Interactions of Cellular Energetic Gene Clusters in the Alzheimer's Mouse Brain.

Alzheimer's disease (AD) is the most common cause of dementia in the aging population. The pathological characteristics include extracellular senile plaques and intracellular neurofibrillary tangles. In addition, mitochondrial dysfunction, oxidative stress, and neuroinflammation contribute to AD pathogenesis. In this study, we sought to determine the crosstalk between different pathways in the brain of 5XFAD mice, a mouse model for amyloid pathology, by RNA-seq analysis. We observed significant changes in the expression of genes (1288 genes; adj p value < 0.05; log2-fold > 1 and < 1) related to pathways including oxidation-reduction, oxidative phosphorylation, innate immune response, ribosomal protein synthesis, and ubiquitin proteosome system. The most striking feature was the downregulation of genes related to oxidation-reduction process with changes in the expression of a large number of mitochondrial genes. We also observed an upregulation of several immune response genes. Gene interaction network of oxidation-reduction related genes further confirmed a tight cluster of mitochondrial genes. Furthermore, gene interaction analysis of all the 1288 genes showed at least three distinct interaction clusters, with the predominant one relating to cellular energetics. In summary, we identified 1288 genes distinctly different in the 5XFAD brain compared to the WT brain and found cellular energetics to be the most distinct gene cluster in the AD mouse brain.

Advances in Rodent Experimental Models of Sepsis.

In the development of therapeutic strategies for human diseases, preclinical experimental models have a key role. However, the preclinical immunomodulatory therapies developed using rodent sepsis were not successful in human clinical trials. Sepsis is characterized by a dysregulated inflammation and redox imbalance triggered by infection. Human sepsis is simulated in experimental models using methods that trigger inflammation or infection in the host animals, most often mice or rats. It remains unknown whether the characteristics of the host species, the methods used to induce sepsis, or the molecular processes focused upon need to be revisited in the development of treatment methods that will succeed in human clinical trials. Our goal in this review is to provide a survey of existing experimental models of sepsis, including the use of humanized mice and dirty mice, and to show how these models reflect the clinical course of sepsis. We will discuss the strengths and limitations of these models and present recent advances in this subject area. We maintain that rodent models continue to have an irreplaceable role in studies toward discovering treatment methods for human sepsis.

Regulating mitochondrial metabolism by targeting pyruvate dehydrogenase with dichloroacetate, a metabolic messenger.

Dichloroacetate (DCA) is a naturally occurring xenobiotic that has been used as an investigational drug for over 50 years. Originally found to lower blood glucose levels and alter fat metabolism in diabetic rats, this small molecule was found to serve primarily as a pyruvate dehydrogenase kinase inhibitor. Pyruvate dehydrogenase kinase inhibits pyruvate dehydrogenase complex, the catalyst for oxidative decarboxylation of pyruvate to produce acetyl coenzyme A. Several congenital and acquired disease states share a similar pathobiology with respect to glucose homeostasis under distress that leads to a preferential shift from the more efficient oxidative phosphorylation to glycolysis. By reversing this process, DCA can increase available energy and reduce lactic acidosis. The purpose of this review is to examine the literature surrounding this metabolic messenger as it presents exciting opportunities for future investigation and clinical application in therapy including cancer, metabolic disorders, cerebral ischemia, trauma, and sepsis.

BACH1-Hemoxygenase-1 axis regulates cellular energetics and survival following sepsis.

Sepsis is a complex disease due to dysregulated host response to infection. Oxidative stress and mitochondrial dysfunction leading to metabolic dysregulation are among the hallmarks of sepsis. The transcription factor NRF2 (Nuclear Factor E2-related factor2) is a master regulator of the oxidative stress response, and the NRF2 mediated antioxidant response is negatively regulated by BTB and CNC homology 1 (BACH1) protein. This study tested whether Bach1 deletion improves organ function and survival following polymicrobial sepsis induced by cecal ligation and puncture (CLP). We observed enhanced post-CLP survival in Bach1-/- mice with a concomitantly increased liver HO-1 expression, reduced liver injury and oxidative stress, and attenuated systemic and tissue inflammation. After sepsis induction, the liver mitochondrial function was better preserved in Bach1-/- mice. Furthermore, BACH1 deficiency improved liver and lung blood flow in septic mice, as measured by SPECT/CT. RNA-seq analysis identified 44 genes significantly altered in Bach1-/- mice after sepsis, including HMOX1 and several genes in lipid metabolism. Inhibiting HO-1 activity by Zinc Protoporphyrin-9 worsened organ function in Bach1-/- mice following sepsis. We demonstrate that mitochondrial bioenergetics, organ function, and survival following experimental sepsis were improved in Bach1-/- mice through the HO-1-dependent mechanism and conclude that BACH1 is a therapeutic target in sepsis.

Juvenile Plasma Factors Improve Organ Function and Survival following Injury by Promoting Antioxidant Response.

Studies have shown that factors in the blood of young organisms can rejuvenate the old ones. Studies using heterochronic parabiosis models further reinforced the hypothesis that juvenile factors can rejuvenate aged systems. We sought to determine the effect of juvenile plasma-derived factors on the outcome following hemorrhagic shock injury in aged mice. We discovered that pre-pubertal (young) mice subjected to hemorrhagic shock survived for a prolonged period, in the absence of fluid resuscitation, compared to mature or aged mice. To further understand the mechanism of maturational dependence of injury resolution, extracellular vesicles isolated from the plasma of young mice were administered to aged mice subjected to hemorrhagic shock. The extracellular vesicle treatment prolonged life in the aged mice. The treatment resulted in reduced oxidative stress in the liver and in the circulation, along with an enhanced expression of the nuclear factor erythroid factor 2-related factor 2 (Nrf2) and its target genes, and a reduction in the expression of the transcription factor BTB and CNC homology 1 (Bach1). We propose that plasma factors in the juvenile mice have a reparative effect in the aged mice in injury resolution by modulating the Nrf2/Bach1 axis in the antioxidant response pathway.

Regulation of NAD+ metabolism in aging and disease.

More than a century after discovering NAD+, information is still evolving on the role of this molecule in health and diseases. The biological functions of NAD+ and NAD+ precursors encompass pathways in cellular energetics, inflammation, metabolism, and cell survival. Several metabolic and neurological diseases exhibit reduced tissue NAD+ levels. Significantly reduced levels of NAD+ are also associated with aging, and enhancing NAD+ levels improved healthspan and lifespan in animal models. Recent studies suggest a causal link between senescence, age-associated reduction in tissue NAD+ and enzymatic degradation of NAD+. Furthermore, the discovery of transporters and receptors involved in NAD+ precursor (nicotinic acid, or niacin, nicotinamide, and nicotinamide riboside) metabolism allowed for a better understanding of their role in cellular homeostasis including signaling functions that are independent of their functions in redox reactions. We also review studies that demonstrate that the functional effect of niacin is partially due to the activation of its cell surface receptor, GPR109a. Based on the recent progress in understanding the mechanism and function of NAD+ and NAD+ precursors in cell metabolism, new strategies are evolving to exploit these molecules' pharmacological potential in the maintenance of metabolic balance.

Rescuing mitochondria in traumatic brain injury and intracerebral hemorrhages - A potential therapeutic approach.

Mitochondria are dynamic organelles responsible for cellular energy production. Besides, regulating energy homeostasis, mitochondria are responsible for calcium homeostasis, signal transmission, and the fate of cellular survival in case of injury and pathologies. Accumulating reports have suggested multiple roles of mitochondria in neuropathologies, neurodegeneration, and immune activation under physiological and pathological conditions. Mitochondrial dysfunction, which occurs at the initial phase of brain injury, involves oxidative stress, inflammation, deficits in mitochondrial bioenergetics, biogenesis, transport, and autophagy. Thus, development of targeted therapeutics to protect mitochondria may improve functional outcomes following traumatic brain injury (TBI) and intracerebral hemorrhages (ICH). In this review, we summarize mitochondrial dysfunction related to TBI and ICH, including the mechanisms involved, and discuss therapeutic approaches with special emphasis on past and current clinical trials.

Pulmonary function changes in older adults with and without metabolic syndrome.

The low-grade inflammation associated with metabolic syndrome (MS) triggers functional and structural alterations in several organs. Whereas lung function impairment is well reported for older adult population, the effect of MS on functional and immunological responses in the lungs remains unclear. In this cross-sectional study we determined whether MS alters pulmonary function, and immunological responses in older adults with MS. The study sample consisted of older adults with MS (68 ± 3 years old; n = 77) and without MS (67 ± 3 years old; n = 77). Impulse oscillometry was used to evaluate airway and tissue resistance, and reactance. Biomarkers of inflammation and fibrosis were assessed in the blood and in breath condensate. The total resistance of the respiratory system (R5Hz; p < 0.009), and the resistance of the proximal (R20Hz; p < 0.001) and distal (R5Hz-R20Hz; p < 0.004) airways were higher in MS individuals compared to those without MS. Pro-inflammatory (leptin, IL-1beta, IL-8, p < 0.001; TNF-alpha, p < 0.04) and anti-inflammatory cytokines (adiponectin, IL-1ra, IL-10, p < 0.001), anti-fibrotic (relaxin 1, relaxin 3, Klotho, p < 0.001) and pro-fibrotic (VEGF, p < 0.001) factors were increased in sera and in breath condensate individuals with MS. The results show that MS adversely affect lung mechanics, function, and immunological response in older adults. The data offer a metabolic basis for the inflammaging of the lungs and suggest the lungs as a potential therapeutic target for controlling the immune response and delaying the onset of impaired lung function in older adults with MS.

Dysregulation of cellular energetics in Gulf War Illness.

Gulf War Illness (GWI) is estimated to have affected about one third of the Veterans who participated in the first Persian Gulf War. The symptoms of GWI include chronic neurologic impairments, chronic fatigue syndrome, as well as fibromyalgia and immune system disorders, collectively referred to as chronic multi-symptom illness. Thirty years after the war, we still do not have an effective treatment for GWI. It is necessary to understand the molecular basis of the symptoms of GWI in order to develop appropriate therapeutic strategies. Cellular energetics are critical to the maintenance of cellular homeostasis, a process that is highly dependent on intact mitochondrial function and there is significant evidence from both human studies and animal models that mitochondrial impairments may lead to GWI symptoms. The available clinical and pre-clinical data suggest that agents that improve mitochondrial function have the potential to restore cellular energetics and treat GWI. To date, the experiments conducted in animal models of GWI have mainly focused on neurobehavioral aspects of the illness. Additional studies to address the fundamental biological processes that trigger the dysregulation of cellular energetics in GWI are warranted to better understand the underlying pathology and to develop new treatment methods. This review highlights studies related to mitochondrial dysfunction observed in both GW veterans and in animal models of GWI.

Rapid senescence-like response after acute injury.

Cellular senescence is a state of irreversible growth arrest. Short-term programmed senescence such as in embryonic development and slowly progressing senescence as in aging are both well described. However, acute senescence in living organisms is not well understood. We hypothesized that hemorrhagic shock injury (HI) caused by whole body hypoxia and nutrient deprivation, resulting in organ dysfunction due to severe blood loss, could lead to acute senescence in vivo. Our experiments, for the first time, demonstrate a rapidly emerged, senolytics-responsive, senescence-like response in the rat liver in less than five hr following hemorrhagic shock. We conclude that the senescence, or pseudosenescence, observed is necessary to maintain tissue homeostasis following the injury.

NLRX1 Regulation Following Acute Mitochondrial Injury.

Several metabolic, cardiovascular, and neurological disorders are characterized by mitochondrial dysfunction followed by dysregulation of cellular energetics. Mitochondria play an important role in ATP production and cell death regulation. NLRX1, a mitochondria-targeted protein, is known to negatively regulate innate immunity, and cell death responses. However, the role of this protein in cellular homeostasis following mitochondrial injury is not well-understood. To understand the mechanisms underlying the effect of acute injury in regulating NLRX1 signaling pathways, we used an in vitro model of mitochondrial injury wherein, rat pulmonary microvascular endothelial cells were subjected to sodium azide treatment or glucose starvation. Both sodium azide and glucose starvation activated NF-κB and TBK1 associated innate immune response. Moreover, increased TBK1, IKK, IκB, and TRAF6 were recruited to mitochondria and interacted with NLRX1. Depletion of endogenous NLRX1 resulted in exacerbated NF-κB and TBK1 associated innate immune response and apoptosis. Our results suggest that NLRX1 participates in the regulation of innate immune response in mitochondria, and plays an important role in the maintenance of cellular homeostasis following acute mitochondrial injury. We propose that the mitochondrial recruitment of inflammatory mediators and their interaction with NLRX1 are protective responses to maintain cellular homeostasis following injury.

MicroRNA-34a (miR-34a) Mediates Retinal Endothelial Cell Premature Senescence through Mitochondrial Dysfunction and Loss of Antioxidant Activities.

Stress-associated premature senescence (SAPS) is involved in retinal microvascular injury and diabetic retinopathy. We have investigated the role and mode of action of miR-34a in retinal endothelial cells senescence in response to glucidic stress. Human retinal microvascular endothelial cells (HuREC) were exposed to glucidic stress (high glucose (HG) = 25 mM d-glucose) and compared to cells exposed to normal glucose (NG = 5 mM) or the osmotic control l-glucose (LG = 25 mM). HG stimulation of HuREC increased the expression of miR-34a and induced cellular senescence. HG also increased the expression of p16ink4a and p21waf1, while decreasing the histone deacetylase SIRT1. These effects were associated with diminished mitochondrial function and loss of mitochondrial biogenesis factors (i.e., PGC-1α, NRF1, and TFAM). Transfection of the cells with miR-34a inhibitor (IB) halted HG-induced mitochondrial dysfunction and up-regulation of senescence-associated markers, whereas miR-34a mimic promoted cellular senescence and mitochondrial dysfunction. Moreover, HG lowered levels of the mitochondrial antioxidants TrxR2 and SOD2, an effect blunted by miR-34a IB, and promoted by miR-34a mimic. 3'-UTR (3'-untranslated region) reporter assay of both genes validated TrxR2 as a direct target of miR-34a, but not SOD2. Our results show that miR-34a is a key player of HG-induced SAPS in retinal endothelial cells via multiple pathways involved in mitochondrial function and biogenesis.

Deficiency of metabolite sensing receptor HCA2 impairs the salutary effect of niacin in hemorrhagic shock.

Inflammation and cellular energetics play critical roles in organ dysfunction following hemorrhagic shock. Recent studies suggest a putative role for sirtuin 1 (SIRT1) in potentiating mitochondrial function and improving organ function following hemorrhagic shock in animal models. SIRT1 is an NAD+ dependent protein deacetylase and increased availability of NAD+ has been shown to augment SIRT1 activity. As niacin is a precursor of NAD+, in this study, we tested whether niacin can improve survival following hemorrhagic shock. However niacin also mediates its biological action by binding to its receptor, hydroxyl-carboxylic acid receptor 2 (HCA2 or Gpr109a); so we examined whether the effect of niacin is mediated by binding to Gpr109a or by increasing NAD+ availability. We found that niacin administered intravenously to rats subjected to hemorrhagic injury (HI) in the absence of fluid resuscitation resulted in a significantly prolonged duration of survival. However, treatment of rats with similar doses of nicotinamide mononucleotide (NMN), a precursor to NAD+ that does not bind Gpr109a, did not extend survival following HI. The duration of survival due to niacin treatment was significantly reduced in Gpr109a-/- mice subjected to HI. These experiments demonstrated that the Gpr109a receptor-mediated pathway contributed significantly to niacin mediated salutary effect. Further studies showed improvement in markers of cellular energetics and attenuation of inflammatory response with niacin treatment. In conclusion, we report that Gpr109a-dependent signalling is important in restoring cellular energetics and immunometabolism following hemorrhagic shock.

A Combination Treatment Strategy for Hemorrhagic Shock in a Rat Model Modulates Autophagy.

Hemorrhagic shock leads to whole body hypoxia and nutrient deprivation resulting in organ dysfunction and mortality. Previous studies demonstrated that resveratrol, dichloroacetate, and niacin improve organ function and survival in rats following hemorrhagic shock injury (HI). We hypothesized that a combinatorial formula that collectively promotes survival will decrease the dose of individual compounds toward effective therapy for HI. Sprague-Dawley rats were subjected to HI by withdrawing 60% blood volume. NiDaR (Niacin-Dichloroacetate-Resveratrol; 2 mg/kg dose of each) or vehicle was administered following the shock in the absence of fluid resuscitation, and survival monitored. In order to study alterations in molecular mediators, separate groups of rats were administered NiDaR or vehicle along with resuscitation fluid, following HI. We observed significant improvement (p < 0.05) in survival following HI in animals that received NiDaR, in the absence of fluid resuscitation. In NiDaR treated animals that received resuscitation fluid, MAP was significantly increased compared to Veh-treated rats. HI-induced increase in systemic IL-6 levels and tissue expression of IL-6, IL-10, IL-1ß, and IL-18 genes in the heart were attenuated with NiDaR treatment. NiDaR promoted autophagy following HI as demonstrated by reduced p-mTOR, increased p-ULK1 and p-Beclin. The combinatorial formula, NiDaR, reduced inflammation, promoted autophagy, and reduced doses of individual compounds used, and may be more effective in genetically heterogeneous population. In conclusion our experiments demonstrated that the combinatorial drug treatment has salutary effect in rats following HI.

Effect of plasma-derived extracellular vesicles on erythrocyte deformability in polymicrobial sepsis.

Sepsis affects microcirculation and tissue perfusion leading to tissue hypoxia and multiple organ dysfunction. Red blood cells (RBCs; erythrocytes) are typically biconcave in shape, transport hemoglobin-bound oxygen and are reversibly deformable facilitating trafficking through capillaries. Decreased deformability of RBCs adversely affects tissue oxygenation. The purpose of this project was to determine RBC deformability in a murine model of polymicrobial sepsis by a method that utilizes laser diffraction and microfluidics, and to identify the causative factors in the plasma that may contribute to loss in RBC deformability. Blood samples from mice subjected to cecal ligation and puncture (CLP) model of sepsis were used. RBC deformability was tested using Rheoscan-AnD 300 under shear stress range of 0-20 Pascal (Pa) that depicts the common rheological behavior of RBCs flowing through blood vessels ranging from major vessels to capillaries. Normal RBCs were treated with plasma-derived extracellular vesicles (EVs) and their effect on RBC deformability was also tested. The experiments demonstrated a significant decrease in RBC deformability following sepsis. RBC deformability recovered in sham-operated animals by the third day, whereas animals with sepsis continued to show decreased levels of deformability. EVs isolated from the plasma of animals from the sepsis group significantly decreased deformability of RBCs ex vivo. Analysis of miRNA cargo in EVs showed distinct molecular profiles for sham-operated and sepsis-induced mice. In summary, sepsis induced a decrease in RBC deformability and the acquired rigidity may have adverse effect on microcirculation, tissue perfusion, and organ function.

Kidney-targeted inhibition of protein kinase C-α ameliorates nephrotoxic nephritis with restoration of mitochondrial dysfunction.

To investigate the role of protein kinase C-α (PKC-α) in glomerulonephritis, the capacity of PKC-α inhibition to reverse the course of established nephrotoxic nephritis (NTN) was evaluated. Nephritis was induced by a single injection of nephrotoxic serum and after its onset, a PKC-α inhibitor was administered either systemically or by targeted glomerular delivery. By day seven, all mice with NTN had severe nephritis, whereas mice that received PKC-α inhibitors in either form had minimal evidence of disease. To further understand the underlying mechanism, label-free shotgun proteomic analysis of the kidney cortexes were performed, using quantitative mass spectrometry. Ingenuity pathway analysis revealed 157 differentially expressed proteins and mitochondrial dysfunction as the most modulated pathway. Functional protein groups most affected by NTN were mitochondrial proteins associated with respiratory processes. These proteins were down-regulated in the mice with NTN, while their expression was restored with PKC-α inhibition. This suggests a role for proteins that regulate oxidative phosphorylation in recovery. In cultured glomerular endothelial cells, nephrotoxic serum caused a decrease in mitochondrial respiration and membrane potential, mitochondrial morphologic changes and an increase in glycolytic lactic acid production; all normalized by PKC-α inhibition. Thus, PKC-α has a critical role in NTN progression, and the results implicate mitochondrial processes through restoring oxidative phosphorylation, as an essential mechanism underlying recovery. Importantly, our study provides additional support for targeted therapy to glomeruli to reverse the course of progressive disease.

Mitochondrial dysfunction in rat splenocytes following hemorrhagic shock.

The regulation of mitochondrial function is critical in cellular homeostasis following hemorrhagic shock. Hemorrhagic shock results in fluid loss and reduced availability of oxygen and nutrients to tissues. The spleen is a secondary lymphoid organ playing a key role in 'filtering the blood' and in the innate and adaptive immune responses. To understand the molecular basis of hemorrhagic shock, we investigated the changes in splenocyte mitochondrial respiration, and concomitant immune and metabolic alterations. The hemorrhagic injury (HI) in our rat model was induced by bleeding 60% of the total blood volume followed by resuscitation with Ringers lactate. Another group of animals was subjected to hemorrhage, but did not receive fluid resuscitation. Oxygen consumption rate of splenocytes were determined using a Seahorse analyzer. We found a significantly reduced oxygen consumption rate in splenocytes following HI compared to sham operated rats. The mitochondrial stress test revealed a decreased basal oxygen consumption rate, ATP production, maximal respiration and spare respiratory capacity. The mitochondrial membrane potential, and citrate synthase activity, were also reduced in the splenocytes following HI. Hypoxic response in the splenocyte was confirmed by increased gene expression of Hif1α. Elevated level of mitochondrial stress protein, hsp60 and induction of high mobility group box1 protein (HMGB1) were observed in splenocytes following HI. An increased inflammatory response was demonstrated by significantly increased expression of IL-6, IFN-ß, Mip-1α, IL-10 and NFκbp65. In summary, we conclude that splenocyte oxidative phosphorylation and metabolism were severely compromised following HI.

Mitochondrial targeting by dichloroacetate improves outcome following hemorrhagic shock.

Hemorrhagic shock is a leading cause of death in people under the age of 45 and accounts for almost half of trauma-related deaths. In order to develop a treatment strategy based on potentiating mitochondrial function, we investigated the effect of the orphan drug dichloroacetate (DCA) on survival in an animal model of hemorrhagic shock in the absence of fluid resuscitation. Hemorrhagic shock was induced in rats by withdrawing 60% of the blood volume and maintaining a hypotensive state. The studies demonstrated prolonged survival of rats subjected to hemorrhagic injury (HI) when treated with DCA. In separate experiments, using a fluid resuscitation model we studied mitochondrial functional alterations and changes in metabolic networks connected to mitochondria following HI and treatment with DCA. DCA treatment restored cardiac mitochondrial membrane potential and tissue ATP in the rats following HI. Treatment with DCA resulted in normalization of several metabolic and molecular parameters including plasma lactate and p-AMPK/AMPK, as well as Ach-mediated vascular relaxation. In conclusion we demonstrate that DCA can be successfully used in the treatment of hemorrhagic shock in the absence of fluid resuscitation; therefore DCA may be a good candidate in prolonged field care following severe blood loss.