Search

Methyl-CpG-binding protein 2 (MECP2) is a chromatin-associated protein that can both activate and repress transcription, playing an essential role in neuronal maturation. MECP2 is encoded by an X-linked gene (chromosome Xq28) with high expression in the brain, including the hypothalamic region. Loss-of-function mutations in MECP2 are usually associated with Rett syndrome, a rare genetic disorder characterized by normal early development followed by regression of acquired skills, such as purposeful hand movements and the ability to communicate, repetitive hand stereotypies, slowed brain growth, seizures, and intellectual disability with female predominance. Notably, early pubertal development (early thelarche, pubarche and, less often, menarche) has been demonstrated in girls with Rett syndrome. In this original study, a total of 329 CPP patients (308 girls and 21 boys) were investigated for MECP2 defects. Familial cases were identified in 38% of this CPP cohort. First, 129 CPP patients were enrolled for multigene sequencing studies (whole-exome sequencing n=58; targeted gene sequencing n=71) as part of genetic investigations based on large-scale approaches. Three rare heterozygous sequence variants predicted to be deleterious in MECP2 gene were identified in 5 girls from 4 unrelated families with CPP: the p.Arg97Cys de novo missense variant in two monozygotic twin sisters with CPP and microcephaly; the p.Ser176Arg de novo missense variant in one girl with sporadic CPP, obesity and autism; and the p.Ala6_Ala8dup insertion in two unrelated girls with sporadic CPP (one with a maternally inherited variant). To expand the investigation for potentially damaging sequence variants of MECP2, a larger multiethnic cohort of 200 CPP patients was further analyzed through Sanger sequencing. One rare heterozygous 3'UTR insertion in MECP2 (c.*36_*37insT) was identified in two unrelated girls with sporadic CPP (one with a maternally inherited variant and other with a de novo variant). None of these MECP2 variants have been previously reported in Rett syndrome cases. The girls with CPP associated with MECP2 defects did not manifest typical Rett syndrome. The three patients with more deleterious MECP2 variants, according to in silico prediction tools, had neurobehavioral phenotypes, likely corresponding to a genotype-phenotype correlation. Immunohistochemistry and immunofluorescence studies were performed in pubertal female mice. Abundant staining for Mecp2 was demonstrated in multiple hypothalamic nuclei (arcuate, suprachiasmatic, and paraventricular) and in the median eminence. Co-localization of Mecp2 and GnRH within GnRH neurons was suggested in double labelling experiments. Hence, Mecp2 expression was demonstrated in key hypothalamic nuclei responsible for GnRH regulation in female mice. In conclusion, we have identified rare dominant MECP2 defects in multiple unrelated girls with CPP with or without mild neurodevelopmental abnormalities, revealing a new X-linked form of premature reactivation of the hypothalamic-pituitary-gonadal axis. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:30 p.m. - 12:35 p.m.

Gonadotropin releasing hormone (GnRH) deficiency is a disorder characterized by absent or delayed puberty, with largely unknown genetic causes. The purpose of this study was to obtain and exploit gene expression profiles of GnRH neurons during development to unveil novel biological mechanisms and genetic determinants underlying GnRH deficiency (GD). Here, we combined bioinformatic analyses of primary embryonic and immortalized GnRH neuron transcriptomes with exome sequencing from GD patients to identify candidate causative genes. Among differentially expressed and filtered transcripts, we found loss-of-function (LoF) variants of the autism-linked Neuroligin 3 (NLGN3) gene in two unrelated patients co- presenting with GD and neurodevelopmental traits. We demonstrated that NLGN3 is upregulated in maturing GnRH neurons and that NLGN3 wild type, but not mutant proteins, promotes neuritogenesis when overexpressed in developing GnRH cells. Our data represent proof-of-principle that this complementary approach can identify novel candidate GD genes and demonstrate that LoF NLGN3 variants may contribute to GD. This novel genotype- phenotype correlation implies common genetic mechanisms underlying neurodevelopmental disorders, such as GD and autistic spectrum disorder.

Introduction: Gonadotropin-releasing hormone (GnRH) deficiency causes hypogonadotropic hypogonadism (HH), a rare genetic disorder that impairs sexual reproduction. HH can be due to defective GnRH-secreting neuron development or function and may be associated with other clinical signs in overlapping genetic syndromes. With most of the cases being idiopathic, genetics underlying HH is still largely unknown. Objective: To assess the contribution of mutated Semaphorin 3G (SEMA3G) in the onset of a syndromic form of HH, characterized by intellectual disability and facial dysmorphic features. Method: By combining homozygosity mapping with exome sequencing, we identified a novel variant in the SEMA3G gene. We then applied mouse as a model organism to examine SEMA3Gexpression and its functional requirement in vivo. Further, we applied homology modelling in silico and cell culture assays in vitro to validate the pathogenicity of the identified gene variant. Results: We found that (i) SEMA3G is expressed along the migratory route of GnRH neurons and in the developing pituitary, (ii) SEMA3G affects GnRH neuron development, but is redundant in the adult hypothalamic-pituitary-gonadal axis, and (iii) mutated SEMA3G alters binding properties in silico and in vitro to its PlexinA receptors and attenuates its effect on the migration of immortalized GnRH neurons. Conclusion: In silico, in vitro, and in vivo models revealed that SEMA3G regulates GnRH neuron migration and that its mutation affecting receptor selectivity may be responsible for the HH-related defects.

The positive regulatory (PR) domain containing 13 (PRDM13) putative chromatin modifier and transcriptional regulator functions downstream of the transcription factor PTF1A, which controls GABAergic fate in the spinal cord and neurogenesis in the hypothalamus. Here, we report a recessive syndrome associated with PRDM13 mutation. Patients exhibited intellectual disability, ataxia with cerebellar hypoplasia, scoliosis, and delayed puberty with congenital hypogonadotropic hypogonadism (CHH). Expression studies revealed Prdm13/PRDM13 transcripts in the developing hypothalamus and cerebellum in mouse and human. An analysis of hypothalamus and cerebellum development in mice homozygous for a Prdm13 mutant allele revealed a significant reduction in the number of Kisspeptin (Kiss1) neurons in the hypothalamus and PAX2+ progenitors emerging from the cerebellar ventricular zone. The latter was accompanied by ectopic expression of the glutamatergic lineage marker TLX3. Prdm13-deficient mice displayed cerebellar hypoplasia and normal gonadal structure, but delayed pubertal onset. Together, these findings identify PRDM13 as a critical regulator of GABAergic cell fate in the cerebellum and of hypothalamic kisspeptin neuron development, providing a mechanistic explanation for the cooccurrence of CHH and cerebellar hypoplasia in this syndrome. To our knowledge, this is the first evidence linking disrupted PRDM13-mediated regulation of Kiss1 neurons to CHH in humans.

PRDM13 (PR Domain containing 13) is a putative chromatin modifier and transcriptional regulator that functions downstream of the transcription factor PTF1A, which in turn controls GABAergic fate in the spinal cord and neuronal development in the hypothalamus. Here, we report a novel, recessive syndrome associated with PRDM13 mutation. Patients exhibited intellectual disability, ataxia with cerebellar hypoplasia, scoliosis and delayed puberty with congenital hypogonadotropic hypogonadism (CHH). Expression studies revealed Prdm13/PRDM13 transcripts in the developing hypothalamus and cerebellum in mouse and human. We investigated the development of hypothalamic neurons and the cerebellum in mice homozygous for a Prdm13 mutant allele.A significant reduction in the number of Kisspeptin (Kiss1) neurons in the hypothalamus and PAX2+ progenitors emerging from the cerebellar ventricular zone were observed. The latter was accompanied by ectopic expression of the glutamatergic lineage marker TLX3. Phenotypically, mice lacking PRDM13 displayed cerebellar hypoplasia, normal gonadal structure, but delayed pubertal onset. Together, these findings identify PRDM13 as a critical regulator of GABAergic cell fate in the cerebellum and of kisspeptin neuron development in the hypothalamus, providing a mechanistic explanation for the co-occurrence of CHH and cerebellar hypoplasia in this syndrome. To our knowledge, this is the first evidence linking disrupted PRDM13-mediated regulation of Kiss1 neurons to CHH in humans.

In mammals, fertility critically depends on the pulsatile secretion of gonadotropin-releasing hormone (GnRH) by scattered hypothalamic neurons (GnRH neurons). During development, GnRH neurons originate in the nasal placode and migrate first into the nasal compartment and then through the nasal/forebrain junction, before they reach their final position in the hypothalamus. This neurodevelopmental process, which has been extensively studied in mouse models, is regulated by a plethora of factors that might control GnRH neuron migration or survival as well as the fasciculation/targeting of the olfactory/vomeronasal axons along which the GnRH neurons migrate. Defects in GnRH neuron development or release can lead to isolated GnRH deficiency, with the underlying genetic causes still being partially unknown. Recently, semaphorins and their receptors neuropilins and plexins, a large family of molecules implicated in neuronal development and plasticity, are emerging as key regulators of GnRH neuron biology and deficiency. Specifically, semaphorins have been shown to play different roles in GnRH neuron biology by regulating migration and survival during embryonic development as well as secretion in adulthood.

Anti-Müllerian hormone (AMH) is secreted by Sertoli or granulosa cells. Recent evidence suggests that AMH may play a role in the pathogenesis of hypogonadotropic hypogonadism (HH) and that its serum levels could help to discriminate HH from delayed puberty. Moreover, the growth hormone (GH)/insulin-like growth factor 1 (IGF1) system may be involved in the function of gonadotropin-releasing hormone (GnRH) neurons, as delayed puberty is commonly found in patients with GH deficiency (GHD) or with Laron syndrome, a genetic form of GH resistance. The comprehension of the stimuli enhancing the migration and secretory activity of GnRH neurons might shed light on the causes of delay of puberty or HH. With these premises, we aimed to better clarify the role of the AMH, GH, and IGF1 on GnRH neuron migration and GnRH secretion, by taking advantage of previously established models of immature (GN11 cell line) and mature (GT1-7 cell line) GnRH neurons. Expression of Amhr, Ghr, and Igf1r genes was confirmed in both cell lines. Cells were then incubated with increasing concentrations of AMH (1.5–150 ng/mL), GH (3–1000 ng/mL), or IGF1 (1.5–150 ng/mL). All hormones were able to support GN11 cell chemomigration. AMH, GH, and IGF1 significantly stimulated GnRH secretion by GT1-7 cells after a 90-min incubation. To the best of our knowledge, this is the first study investigating the direct effects of GH and IGF1 in GnRH neuron migration and of GH in the GnRH secreting pattern. Taken together with previous basic and clinical studies, these findings may provide explanatory mechanisms for data, suggesting that AMH and the GH-IGF1 system play a role in HH or the onset of puberty.

Protein kinase CK2 (CK2) is a highly conserved and ubiquitous kinase is involved in crucial biological processes, including proliferation, migration, and differentiation. CK2 holoenzyme is a tetramer composed by two catalytically active (&alpha, /&alpha, &rsquo, ) and two regulatory (&beta, ) subunits and exerts its function on a broad range of targets. In the brain, it regulates different steps of neurodevelopment, such as neural differentiation, neuritogenesis, and synaptic plasticity. Interestingly, CK2 mutations have been recently linked to neurodevelopmental disorders, however, the functional requirements of the individual CK2 subunits in neurodevelopment have not been yet investigated. Here, we disclose the role of CK2 on the migration and adhesion properties of GN11 cells, an established model of mouse immortalized neurons, by different in vitro experimental approaches. Specifically, the cellular requirement of this kinase has been assessed pharmacologically and genetically by exploiting CK2 inhibitors and by generating subunit-specific CK2 knockout GN11 cells (with a CRISPR/Cas9-based approach). We show that CK2&alpha, subunit has a primary role in increasing cell adhesion and reducing migration properties of GN11 cells by activating the Akt-GSK3&beta, axis, whereas CK2&alpha, subunit is dispensable. Further, the knockout of the CK2&beta, regulatory subunits counteracts cell migration, inducing dramatic alterations in the cytoskeleton not observed in CK2&alpha, knockout cells. Collectively taken, our data support the view that the individual subunits of CK2 play different roles in cell migration and adhesion properties of GN11 cells, supporting independent roles of the different subunits in these processes.

International audience; Cancer stem cells (CSCs) contribute to disease progression and treatment failure in human cancers. The balance among self-renewal, differentiation, and senescence determines the expansion or progressive exhaustion of CSCs. Targeting these processes might lead to novel anticancer therapies. Here, we uncover a novel link between BRD4, mitochondrial dynamics, and self-renewal of prostate CSCs. Targeting BRD4 by genetic knockdown or chemical inhibitors blocked mitochondrial fission and caused CSC exhaustion and loss of tumorigenic capability. Depletion of CSCs occurred in multiple prostate cancer models, indicating a common vulnerability and dependency on mitochondrial dynamics. These effects depended on rewiring of the BRD4-driven transcription and repression of mitochondrial fission factor (Mff). Knockdown of Mff reproduced the effects of BRD4 inhibition, whereas ectopic Mff expression rescued prostate CSCs from exhaustion. This novel concept of targeting mitochondrial plasticity in CSCs through BRD4 inhibition provides a new paradigm for developing more effective treatment strategies for prostate cancer.

Gonadotropin-releasing hormone (GnRH) neurons regulate puberty onset and sexual reproduction by secreting GnRH to activate and maintain the hypothalamic-pituitary-gonadal axis. During embryonic development, GnRH neurons migrate along olfactory and vomeronasal axons through the nose into the brain, where they project to the median eminence to release GnRH. The secreted glycoprotein SEMA3A binds its receptors neuropilin (NRP) 1 or NRP2 to position these axons for correct GnRH neuron migration, with an additional role for the NRP co-receptor PLXNA1. Accordingly, mutations in SEMA3A, NRP1, NRP2 and PLXNA1 have been linked to defective GnRH neuron development in mice and inherited GnRH deficiency in humans. Here, we show that only the combined loss of PLXNA1 and PLXNA3 phenocopied the full spectrum of nasal axon and GnRH neuron defects of SEMA3A knockout mice. Together with Plxna1, the human orthologue of Plxna3 should therefore be investigated as a candidate gene for inherited GnRH deficiency.

Cancer of the prostate is one of the most common malignancies and the second leading cause of cancer death in men in developed countries. There is increasing evidence that cancer stem-like cells (CSCs) are implicated in CRPC disease progression and treatment resistance. Transcriptional, epigenetic and metabolic reprogramming are key features for the acquisition and maintenance of stem-like properties. Understanding the factors regulating self-renewal and survival of prostate CSCs may offer novel targets for innovative therapeutic strategies. LSD1/KDM1A is a lysine demethylase for histone and non-histone proteins and functions as transcriptional corepressor or coactivator depending on the binding partners and substrates. LSD1 is a key epigenetic modifier controlling the fate of pluripotent stem cells in several tissues. Overexpression of LSD1 is found in many human cancers and has been linked to tumorigenic and CSC-like features. The involvement of LSD1 in stem cell pluripotency and differentiation suggests that LSD1 inhibitors, such as INCB059872, might be useful to target the prostate CSC subpopulation. Here, we examined the effects of INCB059872 on the growth properties, self-renewal and tumorigenic capability of prostate CSCs derived from human cell lines and the Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG (ERG/PTEN) mice, which develop highly invasive prostate adenocarcinomas. In ex vivo tumor-sphere assays, INCB059872 significantly suppressed the growth of tumor-initiating stem-like cells isolated from prostatic tumors generated in ERG/PTEN mice. Furthermore, treatment with INCB059872 inhibited colony and tumor-sphere formation by human prostate cancer cells. Conversely, the effects on proliferation and viability of bulk tumor cells were limited and required long-term exposure to the drug. These effects were observed in multiple prostate cancer cell lines, irrespective of the distinctive genetic features and AR status. Importantly, genetic knockdown of LSD1 by siRNAs and shRNAs recapitulated the effects of INCB059872. Both transient and stable knockdown of LSD1 had limited effects on proliferation and viability of bulk tumor cells, whereas they significantly reduced growth of colony and tumor-sphere forming stem-like cancer cells. Furthermore, LSD1 knockdown drastically reduced tumor growth and tumorigenic stem-like cells in subcutaneous xenografts of human prostate cancer cells in nude mice. These results support the hypothesis that LSD1 has a major role in sustaining the stem-like and tumorigenic subpopulation in prostate tumors and its inhibition by chemical or genetic approaches prevents survival and self-renewal capability of prostate CSCs. These data suggest that INCB059872 could be a valid addition to the current treatment strategies for prostate cancer. INCB059872 is currently in phase1/2 clinical studies Citation Format: Gianluca Civenni, Giada Zoppi, Ramiro Vazquez, Dhreeraj Shinde, Alyssa Paganoni, Aleksandra Kokanovic, Sang Hyun Lee, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. INCB059872, a novel FAD-directed LSD1 Inhibitor, is active in prostate cancer models and impacts prostate cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1379.