Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

BACKGROUND: Numerous current studies have suggested that human telomerase reverse transcriptase (hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE: To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 (A beta(25-35)). DESIGN, TIME AND SETTING: The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS: AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Cdk5 and mouse anti-p16 monoclonal antibodies (Lab Vision, USA), and mouse anti-hTERT monoclonal antibody (Epitomics, USA), were used in this study. METHODS: (1) Recombinant adenovirus vectors, encoding hTERT (Ad-hTERT) and green fluorescent protein (Ad-GFP), were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. (2) Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 mu mol/L A beta(25-35) for 24 hours. Normal human embryonic cortical neurons treated with 10 mu mol/L A beta(25-35) for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with A beta(25-35). MAIN OUTCOME MEASURES: Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol (TRAP) ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS: Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups (P < 0.01). Compared with the control group, cell activity was significantly decreased (P < 0.05), and cell apoptotic rate, Cdk5 and p16 expression were significantly increased (P < 0.01) in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection (P < 0.05). Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased (P < 0.01). CONCLUSION: Expression of hTERT in human embryonic cortical neurons can relieve A beta(25-35)-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.