There are over 20 different isoforms of PLA2, including cytosolic, Ca+2-dependent, Ca+2-independent, secretory and mitochondrial isoforms. have been reported in several central nervous system (CNS) diseases, including Alzheimers disease (AD) and Parkinsons disease (PD); however, it remains largely unknown how the suppression of individual ETS complex function could lead to specific dysfunction in different cell types, resulting in distinct disease phenotypes. Our results suggest that the inhibition of each of the five ETS complexes might differentially regulate phospholipase activities within choline metabolic pathways in neuronal cells, which could contribute to the overall understanding of mitochondrial diseases. Keywords:NMR, Pattern recognition, Metabolomics, SH-SY5Y, Mitochondrial disease == 1 Introduction == Choline (Cho) and related lipid metabolites are vital for many aspects of cellular structure and function. In addition to being an important precursor for the synthesis of acetylcholine, a neurotransmitter, it is also required for the synthesis of phosphatidylcholine, sphingosylphosphorylcholine and other phospholipids, which are important for maintaining cell membrane integrity and facilitating signal transduction. Furthermore, Cho is an important methyl-group donor for the synthesis of S-adenosine methionine, an essential metabolite for the methylation of DNA and protein molecules. It is now clear that the deregulation of Cho metabolic pathways has a profound effect on cellular physiology. Abnormal Cho metabolism has been reported in several diseases where mitochondrial dysfunction is implicated (Farber et al. 2000;Michel et al. 2006). In many cases such alterations are characterized by an increase in the amount of total choline metabolite (tCho), which is increasingly utilized as one of the biomarkers for mitochondrial dysfunction (Jenkins et al. 2005;Meyerhoff et al. 1994). For instance, brain cells in AD show membrane defects associated with increased phospholipid turnover, which is characterized by a decrease in Cho and phosphorylcholine (PC) andan increase in glycerophosphorylcholine (GPC) and cytidine diphosphate choline (CDP-Cho) (Farber et al. 2000). The exact relationship between Cho metabolism and mitochondrial dysfunction has not been well understood, but there is increasing evidence that they are functionally connected. Clinical studies using MRS have reported elevations in both tCho and PC in numerous human solid tumors (Negendank 1992;Podo 1999) and in animal models including breast, ovarian (Aboagye and Bhujwalla 1999) and brain tumors (Barker et al. 1993). Mitochondrial functional adaptation to hypoxia has been well documented in various cancer models (Glunde et al. 2006). Recently, hypoxia inducible factor-1 (HIF-1) was shown to induce choline kinase, a key enzyme responsible for the synthesis of PC from Cho in metastatic cancer cells (Glunde et al. 2008). In addition to cancer, MRS studies have also reported changes in Cho metabolites in several CNS diseases associated with mitochondrial abnormalities. Higher tCho/creatine ratios were reported in the posterior mesial gray matter in AD versus control patients (MacKay et al. 1996). Similarly, a significant increase in tCho metabolites was also observed in basal ganglia (Clarke and Lowry 2000). Furthermore, tCho metabolites were found to Roquinimex be increased in a rodent model of Huntingtons disease (HD) (Jenkins et al. 1993). In order to understand the functional significance of Cho metabolism in mitochondrial dysfunction, we embarked on a metabolomics study of SH-SY5Y neuroblastoma cells treated with specific mitochondrial ETS complex inhibitors. This work stems from the fact that complex-specific ETS inhibitors have been shown to induce symptoms in animal models resembling those observed in different Roquinimex human CNS diseases (Brouillet et al. 1999;Kanthasamy et al. 1994;Tetrud and Langston 1989). For example, Complex I inhibition within dopaminergic neurons has been associated with the development of PD (Schmidt and Ferger 2001;Sherer et al. 2002); while a decrease in Complexes Roquinimex II/III activities have been seen in select populations of HD patients (Beal 1998). In addition, Complex IV defects have been observed in animal models of AD (Ohta and Ohsawa 2006). We report here that inhibition of mitochondrial ETS complexes alters Cho catabolic and anabolic pathways, producing NMR spectral patterns similar to those observed by MRS in vivo. In Roquinimex addition, distinct metabolomic patterns following inhibition of individual ETS complexes can be differentiated using1H-NMR analysis of cellular metabolites. In particular, we found that the relative levels of three Cho metabolites, Cho, PC and GPC, were among the Roquinimex most dramatically affected cellular metabolites Rabbit polyclonal to Tyrosine Hydroxylase.Tyrosine hydroxylase (EC 1.14.16.2) is involved in the conversion of phenylalanine to dopamine.As the rate-limiting enzyme in the synthesis of catecholamines, tyrosine hydroxylase has a key role in the physiology of adrenergic neurons. by the inhibition of individual ETS complex. Differential induction of a choline-metabolizing lipase, choline kinase, has been detected following the inhibition of specific ETS complexes. Finally, we discuss a possible relationship between Cho turnover and mitochondrial dysfunction via phospholipase modulation. == 2 Materials and methods == == 2.1 Materials == A.