J. Andrew Pospisilik

Targeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes

Type-2 diabetes results from the development of insulin resistance and a concomitant impairment of insulin secretion. Recent studies place altered mitochondrial oxidative phosphorylation (OxPhos) as an underlying genetic element of insulin resistance. However, the causative or compensatory nature of these OxPhos changes has yet to be proven. Here, we show that muscle- and liver-specific AIF ablation in mice initiates a pattern of OxPhos deficiency closely mimicking that of human insulin resistance, and contrary to current expectations, results in increased glucose tolerance, reduced fat mass, and increased insulin sensitivity. These results are maintained upon high-fat feeding and in both genetic mosaic and ubiquitous OxPhos-deficient mutants. Importantly, the effects of AIF on glucose metabolism are acutely inducible and reversible. These findings establish that tissue-specific as well as global OxPhos defects in mice can counteract the development of insulin resistance, diabetes, and obesity.

Genome-wide RNAi screen identifies genes involved in intestinal pathogenic bacterial infection.

Innate Immunity in the Fly Gut Drosophila melanogaster is an important model system to study innate immunity, being both easy to manipulate and lacking an adaptive immune system. In order to identify genes that regulate innate immunity, Cronin et al. (p. 340; published online 11 June) performed an RNA interference screen on flies infected with the oral bacterial pathogen, Serratia marcescens. Genes involved in intestinal immunity and regulation of hemocytes, macrophage-like cells critical for phagocytosis and killing of the bacteria, were identified. Several hundred genes conferred either enhanced susceptibility or resistance to bacterial infection. Furthermore, the JAK/STAT signaling pathway was activated in intestinal stem cells after bacterial infection, resulting in enhanced susceptibility to infection, most likely through regulation of intestinal stem cell homeostasis. In vivo RNA interference screen reveals regulators of innate immunity in Drosophila. Innate immunity represents the first line of defense in animals. We report a genome-wide in vivo Drosophila RNA interference screen to uncover genes involved in susceptibility or resistance to intestinal infection with the bacterium Serratia marcescens. We first employed whole-organism gene suppression, followed by tissue-specific silencing in gut epithelium or hemocytes to identify several hundred genes involved in intestinal antibacterial immunity. Among the pathways identified, we showed that the JAK-STAT signaling pathway controls host defense in the gut by regulating stem cell proliferation and thus epithelial cell homeostasis. Therefore, we revealed multiple genes involved in antibacterial defense and the regulation of innate immunity.

Drosophila Genome-wide Obesity Screen Reveals Hedgehog as a Determinant of Brown versus White Adipose Cell Fate

Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. We performed a systems-level genetic dissection of adiposity regulation using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo and revealed hedgehog signaling as the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, we generated fat-specific hedgehog-activation mutant mice. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals.

The sedoheptulose kinase CARKL directs macrophage polarization through control of glucose metabolism.

Summary Immune cells are somewhat unique in that activation responses can alter quantitative phenotypes upwards of 100,000-fold. To date little is known about the metabolic adaptations necessary to mount such dramatic phenotypic shifts. Screening for novel regulators of macrophage activation, we found nonprotein kinases of glucose metabolism among the most enriched classes of candidate immune modulators. We find that one of these, the carbohydrate kinase-like protein CARKL, is rapidly downregulated in vitro and in vivo upon LPS stimulation in both mice and humans. Interestingly, CARKL catalyzes an orphan reaction in the pentose phosphate pathway, refocusing cellular metabolism to a high-redox state upon physiological or artificial downregulation. We find that CARKL-dependent metabolic reprogramming is required for proper M1- and M2-like macrophage polarization and uncover a rate-limiting requirement for appropriate glucose flux in macrophage polarization.

A Genome-wide Drosophila Screen for Heat Nociception Identifies α2δ3 as an Evolutionarily Conserved Pain Gene

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.

A Global In Vivo Drosophila RNAi Screen Identifies NOT3 as a Conserved Regulator of Heart Function

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.

Paternal Diet Defines Offspring Chromatin State and Intergenerational Obesity

The global rise in obesity has revitalized a search for genetic and epigenetic factors underlying the disease. We present a Drosophila model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR) and identify genes required for its encoding in offspring. Intriguingly, we find that as little as 2 days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, desilencing chromatin-state-defined domains in both mature sperm and in offspring embryos. We identify requirements for H3K9/K27me3-dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, we find evidence that a similar system may regulate obesity susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for our understanding of phenotypic variation and evolution.

A new fate mapping system reveals context-dependent random or clonal expansion of microglia

Microglia constitute a highly specialized network of tissue-resident immune cells that is important for the control of tissue homeostasis and the resolution of diseases of the CNS. Little is known about how their spatial distribution is established and maintained in vivo. Here we establish a new multicolor fluorescence fate mapping system to monitor microglial dynamics during steady state and disease. Our findings suggest that microglia establish a dense network with regional differences, and the high regional turnover rates found challenge the universal concept of microglial longevity. Microglial self-renewal under steady state conditions constitutes a stochastic process. During pathology this randomness shifts to selected clonal microglial expansion. In the resolution phase, excess disease-associated microglia are removed by a dual mechanism of cell egress and apoptosis to re-establish the stable microglial network. This study unravels the dynamic yet discrete self-organization of mature microglia in the healthy and diseased CNS.

Trim28 Haploinsufficiency Triggers Bi-stable Epigenetic Obesity

Summary More than one-half billion people are obese, and despite progress in genetic research, much of the heritability of obesity remains enigmatic. Here, we identify a Trim28-dependent network capable of triggering obesity in a non-Mendelian, “on/off” manner. Trim28+/D9 mutant mice exhibit a bi-modal body-weight distribution, with isogenic animals randomly emerging as either normal or obese and few intermediates. We find that the obese-“on” state is characterized by reduced expression of an imprinted gene network including Nnat, Peg3, Cdkn1c, and Plagl1 and that independent targeting of these alleles recapitulates the stochastic bi-stable disease phenotype. Adipose tissue transcriptome analyses in children indicate that humans too cluster into distinct sub-populations, stratifying according to Trim28 expression, transcriptome organization, and obesity-associated imprinted gene dysregulation. These data provide evidence of discrete polyphenism in mouse and man and thus carry important implications for complex trait genetics, evolution, and medicine. Video Abstract

The stress kinase MKK7 couples oncogenic stress to p53 stability and tumor suppression

Most preneoplastic lesions are quiescent and do not progress to form overt tumors. It has been proposed that oncogenic stress activates the DNA damage response and the key tumor suppressor p53, which prohibits tumor growth. However, the molecular pathways by which cells sense a premalignant state in vivo are largely unknown. Here we report that tissue-specific inactivation of the stress signaling kinase MKK7 in KRasG12D-driven lung carcinomas and NeuT-driven mammary tumors markedly accelerates tumor onset and reduces overall survival. Mechanistically, MKK7 acts through the kinases JNK1 and JNK2, and this signaling pathway directly couples oncogenic and genotoxic stress to the stability of p53, which is required for cell cycle arrest and suppression of epithelial cancers. These results show that MKK7 functions as a major tumor suppressor in lung and mammary cancer in mouse and identify MKK7 as a vital molecular sensor to set a cellular anti-cancer barrier.