Virtual reality-empowered deep-learning analysis of brain cells.

Automated detection of specific cells in three-dimensional datasets such as whole-brain light-sheet image stacks is challenging. Here, we present DELiVR, a virtual reality-trained deep-learning pipeline for detecting c-Fos+ cells as markers for neuronal activity in cleared mouse brains. Virtual reality annotation substantially accelerated training data generation, enabling DELiVR to outperform state-of-the-art cell-segmenting approaches. Our pipeline is available in a user-friendly Docker container that runs with a standalone Fiji plugin. DELiVR features a comprehensive toolkit for data visualization and can be customized to other cell types of interest, as we did here for microglia somata, using Fiji for dataset-specific training. We applied DELiVR to investigate cancer-related brain activity, unveiling an activation pattern that distinguishes weight-stable cancer from cancers associated with weight loss. Overall, DELiVR is a robust deep-learning tool that does not require advanced coding skills to analyze whole-brain imaging data in health and disease.

Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Whole-mouse clearing and imaging at the cellular level with vDISCO.

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.

Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

Adipocyte STAT5 deficiency does not affect blood glucose homeostasis in obese mice.

The aim of this study was to investigate whether the lack of signal transducer and activator of transcription 5 (STAT5) in mature adipocytes of obese mice (Stat5Adipoq mice) improves glucose and lipid metabolism as previously observed in lean mice. Male Stat5Adipoq mice and their wild type (WT) littermates were fed high-fat diet (HFD). Effects of adipocyte STAT5 deficiency on adiposity as well as on glucose and lipid metabolism were determined under ad libitum feeding and after weight loss induced by calorie restriction. Compared to WT mice, obese Stat5Adipoq mice showed modestly accelerated weight gain and blunted depletion of fat stores under calorie restriction (reduction in % body fat after 3 weeks: WT, -9.3±1.1, vs Stat5Adipoq, -5.9±0.8, p = 0.04). No differences were observed between Stat5Adipoq and WT mice with regard to parameters of glucose and lipid metabolism including basal glycaemia, glucose tolerance, and plasma triglycerides. In conclusion, STAT5 deficiency in the adipocyte of HFD-fed obese mice was associated with increased fat accumulation. In contrast to previous findings in lean mice, however, lipid accumulation was not associated with any improvement in glucose and lipid metabolism. Our results do not support adipocyte STAT5 as a promising target for the treatment of obesity-associated metabolic derangements.

Association of circulating PLA2G7 levels with cancer cachexia and assessment of darapladib as a therapy.

BACKGROUND: Cancer cachexia (CCx) is a multifactorial wasting disorder characterized by involuntary loss of body weight that affects many cancer patients and implies a poor prognosis, reducing both tolerance to and efficiency of anticancer therapies. Actual challenges in management of CCx remain in the identification of tumour-derived and host-derived mediators involved in systemic inflammation and tissue wasting and in the discovery of biomarkers that would allow for an earlier and personalized care of cancer patients. The aim of this study was to identify new markers of CCx across different species and tumour entities. METHODS: Quantitative secretome analysis was performed to identify specific factors characteristic of cachexia-inducing cancer cell lines. To establish the subsequently identified phospholipase PLA2G7 as a marker of CCx, plasma PLA2G7 activity and/or protein levels were measured in well-established mouse models of CCx and in different cohorts of weight-stable and weight-losing cancer patients with different tumour entities. Genetic PLA2G7 knock-down in tumours and pharmacological treatment using the well-studied PLA2G7 inhibitor darapladib were performed to assess its implication in the pathogenesis of CCx in C26 tumour-bearing mice. RESULTS: High expression and secretion of PLA2G7 were hallmarks of cachexia-inducing cancer cell lines. Circulating PLA2G7 activity was increased in different mouse models of CCx with various tumour entities and was associated with the severity of body wasting. Circulating PLA2G7 levels gradually rose during cachexia development. Genetic PLA2G7 knock-down in C26 tumours only partially reduced plasma PLA2G7 levels, suggesting that the host is also an important contributor. Chronic treatment with darapladib was not sufficient to counteract inflammation and tissue wasting despite a strong inhibition of the circulating PLA2G7 activity. Importantly, PLA2G7 levels were also increased in colorectal and pancreatic cancer patients with CCx. CONCLUSIONS: Overall, our data show that despite no immediate pathogenic role, at least when targeted as a single entity, PLA2G7 is a consistent marker of CCx in both mice and humans. The early increase in circulating PLA2G7 levels in pre-cachectic mice supports future prospective studies to assess its potential as biomarker for cancer patients.

Aging Aggravates Cachexia in Tumor-Bearing Mice.

BACKGROUND: Cancer is primarily a disease of high age in humans, yet most mouse studies on cancer cachexia are conducted using young adolescent mice. Given that metabolism and muscle function change with age, we hypothesized that aging may affect cachexia progression in mouse models. METHODS: We compare tumor and cachexia development in young and old mice of three different strains (C57BL/6J, C57BL/6N, BALB/c) and with two different tumor cell lines (Lewis Lung Cancer, Colon26). Tumor size, body and organ weights, fiber cross-sectional area, circulating cachexia biomarkers, and molecular markers of muscle atrophy and adipose tissue wasting are shown. We correlate inflammatory markers and body weight dependent on age in patients with cancer. RESULTS: We note fundamental differences between mouse strains. Aging aggravates weight loss in LLC-injected C57BL/6J mice, drives it in C57BL/6N mice, and does not influence weight loss in C26-injected BALB/c mice. Glucose tolerance is unchanged in cachectic young and old mice. The stress marker GDF15 is elevated in cachectic BALB/c mice independent of age and increased in old C57BL/6N and J mice. Inflammatory markers correlate significantly with weight loss only in young mice and patients. CONCLUSIONS: Aging affects cachexia development and progression in mice in a strain-dependent manner and influences the inflammatory profile in both mice and patients. Age is an important factor to consider for future cachexia studies.

High levels of modified ceramides are a defining feature of murine and human cancer cachexia.

BACKGROUND: Cancer cachexia (CCx) is a multifactorial energy-wasting syndrome reducing the efficiency of anti-cancer therapies, quality of life, and survival of cancer patients. In the past years, most studies focused on the identification of tumour and host-derived proteins contributing to CCx. However, there is still a lack of studies addressing the changes in bioactive lipids. The aim of this study was to identify specific lipid species as a hallmark of CCx by performing a broad range lipid analysis of plasma from well-established CCx mouse models as well as cachectic and weight stable cancer patients. METHODS: Plasma from non-cachectic (PBS-injected mice, NC26 tumour-bearing mice), pre-cachectic and cachectic mice (C26 and LLC tumour-bearing mice, ApcMin/+ mutant mice), and plasma from weight stable and cachectic patients with gastrointestinal cancer, were analysed using the Lipidyzer™ platform. In total, 13 lipid classes and more than 1100 lipid species, including sphingolipids, neutral and polar glycerolipids, were covered by the analysis. Correlation analysis between specific lipid species and readouts of CCx were performed. Lipidomics data were confirmed by gene expression analysis of metabolic organs to analyse enzymes involved in sphingolipid synthesis and degradation. RESULTS: A decrease in several lysophosphatidylcholine (LPC) species and an increase in numerous sphingolipids including sphingomyelins (SMs), ceramides (CERs), hexosyl-ceramides (HCERs) and lactosyl-ceramides (LCERs), were mutual features of CCx in both mice and cancer patients. Notably, sphingolipid levels gradually increased during cachexia development. Key enzymes involved in ceramide synthesis were elevated in liver but not in adipose, muscle, or tumour tissues, suggesting that ceramide turnover in the liver is a major contributor to elevated sphingolipid levels in CCx. LPC(16:1), LPC(20:3), SM(16:0), SM(24:1), CER(16:0), CER(24:1), HCER(16:0), and HCER(24:1) were the most consistently affected lipid species between mice and humans and correlated negatively (LPCs) or positively (SMs, CERs and HCERs) with the severity of body weight loss. CONCLUSIONS: High levels of sphingolipids, specifically ceramides and modified ceramides, are a defining feature of murine and human CCx and may contribute to tissue wasting and skeletal muscle atrophy through the inhibition of anabolic signals. The progressive increase in sphingolipids during cachexia development supports their potential as early biomarkers for CCx.

STAT5 is required for lipid breakdown and beta-adrenergic responsiveness of brown adipose tissue.

OBJECTIVE: Increasing energy expenditure through activation of brown adipose tissue (BAT) thermogenesis is an attractive approach to counteract obesity. It is therefore essential to understand the molecular mechanisms that control BAT functions. Until now several members of the Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway have been implicated as being relevant for BAT physiology. However, whether the STAT family member STAT5 is important for the thermogenic property of adipose tissues is unknown. Therefore, we have investigated the role of STAT5 in thermogenic fat in this paper. METHODS: We performed metabolic and molecular analyses using mice that harbor an adipocyte-specific deletion of Stat5a/b alleles. RESULTS: We found that STAT5 is necessary for acute cold-induced temperature maintenance and the induction of lipid mobilization in BAT following ß3-adrenergic stimulation. Moreover, mitochondrial respiration of primary differentiated brown adipocytes lacking STAT5 was diminished. Increased sensitivity to cold stress upon STAT5 deficiency was associated with reduced expression of thermogenic markers including uncoupling protein 1 (UCP1), while decreased stimulated lipolysis was linked to decreased protein kinase A (PKA) activity. Additionally, brown remodeling of white adipose tissue was diminished following chronic ß3-adrenergic stimulation, which was accompanied by a decrease in mitochondrial performance. CONCLUSION: We conclude that STAT5 is essential for the functionality and the ß-adrenergic responsiveness of thermogenic adipose tissue.

Hepatic growth hormone - JAK2 - STAT5 signalling: Metabolic function, non-alcoholic fatty liver disease and hepatocellular carcinoma progression.

The rising prevalence of obesity came along with an increase in associated metabolic disorders in Western countries. Non-alcoholic fatty liver disease (NAFLD) represents the hepatic manifestation of the metabolic syndrome and is linked to primary stages of liver cancer development. Growth hormone (GH) regulates various vital processes such as energy supply and cellular regeneration. In addition, GH regulates various aspects of liver physiology through activating the Janus kinase (JAK) 2- signal transducer and activator of transcription (STAT) 5 pathway. Consequently, disrupted GH - JAK2 - STAT5 signaling in the liver alters hepatic lipid metabolism and is associated with NAFLD development in humans and mouse models. Interestingly, while STAT5 as well as JAK2 deficiency correlates with hepatic lipid accumulation, recent studies suggest that these proteins have unique ambivalent functions in chronic liver disease progression and tumorigenesis. In this review, we focus on the consequences of altered GH - JAK2 - STAT5 signaling for hepatic lipid metabolism and liver cancer development with an emphasis on lessons learned from genetic knockout models.

Crosstalk between inflammatory mediators and endoplasmic reticulum stress in liver diseases.

An excessive inflammatory response is frequently associated with cellular dysfunction and cell death. The latter may cause single and multiple organ failure. The most susceptible organs are liver, lung, kidney, heart and intestine. This review will focus on the liver as a target organ for an excessive inflammatory response. It is commonly accepted that organ failure is caused by the action of inflammatory cytokines released in excess during the inflammatory response. It has been suggested that inflammation mediated liver failure is not due to an increased death rate of parenchymal cells, but due to an intracellular metabolic disorder. This metabolic disorder is associated with mitochondrial and endoplasmic reticulum (ER) dysfunction during the acute phase response elicited by systemic inflammation. An overproduction of acute phase proteins in the liver as well as elevated reactive oxygen species (ROS) generation induce ER stress, triggering the unfolded protein response (UPR), which may initiate or aggravate inflammation. It is known that certain inflammatory mediators, such as the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α induce ER stress. These findings suggest that ER stress and the subsequent UPR on the one hand, and the inflammatory response on the other create a kind of feed forward loop, which can be either beneficial (e.g., elimination of the pathogen and restoration of tissue homeostasis) or deleterious (e.g., excessive cell dysfunction and cell death). This review aims to unfurl the different pathways contributing to this loop and to highlight the relevance of UPR signaling (IRE1α, ATF6, and PERK) and mediators of the inflammatory response (NF-κB, STAT3, IL-1ß, IL-6, TLR) which have a particular role as pathophysiological triggers in the liver.

STAT5 deficiency in hepatocytes reduces diethylnitrosamine-induced liver tumorigenesis in mice.

Chronic liver diseases and the development of hepatocellular carcinoma are closely linked and pose a major medical challenge as treatment options are limited. Animal studies have shown that genetic deletion of the signal transducer and activator of transcription (STAT) 5 in liver is associated with higher susceptibility to fatty liver disease, fibrosis and cancer, indicating a protective role of hepatic STAT5 in mouse models of chronic liver disease. To investigate the role of STAT5 in the etiology of liver cancer in more detail, we applied the chemical carcinogen diethylnitrosamine (DEN) to mice harboring a hepatocyte-specific deletion of Stat5 (S5KO). At 8 months after DEN injections, tumor formation in S5KO was significantly reduced. This was associated with diminished tumor frequency and less aggressive liver cancer progression. Apoptosis and inflammation markers were not changed in S5KO livers suggesting that the reduced tumor burden was not due to impaired inflammatory response. Despite reduced mRNA expression of the DEN bio-activator cytochrome P450 2e1 (Cyp2e1) in S5KO livers, protein levels were similar. Yet, delayed tumor formation in S5KO mice coincided with decreased activation of c-Jun N-terminal Kinase (JNK). Taken together, while STAT5 has a protective role in fatty liver-associated liver cancer, it exerts oncogenic functions in DEN-induced liver cancer.

Life Under Hypoxia Lowers Blood Glucose Independently of Effects on Appetite and Body Weight in Mice.

Blood glucose and the prevalence of diabetes are lower in mountain than lowland dwellers, which could among other factors be due to reduced oxygen availability. To investigate metabolic adaptations to life under hypoxia, male mice on high fat diet (HFD) were continuously maintained at 10% O2. At variance to preceding studies, the protocol was designed to dissect direct metabolic effects from such mediated indirectly via hypoxia-induced reductions in appetite and weight gain. This was achieved by two separate control groups on normal air, one with free access to HFD, and one fed restrictedly in order to obtain a weight curve matching that of hypoxia-exposed mice. Comparable body weight in restrictedly fed and hypoxic mice was achieved by similar reductions in calorie intake (-22%) and was associated with parallel effects on body composition as well as on circulating insulin, leptin, FGF-21, and adiponectin. Whereas the effects of hypoxia on the above parameters could thus be attributed entirely to blunted weight gain, hypoxia improved glucose homeostasis in part independently of body weight (fasted blood glucose, mmol/l: freely fed control, 10.2 ± 0.7; weight-matched control, 8.0 ± 0.3; hypoxia, 6.8 ± 0.2; p < 0.007 each; AUC in the glucose tolerance test, mol/l*min: freely fed control, 2.54 ± 0.15; weight-matched control, 1.86 ± 0.08; hypoxia, 1.67 ± 0.05; p < 0.05 each). Although counterintuitive to lowering of glycemia, insulin sensitivity appeared to be impaired in animals adapted to hypoxia: In the insulin tolerance test, hypoxia-treated mice started off with lower glycaemia than their weight-matched controls (initial blood glucose, mmol/l: freely fed control, 11.5 ± 0.7; weight-matched control, 9.4 ± 0.3; hypoxia, 8.1 ± 0.2; p < 0.02 each), but showed a weaker response to insulin (final blood glucose, mmol/l: freely fed control, 7.0 ± 0.3; weight-matched control, 4.5 ± 0.2; hypoxia, 5.5 ± 0.3; p < 0.01 each). Furthermore, hypoxia weight-independently reduced hepatic steatosis as normalized to total body fat, suggesting a shift in the relative distribution of triglycerides from liver to fat (mg/g liver triglycerides per g total fat mass: freely fed control, 10.3 ± 0.6; weight-matched control, 5.6 ± 0.3; hypoxia, 4.0 ± 0.2; p < 0.0004 each). The results show that exposure of HFD-fed mice to continuous hypoxia leads to a unique metabolic phenotype characterized by improved glucose homeostasis along with evidence for impaired rather than enhanced insulin sensitivity.

Adipocyte Glucocorticoid Receptor Deficiency Attenuates Aging- and HFD-Induced Obesity and Impairs the Feeding-Fasting Transition.

Glucocorticoids (GCs) are important regulators of systemic energy metabolism, and aberrant GC action is linked to metabolic dysfunctions. Yet, the extent to which normal and pathophysiological energy metabolism depend on the GC receptor (GR) in adipocytes remains unclear. Here, we demonstrate that adipocyte GR deficiency in mice significantly impacts systemic metabolism in different energetic states. Plasma metabolomics and biochemical analyses revealed a marked global effect of GR deficiency on systemic metabolite abundance and, thus, substrate partitioning in fed and fasted states. This correlated with a decreased lipolytic capacity of GR-deficient adipocytes under postabsorptive and fasting conditions, resulting from impaired signal transduction from ß-adrenergic receptors to adenylate cyclase. Upon prolonged fasting, the impaired lipolytic response resulted in abnormal substrate utilization and lean mass wasting. Conversely, GR deficiency attenuated aging-/diet-associated obesity, adipocyte hypertrophy, and liver steatosis. Systemic glucose tolerance was improved in obese GR-deficient mice, which was associated with increased insulin signaling in muscle and adipose tissue. We conclude that the GR in adipocytes exerts central but diverging roles in the regulation of metabolic homeostasis depending on the energetic state. The adipocyte GR is indispensable for the feeding-fasting transition but also promotes adiposity and associated metabolic disorders in fat-fed and aged mice.

Adipocyte STAT5 deficiency promotes adiposity and impairs lipid mobilisation in mice.

AIMS/HYPOTHESIS: Dysfunction of lipid metabolism in white adipose tissue can substantially interfere with health and quality of life, for example in obesity and associated metabolic diseases. Therefore, it is important to characterise pathways that regulate lipid handling in adipocytes and determine how they affect metabolic homeostasis. Components of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway are involved in adipocyte physiology and pathophysiology. However, the exact physiological importance of the STAT family member STAT5 in white adipose tissue is yet to be determined. Here, we aimed to delineate adipocyte STAT5 functions in the context of lipid metabolism in white adipose tissue. METHODS: We generated an adipocyte specific knockout of Stat5 in mice using the Adipoq-Cre recombinase transgene followed by in vivo and in vitro biochemical and molecular studies. RESULTS: Adipocyte-specific deletion of Stat5 resulted in increased adiposity, while insulin resistance and gluconeogenic capacity was decreased, indicating that glucose metabolism can be improved by interfering with adipose STAT5 function. Basal lipolysis and fasting-induced lipid mobilisation were diminished upon STAT5 deficiency, which coincided with reduced levels of the rate-limiting lipase of triacylglycerol hydrolysis, adipose triglyceride lipase (ATGL, encoded by Pnpla2) and its coactivator comparative gene identification 58 (CGI-58). In a mechanistic analysis, we identified a functional STAT5 response element within the Pnpla2 promoter, indicating that Pnpla2 is transcriptionally regulated by STAT5. CONCLUSIONS/INTERPRETATION: Our findings reveal an essential role for STAT5 in maintaining lipid homeostasis in white adipose tissue and provide a rationale for future studies into the potential of STAT5 manipulation to improve outcomes in metabolic diseases.

Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice.

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage.