Overexpression Of TDAG51 Induced Apoptosis In PC12 Cells

At first TDAG51 gene was cloned from T cells and considered as a apoptosis-associated gene and was essencial for the expression of Fas(CD95) in activated-T cells. In the TDAG51-/- T cells Fas (CD95) expression is deficient and T cells is resistant to activation-induced cell death(AICD).So it is considered that TDAG51 promote the apoptosis of activated-T cells and plays a very important role in the immune homeostasis. Expression of TDAG51 gene almost exists in every tissue in our body, but how TDAG51 gene functions is still not very clear. Now study on TDAG51 gene catches more people's attention. TDAG51 protein is rich in PQ(praline-glutamine) motif at the C terminal. It's reported that protein rich in PQ motif probably promote the apoptosis of hippocampal and septal cells independently of Fas. Some study group sort TDAG51 gene as a anti-oncogene to a gene family including TSSC3 and Tih gene. And several studies have identified the abundant expression of TDAG51 gene in brain tissues. But it is not clear whether TDAG51 still acts as an apoptosis-promoting factor in brain tissues and as regard as this there are few study papers.Abnormal apoptosis of neurons occurs under many circumstances and causes many neurodegenerative diseases, such as Alzheimer's disease and Parkinson' disease. Although we know the danger to our health , at present there are no treatments that can stop or reverse the inexorable neurodegenerative process. However, well understanding the cellular and molecular alternations that are responsible for the neuron' s demise may soon help in developing effective preventative and therapeutic strategies.Our experiments are designed to study the functions of TDAG51 gene in neuronal system using PC12 cells as a model in vitro to see whether TDAG51 gene still functions as an apoptosis-associated gene in PC12 cells as in T cells.Method: Transfect PC12 with expression clone pEGFP-TDAG51 or pEGFP-C₁ or nothing using transfection kit. PC12 cells transfected with pEGFP-TDAG51 are setted as test cells; PC12 cells transfected with pEGFP-C₁ or nothing are seen ascontrol cells. Western blotting is employed to detect the expression of TDAG51 after transfection. Phase contrast microscope is used to see the change of the morphology of cultured PCI 2 between test group and control. Use Hoechst33258 for staining the nucleus at 48h after transfection and then observe the nuclear morphological difference between tests and control using fluorescent microscope. Under high-magnification fluorescent microscope randomly select 10 sight fields and count the apoptotic cells. Then analyze the data statistically. At 18h after transfection change the medium with medium containing lOuM caspase-9- specific inhibitor and continue to incubate to 48h. Then analyze the change of apoptotic cells number statistically between tests and control.Result: Our results indicate that constitutive expression of TDAG51 exists, but in the test cells expression of TDAG51 is higher than that in control. Under phase-contrast microscope the test PC12 cells become round and lose neurite growth compared with control. Under fluorescent microscope the test PC12 cells stained with Hoechst33258 exhibit nuclear condensation, chromatin aggregation and nuclear fragmentation characteristic of apoptosis which are not seen in control. Statistic analysis results show that at 48h after transfection the test PCI 2 cells go to apoptosis significantly in contrast with control. Caspase-9 inhibitor partly inhibits PC12 cells apoptosis induced by overexpression of TDAG51.Conclusion: PC12 cells express TDAG51 gene constitutively. Overexpression of TDAG51 induced PC12 cells apoptosis. Intrinsic apoptosis pathway mediated by caspase-9 takes part in the PC12 cells apoptosis induced by overexpression of TDAG51.