The Regulation Of Aβ Precursor Protein (APP) By Pur α And Egr1

Objective:Pur α is one of the three members of the purinerichelement binding protein(PUR) family, mainly binding to sequences like(GGNGGN)n. It plays a significant role in neural development,neurodegenerative diseases such as Fragile X Syndrome,DNA replication and repair,RNA transport and so on.Egr1 is one of inducible immediate early genes which is essential for the formation of long term memory,so it is very important in the neural plasiticity. APP is a single tranmemorane protein and is the precursor of A βwhich is the core of Alzheirmer disease genosis. The processing of APP into Aβis widely reported,but the trancriptinal regulation of APP is little understood.Our published data show that the Pur α negatively regulate the expression of APP,and we found that the APP promoter activity is up-regulated by Egr1. So our corrent work focus on the interaction of Pur α with Egr1 to regulate the expression of APP。Method: To elucidate the mechanisms by which Pur α and Egr1 interact to regulate the expression of APP, we screen the binding sites of Pur α and Egr1 on the APP promoter region with Genome Informatics method. We found that Pur α and Egr1 can all bind to the 63-82 bp downstream of the transcriptional starting site of APP promoter. To make sure the regulating mechanism, we clone the promoter of-100bp- +147bp region with or without the 63-82 bp sequence of the APP promoter. The fragments were then subcloned in to the PGL3 BASIC promoter region. We carried on the experiments listed below:(1) Western blot the APP expression in the Egr1 or Pur α overexpression u251 MG cell cultures.(2) Dual reporter assay with the promoter as described above.(3) CHIP assay to detect the physical interaction of Pur α and Egr1 with APP promoter.(4)EMSA to precisely describe the detailed physical interaction of Pur α and Egr1 with APP promoter region.Result: In our 1st part of work, we found that :(1) Pur α down regulated the expression of APP, wheals Egr1 up regulated the APP expression.(2) Pur α down regulated the promoter activity of-170-+147 and-100-+147 regions of APP promoter in the dual reporter assay. But when the 63-82 bps downstream of SST is deleted, both of Pur α and Egr1 failed to alter the promoter activity in the dual reporter assay.Conclusion: Egr1 and Pur α regulates the expression of APP by competitively occupying the same binding site. Object: Status epilepticus is defined as that the seizure continuing for at least 30 min. Status epilepticus is a neurological emergency with high mortality. But the molecular mechnisms contributing to Status epilepticus is little understood. Recent report shows that the GABA signaling may play a significant role in Status epilepticus. And many GABA receptors are reported to be involved in the genosis and evolution of Status epilepticus. But how the rate limiting enzyme of GABA GAD67 act in the Status epilepticus remain unkown. Here we are exploring the epigenetic upregulation of GAD67.Method:In the second part of our work, we carried on the experiments as below(1) We induced seizure with Kainic Acid to western blot the expression of GAD67,G9 a and histone 3 lysine 9 dimethylation.(2) We treated the cultured neurons with G9 a inhibitor and blot the GAD67 expression and detect the m RNA expression.(3) We detect the physical interaction of G9 a with GAD67 promoter region by CHIP assay.Result: In our 2nd part of our work, we found that:(1):Seizures induced by Kainic Acid is sufficient to down regulate H3K9 dimethylation via G9 a and up regulate the expression of GAD67.(2) Inhibition of G9 a by BIX01294 up regulated the expression of GAD67.(3) G9 a can bind to the promoter region of GAD67.Conclusion: For the second part of our work, seizures up regulate the expression of GAD67 via changing the H3K9 methylation status of their promoter regions.