α7 Nicotinic acetylcholine receptors regulate translocation of HIF-1α to the cell nucleus and mitochondria upon hypoxia.

Nicotinic acetylcholine receptors (nAChRs), initially characterized as ligand-gated ion channels mediating fast synaptic transmission, are now found in many non-excitable cells and mitochondria where they function in ion-independent manner and regulate vital cellular processes like apoptosis, proliferation, cytokine secretion. Here we show that the nAChRs of α7 subtype are present in the nuclei of liver cells and astrocytoma U373 cell line. As shown by lectin ELISA, the nuclear α7 nAChRs are mature glycoproteins that follow the standard rout of post-translational modifications in Golgi; however, their glycosylation profile is non-identical to that of mitochondrial nAChRs. They are exposed on the outer nuclear membrane and are found in combination with lamin B1. The nuclear α7 nAChRs are up-regulated in liver within 1 h after partial hepatectomy and in H2O2-treated U373 cells. As shown both in silico and experimentally, the α7 nAChR interacts with hypoxia-inducible factor HIF-1α and this interaction is impaired by α7-selective agonists PNU282987 and choline or type 2 positive allosteric modulator PNU120596, which prevent HIF-1α accumulation in the nuclei. Similarly, HIF-1α interacts with mitochondrial α7 nAChRs in U373 cells treated with dimethyloxalylglycine. It is concluded that functional α7 nAChRs influence HIF-1α translocation into the nucleus and mitochondria upon hypoxia.

Immunization with 674-685 fragment of SARS-Cov-2 spike protein induces neuroinflammation and impairs episodic memory of mice.

COVID-19 is accompanied by strong inflammatory reaction and is often followed by long-term cognitive disorders. The fragment 674-685 of SARS-Cov-2 spike protein was shown to interact with α7 nicotinic acetylcholine receptor involved in regulating both inflammatory reactions and cognitive functions. Here we show that mice immunized with the peptide corresponding to 674-685 fragment of SARS-Cov-2 spike protein conjugated to hemocyanin (KLH-674-685) demonstrate decreased level of α7 nicotinic acetylcholine receptors, increased levels of IL-1ß and TNFα in the brain and impairment of episodic memory. Choline injections prevented α7 nicotinic receptor decline and memory loss. Mice injected with immunoglobulins obtained from the blood of (KLH-674-685)-immunized mice also demonstrated episodic memory decline. These data allow suggesting that post-COVID memory impairment in humans is related to SARS-Cov-2 spike protein-specific immune reaction. The mechanisms of such effect are being discussed.

SARS-Cov-2 spike protein fragment 674-685 protects mitochondria from releasing cytochrome c in response to apoptogenic influence.

In spite of numerous studies, many details of SARS-Cov-2 interaction with human cells are still poorly understood. The 674-685 fragment of SARS-Cov-2 spike protein is homologous to the fragment of α-cobratoxin underlying its interaction with α7 nicotinic acetylcholine receptors (nAChRs). The interaction of 674-685 peptide with α7 nAChR has been predicted in silico. In the present paper we confirm this prediction experimentally and investigate the effect of SARS-Cov-2 spike protein peptide on mitochondria, which express α7 nAChRs to regulate apoptosis-related events. We demonstrate that SARS-Cov-2 spike protein peptide 674-685 competes with the antibody against 179-190 fragment of α7 nAChR subunit for the binding to α7-expressing cells and mitochondria and prevents the release of cytochrome c from isolated mitochondria in response to 0.5 mM H2O2 but does not protect intact U373 cells against apoptogenic effect of H2O2. Our data suggest that the α7 nAChR-binding portion of SARS-Cov-2 spike protein prevents mitochondria-driven apoptosis when the virus is uncoated inside the cell and, therefore, supports the infected cell viability before the virus replication cycle is complete.

Unusual properties of α7 nicotinic acetylcholine receptor ion channels in B lymphocyte-derived SP-2/0 cells.

This study demonstrates the presence of α7 nicotinic acetylcholine receptors (nAChR) in B lymphocyte-derived SP-2/0 cells by means of flow cytometry and immunocytochemistry. According to lectin and sandwich ELISA, the α7 subunits expressed in SP-2/0 cells are more glycosylated compared to those expressed in the brain or normal B lymphocytes and are combined with ß2 subunits. At zero and negative pipette potentials, either acetylcholine or α7-specific agonist PNU282987 stimulated the ion channel activity in SP-2/0 cells revealed by single channel patch-clamp recordings. The conductivity was within the range of 19 to 39 pS and reversal potential was between -17 mV and +28 mV, the currents were potentiated by α7-specific positive allosteric modulator PNU120596 and were partially blocked by α7-specific antagonist methyllicaconitine (MLA). However, they were oriented downwards suggesting that the channels mediated the cation outflux rather than influx. As shown by Ca2+ imaging studies, PNU282987 did not stimulate immediate Ca2+ influx into SP-2/0 cells. Instead, Ca2+ influx through Ca-release-activated channels (CRACs) was observed within minutes after either PNU282987 or MLA application. It is concluded that SP-2/0 express α7ß2 nAChRs, which mediate the cation outflux under negative pipette potentials applied, possibly, due to depolarized membrane or negative surface charge formed by carbohydrate residues. In addition, α7ß2 nAChRs may influence CRACs in ion-independent way.

Intravenously Injected Mesenchymal Stem Cells Penetrate the Brain and Treat Inflammation-Induced Brain Damage and Memory Impairment in Mice.

Neuroinflammation is regarded as one of the pathogenic factors of Alzheimer disease (AD). Previously, we showed that mice regularly injected with bacterial lipopolysaccharide (LPS) possessed the AD-like symptoms like episodic memory decline, elevated amounts of amyloid beta (Aß) peptide (1-42), and decreased levels of nicotinic acetylcholine receptors (nAChRs) in the brain. The use of mesenchymal stem cells (MSCs), which can differentiate into multiple cell types, including neurons, is an attractive idea of regenerative medicine, in particular, for neurodegenerative disorders like AD. In the present study, we aimed to investigate whether pathogenic effect of LPS on the brain and behavior of mice can be prevented or treated by injection of MSCs or MSC-produced soluble factors. Fluorescently-labeled MSCs, injected intravenously, were found in the brain blood vessels of LPS-treated mice. Mice co-injected with LPS and MSCs did not demonstrate episodic memory impairment, Aß (1-42) accumulation, and nAChR decrease in the brain and brain mitochondria. Their mitochondria released less cytochrome c under the effect of Ca2+ compared to mitochondria of LPS-only-treated mice. Moreover, MSCs could reverse the pathogenic symptoms developed 3 weeks after LPS injection. Cultured MSCs produced IL-6 in response to LPS and MSCs effect in vivo was accompanied by additional stimulation of both micro- and macroglia. Xenogeneic (human) MSCs were almost as efficient as allogeneic (mouse) ones and regular injections of human MSC-conditioned medium also produced positive effect. These data allow suggesting MSCs as a potential therapeutic tool to cure neuroinflammation-related cognitive pathology.

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.

Nicotinic acetylcholine receptors alpha4beta2 and alpha7 regulate myelo- and erythropoiesis within the bone marrow.

Nicotine consumed upon smoking affects numerous physiological processes through nicotinic acetylcholine receptors, which mediate cholinergic regulation by the neuronal and endogenous acetylcholine. Consequently, nicotinic receptors are expressed in many non-excitable tissues including the blood. In spite of the documented effect of nicotine on hematopoiesis, little is known about the expression and role of nicotinic receptors in the course of blood cell differentiation. The aim of the present study was to investigate whether and how nicotinic receptors are involved in the development of myeloid and erythroid cells within the bone marrow. The presence of nicotinic receptors containing alpha4(beta2) and alpha7 subunits in the bone marrow cells of C57Bl/6 mice was shown by the binding of [125I]-alpha-bungarotoxin or [3H]-Epibatidine and by flow cytometry with subunit-specific antibodies or fluorescein-labeled alpha-cobratoxin. Both TER119+ (erythroid) and CD16+CD43med (myeloid) progenitor cells bound more alpha4-specific antibodies than their mature forms, while the binding of alpha-cobratoxin and alpha7-specific antibodies was also high in mature cells. According to morphological analysis, either the absence of alpha7-containing nicotinic receptors in knockout mice or their desensitization in mice chronically treated with nicotine decreased the number of myeloid and erythroid progenitors and junior cells. In contrast, the absence of beta2-containing receptors favored myelocyte generation and erythroid cell maturation. It is concluded that the development of both myeloid and erythroid cell lineages is regulated by endogenous cholinergic ligands and can be affected by nicotine through alpha7- and alpha4beta2-containing nicotinic receptors, which play different roles in the course of the cell maturation.

α7 nicotinic acetylcholine receptors are involved in suppression of the antibody immune response.

This study demonstrates that α7 nicotinic acetylcholine receptors (nAChRs) regulate mouse B lymphocyte proliferation and IgM production in ion-independent manner. The high α7 nAChR levels were found in CD5+ and Foxp3+ B cells; induction of Foxp3+ cells in vitro was attenuated in the absence or upon inhibition of α7 nAChRs. The adoptively transferred B lymphocytes, stimulated in presence of methyllicaconitine, decreased the IgM response and abolished the IgG response in the host. The data obtained demonstrate the importance of cholinergic regulation for the antibody immune response and immunosuppression.

N-Stearoylethanolamine protects the brain and improves memory of mice treated with lipopolysaccharide or immunized with the extracellular domain of α7 nicotinic acetylcholine receptor.

Neuroinflammation is an important risk factor for neurodegenerative disorders like Alzheimer's disease. Nicotinic acetylcholine receptors of α7 subtype (α7 nAChRs) regulate inflammatory processes in various tissues, including the brain. N-stearoylethanolamine (NSE) is a biologically active cell membrane component with anti-inflammatory and membrane-protective properties. Previously we found that mice injected with bacterial lipopolysaccharide (LPS) or immunized with recombinant extracellular domain (1-208) of α7 nAChR subunit possessed decreased α7 nAChR levels, accumulated pathogenic amyloid-beta peptide Aß(1-42) in the brain and demonstrated impaired episodic memory compared to non-treated mice. Here we studied the effect of NSE on behavior and brain components of LPS- treated or α7(1-208)-immunized mice. NSE, given per os, non-significantly decreased LPS-stimulated interleukin-6 elevation in the brain, slowed down the α7(1-208)-specific IgG antibody production and prevented the antibody penetration into the brain of mice. NSE prevented the loss of α7 nAChRs and accumulation of α7-bound Aß(1-42) in the brain and brain mitochondria of LPS-treated or α7(1-208)-immunized mice and supported mitochondria resistance to apoptosis by attenuating Ca2+-stimulated cytochrome c release. Finally, NSE significantly improved episodic memory of mice impaired by either LPS treatment or immunization with α7(1-208). The results of our study demonstrate a therapeutic potential of NSE for prevention of cognitive disfunction caused by neuroinflammation or autoimmune reaction that allows suggesting this drug as a candidate for the treatment or prophylaxis of Alzheimer's pathology.

The role of carbohydrate component of recombinant α7 nicotinic acetylcholine receptor extracellular domain in its immunogenicity and functional effects of resulting antibodies.

Nicotinic acetylcholine receptors of α7 subtype (α7 nAChRs) attenuate the inflammatory cytokines production by macrophages and are involved in pathogenesis of Alzheimer disease by directly influencing the processing of amyloid-beta (Aß) precursor protein in the brain. Previously we found that regular injections of bacterial lipopolysaccharide (LPS) decreased the level of α7 nAChRs and stimulated accumulation of Aß peptide (1-42) in the brain of mice resulting in memory impairment. Similar effects were observed in mice immunized with recombinant extracellular domain (1-208) of α7 nAChR subunit. However, the mechanism of inflammation-like effect of α7-specific antibodies remained unclear. The aim of the present study was to reveal the impact of carbohydrate component of recombinant α7(1-208) produced in yeast in the functional effect of resulting antibodies. For this purpose, C57Bl/6 mice were immunized with either initial α7(1-208) or with that pre-treated with endoglycosidase. Control groups of mice obtained injections of either LPS or complete Freund's adjuvant. Mice were tested for memory performance, their blood sera were examined for the presence and fine specificity of α7(1-208)-specific antibodies and the brain preparations were studied for the levels of α7 nAChR, Aß(1-42) and interleukin-6. It was found that the original α7(1-208) was more immunogenic than the deglycosylated one, and their epitopes were recognized with different efficiency. In contrast to LPS and original α7(1-208), deglycosylated α7(1-208) did not stimulate interleukin-6 elevation in the brain, i.e. had no pro-inflammatory effect. Nevertheless, immunizations with either the original or deglycosylated α7(1-208) resulted in similar decrease of α7 nAChRs, accumulation of Aß(1-42) in the brain and significant episodic memory decline, comparable to those exerted by LPS injections. We conclude that the decrease of α7 nAChR density, caused by α7(1-208)-specific antibody, is critical for Aß(1-42) accumulation and episodic memory impairment, while pro-inflammatory capacity of α7(1-208)-specific antibody plays a secondary role for the development of Alzheimer-like symptoms.

Molecular Mechanisms Regulating LPS-Induced Inflammation in the Brain.

Neuro-inflammation, one of the pathogenic causes of neurodegenerative diseases, is regulated through the cholinergic anti-inflammatory pathway via the α7 nicotinic acetylcholine receptor (α7 nAChR). We previously showed that either bacterial lipopolysaccharide (LPS) or immunization with the α7(1-208) nAChR fragment decrease α7 nAChRs density in the mouse brain, exacerbating chronic inflammation, beta-amyloid accumulation and episodic memory decline, which mimic the early stages of Alzheimer's disease (AD). To study the molecular mechanisms underlying the LPS and antibody effects in the brain, we employed an in vivo model of acute LPS-induced inflammation and an in vitro model of cultured glioblastoma U373 cells. Here, we report that LPS challenge decreased the levels of α7 nAChR RNA and protein and of acetylcholinesterase (AChE) RNA and activity in distinct mouse brain regions, sensitized brain mitochondria to the apoptogenic effect of Ca(2+) and modified brain microRNA profiles, including the cholinergic-regulatory CholinomiRs-132/212, in favor of anti-inflammatory and pro-apoptotic ones. Adding α7(1-208)-specific antibodies to the LPS challenge prevented elevation of both the anti-inflammatory and pro-apoptotic miRNAs while supporting the resistance of brain mitochondria to Ca(2+) and maintaining α7 nAChR/AChE decreases. In U373 cells, α7-specific antibodies and LPS both stimulated interleukin-6 production through the p38/Src-dependent pathway. Our findings demonstrate that acute LPS-induced inflammation induces the cholinergic anti-inflammatory pathway in the brain, that α7 nAChR down-regulation limits this pathway, and that α7-specific antibodies aggravate neuroinflammation by inducing the pro-inflammatory interleukin-6 and dampening anti-inflammatory miRNAs; however, these antibodies may protect brain mitochondria and decrease the levels of pro-apoptotic miRNAs, preventing LPS-induced neurodegeneration.

α7 Nicotinic acetylcholine receptor-specific antibody induces inflammation and amyloid ß42 accumulation in the mouse brain to impair memory.

Nicotinic acetylcholine receptors (nAChRs) expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of ß-amyloid (Aß42), memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208) or injected with bacterial lipopolysaccharide (LPS) for 5 months. We studied their behavior and the presence of α3, α4, α7, ß2 and ß4 nAChR subunits, Aß40 and Aß42 and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208) resulted in region-specific decrease of α7 and α4ß2 and increase of α3ß4 nAChRs, accumulation of Aß42 and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1) neuroinflammation is sufficient to provoke the decrease of α7 and α4ß2 nAChRs, Aß42 accumulation and memory impairment in mice and (2) α7(1-208) nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease.

Mitochondria express several nicotinic acetylcholine receptor subtypes to control various pathways of apoptosis induction.

Nicotinic acetylcholine receptors control survival, proliferation and cytokine release in non-excitable cells. Previously we reported that α7 nicotinic receptors were present in the outer membranes of mouse liver mitochondria to regulate mitochondrial pore formation and cytochrome c release. Here we used a wide spectrum of nicotinic receptor subunit-specific antibodies to show that mitochondria express several nicotinic receptor subtypes in a tissue-specific manner: brain and liver mitochondria contain α7ß2, α4ß2 and less α3ß2 nicotinic receptors, while mitochondria from the lung express preferentially α3ß4 receptor subtype; all of them are non-covalently connected to voltage-dependent anion channels and control cytochrome c release. By using selective ligands of different nicotinic receptor subtypes (acetylcholine (1 µM) or dihydro-ß-erythroidine (1 µM) for α4ß2), conotoxin MII (1 nM) for α3ß2, MLA (50 nM) for α7ß2 and acetylcholine (10 µM) for all subtypes) and apoptogenic agents triggering different mitochondrial signaling pathways (1 µM wortmannin, 90 µM Ca(2+) or 0.5 mM H2O2) it was found that α7ß2 receptors affect mainly PI3K/Akt pathway, while α3ß2 and α4ß2 nAChRs also significantly influence CaKMII- and Src-dependent pathways. It is concluded that cholinergic regulation in mitochondria is realized through multiple nicotinic receptor subtypes, which control various pathways inducing mitochondrial type of apoptosis.

Mitochondria express α7 nicotinic acetylcholine receptors to regulate Ca2+ accumulation and cytochrome c release: study on isolated mitochondria.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate synaptic transmission in the muscle and autonomic ganglia and regulate transmitter release in the brain. The nAChRs composed of α7 subunits are also expressed in non-excitable cells to regulate cell survival and proliferation. Up to now, functional α7 nAChRs were found exclusively on the cell plasma membrane. Here we show that they are expressed in mitochondria and regulate early pro-apoptotic events like cytochrome c release. The binding of α7-specific antibody with mouse liver mitochondria was revealed by electron microscopy. Outer membranes of mitochondria from the wild-type and ß2-/- but not α7-/- mice bound α7 nAChR-specific antibody and toxins: FITC-labeled α-cobratoxin or Alexa 555-labeled α-bungarotoxin. α7 nAChR agonists (1 µM acetylcholine, 10 µM choline or 30 nM PNU-282987) impaired intramitochondrial Ca(2+) accumulation and significantly decreased cytochrome c release stimulated with either 90 µM CaCl(2) or 0.5 mM H(2)O(2). α7-specific antagonist methyllicaconitine (50 nM) did not affect Ca(2+) accumulation in mitochondria but attenuated the effects of agonists on cytochrome c release. Inhibitor of voltage-dependent anion channel (VDAC) 4,4'-diisothio-cyano-2,2'-stilbene disulfonic acid (0.5 µM) decreased cytochrome c release stimulated with apoptogens similarly to α7 nAChR agonists, and VDAC was co-captured with the α7 nAChR from mitochondria outer membrane preparation in both direct and reverse sandwich ELISA. It is concluded that α7 nAChRs are expressed in mitochondria outer membrane to regulate the VDAC-mediated Ca(2+) transport and mitochondrial permeability transition.

The presence and origin of autoantibodies against α4 and α7 nicotinic acetylcholine receptors in the human blood: possible relevance to Alzheimer's pathology.

Alzheimer's disease (AD) is characterized by a loss of α4ß2 and α7 nicotinic acetylcholine receptors (nAChRs) in the brain and severe memory impairments. Previously, we found that antibodies elicited against extracellular domain of α7 nAChR subunit decreased the number of α7 nAChRs in the brains of mice and impaired episodic memory. Here we show that antibodies capable to bind α7(1-208) are present in the blood of both healthy humans and AD patients. In healthy individuals, their capacity to compete with [(125)-I]-α-bungarotoxin for the binding to α7(1-208) increased with age. The level of such antibodies was significantly elevated in children with severe form of obstructive bronchitis and in mice injected with Lewis lung carcinoma cells expressing both α4ß2 and α7 nAChRs. Elevated antibody levels were accompanied with decreased surface nAChRs on the blood lymphocytes of children and of mice immunized with α7(1-208). Among AD patients, the level of α7 nAChR-specific antibodies was significantly larger in people 62.5 ± 1.5 years old with moderate or severe AD stages (15.2 ± 1.3 MMSE scores) compared to those of 76 ± 1.5 years old with the mild (22.7 ± 0.1 MMSE scores) AD stage. We concluded that α7(1-208) nAChR-specific antibodies found in the human blood are formed as a result of common infections accompanied with the destruction of respiratory epithelium. Elevated blood plasma levels of α7(1-208) nAChR-specific antibodies are characteristic for the early-onset AD and, therefore, are suggested as one of the risk factors for the development of this form of the disease.

Antibodies against extracellular domains of α4 and α7 subunits alter the levels of nicotinic receptors in the mouse brain and affect memory: possible relevance to Alzheimer's pathology.

Nicotinic acetylcholine receptors (nAChRs) of α4ß2 and α7 subtypes expressed in the brain neurons are involved in regulating memory and cognition. Their level is decreased upon several neurodegenerative disorders including Alzheimer's disease (AD), although the reasons for such a decrease are not completely understood. To test whether the nAChR-specific antibodies can affect the brain nAChRs and influence the behavior, we either immunized mice with recombinant extracellular domains of α4 and α7, subunits α4(1-209) and α7(1-208), or injected them with α7(1-208)-specific antibodies. A decrease of α4ß2- and α7-nAChRs accompanied with an increase of α4ß4-nAChRs in brain membranes of immunized mice was observed. Both α4(1-209)- and α7(1-208)-specific antibodies were detected in the brain membrane lysates of immunized mice. Antibody injection resulted in brain nAChR decrease only if mice were co-injected intraperitoneally with bacterial lipopolysaccharide. Brain sections of immunized mice were analyzed for the binding of [125I]-α-bungarotoxin and [125I]-epibatidine. A decrease in α-bungarotoxin binding in striatum (nucleus accumbens and caudate putamen) accompanied with an increase of epibatidine binding in the forebrain and caudate putamen was observed in mice immunized with either α4 or α7 nAChR domains compared to those immunized with BSA. Mice immunized with α7(1-208) demonstrated significantly worse episodic memory measured in a novel object recognition task compared to non-immunized animals but did not differ from the controls in locomotor or anxiety-related tests. These results suggest that nAChR-specific antibodies are able to penetrate the brain upon inflammation with resulting decreases of brain nAChRs and worsening episodic memory.

Differential involvement of α4ß2, 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 α4ß2 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. α4ß2 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 α4ß2 nAChR is coupled to IgM. B lymphocytes of both α7(-/-) and ß2(-/-) mice responded to anti-CD40 stronger than those of the wild-type mice, whereas the cells of ß2(-/-) 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 α4ß2 nAChRs with epibatidine increased the IgM-mediated proliferation of the wild-type and α7(-/-), but not ß2(-/-) 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, α4ß2 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.

Functional effects of antibodies against non-neuronal nicotinic acetylcholine receptors.

Non-neuronal nicotinic acetylcholine receptors (nAChRs) are expressed in the spleen and regulate B lymphocyte propagation and activation. The aim of the present study was to investigate the cellular and physiological effects of antibodies against alpha4(1-209) and alpha7(1-208) nAChR extracellular domains. The antibodies, added in vitro, produced in vivo or injected, specifically bound mouse spleen B lymphocytes. Immunization with nAChR extracellular domains resulted in connective tissue overgrowth and infiltration of segmented neutrophils in the spleen, as well as in decreased body weight compared to mice immunized with BSA. In spite of certain cross-reactivity of alpha4(1-209)- and alpha7(1-208)-specific antibodies, all observed effects were more pronounced upon immunization with alpha7 extracellular domain. Spleens of mice injected with alpha7(1-208)-specific antibody contained decreased numbers of Annexin V-positive B lymphocytes compared to mice injected with non-specific IgG. It is concluded that alpha7 nAChRs are involved in regulating the lymphocyte survival, neutrophil migration, connective tissue overgrowth and body weight accumulation. The antibody binding triggers alpha7 nAChR signaling supporting the idea of non-channel mode of nAChR functioning in B lymphocytes.