Aß1-16 controls synaptic vesicle pools at excitatory synapses via cholinergic modulation of synapsin phosphorylation.

Amyloid beta (Aß) is linked to the pathology of Alzheimer's disease (AD). At physiological concentrations, Aß was proposed to enhance neuroplasticity and memory formation by increasing the neurotransmitter release from presynapse. However, the exact mechanisms underlying this presynaptic effect as well as specific contribution of endogenously occurring Aß isoforms remain unclear. Here, we demonstrate that Aß1-42 and Aß1-16, but not Aß17-42, increased size of the recycling pool of synaptic vesicles (SV). This presynaptic effect was driven by enhancement of endogenous cholinergic signalling via α7 nicotinic acetylcholine receptors, which led to activation of calcineurin, dephosphorylation of synapsin 1 and consequently resulted in reorganization of functional pools of SV increasing their availability for sustained neurotransmission. Our results identify synapsin 1 as a molecular target of Aß and reveal an effect of physiological concentrations of Aß on cholinergic modulation of glutamatergic neurotransmission. These findings provide new mechanistic insights in cholinergic dysfunction observed in AD.

Mesenchymal Stem Cells or Interleukin-6 Improve Episodic Memory of Mice Lacking α7 Nicotinic Acetylcholine Receptors.

Nicotinic acetylcholine receptors of α7 subtype (α7 nAChRs) are involved in regulating cognition, inflammation and cell survival. Neuroinflammation is accompanied by the decrease of α7 nAChRs in the brain and impairment of memory. We show here that α7-/- mice possess pro-inflammatory phenotype and demonstrate worse episodic memory compared to wild-type mice. Previously we reported that mesenchymal stem cells (MSCs) restored episodic memory of lipopolysaccharide-treated wild-type mice. The aim of this study was to examine if MSCs or their soluble factors improve memory of α7-/- mice. The α7-specific signal (ELISA) and α7+ cells (IHC) were found in the brain of α7-/- mice on days 7 and 14 after intravenous injection of α7+ MSCs from either human umbilical cord (hMSCs) or mouse placenta (mMSCs). The intravenously injected MSCs or intraperitoneally injected hMSCs-conditioned medium transiently improved episodic memory of α7-/- mice and decreased cytochrome c release from their brain mitochondria under the effect of Ca2+. Either MSCs or conditioned medium stimulated an IL-6 increase in the brain, which coincided with the improvement of episodic memory. Injections of recombinant IL-6 also improved episodic memory of α7-/- mice accompanied by the up-regulation of α3, α4, ß2 and ß4 nAChR subunits in the brain. It is concluded that MSCs, injected intravenously, penetrate the brain of α7-/- mice and persist there for at least 2 weeks. They improve episodic memory of mice and make their mitochondria more resistant to apoptogenic influence. One of the soluble factors responsible for the memory improvement is IL-6.

Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins.

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity α-bungarotoxin (α-bgtx) binding protein in the mammalian CNS. α7-nAChRs associate with proteins that can modulate its properties. The α7-nAChR interactome is the summation of proteins interacting or associating with α7-nAChRs in a protein complex. To identify an α7-nAChR interactome in neural tissue, we isolated α-bgtx-affinity protein complexes from wild-type and α7-nAChR knockout (α7 KO) mouse whole brain tissue homogenates using α-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the α7-nAChR specific ligand, α-bgtx, were determined to be α7-nAChR associated proteins. The α7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to α-bgtx in the absence of α7-nAChR expression, thereby identifying nonspecific proteins for α7-nAChR investigations using α-bgtx enrichment. These results expand on our previous investigations of α7-nAChR interacting proteins using α-bgtx-affinity bead isolation by controlling for differences between α7-nAChR and α-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The α7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of α7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease.

Nicotine withdrawal-induced inattention is absent in alpha7 nAChR knockout mice.

RATIONALE: Smoking is the leading cause of preventable death in the USA, but quit attempts result in withdrawal-induced cognitive dysfunction and predicts relapse. Greater understanding of the neural mechanism(s) underlying these cognitive deficits is required to develop targeted treatments to aid quit attempts. OBJECTIVES: We examined nicotine withdrawal-induced inattention in mice lacking the α7 nicotinic acetylcholine receptor (nAChR) using the five-choice continuous performance test (5C-CPT). METHODS: Mice were trained in the 5C-CPT prior to osmotic minipump implantation containing saline or nicotine. Experiment 1 used 40 mg kg-1 day-1 nicotine treatment and tested C57BL/6 mice 4, 28, and 52 h after pump removal. Experiment 2 used 14 and 40 mg kg-1 day-1 nicotine treatment in α7 nAChR knockout (KO) and wildtype (WT) littermates tested 4 h after pump removal. Subsets of WT mice were killed before and after pump removal to assess changes in receptor expression associated with nicotine administration and withdrawal. RESULTS: Nicotine withdrawal impaired attention in the 5C-CPT, driven by response inhibition and target detection deficits. The overall attentional deficit was absent in α7 nAChR KO mice despite response disinhibition in these mice. Synaptosomal glutamate mGluR5 and dopamine D4 receptor expression were reduced during chronic nicotine but increased during withdrawal, potentially contributing to cognitive deficits. CONCLUSIONS: The α7 nAChR may underlie nicotine withdrawal-induced deficits in target detection but is not required for response disinhibition deficits. Alterations to the glutamatergic and dopaminergic pathways may also contribute to withdrawal-induced attentional deficits, providing novel targets to alleviate the cognitive symptoms of withdrawal during quit attempts.

The nicotinic acetylcholine receptor α7 subunit is an essential negative regulator of bone mass.

The nicotinic receptor α7nAchR reportedly regulates vagal nerve targets in brain and cardiac tissue. Here we show that nAchR7-/- mice exhibit increased bone mass due to decreased osteoclast formation, accompanied by elevated osteoprotegerin/RANKL ratios in serum. Vagotomy in wild-type mice also significantly increased the serum osteoprotegerin/RANKL ratio, and elevated bone mass seen in nAchR7-/- mice was reversed in α7nAchR/osteoprotegerin-doubly-deficient mice. α7nAchR loss significantly increased TNFα expression in Mac1-positive macrophages, and TNFα increased the osteoprotegerin/RANKL ratio in osteoblasts. Targeting TNFα in nAchR7-/- mice normalized both serum osteoprotegerin/RANKL ratios and bone mass. Administration of nicotine, an α7nAchR ligand, to wild-type mice increased serum RANKL levels. Thus, vagal nerve stimulation of macrophages via α7nAchR regulates bone mass by modulating osteoclast formation.

Hepatic vagus nerve regulates Kupffer cell activation via α7 nicotinic acetylcholine receptor in nonalcoholic steatohepatitis.

BACKGROUND: Nonalcoholic fatty liver disease ranges from simple steatosis to nonalcoholic steatohepatitis (NASH). Kupffer cells play a central role in promoting hepatic inflammation, which leads to the development of NASH. We investigated the anti-inflammatory effect of hepatic vagus-mediated stimulation of the α7 nicotinic acetylcholine receptor (α7nAChR) on Kupffer cells in NASH pathogenesis. METHODS: Wild-type (WT) mice undergoing hepatic vagotomy (HV) were fed a methionine- and choline-deficient (MCD) diet for 1 week. α7nAChR knockout (α7KO) chimeric mice were generated by transplanting α7KO bone marrow cells into irradiated and Kupffer cell-deleted WT recipients. Kupffer cells were isolated from WT mice and treated with α7nAChR agonist under stimulation by lipopolysaccharide and/or palmitic acid. RESULTS: HV aggravated MCD diet-induced NASH in both steatosis and inflammation. The hepatic inflammatory response, including the upregulation of tumor necrosis factor alpha (TNFα), interleukin (IL)-12, and monocyte chemoattractant protein 1 (MCP-1), was accelerated in HV mice, accompanied by the downregulation of PPARα pathway genes. Kupffer cells were highly activated via the phosphorylation and nuclear translocation of nuclear factor-kappa B (NF-κB) in MCD diet-fed HV mice. The α7nAchR agonist suppressed the inflammatory response of primary Kupffer cells induced by lipopolysaccharide and palmitic acid by attenuating the NF-κB cascade. α7KO chimeric mice fed an MCD diet for 1 week developed advanced NASH with highly activated Kupffer cells. The hepatic expression of TNFα, IL-12, and MCP-1 was upregulated in α7KO chimeric mice, accompanied by abnormal lipid metabolism. CONCLUSIONS: Hepatic vagus activity regulates the inflammatory response of Kupffer cells via α7nAChR in NASH development.

Nicotine Accelerates Atherosclerosis in Apolipoprotein E-Deficient Mice by Activating α7 Nicotinic Acetylcholine Receptor on Mast Cells.

OBJECTIVE: Cigarette smoking is an independent risk factor for atherosclerosis. Nicotine, the addictive component of cigarettes, induces mast cell (MC) release and contributes to atherogenesis. The purpose of this study was to determine whether nicotine accelerates atherosclerosis through MC-mediated mechanisms and whether MC stabilizer prevents this pathological process. APPROACH AND RESULTS: Nicotine administration increased the size of atherosclerotic lesions in apolipoprotein E-deficient (Apoe-/-) mice fed a fat-enriched diet. This was accompanied by enhanced intraplaque macrophage content and lipid deposition but reduced collagen and smooth muscle cell contents. MC deficiency in Apoe-/- mice (Apoe-/-KitW-sh/W-sh) diminished nicotine-induced atherosclerosis. Nicotine activated bone marrow-derived MCs in vitro, which was inhibited by a MC stabilizer disodium cromoglycate or a nonselective nicotinic acetylcholine receptor blocker mecamylamine. Further investigation revealed that α7 nicotinic acetylcholine receptor was a target for nicotine activation in MCs. Nicotine did not change atherosclerotic lesion size of Apoe-/-KitW-sh/W-sh mice reconstituted with MCs from Apoe-/-α7nAChR-/- animals. CONCLUSIONS: Activation of α7 nicotinic acetylcholine receptor on MCs is a mechanism by which nicotine enhances atherosclerosis.

Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor.

Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity.

Hematopoietic α7 nicotinic acetylcholine receptor deficiency increases inflammation and platelet activation status, but does not aggravate atherosclerosis.

BACKGROUND: The autonomic nervous system attenuates inflammation through activation of the α7 nicotinic acetylcholine receptor (α7nAChR), a pathway termed the cholinergic anti-inflammatory reflex. Interestingly, α7nAChR is expressed on immune cells and platelets, both of which play a crucial role in the development of atherosclerosis. OBJECTIVE: To investigate the role of hematopoietic α7nAChR in inflammation and platelet function in atherosclerotic ldlr(-/-) mice and to identify its consequences for atherosclerotic lesion development. METHODS: Bone marrow from α7nAChR(-/-) mice or wild-type littermates was transplanted into irradiated ldlr(-/-) mice. After a recovery period of 8 weeks, the mice were fed an atherogenic Western-type diet for 7 weeks. RESULTS: Hematopoietic α7nAChR deficiency clearly increased the number of leukocytes in the peritoneum (2.6-fold, P < 0.001), blood (2.9-fold; P < 0.01), mesenteric lymph nodes (2.0-fold; P < 0.001) and spleen (2.2-fold; P < 0.01), indicative of an increased inflammatory status. Additionally, expression of inflammatory mediators was increased in peritoneal leukocytes (TNFα, 1.6-fold, P < 0.01; CRP, 1.8-fold, P < 0.01) as well as in the spleen (TNFα, 1.6-fold, P < 0.01). The lack of α7nAChR on platelets from these mice increased the expression of active integrin αIIb ß3 upon stimulation by ADP (1.9-fold, P < 0.01), indicating increased activation status, while incubation of human platelets with an α7nAChR agonist decreased aggregation (-35%, P < 0.05). Despite the large effects of hematopoietic α7nAChR deficiency on inflammatory status and platelet function, it did not affect atherosclerosis development or composition of lesions. CONCLUSIONS: Hematopoietic α7nAChR is important for attenuation of inflammatory responses and maintaining normal platelet reactivity, but loss of hematopoietic α7nAChR does not aggravate development of atherosclerosis.

α7 Nicotinic acetylcholine receptor is expressed in human atherosclerosis and inhibits disease in mice--brief report.

OBJECTIVE: Cholinergic pathways of the autonomic nervous system are known to modulate inflammation. Because atherosclerosis is a chronic inflammatory condition, we tested whether cholinergic signaling operates in this disease. We have analyzed the expression of the α7 nicotinic acetylcholine receptor (α7nAChR) in human atherosclerotic plaques and studied its effects on the development of atherosclerosis in the hypercholesterolemic Ldlr(-/-) mouse model. APPROACH AND RESULTS: α7nAChR protein was detected on T cells and macrophages in surgical specimens of human atherosclerotic plaques. To study the role of α7nAChR signaling in atherosclerosis, male Ldlr(-/-) mice were lethally irradiated and reconstituted with bone marrow from wild-type or α7nAChR-deficient animals. Ablation of hematopoietic cell α7nAChR increased aortic atherosclerosis by 72%. This was accompanied by increased aortic interferon-γ mRNA, implying increased Th1 activity in the absence of α7nAChR signaling. CONCLUSIONS: The present study shows that signaling through hematopoietic α7nAChR inhibits atherosclerosis and suggests that it operates by modulating immune inflammation. Given the observation that α7nAChR is expressed by T cells and macrophages in human plaques, our findings support the notion that cholinergic regulation may act to inhibit disease development also in man.

Influenza virus-induced lung inflammation was modulated by cigarette smoke exposure in mice.

Although smokers have increased susceptibility and severity of seasonal influenza virus infection, there is no report about the risk of 2009 pandemic H1N1 (pdmH1N1) or avian H9N2 (H9N2/G1) virus infection in smokers. In our study, we used mouse model to investigate the effect of cigarette smoke on pdmH1N1 or H9N2 virus infection. Mice were exposed to cigarette smoke for 21 days and then infected with pdmH1N1 or H9N2 virus. Control mice were exposed to air in parallel. We found that cigarette smoke exposure alone significantly upregulated the lung inflammation. Such prior cigarette smoke exposure significantly reduced the disease severity of subsequent pdmH1N1 or H9N2 virus infection. For pdmH1N1 infection, cigarette smoke exposed mice had significantly lower mortality than the control mice, possibly due to the significantly decreased production of inflammatory cytokines and chemokines. Similarly, after H9N2 infection, cigarette smoke exposed mice displayed significantly less weight loss, which might be attributed to lower cytokines and chemokines production, less macrophages, neutrophils, CD4+ and CD8+ T cells infiltration and reduced lung damage compared to the control mice. To further investigate the underlying mechanism, we used nicotine to mimic the effect of cigarette smoke both in vitro and in vivo. Pre-treating the primary human macrophages with nicotine for 72 h significantly decreased their expression of cytokines and chemokines after pdmH1N1 or H9N2 infection. The mice subcutaneously and continuously treated with nicotine displayed significantly less weight loss and lower inflammatory response than the control mice upon pdmH1N1 or H9N2 infection. Moreover, α7 nicotinic acetylcholine receptor knockout mice had more body weight loss than wild-type mice after cigarette smoke exposure and H9N2 infection. Our study provided the first evidence that the pathogenicity of both pdmH1N1 and H9N2 viruses was alleviated in cigarette smoke exposed mice, which might partially be attributed to the immunosuppressive effect of nicotine.