Neuroprotective And Immunomodulatory Action Of The Endocannabinoid System Under Neuroinflammation Part 3

The development of LTP or LTD depends on whether the frequency of the stimulation is higher than the threshold frequency [99], with the postsynaptic rise of Ca2+ as a main determinant of the development of LTD or LTP. 

Development and memory are two closely related aspects, and the relationship between them is very important. A person's body and brain need to develop continuously during the growth process, and memory is also improved and developed in this process.

In the process of a person's growth, physical development precedes brain development. Children need to get rid of their dependence on adults and learn to walk, stand, and even play ball on their own. These activities comprehensively promote the growth and development of all aspects of the body, and further, they also affect the development of the brain. When we exercise, the body releases many growth hormones, which are not only beneficial to the growth of the body but also promote the development and connection of brain neurons. In addition, physical exercise can improve people's immunity and endurance, which helps to better cope with various pressures and challenges.

In addition to physical development, memory is also a very important aspect of children's growth. Children's brains are constantly learning new knowledge as they grow, from basic colors, shapes, and numbers to language, social skills, and subject knowledge, all of which need to be constantly remembered and used. Studies have shown that when children learn new knowledge, their brains will generate new neurons and connections, which will continue to strengthen and consolidate, thereby improving memory and learning efficiency.

To improve children's memory, physical exercise is an indispensable factor. Appropriate physical exercise can strengthen the connection between the body and the brain, improve blood circulation and oxygen supply, and promote brain neuron activity. In addition, concentration, active learning and application of new knowledge, good living habits, and adequate sleep can improve memory, which are all factors to pay attention to.

In the process of children's growth, development and memory are both key points. The mutual promotion between the two and positive living habits can help to better realize the potential of children. Therefore, we need to pay attention to and attach importance to the comprehensive development of children's bodies and brains, give them more attention and support, and let them thrive in a better environment. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche deserticola can improve blood flow and promote oxygen delivery, which can ensure that the brain obtains adequate nutrition and energy, thereby improving brain vitality and endurance.

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LTD and LTP are well-characterized molecular mechanisms underlying learning and memory formation and are induced in experiments with high- or low-frequency stimulations (repetition of hundreds of pre- or postsynaptic spikes). 

In the striatum of rodents, spike-timing-dependent potentiation (STDP), a phenomenon describing the dependence of the strength of synaptic transmission on the timing between the neuron's output and input action potentials (spikes), is observed for 75–100 pairings, disappears for 25–50 pairings and re-emerges for 5–10 pairings. 

STDP that is induced by very few pairings is independent of NMDA receptors but mediated by 2-AG and AEA, acting on both CB1 and TRPV1 [100].

Figure 5. The stimuli involved in the establishment of synaptic plasticity and retrograde eCB signaling in the glutamatergic synapse. Glutamate release from the presynaptic terminal activates ionotropic (NMDA, AMPA, and kainate) and metabotropic glutamate receptors (mGluRs), which together with the Ca2+ influx through VGCC are potent triggers for eCB production (indicated in green). 

Ca2+ influx through NMDA receptors is involved in the regulation of AMPA receptor trafficking, spine enlargement, and plastic changes in the strength of the synaptic transmission (indicated in purple). AC, adenylate cyclase; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; CaM, calmodulin; CaMKII, calcium-calmodulin dependent protein kinase II; Cdc42, cell division control protein 42 homolog; DAGL, diacylglycerol lipase; EAATs, excitatory amino acid transporters; eCBs, endocannabinoids; Glu, glutamic acid; IP3, inositol 3-phosphate; Kir, inward-rectifier potassium channel; MAPK, mitogen-activated protein kinase; mGluRs, metabotropic glutamate receptors; NAT, N-acyltransferase; NMDA, N-methyl-D-aspartate receptor; PI3K, phosphatidylinositol 3-kinase; PL, phospholipase; RhoA, Ras homolog family member A; VGCC, voltage-gated calcium channels.

5.2. Endocannabinoid-Mediated Synaptic Plasticity

Stimulation of postsynaptic neurotransmitter receptors and sustained Ca?+ influx is the potent trigger for the production of eCBs and their congeners 78,101] in a neuronal activity-dependent manner. 

The released neurotransmitter activates ionotropic neurotransmitter receptors, Gg-coupled metabotropic receptors (group I of metabotropic glutamate receptors,mGluRs, dopamine receptors D2, M1, and M3 muscarinic acetylcholine receptors, M1/M3 mAChRs), and/or voltage-gated calcium channels. This induces a burst of Ca2+ in the postsynapse and the production of eCBs, primarily AEA and 2-AG, which via CB1 and CB2 receptors modulate the presynaptic release of neurotransmitters. 

Thus, the generation of AEA and 2-AG in the brain shows spatial variations and depends on the complement of neurotransmitter receptors on certain neurons. Endocannabinoid-mediated synaptic potentiation can be realized in neurons receiving/sending inputs via taken-activated synapses (homosynaptic) or in other neurons that do not directly contact taken-activated synapses (heterosynaptic). 

Endocannabinoid-mediated long-term depression (eCB-LTD). CB1 and CB2 are Gi/0 protein-coupled receptors that, upon ligand binding, inhibit adenylate cyclase activity and cAMP production, negatively regulate voltage-gated calcium channels (VGCC), and activate inwardly rectifying potassium (Kir) channels and MAP kinase. 

This decreases the Ca2+ influx into the presynaptic terminal, lowers the probability of Ca2+-dependent fusion of synaptic vesicles, and attenuates the presynaptic neurotransmitter release. 

Synaptic vesicle recycling is a highly dynamic multistep process mediated by SNARE-proteins and other regulatory proteins, with Ca2+ influx being a key factor for docked and primed synaptic vesicles to enter the fusion step [102]. Together with the activity-dependent production of eCBs and activation of CB1, presynaptic activity is essential for this type of plasticity as it determines the afferents in which eCB-LTD will be induced [103]. Presynaptic activity dependence is mediated by presynaptic NMDA autoreceptors that detect the release of glutamate. 

Thus, timing-dependent-LTD is induced only under coincident activation of presynaptic NMDA and CB1 receptors [104]. Unlike LTP, which lasts from minutes to hours, short-term plasticity (STP) is maintained for tens of milliseconds to a few minutes at a maximum. Depolarization-induced suppression of inhibition (DSI)/depolarization-induced suppression of excitation (DSE). 

DSI/DSE is a form of STP realized in the inhibitory (GABAergic) and excitatory (glutamatergic) synapses, respectively, and is induced by depolarization of the postsynaptic terminal and Ca2+ influx through VGCC. Pharmacological experiments favor a role for 2-AG rather than AEA or Aladin ether (2-arachidonic glyceryl ether) as the relevant endocannabinoid to elicit DSE [105]. 

Glutamate spillover may profoundly affect network excitability by shifting the duration of eCB-mediated inhibition at GABA synapses. Metabotropic glutamate receptors are involved in the control of the duration of DSI, most likely through heterologous desensitization of CB1 [106]. Thus, glutamate-mediated excitotoxicity can significantly modify the establishment of various forms of synaptic plasticity and balance in glutamate-/GABAergic signaling [47]. 

Synaptically-evoked (or metabotropic-induced) suppression of inhibition/excitation (SSE/SSI). SSE/SSI is a form of STP, driven by activation of postsynaptic metabotropic (Gq/11-coupled) neurotransmitter receptors, subsequent activation of phospholipase C (PLC) and DAGL with generation of 2-AG and suppression of neurotransmitter release via presynaptic CB1 receptors [107]. 

If the synaptic stimulation is profound, produced eCBs can reach more distant sites and mediate the plasticity heterosynaptically. TRPV1-mediated synaptic plasticity. TRPV1-mediated synaptic plasticity demonstrates the interplay between the eCB and endo vanilloid system. This type of synaptic plasticity is mediated by the binding of AEA to TRPV1 at the postsynapse and results in Ca2+ - calcineurin and clathrin-dependent internalization of AMPA receptors, which provoke LTD of excitatory transmission [108,109]. 

TRPV1 integrates the sensation of physical and chemical stimuli and is activated by temperatures greater than 43 ◦C, acidic conditions, vanilloids like capsaicin, or endocannabinoids such as AEA, N-arachidonoyl dopamine, and N-oleoyl dopamine. TRPV1 is well known for its role in the transmission of neuropathic (inflammatory) pain. 

At the same time, TRPV1 mediates LTD in the hippocampus, and 12- (S)-hydroperoxy eicosatetraenoic acid (12-(S)-HPETE), an endogenous eicosanoid released during synaptic stimulation, acts at TRPV1 receptors to trigger LTD [110].

It is hypothesized that eCB-mediated LTP is induced only when massive Ca2+ rise is observed and high levels of 2-AG are produced (i.e., following simultaneous activation of several postsynaptic neurotransmitter receptors, TRPV1, VGCC) [100]. 

The observations that2-AG and AEA mediate different forms of plasticity with the involvement of CB1, TRPV1, and mGluRs receptors depending on the brain region, characterize the eCB system as a polymodal signal integrator that allows the diversification of synaptic plasticity in a single neuron [111]. 

The interference of NAEs and other eCB congeners with enzymatic degradation or endocannabinoid signaling suggests their role in tuning the activity of primary eCBs. NAEs, monoacylglycerols, and certain N-acyl neurotransmitters compete with AEA or 2-AG for FAAH-mediated degradation, thus extending their lifetimes [112] and their ability to interact with cannabinoid receptors. 

This "entourage effect" of the non-cannabinoid 2-acylglycerols and NAEs may serve as an additional fine regulator of cannabinoid activity. The overall interplay and metabolism of endogenous ligands of CB1/2, TRPV1, GPR55, and GPR18 is now integrated into the "endocannabinoidome" and demonstrates that this system is an essential player not only in many aspects of behavior, cognition, and memory but also mediates inherent protective mechanisms of the neuro-immune interface.

6. Neuroinflammation-Induced Synaptopathy and Neurodegenerative Diseases

Neuroinflammation is a common feature of acute and chronic neurodegenerative disorders such as Alzheimer's and Parkinson's disease, viral infections of the CNS, stroke, paraneoplastic disorders, traumatic brain injury, and multiple sclerosis. 

Neuroinflammation is typically characterized by the activation of immunocompetent glial cells (microglia and astroglia), the release of cytokines, prostaglandins, and reactive oxygen species, the impairment of the blood-brain-barrier (BBB) integrity and resultant infiltration of peripheral immune cells.

6.1. Microglia

Microglia are residential innate immune cells that perform primary immune surveillance and macrophage-like activities of the CNS. In a non-stimulated state, microglia contribute to CNS development and maintain tissue homeostasis by supporting neuronal survival, cell death, and synaptogenesis [113]. However, microglial cells can be activated by various pathological stimuli during infections, brain trauma, stroke, and neurodegeneration [114]. 

Activated microglia are characterized by increased proliferation and the production and secretion of a wide spectrum of immune mediators such as cytokines, chemokines, prostaglandins, and reactive oxygen intermediates [115–118]. The production of cytokines and chemokines can facilitate the recruitment of peripheral leukocytes into the brain [119]. 

During neuroinflammation, activated microglia migrate to the site of injury or infection and perform pivotal immunological functions such as phagocytosis of invading microorganisms and removal of dead or damaged cells [120,121]. 

However, chronic activation of microglia is generally considered to be detrimental to neuronal health [122]. In response to activation, microglia can polarize to either a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype, although microglial activation states have been recognized to be more complex [123]. 

Various stimuli induce the classical M1 activation state of microglia such as LPS, interferon (IFN)-γ, amyloid β (Aβ), and α-synuclein [118,124–126]. Toll-like receptor 4 (TLR4), a member of the pattern recognition receptor family, mediates innate immunity and is abundantly expressed in microglia [127]. TLR4-dependent microglial activation has been observed in various neurodegenerative diseases like Alzheimer's disease (AD) and Parkinson's disease (PD) [128,129]. 

In addition, TLR4 is also responsible for chronic neuroinflammation after stroke and spinal cord injury leading to brain damage [130]. TLR4 can be activated by multiple pathogen-associated molecular patterns (PAMPs), such as LPS which is a major component of the outer membrane of Gram-negative bacteria [130]. 

LPS is one of the most extensively studied TLR4 ligands to understand the mechanism of microglial activation in neurodegeneration [127]. In addition to the above-mentioned pro-inflammatory response, microglia can also adopt an anti-inflammatory M2 phenotype. 

After M1 microglia attack invading organisms to limit tissue damage, anti-inflammatory M2 microglia are involved in phagocytosis of cellular debris and wound healing [123]. Another more immuno-suppressive phenotype is induced by the cytokines IL-10 and TGF-β, or by apoptotic cells [123]. Microglia are a crucial source of AEA and 2-AG under basal conditions and during neuroinflammation [75–77]. 

When stimulated with ATP (released from damaged tissue) microglia produce 2-AG themselves [77]. 

CB2 receptor expression is upregulated in microglia stimulated with pro-inflammatory cytokines [131], indicating a significant role of CB2 in the regulation of neuroinflammatory states. Further, it was demonstrated that 2-AG and PEA affect microglial cells and lead to a decrease in the number of damaged neurons after excitotoxic lesions in organotypic hippocampal slice cultures. 

2-AG activated the abnormal cannabidiol (ABN-CBD) receptor and PEA was shown to mediate neuroprotection via PPAR-α [132]. The CB2 agonist AM1241 has also been shown to attenuate microglia activation by reducing the expression of the inducible nitric oxide synthase and shifting their phenotype from M1 to M2 [133]. 

Microglia constantly scan their environment and due to the long processes directed towards synapses can monitor and respond to the functional status of synapses. 

Microglia contribute to neuronal circuit maturation and are involved in both synapse induction and elimination. 

During neuroinflammation, prolonged activation of microglial cells attracted to lesion sites can exacerbate neuronal damage. It is anticipated that 2-AG, produced by overstimulated neurons, induces microglial migration [76] and proliferation [134].

6.2. Astrocytes

Astrocytes are the largest and most abundant group of glial cells in the CNS and play a vital role in regulating CNS homeostasis, synaptic transmission and plasticity, and neuroprotective effects. Glia responds to neuronal activity with the elevation of their internal Ca2+ concentration, which triggers the release of mediators of glial origin. 

The term 'tripartite synapse' describes the bidirectional communication between astrocytes and neurons, where nearby astrocytes respond to synaptic activity and, vice versa, regulate synaptic transmission and plasticity [135]. 

Perisynaptic Schwann cells and synaptically associated astrocytes are viewed as integral modulatory elements of tripartite synapses. CB1 activation in astrocytes amplifies Ca2+ influx and promotes the release of gliotransmitters, like glutamate (gliotransmission), which modulate the target response at pre and postsynaptic sites [24]. 

Impairment of spatial working memory and in vivo long-term depression (LTD) of synaptic strength at hippocampal CA3-CA1 synapses, induced by acute exposure to exogenous cannabinoids, is due to the activation of astroglial CB1 and is associated with astroglia-dependent hippocampal LTD [136]. 

Due to their proximity to blood vessels and other resident cells within the CNS such as neurons, microglia, and oligodendrocytes, astrocytes play a crucial role in BBB maintenance and permeability. In addition, astrocytes are involved in modulating the innate immune response by regulating inflammatory factors, such as cytokines, chemokines, complement fragments, reactive oxygen, or reactive nitrogen species. 

However, dysfunctional astrocytes seem to play an important role in the onset of neurodegenerative diseases such as AD and ALS (reviewed in [137]). Astrocytes become activated or reactivated during various pathological conditions such as stroke, trauma, tumor growth, and neurodegenerative diseases. Following the M1/M2 phenotype classification of microglia and macrophages, neuroinflammation can induce two types of reactive astrocytes, termed A1 and A2 [138]. 

In A1 reactive astrocytes the pro-inflammatory NF-κB pathway is upregulated leading to the release of complement factors [139] that are destructive to synapses and to the secretion of neurotoxins and pro- and anti-inflammatory mediators such as PGD2, IFN-γ, TNF-α, IL-1-β, and TGF-β, respectively [140–142]. 

In A2 reactive astrocytes enhanced STAT3 activity has been observed [143]. Moreover, A2 reactive astrocytes can upregulate many neurotrophic factors such as thrombospondins [144] and brain-derived neurotrophic factor (BDNF) [145,146], which promote either survival and growth of neurons or synaptic repair. Accumulating evidence supports the role of astrocytes as a source of eCBs. 

It has been shown that astrocytes have the potential to produce 2-AG in response to ATP [147], endothelin [148], and CB1 receptor activation [75,149]. Furthermore, the secretion of AEA, homo-gamma-linolenylethanolamide (HEA), and docosatetraenoylethanolamide (DEA) by activated mouse astrocytes has been confirmed [75]. 

Of note, not only microglial cannabinoid receptors but also astroglial CB1 and CB2 receptors play critical roles in the response to neuroinflammation [33]. Activation of astroglial CB1 receptors protects against ceramide-induced oxidative stress and apoptosis [150,151] and activation of both CB1 and CB2 receptors seems to prevent LPS-induced nitric oxide (NO) release by cultured astrocytes [152]. 

Accordingly, 2-AG has been shown to maintain glutamine synthase expression in astrocytes in a MAPK-dependent manner and thus to protect astrocytes from LPS exposure [153]. 

2-AG also seems to reduce the astrocytic production of chondroitin sulfate proteoglycan, which accumulates in MS lesions and is thought to be linked to the failure to regenerate, impeding oligodendrocyte precursor cell differentiation, and neuronal growth [154]. 

Moreover, 2-AG protects astrocytes exposed to oxygen-glucose deprivation through a blockade of NDRG2 signaling and STAT3 phosphorylation [155]. 2-AG and PEA have been shown to attenuate amyloid β-induced astrocyte activation and PEA increased 2-AG production in astrocytes [156–158]. PEA is also suggested to improve neuronal survival by possibly counteracting reactive astrogliosis [159,160]. 

PEA- and OEA-mediated inhibition of astrocyte activation seems to involve PPAR-α [161,162]. Furthermore, AEA has been shown to elicit glutamate release through astrocytic CB1 receptor activation in the core of the nucleus accumbens in rats [163]. 

Astrocytes isolated from mice with acute experimental autoimmune encephalomyelitis (EAE) exhibited reductions in all endocannabinoid metabolism-associated genes, except Faah, which persisted in chronic disease and was associated with reduced Cnr1 transcript levels both at acute and recovery phases [164]. 

Astrocytic- together with neuronal MAGL seems to be responsible for converting 2-AG to prostaglandins and thus protects the nervous system from excessive CB1 receptor activation [165].

6.3. Cytokines Involved in Neuroinflammation

Besides microglia and astrocytes, endothelial cells and other glial cells, may produce cytokines and chemokines. Common cytokines that are produced in response to brain injury or during neurodegenerative diseases able to induce neuronal cytotoxicity are IL-6, IL-1β, and TNF-α [166]. Moreover, sustained release of these cytokines leads to a compromised BBB [167]. 

Subsequently, peripheral immune cells such as macrophages, neutrophils, monocytes, T cells, and B cells can migrate into the brain. This process exacerbates and contributes to chronic neuroinflammation and neurodegeneration. 

For instance, following traumatic brain injury, IL-1β induces neuronal apoptosis, BBB breakdown, and recruitment of immune cells, as well as the production of pro-inflammatory mediators [168]. Moreover, during spinal cord injury, the secretion of pro-inflammatory mediators including IL-1β, inducible nitric oxide synthase (iNOS), IFN-γ, IL-6, IL-23, and TNF-α is followed by the activation of local microglia and attraction of various immune cells such as naive bone-marrow derived macrophages [169]. 

Upon infiltration of the injured site, macrophages undergo phenotype switching from M2 phenotype to M1-like phenotype. Noteworthy, normal aging is often associated with an increased number of activated microglia in the brain which are involved in altered synaptic plasticity mechanisms in the hippocampus, including LTP and thereby reduce memory performance [170]. 

Moreover, aged brains show homeostatic imbalance between anti-inflammatory and pro-inflammatory cytokines increasing the risk for neurodegenerative diseases such as AD. 

Although the pro-inflammatory cytokines may cause cell death and tissue damage, they are also involved in tissue repair [171]. For example, TNF-α causes neurotoxicity at an early stage but contributes to tissue growth at later stages of neuroinflammation. 

Several studies have highlighted the regulatory effects of the ECS on neuroinflammatory conditions by modulating the production of cytokines. For instance, 2-AG was shown to prevent the overexpression of TNF-α, IL-1β, and iNOS in a murine model of SO2-induced brain inflammation [172]. 

MAGL deficiency leading to increased 2-AG levels also reduced brain PGE2 and pro-inflammatory cytokine levels following peripheral LPS administration in mice [173]. 

Selective pharmacologic inhibition of ABHD6 diminished cytokine and chemokine production in a murine model of neuropathic pain [174], and the MAGL inhibitor CPD-4645 significantly reduced IL-1β and IL-6 brain levels after systemic LPS challenge [175]. 

The FAAH inhibitor URB597 attenuated increased TNF-α and IL-1β levels in the hippocampi of aged mice [176] and decreased Iba-1, TNF-α, IL-6, and monocyte chemoattractant protein-1 (MCP-1) levels in the hippocampus of ethanol-exposed rats [177]. 

The FAAH inhibitor PF3845 increased levels of AEA, OEA, and PEA in the frontal cortex and hippocampus of rats [178]. Furthermore, this increase in FAAH substrate levels was associated with a robust attenuation in TNF-α, IL-6, and IL-1β levels in the prefrontal cortex. 

PEA has been shown to reduce pro-inflammatory cytokines after traumatic spinal cord [179] and brain injury [180,181], in a model of sciatic nerve crush [182], in Parkinson's disease models [183,184] and MS patients [185]. Further, OEA administration significantly reduced plasma and brain TNF-α levels after LPS application [186] and SEA was recently shown to suppress increased TNF-α and TGF-β1 levels in the prefrontal cortex of LPS-challenged mice [47]. CB2 receptor agonism has been shown to decrease brain levels of pro-inflammatory cytokines induced by LPS application [187], intracerebral hemorrhages [188], or surgery [189] as well as in a model of PD [190]. 

At the same time, the CB1 receptor inverse agonist SR141716A (rimonabant) and the CB2 receptor antagonist SR144528 significantly reduced LPS-induced IL-1β production in the brain [191] whereas SR141716A was also shown to increase pro-inflammatory cytokines in an EAE model [192]. The neuroprotective effect of SR141716A was shown in the retinal degeneration model [193] and permanent photothrombotic cerebral ischemia [194]. 

These findings indicate that manipulation of CB1 or CB2 receptors may have therapeutic value in neuroinflammation; however, due to the complexity of the ECS, this concept remains to be carefully considered.

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