Cholinergic deficiency in the cholinergic system as a pathogenetic link in the formation of various syndromes in COVID-19

Sergey Petrovich Lysenkov; Dmitriy Vitalevich Muzhenya; Aminat Ramazanovna Tuguz; Tamara Ur'evna Urakova; Dmitriy Sergeevich Shumilov; Ibragim Askarbievich Thakushinov; Rustem Askarbievich Thakushinov; Elena Anatolevna Tatarkova; Diana Muratovna Urakova

doi:10.4103/cjop.CJOP-D-22-00072

REVIEW ARTICLE

Year : 2023 | Volume : 66 | Issue : 1 | Page : 1-13

Cholinergic deficiency in the cholinergic system as a pathogenetic link in the formation of various syndromes in COVID-19

Sergey Petrovich Lysenkov¹, Dmitriy Vitalevich Muzhenya¹, Aminat Ramazanovna Tuguz², Tamara Ur'evna Urakova¹, Dmitriy Sergeevich Shumilov², Ibragim Askarbievich Thakushinov¹, Rustem Askarbievich Thakushinov¹, Elena Anatolevna Tatarkova², Diana Muratovna Urakova¹
¹ FSBEI HE “Maikop State Technological University”, Medical Institute, Maikop, Republic of Adygeya, Russia
² FSBEI HE “Adyghe State University”, Immunogenetic Laboratory of the Research Institute of Complex Problems, Maikop, Republic of Adygeya, Russia

Date of Submission	04-Aug-2022
Date of Decision	18-Oct-2022
Date of Acceptance	27-Oct-2022
Date of Web Publication	17-Jan-2023

Correspondence Address:
Asst. Prof. Dmitriy Vitalevich Muzhenya
Department of Pathomorphology and Clinical Pathophysiology, Medical Institute, FSBEI HE “Maikop State Technological University”, Maikop, Republic of Adygeya
Russia

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjop.CJOP-D-22-00072

Abstract

According to recent data, several mechanisms of viral invasion of the central nervous system (CNS) have been proposed, one of which is both direct penetration of the virus through afferent nerve fibers and damage to the endothelium of cerebral vessels. It has been proven that the SARS-CoV-2 virus affects pathologically not only the human cardiorespiratory system but is also associated with a wide range of neurological diseases, cerebrovascular accidents, and neuromuscular pathologies. However, the observed post-COVID symptom complex in patients, manifested in the form of headache, “fog in the head,” high temperature, muscle weakness, lowering blood pressure, does it make us think about the pathophysiological mechanisms that contribute to the development of this clinical picture? One possible explanation is a disruption in the signaling of the acetylcholine system (AChS) in the body. Viral invasions, and in particular COVID-19, can negatively affect the work of the AChS, disrupting its coordination activities. Therefore, the main goal of this literature review is to analyze the information and substantiate the possible mechanisms for the occurrence of post-COVID syndrome in people who have had COVID-19 from the standpoint of AChS dysfunctions.

Keywords: Acetylcholine, brain, COVID-19, heart, pathophysiology, post-COVID syndrome, signaling disorder, synapse

How to cite this article:
Lysenkov SP, Muzhenya DV, Tuguz AR, Urakova TU, Shumilov DS, Thakushinov IA, Thakushinov RA, Tatarkova EA, Urakova DM. Cholinergic deficiency in the cholinergic system as a pathogenetic link in the formation of various syndromes in COVID-19. Chin J Physiol 2023;66:1-13

How to cite this URL:
Lysenkov SP, Muzhenya DV, Tuguz AR, Urakova TU, Shumilov DS, Thakushinov IA, Thakushinov RA, Tatarkova EA, Urakova DM. Cholinergic deficiency in the cholinergic system as a pathogenetic link in the formation of various syndromes in COVID-19. Chin J Physiol [serial online] 2023 [cited 2023 Sep 28];66:1-13. Available from: https://www.cjphysiology.org/text.asp?2023/66/1/1/370013

Introduction

According to published data in the international literature, disorders in the functioning of the nervous system, both central and peripheral, are increasingly being detected in patients who brought COVID-19. Based on the results obtained, the virus can enter the central nervous system (CNS) structures, most likely through two main mechanisms: through the olfactory nerve, using presumably axonal transport, and the second way, is hematogenous.^[1],[2],[3]

Microglia, macrophages, and astrocytes initially infected with the virus can activate glial cells, and induce local pro-inflammatory cytokines. The anti-inflammatory cholinergic function of the parasympathetic system is depleted under conditions of active cytokine production. Its effectiveness in inhibiting the synthesis and release of tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-6, IL-7, IL-10, and other cytokines in macrophages become untenable. Cytokines, peptides, and immunomodulators synthesized in excess by lymphocytes do not provide adequate feedback between the organs of the immune system and the brain, which significantly aggravates the pathological process in the CNS.^{[4],[5],[6],[7],[8]}

Activation of the inflammatory response in the brain cells provokes its penetration from the cerebral vessels of T-lymphocytes and granulocytes into the subarachnoid space. Ultimately, these activated immune cells and T cells induce other immune cells, leading to neuronal damage, apoptosis, and demyelination.^[4],[9]

”Cytokine storm” is a manifestation of an unfavorable hyperimmune reaction of the body to the introduction of an infection (antigen). Given the close relationship with the acetylcholine system (AChS) and the vagus nerve (n. vagus) system, the “cytokine storm” should be considered as an imbalance between the humoral cellular and the nervous link in the regulation of inflammation.

The AChS works as a powerful cooperative apparatus, having both multiple information inputs from numerous brain structures and outputs. To date, it is known that a large number of acetylcholine (ACh) neurons, sending numerous processes to the cortex and subcortical formations, are located in the brain, especially in the forebrain and brainstem. The presence of the AChS in the brain is an important component of the proper development of the brain and its functions, which provides the synaptic plasticity necessary for the formation of memory and learning. AChS plays a special role in the hippocampus and prefrontal cortex, which are responsible for these functions, where a huge number of ACh synapses and corresponding processes have been found.^{[10],[11],[12]} The activation of ACh receptors (AChRs) in these regions of the brain increases the generation of theta and gamma rhythms. These rhythms are responsible for cognitive functions.^{[12],[13],[14],[15]} It has been proven that not only memory capacity and intelligence play a role in the learning process, but also attention.^{[16],[17],[18],[19]} Experiments have shown that damage to deep ACh structures of the brain disrupts attention processes.^{[20],[21],[22],[23]}

On the other hand, AChS plays an important role in various physiological and pathophysiological processes, including the regulation of the muscular system, immune and inflammatory reactions, wound healing, the development of cardiovascular, respiratory, and other diseases.^{[24],[25],[26],[27],[28],[29]}

The use of synaptic transmission through a chemical synapse is the main way information is transmitted in the nervous system [Figure 1]. The neuromuscular synapse and central synapse include the presynaptic region of the nerve ending, the synaptic cleft, and the postsynaptic region (motor end plate).^[30]

Figure 1: Mechanism of synaptic signal transmission in normal conditions with the participation of NO.

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The nerve fiber terminal contains ACh in vesicles with a diameter of 35–50 nm. Dense strips-active zones through which ACh is released into the synaptic cleft are important formations. ACh release occurs in a special zone of the presynaptic membrane after Ca²⁺ ions enter through voltage-gated Ca²⁺ channels.^{[31],[32],[33],[34],[35]} The fusion of vesicles occurs with the help of the proteins synaptobrevin, syntaxin, SNAP-25, and cholesterol is regulated by binding proteins: (synaptotagmin) and active molecules (NADPH oxidase). A large amount of cholesterol (40% of all lipids) is contained in synaptic vesicles.^{[36],[37],[38],[39],[40]} The process of fusion of protein and lipid components is a stimulus for neuromediators exocytosis, and the delivery of vesicles to excretion sites depends on the function of actin and synapsins.^[38],[39]

ACh activates the Na⁺/K⁺ conductance of the postsynaptic membrane (possibly Na⁺/Ca²⁺) or the end plate to generate a propagating action potential. It should be noted that the number of receptors on the postsynaptic membrane is controlled by small GTPases (Rab II) and protein kinases (Cds42, GSK3β, and phosphoinositol-3-kinase). A decrease in the level of cholesterol in pre- and postsynaptic membranes can cause “scattering” of Ca²⁺ channels over the surface of the plasma membrane and inhibition of neurosecretion.^{[40],[41],[42],[43]} These processes can disrupt synaptic transmission. Recently, the leading role of cholesterol in maintaining the function of Na⁺/K⁺-ATPase in the purinergic synapse has been shown.^{[44],[45],[46]} Similar mechanisms can be assumed in the ACh synapse.

ACh is degraded by acetylcholinesterase (AChE) after interaction with the AChR rapidly; the most part of the degradation products comes back to the presynaptic region, and the other part diffuses into the extrasynaptic regions.^[46],[47] Other neuropeptides are also found in the nerve endings of ACh synapses: calcitonin gene-related peptide, and vasoactive intestinal peptide. They are also involved in synaptic transmission and are part of the parasympathetic nervous system, called the “non-choline system,” located predominantly in the brain.

Interneuronal synapses are similar in the mechanism of functioning to neuromuscular synapses, but the supply of vesicles with AChR is less in central synapses; the synapses are smaller and the postsynaptic membrane does not form folds. However, the principles of the neuromuscular synapse may well be transferred to interneuronal synapses.^[48]

Folds 50–100 nm wide are present on the postsynaptic membrane; there are 7–8 such folds in human, which increases the area of the contact with the mediator. Nicotinic ACh receptors with a density of 100,000/μm² are located in these folds and especially in the area of deepening.^[49] Caveolin, consisting of caveolin proteins (there are 6 varieties of them), is also formed in the postsynaptic membrane of the neuromuscular synapse. Caveola is a kind of bilayer pocket, but they contain sphingolipids, cholesterol, and NO-synthase in high concentration.^[50],[51] Cavelin-3 protein is found in muscle cells, as well as in glial cells and peripheral nerve fibers that have AChR.^[52] Caveolin contains protein kinases, growth factor receptors, endothelial NO synthase, phospholipases, G proteins, ion channels and transporters, receptors.^[53]

Before considering the issue of the post-COVID period, it is logical to consider their role in physiological processes, given the important role of ACh receptors in synaptic transmission. AChRs are mainly localized in the region of the orifices of the subneural grooves, where the bulk of ACh is released. The receptor is represented by a protein complex consisting of five protein subunits (alpha, beta, delta, and gamma proteins), which are located on the postsynaptic membrane opposite the “dense stripes” of the presynaptic membrane.^{[50],[51],[52],[53],[54]}

ACh receptors are surrounded by a belt of lipids (minimum of 45), which determines ion permeability. Cholesterol maintains the functional and structural position of the receptor, and fatty acids can disrupt this functional state.^[14],[55] Fixation of AChR on the postsynaptic membrane occurs with the help of cytoskeletal, scaffold, and microtubule proteins.^[56]

According to the literature, cholesterol plays an important role in the formation of special structures on the membrane rafts (lipid rafts) in synapse membranes. Rafts are organized structures enriched in cholesterol, sphingolipids, and proteins, which can create various combinations. Rafts are very dynamic structures. Therefore, to get a response of the postsynaptic membrane to a mediator stimulus, it is necessary to merge the rafts, concentrate the neurotransmitter, after which the signal cascades are triggered. In turn, disruption of the processes of raft formation, for example, under the action of strong oxidizing agents, such as peroxynitrite, changes the processes of synaptic transmission, especially in the CNS.^[57],[58]

At the moment of attachment of two molecules of ACh to the corresponding receptor, conformational changes occur, and the ACh channel opens, which allows sodium, potassium, and calcium ions to pass through the mouth of the channel. Na⁺ ions have an advantage for passing inside the muscle fiber, which is ensured by its high concentration in the extracellular space and the negative charge at the mouth of the receptor. Active input of sodium (and possibly calcium) generates an end plate potential and an action potential.^[59] The action potential initiates a chain of successive reactions, culminating in contraction and relaxation of the muscle. It should be noted that NO synthase is concentrated in these places. Currently, it has been proven that muscle fibers are able to actively express NO synthase.^[60],[61] This process occurs in the neuromuscular synapse especially.

NO synthase in the region of the neuromuscular junction of mammals is concentrated in the region of NMDA receptors (NMDARs) on the postsynaptic membrane.^[62],[63] Activation of these receptors is accompanied by the entry of Ca²⁺ ions, which is necessary for the synthesis of NO. Nitric oxide can influence the release of calcium ions from the endoplasmic reticulum, as well as accelerate the process of exocytosis of neurotransmitters on the presynaptic membrane by increasing the activity of calcium channels.^[64],[65]

A variety of synaptic proteins can be targets for NO. The most studied in this regard is soluble guanylate cyclase, which can be activated by NO and reversibly desensitized.^[66] In the work of Proskurina, convincingly showed that endogenous nitric oxide is able to inhibit AChE in the neuromuscular synapse of mammals (rats).^[67] This is extremely important with the excess production of nitric oxide. The regulation of synaptic transmission is carried out through the NMDA-receptor-NO-synthase complex by enhancing the synthesis of NO by incoming calcium ions, or by a delayed reaction through dephosphorylation of serine-threonine-protein phosphatase.

Based on the above, we can conclude that the structure of the synapse and the synaptic transmission itself are normally complex physiological processes. However, the issue of changing their work under conditions of viral invasion has not been practically studied. Therefore, the main goal of this literature review is to analyze the information and substantiate the possible mechanisms for the occurrence of post-COVID syndrome in people who have had COVID-19 from the standpoint of AChS dysfunctions.

Pathophysiological Change in Acetylcholine System in COVID-19 Survivors

There is a large amount of research on the impact of COVID-19 on various body systems, such as respiratory,^[68],[69] cardiovascular,^[70],[71] and endocrine.^[72] However, the question of the effect of a viral infection on the nervous system and the effectiveness of signal transmission along nerve fibers, as well as the consequences of such influences, remains poorly understood.

It should be noted that even after recovery, people who have had COVID-19 experience headache, brain fog, periodic fever, muscle weakness, and memory impairment might last for a long time. One of the possible causes for the development of this symptomatology, in our opinion, is a disorder of the AChS in the body. As shown in [Figure 2], the release of pro-inflammatory cytokines IL-1, IL-2, IL-6, TNF-α, γ stimulates the production of nitric oxide, and activation of calcium channels, which leads to an increase in ACh exocytosis. A high concentration of nitric oxide in the synaptic space leads to the inhibition of AChE, which increases the residence time of ACh in the synaptic cleft. On the other hand, the occurrence of a “cytokine storm” activates the synthesis of all forms of nitric oxidesynthase (NOS), inducing the production of a large amount of NO, which can be oxidized to the toxic product – peroxynitrite. The latter reacts with lipids of pre-synaptic and postsynaptic membranes and disrupts the processes of endo- and exocytosis of ACh. In addition, oxidation and denaturation of protein-lipid complexes of ACh, Na⁺, Ca²⁺ channels occur. It should also be noted that a disorder of calcium metabolism leads to an increase in its concentration and a significant change in the potentiation time, and, as a result, depletion of ATP reserves, as well as disruption of the actin-myosin coupling and relaxation. Increasing the concentration of calcium leads to disruption of the mitochondria and the accumulation of products of reactive oxygen species (ROS). The possible causes of these changes due to the emergence of COVID-19 are discussed later in this article in more detail.

Figure 2: Proposed processes of inhibition of synaptic transmission in the neuromuscular synapse during the acute period of COVID infection. Notes: (1) Increase in the amplitude and frequency of the action potential in response to the appearance of IL-1, IL-2, IL-6, TNF-α, γ; activation of calcium channels; (2) Stimulation of ACh exocytosis; (3) Increase in the amount of ACh in the synapse; (4) Decreased amplitude and increased time of action of ACh on the postsynaptic membrane; (5) Modification of ACh, Na⁺ receptors, phospholipids under the action of peroxynitrite; activation of autoimmune inflammation; (6) Oxidation of membrane cholesterol; (7) Violation of actin-mysional conjugation and relaxation. TNF-α: Tumor necrosis factor-alpha, IL-1: Interleukin-1, ACh: Acetylcholine.

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Suppression of the SARS-CoV-2 Acetylcholine System

A key component in the body's autonomic regulation is the n. vagus, which mediates most of both physiological and immunological processes. For example, the nervous system, using the vagus nerve, can rapidly inhibit the release of TNF from macrophages and reduce systemic inflammation.^[73] Therefore, in our opinion, viral invasion can significantly change the functional activity of the vagus nerve.

It is known that ACh and its nicotinic receptors (nAChR) are one of the key components of the CNS, and the cholinergic pathway plays an important role in modulating the inflammatory response.^[74],[75] It has been established that stimulation of the homopentameric α7 nAChR present on the surface of tissue macrophages blocks the expression of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6.^[76],[77] Initiation of vagus nerve activity and its anti-inflammatory mechanism before viremia with a nicotinic receptor activator (nicotine) may have a modulating effect on macrophage activity. However, dysfunction of the cholinergic anti-inflammatory mechanism can lead to a cytokine storm and failure of the immune response to return to homeostasis.^[78] This fact is confirmed in the work of Alexandris et al., who conducted molecular modeling of the interaction of SARS-CoV-2 with nAChRs, as well as using a series of complexes with cholinergic agonists.^[79] Based on these results, the authors suggested that one of the causes for the disruption of the AChS is that the SARS-CoV-2 glycoprotein spike, which carries a “toxin-like” sequence in its receptor-binding domain, can bind using the α-subunits of nicotinic receptors (nAChRs), in particular α7 nAChR, by inhibiting AChS activity.

In their work, they also note the interesting fact that smoking can be a prophylactic for covid infection. The pure nicotine found in cigarettes can moderate the course of the disease. For example, Lakhan and Kirchgessner^[80] found that smokers have a lower incidence of certain inflammatory diseases, including ulcerative colitis. Perhaps this is due precisely to the competition for nAChRs and their moderate stimulation. The interaction of nicotine entering the body through smoking blocks these receptors and prevents SARS-CoV-2 from binding to them, acting, in its own way, as a protective mechanism, but this fact requires further study.^[81],[82]

It should be noted an interesting fact discovered by Kopańska et al. that nicotine and caffeine are able to bind angiotensin-converting enzyme epitopes 2 (ACE 2), which are recognized by SARS-CoV-2, which can be used to inhibit the ACE 2/SARS-CoV-2 complex in the future. The authors also note that blocking is enhanced when nicotine and caffeine are used together with antivirals. This is evidence that nAChR agonists can be used along with antivirals in the treatment of COVID-19.^[83]

Change in Acetylcholine System Receptor Activity in COVID-19

According to recent studies, the number of ACh receptors is maintained both by the insertion of newly synthesized AChRs and by their reuse through the so-called AChR recycling.^[84],[85]

Thus, the rate and balance between synthesis and destruction of receptors play an important role in maintaining the normal functioning of the synapse. However, this process can be disturbed during the development of pathological diseases and cause negative consequences. For example, in “myasthenia gravis” it was found that the formed antibodies to nAChRs on the surface of muscle cells cause the internalization and degradation of nAChR. Internalized and degraded receptors are not replaced by increased synthesis of new nAChRs, so an overall decrease in nAChRs occurs at neuromuscular junctions, with subsequent loss of synaptic efficiency.^[86],[87]

On the other hand, Bruneau and Akaaboune showed that the rate of removal of reducible receptors increases with muscle denervation and pharmacological nerve blockade in a rat neuromuscular preparation, and the rate of formation of new receptors decreases.^[88] It was shown as a result experiment on a rat neuromuscular preparation that the half-life of recycled receptors was 28 h in innervated synapses, and the half-life of pre-existing receptors was 102 h. However, after denervation, the half-life of the restored receptors was 15 h and that of the pre-existing receptors was 48 h. According to the authors, the recovery time of new receptors after denervation was 9 days. From a practical point of view, it is important that direct stimulation of the denervated muscle prevents the loss of newly formed ACh receptors.

However, the mechanism of synaptic transmission disorders, that occurs with COVID-19, is likely to differ from those described above.^{[84],[85],[86],[87],[88]} We believe that one of the causes of the disorders may be associated with the hyperproduction of nitric oxide and the formation of toxic compounds in synapses, such as peroxynitrite. In our opinion, the toxic effect of peroxynitrite during a cytokine storm can be one of the main causes for the development of failures in signal transmission due to the fact that it can oxidize receptors and other synapse structures, as well as disrupt the formation of new ones. As a result, we can get a similar effect observed with surgical or pharmacological nerve denervation. Another important question remains open: “Will newly formed receptors be subjected to immunological attack?” Newly synthesized receptors may no longer carry the epitopes in their structure necessary to trigger the autoimmune process. If this is the case, then methods that accelerate the formation of new receptors are likely to have a clinical effect in restoring the function of the neuromuscular synapse.

Hyperproduction of Nitric Oxide as a Factor Contributing to the Disruption of the Normal Functioning of the Synapse in COVID-19

Reactions that disrupt normal physiological processes are possible with a viral infection. Currently, the role of nitric oxide in the formation of the inflammatory response in COVID infection is shown here. On this issue, we have already published an article,^[89] in which we tried to substantiate this thesis. Acting as an element of antiviral protection, nitric oxide translates the inflammatory response into the pathological nature of inflammation. An increase in NO synthesis during viremia and excessive release of pro-inflammatory cytokines (IL-1, IL-2, IL-6; TNF-γ, etc.) disrupts NO utilization processes. Calcium channels on the presynaptic membrane, which promote the release of ACh from vesicles into the synaptic cleft, are simultaneously activated.

It is known that NO inhibits the synthesis and action of AChE, which manifests itself in a longer activation of ACh receptors, opening of sodium and calcium channels and slowing down their inactivation. It was determined by analogy with this process that changes in the amplitude-time parameters, distinguishing for AChE inhibition, are observed on the activation of NMDARs by glutamate and glycine. Conductivity in the synapse is disturbed; muscle activity is inhibited.^{[67],[90],[91]} Therefore, it can be assumed that similar mechanisms of AChE regulation may also lie in the central synapses. Evidence for this was obtained in the works of Udayabanu et al., Hua et al. using the nitric oxide donor Spermine NONOate.^[92],[93] Hyperproduction of nitric oxide is accompanied by the formation of a very aggressive oxidizing agent – peroxynitrite (and a number of aggressive nitro compounds). Peroxynitrite reacts with lipids of presynaptic and postsynaptic membranes, disrupts the processes of endo- and exocytosis of ACh. In addition, oxidation and denaturation of protein-lipid complexes of ACh, Na⁺, Ca²⁺ channels occur.^[89],[94] All these compounds begin to acquire the properties of antigens. An autoimmune reaction is formed with the production of appropriate antibodies that disrupt the functioning of ACh synapses (and possibly other synapses).

Indeed, antibodies to AChR have been detected in a number of studies in the post-COVID period. In this regard, in the second phase of the infectious process, ACh receptors can be partially blocked by self-antibodies after a cytokine storm. There is one of the variants of myasthenia gravis.^[95],[96] Its symptoms can appear for a long time, and their severity depends on the concentration of antibodies to AChR. The produced antibodies activate the complement system, which is accompanied by damage to the postsynaptic membrane.^{[67],[75],[76],[77],[79],[83],[84],[85]} In addition, antibodies to muscle-specific tyrosine kinase (MuSK) can be produced, but they form a reaction without the participation of the complement system but block the interaction of MuSK with a protein similar to the low-density lipoprotein receptor 4. This leads to a reduction in the AChR clustering process.^[97] It is quite possible to allow the production of antibodies to proteins of the ryanodine receptor (RyR), tantin, cortactin, collagen Q, voltage-gated sodium channel (Kv1.4), voltage-gated sodium channel (VGSC) (similar to myasthenia gravis). The presence of some antibodies has already been proven; the presence of other antibodies in the post-COVID period requires confirmation. Clinically, this manifests itself in rapid muscle fatigue and a decrease in physical endurance.

We have already drawn attention to the fact that endothelial NO synthase is contained in caveolae,^[50],[51] which is activated by pro-inflammatory cytokines with the formation of excess amounts of nitric oxide and peroxynitrite.^[89] Activation of the process of lipid peroxidation plays a role in maintaining viral inflammation. A decrease in the concentration of cholesterol as a result of the action of peroxynitrite leads to a flattening of the structure of caveolae or their disappearance. It should be assumed that under the influence of an active inflammatory process, the processes with the activation of AP generation on the postsynaptic membrane and the operation of the K⁺/Ca²⁺ pump are disrupted.

Thus, partial removal of cholesterol in an experiment on a frog neuromuscular preparation leads to disruption of the process of endocytosis, recycling of synaptic vesicles with ACh, and depletion of the vesicle population during synaptic activity. Cholesterol depletion enhances the production of ROS, causing the activation of lipid peroxidation in the synaptic region.^[98],[99]

The resulting oxysterols are able to interact with oxysterol-binding proteins and receptors.^[99],[100] In addition, oxysterol destroys lipid rafts in the synaptic zone and inhibits the release of the ACh mediator in the neuromuscular preparation during low- and high-frequency stimulation.^[98]

Moreover, it was shown that cholesterol oxidation products 5L-cholestan-3-OH, without affecting the spontaneous release of ACh, inhibited ACh secretion during low- and high-frequency stimulation and disrupted short-term synaptic plasticity.^[101],[102] In turn, the activity of 5L-cholestan-3-OH depended on the content of membrane cholesterol. In an experiment on mice by Grajales-Reyes et al., it was found that exposure to statins leads to a decrease in cholesterol synthesis and disruption of the AChR, which leads to the development of myasthenia gravis.^[103] These processes are aggravated by tissue hypoxia. In view of the above, we believe that peroxidation is one of the mechanisms for the development of post-COVID myasthenia gravis, but not the only one.

Similar changes occur in the synapses of the CNS, but clinically, this manifests itself in impaired functioning of the AChS of the brain: memory disorders and sensory aphasia. Symptoms such as “fog in the head,” reduced attention, impaired short-term memory, impaired orientation in space, impaired memory for names, and names of objects are characteristic of CNS synapses. In the post-COVID period, partial blockade of AChR significantly weakens the anti-inflammatory function of the parasympathetic system, and the main representative of this system – n. vagus.

Deterioration of Calcium Transport in the Synapse in COVID-19

It must be assumed that a huge number of modulating influences and especially biologically active compounds, that act as neurotransmitters and neuromodulators (norepinephrine, somatostatin, calcitonin, and serotonin), have an effect on the acetylcholine system. One of the important participants in the mechanisms of synaptic transmission is calcium ions. For example, the role of calcium ions in the progression of a viral infection is confirmed by the use of calcium channel blockers (amlodipine) as a therapeutic agent that improves the course of the process in COVID-19.^{[104],[105],[106]}

Moreover, it turned out that the process of cooperation of the S-protein of the virus with the host ACE2 receptors occurs with the obligatory participation of the activation (or formation) of the calcium channel. Coronavirus produces a special protein that can assemble as Ca²⁺ conductor viroporins, which facilitates the entry of the virus into the cell. The accumulation of calcium ions in the cell leads to a deviation of the concentration balance and, as a result, can cause disturbances in the work of mitochondria. According to the results presented by Ramadan et al., mitochondrial metabolism and cytosolic calcium have a dynamic relationship.^[106] Increasing Ca²⁺ inside the cell stimulates its absorption by mitochondria, in this case, they act as a kind of buffer system of the cell; however, calcium overload in mitochondria can lead to activation of cell death pathways. Active absorption of calcium ions by mitochondria through the pores in the membrane causes osmotic changes that lead to the swelling of mitochondria and an increase in the production of ROS.^{[107],[108],[109]}

If we turn to the neuromuscular synapse, then the accumulation of Ca²⁺ in mitochondria and ATPase deficiency in the sarcoplasmic reticulum form the syndrome of mitochondrial myopathy. On the other hand, as we noted above, a change in synaptic transmission due to CNS viral invasion can also lead to disruption of muscle work throughout the body. In severe cases, the process of necrosis can cause an increase in the level of K⁺, destruction of the sarcolemma, and myoglobin, followed by heart rhythm disorder and the formation of multiple organ failure.

It is known that for adequate muscle contraction it is necessary that the calcium concentration between myofibrils increases 500 times (up to 2 × 10^-4 mmol/l). After actin-myosin interaction, calcium is “pumped out” into the sarcoplasmic reticulum. It should be noted that the process of “pumping out” of Ca²⁺ into the sarcoplasmic reticulum is energy-consuming and is carried out with the participation of ATPase. If there is a deficiency in the synthesis of ATPase, then this process slows down. In advanced cases, this can cause prolonged contraction (spasm) of the muscles.^{[110],[111],[112],[113]}

The appearance of a whole group of trigger activators of Ca²⁺ channels in COVID infection is accompanied by a sharp increase in Ca²⁺ in the sarcoplasm and active hydrolysis of ATPase. With the consumption of ATPase, there is a slowdown in the work of Ca-ATPase, which causes a slowdown in the relaxation of skeletal muscles and, at the same time, the start of anaerobic respiration in the muscles.^[112],[113] This may be accompanied by an increase in nonshivering (and partially contractile) thermogenesis, an increase in oxygen debt and an accumulation of lactic acid. It is possible that thermogenesis in muscles during COVID infection involving RyR1s is parasympathetically controlled through AChR,^{[114],[115],[116]} as is the case in fish in the heating organ.^[117] Clinically, this is manifested by rapid muscle fatigue. Attempts to make voluntary movements are accompanied by the depletion of the recirculating ACh, and, subsequently, the reserve pool. In addition, the formation of vesicles is impaired in parallel with a decrease in the concentration of membrane cholesterol. The described situation is dangerous for sarcomeres, which can partially go into the stage of apoptosis and necrosis due to hypoxia and calcium overload.

Systemic endotheliitis in the “cytokine storm” causes disturbances in blood flow in muscle tissue, the development of circulatory and tissue hypoxia, metabolic acidosis and impaired resynthesis of ATPase. All these factors are components of neuromuscular transmission disorders.^[118] The concentration of ions in the blood has a significant impact on the activity of the calcium channel. There is still no clarity on this issue, although hypocalcemia has been recorded in 60% of patients admitted to the clinic.^{[119],[120],[121]}

A nonspecific increase in the level of procalcitonin in the blood during a viral infection may indirectly indicate an increase in synthesis not only in nonspecialized cells but also in specialized C-cells of the thyroid gland. The result of the action of calcitonin is a decrease in ionized calcium in the blood.^[120],[121] Given the ability of Ca²⁺ to induce the penetration of the virus into the cell, hypocalcemia can be regarded as the host's evolutionary defense against the virus.

This reaction turned out to be universal since the mechanism of virus penetration into the cell turned out to be common for such viruses as swine coronavirus, recovirus, hepatitis B virus, West Nile virus, herpes virus, influenza A, rotovirus, cytomegalovirus, and coronavirus COVID-19.^[106] The concentration of calcium in most cells is 10⁻⁸–10⁻⁷ mol/L. As soon as the level of 10⁻⁶–10⁻⁵ mol/L is reached, calmodulin shows its effects.^{[118],[121],[122]} However, the correction of hypocalcemia is recognized as an unreasonable tactic in clinical practice today.^[106] The issue of whether such a reaction is biologically appropriate for the host has not been resolved, although there are more arguments in favor of conditional host protection from the virus.

The issue of participation of calcium ions, calcium channels in the genesis of virus replication, modulation of the inflammatory and anti-inflammatory mechanism of the AChS, disruption of the mechanisms of synaptic transmission of information, both in the acute period of viral infection and in the long-term, deserves further study.

Acetylcholine System Disorders and Effects on the Cardiovascular System

Many post-COVID patients often complain about pathologies of the cardiovascular system: cardiac arrhythmias and orthostatic collapses. It is noted in many studies, that 1/3 or more of those with COVID have a lesion of the cardiovascular system. However, as the authors of the studies themselves note, damage to the cardiovascular system in most cases is not directly related to viral invasion, and the nature of the pathologies is not entirely clear.^{[70],[71],[123],[124]}

We believe that after suffering from COVID-19 disease, disturbances in the autonomic nervous system can significantly affect the functions of the cardiovascular system, expressed in the inadequacy of orthostatic,^[125] due to impaired sympathetic control of tone, vessels during the redistribution of blood volume under the influence of gravity.^[126],[127] The innervation of resistive vessels is provided through the sympathetic ganglia, in which ACh acts as a mediator. The loss of sympathetic tone is probably due to a deterioration of neuromuscular transmission in adrenergic synapses of vascular smooth muscles.^[128],[129] The events that we described for the ACh synapse could also occur in this region. The basis for this statement is the data that one of the leading factors in the progression of the disease is inflammation of the endothelium (systemic endotheliitis) in COVID infection. Under these conditions, energy processes are disrupted in all underlying subendothelial areas, including vascular muscles and synapses.

On the other hand, despite the strong influence of the central sections of the CNS on the work of the heart through the parasympathetic and sympathetic sections, it is necessary to take into account the fact that there are intracardiac reflex arcs, which include Dogel cells (1, 2, 3 orders) with the mediator ACh. These reflexes normally provide a rapid restructuring of the heart, after which the mechanisms of systemic regulation are activated through the vasomotor and cardioinhibitory centers.^[122] Loss of control from the vagus nerve (its afferent and efferent links) may be accompanied by attacks of tachycardia and the appearance of ectopic foci in the myocardium. Similar reactions were noted in the elderly. It has been shown that with age the efficiency of cardiovagal baroreceptor regulation decreases due to modification of the receptors of the aortic arch, carotid sinus, cardiopulmonary, and other reflexogenic zones.^{[130],[131],[132]} Under physiological conditions, lowering blood pressure (BP) includes baroreflex regulation of cardiac activity and blood vessels.^[131],[132] It consists of a narrowing of resistive vessels, tachycardia, and an increase in BP, as well as due to the release of adrenaline from the adrenal medulla in response to a decrease in BP. However, it can be assumed that reflex responses in some cases after exposure to the virus will be inadequate and delayed in some patients due to a malfunction of the ACh synapse that innervates this area.

The classic work of Nemecek showed that postganglionic sympathetic fibers innervating sweat glands, skin vessels, and striated muscles use ACh as a mediator.^[133]

A more significant evidence of the failure of nervous regulation is orthostatic hypotension, accompanied by a decrease in venous return to the heart at the time of taking a vertical position, and in some cases, the inclusion of the so-called “reverse” Bainbridge reflex with the development of bradycardia and hypotension. The physiological expediency of this reflex is not entirely clear, and, most likely, this is a manifestation of pathology.^[131],[133]

In rare cases, the manifestation of the Bezold-Jarisch reflex with the development of bradycardia, hypotension, and apnea is possible. The starting point of this reflex is the increased activity of the afferent link (atrial stretch receptors) of the vagus nerve with excessive contraction of the “empty” ventricles. The reflex is consonant with the “reverse” Bainbridge reflex. In all cases (except others), there is a disruption in the functioning of the AChS and its interaction with other regulatory systems.^{[130],[134],[135]}

Conclusion

Based on the literature analysis, the authors propose to consider as a discussion the mechanism of disruption of synaptic transmission of information in the central and peripheral nervous system using the ACh synapse as an example. These mechanisms may underlie the formation of pathological reactions in the acute period of infection, as well as in the late post-COVID period.

According to the authors of the article, which is based on the analysis of modern literature, AChS failure, caused by hyperproduction of pro-inflammatory interleukins and nitric oxide, is formed in conditions of severe COVID infection. The latter, through stimulation of MNDA receptors and calcium channels, stimulates an active release of ACh into synapses, with a sharp inhibition of acetylcholinesterase activity, prolonged activation of Na channels, followed by a deterioration of signal transmission in the neuromuscular, nerve and neuroimmune synapse. An important element is the effect of the COVID-19 virus to form viroporins, their own calcium channels, through which the virus enters the cell and replicates in it. At the same time, the level of plasma calcium decreases, but the mechanism of hypocalcemia remains debatable.

Hyperproduction of nitric oxide is accompanied by the formation of a powerful oxidizing agent, peroxynitrite, which causes modification of the protein and lipid components of the AChR, Ca²⁺, and Na channels, which even more disrupts the transmission of signals from the presynaptic to the postsynaptic membrane. Clinically, this is manifested by severe muscle fatigue in patients, the development of a “cytokine storm” and various neuropsychiatric disorders. The modification of AChR protein-lipid compounds, protein-lipid formations of Na⁺-, Ca²⁺-channels is accompanied by an immune response to self-antigens. Cholinergic deficiency develops after a period of activation of the release of ACh into the synapse, especially during the cytokine storm at the stage of the process subsiding. An autoimmune chronic process is formed with periodic manifestation of one or another syndrome (arthritis, artalgia, myalgia, myasthenia gravis, diabetes mellitus, synovitis, and Guillain–Barré syndrome). The phasic nature of the functioning of the acetylcholine system in viral infection dictates the need for a differentiated approach to treatment at different stages of the inflammatory response.

The functioning of the efferent link, as we noted, largely depends on the state of the “Ca²⁺-channel-ACh-receptors-Acetylcholinesterase system.” Understanding these processes may open up prospects for the treatment and rehabilitation of patients with COVID infection.

Recommendations

In connection with these data, a number of conclusions and assumptions can be made:

Activation of calcium channels, as a result of the action of the virus in the initial period of COVID-19 infection, activates the inflammatory process and contributes to the development of a cytokine storm;
An increase in ACh production and a decrease in AChE activity during the cytokine storm leads to disruption of synaptic transmission in immune cells, neuromuscular synapses, and CNS synapses. The appearance of many symptoms is the result of dysfunction of the autonomic nervous system.
Activation of NO-synthases (all three forms) is accompanied by increased NO synthesis, which significantly reduces cholinesterase function, and increased formation of peroxynitrite. The latter, being the most powerful oxidizing agent, disrupts the lipid bases of the synaptic junction, protein components, and ion channel receptors (AChR; Na⁺, Ca²⁺, K⁺ channels). An additional factor of damage to the channels is hypoxia.
Modified structures of receptors and synaptic junction components cause activation of antibody production and modeling of autoimmune processes.
Autoimmune replenishment forms a chronic inflammatory process with its periodic activation.

Based on the above hypothesis, several principal approaches can be proposed for correcting AChS dysfunction:

Taking into account the role of peroxynitrite in the deterioration of AChS, it is advisable to use antioxidant drugs, as well as compounds that neutralize peroxynitrite. Based on the fact that inflammation in the post-COVID period can take on a chronic long-term course, the use of antioxidants should be long-term as well.
To reduce the concentration of autoimmune complexes, the use of plasmapheresis and therapeutic fasting to ACh receptors, Na/K proteins, Ca-channels is justified. It has been proven that these methods in clinical practice reduce the concentration of immune complexes in the blood.
Based on experimental animal models, it would be logical to test the method of electrical and magnetic muscle stimulation to accelerate AChR recirculation and regeneration. To restore the activity of the central synapses, transcranial magnetic, or electrical stimulation of the brain could be used. It is important that these methods of treatment have already been tested and approved for use in medical practice.
To improve the function of ACh-synapses in the post-COVID period, the use of anticholinesterase drugs is pathogenetically justified.

Some of these proposals require clinical testing, but according to the authors, some of them may in the future be included in rehabilitation programs in the post-COVID period, while the other part can already be used in rehabilitation (plasmapheresis, therapeutic fasting, magnetic and electrical stimulation of the brain and muscles, use of antioxidants).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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