Primary Research Of Caspase 3 In The Hippocampal Neuronal Apoptosis Of Epileptic Model

Epilepsy is one of the common neurological diseases with 6,000,000 patients in Chinese population.Temporal lobe epilepsy(TLE) is one of the most common subtype in partial epilepsy,accounting for 20-30% of all epileptic popularion.Most of TLE will progress to intractable and refractory to medical treatment, so it is harmful to the community.TLE is frequently combined with hippocampal atrophy which is considered to come out of hippocampal neuronal loss which partly in apoptosis and evidences suggest that caspase 3 may play a significant role in the mechanism by which neurons die following seizures induced by systemic kainic acid. Caspase 3 cleaves ICAD in neuronal apoptosis of epileptic model animal,which lead to DNA fragmentation.New genes as well as new proteins is necessary for cells to commit apoptosis,so it is possible that some new genes and new ptotein expressing specially in hippocampal neuronal apoptosis of TLE model, there may be novel substrates for caspase 3 that have never been found out before. Eukaryotic Translation Initiation Factor 2(eIF2) plays an important role in the initiation of translation.It was reported that the α subunit of eIF2 is cleaved by caspase 3 in apoptotic cell. The cleavage decreases the ability of eIF2 to regulate the translational initiation. Apoptotic process variates in different tissues under different conditions.It is still unclear wehether eIF2α is cleaved by caspase 3 in the apoptosis of hippocampal neurons in TLE model either. PART 1 Experimental research in eIF2α, the substrate of caspase 3 in hippocampal neuron of epileptic modelObjective: This investigation is aimed to explore the possible role of caspase 3 and eIF2α in the apoptosis of hippocampal neuron of epileptic model. Methods: Firstly,the kainic acid epileptic model is established; then TUNEL staining is used to detect the apoptosis of hippocampal neurons of the model rats. The Western blot is used to detect the change of eIF2α in hippocampal neuron of epileptic model rats. Results: The apoptotic rate reachs a peak at 72 hours after epileptic discharge. The cleavage of eIF2α by caspase 3 is detected in hippocampal neurons of epileptic model.Conclusion: Recurrently epileptic discharge leads neurons to apoptosis ; the cleavage of eIF2α by caspase 3 accompanies the apoptosis and may play important role in the neuronal apoptosis .PART 2 Cloning of PIAS1 and MIZ1, the novel substrates of caspase 3 in the hippocampus of epilepsy model rat . Objective: To screen the hippocampal cDNA library of epilepsy model rat for novel substrates of Caspase 3. Methods: Construct the yeast three-hybrid cDNA library with hippocampus of epileptic model rats. PCR from the library to get the small subunit of P12 and big subunit of P17 with point mutation of caspase 3. Subclone the two fragments into the Multiclone Sites (MCS) of pBridge vector for using yeast three-hybrid system to screen the caspase 3 interacting proteins. Verify the feasibility of the bait vector with eIF2α. Results: Constructed the yeast three-hybrid cDNA library with hippocampus of epileptic model rat successfully. Construction of the bait vector for caspase 3 in yeast three-hybrid system was accomplished. Obtained two novel substrates of caspase 3,PIAS1 and MIZ1.Discussions: The bait vector is feasible. With the bait vector we verify that caspase 3 can interact with eIF2α. Obtain PIAS1 and MIZ1 as novel substrates of caspase 3 in the hippocampal neuronal apoptosis of epileptic model rat. PIAS1 and MIZ1 may play important role in neuronal apoptosis after status epilepsy. We report the establishment of Kainic-Acid-induced epileptic rat model, the cleavage of eIF2α by caspase 3 and its correlation with hippocampal neuronal apoptotic process.And then we describe the use of yeast three-hybrid approach in screening for novel substrates for caspase 3.Our results make it possible for further understanding of the role that caspase 3 plays in the hipocampal neuronal apoptosis in TLE.