Tunicamycin-induced unfolded protein response in the developing mouse brain

Highlights

• Tunicamycin caused a development-dependent UPR in the mouse brain.

• Immature brain was more susceptible to tunicamycin-induced endoplasmic reticulum stress.

• Tunicamycin caused more neuronal death in immature brain than mature brain.

• Tunicamycin-induced neuronal death is region-specific.

Abstract

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, resulting in the activation of the unfolded protein response (UPR). ER stress and UPR are associated with many neurodevelopmental and neurodegenerative disorders. The developing brain is particularly susceptible to environmental insults which may cause ER stress. We evaluated the UPR in the brain of postnatal mice. Tunicamycin, a commonly used ER stress inducer, was administered subcutaneously to mice of postnatal days (PDs) 4, 12 and 25. Tunicamycin caused UPR in the cerebral cortex, hippocampus and cerebellum of mice of PD4 and PD12, which was evident by the upregulation of ATF6, XBP1s, p-eIF2α, GRP78, GRP94 and MANF, but failed to induce UPR in the brain of PD25 mice. Tunicamycin-induced UPR in the liver was observed at all stages. In PD4 mice, tunicamycin-induced caspase-3 activation was observed in layer II of the parietal and optical cortex, CA1–CA3 and the subiculum of the hippocampus, the cerebellar external germinal layer and the superior/inferior colliculus. Tunicamycin-induced caspase-3 activation was also shown on PD12 but to a much lesser degree and mainly located in the dentate gyrus of the hippocampus, deep cerebellar nuclei and pons. Tunicamycin did not activate caspase-3 in the brain of PD25 mice and the liver of all stages. Similarly, immature cerebellar neurons were sensitive to tunicamycin-induced cell death in culture, but became resistant as they matured in vitro. These results suggest that the UPR is developmentally regulated and the immature brain is more susceptible to ER stress.

Introduction

The endoplasmic reticulum (ER) is a subcellular organelle responsible for posttranslational protein processing and transport. Approximately one third of all cellular proteins are translocated into the lumen of the ER where posttranslational modification, folding and oligomerization occur. The ER is also the site for biosynthesis of steroids, cholesterol and other lipids. Cellular stress conditions, such as perturbed calcium homeostasis or redox status, elevated secretory protein synthesis rates, altered glycosylation levels and cholesterol overloading, can interfere with oxidative protein folding, leading to the accumulation of unfolded or misfolded proteins in the ER lumen. This causes ER stress and activates a compensatory mechanism, called the unfolded protein response (UPR) (Hetz et al., 2013, Wang and Kaufman, 2012). UPR attempts to relieve ER stress by two major pathways: the first is to halt the translation of unfolded proteins and enhance endoplasmic reticulum-associated degradation (ERAD) of unfolded or misfolded proteins; the second is to increase the expression of molecular chaperones to facilitate proper protein folding (Logue et al., 2013, Walter and Ron, 2011). However, when sustained or severe ER stress surpasses the capacity of UPR, apoptotic cell death occurs (Hetz et al., 2013, Logue et al., 2013).

ER stress and UPR participate in various physiological processes such as lipid and cholesterol metabolism, energy homeostasis, circadian function, cell surface signaling, development and cell differentiation (Hetz, 2012, Rutkowski and Hegde, 2010, Walter and Ron, 2011). ER stress and UPR are also involved in many human diseases and disorders, such as inflammation, metabolic disorders, cardiovascular diseases, diabetes, obesity and cancer (Hetz et al., 2013, Wang and Kaufman, 2012, Yamamoto et al., 2010). ER stress has been shown to play an important role in the pathogenesis of various neurological diseases (Cornejo and Hetz, 2013, DeGracia and Montie, 2004, Endres and Reinhardt, 2013, Scheper and Hoozemans, 2009, Vidal et al., 2011, Xu and Zhu, 2012) and have been implicated in neurodegenerative processes in brain ischemia (Tajiri et al., 2004), Alzheimer's disease (AD) (Katayama et al., 2004), Parkinson's disease (PD) (Chen et al., 2004, Silva et al., 2005, Smith et al., 2005), Huntington's disease (HD) (Hirabayashi et al., 2001) and amyotrophic lateral sclerosis (ALS) (Turner and Atkin, 2006).

The developing brain is particularly susceptible to various environmental insults, e.g., exposure to pollutants, infectious pathogens, heavy metals, drugs, alcohol, physical stress and malnutrition. These environmental factors often cause ER stress and induce UPR (Hettiarachchi et al., 2008, Ji, 2014, Kalinec et al., 2014, Ke et al., 2011, Kitamura, 2013, Oh et al., 2012, Pavlovsky et al., 2013, Qian and Tiffany-Castiglioni, 2003, Shin et al., 2007) and ER stress may account for some of their detrimental effects. However, the mechanisms underlying CNS damage caused by these environmental insults are complex; it may be mediated by the interplay of multiple factors, such as direct toxicity, oxidative stress or disruption of cellular metabolism. To evaluate the impact of ER stress on the developing brain, we need a model system which allows more specific induction of ER stress. Tunicamycin is an N-linked glycosylation inhibitor and is commonly used to induce ER stress experimentally. In this study, we evaluated tunicamycin-induced ER stress in the postnatal development of the mouse brain. We also studied tunicamycin-mediated neuroapoptosis.