Deletion of Long Isoform of Eukaryotic Elongation Factor 1Bδ Leads to Audiogenic Seizures and Aversive Stimulus-Induced Long-Lasting Activity Suppression in Mice

Alternative splicing enables a gene to give rise to diverse protein products. The Eef1d gene produces two isoforms: a short isoform that encodes translation elongation factor 1Bδ (eEF1Bδ1), and a long isoform that encodes the heat shock-responsive transcription factor eEF1BδL. Previously, we found that eEF1BδL was a splice variant that was specific to the brain and testis, and the protein encoded is thought to have a function in the central nervous system. In this study, we generated knockout (KO) mice of C57BL/6J background that selectively lacked a specific exon in Eef1d for the long isoform. These KO mice lacked eEF1BδL, but not eEF1Bδ1, in the brain. Although the KO mice showed normal anxiety-related and learning behavior in behavioral tests, some showed severe seizures in response to loud sounds (90 dBA), an audiogenic seizures (AGS) response. Furthermore, after the KO mice had been subjected to the fear conditioning test, they showed remarkably decreased locomotor activity in their home cage and in the open-field and elevated plus-maze tests. After the fear conditioning test, a significant decrease in brain weight, atrophy of the hippocampus and midbrain, and reduced cortical layer thickness were observed in the KO mice. We also found a compensatory increase in the eEF1Bδ1 level and elevated protein synthesis with the induction of endoplasmic reticulum stress markers in these mice. Our results suggest that eEF1BδL has an important role in normal brain function especially when exposed to external stimuli

CaM kinase Iα–induced phosphorylation of Drp1 regulates mitochondrial morphology

Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKIα is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types

Regulation of Hypoxic Signaling and Oxidative Stress via the MicroRNA–SIRT2 Axis and Its Relationship with Aging-Related Diseases

The sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylase and ADP-ribosyl transferases plays key roles in aging, metabolism, stress response, and aging-related diseases. SIRT2 is a unique sirtuin that is expressed in the cytosol and is abundant in neuronal cells. Various microRNAs were recently reported to regulate SIRT2 expression via its 3′-untranslated region (UTR), and single nucleotide polymorphisms in the miRNA-binding sites of SIRT2 3′-UTR were identified in patients with neurodegenerative diseases. The present review highlights recent studies into SIRT2-mediated regulation of the stress response, posttranscriptional regulation of SIRT2 by microRNAs, and the implications of the SIRT2–miRNA axis in aging-related diseases

Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells

Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies

Regulation of Translation Factor EEF1D Gene Function by Alternative Splicing

Alternative splicing is an exquisite mechanism that allows one coding gene to have multiple functions. The alternative splicing machinery is necessary for proper development, differentiation and stress responses in a variety of organisms, and disruption of this machinery is often implicated in human diseases. Previously, we discovered a long form of eukaryotic elongation factor 1Bδ (eEF1Bδ; this long-form eEF1Bδ results from alternative splicing of EEF1D transcripts and regulates the cellular stress response by transcriptional activation, not translational enhancement, of heat-shock responsive genes. In this review, we discuss the molecular function of EEF1D alternative splicing products and the estimated implication of human diseases

Response to Stimulations Inducing Circadian Rhythm in Human Induced Pluripotent Stem Cells

Regenerative medicine and disease modeling are expanding rapidly, through the development of human-induced pluripotent stem cells (hiPSCs). Many exogeneous supplements are often used for the directed differentiation of hiPSCs to specific lineages, such as chemicals and hormones. Some of these are known to synchronize the circadian clock, like forskolin (Frk) and dexamethasone (Dex); however, the response to these stimulations has not been fully elucidated for hiPSCs. In this study, we examined the response of clock genes to synchronizing stimulation, and compared it with fully differentiated cells, U2OS, and fibroblasts. The expression of clock genes did not show circadian rhythms in hiPSCs with Frk and Dex, which could be due to the significantly low levels of BMAL1. On the other hand, a circadian-like rhythm of D-box binding protein (DBP) expression was observed in hiPSCs by culturing them in an environment with a simulated body temperature. However, the inhibition of temperature-inducible factors, which are involved in temperature rhythm-induced synchronization, could not repress the expression of such rhythms, while the inhibition of HIF-1α significantly repressed them. In summary, we suggest that clock genes do not respond to the synchronizing agents in hiPSCs; instead, a unique circadian-like rhythm is induced by the temperature rhythm

TRPM7 Kinase Inactivation Results in Macrophage Alkalinization and Increased Phagocytic Activity

Transient receptor potential melastatin 7 (TRPM7) is a unique protein that is both an ion channel and a cytosolic protein kinase. Normally, TRPM7 channels are tonically inhibited by cytoplasmic Mg2+, polyamines and acidic pH. Channel basal activity can be increased by alkalinization or Mg2+ depletion. We have used the TRPM7 kinase-dead (KD K1646R) mouse to investigate the consequences of kinase inactivation in immune cell function. It also serves as a convenient system to elucidate kinase-channel interactions at a functional level. CD4 and CD8 T lymphocytes isolated from the spleens of TRPM7 KD mice have defects in blastogenesis, proliferation and reduced store-operated calcium entry (SOCE) without a change in TRPM7 current magnitudes. Here we have investigated the effect of kinase inactivation in peritoneal and splenic macrophages. We examined phagocytosis using multiple approaches, finding that KD splenic macrophages phagocytize latex beads, zymosan particles and opsonized red blood cells more efficiently compared to WT. We observed somewhat higher basal cytosolic Ca2+ levels in KD macrophages compared to WT, but no differences in SOCE were apparent. Addition of calcium ionophore ionomycin overnight did not affect phagocytosis rates. By contrast, we found that cytosolic pH was alkalinized in KD mouse cells. In order to test if the KD effects on phagocytosis and pH were linked, we incubated the macrophages in presence of sodium-hydrogen exchanger 1 (NHE1) blockers and found that both pH and phagocytosis returned to normal, WT, levels. Moreover, the basal TRPM7 channel activity (current magnitude at break-in) was also reduced. WT and KD channel sensitivity to Mg2+ was similar. In summary, we have identified a new role for TRPM7 kinase as a regulator of cellular pH and phagocytosis

TRPM7 Kinase Inactivation Results in Macrophage Alkalinization and Increased Phagocytic Activity

Transient receptor potential melastatin 7 (TRPM7) is a unique protein that is both an ion channel and a cytosolic protein kinase. Normally, TRPM7 channels are tonically inhibited by cytoplasmic Mg2+, polyamines and acidic pH. Channel basal activity can be increased by alkalinization or Mg2+ depletion. We have used the TRPM7 kinase-dead (KD K1646R) mouse to investigate the consequences of kinase inactivation in immune cell function. It also serves as a convenient system to elucidate kinase-channel interactions at a functional level. CD4 and CD8 T lymphocytes isolated from the spleens of TRPM7 KD mice have defects in blastogenesis, proliferation and reduced store-operated calcium entry (SOCE) without a change in TRPM7 current magnitudes. Here we have investigated the effect of kinase inactivation in peritoneal and splenic macrophages. We examined phagocytosis using multiple approaches, finding that KD splenic macrophages phagocytize latex beads, zymosan particles and opsonized red blood cells more efficiently compared to WT. We observed somewhat higher basal cytosolic Ca2+ levels in KD macrophages compared to WT, but no differences in SOCE were apparent. Addition of calcium ionophore ionomycin overnight did not affect phagocytosis rates. By contrast, we found that cytosolic pH was alkalinized in KD mouse cells. In order to test if the KD effects on phagocytosis and pH were linked, we incubated the macrophages in presence of sodium-hydrogen exchanger 1 (NHE1) blockers and found that both pH and phagocytosis returned to normal, WT, levels. Moreover, the basal TRPM7 channel activity (current magnitude at break-in) was also reduced. WT and KD channel sensitivity to Mg2+ was similar. In summary, we have identified a new role for TRPM7 kinase as a regulator of cellular pH and phagocytosis

Regulation of TRPM7 by Cytosolic Mg2+ and pH: Insights from VSP Expression

TRPM7 is an ion channel/protein kinase belonging to TRP melastatin and eEF2 kinase families. Under physiological conditions, most native TRPM7 channels are inactive, due to inhibition by cytoplasmic Mg2+, protons and polyamines. ITRPM7 is strongly potentiated when cell cytosol is depleted of Mg2+ or alkalinized. In Jurkat T cells, Mg2+ inhibition involves a high and a low affinity inhibitor sites, whereas proton inhibition involves only one site. Like many other TRP channels, TRPM7 is activated by PI(4,5)P2 and suppressed by its hydrolysis. Here we examined Mg2+ and pH inhibition of native TRPM7 channels in HEK293 cells overexpressing voltage-sensitive phospholipid phosphatase (VSP) or its catalytically inactive C363S mutant. Phosphoinositide depletion by VSP increased the sensitivity of channels to pH and high Mg2+. Specifically internal pH values that were stimulatory when C363S was expressed (pH 8.2) became inhibitory in the wildtype VSP-expressing cells. 150 uM Mg2+ or pHi 6.5 inhibited ITRPM7 both in wildtype and C363S VSP-expressing cells but with a faster time course in the former group. Both basal and maximum currents were reduced in VSP expressing cells while the mean time to reach maximum amplitude was shortened. In order to prevent the activation of VSP by voltages reaching +85 mV used for recording TRPM7 currents, we tested the effects of VSP on inward currents in divalent cation free solutions by applying voltage ramps reaching only +20 mV. Surprisingly, this command voltage protocol produced results similar to those obtained from ramps reaching +85 mV. These observations suggest that in HEK293 cells, VSP may have significant basal activity even prior to application of depolarizing voltage pulses, possibly due to the depolarized resting membrane potential of these cells. Consistent with this scenario, a subpopulation of wildtype VSP transfected cells behaved like C363S-transfected cells, likely because their resting potential was more negative. Growing HEK cells in 25 mM KCl instead of 5 mM to shift K+ equilibrium potential by ∼+40 mV did not result in increased basal VSP activity, however. In summary, our experiments suggest that voltage-independent Mg2+ and pH inhibition of TRPM7 channels is not direct but, rather, reflects electrostatic screening and disruption of PI(4,5)P2-channel interactions