐induced lung cancer. In this study, we explored the underlying mechanisms linking the α5‐nAChR‐Jab1/Csn5 axis with lung cancer epithelial‐mesenchymal transition (EMT) and metastasis, which may provide potential therapeutic targets for future lung cancer treatments. Our results demonstrated that the expression of α5‐nAChR was correlated with the expression of Jab1/Csn5 in lung cancer tissues and lung cancer cells. α5‐nAChR expression is associated with Jab1/Csn5 expression in lung tumour xenografts in mice. In vitro, the expression of α5‐nAChR mediated Stat3 and Jab1/Csn5 expression, significantly regulating the expression of the EMT markers, N‐cadherin and Vimentin. In addition, the down‐regulation of α5‐nAChR or/and Stat3 reduced Jab1/Csn5 expression, while the silencing of α5‐nAChR or Jab1/Csn5 inhibited the migration and invasion of NSCLC cells. Mechanistically, α5‐nAChR contributes to EMT and metastasis by regulating Stat3‐Jab1/Csn5 signalling in NSCLC, suggesting that α5‐nAChR may be a potential target in NSCLC diagnosis and immunotherapy.     

Sensorimotor gating and spatial learning in α7‐nicotinic receptor knockout mice

The role of acetylcholine and specific nicotinic receptors in sensorimotor gating and higher cognitive function has been controversial. Here, we used a commercially available mouse with a null mutation in the Chrna7^tm1Bay gene [α7‐nicotinic acetylcholine receptor (nAChR) knockout (KO) mouse] in order to assess the role of the α7‐nAChR in sensorimotor gating and spatial learning. We examined prepulse inhibition (PPI) of startle and nicotine‐induced enhancement of PPI. We also tested short‐ and long‐term habituation of the startle response as well as of locomotor behaviour in order to differentiate the role of this receptor in the habituation of evoked behaviour (startle) vs. motivated behaviour (locomotion). To address higher cognition, mice were also tested in a spatial learning task. Our results showed a mild but consistent PPI deficit in α7‐nAChR KO mice. Furthermore, they did not show nicotine‐induced enhancement of startle or PPI. Short‐ and long‐term habituation was normal in KO mice for both types of behaviours, evoked or motivated, and they also showed normal learning and memory in the Barnes maze. Thorough analysis of the behavioural data indicated a slightly higher degree of anxiety in α7‐nAChR KO mice; however, this could only be partially confirmed in an elevated plus maze test. In summary, our data suggest that α7‐nAChRs play a minor role in PPI, but seem to mediate nicotine‐induced PPI enhancement. We found no evidence to suggest that they are important for habituation or spatial learning .     

Age‐related loss of neuronal nicotinic receptor expression in the aging mouse hippocampus corresponds with cyclooxygenase‐2 and PPARγ expression and is altered by long‐term NS398 administration

Age‐related changes in the mammalian dorsal hippocampus are associated with diminished expression of neuronal nicotinic acetylcholine receptors (nAChR), which is particularly severe in pathologies such as those associated with dementias, including Alzheimer's disease. Because the mouse is a useful model for age‐related decline in nAChR expression in the basal forebrain and limbic system, we used immunohistochemistry to examine the influence of long‐term (12‐month) oral administration of nicotine and/or the cyclooxygenase‐2 (COX‐2) preferring non‐steroidal anti‐inflammatory drug (NSAID) NS398 on nAChRα4, α5, α7, and β4 expression in the C57BL/6 mouse. Inhibitory neurons of the dorsal hippocampus that express nAChRs also constitutively express COX‐2 and the peroxisome proliferator‐antagonist receptor subtype gamma‐2 (PPARγ2) which is also a target of NS398. Administration of NS398 correlated with retention of nAChRα4 and to a lesser extent nAChRβ4, but not nAChRα5 or α7, but nicotine exhibited no similar effect. Nicotine and NS398 co‐administration abolished the NS398‐related effect on nAChRα4 retention. These results provide evidence that the interaction during aging between oral administration of nicotine and NSAIDs are not straightforward and could even be antagonistic when combined. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

The potential subunits involved in two subtypes of α-Bgt-resistant nAChRs in cockroach dorsal unpaired median (DUM) neurons

The american cockroach (Periplaneta americana) dorsal unpaired median (DUM) neurons provide an native tool to analyze the functional and pharmacological properties of ion channels and membrane receptors, such as nicotine acetylcholine receptors (nAChRs). Here the imidacloprid-activated nAChR subtypes were examined in DUM neurons by the patch-clamp technique and the potential subunits involved in important subtypes were analyzed by combining with RNA interference (RNAi) technique. Imidacloprid exerted agonist activities on one subtype in α-Bgt-sensitive nAChRs and another subtype in α-Bgt-resistant nAChRs, in which the α-Bgt-resistant subtype showed much higher sensitivity to imidacloprid than the α-Bgt-sensitive subtype, with the difference close to 200-fold. In α-Bgt-resistant nAChRs, nicotine exerted the agonist activity on two subtypes (nAChR1 and nAChR2), although imidacloprid only activated nAChR1. RNAi against Paα3, Paα8 and Paβ1 significantly reduced both imidacloprid- and nicotine-activated currents on nAChR1. In contrast, RNAi against Paα1, Paα2 and Paβ1 decreased nicotine-activated currents on nAChR2. The results indicated that, in α-Bgt-resistant nAChRs, Paα3, Paα8 and Paβ1 might be involved in the subunit composition of nAChR1, and Paα1, Paα2 and Paβ1 in nAChR2. In summary, from the present study and previous reports, we deduced that there are at least three nAChR subtypes that are sensitive to imidacloprid in the cockroach DUM neurons.     

Effect of rpoS mutations on stress-resistance and invasion of brain microvascular endothelial cells in Escherichia coli K1

Escherichia coli K1 strains are predominant in causing neonatal meningitis. We have shown that invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for E. coli K1 crossing of the blood-brain barrier. BMEC invasion by E. coli K1 strain RS218, however, has been shown to be significantly greater with stationary-phase cultures than with exponential-phase cultures. Since RpoS participates in regulating stationary-phase gene expression, the present study examined a possible involvement of RpoS in E. coli K1 invasion of BMEC. We found that the cerebrospinal fluid isolates of E. coli K1 strains RS218 and IHE3034 have a nonsense mutation in their rpoS gene. Complementation with the E. coli K12 rpoS gene significantly increased the BMEC invasion of E. coli K1 strain IHE3034, but failed to significantly increase the invasion of another E. coli K1 strain RS218. Of interest, the recovery of E. coli K1 strains following environmental insults was 10-100-fold greater on Columbia blood agar than on LB agar, indicating that growing medium is important for viability of rpoS mutants after environmental insults. Taken together, our data suggest that the growth-phase-dependent E. coli K1 invasion of BMEC is affected by RpoS and other growth-phase-dependent regulatory mechanisms.     

α7 nicotinic acetylcholine receptor (α7nAChR) expression in bone marrow-derived non-T cells is required for the inflammatory reflex

The immune response to infection or injury coordinates host defense and tissue repair, but also has the capacity to damage host tissues. Recent advances in understanding protective mechanisms have found neural circuits that suppress release of damaging cytokines. Stimulation of the vagus nerve protects from excessive cytokine production and ameliorates experimental inflammatory disease. This mechanism, the inflammatory reflex, requires the α7 nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel expressed on macrophages, lymphocytes, neurons and other cells. To investigate cell-specific function of α7nAChR in the inflammatory reflex, we created chimeric mice by cross-transferring bone marrow between wild-type (WT) and α7nAChR-deficient mice. Deficiency of α7nAChR in bone marrow-derived cells significantly impaired vagus nerve-mediated regulation of tumor necrosis factor (TNF), whereas α7nAChR deficiency in neurons and other cells had no significant effect. In agreement with recent work, the inflammatory reflex was not functional in nude mice, because functional T cells are required for the integrity of the pathway. To investigate the role of T-cell α7nAChR, we adoptively transferred α7nAChR-deficient or WT T cells to nude mice. Transfer of WT and α7nAChR-deficient T cells restored function, indicating that α7nAChR expression on T cells is not necessary for this pathway. Together, these results indicate that α7nAChR expression in bone marrow-derived non-T cells is required for the integrity of the inflammatory reflex.     

Lack of modulation of nicotinic acetylcholine alpha-7 receptor currents by kynurenic Acid in adult hippocampal interneurons

Kynurenic acid (KYNA), a classical ionotropic glutamate receptor antagonist is also purported to block the α7-subtype nicotinic acetylcholine receptor (α7* nAChR). Although many published studies cite this potential effect, few have studied it directly. In this study, the α7*-selective agonist, choline, was pressure-applied to interneurons in hippocampal subregions, CA1 stratum radiatum and hilus of acute brain hippocampal slices from adolescent to adult mice and adolescent rats. Stable α7* mediated whole-cell currents were measured using voltage-clamp at physiological temperatures. The effects of bath applied KYNA on spontaneous glutamatergic excitatory postsynaptic potentials (sEPSC) as well as choline-evoked α7* currents were determined. In mouse hilar interneurons, KYNA totally blocked sEPSC whole-cell currents in a rapid and reversible manner, but had no effect on choline-evoked α7* whole-cell currents. To determine if this lack of KYNA effect on α7* function was due to regional and/or species differences in α7* nAChRs, the effects of KYNA on choline-evoked α7* whole-cell currents in mouse and rat stratum radiatum interneurons were tested. KYNA had no effect on either mouse or rat stratum radiatum interneuron choline-evoked α7* whole-cell currents. Finally, to test whether the lack of effect of KYNA was due to unlikely slow kinetics of KYNA interactions with α7* nAChRs, recordings of a7*-mediated currents were made from slices that were prepared and stored in the presence of 1 mM KYNA (>90 minutes exposure). Under these conditions, KYNA had no measurable effect on α7* nAChR function. The results show that despite KYNA-mediated blockade of glutamatergic sEPSCs, two types of hippocampal interneurons that express choline-evoked α7* nAChR currents fail to show any degree of modulation by KYNA. Our results indicate that under our experimental conditions, which produced complete KYNA-mediated blockade of sEPSCs, claims of KYNA effects on choline-evoked α7* nAChR function should be made with caution.     

Nicotine-induced cellular stresses and autophagy in human cancer colon cells: A supportive effect on cell homeostasis via up-regulation of Cox-2 and PGE2 production

Nicotine, one of the active components in cigarette smoke, has been described to contribute to the protective effect of smoking in ulcerative colitis (UC) patients. Furthermore, the nicotinic acetylcholine receptor α7 subunit (α7nAChR) expressed on immune cells, is an essential regulator of inflammation. As intestinal epithelial cells also express α7nAChR, we investigated how nicotine could participate in the homeostasis of intestinal epithelial cells. First, using the human adenocarcinoma cell line HT-29, we revealed that nicotine, which triggers an influx of extracellular Ca²⁺ following α7nAChR stimulation, induces mitochondrial reactive oxygen species (ROS) production associated with a disruption of the mitochondrial membrane potential and endoplasmic reticulum stress. This results in caspase-3 activation, which in turn induces apoptosis. Additionally, we have shown that nicotine induces a PI3-K dependent up-regulation of cyclooxygenase-2 (Cox-2) expression and prostaglandin E2 (PGE₂) production. In this context, we suggest that this key mediator participates in the cytoprotective effects of nicotine against apoptosis by stimulating autophagy in colon cancer cells. Our results provide new insight into one potential mechanism by which nicotine could protect from UC and suggest an anti-inflammatory role for the cholinergic pathway at the epithelial cell level.     

Augmented inducible nitric oxide synthase expression and increased NO production reduce sepsis-induced lung injury and mortality in myeloperoxidase-null mice

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO(-/-) mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO(-/-) mice unexpectedly had improved survival compared with wild-type (WT) mice within 5-12 h after intraperitoneal E. coli challenge. Lungs of MPO(-/-) mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO(-/-) mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO(-/-) mice. Inhibition of iNOS in MPO(-/-) mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO(-/-) mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.     

Inflammation has synergistic effect with nicotine in periodontitis by up‐regulating the expression of α7 nAChR via phosphorylated GSK‐3β

Periodontitis is the leading cause of adult tooth loss, and those who smoke are at an increased risk of developing periodontitis. α7 nicotinic acetylcholine receptor (α7 nAChR) is proposed to mediate the potential synergistic effect of nicotine and inflammation in smoking‐related periodontitis. However, this has not been experimentally demonstrated. We isolated and cultured human periodontal ligament stem cells (PDLSCs) from healthy and inflamed tissues. PDLSCs were treated with either inflammatory factors or nicotine. We measured expression of genes that are associated with osteogenic differentiation and osteoclast formation using RT‐qPCR and Western blot analyses. Besides, immunohistochemical staining, micro‐CT analysis and tartaric acid phosphatase staining were used to measure α7 nAChR expression and function. Inflammation up‐regulated α7 nAChR expression in both periodontal ligament tissues and PDLSCs. The up‐regulated α7 nAChR contributed to the synergistic effect of nicotine and inflammation, leading to a decreased capability of osteogenic differentiation and increased capability of osteoclast formation‐induction of PDLSCs. Moreover, the inflammation‐induced up‐regulation of α7 nAChR was partially dependent on the level of phosphorylated GSK‐3β. This study provides experimental evidence for the pathological development of smoking‐related periodontitis and sheds new light on developing inflammation and α7 nAChR‐targeted therapeutics to treat and prevent the disease.     

Type I and II positive allosteric modulators differentially modulate agonist-induced up-regulation of α7 nicotinic acetylcholine receptors

Long‐term treatment with nicotine or selective α7 nicotinic acetylcholine receptor (nAChR) agonists increases the number of α7 nAChRs and this up‐regulation may be involved in the mechanism underlying the sustained procognitive effect of these compounds. Here, we investigate the influence of type I and II α7 nAChR positive allosteric modulators (PAMs) on agonist‐induced α7 nAChR up‐regulation. We show that the type II PAMs, PNU‐120596 (10 μM) or TQS (1 and 10 μM), inhibit up‐regulation, as measured by protein levels, induced by the α7 nAChR agonist A‐582941 (10 nM or 10 μM), in SH‐EP1 cells stably expressing human α7 nAChR, whereas the type I PAMs AVL‐3288 or NS1738 do not. Contrarily, neither type I nor II PAMs affect 10 μM nicotine‐induced receptor up‐regulation, suggesting that nicotine and A‐582941 induce up‐regulation through different mechanisms. We further show in vivo that 3 mg/kg PNU‐120596 inhibits up‐regulation of the α7 nAChR induced by 10 mg/kg A‐582941, as measured by [¹²⁵I]‐bungarotoxin autoradiography, whereas 1 mg/kg AVL‐3288 does not. Given that type II PAMs decrease desensitization of the receptor, whereas type I PAMs do not, these results suggest that receptor desensitization is involved in A‐582941‐induced up‐regulation. Our results are the first to show an in vivo difference between type I and II α7 nAChR PAMs, and demonstrate an agonist‐dependent effect of type II PAMs occurring on a much longer time scale than previously appreciated. Furthermore, our data suggest that nicotine and A‐582941 induce up‐regulation through different mechanisms, and that this confers differential sensitivity to the effects of α7 nAChR PAMs. These results may have implications for the clinical development of α7 nAChR PAMs.