The Role Of Caspase-2 In Neuronal Apoptotsis

Apoptosis is a genetically programmed mode of cell death that exists in all multicellular species. Its major function is to facilitate the survival of the organism by the controlled elimination of damaged or superfluous cells. Cycteinyl aspartate specific proteases, known as Caspases, are believed to be essential mediators of many of the pathways involved in executing the apoptotic program. Caspase have been widely implicatd as essential mediators of many types of neuronal apoptosis, including β -amyloid peptide -mediated cell death. However, considerable controversy exists in the literature as to what mechanism is involved.Caspase-2 is one special member of Caspases family. It shows structure and function similarity to both initiator Caspases and effector Caspases. Caspase-2 is involved in neuronal apoptosis. Further studies are necessary to understand the function and signal pathway of the effects of Caspase-2.In the present study, we have extensively studied the function of Caspase-2 in neuronal apoptosis in SH-Sy5Y cells overexpressing Caspase-2 and the effect of Caspase-2 overexpression on neuronal apoptosis induced by β -amyloid peptide.1. Caspase-2 induced SH-Sy5Y cells apoptosisPCR has been perfomed to clone rat Caspase-2 cDNA. The PCR product was subcloned into pcDNA3.1(+) vector after DNA sequence confirmation of the clone. The pcDNA3.1/Caspase-2 vector was transfected into SH-Sy5Y. The expression of Caspase-2 and Caspase-3 was determined by fluorescence real-time PCR. Enzymatic activity assay was performed to measure the activity of these Caspases. SH-Sy5Y cell apoptosis was determined by Hochest33342 staining and Annexin v-biotin staining. The results showed that Caspase-2 induced SH-Sy5Y cells apoptosis. Furthermore, both expression level and enzymatic activity of Caspase-3 were elevated in SH-Sy5Y cells overexpressing Caspase-2.2. β -amyloid peptide induced SH-Sy5Y cell apoptosisSH-Sy5Y cells were incubated with different concentrations of A 3 42 for 24h. The cell viability was measured by MTT assay. Apoptosis was analyzed with Hochest 33342 staining and Annexin v-biotin staining. The expression of Caspase-2 was determined by fluorescence real-time PCR. Enzymatic activity assay was performed to measure the activity of Caspase-3. The results showed that the viability ofSH-Sy5Y cells was significantly decreased after the treatment of A β_ 42. We have demonstrated that the level of Caspase-2 mRNA was increased significantly. Surprisely, Caspase-3 activity was decreased significantly after A β_ 42 treatment. 3.The effect of Caspase-2 overexpression on neuronal apoptosis induced by A β_ 42SH-Sy5Y cells overexpressing Caspase-2 was used to investigate the effects of A β_ 42 on neuronal apoptosis. Apoptosis was analyzed with Hochest 33342 staining and Annexin v-biotin staining. The expression of Caspase-2 was determined by fluorescence real-time PCR. Enzymatic activity assay was performed to measure the activity of Caspase-3. The expression of Caspase-3 was measured by Western blotting analysis. We has demonstrated that the apoptotic ratio of cells overexpressing Caspase-2 had no significant changes after treated with A β_ 42 for 24h. Furthermore, A β_ 42 suppressed the overexpression of Caspase-2 and down-regulated Caspase-3 expression and inhibited its activity.