Universal nature of cholinergic regulation demonstrated with nicotinic acetylcholine receptors

Highlights

• •Nicotinic acetylcholine receptors regulate B lymphocyte development and activation.
• •Nicotinic acetylcholine receptors regulate mitochondria apoptosis pathway.
• •Nicotinic acetylcholine receptors regulate synaptic vesicles fusion and re-filling.
• •Nicotinic acetylcholine receptors are involved in Alzheimer disease and COVID-19.
• •Nicotinic acetylcholine receptors function in both ionotropic and metabotropic ways.

Abstract

Mammalian nicotinic acetylcholine receptors (nAChRs) were initially discovered as ligand-gated ion channels mediating fast synaptic transmission in the neuro-muscular junctions and autonomic ganglia. They were further found to be involved in a wide range of basic biological processes within the brain and in non-excitable tissues. The present review summarizes the data obtained in our laboratory during last two decades. Investigation of autonomic ganglia with the nAChR subunit-specific antibodies was followed by identification of nAChRs in B lymphocytes, discovery of mitochondrial nAChRs and their role in mitochondrial apoptosis pathway, and revealing the role of α7 nAChRs and α7-specific antibodies in neuroinflammation-related Alzheimer disease and COVID-19. The data obtained demonstrate the involvement of nAChRs in cell survival, proliferation, cell-to-cell communication and inflammatory reaction. Together with the ability of nAChRs to function in both ionotropic and metabotropic way, these data illustrate the universal nature of cholinergic regulation mediated by nAChRs.

1. Introduction

Nicotinic acetylcholine receptors (nAChRs) were among the first cellular receptors investigated in the middle of the 20^th century. They were initially discovered in electric organs of sea eel Torpedo and were further found in the neuromuscular junctions of mammals. The high density of nAChRs found in electric organs facilitated their purification and structure determination. The nAChRs were classified as ligand–gated ion channels, which mediate the mono-valent ion currents and create significant electric potentials needed to either kill the pray (in Torpedo) or stimulate muscle contraction (in mammals). They were shown to be activated by acetylcholine released from the nerve endings and to be blocked by certain snake toxins like α-bungarotoxin. This is α-bungarotoxin which enabled affinity purification of preparative amounts of nAChRs for further structural investigation. The nAChR molecule was shown to be a pentamer composed of two identical (called α) and three different (called β, γ, δ or ε) subunits permeating the cell membrane and surrounding the ion channel. In contrast to other acetylcholine receptors activated by muscarine, they could be activated by nicotine and, therefore, were called “nicotinic acetylcholine receptors” [1].

Further studies demonstrated that the nAChRs mediate ionic signaling not only between nerve and muscle cells, but also between the neurons of autonomic ganglia. Structural studies revealed that ganglionic nAChRs possess the structure similar to muscular ones but are composed of different types of subunits. Six additional alpha subunits (α2 to α7) and three beta subunits (β2 to β4) were identified. They were shown to be combined in certain established combinations (eg. (α3)₂(β4)₃ or (α4)₂α5(β2)₂) and to be non-identically distributed between various autonomic ganglia [1,2]. In addition to heteropentamers, the nAChRs composed of α7 subunits were shown to be homopentamers permeable to not only monovalent but also divalent cations like Ca²⁺ [1]. Further, the nAChRs were found in the brain where, surprisingly, appeared to be located mainly extrasynaptically to regulate the release of neurotransmitters like dopamine or glutamate [3]. These studies for the first time demonstrated that the nAChRs are much more diverse, both structurally and functionally, that could be expected before. Additional alpha subunits (α8 to α10) were discovered; the α9 subunits were also found to combine as homopentamers [4]. It has been agreed to call the nAChRs found in muscle and electric organ as “muscular” and those found in the brain and autonomic ganglia as “neuronal”.

The next important step was the discovery of “neuronal” nAChRs in non-excitable cells and tissues like skin, respiratory epithelium, vascular endothelium and blood cells [5], [6], [7], [8]. These studies were initiated to investigate the effect of nicotine inhaled by smokers on different tissues and, unexpectedly, revealed that the nAChRs are found in almost all systems of the organism. Those nAChRs do not create significant electric potentials but either mediate small currents to affect the neighboring receptors or influence intracellular signaling pathways in ion-independent way: by activating intracellular kinases either directly or through G-proteins [9,10]. Therefore, it appeared that the nAChRs can signal in both ionotropic and metabotropic manner.

The new trend in understanding cholinergic regulation was established at the beginning of 2000ies when the concept of cholinergic anti-inflammatory pathway has been formulated. It was found that stimulating α7 nAChRs in monocytic cells attenuates the production of pro-inflammatory cytokines and, therefore, can regulate inflammation at the whole-organism level [11,12].

The diversity of nAChR subunits stimulated studies of their evolutionary origin, which showed that the most evolutionary ancient alpha subunits are those forming homopentameric receptors (α7 and α9), while those forming heteropentamers appeared much later as a result of ancestor gene duplication [13]. The nucleotide sequences of nAChR-like subunits were found in all animal species starting from insects and nematodes. Moreover, the early nAChR ancestor was found in bacteria [14]. Taken together with the evidence on the very early appearance of acetylcholine these data indicated that cholinergic regulation is an ancient pathway which changed its targets along the evolution and which fulfills multiple functions due to diversity and plasticity of receptors to acetylcholine.

Probably, a lot from mentioned above can also be related to muscarinic AChRs. Our studies concerned nicotinic AChRs and here I will try to demonstrate how the initially very special aim has lead us to understanding the universal character of cholinergic regulation.

2. Generation of nAChR subunit-specific antibodies

The initial idea of these studies was born in collaboration with the laboratory of Vladimir Skok, who studied the nAChRs of autonomic ganglia in Bogomoletz Institute of Physiology, Kyiv, and was inspired by the studies of Jon Lindstrom from the University of Philadelphia, who obtained a panel of monoclonal antibodies against various nAChR epitopes [15]. The nAChR subunit-specific antibodies were usually generated against cytoplasmic portions of subunits, which are mostly variable. Our idea was to create subunit-specific antibodies, which would bind the extracellular portion of the nAChR molecule close to the agonist-binding cite and, therefore, could be used as subunit-specific nAChR blockers. For this purpose, we performed a careful analysis of amino acid sequences of α3, α4, α5 and α7 nAChR subunits and revealed fragments, which bordered Cys pair 192-193 known to be critical for ACh binding and were quite different between subunits (Table 1). The peptides corresponding to these fragments were synthesized in the laboratory of Socrates Tzartos in Hellenic Pasteur Institute in Athens. We conjugated these peptides to protein carriers, immunized animals and obtained monoclonal mouse α3(181-192)-specific (1D6) and α5(180-191)-specific (4E8) antibodies and polyclonal rabbit α4(181-192)- and α7(179-190)-specific antibodies [16]. NMR studies performed in collaboration with Michel Marraud from Polytechnical School in Nancy, France, revealed that 1D6 antibody stabilized the central part of α3(181-192) peptide in solution [17]. The antibodies stained the neurons of various autonomic ganglia in immunocytochemistry and blocked acetylcholine-induced membrane currents in whole-cell patch-clamp experiments [16]. The antibody against α7 subunit fragment competed with methyllicaconitine (MLA), an established α7-specific competitive antagonist, for the binding to the neurons of submucosal plexus of the guinea-pig that confirmed its binding within the agonist-binding site [18]. Inspired by these results, we further generated rabbit antibodies against β2(190-200) and β4(189-199) fragments of beta subunits [20] and against N-terminal fragments of α9 and α10 subunits [19] synthesized in the laboratory of Victor Tsetlin from Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow (Table 1). The specificity of anti-beta antibodies was confirmed by electrophysiological experiments in the inferior mesenteric ganglion of the guinea pig where the β4- but not β2-specific antibody decreased the amplitude of excitatory post-synaptic potentials (EPSP) stimulated by the irritation of pre-ganglionic nerve [20]. The specificity of α7-, α9, α10 and β2-specific antibodies was confirmed with the preparations of corresponding mutant (knockout) mice lacking corresponding subunits [21,22] and in comparison with the α-conotoxin PeAI (for α9 subunits) [22].

Table 1.

Amino acid sequences of the nAChR subunit fragments used for the antibody generation (UniProtKB/Swiss-Prot data base [23]).

α3 (181-192)	APGYKHEIKYNC
α4 (181-192)	AVGTYNTRKYDC
α5 (180-191)	AMGSKGNRTDSC
α7 (179-190) β2 (190-200) β4 (189-199) α9 (11-23) α10 (10-23)	IPGKRNEKFYEC GRRNENPDDST GRRTVNPQDPS SDLFEDYSSALRP DLFANYTSALRP

The selectivity of nAChR subunit-specific antibodies is often put in doubt and limits their application [24]. According to our experience, the antibody selectivity (or its absence) often depends on the method used. In our hands, the antibodies are often cross-reactive or non-specific upon direct binding in immunocytochemistry or flow cytometry. However, using sandwich ELISA, as a rule, demonstrates very clear difference between the preparations of the wild-type or knockout animals lacking certain nAChR subunits [21,22]. In this approach, the immunoplates are coated with the antibody against the whole extracellular domain (1-208) of α7 subunit (the recombinant α7(1-208) for immunization was kindly provided by Socrates Tzartos). Due to substantial homology of extracellular portions of all α and β nAChR subunits this antibody can capture any of them present in the preparation under study. The second biotinylated antibody is subunit-specific and reveals only the subunits of interest. This approach is analogous to Western blot where the protein is at first immunoprecipitated and then revealed in the gel. Sandwich ELISA is much easier and immediately gives a quantitative result, although it does not provide evidence on the molecular weight of the substance revealed.

3. Study of nAChRs in autonomic ganglia

Application of the antibodies generated in our laboratory allowed us to obtain important information on the subunit composition of nAChRs in different autonomic ganglia and plexuses of the rat and guinea-pig. The main subunits identified were α3, α5, α7 and β4 that corresponded to the data on ganglionic nAChRs published by that time [25]. By means of immunocytochemistry, it was shown that the neurons of different ganglia (superior cervical, inferior mesenteric or submandibular) were stained in different ways, i.e. expressed different sets of nAChRs and with different density [16,18,20]. Moreover, different types of cells within one ganglion were stained non-identically, as well as their somata and dendrites. The double and sequential staining with two different antibodies allowed identifying subunits within the same or neighboring receptors [26]. The use of two electrophysiological approaches: whole-cell patch-clamp or irritation of pre-ganglionic nerve allowed distinguishing the antibody effects on all membrane nAChRs (patch-clamp) or exclusively on synaptic ones (EPSPs). In these experiments, it was found that synaptic transmission in inferior mesenteric ganglia of the guinea-pig is mediated by α3α5-containing nAChRs, whereas α7 nAChRs are localized extra-synaptically in specialized cells and fulfill regulatory function [20]. The data obtained demonstrated that one cell within the ganglion can express several nAChR subtypes and allowed suggesting that the set of nAChR subtypes determines the functional peculiarities of each ganglion within the autonomic network and possibly depends on the ways of their origin in the course of embryogenesis.

The data obtained in autonomic ganglia demonstrated subunit specificity and functional potency of the antibodies obtained against extracellular fragments of nAChR subunits that created a basis for their application to different types of cells.

4. Study of nAChRs in B lymphocytes

In the course of the nAChR-specific antibody production, we relatively easily generated hybridomas and obtained monoclonal antibodies against α3 and α5 subunits. In contrast, generation of α7-specific hybridoma appeared to be problematic because the initially formed α7-specific clones did not survive. This data allowed suggesting that α7-specific antibodies influence hybridoma cells viability and attracted our attention to a possible expression of nAChRs in B lymphocytes.

By that time (beginning of 2000-ies) the presence of cholinergic system components, including nAChRs, was clearly shown in T lymphocytes by the works from Koichiro Kawashima laboratory (Kitasato University School of Pharmaceutical Sciences, Tokyo) [27], while, the presence and role of nAChRs in B lymphocytes was under doubt. We performed a set of studies in collaboration with the laboratory of Jean-Pierre Changeux from Pasteur Institute in Paris to show that normal mouse B lymphocytes, as well as B-lymphocyte-derived cell lines bind ¹²⁵I-α-bungarotoxin and ³H-epibatidine and our nAChR subunit-specific antibodies suggesting the presence of α7 and α4β2 nAChRs [28,29]. The use of knockout mice lacking α4, α7 or β2 nAChR subunits and chimera mice, which lacked α7 or β2 subunits in the blood cells only demonstrated that both α4β2 and α7 nAChRs are involved in B lymphocyte differentiation within the bone marrow and antibody production in mature state [29,30]. Studies with chimera mice demonstrated that both α4β2 and α7 nAChRs regulate the B and T lymphocytes development in the primary lymphoid organs (bone marrow and thymus), while in the periphery (spleen) only α7 nAChRs are involved [30]. Since the bone marrow, but not the spleen or lymph nodes, is innervated by cholinergic nerves [31,32], we suggested that α4β2 nAChRs expressed in lymphocytes may respond to acetylcholine released from the nerve endings, while α7 nAChRs are mainly responsive to endogenous acetylcholine produced within the spleen.

The B lymphocyte activation is stimulated upon interaction with the antigen and helper T lymphocytes and requires signaling through both antigen-specific receptor (BCR) and co-stimulatory molecule CD40. We demonstrated that α7 nAChR is coupled to CD40 and performs an inhibitory mitogenic function, while α4β2  nAChR is coupled to BCR and produces a stimulatory effect. In these experiments, we showed that, in addition to α4β2 and α7 nAChRs, B lymphocytes also express α9α10 nAChR subtype, which is up-regulated and fulfills a "compensatory" function in α7^−/− B-cells (summarized in Fig. 1). This finding additionally demonstrated the importance of cholinergic regulation through nAChRs in B lymphocytes [19]. In confocal microscopy experiments performed in collaboration with Antonella Viola from University of Padova, it was found that α7 nAChRs are recruited to immune synapse formed between T and B lymphocytes upon their activation. Since the nAChRs moved to immune synapse from both cells, this data suggested that cholinergic communication is bi-directional assuming that both B and T lymphocytes produce acetylcholine [19]. The literature data indicated that acetylcholine is produced mainly by T lymphocytes [33]. We showed that acetylcholine esterase inhibitors prozerin, piridostigmine or substance 547 (kindly provided by Eugeniy Nikolsky from Kazan University) dose-dependently inhibited CD40-stimulated B lymphocyte proliferation in the absence of T lymphocytes [34]. This data indicated that activated B lymphocytes do produce endogenous acetylcholine, which negatively regulates their CD40-stimulated proliferation.

Fig. 1.

The suggested scheme of nAChR subtypes participation in B lymphocyte activation. ACh – acetylcholine, BCR – B cell receptor.

We also found that α7 nAChRs are necessary for the formation, induction and functioning of regulatory CD5+ Foxp3+ B lymphocytes, which, together with regulatory T lymphocytes, represent a negative regulatory branch of immunity [34].

The nAChRs concentrated in the post-synaptic membrane of nerve and muscle cells function as ligand-gated ion channels and mediate significant membrane currents. The α7 nAChRs are located mainly extrasynaptically and can function in metabotropic ion-independent manner [35]. In the neurons of submucosal plexus of the guinea-pig, acetylcholine induced membrane currents, which were blocked by MLA demonstrating the activity of α7 nAChR ion channels [18]. In order to reveal if α7 nAChRs expressed in the cells of B lymphocyte origin are valuable ion channels, we performed a set of electrophysiological experiments in SP-2/0 hybridoma cells using single channel patch-clamp recordings. It was found that either acetylcholine or α7-specific agonist PNU282987 stimulated the ion channel activity in SP-2/0 cells; the currents were potentiated by α7-specific positive allosteric modulator PNU120596 and were partially blocked by MLA. However, the orientation of recorded currents suggested that the channels mediated the cation outflux rather than influx. 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. We suggested that unusual character of α7-mediated currents in SP-2/0 cells may be due to peculiarities of their membrane, in particular, the negative surface charge formed by carbohydrate residues [36].

In accordance with this finding, Ca²⁺ imaging studies performed in collaboration with Piotr Bregestovskiy in Aix University of Marseille, demonstrated that α7-specific agonist PNU282987 did not stimulate immediate Ca²⁺ influx into SP-2/0 cells. Instead, Ca²⁺ influx was observed within minutes after application of either PNU282987 or MLA and was blocked by the inhibitor of Ca-release-activated channels (CRACs). It was concluded that α7 nAChRs expressed in SP-2/0 cells influence CRACs in ion-independent manner, i.e. through conformational changes of the nAChR molecule caused by the binding of either agonist or competitive antagonist [36]. Such unusual type of the nAChR signaling was further observed by us in mitochondrial nAChRs that will be described and discussed below.

Taken together, the data obtained demonstrated the expression of several “neuronal” nAChR subtypes involved in regulation of B cell differentiation and activation. Both α4β2 and α7 nAChRs support the survival of B lymphocyte precursors in the course of differentiation within the bone marrow. The α7 nAChRs, which represent the main nAChR subtype in mature B lymphocytes, influence Ca²⁺ influx and negatively regulate CD40-mediated B cell activation. They are recruited to immune synapse formed between interacting T and B lymphocytes to mediate bidirectional paracrine stimulation by endogenous acetylcholine and are present in regulatory B lymphocytes to influence the inhibitory branch of immunity. They form valuable ion channels but demonstrate an unusual activity (at least in a tumor cell line), possibly, due to their post-translational modifications (glycosylation).

The nAChRs were found not only in B lymphocytes, but also in other blood cells of both erythroid and myeloid lineages. The presence of nAChRs containing α4β2 and α7 subunits in the bone marrow cells was shown by the binding of [¹²⁵I]-α-bungarotoxin or [³H]-Epibatidine and by flow cytometry with subunit-specific antibodies or fluorescein-labeled α-cobratoxin. According to morphological analysis, either the absence of α7-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 β2-containing receptors favored myelocyte generation and erythroid cell maturation. It was concluded that the development of both myeloid and erythroid cell lineages is regulated by cholinergic ligands and can be affected by nicotine through α7- and α4β2 nicotinic receptors, which play different roles in the course of the cell maturation [37].

We also found the α7 nAChRs in mesenchymal stem cells separated from either the human umbilical cord or mouse placenta [38]. Their quantity per cell increased upon the cell passages in vitro along with the increase of the cell proliferation potential.

5. Discovery of mitochondrial nAChRs

The studies with knockout and chimera mice demonstrated that the absence of either β2* nAChRs or α7* nAChRs within the bone marrow and the absence of α7* nAChRs in the spleen resulted in the decrease of the size of B lymphocyte populations and increased the number of Annexin5+ cells [29]. This data indicated that the nAChRs are somehow involved in regulation of cell apoptosis and survival during maturation and differentiation. Literature data also suggested the role of α7 nAChRs in regulating apoptosis [39]. Another line of evidence obtained by the 1^st decade of 2000ies demonstrated the presence of nAChR-like receptors in bacteria [14]. These data pushed us to investigate the possible presence of nAChRs in mitochondria, which derive from the ancient bacteria and are involved in initiating the “internal” apoptosis pathway. The general concept accepted by that time was that, in spite of the presence of substantial intracellular pool, functional nAChRs are located only on the cell membrane [40]. We demonstrated the presence of α7 nAChRs in the outer membrane of mouse liver mitochondria by both flow cytometry and ELISA using our α7-specific antibodies along with fluorescently labeled α-cobratoxin and α-bungarotoxin kindly provided by Victor Tsetlin from Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow [41]. We also found that the ligands of α7 nAChRs, choline or PNU282987, attenuated cytochrome c release from isolated mitochondria stimulated by either Ca²⁺ or H₂O₂ [41,42]. Cytochrome c released from mitochondria is a signal for apoptosome formation and further caspase activation leading to apoptosis [43]. Therefore, signaling of mitochondrial α7 nAChRs seemed to be anti-apoptotic. Further experiments demonstrated that mitochondrial nAChRs function in ion-independent manner because the effect on cytochrome c release could be achieved with agonists (choline or PNU282987), antagonists (MLA,) type II positive allosteric modulators (PNU120596, 4BP-TQS or PAM-2) or even α7-specific antibodies [42,44]. Studies performed in collaboration with Uwe Maskos and Pierre-Jean Corringer from Pasteur Institute, Paris, and with Hugo Arias from California Northstate University College of Medicine revealed that the ligand-evoked α7* nAChR intra-mitochondrial signaling can be triggered through orthosteric (agonist) and transmembrane intrasubunit (type II PAM) sites, but not through extracellular vestibular sites (type I PAMs) [44]. We also found that α7 is not the only nAChR subtype located in mitochondria; the set of nAChR subunits present in mitochondria is tissue-specific and probably depends on the nAChR subtypes expressed in a given cell [45]. The binding of subtype-specific ligands resulted in activation or inhibition of intramitochondrial kinases in subunit-specific manner [45,46].

Discovery of functional intracellular nAChRs naturally put a question on the targeting signals directing the new-synthesized nAChRs to either the plasma membrane or mitochondria. Our experiments have demonstrated that targeting nAChR to mitochondria may depend on its special type of glycosylation in Golgi [47].

To reveal the mechanism by which the nAChR affect cytochrome c release we studied its interaction with apoptosis-related proteins within the mitochondria outer membrane. By using molecular modeling in silico, it was shown that pro-apoptotic protein Bax and voltage-dependent anion channel (VDAC1), both involved in mitochondria channel formation, can bind within the 4^th transmembrane portion (M4) of nAChR subunits. Experimentally, α7 nAChR-Bax and α7 nAChR-VDAC1 complexes were identified by sandwich ELISA in mitochondria isolated from astrocytoma U373 cells. Stimulating apoptosis of U373 cells by H₂O₂ disrupted α7-VDAC complexes and favored formation of α7-Bax complexes accompanied by cytochrome c release from mitochondria. α7-selective agonist PNU282987 or type 2 positive allosteric modulator PNU120596 disrupted α7-Bax and returned α7 nAChR to complex with VDAC1 resulting in attenuation of cytochrome c release [48]. These data indicated that mitochondrial nAChRs regulate apoptosis-induced mitochondrial channel formation by modulating the interplay of apoptosis-related proteins in mitochondria outer membrane. Similarly to B lymphocytes, the absence of α7 nAChRs in knockout mice was compensated by increased expression of α9-containing nAChRs. The latter also appeared in the liver mitochondria soon after partial hepatectomy to support the survival of remaining liver cells before regeneration process begins [49]. These data clearly demonstrated the role of mitochondrial nAChRs for supporting the cell viability (Fig. 2, summarized in [50]).

Fig. 2.

The suggested scheme of α7 nAChR involvement in mitochondrial channel opening and cytochrome c release. OM – outer mitochondria membrane, VDAC1 – voltage-dependent anion channel, Cyt c – cytochrome c. For simplicity, the release of Cyt c is shown through VDAC1, although the exact composition of mitochondria apoptosis channel is still under discussion (modified from [48]).

A separate set of experiments demonstrated that mitochondria is not the only intracellular structure bearing functional nAChRs. We demonstrated the presence of α4β2, α7 and α7β2 nAChRs in synaptic vesicles transporting neurotransmitter to synaptic cleft within the brain. The α7* nAChRs were co-localized with synaptic vesicle glycoprotein 2A and influenced Ca²⁺-dependent fusion of vesicles with the plasma membranes, whereas α4β2 nAChRs regulated proton influx and neurotransmitter refilling. These data suggested an active involvement of vesicular nAChRs in neurotransmitter release [51].

6. The nAChRs and neuroinflammation

A new chapter in the non-neuronal nAChR studies was opened in 2000 with the papers from Kevin Tracey's lab showing that vagus nerve stimulation inhibits the release of pro-inflammatory cytokines by peritoneal macrophages under the effect of bacterial endotoxin through α7 nAChRs [11]. The subsequent studies allowed formulating the concept of Cholinergic Anti-inflammatory Pathway, which describes the effect of cholinergic innervation on inflammatory reactions in the periphery [12]. Since there are no direct synapses of cholinergic nerves with the monocytes, it was suggested that innervation stimulates endogenous acetylcholine secretion by splenic T lymphocytes, which finally affects macrophages [52]. We used this concept to study the role of α7 nAChRs in the brain upon neuroinflammation.

Neuroinflammation, the inflammatory process developing in the central nervous system (the brain and spinal cord), accompanies and is considered to be the cause of various neurodegenerative disorders like Parkonson's and Alzheimer's diseases [53,54]. The α7 nAChRs were shown to interact directly with amyloid-β peptides responsible for the formation of senile plaques upon Alzheimer disease [55] and to be involved in learning and memory processes [56]. We found that neuroinflammation stimulated by injections of bacterial lipopolysaccharide was accompanied by the decrease of α7 nAChRs in the brain of mice and, vice versa, the decrease of α7 nAChRs caused by α7-specific antibodies resulted in the raise of pro-inflammatory cytokines, accumulation of amyloid-β peptides in the brain of mice and impairment of episodic memory. Moreover, α7-specific antibodies stimulated the production of pro-inflammatory interleukin-6 by cultured astrocytoma cell line [57]. The decrease of α7 nAChRs in the brain mitochondria made mitochondria more susceptible to the effect of apoptogenic agents that, in a long term, resulted in the decrease of cell numbers in striatum and hippocampus brain areas, i.e. neurodegeneration (reviewed in [58]). In parallel, the increased level of α7-specific antibodies was found in the blood of Alzheimer patients suggesting the involvement of α7 nAChRs in Alzheimer's pathology [59]. These data allowed us to suggest several therapeutic approaches to avoid or cure α7-dependent pathological process. Among these approaches are direct activation of the brain α7 nAChRs with selective agonist [60], the use of anti-inflammatory and membrane-stabilizing lipid N-stearoylethanolamine [61] and injections of mesenchymal stem cells [62]. The suggested scheme of α7 nAChR involvement in pathogenesis of Alzheimer disease is presented in Fig. 3.

Fig. 3.

The suggested scheme of α7 nAChR involvement in pathogenesis of Alzheimer disease and therapeutic approaches used. AD – Alzheimer disease, NSE – N-stearoylethanolamine, MSCs – mesenchymal stem cells, LPS – lipopolysaccharide) (modified from [58]).

7. The nAChRs upon COVID-19

The COVID-19 era provided new challenges and opened new perspectives in the studies of cholinergic regulation. The COVID pathology is accompanied by severe inflammation (“cytokine storm”) that immediately attracted attention to a possible involvement and therapeutic significance of α7 nAChRs.

It was noticed that the fragment 674-685 of SARS-Cov-2 spike protein (YQTQTNSPRRAR) possesses certain homology with the fragments of established α7 nAChR ligands: α-cobratoxin, α-bungarotoxin and rabies virus [63]: it contains a cluster of positively charged amino acid residues (RRAR), which can mimic a quaternary nitrogen of choline and acetylcholine. This fragment is naked upon the virus spike protein cleavage by cellular protease and is considered important for the binding to neuropilin that favors the virus penetration into the cell [64]. We have shown that synthetic peptide corresponding to 674-685 spike protein fragment did bind the α7 nAChR: it competed with α7-specific antibodies for the binding with α7 fragment (179-190). Moreover, when added to mitochondria, the 674-685 peptide behaved similarly to α7-selective ligands: it prevented cytochrome c release from mitochondria stimulated by Ca²⁺ or H₂O₂. These data allowed us to suggest that the fragment of SARS-Cov-2 formed upon spike protein cleavage inside the cell affects mitochondria and supports the infected cell survival until the virus replication cycle is completed [65]. However, no evidence that 674-685 peptide (or the whole spike protein) influences inflammation directly has been obtained.

The “cytokine storm” develops in COVID-19 patients not immediately but after some period post-infection. Similarly, cognitive impairment, in particular, memory decline is observed in the course of so called post-COVID syndrome or “long COVID” when the virus is already eliminated, while the immune reaction to it is still present. We have shown that immunization of mice with 674-685 peptide conjugated to a protein carrier provoked an episodic memory decline in immunized mice that coincided with the appearance of 674-685-specific antibodies. Moreover, passive transfer of immunoglobulins purified from the blood of (674-685)-immunized mice resulted in a quick memory decline in recipient mice. The antibodies of (674-685)-immunized mice were shown to penetrate their brain, to induce pro-inflammatory cytokines production and to decrease the level of α7 nAChRs in the brain. Accompanying injections of choline (α7-specific agonist) prevented both α7 loss and memory decline [66]. Additional experiments demonstrated that appearance of (674-685)-specific antibodies was accompanied by the production of α7-specific antibodies, which could be responsible for the effects observed. Both 674-685-specific and α7-specific antibodies were found in the blood of post-COVID patients, as well as in the blood of people vaccinated against COVID-19. The origin of α7-specific antibodies and their possible involvement in post-COVID and post-vaccination complications is under intensive investigation now. Anyway, the data obtained demonstrate an important role of α7 nAChRs upon COVID-19.

8. Concluding remarks

Taken together, the data summarized here show the presence of nAChRs in multiple cells, tissues and even intracellular compartments and their involvement in a wide range of basic biologicals processes like cell survival, cell activation, cell-to-cell communication and inflammation. When expressed in neurons or muscle cells, the nAChRs are clustered in synapses, function as ligand-gated ion channels and mediate membrane currents sufficient to initiate the ion-dependent cell-to-cell communication. In contrast, in non-excitable cells, they are randomly distributed on the surface and are re-localized to the sites of cell-to-cell interaction (eg. immune synapses) to provide either small membrane currents or to influence the adjacent cell membrane components in ion-independent manner. The intracellular (mitochondrial) nAChRs probably function exclusively ion-independently, since there are no discrete pulses of acetylcholine, but only the changes in intracellular choline concentration and, possibly, occasional formation of acetylcholine by mitochondrial choline-acetyltransferase [67].

Such dual manner of nAChR signaling is possible due to a special, labile structure of its molecule. The nAChR pentamer undergoes substantial conformational changes upon binding the agonist resulting in the ion channel opening (“gating”) [68]. Such conformational (allosteric) movements also change the position of transmembrane and cytoplasmic portions of nAChR subunits enabling their interactions with neighboring membrane components and recruiting kinases or G-proteins. Moreover, the allosteric movements can be stimulated not only by the agonists, but also by other specific ligands binding either close to orthosteric binding site (competitive antagonists, antibodies specific to α7(179-190) or SARS peptide (674-685)) or in allosteric transmembrane site (type II PAMs). Probably, these areas are mostly potent to trigger conformational movements of the pentamer.

Therefore, the nAChRs can function in both ionotropic and metabotropic ways. The metabotropic way of signaling seems to be characteristic for the most ancient nAChR subtypes containing α7 or α9 subunits. The reported presence of nAChR-like receptors in bacteria, as well as discovery of nAChRs in mitochondria, together with the early appearance of acetylcholine, demonstrate the very ancient evolutionary origin of nAChRs, much earlier than the development of nerve and muscle systems. The ancient nAChRs, represented now in mammals by α7- and α9-containing subtypes, were initially intended to regulate the activity of other receptors and membrane components. The more “advanced” α7 nAChRs are widely distributed now, probably, because of their fast desensitization suitable for metabotropic signaling, while the α9-containing nAChRs seem to be a “hidden”, compensatory nAChR subtype; expression of α9 subunits is switched on either in the absence of α7 nAChRs or in urgent situations, like the loss of a part of the organ upon hepatectomy. Upon the development of nerve and muscle cells, the new nAChR subunits/subtypes became specialized in mediating significant ion currents in the synapses of autonomic ganglia (α3β4) and neuromuscular junctions (α1β1γδ/ε), as well as small currents in the brain (α4β2), while the ancient α7-containing nAChRs remained located extrasynaptically and in many (if not all) non-excitable cells and mitochondria. The initially created nAChR structure, capable to various modes of signaling, appeared to be so successful/universal that was further used for different purposes in different cells and intracellular organelles. Our data obtained with the autonomic ganglia, brain, synaptic vesicles, blood cells and mitochondria illustrate the universal character of cholinergic regulation mediated by mammalian nAChRs. Future experiments will probably discover more unexpectable nAChR functions.

Declaration of Competing Interest

The author declare that she has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• Data will be made available on request.