Neuropilin-1 expression in GnRH neurons regulates prepubertal weight gain and sexual attraction.

Hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH), the "master molecule" regulating reproduction and fertility, migrate from their birthplace in the nose to their destination using a system of guidance cues, which include the semaphorins and their receptors, the neuropilins and plexins, among others. Here, we show that selectively deleting neuropilin-1 in new GnRH neurons enhances their survival and migration, resulting in excess neurons in the hypothalamus and in their unusual accumulation in the accessory olfactory bulb, as well as an acceleration of mature patterns of activity. In female mice, these alterations result in early prepubertal weight gain, premature attraction to male odors, and precocious puberty. Our findings suggest that rather than being influenced by peripheral energy state, GnRH neurons themselves, through neuropilin-semaphorin signaling, might engineer the timing of puberty by regulating peripheral adiposity and behavioral switches, thus acting as a bridge between the reproductive and metabolic axes.

Brain endothelial cells control fertility through ovarian-steroid-dependent release of semaphorin 3A.

Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3aloxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction.

SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome.

Kallmann syndrome (KS) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. The genetics of KS involves various modes of transmission, including oligogenic inheritance. Here, we report that Nrp1(sema/sema) mutant mice that lack a functional semaphorin-binding domain in neuropilin-1, an obligatory coreceptor of semaphorin-3A, have a KS-like phenotype. Pathohistological analysis of these mice indeed showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in increased mortality of newborn mice and reduced fertility in adults. We thus screened 386 KS patients for the presence of mutations in SEMA3A (by Sanger sequencing of all 17 coding exons and flanking splice sites) and identified nonsynonymous mutations in 24 patients, specifically, a frameshifting small deletion (D538fsX31) and seven different missense mutations (R66W, N153S, I400V, V435I, T688A, R730Q, R733H). All the mutations were found in heterozygous state. Seven mutations resulted in impaired secretion of semaphorin-3A by transfected COS-7 cells (D538fsX31, R66W, V435I) or reduced signaling activity of the secreted protein in the GN11 cell line derived from embryonic GnRH cells (N153S, I400V, T688A, R733H), which strongly suggests that these mutations have a pathogenic effect. Notably, mutations in other KS genes had already been identified, in heterozygous state, in five of these patients. Our findings indicate that semaphorin-3A signaling insufficiency contributes to the pathogenesis of KS and further substantiate the oligogenic pattern of inheritance in this developmental disorder.

Kisspeptin-GPR54 signaling in mouse NO-synthesizing neurons participates in the hypothalamic control of ovulation.

Reproduction is controlled in the brain by a neural network that drives the secretion of gonadotropin-releasing hormone (GnRH). Various permissive homeostatic signals must be integrated to achieve ovulation in mammals. However, the neural events controlling the timely activation of GnRH neurons are not completely understood. Here we show that kisspeptin, a potent activator of GnRH neuronal activity, directly communicates with neurons that synthesize the gaseous transmitter nitric oxide (NO) in the preoptic region to coordinate the progression of the ovarian cycle. Using a transgenic Gpr54-null IRES-LacZ knock-in mouse model, we demonstrate that neurons containing neuronal NO synthase (nNOS), which are morphologically associated with kisspeptin fibers, express the kisspeptin receptor GPR54 in the preoptic region, but not in the tuberal region of the hypothalamus. The activation of kisspeptin signaling in preoptic neurons promotes the activation of nNOS through its phosphorylation on serine 1412 via the AKT pathway and mimics the positive feedback effects of estrogens. Finally, we show that while NO release restrains the reproductive axis at stages of the ovarian cycle during which estrogens exert their inhibitory feedback, it is required for the kisspeptin-dependent preovulatory activation of GnRH neurons. Thus, interactions between kisspeptin and nNOS neurons may play a central role in regulating the hypothalamic-pituitary-gonadal axis in vivo.

Nitric oxide as key mediator of neuron-to-neuron and endothelia-to-glia communication involved in the neuroendocrine control of reproduction.

Nitric oxide (NO) is a peculiar chemical transmitter that freely diffuses through aqueous and lipid environments and plays a role in major aspects of brain function. Within the hypothalamus, NO exerts critical effects upon the gonadotropin-releasing hormone (GnRH) network to maintain fertility. Here, we review recent evidence that NO regulates major aspects of the GnRH neuron physiology. Far more active than once thought, NO powerfully controls GnRH neuronal activity, GnRH release and structural plasticity at the neurohemal junction. In the preoptic region, neuronal nitric oxide synthase (nNOS) activity is tightly regulated by estrogens and is found to be maximal at the proestrus stage. Natural fluctuations of estrogens control both the differential coupling of this Ca²+-activated enzyme to glutamate N-methyl-D-aspartic acid receptor channels and phosphorylation-mediated nNOS activation. Furthermore, NO endogenously produced by neurons expressing nNOS acutely and directly suppresses spontaneous firing in GnRH neurons, which suggests that neuronal NO may serve as a synchronizing switch within the preoptic region. At the median eminence, NO is spontaneously released from an endothelial source and follows a pulsatile and cyclic pattern of secretion. Importantly, GnRH release appears to be causally related to endothelial NO release. NO is also highly involved in mediating the dialogue set in motion between vascular endothelial cells and tanycytes that control the direct access of GnRH neurons to the pituitary portal blood during the estrous cycle. Altogether, these data raise the intriguing possibility that the neuroendocrine brain uses NO to coordinate both GnRH neuronal activity and GnRH release at key stages of reproductive physiology.

Cyclin dependent kinase 5 (Cdk5) mediated inhibition of the MAP kinase pathway results in CREB down regulation and apoptosis in PC12 cells.

Cyclin dependent kinase 5 (cdk5) is active mainly in postmitotic cells like neurons and regulates important cellular functions by phosphorylating a wide variety of targets. Nerve growth factor stimulates the MEK-ERK MAP kinase pathway and causes neuronal differentiation and survival. It was reported previously that Cdk5 inhibits the MAP kinase pathway by phosphorylating Map kinase kinase-1 (MEK1) [1]. We have delineated the functional consequence of this cross talk and found that the cdk5 mediated inhibition of MEK1 results in apoptosis. We also demonstrate that the activity of transcription factor CREB, which is known to play pro-survival roles in neuronal cells, is attenuated as a result of this cross-talk.