Immunotherapy-related cognitive impairment after CAR T cell therapy in mice.

Persistent central nervous system (CNS) immune dysregulation and consequent dysfunction of multiple neural cell types is central to the neurobiological underpinnings of a cognitive impairment syndrome that can occur following traditional cancer therapies or certain infections. Immunotherapies have revolutionized cancer care for many tumor types, but the potential long-term cognitive sequelae are incompletely understood. Here, we demonstrate in mouse models that chimeric antigen receptor (CAR) T cell therapy for both CNS and non-CNS cancers can impair cognitive function and induce a persistent CNS immune response characterized by white matter microglial reactivity and elevated cerebrospinal fluid (CSF) cytokines and chemokines. Consequently, oligodendroglial homeostasis and hippocampal neurogenesis are disrupted. Microglial depletion rescues oligodendroglial deficits and cognitive performance in a behavioral test of attention and short-term memory function. Taken together, these findings illustrate similar mechanisms underlying immunotherapy-related cognitive impairment (IRCI) and cognitive impairment following traditional cancer therapies and other immune challenges.

Glioma synapses recruit mechanisms of adaptive plasticity.

The role of the nervous system in the regulation of cancer is increasingly appreciated. In gliomas, neuronal activity drives tumour progression through paracrine signalling factors such as neuroligin-3 and brain-derived neurotrophic factor1-3 (BDNF), and also through electrophysiologically functional neuron-to-glioma synapses mediated by AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors4,5. The consequent glioma cell membrane depolarization drives tumour proliferation4,6. In the healthy brain, activity-regulated secretion of BDNF promotes adaptive plasticity of synaptic connectivity7,8 and strength9-15. Here we show that malignant synapses exhibit similar plasticity regulated by BDNF. Signalling through the receptor tropomyosin-related kinase B16 (TrkB) to CAMKII, BDNF promotes AMPA receptor trafficking to the glioma cell membrane, resulting in increased amplitude of glutamate-evoked currents in the malignant cells. Linking plasticity of glioma synaptic strength to tumour growth, graded optogenetic control of glioma membrane potential demonstrates that greater depolarizing current amplitude promotes increased glioma proliferation. This potentiation of malignant synaptic strength shares mechanistic features with synaptic plasticity17-22 that contributes to memory and learning in the healthy brain23-26. BDNF-TrkB signalling also regulates the number of neuron-to-glioma synapses. Abrogation of activity-regulated BDNF secretion from the brain microenvironment or loss of glioma TrkB expression robustly inhibits tumour progression. Blocking TrkB genetically or pharmacologically abrogates these effects of BDNF on glioma synapses and substantially prolongs survival in xenograft models of paediatric glioblastoma and diffuse intrinsic pontine glioma. Together, these findings indicate that BDNF-TrkB signalling promotes malignant synaptic plasticity and augments tumour progression.

Distinguishing features of long COVID identified through immune profiling.

Post-acute infection syndromes may develop after acute viral disease1. Infection with SARS-CoV-2 can result in the development of a post-acute infection syndrome known as long COVID. Individuals with long COVID frequently report unremitting fatigue, post-exertional malaise, and a variety of cognitive and autonomic dysfunctions2-4. However, the biological processes that are associated with the development and persistence of these symptoms are unclear. Here 275 individuals with or without long COVID were enrolled in a cross-sectional study that included multidimensional immune phenotyping and unbiased machine learning methods to identify biological features associated with long COVID. Marked differences were noted in circulating myeloid and lymphocyte populations relative to the matched controls, as well as evidence of exaggerated humoral responses directed against SARS-CoV-2 among participants with long COVID. Furthermore, higher antibody responses directed against non-SARS-CoV-2 viral pathogens were observed among individuals with long COVID, particularly Epstein-Barr virus. Levels of soluble immune mediators and hormones varied among groups, with cortisol levels being lower among participants with long COVID. Integration of immune phenotyping data into unbiased machine learning models identified the key features that are most strongly associated with long COVID status. Collectively, these findings may help to guide future studies into the pathobiology of long COVID and help with developing relevant biomarkers.

Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1.

Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation.

COVID survivors frequently experience lingering neurological symptoms that resemble cancer-therapy-related cognitive impairment, a syndrome for which white matter microglial reactivity and consequent neural dysregulation is central. Here, we explored the neurobiological effects of respiratory SARS-CoV-2 infection and found white-matter-selective microglial reactivity in mice and humans. Following mild respiratory COVID in mice, persistently impaired hippocampal neurogenesis, decreased oligodendrocytes, and myelin loss were evident together with elevated CSF cytokines/chemokines including CCL11. Systemic CCL11 administration specifically caused hippocampal microglial reactivity and impaired neurogenesis. Concordantly, humans with lasting cognitive symptoms post-COVID exhibit elevated CCL11 levels. Compared with SARS-CoV-2, mild respiratory influenza in mice caused similar patterns of white-matter-selective microglial reactivity, oligodendrocyte loss, impaired neurogenesis, and elevated CCL11 at early time points, but after influenza, only elevated CCL11 and hippocampal pathology persisted. These findings illustrate similar neuropathophysiology after cancer therapy and respiratory SARS-CoV-2 infection which may contribute to cognitive impairment following even mild COVID.

GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas.

Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal paediatric tumours of the central nervous system1. We have previously shown that the disialoganglioside GD2 is highly expressed on H3K27M-mutated glioma cells and have demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human phase I clinical trial (NCT04196413). Because CAR T cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutated DIPG or spinal cord DMG treated with GD2-CAR T cells at dose level 1 (1 × 106 GD2-CAR T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T cell infusions administered intracerebroventricularly3. Toxicity was largely related to the location of the tumour and was reversible with intensive supportive care. On-target, off-tumour toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Pro-inflammatory cytokine levels were increased in the plasma and cerebrospinal fluid. Transcriptomic analyses of 65,598 single cells from CAR T cell products and cerebrospinal fluid elucidate heterogeneity in response between participants and administration routes. These early results underscore the promise of this therapeutic approach for patients with H3K27M-mutated DIPG or spinal cord DMG.

Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication.

The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

Mild respiratory SARS-CoV-2 infection can cause multi-lineage cellular dysregulation and myelin loss in the brain.

Survivors of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection frequently experience lingering neurological symptoms, including impairment in attention, concentration, speed of information processing and memory. This long-COVID cognitive syndrome shares many features with the syndrome of cancer therapy-related cognitive impairment (CRCI). Neuroinflammation, particularly microglial reactivity and consequent dysregulation of hippocampal neurogenesis and oligodendrocyte lineage cells, is central to CRCI. We hypothesized that similar cellular mechanisms may contribute to the persistent neurological symptoms associated with even mild SARS-CoV-2 respiratory infection. Here, we explored neuroinflammation caused by mild respiratory SARS-CoV-2 infection - without neuroinvasion - and effects on hippocampal neurogenesis and the oligodendroglial lineage. Using a mouse model of mild respiratory SARS-CoV-2 infection induced by intranasal SARS-CoV-2 delivery, we found white matter-selective microglial reactivity, a pattern observed in CRCI. Human brain tissue from 9 individuals with COVID-19 or SARS-CoV-2 infection exhibits the same pattern of prominent white matter-selective microglial reactivity. In mice, pro-inflammatory CSF cytokines/chemokines were elevated for at least 7-weeks post-infection; among the chemokines demonstrating persistent elevation is CCL11, which is associated with impairments in neurogenesis and cognitive function. Humans experiencing long-COVID with cognitive symptoms (48 subjects) similarly demonstrate elevated CCL11 levels compared to those with long-COVID who lack cognitive symptoms (15 subjects). Impaired hippocampal neurogenesis, decreased oligodendrocytes and myelin loss in subcortical white matter were evident at 1 week, and persisted until at least 7 weeks, following mild respiratory SARS-CoV-2 infection in mice. Taken together, the findings presented here illustrate striking similarities between neuropathophysiology after cancer therapy and after SARS-CoV-2 infection, and elucidate cellular deficits that may contribute to lasting neurological symptoms following even mild SARS-CoV-2 infection.

Disruption of Oligodendrogenesis Impairs Memory Consolidation in Adult Mice.

The generation of myelin-forming oligodendrocytes persists throughout life and is regulated by neural activity. Here we tested whether experience-driven changes in oligodendrogenesis are important for memory consolidation. We found that water maze learning promotes oligodendrogenesis and de novo myelination in the cortex and associated white matter tracts. Preventing these learning-induced increases in oligodendrogenesis without affecting existing oligodendrocytes impaired memory consolidation of water maze, as well as contextual fear, memories. These results suggest that de novo myelination tunes activated circuits, promoting coordinated activity that is important for memory consolidation. Consistent with this, contextual fear learning increased the coupling of hippocampal sharp wave ripples and cortical spindles, and these learning-induced increases in ripple-spindle coupling were blocked when oligodendrogenesis was suppressed. Our results identify a non-neuronal form of plasticity that remodels hippocampal-cortical networks following learning and is required for memory consolidation.

Therapeutic strategies for diffuse midline glioma from high-throughput combination drug screening.

Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi-histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD+) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG.

Electrical and synaptic integration of glioma into neural circuits.

High-grade gliomas are lethal brain cancers whose progression is robustly regulated by neuronal activity. Activity-regulated release of growth factors promotes glioma growth, but this alone is insufficient to explain the effect that neuronal activity exerts on glioma progression. Here we show that neuron and glioma interactions include electrochemical communication through bona fide AMPA receptor-dependent neuron-glioma synapses. Neuronal activity also evokes non-synaptic activity-dependent potassium currents that are amplified by gap junction-mediated tumour interconnections, forming an electrically coupled network. Depolarization of glioma membranes assessed by in vivo optogenetics promotes proliferation, whereas pharmacologically or genetically blocking electrochemical signalling inhibits the growth of glioma xenografts and extends mouse survival. Emphasizing the positive feedback mechanisms by which gliomas increase neuronal excitability and thus activity-regulated glioma growth, human intraoperative electrocorticography demonstrates increased cortical excitability in the glioma-infiltrated brain. Together, these findings indicate that synaptic and electrical integration into neural circuits promotes glioma progression.

Loss of Adaptive Myelination Contributes to Methotrexate Chemotherapy-Related Cognitive Impairment.

Activity-dependent myelination is thought to contribute to adaptive neurological function. However, the mechanisms by which activity regulates myelination and the extent to which myelin plasticity contributes to non-motor cognitive functions remain incompletely understood. Using a mouse model of chemotherapy-related cognitive impairment (CRCI), we recently demonstrated that methotrexate (MTX) chemotherapy induces complex glial dysfunction for which microglial activation is central. Here, we demonstrate that remote MTX exposure blocks activity-regulated myelination. MTX decreases cortical Bdnf expression, which is restored by microglial depletion. Bdnf-TrkB signaling is a required component of activity-dependent myelination. Oligodendrocyte precursor cell (OPC)-specific TrkB deletion in chemotherapy-naive mice results in impaired cognitive behavioral performance. A small-molecule TrkB agonist rescues both myelination and cognitive impairment after MTX chemotherapy. This rescue after MTX depends on intact TrkB expression in OPCs. Taken together, these findings demonstrate a molecular mechanism required for adaptive myelination that is aberrant in CRCI due to microglial activation.

Methotrexate Chemotherapy Induces Persistent Tri-glial Dysregulation that Underlies Chemotherapy-Related Cognitive Impairment.

Chemotherapy results in a frequent yet poorly understood syndrome of long-term neurological deficits. Neural precursor cell dysfunction and white matter dysfunction are thought to contribute to this debilitating syndrome. Here, we demonstrate persistent depletion of oligodendrocyte lineage cells in humans who received chemotherapy. Developing a mouse model of methotrexate chemotherapy-induced neurological dysfunction, we find a similar depletion of white matter OPCs, increased but incomplete OPC differentiation, and a persistent deficit in myelination. OPCs from chemotherapy-naive mice similarly exhibit increased differentiation when transplanted into the microenvironment of previously methotrexate-exposed brains, indicating an underlying microenvironmental perturbation. Methotrexate results in persistent activation of microglia and subsequent astrocyte activation that is dependent on inflammatory microglia. Microglial depletion normalizes oligodendroglial lineage dynamics, myelin microstructure, and cognitive behavior after methotrexate chemotherapy. These findings indicate that methotrexate chemotherapy exposure is associated with persistent tri-glial dysregulation and identify inflammatory microglia as a therapeutic target to abrogate chemotherapy-related cognitive impairment. VIDEO ABSTRACT.

Bad wrap: Myelin and myelin plasticity in health and disease.

Human central nervous system myelin development extends well into the fourth decade of life, and this protracted period underscores the potential for experience to modulate myelination. The concept of myelin plasticity implies adaptability in myelin structure and function in response to experiences during development and beyond. Mounting evidence supports this concept of neuronal activity-regulated changes in myelin-forming cells, including oligodendrocyte precursor cell proliferation, oligodendrogenesis and modulation of myelin microstructure. In healthy individuals, myelin plasticity in associative white matter structures of the brain is implicated in learning and motor function in both rodents and humans. Activity-dependent changes in myelin-forming cells may influence the function of neural networks that depend on the convergence of numerous neural signals on both a temporal and spatial scale. However, dysregulation of myelin plasticity can disadvantageously alter myelin microstructure and result in aberrant circuit function or contribute to pathological cell proliferation. Emerging roles for myelin plasticity in normal neurological function and in disease are discussed. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 123-135, 2018.

Neural Versus Gonadal GnIH: Are they Independent Systems? A Mini-Review.

Based on research in protochordates and basal vertebrates, we know that communication across the first endocrine axes likely relied on diffusion. Because diffusion is relatively slow, rapid responses to some cues, including stress-related cues, may have required further local control of axis outputs (e.g., steroid hormone production by the gonads). Despite the evolution of much more efficient circulatory systems and complex nervous systems in vertebrates, production of many "neuro"transmitters has been identified outside of the hypothalamus across the vertebrate phylogeny and these neurotransmitters are known to locally regulate endocrine function. Our understanding of tissue-specific neuropeptide expression and their role coordinating physiological/behavioral responses of the whole organism remains limited, in part, due to nomenclature and historic dogma that ignores local regulation of axis output. Here, we review regulation of gonadotropin-inhibitory hormone (GnIH) across the reproductive axis in birds and mammals to bring further attention to context-dependent disparities and similarities in neuropeptide production by the brain and gonads. We find that GnIH responsiveness to cues of stress appears conserved across species, but that the response of specific tissues and the direction of GnIH regulation varies. The implications of differential regulation across tissues remain unclear in most studies, but further work that manipulates and contrasts function in different tissues has the potential to inform us about both organism-specific function and endocrine axis evolution.

The Role of RFamide-Related Peptide-3 in Age-Related Reproductive Decline in Female Rats.

Reproductive senescence, the point in time when females cease to show estrous cyclicity, is associated with endocrine changes in the hypothalamus, pituitary, and gonads. However, the mechanisms triggering this transition are not well understood. To gain a better understanding of the top-down control of the transition from reproductive competence to a state of reproductive senescence, we investigated middle-aged female rats exhibiting varying degrees of reproductive decline, including individuals with normal cycles, irregular cycles, and complete cessation of cycles. We identified hormonal changes in the brain that manifest before ovarian cycles exhibit any deterioration. We found that females exhibit an increase in RFamide-related peptide-3 (RFRP3) mRNA expression in the hypothalamus in middle age prior to changes in estrous cycle length. This increase is transient and followed by subsequent decreases in kisspeptin (KiSS1) and gonadotropin-releasing hormone (GnRH) mRNA expression. Expression of RFRP3 and its receptor also increased locally in the ovaries with advancing age. While it is well known that aging is associated with decreased GnRH release and downstream disruption of the hypothalamic-pituitary-gonadal (HPG) axis, herein, we provide evidence that reproductive senescence is likely triggered by alterations in a network of regulatory neuropeptides upstream of the GnRH system.

Glucocorticoid Regulation of Reproduction.

It is well accepted that stress, measured by increased glucocorticoid secretion, leads to profound reproductive dysfunction. In times of stress, glucocorticoids activate many parts of the fight or flight response, mobilizing energy and enhancing survival, while inhibiting metabolic processes that are not necessary for survival in the moment. This includes reproduction, an energetically costly procedure that is very finely regulated. In the short term, this is meant to be beneficial, so that the organism does not waste precious energy needed for survival. However, long-term inhibition can lead to persistent reproductive dysfunction, even if no longer stressed. This response is mediated by the increased levels of circulating glucocorticoids, which orchestrate complex inhibition of the entire reproductive axis. Stress and glucocorticoids exhibits both central and peripheral inhibition of the reproductive hormonal axis. While this has long been recognized as an issue, understanding the complex signaling mechanism behind this inhibition remains somewhat of a mystery. What makes this especially difficult is attempting to differentiate the many parts of both of these hormonal axes, and new neuropeptide discoveries in the last decade in the reproductive field have added even more complexity to an already complicated system. Glucocorticoids (GCs) and other hormones within the hypothalamic-pituitary-adrenal (HPA) axis (as well as contributors in the sympathetic system) can modulate the hypothalamic-pituitary-gonadal (HPG) axis at all levels-GCs can inhibit release of GnRH from the hypothalamus, inhibit gonadotropin synthesis and release in the pituitary, and inhibit testosterone synthesis and release from the gonads, while also influencing gametogenesis and sexual behavior. This chapter is not an exhaustive review of all the known literature, however is aimed at giving a brief look at both the central and peripheral effects of glucocorticoids on the reproductive function.

Knockdown of hypothalamic RFRP3 prevents chronic stress-induced infertility and embryo resorption.

Whereas it is well established that chronic stress induces female reproductive dysfunction, whether stress negatively impacts fertility and fecundity when applied prior to mating and pregnancy has not been explored. In this study, we show that stress that concludes 4 days prior to mating results in persistent and marked reproductive dysfunction, with fewer successful copulation events, fewer pregnancies in those that successfully mated, and increased embryo resorption. Chronic stress exposure led to elevated expression of the hypothalamic inhibitory peptide, RFamide-related peptide-3 (RFRP3), in regularly cycling females. Remarkably, genetic silencing of RFRP3 during stress using an inducible-targeted shRNA completely alleviates stress-induced infertility in female rats, resulting in mating and pregnancy success rates indistinguishable from non-stress controls. We show that chronic stress has long-term effects on pregnancy success, even post-stressor, that are mediated by RFRP3. This points to RFRP3 as a potential clinically relevant single target for stress-induced infertility.

Stress increases putative gonadotropin inhibitory hormone and decreases luteinizing hormone in male rats.

The subjective experience of stress leads to reproductive dysfunction in many species, including rodents and humans. Stress effects on reproduction result from multilevel interactions between the hormonal stress response system, i.e., the hypothalamic-pituitary-adrenal (HPA) axis, and the hormonal reproductive system, i.e., the hypothalamic-pituitary-gonadal (HPG) axis. A novel negative regulator of the HPG axis known as gonadotropin-inhibitory hormone (GnIH) was recently discovered in quail, and orthologous neuropeptides known as RFamide-related peptides (RFRPs) have also been identified in rodents and primates. It is currently unknown, however, whether GnIH/RFRPs influence HPG axis activity in response to stress. We show here that both acute and chronic immobilization stress lead to an up-regulation of RFRP expression in the dorsomedial hypothalamus (DMH) of adult male rats and that this increase in RFRP is associated with inhibition of downstream HPG activity. We also show that adrenalectomy blocks the stress-induced increase in RFRP expression. Immunohistochemistry revealed that 53% of RFRP cells express receptors for glucocorticoids (GCs), indicating that adrenal GCs can mediate the stress effect through direct action on RFRP cells. It is thought that stress effects on central control of reproduction are largely mediated by direct or indirect effects on GnRH-secreting neurons. Our data show that stress-induced increases in adrenal GCs cause an increase in RFRP that contributes to hypothalamic suppression of reproductive function. This novel insight into HPA-HPG interaction provides a paradigm shift for work on stress-related reproductive dysfunction and infertility, and indicates that future work on stress and reproductive system interactions must include investigation of the role of GnIH/RFRP.