The Role Of DPP4 In Febrile Seizures And Its Underlying Mechanisms

Backgroud:Febrile seizures(FS)is one of the common abnormal syndromes in infants and young children aged from 5 months to 6 years,accompanied by high incidence rate of epilepsy in life later,but little is known about the molecular mechanisms in FS.Previous results showed Dipeptidyl peptidase-IV(DPP4)has been implicated in FS generation,yet precisely how DPP4 affects FS remains unclear.DPP4,which is known as an important member of the dipeptidyl peptidase family,is expressed widely in many organs and tissues,including the brain,liver.DPP4 degrades some substrates,such as Glucagon-like peptide-1(GLP-1)to activate intercellular signaling.Previous result revealed that DPP4 was expressed in hippocampus and cortex of rats,involving in development of neuronal diseases.Clarifying these mechanisms will contribute to better understanding the pathogenesis of FS and uncovering a previously unrecognized target for DPP4 in preventing the development of febrile seizures and and subsequent epilepsy.Objective:To identify the expression pattern of DPP4 in FS and explore the molecular mechanism by which DPP4 participates in FS developmentMethods:(1)Repetitive seizures were induced by hyperthermia and scored according to Racine criterion after DPP4 inhibitor sitagliptin i.c.v.injection in rats.qRT-RCR and western blot analysis was performed to examine the expression of DPP4 and GLP-1R after sitagliptin treatment in rats,and ELISA was used to examine the levels of GLP-1 and GABA in FS rat.Immunofluorescence was used to determine the distribution.(2)To further confirm these results,DPP4 expression was examined and its downstream signaling was determined after DPP4 inhibition by sitagliptin and siRNA in primary cultured neurons using western blot.First,the expression of DPP4 after hyperthermia induction and GLP-1R expression after DPP4 inhibitor sitagliptin and siRNA were examined.Next,the effect of DPP4 inhibition by sitagliptin on neuronal excitation was detected using patch clamp in primary cultured neurons.(3)To study the role of DPP4 in regulating GABAergic synaptic transmission in primary cultured neurons,pacth-clamp recording was performed to record the sIPSC and mIPSC in primary cultured hippocampal neurons expressing siRNA-DPP4-FAM or after sitagliptin treatment.Furthermore,the effect of GLP-1R inhibition by exendin9-39 and siRNA-GLP-1R-FAM on sIPSC and mIPSC was also examined using patch-clamp recording.(4)To study the modulation of TRPV1 activity by GLP-1R in primary cultured neurons,hippocampal neurons were transfected with either FAM-modified siRNA-GLP-1R or scrambled siRNA,The expression GLP-1R and TRPV1 was examined using western blot after transfection,and capsaicin-induced TRPV1 current was recorded using whole-cell patch-clamp in cultured neurons showing green fluorescence under an inverted fluorescence microscope.Results:(1)Expression of DPP4 mRNA and protein was significantly increased after hyperthermia induction in hippocampus of rats.Increased in GLP-1R expression and GLP-1 and GABA levels were observed after sitaglipstin treatment in rats.DPP4 immunofluorescent staining was observed in the neuronal membranes in the rat hippocampus.These results show that increased DPP4 expression is associated with FS generation.(2)DPP4 expression was significantly increased in primary cultured neurons submitted to hyperthermia.There was an increase in expression of GLP-1 R after sitagliptin treatment and DPP4 knockdown in cultured neurons.Patch clamp recording revealed that DPP4 inhibitor decreased neuronal firing activity.These data suggest that DPP4 inhibitor produces anti-seizure effect and that GLP-1R signaling is required for DPP4 regulation of FS.(3)DPP4 inhibition by sitagliptin and siRNA significantly enhanced GABA-mediated synaptic transmission by increasing the amplitude and frequency of spontaneous inhibitory post-synaptic currents(sIPSCs)and the frequency of miniature inhibitory post-synaptic currents(mIPSCs)without influencing the amplitude of mIPSCs.DPP4 inhibitor suppresses neuronal firing dependent on GLP-1R pathway.GLP-1R knockdown by siRNA and its antagonist exendin9-39 significantly decreased GABA-mediated synaptic transmission by reducing the amplitude and frequency of sIPSCs.These results show that DPP4 inhibition may act presynaptically to promote GABA release from presynaptic nerve terminals,and GLP-1R signaling is involved in DPP4 regulation of GABAergic neurotransmission.(4)A significant increase in TRPV1 expression was observed in cultured neurons transfected with siRNA-mediated GLP-1R knockdown.Results of patch clamp recording revealed that GLP-1R knockdown enhanced TRPV1 activity by increasing capsaicin(1?M)induced TRPV1 current amplitude,promoting presynaptic terminal GABA release.These data indicate that TRPV1 is required for GLP-1R modulation of GABAergic synaptic signaling.DPP4 modulates FS generation via the TRPV1 signaling.Conclusion:In this study,we have demonstrated that DPP4 expression was increased in hyperthermia induced seizures and DPP4 inhibition or knockdown attenuated seizures through activation of the GLP-1/GLP-1R signaling pathway,further to enhance GABAertic synaptic transmission.Furthermore,TRPV1 was required for GLP-1R signaling regulation of GABA-mediated synaptic transmission,involving in DPP4 modulation of FS.It has demonstrated,for the first time,that the key role of DPP4 in regulation of FS,which may be a promising target in preventing the development of febrile seizures and subsequent epilepsy.