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.

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.

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.

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.

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 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.

Deletion of N-myc downstream-regulated gene 2 attenuates reactive astrogliosis and inflammatory response in a mouse model of cortical stab injury.

N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the CNS. In this study, we examined the expression and the role of Ndrg2 after cortical stab injury. We observed that Ndrg2 expression was elevated in astrocytes surrounding the wounded area as early as day 1 after injury in wild-type mice. Deletion of Ndrg2 resulted in lower induction of reactive astroglial and microglial markers in the injured cortex. Histological analysis showed reduced levels of hypertrophic changes in astrocytes, accumulation of microglia, and neuronal death in Ndrg2(-/-) mice after injury. Furthermore, activation of the IL-6/signal transducer and activator of transcription 3 (STAT3) pathway, including the expression of IL-6 family cytokines and phosphorylation of STAT3, was markedly reduced in Ndrg2(-/-) mice after injury. In a culture system, both of Il6 and Gfap were up-regulated in wild-type astrocytes treated with forskolin. Deletion of Ndrg2 attenuated induction of these genes, but did not alter proliferation or migration of astrocytes. Adenovirus-mediated reexpression of Ndrg2 rescued the reduction of IL-6 expression after forskolin stimulation. These findings suggest that Ndrg2 plays a key role in reactive astrogliosis after cortical stab injury through a mechanism involving the positive regulation of IL-6/STAT3 signaling.

Negative correlation between Per1 and Sox6 expression during chondrogenic differentiation in pre-chondrocytic ATDC5 cells.

Pre-chondrocytes undergo cellular differentiation stages during chondrogenesis under the influence by different transcription factors such as sry-type high mobility group box-9 (Sox9) and runt-related transcription factor-2 (Runx2). We have shown upregulation by parathyroid hormone (PTH) of the clock gene Period-1 (Per1) through the cAMP/protein kinase A signaling pathway in pre-chondrocytic ATDC5 cells. Here, we investigated the role of Per1 in the suppression of chondrogenic differentiation by PTH. In ATDC5 cells exposed to 10 nM PTH, a drastic but transient increase in Per1 expression was seen only 1 h after addition together with a prolonged decrease in Sox6 levels. However, no significant changes were induced in Sox5 and Runx2 levels in cells exposed to PTH. In stable Per1 transfectants, a significant decrease in Sox6 levels was seen, with no significant changes in Sox5 and Sox9 levels, in addition to the inhibition of gene transactivation by Sox9 allies. Knockdown of Per1 by siRNA significantly increased the Sox6 and type II collagen levels in cells cultured for 24 - 60 h. These results suggest that Per1 plays a role in the suppressed chondrocytic differentiation by PTH through a mechanism relevant to negative regulation of transactivation of the Sox6 gene during chondrogenesis.

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.

Osteoclastogenesis is negatively regulated by D-serine produced by osteoblasts.

We have shown the functional expression by chondrocytes of serine racemase (SR) which is responsible for the synthesis of D-serine (Ser) from L-Ser in cartilage. In this study, we evaluated the possible functional expression of SR by bone-forming osteoblasts and bone-resorbing osteoclasts. Expression of SR mRNA was seen in osteoblasts localized at the cancellous bone surface in neonatal rat tibial sections and in cultured rat calvarial osteoblasts endowed to release D-Ser into extracellular medium, but not in cultured osteoclasts differentiated from murine bone marrow progenitor cells. Sustained exposure to D-Ser failed to significantly affect alkaline phosphatase activity and Ca(2+) accumulation in cultured osteoblasts, but significantly inhibited differentiation and maturation in a concentration-dependent manner at a concentration range of 0.1-1 mM without affecting cellular survival in cultured osteoclasts. By contrast, L-Ser promoted osteoclastic differentiation in a manner sensitive to the inhibition by D-Ser. Matured osteoclasts expressed mRNA for the amino acid transporter B(0,+) (ATB(0,+) ) and the system alanine, serine, and cysteine amino acid transporter-2 (ASCT2), which are individually capable of similarly incorporating extracellular L- and D-Ser. Knockdown of these transporters by siRNA prevented both the promotion by L-Ser and the inhibition by D-Ser of osteoclastic differentiation in pre-osteoclastic RAW264.7 cells. These results suggest that D-Ser may play a pivotal role in osteoclastogenesis through a mechanism related to the incorporation mediated by both ATB(0,+) and ASCT2 of serine enantiomers in osteoclasts after the synthesis and subsequent release from adjacent osteoblasts.

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.

Suppression of expression of endoplasmic reticulum chaperones by Helicobacter pylori and its role in exacerbation of non-steroidal anti-inflammatory drug-induced gastric lesions.

Both the use of non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, and infection with Helicobacter pylori are major causes of gastric ulcers. Although some clinical studies suggest that infection with H. pylori increases the risk of developing NSAID-induced gastric lesions, the molecular mechanism governing this effect is unknown. We recently found that in cultured gastric cells, expression of endoplasmic reticulum (ER) chaperones (such as 150-kDa oxygen-regulated protein (ORP150) and glucose-regulated protein 78 (GRP78)) is induced by NSAIDs and confers protection against NSAID-induced apoptosis, which is important in the development of NSAID-induced gastric lesions. In this study we have found that co-culture of gastric cells with H. pylori suppresses the expression of ER chaperones. This suppression was regulated at the level of transcription and accompanied by a reduction in the level of activating transcription factor 6 (ATF6), one of the transcription factors for ER chaperone genes. In vivo, inoculation of mice with H. pylori suppressed the expression of ER chaperones at gastric mucosa both with and without administration of indomethacin. Inoculation with H. pylori also stimulated formation of indomethacin-induced gastric lesions and mucosal cell death. In addition, we found that heterozygous ORP150-deficient mice are sensitive to the development of indomethacin-induced gastric lesions and mucosal cell death. The results of this study suggest that H. pylori exacerbates NSAID-induced gastric lesions through suppression of expression of ER chaperones, which stimulates NSAID-induced mucosal cell death.

Glutamate preferentially suppresses osteoblastogenesis than adipogenesis through the cystine/glutamate antiporter in mesenchymal stem cells.

We have shown that glutamate (Glu) signaling machineries, such as receptors (GluR) and transporters, are functionally expressed by mesenchymal stem cells, in addition to by their progeny cells such as osteoblasts and chondrocytes. Sustained exposure to Glu induced significant decreases in alkaline phosphatase (ALP) staining and osteoblastic marker gene expression in the mesenchymal C3H10T1/2 stem cells infected with runt-related transcription factor-2 (Runx2) adenovirus, without markedly affecting Oil Red O staining for adipocytes in cells cultured with adipogenic inducers. In cells with Runx2 adenovirus, the cystine/Glu antiporter substrate cystine significantly prevented the decreases by Glu in both ALP staining and osteoblastic marker gene expression, with GluR agonists being ineffective. In cells with Runx2 adenovirus, Glu significantly decreased [14C]cystine uptake, intracellular glutathione (GSH) level, Runx2 recruitment to osteocalcin promoter and nuclear Runx2 protein level, respectively. Cystine again significantly prevented the decreases by Glu in both GSH levels and Runx2 recruitment. In mouse bone marrow stromal cells, Glu and a GSH depleter significantly decreased ALP staining without affecting Oil Red O staining. Knockdown of the cystine/Glu antiporter led to markedly decreased ALP staining and GSH levels, with concomitant prevention of the decrease by Glu, in cells with Runx2 adenovirus. These results suggest that Glu may play a role as a negative regulator at an early differentiation stage into osteoblasts than adipocytes through a mechanism relevant to nuclear translocation of Runx2 after regulation of intracellular GSH levels by the cystine/Glu antiporter expressed in mesenchymal stem cells.

Negative regulation of osteoblastogenesis through downregulation of runt-related transcription factor-2 in osteoblastic MC3T3-E1 cells with stable overexpression of the cystine/glutamate antiporter xCT subunit.

We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through a mechanism associated with intracellular GSH depletion mediated by the bidirectional cystine/Glu antiporter in osteoblastic MC3T3-E1 cells cultured in the absence of differentiation inducers. To further evaluate the possible role of the antiporter in osteoblastogenesis, in this study, we have established stable transfectants of the xCT subunit of the antiporter in MC3T3-E1 cells. Stable overexpression led to a significant facilitation of cellular proliferation determined by different indices with increased GSH levels and decreased ROS generation in addition to promoted [(14)C]cystine incorporation, while Glu failed to significantly inhibit cellular proliferation in stable xCT transfectants. In stable transfectants cultured under differentiation conditions, drastic decreases were invariably seen in Ca(2+) accumulation, alkaline phosphatase activity and several osteoblastic marker gene expressions, in addition to downregulation of mRNA and corresponding protein for runt-related transcription factor-2 (Runx2). Runx2 promoter activity was significantly promoted by the introduction of Runx2 expression vector in a manner sensitive to the prevention by the co-introduction of xCT expression vector in MC3T3-E1 cells. In both MC3T3-E1 cells and murine calvarial osteoblasts cultured with differentiation inducers, transient transfection with xCT siRNA significantly increased Runx2 protein expression along with decreases in xCT mRNA expression and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction. These results suggest that the cystine/Glu antiporter plays a pivotal role in cellular differentiation through a mechanism related to the regulation of transactivation of Runx2 essential for osteoblastogenesis toward maturation in osteoblastic cells.

Deletion of Herp facilitates degradation of cytosolic proteins.

Although intracellular stresses are believed to be involved in the process of neurodegeneration, it is not fully understood how one stress/stress response affects another. Herp is an endoplasmic reticulum (ER)-located membrane protein proposed to function in ER-associated degradation (ERAD). Herp is strongly induced by ER stress but rapidly degraded by proteasome. To elucidate the effect of Herp expression on proteolytic stress caused by impairment of the ubiquitin-proteasome system (UPS), we utilized 293T Herp knockdown (KD) cells and F9 Herp knockout cells. Knockdown of Herp gene unexpectedly facilitated the degradation of Parkinson's disease-associated cytosolic proteins such as alpha-synuclein and its binding partner, synphilin-1, and improved cell viability during proteasomal inhibition. A similar tendency was observed in F9 Herp knockout cells transfected with synphilin-1. Herp temporarily bound to alpha-synuclein, synphilin-1 and the E3 ligase SIAH1a during proteolytic stress but not during ER stress. Furthermore, deletion of Herp enhanced the amount of ubiquitinated protein in the cytosol during proteasomal inhibition, although it did not affect the activity or expression of proteasome. These results suggest that ERAD molecule Herp may delay the degradation of cytosolic proteins at the ubiquitination step.

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.