Strategy for searching potential partner genes of Drosophila melanogaster Sws gene

Aim. The Drosophila swiss cheese (sws) gene is a mammalian ortholog of neuropathy target esterase (NTE or PNPLA6). This protein is involved in the functioning of the nervous system and causes some kinds of polyneuropathy with the mechanism unknown. Genes-interactors of the sws gene should be found to investigate the cellular mechanism of SWS/NTE-dependent neuropathy formation and to find out what signaling pathways SWS is involved in. Methods. The search was done in the Drosophila database – Flybase, in the NCBI (Gene) database to search for orthologs, and the String web resource was used in the case of potential protein interactions. Results. Only four experimentally identified genes-interactors were listed in the Flybase. Based on the expression changes data of 28 genes at PNPLA6 silencing, a theoretical search for the corresponding orthologs in Drosophila was done. The in silico assay predicted 10 proteins that are probably functioning within one metabolic pathway. Conclusions. The methods applied gave us a wide list of genes that can interact with sws at different levels: from the expression pattern to the phenotype. Therefore, 25 potentially partner genes were selected to verify the genetic interaction of these genes in vivo. Keywords: gene swiss cheese, neuropathy target esterase, neurodegeneration, in silico analysis, genes interaction, Drosophila melanogaster.

SWS/NTE-dependent neuropathy is the model system to study neurodegeneration

Today there is many described neurodegenerative D. melanogaster mutants, which characterized by development of degenerative changes in brain. One of them are a swiss cheese (sws) gene mutants. Mutations in this gene causes apoptosis of neurons and hyperwrapping of their somas by the glial cells, reducing of life expectancy and decrease of locomotion. The sws gene is the ortholog of mammal’s neuropathy target esterase (NTE / PNPLA6). NTE is s neuronal, transmembrane protein, that possesses serinesterase activity, and can be the target for neurotoxic organophosphorus compounds activity. Mutations in PNPLA6 gene cause number hereditary neurodegenerative disorders, which nowadays are incurable. The search for therapeutic agents require use of model objects because researches on humans have both methodical and ethical limitations. During two last decades D. melanogaster has proven itself as a good model for study of neurodegenerative diseases. In this review, we described general characteristics of D. melanogaster gene sws, consequences of its mutations and provided evidences of high conservatism of gene product. Keywords: gene swiss cheese, neuropathy target esterase, neurodegeneration, brain, life span.

Changes of Neuropathy Target Esterase Affect Phospholipid Homeostasis in SK-N-SH Cells

Neuropathy target esterase (NTE), is a membrane protein located in the endoplasmic reticulum (ER). NTE has the activity of phospholipase B and can catalyze the deacylation of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) to glycerylcholine (GPC). It is phosphorylated and aged by organophosphorus compounds (OPs), that induce delayed neuropathy in humans and sensitive animals. Our previous study has reported that the disruption of ER phospholipid homeostasis caused by the NTE inhibition may contribute to the initiation of the organophosphate-induced delayed neurotoxicity (OPIDN), while it is unknown how the disturbed phospholipid homeostasis initiates OPIDN. It is difficult to change phospholipids in in vivo experiments. Therefore, an in vitro model is urgently needed to explain the role of phospholipid homeostasis disorders in OPIDN. In this study, we altered the expression of NTE in SK-N-SH cells and determined its phospholipid component by using HPLC-MS. Our results showed that the changes of NTE affected the levels of PC, sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylserine (PS), lysophosphatidylserine (LPS), phosphatidyl-glycerol (PG), and phosphatidylinositol (PI). Our results were consistent with the in vivo results. Furthermore, our findings indicate that the SK-N-SH cell model is a significantly useful method for the further research on how the changes of phospholipid homeostasis initiate the OPIDN, which is easier than the in vivo experiments in practice.

Characterization of the Interaction of Neuropathy Target Esterase with the Endoplasmic Reticulum and Lipid Droplets

Neuropathy target esterase (NTE) is an endoplasmic reticulum (ER)-localized phospholipase that deacylates phosphatidylcholine (PC) and lysophosphatidylcholine (LPC). Loss-of-function mutations in the human NTE gene have been associated with a spectrum of neurodegenerative disorders such as hereditary spastic paraplegia, ataxia and chorioretinal dystrophy. Despite this, little is known about structure–function relationships between NTE protein domains, enzymatic activity and the interaction with cellular organelles. In the current study we show that the C-terminal region of NTE forms a catalytically active domain that exhibits high affinity for lipid droplets (LDs), cellular storage organelles for triacylglycerol (TAG), which have been recently implicated in the progression of neurodegenerative diseases. Ectopic expression of the C domain in cultured cells decreases cellular PC, elevates TAG and induces LD clustering. LD interactions of NTE are inhibited by default by a non-enzymatic regulatory (R) region with three putative nucleotide monophosphate binding sites. Together with a N-terminal TMD the R region promotes proper distribution of the catalytic C-terminal region to the ER network. Taken together, our data indicate that NTE may exhibit dynamic interactions with the ER and LDs depending on the interplay of its functional regions. Mutations that disrupt this interplay may contribute to NTE-associated disorders by affecting NTE positioning.

Down-Regulation of Neuropathy Target Esterase in Preeclampsia Placenta Inhibits Human Trophoblast Cell Invasion via Modulating MMP-9 Levels

Background/Aims: Neuropathy target esterase (NTE, also known as neurotoxic esterase) is proven to deacylate phosphatidylcholine (PC) to glycerophosphocholine as a phospholipase B. Recently; studies showed that artificial phosphatidylserine/PC microvesicles can induce preeclampsia (PE)-like changes in pregnant mice. However, it is unclear whether NTE plays a key role in the pathology of PE, a pregnancy-related disease, which was characterized by deficient trophoblast invasion and reduced trophoblast-mediated remodeling of spiral arteries. The aim of this study was to investigate the expression pattern of NTE in the placenta from women with PE and normal pregnancy, and the molecular mechanism of NTE involved in the development of PE. Methods: NTE expression levels in placentas from 20 pregnant women with PE and 20 healthy pregnant women were detected using quantitative PCR and immunohistochemistry staining. The effect of NTE on trophoblast migration and invasion and the underlying mechanisms were examined in HTR-8/SVneo cell lines by transfection method. Results: NTE mRNA and protein expression levels were significantly decreased in preeclamptic placentas than normal control. Over-expression of NTE in HTR-8/SVneo cells significantly promoted trophoblast cells migration and invasion and was associated with increased MMP-9 levels. Conversely, shRNA-mediated down-regulation of NTE markedly inhibited the cell migration and invasion. In addition, silencing NTE reduced the MMP-9 activity and phosphorylated Erk1/2 and AKT levels. Conclusions: Our results suggest that the decreased NTE may contribute to the development of PE through impairing trophoblast invasion by down-regulating MMP-9 via the Erk1/2 and AKT signaling pathway.

Investigation of the Esterase Status as a Complex Biomarker of Exposure to Organophosphorus Compounds

Development of biomarkers of human exposures to organophosphorus compounds OPCs and their quantification is a vital component of a system of prediction and early diagnostics of OPC-induced diseases. Our study was focused on investigation of esterase status as a complex biomarker of exposure to OPCs and an aid in accurate diagnosis. We suggest that this complex biomarker should be more effective and informative than standard assays of plasma butyrylcholinesterase (BChE), erythrocyte acetylcholinesterase (RBC AChE), and lymphocyte neuropathy target esterase (NTE). It will help: 1) to assess an exposure as such and to confirm the nonexposure of individuals suspected to have been exposed; 2) to determine if the exposure was to agents expected to produce acute and/or delayed neurotoxicity; 3) to perform dosimetry of the exposure, which provides valuable information for medical treatment. To confirm this hypothesis, we have examined the changes in activity of blood AChE, NTE, BChE and carboxylesterase (CaE) 1 h after i.p. administration of increasing doses of three OPCs with different esterase profiles: the known neuropathic compound O,O-dipropyl-O-dichlorovinyl phosphate (C3H 7O)2P(O)OCH=CCl2 (diPr-DClVP) as the control compound and two model dialkylphosphates (C2H5O)2P(O)OCH(CF3)2 (diEt-PFP) and (C4H9O)2P(O)OCH(CF3)2 (diBu-PFP). The esterases assay was performed in hemolysed blood by spectrophotometric (AChE, BChE, CaE) and biosensor (NTE) methods. Analysis of the obtained dose-dependences for blood esterases inhibition showed that blood BChE and CaE were the most sensitive biomarkers, allowing detection of low doses. Inhibition of blood NTE and AChE can be used to assess the likelihood that an exposure to OPC would produce cholinergic and/or delayed neuropathic effects.