Microglial Adenosine Receptors: From Preconditioning To Modulating The M1/M2 Balance in Activated Cells Part 3

It is well known that the development of the nervous system requires the programmed death of a significant number of neurons [137]. Less well-known is that neuronal death is a lifelong physiological process. 

The nervous system is an important part of the human body. It is responsible for controlling various physiological and psychological activities of the human body, one of which is memory. Human memory is very important, it affects our life and work. The relationship between the nervous system and memory is also increasingly valued.

The role of the nervous system in memory is mainly reflected in two aspects:

First, neurons form complex neural networks by continuously connecting and transmitting information. These networks play an important role in the formation, storage, and retrieval of memories. Memory is a form of neural network. When we learn new knowledge or experience new things, the connections and signal transmission between neurons will change. This change allows the information to be better stored and retrieved at any time. come out. Therefore, the health of neural networks is critical to ensure that our memory can continue to function.

Secondly, the nervous system also affects our memory through the action of neurotransmitters. There are many types of neurotransmitters in the human body, such as acetylcholine, dopamine, etc., and their release and action will directly affect our memory. For example, acetylcholine is an important neurotransmitter that affects memory. Its release will make the connections between neurons closer, thereby increasing the storage and retrieval ability of memory information.

In addition, some studies have shown that a healthy nervous system can also prevent Alzheimer's disease. Alzheimer's disease is mainly caused by the death of neurons in the brain and the breakdown of connections between neurons. The health of the nervous system can slow down this process and protect the memory of the elderly.

In short, the relationship between the nervous system and memory is very close. The connections between neurons and the action of neurotransmitters are key factors in building memory networks and affecting memory. Therefore, we should pay attention to protecting the health of the nervous system, participate in more exercises, and maintain good living habits to maintain our good memory and overall quality of life. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors. These substances are very important for memory and learning. In addition, Cistanche deserticola can also improve blood flow and promote oxygen delivery, which can ensure that the brain receives sufficient nutrients and energy, thereby improving brain vitality and endurance.

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Indeed, individuals with epileptic seizures do lose neurons in each episode [138,139]. But, also, healthy individuals seemingly lose neurons upon aging. Fortunately, loss of neurons per se does not lead to disease, either because of the redundancy in neural circuits or because neuronal death is not focused on a specific region. 

Redundancy is also observed in the motor control circuits of the basal ganglia since it is estimated that clinical symptoms in Parkinsonian patients appear when the number of nigral cells lost is 70%. 

Age is the main risk factor in the most prevalent CNS neurodegenerative diseases, Parkinson's and Alzheimer's. Accordingly, the progressive loss of neurons in the CNS of the aged human does not lead to disease in physiological aging but may lead to neurodegenerative diseases for which no cure exists. There are few non-optimal therapies to combat Alzheimer's or Huntington's diseases. 

In the case of Parkinsonism, the work and wisdom of Hornykiewicz and colleagues allowed the detection of a loss of dopamine in certain brain areas of the patients. They noticed the poor brain penetrance of dopamine and suggested a treatment with the precursor of the neurotransmitter, levodopa (L-DOPA). L-DOPA can cross the blood–brain barrier and is readily processed to dopamine in the CNS [140–144]. 

L-DOPA is still used today to treat PD symptoms but, unfortunately, it does not delay disease progression. The issue is, therefore, how to afford neuroprotection in neurodegenerative diseases and, eventually, in long-lived healthy individuals. 

Here we will discuss how microglia may have a neuroprotective role in both physiological and pathological aging. The difficulties in demonstrating the efficacy of neuroprotection interventions in humans (see [75]) is a hot topic whose discussion is out of the scope of the present paper.6. Ischemic Preconditioning after Brain Ischemia

Preconditioning is a mechanism by which exposure to an insult prepares the whole system to better respond to a second similar insult. To our knowledge, it was first discovered in the cardiovascular system. 

Upon survival of a heart infarction, the cardiovascular system is better suited to respond to a second one. This preconditioning is mechanistically complex but adenosine receptors (AR) are key players. This is probably because, in glucose and/or oxygen deprivation, ATP is readily converted into adenosine, whose concentration increases in the blood and any (local) extracellular environment. 

The first results linking AR to preconditioning in the ischemic (rabbit) heart appeared in the nineties [145]. The A1 type was presented as the most important receptor in preconditioning [146] but this was probably due to neglecting for decades the relevant role of other AR types in heart function. 

In a model of ischemia-reperfusion, the synergistic action of A1R and A2AR agonists on cardioprotection was reported in 2010 [147]. Ischemic preconditioning in the cardiovascular system prompted scientists to focus on the occurrence of a similar mechanism in the ischemic brain. The protection of hippocampal cell death afforded by sub-lethal ischemia is among the earlier findings in this issue [148]. 

Soon afterward, it was reported that ARs were involved in the preconditioning mechanisms [149–152]. The question relevant to the present article is whether microglial ARs play a role in preconditioning. First of all, it was soon known that both microglia and astroglia play a significant role in ischemic preconditioning [153,154]. Despite the relevant role of AR in modulating microglial function, studies aimed at answering the question of the involvement of microglial AR in preconditioning after brain hypoxia are scarce [131,155]. 

Either microglial ARs are not important for brain ischemic preconditioning or work related to the ischemic brain has focused on neurons, as the focus in the ischemic heart was placed on cardiomyocytes. 

In brain ischemia-reperfusion injury, the neuroprotective role of targeting ARs has been demonstrated, although activation of the A1R receptor is neuroprotective, drugs that activate these receptors have cardiovascular side effects; thus the alternative consists of blocking the effect induced by A2AR through the use of antagonists that, in general, are very safe [156]. In addition, the expression of ARs may be modified after an ischemic insult [105,157–162]. 

In summary, ARs are likely relevant for the functionality and fate of microglia that become activated in ischemia [97,163–167]. Abbracchio and Cattabeni, already in 1999, suggested that antagonists of the A2AR could be useful in neuroprotection by both reducing the neuronal release of glutamate, an excitatory neurotransmitter, and regulating the activation of microglial cells [105].

7. Microglia in Aging and Neurodegenerative Diseases, Friend or Foe?

Microglia are instrumental in the events causing neuronal death during the development of the nervous system and, also, in the clean-up after such neuronal death. It would be naïve to think that, in the absence of any event resulting in clinical symptoms, i.e., in a lifelong physiological/healthy brain, microglia remain static. 

Although the data are scarce, neuronal death occurs throughout the individual's life, although at a much slower rate than during the development of the nervous system. A seminal review in 2007 [168] highlights that the cross-talk between microglia and neurons in developmental stages encompass, among other, Purkinje cell death via microglia-induced respiratory burst, release by microglia of factors that lead to neuronal apoptosis and microglia-induced synaptogenesis and synaptic properties. The role of microglia in maintaining CNS homeostasis in a healthy brain is less known. 

In the words of Graeber, it refers to microglia as: "analogous to electricians, they are capable of removing defunct axon terminals, thereby helping neuronal connections to stay intact" [169]. 

Apart from the role in removing cells that are targeted to die along development, the hypothesis is that, in adult stages, microglia help remove cells that are targeted to die, e.g., those that are not very active and die, to reinforce the synaptic connections of the surviving cells and firm up those neural circuits that seem more necessary.

The evaluation of neuronal death is usually aimed at detecting an underlying pathology. 

In our opinion, this should be questioned, as neuronal death cannot be ruled out in a healthy brain. It is tempting to speculate that physiological aging correlates with neuronal loss but strengthens the synaptic connections that the individual most needs in their daily life. A few years ago it was noted that neurons can die in several ways: "intrinsic and extrinsic apoptosis, necrosis, necroptosis, parthanatos, ferroptosis, apoptosis, autophagic cell death, autos, autolysis, proptosis, pyroptosis, apoptosis, and mitochondrial permeability transition" [115]. 

Some of those may likely be operating in the brain of a healthy aged individual, i.e., not only in patients suffering from neurodegenerative diseases or in patients suffering from a stroke. Despite the difficulties in assessing neuronal death and neuron-microglia cross-talk in the adult brain, future work is required to confirm the bidirectional interactions and decipher the underlying mechanisms.

8. Skewing the M1/M2 Balance towards the Neuroprotective M2 Phenotype

The real state of microglia in the aged brain is not fully elucidated. However, it is suggested that senescent microglia may contribute to age-related neurological diseases. The reduction of phagocytosis in senescent microglia probably prevents the adequate elimination of debris and the predisposition to be activated through the M1 pathway, while the difficulty of developing an M2 phenotype may impede the physiological function of protecting neurons from death [170–172]. In any case, avoiding the senescence in microglia appears as a good strategy to decrease the risk of neurodegenerative diseases. 

In other words, reduced microglia senescence may underlie physiological aging. It should be noted that a portion of microglial cells are activated in physiological aging. Using a marker of activated microglia, (R)-[11C]PK11195, positron emission tomography (PET) brain scans of healthy subjects aged 19 to 79 showed an increased activation upon aging. Authors conclude that "activated microglia appear in several cortical and subcortical areas during healthy aging, suggesting widespread neuronal loss" [173]. 

Working on the expression and function of cannabinoid receptors in resting and activated microglia we found that the expression of cannabinoid CB1 and CB2 receptors in microglia (resting) from a transgenic rodent model of AD was similar to that observed upon activation of microglia from wild-type mice. As the cognition deficits in AD animal models are only evident upon aging, it was tempting to speculate that a certain degree of chronic activation was neuroprotective. It is assumed that such activation is constituted by cells skewed to the M2 phenotype [174]. 

GPCR function is modulated by interaction with other members of the superfamily. We have found interesting results with cannabinoid receptors. There are two types of cannabinoid receptors, CB1 and CB2, and both are capable of interacting with AR. In microglia, the A2AR may directly interact with the CB2R and the structure of the resulting complex is such that the blockade of the A2AR by a selective antagonist increases signaling through CB2R [54]. 

A2AR antagonists appear, once more, as beneficial; in this case, by increasing the action of a receptor that, expressed in glial cells, is considered to be neuroprotective [175–177]. Cannabinoid receptors are now considered promising therapeutic targets for fighting neurodegenerative diseases [178–180]. A review of the role of A2AR-containing heteromers in neurodegenerative events and microglia activation is provided in [57]. The A2AR regulates several functions derived from microglial activation. 

First of all, A2AR activation modulates microglial motility [181]. Furthermore, in mixed glial cultures (astrocytes/microglia) we found that activation of the A2AR results in potentiating the release of nitric oxide by activated microglia. The effect was dependent on the presence of astroglia although both A2AR expression and NO synthase-II immunoreactivity were only observed in microglia. 

These actions, which were not detected in cocultures obtained from A2AR KO animals, suggest that the neuroprotection provided by A2AR blockade comes, at least in part, from effects mediated by receptors expressed in activated microglia [129]. Another action of A2AR antagonists results from negative crosstalk when A2A and CB2 receptors are expressed as heteromers [54,182]. 

By interprotomer interactions within the heteromer, activation of A2AR partially blocks CB2R-mediated signaling, which in microglia leads to the production of neuroprotective factors. Therefore, blocking A2AR would reduce the expression of pro-inflammatory mediators (via the A2AR) and release the brake for CB2R activation, leading to the production of neuroprotective molecules (Figure 2B). Studies in the hippocampus also identified A2AR as modulating the recruitment and activation of microglia [102]. In experiments performed in a microglial cell line, A2AR antagonists decrease the proliferation of activated microglia and the release by these cells of brain-derived neurotrophic factor (BDNF) [182]. 

A review of the potential of targeting microglial A2AR to combat neurodegenerative diseases is found in [183]. Other AR types may participate in adjusting the activation of microglia related to neurodegenerative diseases but they seem of less relevance than the A2AR. Whereas the A3R is expressed in microglial cells [184], a recent paper shows the action of A2AR antagonists and A1R agonists on the production of pro-inflammatory cytokines [185]. 

What is now necessary is to address the expression of AR types in resting, and in activated M1 and M2 microglia and to address the mechanisms of skewing to the M2 phenotype targeting AR and AR-containing heteromers.

Author Contributions: R.F. and G.N. designed the paper. I.R.-R., A.L., and R.R.-S. scanned databases to select ad hoc papers and group them according to the title of the different sections. R.F. wrote sections 1 to 5 and G.N. 6 to 8. All authors edited the manuscript and approved the final version. All authors have read and agreed to the published version of the manuscript.

Funding: This work was in part supported by MCIU/AEI grant #RTI2018-094204-B-I00 and SAF2017- 84117-R from the Spanish "Ministerio de Ciencia, Universidades e Investigación" and Spanish "Agencia Estatal de Investigación" (it includes EU FEDER funds). The research group of the University of Barcelona is considered of excellence (group consolidate #2017 SGR 1497) by the Regional Catalonian Government, which does not provide any specific funding for reagents or payment of services or Open Access fees).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Data in Table 1 is directly retrievable from https://www.proteinatlas. org/search/adenosine+receptor (Accessed on 12 April 2021).

Conflicts of Interest: Authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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