Comparative genomics of N-acetyl-5-methoxytryptamine members in four Prunus species with insights into bud dormancy and abiotic stress responses in Prunus avium.

KEY MESSAGE: This study provides novel insights into the evolution, diversification, and functions of melatonin biosynthesis genes in Prunus species, highlighting their potential role in regulating bud dormancy and abiotic stresses. The biosynthesis of melatonin (MEL) in plants is primarily governed by enzymatic reactions involving key enzymes such as serotonin N-acetyltransferase (SNAT), tryptamine 5-hydroxylase (T5H), N-acetylserotonin methyltransferase (ASMT) and tryptophan decarboxylase (TDC). In this study, we analyzed Melatonin genes in four Prunus species such as Prunus avium (Pavi), Prunus pusilliflora (Ppus), Prunus serulata (Pser), and Prunus persica (Pper) based on comparative genomics approach. Among the four Prunus species, a total of 29 TDCs, 998 T5Hs, 16 SNATs, and 115 ASMTs within the genome of four Prunus genomes. A thorough investigation of melatonin-related genes was carried out using systematic biological methods and comparative genomics. Through phylogenetic analysis, orthologous clusters, Go enrichment, syntenic relationship, and gene duplication analysis, we discovered both similarities and variations in Melatonin genes among these Prunus species. Additionally, our study revealed the existence of unique subgroup members in the Melatonin genes of these species, which were distinct from those found in Arabidopsis genes. Furthermore, the transcriptomic expression analysis revealed the potential significance of melatonin genes in bud dormancy regulation and abiotic stresses. Our extensive results offer valuable perspectives on the evolutionary patterns, intricate expansion, and functions of PavMEL genes. Given their promising attributes, PavTDCs, PavT5H, PavNAT, and three PavASMT genes warrant in-depth exploration as prime candidates for manipulating dormancy in sweet cherry. This was done to lay the foundation for future explorations into the structural and functional aspects of these factors in Prunus species. This study offers significant insights into the functions of ASMT, SNAT, T5H, and TDC genes and sheds light on their roles in Prunus avium. Moreover, it established a robust foundation for further exploration functional characterization of melatonin genes in fruit species.

N-acetylserotonin alleviates retinal ischemia-reperfusion injury via HMGB1/RAGE/NF-κB pathway in rats.

AIM: To observe the effects of N-acetylserotonin (NAS) administration on retinal ischemia-reperfusion (RIR) injury in rats and explore the underlying mechanisms involving the high mobility group box 1 (HMGB1)/receptor for advanced glycation end-products (RAGE)/nuclear factor-kappa B (NF-κB) signaling pathway. METHODS: A rat model of RIR was developed by increasing the pressure of the anterior chamber of the eye. Eighty male Sprague Dawley were randomly divided into five groups: sham group (n=8), RIR group (n=28), RIR+NAS group (n=28), RIR+FPS-ZM1 group (n=8) and RIR+NAS+ FPS-ZM1 group (n=8). The therapeutic effects of NAS were examined by hematoxylin-eosin (H&E) staining, and retinal ganglion cells (RGCs) counting. The expression of interleukin 1 beta (IL-1ß), HMGB1, RAGE, and nod-like receptor 3 (NLRP3) proteins and the phosphorylation of nuclear factor-kappa B (p-NF-κB) were analyzed by immunohistochemistry staining and Western blot analysis. The expression of HMGB1 protein was also detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: H&E staining results showed that NAS significantly reduced retinal edema and increased the number of RGCs in RIR rats. With NAS therapy, the HMGB1 and RAGE expression decreased significantly, and the activation of the NF-κB/NLRP3 pathway was antagonized along with the inhibition of p-NF-κB and NLRP3 protein expression. Additionally, NAS exhibited an anti-inflammatory effect by reducing IL-1ß expression. The inhibitory of RAGE binding to HMGB1 by RAGE inhibitor FPS-ZM1 led to a significant decrease of p-NF-κB and NLRP3 expression, so as to the IL-1ß expression and retinal edema, accompanied by an increase of RGCs in RIR rats. CONCLUSION: NAS may exhibit a neuroprotective effect against RIR via the HMGB1/RAGE/NF-κB signaling pathway, which may be a useful therapeutic target for retinal disease.

Polycystic Ovary Syndrome Pathophysiology: Integrating Systemic, CNS and Circadian Processes.

The conceptualization of polycystic ovary syndrome (PCOS) has primarily focused on hormonal alterations driven by changes within the hypothalamus and ovarian granulosa cells, with treatment by the contraceptive pill and weight loss. However, a growing body of data implicates wider systemic and central nervous system (CNS) changes in the pathoetiology and pathophysiology of PCOS, with consequent implications for targeted treatments. It is proposed that there is a significant role for night-time interactions of factors acting to regulate whether the rising level of cortisol over the night and during the morning cortisol awakening response (CAR) is able to induce the nuclear translocation of the glucocorticoid receptor (GR), thereby influencing how the immune and glial systems regulate cellular function in preparation for the coming day. Factors affording protection in PCOS also inhibit GR nuclear translocation including gut microbiome-derived butyrate, and pineal/local melatonin as well as melatonin regulated bcl2-associated athanogene (BAG)-1. A significant pathophysiological role in PCOS is attributed to the aryl hydrocarbon receptor (AhR), which shows heightened levels and activity in PCOS. The AhR is activated by ligands of many systemic processes, including white adipocyte-derived kynurenine, implicating obesity in the pathophysiological changes occurring in the hypothalamus and ovaries. AhR activation has consequences for the physiological function in the hypothalamic paraventricular nucleus, granulosa cells and adipocytes, partly mediated by AhR upregulation of the mitochondrial N-acetylserotonin/melatonin ratio, thereby decreasing melatonin availability whilst increasing local stress plasticity in the paraventricular nucleus. This article reviews in detail the wider systemic and CNS changes in PCOS highlighting interactions of local and pineal melatonergic pathway, gut microbiome-derived butyrate, white adipocyte-derived kynurenine, the hypothalamic paraventricular nucleus tanycytes/astrocytes, and the hypothalamus-pituitary-adrenal (HPA) axis driven glucocorticoid receptor activation in PCOS pathophysiology. This integrates a wide array of previously disparate data on the biological underpinnings of PCOS, including how PCOS associates with many other currently classified medical conditions, such as depression, bipolar disorder, type 1 diabetes mellitus and the autism spectrum. Numerous future research and treatment implications are detailed.

Combination Therapy with N-Acetylserotonin and Aflibercept Activated the Akt/Nrf2 Pathway to Inhibit Apoptosis and Oxidative Stress in Rats with Retinal Ischemia-Reperfusion Injury.

PURPOSE: N-acetylserotonin (NAS) can reduce retinal ischemia-reperfusion injury (RIRI) by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway. Aflibercept is an anti-VEGF drug used to treat a variety of eye diseases. This study was performed to investigate the effect of combination therapy with N-acetylserotonin and aflibercept on RIRI and its mechanism. METHODS: The RIRI model was established by elevating the intraocular pressure. H&E staining was used to observe the pathological changes in the retinal tissue. Cell apoptosis was evaluated by TUNEL. The expression of cleaved caspase-3 in the retina was detected by immunofluorescence and western blotting. The levels of SOD, GSH-Px, and MDA in retinal tissue were measured by ELISA. The protein expression of cytoplasmic Nrf2, nuclear Nrf2, HO-1, Akt, and p-Akt was determined by western blotting. RESULTS: The results showed that combination therapy with NAS and aflibercept significantly alleviated retinal histopathological damage, decreased retinal thickness (from 335.49 ± 30.50 µm to 226.16 ± 17.20 µm, p < 0.001) and the rate of retinal apoptosis (from 28.27 ± 0.39% to 7.87 ± 0.19%, p < 0.001), and downregulated protein expression (from 2.42 ± 0.03 to 1.39 ± 0.03, p < 0.001) and positive expression (from 31.88 ± 0.52 to 25.36 ± 0.58, p < 0.001) of cleaved caspase-3. In addition, combination therapy with NAS and aflibercept also upregulated the levels of SOD (from 20.31 ± 0.18 to 29.66 ± 0.83, p < 0.001) and GSH-Px (from 13.62 ± 0.36 to 19.31 ± 0.82, p < 0.001) and downregulated the level of MDA (from 0.51 ± 0.01 to 0.41 ± 0.01, p < 0.001) to inhibit oxidative stress. Finally, combination therapy with NAS and aflibercept increased the protein expression of cytoplasmic Nrf2 (from 0.10 ± 0.002 to 0.85 ± 0.01, p < 0.001), nuclear Nrf2 (from 0.43 ± 0.01 to 0.88 ± 0.04, p < 0.001), and HO-1 (from 0.45 ± 0.03 to 0.91 ± 0.04, p < 0.001) and the p-Akt/Akt ratio (from 0.45 ± 0.02 to 0.81 ± 0.07, p < 0.001). CONCLUSIONS: Overall, combination therapy with NAS and aflibercept attenuated RIRI, and its mechanism may be related to inhibiting apoptosis and oxidative stress and activating the Akt/Nrf2 pathway.

Melatonin, BAG-1 and cortisol circadian interactions in tumor pathogenesis and patterned immune responses.

A dysregulated circadian rhythm is significantly associated with cancer risk, as is aging. Both aging and circadian dysregulation show suppressed pineal melatonin, which is indicated in many studies to be linked to cancer risk and progression. Another independently investigated aspect of the circadian rhythm is the cortisol awakening response (CAR), which is linked to stress-associated hypothalamus-pituitary-adrenal (HPA) axis activation. CAR and HPA axis activity are primarily mediated via activation of the glucocorticoid receptor (GR), which drives patterned gene expression via binding to the promotors of glucocorticoid response element (GRE)-expressing genes. Recent data shows that the GR can be prevented from nuclear translocation by the B cell lymphoma-2 (Bcl-2)-associated athanogene 1 (BAG-1), which translocates the GR to mitochondria, where it can have diverse effects. Melatonin also suppresses GR nuclear translocation by maintaining the GR in a complex with heat shock protein 90 (Hsp90). Melatonin, directly and/or epigenetically, can upregulate BAG-1, suggesting that the dramatic 10-fold decrease in pineal melatonin from adolescence to the ninth decade of life will attenuate the capacity of night-time melatonin to modulate the effects of the early morning CAR. The interactions of pineal melatonin/BAG-1/Hsp90 with the CAR are proposed to underpin how aging and circadian dysregulation are associated with cancer risk. This may be mediated via differential effects of melatonin/BAG-1/Hsp90/GR in different cells of microenvironments across the body, from which tumors emerge. This provides a model of cancer pathogenesis that better integrates previously disparate bodies of data, including how immune cells are regulated by cancer cells in the tumor microenvironment, at least partly via the cancer cell regulation of the tryptophan-melatonin pathway. This has a number of future research and treatment implications.

Gut Microbiome and Circadian Interactions with Platelets Across Human Diseases, including Alzheimer's Disease, Amyotrophic Lateral Sclerosis, and Cancer.

Platelets have traditionally been investigated for their role in clot formation in the course of cardiovascular diseases and strokes. However, recent work indicates platelets to be an integral aspect of wider systemic processes, with relevance to the pathophysiology of a host of diverse medical conditions, including neurodegenerative disorders and cancer. This article reviews platelet function and interactions with the gut microbiome and circadian systems, highlighting the role of the platelet mitochondrial melatonergic pathway in determining platelet activation, fluxes and plasticity. This provides a number of novel conceptualizations of platelet function and mode of interaction with other cell types, including in the pathoetiology and pathophysiology of diverse medical conditions, such as cancer, Alzheimer's disease, and amyotrophic lateral sclerosis. It is proposed that a platelet-gut axis allows platelets to contribute to many of the pathophysiological processes linked to gut dysbiosis and gut permeability. This is at least partly via platelet sphingosine- 1-phosphate release, which regulates enteric glial cells and lymphocyte chemotaxis, indicating an etiological role for platelets in a wide array of medical conditions linked to alterations in the gut microbiome. Platelets are also an important regulator of the various microenvironments that underpin most human medical conditions, including the tumor microenvironment, neurodegenerative diseases, and autoimmune disorders. Platelet serotonin release regulates the availability of the mitochondrial melatonergic pathway systemically, thereby being an important determinant of the dynamic metabolic interactions occurring across cell types that underpin the pathoetiology of many medical conditions. In addition, a number of novel and diverse future research directions and treatment implications are proposed.

The cutaneous stress response system in three-spined stickleback and European flounder exposed to oxidative stress: Different mode of action.

In fish, the skin is directly exposed to multiple environmental stressors and provides the first line of defense against harmful external factors. It turned out that cortisol and melatonin (Mel) are involved in fish cutaneous stress response system (CSRS) similar to mammalian. This study investigates the mode of action of CSRS in two teleost species of different biology and skin characteristics, the three-spined stickleback and the European flounder, after exposure to oxidative stress induced by a potassium dichromate solution. The cutaneous stress response system presents different ways of action in two studied species: Mel concentration increases in the skin of both species, but cortisol concentration increases in the skin only in sticklebacks. Data suggest that stickleback skin cells can produce cortisol. However, cortisol is not involved in the response to oxidative stress in flounders. In stickleback skin, two genes encoding AANAT and ASMT/HIOMT (enzymes involved in Mel synthesis), aanat1a and asmt2, are expressed, but in flounder skin, only one, asmtl. Because gene expression does not change in stickleback skin after exposure to stress, the source of increased Mel is probably outside the skin. A lack of expression of the gene encoding AANAT in flounder skin strongly suggests that Mel is transported to the skin by the bloodstream from other sites of synthesis. Pigment dispersion in the skin after exposure to oxidative stress is found only in sticklebacks.

The Role of the PAA1 Gene on Melatonin Biosynthesis in Saccharomyces cerevisiae: A Search of New Arylalkylamine N-Acetyltransferases.

Recently, the presence of melatonin in fermented beverages has been correlated with yeast metabolism during alcoholic fermentation. Melatonin, originally considered a unique product of the pineal gland of vertebrates, has been also identified in a wide range of invertebrates, plants, bacteria, and fungi in the last two decades. These findings bring the challenge of studying the function of melatonin in yeasts and the mechanisms underlying its synthesis. However, the necessary information to improve the selection and production of this interesting molecule in fermented beverages is to disclose the genes involved in the metabolic pathway. So far, only one gene has been proposed as involved in melatonin production in Saccharomyces cerevisiae, PAA1, a polyamine acetyltransferase, a homolog of the vertebrate's aralkylamine N-acetyltransferase (AANAT). In this study, we assessed the in vivo function of PAA1 by evaluating the bioconversion of the different possible substrates, such as 5-methoxytryptamine, tryptamine, and serotonin, using different protein expression platforms. Moreover, we expanded the search for new N-acetyltransferase candidates by combining a global transcriptome analysis and the use of powerful bioinformatic tools to predict similar domains to AANAT in S. cerevisiae. The AANAT activity of the candidate genes was validated by their overexpression in E. coli because, curiously, this system evidenced higher differences than the overexpression in their own host S. cerevisiae. Our results confirm that PAA1 possesses the ability to acetylate different aralkylamines, but AANAT activity does not seem to be the main acetylation activity. Moreover, we also prove that Paa1p is not the only enzyme with this AANAT activity. Our search of new genes detected HPA2 as a new arylalkylamine N-acetyltransferase in S. cerevisiae. This is the first report that clearly proves the involvement of this enzyme in AANAT activity.

Escherichia coli RimI Encodes Serotonin N-Acetyltransferase Activity and Its Overexpression Leads to Enhanced Growth and Melatonin Biosynthesis.

Serotonin N-acetyltransferase (SNAT) functions as the penultimate or final enzyme in melatonin biosynthesis, depending on the substrate. The Escherichia coli orthologue of archaeal SNAT from Thermoplasma volcanium was identified as RimI (EcRimI), with 42% amino acid similarity to archaeal SNAT. EcRimI has been reported to be an N-acetyltransferase enzyme. Here, we investigated whether EcRimI also exhibits SNAT enzyme activity. To achieve this goal, we purified recombinant EcRimI and examined its SNAT enzyme kinetics. As expected, EcRimI showed SNAT activity toward various amine substrates including serotonin and 5-methoxytryptamine, with Km and Vmax values of 531 µM and 528 pmol/min/mg protein toward serotonin and 201 µM and 587 pmol/min/mg protein toward 5-methoxytryptamine, respectively. In contrast to the rimI mutant E. coli strain that showed no growth defect, the EcRimI overexpression strain exhibited a 2-fold higher growth rate than the control strain after 24 h incubation in nutrient-rich medium. The EcRimI overexpression strain produced more melatonin than the control strain in the presence of 5-methoxytryptamine. The enhanced growth effect of EcRimI overexpression was also observed under cadmium stress. The higher growth rate associated with EcRimI expression was attributed to increased protein N-acetyltransferase activity, increased synthesis of melatonin, or the combined effects of both.

Redefining Autoimmune Disorders' Pathoetiology: Implications for Mood and Psychotic Disorders' Association with Neurodegenerative and Classical Autoimmune Disorders.

Although previously restricted to a limited number of medical conditions, there is a growing appreciation that 'autoimmune' (or immune-mediated) processes are important aspects of a wide array of diverse medical conditions, including cancers, neurodegenerative diseases and psychiatric disorders. All of these classes of medical conditions are associated with alterations in mitochondrial function across an array of diverse cell types. Accumulating data indicate the presence of the mitochondrial melatonergic pathway in possibly all body cells, with important consequences for pathways crucial in driving CD8+ T cell and B-cell 'autoimmune'-linked processes. Melatonin suppression coupled with the upregulation of oxidative stress suppress PTEN-induced kinase 1 (PINK1)/parkin-driven mitophagy, raising the levels of the major histocompatibility complex (MHC)-1, which underpins the chemoattraction of CD8+ T cells and the activation of antibody-producing B-cells. Many factors and processes closely associated with autoimmunity, including gut microbiome/permeability, circadian rhythms, aging, the aryl hydrocarbon receptor, brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB) all interact with the mitochondrial melatonergic pathway. A number of future research directions and novel treatment implications are indicated for this wide collection of poorly conceptualized and treated medical presentations. It is proposed that the etiology of many 'autoimmune'/'immune-mediated' disorders should be conceptualized as significantly determined by mitochondrial dysregulation, with alterations in the mitochondrial melatonergic pathway being an important aspect of these pathoetiologies.

N-Acetylserotonin Protects Rat Nucleus Pulposus Cells Against Oxidative Stress Injury by Activating the PI3K/AKT Signaling Pathway.

BACKGROUND: Current studies suggest that the pathogenesis of intervertebral disc degeneration (IDD) is related to oxidative stress damage in nucleus pulposus cells (NPCs). N-acetylserotonin (NAS) is an effective scavenger of reactive oxygen species, but its role in IDD and its underlying mechanisms are not yet clear. Therefore, the aim of this study was to investigate the effect of NAS on oxidative stress injury in NPCs and its mechanism. METHODS: NP tissue of rat intervertebral disc was collected and NPCs were isolated. NPCs were treated with H2O2 to simulate the state of oxidative stress. The effects of NAS on cell viability, apoptosis, senescence, extracellular matrix (ECM), redox status and PI3K/AKT signal pathway were evaluated by cell counting kit-8, western blot, immunofluorescence, flow cytometry and SA-ß-gal staining. Finally, the changes of the above indexes were further observed after the inhibition of PI3K pathway by LY294002. RESULTS: Flow cytometry showed that NAS reduced H2O2-induced apoptosis of NPCs. SA-ß-Gal staining showed that H2O2-induced senescence of NP cells was reversed by NAS. Immunofluorescence staining showed that NAS inhibited H2O2-induced ECM degradation. Western blotting analysis revealed that NAS significantly decreased apoptosis, senescence and ECM degradation. Further analysis showed that NAS treatment activated the PI3K/AKT pathway in H2O2-stimulated NPCs. However, these protected effects were inhibited after LY294002 treatment. CONCLUSIONS: The results of the present study suggest that NAS inhibits H2O2-induced NPCs degeneration by activating PI3K/AKT pathway, suggesting that NAS has the potential to treat IDD.

In vitro deacetylation of N-acetylserotonin by arylacetamide deacetylase.

Arylacetamide deacetylase (AADAC) is a deacetylation enzyme present in the mammalian liver, gastrointestinal tract, and brain. During our search for mammalian enzymes capable of metabolizing N-acetylserotonin (NAS), AADAC was identified as having the ability to convert NAS to serotonin. Both human and rodent recombinant AADAC proteins can deacetylate NAS in vitro, although the human AADAC shows markedly higher activity compared with rodent enzyme. The AADAC-mediated deacetylation reaction can be potently inhibited by eserine in vitro. In addition to NAS, recombinant hAADAC can deacetylate melatonin (to form 5-methoxytryptamine) and N-acetyltryptamine (NAT) (to form tryptamine). In addition to the in vitro deacetylation of NAS by the recombinant AADAC proteins, liver (mouse and human) and brain (human) extracts were able to deacetylate NAS; these activities were sensitive to eserine. Taken together, these results demonstrate a new role for AADAC and suggest a novel pathway for the AADAC-mediated metabolism of pineal indoles in mammals.

Identification of a serotonin N-acetyltransferase from Staphylococcus pseudintermedius ED99.

Serotonin N-acetyltransferase (SNAT) catalyzes the biosynthesis of N-acetylserotonin (NAS) and N-acetyltryptamine (NAT), two pleiotropic molecules with neurotransmitter functions. Here, we report the identification of a SNAT protein in the genus Staphylococcus. The SNAT gene identified in Staphylococcus pseudintermedius ED99, namely SPSE_0802, encodes a 140 residues-long cytoplasmic protein. The recombinant protein SPSE_0802 was expressed in E. coli BL21 and found to acetylate serotonin (SER) and tryptamine (TRY) as well as other trace amines in vitro. The production of the neuromodulators NAS and NAT was detected in the cultures of different members of the genus Staphylococcus and the role of SPSE_0802 in this production was confirmed in an ED99 SPSE_0802 deletion mutant. A search for SNAT homologues showed that the enzyme is widely distributed across the genus which correlated with the SNAT activity detected in 22 out of the 40 Staphylococcus strains tested. The N-acetylated products of SNAT are precursors for melatonin synthesis and are known to act as neurotransmitters and activate melatonin receptors, among others, inducing various responses in the human body. The identification of SNAT in staphylococci could contribute to a better understanding of the interaction between those human colonizers and the host peripheral nervous system.

Neuroprotective Effects of N-acetylserotonin and Its Derivative.

N-acetylserotonin (NAS) is a chemical intermediate in melatonin biosynthesis. NAS and its derivative N-(2-(5-hydroxy-1H-indol-3-yl) ethyl)-2-oxopiperidine-3-carboxamide (HIOC) are potential therapeutic agents for traumatic brain injury, autoimmune encephalomyelitis, hypoxic-ischemic encephalopathy, and other diseases. Evidence shows that NAS and its derivative HIOC have neuroprotective properties, and can exert neuroprotective effects by inhibiting oxidative stress, anti-apoptosis, regulating autophagy dysfunction, and anti-inflammatory. In this review, we discussed the neuroprotective effects and related mechanisms of NAS and its derivative HIOC to provide a reference for follow-up research and applications.

Human Naa50 Shows Serotonin N-Acetyltransferase Activity, and Its Overexpression Enhances Melatonin Biosynthesis, Resulting in Osmotic Stress Tolerance in Rice.

A new clade of serotonin N-acetyltransferase (SNAT), the penultimate enzyme in the melatonin biosynthetic pathway, has been reported in the archaeon Thermoplasma volcanium. The closest homolog of archaea SNAT in human was an N-alpha-acetyltransferase50 (Naa50). To determine whether human Naa50 (hNaa50) shows SNAT enzyme activity, we chemically synthesized and expressed the hNaa50 gene in Escherichia coli, followed by Ni2+ affinity purification. Purified recombinant hNaa50 showed SNAT activity (Km and Vmax values of 986 µM and 1800 pmol/min/mg protein, respectively). To assess its in vivo function, hNaa50 was overexpressed in rice (hNaa50-OE). The transgenic rice plants produced more melatonin than nontransgenic wild-type rice, indicating that hNaa50 is functionally coupled with melatonin biosynthesis. Due to its overproduction of melatonin, hNaa50-OE had a higher tolerance against osmotic stress than the wild type. Enhanced expression of the chaperone genes BIP1 and CNX in hNaa50-OE plants was responsible for the increased tolerance. It is concluded that hNaa50 harbors serotonin N-acetyltransferase enzyme activity in addition to its initial N-alpha-acetyltransferase, suggesting the bifunctionality of the hNaa50 enzyme toward serotonin and protein substrates. Consequently, ectopic overexpression of hNaa50 in rice enhanced melatonin synthesis, indicating that hNaa50 is in fact involved in melatonin biosynthesis.

Type I Diabetes Pathoetiology and Pathophysiology: Roles of the Gut Microbiome, Pancreatic Cellular Interactions, and the 'Bystander' Activation of Memory CD8+ T Cells.

Type 1 diabetes mellitus (T1DM) arises from the failure of pancreatic ß-cells to produce adequate insulin, usually as a consequence of extensive pancreatic ß-cell destruction. T1DM is classed as an immune-mediated condition. However, the processes that drive pancreatic ß-cell apoptosis remain to be determined, resulting in a failure to prevent ongoing cellular destruction. Alteration in mitochondrial function is clearly the major pathophysiological process underpinning pancreatic ß-cell loss in T1DM. As with many medical conditions, there is a growing interest in T1DM as to the role of the gut microbiome, including the interactions of gut bacteria with Candida albicans fungal infection. Gut dysbiosis and gut permeability are intimately associated with raised levels of circulating lipopolysaccharide and suppressed butyrate levels, which can act to dysregulate immune responses and systemic mitochondrial function. This manuscript reviews broad bodies of data on T1DM pathophysiology, highlighting the importance of alterations in the mitochondrial melatonergic pathway of pancreatic ß-cells in driving mitochondrial dysfunction. The suppression of mitochondrial melatonin makes pancreatic ß-cells susceptible to oxidative stress and dysfunctional mitophagy, partly mediated by the loss of melatonin's induction of PTEN-induced kinase 1 (PINK1), thereby suppressing mitophagy and increasing autoimmune associated major histocompatibility complex (MHC)-1. The immediate precursor to melatonin, N-acetylserotonin (NAS), is a brain-derived neurotrophic factor (BDNF) mimic, via the activation of the BDNF receptor, TrkB. As both the full-length and truncated TrkB play powerful roles in pancreatic ß-cell function and survival, NAS is another important aspect of the melatonergic pathway relevant to pancreatic ß-cell destruction in T1DM. The incorporation of the mitochondrial melatonergic pathway in T1DM pathophysiology integrates wide bodies of previously disparate data on pancreatic intercellular processes. The suppression of Akkermansia muciniphila, Lactobacillus johnsonii, butyrate, and the shikimate pathway-including by bacteriophages-contributes to not only pancreatic ß-cell apoptosis, but also to the bystander activation of CD8+ T cells, which increases their effector function and prevents their deselection in the thymus. The gut microbiome is therefore a significant determinant of the mitochondrial dysfunction driving pancreatic ß-cell loss as well as 'autoimmune' effects derived from cytotoxic CD8+ T cells. This has significant future research and treatment implications.

N-Acetylserotonin is an oxidation-responsive activator of Nrf2 ameliorating colitis in rats.

N-Acetylserotonin (NAS) is an intermediate in the melatonin biosynthetic pathway. We investigated the anti-inflammatory activity of NAS by focusing on its chemical feature oxidizable to an electrophile. NAS was readily oxidized by reaction with HOCl, an oxidant produced in the inflammatory state. HOCl-reacted NAS (Oxi-NAS), but not NAS, activated the anti-inflammatory nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase (HO)-1 pathway in cells. Chromatographic and mass analyses demonstrated that Oxi-NAS was the iminoquinone form of NAS and could react with N-acetylcysteine possessing a nucleophilic thiol to form a covalent adduct. Oxi-NAS bound to Kelch-like ECH-associated protein 1, resulting in Nrf2 dissociation. Moreover, rectally administered NAS increased the levels of nuclear Nrf2 and HO-1 proteins in the inflamed colon of rats. Simultaneously, NAS was converted to Oxi-NAS in the inflamed colon. Rectal NAS mitigated colonic damage and inflammation. The anticolitic effects were significantly compromised by the coadministration of an HO-1 inhibitor.

A More Holistic Perspective of Alzheimer's Disease: Roles of Gut Microbiome, Adipocytes, HPA Axis, Melatonergic Pathway and Astrocyte Mitochondria in the Emergence of Autoimmunity.

Alzheimer's disease is widely regarded as poorly treated due to poor conceptualization. For 40 years, Alzheimer's disease pathophysiology has focused on two culprits, amyloid-ß induced plaques and hyperphosphorylated tau associated tangles, with no significant treatment advance. This is confounded by data showing amyloid-ß to be an endogenous antimicrobial that is increased in a wide array of diverse medical conditions associated with heightened inflammation. This article reviews the wider bodies of data pertaining to Alzheimer's disease pathophysiology, highlighting the role of suppressed astrocyte mitochondrial function and mitochondrial melatonergic pathway as a core hub in driving neuronal loss in dementia. It is proposed that astrocyte function over aging becomes dysregulated, at least partly mediated by systemic processes involving the 10-fold decrease in pineal melatonin leading to the attenuated capacity of night-time melatonin to dampen residual daytime inflammation. Suppressed pineal melatonin also attenuates melatonin's inhibition of glucocorticoid receptor nuclear translocation, thereby changing not only stress/hypothalamus-pituitary-adrenal (HPA) axis consequences but also the consequences of the cortisol awakening response, which 'primes the body for the coming day'. Gut microbiome-derived butyrate also inhibits glucocorticoid receptor nuclear translocation, as well as inducing the mitochondrial melatonergic pathway. It is proposed that the loss of astrocyte melatonin prevents the autocrine and paracrine effects of melatonin in limiting amyloid-ß levels and effects. Suppressed astrocyte melatonin production also attenuates the melatonin induction of astrocyte lactate, thereby decreasing neuronal mitochondrial metabolism and the neuronal mitochondrial melatonergic pathway. The loss of astrocyte lactate and melatonin, coupled to the suppression of neuronal mitochondrial metabolism and melatonin production decreases mitophagy, leading to the induction of the major histocompatibility complex (MHC)-1. MHC-1 initiates the chemoattraction of CD8+ t cells, leading to neuronal destruction in Alzheimer's disease being driven by 'autoimmune'/'immune-mediated' processes. Alzheimer's disease may therefore be conceptualized as being initiated by systemic processes that act on astrocytes as a core hub, with the suppression of the astrocyte melatonergic pathway leaving neurons deplete of appropriate metabolic substrates and co-ordinated antioxidants. This culminates in an 'immune-mediated' cell death. Future research and treatment/prevention implications are indicated.

Two-Dimensional Thin Layer Chromatography of Melatonin and Related Compounds.

Two-dimensional thin layer chromatography has been used by workers in the field to separate radiolabeled serotonin derivatives from complex mixtures of culture media and homogenates of glands. The compounds resolved include N-acetylserotonin, melatonin, hydroxytryptophol, methoxytryptophol, hydroxyindole acetic acid, and methoxyindole acetic acid. The method requires either radiolabeled tryptophan or serotonin, if an investigator wants to study conversion. It is also useful in the chemical synthesis of serotonin metabolites because it is relatively fast. It pointed to the enzyme that converts serotonin to N-acetylserotonin as being key in controlling the nocturnal increase in vertebrate melatonin production. This enzyme, arylalkylamine N-acetyltransferase (E.C. 2.3.1.87), has been the focus of hundreds of papers which probed its biology, biochemistry, molecular biology, structural biology, neural regulation, development, evolution, and genetics.

Brain Damage-linked ATP Promotes P2X7 Receptors Mediated Pineal N-acetylserotonin Release.

The pineal gland is a key player in surveillance and defense responses. In healthy conditions, nocturnal circulating melatonin (MEL) impairs the rolling and adhesion of leukocytes to the endothelial layer. Fungi, bacteria, and pro-inflammatory cytokines block nocturnal pineal MEL synthesis, facilitating leukocyte migration to injured areas. ATP is a cotransmitter of the noradrenergic signal and potentiates noradrenaline (NAd)-induced MEL synthesis via P2Y1 receptor (P2Y1R) activation. Otherwise, ATP low-affinity P2X7 receptor (P2X7R) activation impairs N-acetylserotonin (NAS) into MEL conversion in NAd incubated pineals. Here we mimicked a focal increase of ATP by injecting low (0.3 and 1.0 µg) and high (3.0 µg) ATP in the right lateral ventricle of adult rats. Nocturnal pineal activity mimicked the in culture data. Low ATP doses increased MEL output, while high ATP dose and the P2X7R agonist BzATP (15.0-50.0 ng) increased NAS pineal and blood content. In the brain, the response was structure-dependent. There was an increase in cortical and no change in cerebellar MEL. These effects were mediated by changes in the expression of coding genes to synthetic and metabolizing melatonergic enzymes. Thus, the pineal gland plays a role as a first-line structure to respond to the death of cells inside the brain by turning NAS into the darkness hormone.