TRPA1 channel mediates organophosphate-induced delayed neuropathy

OBJECTIVE We want to investigate the mechanism of organophosphate-induced delayed neuropathy(OPIDN) and find appropriate therapeutic medicine.OPIDN,often leads to paresthesias,ataxia and paralysis,occurs in the late-stage of acute poisoning or after repeated exposures to organophosphate(OP) insecticides or nerve agents,and may contribute to the Gulf War Syndrome.METHODS FDSS Ca2^(+)-influx assays,single-cell calcium imaging and patch-clamp electrophysiology were the major testing techniques.Transfected HEK293 cells and dorsal root ganglion(DRG) neurons were used to evaluate the effects of compounds.Wild type and trpa1 knockout mice and adult hyline brown hens were used to evaluate the neuropathological damages caused by the OPs.Transmission electron microscopy imaging was used to observe the nerve injuries ultrastructurally.High-throughput screen for TRPA1 inhibitors was accomplished by Ion Works Barracuda(IWB) automated electrophysiology assay.RESULTS TRPA1(Transient receptor potential cation channel,member A1) channel mediates OPIDN.A variety of OPs,exemplified by malathion,activates TRPA1 but not other neuronal TRP channels.Malathion increases the intracellular calcium levels and upregulates the excitability of mouse DRG neurons in vitro.Mice with repeated exposures to malathion also develop local tissue nerve injuries and pain-related behaviors,which resembles the early symptoms of OPIDN.Both the neuropathological changes and the nocifensive behaviors can be attenuated by treatment of TRPA1 antagonist HC030031 or abolished by knockout of Trpa1 gene.In the classic hens OPIDN model,malathion causes nerve injuries and ataxia to a similar level as the positive inducer tri-ortho-cresyl phosphate(TOCP),which also activates TRPA1 channel.Treatment with HC030031 reduces the damages caused by malathion or TOCP.Duloxetine and Ketotifen,two commercially available drugs exhibiting TRPA1 inhibitory activity,show neuroprotective effects against OPIDN and might be used in emergency situations.CONCLUSION TRPA1 is the major mediator of OPIDN and targeting TRPA1 is an effective way for the treatment of OPIDN.

MLKL forms cation channels

OBJECTIVE To investigate how MLKL functions on the membrane and explore its electrophysiological characters and structure.METHODS The full-length human MLKL were expressed in SF21 cells and purified using glutathione-sepharose affinity chromatography.The currents of purified MLKL proteins were recorded in avoltage-clamp mode using a Warner BC-535 bilayer clamp amplifier.The currents were digitized using p CLAMP 10.2 software.HEK293 cells were cultured and transfected with MLKL plasmid.Cell viability was examined using the Cell Titer-Glo Luminescent Cell Viability Assay kit.RESULT MLKL forms cation channels that are permeable preferentially to Mg2+rather than Ca2+in the presence of Na+and K+.Moreover,each MLKL monomer contains five transmembrane helices:H1,H2,H3,H5 and H6 of the N-terminal domain which is sufficient to form channels.Finally,MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity.

Development of SV2A Ligands for Epilepsy Treatment:A Review of Levetiracetam,Brivaracetam,and Padsevonil

Epilepsy is a common neurological disorder that is primarily treated with antiseizure medications(ASMs).Although dozens of ASMs are available in the clinic,approximately 30%of epileptic patients have medically refractory seizures;other limitations in most traditional ASMs include poor tolerability and drug-drug interactions.Therefore,there is an urgent need to develop alternative ASMs.Levetiracetam(LEV)is a first-line ASM that is well tolerated,has promising efficacy,and has little drug-drug interaction.Although it is widely accepted that LEV acts through a unique therapeutic target synaptic vesicle protein(SV)2A,the molecular basis of its action remains unknown.Even so,the next-generation SV2A ligands against epilepsy based on the structure of LEV have achieved clinical success.This review highlights the research and development(R&D)process of LEV and its analogs,brivaracetam and padsevonil,to provide ideas and experience for the R&D of novel ASMs.