Erythropoietin prevents PC12 cells from beta-amyloid-induced apoptosis via PI3K⁄Akt pathway

Background Several studies indicated that Erythropoietin (Epo) may provide remarkable neuroprotection in some neurological diseases. It also showed the significant decrease of Epo immunoreactivity in the cerebral cortex and hippocampus in aged rats, suggesting the role of Epo in the pathogenesis of age-related neurodegenerative diseases such as AD. Methods The protective effect of Epo was studied in differentiated PC12 cells treated with Abeta. The viability of the cells, the apoptosis of the cells and the level of Bax, Bcl-2, cleaved caspase-3 and cleaved PARP expression were detected by MTT, Hoechst 33258 staining and Western blotting respectively. Results 20 μM Abeta (25-35) could induce a decreased viability and a increased apoptosis in PC12 cell in a time-dependent manner. However, 20 μM Abeta (35-25) had no effect on cell viability and apoptosis. Western blot analysis also showed that Abeta(25-35) treatment could decrease the expression of Bcl-2 (P < 0.05) and increase the expression of Bax (P < 0.05), Cleaved casapase-3 (P < 0.05), and Cleaved PARP (P < 0.05). The pretreatment of Epo could effectively reverse all the above changes induced by Abeta(25-35) (P < 0.05). Furthermore, the protective effect of Epo could be blocked by PI3K inhibitor LY294002 (P < 0.05). Conclusions Epo prevented cell injuries in PC12 cells exposed to the Abeta(25-35) and this effect may depend on the PI3K⁄Akt pathway. Our study provided an important evidence for the potential application of Epo in the therapy of Alzheimer's disease.


Background
Apoptosis is a particular type of programmed cell death controlled by precise intrinsic genetic programme in order to regulate cell population. Among the mechanisms of cell death, apoptosis has been proposed to explain the cell loss observed in many neurodegenerative disorders including Alzheimer's disease (AD) [1][2][3]. AD is a neurodegenerative disorder of the central nervous system (CNS), which correlate with the appearance of neurofibrillary tangles (NFTs) and senile plaques (SPs) [4]. The major component of SPs is beta-amyloid peptide (Abeta), which is believed to be the most probable cause of AD [3,5]. Many studies have shown that Abeta can directly induce neuronal death via apoptosis [2,6,7].
Erythropoietin (Epo) was originally characterized as the principal regulator of erythropoiesis [8]. Many experimental studies have shown that both Epo and its specific receptor (erythropoietin receptor, EpoR) expressing in the CNS, provide remarkable neuroprotection in many neurological diseases [9][10][11][12][13]. Recent research has demonstrated significant decreases in Epo immunoreactivity in the cerebral cortex and hippocampus of aged rats [14] which suggested the role of Epo in the pathogenesis of age-related neurodegenerative diseases such as AD. Therefore, we studied the possible relationship between Epo and Abeta-induced cell apoptosis. In the present study, we observed that Abeta (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) peptide at 20-μM concentrations could induce apoptosis in PC12 cells and Epo could reverse these changes through PI3K/Akt signaling pathway. Our results identifed a potential molecular targets for AD therapy.

Hoechst 33258 staining
For Hoechst 33258 staining, cells were fixed with 4% paraformaldehyde. Cell nuclei were stained with fluorescent dye Hoechst 33258 (Sigma, St. Louis, MO) at a final concentration of 5 μg/ml in PBS, for 20 min at room temperature in a dark chamber, and then observed in a fluorescence microscope (OLYMPUS 1 × 70, Japan) and photographed.

Western blotting
The Western blotting analysis procedure was conducted as previously reported [16]. After the treatment, cells were washed twice with cold phosphate buffered saline and lysed on ice with cell lysis buffer(10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-X 100, 10% glycerol, 1 mM PMSF (made from a 0.3 M stock in DMSO), 60 μg/mL aprotinin, 10 μg/mL leupeptin, 1 μg/mL pepstatin) for 30 mininutes. The soluble fraction was obtained by centrifugation at 14000 g for 20 min at 4°C. The concentration of the protein was determined by the BCA assay (Pierce Biotechnology, Rockford, IL). Equal amounts of the protein (20 μg) were separated in an 8-10% SDS-polyacrylamide gel; the resolved proteins were electrotransferred onto PVDF or nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were subsequently blocked with 5% nonfat milk in TBST for 1 h at room temperature and incubated with appropriate concentrations of primary antibody (1:200 for Bax and Bcl-2 (Santa Cruz Biotechnology, Inc, CA, USA), 1:5000 for beta-actin (Sigma-Aldrich, St. Louis, MO), 1:1000 for Cleaved caspase-3 and PARP (Cell Signaling Technology, Beverly, MA)) at 4°C overnight. The membranes were then washed 3 times with TBST and probed with the corresponding secondary antibodies conjugated with HRP (Cell Signaling Technology, Beverly, MA) at room temperature for 1 h. After washing, the signals were developed using the ECL Advanced Western Blotting Detection kit (Amersham, UK). Band intensities were quantified by densitometric analysis by using an AxioCam digital camera (ZEISS, Germany) and the KS400 photo analysis system (Ver. 3.0).

Statistics
Data are expressed as mean ± standard deviation (S.D.) and were analyzed using SPSS 11.0 statistical software (SPSS Inc., Chicago, IL, USA). Each procedure was performed in duplicate in 3~5 independent experiments. Statistical analyses were performed using one-way ANOVA, followed by the two-tailed Student's t test. Multiple comparison tests were applied when appropriate, and statistical significance was assumed at P < 0.05.
Caspases are a family of cysteine proteases and are critical mediators of cell apoptosis, which play an important role in the apoptotic process [42]. Caspase-3 acts as an apoptotic executor, it can activate DNA fragmentation factor, which in turn activate endonucleases to cleave nuclear DNA, and ultimately leads to cell death [43,44]. Activation of caspase-3 appears to be a key event in execution of the apoptotic cascade in CNS diseases such as AD and Down's syndrome [45,46]. In this study, we also found 20 μM Abeta (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) exposure could induce an increase of Cleaved caspase-3 expression (Figure 5B), and Epo could effectively attenuate these changes ( Figure 5B).