Neuroprotective role of calreticulin after spinal cord injury in mice.

Accumulating evidence suggests that endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are involved in the pathology of spinal cord injury (SCI). To determine the role of the UPR-target molecule in the pathophysiology of SCI, we analyzed the expression and the possible function of calreticulin (CRT), a molecular chaperone in the ER with high Ca2+ binding capacity, in a mouse SCI model. Spinal cord contusion was induced in T9 by using the Infinite Horizon impactor. Quantitative real-time polymerase chain reaction confirmed increase of Calr mRNA after SCI. Immunohistochemistry revealed that CRT expression was observed mainly in neurons in the control (sham operated) condition, while it was strongly observed in microglia/macrophages after SCI. Comparative analysis between wild-type (WT) and Calr+/- mice revealed that the recovery of hindlimb locomotion was reduced in Calr+/- mice, based on the evaluation using the Basso Mouse Scale and inclined-plane test. Immunohistochemistry also revealed more accumulation of immune cells in Calr+/- mice than in WT mice, at the epicenter 3 days and at the caudal region 7 days after SCI. Consistently, the number of damaged neuron was higher in Calr+/- mice at the caudal region 7 days after SCI. These results suggest a regulatory role of CRT in the neuroinflammation and neurodegeneration after SCI.

Brain injury triggers cell-type-specific and time-dependent endoplasmic reticulum stress responses.

The unfolded protein response (UPR) is a signal transduction network that responds to endoplasmic reticulum (ER) stress by coordinating protein homeostasis to maintain cell viability. The UPR can also trigger cell death when adaptive responses fail to improve protein homeostasis. Despite accumulating evidence suggesting that the UPR plays a role in neurodegenerative diseases and brain insults, our understanding of how ER stress is induced under neuropathological conditions is limited. Here, we investigated the cell- and time-specific patterns of the ER stress response after brain injury using ER stress-activated indicator (ERAI) mice, which enable monitoring of the UPR in vivo via increased fluorescence of a spliced XBP-1 protein fused with the green fluorescent protein (GFP) variant Venus. Following cortical stab injury of ERAI mice, the GFP signal and number of GFP+ cells increased in the ipsilateral cortex throughout the observation period (6 h to 7 days post-injury), confirming the induction of the UPR. GFP signals were observed in injured neurons early (from 6 h) after brain injury. However, non-neuronal cells, mainly endothelial cells followed by astrocytes, accounted for the majority of GFP+ cells after brain injury. Similar results were obtained in a mouse model of focal cerebral ischemia. These findings suggest that activation of the UPR in both neuronal and non-neuronal cells, especially endothelial cells and astrocytes, may play an important role in and could be a potential therapeutic target for acute brain injuries.

The ATF6ß-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity.

While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6ß remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6ß is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca2+-binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b-/- mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6ß deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b-/- neurons were recovered by ATF6ß and CRT overexpressions, or by treatment with Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b-/- and Calr+/- mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6ß-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca2+ homeostasis.

Abnormal social behavior and altered gene expression in mice lacking NDRG2.

N-myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family, has multiple functions in cell proliferation, differentiation, and stress responses, and is predominantly expressed by astrocytes in the central nervous system. Previous studies including ours demonstrated that NDRG2 is involved in various central nervous system pathologies. However, the significance of NDRG2 in neurodevelopment is not fully understood. Here, we investigated the expression profile of NDRG2 during postnatal brain development, the role of NDRG2 in social behavior, and transcriptome changes in the brain of NDRG2-deficient mice. NDRG2 expression in the brain increased over time from postnatal day 1 to adulthood. Deletion of NDRG2 resulted in abnormal social behavior, as indicated by reduced exploratory activity toward a novel mouse in a three-chamber social interaction test. Microarray analysis identified genes differentially expressed in the NDRG2-deficient brain, and upregulated gene expression of Bmp4 and Per2 was confirmed by quantitative PCR analysis. Expression of both these genes and the encoded proteins increased over time during postnatal brain development, similar to NDRG2. Gene expression of Bmp4 and Per2 was upregulated in cultured astrocytes isolated from NDRG2-deficient mice. These results suggest that NDRG2 contributes to brain development required for proper social behavior by modulating gene expression in astrocytes.

N-myc downstream-regulated gene 2 protects blood-brain barrier integrity following cerebral ischemia.

Disruption of the blood-brain barrier (BBB) following cerebral ischemia is closely related to the infiltration of peripheral cells into the brain, progression of lesion formation, and clinical exacerbation. However, the mechanism that regulates BBB integrity, especially after permanent ischemia, remains unclear. Here, we present evidence that astrocytic N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, may function as a modulator of BBB permeability following ischemic stroke, using a mouse model of permanent cerebral ischemia. Immunohistological analysis showed that the expression of NDRG2 increases dominantly in astrocytes following permanent middle cerebral artery occlusion (MCAO). Genetic deletion of Ndrg2 exhibited enhanced levels of infarct volume and accumulation of immune cells into the ipsilateral brain hemisphere following ischemia. Extravasation of serum proteins including fibrinogen and immunoglobulin, after MCAO, was enhanced at the ischemic core and perivascular region of the peri-infarct area in the ipsilateral cortex of Ndrg2-deficient mice. Furthermore, the expression of matrix metalloproteinases (MMPs) after MCAO markedly increased in Ndrg2-/- mice. In culture, expression and secretion of MMP-3 was increased in Ndrg2-/- astrocytes, and this increase was reversed by adenovirus-mediated re-expression of NDRG2. These findings suggest that NDRG2, expressed in astrocytes, may play a critical role in the regulation of BBB permeability and immune cell infiltration through the modulation of MMP expression following cerebral ischemia.

Ndrg2 deficiency ameliorates neurodegeneration in experimental autoimmune encephalomyelitis.

N-myc downstream-regulated gene 2 (NDRG2) is a differentiation- and stress-associated molecule that is predominantly expressed in astrocytes in the central nervous system. In this study, we examined the expression and role of NDRG2 in experimental autoimmune encephalomyelitis (EAE), which is an animal model of multiple sclerosis. Western blot and immunohistochemical analysis revealed that the expression of NDRG2 was observed in astrocytes of spinal cord, and was enhanced after EAE induction. A comparative analysis of wild-type and Ndrg2-/- mice revealed that deletion of Ndrg2 ameliorated the clinical symptoms of EAE. Although Ndrg2 deficiency only slightly affected the inflammatory response, based on the results of flow cytometry, qRT-PCR, and immunohistochemistry, it significantly reduced demyelination in the chronic phase, and, more importantly, neurodegeneration both in the acute and chronic phases. Further studies revealed that the expression of astrocytic glutamate transporters, including glutamate aspartate transporter (GLAST) and glutamate transporter 1, was more maintained in the Ndrg2-/- mice compared with wild-type mice after EAE induction. Consistent with these results, studies using cultured astrocytes revealed that Ndrg2 gene silencing increased the expression of GLAST, while NDRG2 over-expression decreased it without altering the expression of glial fibrillary acidic protein. The effect of NDRG2 on GLAST expression was associated with the activation of Akt, but not with the activation of nuclear factor-kappa B. These findings suggest that NDRG2 plays a key role in the pathology of EAE by modulating glutamate metabolism. Cover Image for this Issue: doi: 10.1111/jnc.14173.

Deletion of SERP1/RAMP4, a component of the endoplasmic reticulum (ER) translocation sites, leads to ER stress.

Stress-associated endoplasmic reticulum (ER) protein 1 (SERP1), also known as ribosome-associated membrane protein 4 (RAMP4), is a Sec61-associated polypeptide that is induced by ER stress. SERP1-/- mice, made by targeted gene disruption, demonstrated growth retardation, increased mortality, and impaired glucose tolerance. Consistent with high levels of SERP1 expression in pancreas, pancreatic islets from SERP1-/- mice failed to rapidly synthesize proinsulin in response to a glucose load. In addition, reduced size and enhanced ER stress were observed in the anterior pituitary of SERP1-/- mice, and growth hormone production was slowed in SERP1-/- pituitary after insulin stimulation. Experiments using pancreatic microsomes revealed aberrant association of ribosomes and the Sec61 complex and enhanced ER stress in SERP1-/- pancreas. In basal conditions, the Sec61 complex in SERP1-/- microsomes was more cofractionated with ribosomes, compared with SERP1+/+ counterparts, in high-salt conditions. In contrast, after glucose stimulation, the complex showed less cofractionation at an early phase (45 min) but more at a later phase (120 min). Although intracellular insulin/proinsulin levels were not significantly changed in both genotypes, these results suggest that subtle changes in translocation efficiency play an important role in the regulation of ER stress and rapid polypeptide synthesis.

The endoplasmic reticulum chaperone improves insulin resistance in type 2 diabetes.

To determine the role of the endoplasmic reticulum (ER) in diabetes, Akita mice, a mouse model of type 2 diabetes, were mated with either heterozygous knockout mice or two types of transgenic mice of 150-kDa oxygen-regulated protein (ORP150), a molecular chaperone located in the ER. Systemic expression of ORP150 in Akita mice improves insulin intolerance, whereas the exclusive overexpression of ORP150 in pancreatic beta-cells of Akita mice did not change their glucose tolerance. Both an insulin tolerance test and hyperinsulinemic-euglycemic clamp revealed that ORP150 enhanced glucose uptake, accompanied by suppression of oxidized protein. Furthermore, ORP150 enhanced the insulin sensitivity of myoblast cells treated with hydrogen peroxide. These data suggest that ORP150 plays an important role in insulin sensitivity and is a potential target for the treatment of diabetes.

Role of Herp in the endoplasmic reticulum stress response.

Application of differential display to cultured rat astrocytes allowed cloning of Herp cDNA. Although Herp was strongly induced by endoplasmic reticulum (ER) stress, it decayed rapidly consequent to proteasome-mediated degradation. To investigate the role of this molecule in terms of the stress response, Herp knockout cells were developed using F9 embryonic carcinoma cells. F9 Herp null cells were more vulnerable to ER stress compared with F9 wild-type cells. In the early period of ER stress (0-8 h after tunicamycin treatment), Herp null cells displayed enhanced ER stress signalling and stabilization of an endogenous ERAD substrate, compared with wild-type cells. In the intermediate period (8-20 h after tunicamycin treatment), Herp null cells displayed reduced ER stress signalling, whereas in the late period (20-40 h after tunicamycin treatment), Herp null cells manifested irreversible cellular changes that lead to apoptotic cell death. Transfection analysis revealed that the N-terminal region, including the ubiquitin-like domain of Herp, was required for the survival of F9 cells under ER stress. These results indicate that Herp is a short-lived Ub-like protein improving the balance of folding capacity and protein loads in the ER and plays crucial roles for the ER stress resistance in F9 cells.

Transmission of cell stress from endoplasmic reticulum to mitochondria: enhanced expression of Lon protease.

The rat homologue of a mitochondrial ATP-dependent protease Lon was cloned from cultured astrocytes exposed to hypoxia. Expression of Lon was enhanced in vitro by hypoxia or ER stress, and in vivo by brain ischemia. These observations suggested that changes in nuclear gene expression (Lon) triggered by ER stress had the potential to impact important mitochondrial processes such as assembly and/or degradation of cytochrome c oxidase (COX). In fact, steady-state levels of nuclear-encoded COX IV and V were reduced, and mitochondrial-encoded subunit II was rapidly degraded under ER stress. Treatment of cells with cycloheximide caused a similar imbalance in the accumulation of COX subunits, and enhanced mRNA for Lon and Yme1, the latter another mitochondrial ATP-dependent protease. Furthermore, induction of Lon or GRP75/mtHSP70 by ER stress was inhibited in PERK (-/-) cells. Transfection studies revealed that overexpression of wild-type or proteolytically inactive Lon promoted assembly of COX II into a COX I-containing complex, and partially prevented mitochondrial dysfunction caused by brefeldin A or hypoxia. These observations demonstrated that suppression of protein synthesis due to ER stress has a complex effect on the synthesis of mitochondrial-associated proteins, both COX subunits and ATP-dependent proteases and/or chaperones contributing to assembly of the COX complex.