Peroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting ERK–Drp1-mediated mitochondrial fragmentation

doi:10.1016/j.freeradbiomed.2015.11.015

Free Radical Biology and Medicine

Volume 90, January 2016, Pages 184-194

https://doi.org/10.1016/j.freeradbiomed.2015.11.015 Get rights and content

Highlights

•
Prx5 expression was upregulated by AβO-induced oxidative stress.
•
Prx5 ameliorated AβO-induced phosphorylation of Drp1-Ser616.
•
AβO-induced phosphorylation of Drp1-Ser616 increased mitochondrial fragmentation.
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Prx5 ameliorated ERK phosphorylation by reducing AβO-induced oxidative stress.
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Apoptotic cell death was decreased by Prx5.

Abstract

Alzheimer's disease (AD), a neurodegenerative disorder, is caused by amyloid-beta oligomers (AβOs). AβOs induce cell death by triggering oxidative stress and mitochondrial dysfunction. A recent study showed that AβO-induced oxidative stress is associated with extracellular signal-regulated kinase (ERK)–dynamin related protein 1 (Drp1)-mediated mitochondrial fission. Reactive oxygen species (ROS) are regulated by antioxidant enzymes, especially peroxiredoxins (Prxs) that scavenge H₂O₂. These enzymes inhibit neuronal cell death induced by various neurotoxic reagents. However, it is unclear whether Prx5, which is specifically expressed in neuronal cells, protects these cells from AβO-induced damage. In this study, we found that Prx5 expression was upregulated by AβO-induced oxidative stress and that Prx5 decreased ERK–Drp1-mediated mitochondrial fragmentation and apoptosis of HT-22 neuronal cells. Prx5 expression was affected by AβO, and amelioration of oxidative stress by N-acetyl-l-cysteine decreased AβO-induced Prx5 expression. Prx5 overexpression reduced ROS as well as RNS and apoptotic cell death but Prx5 knockdown did not. In addition, Prx5 overexpression ameliorated ERK–Drp1-mediated mitochondrial fragmentation but Prx5 knockdown did not. These results indicated that inducible Prx5 expression by AβO plays a key role in inhibiting both ERK–Drp1-induced mitochondrial fragmentation and neuronal cell death by regulating oxidative stress. Thus, Prx5 may be a new therapeutic agent for treating AD.

Graphical abstract

Introduction

Alzheimer's disease (AD) is the most common neurodegenerative disorder and is characterized by the progressive impairment of memory and cognition. Typical pathological hallmarks of AD include accumulation of abnormally folded proteins such as extracellular amyloid-beta (Aβ) plaques within neurons [1], [2]. Aβ induces neuronal dysfunctioning and cell death in AD. The most toxic form of Aβ is Aβ42, which forms different-sized self-aggregates and amyloid fibrils [3]. Aβ oligomers (AβOs) are toxic to neurons and lead to their synaptic degeneration, which involves mitochondria. Aβ accumulates in the mitochondria of brains cells of patients and transgenic mice with AD [4], [5]. Accumulation of Aβ in mitochondria disrupts mitochondrial functions such as electron transport chain and calcium storage and mitochondrial dynamics [6], [7]. However, the specific molecular mechanism underlying the disruption of mitochondrial functions by AβOs remains unclear.

Mitochondria are essential intracellular energy-generating organelles with various cellular functions. Neurons greatly depend on mitochondria for energy supply [8]. Mitochondria are highly dynamic organelles that frequently fuse and divide (referred to as fusion and fission, respectively). Fusion and fission of mitochondria are important for many biological processes, including homeostasis of mitochondrial biogenesis, turnover, subcellular distribution, cell division, and apoptosis [9]. Fusion and fission of mitochondria allow the formation of interconnected mitochondrial networks and fragmented mitochondria, respectively [10]. Balanced mitochondrial dynamics is important for sustaining normal mitochondrial function [10]. Therefore, it is important that therapeutic strategies for treating neurodegenerative diseases should exert protective effects on mitochondria.

Mounting evidence suggest that Aβ increases the fission and decreases the fusion of mitochondria, thus promoting mitochondrial fragmentation and neuronal cell death [4], [11], [12]. AβOs reduce mitochondrial length, which is suggested to increase mitochondrial fragmentation [11], [13]. In mammalian cells, mitochondrial fission is controlled by dynamin-related protein 1 (Drp1) and mitochondrial fusion is controlled by mitofusin 1 and 2 and optic atrophy protein 1 [10]. Patients with AD show an abnormal balance in the levels of mitochondrial dynamics-related proteins [14]. In addition, phosphorylation of Drp1 is increased in the brains of patients with AD [11]. However, the mechanism underlying AβO-induced mitochondrial fragmentation is not understood.

Increasing evidence suggest that mitochondrial dysfunction is an early event and a prominent feature of AD progression. The brains of patients with AD show several molecular abnormalities, including oxidative stress and defective energy metabolism [15], [16]. Oxidative stress precedes Aβ accumulation in the brains of mouse models of AD and other cellular models [17]. Excessive oxidative stress results in immoderate mitochondrial fragmentation [18] and sustained activation of extracellular signal-regulated kinase (ERK, also known as p42/44 MAPK) [19]. Several recent studies suggest that fibrillary Aβ induces ERK activation, thus leading to progressive neuronal cell death [20], [21]. Excessive mitochondrial fragmentation also induces apoptosis [18]. However, the correlation between mitochondrial fragmentation and ERK is unclear.

Oxidative stress occurs when the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) surpasses the antioxidant activity of cellular and mitochondrial antioxidant enzymes [22], [23], [24]. Peroxiredoxins (Prxs) are a family of antioxidant enzymes that scavenge H₂O₂ and regulate the transduction of various cellular signals through intracellular oxidative signaling pathways [25], [26]. Prxs eliminate free radicals to protect various cells, including neurons [27]. Recombinant Prx5 exerts protective effects against excitotoxic brain lesions in newborn mice [26], [28]. Immunolabeling analysis performed in our previous study indicated that Prx5 was present in neurons and was evenly dispersed in the CA3 and CA2 regions of the hippocampus [29]. In particular, Prx5 is also known to eliminate peroxynitrite, a type of RNS [30]. However, the role of Prx5 in AD has been rarely studied to date.

In the present study, we investigated the role of Prx5 against AβO-induced toxicity that triggers apoptotic cell death through oxidative stress and mitochondrial fragmentation. Further, we confirmed whether Prx5 decreases mitochondrial fragmentation by regulating AβO-induced ERK activation. Our results showed that Prx5 may be a potential inhibitor of AβO-induced mitochondrial fragmentation and may exert protective effects against AβO-induced neuronal apoptosis.

Section snippets

Materials

N-acetyl-l-cysteine (NAC) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (MO, St. Louis, USA). U0126 was obtained from Santa Cruz Biotechnology Inc. (TX, Dallas, USA).

Oligomerization of Aβ₄₂

Aβ_1–42 was obtained from AnyGen (Jangseong, Korea) and was dissolved in DMSO to obtain a concentration of 5 mM. The dissolved Aβ_1–42 was sonicated for 5 min and was diluted in Opti-MEM (Gibco, NY, USA) to obtain a final concentration of 100 μM, which was stored at 37 °C until further use.

Cell culture and treatment

HT-22 cells were derived from

AβO was formed in HT-22 cells and induced apoptotic cell death

Western blotting confirmed the formation of AβO in HT-22 cells after incubation with 5 µM AβO. Lysates of HT-22 cells were incubated with the 6E10 antibody against Aβ. Lysates of cells not treated with AβO did not produce any signals. In contrast, lysates of cells treated with AβO had 4-kDa Aβ monomers, 8-kDa Aβ dimers, 12-kDa Aβ trimers, and 16-kDa Aβ tetramers (Fig. 1A). Recent studies have shown that AβO induces apoptotic cell death [3], [4], [33], [34], [35]. We performed a time-based MTT

Discussion

Oxidative stress is implicated in the progression of neurodegenerative diseases such as AD [17], [37], [43], [44]. Excessive oxidative stress is induced by two factors, increased ROS and RNS generation and decreased antioxidant enzyme capacity [22], [37], [39]. Antioxidant enzymes such as Prxs are involved in cell survival and cell proliferation [45], [46], [47], [48]. Previous studies indicate that Prx2 expression was increased in AD brain and overexpression of Prx1 and Prx6 decreases AD

Acknowledgments

This research was supported by grant (NRF-2014RA2A1A11054095) from the National Research Foundation of Korea grant funded by the government of the Republic of Korea, by Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research initiative program (KGM4611512), Republic of Korea.

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Citation Excerpt :
On the other hand, confirmation of the relationship between Parkin and oxidative stress was unexpected. AβO most likely increases the intracellular ROS level of HT-22 cells (Kam et al., 2019a; Shen et al., 2018; Kim et al., 2016), and excessive ROS generation from AβO treatment-induced ER stress (Kam et al., 2019a; Xie et al., 2017). The results showed that alteration of Parkin expression increased cell toxicity but not oxidative stress, even though ER stress increased (Fig. 3D and E).
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¹: These authors contributed equally to work.

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Original ContributionPeroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting ERK–Drp1-mediated mitochondrial fragmentation

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Oligomerization of Aβ42

Cell culture and treatment

AβO was formed in HT-22 cells and induced apoptotic cell death

Discussion

Acknowledgments

Trends Neurosci.

Exp. Neurol.

Neurosci. Lett.

J. Biol. Chem.

Biochim. Biophys. Acta

Eur. J. Med. Chem.

Trends Mol. Med.

Free Radic. Biol. Med.

Neurosci. Lett.

Brain Res.

Biophys. J.

Exp. Neurol.

Exp. Cell Res.

Biochim. Biophys. Acta

J. Biol. Chem.

Mol. Cell

Brain Res.

Neurobiol. Aging

FEBS Lett.

J. Biol. Chem.

Mol. Cell

Mol. Cell

Evidence of oxidative stress in Alzheimer's disease brain and antioxidant therapy: lights and shadows

Ann. N.Y. Acad. Sci.

Alzheimer's disease

N. Engl. J. Med.

The toxic Abeta oligomer and Alzheimer's disease: an emperor in need of clothes

Nat. Neurosci.

Mitochondria-specific accumulation of amyloid beta induces mitochondrial dysfunction leading to apoptotic cell death

PLoS One

Amyloid-beta interaction with mitochondria

Int. J. Alzheimers Dis.

Intracellular amyloid-beta in Alzheimer's disease

Nat. Rev. Neurosci.

A beta oligomers – a decade of discovery

J. Neurochem.

Regulation of mitochondrial transport in neurons

Exp. Cell Res.

Mitochondrial dynamics in cell death and neurodegeneration

Cell. Mol. Life Sci.

Mitochondrial fusion and fission in cell life and death

Nat. Rev. Mol. Cell Biol.

Original Contribution
Peroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting ERK–Drp1-mediated mitochondrial fragmentation

Oligomerization of Aβ₄₂