Probes for INS

The complexity of affected brain regions and cell types is a challenge for Huntington's disease (HD) treatment. Here we use single nucleus RNA sequencing to investigate molecular pathology in the cortex and striatum from R6/2 mice and human HD post-mortem tissue. We identify cell type-specific and -agnostic signatures suggesting oligodendrocytes (OLs) and oligodendrocyte precursors (OPCs) are arrested in intermediate maturation states. OL-lineage regulators OLIG1 and OLIG2 are negatively correlated with CAG length in human OPCs, and ATACseq analysis of HD mouse NeuN-negative cells shows decreased accessibility regulated by OL maturation genes. The data implicates glucose and lipid metabolism in abnormal cell maturation and identify PRKCE and Thiamine Pyrophosphokinase 1 (TPK1) as central genes. Thiamine/biotin treatment of R6/1 HD mice to compensate for TPK1 dysregulation restores OL maturation and rescues neuronal pathology. Our insights into HD OL pathology spans multiple brain regions and link OL maturation deficits to abnormal thiamine metabolism.

The central amygdala (CEA) has been richly studied for interpreting function and behavior according to specific cell types and circuits. Such work has typically defined molecular cell types by classical inhibitory marker genes; consequently, whether marker-gene-defined cell types exhaustively cover the CEA and co-vary with connectivity remains unresolved. Here, we combined single-cell RNA sequencing, multiplexed fluorescent in situ hybridization, immunohistochemistry, and long-range projection mapping to derive a "bottom-up" understanding of CEA cell types. In doing so, we identify two major cell types, encompassing one-third of all CEA neurons, that have gone unresolved in previous studies. In spatially mapping these novel types, we identify a non-canonical CEA subdomain associated with Nr2f2 expression and uncover an Isl1-expressing medial cell type that accounts for many long-range CEA projections. Our results reveal new CEA organizational principles across cell types and spatial scales and provide a framework for future work examining cell-type-specific behavior and function.

We show that the sex of human experimenters affects mouse behaviors and responses following administration of the rapid-acting antidepressant ketamine and its bioactive metabolite (2R,6R)-hydroxynorketamine. Mice showed aversion to the scent of male experimenters, preference for the scent of female experimenters and increased stress susceptibility when handled by male experimenters. This human-male-scent-induced aversion and stress susceptibility was mediated by the activation of corticotropin-releasing factor (CRF) neurons in the entorhinal cortex that project to hippocampal area CA1. Exposure to the scent of male experimenters before ketamine administration activated CA1-projecting entorhinal cortex CRF neurons, and activation of this CRF pathway modulated in vivo and in vitro antidepressant-like effects of ketamine. A better understanding of the specific and quantitative contributions of the sex of human experimenters to study outcomes in rodents may improve replicability between studies and, as we have shown, reveal biological and pharmacological mechanisms.

Scents have been employed for millennia to allay stress, but whether or how they might do so is largely unknown. Fear and stress induce increases in blood stress hormones controlled by hypothalamic corticotropin releasing hormone neurons (CRHNs). Here, we report that two common odorants block mouse stress hormone responses to three potent stressors: physical restraint, predator odor, and male-male social confrontation. One odorant inhibits restraint and predator odor activation of excitatory neurons upstream of CRHNs in the bed nucleus of the stria terminalis (BNSTa). In addition, both activate inhibitory neurons upstream of CRHNs in the hypothalamic ventromedial nucleus (VMH) and silencing of VMH inhibitory neurons hinders odor blocking of stress. Together, these findings indicate that odor blocking can occur via two mechanisms: (1) Inhibition of excitatory neurons that transmit stress signals to CRHNs and (2) activation of inhibitory neurons that act directly or indirectly to inhibit stressor activation of CRHNs.

COVID-19-induced ‘acute respiratory distress syndrome’ (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyzed pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single cell genomics, immunohistology and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not Influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

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.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Sepsis is hallmarked by high plasma cortisol/corticosterone (CORT), low adrenocorticotropic hormone (ACTH), and high pro-opiomelanocortin (POMC). While corticotropin-releasing hormone-(CRH) and arginine-vasopressin (AVP)-driven pituitary POMC expression remains active, POMC processing into ACTH becomes impaired. Low ACTH is accompanied by loss of adrenocortical structure, although steroidogenic enzymes remain expressed. We hypothesized that treatment of sepsis with hydrocortisone (HC) aggravates this phenotype whereas CRH infusion safeguards ACTH-driven adrenocortical structure.In a fluid-resuscitated, antibiotics-treated mouse model of prolonged sepsis, we compared the effects of HC and CRH infusion with placebo on plasma ACTH, POMC, and CORT; on markers of hypothalamic CRH and AVP signaling and pituitary POMC processing; and on the adrenocortical structure and markers of steroidogenesis. In adrenal explants, we studied the steroidogenic capacity of POMC.During sepsis, HC further suppressed plasma ACTH, but not POMC, predominantly by suppressing sepsis-activated CRH/AVP-signaling pathways. In contrast, in CRH-treated sepsis, plasma ACTH was normalized following restoration of pituitary POMC processing. The sepsis-induced rise in markers of adrenocortical steroidogenesis was unaltered by CRH and suppressed partially by HC, which also increased adrenal markers of inflammation. Ex vivo stimulation of adrenal explants with POMC increased CORT as effectively as an equimolar dose of ACTH.Treatment of sepsis with HC impaired integrity and function of the hypothalamic-pituitary-adrenal axis at the level of the pituitary and the adrenal cortex while CRH restored pituitary POMC processing without affecting the adrenal cortex. Sepsis-induced high-circulating POMC may be responsible for ongoing adrenocortical steroidogenesis despite low ACTH.

Brain macrophages include microglia in the parenchyma, border-associated macrophages in the meningeal-choroid plexus-perivascular space, and monocyte-derived macrophages that infiltrate the brain under various disease conditions. The vast heterogeneity of these cells has been elucidated over the last decade using revolutionary multiomics technologies. As such, we can now start to define these various macrophage populations according to their ontogeny and their diverse functional programs during brain development, homeostasis and disease pathogenesis. In this review, we first outline the critical roles played by brain macrophages during development and healthy aging. We then discuss how brain macrophages might undergo reprogramming and contribute to neurodegenerative disorders, autoimmune diseases, and glioma. Finally, we speculate about the most recent and ongoing discoveries that are prompting translational attempts to leverage brain macrophages as prognostic markers or therapeutic targets for diseases that affect the brain.