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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/β. 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.

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/β. 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.

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 geneCADM2, we next performed single-SNP phenome-wide studies (PheWAS) of several of the implicated variants inCADM2in a multi-ancestral 23andMe cohort (N=3,229,317, European; N=579,623, Latin American; N=199,663, African American). Finally, we producedCadm2mutant 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=.20-.50) with other personality traits, and various psychiatric and medical traits. We identified significant associations proximal to genes such asTCF4andPTPRF, and also identified nominal associations proximal toDRD2andCRHR1. PheWAS forCADM2variants 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 ofCADM2in impulsivity and numerous other psychiatric and somatic traits across ancestries and species.

Author(s): Han, Claudia; Li, Rick; Hansen, Emily; Bennett, Hunter; Poirion, Olivier; Buchanan, Justin; Challacombe, Jean; Fixsen, Bethany; Trescott, Samantha; Schlachetzki, Johannes CM; Preissl, Sebastian; Wang, Allen; O’Connor, Carolyn; Warden, Anna; Shriram, Shreya; Kim, Roy; Nguyen, Celina; Schafer, Danielle; Ramirez, Gabriela; Anavim, Samuel; Johnson, Avalon; Sajti, Eniko; Gupta, Mihir; Levy, Michael; Ben-Haim, Sharona; Gonda, David; Laurent, Louise; Glass, Christopher; Coufal, Nicole | Abstract: The fetal period is a critical time for brain development, characterized by neurogenesis, neural migration, and synaptogenesis 1-3 . Microglia, the tissue resident macrophages of the brain, are observed as early as the fourth week of gestation 4 and are thought to engage in a variety of processes essential for brain development and homeostasis 5-11 . Conversely, microglia phenotypes are highly regulated by the brain environment 12-14 . Mechanisms by which human brain development influences the maturation of microglia and microglia potential contribution to neurodevelopmental disorders remain poorly understood. Here, we performed transcriptomic analysis of human fetal and postnatal microglia and corresponding cortical tissue to define age-specific brain environmental factors that may drive microglia phenotypes. Comparative analysis of open chromatin profiles using bulk and single-cell methods in conjunction with a new computational approach that integrates epigenomic and single-cell RNA-seq data allowed decoding of cellular heterogeneity with inference of subtype- and development stage-specific transcriptional regulators. Interrogation of in vivo and in vitro iPSC-derived microglia models provides evidence for roles of putative instructive signals and downstream gene regulatory networks which establish human-specific fetal and postnatal microglia gene expression programs and potentially contribute to neurodevelopmental disorders.

The COVID-19 pandemic was caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs. SARS-CoV-2 proteins, NSP8 and NSP12, bind to specific regions in the RNA viral genome, providing evidence for their central and potential roles in replication, transcription and genome recombination. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4,821 unique host RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 which upregulates mitochondrial and N-linked glycosylation proteins. Furthermore, siRNA knockdown of host genes targeted by the viral proteins in human lung organoid cells demonstrates substantial antiviral effects. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens inflammatory responses such as interleukin-1α/β production. Our extensive 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. Funding Information: The work has been supported by Emergency COVID-19 Research Seed Funding (#R00RG2636) from the University of California Office of the President. This publication includes data generated at the UC San Diego IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S10 OD026929). J.S.X as a visiting fellow is partially supported by Agency for Science, Technology and Research (A*STAR) and Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012. ELVN is supported by the NHGRI (R00HG009530). Declaration of Interests: J.S.X, F.E.T, J.C.S and G.W.Y declare a pending patent application. ELVN is co-founder, member of the Board of Directors, on the SAB, equity holder, and paid consultant for Eclipse BioInnovations. ELVN’s interests have been reviewed and approved by the Baylor College of Medicine in accordance with its conflict of interest policies. The authors declare no other competing interests Ethics Approval Statement: This study protocol was approved by the Institutional Review Board of UCSD's Human Research Protections Program (181180).

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.

Polycystic ovary syndrome (PCOS) is a reproductive disorder in women characterized by hyperandrogenemia, anovulation, cystic ovaries, and LH hyper-pulsatility, but the mechanisms causing the pathophysiology remain incompletely understood. We recently reported a novel mouse model that recapitulates the majority of PCOS phenotypes in adulthood. Females given constant, long-term letrozole to reduce aromatase activity demonstrate PCOS-like phenotypes, including polycystic ovaries, anovulation, elevated circulating testosterone, and increased LH. In vivo LH pulsatile secretion, which is greatly elevated in PCOS women, was not previously studied, nor were possible changes in reproductive neurons known to control GnRH/LH secretion. Here, we used recent technical advances in the field to examine in vivo LH pulse dynamics of freely-moving LET female mice versus control and ovariectomized (OVX) mice. We also studied whether hypothalamic gene expression of several important reproductive regulators, 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. LET mice also had markedly elevated Kiss1, Tac2, and Pdyn expression along with increased Kiss1 neuron activation in the hypothalamic arcuate nucleus. Although elevated, most hyperactive LH pulse parameters and increased arcuate mRNA measures of LET mice were significantly lower than in OVX littermates. Our findings demonstrate that LET mice, like PCOS women, have markedly elevated LH pulsatility which likely drives increased ovarian androgen secretion. Increased arcuate kisspeptin and NKB levels may be fundamental contributors to the enhanced stimulation of LH pulse secretion in this PCOS-like condition, and perhaps, in some PCOS women.

Pituitary adenylate cyclase activating polypeptide (PACAP) 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 PACAPfl/flmice led to delayed puberty onset and impaired reproductive function in female, but not male, mice. We propose a new, sex-specific 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.Disclosure statementThe authors have nothing to disclose.

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.