Nitrosative stress and combinatorial stresses in the pathogen Candida albicans

[0664] Mitochondrial aspartyl-tRNA synthetase (DARS2) deficiency in mice S.A. Dogan*1, C. Pujol1, A. Trifunovic1 ,2 1 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Germany, 2Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institutet, Sweden Mitochondrial dysfunction plays a critical role in neurodegeneration, likely owing to the high-energy demand of the central nervous system and its sole reliance on oxidative metabolism. One of the recent diseases added to the ever-growing list of mitochondriarelated neurological diseases is ‘leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation'. LBSL is clinically characterized by slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction and is caused by mutations in the DARS2 gene, which encodes for the mitochondrial aspartyl-tRNA synthetase. To functionally characterize the effect of DARS2 deficiency in a tissue specific manner, we have generated a mouse model with conditional potential. Whole body DARS2 deficiency causes an early developmental arrest between embryonic day 7.5-8.5. In order to elucidate the role of defective mitochondrial translation due to complete loss of DARS2 gene, we are now focusing on three different models for tissue specific DARS2 deficiency: (1) in heart and skeletal muscle (Ckmm-Cre), (2) in adult forebrain neurons (CaMKIICre), and (3) in myelin producing oligodendrocytes and Schwann cells (inducible Plp-Cre). Loss of DARS2 by Ckmm-Cre mediated deletion causes dramatically shortened life span with a maximal longevity of 6 weeks and a gradual increase in heart/body weight Loxp/Loxp, +/Ckmm-cre mice show marked ratio. DARS2 reduction in levels of all assembled respiratory chain complexes, except Complex II. A progressive deterioration in steady-state levels of both mtDNA- and nucleus-encoded respiratory chain subunits, respiratory chain enzyme activities and mitochondrial ATP production rate in isolated mitochondria is apparent. Interestingly, we detected a severe mitochondrial dysfunction in both, heart and skeletal muscle of DARS2Loxp/Loxp, +/Ckmm-cre mice, contrary to other mitochondrial mutants generated using Ckmm-Cre where mice do not present skeletal muscle phenotypes. Our data suggest that major mechanism for skeletal muscle resistance to mitochondrial replication/transcription defects could be the increase in stability of mitochondrial transcripts.

Keywords: Mitochondrial aspartyl-tRNA DARS2, LBSL, mitochondrial dysfunction

synthetase,

doi:10.1016/j.freeradbiomed.2012.08.384 Posters - Nitric Oxide and its Interactions [0038] Nitrosative stress and combinatorial stresses in the pathogen Candida albicans A.T. Tillmann*1, B. Yun2, G. Loake2, M. Gustin3, N. Gow1, A. Brown1 1 University of Aberdeen, UK, 2University of Edinburgh, UK, 3Rice University, USA Candida albicans is a major fungal pathogen that causes frequent mucosal infections in otherwise healthy individuals, and life-threatening systemic infections in immune-compromised patients. The first line of defence against Candida albicans is the release of reactive nitrogen species (RNS), reactive oxygen species (ROS) and cationic fluxes by macrophages and neutrophils. As a result robust responses to RNS, ROS and cationic stresses play major roles in the virulence of C. albicans. This fungus is exposed to all of these stresses simultaneously following phagocytosis. Consequently in this study we examined the responses of this pathogen to combinations of nitrosative, oxidative and cationic stress. The roles of the Cta4-Yhb1, Cap1, Hog1 and Mkc1 signalling pathways, which activate responses to individual nitrosative, oxidative, cationic and cell wall stresses, respectively, were examined. The sensitivity of null mutants to individual and combinatorial stresses was examined, and the activation of these pathways was confirmed by assaying the phosphorylation levels of the key protein kinases. Further evidence of the pathway activation was provided by transcription factor localisation and the expression of downstream target genes. We show that combinatorial nitrosative, oxidative and/or cationic stresses exert unexpected effects upon the classical stress signalling pathways. The relevance to C. albicans infections is discussed. Keywords: Nitrosative stress, RNS combinatorial stress, signalling pathways

and

ROS,

doi:10.1016/j.freeradbiomed.2012.08.385 [0154] Release of reactive oxygen species and production of nitric oxide in the cultures of peripheral blood leukocytes of young athletes

S183