Paraptosis triggers mitochondrial pathway-mediated apoptosis in Alzheimer's disease

Retraction in: /10.3892/etm.2021.10715

  • Authors:
    • Dong‑Pei Jia
    • Song Wang
    • Bao‑Chao Zhang
    • Fang Fang
  • View Affiliations

  • Published online on: May 29, 2015     https://doi.org/10.3892/etm.2015.2531
  • Pages: 804-808
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Abstract

In previous years, increasing evidence has indicated that paraptosis and mitochondrial-mediated apoptosis may be associated with Alzheimer's disease (AD). However, the association between paraptosis and mitochondrial‑mediated apoptosis, and the pathological processes underlying AD, remain elusive. In the present study, the β‑amyloid precursor protein gene, and the gene mutations PS1M146L and L286V, were transfected to an SH‑SY5Y cell line to establish an AD cell model. Subsequently, an MTT assay was used to examine the cell viability of the AD cell model, while a TUNEL assay was employed to observe the number of positively stained apoptotic cells. Cytoplasmic vacuolization was examined using light microscopy and images were photographed. Furthermore, western blot analysis was utilized to detect the expression of golden biomarkers of the mitochondrial pathway, including Bcl‑2 and Bax. The paraptosis inhibitor, cycloheximide, was selected to treat the AD model cells in order to observe the association between paraptosis and mitochondrial‑mediated apoptosis. The results indicated that the decrease in the cell viability of the AD cells was initiated at 24 h, as compared with the normal cells (P<0.05). TUNEL‑positive stained cells were observed at 48 h, which was later compared with the cell death initiation. In addition, examination of cytoplasmic vacuolization using microscopy indicated that there were a small number of paraptosis cells present at 24 h. The expression levels of Bcl‑2 was significantly decreased, while Bax was significantly increased at 48 h. Furthermore, cycloheximide treatment was demonstrated to significantly increase Bcl‑2 expression, while decreasing Bax expression (P>0.05). In conclusion, the occurrence of paraptosis was demonstrated in the early pathological stages of AD, which may subsequently damage the mitochondria and trigger mitochondrial pathway‑mediated apoptosis. Thus, paraptosis may trigger programmed cell death directly, or indirectly through the regulation of Bcl-2 and Bax protein expression.

Introduction

Alzheimer's disease (AD), known to be the leading cause of dementia in elderly populations in clinical practice, is a neurodegenerative disease that is characterized by a progressive loss of memory and cognitive function (1). The main characteristic of AD is the formation of extracellular senile plaques, which include β-amyloid precursor protein (APP) and intracellular neurofibrillary tangles (2,3). The pathological mechanism underlying AD has been investigated for a number of years; however, the essential cause of the disease has not been fully elucidated. In previous years, increasing evidence has indicated that the mitochondrial pathway may trigger the apoptosis of cells in AD patients (13).

Previous studies have been indicated that AD could induce the formation of vacuoles (4), and the formed vacuoles may be associated with cell death. Cell death primarily comprises two main types, including programmed cell death (PCD) and passive (necrotic) cell death (5). Under physiological conditions, apoptosis and autophagy are the two main types of PCD. Furthermore, non-lysosomal vacuolated degeneration (also known as paraptosis) is a novel type of PCD, which is characterized by cytoplasmic vacuolization derived from 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 were a number of necessary processes prior to cells undergoing mitochondrial pathway-mediated apoptosis. Thus, the aim of the present study was to investigate the association between mitochondrial pathway meditated-apoptosis and paraptosis.

Materials and methods

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 transfection of the above genes and mutations was performed using Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). According to the identification of amyloid-β, the results demonstrated that the AD cell model had been established successfully.

All animal experiments were performed in accordance with the guidelines of the Laboratory Animal Ethical Standards of Nanyang City Center Hospital (Nanyang, China).

MTT assay

AD cell models were seeded and cultured in 96-well plates at a density of 2×104 cells/ml in complete medium and incubated overnight. The cell viability was detected using an MTT assay (Sigma-Aldrich, St. Louis, MO, USA), as described previously (6). The MTT assay was performed at different time points, which included 24, 48 and 72 h.

Sample treatment and western blot analysis

AD model cells were homogenized gently in an isolation buffer containing 0.25 M sucrose, 10 mM HEPES-NaOH, (pH 7.4) and 1 mM EDTA. Subsequently, the samples were homogenized in a prechilled lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 mg/ml leupeptin, 1 mM Na3VO4 and 1 mM NaF) overnight at 4°C. The homogenates were collected and centrifuged at 12,000 × g for 20 min. The brain samples were subjected to 15% SDS-PAGE, and the obtained proteins in the gels were transferred to polyvinylidene difluoride membranes (Millipore Corporation, Temecula, CA, USA). The membranes were incubated with the following primary antibodies, mouse anti-human Bax monoclonal (1:3,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse anti-human Bcl-2 monoclonal (1:4,000; Santa Cruz Biotechnology, Inc.) and mouse anti-β-actin (1:4,000; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Subsequently, the membranes were washed and incubated with a horseradish peroxidase-conjugated secondary rabbit anti-mouse antibody (1:3,000; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Immunoreactive bands were visualized with the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL, USA) using ChemDoc XRS with Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Detection of cytoplasmic vacuolization

AD cells were cultured on 24-well plates. At the time points of 24, 48 and 72 h, the cells were visualized using a CX31 light microscope (Olympus Corporation, Tokyo, Japan) and images were photographed. AD and normal cells were analyzed using a washout assay and phase contrast microscopy, according to the protocol described in a previous study (4). Light and fluorescent microscopic images were selected and recorded from the representative fields of the cells plates at the different time points. All the experiments were performed a minimum of three times independently, and all the images were photographed in the same manner.

Transmission electron microscopy analysis of cytoplasmic vacuolization

The structure of the brain cells was observed utilizing a transmission electron microscope (H-600IV; Hitachi, Ltd., Tokyo, Japan), as reported in a previous study (7).

Statistical analysis

Every experiment was repeated a minimum of three times. The average value of the repeated data was expressed as the mean ± standard error of the mean. Statistical comparisons were performed using the Student's t-test with SPSS 19.0 software (SPSS IBM, Armonk, NY, USA), where P<0.05 was considered to indicate a statistically significant difference.

Results

Cell viability of the AD cell model and normal cells

In order to investigate the effects of double transgenesis on the viability of the AD cell model, the cell viability was examined using an MTT assay. The MTT results indicated that a decrease in the cell viability of the AD cells was observed initially at 24 h (Fig. 1A). However, the cell viability of the AD cells at 48 and 72 h was significantly decreased when compared with the normal cells (Fig. 1A; P<0.05).

Apoptosis observations in the AD cell model

A TUNEL assay was performed to detect the rate of apoptosis in the AD cells and normal cells. The results indicated that TUNEL-positive stained cells were observed at 48 h, which was later compared with the occurrence of cell death (Fig. 1B). In addition, the number of TUNEL-positive cells at 72 h was significantly increased when compared with the number at 24 h (Fig. 1B; P<0.05). However, no TUNEL-positive cells were observed in the normal cell samples at any of the time points.

Paraptosis observations during the early growth stages of the AD cells

In order to investigate the specific pathological mechanism underlying the apoptosis of AD cells, the AD cells were visualized using light microscopy and images were photographed. The results revealed that there were a small number of paraptosis cells observed under microscopy at the 24-h time point (Fig. 2). Furthermore, the number of paraptosis cells was shown to increase with an increase in culture time, with significantly increased numbers observed at 48 or 72 h when compared with the number at 24 h (Fig. 2). However, there were no paraptosis cells observed in any of the normal cell samples cultured at the different time points.

Changes in the expression of Bcl-2 and Bax during the late growth stage of AD cells

The role of mitochondrial pathway-mediated apoptosis in AD was also investigated by analyzing the protein expression levels of Bcl-2 and Bax using western blot analysis. The results indicated that the expression levels of Bcl-2 at 48 h (Fig. 3A; P<0.05) and 72 h (Fig. 3; P<0.01) were significantly decreased when compared with the level at 24 h. With regard to Bax protein expression, the levels were significantly increased at 48 h (Fig. 3A; P<0.01) and 72 h (Fig. 3; P<0.01) when compared with the expression level at 24 h.

Paraptosis inhibitor blocks mitochondrial pathway-mediated apoptosis

In order to confirm the induction of mitochondrial pathway-mediated apoptosis with paraptosis as the trigger, an inhibitor of paraptosis, namely cycloheximide (Sigma-Aldrich), was added to the cell cultures. The results revealed that cycloheximide treatment significantly increased the expression levels of Bcl-2, while decreasing Bax expression at 48 or 72 h when compared with the expression levels at 24 h (Fig. 3B; P>0.05).

Discussion

Previous studies have reported a number of mechanisms for the cell death and apoptosis of AD cells (24). However, the specific processes of apoptosis in AD have not been fully elucidated. Therefore, the present study investigated the details of the apoptotic pathway in AD pathology.

Paraptosis is a recently defined as a form of PCD (7); however, the underlying mechanism has not been fully investigated. To the best of our knowledge, there have been no previous studies investigating the interactions between paraptosis and other types of PCD in the pathogenesis of AD disease. Thus, the aim of the present study was to investigate the association between paraptosis and mitochondrial pathway-mediated apoptosis in the pathogenic processes underlying AD.

Previous studies have indicated that extensive cytoplasmic vacuolization may be associated with necrosis and necroapoptosis (8,9). Damage to the cell membrane is one of the characteristics of paraptosis (9). Paraptosis is defined as a form of cell death, with the characteristics of cytoplasmic vacuolization, cellular swelling, membrane blebbing and increased membrane permeability (10,11). Wang et al (12) demonstrated that paraptosis triggers retinal ganglion cell death via the production of reactive oxygen species, and hypothesized that paraptosis may be associated with mitochondrial damage.

Bcl-2 and Bax proteins are members of the Bcl family of proteins (13,14). A number of studies have reported that the Bcl-2 family proteins participate in AD, and are associated with cell apoptosis (15,16). In particular, Bcl-2 protein is known to inhibit a variety of apoptotic pathways, and in the majority of cases, Bcl-2 is considered to function through the inhibition of Bax protein (17). Therefore, the association between paraptosis and mitochondrial pathway-mediated apoptosis was investigated with the aim to further elucidate the pathogenic processes underlying AD.

In the present study, the cell viability of the AD cell model was found to significantly decrease when compared with the normal cells. However, the decrease in cell viability was initiated after culture for 24 h. In order to investigate the reasons underlying the decrease in cell viability for the AD cells, the extent of apoptosis was detected using a TUNEL assay. However, the TUNEL assay results indicated that apoptosis was initiated until the 48-h time point. Thus, the MTT and TUNEL results demonstrated that the apoptosis of the cells may occur prior to necrosis. Therefore, in the following experiments, the extent of paraptosis and mitochondrial pathway mediated-apoptosis was investigated.

The results of the present study indicated that paraptosis was initiated after 24 h of culture in the AD model cells. However, mitochondrial pathway-mediated apoptosis was initiated after 48 h of cell culture. Therefore, the PCD that occurs during the early stages of AD (24 h) was hypothesized to not be the result of mitochondrial mediated-apoptosis, but the result of paraptosis. In addition, the results indicated that paraptosis may trigger mitochondrial pathway-mediated apoptosis. In order to confirm this hypothesis, an inhibitor of paraptosis, namely cycloheximide (18), was applied as treatment to the AD cells. Following treatment of the AD cells with cycloheximide, the expression of Bcl-2 was shown to increase, while the protein expression of Bax protein was found to decrease, with results similar to those observed in the normal cells. In future studies, cycloheximide may be applied in animals to investigate its effects on the rate of paraptosis in vivo.

Therefore, according to the aforementioned results, a hypothesis was established with regard to the pathogenic process underlying AD. In the early pathological stages of AD, paraptosis occurs, which may damage the mitochondria with disease progression. Subsequently, the damaged mitochondria induce a decrease in Bcl-2 expression and an increase in Bax expression, which are key biomarkers for mitochondrial pathway-mediated apoptosis. Furthermore, the paraptosis inhibitor (8), cycloheximide, was demonstrated to block paraptosis, and consequently inhibit the changes in Bcl-2 and Bax protein expression (Fig. 4).

In conclusion, paraptosis was demonstrated to occur during the early pathological stages of AD, which subsequently damaged the mitochondria and triggered mitochondrial pathway-mediated apoptosis. Therefore, paraptosis was shown to trigger PCD directly, or indirectly through the regulation of Bcl-2 and Bax protein expression.

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Spandidos Publications style
Jia DP, Wang S, Zhang BC and Fang F: Paraptosis triggers mitochondrial pathway-mediated apoptosis in Alzheimer's disease Retraction in /10.3892/etm.2021.10715. Exp Ther Med 10: 804-808, 2015
APA
Jia, D., Wang, S., Zhang, B., & Fang, F. (2015). Paraptosis triggers mitochondrial pathway-mediated apoptosis in Alzheimer's disease Retraction in /10.3892/etm.2021.10715. Experimental and Therapeutic Medicine, 10, 804-808. https://doi.org/10.3892/etm.2015.2531
MLA
Jia, D., Wang, S., Zhang, B., Fang, F."Paraptosis triggers mitochondrial pathway-mediated apoptosis in Alzheimer's disease Retraction in /10.3892/etm.2021.10715". Experimental and Therapeutic Medicine 10.2 (2015): 804-808.
Chicago
Jia, D., Wang, S., Zhang, B., Fang, F."Paraptosis triggers mitochondrial pathway-mediated apoptosis in Alzheimer's disease Retraction in /10.3892/etm.2021.10715". Experimental and Therapeutic Medicine 10, no. 2 (2015): 804-808. https://doi.org/10.3892/etm.2015.2531