The Expression Of Dipeptidyl Peptidase 6 In Intractable Temporal Lobe Epilepsy Patients And Pilocarpine-Induced Rat Model

Objectives:Epilepsy is a disorder of the brain characterized by an enduring predisposition to generate epileptic seizures and by the neurobiologic, cognitive, psychological, and social consequences of this condition. Epilepsy is estimated to affect approximately 50 million people worldwide. Despite complying scrupulously with the prescribed regimen, up to 30% of patients with epilepsy still undergo failure of two antiepileptic drugs (AEDs), qualified as having "medically intractable epilepsy". Temporal Lobe Epilepsy (TLE) is the most common type of refractory epilepsy. However, the pathogenesis underlying the refractory TLE is not fully elucidated Dipeptidyl peptidase 6 (DPP6) is a single transmembrane glycoprotein. DPP6 plays an important role in the formation and stability of dendritic filopodia during early hippocampal neurons development. Here we investigate the expression of DPP6 in intractable temporal lobe epilepsy (TLE) patients and a rat model and discuss its role in the formation of intractable epilepsy, representing a novel therapeutic target for epilepsy.Methods:Twenty seven human brain samples were selected randomly from our epilepsy brain tissue bank. For comparison, we obtained 15 histologically normal temporal neocortex samples from individuals treated for severe brain trauma. DPP6 expression was detected by western blotting and immunohistochemistry. In addition, the dynamic changes of DPP6 expression were evaluated in the hippocampus and adjacent cortex of pilocarpine-induced epilepsy rats at different times (6h,24h,72h,1w,2w,1m,2 m) and controls by western blotting, immunohistochemistry and double-label immunofluorescence.Results:1 TLE patients and controlsclinical characteristics:In our study, the 27 patients in the TLE group had a mean disease course of 12.11±1.42 years.92.6% of patients had a disease course for more than 5 years, and 55.6% of patients had at least a 10-year clinical history. There was no significant difference in sex distribution between the TLE patients and the control subjects.Western blotting analysis showed the expression levels of DPP6 protein were higher in the samples of the TLE patients than in the control samples. The expression of DPP6 was normalized by calculating the ratio of the optical density (OD) of the bands for DPP6 and GAPDH. The mean OD ratio between the TLE group (0.86±0.04) and the control group (0.37±0.02) was statistically significant.Immunoreactivity assays for DPP6 were performed in the temporal neocortex of all the subjects. DPP6 was expressed in the membrane of neurons in temporal neocortex of control subjects and TLE patients, respectively. The mean OD values between the TLE group (0.50±0.02) and the control group (0.21±0.03) was statistically significant.2 Animal models:Western Blotting showed that in the temporal cortices of pilocarpine-treated rats, DPP6 increased along with the time and maintained at significant high levels at 30 days, while the expression of hippocampal DPP6 increased in acute stage and chronic stage, and decreased in latent stage compared to acute stage. The mean OD ratio of DPP6 was statistically significant between the control group and epileptic groups at each time point after seizures (P<0.001).Immunohistochemistry staining showed that DPP6 protein was mainly focused in the membrane of neurons in the hippocampus and adjacent cortex in epileptic and control rats. Strong DPP6 staining was observed in the temporal cortex and CA1 pyramidal cell layer in epileptic rats, as compared to the control rats. The changes of DPP6 expression level in epileptic and control rats showed a similar expression profile as was detected by western blotting analysis.Double immunofluorescence labeling of the temporal lobe and hippocampus from epileptic rats showed that DPP6 was co-expressed with MAP2 (microtubule-associated protein-2) in neurons. In contrast, DPP6 did not colocalize with GFAP (glial fibrillary acidic proteinin astrocytes. These results indicated that DPP6 protein was mainly expressed in the neurons but not in glial cells.Conclusions:1. The expression of DPP6 in the brain tissues of epileptic patients and pilocarpine-induced epilepsy rats were both higher than in the controls, respectively.2. DPP6 was found to be expressed mainly in the membrane of neurons, but not expressed in astrocytes.3. DPP6 may contribute to epileptogenesis and represent a novel therapeutic target.