The Journal of Clinical Investigation COMMENTARY Makorin rings the kisspeptin bell to signal pubertal initiation Ali Abbara and Waljit S. Dhillo Section of Endocrinology and Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital, London, United Kingdom. Key players in the The signals maintaining quiescence of the reproductive endocrine axis neuroendocrine control during childhood before its reawakening at puberty had been enigmatic. of puberty Revelation of the central actors respon- Studies in patients with abnormal puberty have illuminated the identity sible for pubertal initiation has predomi- of the signals; kisspeptin has emerged as a major stimulator of puberty, nantly emanated from studies in patients and makorin RING finger protein 3 (MKRN3) as an inhibitory signal with disordered puberty, i.e., precocious that prevents premature initiation of puberty. In this issue of the JCI, (early) or delayed (late)/absent puberty. Abreu et al. investigated the mechanism by which MKRN3 regulates Many of these discoveries have not only pubertal onset. The authors found that a reduction in MKRN3 alleviated transformed our understanding of the the constraint on kisspeptin-expressing neurons to allow pubertal signals regulating puberty, but also more initiation, a phenomenon observed across species, including nonhuman widely of the physiological regulation of primates. Further, the ubiquitinase activity of MKRN3 required its the endocrine HPG axis. RING finger domain, in order to repress the promoter activity of In 2003, loss-of-function variants in genes encoding kisspeptin and neurokinin B. These data advance our the gene encoding the kisspeptin receptor understanding of the regulation of kisspeptin-expressing neurons by were reported to result in a failure of puber- MKRN3 to initiate puberty. tal maturation (6, 7). Conversely, activat- ing variants of the kisspeptin receptor were found to cause central precocious puberty (CPP) (8, 9). Accordingly, hypothalamic kisspeptin expression is increased at the What initiates puberty? axis is believed to be important for prim- time of puberty, as is the number of kiss- Activation of the hypothalamic-pituitary- ing reproductive organs to fully respond peptin-responsive gonadotropin-releasing gonadal (HPG) axis to initiate puberty to gonadotropins during puberty. Mini- hormone (GnRH) neurons (10). These and acquire competence for adult repro- puberty is especially important in boys, data first indicated the crucial role of kis- ductive health is a fundamental phys- whereby the proliferation of immature speptin neurons as putative gatekeepers iological process. Conspicuously, the Sertoli cells impacts the success of future of pubertal maturation. Moreover, the timing of the pubertal transition varies spermatogenesis (3). Nevertheless, from function of kisspeptin extends far beyond across the population and is contingent the age of 6 months in boys and 3–4 years its critical role in pubertal initiation to the on a complex interplay between genetic, in girls, the HPG axis is inhibited and regulation of hypothalamic GnRH func- environmental, and metabolic factors remains quiescent until its reactivation tion in both health and disease. Kisspeptin (1). This genetic-environmental interplay to initiate puberty (2, 3). However, what neurons integrate feedback from sex ste- is highlighted by a reduction in the age instigates this reactivation of the HPG roids and metabolic and nutritional signals of pubertal onset over the last century, axis has remained an intriguing mystery to regulate GnRH neuronal secretion. which has been attributed partly to met- for many years; indeed, “What triggers A second neuropeptide discovered abolic factors such as obesity, as well as puberty?” was one of the top unanswered in 2009 to play an important role in the environmental factors such as endocrine questions in the scientific field published pubertal transition is neurokinin B, encod- disruptors (2–4). in 2005 (5). Since then, our knowledge of ed by the TAC3 gene in humans (11). Stud- However, puberty is not the first acti- the key signals that initiate puberty has ies in animal models have revealed that vation of the HPG axis during the lifes- advanced substantially with some nota- neurokinin B is coexpressed with kiss- pan; indeed, it is active in fetal and ear- ble discoveries, of which MKRN3 is a fur- peptin and dynorphin in kisspeptin/neuro- ly neonatal life, termed “minipuberty.” ther major breakthrough that sheds light kinin B/dynorphin (KNDy) neurons in the This early activation of the endocrine on this mystery. arcuate nucleus (12–15). These neuropep- tides are believed to act in concert and in an autocrine manner to regulate the release of Related Article: p. 4486 kisspeptin and thus determine the pulsatile nature of GnRH secretion. Consequently, Conflict of interest: AA and WSD have undertaken consultancy work for Myovant Sciences Ltd. Copyright: © 2020, American Society for Clinical Investigation. KNDy neurons are regarded as vital com- Reference information: J Clin Invest. 2020;130(8):3957–3960. https://doi.org/10.1172/JCI139586. ponents of the GnRH pulse generator. jci.org Volume 130 Number 8 August 2020 3957 COMMENTARY The Journal of Clinical Investigation hypothalamus (19), consistent with its cen- tral role in the neuroendocrine control of puberty. More specifically, though MKRN3 was expressed in a number of hypothalam- ic nuclei, it was most strongly expressed in the arcuate and ventromedial nuclei (19). Moreover, MKRN3 colocalized with kisspeptin-expressing neurons, and its expression decreased during the lead up to puberty (19), consistent with the working hypothesis that the reduction in MKRN3 alleviates the constraint on these neurons to allow the initiation of puberty (Figure 1). The investigators revealed further detail as to how MKRN3 interacted with KNDy neu- rons, specifically that MKRN3 repressed the promoter activity of genes encoding Figure 1. A model for how the transition to puberty is controlled by the effect of MKRN3 on kiss- kisspeptin and neurokinin B (19). Finally, peptin-expressing neurons in the hypothalamus. Abreu et al. showed that MKRN3 repressed the pro- moter activity of genes encoding kisspeptin and neurokinin B (19). The reduction in MKRN3 in the lead the authors demonstrated that MKRN3 up to puberty alleviates the constraint on kisspeptin/neurokinin B/dynorphin (KNDy) neurons to allow achieved inhibition of KNDy neurons reactivation of the hypothalamic-pituitary-gonadal axis and the initiation of puberty. NKB, neurokinin through its action as an E3 ubiquitin ligase. B; FSH, follicle-stimulating hormone. Ubiquitin is a small regulatory protein, which as its name suggests, is ubiquitously present in most eukaryotic cells. Ubiquiti- nation refers to the process by which ubiq- Overall, activation of these KNDy neurons the epigenetic phenomenon that results in uitin binds to a substrate protein to modify plays a key role in initiating puberty, but selective expression of genes depending its function, or to target it for degradation. what constrains KNDy neuronal activity on whether they are inherited from the Ubiquitination involves 3 sequential steps following minipuberty throughout child- maternal or paternal parent. For instance, that are catalyzed by specific groups of hood had remained an enigma. while paternal inheritance of variants in the enzymes, namely activation (E1), conjuga- In 2013, a landmark study reported that PWS critical region results in PWS (hypoto- tion (E2), and ligation (E3). E3 enzymes can loss-of-function variants in the MKRN3 nia, obesity, and hypogonadism), maternal also undergo autoubiquitination, whereby gene (previously known as ZNF127), which inheritance of variants in the same region they catalyze the addition of polyubiquitin encodes makorin RING finger protein 3, results in Angelman syndrome (seizures, to themselves, a feature that can be used to resulted in CPP (16). Fittingly, RING in this developmental and neurological disorders). monitor E3 enzymatic activity. Abreu et al. context does not indicate that the protein In a genome-wide association study to demonstrated that the RING finger domain has a circular structure but rather denotes identify genes associated with the natural of MKRN3 was obligatory for its ubiquiti- really interesting new gene. Indeed, this variation in pubertal onset, MKRN3 had the nase activity, which in turn was required to was the first reported loss-of-function greatest association with menarche when repress kisspeptin and neurokinin B gene variant causing CPP and remains the com- paternally inherited (1). promoters (19). MKRN3 is an E3 ubiquitin monest genetic cause of CPP, enabling the ligase, and methyl-CpG-DNA binding pro- reclassification of many children previous- How does MKRN3 interact with tein (MBD3) has recently been identified ly labeled as having idiopathic CPP (17). A kisspeptin neurons? as a substrate for its E3 ligase activity (20). recent meta-analysis revealed that MKRN3 Abreu and Kaiser published the seminal Additionally, MKRN3-mediated ubiquiti- variants are found in 9% (95%CI 4%–15%) report in 2013 demonstrating that loss-of- nation has been reported to disrupt inter- of children with CPP, and more frequently function variants in MKRN3 cause CCP actions between MBD3 and the GNRH1 in familial CPP (19%) (18). Although typi- (2). In this issue of the JCI, Abreu and promoter to cause epigenetic silencing of cally CPP occurs