Probes for INS

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive PACAP knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in wild-type mice that do not occur in knock-out mice (aPRGs). Comparing constitutive PACAP knock-out mice to a variety of temporally and regionally specific PACAP knock-outs, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knock-out mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.

Infant avoidance and aggression are promoted by activation of the Urocortin-3 expressing neurons of the perifornical area of hypothalamus (PeFAUcn3) in male and female mice. PeFAUcn3 neurons have been implicated in stress, and stress is known to reduce maternal behavior. We asked how chronic restraint stress (CRS) affects infant-directed behavior in virgin and lactating females and what role PeFAUcn3 neurons play in this process. Here we show that infant-directed behavior increases activity in the PeFAUcn3 neurons in virgin and lactating females. Chemogenetic inhibition of PeFAUcn3 neurons facilitates pup retrieval in virgin females. CRS reduces pup retrieval in virgin females and increases activity of PeFAUcn3 neurons, while CRS does not affect maternal behavior in lactating females. Inhibition of PeFAUcn3 neurons blocks stress-induced deficits in pup-directed behavior in virgin females. Together, these data illustrate the critical role for PeFAUcn3 neuronal activity in mediating the impact of chronic stress on female infant-directed behavior.

Seasonal changes in day length (photoperiod) affect numerous physiological functions. The suprachiasmatic nucleus (SCN)-paraventricular nucleus (PVN) axis plays a key role in processing photoperiod-related information. Seasonal variations in SCN and PVN neurotransmitter expression have been observed in humans and animal models. However, the molecular mechanisms by which the SCN-PVN network responds to altered photoperiod is unknown. Here, we show in mice that neuromedin S (NMS) and vasoactive intestinal polypeptide (VIP) neurons in the SCN display photoperiod-induced neurotransmitter plasticity. In vivo recording of calcium dynamics revealed that NMS neurons alter PVN network activity in response to winter-like photoperiod. Chronic manipulation of NMS neurons is sufficient to induce neurotransmitter switching in PVN neurons and affects locomotor activity. Our findings reveal previously unidentified molecular adaptations of the SCN-PVN network in response to seasonality and the role for NMS neurons in adjusting hypothalamic function to day length via a coordinated multisynaptic neurotransmitter switching affecting behavior.

Oligodendrocytes are glial cells that support and insulate axons in the central nervous system through the production of myelin. Oligodendrocytes arise throughout embryonic and early postnatal development from oligodendrocyte precursor cells (OPCs), and recent work demonstrated that they are a transcriptional heterogeneous cell population, but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of the corpus callosum, cingulate, motor, and somatosensory cortex in the brain, as well as gray matter (GM) and white matter (WM) regions in the spinal cord, at postnatal (P10), juvenile (P20), and young adult (P60) stages. We systematically compare the abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations.We observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain, corroborating with previous studies. We found myelination still ongoing in the adult corpus callosum while it was more advanced in the cortex. Interestingly, we also observed a lateral-to-medial gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. The ISS experiments also exposed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM, and we found that neighboring preferences of some oligodendroglia populations are altered from the juvenile to the adult CNS.Overall, our ISS experiments reveal spatial heterogeneity of oligodendrocyte lineage progression in the brain and spinal cord and uncover differences in the timing of oligodendrocyte differentiation and myelination, which could be relevant to further investigate functional heterogeneity of oligodendroglia, especially in the context of injury or disease.

Interleukin-6 (IL-6) has been long considered a key player in cancer-associated cachexia 1-15 . It is believed that sustained elevation of IL-6 production during cancer progression causes brain dysfunctions, which ultimately result in cachexia 16-20 . However, how peripheral IL-6 influences the brain remains poorly understood. Here we show that neurons in the area postrema (AP), a circumventricular structure in the hindbrain, mediate the function of IL-6 in cancer-associated cachexia in mice. We found that circulating IL-6 can rapidly enter the AP and activate AP neurons. Peripheral tumor, known to increase circulating IL-6 1-5,15,18,21-23 , leads to elevated IL-6 and neuronal hyperactivity in the AP, and causes potentiated excitatory synaptic transmission onto AP neurons. Remarkably, neutralization of IL-6 in the brain of tumor-bearing mice with an IL-6 antibody prevents cachexia, reduces the hyperactivity in an AP network, and markedly prolongs lifespan. Furthermore, suppression of Il6ra , the gene encoding IL-6 receptor, specifically in AP neurons with CRISPR/dCas9 interference achieves similar effects. Silencing of Gfral-expressing AP neurons also ameliorates the cancer-associated cachectic phenotypes and AP network hyperactivity. Our study identifies a central mechanism underlying the function of peripheral IL-6, which may serve as a target for treating cancer-associated cachexia.

Interferon regulatory factor 8 (IRF8) is a transcription factor necessary for the maturation of microglia, as well as other peripheral immune cells. It also regulates the transition of microglia and other immune cells to a pro-inflammatory phenotype. Irf8 is also a known risk gene for multiple sclerosis and lupus and it has recently been shown to be downregulated in schizophrenia. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. Here, we show by RNAseq from Irf8-/- male and female mouse brains that several genes involved in regulation of neural activity are dysregulated. We then show these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to lethal seizures in male and female Irf8-/- mice. Finally, we identify that TNF-α is elevated specifically in microglia in the CNS and genetic or acute pharmacological blockade of TNF-α in the Irf8-/- central nervous system (CNS) rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. Our data further suggest that neuronal function is impacted by loss of IRF8, a factor involved in neuropsychiatric and neurodegenerative diseases.SIGNIFICANCE STATEMENTHere, we identify a previously unknown and key role for Interferon regulator factor 8 (IRF8) in regulating neural excitability and seizures. We further determine these effects on neural circuits are through elevated TNF-α in the CNS. As IRF8 has most widely been studied in the context of regulating the development and inflammatory signaling in microglia and other immune cells, we have uncovered a novel function. Further, IRF8 is a risk gene for multiple sclerosis (MS) and lupus, IRF8 is dysregulated in schizophrenia, and elevated TNF-α has been identified in a multitude of neurological conditions. Thus, elucidating these IRF8 and TNF-α-dependent effects on brain circuit function have profound implications for understanding underlying, therapeutically-relevant mechanisms of disease.

The germinal center (GC) plays a central role in the generation of antigen-specific B cells and antibodies. Tight regulation of the GC is essential due to the inherent risks of tumorigenesis and autoimmunity posed by inappropriate GC B cell processes. Gammaherpesviruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) utilize numerous armaments to drive infected naïve B cells, independent of antigen, through GC reactions to expand the latently infected B cell population and establish a stable latency reservoir. We previously demonstrated that the MHV68 microRNA (miRNA) mghv-miR-M1-7-5p represses host EWSR1 (Ewing sarcoma breakpoint region 1) to promote B cell infection. EWSR1 is a transcription and splicing regulator that is recognized for its involvement as a fusion protein in Ewing sarcoma. A function for EWSR1 in B cell responses has not been previously reported. Here, we demonstrate that 1) B cell-specific deletion of EWSR1 had no effect on generation of mature B cell subsets or basal immunoglobulin levels in naïve mice, 2) repression or ablation of EWSR1 in B cells promoted expansion of MHV68 latently infected GC B cells, and 3) B cell-specific deletion of EWSR1 during a normal immune response to nonviral antigen resulted in significantly elevated numbers of antigen-specific GC B cells, plasma cells, and circulating antibodies. Notably, EWSR1 deficiency did not affect the proliferation or survival of GC B cells but instead resulted in the generation of increased numbers of precursor GC B cells. Cumulatively, these findings demonstrate that EWSR1 is a negative regulator of B cell responses.

During infection, animals exhibit adaptive changes in physiology and behaviour aimed at increasing survival. Although many causes of infection exist, they trigger similar stereotyped symptoms such as fever, warmth-seeking, loss of appetite and fatigue1,2. Yet exactly how the nervous system alters body temperature and triggers sickness behaviours to coordinate responses to infection remains unknown. Here we identify a previously uncharacterized population of neurons in the ventral medial preoptic area (VMPO) of the hypothalamus that are activated after sickness induced by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid. These neurons are crucial for generating a fever response and other sickness symptoms such as warmth-seeking and loss of appetite. Single-nucleus RNA-sequencing and multiplexed error-robust fluorescence in situ hybridization uncovered the identity and distribution of LPS-activated VMPO (VMPOLPS) neurons and non-neuronal cells. Gene expression and electrophysiological measurements implicate a paracrine mechanism in which the release of immune signals by non-neuronal cells during infection activates nearby VMPOLPS neurons. Finally, we show that VMPOLPS neurons exert a broad influence on the activity of brain areas associated with behavioural and homeostatic functions and are synaptically and functionally connected to circuit nodes controlling body temperature and appetite. Together, these results uncover VMPOLPS neurons as a control hub that integrates immune signals to orchestrate multiple sickness symptoms in response to infection.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with short overall survival; the standard of care (SoC) is chemotherapy. Immunotherapies in development aim to remodel the stroma by depleting immunosuppressive cell types or using T-cell redirection to kill tumor cells. To date, none of these methods have improved overall survival beyond SoC. Next generation immunotherapies that employ histopathology and molecular subtyping1 for target and patient selection may succeed. Here we leverage a spatial transcriptomics platform (Nanostring Digital Spatial Profiling, DSP) to reveal molecular signaling in tumoral and stromal cells in 57 PDAC patients using tumor microarrays (TMAs). This approach is rapid and clinically relevant as molecular and histology data can be easily bridged.MethodsTMAs generated from surgical resection tissue were commercially sourced. DSP was performed using the CTA RNA panel (1,800 target genes) using PanCK fluorescence for tumor/stroma segmentation. In parallel, slides were chromogenically stained for T-cells (CD3) and macrophages (CD68/CD163). Differential gene expression, gene signature and gene co-expression network analysis was performed using linear models in R.2 3ResultsDifferential gene expression analysis and correlation to IHC confirmed the DSP platform successfully profiled tumor and stromal compartments (figure 1). Immune cell signatures4 and pathway analysis revealed a heterogenous stromal environment. Using a fibroblast gene signature derived from single-cell RNAseq5 we found fibroblast density was positively correlated to PDGFR signaling and MHC-II expression but negatively correlated to B, CD4+ T and neutrophil cell levels (figure 2a). This finding supports the idea that atypical antigen presentation in cancer associated fibroblasts (CAFs) may be exploitable for immunotherapies.6 We constructed a co-expression network from in-situ stromal gene expression and used it to identify receptors coordinately expressed with the immunosuppressive macrophage marker CSF1R as a bispecific antibody partner (figure 2b).7 Classical macrophage markers were identified but also receptors with lesser-known functions in macrophages (TIM3/HAVCR2, FPR3, MS4A6A, LILRB4). Surveying target pairs in this method allows rapid, patient-specific confirmation in serial TMA sections with singleplex IHC or RNAscope.Abstact 928 Figure 1Segmentation strategy and validation of DSP (A) PanCK, CD68 and CD3 staining from two representative tumor cores; (B, C) correlation of gene transcripts in stroma to cell counts from chromogenic staining; (D) heatmap of selected genes differentially expressed in tumor and stroma (n=57 patients).Abstract 928 Figure 2Exploration of the stromal compartment in PDAC TMAs. (A) Heatmap of selected cell type and gene signatures from gene expression in the stroma, color represents single sample enrichment score using GSVA method; (B) a gene co-expression subnetwork in the stroma centered on CSF1R, edge thickness represents strength of correlation, green nodes have evidence for cell surface expression based on proteomic profiling.7ConclusionsIn this study we were able to recapitulate known PDAC biology using very small samples of primary tumors. The combination of TMAs and DSP enables a rapid validation of targets and hypothesis generation for bispecific parings. Further analysis of untreated (n=14) and post-adjuvant chemotherapy (n=26) patients using RNA DSP, IHC and bulk RNAseq is under way. Results from this cohort will enable an integrated histopathology and molecular approach to developing next-generation immunotherapies.ReferencesCollisson EA, Bailey P, Chang DK, Biankin AV. Molecular subtypes of pancreatic cancer. Nat Rev Gastroenterol Hepatol 2019 April;16(4):207-220.Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK (2015). “limma powers differential expression analyses for RNA-sequencing and microarray studies.” Nucleic Acids Research 43(7):e47.Hänzelmann S, Castelo R, Guinney J (2013). “GSVA: gene set variation analysis for microarray and RNA-Seq data.” BMC Bioinformatics 14,7.Charoentong P, Finotello F, Angelova M, Mayer C, Efremova M, Rieder D, Hackl H, Trajanoski Z. Pan-cancer immunogenomic analyses reveal genotype-immunophenotype relationships and predictors of response to checkpoint blockade. Cell Rep 2017 January 3;18(1):248-262.Tirosh I, Izar B, Prakadan SM, Wadsworth MH 2nd, Treacy D, Trombetta JJ, Rotem A, Rodman C, Lian C, Murphy G, Fallahi-Sichani M, Dutton-Regester K, Lin JR, Cohen O, Shah P, Lu D, Genshaft AS, Hughes TK, Ziegler CG, Kazer SW, Gaillard A, Kolb KE, Villani AC, Johannessen CM, Andreev AY, Van Allen EM, Bertagnolli M, Sorger PK, Sullivan RJ, Flaherty KT, Frederick DT, Jané-Valbuena J, Yoon CH, Rozenblatt-Rosen O, Shalek AK, Regev A, Garraway LA. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 2016 April 8;352(6282):189-96.Elyada E, Bolisetty M, Laise P, Flynn WF, Courtois ET, Burkhart RA, Teinor JA, Belleau P, Biffi G, Lucito MS, Sivajothi S, Armstrong TD, Engle DD, Yu KH, Hao Y, Wolfgang CL, Park Y, Preall J, Jaffee EM, Califano A, Robson P, Tuveson DA. Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts. Cancer Discov 2019 August;9(8):1102-1123. Bausch-Fluck D, Hofmann A, Bock T, Frei AP, Cerciello F, Jacobs A, Moest H, Omasits U, Gundry RL, Yoon C, Schiess R, Schmidt A, Mirkowska P, Härtlová A, Van Eyk JE, Bourquin JP, Aebersold R, Boheler KR, Zandstra P, Wollscheid B. A mass spectrometric-derived cell surface protein atlas. PLoS One 2015 April 20;10(3):e0121314.Ethics ApprovalSpecimens were harvested from unused tissue after a surgical tumor resection procedure. A discrete legal consent form from both hospital and individuals was obtained by the commercial tissue vendor BioMax US for all samples analyzed in this abstract. All human tissues are collected under HIPPA approved protocols.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Obesity is a global pandemic that is causally linked to many life-threatening diseases. Apart from some rare genetic conditions, the biological drivers of overeating and reduced activity are unclear. Here, we show that neurotensin-expressing neurons in the mouse interstitial nucleus of the posterior limb of the anterior commissure (IPAC), a nucleus of the central extended amygdala, encode dietary preference for unhealthy energy-dense foods. Optogenetic activation of IPACNts neurons promotes obesogenic behaviors, such as hedonic eating, and modulates food preference. Conversely, acute inhibition of IPACNts neurons reduces feeding and decreases hedonic eating. Chronic inactivation of IPACNts neurons recapitulates these effects, reduces preference for sweet, non-caloric tastants and, furthermore, enhances locomotion and energy expenditure; as a result, mice display long-term weight loss and improved metabolic health and are protected from obesity. Thus, the activity of a single neuronal population bidirectionally regulates energy homeostasis. Our findings could lead to new therapeutic strategies to prevent and treat obesity.