Regional specific activations of ERK1/2 and CDK5 differently regulate astroglial responses to ER stress in the rat hippocampus following status epilepticus
Graphical abstract
Introduction
Endoplasmic reticulum (ER) is one of organelles, which play roles in protein synthesis, lipid metabolism, detoxification and intracellular Ca2+ homeostasis (Martin-Jiménez et al., 2017). Disturbances in ER function result in ER stress due to the accumulation of unfolded proteins or changes in Ca2+ homeostasis (Nakagawa et al., 2000, Verkhratsky, 2005). Under mild ER stress conditions, the cells develop a protective mechanism, which is mediated by the expressions of chaperones, attenuation of protein translation and activation of ER-associated degradation through the activation of ER sensor proteins (Bertolotti et al., 2000). However, more aggressive conditions up-regulate the expression of pro-apoptotic factors and autophagic molecules leading to cell death (Nakagawa et al., 2000, Yorimitsu and Klionsky, 2007).
Astrocytes play a pivotal role in the maintenance of microenvironment of brain, metabolic homeostasis and blood brain barrier (BBB) integrity (Kim et al., 2010, Kim et al., 2010, Kim et al., 2013, Kimelberg and Nedergaard, 2010). After brain damage, astrocytes show hyperplasia, hypertrophy, increase in glial fibrillary acidic protein (GFAP) expression and proliferation, so-called reactive astrogliosis (Ridet et al., 1997, Di Giovanni et al., 2005). However, a number of studies have also demonstrated acute regional-specific astroglial responses following brain injury (Kang et al., 2006, Kim et al., 2008, Kim et al., 2010, Kim et al., 2010, Kim et al., 2011, Kim et al., 2014). Briefly, apoptotic astroglial death is observed in the molecular layer (not the hilus) of the dentate gyrus, while reactive astrogliosis is detected within the stratum radiatum of the CA1 region (Sugawara et al., 2002, Kim et al., 2008, Kim et al., 2011). We have reported that ER stress triggers reactive astrogliosis as well as astroglial apoptosis in a regional specific manner following status epilepticus (SE, a prolonged seizure activity; Ko et al., 2015a, Ko et al., 2015b). Although ER stress be most likely leading to regional specific astroglial responses, the epiphenomena/downstream effecters for ER stress and the mechanism of ER stress signaling in astroglial apoptosis have not been fully understood.
Recently, our previous study demonstrated that cyclin-dependent kinase 5 (CDK5) activation evokes reactive astrogliosis in the CA1 region as well as astroglial apoptosis in the molecular layer of the dentate gyrus. Furthermore, roscovitine (a CDK5 inhibitor) mitigate reactive astrogliosis and apoptotic astroglial death (Hyun et al., 2017). Thus, implication of CDK5 in ER stress would be one of the possible mechanisms for regional specific astroglial responses induced by SE, although the up-stream effector of CDK5 activation has been unknown.
Extracellular signal-activated protein kinase 1/2 (ERK1/2) is one of responsive down-stream molecules to ER stress, and participates the defensive effects against ER stress (Kurita et al., 2016). Indeed, ERK1/2 activation is necessary for induction of glucose-regulated protein 78 (GRP78), an important sensor of ER stress, that protects cells against apoptosis submitted to ER stress (Zhang et al., 2009). Furthermore, CDK5-serine (S) 159 site phosphorylation is regulated by ERK1/2 pathway (Zhang et al., 2014). However, the roles of ERK1/2 and CDK5 activation in the molecular mechanisms underlying ER stress-mediated reactive astrogliosis and astroglial damage have not been examined, and this issue is the focus of the present study.
Here, we demonstrate that tunicamycin-induced ER stress resulted in reactive astrogliosis-like events showing astroglial hypertrophy with the elevated ERK1/2 and CDK5 phosphorylations in the CA1 region. However, tunicamycin could not induce astroglial GRP78 expression, ERK1/2 phosphorylation and astroglial apoptosis in the molecular layer of the dentate gyrus. U0126 (an ERK1/2 inhibitor) reversed these effects of tunicamycin in the CA1 region. Roscovitine suppressed astroglial hypertrophy in the molecular layer of the dentate gyrus and the CA1 region, although it did not affect tunicamycin-induced ERK1/2 phosphorylation in the CA1 region. Furthermore, salubrinal (an ER stress inhibitor) abrogated activations of ERK1/2 and CDK5, and attenuated reactive astrogliosis in the CA1 region and astroglial apoptosis in the molecular layer of the dentate gyrus following SE. These findings indicate that ER stress may induce reactive astrogliosis via ERK1/2-mediated CDK5 activation in the CA1 region. In the molecular layer of the dentate gyrus, however, ER stress may participate in astroglial apoptosis through ERK1/2-independent CDK5 activation following SE.
Section snippets
ER stress leads to the distinct astroglial responses in the hippocampus
First, we applied tunicamycin to elucidate the role of ER stress in the properties of naïve astrocytes within the rat hippocampus. Since PERK and eIF2A are ER stress responsive molecules (Travers et al., 2000, Harding et al., 2003, Yamamoto et al., 2007), we investigated the effect of tunicamycin on PERK and eIF2A phosphorylations in astrocytes. In the CA1 region and the molecular layer of the dentate gyrus, astroglial PERK and eIF2A phosphorylation levels were undetectable in vehicle-treated
Discussion
ER stress is one of the cellular protective responses, so-called the unfolded protein response, which are mediated by induction of ER chaperones and suppression of translation activity (Rutkowski and Kaufman, 2004). However, the sustained ER stress triggers the cell death executive process including activations of pro-apoptotic factors such as C/EBP homologues protein (CHOP/GADD153) and caspases (Fang and Weissman, 2004). ER stress activates PERK, which phosphorylates the eIF2A thereby
Experimental animals and chemicals
Adult male Sprague-Dawley (SD) rats (weight 250–280 g) were used in the study. Animals were kept under controlled environmental conditions (23–25 °C, 12 h light/dark cycle) with free access to water and food. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Hallym University (Chuncheon, South Korea, Hallym R1-2013-107, 3rd December 2015 and Hallym 2018-3, 30th April 2018). All reagents were obtained from Sigma-Aldrich (USA), unless otherwise noted.
ER stress induction
Funding
This study was supported by a grant of National Research Foundation of Korea (NRF) grant (No. 2018R1C1B6005216). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author contributions
J-EK designed and supervised the project. D-SL and J-EK performed the experiments described in the manuscript. J-EK analyzed the data, and wrote the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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