Bacterial reprogramming of tick metabolism impacts vector fitness and susceptibility to infection.

Arthropod-borne pathogens are responsible for hundreds of millions of infections in humans each year. The blacklegged tick, Ixodes scapularis, is the predominant arthropod vector in the United States and is responsible for transmitting several human pathogens, including the Lyme disease spirochete Borrelia burgdorferi and the obligate intracellular rickettsial bacterium Anaplasma phagocytophilum, which causes human granulocytic anaplasmosis. However, tick metabolic response to microbes and whether metabolite allocation occurs upon infection remain unknown. Here we investigated metabolic reprogramming in the tick ectoparasite I. scapularis and determined that the rickettsial bacterium A. phagocytophilum and the spirochete B. burgdorferi induced glycolysis in tick cells. Surprisingly, the endosymbiont Rickettsia buchneri had a minimal effect on bioenergetics. An unbiased metabolomics approach following A. phagocytophilum infection of tick cells showed alterations in carbohydrate, lipid, nucleotide and protein metabolism, including elevated levels of the pleiotropic metabolite ß-aminoisobutyric acid. We manipulated the expression of genes associated with ß-aminoisobutyric acid metabolism in I. scapularis, resulting in feeding impairment, diminished survival and reduced bacterial acquisition post haematophagy. Collectively, we discovered that metabolic reprogramming affects interspecies relationships and fitness in the clinically relevant tick I. scapularis.

Longitudinal Biochemical and Behavioral Alterations in a Gyrencephalic Model of Blast-Related Mild Traumatic Brain Injury.

Blast-related traumatic brain injury (bTBI) is a major cause of neurological disorders in the U.S. military that can adversely impact some civilian populations as well and can lead to lifelong deficits and diminished quality of life. Among these types of injuries, the long-term sequelae are poorly understood because of variability in intensity and number of the blast exposure, as well as the range of subsequent symptoms that can overlap with those resulting from other traumatic events (e.g., post-traumatic stress disorder). Despite the valuable insights that rodent models have provided, there is a growing interest in using injury models using species with neuroanatomical features that more closely resemble the human brain. With this purpose, we established a gyrencephalic model of blast injury in ferrets, which underwent blast exposure applying conditions that closely mimic those associated with primary blast injuries to warfighters. In this study, we evaluated brain biochemical, microstructural, and behavioral profiles after blast exposure using in vivo longitudinal magnetic resonance imaging, histology, and behavioral assessments. In ferrets subjected to blast, the following alterations were found: 1) heightened impulsivity in decision making associated with pre-frontal cortex/amygdalar axis dysfunction; 2) transiently increased glutamate levels that are consistent with earlier findings during subacute stages post-TBI and may be involved in concomitant behavioral deficits; 3) abnormally high brain N-acetylaspartate levels that potentially reveal disrupted lipid synthesis and/or energy metabolism; and 4) dysfunction of pre-frontal cortex/auditory cortex signaling cascades that may reflect similar perturbations underlying secondary psychiatric disorders observed in warfighters after blast exposure.

Metabolic disruption impacts tick fitness and microbial relationships.

Arthropod-borne microbes rely on the metabolic state of a host to cycle between evolutionarily distant species. For instance, arthropod tolerance to infection may be due to redistribution of metabolic resources, often leading to microbial transmission to mammals. Conversely, metabolic alterations aids in pathogen elimination in humans, who do not ordinarily harbor arthropod-borne microbes. To ascertain the effect of metabolism on interspecies relationships, we engineered a system to evaluate glycolysis and oxidative phosphorylation in the tick Ixodes scapularis. Using a metabolic flux assay, we determined that the rickettsial bacterium Anaplasma phagocytophilum and the Lyme disease spirochete Borrelia burgdorferi, which are transstadially transmitted in nature, induced glycolysis in ticks. On the other hand, the endosymbiont Rickettsia buchneri, which is transovarially maintained, had a minimal effect on I. scapularis bioenergetics. Importantly, the metabolite ß-aminoisobutyric acid (BAIBA) was elevated during A. phagocytophilum infection of tick cells following an unbiased metabolomics approach. Thus, we manipulated the expression of genes associated with the catabolism and anabolism of BAIBA in I. scapularis and detected impaired feeding on mammals, reduced bacterial acquisition, and decreased tick survival. Collectively, we reveal the importance of metabolism for tick-microbe relationships and unveil a valuable metabolite for I. scapularis fitness.

Induction of endothelial barrier dysfunction by serum factors in rats subjected to traumatic brain injury and hemorrhagic shock.

Traumatic brain injury (TBI) has been associated with the development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanisms linking TBI with the development of ARDS, we characterized the effects of serum factors released following TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS. We found that serum samples from animals exposed to both controlled cortical impact (CCI) and HS, but not from sham-operated rats induced significant barrier dysfunction in human pulmonary artery EC monolayers at 2 days post injury. Thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of the serum-induced trans-endothelial resistance (TER) decline caused by CCI-HS serum, but not in later time points. However, both the early and late phases of CCI-HS-induced EC permeability were inhibited by heparin. The barrier disruptive effects of CCI-HS serum were also prevented by serum preincubation with heparin-sepharose. Pulmonary EC treated for 3 h with serum from CCI-HS rats demonstrated a significant decline in expression of EC junctional protein, VE-Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE-cadherin antibody. These results suggest that exposure to CCI-HS causes early and late-phase barrier disruptive effects in vascular endothelium. While thrombin-PAR1 signaling has been identified as a mechanism of acute EC permeability increase by CCI-HS serum, the factor(s) defining long-term EC barrier disruption in CCI-HS model remains to be determined.

Metabolic imaging of energy metabolism in traumatic brain injury using hyperpolarized [1-13C]pyruvate.

Traumatic brain injury (TBI) is known to cause perturbations in the energy metabolism of the brain, but current tests of metabolic activity are only indirect markers of energy use or are highly invasive. Here we show that hyperpolarized 13C magnetic resonance spectroscopic imaging (MRSI) can be used as a direct, non-invasive method for studying the effects of TBI on energy metabolism. Measurements were performed on rats with moderate TBI induced by controlled cortical impact on one cerebral hemisphere. Following injection of hyperpolarized [1-13C]pyruvate, the resulting 13C-bicarbonate signal was found to be 24 ± 6% lower in the injured hemisphere compared with the non-injured hemisphere, while the hyperpolarized bicarbonate-to-lactate ratio was 33 ± 8% lower in the injured hemisphere. In a control group, no significant difference in signal was found between sides of the brain. The results suggest an impairment in mitochondrial pyruvate metabolism, resulting in a decrease in aerobic respiration at the location of injury following TBI.

The RUNX2 Transcription Factor Negatively Regulates SIRT6 Expression to Alter Glucose Metabolism in Breast Cancer Cells.

Activation of genes promoting aerobic glycolysis and suppression of mitochondrial oxidative phosphorylation is one of the hallmarks of cancer. The RUNX2 transcription factor mediates breast cancer (BC) metastasis to bone and is regulated by glucose availability. But, the mechanisms by which it regulates glucose metabolism and promotes an oncogenic phenotype are not known. RUNX2 expression in luminal BC cells correlated with lower estrogen receptor-α (ERα) levels, anchorage-independent growth, expression of glycolytic genes, increased glucose uptake, and sensitivity to glucose starvation, but not to inhibitors of oxidative phosphorylation. Conversely, RUNX2 knockdown in triple-negative BC cells inhibited mammosphere formation and glucose dependence. RUNX2 knockdown resulted in lower LDHA, HK2, and GLUT1 glycolytic gene expression, but upregulation of pyruvate dehydrogenase-A1 (PDHA1) mRNA and enzymatic activity, which was consistent with lower glycolytic potential. The NAD-dependent histone deacetylase, SIRT6, a known tumor suppressor, was a critical regulator of these RUNX2-mediated metabolic changes. RUNX2 expression resulted in elevated pAkt, HK2, and PDHK1 glycolytic protein levels that were reduced by ectopic expression of SIRT6. RUNX2 also repressed mitochondrial oxygen consumption rates (OCR), a measure of oxidative phosphorylation (respiration). Overexpression of SIRT6 increased respiration in RUNX2-positive cells, but knockdown of SIRT6 in cells expressing low RUNX2 decreased respiration. RUNX2 repressed SIRT6 expression at both the transcriptional and post-translational levels and endogenous SIRT6 expression was lower in malignant BC tissues or cell lines that expressed high levels of RUNX2. These results support a hypothesis whereby RUNX2-mediated repression of the SIRT6 tumor suppressor regulates metabolic pathways that promote BC progression.

Brain diabetic neurodegeneration segregates with low intrinsic aerobic capacity.

OBJECTIVES: Diabetes leads to cognitive impairment and is associated with age-related neurodegenerative diseases including Alzheimer's disease (AD). Thus, understanding diabetes-induced alterations in brain function is important for developing early interventions for neurodegeneration. Low-capacity runner (LCR) rats are obese and manifest metabolic risk factors resembling human "impaired glucose tolerance" or metabolic syndrome. We examined hippocampal function in aged LCR rats compared to their high-capacity runner (HCR) rat counterparts. METHODS: Hippocampal function was examined using proton magnetic resonance spectroscopy and imaging, unbiased stereology analysis, and a Y maze. Changes in the mitochondrial respiratory chain function and levels of hyperphosphorylated tau and mitochondrial transcriptional regulators were examined. RESULTS: The levels of glutamate, myo-inositol, taurine, and choline-containing compounds were significantly increased in the aged LCR rats. We observed a significant loss of hippocampal neurons and impaired cognitive function in aged LCR rats. Respiratory chain function and activity were significantly decreased in the aged LCR rats. Hyperphosphorylated tau was accumulated within mitochondria and peroxisome proliferator-activated receptor-gamma coactivator 1α, the NAD(+)-dependent protein deacetylase sirtuin 1, and mitochondrial transcription factor A were downregulated in the aged LCR rat hippocampus. INTERPRETATION: These data provide evidence of a neurodegenerative process in the hippocampus of aged LCR rats, consistent with those seen in aged-related dementing illnesses such as AD in humans. The metabolic and mitochondrial abnormalities observed in LCR rat hippocampus are similar to well-described mechanisms that lead to diabetic neuropathy and may provide an important link between cognitive and metabolic dysfunction.

Monoallelic loss of tumor suppressor GRIM-19 promotes tumorigenesis in mice.

Gene-associated with retinoid-interferon induced mortality-19 (GRIM-19), a STAT3-inhibitory protein, was isolated as a growth-suppressive gene product using a genome-wide expression knockdown screen. We and others have shown a loss of expression and occurrence of mutations in the GRIM-19 gene in a variety of primary human cancers, indicating its potential role as tumor suppressor. To help investigate its role in tumor development in vivo, we generated a genetically modified mouse in which Grim-19 can be conditionally inactivated. Deletion of Grim-19 in the skin significantly increased the susceptibility of mice to chemical carcinogenesis, resulting in development of squamous cell carcinomas. These tumors had high Stat3 activity and an increased expression of Stat3-responsive genes. Loss of Grim-19 also caused mitochondrial electron transport dysfunction resulting from failure to assemble electron transport chain complexes and altered the expression of several cellular genes involved in glycolysis. Surprisingly, the deletion of a single copy of the Grim-19 gene was sufficient to promote carcinogenesis and formation of invasive squamous cell carcinomas. These observations highlight the critical role of GRIM-19 as a tumor suppressor.

A role for mitochondrial dysfunction in perpetuating radiation-induced genomic instability.

Radiation-induced genomic instability (RIGI) manifests as a heritable increased rate of genetic alterations in the progeny of irradiated cells generations after the initial insult. The progeny can show an increased frequency of chromosomal translocations, deletions, mutations, micronuclei, and decreased plating efficiency. What perpetuates RIGI is unclear; however, persistently increased levels of reactive oxygen species (ROS) are frequently associated with genomically unstable clones. Furthermore, addition of free radical scavengers (e.g., DMSO, glycerol, and cationic thiol cysteamine) reduces the incidence of instability after irradiation, implicating a ROS-mediated role in RIGI induction. Because mitochondria are a major natural cellular source of ROS, we tested the hypothesis that mitochondrial dysfunction has a role in maintaining the elevated ROS levels in our irradiated, genetically unstable GM10115 Chinese hamster ovary cells. Amplex Red fluorometry measurements indicate that the relative contribution of uncoupler-sensitive mitochondrial hydrogen peroxide production to total cellular hydrogen peroxide generation is greater in unstable cells. Measurements of mitochondrial DNA levels and cell cytometric fluorescent measurements of Mitotracker Green FM indicate that differences in mitochondrial ROS production are not due to varying mitochondrial levels. However, mitochondrial respiration measured in digitonin-permeabilized cells is impaired in unstable clones. In addition, manganese superoxide dismutase, a major mitochondrial antioxidant enzyme, exhibits increased immunoreactivity but decreased enzyme activity in unstable clones, which along with decreased respiration rates may explain the increased levels of cellular ROS. These studies show that mitochondria from unstable cells are abnormal and likely contribute to the persistent oxidative stress in the unstable clones.