Differential Expression Of Proteins Related To Organophosphorus-induced Delayed Neuropathy And Corresponding Mechanisms In Hens

Background and ObjectiveOrganophosphate-induced delayed neuropathy （OPIDN） is a well-recognizedserious neuropathy induced by organophosphorus compounds （OPs） during2–3weeksafter acute exposure. Tri-ortho-cresyl-phosphate （TOCP） is widely used as additive orsoftener and has been believed to be associated with OPIDN. The mechanism ofOPIDN is still unclear, so OPIDN has been increasingly concerned.Because enoughdoses of TOCP administered to the laboratory animals not only promoted the initiationof OPIDN but also induced immediate neurotoxic action at the same time, it is difficultto identify the biochemical changes resulted from acute toxicity with those from OPIDN.It has been documented that pretreatment of Phenylmethylsulfonyl fluoride （PMSF）prior to OPs administration could prevent the signs and symptoms of the delayedneuropathy in hens. The biochemical changes both related to OPIDN and acute toxicityoccur when only TOCP is administered to hens, but the biochemical changes related toacute toxicity occur alone when hens exposed to TOCP followed by PMSF. In thepresent study, to screen the changed protein related to OPIDN and help researchersfurther explore the molecule mechanism of OPIDN, Two-dimensional polyacrylamidegel electrophoresis （2D-PAGE） and mass spectrometry （MS） were used to investigatethe disturbance of proteins in the brain of hens in OPIDN. As the result of ourproteomic analysis, we found that glutamine synthetase （GS） was showed dramaticallydecreased expression in OPIDN. Because GS play an important role in intracellularCa²⁺homeostasis, we further studied the expression of GS by real time RT-PCR andELISA, and the expression of another protein glutaminase （GLS）, which were notidentified by our2D-PAGE but was thought to be closely related to the physiologicalfunction of GS. At last, we also examined the levels of glutamate （Glu） and glutamine （Gln）, and the concentration of intracellular Ca²⁺.Methods1. Animal model of OPIDN: The hens were randomly divided into four groups.TOCP group was treated with TOCP by gavage at a single dosage of1000mg/kg and750mg/kg respectively, and control group was given an equivalent volume vehicle bygavage while hens in the PMSF+TOCP group were subcutaneously injected with40mg/kg PMSF followed by1000mg/kg TOCP24h later.2. Neurological behavior scores: OPIDN neurological signs were assessed by asix-point graded scale.3. The ultrastructural alterations in OPIDN: The samples cut into50nm thicksections and examined by transmission electron microscopy.4. Two-dimensional electrophoresis and image analysis and Mass SpectrometryAnalysis.5. Bioinformatics analysis: The GO category and pathway analysis were carriedout statistical analysis tools of DAVID.6. Real-time RT-PCR was used to determination the expression of the proteinsrelated OPIDN and GLS.7. ELISA was used to determination the expression of GS and GLS.8. Measurement of the activity of GS and the level of Glu and Gln.9. Measurement of intracellular Ca²⁺level.Results1. Delayed neurotoxic symptoms: Abnormal gaits progressed in severity with timeand hens were nearly or completely paralyzed by the end of21-day experimental period.However, no clinical signs of delayed neuropathy were observed in hens of the othertwo groups during the whole experiment period.2. Effects of TOCP on morphological changes in OPIDN: The changes includedswelling and vacuolation of mitochondria, the fragmentation of microtubule andneurofilaments in axon, etc.3.2D-PAGE combined MS analysis of protein expression in hens:（1）2D-PAGE analysis of protein expression in hensIn the brain:102spots of the differentially expressed proteins closely related toOPIDN were found in the TOCP group on day5, which63spots were upregulated and39spots were downregulated. On day21,150protein spots were found, which81spotswere upregulated and69spots were downregulated. In the spinal cord:43spots of the differentially expressed proteins closely relatedto OPIDN were found in the TOCP group on day5, which24spots were upregulatedand19spots were downregulated. On day21,86protein spots were found, which46spots were upregulated and40spots were downregulated.（2） Identification of proteins closely related to the OPIDN in brains of hensIn the brain: The upregulated proteins were EIF5A2, DSTN, PSMA1and CKB.And the dowregulated proteins were GS, LDHB, HOMER1, VDAC2, Hsc70andGSTA.In the spinal cord: The upregulated protein was NEFM. And the dowregulatedproteins were HSPB1，LMW-PTP, ETFa, STMN1, MDH1, THAP5, MBP, SNCA andVAT1.（3） The results of GO category: The main GO categories are GO:0005737^cytoplasm, GO:0005739^mitochondrion, GO:0043244^regulation of proteincomplex disassembly, GO:0051493^regulation of cytoskeleton organization, GO:0006519^cellular amino acid and derivative metabolic process, GO:0030834^regulation of actin filament depolymerization and GO:0016491^oxidoreductase activity.（4） Gene expression of the identified proteinLDHB，HSPA8，CKB，PSMA1，STMN1，THAP5，MBP，NEFM，SNCA werechosen to study whether the differential level of proteins was related to the differentmRNA level and the results were consistent with those from2-DE.4. Impact of TOCP exposure on the expression of GS and GLS, and theglutamate–glutamine cycle:（1）Expressions of GS and GLS after TOCP exposed: the gene and proteinexpression of GS in the CNS of hens decreased significantly in the TOCP groupcompared to control group or to PMSF+TOCP group on day5. However, there was nosignificant difference in the expression of GS between control group and PMSF+TOCPgroup on day21, and there was no significant difference in the expression of GLSbetween control group and PMSF+TOCP group on day5or day21.（2） The activity of GS after TOCP exposed: The activity of GS in the CNS of hensdecreased significantly in the TOCP group compared to control group on day5.However, there was no significant difference in the expression of GS between controlgroup and PMSF+TOCP group on day21.（3） The levels of Glu and Gln after TOCP exposed: On day5, Glu level in CNS of hens was significantly increased in TOCP group compared to control and PMSF+TOCPgroups. There were no significant differences in the Glu level among the three groupson day21. In contrast, Gln level in CNS of hens was significantly in TOCP groupcompared to control and PMSF+TOCP groups. However, there was no significantdifference in the Gln level between control group and PMSF+TOCP group on day5orday21.（4） The level of the intracellular Ca²⁺in brains of hens: Intracellular level of Ca²⁺in the CNS of hens was significantly increased in TOCP group compared to controlgroup. However, there was no significant difference in the intracellular level of Ca²⁺inthe brains of hens among the three groups on day21.Conclusions1. Ten proteins were identified in the brain, which were EIF5A2, DSTN, PSMA1,CKB, GS, LDHB, HOMER1, VDAC2, Hsc70and GSTA.2. Ten proteins were identified in the spinal cord, which were NEFM, HSPB1，LMW-PTP, ETFa, STMN1, MDH1, THAP5, MBP, SNCA and VAT1.3. The results of GO category indicated that proteins related OPIDN were mainlyin cytoplasm and mitochondrion, and involved in regulation of protein complexdisassembly, regulation of cytoskeleton organization, regulation of actin filamentdepolymerization and oxidoreductase activity;4. The expression and the activity of GS were down regulation in the early stageafter TOCP exposure;5. The level of Glu was up regulation in the early stage after TOCP exposure;6. The level of Gln was down regulation in the early stage after TOCP exposure;7. The intracellular Ca²⁺homeostasis was up regulation in the early stage afterTOCP exposure.8. TOCP exposure might induce downregulation of GS and increase in glutamatelevels in the brains of hens in the early stage after administration.9. The increased extracellular glutamate level in brain of hens exposed to TOCPmay be associated with the downregulated expression of GS.