REM sleep and its Loss-Associated Epigenetic Regulation with Reference to Noradrenaline in Particular.

Sleep, an essential physiological process, has been divided into rapid eye movement sleep (REMS) and non-REMS (NREMS) in higher animals. REMS is a unique phenomenon that unlike other sleep-waking states is not under voluntary control. Directly or indirectly it influences or gets influenced by most of the physiological processes controlled by the brain. It has been proposed that REMS serves house-keeping function of the brain. Extensive research has shown that during REMS at least noradrenaline (NA)-ergic neurons must cease activity and upon REMS loss, there is increased level of NA in the brain, which then induces many of the REMS loss associated acute and chronic effects. The NA level is controlled by many bio-molecules, which are regulated at the molecular and transcriptional levels. Similarly, NA may also directly or indirectly modulate synthesis and levels of many molecules, which in turn may affect physiological processes. The burgeoning field of behavioral neuro-epigenetics has gained importance in recent years and explains the regulatory mechanisms underlying several behavioral phenomena. As REMS and its loss associated changes in NA modulates several pathophysiological processes, in this review we have attempted to explain on one hand how the epigenetic mechanisms regulating the gene expression of factors like tyrosine hydroxylase (TH), monoamine oxidase (MAO), noradrenaline transporter (NAT) control NA levels and on the other hand, how NA per se may affect other molecules in neural circuitry at epigenetic level resulting in behavioral changes in health and diseases. An understanding of these events will expose the molecular basis of REMS and its loss-associated pathophysiological changes; which are presented as testable hypothesis for confirmation.

INTRODUCTION

Basic rest and activity cycle (BRAC) is ubiquitously present in every life form and is associated with normal well being of an individual. Sleep-wakefulness in higher order animals is an evolved form of this BRAC. The evolutionary significance of sleep lies in the fact that it saves energy and is crucial for adjustment of the animal to ecological and environmental factors [1]. Sleep and wakefulness are behavioral phenomena, which have been objectively identified, defined, and classified on the basis of electrophysiological signals recorded from the brain the electroencephalogram (EEG), the muscles, the electromyogram (EMG) and the eye movement, the electrooculogram (EOG). Based on these characteristic electrophysiological parameters sleep has been broadly classified into rapid eye movement sleep (REMS) and non-REMS (NREMS). REMS is a reversible, unique physiological process characterized by simultaneous EEG desynchronization, rapid eye movements in the EOG and complete loss of muscle tone (atonia) in EMG. Since EEG is the most important characteristic feature for identification of REMS, it is natural that this state has been identified in species higher in evolution having a reasonably well developed and evolved brain. However, its presence or absence in lower species could not be confirmed as yet primarily due to lack of identification of a more fundamental characteristic marker.

Ontogenetic studies have shown that REMS is expressed maximum in newborn babies and its quantity reduces with ageing, however, it is never absent in life [2, 3]. Its role in brain development has been postulated by the fact that the period spent in REMS is higher in newborn and in babies than in the adults and it is more in babies who are born immature [4]. It is also important to note that this stage does not have voluntary regulation. Its fundamental regulation is done by the neurons in the brain stem, the site for the neural control of other life- sustaining autonomic physiological processes viz. cardiovascular and respiratory regulation. Phylogenetic studies across species suggest that the brain stem activation is the initial element in the REMS evolution [5]. REMS serve several crucial functions and its loss affects various pathophysiological states and processes [6, 7]. It is affected in most pathological conditions, including neurodegenerative diseases, e.g. Parkinson’s, Alzheimer’s, narcolepsy, epilepsy and psychiatric disorders [8-11]. Experimental deprivation of REMS in humans and in animals reported elevated aggressiveness, irritability, confusion, hypersexuality, loss of concentration, impairment of memory processing and memory consolidation [7, 12, 13].

The development and establishment/maturation of any behavioral phenomenon are influenced by several environmental factors like nutrition, social experiences, hormones, etc. [14] and sleep is no exception to it. One of the important factors (if not the most important factor) within the biological system that exerts sustained biological and neurobehavioral manifestations is through the chemical modifications of DNA and histone protein molecules within the cells together known as epigenetic modifications [15]. The field of biology studying the interplay between gene and environmental signals that trigger molecular changes in cells is known as behavioral epigenetics [14]. Epigenetic mechanisms decide the pattern in which environment regulates/influences the genomic organization of living beings. Increasing evidence (mostly indirect though) suggests that epigenetic changes are crucial for chronic or accumulated sleep-loss associated disorders including behavioral changes and also possibly in the regulation of sleep-wake states [16]. Genomic imprinting, which is established by epigenetic processes, also extends its effects to sleep–wake regulation [17]. REMS and NREMS are regulated by separate sets of imprinted genes and these genes are differentially expressed in brain regions [18]. In support, it has been shown that the maternally expressed imprinted gene, Gnas for example, modulates the expression of sleep-wake states [19]. Epigenetic changes have therefore attracted great attention in recent years as researchers are exploring the molecular circuitry underlying several behavioral phenomena including those associated with sleep and its loss. In this review, we have attempted to gain insights into the role of epigenetic changes in the regulation of REMS in particular and its loss-associated disorders/dysfunctions with particular emphasis on noradrenaline (NA).

REMS REGULATION AND NORADRENALINE

It has been recently proposed that REMS serves housekeeping function of the brain [20]. The locus coeruleus (LC) possesses mostly the NA-ergic neurons, which project throughout the brain. The NA-ergic neurons in LC cease activity during REMS and are known as REM-OFF neurons while presumably cholinergic REM-ON neurons increase activity during REMS and are located in the dorsolateral pontine region. The REM-OFF neurons, which normally cease activity during REMS, continue firing upon REMS deprivation (REMSD) [21] causing increased levels of NA in the brain [22]. The elevated levels of NA associated with REMS loss have been correlated with many patho-physiological conditions leading to expression of altered behavior and symptoms associated with various disorders [20, 23]. The increased NA also leads to decreased intracellular [Ca2+] which in turn increases Na-K ATPase activity, which would alter brain excitability [20]. Therefore, understanding of the factors regulating the activity of NA-ergic neurons, which in turn would modulate the levels of NA in the brain, is of great significance. The NA levels in the brain may be modulated by a) rate and period of LC neuronal activity which are modulated by various inputs and neurotransmitters, and b) synthesis, release and effective removal of NA from released sites (i.e. projections mostly from the LC neurons).

NA Neuronal Activity Regulation

REMS is generated and maintained by the neurons located in the core of the brain stem; the neurons from different other brain regions modulate REMS through the former [24]. The LC neurons (the major source of NA in the brain) show a progressive decrease in their activities as the subject moves from wakefulness to sleep and they remain virtually silent during REMS [25]. In rats, when LC neurons were activated using continuous low frequency, mild electrical stimulation [26] or by blocking gamma amino butyric acid (GABA) -ergic receptors [27-29] or by neutralizing Na-K ATPase inhibitors by application of antisera for endoubain [30], REMS was reduced and there was a rebound increase in REMS upon recovery. These, along with other supporting evidence suggested that cessation of LC neurons is a pre-requisite for REMS occurrence and their activation results in REMS loss [31]. The recent finding that optogenetic stimulation of LC neurons enhanced wakefulness and reduced REMS as well as NREMS [32] also supports our contention.

Maintenance of Effective NA Levels in the Brain

The process of biosynthesis, release, reuptake and degradation is responsible for maintenance of neuro-transmitter levels at the synaptic cleft and neuronal surroundings in the brain. Inputs from various regions of the brain on the LC-NA-ergic neurons exert excitatory and inhibitory influences [33] and accordingly have facilitatory and inhibitory effects, respectively, on NA release from the NA-ergic neurons at the target sites. Tyrosine hydroxylase (TH), dopamine β-hydroxylase, monoamine oxidase-A (MAO-A), noradrenaline transporter (NAT) are some of the important molecules directly modulating effective NA levels in the brain. REMSD has been reported to modulate many of these molecules and their activities, which in turn are likely to affect NA levels in the brain. For example, TH level was elevated [22] while MAO-A was decreased [34] after REMSD and the excess of catecholamines is also known to inhibit the activity of TH by negative feedback regulation.

After NA release and activation of its receptors at the synaptic cleft, part of the unused NA is re-uptaken from the extracellular space into the presynaptic terminal by NAT. The transporter also helps preventing excessive build-up of NA concentration at the synaptic cleft. NAT is a monoamine transporter responsible for sodium–chloride dependent re-uptake of NA. This re-uptake is essential for maintenance of NA level at the synaptic cleft and its biological effects, including adrenergic neurotransmission in the brain, heart and peripheral organs [35]. Various adrenoreceptor isoforms e.g. α1, α2, β are expressed on neurons in different brain areas and are responsible for NA mediated effects including during REMSD. The following may be cited in support as example; adrenergic receptor agonist and antagonist injected intraperitoneally or locally in the brain have been reported to modulate REMS [36, 37]; monoamine re-uptake blockers suppress REMS possibly due to elevated NA levels at the synaptic sites [38]; and elevated NAT mRNA has been reported during REMSD [39].

MOLECULAR FACTORS REGULATING NA AT THE GENE LEVEL AND ITS RELEVANCE TO REMS

REMS is an essential phenomenon necessary for the maintenance of normal brain function. However, the detailed mechanisms regulating REMS are yet to be completely understood. Nevertheless, it is known that NA-ergic neurons must cease activity for the generation of REMS [33] and NA levels increase upon REMSD [22]. Independent studies have shown that the bio-molecules (factors) controlling NA levels and its action in the brain, for instance, TH [40-43], α1 adrenergic receptor [44, 45] and MAO [46] are transcriptionally regulated. These factors are encoded by specific genes at the molecular level; however, the details of their modulation and transcriptional regulation in association with REMS and its loss, if any, are still lacking. In addition, the binding of specific transcription factors (TFs) to the specific gene promoter is essential for the homeostatic maintenance of NA level. Therefore, for a comprehensive understanding of the regulation of levels of NA in the brain it is essential to understand the mechanisms controlling transcription of the genes regulating the synthesis and degradation of NA including in relation to REMS, its loss and associated pathophysiological states.

Regulation of gene expression at the transcriptional level involves several processes; the epigenetic modification is among the early ones and also for sustained effects. Epigenetic regulation involves DNA methylation and chromatin remodeling through histone modifications. These regulate chromatin uncoiling and thus allow access to TFs and activation of the transcriptional machinery. In a recent study, it was found that DNA methylation modulates the transcriptional and synaptic responses of neuron to sleep loss [47]. The upcoming field of epigenetics has broadened our basic understanding about the regulation of several essential physiological phenomenon and is likely to serve as the much needed fundamental background mechanism regulating innumerable cellular processes. In this review, an attempt has been made to discuss important findings related to epigenetic changes in the neuronal system associated with REMS loss and its related symptoms. Also, the possibilities of epigenetic changes after REMS loss, especially in the regulation of NA-ergic machinery in the brain have been discussed. Below we explain epigenetic modifications and how they may be useful to our understanding of sleep research and sleep-loss associated symptoms.

EPIGENETIC MODIFICATIONS

The term epigenetics was first coined by Conrad Waddington (1942), who defined the term as “the fundamental interactions between genes and their products, which brings the phenotype into being”. It includes the processes involving chemical modifications of genomic DNA and histone proteins without affecting the DNA sequence [48]. The non-coding RNA (ncRNA) based epigenetic modifications have been described recently, which as yet is the least understood [49]. Below first we discuss, in brief, the possible mechanisms of epigenetic modifications.

DNA Methylation

DNA methylation occurs predominantly across regions of genome having a high frequency of –cytosine–phosphate–guanine– (CpG) sequence, known as CpG island, often found in the promoter region of a gene [50-51]. It constitutes the covalent modification of cytosine residues (5’-position) in the CpG dinucleotide sequence resulting in the formation of 5-methylcytosine as shown in Fig. . The enzymes that catalyze the transfer of the methyl group from S-adenosyl methionine to cytosine residues are known as DNA methyl transferases (DNMT) [52]. Another regulatory protein molecule, the methyl CpG-binding-domain (MBD or MeCP2), recognizes and binds to the methylated DNA. The MeCP2 further recruits additional epigenetic modulatory factors, including transcriptional co-repressors and histone deacetylases (HDAC) [53, 54]. As a result, the chromatin becomes densely packed which hinders the accessibility of TFs and other regulatory molecules, which results in the switching off the gene expression [55]. The DNA methylation is a dynamic process and continues to coordinate the transcriptional programming throughout the lifespan of a cell [56].

Histone Modifications

The fundamental structural unit of eukaryotic chromatin is nucleosome, which comprises of an octamer of the four core histones- H2A, H2B, H3 and H4 wrapped with 147 bp of genomic DNA. The nucleosome assembles in chromatin in varying degree of condensation that plays a key role in controlling accessibility of the DNA to the replication and transcriptional machinery and thus regulates gene expression. In loosely packed chromatin, the DNA sequences are easily accessible to a wide range of molecules, including the TFs and other regulatory molecules [57]. The uncoiling of chromatin and binding of TFs to DNA are regulated by two interlinked processes a) chromatin remodeling, which involves ATP-dependent mobilization of nucleosome and exposure of genomic DNA wrapped around the histone proteins in the nucleosome core; and b) specific histone tail modifications [58]. The histone modifying enzymes catalyze the post-translational modifications, including acetylation, methylation, phosphorylation, SUMOylation, ubiquitylation, ADP-ribosylation, etc. at N-terminal domains of histone protein. Each of these modifications affects the interaction of DNA with histone proteins possibly in a unique manner resulting into distinct transcriptional and downstream events culminating in functional implications [59].

Acetylation occurs at specific lysine residues in the N-terminal tails of the histone H3 and H4 and neutralizes their positive charges. This disrupts the interaction of histones with the negatively charged DNA molecules, which results in a more relaxed and accessible chromatin structure [59, 60] (Fig. ). Deacetylation of these residues results in condensation of chromatin and heterochromatin formation. The acetylation and deacetylation reactions are catalyzed by histone acetyltransferases (HAT) and HDAC. Different histone modifications have differential effects on gene transcription. In comparison to acetylation, histone methylation can have activating or inhibiting effect(s) on the gene expression and it depends exclusively on the localization of the lysine residue that is being covalently modified. For example, methylation of lysine 4 and 36 in histone H3 (H3K4, H3K36) is associated with decondensed chromatin and active gene expression, while methylation of the lysine 9 and 27 in histone H3 results in compaction of chromatin. Histone lysine residues can carry up to three methyl groups [61]. The cumulative effect of all these histone modifications work in concert and bring about a particular effect on transcription. The cooperative interactions amongst different histone modifications establish functional domains in the nucleus referred to as “histone code” [62]. The histone modifications and DNA methylation work hand in hand to ensure efficient gene transcription or its silencing. An intricate balance between these finally regulates gene expression leading to cellular to systemic and behavioral expressions in health and diseases.

EPIGENETIC MODIFICATIONS MODULATING BRAIN FUNCTIONING

Neuroepigenetics or behavioral epigenetics has recently gained recognition and involves “unique mechanisms that allow dynamic experience-dependent regulation of the neuronal epigenome” [63]. Epigenetic regulation allows living cells, including neurons, individually as well as in system to adjust transcriptional changes in response to specific inputs arising from internal as well as external environment from birth until death, through the development, growth and maintenance phases [64]. The synthesis of neurotransmitters and other molecules for the growth, development and sustenance of neural circuitry in response to internal and external signals dynamically are controlled at multiple levels. The crucial neurobiological processes viz. development, neural stem cell maintenance, differentiation generating neural cell identity, neural network connectivity and plasticity have been suggested to be mediated through epigenetic modifications [65]. Thus, the epigenetic regulation constitutes an essential fundamental molecular step, which is necessary for brain functions. Therefore, it is reasonable and timely to understand epigenetic modifications associated with fundamental physiological processes in higher animals, including those in relation to the REMS and its loss.

EMERGING ROLE OF EPIGENETIC MODIFICATIONS IN NEUROLOGICAL DISORDERS AND COGNITIVE PROCESSES, ESPECIALLY ASSOCIATED WITH REMS LOSS

Sleep disorders have been classified broadly into conditions of excessive wakefulness (insomnia) and/or excessive sleepiness (narcolepsy, shift work disorder, jet lag) [66]. It has been observed that patients with psychosomatic disorders suffering from depression, Parkinson’s and Alzheimer’s diseases experience co-morbid sleep disturbances [67-69]. In the following section, we provide an account of the role of epigenetic modifications in the regulation of gene expressions in some representative sleep-loss associated pathophysiological and neurobehavioral dysfunctions.

Alzheimer’s Disease

Alzheimer’s disease (AD) is possibly the most common form of dementia and is characterized by severe memory loss, confusion, depression, apathy, agitation, anxiety and abnormal motor behavior [70]. The brain histology of patients with AD shows two major characteristics viz. neuritic plaques and neurofibrillary tangles due to amyloid β-peptide, amyloid precursor protein and hyper-phosphorylated tau protein, respectively [71, 72]. Epigenetic changes have been correlated with the pathophysiology of AD [73]. In the human cerebral cortex, the age- related alterations in DNA methylation of amyloid precursor protein and microtubule-associated tau protein gene promoter region influence the transcriptional activity of these genes [74, 75]. The amyloid precursor protein is known to form a complex with HAT enzyme, the lysine acetyltransferase 5 (KAT5/Tip60) [76]. Presenilin 1, the gene involved in β-amyloid processing, affects the function of another HAT enzyme, the cAMP response element -binding protein (CREB) -binding protein (CBP/p300) [77].

Further, significantly higher amyloid β- peptide plaque deposition in multiple sub-regions of the cortex was observed after daily sleep restriction for 20 hrs for 21 days [78]. This suggested that sleep disturbances also might be associated with the pathogenesis of AD. Independent studies showed increased apoptosis of neurons by NA after REMSD [79], degeneration of NA-ergic neurons in the LC in AD [23, 80, 81], reduced NAT level in the LC neurons in AD [82] and reduced tissue level of NA in a transgenic mouse model of AD [83] indicate the possibility of involvement of NA in the pathogenesis of AD. However, how at the molecular level the epigenetic modifications affect these changes, which directly or indirectly affect the NA levels in the brain, especially in relation to REMS and its loss, are still unknown.

Depression

Epigenetic modifications of many genes in neurons have been associated with depression [84]. Transcription of brain-derived neurotrophic factor (BDNF), a product of memory-related gene, is decreased in patients with major depression disorder [85] and DNA methylation has been shown to regulate its gene expression [86]. BDNF is also known to regulate neuronal differentiation and growth [87, 88] and its decreased expression in depressed patients may lead to reduced hippocampal volume [85]. Furthermore, histone acetylation (H3K14Ac) is transiently decreased and then consistently increased in the nucleus accumbens after chronic social defeat stress in depressed patients [89]. Decreased acetylation (H4K12Ac) and phosphoacetylation (H3K9S10) were seen in CA3 and dentate gyrus region in the rat depression model [90].

Depression with or without stress disorder, which may affect the hypothalamic- pituitary-adrenal axis, is also associated with the dysregulation of the NA-ergic LC system [23, 91]. Interestingly, sleep deprivation (SD), including REMSD, reduces the symptoms of depression and improves mood disorder [92]. It appears that increased LC neuronal activity resulting in elevated NA is involved in the antidepressant effects of SD. Our contention may be supported by the fact that dysregulation of NAT has been described in depression and antidepressant drugs are known to inhibit the NAT and increase the NA levels [93, 94]. However, the role of epigenetic modifications in regulating the antidepressant effects of SD needs detailed study.

Schizophrenia

It is a complex neuropsychiatric disorder characterized by hallucinations, delusions and working memory deficits [95]. A deficit in GABA-ergic local interneurons is thought to be one of the important correlates in this disease [96, 97]. Decreased expression of glutamic acid decarboxylase 1 (GAD1), which encodes the 67-kDa glutamate decarboxylase, the GABA synthesizing enzyme is commonly seen in schizophrenic postmortem brains [98, 99].

It has been reported that in the normal brain there is a progressive increase in H3K4 methylation at GABA-ergic gene promoters, while in the schizophrenic patients there is a decrease in histone methylation [100]. Changes in acetylation levels across histone proteins have been correlated with gene expression of several schizophrenia-related genes [101], including GAD1. Treatment of the rat with methionine for 15 days decreased GAD1 mRNA and its protein expression. This suggests that GAD1 promoter methylation is involved in the transcriptional repression of this gene [102]. It is pertinent to highlight that REMS latency is reduced in schizophrenia [103], GABA levels change through sleep-waking-REMS [104, 105] as well as upon REMSD (our unpublished data) and GAD levels also change upon REMSD [106]. The above correlation studies, although indicate REMSD associated epigenetic modifications causing changes in GAD, it needs to be studied for confirmation. Further, as NA is increased during REMSD, it may influence GAD or vice versa through GABA (a product of the GAD), particularly in relation to REMS and its loss i.e. REMSD.

Cognitive Processes

An understanding of the cellular and molecular mechanisms of learning and memory processes has been an important area of molecular neuroscience research. It is only recently that epigenetic changes have gained attention as regulators of memory formation [107]; any alteration in these processes is likely to affect (directly or indirectly) various cognitive disorders [108, 109]. DNA methylation was proposed to be the fundamental mechanism required to propagate memory over generations [110]. An increased de novo DNMT expression and methylation across memory–suppressor gene, protein phosphatase I (PPI) was observed in the hippocampus in response to contextual fear conditioning [111]. The PPI is reported to be regulated by NA [112], interacts with HDACs and histone demethylases to increase their activities and thus favors transcriptional silencing. Nuclear PPI promotes memory suppression through dephosphorylation of serine 10 on histone H3 [113]. Learning-induced changes in DNA methylation of BDNF, arc and calcineurin genes are important for formation and maintenance of memory [114, 115]. Hippocampus-dependent tasks were seen to be associated with the global increase of euchromatin-related post-translational modifications of histones. During memory consolidation, H3S10 phosphorylation, H3K14 and H4K5 acetylation, H3K36 trimethylation are rapidly and transiently activated in the hippocampus, whereas they occur with a delay and persist longer in the prefrontal cortex [116].

Sleep including REMS, has been implicated in neuronal plasticity in the brain that underlies the basic mechanism of learning and memory consolidation [117]. Indications are plenty that sleep participates in the consolidation of fresh memory traces arising out of a wide range of experimental conditions [118].NA is an essential modulator of memory formation because of its ability to regulate synaptic plasticity [112]. It is released during arousal and has a central role to play in the emotional regulation of memory [119]. The levels of NA decrease during REMS and is increased upon REMSD [13, 22] and this elevated NA induces many of the REMSD associated molecular changes [20].

In addition to the above, many other neurological disorders, including ageing, attention deficit/hyperactivity disorder, anxiety and post-traumatic stress disorder, are also associated with REMS loss or its dysregulation. A closer look revealed that NA and LC-NA-ergic system are common factors to be dysregulated during these neurological disorders. However, compelling direct evidences relating the role of epigenetic modifications of the NA-ergic system in these neurological disorders, especially in relation to REMS-loss or -dysfunction, are lacking. Nevertheless, it may be summarized that the findings from isolated, independent studies are suggestive of epigenetic modifications in the regulation of expressions of specific genes or bio-molecules associated with neurobehavioral disorders, which are directly or indirectly related to total sleep or REMS disruption and associated imbalance in neurotransmitter levels, particularly that of NA (Fig. ). REMS loss is reported to modulate several neurotransmitters, NA being a prominent and most studied one. As NA has been shown to affect many REMSD-associated cellular, molecular and physiological processes [20], we propose a testable hypothesis and a model explaining the role of epigenetic modifications responsible for changes in NA levels and its role in modulating the expressions of genes or bio-molecules (factors) which regulate REMS or are affected during REMS-loss and are possibly responsible for REMSD-associated changes.

A PROPOSED MODEL FOR REMS AND ITS LOSS ASSOCIATED EPIGENETIC REGULATION

So far we have discussed that neural regulation of REMS is a complex process involving several neurotransmitters and other bio-molecules. We now know that the NA-ergic neurons in the LC must cease activity for the appearance of REMS, their continuous activation causes REMS loss [33] and elevated NA levels due to REMS loss is a key factor for inducing many of the REMS loss associated symptoms [20]. Therefore, we propose that REMS is affected by several bio-molecules (factors) which are regulated by the epigenetic changes and in turn modulate i) the levels of NA at the synaptic site, and ii) NA-induced pathophysiological changes. We now discuss the possible epigenetic regulation of the bio-molecules.

Epigenetic Control of Modulation of NA Levels at the Synaptic Site

The maintenance of levels of neurotransmitters, including NA at the synaptic site is a dynamic state. The NA levels at the synaptic site would depend on its release from the presynaptic terminal, which in turn is dependent on the various inputs on the LC-NA-ergic neurons. The NA levels and its physiological actions are dependent on its synthesis, degradation, re-uptake and the density of NA-receptors at the pre- and post-synaptic sites. Each of these processes is dependent on various enzymes and transporters like TH, MAO, NAT and NA-ergic receptor; a common factor being the transcriptional regulation of synthesis of these molecules. For example, as explained earlier, epigenetic modulation of TH [120], NAT [121, 122] and NA-ergic receptors [44, 123, 124] have been reported; however their relationship with REMS and its loss needs to be studied. We propose that factors directly or indirectly modulating transcription and effectiveness of these molecules may affect REMS, or conversely REMS and its loss may modulate some factors, which in turn may affect these molecules and induce REMS-loss associated changes.

Neuronal activity and release of neurotransmitters are dependent on depolarization of the neurons. In response to depolarization of neurons, voltage gated Ca2+ channels in the neuronal membranes open [125] and increase Ca2+ influx into the intracellular space. This cytosolic Ca2+ plays a significant role in regulating many intracellular signaling processes [126, 127], including neuronal activity-dependent gene expressions [128, 129]. The Ca2+ stimulates secondary messengers causing activation and translocation of kinase pathway, including CREB kinase into the nucleus, which results in phosphorylation and activation of CREB [127]. The Ca2+ influx into the neurons is known to recruit calmodulin kinase (CaMK) IV and it is important to note that like N-methyl D- aspartate receptor, Ca2+ is equally potent in causing nuclear transport and activation of HAT protein p300/CBP mediated gene expression [126]. The activation and binding of HAT protein can also mediate the export of HDAC2 and HDAC5 from the nucleus [130]. It is known that pCREB binds to the promoter regions of NA regulated genes [120, 132] and mediates the downstream signaling pathway of NA via PKA/PKC pathway [131]; it also binds to the NAT promoter region [133]. It has been reported that upon REMSD there are alterations in the activity of the NA-ergic neurons [21], the intracellular Ca2+-level [134], TH level [106], ATPase [135] and MAO [34] activities. Thus, upon REMS loss it is likely that the elevated intracellular [Ca2+] concentration would recruit CaMK IV, which would phosphorylate and activate CBP and possibly other factors as well (Fig. ). The CBP acts as both CREB coactivator as well as HAT protein, which gets activated after phosphorylation by CaMK IV [136]. The p300 is also a HAT protein involved in the chromatin uncoiling. The activated CBP and p300 cause relaxation of the chromatin structure through their intrinsic HAT activity, that increase accessibility of the TF, like CREB and cofactors to the TH genomic DNA and thus upregulates the gene transcription.

The proposition given above may be supported by the fact that epigenetic regulation of TH gene promoter through methylation has been observed in the hypothalamic region of the brain [137]. Synaptic activity dependent Ca2+ signaling can also deactivate the MeCP2 protein via its phosphorylation by CaMKs [138]. The MeCP2 protein also recruits HDAC to inhibit gene expression [139]. Thus, MeCP2 suppression through phosphorylation by CaMKs will prevent its interaction with CpG residues across the TH gene promoter which further would augment TH gene expression. DNA methylation was seen to alter the function of many elements linked to sleep need and synaptic partners of neuroligin, which are regulators of sleep intensity following SD [47].

Thus, epigenetic modulation within the NA-ergic neurons provide an environment for co-repressors and co-activators to bind to the modified histone tails and regulate the transcription of one or more genes (e.g. TH, MAO, NAT) involved in the synthesis, release, degradation and re-uptake of NA in the LC-NA-ergic REM-OFF neurons. Our model may further be supported by the observation that sleep loss has been shown to up- or down-regulate several genes [140] and it has also been proposed to affect microRNA levels in the brain [141, 142]. Therefore, epigenetic modulation in NA-ergic neurons in response to synaptic activity, specifically for TH regulation, due to altered HATs activity and DNA methylation across the TH gene promoter would open chromatin template. This will make the DNA accessible for the binding of TFs and other co-activator proteins required for altered TH gene expression leading to modulation of NA synthesis. Similarly, expressions of NAT, MAO, adrenoceptors also might get modulated. However, the specific changes taking place due to REMS loss need to be studied.

REMS Loss Associated NA-induced Epigenetic Modification

We have argued above that how modulation of various factors may alter the synthesis and release of NA. The altered levels of NA may then modulate gene expression in the projected neurons or in itself due to the presence of collateral inputs onto itself [143, 144]. Increased NA-ergic signaling would activate adrenoceptor and mediate signaling by activation or inhibition of various TFs. For example, the NA induces phosphorylation of glutamate receptor1 (GluR1) by inhibiting PPI [119] or activation of pCREB following the PKA pathway [131]. Similarly, increased NA can modulate the intracellular Ca2+ [20, 145, 146] and induce epigenetic modifications (as explained above) to modulate GluR1- and PPI-gene expressions. Thus, the altered NA levels, including upon REMS loss, may modulate the chromatin uncoiling, phosphorylation and expression of many genes including that of GluR1.

Such changes may directly or indirectly modulate expressions of many molecules including neurotransmitters, which are known to affect REMS and its loss-associated memory impairment [117], psycho-behavioral changes e.g. AD, mood disorders, depression and apoptosis [79]. Modulation of the gene expression e.g. by DNMTs and HDAC inhibitors, which may precipitate or predispose to psychiatric disorders, are potential targets of epigenetic drug therapy. For example, HDAC inhibitors like valproic acid and sodium butyrate have been used in the treatment of sleep loss associated disorders like depression [147], learning and memory [148], epilepsy [121], schizophrenia [149]. Also, ‘MS-275’, which is known to cross blood brain barrier and has selectivity for HDAC1, has been developed as one such potential drug molecule [149].

CONCLUSION

REMS loss is reported to elevate the levels of NA in the brain. The modulation of the levels of NA at the synaptic cleft is a dynamic process and it is regulated by many biomolecules, which are transcriptionally regulated. The NA per se may modulate transcription of many molecules, which then may be responsible for REMS loss associated patho-physio-psycho-behavioral changes. Obviously, dynamicity and equilibrium of these processes are very complex and diverse and therefore, studying REMS loss associated changes have been difficult. Although the investigation of epigenetic mechanisms in REMS regulation is still at its infancy, using modern technology, these molecular mechanisms need to be explored for better understanding of REMS regulation in normal and diseased conditions.