Tachykinin signaling in the control of puberty onset.

Abstract

The tachykinin family of peptides has emerged as a critical component of the central control of the reproductive axis. Mounting evidence suggests that neurokinin B (NKB) plays an essential role in sexual maturation and fertility by directly stimulating the release of kisspeptin, with the contribution of additional tachykinins (neurokinin A [NKA] and substance P [SP]) in the fine tuning of the activity of Kiss1 neurons. The expression of tachykinins increases in the hypothalamus before puberty and, therefore, they are considered as initiators of pubertal development by stimulating the awakening of Kiss1 neurons. This is supported by studies showing delayed or absent puberty onset in humans and mice devoid of tachykinin signaling, and the advancement of puberty onset in rodents subjected to chronic activation of tachykinin receptors. This review compiles the current knowledge on the role of tachykinins in the control of puberty onset.

1. What are tachykinins?

Tachykinins are a family of peptides that includes substance P (SP), neurokinin A (NKA), and neurokinin B (NKB)¹. While neurokinins are mostly restricted to the central nervous system, SP is also present in a number of peripheral tissues¹. These tachykinins bind preferentially to NK1R, NK2R and NK3R, respectively, although a large degree of cross-activation has been documented for all three ligand-receptor tachykinin systems^1,2. A number of neuropathophysiological processes have been related to the misfunction of tachykinin systems (mostly SP and NKB) in anxiety, learning, fear conditioning or cognitive behavior¹. However, a less characterized role of tachykinins pertained to their action within the hypothalamic-pituitary-gonadal (HPG) axis. A seminal publication in 2009 reporting that the loss of function of the genes encoding for NKB or NK3R in humans (TAC3/TACR3) led to hypogonadotropic hypogonadism, with a severe impairment in these patients to achieve puberty onset and fertility³, rapidly placed tachykinins in the spotlight for reproductive biologists.

Over the last decade, the characterization of the reproductive role of tachykinins has grown exponentially^4,5. Because of the human studies cited above, the NKB/NK3R system has received most of the attention in this area. This interest was further increased by the observation that NKB and its receptor, NK3R, co-localize in the majority of Kiss1 neurons of the arcuate nucleus of most studied mammalian species, including sheep, mice, primates and humans^6–9. The importance of this finding lies in the critical role of Kiss1 neurons for reproductive function.

Kiss1 neurons are located mostly in two distinct hypothalamic areas, the arcuate nucleus (ARH) (infundibular nucleus in humans) and the anteroventral periventricular area (AVPV/PeN)-almost exclusive to the female brain¹⁰. Studies from our lab and others have documented that while the co-expression of NKB and its receptor in Kiss1^ARH neurons is >90%, NKB is absent in Kiss1^AVPV/PeN neurons, with only a fourth of this population expressing NK3R^8,11. This distribution is indicative of the role of tachykinins in reproduction. Kiss1^ARH are considered the master regulatory component of the HPG axis⁴, particularly in the mediation of the negative feedback of sex steroids and the generation of the tonic (pulsatile) release of gonadotropin-releasing hormone (GnRH)⁴ from GnRH neurons. Regarding SP and NKA, because they are encoded by the same gene (Tac1), both peptides are cleaved from a precursor peptide in the same neurons, which are different from any of the Kiss1 neuron populations^4,11. Rather, Tac1 neurons are sparse within the ARH but abundant in the adjacent ventral hypothalamic area (VMH)¹¹. While evidence that the VMH is the source of the SP and NKA that control GnRH release is currently missing, given the proximity to the ARH and their regulation by sex steroids in a similar manner to Kiss1^ARH neurons¹¹, i.e. inhibited, it is likely that the reproductive role of Tac1 is played by, at least, VMH neurons. Importantly, ~50% of Kiss1^ARH neurons and ~25% of Kiss1^AVPV/PeN neurons express NK1R, while none of them express NKA’s receptor (NK2R)¹¹, suggesting a) that SP likely plays a direct role on Kiss1 neurons, and b) the existence of yet unknown intermediate neurons in the action of NKA in reproduction. An important factor in the overall reproductive role of tachykinins is the limited expression of their receptors in GnRH neurons. Only a small fraction of these neurons express NK1R and NK3R¹¹ which, together with the absence of any effect on gonadotropin release in the absence of kisspeptin signaling^11–13, strongly supports a role for all tachykinins on, or above, Kiss1^ARH neurons.

In this context, these three tachykinins have been reported to have an overall stimulatory action on LH release in several species^9,14–21. However, the gonadotropic stimulatory action of NKA and NKB (but not SP) is dependent on the existence of circulating sex steroids, which turns inhibitory in their absence, e.g. after gonadectomy, when the central components of the HPG axis are hyper-stimulated by the absence of the inhibitory action of sex steroids^5,11,21,22. This phenomenon appears to be mediated by the increase in the inhibitory tone of dynorphin A^5,21,23. NKB appears to play the most fundamental role of all tachykinins by serving as the “accelerator” of the GnRH pulse generator within Kiss1^ARH neurons in a process that is dependent on the level of sex steroids. NKB acts autosynaptically on Kiss1^ARH neurons to induce the release of a kisspeptin pulse. Every pulse is then ceased by the inhibitory action of dynorphin A on the same neurons. A number of chemo and opto-genetic studies have confirmed this role of Kiss1^ARH neurons as the GnRH pulse generator^24–26, which is critical for the attainment and maintenance of reproductive function, since it is highly dependent on the pattern of pulsatile GnRH release.

Given this significant regulatory action of tachykinins on gonadotropin release and the fact that humans and mouse models with inactivating mutations in one of these genes have delayed or absent puberty onset^3,27–29, there is currently a general consensus that tachykinin action is necessary for the proper maturation of the reproductive axis (i.e. puberty onset), however, the underlaying mechanisms and overall contribution of tachykinins to this developmental process remain largely unknown.

2. What drives puberty onset?

Unraveling the molecular and physiological mechanisms that trigger puberty onset has been a major goal for neurobiologists and pediatricians for the last decades. Important advances have been made since the identification of the “reawakening” of GnRH pulsatility during the late juvenile period as the main event to kickstart sexual maturation³⁰. Since then, an extensive number of publications have characterized an ever-growing number of excitatory signals that get activated, and inhibitory signals that reduce their expression and tone on the HPG axis, either right before puberty onset, or during earlier developmental periods through the induction of epigenetic modifications that affect pubertal progression³¹.

As mentioned earlier, GnRH neurons are devoid of most of the necessary regulatory elements and receptors to accurately determine the time of puberty onset³⁰. Therefore, the existing consensus is that GnRH neurons are surrogated to the action of Kiss1 neurons during this process. So, what determines the timing of the activation of Kiss1 neurons? It is worth mentioning that while Kiss1 neurons are necessary for puberty onset, there is no clear evidence as to which population (ARH vs AVPV/PeN) plays a more determinant role in this process and even reports of puberty onset in the absence of Kiss1 neurons have been described³². In this context, it is important to highlight the fact that the population of Kiss1 neurons is highly redundant and as few as 5% of these neurons are sufficient to drive sexual maturation³³, which further emphasizes the need for a complete characterization of the role of each Kiss1 neuron population in this process. In rodents, within the whole hypothalamus, Kiss1 expression increases as the timing of puberty onset approaches³⁴, however, more precise studies investigating the ARH vs the AVPV/PeN have demonstrated that Kiss1 expression in the ARH does not vary significantly across puberty³⁵, while it increases in the preoptic area³⁶. Yet, this increase in Kiss1 expression in the preoptic area may be a secondary effect of the rise in circulating sex steroids. In any case, it is possible that the increase in the kisspeptidergic tone on GnRH neurons happens only at the level of kisspeptin release without significant changes in the expression of the gene in the ARH. This hypothesis remains to be investigated.

3. What is the role of tachykinins in puberty onset?

Despite the lack of discernable changes in Kiss1 expression in the ARH across puberty, that of Tac1 and Tac2 (encoding NKB in rodents) in the ARH have been documented to increase^28,35, leading to the hypothesis that tachykinins impose the initial activation of Kiss1 neurons to ramp up the pulsatile release of kisspeptin (and therefore GnRH and LH). This role would explain why NKB signaling deficient patients fail to undergo proper pubertal transition^3,27. However, unlike in the absence of kisspeptin signaling, where the hypogonadism prevents any possibility of reproduction in humans and mice^37–39, the reproductive phenotype of NKB signaling deficiency in these species is milder^29,40. First, a number of the human patients described above experience sporadic activation of the HPG axis that, in some cases, may even allow successful pregnancies⁴¹. Second, Tac2 knockout (KO) and Tacr3KO mice display delayed puberty onset but overall preserved fertility^29,40. Together, these studies suggest that NKB signaling may be critical for the acute timing of puberty onset but, in its absence, compensatory mechanisms take place to overcome its deficit. However, there is evidence that specific NK3R antagonists inhibit the tonic release of LH release in male and female monkeys as well as in normal men and pre- and post-menopausal women^42–46, indicating that in these species compensation to the acute blockade of NKB signaling does not take place. While species differences between the primate and rodent models cannot be excluded, these data suggest that compensation for the absence of NKB signaling in primates only occurs developmentally after congenital deficiency.

In fact, we have observed that humans and mice with NKB deficiency retain the ability to release LH pulses^47,48, suggesting that Kiss1 neurons are still able to produce kisspeptin pulses in the face of NKB deficiency. Interestingly, Tac2KO mice present a higher frequency and amplitude of pulses in their gonad intact state and lower frequency and amplitude after gonadectomy (unpublished data). This reversed regulation of the LH pulse frequency (i.e. faster frequency in intact NKB deficient individuals) is similarly observed in TAC3 mutant patients^41,47. Therefore, in situations of low sex steroid milieu, such as prepubertally or after gonadectomy, and absent NKB signaling, the GnRH pulse generator manifests a deceleration that leads to delayed puberty onset and delayed compensatory rise of LH after gonadectomy, at least in the mouse^21,29,48.

A 2019 optogenetic study in mice further validated the pivotal role of NKB and Dyn in the initiation and termination, respectively, of each kisspeptin/GnRH/LH pulse²⁶. This study demonstrated that NKB is sufficient to induce a kisspeptin pulse in the presence of sufficient basal activity of the neuron. However, it also presented the possibility that additional factors, from outside of Kiss1^ARH neurons, could modify this basal activity and, therefore, modulate the response of the neuron to NKB. Among these factors, we can include SP and NKA.

Because SP and NKA can stimulate gonadotropin release through the action on NK1R and NK2R, respectively, and through the documented cross-reactivity of the ligand-receptor systems (e.g. SP can also bind NK3R)², a plausible hypothesis is that NKB deficient patients and mice attained reproductive capabilities via the compensatory action of the remaining tachykinin systems. Interestingly, through the generation of double Tac1/Tac2KO mice, our lab recently documented that under the complete absence of tachykinin signaling, the profile of LH release closely resembles that of Tac2KO mice, i.e. the presence of rudimentary LH pulses^47,48. Nonetheless, these pulses are sufficient to induce (delayed) puberty onset in males (preputial separation) and females (vaginal opening)^29,48. These makers suggest the existence of rising sex steroid levels, typical of pubertal progression. However, females failed to exhibit first estrus, which indicates a central deficit in pubertal transition⁴⁸. Altogether, these data suggest that without tachykinin signaling, compensatory mechanisms develop to allow a basal level of activity in Kiss1 neurons that leads to sufficient GnRH pulses to activate the HPG axis, albeit with a delay in timing compared to controls. Moreover, tachykinins may also play a role in the induction of ovulation and likely menarche in humans, which may be compensated between the different tachykinin systems, and therefore only exposed after the complete removal of all tachykinins⁴⁸.

4. Summary and conclusions

Overall, and despite initial expectations of the pivotal role of NKB (and by extension the other tachykinins) as a fundamental piece of the GnRH pulse generator, recent evidence from humans and rodent genetic models points to a less fundamental role of tachykinins than what surmises from the initial publications. While it is true that the reproductive impairments in human patients bearing NKB signaling deficiency are significantly more severe than in the equivalent mouse models, reversal of this phenotype is relatively frequent^41,47, and mice largely preserve fertility in the absence of NKB signaling^29,40. On one hand, this suggests that either compensatory mechanisms develop (which are independent of tachykinins, at least for the tonic release of GnRH) or that Kiss1 neurons are equipped with an intrinsic pulsatile mechanism that allows for a basal tone of GnRH release, sufficient to eventually induce gonadal activity. This latter contention is supported by the fact that Kiss1 neurons present a pattern of activity that is consistent with a pacemaker type of neuron⁴⁹. On the other hand, this ability of the central elements of the HPG axis to overcome the absence of a potent factor in the fine tuning of the activity of the Kiss1 and, by extension, GnRH neurons, speaks to the evolutionary forces that have led to the existence of a number of failsafe mechanisms to ensure the successful attainment of reproduction, crucial for the perpetuation of the species. Moreover, this “wiggle room” in the action (and activation) of the different tachykinin systems between them and the participation of unknown factors that maintain the activity of Kiss1 neurons in their absence, opens the door to the development of novel approaches to treat reproductive impairments of central origin, offering new strategies to eventually modify the output of kisspeptin and GnRH release.