Development And Regulation Of The Gonadotropin Releasing Hormone System

National Institute Of Neurological Disorders And Stroke

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Abstract

GnRH neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain where they become integral members of the hypothalamic-pituitary-gonadal axis. We study mechanism(s) underlying GnRH neuronal differentiation, migration and axonal targeting in normal/transgenic animals, and nasal explants. Using these same models, our work also addresses the mechanisms regulating (intrinsic and trans-synaptic) GnRH gene expression, peptide synthesis and secretion in GnRH neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH neurons to their final location in the CNS. These include differential screening of libraries obtained from migrating versus non-migrating cells, examination of molecules differentially expressed at key locations along the migratory route, morphological examination of the development of the GnRH system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH neuronal movement. As GnRH neurons migrate, they also mature, and the two processes may in fact be linked. To investigate the maturation of GnRH neurons, we use calcium imaging, electrophysiology and biochemical measures to examine GnRH neuronal activity and peptide secretion. In addition, we collaborate with labs performing human genetic screening of Kallman patients. Once a mutation is identified, we analyze the expression pattern in mice and perform biological assays to determine the outcome of the mutated gene on GnRH development. Over the past year, four articles were published. In vertebrates, Gonadotropin releasing hormone-1 (GnRH) neuroendocrine cells originate in the olfactory placode and migrate into the forebrain where they regulate reproduction. Two publications focused on the development of the GnRH system and two focused on regulation of GnRH relevant for reproductive function. Developmental papers: Our publication GLIA (Saglam, Calof, Wray, 2020), addresses the plasticity of olfactory ensheathing cells (OECs), cells that are in intimate contact with GnRH cells during embryonic development. OECs have been shown to moderate astrocyte reactivity, creating an environment conducive to regeneration. In this study, we screened primary and immortalized OEC lines to identify factors that attenuate neurotoxicity genes, and discovered that Alpha B-crystallin (CryAB), an anti-inflammatory protein, is secreted by OECs via exosomes, coordinating an intercellular immune response. Our results indicate that CryAB, as well as other factors secreted by OECs, are potential agents that can ameliorate, or even reverse, the growth-inhibitory environment created by neurotoxic reactive astrocytes following CNS injuries;OECs are likely important players in regenerative properties of the olfactory system. In Shan, Farmer, and Wray (2021), appearing in PNAS, we address how chemokines and cytoskeletal components interact during neuronal migration. We reveal a novel mechanism whereby the chemokine receptor (CXCR4), after being activated by its ligand, SDF-1, interacts directly with the cytoskeleton compartment via drebrin, an actin-binding protein that interacts with microtubule plus-endbinding proteins. The interaction between activated CXCR4/drebrin promotes neuronal migration. The study provides insights on the relationship between extracellular cues and intracellular components. Regulation of GnRH: Our Endocrinology paper (Constantin et al, 2021) addresses modulation of GnRH neuronal activity by RFamide-related peptides (RFRPs, mammalian orthologs of gonadotropin-inhibitory hormone) which are known to convey circadian, seasonal and social cues to the reproductive system. We show that they regulate gonadotropin secretion by modulating GnRH neurons via the RFRP receptor. In addition we found 1) NPFF inhibits GnRH neuron excitability via the RFRP receptor and its canonical signaling pathway (Gi/o protein and G protein coupled inwardly-rectifying potassium channels), 2) NPFF-like fibers in the vicinity of GnRH neurons coexpress neuropeptide Y, 3) the majority of NPFF-like cell bodies in the NTS also coexpress neuropeptide Y, and 4) acute fasting increased NPFF-like immunoreactivity in the NTS. Together these data indicate that NPFF neurons within the NTS inhibit GnRH neurons, and thus reproduction, during fasting but prior to the energy deficit. Appearing in PNAS (Constantin et al, 2021) examined pulsatile release of GnRH peptide, which is necessary for physiological function. While it is clear that kisspeptin neurons trigger GnRH activity and pulses, how GnRH neurons return to baseline activity before the next stimulation is unknown. Here, we show a) that the long-lasting response of GnRH neurons to kisspeptin depends on the degradation of phosphatidylinositol 4,5-bisphosphate (PIP2) and its slow resynthesis keeping canonical transient receptor potential channels opened and b) a mechanism whereby nitric oxide facilitates PIP2 resynthesis and GnRH neuron recovery from kisspeptin activation. Defining the overlap in kisspeptin and nitric oxide signaling pathways may provide an avenue for fertility treatment.