Elsevier

Brain Research

Volume 1662, 1 May 2017, Pages 57-64
Brain Research

Research report
Mutant presenilin2 promotes apoptosis through the p53/miR-34a axis in neuronal cells

https://doi.org/10.1016/j.brainres.2017.01.034Get rights and content

Highlights

  • Mutant PS-2 activates p53 and miR-34a expression in PC12 cells.

  • Mutant PS-2 promotes PC12 cell apoptosis through triggering the p53/miR-34a axis.

  • Mutant PS-2 reduced the expression of miR-34a target gene Sirt1 and Bcl-2.

Abstract

Neurodegenerative disorders have attracted attention in last decades due to their high incidence in the world. The p53/miR-34a axis triggers apoptosis and suppresses viability in multiple types of cells, but little is known about its role in neurodegenerative diseases. In this study, we showed that presenilin (PS)-2, a major gene associated with familial Alzheimer’s disease (AD) could trigger the apoptosis through the p53/miR-34a axis in PC12 cells. First we found that PC12 cell viability was downregulated by PS-2 and mutant PS-2 overexpression, especially by mutant PS-2 overexpression. Then, we established a mutant PS-2-overexpressing PC12 cell line and confirmed that mutant PS-2 induced not only p53 but also miR-34a expression. The transfection of miR-34a inhibitor reversed PS-2-induced effects on cellular viability and apoptosis. Mutant PS-2 overexpression promoted caspase-3 expression, reduced Sirt1 and Bcl-2 expression, all of which were miR-34a downstream genes related with cell apoptosis. Moreover, mutant PS-2 also activated the p53/miR-34a axis and induced apoptosis in AD transgenic mice brain. These results implied that mutant PS-2 might promote the apoptosis of neuronal cells through triggering the p53/miR-34a axis. Altogether our results provide a novel insight into neurodegenerative disease and deepen our understandings of AD pathogenic processes.

Introduction

Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders characterized by cognitive impairment, neocortical neurofibrillary tangles and neocortical neuritic amyloid plaques (Crews and Masliah, 2010, McAvinchey and Burns, 2009, Serrano-Pozo et al., 2011). Some apoptosis-related genes, might cause massive neuronal death and be considered as one of the most common characteristic in the AD brains (Behl, 2000, LeBlanc, 2005). Common genes involved in the signaling transduction of apoptosis were found including c-Jun N-terminal kinases (JNK), caspsase-3 and p53 (Hashimoto et al., 2003, Marchal et al., 2014). P53, a well-known tumor suppressor, is also associated with AD which is up-regulated in the central nervous system of AD patients and contributes to the cell death in brains (Hooper et al., 2007). Based on previous studies, the cause of neuron death in AD is rather complicated (Selkoe and Schenk, 2003, Wilquet and De Strooper, 2004).

In the recent decade, the identification of genetic mutations linked to familial AD and the development of engineered mouse models significantly contributed to advancing the understanding of AD. Mutations in the presenilin (PS) genes are the main causes of early onset familial AD. PS has been reported to regulate several apoptosis genes, including Bcl-2 and caspase-3 (Alberici et al., 1999, Miyoshi et al., 2009). Moreover, it has been confirmed that apoptosis is associated with AD neuropathology and that PSs (especially PS-2) are modulators of apoptosis (Monteiro, 2005). However, the molecular mechanism by which PS-2 induces neuron apoptosis remains unclear.

MicroRNAs (miRNAs) are a large family of highly conserved small non-coding RNAs which regulate diverse biological process by modulating gene expression at the posttranscriptional level (He and Hannon, 2004). Several studies have found that miRNAs play a crucial role in regulating differentiation, proliferation, and the death of cells in neurodegenerative disorder (Saito and Saito, 2012). Recently, independent neurological research laboratories have provided evidence for the up-regulation of a small family of six inducible, pathogenic miRNAs in AD: miRNA-7, miRNA-9, miRNA-125b, miRNA-146a, miRNA-155 and miRNA-34a (Bhattacharjee et al., 2014, Devier et al., 2015, Saba et al., 2014). In this study, we chose PS-2 (N141I) mutant transgenic mice and PC12 cells as in vivo and in vitro models respectively. We tested whether PS-2 might function as an upstream regulator of the p53/miR-34a axis and might promote the apoptosis in neuronal cells.

Section snippets

The effect of PS-2 mutation on the viability and apoptosis of PC12 cells

To investigate the effect of PS-2 mutation on PC12 cells, pcDNA3.1/PS-2/PS-2 mutant was transfected into PC12 cells, and then the cell viability and apoptosis was determined in response to PS-2 overexpression. PS-2 or PS-2 mutant overexpression was verified using Immunoblotting (Fig. 1A). Results from MTT assays suggested that cell viability was inhibited by PS-2 or PS-2 mutant overexpression, and more intensely inhibited by PS-2 mutant (Fig. 1B). Then cell apoptosis was determined by flow

Discussion

The suppressive role of p53/miR-34a in several kinds of cells has been extensively studied in recent years. It regulates cell cycle and apoptosis through transcriptional regulation of a variety of genes. The progress of AD is associated with the mutation of PS-1 or PS-2, which cause the altered expression of apoptosis related genes. PS-2 transgenic mice, especially in cases of mutant PS-2 transgenic ones also confirmed the involvement of PS-2 gene in the neurodegeneration of AD (da Costa et

Ethics statement

This study was performed in strict accordance with animal use protocols approved by the Committee for Ethics of Animal Experiments of Third Xiangya Hospital of Central South University. All efforts were made to minimize animal suffering.

Animal preparation and extraction of tissues

The PS-2 mutant (N141I) transgenic mice were purchased from the Model Animal Research Center of Nanjing University. PS-2 (N141I) mutant transgenic mice have higher Aβ42 levels compared with wild type PS-2 transgenic mice and they exhibit phenotypes that mimics

Acknowledgments

This project is supported by Hunan Provincial Science and Technology Foundation (2011WK3046), Hunan Provincial Natural Science Foundation of China (12JJ2051) and National Natural Science Foundation of China (81671398).

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