NOVEL INSIGHTS INTO GENETIC AND ENVIRONMENTAL DETERMINANTS SHAPING GNRH NEURON BIOLOGY AND RELATED REPRODUCTIVE DISORDERS
The hypothalamic-pituitary-gonadal (HPG) axis is a conserved neuroendocrine system essential for reproductive function in vertebrates. Central to this axis is the secretion of gonadotropin-releasing hormone (GnRH), a neuropeptide that stimulates the release of gonadotropins from the pituitary, driving gametogenesis and sex steroid production. GnRH-secreting neurons originate from the olfactory placode during embryogenesis and migrate into the forebrain to reach the hypothalamus, where they integrate into the reproductive axis. This migratory journey depends on tightly regulated molecular mechanisms involving transcription factors, guidance cues, adhesion molecules, and extracellular matrix components. Disruption of any of these pathways can impair GnRH neuron development and function, leading to isolated GnRH deficiency (IGD), a rare congenital disorder characterized by absent or delayed puberty and infertility. While once considered a monogenic condition, IGD is now recognized as genetically heterogeneous, with oligogenic inheritance frequently observed. Although over 44 genes have been implicated in IGD, only about 50% of cases are genetically explained, highlighting the likely involvement of additional unidentified genetic contributors and environmental factors. A major focus of this thesis is CHARGE syndrome (CS), a multisystem disorder clinically overlapping with IGD and primarily caused by heterozygous loss-of-function variants in the CHD7 gene, encoding a chromatin remodeler essential for neural crest and olfactory system development. CS is characterized by a range of anomalies, including craniofacial malformations, sensory deficits, and hypogonadotropic hypogonadism. Despite CHD7 mutations being the major cause, the broad clinical variability observed among patients suggests the involvement of genetic modifiers. Recent evidence highlights the Semaphorin (SEMA) signalling pathway as a candidate downstream effector of CHD7. To investigate this hypothesis and explore therapeutic avenues for CS, this thesis developed a dual disease-model screening platform comprising a Caenorhabditis elegans (C. elegans) chd-7 mutant and a CRISPR-engineered Chd7-depleted mouse cell line modelling immature GnRH neurons. Transcriptomic profiling of the Chd7-depleted cells and in vitro assays revealed cellular and genetic signatures linked to defective cell proliferation and impaired migration, alongside dysregulation of SEMA genes, which are crucial for axon guidance and GnRH development. In parallel, a targeted screen of 234 chromatin-modifying compounds in the C. elegans model identified hits capable of rescuing chd-7 mutant reproductive defects. These compounds were then validated in our newly generated murine cellular model of CS, enabling the identification of candidate small molecules rescuing disease-relevant phenotypes through restoration of SEMA gene expression. These findings support the therapeutic potential of targeting SEMA signalling to bypass CHD7 haploinsufficiency and address CS-associated neurological and neurodevelopmental defects. In a second line of investigation, this thesis explored the role of HS6ST1 and HS6ST2, enzymes that modify heparan sulfate proteoglycans (HSPGs) and thereby regulate the binding of axon guidance molecules like VEGF and SEMA3A. While mutations in HS6ST1 have previously been reported in IGD patients, the role of HS6ST2 remained uncharacterized. Using a murine model, it was demonstrated that combined, but not single, loss of Hs6st1 and Hs6st2 leads to increased apoptosis and defective brain entry of GnRH neurons, mimicking phenotypes observed in mice with defective Sema3a and Vegfa/Nrp1 signalling. These results identify HS6ST2 as a novel candidate gene for IGD and underscore the critical role of HS6ST-mediated HSPG sulfation in ensuring proper ligand-receptor interactions essential for neuronal migration and survival. A final part of this thesis addressed the emerging role of environmental factors in GnRH neuron dysfunction. Specifically, we examined the effects of polystyrene nanoplastics (PS-NPs), an increasingly prevalent environmental contaminant, on GnRH neuron biology. Using two in vitro models, GT1-7 (secreting) and GN11 (migrating) GnRH neuronal cells, PS-NPs were shown to enter cells via non-classical endocytosis and impair key cellular processes. In GT1-7 cells, PS-NPs altered GnRH neuropeptide production, while in GN11 cells, migration was significantly impaired. Transcriptomic profiling of NP-exposed GN11 cells revealed changes in genes critical to GnRH neuron development. Notably, integration of these data with exome sequencing from IGD patients identified a rare NPAS2 variant in an individual with severe delayed puberty, suggesting potential gene-environment interactions and highlighting NPs as novel endocrine disruptors. Together, these complementary lines of research reinforce the concept that GnRH neuron development is governed by a highly intricate network of genetic, epigenetic, and environmental factors. Indeed, the findings presented in this thesis highlight HS6ST2 as a previously unrecognized IGD susceptibility gene, identify environmental pollutants like NPs as disruptors of reproductive neurobiology, and demonstrate the therapeutic potential of SEMA pathway modulation in CHD7-deficiency disorders. Collectively, this body of work advances our understanding of the mechanisms underlying IGD and related syndromes, laying the groundwork for future therapeutic strategies aimed at restoring reproductive function in individuals affected by congenital or environmentally induced forms of GnRH deficiency.
GnRH, HPG axis, reproductive disorders, Isolated GnRH deficiency, CHARGE Syndrom, CHD7, Semaphorin, HS6ST, Nanoplastics, Settore BIOS-10/A - Biologia cellulare e applicata
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