Neuroprotective And Immunomodulatory Action Of The Endocannabinoid System Under Neuroinflammation Part 2

N-acyl neurotransmitters and lipo amino acids represent a separate cluster within the endocannabinoidome, that bears the potential for the identification of novel receptor targets in this system. 

Acyl neurotransmitters are a type of neurotransmitter synthesized from glutamate and acetyl coenzyme A. They are widely distributed in the brain and are involved in a variety of physiological and pathological processes, such as cognitive function, attention, inhibition, etc. In recent years, more and more studies have shown that acyl neurotransmitters are closely related to memory.

First, acyl neurotransmitters play an important role in the memory process. It participates in synaptic transmission between neurons and regulates the excitability and inhibition of neurons, playing an important role in memory formation, storage, and retrieval. Studies have found that the level of acyl neurotransmitters is positively correlated with memory, and when its level increases, memory also improves.

Secondly, acyl neurotransmitters are closely related to cognitive function. In cognitive tasks, acyl neurotransmitters regulate the excitability and inhibition of neurons by regulating calcium ion channels in the presynaptic membrane and calcium ion transmission in the postsynaptic membrane, thereby affecting the performance of cognitive function. Studies have shown that when the level of acyl neurotransmitters decreases, cognitive function also decreases.

In addition, acyl neurotransmitters are also related to emotional regulation. Studies have shown that acyl neurotransmitters can affect emotional states and emotional regulation by regulating the levels of neurotransmitters such as dopamine, serotonin, and norepinephrine. When the level of acyl neurotransmitters increases, emotional stability will also increase.

In short, acyl neurotransmitters are closely related to memory, cognitive function, and emotional regulation. We can increase the level of acyl neurotransmitters through proper diet, exercise, and rest, thereby promoting the improvement of memory and cognitive function, and maintaining emotional stability and a happy state of mind. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidative and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

Click now improving brain function

Among these compounds, N-acyldopamines are CB1 and TRPV1 agonists, while N-acylserotonines are TRPV1 antagonists and N-arachidonoyl-γ-aminobutyric acid (NAGABA) activates the GPR92 receptor. The liberation of free neurotransmitters following the degradation of N-acyl neurotransmitters makes their action more complex.

3.3. Involvement of Endocannabinoid System in Response to Neuropathology

Endocannabinoids and related NAEs are produced on demand and play a crucial regulatory role in metabolic processes, behavior, and immunity. Under healthy conditions, these lipid mediators are most abundant in the brain and barely found in circulation and peripheral tissues [55]. 

During various pathological conditions of the CNS, the profiles of eCBs and their congeners undergo significant changes, which are associated with the inflammation-modulating, analgesic, and neuroprotective activity of these compounds (summarized in Table 2).

Figure 3. Signaling pathway and anti-inflammatory actions of NAEs. Arachidonoylethanolamide (AEA) signals via the cannabinoid receptors CB1/2, the non-cannabinoid G protein-coupled receptor (GPR)55, the transient receptor potential cation channel subfamily V member 1 (TRPV1), and the peroxisome proliferator-activated receptor (PPAR)-α and γ. 

Palmitoylethanolamide (PEA) has been shown to inhibit the fatty acid amide hydrolase (FAAH) and to signal via GPR55, TRPV1, and PPAR-α, while CB1/2 binding is still controversial. 

Oleoylethanolamide (OEA) activates TRPV1 and PPAR-α, while stearoylethanolamide (SEA) inhibits FAAH and activates PPAR-α. Linoleylethanolamide (LEA) was shown to activate TRPV1, GPR119, and to inhibit FAAH, while docosahexaenoylethanolamide (DHEA) signaling involves the activation of GPR110.

4. Glutamate Receptor-Mediated Neurotoxicity

4.1. Glutamate as a Major Excitatory Neurotransmitter in Mammals and Potential Neurotoxin

Activation of postsynaptic neurotransmitter receptors and Ca2+ influx into the postsynaptic terminal induce the synthesis of eCBs and related compounds. This activity-dependent production of eCBs is essential for the fine regulation of neurotransmission. 

In the mammalian brain, glutamate is the main excitatory neurotransmitter implicated in learning and memory formation. Glutamatergic neurotransmission mediates synaptic plasticity, whereby ionotropic and metabotropic (mGluRs) glutamate receptors play a primary role. 

Between the quantal neurotransmitter releases, the level of glutamate in the synaptic cleft is estimated to be <1 µM. This low basal level is maintained by rapid reuptake of glutamate from the extracellular space into the cytosol by high-affinity glutamate transporters EAATs (excitatory amino acid transporters). EAATs are localized on neurons (primarily EAAT4 and EAAT3 (EAAC1, Excitatory Amino Acid Carrier)) and astrocytes (primarily glutamate transporter GLT-1 and glutamate-aspartate transporter GLAST), and co-transport one molecule of L-glutamate (or L-/D-aspartate) with 3Na+ and 1H+ in exchange of 1K+ [66]. 

The dependence of this transport system on Na+ and K+ gradients across the plasma membrane makes it highly vulnerable to ATP depletion with subsequent inhibition of glutamate uptake or reversal of transporters [67]. 

Another factor affecting the efficiency of glutamate removal from the synaptic cleft is translational control of EAATs or post-translational modification of the transporter molecules, which affects the level of active transporters. Glutamate is further accumulated in the synaptic vesicles via vesicular glutamate transporters VGLUTs using the ∆µH + gradient. 

Considering the high energy-dependent compartmentalization of glutamate and its gradient across the synaptic bouton, i.e., from synaptic vesicles (~200 mM [68]) to the synaptic cleft (<1 µM between release events, around 1 mM during the peak of SV release [69]), any factors affecting the efficiency of high-affinity glutamate uptake represent a potential risk of neurotoxic neuronal damage. 

The pathophysiological conditions underlying the long-term glutamate rise in the synaptic cleft and extrasynaptic glutamate spillover are traumatic brain injury, ischemia, and other causes of hypoxia, stroke, and oxidative stress lead to the transition of the significant portions of EAATs to the reverse mode when glutamate is released from the cytosol to the extracellular space. Glutamate-mediated neurotoxicity originates from overstimulation of ionotropic glutamate receptors, primarily N-methyl-D-aspartate (NMDA) receptors, and massive Ca2+ flux to the postsynaptic terminal. 

High extracellular concentrations of glutamate lead to the prolonged co-activation of synaptic and extrasynaptic (localized to non-synaptic sites) NMDA receptors, glutamate-mediated neurotoxicity [70], and are involved in the pathogenesis of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease. Due to Ca2+ permeability and high affinity for glutamate, NMDA receptors are among the primary molecular targets implicated in the pathogenesis of excitotoxicity. 

At resting membrane potential, the current through channels of NMDA receptors is almost fully blocked by Mg2+ preventing the conductance between the stimuli. During quantal neurotransmitter release, two conditions for Ca2+ influx through NMDA receptors are met: * glutamate concentrations rise rapidly, and ** the depolarization of synaptic membranes removes the Mg2+ block from NMDA receptor channels. 

Therapeutic concentrations (1–10 µM) of the NMDA receptor antagonist memantine, used for the treatment of Alzheimer's disease, preferentially block extrasynaptic rather than synaptic currents through NMDA receptors in the same neuron [71]. 

The mode of memantine action enables effective prevention of excessive extrasynaptic NMDA receptor stimulation, with much less effect on NMDA receptor-mediated synaptic activity when glutamate is elevated for only milliseconds [72]. 

Under the prominent rise of intracellular Ca2+ levels, vesicular glutamate release is another factor contributing to elevated extracellular glutamate concentration and excitotoxic damage. 

In rats and mice, ischemic conditions, followed by release of axonal vesicular glutamate into the peri-axonal space under the myelin sheath, trigger activation of myelinic GluN2C/D-containing NMDA receptors [73], which are generally extrasynaptic [74].

4.2. Excitotoxicity as a Prerequisite and Consequence of Neuroinflammation and Neurodegeneration

Due to the ability of Ca2+ to activate a range of enzymes, glutamate receptor-mediated excitotoxicity provokes necrotic and apoptotic neuronal death. The massive influx of Ca2+ overloads the intracellular buffer systems for this ion, provokes mitochondrial dysfunction, and activation of a range of proteases, including caspases and calpain, leading to the subsequent degradation of components of the neuronal cytoskeleton and the release of apoptotic factors. One example of the active involvement of the ECS in mediating cellular communication is the functional coupling of microglia and synapses during normal synaptic activity as well as excitotoxic injury. 

As previously suggested, microglia are a crucial source of de novo-produced AEA and 2-AG under basal conditions and during neuroinflammation [75–77]; however, high glutamate application induces a prominent 2-AG overproduction in neurons [76,78]. 

Under these conditions, the neuronal production of AEA increases only slightly, while the production of two putative endocannabinoids, homogamma-linolenylethanolamide and docosatetraenylethanolamide remains unchanged [76]. 

LPS-induced systemic inflammation in mice is accompanied by an increase in basal glutamate levels in the prefrontal cortex [47]. Elevated glutamate may originate from both inflammation-associated decrease in uptake, and neuronal and non-neuronal (from astrocytes and microglia) glutamate release. Glutamate flow favors the spatial cooperation between dendritic spines and ramified microglial cells and induces microglial process extension toward neurons (Figure 4). 

2-AG induces chemokinesis (random motion increased by a chemical stimulus) and chemotaxis, (directed cell migration along a chemical gradient) in microglial cells [76,79]. 

In line with this, activated microglia express CB2 receptors at the leading edge of their motile protrusions [76]. Arachidonylcyclopropylamide (ACPA)-induced migration of BV-2 microglia could be blocked by the highly selective CB2 antagonist SR145528 [80]. Similarly, the migratory responses towards 2-AG and the synthetic cannabinoid CP 55,940 were inhibited by CB2 receptor antagonism [76,79]. 

We hypothesize that eCBs released at sites of synaptic activity (or injury) may act as chemoattractants to recruit microglia in a CB2-dependent manner, toward neuroinflammatory lesion sites. Moreover, in organotypic hippocampal slice cultures, 2-AG mediated neuroprotection against NMDA-induced excitotoxicity by acting explicitly on abnormal-cannabidiol (ABN-CBD)-sensitive receptor, putative GPR18, on microglial cells [81].

cannabinoids produced in the glutamatergic synapse under excitotoxic conditions attract microglial cells into proximity to spines. By adopting a pro-inflammatory or pro-survival phenotype microglia largely define the fate of the injured cells and spines. eCB signaling decreases the presynaptic neurotransmitter release, supports the glutamate-glutamine cycle, and balances glutamate/GABAergic transmission. 

AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; BDNF, brain-derived neurotrophic factor; CB, cannabinoid receptor; EAATs, excitatory amino acid transporters; eCBs, endocannabinoids; ER, endoplasmic reticulum; GDNF, glial cell line-derived neurotrophic factor; Gln, glutamine; Glu, glutamic acid; IFNγ, interferon-gamma; IL, interleukin; mGluRs, metabotropic glutamate receptors; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate receptor; NO, nitrogen monoxide; PGD2, prostaglandin D2; QUIN, quinolinic acid; Figure 4. 

The development of synaptic dysfunction and the involvement of the ECS in neuroprotective responses. Endocannabinoids produced in the glutamatergic synapse under excitotoxic conditions attract microglial cells into proximity to spines. 

By adopting a pro-inflammatory or pro-survival phenotype microglia largely define the fate of the injured cells and spines. eCB signaling decreases the presynaptic neurotransmitter release, supports the glutamate-glutamine cycle, and balances glutamate/GABAergic transmission. 

AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; BDNF, brain-derived neurotrophic factor; CB, cannabinoid receptor; EAATs, excitatory amino acid transporters; eCBs, endocannabinoids; ER, endoplasmic reticulum; GDNF, glial cell line-derived neurotrophic factor; Gln, glutamine; Glu, glutamic acid; IFNγ, interferon-gamma; IL, interleukin; mGluRs, metabotropic glutamate receptors; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate receptor; NO, nitrogen monoxide; PGD2, prostaglandin D2; QUIN, quinolinic acid; ROS, reactive oxygen species; TGFβ, transforming growth factor-beta; TNFα, tumor necrosis factor alpha; VGCC, voltage-gated calcium channels.

There is an activity-dependent modification of microglia–synapse contacts in vivo. The ischemic brain is characterized by the markedly prolonged contact time between microglial processes and synaptic structures and the wrapping of microglial processes around the synapse, followed by the disappearance of presynaptic boutons [82]. 

One of the mechanisms by which microglia eliminate presynaptic boutons and axons is trogocytosis [83], a process described in the immune system as a non-apoptotic mechanism for the capture of membrane components that differs from phagocytosis and involves the engulfment and clearance of cellular structures larger than 1 µm [84]. 

In a mouse model of cortical multiple sclerosis in vivo imaging demonstrated that cortical inflammation disrupts circuit activity, which coincides with a widespread, but reversible, loss of dendritic spines. Under these circumstances, spines displaying local calcium accumulations are eliminated by invading macrophages or resident-activated microglia [85]. 

Acute microglia activation is accompanied by the release of glutamate, quinolinic acid, proinflammatory cytokines (IL-1β, TNF-α, IL-2, IL-6), chemokines-macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1), and free arachidonic acid. 

Quinolinic acid produced exclusively in activated microglia and macrophages, is a NMDA receptor agonist and mediates excitotoxicity during immune response. 

By contributing to the destabilization of the cytoskeleton in astrocytes and endothelial cells, quinolinic acid decreases the integrity of the neurovascular unit and increases the influx of BBB impermeable quinolinic acid from the periphery [86]. 

Produced excitotoxic molecules and proinflammatory cytokines intensify free radical generation and lipid peroxidation, which provoke mitochondrial dysfunction and further exacerbate excitotoxicity. 

Microglia largely define the fate of damaged synaptic contacts and cells and promote the resolution of neuroinflammation and regeneration by releasing brain-derived neurotrophic factor (BDNF) and cytokines with dual (pro- and anti-inflammatory) potential, like TGF-β and IL-10.

5. The Role of Retrograde Endocannabinoid Signaling in the Tuning of Synaptic Strength

5.1. Synaptic Plasticity in Glutamatergic Synapses

When glutamate levels reach a certain concentration in the synaptic cleft, it binds to AMPA receptors and induces Na+ influx, which is registered as excitatory postsynaptic potentials (EPSP) of certain amplitudes. 

Due to the presence of the GluR2 subunit, the majority of AMPA receptors in the CNS are impermeable to Ca2+ [87] and postsynaptic Ca2+ influx triggered by glutamate is mainly mediated by NMDA receptors. The influx of Ca2+ through NMDA receptor channels activates a range of kinases, primarily Ca2+/calmodulin-dependent protein kinase II (CaMKII) [88,89], which in turn activates Rho GTPases, Cdc42, and RhoA [90]. 

This reorganizes the postsynaptic density via * remodeling of the actin cytoskeleton and transient (~5 min) enlargement of the spine (Figure 5); ** enhanced trafficking of AMPA receptors to post-synaptic sites as a result of their redistribution from recycling endosome to the plasma membrane [91,92], and *** increased single-channel conductance of AMPA receptors as a result of direct phosphorylation [93]. 

Synaptic recruitment of Ca2+-permeable AMPA receptors via CaMKI is also suggested to contribute to signaling pathways that drive spine enlargement via actin polymerization [94]. 

Thus, following the repeated cycles of activation, the amplitude of evoked EPSC increases, i.e., is potentiated (long-term potentiation, LTP). This effect is typical for excitatory neurotransmission and persists in synapses depending on the stimulus mode. In contrast, long-term depression (LTD) is a long-lasting drop in the efficiency of synaptic transmission as a result of a decrease in postsynaptic receptor density and/or presynaptic neurotransmitter release. While for the development of LTP, the activation of certain protein kinases is essential, LTD induction is dependent on protein phosphatase activity and target dephosphorylation. 

Prolonged 1 Hz stimulation leads to Ca2+ rise and calmodulin-dependent activation of calcineurin (protein phosphatase 2B, PP2B) [95], which via serine/threonine protein phosphatases PP1 or PP2A, results in the dephosphorylation of AMPA receptors [96], decrease of their channel conductance, and arrest of their recycling [97]. 

Dephosphorylation of the transcription factor cAMP response element binding protein (CREB) in the hippocampal area CA1 in vivo is suggested to be one of the mechanisms through which these protein phosphatases contribute to the prolonged maintenance of LTD [98].

For more information:1950477648nn@gamil.com