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Review
. 2023 Oct 10;12(10):1844.
doi: 10.3390/antiox12101844.

Non-Excitatory Amino Acids, Melatonin, and Free Radicals: Examining the Role in Stroke and Aging

Victoria Jiménez Carretero  1 , Eva Ramos  2 , Pedro Segura-Chama  3 , Adan Hernández  4 , Andrés M Baraibar  5 , Iris Álvarez-Merz  1 , Francisco López Muñoz  6   7 , Javier Egea  8 , José M Solís  9 , Alejandro Romero  2 , Jesús M Hernández-Guijo  1   10
Affiliations
Review

Non-Excitatory Amino Acids, Melatonin, and Free Radicals: Examining the Role in Stroke and Aging

Victoria Jiménez Carretero et al. Antioxidants (Basel). .

Abstract

The aim of this review is to explore the relationship between melatonin, free radicals, and non-excitatory amino acids, and their role in stroke and aging. Melatonin has garnered significant attention in recent years due to its diverse physiological functions and potential therapeutic benefits by reducing oxidative stress, inflammation, and apoptosis. Melatonin has been found to mitigate ischemic brain damage caused by stroke. By scavenging free radicals and reducing oxidative damage, melatonin may help slow down the aging process and protect against age-related cognitive decline. Additionally, non-excitatory amino acids have been shown to possess neuroprotective properties, including antioxidant and anti-inflammatory in stroke and aging-related conditions. They can attenuate oxidative stress, modulate calcium homeostasis, and inhibit apoptosis, thereby safeguarding neurons against damage induced by stroke and aging processes. The intracellular accumulation of certain non-excitatory amino acids could promote harmful effects during hypoxia-ischemia episodes and thus, the blockade of the amino acid transporters involved in the process could be an alternative therapeutic strategy to reduce ischemic damage. On the other hand, the accumulation of free radicals, specifically mitochondrial reactive oxygen and nitrogen species, accelerates cellular senescence and contributes to age-related decline. Recent research suggests a complex interplay between melatonin, free radicals, and non-excitatory amino acids in stroke and aging. The neuroprotective actions of melatonin and non-excitatory amino acids converge on multiple pathways, including the regulation of calcium homeostasis, modulation of apoptosis, and reduction of inflammation. These mechanisms collectively contribute to the preservation of neuronal integrity and functions, making them promising targets for therapeutic interventions in stroke and age-related disorders.

Keywords: aging; free radicals; melatonin; non-excitatory amino acids; stroke.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Positive correlation between fEPSP potentiation and amino acid accumulation represented by the solid straight line of linear regression (R2 = 0.45; p < 0.001). The dashed line indicates the total amino acid content of control slices. Adapted with permission from ref. [157].
Figure 2
Figure 2
Amino acid content of control slices (white boxes) and after the application of AGHQSTU (alanine, glutamine, glycine, histidine, serine, taurine, and threonine; grey boxes). The total amino acid content has been determined as the sum of all amino acids quantified and reflected in this figure, including tyrosine. * p < 0.05; ** p < 0.01; and *** p < 0.001. One-way ANOVA, followed by Tukey’s test for multiple comparisons of parametric data and Kruskal–Wallis test followed by Dunn’s test for multiple comparisons of nonparametric data. The numbers in parentheses indicate the number of slices. Adapted with permission from ref. [165].
Figure 3
Figure 3
The hypoxia-induced depression of synaptic potentials becomes irreversible in the presence of AGHQSTU (alanine, glutamine, glycine, histidine, serine, taurine, and threonine; black symbols). Time course of changes in fEPSP (left panel) and FV (right panel) elicited by a 40 min period of hypoxia (indicated by the horizontal bar). LCRA: artificial cerebrospinal fluid. Adapted with permission from ref. [165].
Figure 4
Figure 4
Graphical representation of the proposed model. (1) During hypoxia, adenosine release inhibits glutamatergic transmission via its A1 receptors. (2) Uptake of non-excitatory amino acids by glial cells (via transporters such as those of the N-system and ASCT2) leads to an increase in cell volume. The activation of volume-regulated anion channels (VRAC) releases excitotoxins (such as glutamate, aspartate, and D-serine) into the already reduced extracellular space. This glutamate is in addition to glutamate released by other pathways during ischemia (synaptic glutamate, reversal of its transporters, etc.). (3) Glutamate and D-serine released into the reduced interstitial space leads to a progressive increase in their extracellular concentration and the activation of NMDA receptors, triggering excitotoxicity phenomena that result in increased cell volume and dendritic beading. Glu: glutamate, Gln: glutamine, D-Ser: D-serine, AA: other amino acids. Image designed with BioRender.com.
Figure 5
Figure 5
Schematic view showing melatonin-mediated actions in stroke. In neuronal cells, glutamate (GLU) interacts with the NMDA receptor (NMDAR), boosting a high intracellular Ca2+ and NO• levels by activating nitric oxide synthase (NOS), which causes a reactive oxygen and nitrogen species (RONS) flux overflow within the cell. Subsequently, melatonin’s ability to easily diffuse allows it to enter neuronal cells, where it binds to calmodulin with high affinity inhibiting CaMKII (Ca2+/calmodulin dependent protein kinase-II), a major mediator of neuronal cell death involved in pathological glutamate signaling, thereby mitigating excitotoxicity damage. Furthermore, radicals are generated as a result of electron leakage from the inner mitochondrial membrane’s electron transport chain; the rogue electrons chemically convert adjacent oxygen molecules to produce the superoxide anion radical (O2•). This reactant either combines with nitric oxide to form the highly oxidizing peroxynitrite anion (ONOO−) or is immediately dismutated by superoxide dismutase 2 (SOD2) to hydrogen peroxide (H2O2). Both the Fenton reaction and the kinetically sluggish Haber–Weiss reaction require a transition metal, such as ferrous iron (Fe2+), to convert H2O2 to the hydroxyl radical (•OH). The •OH damages molecules along with other oxidants, which promotes neuronal cell death. In this regard, melatonin acts as a direct radical scavenger (directly neutralizes •OH and the ONOO−), regulating RONS overload, as well as an indirect agent, boosting antioxidant enzymes such as glutathione peroxidase (GPx) and SOD2, preserving mitochondrial homeostasis and, as a result, enhancing cellular energy efficiency. In addition, melatonin attenuates oxidative stress by stimulating Nuclear erythroid-related factor 2 (Nrf2), the principal transcription factor that regulates antioxidant response element (ARE)-mediated expression of phase II detoxifying antioxidant enzymes. Under normal conditions, Nrf2 is sequestered in the cytoplasm by an actin-binding (Kelch-like) protein (Keap1); on exposure of cells to oxidative stress, Nrf2 dissociates from Keap1, translocates into the nucleus, binds to ARE, and transactivates phase II detoxifying and antioxidant genes. Among the spectrum of antioxidant genes controlled by Nrf2 are catalase, SOD, hemoxigenase-1 (HO-1), and GPx. Melatonin increases Nrf2 gene expression. Melatonin also functions as an anti-inflammatory. Thus, DAMPs, released from damaged or dying cells, promote pathological inflammatory response through toll-like receptors (TLR2/4). In this context, TNF-α acts by binding to its receptor TNFR (TNF receptor), which recruits TRADD (TNF Receptor-Associated Death Domain). This protein binds to TRAF2 (TNF Receptor-Associated Factor-2) to phosphorylate and activate the IKK (I-KappaB-Alpha kinase complex). Then, the IKK complex phosphorylates IKBα, resulting in the translocation of NF-κβ (Nuclear Factor-κβ) to the nucleus where it targets many coding genes for mediators of inflammatory responses. Subsequently, in response to cytokine receptor stimulation STAT3 (Signal Transducers and Activators of Transcription) following tyrosine and JAK2 (Janus-family tyrosine kinases) phosphorylation, dimerize and translocate to the nucleus. In addition, melatonin reverts these pro-inflammatory effects by inhibiting the JAK2/STAT3 signaling pathway and NF-κβ translocation. Additionally, melatonin-mediated signaling through MT1 receptors promotes PI3K/Akt phosphorylation. Akt coactivator-1-α (PGC-1α) complex dimerize and translocate to the nucleus where estrogen-related receptor-α (ERRα) codes to sirtuin 3 (SIRT3) gene, upregulating the expression and activity of SOD2. In this regard, melatonin is able to activate the multifaceted regulator SIRT1, which enhances the stress response by repressing p53 and promoting DNA repair. Stimulation (blue colored) or inhibition (red colored) by melatonin are also shown.
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Grants and funding

This work was supported by MICIU (grant number PID2021-128133NB-I00/AEI/FEDER10.13039/501100011033) to J.M.H.-G. and V.J.C. enjoys a contract from the CAM “Investigo” program (PIP/2022-09971). A.R. thanks UCJC (INFLAMAMEL 2022-07 project) for its continued support.

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