Yoshiko Nagaoka

Go2 G protein mediates galanin inhibitory effects on insulin release from pancreatic β cells

The neuropeptide galanin regulates numerous physiological activities in the body, including feeding and metabolism, learning and memory, nociception and spinal reflexes, and anxiety and related behaviors. Modulation of blood glucose levels by suppressing insulin release was the first reported activity for galanin. This inhibition was mediated by one or more pertussis toxin-sensitive G proteins of the Gi/o subfamily. However, the molecular identities of the specific G protein(s) and intracellular effectors have not been fully revealed. Recently, we demonstrated that mice lacking Go2, but not other members of the Gi/o protein family, secrete more insulin than controls upon glucose challenge, indicating that Go2 is a major transducer for the inhibitory regulation of insulin secretion. In this study, we investigated galanin signaling mechanisms in β cells using cell biological and electrophysiological approaches. We found that islets lacking Go2, but not other Gi/o proteins, lose the inhibitory effect of galanin on insulin release. Potentiation of ATP-sensitive potassium (KATP) and inhibition of calcium currents by galanin were disrupted by anti-Go2α antibodies. Galanin actions on KATP and calcium currents were completely lost in Go2−/− β cells. Furthermore, the hyperglycemic effect of galanin is also blunted in Go2−/− mice. Our results demonstrate that Go2 mediates the inhibition of insulin release by galanin by regulating both KATP and Ca2+ channels in mice. Our findings provide insight into galanins action in glucose homeostasis. The results may also be relevant to the understanding of galanin signaling in other biological systems, especially the central nervous system.

Reassessing the Role of APOBEC3G in Human Immunodeficiency Virus Type 1 Infection of Quiescent CD4+ T-Cells

HIV-1 is restricted for infection of primary quiescent T-cells. After viral entry, reverse transcription is initiated but is not completed. Various hypotheses have been proposed for this cellular restriction including insufficient nucleotide pools and cellular factors, but none have been confirmed as the primary mechanism for restriction. A recent study by Chiu et al. implicates APOBEC3G, an anti-retroviral cytidine deaminase, as the cellular restriction factor. Here, we attempted to confirm these findings using the same strategy as reported by Chiu et al. of siRNA targeting knock-down of APOBEC3G expression. In contrast to the published study, our results do not support a role for APOBEC3G in restriction of HIV-1 in quiescent CD4+ T-cells. In our study, we tested the same siRNA as reported by Chiu et al. as well as two additional siRNAs targeting APOBEC3G, one of which showed 2-fold greater knock-down of APOBEC3G mRNA. However, none of the three siRNAs tested had a discernable effect on enhancing infection by HIV-1 in quiescent CD4+ T-cells. Therefore, we conclude that the primary mechanism of HIV-1 restriction in quiescent CD4+ T-cells remains to be elucidated.

Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of Endogenous microRNAs

Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs (miR-302a and miR-302d) and mCherry conjugated to the targets of differentiated cells specific miRNAs (miR-142-3p, miR-155, and miR-223). The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors (OCT4, SOX2, KLF4 and cMYC) were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells.

Augmented glucose-induced insulin release in mice lacking G(o2), but not G(o1) or G(i) proteins.

Insulin secretion by pancreatic β cells is a complex and highly regulated process. Disruption of this process can lead to diabetes mellitus. One of the various pathways involved in the regulation of insulin secretion is the activation of heterotrimeric G proteins. Bordetella pertussis toxin (PTX) promotes insulin secretion, suggesting the involvement of one or more of three Gi and/or two Go proteins as suppressors of insulin secretion from β cells. However, neither the mechanism of this inhibitory modulation of insulin secretion nor the identity of the Gi/o proteins involved has been elucidated. Here we show that one of the two splice variants of Go, Go2, is a key player in the control of glucose-induced insulin secretion by β cells. Mice lacking Go2α, but not those lacking α subunits of either Go1 or any Gi proteins, handle glucose loads more efficiently than wild-type (WT) mice, and do so by increased glucose-induced insulin secretion. We thus provide unique genetic evidence that the Go2 protein is a transducer in an inhibitory pathway that prevents damaging oversecretion of insulin.

Generation of Human Induced Pluripotent Stem Cells Bearing an Anti-HIV Transgene by a Lentiviral Vector Carrying an Internal Murine Leukemia Virus Promoter

The recent development of induced pluripotent stem cells (iPSCs) by ectopic expression of defined reprogramming factors offers enormous therapeutic opportunity. To deliver these factors, murine leukemia virus (MLV)-based vectors have been broadly used in the setting of hematopoietic stem cell transplantation. However, MLV vectors have been implicated in malignancy induced by insertional mutagenesis, whereas lentiviral vectors have not. Furthermore, the infectivity of MLV vectors is limited to dividing cells, whereas lentiviral vectors can also transduce nondividing cells. One important characteristic of MLV vectors is a self-silencing property of the promoter element in pluripotent stem cells, allowing temporal transgene expression in a nonpluripotent state before iPSC derivation. Here we test iPSC generation using a novel chimeric vector carrying a mutant MLV promoter internal to a lentiviral vector backbone, thereby containing the useful properties of both types of vectors. Transgene expression of this chimeric vector was highly efficient compared with that of MLV vectors and was silenced specifically in human embryonic stem cells. Human fetal fibroblasts transduced with the vector encoding each factor were efficiently reprogrammed into a pluripotent state, and these iPSCs had potential to differentiate into a variety of cell types. To explore the possibility of iPSCs for gene therapy, we established iPSC clones expressing a short hairpin RNA (shRNA) targeting chemokine receptor 5 (CCR5), the main coreceptor for HIV-1. Using a reporter construct for CCR5 expression, we confirmed that CCR5 shRNA was expressed and specifically knocked down the reporter expression in iPSCs. These data indicate that our chimeric lentiviral vector is a valuable tool for generation of iPSCs and the combination with vectors encoding transgenes allows for rapid establishment of desired genetically engineered iPSC lines.

Human immunodeficiency virus type 1 Vpr binds to the N lobe of the Wee1 kinase domain and enhances kinase activity for CDC2.

ABSTRACT Human immunodeficiency virus type 1 Vpr is a virion-associated accessory protein that has multiple activities within an infected cell. One of the most dramatic effects of Vpr is the induction of cell cycle arrest at the G2/M boundary, followed by apoptosis. This effect has implications for CD4+ cell loss in AIDS. In normal cell cycle regulation, Wee1, a key regulator for G2-M progression, phosphorylates Tyr15 on Cdc2 and thereby blocks the progression of cells into M phase. We demonstrate that Vpr physically interacts with Wee1 at the N lobe of the kinase domain analogous to that present in other kinases. This interaction with Vpr enhances Wee1 kinase activity for Cdc2. Overexpression of Wee1 kinase-deficient mutants competes for Vpr-mediated cell cycle arrest, and deletion of the region of Wee1 that binds Vpr abrogates that competition. However, the Vpr mutants I74P and I81P, which fail to induce G2 arrest, can bind to and increase the kinase activity of Wee1 to the same extent as wild-type Vpr. Therefore, we conclude that the binding of Vpr to Wee1 is not sufficient for Vpr to activate the G2 checkpoint, and it may reflect an independent function of Vpr.

Transgenic overexpression of hexose-6-phosphate dehydrogenase in adipose tissue causes local glucocorticoid amplification and lipolysis in male mice

The prereceptor activation of glucocorticoid production in adipose tissue by NADPH-dependent 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) has emerged as a potential mechanism in the pathogenesis of visceral obesity and metabolic syndrome. Hexose-6-phosphate dehydrogenase (H6PDH) is an endoplasmic reticulum lumen-resident enzyme that generates cofactor NADPH and thus mediates 11β-HSD1 activity. To determine the role of adipose H6PDH in the prereceptor modulation of 11β-HSD1 and metabolic phenotypes, we generated a transgenic (Tg) mouse model overexpressing H6PDH under the control of the enhancer-promoter region of the adipocyte fatty acid-binding protein (aP2) gene (aP2/H6PDH Tg mice). Transgenic aP2/H6PDH mice exhibited relatively high expression of H6PDH and elevated corticosterone production with induction of 11β-HSD1 activity in adipose tissue. This increase in corticosterone production in aP2-H6PDH Tg mice resulted in mild abdominal fat accumulation with induction of C/EBP mRNA expression and slight weight gain. Transgenic aP2/H6PDH mice also exhibited fasting hyperglycemia and glucose intolerance with insulin resistance. In addition, the aP2/H6PDH Tg mice have elevated circulating free fatty acid levels with a concomitant increased adipose lipolytic action associated with elevated HSL mRNA and Ser(660) HSL phosphorylation within abdominal fat. These results suggest that increased H6PDH expression specifically in adipose tissue is sufficient to cause intra-adipose glucocorticoid production and adverse metabolic phenotypes. These findings suggest that the aP2/H6PDH Tg mice may provide a favorable model for studying the potential impact of H6PDH in the pathogenesis of human metabolic syndrome.

Purification of BAC DNA for high-efficiency transgenesis

An unresolved bottleneck in bacterial artificial chromosome (BAC) transgenesis is low efficiency generation of founder mice because of suboptimal quality of the manipulated BAC DNA. Using mini-gel electrophoresis and electro-elution that circumvents CsCl(2) centrifugation, column chromatography, and resin purifications, we have used RECOCHIP, a commercially available dialysis cassette for the purification of BAC DNA that generates transgenic founders with up to 80% efficiency.

CCR5 is a suppressor for cortical plasticity and hippocampal learning and memory

Although the role of CCR5 in immunity and in HIV infection has been studied widely, its role in neuronal plasticity, learning and memory is not understood. Here, we report that decreasing the function of CCR5 increases MAPK/CREB signaling, long-term potentiation (LTP), and hippocampus-dependent memory in mice, while neuronal CCR5 overexpression caused memory deficits. Decreasing CCR5 function in mouse barrel cortex also resulted in enhanced spike timing dependent plasticity and consequently, dramatically accelerated experience-dependent plasticity. These results suggest that CCR5 is a powerful suppressor for plasticity and memory, and CCR5 over-activation by viral proteins may contribute to HIV-associated cognitive deficits. Consistent with this hypothesis, the HIV V3 peptide caused LTP, signaling and memory deficits that were prevented by Ccr5 knockout or knockdown. Overall, our results demonstrate that CCR5 plays an important role in neuroplasticity, learning and memory, and indicate that CCR5 has a role in the cognitive deficits caused by HIV. DOI: http://dx.doi.org/10.7554/eLife.20985.001

HSF4 Mutation p.Arg116His Found in Age-Related Cataracts and in Normal Populations Produces Childhood Lamellar Cataract in Transgenic Mice

The p.Arg116His mutation in the heat shock transcription factor‐4 (HSF4) has been associated with age‐related cataracts, but it is also seen in 2% of the normal population, indicating either reduced penetrance or that the normal subjects were not old enough to express the phenotype. Based on the proximity of p.Arg116His to two known mutations in the DNA‐binding domain of HSF4, namely, p.Leu114Pro and p.Arg119Cys, which segregate with childhood lamellar cataract, we tested the possibility that this phenotype may have been missed by the ophthalmologist and/or that it did not spread to the visual axis so as to affect vision significantly. Here, we demonstrate via BAC (bacterial artificial chromosome) transgenesis that p.Arg116His recreates the childhood lamellar cataract in mice suggesting that incomplete penetrance associated with early cataracts may not be an absence but a limitation of the detection of the phenotype.