Myths and Evidence Regarding Melatonin Supplementation for Occasional Sleeplessness in the Pediatric Population.

Occasional sleeplessness in children is common, with as many as 25% of all healthy children experiencing a problem sleeping at some point over the course of their childhood. Occasional sleeplessness is poorly understood, has a significant impact on quality of life in children and their families, and is often challenging to manage. There is substantial evidence supporting the safe and effective use of the widely available dietary supplement melatonin for children with chronic conditions. This article summarizes the views expressed in a recent Consensus Panel meeting convened to evaluate the use of melatonin in children, as well as the published scientific literature related to the effectiveness and safety of melatonin, with a focus on occasional sleeplessness in healthy children. We provide an evidence-based framework for the implementation of a standard process to effectively manage occasional sleeplessness in children and adolescents. Unsubstantiated concerns in the past may have limited melatonin's use in children with conditions for which the supplement may support a better sleep pattern and, by doing so, may help to improve quality of life. Melatonin dietary supplements using high quality standards may be provided to children together with cognitive-behavioral therapy after proper sleep evaluation and after improved sleep hygiene, family education, and sleep diary activities have failed to resolve sleep difficulties. [Pediatr Ann. 2021;50(9):e391-e395.].

Mesenchymal stem cell-derived extracellular vesicles ameliorate Alzheimer's disease-like phenotypes in a preclinical mouse model.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects more than 44 million people worldwide. Despite the high disease burden, there is no effective treatment for people suffering from AD. Mesenchymal stem cells (MSCs) are multipotent stromal cells that have been widely studied due to their therapeutic potential. However, administration of cells has been found to have a multitude of limitations. Recently, extracellular vesicles (EVs) derived from MSCs have been studied as a therapeutic candidate, as they exhibit similar immunoprotective and immunomodulatory abilities as the host human MSCs. Methods: To test the potential therapeutic effects of MSC EVs, human bone-marrow derived MSCs were grown in three-dimensional (3D) cell culture, and small EVs were harvested using differential ultracentrifugation. These small EVs were given to non-transgenic (NT) or 5XFAD (5 familial Alzheimer's disease mutations) mice intranasally (IN) every 4 days for 4 months. The mice were then required to perform a variety of behavioral assays to measure changes in learning and memory. Afterwards, immunohistochemistry was performed on brain slices to measure amyloid beta (Aß) and glial fibrillary acidic protein (GFAP) levels. Results: The data revealed that 5XFAD mice that received hMSC-EV treatment behaved significantly better in cognitive tests than saline treated 5XFAD mice, with no significant change between EV-treated 5XFAD mice and NT mice. Additionally, we found lower Aß plaque load in the hippocampus of the EV-treated mice. Finally, less colocalization between GFAP and Aß plaques was found in the brain of EV-treated mice compared to saline. Conclusions: Taken together, these data suggest that IN administration of MSC-derived EVs can slow down AD pathogenesis.

Melatonin and Female Reproduction: An Expanding Universe.

For more than a half century the hormone melatonin has been associated with vertebrate reproduction, particularly in the context of seasonal breeding. This association is due in large measure to the fact that melatonin secretion from the pineal gland into the peripheral circulation is a nocturnal event whose duration is reflective of night length, which of course becomes progressively longer during winter months and correspondingly shorter during the summer months. The nocturnal plasma melatonin signal is conserved in essentially all vertebrates and is accessed not just for reproductive rhythms, but for seasonal cycles of metabolic activities, immune functions, and behavioral expression. A vast literature on melatonin and vertebrate biology has accrued over the past 60 years since melatonin's discovery, including the broad topic of animal reproduction, which is far beyond the scope of this human-focused review. Although modern humans in the industrialized world appear in general to have little remaining reproductive seasonality, the relationships between melatonin and human reproduction continue to attract widespread scientific attention. The purpose of this chapter is to draw attention to some newer developments in the field, especially those with relevance to human fertility and reproductive medicine. As the vast majority of studies have focused on the female reproductive system, a discussion of the potential impact of melatonin on human male fertility will be left for others.

Extraction of Extracellular Vesicles from Whole Tissue.

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Relationship between endogenous melatonin concentrations and uterine contractions in late third trimester of human pregnancy.

In humans, circulating levels of the hormone melatonin and the initiation of spontaneous labor are both higher at night than during the day. Since activation of uterine melatonin receptors can stimulate human in vitro uterine contractions and these receptors are only expressed on the uterine tissue of women in labor, we hypothesized that circulating melatonin concentrations would affect uterine contractions in vivo. We evaluated the impact of light-induced modulation of melatonin secretion on uterine contractions in women during late third trimester (~36-39 weeks) of pregnancy in two inpatient protocols. We found a significant (P < 0.05) positive linear association between circulating melatonin concentrations and the number of uterine contractions under both protocols. On average, uterine contractions increased between 1.4 and 2.1 contractions per 30 minutes for every 10 pg/mL*h increase in melatonin concentration. These findings have both basic science and clinical implications for pregnant women, since endogenous melatonin levels and melatonin receptor activity can be altered by light and/or pharmaceutical agents.

An optimized method for enrichment of whole brain-derived extracellular vesicles reveals insight into neurodegenerative processes in a mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the major cause of dementia that has increased dramatically in prevalence over the past several decades. Yet many questions still surround the etiology of AD. Recently, extracellular vesicles (EVs) that transport protein, lipid, and nucleic acids from cell to cell have been implicated in the clearance and propagation of misfolded proteins. Investigation of EVs in AD progression, and their potential diagnostic utility may contribute to understanding and treating AD. However, the challenges of isolating brain-derived EVs have in part hindered these studies. NEW METHOD: Here, we provide an optimized method for the enrichment of brain-derived EVs by iodixanol floatation density gradient for mass spectrometry analysis. RESULTS: We demonstrate the isolation of these vesicles and the enrichment of EV proteins compared to sedimentation gradient isolation of vesicles. Moreover, comparative proteomic analysis of brain-derived EVs from healthy and AD mouse brains revealed differences in vesicular content including proteins involved in aging, immune response, and oxidation-reduction maintenance. These changes provide insight into AD-associated neurodegeneration and potential biomarkers of AD. Comparison with existing methods: Recent techniques have used sedimentation sucrose gradients to isolate EVs from brain tissue. However, here we demonstrate the advantages of floatation iodixanol density gradient isolation of small EVs, and provide evidence of EV enrichment by electron microscopy, immunoblot analysis, and quantitative mass spectrometry. CONCLUSIONS: Together these findings offer a rigorous technique for enriching whole tissue-derived EVs for downstream analyses, and application of this approach to uncovering molecular changes in AD progression and other neurological conditions.

Combinatorial Treatment Effects in a Cell Culture Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the leading cause of dementia, and as its prevalence increases, so does its detrimental impact on society. The currently available therapies have limited efficacy, leaving AD patients on an irrevocably fatal path of this disease. OBJECTIVE: The purpose of this study was to test efficacy of a novel combinatorial treatment approach to alleviate AD-like pathology. METHODS: We selected four naturally occurring compounds and used them in different combinations to test their effect on AD-like pathology. Employing a well-established cell culture AD model system, we evaluated levels of several diverse biomarkers associated with a number of cellular pathways associated with AD. The readouts included: amyloid-ß peptides, anti-inflammatory and anti-apoptotic proteins, oxidative enzymes, and reactive oxygen species. RESULTS: Using this approach, we demonstrated that the compounds delivered in combination had higher efficacy than individual treatments. Specifically, we observed significant reduction in levels of the amyloid-ß peptides, as well as pro-inflammatory proteins and reactive oxygen species. Similarly, delivery of compounds in combination resulted in an increased expression of anti-apoptotic proteins and anti-oxidative enzymes. Collectively, these modifications in AD pathology biomarkers reflect a promising therapeutic and preventive strategy to combat this disease. CONCLUSION: The above findings support a novel therapeutic approach to address a currently unmet medical need, which would benefit not only AD patients and their caregivers, but also society as a whole.

Hypothalamic-Pituitary Function in Brain Death: A Review.

The Uniform Determination of Death Act (UDDA) states that an individual is dead when "all functions of the entire brain" have ceased irreversibly. However, it has been questioned whether some functions of the hypothalamus, particularly osmoregulation, can continue after the clinical diagnosis of brain death (BD). In order to learn whether parts of the hypothalamus can continue to function after the diagnosis of BD, we performed 2 separate systematic searches of the MEDLINE database, corresponding to the functions of the posterior and anterior pituitary. No meta-analysis is possible due to nonuniformity in the clinical literature. However, some modest generalizations can reasonably be drawn from a narrative review and from anatomic considerations that explain why these findings should be expected. We found evidence suggesting the preservation of hypothalamic function, including secretion of hypophysiotropic hormones, responsiveness to anterior pituitary stimulation, and osmoregulation, in a substantial proportion of patients declared dead by neurological criteria. We discuss several possible explanations for these findings. We conclude by suggesting that additional clinical research with strict inclusion criteria is necessary and further that a more nuanced and forthright public dialogue is needed, particularly since standard diagnostic practices and the UDDA may not be entirely in accord.

Circadian clock regulation of melatonin MTNR1B receptor expression in human myometrial smooth muscle cells.

Circadian genes are expressed in virtually all cells and tissues, and circadian rhythms influence many bodily processes, including reproductive physiology. The expression of hMTNR1B is suppressed during pregnancy until late in term (much like the oxytocin receptor), at which time it is up-regulated to allow for the nocturnal melatonin/oxytocin synergy, which promotes strong nocturnal contractions. Little is currently known about the regulation of hMNTR1b, nor about its functional significance in the myometrium. We, therefore, aimed to elucidate some of the transcription factors that regulate hMNTR1b gene expression in the human myometrium and to determine if hMNTR1b is under circadian control. In this study, we used immortalized and primary myometrial cells that were assessed for circadian gene expression rhythms using real-time bioluminometry and quantitative PCR. Chromatin immunoprecipitation examined the binding of the clock gene product brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) to the promoter of the hMTNR1B gene. Overexpression studies tested the role of circadian locomotor output cycles kaput (CLOCK) and its partner BMAL1 in regulating hMTNR1B expression. We confirmed circadian clock gene expression in both immortalized human myometrial cells and primary myometrial cell cultures. We further showed that the hBMAL1 protein binds to an E-box motif in the proximal promoter of the hMTNR1B gene. Overexpression studies demonstrated that the BMAL1/CLOCK complex activates expression of hMTNR1B leading to a circadian rhythm in phase with the E-box driven clock gene hPER2 (Period 2). These results indicate, for the first time, the presence of a functional circadian clock in the human myometrium with the hMTNR1B gene as a clock controlled target. Further investigations could open new vistas for understanding the regulation of uterine contractions and the timing of human labor.

Clinical significance of melatonin receptors in the human myometrium.

OBJECTIVE: To review and update the research on melatonin receptor expression in the human myometrium, in particular as it pertains to uterine contractility at labor. DESIGN: Summary of previous studies with the addition of new data on the transcriptional regulation of melatonin receptor expression in human myometrial cells. SETTING: Not applicable. PATIENT(S): Late-term pregnant volunteers. INTERVENTION(S): Biopsy collection for in vitro analyses provided the original data. More recently, uterine contractions in late-term pregnant volunteers were assessed before, during, and after acute white-light exposure. MAIN OUTCOME MEASURE(S): Melatonin receptor signaling in myometrial cells and uterine contractions in late-term pregnant volunteers. RESULT(S): Melatonin acts through the MTNR1B melatonin receptor that is expressed in the myometrium at late term to synergistically enhance oxytocin-dependent signaling and contractions. Acute inhibition of endogenous melatonin levels with light reversibly suppresses uterine contractions. CONCLUSION(S): These results point to a significant role for circulating melatonin in the timing and degree of uterine contractions in late-term pregnancy. Understanding the regulation of melatonin receptors remains a future objective.

Melatonin and the circadian timing of human parturition.

Although the onset of spontaneous human parturition has long been known to occur preferentially during the nighttime and early morning hours, no convincing physiological explanation for this pattern has yet been proposed. This review focuses on the circadian timing of mammalian parturition, particularly in the human. It is proposed that differences in the phasing of parturition among different species are likely a function of opposite uterine responses to humoral cues, in particular those coding for time of day. The brain hormone melatonin fulfills many of the prerequisites to serve as a circadian signal for initiating uterine contractions that lead to human parturition. These encompass direct actions of melatonin on myometrial smooth muscle cells that are synergistic with oxytocin in facilitating greater uterine contractions at night. This may not only help to explain the nocturnal phasing of human parturition but also open new avenues for the management of term and preterm labor.

Circadian aspects of mammalian parturition: a review.

The identification of circadian clocks in endocrine tissues has added considerable depth and complexity to our understanding of their physiology. A growing body of research reveals circadian clock gene expression in the uterus of non-pregnant and pregnant rodents. This review will focus on the mammalian uterus and its rhythmicity, particularly as it pertains to the circadian timing of parturition. This key event in the reproductive axis shows dramatic species-specific differences in its circadian phase. It is proposed here that these differences in the phasing of mammalian parturition are likely a function of opposite uterine cell responses to humoral cues. The argument will be made that melatonin fulfills many of the criteria to serve as a circadian signal in the initiation of human parturition, including specific actions on uterine smooth muscle cells that are consistent with a role for this hormone in the circadian timing of parturition.

Melatonin treatment restores mitochondrial function in Alzheimer's mice: a mitochondrial protective role of melatonin membrane receptor signaling.

Mitochondrial dysfunction is a hallmark of Alzheimer's disease (AD) and is observed in mutant amyloid precursor protein (APP) transgenic mouse models of familial AD. Melatonin is a potent antioxidant, can prevent toxic aggregation of Alzheimer's beta-amyloid (Aß) peptide and, when taken long term, can protect against cognitive deficits in APP transgenic mice. To study the effects of melatonin on brain mitochondrial function in an AD model, APP/PS1 transgenic mice were treated for 1 month with melatonin. Analysis of isolated brain mitochondria from mice indicated that melatonin treatment decreased mitochondrial Aß levels by two- to fourfold in different brain regions. This was accompanied by a near complete restoration of mitochondrial respiratory rates, membrane potential, and ATP levels in isolated mitochondria from the hippocampus, cortex, or striatum. When isolated mitochondria from untreated young mice were given melatonin, a slight increase in respiratory rate was observed. No such effect was observed in mitochondria from aged mice. In APP-expressing neuroblastoma cells in culture, mitochondrial function was restored by melatonin or by the structurally related compounds indole-3-propionic acid or N(1)-acetyl-N(2)-formyl-5-methoxykynuramine. This restoration was partially blocked by melatonin receptor antagonists indicating melatonin receptor signaling is required for the full effect. Therefore, treatments that stimulate melatonin receptor signaling may be beneficial for restoring mitochondrial function in AD, and preservation of mitochondrial function may an important mechanism by which long term melatonin treatment delays cognitive dysfunction in AD mice.

International Union of Basic and Clinical Pharmacology. LXXV. Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors.

The hormone melatonin (5-methoxy-N-acetyltryptamine) is synthesized primarily in the pineal gland and retina, and in several peripheral tissues and organs. In the circulation, the concentration of melatonin follows a circadian rhythm, with high levels at night providing timing cues to target tissues endowed with melatonin receptors. Melatonin receptors receive and translate melatonin's message to influence daily and seasonal rhythms of physiology and behavior. The melatonin message is translated through activation of two G protein-coupled receptors, MT(1) and MT(2), that are potential therapeutic targets in disorders ranging from insomnia and circadian sleep disorders to depression, cardiovascular diseases, and cancer. This review summarizes the steps taken since melatonin's discovery by Aaron Lerner in 1958 to functionally characterize, clone, and localize receptors in mammalian tissues. The pharmacological and molecular properties of the receptors are described as well as current efforts to discover and develop ligands for treatment of a number of illnesses, including sleep disorders, depression, and cancer.

Melatonin sensitizes human myometrial cells to oxytocin in a protein kinase C alpha/extracellular-signal regulated kinase-dependent manner.

CONTEXT: Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night. OBJECTIVE: The purpose of this study was to determine the signaling pathway underlying the effects of MEL on contractility and the contractile machinery in immortalized human myometrial cells. DESIGN: To ascertain the signaling pathway of MEL leading to its effects on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodo-MEL (I-MEL) with or without oxytocin and the Rho kinase inhibitor Y27632. I-MEL effects on inositol trisphosphate (IP(3))/diacylglycerol (DAG)/protein kinase C (PKC) signaling were also investigated. Additionally, we assayed for caldesmon phosphorylation and ERK1/2 activation. RESULTS: I-MEL was found to activate PKC alpha via the phospholipase C/IP(3)/DAG signaling pathway, which was confirmed by PKC enzyme assay. I-MEL did not affect myosin light chain phosphatase activity, and its effects on contractility were insensitive to Rho kinase inhibition. I-MEL did increase phosphorylation of ERK1/2 and caldesmon, which was inhibited by the MAPK kinase inhibitor PD98059 or the PKC inhibitor C1. CONCLUSIONS: MEL sensitizes myometrial cells to subsequent procontractile signals in vitro through activation of the phospholipase C/IP(3)/DAG signaling pathway, resulting in specific activation of PKC alpha and ERK1/2, thereby phosphorylating caldesmon, which increases actin availability for myosin binding and cross-bridging. In vivo, this sensitization would provide a mechanism for the increased nocturnal uterine contractility and labor that has been observed in late-term human pregnancy.

Protection against cognitive deficits and markers of neurodegeneration by long-term oral administration of melatonin in a transgenic model of Alzheimer disease.

The neurohormone melatonin has been reported to exert anti-beta-amyloid aggregation, antioxidant, and anti-inflammatory actions in various in vitro and animal models. To comprehensively determine the potential for long-term melatonin treatment to protect Alzheimer's transgenic mice against cognitive impairment and development of beta-amyloid (Abeta) neuropathology, we administered melatonin (100 mg/L drinking water) to APP + PS1 double transgenic (Tg) mice from 2-2.5 months of age to their killing at age 7.5 months. A comprehensive behavioral battery administered during the final 6 weeks of treatment revealed that Tg mice given melatonin were protected from cognitive impairment in a variety of tasks of working memory, spatial reference learning/memory, and basic mnemonic function; Tg control mice remained impaired in all of these cognitive tasks/domains. Immunoreactive Abeta deposition was significantly reduced in hippocampus (43%) and entorhinal cortex (37%) of melatonin-treated Tg mice. Although soluble and oligomeric forms of Abeta1-40 and 1-42 were unchanged in the hippocampus and cortex of the same melatonin-treated Tg mice, their plasma Abeta levels were elevated. These Abeta results, together with our concurrent demonstration that melatonin suppresses Abeta aggregation in brain homogenates, are consistent with a melatonin-facilitated removal of Abeta from the brain. Inflammatory cytokines such as tumor necrosis factor (TNF)-alpha were decreased in hippocampus (but not plasma) of Tg+ melatonin mice. Finally, the cortical mRNA expression of three antioxidant enzymes (SOD-1, glutathione peroxidase, and catalase) was significantly reduced to non-Tg levels by long-term melatonin treatment in Tg mice. Thus, melatonin's cognitive benefits could involve its anti-Abeta aggregation, anti-inflammatory, and/or antioxidant properties. Our findings provide support for long-term melatonin therapy as a primary or complementary strategy for abating the progression of Alzheimer disease.

Melatonin synergizes with oxytocin to enhance contractility of human myometrial smooth muscle cells.

CONTEXT: Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night. OBJECTIVE: The purpose of this study was to determine the effects of MEL on contractility and the contractile machinery in telomerase-immortalized human myometrial cells. DESIGN: To ascertain the effect of MEL on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodomelatonin +/- oxytocin (OT). The effects of iodomelatonin on gap junctions were also investigated. Additionally, expression levels of the type 2 MEL receptor (MT2R) were assessed in myometrial biopsies from term pregnant women with or without labor. RESULTS: MEL was found to synergistically enhance OT-induced contractility via the MT2R, which is coupled to a protein kinase C-dependent increase in phosphorylation of the myosin light chain protein. MT2R expression was markedly elevated in samples from pregnant women who had entered labor, as compared to matched nonlaboring pregnant women. MEL increased expression of the gap junction protein, connexin 43. In vitro dye spread assays showed that MEL-treated cells displayed substantially increased intercellular coupling. Increases in connexin 43 mRNA and cell to cell coupling were also found to be mediated via the MT2R in a protein kinase C-dependent manner. CONCLUSIONS: MEL synergizes with OT to promote myometrial cell contractions and to facilitate gap junction activity in vitro. Such a synergy in vivo would promote coordinated and forceful contractions of the late term pregnant uterus necessary for parturition.

Transcriptional inhibition of oxytocin receptor expression in human myometrial cells by melatonin involves protein kinase C signaling.

CONTEXT: Our laboratory recently characterized the expression of the melatonin receptors in the human myometrium and showed that the expression of these receptors is suppressed during late pregnancy. OBJECTIVE: In an effort to understand better the significance of melatonin in the human myometrium, we explored the mechanisms through which this hormone influences the expression of the oxytocin receptor in vitro. DESIGN: The stable melatonin analog iodomelatonin was presented to cultured telomerase-immortalized myometrial cells of the human telomerase reverse transcriptase line under physiological doses and durations. Pharmacological inhibitors of melatonin binding, gene transcription, phospholipase C, and protein kinase C signaling were used to define the mechanism of melatonin action. RESULTS: Our results reveal that melatonin significantly inhibits oxytocin receptor mRNA expression primarily via the melatonin 2 receptor. The melatonin-dependent decrease in oxytocin receptor transcripts involves suppression of gene transcription rather than enhanced rates of transcript degradation. Melatonin effects were abolished by pretreating the cells with the phospholipase C inhibitor U73122 or the protein kinase C inhibitor C1. CONCLUSIONS: Melatonin, like oxytocin, can negatively regulate oxytocin receptor transcription in human myometrial cells via modulation of protein kinase C signaling. This is consistent with the hypothesis that the reduced melatonin receptor expression during late pregnancy, which occurs at a time when oxytocin receptors are up-regulated, may be physiologically important for the subsequent timing of labor.