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.

Nicotinic acetylcholine receptor subunit α7-knockout mice exhibit degraded auditory temporal processing.

The CHRNA7 gene that encodes the α7-subunit of the nicotinic acetylcholine receptor (α7-nAChR) has been associated with some autism spectrum disorders and other neurodevelopmental conditions characterized, in part, by auditory and language impairment. These conditions may include auditory processing disorders that represent impaired timing of neural activity, often accompanied by problems understanding speech. Here, we measure timing properties of sound-evoked activity via the auditory brainstem response (ABR) of α7-nAChR knockout mice of both sexes and wild-type colony controls. We find a significant timing delay in evoked ABR signals that represents midbrain activity in knockouts. We also examine spike-timing properties of neurons in the inferior colliculus, a midbrain nucleus that exhibits high levels of α7-nAChR during development. We find delays of evoked responses along with degraded spiking precision in knockout animals. We find similar timing deficits in responses of neurons in the superior paraolivary nucleus and ventral nucleus of the lateral lemniscus, which are brainstem nuclei thought to shape temporal precision in the midbrain. In addition, we find that other measures of temporal acuity including forward masking and gap detection are impaired for knockout animals. We conclude that altered temporal processing at the level of the brainstem in α7-nAChR-deficient mice may contribute to degraded spike timing in the midbrain, which may underlie the observed timing delay in the ABR signals. Our findings are consistent with a role for the α7-nAChR in types of neurodevelopmental and auditory processing disorders and we identify potential neural targets for intervention.NEW & NOTEWORTHY Disrupted signaling via the α7-nicotinic acetylcholine receptor (α7-nAChR) is associated with neurodevelopmental disorders that include impaired auditory processing. The underlying causes of dysfunction are not known but a common feature is abnormal timing of neural activity. We examined temporal processing of α7-nAChR knockout mice and wild-type controls. We found degraded spike timing of neurons in knockout animals, which manifests at the level of the auditory brainstem and midbrain.

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 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.

Effects of neuromuscular presynaptic muscarinic M1 receptor blockade on rocuronium-induced neuromuscular blockade in immobilized tibialis anterior muscles.

This in vivo study tested the hypothesis that the modulation of acetylcholine (ACh) release by the M1 muscarinic receptor (mAChR) in the neuromuscular junction of disused muscles may affect the tensions of the muscles during the neuromuscular monitoring of a rocuronium-induced neuromuscular block and compared the results with those obtained from normal muscles. A total of 20 C57BL/6 (wild-type) and 10 α7 knock out (α7KO) mice were used in this experiment. As a pre-experimental procedure, knee and ankle joints of right hind limbs were fixed by needle pinning at the 90° flexed position. After 2 weeks, the main experiment was performed. Both tendons of the tibialis anterior (TA) muscles were obtained, and the muscle tensions were recorded while the dose-responses of rocuronium were measured three times in the same mouse by the serial administration of pirenzepine (0, 0.001 and 0.01 µg/g). Weight losses were observed after 2 weeks of immobilization in both groups, and a decrease in the mass of TA muscles at the immobilized side was observed compared to those of the contralateral nonimmobilized side. Tension depression of the TA muscles at immobilized side of the α7KO group was faster than those of the wild-type group, but these differences decreased after the administration of pirenzepine. The tension depressions were similar regardless of the pirenzepine doses at the same side in the group. Tension depression may become more rapid in the α7 AChR-expressed disused muscles by the decreased release of ACh release upon neuronal firing by the blockade of facilitatory M1 mAChR.

Deficiency of α7 nicotinic acetylcholine receptor attenuates bleomycin-induced lung fibrosis in mice.

α7 nicotinic acetylcholine receptor (α7 nAChR, coded by Chrna7) is indispensible in dampening proinflammatory responses. However, whether α7 nAChR would play a role in regulating bleomycin (BLM)-induced lung fibrosis is less investigated. Here, we intratracheally challenged wildtype and Chrna7-/- mice with BLM to elicit lung fibrosis. Taken advantage of this model, we measured body weight loss, lung fibrogenic genes (Acta2, Col1a1, Fsp1, and Fstl1), histology, Masson's trichrome staining, hydroxyproline levels, and expression of α-SMA at protein levels in the BLM-challenged lung for evaluating severity of lung fibrosis. We also pretreated human fibroblasts (MRC5 cell line) and isolated mouse lung fibroblasts with GTS-21 (an α7 nAChR agonist) to study its effects on TGF-ß-stimulated profibrotic profiles. We found that lung Chrna7 expression and CD4+CHAT+ (Choline acetyltransferase, an enzyme for local acetylcholine synthesis) cells were 12-fold and 4.5-fold respectively elevated in the early stage of lung fibrosis. Deletion of Chrna7 prevented body weight loss and reduced lung fibrogenic genes (Acta2, Col1a1, Fsp1, and Fstl1) and Arg 1 (coding arginase 1). Deletion of Chrna7 attenuated lung arginase 1+Ly6C+ cells, Masson's trichrome staining, hydroxyproline levels, and expression of α-SMA at protein levels in BLM-challenged mice. Mechanistically, activation of α7 nAChR in human fibroblasts increased TGF-ß-induced phosphorylation of Smad2/3 and transcription of fibrogenic genes (Acta2, Col1a1). In isolated mouse lung fibroblasts, activation of α7 nAChR also enhanced TGF-ß induced-transcription of fibrogenic genes; however, deletion of Chrna7 diminished these effects. Taken together, deficiency of α7 nAChR could suppress the development of BLM-induced lung fibrosis. Thus, α7 nAChR might be a novel therapeutic target for treating lung fibrosis.

α7 Nicotinic Acetylcholine Receptor Relieves Angiotensin II-Induced Senescence in Vascular Smooth Muscle Cells by Raising Nicotinamide Adenine Dinucleotide-Dependent SIRT1 Activity.

OBJECTIVE: α7 nicotinic acetylcholine receptor (α7nAChR) is a subtype of nAChR and has been reported to be involved in hypertension end-organ damage. In this study, we tested the role of α7nAChR in angiotensin II (Ang II)-induced senescence of vascular smooth muscle cells (VSMCs). APPROACH AND RESULTS: Expression of α7nAChR was not influenced by Ang II. Ang II induced remarkable senescent phenotypes in rodent and human VSMCs, including increased senescence-associated ß-galactosidase activity, phosphorylation of H2A.X(Ser139), phosphorylation of Chk1(Ser317), reduced replication, and downregulation of proliferating cell nuclear antigen. Activation of α7nAChR with a selective agonist PNU-282987 blocked Ang II-induced senescence in cultured VSMCs. Moreover, PNU-282987 treatment attenuated the Ang II infusion-induced VSMC senescence in wild-type but not in α7nAChR(-/-) mice. PNU-282987 reduced the Ang II-enhanced reactive oxygen species, lipid peroxidation, and the expression of NADPH oxidase 1, NADPH oxidase 4, and p22(phox) in cultured VSMCs isolated from wild-type but not in α7nAChR(-/-) mice. Furthermore, PNU-282987 diminished Ang II-induced prosenescence signaling pathways, including p53, acetyl-p53, p21, and p16(INK4a). Finally, although α7nAChR activation by PNU-282987 did not affect the Ang II-induced downregulation of sirtuin 1 (SIRT1), it significantly increased intracellular NAD(+) levels, and thereby enhanced SIRT1 activity in an AMP-dependent protein kinase-independent manner. Depletion of SIRT1 by knockdown or SIRT1 inhibitor EX527 abrogated the antisenescence effect of α7nAChR against Ang II. CONCLUSIONS: Our results demonstrate that activation of α7nAChR alleviates Ang II-induced VSMC senescence through promoting NAD(+)-SIRT1 pathway, suggesting that α7nAChR may be a potential therapeutic target for the treatment of Ang II-associated vascular aging disorders.

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.

Cortical parvalbumin GABAergic deficits with α7 nicotinic acetylcholine receptor deletion: implications for schizophrenia.

Dysfunction of cortical parvalbumin (PV)-containing GABAergic interneurons has been implicated in cognitive deficits of schizophrenia. In humans microdeletion of the CHRNA7 (α7 nicotinic acetylcholine receptor, nAChR) gene is associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia while in mice similar deletion causes analogous abnormalities including impaired attention, working-memory and learning. However, the pathophysiological roles of α7 nAChRs in cortical PV GABAergic development remain largely uncharacterized. In both in vivo and in vitro models, we identify here that deletion of the α7 nAChR gene in mice impairs cortical PV GABAergic development and recapitulates many of the characteristic neurochemical deficits in PV-positive GABAergic interneurons found in schizophrenia. α7 nAChR null mice had decreased cortical levels of GABAergic markers including PV, glutamic acid decarboxylase 65/67 (GAD65/67) and the α1 subunit of GABAA receptors, particularly reductions of PV and GAD67 levels in cortical PV-positive interneurons during late postnatal life and adulthood. Cortical GABAergic synaptic deficits were identified in the prefrontal cortex of α7 nAChR null mice and α7 nAChR null cortical cultures. Similar disruptions in development of PV-positive GABAergic interneurons and perisomatic synapses were found in cortical cultures lacking α7 nAChRs. Moreover, NMDA receptor expression was reduced in GABAergic interneurons, implicating NMDA receptor hypofunction in GABAergic deficits in α7 nAChR null mice. Our findings thus demonstrate impaired cortical PV GABAergic development and multiple characteristic neurochemical deficits reminiscent of schizophrenia in cortical PV-positive interneurons in α7 nAChR gene deletion models. This implicates crucial roles of α7 nAChRs in cortical PV GABAergic development and dysfunction in schizophrenia and other neuropsychiatric disorders.

Cortical synaptic NMDA receptor deficits in α7 nicotinic acetylcholine receptor gene deletion models: implications for neuropsychiatric diseases.

Microdeletion of the human CHRNA7 gene (α7 nicotinic acetylcholine receptor, nAChR) as well as dysfunction in N-methyl-d-aspartate receptors (NMDARs) have been associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia. However, the pathophysiological roles of synaptic vs. extrasynaptic NMDARs and their interactions with α7 nAChRs in cortical dysfunction remain largely uncharacterized. Using a combination of in vivo and in vitro models, we demonstrate that α7 nAChR gene deletion leads to specific loss of synaptic NMDARs and their coagonist, d-serine, as well as glutamatergic synaptic deficits in mouse cortex. α7 nAChR null mice had decreased cortical NMDAR expression and glutamatergic synapse formation during postnatal development. Similar reductions in NMDAR expression and glutamatergic synapse formation were revealed in cortical cultures lacking α7 nAChRs. Interestingly, synaptic, but not extrasynaptic, NMDAR currents were specifically diminished in cultured cortical pyramidal neurons as well as in acute prefrontal cortical slices of α7 nAChR null mice. Moreover, d-serine responsive synaptic NMDAR-mediated currents and levels of the d-serine synthetic enzyme serine racemase were both reduced in α7 nAChR null cortical pyramidal neurons. Our findings thus identify specific loss of synaptic NMDARs and their coagonist, d-serine, as well as glutamatergic synaptic deficits in α7 nAChR gene deletion models of cortical dysfunction, thereby implicating α7 nAChR-mediated control of synaptic NMDARs and serine racemase/d-serine pathways in cortical dysfunction underlying many neuropsychiatric and neurodevelopmental disorders, particularly those associated with deletion of human CHRNA7.

Acetylcholine receptors in the retinas of the α7 nicotinic acetylcholine receptor knockout mouse.

PURPOSE: The α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse. METHODS: To examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes. RESULTS: In the whole retina, there was a statistically significant upregulation of α2, α9, α10, ß4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while ß3 and ß4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, ß4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), ß2, ß4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3-α5 and α9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species. CONCLUSIONS: Rather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.

Endogenous α7 nAChR Agonist SLURP1 Facilitates Escherichia coli K1 Crossing the Blood-Brain Barrier.

Alpha 7 nicotinic acetylcholine receptor (α7 nAChR) is critical for the pathogenesis of Escherichia coli (E. coli) K1 meningitis, a severe central nervous system infection of the neonates. However, little is known about how E. coli K1 manipulates α7 nAChR signaling. Here, through employing immortalized cell lines, animal models, and human transcriptional analysis, we showed that E. coli K1 infection triggers releasing of secreted Ly6/Plaur domain containing 1 (SLURP1), an endogenous α7 nAChR ligand. Exogenous supplement of SLURP1, combined with SLURP1 knockdown or overexpression cell lines, showed that SLURP1 is required for E. coli K1 invasion and neutrophils migrating across the blood-brain barrier (BBB). Furthermore, we found that SLURP1 is required for E. coli K1-induced α7 nAChR activation. Finally, the promoting effects of SLURP1 on the pathogenesis of E. coli K1 meningitis was significantly abolished in the α7 nAChR knockout mice. These results reveal that E. coli K1 exploits SLURP1 to activate α7 nAChR and facilitate its pathogenesis, and blocking SLURP1-α7 nAChR interaction might represent a novel therapeutic strategy for E. coli K1 meningitis.

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.

α7nAChR Deletion Aggravates Myocardial Infarction and Enhances Systemic Inflammatory Reaction via mTOR-Signaling-Related Autophagy.

Alpha7 nicotinic acetylcholine receptor (α7nAChR) has been previously reported to play an alleviative role in myocardial infarction (MI). In this study, we investigated its specific mechanism. α7nAChR-/- mice and its control (α7nAChR+/+) were used for the study of α7nAChR. Left anterior descending coronary artery occlusion was conducted for the creation of mice MI model and lipopolysaccharide (LPS) was used as inflammatory stressor in murine peritoneal macrophages. Triphenyltetrazolium chloride (TTC) staining and echocardiography was used for the detection of infarct size and cardiac function, respectively. Western blot was conducted for the testing of autophagy-related proteins and enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) was used for the testing of proinflammatory cytokines. Rapamycin was used for the induction of autophagy through inhibiting mammalian target of rapamycin (mTOR)-related signaling. We found that knocking out α7nAChR enhanced the cardiac infarct size and damaged cardiac function in MI. α7nAChR deficiency increased the levels of several proinflammatory cytokines in serum and spleen from MI mice as well as murine macrophages under inflammatory stress. α7nAChR deletion decreased the level of autophagy in spleen from MI mice and macrophages under inflammatory stress. Rapamycin alleviated the cardiac function and systemic inflammatory reaction in MI mice as well as inflammatory reaction in macrophages under inflammatory stress, which was attenuated by knocking out α7nAChR. Our current study investigated the mechanism of α7nAChR-mediated cardio-protective and anti-inflammatory effect related to mTOR-related autophagy, which might provide a novel insight in the treatment of MI.

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.

Chrna7 deficient mice manifest no consistent neuropsychiatric and behavioral phenotypes.

The alpha7 nicotinic acetylcholine receptor, encoded by the CHRNA7 gene, has been implicated in various psychiatric and behavioral disorders, including schizophrenia, bipolar disorder, epilepsy, autism, Alzheimer's disease, and Parkinson's disease, and is considered a potential target for therapeutic intervention. 15q13.3 microdeletion syndrome is a rare genetic disorder, caused by submicroscopic deletions on chromosome 15q. CHRNA7 is the only gene in this locus that has been deleted entirely in cases involving the smallest microdeletions. Affected individuals manifest variable neurological and behavioral phenotypes, which commonly include developmental delay/intellectual disability, epilepsy, and autism spectrum disorder. Subsets of patients have short attention spans, aggressive behaviors, mood disorders, or schizophrenia. Previous behavioral studies suggested that Chrna7 deficient mice had attention deficits, but were normal in baseline behavioral responses, learning, memory, and sensorimotor gating. Given a growing interest in CHRNA7-related diseases and a better appreciation of its associated human phenotypes, an in-depth behavioral characterization of the Chrna7 deficient mouse model appeared prudent. This study was designed to investigate whether Chrna7 deficient mice manifest phenotypes related to those seen in human individuals, using an array of 12 behavioral assessments and electroencephalogram (EEG) recordings on freely-moving mice. Examined phenotypes included social interaction, compulsive behaviors, aggression, hyperactivity, anxiety, depression, and somatosensory gating. Our data suggests that mouse behavior and EEG recordings are not sensitive to decreased Chrna7 copy number.

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.

Involvement of arterial baroreflex and nicotinic acetylcholine receptor α7 subunit pathway in the protection of metformin against stroke in stroke-prone spontaneously hypertensive rats.

Stroke is a leading cause of mortality and disability worldwide. There is growing evidence that metformin (Met) has potent neuroprotective effects; however, its mechanisms remain unclear. We examined the role of the arterial baroreflex and cholinergic-α7 nicotinic acetylcholine receptor (α7nAChR) anti-inflammory pathway in the beneficial effects of Met against stroke. Stroke-prone spontaneously hypertensive rats (SHRSP) were used to observe stroke development indicated by lifespan of SHRSP and the ischemic injury induced by permanent middle cerebral artery occlusion (MCAO). Sinoaortic denervation was used to inactivate the arterial baroreflex. MCAO were also performed in α7nAChR knockout (KO) mice. Briefly, Met increased the life span of SHRSP and reduced the infarct area induced by MCAO. Met also improved the function of arterial baroreflex. The beneficial effects of Met on stroke were markedly attenuated by blunting the arterial baroreflex. Met up-regulated the expression of vesicular acetylcholine transporter (VAChT) and α7nAChR, down-regulated the level of pro-inflammtory cytokines in serum and peri-infarct of ischemic brain. Arterial baroreflex dysfunction decreased the expression of VAchT and α7nAChR, showed upward tendency in the level of pro-inflammtory cytokines. Most importantly, arterial baroreflex dysfunction nearly abolished such effect of Met on cholinergic signaling. In addition, the α7nAChR KO mice also had significantly worse ischemic damage induced by MCAO, and neuroprotection of Met disappeared in α7nAChR KO mice. In conclusion, Met improved the arterial baroreflex function, and then enhancing cholinergic anti-inflammatory pathway in an α7nAChR-dependent manner, thereby effectively prevent ischemic induced brain injury and delayed stroke onset in SHRSP.

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.

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.