Stress Granule Assembly Disrupts Nucleocytoplasmic Transport.

Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.

A novel ligand on astrocytes interacts with natural cytotoxicity receptor NKp44 regulating immune response mediated by NK cells.

NK cells play important role in immunity against pathogens and cancer. NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. NK cells were shown to be recruited to the CNS following several pathological conditions. NK cells could impact CNS physiology by killing glial cells and by secreting IFN-γ. Astrocytes are intimately involved in immunological and inflammatory events occurring in the CNS and reactive astrogliosis is a key feature in HIV-associated neurocognitive disorders. There is little data on NK-astrocyte interactions and ligands expressed on astrocytes that could impact NK cell function. Natural cytotoxicity receptors (NCRs) play a critical role in the cytolytic function of NK cells. Among the NCRs, NKp44 is unique in expression and signal transduction. NKp44 is expressed only upon activation of NK cells and it can mediate both activating and inhibitory signals to NK cells. Here, we have studied the expression and function of natural cytotoxicity receptor NKp44 upon NK-astrocytes interactions in the presence or absence of an HIV peptide (HIV-3S peptide) shown to induce NK cell killing of CD4+ T cells during HIV-infection. Using a fusion protein consisting of the extracellular domain of NKp44 fused to Fc portion of human IgG, we determined the expression of a novel ligand for NKp44 (NKp44L) on astrocytes. Incubation of astrocytes with HIV-3S peptide downregulated NKp44L expression on astrocytes implicating protection from NK mediated killing. Thus, our study showed that NKp44 have a protective effect on astrocytes from NK cell mediated killing during HIV infection and impact astrocyte role in HAND.

Pyruvate metabolism of perfused rat lungs after exposure to 100% oxygen.

Previous studies with lung homogenates have suggested that pulmonary O2 toxicity is in part a result of inhibited mitochondrial energy metabolism. In this study, mitochondrial metabolism was determined by measurements of 14CO2 production from [1-14C]-pyruvate in perfused lungs, isolated after 0, 3, 6, 12, and 24 h of exposure to 100% O2. Measurements were made under normal and stimulated conditions brought about by uncoupling oxidative phosphorylation with 2,4-dinitrophenol (DNP). Lungs were ventilated with 5% CO2 in O2 and perfused for 100 min with 12.5 mM 14C labeled pyruvate. Unexposed lungs gave a linear rate of 14CO2 production of 121 +/- 16 mumol/h/g dry wt (n = 5), which was maximally stimulated 84% by perfusion with 0.8 mMDNP. Twenty-four hours of exposure to 100% O2 did not significantly affect 14CO2 production. In contrast, DNP failed to significantly stimulate pyruvate metabolism to CO2 in lungs exposed for greater than 3 h to 100% O2. These latter data suggested that O2 exposure makes lung mitochondria unable to respond to increased ATP demands associated with DNP uncoupling. Compromised energy metabolism is therefore an important early event in O2 toxicity.

Rat lung glucose metabolism after 24 h of exposure to 100% oxygen.

Previous studies with lung homogenates and isolated cells have suggested oxygen cell injury results from the inhibition of key enzymes involved in both cytosolic and mitochondrial energy generation. In this study, the extent and pattern of metabolism of D-[U-14C, 5-3H]glucose was examined in perfused lungs isolated from rats before and after 24 h of in vivo exposure to 100% O2. Lung ATP levels after O2 exposure were maintained by a 53% increase in glucose utilization from an unexposed control value of 18.0 +/- 3.2 to 27.5 +/- 3.0 mumol 3H2O.h-1.g dry wt-1, accounted for by an enhanced rate of lactate plus pyruvate production from 15.7 +/- 2.0 to 32.7 +/- 4.1 mumol.h-1.g dry wt-1 with no alteration in lactate-to-pyruvate ratio. CO2 production was unaltered from a control rate of 27.5 +/- 4.0 14CO2 mumol.h-1.g dry wt-1. Maximal rates of glucose metabolism were determined by perfusion with 0.8 mM dinitrophenol, giving for air-exposed lungs a rate of 53.5 +/- 5.0 mumol 3H2O.h-1.g dry wt-1 and increased lactate plus pyruvate and 14CO2 production rates of 46.5 +/- 6.5 and 128.3 +/- 19.6 mumol.h-1.g dry wt-1, respectively. Although this maximal rate of glucose utilization was unaltered in oxygen-exposed lungs, lactate plus pyruvate production was further increased to 80.0 +/- 9.1 mumol.h-1.g dry wt-1 with a concomitant decrease in the dinitrophenol-induced rate of 14CO2 production to 81.5 +/- 9.2 mumol.h-1.g dry wt-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat lung metabolism after 3 days of continuous exposure to 0.6 ppm ozone.

Continuous exposure of rats to low concentrations of ozone has previously been associated with enhanced metabolic enzyme activities, when measured in lung homogenates. In this study, metabolic rates were measured in intact perfused lungs with altered pathology brought about by 3 days continuous exposure to 0.6 ppm ozone. Increased metabolism of ozone-exposed lungs was indicated by a twofold enhancement in glucose utilization, associated with a 62% increase in lactate formation and a 166% increase in the rate of 14CO2 production from D-[U-14C]glucose from control values of 5.2 +/- 0.5 mumol lactate and 4.4 +/- 0.6 mumol 14CO2/h per lung (+/- SE, n = 4), respectively. Mitochondrial metabolism was separately assessed by measurements of 14CO2 production from [U-14C]-pyruvate, which was found not to be significantly altered by ozone exposure, although homogenate oxygen uptake in the presence of succinate was significantly enhanced by 57%. These changes in intermediary metabolism could be correlated with increased glucose carbon incorporation into lipid and elevated activity of glucose-6-phosphate dehydrogenase. The observed elevated metabolic rates were consistent with the energy and synthetic needs of a lung during repair of ozone-induced damage.

Adaptation of a perfused rat hemidiaphragm preparation to the study of intermediary metabolism.

Glucose catabolism of a vascular perfused rat hemidiaphragm was determined at rest and during stimulation of the phrenic nerve with trains of either 5 (T5) or 15 (T15) pulses (20 msec intervals) per second. Tissues were perfused and bathed in HEPES-buffered medium containing 11 mM D-[U-14C, 5-3H]glucose, equilibrated with 100% O2. Resting glucose catabolism via the Emden-Meyerhof pathway was indicated by a 3H2O production rate per hemidiaphragm of 1.45 +/- 0.07 mumol/h, of which 47% was recovered as [14C]lactate with the remainder assumed to be metabolised by mitochondria. During the first 30 min of T5 and T15 stimulation, peak isometric tension declined from an initial value of 105 +/- 8 g by 54% and 79%, respectively. The resulting peak tensions of 48 and 22 g remained constant for the next 60 min. These tensions were associated with linear rates of 3H2O production of 2.93 +/- 0.41 and 2.84 +/- 0.25 mumol/h. Stimulation by T5 and T15 increased mitochondrial metabolism of glucose by 64% and 95%, respectively, with no significant alterations in lactate formation from either exogenous or endogenous sources. The results suggest that the initial decline in tension is due to fatigue of the fast anaerobic myofibers; whereas, the sustained force beyond 30 min is attributable to the mitochondrial-rich slow myofibers.

Rat lung benzo(a)pyrene metabolism following three days continuous exposure to 0.6 ppm ozone.

Changes in the extent and pattern of benzo(a)pyrene metabolism were investigated in lungs isolated from rats following ozone exposures that are associated with the proliferation of alveolar and bronchiolar epithelia. Radiolabel incorporation into metabolic products were determined at the end of 60 min perfusions with 50-55 nmol of [6-3H] [7, 10-14C] benzo(a)pyrene (BaP), which in unexposed lungs resulted in a total BaP utilization of 0.77 +/- 0.05 nmol [14C] BaP/h/lung, recovered bound to tissue macromolecules (12%), as tissue and perfusate ethyl acetate-soluble products (59%), and as perfusate water-soluble conjugates (29%). Total metabolism at the sixth position of the BaP molecule was indicated by a 3H2O production of 0.07 +/- 0.01 nmol BaP/h per lung, that resulted in the formation of quinones (33%), acid-hydrolysable (40%) and acid-resistant (27%) water-soluble products, indicated by 14C- minus 3H-labelling. Ozone-exposed lungs demonstrated an increased total [14C] BaP utilization to 3.05 +/- 0.05 nmol/h/lung. Although BaP metabolism to all products was increased, the proportion of metabolism involving the 6th position was enhanced from 10% to 25% of total BaP utilization, which was accounted for by relative increases in tissue retained quinones and in perfusate acid-hydrolysable conjugates. These data demonstrated that quinone formation represents a major pathway of lung polycyclic aromatic hydrocarbon metabolism that is greatly enhanced in lungs with proliferating epithelia associated with oxidant exposure.

Rat lung recovery from 3 days of continuous exposure to 0.75 ppm ozone.

The present study investigated the inflammatory responses and enzyme levels in lungs isolated from male Wistar rats after 3 d of continuous exposure to 0.75 ppm ozone and following 4 d of recovery in air. These times are associated with maximal proliferation of the alveolar type II epithelium and their subsequent transformation to new type I cells. Immediately following ozone exposure, bronchoalveolar lavage demonstrated neutrophil accumulation that was no longer present 4 d later. The number of lavaged macrophages was also found to be increased immediately following ozone exposure, and remained elevated at 4 d postexposure. Whole-lung determinations of key enzymes involved in energy generation (succinate oxidase) and maintenance of lung NADPH and reduced glutathione were corrected for changes in cell number, by use of lung DNA measurements. Immediately following ozone exposure succinate oxidase (SOX), glucose-6-phosphate (G6PD), and 6-phosphogluconate (6PGD) dehydrogenase activities per milligram DNA were significantly enhanced by 76%, 48%, and 21%, respectively. These data suggested that ozone-exposed lungs had cells with increased mitochondria and NADPH-generating capability consistent with the increased metabolic needs of a proliferating epithelium. At 4 d postexposure, only G6PD activity per milligram DNA remained higher by 22% than air-exposed controls. Although both glutathione reductase (GSSG-R) and peroxidase (GSH-Px) activities per lung were elevated in lungs immediately following exposure and 4 d later, when corrected for DNA only GSH-Px activity was significantly increased by 29% in lungs after the postexposure period. Lungs 4 d postexposure therefore had cells relatively enriched in G6PD and GSH-Px that might account for the increased ozone tolerance that has previously been associated with the formation of new type I epithelium.

A reversible model of acute lung injury based on ozone exposure.

In this study inflammatory responses were determined in rat lungs 0, 1, 3, and 8 days following single 2- and 4-hr exposures to 1.8 ppm ozone. Analysis of lavage fluid immediately following exposure demonstrated enhanced lactate dehydrogenase activity and decreased numbers of lavageable macrophages but no alterations in albumin content. Similar analyses at one day postexposure demonstrated 282% and 456% increases in albumin content and enhanced numbers of lavageable neutrophils from a control value of 0.01 +/- 0.01 to 0.27 +/- 0.10 and 0.78 +/- 0.11 million cells per lung for 2-hr and 4-hr exposures, respectively. The observed increased levels of albumin were also present at 3 days, at which time the number of lavageable neutrophils was not significantly different than control. At both one and 3 days postexposure, lavageable lymphocytes were significantly increased 10-fold from a control value of 0.03 +/- 0.01 million cells per lung. However, the number of lavageable macrophages was unaltered on day 1, but enhanced on day 3, giving values of 0.67 +/- 0.05 (control), 2.25 +/- 0.46 (2 hr), and 2.70 +/- 1.05 (4 hr) million cells per lung. By 8 days both inflammatory cell numbers and albumin levels had returned to control values. Since these data demonstrated different time courses for each inflammatory cell type, this reversible model of acute lung injury should be useful for establishing possible involvement of these cells in processes of lung injury.