Microglia regulate sleep through calcium-dependent modulation of norepinephrine transmission.

Sleep interacts reciprocally with immune system activity, but its specific relationship with microglia-the resident immune cells in the brain-remains poorly understood. Here, we show in mice that microglia can regulate sleep through a mechanism involving Gi-coupled GPCRs, intracellular Ca2+ signaling and suppression of norepinephrine transmission. Chemogenetic activation of microglia Gi signaling strongly promoted sleep, whereas pharmacological blockade of Gi-coupled P2Y12 receptors decreased sleep. Two-photon imaging in the cortex showed that P2Y12-Gi activation elevated microglia intracellular Ca2+, and blockade of this Ca2+ elevation largely abolished the Gi-induced sleep increase. Microglia Ca2+ level also increased at natural wake-to-sleep transitions, caused partly by reduced norepinephrine levels. Furthermore, imaging of norepinephrine with its biosensor in the cortex showed that microglia P2Y12-Gi activation significantly reduced norepinephrine levels, partly by increasing the adenosine concentration. These findings indicate that microglia can regulate sleep through reciprocal interactions with norepinephrine transmission.

Brown adipose tissue CoQ deficiency activates the integrated stress response and FGF21-dependent mitohormesis.

Coenzyme Q (CoQ) is essential for mitochondrial respiration and required for thermogenic activity in brown adipose tissues (BAT). CoQ deficiency leads to a wide range of pathological manifestations, but mechanistic consequences of CoQ deficiency in specific tissues, such as BAT, remain poorly understood. Here, we show that pharmacological or genetic CoQ deficiency in BAT leads to stress signals causing accumulation of cytosolic mitochondrial RNAs and activation of the eIF2α kinase PKR, resulting in activation of the integrated stress response (ISR) with suppression of UCP1 but induction of FGF21 expression. Strikingly, despite diminished UCP1 levels, BAT CoQ deficiency displays increased whole-body metabolic rates at room temperature and thermoneutrality resulting in decreased weight gain on high-fat diets (HFD). In line with enhanced metabolic rates, BAT and inguinal white adipose tissue (iWAT) interorgan crosstalk caused increased browning of iWAT in BAT-specific CoQ deficient animals. This mitohormesis-like effect depends on the ATF4-FGF21 axis and BAT-secreted FGF21, revealing an unexpected role for CoQ in the modulation of whole-body energy expenditure with wide-ranging implications for primary and secondary CoQ deficiencies.

Gene Expression Signatures of Contact Lens-Induced Myopia in Guinea Pig Retinal Pigment Epithelium.

Purpose: To identify key retinal pigment epithelium (RPE) genes linked to the induction of myopia in guinea pigs. Methods: To induce myopia, two-week-old pigmented guinea pigs (New Zealand strain, n = 5) wore -10 diopter (D) rigid gas-permeable contact lenses (CLs), for one day; fellow eyes were left without CLs and served as controls. Spherical equivalent refractive errors (SE) and axial length (AL) were measured at baseline and one day after initiation of CL wear. RNA sequencing was applied to RPE collected from both treated and fellow (control) eyes after one day of CL-wear to identify related gene expression changes. Additional RPE-RNA samples from treated and fellow eyes were subjected to quantitative real-time PCR (qRT-PCR) analysis for validation purposes. Results: The CLs induced myopia. The change from baseline values in SE was significantly different (P = 0.016), whereas there was no significant difference in the change in AL (P = 0.10). RNA sequencing revealed significant interocular differences in the expression in RPE of 13 genes: eight genes were significantly upregulated in treated eyes relative to their fellows, and five genes, including bone morphogenetic protein 2 (Bmp2), were significantly downregulated. The latter result was also confirmed by qRT-PCR. Additional analysis of differentially expressed genes revealed significant enrichment for bone morphogenetic protein (BMP) and TGF-ß signaling pathways. Conclusions: The results of this RPE gene expression study provide further supporting evidence for an important role of BMP2 in eye growth regulation, here from a guinea pig myopia model.

Estrogen Receptor ß as a Candidate Regulator of Sex Differences in the Maternal Immune Activation Model of ASD.

Interestingly, more males are diagnosed with autism spectrum disorder (ASD) than females, yet the mechanism behind this difference is unclear. Genes on the sex chromosomes and differential regulation by sex steroid hormones and their receptors are both candidate mechanisms to explain this sex-dependent phenotype. Nuclear receptors (NRs) are a large family of transcription factors, including sex hormone receptors, that mediate ligand-dependent transcription and may play key roles in sex-specific regulation of immunity and brain development. Infection during pregnancy is known to increase the probability of developing ASD in humans, and a mouse model of maternal immune activation (MIA), which is induced by injecting innate immune stimulants into pregnant wild-type mice, is commonly used to study ASD. Since this model successfully recaptures the behavioral phenotypes and male bias observed in ASD, we will discuss the potential role of sex steroid hormones and their receptors, especially focusing on estrogen receptor (ER)ß, in MIA and how this signaling may modulate transcription and subsequent inflammation in myeloid-lineage cells to contribute to the etiology of this neurodevelopmental disorder.

Tumor-induced disruption of the blood-brain barrier promotes host death.

Cancer patients often die from symptoms that manifest at a distance from any tumor. Mechanisms underlying these systemic physiological perturbations, called paraneoplastic syndromes, may benefit from investigation in non-mammalian systems. Using a non-metastatic Drosophila adult model, we find that malignant-tumor-produced cytokines drive widespread host activation of JAK-STAT signaling and cause premature lethality. STAT activity is particularly high in cells of the blood-brain barrier (BBB), where it induces aberrant BBB permeability. Remarkably, inhibiting STAT in the BBB not only rescues barrier function but also extends the lifespan of tumor-bearing hosts. We identify BBB damage in other pathological conditions that cause elevated inflammatory signaling, including obesity and infection, where BBB permeability also regulates host survival. IL-6-dependent BBB dysfunction is further seen in a mouse tumor model, and it again promotes host morbidity. Therefore, BBB alterations constitute a conserved lethal tumor-host interaction that also underlies other physiological morbidities.

Mitohormesis reprogrammes macrophage metabolism to enforce tolerance.

Macrophages generate mitochondrial reactive oxygen species and mitochondrial reactive electrophilic species as antimicrobials during Toll-like receptor (TLR)-dependent inflammatory responses. Whether mitochondrial stress caused by these molecules impacts macrophage function is unknown. Here, we demonstrate that both pharmacologically driven and lipopolysaccharide (LPS)-driven mitochondrial stress in macrophages triggers a stress response called mitohormesis. LPS-driven mitohormetic stress adaptations occur as macrophages transition from an LPS-responsive to LPS-tolerant state wherein stimulus-induced pro-inflammatory gene transcription is impaired, suggesting tolerance is a product of mitohormesis. Indeed, like LPS, hydroxyoestrogen-triggered mitohormesis suppresses mitochondrial oxidative metabolism and acetyl-CoA production needed for histone acetylation and pro-inflammatory gene transcription, and is sufficient to enforce an LPS-tolerant state. Thus, mitochondrial reactive oxygen species and mitochondrial reactive electrophilic species are TLR-dependent signalling molecules that trigger mitohormesis as a negative feedback mechanism to restrain inflammation via tolerance. Moreover, bypassing TLR signalling and pharmacologically triggering mitohormesis represents a new anti-inflammatory strategy that co-opts this stress response to impair epigenetic support of pro-inflammatory gene transcription by mitochondria.

Lytic Cell Death in Specific Microglial Subsets Is Required for Preventing Atypical Behavior in Mice.

Microglial cells are known to contribute to brain development and behaviors, but the mechanisms behind such functions are not fully understood. Here, we show that mice deficient in inflammasome regulators, including caspase-1 (Casp1), NLR family pyrin domain containing 3 (Nlrp3), IL-1 receptor (Il-1r), and gasdermin D (Gsdmd), exhibit behavior abnormalities characterized by hyperactivity and low anxiety levels. Furthermore, we found that expression of Casp1 in CX3CR1+ myeloid cells, which includes microglia, is required for preventing these abnormal behaviors. Through tissue clearing and 3D imaging, we discovered that small numbers of Cx3cr1-GFP+ fetal microglial cells formed clusters and underwent lytic cell death in the primitive thalamus and striatum between embryonic day (E)12.5 and E14.5. This lytic cell death was diminished in Casp1-deficient mice. Further analysis of the microglial clusters showed the presence of Pax6+ neural progenitor cells (NPCs); thus, we hypothesized that microglial lytic cell death is important for proper neuronal development. Indeed, increased numbers of neurons were observed in the thalamic subset in adult Casp1-/- brains. Finally, injection of drug inhibitors of NLRP3 and CASP1 into wild-type (WT) pregnant mice from E12.5 to E14.5, the period when lytic cell death was detected, was sufficient to induce atypical behaviors in offspring. Taken together, our data suggests that the inflammasome cascade in microglia is important for regulating neuronal development and normal behaviors, and that genetic or pharmacological inhibition of this pathway can induce atypical behaviors in mice.

Activity-Based Sensing with a Metal-Directed Acyl Imidazole Strategy Reveals Cell Type-Dependent Pools of Labile Brain Copper.

Copper is a required nutrient for life and particularly important to the brain and central nervous system. Indeed, copper redox activity is essential to maintaining normal physiological responses spanning neural signaling to metabolism, but at the same time copper misregulation is associated with inflammation and neurodegeneration. As such, chemical probes that can track dynamic changes in copper with spatial resolution, especially in loosely bound, labile forms, are valuable tools to identify and characterize its contributions to healthy and disease states. In this report, we present an activity-based sensing (ABS) strategy for copper detection in live cells that preserves spatial information by a copper-dependent bioconjugation reaction. Specifically, we designed copper-directed acyl imidazole dyes that operate through copper-mediated activation of acyl imidazole electrophiles for subsequent labeling of proximal proteins at sites of elevated labile copper to provide a permanent stain that resists washing and fixation. To showcase the utility of this new ABS platform, we sought to characterize labile copper pools in the three main cell types in the brain: neurons, astrocytes, and microglia. Exposure of each of these cell types to physiologically relevant stimuli shows distinct changes in labile copper pools. Neurons display translocation of labile copper from somatic cell bodies to peripheral processes upon activation, whereas astrocytes and microglia exhibit global decreases and increases in intracellular labile copper pools, respectively, after exposure to inflammatory stimuli. This work provides foundational information on cell type-dependent homeostasis of copper, an essential metal in the brain, as well as a starting point for the design of new activity-based probes for metals and other dynamic signaling and stress analytes in biology.

Author Correction: A monocyte gene expression signature in the early clinical course of Parkinson's disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Blockade of IL-17 signaling reverses alcohol-induced liver injury and excessive alcohol drinking in mice.

Chronic alcohol abuse has a detrimental effect on the brain and liver. There is no effective treatment for these patients, and the mechanism underlying alcohol addiction and consequent alcohol-induced damage of the liver/brain axis remains unresolved. We compared experimental models of alcoholic liver disease (ALD) and alcohol dependence in mice and demonstrated that genetic ablation of IL-17 receptor A (IL-17ra-/-) or pharmacological blockade of IL-17 signaling effectively suppressed the increased voluntary alcohol drinking in alcohol-dependent mice and blocked alcohol-induced hepatocellular and neurological damage. The level of circulating IL-17A positively correlated with the alcohol use in excessive drinkers and was further increased in patients with ALD as compared with healthy individuals. Our data suggest that IL-17A is a common mediator of excessive alcohol consumption and alcohol-induced liver/brain injury, and targeting IL-17A may provide a novel strategy for treatment of alcohol-induced pathology.

The Endocannabinoid System as a Window Into Microglial Biology and Its Relationship to Autism.

Microglia are the resident, innate immune cells of the central nervous system (CNS) and are critical in managing CNS injuries and infections. Microglia also maintain CNS homeostasis by influencing neuronal development, viability, and function. However, aberrant microglial activity and phenotypes are associated with CNS pathology, including autism spectrum disorder (ASD). Thus, improving our knowledge of microglial regulation could provide insights into the maintenance of CNS homeostasis as well as the prevention and treatment of ASD. Control of microglial activity is in part overseen by small, lipid-derived molecules known as endogenous cannabinoids (endocannabinoids). Endocannabinoids are one component of the endocannabinoid system (ECS), which also includes the enzymes that metabolize these ligands, in addition to cannabinoid receptor 1 (CB1) and 2 (CB2). Interestingly, increased ECS signaling leads to an anti-inflammatory, neuroprotective phenotype in microglia. Here, we review the literature and propose that ECS signaling represents a largely untapped area for understanding microglial biology and its relationship to ASD, with special attention paid to issues surrounding the use of recreational cannabis (marijuana). We also discuss major questions within the field and suggest directions for future research.

Generation of a human induced pluripotent stem cell line, BRCi001-A, derived from a patient with mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a rare inherited metabolic disorder caused by defects in alpha-L-iduronidase (IDUA), a lysosomal protein encoded by IDUA gene. MPS I is a progressive multisystemic disorder with a wide range of symptoms, including skeletal abnormalities and cognitive impairment, and is characterized by a wide spectrum of severity levels caused by varied mutations in IDUA. A human iPSC line was established from an attenuated MPS I (Scheie syndrome) patient carrying an IDUA gene mutation (c.266G > A; p.R89Q). This disease-specific iPSC line will be useful for the research of MPS I.

A monocyte gene expression signature in the early clinical course of Parkinson's disease.

Microglia are the main immune cells of the brain and express a large genetic pattern of genes linked to Parkinson's disease risk alleles. Monocytes like microglia are myeloid-lineage cells, raising the questions of the extent to which they share gene expression with microglia and whether they are already altered early in the clinical course of the disease. To decipher a monocytic gene expression signature in Parkinson's disease, we performed RNA-seq and applied the two-sample Kolmogorov-Smirnov test to identify differentially expressed genes between controls and patients with Parkinson's disease and changes in gene expression variability and dysregulation. The gene expression profiles of normal human monocytes and microglia showed a plethora of differentially expressed genes. Additionally, we identified a distinct gene expression pattern of monocytes isolated from Parkinson's disease patients at an early disease stage compared to controls using the Kolmogorov-Smirnov test. Differentially expressed genes included genes involved in immune activation such as HLA-DQB1, MYD88, REL, and TNF-α. Our data suggest that future studies of distinct leukocyte subsets are warranted to identify possible surrogate biomarkers and may lead to the identification of novel interventions early in the disease course.

Versatile Histochemical Approach to Detection of Hydrogen Peroxide in Cells and Tissues Based on Puromycin Staining.

Hydrogen peroxide (H2O2) is a central reactive oxygen species (ROS) that contributes to diseases from obesity to cancer to neurodegeneration but is also emerging as an important signaling molecule. We now report a versatile histochemical approach for detection of H2O2 that can be employed across a broad range of cell and tissue specimens in both healthy and disease states. We have developed a first-generation H2O2-responsive analogue named Peroxymycin-1, which is based on the classic cell-staining molecule puromycin and enables covalent staining of biological samples and retains its signal after fixation. H2O2-mediated boronate cleavage uncages the puromycin aminonucleoside, which leaves a permanent and dose-dependent mark on treated biological specimens that can be detected with high sensitivity and precision through a standard immunofluorescence assay. Peroxymycin-1 is selective and sensitive enough to image both exogenous and endogenous changes in cellular H2O2 levels and can be exploited to profile resting H2O2 levels across a panel of cell lines to distinguish metastatic, invasive cancer cells from less invasive cancer and nontumorigenic counterparts, based on correlations with ROS status. Moreover, we establish that Peroxymycin-1 is an effective histochemical probe for in vivo H2O2 analysis, as shown through identification of aberrant elevations in H2O2 levels in liver tissues in a murine model of nonalcoholic fatty liver disease, thus demonstrating the potential of this approach for studying disease states and progression associated with H2O2. This work provides design principles that should enable development of a broader range of histochemical probes for biological use that operate via activity-based sensing.

Actomyosin-Mediated Tension Orchestrates Uncoupled Respiration in Adipose Tissues.

The activation of brown/beige adipose tissue (BAT) metabolism and the induction of uncoupling protein 1 (UCP1) expression are essential for BAT-based strategies to improve metabolic homeostasis. Here, we demonstrate that BAT utilizes actomyosin machinery to generate tensional responses following adrenergic stimulation, similar to muscle tissues. The activation of actomyosin mechanics is critical for the acute induction of oxidative metabolism and uncoupled respiration in UCP1+ adipocytes. Moreover, we show that actomyosin-mediated elasticity regulates the thermogenic capacity of adipocytes via the mechanosensitive transcriptional co-activators YAP and TAZ, which are indispensable for normal BAT function. These biomechanical signaling mechanisms may inform future strategies to promote the expansion and activation of brown/beige adipocytes.

Histone demethylase LSD1 regulates hematopoietic stem cells homeostasis and protects from death by endotoxic shock.

Hematopoietic stem cells (HSCs) maintain a quiescent state during homeostasis, but with acute infection, they exit the quiescent state to increase the output of immune cells, the so-called "emergency hematopoiesis." However, HSCs' response to severe infection during septic shock and the pathological impact remain poorly elucidated. Here, we report that the histone demethylase KDM1A/LSD1, serving as a critical regulator of mammalian hematopoiesis, is a negative regulator of the response to inflammation in HSCs during endotoxic shock typically observed during acute bacterial or viral infection. Inflammation-induced LSD1 deficiency results in an acute expansion of a pathological population of hyperproliferative and hyperinflammatory myeloid progenitors, resulting in a septic shock phenotype and acute death. Unexpectedly, in vivo administration of bacterial lipopolysaccharide (LPS) to wild-type mice results in acute suppression of LSD1 in HSCs with a septic shock phenotype that resembles that observed following induced deletion of LSD1 The suppression of LSD1 in HSCs is caused, at least in large part, by a cohort of inflammation-induced microRNAs. Significantly, reconstitution of mice with bone marrow progenitor cells expressing inhibitors of these inflammation-induced microRNAs blocked the suppression of LSD1 in vivo following acute LPS administration and prevented mortality from endotoxic shock. Our results indicate that LSD1 activators or miRNA antagonists could serve as a therapeutic approach for life-threatening septic shock characterized by dysfunction of HSCs.

CLO: The cell line ontology.

BACKGROUND: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. CONSTRUCTION AND CONTENT: Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as 'cell line', 'cell line cell', 'cell line culturing', and 'mortal' vs. 'immortal cell line cell'. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. UTILITY AND DISCUSSION: The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO's utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development.

25-Hydroxycholesterol activates the integrated stress response to reprogram transcription and translation in macrophages.

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

Amyloid arthropathy associated with multiple myeloma: a systematic analysis of 101 reported cases.

OBJECTIVE: Amyloid deposition in multiple myeloma (MM) may lead to an arthropathy resembling rheumatoid arthritis (RA). Since a systematic description of its natural history is lacking, we have performed a systematic analysis of all published cases. METHODS: Literature review featuring backward and forward database searches and direct inspection of reference lists. Inclusion criteria were as follows: publication between 1931 and 2012, diagnosis of multiple myeloma, and demonstration of light chain amyloid (AL) in any organ or in synovial fluid, arthritis, or synovitis. RESULTS: Overall, 101 cases were identified. Median age was 59 years and the male-to-female ratio was 1:1. A systemic manifestation of MM was reported in 88 cases. In 53 of these, characteristic physical findings (carpal tunnel syndrome, macroglossia, shoulder pad, and soft tissue swelling/masses) were present. Arthritis manifested before the diagnosis of MM in 63 cases, with 33 cases initially misdiagnosed as RA. There were 72 cases of poly-, 17 of oligo-, and three of monoarthritis. The shoulder joint was most commonly affected, followed by knees and small hand joints. Median synovial fluid leukocyte count was 2460 cells/mm(3), and was normal in seven cases. Synovial histopathology often featured mild synovitis without plasma cell infiltration. Imaging revealed articular or periarticular inflammation in many cases and bone lesions near 22% of affected joints. Treatments varied but led to some improvement in the majority of cases. CONCLUSIONS: These results solidify previous experience that MM arthropathy tends to feature a symmetric RF-negative nonerosive polyarthritis. However, the results also highlight the diversity of its presentations and stress the importance of arthropathy as a potentially under-recognized presenting manifestation of MM.

Regulation of microglia activation and deactivation by nuclear receptors.

Microglia cells function as sentinels for innate immunity in the central nervous system (CNS). To perform this function, microglia express a diverse set of pattern recognition receptors (PRRs) for pathogen-associated molecular patterns (PAMPs) that include Toll-like receptors (TLRs) and inflammasomes. Several members of the TLR and inflammasome family also recognize endogenously derived molecules that are generated as a consequence of tissue injury or other pathological processes. Recognition of PAMPs or endogenous ligands by PRRs in microglia induces the robust activation of innate immune responses leading to the production of proinflammatory mediators and the activation of adaptive immunity. Activation of microglia is essential for clearance of infection and repair of tissue injury. However, uncontrolled inflammatory responses of microglia are also thought to contribute to the severity of many neurodegenerative diseases. Thus, activation of microglia must be properly and tightly regulated to maintain normal tissue homeostasis. Several mechanisms have been identified that appear to function in the active maintenance of quiescence under normal conditions and/or re-establish this state following resolution of infection or injury. These mechanisms involve communication with neurons and other glia through secreted molecules or surface expressing receptors as well as actions of members of the nuclear receptor (NR) superfamily of transcription factors. Here, we review recent advances in our understanding of the regulation of microglia activation and deactivation with a focus on counter-regulation of microglia activation by nuclear receptors.