Novel oxindole derivatives prevent oxidative stress-induced cell death in mouse hippocampal HT22 cells.

The current medical and surgical therapies for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease offer symptomatic relief but do not provide a cure. Thus, small synthetic compounds that protect neuronal cells from degeneration are critically needed to prevent and treat these. Oxidative stress has been implicated in various pathophysiological conditions, including neurodegenerative diseases.

Elucidating post-translational regulation of mouse CREB3 in Neuro2a cells.

CREB3 is an ER membrane-bound transcription factor; however, post-translational regulation of CREB3, including expression, processing, and activation, is not fully characterized. We therefore constructed several types of mouse CREB3 expression genes and elucidated their expression in Neuro2a cells by treatment with stimuli and co-transfection with genes associated with ER-Golgi homeostasis, such as mutant Sar1 [H79G], GRP78, and KDEL receptor 1 (KDELR1). Interestingly, treatment of Neuro2a cells expressing Flag-tagged full-length CREB3 with monensin and nigericin induced the expression of the approximately 50 kDa N-terminal fragment; however, its cleavage was not parallel to the levels of GADD153 and LC3-II.

Prevention of oxytosis-induced c-Raf down-regulation by (arylthio)cyclopentenone prostaglandins is neuroprotective.

Prolonged exposure to high concentrations of glutamate leads to cell type specific glutathione depletion and resulting oxidative stress, known as oxytosis. As a result of glutathione depletion, accumulation of reactive oxygen species and Ca influx are increased; however, the specific target of oxytosis has yet to be identified. In the present study, we focused on the effect of glutamate-induced oxidative stress on the extracellular-regulated protein kinase (ERK) pathway using the murine hippocampal HT22 cell line.

SOD1 dimerization monitoring using a novel split NanoLuc, NanoBit.

In the present study, we applied a highly sensitive NanoLuc-based technology to understand the status of superoxide dismutase 1 (SOD1) within mammalian cells. Two fragments of NanoLuc (NanoBit), large N-terminal and small C-terminal regions, were fused with wild-type (wt) and mutant human SOD1 (hSOD1) genes and transfected into cells. Luciferase activity through NanoBit assembly was only detected in NanoBit-tagged wtSOD1-expressing cells.

Application of NanoLuc to monitor the intrinsic promoter activity of GRP78 using the CRISPR/Cas9 system.

In this study, we applied a highly sensitive small luciferase, NanoLuc, to establish a knock-in cell line using the CRISPR/Cas9 system and characterized the endogenous promoter activity of the glucose-regulated protein 78 (GRP78) gene. The N-terminal region of the human GRP78 gene was fused to the NanoLuc gene and aligned with the puromycin-resistant gene through the 2A peptide sequence and used as a knock-in vector. The selected cells responded to both pharmacological and genetic ER stress and show NanoLuc-based CRISPR/Cas9 system is a very useful tool to isolate gene-edited cells and to characterize the endogenous promoter activity for genes of interest.

Elucidation of a novel phenformin derivative on glucose-deprived stress responses in HT-29 cells.

Recently, we developed a variety of phenformin derivatives as selective antitumor agents. Based on previous findings, this study evaluated a promising compound, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on the basis of stress responses in the human colon cancer cell line HT-29 under a serum- and glucose-deprived condition. 2-Cl-Phen triggered morphological changes such as shrinkage and plasma membrane disintegration, as well as a decrease in mitochondrial activity and an increase in LDH leakage.

Translational and post-translational regulation of mouse cation transport regulator homolog 1.

Cation transport regulator homolog 1 (Chac1) is an endoplasmic reticulum (ER) stress inducible gene that has a function as a γ-glutamyl cyclotransferase involved in the degradation of glutathione. To characterize the translation and stability of Chac1, we found that the Kozak-like sequence present in the 5' untranslated region (5'UTR) of the Chac1 mRNA was responsible for Chac1 translation. In addition, the short form (ΔChac1), which translated from the second ATG codon, was generated in the absence of the 5'UTR.

A Comparative Analysis of the Molecular Features of MANF and CDNF.

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system.

A highly sensitive assay of IRE1 activity using the small luciferase NanoLuc: Evaluation of ALS-related genetic and pathological factors.

Activation of inositol-requiring enzyme 1 (IRE1) due to abnormal conditions of the endoplasmic reticulum (ER) is responsible for the cleavage of an unspliced form of X-box binding protein 1 (uXBP1), producing its spliced form (sXBP1). To estimate IRE1 activation, several analytical procedures using green fluorescence protein and firefly luciferase have been developed and applied to clarify the roles of IRE1-XBP1 signaling pathways during development and disease progression. In this study, we established a highly sensitive assay of IRE1 activity using a small luciferase, NanoLuc, which has approximately 100-fold higher activity than firefly luciferase.

Characterization of the Role of MANF in Regulating the Secretion of CRELD2.

We recently demonstrated that the secretion of two novel endoplasmic reticulum (ER) stress-inducible proteins, cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) and mesencephalic astrocyte-derived neurotrophic factor (MANF), are oppositely regulated by the overexpression of 78 kDa glucose-regulated protein (GRP78). In the present study, we found that the co-transfection of CRELD2 and MANF remarkably enhanced the secretion of CRELD2 without affecting the expression level of GRP78. To identify the structural features of CRELD2 and MANF involved in this process, we generated several CRELD2 and MANF expression constructs.

Transcriptional and post-transcriptional regulation of transmembrane protein 132A.

Transmembrane protein 132A (TMEM132A) was first isolated from rat brain using PCR-selected cDNA subtraction, and it was found to be predominantly expressed in the brain. However, the transcriptional regulation of the TMEM132A gene has not been fully characterized. In this study, we characterized the promoter activity of the 880-bp region upstream of the mouse TMEM132A, identifying several putative sites recognized by transcription factors, which are highly conserved between the mouse and human TMEM132A genes.

A sensitive assay for the biosynthesis and secretion of MANF using NanoLuc activity.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to prevent neuronal cell death caused by certain stimuli. Accordingly, the molecular features of MANF have been intensively investigated since the reporting of its cytoprotective actions. In addition to the characterization of the transcriptional regulation of MANF under pathophysiological conditions, it is important to understand its intracellular transport and secretion after translation.

Characterization of V-ATPase inhibitor-induced secretion of cysteine-rich with EGF-like domains 2.

We previously demonstrated that cysteine-rich with EGF-like domains 2 (CRELD2), a novel ER stress-inducible factor, is a secretory glycoprotein; however, the stimuli that induce CRELD2 secretion have not yet been characterized. In this study, we found that the perturbation of intravesicular acidification of cytoplasmic organelles in HEK293 cells stably expressing wild-type (wt) CRELD2 induced its secretion. In particular, Concanamycin A (CMA) and Bafilomycin A1 (Baf), inhibitors of vacuolar ATPase (V-ATPase), increased the secretion of CRELD2 without relying on its C-terminal structure.

Transcriptional regulation of mouse mesencephalic astrocyte-derived neurotrophic factor in Neuro2a cells.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel type of trophic factor. Recent studies indicate that the MANF gene is induced in response to endoplasmic reticulum (ER) stress through ER stress response element II (ERSE-II) in its 5'-flanking region. In this study, we evaluated the roles of six ER stress response transcription factors in the regulation of the promoter activities of the mouse MANF gene via ERSE-II using various types of mutant MANF luciferase reporter constructs.

Characterization of the 5'-flanking region of the mouse asparagine-linked glycosylation 12 homolog gene.

Recently, we characterized multiple roles of the endoplasmic reticulum stress responsive element (ERSE) in the promotion of a unique head-to-head gene pair: mammalian asparagine-linked glycosylation 12 homolog (ALG12) and cysteine-rich with EGF-like domains 2 (CRELD2). This bidirectional promoter, which consists of fewer than 400 base pairs, separates the two genes. It has been demonstrated that the ALG12 promoter shows less transcriptional activity through ERSE, but its basic regulatory mechanism has not been characterized.

Transcriptional and post-translational regulation of mouse cation transport regulator homolog 1.

Recently, cation transport regulator homolog 1 (Chac1) has been identified as a novel pro-apoptotic factor in cells under endoplasmic reticulum (ER) stress. Of the three major ER stress sensors, it is suggested that ATF4 participates in the transcriptional regulation of Chac1 gene expression. The precise characterization of the Chac1 promoter, however, has not yet been elucidated.

Neuroprotective cyclopentenone prostaglandins up-regulate neurotrophic factors in C6 glioma cells.

In a previous study, we developed newly synthesized arylthio derivatives of cyclopentenone prostaglandins (GIF-0642, GIF-0643, GIF-0644, GIF-0745 and GIF-0747), which are neuroprotective against both manganese toxicity in PC12 cells and glutamate toxicity in HT22 cells. In the present study, we showed that these compounds and their lead compound, NEPP11, are potent inducers of glial cell line-derived neurotrophic factor (GDNF) expression in C6 glioma cells and primary astrocytes. These neuroprotective cyclopentenone prostaglandins also induced the gene expression of nerve growth factor and, to a lesser extent, brain-derived neurotrophic factor.

Manganese regulates caspase-3 gene promoter activity by inducing Sp1 phosphorylation in PC12 cells.

Chronic manganese exposure causes selective toxicity to the dopaminergic system, resulting in a parkisonian-like neurological condition known as manganism. Manganese causes a typical apoptosis, which includes activation of the caspase cascade and DNA fragmentation in PC12 cells. Caspase-3 is a major contributor to the execution of neuronal apoptosis.

Characterization of the expression and cell-surface localization of transmembrane protein 132A.

Transmembrane protein 132A (TMEM132A, KIAA1583) was first isolated as a novel gene that is enriched during the embryonic and postnatal stages of rat brain development and interacts with GRP78. However, the biological functions of TMEM132A are scarcely characterized because the protein does not contain any known structural domains. Using a cell-surface biotinylation assay and immunocytochemical staining, we found that TMEM132A is a transmembrane glycoprotein consisting of a large extracellular domain in the N-terminal region and a small cytosolic domain in the C-terminal region.

Characterization of 3'-untranslated region of the mouse GDNF gene.

Background: Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for many cell types, and its expression is widespread both within and outside of the nervous system. The regulation of GDNF expression has been extensively investigated but is not fully understood.

Results: Using a luciferase reporter assay, we identified the role of the 3'-untranslated region (3'-UTR) of the mouse GDNF gene in the regulation of gene expression.

Differential effects of Akt pathway inhibitors on IL-1β-induced protein phosphorylation in human fibroblast-like synoviocytes.

Context: Interleukin (IL)-1β activates various signal transduction pathways including p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt in human fibroblast-like synoviocytes (HFLS).

Objective: We investigated the effects of an Akt inhibitor, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and Akt RNAi knockdown on IL-1β-induced protein phosphorylation in HFLS to clarify the role of the PI3K/Akt signaling pathway in the phosphorylation of the inhibitor of κB (IκB)α and heat shock protein 27 (HSP27).

Materials And Methods: A multiplex suspension array system was used for the detection of phosphorylated proteins.

Intracellular trafficking and secretion of mouse mesencephalic astrocyte-derived neurotrophic factor.

Recently, mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to prevent cell death under some pathophysiological conditions. MANF, also referred to as arginine rich, mutated in early stage of tumors (Armet), was identified as an endoplasmic reticulum (ER) stress-inducible factor. Using RT-PCR, we found two variants of MANF mRNA: wild type, which contains exon 1 (wt-MANF), and one lacking exon 1, which is presumably not secreted (ΔΝ-MANF) in Neuro2a cells.

Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor.

Chloroquine, a widely used anti-malarial and anti-rheumatoid agent, has been reported to induce apoptotic and non-apoptotic cell death. Accumulating evidence now suggests that chloroquine can sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis by inhibiting autophagy. However, chloroquine is reported to induce GM1 ganglioside accumulation in cultured cells at low μM concentrations and prevent damage to the blood brain barrier in mice.

Biosynthesis and secretion of mouse cysteine-rich with EGF-like domains 2.

In this study, we found that Cysteine-rich with EGF-like domains 2 (CRELD2), a novel endoplasmic reticulum stress-inducible protein, is not only localized in the ER-Golgi apparatus but also spontaneously secreted. Deletion of four C-terminal amino acids from mouse CRELD2 or addition of tag-peptides to its C-terminus dramatically enhanced CRELD2 secretion. Intra- and extra-cellular CRELD2 is differentially glycosylated and its spontaneous secretion was significantly prevented by overexpression of a dominant negative mutant Sar1 and treatment with brefeldin A.

Expression of neutral endopeptidase activity during clinical and experimental acute lung injury.

Background: Neutral endopeptidase (NEP), an enzyme that cleaves inflammatory bioactive peptides, may play a protective role in the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, its low extracellular activity hinders the precise measurement of changes that take place during ALI/ARDS. The main objective of this study was to clarify the regulation of NEP activity and its expression during ALI/ARDS.