Elsevier

Neurobiology of Aging

Volume 35, Issue 2, February 2014, Pages 421-430
Neurobiology of Aging

Regular article
Uncoupling protein 2 deficiency aggravates astrocytic endoplasmic reticulum stress and nod-like receptor protein 3 inflammasome activation

https://doi.org/10.1016/j.neurobiolaging.2013.08.015Get rights and content

Abstract

Astrocytes play crucial roles in determining the susceptibility to oxidative stress in the brain, and uncoupling protein 2 (UCP2) has been demonstrated to regulate reactive oxygen species (ROS) production. However, it is unclear whether UCP2 is expressed in astrocytes, and whether it participates in the regulation of astrocytic functions. Here we show that UCP2 knockout exacerbated dopaminergic neuron loss in a murine model of 1,2,3,6-methyl-phenyl-tetrahydropyridine (MPTP)–induced Parkinson's disease (PD), accompanied by overactivation of astrocytes. We further detected expression of UCP2 in primary cultures of mesencephalic astrocytes. UCP2 knockout increased intracellular ROS production and induced oxidative stress in response to l-methyl-4-phenylpyridinium (MPP+) treatment. Subsequently, UCP2 deficiency exacerbated endoplasmic reticulum (ER) stress, as evidenced by the upregulations of C/EBP homologous protein (CHOP), cleavage of caspase-12, and aggravated neuroinflammation via the activation of nod-like receptor protein 3 (NLRP3) inflammasomes in astrocytes. Collectively, our study indicates that UCP2 expressed in astrocytes modulates ER stress and neuroinflammation, and is crucial for the survival of dopaminergic neuron in the pathogenesis of PD. These findings gives us insights into the potential of UCP2 as a novel therapeutic avenue for PD treatment.

Introduction

Uncoupling protein 2 (UCP2) is located in the inner membrane of mitochondria and was discovered because of its sequence homology to the brown fat UCP1 (Mattiasson and Sullivan, 2006). The primary function of UCP2 is recognized to be the translocation of protons from intermembrane space to the matrix of mitochondria. Accumulating evidence supports that UCP2 does not contribute to adaptive thermogenesis (Azzu and Brand, 2010, Brand and Esteves, 2005, Yonezawa et al., 2009), but participates in the control of mitochondria-derived reactive oxygen species (ROS) (Echtay et al., 2001, Mailloux and Harper, 2011, Pi and Collins, 2010). For example, UCP2 knockout increased free radical production of macrophages in mice, whereas overexpression of UCP2 in vitro (immortalized β-cells) and in vivo (neurons) is protective against oxidative damage (Andrews et al., 2005a, Arsenijevic et al., 2000). In addition, oxidative stress induced by H2O2, lipopolysaccharide and irradiation can activate transcription, translation, and protein activity of UCP2 (Arsenijevic et al., 2000, Emre et al., 2007, Rousset et al., 2006). Thus, the major physiological function of UCP2 is to attenuate mitochondrial production of ROS. Activation and/or upregulation of UCP2 may function as an adaptive response to oxidative stress.

A growing body of evidence indicates that mitochondrial oxidative stress is critical in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD) (Bueler, 2009, Gao et al., 2003, Iravani et al., 2002, Knott et al., 2000, McGeer and McGeer, 2004, Van Laar and Berman, 2009). Oxidative stress may account for the nigral defect of complex I, which is highly vulnerable to oxidative damage. Inhibition of complex I leads to increased ROS formation in the pathogenesis of PD (Lemasters, 2007). It has been reported that UCP2 knockout increased the loss of nigral dopamine neurons in 1,2,3,6-methyl-phenyl-tetrahydropyridine/probenecid (MPTP/p) PD model mice because of increased ROS production (Andrews et al., 2005b). Excessive ROS not only induces oxidative injury but also results in endoplasmic reticulum (ER) stress. Increasing evidence demonstrates that ER stress, in conjunction with abnormal protein degradation, can contribute to the pathophysiology of PD (Egawa et al., 2011, Ryu et al., 2002). ER stress activates the unfolded protein response that induces ER-associated protein degradation as a self-protective mechanism, thereby leading to rescue or adaptive responses (Hara et al., 2011, Rao et al., 2004). Nevertheless, excessive ER stress long term may result in cell apoptosis or even necrosis. In addition to ER stress, production of numerous inflammatory factors, such as interleukin-1β (IL-1β), has been considered to play an important role in neurodegenerative disorders including PD (Teismann and Schulz, 2004, Wen et al., 2012). Emerging evidence indicates that accumulated ROS leads to glial inflammation, including the activation of nod-like receptor protein 3 (NLRP3) inflammasome, which modulates the production of mature IL-1β (Wen et al., 2012). However, it remains unknown whether UCP2 regulates ER stress and NLRP3 inflammasome-initiated inflammation by controlling ROS production in astrocytes.

Astrocytes are the primary site for modulation of redox homeostasis, and play crucial roles in determining susceptibility of neuron to oxidative stress in the brain (Simpson et al., 2010); it is unclear, however, whether UCP2 is expressed in astrocytes and regulates astrocytic functions. Therefore, the primary goal of the present study was to identify UCP2 expression in primary cultured astrocytes. Thereafter, UCP2 knockout mice were used to establish PD models to investigate whether UCP2 participates in the pathogenesis of PD via modulating astrocytic ER stress and NLRP3 inflammasome activation.

Section snippets

Animals and reagents

UCP2 knockout (KO) mice (on a C57/B6 background) were obtained from Prof. Zhang Chenyu (Zhang et al., 2001) (School of Life Science, Nanjing University). Mice were bred and maintained in the Animal Resource Centre of the Faculty of Medicine, Nanjing Medical University. Three-month-old male C57BL/6 mice were used as wild-type (WT) controls. The WT and KO mice were housed with free access to food and water in a room with an ambient temperature of 22 °C ± 2 °C and a 12:12-hour light/dark cycle.

UCP2 knockout aggravates loss of dopaminergic neuron in association with astrocytic activation in SNc of MPTP/p PD model mice

Stereological counts of SNc and VTA dopaminergic neurons, defined by TH staining, showed no difference in SNc and VTA TH cell number between mice of both genotypes. MPTP treatment decreased TH-positive cells by 46% in SNc of WT mice, but decreased TH positive cells by 74% in UCP2 KO mice (2-way ANOVA, MPTP: F1,20 = 1146.18, p < 0.001; genotype: F1,20 = 32.483, p < 0.001; interaction: F1,20 = 71.976, p < 0.001). Meanwhile, MPTP treatment reduced TH-positive cells by 26% in VTA of WT mice, and by

Discussion

Our study findings demonstrate that UCP2 knockout exacerbated the loss of DA neurons in the substantia nigra of MPTP/p PD model mice, accompanied with the overactivation of astrocytes and neuroinflammation. In addition, we found that primary cultured mouse astrocytes expressed UCP2, and that its deficiency enhanced the vulnerability of astrocytes in response to MPP+ treatment. Furthermore, UCP2 deficiency increased ROS production, exacerbated MPP+-induced ER stress as evidenced by CHOP

Disclosure statement

The authors declare no conflicts of interest.

Acknowledgements

The authors thank Prof. Zhang Chenyu (School of Life Sciences, Nanjing University, P. R. China) for his generosity in providing UCP2 knockout mice. This study was supported by the grants from the National Key Basic Research Program of China (Nos.2011CB504103 and 2009CB521906), the National Natural Science Foundation of China (No. 81030060 and 81202514) and the National Science and Technology Major Project (No. 2012ZX09304-001).

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