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.

Postnatal expression of CD38 in astrocytes regulates synapse formation and adult social memory.

Social behavior is essential for health, survival, and reproduction of animals; however, the role of astrocytes in social behavior remains largely unknown. The transmembrane protein CD38, which acts both as a receptor and ADP-ribosyl cyclase to produce cyclic ADP-ribose (cADPR) regulates social behaviors by promoting oxytocin release from hypothalamic neurons. CD38 is also abundantly expressed in astrocytes in the postnatal brain and is important for astroglial development. Here, we demonstrate that the astroglial-expressed CD38 plays an important role in social behavior during development. Selective deletion of CD38 in postnatal astrocytes, but not in adult astrocytes, impairs social memory without any other behavioral abnormalities. Morphological analysis shows that depletion of astroglial CD38 in the postnatal brain interferes with synapse formation in the medial prefrontal cortex (mPFC) and hippocampus. Moreover, astroglial CD38 expression promotes synaptogenesis of excitatory neurons by increasing the level of extracellular SPARCL1 (also known as Hevin), a synaptogenic protein. The release of SPARCL1 from astrocytes is regulated by CD38/cADPR/calcium signaling. These data demonstrate a novel developmental role of astrocytes in neural circuit formation and regulation of social behavior in adults.

Sympathetic Nervous Hyperactivity Impairs Microcirculation Leading to Early Brain Injury After Subarachnoid Hemorrhage.

BACKGROUND: Although early brain injury (EBI) is recognized as a critical step following subarachnoid hemorrhage (SAH), its pathophysiology and underlying mechanisms remain poorly understood. Herein, we investigated the role of cerebral circulation in the acute phase using patient data and a mouse SAH model and evaluated its regulation via the sympathetic nervous system. METHODS: The cerebral circulation time and neurological outcomes in the human body were retrospectively examined in 34 SAH cases with ruptured anterior circulation aneurysms and 85 cases with unruptured anterior circulation cerebral aneurysms at Kanazawa University Hospital from January 2016 to December 2021. In a mouse study, a SAH model was created via endovascular perforation, and India-ink angiography was performed over time. Additionally, bilateral superior cervical ganglionectomy was performed immediately before surgery, and neurological scores and brain water content were evaluated after SAH. RESULTS: Cerebral circulation time was prolonged in the acute phase of SAH compared with that in the unruptured cerebral aneurysm group, especially in those with electrocardiographic changes. Furthermore, it was more prolonged in the poor prognosis group (modified Rankin Scale scores 3-6) than in the good prognosis group (modified Rankin Scale scores 0-2) at discharge. In mice, cerebral perfusion was significantly reduced at 1 and 3 hours after SAH and recovered at 6 hours. superior cervical ganglionectomy improved cerebral perfusion without altering the diameter of the middle cerebral artery at 1 hour and improved neurological outcomes at 48 hours after SAH. Consistently, brain edema, quantified by brain water content, was improved by superior cervical ganglionectomy 24 hours after SAH. CONCLUSIONS: Sympathetic hyperactivity may play a critical role in the development of EBI by impairing cerebral microcirculation and edema in the acute phase following SAH.

ATF6ß Deficiency Elicits Anxiety-like Behavior and Hyperactivity Under Stress Conditions.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6ß, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca2+-binding capacity. In the present study, we demonstrate that ATF6ß deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b-/- mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b-/- mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b-/- mice. These findings suggest that ATF6ß deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6ß could play a role in psychiatric conditions in the emotional centers of the brain.

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.

Isolation of ferret astrocytes reveals their morphological, transcriptional, and functional differences from mouse astrocytes.

Astrocytes play key roles in supporting the central nervous system structure, regulating synaptic functions, and maintaining brain homeostasis. The number of astrocytes in the cerebrum has markedly increased through evolution. However, the manner by which astrocytes change their features during evolution remains unknown. Compared with the rodent brain, the brain of the ferret, a carnivorous animal, has a folded cerebral cortex and higher white to gray matter ratio, which are common features of the human brain. To further clarify the features of ferret astrocytes, we isolated astrocytes from ferret neonatal brains, cultured these cells, and compared their morphology, gene expression, calcium response, and proliferating ability with those of mouse astrocytes. The morphology of cultured ferret astrocytes differed from that of mouse astrocytes. Ferret astrocytes had longer and more branched processes, smaller cell bodies, and different calcium responses to glutamate, as well as had a greater ability to proliferate, compared to mouse astrocytes. RNA sequencing analysis revealed novel ferret astrocyte-specific genes, including several genes that were the same as those in humans. Astrocytes in the ferret brains had larger cell size, longer primary processes in larger numbers, and a higher proliferation rate compared to mouse astrocytes. Our study shows that cultured ferret astrocytes have different features from rodent astrocytes and similar features to human astrocytes, suggesting that they are useful in studying the roles of astrocytes in brain evolution and cognitive functions in higher animals.

[Astrocytes in the pathogenesis of multiple sclerosis].

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), and is designated as an intractable disease in Japan. It is characterized by dissemination of plaque-like sclerosis in space and time, accompanied with various symptoms corresponding to the CNS lesion site. Typically, neurological symptoms chronically progress accompanied with relapses and remissions, and there is still no curative therapy. A number of studies using MS specimen and the animal MS model experimental autoimmune encephalomyelitis (EAE) have shown that MS is an autoimmune disease that targets myelin sheath in the CNS. Autoreactive T cells and B cells play a central role in pathogenesis of MS. MS comprise relapsing-remitting MS and progressive MS, the latter accumulates clinical disability without relapse. Based on the importance of adaptive immunity, various disease-modifying drugs have been developed to treat relapsing-remitting MS. On the other hand, an effective treatment for progressive MS has not yet been established. Increasing evidence have been recognized glial cells as key components of MS immunopathology, in addition to innate immunity and adaptive immunity. However, molecular mechanisms of crosstalk between immune cells, glial cells and neurons remain to be elucidated. Here, we review MS pathology and recent advances in the disease-modifying therapy that efficiently reduce disease activity in relapsing-remitting MS and introduce an update of recent evidence that astrocyte is involved in the MS pathology with including our research analyzed in mouse EAE model.

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.

Inhibition of CD38 and supplementation of nicotinamide riboside ameliorate lipopolysaccharide-induced microglial and astrocytic neuroinflammation by increasing NAD.

Neuroinflammation is initiated by activation of the brain's innate immune system in response to an inflammatory challenge. Insufficient control of neuroinflammation leads to enhanced or prolonged pathology in various neurological conditions including multiple sclerosis and Alzheimer's disease. Nicotinamide adenine dinucleotide (NAD+ ) plays critical roles in cellular energy metabolism and calcium homeostasis. Our previous study demonstrated that deletion of CD38, which consumes NAD+ , suppressed cuprizone-induced demyelination, neuroinflammation, and glial activation. However, it is still unknown whether CD38 directly affects neuroinflammation through regulating brain NAD+ level. In this study, we investigated the effect of CD38 deletion and inhibition and supplementation of NAD+ on lipopolysaccharide (LPS)-induced neuroinflammation in mice. Intracerebroventricular injection of LPS significantly increased CD38 expression especially in the hippocampus. Deletion of CD38 decreased LPS-induced inflammatory responses and glial activation. Pre-administration of apigenin, a flavonoid with CD38 inhibitory activity, or nicotinamide riboside (NR), an NAD+ precursor, increased NAD+ level, and significantly suppressed induction of cytokines and chemokines, glial activation and subsequent neurodegeneration after LPS administration. In cell culture, LPS-induced inflammatory responses were suppressed by treatment of primary astrocytes or microglia with apigenin, NAD+ , NR or 78c, the latter a specific CD38 inhibitor. Finally, all these compounds suppressed NF-κB signaling pathway in microglia. These results suggest that CD38-mediated neuroinflammation is linked to NAD+ consumption and that boosting NAD+ by CD38 inhibition and NR supplementation directly suppress neuroinflammation in the brain.

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.

Roles of N-myc downstream-regulated gene 2 in the central nervous system: molecular basis and relevance to pathophysiology.

N-myc downstream-regulated gene 2 (NDRG2) is a member of the NDRG family, whose members have multiple functions in cell proliferation, differentiation, and stress responses. NDRG2 is widely distributed in the central nervous system and is uniquely expressed by astrocytes; however, its role in brain function remains elusive. The clinical relevance of NDRG2 and the molecular mechanisms in which it participates have been reported by studies using cultured cells and specimens of patients with neurological disorders. In recent years, genetic tools, including several lines of Ndrg2-knockout mice and virus-mediated gene transfer, have improved understanding of the roles of NDRG2 in vivo. This review aims to provide an update of recent growing in vivo evidence that NDRG2 is involved in brain function, focusing on research of Ndrg2-knockout mice with neurological disorders such as brain tumors, chronic neurodegenerative diseases, and acute brain insults including brain injury and cerebral stroke. These studies demonstrate that NDRG2 plays diverse roles in the regulation of astrocyte reactivity, blood-brain barrier integrity, and glutamate excitotoxicity. Further elucidation of the roles of NDRG2 and their molecular basis may provide novel therapeutic approaches for various neurological disorders.

Deletion of CD38 Suppresses Glial Activation and Neuroinflammation in a Mouse Model of Demyelination.

CD38 is an enzyme that catalyzes the synthesis of cyclic adenosine diphosphate-ribose from nicotinamide adenine dinucleotide (NAD+). We recently reported that this molecule regulates the maturation and differentiation of glial cells such as astrocytes and oligodendrocytes (OLs) in the developing brain. To analyze its role in the demyelinating situation, we employed cuprizone (CPZ)-induced demyelination model in mice, which is characterized by oligodendrocyte-specific apoptosis, followed by the strong glial activation, demyelination, and repopulation of OLs. By using this model, we found that CD38 was upregulated in both astrocytes and microglia after CPZ administration. Experiments using wild-type and CD38 knockout (KO) mice, together with those using cultured glial cells, revealed that CD38 deficiency did not affect the initial decrease of the number of OLs, while it attenuated CPZ-induced demyelination, and neurodegeneration. Importantly, the clearance of the degraded myelin and oligodendrocyte repopulation were also reduced in CD38 KO mice. Further experiments revealed that these observations were associated with reduced levels of glial activation and inflammatory responses including phagocytosis, most likely through the enhanced level of NAD+ in CD38-deleted condition. Our results suggest that CD38 and NAD+ in the glial cells play a critical role in the demyelination and subsequent oligodendrocyte remodeling through the modulation of glial activity and neuroinflammation.

Microglial activation in the cochlear nucleus after early hearing loss in rats.

OBJECTIVE: Microglia are highly specialized tissue macrophages in the central nervous system. Their activation in the auditory system has been reported in adult hearing loss models, but their status in the developing auditory system is less understood. Therefore, we investigated microglial status in the cochlear nucleus (CN) during normal developing periods and after exposing rats to amikacin, a potent ototoxin, around the time of hearing onset. METHODS: To develop the deafness model, rats were administered with a daily intraperitoneal injection of amikacin (500 mg/kg) from postnatal day 7 (P7) to P15. To evaluate the expression of ionized calcium binding adaptor molecule 1 (Iba1), we performed immunohistochemical analysis using rat brains from P10-60. To compare the expression of microglia-related gene, reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis were performed. RESULTS: Immunohistochemical analysis revealed that, under normal conditions, microglia had relatively large cell bodies with several extended processes that surrounded other cells at P10, while the sizes and number of these cells gradually decreased afterward. In contrast, when amikacin was administered from P7 to P15, microglia maintained large cell bodies with relatively shorter processes at both P15 and P21. Furthermore, RT-qPCR analysis revealed upregulation of genes including phagocytotic and anti-inflammatory markers after amikacin administration. CONCLUSION: These results suggest that microglia are activated in the CN, and they may contribute to tissue remodeling after early hearing loss in the developing auditory system.

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.

CD38 positively regulates postnatal development of astrocytes cell-autonomously and oligodendrocytes non-cell-autonomously.

Glial development is critical for the function of the central nervous system. CD38 is a multifunctional molecule with ADP-ribosyl cyclase activity. While critical roles of CD38 in the adult brain such as oxytocin release and social behavior have been reported, those in the developing brain remain largely unknown. Here we demonstrate that deletion of Cd38 leads to impaired development of astrocytes and oligodendrocytes in mice. CD38 is highly expressed in the developing brains between postnatal day 14 (P14) and day 28 (P28). In situ hybridization and FACS analysis revealed that CD38 is expressed predominantly in astrocytes in these periods. Analyses of the cortex of Cd38 knockout (Cd38-/- ) mice revealed delayed development of astrocytes and subsequently delayed differentiation of oligodendrocytes (OLs) at postnatal stages. In vitro experiments using primary OL cultures, mixed glial cultures, and astrocytic conditioned medium showed that astrocytic CD38 regulates the development of astrocytes in a cell-autonomous manner and the differentiation of OLs in a non-cell-autonomous manner. Further experiments revealed that connexin43 (Cx43) in astrocytes plays a promotive role for CD38-mediated OL differentiation. Finally, increased levels of NAD+ , caused by CD38 deficiency, are likely to be responsible for the suppression of astrocytic Cx43 expression and OL differentiation. Our data indicate that CD38 is a positive regulator of astrocyte and OL development.

Atf6α deficiency suppresses microglial activation and ameliorates pathology of experimental autoimmune encephalomyelitis.

Accumulating evidence suggests a critical role for the unfolded protein response in multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we investigated the relevance of activating transcription factor 6α (ATF6α), an upstream regulator of part of the unfolded protein response, in EAE. The expressions of ATF6α-target molecular chaperones such as glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94) were enhanced in the acute inflammatory phase after induction of EAE. Deletion of Atf6α suppressed the accumulation of T cells and microglia/macrophages in the spinal cord, and ameliorated the clinical course and demyelination after EAE induction. In contrast to the phenotypes in the spinal cord, activation status of T cells in the peripheral tissues or in the culture system was not different between two genotypes. Bone marrow transfer experiments and adoptive transfer of autoimmune CD4+ T cells to recipient mice (passive EAE) also revealed that CNS-resident cells are responsible for the phenotypes observed in Atf6α-/- mice. Further experiments with cultured cells indicated that inflammatory response was reduced in Atf6α-/- microglia, but not in Atf6α-/- astrocytes, and was associated with proteasome-dependent degradation of NF-κB p65. Thus, our results demonstrate a novel role for ATF6α in microglia-mediated CNS inflammation. We investigated the relevance of ATF6α, an upstream regulator of part of the UPR, in EAE. Deletion of Atf6α suppressed inflammation, and ameliorated demyelination after EAE. Bone marrow transfer experiments and adoptive transfer of autoimmune CD4+ T cells revealed that CNS-resident cells are responsible for the phenotypes in Atf6α-/- mice. Furthermore, inflammatory response was reduced in Atf6α-/- microglia, and was associated with degradation of NF-κB p65. Our results demonstrate a novel role for ATF6α in microglia-mediated inflammation. Cover image for this issue: doi: 10.1111/jnc.13346.

Deletion of Herpud1 Enhances Heme Oxygenase-1 Expression in a Mouse Model of Parkinson's Disease.

Herp is an endoplasmic reticulum- (ER-) resident membrane protein that plays a role in ER-associated degradation. We studied the expression of Herp and its effect on neurodegeneration in a mouse model of Parkinson's disease (PD), in which both the oxidative stress and the ER stress are evoked. Eight hours after administering a PD-related neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to mice, the expression of Herp increased at both the mRNA and the protein levels. Experiments using Herpud1 (+/+) and Herpud1 (-/-) mice revealed that the status of acute degeneration of nigrostriatal neurons and reactive astrogliosis was comparable between two genotypes after MPTP injection. However, the expression of a potent antioxidant, heme oxygenase-1 (HO-1), was detected to a higher degree in the astrocytes of Herpud1 (-/-) mice than in the astrocytes of Herpud1 (+/+) mice 24 h after MPTP administration. Further experiments using cultured astrocytes revealed that the stress response against MPP(+), an active form of MPTP, and hydrogen peroxide, both of which cause oxidative stress, was comparable between the two genotypes. These results suggest that deletion of Herpud1 may cause a slightly higher level of initial damage in the nigrastrial neurons after MPTP administration but is compensated for by higher induction of antioxidants such as HO-1 in astrocytes.

Deletion of Atf6α impairs astroglial activation and enhances neuronal death following brain ischemia in mice.

To dissect the role of endoplasmic reticulum (ER) stress and unfolded protein response in brain ischemia, we investigated the relevance of activating transcription factor 6α (ATF6α), a master transcriptional factor in the unfolded protein response, after permanent middle cerebral artery occlusion (MCAO) in mice. Enhanced expression of glucose-regulated protein78, a downstream molecular chaperone of ATF6α, was observed in both neurons and glia in the peri-infarct region of wild-type mice after MCAO. Analysis using wild-type and Atf6α(-/-) mice revealed a larger infarct volume and increased cell death in the peri-ischemic region of Atf6α(-/-) mice 5 days after MCAO. These phenotypes in Atf6α(-/-) mice were associated with reduced levels of astroglial activation/glial scar formation, and a spread of tissue damage into the non-infarct area. Further analysis in mice and cultured astrocytes revealed that signal transducer and activator of transcription 3 (STAT3)-glial fibrillary acidic protein signaling were diminished in Atf6α(-/-) astrocytes. A chemical chaperone, 4-phenylbutyrate, restored STAT3-glial fibrillary acidic protein signaling, while ER stressors, such as tunicamycin and thapsigargin, almost completely abolished signaling in cultured astrocytes. Furthermore, ER stress-induced deactivation of STAT3 was mediated, at least in part, by the ER stress-responsive tyrosine phosphatase, TC-PTP/PTPN2. These results suggest that ER stress plays critical roles in determining the level of astroglial activation and neuronal survival after brain ischemia.