Kana Namiki

Arginine Methylation of FOXO Transcription Factors Inhibits Their Phosphorylation by Akt

Forkhead box O (FOXO) transcription factors, the key regulators of cell survival, are negatively controlled through the PI3K-Akt signaling pathway. Phosphorylation of FOXO by Akt leads to cytoplasmic localization and subsequent degradation via the ubiquitin-proteasome system. Here we show a paradigm of FOXO1 regulation by the protein arginine methyltransferase PRMT1. PRMT1 methylated FOXO1 at conserved Arg248 and Arg250 within a consensus motif for Akt phosphorylation; this methylation directly blocked Akt-mediated phosphorylation of FOXO1 at Ser253 in vitro and in vivo. Silencing of PRMT1 by small interfering RNA enhanced nuclear exclusion, polyubiquitination, and proteasomal degradation of FOXO1. PRMT1 knockdown led to a decrease in oxidative-stress-induced apoptosis depending on the PI3K-Akt signaling pathway. Furthermore, stable expression of enzymatic inactive PRMT1 mutant increased resistance to apoptosis, whereas this effect was reversed by expression of phosphorylation-deficient FOXO1. Our findings predict a role for arginine methylation as an inhibitory modification against Akt-mediated phosphorylation.

ScaleS: an optical clearing palette for biological imaging

Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimers disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimers disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.

Involvement of p38α Mitogen-activated Protein Kinase in Lung Metastasis of Tumor Cells

To study the role of p38 mitogen-activated protein kinase (p38) activity during the process of metastasis, p38α+/- mice were subjected to an in vivo metastasis assay. The number of lung colonies of tumor cells intravenously injected in p38α+/- mice was markedly decreased compared with that in wild-type (WT) mice. On the other hand, the time-dependent increase in tumor volume after subcutaneous tumor cells transplantation was comparable between WT and p38α+/- mice. Platelets of p38α+/- mice were poorly bound to tumor cells in vitro and in vivo compared with those of WT mice. E- and P-selectin mRNAs were markedly induced in the lung after intravenous injection of tumor cells. However, the induction of these selectin mRNAs in p38α+/- mice was weaker than that in WT mice. Furthermore, the resting expression levels of E-selectin in lung endothelial cells and P-selectin in platelets of p38α+/- mice were suppressed compared with those of WT mice. The number of tumor cells attached on lung endothelial cells of p38α+/- mice was significantly reduced compared with that of WT mice. The transmigrating activity of tumor cells through lung endothelial cells of p38α+/- mice was similar to that of WT mice. These results suggest that p38α plays an important role in extravasation of tumor cells, possibly through regulating the formation of tumor-platelet aggregates and their interaction with the endothelium involved in a step of hematogenous metastasis.

N-type Calcium Channel in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca2+ channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca2+ channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca2+ channel α1B-deficient (α1B−/−) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35–55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α1B−/− mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α1B−/− mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α1B−/− mice and was significantly inhibited by a selective N-type Ca2+ channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca2+. These results suggest that the N-type Ca2+ channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.

Mechanism for p38α-mediated experimental autoimmune encephalomyelitis

Background: p38 signaling pathway plays a key role in inflammatory diseases. Results: A single copy disruption of the p38α gene or a p38α inhibitor markedly reduced the pathogenesis of EAE by decreasing IL-17 production. Conclusion: p38α regulates the pathogenesis of EAE through transcriptional regulation of IL-17 production. Significance: Anti-p38α strategy achieves therapeutic benefit for the treatment of multiple sclerosis. One of the mitogen-activated protein kinases, p38, has been found to play a crucial role in various inflammatory responses. In this study, we analyzed the roles of p38α in multiple sclerosis, using an animal model, experimental autoimmune encephalomyelitis (EAE). p38α+/− mice (p38α−/− showed embryonic lethality) showed less severe neurological signs than WT mice. Adoptive transfer of lymph node cells (LNC) from sensitized WT mice with MOG(35–55) to naive WT-induced EAE was much more severe compared with the case using LNC from sensitized p38α+/− mice. Comprehensive analysis of cytokines from MOG(35–55)-challenged LNC by Western blot array revealed that production of IL-17 was significantly reduced by a single copy disruption of the p38α gene or a p38 inhibitor. Likewise, by a luciferase reporter assay, an electrophoresis mobility shift assay, and characterization of the relationship between p38 activity and IL-17 mRNA expression, we confirmed that p38 positively regulates transcription of the Il17 gene. Furthermore, oral administration of a highly specific p38α inhibitor (UR-5269) to WT mice at the onset of EAE markedly suppressed the progression of EAE compared with a vehicle group. These results suggest that p38α participates in the pathogenesis of EAE through IL-17 induction.

Involvement of p38α in Kainate-Induced Seizure and Neuronal Cell Damage

We investigated how p38α mitogen-activated protein kinase (p38) is related to kainate-induced epilepsy and neuronal damages, by using the mice with a single copy disruption of the p38 α gene (p38α+/−). Mortality rate and seizure score of p38α+/− mice administered with kainate were significantly reduced compared with the case of wild-type (WT) mice. This was clearly supported by the electroencephalography data in which kainate-induced seizure duration and frequency in the brain of p38α+/− mice were significantly suppressed compared to those of WT mice. As a consequence of seizure, kainate induced delayed neuronal damages in parallel with astrocytic growth in the hippocampus and ectopic innervation of the mossy fibers into the stratum oriens in the CA3 region of hippocampus in WT mice, whose changes were moderate in p38α+/− mice. Likewise, kainate-induced phosphorylation of calcium/calmodulin-dependent kinase II in the hippocampus of p38α +/− mice was significantly decreased compared to that of WT mice. These results suggest that p38α signaling pathway plays an important role in epileptic seizure and excitotoxicity.

PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination

PRMT8 directly hydrolyzes phosphatidylcholine, which is important for brain functions. The development of vertebrate neurons requires a change in membrane phosphatidylcholine (PC) metabolism. Although PC hydrolysis is essential for enhanced axonal outgrowth mediated by phospholipase D (PLD), less is known about the determinants of PC metabolism on dendritic arborization. We show that protein arginine methyltransferase 8 (PRMT8) acts as a phospholipase that directly hydrolyzes PC, generating choline and phosphatidic acid. We found that PRMT8 knockout mice (prmt8−/−) displayed abnormal motor behaviors, including hindlimb clasping and hyperactivity. Moreover, prmt8−/− mice and TALEN-induced zebrafish prmt8 mutants and morphants showed abnormal phenotypes, including the development of dendritic trees in Purkinje cells and altered cerebellar structure. Choline and acetylcholine levels were significantly decreased, whereas PC levels were increased, in the cerebellum of prmt8−/− mice. Our findings suggest that PRMT8 acts both as an arginine methyltransferase and as a PC-hydrolyzing PLD that is essential for proper neurological functions.

Single-cell bioluminescence imaging of deep tissue in freely moving animals

Improved spy tactics for single cells Bioluminescence imaging is a tremendous asset to medical research, providing a way to monitor living cells noninvasively within their natural environments. Advances in imaging methods allow researchers to measure tumor growth, visualize developmental processes, and track cell-cell interactions. Yet technical limitations exist, and it is difficult to image deep tissues or detect low cell numbers in vivo. Iwano et al. designed a bioluminescence imaging system that produces brighter emission by up to a factor of 1000 compared with conventional technology (see the Perspective by Nasu and Campbell). Individual tumor cells were successfully visualized in the lungs of mice. Small numbers of striatal neurons were detected in the brains of naturally behaving marmosets. The ability of the substrate to cross the blood-brain barrier should provide important opportunities for neuroscience research. Science, this issue p. 935; see also p. 868 A bioengineered light source allows in vivo imaging of individual cells. Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.

Therapeutic effect of lung mixed culture-derived epithelial cells on lung fibrosis

Cell-based therapy is recognized as one of potential therapeutic options for lung fibrosis. However, preparing stem/progenitor cells is complicated and not always efficient. Here, we show easily prepared cell populations having therapeutic capacity for lung inflammatory disease that are named as ‘lung mixed culture-derived epithelial cells’ (LMDECs). LMDECs expressed surfactant protein (SP)-C and gave rise to type I alveolar epithelial cells (AECs) in vitro and in vivo that partly satisfied type II AEC-like characteristics. An intratracheal delivery of not HEK 293 cells but LMDECs to the lung ameliorated bleomycin (BLM)-induced lung injury. A comprehensive analysis of bronchoalveolar fluid by western blot array revealed that LMDEC engraftment could improve the microenvironment in the BLM-instilled lung in association with stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 signaling axis. SDF-1 enhanced both migration activity and differentiating efficiency of LMDECs. Further classification of LMDECs by flow cytometric study showed that a major population of LMDECs (LMDECMaj, 84% of total LMDECs) was simultaneously SP-C+, CD44+, CD45+, and hematopoietic cell lineage+ and that LMDECs included bronchioalveolar stem cells (BASCs) showing SP-C+Clara cell secretory protein+stem cell antigen (Sca)1+ as a small population (1.8% of total LMDECs). CD44+-sorted LMDECMaj and Sca1+-sorted LMDECs equally ameliorated fibrosis induced by BLM like LMDECs did. However, infiltrated neutrophils were observed in Sca1+-sorted LMDEC-treated alveoli that was not typical in LMDECMaj- or LMDEC-treated alveoli. These findings suggest that the protective effect of LMDECs against BLM-induced lung injury depends greatly on that of LMDECMaj. Furthermore, the cells expressing both alveolar epithelial and hematopoietic cell lineage markers (SP-C+CD45+) that have characteristics corresponding to LMDECMaj were observed in the alveoli of lung and increased approximately threefold in response to BLM instillation. Taken together, LMDECs newly classified in the present study are easily culture expanded and have a potential role in future regenerative cell therapy for pulmonary fibrosis.

p38α controls self-renewal and fate decision of neurosphere-forming cells in adult hippocampus

Neural stem cells (NSC) from the adult hippocampus easily lose their activityin vitro. Efficientin vitro expansion of adult hippocampus‐derived NSC is important for generation of tools for research and cell therapy. Here, we show that a single copy disruption or pharmacological inhibition of p38α enables successful long‐term neurosphere culture of adult mouse hippocampal cells. Expanded neurospheres with high proliferative activity differentiated into the three neuronal lineages under differentiating conditions. Thus, inhibition of p38α can maintain adult hippocampal NSC activityin vitro.