Simplified drug efficacy evaluation system for vasopressin neurodegenerative disease using mouse disease-specific induced pluripotent stem cells.

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins.

Repositioning of mifepristone as an integrated stress response activator to potentiate cisplatin efficacy in non-small cell lung cancer.

Lung cancer, primarily non-small-cell lung cancer (NSCLC), is a significant cause of cancer-related mortality worldwide. Cisplatin-based chemotherapy is a standard treatment for NSCLC; however, its effectiveness is often limited due to the development of resistance, leading to NSCLC recurrence. Thus, the identification of effective chemosensitizers for cisplatin is of paramount importance. The integrated stress response (ISR), activated by various cellular stresses and mediated by eIF2α kinases, has been implicated in drug sensitivity. ISR activation globally suppresses protein synthesis while selectively promoting the translation of ATF4 mRNA, which can induce pro-apoptotic proteins such as CHOP, ATF3, and TRIB3. To expedite and economize the development of chemosensitizers for cisplatin treatment in NSCLC, we employed a strategy to screen an FDA-approved drug library for ISR activators. In this study, we identified mifepristone as a potent ISR activator. Mifepristone activated the HRI/eIF2α/ATF4 axis, leading to the induction of pro-apoptotic factors, independent of its known role as a synthetic steroid. Our in vitro and in vivo models demonstrated mifepristone's potential to inhibit NSCLC re-proliferation following cisplatin treatment and tumor growth, respectively, via the ISR-mediated cell death pathway. These findings suggest that mifepristone, as an ISR activator, could enhance the efficacy of cisplatin-based therapy for NSCLC, highlighting the potential of drug repositioning in the search for effective chemosensitizers.

The multifaceted role of ATF4 in regulating glucose-stimulated insulin secretion.

Stress-inducible transcription factor ATF4 is essential for survival and identity of ß-cell during stress conditions. However, the physiological role of ATF4 in ß-cell function is not yet completely understood. To understand the role of ATF4 in glucose-stimulated insulin secretion (GSIS), ß-cell-specific Atf4 knockout (ßAtf4KO) mice were phenotypically characterized. Insulin secretion and mechanistic analyses were performed using islets from control Atf4f/f and ßAtf4KO mice to assess key regulators for triggering and amplifying signals for GSIS. ßAtf4KO mice displayed glucose intolerance due to reduced insulin secretion. Moreover, ßAtf4KO islets exhibited a decrease in both the insulin content and first-phase insulin secretion. The analysis of ßAtf4KO islets showed that ATF4 is required for insulin production and glucose-stimulated ATP and cAMP production. The results demonstrate that ATF4 contributes to the multifaceted regulatory process in GSIS even under stress-free conditions.

Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophages.

Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues, in contrast to the limited regenerative abilities of human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA sequencing during leg regeneration. Of the 11 Toll genes in the Gryllus genome, expression of Toll2-1, Toll2-2 and Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Toll, Toll2-1, Toll2-2, Toll2-3 or Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Toll2-2 led to a decrease in the ratio of S- and M-phase cells, reduced expression of JAK/STAT signalling genes, and reduced accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, as well as fewer proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway.

ATF4-mediated transcriptional regulation protects against ß-cell loss during endoplasmic reticulum stress in a mouse model.

OBJECTIVE: Activating transcription factor 4 (ATF4) is a transcriptional regulator of the unfolded protein response and integrated stress response (ISR) that promote the restoration of normal endoplasmic reticulum (ER) function. Previous reports demonstrated that dysregulation of the ISR led to development of severe diabetes. However, the contribution of ATF4 to pancreatic ß-cells remains poorly understood. In this study, we aimed to analyze the effect of ISR enhancer Sephin1 and ATF4-deficient ß-cells to clarify the role of ATF4 in ß-cells under ER stress conditions. METHODS: To examine the role of ATF4 in vivo, ISR enhancer Sephin1 (5 mg/kg body weight, p.o.) was administered daily for 21 days to Akita mice. We also established ß-cell-specific Atf4 knockout (ßAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, ß-cell function, and morphology of the islets. To identify the downstream factors of ATF4 in ß-cells, the islets of ßAtf4-KO mice were subjected to cDNA microarray analyses. To examine the transcriptional regulation by ATF4, we also performed in situ PCR analysis of pancreatic sections from mice and ChIP-qPCR analysis of CT215 ß-cells. RESULTS: Administration of the ISR enhancer Sephin1 improved glucose metabolism in Akita mice. Sephin1 also increased the insulin-immunopositive area and ATF4 expression in the pancreatic islets. Akita/ßAtf4-KO mice exhibited dramatically exacerbated diabetes, shown by hyperglycemia at an early age, as well as a remarkably short lifespan owing to diabetic ketoacidosis. Moreover, the islets of Akita/ßAtf4-KO mice presented increased numbers of cells stained for glucagon, somatostatin, and pancreatic polypeptide and increased expression of aldehyde dehydrogenase 1 family member 3, a marker of dedifferentiation. Using microarray analysis, we identified atonal BHLH transcription factor 8 (ATOH8) as a downstream factor of ATF4. Deletion of ATF4 in ß-cells showed reduced Atoh8 expression and increased expression of undifferentiated markers, Nanog and Pou5f1. Atoh8 expression was also abolished in the islets of Akita/ßAtf4-KO mice. CONCLUSIONS: We conclude that transcriptional regulation by ATF4 maintains ß-cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes.

Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-mediated heme-regulated inhibitor (HRI) activation.

The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Four eIF2α kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2α kinase is activated by nsPEF treatment. To assess the responsible eIF2α kinase, we used previously established eIF2α kinase quadruple knockout (4KO) and single eIF2α kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2α phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2α phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2α phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2α kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS.

Cell-based HTS identifies a chemical chaperone for preventing ER protein aggregation and proteotoxicity.

The endoplasmic reticulum (ER) is responsible for folding secretory and membrane proteins, but disturbed ER proteostasis may lead to protein aggregation and subsequent cellular and clinical pathologies. Chemical chaperones have recently emerged as a potential therapeutic approach for ER stress-related diseases. Here, we identified 2-phenylimidazo[2,1-b]benzothiazole derivatives (IBTs) as chemical chaperones in a cell-based high-throughput screen. Biochemical and chemical biology approaches revealed that IBT21 directly binds to unfolded or misfolded proteins and inhibits protein aggregation. Finally, IBT21 prevented cell death caused by chemically induced ER stress and by a proteotoxin, an aggression-prone prion protein. Taken together, our data show the promise of IBTs as potent chemical chaperones that can ameliorate diseases resulting from protein aggregation under ER stress.

Atovaquone is active against AML by upregulating the integrated stress pathway and suppressing oxidative phosphorylation.

Atovaquone, a US Food and Drug Administration-approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 <30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.

Molecular mechanisms of limb regeneration: insights from regenerating legs of the cricket Gryllus bimaculatus.

This review summarizes recent advances in leg regeneration research, focusing on the cricket Gryllus bimaculatus. Recent studies have revealed molecular mechanisms on blastema formation, establishment of positional information, and epigenetic regulation during leg regeneration. Especially, these studies have provided molecular bases in classical conceptual models such as the polar coordinate model, the intercalation model, the boundary model, the steepness model, etc., which were proposed to interpret regeneration processes of the cockroach legs. When a leg is amputated, a blastema is formed through the activation of the Janus-kinase (Jak)/Signal-Transduction-and-Activator-of-Transcription (STAT) pathway. Subsequently, the Hedgehog/Wingless/Decapentaplegic/Epidermal-growth-factor pathways instruct distalization in the blastema, designated as the molecular boundary model. Downstream targets of this pathway are transcription factors Distal-less (Dll) and dachshund (dac), functioning as key regulators of proximodistal pattern formation. Dll and dac specify the distal and proximal regions in the blastema, respectively, through the regulation of tarsal patterning genes. The expression of leg patterning genes during regeneration may be epigenetically controlled by histone H3K27 methylation via Enhancer-of-zeste and Ubiquitously-transcribed-tetratricopeptide-repeat-gene-X-chromosome. For the molecular mechanism of intercalation of the missing structures between the amputated position and the most distal one, Dachsous/Fat (Ds/Ft) steepness model has been proposed, in which the Ds/Ft pathway maintains positional information and determines leg size through dac expression. This model was theoretically verified to interpret the experimental results obtained with cricket legs. Availability of whole-genome sequence information, regeneration-dependent RNA interference, and genome editing technique will have the cricket be an ideal model system to reveal gene functions in leg regeneration.

Enhancer of zeste plays an important role in photoperiodic modulation of locomotor rhythm in the cricket, Gryllus bimaculatus.

INTRODUCTION: Insects show daily behavioral rhythms controlled by an endogenous oscillator, the circadian clock. The rhythm synchronizes to daily light-dark cycles (LD) and changes waveform in association with seasonal change in photoperiod. RESULTS: To explore the molecular basis of the photoperiod-dependent changes in circadian locomotor rhythm, we investigated the role of a chromatin modifier, Enhancer of zeste (Gb'E(z)), in the cricket, Gryllus bimaculatus. Under a 12 h:12 h LD (LD 12:12), Gb'E(z) was constitutively expressed in the optic lobe, the site of the biological clock; active phase (α) and rest phase (ρ) were approximately 12 h in duration, and α/ρ ratio was approximately 1.0. When transferred to LD 20:4, the α/ρ ratio decreased significantly, and the Gb'E(z) expression level was significantly reduced at 6 h and 10 h after light-on, as was reflected in the reduced level of trimethylation of histone H3 lysine 27. This change was associated with change in clock gene expression profiles. The photoperiod-dependent changes in α/ρ ratio and clock gene expression profiles were prevented by knocking down Gb'E(z) by RNAi. CONCLUSIONS: These results suggest that histone modification by Gb'E(z) is involved in photoperiodic modulation of the G. bimaculatus circadian rhythm.

Leg regeneration is epigenetically regulated by histone H3K27 methylation in the cricket Gryllus bimaculatus.

Hemimetabolous insects such as the cricket Gryllus bimaculatus regenerate lost tissue parts using blastemal cells, a population of dedifferentiated proliferating cells. The expression of several factors that control epigenetic modification is upregulated in the blastema compared with differentiated tissue, suggesting that epigenetic changes in gene expression might control the differentiation status of blastema cells during regeneration. To clarify the molecular basis of epigenetic regulation during regeneration, we focused on the function of the Gryllus Enhancer of zeste [Gb'E(z)] and Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (Gb'Utx) homologues, which regulate methylation and demethylation of histone H3 lysine 27 (H3K27), respectively. Methylated histone H3K27 in the regenerating leg was diminished by Gb'E(z)(RNAi) and was increased by Gb'Utx(RNAi). Regenerated Gb'E(z)(RNAi) cricket legs exhibited extra leg segment formation between the tibia and tarsus, and regenerated Gb'Utx(RNAi) cricket legs showed leg joint formation defects in the tarsus. In the Gb'E(z)(RNAi) regenerating leg, the Gb'dac expression domain expanded in the tarsus. By contrast, in the Gb'Utx(RNAi) regenerating leg, Gb'Egfr expression in the middle of the tarsus was diminished. These results suggest that regulation of the histone H3K27 methylation state is involved in the repatterning process during leg regeneration among cricket species via the epigenetic regulation of leg patterning gene expression.

Analysis of RNA-Seq data reveals involvement of JAK/STAT signalling during leg regeneration in the cricket Gryllus bimaculatus.

In the cricket Gryllus bimaculatus, missing distal parts of the amputated leg are regenerated from the blastema, a population of dedifferentiated proliferating cells that forms at the distal tip of the leg stump. To identify molecules involved in blastema formation, comparative transcriptome analysis was performed between regenerating and normal unamputated legs. Components of JAK/STAT signalling were upregulated more than twofold in regenerating legs. To verify their involvement, Gryllus homologues of the interleukin receptor Domeless (Gb'dome), the Janus kinase Hopscotch (Gb'hop) and the transcription factor STAT (Gb'Stat) were cloned, and RNAi was performed against these genes. Gb'dome(RNAi), Gb'hop(RNAi) and Gb'Stat(RNAi) crickets showed defects in leg regeneration. Blastema expression of Gb'cyclinE was decreased in the Gb'Stat(RNAi) cricket compared with that in the control. Hyperproliferation of blastema cells caused by Gb'fat(RNAi) or Gb'warts(RNAi) was suppressed by RNAi against Gb'Stat. The results suggest that JAK/STAT signalling regulates blastema cell proliferation during leg regeneration.

Lowfat, a mammalian Lix1 homologue, regulates leg size and growth under the Dachsous/Fat signaling pathway during tissue regeneration.

In the cricket Gryllus bimaculatus, missing distal parts of amputated legs are regenerated from blastemas based on positional information. The Dachsous/Fat (Ds/Ft) signaling pathway regulates blastema cell proliferation and positional information along the longitudinal axis during leg regeneration. Herein, we show that the Gryllus homologue of Lowfat (Gb'Lft), which modulates Ds/Ft signaling in Drosophila, is involved in leg regeneration. Gb'lft is expressed in regenerating legs, and RNAi against Gb'lft (Gb'lft(RNAi)) suppressed blastema cell hyperproliferation caused by Gb'ft(RNAi) or Gb'ds(RNAi) but enhanced that caused by Gb'kibra(RNAi) or Gb'warts(RNAi). In Gb'lft(RNAi) nymphs, missing parts of amputated legs were regenerated, but the length of the regenerated legs was shortened depending on the position of the amputation. Both normal and reversed intercalary regeneration occurred in Gb'lft(RNAi) nymphs, suggesting that Gb'Lft is involved in blastema cell proliferation and longitudinal leg regeneration under the Ds/Ft signaling pathway, but it is not required for intercalary regeneration.

Aging-related changes of QT and RR intervals in conscious guinea pigs.

INTRODUCTION: Although guinea pigs are suitable for in vivo QT assessment of newly discovered drugs at the pre-clinical stage because of the similarity of the ion channels between the guinea pig heart and the human, there is limited data available regarding the characteristics of QT interval in conscious guinea pigs. Aging is one of several factors which have been shown to affect the QT interval in humans and animals. In the present study, we examined the influence of age on QT and RR intervals in conscious guinea pigs. METHODS: Electrocardiograms were recorded from female Hartley guinea pigs at the age of 6 weeks (young; n=6) and 23 months (old; n=4) via a telemetry system. The QT and RR intervals were measured during daytime and nighttime, and following intravenous bolus injection of E-4031 (0.1 mg/kg) or terfenadine (4 mg/kg). Comparisons were made to determine group differences in: (1) the normal values of the QT and RR intervals, (2) the best-fit QT-correction formula, (3) the circadian rhythm of QT and RR intervals, and (4) drug effects on repolarization. RESULTS: The normal values of QT and RR intervals in the old group were significantly longer than those in the young group. The best-fit formula for correcting QT interval was a modified Bazett's formula for both young and old groups. The old group did not show the nocturnal variation of either QT or RR interval. Terfenadine caused significantly greater QTc prolongation in the old group compared to the young. DISCUSSION: Aging affects resting QT and RR intervals in conscious female guinea pigs, a factor which should be considered when examining the effects of compounds on cardiac repolarization. Also, the present study suggests a possibility that age can affect QTc prolongation induced by some IKr blockers.

Changes of micro-RNA expression in rat liver treated by acetaminophen or carbon tetrachloride--regulating role of micro-RNA for RNA expression.

Recently, microRNAs, involved in RNA interference, were discovered as a new gene regulation, with little is known in the filed of toxicology. In this study, a toxic dose of acetaminophen or carbon tetrachloride was administered singly to male rats, and microarry analysis using mirVana miRNA bioarray was performed. Partial least squares-discriminant analysis of the microarray data revealed that microRNAs expression was specifically changed by treatments at 6 hr after dosing. Furthermore, we focused on miR298 and miR370 among the microRNAs commonly affected by hepatotoxicants, because they were speculated to regulate an oxidative stress-related gene. From real-time RT-PCR analysis, microRNAs expression was suppressed by hepatotoxicants at 6 and 24 hr. Regarding acetaminophen, the decreases were found even though there were no morphological changes in the liver at 6 hr. To investigate these 2 microRNAs in more detail, we measured their expression, WST-1 for mitochondrial function and LDH release for cell collapse in primary cultured hepatocytes exposed to several concentrations of acetaminophen for 3 hr. At more than 5 mM, the microRNA expression and WST-1 decreased, whereas LDH was unchanged. Therefore, the change in microRNA expression occurred at the time when mitochondrial function was damaged prior to cell collapse. From all the above findings, we conclude that microRNAs were affected by hepatotoxicants and that the changes were found in the early phase of toxicity. Thus, our data suggest microRNAs have an important role for toxicological mechanism and we proposed that the changes in microRNA expression might be key molecules for toxicity expression.

Simultaneous measurement of nucleated cell counts and cellular differentials in rat bone marrow examination using flow cytometer.

The purpose of this study was to establish the simultaneous measurement of nucleated cell counts and cellular differentials in rat bone marrow examination. The bone marrow cells were stained with an anthraquinone fluorescent DNA stain (DRAQ5) and fluorescence-labeled antibodies, and were analyzed quantitatively using a flow cytometer in the presence of internal standard beads. DRAQ5 distinguished populations of nucleated cells. The absolute counts of nucleated cells were determined using an internal standard, and were equivalent to that measured by the electrical resistance method. The population of nucleated cells was classified into myeloids and erythroids by labeling with CD11b/c and CD71 antibodies, respectively. In a separate examination, T- and B-lymphocytes were also classified by labeling with CD3 and CD45RA antibodies, respectively. The classification of each cell lineage was identical with that of the alternative flow-cytometric method in which cells were differentiated according to cellular size and the fluorescence of a peroxidase indicator, 2',7'-dichlorofluorescin. The ratios of cell lineage, together with myeloid/erythroid ratio (ME), were the same as those obtained by a manual microscopic method. The present flow cytometric method enables the simultaneous measurement of the total nucleated cell counts and cellular differentials of rat bone marrow cells, allowing for rapid and highly quantitative bone marrow examination in rats.