COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

Obesity-Linked PPARγ S273 Phosphorylation Promotes Insulin Resistance through Growth Differentiation Factor 3.

The thiazolidinediones (TZDs) are ligands of PPARγ that improve insulin sensitivity, but their use is limited by significant side effects. Recently, we demonstrated a mechanism wherein TZDs improve insulin sensitivity distinct from receptor agonism and adipogenesis: reversal of obesity-linked phosphorylation of PPARγ at serine 273. However, the role of this modification hasn't been tested genetically. Here we demonstrate that mice encoding an allele of PPARγ that cannot be phosphorylated at S273 are protected from insulin resistance, without exhibiting differences in body weight or TZD-associated side effects. Indeed, hyperinsulinemic-euglycemic clamp experiments confirm insulin sensitivity. RNA-seq in these mice reveals reduced expression of Gdf3, a BMP family member. Ectopic expression of Gdf3 is sufficient to induce insulin resistance in lean, healthy mice. We find Gdf3 inhibits BMP signaling and insulin signaling in vitro. Together, these results highlight the diabetogenic role of PPARγ S273 phosphorylation and focus attention on a putative target, Gdf3.

Adipocytes fail to maintain cellular identity during obesity due to reduced PPARγ activity and elevated TGFß-SMAD signaling.

OBJECTIVE: Obesity due to overnutrition causes adipose tissue dysfunction, which is a critical pathological step on the road to type 2 diabetes (T2D) and other metabolic disorders. In this study, we conducted an unbiased investigation into the fundamental molecular mechanisms by which adipocytes transition to an unhealthy state during obesity. METHODS: We used nuclear tagging and translating ribosome affinity purification (NuTRAP) reporter mice crossed with Adipoq-Cre mice to determine adipocyte-specific 1) transcriptional profiles (RNA-seq), 2) promoter and enhancer activity (H3K27ac ChIP-seq), 3) and PPARγ cistrome (ChIP-seq) profiles in mice fed chow or a high-fat diet (HFD) for 10 weeks. We also assessed the impact of the PPARγ agonist rosiglitazone (Rosi) on gene expression and cellular state of adipocytes from the HFD-fed mice. We integrated these data to determine the transcription factors underlying adipocyte responses to HFD and conducted functional studies using shRNA-mediated loss-of-function approaches in 3T3-L1 adipocytes. RESULTS: Adipocytes from the HFD-fed mice exhibited reduced expression of adipocyte markers and metabolic genes and enhanced expression of myofibroblast marker genes involved in cytoskeletal organization, accompanied by the formation of actin filament structures within the cell. PPARγ binding was globally reduced in adipocytes after HFD feeding, and Rosi restored the molecular and cellular phenotypes of adipocytes associated with HFD feeding. We identified the TGFß1 effector protein SMAD to be enriched at HFD-induced promoters and enhancers and associated with myofibroblast signature genes. TGFß1 treatment of mature 3T3-L1 adipocytes induced gene expression and cellular changes similar to those seen after HFD in vivo, and knockdown of Smad3 blunted the effects of TGFß1. CONCLUSIONS: Our data demonstrate that adipocytes fail to maintain cellular identity after HFD feeding, acquiring characteristics of a myofibroblast-like cell type through reduced PPARγ activity and elevated TGFß-SMAD signaling. This cellular identity crisis may be a fundamental mechanism that drives functional decline of adipose tissues during obesity.

Warming Induces Significant Reprogramming of Beige, but Not Brown, Adipocyte Cellular Identity.

Beige and brown adipocytes generate heat in response to reductions in ambient temperature. When warmed, both beige and brown adipocytes exhibit morphological "whitening," but it is unknown whether or to what extent this represents a true shift in cellular identity. Using cell-type-specific profiling in vivo, we uncover a unique paradigm of temperature-dependent epigenomic plasticity of beige, but not brown, adipocytes, with conversion from a brown to a white chromatin state. Despite this profound shift in cellular identity, warm whitened beige adipocytes retain an epigenomic memory of prior cold exposure defined by an array of poised enhancers that prime thermogenic genes for rapid response during a second bout of cold exposure. We further show that a transcriptional cascade involving glucocorticoid receptor and Zfp423 can drive warm-induced whitening of beige adipocytes. These studies identify the epigenomic and transcriptional bases of an extraordinary example of cellular plasticity in response to environmental signals.

Simultaneous Transcriptional and Epigenomic Profiling from Specific Cell Types within Heterogeneous Tissues In Vivo.

Epigenomic mechanisms direct distinct gene expression programs for different cell types. Various in vivo tissues have been subjected to epigenomic analysis; however, these studies have been limited by cellular heterogeneity, resulting in composite gene expression and epigenomic profiles. Here, we introduce "NuTRAP," a transgenic mouse that allows simultaneous isolation of cell-type-specific translating mRNA and chromatin from complex tissues. Using NuTRAP, we successfully characterize gene expression and epigenomic states of various adipocyte populations in vivo, revealing significant differences compared to either whole adipose tissue or in vitro adipocyte cell lines. We find that chromatin immunoprecipitation sequencing (ChIP-seq) using NuTRAP is highly efficient, scalable, and robust with even limited cell input. We further demonstrate the general utility of NuTRAP by analyzing hepatocyte-specific epigenomic states. The NuTRAP mouse is a resource that provides a powerful system for cell-type-specific gene expression and epigenomic profiling.

Nuclear Mechanisms of Insulin Resistance.

Insulin resistance is a sine qua non of type 2 diabetes and is associated with many other clinical conditions. Decades of research into mechanisms underlying insulin resistance have mostly focused on problems in insulin signal transduction and other mitochondrial and cytosolic pathways. By contrast, relatively little attention has been focused on transcriptional and epigenetic contributors to insulin resistance, despite strong evidence that such nuclear mechanisms play a major role in the etiopathogenesis of this condition. In this review, we summarize the evidence for nuclear mechanisms of insulin resistance, focusing on three transcription factors with a major impact on insulin action in liver, muscle, and fat.

Charting a dynamic DNA methylation landscape of the human genome.

DNA methylation is a defining feature of mammalian cellular identity and is essential for normal development. Most cell types, except germ cells and pre-implantation embryos, display relatively stable DNA methylation patterns, with 70-80% of all CpGs being methylated. Despite recent advances, we still have a limited understanding of when, where and how many CpGs participate in genomic regulation. Here we report the in-depth analysis of 42 whole-genome bisulphite sequencing data sets across 30 diverse human cell and tissue types. We observe dynamic regulation for only 21.8% of autosomal CpGs within a normal developmental context, most of which are distal to transcription start sites. These dynamic CpGs co-localize with gene regulatory elements, particularly enhancers and transcription-factor-binding sites, which allow identification of key lineage-specific regulators. In addition, differentially methylated regions (DMRs) often contain single nucleotide polymorphisms associated with cell-type-related diseases as determined by genome-wide association studies. The results also highlight the general inefficiency of whole-genome bisulphite sequencing, as 70-80% of the sequencing reads across these data sets provided little or no relevant information about CpG methylation. To demonstrate further the utility of our DMR set, we use it to classify unknown samples and identify representative signature regions that recapitulate major DNA methylation dynamics. In summary, although in theory every CpG can change its methylation state, our results suggest that only a fraction does so as part of coordinated regulatory programs. Therefore, our selected DMRs can serve as a starting point to guide new, more effective reduced representation approaches to capture the most informative fraction of CpGs, as well as further pinpoint putative regulatory elements.

Drosophila tao controls mushroom body development and ethanol-stimulated behavior through par-1.

In both mammalian and insect models of ethanol-induced behavior, low doses of ethanol stimulate locomotion. However, the mechanisms of the stimulant effects of ethanol on the CNS are mostly unknown. We have identified tao, encoding a serine-threonine kinase of the Ste20 family, as a gene necessary for ethanol-induced locomotor hyperactivity in Drosophila. Mutations in tao also affect behavioral responses to cocaine and nicotine, making flies resistant to the effects of both drugs. We show that tao function is required during the development of the adult nervous system and that tao mutations cause defects in the development of central brain structures, including the mushroom body. Silencing of a subset of mushroom body neurons is sufficient to reduce ethanol-induced hyperactivity, revealing the mushroom body as an important locus mediating the stimulant effects of ethanol. We also show that mutations in par-1 suppress both the mushroom body morphology and behavioral phenotypes of tao mutations and that the phosphorylation state of the microtubule-binding protein Tau can be altered by RNA interference knockdown of tao, suggesting that tao and par-1 act in a pathway to control microtubule dynamics during neural development.

Drosophila, a genetic model system to study cocaine-related behaviors: a review with focus on LIM-only proteins.

In the last decade, the fruit fly Drosophila melanogaster, highly accessible to genetic, behavioral and molecular analyses, has been introduced as a novel model organism to help decipher the complex genetic, neurochemical, and neuroanatomical underpinnings of behaviors induced by drugs of abuse. Here we review these data, focusing specifically on cocaine-related behaviors. Several of cocaine's most characteristic properties have been recapitulated in Drosophila. First, cocaine induces motor behaviors in flies that are remarkably similar to those observed in mammals. Second, repeated cocaine administration induces behavioral sensitization a form of behavioral plasticity believed to underlie certain aspects of addiction. Third, a key role for dopaminergic systems in mediating cocaine's effects has been demonstrated through both pharmacological and genetic methods. Finally, and most importantly, unbiased genetic screens, feasible because of the simplicity and scale with which flies can be manipulated in the laboratory, have identified several novel genes and pathways whose role in cocaine behaviors had not been anticipated. Many of these genes and pathways have been validated in mammalian models of drug addiction. We focus in this review on the role of LIM-only proteins in cocaine-induced behaviors.

Distinct behavioral responses to ethanol are regulated by alternate RhoGAP18B isoforms.

In most organisms, low ethanol doses induce increased activity, while high doses are sedating. To investigate the underlying mechanisms, we isolated Drosophila mutants with altered ethanol responsiveness. Mutations in white rabbit (whir), disrupting RhoGAP18B, are strongly resistant to the sedating effects of ethanol. This resistance can be suppressed by reducing the levels of Rho1 or Rac, implicating these GTPases in the behavioral response to ethanol. Indeed, expression of constitutively active forms of Rho1 or Rac1 in adult flies results in ethanol resistance similar to that observed in whir mutants. The whir locus produces several transcripts, RA-RD, which are predicted to encode three distinct RhoGAPs that share only the GAP domain. The RC transcript mediates the sedating effects of ethanol, while the RA transcript regulates its stimulant effects. Thus, distinct RhoGAPs, encoded by the same gene, regulate different manifestations of acute ethanol intoxication.

moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila.

We identified moody in a genetic screen for Drosophila mutants with altered cocaine sensitivity. Hypomorphic mutations in moody cause an increased sensitivity to cocaine and nicotine exposure. In contrast, sensitivity to the acute intoxicating effects of ethanol is reduced. The moody locus encodes two novel GPCRs, Moody-alpha and Moody-beta. While identical in their membrane-spanning domains, the two Moody proteins differ in their long carboxy-terminal domains, which are generated by use of alternative reading frames. Both Moody forms are required for normal cocaine sensitivity, suggesting that they carry out distinct but complementary functions. Moody-alpha and Moody-beta are coexpressed in surface glia that surround the nervous system, where they are actively required to maintain the integrity of the blood-brain barrier in the adult fly. We propose that a Moody-mediated signaling pathway functions in glia to regulate nervous system insulation and drug-related behaviors.

Lmo mutants reveal a novel role for circadian pacemaker neurons in cocaine-induced behaviors.

Drosophila has been developed recently as a model system to investigate the molecular and neural mechanisms underlying responses to drugs of abuse. Genetic screens for mutants with altered drug-induced behaviors thus provide an unbiased approach to define novel molecules involved in the process. We identified mutations in the Drosophila LIM-only (LMO) gene, encoding a regulator of LIM-homeodomain proteins, in a genetic screen for mutants with altered cocaine sensitivity. Reduced Lmo function increases behavioral responses to cocaine, while Lmo overexpression causes the opposite effect, reduced cocaine responsiveness. Expression of Lmo in the principal Drosophila circadian pacemaker cells, the PDF-expressing ventral lateral neurons (LN(v)s), is sufficient to confer normal cocaine sensitivity. Consistent with a role for Lmo in LN(v)function,Lmomutants also show defects in circadian rhythms of behavior. However, the role for LN(v)s in modulating cocaine responses is separable from their role as pacemaker neurons: ablation or functional silencing of the LN(v)s reduces cocaine sensitivity, while loss of the principal circadian neurotransmitter PDF has no effect. Together, these results reveal a novel role for Lmo in modulating acute cocaine sensitivity and circadian locomotor rhythmicity, and add to growing evidence that these behaviors are regulated by shared molecular mechanisms. The finding that the degree of cocaine responsiveness is controlled by the Drosophila pacemaker neurons provides a neuroanatomical basis for this overlap. We propose that Lmo controls the responsiveness of LN(v)s to cocaine, which in turn regulate the flies' behavioral sensitivity to the drug.

High-resolution analysis of ethanol-induced locomotor stimulation in Drosophila.

Understanding how ethanol influences behavior is key to deciphering the mechanisms of ethanol action and alcoholism. In mammals, low doses of ethanol stimulate locomotion, whereas high doses depress it. The acute stimulant effect of ethanol has been proposed to be a manifestation of its rewarding effects. In Drosophila, ethanol exposure transiently potentiates locomotor activity in a biphasic dose- and time-dependent manner. An initial short-lived peak of activity corresponds to an olfactory response to ethanol. A second, longer-lasting period of increased activity coincides with rising internal ethanol concentrations; these closely parallel concentrations that stimulate locomotion in mammals. High-resolution analysis of the walking pattern of individual flies revealed that locomotion consists of bouts of activity; bout structure can be quantified by bout frequency, bout length, and the time spent walking at high speeds. Ethanol exposure induces both dramatic and dynamic changes in bout structure. Mutants with increased ethanol sensitivity show distinct changes in ethanol-induced locomotor behavior, as well as genotype-specific changes in activity bout structure. Thus, the overall effect of ethanol on locomotor behavior in Drosophila is caused by changes in discrete quantifiable parameters of walking pattern. The effects of ethanol on locomotion are comparable in flies and mammals, suggesting that Drosophila is a suitable model system to study the underlying mechanisms.