Orphan GPCRs in the regulation of sleep and circadian rhythm


The hypothalamus is the brain center that is most important in regulating sleep duration and circadian rhythm. Some 75 years ago, it was predicted that the rostral hypothalamus contains the sleep-promoting neurons, while the posterior hypothalamus contains wakefulness-promoting neurons [1]. Sleep is controlled by two processes, a homeostatic and a circadian timing process, which together determine the propensity, length, and incidence of episodes and intensity of sleep [2]. Sleep itself is divided into two major phases, nonrapid eye movement (NREM) and rapid eye movement (REM) sleep [3]. Modulation of these phases and of the circadian rhythms relies on several transmitters which together generate and maintain sleep [4].

The sleep-producing neurons are γ-aminobutyrate(GABA)-ergic cells [3,4]. They induce sleep by inhibiting cells that are involved in arousal functions. The wakefulness-promoting neurons on the other hand, rely on several transmitters (Fig. 1). The peduncolopontine and laterodorsal tegmental (PPT–LDT) nuclei rely on acetylcholine and fire rapidly during wakefulness and REM sleep but become inactive during NREM sleep. It projects to the thalamus, in particular to the reticular nucleus, which is thought to be critical in activating thalamocortical transmission. Three other distinct nuclei exhibit similar activity during the wake-sleep phases. The tuberomammilary nucleus (TMN) is a histaminergic nucleus and plays a major role in the maintenance of wakefulness. Inhibition of their activity by GABAergic cells appears to be closely linked to sleepiness, as evidenced by the drowsiness elicited by antihistamine drugs. The locus coeruleus (LC), center of norepinephrine production, is active during wakefulness, displays low activity during NREM sleep and is inactive during REM sleep. The raphe nucleus, which relies on serotonin as transmitter, is also inactive during sleep, in particular during REM sleep. Its inactivity allows electrical activity to propagate from the pons to the thalamus and cortex inducing eye movement and twitches. Neurons from the TMN, LC and dorsal raphe fire fastest during wakefulness, slow down during NREM and nearly stop firing during REM sleep.

Figure 1.

Sleep architecture and its transmitters. In green are the traditional transmitters: GABA, 4-aminobutyrate; Hist, histamine; NA, norepinephrine; Ach, acetylcholine. The new tranmitters and their sleep-related sites of synthesis: TMN, tuberomammilary nucleus; Raphe, raphe nucleus; LC, locus coeruleus; PPT/LTD, peduncolopontine and laterodorsal tegmental. The novel transmitters are in red: HCRT, hypocretin/orexin; PK2, prokineticin 2; NPS, neuropeptide S; UII, urotensin II. - - - - - , sleep-producing pathways; ----, wakefulness-producing pathway.

While most sleep research has focused on the actions of neurotransmitters, an increasing amount of data point at neuropeptides as being important in modulating the sleep-wake cycle. Neuropeptides exert their actions by activating G-protein coupled receptors (GPCRs). Interestingly, most of these sleep-regulating neuropeptides were discovered recently, not for their activities at regulating sleep, but as the natural ligands of orphan GPCRs.

The human genome expresses some 800 GPCRs of which some 360 are activated by transmitters. Ten years ago, half of these GPCRs had not been matched to any known transmitters; they became to be known as orphan GPCRs [5]. Because of their intrinsic receptor nature, a strategy was developed to use the orphan GPCRs as targets in the discovery of novel transmitters. In short, orphan GPCRs are heterologously expressed in cells in culture and subjected to tissue extracts expected to contain their natural transmitters. Activation of the orphan GPCRs are monitored through their second messenger responses. This strategy, first reported in 1995, has led to the discovery of 10 novel neuropeptides. It has also allowed matching of several orphan GPCRs to neuropeptides that had been described previously [5].

Novel neuropeptides discovered as ligands of orphan GPCRs have unknown functions. Finding these requires experimental studies that are predominantly directed by the anatomical localization analyses of the sites of synthesis and the sites of action of the novel neuropeptides in the central nervous system (CNS). But ultimately, it is the administration of the novel neuropeptide to animals and/or the engineering of knockout mouse strains that reveals the function of the novel neuropeptide system. Surprisingly a series of novel neuropeptide systems were found to be implicated in sleep and wakefulness.

The first deorphanized GPCR system to be shown to modulate sleep was the hypocretin/orexin (Hcrt/orx) system. This system relies on the action of two closely related neuropeptides at two sequentially similar GPCRs. This system originates in the lateral hypothalamus and projects to throughout the whole brain but in particular to the LC, dorsal raphe and PPT–LDT. That the Hcrt/orx system is a major modulator of sleep has been demonstrated by the discovery that animals, in which one of the Hcrt receptors is inactive (Hcrt2), are narcoleptic and that most human narcoleptic patients have no detectable circulating Hcrt and exhibit a reduced number of Hcrt neurons. This system is reviewed in this series by de Lecea & Sutcliffe.

Another novel neuropeptide system that modulates sleep is the neuropeptide S (NPS) system. NPS is synthesized in several parts of the CNS but in particular in a nucleus anatomically associated with the LC. Activation of the NPS system promotes wakefullness by decreasing the NREM and REM stages of sleep. This system is reviewed in this series by Reinscheid & Xu.

The urotensin II (UII) receptor, a GPCR deorphanized in 1999, has been shown to be selectively expressed in the cholinergic LDT–PPT neurons in the CNS. These neurons fire during REM sleep. The UII receptor acts as a presynaptic receptor in the LDT–PPT and consequently activation of this system has been shown to increase REM sleep. This system is reviewed by Nothacker & Clark.

However, there are other orphan GPCR systems that impact sleep. The prolactin-releasing peptide (PrRP) system is one of these. In the CNS, the PrRP receptor is predominantly expressed in the reticular nucleus of the thalamus. This is the thalamic relay nucleus of the cholinergic LDT–PPT and it has been shown that activation of this system induces sleep [6].

Sleep regulation is directly linked to the circadian rhythm. The circadian rhythm is orchestrated in the suprachiasmatic nucleus (SCN) which provides the genetically based clock. The SCN clock relies on positive and negative feedback loops involving the time-dependent transcription of a series of genes [7]. The factors responsible for the output of the SCN clock however, were unknown until recently when prokineticin 2 (PK2) was shown to be such a factor. PK2 was discovered as the natural ligand of an orphan GPCR and is reviewed in this series by Zhou.

This minireview series is intended to present the impact that novel neuropeptides have on our understanding of the sleep-wake cycle. This is not the only field on which novel neuropeptides have had an impact. Most notably, our understanding of the regulation of feeding has greatly gained from the discoveries of novel neuropeptides. These reviews therefore serve as examples of the importance of the emerging field of the natural ligands of orphan GPCRs.

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