The dynamic changes of endoplasmic reticulum stress pathway markers GRP78 and CHOP in the hippocampus of diabetic mice

Highlights

• ER stress markers GRP78 and CHOP change dynamically in hippocampus of diabetic mice.

• Pro-survival chaperone GRP78 up-regulates in hippocampus soon after STZ injection.

• Proapoptotic transcriptional regulator CHOP increased following diabetes progress.

• Neuronal apoptosis in hippocampus of diabetic mice is associated with ER stress.

• ER stress plays an important role in the pathogenesis of diabetic encephalopathy.

Abstract

Diabetic encephalopathy has recently been recognized late complication of diabetes resulting in progressive cognitive deficits. Emerging evidence has indicated that endoplasmic reticulum (ER) stress-mediated apoptosis is involved in the pathogenesis of diabetic eye and kidney as well as non-diabetic neurodegeneration. However, there was little direct evidence for the involvement of ER stress in diabetic encephalopathy up to now. In the present work, we investigated the role of ER stress in the pathogenesis of diabetic encephalopathy. Our results have demonstrated the existence of ER stress in the hippocampus of streptozotocin (STZ)-induced diabetic mice. STZ injection i.p. rapidly induced up-regulation of the ER stress marker, the prosurvival chaperone glucose-regulated protein 78 (GRP78), as early as 6–24 h and persisted at least for up to 72 h in the hippocampus of mice, indicating the UPR activation soon after STZ administration. The increased expression of GRP78 in hippocampal cells is to relieve the ER stress. With the development of diabetes, the expression of GRP78 decreases while the expression of UPR-associated proapoptotic transcriptional regulator C/EBP homologous protein (CHOP) increases significantly in the hippocampal neurons of diabetic mice from 1 week after STZ administration to 12 weeks/the end of the study. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells in the hippocampus of diabetic mice were largely colocalized with NeuN- and CHOP-positive cells, indicating that the up-regulation of CHOP in hippocampal neurons of diabetic mice may promote neuronal apoptosis and account for the damaged learning and memory ability of diabetic mice. Therefore, our study provides evidence that ER stress may play an important role in the pathogenesis of neuronal degeneration and may contribute to cognitive dysfunction of diabetic encephalopathy.

Introduction

Endoplasmic reticulum (ER) is one of the major compartments for the biosynthesis of lipids as well as the proper synthesis, maturation and folding of proteins. Perturbation of ER homeostasis such as imbalance in ER calcium levels, accumulation of misfolded proteins in ER, glucose and energy deprivation, hypoxia can all cause ER stress (Rao et al., 2004). ER stress triggers an evolutionarily conserved cellular stress response termed the unfolded protein response (UPR) which intended to protect the cell against the toxic aggregated proteins. The activation of UPR results in an overall decrease in translation, increase clearance of misfolded proteins in ER lumen and increased levels of ER chaperones, including glucose-regulated protein 78 (GRP78), which consequently increases the protein folding capacity of ER (Kim et al., 2008, Xu et al., 2005). In general, cells may return to normal ER homeostasis under ER stress. However, cells may also continue toward apoptosis under prolonged ER stress if homeostasis is not restored. This apoptotic event is mediated by induced the expression of the UPR-associated proapoptotic transcriptional regulator C/EBP homologous protein (CHOP) and others, which participate in the control of cellular redox status and cell death (Oyadomari and Mori, 2004, Paz Gavilán et al., 2006). GRP78 and CHOP are the markers when ER stress occurred. The balance between the prosurvival chaperone GRP78 and CHOP drives the cell destiny following ER stress.

Diabetic encephalopathy is now accepted complications of diabetes mellitus (DM). The patients of diabetes have impairment of cognitive function (Sima, 2010, Umegaki et al., 2013). In type 1 diabetes, perturbed cognitive function involves intelligence, attention, processing speed, spatial learning, and long-term memory, whereas learning abilities per se appear to be spared (Brands et al., 2005). In type 2 diabetes, neuropsychological deficits tend to involve verbal and recent memory and information processing (Awad et al., 2004). Studies in streptozotocin (STZ)-induced diabetes in rats and mice have demonstrated neurobehavioral deficits using the Morris water maze paradigm, associated with impaired hippocampal long-term potentiation (Biessels et al., 1996, Gispen and Biessels, 2000, Zhao et al., 2003). STZ-rat was reported loss of neocortical neurons (Jakobsen et al., 1987). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive neurons as well as nucleosomal DNA fragmentation were increased in the hippocampus and frontal cortex of 8-month diabetic rats, in association with significant neuronal loss in hippocampal CA1 and CA2 (Li et al., 2002, Sima and Li, 2005), suggesting increased apoptotic activity and neuronal death with duration of diabetes. However, the precise mechanism(s) for the onset and progression of diabetes encephalopathy has still not been determined. Our previous study showed that the Ser199/Ser202 sites of microtubule-associated tau protein were hyperphosphorylated and the expression of beta-amyloid (Aβ) was increased in the hippocampus of STZ-diabetic mice, indicating that many proteins metabolism was disturbed in the brain of diabetic mice (Zhao et al., 2003, Shuli et al., 2001). Aβ and phospho-tau accumulation is also found in type 1 diabetic BB/Wor rats and type 2 diabetic BBZDR/Wor rats as well as type 2 db/db mouse models (Li et al., 2007, Kim et al., 2009), indicating that abnormal metabolism of proteins exists in the ER of the brain of diabetic mice which may disrupt ER function. ER becomes swollen in the diabetic brains under ultrastructural examination (Ai et al., 2010, Dheen et al., 1994), suggesting the disorder of the ER under diabetic condition. ER-stress mediated cell death may play an important role in the pathogenesis of diabetic encephalopathy. Emerging evidence has indicated that ER stress-mediated apoptosis is involved in the pathogenesis of diabetic eye and kidney as well as non-diabetic neurodegeneration, such as Alzheimer's disease and Parkinson's disease (Oyadomari and Mori, 2004, Cornejo and Hetz, 2013, Abisambra et al., 2013). However, there was little direct evidence for the involvement of ER stress in diabetic encephalopathy up to now.

In order to investigate the molecular basis of diabetic encephalopathy, we have studied whether ER stress is involved in the mechanism underlying the neurons death in the brain of diabetic mice induced by STZ. We have examined the cognitive performance of diabetic mice and the expression of ER stress markers GRP78 and CHOP in the hippocampus of diabetic mice sequentially in different time, as well as the correlation between CHOP expression and hippocampal neuronal apoptosis, by using immunohistochemisty, double immunofluorescence, Western blotting analysis and TUNEL staining, so as to understand the mechanism underlying diabetic encephalopathy.