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Paraptosis Triggers Mitochondrial Pathway-Mediated Apoptosis in Alzheimer's Disease

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Paraptosis Triggers Mitochondrial Pathway-Mediated Apoptosis in Alzheimer's Disease 804 EXPERIMENTAL AND THERAPEUTIC MEDICINE 10: 804-808, 2015 Paraptosis triggers mitochondrial pathway-mediated apoptosis in Alzheimer's disease DONG-PEI JIA*, SONG WANG*, BAO-CHAO ZHANG and FANG FANG Department of Neurological Rehabilitation, Nanyang City Center Hospital, Nanyang, Henan 473003, P.R. China Received March 8, 2015; Accepted May 8, 2015 DOI: 10.3892/etm.2015.2531 Abstract. In previous years, increasing evidence has indicated stages of AD, which may subsequently damage the mitochon- that paraptosis and mitochondrial-mediated apoptosis may dria and trigger mitochondrial pathway-mediated apoptosis. be associated with Alzheimer's disease (AD). However, the Thus, paraptosis may trigger programmed cell death directly, association between paraptosis and mitochondrial-mediated or indirectly through the regulation of Bcl-2 and Bax protein apoptosis, and the pathological processes underlying AD, expression. remain elusive. In the present study, the β-amyloid precursor protein gene, and the gene mutations PS1M146L and L286V, Introduction were transfected to an SH-SY5Y cell line to establish an AD cell model. Subsequently, an MTT assay was used to examine Alzheimer's disease (AD), known to be the leading cause the cell viability of the AD cell model, while a TUNEL assay of dementia in elderly populations in clinical practice, is a was employed to observe the number of positively stained neurodegenerative disease that is characterized by a progres- apoptotic cells. Cytoplasmic vacuolization was examined using sive loss of memory and cognitive function (1). The main light microscopy and images were photographed. Furthermore, characteristic of AD is the formation of extracellular senile western blot analysis was utilized to detect the expression of plaques, which include β-amyloid precursor protein (APP) and golden biomarkers of the mitochondrial pathway, including intracellular neurofibrillary tangles (2,3). The pathological Bcl-2 and Bax. The paraptosis inhibitor, cycloheximide, was mechanism underlying AD has been investigated for a number selected to treat the AD model cells in order to observe the of years; however, the essential cause of the disease has not association between paraptosis and mitochondrial-mediated been fully elucidated. In previous years, increasing evidence apoptosis. The results indicated that the decrease in the cell has indicated that the mitochondrial pathway may trigger the viability of the AD cells was initiated at 24 h, as compared with apoptosis of cells in AD patients (1‑3). the normal cells (P<0.05). TUNEL-positive stained cells were Previous studies have been indicated that AD could induce observed at 48 h, which was later compared with the cell death the formation of vacuoles (4), and the formed vacuoles may initiation. In addition, examination of cytoplasmic vacuoliza- be associated with cell death. Cell death primarily comprises tion using microscopy indicated that there were a small number two main types, including programmed cell death (PCD) and of paraptosis cells present at 24 h. The expression levels of passive (necrotic) cell death (5). Under physiological condi- Bcl‑2 was significantly decreased, while Bax was significantly tions, apoptosis and autophagy are the two main types of increased at 48 h. Furthermore, cycloheximide treatment was PCD. Furthermore, non-lysosomal vacuolated degeneration demonstrated to significantlyRETRACTED increase Bcl‑2 expression, while (also known as paraptosis) is a novel type of PCD, which is decreasing Bax expression (P>0.05). In conclusion, the occur- characterized by cytoplasmic vacuolization derived from rence of paraptosis was demonstrated in the early pathological endoplasmic reticulum and mitochondria swelling; however, there is a shortage of apoptotic morphology (4). In a preliminary study, brain cells were not found to be apoptotic prior to mitochondrial-triggered apoptosis under TUNEL analysis. Therefore, it was hypothesized that there Correspondence to: Dr Bao-Chao Zhang, Department of were a number of necessary processes prior to cells undergoing Neurological Rehabilitation, Nanyang City Center Hospital, mitochondrial pathway-mediated apoptosis. Thus, the aim of 312 Gongnong Road, Nanyang, Henan 473003, P.R. China the present study was to investigate the association between E-mail: [email protected] mitochondrial pathway meditated-apoptosis and paraptosis. *Contributed equally Materials and methods Key words: Alzheimer's disease, paraptosis, mitochondria-mediated apoptosis, cycloheximide, Bcl-2 Establishment of an AD cell model. The APP gene of (Swedish, Florida, London) strain and PS1M146L and L286V mutations were transfected into a SH-SY5Y cell line (American Type Culture Collection, Manassas, VA, USA). The transfec- JIA et al: PARAPTOSIS TRIGGERS MITOCHONDRIA-MEDIATED APOPTOSIS IN AD 805 tion of the above genes and mutations was performed using Hitachi, Ltd., Tokyo, Japan), as reported in a previous Lipofectamine 2000 transfection reagent (Invitrogen Life study (7). Technologies, Carlsbad, CA, USA). According to the identifi- cation of amyloid-β, the results demonstrated that the AD cell Statistical analysis. Every experiment was repeated a model had been established successfully. minimum of three times. The average value of the repeated All animal experiments were performed in accordance with data was expressed as the mean ± standard error of the mean. the guidelines of the Laboratory Animal Ethical Standards of Statistical comparisons were performed using the Student's Nanyang City Center Hospital (Nanyang, China). t‑test with SPSS 19.0 software (SPSS IBM, Armonk, NY, USA), where P<0.05 was considered to indicate a statistically MTT assay. AD cell models were seeded and cultured in significant difference. 96‑well plates at a density of 2x104 cells/ml in complete medium and incubated overnight. The cell viability was Results detected using an MTT assay (Sigma-Aldrich, St. Louis, MO, USA), as described previously (6). The MTT assay was Cell viability of the AD cell model and normal cells. In order performed at different time points, which included 24, 48 and to investigate the effects of double transgenesis on the viability 72 h. of the AD cell model, the cell viability was examined using an MTT assay. The MTT results indicated that a decrease in Sample treatment and western blot analysis. AD model cells the cell viability of the AD cells was observed initially at 24 h were homogenized gently in an isolation buffer containing (Fig. 1A). However, the cell viability of the AD cells at 48 and 0.25 M sucrose, 10 mM HEPES-NaOH, (pH 7.4) and 1 mM 72 h was significantly decreased when compared with the EDTA. Subsequently, the samples were homogenized in a normal cells (Fig. 1A; P<0.05). prechilled lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS, Apoptosis observations in the AD cell model. A TUNEL assay 1 mM PMSF, 10 mg/ml leupeptin, 1 mM Na3VO4 and 1 mM was performed to detect the rate of apoptosis in the AD cells NaF) overnight at 4˚C. The homogenates were collected and and normal cells. The results indicated that TUNEL-positive centrifuged at 12,000 x g for 20 min. The brain samples stained cells were observed at 48 h, which was later compared were subjected to 15% SDS-PAGE, and the obtained with the occurrence of cell death (Fig. 1B). In addition, the proteins in the gels were transferred to polyvinylidene number of TUNEL‑positive cells at 72 h was significantly difluoride membranes (Millipore Corporation, Temecula, CA, increased when compared with the number at 24 h (Fig. 1B; USA). The membranes were incubated with the following P<0.05). However, no TUNEL-positive cells were observed in primary antibodies, mouse anti-human Bax monoclonal the normal cell samples at any of the time points. (1:3,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse anti-human Bcl-2 monoclonal (1:4,000; Santa Cruz Paraptosis observations during the early growth stages of Biotechnology, Inc.) and mouse anti-β-actin (1:4,000; Santa the AD cells. In order to investigate the specific pathological Cruz Biotechnology, Inc.) at 4˚C overnight. Subsequently, the mechanism underlying the apoptosis of AD cells, the AD membranes were washed and incubated with a horseradish cells were visualized using light microscopy and images were peroxidase-conjugated secondary rabbit anti-mouse antibody photographed. The results revealed that there were a small (1:3,000; Santa Cruz Biotechnology, Inc.) for 2 h at room number of paraptosis cells observed under microscopy at the temperature. Immunoreactive bands were visualized with the 24-h time point (Fig. 2). Furthermore, the number of paraptosis SuperSignal West Pico Chemiluminescent Substrate (Pierce cells was shown to increase with an increase in culture time, Biotechnology, Inc., Rockford, IL, USA) using ChemDoc with significantly increased numbers observed at 48 or 72 h XRS with Quantity One software (Bio-Rad Laboratories, Inc., when compared with the number at 24 h (Fig. 2). However, Hercules, CA, USA). RETRACTEDthere were no paraptosis cells observed in any of the normal cell samples cultured at the different time points. Detection of cytoplasmic vacuolization. AD cells were cultured on 24-well plates. At the time points of 24, 48 and Changes in the expression of Bcl‑2 and Bax during the 72 h, the cells were visualized using a CX31 light microscope late growth stage of AD cells. The role of mitochondrial (Olympus Corporation, Tokyo, Japan) and images were pathway-mediated apoptosis in AD was also investigated by photographed. AD and normal cells were analyzed using analyzing the protein expression levels of Bcl-2 and Bax using a washout assay and phase contrast microscopy, according western blot analysis. The results indicated that the expres- to the protocol described in a previous study (4). Light and sion levels of Bcl‑2 at 48 h (Fig. 3A; P<0.05) and 72 h (Fig. 3; fluorescent microscopic images were selected and recorded P<0.01) were significantly decreased when compared with the from the representative fields of the cells plates at the different level at 24 h.
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