Targeted inhibition of kisspeptin neurons reverses hyperandrogenemia and abnormal hyperactive LH secretion in a preclinical mouse model of polycystic ovary syndrome.

STUDY QUESTION: Do hyperactive kisspeptin neurons contribute to abnormally high LH secretion and downstream hyperandrogenemia in polycystic ovary syndrome (PCOS)-like conditions and can inhibition of kisspeptin neurons rescue such endocrine impairments? SUMMARY ANSWER: Targeted inhibition of endogenous kisspeptin neuron activity in a mouse model of PCOS reduced the abnormally hyperactive LH pulse secretion and hyperandrogenemia to healthy control levels. WHAT IS KNOWN ALREADY: PCOS is a reproductive disorder characterized by hyperandrogenemia, anovulation, and/or polycystic ovaries, along with a hallmark feature of abnormal LH hyper-pulsatility, but the mechanisms underlying the endocrine impairments remain unclear. A chronic letrozole (LET; aromatase inhibitor) mouse model recapitulates PCOS phenotypes, including polycystic ovaries, anovulation, high testosterone, and hyperactive LH pulses. LET PCOS-like females also have increased hypothalamic kisspeptin neuronal activation which may drive their hyperactive LH secretion and hyperandrogenemia, but this has not been tested. STUDY DESIGN, SIZE, DURATION: Transgenic KissCRE+/hM4Di female mice or littermates Cre- controls were treated with placebo, or chronic LET (50 µg/day) to induce a PCOS-like phenotype, followed by acute (once) or chronic (2 weeks) clozapine-N-oxide (CNO) exposure to chemogenetically inhibit kisspeptin cells (n = 6 to 10 mice/group). PARTICIPANTS/MATERIALS, SETTING, METHODS: Key endocrine measures, including in vivo LH pulse secretion patterns and circulating testosterone levels, were assessed before and after selective kisspeptin neuron inhibition and compared between PCOS groups and healthy controls. Alterations in body weights were measured and pituitary and ovarian gene expression was determined by qRT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Acute targeted inhibition of kisspeptin neurons in PCOS mice successfully lowered the abnormally hyperactive LH pulse secretion (P < 0.05). Likewise, chronic selective suppression of kisspeptin neuron activity reversed the previously high LH and testosterone levels (P < 0.05) down to healthy control levels and rescued reproductive gene expression (P < 0. 05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ovarian morphology was not assessed in this study. Additionally, mouse models can offer mechanistic insights into neuroendocrine processes in PCOS-like conditions but may not perfectly mirror PCOS in women. WIDER IMPLICATIONS OF THE FINDINGS: These data support the hypothesis that overactive kisspeptin neurons can drive neuroendocrine PCOS-like impairments, and this may occur in PCOS women. Our findings complement recent clinical investigations using NKB receptor antagonists to lower LH in PCOS women and suggest that pharmacological dose-dependent modulation of kisspeptin neuron activity may be a valuable future therapeutic target to clinically treat hyperandrogenism and lower elevated LH in PCOS women. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by NIH grants R01 HD111650, R01 HD090161, R01 HD100580, P50 HD012303, R01 AG078185, and NIH R24 HD102061, and a pilot project award from the British Society for Neuroendocrinology. There are no competing interests.

Decoding protein-RNA interactions using CLIP-based methodologies.

Protein-RNA interactions are central to all RNA processing events, with pivotal roles in the regulation of gene expression and cellular functions. Dysregulation of these interactions has been increasingly linked to the pathogenesis of human diseases. High-throughput approaches to identify RNA-binding proteins and their binding sites on RNA - in particular, ultraviolet crosslinking followed by immunoprecipitation (CLIP) - have helped to map the RNA interactome, yielding transcriptome-wide protein-RNA atlases that have contributed to key mechanistic insights into gene expression and gene-regulatory networks. Here, we review these recent advances, explore the effects of cellular context on RNA binding, and discuss how these insights are shaping our understanding of cellular biology. We also review the potential therapeutic applications arising from new knowledge of protein-RNA interactions.

Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion.

RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification-mass spectrometry to investigate a subset of candidates with no prior association with splicing. Integrative analysis of these assays indicates surprising roles for TRNAU1AP, SCAF8 and RTCA in the modulation of hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using these identified domains, we engineered programmable fusion proteins that outperform current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. This tethering approach characterizes the ability of RBPs to induce exon inclusion and yields new molecular parts for programmable splicing control.

Human microglia maturation is underpinned by specific gene regulatory networks.

Microglia phenotypes are highly regulated by the brain environment, but the transcriptional networks that specify the maturation of human microglia are poorly understood. Here, we characterized stage-specific transcriptomes and epigenetic landscapes of fetal and postnatal human microglia and acquired corresponding data in induced pluripotent stem cell (iPSC)-derived microglia, in cerebral organoids, and following engraftment into humanized mice. Parallel development of computational approaches that considered transcription factor (TF) co-occurrence and enhancer activity allowed prediction of shared and state-specific gene regulatory networks associated with fetal and postnatal microglia. Additionally, many features of the human fetal-to-postnatal transition were recapitulated in a time-dependent manner following the engraftment of iPSC cells into humanized mice. These data and accompanying computational approaches will facilitate further efforts to elucidate mechanisms by which human microglia acquire stage- and disease-specific phenotypes.

CADM2 is implicated in impulsive personality and numerous other traits by genome- and phenome-wide association studies in humans and mice.

Impulsivity is a multidimensional heritable phenotype that broadly refers to the tendency to act prematurely and is associated with multiple forms of psychopathology, including substance use disorders. We performed genome-wide association studies (GWAS) of eight impulsive personality traits from the Barratt Impulsiveness Scale and the short UPPS-P Impulsive Personality Scale (N = 123,509-133,517 23andMe research participants of European ancestry), and a measure of Drug Experimentation (N = 130,684). Because these GWAS implicated the gene CADM2, we next performed single-SNP phenome-wide studies (PheWAS) of several of the implicated variants in CADM2 in a multi-ancestral 23andMe cohort (N = 3,229,317, European; N = 579,623, Latin American; N = 199,663, African American). Finally, we produced Cadm2 mutant mice and used them to perform a Mouse-PheWAS ("MouseWAS") by testing them with a battery of relevant behavioral tasks. In humans, impulsive personality traits showed modest chip-heritability (~6-11%), and moderate genetic correlations (rg = 0.20-0.50) with other personality traits, and various psychiatric and medical traits. We identified significant associations proximal to genes such as TCF4 and PTPRF, and also identified nominal associations proximal to DRD2 and CRHR1. PheWAS for CADM2 variants identified associations with 378 traits in European participants, and 47 traits in Latin American participants, replicating associations with risky behaviors, cognition and BMI, and revealing novel associations including allergies, anxiety, irritable bowel syndrome, and migraine. Our MouseWAS recapitulated some of the associations found in humans, including impulsivity, cognition, and BMI. Our results further delineate the role of CADM2 in impulsivity and numerous other psychiatric and somatic traits across ancestries and species.

Tools for studying human microglia: In vitro and in vivo strategies.

Microglia may only represent 10% of central nervous system (CNS) cells but they perform critical roles in development, homeostasis and neurological disease. Microglia are also environmentally regulated, quickly losing their transcriptomic and epigenetic signature after leaving the CNS. This facet of microglia biology is both fascinating and technically challenging influencing the study of the genetics and function of human microglia in a manner that recapitulates the CNS environment. In this review we provide a comprehensive overview of existing in vitro and in vivo methodology to study human microglia, such as immortalized cells lines, stem cell-derived microglia, cerebral organoids and xenotransplantation. Since there is currently no single method that completely recapitulates all hallmarks of human ex vivo adult homeostatic microglia, we also discuss the advantages and limitations of each existing model as a practical guide for researchers.

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

Hyperactive LH Pulses and Elevated Kisspeptin and NKB Gene Expression in the Arcuate Nucleus of a PCOS Mouse Model.

Polycystic ovary syndrome (PCOS), a common reproductive disorder in women, is characterized by hyperandrogenemia, chronic anovulation, cystic ovarian follicles, and luteinizing hormone (LH) hyper-pulsatility, but the pathophysiology isn't completely understood. We recently reported a novel mouse model of PCOS using chronic letrozole (LET; aromatase inhibitor). Letrozole-treated females demonstrate multiple PCOS-like phenotypes, including polycystic ovaries, anovulation, and elevated circulating testosterone and LH, assayed in "one-off" measures. However, due to technical limitations, in vivo LH pulsatile secretion, which is elevated in PCOS women, was not previously studied, nor were the possible changes in reproductive neurons. Here, we used recent technical advances to examine in vivo LH pulse dynamics of freely moving LET female mice versus control and ovariectomized (OVX) mice. We also determined whether neural gene expression of important reproductive regulators such as kisspeptin, neurokinin B (NKB), and dynorphin, is altered in LET females. Compared to controls, LET females exhibited very rapid, elevated in vivo LH pulsatility, with increased pulse frequency, amplitude, and basal levels, similar to PCOS women. Letrozole-treated mice also had markedly elevated Kiss1, Tac2, and Pdyn expression and increased Kiss1 neuronal activation in the hypothalamic arcuate nucleus. Notably, the hyperactive LH pulses and increased kisspeptin neuron measures of LET mice were not as elevated as OVX females. Our findings indicate that LET mice, like PCOS women, have markedly elevated LH pulsatility, which likely drives increased androgen secretion. Increased hypothalamic kisspeptin and NKB levels may be fundamental contributors to the hyperactive LH pulse secretion in the LET PCOS-like condition and, perhaps, in PCOS women.

Sex- and sub region-dependent modulation of arcuate kisspeptin neurones by vasopressin and vasoactive intestinal peptide.

A population of kisspeptin neurones located in the hypothalamic arcuate nucleus (ARN) very likely represent the gonadotrophin-releasing hormone pulse generator responsible for driving pulsatile luteinising hormone secretion in mammals. As such, it has become important to understand the neural inputs that modulate the activity of ARN kisspeptin (ARNKISS ) neurones. Using a transgenic GCaMP6 mouse model allowing the intracellular calcium levels ([Ca2+ ]i ) of individual ARNKISS neurones to be assessed simultaneously, we examined whether the circadian neuropeptides vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) modulated the activity of ARNKISS neurones directly. To validate this methodology, we initially evaluated the effects of neurokinin B (NKB) on [Ca2+ ]i in kisspeptin neurones residing within the rostral, middle and caudal ARN subregions of adult male and female mice. All experiments were undertaken in the presence of tetrodotoxin and ionotropic amino acid antagonists. NKB was found to evoke an abrupt increase in [Ca2+ ]i in 95%-100% of kisspeptin neurones throughout the ARN of both sexes. By contrast, both VIP and AVP were found to primarily activate kisspeptin neurones located in the caudal ARN of female mice. Although 58% and 59% of caudal ARN kisspeptin neurones responded to AVP and VIP, respectively, in female mice, only 0%-8% of kisspeptin neurones located in other ARN subregions responded in females and 0%-12% of cells in any subregion in males (P < 0.05). These observations demonstrate unexpected sex differences and marked heterogeneity in functional neuropeptide receptor expression amongst ARNKISS neurones organised on a rostro-caudal basis. The functional significance of this unexpected influence of VIP and AVP on ARNKISS neurones remains to be established.

PACAP neurons in the ventral premammillary nucleus regulate reproductive function in the female mouse.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) is a neuromodulator implicated in anxiety, metabolism and reproductive behavior. PACAP global knockout mice have decreased fertility and PACAP modulates LH release. However, its source and role at the hypothalamic level remain unknown. We demonstrate that PACAP-expressing neurons of the ventral premamillary nucleus of the hypothalamus (PMVPACAP) project to, and make direct contact with, kisspeptin neurons in the arcuate and AVPV/PeN nuclei and a subset of these neurons respond to PACAP exposure. Targeted deletion of PACAP from the PMV through stereotaxic virally mediated cre- injection or genetic cross to LepR-i-cre mice with Adcyap1fl/fl mice led to delayed puberty onset and impaired reproductive function in female, but not male, mice. We propose a new role for PACAP-expressing neurons in the PMV in the relay of nutritional state information to regulate GnRH release by modulating the activity of kisspeptin neurons, thereby regulating reproduction in female mice.