Effect Of Anti-apoptotic Protein Bcl-2 On Neurodegeneration Induced By Okadaic Acid

Apoptosis involves in progressive neurodegeneration in AD. Bcl-2 family plays important role in apoptotic signal transduction pathway. The family members are classified as two kinds according to their opposite functions, pro-apoptotic or anti-apoptotic activity. Bcl-2 and Bax are representative of anti-apoptotic and pro-apoptotic proteins, respectively. The anti-apoptotic activity of Bcl-2 associates closely with the pro-apoptotic activity of Bax. Occurrence of cell apoptosis is decided by the ratio of Bcl-2 versus Bax under certain physiological and pathological conditions. Okadaic acid (OA), a protein phosphatase inhibitor, induces microtubule-associated protein tau hyperphosphorylation both in vitro and in vivo, which is considered to associate with pathogenesis of neurodegeneration. Apoptosis is responsible for neurodegeneration induced by OA in vitro. It has also been found that apoptosis-like DNA double strand damage appeared in OA-induced neurodegeneration in vivo. In the present study, microinjection of OA into rat frontal cortex was used to explore the possible roles of Bcl-2-related apoptotic regulatory mechanism in OA-induced neurodegeneration process in vivo. Immunohistochemistry was applied to observe alteration of Bcl-2 and Bax expression following OA injection. Triple immuno-fluorescent labeling combined with cofocal laser scanning microscopic analysis was performed to illustrate the topographic relationship among tau hyperphosphorylation, Bcl-2 expression and Bax expression induced by OA. Furthermore, human SH-SY5Y neuroblastoma cells were applied to investigate the influence of exogenous Bcl-2 overexpression on apoptotic neuronal death induced by OA. The results are as follows:Experiment in vivo Effect of Bcl-2 on OA-induced neurodegeneration in rat brainBcl-2 immunostaining showed that (1) the number of Bcl-2 positive staining cells significantly increased at 12 h, peaked at 1 d, then decreased at 3 d after injection of 20 ng OA compared with the number of Bcl-2-positive cells after vehicleinjection (p<0.001); 12 h, 1 d and 3 d of 20 ng OA treatment significantly increased the number of Bcl-2-positive cells compared with 3 h, 6 h and 7 d of 20 ng OA treatment (12 h, p<0.01; 1 d, 3 d, p<0.05); (2) compared with vehicle injection, injection of, 20 ng, 50 ng and 100 ng OA significantly increased the number of Bcl-2-positive cells at 1 d (20 ng,p<0.01; 50 ng, 100 ng,p<0.05); compared with 10 ng OA injection, injection of 20 ng and 100 ng OA also significantly increased the number of Bcl-2-positive cells at 1 d (p<0.05)Bax immunostaining showed that (1) the number of Bax-positive cells also significantly increased at 12 h, peaked at 1 d, then decreased at 3 d after injection of 20 ng OA compared with the number of Bax-positive cells after vehicle injection (p<0.01); (2) injection of 50 ng and 100 ng OA tended to elevate the number of Bax-positive cells at 1 d, but not reach statistical level.Immunohistochemistry of adjacent brain slices from the same rat delineated that the distributional profiles of AT-8-, Bcl-2- and Bax-positive cells were similar. However, the distributional range and the number of these positive cells were obviously different from one another. Among them, the range of Bcl-2-positive cells was most widely and the number was largest, the range of Bax-positive cells was least widely and the number was smallest and these two indices of AT-8-positive cells were in the middle.Triple immuno-fluorescent staining delineated that Bcl-2 expression co-stained with AT-8 and Bax expression or only co-stained with AT-8 expression in the affected area more adjacent to OA injected site. But in the affected area less neighboring to OA injected site, only Bcl-2 immunoreactivity was observed, but neither AT-8 nor Bax immunoreactivity.Experiment in vitro Effect of Bcl-2 overexpression on OA-induced cell death in SH-SY5Y cellsOA decreased cell viability of SH-SY5Y cells in dose dependent manner. 60 nM, 80 nM and 100 nM OA significantly reduced cell viability after 24 h of treatment (60 nM, 80 nM,p<0.05; 100 nM,p<0.01). Hoechst staining showed that a lot of chromatin condensation and fragmentation typical of apoptosis appeared following 24 h of 80 nM OA treatment.The transfection efficiency of recombinant plasmid pEGFP-N1-Bcl-2 and empty plasmid pEGFP-N1 was 2.01% and 2.18% respectively. Exogenous Bcl-2-GFP induced by recombinant plasmid transfection mainly resided in peri-nucleus cell bodies, while Exogenous GFP induced by vector transfection located in cytosol, nuclei and processes. Hoechst staining showed that apoptotic Bcl-2 transfected cells were markedly less than apoptotic vector transfected cells after 24 h of 200 nM staurosporine (STS) exposure (p<0.01).Hoechst staining further revealed that apoptotic Bcl-2 transfected cells were markedly less than apoptotic vector transfected cells after 24 h of 80 nM OA exposure (p<0.05).Conclusion1. Microinjection of OA into rat frontal cortex upregulated Bcl-2 and Bax protein expression in affected brain area. Moreover, induction of Bcl-2 and Bax expression by OA had temporal change and dose response feature. The upregulation of these two apoptotic regulatory protein probably attributed to tau hyperphosphorylation stimulated by OA. In affected cortex by OA, AT-8, Bcl-2 and Bax expression co-stained or AT-8 and Bcl-2 expression co-stained, suggestive of Bcl-2 family actively participated in OA-induced neurodegeneration process.2. Exogenous Bcl-2 overexpression partially inhibited OA-induced apoptosis in SH-SY5Y neuronal cells, suggesting that Bcl-2 related apoptotic regulatory mechanism involved in OA-induced neurodegenerative pathway and that Bcl-2 had neuronal protective role in it.3. Taken together, the results in vivo and in vitro indicated that Bcl-2 related apoptotic regulatory mechanism participated in OA-induced neurodegeneration and that the upregulation of Bcl-2 protein expression probably represented neuronal compensatory mechanism in response to OA toxicity to neuronal cytoskeleton.