Nuevos mecanismos reguladores del control central de la pubertadanálisis del papel de la señalización de ceramidas y el sistema mkrn3/mir-30b

Resumo

1. Introduction Puberty is a crucial and complex developmental event characterized by the acquisition of reproductive capacity and sexual and somatic maturation1. Nowadays, it is well established that pubertal timing is under the control of sophisticated regulatory mechanisms highly sensitive to metabolic and environmental factors. However, our knowledge about these mechanisms and their potential involvement in pubertal alterations is still incomplete. The importance to unveil new molecular and neuroendocrine pathways for the central control of puberty is illustrated by recent epidemiological studies conducted in Europe and USA that have documented a trend for advanced puberty; a phenomenon related to higher risk of suffering other cardiovascular and metabolic diseases (e.g. hypertension, obesity, and diabetes). On the above basis, it is important to notice that the activation of the reproductive axis at puberty depends on the degree of energy reserves of the organism2. In the last few years, the mechanisms involved in the integrative control of the energy balance and pubertal development have been extensively studied. These mechanisms seemingly involve a large array of metabolic hormones and neuropeptides, which impinge and integrate at the hypothalamic centers controlling the reproductive axis3,4. Among those regulatory elements, the metabolic hormones leptin and ghrelin, as stimulatory and inhibitory signals of puberty onset, respectively, and the puberty-activating neuropeptide, kisspeptin, as a central conduit for transmitting the pubertal actions of different metabolic hormones to the reproductive brain, have proven to be especially relevant3,5-7. Yet, our knowledge about the mechanism(s) of action of these (and others) regulatory factors in the central control of puberty and their potential contribution to alterations in the timing of puberty is still incomplete. Interestingly, ceramides, a family of sphingolipids of ubiquitous nature involved in different cellular processes, have been recently proposed as hypothalamic mediators in the control of energy homeostasis and metabolic disorders8-10. In particular, high hypothalamic levels of ceramides have been reported to block the anorexigenic effects of leptin11, while they might mediate the orexigenic roles of ghrelin in the control of food intake and energy balance12. However, the potential role of hypothalamic ceramides in the central control of puberty onset, as well as their putative interactions with other relevant neuroendocrine factors, such as kisspeptins or leptin, in this context, remain fully unexplored. On the other hand, it is worth to note that novel targets and regulatory mechanisms have been recently identified in the context of the central control of puberty. Among them, the maternally imprinted gene encoding the makorin RING-finger protein 3, Mkrn3, as a novel target, and miRNAs, as a novel regulatory mechanism, seems to have a relevant role13-15. Recent evidence has suggested that Mkrn3 may act as a potential repressor of puberty onset. This is based on (i) the association of deleterious mutations of MKRN3 with central precocious puberty in boys and girls16-22; (ii) the decrease in circulating levels of Mkrn3 detected in both sexes before puberty onset23-25; and (iii) the significant reduction of the hypothalamic Mkrn3 expression observed during the juvenile-pubertal transition in rodents16,26. Despite such evidence, the regulatory mechanisms whereby Mkrn3 is precisely controlled during postnatal/pubertal maturation and their biological actions in normal and altered puberty are totally unknown. Interestingly, bioinformatic analyses conducted in our group have revealed that the microRNA miR-30b shows three predicted and conserved binding sites at the 3’ untranslated region (3’-UTR) of Mkrn3. Whether such miRNA contributes to the regulation of Mkrn3 expression in the central control of puberty has not been explored so far. Based on the above, the main objective of this Doctoral Thesis is to characterize novel regulatory mechanisms involved in the physiological control of puberty, such as central ceramide signaling and the miR-30b/Mkrn3 pathway, and to elucidate their potential involvement in the alterations of pubertal development frequently linked to unfavourable (mainly metabolic) conditions. 2. Research contents In the experimental set 1#, a series of studies were implemented to analyze the putative role of central ceramide signaling in the metabolic control of puberty onset, using prepubertal female rats in normal conditions or subjected to obesogenic insults promoting precocious puberty (i.e. early overfeeding). Our initial studies documented a significant increase in the hypothalamic levels of ceramides in early overfed female rats (ON) with precocious puberty. Based on these data, we decided to evaluate the involvement of perturbed central ceramide signaling in the etiopathogenesis of precocious puberty linked to early overfeeding. For this purpose, prepubertal female rats were intracerebroventricularly injected with a cell-penetrating precursor of the de novo ceramides synthesis, called CER C6, resulting in advanced puberty onset. In contrast, the chronic inhibition of central ceramide synthesis with myriocin (MYR) caused a significant delay of puberty. Importantly, none of the two treatments altered body weight, food intake or gonadotropin levels. Next, we analyzed the potential interaction between central ceramide signaling and kisspeptin or leptin in the timing of puberty in a model of delayed puberty induced by chronic undernutrition (25%), in which endogenous kisspeptin and leptin levels are suppressed, and hence the effects of “rescue” experiments by administration of the exogenous factors can be readily detected. Treatment with either kisspeptin or leptin partially rescued delayed puberty. Conversely, such stimulatory effects, especially those derived from kisspeptin, were largely prevented by co-administration with the ceramide inhibitor, MYR, thus suggesting that central ceramide signaling mediates part of the stimulatory effects of kisspeptin and, to a lesser degree, leptin on puberty onset. However, the fact that MYR did not alter Kiss1 mRNA expression in two relevant hypothalamic populations for the reproductive actions of kisspeptin, such as the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), or did it attenuate kisspeptin-induced GnRH/LH responses ex vivo or in vivo, respectively, suggested that the pubertal actions of central ceramides might require the involvement of an alternative non-neuroendocrine pathway. In this context, additional studies were focused in deciphering the potential role of central ceramide signaling in the modulation of an alternative Paraventricular (PVN)-ovarian sympathetic pathway that might lead to the onset of puberty and, eventually, contributes to obesity-induced precocious puberty. Our results suggest that early-onset obesity advanced the maturation of the ovarian sympathetic tone in immature female rats (PND25), as evidenced by increased levels of relevant markers of sympathetic activity, such as noradrenaline (NE) and 3-Methoxy-4-Hydroxyphenylglycol (MHPG) in the celiac ganglia and the ovary, as well as Ngf/Ngfr ovarian signaling. Interestingly, the blockade of central ceramide synthesis with MYR in early overfed female rats resulted in a partial normalization of the timing of puberty, in terms of vaginal opening and first estrus, which are considered external markers of puberty onset and ovulation, respectively. Furthermore, the ovarian sympathetic activity was partially normalized in those animals, which showed lower levels of NE in the celiac ganglia and the ovary, as well as decreased ovarian Ngf/Ngfr signaling. Remarkably, we obtained neuroanatomical evidence that supports the potential role of ceramides synthesis at the PVN, considered as the hypothalamic start-point of the ovarian sympathetic pathway, in the precocious puberty linked to early-onset obesity. In particular, the expression of serine palmitoyltransferase (SPTLC-1), the gene encoding the first enzyme of the de novo synthesis of ceramides, was significantly increased in the PVN of early overfed rats with advanced puberty. Additionally, our data show that the number of kisspeptin-ir fibers is significantly reduced in the PVN of those animals, thus suggesting its potential relevance in such context. In the experimental set 2#, different studies were performed in rodent models to evaluate the putative physiological role of miR-30b/Mkrn3 system in the central control of puberty. Hypothalamic expression profiles of Mkrn3 and miR-30b were analyzed during normal postnatal maturation, and in preclinical models of altered puberty, such as early (neonatal and/or infantile) manipulation of feeding (20 pups/litter), sex steroid milieu, and photoperiod (constant darkness during 5-10 days). At the hypothalamic level, our data document a decrease in Mkrn3 expression along postnatal maturation. Conversely, miR-30b levels displayed an opposite expression pattern, with minimal neonatal levels and progressive increases along postnatal development. In addition, our results show that neonatal estrogenization and early postnatal underfeeding, two models of perturbed puberty, alter the hypothalamic ratios of miR-30b/Mkrn3 at the expected time of puberty and the early infantile period, respectively, in female rats. Furthermore, in vitro assays, based on heterologous expression of a reporter vector harboring the 3’-UTR of mouse Mkrn3 in HEK-293 cells, showed that miR-30b represses the transcriptional activity of Mkrn3. In the same vein, timely (juvenile) blockade of miR-30b binding to its seeds regions at the 3’-UTR of Mkrn3 in vivo, by central infusion of tailored target site blockers, reversed the prepubertal down-regulation of hypothalamic Mkrn3 protein and delayed female puberty. 3. Conclusions The main conclusions of our studies are the following: 1. Ceramide signaling constitutes a novel pathway for the central control of pubertal timing, which mediates at least part of the regulatory actions of kisspeptins (and to a lesser extent leptin), likely via a GnRH-independent pathway, involving the PVN and ovarian sympathetic innervation. 2. This kisspeptin-ceramide pathway at the PVN plays a relevant pathophysiological role in the generation of pubertal precocity associated to early-onset obesity. 3. The miRNA, miR-30b, is a novel central regulator of the puberty-repressing factor, Mkrn3, acting at highly conserved regions at the 3’-UTR of this gene. 4. This miR-30b/Mkrn3 pathway seemingly plays a distinct role in the physiological control of the timing of puberty, and its perturbations in conditions of early nutritional or hormonal alterations. 4. Bibliography 1. Parent, A.S. et al. The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration. Endocr Rev 24, 668-93 (2003). 2. Roa, J. et al. Metabolic control of puberty onset: new players, new mechanisms. Mol Cell Endocrinol 324, 87-94 (2010). 3. Castellano, J.M. & Tena-Sempere, M. Metabolic control of female puberty: potential therapeutic targets. 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