Novel Insights into Minipuberty and GnRH: Implications on Neurodevelopment, Cognition and Covid-19 Therapeutics

Abstract

In humans, the first 1000 days of life are pivotal for brain and organism development. Shortly after birth, gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus are activated, a phenomenon known as minipuberty. This phenomenon, observed in all mammals studied, influences the postnatal development of the hypothalamic-pituitary-gonadal (HPG) axis and reproductive function. This review will put into perspective the results of recent studies showing that the impact of minipuberty extends beyond reproductive function, influencing sensory and cognitive maturation. Studies in mice have revealed the role of nitric oxide (NO) in regulating minipuberty amplitude, with NO deficiency linked to cognitive and olfactory deficits. Additionally, findings indicate that cognitive and sensory defects in adulthood in a mouse model of Down syndrome are associated with an age-dependent decline of GnRH production, whose origin can be traced back to minipuberty, and point to the potential therapeutic role of pulsatile GnRH administration in cognitive disorders. Furthermore, this review delves into the repercussions of COVID-19 on GnRH production, emphasizing potential consequences for neurodevelopment and cognitive function in infected individuals. Notably, GnRH neurons appear susceptible to SARS-CoV-2 infection, raising concerns about potential long-term effects on brain development and function. In conclusion, the intricate interplay between GnRH neurons, GnRH release, and the activity of various extrahypothalamic brain circuits reveals an unexpected role for these neuroendocrine neurons in the development and maintenance of sensory and cognitive functions, supplementing their established function in reproduction. Therapeutic interventions targeting the HPG axis, such as inhaled NO therapy in infancy and pulsatile GnRH administration in adults, emerge as promising approaches for addressing neurodevelopmental cognitive disorders and pathological aging.

In humans, the first 1000 days of life, from conception to 2 years of age, play a particularly critical role in the development of the brain and the rest of the organism. A significant part of morphological development and tissue differentiation occurs during this period through dynamic processes modulated by environmental stimuli. One of these processes, which starts during the second week of extrauterine life, is a phenomenon known as minipuberty, when gonadotropin-releasing hormone (GnRH) neurons of the hypothalamus, a brain region that regulates vital functions (appetite, growth, sleep, reproduction, etc.), are activated, leading to the first activation of the hypothalamic-pituitary-gonadal (HPG) or reproductive axis.

GnRH is a peptidergic neurohormone synthesized and released by a small population of neurons (2000 neurons out of the hundred billion neurons in the human brain, 700 neurons in mice) (1, 2). These neurons, which ensure the survival of the species, are unique to vertebrates. They originate in the nose during embryogenesis, and migrate to the brain during fetal life (1, 2). Distributed principally in the preoptic area of the hypothalamus, they project to the median eminence, where they secrete GnRH into the hypothalamic-pituitary portal circulation connecting the neuroendocrine hypothalamus to the anterior pituitary. This secretion is finely regulated by communication between GnRH neurons, endothelial cells, and specialized glial cells known as tanycytes, which line the floor of the third ventricle (3). Once carried by the blood to the anterior pituitary, GnRH stimulates the activity of gonadotropin-producing cells, influencing the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by acting on its cognate receptor, GnRHR (4). LH and FSH are subsequently released into the bloodstream, where they stimulate gonadal growth, gametogenesis, and the synthesis of gonadal steroids. In males, this includes testosterone production, while in females, it involves the production of estrogens. These gonadal steroids, in turn, form a positive or negative feedback loop at different levels of the HPG axis, modulating pulsatile GnRH/LH secretion in both sexes and controlling the onset of the preovulatory GnRH/LH surge in females (5–7).

Within the hypothalamus, kisspeptin and neuronal nitric oxide synthase (Nos1)- expressing neurons are two key targets of gonadal steroids, playing crucial roles in the upstream control of the GnRH population (8–11). Kisspeptin, a neuropeptide that acts through the kisspeptin receptor (Kiss1R) on GnRH neurons, positively influences their activity, leading to the stimulation of GnRH release (12, 13). In contrast, neuronal nitric oxide (NO), a highly diffusible neuromodulator produced by Nos1-neurons in the brain, acts as a negative regulator of GnRH activity and release (10, 14–16). Recent studies suggest that kisspeptin-expressing neurons and Nos1-expressing neurons form a microcircuit that delicately balances excitatory and inhibitory signals, contributing to the fine-tuning of GnRH activity and release (16, 17).

Minipuberty

Minipuberty, which occurs during a critical period for growth and brain development (18), could, according to preclinical data, play an important role in the maturation of the HPG axis and reproductive function in both sexes (10, 19). However, the specific impacts of minipuberty on reproductive health in female infants remain unclear, while it induces changes in the cellular composition of the testes in boys (18). During minipuberty in infants, circulating FSH levels peak around 1 to 3 months of extra-uterine life, yet in boys, unlike in girls, LH levels predominate (20). In mice and rats, minipuberty occurs at postnatal day 12 for females and approximately 10 days later for males, concluding before the emergence of the first external signs of sexual maturation, such as the vaginal opening and the balanopreputial separation (around P30) (10, 21, 22). This early postnatal rise in gonadotropins activates gonadal steroidogenesis, leading to increased aromatase activity in the immature ovary and testes, and the synthesis of estradiol (23–28). Estradiol then provides feedback influencing the maturation and programming of estrogen-sensitive neuronal circuits in the hypothalamus (10, 22) and throughout the brain (29) (Figure 1). It is noteworthy that in short-lived species like mice and rats, sexual maturation progresses continuously from minipuberty to puberty onset (Figure 2), while in long-lived species such as humans, the HPG system remains inactive for several years after minipuberty before reactivation during adolescence (18, 19, 30).

Figure 1. Minipuberty is a critical period for brain and body development.

During minipuberty, a critical period of infantile development that lasts only a few days in rodents and a few months in humans, there are major changes in the control of Gnrh expression and thus the activity of the reproductive axis that are vital to prime the gonad for future reproductive function after puberty. Minipuberty results in the activation of GnRH-synthesizing neurons and the induction of a peak in LH and FSH, which trigger steroidogenesis in the infantile gonads. This results in an increase in circulating gonadal steroids, which activate brain neurons expressing their receptors and, in particular, hypothalamic neurons producing nitric oxide (NO), which control both the amplitude and completion of minipuberty. Failure of infantile activation of NO-synthesizing neurons leads to impaired minipuberty and reproductive, metabolic, sensory and cognitive co-morbidities later in life. These can be prevented by infantile exposure to inhaled NO. Adapted from (47) with authorization.

Figure 2. Postnatal development of the neuronal microcircuit and miRNA-gene network in hypothalamic GnRH neurons leading to the onset of puberty and adult fertility in mice.

Schematic diagrams illustrating the potential contribution of the miR-155 and miR-200 families and their target genes to the increase in GnRH gene expression at minipuberty, and how these events could be intertwined with the integration of postmigratory GnRH neurons into the neural network responsible for regulating the timely onset of puberty. GnRH neuroglial network maturation (top panels). Although the morphological development of the hypothalamus is almost complete at birth, axons of the neurons located in the arcuate nucleus of the hypothalamus (ARH), which are thought to mediate at least part of the effects of gonadal steroid on the HPG axis, first reach the preoptic region during the infantile period, when astrogliogenesis, synaptogenesis, and dendritic pruning are also thought to occur. These maturational events are well positioned to trigger the miRNA-evoked changes in the transcription factor-gene network controlling GnRH promoter activity during the infantile period. Hormonal profiles (bottom panel). The hormonal profiles illustrated in the schematic are those for a female mouse. In females, the first postnatal activational period of the HPG axis at minipuberty coincides with the arrival of ARH fibers in the preoptic region, which results in an infantile surge of FSH that triggers steroidogenesis and the subsequent growth of the first pool of ovarian follicles, destined to ovulate at puberty, as well as the sporadic elevation of LH levels that contributes to their maturation. The second activational period occurs during the peripubertal period when a diurnal rhythm of LH release that accelerates the functional development of the ovaries is established. A third and final activational period coincides with the moment when ovarian follicles reach full maturity, i.e. the Graafian stage, and release massive amounts of ovarian steroids, specifically estrogens, which exert a positive-feedback effect on the HPG, coordinating the onset of the first preovulatory GnRH and LH/FSH surge and triggering the first ovulation, thus conferring fertility on the individual. In males, as in females, the primary events that initiate the onset of puberty originate within the hypothalamus. From (34) with permission.

While the initiation of minipuberty seems to be related to the wiring of post-migratory GnRH neurons to the regulatory neuron network, enabling them to perceive information about the internal and environmental conditions continuously (31), the regulation of its amplitude and duration appears to be linked to the awakening of a population of neurons secreting NO, through the first feedback loop exerted by estrogens on the hypothalamus (10, 22). Preclinical studies show that individuals lacking Nos1 exhibit exacerbated minipuberty (10), a phenomenon that occurs naturally in prematurely born girls (32), increasing the risk of delayed puberty and altered estrous cyclicity in adulthood (10, 33). Conversely, preclinical models have demonstrated that inhaled NO supplementation during infancy corrects these abnormally high minipubertal FSH levels and prevents the deleterious consequences of exacerbated minipuberty on reproduction (10).

At the molecular level, a key change occurs during minipuberty in the control of GnRH expression by hypothalamic neurons producing this peptide (34). This switch involves microRNAs (miRNAs) that modulate the expression of transcription factors acting as activators or repressors of the promoter of the Gnrh1 gene, which codes for GnRH expression (Figure 1). Two species of miRNA are particularly involved in this process: the miR-200/429 family, whose expression is enriched in GnRH neurons and increases during this critical period, and miR-155, whose expression also increases in this population of hypothalamic neurons during minipuberty (34). Alteration of the activation of these miRNA/transcription factor gene micro-networks, which are involved in the control of Gnrh1 promoter activity during minipuberty, can lead to the extinction of the expression of the gene and, consequently, the loss of GnRH production later in life (34). Among the transcription factors directly targeted by miR-155 and miR-200/429 are Cebpb and Zeb1 (35, 36), which are repressors of the Gnrh1 promoter, but also other transcription factors that activate this promoter, such as Otx2 (34, 37, 38). Notably, Cebpb codes for the CAAT/enhancer-binding protein (C/EBPβ), which requires the presence of NO to exert its repressive effect on the GnRH promoter (39). Interestingly, in addition to their direct control of the Gnrh promoter, Zeb1, and Cebpb could repress Gnrh gene transcription indirectly by blocking kisspeptidergic signaling through Kiss1R (34), which is known to increase Gnrh transcription by promoting the nuclear translocation of the Gnrh promoter activator Otx2 (40) (Figure 2).

At the neural circuit level, kisspeptin-containing fibers originating from the arcuate nucleus of the hypothalamus, which develop postnatally, extend to the preoptic region around P12 and achieve their final distribution by the end of the infantile period in both males and females (41, 42). The subsequent formation of new functional synaptic contacts with GnRH cell bodies favored by local astrogenesis (31) and dendritic pruning (43) could serve as triggers for the miRNA-mediated switch in controlling Gnrh promoter activity during minipuberty (Figure 2). It is noteworthy that the wiring of the GnRH neurons with the kisspeptin neurons of the anteroposterior periventricular nucleus, which establishes its projections to the medial preoptic nucleus during embryogenesis (41, 42), may also occur during minipuberty. Moreover, the estrogen-induced release of minipubertal NO, known for its role in synapse formation, elimination, and efficacy (44, 45), may not only control GnRH expression through modulation of the miRNA-gene network in GnRH neurons, but also influence the activity of newly established kisspeptin afferences. This interaction could potentially initiate rhythmic GnRH gene transcription and pulsatile GnRH secretion (46). Therefore, the tripartite kisspeptin/nNOS/GnRH (KiNG) neuronal network, activated by developmental and physiological cues during the infantile period, may play a pivotal role in the GnRH-driven onset of puberty and adult fertility (17).

A Role for Minipuberty in Neurodevelopment

Recent research suggests that minipuberty may affect functional brain development, particularly in the maturation of sensory functions such as olfaction and hearing, as well as cognitive functions (10), and their maintenance throughout life (47). In addition to reproductive alterations, the loss of expression of the Nos1 gene in mutant mice, or the presence of deleterious NOS1 mutations in patients with congenital hypogonadotropic hypogonadism, leads to olfactory, auditory and cognitive co-morbidities (10) (Figure 1). However, in Nos1-deficient animals, compensation for NO deficiency during infancy by inhalation of this gas restores olfactory and cognitive functions in adulthood (10), in addition to the normalization of sexual maturation as mentioned earlier. These findings underline the critical role of Nos1 activity during infancy for the development of the organism, including the brain.

Around one child in ten is born prematurely, i.e. before the end of the 37th week of gestation. According to the World Health Organization, these children are predisposed to certain neurological disorders (behavioral and learning impairments, autism), as well as certain metabolic disorders (diabetes, obesity), fertility disorders and cardiovascular disorders (48–50). In these infants, minipuberty is characterized by abnormally high levels of circulating FSH (20, 32). Given the importance of minipuberty for the development of the central nervous system in mice (10, 51), and the beneficial effect of normalizing this period with inhaled NO (10), a treatment already administered to prematurely born children to promote maturation and pulmonary vascularization (52, 53), prolonging this treatment in these children during the minipubertal period could potentially have a beneficial effect on their brain development. This hypothesis is currently being tested in a clinical trial conducted jointly at the Lille University Hospital and Athens Paediatric Hospital (Greece) as part of the European miniNO project (https://minino-project.com). The unexpected discovery of the involvement of minipuberty in the establishment of sensory and cognitive functions in animal models, and the current testing of its relevance to human health, offer hope for improving the management of prematurely born individuals and certain neurodevelopmental disorders.

Minipuberty, Down Syndrome and Adult Cognition

The presence of the gene encoding miR-155 on the human chromosome 21 and the mouse chromosome 16 (which carries most of the genes homologous to the human chromosome 21) suggests the possibility of altered expression of GnRH in Down syndrome, caused by trisomy 21, and murine models of this chromosomal anomaly, such as the trisomic Ts65Dn mouse line (54). Exploring this lead showed that Ts65Dn mice not only exhibited infertility or sexually dimorphic subfertility, similar to that found in men and women with Down syndrome (55, 56), but also the progressive onset of olfactory and cognitive deficits with age (51, 57). These neurological symptoms, independent of reproductive function, are closely associated with a loss of GnRH production and its transport along neuronal fibers, particularly those projecting to extrahypothalamic regions, including the hippocampus and cerebral cortex (51). These cortical brain structures harbor GnRH-receptor-expressing neuronal populations specifically involved in the maintenance of sensory and cognitive function (51). Intriguingly, the GnRH-containing extrahypothalamic projections do not stem from a separate group of GnRH neurons, but rather from the same neuroendocrine GnRH neurons that project to the median eminence (51). In Ts65Dn mice, this loss of GnRH production results in changes in the levels and mode of release of LH into the blood (51).

It is interesting to note that in Ts65Dn mice, the decrease in GnRH expression in adulthood is accompanied by an imbalance in the complex network of microRNAs and transcription factors that control GnRH expression (51). Quite unexpectedly, miR-155 expression is not increased in the preoptic region of the hypothalamus in Ts65Dn mice but rather tends to be decreased and preoptic Cebpb expression is increased. Additionally, the expression of all members of the miR-200 family (not present on mouse chromosome 16) are greatly reduced, resulting in increased expression of Zeb1 and decreased expression of Otx2 in GnRH neurons from the minipubertal period, well before the onset of cognitive or olfactory deficits (51) (Figure 3). The hypothalamic alteration of miR-200 seems to lead to a modification of communication between the preoptic region of the hypothalamus and the hippocampus. This results in changes in the expression of a number of genes in the hippocampus, including those involved in myelination and synaptic transmission, as well as a modification of neuronal connectivity between the left and right hippocampus in vivo (51). The overexpression of miR-200b in the preoptic region of the adult hypothalamus through gene therapy reverses both gene expression modifications and neuronal transmission deficits in the hippocampus, correcting olfactory and cognitive impairments in Ts65Dn mice (Figure 3) (51). This therapy not only reactivates GnRH expression at the transcriptional level during adulthood but also enhances the proportion of neurons expressing the transcriptional activator Otx2. Otx2 is known to regulate the timing of other crucial periods of brain maturation (58). Confirming the link to the restoration of a physiological GnRH secretion rhythm, cell therapy using wild-type hypothalamic neurons, along with chemogenetic and pharmacological interventions to induce GnRH at physiological levels and in a pulsatile pattern, also significantly improved olfactory and cognitive deficits in adult Ts65Dn mice (51). Finally, since pulsatile GnRH administration is already used as a treatment to restore fertility in human patients (59), a pilot clinical study was conducted in adult men with Down syndrome. The 6-month treatment with a GnRH pump was well-tolerated and improved both intellectual performance and resting-state functional connectivity, visualized by functional MRI, in certain circuits known to be altered in this syndrome (51). Notably, pulsatile GnRH therapy decreased resting neuronal connectivity between the hippocampus and amygdala, known to be altered in individuals with Down syndrome and involved in anxiety phenomena (60), while it increased resting functional connectivity in large regions of the cerebral cortex, including networks connecting visual and sensorimotor areas, which are known to be less active in this syndrome (61), to levels close to those observed in the general population (51).

Figure 3. GnRH supplementation improves cognition in Ts65Dn mice, a model of trisomy 21, and in people with Down syndrome.

Individuals with Down's syndrome suffer from olfactory impairment and cognitive problems in addition to intellectual disability and impaired maturation of reproductive function. (a) Some of the neuroendocrine neurons that synthesize GnRH, the hormone that controls the endocrine axis of reproduction, project their axons to areas of the brain involved in cognition, such as the hippocampus. Gnrh expression is tightly controlled by a miRNA-transcription-factor micronetwork involving the miR200 family and repressors (Zeb1) and activators (Otx2) of the activity of the Gnrh promoter. (b) In Ts65Dn mice, whose pathological phenotype is similar to that of patients with trisomy 21, the expression of GnRH by hypothalamic neurons decreases progressively during postnatal development due to an alteration of the miRNA-transcription-factor micronetwork regulating its transcription. (c) Overexpression of miR200b in the preoptic region and subcutaneous implantation of a programmable minipump enabling pulsatile administration of GnRH in adult Ts65Dn mice both rescue cognitive and olfactory performances in these mice modeling Down syndrome 3 months and two weeks after the initiation of the treatment, respectively. (d) Pulsatile GnRH therapy in adult volunteers with trisomy 21 is seen to improve functional brain connectivity and cognition after 6 months of treatment. LH: luteinizing hormone; POA: preoptic area; OVLT: vascular organ of the terminal lamina. Figure created in part with BioRender.com.

GnRH and brain aging

Some forms of dementia, such as Alzheimer's disease (AD), are associated with an imbalance in the expression of myelination genes (62), a pattern shared with Ts65Dn mice (51). Conversely, genes associated with GnRH signaling show a significant down-regulation in specific cortical regions in postmortem brains from AD patients (63), and a similar dysregulation is observed in the hippocampus of the Thy::Tau22 mouse AD model (64). This model is characterized by progressive hippocampal Tau pathology and cognitive deficits (64, 65). In 12-month-old male Thy::Tau22 mice, LH pulse frequency remains normal, while LH pulse amplitude decreases (Figure 4a), mirroring LH pulsatile pattern alterations in adult Ts65Dn mice (51). The infusion of pulsatile GnRH rescues odor discrimination and object recognition memory in these mice (Figure 4a) (51), suggesting that the loss of GnRH might contribute to olfactory and cognitive deficits in various neurodegenerative disorders, and that such deficits can potentially be reversed through GnRH replacement, possibly via the same molecular pathways (66) (Figure 4b). By drawing parallels between Down syndrome and Alzheimer's disease (AD), we find that the down-regulation of Afg3l2, a mitochondrial gene, in the hippocampus of Ts65Dn mice treated with miR-200b overexpression in the preoptic region, is linked to the prevention of demyelination and Tau hyperphosphorylation, traits reminiscent of the AD-like phenotype observed in Down syndrome (67, 68). Additionally, DUSP6, mutations of which have been found in individuals with hypogonadotropic hypogonadism (69), shows hypermethylation in AD, thereby inhibiting Tau hyperphosphorylation (70, 71). These gene alterations in Ts65Dn mice provide a link between reproductive dysfunction and AD-like cognitive and neurodegenerative changes, resembling the phenotype observed in Down syndrome patients.

Figure 4. Pulsatile GnRH treatment rescues olfaction and cognition in a mouse model of Alzheimer’s disease.

(a) LH pulsatility, olfaction, and cognition are impaired in 12-month-old mice expressing the pathogenic TAU22 under the control of the neuronal THY promoter that models some aspects of Alzheimer disease. Left panel. Assessment of pulsatile LH release by serial blood sampling in THY::TAU22 mice showed that they had a significantly decreased LH pulse amplitude. Right panels. Pulsatile GnRH (Lutrelf®) rescues the capacity of THY::TAU22 mice to discriminate between different odors (middle panel) and objects (right panel). From (51) with permission. (b) Alterations in the pattern of hypothalamic GnRH release can impair cognitive reserves during aging, a phenomenon that could potentially be reversed with pulsatile GnRH therapy, even in the context of Alzheimer disease. Adapted from (66) with permission.

These unforeseen discoveries regarding the cognitive impact of GnRH hold considerable promise (47, 72, 73). For instance, in menopause, women commonly report experiencing "brain fog," a symptom linked to hippocampal changes associated with lack of the sex steroid estradiol (74). Given that estradiol production depends on GnRH regulation of gonadotropin release, and that menopause decelerates GnRH pulse frequency over age (75), it is conceivable that normalizing GnRH pulsatile release through implantable minipumps could prevent menopause-related memory deficits and "brain fog".

Diminished reproductive function in later stages of life is not limited to women, and men also undergo altered gonadotropin release with age (76). This suggests that aging men may experience changes in GnRH release patterns, affecting pulse amplitude and frequency. If GnRH neurons projecting to cognitive centers in the brain lose their ability to release GnRH at appropriate levels or patterns in older adults, pulsatile GnRH administration could represent a promising therapeutic approach to enhance cognitive function in various conditions marked by cognitive decline, all of which are characterized by impaired GnRH neuron function (77), with minimal anticipated side effects.

COVID-19-induced GnRH deficiency

COVID-19 infection seems to be linked to accelerated aging and an elevated risk of neurodegenerative conditions like Alzheimer's disease in affected individuals (78–82). Furthermore, amidst the ongoing emergence of new variants of SARS-CoV-2, the virus causing COVID-19, the focus is shifting towards "long COVID" or "post-COVID-19 syndrome" rather than acute infections, which is a major concern from both healthcare and economic perspectives. Although definitions of "long COVID" vary, a noteworthy number of individuals who have had SARS-CoV-2 infections continue to exhibit symptoms consistent with reports of the virus invading the nervous system. These symptoms include fatigue, cognitive difficulties or "brain fog," headaches, and persistent anosmia, persisting for several months to over a year after the initial infection (83–86). Of note, a significant proportion of male COVID-19 patients also experience low testosterone levels that can endure for months after recovering from infection, resembling the absence or alteration of GnRH production or secretion and the dysfunction of the HPG axis (87, 88).

Our work indicates that persistent hypotestosteronemia in certain men may have a hypothalamic origin, potentially contributing to cognitive or neurological symptoms post-COVID (89). The infection of olfactory sensory neurons and tanycytes suggests at least two plausible routes for neuroinvasion: the olfactory and the blood-borne routes. Additionally, GnRH neurons, which were themselves infected (Figure 5a), were found to be sick or dying in all patient brains examined, leading to a significant reduction in GnRH expression in the tuberal region of the hypothalamus (Figure 5c) (89).

Figure 5. SARS-CoV-2 infects GnRH neurons and leads to their death in COVID-19 patients.

(a) Immunolabeling for GnRH (red), ACE2 (white), and S-protein (green) in hypothalamic GnRH neurons in a COVID-19 patient. Arrowheads show a triple-labelled GnRH neuron, white arrows show a GnRH-immunoreactive process that does not express ACE2, and empty arrows show an ACE2-immunoreactive neuron-like process that does not express GnRH. Blue: DAPI. Scale bar: 50 □m. (b) Immunolabeling of the SARS-CoV-2 S-protein in human GnRH-secreting FNC-B4 cells. Scale bar: 20 µm. (c) Immunolabeling for GnRH (red) and cleaved caspase 3 (green) in the infundibular nucleus (Inf) - median eminence (ME) area of the hypothalamus of a COVID-19 patient. Blue: DAPI. Scale bars: 500 μm (inset 30 μm). (d) Schematic representation of a horizontal section through the nose and brain of a gestational week (GW) 14 human fetus, showing region immunolabeled in (e-g). (e-g) TMPRSS2 (e,f, red) and ACE2 (g, red) immunolabeling in the olfactory epithelium (OE), vomeronasal organ (VNO) and olfactory nerve (ON) of a GW 14 fetus. Blue: DAPI. Scale bars: 1 mm in e and 100 μm in c-d. (h) In a GW 11 human fetus, many GnRH neurons (white) migrating out of the VNO also express NRP1 (green) and/or ACE2 (red), host cell proteins that mediate SARS-CoV-2 infection (white arrows), while NRP1 and ACE2 are also expressed by some olfactory and vomeronasal nerve axons that form the scaffold for GnRH neurons. Blue: DAPI. Scale bar: 40 µm. From (89) with permission.

The presence of an olfactory route, coupled with the susceptibility of GnRH neurons to SARS-CoV-2, evokes the possibility that these neurons, which originate in the nasal region, could be infected during embryonic development or early childhood. Examination of the olfactory epithelium in human fetuses aged 7, 11, and 14 weeks revealed notable expression of two receptors crucial for SARS-CoV-2 infection, namely angiotensin-converting enzyme 2 (ACE2) and the priming enzyme for the viral spike protein, transmembrane protease, serine 2 (TMPRSS2) (90). This expression was observed in both olfactory sensory neurons and their axons that extend into the olfactory bulb (Figure 5d-e) (89). This discovery corresponds with previous findings indicating the presence of these susceptibility factors in various cell populations within the olfactory epithelium (91). Notably, neuropilin 1 (NRP1) (92, 93), a SARS-CoV-2 co-receptor (94–96), was also expressed in addition to ACE2 by GnRH neurons within the putative vomeronasal organ, their birthplace, as well as by the axonal tracts along which they migrate into the brain (Figure 5h) (89). This suggests a potential enhancement of SARS-CoV-2 cell entry and infection within this critical neuronal population.

To directly investigate the susceptibility of human fetal GnRH neurons to SARS-CoV-2 infection, experiments were conducted using pseudotyped viral particles expressing the full-length SARS-CoV-2 spike protein and the ZsGreen reporter gene, as well as the native SARS-CoV-2 virus (Figure 5b), on a human fetal GnRH-expressing cell line, FNC-B4 (89, 97). Notably, during the differentiation into GnRH neurons, cells not only expressed ACE2, unlike their non-GnRH expressing counterparts, but also exhibited infection by the pseudovirus or the native SARS-CoV-2 virus (89). These experiments strongly suggest the potential for at least some GnRH neurons in human fetuses or newborns being infected by SARS-CoV-2 in case of vertical transmission from infected mothers (see, for example, (98, 99)), potentially leading to long-term mental and non-mental consequences later in life.

In light of the unique vulnerability of fetal GnRH neurons, it is crucial to carefully consider the implications of maternal or perinatal COVID-19 infection on neonates (100). The emerging evidence that some neonates born to infected mothers may test positive for COVID-19 is particularly worrisome (101). This concern arises because minipuberty, as mentioned in the previous sections, plays a crucial role in the subsequent maturation of the reproductive system (31, 34) and likely also in broader aspects of brain development (10). The disruption of minipuberty, for instance due to premature birth, may be associated with the occurrence of various age-related noncommunicable diseases or metabolic dysfunctions (48, 49). Early reports also suggest that antenatal or neonatal exposure to SARS-CoV-2 could result in neurodevelopmental delays (102). Studies that follow cohorts of babies born during the pandemic, such as the mini-COVID study conducted by the European miniNO consortium (https://www.minino-project.com; ClinicalTrials.gov ID NCT04952870), are therefore crucial to fully understanding the repercussions of these often-asymptomatic infections on healthy aging. Such studies are essential for the implementation of proactive measures to mitigate any potential negative impacts.

All in all, the control and maintenance of GnRH production by the hypothalamus after birth appear to play an essential role in the development and maturation of the HPG axis, but also of the brain in general, including the development of sensory and cognitive functions. Viral infections, such as SARS-CoV-2, which can impact both humans and animals, may pose a risk to the course of minipuberty and consequently contribute to infertility as well as sensory and cognitive disorders later in life (103). Conversely, inhaled NO therapy in infants and pulsatile GnRH administration in adults, respectively, appear to be promising for improving neurodevelopment and reversing cognitive deficits. Intriguingly, some pre-clinical data suggest that pulsatile GnRH could also be used to mobilize the cognitive reserve in certain dementias, such as Alzheimer's disease.