Research progress on adenosine in central nervous system diseases

Abstract As an endogenous neuroprotectant agent, adenosine is extensively distributed and is particularly abundant in the central nervous system (CNS). Under physiological conditions, the concentration of adenosine is low intra‐ and extracellularly, but increases significantly in response to stress. The majority of adenosine functions are receptor‐mediated, and primarily include the A1, A2A, A2B, and A3 receptors (A1R, A2AR, A2BR, and A3R). Adenosine is currently widely used in the treatment of diseases of the CNS and the cardiovascular systems, and the mechanisms are related to the disease types, disease locations, and the adenosine receptors distribution in the CNS. For example, the main infarction sites of cerebral ischemia are cortex and striatum, which have high levels of A1 and A2A receptors. Cerebral ischemia is manifested with A1R decrease and A2AR increase, as well as reduction in the A1R‐mediated inhibitory processes and enhancement of the A2AR‐mediated excitatory process. Adenosine receptor dysfunction is also involved in the pathology of Alzheimer's disease (AD), depression, and epilepsy. Thus, the adenosine receptor balance theory is important for brain disease treatment. The concentration of adenosine can be increased by endogenous or exogenous pathways due to its short half‐life and high inactivation properties. Therefore, we will discuss the function of adenosine and its receptors, adenosine formation, and metabolism, and its role for the treatment of CNS diseases (such as cerebral ischemia, AD, depression, Parkinson's disease, epilepsy, and sleep disorders). This article will provide a scientific basis for the development of novel adenosine derivatives through adenosine structure modification, which will lead to experimental applications.

Adenosine has strong effects in the coronary artery, and anti-epilepsy, which is often used to treat cerebrovascular disorders, apoplexy sequelae, coronary insufficiency, angina, arteriosclerosis, and primary hypertension. Since energy utilization is ubiquitous, 1 adenosine can be produced both intra-and extracellularly in tissues throughout the body, including the brain and heart. Adenosine is widely distributed in the central nervous system (CNS). Adenosine can be considered as a central excitatory and inhibitory neurotransmitter in the brain. Under ischemia, hypoxia, tissue damage, and other pathologic conditions, the degradation of ATP is increased. As a signaling nucleoside, adenosine plays a protective role by interacting with adenosine receptors when its extracellular concentration is increased. 2

| ADENOS INE S TRUC TURE AND ITS MODIFIC ATION
Adenosine contains multiple reactive sites of nucleoside molecules and thus has a strong "plasticity." According to the classification of types of modification, modifications can be divided into base modification, glycosyl modification, and simultaneous basic-glycosyl modification, such as the addition of one site, the substitution of two sites, the substitution or elimination reaction of six sites on the base, and the introduction of different groups on the 2′ site of the glycosyl (Figure 1). The main introduction sites are 2, 6, and 5′ on the bases and glycosyl in the treatment of central nervous diseases. The modification of two sites in bases is usually a substitution reaction to produce 2-halogen products, such as 2-chloro-adenosine. The six site amino derivatives can be deaminated by deamination or hydrolyzed by HNO 2 diazotization to form hydroxyl substitution derivatives, such as inosine.
2′ site derivatives in glycosyl with protective effects on nerve cells were obtained by conversion, substitution and elimination, such as adenosine cobalt amine. Other derivatives are listed in Table 1.

| ADENOS INE PRODUC TION AND ME TABOLIS M
Intracellular adenosine is produced mainly via three pathways: (a) When energy consumption increases, or the energy supply is relatively insufficient, ATP loses two phosphate groups and becomes AMP, which is converted into adenosine by removing phosphate groups under the action of the 5-nucleotidase enzyme (5′-N). (b) Adenine reacts with 1-phosphate ribose to form phosphoric acid and adenosine. 11 (c) S-adenosyl-l-homocysteine (SAH) can be hydrolyzed into homocysteine and adenosine. Extracellular adenosine occurs mainly due to the transport of intracellular adenosine and the hydrolysis of extracellular adenine nucleoside. First, intracellular adenosine rapidly passes through the nucleoside transporters to maintain the extracellular adenosine concentration at a certain level. Second, ATP or ADP turns into AMP through CD39 or E-NTPDase and then is hydrolyzed into adenosine by CD73 or 5′nucleotidase. In addition, the extracellular adenosine can be released by nerve endings or gliocytes ( Figure 2). The physiological base level of extracellular adenosine is generally maintained at 0.04-0.2 μmol/L. 12 There are two main metabolic pathways of adenosine: (a) it becomes inosine under the action of adenosine deaminase (ADA) then generates hypoxanthine and hypoxanthine nucleotides through nucleosidases and finally becomes uric acid, which is an end product of purine derivatives in human metabolism. 13 (b) Most adenosine enters the cell via a bidirectional balance transporter, and the adenosine kinase (ADK), which generates AMP by phosphorylation in central cells, only exists in the astrocytes. Adenosine kinase then forms ATP and completes the recycling of adenosine, which is also the main way to metabolise adenosine. Adenosine decomposition is carried out in the cell, and the extracellular adenosine must pass through the cell membrane into the cell through the above pathways of catabolism. Nitrobenzylthioinosine (NBTI) is an inhibitor of adenosine transport in cell membranes 14 (Figure2).
Because of the rapid uptake and metabolism of adenosine, its balance is inefficient during times of stress. Adenosine does not exceed 1 μmol kg −1 in the brain under normal circumstances. 15 However, adenosine degrades at a slower rate than those by which it is produced during ischemia, trauma, inflammation, and some other pathological conditions. This imbalance leads to a rapid increase in extracellular adenosine concentrations. However, adenosine has a short half-life and this concentration change is temporary, and it is difficult to sustain as a protective mechanism against subsequent pathological conditions. F I G U R E 1 The modifying sites of adenosine. Adenosine derivatives are obtained by base modification, glycosyl modification, and simultaneous modification at multiple reaction sites of adenosine, which have different roles in central nervous system diseases

| ADENOS INE RECEP TOR S
Many physiological functions of adenosine are achieved through receptor mediation. The adenosine receptors belong to the G protein-coupled receptor (GPCR) family. This family mainly includes four kinds of receptors, A1, A2A, A2B, and A3, of which A1R and A3R belong to the Gi family of G proteins while A2AR and A2BR are part of the Gs family.
A1R is a glycoprotein containing 326 amino acids with a relative molecular weight of 36 600. A1R has the highest affinity for adenosine, is coupled with pertussis sensitive G protein (Gi1~3,Go) and exists in all systems 16 . However, the expression of A1R is highest in the CNS, mainly distributed in the cerebral cortex, thalamus, hippocampus, basal forebrain, lateral hypothalamus, medulla oblongata, olfactory bulb and cerebellum, etc. The A1R is activated in the presynaptic membrane and can inhibit the activity of adenylyl cyclase (AC), reduce the content of cyclic adenosine-3,5 monophosphate (cAMP), and regulate cAMP-dependent protein kinase activity. In addition, A1R can also activate phospholipase C (PLC), and the latter can adjust the inositol phosphate metabolism of the cell membrane, increasing the content of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Among these, IP3 can stimulate the release of Ca 2+ from the intracellular calcium store and inhibit the N-, Q-, and P-type calcium channels, which leads to the reduction in Ca 2+ influx, inhibits the release of glutamate, and reduces the excitability of nerve conduction. 1 In the postsynapse membrane, A1R becomes activated to open potassium channels and increase the outflow of K + , resulting in hyperpolarization of the membrane, thus reducing excitability and protecting neurons. 17 In addition, A1R, when activated, can also open the ATP sensitive potassium channel (KATP) of neurons in the substantia nigra, increasing the outward current and decreasing the excitability of the membrane. 18 The relative molecular weight of A2AR is 45 000, and it is mainly distributed in the brain areas rich in dopamine, such as the striatum globus pallidus, nucleus accumbens, olfactory tubercle, bulbus olfactorius, and nucleus nervi acustici. A2AR is also expressed in the cerebral cortex, amygdala, hypothalamus, hippocampus, thalamus, and cerebellum. When A2AR is activated, it is coupled with Gs protein in the peripheral tissues or Golf protein in the brain, and the protein kinase A (PKA) pathway is activated. Protein kinase A is also known as cyclic adenosine dependent protein kinase. Only the second messenger cAMP can activate PKA. Meanwhile, through the cAMP responsive element binding protein (CREB) known as cAMP-PKA phosphorylated transcription factor, PKA interferes with nuclear factors-activated-κB (NF-κB) and regulates gene expression. 19

A2AR
can also activate the mitogen-activated protein kinases (MAPK), increase the production of collagen, and inhibit the peroxidation of neutrophils. Unlike A1R, adenosine promotes excitatory transmitter release through A2AR activation. In terms of blood vessels, A2AR also mediate vasodilatation and A2BR lead to a weak vasodilatation. Inhibition of platelet aggregation is mainly related to the A2A receptor. 20 TA B L E 1 Structural modification of adenosine and its derivatives

Structural modification Pharmacological action References
Adenosine The damage of primary cultured cortical or hippocampal neurons was significantly reduced under the condition of oxygen-glucose deprivation 3 2-chloro-adenosine R 1 =Cl,R 2 =NH 2 ,R 3 =OH,R 4 =OH It induces astrocytes to apoptosis 4 Inosine R 1 =H,R 2 =OH 2 ,R 3 =OH,R 4 =OH Inosine regulates depression-like behavior, and binds to adenosine receptors to activate the intracellular ERK-CREB signaling system OH,R4=OH Antiplatelet aggregation activity "energy currency," as a signal molecule in the information delivering between nerve cells, it can improve the body's metabolism and energy source. ATP is a key factor that regulates neuronal support for axonal regeneration 8,9 Cobamamide R1=H,R2=NH 2 ,R3=OH, R4=cobalamin To reduce cerebral tissue damage and neuronal apoptosis induced by ischemia and anoxia injury has different effects at different doses and administration times.
Activation of A3R has both neuroprotective and neurotraumatic effects ( Figure 3).
During the pathological circumstances of slight and short-term ischemia, trauma, and inflammation, it is possible to improve the protein expression level of AR and increase the synthesis of receptor proteins; perhaps the membrane "spare receptor" is activated, or the transport of receptor proteins from the cytoplasm to the cell membrane is accelerated. 22 The change in AR is sustained over a long duration of approximately 1-3 or 7 days, during which adenosine is more likely to bind to its receptors that are synthesized by brain cells.

| Adenosine and cerebral ischemia
Cerebral ischemia is a common acute cerebrovascular disease

| Excitatory amino acid (EAA)
The toxicity of excessive EAAs, such as glutamic acid and aspartate, is an important cause of neuronal death in cerebral ischemia. By activating A1R, the release of EAA can be inhibited to protect nerve cells, and the mechanism may be as follows: In presynaptic membranes, A1R reduces calcium influx by inhibiting N or Q-type Ca  Therefore, adenosine can inhibit the release of glutamate and aspartate after ischemia and reduce the cytotoxic effects of EAA, thereby protecting neurons.

| Reactive astrogliosis
Reactive astrogliosis is a repair response that is characterized with astrocytes hypertrophy, protuberant elongation, glial fibrillary acidic protein (GFAP) expression and astrocyte proliferation after brain damage. The activation of different adenosine receptors has different effects on reactive gliosis. A1R and A3R have inhibitory effects on reactive gliosis, while A2R promotes glia reactions. A1R can accelerate the apoptosis of RCR-1 astrocytoma in rats, 28 and A3R can also inhibit reactive gliosis by inducing apoptosis of astrocytes. Since the adenosine analogue 2-chloro-adenosine (2-CA) is not sensitive to A1R and A2R antagonists, this may be mediated by A3R through cysteine kinase-3 pathways, or it may be related to the decrease in the concentration ratio between SAH and S-adenosyl methionine (SAM), which triggers apoptosis. 29 A2R may indirectly promote reactive gliosis by interacting with other cytokines, such as tumor necrosis factor-alpha (TNF-α), basic fibroblast growth factor (bFGF).

| Trophic action of nerves
Astrocytes can secrete a large number of neurotrophic factors and cytokines with neuroprotective effects, including nerve growth factor (NGF), S-100B, neurotrophic factor 3 and 4 (NT3 and NT4), brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF), IL-6 and selective chemokines (CCL2). Nerve growth factor plays a neuroprotective role in cerebral ischemia, it is secreted mainly by astrocytes next is microglia, and is related to A1R and A2aR, respectively. As one of the factors in the S-100 family, S-100B is a Ca 2+ binding protein mainly secreted by astrocytes and plays important roles in cell proliferation, differentiation, and protein phosphorylation, which is mediated by adenosine A1R. After binding to its receptor, S-100B can induce NF-κB nuclear translocation, stimulate the expression of Bcl 2, and promote the recovery of neurons and the growth of axons. During the pathogenic process of AD, the balance of adenosine receptors is disrupted, and mainly manifested as A1R expression decrease and A2AR expression increase, as well as disruption in inhibition and excitation processes, which eventually leads to cognitive dysfunction. 31 Therefore, the balance of adenosine receptors can play a neuroprotective role by activating A1 receptors, 32 inhibiting A2a receptors, 33,34 restoring the function of the cholinergic system, and improving the hippocampal synaptic plasticity and neurotrophic factors. 35 Alzheimer's disease starts with the lack of synaptic function, and adenosine A2AR is mainly located in the synapses that control synaptic plasticity. So A2AR antagonists can repair the early synaptic and memory dysfunction of AD. 36  In adulthood, new neurons are created in the dentate gyrus of the hippocampus and subventricular zone, two areas of the brain associated with AD. Disruption of this process can lead to neurodegenerative diseases, including AD. Based on a genetic correlation analysis, adenosine A2AR was significantly correlated with hippocampal volume, adenosine rs9608282 small alleles being associated with a greater hippocampal volume and improved memory. 41 In terms of other receptors, caffeine has a protective effect on the amyloid process by inhibiting the A3R-mediated internalization of the β-amyloid precursor. 42 Thus, the adenosine receptor balance theory has been increasingly accepted in AD.

| Adenosine and epilepsy
Epilepsy is a chronic and recurrent brain dysfunction syndrome that is caused by the abnormal discharge of neurons in the brain. Epilepsy is clinically manifested as a whole body tonic clonus attack, and during the seizure, the EEG shows typical explosive multi-spinous waves, and spine-slow waves. The main pathogenesis is glial cell proliferation, adenosine dysfunction, abnormal nerve conduction pathways, selective neuron cell loss, nerve inflammation, and mossy fiber bud phenomenon.

| Astrocyte
Astrocytes are large glial cells that are the most widely distributed glial cells in the brain of mammals. Astrocytes also make up the larg-

| Adenosine steady-state
Adenosine kinase converts adenosine into AMP in astrocytes, and the intracellular adenosine concentration then decreases.

| Glutamic acid
Glutamate has the highest content of amino acids in the CNS and is one of the explicit EAAs. Glutamate has a wide and strong excitatory effect on the cerebral cortex and is closely related to the triggering and spreading of epileptic discharges. Ca 2+ is an important second messenger, and calcium signaling in astrocytes is mainly controlled by receptors. The release of glutamate is related to adenosine binding to its receptor. When adenosine binds to A1R, it inhibits the release of excitatory transmitter glutamate by inhibiting the Ntype voltage-gated calcium ion channel. When adenosine binds to A2AR, it increases intracellular calcium ion concentration through the protein kinase A pathway, thereby stimulating the release of glutamate. 59 Therefore, activation of adenosine A1R or inhibition of adenosine A2AR can inhibit the release of glutamate.

| Epigenetic modification
Epigenetic modifications include changes in DNA methylation that lead to the change in gene expression. Therefore, there may be a basis of epileptogenesis by inducing permanent changes in neural excitability. 60 The main mechanism is that the methyl group is provided by S-adenosyl-l-methionine(SAMe) and selectively added by cytosine of C and G nucleotides of DNA under the catalysis of DNA methyltransferase(DNMTs), and s-adenosine homocysteine (SAH) produces adenosine and homocysteine by hydrolytic enzyme. 61 Therefore, adenosine can inhibit or reverse these changes, such as adenosine not being cleared in time from the synaptic cleft, and the treatment of exogenous adenosine can inhibit the process of DNA methylation and thus inhibit the occurrence of epilepsy. The epigenetic function of endogenous adenosine was determined by inducing DNA hypomethylation due to biochemical interference into the methylation pathway. 62 Exogenous adenosine can reverse the DNA methylation process in temporal lobe epilepsy of rats, inhibit the mossy fiber sprouting in the hippocampus, and control the occurrence of epilepsy.

| Adenosine and sleep disorders
The "sleep substance," adenosine, is an effective endogenous sleeppromoting factor which accumulates in the brain during wakefulness and induces physiological sleep. 65 Among the four adenosine receptors, the role of A2AR is predominant in sleep regulation, whereas A1R contributes to sleep induction in a region-dependent manner but may not be absolutely necessary for sleep homeostasis. 66 In nor-

| ADENOS INE TRE ATMENT
Manipulation of endogenous adenosine level is used in the treatment of CNS disorders. For example, adenosine A1R agonists or ADK inhibition can effectively prevent or reduce the onset of CNS diseases.
However, exogenous adenosine therapy is not the main treatment method, 69 due to the side effects of adenosine in the cardiovascular system and the hepatotoxicity of ADK inhibitors, as well as the fact that exogenous adenosine is quickly absorbed and metabolized after entering the human body by the high affinity nucleoside transporters located on red blood cells, capillary endothelial cells, and smooth muscle cells. 70 However, the ultimate goal of these approaches is to The mechanism of action of adenosine in epilepsy. Adenosine therapy for epilepsy was summarized from the aspects of astrocytes, ADK induced the changes of adenosine concentration, changes in DNA methylation, GABAAR increase local adenosine levels in the brain while avoiding side effects and damage to other organs. Therefore, adenosine metabolic balance has become the developmental direction of adenosine synergistic therapy in brain.

| Silk that controls adenosine release
The continuous release of adenosine can be controlled by planting silk membranes wrapped with adenosine around the hippocampus, which can effectively control the onset of epilepsy. 71

| ADK expression control
Adenosine can be released from human mesenchymal stem cells and mouse embryonic stem cells with endogenous ADK gene knocked out after being implanted into the hippocampus. 72 Theofilas used an adenovirus eight gene expression system to knock out the ADK gene, the ADK level of glial cells in the hippocampus decreased, and the occurrence of epilepsy was controlled. 73

| Adenosine receptors
When the adenosine A1R gene was knocked out in mice, a ketogenic diet could not exert effects on epilepsy, indicating that a ketogenic diet inhibited the generation of epilepsy through the excitatory effect of adenosine A1R. 74 Yang et al 75 also demonstrated that a ketogenic diet significantly increased the tolerance to cerebral ischemia in mice, which may be achieved by increasing the level of extracellular adenosine in the ischemic area and may be mediated by A1 receptors. Researchers also found that the c-fos immune response activity in KO mice was higher than in WT mice by knocking out adenosine A2A receptors in the anterior cingulate and amygdala, thus causing defects in social active behavior (social withdrawal), such as in autism and depression. 76

| Trophic action of nerve
As a metabolite of adenosine, inosine can promote axonal regeneration, mainly by activating intracellular serine/threonine kinases, thus upregulating the key factor GAP-43 protein required for axonal regrowth and acting as a neural growth factor. 77

| Improve blood rheology
After the knockout of the A2AR gene in mice, platelet aggregation was enhanced. Therefore, the activation of A2AR could improve haemorheology, inhibit platelet aggregation, and promote thrombolysis. 78

| Excitatory cytotoxicity and Ca 2+ alleviation
Low and medium concentrations of adenosine can activate adenosine A1R and inhibit the release of glutamate. Medium and high concentrations of adenosine can activate adenosine A2AR and promote the release of glutamate. Adenosine A2AR has an inhibitory effect on A1R at medium and high concentrations, and the inhibitory effect increases with concentration. 79

| S IDE EFFEC TS OF ADENOS INE
Adenosine has a few side effects. The most common side effects are flushing, discomfort in the throat, neck, jaw, upper limbs and gastrointestinal tract, breathing difficulties, and dizziness. Because adenosine has a very short half-life, these side effects usually do not require intervention and disappear quickly, which are easily accepted by patients. Adenosine is applicable to patients who cannot exercise for various reasons or cannot achieve the target amount of exercise.

| D ISCUSS I ON
In However, the concentration of adenosine is only temporarily elevated and is quickly metabolized. At the same time, a high concentration of adenosine activates A2AR, and the concentration of EAA also rises sharply, initiating a series of pathophysiological processes that eventually lead to the necrosis or apoptosis of nerve cells. 83 As low concentrations of adenosine can activate A1R and inhibit the release of EAAs, A2AR is conversely activated with an increase in adenosine concentration, and A2AR can block heteromeric A1R through a receptor-receptor allosteric trans-inhibition. 79 Therefore, adenosine A2AR antagonists are very important for the protection of the CNS.
In the pathological conditions of ischemia, hypoxia, trauma, and inflammation, the extracellular adenosine concentration increases rapidly, resulting in an imbalance of intracellular and extracellular concentrations of adenosine. Adenosine has a short half-life, adenosine concentration is difficult to sustain the protective effect during pathological conditions. There are many ways to manipulate the endogenous adenosine concentration including adenosine agonists administration, ADK, ADA and extracellular enzymes (such as CD39 and CD73) regulation and the use of dipyridamole to inhibit the adenosine transporter. 84 Exogenous adenosine can also be used to increase the adenosine concentration, 85 Yuan et al 86 found that the effect of preconditioning with adenosine on brain ischemic tolerance in rats. Perrier et al 87 think that the main effects of adenosine were to decrease neurotransmitter release probability and to attenuate short-term depression mechanisms. But this use is relatively rare, and it needs to be further examined. At the same time, whether adenosine receptor balance or intracellular and extracellular adenosine concentration balance has a key role in the treatment of CNS diseases remains unclear. The specific mechanisms remain to be further studied.

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.