Final Report Summary - META-GNRH (Metabolic Targeting of GnRH Neurons: Molecular Mechanisms and Neuropeptide Pathways)
Reproduction, an essential function for the perpetuation of the species, is under the control of a sophisticated network of regulatory signals. Notably, regulation of reproductive capacity is closely linked to that of other relevant body functions, including energy balance and body weight homeostasis. Indeed, conditions of metabolic stress and sustained energy insufficiency (e. g., during starvation or anorexia nervosa) are frequently coupled to disturbed reproductive maturation and/or infertility. Likewise, obesity is also commonly linked to altered puberty onset, hypogonadism and reproductive dysfunction. Elucidation of the mechanisms for such a metabolic/reproductive interplay might have translational relevance, as it should help to define the rational basis for novel therapeutic strategies in the management of reproductive disorders, especially, but not exclusively, in the context of metabolic diseases. In this scenario, our understanding of how hormones and neuropeptide signals participate in the metabolic targeting of the GnRH system is still fragmentary.
In the above context, this project aimed to gain novel insights into the neuroendocrine and molecular mechanisms responsible for the metabolic regulation of puberty and fertility, as a means to define their eventual alterations in conditions of disturbed energy balance (from energy insufficiency to obesity) as causative etiological factor for various reproductive disorders, whose frequency is likely increasing in European countries. As specific objectives, the present project intended
(a) To analyze the putative role of the sensor of intracellular energy status, the mammalian target of Rapamycin (mTOR), in mediating the actions of the above signals on GnRH neurons, either directly or via Kiss1 neurons, using electrophysiological and calcium imaging recordings in acute brain slices;
(b) To test the effects of selected metabolic neuropeptides (NPY, AgRP, alpha-MSH, CART, beta-Endorphin) on the patterns of GnRH neuronal activation, using mainly electrophysiological recordings in living GnRH neurons; and
(c) To study the possible role of Kisspeptin/GPR54 signaling on POMC neurons, with key roles in metabolism and body weight homeostasis, by ablation of GPR54 in this neuronal population using Cre-loxP strategies in mouse models.
The effects of mTOR signaling upon the activity of GnRH neurons have been assessed. Calcium-imaging experiments demonstrated that blockade of mTOR resulted in acute suppression of calcium transients in approximately 35 % of GnRH neurons located in the rostral preoptic area (POA), whereas mTOR activation by L-Leucine treatment increased the electrical firing rate of approx. 30 % of GnRH neurons in this area. However, dual-label immune-histochemistry (IHC) documented the absence of pS6, the putative downstream target of mTOR, in GnRH neurons, suggesting this to be an indirect action. Of note, by the use of IHC, Kiss1 neurons, as major afferent pathway to GnRH neurons were also shown to be devoid of expression of pS6, thus pointing out a primary action of mTOR pathways upstream Kiss1 and GnRH neurons to modulate the HPG axis.
In addition, electrophysiological analyses of GnRH neuronal firing in response to an array of selected metabolic neuropeptides, produced by key hypothalamic populations in the control of body weight, namely POMC or NPY neurons, documented predominant direct stimulatory effects of alpha-MSH (product of POMC neurons) on GnRH neurons, whereas other co-transmitters, such as CART and beta-Endorphin, elicited excitatory (CART) or inhibitory (beta-Endorphin) responses in a much smaller subset of GnRH neurons. In addition, our studies have characterised a complex pattern of response of GnRH neurons to changes in NPY signaling depending on the predominant receptor activated, with presumable inhibitory actions being mediated via Y1 receptor and possible excitatory actions conducted by Y4. In addition, the other major product of NPY neurons, AgRP, was shown to induce modest inhibitory responses in a minute fraction of GnRH neurons, while it also evoked discernible excitatory responses in approx. one-forth of GnRH neurons. Thus, our analyses have contributed to surface the complex patterns of electrical responses of GnRH neurons to a variety of key metabolic neuropeptides; a phenomenon that illustrates the intricate basis for the dynamic interplay between energy homeostasis and reproduction.
As complement to our electrophysiological studies, in vivo testing of the effects of specific agonists and antagonists of melanocortin (alpha-MSH) pathways has been also conducted in this project. Our analyses, using combined and specific agonists of the major receptors for alpha-MSH, namely MC3R and MC4R, revealed a potent stimulatory effect of MC4R activation of LH secretion (as surrogate in vivo marker for GnRH neuronal activation) in pubertal rats, whereas specific stimulation of MC3R resulted in modest LH inhibition. In keeping with a predominant stimulatory role of alpha-MSH signaling on the gonadotropic axis, blockade of MC3/4R during the pubertal transition in female rats resulted in delayed puberty, as revealed by a combination of phenotypic and hormonal indices of pubertal maturation. These data are compatible and fully back-up our initial electrophysiological results and support a relevant physiological of MSH signaling in the metabolic control of GnRH neurons and puberty.
Finally, the demonstration of a putative role of GPR54 signaling in POMC neurons, as reported recently, has raised the possibility of direct effects of kisspeptins in the control of body weight homeostasis. Specific analyses in this sense have been conducted in this project, by the use of a genetically modified animal model where GPR54, and hence kisspeptin action, has been selectively eliminated from POMC neurons. Due to the complex breeding of this conditional knock-out mouse line, phenotypic, hormonal and metabolic analyses of this animal model are still in progress in order to elucidate the actual physiologic roles of kisspeptins in the functional modulation of POMC pathways and, therefore, the relevance, if any, of Kiss1 signaling in the direct regulation of body weight and its coupling with reproductive parameters.
In sum, we believe that this project has addressed relevant research questions, not only of fundamental (physiologic) importance, but also of translatable interest. Disorders of puberty and fertility are becoming increasingly common in Europe and other developed countries, and some of those conditions may be, at least partially, linked to the rapid increase in metabolic disorders. Thus, the knowledge obtained here pertaining to the physiological mechanisms and pathways coupling energy status and reproduction is likely to provide essential information for the rational management of different reproductive disorders that have attracted interest and concern not only from the scientific community but also of the lay public. In addition, the project has been endowed with very important formative contents, which have helped the fellow to expand his skills and methodological abilities in the area of Neurophysiology and Neuro-endocrinology in particular, and of experimental Biosciences in general.
In the above context, this project aimed to gain novel insights into the neuroendocrine and molecular mechanisms responsible for the metabolic regulation of puberty and fertility, as a means to define their eventual alterations in conditions of disturbed energy balance (from energy insufficiency to obesity) as causative etiological factor for various reproductive disorders, whose frequency is likely increasing in European countries. As specific objectives, the present project intended
(a) To analyze the putative role of the sensor of intracellular energy status, the mammalian target of Rapamycin (mTOR), in mediating the actions of the above signals on GnRH neurons, either directly or via Kiss1 neurons, using electrophysiological and calcium imaging recordings in acute brain slices;
(b) To test the effects of selected metabolic neuropeptides (NPY, AgRP, alpha-MSH, CART, beta-Endorphin) on the patterns of GnRH neuronal activation, using mainly electrophysiological recordings in living GnRH neurons; and
(c) To study the possible role of Kisspeptin/GPR54 signaling on POMC neurons, with key roles in metabolism and body weight homeostasis, by ablation of GPR54 in this neuronal population using Cre-loxP strategies in mouse models.
The effects of mTOR signaling upon the activity of GnRH neurons have been assessed. Calcium-imaging experiments demonstrated that blockade of mTOR resulted in acute suppression of calcium transients in approximately 35 % of GnRH neurons located in the rostral preoptic area (POA), whereas mTOR activation by L-Leucine treatment increased the electrical firing rate of approx. 30 % of GnRH neurons in this area. However, dual-label immune-histochemistry (IHC) documented the absence of pS6, the putative downstream target of mTOR, in GnRH neurons, suggesting this to be an indirect action. Of note, by the use of IHC, Kiss1 neurons, as major afferent pathway to GnRH neurons were also shown to be devoid of expression of pS6, thus pointing out a primary action of mTOR pathways upstream Kiss1 and GnRH neurons to modulate the HPG axis.
In addition, electrophysiological analyses of GnRH neuronal firing in response to an array of selected metabolic neuropeptides, produced by key hypothalamic populations in the control of body weight, namely POMC or NPY neurons, documented predominant direct stimulatory effects of alpha-MSH (product of POMC neurons) on GnRH neurons, whereas other co-transmitters, such as CART and beta-Endorphin, elicited excitatory (CART) or inhibitory (beta-Endorphin) responses in a much smaller subset of GnRH neurons. In addition, our studies have characterised a complex pattern of response of GnRH neurons to changes in NPY signaling depending on the predominant receptor activated, with presumable inhibitory actions being mediated via Y1 receptor and possible excitatory actions conducted by Y4. In addition, the other major product of NPY neurons, AgRP, was shown to induce modest inhibitory responses in a minute fraction of GnRH neurons, while it also evoked discernible excitatory responses in approx. one-forth of GnRH neurons. Thus, our analyses have contributed to surface the complex patterns of electrical responses of GnRH neurons to a variety of key metabolic neuropeptides; a phenomenon that illustrates the intricate basis for the dynamic interplay between energy homeostasis and reproduction.
As complement to our electrophysiological studies, in vivo testing of the effects of specific agonists and antagonists of melanocortin (alpha-MSH) pathways has been also conducted in this project. Our analyses, using combined and specific agonists of the major receptors for alpha-MSH, namely MC3R and MC4R, revealed a potent stimulatory effect of MC4R activation of LH secretion (as surrogate in vivo marker for GnRH neuronal activation) in pubertal rats, whereas specific stimulation of MC3R resulted in modest LH inhibition. In keeping with a predominant stimulatory role of alpha-MSH signaling on the gonadotropic axis, blockade of MC3/4R during the pubertal transition in female rats resulted in delayed puberty, as revealed by a combination of phenotypic and hormonal indices of pubertal maturation. These data are compatible and fully back-up our initial electrophysiological results and support a relevant physiological of MSH signaling in the metabolic control of GnRH neurons and puberty.
Finally, the demonstration of a putative role of GPR54 signaling in POMC neurons, as reported recently, has raised the possibility of direct effects of kisspeptins in the control of body weight homeostasis. Specific analyses in this sense have been conducted in this project, by the use of a genetically modified animal model where GPR54, and hence kisspeptin action, has been selectively eliminated from POMC neurons. Due to the complex breeding of this conditional knock-out mouse line, phenotypic, hormonal and metabolic analyses of this animal model are still in progress in order to elucidate the actual physiologic roles of kisspeptins in the functional modulation of POMC pathways and, therefore, the relevance, if any, of Kiss1 signaling in the direct regulation of body weight and its coupling with reproductive parameters.
In sum, we believe that this project has addressed relevant research questions, not only of fundamental (physiologic) importance, but also of translatable interest. Disorders of puberty and fertility are becoming increasingly common in Europe and other developed countries, and some of those conditions may be, at least partially, linked to the rapid increase in metabolic disorders. Thus, the knowledge obtained here pertaining to the physiological mechanisms and pathways coupling energy status and reproduction is likely to provide essential information for the rational management of different reproductive disorders that have attracted interest and concern not only from the scientific community but also of the lay public. In addition, the project has been endowed with very important formative contents, which have helped the fellow to expand his skills and methodological abilities in the area of Neurophysiology and Neuro-endocrinology in particular, and of experimental Biosciences in general.